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.

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.

Prevalence and drivers of false-positive rifampicin-resistant Xpert MTB/RIF results: a prospective observational study in Rwanda.

BACKGROUND: The Xpert MTB/RIF (Xpert) assay is used globally to rapidly diagnose tuberculosis and resistance to rifampicin. We investigated the frequency and predictors of false-positive findings of rifampicin resistance with Xpert. METHODS: We did a prospective, observational study of individuals who were enrolled in a Rwandan nationwide diagnostic cohort study (DIAMA trial; NCT03303963). We included patients identified to have rifampicin resistance on initial Xpert testing. We did a repeat Xpert assay and used rpoB Sanger and deep sequencing alongside phenotypic drug susceptibility testing (pDST) to ascertain final rifampicin susceptibility status, with any (hetero)resistant result overriding. We used multivariable logistic regression to assess predictors of false rifampicin resistance on initial Xpert testing, adjusted for HIV status, tuberculosis treatment history, initial Xpert semi-quantitative bacillary load, and initial Xpert probe. FINDINGS: Between May 4, 2017, and April 30, 2019, 175 people were identified with rifampicin resistance at initial Xpert testing, of whom 154 (88%) underwent repeat Xpert assay. 54 (35%) patients were confirmed as rifampicin resistant on repeat testing and 100 (65%) were not confirmed with resistance. After further testing and sequencing, 121 (79%) of 154 patients had a final confirmed status for rifampicin susceptibility. 57 (47%) of 121 patients were confirmed to have a false rifampicin resistance result and 64 (53%) had true rifampicin resistance. A high pretest probability of rifampicin resistance did not decrease the odds of false rifampicin resistance (adjusted odds ratio [aOR] 6·0, 95% CI 1·0-35·0, for new tuberculosis patients vs patients who needed retreatment). Ten (16%) of the 64 patients with true rifampicin resistance did not have confirmed rifampicin resistance on repeat Xpert testing, of whom four had heteroresistance. Of 63 patients with a very low bacillary load on Xpert testing, 54 (86%) were falsely diagnosed with rifampicin-resistant tuberculosis. Having a very low bacillary load on Xpert testing was strongly associated with false rifampicin resistance at the initial Xpert assay (aOR 63·6, 95% CI 9·9-410·4). INTERPRETATION: The Xpert testing algorithm should include an assessment of bacillary load and retesting in case rifampicin resistance is detected on a paucibacillary sputum sample. Only when rifampicin resistance has been confirmed on repeat testing should multidrug-resistant tuberculosis treatment be started. When rifampicin resistance has not been confirmed on repeat testing, we propose that patients should be given first-line anti-tuberculosis drugs and monitored closely during treatment, including by baseline culture, pDST, and further Xpert testing. FUNDING: The European & Developing Countries Clinical Trials Partnership 2 programme, and Belgian Directorate General for Development Cooperation.

Performance of OMNIgene•SPUTUM (DNA Genotek) and cetylpyridinium chloride for sputum storage prior to mycobacterial culture.

Purpose. The aim was to assess the performance of both cetylpyridinium chloride (CPC) and OMNIgene•SPUTUM (OMNI) reagents for the maintenance of Mycobacterium tuberculosis viability in sputum prior to recovery by culture.Methodology. Using 312 sputa, we evaluated the performance of the two reagents using culture on Löwenstein-Jensen medium after sputum storage in CPC or OMNI for up to 28 days. In addition, the viability of M. tuberculosis isolates stored in both reagents was assessed.Results. The contamination rates for freshly processed samples and those stored in CPC were not statistically different, while the contamination rate for OMNI was significantly lower than that for fresh sputa (P=0.026 for 8 days and P=0.002 for 28 days of storage). The culture positivity for fresh sputa (81.7 %) was similar to that for samples stored in CPC, regardless of the storage time (89.8 % for CPC-8 and 73.0 % for CPC-28). For OMNI-preserved samples, the culture positivity was similar after 8 days of storage (84.2 %), but decreased significantly after 28 days (42.7 %; P<0.0001) compared to fresh sputa, CPC-8, CPC-28 and OMNI-8. There was a significant loss of viability for the H37Rv strain when it was stored in OMNI at room temperature beyond 8 days compared to CPC, but storage at 37 °C decreased recovery from both CPC- and OMNI-stored suspensions.Conclusion. Culture from sputum stored for 8 days at room temperature in OMNI or CPC gave comparable culture positivity rates to culture from fresh sputum, but after 28 days of storage the performance of OMNI decreased significantly compared to CPC.

First Insight into a Nationwide Genotypic Diversity of Mycobacterium tuberculosis among Previously Treated Pulmonary Tuberculosis Cases in Benin, West Africa.

BACKGROUND: Molecular studies on tuberculosis (TB) are rare in low-resource countries like Benin, where data on molecular study on previously treated TB cases is unavailable. MATERIALS AND METHODS: From January to December 2014, all smear- and culture-positive previously treated pulmonary TB patients from all TB clinics were systematically recruited. Drug susceptibility testing and spoligotyping were performed on all isolates. RESULTS: Of the 100 patients recruited, 71 (71.0%) were relapse cases and 24 (24.0%) were failure cases, while 5 (5.0%) were default cases. Resistance rate to any first-line drug was 40.0%, while 12.0% of strains were multidrug-resistant (MDR) and no strain was extensively drug-resistant (XDR). A total of 40 distinct spoligotypes were found to be corresponding to a genotypic diversity of 40.0%. ST61 was the most predominant spoligotype with prevalence of 33.0%. In all, 31 single spoligotypes and nine clusters were observed with 2 to 33 strains per cluster giving a clustering rate of 69.0%. Euro-American (Lineage 4) was the most prevalent lineage (74.0%) and Lineage 2 was associated with resistance to streptomycin. CONCLUSION: This first insight into genetic diversity of previously treated pulmonary TB patients in Benin showed a relatively high genetic diversity of Mycobacterium tuberculosis.

Rapid detection of extended-spectrum-ß-lactamase-producing Enterobacteriaceae in blood cultures using the ESBL NDP test in Cotonou, Benin.

PURPOSE: Rapid and inexpensive tests for detecting extended-spectrum-ß-lactamase (ESBL)-producing Enterobacteriaceae are needed, particularly in low-resource countries where infections with these bacteria constitute a major public health issue. The recently described ESBL NDP test performed well in developed countries. This study was designed to assess performance, cost and feasibility of this test in positive blood cultures, in Cotonou, Benin (West Africa). METHODOLOGY: The test was performed on 175 positive Bactec broth blood cultures containing Enterobacteriaceae, and blindly compared with the double-disc synergy test (DDST) for the phenotypic detection of ESBL producers. RESULTS: There was a complete agreement between the ESBL NDP test and the DDST. On average, the time to give results was 37 min for a sample and the cost was US$ 7.3. CONCLUSION: The ESBL NDP test is rapid, relatively affordable and performed well in our setting.