Genotypic diversity of Mycobacterium tuberculosis in Pretoria

Genotypic diversity of Mycobacterium tuberculosis in Pretoria P Hove, J Molepo, S Dube, M Nchabeleng Prisca Hove, HBMLS, MSc(Med)Micro, Postgraduate Student Julitha Molepo, NDMedTech, BSc (Hons), MSc, PhD, Senior Scientist Samukeliso Dube, MBCHB, MPH, Senior Lecturer, Research Physician Maphoshane Nchabeleng, MBChB, MMed(Micro), Dip HIVMan, Postgrad Diploma(Inf PrevControl), Head of Department Department of Microbiological Pathology, University of Limpopo (Medunsa Campus) E-mail: molepo@ul.ac.za Keywords: molecular epidemiology, spoligotyping, Mycobacterium tuberculosis, drug susceptibility, genotypic diversity, Pretoria Tuberculosis is a global health problem. Continuous efforts are needed to understand the genetic diversity and geographical distribution of Mycobacterium tuberculosis. The objective of this study was to determine the genetic diversity of M. tuberculosis strains in Soshanguve, Pretoria. Eighty-nine isolates that were sputum culture-positive in Mycobacterium Growth Indicator Tube 960, and positively identified by Accuprobe Probe assay as M. tuberculosis complex, were used in the study. The samples were sub-cultured on Lowenstein-Jensen (L-J) slants to ensure purity. Spoligotyping was performed, with slight modifications according to the manufacturer s specifications. Genotypic data were compared to the international spoligotyping database 4 (SpolDB4) and that suggested by Streicher et al. Spoligotyping identified 12 genotypes. Of the 89 isolates studied, 75 could be grouped into 11 clusters. The Beijing genotype family formed the largest group, with 21 isolates (28%). The remaining isolates were distributed among the Latino-American-Mediterranean (LAM) family: LAM3 (13%), LAM4 (4%), LAM9 (3%); the T family: T1 (23%), T2 (9%), and T3 (3%); the S family (8%); the X3 family (4%); CAS1-KILI (3%); and LAM11-ZWE (3%). Fourteen (16%) of the isolates had spoligotypes that did not match any of the spoligotype patterns deposited in the SpolDB4 database. Beijing was the most common genotype family, identified in 28% of the cases, followed by T1 (23%). The high prevalence of Beijing and T1 in this study reflects transmission of these genotype families within this community. The results of this study showed the dire need for more robust prevention strategies in tuberculosis control programmes. Other genotyping methods with a higher discriminatory power, such as restriction fragment length polymorphism, will be useful in defining the transmission patterns in this community. Peer reviewed. (Submitted: 2011-03-18. Accepted: 2011-09-05.) SAJEI 2012;27(2):77-83 Introduction Despite the availability, for over 50 years, of effective antibiotic treatment of tuberculosis, every 15 seconds someone dies of the disease. 1 The incidence of tuberculosis has escalated from less than 200/100 000 persons per year in 1990, to more than 500/100 000 persons per year in 2006 in Africa s human immunodeficiency virus (HIV) burdened regions, which include sub-saharan Africa. 2,3 South Africa is among the 22 highest burdened countries affected by tuberculosis, with a very high number of cases relative to the total population. 4 The population of South Africa makes up 0.7% of the world s population, and yet contributes 28% of the number of HIVpositive tuberculosis cases worldwide, and 33% of HIV-positive tuberculosis cases in Africa. 3 The incidence of tuberculosis in South Africa is estimated to be more than 940/100 000 persons per year, while the mortality rate is estimated to be 135/100 000 persons per year. 5 The World Health Organization 2008 report showed a mortality rate of 218/100 000 persons per year, which is a serious cause for concern. The emergence of multidrug-resistant (MDR) tuberculosis and extensively drug-resistant (XDR) tuberculosis (defined as resistant to Isoniazid and Rifampicin, in addition to any fluoroquinolones, and at least one injectable antituberculosis drug), 6 has worsened tuberculosis morbidity and mortality. 2 Molecular epidemiological techniques, such as IS6110- based restriction fragment length polymorphism (RFLP) and spoligotyping used for typing Mycobacterium tuberculosis, have expanded our ability to investigate and understand the tuberculosis epidemic. 7 The use of the IS6110 RFLPtyping method for differentiating M. tuberculosis strains is well documented. 8-12 However, RFLP analysis is labourintensive, has poor inter-laboratory reproducibility, and requires weeks of incubation for sufficient quantities of deoxyribonucleic acid (DNA). Thus, time remains a limiting factor in obtaining a highly effective method for epidemiological surveys, and preventing the spreading of disease. 77 2012;27(2)

Spoligotyping, a method for simultaneous detection and typing of M. tuberculosis complex bacteria, has been developed, 13-15 and is used as a marker to study the phylogeny of the M. tuberculosis complex. 16 This technique has been described as a highly practical, fast, and precise polymerase chain reaction- (PCR) based method, which exploits the DNA polymorphism in the direct repeat (DR) region of M. tuberculosis complex strains. Strains vary in the number of DRs, and in the presence or absence of particular spacers. These advantages make it an ideal molecular epidemiological tool. 13 The discriminatory power of spoligotyping is reported to be lower than that of RFLP. 17 However, despite this limitation, spoligotyping remains an important tool in genotyping clinical isolates in different epidemiological settings. 18,19 The Beijing strain of M. tuberculosis, which is thought to be virulent, has been associated with drug resistance. 20-25 If these strains have an enhanced capacity to gain resistance, this will have serious consequences for the treatment of tuberculosis. It is relevant to know whether specific genotype families are overrepresented, and whether these strains are successfully transmitted within the community. The purpose of this study was to determine the genetic diversity of M. tuberculosis strains in Soshanguve, Pretoria. Method Sample collection Convenience sampling was carried out, and 89 consecutive M. tuberculosis isolates from sputum cultures were collected, regardless of patient type, from the Dr George Mukhari National Health Laboratory Services mycobacteriology laboratory between June-August 2008, and included in the study. The isolates were collected from Mycobacterium Growth Indicator Tube (MGIT) 960 tubes after positive identification as M. tuberculosis complex using Accuprobe assay (Gene-probe, San Diego, California, USA). The study was approved by the University of Limpopo (Medunsa Campus) research and ethics committee. The samples were recovered by subculturing 0.4 ml of the MGIT culture on L-J slants to rule out contamination, and to ensure the availability of samples for further testing. The samples were inoculated in duplicate to ensure maximum recovery. The slants were placed on their sides, such that the solid medium was covered by the MGIT culture, and incubated for 24 hours at 37ºC. The cultures were then incubated for up to six weeks, or until confluent growth was observed. Spoligotyping DNA was extracted from the M. tuberculosis isolates as previously described. 26 Spoligotyping was performed with slight modifications according to the manufacturer s instructions (Isogen Biosolutions, Ijsseltein, the Netherlands), and as previously described. 12 Briefly, the DR region of the tuberculosis genome was amplified using primers, DRa and DRb, and the amplified biotinylated products were hybridised to a set of 43 oligonucleotides covalently bound to a membrane. The hybridised PCR products were then incubated with a streptavidin-peroxidase conjugate, and the membrane was then exposed to chemiluminescence (Amersham Buster, Braunschweig, Germany), and exposed on an X-ray film (Amersham Hyperfilm ECL, Braunschweig, Germany) according to the manufacturer s instructions. The X-ray film was developed using standard photochemical procedures. DNA extracts of M. tuberculosis H37Rv and M. bovis BCG were used as controls. Drug susceptibility Susceptibility testing was carried out, using the Middlebrook 7H9 (Becton Dickinson, Johannesburg, South Africa) proportion method, according to the manufacturer s instructions, and Clinical and Laboratory Standards Institute guidelines. First-line drugs, Streptomycin, Rifampicin, Isoniazid and Ethambutol were tested. Statistical analyses All patients included in this study were classified into two groups, characterised by clustered and nonclustered M. tuberculosis isolates. A cluster was defined as two or more patients strains with identical genetic patterns, and patients strains with unmatched genetic profiles were considered to be nonclustered. If the isolate did not have a matching pattern in the SpolDB4, it was defined as an orphan. Clusters were assumed to have arisen from recent transmission, and the clustering rate was used to determine the amount of recent transmission in this population. 12 The patients strains with the same genetic pattern represented an epidemiologically linked cluster. The association between clustering and drug resistance was tested in a bivariate analysis in order to test for independent risk factors for recent transmission. The data were analysed using EPI Info version 6.0 and SPSS version 17.0 (SigmaStat, SPSS, Chicago, Illinois, USA). Results Spoligotyping Isolates were assigned to specific genotype families according to their spoligotype signature, using the SpolDB4 database. 27 The following 11 genotype families were identified: Beijing, T1, T2, T3, LAM3, LAM4, LAM9, LAM11-ZWE, CAS1-KILI, H1 and X3 (Table I). Fourteen (16%) of the patterns were orphans, one of which was classified as F15, 39 according to Streicher et al (Table II). Spoligotype clusters among the study population Of the 89 isolates studied, 75 could be grouped into 13 clusters. The Beijing genotype family formed the largest group, with 21 isolates (28% of the clustered isolates). The cluster sizes ranged from 2-21 patterns per cluster. The isolates were 78 2012;27(2)

Table I: Spoligotype clusters observed in the study population (n = 75) Spoligotype family n (%) clusters Spoligopattern SIT a LAM3 10 (13%) 33 Beijing 21 (28%) 1 T2 7 (9%) 52 S 6 (8%) 34 LAM9 2 (3%) 1 530 T1 17 (23%) 53 X3 3 (4%) 92 LAM4 3 (4%) 60 CAS1-KILI 2 (3%) 21 T3 2 (3%) 37 LAM11-ZWE 2 (3%) 59 Unique 2 (3%) Orphan Total 75 a = shared international types Table II: Spoligopatterns of isolates with no match in the international spoligotyping database 4 n Spoligotype description binary SpolDB4 a Streicher et al 39 2 Not in SpolDB4 F15 a = international spoligotyping database 4 distributed among the Latino-American-Mediterranean (LAM) family: LAM3 (13%), LAM4 (4%), LAM9 (3%); the T family: T1 (23%), T2 (9%) and T3 (3%); the S family (8%); the X3 family (4%); CAS1-KILI (3%); and LAM11-ZWE (3%); and one cluster was an orphan (3%) (Table II). Drug susceptibility All isolates belonging to the LAM3, S, LAM9, X3, LAM4, CAS1-KILI and LAM11-ZWE families were susceptible to all drugs. Of the 21 isolates belonging to the Beijing family, two (10%) were resistant to Isoniazid (with one being monoresistant), one (5%) to Rifampicin, and three (14%) to Ethambutol. Only one (5%) of these isolates was MDRtuberculosis. In the T1 family, one (6%) of the 17 isolates was MDR-tuberculosis, one (6%) was resistant to Ethambutol, and one (6%) was resistant to Streptomycin. One of the two isolates belonging to T3 was resistant to Isoniazid, and both were resistant to Ethambutol. Seven isolates belonging to T2 had two (29%) resistant to Isoniazid, one (14%) resistant to Rifampicin, and two (29%) resistant to Ethambutol (Table I). Discussion The genotypic diversity of M. tuberculosis strains in an urban area of Soshanguve in Pretoria was described. The results of this study were compared with those of other South African studies where the distribution of M. tuberculosis genotypes was detailed. 20,25,28 79 2012;27(2)

Table III: Drug susceptibility results among isolates belonging to different families Spoligotype family n (%) clusters S a I b R c E d LAM3 10 (13%) S S S S Beijing 21 (28%) S R (10%) R (5%) R (14%) T2 7 (9%) S R (29%) R (14%) R (29%) S 6 (8%) S S S S LAM9 2 (2.5%) S S S S T1 17 (22%) R (6%) R (6%) R (6%) R (6%) X3 3 (4%) S S S S LAM4 3 (4%) S S S S CAS1-KILI 2 (2.5%) S S S S T3 2 (2.5%) S R (50%) S R (100%) LAM11-ZWE 2 (2.5%) S S S S a = Streptomycin, b = Isonioazid, c = Rifampicin, d = Ethambutol, e = susceptible, f = resistant In the present study, the Beijing strain was found to be the most common genotype family, identified in 28% of cases. This finding is similar to that reported in several previous overseas 29-33 and South African studies. 20,25 In a South African study by Nicol et al, 28 LAM3 was found to be the highest genotype (31%), followed by Beijing (23%). However, other studies have reported a lower Beijing strain prevalence. 34-38 In the Western Cape, the F11 strain has been found to be the dominant strain. 39 The high rate of Beijing genotype strains reported here poses a cause for concern, as this family is implicated in drug resistance and is reported to be highly virulent. 40 Most of the isolates in the study clustered into the Beijing family, and this association of the Beijing genotype with clustering suggests that these strains are being transmitted throughout the community. The results may have been overestimated due to the low discriminatory power of spoligotyping which was the only genotyping method used in this study. In this investigation, 89% of the isolates were grouped into clusters. The presence of this high clustering rate suggests that there is ongoing transmission in the study population, and these results are consistent with previous reports. 22,41 The clustering rate in the present study is considerably higher than that reported in a study conducted in Botswana, which had a clustering rate of approximately 42%, 42 although these investigators used RFLP, which has a higher discriminatory power, compared to spoligotyping. 17 Only five per cent of the 21 isolates belonging to the Beijing family were MDR-tuberculosis (see Table I), and this is in contrast to reports from previous studies, where a high association between drug resistance and the Beijing genotype was reported. 24,25,29,43 Marais et al 44 also found the Beijing strain to be overrepresented in children with drug-resistant tuberculosis in the Western Cape. In the present study, the Beijing genotype was found in ages ranging from 15-60 years (data not shown). This is in contrast to studies from Vietnam and Russia, where the Beijing genotype was associated with a young age in adult studies, 33 suggesting a more recent spread. The T1 family was the second most common genotype family in the present study (23%). This result is in contrast to a report by a recent South African study, where drug-resistant isolates belonging to the T lineage occurred with the highest frequency in two provinces (32% and 40%). 45 In that study, the Beijing isolates accounted for 10.3% of the total number of isolates tested, and had the second most prevalent genotype after T1, which was in concordance with numbers reported in other studies. 27,46 The remaining isolates were distributed among the LAM family: LAM3 (13%), LAM4 (4%), LAM9 (3%); the T family: T1 (23%), T2 (9%), and T3 (3%); the S family (8%); the X3 family (4%); CAS1-KILI (3%); and LAM11-ZWE (3%). T2 had the fourth highest frequency in this study (9%). This strain was also found in a study in Cameroon patients with pulmonary tuberculosis 47 and in a study in Cape Town. 20 The LAM3 strain is widely distributed worldwide and has been implicated in the tuberculosis epidemic in South Africa, and particularly the Western Cape province. 48 LAM4 and LAM9 have been found previously in XDR-tuberculosis patients in South Africa, 25 and also in studies conducted in Zimbabwe, Zambia and the Western Cape, South Africa. 49 The limitation of this study was the sole use of the spoligotyping method for the genotyping of strains, which has been reported to have a low discriminatory power than that of RFLP. 17 If both methods were used, different results may have been obtained. Conclusion Beijing was the most common genotype family, identified in 28% of the cases, followed by T1 (23%). The high prevalence of Beijing and T1 in this study, and the high clustering rates, may reflect that there is ongoing transmission of 80 2012;27(2)

these genotype families within this community. However, a conclusion cannot be made due to the low discriminatory power of spoligotyping. If confirmatory genotyping methods, with a higher discriminatory power such as RFLP IS6110 were also used in the study, different results may have been obtained. The results of this study emphasise the need for continuous surveillance programmes to monitor tuberculosis control efforts, especially in tuberculosis- and HIV-endemic areas, such as the area that was studied. Acknowledgements The authors are thankful to Mr Tom Letswalo of the Microbiological Pathology Laboratory, National Health Laboratory Services, Dr George Mukhari Laboratory, Pretoria, for assistance in the collection of samples. The authors extend their thanks to Prof Rob Warren of the Centre of Excellence for Biomedical Tuberculosis Research, Stellenbosch University, and his team for providing guidance and resources for the spoligotyping. 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