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We compared cetylpyridinium chloride (CPC), ethanol (ETOH), and OMNIgene.SPUTUM (OMNI) for 28-day storage of sputum at ambient temperature before molecular tuberculosis diagnostics. Three sputum samples were collected from each of 133 smear-positive tuberculosis (TB) patients (399 sputum samples). Each patient's sputum was stored with either CPC, ETOH, or OMNI for 28 days at ambient temperature, with subsequent rpoB amplification targeting a short fragment (81 bp, GeneXpert MTB/RIF [Xpert]) or a long fragment (1,764 bp, in-house nested PCR). For 36 patients, Xpert was also performed at baseline on all 108 fresh sputum samples. After the 28-day storage (D28), Xpert positivity did not significantly differ between storage methods. In contrast, higher positivity for rpoB nested PCR was obtained with OMNI ( n = 125, 94%) than with ETOH ( n = 114, 85.7%; P = 0.001). Smears with scanty acid-fast bacilli (AFB) had lower rpoB PCR positivity with ETOH storage ( n = 10, 41.7%) than with CPC ( n = 16, 66.7%; difference, 25%; 95% confidence interval [CI], 3.5 to 46.5; P = 0.031) or OMNI ( n = 16, 69.6%; difference, 26.1%; 95% CI, 3.8 to 48.4; P = 0.031), with no difference between CPC and OMNI. Poststorage, the threshold cycle ( C T ) values significantly decreased compared to those prestorage with ETOH (difference, -1.1; 95% CI, -1.6 to -0.6; P = 0.0001) but not with CPC ( P = 0.915) or OMNI ( P = 0.33). For one patient's ETOH- and CPC-stored specimens with a C T of <10, Xpert gave results of rifampin false resistant at D28, which was resolved by repeating Xpert on a 1/100 diluted specimen. In conclusion, 28-day storage of sputum in OMNI, CPC, or ETOH at ambient temperature does not impact short-fragment PCR (Xpert), including for low smear grades. However, for long-fragment PCR, ETOH yielded a lower PCR positivity for low smear grades, while the performance of OMNI and CPC was excellent for all smear grades. (The study has been registered at ClinicalTrials.gov under registration number NCT02744469.)., (Copyright © 2019 Sanoussi et al.)

A balanced perspective is advocated for the assessment and application of the most recent and the oldest diagnostic methods for pulmonary tuberculosis (TB)-the molecular Xpert ® MTB/RIF assay and microscopy for acid-fast bacilli. We discuss their respective merits and shortcomings and identify threats that may hamper their use in TB control. Neither test on its own provides all the information needed for diagnosis and treatment monitoring. Considering all aspects important for both individual patient care and disease control, neither seems 'better' than the other. The required advancement of microscopy had already been hampered before the introduction of the GeneXpert technology by unsuccessful and probably misguided attempts to decentralise culture-based diagnosis and drug susceptibility testing. It seems evident that systematic replacement of microscopy by Xpert is not a viable option for the foreseeable future. Instead, the two methods should complement each other to arrive at a comprehensive, accessible and continuous service for a maximum number of patients. This will intrinsically prioritise targeting the most potent transmitters with the worst prognosis, simultaneously offering optimised prospects for efficient TB control. New microscopy and Xpert applications are expected to ultimately make control programmes independent of culture-based methods in diagnosis, treatment monitoring and outcome assessment.

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.

Setting: The main tuberculosis (TB) centre in Benin, West Africa, where only 2% of adult pulmonary TB cases are sputum smear-negative, all other pulmonary cases being smear-positive., Objectives: To assess the burden of smear-negative, culture-positive pulmonary TB among TB suspects in Cotonou, and to estimate the total number of non-smear-positive TB cases at country level., Design: For 1 year, one morning sputum culture was performed for every TB suspect (cough lasting >3 weeks, as defined in Benin's national guidelines) with three negative sputum smears (fluorescence technique)., Results: Of 214 TB suspects for whom culture was performed, only 22 smear-negative, culture-positive cases were identified. During the same period, 831 sputum smear-positive cases were diagnosed. Culture therefore contributed only 2.6% of the total number of bacteriologically proven cases., Conclusion: These results show the relatively low input of culture in TB diagnosis among chronic coughers in Cotonou, Benin, and demonstrates that the expected number of non-smear-positive TB cases in Benin is probably much lower than the World Health Organization's current annual estimates.

Setting: National Reference Laboratory, Benin., Objectives: To compare the performance of Fraen FluoLED and LW Lumin light-emitting diode (LED) fluorescence microscopy modules., Design: Acid-fast bacilli (AFB) smears, routinely examined with a classical fluorescence microscope, were blindly re-read with both LED systems at 200x magnification. Smears with discordant results were rechecked on all systems at 200x, and 100 randomly chosen smears were read again at 400x. Confirmed presence of AFB with any system was accepted as a true positive., Results: A total of 1937 smears were examined by all systems. The Fraen and LW detected 895 (46.2%) and 817 (42.2%) positive and scanty positive smears. After rechecking 201 smears, 15 false-positive and 61 false-negative results were declared for Fraen, against 11 and 135 for LW. The systems had similar false-positive rates (1.7% for Fraen and 1.4% for LW), but differed significantly regarding detection of confirmed microscopy positives (93.5% and 85.6% respectively, P < 0.00001). A high correlation between both LED systems was found at 400x magnification., Conclusions: The Fraen LED fluorescence microscopy module performed significantly better than the LW LED at the most efficient 200x magnification. It was also more appreciated by all users. The LW module may perform equally well at higher magnification.

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.

Despite a theoretical risk of transfer of bacilli from a positive to a negative smear, bulk staining is routinely performed in many laboratories. To assess this risk in our laboratory, two smears were made from each sputum specimen and stained with auramine: one smear was stained on a rack and the second using the bulk method. Smears were read blind using a fluorescence microscope. A total of 811 sputum specimens were analysed. No acid-fast bacilli transfer was observed even when staining solution jars had not been renewed for 3 days. Bulk staining is rapid and cheap, and could be used in laboratories with a high workload in low-resource settings.

The aim of this study was to evaluate a nitrate reductase assay (NRA) performed on smear-positive sputa for the direct detection of rifampin resistance in Mycobacterium tuberculosis. A total of 213 smear-positive sputa with a positivity score of 1+ or more (>1 acid-fast bacillus per field by fluorescence microscopy) were used in the study. The samples were decontaminated using the modified Petroff method, and portions of the resulting suspension were used to perform the NRA. The NRA results were compared with the reference indirect proportion method for 177 specimens for which comparable results were available. NRA results were obtained at day 10 for 15 specimens (9%), results for 88 specimens (50%) were obtained at day 14, results for 66 specimens (37%) were obtained at day 18, and results for the remaining 8 specimens (4%) were obtained at day 28. Thus, 96% of NRA results were obtained in 18 days. Of the 177 specimens, there was only one discrepancy (susceptible according to the NRA and resistant according to the indirect proportion method). NRA is simple to perform and provides a rapid, accurate, and cost-effective means for the detection of rifampin resistance in M. tuberculosis isolates.

Abstract: Objectives: The authors had for aim to assess the management of tuberculosis and HIV co-infection in Cotonou, Benin. Patients and methods: We made a cross-sectional, retrospective, and descriptive study comparing the clinical presentation and outcome of patients with tuberculosis and HIV co-infection versus patients with tuberculosis alone, all managed at the National Pneumophtisiology Center in Cotonou, Benin, in 2009. Results: The rate of HIV screening in TB patients was 99%. One thousand and eighty-six TB patients were included and 259 were HIV positive. The mean age of co-infected patients was 36 years, versus 34 for TB mono-infected patients. The sex ratio among co-infected was 1.15 versus 2.25 among TB patients. Positive pulmonary sputum was less frequent with co-infection. Two hundred and fifty-seven over 259 patients were treated with cotrimoxazole. One hundred and eighty-five over 234 (79.05%) had CD4 counts<350. Eighty-five (46%) of the 185 patients with CD4<350, were given antiretroviral therapy. Treatment success rate was lower for co-infected (75%) than for patients with TB alone (86%), and death rates were higher in co-infected patients (10% vs. 3%). Conclusion: High death rate and high rate of lost to follow-up are arguments for systematic antiretroviral treatment of co-infected patients. Early screening for TB and HIV, and reviewing the current national recommendations, as well as an increased governmental effort to provide medicines to all patients in need of ARV are mandatory. [Copyright &y& Elsevier]