Dr Mallorie Hide

Dr Jonathan Hoffmann

Marie Gauthier

Dr Carole Chedid

Quang Huy

Mrs Thi Thuong Nguyen

Dr Phetsavanh Vu

Dr Inthalaphone Chanthavilay

Mr Johney Keovichit

Khamixay

Guérin Calmette

Bdq

/ Kan

Kanamycin Km

Kit

Paboriboune

Thi Van

Anh Nguyen

Anne-Laure Bañuls

High-dose isoniazid IDU Injection Drug Use INH/H Isoniazid 12 HIV Human immunodeficiency virus IQR Interquartile range

Keywords: fluoroquinolone, SLID, Second line injectable drug, Ofx = Ofloxacin

Epidémiologie moléculaire de Mycobacterium tuberculosis et sa résistance aux antibiotiques en RDP Lao RÉSUMÉ La tuberculose (TB) reste parmi les 10 premières causes de décès dans le monde l'émergence/réémergence de la TB résistante aux antituberculeux aggrave la situation et représente un défi majeur pour l'éradication de la TB. Le Laos est entouré par des pays fortement touchés par la TB et la TB multi-résistante (MDR) et cette maladie représente une priorité en termes de santé publique dans ce pays. Il n'existe encore aucune donnée sur la structure génétique et la résistance aux antibiotiques de la population de M. tuberculosis au Laos. Dans ce contexte, ce travail avait pour but d'analyser la diversité génétique et la structure des populations de M. tuberculosis ainsi que les déterminants génétiques associés à la résistance à partir d'échantillons collectés lors de l'enquête de prévalence nationale de la Tuberculose (TBPS) 2010-2011, l'enquête de résistance aux antituberculeux (DRS) 2016-2017 et chez les cas suspects de MDR-TB au Laos (2010-2014). Plusieurs techniques d'analyses ont été utilisées, comprenant les tests de sensibilité aux médicaments (phénotypique et génotypique), le séquençage et le génotypage par spoligotypage et MIRU-VNTR. Les données ont été analysées par des méthodes statistiques et phylogénétiques. Premièrement, ce travail s'est focalisé sur la diversité des familles de M. tuberculosis circulant au Laos. Les familles EAI et Beijing (76.7% et 14.4% respectivement) ont été principalement observées dans les échantillons de TBPS, alors que la famille Beijing était plus fréquente dans les échantillons de DRS et chez les patients suspectés de MDR-TB (34.9% et 41.0% respectivement). La transmission récente était non-négligeable avec un taux de « clustering » global de 11.9%, et des taux pour Beijing de 20.7 % et EAI de 11.0 %. Deuxièmement, les résultats ont révélé des profils de résistance très diverses allant de la monorésistance jusqu'à la pré-XDR (ultrarésistance). Les mutations associées aux profils de résistance ont montré une grande diversité, avec cependant certaines mutations majeures dans les gènes rpoB, katG, et rpsL. Le gène pncA a montré un pattern différent avec de la diversité Molecular epidemiology of Mycobacterium tuberculosis and antibiotic resistance in Lao PDR SUMMARY Tuberculosis (TB) is still one of the top 10 leading causes of death worldwide; the emergence/re-emergence of drug resistant TB aggravates the situation globally and challenges the prospect of ending TB by 2035. Lao PDR is surrounded by TB and MDR-TB high burden countries and TB continues to be one of the priority infection diseases in this country. The prevalence of TB in 2010 was almost twice as high than previous estimates and little is known about drug resistance. Up to now, M. tuberculosis population data regarding drug resistance and genetic structure are totally absent. In this context, we aimed to study the diversity and the structure of M. tuberculosis population and the genetic determinants associated to drug resistance using clinical samples collected from the TB prevalence survey (TBPS), 2010-2011; from the Drug resistance survey (DRS), 2016-2017 and from presumptive MDR-TB cases in Lao PDR (2010-2014). Various methods and analyses were used, including drug susceptibility testing (phenotypic and genotypic), DNA sequencing and genotyping of M. tuberculosis using spoligotyping and MIRU-VNTR. The data were analyzed by statistical and phylogenetic analyses.

Firstly, this work was focused on the diversity of M. tuberculosis families circulating in Lao PDR.

According to the result form TBPS, EAI and Beijing family (76.7% and 14.4% respectively) were mainly observed, while Beijing family was more observed in DRS, and presumptive MDR-TB cases (34.9% and 41.0% respectively). The level of recent transmission in Lao PDR was nonnegligible with a global clustering rate of 11.9% and in Beijing and EAI of 20.7% and 11.0%, respectively. Secondly, the results demonstrated the diversity of drug resistant patterns from mono-resistance to pre-extensively drug resistance (pre-XDR). A high diversity of mutations associated with drug resistance was also observed, however common mutations were mainly found (e.g: mutations in rpoB gene, katG and rpsL). The pattern was different for pncA gene, we observed a diversity of mutations without preeminent ones. Besides the number of known and unknown mutations associated with anti-TB drug resistance, deletion and insertion of bases First and foremost, I would like to extend my sincere gratitude to my advisor Dr. Anne-Laure Bañuls for introducing me to this exciting field of research and for her dedicated help, advice, inspiration, encouragement and continuous heart-warming support, throughout my PhD.

sans mutations prééminentes. En plus des mutations détectées, des délétions et insertions de bases ont été également observées. Le séquençage a montré son utilité pour la détection de la résistance aux antibiotiques dans les trois échantillons à l'étude. Enfin, la famille Beijing, famille la plus problématique au niveau mondial en termes de résistance et de transmissibilité, a été identifiée de manière significative dans le groupe de patients <35 ans, principalement dans les provinces du Nord, dans les cas de transmissions récentes et chez les isolats très résistants.

Tous ces points suggèrent un risque d'émergence de la MDR-TB accrue au Laos dû à la famille Beijing.

En conclusion, cette étude permet d'avoir pour la première fois un aperçu de la structure des populations de M. tuberculosis au Laos. Les résultats soulignent le risque d'augmentation du nombre de cas infectés par la famille Beijing et donc des cas de résistance. Pour empêcher une dégradation de la situation, il est essentiel d'améliorer les stratégies pour le dépistage des résistances et de développer des tests moléculaires capables de couvrir un large nombre de mutations qui soit simple à implémenter dans les pays à ressources limités. Les résultats de ce travail serviront de base en termes de famille/sous-famille/génotype et de mutations associées à la résistance au Laos. Ces données pourront être comparées avec de futures études/analyses pour étudier l'évolution de la TB et de la TB résistante et ainsi d'évaluer l'efficacité des politiques de contrôle mises en place. La description des mutations associées aux résistances est utilisée pour créer une base de données régionale en collaboration avec le Vietnam et le Cambodge pour développer un outil de diagnostic basé sur la technologie des puces à ADN pour améliorer la détection de la résistance dans la région.

Les mots clés : Epidémiologie moléculaire, Mycobacterium tuberculosis, Résistance aux antibiotiques, RDP Lao, Asie du Sud-est, Déterminant génétique, Beijing, EAI, transmission récente, Séquençage de gènes ciblés.

were also observed. The sequencing showed its usefulness for drug resistance detection. Lastly, Beijing family, which is the more problematic family in the world in terms of resistance and transmissibility, was observed on a significant manner in young age group, mainly in the northern provinces, in recent transmission cases and among highly drug resistant isolates, suggesting an increasing risk of highly drug resistance TB due to highly transmissible Beijing strains in Lao PDR.

In conclusion, this study provides the first genetic insights into the M. tuberculosis population in Lao PDR. The results underline the risk of increase of Beijing and drug resistant TB in the country. In order to prevent a more serious situation in the future regarding drug resistance as observed in neighboring countries, there is an urgent need of effective strategy improvement for drug resistance screening and the development of rapid molecular tests that cover a large number of drug resistance simultaneously with a feasible implementation in the limited resource countries. The results of genotyping from our study will be the baseline of families/subfamilies/genotype of M. tuberculosis population and of the mutations associated with drug resistance in Lao PDR. These data will be compared with further study/analysis to evaluate the trend of TB and drug resistant TB in the country and to determine if the drug resistance is under control after the set-up of new policies. The data of drug resistance associated mutations are used to build a regional database in collaboration with Vietnam and Cambodia in order to develop a diagnostic tool based on DNA chip technology to improve the drug resistance detection in the region.

Key words: Molecular epidemiology, Mycobacterium tuberculosis, Antibiotic resistance, Lao PDR, Southeast Asia, Genetic determinant, Beijing, EAI, Recent transmission, Target genes sequencing.

Laboratory:

1. UMR MIVEGEC (224 IRD-5290 CNRS Université de Montpellier), Montpellier, France 2. Centre d'Infectiologie Lao-Christophe Mérieux, Ministère de la santé, Vientiane, RDP Lao 3. Laboratoire des Pathogènes Emergents, Fondation Mérieux, Lyon, France and appreciation to all those who made this PhD thesis possible. And my special regards to the "Allocations de Recherche pour une Thèse au Sud (ARTS) -IRD-Fondation Mérieux program" for the fully funded PhD studentship, the JEAI "Mycobaterium tuberculosis in Southeast Asia (MySA) and the LMI "Drug Resistance in South East Asia" (DRISA) projects and the Young Scientist grand from GABRIEL network and Fondation Merieux for the research funding support.

I would also like to acknowledge Université de Montpellier, CBS2 doctoral school for their kind support and their assistance during my PhD study. And I would like to thank also Professeurs of Montpellier University and the thesis committee for their insightful comments and encouragement My deepest thanks is reserved for my brothers, sisters, and the whole family of SOMPHAVONG and IEM, as well as my uncle and auntie for their heart-warming support and encouragement.

I express my heart-felt thanks to my husband (Vibol) who was a great co-worker for professional life and a moral supporter for personal life. And to my son (Ryan) who was coming at the right time to share the greatest moment of my life.

My last heart-felt thanks go to my beloved parents (Phor Khampheuane and Mae Phan Somphavong) for always trusting me, for giving me all your love and inspiration. List of Tables TABLE 1.

List of Figures

Introduction and objectives

Tuberculosis (TB) remains a public health problem worldwide and the ninth leading cause of death due to a single agent, ranking above human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) [START_REF]Global tuberculosis report[END_REF]. In 2016, the estimation of TB incidence was 10.4 million, 56 % of the incidence were in five countries located in Asia: India, Indonesia, China, the Philippines and Pakistan. The incidence of multidrug-resistant TB (MDR-TB, resistant to at least both isoniazid (INH) and rifampicin (RIF)), rifampicin resistant TB (RR-TB) and extensively drugresistant TB (XDR-TB, MDR plus resistance to at least one fluoroquinolone (FQ) and one second line injectable drug (SLID)) is continuously increasing and is defined as a major threat to TB control. Moreover, only one-fourth of the 600,000 incident MDR-TB/RR-TB cases were

detected in 2016 and the successful rate of treatment in MDR/RR-TB patients was 54 % and only 30 % in XDR-TB patients [START_REF]Global tuberculosis report[END_REF].

Despite the huge problem of public health that TB represents in the world, in many countries and especially in low-income countries such as in Lao people's democratic republic (Lao PDR) still little is known in terms of epidemiology of TB and drug resistance. This country is not notified as high TB burden countries but it is a landlocked country in Southeast Asia, surrounded by five of the 30 high TB burden countries in the world (China, Myanmar, Cambodia, Vietnam and Thailand) [START_REF]Global tuberculosis report[END_REF]. Despite the establishment in 1995 of the Directly Observed Treatment Short-Course (DOTS), many people have still no access to quality TB diagnosis and treatment services and many cases remain undiagnosed [START_REF] Law | The first national tuberculosis prevalence survey of Lao PDR (2010-2011)[END_REF]. The first TB prevalence survey (TBPS) in 2010-2011 showed that the prevalence of TB in Lao PDR is two times higher than the WHO estimates [START_REF] Law | The first national tuberculosis prevalence survey of Lao PDR (2010-2011)[END_REF]. And after the first national TBPS, WHO re-estimated the prevalence of all TB forms at 540/100,000 populations [START_REF] Law | The first national tuberculosis prevalence survey of Lao PDR (2010-2011)[END_REF][START_REF]Global tuberculosis report[END_REF]. Regarding drug resistant TB still little is known in this country. The conventional culture-based drug susceptibility testing (DST) is available only at National Reference laboratory (NRL). Besides, the use of available molecular tests for detection of first line drug (FLD) and second line drug (SLD) resistance is limited that leads to a lack of knowledge concerning the genetic determinants linked to drug resistance in this country. Only data of resistance to INH and RIF was explored by a muticentricstudy in three regional hospitals [START_REF] Iem | Resistance of Mycobacterium tuberculosis to antibiotics in Lao PDR: first multicentric study conducted in 3 hospitals[END_REF]. The resistance to the other FLDs (ethambutol (EMB), streptomycin (STR) and pyrazinamide (PZA)) has not been screened; the extent threat of the resistance and the associated genetic determinants to these drugs cannot be evaluated.

Nevertheless, it is essential to detect the resistance and the associated mechanisms for both FLD and SLD in order to better know the processes of drug resistance emergence and spread.

In addition, the analysis of specific genomic regions of M. tuberculosis known to be associated with anti-TB drug resistance is more and more considered as valuable tool for drug resistance detection, surveillance, providing new opportunities to monitor drug resistance in TB in resource-poor countries [START_REF] Zignol | Genetic sequencing for surveillance of drug resistance in tuberculosis in highly endemic countries: a multi-country population-based surveillance study[END_REF].

In addition, data on population genetic structure are totally absent in Lao PDR, although they are essential to evaluate the risk of highly drug resistance emergence, such as XDR-TB, and its spread in the population. As example, significant associations were frequently observed between M. tuberculosis genotypes and drug resistance [START_REF] Ramazanzadeh | Prevalence of Beijing family in Mycobacterium tuberculosis in world population: Systematic Review and Meta-Analysis[END_REF]. More specifically, Beijing family is associated with epidemics and drug resistance in many countries all over the world [START_REF] Ramazanzadeh | Prevalence of Beijing family in Mycobacterium tuberculosis in world population: Systematic Review and Meta-Analysis[END_REF][START_REF] Dou | Molecular epidemiology and evolutionary genetics of Mycobacterium tuberculosisin Taipei[END_REF][START_REF] Faksri | Epidemiological trends and clinical comparisons of Mycobacterium tuberculosis lineages in Thai TB meningitis[END_REF][START_REF] Anh | Mycobacterium tuberculosis Beijing genotype emerging in Vietnam[END_REF][START_REF] Phyu | Predominance of Mycobacterium tuberculosis EAI and Beijing Lineages in Yangon, Myanmar[END_REF]. Up to now, despite the numerous studies carried out in neighbouring countries like Vietnam, China, Thailand and Myanmar [START_REF] Dou | Molecular epidemiology and evolutionary genetics of Mycobacterium tuberculosisin Taipei[END_REF][START_REF] Faksri | Epidemiological trends and clinical comparisons of Mycobacterium tuberculosis lineages in Thai TB meningitis[END_REF][START_REF] Anh | Mycobacterium tuberculosis Beijing genotype emerging in Vietnam[END_REF][START_REF] Phyu | Predominance of Mycobacterium tuberculosis EAI and Beijing Lineages in Yangon, Myanmar[END_REF][START_REF] Nguyen | High Prevalence of Beijing and EAI4-VNM Genotypes among M. tuberculosis Isolates in Northern Vietnam: Sampling Effect, Rural and Urban Disparities[END_REF][START_REF] Dong | Spoligotypes of Mycobacterium tuberculosis from Different Provinces of China[END_REF], no information is available yet in Lao PDR.

In this context and in order to acquire the first insight on genetic basis of M. tuberculosis

Global tuberculosis disease burden

Tuberculosis (TB) is an ancient disease, caused by bacteria called "Mycobacterium tuberculosis". It has affected humankind throughout known history and human prehistory [START_REF] Daniel | The history of tuberculosis[END_REF].

TB has surged in great epidemics and continues to be a very significant global health problem.

TB occurs in every part of the world, in 2016, the largest number of new TB cases occurred in South-East Asia and Western Pacific regions, with 62% of new cases, followed by the African region, with 25% of new cases [START_REF]Global tuberculosis report[END_REF]. The Figure 1.1 shows the TB incidence rates in 2016. Most of the estimated number of incident cases in 2016 occurred in the WHO South-East Asia Region (45%), the WHO African Region (25%) and the WHO Western Pacific Region (17%); smaller proportions of cases occurred in the WHO Eastern Mediterranean Region (7%), the WHO European Region (3%) and the WHO Region of the Americas (3%) [START_REF]Global tuberculosis report[END_REF].

Globally, the TB mortality rate is falling at about 3% per year and TB incidence is falling at about 2% per year; this needs to improve to 4-5% per year by 2020 to reach the first milestones of the End TB Strategy [START_REF]Global tuberculosis report[END_REF]. Regionally, the fastest declines in the TB mortality rate are in the WHO European Region and the WHO Western Pacific Region (6.0% and 4.6% per year, respectively, since 2010) [START_REF]Global tuberculosis report[END_REF]. High TB burden countries with rates of decline exceeding 6% per year since 2010 include Ethiopia, the Russian Federation, the United Republic of Tanzania, Viet Nam and Zimbabwe [START_REF]Global tuberculosis report[END_REF]. And the fastest decline in TB incidence is in the WHO European Region (4.6 % from 2015 to 2016). The decline since 2010 has exceeded 4% per year in several high TB burden countries, including Ethiopia, Kenya, Lesotho, Namibia, the Russian Federation, the United Republic of Tanzania, Zambia and Zimbabwe [START_REF]Global tuberculosis report[END_REF]. Nevertheless, despite the decline in TB incidence and TB deaths, alongside HIV, it remains a top of cause of deaths from an infectious disease [START_REF]Global tuberculosis report[END_REF].

Global situation of drug-resistant tuberculosis

Drug-resistant TB threatens global TB care and prevention, and remains a major public health concern in many countries. Three major categories are used for global surveillance and treatment MDR-TB, RR-TB and XDR-TB [START_REF]Global tuberculosis report[END_REF]. Globally in 2016, an estimated There were an estimated 600,000 (range, 540,000-660,000) incident cases of MDR/RR-TB in 2016, with cases of MDR-TB accounting for 82% (490,000) of the total [START_REF]Global tuberculosis report[END_REF]. There were about 240,000 (range, 140,000-340,000) deaths from MDR/RR-TB in 2016 [START_REF]Global tuberculosis report[END_REF].

Despite the increase in testing, the number of MDR/RR-TB cases detected in 2016 only reached 153,000. In 2016, 8,000 patients with XDR-TB were reported worldwide, 123

countries have reported at least one XDR-TB case. On average, an estimated 6.2% of people with MDR-TB have XDR-TB [START_REF]Global tuberculosis report[END_REF] . In 2016, 130,000 patients were enrolled on MDR-TB treatment, equivalent to about 22% of the 600,000 incident MDR/RR-TB cases that year.

Enrolments have increased over time and in several countries, the gap between detecting MDR/RR-TB cases and starting them on treatment has narrowed. In 2016, 8,500 patients with XDR-TB were enrolled in treatment, with 17% increase over 2015. However, the treatment success in MDR/RR-TB and XDR-TB patients were only 54 % and 30%

respectively [START_REF]Global tuberculosis report[END_REF].

Beside the MDR/RR-TB and XDR-TB, drug resistance surveys have shown that mono-and poly-resistant TB (drug-resistant TB other than MDR-TB) are actually more common than MDR-TB. The global prevalence of MDR-TB in new cases is around 3% while the prevalence of mono-and poly-resistant strains is almost 17% [START_REF] Wright | Anti-tuberculosis drug resistance in the world: fourth global report: the World Health Organization/International Union Against Tuberculosis and Lung Disease (WHO/UNION) Global Project on Anti-Tuberculosis Drug Resistance Surveillance, 2002-2007[END_REF]. Many of these cases contribute to the increase of resistance and, eventually, can lead to MDR if they are not properly managed [START_REF]WHO | Guidelines for the programmatic management of DR-TB. Companion Handbook to the WHO Guidelines for the Programmatic Management of Drug-Resistant Tuberculosis[END_REF]. The forms, mono-resistant and poly-resistant TB, often remain undiagnosed in resource-limited settings because they are not considered as priority.

However, the risk of failure or relapse is well described [START_REF]WHO | Guidelines for the programmatic management of DR-TB. Companion Handbook to the WHO Guidelines for the Programmatic Management of Drug-Resistant Tuberculosis[END_REF]. INH is one of the most two powerful anti-TB drugs, used in all six months long for standard treatment regimen, used in some MDR-TB regimen and used as preventive therapy for people living with HIV [START_REF]WHO | Guidelines for the programmatic management of DR-TB. Companion Handbook to the WHO Guidelines for the Programmatic Management of Drug-Resistant Tuberculosis[END_REF][START_REF] Care | Guidelines for Treatment of Drugsusceptible Tuberculosis and Patient Care[END_REF][START_REF] Malhotra | World Health Organization, Department of HIV/AIDS, Stop TB Initiative (World Health Organization). Guidelines for intensified tuberculosis case-finding and isoniazid preventative therapy for people living with HIV in resource-constrained settings[END_REF].

However, INH mono resistance is the most common form of mono resistance, with estimated prevalence ranges between 0 to.-9.5% (0-12.8% among new cases and 0-30.8% among retreated cases) [START_REF] Wright | Anti-tuberculosis drug resistance in the world: fourth global report: the World Health Organization/International Union Against Tuberculosis and Lung Disease (WHO/UNION) Global Project on Anti-Tuberculosis Drug Resistance Surveillance, 2002-2007[END_REF].

Through evidences that relied on simulations from modeling work, performing DST in all patients before treatment using a rapid test that detects resistance to INH and RIF would be the most cost-effective strategy for averting deaths and preventing acquired MDR-TB [START_REF] Oxlade | The impact and cost-effectiveness of strategies to detect drug-resistant tuberculosis[END_REF].

Performing rapid DST to INH and RIF at the start of treatment would help identify many more cases of mono-and poly-resistant TB. Clinicians should therefore expect to see more cases of mono-and poly-resistant TB in the future as rapid drug resistance diagnostic becomes more commonly used.

Diagnosis of tuberculosis and drug resistance

The diagnosis of TB still relies primarily on the identification of Acid-Fast Bacilli (AFB) in sputum smears using a conventional light microscope in high burden countries [START_REF] Parsons | Laboratory Diagnosis of Tuberculosis in Resource-Poor Countries: Challenges and Opportunities[END_REF]. The sputum specimens are smeared directly onto the slides (direct smears) and subjected to Ziehl-Neelsen (ZN) staining. This method, first developed in the 1880s and basically unchanged today, has the advantage of being simple, but is hampered by very low sensitivity. It may only detect half of all cases with active infection [START_REF] Isenberg | Clinical Microbiology Procedures Handbook[END_REF] and its usefulness is questionable for patients with reduced pulmonary cavity formation or reduced sputum bacillary load, such as children and HIVcoinfected patients. Moreover, this method cannot distinguish between drug-susceptible and drug-resistant M. tuberculosis or between different species of mycobacteria such as non-tuberculous mycobacteria (NTM). Currently, WHO recommends the use of fluorescent lightemitting diode (LED) microscopy as alternative technique because it is more sensitive (10%) than ZN method [START_REF]Implementing tuberculosis diagnostics: policy framework[END_REF]. Nevertheless, the use of LED microscopy is limited because of its high cost. Therefore only 7% of TB centers worldwide used this technology in 2014. Now a day, several methods can be used for determining the AFB, either culture based methods or Non culture based methods.

The culture-based method remains the gold standard for both diagnosis and drug sensitivity testing (DST). The detection rate often increases of 30-50% compared to microscopy [START_REF]Implementing tuberculosis diagnostics: policy framework[END_REF]. However, this method is more complex and expensive than microscopy, time consuming (4-8 weeks by solid culture) and requires strict biosafety measures [START_REF]Implementing tuberculosis diagnostics: policy framework[END_REF]23,[START_REF]WHO | Guidelines for surveillance of DR in TB -5th edit. WHO | Guidelines for surveillance of drug resistance in tuberculosis -5th edition[END_REF]. Besides, liquid culture media is a more sensitive and faster culture system but does not permit to identify contamination by other bacteria [START_REF]Implementing tuberculosis diagnostics: policy framework[END_REF]23,[START_REF]WHO | Guidelines for surveillance of DR in TB -5th edit. WHO | Guidelines for surveillance of drug resistance in tuberculosis -5th edition[END_REF]. The reliability of DST varies with the anti-TB drugs.

DST is more accurate in detecting susceptibility to INH, RIF, FQ and SLIDs, but results are less reliable and reproducible for EMB, STR, PZA and for drugs of groups 4 and 5 [START_REF]Implementing tuberculosis diagnostics: policy framework[END_REF][START_REF]WHO | Guidelines for surveillance of DR in TB -5th edit. WHO | Guidelines for surveillance of drug resistance in tuberculosis -5th edition[END_REF][START_REF] Ängeby | Challenging a dogma: antimicrobial susceptibility testing breakpoints for Mycobacterium tuberculosis[END_REF].

The development of molecular methods showed considerable advantages in M.

tuberculosis and drug resistance detection. In order to rapid first-step identification of RR-TB, and MDR-TB, the two main molecular tools endorsed by WHO in 2008 and 2010 are Line Probe Assays (LPA) and Xpert MTB/RIF respectively [START_REF]molecular line probe assays for rapid screening of patients at risk of multidrug-resistant tuberculosis[END_REF][START_REF]WHO-Xpert mtb/rif implementation manual. Xpert mtb/rif implementation manual: technical and operational "how-to[END_REF]. LPA allows rapid detection of M.

tuberculosis and RIF resistance alone (INNOLiPA®Rif.TB assay, Innogenetics, Ghent, Belgium) or in combination with INH (GenoType® MTBDR assay, Hain Lifescience, Nehren, Germany) within 24 hours [START_REF]Implementing tuberculosis diagnostics: policy framework[END_REF]. LPA is suitable for both AFB smear-positive sputum specimens and M.

tuberculosis isolates grown by conventional culture method. This test showed high sensitivity for detection of RIF resistance (over 94%), but is less sensitive for the detection of INH resistance (approximately 85%) [START_REF]Implementing tuberculosis diagnostics: policy framework[END_REF][START_REF] Tomasicchio | The diagnostic accuracy of the MTBDRplus and MTBDRsl assays for drug-resistant TB detection when performed on sputum and culture isolates[END_REF]. Therefore, it may underestimate the number of MDR cases [START_REF]Implementing tuberculosis diagnostics: policy framework[END_REF].

The Xpert MTB/RIF assay allows identifying M. tuberculosis complex and the RIF resistance directly from sputum specimens in less than two hours [START_REF]Implementing tuberculosis diagnostics: policy framework[END_REF][START_REF]WHO-Xpert mtb/rif implementation manual. Xpert mtb/rif implementation manual: technical and operational "how-to[END_REF]. The assays had similar sensitivity, specificity and accuracy as culture on solid media and this tool has been recommended by WHO as initial diagnostic test for persons with a risk of MDR-TB and HIV.

However, the tests for detection of RIF resistance alone cannot accurately predict RIF resistance and MDR-TB since about 10% of RIF resistant isolates were sensitive to INH [START_REF]Implementing tuberculosis diagnostics: policy framework[END_REF].

Besides, despite the overall high sensitivity (99%), Xpert MTB/RIF sensitivity was 60-88% compared with liquid culture and false results have been reported [START_REF] Sanchez-Padilla | Detection of Drug-Resistant Tuberculosis by Xpert MTB/RIF in Swaziland[END_REF]. Recently, a new version called Xpert Ultra showed similar performance as liquid culture and is able to detect M.

tuberculosis in specimens with low numbers of bacilli, especially in smear-negative, culturepositive specimens (such as those from persons with HIV co-infection), in pediatric specimens and in extra-pulmonary specimens (notably cerebrospinal fluid). The WHO issued a recommendation in which Ultra can be used as an alternative to the existing Xpert MTB/RIF test in all settings [START_REF]WHO Meeting Report of a Technical Expert Consultation[END_REF] For the rapid identification of resistance to SLDs or XDR-TB, MTBDRsl assay version 1.0 was developed in 2009, followed by the MTBDRsl version 2.0 in 2015. The assays detect the mutations associated with FQs and SLID resistance. Once a diagnosis of RR-TB or MDR-TB has been established, MTBDRsl can be used to detect additional resistances to SLDs [START_REF]WHO | The use of molecular line probe assay for the detection of resistance to second-line anti-tuberculosis drugs[END_REF].

However, the moderate sensitivity (69%) for XDR-TB detection leads to an underestimation of XDR-TB cases [START_REF]Global tuberculosis report[END_REF]. The accuracy of MTBDRsl by indirect testing for the detection of FQ resistance in patients with RIF-resistant or MDR-TB was 86% of sensitivity and 99% of specificity [START_REF]WHO | The use of molecular line probe assay for the detection of resistance to second-line anti-tuberculosis drugs[END_REF]. For detection of SLID resistance by indirect testing, this test showed 77% of sensitivity and 99% of specificity [START_REF]WHO | The use of molecular line probe assay for the detection of resistance to second-line anti-tuberculosis drugs[END_REF]. Therefore, the results obtained by MTBDRsl may be used as initial test for detection of SLD resistance but cannot be used to properly guide the choice of SLIDs for the MDR-TB treatment [START_REF]Implementing tuberculosis diagnostics: policy framework[END_REF][START_REF]WHO | Guidelines for surveillance of DR in TB -5th edit. WHO | Guidelines for surveillance of drug resistance in tuberculosis -5th edition[END_REF][START_REF]WHO | The use of molecular line probe assay for the detection of resistance to second-line anti-tuberculosis drugs[END_REF].

Nowadays, many studies in different settings showed the potential use of whole genome sequencing (WGS) for getting rapid and full drug resistant-TB pattern [START_REF] Chaidir | Use of whole genome sequencing to predict Mycobacterium tuberculosis drug resistance in Indonesia[END_REF][START_REF] Aung | Whole-genome sequencing of multidrug-resistant Mycobacterium tuberculosis isolates from Myanmar[END_REF][START_REF] Ssengooba | Whole genome sequencing to complement tuberculosis drug resistance surveys in Uganda[END_REF][START_REF] Ruesen | Linking minimum inhibitory concentrations to whole genome sequence-predicted drug resistance in Mycobacterium tuberculosis strains from Romania[END_REF][START_REF] Witney | Clinical Application of Whole-Genome Sequencing To Inform Treatment for Multidrug-Resistant Tuberculosis Cases[END_REF]. Furthermore, WHO conducted a multi-country population-based surveillance study of drug resistance in TB in highly endemic countries, using sequencing (either through WGS or targeted gene sequencing)

and the study demonstrated that sequencing can be a valuable tool for surveillance of drug resistance, providing new opportunities to monitor drug resistance in TB in resource-poor countries. [START_REF] Zignol | Genetic sequencing for surveillance of drug resistance in tuberculosis in highly endemic countries: a multi-country population-based surveillance study[END_REF] Overall, the TB case detection remains low. In 2016, only 6.6 million (63%) out of the 10.4 million estimated TB cases by WHO were reported [START_REF]Global tuberculosis report[END_REF]. Furthermore, only 59 % of estimated MDR/RR-TB were notified in 2016, and still only 16% of new TB cases and 60% of previously treated TB cases were examined for drug susceptibility [START_REF]Global tuberculosis report[END_REF]. As a result, many TB patients with undetected MDR were not potentially correctly treated, leading to treatment failure and an increased risk of MDR transmission in the community. Moreover, among the 1,694,000 MDR-TB patients enrolled in MDR-TB treatment according to WHO standards, only 35.5%

received DST for both FQs and SLIDs [START_REF]Global tuberculosis report[END_REF] This suggests that many XDR-TB cases are never diagnosed. These data show the depth of the challenge for the management of MDR-TB and emergence of XDR-TB worldwide.

Characteristics and worldwide distribution of M. tuberculosis

A.

Characteristics of M. tuberculosis

On 24 March 1882, the German doctor Robert Koch discovered the microorganism responsible for the deadly pulmonary TB [START_REF] Cambau | Steps towards the discovery of Mycobacterium tuberculosis by Robert Koch, 1882[END_REF]. It was in 1883 that the TB agent was named Mycobacterium tuberculosis. Further molecular analysis of these first isolates confirmed the identification of M. tuberculosis and indicated that Koch's isolates belong to the "modern" lineage of M. tuberculosis [START_REF] Cambau | Steps towards the discovery of Mycobacterium tuberculosis by Robert Koch, 1882[END_REF]. This bacteria belongs to the slow-growing bacterial group, characterized by one division every 18-24 hours [START_REF] Gengenbacher | Mycobacterium tuberculosis: Success through dormancy[END_REF]. Consequently, the growth on Löwenstein-Jensen (LJ) medium requires at least 3-4 weeks [START_REF] Gengenbacher | Mycobacterium tuberculosis: Success through dormancy[END_REF]. Faster results can be obtained using solid Middlebrook medium with growth supplement (OADC) or liquid medium (BACTEC) [START_REF] Tortoli | Use of BACTEC MGIT 960 for Recovery of Mycobacteria from Clinical Specimens: Multicenter Study[END_REF][START_REF] Somoskövi | Comparison of the Mycobacteria Growth Indicator Tube with MB Redox, Löwenstein-Jensen, and Middlebrook 7H11 Media for Recovery of Mycobacteria in Clinical Specimens[END_REF][START_REF] Naveen | Comparison of the Lowenstein-Jensen Medium, the Middlebrook 7H10 Medium and MB/BacT for the Isolation of Mycobacterium Tuberculosis (MTB) from Clinical Specimens[END_REF][START_REF] Adler | Comparison of BacT/ALERT 3D, Lowenstein-Jensen medium and Middlebrook 7H10/7H11 biplate for recovering mycobacteria from clinical specimens[END_REF]. M.

tuberculosis is non-pigmented, rough, dry colonies and forms a cord-like structure on LJ Nevertheless, the membrane characteristics do not correspond to Gram-positive ones. Indeed, the bacteria does not retain the crystal violet dye as expected for Gram-positive bacteria, sometimes resulting in "ghost" appearance after washing with alcohol or acetone. M.

tuberculosis has a specialized cell wall complex, which consists of four major components, mycoside, mycolic acids, arabinogalactan and peptidoglycan. Mycolic acids, the major lipids of the cell wall of mycobacteria in general, are major components of the outer permeability barrier and are responsible for the "acid-fastness" of this group of microorganisms [START_REF] Kaur | Biogenesis of the cell wall and other glycoconjugates of Mycobacterium tuberculosis[END_REF]. Furthermore, the fatty acids are linked with carbohydrate components that form a unique envelope which inhibits phagolysosome fusion [START_REF] Gengenbacher | Mycobacterium tuberculosis: Success through dormancy[END_REF]. Like many other bacteria, M. tuberculosis does not form spores but has the capacity to become dormant, a non-replicating state characterized by low metabolic activity and prolonged persistence [START_REF] Gengenbacher | Mycobacterium tuberculosis: Success through dormancy[END_REF]. [START_REF] Cole | Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence[END_REF]. Recently, thanks to the progresses in whole genome sequencing (WGS) technology, the sequences of many genomes have been determined covering all types of drug resistance from pan-drug sensitive to MDR and XDR strains [START_REF] Walker | Whole-genome sequencing to delineate Mycobacterium tuberculosis outbreaks: a retrospective observational study[END_REF][START_REF] Fleischmann | Whole-Genome Comparison of Mycobacterium tuberculosis Clinical and Laboratory Strains[END_REF][START_REF] Merker | Whole Genome Sequencing Reveals Complex Evolution Patterns of Multidrug-Resistant Mycobacterium tuberculosis Beijing Strains in Patients[END_REF][START_REF] Ilina | Comparative Genomic Analysis of Mycobacterium tuberculosis Drug Resistant Strains from Russia[END_REF][START_REF] Casali | Microevolution of extensively drug-resistant tuberculosis in Russia[END_REF] . M. tuberculosis is described as a clonal bacteria and the genome is highly conserved [START_REF] Cole | Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence[END_REF][START_REF] Coscolla | Consequences of genomic diversity in Mycobacterium tuberculosis[END_REF][START_REF] Vultos | Evolution and Diversity of Clonal Bacteria: The Paradigm of Mycobacterium tuberculosis[END_REF]. The pathogen is characterized by a low mutation rate about 10 -9 mutation/bacterium/cell division. The genome evolutionary rate is very low, estimated between 0.4 -0.5 SNP/genome/year [START_REF] Walker | Whole-genome sequencing to delineate Mycobacterium tuberculosis outbreaks: a retrospective observational study[END_REF][START_REF] Roetzer | Whole Genome Sequencing versus Traditional Genotyping for Investigation of a Mycobacterium tuberculosis Outbreak: A Longitudinal Molecular Epidemiological Study[END_REF] [START_REF] Gagneux | Variable host-pathogen compatibility in Mycobacterium tuberculosis[END_REF][START_REF] Firdessa | Mycobacterial Lineages Causing Pulmonary and Extrapulmonary Tuberculosis, Ethiopia[END_REF][START_REF] Tessema | Molecular epidemiology and transmission dynamics of Mycobacterium tuberculosis in Northwest Ethiopia: new phylogenetic lineages found in Northwest Ethiopia[END_REF] . From several detailed phylogenetic analyses, the lineages 1, 5 and 6 were defined as ancient lineages with M. canetti as the more ancestral branch; lineages 2, 3, 4 as modern lineages and lineage 7

appears to be intermediate between the ancient and modern lineages [START_REF] Coscolla | Consequences of genomic diversity in Mycobacterium tuberculosis[END_REF][START_REF] Brosch | A new evolutionary scenario for the Mycobacterium tuberculosis complex[END_REF][START_REF] Comas | Out-of-Africa migration and Neolithic co-expansion of Mycobacterium tuberculosis with modern humans[END_REF][START_REF] Galagan | Genomic insights into tuberculosis[END_REF].

Detailed phylogenetic analyses suggested that during these migration events, the M.

tuberculosis lineages would have adapted to different human populations [START_REF] Coscolla | Consequences of genomic diversity in Mycobacterium tuberculosis[END_REF][START_REF] Comas | Out-of-Africa migration and Neolithic co-expansion of Mycobacterium tuberculosis with modern humans[END_REF]. Indeed, the lineages are strongly associated with geographical areas, their names reflecting the geographical origin of the M. tuberculosis population (Figure 1.6) [START_REF] Coscolla | Consequences of genomic diversity in Mycobacterium tuberculosis[END_REF] The ancient lineage 1 (Indo-Oceanic, mainly EAI family) is reported in East Africa, but also spread all around the Indian Ocean and is frequently reported in Southeastern and Southern Asia, accounting for over 33-73% of total cases [START_REF] Demay | SITVITWEB -A publicly available international multimarker database for studying Mycobacterium tuberculosis genetic diversity and molecular epidemiology[END_REF][START_REF] Brudey | Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology[END_REF]. This family is also prevalent in Northern Europe, Middle East and Central Asia, and in Oceania (22 -25% of total cases) [START_REF] Demay | SITVITWEB -A publicly available international multimarker database for studying Mycobacterium tuberculosis genetic diversity and molecular epidemiology[END_REF][START_REF] Brudey | Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology[END_REF] .

Lineage 2 (East-Asian, mainly Beijing family) is one of the most virulent M. tuberculosis lineages and is spreading all over the world [START_REF] Merker | Whole Genome Sequencing Reveals Complex Evolution Patterns of Multidrug-Resistant Mycobacterium tuberculosis Beijing Strains in Patients[END_REF][START_REF] Demay | SITVITWEB -A publicly available international multimarker database for studying Mycobacterium tuberculosis genetic diversity and molecular epidemiology[END_REF][START_REF] Brudey | Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology[END_REF] . More specifically, the Beijing family is predominant in East and South East Asia and in the countries of former Soviet Union, accounting for over 50 -85% of total cases. This family is also highly prevalent in Oceania, Africa (except the West) and in North America (more than 17% of total cases) [START_REF] Demay | SITVITWEB -A publicly available international multimarker database for studying Mycobacterium tuberculosis genetic diversity and molecular epidemiology[END_REF][START_REF] Brudey | Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology[END_REF].

However, this lineage is less detected in the other regions of the world, such as in Northern Europe, India, Central and South America, and in Middle East (less than 10% of total cases) [START_REF] Demay | SITVITWEB -A publicly available international multimarker database for studying Mycobacterium tuberculosis genetic diversity and molecular epidemiology[END_REF][START_REF] Brudey | Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology[END_REF]. The Beijing family was found at very high frequency with more than 85% in the Beijing region of China [START_REF] Qian | Retrospective Analysis of the Beijing Family of Mycobacterium tuberculosis in Preserved Lung Tissues[END_REF]. This family is also found in high proportion in Mongolia, South Korea, Hong Kong, Taiwan, Vietnam, Thailand, and Malaysia [START_REF] Demay | SITVITWEB -A publicly available international multimarker database for studying Mycobacterium tuberculosis genetic diversity and molecular epidemiology[END_REF][START_REF] Brudey | Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology[END_REF][START_REF] Van Soolingen | Predominance of a single genotype of Mycobacterium tuberculosis in countries of east Asia[END_REF]. Overall, this lineage accounted for 13% of all M. tuberculosis lineages [START_REF] Brudey | Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology[END_REF].

The lineage 3 (East-African-Indian, mainly CAS family) is essentially localized in the Southern and Western Asia (9-30% of total cases, mainly in the South). This lineage is also found in the Eastern and Northern Africa (7 -12% of total cases, mainly in the East). In several other regions including Central and North America, Europe, Far-East-Asia, and Oceania, this lineage is less frequent (0.1 -5% of total cases). This lineage is predominant in India, Iran, and Pakistan, accounting for over 50% of total cases [START_REF] Demay | SITVITWEB -A publicly available international multimarker database for studying Mycobacterium tuberculosis genetic diversity and molecular epidemiology[END_REF][START_REF] Brudey | Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology[END_REF].

The lineage 4 (Euro-American) consists of 10 different families, in which 5 main families LAM, T, X, H and S that are widespread throughout the world [START_REF] Demay | SITVITWEB -A publicly available international multimarker database for studying Mycobacterium tuberculosis genetic diversity and molecular epidemiology[END_REF][START_REF] Brudey | Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology[END_REF]. Molecular epidemiology data showed that this lineage is the most frequent in Europe and Americas, but is also dominant in North Africa, Middle East and Oceania [START_REF] Comas | Out-of-Africa migration and Neolithic co-expansion of Mycobacterium tuberculosis with modern humans[END_REF][START_REF] Demay | SITVITWEB -A publicly available international multimarker database for studying Mycobacterium tuberculosis genetic diversity and molecular epidemiology[END_REF][START_REF] Brudey | Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology[END_REF]. The distribution of these specific families varies according to the regions. The T family was found in all continents, accounting for 20 -35% of total cases [START_REF] Demay | SITVITWEB -A publicly available international multimarker database for studying Mycobacterium tuberculosis genetic diversity and molecular epidemiology[END_REF][START_REF] Brudey | Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology[END_REF]. The LAM family is the most represented in Americas (20 -50% of total cases, mainly in the South), in Oceania (20% of total cases) and in all sub-regions of Africa (37% of total cases, except the West). The H family is the most represented family in Europe (24% of total cases) and in America (15 -25% of total cases, mainly in the Caribbean region), while the X family is prevalent in Americas (8 -21% of total cases, mainly in the North). Finally, the S family is found in Africa (5 -8% of total cases, mainly in the North) and in Southern Europe (5.8% of total cases) [START_REF] Demay | SITVITWEB -A publicly available international multimarker database for studying Mycobacterium tuberculosis genetic diversity and molecular epidemiology[END_REF][START_REF] Brudey | Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology[END_REF].

In summary, the molecular epidemiology and clinical studies showed that the most geographically widespread lineages, which are the lineages 2 and 4, are the more virulent ones [START_REF] Coscolla | Consequences of genomic diversity in Mycobacterium tuberculosis[END_REF][START_REF] Reiling | Clade-specific virulence patterns of Mycobacterium tuberculosis complex strains in human primary macrophages and aerogenically infected mice[END_REF][START_REF] Brites | Co-evolution of Mycobacterium tuberculosis and Homo sapiens[END_REF][START_REF] Gagneux | Host-pathogen coevolution in human tuberculosis[END_REF]. In particular, Beijing family has been reported to be associated with young people, high virulence, drug resistance and MDR, relapses and treatment failures in many countries in the world [START_REF] Merker | Whole Genome Sequencing Reveals Complex Evolution Patterns of Multidrug-Resistant Mycobacterium tuberculosis Beijing Strains in Patients[END_REF][START_REF] Coscolla | Consequences of genomic diversity in Mycobacterium tuberculosis[END_REF][START_REF] Huyen | Tuberculosis Relapse in Vietnam Is Significantly Associated With Mycobacterium tuberculosis Beijing Genotype Infections[END_REF][START_REF] Casali | Evolution and transmission of drug resistant tuberculosis in a Russian population[END_REF][START_REF] Ribeiro | Mycobacterium tuberculosis Strains of the Modern Sublineage of the Beijing Family Are More Likely To Display Increased Virulence than Strains of the Ancient Sublineage[END_REF][START_REF] Klopper | Emergence and Spread of Extensively and Totally Drug-Resistant Tuberculosis, South Africa[END_REF]]

The tuberculosis situation in Lao PDR

A. TB and HIV epidemiological situation

Lao PDR (6.9 M population in 2017) TB burden remains considerable with incidence (include TB-HIV) at 168/100,000 (12,000 cases) and TB mortality 37/100,000, with the greater number in age above 14 years old (10,000 cases) [73].

The National TB Center (NTC) conducted the first National TB prevalence survey (TBPS)

with WHO assistance in 2010-2011. The survey found that the prevalence of TB (≥15 yearsold) is likely to be two times higher than the previous estimates (WHO re-estimated TB all form 540 per 100,000). The survey also remarked that case detection efforts remain the primary goal of NTCP with case notifications being very low in comparison with the estimated number of prevalent cases [START_REF] Law | The first national tuberculosis prevalence survey of Lao PDR (2010-2011)[END_REF].

Prevalence of HIV is reported low (0.2% prevalence in population the correct treatment. One single type of pill is also easier to order, distribute and manage than 4 separate drugs. Most importantly, a patient taking 4FDC tablets always takes 4 drugs at the time. This will contribute to the prevention of drug resistance.

Loose pills still need to be available at the provincial and central hospitals to treat patients presenting with drug side effects. The dosage of the drugs depends on the weight of the patient. In order to make the drug administration as easy as possible, treatment prescription has been standardized according to weight categories. These categories are very broad for adults, but are more precise for children.

The standard 6-month treatment regimen of 2HRZE/4HR (Table 1.2) is recommended by the WHO for the treatment of patients with drug-susceptible pulmonary TB disease. Intensive phase of 2 months' (60 days) duration with 4 drugs (R, H, Z, E) daily followed by a continuation phase of 4 months' (120 days) duration with 2 drugs (R, H) daily. In patients with severe EP TB (TB meningitis, miliary TB, pericardial TB or osteo-articular TB) the continuation phase is extended to 10 months.

Ø

Treatment regimens for RR/MDR-TB patients eligible for the 9 months regimen

The 2016 WHO conditional recommendation indicated that a shorter MDR-TB regimen of 9-12 months may be used instead of longer MDR-TB regimens. This regimen is specifically designed for RR/MDR-TB patients who do not present resistance to the FQs or SLIDs. In Lao PDR, all patients who, after a RR Xpert result, present no resistance on the MTBDRsl test, will receive the 9-month regimen, but women of child bearing age should receive contraception.

The 9-month regimen should not be prescribed in the following situations:

• The MTBDRsl test shows resistance to FQs and/or SLIDs

• Pregnant woman in the first trimester of pregnancy

• Patient with MDR-TB meningitis

• Patient who is unable or unwilling to comply with the treatment

• Patient with a known allergy to any of the drugs used in the 9-month regimen

• Patient taking any medications contraindicated in combination with the drugs used in the 9month regimen

The 9-month regimen consists of an intensive phase of 4 months with 7 drugs, followed by a continuation phase of 5 months with 4 drugs (Table 1.3). The intensive phase will be extended until smear conversion if smear conversion is not achieved within 4 months, with a maximum of 6 months:

Table 1. 3 Shorter MDR-TB regimen

Anti-TB drugs Intensive phase Continuation phase 4 (+1 or 2) months 5 months

Kanamycin X Prothionamide X Isoniazid high-dose X Moxifloxacin high-dose X X Clofazimine X X Pyrazinamide X X Ethambutol X X

Ø

Treatment regimens for RR/MDR-TB patients non eligible for the 9 months regimen

An individualized long-course regimen must be prescribed whenever it is not possible to administer the 9-month regimen. WHO has released a rapid communication with key changes to treatment of MDR and RR-TB in August 2018. The individualized MTB-TB regimen is established on a stepwise approach and the drugs are selected according to a cascade of sequence.

In patients with RR-TB or MDR-TB, a regimen with at least five effective TB medicines during the intensive phase is recommended, including PZA and 4 core SLDsone chosen from Group A, one from Group B, and at least two from Group C, based on the latest evidence about the balance of effectiveness to safety:

-Group A: Medicines to be prioritized: levofloxacin/moxifloxacin, bedaquiline and linezolid -Group B: Medicines to be added next: clofazimine, cycloserine/terizidone Group C: Medicines to be included to complete the regimens and when agents from Groups

A and B cannot be used: ethambutol, delamanid, pyrazinamide, imipenem-cilastatin, meropenem, amikacin (streptomycin), ethionamide/prothionamide, p-aminosalicylic acid;

Kanamycin and capreomycin are no longer recommended, given increased risk of treatment failure and relapse associated with their use in longer MDR-TB regimens.

In summary, TB continues to be one of the priority infection diseases to combat in Lao PDR. The screening of RR is routinely performed on presumptive TB and MDR-TB cases, however the resistance to other FLDs (INH, EMB and PZA) are not initially screened; the extent threat of the resistance to these drugs cannot be evaluated. The use of available molecular tests (XpertMTB/RIF and Hain tests) for detection of FLD and SLD resistance is limited and these tests target only some mutations associated with drug resistance in a limited number of genes or genomic regions. The conventional phenotypic DST method is available only at the National Reference laboratory (NRL), but the method is labor-intensive, time-consuming and requires competent staff and a biosafety Level 3 laboratory. Up to now, genetic data, regarding the complete antibiotic resistance profiles or the overall genotype of the strain, were totally absent in Lao PDR.

Treatment regimens for the mono-resistant and poly-resistant TB (drugresistant TB other than MDR-TB) (WHO recommendations)

Mono-resistance cases in this section refer to resistance to a single first-line drug, and poly resistance cases refer to resistance to two or more first-line drugs but not to both isoniazid and rifampicin (i.e. not MDR-TB). WHO recommendations for building of treatment regimens according to individual drug resistant pattern (not currently used in Lao PDR) (Table 1.4)

Anti-tuberculosis drugs

A.

Classes of anti-TB drugs

The classes of anti-TB drugs have traditionally been divided into first-and second-line anti-TB drugs with isoniazid (INH), rifampicin (RIF), pyrazinamide (PZA), ethambutol (EMB) and streptomycin (STR) being the primary first-line anti-TB drugs. While this classification is used in this document, it also uses a system that classifies the drugs into five different groups. The fivegroup system is based on efficacy, experience of use, safety and drug class [START_REF]WHO | Guidelines for the programmatic management of DR-TB. Companion Handbook to the WHO Guidelines for the Programmatic Management of Drug-Resistant Tuberculosis[END_REF]. WHO recommended the drugs for the treatment of RR-TB and MDR-TB in the 2016 update [START_REF]WHO treatment guidelines for drug-resistant tuberculosis: 2016 update[END_REF]. The different groups are shown in Table 1.5. The drugs in the same group do not come from the e HIV-status must be confirmed to be negative before thioacetazone is started

B. Mechanisms of action and resistance of anti-TB drugs

Tuberculosis drugs target various aspects of M. tuberculosis biology, including inhibition of cell wall synthesis, protein synthesis or nucleic acid synthesis. Table 1.6 summarizes the mechanisms of action and main mechanisms of resistance to FLDs and main SLDs. Figures 1.9 Regarding drug resistance, there are two main mechanisms: 1). primary or transmitted drug resistance, occurs when resistant strains are transmitted to a new host, and 2). secondary or acquired drug resistance, which occurs through the acquisition of drug resistance mutations to one or more drugs [START_REF]WHO treatment guidelines for drug-resistant tuberculosis: 2016 update[END_REF][START_REF] Palomino | Drug Resistance Mechanisms in Mycobacterium tuberculosis[END_REF][START_REF] Da Silva | Molecular basis and mechanisms of drug resistance in Mycobacterium tuberculosis: classical and new drugs[END_REF]. The acquisition of mutations in genes that code for drug targets or drug-activating enzymes is the primary vehicle driving drug resistance in M. tuberculosis. These are mainly in the form of SNPs, insertions or deletions and to a lesser extent, large deletions.

Unlike other bacteria, resistance is not acquired via horizontal gene transfer by mobile genetic elements [START_REF] Gillespie | Evolution of Drug Resistance in Mycobacterium tuberculosis: Clinical and Molecular Perspective[END_REF]. Other mechanisms of drug resistance in M. tuberculosis include compensatory mechanism, efflux-mediated resistance and deficient DNA repair mechanisms. Compensatory mechanism: The presence of co-occurrence of secondary mutations that act as compensatory mechanisms for the impaired fitness of the pathogen. These compensatory mutations are believed to occur in genes encoding the same protein or genes involved in similar metabolic pathways [START_REF] Fonseca | The complex evolution of antibiotic resistance in Mycobacterium tuberculosis[END_REF]; Efflux-mediated resistance: Efflux pump systems are involved in expelling drugs from the bacterial cell, enabling acquisition of resistance mutations in the bacterial genome. The overexpression of efflux pumps is believed to mediate the build-up of resistance mutations, which confers high-level drug resistance allowing M. tuberculosis to survive and persist at clinically relevant drug concentrations. The ability of the efflux pumps to extrude a diversity of compounds allows them to expel multiple drugs leading to the MDR phenotype [START_REF] Silva Pea Da | Efflux as a mechanism for drug resistance in Mycobacterium tuberculosis[END_REF][START_REF] Louw | A Balancing Act: Efflux/Influx in Mycobacterial Drug Resistance[END_REF];

Deficient DNA repair mechanisms: Mutations occurring in DNA repair systems alter the ability of such systems to repair efficiently the damaged DNA, thereby increasing mutation rates. This provides a selective advantage to bacteria that bear resistance-conferring mutations [START_REF] Chopra | The role of mutators in the emergence of antibiotic-resistant bacteria[END_REF][START_REF] Martinez | Mutation Frequencies and Antibiotic Resistance[END_REF]. [START_REF] Ramaswamy | Single Nucleotide Polymorphisms in Genes Associated with Isoniazid Resistance in Mycobacterium tuberculosis[END_REF][START_REF] Hazbón | Population Genetics Study of Isoniazid Resistance Mutations and Evolution of Multidrug-Resistant Mycobacterium tuberculosis[END_REF][START_REF] Vilchèze | The Mechanism of Isoniazid Killing: Clarity Through the Scope of Genetics[END_REF][START_REF] Banerjee | inhA, a gene encoding a target for isoniazid and ethionamide in Mycobacterium tuberculosis[END_REF][START_REF] Larsen | Overexpression of inhA, but not kasA, confers resistance to isoniazid and ethionamide in Mycobacterium smegmatis, M. bovis BCG and M. tuberculosis[END_REF] Rifampicin (RIF)

Table 1. 6 Mechanisms of action and main mechanisms of resistance to FLDs and SLDs

Anti -TB drugs Mechanisms of action Mains mechanisms of resistance

-RIF effective against actively metabolizing and slow-metabolizing bacilli. -Binding to the β subunit of the RNA polymerase, resulting in the inhibition of elongation of mRNA [START_REF] Palomino | Drug Resistance Mechanisms in Mycobacterium tuberculosis[END_REF][START_REF] Da Silva | Molecular basis and mechanisms of drug resistance in Mycobacterium tuberculosis: classical and new drugs[END_REF].

-Resistance to RIF is mediated by mutations clustered in RRDR (codons 507-533) of the gene coding for the RNA polymerase β subunit (rpoB) [START_REF] Palomino | Drug Resistance Mechanisms in Mycobacterium tuberculosis[END_REF][START_REF] Da Silva | Molecular basis and mechanisms of drug resistance in Mycobacterium tuberculosis: classical and new drugs[END_REF] Pyrazinamide (PZA)

-Iinhibit semi-dormant bacilli located in acidic environments [START_REF] Mitchison | Basic Mechanisms of Chemotherapy[END_REF] -Activated by the pyrazinamidase/ nicotinamidase (PZase) enzyme, encoded by the pncA gene [89] -Disrupts the bacterial membrane energetics, inhibiting membrane transport and damage cell [START_REF] Zhang | The curious characteristics of pyrazinamide: a review[END_REF] -Mutations in the pncA (promoter and gene coding), the most common mechanism mediating pyrazinamide resistance [91] -Diversity of mutations (600 unique mutations in 400 positions) [START_REF] Whitfield | A Global Perspective on Pyrazinamide Resistance: Systematic Review and Meta-Analysis[END_REF] Ethambutol (EMB)

-Active against actively multiplying bacilli, disrupting the biosynthesis of the arabinogalactan in the cell wall. -The embCAB operon encodes the mycobacterial arabinosyl transferase enzyme -Resistance to EMB is mediated via mutations in the embB gene [START_REF] Takayama | Inhibition of synthesis of arabinogalactan by ethambutol in Mycobacterium smegmatis[END_REF][START_REF] Mikusová | Biogenesis of the mycobacterial cell wall and the site of action of ethambutol[END_REF] -Alteration in codon 306 of the embB gene, the most common resistance mechanism [START_REF] Telenti | The emb operon, a gene cluster of Mycobacterium tuberculosis involved in resistance to ethambutol[END_REF][START_REF] Sreevatsan | Ethambutol resistance in Mycobacterium tuberculosis: critical role of embB mutations[END_REF] Streptomycin (STR)

-Active against slow-growing bacilli and acts by irreversibly binding to the ribosomal protein S12 and 16S rRNA, which are the components of the 30S subunit of the bacterial ribosome. -Blocks translation thereby inhibiting protein synthesis [START_REF] Moazed | Interaction of antibiotics with functional sites in 16S ribosomal RNA[END_REF][START_REF] Finken | Molecular basis of streptomycin resistance in Mycobacterium tuberculosis: alterations of the ribosomal protein S12 gene and point mutations within a functional 16S ribosomal RNA pseudoknot[END_REF] -The main mechanism of resistance to STR is believed to be mediated via mutations in the rpsL and rrs genes, encoding the ribosomal protein S12 and the 16S rRNA, respectively [START_REF] Gillespie | Evolution of Drug Resistance in Mycobacterium tuberculosis: Clinical and Molecular Perspective[END_REF] Second-line injectable agents:

-Aminoglycosides ( KAN, Am) -Cyclic polypeptide (CAP) Source: National Institute of Allergy and Infectious Diseases (NIAID). https://www.niaid.nih.gov/diseases-conditions/tbdrugs

-

C. Common genes involved in resistance of Mycobacterium tuberculosis

There are two types of drug resistance in M. tuberculosis: genetic resistance and phenotypic resistance. Genetic drug resistance is due to mutations in chromosomal genes in growing bacteria, while phenotypic resistance or drug tolerance is due to epigenetic changes in gene expression and protein modification that cause tolerance to drugs in non-growing persister

Ethic approval of research

This study proposal was approved by the National Ethics Committee of Health Research of Lao PDR. Written informed consents were obtained from all study participants.

Methods

Xpert MTB/RIF testing

The Xpert MTB/RIF assay is almost fully automated cartridge-based system, utilizing analysis. For the use of GenoLyse kit, the subcultures were scraped from media slopes and resuspended in 100 µL of lysis buffer. The samples were then incubated for 5 min at 95°C (lysis under alkaline conditions). Then we added 100 µl of neutralization buffer, vortex sample, spin down and the supernatants were transfered to a new tube and stored at -80 °c.

GenoType® Mycobacteria Series (GenoType® MTBDRplus, MTBDRsl and Mycobacterium CM)

The GenoType® MTBDRplus ver. and AP and an AP mediated staining reaction) and 4). Evaluation and interpretation of results.

The MTBDRplus and MTBDRsl were firstly evaluated by the presence of three control bands (conjugate control, amplification control and M. tuberculosis complex control band) and each locus control (Figure 2.3). Positive results for all wild type probes of a gene and absence of positive signal for mutation probes suggest strain sensitivity for the considered antibiotic. The absence of signal for at least one of the wild type probes (with or without the presence of mutation probes), hence indicates resistance of tested strain to the considered antibiotic (Figure 2.3).

Spoligotyping

Spoligotyping is an amplification-based genotyping method that assesses the genetic In this study, the classical 43-spacer format of spoligotyping was performed as previously described [114,116]. DNA samples of the M. tuberculosis H37Rv and Mycobacterium bovis BCG strains were included as positive controls. Molecular biology-grade water was used as a negative control. The spoligotypes (presence and absence of spacers)

were then recorded in 43-digit binary format and compared with those recorded in the SpolDB4 database (http://www.pasteur-guadeloupe.fr:8081/ SITVIT_ONLINE/) to identify the Spoligotype International Type (SIT) and family [START_REF] Demay | SITVITWEB -A publicly available international multimarker database for studying Mycobacterium tuberculosis genetic diversity and molecular epidemiology[END_REF]. For the spoligotypes that matched the SITs, but could not be related to any family (i.e., unknown), and for the spoligotypes that were not present in the SpolDB4 database (e.g., orphan), the SPOTCLUST program, which was built from the spolDB3 database (http://tbinsight.cs.rpi.edu/run_spotclust.html) [117], was used to search for M.

tuberculosis family similarity. In the SPOTCLUST analyses, the family assignation was retained when the probability was ≥90%. Nevertheless, the final designation of families and subfamilies was also based on the MIRU-VNTR data (see below). Results: Among the 222 M. tuberculosis isolates, 11 were mono-resistant to isoniazid and 2

were resistant to isoniazid and rifampicin (MDR-TB), using the GenoType MTBDRplus test.

Among the 202 genetically characterized isolates, the East African-Indian (EAI) family was predominant (76.7%) followed by the Beijing (14.4%) and T (5.5%) families. EAI isolates came from all the country provinces, whereas Beijing isolates were found mainly in the northern and WHO re-estimated the prevalence of all TB forms at 540/100,000, 1.9 times higher than previous estimates (Law et al. 2015). Moreover, little is known about anti-TB drug resistance.

The only available cross-sectional study (n=87 TB isolates) conducted in three hospitals in 2010

showed that 8% of isolates were resistant to isoniazid (INH) and 1.2% caused XDR-TB (Iem et al. 2013). Similarly, the M. tuberculosis population in Lao PDR is still unknown. Many studies have reported that the Beijing and East African-Indian (EAI) families are predominant in Asian countries [START_REF] Chen | Genetic diversity of the Mycobacterium tuberculosis East African-Indian family in three tropical Asian countries[END_REF][START_REF] Ismail | Study of Mycobacterium Tuberculosis Complex Genotypic Diversity in Malaysia Reveals a Predominance of Ancestral East-African-Indian Lineage with a Malaysia-Specific Signature[END_REF][START_REF] Yu | Genetic Diversity of Mycobacterium Tuberculosis Isolates from Inner Mongolia, China[END_REF]Phyu et al. 2009;Zhang et al. 2011;Nguyen et al. 2012). In Vietnam, the Beijing family is currently invading the country and is more likely to be drug resistant than the EAI family [START_REF] Phyu | Predominance of Mycobacterium tuberculosis EAI and Beijing Lineages in Yangon, Myanmar[END_REF][START_REF] Nguyen | High Prevalence of Beijing and EAI4-VNM Genotypes among M. tuberculosis Isolates in Northern Vietnam: Sampling Effect, Rural and Urban Disparities[END_REF]( Nguyen et al. 2012Nguyen et al. , 2016)). In this context, the main objective was to study the genetic characteristics and drug resistance of M. tuberculosis families and drug resistant patterns

Methods

Study population

The TB isolates used in this study were collected during the first national TB prevalence survey in Lao PDR (July 2010-December 2011). The survey design and sample size were determined according to the WHO recommendations and has been described in Law et al. (2015). During this survey that covered the 17 provinces of Lao PDR (organized in three main regions, North [START_REF]Global tuberculosis report[END_REF][START_REF] Law | The first national tuberculosis prevalence survey of Lao PDR (2010-2011)[END_REF][START_REF]Global tuberculosis report[END_REF][START_REF] Iem | Resistance of Mycobacterium tuberculosis to antibiotics in Lao PDR: first multicentric study conducted in 3 hospitals[END_REF][START_REF] Zignol | Genetic sequencing for surveillance of drug resistance in tuberculosis in highly endemic countries: a multi-country population-based surveillance study[END_REF][START_REF] Ramazanzadeh | Prevalence of Beijing family in Mycobacterium tuberculosis in world population: Systematic Review and Meta-Analysis[END_REF][START_REF] Dou | Molecular epidemiology and evolutionary genetics of Mycobacterium tuberculosisin Taipei[END_REF][START_REF] Faksri | Epidemiological trends and clinical comparisons of Mycobacterium tuberculosis lineages in Thai TB meningitis[END_REF][START_REF] Anh | Mycobacterium tuberculosis Beijing genotype emerging in Vietnam[END_REF], Center [START_REF] Phyu | Predominance of Mycobacterium tuberculosis EAI and Beijing Lineages in Yangon, Myanmar[END_REF][START_REF] Nguyen | High Prevalence of Beijing and EAI4-VNM Genotypes among M. tuberculosis Isolates in Northern Vietnam: Sampling Effect, Rural and Urban Disparities[END_REF][START_REF] Dong | Spoligotypes of Mycobacterium tuberculosis from Different Provinces of China[END_REF][START_REF] Daniel | The history of tuberculosis[END_REF] and South [START_REF] Wright | Anti-tuberculosis drug resistance in the world: fourth global report: the World Health Organization/International Union Against Tuberculosis and Lung Disease (WHO/UNION) Global Project on Anti-Tuberculosis Drug Resistance Surveillance, 2002-2007[END_REF][START_REF]WHO | Guidelines for the programmatic management of DR-TB. Companion Handbook to the WHO Guidelines for the Programmatic Management of Drug-Resistant Tuberculosis[END_REF][START_REF] Care | Guidelines for Treatment of Drugsusceptible Tuberculosis and Patient Care[END_REF][START_REF] Malhotra | World Health Organization, Department of HIV/AIDS, Stop TB Initiative (World Health Organization). Guidelines for intensified tuberculosis case-finding and isoniazid preventative therapy for people living with HIV in resource-constrained settings[END_REF], see Figure 1) , at least one sputum specimen was collected from 6,290 (99.1%) of the 6,346 participants suspected to have TB on the basis of clinical data (chronic cough and/or hemoptysis and/or chest X-ray abnormalities).

Finally, TB was confirmed in 237 participants, according to the study case definition (Law et al. 2015). Among these 237 patients, 94 had at least one smear-positive sputum and cultureconfirmed M. tuberculosis (definite cases), 13 had at least one smear-positive sputum and chest X-ray (CXR) findings suggestive of TB with negative culture (probable cases), and 130 had smear-negative but culture-positive specimens. In summary, the presence of M. tuberculosis was confirmed by culture in 224 isolates and 222 isolates of these isolates (corresponding to 222 different patients) could be included in this study. The collected sputum specimens were decontaminated with 4% sodium hydroxide and then they were inoculated on two slopes of solid Kudoh-modified Ogawa medium without centrifugation [START_REF] Hans | Priorities for Tuberculosis Bacteriology Services in Low-Income Countries[END_REF]). All subcultures were sent by the National Tuberculosis Reference Laboratory (NRL) to the Center of Infectiology Lao-Christophe Mérieux (CILM) for species identification and genetic characterization. Colonies were scraped from the medium slopes and resuspended in 300µL of distilled water, heated at 95°C for 20 min, and centrifuged at 13000 g for 5 min. Then, the DNA-containing supernatant was transferred into a new tube and stored at -80°C.

The patients' demographic, epidemiologic, and clinical data were collected using a questionnaire, including residence, sex, age, TB history, TB symptoms, and CXR findings.

Mycobacterium tuberculosis complex identification and drug resistance testing

The GenoType® MTBDRplus test (Hain Lifescience GmbH), a DNA STRIP®-based technology, was used according to the manufacturer's instructions to identify the M. tuberculosis complex and resistance to RIF and/or INH ("GenoTypeMTBDRplusver1.Pdf," n.d.).

Spacer Oligonucleotide Typing (Spoligotyping)

Spoligotyping (the classical 43-spacer format) was performed as previously described (Filliol et al. 2003;Kamerbeek et al. 1997). DNA samples of the M. tuberculosis H37Rv and Mycobacterium bovis BCG strains were included as positive controls. Molecular biology-grade water was used as a negative control. The spoligotypes were then recorded in 43-digit binary format and compared with those recorded in the SpolDB4 database (http://www.pasteurguadeloupe.fr:8081/ SITVIT_ONLINE/) to identify the Spoligotype International Type (SIT) and family (Demay et al. 2012). For the spoligotypes that matched the SITs, but could not be related to any family (i.e., unknown), and for the spoligotypes that were not present in the SpolDB4 database (i.e., orphan), the SPOTCLUST program, which was built from the spolDB3 database (http://tbinsight.cs.rpi.edu/run_spotclust.html) [START_REF] Vitol | Identifying Mycobacterium Tuberculosis Complex Strain Families Using Spoligotypes[END_REF], was used to search for M.

tuberculosis family similarity. In the SPOTCLUST analyses, the family assignation was retained when the probability was ≥90%. Nevertheless, the final designation of families and subfamilies was also based on the MIRU-VNTR data (see below). The final designation of family/subfamily was revised using the MIRU-VNTRplus website (MIRU-VNTRplus.org) based on the family results for each isolate and the MIRU-VNTR/spoligotyping phylogenetic tree.

Mycobacterial Interspersed

Data analysis

A cluster was defined as two or more isolates with identical genotype by spoligotyping and MIRU-VNTR typing. Recent transmission was estimated by calculating the clustering rate as follows: CR = (nc-c)/n, where CR is the clustering rate, nc is the total number of clustered isolates, c is the total number of clusters, and n is the total number of isolates (van Deutekom et al. 2004). The patients' age was shown as median and interquartile range (IQR). Associations between M. tuberculosis families, patient data and overall drug resistance status were assessed using the Chi-square or Fisher's exact test, when the sample size was lower than 5. The statistical analysis was not performed for RIF and INH resistance independently due to the small number of resistant isolates. A P-value <0.05 was considered statistically significant. Statistical analyses were done using Stata (v12.1, Stata Corporation, USA).

Ethics approval and consent to participate

This retrospective genotyping study was approved by the National Ethics Committee of Health Research of Lao PDR. A written informed consent was obtained from all study participants during the national tuberculosis prevalence survey (2010-2011).

Results

M. tuberculosis complex identification and epidemiological data

The GenoType® MTBDRplus test allowed confirming that the 222 isolates included in the study belonged to the M. tuberculosis complex. The patients' median age was 56 years (IQR: , with a men to women ratio of 2:1. Patients were mainly from rural areas (83.3% vs 16.7% from urban areas), and the number of M. tuberculosis isolates across the 17 provinces varied from 0 to 34 (mean number= 13) (Table S1).

Characterization of anti-TB drug resistance (GenoType® MTBDRplus test)

Analysis of the RIF and INH resistance profile of 222 isolates with the GenoType® MTBDRplus test showed that 209 isolates (94.1%) were sensitive to both drugs, 11 (5%) were resistant only to INH, and 2 (0.9%) were resistant to both INH and RIF (MDR-TB). Among the 13 INH-resistant isolates, 10 (76.9%) had mutations in the katG gene (S315T in all isolates) and 3 (23.1%) had mutations in inhA promoter region (C15T in two isolates and T8C in one). The two RIF-resistant isolates carried the D516V mutation in rpoB gene.

Identification of the M. tuberculosis families/subfamilies

Spoligotyping and 24-locus MIRU-VNTR typing were performed on 206 of the 222 isolates (Table 1, Figure S1). The M. tuberculosis family/subfamily identifications were determined using SITVITWEB (SpolDB4 database), SPOTCLUST and MIRU-VNTRplus. The patterns of four isolates reflected either clonal variants (with double alleles at a single MIRU-VNTR locus) or mixed infections (with double alleles at two MIRU-VNTR loci) [START_REF] Shamputa | Genotypic and Phenotypic Heterogeneity among Mycobacterium Tuberculosis Isolates from Pulmonary Tuberculosis Patients[END_REF]) (Table S1). These four isolates were removed from the analysis to avoid incorrect designation. Only one orphan and one unknown isolate could not be identified. Within the EAI family, the most frequent subfamily was EAI5 (53.0%, n=107), followed by EAI1-SOM (8.9%, n=18) and EAI2-Nonthaburi (6.4%, n=13) (Table 1). The subfamily EAI4-VNM, which is found specifically in Vietnam, was poorly represented in our sampled strains (4.5%, n=9) (Table 1). In the southern provinces (N. [START_REF] Wright | Anti-tuberculosis drug resistance in the world: fourth global report: the World Health Organization/International Union Against Tuberculosis and Lung Disease (WHO/UNION) Global Project on Anti-Tuberculosis Drug Resistance Surveillance, 2002-2007[END_REF][START_REF]WHO | Guidelines for the programmatic management of DR-TB. Companion Handbook to the WHO Guidelines for the Programmatic Management of Drug-Resistant Tuberculosis[END_REF][START_REF] Care | Guidelines for Treatment of Drugsusceptible Tuberculosis and Patient Care[END_REF][START_REF] Malhotra | World Health Organization, Department of HIV/AIDS, Stop TB Initiative (World Health Organization). Guidelines for intensified tuberculosis case-finding and isoniazid preventative therapy for people living with HIV in resource-constrained settings[END_REF] where only the EAI family was represented (Figure 1), the EAI5 subfamily was the most common (65.4%, n=34), followed by EAI1-SOM (19.2%, n=10), whereas EAI4-VNM was absent. Unlike the EAI family, which was present in all regions of Lao PDR, the Beijing family was predominantly observed in the northern (58.6%, n=17) and central provinces (41.4%, n=12), and was absent in the southern provinces (Figure 1).

202

*Spoligotype defined by SPOTCLUST (probability ≥0.9)

a One isolate with double allele on ETRA and one isolate with double allele on QUB26 were removed from the analysis b One isolate with double allele on ETRA was removed from the analysis c One isolate with hybridization for all 43 spacers + double alleles on ETRA and Mtub29 was removed from the analysis

The distribution of the M. tuberculosis EAI and Beijing families varies according to age, geographical origin and drug-resistance

The M. tuberculosis family (EAI or Beijing) distribution in the three age groups (15-34, 35-64, and ≥65 years of age) was significantly different (p=0.002, Table 3). Specifically, the percentage

24-locus MIRU-VNTR typing and cluster analysis

24-locus MIRU-VNTR patterns

The 206 isolates that underwent spoligotyping were also typed by 24-locus MIRU-VNTR typing. In 182 isolates (88.4%), all 24 loci could be amplified, whereas in 24 (11.7%) at least one locus could not be amplified (repeated three times). ETRA was the most frequently nonamplified locus (9/206 isolates), followed by QUB4156 (6/206) and QUB11b (5/206). These results were treated as missing data. The four isolates with double alleles (three had double alleles at only one locus and one at two loci (Table S1)) were removed from the global analysis.

Thus, the analyses were performed on 202 isolates. By using the results of the 24-locus MIRU-VNTR technique alone, the 202 isolates generated 173 profiles (152 unique profiles and 21 clusters). The 21 clusters contained 50 isolates (2-4 isolates per cluster; average: 2.4). Two clusters included four isolates, four clusters contained three isolates, and 15 were composed by two isolates.

Phylogenetic tree and cluster analysis

The NJ tree built by combining the MIRU-VNTR and spoligotyping data for the 202 isolates clearly differentiated the Beijing clade from the other families (Figure S1). Nineteen clusters including 43 isolates (2 to 4 isolates per cluster; average: 2.3 isolates per cluster) were showed (see Figure 2 and Table S1). The EAI, Beijing and T families were present in these clusters, accounted for 32, 9 and 2 isolates respectively (Table 3) and were grouped in 15, 3 and 1 cluster respectively. 13 out of 15 EAI clusters and all 3 Beijing clusters could be geographically linked (isolates were either from patients living in the same village or district or provinces) (see Figure 2 and Table S1). Regarding drug resistant isolates, only one cluster of Beijing family (CN.18, Figure 2) contained three INH-resistant isolates.

Finally, these data allowed calculating the overall clustering rate (11.9%) and the clustering rate for the Beijing, EAI and T families (Table 3). and they are distributed all over the country. These data could partly explain the distribution of the Beijing and EAI4-VNM subfamilies in Lao PDR and raise the question of the risk of a progressive invasion by Beijing strains, as previously observed in Vietnam (Nguyen et al. 2012). were geographically linked and one of the three Beijing clusters included drug-resistant isolates (see Figure 2 andTableS1). These findings underline the risk of Beijing strain expansion in Lao PDR and consequently the increasing risk of primary drug resistance in recent transmission.

Genetic diversity and transmission of M. tuberculosis families in Lao PDR

Conclusion

This Beijing, EAI and other families of M. tuberculosis were observed among all presumptive MDR-TB (41 %, 38 % and 21 % respectively). Beijing was significantly higher than EAI and other families among drug resistant isolates (p=0.005) and only Beijing was observed among pre-XDR cases. The proportion of failures/late smear conversions was also higher than relapses among drug resistant isolates (p=0.01).

Conclusions: DNA sequencing revealed a large panel of mutations associated with drug resistance reflecting various patterns from mono-resistance to pre-XDR. The results show that the screening of resistance to both FLD and SLD by molecular method could be extremely useful to rapidly determine the optimal treatment regimen. As expected, Beijing family is associated with drug resistance and this is the future concern in terms of MDR-TB increase risk in Lao PDR. Efforts for accurate and rapid detection of drug resistance is urgently needed for all TB patients in order to prescribe appropriate treatment and limit the transmission of drug resistant TB in Laos. [START_REF] Paudel | Risk Factors of Multidrug-Resistant Tuberculosis[END_REF][START_REF] Marahatta | Risk Factors of Multidrug Resistant Tuberculosis in Central Nepal: A Pilot Study[END_REF][START_REF] Faustini | Risk Factors for Multidrug Resistant Tuberculosis in Europe: A Systematic Review[END_REF][START_REF] Ahmad | Risk Factors for Multidrug-Resistant Tuberculosis in Urban Pakistan: A Multicenter Case-Control Study[END_REF][START_REF] Mulisa | Multidrug-Resistant Mycobacterium tuberculosis and Associated Risk Factors in Oromia Region of Ethiopia[END_REF][START_REF] Balabanova | Risk Factors for Drug-Resistant Tuberculosis Patients in Lithuania, 2002-2008[END_REF]. Furthermore, young people and males are more likely to have MDR-TB [START_REF] Paudel | Risk Factors of Multidrug-Resistant Tuberculosis[END_REF][START_REF] Faustini | Risk Factors for Multidrug Resistant Tuberculosis in Europe: A Systematic Review[END_REF][START_REF] Ahmad | Risk Factors for Multidrug-Resistant Tuberculosis in Urban Pakistan: A Multicenter Case-Control Study[END_REF]. Another key factor is the poor adherence to anti-TB drug treatment [START_REF] Paudel | Risk Factors of Multidrug-Resistant Tuberculosis[END_REF]).

Key words:

Regarding M. tuberculosis families, Beijing family was reported to be associated with MDR-TB in many parts of the world, especially in Asian countries [START_REF] Drobniewski | Drug-Resistant Tuberculosis, Clinical Virulence, and the Dominance of the Beijing Strain Family in Russia[END_REF][START_REF] Mokrousov | At Baltic Crossroads: A Molecular Snapshot of Mycobacterium tuberculosis Population Diversity in Kaliningrad, Russia[END_REF][START_REF] Krüüner | Spread of Drug-Resistant Pulmonary Tuberculosis in Estonia[END_REF]Tracevska et al. 2003;Glynn et al. 2002;[START_REF] Bifani | Identification of a W Variant Outbreak of Mycobacterium tuberculosis via Population-Based Molecular Epidemiology[END_REF][START_REF] Zhang | A first assessment of the genetic diversity of Mycobacterium tuberculosis complex in Cambodia[END_REF]Pang et al. 2012;Phyu et al. 2009;[START_REF] Lisdawati | Molecular Epidemiology Study of Mycobacterium tuberculosis and Its Susceptibility to Anti-Tuberculosis Drugs in Indonesia[END_REF][START_REF] Cheunoy | Drug Resistance and Genotypic Analysis of Mycobacterium tuberculosis Strains from Thai Tuberculosis Patients[END_REF]Anh et al. 2000;An et al. 2009;[START_REF] Buu | The Beijing Genotype Is Associated with Young Age and Multidrug-Resistant Tuberculosis in Rural Vietnam[END_REF]Nguyen et al. 2016).

Regarding the drug resistance detection, the culture-based drug susceptibility testing (DST) is the gold standard for MDR-TB diagnosis; however, the method is labor and time Ø 43-spacer oligotyping (Spoligotyping)

Spoligotyping was performed as previously described (Filliol et al. 2003;Kamerbeek et al. 1997). DNA of H37Rv strain and M. bovis BCG were included as positive controls. Biological molecular grade water was used as a negative control. The spoligotypes were then recorded in 43-digit binary format and compared with those recorded in the SpolDB4 database (http://www.pasteur-guadeloupe.fr:8081/ SITVIT_ONLINE/) in order to identify the SIT (Spoligotype International Type) and the families (Demay et al. 2012). For the spoligotypes which matched to Spoligotype International Types (SITs) but could not be related to any family (unknown) and for the spoligotypes which were absent from SpolDB4 (orphan), SPOTCLUST (http:// tbinsight.cs.rpi.edu/run_spotclust.html) [START_REF] Vitol | Identifying Mycobacterium Tuberculosis Complex Strain Families Using Spoligotypes[END_REF]) was used to search M.

tuberculosis family similarity. For SPOTCLUST analyses, we considered the family assignation when the probability was equal or higher than 90 %. Nevertheless, the final designation of families and subfamilies was also based on the MIRU-VNTR data (see below).

CILM are located. The number of patients was variable according to the presumptive MDR-TB criteria with a majority of relapses (53.7 %) and failures (23.5 %) (Table 1)

GenoType® Mycobacterium CM and MTBDRplus

Among the 155 isolates, the MTBDRplus test identified 139 (89.7 %) M. tuberculosis complex (MTBc) isolates. The 16 (10.3 %) and M. fortuitum (N=1). Four NTM isolates could not be identified and one isolate was determined as Gram positive bacteria (Figure 1).

Among the 139 confirmed MTBc, 96 (69.1 %) were susceptible and 43 (30.9 %) were resistant to at least one anti-TB drug (RIF and/or INH). Among the 43 drug resistant isolates, 17 (12.2 %) were MDR, seven (5.0 %) were mono-resistant to RIF and 19 (13.7 %) were monoresistant to INH (Figure 1). Of 43 resistant isolates, 25 (58.1 %) were relapse cases, 14 (32.6 %) failure/late smear conversion cases, two (4.7 %) return after loss to follow up cases, one (2.3 %) TB-HIV co-infected and one (2.3 %) missing data. Of 17 MDR-TB cases, nine were relapse cases; seven failure/late smear conversions and one missing data (Table 2). According to MTBDRplus, 23 (54.8 %) isolates had mutations in rpoB gene, 29 (67.4 %) isolates had mutations only in katG, six (14.0 %) isolates had mutations only in inhA-promoter and one (2.3 %) isolate had mutations in both katG and inhA-promoter (Table S 2.2). 2 andS 2.1) were detected by sequencing leading to a concordance between MTBDRplus and the sequencing results of 92 % for rpoB (RIF resistance) and 90 % for katG and inhA genes and inhA-promoter together (INH resistance). Of 42 isolates,25 (59.5 %) had mutations in rpoB gene, the majority (n=23, 92 %) of them were found only within the 81-bp rifampicin resistance determining region (RRDR). The most common mutations were Ser531Leu (n=6, 24 %); His526Tyr (n=5, 20 %) and His526Arg (n=5, 20 %), these mutations have generally been reported as conferring high level resistance to RIF [START_REF] Lee | High Frequency of Mutations in the RpoB Gene in Rifampin-Resistant Clinical Isolates of Mycobacterium tuberculosis from Singapore[END_REF]Campbell et al. 2011). One isolate had combination of two mutations, one mutation located in the RRDR (Met515Leu) and one located outside the RRDR (Ile572Phe) (Table S 2.1). The rpoB mutant (ID: MR20; see Tables S1 andS2) detected only by sequencing showed Leu511Pro mutation. One isolate revealed a mutation out of the RRDR (Met655Thr) combined with an insertion at the codon 514 (TGCCAA, CysGln) (Table S 2.

1).

Thirty-nine out of the 42 isolates (92.9 %) revealed non-synonymous mutations or deletion or insertion of bases in katG and/or in inhA-promoter and/or in inhA-coding gene. A total of 33 (84.6 %) isolates had mutations in katG gene, 29 out of 33 (87.9%) carried a mutation at codon 315 (the most prevalent associated-INH resistant codon). The most frequent mutation was Ser315Thr (n=25,75.8 %). This mutation was found alone or in combination with a mutation in inhA-promoter or in inhA coding gene (Table S 2.1). One non-synonymous mutation not known to be associated to INH resistance was detected at the codon Pro100Thr. Another mutation, Asp189Gly, recently associated with INH resistance [START_REF] Brossier | Comparative Study of Enzymatic Activities of New KatG Mutants from Low-and High-Level Isoniazid-Resistant Clinical Isolates of Mycobacterium tuberculosis[END_REF]) was observed. One isolate showed an insertion of T at the codon 630 leading to a frameshift. This frameshift mutation was previously found to be likely associated with INH resistance [START_REF] Kandler | Validation of Novel Isoniazid Resistance Mutations Not Detectable by Common Molecular Tests[END_REF]. One isolate showed a C deletion at the codon 32 leading to a frameshift in the sequence; no information is available regarding INH resistance for this mutation. Mutations in inhA-promoter were identified in seven (17.9 %) isolates. The position (-) 15CT was the most prevalent (n=6). Three (7.7 %) isolates had mutations in inhA coding region, all these mutations were found in combination either with katG mutation or with inhA-promoter mutation (Table S 2.1). In total, four isolates (10.3%) showed a combination of mutations in different genes: one isolate carried katG and inhA-promoter mutations (Ser315Thr/-15(CT)), one carried inhApromoter and inhA coding region (-15(CT)/Ile21Val), two carried mutations in katG and inhAcoding gene (Ser315Thr/Asp335Asn and Ser315Thr/Ile144Val) (Tables S 2.1). It is worth noting that we did not include the mutation Arg463Leu of katG gene, described as phylogenetic marker and not as drug resistant determinant (Sreevatsan et al. 1997;[START_REF] Torres | Novel katG mutations causing isoniazid resistance in clinical M. tuberculosis isolates[END_REF]. This mutation was observed in 38 out of 42 isolates.

Regarding embB gene linked to EMB resistance, 14 (33.3%) isolates revealed seven different non-synonymous mutations (Table S 2.1). The most frequent mutations were at codon 306 (Met306Val, n=4, Met306Ile, n=3), followed by the mutation at codon 360 (Val360Met, n=3).

Twelve out of 42 isolates carried the Glu378Ala mutation. This mutation was not included in the analysis because it was considered as phylogenetic marker and was not associated with EMB resistance (Köser et al. 2014).

PncA mutations linked to PZA resistance were found only in coding region of four (9.5 %) isolates, including non-synonymous mutations (Cys72Arg and Phe106Val) and base deletion (C deletion at codon 19 and A deletion at codon 160) (Table S 2.1).

Mutations in gyrA gene (FQ resistance) were observed also in four (9.5 %) isolates.

These mutations detected in codons 88, 90, 91 and 94 were previously associated with drug resistance (Table S 2.1). We excluded the Glu21Gln and Ser95Thr mutations (found in 41/42 and 40/42 isolates respectively), described as lineage genetic markers (Table S 2.2) (Farhat et al. 2016;[START_REF] Miotto | Drug Resistance InMycobacterium tuberculosis: Molecular Mechanisms Challenging Fluoroquinolones and Pyrazinamide Eff Ectiveness[END_REF]. No mutation was found in gyrB (FQ resistance) and in rrs-F2 (SLID resistance).

Regarding mutations associated to STR resistance, a total of 26 (61.9%) of 42 isolates revealed non-synonymous mutations either in rrs F1 and/or rpsL. Mutations in rrs-F1 were observed in 5 (11.9%) isolates with the most common mutation at 517(CT), while rpsL mutations were found in 22 (52.4 %) isolates, with the most common mutation, Lys43Arg (Table S 2.

1).

Finally, according to the non-synonymous mutations patterns (including nucleotide deletion and insertion), we could determine a multi-genic pattern for each isolate (Table 3). Nine isolates (21.4%) showed mutations linked to resistance to only one anti-TB drug in agreement with a mono-resistance pattern (Box 1); 11 (26.2 %) showed drug resistance-associated mutations in genes or DNA region in agreement with a poly-drug resistance pattern (Box 1); 10 (23.0%) showed at least mutations linked to resistance to RIF and INH corresponding to MDR pattern; 9 (21.4 %) showed mutations linked to resistance to four FLDs in agreement with a QDR pattern (Box 1) and 3 (7.1 %) revealed mutations linked to RIF, INH and FQ resistances, suggesting pre-XDR. Finally, among the 42 isolates either mono RIF resistant or mono INH resistant or MDR detected by MTBDRplus, 28 (66.6 %) isolates showed additional mutations conferring resistances to EMB, PZA, FQ or STR (Tables 3 andS 2

T2 2 (1.4) 1 (1.0) 1 (2.3) T3 2 (1.4) 2 (2.1) 0 H1 1 (0.7) 0 1 (2.3) H3 2 (1.4) 1 (1.0) 1 (2.3) Manu-Ancestor 1 (0.7) 1(1.0) 0 orphan/unknown 7 (5.0) 6 (6.3) 1 (2.
3) 373 374 24-locus MIRU-VNTR typing 5.

375

In order to explore the transmission, the 42 drug resistant isolates were typed by 24-locus 376 MIRU-VNTR typing, resulting in 42 different patterns (Figure 2). The figure 2 illustrates the 377 absence of clusters in this sample and the clear differentiation between Beijing and EAI families. 378

The tree highlights the higher frequency of Beijing in overall drug resistant, poly-drug resistant 379

data in overall drug resistant patterns

The different proportions of gender, age, regions of residence, types of presumptive MDR-TB and M. tuberculosis families were assessed among susceptible and resistant isolates (defined by MTBDRplus). Age of patients was also grouped according to independent (15-64 years old) and dependent age group (from 65 years old) and for better distribution, the independent age group therefore was subdivided into 2 groups 15 to 34 and 35 to 64 years old.

103

For the types of presumptive MDR-TB, contact with proven RR/MDR case was not included in the analysis due to only one observed case. The analysis showed that there were significant different proportions of drug resistant isolates according to the types of presumptive MDR-TB and the M. tuberculosis families (p=0.01 and p=0.005 respectively) (Table 6). Among different types of presumptive MDR-TB, the proportion of failure/late smear conversion cases having drug resistant isolates (46.7 %) was significantly higher than relapses, returns after loss follow up and TB-HIV cases (46.7 % > 33.3 % > 18.2 % > 5.6 %). However, there were no significant different proportions of drug resistant isolates within gender, age group or regions of residence. Regarding the M. tuberculosis families, the distribution was significantly different in the three age groups (p=0.01, Table 7). Specifically, the percentage of Beijing family was higher in the " [START_REF]WHO | Guidelines for the programmatic management of DR-TB. Companion Handbook to the WHO Guidelines for the Programmatic Management of Drug-Resistant Tuberculosis[END_REF][START_REF] Care | Guidelines for Treatment of Drugsusceptible Tuberculosis and Patient Care[END_REF][START_REF] Malhotra | World Health Organization, Department of HIV/AIDS, Stop TB Initiative (World Health Organization). Guidelines for intensified tuberculosis case-finding and isoniazid preventative therapy for people living with HIV in resource-constrained settings[END_REF][START_REF] Oxlade | The impact and cost-effectiveness of strategies to detect drug-resistant tuberculosis[END_REF][START_REF] Parsons | Laboratory Diagnosis of Tuberculosis in Resource-Poor Countries: Challenges and Opportunities[END_REF][START_REF] Isenberg | Clinical Microbiology Procedures Handbook[END_REF][START_REF]Implementing tuberculosis diagnostics: policy framework[END_REF][22][23][START_REF]WHO | Guidelines for surveillance of DR in TB -5th edit. WHO | Guidelines for surveillance of drug resistance in tuberculosis -5th edition[END_REF][START_REF] Ängeby | Challenging a dogma: antimicrobial susceptibility testing breakpoints for Mycobacterium tuberculosis[END_REF][START_REF]molecular line probe assays for rapid screening of patients at risk of multidrug-resistant tuberculosis[END_REF][START_REF]WHO-Xpert mtb/rif implementation manual. Xpert mtb/rif implementation manual: technical and operational "how-to[END_REF][START_REF] Tomasicchio | The diagnostic accuracy of the MTBDRplus and MTBDRsl assays for drug-resistant TB detection when performed on sputum and culture isolates[END_REF][START_REF] Sanchez-Padilla | Detection of Drug-Resistant Tuberculosis by Xpert MTB/RIF in Swaziland[END_REF][START_REF]WHO Meeting Report of a Technical Expert Consultation[END_REF][START_REF]WHO | The use of molecular line probe assay for the detection of resistance to second-line anti-tuberculosis drugs[END_REF][START_REF]Global tuberculosis report[END_REF][START_REF] Chaidir | Use of whole genome sequencing to predict Mycobacterium tuberculosis drug resistance in Indonesia[END_REF][START_REF] Aung | Whole-genome sequencing of multidrug-resistant Mycobacterium tuberculosis isolates from Myanmar[END_REF]56.3 % vs 34.8 %,43.7 % respectively), and the percentage of EAI family higher in the "> 64" group compared to Beijing (73.9 % vs 26.1 %). However, their gender and geographical distribution were not significantly different (Table 7). Regarding presumptive MDR-TB types, the distribution of Beijing family was higher among failure/late smear conversion cases than EAI, the proportion was significantly different (p=0.02) (75.0 % vs 25.0 %) (Table 7) a four missing data of presumptive MDR-TB type and one case of new patient in contact with proven RR/MDR was not included

Discussion

Identification of drug resistant TB and Non-tuberculous mycobacteria among presumptive MDR-TB by MTBDRplus

Beside M. tuberculosis, the Non-tuberculous mycobacteria (NTM) were identified in 12 (8.6 %) presumptive MD-TB cases, which is higher than in China (3.4 %) [START_REF] Shao | The Epidemiology and Geographic Distribution of Nontuberculous Mycobacteria Clinical Isolates from Sputum Samples in the Eastern Region of China[END_REF].

Moreover, the four species (M. scrofulaceum, M. abscessus, M. chelonae and M. fortuitum)

found in our study were reported to be the cause of pulmonary infections [START_REF] Griffith | An Official ATS/IDSA Statement: Diagnosis, Treatment, and Prevention of Nontuberculous Mycobacterial Diseases[END_REF]Stout, Koh, and Yew 2016). This finding underlines the need to also detect the NTM species in patients with TB-like symptoms but with MTBc negative results in order to give the appropriate treatment.

According to MTBDRplus results, mono RIF resistance rate (5.0 %) was higher than the one in Cambodia (2.4 %), but lower than in India and Ethiopia (13.0 % and 10.7 % respectively).

Conversely, the mono INH resistance rate (13.7 %) was higher compared to Cambodia, India and Ethiopia (7.3 %, 8.0 % and 7.3% respectively) [START_REF] Khann | Linkage of Presumptive Multidrug Resistant Tuberculosis (MDR-TB) Patients to Diagnostic and Treatment Services in Cambodia[END_REF][START_REF] Pradhan | Patterns of TB Drug-Resistance in a Tertiary Care Facility in Pune, India[END_REF]Tesfay et al. 2016). Among INH resistant, 30 (69.8 %) isolates had mutations in katG gene (codon 315), which is known to be associated with a high level of INH resistance [START_REF] Jagielski | Detection of Mutations Associated with Isoniazid Resistance in Multidrug-Resistant Mycobacterium tuberculosis Clinical Isolates[END_REF];[START_REF] Lempens | Isoniazid Resistance Levels of Mycobacterium tuberculosis Can Largely Be Predicted by High-Confidence Resistance-Conferring Mutations[END_REF]) and high level of INH resistance cannot be overcome by the use of high-dose INH in the treatment [START_REF] Domínguez | Clinical Implications of Molecular Drug Resistance Testing for Mycobacterium tuberculosis: A TBNET/RESIST-TB Consensus Statement[END_REF]. This is an important concern for TB treatment in Laos, since INH resistance is not screened in routine. The patients with mono INH resistance will be treated by the standard regimen (2HREZ/4HR), these patients are at risk for treatment failure, relapse or acquisition of additional drug resistances, especially the acquisition of additional resistance to RIF due to inadequate treatment of INH resistant TB ("FAQ_TB_policy_recommendations_guidelines.Pdf" n.d.). At last, the rate of MDR-TB (12.2 %) is lower than those obtained in Asian and African countries like Cambodia, India and Ethiopia (26.0 %, 53.0 % and 54.6 % respectively) [START_REF] Khann | Linkage of Presumptive Multidrug Resistant Tuberculosis (MDR-TB) Patients to Diagnostic and Treatment Services in Cambodia[END_REF][START_REF] Pradhan | Patterns of TB Drug-Resistance in a Tertiary Care Facility in Pune, India[END_REF]Tesfay et al. 2016).

Genetic determinants associated with anti-TB drug resistance

The specific genes or DNA regions selected in the study are known to be associated with anti-TB drug resistance (Dookie et al. 2018). It is worth noting that the sequencing and MTBDRplus data showed an agreement of 92 % for the detection of RIF resistance and of 90 % for INH resistance. [START_REF] Horne | Diagnostic Accuracy and Reproducibility of WHO-Endorsed Phenotypic Drug Susceptibility Testing Methods for First-Line and Second-Line Antituberculosis Drugs[END_REF]Madison et al. 2002;Campbell et al. 2011) The conventional DST for the detection of PZA resistance is also challenging and problematic due to the poor growth of M. tuberculosis under the acidic conditions (pH 5.5 to 6.0) required for optimal drug activity [START_REF] Mackaness | The Intracellular Activation of Pyrazinamide and Nicotinamide[END_REF]McDERMOTT and Tompsett 1954). On a molecular point of view, the acquisition of mutations in pncA gene is the main mechanism associated with PZA resistance. It is worth noting that mutations in pncA are very diverse and widely dispersed throughout the gene (Morlock et al. 2000). In our analysis, two isolates carrying mutations in pncA (Cys72Arg and Phe106Val) and two had base deletion (C deletion at codon 19 and A deletion at codon 160, leading to frameshift). The Cys72Arg and the deletion of C at codon 19 were identified among PZA resistant isolates in recent studies (Wade et al. 2004;Q. H. Nguyen et al. 2017). The deletion of A at codon 160 has not been specifically previously described to be associated with PZA resistance, but mutation occurring at codon 160 (Thr160Lys/ACG160AAG) was already found to be associated with PZA resistance (Cuevas-Córdoba et al. 2013). At last, the Phe106Val has not been previously described to be associated with PZA resistance but the Phe106Ser mutation was recently described [START_REF] Maningi | Multi-and Extensively-Drug Resistant Mycobacterium tuberculosis in South Africa: A Molecular Analysis of Historical Isolates | Request PDF[END_REF] . Thus all the isolates with mutations could not be firmly associated with PZA resistance. Further studies will be necessary to explore the link between the A deletion at codon 160 and the Phe106Val mutation and the PZA resistance. Since the culture-based DST of EMB and PZA are still challenging, the DNA sequencing could bring an indubitable advantage. The presence of these mutations led to one poly-drug resistant (HFS) and three pre-XDR TB isolates (RHEFS, RHZFS and RHZEFS, see Table 3). The pre-XDR TB accounted for 2.2 % of all presumptive MDR-TB. This underlines that it is extremely necessary to look for pre-XDR in presumptive MDR isolates in Laos in order to prescribe the appropriate treatment regimen

Regarding STR resistance, the number of resistant isolates with mutations associated with STR resistance was quite high in our sample (61.9 %) and only two mutations were observed (Lys43Arg and Lys88Thr). These patterns fully justify that STR is no longer used in Laos according to WHO recommendation.

Among the 42 drug resistant isolates, no mutation was observed in rrs-F2 (resistance to injectable drugs) reflecting the absence of XDR-TB in presumptive MDR-TB cases under study.

This shows that XDR is still little detected and thus it is urgent to develop efficient TB and DR-TB detection in Lao PDR to preclude the emergence and spread of XDR strains.

In total, the sequencing revealed 66.6 % (28/42) of drug resistant isolates detected by the MTBDRplus with additional mutations conferring resistance to EMB, PZA, FQ or STR. The patterns of drug resistance include mono-resistance, poly-resistance, MDR, QDR and pre-XDR.

The determination of these patterns is critical for the prescription of appropriate treatment. PDR. This finding is in agreement with previous studies worldwide, especially in Asian countries [START_REF] Cheunoy | Drug Resistance and Genotypic Analysis of Mycobacterium tuberculosis Strains from Thai Tuberculosis Patients[END_REF]An et al. 2009;[START_REF] Cox | The Beijing Genotype and Drug Resistant Tuberculosis in the Aral Sea Region of Central Asia[END_REF] Regarding the type of presumptive MDR-TB cases, the proportion of failure/late smear conversion cases was significantly higher than relapses, returns after loss follow up and TB-HIV among drug resistant isolates (46.7 %> 33.3 % > 18.2 % > 5.6 % respectively) (p=0.01). This group has thus a higher potential to develop drug resistant TB than the other groups and required full investigation for identifying disease etiology. It is essential to notice that the proportion of Beijing was also higher than EAI among failure/late smear conversion cases (p=0.02). Moreover, Beijing appears as a factor of failures and relapses in TB patients as previously observed in many settings (Ramazanzadeh and Sayhemiri 2014).

Characteristics of M. tuberculosis

Nevertheless, these DR isolates do not seem to spread in the country. Indeed, out of the The data was stored in google drive, please follow the link bellow:

https://drive.google.com/drive/folders/1tiqNwVjTM5OQAF0lJn-rxxu-R678QPIa

Methods

The methods used on these samples are described in "Material and Methods" section (see page 49)

Drug resistant patterns from the drug resistance survey

Of 1006 patients included in the survey, 820 patients had DST results of all drugs.

Based on the DST results, the prevalence of any resistance to FLD and SLD was 9.1 % , which represents 9.0 % among new cases and previously treated cases, respectively. The prevalence of any resistance to FLD was 8.7 % (95 % CI: 6.8-10.8) and any resistance to SLD was 0.9 % (95 % CI: 0.3-1.8).

The prevalence of mono drug resistance, poly drug resistance and multi drug resistance (Box 1, p.12) were 6.8 % (95 % CI: 5.2-8.8), 1.7 % (95 % CI: 0.9-2.8) and 0.6 % (95 % CI: 0.2-1.4) respectively (Table 3. 2). Five MDR-TB cases were identified, of which two cases had additional resistance to STR (RHS); three cases had additional resistance to STR and EMB (RHSE), referring to QDR (Box 1, p.12). The MDR-TB prevalence among new cases was 0.6 % (95 %

Mutations in genes or regions of M. tuberculosis associated with anti-TB drug resistance

The Sanger sequencing of specific genes or regions involved in drug resistance to FLDs and SLDs could be performed on 74 isolates (60 any drug resistant and 14 susceptible).

However, the sequencing result could not be obtained for some isolates, due to the absence of amplification or poor quality of sequence. The results of these isolates were notified as "Invalid" (Table 3.3).

Rifampicin resistance and rpoB gene:

Based on available sequencing results, the majority (n=8/9, 88.9 %) of RIF resistant isolates harbored at least one mutation within the 81-bp Rifampicin resistance-determining region (RRDR), while one out of 9 (11.1 %) did not harbor any mutation in rpoB region under study. The most common mutations in rpoB were His526Tyr, His526Asp and Ser531Leu (27.3%, 18.2% and 18.2% respectively) (Table 3.3).

Among RIF susceptible isolates, one out of 54 (1.8%) isolates with valid sequencing result harbored His526Ser (CAC526TCC) mutation (ID: 635, Table S 3.1). A total of five isolates harbored the Met736Thr mutation outside the RRDR. This mutation was found in combination with His526Tyr (His526Tyr/Met736Thr) in one RIF resistant isolate and alone in four susceptible isolates (Table 3.3).

Isoniazid resistance and

Injectable drug resistance (Kanamycin (KAN), Capreomycin (CAP)) and rrs-F2:

There was one KAN resistant isolate for which the sequencing analysis was invalid due to the poor quality of sequencing result. Among the four CAP resistant isolates, sequencing results showed no mutation in rrs-F2 for three isolates while result was invalid for the remaining isolate.

Pyrazinamide resistance and pncA: DST was not performed for detection of PZA resistance. We evaluated the resistance to PZA by sequencing of pncA gene. In our study, five unique mutations were observed ((-)13GT and (-)7TG) in the promoter, Pro54ser, Ile5Thr and Cys72Stop codon in the coding gene).

Identification of M. tuberculosis population by Spoligotyping and MIRU-VNTR

Ø

Spoligotyping patterns

Spoligotyping and 24-locus MIRU-VNTR typing were performed on 74 isolates. The patterns of 11 (14.9%) isolates reflected either clonal variants (with double alleles at a single MIRU-VNTR locus) or mixed infections (with double alleles at least at two MIRU-VNTR loci)

[142] (Table 3. 5 and Table S 3.1). These 11 isolates were removed from the family definition analysis to avoid incorrect designation. Thus, the analysis was performed on 63

isolates. Spoligotyping generated 34 different profiles, of which 27 were unique and seven represented clusters (36 isolates). Each cluster contained 2 to 19 isolates (average = 5).

Forty-one out of 63 (65.1%) isolates were assigned to 15 SITs and four families reported in the SpoIDB4 database; 1 (1.6%) was unknown (the spoligotype matched the SIT but could not be related to any family); and 21 (33.3%) were orphans (not match with any SIT in SPOLDB4 database). The 21 orphan and the one unknown isolate were then compared using SPOTCLUST (Table 3. 5). Finally, the 63 isolates could be classified in four M.

tuberculosis families (EAI, Beijing, T and Haarlem) and 9 subfamilies, three isolates remained orphan and one unknown (Table 3. 5 and Table S 3.1). EAI was the predominant family (47.6 %, n=30), followed by Beijing (34.9 %, n=22). T and H families were present in small proportion (7.9 %, n=5 and 3.2 %, n=2 respectively). Within the EAI family, the most frequent subfamily was EAI5 (41.3 %, n=26), followed by EAI2-Nonthaburi (4.8 %, n=3), only one EAI1-SOM was observed. When we compared the proportions between EAI and

Beijing and other families among overall drug resistant and susceptible isolates, Beijing showed significant (p=0.01) higher proportion than EAI and other families among drug resistant isolates (95.4 %, 76.7 % and 54.5 % respectively) (Table 3. 6) One orphan isolate was related with beijing. it is worth noting that MIRU-VNTR typing revealed occurrence of mixed infections and/or clonal (Table 3. 3). This mutation was never described as RIF resistance associated mutation. The sensitivity of sequencing for detection of INH resistance was higher than MTBDRplus (95.5 % vs 60.7 %), while the specificity was lower (78.4 % vs 86.7 %). This was due to the detection of (-)15CT (this mutation is known to be associated with low level of INH resistance) in susceptible isolates and the detection of unknown mutations in katG and inhA-coding gene (Table 3. 3). The small number of resistant isolates of EMB, OFX, KAN and CAP (n=2,[START_REF]Global tuberculosis report[END_REF][START_REF]Global tuberculosis report[END_REF]and 4 respectively) did not allow to give a strong evaluation of sequencing performance for these drug resistance.

Regarding STR resistance, sequencing showed high value of sensitivity and specificity, 89.3 % and 94.6 % respectively.

Finally, from this study, we can state that the targeted gene sequencing can be an extremely powerful tool for drug resistance detection. Nevertheless, a first step of evaluation is necessary to establish the link between each mutation present in Lao PDR and the corresponding drug resistance in order to increase the specificity and the sensibility for each resistance. As example, the tlyA should be added in the list of genes and the Met736Thr mutation should not be considered as drug resistance associated mutation. Furthermore, the "disputed" mutations should be used and linked to drug resistance with an estimated probability to orientate the clinical making decision and the treatment prescription.

Conclusion

Globally this study revealed a very low MDR prevalence in Lao PDR with 0.6 % in new cases and the RR-TB 1.2 % among new cases and 4.1 % among previously retreated cases.

The situation of Lao PDR is particular since despite a relatively high TB incidence 168/100,000 in this country, the drug resistance is still low. It is thus urgent to increase the TB and drug resistance detection in order to preclude the rapid emergence of highly drug resistance strains in the country as we observed in the neighboring countries. Furthermore, Beijing family is present in Laos and linked to drug resistance, it is thus essential from now to stop its unavoidable progression that will occur if the TB control is not rapidly improved.

Some main risk factors associated with drug resistance could be identified such as the north part of the country and the Beijing family. Surprisingly, a low number of retreated cases were included in the survey suggesting a basic problem of patient recruitment. This aspect needs to be also rapidly improved in Lao PDR.

Besides, the indispensable need to improve capacity building for a better TB patient detection and care, the drug resistance diagnostic is also a serious concern. This study showed that the sequencing and especially the targeted gene sequencing can really improve the drug resistance diagnostic in terms of time and quality (detection of large drug resistant patterns).

Vietnam, Cambodia and Myanmar showed lower proportion of EAI than that observed in Laos (0.03%, 38.5%, 60%, and 48.4% respectively) but higher proportion of Beijing (74.1% , 38.5 %, 30 % and 31.9% respectively) [10-12, 177]. As previously described, EAI is one of the most ancient and predominant M. tuberculosis family in many Asian countries [START_REF] Gagneux | Variable host-pathogen compatibility in Mycobacterium tuberculosis[END_REF]178]. This family was associated with older population, lower rates of TB transmission and mildly virulent [START_REF] Nguyen | High Prevalence of Beijing and EAI4-VNM Genotypes among M. tuberculosis Isolates in Northern Vietnam: Sampling Effect, Rural and Urban Disparities[END_REF]179]. In contrast, Beijing family was associated with young people, urban areas [START_REF] Nguyen | High Prevalence of Beijing and EAI4-VNM Genotypes among M. tuberculosis Isolates in Northern Vietnam: Sampling Effect, Rural and Urban Disparities[END_REF]180],

greater virulence, drug resistance and highly transmissible [181][182][183]. The findings in Laos are in favor of the endemicity of EAI and the circulation of Beijing family still at low level unlike

Vietnam [184]. This could be explained by the low population density (27 people per km2) of

Lao PDR [185]. Nevertheless, the data can be biased since the participants included in the TBPS were mainly from rural areas with a majority of TB cases collected from older age group [START_REF] Law | The first national tuberculosis prevalence survey of Lao PDR (2010-2011)[END_REF]. EAI family is generally associated to these groups of people as demonstrated in Vietnam but also to the reactivation of latent TB as observed in our study (low rate of recent transmission) [START_REF] Nguyen | High Prevalence of Beijing and EAI4-VNM Genotypes among M. tuberculosis Isolates in Northern Vietnam: Sampling Effect, Rural and Urban Disparities[END_REF] Conversely to population based sampling (TBPS), the Beijing family was more preponderant in hospital based sampling (DRS 2016(DRS -2017)), and presumptive MDR-TB cases (2010-2014)

35 % and 41 % respectively. This finding highlights the association of Beijing family with drug resistant TB and presumptive MDR-TB cases and thus the risk of increase and spread of this family in the country.

Regarding social-geographic distribution, the TBPS sampling showed higher proportion of Beijing in Northern provinces compared to the other provinces, whereas EAI is distributed all over the country. This finding suggests an introduction of Beijing family in the country from the North (N=0 in the South). This result is confirmed by the origin of the DRS isolates and presumptive MDR-TB cases (data not shown). Furthermore, the EAI family was more detected in age group ³ 35 years old, whereas Beijing family was also strongly present in younger age group (lower than 35 years old, 34.5% of Beinjing isolates). Furthermore, the analysis carried out in the framework of the TBPS showed 11.9% of recent transmission with a higher clustering rate in Beijing family compared to EAI (11% versus 20.7%, respectively). These findings suggest that the TB epidemiological pattern in Lao PDR can shortly change with a high risk of increase of Beijing family in the country. This hypothesis is supported by numerous studies that showed the high capacity to produce outbreaks and the epidemiogenic properties of the Beijing family [186][187][188]. This highlights the urgent need to improve the TB detection in the north and to follow the Beijing infection cases to preclude outbreaks and the potential spread all the country scale. It is worth noting that the two molecular typing techniques (Spoligotyping and MIRU-VNTR) used in this study showed their usefulness for molecular epidemiology study. The regular use of these methods will permit to follow the trend of TB and drug resistant TB in Lao PDR and to evaluate the efficacy of the TB control over time.

Ø Link between M. tuberculosis families and drug resistance and the drug resistance transmission in Lao PDR.

The drug resistant isolates identified during our study belong to different M. tuberculosis families (EAI, Beijing, T, H and others). However, the EAI and Beijing were still the most prevalent in all three samplings. Nevertheless, conversely to the distribution in the whole M.

tuberculosis, the proportion of Beijing family was higher in the drug resistant population compared to EAI and other families, and especially among highly drug resistant patterns (such as QDR and pre-XDR cases). Thus in addition to the epidemiogenic properties of the Beijing family demonstrated in the TBPS study, we also demonstrated that this species is specifically associated with drug resistance and highly drug resistance. This highlights a high risk of drug resistance increase due to the highly transmissible Beijing family in Lao PDR. This point is reinforced by the observation of genetic clusters of drug resistant isolates in the TBPS study (a cluster of three INH resistant isolates was found in the Northern Province with epidemiological data link). No cluster was observed in DRS and presumptive MDR-TB cases but for these two used as a frontline test for all presumptive TB and MDR-TB, leading to routinely undiagnosed mono-resistant and poly-resistant TB. This means that undiagnosed mono-and poly-resistant TB are likely often treated with standardized first line drug regimen (2RHEZ/4RH). Some of these patients may experience transient clinical and bacteriological improvement but are at risk for failure or relapse, often with higher resistance patterns. Appropriate treatment of mono-and poly-resistant TB can therefore prevent the development of MDR-TB.

Concerning INH resistance, the overall rate (among TBPS, DRS and presumptive MDR-TB were 6 %, 5 % and 26 % respectively. Furthermore, the rate of mono INH resistance was 3 %, 3 % and 4 % respectively. As globally observed, the mono INH resistance is the most common form of mono resistance, with estimated prevalences ranging between 0 to.-9.5% (0-12.8%

among new cases and 0-30.8% among retreated cases) [START_REF] Wright | Anti-tuberculosis drug resistance in the world: fourth global report: the World Health Organization/International Union Against Tuberculosis and Lung Disease (WHO/UNION) Global Project on Anti-Tuberculosis Drug Resistance Surveillance, 2002-2007[END_REF]. In our study, the distribution of mono INH resistant among presumptive MDR-TB was 57.9 %, 31.6 %, 10.5 % respectively among relapse, failure and return after loss follow up cases. The NTCP needs to consider these data since they can lead to TB treatment failure as previously observed [190]. Moreover when INH is used in combination with RIF during the 4 months of continuous phase for standard regimen, if INH resistance is undiagnosed, RIF is thus effectively used alone corresponding to mono drug therapy. There is thus a high risk of acquisition of RIF resistance as described by Hobby and Lenert 1979 [191].

Besides, the mono resistance to OFX and CAP were identified among new cases from DRS sampling. Since OFX is recently used in TB regimen for MDR-TB treatment (late 2013) and CAP was never used in TB regimen before. The question of acquisition of these resistances arises. The OFX resistance might be due to the extensive use of this antibiotic for the treatment of respiratory diseases other than TB. Nevertheless, the reason for CAP resistance is still unclear. Whatever the etiology of drug resistance acquisition, the detection of this monoresistance underlines a real risk of pre-XDR and XDR emergence in the country. The best management of these OFX and CAP resistance cases is to strictly follow up of patients to prevent the failure of standard treatment regimen which could lead to pre-XDR and XDR emergence in the future.

PZA is one of the core drugs used in the standard first regimen and the second line treatment. Nevertheless, since the conventional DST for PZA is not yet standardized [168,189],

the PZA resistance was neither assessed in the first DSR nor in routinely diagnosis. The sequencing that we carried out in this work showed several mutations in pncA gene (nine in total). The majority of them were reported associated with PZA resistance. One third of these mutations were identified among highly drug resistant patterns (QDR, pre-XDR). This is alarming since critical treatment outcomes were previously observed consequently to PZA resistance [192,193].

Additional resistances to MDR-TB were identified in our study (the sampling of presumptive MDR-TB cases), leading to QDR and pre-XDR TB forms (n=9/17 QDR and 3/17 pre-XDR). The patients infected by FQ resistant are non-eligible for the short course nine-month regimen for MDR-TB. As resistance to both FLDs and SLDs was identified in Lao PDR in our study, the most important questions are how to control and prevent the transmission of such deadly drug resistant strains and what combination of drugs has to be used for an effective treatment. These findings underline an urgent need to screen the resistance to all drugs used for TB and MDR-TB treatment in Laos. All presumptive TB and MDR-TB cases need to have access to the full drug resistance testing, since it is critical for the prescription of appropriate treatment. It is first to increase the cure rate among patients but also to limit the spread of TB and drug resistant TB.

Ø Genetic determinants associated with anti-TB drug resistance and molecular method use for rapid diagnosis of drug resistant TB

The sequencing of the genes involved in drug resistance showed a diversity of mutations associated with drug resistance. However, the most common mutations were mainly observed as previously described: mutations in rpoB gene at codons 526 and 531 [152,171,[194][195][196]; methods such as MICs, targeted gene sequencing or WGS or to find a new diagnostic tool to complete the diagnosic of drug resistance for all the drugs used.

Conclusion

This study provides the first genetic insights into the M. tuberculosis population in Lao PDR. The presence of the main families detected in neighboring countries was determined, mainly EAI and Beijing families. The 11% of recent transmission rate and mini outbreak of drug resistant isolates represent a challenge in Lao PDR. EAI family is predominant in Laos, more prevalent in rural area and in older people but associated with a lower rate of TB transmission and known to be mildly virulent. Conversely, Beijing family, known to be highly transmissible and drug resistant, represents a low frequency in the global population of the TBPS sampling.

Nevertheless, this family was more detected among hospital based sampling collected in the framework of DRS and in the presumptive MDR-TB samplings. It is worth noting that Beijing was especially observed in highly drug resistant patterns such as in QDR and pre-XDR isolates (7/10 QDR and 3/3 pre-XDR isolates respectively). Moreover, this family was significantly observed in younger age group (<35 yrs), and also involved in recent transmission, suggesting a risk of rapid spread in the country from north to south. This situation could change the TB epidemiology in the near future in Lao PDR. This underlines the need to reinforce the efforts to maintain an efficient TB control and surveillance system in order to prevent the emergence of highly transmissible and drug-resistant strains in Lao PDR, as observed in neighboring countries.

Even though the MDR-TB prevalence in Lao PDR is still low compared to neighboring countries, the overall rate and variety of patterns of drug resistance have to attract the attention of NTCP. If mono-and poly-drug resistances are undiagnosed and inappropriately treated, this could lead to critical treatment outcome and amplify the risk of MDR, pre-XDR and XDR emergence in the future. Moreover, our data showed that the MDR-TB cases had additional resistances to other drugs, leading to QDR and pre-XDR-TB. The NTCP should take into account these information since these patients are non-eligible for the shorter 9 month regimen and present a risk to develop XDR that could be then transmitted in the population.

Every single drug resistance needs to be identified, even mono or poly-resistances, in order to monitor on the best way the treatment course and the treatment outcome. The full drug resistance testing is critical for the prescription of appropriate treatment. The consequence it is not just to increase the cure rate among patients but also to limit the spread of TB and drug resistance TB in the community.

The DNA sequencing provided crucial information on mutations associated with FLD and SLDs drug resistance. The result revealed various mutations reflecting the diversity of drug resistant mechanisms in M. tuberculosis in Lao PDR. These findings showed that the surveillance of drug resistance is essential in order to follow the evolution of drug resistance in the country and to prescribe the most appropriate treatment for the patient care. Nevertheless, the available molecular tests target only single or few loci limits the ability for accuracy diagnosis. In order to detect more MDR-TB cases, it is necessary to intensify case finding able to screen MDR-TB among both new and previously treated cases. Furthermore, to complete the knowledge on drug resistance to FLDs and SLDs, molecular methods, especially DNA sequencing should be considered to be applied for all TB cases. Indeed, there is a current need for the development of rapid molecular tests that detect mutations associated with drug resistance in strains of M. tuberculosis with a feasible implementation in the limited resources countries.

Perspectives

This first molecular epidemiology study reflects the extent threat of TB. The data demonstrated the need to emphasize TB control in general, but also in specific areas (high risk provinces such as Northern provinces invaded by Beijing transmission and drug resistance emergence). The data obtained will be the baseline of the families/subfamilies/genotypes of M.

tuberculosis population and of the mutations associated with drug resistance in Lao PDR. These data could be the used to explore the evolution of TB and drug resistant TB in the country by comparison with further analysis. This follow up will permit to evaluate the impact of control improvement and new strategy development set up by the NTCP. The data on mutations associated with drug resistance and the diversity of mutations will be used to complete the database of drug resistant mutations in the region (Laos, Vietnam and Cambodia). These data will be used to develop a diagnostic tool based on DNA chip technology to improve the drug resistance detection in the region. Patients, with mono-and poly-resistant strains identified during our study, who underwent for standard regimen of treatment need to be followed up and assessed for treatment outcome in order to evaluate the efficiency of standard regimen in these patients and the risk of MDR or pre-XDR emergence. This will help NTCP to make appropriate decision concerning the regimen of each patient. The strain with discordant result between phenotypic DST and sequencing, as well as uncommon mutations will be further studied by WGS in order to determine the mechanisms of resistance. These data regarding molecular epidemiology and drug resistance (mono/poly/MDR/pre-XDR) will be reported to NTCP and

MoH. These kind of data are an essential source of information to guide the national making decision towards better strategy for TB control, to adapt the algorithm for diagnosis of TB and drug resistant TB, and to implement the best treatment strategies which combine the effective anti-TB drugs and the appropriate treatment according to the individual drug resistant patterns.

The final goal is to limit the increase of drug resistant TB in Lao PDR. For this, the next step is to implement a molecular diagnostic test based on targeted gene sequencing to get rapidly the full drug resistant patterns and to permit a rapid clinical making decision for the determination of the appropriate treatment regimen for each patient.

2. 4 .

 4 Data analysis ………………………………………………………………………………… 62 Chapter 3 RESULTS AND DISCUSSIONS ………………………………………………………… 63 3.1. Result1 (Paper 1): First insights into the genetic characteristics and drug resistance of Mycobacterium tuberculosis population collected during the first National Tuberculosis Prevalence Survey of Lao PDR (2010-2011) …………………………………………………… 63 3.2. Result 2 (Paper 2): Genetic characterization of Mycobacterium tuberculosis from presumptive MDR-TB in Lao PDR ………………………………………………………………… 85 3.3. Result 3: Molecular analysis of drug resistance in Mycobacterium tuberculosis population collected during the first national anti-tuberculosis Drug Resistance Survey in Lao PDR (2016-2017) ………………………………………………………………………………….. 119 Chapter 4 GENERAL DISCUSSION, CONCLUSION AND PERSPECTIVES ………………… 144 4.1. General discussion ………………………………………………………………………… 144 4.2. Conclusion …………………………………………………………………………………. 151 4.3. Perspectives ……………………………………………………………………………….. 152 ANNEX ………………………………………………………………………………………………… 154 References …………………………………………………………………………………………… 154

  1 RR/MDR-TB NOTIFICATION AND TREATMENT RESULTS (2011-2016) IN LAO PDR ...... 36 TABLE 1. 2 STANDARD 6-MONTH TREATMENT REGIMEN OF 2HRZE/4HR ....................................... 37 TABLE 1. 3 SHORTER MDR-TB REGIMEN .............................................................................................. 38 TABLE 1. 4 TREATMENT REGIMENS FOR THE MANAGEMENT OF MONO-AND POLY-RESISTANT* ............................................................................................................................................................ 40 TABLE 1. 5 ANTI-TB DRUGS AND MEDICINES RECOMMENDED FOR THE TREATMENT OF RR/MDR-TB* ....................................................................................................................................... 41 TABLE 1. 6 MECHANISMS OF ACTION AND MAIN MECHANISMS OF RESISTANCE TO FLDS AND SLDS ................................................................................................................................................... 43 TABLE 1. 7 COMMON GENES INVOLVED IN RESISTANCE OF M. TUBERCULOSIS TO CLASSICAL, NEW AND REPURPOSED ANTI-TB DRUGS* .................................................................................. 47 TABLE 2. 1 PRIMERS USED FOR DNA AMPLIFICATION AND SEQUENCING OF GENES INVOLVED IN ANTI-TB DRUG RESISTANCE ...................................................................................................... TABLE 2. 2 PCR CYCLE AND TEMPERATURE CONDITIONS WITH HOTSTARTAQ ........................... TABLE 3. 1 CHARACTERISTICS OF PATIENTS INCLUDED IN THE DRUG RESISTANCE SURVEY AND MOLECULAR ANALYSIS ......................................................................................................... TABLE 3. 2 DRUG RESISTANCE PATTERNS TO FLD AND SLD BASED ON DRUG SUSCEPTIBILITY TESTING ........................................................................................................................................... TABLE 3. 3 CUMULATIVE FREQUENCY OF ALL MUTATIONS AMONG THE 60 DRUG RESISTANT ISOLATES AND THE 14 SUSCEPTIBLE ISOLATES. ..................................................................... TABLE 3. 4 PERFORMANCE OF XPERT MTB/RIF; MTBDRPLUS/MTBDRSL AND SEQUENCING FOR THE DETECTION OF FLD AND SLD RESISTANCE COMPARED TO DST RESULTS ................. TABLE 3. 5 SPOLIGOTYPING PATTERNS ............................................................................................. TABLE 3. 6 DRUG RESISTANT PATTERNS BASED ON DST ACCORDING TO M. TUBERCULOSIS FAMILIES .......................................................................................................................................... TABLE 3. 7 CHARACTERISTIC OF PATIENTS WITH RESISTANT AND SUSCEPTIBLE M. TUBERCULOSIS ISOLATES ............................................................................................................

FIGURE 1 .

 1 FIGURE 1. 1 ESTIMATED TB INCIDENCE RATES, 2016 (WHO, GLOBAL TB REPORT 2017) .............

  FIGURE 1. 1 ESTIMATED TB INCIDENCE RATES, 2016 (WHO, GLOBAL TB REPORT 2017) ............. FIGURE 1. 2 PERCENTAGE OF NEW TB CASES WITH MDR/RR-TB (WHO, GLOBAL TB REPORT 2017) ................................................................................................................................................... FIGURE 1. 3 PERCENTAGE OF PREVIOUSLY TREATED TB CASES WITH MDR/RR-TB (WHO, GLOBAL TB REPORT 2017) .............................................................................................................. FIGURE 1. 4 M. TUBERCULOSIS COLONIES ON LOWENSTEIN-JENSEN MEDIUM (A); M. TUBERCULOSIS STAINED BY ZIEHL-NEELSEN METHOD (B) AND M. TUBERCULOSIS SCANNING ELECTRON MICROSCOPY (C) ..................................................................................... FIGURE 1. 5 CIRCULAR MAP OF THE CHROMOSOME OF M. TUBERCULOSIS H37RV ....................FIGURE 1. 6 PHYLOGENY OF THE MTBC AND DISTRIBUTION OF THE 7 MAIN M. TUBERCULOSIS LINEAGES ..........................................................................................................................................

FIGURE 1 .

 1 FIGURE 1. 7 TB CASE DETECTION RATE, LAOS, 1995-2017................................................................

FIGURE 1 .

 1 FIGURE 1. 8 EXTERNAL QUALITY ASSESSMENT OF AFB MICROSCOPY (ZIEHL NIELSEN) IN LAO PDR, 2004-2017 .................................................................................................................................

FIGURE 1 .

 1 FIGURE 1. 9 MECHANISMS OF ACTION OF CURRENT ANTI-TB DRUGS ........................................... FIGURE 1. 10 MECHANISMS OF ACTION OF ANTI-TB DRUGS UNDER DEVELOPMENT ..................

FIGURE 2 . 1 FLOWCHART

 21 FIGURE 2. 1 FLOWCHART OF THE STUDY PRESENTING THE THREE DIFFERENT SAMPLINGS USED IN THE STUDY AND THE METHODS APPLIED ON EACH SAMPLING ...............................

FIGURE 2 .

 2 FIGURE 2. 2 XPERT MTB/RIF TESTING ..................................................................................................

FIGURE 2 .

 2 FIGURE 2. 3 MTBDRPLUS VER.1 AND MTBDRSL VER.1 EXAMPLE OF BANDING PATTERNS FOR SENSITIVE AND RESISTANT SAMPLES .........................................................................................

FIGURE 2 .

 2 FIGURE 2. 4 GENOTYPE MYCOBACTERIUM INTERPRETATION CHART ..........................................

FIGURE 2 .

 2 FIGURE 2. 5 DIRECT REPEAT LOCUS AND SCHEMA OF SPOLIGOTYPING ......................................

FIGURE 2 .

 2 FIGURE 2. 6 TANDEM REPEAT VARIABILITY .........................................................................................

FIGURE 2 .

 2 FIGURE 2. 7 CHROMOSOME OF M. TUBERCULOSIS HYPOTHETICAL STRAIN X AND GENOTYPING OF M. BOVIS BCG, THE M. TUBERCULOSIS LABORATORY STRAIN H37RV, AND STRAIN X ON THE BASIS OF IS6110 INSERTION SEQUENCES AND MYCOBACTERIAL INTERSPERSED REPETITIVE UNITS (MIRUS). ...........................................................................................................

FIGURE 3 .

 3 FIGURE 3. 1 ALGORITHM OF THE STUDY ............................................................................................

FIGURE 3 . 1

 31 FIGURE 3. 2 DENDROGRAM BASED ON MIRU-VNTR AND SPOLIGOTYPES PROFILES FROM THE 63 ISOLATES ....................................................................................................................................

  and drug resistance in Lao PDR, this work aimed to study the diversity, the population structure and the genetic determinants of drug resistance in M. tuberculosis clinical samples collected from three different samplings: 1). a population based sampling (First National TB prevalence survey (TBPS), 2010-2011); 2). a routinely consecutive collection of patients with high risk of MDR-TB (Presumptive MDR-TB), 2010-2014 and 3). a hospital based sampling (First national anti-TB drug resistance survey (DRS), 2016-2017) focused on drug resistant isolates. By analysing isolates from these three samplings, we expect to better understand the epidemiology and the extent threat of TB and drug resistant TB in the country; to define what are the different M. tuberculosis families involved in recent transmission and acquisition of drug resistance; what are the genetic determinants of drug resistance in our settings; and what kind of molecular methods can be used for detection of drug resistance TB in Lao PDR. Finally, the results from these studies will be crucial information for National Tuberculosis Control Program (NTCP) and Ministry of Health (MoH) in order to improve the TB control strategy and limit the increase of drug resistance in Lao PDR. In this context, the specific objectives are as follows: 1. To determine the family/subfamily/genotype of M. tuberculosis population circulating in Lao PDR, using 43-spacer oligonucleotide typing (spoligotyping) and 24-locus MIRU-VNTR on M. tuberculosis isolates collected from three different samplings: 1). TBPS (2010-2011); 2). Presumptive MDR-TB (2010-2014) and 3).DRS (2016-2017). 2. To determine the transmission (recent versus ancient transmission) according to M. tuberculosis family and drug resistant patterns by estimation of the clustering rate in the different M. tuberculosis families present in Lao PDR. 3. To describe the structure of M. tuberculosis population by exploring the link between genetic diversity and epidemiological data and drug resistant patterns. 4. To determine the mutations in genes/regions of M. tuberculosis associated with first and second line drug resistance by DNA sequencing 5. To characterize drug resistance patterns and evaluate level of drug resistance by both methods phenotypic and genotypic drug susceptibility testing. 6. To evaluate the performance of different molecular methods of DNA sequencing compared to Xpert MTB/RIF, Genotypes MTBDRplus/Genotype MTBDRsl for detection of first and second line anti-TB drug resistance

Figure 1 . 1

 11 Figure 1. 1 Estimated TB incidence rates, 2016 (WHO, Global TB report 2017)

Figure 1 . 2 Figure 1 . 3

 1213 Figure 1. 2 Percentage of new TB cases with MDR/RR-TB (WHO, Global TB report 2017)

medium (Figure 1 . 4 A

 14 ). Using Ziehl-Neelsen staining or acid-fast staining method, the tubercle bacteria are rod-shaped and bright red (Figure1.4 B). Under electron microscope, the bacteria are about 2 -4 μm in length and 0.2-0.5 μm in width (Figure1.4 C). M. tuberculosis is classified as acid-fast Gram-positive bacteria due to their lack of outer cell membrane.

Figure 1 . 4 M

 14 Figure 1. 4 M. tuberculosis colonies on Lowenstein-Jensen medium (A); M. tuberculosis stained by Ziehl-Neelsen method (B) and M. tuberculosis scanning electron microscopy (C) (Source from http://textbookofbacteriology.net/tuberculosis.html)

Figure 1 . 5

 15 Figure 1. 5 Circular map of the chromosome of M. tuberculosis H37Rv (Source from Cole et al 1998) [45] The outer circle shows the scale in megabases, with 0 representing the origin of replication. The first ring from the exterior denotes the positions of stable RNA genes (tRNAs are blue, and others Circular map of the chromosome of M. tuberculosis H37Rv. The outer circle shows the scale in megabases, with 0 representing the origin of replication. The first ring from the exterior denotes the positions of stable RNA genes (tRNAs are blue, and others are pink) and the direct-repeat region (pink cube); the second ring shows the coding sequence by strand (clockwise, dark green; anticlockwise, light green); the third ring depicts repetitive DNA (insertion sequences, orange; 13E12 REP family, dark pink; prophage, blue); the fourth ring shows the positions of the PPE family members (green); the fifth ring shows the positions of the PE family members (purple, excluding PGRS); and the sixth ring shows the positions of the PGRS sequences (dark red). The histogram (center) represents the G+C content, with <65% G+C in yellow and >65% G+C in red

Figure 1 . 6

 16 Figure 1. 6 Phylogeny of the MTBC and distribution of the 7 main M. tuberculosis lineages (Source from Coscolla and Gagneux 2014) [51].

(

  A) Node support after 1000 bootstrap replications is shown on branches and the tree is rooted by the outgroup M. canettii. Large Sequence Polymorphisms (LSPs) are indicated along branches. Scale bar indicates the number of nucleotide substitutions per site. (B-D) Dominant MTBC lineages per country. Each dot corresponds to 1 of 80 countries represented in the 875 MTBC strains from the global strain collection analyzed by Gagneux et al 2006. [55]. The yellow dot represents the Lineage 7 in Ethiopia and the orange one the extinct MTBC strains from Peru, respectively. Panel (B) shows the most geographically widespread lineages, panel (C) the intermediately distributed lineages, and panel (D) the most geographically restricted lineages.

  same "drug class" or have the same efficacy or safety. The individual description of each group is described in the Companion Handbook to the WHO Guidelines for the Programmatic Management of Drug-Resistant Tuberculosis 2014[START_REF]WHO | Guidelines for the programmatic management of DR-TB. Companion Handbook to the WHO Guidelines for the Programmatic Management of Drug-Resistant Tuberculosis[END_REF] the companion handbook to the WHO Guidelines for the programmatic management of drug resistant tuberculosis, 2014 and the WHO treatment guidelines for drug resistant tuberculosis, 2016 update a This regrouping is intended to guide the design of longer regimens; the composition of the recommended shorter MDR-TB regimen is standardized b Medicines in Groups A and C are shown by decreasing order of usual preference for use c Refer to the text for the conditions under which streptomycin may substitute other injectable agents, Resistance to streptomycin alone does not qualify for the definition of XDR-TB d Carbapenems and clavulanate are meant to be used together; clavulanate is only available in formulations combined with amoxicillin.

and 1 .

 1 10 illustrate briefly the mechanisms of action of current TB drugs and drugs under development respectively.

--Figure 1 . 9 Figure 1 .

 191 Figure 1. 9 Mechanisms of action of current anti-TB Drugs

Figure 2 . 1

 21 Figure 2. 1 Flowchart of the study presenting the three different samplings used in the study and the methods applied on each sampling

2. 3 . 1 .

 31 Drug susceptibility testing (DST)Drug susceptibility testing (DST) was recently available at NRL (2015). Among the three sampling of this thesis, onlyDRS (2016DRS ( -2017) ) had available DST results. The proportion method on solid Löwenstein-Jensen culture was used. A loop of colonies was scrapped from the subculture, transferred into a tube containing glass beads and vortexed to separate the colonies. The concentration of bacilli was adjusted to a Mac Farland No 1 standard. Tenfold dilution was realized until the 10-5 dilution. One hundred µl of the 10-3 dilution was inoculated onto 2 LJ drug free media and all drug containing LJ media. One hundred µl of the 10-5 dilution was inoculated onto 2 LJ drug free media. The minimal inhibitory concentration (MIC) of each drug was chosen following WHO's recommendation: 0.2 µg/ml for INH, 40 µg/ml for RIF, 4.0µg/ml for STR, 2.0 µg/ml for EMB, 30 µg/ml for KAN, 40 µg/ml for CAP and 40 µg/ml for OFX. The interpretation of the resistance was determined according to the proportion method principle based on the number of colonies observed[105, 106] .

  real-time PCR technology to both diagnose TB and detect rifampicin resistance in less than 2 hours. The assay uses molecular beacon technology[107, 108] to detect DNA sequences amplified in a hemi-nested real time-PCR assay. Five different nucleic acid hybridization probes are used in the same multiplex reaction[109, 110]. Each probe is complementary to a different target sequence within the rpoB gene of rifampicin-susceptible M. tuberculosis and is labeled with a differently colored fluorophore. Together, these overlapping probes span the entire 81 base pairs core region of the rpoB gene. M. tuberculosis is identified when at least two of the five probes give positive signals with a cycle threshold (CT) of ≤38 cycles[111, 112]. The system reports resistance, when the difference between the first (early CT) and the last (late CT) M. tuberculosis-specific beacon (ΔCT) was >3.5 cycles (e.g: the first probe CT is >34.5 cycles and the last probe has a CT of >38 cycles). If ΔCT is ≤3.5 cycles, the system reports sensitivity. A semi-quantitative estimate of the concentration of bacilli is defined by the CT range(high, <16; medium, 16-22; low, 22-28; very low, >28).During DRS (2016-2017), Xpert MTB/RIF was performed in parallel with AFB smear microscopic. Specimens analyzed by Xpert MTB/RIF were not treated with CPC. Xpert MTB/RIF testing was performed following Cepheid instructions. Two volumes of the "Sample Reagent" were added to one volume of sputum. The sample was shaken vigorously 10-20 times. After 10 minutes of incubation at room temperature, the sample was shaken again 10-20 times then left at room temperature for another 5 minutes. The sample was then transferred into the cartridge and ready to be loaded in the Xpert MTB/RIF module (Figure2.2)[113].

Figure 2 . 2

 22 Figure 2. 2 Xpert MTB/RIF testing (Source: Xpert MTB/RIF Brochure -Cepheid) A. Five molecular probes overlapping the entire 81 bp core region of the rpoB gene B. The three easy steps of Xpert MTB/RIF testing C. GeneXpert Dx System-Privileged User View Results window, MTB Detected Low, RIF resistance detected

Figure 2 . 3

 23 Figure 2. 3 MTBDRplus ver.1 and MTBDRsl ver.1,example of banding patterns for sensitive and resistant samples For evaluation and interpretation of Mycobacterium CM result, the three control bands (Conjugate Control (CC), Universal Control (UC) and Genus Control (GC)) must be present. Determine species with the help of the interpretation chart (Figure 2.4)

Figure 2 . 4 Figure 2 . 3

 2423 Figure 2. 4 GenoType Mycobacterium interpretation chart

  diversity of direct repeat (DR) locus [114]. The DR locus contains multiple 36-base pair (bp) DRs that are separated by 37 to 41 bp unique spacer sequences (Figure 2.5) [115]. The 43 spacers are commonly used for genotyping [114]. Classical spoligotyping is performed by reverse line blot hybridization of biotinylated PCR products to a membrane with 43 covalently bound synthetic oligonucleotides representing the different spacers selected from M. tuberculosis H37Rv (spacers 1-19, 22-32, and 37-43) and M. bovis BCG (spacers 20-21 and 33-36) (Figure 2.5) [115]. The presence and absence of each spacer is specific for each individual and used for genotyping.

Figure 2 . 5

 25 Figure 2. 5 Direct repeat locus and Schema of spoligotyping

Figure 2 . 6

 26 Figure 2. 6 Tandem repeat variability

Figure 2 . 7

 27 Figure 2. 7 Chromosome of M. tuberculosis hypothetical strain X and Genotyping of M. bovis BCG, the M. tuberculosis laboratory strain H37Rv, and Strain X on the Basis of IS6110 insertion sequences and Mycobacterial Interspersed Repetitive Units (MIRUs).

  (the anonymity of the patients was maintained) was registered and cross-checked by research team before being imported to Stata (v12.1, Stata Corporation, USA) for statistical analyses. Median and interquartile were calculated for age of patients.Comparisons between proportions (e.g. Gender, Age group, Strata, Regions, M. tuberculosis families, Drug resistant patterns) were performed using chi-square analysis and Fisher's exact test; when the sample size was lower than 5. Statistical significance was defined as a P value of <0.05. A genetic cluster was defined as two or more isolates with identical genotype by 43spacer spoligotyping and 24-locus MIRU-VNTR typing. Recent transmission was estimated by calculating the clustering rate as follows: CR = (nc-c)/n, where CR is the clustering rate, nc is the total number of clustered isolates, c is the total number of clusters, and n is the total number of isolates[135]. A Neighbor-Joining (NJ) tree based on categorical distances was built by combining the spoligotyping and MIRU-VNTR results, using MIRU-VNTRplus (http://www.miruvntrplus.org), a freely accessible web-based program [132]. The performances of molecular tests (Xpert MTB/RIF, GenoType MTBDRplus/MTBDRsl and DNA sequencing) were compared to that of a conventional DST for the detection of anti-TB drug resistance. The sensitivity, specificity, predictive positive value (PPV) and Negative predictive value (NPV) was calculated using online tool (https://www.medcalc.org/calc/diagnostic_test.php) population collected during the first National TB Prevalence Survey of Lao PDR (2010-2011) in order to better understand the TB epidemiology in this country. Two hundred and twenty two isolates were analyzed with the GenoType MTBDRplus test for M. tuberculosis identification and drug resistance detection. Then, 206 of the 222 isolates were characterized by spoligotyping and MIRU-VNTR typing.

  central provinces. A higher proportion of Beijing isolates was observed in people younger than 35 years compared to EAI. Moreover, the percentage of drug resistance was higher among Beijing(17.2%) than EAI (5.2%) isolates, and the two MDR-TB isolates belonged to the Beijing family. Combined analysis of the MIRU-VNTR and spoligotyping results (n=202 isolates) revealed an estimated clustering rate of 11% and the occurrence of mini-outbreaks of drugresistant TB caused by Beijing genotypes.Conclusions:The EAI family (the ancient and endemic family in Asia) is predominant in Lao PDR whereas the prevalence of Beijing, the most harmful M. tuberculosis family for humans, is still low, differently from neighboring countries. However, its involvement in recent transmission, its association with drug resistance, and its presence in young patients suggest that the Beijing family could change TB epidemiological pattern in Lao PDR. Therefore, efficient TB control and surveillance systems must be maintained and reinforced to prevent the emergence of highly transmissible and drug-resistant strains in Lao PDR, as observed in neighboring countries. Key words: Molecular epidemiology, Mycobacterium tuberculosis family, Drug-resistant tuberculosis, Lao PDR. Background Tuberculosis (TB) remains a major public health problem. Although the number of TB deaths fell by 22% between 2000 and 2015, TB still was one of the top 10 causes of death worldwide in 2015 (World Health Organization 2016), with an estimated 10.4 million of new TB cases worldwide. Six countries account for 60% of all new cases (India, Indonesia, China, Nigeria, Pakistan and South Africa) and more than half of these cases were in Asia. The emergence of drug-resistance is a global issue for TB control. In 2015, the number of new cases of multidrug-resistant TB (MDR-TB) was estimated at 480 000, with an additional 100,000 people with rifampicin (RIF)-resistant TB who are eligible for MDR-TB treatment. India, China and the Russian Federation accounted for 45% of all these cases. In Southeast Asian countries, TB is one of the top ten communicable diseases with an increasing emergence of MDR-TB and extensively drug-resistant TB (XDR-TB) (Coker et al. 2011). Lao People's democratic Republic (PDR) (population of 6.8 million in 2015) is not among the high TB burden countries. However, this landlocked country is surrounded by China, Myanmar, Cambodia, Vietnam and Thailand that are among the 30 high TB burden countries in the world. The National Tuberculosis Control Program (NTCP) of Lao PDR started the Directly Observed Treatment Short (DOTS) course strategy in 1995 with the support of the Damien Foundation Belgium (DFB) and WHO. After the first national TB prevalence survey (2010-2011),

  tuberculosis population collected during the first National TB Prevalence Survey of Lao PDR (2010-2011) in order to better understand the M. tuberculosis population structure and the TB epidemiology in this country. The specific objectives were: a) to characterize using different molecular methods M. tuberculosis isolates collected during the first national survey; b) to describe the spatial distribution of families and drug resistance; c) to explore the link between genetic diversity and demographical data; d) to estimate the clustering rate according to the M.

  typing was performed as previously described(Supply et al. 2006; Gauthier et al. 2015) and the full set of 24 MIRU-VNTR loci was used for isolate characterization. The patterns obtained for the 24 loci were used to create a 24-digit allelic profile for each isolate.The MIRU-VNTR typing results were analyzed using MIRU-VNTRplus (http://www.miruvntrplus.org), a freely accessible web-based program(Allix-Béguec et al. 2008). A Neighbor-Joining (NJ) tree based on categorical distances was built by combining the spoligotyping and MIRU-VNTR results.

Figure 1 .

 1 Figure 1. Distribution of M. tuberculosis families in the different provinces of Lao PDR (PDF) The numbers on the map (1 to 17) correspond to the provinces divided in three regions (North, Center, and South). The numbers in the pie charts indicate the number of isolates found in each province. Each M. tuberculosis family is represented by a different color (see color code in figure)

Figure 1

 1 Figure 1 Neighbor-joining tree based on the MIRU-VNTR and spoligotyping data for 43 clustered isolates From left to right: i) Neighbor-joining tree based on the 24-locus MIRU-VNTR and spoligotyping data for the 43 isolates grouped in 19 clusters (built using the MIRU-VNTRplus analysis tool; ii) Number of repetitions of each VNTR according to the nomenclature by Supply et al (2006); and iii) 43-spacer spoligotypes: black spots represent the presence and white spot represent the absence of 1-43 spacers(according to the numbering by[START_REF] Van Embden | Genetic Variation and Evolutionary Origin of the Direct Repeat Locus of Mycobacterium tuberculosis Complex Bacteria[END_REF]. Yellow squares, Beijing clusters; orange squares, EAI clusters; dark pink, T clusters.

  study provides the first genetic insights into the M. tuberculosis population in Lao PDR. The presence of the main families detected in neighboring countries, particularly the EAI and Beijing families, and the 11% of recent transmission rate show that TB represents a challenge in Lao PDR. Although, the EAI family is predominant, the diversity of families observed in big cities (Vientiane, Luang Prabang, Khammuane and Savannhaket) highlights the risk of transmission of other families than EAI in the country. Although the Beijing family prevalence is still low, its presence mainly in the northern and central provinces, its association with drug resistance and its involvement in recent transmission (clustering rate = 20% based on rifampicin resistant, 19 isoniazid resistant and 17 MDR). DNA sequencing of 42 available isolates revealed the mutations associated with anti-TB drugs allowing the identification of multigenic patterns and the probable drug resistant profiles (nine mono drug resistant, 11 poly drug resistant, 10 MDR, nine QDR (Quadruple resistance to first line drugs) and three pre-XDR (MDR plus resistance either to one fluoroquinolone or to one second line injectable drug).

  Presumptive MDR-TB, Mycobacterium tuberculosis, Genetic charaterisation, Drugresistant tuberculosis, Lao PDR. Background Tuberculosis (TB) is the ninth leading cause of death worldwide due to a single agent, ranking above HIV/AIDS (Human immunodeficiency virus/Acquired immunodeficiency syndrome). The emergence/re-emergence of drug resistant TB aggravates the situation worldwide and challenges the prospect of ending TB by 2035. Even though the number of multidrug-resistant TB (MDR-TB, Box 1) and rifampicin resistant TB (RR-TB, Box 1) is only 5% of TB incidence, the number of deaths among MDR/RR-TB is almost threefold higher than among susceptible TB (40% vs 16%) . In addition, about 6.2% of MDR-TB cases are extensively drug-resistant (XDR-TB, Box 1). The treatment success rate for these forms is only 30% (World Health Organization 2017). Globally, 4.1% of new cases and 19% of previously treated TB cases was estimated to be RR or MDR-TB. Many studies have identified the factors associated with MDR-TB, including social-demographic, clinic and genetic factors. The common risk factors for MDR-TB are having TB history, contact with confirmed TB patients, TB/HIV coinfection

1 . 2 .

 12 consuming and requires high biosafety level. Nowadays, molecular-based tests like XpertMaterials and Methods Study settings and populationDuring 2010-2014, the specimens of presumptive MDR-TB were collected and sent by health facilities of NTCP network to NRL located in Vientiane capital to perform the cultures.Fresh subcultures were then sent to CILM for molecular testing to identify M. tuberculosis complex (MTBc) and its resistance to RIF and/or INH by the line probe assay (MTBDRplus, Hain Lifescience GmbH, Nehren, Germany). Each isolate was sent with the patient's request form, including socio-demographic and clinical data. The study population included only patients aged ³ 15 years old, who met the 12 presumptive MDR-TB criteria as defined in the guidelines of NTCP established in 2010[START_REF] Ntcp | Final National Technical Guidelines 3rd Edition[END_REF]. In this study, the types of presumptive MDR-TB cases were grouped into 1). Relapse after treated with FLD/SLD; 2). Failure/late smear conversion; 3). Return after loss to follow up; 4). New patient with TB-HIV co-infection; 5). New patient in contact with proven RR /MDR-TB Box 2. Definitions of drug resistance terms used in this paper Mono-resistance: resistance to only one anti-TB drug (First Line Drugs (FLDs) or Second Line Drugs (SLDs)) Poly-resistance: resistance to more than one anti-TB drug (FLD and/or SLD) other than both isoniazid (INH) and rifampicin (RIF) Multidrug resistance (MDR): resistance to at least both INH and RIF Quadruple drug resistance (QDR): MDR plus resistance to at least 2 more FLDs. Pre-Extensive drug resistance (pre-XDR): MDR plus resistance to any fluoroquinolone (FQ) or to any second-line injectable drugs (SLIDs) Extensive drug resistance (XDR): MDR resistance plus resistance to any FQ and at least one of the three SLIDS, capreomycin (CAP), kanamycin (KAN) and amikacin (AMK) Molecular characterization of Mycobacterium tuberculosis Ø GenoType® MTBDRplus and GenoType® Mycobacterium CM kits The GenoType® MTBDRplus ver.1.0 test permitted to identify M. tuberculosis complex and its resistance to R and/or H. The GenoType® Mycobacterium CM ver.1.0 test permitted the identification of the common NonTuberculous mycobacteria (NTM). The subcultures sent by NRL were scraped from media slopes and resuspended in 300µL of distilled water, heated for 20 min at 95°C, centrifuged for 5 minutes at 13000 g. Then the supernatant containing DNA was transferred into a new tube and stored at -80 °c. The GenoType® MTBDRplus ver.1.0 and the GenoType® Mycobacterium CM ver.1.0 tests, a DNA STRIP® based technology, were performed according to the instructions of the manufacturer (Hain Lifescience GmbH).

  remaining isolates did not belong to MTBc. The 16 non MTBc isolates were more observed among failures/late smear conversions (n=5, 31.3 %), relapses (n=5, 31.3%) and HIV infection (n=3, 18.8 %). Thirteen non MTBc isolates could be tested by the GenoType® Mycobacterium CM test. Four different Non tuberculous mycobacteria (NTM) species were identified, M. scrofulaceum (N=4), M. abscessus (N=2), M. chelonae (N=1)

Figure 1

 1 Figure 1 Identification of mycobacteria and resistance to isoniazid (INH) and/or rifampicin (RIF) among presumptive MDR-TB cases a The GenoType® Mycobacterium CM could not identify the NTM species for 4 isolates

  .1).

  had a combination of rrs-F2/rpsL (274GA/ Lys43Arg) b one isolate had a combination of katG/inhA-coding (Ser315Thr/Ile144Val) and one inhA-promoter/inhAcoding ((-15)CT/ Ile21Val) c one mutant rpoB (Leu511Pro) detected by sequencing, this isolate had a combination of katG/inhAcoding (Ser315Thr/Asp335Asn) d one insertion (TGCCAA) in rpoB detected by sequencing

Figure 2

 2 Figure 2 MIRU-VNTR and spoligotyping profiles among 42 resistant isolates

  right: i) Neighbor-joining tree based on the 24-loci MIRU-VNTR and 43-spacer spoligotyping data for the 42 isolates; ii) Number of repetitions of each VNTR according to the nomenclature by Supply et al (2006) (Supply et al. 2006) and iii) 43-spacer spoligotypes: black spots represent the presence and white spot represent the absence of 1-43 spacers (according to the numbering by Van Embden et al. 2000) (Embden et al. 2000). Yellow squares = Beijing isolates; orange squares = EAI; Green square = others (T, H, Orphan Each isolate is represented by the family, the isolate code and the resistant based on sequencing : H = isoniazid, R = rifampicin, E = ethambutol, S = streptomycin, Z = pyrazinamide, F = fluoroquinolone.

Fluoroquinolones(

  FQs) are important bacterial antibiotics currently used as second line treatment for MDR-TB. FQ resistance in M. tuberculosis is mainly due to the acquisition of mutations within the quinolone resistance-determining region (QRDR) of the gyrA gene (codons 74 to 113), globally accounting for nearly 90 % of FQ resistance in M. tuberculosis.Codons 90,[START_REF] Scorpio | Characterization of pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis[END_REF][START_REF] Mikusová | Biogenesis of the mycobacterial cell wall and the site of action of ethambutol[END_REF] are the most mutated sites[START_REF] Miotto | Drug Resistance InMycobacterium tuberculosis: Molecular Mechanisms Challenging Fluoroquinolones and Pyrazinamide Eff Ectiveness[END_REF][START_REF] Maruri | A Systematic Review of Gyrase Mutations Associated with Fluoroquinolone-Resistant Mycobacterium tuberculosis and a Proposed Gyrase Numbering System[END_REF][START_REF] Lau | Molecular Characterization of Fluoroquinolone Resistance in Mycobacterium tuberculosis: Functional Analysis of GyrA Mutation at Position 74[END_REF]. Our study identified four mutations at codons 88, 90, 91 and 94 of the gyrA gene.

  the algorithm of sampling during drug resistance survey (2016-2017) and the selected samples for molecular analysis

  , H = isoniazid; E = ethambutol; S = Streptomycin; FQ = fluoroquinolone; Ofx = Ofloxacin; Km = Kanamycin; Cm= Capreomycin; MDR = multidrug resistance; QDR = quadruple drug resistance. Ø MIRU-VNTR typing By using the 24-locus MIRU-VNTR data alone, the 63 isolates generated only unique profile which corresponded to 63 different patterns. These findings underlined the absence of clusters and thus no case of recent transmission in our sample (Figure 3.2). The tree based on the combination of spoligotyping and MIRU-VNTR globally differentiate correctly beijing from EAI.

Figure 3 . 2

 32 Figure 3. 2 Dendrogram based on MIRU-VNTR and spoligotypes profiles from the 63 isolates From left to right: i) Neighbor-joining tree based on the 24-locus MIRU-VNTR and spoligotyping data from the 63 isolates included in the analysis; ii) Number of repetitions of each VNTR according to the nomenclature by Supply et al. [130] and iii) 43-spacer spoligotypes: black spots represent the presence and white spot represent the absence of 1-43 spacers (according to the numbering of Van Embden et al.)[143]. Yellow squares = Beijing isolates; orange squares = EAI; green square = other families

B. M. tuberculosis lineages and families

	M. tuberculosis is a member of the M. tuberculosis complex (MTBC), which comprises
	three human-adapted species (including 8 lineages), M. tuberculosis (5 lineages),
	Mycobacterium africanum (2 lineages) and Mycobacterium canettii and several animal-sourced
	lineages including Mycobacterium bovis (mainly pathogen of cattle), Mycobacterium caprae
	(pathogen of sheep and goats), Mycobacterium microti (pathogen of voles) and Mycobacterium
	pinnipedii (pathogen of seals and sea lions) (Figure 1.6) [54-59].
	Among the eight human-adapted lineages, the 5 lineages belonging to M. tuberculosis
	are M. tuberculosis lineage 1 (The Philippines and Indian-Ocean), M. tuberculosis lineage 2
	(East-Asian), M. tuberculosis lineage 3 (East-African-Indian), M. tuberculosis lineage 4 (Euro-
	American) and M. tuberculosis lineage 7 (Ethiopia) (Figure 1.6)

Figure 1. 7 TB case detection rate, Laos, 1995-2017 (Source: NTCP Laos) C. National TB Reference Laboratory (NRL) and laboratory network

	B. proportion of bacteriology positive among patients examined was 9.5% in 2013 and 9.2% in National Tuberculosis Center (NTC)
	2014.
	The NTC started directly observed treatment short-course (DOTS) in 1995 with the
	support of WHO and Damien Foundation Belgium (DFB). TB services are integrated in the primary health cares in five central, 18 provincial and 148 districts public hospitals since and currently in more than 1000 health centres. The DOTS has been reached full country coverage from central to village level since 2005 with high treatment success rates since then (treatment success reached 87% among new TB cases in 2016). Despite decades of TB 5% 5% 50 8% 11% 12% 10% 100 14% 13% 15% 16% 17% 20% 24% 27% 27% 28% 27% 29% 32% 31% 32% 35% 49% 45% 50% 450 Estimated TB incidence/100k (WHO) 400 42% 40% 350 37% 15% 20% 25% 30% 35% 150 200 250 300 TB notification rate/100k Rate/100,000 Treatment coverage %
	-	0%
	control, and a 100% DOTS coverage, many people have no access to quality TB diagnosis and
	treatment services; Case notification has stagnated and many cases remain undiagnosed
	Treatment coverage (% notified TB patients among estimated incident new TB cases) was 15%
	in 2000. Since then, thanks to Global Fund continued support, a progressive scaling-up of
	15-49-year-old in 2011) GeneXpert testing capacity among all presumptive TB since end of 2013, an accelerated
	implementation of systematic TB screening (using chest Xray) among TB contacts and other with concentration in particular geographical areas and sub-populations, 6238 HIV cases in 2013 (53, 8 % M and 46.2% F), 3781 AIDS cases (58% men) and 1508 death (61% men) in high-risk groups (22,295 people at higher risk in 8 prisons and 46 other high TB burden areas Ø Microscopy
	2013. Transmission is heterosexual 88%, mother to child 4.9%, homosexual and injection drug representing 1,040 additional new TB cases in 2017). TB treatment coverage has increased to 164 TB laboratories perform quality assured AFB microscopy (ZN) at central, provincial
	use (IDU) 4%, 11% among military and police. Among estimated 11,556 people living with HIV 42% in 2016 and up to 50% in 2017 (Figure 1.7) of the estimated incidence in year 2017 (WHO and district levels. All laboratories participate in the External Quality Assessment (EQA)
	(PLHIV), 4730 are reported alive, 2787 (56%) are under antiretroviral therapy (ART), 2068 with country profile 2018). NTC outreach teams (5 teams in NTC Vientiane and one team in microscopy. Province laboratories re-read blindly a random sample of 25 district slides each
	access to viral load (VL) and 1954 VL <1000 copies. ART coverage is low among pregnant Khamouane province) screened for TB, 11,738 high risk people in 30 sites and notified quarter and NRL re-read samples of provincial and central levels laboratories. NRL confirms
	women HIV positive. Prevention of mother-to-child transmission (PMTCT) was 76% in 2013 additional new TB cases in the first 6 months of 2018 (vs. 1,040 in all year 2017). The National discrepant results (second level). Average 3% of all TB laboratories showed at least one major
	(HIV Epi Review and impact analysis for Lao PDR, June 2014). 93% of the TB patients had an TB Strategic Plan 2017-2020 has set the target of 70% treatment coverage by the end of error in 2017 (Figure 1.8).
	HIV test result available and 80% among 301 TB-HIV patients received ART during their TB in order to achieve a 20% decline in incidence between 2015 and 2020, in line with End TB
	treatment. targets. However, only half of estimated cases was notified (n= 5,934 cases), of which 93 %
	were pulmonary TB [73].
	164 TB laboratories examined 45,356 presumptive TB patients, including 22,521 (50%) by
	Xpert MTB/RIF in 2017, in progress from respectively 41,314 and 19,450 (47%) in 2016. The

Figure 1. 8 External Quality Assessment of AFB microscopy (Ziehl Nielsen) in Lao PDR, 2004-2017 (Source: NTCP Laos) Ø Xpert MTB/RIF system

	26.6% SLDs on strains send by NRL and conducts regular on-site visit (two times per year) to the NRL. 30.0% 160
	104 123 At the NRL, samples processed for culture are inoculated onto a solid culture medium 151 154 154 155 153 155 157 158 159 157 160 25.0% 120 140 No. of participating 20.0% 100 laboratories 13.0% 10.3% 15.0% 80 (average by (Löwenstein-Jensen). If no colony has appeared after 8 weeks, the culture is considered
	51 negative. If bacilli are present in the sample, colonies start to grow on the medium after 4 to 6 5.9% 4.5% 6.0% 4.5% 5.9% 4.0% 6.4% 4.5% 3.9% 4% 3% 0.0% 60 year) 10.0% 40 % lab. with 5.0% 0 20 at least one major error weeks. The colony growth will be graded according to the number of colonies observed: 1-19
		(average by
	year) colonies (Exact number counted), 20-100(1+), 101-200(2+), 201-500(3+), more than
	500/confluent (4+). DST is performed following the growth of the culture and takes another 4
	weeks. The following drugs are tested at NRL: INH (concentration tested 0,2 and 1 µg/ml ), RIF
	(40 µg/ml), STR (4 and 10 µg/ml), EMB (2 µg/ml), these drugs were tested since 2015. And the
	By the end of 2013, the use of Xpert MTB/RIF testing was started in the central level (NRL following were started testing in late 2017 : KAN (30 µg/ml), CAP (40 µg/ml), Ethionamid (40
	and CILM) and Savannakhet provincial hospital in 2014. In 2018, there are total 20 Xpert µg/ml), Cycloserine (30 µg/ml), Para-Amino Salicylic acid (1 µg/ml), Ofloxacin (4 µg/ml),
	MTB/RIF machine in the country. These machines are also distributed in provincial level for Moxifloxacin (2 and 1 µg/ml), Amykacin (30 µg/ml), Levofloxacin (2 µg/ml), Rifabutin (20 µg/ml),
	routine diagnosis and are used for active case finding (ACF) among high-risk groups. Linezolid (2 µg/ml). However, no DST is performed for pyrazinamide.
		The current situation towards Xpert MTB/RIF availabilities in 2018: D.
		• NRL is equipped with 3 Xpert MTB/RIF machines for routine activities
		• CILM (central level) is equipped with 1 Xpert MTB/RIF machine
		• 10 provinces laboratories are equipped with one Xpert MTB/RIF machine
		• 1 provincial laboratory (Champasack) is equipped with 2 machines for routine diagnosis
		• Active case finding (ACF) : 2 machines are kept at central level and 1 is kept at
		Khammouane province
		• 1 machine is kept as spare part at central level
	Ø	Culture and DST
		The National TB Reference Laboratory (NRL) is equipped with BSL-3 room for culture and
	drug sensitivity testing (DST). The Korean Institute of Tuberculosis (KIT) supra national
	reference laboratory (SRL) performs DST external quality assessment (EQA) for FLDs and

Programmatic Management of Drug-Resistant TB

	have access to rapid test for RR using Xpert MTB/RIF test. Once RR is detected, patients are
	referred to the MDR treatment unit, for sample collection for culture and DST; for
	fluoroquinolones (FQs) and second line injectable drugs (SLIDs) resistance testing (using
	GenoType MTBDRsl test) and other examinations (liver and renal function, EKG) before
	initiating a SLD treatment. MDR-TB units are set up in Setthatthirath Hospital, Vientiane capital
	since 2011 and in Khammuane and Luangprabang provincial hospitals since 2016.
	The estimation of total MDR-TB/RR-TB was 2.5 per 100,000 (170 cases) and 76 cases
	among notified pulmonary TB, while 38 MDR-TB/RR-TB cases were confirmed. There were
	about half (56 %) of new cases and only one third (36 %) of previously treated cases were
	tested for drug resistance (RR) [73].
	Active case finding and search of MDR among new cases contribute to an increasing
	number of MDR-TB cases detected and started on SLD treatment. However early enrolment
	and MDR-TB patients management need to be improved. Indeed, the number of MDR-TB
	patients notified and enrolled in treatment increased from 14 in 2013, 25 in 2014 to 34 in 2017.
	-Thirty two among 38 MDR-TB patients diagnosed were enrolled on treatment in 2015 and
	Regarding drug resistant TB little is known. Only data of resistance to INH and rifampicin 27 MDR-TB patients (84%) were treated successfully in 2015 while 33/38 MDR-TB patients
	RIF was explored by a multicentric-study in three regional hospitals in Laos conducted in 2010. were enrolled and 27 (82%) were treated successfully in 2016 (Table 1.1). 34 MDR-TB
	The study has shown that out of 87 MTBc isolates, seven (8 %) cases were INH mono-resistant patients started a shorter 9-month MDR treatment in 2017 and 24 during the first 6 months
	(6.8 % (5/73) among new cases and 14.3 % (2/14) among previously treated cases), one (1.1 of 2018. MDR treatment success was 82% in the 2016 cohort.
	%) was MDR (7.1 % (1/14) among previously treated cases) [1]. National TB Control Program -Most of the RR/MDR-TB patients are tested sensitive to FQs and SLIDs by LPA and can
	(NTCP) started the programmatic management of drug resistant TB in 2011 with 24 month receive shorter 9-month MDR treatment. However, few RR/MDR-TB patients require an
	treatment regimen and switched to shorter 9-month MDR treatment (4Km-Mfx-Pto-Cfz-H-E-adjusted regimen due to either resistance or intolerance to some of the SLDs. Currently the
	Z/5Mfx-Cfz-E-Z) in 2013, which has become the standard MDR-TB treatment for the patients programme has no direct rapid access to Bedaquilin or other SLDs. NTC has been reporting
	sensitive to FQs and SLIDs after LPA testing by GenoType MTBDRsl test (HainLife Science) in a relatively small number of treatment failure each year over the past ten years
	CILM.
	To date, the current national guidelines allow all presumptive TB and MDR-TB cases to

Table 1 . 1 RR/MDR-TB notification and treatment results (2011-2016) in Lao PDR

 1

	(Source: NTCP and MDR-TB treatment centre, *start of 9Month MDR-TB regimen)
	E.	TB treatment in Lao PDR
	TB treatment in Laos is based on three main groups of patients: 1). Patients infected
	with drug susceptible TB, 2). Patients infected with RR/MDR-TB eligible for the short 9 month
	regimen, 3). Patients infected with RR/MDR-

TB not eligible for the short 9 month regimen Ø Treatment regimens for drug-susceptible tuberculosis

	MDR-TB Lao PDR	2011	2012	2013*	2014	2015	2016
	Diagnosed RR/MDR	8	18	13	27	38	38
	Died before treatment	3	1	1	-	1	3
	Other treatment	1	2	1	-	-	1
	Refused treatment	-	-	3	2	3	1
	No drug resistance	-	2	1	-	-	-
	Transferred out	-	-	-	1	2	0
	Started on MDR treatment	4	13	7	24	32	33
	Cured	3	6	5	18	25	25
	Complete	-	-	-	-	2	2
	Died	1	5	1	5	2	4
	Lost of follow up	-	2	1	1	3	2
	% successfully treated	75%	46%	71.4%	75%	84%	82%
	TB drugs treatment can be administrated in 2 forms: it can be a fixed-dose combination
	tablet or a separate drug formulation treatment. However, the WHO recommends the use of
	4FDC (4-drug fixed dose combinations) tablets over the loose pills. FDCs may provide
	programme benefits by making the ordering of medication easier, simplifying supply chain
	management, reducing the occurrence of stock-outs, and facilitating drug delivery and
	prescription preparation. If at all possible, always use RHZE (4FDC) tablets in adult patients.
	4FDC tablets are simple and easy to use. They make it easier for the health worker to prescribe

Table 1 . 2 Standard 6-month treatment regimen of 2HRZE/4HR Prescription using 4FDC tablets

 1

	Treatment phase	Months	Drug	Administration	Weight in kg (adult) 25-37 38-54 >54
	Intensive	1 st to 2 nd	RHZE (4FDC)	daily	2	3	4
	Continuation	3 rd to 6 th	RH 150/75	daily	2	3	4
		Prescription using loose pills
	Treatment phase	Months	Drug	Administration	Weight in kg (adult) 25-37 38-54 >54
			RH 150/75		2	3	4
	Intensive	1 st to 2 nd	Z 400	daily	2	3	4
			E 400		1.5	2	3
	Continuation	3 rd to 6 th	RH 150/75	daily	2	3	4

Table 1 . 4 Treatment regimens for the management of mono-and poly-resistant*

 1 Table from the companion handbook to the WHO Guidelines for the programmatic management of drug resistant tuberculosis, 2014[START_REF]WHO | Guidelines for the programmatic management of DR-TB. Companion Handbook to the WHO Guidelines for the Programmatic Management of Drug-Resistant Tuberculosis[END_REF]

	Pattern of drug resistance	Suggested regimen	Minimum duration of treatment	Comments
			(months)
	INH (± SM)	RIF, PZA, and EMB (± fluoroquinolone)	6-9 months
	INH and EMB	RIF, PZA, and fluoroquinolone	6-9 months	A longer duration of treatment should be used for patients with extensive disease.
	INH and PZA	RIF, EMB, and fluoroquinolone	9-12 months	A longer duration of treatment should be used for patients with extensive disease.
		RIF, fluoroquinolone,
	INH, EMB, PZA (± SM)	plus an oral second-line agent, plus an injectable agent for the first 2-3	9-12 months	A longer course (6 months) of the injectable may strengthen the regimen for patients with extensive disease.
		months
		INH, EMB,
	RIF	fluoroquinolone, plus at	12-18 months
		least 2 months of PZA
		INH, PZA,
	RIF and EMB (± SM)	fluoroquinolone, plus an injectable agent for at least the first 2-3	12-18 months	A longer course (6 months) of the injectable may strengthen the regimen for patients with extensive disease.
		months
		INH, EMB,
	RIF and PZA (± SM)	fluoroquinolone, plus an injectable agent for at least the first 2-3	18 months	A longer course (6 months) of the injectable may strengthen the regimen for patients with extensive disease.
		months
	PZA	INH, RIF	9 months	Most commonly seen in M. bovis infections.

A fluoroquinolone may strengthen the regimen for patients with extensive disease. For additional options, see section: Isolated resistance to INH in

[START_REF]WHO | Guidelines for the programmatic management of DR-TB. Companion Handbook to the WHO Guidelines for the Programmatic Management of Drug-Resistant Tuberculosis[END_REF]

.

An injectable drug may strengthen the regimen for patients with extensive disease. For additional options, see section: Isolated resistance to RIF.

[START_REF]WHO | Guidelines for the programmatic management of DR-TB. Companion Handbook to the WHO Guidelines for the Programmatic Management of Drug-Resistant Tuberculosis[END_REF]

*

Table 1 . 7 Common genes involved in resistance of M. tuberculosis to classical, new and repurposed anti-TB drugs*

 1 bacteria[103]. At present, there are several mutations (SNPs, insertions or deletions of bases) in genes or genomic regions of M. tuberculosis described associated with anti-TB drug resistance (Table1.7); however the most studies are the ones related to the FLD and the core SLD resistance, such as katG gene, inhA gene coding and the inhA promoter (INH resistance);

	rpoB gene (RIF resistance); rpsL gene and rrs-F1 fragment (STR resistance); embB gene (EMB
	resistance); pncA gene and its promoter (PZA resistance), gyrA and gyrB genes (FQ
	resistance), rrs-F2 fragment (SLID resistance).
	Drug/gene	Associated MIC (mg/L)	Mutation frequency among resistant isolates (%)	Compensatory mechanisms
	Isoniazid
	katG	0.02-0.2	70	oxyR0 and ahpC
	inhA		10
	kasA		10
	Rifampicin
	rpoB	0.05-1	95	rpoA and rpoC
	Ethambutol
	embB	1-5	70	unknown
	ubiA		45, occurs with embB mutations
	Pyrazinamide
	pncA	16-100	99	unknown
	rpsA		no clinical evidence
	panD		no clinical evidence
	Streptomycin
	rpsL	2-8	6	unknown
	rrs		<10
			clinical relevance to be
	gidB		determined
	Fluoroquinolones
				gyrA (T80A and
	gyrA	0.5-2.5	90	A90G)
	gyrB		<5	putative gyrB

The figure 2.1 presents the flowchart of the study. Each sampling (the first national tuberculosis prevalence survey in Lao PDR (TBPS) 2010-2011, the presumptive MDR-TB 2010-2014 and the first national anti-tuberculosis drug resistance survey (DRS) 2016-2017)

is detailed in "Results" section.

rpoB, katG, inhA, embB, pncA, gyrA, gyrB, rrs (F2), rpsL, rrs (F1) rpoB, katG, inhA, embB, pncA, gyrA, gyrB, rrs (F2)

, rpsL, rrs (F1) Drug susceptibility testing:

Proportional method on LJ culture

Table 2 . 1 Primers used for DNA amplification and sequencing of genes involved in anti-TB drug resistance

 2

	Drug(s)	Gene/gene promoter	Primer sequence	Annealing T °C	Length (bp)	Target region
	RIF	rpoB-F1	F-rpoB1: 5'-GTCGACGCTGACCGAAGAAG-3' R-rpoB1:	62	1148	Clusters I (including RRDR), II, III

  The other 202 isolates had 58 different spoligotype profiles among which 41 spoligotypes were unique and 17 patterns allowed the clustering of 161 isolates. Each cluster contained 2 to 40 isolates (average = 9). Moreover, 165 isolates (81.68% of 202) were assigned to 29 SITs and seven families present in the SpoIDB4 database; two (1.0%) were unknown; and 35(17.3%) were orphans. The 35 orphan and the two unknown isolates were then compared using SPOTCLUST. Finally, isolates could be classified in seven M. tuberculosis families and ten subfamilies (Table1). EAI was the predominant family (76.7%, n=155 isolates), followed by

Beijing

(14.4%, n=29)

and T

(5.5%, n=11)

. Five isolates (2.5%) belonged to other families, such as Haarlem (H), Central Asian Strain (CAS), Latin American-Mediterranean (LAM), and Manu.

Table 1 . Distinct spoligotyping patterns obtained for the 206 M. tuberculosis isolates under study 201

 1

	N	Spoligotype 43-spacer patterns	SITs	SPOLDB4 family/subfamily	SPOTCLUST (probability)*	Final defined family/subfamily	N. of isolates (%)
	1	¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢oooo¢o¢¢¢¢¢¢¢¢¢	236	EAI5		EAI5	42 (20.4) a
	2	oooooooooooooooooooooooooooooooooo¢¢¢¢¢¢¢¢¢	1	Beijing		Beijing	26 (12.6)
	3	¢¢¢¢¢¢¢oooo¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢oooo¢o¢¢¢¢¢¢¢¢¢	951	EAI5		EAI5	19 (9.2)
	4	¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢oooo¢o¢¢¢¢¢o¢¢¢	48	EAI1-SOM		EAI1-SOM	12 (5.8)
	5	¢¢o¢¢¢¢oooooooooooooooooo¢¢¢oooo¢o¢¢¢¢¢¢¢¢¢	89	EAI2-Nonthaburi		EAI2-Nonthaburi	12 (5.8)
	6	¢¢¢¢¢¢¢o¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢oooo¢o¢¢¢¢¢¢¢¢¢	939	EAI5		EAI5	11 (5.3)
	7	¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢oo¢oooo¢o¢¢¢¢¢¢¢¢¢	139	EAI4-VNM		EAI4-VNM	(3.9)
	8	¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢oooo¢¢¢¢¢¢¢	53	T1		T1	(2.9)
	9	¢¢¢¢¢¢¢¢¢¢o¢¢¢¢¢¢¢¢¢¢¢o¢¢¢¢¢oooo¢o¢¢¢¢¢¢¢¢¢	1513	EAI6-BGD1		EAI6-BGD1	(2.9)
	10 ¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢oooo¢o¢¢¢¢o¢¢¢¢	256	EAI5		EAI5	(1.5)
	11 ¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢oooo¢o¢¢¢¢o¢¢¢o	1801	EAI1-SOM		EAI1-SOM	(1.0)
	12 ¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢o¢¢¢¢¢¢¢¢¢¢oooo¢o¢¢¢¢o¢¢¢¢	735	EAI1-SOM		EAI1-SOM	(1.0)
	13 ¢¢¢o¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢oooo¢o¢¢¢¢o¢¢¢¢	711	EAI1-SOM		EAI1-SOM	(1.0)
	14 ¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢o¢¢¢¢¢¢¢¢¢oooo¢o¢¢¢¢¢¢¢¢¢	2671	EAI5		EAI5	2(1.0) b
	15 ¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢oooo¢o¢¢ooo¢¢¢¢	934	EAI5		EAI5	(0.5)
	16 ¢¢¢¢¢¢¢¢¢¢¢¢o¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢oooo¢o¢¢¢¢¢¢¢¢¢	618	EAI5		EAI5	(0.5)
	17 ¢¢¢¢¢oo¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢oooo¢o¢¢¢¢¢¢¢¢¢	792	EAI5		EAI5	(0.5)
	18 ¢¢¢o¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢oooo¢o¢¢¢¢¢¢¢¢¢	204	EAI5		EAI5	(0.5)
	19 ¢o¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢oooo¢o¢¢¢¢¢¢¢¢¢	380	EAI5		EAI5	(0.5)
	20 oo¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢oooo¢o¢¢¢¢¢¢¢¢¢	470	EAI5		EAI5	(0.5)
	21 ¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢o¢¢¢¢¢oooo¢o¢¢o¢¢¢¢¢¢	292	EAI6-BGD1		EAI6-BGD1	(0.5)
	22 ¢¢¢¢¢¢¢¢¢¢¢¢o¢¢¢¢¢¢¢¢¢¢¢¢oo¢oooo¢o¢¢¢¢¢¢¢¢¢	564	EAI4-VNM		EAI4-VNM	(0.5)
	23 ¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢oooo¢¢¢o¢¢¢	52	T2		T2	(0.5)
	24 ¢¢¢¢¢¢¢¢¢¢¢¢oooo¢¢¢¢¢¢o¢¢¢¢¢¢¢¢¢oooo¢¢¢¢¢¢¢	214	T5		T5	(0.5)
	25 ¢¢¢o¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢oooo¢¢¢¢¢¢¢¢oooo¢¢¢¢¢¢¢	388	LAM9		LAM9	(0.5)
	26 ¢¢¢oooooooooooooooooooooooooooooooooooooooo	2148	CAS		CAS	(0.5)
	27 ¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢oo¢¢¢¢¢¢¢¢¢	54	Manu2		Manu2	(0.5)
	28 ¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢	523	Manu ancestor		Manu ancestor	(0.5) c
	29 ooooooooooooooooooooooooooooooooooooo¢¢¢¢¢¢	250	Beijing-like		Beijing	(0.5)
	30 oooooooooooooooooooooooooooooooooo¢¢o¢¢¢o¢¢	Orphan	Orphan	Beijing* (0.99)	Beijing*	(1.0)
	31 ¢¢¢¢¢¢¢o¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢oooo¢o¢¢¢oo¢¢¢¢	Orphan	Orphan	EAI5* (0.99)	EAI5*	(1.9)
	32 ¢¢¢¢¢¢¢o¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢oooo¢oo¢¢¢¢¢¢¢¢	Orphan	Orphan	EAI5* (0.99)	EAI5*	(1.9)
	33 ¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢o¢¢¢¢¢¢¢¢¢¢oooo¢o¢¢¢¢¢¢¢¢¢	Orphan	Orphan	EAI5* (0.98)	EAI5*	(1.0)
	34 ¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢oooo¢o¢¢oo¢¢¢¢¢	Orphan	Orphan	EAI5* (0.98)	EAI5*	(0.5)
	35 ¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢oooo¢ooo¢¢¢o¢¢¢	Orphan	Orphan	EAI5* (0.96)	EAI5*	(0.5)
	36 ¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢oo¢¢¢¢¢oooo¢o¢¢oooo¢¢¢	Orphan	Orphan	EAI5* (0.99)	EAI5*	(0.5)
	37 ¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢o¢¢¢¢¢¢¢¢¢¢oooo¢o¢¢¢¢¢o¢¢o	Orphan	Orphan	EAI5* (0.99)	EAI5*	(0.5)
	38 ¢¢¢¢¢¢¢¢¢¢¢¢¢¢oo¢¢¢¢¢¢¢¢¢¢¢¢oooo¢o¢¢¢¢¢¢¢¢¢	Orphan	Orphan	EAI5* (0.98)	EAI5*	(0.5)
	39 ¢¢¢¢¢¢¢¢¢¢¢o¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢oooooo¢¢¢¢¢¢¢¢¢	Orphan	Orphan	EAI5* (0.95)	EAI5*	(0.5)
	40 ¢¢¢¢¢¢¢¢¢¢¢oooo¢¢¢¢¢¢¢¢¢¢o¢¢oooo¢o¢¢¢¢¢¢¢¢¢	Orphan	Orphan	EAI5* (0.99)	EAI5*	(0.5)
	41 ¢¢¢¢¢¢¢¢¢¢oooo¢¢¢¢¢¢¢¢¢¢¢¢¢¢oooo¢o¢¢¢¢¢o¢¢¢	Orphan	Orphan	EAI5* (0.99)	EAI5*	(0.5)
	42 ¢¢¢¢¢¢¢o¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢oooo¢o¢¢¢ooo¢¢¢	Orphan	Orphan	EAI5* (0.99)	EAI5*	(0.5)
	43 ¢¢¢¢¢¢¢o¢¢¢¢¢¢¢¢o¢¢¢¢¢¢¢¢¢¢¢oooo¢o¢¢¢¢¢¢oo¢	Orphan	Orphan	EAI5* (0.93)	EAI5*	(0.5)
	44 ¢¢¢¢¢¢¢o¢¢¢¢¢¢¢¢ooo¢¢¢¢¢¢¢¢¢oooo¢o¢¢¢¢¢o¢¢¢	Orphan	Orphan	EAI5* (0.99)	EAI5*	(0.5)
	45 ¢¢¢¢¢¢¢ooo¢¢¢¢¢¢¢¢o¢¢¢¢¢¢¢¢¢oooo¢o¢¢¢¢¢o¢¢¢	Orphan	Orphan	EAI5* (0.99)	EAI5*	(0.5)
	46 ¢¢¢¢¢o¢oooo¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢oooo¢o¢¢¢¢¢¢¢¢¢	Orphan	Orphan	EAI5* (0.99)	EAI5*	(0.5)
	47 ¢¢¢o¢¢¢o¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢oooo¢o¢¢¢oo¢¢¢¢	Orphan	Orphan	EAI5* (0.99)	EAI5*	(0.5)
	48 o¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢oooo¢o¢¢¢¢¢¢¢¢¢	Orphan	Orphan	EAI5* (0.98)	EAI5*	(0.5)
	49 oooo¢¢o¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢oooo¢o¢¢¢¢¢¢¢¢¢	Orphan	Orphan	EAI5* (0.99)	EAI5*	(0.5)
	50 ¢o¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢o¢¢¢¢¢oooooo¢¢¢¢¢¢¢¢¢	1436	Unknown	EAI5* (0.98)	EAI5*	(0.5)
	51 ¢oo¢¢¢¢oooooooooooooooooo¢¢¢oooo¢o¢¢¢¢¢¢¢¢¢	Orphan	Orphan	EAI2* (0.90)	EAI2*	(0.5)
	52 ¢oooo¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢oooooooooooo¢¢¢	Orphan	Orphan	EAI3* (0.94)	EAI3*	(0.5)
	53 ¢¢¢¢¢¢¢o¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢oooooooooo¢¢¢¢¢¢¢	Orphan	Orphan	Haarlem1*(0.97)	Haarlem1*	(0.5)
	54 oo¢¢¢¢¢¢¢o¢¢¢¢¢¢¢¢¢¢¢¢¢¢ooooooo¢oooo¢¢¢¢¢¢¢	Orphan	Orphan	Haarlem1*(0.99)	Haarlem1*	(0.5)
	55 ¢¢¢¢¢¢¢¢¢¢¢o¢ooooooooooo¢¢ooooooooooo¢¢¢¢¢¢	Orphan	Orphan	T4* (0.99)	T4*	(0.5)
	56 ¢¢¢¢¢¢¢¢¢¢¢¢o¢¢o¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢oooo¢¢¢¢¢¢¢	Orphan	Orphan	T1* (0.99)	T1*	(0.5)
	57 ¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢o¢¢¢¢¢¢¢¢¢oooo¢¢¢o¢¢¢	943	Ambiguous: T2 T5	T1* (0.99)	T1*	(0.5)
	58 ¢¢¢¢¢¢¢¢¢¢¢¢ooooooooooooooooooooooo¢¢¢¢o¢¢¢	1083	Unknown	Unknown	Unknown	(0.5)
	59 ¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢¢oooo¢¢¢¢¢¢oooo¢o¢¢¢¢¢¢¢¢¢	Orphan	Orphan	Orphan	Orphan	(0.5)

Table 2 . Characteristics of the patients infected with EAI (76.7%) or Beijing isolates (14.4%)

 2 of Beijing family was higher in the "15-34" group compared to EAI (34.5%, 10/29 vs 10.3%, 16/155), and the percentage of EAI family higher in the "

	(54.8%, 85/155 vs 34.5%, 10/29). Their geographical distribution also was significantly different
	(p=0.001, Table 3). In the North and Center, the percentage of Beijing isolates was higher than
	that of EAI isolates (58.6% and 41.4% vs 37.4% and 29.0% respectively), whereas the Beijing
	family was not observed in the South. Similarly, drug-resistance was higher in the Beijing than
	EAI family (p=0.03): 17.2% (5/29) of Beijing isolates were resistant to RIF and/or INH compared
	with 5.2% (8/155) of EAI isolates. Conversely, the proportion of Beijing and EAI isolates was not
	significantly different when patients were divided according to sex and strata (urban versus
	rural) (Table 3).
	Characteristics	Patients infected with EAI, n=155 (%)	Patients infected with Beijing, n=29 (%)	p-value
	Age group (years)
	15-34 35-64	16 (10.3) 85 (54.8)	10 (34.5) 10 (34.5)	0.002
	≥65	54 (34.8)	9 (31.0)
	Sex
	Men Women	105 (67.7) 50 (32.3)	15 (51.7) 14 (48.3)	0.09
	Strata
	Rural Urban	134 (86.5 ) 21 (13.5 )	22(75.9) 7(24.1)	0.14
	Regions
	North	58 (37.4)	17(58.6)
	Centre	45 (29.0)	12(41.4)	<0.001
	South	52 (33.6)	0
	Anti-Drug resistance status	a
	Sensitive Resistant b C	147 (94.8) 8 (5.2)	24(82.8) 5 d (17.2)	0.03

a Tested with the MTBDRplus test for Rifampicin (RIF) and isoniazid (INH) resistance. b Sensitive to INH and RIF c Isolates were considered resistant when they were INH and/or RIF-resistant. d Contains two isolates resistant to both INH and RIF (MDR-TB).

Table 3 . Estimation of the clustering rate for the EAI, Beijing and T families

 3

	Characteristics	EAI	Beijing	T
	Total number of isolates	155	29	11
	Unique isolates	123	20	9
	Clustered isolates	32	9	2
	N. of clusters	15	3	1
	Clustering rate	11.0%	20.7%	9.1%

(Dong et al. 2010)

. Conversely, the proportion of isolates belonging to minor families (T, H, CAS, LAM, and MANU) was lower in Lao PDR than in Vietnam and Myanmar

(7.9% vs 23% and 15%, respectively)

(Phyu et al. 2009; Nguyen et al. 2012)

. Moreover, only one isolate belonged to the CAS family, which is totally absent in Cambodia and Vietnam

(Zhang et al. 2011; Nguyen et al. 2012

). This result is in agreement with the reported low prevalence of CAS isolates in Southeast Asia, differently from South-Central Asia (56.5% in Pakistan, 26% in India)

[START_REF] Ali | Mycobacterium Tuberculosis Central Asian Strain (CAS) Lineage Strains in Pakistan Reveal Lower Diversity of MIRU Loci than Other Strains[END_REF][START_REF] Gutierrez | Predominance of Ancestral Lineages of Mycobacterium Tuberculosis in India[END_REF]

.

Our findings indicate that the M. tuberculosis population in Lao PDR is mainly composed of strains belonging to the EAI (76.7%) and Beijing (14.4%) families, similarly to neighboring countries but in different proportions. Indeed, in Cambodia and Myanmar, the EAI family is predominant (60% and 48.4% respectively), but the Beijing family also is highly prevalent (30%, and 31.9%)

(Phyu et al. 2009; Zhang et al. 2011)

. In Vietnam, the Beijing and EAI families represent 38.5%/each of the M. tuberculosis population (Beijing isolates were found particularly in urban areas with high population density, such as Hanoi and Ho Chi Minh)

(Nguyen et al. (30.6%)

. On the other hand, EAI4-VNM, which was mainly identified in Vietnam (65.9%), was less frequent (4.5%) and found only in the central provinces. These data suggest that EAI5 is the most ancient M. tuberculosis family circulating in Lao PDR. The long history of socialeconomic exchange with neighboring countries has undoubtedly favored the spread of specific genotypes in the country. The "4th Population and Housing Census" (PHC) of 2015 estimated the global number of migrants at 42,000 (Lao PDR-Population and Housing Census 2015). Most of them came from Thailand (37%), Vietnam (26%), China (23%), Myanmar (6%) and Cambodia (1%). Currently, Vientiane Capital hosts the largest proportion of migrants, and this could explain the high diversity of M. tuberculosis families (n=5) observed in this province compared with most of the other provinces (0 to 4 families) (Figure

1

and Table

S1

). Migrants from China and Myanmar live mostly in northern provinces, those from Thailand are mainly in the central part of the country, and migrants from Vietnam are found in the center and in Attapeu province in the South (Lao PDR-Population and Housing Census 2015). The number of migrants from Cambodia (1%) is very low compared with those from other neighboring countries

  To explore the genetic diversity of M. tuberculosis population in Lao PDR, 202 isolates observed in the capital city. This could be explained by the global low population density in cities and the higher patients' recruitment in rural areas than in urban areas in our study.The proportion of the two main families was significantly different in function of the age group, region of origin and drug-resistant status. The proportion of isolates belonging to the EAI family was higher in the 35-64 age group, as observed in Cambodia, Vietnam and Myanmar, reflecting the endemic circulation of EAI in this part of the world. On the other hand, in Lao PDR the proportion of Beijing isolates in the 15-34 and 35-64 age groups was similar, whereas in Vietnam the proportion of Beijing isolates decreases with age(Nguyen et al. 2012).

	were characterized by spoligotyping and MIRU-VNTR typing. The results revealed 178
	genotypes, a result similar to the one reported for Cambodia (91 patterns in 105 isolates) and
	Epidemiological consideration and drug resistant TB
	higher than that for Vietnam (153 genotypes for 221 isolates) (Zhang et al. 2011; Nguyen et al.
	2012). As expected, the EAI family was more diverse than the Beijing family (138 genotypes for
	155 isolates vs 23 genotypes for 29 isolates). The 19 clusters grouped 43 isolates that belonged
	only to the three main families (EAI, Beijing and T). The overall clustering rate was 11.9%,
	hypothesis that Beijing, as demonstrated in many studies, is more involved in recent
	transmission than the other families in Laos. EAI isolate predominance, higher diversity and
	lower clustering rate compared with the Beijing family reinforce the hypothesis that the EAI
	family (specifically the EAI5 sub-family) is the more ancient M. tuberculosis family in Lao PDR.
	Most isolates in clusters (16 of the 19 clusters, and 37 of the 43 clustered isolates) were
	geographically linked, reflecting the occurrence of recent transmissions. Clusters were mainly

reflecting a non-negligible level of recent transmission compared with high TB burden countries, such as Vietnam (16.3%)

(Nguyen et al. 2012)

and China (18.4%)

[START_REF] Yang | Transmission of Mycobacterium Tuberculosis in China: A Population-Based Molecular Epidemiologic Study[END_REF]

. Moreover, the Beijing family clustering rate was higher than the clustering rates of the other families (20.7% for Beijing vs 11.0% for EAI vs 9.1% for T), suggesting a higher involvement of the Beijing family in recent transmission cases, as demonstrated in many studies

(Nguyen et al. 2012;[START_REF] Wang | Genotypes and Characteristics of Clustering and Drug Susceptibility of Mycobacterium Tuberculosis Isolates Collected in Heilongjiang Province, China[END_REF][START_REF] Iwamoto | Genetic Diversity and Transmission Characteristics of Beijing Family Strains of Mycobacterium Tuberculosis in Peru[END_REF][START_REF] Niemann | Mycobacterium Tuberculosis Beijing Lineage Favors the Spread of Multidrug-Resistant Tuberculosis in the Republic of Georgia[END_REF]

). Nevertheless, it is worth noting that the combination of 24 Loci MIRU-VNTR and spoligotyping can lack discrimination (only the whole genome sequencing can give us the real genotype of each isolate) making possible that some clusters include slightly different genotypes. This lack of discrimination can lead to a global overestimated clustering rate in our study. However, the large difference observed between the families (20.7% for Beijing vs 11.0% for EAI vs 9.1% for T) supports the observed in the northern and southern provinces, and mostly in rural area. Surprisingly, no cluster was Finally, despite the low prevalence of drug resistance in Lao PDR, the Beijing family was more represented among drug-resistant isolates, as previously reported in Cambodia, Vietnam, and China

(Zhang et al. 2011; Nguyen et al. 2012; Pang et al. 2012)

. The Beijing isolates in clusters

Table 1 Socio-demographic and characterization of presumptive MDR-TB patients

 1

	Characteristics of presumptive MDR-TB	N (%)
	Median age (N=154	a )		52 (IQR: 36-60)
	Age groups (in year)		15-24	12 (7.8)
				25-34	20 (13.0)
				35-44	27 (17.5)
				45-54	28 (18.2)
				55-64	36 (23.4)
				≥65	31 (20.1)
	Gender (N=155)			Female	47 (30.3)
				Male	108 (69.7)
	Regions of residence (N=155)		North	60 (38.7)
				Centre	80 (51.6)
				South	15 (9.7)
	Presumptive MDR-TB type (N=149	b )	Relapse after treated with FLD/SLD	80 (53.7)
				Failure/late smear conversion	35 (23.5)
				Return after loss to follow up	12 (8.1)
				New patient with TB-HIV co-infection	21 (14.1)
				New patient in contact with proven RR /MDR case	1 (0.7)

a one missing data ;

b six missing data Identification

of Mycobacteria species and resistance to rifampicin and isoniazid by 2.

Table 2 Distribution of drug susceptible and drug resistant isolates among presumptive MDR-TB

 2 The Sanger sequencing of specific genes or regions involved in drug resistance to FLDs and SLDs could be performed on 42 out of the 43 resistant isolates determined by the MDRTBplus test. Based on MTBDRplus test, 16 out of 42 isolates were MDR, seven were mono RIF resistant, and 19 were mono INH resistant. The sequencing was able to detect mutations in all the 42 drug resistant isolates. Two more rpoB mutant isolates and four more katG mutant isolates (Tables

		Presumptive MDR-TB	Total, N=139 (%)	Susceptible, N= 96 (%)	Resistant, N=43 (%) MDR-TB, Mono RIF, Mono INH, N=17 N=7 N=19
	Relapse after treated with FLD/SLD	75 (54.0)	50 (52.1)	9 (52.9)	5 (71.4)	11 (57.9)
	Failure/late smear conversion	30 (21.6)	16 (16.7)	7 (41.2)	1 (14.3)	6 (31.6)
	Return after loss to follow up	11 (7.9)	9 (9.4)	0	0	2 (10.5)
	New patient with TB-HIV	18 (13.0)	17 (17.7)	0	1 (14.3)	0
	New patient in contact with proven RR/MDR	1 (0.7)	1 (1.0)	0	0	0
	Missing data	4 (2.9)	3 (3.1)	1 (5.9)	0	0
	3.	Sanger sequencing of drug resistance genes or regions

Table 3 Probable anti-TB drug resistant patterns based on sequencing data among the 42 drug resistant isolates determined by GenoType® MTBDRplus and according to the M. tuberculosis family.

 3

	Drug resistance patterns	Total, N=42 (%) Beijing, N=25 (%) EAI, N=14 (%) Others, N=3 (%)
	Mono-Resistance	9 (21.4)	4(16.0)	5 (35.7)	0
	H	6 (14.3)	2 (8.0)	4 (28.6)	0
	R	3 (7.1)	2 (8.0)	1 (7.1)	0
	Poly-Drug resistance	11 (26.2)	9 (36.0)	1 (7.1)	1 (33.3)
	HS	8 a (19.1)	6 (24.0)	1(7.1)	1 (33.3)
	HES	2 (4.8)	2 (8.0)	0	0
	HFS	1 (2.4)	1 (4.0)	0	0
	MDR	10 (23.8)	3 (12.0)	6 (42.7)	1 (33.3)
	RH	5 b (11.9)	1 (4.0)	3 (21.4)	1 (33.)
	RHE	2 c (4.8)	0	2 (

Table 4 Determination of M. tuberculosis families/subfamilies by spoligotyping

 4

	N	Spoligo patterns	DB4-SIT	DB4-Clade	SPOTCLUST (probability)*	Final characterisation	N (%)
	1	□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□■■■■■■■■■	1	Beijing		Beijing	53(38.1
	2	■■■■■■■■■■■■■■■■■■■■■■■■■■■■□□□□■□■■■■■■■■■	236	EAI5		EAI5	) 13(9.4)
	3	■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■□□□□■■■■■■■	53	T1		T1	10(7.2)
	4	■■■■■■■■■■■■■■■■■■■■■■■■■■■■□□□□■□■■■■■□■■■	48	EAI1-Som		EAI1-Som	7(5)
	5	■■□■■■■□□□□□□□□□□□□□□□□□□■■■□□□□■□■■■■■■■■■	89	EAI2-		EAI2-Nonthaburi	6(4.3)
	6	■■■■■■■■■■■■■■■■■■■■■■■■■■□■□□□□■□■■■■■■■■■	152	Nonthaburi EAI5		EAI5	4(2.9)
	7	■■■■■■■■■■□■■■■■■■■■■■□■■■■■□□□□■□■■■■■■■■■	1513	EAI6-BGD1		EAI6-BGD1	3(2.2)
	8	■■■■■■■□■■■■■■■■■■■■■■■■■■■■□□□□■□■■■■■■■■■	939	EAI5		EAI5	3(2.2)
	9	□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□■■■■■■	250	Beijing		Beijing	3(2.2)
		■■■■■■■■■■■■■■■■■■■■■■■■■■■■□□□□■□■■■■□■■□□	139	EAI4-VNM		EAI4-VNM	2(1.4)
		■■■■■■■■■■■■■■■■■■■■■■■■■□□■□□□□■□■■■■■■■■■	564	EAI4-VNM		EAI4-VNM	2(1.4)
		■■■■■■■■■■■■□■■■■■■■■■■■■□□■□□□□■□■■■■■■■■■	523	Manu-Ancestor		Manu-Ancestor	1(0.7)
		■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■	Orphan	Orphan		orphan	2(1.4)
		■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■□■■□■■■	1149	unknown		unknown	1(0.7)
		■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■□□□□■■■■■■	Orphan	Orphan	T1*(0.97)	T1*	1(0.7)
		■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■□□□□■■■□■■■	52	T2		T2	1(0.7)
		■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■□■□□□□■■■■■■■	50	H3		H3	1(0.7)
		■■■■■■■■■■■■■■■■■■■■■■■■■■■■□□□□■□■■■■□□■■■	947	EAI5		EAI5	1(0.7)
		■■■■■■■■■■■■■■■■■■■■■■■■■■■■□□□□■□■□■■■■■■■	Orphan	Orphan		orphan	1(0.7)
		■■■■■■■■■■■■■■■■■■■■■■□■■■■■■■■■■■■■■■■■■■■	623	unknown		unknown	1(0.7)
		■■■■■■■■■■■■■■■■■■■■■□■■■■■■□□□□■□■■■■■■■■■	1495	EAI5		EAI5	1(0.7)
		■■■■■■■■■■■■■■■■■■■□□□□□□□□□□□□□□□□□□□□□□□□	Orphan	Orphan		orphan	1(0.7)
		■■■■■■■■■■■■■■■■■■□■■■■■■■■■■■□■□□□□■■■■■■■	183	H3		H3	1(0.7)
		■■■■■■■■■■■■■■■■■■□■■■■■■■■■□□□□■□■■■■■■■■■	2671	EAI5		EAI5	1(0.7)
		■■■■■■■■■■■■■■■■■□■■■■■■■■■■□□□□■□■■■■■■■■■	Orphan	Orphan	EAI5*(0.98)	EAI5*	1(0.7)
		■■■■■■■■■■■■□■■■■■■■■■■■■■■■■■■■□□□□■■■■■■■	37	T3		T3	1(0.7)
		■■■■■■■■■■■■□■■■■■■■■■■■□■■■■■■■□□□□■■■■■■■	Orphan	Orphan	T1*(0.99)	T1*	1(0.7)
		■■■■■■■■■■■■□■■□■■■■■■■■■■■■■■■■□□□□■■■■■■■	Orphan	Orphan	T1*(0.99)	T1*	1(0.7)
		■■■■■■■■■■■□■■■■■■■■■■■■■■■■■■■■□□□□■■■□■■■	2672	T2		T2	1(0.7)
		■■■■■■■■■■■□□□□□□□□□□□□□□□□□□□□□□□□□□□□■■■■	Orphan	Orphan		orphan	1(0.7)
		■■■■■■■■□□□■■■■■■■■■■■■■■■■■□□□□■□■■■■■□■■■	Orphan	Orphan	EAI5*(0.99)	EAI5*	1(0.7)
		■■■■■■■■□□□□■■■■■■■■■■■■■■■■□□□□■□■■■■■□■■■	Orphan	Orphan	EAI5*(0.99)	EAI5*	1(0.7)
		■■■■■■■□■■■■■■■■■■■■■■■■■■■■□□□□■□■■■□□■■■■	Orphan	Orphan	EAI5*(0.99)	EAI5*	1(0.7)
		■■■■■■■□■■■■■■■■■□■■■■■■■■■■□□□□■□■■■□□■■■■	Orphan	Orphan	EAI5*(0.99)	EAI5*	1(0.7)
		■■■■■■■□□□□■■■■■■■■■■■■■■■■■□□□□■□■■■■■■■■■	951	EAI5		EAI5	1(0.7)
		■■■■■■■□□□□□□□□□□□□■■■□■□□□□□□□■□□□□■■■■■■■	Orphan	Orphan	T3*(0.99)	T3*	1(0.7)
		■■■□□■■■■■■■■■■■■■■■■■■■■■■■□□□□■□■■■■■■■■■	Orphan	Orphan	EAI5*(0.99)	EAI5*	1(0.7)
		■■■□□□□□□□□□□□□□■■■■■■■■■■■■□□□□■□■■■■■■■■■	Orphan	Orphan	EAI5*(0.99)	EAI5*	1(0.7)
		■■□■■■■■■■■■■■■■■■■□□■■■■■■■□□□□■□■■■■■■■■■	19	EAI2-Manila		EAI2-Manila	1(0.7)
		■□■■■■■■■■■■■■■■■■■■■■■■■■■■□□□□■□■■■■■■■■■	380	EAI5		EAI5	1(0.7)
		■□■■■■■■■■■■■■■■■■■■■■■■■■■□□□■■□□□□■■■□■■■	Orphan	Orphan	T1*(0.99)	T1*	1(0.7)
		■□□■■■■■■■■■■■■■■■■■■■■■□□□□□□□□□□□□■■■■■■■	Orphan	Orphan	Haarlem1*(0.99)	Haarlem1*	1(0.7)
		□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□■■■■■■■□■	1674	Beijing		Beijing	1(0.7)

Table 5 Families and subfamilies of M. tuberculosis identified among presumptive MDR-TB cases, drug 371 susceptible and drug resistant isolates. 372

 5

	Families and subfamilies	Total isolates, N=139 (col. %)	Susceptible, N=96 (col. %)	Resistant, N=43 (col. %)
	Beijing	57 (41.0)	31 (32.3)	26 (60.5)
	EAI	53 (38.1)	40 (41.7)	13 (32.2)
	EAI5	32 (23.0)	25 (26.0)	7 (16.3)
	EAI1-Som	7 (5.0)	3 (3.1)	4 (9.3)
	EAI2-Nonthaburi	6 (4.3)	5 (5.2)	1 (2.3)
	EAI4-VNM	4 (2.9)	4 (4.2)	0
	EAI6-BGD1	3 (2.2)	3 (3.1)	0
	EAI2-Manila	1 (0.7)	0	1 (2.3)
	Other families	29 (20.9)	25 (26.0)	4 (9.39)
	T1	14 (10.1)	14 (14.6)	0

Table 6 Characteristic of patients infected with susceptible and resistant M. tuberculosis isolates

 6

	Characteristics of presumptive MDR-TB	Total presumptive MDR-TB, N=139	Susceptible, N=96 (69.1%)	Resistant, N=43 (30.9%)	p-value
	Gender (N=139)	Female	38	27 (71.0)	11 (29.0)	0.7
		Male	101	69 (68.3)	32 (31.7)
	Age (N=138)	15-34	31	24 (77.4)	7 (22.6)	0.2
		35-64	81	56 (69.1)	25 (30.9)
		>=65	26	15 (57.7)	11 (42.3)
	Regions (N=139)	North	52	33 (63.5)	19 (36.5)	0.5
		Center	73	53 (72.6)	20 (27.4)
		South	14	10 (71.4)	4 (28.6)
	Types of presumptive MDR-TB
	Relapse after treated with
	FLDs/SLDs		75	50 (66.7)	25 (33.3)	0.01
	Failure/late smear conversion	30	16 (53.3)	14 (46.7)
	Return after loss follow up	11	9 (81.8)	2 (18.2)
	New patient with TB-HIV	18	17 (94.4)	1 (5.6)
	M. tuberculosis families
	Beijing		57	31 (54.4)	26 (45.7)	0.005
	EAI		53	40 (75.5)	13 (24.5)
	others		29	25 (86.2)	4 (13.8)

Table 7 Characteristics of the patients infected with EAI or Beijing isolates

 7

	Characteristics	Total, N=110	Infected with EAI, n=53 (48.2%)	Infected with Beijing, n=57 (51.8%)	p-value
	Age group (years)
	15-34		23	8 (34.8)	15 (65.2)
	35-64		64	28 (43.7)	36 (56.3)	0.01
	≥65		23	17 ( 73.9)	6 (26.1)
	Sex
	Men Women		80 31	42 (52.5) 12 (38.7)	38 (47.5) 19 (61.3)	0.1
	Regions
	North		39	16 (41.0)	23 (59.0)
	Centre		60	30 (50.0)	30 (50.0)	0.3
	South		12	8 (66.7)	4 (33.3)
	Presumptive MDR-TB	a
	Relapse after treated with FLDs/SLDs	61	33 (54.1)	28 (45.9)
	Failure/late smear conversion	24	6 (25.0)	18 (75.0)	0.02
	Return after loss follow up	8	5 (62.5)	3 (37.5)
	New patient with TB-HIV	13	9 (69.2)	4 (30.7)

  . On molecular point of view, the mutations in embB at codon 306 are the most commonly detected point mutations of EMB TableS 2.1). These mutations were identified among EMB resistant in previous studies (Q. H.Nguyen 2016;[START_REF] Plinke | EmbCAB Sequence Variation among Ethambutol-Resistant Mycobacterium tuberculosis Isolates without EmbB306 Mutation[END_REF] Campbell et al. 2011)

	resistant strains (from 30% to 70%) (Y. Zhang and Yew 2009; Z. Zhang et al. 2014). However,
	diversity of mutations in embB associated with EMB resistance was reported, consisting of 16
	unique mutation within 11 different codons, two mutations Met306Val and Met306Ile accounted
	for 77 % (53 % and 24 % respectively), other mutations included Met306Leu, Tyr319Cys,
	Asp328Tyr, Arg351His, Asp354Ala, Gly406Asp, etc. (Campbell et al. 2011). In our study, the
	two mutations at Met306Val and Met306Ile were also prevalent, accounting for 50 % (29 % and
	21 % respectively), another 50 % carried five different mutations (Val360Met, Asp354Ala,
	Pro404Ser, Gly406Asp, Gln497Lys) (

population among presumptive MDR-TB and drug resistant TB

	Spoligotyping identified Beijing and EAI as the two most predominant M. tuberculosis
	families among presumptive MDR-TB. It is worth noting that the proportion of Beijing was higher
	than EAI (41 % vs 38 %), although EAI is predominant in the global M. tuberculosis population
	of Laos (Somphavong et al. unpublished data). Indeed, the previous population-based study
	performed on the samples collected in the framework of the first National TB Prevalence Survey
	(TBPS) showed a low proportion of Beijing compared to EAI (14 % vs 76 %). In addition, both
	studies showed that Beijing is significantly more detected than EAI among drug resistant
	isolates (TBPS: 17 % DR-Beijing vs 5 % DR-EAI (p=0.03); Presumptive MDR-TB: 46 % DR-
	Beijing vs 25 % DR-EAI (p=0.005)). This result indicates that Beijing could be a factor
	associated with drug resistance and highly drug resistance (Beijing family was observed among
	pre-XDR isolates) and thus is more frequently observed in presumptive MDR-TB cases in Lao

Table S 2.2. Complete data (clinical, epidemiological, demographic and genetic data) for the 155 isolates from presumptive MDR-TB in Lao PDR (2010-2014) (xlsx)

 S 42 DR isolates, MIRU-VNTR typing generated 42 unique patterns, indicating the absence of recent transmission among the resistant isolates among the presumptive MDR-TB samples.

	Pyrazinamide
	pncA (n=4/42, 9.5%)		N=4 (%)
	Anti-TB Drug/Gene (N. of mutated isolates, %) a Rifampicin rpoB (n=25/42, 59.5%)	C deletion at codon 19 → Frameshift Observed mutations A deletion at codon 160 → Frameshift Cys72Arg Phe106Val	1 (25.0) Total, N (%) 1 (25.0) 1 (25.0) N=25 (%) 1 (25.0)
	Fluoroquinolone	Ser531Leu	6 (24.0)
	gyrA (n=4/42, 9.5%)	His526Arg	5 (20.0) N=4 (%)
			His526Tyr Gly88Ala	5 (20.0) 1 (25.0)
			Leu511Pro Ala90Val	(8.0) 1 (25.0)
			Asp516Val Ser91Pro	(8.0) 1 (25.0)
			His526Asp Asp94Gly	(8.0) 1 (25.0)
	gyrB (n=0/42)	His526Leu	(4.0)
			Met515Leu & Ile572Phe -	(4.0) -
	Injectable drugs (AG/CP) rrs-F2 (n=0/42)	insert TGCCAA (CysGln) at 514 & Met655Thr	(4.0)
	Isoniazid	b (n=39, 92.9)	-	-
	katG (n=33/42, 78.6 %) Streptomycin c (n=26/42,		N=33 (%)
	61.9%)	Ser315Thr	24 (72.7)
	rrs-F1 (n=5/42, 11.9%)	Ser315Asn	(9.1) N=5 (%)
			Ser315Arg & Glu582Lys 517(CT)	(3.0) 2 (40.0)
			Ser315Thr 514(AC)	(3.0) 1 (20.0)
			Asp189Gly 514(AT) Pro100Thr 274(GA)	1 (20.0) (3.0) (3.0) 1 d (20.0)
	rpsL (n=22/42, 52.4%)	C deletion at codon 32 → Frameshift	(3.0) N=22 (%)
			T insertion at codon 630 → Frameshift Lys43Arg	(3.0) 16 (72.7)
	inhA-promoter (n=7/42, 16.7%)	Lys88Thr	N=7 (%) 6 (27.3)
			-15 (CT)	6 (85.7)
	-17 (GT) a One isolate was not detected by MTBDRplus (Leu511Pro) inhA coding region (n=3/42, 7.1%) b One isolate showed a combination of mutations in katG/inhA-promoter (Ser315Thr/(-)15CT); two isolates 1 (14.3) N=3 (%) showed a combination in katG/inhA-coding (Ser315Thr/Asp335Asn and Ser315Thr/ Ile144Val); and One
	Ile21Val isolate showed a combination in inhA-promoter/inhA-coding ((-15)CT/ Ile21Val)	1 (33.3)
	Asp335Asn c One isolate showed a combination in rrs-F1/ rpsL (274GA/ Lys43Arg)	1 (33.3)
			Ile144Val	1 (33.3)
	Ethambutol
	embB (n=14/42, 33.3%)		N=14 (%)
			Met306Val	4 (28.6)
			Met306Ile	3 (21.5)
			Val360Met	3 (21.5)
			Asp354Ala	(7.1)
			Pro404Ser	(7.1)
			Gly406Asp	(7.1)
			Gln497Lys	(7.1)

Table 3 . 1 Characteristics of patients included in the drug resistance survey and molecular analysis

 3

	Characteristics	AFB smear+ eligible, N=1006	M. tuberculosis culture+, N=820	Any drug resistance, N=75	Molecular analysis, N=74
	Median age	50 (IQR: 36-61)	50 (IQR: 36-61)	48 (IQR: 36-60)	45 (IQR: 30-58)
	Age group,
	years
	0-24	87 (8.7)	67 (8.2)	3 (4.0)	4 (5.4)
	25-34	130 (12.9)	106 (12.9)	15 (20.0)	17 (23.0)
	35-44	161 (16.0)	136 (16.6)	9 (12.0)	13 (17.6)
	45-54	208 (20.7)	171 (20.9)	23 (30.7)	20 (27.0)
	55-64	212 (21.1)	177 (21.6)	12 (16.0)	8 (10.8)
	≥65	208 (20.7)	163 (19.9)	13 (17.3)	12 (16.2)
	Gender
	Male	685 (68.1)	558 (68.1)	56 (74.7)	52 (70.3)
	Female	321 (31.9)	262 (32.0)	19 (25.3)	22 (29.7)
	Region of
	residence
	North	240 (23.9)	190 (23.2)	26 (34.7)	23 (31.1)
	Center	510 (50.7)	433 (52.8)	41 (54.7)	40 (54.1)
	South	256 (25.5)	197 (24.0)	8 (10.7)	11 (14.9)
	TB patient
	category
	New cases	948 (94.2)	776 (94.6)	70 (93.3)	69 (93.2)
	Previously cases	58 (5.8)	44 (5.4)	5 (6.7)	5 (6.8)

katG gene and inhA (promoter and coding region): Of 29

	isolates had mutations in either katG or inhA (both promoter and coding region) (Table 3. 3),
	including one isolate with Pro232Ser of katG; four isolates with (-15CT) of inhA-promoter
	and three isolates with Ile144Val of inhA-coding region. It is worth noting that we did not
	include the mutation Arg463Leu of katG gene, described as a phylogenetic marker and not
	as a drug resistant determinant [136, 137] . This mutation was observed in 51 out of 58
	isolates with valid sequencing results.
	Ethambutol resistance and embB: In our study, two isolates were EMB resistant; one
	had Asp354Ala and one had Met306Ile mutations in embB gene. The sequencing detected
	Val360Met mutation in three EMB susceptible isolates whereas, this mutation was reported
	to be associated with EMB resistant [138]. Twenty seven out of 56 isolates carried the
	Glu378Ala mutation. This mutation previously defined as phylogenetic marker was not
	included in the analysis [139].
	Streptomycin resistance and rpsL and rrs-F1 fragment: Among 31 STR resistant
	isolates, mutations in rpsL were more observed than in rrs-F1 (72.2 % vs 6.5 %). The most
	common mutation in rpsL was Lys43Arg (54.8 %) and in rrs-F1 fragment was (-)517CT. No
	mutation was detected for three STR resistant isolates neither in rpsL nor rrs-F1. The
	Val8Phe mutation of rpsL and (-)87AG mutation of rrs-F1 were identified, however they are
	found only in STR susceptible isolates.
	INH resistant isolates, 16 (55.2%) isolates had mutation only in katG gene (single and
	double mutations) (Table 3. 3), five (17.2%) isolates had mutation only in inhA-promoter,
	one (3.4 %) had mutation only in inhA coding and one (3.4%) had triple mutations in KatG
	(Ala480Ser) & inhA-promoter (-15CT) and inhA-coding region (Ile21Val). No mutation was
	found in one resistant isolate (wild type) and five isolates had invalid results (Table 3. 3 and
	Table S 3.1). The most common mutations were Ser315Thr (48.3%) in katG and (-)15CT
	(13.8%) in inhA-promoter. Among 45 INH susceptible isolates, a total of eight (17.8%)

Ofloxacin resistance and gyrA, gyrB:

  Of three OFX resistant isolates, two had Ala90Val mutation in gyrA, while no mutation was detected neither in gyrA nor gyrB for one OFX resistant isolate. Among the susceptible isolates, there was no mutation observed neither in gyrA nor in gyrB. We excluded the Glu21Gln and Ser95Thr mutations (found in

	57/60 and 56/60 isolates respectively), since they were described as lineage genetic
	markers [140, 141]

Table 3 . 3 Cumulative frequency of all mutations among the 60 drug resistant isolates and the 14 susceptible isolates.

 3 DST was not performed for detection of PZA resistance a combination of KatG (Ala480Ser) & inhA-promoter (-15CT) & inhA-coding region (Ile21Val) b three isolates had mutation in inhA-promoter, one had mutation in inhA-coding and one isolate with no mutation c sixteen isolates had mutations in katG only, one had mutation in inhA-coding gene only. One isolate had no mutation and five isolates had wild type for inhA-promoter but invalid results of katG and inhA-coding gene. d sixteen isolates had mutations in katG only, five had mutation in inhA-promoter only. One isolate had no mutation and two isolates had wild type for inhA-promoter and coding gene but had invalid results for katG. e two isolates had mutations in rrs-F1, three had no mutation, one had wild type for rpsL but invalid results for rrs-F1 f twenty isolates had mutations in rpsL, three had no mutation g two isolates had mutation in gyrA , one had no mutation

	Drugs/Genes	DNA Sequencing Mutations	DST using LJ proportion method N. of resistant N. of susceptible	Total
	Rifampicin		N=11 (%)	N=63 (%)	N=74 (%)
	rpoB	His526Tyr	2 (18.2)	0		2 (2.7)
		His526Tyr & Met736Thr	1 (9.1)	0		1 (1.4)
		His526Asp	2 (18.2)	0		2 (2.7)
		Ser531Leu	2 (18.2)	0		2 (2.7)
		Leu533Pro	1 (9.1)	0		1 (1.4)
		His526Ser	0	1 (1.6)		1 (1.4)
		Met736Thr	0	4 (6.4)		4 (5.4)
		Wild type	1 (9.1)	49 (77.8)	50 (67.6)
		Invalid	2 (18.2)	9 (14.28)	11 (14.9)
	Isoniazid		N=29 (%)	N=45 (%)	N=74 (%)
	katG only	Ser315Thr	14 (48.3)	0	14 (18.9)
		Ser315Asn	1 (3.5)	0		1 (1.4)
		Ser315Asn & Ile317Val Ala480Ser a	1 (3.5) 1 (3.5)	0 0		1 (1.4) 1 (1.4)
		Pro232Ser Wild type	0 b (17.2) 5	1 (2.2) 36 (80.0)	1 (1.4) 41 (55.4)
		Invalid	7 (24.1)	8 (17.8)	15 (20.0)
	inhA promoter	(-)15CT	4 (13.8)	4 (8.9)	8 (10.8)
		(-)17GT	1 (3.5)	0		1 (1.4)
		(-)34CG Wild type	1 (3.0) 23 c (79.3)	0 41 (91.1)	1 (1.4) 64 (86.5)
		Invalid	0	0		0
	inhA coding region	Ile21Thr	1 (3.5)	0		1 (1.4)
		Ile21Val	1 (3.5)	0		1 (1.4)
		Ile144Val Wild type	0 d (82.8) 24	3(6.7) 40 (88.9)	3 (4.1) 64 (86.5)
		Invalid	3 (10.3)	2 (4.4)		5 (6.8)

*

4. Performance of different molecular methods for detection of drug resistant TB All

  the 74 samples included in our analysis had Xpert MTB/RIF results; the MTBDRplus ver.1 and MTBDRsl ver.1 tests were obtained for 73 isolates. Regarding the sequences, it was variable according to the gene or region under study (Table3. 4). The performance of Xpert MTB/RIF, MTBDRplus/MTBDRsl, and sequencing for detection of anti-TB drug resistance were assessed using the culture based phenotypic DST results as reference. The sensitivity, specificity, predictive positive value (PPV) and Negative predictive value (NPV) were calculated.The value of likelihood ratio positive (LR+) and likelihood ratio negative (LR-) were also presented (Table3. 4). Based on DST results, 60 out of 74 isolates were resistant to at least one anti TB drug (including mono drug resistant, poly drug resistant, MDR and QDR), 14 were susceptible to all the seven tested drugs (RIF, INH, EMB, OFX, KAN, CAP and STR).

	Performance of the Xpert MTB/RIF assay for the detection of RIF resistance: Based on
	phenotypic DST result, 11 isolates were resistant to RIF and 63 were susceptible. Xpert
	MTB/RIF assay detected RIF resistance in 9/11 of RIF resistant isolates and in 1/63 of RIF
	susceptible isolates. Xpert MTB/RIF assay demonstrated a sensitivity of 81.8 % and specificity
	of 98.4 %, with a PPV of 90 % and a NPV of 96.9 % (Table 3. 4).

Performance of MTBDRplus and MTBDRsl for the detection of resistance to RIF, INH, FQ, SLIDS (KAN, CAP) and EMB: MTBDRplus

	ver.1 and MTBDRsl ver.1 results were
	available for 73 isolates. The MTBDRplus test had a sensitivity and specificity of 63.6 % and
	95.2 % for the detection of RIF resistance and 60.7 % and 86.7 % for INH resistance
	respectively. The MTBDRsl test had a sensitivity and specificity of 100 % and 100 % for
	detection of OFX resistance, 100 % and 100 % for detection of KAN resistance, 25.0 % and 100
	% for detection of CAP and 50.0 % and 100 % for detection of EMB resistance respectively
	(Table 3. 4).

Table 3 . 4 Performance of Xpert MTB/RIF; MTBDRplus/MTBDRsl and Sequencing for the detection of FLD and SLD resistance compared to DST results

 3

	Molecular methods (gene/region analyzed)	Drugs	DST results, No. R S	Sensitivity	Performance, % (95% CI) Specificity PPV NPV	LR+	LR-
	Xpert MTB/RIF	RIF-R	9	1	81.8	98.4	90	96.9	51.6	0.9
	(rpoB)	RIF-S	2	62	(48.2-97.7)	(91.5-100)	(55.8-98.5)	(89.8-99.1)
	MTBDRplus	RIF-R	7	3	63.6	95.2	70	93.67	13.2	0.4
	(rpoB)	RIF-S	4	59	(30.8-89.1)	(86.5-99.0)	(41.5-88.5)	(87.1-97.0)
	MTBDRplus (katG+inhA-promoter)	INH-R INH-S	17 11	6 39	60.7 (40.6-78.5)	86.7 (73.2-95.0)	73.9 (56.0-86.3)	78 (68.8-85.1)	4.6	0.5
	MTBDRsl	EMB-R	1	1	50.0	100	100	98.6	NA	0.5
	(embB)	EMB-S	1	70	(1.3-98.7)	(94.9-100)	NA	(94.6-99.6)
	MTBDRsl	OFX-R	3	0	100	100	100	100	NA	0
	(gyrA)	OFX-S	0	70	(29.2-100)	(94.9-100)
	MTBDRsl (rrs-	KAN-R	1	0	100	100	100	100	NA	0
	F2)	KAN-S	0	72	(2.5-100)	(95.0-100)	NA
	MTBDRsl (rrs-	CAP-R	1	0	25	100	100	95.8	NA	0.8
	F2)	CAP-S	3	69	(0.6-80.6)	(94.8-100)	NA	(92.9-97.6)
	Sequencing	RIF-R	8	5	88.9	90.7	61.5	98	9.6	0.1
	(rpoB)	RIF-S	1	49	(51.8-99.7)	(79.7-96.9)	(40.2-79.2)	(88.5-99.7)
	Sequencing (katG+inhA-	INH-R	21	8	95.5	78.4	72.4	96.7	4.4	0.06
	promoter+inhA-codong)	INH-S	1	29	(77.2-99.9)	(61.8-90.2)	(58.5-83.0)	(80.9-99.5)
	Sequencing	EMB-R	2	3	100	94.4	40.0	100	18.0	0
	(embB)	EMB-S	0	51	(15.8-100)	(84.6-98.8)	(18.2-67.0)
	Sequencing	OFX-R	2	0	66.7	100	100	98.4	NA	0.3
	(gyrA+gyrB)	OFX-S	1	61	(9.4-99.2)	(94.1-100)	NA	(92.5-99.7)
	Sequencing	KAN-R	0	0	NA	100	NA	100	NA	1
	(rrs-F2)	KAN-S	0	67	NA	NA	NA	NA
	Sequencing	CAP-R	0	0	0	100	NA	95.5	NA	1
	(rrs-F2)	CAP-S	3	64	NA	NA		(95.5-95.5)
	Sequencing	STR-R	25	2	89.3	94.6	92.6	92.1	16.5	0.1
	(rpsL+rrs-F1)	STR-S	3	35	(71.8-97.7)	(81.8-99.3)	(76.3-98.0)	(80.0-97.2)

Table 3 . 5 Spoligotyping patterns

 3

	N	Spoligotype 43-spacers			SIT	SPOLDB4 Clade	SPOTCLUST (Probability) Family/Subfamily Final definition	N. (%)
	1	oooooooooooooooooooooooooooooooooonnnnnnnnn		1	Beijing		Beijing	21 (28.4) a
	2	nonnonnnnnnnnnonnnnnnonnnnnnoooononnnnnnnnn	Orphan	Orphan	EAI5 (0.99)	EAI5*	(5.4)
	3	nnnnnnnonnnnnnnnnnnnnnnnnnnnoooononnnnnnnnn		939	EAI5		EAI5	(4.1)
	4	nnonnnnoooooooooooooooooonnnoooononnnnnnnnn		89	EAI2-Nonthaburi		EAI2-Nonthaburi	(4.1)
	5	ooooooooooooooooooooooooooooooooooooonnnnnn		250	Beijing		Beijing	(4.1)
	6	nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnoooonnnnnnn		53	T1		T1	(2.7)
	7	nnnnnnnnnnnnnnnnnnnnnnnnnnnnoooononnnnnnnnn		236	EAI5		EAI5	(2.7)
	8	nnnnnnnnnnnnnnnnnnnnnnnnnnnnoooononnnnnonnn		48	EAI1-SOM		EAI1-SOM	2 (2.7) b
	9	nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnoonnnnnnnn	Orphan	Orphan	Family33 (0.95)	mix	1 (1.4) c
	10	nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnonoooonnnnnnn		50	H3		H3	(1.4)
	11	nnnnnnnnnnnnnnnnnnnnnnnnnnnnoooononnnnonnoo	Orphan	Orphan	EAI5 (0.89)	EAI5**	(1.4)
	12	nnnnnnnnnnnnnnnnnnnnnnnnnnonoooononnnnnnnnn		152	EAI5		EAI5	(1.4)
	13	nnnnnnnnnnnnnnnnnnnnnnnnnnoooooooonnnnnnnnn	Orphan	Orphan	EAI5 (0.94)	EAI5*	(1.4)
	14	nnnnnnnnnnnnnnnnnnnnnnnonnnnnnnnoooonnnnnnn		373	T1		T1	(1.4)
	15	nnnnnnnnnnnnnnnnnnnnnnnonnnnoooooonnnnnnnno	Orphan	Orphan	EAI5 (0.95)	EAI5*	(1.4)
	16	nnnnnnnnnnnnnnnnnnnnnnonnnnnnnnnoooonnnonnn		943	T		T	(1.4)
	17	nnnnnnnnnnnnnnnnnnnnnonoooooooonoooonnnnnnn	Orphan	Orphan	Haarlem1 (1)	H1*	(1.4)
	18	nnnnnnnnnnnnnnnnnnnnooooooooooooooooooooooo		402	unknown	Family34	Unknown	(1.4)
	19	nnnnnnnnnnnnnnnnnnonnnnnnnnnnnonoooonnnnnnn		183	H3		mix	1 (1.4) d
	20	nnnnnnnnnnnnnnnnnnonnnnonnnnnnonoooonnnonnn	Orphan	Orphan		Orphan	(1.4)
	21	nnnnnnnnnnnnonnnnnnnnnnnnnnnoooononnnnnnnnn		618	EAI5		EAI5	(1.4)
	22	nnnnnnnnnnnonnnnnnnnnnnnnnnnnnnnoooonnnonnn		2672	T2		T2	(1.4)
	23	nnnnnnnnnnnononononnnnnonnnnoooooonnnnonnnn	Orphan	Orphan	EAI5 (0.96)	EAI5*	(1.4)
	24	nnnnnnnnonnnnnnnnnonnnnonnnnnnonoooonnnonnn	Orphan	Orphan		Orphan	(1.4)
	25	nnnnnnnonnnnnnnnnnnnnnnnnnnnoooononnnoonnnn	Orphan	Orphan	EAI5 (0.99)	EAI5*	(1.4)
	26	nnnnnnnoooonnnnnnnnnnnnnnnnnoooononnnnnnnnn		951	EAI5		EAI5	(1.4)
	27	nnnnnnnoooonnnonnnnnnnnnnnnnoooononnnnnnnnn	Orphan	Orphan	EAI5 (0.99)	EAI5	(1.4)
	28	nnnnnnnooooonnnnnononnoonnnooooooonnnnonnnn	Orphan	Orphan		mix	1 (1.4) e
	29	nnnnnoonnnnnnnnnnnnnnnnnnnnnoooononnnnnnnnn		792	EAI5		EAI5	(1.4)
	30	nnooooooooonnnnnnnnnnnnnnnnnoooononnonnnnnn	Orphan	Orphan	EAI5 (0.99)	EAI5*	(1.4)
	31	nonnonnnnnnnnnonnnnononnnnnnoooononnnnnnnno	Orphan	Orphan	EAI5 (0.99)	mix	(1.4) f
	32	nonnonnnnnnnnnonnonnnonnnnnnoooononnnnnonnn	Orphan	Orphan	EAI5 (0.99)	EAI5*	(1.4)
	33	nonnonnnnnnnononnnnnnonnnnnnoooononnnnnnnnn	Orphan	Orphan	EAI5 (0.99)	EAI5*	(1.4)
	34	nonnonnnnnnoononnnnnnoonnnnnoooononnnnnnnnn	Orphan	Orphan	EAI6 (0.99)	EAI5*	(1.4)
	35	nonnonnonnnnnnonnnnnnonnnnnnoooononnnnnnnnn	Orphan	Orphan	EAI5 (0.99)	EAI5*	(1.4)
	36	nonnonnoooonnnonnnnnnonnnnnnoooononnnnnnnnn	Orphan	Orphan	EAI5 (0.99)	EAI5*	(1.4)
	37	nonnonnoooonnnonnnnononnnnnnoooononnnnnnnnn	Orphan	Orphan	EAI5 (0.99)	EAI5*	(1.4)
	38	noononnnnnnnnnonnnnooonnnnnnoooononnnnnnnnn	Orphan	Orphan	EAI2 (0.99)	mix	1 (1.4) g
	39	nooooooooooonooooooonooonooooooooonnnnnnnnn	Orphan	Orphan		mix	1 (1.4) h
	40	onnoononoooononooooonnooonoonnoooonnnnnnnnn	Orphan	Orphan	Family33 (1)	Orphan	(1.4)
	41	oonoonooooooooooooooonooonoooooooonnnnnnnnn	Orphan	Orphan		mix	(1.4) i
	42	ooooonoooooonooooooooooooooooooooonoooonnon	Orphan	Orphan		mix	(1.4) j
		*defined by SPOTCLUST with probability equal and greater than 0.9
		**defined by NJ tree (MIRU-VNTRplus) and SPOTCLUST probability=0.89
		a two isolates had double alleles at one locus;	b one isolate had double alleles at one locus;	ei each pattern
		had double alleles at one locus;	cdfghjk each pattern had double alleles at least at two loci. All isolates with
		double alleles at least at one locus were removed from M. tuberculosis family designation.

Table 3 . 6 Drug resistant patterns based on DST according to M. tuberculosis families

 3

	Drug resistant status	Total, N=63 (col. %)	M. tuberculosis families Beijing, n=22 EAI, n=30 Others, n=11 (col. %) (col. %) (col. %)
	Susceptible	13 (20.6)	1 (4.6)	7 (23.3)	5 (45.5)
	Resistant	50 (79.4)	21 (95.4)	23 (76.7)	6 (54.5)
	Mono resistance	39 (61.9)	14 (63.6)	19 (63.3)	6 (54.5)
	R	5 (7.9)	3 (13.6)	1 (3.3)	1 (9.1)
	H	14 (22.2)	1 (4.6)	12 (40.0)	1 (9.1)
	S	15 (23.8)	9 (40.9)	5 (16.7)	1 (9.1)
	Ofx	3 (4.7)	1 (4.6)	1 (3.3)	1 (9.1)
	Cm	2 (3.2)	0	0	2 (18.2)
	Poly-resistance	9 (14.3)	6 (27.3)	3 (10.0)	0
	RS	1 (1.6)	1 (4.6)	0	0
	HE	1 (1.6)	0	1 (3.3)	0
	HS	6 (9.5)	4 (18.2)	2 (6.7)	0
	SKmCm	1 (1.6)	1 (4.6)	0	0
	MDR/QDR	2 (3.2)	1 (4.6)	1 (3.3)	0
	RHS	1 (1.6)	0	1 (3.3)	0
	RHSE	1 (1.6)	1 (4.

6. Stratification of social-demographic data in overall drug resistant and susceptible patterns

  The proportions of gender, age group, regions of residence, type of patients and M. tuberculosis families were assessed among susceptible and resistant isolates. Ages of patients were grouped according to independent (15-64 years old) and dependent age group (from 65 years old). For better distribution, the independent age group therefore was subdivided into two groups 15 to 34 and 35 to 64 years old. The total number of isolates with successful designation of M. tuberculosis families was 63, the rest were not included into the analysis due to the detection of mixed infection. The analysis showed significant different proportions of drug resistant isolates according to the regions of residence and M. tuberculosis families (p=0.03 and p=0.01 respectively). The proportion of drug resistance was higher in the northern part than in the central and southern part, while the proportion of drug resistance were similar in the central and southern part (90.9 % , 66.7 % and 66.7 % respectively). Among M. tuberculosis families, the proportion of drug resistance was higher in Beijing family than EAI and other families(95.4 %, 76.7 % and 54.5 % respectively). There was no significant difference of the proportion of drug resistance in gender, age group and type of patients (Table3.7).

Table 3 . 7 Characteristic of patients with resistant and susceptible M. tuberculosis isolates

 3

	Characteristics of patients	Total, N=74	Susceptible, N=14 (30.9%)	Resistant, N=60 (69.1%)	p-value
	Gender	Female Male	22 52	5(22.7) 9 (17.3)	17(77.3) 43 (82.7)	0.7
	Age	15-34	21	5 (23.8)	16 (76.2)
		35-64	33	6 (18.2)	27 (81.8)	0.8
		>=65	20	3 (15.0)	17 (85.0)
	Regions of residences	North Center	44 24	4 (9.1) 8 (33.3)	40 (90.9) 16 (66.7)	0.03
		South	6	2 (33.3)	4 (66.7)
	Type of patients
	New cases Previously retreated cases	69 5	13 (18.8) 1 (20.0)	56 (81.2) 4 (80.0)	1
	M. tuberculosis families a
	EAI		30	7 (23.3)	23 (76.7)
	Beijing		22	1 (4.5)	21 (95.4)	0.01
	Others		11	5 (45.5)	6 (54.5)

a a total of 63 isolates with successful family designation

Discussion Socio-demographic characteristics of the study population

  ) cases in Lao PDR. In terms of geography, despite the fact that the patients transmission of MDR in the country. Surprisingly, mono resistance to OFX and CAP were detected, reflecting a wide use of OFX in the country for the treatment of respiratory diseases other than TB. The circulation of these isolates is of high concern because they have a higher potential to evolve towards pre-XDR and XDR. Furthermore, a variety of drug resistant patterns, including mono and poly-drug resistant patterns other than RIF resistance and MDR were observed. These isolates can also have a negative impact on the treatment outcome and might lead to relapse or treatment failure. Indeed, several studies underlined that drug resistant isolates are more prone to acquire other drug resistances compared to susceptible ones (see for review[148]

	The median age of the 1006 patients with AFB smear positive (50 years old) found in
	this survey was similar to the one observed in the first National TB prevalence survey of Lao
	PDR in 2010-2011 [2]. This study showed that males are more infected than females which is
	also similar to the global observation, especially in Southeast Asia [1]. Regarding TB treatment
	history, 5.8 % of patients recruited were previously treated cases (including relapses) whereas,
	in Vietnam the population of retreated cases in the last drug resistance survey represented 15
	% [144]. This difference can reflect a problem of case detection among previously treated
	(including relapses

Mutations associated with first and second line anti-TB drugs

  Caws et al. 2006). The mutations at codon 315 of katG gene were strictly detected in INH resistant isolates while the (-)15CT mutation of inhA-promoter were observed in both INH resistant and susceptible isolates as single mutation or combined with katG and inhA-coding gene. Overall 17.8 % of INH susceptible isolates had mutations in either katG or inhA, indicating that the mechanism of INH resistance is not yet clearly determined as previously observed [161].Among the two EMB resistant isolates, the sequencing allowed to detect the embB mutations associated with these two isolates. Nevertheless, the Val360Met mutation in embB was detected in three EMB susceptible isolates, whereas this mutation was reported to be associated with EMB resistant [138], however, this mutation need to be confirmed with the a large number of samples. On the other hand, the discordance could be explained by the problem encountered with the conventional culture-based EMB susceptibility testing which could The CAP resistance could be due to other mechanism of resistance, as observed in a recent study. The authors demonstrated that resistance to CAP could be also associated with mutations in tlyA gene[164]. Include the analysis of the tlyA gene could provide a better understanding of CAP resistance mechanism.The number of STR resistant isolates (n=31) was higher than other drug resistance in our study. The main mutations associated with STR resistance were observed in both rpsL and rrs-F1 and 9.7 % of STR resistant isolates lacked mutations in rpsL and rrs-F1. In contrast, 4.7 % of STR susceptible isolates had mutations either in rpsL or rrs-F1. The high rate of STR resistant isolates observed in this survey justifies stopping the use of this antibiotic in Lao PDR in agreement with WHO recommendations.The presence of mutations in pncA is correlated with PZA resistance[165]. A large panel of pncA mutations has been reported[166, 167] . In our study, we found five different mutations which were previously associated with PZA resistance[165][166][167]. Conventional DST of PZA is challenging and problematic due to the poor growth of M. tuberculosis under acidic conditions (pH 5.5 to 6.0) that are required for optimal drug activity[168]. Thus, the good correlation between the presence of pncA mutations and PZA resistance makes DNA sequencing very useful for the PZA resistance detection. Nevertheless, a number of mutations still need to be experimentally tested to confirm their link with PZA resistance through the determination of MIC values or the use of functional genomics.

	recently a study demonstrated an association of this mutation with very low level of RIF resistance but probably clinically relevant RIF resistance [159, 160] Among the INH resistant isolates tested, the proportion of mutations in katG was predominant (55 %), followed by the mutation in inhA-promoter (17 %). In Vietnam, the mutation in katG was found in 70 % of INH resistant isolates and mutation in inhA-promoter was found in 17 % (The

The mutations in the rpoB gene, encoding the β subunit of RNA polymerase, have been shown to be strongly associated with RIF-resistant phenotypes in multiple study populations

[149][150][151]

. The most common mutated codons found in our study (codon 526 and 531) were similar to the data observed in neighboring countries (China and Vietnam)

[152, 153]

. In one RIF resistant isolate, no mutation was detected. Similar observations have been reported in previous studies

[154, 155]

, suggesting that RIF resistance associated mutations could be located outside the region under study. One RIF susceptible isolate had His526Ser mutation (ID: 635, Table

3

. 3). This mutation is a "disputed" mutation (discordant results by DST) and was rarely detected. It was found inconsistently associated with RIF resistance

[156][157][158]

. However, give false-susceptible EMB results

[162]

. The sequencing of embB would substantially improve the diagnostic of EMB resistance. Regarding OFX and the detection of mutations in gyrA and gyrB, of three OFX resistant isolates, two carried Ala90Val mutation in gyrA which is one of the most common resistance-associated mutations to FQ resistance

[100, 163]

. No mutation was detected for one of the three OFX isolates. This profile could be explained by another mechanisms of FQ resistance

[161]

. Regarding injectable drug resistant isolates, all the three CAP resistant isolates did not harbor any mutations in rrs-F2.

Performance of different molecular methods for detection of first line drugs and second line drugs Ø The performance of Xpert MTB/RIF for the detection of RIF resistance:

  According to literature, the overall sensitivity of Xpert MTB/RIF assay for detection of MTBc was different based on settings, sample type, subject age, HIV co-infection and smearpositivity[169]. For the detection of RIF resistance, the pooled sensitivity and specificity was 95 % and 98 % respectively[170]. In our study, the sensitivity was found lower than the one observed in previous studies (81.8 % vs 95 %) but the specificity was similar (98.4 % vs 98 %).The Xpert MTB/RIF missed two isolates among RIF resistant, isolates whereas one more RIF resistant was detected (Table3. 4). The data suggest the presence of rpoB mutations outside RRDR or different mechanisms of RIF resistance or a mixture of mycobacterial subpopulations with different susceptibilities to RIF as previously observed[171, 172]. Regarding the last point,

The top left-hand panel shows the chromosome of hypothetical strain X, as shown by the arrows. The top right-hand panel shows the results of IS6110-based genotyping. The three bottom panels show the results of MIRU-based genotyping.(Source:[START_REF] Barnes | Molecular Epidemiology of Tuberculosis[END_REF]

24-loci MIRU-VNTR43-spacer, spolityping

ACKNOWLEDGMENTS Acknowledgments

We thank the Center for Infectiology Lao-Christophe Mérieux, the Institut de Recherche pour le Dévelopement (IRD), France, the Fondation Mérieux/ Laboratoire des Pathogènes Emergents (LPE), France, and The National Institute of Hygiene and Epidemiology (NIHE), Vietnam, for their support.

We are also grateful to the Ministry of Health, the National TB Control Program, the National reference laboratory, the survey teams, the experts for technical validation, all participants and funders of the first National TB prevalence survey of Lao PDR. We thank Elisabetta Andermarcher for assistance in preparing and editing the manuscript.

This research was carried out in the framework of the JEAI "Mycobaterium tuberculosis in Southeast Asia (MySA) and the LMI "Drug Resistance in South East Asia" (DRISA) projects.

Acknowledgments

We thank the Center for Infectiology Lao-Christophe Mérieux, the Institut de Recherche pour le Développement (IRD), France, the Fondation Mérieux/ Laboratoire des Pathogènes Emergents (LPE), France and the LMI DRISA (Drug Resistance in South East Asia) for their support.

We are also grateful to the Ministry of Health, the National TB Control Program, the National reference laboratory, the central and provincial hospitals of Lao PDR and all participants of this study.

My special words of thanks should definitely go to my research supervisor Mr Jean-Luc Berland for his continuous support, cooperation, encouragement and for facilitating all I am also grateful to Dr Ot Manolin, former director of Centre d'Infectiology Lao-Christophe Mérieux The work presented in this thesis would not have been possible without the support of kindness people from the Institut de Recherche pour le Dévelopement (IRD) France, the Centre d'Infectiology Lao-Christophe Mérieux (CILM) Laos, the Fondation Mérieux/ Laboratoire des Pathogènes Emergents (LPE) France, The National Institute of Hygiene and Epidemiology (NIHE) Vietnam, the Ministry of Health, the National TB Control Program (NTCP) and the National reference laboratory (NRL) Laos. I take this opportunity to extend my sincere gratitude /Bdq Bedaquiline BSL-3 Biosafty level 3 CAP/Cap Reference Laboratory NPV Negative predictive value NTC National TB Center NTCP National Tuberculosis Control Program NTM non-tuberculous mycobacteria OFX/Ofx Ofloxacin PAS/Pas

Funding

"Drug Resistance in South East Asia" (DRISA) project; Fondation Mérieux (FMX); Institut de Recherche pour le Dévelopement (IRD), Center for Infectiology Lao-Christophe Mérieux (CILM).

Silaphet Somphavong was supported by the "Allocations de Recherche pour une Thèse au Sud (ARTS) -IRD-Fondation Mérieux program" for the fully funded PhD studentship. Stout, Jason E., Won-Jung Koh, and Wing Wai Yew. 2016. "Update on Pulmonary Disease Due to Non-Tuberculous Mycobacteria." International Journal of Infectious Diseases 45 (April): 123-34. https://doi.org/10.1016/j.ijid.2016.03.006. Supply, Philip, Caroline Allix, Sarah Lesjean, Mara Cardoso-Oelemann, Sabine Rüsch-Gerdes, Eve Willery, Evgueni Savine, et al. 2006. "Proposal for Standardization of Optimized Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat Typing of Mycobacterium tuberculosis." Journal of Clinical Microbiology 44 (12): 4498-4510. https://doi.org/10.1128/JCM.01392-06. Takawira, Faustinos Tatenda, Racheal Shamiso Dube Mandishora, Zephaniah Dhlamini, Ellen Munemo, and Babill Stray-Pedersen. 2017. "Mutations in RpoB and KatG Genes of Multidrug Resistant Mycobacterium tuberculosis Undetectable Using Genotyping Diagnostic Methods." The Pan African Medical Journal 27 (June). https://doi.org/10.11604/pamj.2017.27.145.10883. Tesfay, Kebede, Shinesh Tesfay, Etsay Nigus, Araya Gebreyesus, Dawit Gebreegziabiher, and Kelemework Adane. 2016. "More than Half of Presumptive Multidrug-Resistant Cases Referred to a Tuberculosis Referral Laboratory in the Tigray Region of Ethiopia Are Multidrug Resistant." International Journal of Mycobacteriology 5 (3): 324-27. https://doi.org/10.1016/j.ijmyco.2016.07.007. Torres, Jessica N, Lynthia V Paul, Timothy C Rodwell, Thomas C Victor, Anu M Amallraja, Afif Elghraoui, Amy P Goodmanson, et al. 2015. "Novel KatG Mutations Causing Isoniazid Resistance in Clinical M. Tuberculosis Isolates." Emerging Microbes & Infections 4 (7): e42. https://doi.org/10.1038/emi.2015.42. Tracevska, T., I. Jansone, V. Baumanis, O. Marga, and T. Lillebaek. 2003. "Prevalence of Beijing Genotype in Latvian Multidrug-Resistant Mycobacterium tuberculosis Isolates." Text. November 2003. http://www.ingentaconnect.com/content/iuatld/ijtld/2003/00000007/00000011/art00014#. Vitol, Inna, Jeffrey Driscoll, Barry Kreiswirth, Natalia Kurepina, and Kristin P. Bennett. 2006. "Identifying Mycobacterium tuberculosis Complex Strain Families Using Spoligotypes." Infection, Genetics and Evolution 6 (6): 491-504. https://doi.org/10.1016/j.meegid.2006.03.003. Wade, Mary Margaret, Dmitriy Volokhov, Mike Peredelchuk, Vladimir Chizhikov, and Ying Zhang. 2004. "Accurate Mapping of Mutations of Pyrazinamide-Resistant Mycobacterium tuberculosis Strains with a Scanning-Frame Oligonucleotide Microarray." Diagnostic Microbiology and Infectious Disease 49 (2): 89-97. https://doi.org/10.1016/j.diagmicrobio.2004.01.001. World Health Organization. 2017. Global Tuberculosis Report 2017. Geneva, Switzerland: WHO/HTM/TB/2017.23. Zaw, Myo T., Nor A. Emran, and Zaw Lin. 2018. "Mutations inside Rifampicin-Resistance Determining Region of RpoB Gene Associated with Rifampicin-Resistance in Mycobacterium tuberculosis." Journal of Infection and Public Health 11 (5): 605-10. https://doi.org/10.1016/j.jiph.2018.04.005. Zhang, Jian, Seiha Heng, Stéphanie Le Moullec, Guislaine Refregier, Brigitte Gicquel, Christophe Sola, and Bertrand Guillard. 2011. "A First Assessment of the Genetic Diversity of Mycobacterium tuberculosis Complex in Cambodia." BMC Infectious Diseases 11 (February): 42. https://doi.org/10.1186/1471-2334-11-42. Zhang, Y., and W. W. Yew. 2009. "Mechanisms of Drug Resistance in Mycobacterium tuberculosis [State of the Art Series. Drug-Resistant Tuberculosis. Edited by C-Y. Chiang. Number 1 in the Series]." Text.

Availability of data and materials

The dataset supporting the conclusions of this article is included within the article and its additional file.

Sanger sequencing

The main genes associated with resistance to first line anti-TB drugs (FLDs) and second line anti-TB drugs (SLDs) were amplified by PCR and sequenced. For the FLD resistance, the following genes and gene fragments were studied: katG gene, inhA gene coding and the inhA promoter (INH resistance); rpoB gene (RIF resistance); rpsL gene and rrs-F1 fragment (STR resistance); embB gene (EMB resistance); pncA gene and its promoter (PZA resistance).

For the SLDs resistance, the following genes and gene fragments were analyzed: gyrA and gyrB genes (FQ resistance), rrs-F2 fragment (SLID resistance). The list of primers (Table 2.1), PCR conditions (Table 2.2) and DNA sequencing are previously described [133,134]. Each sequence was treated independently using the Bioedit software (version7. 1.10). The consensus sequence was generated. Multi-sequence alignment was then performed. Point mutations were identified by comparison with the sequence of the M. tuberculosis H37Rv reference strain available in GenBank (NC.000962.3). To describe the resistanceassociated mutations in rpoB gene, a numbering system based on the Escherichia coli sequence annotation has been used. TB epidemiological pattern in Lao PDR. This underlines the need to continue and reinforce the effort to maintain an efficient TB control and surveillance system in order to prevent the emergence of highly transmissible and drug-resistant strains in Lao PDR, as observed in neighboring countries.

Declarations

Consent for publication: Not applicable

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

Design of the study: ALB, TVAN, SS. Supervision of the study: ALB, TVAN, JLB, PP, GPB. The first XDR-TB case was detected in 2010 in a multicentric study conducted in three regional hospitals. This study showed that 7 (8.0%) out of 87 M. tuberculosis isolates were INH mono resistant and 1 was XDR-TB (Iem et al. 2013)

Repeat (MIRU-VNTR) typing

The full set of 24 MIRU-VNTR loci was used to characterize the isolates. The MIRU-VNTR typing was performed as previously described (Supply et al. 2006;Gauthier et al. 2015).

The patterns obtained for the 24 loci were used to create a 24 digit allelic profile for each isolate.

The results of the MIRU-VNTR typing method were analyzed using MIRU-VNTRplus promoter (pyrazinamide (PZA) resistance). Regarding SLD resistance, the following genes and gene fragments were analyzed: gyrA and gyrB genes (Fluoroquinolone (FQ) resistance), rrs-F2 fragment (second line injectable drug (SLID) resistance). The list of primers, PCR conditions and DNA sequencing were previously described (Nguyen et al. 2015;Nguyen et al. 2017). Each sequence was treated independently using the Bioedit software (version7.1.10) and the consensus sequence was generated. Multi-sequence alignment was then performed. Point mutations were identified by comparison with the sequence of the M. tuberculosis H37Rv reference strain available in GenBank (NC.000962.3). To describe the resistance-associated mutations in rpoB gene, a numbering system based on the Escherichia coli sequence annotation has been used.

Data analysis

Patients' information (the anonymity of the patients was maintained) was registered and cross-checked by CILM research team before being imported to Stata (v12.

Results

Socio-demographic characteristics of presumptive MDR-TB 1.

A total of 155 presumptive MDR-TB culture positive isolates collected between 2010 and 2014 were included in the study. The median age of the patients was 52 years-old (IQR: .

The number of patients increased with age (except for the age range > to 65 years old) (Table 1). There were more men than women (69.7% vs 30.3%), with a men/women ratio of 2.3. More than 50 % of patients were from the center part of Laos (n=80, 51.6 %), where the NRL and

Spoligotyping characterization 4.

Spoligotyping was performed on 139 M. tuberculosis isolates.The M. tuberculosis family/subfamily identifications were determined using SITVITWEB (SpolDB4 database), SPOTCLUST and MIRU-VNTRplus. A total of 43 different patterns were observed including 12 clusters (108 isolates) and 31 unique patterns (Table 4). Each cluster contained 2 to 53 isolates (average 9 isolates per cluster). One hundred and nineteen (85.6%) isolates could be assigned to 24 exiting SITs and five families in SpoIDB4; two (1.4%) were unknown (existing SITs in SpolDB4 but could not be related to any family); and 18 (12.9%) were orphans (absent from SpolDB4). The two unknown and 18 orphans were then compared with SPOTCLUST (SpolDB3) database. Finally, 133 (95.7%) isolates represented five families, consisting of Beijing, East African Indian (EAI), T, Haarlem (H) and Manu, and 7 (5.0%) isolates remained orphans or unknowns (Table 4,5). Among the 139 presumptive MDR-TB, Beijing was the predominant family (41.0 %, n=57), followed by EAI (38.1 %, n=53) and other families (20.9 %, n=29) (Table 5). Moreover, Beijing was more prevalent among the 43 drug resistant isolates (determined by MTBDRplus) than EAI and other families (60.5 %, 32.2 % and 9.3 % respectively) (Table 5) and this was significantly different (p=0.005). The poly-drug resistant and QDR patterns were more prevalent in Beijing isolates and pre-XDR isolates belonged only to Beijing family (Table 3).

According to the sequencing results, the mutations in rpoB were mainly located in the RRDR known to be associated with RIF resistance [START_REF] Hirano | Mutations in the RpoB Gene of Rifampin-Resistant Mycobacterium tuberculosis Strains Isolated Mostly in Asian Countries and Their Rapid Detection by Line Probe Assay[END_REF][START_REF] Zaw | Mutations inside rifampicin-resistance determining region of rpoB gene associated with rifampicin-resistance in Mycobacterium tuberculosis[END_REF]Bahrmand et al. 2009). Besides, one mutation outside RRDR (Ile572Phe) and one insertion (TGCCAA, CysGln) at codon 514 represented 8 % of all rpoB mutations. This mutation and insertion were recently identified in MDR isolates [START_REF] Hirano | Mutations in the RpoB Gene of Rifampin-Resistant Mycobacterium tuberculosis Strains Isolated Mostly in Asian Countries and Their Rapid Detection by Line Probe Assay[END_REF][START_REF] Zaw | Mutations inside rifampicin-resistance determining region of rpoB gene associated with rifampicin-resistance in Mycobacterium tuberculosis[END_REF]Bahrmand et al. 2009;Takawira et al. 2017;Nguyen 2017).

Although MTBDRplus was designed to cover RRDR, one mutant isolate (Leu511Pro) detected by sequencing was misdiagnosed by MTBDRplus. The Leu511Pro mutation was described as a mutation linked to low-level resistance to RIF [START_REF] Jo | Frequency and Type of Disputed RpoB Mutations in Mycobacterium tuberculosis Isolates from South Korea[END_REF][START_REF] Ocheretina | Correlation between Genotypic and Phenotypic Testing for Resistance to Rifampin in Mycobacterium tuberculosis Clinical Isolates in Haiti: Investigation of Cases with Discrepant Susceptibility Results[END_REF]), however, this isolate was identified from patient with relapse after treatment.

INH resistance is mainly mediated by mutations in katG or inhA genes or within the promoter region of inhA (Dookie et al. 2018). Around 80 % of M. tuberculosis isolates with INH resistance revealed mutations in codon 315 of the katG gene or position -15 in the inhApromoter (43 % to 94 % of katG315; 19 % of -15 inhA-promoter) (Seifert et al. 2015). Our results showed similar proportions of katG and inhA-promoter mutations (87.9 % and 18.2 % respectively). However, other uncommon mutations associated with low level and high level of INH resistance were detected (Asp189Gly and G630 leading to a Frameshift) [START_REF] Brossier | Comparative Study of Enzymatic Activities of New KatG Mutants from Low-and High-Level Isoniazid-Resistant Clinical Isolates of Mycobacterium tuberculosis[END_REF][START_REF] Kandler | Validation of Novel Isoniazid Resistance Mutations Not Detectable by Common Molecular Tests[END_REF]. A combination of mutations in inhA-promoter and in inhA-coding (-15(CT)/Ile21Val), previously described to be associated with high level of INH resistance and cross-resistance to ethionamide and it was found in one isolate of our samples [START_REF] Machado | High-Level Resistance to Isoniazid and Ethionamide in Multidrug-Resistant Mycobacterium tuberculosis of the Lisboa Family Is Associated with InhA Double Mutations[END_REF]).

The conventional culture-based DST for EMB resistance is problematic. Indeed, poor and variable agreements between MGIT 960, agar proportion methods or Bactec 460 were observed [START_REF] Horne | Diagnostic Accuracy and Reproducibility of WHO-Endorsed Phenotypic Drug Susceptibility Testing Methods for First-Line and Second-Line Antituberculosis Drugs[END_REF]). This could be due to the narrow range between the MICs of susceptible and resistant isolates of M. tuberculosis. Indeed, the MICs of some isolates were only weakly higher than the critical concentration and could result in false-susceptible results Nevertheless, since the sample is not exhaustive, this result cannot be extended at the national level. Further studies are needed to explore the molecular epidemiology of the MDR M. tuberculosis isolates.

Conclusion

The DNA sequencing provides crucial information on mutations associated with drug resistance among presumptive MDR-TB cases in Laos. The results revealed various mutations reflecting different patterns of resistance from mono-resistance to pre-XDR. This information is essential to help the prescription of appropriate treatment. Regarding the M. tuberculosis families, as expected our data showed that Beijing is significantly associated with drug resistance and more particularly with highly drug resistance patterns (QDR and pre-XDR). Every effort for accurate and rapid detection of causative species and drug resistance patterns is urgently needed for all TB patients in Laos. Molecular methods could be promising and reliable tools for drug resistance detection in order to limit the emergence and spread of MDR, pre-XDR and XDR-TB in the country.

Competing interests

The authors declare that they have no competing interests Selection of isolates for molecular analysis at CILM (7) , N=60 (any drug resistance detected by DST) + 14 susceptible 1 specimen sent for Xpert MTB/RIF at provincial level All sputum specimens sent to NRL (1) for culture [START_REF]Global tuberculosis report[END_REF] NRL: national reference laboratory; (2) Xpert MTB/RIF+: MTB detected [START_REF]Global tuberculosis report[END_REF] Xpert MTB/RIF-: MTB not detected; (4) DST: drug susceptibility testing (5) H: isoniazid, R: rifampicin, S: streptomycin, E: ethambutol, Km: kanamycin, Cm: capreomycin, Ofx: ofloxacin, (6) ), while the previously retreated cases accounted for 6.8 % (n=5). About half of study cases were from center part of the country (Table 3.1).

Supplementary tables 909

Table S 2.1 Frequencies of each mutation among the 42 drug resistant isolates.

CI: 0.2-1.5), whereas the MDR-TB was not identified among previously retreated cases due to negative culture results. However, based on Xpert MTB/RIF results, the rate of RR-TB among new cases was 1.2 % (95 % CI: 0.5-2.0) and among previously retreated cases was 4.1 % ( 95% CI: 0-9.6) (DRS report, June 2018). Regarding FQ and SLIDS, the mono OFX resistance and mono CAP resistance were observed (Table 3. 2). However, no resistance to SLIDs or FQs was detected among MDR-TB patients. Susceptible to all drugs 706 (91.0 [88.7-92.9]) 39 (88.6 [75.4-96.2]) 745 (90.9 [88.7-92.7])

Any resistance to FLD and SLD 70 (9.0 [7.1-11.1]) 5 (11.4 [3.8-24.6]) 75 (9.1 [7.3-11.3])

Any resistance to FLD (R, H, E, S) 64 (8.2 [6.4-10.4]) 5 (11.4 [3.8-24.6]) 71 (8.7 [6.8-10.8])

Any resistance to FQ and SLID (Ofx, Km, Cm)

Mono drug resistance 54 (7.0 [5.3-9.0]) 2 (4.5 [0.6-15.5]) 56 (6.8 [5.2-8.8 19 (2.4 [1.5-3.8]) 1 (2.3 [0.1-12.0]) 20 (2.4 [1.5-3.7

Poly drug resistance

11 (1.4 [0.7-2.5]) 3 (6.8 [1.4-18.7]) 14 (1.7 [0.9-2.8]) RS 2 (0.3 [0.03-0.9]) 1 (2.3 [0.1-12. The performance of sequencing for detection of INH resistance was evaluated by the presence of mutations in katG, inhA-promoter and inhA-coding gene together. For detection of OFX resistance, the mutations in both gyrA and gyrB were considered and for detection of STR resistance, mutations in both rpsL and rrs-F1 were considered (Table 3.4). The sequencing revealed a sensitivity and a specificity of 88.9 % and 90.7 % for the detection of RIF resistance; from the central provinces, including Vientiane capital, represented about half of the study population, the proportion of drug resistant isolates was higher in the northern provinces (p=0.03) (Table 3. 7). It is worth noting that there was also a significant difference of drug resistance proportion according to the M. tuberculosis family (p=0.01). Indeed, the proportion of drug resistance was higher among Beijing isolates than in EAI and other family isolates. This finding suggests that the association of drug resistance with the northern part of Lao PDR is linked to the higher preponderance of Beijing in the North as described in the first National TB prevalence survey (see Chapter 3, Result 1 (Paper 1)).

Prevalence and extent of drug resistant TB in Lao PDR

In this first national anti-tuberculosis drug resistance survey (DRS), the MDR-TB rate based on phenotypic DST is 0. 11% -27% among retreated cases [START_REF]Global tuberculosis report[END_REF]. The low proportion of Beijing family could be an explanation for the low level of drug resistant TB in Laos. However, the data suggest a potential risk of Beijing increase and thus an undibitable risk of drug resistance increase.

While the NTCP mainly focuses on MDR/RR-TB, the mono resistant and poly-resistant TB (drug-resistant TB other than MDR-TB) are actually more common than MDR/RR-TB among our three samplings, TBPS, DRS and presumptive MDR-TB (5 %, 8 %, 12 % respectively). The data was in agreement with the global report. Indeed, the worldwide prevalence of MDR-TB in new cases is around 3% while the prevalence of mono-and poly-resistant strains is almost 17% [START_REF] Wright | Anti-tuberculosis drug resistance in the world: fourth global report: the World Health Organization/International Union Against Tuberculosis and Lung Disease (WHO/UNION) Global Project on Anti-Tuberculosis Drug Resistance Surveillance, 2002-2007[END_REF]. Many of these cases contribute to the amplification of resistance and, eventually, can lead to MDR-TB if they are not properly managed [START_REF]WHO | Guidelines for the programmatic management of DR-TB. Companion Handbook to the WHO Guidelines for the Programmatic Management of Drug-Resistant Tuberculosis[END_REF]. In Lao PDR, the Xpert MTB/RIF has been mutations in katG gene at codon 315 [104,196,197] and mutations in rpsL at codon 43 [198, 199]. These prevailing mutations are strongly selected in the population [200,201]. The pattern is different for pncA gene since we observed a diversity of mutations without preeminent ones as described in many studies (four unique mutations among presumptive MDR-TB cases and 5

unique mutations among DRS). The majority of them were previously correlated with PZA resistance.

The By assessing the performance of molecular methods for the detection of drug resistance, the current GeneXpert MTB/RIF used in the country showed higher sensitivity and specificity for detection of RIF resistance than MTBDRplus, however its sensitivity was lower than DNA sequencing. The data demonstrated that the use of a limited number of targets for the detection of drug resistance is not enough to get the full drug resistant patterns and determine the appropriate treatments. Higher the number of genes/regions will be explored, the more we will be close to the true drug resistance. In this context, NRL needs to either implement new ANNEX Table S 3 The data was stored in google drive, please follow the link below:

https://drive.google.com/drive/folders/1-IzHwzjLL5v69ER6QaELGbPt-zSXqxEB