Abstract
Despite the fact that Nepal is one of the first countries globally to introduce multidrug-resistant tuberculosis (MDR-TB) case management, the number of MDR-TB cases is continuing to rise in Nepal. Rapid molecular tests applicable in this setting to identify resistant organisms would be an effective tool in reversing this trend. To develop such tools, information about the frequency and distribution of mutations that are associated with phenotypic drug resistance in Mycobacterium tuberculosis is required. In the present study, we investigated the prevalence of mutations in rpoB and katG genes and the inhA promoter region in 158 M. tuberculosis isolates (109 phenotypically MDR and 49 non-MDR isolates collected in Nepal) by DNA sequencing. Mutations affecting the 81-bp rifampin (RIF) resistance-determining region (RRDR) of rpoB were identified in 106 of 109 (97.3%) RIF-resistant isolates. Codons 531, 526, and 516 were the most commonly affected, at percentages of 58.7, 15.6, and 15.6%, respectively. Of 113 isoniazid (INH)-resistant isolates, 99 (87.6%) had mutations in the katG gene, with Ser315Thr being the most prevalent (81.4%) substitution. Mutations in the inhA promoter region were detected in 14 (12.4%) INH-resistant isolates. The results from this study provide an overview of the current situation of RIF and INH resistance in M. tuberculosis in Nepal and can serve as a basis for developing or improving rapid molecular tests to monitor drug-resistant strains in this country.
INTRODUCTION
With an estimated 9 million new cases and 2 million deaths every year, tuberculosis (TB) represents one of the most serious infectious diseases worldwide (35). The increasing spread of multidrug-resistant TB (MDR-TB), which is resistant to at least two drugs, including isoniazid (INH) and rifampin (RIF), and the recent emergence of extensively drug-resistant TB (XDR-TB), with additional resistance to a fluoroquinolone (FQ) and at least one of the three injectable second-line drugs, pose a significant threat to tuberculosis control (19, 35). Lack of adequate treatment, often due to irregular drug supply, inappropriate regimens, or poor patient compliance, is associated with the emergence of drug-resistant Mycobacterium tuberculosis (9, 13). In 2008, approximately 440,000 cases of MDR-TB were estimated throughout the world, and 58 nations had reported to World Health Organization (WHO) at least one case of XDR-TB (19, 21, 35). Among the countries listed in the WHO report, India and China had the highest burden of MDR-TB, together accounting for almost half of the world's total cases (19, 35). In Nepal, the incidence of all forms of TB was estimated to be 173/100,000 population, while the incidence of new smear-positive cases was at 77/100,000 in 2008 (14, 35). According to the national drug resistance survey conducted in 2006, the prevalences of MDR-TB in Nepal among new and retreatment cases were 2.9 and 11.7%, respectively (14).
Nepal is a landlocked country in Southeast Asia, bounded to the north by China and to the south by India, sharing an open border with India. Every year, a large number of people of Nepal and India cross the border for various purposes, such as work, study, trade, pilgrimage, cultural visits, and so on. According to the 2001 census of Nepal, 762,181 people were abroad, with 78% in India. The census recorded 116,571 foreign citizens residing in Nepal, 88% of whom were Indians (20). However, this information does not adequately cover the short-term and short-distance mobility that could significantly contribute TB epidemics in Nepal. Since drug resistance rates on one side of the border impact the other side of the border (33), a high proportion of MDR-TB in Nepal may reflect the possible dissemination of infection from surrounding two countries, mainly from India.
Rapid determination of the antimicrobial susceptibility pattern in clinical isolates of M. tuberculosis is important for the early administration of appropriate therapeutic agents for the prevention of additional resistance development (21). In this context, molecular characterization of drug resistance by identifying mutations in associated genes will be applicable for developing a potential rapid molecular drug susceptibility test as an alternative to conventional methods (16, 23).
The collection of data from different countries has indicated that resistance to RIF in >90% of cases is due to mutations resulting in an amino acid substitution within the 81-bp core region of the RNA polymerase β-subunit gene (rpoB), called the RIF resistance-determining region (RRDR) (8, 24, 26, 30). In contrast, INH resistance is mediated by mutations in several genes, most frequently within the katG gene, encoding a catalase-peroxidase which transforms INH into its active form (6, 11, 24), and in the promoter region of inhA, encoding a putative enzyme involved in mycolic acid biosynthesis. An upregulation mutation in the inhA promoter region results in the overexpression of InhA and develops INH resistance via a titration mechanism (24).
In the present study, we sought to determine the prevalence of resistance-associated mutations in three specific genes (rpoB, katG, and the inhA promoter region) of M. tuberculosis isolates in Nepal and to compare the frequency of different mutations with those in isolates circulating in the surrounding countries.
MATERIALS AND METHODS
Isolates.
In total, 109 and 49 samples were randomly selected from MDR and non-MDR clinical isolates, respectively, in isolates bank at the German Nepal Tuberculosis Project (GENETUP) over a 3-year period from 2007 and 2010. The isolates were recovered from 158 patients living in nine different cities of Nepal, six of which have an open border with northern India. Of 109 MDR isolates, the numbers of isolated from each city were as follows: Kathmandu (n = 70), Biratnagar (n = 8), Bhairahawa (n = 8), Pokhara (n = 7), Birgunj (n = 4), Nepalgunj (n = 4), Dhangadi (n = 4), Butwal (n = 3), and Sarlahi (n = 1). Of the non-MDR isolates, 48 were obtained from patients in Kathmandu, and 1 was obtained from Biratnagar. Histories of previous TB treatment were available in 94.5% of the MDR and 42.9% of the non-MDR patients. A drug susceptibility test was performed using Löwenstein-Jensen medium by a conventional proportional method with the following critical drug concentrations of INH, RIF, streptomycin (STR), and ethambutol (EMB): 0.2, 40, 4, and 2 μg/ml, respectively (2).
DNA extraction.
DNAs were prepared for PCR by mechanical disruption, as described previously (29). Briefly, the colonies were suspended in TE buffer consisting of 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA in a 2-ml screw-cap vial, one-fourth of which was filled with 0.5-g glass beads (0.1 mm; BioSpec Products, Inc., OK). Mycobacterial cells were disrupted by shaking with 0.5 ml of chloroform on a cell disrupter (Micro Smash; Tomy Seiko Co., Ltd., Tokyo, Japan) for 1 min. After centrifugation, the DNAs in the upper layer were concentrated by ethanol precipitation and dissolved in 100 μl of TE buffer.
Species differentiation multiplex PCR.
M. tuberculosis species were identified on the isolates by a multiplex PCR with primer pairs designed to amplify three genetic regions (cfp32, RD9, and RD12), as described previously (18).
Sequencing of the rpoB and katG encoding regions and the inhA promoter region.
PCRs were performed in a 20-μl mixture containing 0.25 mM (each) deoxynucleoside triphosphates, 0.5 M betaine, 0.5 μM concentrations of each primer (Table 1), 1 U of GoTaq DNA polymerase (Promega, WI), GoTaq buffer, and 1 μl of DNA template. The reaction was carried out in a thermal cycler (Bio-Rad Laboratories, CA) under the following conditions: denaturation at 96°C for 60 s, followed by 35 cycles of amplification at 96°C for 10 s, 55°C for 10 s, and 72°C for 30 s, with a final extension at 72°C for 5 min. The presence of PCR products was confirmed by agarose gel electrophoresis. PCR products were sequenced according to the manufacturer's protocol with the primers TB rpoB S, TB katG S, and TB inhA S for rpoB, katG, and inhA, respectively, and the BigDye Terminator v3.1 cycle sequencing kit (Life Technologies Corp., CA) using an ABI Prism 3130xl Genetic Analyzer (Life Technologies Corp.). The resulting sequences were compared to wild-type sequences of M. tuberculosis H37Rv using Bio-Edit software (version 7.0.9) (5).
Table 1.
Locus | Primer | Nucleotide sequence (5′–3′) | Target region (position) | Product size (bp) |
---|---|---|---|---|
rpoBa | TB rpoB S | CAGGACGTGGAGGCGATCAC | 1519–1599a | 278 |
TB rpoB AS | GAGCCGATCAGACCGATGTTGG | |||
katG | TB katG S | ATGGCCATGAACGACGTCGAAAC | 823–1140 | 392 |
TB katG AS | CGCAGCGAGAGGTCAGTGGCCAG | |||
inhA | TB inhA S | TCACACCGACAAACGTCACGAGC | −50 to −1 | 231 |
TB inhA AS | AGCCAGCCGCTGTGCGATCGCCA |
Corresponding E. coli numbering was used for rpoB.
RESULTS
Drug susceptibility patterns.
Of the 109 MDR isolates, 102 were resistant to three or more first-line anti-TB drugs (Table 2). Forty-nine non-MDR isolates consisted of 41 fully susceptible and 2, 3, and 1 isolates with monoresistance against INH, STR, and EMB, respectively. Two isolates were resistant to both INH and STR.
Table 2.
Characteristic | Resistance patterna | No. of isolates |
---|---|---|
MDR | INH + RIF | 7 |
INH + RIF + EMB | 6 | |
INH + RIF + STR | 17 | |
INH + RIF + EMB + STR | 79 | |
Non-MDR | None | 41 |
INH | 2 | |
STR | 3 | |
EMB | 1 | |
INH + STR | 2 |
INH, isoniazid; RIF, rifampin; STR, streptomycin; EMB, ethambutol.
Species identification.
All 158 isolates showed three amplified bands corresponding to cfp32, RD9, and RD12 by multiplex PCR and were classified as M. tuberculosis (data not shown).
Mutations in the rpoB gene.
Mutations in the RRDR of the rpoB gene were identified in 106 of 109 RIF-resistant (RIFr) isolates (Table 3). A single nucleotide alteration in codon 531, resulting in the amino acid substitution of Ser to Leu, was most prevalent and observed in 62 isolates (56.9%). The second most affected codons were 516 and 526, which were found in 17 (15.6%) isolates each, and had 3 and 6 types of amino acid substitutions, respectively. Five (4.6%) isolates had a mutation in codon 513, and three (2.8%) had a mutation in codon 533. An insertion of Phe between codons 514 and 515 was observed in two (1.8%) isolates, one of which had an additional point mutation affecting codon 531. Two isolates carried double mutations in two separate codons, i.e., codons 513 and 526 and codons 516 and 533, respectively. No mutations were detected in the remaining 3 (2.8%) RIFr and 49 RIF-susceptible (RIFs) isolates.
Table 3.
Mutated codon(s) | Amino acid change(s)a | Nucleotide change(s) | No. (%) of isolates |
|
---|---|---|---|---|
RIFr (n = 109) | RIFs (n = 49) | |||
511 | Leu→Pro | CTG→CCG | 1 (0.9) | 0 (0.0) |
513 | Gln→Leu | CAA→CTA | 2 (1.8) | 0 (0.0) |
Gln→Lys | CAA→AAA | 2 (1.8) | 0 (0.0) | |
514 | Phe (ins) | TTC→TTCTTC | 1 (0.9) | 0 (0.0) |
516 | Asp→Val | GAC→GTC | 13 (11.9) | 0 (0.0) |
Asp→Phe | GAC→TTC | 2 (1.8) | 0 (0.0) | |
Asp→Tyr | GAC→TAC | 1 (0.9) | 0 (0.0) | |
526 | His→Tyr | CAC→TAC | 5 (4.6) | 0 (0.0) |
His→Arg | CAC→CGC | 4 (3.7) | 0 (0.0) | |
His→Asp | CAC→GAC | 3 (2.8) | 0 (0.0) | |
His→Cys | CAC→TGC | 2 (1.8) | 0 (0.0) | |
His→Gly | CAC→GGC | 1 (0.9) | 0 (0.0) | |
His→Leu | CAC→CTC | 1 (0.9) | 0 (0.0) | |
531 | Ser→Leu | TCG→TTG | 61 (56.0) | 0 (0.0) |
Ser→Gln | TCG→CAG | 1 (0.9) | 0 (0.0) | |
Ser→Val | TCG→GTG | 1 (0.9) | 0 (0.0) | |
533 | Leu→Pro | CTG→CCG | 2 (1.8) | 0 (0.0) |
531 and 514 | Ser→Leu and Phe (ins) | TCG→TTG and TTC→TTCTTC | 1 (0.9) | 0 (0.0) |
513 and 526 | Gln→Lys and His→Asp | CAA→AAA and CAC→GAC | 1 (0.9) | 0 (0.0) |
516 and 533 | Asp→Ala and Leu→Pro | GAC→GCC and CTG→CCG | 1 (0.9) | 0 (0.0) |
Wild typeb | None | None | 3 (2.8) | 49 (100.0) |
Phe (ins), Phe insertion.
That is, there were no mutations in the sequenced region.
Mutations in katG encoding region and inhA promoter region.
Of 113 phenotypically INHr isolates, 99 (87.6%) had katG mutations, the vast majority of which was the commonly described substitution katG(Ser315Thr) (Table 4). Only one isolate had a Ser-to-Asn substitution at katG position 315 (katG 315). katG(Gly299Ser) and katG(Asp329Ala) mutations were detected in two INHr isolates. One isolate showed double mutations in two separate katG codons: Thr275Ala and Ser315Thr. Mutations in the inhA promoter region were observed in 14 (12.4%) INHr isolates; 12 of which had a mutation at −15 in the inhA promoter. Among the isolates with mutation in inhA promoter, three had additional mutation in katG 315, and one each had additional mutations in katG 285, katG 289, and katG 289 plus katG 296. No mutations in either region were identified in 7 (6.2%) INHr and 45 INHs isolates.
Table 4.
Locus | Amino acid change(s) | Nucleotide change(s) | No. (%) of isolates |
|
---|---|---|---|---|
INHr (n = 113) | INHs (n = 45) | |||
katG 315 | Ser→Thr | AGC→ACC | 86 (76.1) | 0 (0.0) |
Ser→Thr | AGC→ACT | 1 (0.9) | 0 (0.0) | |
Ser→Asn | AGC→AAC | 1 (0.9) | 0 (0.0) | |
katG 299 | Gly→Ser | GGC→AGC | 1 (0.9) | 0 (0.0) |
katG 329 | Asp→Ala | GAC→GCC | 1 (0.9) | 0 (0.0) |
katG 341 | Trp→Gly | TGG→GGG | 1 (0.9) | 0 (0.0) |
katG 275 and katG 315 | Thr→Ala and Ser→Thr | ACC→GCC and AGC→ACC | 1 (0.9) | 0 (0.0) |
inhA −15 | NAb | C→T | 6 (5.3) | 0 (0.0) |
inhA −8 | NA | T→C | 1 (0.9) | 0 (0.0) |
katG 285 and inhA −15 | Gly→Asp and NA | GGC→GAC and C→T | 1 (0.9) | 0 (0.0) |
katG 289 and inhA −15 | Glu→Ala and NA | GAG→GCG and C→T | 1 (0.9) | 0 (0.0) |
katG 289, katG 296, and inhA −15 | Glu→Ala, Met→Val, and NA | GAG→GCG, ATG→GTG, and C→T | 1 (0.9) | 0 (0.0) |
katG 315 and inhA −12 | Ser→Thr and NA | AGC→ACC and T→A | 1 (0.9) | 0 (0.0) |
katG 315 and inhA −15 | Ser→Thr and NA | AGC→ACC and C→T | 3 (2.7) | 0 (0.0) |
Wild typea | None | None | 7 (6.2) | 45 (100) |
That is, there were no mutations in sequenced regions of katG and inhA promoter.
NA, not applicable.
DISCUSSION
Antituberculosis drug resistance poses a significant threat to human health, which usually develops due to the alteration of drug targets by mutations in M. tuberculosis chromosomal genes (24, 26). Although a large number of mutations in several genes that confer resistance to M. tuberculosis have been reported from different countries, no study until now has managed to reveal the range of mutation in clinical samples from Nepal, one of the countries with the highest TB prevalence. Hence, in the present study, we attempted to identify the molecular basis of the drug resistance of M. tuberculosis circulating in Nepal.
RIF resistance is often considered as a surrogate marker for checking MDR-TB (7, 24). This hypothesis is supported by the finding in the present study that 100% of the RIFr isolates were MDR. Consistent with previous studies that ca. 95% of RIF-resistant M. tuberculosis isolates worldwide have mutations within the 81-bp core region of the rpoB gene, we found mutations in this region in 97.3% of RIFr isolates. The most frequently mutated codon in our study was codon 531 (58.7%), which was similar to those reported in clinical isolates from India (15, 27, 28), China (4, 10, 12, 36), and other geographical regions (3, 31) (Table 5). Although low frequencies of mutations in codon 516 in clinical isolates have been reported from various parts of China (4, 12, 36), we found a higher frequency of this mutation (15.6%), which was comparable to that of northern India (20.5%) (27).
Table 5.
Mutated codon | % Mutations in different geographic regionsa |
|||||||
---|---|---|---|---|---|---|---|---|
Northern India (n = 93) | India 1b (n = 149) | India 2c (n = 44) | Southern China (n = 60) | Eastern China 1d (n = 242) | Chinae (n = 72) | Eastern China 2 (n = 53) | This study (n = 109) | |
511 | 9.7 | 1.3 | 6.0 | 3.3 | 3.3 | 1.4 | 0.9 | |
513 | 0.7 | 2.0 | 2.6 | 2.9 | 1.4 | 4.6 | ||
516 | 20.5 | 11.5 | 4.0 | 5.0 | 7.4 | 4.2 | 7.5 | 15.6 |
518 | 7.5 | 2.0 | 1.4 | |||||
522 | 5.4 | 2.6 | 1.7 | 2.8 | ||||
526 | 20.4 | 22.0 | 19.0 | 11.6 | 19.4 | 36.1 | 30.2 | 15.6 |
531 | 38.7 | 59.0 | 53.0 | 58.3 | 61.2 | 37.5 | 58.5 | 58.7 |
533 | 4.0 | 2.0 | 5.0 | 5.0 | 1.4 | 2.8 | ||
Others | 10.8 | 1.3 | 13.7 | 2.1 | 4.2 | 1.8 | ||
None | 2.0 | 2.0 | 10.0 | 3.7 | 9.7 | 7.5 | 2.8 |
The values include isolates with mutations at multiple codons. Source references for the various regions were as follows: northern India (27), India 1 (28), India 2 (15), southern China (4), eastern China 1 (12), China (36), and eastern China 2 (10).
Includes northern India (n = 110) and southern India (n = 39).
Includes southern India (n = 35), northern India (n = 6), and western India (n = 3).
Collected only in Shanghai (n = 242).
Includes southern China (n = 26), northern China (n = 16), and eastern China (n = 30).
Phenotypically RIFr isolates with no rpoB mutations in our study were 2.8%, similar to those reported previously (3, 10, 12, 26, 28). Therefore, this finding suggested that majority of RIFr isolates in Nepal could be rapidly detected by screening for the most common genetic alterations in RRDR of the rpoB gene, although the prevalence of isolates lacking mutations also needs to be considered.
Previous studies indicated that INH resistance was mediated by mutations in several genes, most commonly katG, particularly in codon 315, and the promoter region of inhA (6, 11, 16, 24). Accordingly, we found that 87.6 and 12.4% of phenotypically INHr clinical isolates had point mutations in katG and in the inhA promoter region, respectively, and the frequencies were similar to those reported by other researchers (1, 3, 8). However, no deletion or insertion in katG was detected in any isolates in the present study. This result confirmed previous reports from different geographic regions of the rarity of this event in causing INH resistance (4, 8, 10, 11, 12, 16, 22). The seven (6.2%) INHr M. tuberculosis isolates had no resistance-associated alterations in the two targets analyzed, indicating that resistance in these isolates could be due to mutations present outside of the sequenced area or in other genes (e.g., kasA and ndh) (6, 8, 26).
It has been postulated that the amino acid substitution katG(Ser315Thr) is favored by the bacteria because this alteration was elucidated to spoil INH activation and, on the other hand, to retain 30 to 40% of the catalase-peroxidase activity necessary for virulence (25); however, the prevalence of the katG(Ser315Thr) substitution in M. tuberculosis isolates around the world varies, especially with regard to the prevalence of TB. In general, a higher prevalence of this substitution has been observed in high TB burden regions, often with the predominance of Beijing and MDR M. tuberculosis strains, compared to regions where the prevalence of TB is intermediate or low (10, 17). The present study documented the prevalence of the KatG Ser315Thr substitution in 81.4% of INHr isolates, which was not as high as those reported in INHr isolates in northeastern Russia (93.6%) (17) but was comparable to those in Lithuania and Germany (85.7 and 88.4%, respectively) (1, 26). The occurrence of the KatG Ser315Thr alteration among Nepalese isolates was higher than that reported in India (16, 22) and in China (4, 10, 12) (Table 6).
Table 6.
Locus | % Mutations in different geographic regionsa |
|||||
---|---|---|---|---|---|---|
Northern India (n = 121) | Southern India (n = 70) | Southern China (n = 50) | Eastern China 1 (n = 131) | Eastern China 2c (n = 242) | This study (n = 113) | |
katG 315 | 55.4 | 64.3 | 60.0 | 61.8 | 72.7 | 82.3 |
inhA −15 | 25.6 | 11.4 | 8.0 | 21.4 | 8.3 | 10.6 |
Othersb | 27.3 | 28.6 | 36.0 | 18.3 | 21.5 | 10.8 |
Values include isolates with mutations at both loci. Source references for the various regions were as follows: northern India (16), southern India (22), southern China (4), eastern China 1 (10), and eastern China 2 (12).
Includes both other mutations and no mutations.
Collected only in Shanghai (n = 242).
Van Soolingen et al. (32) reported that strains with amino acid substitutions in katG 315 are more likely to develop resistance to other drugs. In this respect, we found a correlation between this alteration and resistance to other drugs: 100% of the isolates with a katG 315 substitution showed resistance to RIF. Meanwhile, this mutation was found among 92 in 109 (84.4%) of MDR and none in four non-MDR INHr isolates. This is consistent with the finding of previous studies in which substitutions in codon 315 of KatG are more common in MDR isolates (6, 26, 31). Several studies from different countries have shown that ca. 10 to 34% of INHr cases have mutations in the inhA promoter region (11, 34). In contrast, we identified mutations in only 12.4% of INHr isolates, the majority of which was a C-to-T mutation at position −15.
Since Nepal shares an open border with northern India, there is a large amount of population movement between these countries (20). Patients from northern India usually come to Nepal because of cheaper TB treatment facilities in Nepal; thus, we postulated the frequent air-born transmission of TB between these points (33). By comparing data with neighboring countries, we observed a similarity between Nepalese and northern Indian RIFr isolates in the occurrence of mutations in codons 531, 526, and 516 of the rpoB gene (Table 5). In contrast, the frequency of katG(Ser315Thr) substitution and C-to-T mutations at position −15 in the inhA promoter between Nepalese and northern Indian INHr isolates showed a significant difference (Table 6). This discrepancy might not suggest transport but the possible emergence of MDR-TB in Nepal. For confirmation, molecular typing of strains circulating in Nepal and northern India seems to be necessary.
In conclusion, we provide here valuable information on mutations occurring at rpoB, the katG gene, and the promoter region of inhA in Nepalese clinical isolates of M. tuberculosis. These findings expand our current knowledge of the molecular mechanisms of drug resistance and also assist in improving current molecular techniques for the diagnosis of MDR-TB in Nepal. Such methods promise rapid detection rates compared to those achieved by methods based solely on culture of the isolates.
ACKNOWLEDGMENTS
This study was supported in part by J-GRID; the Japan Initiative for Global Research Network on Infectious Diseases from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT); the Global Center of Excellence (COE) Program, Establishment of International Collaboration Centers for Zoonosis Control, from MEXT; a grant from U.S.-Japan Cooperative Medical Science Programs to Y.S.; and Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science to Y.S. and C.N.
Footnotes
Published ahead of print 26 March 2012
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