Prevalence and molecular characteristics of drug-resistant tuberculosis in Hunan,

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1 AAC Accepts, published online ahead of print on 14 April 2014 Antimicrob. Agents Chemother. doi: /aac Copyright 2014, American Society for Microbiology. All Rights Reserved Prevalence and molecular characteristics of drug-resistant tuberculosis in Hunan, China Li-li Zhao 1,2#, Yan Chen 1,3#, Zhong-nan Chen 4#, Hai-can Liu 1,2, Pei-lei Hu 4, Qing Sun 1,3, Xiu-qin Zhao 1,2, Yi jiang 1,2, Gui-lian Li 1,2, Yun-hong Tan 4 *, Kang-lin Wan 1 * 1 National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention /State Key Laboratory for Infectious Disease Prevention and Control /National Reference Laboratory of tuberculosis, Beijing , China 2 Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou , China 3 Pathogenic Biology Institute, University of South China, Hengyang, , Hunan Province, China 4 Hunan Provincial Chest Hospital, Tuberculosis Control Institution of Hunan Province, Hunan , China # contribute equally to this work * Corresponding author: Kang-lin Wan, P.O. Box 5, Changping, Beijing , China. Tel and fax: wankanglin@icdc.cn. Yun-hong Tan, Hunan Provincial Chest Hospital, Tuberculosis Control Institution of Hunan Province, Hunan , China, tanyunhong@163.com. Abstract To determine the prevalence and molecular characteristics of drug-resistant tuberculosis in Hunan province, drug susceptibility testing and spoligotyping method were performed among 171 Mycobacterium tuberculosis (M. tuberculosis) isolates. In addition, the mutated characteristics of 12 loci including katg, inha, rpob, rpsl, rrs 1, embb, pnca, tlya, eis, rrs 2, gyra and gyrb among drug-resistant isolates were also analyzed by DNA sequencing. Our results indicated that the prevalence of isoniazid (INH), rifampin (RIF), streptomycin (SM), ethambutol (EMB), pyrazinamide (PZA), capreomycin (CAP), kanamycin (KAN), amikacin (AKM) and ofloxacin (OFX) resistance in Hunan province were 35.7%, 26.9%, 20.5%, 9.9% 15.2%, 2.3%, 1.8%,

2 % and 10.5%, respectively. The previously treated patients presented significantly increased risks for developing drug resistance. The majority of M. tuberculosis isolates belonged to Beijing family. Almost all the drug resistance demonstrated no association with genotype. The most frequent mutations of drug-resistant isolates were katg315, inha-15, rpob531, 526 and 516, rpsl43, rrs 1 514, embb306, pnca96, rrs , gyra94 and 90, respectively. These results contribute to the knowledge of the prevalence of drug resistance in Hunan and also expand the molecular characteristics of drug resistance in China. Key words: Mycobacterium tuberculosis, drug resistance, prevalence, molecular characteristics, genotyping, mechanisms of resistance, The emergence of drug resistant tuberculosis (TB), particularly multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB), has been identified as one of the major obstacles to effective TB control in many countries (1). As one of the 22 high TB-burden countries, the prevalence of drug-resistant TB is also a serious problem in China. The latest data from the national baseline survey on TB indicated that the frequencies of drug-resistant TB, MDR-TB and XDR-TB among pulmonary TB patients in China were 38.25%, 8.32% and 0.68% (2), respectively. The magnitude and pattern of drug resistance varied greatly with regions because of the huge size of the country, the diverse population density, and the unbalanced economic development in China (3). Drug-resistant TB is usually associated with inadequate anti-tb treatment or direct transmission of drug-resistant strains from one individual to another. Knowledge of the clinical characteristics and molecular characteristics of drug-resistant TB is very helpful in their rapid diagnosis and containment. However, previous studies demonstrated that these information varied in different geographical areas (1, 4-7). Hunan province, with a total population of million, is a high TB burden area in the south-central of China. According to the recently updated epidemiological data, the prevalence of smear positive pulmonary TB in this region was estimated at 94 cases per 100,000 inhabitants in 2010 (data not published), higher than the average

3 level of China (66 cases per 100,000 inhabitants, Unfortunately, thus far, the prevalence and molecular characteristics of drug-resistant TB of Hunan province remains unclear and should be explored to facilitate control of the TB epidemic in this region and throughout China. Therefore, in the present study, we investigated the genotypes and current levels of resistance to TB drugs (INH, RIF, SM, EMB, PZA, CAP, KAN, AKM and OFX) among patients with pulmonary TB in Hunan. Moreover, we analyzed 12 genetic loci association with drug resistance, including katg and inha (for resistance to INH), rpob (RIF), rpsl and rrs 1 (SM), embb (EMB), pnca (PZA), tlya (CAP), eis (KAN or AKM), rrs 2 (CAP, KAN or AKM), gyra and gyrb (OFX) among drug-resistant isolates from Hunan. Methods Ethical approval The study obtained approval from the Ethics Committee of National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention. The patients with TB were included in the present research only after receiving informed written consent from themselves or from their parents/ guardians if they were child (<18 years of age). An assent was also obtained from participants between years of age. Mycobacterium tuberculosis isolates This study was carried out between December 2009 and August 2010 at the Hunan Chest Hospital located in the capital of Hunan Province, which serves as the sole specialized TB hospital in Hunan. A total of 171 inpatients with clinically diagnosed pulmonary tuberculosis and positive cultures identified as M. tuberculosis complex (MTC) were interviewed and enrolled during the study period. Strains isolation and identification were performed at the provincial reference laboratory of Hunan Chest Hospital. Only one isolate per patient was collected and tested. Drug susceptibility testing The drug susceptibility testing (DST) was performed using BACTEC MGIT 960

4 system in national tuberculosis reference laboratory (NTRL). The critical concentrations for the DST were 0.1 μg/ ml for INH, 1.0 μg/ ml for RIF, 1.0 μg/ ml for SM, 5.0 μg/ ml for EMB, μg/ ml for PZA, 2.5μg/ ml for CAP, 2.5μg/ ml for KAN, 2.5μg/ ml for AKM, 2.0μg/ ml for OFX, respectively (8). Data collection and definitions The demographic and clinical information of enrolled patients were obtained from the inpatient s records including gender, age, occupation, address, complication and TB treatment history. MDR-TB was defined as resistance to the two first-line drugs, INH and RIF. XDR TB is resistant to INH and RIF, plus OFX and at least one of the three injectable second-line drugs (CAP, KAN or AKM). Pre-XDR TB was defined as TB with resistance to INH and RIF and either OFX or a second-line injectable agent but not both. Migrants were defined as individuals from other provinces of China or other country who moved to Hunan. Residents were defined as persons with a registered permanent residence in Hunan. New or previously treated TB cases were defined as previously described (9). DNA extraction Genomic DNA were prepared by CTAB method as described previously (10) and stored at -20 ºC for further use. Spoligotyping and data analysis Spoligotyping was performed using 43 covalently bound oligonucleotides derived from the spacer sequences of M. tuberculosis H37Rv and Mycobacterium bovis BCG P3 as previously described by Kamerbeek et al (11). The results in binary format were entered in an Excel spreadsheet and compared with the spoligotyping database SpolDB4 ( PCR amplification and sequencing Expected fragment were amplified using primers in Supplemental Table 1. Each 30-μl PCR mixture contained 15 μl 2x Taq Master Mix (Takara), 1 μl of the forward and reverse 5 μm primers, 12 μl distilled H 2 O and 1 μl of genomic DNA. The reaction

5 conditions consisted of a denaturation step of 5 min at 95ºC, followed by 35 cycles of 30 sec at 94 ºC, 30 sec at 60 ºC, 45 sec at 72 ºC, and a final extension step of 5 min at 72 ºC. PCR products were sent for sequencing. All sequence data were manipulated with BioEdit version and were compared the published sequences (GenBank accession number NC_000962). Resolution of discrepant results For the discrepant results between the DST and DNA sequencing, retesting was performed twice using both methods. If the retesting results were in conflict with the original data, a third round of testing was completed, with the final value representing two out of the three cycles. Statistical analysis The data were analyzed with the use of SPSS software 16.0 version. Frequencies, percentages, ranges and confidences intervals (CI) were calculated as appropriated. Chi square test or Fisher s exact tests was used for inter group comparison. A two sided p value <0.05 was considered as statistically significant. Results Demographic information Among the 171 isolates, 129 (75.4%) were from male patients and 42 (24.6%) from female patients. The age of the patients ranged from 15 to 88 years (mean ± SE, 44.8 ± 1.3). Majorities (96.5%, 165/171) of them were Hunan residents and 56.7% (97/171) were newly diagnosed cases. Drug susceptibility patterns In this study, 59.1% (95% CI, ) of the TB cases were resistant to at least one of the five first-line drugs (INH, RIF, SM, EMB and PZA); 11.1% (95% CI, ) were resistant to at least one of the four second-line drugs (CAP, KAN, AKM and OFX) (Table 1). The proportion of MDR, pre-xdr and XDR were 25.2% (95% CI, ), 8.7% (95% CI, ) and 1.8%, respectively (Table 1). In addition, the proportion of drug resistant TB among new cases and previously treated cases were also summarized in Table 1. Factors linked to drug-resistant tuberculosis

6 Table 2 showed that the analysis of risk factors for MDR and drug-resistant (but not MDR) tuberculosis among all the 171 patients. The previously treated patients presented significantly increased risks for developing drug resistance (but not MDR) with an OR = 3.35 (95% CI, ; P = 0.01). Furthermore, the risk increased highly significantly in MDR-cases with an OR = 6.17 (95% CI, ; P = 0.00). Genotype results Among 171 TB isolates, 126 (73.7%) belonged to the Beijing family, while 45 (26.3%) were non-beijing family (Supplemental Table 2). Isolates classified into non-beijing family included 34 isolates (19.9%) from T family, 4 (2.3%) from U family, 1 (0.6%) from CAS1-DELHI family, 1 (0.6%) from MANU2 family, and remaining 5 being orphan (2.9%). Drug susceptibility profiles of different genotype The drug susceptibility profiles of Beijing family and non-beijing family were compared in Supplemental Table 3. The distribution of any drug resistance (apart from PZA) among Beijing family was observed more frequently than among non-beijing family, but there was no significant difference except for SM (P<0.05). Detection of drug-resistance associated mutations by DNA sequencing DNA sequencing could identify 78.7% (48/61) INH resistant isolates, 87.0% (40/46) RIF resistant isolates, 88.6% (31/35) SM resistant isolates, 76.5% (13/17) EMB resistant isolates, 84.6% (22/26) PZA resistant isolates, 75.0% (3/4) CAP resistant isolates, 100.0% (3/3) KAN or (2/2) AKM resistant isolates, and 77.8% (14/18) OFX resistant isolates. All the mutated characteristics of drug-resistant isolates were summarized in Supplemental Table The major mutations were katg315 (67.2%), inha-15 (9.8%), rpob531 (47.8%), 526 (21.7%) and 516 (8.7%), rpsl43 (71.4%), rrs (11.5%), embb306 (70.6%), pnca96 (11.5%), rrs (75.0% or 100.0%), and gyra94 (55.6%) and 90 (22.2%), respectively (Table 4). In addition, no mutations were founded within the target regions of tlya, eis and gyrb among the corresponding drug-resistant isolates. Discussion The present study demonstrated that 41.5% (71/171) patients with pulmonary TB

7 had drug-resistant disease, suggesting that serious epidemic of drug-resistant tuberculosis among patients with pulmonary TB in Hunan. The proportions of MDRand XDR-TB among patients were 25.2% and 1.8%, nearly three times of the data from national baseline survey in 2007 (2). The reason contributing to this obvious difference was probably that the isolates of present study were obtained from the specialized hospital instead of epidemiological survey, causing a distinctly higher proportion of previously treated patients (43.3%) than the data from national baseline survey (22.7%). The proportions of drug-resistant TB in previously treated patients were quite higher than those in new patients (2) and confirmed by this study. A higher risk of drug-resistance was also found among previously treated patients. Hence, some appropriate strategies must be implemented to increase continuity of treatment and reduce the rate of treatment default. Notably, a lower incidence of drug resistance in new patients was observed in this study than that of national baseline survey, implying that drug resistance in new cases varied greatly with regions and the transmission of drug-resistant TB in Hunan is less heavy than some regions. The percentages of second-line injectable drug resistance for CAP, KAN, and AKM were 2.3%, 1.8%, 1.2%, respectively, significantly lower than that of OFX (10.5%). These really low resistant levels were detected also in MDR-TB (9.3% for CAP, 7.0% for KAN and 4.7% for AKM). All these results point out that the second-line injectable drug is excellent for the potential of a successful treatment against TB or MDR-TB. The high rate of OFX in TB isolates, especially in MDR-TB isolates (32.6%) indicated that the OFX have been used extensively in Hunan during the past years to treat drug-resistant TB patients and retreatment patients. In addition, among patients with MDR-TB, 34.9% (15/43) were pre-xdr TB, placing them only one step away from having XDR-TB. Beijing family was the most dominant genotype in Hunan province, which is accordant with the findings from most areas of China (12-14). In addition to Beijing family, other families including T, H, MANU2, U, CAS1-DELHI and Orphan were also identified. Notably, only 1strain belonged to CAS1-DELHI family. This strain was isolated from a patient who was an overseas student from Pakistan, a country

8 with a predominance of CAS family (including CAS1, CAS sub-families and Orphan Pak clusters) (15). Some publications showed that Beijing family was associated with drug resistance (15, 16). However, less association also were reported in other geographic settings (17, 18). Our study suggested that the association with drug resistance was significant for only SM, indicating that the Beijing family is less likely to be associated with the most of drug resistance in Hunan area. DNA sequencing of the hot spot regions in genetic loci demonstrated that the frequencies of mutations occurring among INH resistant isolates were 78.7%, similar to the results reported in Jiangxi (19), but lower than the data from Fujian and Shanghai of China. For the RIF resistant isolates in China, a substantial proportions ( %) of mutations within the rpob gene were observed (19-25). Our results displayed that 87.0% of the RIF-resistant isolates carrying mutations in rpob gene. We presumed it probably that the limited number of RIF-resistant isolates (46 isolates) was analyzed in our study. Furthermore, the mutated profiles of M. tuberculosis isolates from different areas might have somewhat geographical difference. The mutated frequencies in SM, EMB, PZA and OFX resistant isolates were 88.6%, 76.5%, 84.6% and 77.8%, respectively, which were agreement with the findings from many investigations (19, 26-28). However, the mutated frequencies in CAP and KAN/AKM resistant isolates (75.0% and 100.0%) were inconsistent with previous reports (19, 27-30). One possible reason was that the account of CAP, KAN/AKM resistant isolates in our study was very small. There were still some resistant-isolates harboring no mutation within the sequenced regions. Although retesting was performed using DST and DNA sequencing, the retesting results remained same. This implied that these isolates probably harbored mutations outside the sequenced area, or the resistance may be caused by other mechanisms, such as efflux pumps (31). In addition, TB cultures might also comprised a mixed population of resistant and susceptible bacteria (32), and thus, ordinary PCR-based DNA sequencing could not detect the low proportion of drug-resistant TB among the predominant wild-type TB, leading to a falsely low

9 detection rate. The most common mutations among these drug-resistant isolates were katg315, inha-15, rpob531, 526 and 516, rpsl43, rrs 1 514, embb306, rrs , gyra94 and 90, as previously reported (19, 27-30, 33). For the pnca gene, although codon 96 was the major mutation, it was found in only 3 PZA-resistant isolates (11.5%). Next were the codon 120, 141, 142 and 177, which were indentified in 2 PZA-resistant isolates, respectively. Other mutations consisted of 11 unique alterations distributed throughout the whole gene. These supported that the mutations harboring pnca was tremendous diverse and scattered along the whole gene (27, 34, 35). There was no any mutation within tlya, eis and gyrb conferring drug resistance in the present study, which were probably due to the limited number of resistant isolates for CAP, KAN/AKM and OFX. The limitations of the current study should be address. First, this study was carried out in only one specialized TB hospital in Hunan. Although this hospital is the largest specialized hospital for TB in the region, the results based on inpatients from only one hospital might not reflect overall situation in the region. Then, the specialized hospital might have higher rates of serious TB cases than other hospitals in the region, probably leading to an overestimation of drug resistant tuberculosis. Next, the number of second-line drugs resistant isolates, especially for CAP (n=4), KAN (n=3), and AKM-resistant isolates (n=2) in our research was relatively small. This might have limited the detection of the variety of gene variations. Moreover, since the sequencing data of drug-susceptible isolates were not included in our study, the specificities of different resistance related mutations were not evaluated. Therefore, additional studies containing substantial panel of drug-resistant and susceptible isolates will be required in the future. In summary, our results indicated that the prevalence of INH, RIF, SM, EMB, PZA, CAP, KAN, AKM and OFX resistance in Hunan province were 35.7%, 26.9%, 20.5%, 9.9%, 15.2%, 2.3%, 1.8%, 1.2% and 10.5%, respectively. The majority of M. tuberculosis belonged to Beijing family. Almost all the drug resistance demonstrated no association with genotype. The most frequent mutations were katg315, inha-15,

10 rpob531, 526 and 516, rpsl43, rrs 1 514, embb306, pnca96, rrs , gyra94 and 90, respectively. These results were very helpful to generate the appropriate TB control policy and establish the rapid molecular diagnostic methods which will be implemented in Hunan province and throughout China. Funding: This study was supported by the projects of National Natural Science Foundation of China (Grant No ) and the National Key Program of Mega Infectious Diseases (Grant No. 2013ZX ). The funder had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Conflict of interest statements: All authors have no competing interests. Reference: 1. Gandhi NR, Nunn P, Dheda K, Schaaf HS, Zignol M, van Soolingen D, Jensen P, Bayona J Multidrug-resistant and extensively drug-resistant tuberculosis: a threat to global control of tuberculosis. Lancet 375: Zhao Y, Xu S, Wang L, Chin DP, Wang S, Jiang G, Xia H, Zhou Y, Li Q, Ou X, Pang Y, Song Y, Zhao B, Zhang H, He G, Guo J, Wang Y National survey of drug-resistant tuberculosis in China. N Engl J Med 366: He GX, Zhao YL, Jiang GL, Liu YH, Xia H, Wang SF, Wang LX, Borgdorff MW, van der Werf MJ, van den Hof S Prevalence of tuberculosis drug resistance in 10 provinces of China. BMC Infect Dis 8: Afanas'ev MV, Ikryannikova LN, Il'ina EN, Sidorenko SV, Kuz'min AV, Larionova EE, Smirnova TG, Chernousova LN, Kamaev EY, Skorniakov SN, Kinsht VN, Cherednichenko AG, Govorun VM Molecular characteristics of rifampicin- and isoniazid-resistant Mycobacterium tuberculosis isolates from the Russian Federation. J Antimicrob Chemother 59: Filliol I, Driscoll JR, Van Soolingen D, Kreiswirth BN, Kremer K, Valetudie G, Anh DD, Barlow R, Banerjee D, Bifani PJ, Brudey K, Cataldi A, Cooksey RC, Cousins DV, Dale JW, Dellagostin OA, Drobniewski F, Engelmann G, Ferdinand S, Gascoyne-Binzi D, Gordon M, Gutierrez MC, Haas WH, Heersma H, Kallenius G, Kassa-Kelembho E, Koivula T, Ly HM, Makristathis A, Mammina C, Martin G, Mostrom P, Mokrousov I, Narbonne V, Narvskaya O, Nastasi A, Niobe-Eyangoh SN, Pape JW, Rasolofo-Razanamparany V, Ridell M, Rossetti ML, Stauffer F, Suffys PN, Takiff H, Texier-Maugein J, Vincent V, De Waard JH, Sola C, Rastogi N Global distribution of Mycobacterium tuberculosis spoligotypes. Emerg Infect Dis 8: Ahmed N, Alam M, Rao KR, Kauser F, Kumar NA, Qazi NN, Sangal V, Sharma VD, Das R, Katoch VM, Murthy KJ, Suneetha S, Sharma SK, Sechi LA, Gilman RH, Hasnain SE

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13 : Barco P, Cardoso RF, Hirata RD, Leite CQ, Pandolfi JR, Sato DN, Shikama ML, de Melo FF, Mamizuka EM, Campanerut PA, Hirata MH pnca mutations in pyrazinamide-resistant Mycobacterium tuberculosis clinical isolates from the southeast region of Brazil. J Antimicrob Chemother 58: Mphahlele M, Syre H, Valvatne H, Stavrum R, Mannsaker T, Muthivhi T, Weyer K, Fourie PB, Grewal HM Pyrazinamide resistance among South African multidrug-resistant Mycobacterium tuberculosis isolates. J Clin Microbiol 46: Downloaded from on October 17, 2018 by guest

14 Table 1 Drug susceptibility patterns of 171 clinical M. tuberculosis isolates Susceptibility or resistance Total patients (n=171) New patients (n=97) Previously treated patients (n=74) No. % (95% CI) No. % (95% CI) No. % (95% CI) Susceptibility to overall first-line drugs ( ) ( ) ( ) Overall first-line drugs resistance ( ) ( ) ( ) INH ( ) ( ) ( ) RIF ( ) ( ) ( ) SM ( ) ( ) ( ) EMB ( ) 2 2.1* ( ) PZA ( ) 3 3.1* ( ) overall MDR ( ) ( ) ( ) INH+RIF ( ) 1 1.0* ( ) INH+RIF+SM ( ) 2 2.1* 5 6.8* INH+RIF+EMB * * INH+RIF+PZA ( ) ( ) INH+RIF+SM+EMB * * INH+RIF+SM+PZA * 1 1.0* * INH+RIF+EMB+PZA * * INH+RIF+SM+EMB+PZA ( ) 2 2.1* 5 5.4* Susceptibility to all second-line drugs ( ) * ( ) Overall second-line drugs resistance ( ) 2 2.1* ( ) CAP * * KAN * * AKM * * OFX ( ) 2 2.1* ( ) Overall pre-xdr ( ) 1 1.0* ( )

15 CAP * * KAN * * AKM * OFX ( ) 1 1.0* ( ) Overall XDR * * CAP+OFX * * CAP+KAN+AKM+OFX * * *, 95% CI was not determined. Downloaded from on October 17, 2018 by guest

16 Factors Table 2 Factors associated with drug-resistant tuberculosis Pan-susceptible TB No. (%) isolates drug-resistant, but not MDR TB MDR TB drug-resistant, but not MDR TB vs. Pan-susceptible TB Odds Ratio (95% CI) P value MDR TB vs. drug-resistant, but not MDR TB Odds Ratio (95% CI) P value Sex Male reference reference Female ( ) ( ) 0.62 Age group <30 yr reference reference yr ( ) ( ) yr ( ) ( ) 1.00 Occupation Farmer reference reference Others ( ) ( ) 0.97 Treatment New cases reference reference Previously treated cases ( ) 0.01* 6.17 ( ) 0.00** CI, confidence interval. *, P<0.05 (significant). **, P<0.01 (highly significant)

17 Table 3 Most frequent identified mutations within twelve drug-resistant associated loci among drug-resistant M. tuberculosis Drug (drug-resistant Relative Locus Mutated position No. (%) of isolates isolates) frequency (%) a INH (61) KatG inha RIF (46) rpob SM (35) rpsl rrs EMB (17) embb PZA (26) pnca CAP (4) rrs tlya none NA b NA KAN (3) rrs eis none NA NA AKM (2) rrs eis none NA NA OFX (18) gyra a Compared to the total number of isolates resistant to the drug of interest. b NA, not applicable Downloaded from on October 17, 2018 by guest