Introduction

The implementation of rapid molecular drug resistance testing for tuberculosis (TB) has greatly improved the diagnosis of drug resistant TB (DR-TB). Although molecular tests have replaced phenotypic drug susceptibility testing (DST) in the initial diagnosis of DR-TB, the latter remains the gold standard. Consequently, soon after the implementation of molecular tests, reports began to emerge showing M. tuberculosis isolates with rpoB mutations that were phenotypically susceptible to rifampicin (herein referred to as rifampicin discordant) [1,2]. Later, it became apparent that rifampicin discordant isolates were not uncommon, accounting for 10–20% of M. tuberculosis isolates with rpoB mutations [3,4].

The specific mutations responsible for this rifampicin discordance have been identified and proven to be different to those responsible for high level rifampicin resistance (rifampicin concordant). The most commonly described rifampicin discordant mutations include; L430P, D435Y, H445L, H445N and L452P while the most common concordant mutations include S450L, D435V, H445Y and H445D [5–7]. In addition, while it is well known that rifampicin resistance is associated with resistance to other rifamycins (rifabutin and rifapentin) as well as isoniazid and pyrazinamide [5,8–10], recent studies have shown that the majority of rifampicin discordant isolates remain susceptible to rifabutin and a significant amount is still susceptible to isoniazid [11,12]. However, susceptibility of rifampicin discordant isolates against pyrazinamide has not been studied.

Pyrazinamide is a critical drug in the treatment of both drug susceptible and DR-TB. Although the exact mechanism of action is not known, it requires activation to an active form called pyrazinoic acid [13,14]. This activation is performed by the pyrazinamidase enzyme encoded by the pncA gene. Consequently, mutations in the pncA gene are the main cause of resistance against pyrazinamide, accounting for about 90% of resistance [15].

Pyrazinamide is unique to other TB drugs because it only acts under acidic conditions and it targets the slowly multiplying Mycobacteria that persist in these environments including those located inside the macrophages [16]. It was this unique activity that led to the reduction of duration of therapy for drug susceptible TB from nine months to six months. However, the need for an acidic environment has complicated the in vitro susceptibility testing as acidic conditions that are required for testing are also detrimental to Mycobacteria [17]. As a result, pyrazinamide DST is not routinely performed. The rifamycins, isoniazid and pyrazinamide are the crucial drugs in the standard first line TB treatment regimen.

Despite rifampicin discordant M. tuberculosis being a distinct entity from rifampicin resistant (concordant) TB as evidenced by different rpoB mutations and antimicrobial susceptibility profile, these patients continue to be managed the same way. This is due to lack of evidence on alternative treatment regimens for rifampicin discordant TB. The second-line treatment regimen is known to be inferior, longer and more toxic compared to the first line treatment. In this study we examined the susceptibility of rifampicin discordant M. tuberculosis isolates against pyrazinamide in order to determine the appropriate treatment for the affected patients. In addition, we compared the susceptibility of pyrazinamide to that of isoniazid.

Materials and methods

Results

Among the 80 rifampicin discordant isolates tested for pyrazinamide susceptibility, six (7.5%) were found to be resistant to pyrazinamide (Table 1). Notably, out of these six isolates, only one was among the 36 (2.8%) which remained discordant after applying the new rifampicin critical concentration of 0.5 μg/mL. Of the 80 rifampicin discordant isolates that were tested for pyrazinamide susceptibility, 48 (60%) were susceptible to isoniazid while 32 (40%) were resistant to isoniazid. Importantly, all six pyrazinamide resistant isolates were among those that were also resistant to isoniazid. Overall, pyrazinamide resistance was associated with isoniazid resistance (p = 0.001). All 25 susceptible isolates were also susceptible to pyrazinamide.

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Table 1. Pyrazinamide phenotypic and genotypic drugs susceptibility results.

https://doi.org/10.1371/journal.pone.0274688.t001

Out of the 80 rifampicin discordant isolates, sequencing of the pncA gene revealed a polymorphism is five isolates. This included one deletion in nucleotide position 214 of one isolate and four unique nonsynonymous mutations (T47A, H51P, P54S and S104R) in other four isolates, but sequencing failed on one phenotypically resistant isolate (Table 1). As indicated in Table 1, three of these mutations (T47A, H51Pand S104R have been classified as associated with resistance according to the recently released WHO catalogue of mutations in M. tuberculosis [22].

Pyrazinamide phenotypic MGIT DST of 80 rifampicin borderline resistant isolates demonstrated resistance in all but one genotypically resistant isolate. This one isolate was difficult to grow and the MGIT DST revealed growth on the drug containing tubes three days before the control tubes on all three repeat occasions. Therefore, the phenotypic test was regarded as unsuccessful (failed). Among the 25 susceptible isolates, no pyrazinamide resistance was detected by both phenotypic MGIT DST as well as sequencing of the pncA gene.

Discussion

In this study, we present for the first time, the prevalence of pyrazinamide resistance among rifampicin discordant M. tuberculosis isolates. We found a 7.5% prevalence of pyrazinamide resistance which is much lower than the reported resistance of 31 to 89% in MDR-TB [23]. Pyrazinamide resistance was associated with isoniazid resistance and no pyrazinamide resistance was found among drug susceptible TB isolates.

The prevalence of pyrazinamide resistance is known to increase with increasing resistance against other TB drugs [23–26]. A systemic review with meta-analysis reported a median prevalence of pyrazinamide resistance of 51% (31% to 89%) in multidrug-resistant TB [23]. In a study conducted by Allana et al in the KZN province, pncA polymorphisms was found in 68% of MDR-TB and 96% of XDR-TB isolates [25]. Additionally, in a TB drug resistance survey conducted in South Africa between 2012–2014, the prevalence of pyrazinamide resistance among MDR-TB cases was 59·1% while it was 13.9 among rifampicin mono-resistant cases [26].

In contrast to drug resistant TB, pyrazinamide resistance in drug susceptible TB is reportedly low, ranging from 0 to 10% [23,26,27]. In the South African drug resistance survey, the overall pyrazinamide resistance was 3.7% [26]. With a resistance rate of 7.5%, our study shows that pyrazinamide resistance among discordant isolates was more in keeping with that of drug susceptible TB. Furthermore, when we only considered those isolates that remained discordant at the new WHO reduced rifampicin critical concentration of 0.5 μg/mL, pyrazinamide resistance was even much lower at 2.8%. This underscores the important role of this drug in the treatment of patients with TB disease caused by rifampicin discordant M. tuberculosis.

The appropriate diagnosis and treatment of rifampicin discordant TB has not been established. As a result, the WHO recommends standard treatment for RR/MDR-TB until new evidence emerges. Although small retrospective observational studies have reported unfavourable treatment outcomes with standard first line therapy, these studies involved patients who were already resistant to other first line drugs especially isoniazid [2,7]. In contrast, other small studies have reported favourable response to first line therapy in patients who were still susceptible to isoniazid [3,28]. In our study, isoniazid susceptibility was associated with pyrazinamide susceptibility. However, this finding still need to be confirmed in larger multicenter studies. Previous studies have shown that rifampicin discordant M. tuberculosis isolates may remain susceptible to rifabutin [11,12,29]. In a recent South African study as many as 33.2% of rpoB polymorphisms, were found to be susceptible to rifabutin, the majority of which belonged to the rifampicin discordant mutations [29]. Given the high susceptibility to pyrazinamide shown in this study, as well as the high susceptibility of these strains to rifabutin and isoniazid published elsewhere, it would seem that a significant proportion of patients with rifampicin discordant TB could benefit from rifabutin based first line therapy [11,12]. There is therefore a need for clinical studies investigating the use of such treatment combinations.

Both genotypic and phenotypic pyrazinamide susceptibility testing have limitations. The MGIT method may overcall resistance due to an inoculum effect. A study by Zhang et al showed that high inoculum sizes caused an increase in pH over time which leads to loss of acid inhibition, allowing growth of organisms in the presence of the drug [17]. It was however reassuring that all the susceptible isolates in our study were susceptible by the phenotypic method and no pncA mutations were detected. Nonetheless, due to the challenges with phenotypic DST, the WHO now recommends genotypic DST for detection of pyrazinamide resistance [30].

Developing a rapid molecular test for pyrazinamide DST has proven difficult as published literature shows lack of hot spot region for pncA gene mutations [25]. Our study findings also confirm this, with four unique mutations and one deletion detected covering a wide range of the pncA gene. These mutations are reported to confer resistance to pyrazinamide [22,31]. The WHO recently endorsed the GenoScholar PZA-TB test (Nipro, Osaka, Japan) which is a reverse hybridization molecular assay for detection of pyrazinamide resistance [32]. This assay has a large number of hybridization probes which cover the whole pncA gene, making it more complex than the currently used reverse hybridization molecular assays. Implementation of this assay may prove to be more challenging high TB endemic regions. Nevertheless, where pyrazinamide susceptibility testing is not feasible, our study shows that pyrazinamide is most likely to be susceptible in rifampicin discordant TB, even more so if isoniazid susceptible as no pyrazinamide resistance was found in these patients.

This study was not without any limitations. We used manual MGIT for incubation and reading of pyrazinamide phenotypic DST instead of the preferred automated method. This highlights the gap in susceptibility testing of pyrazinamide as this test is not offered in many high TB burdened countries including our laboratory. Because we used both phenotypic and genotypic methods to determine the prevalence of pyrazinamide resistance, we were able to mitigate this limitation. Even the one isolate that failed phenotypic DST had a known pyrazinamide resistance conferring mutation [22,31].

Conclusion

The current routinely used rapid molecular tests do not differentiate between high level rifampicin resistant mutations and rifampicin discordant mutations. Although the latter constitutes a smaller fraction of the overall DR-TB, the fact that it has unique rpoB mutations, and has high susceptibility to pyrazinamide, isoniazid and rifabutin suggests that they may require an alternative first line based treatment regimen. However, a clearer understanding of their clinical significance and their relative contribution to clinical outcomes remains a prerequisite to finding appropriate treatment for these patients.

Acknowledgments

The authors would like to acknowledge Partson Tinawo for performing statistical analysis for this study.