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.

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.

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.

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.

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.