Abstract
This study aimed to investigate the prevalence of resistance to second-line antituberculosis (anti-TB) drugs and its association with resistance-related mutations in Mycobacterium tuberculosis isolated in China. In the present study, we collected 380 isolates from a population-based study in China and tested the drug susceptibility to first- and selected second-line drugs. These results were compared with polymorphisms in the DNA sequences of genes associated with drug resistance and MIC values of the studied second-line drugs. Of 43 multidrug-resistant M. tuberculosis isolates, 13 showed resistance to fluoroquinolones or injectable second-line drugs (preextensively drug-resistant TB [pre-XDR-TB]), and 4 were resistant to both and thus defined as extensively drug-resistant TB (XDR-TB). Age and previous TB therapy, including use of second-line drugs, were two independent factors associated with increased resistance to both first- and second-line drugs. Molecular analysis identified the most frequent mutations in the resistance-associated genes: D94G in gyrA (29.1%) and A1401G in rrs (30.8%). Meanwhile, all 4 XDR-TB isolates had a mutation in gyrA, and 3 of them carried the A1401G mutation in rrs. Mutations in gyrA and rrs were associated with high-level resistance to fluoroquinolones and the second-line injectable drugs. In addition to the identification of resistance-associated mutations and development of a rapid molecular test to diagnose the second-line drug resistance, it should be a priority to strictly regulate the administration of second-line drugs to maintain their efficacy to treat multidrug-resistant TB.
INTRODUCTION
Multidrug-resistant tuberculosis (MDR-TB) is a public health concern that threatens the success of global TB control programs (1). An injectable agent and a fluoroquinolone (FQ) are the core substances for MDR-TB treatment. However, the emergence of extensively drug-resistant TB (XDR-TB)—defined as MDR-TB with additional resistance to FQ and an injectable second-line agent—has been identified in 84 countries worldwide (2) and is of great concern, since few treatment options remain against such highly drug-resistant strains and the outcome for patients with XDR-TB is in most cases poor (3). Timely detection of resistance to second-line drugs is of key importance to optimize treatment and to direct infection control measures to block transmission of MDR-TB.
China is one of the countries with the highest incidence of TB and MDR-TB (1). A recent national survey of drug-resistant TB in China estimated that 5.7% of new cases and 25.6% of previously treated cases had MDR-TB. In 2007, there were 110,000 cases of MDR-TB and 8,200 cases of XDR-TB in China (4). Previously, studies showed the proportion of XDR among MDR-TB varies from 6.5% in Shanghai (5) to 20% in Shandong Province (6). The longitudinal trend study of drug-resistant TB in one Beijing hospital demonstrated an increasing proportion of XDR among MDR-TB cases (7). However, these studies were performed in an urban setting or based in hospitals, while the percentage of MDR-TB cases resistant to second-line drugs is generally not known in rural areas of China, where the burden of MDR-TB is high and the ability of laboratory demonstration of drug resistance is poor since bacterial culture and drug susceptibility tests (DST) were not routinely performed in the local county TB dispensaries in rural China. Given the high prevalence of TB and MDR-TB and the broad use of second-line anti-TB drugs, especially of FQ, the existence of second-line drug-resistant TB in rural China seems inevitable.
The molecular mechanisms of action for the major second-line antituberculosis drugs and the resistance to these drugs have been elucidated (8). Resistance to FQ, such as ofloxacin (OFX), commonly used to treat MDR-TB, is thought to be mediated by mutations (single-nucleotide polymorphisms [SNPs]) in a short, discrete region of the target gene gyrA and, less frequently, in gyrB, termed the quinolone resistance-determining region (QRDR) (9), which encode the respective subunits of the DNA topoisomerase gyrase (10). Resistances to amikacin (AMK), kanamycin (KM), and capreomycin (CAP) are associated with SNPs in the 16S rRNA gene (rrs), especially in the region between nucleotides 1400 and 1500 (11, 12). Resistance to CAP is thought to be additionally mediated by mutations located anywhere in the tlyA gene, which encodes 2-O-methyltransferase (13, 14). Limited available data suggest that up to 70% of FQ and up to 87% of KM/AMK resistance worldwide can be attributed to the amino acid substitution within the QRDR of gyrA and the nucleotide substitution in rrs, respectively (9). Since resistance-associated mutations vary geographically (15, 16), we stress the need for increased information concerning the prevalence of mutations associated with second-line drug resistance in local settings.
For this purpose, we performed a community-based multicenter study to determine the prevalence of second-line drug-resistant TB and especially XDR-TB in rural China. Additionally, we aimed to determine if mutations of target genes (gyrA, gryB, rrs, eis, and tlyA) were related to resistance to second-line drugs (FQ, KM, CAP, AMK) among pulmonary TB patients in the high-incidence settings from rural China.
MATERIALS AND METHODS
Settings.
Five rural counties (DQ, LY, GY, YA, YI) in Zhejiang and Jiangsu Provinces, China, were selected as study sites. Together, these regions host a population of about 4.2 million people. The registration rate of smear-positive TB cases ranged from 30/100,000 to 37/100,000 in the five studied counties in 2008. The directly observed treatment, short-course (DOTS) strategy has been implemented since the early 1990s in all study sites, except for YA, where it began in 2003. In all the counties, the county TB dispensary is the only designated health facility for TB diagnosis, treatment, and case management. As recommended by the DOTS strategy, TB diagnosis is based on sputum smear microscopy, and a standardized 6- to 8-month treatment course using first-line anti-TB drugs is routinely applied.
Study design.
We performed a cross-sectional study using the data and specimens from TB patients who were diagnosed at the TB dispensaries during the period of January 2008 through December 2008. According to the study protocol, all suspected pulmonary TB cases detected in general hospitals or community health centers were referred to a specialized TB dispensary for further testing, such as sputum smear microscopy, culture, and chest radiography. All the eligible patients were investigated using a structured questionnaire by a trained interviewer to acquire their sociodemographic and clinical information. All the investigation protocols in this study were approved by the ethics committee of the School of Public Health, Fudan University, and informed consent was obtained from all patients for the information to be used in scientific investigations.
Species identification and drug susceptibility testing.
Sputum samples were decontaminated and digested with 2% NaOH. The mixture was then concentrated by centrifugation and inoculated on LJ medium. Species identification of mycobacteria was performed by conventional biochemical tests (17).
DST for first-line anti-TB drugs was performed using the proportion method (18) on egg-based LJ medium with the following drug concentrations: isoniazid (INH), 0.2 mg/liter; rifampin (RIF), 40.0 mg/liter; streptomycin (STR), 4.0 mg/liter; and ethambutol (EMB), 2.0 mg/liter. Furthermore, we used the WHO Guidelines for DST of second-line drugs with the agar proportion method on Middlebrook 7H10 or 7H11 agar. We included five important second-line drugs, widely used for MDR-TB treatment in China, and performed DST using the following concentrations: ofloxacin (OFX), 2 mg/liter; levofloxacin (LEV), 1 mg/liter; kanamycin (KM), 30 mg/liter; capreomycin (CAP), 40 mg/liter; and amikacin (AMK), 40 mg/liter. All drug compounds were obtained from Sigma Life Science Company.
For quality assurance, the DST was repeated for 10% of the isolates by an external technician from Shanghai municipal CDC who had passed the WHO's external quality control assurance with a consistency of over 95%. MDR was defined as resistant to at least INH and RIF. XDR was defined as MDR-TB with additional resistance to FQ and injectable second-line drugs (19). Preextensively drug-resistant TB (pre-XDR-TB) was defined as an MDR strain resistant to either FQ or a second-line injectable drug (20). The first- and second-line drug resistance was defined as the combined drug resistance to the first- and second-line drugs studied. In the present study, new cases were defined as TB patients who denied having had any prior anti-TB treatment or who received anti-TB treatment for up to 30 days. Previously treated cases were TB patients who reported having been treated for tuberculosis for at least 30 days or who had documented evidence of prior treatment in the case report form or surveillance database.
We established MIC determinations on Middlebrook 7H10 agar containing 10% oleic acid-albumin-dextrose-catalase (OADC) and 5‰ glycerol for CAP, AMK, and KM for all clinical isolates as previously described (21). Briefly, bacterial suspensions were transferred to Middlebrook 7H10 agar plates with serial 1:2 dilutions from 1 to 32 mg/liter for OFX and LEV and from 0.5 to 256 mg/liter for CAP, AMK, and KM by using a 96-stick replicator. Control plates without any antibiotic were inoculated with undiluted and 1:100-diluted bacterial suspensions. All plates were incubated at 37°C for 3 to 4 weeks. The MIC was defined as the first antibiotic concentration that showed less growth than the 1:100-diluted control of the corresponding strain, i.e., the lowest concentration of the drug that inhibited more than 99% of the bacterial population. Duplicates of the M. tuberculosis H37Rv reference strain were included in each run as inter- and intrareplication quality controls. The MIC determination was also repeated twice for 10% of in vitro selected mutants to ensure reproducibility.
DNA isolation, amplification, and sequencing of loci.
The loci were chosen on the basis of documented association with second-line drug resistance and included gyrA (FQ) and rrs, eis, and tlyA (second-line injectable drugs). DNA was isolated from 7H9 subcultures by mechanical cell disruption, as previously described (21). The following four loci were amplified by PCR with locus-specific primers as earlier described: gyrA and gyrB (FQ), rrs (KM, CAP, and AMK), eis (KM), and tlyA (CAP) (22). Sequence data produced by the ABI 3130xl genetic analyzer were reviewed for confidence levels with an ABI sequence scanner, and chromatograms were analyzed for the presence or absence of mutations by comparison with published sequences of H37Rv using the SeqMan alignment application of the DNAStar Lasergene (version 8.0) program. Genotypic data for each isolate at a particular locus were recorded in a Microsoft Office Excel 2003 spreadsheet.
Data collection and analysis.
Data on demographics as well as on previous and current anti-TB treatment were recorded routinely during the clinical assessment by a standardized questionnaire interview. Data on previous TB treatment and use of second-line drugs were additionally verified among patients starting treatment through a medical record review. All data were entered on a database using Epidata (http://www.epidata.dk). Data analysis was conducted with SPSS 11.0 (Chicago, IL). A χ2 test and Fisher's exact test (if any expected counts are less than 5) were used to compare the proportions of first- and second-line drug susceptibilities between isolates from new and previously treated TB cases. For the MIC ranked data, the nonparametric Mann-Whitney U test was used to compare the MIC values between the second-line drug-resistant M. tuberculosis isolates with and without the specific genetic mutation. To identify the association between drug use and drug resistance, drug-resistant rates were compared between the subjects with and without a history of the specific drug use. The odds ratios (OR) and 95% confidence intervals (CI) were calculated using logistic regression to assess potential factors associated with second-line drug resistance and combined first- and second-line drug resistance. P values of less than 0.05 were considered statistically significant.
RESULTS
Demographic and clinical characteristics of the subjects.
From January 2008 to December 2008, a total of 415 patients were diagnosed with pulmonary TB and registered in the local TB dispensaries. Of these, 390 (94.0%) patients were culture positive for M. tuberculosis. We excluded 10 culture-positive TB patients without DST results. The remaining 380 patients with culture-confirmed TB and known drug susceptibility were included for analysis. Among them, 267 (70.3%) patients were male, with an average age of 45 years and a range of 15 to 90 years. A total of 59 patients (15.5%) were previously treated with first-line drugs. Additionally, 31.3% of subjects reported to have used the second-line drugs before the diagnosis of TB.
Overall, the proportions for the resistance to first-line drugs and MDR-TB were 25.9% (83/321) and 9.0% (29/321) among new cases, while they were 59.3% (35/59) and 23.7% (14/59) in previously treated cases. Statistically significant differences were observed between new and previously treated TB cases regarding MDR-TB (9.0% versus 23.7%; P = 0.001), INH resistance combined with STR and/or EMB resistance (6.2% versus 15.3%; P = 0.016), and mono-STR resistance (5.0% versus 11.9%; P = 0.04) (Table 1).
Table 1.
First-line drug susceptibility profile of the M. tuberculosis isolates in the present study
| Drug-resistant profilec | New cases (n = 321) |
Previously treated cases (n = 59) |
χ2 | P | ||
|---|---|---|---|---|---|---|
| No. | % | No. | % | |||
| Monodrug resistance | ||||||
| INH | 10 | 3.1 | 3 | 5.1 | 0.59 | 0.44 |
| RIF | 1 | 0.3 | 1 | 1.7 | 0.29a | |
| STR | 16 | 5.0 | 7 | 11.9 | 4.15 | 0.04b |
| EMB | 2 | 0.6 | 0 | 0 | 0.71a | |
| Multidrug resistance | ||||||
| INH+RIF | 10 | 3.1 | 6 | 10.2 | 6.15 | 0.013b |
| INH+RIF+STR/+EMB | 19 | 5.9 | 8 | 13.6 | 4.41 | 0.036b |
| Poly-drug resistance | ||||||
| INH+STR/+EMB | 20 | 6.2 | 9 | 15.3 | 5.76 | 0.016b |
| RIF+STR/+EMB | 4 | 1.2 | 1 | 1.7 | 0.57a | |
| STR+EMB | 1 | 0.3 | 0 | 0 | 0.84a | |
P value from Fisher's exact test.
P < 0.05.
INH, isoniazid; RIF, rifampin; STR, streptomycin; EMB, ethambutol; +, and; /, or.
Second-line antituberculosis drug resistance.
Of the 380 isolates tested for the susceptibility to second-line drugs, 55 (14.5%) were resistant to FQ, including 48 (12.6%) resistant to OFX and 41 (10.8%) resistant to LEV. Furthermore, the cross-resistance to LEV was observed among the 34 (70.8%) OFX-resistant isolates. As for the second-line injectable drugs, the drug resistances to KM, AMK, and CAP were observed in 57 (15%), 44 (11.6%), and 41 (10.8%) of the isolates, respectively. A high proportion (75.4%; 43/57) of KM-resistant isolates was resistant to AMK. A high rate of cross-resistance was also observed between KM and CAP.
Furthermore, the susceptibilities to second-line drugs were compared between the first-line drug-sensitive, other first-line drug-resistant, and MDR-TB groups. Among the 43 MDR-TB cases, 13 (30.2%) were found to have additional second-line drug resistance, including 8 with resistance to FQ and 5 with resistance to at least one of the second-line injectable agents. Another 4 isolates were simultaneously resistant to FQ and at least one of the injective drugs; thus, these were classified as XDR-TB. Some second-line resistance was also observed among non-MDR strains, with either no first-line resistance or with mono- and poly-resistance to first-line drugs. The proportion of FQ resistance was significantly higher in MDR-TB than in non-MDR isolates (30.2% versus 12.4%; χ2 = 9.73, P = 0.002). On the contrary, there was no statistically significant difference of resistance to injectable drugs between MDR and non-MDR isolates (Table 2).
Table 2.
Second-line drug susceptibility profile in different first-line drug susceptibility groups
| Drugb | First-line drug susceptibility |
χ2 | P | |||||
|---|---|---|---|---|---|---|---|---|
| Sensitive isolates (n = 262) |
MDR-TB isolates (n = 43) |
Other drug-resistant isolates (n = 75) |
||||||
| No. | % | No. | % | No. | % | |||
| FQ | ||||||||
| OFX | 26 | 10.0 | 12 | 27.9 | 10 | 13.3 | 10.87 | 0.004a |
| LEV | 21 | 8.0 | 10 | 23.3 | 10 | 13.3 | 9.542 | 0.008a |
| Total | 31 | 11.8 | 13 | 30.2 | 11 | 14.7 | 9.071 | 0.011a |
| Injective drugs | ||||||||
| KM | 38 | 14.5 | 8 | 18.6 | 11 | 14.7 | 0.495 | 0.781 |
| AMK | 28 | 10.7 | 7 | 16.3 | 9 | 12.0 | 0.441 | 0.802 |
| CM | 26 | 10.0 | 6 | 14.0 | 9 | 12.0 | 1.039 | 0.595 |
P < 0.05.
FQ, fluoroquinolones; OFX, ofloxacin; LEV, levofloxacin; KM, kanamycin; CAP, capreomycin; AMK, amikacin.
Association between treatment history and resistance to second-line drugs.
The subjects' sociodemographic and clinical characteristics were further analyzed to identify risk factors for second-line drug resistance and pre-XDR-TB and XDR-TB. Age was significantly associated with increased risk of resistance to second-line drugs (OR, 1.01; 95% CI, 1.001 to 1.024), first- and second-line drug resistance (OR, 1.03; 95% CI, 1.007 to 1.043), and pre-XDR-TB/XDR-TB (OR, 1.02; 95% CI, 1.010 to 1.059). Meanwhile, a significantly higher proportion of the population previously treated with first-line drugs showed first- and second-line drug resistance (10.2% versus 5.6%; OR, 2.33; 95% CI, 1.058 to 5.145), whereas no significant difference between them was observed in the second-line drug resistance and pre-XDR-TB/XDR-TB. In addition, patients having used anti-TB drugs previously were more likely to have second-line drug resistance (37.8% versus 26.4%; OR, 1.60; 95% CI, 1.076 to 2.920), first- and second-line drug resistance (25.6% versus 4.1%; OR, 8.05; 95% CI, 2.898 to 21.35), and pre-XDR-TB/XDR-TB (18.9% versus 2.9%; OR, 7.04; 95% CI, 2.129 to 21.49) compared to those without a history of second-line drug use. However, the patient with different clinical characteristics (cavities, sputum smear results) had a similar risk (Table 3).
Table 3.
Sociodemographic and clinical characteristics associated with second-line drug resistance among the studied M. tuberculosis isolates
| Characteristic | No. and proportion of isolates with resistance to: |
||||||||
|---|---|---|---|---|---|---|---|---|---|
| Second-line drugs |
First- and second-line drugs |
Pre-XDR-TB/XDR-TB |
|||||||
| Total | No. (%) | OR (95% CI)a | Total | No. (%) | OR (95% CI)a | Total | No. (%) | OR (95% CI)a | |
| Age | 1.01 (1.001–1.024)b | 1.03 (1.007–1.043)b | 1.02 (1.010–1.059)b | ||||||
| Sex | |||||||||
| Female | 113 | 31 (27.4) | 113 | 6 (5.3) | 1 | 113 | 4 (3.5) | 1 | |
| Male | 267 | 83 (31.1) | 1.082 (0.665–1.761) | 267 | 18 (6.7) | 1.29 (0.474–4.093) | 267 | 13 (4.9) | 1.39 (0.418–5.999) |
| Anti-TB treatment history | |||||||||
| New | 321 | 93 (29.0) | 321 | 18 (5.6) | 1 | 321 | 12 (3.7) | 1 | |
| Previously treated | 59 | 21 (35.6) | 1.44 (0.626–3.090) | 59 | 6 (10.2) | 2.33 (1.058–5.145)b | 59 | 5 (8.4) | 2.38 (0.630–7.616) |
| Cavity | |||||||||
| No | 257 | 71 (27.6) | 1 | 257 | 16 (6.2) | 1 | 257 | 11 (4.3) | 1 |
| Yes | 123 | 43 (35.0) | 1.131 (0.893–1.431) | 123 | 8 (6.5) | 1.04 (0.377 −2.687) | 123 | 6 (4.9) | 1.15 (0.340–3.480) |
| Previous uses of second-line drugc | |||||||||
| No | 261 | 69 (26.4) | 1 | 341 | 14 (4.1) | 1 | 343 | 10 (2.9) | 1 |
| Yes | 119 | 45 (37.8) | 1.60 (1.076–2.920)b | 39 | 10 (25.6) | 8.05 (2.898–21.35)b | 37 | 7 (18.9) | 7.04 (2.129–21.49)b |
OR and 95% CI were calculated by comparing the drug-resistant rates between subjects with different sociodemographic and clinical features in the binary logistic regression model.
P < 0.05.
The association between previous uses of second-line drugs and drug resistance was analyzed by comparing the groups with previous use of the specific drug combinations (any second-line drugs, first- and second-line drugs, INH and RIF and any second-line drug) and that without use of the drug combinations.
Molecular characterization of second-line drug-resistant M. tuberculosis.
To determine the molecular basis of resistance to FQ and the injectable agents, the gyrA, rrs, eis, and tlyA regions were sequenced both in drug-resistant and -susceptible strains. Table 4 shows the mutations in the resistance-determining region of gyrA, eis, rrs, and tlyA as well as the corresponding resistance phenotypes. Mutations were observed in the QRDRs of gyrA in 41 (74.5%) FQ-resistant isolates, with mutations predominantly occurring at codon 94, while none of the FQ-susceptible isolates displayed mutations. The most frequent single mutation detected was the substitution from adenine to guanine in gyrA at codon 94, which led to an amino acid change from aspartic acid to glycine (16/55 isolates, 29.1%). Additionally, two FQ-resistant isolates with the mutation at codon 94 also carried a mutation at codon 74. Furthermore, in gyrB, only two FQ-resistant isolates had a change from cytosine to thymine at codon 543, but these isolates also had a mutation in gyrA (one with gyrA94Gly and the other with gyrA91Pro). Sequence analyses of gyrA showed a sensitivity and a specificity of 74.5% and 100%, respectively, for the detection of FQ resistance among the isolates analyzed.
Table 4.
Genetic characteristics and diagnostic performance of second-line drug resistance
| Drugc | Gene and mutation/amino change | No. of isolates |
Accuracy value |
||||
|---|---|---|---|---|---|---|---|
| Resistant |
Susceptible |
Sensitivitya | Specificityb | ||||
| With mutation | Without mutation | With mutation | Without mutation | ||||
| FQ | gyrA | 41 | 14 | 0 | 325 | 74.5 | 100 |
| gyrA90, Val(GTG) | 8 | 47 | 0 | 325 | 14.5 | 100 | |
| gyrA94, Gly(GGC) | 16 | 39 | 0 | 325 | 29.1 | 100 | |
| gyrA94, Asn(AAC) | 10 | 45 | 0 | 325 | 18.2 | 100 | |
| gyrA91, Pro(CCG) | 6 | 49 | 0 | 325 | 10.9 | 100 | |
| gyrA74, Ser(TCC) | 3 | 52 | 0 | 325 | 5.5 | 100 | |
| KM | rrs | 41 | 16 | 1 | 322 | 71.9 | 99.7 |
| rrs1401, G | 41 | 16 | 1 | 322 | 71.9 | 99.7 | |
| eis | 14 | 43 | 3 | 320 | 24.6 | 99.1 | |
| eis, G(−10)A | 8 | 49 | 2 | 321 | 14.0 | 99.4 | |
| eis, C(−14)T | 6 | 51 | 1 | 322 | 10.5 | 99.7 | |
| rrs and/or eis | 53 | 4 | 4 | 319 | 93.0 | 98.8 | |
| AMK | rrs | 38 | 6 | 4 | 332 | 86.4 | 98.8 |
| rrs1401, G | 38 | 6 | 4 | 332 | 86.4 | 98.8 | |
| CAP | rrs | 29 | 12 | 15 | 324 | 70.7 | 95.6 |
| rrs1401, G | 27 | 14 | 15 | 324 | 65.9 | 95.6 | |
| rrs1402, T | 2 | 39 | 0 | 339 | 4.9 | 100 | |
| tlyA | 4 | 37 | 4 | 335 | 9.8 | 98.8 | |
| tlyA, C14T | 1 | 40 | 3 | 336 | 2.4 | 99.1 | |
| tlyA, T708G | 3 | 38 | 1 | 338 | 7.3 | 99.7 | |
| rrs and/or tlyA | 30 | 11 | 17 | 322 | 73.2 | 95.0 | |
| Pre-XDR-TB | gyrA | 7 | 6 | 34 | 333 | 53.8 | 90.7 |
| rrs | 5 | 8 | 37 | 330 | 38.5 | 89.9 | |
| gyrA and/or rrs | 9 | 4 | 16 | 351 | 69.2 | 95.6 | |
Sensitivity = no. of drug-resistant isolates with mutations/(no. of drug-resistant isolates with mutations + no. of drug-resistant isolates without mutations).
Specificity = no. of drug-susceptible isolates without mutations/(no. of drug-susceptible isolates with mutations + no. of drug-susceptible isolates without mutations).
FQ, fluoroquinolones; KM, kanamycin; CAP, capreomycin; AMK, amikacin; Pre-XDR-TB, preextensively drug-resistant tuberculosis.
As for isolates with resistance to second-line injectable drugs, the most common mutation was the change from adenine to guanine at position 1401 of the rrs gene, observed in 71.9% (41/57) of KM-resistant isolates, 86.4% (38/44) of AMK-resistant isolates, and 65.9% (27/41) of CAP-resistant isolates. This mutation was found in 86.0% of the isolates (37/43) resistant to both AMK and KM. Two additional isolates that were resistant only to KM displayed a C1402T mutation in rrs. Four CAP-resistant isolates showed the mutations in tlyA, with one at codon 14 and three at codon 708. Among isolates susceptible to KM, AMK, and CAP, 4 (1.2%), 4 (1.2%), and 17 (5%), respectively, did carry a mutation in rrs, tlyA, or eis. Thus, the DNA sequencing was able to detect 93.0% of the resistance to KM, 86.4% of the resistance to AMK, and 73.2% of the resistance to CAP. Additionally, sequencing the gyrA gene alone to identify the pre-XDR-TB strain, the sensitivity and specificity were 53.8% and 90.7%, respectively. If jointly using gyrA and rrs, the sensitivity and specificity for identifying XDR-TB would be 69.2% and 95.6%, respectively.
MIC related to second-line drug resistance.
Among the 41 FQ-resistant isolates with a gyrA mutation, 53.6% were highly resistant to OFX (MIC ≥ 16 mg/liter) and 26.8% were highly resistant to LEV (MIC ≥ 16 mg/liter). High resistances to AMK, KM, and CAP (MIC ≥ 100 mg/liter) were observed, respectively, in 63.4%, 57.9%, and 31% of resistant isolates with an A1401G rrs mutation. However, 71.4% of the resistant mutants with a C1402T mutation in rrs were borderline resistant to KM, and the 66.7% of clinical isolates with tlyA mutations were low-level or borderline resistant to CAP. In contrast, the resistant mutants with the mutation in gyrA were higher-level resistant to FQ than those without an identified mutation in gyrA, as was observed in the rrs mutant with high-level resistance to the second-line injective drugs (Fig. 1).
Fig 1.
The MIC value of second-line drugs in isolates with genetic mutation related to the drug resistance. OFX, ofloxacin; LEV, levofloxacin; KM, kanamycin; CAP, capreomycin; AMK, amikacin. The distribution of MIC values is presented by symbols for each isolate, with the dotted line indicating the high level of drug resistance. The MIC values were compared between a specific genetic mutant and wild type of second-line drug-resistant isolates using the Mann-Whitney U test. a, P < 0.05.
Genetic characterization and MIC values for the extensively drug-resistant isolates.
All four XDR-TB isolates in this study showed a mutation in gyrA and a predominant rrs gene mutation at nucleotide 1401. Of the isolates with a gyrA mutation, all showed high-level resistance to both OFX and LEV. The three XDR-TB isolates with the rrs A1401G mutation showed high-level resistance to either AMK, KM, or CAP, while two of them showed low-level resistance to CAP (Table 5).
Table 5.
Genetic mutation and second-line drug MICs for 4 extensively drug-resistant isolates
| Isolate | Mutation |
MIC (mg/liter)a |
||||||
|---|---|---|---|---|---|---|---|---|
| gyrA | rrs | eis | OFX | LEV | CAP | AMK | KAN | |
| LY148 | D94G | A1401G | None | 16 | 32 | 64 | 64 | 128 |
| GA060 | D94G | A1401G | None | 16 | 16 | 128 | 64 | 64 |
| DQ096 | S91P | A1401G | None | 32 | 32 | 64 | 128 | 128 |
| YI207 | S91P | None | G-10A | 16 | 16 | 8 | 32 | 32 |
OFX, ofloxacin; LEV, levofloxacin; KM, kanamycin; CAP, capreomycin; AMK, amikacin.
DISCUSSION
China has been described as a global “hot spot” for drug-resistant TB. The present study attempted to systematically investigate the prevalence of second-line drug resistance and to identify the resistance-related mutations in the M. tuberculosis strains from TB patients in rural settings in China. The increased knowledge of second-line drug resistance could be valuable for development of rapid diagnostics molecular tools for timely detection of pre-XDR-TB/XDR-TB as well as for selection of an effective combination of second-line drugs for treatment of MDR-TB.
A small minority of the MDR-TB patients (9.3%) was resistant also to FQ and injectable drugs and identified as XDR. This can be compared to earlier information from Shanghai, China (10%) (5), the United states (5%) (23), and Latvia (19%) (24). On the other hand, the proportion of FQ resistance was significantly higher in MDR-TB isolates than in drug susceptibility isolates, whereas resistances to injectable drugs were similarly low in MDR and non-MDR isolates. Based on these results, there was concern about the increasing FQ resistance in MDR-TB and the possible reduced efficacy of drug combinations including FQ in treating MDR-TB. Additionally, due to the extent of high-level resistance to second-line drugs in most studied XDR-TB isolates, the treatment options for XDR-TB are limited, and new effective drug combinations are urgently needed.
Several sociodemographic and clinical characteristics were associated with second-line drug resistance. The elderly TB patient was at a high risk of having pre-XDR-TB and XDR-TB. This might be the result of the accumulation of drug resistance due to aging. Therefore, more careful consideration should be given to the treatment of TB among the elderly (25). Meanwhile, patients with a treatment history of first- and second-line drugs were more likely to have isolates resistant to second-line drugs. This association, to some degree, could reflect the poor administration of drugs in health facilities, where some second-line drugs, like FQ, are easily and extensively prescribed for respiratory infections and other bacterial infections and in some cases even available without a prescription in local drug stores. Easy access and inappropriate use of these drugs increase the risk for the emergence of drug-resistant TB (25). Meanwhile, this highlights the utmost importance of strict regulation of the drug use in the therapy of TB and drug-resistant TB.
Molecular methods to identify mutations associated with drug resistance can significantly decrease diagnostic delay and, in some cases, may prove to be more specific than phenotypic DST. Prior to broad implementation of molecular methods in detecting the second-line drug resistance, it is necessary to identify the genes and loci associated with drug resistance among isolates circulating in any specific setting. We observed that the mutation frequency of D94G (29.0%) in the gyrA gene in our study was lower than earlier reported, ranging from 33.3% to 41.5% (26–28). The sensitivity to detect FQ resistance in our study (74.5%) was lower than results reported in Germany (90.6%, n = 32 cases) (27) and France (87.5%, n = 24 cases) (26) but similar to what was seen in Vietnam (75.6%, n = 41 cases) (29). Therefore, the study of the mutations in other genes, such as mfpA (Rv3361c) (30), or the active efflux pump Rv2686c-Rv2687c-Rv2688c operon (31, 32) might help to better understand the different mechanisms of FQ resistance and could improve the performance of molecular diagnosis for the resistance to FQ.
We also studied mutations in genes responsible for resistance to second-line injectable drugs in China. The A1401G mutation appeared in the majority of the KM- and AMK-resistant isolates, with 76.9% and 86.4% sensitivity, respectively, compared to the DST test, and this mutation provided a better marker for AMK resistance than for KM resistance. It is possible that KM resistance is caused by a mutation in another gene, such as the eis promoter region (33). In fact, we observed that 21.1% of our KM-resistant but AMK-susceptible isolates had a mutation in the eis gene. Therefore, the eis gene should be included in the molecular analysis of KM resistance. However, we identified 15 CAP-susceptible isolates with the mutations in the rrs gene. Meanwhile, the tlyA mutation was, however, also observed in CAP-susceptible isolates. These data might suggest that using rrs and tlyA to detect CAP resistance could lead to a certain amount of false positivity. Additionally, as previously reported, we observed a significant cross-resistance between KM and AMK associated with the A1401G mutation of the rrs gene (26, 34). Therefore, an A1401G mutation might predict the cross-resistance among these agents.
Our study was limited to patients in eastern rural China and cannot represent the overall situation in the whole country. It is likely that the situation is even more severe in rural areas where second-line drugs have been extensively used to treat MDR-TB. Additionally, we attempted to achieve a clear view of the prevalence of second-line drug-resistant TB and the corresponding resistance-related mutations based in rural China. But still some part of second-line drug resistance could not be explained, i.e., the resistance to FQ and CAP. Whole-genome sequencing might help to find more loci associated with the resistance to second-line drugs and improve the performance of the molecular diagnosis.
In conclusion, in our study period, 5.6% of the TB cases in eastern rural China had MDR, and 39.5% of these isolates were resistant also to at least one of the key second-line drugs. Association between second-line drug resistance and previous use of second-line drugs indicate that an improper use of second-line drugs for other lung infections is a real threat to effective MDR-TB treatment. Detecting mutations in the resistance-related genes could offer timely detection of MDR-TB and XDR-TB and could assist in controlling these severe forms of TB.
ACKNOWLEDGMENTS
This work was supported by an NIH grant (R01AI075463) and a grant from National Natural Science Foundation of China (no. 30771843).
Footnotes
Published ahead of print 3 June 2013
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