Beijing and Haarlem Genotypes Are Overrepresented among Children with Drug-Resistant Tuberculosis in the Western Cape Province of South Africa

Abstract

Drug resistance among children with culture-confirmed tuberculosis (TB) provides an accurate measure of transmitted drug resistance within the community. We describe the genotype diversity in children with culture-confirmed TB and investigate the relationship between genotype and drug resistance. A prospective study was conducted from March 2003 through August 2005 at Tygerberg Children's Hospital, in the Western Cape Province of South Africa. All children (<13 years of age) diagnosed with culture-confirmed TB were included. Genotype analysis and phenotypic drug susceptibility testing were performed on the first culture-positive isolate from each patient. Mutation analysis was performed on all drug-resistant isolates. Spoligotyping was successfully performed on isolates from 391/399 (98%) children diagnosed with culture-confirmed TB. Drug susceptibility testing was also performed on 391 isolates; 49 (12.5%) were resistant to isoniazid, and 20 (5.1%) of these were resistant to both isoniazid and rifampin. Beijing was the most common genotype family, identified in 130/391 (33.2%) cases, followed by LAM in 114/391 (29.2%) cases. The presence of both Beijing and Haarlem genotype families was significantly associated with drug resistance (26/49 [53.1%] versus 113/342 [33.0%]; odds ratio, 1.7; 95% confidence interval, 1.0 to 2.9). The high prevalence of Beijing and LAM in children with culture-confirmed TB reflects considerable transmission of these genotype families within the community. The overrepresentation of Beijing and Haarlem genotype families in children with drug-resistant TB demonstrates their contribution to transmitted drug resistance and their potential importance in the emergent drug-resistant TB epidemic.

At a molecular level, the global tuberculosis (TB) epidemic consists of multiple genotype-specific subepidemics. Over time, the host-pathogen interaction, as well as the environment, selects the most “successful” genotypes within particular geographic locations (10). Different Mycobacterium tuberculosis (M. tuberculosis) genotypes can be identified by variation in certain well-characterized repeat sequences, such as the IS6110 transposable element and the direct repeat region (33).

Host-related selective forces include variability in the immune response to M. tuberculosis infection and the provision of anti-TB chemotherapy to TB patients. The immune response of the host is influenced by genetic predisposition and acquired factors (6, 10), including bacillus Calmette-Guerin (BCG) vaccination, previous M. tuberculosis infection, exposure to environmental mycobacteria, and human immunodeficiency virus (HIV) infection. Genotype-specific differences in the host-pathogen interaction are well documented in animal models, where infection with different M. tuberculosis genotypes evokes markedly different immune responses (14). In addition, BCG vaccination provides variable genotype-specific protection; infection with strains from the Beijing genotype family in particular has been associated with decreased protection following BCG vaccination in mice (14).

The Beijing genotype family is well recognized as a distinct genetic lineage, and it is genetically highly conserved (11), although large sequence polymorphisms identify four monophyletic subgroups (30). It has a worldwide distribution but predominates in certain geographic areas, particularly in parts of Asia (13, 31) and Russia (7). Based on its association with young age in adult studies from Vietnam and Russia (7), suggesting more recent spread, it has been proposed that Beijing should be regarded as an emerging genotype family (2).

The association between drug resistance and the Beijing genotype is well documented in adults (1, 4, 13, 23, 29, 30). The geographic variability observed in this association (30), the frequent clustering of resistant genotypes, and their successful spread within the Russian prison system (7, 29) suggest recent clonal expansion. This is supported by evidence that some strains of the Beijing genotype family retain fitness despite the acquisition of drug resistance (28). The Haarlem genotype family has also been associated with drug resistance and rapid clonal expansion (8, 19). The association of these genotype families with drug-resistant outbreaks clearly demonstrates their epidemic potential (19, 21). From a TB control point of view, it is relevant to know whether specific genotype families are overrepresented among drug-resistant cases, and in particular if these resistant strains are successfully transmitted within the community.

Children provide a valuable epidemiologic perspective, as the burden of childhood TB reflects the level of epidemiologic control achieved within a particular community (18). Because children usually progress to disease within 12 months of primary infection (15, 16), genotype analysis of isolates collected from children reflects current transmission patterns within the community (35). Children tend to develop pauci-bacillary disease and are therefore unlikely to acquire drug resistance (15, 26). Consequently, drug resistance patterns among children with culture-confirmed TB serve as a good indicator of transmitted (primary) drug resistance within the community (26).

Genotype diversity, and in particular the relation between genotype and drug resistance, are poorly documented in children. The aims of this study were to describe the genotype diversity in children with culture-confirmed TB and to investigate the relationship between genotype and drug resistance.

MATERIALS AND METHODS

Study population and setting.

Isolates were prospectively collected from March 2003 through August 2005 at Tygerberg Children's Hospital, a pediatric referral hospital in the Western Cape Province of South Africa. All children (<13 years of age) diagnosed with culture-confirmed tuberculosis were included. The Western Cape Province reported a TB incidence of 678/100,000 in 2003 (3), and the incidence of childhood TB in areas adjacent to Tygerberg Children's Hospital was 407/100,000 in the same period, representing 13.7% of all treated cases (17).

Laboratory procedures.

Diagnostic cultures were performed using Middlebrook 7H9 broth-based Mycobacteria Growth Indicator Tubes (MGIT; Becton Dickinson, Sparks, MD). M. tuberculosis was confirmed by routine PCR-based speciation of the M. tuberculosis complex (5). The first culture-positive isolate from each patient was sent to the regional diagnostic laboratory for phenotypic drug susceptibility testing, using the indirect proportional method. Drug resistance was defined according to international criteria (≥1% bacterial growth on drug-containing media compared to a non-drug-containing control) using critical concentrations of 0.2 μg/ml isoniazid (INH) or 30 μg/ml rifampin (RMP) (26). In-house quality assurance checks were performed after every batch, with external review by the national reference TB laboratory on a quarterly basis.

Genotypic testing for specific mutations in the katG, inhA, and rpoB genes that may confer resistance to INH and/or RMP was performed on all isolates with phenotypic drug resistance. DNA sequencing included the following regions: position 15 in the inhA promoter region, codon 315 of the katG gene, and codons 526 and 531 of the rpoB gene. An equal number of drug-sensitive specimens was tested as controls.

Mycobacterial subcultures made on standard Löwenstein-Jensen slants were used for genotyping. Strict protocols were in place to prevent cross-contamination; subcultures were prepared within a negative-pressure extraction hood, using sterile equipment and containers, by direct aspiration from the liquid growth media. Genotype determination was done using standardized spoligotyping methodology (33). Isolates were assigned to specific genotype families according to their spoligotype signature, using the spoldb3 database (www.pasteur-guadeloupe.fr/tb/spoldb3). The following internationally recognized genotype families were identified: Beijing, LAM (Latin American and Mediterranean family), X (a European clade of IS6110 low banders), and Haarlem (9), while Family 28 was identified as a “stand alone” genotype family (E. Streicher, personal communication). Genotype families with a low frequency (<10 cases) were combined and categorized as “Other.”

Some isolates could not be assigned to an internationally recognized genotype family, and these specimens were further characterized by using a novel PCR-based technique. PCR primers were designed to identify the presence of an IS6110 insertion (position 932204 according to the whole H37RV genome sequence), which is unique to all members of the LAM family (25). DNA was PCR amplified in a total volume of 25 μl, containing 1 μl DNA template, 1× Q-Buffer, 1× enzyme buffer, 3.5 mM MgCl₂, 1.6 mM deoxynucleoside triphosphates, 25 pmol of each primer (LAM F, TAG CCC ACC ACC ACA GCT TC; LAM R, ACC ACC CTG CCT AAC CAA TTC; Xho1, TTC AAC CAT CGC CGC CTC TAC), and 0.25 U HotStarTaq DNA polymerase (QIAGEN, Germany). Amplification was initiated by incubation at 95°C for 15 min, followed by 40 cycles at 94°C for 1 min, 62°C for 1 min, and 72°C for 1 min. After the last cycle, isolates were incubated at 72°C for 10 min. PCR amplification products were electrophoretically fractionated in 3.0% agarose in 1× Tris-borate-EDTA, pH 8.3, at 3.5 V/cm for 4 h and visualized by staining with ethidium bromide. The presence of a LAM strain was represented by a band of 205 base pairs, while a non-LAM strain was represented by a band of 141 base pairs. The specificity of amplification was determined by amplification of a panel of genetically unrelated and related strains and was shown to be 100% (95% confidence interval [CI], 85 to 100%) compared to the gold standard of IS6110 DNA fingerprinting. To minimize the risk of laboratory cross-contamination during PCR amplification, each procedure (preparation of the PCR mixes, the addition of the DNA, the PCR amplification, and the electrophoretic fractionation) was conducted in physically separated rooms. Negative controls (water) were included to control for reagent contamination.

After performing the LAM-specific PCR test, some isolates could still not be assigned to an internationally recognized genotype family, and these isolates were categorized as “unclassified.”

Children were tested for human immunodeficiency virus (HIV) infection, using the Determine HIV1/2 rapid test (Abbot, Tokyo, Japan) as a screening test and an enzyme-linked immunosorbent assay (DNA PCR in children <18 months of age) for confirmation. The study was approved by the Ethics Committee, Faculty of Health Sciences, Stellenbosch University.

Data analysis.

Descriptive statistical analysis was done with SPSS, version 13 (SPSS Inc., Chicago, IL) on anonymous unlinked data. Categorical data were analyzed with the χ² test, using Epi-Info version 6.04. We report the relative proportion of different genotype families, comparing drug-susceptible to drug-resistant isolates, and describe the specific mutations associated with resistance.

RESULTS

During the study period, 399 children were diagnosed with culture-confirmed TB. Patient characteristics and drug susceptibility test (DST) results are reflected in Table 1. Spoligotyping and DST analysis were performed on 391 isolates; 8 (2%) isolates developed bacterial contamination or lost viability during drug susceptibility testing. Of the isolates tested, 49 (12.5%) were resistant to INH, of which 20 (5.1%) were resistant to both INH and RMP (multidrug resistant [MDR]). No case of RMP monoresistance was recorded.

TABLE 1.

Patient characteristics and drug susceptibility test results of children diagnosed with culture-confirmed tuberculosis

Patient group and characteristic	Valuea
All patients with culture-confirmed TB (n = 399)
Boys (no. of patients)	210 (52.6)
Age (yr)
Median	2
Range	0-12
HIV testing (no. of patients)
HIV test performed	297 (74.4)
HIV infected	73 (18.3)
DST result available (no. of patients)	391 (98.0)
All patients with a DST result (n = 391)
Drug susceptible	342 (87.5)
All INH resistance	49 (12.5)
INH monoresistance	29 (7.4)
MDR	20 (5.1)

^aValues in parentheses are percentages.

Table 2 reflects the relative proportion of different genotype families, comparing drug-susceptible to drug-resistant isolates. Beijing was the most prevalent genotype family, identified in 130/391 (33.2%) isolates, followed by LAM, identified in 114/391 (29.2%) isolates. Children infected with either the Beijing or Haarlem strain families were more likely to have drug-resistant disease compared to those infected with other strain families (26/139 [18.7%] versus 23/252 [9.1%]; odds ratio [OR], 2.29; 95% confidence interval [CI], 1.2 to 4.4). The Beijing and Haarlem families combined were also more prevalent in the drug-resistant compared to the drug-susceptible group (26/49 [53.1%] versus 113/342 [33.0%]; OR, 1.7; 95% CI, 1.0 to 2.9).

TABLE 2.

Mycobacterial genotype and drug resistance in children with culture-confirmed tuberculosis

Genotype family designationa	No. (%) of isolates	No. (%) of isolates with DST result available		Odds ratio (95% CI)
		Drug susceptible	Drug resistant
Beijing	130 (32.6)	105 (30.7)	22 (44.9)	1.5 (0.8-2.6)
LAM	114 (28.6)	99 (28.9)	13 (26.5)	0.9 (0.4-1.8)
X	25 (6.3)	22 (6.4)	3 (6.1)	1.0 (0.2-3.5)
Family 28	25 (6.3)	24 (7.1)	1 (2.0)	0.3 (0.0-1.8)
Haarlem	12 (3.0)	8 (2.3)	4 (8.2)	3.7 (0.8-14.5)
Other	7 (2.0)	7 (2.0)	0
Unclassified	78 (19.5)	71 (20.8)	5 (10.3)	0.4 (0.1-1.1)
Not done	8 (2.0)	6 (1.8)	1 (2.0)	1.2 (0.0-9.9)
Total	399 (100.0)	342 (100)	49 (100)

^aOther, all genotype families with a frequency of less than 10 cases; X, European clade of IS6110 low banders, identified by spoligotyping (9); unclassified, no internationally recognized genotype family assigned, based on the spoldb3 spoligotype database (9), and negative for the LAM-specific PCR test.

Table 3 reflects specific mutations in the inhA promoter region and in the katG gene. Resistance mutations were detected in 31/49 (64.6%) isolates with phenotypic INH resistance. The two most common mutations detected were −15 in the inhA promoter region (18/49; 36.7%) and 315ACC in the katG gene (16/49; 32.7%). Among Beijing isolates, the −15 inhA promoter region mutation occurred most frequently (13/22; 59.1%) and was exclusively detected in the Beijing 220 subgroup. The Beijing 220 subgroup is a well-recognized drug-resistant strain in the Western Cape Province (T. C. Victor, personal communication). Mutations found in the MDR isolates included −15 in the inhA promoter region (7/20; 35%) as well as 315ACC (10/20; 50%) and 315ACA in the katG gene (1/20; 5%). No INH resistance-associated mutation was detected in 2/20 (10%) MDR isolates, using the limited mutation analysis described, and no multiple mutations were detected in the same isolate. No resistance-associated mutations, either for INH or RMP, were identified in any of the drug-sensitive control isolates.

TABLE 3.

Specific resistance mutations in children with INH-resistant tuberculosis

Genotype family designationa	No. of strains with resistance mutation:			No. of wild-type strains	Total no. of strains
	inhA −15	katG 315ACA	katG 315ACC
Beijing	13	0	5	4	22
LAM	3	0	4	6	13
X	0	1	1	1	3
Family 28	1	0	0	0	1
Haarlem	0	0	3	1	4
Other	0	0	0	0	0
Unclassified	0	0	3	2	5
Not done	1	0	0	0	1
Total	18	1	16	14	49

^aOther, all genotype families with a frequency of less than 10 cases; X, European clade of IS6110 low banders, identified by spoligotyping (9); unclassified, no internationally recognized genotype family assigned, based on the spoldb3 spoligotype database (9), and negative for the LAM-specific PCR test.

Table 4 reflects specific mutations in the rpoB gene in children with RMP-resistant TB. Resistance mutations were detected in all isolates with phenotypic RMP resistance. The most common rpoB gene mutation was 531TTG (13/20; 65%). This mutation was identified in all (8/8) multidrug-resistant Beijing isolates.

TABLE 4.

Specific rpoB mutations in children with MDR^a tuberculosis

Genotype family designationb	No. of strains with resistance mutation:			No. of wild-type strains	Total no. of strains
	526	531 CAG	531 TTG
Beijing	0	0	8	0	8
LAM	1	1	3	0	5
X	0	0	1	0	1
Family 28	1	0	0	0	1
Haarlem	0	0	2	0	2
Other	0	0	0	0	0
Unclassified	2	0	1	0	3
Total	4	1	15	0	20

^aMDR, multidrug resistant (resistant to both INH and rifampin); all rifampin-resistant isolates were resistant to isoniazid as well.

^bOther, all genotype families with a frequency of less than 10 cases; X, European clade of IS6110 low banders, identified by spoligotyping (9); unclassified, no internationally recognized genotype family assigned, based on the spoldb3 spoligotype database (9), and negative for the LAM-specific PCR test.

DISCUSSION

To our knowledge this is the first report of genotype diversity in relation to phenotypic and genotypic drug resistance in children with TB, and it represents one of the largest cohorts of children with DST results reported to date. As such, the report provides an estimate of genotype-specific transmission patterns at the community level, both for drug-susceptible and drug-resistant organisms. The association found between Beijing and Haarlem genotype families and drug-resistant TB is consistent with previous reports on adults (4, 7, 8, 19, 23, 29).

The predominance of specific mutations such as the −15 inhA promoter region mutation and the 531TTG rpoB mutation in the Beijing genotype family is an interesting observation. The frequency with which these specific mutations were detected may reflect random mutation and subsequent clonal expansion. Alternatively, certain genotype families may preferentially develop resistance mutations at particular locations in so-called mutation hotspots. Unique polymorphisms in the genes involved in DNA repair (mut genes) have been demonstrated in the Beijing genotype family, which may explain the ability of this clonal family to rapidly adapt to selective pressure (24); however, functional correlates of the “mutator phenotype” hypothesis have not been demonstrated.

In a comparative RMP resistance study, Beijing genotypes did not acquire rpoB mutations more rapidly than the reference H37Rv strain (34). However, the Beijing genotype family is not a homogeneous group and demonstrates a deeper population structure than previously appreciated (30). Therefore, the reported comparable acquisition of RMP resistance may not necessarily apply to the Beijing genotype family as a whole. In addition, the ability to acquire INH resistance may be the most important step in the acquisition of MDR disease. RMP monoresistance is rare. Due to the exceptional early bactericidal activity of INH (20), the development of monoresistance to INH is probably a more relevant event in the acquisition of multidrug resistance. In this regard, the specific −15 inhA promoter region mutation, documented in children infected with the particular Beijing 220 subgroup, is of particular interest and warrants further investigation. The substantial pool of INH monoresistant isolates identified may represent a source of future MDR disease (27).

Similar to a recent report from an adjacent study setting in Cape Town, South Africa (22), this study demonstrates that Beijing is the dominant genotype family in children with culture-confirmed TB. An important finding is that the prevalence of the Beijing genotype in children seems to differ from that described in adults from the same geographic area. LAM (which includes Family 11) has been reported as the dominant genotype family among adults in Cape Town (32). This discrepancy may reflect an important “age-shift,” similar to adult data from Vietnam (2), indicating emergence of the Beijing genotype family in the Western Cape Province of South Africa. However, the validity of comparisons with adult data is limited, as adult and child data were not collected from the same geographic area or the same period of time.

In Vietnam, the prevalence of the Beijing genotype family was found to be associated with the presence of a BCG scar (2). Although this was not significant after correcting for the cohort effect (2), it lends preliminary support to observations in animal studies that BCG provides less efficient protection against Beijing (14). This may provide a selective advantage to the Beijing genotype family in countries with universal BCG vaccination policies, such as South Africa.

This study has several limitations. Limiting selection bias presents a huge challenge when studying children with culture-confirmed TB, as culture confirmation is rarely achieved at the primary health care level, and hospital-based studies are inherently limited by selection bias. However, a recent report that compared drug resistance patterns between hospital- and community-recruited children from the same cohort found no significant differences between the two groups (26).

When investigating genotype diversity, an important consideration that is often overlooked is the potential selection bias imposed by the variable culturability of different genotypes. If particular genotypes are easier to culture in vitro, particularly in children with pauci-bacillary disease, this may introduce a significant bias. In vivo data demonstrate that variation in strain-specific culturability is a reality that requires further exploration (12). Finally, the spoligotype fingerprint of the Beijing genotype family is short and distinct. Therefore, the probability of losing the spoligotype fingerprint of other strains through mutational events is greater. As a result, most of the unclassified strains would represent non-Beijing genotypes. We used a novel PCR-based test to reduce the number of unclassified isolates, and during analysis, all unclassified strains were regarded as non-Beijing to limit potential bias.

In conclusion, the high prevalence of Beijing and LAM in children with culture-confirmed TB reflects considerable transmission of these genotype families within the community. The overrepresentation of Beijing and Haarlem genotype families in children with drug-resistant TB demonstrate their contribution to transmitted drug resistance and their potential importance in the emergent drug-resistant TB epidemic.