There has been a gradual decline in the global tuberculosis (TB) incidence and mortality rates over time, by approximately 2% and 3% per year, respectively.1 Despite this trend, TB remains a leading cause of death, and Mycobacterium tuberculosis drug resistance continues to be a major public health issue. Globally, isoniazid (INH)-resistant, rifampicin (RIF)-susceptible TB (Hr-TB) is the most common drug-resistant form of disease.2 In 2017, the rates of Hr-TB were 7.1% among patients with new TB diagnoses and 7.9% in patients who had previously undergone treatment for TB.1 In Hong Kong, the respective rates were 5.3% and 9.5%, with a combined rate of 5.7% in 2016.3 The Hr-TB is associated with worse clinical outcome and development of multidrug resistance (MDR),4 5 although the findings have been inconsistent.6 7

Isoniazid constitutes a key component in the treatment of drug-susceptible TB, through its inhibition of mycolic acid biosynthesis in the mycobacterial cell wall. Over 85% of INH resistance is conferred by mutations residing in the katG gene and inhA promoter region,8 leading to high and low levels of resistance, respectively. These mutations can readily be detected by commercial molecular line-probe assays. The level of resistance can also vary according to the co-occurrences of less common mutations in other regions.

For the treatment of Hr-TB, current World Health Organization (WHO) guidelines recommend 6 months of RIF, pyrazinamide (PZA), ethambutol, and levofloxacin (LVX). The guidelines also recommend examination of resistances towards fluoroquinolones and PZA, prior to treatment.9

In our locality, routine testing of LVX and PZA susceptibility has not been implemented for the treatment of Hr-TB. To determine the most cost-effective testing strategy, this study reviewed an annual collection of Hr-TB isolates, with the aim of determining the prevalences of LVX resistance and pncA mutations (as a molecular marker of PZA resistance in Hr-TB) and identifying factors associated with these resistances.

Data were reviewed from the TB Reference Laboratory of the Department of Health. This laboratory processes local M tuberculosis strains from specimens collected in out-patient clinics and in-patient hospitals in both public and private sectors. All phenotypic Hr-TB isolates in 2018 were identified and de-duplicated using unique patient identifiers. The following data were included in this analysis: basic patient demographics, phenotypic susceptibility results of first-line drugs (INH at critical concentrations 0.1 μg/mL and 0.4 μg/mL as recommended by the WHO, RIF, streptomycin, and ethambutol), and genotypic susceptibility results (inhA promoter region, katG codon 315, and rpoB hotspot region) from the GenoType MTBDRplus assay, version 2.0 (Hain Lifescience). Any discrepant or unsuccessful test results were resolved by the agar proportion method and/or DNA sequencing; when applicable, these additional data were reported as the final results. All available data from the Laboratory Information System were reviewed for each patient to determine any history of TB, fluorescence microscopy results (according to Global Laboratory Initiative grading10), site of isolation (pulmonary versus extrapulmonary), any documented sputum culture conversion and the duration (ie, time between the date of first positive culture for current infection and the date of consistently negative culture), and microbiological outcome.

Selected M tuberculosis strains were retrieved from the laboratory archive and sub-cultured, then subjected to LVX susceptibility testing using the BD BACTEC MGIT 960 system (Becton, Dickson and Company), in accordance with the manufacturer’s instructions. The LVX critical concentration at 1.0 μg/mL was used as recommended by the WHO. DNA extraction was performed using GenoLyse (Hain Lifescience). The pncA gene was amplified with the primers pncA-1F (5′-CGCTCAGCTGGTCATGTTC-3′) and pncA-1R (5′-CCCACCGGGTCTTCGAC-3′) to produce an amplicon of 798 bp, using the GeneAmp PCR System 9700 (Applied Biosystems). Each 25-μL reaction mixture contained 12.5 μL of GoTaq G2 Hot Start Colorless Master Mix (Promega) [1×], 0.25 μL of each primer (1.0 pM/μL), 9.5 μL PCR-grade water, and 2.5 μL of template DNA. Reaction mixtures were amplified using the following protocol: 2 minutes at 95°C; 35 cycles of 1 min at 95°C, 1 minute at 65°C, and 1 minute at 72°C; and 10 minutes at 72°C. Sequencing was performed using the 3730 × l DNA Analyzer (Applied Biosystems) with the same primers. Resulting sequences were compared with the sequence of wild-type M tuberculosis H37Rv for detection of pncA mutations. Strains with detected pncA mutations were subjected to further analysis of PZA susceptibility via the MGIT 960 system in accordance with the manufacturer’s instructions, with a critical concentration of 100 μg/mL.

Univariate analysis comprised odds ratio calculations and Fisher’s exact test. Firth logistic regression was employed for multivariable analysis because of the low outcome frequency. IBM SPSS Statistics Subscription (Windows version, IBM Corp, Armonk [NY], United States) was used for data analysis. A P value of <0.05 was considered statistically significant. This article complies with the STROBE Statement reporting guidelines.

In total, 8865 M tuberculosis isolates from 3411 patients were processed in 2018. Susceptibility profiles were available for all except repeated strains collected from the same patient within 3 months. De-duplication of 393 isolates yielded 160 patients with phenotypic Hr-TB, amounting to a case rate of 4.7%. rpoB hotspot mutations were identified in five cases by GenoType MDRTBplus, three of which were confirmed to confer RIF resistance according to rpoB sequencing results. These three cases were considered to be RIF-resistant and excluded from further analysis. The mean age of the patients in the remaining 157 cases was 61.3 years (range, 15-95 years); the male to female ratio was 1.8. Pulmonary TB was present in 90.4% of affected patients (n=142), while 9.6% of affected patients (n=15) had extrapulmonary involvement. There was a documented history of TB by positive culture in 4.5% of affected patients (n=7). Microscopy results were available for cases in which direct specimens were received by our laboratory at the time of initial diagnosis (n=45). The majority of specimens was acid-fast bacillus smear-negative (n=33), followed by 1-4 acid-fast bacilli per length (n=4), scanty (n=4), 1+ (n=2), and 2+ and 3+ (n=1 each). In terms of microbiological outcome, 76.4% of affected patients (n=120) had documented sputum culture conversion without recurrence after follow-up of at least 9 months, among which 106 attained conversion within 5 months after diagnosis. The median interval required for culture conversion was 72.5 days (range, 9-622 days). No clearance was documented for patients in the remaining 37 cases, for whom no follow-up specimens were received for >1 year.

Table 1 shows the patterns of resistance among first-line drugs and distributions of cases with LVX resistance and pncA mutations. Table 2 shows the patterns of mutations detected. With respect to INH, 45.2% (n=71) of the isolates exhibited low-level resistance (minimum inhibitory concentration 0.1-0.4 μg/mL) and 54.8% (n=86) of the isolates exhibited high-level resistance (minimum inhibitory concentration >0.4 μg/mL).

For LVX, only one strain was resistant, while 155 were sensitive (one isolate failed to be recovered). The number of resistant cases was insufficient for correlation analysis. pncA mutations were detected in 4.5% (7/157 cases), and all detected mutations were previously identified as PZA resistance–related. All isolates with pncA mutations were phenotypically resistant to PZA. Univariate analysis showed that pncA mutations were associated with documented history of TB in the affected patient (odds ratio [OR]=11.60, 95% confidence interval [CI]=1.79-75.00; P=0.03) and INH poly-resistance (OR=19.06, 95% CI=1.07-339.78; P=0.04) but not mono-resistance, as shown in Table 3. In multivariable logistic regression, pncA mutations were associated with documented history of TB in the affected patient (OR=18.03, 95% CI=2.25-153.85; P=0.008), INH triple resistance (OR=409.11, 95% CI=6.52 to >1000; P<0.001), and INH double resistance (OR=13.50, 95% CI=1.43 to >1000; P=0.019).

In this study, the frequency of LVX resistance was very low in Hr-TB. Levofloxacin is an important drug in the management of drug-resistant TB. In a 2009 study in Shanghai, Xu et al11 estimated the rates of LVX resistance to be 1.9% in strains pan-susceptible to first-line drugs, 6.7% in INH mono-resistant strains, and ≤25% in MDR-TB. Independent risk factors associated with LVX resistance included MDR, RIF mono-resistance, poly-resistance (resistance to ≥2 first-line drugs, but not MDR), age ≥46 years, and TB re-treatment, but not INH mono-resistance. These findings were consistent with the results of a 2017 study in Ningbo (a city near Shanghai), whereby Che et al12 revealed no LVX resistance in strains pan-susceptible to first-line drugs, 3% in strains with any resistance to first-line drugs except MDR, and ≤30% in MDR-TB. Most fluoroquinolone resistance was present in the MDR group. Che et al12 also found that prevalence of LVX resistance in MDR-TB increased with duration of inappropriate treatment before LVX; this relationship was stronger than any relationships with previous fluoroquinolone exposures. Further studies are needed to determine whether this finding also applies to Hr-TB. Levofloxacin resistance is uncommon in non-MDR strains, especially in the present study. Since 2015, our laboratory has performed LVX susceptibility testing on 640 Hr-TB strains, identifying only two resistant cases (0.31%; unpublished data), including the one in this study. Clinicians could treat affected patients empirically, in accordance with WHO recommendations; further testing could be arranged based on clinical, radiological, and microbiological responses during early follow-up. In patients with Hr-TB that exhibits LVX resistance or poly-resistance to other primary agents, individualised adjustment of the treatment regimen is recommended in accordance with WHO guidelines9 (eg, exclusion of LVX or inclusion of second-line TB medicines). In our single case with LVX resistance, the patient had no history of TB. His sputum acid-fast bacillus smear result was 2+. The Hr-TB strain isolated was initially LVX-sensitive without mutations in rpoB, gyrA, or gyrB on diagnosis and follow-up at 8 months. The patient continued to exhibit sputum culture-positive results after 15 months of treatment; further analysis revealed that the isolate had acquired gyrA mutation (detected by line-probe assays) and LVX phenotypic resistance. It then developed into an MDR strain; the patient eventually achieved culture conversion at approximately 18 months after diagnosis. Underlying hetero-resistance was a possible contributing factor.

Phenotypic PZA susceptibility testing has often been problematic. pncA mutations constitute the most common and primary determinant of PZA resistance, with reported sensitivity of approximately 80% to 95% and specificity of approximately 85% to 100%.13 14 Resistance-conferring mutations are known to be diverse and scattered over the gene’s full length. Our seven isolates all had unique mutations (Gln10Arg, His51Tyr, Trp68Stop, Thr47Pro, Ala102Thr, Asp63Gly, and Val139Ala), which were associated with PZA resistance at a high probability of 0.985 according to available literature15 (except Val139Ala, probability of resistance=0.783). These seven isolates were also confirmed to be phenotypically resistant to PZA in our study.

Thus far, investigations of phenotypic and genotypic PZA resistance have generally focused on MDR-TB. Concerning non-MDR-resistant strains, there is considerable geographical variation in the reported rates, from 2.92% to 4.8% in the United States,16 17 to 24% in the Western Pacific and 75% in South East Asia.18 For Hr-TB in particular, phenotypic PZA resistance was reported in 2% of INH mono-resistant strains in South Africa,19 no PZA resistance was detected (phenotypic or pncA mutation) in Georgia (Eastern Europe) among Hr-TB strains,20 and ≤14.9% of Hr-TB strains exhibited pncA mutations in Vietnam.21 In our study, we observed that 4.5% of Hr-TB isolates had pncA mutations. Such variation may be related to multiple factors, including differences in inclusion criteria, testing method, treatment, infection control practice, and disease burden. In terms of risk factors, we found that history of TB in the affected patient and poly-resistance (but not mono-resistance) were significantly associated with pncA mutations. These results are consistent with findings from previous studies, which also showed an association with MDR.21 22 23 Clinicians are advised to consider the possibility of PZA resistance in patients with Hr-TB and these risk factors. Routine testing for LVX or PZA susceptibility in patients with INH mono-resistant TB alone (ie, no other risk factors) is not considered cost-effective, based on our findings.

In our study, most patients with Hr-TB had a satisfactory microbiological outcome without recurrence. However, the outcome was unknown in 23.6% of cases. Most of these involved patients were diagnosed and managed in hospitals, where only positive isolates were subjected to reference testing at our laboratory. Based on the recommended management regimen for TB in Hong Kong, these patients would have been followed up until sputum culture conversion. Considering the time elapsed since the latest positive laboratory result, the patients in most cases with unknown outcomes likely already had culture conversion.

In general, Hr-TB is presumably associated with higher rates of treatment failure and MDR acquisition, compared with drug-susceptible strains.4 5 Important considerations for disease control include recognition of risk factors associated with Hr-TB (eg, history of TB treatment, incarceration in prisons, and homelessness7 24), early detection of INH resistance, exclusion of RIF resistance by appropriate molecular methods (eg, line-probe assays),2 and compliance with current treatment guidelines. With the maturation of whole-genome sequencing, the technology is becoming integrated into the local routine drug susceptibility testing protocol for all M tuberculosis isolates; multiple and uncommon molecular markers of drug resistance might thus be identified earlier in a single set of assays for proper treatment guidance.

Because our laboratory serves as a local TB reference laboratory, this study was able to use a representative M tuberculosis collection that reflected the status of Hr-TB in Hong Kong. Furthermore, because our laboratory serves as a supranational reference laboratory, we were able to perform an array of confirmatory tests in accordance with WHO recommendations, thus ensuring reliable results. However, this study had some limitations. First, it lacked data regarding microscopy results, drug treatment, and clinical outcome, thus preventing a more comprehensive assessment. Second, the small sample size and low outcome frequencies of LVX and PZA non-susceptibilities led to limited correlation analysis. Third, less common mutations mediating INH resistance were not examined to determine their prevalences; Hr-TB cases with these mutations would only be identified on the basis of phenotypic results. A substantial proportion of cases (22.3%; n=35) had negative GenoType MDRTBplus results. Because this test only detects the most common mutations at the inhA promoter region and katG codon 315, other relevant mutations might have been missed. Furthermore, in 3.2% (n=5) of cases with high-level INH resistance, mutations were detected in the inhA promoter region alone. The availability of preliminary information before receiving the INH phenotypic susceptibility results might lead to mismanagement of patient treatment (eg, INH inclusion in the therapeutic regimen). Fourth, low-level RIF resistance mediated by mutations outside the rpoB hotspot region and undetected by phenotypic testing was not ruled out in this study. Inclusion of such isolates might have led to overestimation of true resistance in Hr-TB. Fifth, PZA resistance could be mediated by other less common mutations, which were not examined by performing phenotypic PZA susceptibility testing on all Hr-TB isolates; thus, we may have underestimated its prevalence. Our laboratory has found that all PZA-resistant M tuberculosis isolates thus far could be confirmed through pncA mutation analysis. Nevertheless, we presume that these limitations do not greatly affect the reliability and overall interpretation of our findings.

In Hong Kong, the rate of Hr-TB was 4.7% among active TB cases in 2018. Levofloxacin and PZA resistances among Hr-TB were uncommon (0.6% and 4.5%, respectively). History of TB in the affected patient and INH poly-resistance, including both double and triple resistances, were independent risk factors for pncA mutations. The findings from this study do not support routine susceptibility testing for these two drugs in patients with INH mono-resistant TB who lack additional risk factors.

Concept or design: KKM Ng.
Acquisition of data: All authors.
Analysis or interpretation of data: All authors.
Drafting of the manuscript: KKM Ng.
Critical revision of the manuscript for important intellectual content: KKM Ng.

All authors had full access to the data, contributed to the study, approved the final version for publication, and take responsibility for its accuracy and integrity.

All authors have disclosed no conflicts of interest.

The authors acknowledge the excellent work and contribution by staff, especially Mr Steven CW Lui and Ms Angela WL Lau, at the Tuberculosis Laboratory and Special Investigation Laboratory of Public Health Laboratory Services Branch, Centre for Health Protection, Department of Health, Hong Kong SAR Government.

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Ethics approval for this study was obtained from the Ethics Committee of the Department of Health, Hong Kong SAR Government (Ref: LM 425/2019). All included patients consented to testing for tuberculosis.

1. World Health Organization. Global tuberculosis report 2018. Available from: https://www.who.int/tb/publications/global_report/en/. Accessed 19 Sep 2019.

2. Romanowski K, Campbell JR, Oxlade O, Fregonese F, Menzies D, Johnston JC. The impact of improved detection and treatment of isoniazid resistant tuberculosis on prevalence of multi-drug resistant tuberculosis: a modelling study. PLoS One 2019;14:e0211355. Crossref

3. Tuberculosis & Chest Service of Department of Health, Hong Kong SAR Government. Annual report 2016. Available from: https://www.info.gov.hk/tb_chest/doc/Annual_Report_2016.pdf. Accessed 19 Sep 2019.

4. Gegia M, Winters N, Benedetti A, van Soolingen D, Menzies D. Treatment of isoniazid-resistant tuberculosis with first-line drugs: a systematic review and meta-analysis. Lancet Infect Dis 2017;17:223-34. Crossref

5. Karo B, Kohlenberg A, Hollo V, et al. Isoniazid (INH) mono-resistance and tuberculosis (TB) treatment success: analysis of European surveillance data, 2002 to 2014. Euro Surveill 2019;24:1800392. Crossref

6. Salindri AD, Sales RF, DiMiceli L, Schechter MC, Kempker RR, Magee MJ. Isoniazid monoresistance and rate of culture conversion among patients in the State of Georgia with confirmed tuberculosis, 2009-2014. Ann Am Thorac Soc 2018;15:331-40. Crossref

7. Cattamanchi A, Dantes RB, Metcalfe JZ, et al. Clinical characteristics and treatment outcomes of isoniazid-monoresistant tuberculosis. Clin Infect Dis 2009;48:179-85. Crossref

8. Seifert M, Catanzaro D, Catanzaro A, Rodwell TC. Genetic mutations associated with isoniazid resistance in Mycobacterium tuberculosis: a systematic review. PLoS One 2015;10:e0119628. Crossref

9. World Health Organization. WHO consolidated guidelines on drug-resistant tuberculosis treatment. Available from: https://www.who.int/tb/publications/2019/consolidated-guidelines-drug-resistant-TB-treatment/en/. Accessed 19 Sep 2019. Crossref

10. Global Laboratory Initiative. Mycobacteriology Laboratory Manual. 1st ed. Global Laboratory Initiative; 2014

11. Xu P, Li X, Zhao M, et al. Prevalence of fluoroquinolone resistance among tuberculosis patients in Shanghai, China. Antimicrob Agents Chemother 2009;53:3170-2. Crossref

12. Che Y, Song Q, Yang T, Ping G, Yu M. Fluoroquinolone resistance in multidrug-resistant Mycobacterium tuberculosis independent of fluoroquinolone use. Eur Respir J 2017;50:1701633. Crossref

13. Streicher EM, Maharaj K, York T, et al. Rapid sequencing of the Mycobacterium tuberculosis pncA gene for detection of pyrazinamide susceptibility. J Clin Microbiol 2014;52:4056-7. Crossref

14. Khan MT, Malik SI, Ali S, et al. Pyrazinamide resistance and mutations in pncA among isolates of Mycobacterium tuberculosis from Khyber Pakhtunkhwa, Pakistan. BMC Infect Dis 2019;19:116. Crossref

15. Miotto P, Cabibbe AM, Feuerriegel S, et al. Mycobacterium tuberculosis pyrazinamide resistance determinants: a multicenter study. mBio 2014;5:e01819-4.Crossref

16. Kurbatova EV, Cavanaugh JS, Dalton T, Click ES, Cegielski JP. Epidemiology of pyrazinamide-resistant tuberculosis in the United States, 1999-2009. Clin Infect Dis 2013;57:1081-93. Crossref

17. Budzik JM, Jarlsberg LG, Higashi J, et al. Pyrazinamide resistance, Mycobacterium tuberculosis lineage and treatment outcomes in San Francisco, California. PLoS One 2014;9:e95645. Crossref

18. Whitfield MG, Soeters HM, Warren RM, et al. A global perspective on pyrazinamide resistance: systematic review and meta-analysis. PLoS One 2015;10:e0133869. Crossref

19. Whitfield MG, Streicher EM, Dolby T, et al. Prevalence of pyrazinamide resistance across the spectrum of drug resistant phenotypes of Mycobacterium tuberculosis. Tuberculosis (Edinb) 2016;99:128-30. Crossref

20. Sengstake S, Bergval IL, Schuitema AR, et al. Pyrazinamide resistance-conferring mutations in pncA and the transmission of multidrug resistant TB in Georgia. BMC Infect Dis 2017;17:491. Crossref

21. Huy NQ, Lucie C, Hoa T, et al. Molecular analysis of pyrazinamide resistance in Mycobacterium tuberculosis in Vietnam highlights the rate of pyrazinamide resistance-associated mutations in clinical isolates. Emerg Microbes Infect 2017;6:e86. Crossref

22. Li D, Hu Y, Werngren J, et al. Multicenter study of the emergence and genetic characteristics of pyrazinamide-resistant tuberculosis in China. Antimicrob Agents Chemother 2016;60:5159-66. Crossref

23. Chiu YC, Huang SF, Yu KW, Lee YC, Feng JY, Su JY. Characteristics of pncA mutations in multidrug-resistant tuberculosis in Taiwan. BMC Infect Dis 2011;11:240. Crossref

24. Smith CM, Trienekens SC, Anderson C, et al. Twenty years and counting: epidemiology of an outbreak of isoniazid-resistant tuberculosis in England and Wales, 1995 to 2014. Euro Surveill 2017;22:30467. Crossref