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.

Follow-up and tracing of tuberculosis patients who fail to attend their scheduled appointments in Cotonou, Benin: a retrospective cohort study.

BACKGROUND: In the "Centre National Hospitalier de Pneumo-Phtisiologie" of Cotonou, Benin, little is known about the characteristics of patients who have not attended their scheduled appointment, the results of tracing and the possible benefits on improving treatment outcomes. This study aimed to determine the contribution of tracing activities for those who missed scheduled appointments towards a successful treatment outcome. METHODS: A retrospective cohort study was carried out among all smear-positive pulmonary tuberculosis patients treated between January and September 2013. Data on demographic and diagnostic characteristics and treatment outcomes were accessed from tuberculosis registers and treatment cards. Information on those who missed their scheduled appointments was collected from the tracing tuberculosis register. A univariate analysis was performed to explore factors associated with missing a scheduled appointment. RESULTS: Of 457 patients (410 new smear-positive and 47 retreatment tuberculosis), 37 (8%) missed one or more of their appointments with a total of 44 episodes of missed appointments. The 3.5th (32%) and 5th (43%) month appointments were the ones most likely to be missed. Being male was associated with a higher risk of missing appointments (RR = 4.2; 95% CI = 1.5-11.8, p = 0.004) while having HIV infection was associated with a lower risk (RR = 0.3, 95% CI = 0.1-0.9, p = 0.03). Principal reasons for missed appointments were travelling outside Cotonou (34%) and feeling better (21%). In 24 (55%) of these 44 episodes of missed appointments, contact was made with the patient who returned to the programme. These follow-up activities increased the treatment success by 4%. CONCLUSION: In Cotonou, Benin, less than 10% of tuberculosis patients miss at least one of their scheduled appointments. Tracing activities increase the treatment success rate by 4% and current on-going practices in the Programme need to be endorsed and encouraged.

Assessing the economic burden of illness for tuberculosis patients in Benin: determinants and consequences of catastrophic health expenditures and inequities.

OBJECTIVES: To inform policy-making, we measured the risk, causes and consequences of catastrophic expenditures for tuberculosis and investigated potential inequities. METHODS: Between August 2008 and February 2009, a cross-sectional study was conducted among all (245) smear-positive pulmonary tuberculosis patients of six health districts from southern Benin. A standardised survey questionnaire covered the period of time elapsing from onset of tuberculosis symptoms to completion of treatment. Total direct cost exceeding the conventional 10% threshold of annual income was defined as catastrophic and used as principal outcome in a multivariable logistic regression. A sensitivity analysis was performed while varying the thresholds. RESULTS: A pure gradient of direct costs of tuberculosis in relation to income was observed. Incidence (78.1%) and intensity (14.8%) of catastrophic expenditure were high; varying thresholds was insensitive to the intensity. Incurring catastrophic expenditure was independently associated with lower- and middle-income quintiles (adjusted odd ratio (aOR) = 36.2, 95% CI [12.3-106.3] and aOR = 6.4 [2.8-14.6]), adverse pre-diagnosis stage (aOR = 5.4 [2.2-13.3]) and less education (aOR = 4.1[1.9-8.7]). Households incurred important days lost due to TB, indebtedness (37.1%), dissaving (51.0%) and other coping strategies (52.7%). CONCLUSIONS: Catastrophic direct costs and substantial indirect and coping costs may persist under the 'free' tuberculosis diagnosis and treatment strategy, as well as inequities in financial hardship.

Assessment of HIV-related risky behaviour: a comparative study of face-to-face interviews and polling booth surveys in the general population of Cotonou, Benin.

OBJECTIVES: During the 2008 HIV prevalence survey carried out in the general population of Cotonou, Benin, face-to-face interviews (FTFI) were used to assess risky behaviours for HIV and other sexually transmitted infections (STI). We compared sexual behaviours reported in FTFI with those reported in polling booth surveys (PBS) carried out in parallel in an independent random sample of the same population. METHODS: In PBS, respondents grouped by gender and marital status answered simple questions by putting tokens with question numbers in a green box (affirmative answers) or a red box (negative answers). Both boxes were placed inside a private booth. For each group and question, data were gathered together by type of answer. The structured and gender-specific FTFI guided by trained interviewers included all questions asked during PBS. Pearson χ2 or Fisher's exact test was used to compare FTFI and PBS according to affirmative answers. RESULTS: Overall, respondents reported more stigmatised behaviours in PBS than in FTFI: the proportions of married women and men who reported ever having had commercial sex were 17.4% and 41.6% in PBS versus 1.8% and 19.6% in FTFI, respectively. The corresponding proportions among unmarried women and men were 16.1% and 25.5% in PBS versus 3.9% and 13.0% in FTFI, respectively. The proportion of married women who reported having had extramarital sex since marriage was 23.6% in PBS versus 4.6% in FTFI. CONCLUSIONS: PBS are suitable to monitor reliable HIV/STI risk behaviours. Their use should be expanded in behavioural surveillance.

Effects of decontamination, DNA extraction, and amplification procedures on the molecular diagnosis of Mycobacterium ulcerans disease (Buruli ulcer).

We compared two DNA extraction methods (a semiautomated method using a Maxwell kit and a modified Boom method) and three amplification procedures (a single-step PCR, a nested PCR, and a real-time quantitative PCR) on 74 surgical tissue specimens from patients with clinically suspected Buruli ulcer. All of these procedures were compared before and after decontamination. We observed that, among the procedures tested, real-time PCR after the modified Boom extraction method or a single-run PCR assay after the Maxwell 16 extraction method, performed on nondecontaminated suspensions, are the best for the molecular diagnosis of Mycobacterium ulcerans disease.

Antibiotic Usage in Patients Having Undergone Caesarean Section: A Three-Level Study in Benin.

The intense use and misuse of antibiotics is undoubtedly the main factor associated with the high numbers of antibiotic-resistant pathogenic and commensal bacteria worldwide. In low-income countries, this misuse and overuse is widespread, with great consequences at the personal and global levels. In the context of user fee exemptions in caesarean sections, we performed a descriptive study in women to assess the use of antibiotics on three levels-antenatal, during caesarean section, and postpartum-in four Beninese hospitals. Out of the 141 women included, 56.7% were using antibiotics. More than the half (71.3%) were taking more than one antibiotic, either for a long time or in acute treatment. In prophylaxis, the timing, dose, and duration of administration were not correctly achieved. Only 31.2% of women received optimal antibiotic prophylaxis. Various antibiotics including broad-spectrum molecules were used in the patients after caesarean section. The use of antibiotics was improper on the three levels studied. The high rate of self-administered antibiotics, the poor achievement of antibiotic prophylaxis, and the postpartum overuse of antibiotics showed a poor quality of care provided in pregnancy. A national policy is essential to improve the use of antibiotics by the general public as well as by professionals.

Healthcare Professionals' Knowledge and Beliefs on Antibiotic Prophylaxis in Cesarean Section: A Mixed-Methods Study in Benin.

A low adherence to recommendations on antibiotic prophylaxis has been reported worldwide. Since 2009, cesarean sections have been performed under user fee exemption in Benin with a free kit containing the required supplies and antibiotics for prophylaxis. Despite the kit, the level of antibiotic prophylaxis achievement remains low. We conducted a convergent parallel design study in 2017 using a self-administered questionnaire and interviews to assess the knowledge and explore the beliefs of healthcare professionals regarding antibiotic prophylaxis in three hospitals. Of the 35 participants, 33 filled out the questionnaire. Based on the five conventional criteria of antibiotic prophylaxis, the mean level of knowledge was 3.3 out of 5, and only 15.2% scored 5 out of 5. From the verbatim of 19 interviewees, determinants such as suboptimal patient status health, low confidence in antibiotics, some disagreement with the policy, inappropriate infrastructures and limited financial resources in hospitals, poor management of the policy in the central level, and patient refusal to buy antibiotics can explain poor practices. Because of the dysfunction at these levels, the patient becomes the major determinant of adequate antibiotic prophylaxis. Policymakers have to consider these determinants for improving antibiotic prophylaxis in a way that ensures patient safety and reduces the incidence of antimicrobial resistance.

Buruli ulcer: a review of in vitro tests to screen natural products for activity against Mycobacterium ulcerans.

Buruli ulcer (BU), caused by Mycobacterium ulcerans, has recently been recognized by the World Health Organization (WHO) as an important emerging disease. It is largely a problem of the poor in remote rural areas and has emerged as an important cause of human suffering. While antimycobacterial therapy is often effective for the earliest nodular or ulcerative lesions, for advanced ulcerated lesions, surgery is sometimes necessary. Antimycobacterial drugs may also prevent relapses or disseminated infections. Efficient alternatives different from surgery are presently explored because this treatment deals with huge restrictive factors such as the necessity of prolonged hospitalization, its high cost, and the scars after surgery. Traditional treatment remains the first option for poor populations of remote areas who may have problems of accessibility to synthetic products because of their high cost. The search for efficient natural products active on M. ulcerans should then be encouraged because they are part of the natural heritage of these populations; they are affordable financially and can be used at the earliest stage. This review provides a number of tests that will help to evaluate the antimycobacterial activity of natural products against M. ulcerans, which are adapted to its slow growing rate, and lists active extracts published up to now in Medline.

Effectiveness and cost-effectiveness of a health systems intervention for latent tuberculosis infection management (ACT4): a cluster-randomised trial.

BACKGROUND: Reaching the UN General Assembly High-Level Meeting on Tuberculosis target of providing tuberculosis preventive treatment to at least 30 million people by 2022, including 4 million children under the age of 5 years and 20 million other household contacts, will require major efforts to strengthen health systems. The aim of this study was to evaluate the effectiveness and cost-effectiveness of a health systems intervention to strengthen management for latent tuberculosis infection (LTBI) in household contacts of confirmed tuberculosis cases. METHODS: ACT4 was a cluster-randomised, open-label trial involving 24 health facilities in Benin, Canada, Ghana, Indonesia, and Vietnam randomly assigned to either a three-phase intervention (LTBI programme evaluation, local decision making, and strengthening activities) or control (standard LTBI care). Tuberculin and isoniazid were provided to control and intervention sites if not routinely available. Randomisation was stratified by country and restricted to ensure balance of index patients with tuberculosis by arm and country. The primary outcome was the number of household contacts who initiated tuberculosis preventive treatment at each health facility within 4 months of the diagnosis of the index case, recorded in the first or last 6 months of our 20-month study. To ease interpretation, this number was standardised per 100 newly diagnosed index patients with tuberculosis. Analysis was by intention to treat. Masking of staff at the coordinating centre and sites was not possible; however, those analysing data were masked to assignment of intervention or control. An economic analysis of the intervention was done in parallel with the trial. ACT4 is registered at ClinicalTrials.gov, NCT02810678. FINDINGS: The study was done between Aug 1, 2016, and March 31, 2019. During the first 6 months of the study the crude overall proportion of household contacts initiating tuberculosis preventive treatment out of those eligible at intervention sites was 0·21. After the implementation of programme strengthening activities, the proportion initiating tuberculosis preventive treatment increased to 0·35. Overall, the number of household contacts initiating tuberculosis preventive treatment per 100 index patients with tuberculosis increased between study phases in intervention sites (adjusted rate difference 60, 95% CI 4 to 116), while control sites showed no statistically significant change (-12, -33 to 10). There was a difference in rate differences of 72 (95% CI 10 to 134) contacts per 100 index patients with tuberculosis initiating preventive treatment associated with the intervention. The total cost for the intervention, plus LTBI clinical care per additional contact initiating treatment was estimated to be CA$1348 (range 724 to 9708). INTERPRETATION: A strategy of standardised evaluation, local decision making, and implementation of health systems strengthening activities can provide a mechanism for scale-up of tuberculosis prevention, particularly in low-income and middle-income countries. FUNDING: Canadian Institutes of Health Research.

Possible outbreak of streptomycin-resistant Mycobacterium tuberculosis Beijing in Benin.

Using geographic information system and molecular tools, we characterized a possible outbreak of tuberculosis caused by Mycobacterium tuberculosis Beijing strain in 17 patients in Cotonou, Benin, during July 2005-October 2006. Most patients lived or worked in the same area and frequented the same local drinking bar. The isolates were streptomycin resistant.

Rapid and inexpensive detection of multidrug-resistant Mycobacterium tuberculosis with the nitrate reductase assay using liquid medium and direct application to sputum samples.

For rapid and low-cost detection of multidrug-resistant (MDR) Mycobacterium tuberculosis, we applied the nitrate reductase assay (NRA) using a liquid medium directly to sputum samples. A total of 179 sputum samples were analyzed by the NRA, and results were compared to those obtained by the indirect proportion method (IPM) as a standard reference. Out of 144 specimens for which comparable results were available, only one discrepant result was obtained: MDR by NRA but susceptible by the IPM. In total 56% of the results were obtained in 10 days by the NRA. NRA performed in liquid medium is rapid and inexpensive and can be easily implemented in low-income countries.

Setting up a national reference laboratory for Buruli ulcer: the case of Benin.

OBJECTIVE: To report the experience of Benin, where Buruli ulcer (BU) is endemic, in the implementation of diagnostic laboratory services. METHODS AND RESULTS: There has been a gradual introduction of biologic diagnostic activities for BU comprising (1) training of a laboratory technician in a highly experienced reference laboratory; (2) acquiring indispensable laboratory start-up materials; (3) progressive development of diagnostic laboratory activities; (4) regular external quality assessment with an experienced reference laboratory and (5) decentralization of activities to various clinical diagnostic and treatment centres for BU in Benin. CONCLUSION: Setting up a reference laboratory for BU is a continuous process, which necessitates motivated personnel and the cooperation of an experienced external reference laboratory.

Effects of grinding surgical tissue specimens and smear staining methods on Buruli ulcer microscopic diagnosis.

To optimize Buruli ulcer (BU) microscopic diagnosis, we compared two smear preparation methods from tissue specimens: smears made with tissue suspension after grinding and smears made directly with unground tissue. We also compared two smear staining methods: auramine and Ziehl-Neelsen (ZN). IS 2404-PCR was used as reference method. One hundred and thirty-one surgical tissue specimens from patients suspected of having BU were analyzed. Both smear preparation methods and both staining methods were equivalent in any combination. Thus we recommend ZN stained smears of unground tissue for peripheral treatment centres.

Rapid detection of multidrug-resistant Mycobacterium tuberculosis in Cotonou (Benin) using two low-cost colorimetric methods: resazurin and nitrate reductase assays.

We have evaluated two simple, rapid and low-cost colorimetric methods for the detection of multidrug-resistant Mycobacterium tuberculosis. A total of 151 M. tuberculosis strains were tested for resistance to rifampicin (RMP) and isoniazid by resazurin microplate assay (REMA) and nitrate reductase assay (NRA) in comparison with the conventional proportion method (PM) on Löwenstein-Jensen medium. A complete agreement was found between NRA and PM, while one false RMP-susceptible result was found by REMA. REMA and NRA tests are rapid and inexpensive, and could be good alternatives to the conventional PM in low-resource countries.

Low sensitivity of the MPT64 identification test to detect lineage 5 of the Mycobacterium tuberculosis complex.

PURPOSE: Differentiation of the Mycobacterium tuberculosis complex (MTBc) from non-tuberculous mycobacteria (NTM) is important for tuberculosis diagnosis and is a prerequisite for reliable phenotypic drug-resistance testing. We evaluated the performance of the rapid MPT64 antigen identification test for the detection of Mycobacterium africanum lineage 5 (MAF L5). METHODOLOGY: Smear-positive tuberculosis patients' sputa were included prospectively. Culture was performed on Löwenstein-Jensen medium and, when positive, the MPT64 test and the classical para-nitro benzoic acid susceptibility and heat-labile catalase (PNB/catalase) identification tests were performed. The MPT64 test was repeated 14 days after an initially negative first testing. Direct spoligotyping was performed for MTBc lineage determination. RESULTS: In total, 333 isolates were tested for all of the methods. Three hundred and twenty-two (96.7 %) were pure MTBc, by agreement between spoligotyping and PNB/catalase, and 11 were NTM or a mixture of MTBc/NTM. The MPT64 test conducted on day zero of culture-positivity correctly identified most of the pure MTBc isolates (93.2 %, 300/322), but it failed to detect 24 % of the L5 isolates (18/75) versus 2 % (4/202) of the L4 ones [OR=15.6 (5.3-45.8), P<0.0001], with improved sensitivity for L5 detection on repeat testing after 14 days. The L5-wide non-synonymous single-nucleotide polymorphism in the mpt64 gene may explain the poor performance of the MPT64 test for L5. CONCLUSION: The MPT64 test has a lower sensitivity for detecting L5 isolates of the MTBc, and can be considered as a first-screening test that should be confirmed by another identification method when it produces negative results in countries with L5. Given the microbiological bias in both the isolation and identification of MAF lineages, diagnostics with high sensitivity for direct testing on clinical material are preferable.

Genotypic characterization directly applied to sputum improves the detection of Mycobacterium africanum West African 1, under-represented in positive cultures.

BACKGROUND: This study aimed to compare the prevalence of Mycobacterium tuberculosis complex (MTBc) lineages between direct genotyping (on sputum) and indirect genotyping (on culture), to characterize potential culture bias against difficult growers. METHODOLOGY/PRINCIPAL FINDINGS: Smear-positive sputa from consecutive new tuberculosis patients diagnosed in Cotonou, (Benin) were included, before patients had started treatment. An aliquot of decontaminated sputum was used for direct spoligotyping, and another aliquot was cultured on Löwenstein Jensen (LJ) medium (90 days), for indirect spoligotyping. After DNA extraction, spoligotyping was done according to the standard method for all specimens, and patterns obtained from sputa were compared versus those from the derived culture isolates. From 199 patient's sputa, 146 (73.4%) yielded a positive culture. In total, direct spoligotyping yielded a pattern in 98.5% (196/199) of the specimens, versus 73.4% (146/199) for indirect spoligotyping on cultures. There was good agreement between sputum- and isolate derived patterns: 94.4% (135/143) at spoligotype level and 96.5% (138/143) at (sub)lineage level. Two of the 8 pairs with discrepant pattern were suggestive of mixed infection in sputum. Ancestral lineages (Lineage 1, and M. africanum Lineages 5 and 6) were less likely to grow in culture (OR = 0.30, 95%CI (0.14 to 0.64), p = 0.0016); especially Lineage 5 (OR = 0.37 95%CI (0.17 to 0.79), p = 0.010). Among modern lineages, Lineage 4 was over-represented in positive-culture specimens (OR = 3.01, 95%CI (1.4 to 6.51), p = 0.005). CONCLUSIONS/ SIGNIFICANCE: Ancestral lineages, especially M. africanum West African 1 (Lineage 5), are less likely to grow in culture relative to modern lineages, especially M. tuberculosis Euro-American (Lineage 4). Direct spoligotyping on smear positive sputum is effective and efficient compared to indirect spoligotyping of cultures. It allows for a more accurate unbiased determination of the population structure of the M. tuberculosis complex. TRIAL REGISTRATION: ClinicalTrials.gov NCT02744469.

Operational research within the national tuberculosis control programme in Benin.

OBJECTIVE: To document whether the placement of operational research (OR) fellows within disease control programmes in low and middle income countries leads to the implementation of operational research and improvements in policy and practice. RESULT: In 2012, an OR fellow was placed within the National TB Programme, Benin, to strengthen the implementation of operational research. From 2012 to 2015, eight OR projects were implemented, of which three contributed to changes in programme practice and five provided information which was not previously available from quarterly/annual reports. Two of these projects-one on the burden and treatment outcomes of childhood TB and one on tracing patients who had discontinued treatment-are discussed in more detail. OR should be strongly encouraged within national TB programme settings and an OR fellow facilitates this process.

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.

Tuberculosis Case Finding in Benin, 2000-2014 and Beyond: A Retrospective Cohort and Time Series Study.

Objective. To determine any changes in tuberculosis epidemiology in the last 15 years in Benin, seasonal variations, and forecasted numbers of tuberculosis cases in the next five years. Materials and Methods. Retrospective cohort and time series study of all tuberculosis cases notified between 2000 and 2014. The "R" software version 3.2.1 (Institute for Statistics and Mathematics Vienna Austria) and the Box-Jenkins 1976 modeling approach were used for time series analysis. Results. Of 246943 presumptive cases, 54303 (22%) were diagnosed with tuberculosis. Annual notified case numbers increased, with the highest reported in 2011. New pulmonary bacteriologically confirmed tuberculosis (NPBCT) represented 78% ± SD 2%. Retreatment cases decreased from 10% to 6% and new pulmonary clinically diagnosed cases increased from 2% to 8%. NPBCT notification rates decreased in males from 2012, in young people aged 15-34 years and in Borgou-Alibori region. There was a seasonal pattern in tuberculosis cases. Over 90% of NPBCT were HIV-tested with a stable HIV prevalence of 13%. The ARIMA best fit model predicted a decrease in tuberculosis cases finding in the next five years. Conclusion. Tuberculosis case notifications are predicted to decrease in the next five years if current passive case finding is used. Additional strategies are needed in the country.