The Second Zambian National Tuberculosis Drug Resistance survey - a comparison of conventional and molecular methods.

OBJECTIVE: The prevalence of MDR-TB in Zambia was estimated to be 1.8% in 2001. A second drug resistance survey was conducted in 2008 to determine trends; the use of the Genotype MTBDRplus assay was applied to compare results to the gold standard. METHOD: A two-stage cluster sampling, with health facilities as primary sampling units. Processed sputum specimens were inoculated on solid media for culture; heat-inactivated bacterial suspensions from sputum samples were tested on a commercial line probe assay for the identification of rifampicin and isoniazid resistance. RESULTS: A total of 917 patients with TB were enrolled and 883 (96.3%) analysed. A total of 574 (65%) had LJ results and 824 (93.3%) had results from MTBDRplus assay. The median age was 32, and 63.3% were males. MDR-TB according to LJ-based DST was 1.1% (CI 0.1-2.4) whereas according to MDTBDRplus assay was 1.6% (CI 0.6-2.6). Isoniazid monoresistance in new cases was 2.4% (CI 0.613-4.26) based on LJ results and 5.0% (CI 3.2-6.7) based on the MTBDRplus; in retreatment cases, it was 4.4% (CI 0.3-8.6) and 2.40% (CI <0.1-5.1) on LJ and MTBDRplus, respectively. Rifampicin monoresistance in new cases was 0.1% (CI <0.1-0.4) based on LJ and 0.6% (CI 0.01-1.1) based on the MTBDRplus; in retreatment cases, it was 0% (CI 0-3.8) and 1.8% (CI <0.1-4.0) on LJ and MTBDRplus, respectively. There were no XDR-TB cases found and no association between MDR-TB and HIV. CONCLUSION: There was no increase in MDR-TB prevalence in Zambia from 2001 to 2008; results from the two methods were similar. Molecular methods were quicker and simpler to use.

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.

Characterization of Mutations Associated with Streptomycin Resistance in Multidrug-Resistant Mycobacterium tuberculosis in Zambia.

Streptomycin (STR) is recommended for the management of multidrug-resistant tuberculosis (MDR-TB). Streptomycin resistance-conferring mutation types and frequency are shown to be influenced by genotypes of circulating strains in a population. This study aimed to characterize the mutations in MDR-TB isolates and examine their relationship with the genotypes in Zambia. A total of 138 MDR-TB isolates stored at the University Teaching Hospital Tuberculosis Reference Laboratory in Zambia were analyzed using spoligotyping and sequencing of STR resistance-associated genes. Streptomycin resistance was observed in 65.9% (91/138) of MDR-TB isolates. Mutations in rpsL, rrs, and gidB accounted for 33%, 12.1%, and 49.5%, respectively. Amino acid substitution K43R in rpsL was strongly associated with the CAS1_Kili genotype (p < 0.0001). The combination of three genes could predict 91.2% of STR resistance. Clustering of isolates based on resistance-conferring mutations and spoligotyping was observed. The clustering of isolates suggests that the increase in STR-resistant MDR-TB in Zambia is largely due to the spread of resistant strains from inadequate treatment. Therefore, rapid detection of STR resistance genetically is recommended before its use in MDR-TB treatment in Zambia.

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.

Genotypic characterization of pyrazinamide resistance in Mycobacterium tuberculosis isolated from Lusaka, Zambia.

Pyrazinamide forms a core part of treatment for all types of tuberculosis (TB) in Zambia. Due to challenges associated with pyrazinamide testing, little information is available to indicate the frequency of resistance to this drug in Zambia. To determine the frequency of pyrazinamide (PZA) resistance and its correlation with mutation in pncA in Mycobacterium tuberculosis isolated from patients in Lusaka, Zambia, BACTEC MGIT M960 was used for phenotypic PZA susceptibility testing while sequencing was used to determine resistance-conferring mutations in the pncA. Of the 131 isolates analyzed, 32 were phenotypically resistant to PZA. Among multidrug-resistant (MDR) M. tuberculosis isolates, the frequency of PZA resistance was 21 of 35 (58.3%). And 27 of 32 PZA resistant isolates had mutations in the pncA that seem to confer resistance. With BACTEC MGIT 960 as the reference standard, gene sequencing showed 84.4% sensitivity and 100% specificity. Nine new mutations were identified and the single nucleotide substitution T104G and C195T were the most frequent mutations. However, they were observed in both susceptible and resistant strains and indicating that they are non-resistance conferring mutations. This study has demonstrated that PZA susceptibility testing is necessary especially in patients suffering from MDR-TB as approximately half of the patients have PZA resistant TB. Similar studies will have to be carried out in other provinces to get an accurate estimate of PZA resistance in Zambia. Mutations in pncA were the major mechanism of PZA resistance with no involvement of rpsA and panD genes. However, the presence of mutations among phenotypically PZA susceptible M. tuberculosis isolates makes it challenging to independently use genotyping method for the determination of PZA resistance.

The Prevalence of Tuberculosis in Zambia: Results from the First National TB Prevalence Survey, 2013-2014.

BACKGROUND: Tuberculosis in Zambia is a major public health problem, however the country does not have reliable baseline data on the TB prevalence for impact measurement; therefore it was among the priority countries identified by the World Health Organization to conduct a national TB prevalence survey. OBJECTIVE: To estimate the prevalence of tuberculosis among the adult Zambian population aged 15 years and above, in 2013-2014. METHODS: A cross-sectional population-based survey was conducted in 66 clusters across all the 10 provinces of Zambia. Eligible participants aged 15 years and above were screened for TB symptoms, had a chest x-ray (CXR) performed and were offered an HIV test. Participants with TB symptoms and/or CXR abnormality underwent an in-depth interview and submitted one spot- and one morning sputum sample for smear microscopy and liquid culture. Digital data collection methods were used throughout the process. RESULTS: Of the 98,458 individuals who were enumerated, 54,830 (55.7%) were eligible to participate, and 46,099 (84.1%) participated. Of those who participated, 45,633/46,099 (99%) were screened by both symptom assessment and chest x-ray, while 466/46,099 (1.01%) were screened by interview only. 6,708 (14.6%) were eligible to submit sputum and 6,154/6,708 (91.7%) of them submitted at least one specimen for examination. MTB cases identified were 265/6,123 (4.3%). The estimated national adult prevalence of smear, culture and bacteriologically confirmed TB was 319/100,000 (232-406/100,000); 568/100,000 (440-697/100,000); and 638/100,000 (502-774/100,000) population, respectively. The risk of having TB was five times higher in the HIV positive than HIV negative individuals. The TB prevalence for all forms was estimated to be 455 /100,000 population for all age groups. CONCLUSION: The prevalence of tuberculosis in Zambia was higher than previously estimated. Innovative approaches are required to accelerate the control of TB.

Isolation and molecular characterization of Mycobacterium tuberculosis from humans and cattle in Namwala District, Zambia.

Mycobacterium tuberculosis, the causative agent of tuberculosis in humans, is considered primarily a human pathogen. It has, however, been reported in a wide range of domestic and wild animals, often living in close prolonged contact with humans. Sputum samples in which acid fast bacteria were detected in smears were collected from patients at three health facilities in Namwala district, Zambia. Samples from cattle presenting gross lesions compatible with bovine tuberculosis were collected at a local abattoir in the same district. Isolated mycobacteria were identified and genotyped using classical molecular methods. From a total of 33 isolates of M. tuberculosis detected (30 from humans and 3 from cattle), two cattle isolates shared the same spoligotype and MIRU-VNTR pattern with a human patient. This study has for the first time documented the isolation of M. tuberculosis from cattle in Zambia and provides molecular evidence of an epidemiological link between M. tuberculosis isolates from humans and cattle in Namwala district. A possible spill back of M. tuberculosis to humans cannot be excluded and therefore further studies documenting to what extent M. tuberculosis is shed in cattle milk are needed. This finding further suggests that veterinary public health measures to control human TB, should also take into account the bovine reservoir.

Characterization of Mycobacterium bovis from Humans and Cattle in Namwala District, Zambia.

Tuberculosis remains a major public health problem in Zambia. While human to human transmission of Mycobacterium tuberculosis is of major importance in driving the tuberculosis epidemic, the impact of Mycobacterium bovis transmission from infected cattle is largely unknown. This cross-sectional study aimed at molecular characterization of M. bovis in humans and cattle. A total of 100 human sputum samples and 67 bovine tissues were collected and analyzed for the presence of mycobacteria. Of 65 human samples that harbored acid fast bacteria (AFB), 55 isolates were obtained of which 34 were identified as M. tuberculosis and 2 as M. bovis. AFB-positive bovine samples (n = 67) yielded 47 mycobacterial isolates among which 25 were identified as M. bovis and no M. tuberculosis was found. Among the M. bovis isolates, spoligotyping revealed a high homogeneity in genotypes circulating in Namwala district. Human and cattle isolates shared identical MIRU-VNTR genotypes, suggesting that transmission between the two hosts may occur. Therefore, this study has documented zoonotic TB in human patients in Namwala district of Zambia. However, further molecular epidemiological studies in the study area are recommended.