N Pham Barthélémy

Dang

M.D. Candice Delbet-Dupas

Hospital Practitioner Laurent Devoize

Professor, Department Head Arnaud Depeyre

Hospital Practitioner Aurélien Mulliez

Biostatisticien Isabelle Barthélémy

Professor, Department Head Nathalie Pham Dang

ARE ROUTINE MICROBIOLOGICAL SAMPLINGS IN ACUTE DENTAL INFECTIONS JUSTIFIED? OUR 10 YEAR'S REAL-LIFE EXPERIENCE

Keywords: Odontogenic, infections, antibiotics, microbiology, sensitivity test

HAL is

INTRODUCTION

With the advent of antibiotics such as penicillin and better dental care, odontogenic infection is now considered to be an easily treatable condition. However, despite the improvement in living standards, health and quality of life, odontogenic infections are still a common occurrence. North American and European studies report increasing rates of emergency department visits and hospitalizations for acute dental infections [START_REF] Connors | Acute dental infections managed in an outpatient parenteral antibiotic program setting: prospective analysis and public health implications[END_REF]. Severe odontogenic infection is a disease entity with potentially systemic complications. Treatment guidelines are surgical incision and drainage of the purulent collections in combination with extraction of the tooth concerned or root canal treatment, oral cavity rehabilitation, probabilistic parenteral antibiotic therapy and hospitalization 2 . In France, non-severe odontogenic infections are managed by the dentist while hospitals deal with all severe cases involving trismus, extensive cellulitis, dysphagia, dysphonia, dyspnoea and refractory pain. Various reports state that odontogenic infection is due to the interdependent and synergistic metabolism of a variety of mixed aerobic and anaerobic microorganisms whose collection and culture are complex [START_REF] Bahl | Odontogenic infections: Microbiology and management[END_REF][START_REF] Farmahan | The clinical relevance of microbiology specimens in head and neck space infections of odontogenic origin[END_REF][START_REF] Flynn | Severe odontogenic infections, part 2: prospective outcomes study[END_REF][START_REF] Flynn | Severe odontogenic infections, part 1: prospective report[END_REF][START_REF] Flynn | Severe odontogenic infections, part 1: prospective report[END_REF][START_REF] Jundt | Characteristics and cost impact of severe odontogenic infections[END_REF][START_REF] Opitz | Incidence and management of severe odontogenic infections-A retrospective analysis from 2004 to 2011[END_REF][START_REF] Rastenienė | Odontogenic Maxillofacial Infections: A Ten-Year Retrospective Analysis[END_REF][START_REF] Sánchez | Severe odontogenic infections: epidemiological, microbiological and therapeutic factors[END_REF][START_REF] Sato | Eight-year retrospective study of odontogenic origin infections in a postgraduation program on oral and maxillofacial surgery[END_REF][START_REF] Seppänen | Analysis of systemic and local odontogenic infection complications requiring hospital care[END_REF][START_REF] Storoe | The changing face of odontogenic infections[END_REF][START_REF] Storoe | The changing face of odontogenic infections[END_REF][START_REF] Wang | A five-year retrospective study of odontogenic maxillofacial infections in a large urban public hospital[END_REF][START_REF] Wang | A five-year retrospective study of odontogenic maxillofacial infections in a large urban public hospital[END_REF][START_REF] Ylijoki | Differences between patients with or without the need for intensive care due to severe odontogenic infections[END_REF][START_REF] Zirk | Empiric systemic antibiotics for hospitalized patients with severe odontogenic infections[END_REF]. Empirically and in our everyday experience, we have observed that the probabilistic antibiotic therapy is rarely modified during a hospital stay. The objective of this prospective study was to determine if bacteriological sampling provides relevant information for the management of patients with acute dental infections and whether this procedure could be reserved for a specific population.

MATERIAL AND METHODS

Recruitment

We performed an ancillary observational study prospectively between January 2004 and December 2014 in the Department of Oral and Maxillofacial Surgery, CHU Clermont-Ferrand, France, in accordance with the ethical principles of the World Medical Association Declaration of Helsinki for medical research involving human subjects (CE-CIC-GREN-12-

08).

All patients enrolled in the study had an acute dental infection as defined by the following criteria: hospital admission, intravenous antibiotic treatment and tooth extraction with incision and drainage under general anaesthesia. Patients who could be managed under local anaesthesia were directly referred to the Department of Odontology.

Data collection

The following data were collected:

-socio-demographic data: age, sex, associated medical conditions such as penicillin allergy, smoking status, alcohol consumption, psychiatric disorder, immunodepression [START_REF] Chinen | Secondary immunodeficiencies, including HIV infection[END_REF] (diabetes mellitus, obesity [START_REF] Dobner | Body mass index and the risk of infection -from underweight to obesity[END_REF],chemotherapy, HIV and hepatitis B or C infection, transplant surgery), asthma, non-mellitus diabetes -number and type of spaces involved by the infection -treatment on admission: ongoing antibiotic and/or anti-inflammatory treatment -duration of hospitalization and number of surgical procedures -bacteriological results, results of culture and sensitivity testing -antibiotic adaptation

Patient management

On admission, a medical history was taken and results of clinical examination, C-reactive protein (CRP) assay and white blood count were recorded. The biological results provided no relevant information and hence are not given in the text. Dental panoramic radiography was systematically performed and in case of symptoms such as dyspnoea or dysphagia a cervicofacial CT-scan. After the airway had been secured, the causal tooth was extracted under general anaesthesia, with incision and drainage via the intraoral and/or extraoral sites.

Debridement was performed of all cervical facial spaces involved by infection. All patients received intravenous probabilistic antibiotic treatment against oral mucosal flora. The French Health Products Safety Agency (ANSM) recommends amoxicillin and clavulanic acid (1 gram every 8 hours), and clindamycin (300 mg every 8 hours) and metronidazole (500 mg every 8 hours) in case of penicillin allergy. If necessary, antibiotics were adapted according to bacteriological results (Recommendations of the French Health Products Safety Agency,

2003).

Microbiological culture and sensitivity

Pus was sampled in patients at the first surgical procedure and in the event of repeat surgeries.

Bacteriological diagnosis began with direct examination, after which the swab was fixed and Gram-stained. Culture was then performed under aerobic and anaerobic conditions. Samples were seeded on Columbia blood agar plus colistin and nalidixic acid (CNA), and on chocolate agar plus PolyViteX (bioMérieux, France) incubated at 37 •C in CO2-enriched atmosphere at 5%. The dishes were checked daily for 3 days. Anaerobic bacteria and anaerobic agar (Oxoid, France) were seeded and incubated at 37 •C using jars and packets generating an anaerobic atmosphere (Anaerogen Oxoid, France) for at least 5 days. Dishes were checked daily after 48 hours of culture. The bacterial species were identified with the API 20 A (bioMérieux) and Rapid ID 32A test kits.

The cost of microbiological culture and sensitivity testing of an abscess sample in France is 54 euros.

Statistical analysis

Descriptive statistics are expressed as mean and range (min-max), according to their distribution, and number of patients (%) for categorical data. Statistics were computed with STATA V12 (Stata Corp, College Station, Texas, USA). Tests were two-sided and a p-value<0.05 was considered statistically significant.

RESULTS

Data collection

During the period of the study, 653 patients were enrolled: 386 (59%) were male and 267 (41%) female, with a mean age of 37 years (range 8 -88), 375 (57%) were smokers, 78 (12%) regular drinkers, 54 (8%) addicted to drugs, 23 (3.5%) non-mellitus diabetics, 25 (3.8%) immunosuppressed, 33 (5%) asthmatics and 62 (9.5%) had psychiatric disorders. Forty seven patients (7.2%) were allergic to penicillin. During surgery, 516 patients (80%) had a single facial space infected by pus, 95 (15%) patients 2 spaces, 28 (4%) patients 3 spaces, 5 patients (1%) 4 spaces and 1 patient had 6 spaces involved. The submandibular space 32% (n=209) and the vestibular space 28% (n=182) were the most frequently involved.

A total of 378 (58%) patients had been receiving oral antibiotics before admission to hospital, for a mean duration of 4.1 days (range 1 -30). Most patients (231: 61%) had been prescribed amoxicillin or amoxicillin and clavulanic acid, 75 (20%) had a combination of spiramycin and metronidazole, 37 (10%) pristinamycin, 36 (9.5%) metronidazole and 2 (0.5%) clindamycin.

Microbiological culture and sensitivity testing (Fig.1)

A total of 535 (81.9%) patients had swabs taken during surgery, 118 (18.1%) were excluded from the study because bacteriological sampling was not performed. It is noteworthy that there was no adverse clinical outcome for any of these 118 patients. In 58 patients (10.9%) Gram-staining identified no microorganism. In 100 of the 477 (89.1%) patients with positive examination, one to three non-commensal or pathogenic bacterial species were isolated. In the remaining 377 no predominant bacteria were isolated (polymorphic oropharyngeal flora).

Antibiotic sensitivity testing was performed in the group of 100 patients (18.7%) with one to three bacteria isolated on culture. In 77 patients, the probabilistic antibiotic initially prescribed was active against the bacteria isolated. Antibiogram results led to antibiotic therapy adaptation in the remained 23 patients, of whom 10 were discharged because of favourable clinical evolution before results were available. About 20% of patients under antibiotic therapy before admission had a germ identification and 17% of patients without any antibiotic treatment before sampling, with no significant statistical difference.

Analysis of swab samples showed that 107 (55.7%) patients had aerobic flora, 79 (37.7%) anaerobic flora and 14 (6.6%) mixed aerobic-anaerobic flora. Gram-positive cocci were isolated in 103 (19.2%) cultures. The most common bacteria isolated were group F Streptococcus (22 swabs), α-hemolytic Streptococcus (18 swabs), and Streptococcus constellatus (16 swabs). (Tab. 1). Among the antibiograms performed, 23% contained bacteria resistant or with intermediate sensitivity to amoxicillin-clavulanic acid, 78% resistant to gentamycin, 36% to clindamycin and 2% to pristinamycin. Forty-two patients (6.4%) had more than one surgical procedure to drain collections. All had undergone new sampling during second-look surgery. The mean duration of their hospital stay was 17.3 days (range 5-37 days). Five of them (12%) were immunocompromised and therefore had a statistically significant greater risk of developing complications than nonimmunocompromised patients (p= 0.001); 13 (30.9%) patients needed antibiotic adaptation subsequent to the first or other samplings.

DISCUSSION

Our study showed that 83% of patients had no probative microbiological results, either because there was no germ growth or because polymorphic oropharyngeal flora was present. Culture and sensitivity testing was possible for only 18.6% patients. Antibiogram results led to antibiotic adaptation in 23 patients (4.3%). In only 13 patients (2.4%) did it improve clinical symptoms, all of them were in the group of patient with several surgeries et complex evolution.. Clinicians are aware of the difficulties in taking samples for qualitative microbiological testing [START_REF] Chovanec | Comparison of two non-invasive methods of microbial analysis in surgery practice: incision swabbing and the indirect imprint technique[END_REF]. In our study, patient management was always performed in the emergency department by different surgeons, of whom some had forgotten to perform sampling and others thought that it was unnecessary. Two different sampling techniques exist, swabbing and aspiration. Aspiration is more effective: it recovers more anaerobes than swabbing [START_REF] Poeschl | Endodontic pathogens causing deep neck space infections: clinical impact of different sampling techniques and antibiotic susceptibility[END_REF] and decreases the risk of contamination from the skin, oral mucosa and saliva. Bacteria and more specifically anaerobes are sensitive to milieu change in the time between sampling and transport to the laboratory [START_REF] Bahl | Odontogenic infections: Microbiology and management[END_REF][START_REF] Poeschl | Endodontic pathogens causing deep neck space infections: clinical impact of different sampling techniques and antibiotic susceptibility[END_REF]. Anaerobic strains need more time in culture to grow [START_REF] Brook | Anaerobic bacteria in upper respiratory tract and other head and neck infections[END_REF].

Hence, isolating and identifying a bacterium are difficult and the isolated microorganism may not be representative of the pathogenic bacteria.

Moreover, in the study, 58% of patients had antibiotic therapy before their hospitalisation in our Department, which could prevent or delay bacteria identification. When culture enabled us to isolate bacteria, the most commonly observed in the last four decades, as in other studies, was Streptococcus alpha-haemolyticus [START_REF] Opitz | Incidence and management of severe odontogenic infections-A retrospective analysis from 2004 to 2011[END_REF][START_REF] Rastenienė | Odontogenic Maxillofacial Infections: A Ten-Year Retrospective Analysis[END_REF][START_REF] Zirk | Empiric systemic antibiotics for hospitalized patients with severe odontogenic infections[END_REF][START_REF] Brook | Anaerobic bacteria in upper respiratory tract and other head and neck infections[END_REF][START_REF] Kouassi | Microbiology of facial cellulitis related to dental infection[END_REF] and more specifically, Streptococcus Viridans [START_REF] Farmahan | The clinical relevance of microbiology specimens in head and neck space infections of odontogenic origin[END_REF]. Obviously, probabilistic antibiotic therapy must target it. Our results show that 23% of the bacteria were resistant to combined amoxicillin and clavulanic acid and 36% to clindamycin. In the literature, the rate of susceptibility to penicillin varies from 100% [START_REF] Poeschl | Endodontic pathogens causing deep neck space infections: clinical impact of different sampling techniques and antibiotic susceptibility[END_REF][START_REF] Kuriyama | An outcome audit of the treatment of acute dentoalveolar infection: impact of penicillin resistance[END_REF] to 70% [START_REF] Farmahan | The clinical relevance of microbiology specimens in head and neck space infections of odontogenic origin[END_REF][START_REF] Rastenienė | Odontogenic Maxillofacial Infections: A Ten-Year Retrospective Analysis[END_REF]. In the study of Farmahan et al., resistance to amoxicillin was 26.6%, and 18.7% to a combination of amoxicillin and metronidazole [START_REF] Farmahan | The clinical relevance of microbiology specimens in head and neck space infections of odontogenic origin[END_REF]. The incidence of resistance to antibiotics routinely used in deep space head and neck infection is 18% for clindamycin, 14% for macrolides and 7% for penicillin G in the aerobic spectrum, and 11% for clindamycin, 6% for metronidazole and 8% for penicillin G in the anaerobes [START_REF] Poeschl | Antibiotic susceptibility and resistance of the odontogenic microbiological spectrum and its clinical impact on severe deep space head and neck infections[END_REF].

However, in the study of Poeschl et al. [START_REF] Poeschl | Antibiotic susceptibility and resistance of the odontogenic microbiological spectrum and its clinical impact on severe deep space head and neck infections[END_REF] no clinical antibiotic failure was observed in patients treated by an association of amoxicillin and clavulanic acid. Warnke et al. [START_REF] Warnke | Penicillin compared with other advanced broad spectrum antibiotics regarding antibacterial activity against oral pathogens isolated from odontogenic abscesses[END_REF] found that amoxicillin with clavulanic acid would have been sufficient as a single agent in only one third of the patients. However, these in vitro results do not reflect clinical success because in the study an absolute majority of patients treated with penicillin and abscess drainage had a rapid improvement in clinical symptoms [START_REF] Kuriyama | An outcome audit of the treatment of acute dentoalveolar infection: impact of penicillin resistance[END_REF]. The clinical efficacy of penicillin could be the result of the vulnerability of the dominant strains to the antibiotic [START_REF] Farmahan | The clinical relevance of microbiology specimens in head and neck space infections of odontogenic origin[END_REF][START_REF] Rastenienė | Odontogenic Maxillofacial Infections: A Ten-Year Retrospective Analysis[END_REF][START_REF] Warnke | Penicillin compared with other advanced broad spectrum antibiotics regarding antibacterial activity against oral pathogens isolated from odontogenic abscesses[END_REF], coupled to collection drainage. The relation between probabilistic antibiotic treatment and bacterial resistance is unclear.

At least 24 to 48 hours are required to isolate a bacterium, a period that can be extended by 48 hours to isolate a dominant bacterium when there are polymorphic flora or in the event of antibiotic treatment before sampling (which was the case for 58% of patients in our study). About 4 to 6 days are needed to obtain an antibiogram if a bacterium is isolated. In our study, 493 (92%) patients were discharged after 5.4 days on average and successfully treated before the results of culture and sensitivity testing were available.

Based on our findings, 83% of patients had no probative microbiological results and 92% are discharged home before results are available; this suggest that bacteriological analysis had no impact on their evolution.

In light of these results and those from the literature, pus sampling in the management of patients with severe odontogenic infection could be restricted to a well-defined type of patient. On the right side are reported the number of patients for whom a bacteriological analysis was performed including sampling, direct observation, culture and sensibility and finally the clinical relevance. The light grey coloration correspond to patients with a positive result. The percentage are calculated by reference to the 535 for whom a sampling was performed.

Figure 1 :

 1 Figure 1: From sampling to clinical relevance.

Figure 2 :

 2 Figure 2: Decisional tree

Informed consent:

For this type of study, formal consent is required.

Informed consent was obtained from all individual participants included in the study.

grant from funding agencies in the public, commercial, or not-for-profit sectors.

Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Ethical approval:

This article does contain studies with human participants.

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Microorganisms


Van-Thuan Hoang

Mohammed Meftah

Tran Duc

Anh Ly

Tassadit Drali

Saber Yezli

Badriah Alotaibi

Didier Raoult

Philippe Parola

Vincent Pommier De Santi

Philippe Gautret
email: philippe.gautret@club-internet.fr

Bacterial respiratory carriage in French Hajj pilgrims and the effect of pneumococcal vaccine and other individual preventive measures: A prospective cohort survey

Keywords: Hajj, respiratory infections, prevention, pneumococcal vaccine, bacteria

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

Introduction

The Hajj is one of the largest annual religious mass gatherings in the world. Each year, Saudi Arabia attracts over 2 million pilgrims from more than 180 countries including about 2,000 from Marseille, France [START_REF] Gautret | Pilgrims from Marseille, France, to Mecca: demographics and vaccination status[END_REF]. The Hajj presents major challenges in public health and infection control and the crowding conditions favor the acquisition, dissemination and transmission of pathogenic microorganisms [START_REF] Memish | Hajj: infectious disease surveillance and control[END_REF]. Respiratory diseases are particularly common during the pilgrimage, representing a significant cause of morbidity and a major cause of hospitalization with community-acquired pneumonia being a leading cause of serious illness among pilgrims [START_REF] Al-Tawfiq | Mass Gatherings and the Spread of Respiratory Infections. Lessons from the Hajj[END_REF]. The etiology of respiratory tract infection is multifactorial and complex. Whereas viruses are likely responsible for mild upper respiratory tract infections [START_REF] Gautret | Hajj-associated viral respiratory infections: A systematic review[END_REF], some studies have identified Streptococcus pneumoniae as one of the main etiologic agents associated with severe respiratory infections in Hajj pilgrims [START_REF] Al-Tawfiq | Prevention of pneumococcal infections during mass gathering[END_REF].

Predisposing factors for pneumococcal infections at the Hajj include older age, chronic diseases, overcrowding conditions and environmental pollution [START_REF] Ridda | Pneumococcal infections at Hajj: current knowledge gaps[END_REF]. The natural route of pneumococcal infection is initiated by nasopharyngeal colonization and may progress towards an invasive disease, especially if natural immunological barriers are crossed [START_REF] Bogaert | Colony blot assay: a useful method to detect multiple pneumococcal serotypes within clinical specimens[END_REF]. The reported rates of S. pneumoniae nasopharyngeal carriage depend on age, geographical area, genetic heritage and socio-economic conditions [START_REF] Bogaert | Colony blot assay: a useful method to detect multiple pneumococcal serotypes within clinical specimens[END_REF]. This nasopharyngeal carriage is considered as the principal source of person-toperson transmission of S. pneumoniae [START_REF] Bogaert | Colony blot assay: a useful method to detect multiple pneumococcal serotypes within clinical specimens[END_REF]. In addition to S. pneumoniae other bacterial pathogens such as Haemophilus influenzae, Klebsiella pneumoniae and Staphylococcus aureus were frequently isolated from Hajj pilgrims suffering from pneumonia [START_REF] Mandourah | Clinical and temporal patterns of severe pneumonia causing critical illness during Hajj[END_REF][START_REF] Shirah | Mass gathering medicine (Hajj Pilgrimage in Saudi Arabia): The clinical pattern of pneumonia among pilgrims during Hajj[END_REF][START_REF] Memish | Etiology of severe community-acquired pneumonia during the 2013 Hajj-part of the MERS-CoV surveillance program[END_REF]. However, available data about the carriage of S. pneumoniae and other respiratory bacteria in the overall population of Hajj pilgrims is limited [START_REF] Al-Tawfiq | Mass Gatherings and the Spread of Respiratory Infections. Lessons from the Hajj[END_REF][START_REF] Al-Tawfiq | Prevention of pneumococcal infections during mass gathering[END_REF].

The objective of this study was to investigate the prevalence of pharyngeal carriage of respiratory bacteria including S. pneumoniae, H. influenzae, K. pneumoniae and S. aureus in French pilgrims, before and after the Hajj in 2015.

Methods

Participants and study design

Pilgrims participating in the 2015 Hajj (from September 21 st to 26 th , 2015) were recruited in August 2015, at a private specialized travel agency in Marseille, France, which organizes trips to Mecca, Saudi Arabia. Potential adult participants were asked to participate in the study on a voluntary basis. They were included and followed up before departing from France and immediately before leaving Saudi Arabia by a medical bilingual (Arabic and French) doctor who traveled with the group. Upon inclusion (August 23 rd to 30 th , 2015), the participants were interviewed using a standardized pre-Hajj questionnaire that collected information on demographics, immunization status and medical history. Based on French recommendations [12][13], pilgrims with risk factors for invasive pneumococcal disease (IPD) such as age superior or equal to 60 years, diabetes mellitus, chronic respiratory disease, chronic heart disease, chronic kidney disease and immunodeficiency, were vaccinated with the 13-valent conjugate pneumococcal vaccine (PCV-13) unless they received the vaccine in the past 5 years. A post-Hajj questionnaire that collected clinical data and information on compliance with face masks use as well as hand washing, use of disinfectant hand gel and disposable handkerchiefs, was completed during a face-to-face interview, 2 days before the pilgrims' return to France. Pharyngeal swabs were collected upon inclusion on August 30 th (pre-Hajj specimens) and 2 days prior to the return of the pilgrims on September 30 th , 2015 (post-Hajj specimens). Influenza-like illness (ILI) was defined as the presence of cough, sore throat and subjective fever [START_REF] Rashid | Influenza and the Hajj: defining influenza-like illness clinically[END_REF]. The protocol was approved by the Aix-Marseille University institutional review board (July 23 rd , 2013; reference no. 2013-A00961-44). The study was performed in accordance with the good clinical practices recommended by the Declaration of Helsinki and its amendments. All participants provided written consent.

Respiratory specimen. Pharyngeal swabs were collected from each participant, transferred to Sigma-Virocult ® medium and stored at -80°C.

Identification of respiratory bacteria. The DNA extractions were concurrently performed using the EZ1 Advanced XL (Qiagen, German) with the DNA Kit v2.0 (Qiagen). Real-time PCR amplifications were carried out by using LightCycler ® 480 Probes Master kit (Roche diagnostics, France) according to the manufacturer's recommendations. All real-time PCR reactions were performed using a C1000 Touch™ Thermal Cycle (Bio-Rad, USA). Positive results of bacteria amplification were defined as those with a cycle threshold (CT) value ≤35. The SDD gene of H. influenzae, phoE gene of K. pneumoniae, nucA gene of S. aureus and lytA gene of S. pneumoniae were amplified with internal DNA extraction controls TISS, as previously described [START_REF] Memish | Mass gathering and globalization of respiratory pathogens during the 2013 Hajj[END_REF].

Statistical analysis

Differences in the proportions were tested by Pearson's chi-square or Fisher's exact tests when appropriate. To evaluate the potential acquisition of respiratory pathogens in Saudi Arabia, we used the McNemar's Test to compare their prevalence before leaving France and in Saudi Arabia (during and after the Hajj). Percentages and odds ratio (OR) with 95% confidence interval (CI) estimations and comparisons were carried out using STATA 11.1 (Copyright 2009 StataCorp LP, http://www.stata.com). A p value of ≤0.05 was considered significant.

Results

Characteristics of study participants

A total of 119 persons of the 131 pilgrims who traveled with the travel agency (90.8%) agreed to participate and answered the pre-and post-travel questionnaires; 12 pilgrims were excluded because they joined the group in Saudi Arabia one week after the main group arrived. 62 pilgrims were male (52.1%) and 57 female (47.9%) with a gender ratio of 1:1. The median age was 61 years of age (ranging from 24 to 81 years). Fifty-nine (50.9%) of the 119 participants were over 60 years of age.

Sixty-five pilgrims (54.6%) had an indication for vaccination against IPD according to French recommendation at the time of inclusion. Diabetes was the most common comorbidity (Table 1).

Regarding preventive measures, 4.2% (5/119) of pilgrims reported pneumococcal vaccination (PCV-13) in the past 5 years and 57 were vaccinated upon inclusion (three refused), so 62/119 (52.1%) of them were immunized. Ninety-eight pilgrims (82.4%) reported using face masks during the Hajj, including 68.1% (81/119) who reported using masks sometimes and 14.3% frequently (17/119). 47.1% (56/119) declared washing their hands during the Hajj more often than usual, 37.0% (44/119) used hand gel and 41.2% (49/119) used disposable handkerchiefs during their stay in Saudi Arabia.

Clinical features

During the three weeks of their stay in Saudi Arabia, 91 (76.5%) pilgrims reported at least one respiratory symptom with cough being the most frequent (70.6%, 84/119), followed by sore throat (44.5%, 53/119), rhinitis (37.0%, 44/119), voice failure (15.1%, 18/119) and dyspnea (6.7%, 8/119).

38.7% (46/119) reported fever and 20.2% (24/119) ILI. A proportion of 42.0% (50/119) received antibiotics for respiratory tract infection symptoms. The highest antibiotic consumption was βlactams (42/50, 84.0%), followed by macrolides (5/50, 10.0%). None of the pilgrims suffered from pneumonia or other invasive pneumococcal diseases and none was hospitalized.

Overall, respiratory symptoms, ILI and antibiotic intake were more frequent in pilgrims vaccinated against IPD compared to those who were unvaccinated (Table 2). These differences were statistically significant for cough, sore throat and antibiotic intake. Prevalence of cough, fever and antibiotic intake was higher among those who reported using face masks.

Detection of respiratory bacteria

Pre-and post-Hajj specimens were collected from 109 (91.6%) and 112 (94.1%) participants, respectively. The prevalence of bacterial carriage significantly increased after participation in Hajj, compared to pre-Hajj status. S. pneumoniae carriage increased from 1.8% pre-Hajj to 9.8% post-Hajj (p = 0.01). The prevalence of H. influenzae carriage increased from 0.9% pre-Hajj to 45.4% post-Hajj (p <0.0001). The prevalence of K. pneumoniae increased from 2.8% pre-Hajj to 9.8% post-Hajj (p = 0.02). S. aureus carriage increased from 29.4% in pre-Hajj samples to 33.0% in post-Hajj samples but the difference was not statistically significant with p = 0.46 (Figure 2). Pilgrims vaccinated against IPD were seven time less likely to harbor S. pneumoniae after the Hajj compared to unvaccinated pilgrims (3.2% vs. 18.0%, OR = 0.15; 95% CI [0.03 -0.74], p = 0.02).

Using disinfectant gel was significantly associated with an increased S. aureus carriage after the Hajj. Pilgrims using disposable handkerchiefs were 3 times less likely to be positive for H. influenzae in post-Hajj samples. Antibiotic intake was significantly associated with increased carriage of K. pneumoniae in post-Hajj samples (Table 3). We also compared the carriage rate of the four respiratory bacteria in pre-Hajj samples according to adherence to preventive measures during the Hajj for potential confounding factors and found no significant difference (data not shown).

When analyzing the relationship between the carriage of the four respiratory bacteria in post-Hajj samples and the prevalence of clinical symptoms, no significant differences were observed.

Discussion

We confirm here that upper respiratory tract symptoms are very frequent in French Hajj pilgrims, leading to an excessive consumption of antibiotics [START_REF] Gautret | The inevitable Hajj cough: Surveillance data in French pilgrims, 2012-2014[END_REF]. On the other hand, our study showed a significantly increased carriage of S. pneumoniae, H. influenzae and K. pneumoniae following participation in Hajj, confirming previous results obtained from cohort studies conducted among French pilgrims [START_REF] Benkouiten | Respiratory viruses and bacteria among pilgrims during the 2013 Hajj[END_REF][START_REF] Benkouiten | Acquisition of Streptococcus pneumoniae carriage in pilgrims during the 2012 Hajj[END_REF] and from a large cohort study enrolling pilgrims from 13 countries [START_REF] Memish | Mass gathering and globalization of respiratory pathogens during the 2013 Hajj[END_REF]. Such bacterial acquisition is worrying since these pathogens are frequently responsible for Hajjassociated pneumonia [START_REF] Mandourah | Clinical and temporal patterns of severe pneumonia causing critical illness during Hajj[END_REF][START_REF] Shirah | Mass gathering medicine (Hajj Pilgrimage in Saudi Arabia): The clinical pattern of pneumonia among pilgrims during Hajj[END_REF][START_REF] Memish | Etiology of severe community-acquired pneumonia during the 2013 Hajj-part of the MERS-CoV surveillance program[END_REF] or may be responsible for acute rhinosinusitis [START_REF] Marglani | Acute rhinosinusitis during Hajj season 2014: Prevalence of bacterial infection and patterns of antimicrobial susceptibility[END_REF].

We demonstrate here that vaccination against IPD with a conjugate vaccine had a significant protective effect against pneumococcal carriage following participation in the Hajj. Additionally, none of the pilgrims in our cohort suffered from pneumonia. The impact of vaccination against IPD on the reduction of the acquisition of S. pneumoniae respiratory carriage and on the prevention of pneumococcal disease in adults is well-described in the literature, especially in at-risk patients, including those with bronchial asthma, chronic obstructive pulmonary disease, diabetes mellitus, chronic heart failure, splenectomy or immunodeficiency [START_REF] Bonten | Polysaccharide conjugate vaccine against pneumococcal pneumonia in adults[END_REF]. Consistent local or national official recommendations on the use of pneumococcal vaccine for Hajj pilgrims are lacking [START_REF] Ridda | Pneumococcal infections at Hajj: current knowledge gaps[END_REF][START_REF] Rashid | The potential for pneumococcal vaccination in Hajj pilgrims: expert opinion[END_REF][START_REF] Al-Tawfiq | Expected immunizations and health protection for Hajj and Umrah 2018 -An overview[END_REF][START_REF] Patel | The Hajj and Umrah: Health protection matters[END_REF]. The recent recommendations of the Saudi Thoracic Society for pneumococcal vaccination before the Hajj season are to vaccinate all persons aged 50 years and over with combined pneumococcal conjugate vaccine 13 and pneumococcal polysaccharide vaccine 23 [START_REF] Alharbi | The Saudi Thoracic Society pneumococcal vaccination guidelines-2016[END_REF]. 54.6% of the participants in our study had chronic diseases, making them at risk for IPD, or were aged 60 years and over, which is the age cut-off for recommended pneumococcal vaccination in Hajj pilgrims according to French medical authorities [12]. If the Saudi recommendation were to be applied, this proportion would have been 73.3%. In our study, 32.8% of the participants were diabetic. A similar proportion was found in a study conducted in patients hospitalized for severe pneumonia during the 2013 Hajj with a 34.8% prevalence of diabetes mellitus [START_REF] Shirah | Mass gathering medicine (Hajj Pilgrimage in Saudi Arabia): The clinical pattern of pneumonia among pilgrims during Hajj[END_REF]. Respiratory symptoms and antibiotic consumption were more frequent in pilgrims with indication for vaccination against IPD in our survey. This apparently paradoxical result is likely due to the fact that pilgrims with indication for vaccination against IPD were older, more likely to present with chronic respiratory disease, diabetes or other chronic conditions making them at higher risk for respiratory infections due to S. pneumoniae for which they were protected by vaccination or due other respiratory pathogens for which they were not protected. This finding indicates that criteria used to identify pilgrims at risk for IPD could indeed qualify for pre-screening of pilgrims at higher risk for any respiratory infection and help targeting them for reinforced measures against pulmonary communicable diseases including vaccination against influenza, reinforced hand hygiene practice and use of face masks.

When looking at non-pharmaceutical preventive measures against respiratory infections, our results showed that while a majority of pilgrims used face-masks at least sometimes during their stay in Saudi Arabia, only 14.3% used masks frequently and less than half reported compliance with frequent hand washing, use of disinfectant hand-gel and disposable handkerchiefs. Wearing masks especially when in crowded places, frequent hand washing with soap and water or disinfectant, especially after coughing and sneezing, and the use of disposable handkerchiefs are recommended by the Saudi Ministry of Health [START_REF]Health Requirements and Recommendations for Travelers to Saudi Arabia for Hajj and Umrah -2018/1439H[END_REF]. Only 8.4% of the Hajj pilgrims wore masks during the H1N1 outbreak in 2009 and less than 1% wore masks during the MERS-CoV epidemic in 2013 [START_REF] Al-Tawfiq | Prevention of pneumococcal infections during mass gathering[END_REF]. In our survey, cough, fever and antibiotic consumption were significantly more frequent in individuals using face masks, which likely accounts for the high willingness of pilgrims suffering from febrile cough to wear a face mask, with the aim of avoiding spreading diseases. We have no explanation for the positive association between the use of hand gel and the prevalence of S. aureus carriage in post-Hajj throat samples. The negative association between the use of disposable handkerchief and H. influenzae carriage in post-Hajj throat samples may possibly reflect an increased removal of organisms by cleaning of the nasopharynx. The 6-fold increase in K. pneumoniae carriage in post-Hajj samples in individuals who consumed antibiotics during their stay in Saudi Arabia compared to those who did not may possibly be indicative of the high capacity of K. pneumoniae to acquire antibiotic resistance genes under antibiotic selective pressure [START_REF] Navon-Venezia | Klebsiella pneumoniae: a major worldwide source and shuttle for antibiotic resistance[END_REF].

Our study had several limitations. The sample size was small and only limited numbers of patients infected with bacteria were recruited. The study was conducted in French pilgrims only and our results cannot be extrapolated to all pilgrims. Information regarding observance of prevention measures was self-reported and subjective. Also, qPCR does not distinguish between living and dead microorganisms and does not allow assessing antibiotic susceptibility. Finally, we did not investigate vaccine pneumococcal serotypes.

Conclusions

Hajj pilgrims acquire bacterial pathogens during Hajj, which is a risk for disease especially among the significant population of at-risk individuals among pilgrims. Vaccination with the pneumococcal conjugate vaccine appears to reduce the carriage rate of S. pneumoniae among the vaccinated.

Further studies based on larger numbers of pilgrims are needed in order to confirm our preliminary findings.

Table 1: Socio-demographic characteristics and comorbidities of 119 pilgrims according to vaccination status against invasive pneumococcal disease Table 2: Prevalence of symptoms and antibiotic intake during Hajj according to preventive measures. Table 3. Bacterial carriage in post-Hajj pharyngeal samples according to preventive measures and antibiotic intake IPD: invasive pneumococcal disease, OR: Odds ratio, CI 95%: 95% confidence interval

Figure 1 :

 1 Figure 1: Prevalence of bacterial carriage in pre-and post-Hajj pharyngeal samples

Table 1 :

 1 Socio-demographic characteristics and comorbidities of 119 pilgrims according to vaccination 1

	2	status against invasive pneumococcal disease
				Vaccinated	Unvaccinated
			Total
		Socio-demographic characteristics		pilgrims	pilgrims
			N = 119
		and comorbidities		n =62	n =57
			n (%)	n (%)	n (%)
		Male	62 (52.1)	30 (48.4)	32 (56.1)
		Female	57 (47.9)	32 (51.6)	25 (43.9)
		Median age [IQR] 1 (years)	61 [52 ; 66]	65 [63 ; 69]	52.5 [34 ; 58]
		Age ≥ 60 years 2	59 (50.9)	57 (91.9)	2 (3.7)
		previous Hajj	13 (10.9)	9 (14.5)	4 (7.0)
		Diabetes mellitus	39 (32.8)	36 (58.1)	3 (5.3)
		Chronic respiratory disease	12 (10.1)	12 (19.4)	0 (0)
		Chronic heart disease	8 (6.7)	7 (11.3)	1 (1.8)
		Chronic kidney disease	1 (0.8)	1 (1.6)	0 (0)
		Immunodeficiency	0 (0)	0 (0)	0 (0)
		Indication for vaccination against
			65 (54.6)	62 (100)	3 4 (5.3)
		invasive pneumococcal disease 3
	3	1. Interquartile range
	4	2. Data based on 116 individuals, data from three participants were missing
	5	3. Age of 60 years or over, diabetes mellitus, chronic respiratory disease, chronic heart disease, chronic
	6	kidney disease and immunodeficiency
		4. Refused vaccination

Table 2 :

 2 Prevalence of symptoms and antibiotic intake during Hajj according to preventive measures.

					Cough		Dyspnea		Sore throat	Voice failure		Rhinitis		Fever		ILI	ATB
	Preventive measures	n (%)	OR [CI 95%] p	n (%)	OR [CI 95%] p	n (%)	OR [CI 95%] p	n (%)	OR [CI 95%] p	n (%)	OR [CI 95%] p	n (%)	OR [CI 95%] p	n (%)	OR [CI 95%] p	n (%)	OR [CI 95%] p
	Vaccination against IPD		Yes No	50 (80.6) 34 (59.7)	2.82 [1.24 -6.42] p = 0.01	7 (11.3) 1 (1.8)	7.12 [0.85 -59.85] p = 0.07	33 (53.2) 20 (35.1)	2.1 [1.01 -4.4] p = 0.05	11 (11.7) 7 (12.3)	1.54 [0.55 -4.29] p = 0.41	26 (41.9) 18 (31.6)	1.56 [0.74 -3.32] p = 0.24	29 (46.8) 17 (29.8)	2.07 [0.97 -4.4] p = 0.06	16 (13.4) 8 (6.7)	2.13 [0.83 -5.45] p = 0.12	32 (51.6) 18 (31.6)	2.31 [1.09 -4.88] p = 0.03
	Mask		Yes No	73 (74.5) 11 (52.4)	2.65 [1.01 -7.00] p = 0.05	6 (6.1) 2 (9.5)	0.62 [0.12 -3.31] p = 0.58	46 (46.9) 7 (33.3)	1.77 [0.66 -4.76] p = 0.26	16 (16.3) 2 (9.5)	1.85 [0.39 -8.75] p = 0.44	38 (38.8) 6 (28.6)	1.58 [0.56 -4.44] p = 0.38	42 (42.9) 4 (19.0)	3.19 [1.00 -10.17] p = 0.05	23 (23.5) 1 (0.8)	6.13 [0.78 -48.22] p = 0.08	46 (46.9) 4 (19.0)	3.76 [1.18 -11.98] p = 0.02
	Hand washing	More often than usual As usual	38 (67.9) 46 (73.0)	0.78 [0.35 -1.72] p = 0.54	5 (8.9) 3 (4.8)	1.96 [0.45 -8.61] p = 0.37	23 (41.1) 30 (47.6)	0.77 [0.37 -1.58] p = 0.47	11 (19.6) 7 (11.1)	1.96 [0.7 -5.45] p = 0.2	22 (34.9) 22 (39.3)	1.2 [0.57 -2.54] p = 0.62	20 (35.7) 26 (41.3)	0.79 [0.38 -1.66] p = 0.54	8 (6.7) 16 (13.4)	0.49 [0.19 -1.25] p = 0.14	24 (42.9) 26 (41.3)	1.07 [0.51 -2.21] p = 0.86
	Disinfectant gel	Yes No	32 (72.7) 52 (69.3)	1.18 [0.52 -2.69] p = 0.7	0 (0) 8 (10.8)	NA p = 0.02	22 (50.0) 31 (41.3)	1.42 [0.67 -3] p = 0.36	7 (15.9) 11 (14.7)	1.1 [0.39 -3.08] p = 0.86	17 (38.6) 27 (36.0)	1.12 [0.52 -2.41] p = 0.77	14 (31.8) 32 (42.7)	0.63 [0.29 -1.37] p = 0.24	9 (7.6) 15 (12.6)	1.03 [0.41 -2.6] p = 0.95	18 (40.9) 32 (42.7)	0.93 [0.44 -1.98] p = 0.85
	Disposable handkerchiefs	Yes No	36 (73.5) 48 (68.6)	1.27 [0.56 -2.85] p = 0.56	2 (4.1) 6 (8.6)	0.45 [0.09 -2.35] p = 0.35	21 (42.7) 32 (45.7)	0.89 [0.43 -1.86] p = 0.76	7 (14.3) 11 (15.7)	0.89 [0.32 -2.5] p = 0.83	20 (40.8) 24 (34.3)	1.32 [0.62 -2.81] p = 0.47	23 (46.9) 23 (32.9)	1.81 [0.85 -3.83] p = 0.12	10 (8.4) 14 (11.8)	1.02 [0.41 -2.54] p = 0.96	22 (44.9) 28 (40.0)	1.22 [0.58 -2.56] p = 0.59
		NA: not applicable
		IPD: invasive pneumococcal disease, OR: Odds ratio, CI 95%: 95% confidence interval; ILI: influenza like illness, ATB: Antibiotic treatment

Table 3 .

 3 Bacterial carriage in post-Hajj pharyngeal samples according to preventive measures and antibiotic intake

			Staphylococcus aureus		Streptococcus pneumoniae	Haemophilus influenzae		Klebsiella pneumoniae
	Preventive measures	n (%)	OR	p	n (%)	OR	p	n (%)	OR	p	n (%)	OR	p
				CI 95%			CI 95%			CI 95%			CI 95%
	Vaccination against IPD	Yes No	18 (29.0) 19 (38.0)	0.67 [0.30 -1.47]	0.32	2 (3.2) 9 (18)	0.15 [0.03 -0.74]	0.02	26 (41.9) 25 (50.0)	0.72 [0.34 -1.53]	0.40	7 (11.3) 4 (8.0)	1.46 [0.40 -5.31]	0.56
	Mask	Yes No	35 (36.1) 2 (13.3)	3.67 [0.78 -17.21]	0.10	8 (8.2) 3 (20.0)	0.36 [0.08 -1.54]	0.17	45 (46.4) 6 (40.0)	1.30 [0.43 -3.93]	0.64	9 (9.3) 2 (13.3)	0.66 [0.13 -3.42]	0.62
	Hand washing	Much more As usual	20 (35.7) 17 (30.4)	1.27 [0.58 -2.81]	0.55	6 (10.7) 5 (8.9)	1.22 [0.35 -4.27]	0.75	27 (48.2) 24 (42.9)	1.24 [0.59 -2.61]	0.57	7 (12.5) 4 (7.1)	1.86 [0.51 -6.74]	0.94
	Disinfectant gel	Yes No	19 (44.2) 18 (26.1)	2.24 [1.01 -5.03]	0.05	7 (16.3) 4 (5.8)	3.16 [0.87 -11.53]	0.08	17 (39.5) 34 (49.3)	0.67 [0.31 -1.46]	0.32	6 (14.0) 5 (7.2)	2.08 [0.59 -7.27]	0.25
	Disposable handkerchiefs	Yes No	12 (24.5) 25 (39.7)	0.49 [0.22 -1.12]	0.09	3 (6.1) 8 (12.7)	0.45 [0.11 -1.79]	0.26	16 (32.6) 35 (55.6)	0.39 [0.18 -0.84]	0.02	6 (12.2) 5 (7.9)	1.62 [0.46 -5.65]	0.45
	Antibiotic treatment	Yes No	18 (36.0) 19 (30.6)	1.27 [0.58 -2.81]	0.55	4 (8.0) 7 (11.3)	0.68 [0.19 -2.48]	0.56	22 (44.0) 29 (46.8)	0.89 [0.42 -1.89]	0.77	9 (18.0) 2 (3.2)	6.58 [1.35 -32.06]	0.02


Vincent Bérot
email: vincent.berot@aphp.fr

Ahmed-Fateh Ousser

Wladimir Sougakoff

Gabriel Lecso

Gentiane Monsel

Eric Caumes

A patient from Mali with Actinomadura bangladeshensis-induced foot mycetoma: A diagnostic challenge

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.

Text

A 51-year-old man from Mali, living in France since 1990 and travelling yearly to Kayes area in Southwestern Mali, presented in July 2018 with a painless left-foot firm swelling (Fig. 1A) of 8-year duration. Foot skin was covered by deep fistulas. Diagnosis of actinomycetoma was confirmed by identifying in the pus discharged from fistulas white grains (Fig. 1B) containing filamentous branched and ramified Gram-positive bacilli (Fig. 1C, Gram staining, 100-fold magnification). Bacilli were identified by 16S rRNA gene sequencing as Actinomadura bangladeshensis (99.2% nucleotide identity over 493 bp in the GenBank database, strain 3-46-b(3), NCBI Reference Sequence NR_041618.1). Culture returned positive after 22 days but due to low inoculum and slow growth speed of these bacteria, no in vitro susceptibility testing was possible.

X-Ray and MRI showed cortical erosions on first metatarsal and navicular bones (Fig. 1D).

Treatment with trimethoprim-sulfamethoxazole was started and is still pursued for at least one year with possibility of amputation in case of failure.

Mycetoma is a chronic soft tissue infection that creates sinusoidal cavities, fistulized to the skin and producing grains. Grains correspond to the aggregation of filamentous pathogens surrounded by biofilm, either bacteria in actinomycetomas or fungi in eumycetomas. Grain colour may orient towards fungal or bacterial origin. Mycetomas remain endemic in tropical countries and are considered by WHO as a neglected tropical disease. They are sporadic in developed countries and reported almost only in migrants from countries between 30 th northern and 15 th southern parallels, also known as "Mycetoma Belt". However, this term should no more be used because mycetomas are described elsewhere as in Southern Europe.