Genomic surveillance and antimicrobial resistance determinants in Neisseria gonorrhoeae isolates from Uganda, Malawi and South Africa, 2015-20.

OBJECTIVES: Global antimicrobial resistance (AMR) surveillance in Neisseria gonorrhoeae is essential. In 2017-18, only five (10.6%) countries in the WHO African Region reported to the WHO Global Gonococcal Antimicrobial Surveillance Programme (WHO GASP). Genomics enhances our understanding of gonococcal populations nationally and internationally, including AMR strain transmission; however, genomic studies from Africa are extremely scarce. We describe the gonococcal genomic lineages/sublineages, including AMR determinants, and baseline genomic diversity among strains in Uganda, Malawi and South Africa, 2015-20, and compare with sequences from Kenya and Burkina Faso. METHODS: Gonococcal isolates cultured in Uganda (n = 433), Malawi (n = 154) and South Africa (n = 99) in 2015-20 were genome-sequenced. MICs were determined using ETEST. Sequences of isolates from Kenya (n = 159), Burkina Faso (n = 52) and the 2016 WHO reference strains (n = 14) were included in the analysis. RESULTS: Resistance to ciprofloxacin was high in all countries (57.1%-100%). All isolates were susceptible to ceftriaxone, cefixime and spectinomycin, and 99.9% were susceptible to azithromycin. AMR determinants for ciprofloxacin, benzylpenicillin and tetracycline were common, but rare for cephalosporins and azithromycin. Most isolates belonged to the more antimicrobial-susceptible lineage B (n = 780) compared with the AMR lineage A (n = 141), and limited geographical phylogenomic signal was observed. CONCLUSIONS: We report the first multi-country gonococcal genomic comparison from Africa, which will support the WHO GASP and WHO enhanced GASP (EGASP). The high prevalence of resistance to ciprofloxacin (and empirical use continues), tetracycline and benzylpenicillin, and the emerging resistance determinants for azithromycin show it is imperative to strengthen the gonococcal AMR surveillance, ideally including genomics, in African countries.

Evaluation of the impact of COVID-19 in people coinfected with HIV and/or tuberculosis in low-income countries: study protocol for mixed methods research in Burkina Faso.

BACKGROUND: An issue of particular concern is the impact of the 2019 novel coronavirus (2019 nCOV) on the people coinfected with the Human Immuno-deficiency Virus (HIV) and/or tuberculosis (TB). Unfortunately, this interaction has not been well explored in African despite the large proportion of these risk populations living with HIV and/or patients and/or tuberculosis (TB) in the African region. This study aims to design a research protocol for assessment of the impact of coronavirus disease 2019 (COVID-19) on these risk populations in response to COVID-19 strategic plans in Burkina Faso by generating serological, epidemiological, virological, clinical and socio-anthropological evidence-based data. METHODS: A multidisciplinary research will be conducted in the city of Bobo-Dioulasso, Burkina Faso using mixed methods. Data will be collected from a cohort of people living with HIV and/or TB patients in the city (i) to determine the proportion of people with specific antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using retrospective data ; (ii) to determine the proportion of people infected with Covid-19 and the dynamics of viral loads and antibodies in these people based on prospective data; (iii) to identify circulating SARS-COV-2 variants and novel biomarkers using prospective data ; (iv) to analyze perceptions, community experiences and response strategies during the public health emergencies imposed by COVID-19 through a qualitative study. DISCUSSION: This study will generate factual and comprehensive data that will contribute in improving response strategies to COVID-19 and the other possible emerging diseases with keen interest on the risk populations living with HIV and/or TB infected patients.

Determination of the Optimal Bacterial DNA Extraction Method to Explore the Urinary Microbiota.

Recent advances in molecular biology have been successfully applied to the exploration of microbiota from various fluids. However, the urinary microbiota remains poorly explored, as its analysis requires specific technical considerations. Indeed, urine is a low microbial biomass environment, in which the representativity of each bacterium must be respected to obtain accurate data. Thus, sensitive extraction methods must be used to obtain good quality DNA while preserving the proportions between species. To address this, we compared the efficiency of five extraction methods on artificial urine samples spiked with low amounts of four bacteria species. The quality of the DNA obtained was further evaluated by different molecular biology approaches, including quantitative PCR and amplicon-based next-generation sequencing (NGS). Although two extraction methods allowed DNA of sufficient quality for NGS analysis to be obtained, one kit extracted a larger amount of DNA, which is more suitable for the detection of low-abundant bacteria. Results from the subsequent assessment of this kit on 29 human clinical samples correlated well with results obtained using conventional bacterial urine culture. We hope that our work will make investigators aware of the importance of challenging and adapting their practice in terms of the molecular biology approaches used for the exploration of microbiota.

Fecal Excretion of Mycobacterium leprae, Burkina Faso.

Mycobacterium leprae was detected by optical microscopy, fluorescent in situ hybridization, and molecular detection in feces collected for the diagnosis of Entamoeba coli enteritis in a leprosy patient in Burkina Faso. This observation raises questions about the role of fecal excretion of M. leprae in the natural history and diagnosis of leprosy.

Lower mgpB diversity in macrolide-resistant Mycoplasma genitalium infecting men visiting two sexually transmitted infection clinics in Montpellier, France.

OBJECTIVES: Men engaged in high-risk sexual behaviour, such as MSM, are likely to be infected by resistant Mycoplasma genitalium strains. Understanding the transmission dynamics is challenging. We aimed to investigate the molecular epidemiology of M. genitalium in men visiting sexually transmitted infection (STI) clinics. PATIENTS AND METHODS: Between June 2017 and February 2018, 95 M. genitalium-positive specimens from 78 men, including 76.9% MSM, visiting two STI clinics in Montpellier, France, were analysed for SNPs in the mgpB adhesin gene and number of tandem repeats in the MG_309 gene. Macrolide and fluoroquinolone resistance were determined. Typing results were compared with antibiotic resistance, sexual behaviour, sampling site, HIV pre-exposure prophylaxis (PrEP) usage and HIV status. RESULTS: Thirty-eight mgpB STs were identified, including 23 new STs, with ST4 being most prevalent. The mgpB/MG_309 typing method identified 52 genetic profiles, resulting in a discriminatory index of 0.979. Macrolide and fluoroquinolone resistance-associated mutations were detected in 58.3% and 10.8% of patients, respectively. The macrolide resistance rate was higher among MSM than among men who have sex with women only (68.4% versus 9.1%; adjusted OR, 1.57; 95% CI, 1.13-2.18; P = 0.007). A lower mgpB diversity of 0.870 was found among macrolide-resistant strains in comparison with 0.978 in macrolide-susceptible strains, with an over-representation of mgpB ST62 and ST153. CONCLUSIONS: Although macrolide resistance spread appears polyclonal in M. genitalium, the lower diversity of mgpB types among macrolide-resistant strains may reflect the easier spread of a few specific mgpB types or the occurrence of sexual networks among MSM.

Management of patients with pulmonary mycobacteriosis in France: a multicenter retrospective cohort study.

BACKGROUND: Recent studies report very low adherence of practitioners to ATS/IDSA recommendations for the treatment of nontuberculous mycobacteria pulmonary disease (NTM-PD), as well as a great variability of practices. Type of management could impact prognosis. METHODS: To evaluate management and prognosis of patients with NTM-PD cases with respect to ATS recommendations, we conducted a multicenter retrospective cohort study (18 sentinel sites distributed throughout France), over a period of six years. We collected clinical, radiological, microbiological characteristics, management and outcome of the patients (especially death or not). RESULTS: 477 patients with NTM-PD were included. Respiratory comorbidities were found in 68% of cases, tuberculosis sequelae in 31.4% of patients, and immunosuppression in 16.8% of cases. The three most common NTM species were Mycobacterium avium complex (60%), M. xenopi (20%) and M. kansasii (5.7%). Smear-positive was found in one third of NTM-PD. Nodulobronchiectatic forms were observed in 54.3% of cases, and cavitary forms in 19.1% of patients. Sixty-three percent of patients were treated, 72.4% of patients with smear-positive samples, and 57.5% of patients with smear-negative samples. Treatment was in adequacy with ATS guidelines in 73.5%. The 2-year mortality was 14.4%. In the Cox regression, treatment (HR = 0.51), age (HR = 1.02), and M. abscessus (3.19) appeared as the 3 significant independent prognostic factors. CONCLUSION: These findings highlight the adequacy between French practices and the ATS/IDSA guidelines. Treatment was associated with a better survival.

Impact of systematic early tuberculosis detection using Xpert MTB/RIF Ultra in children with severe pneumonia in high tuberculosis burden countries (TB-Speed pneumonia): a stepped wedge cluster randomized trial.

BACKGROUND: In high tuberculosis (TB) burden settings, there is growing evidence that TB is common in children with pneumonia, the leading cause of death in children under 5 years worldwide. The current WHO standard of care (SOC) for young children with pneumonia considers a diagnosis of TB only if the child has a history of prolonged symptoms or fails to respond to antibiotic treatments. As a result, many children with TB-associated severe pneumonia are currently missed or diagnosed too late. We therefore propose a diagnostic trial to assess the impact on mortality of adding the systematic early detection of TB using Xpert MTB/RIF Ultra (Ultra) performed on nasopharyngeal aspirates (NPA) and stool samples to the WHO SOC for children with severe pneumonia, followed by immediate initiation of anti-TB treatment in children testing positive on any of the samples. METHODS: TB-Speed Pneumonia is a pragmatic stepped-wedge cluster randomized controlled trial conducted in six countries with high TB incidence rate (Côte d'Ivoire, Cameroon, Uganda, Mozambique, Zambia and Cambodia). We will enrol 3780 children under 5 years presenting with WHO-defined severe pneumonia across 15 hospitals over 18 months. All hospitals will start managing children using the WHO SOC for severe pneumonia; one hospital will be randomly selected to switch to the intervention every 5 weeks. The intervention consists of the WHO SOC plus rapid TB detection on the day of admission using Ultra performed on 1 nasopharyngeal aspirate and 1 stool sample. All children will be followed for 3 months, with systematic trial visits at day 3, discharge, 2 weeks post-discharge, and week 12. The primary endpoint is all-cause mortality 12 weeks after inclusion. Qualitative and health economic evaluations are embedded in the trial. DISCUSSION: In addition to testing the main hypothesis that molecular detection and early treatment will reduce TB mortality in children, the strength of such pragmatic research is that it provides some evidence regarding the feasibility of the intervention as part of routine care. Should this intervention be successful, safe and well tolerated, it could be systematically implemented at district hospital level where children with severe pneumonia are referred. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03831906 . Registered 6 February 2019.

Fluorescent Hybridization of Mycobacterium leprae in Skin Samples Collected in Burkina Faso.

Leprosy is caused by Mycobacterium leprae, and it remains underdiagnosed in Burkina Faso. We investigated the use of fluorescent in situ hybridization (FISH) for detecting M. leprae in 27 skin samples (skin biopsy samples, slit skin samples, and skin lesion swabs) collected from 21 patients from Burkina Faso and three from Côte d'Ivoire who were suspected of having cutaneous leprosy. In all seven Ziehl-Neelsen-positive skin samples (four skin biopsy samples and three skin swabs collected from the same patient), FISH specifically identified M. leprae, including one FISH-positive skin biopsy sample that remained negative after testing with PCR targeting the rpoB gene and with the GenoType LepraeDR assay. Twenty other skin samples and three negative controls all remained negative for Ziehl-Neelsen staining, FISH, and rpoB PCR. These data indicate the usefulness of a microscopic examination of skin samples after FISH for first-line diagnosis of cutaneous leprosy. Accordingly, FISH represents a potentially useful point-of-care test for the diagnosis of cutaneous leprosy.

Epidemiology and microbiological features of anaerobic bacteremia in two French University hospitals.

Bacteremia implicating anaerobic bacteria (BIAB) represents 2-6% of all episodes of bacteremia and is associated with high mortality. In this retrospective study from June 2015 to December 2016, we compared BIAB frequency in two hospital centers in Montpellier (France): Montpellier university hospital (MUH) and a center specialized in cancer (ICM). Among the 2465 microbiologically relevant episodes of bacteremia, we identified 144 (5.8%) in which anaerobic bacteria were implicated. BIAB frequency was higher at ICM than MUH (10.4%, vs. 4.9%, p < 0.01). Poly-microbial bacteremia was more frequent among the BIAB episodes (31.9% vs. 11.0% for aerobic-only bacteremia, p < 0.01). Bacteroides and Clostridium were the most frequently identified genera of anaerobic bacteria (64 and 18 episodes, respectively), with the B. fragilis group (BFG) involved in 68/144 episodes. We could perform antibiotic susceptibility typing in 106 of the 144 anaerobic isolates, including 67 BFG isolates. All isolates but one were susceptible to metronidazole. In the BFG, sporadic resistant or intermediate results were found for amoxicillin-clavulanate (5/67), piperacillin-tazobactam (2/67) and imipenem (1/67). BFG isolates were susceptible also to cefoxitin (90.8%), rifampicin (97.0%) and tigecyclin (91.0%). Multidrug resistance in this group (7 isolates) was mostly due to acquired resistance to moxifloxacin, clindamycin and tigecyclin. This study shows that BIAB frequency can vary among hospitals and services. They should especially be taken into account in centers specialized in cancer treatment. However, the implicated bacteria remain frequently susceptible to the most used antibiotics used against anaerobic bacteria, although resistance does exist.

Isolation of Nontuberculous Mycobacteria in Southeast Asian and African Human Immunodeficiency Virus-infected Children With Suspected Tuberculosis.

We enrolled 427 human immunodeficiency virus-infected children (median age, 7.3 years), 59.2% severely immunodeficient, with suspected tuberculosis in Southeast Asian and African settings. Nontuberculous mycobacteria were isolated in 46 children (10.8%); 45.7% of isolates were Mycobacterium avium complex. Southeast Asian origin, age 5-9 years, and severe immunodeficiency were independently associated with nontuberculous mycobacteria isolation. CLINICAL TRIALS REGISTRATION: NCT01331811.

Mycobacterium chimaera Pulmonary Disease in Cystic Fibrosis Patients, France, 2010-2017.

We report Mycobacterium chimaera pulmonary disease in 4 patients given a diagnosis of cystic fibrosis in a university hospital in Montpellier, France. All patients had M. chimaera-positive expectorated sputum specimens, clinical symptoms of pulmonary exacerbation, or a decrease in spirometry test results that improved after specific treatment.

Spread of NDM-5 and OXA-181 Carbapenemase-Producing Escherichia coli in Chad.

We detected for the first time blaNDM-5 and blaOXA-181 in Escherichia coli isolates from hospitalized patients and healthy volunteers in Chad. These resistance genes were located on IncX3 and IncF plasmids. Despite the large diversity of E. coli clones, the identified resistant intestinal isolates belonged mainly to the same sequence type.

High prevalence and characterization of extended-spectrum ß-lactamase producing Enterobacteriaceae in Chadian hospitals.

BACKGROUND: Extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-PE) represent a major problem in the management of nosocomial infections. However, ESBL-PE are not systematically monitored in African countries. The aim of this study was to determine ESBL-PE prevalence in patients from three hospitals in N'Djamena, the capital city of Chad, and to characterize the genetic origin of the observed resistance. METHODS: From January to March 2017, 313 non-duplicate isolates were recovered from various clinical specimens obtained from 1713 patients in the three main hospitals of N'Djamena. Bacterial species were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Susceptibility to 28 antibiotics was tested using the disk diffusion method on Müller-Hinton agar, and ESBL production was confirmed with the double-disc synergy test. The most prevalent ESBL genes associated with the observed resistance were detected using multiplex PCR followed by double-stranded DNA sequencing. RESULTS: Among the 313 isolates, 197 belonged to the Enterobacteriaceae family. The overall ESBL-PE prevalence was 47.72% (n = 94/197), with a higher rate among inpatients compared with outpatients (54.13% vs. 34.37%). ESBL-PE prevalence was highest in older patients (≥60 years of age). E. coli was the most common ESBL-producer organism (63.8%), followed by K. pneumoniae (21.2%). ESBL-PE were mainly found in urine samples (75%). The CTX-M-1 group was dominant (96.7% of the 94 ESBL-PE isolates, CTX-M-15 enzyme), followed by the CTX-M-9 group (4.1%). 86% of resistant isolates harbored more than one ESBL-encoding gene. ESBL production was also associated with the highest levels of resistance to non-ß-lactam drugs. CONCLUSIONS: The prevalence of ESBL-PE harboring resistant genes encoding ESBLs of the CTX-M-1 group was high (48%) among clinical isolates of three main hospitals in Chad, suggesting an alarming spread of ESBL-PE among patients.

Clinical sources and antimicrobial susceptibility of Prevotella timonensis at the university hospital of Montpellier, France.

We describe 84 clinical isolates of Prevotella timonensis recovered between January 2007 and November 2016 at the University Hospital of Montpellier. They were recovered from a variety of clinical samples, mostly of genital and wound origins. All isolates were isolated from a mixed aerobic and anaerobic microbiota. Antimicrobial susceptibility testing of 50 isolates showed 56% of beta-lactamase production and 40% of resistance to clindamycin. One strain was resistant to metronidazole.

Nontuberculous Mycobacteria Infections at a Provincial Reference Hospital, Cambodia.

Prevalence of nontuberculous mycobacteria (NTM) disease is poorly documented in countries with high prevalence of tuberculosis (TB). We describe prevalence, risk factors, and TB program implications for NTM isolates and disease in Cambodia. A prospective cohort of 1,183 patients with presumptive TB underwent epidemiologic, clinical, radiologic, and microbiologic evaluation, including >12-months of follow-up for patients with NTM isolates. Prevalence of NTM isolates was 10.8% and of disease was 0.9%; 217 (18.3%) patients had TB. Of 197 smear-positive patients, 171 (86.8%) had TB confirmed (167 by culture and 4 by Xpert MTB/RIF assay only) and 11 (5.6%) had NTM isolates. HIV infection and past TB were independently associated with having NTM isolates. Improved detection of NTM isolates in Cambodia might require more systematic use of mycobacterial culture and the use of Xpert MTB/RIF to confirm smear-positive TB cases, especially in patients with HIV infection or a history of TB.

Kinetic characterization and molecular docking of novel allosteric inhibitors of aminoglycoside phosphotransferases.

BACKGROUND: Bacterial antibiotic resistance often leads to treatment failure which may have serious consequences, especially in critically sick patients. Resistance to aminoglycosides is mainly due to the expression of antibiotic-modifying enzymes. One important mechanism of aminoglycoside modification is the ATP/GTP-dependent O-phosphorylation catalyzed by aminoglycoside phosphotransferases, APHs. The aim of this study is to identify specific inhibitors of APHs that could restore bacterial susceptibility to aminoglycosides. METHODS: We focused on the search for allosteric inhibitors that bind to small cavities of the protein and block the enzyme function by perturbing its dynamics. RESULTS: From normal mode analysis, a cavity of variable volume belonging to a large groove which splits the protein into two parts was chosen as target. By molecular docking, we screened a large library of commercially available compounds. Seventeen of the highest ranked compounds were tested by in vitro kinetic experiments in order to evaluate their ability to inhibit APHs. Site-directed mutagenesis was carried out with the aim of confirming the inhibition mechanism determined kinetically and the interactions with the protein predicted by in silico studies. These interactions were also confirmed by the use of structurally-related molecules. CONCLUSIONS: Two compounds showed interesting inhibition properties, and one was able to block two different classes of APH. GENERAL SIGNIFICANCE: This study gives new insights into the inhibition of APHs by such allosteric inhibitors, and provides the basis for the future development of combined therapies, antibiotic plus APH inhibitor, which may reverse the resistance to aminoglycosides in a clinical context.

Circulating Cell Free Tumor DNA Detection as a Routine Tool forLung Cancer Patient Management.

Circulating tumoral DNA (ctDNA), commonly named "liquid biopsy", has emerged as a new promising noninvasive tool to detect biomarker in several cancers including lung cancer. Applications involving molecular analysis of ctDNA in lung cancer have increased and encompass diagnosis, response to treatment, acquired resistance and prognosis prediction, while bypassing the problem of tumor heterogeneity. ctDNA may then help perform dynamic genetic surveillance in the era of precision medicine through indirect tumoral genomic information determination. The aims of this review were to examine the recent technical developments that allowed the detection of genetic alterations of ctDNA in lung cancer. Furthermore, we explored clinical applications in patients with lung cancer including treatment efficiency monitoring, acquired therapy resistance mechanisms and prognosis value.

Performance of Xpert MTB/RIF and Alternative Specimen Collection Methods for the Diagnosis of Tuberculosis in HIV-Infected Children.

BACKGROUND: The diagnosis of tuberculosis in human immunodeficiency virus (HIV)-infected children is challenging. We assessed the performance of alternative specimen collection methods for tuberculosis diagnosis in HIV-infected children using Xpert MTB/RIF (Xpert). METHODS: HIV-infected children aged ≤13 years with suspected intrathoracic tuberculosis were enrolled in 8 hospitals in Burkina Faso, Cambodia, Cameroon, and Vietnam. Gastric aspirates were taken for children aged <10 years and expectorated sputum samples were taken for children aged ≥10 years (standard samples); nasopharyngeal aspirate and stool were taken for all children, and a string test was performed if the child was aged ≥4 years (alternative samples). All samples were tested with Xpert. The diagnostic accuracy of Xpert for culture-confirmed tuberculosis was analyzed in intention-to-diagnose and per-protocol approaches. RESULTS: Of 281 children enrolled, 272 (96.8%) had ≥1 specimen tested with Xpert (intention-to-diagnose population), and 179 (63.5%) had all samples tested with Xpert (per-protocol population). Tuberculosis was culture-confirmed in 29/272 (10.7%) children. Intention-to-diagnose sensitivities of Xpert performed on all, standard, and alternative samples were 79.3% (95% confidence interval [CI], 60.3-92.0), 72.4% (95% CI, 52.8-87.3), and 75.9% (95% CI, 56.5-89.7), respectively. Specificities were ≥97.5%. Xpert combined on nasopharyngeal aspirate and stool had intention-to-diagnose and per-protocol sensitivities of 75.9% (95% CI, 56.5-89.7) and 75.0% (95% CI, 47.6-92.7), respectively. CONCLUSIONS: The combination of nasopharyngeal aspirate and stool sample is a promising alternative to methods usually recommended by national programs. Xpert performed on respiratory and stools samples enables rapid confirmation of tuberculosis diagnosis in HIV-infected children. CLINICAL TRIALS REGISTRATION: The ANRS (Agence Nationale de Recherche sur le Sida) 12229 PAANTHER (Pediatric Asian African Network for Tuberculosis and HIV Research) 01 study is registered at ClinicalTrials.gov (NCT01331811).