Volume 35 Issue 5
May  2022
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JIA Qing Jun, ZENG Mei Chun, XIE Li, CHENG Qing Lin, HUANG Yin Yan, LI Qing Chun, WU Yi Fei, AI Li Yun, LU Min, FANG Zi Jian. Molecular Detection of Ofloxacin and Kanamycin Resistance in Patients with MDR and Non-MDR TB from Suburban Districts in Hangzhou, China, 2019–2020[J]. Biomedical and Environmental Sciences, 2022, 35(5): 468-471. doi: 10.3967/bes2022.064
Citation: JIA Qing Jun, ZENG Mei Chun, XIE Li, CHENG Qing Lin, HUANG Yin Yan, LI Qing Chun, WU Yi Fei, AI Li Yun, LU Min, FANG Zi Jian. Molecular Detection of Ofloxacin and Kanamycin Resistance in Patients with MDR and Non-MDR TB from Suburban Districts in Hangzhou, China, 2019–2020[J]. Biomedical and Environmental Sciences, 2022, 35(5): 468-471. doi: 10.3967/bes2022.064

Molecular Detection of Ofloxacin and Kanamycin Resistance in Patients with MDR and Non-MDR TB from Suburban Districts in Hangzhou, China, 2019–2020

doi: 10.3967/bes2022.064
Funds:  This work was sponsored by the Hangzhou Health Science and Technology Projects [A20220558]; Zhejiang General Research Project on Medical Health and Science Technology Plan [2021KY949]; and Medical and Health Science and Technology Project of Zhejiang Province [2020KY238]
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  • Author Bio:

    JIA Qing Jun, male, born in 1990, Master’s Degree, majoring in tuberculosis control and prevention;

    ZENG Mei Chun, female, born in 1992, Master’s Degree, majoring in pathology

  • &These authors contributed equally to this work.
  • Received Date: 2022-01-15
  • Accepted Date: 2022-03-31
  • &These authors contributed equally to this work.
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  • [1] Umar FF, Husain DR, Hatta MM, et al. Molecular characterisation of mutations associated with resistance to first- and second-line drugs among Indonesian patients with tuberculosis. J Taibah Univ Med Sci, 2020; 15, 54−8.
    [2] World Health Organization. Guidelines for the programmatic management of drug-resistant tuberculosis: emergency update 2008. World Health Organization. 2008.
    [3] Zhao YL, Pang Y. Laboratory test procedures for tuberculosis. People’s Health Press, 2015; 1−260. (In Chinese)
    [4] Zhao LL, Chen Y, Chen ZN, et al. Prevalence and molecular characteristics of drug-resistant Mycobacterium tuberculosis in Hunan, China. Antimicrob Agents Chemother, 2014; 58, 3475−80. doi:  10.1128/AAC.02426-14
    [5] Liu Q, Yang DD, Qiu BB, et al. Drug resistance gene mutations and treatment outcomes in MDR-TB: a prospective study in Eastern China. PLoS Negl Trop Dis, 2021; 15, e0009068. doi:  10.1371/journal.pntd.0009068
    [6] Li QL, Wang YL, Li YN, et al. Characterisation of drug resistance-associated mutations among clinical multidrug-resistant Mycobacterium tuberculosis isolates from Hebei Province, China. J Glob Antimicrob Resist, 2019; 18, 168−76. doi:  10.1016/j.jgar.2019.03.012
    [7] Lee JJ, Kang HY, Lee WI, et al. Efflux pump gene expression study using RNA-seq in multidrug-resistant TB. Int J Tuberc Lung Dis, 2021; 25, 974−81. doi:  10.5588/ijtld.21.0117
    [8] Kambli P, Ajbani K, Nikam C, et al. Correlating rrs and eis promoter mutations in clinical isolates of Mycobacterium tuberculosis with phenotypic susceptibility levels to the second-line injectables. Int J Mycobacteriol, 2016; 5, 1−6. doi:  10.1016/j.ijmyco.2015.09.001
    [9] Zignol M, Cabibbe AM, Dean AS, et al. Genetic sequencing for surveillance of drug resistance in tuberculosis in highly endemic countries: a multi-country population-based surveillance study. Lancet Infect Dis, 2018; 18, 675−83. doi:  10.1016/S1473-3099(18)30073-2
    [10] Zeng MC, Jia QJ, Huang YY. The spatiotemporal dynamic distributions of new tuberculosis in Hangzhou, China. Biomed Environ Sci, 2020; 33, 277−81.
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Molecular Detection of Ofloxacin and Kanamycin Resistance in Patients with MDR and Non-MDR TB from Suburban Districts in Hangzhou, China, 2019–2020

doi: 10.3967/bes2022.064
Funds:  This work was sponsored by the Hangzhou Health Science and Technology Projects [A20220558]; Zhejiang General Research Project on Medical Health and Science Technology Plan [2021KY949]; and Medical and Health Science and Technology Project of Zhejiang Province [2020KY238]
  • Author Bio:

  • &These authors contributed equally to this work.
&These authors contributed equally to this work.
JIA Qing Jun, ZENG Mei Chun, XIE Li, CHENG Qing Lin, HUANG Yin Yan, LI Qing Chun, WU Yi Fei, AI Li Yun, LU Min, FANG Zi Jian. Molecular Detection of Ofloxacin and Kanamycin Resistance in Patients with MDR and Non-MDR TB from Suburban Districts in Hangzhou, China, 2019–2020[J]. Biomedical and Environmental Sciences, 2022, 35(5): 468-471. doi: 10.3967/bes2022.064
Citation: JIA Qing Jun, ZENG Mei Chun, XIE Li, CHENG Qing Lin, HUANG Yin Yan, LI Qing Chun, WU Yi Fei, AI Li Yun, LU Min, FANG Zi Jian. Molecular Detection of Ofloxacin and Kanamycin Resistance in Patients with MDR and Non-MDR TB from Suburban Districts in Hangzhou, China, 2019–2020[J]. Biomedical and Environmental Sciences, 2022, 35(5): 468-471. doi: 10.3967/bes2022.064
  • The continuing emergence and spread of drug-resistant Mycobacterium tuberculosis complex (MTBC) poses a challenge to global tuberculosis (TB) control programs. Drug resistance in TB is mainly conferred by specific point mutations in the MTBC genome[1]. Multidrug-resistant TB (MDR-TB), which is caused by MTBC strains with resistance to both isoniazid and rifampicin (RIF), the most important first-line medicines, poses the main threat to TB control and elimination. Fluoroquinolones (FQs) and second-line injectable drugs (SLIDs) are core compounds in current MDR-TB treatment regimens[2]; however, data on the prevalence of resistance to FQs and SLIDs among MTBC strains has been lacking. The rapidly increasing number of DR-TB cases further underscores the need for more intensive surveillance of susceptibility to these important second-line drugs, to minimize further increases in the number of cases of preXDR-TB (MDR-TB with additional resistance to any FQ drug or at least one of the three SLIDs) and XDR-TB (MDR-TB with resistance to both FQs and SLIDs).

    Therefore, in the present study, we investigated the ofloxacin (OFX) and kanamycin (KM) resistance in MDR and non-MDR TB isolates collected from clinical sputum specimens in northwest Zhejiang, China—a rural area with approximately 5,140,000 inhabitants. Mycobacteria were isolated from sputum specimens collected from patients with suspected TB. Sputum smears and GeneXpert MTB/RIF tests were performed simultaneously. Species identification was performed with p-nitrobenzoic acid, and phenotypic drug susceptibility testing to determine the susceptible proportions was performed on Lowenstein–Jensen medium according to the national guidelines[3]. Genomic DNA of inactivated MTBC strains (resistant to OFX or KM) was extracted with a DNA extraction kit (Invitrogen Thermo Fisher Scientific, Waltham, MA, USA). Fragments of the gyrA, gyrB, rrs2, and eis genes were amplified (Supplementary Figure S1, available in www.besjournal.com), and sequencing was performed to identify point mutations in the selected resistant strains as previously reported[4].

    Figure S1.  Agarose gel electrophoresis of PCR assays for the identification of eis, rrs2, gyrA, and gyrB genes. M: DL500 DNA Marker (Takara, Code No. 3590A).

    A total of 1,888 sputum culture-positive specimens of suspected TB from ten designated hospitals were collected from 2019 to 2020. Of these, 289 had putative contamination and were either re-culture failures or repeat samples from some patients, and therefore, were excluded from the study (Figure 1). Six specimens had no records of patient information, and 209 were non-tuberculosis Mycobacterium. Of the remaining 1,384 specimens, 1,124 were susceptible to all six drugs, 44 were MDR-TB, and 58 were resistant to OFX, KM, or both.

    Figure 1.  Flow chart of drug resistance MTBC identified from 1,888 culture-positive specimens of suspected TB patients in rural areas of Hangzhou from 2019 to 2020. MTBC: mycobacterium tuberculosis complex, TB: tuberculosis, NTM: nontuberculosis mycobacterium; MDR-TB: multi-drug resistant TB, XDR: extensively-drug resistant TB, OFX: ofloxacin, KM: kanamycin.

    Thirty-three specimens were resistant only to OFX, and 11 were resistant to KM; 44 of 58 strains were non-MDR, and 14 were MDR (Table 1). Neither gyrA nor gyrB gene mutations were found in eight OFX resistant strains, and neither eis not rrs2 gene mutations were found in three KM resistant strains (Table 1). The most prevalent mutations were located in gyrA at codon 94 (D→G) (29/50; 58%), followed by gyrA at codon 90 (A→V) (12/50; 24%), as previously reported[5]. We identified a novel mutation in the gyrB gene at codon 499 (N→D) (1/50; 2%) in a non-MDR strain that was phenotypically resistant to both OFX and KM. The mutation at codon 499 in the gyrB gene was also reported previously; however, the amino acid change was different[6]. Therefore, continued efforts should be aimed at clarifying how these mutations confer phenotypic OFX resistance. We detected the presence of a mutation in the rrs2 gene at codon 1401 (A→G) (8/11; 72.73%) in phenotypic KM resistant strains, with a higher frequency than reported in earlier studies[6]. The predominance of the gyrA94 (D→G) and rrs21401 (A→G) mutations indicated that mutations in these two genes play a central role in OFX and KM resistance in Hangzhou, China.

    Resistance patternsNo. of strainsMutant alleles (detected/not detected)
    O resistance (n = 50)K resistance (n = 11)
    gyrAgyrBNot detectedeisrrs2
    Susceptible to all drugs1,124
    Non-MDRI62
    R5
    S56
    E3
    O34A90V (10/34)D94G (21/34)3
    IS40
    IE4
    IO3A90V (1/3)D94G (2/3)
    SE2
    EK1A1401G (1/1)
    OK1N499D (1/1)A1401G (1/1)
    ISO22
    ISK1A1401G (1/1)
    IEK11
    ISEK1A1401G (1/1)
    MDRIR11
    IRS8
    IRE2
    IRSE9
    IREO1D94G (1/1)
    IRSEO7A90V (1/7)D94G (4/7)2
    IRSEK41A1401G (3/4)
    IRSEOK2 D94G (1/2) 11 A1401G (1/2)
      Note. MDR, Multidrug resistant tuberculosis; XDR, extensively drug resistant tuberculosis; I, Isoniazid; R, Rifampicin; S, Streptomycin; E, Ethambutol; O, Ofloxacin; K, Kanamycin.

    Table 1.  Genetic mutations of Ofloxacin and Kanamycin resistantce for MDR and non-MDR strains

    In our study, no KM resistant strains had mutations in the eis gene, and we discovered a discordance between the results of phenotypic drug susceptibility testing and genotypic mutations in 18.97% (11/58) of these strains (Table 1)—a proportion much lower than that in previous studies[5]. Nonetheless, our findings may suggest the existence of unexplored resistance mechanisms[7].

    As shown in Table 1, in all mono-drug resistant strains, 21.25% (34/160) had OFX resistance. The proportions of OFX and KM resistant strains were 18.52% (40/216) and 2.31% (5/216) in non-MDR, and 22.73% (10/44) and 13.64% (6/44) in MDR. The rapid increase in KM resistance in MDR might be indicative of poor outcomes and survival among these patients. We found no mutations in the eis promoter gene associated with KM resistance, although its mutations have been reported to be highly associated with KM resistance[8]. We were unable to draw a conclusion regarding the frequencies of mutations in the eis gene, because of the small number of KM resistance strains.

    In addition, to assess the performance of gene sequencing in predicting OFX and KM resistance, we analyzed the phenotypic and genotypic results of 116 strains (Table 2). With phenotypic drug susceptibility testing, the gold standard method, the sensitivity for detection of OFX resistance according to the gyrA gene was 84.0% (95% CI: 70.89% to 92.83%), similarly to data from other countries[9]. However, the sensitivity of the rrs2 gene for detecting KM resistance was 72.73% (95% CI: 39.03% to 93.98%), a value lower than that reported for Hebei province[6]. Both gyrB and eis had lower sensitivity for phenotypic prediction.

    Mutant allelesDrug resistance (n = 58) Drug susceptible (n = 58)Sensitivity (%, 95% CI)Specificity (%, 95% CI)Accuracy (%, 95% CI)
    OK OK
    MNM MNM MNM MNM
    gyrA428    058   84.00
    (70.89−92.83)
    100.00
    (93.84−100.00)
    92.59
    (85.93−96.75)
    gyrB149 058 2.00
    (0.05−10.65)
    100.00
    (93.84−100.00)
    54.63
    (44.76−64.24)
    eis 011 0580.00
    (0.00−28.49)
    100.00
    (93.84−100.00)
    84.06
    (73.26−91.76)
    rrs2 (nt 1158−1674) 83 05872.73
    (39.03−93.98)
    100.00
    (93.84−100.00)
    95.65
    (87.82−99.09)
      Note. "M" means "mutations", and "NM" means "no mutations were found"; O, Ofloxacin; K, Kanamycin

    Table 2.  Accuracy analysis of genotypic for Ofloxacin and Kanamycin resistantce

    We selected samples from seven suburban counties in Hangzhou for the following reasons: 1) the population in these areas has been growing rapidly in recent years, with many migrants and large population flows; 2) data from drug resistant TB surveillance have indicated a recent increase in OFX resistance in these regions; and 3) some counties are lagging in economic development and have a high incidence of drug-resistant TB. Our results showed that the three regions with the highest numbers of TB cases were Yuhang, Jiande, and Xiaoshan, with case numbers of 286, 233, and 255, respectively. The two districts with the highest and lowest incidence rate per 100,000 people were Chun’an and Xiaoshan according to the most recent census data (https://tjgb.hongheiku.com/10425.html), in agreement with the results of our previous studies (Supplementary Figure S2, available in www.besjournal.com)[10].

    Figure S2.  The spatial distributions and TB prevalence rate of seven districts in Hangzhou from 2019 to 2020. Spatial distributions of seven suburban districts within Hangzhou (A); Distributions of TB incidences (n) (B); Incidences of TB cases per 100,000 population by districts (C).

    Out results may aid in establishing rapid molecular diagnostic methods for identifying OFX and KM resistance in Hangzhou, China. However, additional studies including a substantial panel of OFX resistant strains are required to further explore the proportions of the novel mutation in the gyrB at codon 499 (N→D) in this region.

    On the basis of the high prevalence of OFX resistance in these areas, we identified 58 OFX and/or KM resistant strains from 1,384 MTBC. Because resistance to FQs and SLIDs other than OFX and KM was not analyzed, we found no statistical difference in the proportion of resistance to the two second-line drugs between MDR and non-MDR.

    The major conclusions of this study are as follows. First, the most common mutations were single or double amino acid substitutions in gyrA at codon 90 (A→V) and 94 (D→G) for OFX resistance, and mutations in rrs2 at codon 1401 (A→G) for KM resistance. Second, a novel substitution in gyrB at codon 499 (N→D) was also detected. Finally, based on phenotypic drug resistance results, the specificity of genotype for predicting drug resistance was 100.0% (95% CI: 93.84% to 100.0%), the sensitivity of gyrA mutations for predicting OFX resistance was 84.0% (95% CI: 70.89% to 92.83%), and the sensitivity of rrs2 mutations for predicting KM resistance was 72.73% (95% CI: 39.03% to 93.98%).

    JIA Qing Jun and ZENG Mei Chun contributed to the study conception, methods, and writing; XIE Li and CHENG Qing Lin designed the study and performed literature research; LI Qing Chun and HUANG Yin Yan performed the data analysis; WU Yi Fei and AI Li Yun performed the experimental studies; LU Min and FANG Zi Jian performed writing, review and editing. All authors read and approved the final version of the manuscript.

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