The paradox of pyrazinamide: an update on the molecular mechanisms of pyrazinamide resistance in Mycobacteria

Pyrazinamide (PZA) is an important front line anti-tuberculosis drug because of its sterilizing activity against semi-dormant tubercle bacilli. In spite of its remarkable role in shortening the treatment duration from 9 months to 6 months when used in combination with Rifampicin and Isoniazid, PZA remains a difficult paradox because of its incompletely understood mode of action and mechanism of resistance. PZA is a nicotinamide analog prodrug which is converted into the active bactericidal form pyrazinoic acid by the bacterial enzyme pyrazinamidase (PZase). PZA does not appear to have a specific cellular target and instead, exerts its bactericidal effect by disrupting the membrane energetics and acidification of cytoplasm. Majority (72-97%) of PZA-resistant isolates of M. tuberculosis exhibit mutations in their pncA gene or upstream area leading to loss of PZase activity. A wide diversity of pncA mutations scattered along the entire length of pncA gene is unique to PZA resistance. However, PZA resistant isolates with normal PZase activity and wild type pncA sequences have also been reported in several studies which indicate that alternate mechanisms of PZA resistance exist. Investigations into these mechanisms would be useful in developing alternative diagnostic/therapeutic measures. This review presents the update of various mechanisms of PZA resistance in different mycobacteria with special emphasis on mode of action of PZA and mechanisms of resistance in Mycobacterium tuberculosis.     

Characterisation of the pncA gene in Mycobacterium tuberculosis isolates from Gauteng, South Africa.

SETTING: The use of pyrazinamide (PZA) is important for the treatment of Mycobacterium tuberculosis as it is bactericidal to semi-dormant mycobacteria that are not affected by other drugs. The incidence of resistance to PZA and other drugs used in the treatment of M. tuberculosis is increasing in South Africa. OBJECTIVE: To characterise the pncA gene of M. tuberculosis isolates from Gauteng, South Africa, and to develop a rapid diagnostic method. DESIGN: The pncA gene and the putative regulatory gene were characterised by sequence analysis in a total of six PZA susceptible and 15 resistant isolates. The association with classical PZA susceptibility testing and PZase activity was determined. RESULTS: All PZA-resistant isolates were PZase negative as well as resistant to at least one other anti-tuberculosis drugs. Mutations were identified throughout the length of the pncA gene in 10/15 PZA-resistant isolates. Five lacked PZase activity, but the wild type pncA sequence was present. In all six PZase-positive strains, a PZA-susceptible pattern was obtained on BACTEC and the wild type pncA sequence was present. CONCLUSION: Sequencing is an effective means to identify mutations in the pncA gene in M. tuberculosis and therefore resistance to PZA. The fact that some PZA-resistant M. tuberculosis isolates lack mutations in the pncA gene suggests that alternative mechanisms for drug resistance exist. In PZase negative strains with no genetic changes which are resistant to 100 microg/ml and susceptible to 300 microg/ml, 300 microg/ml may be a more reliable breakpoint.     

pncA gene mutations in clinical isolates of tubercle bacillus by polymerase chain reaction-direct sequencing method: in relationship to pyrazinamide resistance]

We screened clinical isolates of tubercle bacillus for mutations in the pncA gene, which encodes pyrazinamidase (PZase), by polymerase chain reaction (PCR)-direct sequencing method. Sixty-eight strains of tubercle bacillus were isolated from 32 patients with pulmonary tuberculosis. The patients were treated with antituberculous agents including pyrazinamide (PZA) for 2 months. Thirty-two of the 68 strains were isolated from sputum samples collected from the patients before treatment; 29 strains and 7 strains were collected after 1 month and 2 months of treatment, respectively. The pncA genes in these strains, were assessed for mutations by direct sequencing of PCR products using an automated sequencer. Similarly, we examined two clinical isolates (ka567 and minami22) of tubercle bacillus, determined to be deficient in PZase activity by the Wayne method. A PZA-sensitive strain (H37Rv, ATCC27294), and a PZA-resistant strain (H37Rv-PZA-R, ATCC35828) were used as negative and positive controls for mutations in the pncA gene, respectively. None of the 68 strains demonstrated any mutations in the pncA gene; however, the 2 PZase-deficient strains had missense mutations in the pncA gene resulting in an amino acid substitution from His82 to Arg in clone ka567, and from Ala171 to Val in clone minami22.     

Pyrazinoic acid efflux rate in Mycobacterium tuberculosis is a better proxy of pyrazinamide resistance

Pyrazinamide is one of the most important drugs in the treatment of latent Mycobacterium tuberculosis infection. The emergence of strains resistant to pyrazinamide represents an important public health problem, as both first- and second-line treatment regimens include pyrazinamide. The accepted mechanism of action states that after the conversion of pyrazinamide into pyrazinoic acid by the bacterial pyrazinamidase enzyme, the drug is expelled from the bacteria by an efflux pump. The pyrazinoic acid is protonated in the extracellular environment and then re-enters the mycobacterium, releasing the proton and causing a lethal disruption of the membrane. Although it has been shown that mutations causing significant loss of pyrazinamidase activity significantly contribute to pyrazinamide resistance, the mechanism of resistance is not completely understood. The pyrazinoic acid efflux rate may depend on multiple factors, including pyrazinamidase activity, intracellular pyrazinamidase concentration, and the efficiency of the efflux pump. Whilst the importance of the pyrazinoic acid efflux rate to the susceptibility to pyrazinamide is recognized, its quantitative effect remains unknown. Thirty-four M.?tuberculosis clinical isolates and a Mycobacterium smegmatis strain (naturally resistant to PZA) were selected based on their susceptibility to pyrazinamide, as measured by Bactec 460TB and the Wayne method. For each isolate, the initial velocity at which pyrazinoic acid is released from the bacteria and the initial velocity at which pyrazinamide enters the bacteria were estimated. The data indicated that pyrazinoic acid efflux rates for pyrazinamide-susceptible M.?tuberculosis strains fell within a specific range, and M.?tuberculosis strains with a pyrazinoic acid efflux rate below this range appeared to be resistant. This finding contrasts with the high pyrazinoic acid efflux rate for M.?smegmatis, which is innately resistant to pyrazinamide: its pyrazinoic acid efflux rate was found to be 900 fold higher than the average efflux rate for M.?tuberculosis strains. No significant variability was observed in the pyrazinamide flux rate. The pyrazinoic acid efflux rate explained 61% of the variability in Bactec pyrazinamide susceptibility, 24% of Wayne activity, and 51% of the Bactec 460TB growth index. In contrast, pyrazinamidase activity accounted for only 27% of the Bactec pyrazinamide susceptibility. This finding suggests that mechanisms other than pncA mutations (reduction of pyrazinamidase activity) are also implicated in pyrazinamide resistance, and that pyrazinoic acid efflux rate acts as a better proxy for pyrazinamide resistance than the presence of pncA mutations. This is relevant to the design of molecular diagnostics for pyrazinamide susceptibility, which currently rely on pncA gene mutation detection.     

Pyrazinamide and pyrazinoic acid activity against tubercle bacilli in cultured human macrophages and in the BACTEC system

Pyrazinamide (PZA) has become an essential component of current 6-month regimens for therapy of tuberculosis. Susceptible strains of tubercle bacilli convert PZA to pyrazinoic acid (POA) through pyrazinamidase (PZase), which resistant strains and Mycobacterium bovis bacille Calmette-Guérin lack. PZA susceptibility results obtained in cultured human macrophages were compared with those in the broth BACTEC system with 7H12 medium at pH 6.0 for strains known to be PZase-positive or -negative. Although added POA was unable to inhibit tubercle bacilli in cultured macrophages, it was able to inhibit them at very high concentrations in the BACTEC broth. Intracellularly formed POA would not be able to escape from the macrophage, and therefore would accumulate sufficiently to lower pH to toxic levels for tubercle bacilli. The results suggest that the cultured macrophages contribute actively or passively to the effectiveness of PZA, such as through the proposed mechanism of low pH generated by PZase in the phagolysosomes.     

pncA mutations in clinical Mycobacterium tuberculosis isolates from Korea

Background  

Pyrazinamide (PZA) is among the first-line drugs for the treatment of tuberculosis. In vitro, it kills semidormant mycobacteria only at low pH. The purpose of this study was to compare PZA resistance with pyrazinamidase (PZase) activity and the genotype to better understand the molecular basis of PZA resistance and to expand the profile of pncA mutations worldwide.     

Frequency and implications of pyrazinamide resistance in managing previously treated tuberculosis patients.

OBJECTIVE: To determine the extent of pyrazinamide (PZA) resistance in isolates from previously treated patients from the Western Cape, South Africa. DESIGN: Drug-resistant isolates, isolates resistant to one or more drugs other than PZA (PZA resistance is not routinely determined) (n = 127), and drug-susceptible (n = 47) clinical isolates of Mycobacterium tuberculosis from previously treated patients from the Western Cape were phenotypically (BACTEC MGIT 960) and genotypically (pncA gene sequencing) analysed for PZA resistance. RESULTS: MGIT analysis found that 68 of the 127 drug-resistant isolates were PZA-resistant. Nearly all (63/68) PZA-resistant isolates had diverse nucleotide changes scattered throughout the pncA gene, and five PZA-resistant isolates had no pncA mutations. Of the 47 phenotypically susceptible isolates, 46 were susceptible to PZA, while one isolate was PZA-monoresistant (OR = 53.0, 95% CI = 7.1-396.5). A pncA polymorphism (Thr114Met) that did not confer PZA resistance was also identified. PZA resistance was strongly associated with multidrug-resistant tuberculosis (MDR-TB). CONCLUSION: An alarmingly high proportion of South African drug-resistant M. tuberculosis isolates are PZA-resistant, indicating that PZA should not be relied upon in managing patients with MDR-TB in the Western Cape. A method for the rapid detection of PZA resistance would be beneficial in managing patients with suspected drug resistance.     

Sterilising action of pyrazinamide in models of dormant and rifampicin-tolerant Mycobacterium tuberculosis.

SETTING: Pyrazinamide (PZA) is an effective sterilising drug in tuberculosis, but its mode of action is controversial. OBJECTIVE: To test the bactericidal activity of 1.56-100 microg/ml PZA in Hu/Coates models of dormant and rifampicin (RMP) tolerant Mycobacterium tuberculosis. METHODS: In model 1, bactericidal activity was tested in pH 5.5 medium against 4-day, 30-day or 100-day static, hypoxic cultures. In models 2 and 3, 100 microg/ml RMP was added to a 100-day culture and PZA was added either during incubation with RMP in model 3, or after resuspension in RMP-free medium in model 2. RESULTS: Model 1: cfu counts on the 100-day and 30-day cultures fell by a maximum of about 1.6 log cfu/ml with increasing culture age, PZA concentration and incubation period, while counts on the 4-day culture showed little change. Model 2: cfu counts at the end of 7 days of recovery showed little bactericidal activity. Model 3: viable bacilli were almost completely eliminated. Bactericidal activity in these models increased with decreasing metabolic bactericidal activity, as measured by the uptake of [3H] uridine into bacterial RNA. CONCLUSION: PZA differs from other anti-tuberculosis drugs in showing greater bactericidal activity the slower the bacillary metabolic activity, hence its great value as a sterilising drug, likely to remain as an effective companion drug with newer sterilising drugs.     

Characterization of pyrazinamide and ofloxacin resistance among drug resistant Mycobacterium tuberculosis isolates from Singapore.

OBJECTIVES: To evaluate rapid molecular approaches for the detection of pyrazinamide (PZA) and ofloxacin resistance, by screening 100 known drug-resistant Mycobacterium tuberculosis isolates. METHODS: Mycobacterium tuberculosis isolates were tested for phenotypic resistance to pyrazinamide and ofloxacin using the BACTEC 460 radiometric method and the E-test, respectively. Mutation screening was done by amplifying the pncA, gyrA, and gyrB genes by the polymerase chain reaction (PCR) and direct automated sequencing. RESULTS: Twelve isolates were PZA-resistant and 8 of 12 (66.7%) isolates had missense mutations or deletions at the pncA gene, suggesting that mutation or deletion at the pncA gene is the major molecular mechanism of PZA resistance among the Singaporean isolates. Using the E-test, 48 isolates were resistant to ofloxacin, with minimum inhibitory concentrations of 4 microg/mL or higher. No mutations were observed at the quinolone resistance-determining region (QRDR) of gyrA in all isolates. At the QRDR of gyrB, mutations were present in 1 of 48 ofloxacin-resistant isolates and 0 of 19 ofloxacin-susceptible isolates. CONCLUSIONS: In Singapore, genotypic analysis of resistance to PZA and ofloxacin is inadequate and should be complemented by conventional methods.     

Discrepant results between pyrazinamide susceptibility testing by the reference BACTEC 460TB method and pncA DNA sequencing in patients infected with multidrug-resistant W-Beijing Mycobacterium tuberculosis strains

BACKGROUND: Mycobacterium tuberculosis strains belonging to the W-Beijing family have received broad clinical and public health attention because of their rapid worldwide spread and their frequent association with outbreaks, multidrug resistance, and treatment failures and relapses. METHODS: The present study examined a large number of multidrug-resistant strain-W isolates (isolates of 29 patients) by susceptibility testing for pyrazinamide (PZA) using the reference BACTEC 460TB method (Becton Dickinson Diagnostic Instrument Systems; Sparks, MD) and also by DNA sequencing of the pncA gene. RESULTS: We found that despite of the presence of a strain W-specific Thr47Ala in the pncA gene, all strains showed susceptibility to PZA in the reference BACTEC 460TB system due to their higher minimum inhibitory concentrations (relative to BACTEC 460TB PZA-susceptible strains). CONCLUSIONS: Our results suggest that the current radiometric reference method cannot reproducibly detect PZA resistance in patients infected with W-Beijing strains. Therefore, PZA susceptibility testing should instead be based on analysis of the pncA gene for resistance-associated mutations.     

结核分支杆菌五种耐药基因检测的临床应用及评价

目的　检测结核分支杆菌rpoBkatG、rpsL、pncA和embB耐药基因 ,评价其临床应用价值。方法　采用聚合酶链反应 单链构象多态性 (PCR SSCP)分析和药敏试验 (比例法 ) ,了解 10 9例肺结核患者结核分支杆菌耐药情况 ,并分析、比较临床治疗效果。结果　 1/ 2以上的肺结核患者至少耐两种抗结核药物 ,对RFP、INH、SM、PZA和EB总耐药率分别为 80 7%、71 5 %、78 8%、5 7 7%和48 6%。rpoB、katG、rpsL、pncA和embB基因突变率分别为 76%、68%、71%、5 1%和 3 0 %。结核分支杆菌耐药基因突变率与耐药水平联系密切 ,多数结核分支杆菌耐药基因突变易发生在高耐药株 ,也有少数基因突变发生在低耐药株。根据药敏试验和耐药基因检测结果 ,6个月耐多药结核治愈率分别达到 5 4 8%和 62 8% ,治疗效果满意 ,两种方法差异无显著性 (P >0 0 5 )。结论　耐药基因检测指导治疗是一种新探索 ,PCR SSCP方法敏感、特异 ,可以快速检测结核分支杆菌rpoB、katG、rpsL、pncA和embB耐药基因突变 ,可能会成为临床指导用药的好方法     

吡嗪酰胺酶活性与耐多药肺结核近期临床疗效的相关性研究

目的 研究吡嗪酰胺酶活性与耐多药肺结核(multidrug-resistant pulmonary tuberculosis,MDR-PTB)近期疗效的相关性,为MDR-PTB患者化学药物治疗的选择提供指导。 方法 采用前瞻性队列研究的方法,将广州市胸科医院2018年7月至2019年12月收治的85例MDR-PTB患者作为研究对象,均接受世界卫生组织推荐的6Am-Mfx-PZA-Pto-Cs/18Mfx-PZA-Pto-Cs(Am:阿米卡星;Mfx:莫西沙星;PZA:吡嗪酰胺;Pto:丙硫异烟胺;Cs:环丝氨酸)治疗方案。85例患者中,剔除9例丢失、中断治疗、失访患者,共76例患者纳入最终研究。以Wayne方法检测吡嗪酰胺酶的活性,以BACTEC MGIT 960方法检测吡嗪酰胺药物敏感性;比较吡嗪酰胺酶阳性患者与阴性患者治疗2、4、6个月末痰菌阴转、病灶吸收和空洞闭合情况。 结果 76例患者中,吡嗪酰胺酶阳性32例(42.1%),吡嗪酰胺酶阴性44例(57.9%);吡嗪酰胺表型耐药36例(47.4%),吡嗪酰胺表型敏感40例(52.6%)。吡嗪酰胺药物敏感性检测与吡嗪酰胺酶活性检测的一致性较高(Kappa=0.687,P=0.000)。吡嗪酰胺酶阳性患者治疗4个月末病灶吸收率(59.4%,19/32)及空洞闭合有效率(65.5%,19/29)均高于吡嗪酰胺酶阴性患者[分别为36.4%(16/44)和36.8%(14/38)],差异均有统计学意义(χ²=3.949,P=0.047;χ²=5.411,P=0.020)。吡嗪酰胺酶阳性患者治疗6个月后的空洞闭合有效率(86.2%,25/29)高于吡嗪酰胺酶阴性患者(63.2%,24/38),差异有统计学意义(χ²=4.447,P=0.035)。 结论 MDR-PTB患者的疗效与吡嗪酰胺酶活性有关,吡嗪酰胺酶阳性患者应用含吡嗪酰胺的MDR-PTB标准方案治疗后,病灶吸收及空洞闭合的效果优于吡嗪酰胺酶阴性患者。     

结核分枝杆菌能量代谢对吡嗪酰胺抗结核作用的影响

目的研究结核分枝杆菌能量代谢对吡嗪酰胺抗结核作用的机制和作用靶点。方法将饥饿3、5和10d的结核分枝杆菌及未经过营养饥饿的结核分枝杆菌分别加入100μg／ml吡嗪酰胺和脂肪酸、苯甲酸和水杨酸进行处理，观察营养饥饿处理过程对于吡嗪酰胺抗结核分枝杆菌作用的影响，以及酸性物质对吡嗪酰胺抗菌活性的影响。同时用流式细胞仪检测结核分枝杆菌膜电位和平均荧光强度，以探讨吡嗪酰胺的作用机制。结果未经过营养饥饿的结核分枝杆菌和饥饿3、5和10d的结核分枝杆菌经吡嗪酰胺处理后，菌落形成单位（CFU）分别减少23．08％、37．75％、82．32％和81．03％；营养饥饿5d后，结核分枝杆菌的膜电位明显降低，经吡嗪酰胺处理后的膜电位则大幅降低，加入能量物质葡萄糖后，因吡嗪酰胺作用而降低的膜电位可得到恢复。脂肪酸、苯甲酸和水杨酸对于正常生长的和营养饥饿后的结核分枝杆菌均有促进吡嗪酰胺抗菌效果的作用，对营养饥饿后的结核分枝杆菌作用更明显。结论饥饿状态和弱酸能增强吡嗪酰胺对结核分枝杆菌的抗菌作用，吡嗪酰胺可能是通过于扰结核分枝杆菌的膜电位及其能量代谢而产生作用。     

分析老年矽肺结核病的耐药水平与临床治疗

目的分析老年矽肺结核病的耐药水平和7种耐药基因突变情况。方法 117例老年矽肺结核患者的临床分离株均做传统梯度药敏试验和聚合酶链反应-单链构象多态性(PCR-SSCP)分析。结果被测菌株传统药敏试验耐药率82.1%。7种耐药基因[链霉素(rpsL)、利福平(rpoB)、异烟肼(katG)、乙胺丁醇(embB)、吡嗪酰胺(pncA)、喹诺酮(gyrA)、卡那霉素(km)]突变率分别为78.1%、65.6%、52%、33.3%、37.5%、54.1%、28.1%。结论传统梯度药敏试验与7种耐药基因联合检测,证实耐药基因突变与耐药水平和治疗关系密切。     

Is pyrazinamide bactericidal against Mycobacterium tuberculosis?

Bactericidal activity of pyrazinamide (PZA) was tested at pH 5.6 in 7H12 broth against drug-susceptible M. tuberculosis strains. The highest tested concentrations of PZA, 500 and 1,000 micrograms/ml, killed no more than 76% of the bacterial population. These concentrations are more than 32 times greater than the minimal inhibitory concentration (MIC) and the achievable in vivo concentrations. Despite high clinical efficacy of PZA and its so-called sterilizing activity in mouse experiments, this drug is much less bactericidal in vitro than any other known antituberculosis drug.     

Absence of the genetic marker IS6110 from a strain of Mycobacterium tuberculosis isolated in Ontario

A 35-year-old female patient from Waterloo, Ontario was diagnosed with pulmonary tuberculosis in June 1995. Records indicated that the patient had emigrated from Laos circa 1990. A culture grown from a bronchoalveolar lavage specimen was identified as Mycobacterium tuberculosis by standard biochemical methods. Drug-susceptibility testing indicated the strain was resistant to pyrazinamide (PZA), and a mutation was detected within pncA, a gene associated with PZA resistance. Sequence data from the 16S rRNA gene and the 16S/23S rRNA gene spacer confirmed that the strain was a member of the M tuberculosis complex, and analysis of the mpcA and pncA genes supported the identification of the strain as M tuberculosis rather than Mycobacterium bovis. However, the insertion element IS6110, which is used for epidemiological tracing of M tuberculosis, was not detected in this strain by either restriction fragment length polymorphism analysis or by polymerase chain reaction. Two other genetic markers associated with the M tuberculosis complex, IS1081 and the direct repeat element, were present. The arrival of immigrants with tuberculosis from southeast Asia, where most strains of M tuberculosis lacking IS6110 have been traced, has important implications for epidemiological studies of tuberculosis in North America.     

结核分支杆菌耐吡嗪酰胺分离株pncA基因突变的研究

目的 了解我国结核分支杆菌耐吡嗪酰胺（ＰＺＡ）分离株ｐｎｃＡ基因突变情况,研究其与耐ＰＺＡ之间的关系。方法 通过聚合酶链反应（ＰＣＲ）－单链构象多态性（ＳＳＣＰ）和ＰＣＲ－循环测序法（ＡＳ）分析７４株结核分支杆菌临床分离株的ｐｎｃＡ基因。以结核分支杆菌Ｈ３７ＲＶ标准株为对照。结果３２株药物敏感株ｐｎｃＡ基因ＳＳＣＰ分析未发现异常。２０株耐非ＰＺＡ药物的分离株中,１６株ｐｎｃＡ基因ＳＳＣＰ泳动正常;     

Pyrazinamide is not active in vitro against Mycobacterium avium complex

Strains of M. tuberculosis that elaborate pyrazinamidase are typically susceptible in vitro and in vivo to pyrazinamide (PZA). However, we found that 33 strains of M. avium complex (MAC), all of which were pyrazinamidase-positive, were resistant in vitro to a high concentration (100 micrograms/ml) of PZA when tested at low pH in 7H12 broth by radiometric (BACTEC) method. The drug was equally ineffective against these bacteria within cultured normal human macrophages. We conclude that the pyrazinamidase test is not suitable for susceptibility studies against M. avium complex. On the basis of our in vitro studies, which support earlier data, we believe that PZA is not appropriate for therapy of MAC disease.     

Pyrazinamide is not active against Mycobacterium tuberculosis residing in cultured human monocyte-derived macrophages.

SETTING: Mycobacteriology Laboratory, National Jewish Medical and Research Center, Denver, Colorado. OBJECTIVE: To evaluate the antimicrobial activity of pyrazinamide against Mycobacterium tuberculosis in cultured human monocyte-derived normal and activated macrophages. DESIGN: Monocytes separated from human blood were incubated in plastic plates for seven days to mature into macrophage monolayers. After activation with TNF-alpha or IFN-gamma or without prior treatment, the macrophages were infected with M. tuberculosis. Various concentrations of pyrazinamide (PZA), morphazinamide (MZA) or isoniazid (INH) were added the next day, and the viable counts of the intracellular bacteria were determined at days 0, 4, and 8. RESULTS: No inhibitory activity of PZA at any concentration was detected, while clear dose-dependent bacteriostatic and bactericidal activities were demonstrated by MZA and INH in the same experimental model. CONCLUSIONS: PZA has neither bacteriostatic nor bactericidal activity against M. tuberculosis persisting or multiplying in cultured monocyte-derived human macrophages, and it might be that the well-known effectiveness of this drug in tuberculosis patients is not related to its supposed activity against intracellular bacterial subpopulations.     

Fluoroquinolone and pyrazinamide resistance in multidrug-resistant tuberculosis

In a study performed in Cambodia, a higher number of tuberculosis (TB) strains with mutations in the pncA gene associated with pyrazinamide resistance (PZA-R) was found in fluoroquinolone-resistant (FQ-R) multidrug-resistant (MDR) strains (93%), compared with 47% in MDR and 3% in non-MDR strains. This emphasises the need for easy and rapid tests for identification of PZA-R for efficient treatment of MDR-TB.