Characterization of embB mutations involved in ethambutol resistance in multi-drug resistant Mycobacterium tuberculosis isolates in Zambia.

BACKGROUND: Ethambutol (EMB) is an important anti-tuberculosis drug used in the management of multi-drug resistant tuberculosis (MDR-TB). Mutations in embB are the major mechanism of resistance. This study investigated embB mutations among MDR-TB isolates and analyzed their correlations with phenotypic drug susceptibility testing (DST) in Zambia. METHOD: A total of 132 MDR-TB isolates were collected from January 2014 to April 2017 and characterized using MGIT 960 systems, embB sequencing, and spoligotyping. RESULTS: Out of 61 phenotypically EMB resistant isolates, 53 had mutations in embB. Among the 71 EMB susceptible isolates, 47 had embB mutations. Sensitivity of embB mutations was 86.9% while specificity was 33.8%. CAS1_Kili (SIT21) had high odds of having embB mutations, particularly, G918A (Met306eIl) (Odds ratio 16.7, p < 0.0001). CONCLUSION: Molecular EMB resistance testing by DNA sequencing can improve detection of EMB resistance among MDR-TB patients in Zambia. Additionally, CAS1_Kili was associated with embB amino acid substitution Met306Ile suggesting transmission. A detailed investigation to track and determine transmission hotspot area for MDR-TB could help optimize control strategies.

Mutations in rpoB and katG genes and the inhA operon in multidrug-resistant Mycobacterium tuberculosis isolates from Zambia.

OBJECTIVES: It is established that resistance to rifampicin (RIF) in 90% of RIF-resistant Mycobacterium tuberculosis isolates is attributable to point mutations in the rpoB gene, whilst 50-95% of M. tuberculosis resistance to isoniazid (INH) is caused by mutations in the katG gene. However, the patterns and frequencies of mutations vary by geographical region. In Zambia, the genetic mechanisms of resistance of M. tuberculosis to RIF and INH were unreported before this study. METHODS: Using gene sequencing, the rpoB, katG and inhA genes of 99 multidrug-resistant M. tuberculosis (MDR-TB) and 49 pan-susceptible M. tuberculosis isolates stored at a tuberculosis reference laboratory from 2013 to 2016 were analysed and were compared with published profiles from other African countries. RESULTS: Of the 99 MDR-TB isolates, 95 (96.0%) carried mutations in both rpoB and katG. No mutations were detected among the pan-susceptible isolates. The most common mutations among RIF- and INH-resistant isolates were in codon 531 of the rpoB gene (55.6%; 55/99) and codon 315 of the katG gene (94.9%; 94/99), respectively. Distinctly, katG mutations were predominantly high among Zambian isolates (96.0%) compared with other countries in the region. CONCLUSION: Resistance-associated mutations to RIF and INH circulating in Zambia are similar to those reported globally, therefore these data validate the applicability of molecular diagnostic tools in Zambia. However, katG mutations were predominantly high among M. tuberculosis isolates in this study compared with other regional countries and might distinguish cross-boundary transmission of MDR-TB from other African nations.

High diversity of multidrug-resistant Mycobacterium tuberculosis Central Asian Strain isolates in Nepal.

OBJECTIVES: Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) poses a major public health problem in Nepal. Although it has been reported as one of the dominant genotypes of MTB in Nepal, little information on the Central Asian Strain (CAS) family is available, especially isolates related to multidrug resistance (MDR) cases. This study aimed to elucidate the genetic and epidemiological characteristics of MDR CAS isolates in Nepal. METHODS: A total of 145 MDR CAS isolates collected in Nepal from 2008 to 2013 were characterized by spoligotyping, mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) analysis, and drug resistance-associated gene sequencing. RESULTS: Spoligotyping analysis showed CAS1_Delhi SIT26 as predominant (60/145, 41.4%). However, by combining spoligotyping and MIRU-VNTR typing, it was possible to successfully discriminate all 145 isolates into 116 different types including 18 clusters with 47 isolates (clustering rate 32.4%). About a half of these clustered isolates shared the same genetic and geographical characteristics with other isolates in each cluster, and some of them shared rare point mutations in rpoB that are thought to be associated with rifampicin resistance. CONCLUSIONS: Although the data obtained show little evidence that large outbreaks of MDR-TB caused by the CAS family have occurred in Nepal, they strongly suggest several MDR-MTB transmission cases.