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J Clin Microbiol. Jun 2009; 47(6): 1848–1856.
Published online Apr 22, 2009. doi:  10.1128/JCM.02167-08
PMCID: PMC2691085

High Diversity of Mycobacterium tuberculosis Genotypes in South Africa and Preponderance of Mixed Infections among ST53 Isolates [down-pointing small open triangle]

Abstract

The reemergence of tuberculosis (TB) has become a major health problem worldwide, especially in Asia and Africa. Failure to combat this disease due to nonadherence or inappropriate drug regimens has selected for the emergence of multiple-drug-resistant (MDR) TB. The development of new molecular genotyping techniques has revealed the presence of mixed Mycobacterium tuberculosis infections, which may accelerate the emergence of drug-resistant strains. There are some studies describing the local distribution of circulating strains in South Africa, but to date, reports describing the frequency and distribution of M. tuberculosis genotypes, and specifically MDR genotypes, across the different provinces are limited. Thus, 252 isolates (of which 109 were MDR) from eight of the nine provinces of South Africa were analyzed by spoligotyping. Spoligotyping showed 10 different lineages, and ST53 (11.1%) and ST1 (10.3%) were the most frequent genotypes. Of the 75 different spoligopatterns observed, 20 (7.9%) were previously unreported. Analysis of the mycobacterial interspersed repetitive units of variable-number tandem repeats of the ST53 and ST1 isolates revealed that ~54% of the ST53 isolates were of mixed M. tuberculosis subpopulations. Drug resistance (defined as resistance to at least isoniazid and/or rifampin) could only be linked to a history of previous anti-TB treatment (adjusted odds ratio, 4.0; 95% confidence interval, 2.27 to 7.10; P = <0.0001). This study describes a high diversity of circulating genotypes in South Africa in addition to a high frequency of mixed M. tuberculosis subpopulations among the ST53 isolates. MDR TB in South Africa could not be attributed to the spread of any single lineage.

Tuberculosis (TB) is a major cause of illness and death worldwide but especially in Asia and Africa (42). Twenty-two countries designated high TB burden countries account for 80% of all new cases worldwide (42). As of 2008, South Africa was ranked fourth among these, with an incidence rate of 940 cases per 100,000 persons (42) (up from 536 in 2005 [41]). Due to nonadherence to drug regimens or the use of inappropriate drug regimens, the TB epidemic has been largely exacerbated by the emergence of multidrug-resistant (MDR) TB (7).

Traditionally, it has been assumed that TB is caused by an infection with a single strain and that recurrences are the result of reactivation of the strain causing the first episode (6, 24, 39). However, it has recently been shown that patients, both human immunodeficiency virus (HIV) positive and HIV negative, in high-incidence settings may have more than one strain in the same sputum sample (24, 39) and that mixed infections may cause complications in the treatment of the disease if a patient is infected with both a sensitive and a resistant Mycobacterium tuberculosis isolate (39). Extensively drug-resistant (XDR) TB strains (defined as resistant to isoniazid [INH] and rifampin [RIF], in addition to any fluoroquinolones and at least one injectable anti-TB drug) (5) were first reported in South Africa in 2006 and later shown to be present in at least 17 countries and on all of the continents (9). Spoligotyping has revealed that the majority of these cases were caused by M. tuberculosis belonging to the KZN family (ST60), which has been known to be prevalent in this area since 1994 (23). Since then, only one other study has been published (19) providing genotypic information on XDR TB strains in South Africa. That study shows that XDR TB strains in South Africa belong to seven different lineages and are present in four of the nine provinces. Such studies highlight the need for standardized and accurate drug susceptibility testing in combination with high-level molecular genotyping in order to carefully monitor new and emerging MDR and XDR TB strains.

The “gold standard” for the typing of M. tuberculosis is currently IS6110-based restriction fragment length polymorphism (RFLP). Combined with spoligotyping, this has proven to be very useful for the study of the transmission, evolution, and phylogeny of M. tuberculosis (18). However, RFLP is laborious, requires a large amount of DNA, and has poor interlaboratory reproducibility. Recently, a new genotyping technique based on PCR amplification of mycobacterial interspersed repetitive units of variable-number tandem repeats (MIRU-VNTR) was introduced (31, 32). This method is much faster than IS6110 RFLP and requires less DNA. Fifteen-locus-based MIRU-VNTR analysis has been shown to have slightly better discriminatory power than IS6110 RFLP, especially when combined with spoligotyping (2, 22). In addition to being a rapid and highly discriminatory genotyping method, MIRU-VNTR can also be used for the detection of mixed subpopulations in a single sputum sample (1, 26). Several studies have investigated the differentiation power of different MIRU-VNTR locus combinations for strains of the Beijing lineage (11, 14, 15). The studies suggest that the choice of appropriate MIRU-VNTR loci requires further investigation in diverse M. tuberculosis lineages in countries with low and high TB endemicity.

Thus, the objectives of this study were to assess the distribution and diversity of MDR M. tuberculosis genotypes across the South African provinces and to determine if there is an association between MDR TB and a particular M. tuberculosis genotype. We also determined the general population structure of South African M. tuberculosis isolates, irrespective of the drug susceptibility pattern. Furthermore, we assessed the ability of the MIRU-VNTR method to discriminate the most frequent genotypes observed.

MATERIALS AND METHODS

Study sample.

The M. tuberculosis isolates used in this study were collected during a national drug surveillance study conducted by the South African Medical Research Council in 2001 and 2002 according to World Health Organization guidelines. During the survey, a total of 5,866 M. tuberculosis isolates were collected from eight (Eastern Cape [EC], Limpopo [NP], North West [NW], Free State [FS], Mpumalanga [ML], Gauteng [GP], KwaZulu-Natal [KN], and Western Cape [WC]) of the nine provinces of South Africa. Drug resistance to any of the four first-line drugs INH, RIF, ethambutol, and streptomycin was found in 9.9% of the isolates. From this collection, 252 M. tuberculosis isolates were selected for genotyping. All available MDR M. tuberculosis isolates (n = 109) from the total collection of 5,866 isolates were selected for genotyping. In addition, from the same collection, a representative sample of 113 fully sensitive isolates (EC, 14; FS, 13; GP, 22; KN, 17; NP, 18; ML, 10; NW, 5; WC, 14), as well as 8 resistant to INH alone, 12 resistant to RIF alone, 8 resistant to other drug combinations, and 3 with unknown resistance), were selected for genotyping. The characteristics of the 252 isolates included in this study are provided in Table Table11.

TABLE 1.
Characteristics of M. tuberculosis isolates from the national drug resistance surveya of 2001 and 2002 included in this study

DNA isolation.

M. tuberculosis isolates were grown on Lowenstein-Jensen medium at 37°C for 3 to 4 weeks. DNA was extracted by a boiling technique in which a 1-μl loopful of bacterial cells was suspended in 200 μl of TE buffer (10 mM Tris-Cl, 1 mM EDTA) and heat killed by incubation at 95°C for 15 to 20 min (9). The supernatant containing the DNA was collected by centrifugation at 12,000 rpm for 7 min.

Spoligotyping.

Spoligotyping was performed with genomic DNA according to the internationally standardized protocol (16). Based on their spoligotype patterns, the isolates were assigned to families in accordance with previously described criteria (4).

MIRU-VNTR genotyping.

Isolates with the two spoligotype patterns most frequently observed in this study were chosen for MIRU-VNTR genotyping by amplification of 15 loci as described by Supply et al. (30). Amplification was performed in a total volume of 20 μl containing 1 μl DNA, 0.04 to 0.4 μM of all 15 primer sets, and HotStart Taq Plus polymerase Master Mix (Qiagen). All reaction mixtures were subjected to 95°C for 5 min; 30 cycles of 30 s at 94°C, 1 min at 55°C, and 1.5 min at 72°C; and 7 min at 72°C. Analysis of the genotyping results was performed by multiplex PCR with a Rox-labeled MapMarker 1000 size standard (PE Applied Biosystems) for sizing of the PCR products. The PCR fragments were analyzed with a capillary-based electrophoresis sequencer (ABI 3700), and sizing of the various VNTR alleles was done with the Peak Scanner Software v1.0 (PE Applied Biosystems). The number of repeats present at each locus was determined, and alleles were assigned numerical values accordingly. Furthermore, isolates with identical MIRU-VNTR genotypes were defined as belonging to the same cluster.

IS6110 RFLP.

Isolates that formed clusters of identical MIRU-VNTR types by 15-locus MIRU-VNTR genotyping were subjected to IS6110 RFLP analysis. IS6110 RFLP analysis was performed with PvuII-restricted DNA according to the standardized procedure described by van Embden et al. (34, 35). The enhanced chemiluminescence direct nucleic acid labeling and detection system kit (Amersham Pharmacia) was used for labeling and detection of the probe. Subsequently, the membranes were autoradiographed with Hyperfilm (Amersham Pharmacia) and the film was developed to visualize the DNA fingerprints.

Statistical analysis.

Multivariate logistic regression analysis and the chi-square exact test were performed to test for associations between lineages and drug resistance (SPSS version 14.0; Softonic). The Breslow-Day test was performed to test for homogeneity of the odds ratios (ORs), and the Mantel-Haenszel test was used to test and estimate the common OR. The Hunter-Gaston discriminatory index (HGDI) of MIRU-VNTR types was calculated as described previously (13) and categorized according to Sola et al. (27).

RESULTS

Spoligotyping.

The characteristics of the 252 isolates included in this study are provided in Table Table1.1. Of the 252 isolates spoligotyped, 231 were assigned to 55 previously described shared types belonging to 10 different lineages, i.e., Latin America and Mediterranean (LAM), T, X, Haarlem, S, MANU2, East African-Indian (EAI), Central and Middle Eastern Asian (CAS), H37Rv, and Beijing, according to a previous report (4) (Fig. (Fig.1).1). The remaining 21 isolates were previously unreported and were termed orphans. Of the 21 orphan isolates, 5 belonged to the LAM lineage, 6 belonged to the T lineage, 5 belonged to the X lineage, 2 belonged to the S lineage, 1 belonged to the Haarlem lineage, and 1 belonged to the EAI lineage (Fig. (Fig.1).1). One orphan isolate was not assigned to any lineage. Lineage T was the most frequent, making up 25% (n = 63) of the isolates, whereas MANU2 was the least frequent, making up 0.8% (n = 2). Seven isolates (including the one orphan spoligotype) could not be assigned to any of the lineages previously described, and one isolate was reported as H37Rv (Fig. (Fig.1).1). MDR was observed in isolates within all of the lineages, except for MANU2 and H37Rv. The chi-square exact test showed an association between a history of previous treatment and drug resistance for three lineages, S, T, and X (Table (Table2),2), but the association between a history of previous treatment for TB and drug resistance did not differ significantly between the lineages (test of homogeneity, P = 0.225). Multivariate logistic regression analysis showed that drug resistance (defined as collective resistance to INH, RIF, or both INH and RIF) could be linked only to a history of previous treatment (adjusted OR, 4.0; 95% confidence interval [CI], 2.27 to 7.10; P = <0.0001) and not to any of the lineages in this study.

FIG. 1.
Spoligotype patterns of the 252 M. tuberculosis isolates used in this study.
TABLE 2.
Association between drug resistance and history of previous anti-TB treatment for each lineage

Two hundred five of the 252 isolates grouped into 28 clusters (a cluster was defined as 2 or more isolates with identical spoligotype patterns), with each cluster consisting of 2 to 28 isolates (Fig. (Fig.1).1). The 47 remaining isolates showed unique spoligotype patterns (Fig. (Fig.1).1). In total, there were 75 different spoligotype patterns, including 20 which were previously unreported (orphan) (Fig. (Fig.1).1). The two largest groups, ST53 (T1 sublineage) and ST1 (Beijing lineage), consisted of 28 and 26 isolates, respectively. In four of the eight provinces (EC, KN, ML, and NW), the LAM lineage was the most common one represented (34.5%, 34.1%, 30.1%, and 30.4%, respectively). In two provinces, FS and GP, isolates belonging to the T lineage occurred with the highest frequency (32% and 40%, respectively), and in NP and WC, the X lineage was the most common (24.2% and 37%, respectively). Four lineages (Beijing, LAM, T, and X) were found in all eight provinces. The NW province showed the highest diversity of strains, with as many as 18 different spoligotype patterns among the 23 isolates examined (78.3%) (Table (Table33).

TABLE 3.
The different spoligopatterns observed among the 252 isolates in this study distributed across the 8 eight South African provinces

MIRU-VNTR genotyping.

Fifty-four isolates belonging to the two lineages most frequently found in this study, the well-defined Beijing lineage (n = 26) and the poorly defined T1 lineage (ST53; n = 28), were analyzed by 15-locus MIRU-VNTR genotyping in order to discriminate these isolates further. MIRU-VNTR genotyping of the 26 Beijing isolates showed that 15 isolates had unique MIRU types, whereas 11 isolates produced five clusters with identical MIRU types (Fig. (Fig.2).2). Eight Beijing isolates (31%) had identical copy numbers for the first 12 loci and could only be discriminated by the loci QUB-11b (ML153, WC158, and NT3) and QUB-26 (NP156 and NT157) (Fig. (Fig.2).2). Sixteen of 26 isolates could only be separated by different copy numbers in one or two loci (Fig. (Fig.2).2). Of the 28 ST53 isolates, 13 had only one allele for each of the 15 loci analyzed. All 13 isolates showed a unique MIRU type and could thus be discriminated by the 15 standard loci (Fig. (Fig.2).2). Of the 15 remaining ST53 isolates, 2 (FS203, WC84) showed two alleles each at the MIRU10 locus, 2 isolates (ML209 and GP205) showed two alleles at the ETR-A and QUB-11b loci, respectively, whereas 1 isolate (NP213) showed triple alleles at the ETR-C locus (Table (Table4).4). One isolate showed two alleles at two loci, two isolates had two alleles at three loci, two isolates had two alleles at four loci, three isolates had two alleles t six loci, and two isolates had two alleles at eight loci (Table (Table4).4). NP212 had triple alleles for ETR-A. HGDI scores for each of the 15 MIRU loci were calculated for the Beijing and ST53 isolates separately (Table (Table5)5) and categorized as highly, moderately, or poorly discriminatory by the criteria suggested by Sola et al. (27). As shown in Table Table5,5, only QUB-11b was considered highly discriminatory for the Beijing isolates. QUB-11b, in addition to MIRU40, Mtub39, and QUB-26, was also highly discriminatory for the 13 ST53 isolates that showed only one allele for each locus. MIRU4 discriminated poorly between isolates of both the Beijing and ST53 lineages.

FIG. 2.
MIRU-VNTR analysis of 26 ST1 and 13 ST53 isolates from South Africa. Five clusters with identical MIRU-VNTR types comprising 11 isolates were observed among the 26 ST1 isolates. No such clusters were observed among the ST53 isolates.
TABLE 4.
Fifteen ST53 isolates showing either clonal variation (two alleles at locus) or mixed M. tuberculosis subpopulationa
TABLE 5.
HGDI scores for 15 MIRU-VNTR locia

IS6110 RFLP.

Eleven isolates belonging to the Beijing lineage that formed five clusters of 100% identical MIRU-VNTR types were analyzed by IS6110 RFLP. IS6110 RFLP could discriminate among five isolates belonging to three different clusters; KN88 differed by one band from ML2 and ML91, GP148 and GP149 also differed by one band, whereas WC158 and NT3 differed by three bands (data not shown). However, six isolates belonging to three clusters (cluster 1, ML2 and ML91; cluster 2, NP156 and NT157; cluster 3, NP155 and EC145) could not be discriminated by IS6110 RFLP and were thus considered to be clonally related. Isolates belonging to cluster 1 were collected from two different districts in the same province (ML); however, a closer epidemiological link could not be determined. Isolates of clusters 2 and 3 were collected from different provinces.

DISCUSSION

The distribution of M. tuberculosis genotypes in some areas of South Africa has been described previously (21, 28); however, apart from a recent study that investigated the spoligotype patterns among 41 XDR M. tuberculosis isolates, there are, to our knowledge, no studies which have mapped the distribution of M. tuberculosis genotypes across the South African provinces. In this study, 252 isolates from eight of the nine provinces of South Africa were genotyped by spoligotyping. Ninety-two percent of the spoligopatterns were previously reported in SpolDB4 (4) and were assigned to 10 different lineages.

The most common lineage observed in this study was the ill-defined T lineage, which made up 25.8% of the isolates and was the most common lineage in two provinces, FS (32.0%) and GP (40.0%). The ST53 genotype (n = 28), which is a member of the T lineage, accounted for 11.1% of the total number of isolates tested and was the most frequently found genotype in this study. This is in concordance with the ranking of this genotype in SpolDB4 (4). However, as shown by 15-locus MIRU-VNTR analysis, 53.6% of the ST53 isolates in this study were of mixed M. tuberculosis subpopulations. Patients (both HIV positive and HIV negative) in high-incidence settings have been shown to be more prone to having more than one strain in the same sputum sample (24, 39), and mixed infections may cause complications in the treatment of TB (39). In this study, of the 15 ST53 isolates that were of mixed subpopulations, 8 isolates were MDR and 2 were monoresistant to RIF. Seven of the 15 patients with mixed infections were HIV positive. For the two most frequent spoligotypes observed in this study, isolates of mixed subpopulations were only seen with the ST53 genotype and not with the ST1 genotype, suggesting that laboratory cross-contamination is unlikely. The ST53 isolates are, by spoligotyping, defined by the lack of spacers 33 to 36 while having all other spacers present (8). Due to this somewhat unstringent definition, it is possible that the presence of two different genotypes in a mixed subpopulation combined may produce the same spoligopattern as the ST53 isolates when analyzed by spoligotyping, thus producing a “false” ST53 result. As previously shown (25), analysis of isolates from a single sputum specimen may underestimate the frequency of mixed infections. For this study, only one sample from each patient was available; thus, MIRU-VNTR analysis of isolates from multiple samples from each patient may give a more precise estimate of the true rate of mixed infections in South Africa. As different M. tuberculosis subpopulations in a mixed infection may have different drug susceptibility patterns, it is possible that failure to detect this heterogeneity will result in the use of an improper combination of anti-TB drugs to treat the infection.

Isolates of the Beijing lineage have been the cause of large outbreaks of TB worldwide, and although some studies have shown an association of isolates of the Beijing lineage with MDR TB (17, 29, 33), others have not found this association (12). The Beijing lineage has been shown to be the most common in the Beijing area of China, accounting for 92% of the strains identified (10), and is also highly frequent in other parts of Southeast Asia (3). In our study, the Beijing isolates accounted for 10.3% of the total number of isolates tested and had the second most prevalent genotype after ST53. This result is in concordance with numbers reported for Africa in other studies (4, 20). Additionally, unlike a study by Streicher et al. (28), our study showed no association between MDR and any single M. tuberculosis lineage.

The 15-loci MIRU-VNTR analysis of the predominant M. tuberculosis lineages in our study (26 ST1 and 13 ST53 single M. tuberculosis isolates) could distinguish between the two dominant genotypes. MIRU-VNTR analysis was highly discriminatory for the ST53 isolates; however, 42.3% of the ST1 genotype isolates could not be differentiated. Secondary analysis by IS6110 RFLP could only discriminate among 5 of the 11 clustered isolates. Our findings therefore support a previous study (11) that suggests that there are discriminatory limitations for all markers in areas where TB is highly endemic.

The LAM lineage, which made up 25% of the total number of isolates, was the most common lineage in four of the eight provinces included in this study. The LAM lineage can be split into sublineages LAM1 to LAM11. One M. tuberculosis genotype belonging to LAM3, F11, has been shown to be at least as successful as the Beijing genotype in contributing to the TB problem in the WC province of South Africa (36). Two of the characteristics of the F11 genotype are a lack of spoligotype spacers 9 to 11, 21 to 24, and 33 to 36 and a unique C-T polymorphism at position 491 of the rrs gene (36-38). In our study, 18 isolates (ST33) matched the spoligotype criteria (Fig. (Fig.1);1); however, analysis of position 491 of the rrs gene was not performed. An additional 17 isolates of the LAM and T lineages and 3 isolates belonging to undesignated lineages showed high similarity in their spoligotype patterns to the ST33/F11 genotype. Of the 18 ST33 isolates, only 1 was from the WC province, whereas the remaining 17 isolates were found in seven of the eight provinces included in this study. The 20 isolates with spoligotype patterns similar to that of ST33/F11 were found in all eight of the provinces included in this study, suggesting that variants of ST33/F11 also contribute to the TB epidemic in other areas of South Africa.

This study shows that drug resistance could only be linked to a previous history of anti-TB treatment and not to any single strain lineage. The large number of different spoligotype patterns observed in this study indicates that there is a high diversity of circulating M. tuberculosis strains in South Africa and that no single genotype dominates the TB epidemic in this area. Furthermore, we observed a high proportion of mixed M. tuberculosis infections among isolates of the most frequent genotype, ST53. The relevance of mixed infections for the patient and for TB control is not completely clear. Mixed infections could potentially accelerate the emergence of MDR TB isolates. Moreover, the high proportions of mixed infections in an area where TB is endemic may have implications for prophylactic approaches, such as new vaccines, for the control of TB.

Acknowledgments

We thank the staff of the South African Medical Research Council for the collection of isolates. We thank Geir Egil Eide for help and discussion on statistical analyses. We also thank Robin Warren, Ben Marais, and Halvor Sommerfelt for valuable comments.

This study was supported by the Norwegian Research Council, University of Bergen, NUFU, and Helse Vest.

Footnotes

[down-pointing small open triangle]Published ahead of print on 22 April 2009.

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