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

Abstract

Every year close to 9 million people contract tuberculosis (TB) and approximately 2 million die from the disease. The highest number of TB cases is in Asia while Africa has the highest incidence rates due to high rates of HIV and malnutrition that weakens the immune systems and speeds up the spread of the disease. The management of TB has faced many challenges in the past but the two most important threats to global TB control are the HIV epidemic and the increasing prevalence of drug resistance. The occurrence and transmission of Mycobacterium tuberculosis (M. tuberculosis) strain families vary by countries or by regions within the same country. Correct identification of M. tuberculosis strain families in a given geographical area is therefore, important for epidemiological investigation. Molecular typing of M. tuberculosis isolates has facilitated the understanding of the epidemiology of TB, its control and prevention. The insertion sequence 6110 restriction fragment length polymorphism (IS6110 RFLP) has been considered the „gold standard‟ in M. tuberculosis genotyping due to its high discriminatory power. However, due to limitations, such as the requirement of large quantities of DNA, several polymerase chain reaction (PCR) based genotyping methods have been developed. These methods include spoligotyping and mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR). Spoligotyping is widely used because of its low cost, high reproducibility, simplicity and ease of interpretation due to its binary results format, while the MIRU-VNTR assay is robust, reliable and easier to perform compared to IS6110 RFLP typing. In South Africa, M. tuberculosis genotyping assays have been applied in only a few provinces, such as the Western Cape and KwaZulu-Natal. The purpose of this study was to determine the prevalence of the M. tuberculosis strains circulating in the Kalafong Hospital in Pretoria, Gauteng province by using spoligotyping and MIRU-VNTR typing methods. Spoligotyping identified 39 distinct spoligotypes of which 36% (14/39) were unreported in the SITVIT2 database. There were three strain families that were found to be represented by most of the isolates in the study („ill-defined‟ T, Beijing and LAM). These strain families fall within the major families of the M. tuberculosis strains (Brudey et al., 2006). The T1 subfamily, 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 of which 79 were unique patterns and the remaining eight were represented by 21 clustered isolates. 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 allelic diversity of 0.743. The combination of spoligotyping and MIRU-VNTR typing data resulted in a 0.998 discriminative power. Combining the two methods proved to result in a higher discriminatory power than using the methods individually. Using these typing methods, the study has identified the most prevalent circulating M. tuberculosis strain families, subfamilies and variants in patients seeking medical attention at the Kalafong Hospital. The study has shown that the use of two molecular genotyping methods improves the discriminatory power of the techniques. Hence, these genotyping methods can be used as an alternative for the IS6110 RFLP typing method to analyse M. tuberculosis strains from clinical settings.