Knowledge, awareness, and risk practices related to bacterial contamination of antiseptics, disinfectants, and hand hygiene products among healthcare workers in sub-saharan Africa: a cross-sectional survey in three tertiary care hospitals (Benin, Burkina Faso, and DR Congo).

BACKGROUND: Antiseptics, disinfectants, and hand hygiene products can be contaminated with bacteria and cause healthcare-associated infections, which are underreported from low- and middle-income countries. To better understand the user-related risk factors, we conducted a knowledge, awareness, and practice survey among hospital staff in sub-Saharan Africa. METHODS: Self-administered questionnaire distributed among healthcare workers in three tertiary care hospitals (Burkina Faso, Benin, Democratic Republic of the Congo). RESULTS: 617 healthcare workers (85.3% (para)medical and 14.7% auxiliary staff) participated. Less than half (45.5%) had been trained in Infection Prevention & Control (IPC), and only 15.7% were trained < 1 year ago. Near two-thirds (64.2%) preferred liquid soap for hand hygiene, versus 33.1% for alcohol-based hand rub (ABHR). Most (58.3%) expressed confidence in the locally available products. Knowledge of product categories, storage conditions and shelf-life was inadequate: eosin was considered as an antiseptic (47.5% of (para)medical staff), the shelf life and storage conditions (non-transparent container) of freshly prepared chlorine 0.5% were known by only 42.6% and 34.8% of participants, respectively. Approximately one-third of participants approved using tap water for preparation of chlorine 0.5% and liquid soap. Most participants (> 80%) disapproved recycling soft-drink bottles as liquid soap containers. Nearly two-thirds (65.0%) declared that bacteria may be resistant to and survive in ABHR, versus 51.0% and 37.4% for povidone iodine and chlorine 0.5%, respectively. Depicted risk practices (n = 4) were ignored by 30 to 40% of participants: they included touching the rim or content of stock containers with compresses or small containers, storing of cotton balls soaked in an antiseptic, and hand-touching the spout of pump dispenser. Filling containers by topping-up was considered good practice by 18.3% of participants. Half (52.1%) of participants acknowledged indefinite reuse of containers. Besides small differences, the findings were similar across the study sites and professional groups. Among IPC-trained staff, proportions recognizing all 4 risk practices were higher compared to non-trained staff (35.9% versus 23.8%, p < 0.0001). CONCLUSIONS: The present findings can guide tailored training and IPC implementation at the healthcare facility and national levels, and sensitize stakeholders' and funders' interest.

Diagnostic accuracy of Xpert MTB/RIF Ultra for childhood tuberculosis in West Africa - a multicenter pragmatic study.

OBJECTIVE: To evaluate the performance of Xpert Mycobacterium Tuberculosis/rifampicin (MTB/RIF) Ultra (Ultra) for diagnosis of childhood tuberculosis (TB) within public health systems. METHODS: In this cross-sectional study, children aged <15 years with presumptive pulmonary TB were consecutively recruited and evaluated for TB at tertiary-level hospitals in Benin, Mali, and Ghana. Bivariate random-effects models were used to determine the pooled sensitivity and specificity of Ultra against culture. We also estimated its diagnostic yield against a composite microbiological reference standard (cMRS) of positive culture or Ultra. RESULTS: Overall, 193 children were included in the analyses with a median (interquartile range) age of 4.0 (1.1-9.2) years, 88 (45.6%) were female, and 36 (18.7%) were HIV-positive. Thirty-one (16.1%) children had confirmed TB, 39 (20.2%) had unconfirmed TB, and 123 (63.7%) had unlikely TB. The pooled sensitivity and specificity of Ultra verified by culture were 55.0% (95% confidence interval [CI]: 28.0-79.0%) and 95.0% (95% CI: 88.0-98.0%), respectively. Against the cMRS, the diagnostic yield of Ultra and culture were 67.7% (95% CI: 48.6-83.3%) and 70.9% (95% CI: 51.9-85.8%), respectively. CONCLUSION: Ultra has suboptimal sensitivity in children with TB that were investigated under routine conditions in tertiary-level hospitals in three West African countries.

A genomic appraisal of invasive Salmonella Typhimurium and associated antibiotic resistance in sub-Saharan Africa.

Invasive non-typhoidal Salmonella (iNTS) disease manifesting as bloodstream infection with high mortality is responsible for a huge public health burden in sub-Saharan Africa. Salmonella enterica serovar Typhimurium (S. Typhimurium) is the main cause of iNTS disease in Africa. By analysing whole genome sequence data from 1303 S. Typhimurium isolates originating from 19 African countries and isolated between 1979 and 2017, here we show a thorough scaled appraisal of the population structure of iNTS disease caused by S. Typhimurium across many of Africa's most impacted countries. At least six invasive S. Typhimurium clades have already emerged, with ST313 lineage 2 or ST313-L2 driving the current pandemic. ST313-L2 likely emerged in the Democratic Republic of Congo around 1980 and further spread in the mid 1990s. We observed plasmid-borne as well as chromosomally encoded fluoroquinolone resistance underlying emergences of extensive-drug and pan-drug resistance. Our work provides an overview of the evolution of invasive S. Typhimurium disease, and can be exploited to target control measures.

Whole-Genome Sequencing-Based Screening of MRSA in Patients and Healthcare Workers in Public Hospitals in Benin.

Methicillin-resistant Staphylococcus aureus (MRSA) constitutes a serious public health concern, with a considerable impact on patients' health, and substantial healthcare costs. In this study, patients and healthcare workers (HCWs) from six public hospitals in Benin were screened for MRSA. Strains were identified as MRSA using conventional microbiological methods in Benin, and confirmed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in Belgium. Whole-genome sequencing (WGS) was used on the confirmed MRSA isolates, to characterize their genomic content and study their relatedness. Amongst the 305 isolates (304 wound swabs and 61 nasal swabs) that were collected from patients and HCWs, we detected 32 and 15 cases of MRSA, respectively. From this collection, 27 high-quality WGS datasets were obtained, which carried numerous genes and mutations associated with antimicrobial resistance. The mecA gene was detected in all the sequenced isolates. These isolates were assigned to five sequence types (STs), with ST8 (55.56%, n = 15/27), ST152 (18.52%, n = 5/27), and ST121 (18.52%, n = 5/27) being the most common. These 27 isolates carried multiple virulence genes, including the genes encoding the Panton-Valentine leukocidin toxin (48.15%, n = 13/27), and the tst gene (29.63%, n = 8/27), associated with toxic shock syndrome. This study highlights the need to implement a multimodal strategy for reducing the risk of the cross-transmission of MRSA in hospitals.

Growth of Gram-Negative Bacteria in Antiseptics, Disinfectants and Hand Hygiene Products in Two Tertiary Care Hospitals in West Africa-A Cross-Sectional Survey.

Antiseptics, disinfectants, and hand hygiene products can act as reservoirs of Gram-negative bacteria causing healthcare-associated infections. This problem is rarely documented in low- and middle-income countries, particularly in sub-Saharan Africa. In a cross-sectional survey, we assessed the bacterial contamination of antiseptics, disinfectants, and hand hygiene products in two university hospitals in Burkina Faso and Benin. During ward visits and staff interviews, in-use products were cultured for the presence of Gram-negative bacteria. The growth of Gram-negative bacteria was absent or rare in alcohol-based products, povidone iodine, and Dakin solution. Contamination was highest (73.9% (51/69)) for liquid soap products (versus antiseptic/disinfectants (4.5%, 7/157) (p < 0.0001)), mostly used in high-risk areas and associated with high total bacterial counts (>10,000 colony-forming units/mL). Contaminating flora (105 isolates) included Enterobacterales and the Vibrio non-cholerae/Aeromonas group (17.1%) and non-fermentative Gram-negative rods (82.8%). Multidrug resistance was present among 9/16 Enterobacterales (Klebsiella and Enterobacter spp.) and 3/12 Acinetobacter spp., including carbapenem resistance (Acinetobacter baumannii: NDM, Pseudomonas stutzeri: VIM). The risk factors for contamination included the type of product (cleaning grade and in-house prepared liquid soap), use of recycled disposable containers and soft drink bottles, absence of labeling, topping-up of containers, dilution with tap water (pharmacy and ward), and poor-quality management (procurement, stock management, expiry dates, and period after opening).

Évaluation des capacités du système de laboratoires biologiques pour la détection des menaces infectieuses au Bénin.

Background : International Health Regulations require countries to establish laboratory systems for rapid and safe confirmation of public health emergencies.Objective : This study assessed the capacity of the National Laboratory System for the detection of infectious threats to global health security in Benin.Method : The study was descriptive, cross-sectional, and evaluative. The targets were laboratories performing the confirmation of infectious threats. The sampling method was non-probabilistic with the reasoned choice of 74 laboratories. Four collection tools were used. The World Health Organization’s laboratory assessment tool for health facilities was used to assess the national public health laboratory. The assessment was based on the capacities of peripheric laboratories and of the national reference laboratory. The capacity was rated good if at least 80% of peripheric laboratories met at least 80% of the criteria and if the national public health laboratory had an average indicator of at least 80%. Otherwise, the capacity was rated insufficient.Results : The national laboratory system capacity was insufficient. Only 54% of peripheric laboratories had good capacity. The national reference laboratory had an average indicator of 71%. In this reference laboratory, specimen collection and transport, laboratory testing performance, consumables and reagents, and public health functions had the best scores, above 80%. Biorisk management, organization and management, and documents had the lowest scores. In peripheric laboratories, the testing performance was the only domain with good capacity.Conclusion : To ensure effective Global Health Security in Benin, a National Laboratory System capacity improvement strategic plan need to be developed and implemented.

GeneXpert or chest-X-ray or tuberculin skin testing for household contact assessment (GXT): protocol for a cluster-randomized trial.

BACKGROUND: The World Health Organization recommends tuberculosis (TB) preventive treatment (TPT) for all people living with HIV (PLH) and household contacts (HHC) of index TB patients. Tests for TB infection (TBI) or to rule out TB disease (TBD) are preferred, but if not available, this should not be a barrier if access to these tests is limited for high-risk people, such as PLH and HHC under 5 years old. There is equipoise on the need for these tests in different risk populations, especially HHC aged over 5. METHODS: This superiority cluster-randomized multicenter trial with three arms of equal size compares, in Benin and Brazil, three strategies for HHC investigation aged 0-50: (i) tuberculin skin testing (TST) or interferon gamma release assay (IGRA) for TBI and if positive, chest X-Ray (CXR) to rule out TBD in persons with positive TST or IGRA; (ii) same as (i) but GeneXpert (GX) replaces CXR; and (iii) no TBI testing. CXR for all; if CXR is normal, TPT is recommended. All strategies start with symptom screening. Clusters are defined as HHC members of the same index patients with newly diagnosed pulmonary TBD. The main outcome is the proportion of HHC that are TPT eligible who start TPT within 3 months of the index TB patient starting TBD treatment. Societal costs, incidence of severe adverse events, and prevalence of TBD are among secondary outcomes. Stratified analyses by age (under versus over 5) and by index patient microbiological status will be conducted. All participants provide signed informed consent. The study was approved by the Research Ethic Board of the Research Institute of the McGill University Health Centre, the Brazilian National Ethical Board CONEP, and the "Comité Local d'Éthique Pour la Recherche Biomédicale (CLERB) de l'Université de Parakou," Benin. Findings will be submitted for publication in major medical journals and presented in conferences, to WHO and National and municipal TB programs of the involved countries. DISCUSSION: This randomized trial is meant to provide high-quality evidence to inform WHO recommendations on investigation of household contacts, as currently these are based on very low-quality evidence. TRIAL REGISTRATION: ClinicalTrials.gov NCT04528823.

Priority Activities in Child and Adolescent Tuberculosis to Close the Policy-Practice Gap in Low- and Middle-Income Countries.

Over the past 15 years, and despite many difficulties, significant progress has been made to advance child and adolescent tuberculosis (TB) care. Despite increasing availability of safe and effective treatment and prevention options, TB remains a global health priority as a major cause of child and adolescent morbidity and mortality-over one and a half million children and adolescents develop TB each year. A history of the global public health perspective on child and adolescent TB is followed by 12 narratives detailing challenges and progress in 19 TB endemic low and middle-income countries. Overarching challenges include: under-detection and under-reporting of child and adolescent TB; poor implementation and reporting of contact investigation and TB preventive treatment services; the need for health systems strengthening to deliver effective, decentralized services; and lack of integration between TB programs and child health services. The COVID-19 pandemic has had a significant negative impact on case detection and treatment outcomes. Child and adolescent TB working groups can address country-specific challenges to close the policy-practice gaps by developing and supporting decentral ized models of care, strengthening clinical and laboratory diagnosis, including of multidrug-resistant TB, providing recommended options for treatment of disease and infection, and forging strong collaborations across relevant health sectors.

Prevalence, acceptability, and cost of routine screening for pulmonary tuberculosis among pregnant women in Cotonou, Benin.

OBJECTIVES: We sought to evaluate the yield, cost, feasibility, and acceptability of routine tuberculosis (TB) screening of pregnant women in Cotonou, Benin. DESIGN: Mixed-methods, cross-sectional study with a cost assessment. SETTING: Eight participating health facilities in Cotonou, Benin. PARTICIPANTS: Consecutive pregnant women presenting for antenatal care at any participating site who were not in labor or currently being treated for TB from April 2017 to April 2018. INTERVENTIONS: Screening for the presence of TB symptoms by midwives and Xpert MTB/RIF for those with cough for at least two weeks. Semi-structured interviews with 14 midwives and 16 pregnant women about experiences with TB screening. PRIMARY AND SECONDARY OUTCOME MEASURES: Proportion of pregnant women with cough of at least two weeks and/or microbiologically confirmed TB. The cost per pregnant woman screened and per TB case diagnosed in 2019 USD from the health system perspective. RESULTS: Out of 4,070 pregnant women enrolled in the study, 94 (2.3%) had a cough for at least two weeks at the time of screening. The average (standard deviation) age of symptomatic women was 26 ± 5 years and 5 (5.3%) had HIV. Among the 94 symptomatic women, 2 (2.3%) had microbiologically confirmed TB for a TB prevalence of 49 per 100,000 (95% CI: 6 to 177 per 100,000) among pregnant women enrolled in the study. The average cost to screen one pregnant woman for TB was $1.12 USD and the cost per TB case diagnosed was $2271 USD. Thematic analysis suggested knowledge of TB complications in pregnancy was low, but that routine TB screening was acceptable to both midwives and pregnant women. CONCLUSION: Enhanced screening for TB among pregnant women is feasible, acceptable, and inexpensive per woman screened, however in this setting has suboptimal yield even if it can contribute to enhance TB case finding.

Multidrug-resistant tuberculosis control in Rwanda overcomes a successful clone that causes most disease over a quarter century.

SUMMARY BACKGROUND: Multidrug-resistant (MDR) tuberculosis (TB) poses an important challenge in TB management and control. Rifampicin resistance (RR) is a solid surrogate marker of MDR-TB. We investigated the RR-TB clustering rates, bacterial population dynamics to infer transmission dynamics, and the impact of changes to patient management on these dynamics over 27 years in Rwanda. METHODS: We analysed whole genome sequences of a longitudinal collection of nationwide RR-TB isolates. The collection covered three important periods: before programmatic management of MDR-TB (PMDT; 1991-2005), the early PMDT phase (2006-2013), in which rifampicin drug-susceptibility testing (DST) was offered to retreatment patients only, and the consolidated phase (2014-2018), in which all bacteriologically confirmed TB patients had rifampicin DST done mostly via Xpert MTB/RIF assay. We constructed clusters based on a 5 SNP cut-off and resistance conferring SNPs. We used Bayesian modelling for dating and population size estimations, TransPhylo to estimate the number of secondary cases infected by each patient, and multivariable logistic regression to assess predictors of being infected by the dominant clone. RESULTS: Of 308 baseline RR-TB isolates considered for transmission analysis, the clustering analysis grouped 259 (84.1%) isolates into 13 clusters. Within these clusters, a single dominant clone was discovered containing 213 isolates (82.2% of clustered and 69.1% of all RR-TB), which we named the "Rwanda Rifampicin-Resistant clone" (R3clone). R3clone isolates belonged to Ugandan sub-lineage 4.6.1.2 and its rifampicin and isoniazid resistance were conferred by the Ser450Leu mutation in rpoB and Ser315Thr in katG genes, respectively. All R3clone isolates had Pro481Thr, a putative compensatory mutation in the rpoC gene that likely restored its fitness. The R3clone was estimated to first arise in 1987 and its population size increased exponentially through the 1990s', reaching maximum size (∼84%) in early 2000 s', with a declining trend since 2014. Indeed, the highest proportion of R3clone (129/157; 82·2%, 95%CI: 75·3-87·8%) occurred between 2000 and 13, declining to 64·4% (95%CI: 55·1-73·0%) from 2014 onward. We showed that patients with R3clone detected after an unsuccessful category 2 treatment were more likely to generate secondary cases than patients with R3clone detected after an unsuccessful category 1 treatment regimen. CONCLUSIONS: RR-TB in Rwanda is largely transmitted. Xpert MTB/RIF assay as first diagnostic test avoids unnecessary rounds of rifampicin-based TB treatment, thus preventing ongoing transmission of the dominant R3clone. As PMDT was intensified and all TB patients accessed rifampicin-resistance testing, the nationwide R3clone burden declined. To our knowledge, our findings provide the first evidence supporting the impact of universal DST on the transmission of RR-TB.

Blood culture surveillance in a secondary care hospital in Benin: epidemiology of bloodstream infection pathogens and antimicrobial resistance.

BACKGROUND: Although global surveillance of antimicrobial resistance (AMR) is considered key in the containment of AMR, data from low- and middle-income countries, especially from sub-Saharan Africa, are scarce. This study describes epidemiology of bloodstream infections and antimicrobial resistance rates in a secondary care hospital in Benin. METHODS: Blood cultures were sampled, according to predefined indications, in BacT/ALERT FA Plus and PF Plus (bioMérieux, Marcy-l'Etoile, France) blood culture bottles (BCB) in a district hospital (Boko hospital) and to a lesser extent in the University hospital of Parakou. These BCB were incubated for 7 days in a standard incubator and twice daily inspected for visual signs of growth. Isolates retrieved from the BCB were processed locally and later shipped to Belgium for reference identification [matrix-assisted laser desorption/ionization time-of-flight spectrometry (MALDI-TOF)] and antibiotic susceptibility testing (disk diffusion and E-tests). RESULTS: From October 2017 to February 2020, 3353 BCB were sampled, corresponding to 3140 blood cultures (212 cultures consisting of  > 1 BCB) and 3082 suspected bloodstream infection (BSI) episodes. Most of these cultures (n = 2471; 78.7%) were sampled in children < 15 years of age. Pathogens were recovered from 383 (12.4%) cultures, corresponding to 381 confirmed BSI. 340 of these pathogens were available and confirmed by reference identification. The most common pathogens were Klebsiella pneumoniae (n = 53; 15.6%), Salmonella Typhi (n = 52; 15.3%) and Staphylococcus aureus (n = 46; 13.5%). AMR rates were high among Enterobacterales, with resistance to third-generation cephalosporins in 77.6% of K. pneumoniae isolates (n = 58), 12.8% of Escherichia coli isolates (n = 49) and 70.5% of Enterobacter cloacae isolates (n = 44). Carbapenemase production was detected in 2 Escherichia coli and 2 Enterobacter cloacae isolates, all of which were of the New Delhi metallo-beta lactamase type. Methicillin resistance was present in 22.4% of S. aureus isolates (n = 49). CONCLUSION: Blood cultures were successfully implemented in a district hospital in Benin, especially among the pediatric patient population. Unexpectedly high rates of AMR among Gram-negative bacteria against commonly used antibiotics were found, demonstrating the clinical and scientific importance of clinical bacteriology laboratories at this level of care.

Tuberculosis screening among ambulatory people living with HIV: a systematic review and individual participant data meta-analysis.

BACKGROUND: The WHO-recommended tuberculosis screening and diagnostic algorithm in ambulatory people living with HIV is a four-symptom screen (known as the WHO-recommended four symptom screen [W4SS]) followed by a WHO-recommended molecular rapid diagnostic test (eg Xpert MTB/RIF [hereafter referred to as Xpert]) if W4SS is positive. To inform updated WHO guidelines, we aimed to assess the diagnostic accuracy of alternative screening tests and strategies for tuberculosis in this population. METHODS: In this systematic review and individual participant data meta-analysis, we updated a search of PubMed (MEDLINE), Embase, the Cochrane Library, and conference abstracts for publications from Jan 1, 2011, to March 12, 2018, done in a previous systematic review to include the period up to Aug 2, 2019. We screened the reference lists of identified pieces and contacted experts in the field. We included prospective cross-sectional, observational studies and randomised trials among adult and adolescent (age ≥10 years) ambulatory people living with HIV, irrespective of signs and symptoms of tuberculosis. We extracted study-level data using a standardised data extraction form, and we requested individual participant data from study authors. We aimed to compare the W4SS with alternative screening tests and strategies and the WHO-recommended algorithm (ie, W4SS followed by Xpert) with Xpert for all in terms of diagnostic accuracy (sensitivity and specificity), overall and in key subgroups (eg, by antiretroviral therapy [ART] status). The reference standard was culture. This study is registered with PROSPERO, CRD42020155895. FINDINGS: We identified 25 studies, and obtained data from 22 studies (including 15 666 participants; 4347 [27·7%] of 15 663 participants with data were on ART). W4SS sensitivity was 82% (95% CI 72-89) and specificity was 42% (29-57). C-reactive protein (≥10 mg/L) had similar sensitivity to (77% [61-88]), but higher specificity (74% [61-83]; n=3571) than, W4SS. Cough (lasting ≥2 weeks), haemoglobin (<10 g/dL), body-mass index (<18·5 kg/m2), and lymphadenopathy had high specificities (80-90%) but low sensitivities (29-43%). The WHO-recommended algorithm had a sensitivity of 58% (50-66) and a specificity of 99% (98-100); Xpert for all had a sensitivity of 68% (57-76) and a specificity of 99% (98-99). In the one study that assessed both, the sensitivity of sputum Xpert Ultra was higher than sputum Xpert (73% [62-81] vs 57% [47-67]) and specificities were similar (98% [96-98] vs 99% [98-100]). Among outpatients on ART (4309 [99·1%] of 4347 people on ART), W4SS sensitivity was 53% (35-71) and specificity was 71% (51-85). In this population, a parallel strategy (two tests done at the same time) of W4SS with any chest x-ray abnormality had higher sensitivity (89% [70-97]) and lower specificity (33% [17-54]; n=2670) than W4SS alone; at a tuberculosis prevalence of 5%, this strategy would require 379 more rapid diagnostic tests per 1000 people living with HIV than W4SS but detect 18 more tuberculosis cases. Among outpatients not on ART (11 160 [71·8%] of 15 541 outpatients), W4SS sensitivity was 85% (76-91) and specificity was 37% (25-51). C-reactive protein (≥10 mg/L) alone had a similar sensitivity to (83% [79-86]), but higher specificity (67% [60-73]; n=3187) than, W4SS and a sequential strategy (both test positive) of W4SS then C-reactive protein (≥5 mg/L) had a similar sensitivity to (84% [75-90]), but higher specificity than (64% [57-71]; n=3187), W4SS alone; at 10% tuberculosis prevalence, these strategies would require 272 and 244 fewer rapid diagnostic tests per 1000 people living with HIV than W4SS but miss two and one more tuberculosis cases, respectively. INTERPRETATION: C-reactive protein reduces the need for further rapid diagnostic tests without compromising sensitivity and has been included in the updated WHO tuberculosis screening guidelines. However, C-reactive protein data were scarce for outpatients on ART, necessitating future research regarding the utility of C-reactive protein in this group. Chest x-ray can be useful in outpatients on ART when combined with W4SS. The WHO-recommended algorithm has suboptimal sensitivity; Xpert for all offers slight sensitivity gains and would have major resource implications. FUNDING: World Health Organization.

Whole-Genome Sequencing-Based Antimicrobial Resistance Characterization and Phylogenomic Investigation of 19 Multidrug-Resistant and Extended-Spectrum Beta-Lactamase-Positive Escherichia coli Strains Collected From Hospital Patients in Benin in 2019.

The increasing worldwide prevalence of extended-spectrum beta-lactamase (ESBL) producing Escherichia coli constitutes a serious threat to global public health. Surgical site infections are associated with high morbidity and mortality rates in developing countries, fueled by the limited availability of effective antibiotics. We used whole-genome sequencing (WGS) to evaluate antimicrobial resistance and the phylogenomic relationships of 19 ESBL-positive E. coli isolates collected from surgical site infections in patients across public hospitals in Benin in 2019. Isolates were identified by MALDI-TOF mass spectrometry and phenotypically tested for susceptibility to 16 antibiotics. Core-genome multi-locus sequence typing and single-nucleotide polymorphism-based phylogenomic methods were used to investigate the relatedness between samples. The broader phylogenetic context was characterized through the inclusion of publicly available genome data. Among the 19 isolates, 13 different sequence types (STs) were observed, including ST131 (n = 2), ST38 (n = 2), ST410 (n = 2), ST405 (n = 2), ST617 (n = 2), and ST1193 (n = 2). The bla CTX-M-15 gene encoding ESBL resistance was found in 15 isolates (78.9%), as well as other genes associated with ESBL, such as bla OXA-1 (n = 14) and bla TEM-1 (n = 9). Additionally, we frequently observed genes encoding resistance against aminoglycosides [aac-(6')-Ib-cr, n = 14], quinolones (qnrS1 , n = 4), tetracyclines [tet(B), n = 14], sulfonamides (sul2, n = 14), and trimethoprim (dfrA17, n = 13). Nonsynonymous chromosomal mutations in the housekeeping genes parC and gyrA associated with resistance to fluoroquinolones were also detected in multiple isolates. Although the phylogenomic investigation did not reveal evidence of hospital-acquired transmissions, we observed two very similar strains collected from patients in different hospitals. By characterizing a set of multidrug-resistant isolates collected from a largely unexplored environment, this study highlights the added value for WGS as an effective early warning system for emerging pathogens and antimicrobial resistance.

Tuberculosis prevalence and associated factors among persons with diabetes mellitus after intensified case finding in three West African countries.

BACKGROUND: To determine the prevalence of tuberculosis (TB) and associated factors in persons with diabetes mellitus (DM) in Benin, Guinea and Senegal. PATIENTS AND METHOD: A cross-sectional study was conducted in the largest DM center in each country. Participants systematically underwent clinical screening and chest radiography. Participants who were symptomatic or with abnormal radiography underwent bacteriological investigations (sputum smear, Xpert MTB/RIF and culture) on sputum. Participants with no TB at enrolment were re-examined for TB six months later. Logistic regression was performed to identify factors associated with TB. RESULTS: There were 5,870 DM patients: 1,881 (32.0%) in Benin, 1,912 (32.6%) in Guinea and 2,077 (35.4%) in Senegal. Out of these, 114 had bacteriologically-confirmed TB, giving a pooled prevalence of 1.9% (95%CI=1.6-2.3). TB prevalence was 0.5% (95%CI=0.3-1.0), 2.4% (95%CI=1.8-3.2) and 2.8% (95%CI=2.2-3.6), respectively, in Benin, Guinea and Senegal. Factors associated with an increased odds of TB diagnosis were a usual residence in Guinea (aOR=2.62;95%CI=1.19-5.77; p=0.016) or in Senegal (aOR=3.73;95%CI=1.85-7.51; p<0.001), the age group of 35-49 years (aOR=2.30;95%CI=1.11-4.79; p=0.025), underweight (aOR=7.34;95%CI=4.65-11.57; p<0.001) and close contact with a TB case (aOR=2.27;95%CI=1.37-3.76; p=0.002). Obesity was associated with lower odds of TB (aOR=0.20; 95%CI=0.06-0.65; p=0.008). CONCLUSION: TB is prevalent among DM patients in Benin, Guinea and Senegal and higher than among the general population. The findings support the need for intensified case finding in DM patients in order to ensure systematic early detection of TB during the routine consultation process.

Spot specimen testing with GeneXpert MTB/RIF results compared to morning specimen in a programmatic setting in Cotonou, Benin.

BACKGROUND: The diagnosis of tuberculosis (TB) using smear microscopy has been based on testing two specimens: one spot and one early morning sputa. Recently, the World Health Organization (WHO) has recommended to replace, whenever possible, microscopy with GeneXpert® MTB/RIF performed on a single specimen. However, as the bacterial load is higher in early morning specimens than in spot specimens, one could expect lower sensitivity of GeneXpert® MTB/RIF performed only on spot specimens. In this study, we compared results of GeneXpert® MTB/RIF on spot specimens versus early morning specimens, under programmatic conditions in Cotonou, Benin. METHODS: From June to September 2018, all sputa received from presumptive TB patients at the Supranational Reference Laboratory for Tuberculosis of Cotonou were included in the study. From each patient, two specimens were collected (one spot and one early morning) and GeneXpert® MTB/RIF was performed on both specimens. RESULTS: In total, 886 participants were included in the study, of whom 737 provided both sputa and 149 (16.8%) gave only the spot specimen. For the 737 participants who provided both sputa, GeneXpert® MTB/RIF was positive for both specimens in 152 participants; for three participants GeneXpert® MTB/RIF was positive on spot specimen but negative on morning specimen while for another three, the test was positive on morning specimen but negative on spot specimen. The overall percentage of agreement was excellent (99.2%) with a positive and negative percent agreement greater than 98%. CONCLUSION: For TB diagnosis under programmatic conditions in Cotonou, GeneXpert® MTB/RIF in spot specimens gave similar results with the test in morning specimens. Performing GeneXpert® MTB/RIF in both specimens did not significantly increase the number of cases detected. To avoid losing patients from the diagnostic cascade, it is preferable to test sputa produced at the time of the first visit at the health center.

Assessment of the Biosafety and Biosecurity in the Reference Veterinary Laboratory of Parakou in Benin.

Optimal biosafety and biosecurity are major requirements of global health security. This study assessed the biorisk management in the reference veterinary laboratory of Parakou (Benin). The study was cross-sectional, descriptive, and evaluative. The non-probability sampling method with the reasoned choice was used. The Food and Agriculture Organization laboratory mapping tool-safety was used to collect information from the laboratory team. Group discussion, working environment observation, and document exploitation were the data collection techniques. The biorisk management was rated good if the average indicator of the laboratory reached at least 80%. Otherwise, the biorisk management was rated insufficient. The overall laboratory biosafety and biosecurity score was insufficient (42.4%). Per area, the scores were 26.7% for engineering, 33.3% for administration, 53.8% for personal protective equipment, and 62.3% for the operational. There was no area or category score that reached 80%. Containment, waste disposal, and personal protective equipment disposal were the best performing categories with a score above 60%. The laboratory has no biosafety and accident prevention program. Its premises require renovation. The standard operating procedures for biosafety are not yet finalized, and the training mechanism is not optimal. Therefore, strong advocacy and implementation of a biorisk management improvement plan appear as urgent corrective actions which are required to help the reference veterinary laboratory of Parakou in its task to protect the livestock and, ultimately, the people of Benin from dangerous diseases and emerging pathogens.

Mycobacterium tuberculosis complex lineage 5 exhibits high levels of within-lineage genomic diversity and differing gene content compared to the type strain H37Rv.

Pathogens of the Mycobacterium tuberculosis complex (MTBC) are considered to be monomorphic, with little gene content variation between strains. Nevertheless, several genotypic and phenotypic factors separate strains of the different MTBC lineages (L), especially L5 and L6 (traditionally termed Mycobacterium africanum) strains, from each other. However, this genome variability and gene content, especially of L5 strains, has not been fully explored and may be important for pathobiology and current approaches for genomic analysis of MTBC strains, including transmission studies. By comparing the genomes of 355 L5 clinical strains (including 3 complete genomes and 352 Illumina whole-genome sequenced isolates) to each other and to H37Rv, we identified multiple genes that were differentially present or absent between H37Rv and L5 strains. Additionally, considerable gene content variability was found across L5 strains, including a split in the L5.3 sub-lineage into L5.3.1 and L5.3.2. These gene content differences had a small knock-on effect on transmission cluster estimation, with clustering rates influenced by the selected reference genome, and with potential overestimation of recent transmission when using H37Rv as the reference genome. We conclude that full capture of the gene diversity, especially high-resolution outbreak analysis, requires a variation of the single H37Rv-centric reference genome mapping approach currently used in most whole-genome sequencing data analysis pipelines. Moreover, the high within-lineage gene content variability suggests that the pan-genome of M. tuberculosis is at least several kilobases larger than previously thought, implying that a concatenated or reference-free genome assembly (de novo) approach may be needed for particular questions.

Mycobacterium tuberculosis precursor rRNA as a measure of treatment-shortening activity of drugs and regimens.

There is urgent need for new drug regimens that more rapidly cure tuberculosis (TB). Existing TB drugs and regimens vary in treatment-shortening activity, but the molecular basis of these differences is unclear, and no existing assay directly quantifies the ability of a drug or regimen to shorten treatment. Here, we show that drugs historically classified as sterilizing and non-sterilizing have distinct impacts on a fundamental aspect of Mycobacterium tuberculosis physiology: ribosomal RNA (rRNA) synthesis. In culture, in mice, and in human studies, measurement of precursor rRNA reveals that sterilizing drugs and highly effective drug regimens profoundly suppress M. tuberculosis rRNA synthesis, whereas non-sterilizing drugs and weaker regimens do not. The rRNA synthesis ratio provides a readout of drug effect that is orthogonal to traditional measures of bacterial burden. We propose that this metric of drug activity may accelerate the development of shorter TB regimens.

Culture media for clinical bacteriology in low- and middle-income countries: challenges, best practices for preparation and recommendations for improved access.

BACKGROUND: Culture media are fundamental in clinical microbiology. In laboratories in low- and middle-income countries (LMICs), they are mostly prepared in-house, which is challenging. OBJECTIVES: This narrative review describes challenges related to culture media in LMICs, compiles best practices for in-house media preparation, gives recommendations to improve access to quality-assured culture media products in LMICs and formulates outstanding questions for further research. SOURCES: Scientific literature was searched using PubMed and predefined MeSH terms. In addition, grey literature was screened, including manufacturer's websites and manuals as well as microbiology textbooks. CONTENT: Bacteriology laboratories in LMICs often face challenges at multiple levels: lack of clean water and uninterrupted power supply, high environmental temperatures and humidity, dust, inexperienced and poorly trained staff, and a variable supply of consumables (often of poor quality). To deal with this at a base level, one should be very careful in selecting culture media. It is recommended to look for products supported by the national reference laboratory that are being distributed by an in-country supplier. Correct storage is key, as is appropriate preparation and waste management. Centralized media acquisition has been advocated for LMICs, a role that can be taken up by the national reference laboratories, next to guidance and support of the local laboratories. In addition, there is an important role in tropicalization and customization of culture media formulations for private in vitro diagnostic manufacturers, who are often still unfamiliar with the LMIC market and the plethora of bacteriology products. IMPLICATION: The present narrative review will assist clinical microbiology laboratories in LMICs to establish best practices for handling culture media by defining quality, regulatory and research paths.