Rapid sequencing of the Mycobacterium tuberculosis pncA gene for detection of pyrazinamide susceptibility.

We developed a pyrazinamidase gene DNA-sequencing method to rapidly identify pyrazinamide resistance-causing mutations in GenoLyse-treated, smear-positive sputum specimens. The sensitivity and specificity were 90.9 and 100%, respectively, compared to those of MGIT drug susceptibility testing, after the exclusion of synonymous mutations and nonsynonymous mutations previously associated with susceptibility to pyrazinamide.

GeneXpert MTB/RIF version G4 for identification of rifampin-resistant tuberculosis in a programmatic setting.

A recent Cochrane review estimated GeneXpert MTB/RIF specificity for rifampin resistance as 98% (95% confidence interval [CI], 97 to 99), based on results from earlier test versions. The measured positive predictive value of the new generation test from programmatic implementation in Cape Town, South Africa, was 99.5% (95% CI, 98.5 to 100), confirming excellent specificity.

Programmatically selected multidrug-resistant strains drive the emergence of extensively drug-resistant tuberculosis in South Africa.

BACKGROUND: South Africa shows one of the highest global burdens of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB). Since 2002, MDR-TB in South Africa has been treated by a standardized combination therapy, which until 2010 included ofloxacin, kanamycin, ethionamide, ethambutol and pyrazinamide. Since 2010, ethambutol has been replaced by cycloserine or terizidone. The effect of standardized treatment on the acquisition of XDR-TB is not currently known. METHODS: We genetically characterized a random sample of 4,667 patient isolates of drug-sensitive, MDR and XDR-TB cases collected from three South African provinces, namely, the Western Cape, Eastern Cape and KwaZulu-Natal. Drug resistance patterns of a subset of isolates were analyzed for the presence of commonly observed resistance mutations. RESULTS: Our analyses revealed a strong association between distinct strain genotypes and the emergence of XDR-TB in three neighbouring provinces of South Africa. Strains predominant in XDR-TB increased in proportion by more than 20-fold from drug-sensitive to XDR-TB and accounted for up to 95% of the XDR-TB cases. A high degree of clustering for drug resistance mutation patterns was detected. For example, the largest cluster of XDR-TB associated strains in the Eastern Cape, affecting more than 40% of all MDR patients in this province, harboured identical mutations concurrently conferring resistance to isoniazid, rifampicin, pyrazinamide, ethambutol, streptomycin, ethionamide, kanamycin, amikacin and capreomycin. CONCLUSIONS: XDR-TB associated genotypes in South Africa probably were programmatically selected as a result of the standard treatment regimen being ineffective in preventing their transmission. Our findings call for an immediate adaptation of standard treatment regimens for M/XDR-TB in South Africa.

Population structure of mixed Mycobacterium tuberculosis infection is strain genotype and culture medium dependent.

BACKGROUND: Molecular genotyping methods have shown infection with more than one Mycobacterium tuberculosis strain genotype in a single sputum culture, indicating mixed infection. AIM: This study aimed to develop a PCR-based genotyping tool to determine the population structure of M. tuberculosis strain genotypes in primary Mycobacterial Growth Indicator Tubes (MGIT) and Löwenstein-Jensen (LJ) cultures to identify mixed infections and to establish whether the growth media influenced the recovery of certain strain genotypes. METHOD: A convenience sample of 206 paired MGIT and LJ M. tuberculosis cultures from pulmonary tuberculosis patients resident in Khayelitsha, South Africa were genotyped using an in-house PCR-based method to detect defined M. tuberculosis strain genotypes. RESULTS: The sensitivity and specificity of the PCR-based method for detecting Beijing, Haarlem, S-family, and LAM genotypes was 100%, and 75% and 50% for detecting the Low Copy Clade, respectively. Thirty-one (15%) of the 206 cases showed the presence of more than one M. tuberculosis strain genotype. Strains of the Beijing and Haarlem genotypes were significantly more associated with a mixed infection (on both media) when compared to infections with a single strain (Beijing MGIT p = 0.02; LJ, p<0.01) and (Haarlem: MGIT p<0.01; LJ, p = 0.01). Strains with the Beijing genotype were less likely to be with "other genotype" strains (p<0.01) while LAM, Haarlem, S-family and LCC occurred independently with the Beijing genotype. CONCLUSION: The PCR-based method was able to identify mixed infection in at least 15% of the cases. LJ media was more sensitive in detecting mixed infections than MGIT media, implying that the growth characteristics of M. tuberculosis on different media may influence our ability to detect mixed infections. The Beijing and Haarlem genotypes were more likely to occur in a mixed infection than any of the other genotypes tested suggesting pathogen-pathogen compatibility.

Genotype MTBDRsl line probe assay shortens time to diagnosis of extensively drug-resistant tuberculosis in a high-throughput diagnostic laboratory.

RATIONALE: Conventional culture-based drug susceptibility testing (DST) for the second-line antituberculosis drugs is slow, leading to diagnostic delay with associated exacerbation of transmission, amplification of resistance, and increased mortality. OBJECTIVES: To assess the diagnostic performance of the GenoType MTBDRsl line probe assay (LPA) for the rapid detection of mutations conferring resistance to ofloxacin (OFX), amikacin (AMK), and ethambutol and to determine the impact of implementation on the turnaround time in a high-throughput diagnostic laboratory. METHODS: Six hundred and fifty-seven direct patient acid-fast bacilli smear-positive specimens resistant to isoniazid, rifampin, or both according to the GenoType MTBDRplus assay were consecutively tested, using the GenoType MTBDRsl LPA. The diagnostic performance was assessed relative to the "gold standard" culture-based method, and the laboratory turnaround times for both methods were determined. MEASUREMENTS AND MAIN RESULTS: A total of 516 of 657 patient specimens had valid results for both tests. The sensitivity for detecting OFX, AMK, and extensive drug resistance, using the GenoType MTBDRsl LPA, was 90.7% (95% confidence interval [CI], 80.1-96.0%), 100% (95% CI, 91.8-100%), and 92.3% (95% CI, 75.9-97.9%), respectively, and the specificity for detection was 98.1% (95% CI, 96.3-99.0%), 99.4% (95% CI, 98.2-99.8%), and 99.6% (95% CI, 98.5-99.9%), respectively. Implementation of this test significantly reduced the turnaround time by 93.3% (P < 0.001), calculated from the date that the specimen was received at the laboratory to reporting second-line results. In addition, a significant increase in diagnostic yield of 20.1% and 19.3% (P < 0.001) for OFX and AMK resistance, respectively, was obtained for isolates that were either contaminated or had lost viability. CONCLUSIONS: The GenoType MTBDRsl LPA is a rapid and reliable DST that can be easily incorporated into the diagnostic algorithm. This assay significantly improved diagnostic yield (P < 0.001) while simultaneously decreasing diagnostic delay for reporting second-line DST. The rapid dissemination of second-line DST results will guide initiation of appropriate treatment, thereby reducing transmission and improving treatment outcome.

Population structure of multi- and extensively drug-resistant Mycobacterium tuberculosis strains in South Africa.

Genotyping of multidrug-resistant (MDR) Mycobacterium tuberculosis strains isolated from tuberculosis (TB) patients in four South African provinces (Western Cape, Eastern Cape, KwaZulu-Natal, and Gauteng) revealed a distinct population structure of the MDR strains in all four regions, despite the evidence of substantial human migration between these settings. In all analyzed provinces, a negative correlation between strain diversity and an increasing level of drug resistance (from MDR-TB to extensively drug-resistant TB [XDR-TB]) was observed. Strains predominating in XDR-TB in the Western and Eastern Cape and KwaZulu-Natal Provinces were strongly associated with harboring an inhA promoter mutation, potentially suggesting a role of these mutations in XDR-TB development in South Africa. Approximately 50% of XDR-TB cases detected in the Western Cape were due to strains probably originating from the Eastern Cape. This situation may illustrate how failure of efficient health care delivery in one setting can burden health clinics in other areas.

Rapid molecular screening for multidrug-resistant tuberculosis in a high-volume public health laboratory in South Africa.

RATIONALE: The dual challenges to tuberculosis (TB) control of HIV infection and multidrug resistance are particularly pressing in South Africa. Conventional methods for detecting Mycobacterium tuberculosis drug resistance take weeks to months to produce results. Rapid molecular testing for drug resistance is available but has not been implemented in high-TB-burden settings. OBJECTIVES: To assess the performance and feasibility of implementation of a commercially available molecular line-probe assay for rapid detection of rifampicin and isoniazid resistance. METHODS: We performed the assay directly on 536 consecutive smear-positive sputum specimens from patients at increased risk of multidrug-resistant (MDR) TB in a busy routine diagnostic laboratory in Cape Town, South Africa. Results were compared with conventional liquid culture and drug susceptibility testing on solid medium. MEASUREMENTS AND MAIN RESULTS: Overall, 97% of smear-positive specimens gave interpretable results within 1-2 days using the molecular assay. Sensitivity, specificity, and positive and negative predictive values were 98.9, 99.4, 97.9, and 99.7%, respectively, for detection of rifampicin resistance; 94.2, 99.7, 99.1, and 97.9%, respectively, for detection of isoniazid resistance; and 98.8, 100, 100, and 99.7%, respectively, for detection of multidrug resistance compared with conventional results. The assay also performed well on specimens that were contaminated on conventional culture and on smear-negative, culture-positive specimens. CONCLUSIONS: This molecular assay is a highly accurate screening tool for MDR TB, which achieves a substantial reduction in diagnostic delay. With overall performance characteristics that are superior to conventional culture and drug susceptibility testing and the possibility for high throughput with substantial cost savings, molecular testing has the potential to revolutionize MDR TB diagnosis.

Improved contamination control for a rapid phage-based rifampicin resistance test for Mycobacterium tuberculosis.

A prospective study was conducted of the rapid FASTPlaque-Response test for determination of rifampicin resistance in Mycobacterium tuberculosis with and without the addition of an antimicrobial supplement containing nystatin, oxacillin and aztreonam (NOA) to control specimen-related contamination. A total of 631 smear-positive sputum specimens was tested. The age of specimens ranged from 0 to 21 days. The NOA antimicrobial was effective at controlling contamination, with 4.1 % of specimens contaminated when the NOA antimicrobial supplement was used compared with 13.9 % contamination without NOA. Overall levels of interpretability of the test with NOA were 87.8 % with specimens of < or =3 days and 79.0 % for all specimens. This compared with 70.1 and 73.8 % readable results, respectively, from conventional culture-based drug susceptibility testing (DST). Sensitivity, specificity and overall accuracy of the FASTPlaque-Response test for rifampicin resistance were 98.1, 96.3 and 96.6 %, respectively, for all specimens with NOA, and 93.2, 96.3 and 95.9 % without NOA, when compared with resolved conventional DST results. Inclusion of the NOA supplement reduced contamination, increased the number of interpretable results and did not adversely affect the performance of the FASTPlaque-Response test. Thus, the use of NOA improves the robustness of the test, facilitating its wider implementation.