Genetic relatedness of Mycobacterium tuberculosis strains obtained from Kalafong Hospital in Pretoria using Spoligotyping and MIRU-VNTR typing

[摘要] Every year close to 9 million people contract tuberculosis (TB) and approximately 2 milliondie from the disease. The highest number of TB cases is in Asia while Africa has the highestincidence rates due to high rates of HIV and malnutrition that weakens the immune systemsand speeds up the spread of the disease. The management of TB has faced many challengesin the past but the two most important threats to global TB control are the HIV epidemic andthe increasing prevalence of drug resistance.The occurrence and transmission of Mycobacterium tuberculosis (M. tuberculosis) strainfamilies vary by countries or by regions within the same country. Correct identification ofM. tuberculosis strain families in a given geographical area is therefore, important forepidemiological investigation. Molecular typing of M. tuberculosis isolates has facilitated theunderstanding of the epidemiology of TB, its control and prevention.The insertion sequence 6110 restriction fragment length polymorphism (IS6110 RFLP) hasbeen considered the „gold standard‟ in M. tuberculosis genotyping due to its highdiscriminatory power. However, due to limitations, such as the requirement of largequantities of DNA, several polymerase chain reaction (PCR) based genotyping methods havebeen developed. These methods include spoligotyping and mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR). Spoligotyping is widelyused because of its low cost, high reproducibility, simplicity and ease of interpretation due toits binary results format, while the MIRU-VNTR assay is robust, reliable and easier toperform compared to IS6110 RFLP typing.In South Africa, M. tuberculosis genotyping assays have been applied in only a fewprovinces, such as the Western Cape and KwaZulu-Natal. The purpose of this study was todetermine the prevalence of the M. tuberculosis strains circulating in the Kalafong Hospital inPretoria, Gauteng province by using spoligotyping and MIRU-VNTR typing methods.Spoligotyping identified 39 distinct spoligotypes of which 36% (14/39) were unreported inthe SITVIT2 database. There were three strain families that were found to be represented bymost of the isolates in the study („ill-defined‟ T, Beijing and LAM). These strain families fallwithin the major families of the M. tuberculosis strains (Brudey et al., 2006). The T1subfamily, which is a member of the „ill-defined‟ T family had the highest number of isolates(19). In the 12 loci based MIRU-VNTR typing analysis, 87 distinct patterns were obtained ofwhich 79 were unique patterns and the remaining eight were represented by 21 clusteredisolates. The 12 MIRU loci included were 02, 04, 10, 16, 20, 23, 24, 26, 27, 31, 39 and 40.The MIRU locus 10 was found to be the most discriminatory among the 12 loci with an allelicdiversity of 0.743. The combination of spoligotyping and MIRU-VNTR typing data resultedin a 0.998 discriminative power. Combining the two methods proved to result in a higherdiscriminatory power than using the methods individually.Using these typing methods, the study has identified the most prevalent circulatingM. tuberculosis strain families, subfamilies and variants in patients seeking medical attentionat the Kalafong Hospital. The study has shown that the use of two molecular genotypingmethods improves the discriminatory power of the techniques. Hence, these genotypingmethods can be used as an alternative for the IS6110 RFLP typing method to analyseM. tuberculosis strains from clinical settings.

[发布机构] University of Pretoria