Fluoroquinolones have long been widely used to treat several infectious diseases and in certain regions are easily accessible even without prescription. Such misuse of FQs has highly contributed to their efficacy in the treatment of TB but also the emergence of FQ-resistance [10].
The current cross-sectional study included presumptive multi-drug-resistant isolates of MTB. High proportions of resistance to rifampicin (RIF) and isoniazid (INH) were observed. Of all samples tested, resistance to first-line drugs (INH and RIF) was identified in 92% (521/562, i.e. 430 MDR and 91 mono-resistant). Among FQ-resistant isolates, eight were RIF-sensitive, eight were mono-resistant to RIF, and 88 were MDR. Isolates having resistance against rifampicin and isoniazid are termed MDR-TB, and if they also develop additional resistance against FQs, are then known as pre-XDR TB [11].
MTB develops resistance against FQs mainly by developing mutations in the drug-targeted proteins. The detection of gyrase mutations in particular can help in predicting the presence and level of FQ resistance [12]. GenoType MTBDRsl assay can detect mutations in the conserved QRDR region of gyrase genes (gyrA and gyrB) which changes the structure of the drug binding pocket (QBP) and results in cross resistance to all FQs. GenoType MTBDRsl assays were used to determine the frequency of FQ resistance in our isolates. A total of 104/562 isolates (18.5%) were found to be resistant to FQs. A high prevalence of FQ resistance was also reported in other provinces of Pakistan [10, 13] and in the neighboring countries of India [14, 15], China [16, 17], and Bangladesh [18, 19].
Short treatment regimens are used to reduce emergence of antimicrobial resistance in MTB. According to the 2019 national guidelines for the control of TB in Pakistan (adopted by the WHO), the anti-TB short regimens for drug-susceptible cases include third- or fourth -generation fluoroquinolones (levofloxacin or moxifloxacin, respectively) for 4 months. These drugs are also given for isoniazid-resistant and previously-treated cases in the initial phase of therapy (2 months). The high proportion of FQ resistance indicates an ineligibility of patients in this population for shorter regimens.
Among participants in this study, the frequency of gyrA mutations was much higher than that for gyrB. Hybridization pattern analysis revealed that most isolates had a mutation in the gyrA gene locus, with substitutions at amino acid 94 (D94G) and 90 (A90V) being more prevalent. Both of these mutations are associated with a high level of resistance to fluoroquinolone antibiotics (Fig. 2). The A90V mutation confers resistance against levofloxacin, but a higher generation of FQ (i.e. moxifloxacin) can be used at a higher dose. However, if the D94G mutation is present, both levofloxacin and moxifloxacin are ineffective [20]. Additional mutations found in our isolates were S91P, D94A, and D94N/Y. Isolates with the first two mutations could be susceptible to moxifloxacin at higher doses but resistant to levofloxacin. However, D94N/Y confers resistance against both levofloxacin and moxifloxacin. These findings correlate with most of the previous studies [6, 21, 22].
This study assessed patient characteristics and type of mutation in two ways. The first approach was to determine the frequency of particular mutations when categorized according to their resistance to first-line drugs, and the second when categorized according to treatment history. In MDR and XRD cases, FQ resistance was most commonly associated with the D94G mutation. This mutation shows high-level resistance to all fluoroquinolones, even the fourth-generation moxifloxacin. Isolates with INH monoresistance exhibited the S91P and D94N/Y mutations, with D94N/Y also being observed in XRD. Newly-diagnosed TB isolates with FQ resistance showed a high proportion of A90V mutations, for which moxifloxacin still remains the drug of choice at higher doses. In contrast, treatment failure and relapse cases commonly exhibited the D94G mutation.
Since GenoType MTBDRsl assay targets only a small region of a gene and supports a limited number of well-known mutations, interpretation in terms of the cross-resistance to FQs that occurs due to particular gyrA mutations is sometimes indistinct. Accordingly, sequence analysis was performed to understand resistance at the genotypic level [23]. Some isolates (4/102) showed co-existence of mutations in a hybridization pattern. The combinations identified were: D94Awith D94H, S91P with D94G, D94G with D94N/Y, and A90V with D94G. Co-existence of mutations was also observed in sequence analysis of gyrA, with the S91T mutation being detected in 95% isolates. However, this mutation is not related to fluoroquinolone resistance, other studies have determined it could be present even in sensitive isolates [24, 25]. All of our mutation results are in line with a study conducted in Pakistan that detected mutations in extensively drug-resistant strains [26].
Interestingly, we found some other hot-spot mutations during hybridization pattern analysis, wherein all wild-type probes were present and co-existed with a mutant probe. In these two isolates, when MUT3C and MUT2 appeared in the presence of the associated wild-type probe, a substitution of Ala with Val was observed and when the MUT2 probe was present but WT3 absent, a mutation of Asp to Ala occurred. Four isolates exhibited our most common pattern, i.e. absence of WT3 and presence of the corresponding mutant probe MUT3C, but one isolate exhibited a mutation of Thr into Ala combined with G/M/R into an undetermined amino acid (X). However, for most isolates that deviated from the pattern of well-known mutations, the affected amino acids mostly occurred at typical sites, i.e. codons 94 and 91. Overall, these findings suggest that mutation patterns can differ according and this can be revealed in the hybridization patterns of wild-type and mutant probes.
The results of this study present a burden of fluoroquinolone resistance in MDR-TB patients regardless of FQ antibiotic therapy. However, further understanding of the relevance of genotypic and phenotypic resistance is important for the accurate prediction of FQ resistance. Although we found genotypes consistent with FQ resistance in susceptible isolates, these results do not reflect the true prevalence of resistance in these patients. The resistance might develop due to the prior use of FQ antibiotics, which information was not included in the study.