Anne Darmon-Curti

François Darmon

Aurélie Hennebique

Thomas Guimard

Guillaume Martin-Blondel

Timothée Klopfenstein

Jean-Philippe Talarmin

Didier Raoult

Max Maurin

Pierre-Edouard Fournier
email: pierre-edouard.fournier@univ-amu.fr

A Case Series of Patients Diagnosed at the National Reference Center for Rickettsioses From 2008 to 2017

Keywords: Tularemia, Francisella tularensis, case series, France, diagnosis

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Introduction

Tularemia is a zoonotic disease caused by Francisella tularensis. This facultative intracellular Gramnegative bacillus was isolated for the first time from flying squirrels in Tulare County in the USA by Mac Coy and Chapin [START_REF] Georges | Further Observations on a Plague-Like Disease of Rodents with a Preliminary Note on the Causative Agent , Bacterium tularense[END_REF] . Tularemia is endemic in North America 2 , Asia [START_REF] Chitadze | Water-borne outbreak of oropharyngeal and glandular tularemia in Georgia: Investigation and follow-up[END_REF] and Europe [START_REF] Hauri | Investigating an airborne tularemia outbreak, Germany[END_REF][START_REF] Kantardjiev | Tularemia outbreak, Bulgaria, 1997-2005[END_REF][START_REF] Reintjes | Tularemia outbreak investigation in Kosovo: case control and environmental studies[END_REF][START_REF] Pérez-Castrillón | Tularemia epidemic in northwestern Spain: clinical description and therapeutic response[END_REF][START_REF] Eliasson | Tularemia: Current Epidemiology and Disease Management[END_REF] .

Francisella tularensis subspecies tularensis (type A), almost restricted to North America, is responsible for the most severe diseases. The lethality may be as high as 30% in untreated pulmonary forms 9 . In contrast, F. tularensis subspecies holarctica (type B) is widely distributed in the Northern Hemisphere, but also in southern Australia, and associated with a lethality rate < 1%. Clinical signs vary according to the geographical area, season and thus mode of contamination [START_REF] Reintjes | Tularemia outbreak investigation in Kosovo: case control and environmental studies[END_REF][START_REF] Pérez-Castrillón | Tularemia epidemic in northwestern Spain: clinical description and therapeutic response[END_REF][START_REF] Eliasson | Tularaemia in an emergent area in Sweden: an analysis of 234 cases in five years[END_REF][11][START_REF] Helvaci | Tularemia in Bursa, Turkey: 205 cases in ten years[END_REF][START_REF] Feldman | An Outbreak of Primary Pneumonic Tularemia on Martha's Vineyard[END_REF] . Six clinical forms are classically recognized: the ulceroglandular (ulcer associated with lymphadenopathy) and glandular (lymphadenopathy) forms, upon skin inoculation after animal contact (most often lagomorphs or small rodents) or by arthropod bite; the oculoglandular (conjunctivitis) form after conjunctival inoculation; the oropharyngeal (sore throat) form upon ingestion of contaminated water or food [START_REF] Maurin | Tularaemia: clinical aspects in Europe[END_REF] ; and two systemic diseases, including the pneumonic (pneumonia) and the typhoidal forms (mimicking symptoms of typhoid). Frequents complications of lymphadenopathies are suppurations and even skin fistulas. Other complications such as sepsis or meningitis [START_REF] Gangat | Cerebral abscesses complicating tularemia meningitis[END_REF] are more rarely reported and usually occur in patients with comorbidities and/or immunosuppression.

Current knowledge of tularemia comes from data reported in highly endemic areas such as North America 11, [START_REF] Matyas | Pneumonic tularemia on Martha's Vineyard: Clinical, epidemiologic, and ecological characteristics[END_REF] and Northern Europe [START_REF] Eliasson | Tularaemia in an emergent area in Sweden: an analysis of 234 cases in five years[END_REF] . In contrast, few data exist for Western Europe [START_REF] Pilo | Identification of Francisella tularensis cluster in central and western Europe[END_REF][START_REF] Mailles | 10 years of surveillance of human tularaemia in France[END_REF][START_REF] Maurin | Human Tularemia in France, 2006-2010[END_REF] .

Here, we present the epidemiological, clinical, diagnostic and treatment data of 177 patients diagnosed with tularemia in the French reference center for rickettsioses, coxiellosis and bartonelloses in Marseille, France, from January 1, 2008 to December 31, 2017.

A c c e p t e d M a n u s c r i p t 5

Methods

Definition of tularemia cases:

All patients with a suspected diagnosis of tularemia were considered as confirmed when they exhibited compatible clinical findings and at least: (1) a positive real-time PCR (RT-PCR) and/or 16S rRNA PCR for F. tularensis; (2) seroconversion; or (3) a four-fold increase in immunofluorescence serological titers, as previously described [START_REF]Tularémie : définition de cas. Saint-Maurice: InVS[END_REF] . Culture was carried out only on PCR-positive samples.

Patients with compatible clinical findings and a single positive serological test were considered probable cases.

Patients and clinical samples:

The present study is a retrospective analysis of epidemiological and clinical data from patients with tularemia. Data had been prospectively collected since 2008 using an anonymized and standardized questionnaire and stored on a secured computer by the French reference center for tularemia.

Patient Consent Statement

The present study was validated by the Ethics Committee of the "Institut hospitalo-universitaire" (IHU) Méditerranée Infection under reference 2017-029.

The consent form to be signed by patients was sent to the clinicians, who obtained the signature before transmitting the data.

Mode of transmission:

We assigned the most likely source of contamination according to the scheme already used by Maille et al. [START_REF] Mailles | 10 years of surveillance of human tularaemia in France[END_REF] :

-Any patient infected with F. tularensis who reported direct contact with hares during the month before the onset of symptoms would have been infected by this exposure; A c c e p t e d M a n u s c r i p t 6 -A tick bite reported during the month before the onset of symptoms would be the route of contamination for any patient unless the patient also reported direct contact with hares; -An occupational exposure during the month before the onset of symptoms would constitute a circumstance of contamination unless the patient also reported direct contact with hares or a tick bite; -Recreational activities resulting in exposure to aerosols or dust in the forest during the month before the onset of symptoms would be circumstances of contamination unless the patient reported direct contact with hares, tick bite or occupational exposure.

Laboratory diagnosis

Serology was performed using indirect immunofluorescence. Immunoglobulin G and M titers were measured. For this purpose, a formalin-inactivated antigen prepared in-house from a biovar I strain of F. tularensis subspecies holarctica was used as previously described [START_REF]Tularémie : définition de cas. Saint-Maurice: InVS[END_REF] .

When lymph node biopsy specimens were available, a F. tularensis-specific RT-PCR assay targeting the yqaB gene (Ftul0541F, Ftul0541R and Ftul0541P), was also used for the detection of the bacterium, as previously described [START_REF] Angelakis | Real-time PCR strategy and detection of bacterial agents of lymphadenitis[END_REF] . In addition, a patient with hip arthritis was detected positive using a broad range PCR assay followed by sequencing the 16S RNA gene with the fD1 and rP2 primers was used [START_REF] Angelakis | Real-time PCR strategy and detection of bacterial agents of lymphadenitis[END_REF] .

Outcome

Therapeutic success was defined as the resolution of symptoms (fever, abscess) one month after appropriate antibiotics (fluoroquinolone, tetracycline, or aminoglycoside) had been discontinued.

We considered as therapeutic failure the persistence of symptoms and/or the occurrence of complication(s) despite appropriate antibiotic.

Surgery was performed either as a diagnostic and/or a curative procedure. Diagnostic surgery was performed in patients presenting with enlarged lymph nodes of unknown or uncertain etiology. Curative surgery was performed in patients with a complicated form such as abscess, prosthesis infection, aortitis.

Statistics

The Fisher's exact test was used to compare the number of therapeutic failures with doxycycline and fluoroquinolones, and assess the correlation between immunosuppression and the need for surgical treatment as well as the correlation between immunosuppression and the occurrence of therapeutic failure.

An ANOVA test was carried out to investigate statistical correlations between the occurrence of therapeutic failure or the need for surgical treatment and the time between the onset of symptoms and effective antibiotic therapy.

Results:

Confirmed and probable cases (Supplementary Figure1): A total of 251 patients were included. For 68 patients, the laboratory criteria were not fulfilled.

Another six patients that had been infected abroad were excluded. Overall, 89 confirmed and 88 probable cases (total 177 patients) were included in the study.

Epidemiological data:

The male-female sex ratio was 2.28 (54 female and 123 male). The mean age of patients was 47.38 +/-17.8 years (range 2 to 89 years).

A total of 173 cases were sporadic, and four cases were clustered in two groups: two were household cases, and two were patients infected during an orientation race in a forest. The geographical distribution of infected patients is depicted in Figure 1.

Exposure factors are detailed in Figure 2. The most common risk factor was a contact with hares, game (including boar, deer and doe) or rodents (21.5%), followed by exposure to aerosols (20.9%).

No specific exposure factor was identified in 18 patients.

A c c e p t e d M a n u s c r i p t 8

Contact with hares, game or rodents occurred more often in the winter season, while tick-bites were mostly reported in Summer (Supplementary Figure 2). Clinical data (Tables 1 and2):

Significant background: Thirteen of 142 patients (9.15%) were immunocompromised: eight patients had diabetes (one type I and seven type II). Four patients were on immunosuppressive therapy: one on mycophenolate mofetil, one on methotrexate, and two on anti-TNFα. The thirteenth patient suffered from liver cirrhosis. One patient had a vascular prosthesis.

The average incubation time of tularemia, calculated from 49 patients' data, was 9.28+/-9.7 days (range 1 to 43 days). Supplementary Figure 3 represents the distribution of the incubation times, which mostly ranged from one to five days. In our series, 7 patients exhibited unusually long incubation delays, ranging from 16 to 43 days.

Clinical forms:

Sixty-one (34.5%), 48 (27.1%), 32 (18%), 14 (7.9%), nine (5.0%) and four (2.3%) patients presented with an ulceroglandular, glandular, pleuropulmonary, typhoidal, oropharyngeal, or oculoglandular forms, respectively. We identified nine (5.1%) atypical forms which are presented in Supplementary Tables 1 and2: a combined oculoglandular and ulceroglandular form, a pre-thyroid abscess, an ulceroglandular form associated with aortitis, a pleuropulmonary form associated with aortitis, a pleuropulmonary form associated with myocarditis, an aortic endocarditis, a total hip prosthesis infection, a pleuropulmonary form complicated by a splenic hematoma, and a pleuropulmonary form complicated by pericarditis.

The hip prosthesis infection occurred in a 49-year-old immunocompromised patient with cirrhosis.

While drunk and walking outside, this male patient fell and suffered both femoral neck fracture and skin lacerations. He had a total hip replacement. In addition to hepato-renal decompensation of his cirrhosis with hepatic encephalopathy, the immediate aftermath of surgery was complicated by a Downloaded from https://academic.oup.com/ofid/advance-article/doi/10.1093/ofid/ofaa440/5908812 by guest on 04 November 2020 A c c e p t e d M a n u s c r i p t 9 hematoma of the hip that had to be surgically drained. 16S rRNA PCR testing of a hematoma sample revealed the presence of F. tularensis. No culture was performed. The evolution of the illness was favorable with a three-month treatment with doxycycline followed by a one-stage exchange arthroplasty and additional antibiotic therapy with ciprofloxacin and gentamicin for three months.

The case of splenic hematoma occurred in a 73-year-old male with a pleuropulmonary form of tularemia. This non-immunocompromised patient had a history of pericarditis, hypothyroidism and coronary stent placement. The diagnosis of tularemia was confirmed by PCR-detection of F. tularensis DNA in a mediastinal lymph node. A spontaneous splenic hematoma developed during hospitalization for tularemia as a complication of a moderate splenomegaly. The evolution of the illness was favorable after 21 days of doxycycline treatment.

We also diagnosed tularemia in a patient who developed a pre-thyroid lodge abscess without having any evident history of exposure to F. tularensis. While performing a mediastinoscopy for a mediastinal lymph node biopsy, the surgeon unexpectedly drained an abscess from the pre-thyroid chamber. The diagnosis of tularemia was made by F. tularensis-specific PCR from the abscess pus.

The evolution of the illness was favorable after surgical drainage and doxycycline therapy.

Symptoms

The patients' symptoms are summarized in Table 2. The main symptoms included fever (85.0%), general impairment, (33.3%), chills (26.4%), myalgia (25.5%), sweats (25.0%) and arthralgia (21.2%).

Location of lymphadenopathies:

The upper limbs were the most frequent location, in 56 patients (31.6%), followed by the chest (46, 30.0%), lower limbs (38, 21.4%), neck (31, 17.5%) and abdomen (2, 1.1%). Twenty-nine patients (16.4%) had enlarged nodes in various anatomical sites. Twenty-nine patients (17.0%) exhibited an inoculation lesion on the upper limbs, 23 (13.5%) on the lower limbs, five (2.9%) on the trunk, three (1.7%) on the scalp, and one (0.6%) patient had both upper limb and facial ulcers.

Secondary dermatological lesions (all dermatological manifestations following the initial inoculation ulcer):

Nineteen patients had secondary dermatological lesions, including seven with lymphangitis ranging from the primary skin lesion to an adenopathy, five had a rash (location and type not specified), three had nodosum, one had cellulitis, one had erythema multiforme, one had pustules, and one had livedo reticularis.

Microbiological results

Initial presumed diagnosis:

For 67/148 patients (45.2%) for whom a suspected diagnosis was specified, tularemia was the main diagnostic request. In the other cases, the main suspected etiologies were: cat-scratch disease (44, 27.7%), Q fever (19, 12.8%), rickettsiosis (12, 7.9%), Whipple's disease (3, 2.0%), ehrlichiosis (2, 1.3%) and anaplasmosis (1, 0.7%).

Confirmation of tularemia diagnosis:

Six cases (6.9%) were confirmed by seroconversion and five (5.8%) by a four-fold rise in antibody titers between two serum samples.

Confirmed diagnosis of tularemia was obtained in 78 patients (86%) by PCR testing of lymph node (64), skin (6), lung (3) or aorta biopsies (2), bronchoalveolar lavage (1) and joint (1) fluid.

In addition, F. tularensis was identified by 16S rRNA PCR-sequencing of a bacterial strain that could not be routinely identified.

Blood tests:

Blood test results are summarized in Supplementary Table 3. Twenty-two out of 91 (24.1%) patients had leukocytosis, 1.1% had leukopenia, 31.3% had liver cytolysis, 6.7% had thrombocytopenia, and 4.0% had thrombocytosis. The mean CRP rate was 81.6 +/-60.5 mg/L (range 4 to 248).

Evolution:

Seventy-eight (53.4%) patients were hospitalized. Sixty (40.5%) developed complications, including lymph node abscesses (39), fistulizations (14), persistent asthenia (2), aortitis (2), and one case each of myocarditis, pericarditis and splenic hematoma.

Antibiotic therapy:

One hundred and seventy-two patients received an antibiotic therapy active against F. tularensis, including 118 who received one antibiotic. The most commonly prescribed antibiotics were doxycycline (106 patients) and an oral fluoroquinolone (42). Two patients were administered a fluoroquinolone and doxycycline. Table 3 and Supplementary Table 4 specify the effectiveness of treatments known to be active against tularemia. The mean duration of antibiotic therapy was 16 (min 7, max 28, SD 4.5) days for doxycycline, and 21 (min 7, max 84, SD 18.3) for fluoroquinolones. There were more therapeutic failures for fluoroquinolones (ciprofloxacin, levofloxacin, ofloxacin and unspecified fluoroquinolones, 14/43 [33%]) than for doxycycline (9/91 [9.9%], p=0.02). The mean duration between the onset of symptoms and the onset of effective antibiotics was 38.6 days (range 5 to 71, SD 16.7) for patients who presented with a therapeutic failure, versus 42.5 days (range 1 to 175, SD 33.4) for patients who recovered (p=0.64).

In contrast, 13 patients recovered without any effective antibiotic therapy, including four who benefited from lymph node excision, six who received an ineffective empirical antibiotic treatment, and three who neither received antibiotics nor underwent surgery. Their characteristics are specified in Supplementary Table 5. None of the thirteen patients was immunocompromised. There were four

Surgery (Supplementary Table 4):

Eighty patients (51.6%) underwent surgery: 31 (20%) had a lymph node puncture, 47 had lymph node removal (30.3%), 1 (0.6%) had an aortic allograft, and 1 (0.6%) had a total hip prosthesis replacement.

Thirty-four patients (21.9%) underwent surgery before effective antibiotic therapy, seventeen (10.9) after, eight (5.1%) at the same time. For twenty-one patients, the date of surgery was unknown. No data about surgery was available for 22 patients.

For patients who underwent surgery, the mean delay between onset of symptoms and effective antibiotic therapy was longer (52.1 days, range 7 to 175, SD 37.9) than for those who did not have surgery (mean 32.1 days, range 1 to 87 SD 19.5, p=0.006). In immunocompromised patients, 5/13 patients (38.5%) benefited from surgery, compared to 21/127 (16.6%) in immunocompetent patients (p=0.06).

Discussion

We describe 177 cases of tularemia diagnosed in the French reference center for rickettsioses, coxiellosis and bartonelloses from January 1 st , 2008 to December 31 st , 2017. During this period, the French reference center for tularemia in Grenoble recorded 317 cases, and Santé Publique France 611 cases. Among our 177 cases, three were previously described in a published series by the French reference center for tularemia [START_REF] Maurin | Human Tularemia in France, 2006-2010[END_REF] and 44 cases included in a study published by Santé Publique France, the French national public health agency [START_REF] Mailles | 10 years of surveillance of human tularaemia in France[END_REF] . Table 1 compares these three French series.

The most affected regions were "Nouvelle Aquitaine", "Pays de la Loire" and "Grand Est" (Figure 1), as previously described [START_REF] Mailles | 10 years of surveillance of human tularaemia in France[END_REF][START_REF] Maurin | Human Tularemia in France, 2006-2010[END_REF] . The annual distribution of cases is similar to that described in the literature [START_REF] Maurin | Human Tularemia in France, 2006-2010[END_REF] : tick-borne contamination occurred more often in Summer, while contamination during hunting or contact with infected animals occurred more often in Winter. The mean incubation time A c c e p t e d M a n u s c r i p t 13 was 9.28 days, which is longer than that usually described (3 to 5 days [START_REF] Matyas | Pneumonic tularemia on Martha's Vineyard: Clinical, epidemiologic, and ecological characteristics[END_REF] ). As expected, the most common forms were ulceroglandular (34.5%) and glandular (27.1%). These forms had a significantly higher therapeutic failure rate than the other forms (19.2% versus 7.6%, p<10 -2 ). We also diagnosed rare forms (Supplementary Tables 1 and4), including one case each of hip prosthesis infection, splenic hematoma and pre-thyroid lodge abscess. To date, few cases of joint prosthetic infections have been reported, affecting the knees [START_REF] Cooper | Chronic Prosthetic Device Infection with Francisella tularensis[END_REF][START_REF] Chrdle | Francisella tularensis Periprosthetic Joint Infections Diagnosed with Growth in Cultures[END_REF] or hips [START_REF] Rawal | Unusual case of prosthetic joint infection caused by Francisella Tularensis[END_REF] . We assume that the hip prosthesis infection complicated a skin wound contamination with F. tularensis. In contrast, we could not find any previous case of spontaneous splenic hematoma complicating a splenomegaly, or pre-thyroid abscess, in the course of tularemia. However, a case of parapharyngeal abscess was reported in the literature [START_REF] Koc | Tularaemia presenting as parapharyngeal abscess: Case presentation[END_REF] .

In addition, we diagnosed two cases of F. tularensis aortitis, one of which had previously been published [START_REF] Briere | Francisella tularensis aortitis[END_REF] , and a case of pericarditis complicating a pleuropulmonary form. Other cases of pericarditis have been described in the literature [START_REF] Cw | Tularemic pericarditis; report of two cases and review of literature[END_REF] , including one previously diagnosed in our laboratory in 2007 [START_REF] Landais | Pericardial effusion as the only manifestation of infection with Francisella tularensis: A case report[END_REF] . We also diagnosed a case of F. tularensis aortic endocarditis [START_REF] Gaci | Francisella tularensis endocarditis: two case reports and a literature review[END_REF] . Only four confirmed cases of F. tularensis endocarditis have been described to date [START_REF] Gaci | Francisella tularensis endocarditis: two case reports and a literature review[END_REF][START_REF] Tancik | Francisella tularensis Endocarditis Trovafloxacin-Induced Acute Hepatitis[END_REF][START_REF] Salit | Tularemia endocarditis from domestic pet exposure[END_REF] . We also described a probable case of myocarditis complicating a pleuropulmonary form. Indeed, the diagnosis was made on the basis of a positive serology and histological findings observed in a mediastinal lymphadenopathy biopsy. To date, only two cases of F. tularensis myocarditis have been described in the literature [START_REF] Franco | Infection due to Francisella tularensis, myocarditits ant dilated myocardiopathy[END_REF][START_REF] Frischknecht | Tularemia : an experience of 13 cases including a rare myocarditis in a referral center in Eastern Switzerland ( Central Europe ) and a review of the literature[END_REF] .

Seventy-eight of the 177 patients (53.4%) required hospitalization. The clinical signs exhibited by patients were not specific (Table 2). In our series, the majority of patients had fever (85%), lymphadenopathy (72.2%) and inoculation lesions (36%). During our study, we did not find a statistical relationship between the time to introduce effective treatment and the occurrence of therapeutic failures. The diagnosis was obtained in 99 patients (56%) by serology, and in 78 patients (44%) by PCR.

The antibiotic that was most commonly used in our series was doxycycline (administered 106 times as monotherapy), followed by a fluoroquinolone alone (42 patients). In our series, the observed A c c e p t e d M a n u s c r i p t 14 therapeutic failure rates for doxycycline and fluoroquinolones (9.9% and 32.5%, respectively) were high. It was previously demonstrated that the earlier effective antibiotic therapy is administered to tularemia patients, the less often therapeutic failure occurs [START_REF] Meric | Evaluation of clinical, laboratory, and therapeutic features of 145 tularemia cases: The role of quinolones in oropharyngeal tularemia[END_REF][START_REF] Gozel | Evaluation of clinical and laboratory findings of pediatric and adult patients with oropharyngeal tularemia in turkey: A combination of surgical drainage and antibiotic therapy increases treatment success[END_REF] . We observed long delays prior to antibiotic therapy onset, which may explain such high therapeutic failure rates. However, the therapeutic failure rate was significantly higher for fluoroquinolones (p = 0.002), which are currently recommended with doxycycline as first line treatment the less severe cases [START_REF] Caspar | Antibiotic susceptibility of Francisella tularensis subsp. holarctica strains isolated from tularaemia patients in France between 2006 and 2016[END_REF] by the World Health Organization (WHO) 38 . In their series, Rojas-Moreno described in their retrospective analysis of 17 patients no case of therapeutic failure under doxycycline for a mean duration of 21 days in addition to surgical treatment [START_REF] Rojas-Moreno | Tetracyclines for Treatment of Tularemia : A Case Series[END_REF] . Castrillon et al. and Eliasson et al. both described more therapeutic failures with doxycycline than with fluoroquinolones (42.8% and 3% therapeutic failures with doxycycline, respectively, and 4.5% and no therapeutic failure with fluoroquinolones [START_REF] Pérez-Castrillón | Tularemia epidemic in northwestern Spain: clinical description and therapeutic response[END_REF][START_REF] Eliasson | Tularaemia in an emergent area in Sweden: an analysis of 234 cases in five years[END_REF] ). In our series, among the 42 patients on fluoroquinolone alone, only 11 patients received ciprofloxacin 500mg 2 times per day (Table 3). We acknowledge the fact that the high failure rate observed for fluoroquinolones may at least in part be explained by the heterogeneity in molecule, dosage and treatment duration used.

Due to its clinical and pathological presentation, tularemia can be confused with cat-scratch disease or tuberculosis [START_REF] Turhan | Differential diagnosis of cervical lymphadenitis mimicking malignancy due to tularemia: our experiences[END_REF][START_REF] Yildirim | Tuberculosis or tularemia? A molecular study in cervical lymphadenitis[END_REF] . In our series, the suspected diagnosis by clinicians was tularemia in only 67 patients (45.2%), and Bartonella infection in 44 patients (27.7%). Our data suggest that tularemia is probably underdiagnosed in France due to its unspecific clinical presentation and insufficient knowledge of the disease by clinicians. We suggest that tularemia should systematically be considered among the differential diagnoses of enlarged lymph nodes or prolonged fever in patients with outdoor activities.

Conclusion

Tularemia is endemic in France in the Nouvelle Aquitaine, Grand Est and Pays de la Loire regions. This infection is probably underdiagnosed. Although the ulceroglandular form was the most common, we identified rare forms of the disease such as splenic hematoma and osteoarticular infection. In endemic areas and in a consistent epidemiological context, diagnosing this disease allows optimized patient management.

A

  c c e p t e d M a n u s c r i p t 11 Cultures were performed for 59 of the 78 PCR-proven specimens, and 6 (1%) were positive.

A

  c c e p t e d M a n u s c r i p t 12 pleuropulmonary forms, three oropharyngeal forms, three glandular forms, two typhoidal forms and one oculoglandular form.

  Downloaded from https://academic.oup.com/ofid/advance-article/doi/10.1093/ofid/ofaa440/5908812 by guest on 04 November 2020

Table 1 :

 1 Comparison of the data in our series with 2 other French series

	Number of patients included Mean age Sex ratio Hunters Incubation time (days) Glandular forms Ulceroglandular forms Pleuropulmonary forms Oropharyngeal forms Oculoglandular forms Typhoidal forms Hospitalization Complications	Maurin et al. 19 101 51.7 1.2 16 (15.8%) 5.5 24 (23 .7%) 34 (33.7%) 10 (10%) 17 (16.8%) 4 (4%) 9 (8.9%) 30 (29.7%) NR	Mailles et al. 18 433 49 1.8 52 (12%) NR 200 (46%) 113 (26%) 42 (10%) 25 (6%) 8 (2%) 45 (10%) 188 (43%) 20	Our series 177 47.38 2.28 39 (21.7) 9.28 48 (27.1%) 61 (34.5%) 32 (18%) 9 (5.0%) 4 (2.3%) 14 (7.9%) 78 (53.4%) 60 (40.8)	Downloaded from https://academic.oup.com/ofid/advance-article/doi/10.1093/ofid/ofaa440/5908812 by guest on 04 November 2020
	Death	1	2	0
	Confirmed cases	94	130	89

Table 2 :

 2 Clinical data of 177 patients in France (2008-2017): Comparison with data from 5 previously reported case series A Downloaded from https://academic.oup.com/ofid/advance-article/doi/10.1093/ofid/ofaa440/5908812 by guest on 04 November 2020

		Ref 11	Ref 8	Ref 40	Ref 7	Ref 19	Our study
	Country	USA	Sweden	Turkey	Spain	France	France
	Number of patients (M/F)	88 (3.5)	234 (1.05)	205 (0.55)	142 (0.59)	101 (1.37)	177 (2.28)
	Study period	1949-1979	2000-2004	1988-1998	1997-1998	2006-2010	2008-2017
	Age ranges (means)	2-82 (NI)	1-88 (48.3)	5-75 (NI)	14-82 (52)	5-95 (52)	2-89 (47.4)
	Main sources of infection	Hares	Mosquitos	Water	Hares	Hares	Hares
	Fever	85	83	66	90.8	67.3	85.0
	Headache	45	32	15	9.9	NA	13.4
	Myalgia	31	NA	6	25.4	20.8	25.5
	Arthralgia	15	NA	NA	14.8	16.8	21.2
	Cough	38	25	NA	24	5.9	9.2
	Sore throat	15	3	58.5	16.9	15.8	7.0
	Nausea and/or vomiting	17	23	NA	24	5.9	2.8
	Diarrhea	10	9	NA	2.1	3.9	6.4
	Inoculation lesion	59	88	NA	61.4	31.7	35.6
	Secondary	5.7	29.9	14	17.6	2.9	11.9

M/F: Male/female sex ratio, NA: no data available A : All data are given as percentages unless otherwise specified.

Table 3 :

 3 Antibiotic therapy administred and therapeutic failure rates Downloaded from https://academic.oup.com/ofid/advance-article/doi/10.1093/ofid/ofaa440/5908812 by guest on 04 November 2020 = number of patients; TF = therapeutic failure; * = number of patients for whom data is available; mg/d = milligram/day; X2/d = two times per day; IV = intravenous 1 If not specified, antibiotics were administred orally.Downloaded from https://academic.oup.com/ofid/advance-article/doi/10.1093/ofid/ofaa440/5908812 by guest on 04 November 2020 Geographical distribution of tularemia cases Downloaded from https://academic.oup.com/ofid/advance-article/doi/10.1093/ofid/ofaa440/5908812 by guest on 04 November 2020 Source of exposure (game include boar, deer and doe) Downloaded from https://academic.oup.com/ofid/advance-article/doi/10.1093/ofid/ofaa440/5908812 by guest on 04 November 2020

		Total number of patients on
	Antibiotic therapy 1	antibiotic therapy
		N (%)	TF /* (%)
	Doxycycline, 200 mg/d	104 (59)	9 /91 (9.9)
	Ciprofloxacin, 500 mg X2/d	21 (12)	7/21 (33)
	Unspecified fluoroquinolone and dosage	11(6.2)	4/9 (44)
	Ofloxacin, 200 mg X2/d	5 (2.8)	1/5 (20)
	Levofloxacin, 500 mg/d	4 (2.3)	2/4 (50)
	Doxycycline, 200mg/d + IV amikacin	2 (1.1)	0/2 (0)
	Ciprofloxacin, 750mg X2/d + IV gentamicin + doxycycline, 200mg/d	1 (0.56)	0 /1(0)
	Unspecified fluoroquinolone + IV gentamicin	1 (0.60)	0 (0)
	Levofloxacin, 500mg X2/d + doxycycline, 200mg/d	1 (0.60)	0/1 (0)
	Ofloxacin, 400 mg X2/d	1 (0.60)	0/1(0)
	IV amikacin	1 (0.60)	0/1 (0)

N

26

FIGURE TABLE AND GRAPHICS

Figure 1:

Downloaded from https://academic.oup.com/ofid/advance-article/doi/10.1093/ofid/ofaa440/5908812 by guest on 04 November 2020


Aurélie Hennebique
email: ahennebique@chu-grenoble.fr

Marie Bidart

Sophie Jarraud

Laëtitia Beraud

Carole Schwebel

Max Maurin

Sandrine Boisset
email: sboisset@chu-grenoble.fr.

Digital PCR for Detection and Quantification of Fluoroquinolone Resistance in Legionella pneumophila

Keywords: digital PCR, antibiotic resistance, fluoroquinolones, gyrA, Legionella pneumophila

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Legionella pneumophila, a Gram-negative, facultative intracellular bacterium, is the causative agent of legionellosis, a severe pneumonia associated with mortality rates ranging from 5% to 25% [START_REF] Mykietiuk | Clinical outcomes for hospitalized patients with Legionella pneumonia in the antigenuria era: the influence of levofloxacin therapy[END_REF]. Diagnosis of this disease mainly relies on urinary antigen test, and culture and PCR testing of respiratory samples. The first-line drugs for treatment of legionellosis are the macrolides and the fluoroquinolones (FQ) [START_REF] Griffin | Macrolides versus quinolones in Legionella pneumonia: results from the Community-Acquired Pneumonia Organization international study[END_REF]. In vitro antibiotic susceptibility testing of Legionella sp. strains is currently not recommended on a routine basis, especially because no standardized method is available from the CLSI (Clinical and Laboratory Standards Institute) or the EUCAST (European Committee on Antimicrobial Susceptibility Testing). However, therapeutic failures and relapses are still reported [START_REF] Kurz | Failure of treatment of legionella pneumonia with ciprofloxacin[END_REF][START_REF] Schindel | Persistent Legionella infection in a patient after bone marrow transplantation[END_REF][START_REF] O'reilly | Persistent culturepositive Legionella infection in an immunocompromised host[END_REF], FQ-resistant mutants of L. pneumophila have been readily selected in vitro [START_REF] Nielsen | Susceptibility of Legionella species to five antibiotics and development of resistance by exposure to erythromycin, ciprofloxacin, and rifampicin[END_REF][START_REF] Linde | The amino acid substitution Asp87Tyr in GyrA of Legionella pneumophila Sg 1 is associated with elevated MICs to nalidixic acid but not to ciprofloxacin or moxifloxacin in vitro[END_REF][START_REF] Jonas | Development and mechanism of fluoroquinolone resistance in Legionella pneumophila[END_REF][START_REF] Almahmoud | Mutational paths towards increased fluoroquinolone resistance in Legionella pneumophila[END_REF], and FQresistant mutants have also recently been found in legionellosis patients [START_REF] Bruin | Isolation of ciprofloxacin-resistant Legionella pneumophila in a patient with severe pneumonia[END_REF][START_REF] Shadoud | Hidden Selection of Bacterial Resistance to Fluoroquinolones In Vivo: The Case of Legionella pneumophila and Humans[END_REF]. Indeed, Shadoud et al. recently characterized the in vivo selection of such FQ-resistant mutants of L. pneumophila in two legionellosis patients treated with these antibiotics [START_REF] Shadoud | Hidden Selection of Bacterial Resistance to Fluoroquinolones In Vivo: The Case of Legionella pneumophila and Humans[END_REF].

Bacterial resistance to quinolones of clinical relevance is most often related to mutations in the genes encoding type II topoisomerases, also called DNA gyrase (encoded by gyrA and gyrB genes) and type IV topoisomerase (encoded by parC and parE genes) [START_REF] Jacoby | Mechanisms of Resistance to Quinolones[END_REF].

Resistance-conferring mutations occur within specific regions of these genes, called the quinolone resistance-determining regions (QRDRs). The in vitro selection of L. pneumophila strains with high-level resistance to quinolones has been mainly related to mutations affecting codons 83 and 87 of gyrA QRDR [START_REF] Linde | The amino acid substitution Asp87Tyr in GyrA of Legionella pneumophila Sg 1 is associated with elevated MICs to nalidixic acid but not to ciprofloxacin or moxifloxacin in vitro[END_REF][START_REF] Jonas | Development and mechanism of fluoroquinolone resistance in Legionella pneumophila[END_REF][START_REF] Almahmoud | Mutational paths towards increased fluoroquinolone resistance in Legionella pneumophila[END_REF], although mutations in gyrB and parC were also described [START_REF] Almahmoud | Mutational paths towards increased fluoroquinolone resistance in Legionella pneumophila[END_REF]. Only mutations at codon position 83 of the gyrA gene have been reported so far in vivo [START_REF] Bruin | Isolation of ciprofloxacin-resistant Legionella pneumophila in a patient with severe pneumonia[END_REF][START_REF] Shadoud | Hidden Selection of Bacterial Resistance to Fluoroquinolones In Vivo: The Case of Legionella pneumophila and Humans[END_REF]. Using a next-generation DNA sequencing (NGS) approach, Shadoud et al. previously demonstrated that a L. pneumophila mutant population harboring the 248C>T (T83I) mutation was rapidly selected in vivo in two legionellosis patients treated with a FQ, representing from 1.05% of the total L. pneumophila lung population at the time of diagnosis up to 94% after a few days of FQ treatment [START_REF] Shadoud | Hidden Selection of Bacterial Resistance to Fluoroquinolones In Vivo: The Case of Legionella pneumophila and Humans[END_REF]. It can be hypothesized that a higher proportion of mutated gyrA sequences at the time of legionellosis diagnosis may represent a higher risk of FQ treatment failure. Also, selection of the gyrA mutated population by the administration of a FQ may occur faster when the proportion of mutants over wild-type strains is high at the time of treatment onset.

The prevalence of the in vivo selection of L. pneumophila FQ-resistant mutants in legionellosis patients treated with these antibiotics remains to be established. It cannot be determined by traditional phenotypic methods for several reasons: 1/ Legionella cultures are negative in most legionellosis patients because of the fastidious nature of this bacterium; 2/ L. pneumophila isolation is even more difficult to obtain in patients under antibiotic treatment, even when patients experience treatment failure or relapses; 3/ a mixture of antibiotic-susceptible and a few resistant strains of L. pneumophila would certainly result in selecting the most susceptible population for antibiotic susceptibility testing; and 4/ it is necessary to evaluate the respective proportion of such susceptible and resistant populations to evaluate the potential risk of treatment failure.

Digital PCR (dPCR) is an innovative PCR technology developed in the 2000s, based in the partition of the sample to be analyzed in thousands to millions of individual PCR reaction. A major advantage of dPCR, compared to real-time quantitative PCR (qPCR) is an increased sensitivity for detection of a few mutants mixed with wild-type DNA sequences [START_REF] Sedlak | The potential advantages of digital PCR for clinical virology diagnostics[END_REF]. dPCR has wide clinical applications in the oncology field [START_REF] Day | Digital PCR strategies in the development and analysis of molecular biomarkers for personalized medicine[END_REF], and ongoing applications in noninvasive prenatal diagnosis [START_REF] Fan | Detection of aneuploidy with digital polymerase chain reaction[END_REF][START_REF] Barrett | Digital PCR analysis of maternal plasma for noninvasive detection of sickle cell anemia[END_REF] and organ transplant rejection monitoring [START_REF] Beck | Digital droplet PCR for rapid quantification of donor DNA in the circulation of transplant recipients as a potential universal biomarker of graft injury[END_REF]. For infectious diseases, dPCR technology has been mainly used for diagnosis of viral diseases [START_REF] Sedlak | The potential advantages of digital PCR for clinical virology diagnostics[END_REF], quantification of bacteria in clinical samples [START_REF] Roberts | Development and evaluation of a nextgeneration digital PCR diagnostic assay for ocular Chlamydia trachomatis infections[END_REF][START_REF] Devonshire | Highly reproducible absolute quantification of Mycobacterium tuberculosis complex by digital PCR[END_REF][START_REF] Talarico | Quantitative Detection and Genotyping of Helicobacter pylori from Stool using Droplet Digital PCR Reveals Variation in Bacterial Loads that Correlates with cagA Virulence Gene Carriage[END_REF][START_REF] Qian | Determination of Bifidobacterium and Lactobacillus in breast milk of healthy women by digital PCR[END_REF], detection and quantification of antibiotic resistance genes [START_REF] Kelley | Detection of methicillin-resistant Staphylococcus aureus by a duplex droplet digital PCR assay[END_REF][START_REF] Pholwat | Digital PCR to detect and quantify heteroresistance in drug resistant Mycobacterium tuberculosis[END_REF], and detection and quantification of bacterial toxins [START_REF] Verhaegen | Comparison of Droplet Digital PCR and qPCR for the Quantification of Shiga Toxin-Producing Escherichia coli in Bovine Feces[END_REF].

The aim of the present study was to develop dPCRgyrA assays for detection and quantification of FQ-resistant mutants of L. pneumophila in respiratory samples collected from legionellosis patients.

MATERIALS AND METHODS:

Bacterial strains and culture conditions

The L. pneumophila strains used in this study are listed in Table 1. The L. pneumophila sg 1 strain Paris CIP107629T (referred to as LPP) was used as a FQ-susceptible control. The FQresistant mutants LPPI1, LPPI4, and LPPI5 were derived from LPP by homologous recombination introducing the three main gyrA mutations previously selected and characterized in vitro, including the mutations 248C>T (T83I) for LPPI1, 248C>T and 259G>A (D87N) for LPPI4, and 248C>T and 259G>C (D87H) for LPPI5 [START_REF] Almahmoud | Mutational paths towards increased fluoroquinolone resistance in Legionella pneumophila[END_REF].

For specificity purposes, 20 reference or clinical strains belonging to bacterial species other than Legionella sp. (Table S1) and 19 strains of Legionella species other than L. pneumophila, provided by the French National Reference Center (NRC) for Legionella (Table S2) were used.

Legionella strains were grown on BMPA medium (Oxoid, Cambridge, UK) at 37°C in 5% CO 2enriched atmosphere for 3 days. FQ-resistant Legionella strains were grown in a biosafety level 3 laboratory. The other bacterial species were grown on Columbia agar supplemented with 5% sheep blood, or on chocolate agar supplemented with 5% PolyVitex® (bioMérieux, Marcy L'Etoile, France) at 37°C in 5% CO 2 -enriched atmosphere for 24 h.

Patients and clinical samples

Firstly, eight respiratory samples from six patients, previously studied with NGS [START_REF] Shadoud | Hidden Selection of Bacterial Resistance to Fluoroquinolones In Vivo: The Case of Legionella pneumophila and Humans[END_REF], were analyzed by dPCR. These samples came from a previous single-center cohort of legionellosis patients admitted to an intensive care unit at Grenoble University Hospital between 2006 and 2011. Two patients previously detected positive for T83I FQ-resistant mutants of L. pneumophila using NGS served as positive controls [START_REF] Shadoud | Hidden Selection of Bacterial Resistance to Fluoroquinolones In Vivo: The Case of Legionella pneumophila and Humans[END_REF]. Four patients with NGS-negative samples served as negative controls [START_REF] Shadoud | Hidden Selection of Bacterial Resistance to Fluoroquinolones In Vivo: The Case of Legionella pneumophila and Humans[END_REF]. These controls were used for validation of the dPCRgyrA assay developed targeting the T83I mutation.

Then 38 respiratory samples from 23 patients with unknown NGS status were retrospectively evaluated for the presence of the T83I mutation. These patients were admitted to an intensive care unit at Grenoble University Hospital (France) or Lyon University Hospital (France) between 2011 and 2016. They included 20 males and three females, with a mean age of 56 years (range, 23-83 years). During hospitalization, lower respiratory tract (LRT) (i.e., bronchoalveolar lavage or bronchial aspirations or tracheal aspiration or sputum) and urine samples were collected for diagnostic purposes. LRT samples were tested using routine bacterial cultures, Legionella cultures, and Legionella qPCR tests (qPCR16S for all Legionella species and qPCRmip for L. pneumophila, as previously described ( 25)), and then frozen at -80°C until use for this study. The 23 patients were selected because of positive Legionella qPCR tests (qPCR16S and qPCRmip) (see Results section), while 21 patients also had a positive urinary antigen test for L. pneumophila sg1, and 18 patients had a positive culture allowing isolation of a L. pneumophila sg 1. All these patients were considered infected with a L. pneumophila strain because of the positive qPCRmip test on LRT samples. Sixteen (69.6%) out of the 23 patients were treated with a FQ.

In addition, to validate the clinical use of the dPCRgyrA targeting T83I mutation, ten respiratory samples from patients suffering from pneumonia other than legionellosis were used. These samples were negative for Legionella qPCR tests (qPCR16S and qPCRmip), while commensal and pathogenic bacterial species were detected by routine bacterial cultures.

DNA extraction

For bacterial strains, colonies were harvested and suspended in Tris-EDTA buffer solution (Sigma-Aldrich, St. Louis, MO, USA). For FQ-resistant Legionella strains, bacteria were inactivated by heating at 95°C for 15 min before DNA extraction. DNA was extracted using the QIAamp DNA Mini kit (Qiagen, Courtaboeuf, France), according to the manufacturer's recommendations.

DNA concentration of LPP, LPPI1, LPPI4, and LPPI5 was assessed using Qubit® Fluorometric Quantitation (Thermo Fisher Scientific, Carlsbad, CA, USA) according to the manufacturer's recommendations and adjusted to 1 ng/µL. Then mixtures of DNA from LPPI1, LPPI4, or LPPI5 with DNA from the wild-type LPP strain were prepared at different mutant:LPP ratios:

1:1, 1:10, 1:100, 1:500, and 1:1000.

DNA concentrations of suspensions of strains belonging to non-Legionella species and

Legionella species other than L. pneumophila were assessed using a NanoDrop® 2000c spectrophotometer (Thermo Fisher Scientific), according to the manufacturer's recommendations.

For the respiratory samples, DNA extraction was performed using either the EZ1® DNA Tissues kit (Qiagen) or the MagNA Pure Compact Nucleic Acid Isolation Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturers' instructions.

Sanger sequencing of the L. pneumophila gyrA QRDR

The mutant:LPP mixtures were tested using a PCR-sequencing approach in order to assess the sensitivity of this technique to detect mutated gyrA QRDR sequences in the presence of wild-type sequences.

A 383-bp fragment of the gyrA QRDR was amplified using the primers LgyrALubS7 (5′-GCGATGAGTGTCATTGTAG-3′) and LgyrALubASR2bis

(5′GTTTCATCATAGTTAGGGCTAAAATCAAC-3′) as previously described [START_REF] Shadoud | Hidden Selection of Bacterial Resistance to Fluoroquinolones In Vivo: The Case of Legionella pneumophila and Humans[END_REF]. Sequencing was performed using the same primers and the dye terminator method, with the DTCS Quick Start Kit (Beckman Coulter, Villepinte, France) on a CEQ 8000 sequencer (Beckman Coulter)

as previously described [START_REF] Almahmoud | Mutational paths towards increased fluoroquinolone resistance in Legionella pneumophila[END_REF]. Amplicons were sequenced from both ends. The sequences obtained were visualized and analyzed with Chromas LITE (version 2.1.1, Technelysium) and aligned with Seaview4 [START_REF] Gouy | SeaView version 4: A multiplatform graphical user interface for sequence alignment and phylogenetic tree building[END_REF].

Primers and probes for qPCRgyrA and dPCRgyrA

We followed the MIQE Guidelines for nomenclature and qPCR protocol development [START_REF] Bustin | The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments[END_REF].

For qPCRgyrA and dPCRgyrA experiments, primers and probes targeting the wild-type and mutated sequences of the gyrA QRDR were designed using the Custom TaqMan® Assay Design Tools (Life Technologies, Thermo Fisher Scientific, Carlsbad, CA, USA). Three assays were developed to detect the wild-type gyrA sequence and the gyrA QRDR mutations previously selected in vitro in L. pneumophila (9): 248C>T (T83I), 259G>A (D87N), and 259G>C (D87H).

Primers and probes were provided by Life Technologies. Each assay mix contained PCR primers and two hydrolysis MGB probes, including a VIC-labeled probe complementary to the wild-type gyrA QRDR sequence and a FAM-labeled probe complementary to one of the three mutated gyrA QRDR sequences (Table 2).

qPCRgyrA validation and study of the mutant:LPP mixtures

Validation of the three qPCRgyrA assays for the specific detection of the wild-type and mutated (T83I, D87N, or D87H) gyrA QRDR sequences was performed by testing the LPP, LPPI1, LPPI4, and LPPI5 strains with the three assays. Each PCR run included no-template controls (NTC) to validate the absence of false-positive results caused by exogenous DNA contamination, autohydrolysis of the probes, or nonspecific hybridization.

Then the mutant:LPP mixtures (1:1, 1:10, 1:100, 1:1000) were tested using the qPCRgyrA assays, in duplicate, in order to assess the sensitivity of this technique to detect mutated gyrA QRDR sequences in the presence of wild-type sequences.

The PCR mixture (25 µL) contained 12.5 µL of TaqMan® Genotyping Master Mix (Life Technologies, Thermo Fisher Scientific), 0.625 µL of 40X TaqMan® assay mix (36 µM of each primer, 8 µM of the wild-type probe, and 8 µM of the mutant probe), and 11.875 µL of diluted DNA (i.e., 5-10 ng of DNA per reaction). The qPCRgyrA assays were performed using a StepOnePlus™ system (Life Technologies, Thermo Fisher Scientific) with an initial denaturation step at 95°C for 10 min, followed by 40 cycles of denaturation at 92°C for 15 s and annealing and extension at 60°C for 1 min, and a final step of incubation at 60°C for 30 s.

The results were analyzed at end-point with the StepOne software v2.3 (Life Technologies, Thermo Fisher Scientific).

dPCRgyrA validation and study of the mutant:LPP mixtures

As above, validation of the three dPCRgyrA assays for the specific detection of the wild-type gyrA QRDR sequence, T83I, D87N, and D87H mutations, was performed by testing the LPP, LPPI1, LPPI4, and LPPI5 strains with the three assays. NTCs were also added to each run.

Then the mutant:LPP mixtures (1:1, 1:10, 1:100, 1:500, and 1:1000) were analyzed in duplicate by the dPCRgyrA assays in order to assess the sensitivity of this technique to detect mutated gyrA QRDR sequences in the presence of wild-type sequences.

dPCRgyrA-T83I assay validation for application to human respiratory samples

Additional specificity testing, evaluation of linearity, and determination of interpretative criteria for a dPCRgyrA result were performed for the dPCRgyrA-T83I assay.

Given that respiratory samples may contain DNA from a number of commensal or pathogenic microorganisms other than L. pneumophila, as well as human DNA, additional specificity testing for the dPCRgyrA-T83I assay was performed. Therefore, 20 DNA extracts from non-Legionella bacterial species commonly isolated from respiratory samples (Table S1) and 19 DNA extracts from Legionella species other than L. pneumophila (Table S2) were tested. In addition, ten DNA extracts from respiratory samples collected in non-legionellosis patients containing commensal or pathogenic bacteria, but no Legionella sp. as determined by negative Legionella sp. qPCR16S and L. pneumophila qPCRmip tests, were tested.

The Legionella DNA concentration in clinical samples is difficult to determine accurately, and thus it can hardly be standardized for dPCR testing. Therefore, it was necessary to evaluate the linearity of the dPCRgyrA-T83I assay and the minimum DNA concentration required in a respiratory sample to obtain interpretable results. For this purpose, a range of L.

pneumophila genome concentrations (from 1.9 to 19,000 genome copies/µL, as determined using the NanoDrop® 2000c spectrophotometer) were tested using the dPCRgyrA-T83I assay as well as the Legionella sp. qPCR16S and L. pneumophila qPCRmip assays.

dPCRgyrA-T83I assay on respiratory samples

The dPCRgyrA-T83I assay was applied to DNA extracts from respiratory samples collected in legionellosis patients.

The manufacturers' instructions specify that for any dPCR reaction the maximum precision is obtained at 1.

RESULTS

Sanger sequencing of the gyrA QRDR of the mutant:LPP mixtures

The mutated gyrA QRDR sequences were all detected at a 1:1 mutant:LPP ratio. In contrast, the mutated gyrA sequences were only occasionally detected at a 1:10 ratio and were not detected at 1:100 and 1:1000 ratios. Figure 1A shows the sequencing chromatograms of the gyrA QRDR sequences for the LPPI5:LPP mixtures. Similar results were obtained for the LPPI1:LPP and LPPI4:LPP mixtures (data not shown). The Sanger sequencing sensitivity was evaluated at a 1:1 ratio (50% of mutated gyrA sequences).

Validation of the qPCRgyrA assays

The results of the experiments conducted for validation of the three qPCRgyrA assays are shown in the additional data (Figures S1,S2, and S3). The three assays were highly specific for their targets, although weak fluorescence signals were detected with the wild-type and mutant probes in the absence of the corresponding targets for the qPCRgyrA-D87N assay, and with the wild-type probe for the qPCRgyrA-T83I assay. An adjustment of the thresholds allowed us to circumvent these nonspecific reactions. No signal was obtained with the NTCs.

Study of the mutant:LPP mixtures using the qPCRgyrA assays

The presence of mutated gyrA QRDR sequences was detected at 1:1 and 1:10 mutant:LPP ratios. In contrast, the mixtures at the 1:100 and 1:1000 ratios were reported as wild-type DNA sequences by qPCRgyrA testing. The results for the LPPI5:LPP mixtures are shown in Figure 1B. Similar results were obtained for the LPPI1:LPP and LPPI4:LPP mixtures (data not shown). The qPCRgyrA sensitivity was evaluated at a 1:10 ratio (10% of mutated gyrA sequences).

Validation of the dPCRgyrA assays

The results of experiments conducted for validation of the dPCRgyrA assays are shown in Table S3. As expected, dPCRgyrA quantified on average 1000 target copies/µL. The three assays were highly specific for their targets, although the weak cross-hybridization reactions detected for qPCRgyrA assays were also visible in dPCRgyrA experiments as a slight background noise. For the dPCRgyrA-T83I assay, the background noise was observed only for the mutant probe at 0.43 target copies/µL. For the dPCRgyrA-D87N assay, the background noise was observed with mutant and wild-type probes up to 0.69 target copies/µL. For the dPCRgyrA-D87H assay, the background noise was also observed with mutant and wild-type probes up to 1.27 target copies/µL. This small background noise was circumvented by fixation of thresholds above these nonspecific signals. No signal was observed with the NTCs.

Study of the mutant:LPP mixtures using the dPCRgyrA assays

The percentages of mutated gyrA QRDR sequences in the mutant:LPP mixtures were calculated using the formula: mutant percentage = [mutant copies/(mutant copies + wild copies)]*100. dPCRgyrA was able to detect the presence of mutated gyrA QRDR sequences up to 1:1000 mutant:LPP ratios. Taking into account the previously defined thresholds, the dPCRgyrA sensitivity was evaluated at a 1:1000 ratio (0.1% of mutated gyrA sequences) for the dPCRgyrA-T83I and dPCRgyrA-D87N assays, and at a 1:500 ratio (0.2% of mutated gyrA sequences) for the dPCRgyrA-D87H assay. The results are shown in Table 3 for all the mixtures and in Figure 1C for the LPPI5:LPP mixtures. Similar results were obtained for the LPPI1:LPP and LPPI4:LPP mixtures (data not shown).

Validation of the dPCRgyrA-T83I assay for application to human respiratory samples

dPCRgyrA-T83I assay specificity was evaluated by testing DNA extracts from other bacterial species and Legionella-negative respiratory samples. The dPCRgyrA-T83I assay showed no cross-reactivity against any of the non-Legionella strains, or against the Legionella nonpneumophila species, except a weak signal with the L. parisiensis strain. No cross-reactivity was observed with the culture-positive but Legionella-free respiratory samples.

The linearity and interpretative criteria of a dPCRgyrA-T83I result were determined by testing a range of a L. pneumophila DNA concentrations (from 1.9 to 19,000 copies/µL as determined with the NanoDrop® 2000c spectrophotometer) using the dPCRgyrA-T83I assay and Legionella sp. qPCR16S and L. pneumophila qPCRmip tests. Experiments conducted to determine the linearity and interpretative criteria of the dPCRgyrA-T83I assay are presented in Table S4. Firstly, for the L. pneumophila DNA concentration at 1.9 DNA copies/µL as determined using the NanoDrop spectrophotometer, the dPCRgyrA-T83I assay quantified 0.49 copies of Legionella DNA/µL. This concentration corresponded to the background noise of the dPCRgyrA-T83I assay (which was evaluated as 0.43 target copies/µL). For this concentration, the Legionella sp. qPCR16S and L. pneumophila qPCRmip Cq (quantification cycle) were close to 36 cycles. Consequently, to obtain a dPCRgyrA-T83I signal above the background noise, we decided that only clinical samples with a Legionella sp. qPCR16S or L.

pneumophila qPCRmip Cq less than or equal to 35 cycles would be considered appropriate for dPCRgyrA-T83I testing. Moreover, we considered that because the proportion of mutant sequences in a mixed mutant:wild-type population of L. pneumophila can be very low, a minimum total DNA concentration would be necessary to obtain a reliable evaluation of the percentage of mutant sequences. Therefore, we decided to set at 4 total DNA copies/µL (as determined by dPCRgyrA-T83I) the minimum total DNA concentration required to obtain accurate analysis of the percentage of L. pneumophila gyrA mutants.

Correlation between dPCRgyrA and NGS results for respiratory samples

The dPCRgyrA-T83I assay was applied to DNA samples obtained from six legionellosis patients previously studied with NGS [START_REF] Shadoud | Hidden Selection of Bacterial Resistance to Fluoroquinolones In Vivo: The Case of Legionella pneumophila and Humans[END_REF]. In this previous work, NGS identified the in vivo selection of a T83I mutation in two patients (patients #2 and #4), with proportions of mutated sequences ranging from 1.05% to 94%. dPCRgyrA confirmed the T83I mutation in all the samples collected in these two patients, in proportions ranging from 0.38% to 99.67%.

Unfortunately, there was not enough remaining material from the sample collected from patient #2 at day 4 (day after legionellosis diagnosis) to be tested by dPCR. For the four remaining patients, NGS found lower percentages of T83I mutations, ranging from 0.023% to 0.19%, which were considered as nonsignificant [START_REF] Shadoud | Hidden Selection of Bacterial Resistance to Fluoroquinolones In Vivo: The Case of Legionella pneumophila and Humans[END_REF]. dPCRgyrA did not find any T83I mutation in these four patients. Table 4 compares the results obtained with the NGS and dPCRgyrA methods.

Analysis of respiratory samples from other legionellosis patients

Using the dPCRgyrA-T83I assay, 38 respiratory samples collected from 23 severe legionellosis patients were retrospectively analyzed. These samples corresponded to the previously defined selection criteria for a dPCRgyrA analysis: a positive Legionella sp.qPCR16S or L.

pneumophila qPCRmip test with a Cq less than or equal to 35 cycles.

dPCRgyrA gave a significant amplification signal (DNA concentration higher than 4 copies/µL, as previously defined) for 34 (89.5%) samples from 20 (87.0%) patients. The results of dPCR for these samples are presented in Table 5. For three samples, mutant sequences in proportions less than the dPCRgyrA-T83I assay sensitivity (0.1%) and/or background noise (0.43 FAM copies/µL) were found; these results were considered uninterpretable. For four samples collected in three patients, low percentages (0.13-1.13%) of T83I mutation were found. These three patients were treated with levofloxacin at the dose of 500mg twice a day from the day of legionellosis diagnosis.

DISCUSSION

It has commonly been assumed that L. pneumophila could not develop FQ resistance.

However, Bruin et al. recently reported the isolation from a legionellosis patient of a L.

pneumophila strain displaying a ciprofloxacin MIC of 2mg/L, higher than the epidemiological cutoff (1mg/L) he previously defined [START_REF] Bruin | Isolation of ciprofloxacin-resistant Legionella pneumophila in a patient with severe pneumonia[END_REF]. Although, no FQ resistance breakpoint has been currently established for Legionella, this strain was considered as FQ-resistant because associated with a poor response to the antibiotic therapy and a prolonged hospitalization of the patient.

More recently, using a NGS approach, Shadoud et al. showed the in vivo selection of a FQ-resistant subpopulation of this pathogen in two legionellosis patients [START_REF] Shadoud | Hidden Selection of Bacterial Resistance to Fluoroquinolones In Vivo: The Case of Legionella pneumophila and Humans[END_REF]. Indeed, NGS allowed the detection over time of increasing percentages (from 1.05% to 94%) of gyrA83 mutated sequences in respiratory samples from these legionellosis patients [START_REF] Shadoud | Hidden Selection of Bacterial Resistance to Fluoroquinolones In Vivo: The Case of Legionella pneumophila and Humans[END_REF]. In these two patients, the most probable situation was the presence of a low level of resistant mutants that could have been missed by phenotypic tests, but that dramatically expanded after administration of a FQ, leading to therapeutic failure [START_REF] El-Halfawy | Antimicrobial heteroresistance: an emerging field in need of clarity[END_REF]. Defining ciprofloxacin resistance from molecular data is challenging. In this study, we considered as FQ-resistant L.

pneumophila strains harboring gyrA83 mutations for three main reasons: first these mutants were previously shown to display an eight-fold increase of ciprofloxacin MIC in comparison with their wild-type parental strain [START_REF] Almahmoud | Mutational paths towards increased fluoroquinolone resistance in Legionella pneumophila[END_REF]; second the strain isolated by Bruin et al. [START_REF] Bruin | Isolation of ciprofloxacin-resistant Legionella pneumophila in a patient with severe pneumonia[END_REF] also harbored a gyrA83 mutation; and third such a mutation is well known to be associated with high-level FQ resistance in many other Gram-negative bacterial species [START_REF] Jacoby | Mechanisms of Resistance to Quinolones[END_REF][START_REF] Lindgren | Mutation rate and evolution of fluoroquinolone resistance in Escherichia coli isolates from patients with urinary tract infections[END_REF][START_REF] Wong | Parallel evolution and local differentiation in quinolone resistance in Pseudomonas aeruginosa[END_REF], and occurs at a high rate [START_REF] Jacoby | Mechanisms of Resistance to Quinolones[END_REF].

Consequently, it is of clinical interest to assess the presence of FQ-resistant mutants in a L. pneumophila population infecting a given legionellosis patient in order to adapt the antibiotic therapy. However, standard culture-based antibiotic susceptibility testing methods are not appropriate for L. pneumophila because of the fastidious nature of this pathogen.

Therefore, the development of molecular techniques allowing detection of FQ-resistant subpopulations of L. pneumophila in respiratory samples is warranted. Shadoud et al.

previously demonstrated the usefulness of NGS for the detection, identification, and quantification of specific gyrA mutants within a L. pneumophila population [START_REF] Shadoud | Hidden Selection of Bacterial Resistance to Fluoroquinolones In Vivo: The Case of Legionella pneumophila and Humans[END_REF]. However, NGS technology remains expensive and data interpretation is complex and time-consuming.

This technique is not yet used on a routine basis in bacteriology laboratories. In contrast, dPCR is less expensive, easier to perform, and can also detect and quantify rare mutant DNA sequences in a mixed mutant-wild-type population.

In this work, we developed dPCRgyrA assays to detect and quantify FQ-resistant subpopulations of L. pneumophila in respiratory samples. As a proof of concept, three dPCRgyrA assays targeting the gyrA QRDR mutations previously characterized as responsible for FQ resistance in L. pneumophila were developed [START_REF] Jonas | Development and mechanism of fluoroquinolone resistance in Legionella pneumophila[END_REF][START_REF] Almahmoud | Mutational paths towards increased fluoroquinolone resistance in Legionella pneumophila[END_REF][START_REF] Shadoud | Hidden Selection of Bacterial Resistance to Fluoroquinolones In Vivo: The Case of Legionella pneumophila and Humans[END_REF]. The dPCRgyrA-T83I assay targeted the 248C>T mutation (T83I substitution), a mutation systematically observed in evolved FQ-resistant clones selected in vitro by Almahmoud et al. [START_REF] Almahmoud | Mutational paths towards increased fluoroquinolone resistance in Legionella pneumophila[END_REF]. It was also described in vitro by Jonas et al. [START_REF] Jonas | Development and mechanism of fluoroquinolone resistance in Legionella pneumophila[END_REF] and in vivo in two legionellosis patients by Shadoud et al. [START_REF] Shadoud | Hidden Selection of Bacterial Resistance to Fluoroquinolones In Vivo: The Case of Legionella pneumophila and Humans[END_REF]. The 248C>T mutation was found alone or combined with other mutations at codon position GyrA87 [START_REF] Almahmoud | Mutational paths towards increased fluoroquinolone resistance in Legionella pneumophila[END_REF]. Thus, we designed two dPCRgyrA assays, the dPCRgyrA-D87N assay and the dPCRgyrA-D87H assay, targeting the mutations 259G>A and 259G>C, respectively.

Validation of the three dPCRgyrA assays showed high sensitivity. dPCRgyrA was able to detect gyrA mutant sequences mixed with wild-type sequences up to mutant:susceptible allele ratios of 1:1000 (0.1%). In comparison, the Sanger sequencing and qPCR tests were less sensitive because they detected mutant sequences only at 1:1 (50%) and 1:10 (10%) ratios, respectively. Consequently, the Sanger sequencing and qPCR tests may not be able to detect antibiotic-resistant subpopulations of L. pneumophila in the early stage of infection in legionellosis patients. The dPCRgyrA sensitivity levels observed in this study are close to those previously reported by Pholwat et al. for detection and quantification of isoniazid (katG315 mutation), rifampicin (rpoB531 mutation), FQ (gyrA94,95 mutations), and aminoglycoside (rrs1401 mutation) resistance in Mycobacterium tuberculosis (sensitivity from 1% to 0.1%) [START_REF] Pholwat | Digital PCR to detect and quantify heteroresistance in drug resistant Mycobacterium tuberculosis[END_REF]. In the literature, the maximum sensitivity reported for dPCR is 0.001% (one mutant sequence for 100,000 wild-type sequences) [START_REF] Heyries | Megapixel digital PCR[END_REF][START_REF] Pekin | Quantitative and sensitive detection of rare mutations using droplet-based microfluidics[END_REF][START_REF] Ma | Quantification of Plasma miRNAs by Digital PCR for Cancer Diagnosis[END_REF]. Such high sensitivity may be obtained either with a higher number of individual reactions (e.g., more than 1,000,000 individual reactions), a different dPCR technique (droplet dPCR versus dPCR on a chip), or both. dPCRgyrA could be considered a highly accurate method because the percentages of mutant sequences quantified into the mixtures were very close to the theoretical proportions. dPCRgyrA also produced highly reproducible results since the quantitation of mutant sequences between the two chip replicates were very close.

Currently, resistance to FQ in L. pneumophila strains infecting humans has only been associated with mutations at codon gyrA83 [START_REF] Bruin | Isolation of ciprofloxacin-resistant Legionella pneumophila in a patient with severe pneumonia[END_REF][START_REF] Shadoud | Hidden Selection of Bacterial Resistance to Fluoroquinolones In Vivo: The Case of Legionella pneumophila and Humans[END_REF]. Therefore, we applied the dPCRgyrA-T83I assay to respiratory samples collected from legionellosis patients. Additional validation steps for the dPCRgyrA-T83I assay were performed beforehand. Specificity was assessed by testing bacterial species belonging to genera other than Legionella and Legionella species other than L. pneumophila. No cross-amplification was observed, except a slight crossreactivity with L. parisiensis. However, only three cases of human infection with this species have been reported so far in the literature [START_REF] Presti | The first clinical isolate of Legionella parisiensis, from a liver transplant patient with pneumonia[END_REF][START_REF] Igel | Isolation and characterization of a nonfluorescent strain of Legionella parisiensis[END_REF][START_REF] Han | Microbiological and Clinical Studies of Legionellosis in 33 Patients with Cancer[END_REF]. We also determined that only respiratory samples with a Legionella sp. qPCR16S or L. pneumophila qPCRmip Cq of 35 cycles or less were considered eligible for a dPCRgyrA analysis because of a sufficient DNA amount. In addition, we considered 4 total DNA copies/µL as a threshold for reliable interpretation of the percentage of gyrA mutant sequences.

We then applied the dPCRgyrA-T83I assay to clinical samples from legionellosis patients previously studied with NGS [START_REF] Shadoud | Hidden Selection of Bacterial Resistance to Fluoroquinolones In Vivo: The Case of Legionella pneumophila and Humans[END_REF]. dPCRgyrA confirmed the in vivo selection of a FQ-resistant mutant in patient #4, with 0.38% T83I mutants at D0 (versus 1.05% for NGS) and 99.67% at D5 (versus 85% for NGS).

We then retrospectively analyzed 38 clinical samples from 23 legionellosis patients using the dPCRgyrA-T83I assay. No sample harbored a majority of gyrA T83I mutants. This result was not unexpected because FQ resistance selection in L. pneumophila seems a rare phenomenon [START_REF] Shadoud | Hidden Selection of Bacterial Resistance to Fluoroquinolones In Vivo: The Case of Legionella pneumophila and Humans[END_REF], and most respiratory samples were collected early in the course of legionellosis for the majority of these patients, precluding visualization of in vivo selection of FQ-resistant mutants under FQ therapy. However, small amounts of gyrA83 mutants (0.13-1.68%) were detected in four (10.5%) samples from three (13.0%) patients. Although higher than the cutoffs determined, these low percentages of mutant gyrA sequences must be interpreted with caution because they may correspond to false-positive results of the dPCRgyrA assays, as well as for NGS [START_REF] Shadoud | Hidden Selection of Bacterial Resistance to Fluoroquinolones In Vivo: The Case of Legionella pneumophila and Humans[END_REF]. Alternatively, they may correspond to the presence of a low level of FQ-resistant mutants in the Legionella population infecting these patients.

We only studied clinical samples collected on a routine basis for diagnostic purposes. The possibility of collecting several respiratory samples in legionellosis patients under FQ therapy would make possible to better monitor the kinetics of the percentage of mutants. Indeed, an increase in the percentage of gyrA mutants would indicate an in vivo emergence of FQresistant mutants of L. pneumophila, and could be an alert on the possibility of the occurrence of a therapeutic failure. A demonstration was done with patient #4 for which dPCRgyrA detected 0.38% T83I mutant at D0 and 81.65% at D2, suggesting that the low level of mutant strain at D0 likely reflected true FQ-resistant mutants rather than false-positive results. This legionellosis patient might have been infected with a mixed L. pneumophila population containing a low level of T83I mutant, or these mutants might have occurred rapidly after infection.

The incidence of in vivo selection of FQ resistance in L. pneumophila is probably underestimated given that only a single FQ-resistant strain of L. pneumophila has been isolated so far in the clinical setting [START_REF] Bruin | Isolation of ciprofloxacin-resistant Legionella pneumophila in a patient with severe pneumonia[END_REF]. dPCR exhibits a sensitivity level as good as that of NGS, but it is more adapted for routine diagnostic use. It is an inexpensive, easy-to-handle, and rapid technology, and data interpretation is simple. Consequently, a good strategy for characterization of antibiotic resistance in fastidious microorganisms would be to use NGS for identification of the gene mutations involved, and then to develop dPCR tests to detect these resistances more easily. Although not found by Shadoud et al. [START_REF] Shadoud | Hidden Selection of Bacterial Resistance to Fluoroquinolones In Vivo: The Case of Legionella pneumophila and Humans[END_REF] using NGS, it would be particularly interesting to look for the gyrA 247A>G mutation (T83A) previously described by Bruin et al. for the FQ-resistant strain isolated in a respiratory sample [START_REF] Bruin | Isolation of ciprofloxacin-resistant Legionella pneumophila in a patient with severe pneumonia[END_REF]. Similarly, other mutations in DNA gyrase (e.g., gyrA83, gyrA87, and gyrB) and in topoisomerase IV encoding genes should be searched for using dPCR, although they were only described in vitro in L. pneumophila [START_REF] Linde | The amino acid substitution Asp87Tyr in GyrA of Legionella pneumophila Sg 1 is associated with elevated MICs to nalidixic acid but not to ciprofloxacin or moxifloxacin in vitro[END_REF][START_REF] Jonas | Development and mechanism of fluoroquinolone resistance in Legionella pneumophila[END_REF][START_REF] Almahmoud | Mutational paths towards increased fluoroquinolone resistance in Legionella pneumophila[END_REF]. It would then be possible to develop multiplex dPCR assays to detect the most common mutations responsible for FQ resistance. Indeed, multiplex dPCR assays have already been developed, for example, in cancerology for KRAS mutation (37).

In conclusion, we developed dPCRgyrA assays to detect and quantify FQ-resistant subpopulations of L. pneumophila harboring mutations in the gyrA QRDR. We demonstrated that dPCR is a really powerful tool displaying high sensitivity, reproducibility, and precision and that this technique is suitable for daily practice in bacteriology laboratories.

  The clinical sample collections of Grenoble and Lyon University Hospitals were declared to the French Ministry of Education and Research (number DC-2008-677 and DC-2008-176 respectively), and an ethics committee (CPP Sud-Est V) gave the authorization to use it for research purposes. Patient information was provided through a hospital medical booklet and only non-opposition on the part of the patients was needed.

Figure 1 .

 1 Figure 1. Comparison of Sanger sequencing, qPCRgyrA and dPCRgyrA for mutant sequences

ba

  As determined by Shadoud et al.[START_REF] Shadoud | Hidden Selection of Bacterial Resistance to Fluoroquinolones In Vivo: The Case of Legionella pneumophila and Humans[END_REF] NP = Not performed. Sampling day is given according to the day of legionellosis diagnosis (D0).b Three samples with mutant sequences in proportions inferior to dPCRgyrA-T83I assay sensitivity (0.1%) and/or background noise (0.43 FAM copies/µL).

  The major clinical interest of this technique was highlighted by confirming the in vivo emergence of FQresistant mutants of L. pneumophila in respiratory samples from a legionellosis patient, as previously characterized using NGS. The dPCR approach would be useful to set up a multicenter study of a larger cohort of patients to further define the prevalence of FQresistant mutants in legionellosis patients and evaluate their potential clinical impact. In case gyrA mutations in L. pneumophila are associated with a worsened prognosis, dPCR would be a useful diagnostic tool to predict the effectiveness of the FQ therapy in legionellosis patients.Multiplex KRASG12/G13 Mutation Testing of Unamplified Cell-Free DNA from the Plasma of Patients with Advanced Cancers Using Droplet Digital Polymerase Chain Reaction. Ann Oncol Off J Eur Soc Med Oncol.

Table 1 . L. pneumophila strains used in this study

 1

				FQ susceptibility
	Strain	GyrA a	gyrA b	(Moxifloxacin	Reference
				MIC c )
	L. pneumophila serogroup 1 strain Paris, CIP107629T (LPP)	Wild-type	Wild-type	Susceptible (MIC = 0.0625 mg/L)	NRC for Legionella (Lyon, France)
	LPPI1	LPP + GyrA83 (T83I)	248C>T	Resistant (MIC = 0.5 mg/L)	(9)
	LPPI4	LPP + GyrA83 (T83I) + GyrA87 (D87N)	248C>T+259G>A	Resistant (MIC = 0.5 mg/L)	(9)
	LPPI5	LPP + GyrA83 (T83I) + GyrA87*(D87H)	248C>T+259G>C	Resistant (MIC = 0.5 mg/L)	(9)

a Change in the amino acid sequence of the corresponding protein. b Change in the nucleotide sequence of the gene. c According to Almahmoud et al. (9).

Table 2 . Primers and probes for qPCRgyrA and dPCRgyrA experiments

 2

	Assay	Primers and probes	Sequences
	Assay T83I	T83I-F	5′-ATGTCATCGGTAAATACCATCCTCAC-3′
		T83I-R	5′-GGTTGGGCCATACGAACAATG-3′
		T83I-wt	5′-VIC-AAACAGCTGTATCCCC-3′
		T83I-mt	5′-FAM-AAACAGCTATATCCCC3′
	Assay D87N	D87N-F	5′-GTCATCGGTAAATACCATCCTCACG-3′
		D87N-R	5′-CGATTAAAAGGTAGCGCATGGAAAA-3′
		D87N-wt	5′-VIC-CAATGGTGTCATAAACA-3′
		D87N-mt	5′-FAM-ACAATGGTGTTATAAACA-3′
	Assay D87H	D87H-F	5′-GTCATCGGTAAATACCATCCTCACG-3′
		D87H-R	5′-CGATTAAAAGGTAGCGCATGGAAAA-3′
		D87H-wt	5′-VIC-CAATGGTGTCATAAACA-3′
		D87H-mt	5′-FAM-CAATGGTGTGATAAACA-3′
	wt=wild-type
	mt = mutant

Table 3 . dPCRgyrA results for the mutant:LPP mixtures

 3 The result of each chip is presented, followed by the mean and the standard deviation (SD) of the duplicate.

	Mutant	Mutant:LPP	Wild copies/µL (VIC)	Mutant copies/µL (FAM)	%
	strain	mixture	Chip 1	Chip 2	Mean	Chip 1 Chip 2 Mean	Mutant
	(mutation)				(SD)			(SD)	Mean
								(SD)
	LPPI1	1:1	660.31 482.67	571.49	655.22 651.94 653.58	53.63
	(T83I)				(125.61)			(2.32)	(5.41)
		1:10	1601.80 1550.60 1576.20	119.84 129.20 124.52	7.33
					(36.20)			(6.62)	(0.52)
		1:100	1393.90 1385.60 1389.75	9.88	6.87	8.38	0.60
					(5.87)			(2.13)	(0.15)
		1:500	1534.70 1369.30 1452.00	4.83	2.31	3.57	0.24
					(116.96)			(1.78)	(0.10)
		1:1000	1249.90 1325.00 1287.45	0.83	1.27	1.05	0.08
					(53.10)			(0.31)	(0.02)
	LPPI4	1:1	434.52 432.43	433.48	365.80 353.98 359.89	45.36
	(D87N)				(1.48)			(8.36)	(0.49)
		1:10	1075.90 1091.20 1083.55	81.92 80.50	81.21	6.97
					(10.82)			(1.00)	(0.14)
		1:100	1353.00 1303.70 1328.35	9.88	8.43	9.16	0.68
					(34.86)			(1.03)	(0.06)
		1:500	1412.70 1450.50 1431.60	1.83	2.66	2.25	0.16
					(26.73)			(0.59)	(0.04)
		1:1000	1462.10 1418.80 1440.45	0.94	0.87	0.91	0.06
					(30.62)			(0.05)	(0.002)
	LPPI5	1:1	633.68 650.10	641.89	510.10 533.82 521.96	44.84
	(D87H)				(11.61)			(16.77)	(0.35)
		1:10	1306.80 1299.30 1303.05	98.79 95.77	97.28	6.95
					(5.30)			(2.14)	(0.12)
		1:100	1099.90 1284.50 1192.20	7.89	8.45	8.17	0.68
					(130.53)			(0.40)	(0.04)
		1:500	1189.40 1112.90 1151.15	2.30	1.82	2.06	0.18
					(54.09)			(0.34)	(0.02)
		1:1000	1311.20 1282.70 1296.95	1.33	0.87	1.10	0.08
					(20.15)			(0.33)	(0.02)

Table 4 . Concordance between dPCRgyrA and NGS on clinical samples Patient Sampling day a Fluoroquinolone treatment Percentage of T83I mutant with NGS b

 4

	Percentage of T83I
	mutant with
	dPCRgyrA-T83I
	assay

a

Sampling day is given according to the day of legionellosis diagnosis.

ACKNOWLEDGEMENTS

This work was supported by the French Centre National de la Recherche Scientifique, the Université Grenoble Alpes, and the Direction de la Recherche Clinique et de l'Innovation (DRCI) Centre Hospitalier Universitaire Grenoble Alpes. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Conception of the work was performed by S.B. and A.H. Experiments were performed by A.H. Analysis of the data was performed by S.B., A.H., and M.B. Clinical informations about the patients were provided by S.J., L.B., and C.S. The manuscript was written by A.H., S.B., and M.M. All authors reviewed the manuscript before submission.

All the authors have no conflict of interest to declare.

We thank Charles Coutton who loaned the StepOnePlus system and Xavier Fonrose for his assistance in the qPCR data interpretation. We also thank Dominique Dewolf, from Thermo Fisher Scientific, for her technical assistance during this study. To finish, we thank Linda Northrup for editing the manuscript.