Multi- and Extensively Drug Resistant Mycobacterium tuberculosis in South Africa: a Molecular Analysis of Historical Isolates.

Modern advances in genomics provide an opportunity to reinterpret historical bacterial culture collections. In this study, genotypic antibiotic resistance profiles of Mycobacterium tuberculosis isolates from a historical 20-year-old multidrug-resistant tuberculosis (MDR-TB) culture collection in South Africa are described. DNA samples extracted from the phenotypically MDR-TB isolates (n = 240) were assayed by Hain line probe assay (LPA) for the confirmation of MDR-TB and by Illumina Miseq whole-genome sequencing (WGS) for the characterization of mutations in eight genes (rpoB, katG, inhA, rpsL, pncA, embB, gyrA, and rrs) that are known to code for resistance to commonly used anti-TB agents. LPA identified 71.3% of the TB isolates as MDR-TB, 18.3% as rifampin (RIF) monoresistant, 2% as isoniazid (INH) monoresistant, and 8.3% as susceptible to both RIF and INH (RIF+INH). In a subset of 42 randomly selected isolates designated as RIF+INH resistant by Löwenstein-Jensen (LJ) culture in 1993, LPA and WGS results confirmed MDR-TB. In all five INH-monoresistant isolates by LPA and in all but one (the wild type) of the 34 successfully sequenced RIF-monoresistant isolates, WGS revealed matching mutations. Only 26% of isolates designated as susceptible by LPA, however, were found to be wild type by WGS. Novel mutations were found in the rpoB (Thr480Ala, Gln253Arg, Val249Met, Val251Tyr, Val251Phe), katG (Trp477STOP, Gln88STOP, Trp198STOP, Trp412STOP), embB (Thr11Xaa, Gln59Pro), and pncA (Thr100Ile, Thr159Ala, Ala134Arg, Val163Ala, Thr153Ile, DelGpos7, Phe106Ser) genes. Three MDR-TB isolates showed mutations in both the gyrA and rrs genes, suggesting that extensively drug-resistant tuberculosis existed in South Africa well before its formal recognition in 2006.

Evaluation of Semiautomated IS6110-Based Restriction Fragment Length Polymorphism Typing for Mycobacterium tuberculosis in a High-Burden Setting.

The manual IS6110-based restriction fragment length polymorphism (RFLP) typing method is highly discriminatory; however, it is laborious and technically demanding, and data exchange remains a challenge. In an effort to improve IS6110-based RFLP to make it a faster format, DuPont Molecular Diagnostics recently introduced the IS6110-PvuII kit for semiautomated typing of Mycobacterium tuberculosis using the RiboPrinter microbial characterization system. This study aimed to evaluate the semiautomated RFLP typing against the standard manual method. A total of 112 isolates collected between 2013 and 2014 were included. All isolates were genotyped using manual and semiautomated RFLP typing methods. Clustering rates and discriminatory indexes were compared between methods. The overall performance of semiautomated RFLP compared to manual typing was excellent, with high discriminatory index (0.990 versus 0.995, respectively) and similar numbers of unique profiles (72 versus 74, respectively), numbers of clustered isolates (33 versus 31, respectively), cluster sizes (2 to 6 and 2 to 5 isolates, respectively), and clustering rates (21.9% and 17.1%, respectively). The semiautomated RFLP system is technically simple and significantly faster than the manual RFLP method (8 h versus 5 days). The analysis is fully automated and generates easily manageable databases of standardized fingerprints that can be easily exchanged between laboratories. Based on its high-throughput processing with minimal human effort, the semiautomated RFLP can be a very useful tool as a first-line method for routine typing of M. tuberculosis isolates, especially where Beijing strains are highly prevalent, followed by manual RFLP typing if resolution is not achieved, thereby saving time and labor.

Field evaluation of a novel preservation medium to transport sputum specimens for molecular detection of Mycobacterium tuberculosis in a rural African setting.

OBJECTIVES: To assess the performance of an innovative method of transporting sputum to centralised facilities for molecular detection of Mycobacterium tuberculosis: using a swab to inoculate sputum in a transport medium, PrimeStore(®) Molecular Transport Medium (PS-MTM). METHODS: Two sputum specimens were obtained from suspected patients with tuberculosis (TB) at rural healthcare facilities in South Africa. A swab was taken from each specimen and placed into PS-MTM, prior to it being processed by either liquid culture or Xpert MTB/RIF assay (Xpert). RESULTS: A total of 141 patients (including 47 with laboratory-confirmed TB) were included in this analysis. M. tuberculosis was detected at 29% by culture and 29% by Xpert, whereas 31% tested positive by IS6110 real-time PCR of PS-MTM from the culture and 36% from the Xpert-paired specimen. Concordance between the method under evaluation with culture was 82% (McNemar, P = 0.55) and 84% (McNemar, P = 0.05) for Xpert. Stratified by culture result, the detection rate by IS6110 real-time PCR of PS-MTM was similar to Xpert for patients with positive culture (P = 0.32), but significantly higher if culture was negative (P = 0.008). CONCLUSIONS: These results suggest that swab collection of sputum into PS-MTM for transport is a promising method for diagnosis of TB in rural healthcare settings, thereby potentially improving the options available for molecular diagnosis of TB in countries incapable of applying decentralised high-tech molecular testing.

Genetic diversity of Mycobacterium tuberculosis strains isolated from spiritual holy water site attendees in Northwest Ethiopia. A cross-sectional study.

Background: The genetic diversity of Mycobacterium tuberculosis complex (MTBC) strains was characterized among isolates from individuals with pulmonary tuberculosis (PTB) symptoms attended holy water sites (HWSs) in the Amhara region, Ethiopia. Methods: A cross-sectional study was done from June 2019 to March 2020 to describe the genetic diversity and drug-resistance profiles of MTBC isolates. Sputum specimens were collected and cultured in the Löwenstein-Jensen culture medium. Line Probe Assay, MTBDRplus VER 2.0, and MTBDRsl VER 2.0 were used to detect first-and second-line anti-TB drug-resistance patterns. A spoligotyping technique was utilized to characterize the genetic diversity. Statistical analysis was performed using STATA 15. Results: Of 560 PTB-symptomatic participants, 122 (21.8%) were culture-positive cases. Spoligotyping of 116 isolates revealed diverse MTBC sublineages, with four major lineages: Euro-American (EA) (Lineage 4), East-African-Indian (EAI) (Lineage 3), Ethiopian (ETH) (Lineage 7), East Asian (EA) (Lineage 2). The majority (96.6%) of the isolates were EA (lineage 4) and EAI, with proportions of 54.3% and 42.2%, respectively. A total of 31 spoligotype patterns were identified, 26 of which were documented in the SITVIT2 database. Of these, there were 15 unique spoligotypes, while eleven were grouped with 2-17 isolates. SIT149/T3-ETH (n = 17), SIT26/CAS1-DELHI (n = 16), SIT25/CAS1-DELHI (n = 12), and SIT52/T2 (n = 11) spoligotypes were predominant. A rare spoligotype pattern: SIT41/Turkey and SIT1/Beijing, has also been identified in North Shewa. The overall clustering rate of sub-lineages with known SIT was 76.4%.Of the 122 culture-positive isolates tested, 16.4% were resistant to rifampicin (RIF) and/or isoniazid (INH). Multidrug-resistant TB (MDR-TB) was detected in 12.3% of isolates, five of which were fluoroquinolones (FLQs) resistant. SIT149/T3-ETH and SIT21/CAS1-KILI sublineages showed a higher proportion of drug resistance. Conclusions: Diverse MTBC spoligotypes were identified, with the T and CAS families and EA (lineage 4) predominating. A high prevalence of drug-resistant TB, with SIT149/T3-ETH and CAS1-KILI sublineages comprising a greater share, was observed. A study with large sample size and a sequencing method with stronger discriminatory power is warranted to understand better the genetic diversity of circulating MTBC in this cohort of study, which would help to adopt targeted interventions.

Molecular characterization and second-line antituberculosis drug resistance patterns of multidrug-resistant Mycobacterium tuberculosis isolates from the northern region of South Africa.

Despite South Africa being one of the high-burden multidrug-resistant tuberculosis (MDR-TB) countries, information regarding the population structure of drug-resistant Mycobacterium tuberculosis strains is limited from many regions of South Africa. This study investigated the population structure and transmission patterns of drug-resistant M. tuberculosis isolates in a high-burden setting of South Africa as well as the possible association of genotypes with drug resistance and demographic characteristics. A total of 336 consecutive MDR-TB isolates from four provinces of South Africa were genotyped using spoligotyping and mycobacterial interspersed repetitive-unit-variable number tandem repeat (MIRU-VNTR) typing. Drug susceptibility testing for ofloxacin, kanamycin, and capreomycin was performed using the agar proportion method. The results showed that 4.8% of MDR-TB isolates were resistant to ofloxacin, 2.7% were resistant to kanamycin, and 4.5% were resistant to capreomycin, while 7.1% were extensively drug resistant (XDR), and the remaining 83.6% were susceptible to all of the second-line drugs tested. Spoligotyping grouped 90.8% of the isolates into 25 clusters, while 9.2% isolates were unclustered. Ninety-one percent of the 336 isolates were assigned to 21 previously described shared types, with the Beijing family being the predominant genotype in the North-West and Limpopo Provinces, while the EAI1_SOM family was the predominant genotype in the Gauteng and Mpumalanga Provinces. No association was found between genotypes and specific drug resistance patterns or demographic information. The high level of diversity and the geographical distribution of the drug-resistant M. tuberculosis isolates in this study suggest that the transmission of TB in the study settings is not caused by the clonal spread of a specific M. tuberculosis strain.

Comparison between the BACTEC MGIT 960 system and the agar proportion method for susceptibility testing of multidrug resistant tuberculosis strains in a high burden setting of South Africa.

BACKGROUND: The increasing problem of multi-drug-resistant (MDR) tuberculosis (TB) [ie resistant to at least isoniazid (INH) and rifampicin (RIF)] is becoming a global problem. Successful treatment outcome for MDR-TB depends on reliable and accurate drug susceptibility testing of first-line and second-line anti-TB drugs. METHOD: Consecutive M. tuberculosis isolates identified as MDR-TB during August 2007 to January 2008 using the BACTEC MGIT 960 systems and the agar proportion method were included in this study. Susceptibility testing of MDR-TB isolates against ethambutol (EMB) and streptomycin (STR) as well as two second-line anti-TB drugs, kanamycin (KAN) and ofloxacin (OFX) was performed using the BACTEC MGIT 960 systems at a routine diagnostic laboratory. The results were compared to those obtained by the agar proportion method. RESULT: The agreement between the BACTEC MGIT 960 system and the agar proportion method was 44% for EMB, 61% for STR and 89% for both KAN and OFX. The sensitivity and specificity of the BACTEC MGIT 960 system using the agar proportion method as a gold standard was 92% and 37% for EMB, 95% and 37% for STR, 27% and 97% for KAN and 84% and 90% for OFX, respectively. CONCLUSIONS: The BACTEC MGIT 960 system showed acceptable sensitivity for EMB, STR, and OFX; however, the BACTEC MGIT 960 system was less specific for EMB and STR and demonstrated a low sensitivity for KAN. The lower agreement found between the two methods suggests the unreliability of the BACTEC MGIT 960 system for the drugs tested. The reasons for the lower agreement between the two methods need to be investigated and further studies are needed in this setting to confirm the study finding.

Evaluation of TBc identification immunochromatographic assay for rapid identification of Mycobacterium tuberculosis complex in samples from broth cultures.

Tuberculosis (TB) is a disease of major public health concern worldwide, especially in developing countries. In addition, the human immunodeficiency virus (HIV) epidemic has increased the incidence of infection with nontuberculous mycobacteria (NTM). Rapid, accurate, and simple methods for differentiation of Mycobacterium tuberculosis complex (MTBC) isolates from NTM is greatly needed for successful control of the TB epidemic. This study was done to evaluate the clinical performance of the BD MGIT TBc identification test (TBc ID) for rapid identification of MTBC in samples from broth cultures. A total of 229 Ziehl-Neelsen (ZN) stain-positive MGIT cultures were tested using the TBc ID test, and the results were compared with those of the AccuProbe MTBC identification test (GenProbe, San Diego, CA). The agreement between the TBc ID test and the AccuProbe assay was 96% (kappa = 0.92; confidence interval [CI] = 0.869 to 0.971). The sensitivity, specificity, and negative and positive predictive values of the TBc ID test compared to the AccuProbe assay were 100%, 92.4%, 100%, and 92.2%, respectively. After additional molecular testing, the agreement between the two methods increased to 97.8% (kappa = 0.96; CI = 0.917 to 0.994), and the specificity and positive predictive value increased to 95.6% and 95.7%, respectively. The TBc ID test is a simple, sensitive, and specific test for identification of MTBC in samples from acid-fast bacillus (AFB) smear-positive cultures. The TBc ID test could be a good alternative to the AccuProbe test in TB diagnostic laboratories.

Whole genome sequencing for drug resistance determination in Mycobacterium tuberculosis.

South Africa remains challenged with a high tuberculosis burden accompanied by an increase in drug resistant cases. We assessed the use of the Illumina MiSeq, a next-generation sequencing platform for whole genome sequencing, followed by bioinformatic analysis using a commercial software package to determine resistance to selected drugs used for Mycobacterium tuberculosis treatment in our setting. Whole genome sequencing shows potential as a diagnostic platform for the detection of drug resistance in Mycobacterium tuberculosis with the provision of information for several drugs simultaneously.

Correlation of rpoB Mutations with Minimal Inhibitory Concentration of Rifampin and Rifabutin in Mycobacterium tuberculosis in an HIV/AIDS Endemic Setting, South Africa.

Treatment of tuberculosis (TB) and HIV co-infections is often complicated by drug-to-drug interactions between anti-mycobacterial and anti-retroviral agents. Rifabutin (RFB) is an alternative to rifampin (RIF) for TB regimens and is recommended for HIV patients concurrently receiving protease inhibitors because of reduced induction of CYP3A4. This study sought to determine the proportion of RFB susceptible isolates among RIF-resistant strains in a high HIV prevalence setting in South Africa. In addition, the study explored the association between rpoB mutations and minimum inhibitory concentrations (MIC) of RIF and RFB. A total of 189 multidrug resistant (MDR) Mycobacterium tuberculosis isolates from the Centre for Tuberculosis repository were analyzed. The MICs were determined using a MYCOTB Sensititre plate method and the rpoB gene was sequenced. Of the 189 MDR isolates, 138 (73%) showed resistance to both RIF and RFB, while 51 (27%) isolates were resistant to RIF but retained susceptibility to RFB. The S531L was the most frequent rpoB point mutation in 105/189 (56%) isolates, followed by H526Y in 27/189 (14%) isolates. Resistance to both RIF and RFB was found predominantly in association with mutations S531L (91/105, 87%), H526Y (20/27, 74%), and H526D (15/19, 79%), while D516V (15/17, 88%), and L533P (3/4, 75%) were found in RIF-resistant, RFB-susceptible isolates. This study has shown that up to 27% of MDR-TB patients in South Africa may benefit from a treatment regimen that includes RFB.

A Novel Molecular Strategy for Surveillance of Multidrug Resistant Tuberculosis in High Burden Settings.

BACKGROUND: In South Africa and other high prevalence countries, transmission is a significant contributor to rising rates of multidrug resistant tuberculosis (MDR-TB). Thus, there is a need to develop an early detection system for transmission clusters suitable for high burden settings. We have evaluated the discriminatory power and clustering concordance of a novel and simple genotyping approach, combining spoligotyping with pncA sequencing (SpoNC), against two well-established methods: IS6110-RFLP and 24-loci MIRU-VNTR. METHODS: A total of 216 MDR-TB isolates collected from January to June 2010 from the NHLS Central TB referral laboratory in Braamfontein, Johannesburg, representing a diversity of strains from South Africa, were included. The isolates were submitted for genotyping, pncA sequencing and analysis to the Centre for Tuberculosis in South Africa and the Public Health Research Institute Tuberculosis Center at Rutgers University in the United States. Clustering rates, Hunter-Gaston Discriminatory Indexes (HGI) and Wallace coefficients were compared between the methods. RESULTS: Overall clustering rates were high by both IS6110-RFLP (52.8%) and MIRU-VNTR (45.8%), indicative of on-going transmission. Both 24-loci MIRU-VNTR and IS6110-RFLP had similar HGI (0.972 and 0.973, respectively), with close numbers of unique profiles (87 vs. 70), clustered isolates (129 vs. 146), and cluster sizes (2 to 26 vs. 2 to 25 isolates). Spoligotyping alone was the least discriminatory (80.1% clustering, HGI 0.903), with 28 unique types. However, the discriminatory power of spoligotyping was improved when combined with pncA sequencing using the SpoNC approach (61.8% clustering, HGI 0.958). A high proportion of MDR-TB isolates had mutations in pncA (68%, n = 145), and pncA mutations were significantly associated with clustering (p = 0.007 and p = 0.0013 by 24-loci MIRU-VNTR and IS6110-RFLP, respectively), suggesting high rates of resistance to pyrazinamide among all MDR-TB cases and particularly among clustered cases. CONCLUSION: We conclude that SpoNC provides good discrimination for MDR-TB surveillance and early identification of outbreaks in South Africa, with 24-loci MIRU-VNTR applied for pncA wild-type strains as needed.

Laboratory evaluation of a specimen transport medium for downstream molecular processing of sputum samples to detect Mycobacterium tuberculosis.

BACKGROUND: Modern molecular-based approaches for the detection of Mycobacterium tuberculosis in sputum samples promise quicker and more accurate detection of cases. However, processing sputum samples at central diagnostic facilities provides a diagnostic approach, but requires a safe and efficient system that is not affected by transport delays and ambient temperature to be feasible. We evaluated the technical properties of PrimeStore®-Molecular Transport Medium (PS-MTM) for its ability to inactivate mycobacteria, ensuring stability of DNA over time at ambient temperatures and to assess the compatibility of the transport medium with DNA extraction systems. METHODS: Assessment of the transport medium for application of sputum samples processed for the detection of M. tuberculosis included the inactivation of M. tuberculosis in spiked sputum samples, compatibility of the medium with three commercial nucleic extraction systems and stability of DNA in the medium at ambient temperature over 28 days. We further performed a clinical laboratory evaluation on 256 sputum specimens sent for tuberculosis investigation. RESULTS: Complete inactivation of M. tuberculosis occurred within 30 min of exposure at a ratio of 1:3 for sputum to PS-MTM. Sputum specimen in PS-MTM showed very good compatibility with automated bead-based extraction systems, producing high DNA output (estimated lower limits of detection: ~170 CFU/ml). Furthermore, PS-MTM samples remained stable over 28 days at ambient temperature displaying no significant change over time in Ct-values (<5% on a mean starting value of 22.47). Of the 256 clinical sputum specimens, 10.2% were culture positive and 11.0% were positive by real-time PCR of PS-MTM samples. CONCLUSIONS: Collecting and transporting sputum from TB suspects in PS-MTM offer safe transport at ambient temperature, DNA stability for extended periods without cooling and specimens directly suitable for molecular testing. This novel approach may support introduction and further scale-up of molecular diagnostics for TB in resource-limited settings.

Analytical performance of the Roche LightCycler® Mycobacterium Detection Kit for the diagnosis of clinically important mycobacterial species.

BACKGROUND: The LightCycler® Mycobacterium Detection Kit based on real-time PCR technology for the detection of Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium kansasii was recently developed. This study evaluated its analytical sensitivity, specificity and reproducibility. METHODOLOGY/PRINCIPAL FINDINGS: Plasmid standards were prepared and used to determine the limit of detection. The assay was also performed against organisms other than mycobacteria, other mycobacterial strains and interfering substances to exclude cross-reactivity and interference. Reference standards were prepared and tested to assess the assay's reproducibility. All PCR assays were performed using the LightCycler® 2.0 Instrument. The detection limit for M. tuberculosis was 28 copies per microlitre. Neither cross-reactivity nor interference occurred with non-mycobacterial organisms and substances tested. Overall reproducibility for consecutive measurements, run-to-run, lot-to-lot, day-to-day and laboratory-to-laboratory achieved a coefficient of variance of less than two percent. SIGNIFICANCE: The LightCycler® Mycobacterium Detection kit has shown to be a robust and accurate assay with the potential to be used as a rapid TB diagnostic test.