Evaluation of a novel line probe assay to detect resistance to pyrazinamide,a key drug used for tuberculosis treatment

ObjectivesThe development of rapid molecular diagnostic assays for pyrazinamide (PZA) resistance is considered technically challenging as mutations are highly diverse, scattered along the full length of the pncA gene and not all are associated with PZA resistance. We evaluated the performance of the novel Genoscholar PZA-TB II line probe assay (PZA-LPA2; NIPRO Corporation, Japan).MethodsTo evaluate the applicability of the PZA-LPA2 in clinical settings, we compared the performance of the PZA-LPA2 to a composite reference standard pncA Sanger and Illumina sequencing plus phenotypic susceptibility testing on a panel of 87 Mycobacterium tuberculosis isolates from World Health Organization (WHO) drug resistance surveys, harbouring mutations previously classified as associated or not associated with resistance according to data from peer-reviewed literature. In addition, the PZA-LPA2 was challenged against a selection of isolates with lineage-specific and non-resistance-associated mutations, for which the frequency among clinical isolates is unknown, and tested directly on 59 sputum extracts.ResultsFor the survey isolates, the PZA-LPA2 reached an overall agreement with the composite reference of 97.6% (80/82) or 94.3% (82/87) excluding or including heteroresistance, respectively. The PZA-LPA2 failed on 8.5% (5/59) of clinical samples; among valid results, 100% (14/14) sensitivity and 100% (7/7) specificity was reached relative to pncA Sanger sequencing.ConclusionsThe PZA-LPA2 represents a valid and rapid alternative for indirect PZA susceptibility testing. Preliminary findings on clinical samples show promise for direct testing. Further studies are needed to assess the clinical risk of missing heteroresistance and falsely detecting lineage-specific, silent and nonassociated mutations.     

Pyrazinamide resistance in multidrug-resistant Mycobacterium tuberculosis isolates in Japan

Thirty-six multidrug-resistant (MDR) Mycobacterium tuberculosis isolates collected in Japan were examined for pyrazinamide susceptibility and pyrazinamidase activity, and analysed by pncA sequencing and a hybridization-based line probe assay (LiPA), which was used to detect pncA mutations for the rapid identification of pyrazinamide-resistant isolates. Pyrazinamide resistance was found in 19 (53%) of them. All pyrazinamide-resistant isolates had no pyrazinamidase activity and at least one mutation in pncA. Among the pncA mutations, 11 had not been previously reported. The results of the LiPA were fully consistent with the DNA sequencing results. A majority of MDR M. tuberculosis isolates in Japan were resistant to pyrazinamide.     

Rapid diagnosis of pyrazinamide-resistant multidrug-resistant tuberculosis using a molecular-based diagnostic algorithm

There is an urgent need for rapid and accurate diagnosis of pyrazinamide-resistant multidrug-resistant tuberculosis (MDR-TB). No diagnostic algorithm has been validated in this population. We hypothesized that pncA sequencing added to rpoB mutation analysis can accurately identify patients with pyrazinamide-resistant MDR-TB. We identified from the Dutch national database (2007-11) patients with a positive Mycobacterium tuberculosis culture containing a mutation in the rpoB gene. In these cases, we prospectively sequenced the pncA gene. Results from the rpoB and pncA mutation analysis (pncA added to rpoB) were compared with phenotypic susceptibility testing results to rifampicin, isoniazid and pyrazinamide (reference standard) using the Mycobacterial Growth Indicator Tube 960 system. We included 83 clinical M. tuberculosis isolates containing rpoB mutations in the primary analysis. Rifampicin resistance was seen in 72 isolates (87%), isoniazid resistance in 73 isolates (88%) and MDR-TB in 65 isolates (78%). Phenotypic reference testing identified pyrazinamide-resistant MDR-TB in 31 isolates (48%). Sensitivity of pncA sequencing added to rpoB mutation analysis for detecting pyrazinamide-resistant MDR-TB was 96.8%, the specificity was 94.2%, the positive predictive value was 90.9%, the negative predictive value was 98.0%, the positive likelihood was 16.8 and the negative likelihood was 0.03. In conclusion, pyrazinamide-resistant MDR-TB can be accurately detected using pncA sequencing added to rpoB mutation analysis. We propose to include pncA sequencing in every isolate with an rpoB mutation, allowing for stratification of MDR-TB treatment according to pyrazinamide susceptibility.     

Rapid Detection of Mycobacterium tuberculosis and Pyrazinamide Susceptibility Related to pncA Mutations in Sputum Specimens through an Integrated Gene-to-Protein Function Approach

Testing the pyrazinamide (PZA) susceptibility of Mycobacterium tuberculosis isolates is challenging. In a previous paper, we described the development of a rapid colorimetric test for the PZA susceptibility of M. tuberculosis by a PCR-based in vitro-synthesized-pyrazinamidase (PZase) assay. Here, we present an integrated approach to detect M. tuberculosis and PZA susceptibility directly from sputum specimens. M. tuberculosis was detected first, using a novel long-fragment quantitative real-time PCR (LF-qPCR), which amplified a fragment containing the whole pncA gene. Then, the positive amplicons were sequenced to find mutations in the pncA gene. For new mutations not found in the Tuberculosis Drug Resistance Mutation Database (www.tbdreamdb.com), the in vitro PZase assay was used to test the PZA resistance. This approach could detect M. tuberculosis within 3 h with a detection limit of 7.8 copies/reaction and report the PZA susceptibility within 2 days. In an initial testing of 213 sputum specimens, the LF-qPCR found 53 positive samples with 92% sensitivity and 97% specificity compared to the culture test for M. tuberculosis detection. DNA sequencing of the LF-qPCR amplicons revealed that 49 samples were PZA susceptible and 1 was PZA resistant. In the remaining 3 samples, with new pncA mutations, the in vitro PZase assay found that 1 was PZA susceptible and 2 were PZA resistant. This integrated approach provides a rapid, efficient, and relatively low-cost solution for detecting M. tuberculosis and PZA susceptibility without culture.     

Role of pncA and rpsA Gene Sequencing in Detection of Pyrazinamide Resistance in Mycobacterium tuberculosis Isolates from Southern China

We sequenced pncA and rpsA genes plus flanking regions of 161 Mycobacterium tuberculosis isolates and found 10 new pncA and 3 novel rpsA mutations in pyrazinamide-resistant strains determined by the Bactec MGIT 960 system. The 3′ end of rpsA might be added as the target of molecular detection of pyrazinamide susceptibility.     

Characterization of pncA mutations of pyrazinamide-resistant Mycobacterium tuberculosis in Korea

Pyrazinamide (PZA) is one of the most important drugs for the treatment of Mycobacterium tuberculosis infection. However, the increasing frequency of PZA-resistant strains limits its effectiveness. In Korea, most PZA-resistant strains also exhibit both isoniazid and rifampin resistance making it essential to identify these resistant strains accurately and rapidly for effective treatment of mycobacterial infection. In this study, the characteristics and frequency of mutations of the pncA gene encoding pyrazinamidase were investigated in PZA-resistant clinical isolates from Korea. Automated DNA sequencing was used to evaluate the usefulness of DNA-based detection of PZA resistance. Among 95 PZA-resistant clinical isolates, 92 (97%) exhibited mutations potentially affecting either the production or the activity of the enzyme. Mutations were found throughout the pncA gene including the upstream region. Single nucleotide replacement appeared to be the major mutational event (69/92), although multiple substitutions as well as insertion and deletion of nucleotides were also identified. The high frequency of pncA mutations observed in this study supports the usefulness of DNA-based detection of PZA-resistant M. tuberculosis. Having verified the scattered and diverse mutational characteristics of the pncA gene, automated DNA sequencing seems to be the best strategy for rapid detection of PZA-resistant M. tuberculosis.     

Evaluation of PCR pncA-restriction fragment length polymorphism and PCR amplification of genomic regions of difference for the identification of M. bovis strains in lymph nodes cultures

BackgroundA rapid accurate identification of Mycobacterium bovis is essential for surveillance purposes.ObjectivesA PCR pncA-Restriction Fragment Length Polymorphism (RFLP) and a multiplex PCR based on the detection of 3 regions of difference (RD-PCR): RD9, RD4 and RD1 were evaluated for the identification of M. bovis in lymph nodes cultures, in Tunisia, during 2013–2015.MethodsEighty-two M. tuberculosis complex strains were identified using the biochemical tests, GenoType MTBC assay, PCR pncA-RFLP and RD-PCR.ResultsThe PCR pncA-RFLP showed that 54 M. bovis strains, identified by GenoType MTBC, had a mutation at position 169 of pncA gene. Twenty-eight strains did not show any mutation at this position 27 M. tuberculosis isolates and one M. caprae. The PCR pncA-RFLP had a sensitivity of 100.0% (95%CI: 93.3 -100.0) and a specificity of 100.0% (95%CI: 87.9–100.0) for identifying M. bovis. The RD-PCR showed that all M. bovis strains had the RD9 and RD4 deleted but presented RD1. RD-PCR also presented high sensitivity and specificity in detecting M. bovis strains (100.0%).ConclusionsPCR pncA-RFLP and RD-PCR represent very accurate and rapid tools to identify M. bovis. They can be easily implemented in each laboratory due to their low cost and easy use.     

Prevention of False Resistance Results Obtained in Testing the Susceptibility of Mycobacterium tuberculosis to Pyrazinamide with the Bactec MGIT 960 System Using a Reduced Inoculum

The susceptibility of 211 clinical isolates of Mycobacterium tuberculosis complex (201 M. tuberculosis and 10 Mycobacterium bovis isolates) to pyrazinamide (PZA) was assessed by the nonradiometric Bactec MGIT 960 system (M960). Detection of PZA resistance was followed by a repeat testing using a reduced inoculum (RI) of 0.25 ml instead of 0.5 ml. According to the first M960 analysis, resistance was observed in 55 samples. In the RI assay, 32 samples turned out to be susceptible and 23 proved to be resistant (58.2% false positivity). The Bactec 460 assay confirmed as resistant those strains detected by the RI assay, while discrepant results were found susceptible. Mutation analysis performed on 13 M. tuberculosis isolates detected pncA mutations in 11 samples. On the basis of our data, we suggest using the RI assay to confirm all PZA resistance results obtained with the standard M960 assay. Further studies are required to confirm our findings.     

Correlation of pncA sequence with pyrazinamide resistance level in BACTEC for 21 mycobacterium tuberculosis clinical isolates

Mutations in the pncA gene, encoding pyrazinamidase, are considered the major mechanism of pyrazinamide (PZA) resistance in Mycobacterium tuberculosis, but resistant strains containing the wild-type gene have been described. The correlation of pncA sequence with PZA resistance level was examined for 21 M. tuberculosis clinical isolates. Susceptibility patterns were determined for 100, 300, and 900 microg/ml concentrations of the drug in BACTEC. Insertions and deletions and a substitution in the putative promoter region led to high-level resistance, whereas substitutions within the open reading frame seemed to confer variable levels of resistance. Variable resistance levels and PZase activities were also observed among isolates lacking pncA mutations. The high-level resistance (900 microg/ml) in pncA wild-type isolates highlights the clinical significance of these isolates. These data also suggest that there may still be more than one alternative mechanism leading to PZA resistance in M. tuberculosis isolates.     

Simultaneous identification and typing of multi-drug-resistant Mycobacterium tuberculosis isolates by analysis of pncA and rpoB

In Mycobacterium tuberculosis there is a strong correlation between in-vitro resistance to rifampicin (RIF) and pyrazinamide (PZA) and mutations in rpoB and pncA, respectively. Approximately 50 mutations associated with resistance have been reported for rpoB and 70 for pncA, and, theoretically, many more are possible. Therefore, the identification of rpoB and pncA mutations in M. tuberculosis might be used for the simultaneous determination of resistance and for typing multi-drug-resistant (MDR) strains during possible outbreaks. The present study examined four sensitive and six MDR isolates of M. tuberculosis from Turkey and eight isolates from a nosocomial MDR tuberculosis (TB) outbreak in the UK. Gene mutations were identified by the Innogenetics LiPA rpoB assay or automated sequencing, or both. All the sensitive isolates had rpoB and pncA wild-type genotypes, whereas all the RIF- and PZA-resistant isolates had rpoB and pncA mutations. All four mutations seen in rpoB, but none of the six in pncA, had been reported previously. The rpoB and pncA mutations seen in the Turkish isolates defined six distinct genotypes amongst the six MDR isolates, while standard IS6110 typing discriminated only four. All isolates from the single strain MDR-TB outbreak had identical genotypes. Rapid genotyping was performed on the sputum from a patient who presented 2 years after the initial MDR-TB outbreak and this showed rpoB and pncA genotypes identical to the other outbreak isolates. This result was available within 36 h. The analysis of rpoB and pncA is a rapid and practical means of simultaneously identifying and typing MDR isolates of M. tuberculosis.     

Development and evaluation of a line probe assay for rapid identification of pncA mutations in pyrazinamide-resistant mycobacterium tuberculosis strains

Resistance of Mycobacterium tuberculosis to pyrazinamide (PZA) derives mainly from mutations in the pncA gene. We developed a reverse hybridization-based line probe assay with oligonucleotide probes designed to detect mutations in pncA. The detection of PZA resistance was evaluated in 258 clinical isolates of M. tuberculosis. The sensitivity and specificity of PZA resistance obtained by this new assay were both 100%, consistent with the results of conventional PZA susceptibility testing. This assay can be used with sputa from tuberculosis patients. It appears to be reliable and widely applicable and, given its simplicity and rapid performance, will be a valuable tool for diagnostic use.     

Validation of pncA gene sequencing in combination with the mycobacterial growth indicator tube method to test susceptibility of Mycobacterium tuberculosis to pyrazinamide

Pyrazinamide is important in the treatment of tuberculosis. Unfortunately, the diagnosis of pyrazinamide resistance is hampered by technical difficulties. We hypothesized that mutation analysis combined with the mycobacterial growth indicator tube (MGIT) phenotypic method would be a good predictor of pyrazinamide resistance. We prospectively analyzed 1,650 M. tuberculosis isolates referred to our tuberculosis reference laboratory in 2008 and 2009. In our laboratory, the MGIT 960 system was used for pyrazinamide resistance screening. If a pyrazinamide-resistant strain was detected, we performed a pncA gene mutation analysis. A second MGIT 960 susceptibility assay was performed afterwards to evaluate the accuracy of the pncA mutation analysis to detect true- or false-positive MGIT results. We observed pyrazinamide resistance in 69 samples using the first MGIT 960 analysis. In a second MGIT 960 analysis, 47 of the 69 samples proved susceptible (68% false positivity). Sensitivity of nonsynonymous pncA mutations for detecting resistant isolates was 73% (95% confidence interval [CI], 61% to 73%), and specificity was 100% (95% CI, 95% to 100%). A diagnostic algorithm incorporating phenotypic and molecular methods would have a 100% positive predictive value for detecting pyrazinamide-resistant isolates, indicating that such an algorithm, based on both methods, is a good predictor for pyrazinamide resistance in routine diagnostics.     

Sequencing of the pncA gene in members of the Mycobacterium tuberculosis complex has important diagnostic applications: Identification of a species-specific pncA mutation in "Mycobacterium canettii" and the reliable and rapid predictor of pyrazinamide resistance

Testing for susceptibility to pyrazinamide (PZA) and analysis of the pncA gene sequences of 423 Mycobacterium tuberculosis complex isolates have revealed a unique silent nucleotide substitution that enables the rapid identification of "M. canettii" (proposed name). Moreover, the lack of a defined mutation within the pncA gene strongly suggests that an alternative mechanism is responsible for PZA resistance. Our results indicate that DNA sequencing of the pncA gene has the potential to shorten the turnaround time and increase the accuracy of PZA susceptibility testing of the M. tuberculosis complex.     

Potential for Erroneous Results Indicating Resistance When Using the Bactec MGIT 960 System for Testing Susceptibility of Mycobacterium tuberculosis to Pyrazinamide

During susceptibility testing of 743 isolates of Mycobacterium tuberculosis to pyrazinamide (PZA) using the Bactec 960 system, 57 (7.7%) isolates showed PZA resistance. Repeat testing of resistant isolates with the Bactec 460 reference method confirmed 33 (4.4%) of these isolates as resistant, and 24 (3.2%) were susceptible. Erroneous results for resistance with the Bactec 960 were confirmed by testing the 24 discordant isolates for pyrazinamidase and mutations in the pncA gene.Pyrazinamide (PZA) is an important component of the multidrug regimen used to treat tuberculosis (TB). With increasing worldwide prevalence of drug-resistant TB, it is vital for laboratories to accurately detect resistance to first-line antimicrobials.The CLSI-recommended method for PZA testing (4) is the Bactec 460TB radiometric system (Becton Dickinson, Sparks, MD). Most laboratories have now replaced the 460TB system with the nonradiometric Bactec MGIT 960 (BT960) system (Becton Dickinson, Sparks, MD). Both methods utilize an acidified Middlebrook broth and a critical concentration of 100 μg/ml.PZA is a prodrug which in Mycobacterium tuberculosis is converted to its active form, pyrazinoic acid (POA), by the enzyme pyrazinamidase (PZase) (5, 7). The absence of a functional PZase enzyme in an M. tuberculosis strain therefore indicates resistance to PZA. The pncA gene coding for PZase in M. tuberculosis has been sequenced, and mutations in this gene have been shown to be responsible for resistance to PZA (5, 8, 12). Tests both for PZase activity and for the detection of mutations in pncA may be utilized as alternative methods for the detection of PZA resistance in M. tuberculosis.All new M. tuberculosis isolates are tested for susceptibility to first-line drugs, including PZA, at the Public Health Laboratory, Toronto. During the report period, any isolate demonstrating PZA resistance by the BT960 was retested using the 460TB. If the 460TB PZA result was discordant, these isolates were further tested for the presence of PZase activity and mutations in the pncA gene, and testing in the BT960 was repeated.PZA susceptibility testing in the BT960 system was performed according to the manufacturer''s instructions (2). Briefly, isolates of M. tuberculosis in Mycobacteria Growth Indicator Tubes (MGITs) were used as the test inocula. A drug-free control tube was inoculated with a 1:10 dilution of the inoculum, and the PZA test tube was inoculated with 0.5 ml of the inoculum and 0.1 ml of PZA. The tubes were monitored with the BT960 instrument until the growth control tube flagged positive. At that time, the instrument read the PZA test tube as either resistant (growth unit [GU] ≥ 100) or susceptible (GU < 100). A blood agar purity plate from the inoculum was incubated for 3 days.PZA testing in the BT 460TB system was performed according to the manufacturer''s instructions (13). Briefly, a drug-free control vial and the PZA test vial were each inoculated with 0.1 ml of inoculum from an actively growing culture. The vials were incubated and were read on the BT460 instrument daily until the growth index (GI) in the control vial was ≥200. At that time, the GI in the test vial was calculated as a percentage of the GI in the control. A result of ≥11% indicated resistance, <9% indicated susceptibility, and from 9 to 11% was borderline. A blood agar purity plate from the inoculum was incubated for 3 days.Isolates with results that were discordant between the two systems were tested for the presence of PZase activity and for mutations in the pncA gene at the National Reference Centre for Mycobacteriology, Winnipeg, Manitoba, Canada, by standard methodologies (12, 14) and with repeat testing in the BT960.During the report period, 743 PZA susceptibility tests were performed using the BT960. Of these, 57 (7.7%) showed PZA resistance. Thirty-three of these isolates were confirmed to be PZA resistant using the Bactec 460 system and were eliminated from the discordant-data analysis. The remaining 24 PZA-resistant isolates (3.2% of the total) were susceptible to PZA with the 460TB and were considered discordant. Follow-up testing of these isolates showed the presence of PZase in all isolates, and all were negative for mutations in the pncA gene. Repeat PZA testing of the discordant isolates with the BT960 gave a second resistant result for 10 isolates and a susceptible result for 14 isolates, indicating lack of reproducibility. The 460TB result was considered the gold standard for reporting results.There are reports citing technical problems with in vitro testing of M. tuberculosis with PZA. None of the methods described give 100% agreement when compared with the 460TB reference method, and most cite problems with false resistance (1, 10, 11, 15).The effects of inoculum concentration, volume, and homogeneity, as well as the lack of reproducibility in BT960 PZA tests, have been cited (1, 5, 6, 9). However, we are not aware of a study where BT960-resistant strains were tested with the 460TB plus PZase and molecular testing.There are several differences in the inocula used for testing PZA in the two systems. First, in the 460TB, the ratio of inoculum to medium is 1:42, whereas in the BT960 system, the ratio is 1:16.6. The concentration of inoculum in the test medium in the BT960 is thus more than 2.5 times greater than that used in the 460TB. The volume of inoculum used in the BT960 is 0.5 ml, versus 0.1 ml of inoculum used in the 460TB. The higher concentration and volume of inoculum used in the BT960 has a higher likelihood of containing organisms resistant to PZA, as it is estimated that in M. tuberculosis, between 1 in 10⁷ and 1 in 10¹⁰ cells are resistant to any drug (9).Second, there is variability in the concentration of the inoculum used in the BT960 test according to the day of test setup. For days one and two after the culture flags positive, there is no dilution of the MGIT seed vial, but for days 3 to 5, the inoculum is diluted 1:5. This may lead to considerable variation in the amount of the organism in the inoculum and could cause the lack of reproducibility found during repeat testing.Third, the inoculation method differs between the two systems. For the 460TB, a fine-needle tuberculin syringe is used, and for the BT960, the inoculum is dispensed with a disposable pipette tip, which may result in uneven distribution of bacilli due to “clumping.”The sensitivity of the PZase assay is reported to vary between 79 and 96% (1, 5, 7). All of the discordant isolates that were tested showed the presence of PZase. Sequencing of the pncA gene that encodes PZase has shown that 74% to 97% of all PZA-resistant strains of M. tuberculosis carry a mutation in several different regions of the gene (7, 8, 12). None of the discordant strains were found to have pncA gene mutations.These results corroborate the 460TB results as PZA susceptible and the BT960 results as falsely resistant. For laboratories which perform a large number of susceptibility tests with the BT960 system, this discordance could lead to a significant number of false resistant and major error (3) results for PZA, as well as therapeutic issues in patient management.Due to the potential for false resistant results during PZA testing with the BT960, laboratories should consider retesting all PZA-resistant isolates with the 460TB reference method before reporting results. Since PZA is considered an essential component of first-line TB therapy, it is important that laboratories find a successful algorithm to provide rapid and accurate susceptibility results for PZA.     

Detection of multidrug resistance in Mycobacterium tuberculosis

We developed a DNA sequencing-based method to detect mutations in the genome of drug-resistant Mycobacterium tuberculosis. Drug resistance in M. tuberculosis is caused by mutations in restricted regions of the genome. Eight genome regions associated with drug resistance, including rpoB for rifampin (RIF), katG and the mabA (fabG1)-inhA promoter for isoniazid (INH), embB for ethambutol (EMB), pncA for pyrazinamide (PZA), rpsL and rrs for streptomycin (STR), and gyrA for levofloxacin, were amplified simultaneously by PCR, and the DNA sequences were determined. It took 6.5 h to complete all procedures. Among the 138 clinical isolates tested, 55 were resistant to at least one drug. Thirty-four of 38 INH-resistant isolates (89.5%), 28 of 28 RIF-resistant isolates (100%), 15 of 18 EMB-resistant isolates (83.3%), 18 of 30 STR-resistant isolates (60%), and 17 of 17 PZA-resistant isolates (100%) had mutations related to specific drug resistance. Eighteen of these mutations had not been reported previously. These novel mutations include one in rpoB, eight in katG, one in the mabA-inhA regulatory region, two in embB, five in pncA, and one in rrs. Escherichia coli isolates expressing individually five of the eight katG mutations showed loss of catalase and INH oxidation activities, and isolates carrying any of the five pncA mutations showed no pyrazinamidase activity, indicating that these mutations are associated with INH and PZA resistance, respectively. Our sequencing-based method was also useful for testing sputa from tuberculosis patients and for screening of mutations in Mycobacterium bovis. In conclusion, our new method is useful for rapid detection of multiple-drug-resistant M. tuberculosis and for identifying novel mutations in drug-resistant M. tuberculosis.     

Pyrazinamide-monoresistant Mycobacterium tuberculosis in the United States

Mycobacterium bovis is naturally resistant to the antituberculosis drug pyrazinamide (PZA). To determine whether all Mycobacterium tuberculosis complex isolates demonstrating PZA monoresistance were truly M. bovis, we examined the phenotype and genotype of isolates reported as PZA monoresistant in five counties in California from January 1996 through June 1999. Isolates reported by local laboratories to be PZA monoresistant were sent to the state reference laboratory for repeat susceptibility testing using the BACTEC radiometric method and to the Centers for Disease Control and Prevention for pncA sequencing and PCR-restriction fragment length polymorphism (RFLP) analysis of the oxyR gene. Of 1,916 isolates, 14 were reported as PZA monoresistant and 11 were available for retesting. On repeat testing, 6 of the 11 isolates were identified as PZA-susceptible M. tuberculosis, 1 was identified as PZA-monoresistant M. bovis, and 1 was identified as M. bovis BCG. The three remaining isolates were identified as PZA-monoresistant M. tuberculosis. Sequencing of the pncA and oxyR genes genotypically confirmed the two M. bovis and the six susceptible M. tuberculosis species. Each of the three PZA-monoresistant M. tuberculosis isolates had different, previously unreported, pncA gene mutations: a 24-bp deletion in frame after codon 88, a base substitution at codon 104 (Ser104Cys), and a base substitution at codon 90 (Ile90Ser). This study demonstrates that PZA monoresistance is not an absolute marker of M. bovis species but may also occur in M. tuberculosis, associated with a number of different mutational events in the pncA gene. It is the first report of PZA-monoresistant M. tuberculosis in the United States.     

Whole-genome sequencing for surveillance of tuberculosis drug resistance and determination of resistance level in China

ObjectivesPhenotypic drug susceptibility testing for prediction of tuberculosis (TB) drug resistance is slow and unreliable, limiting individualized therapy and monitoring of national TB data. Our study evaluated whole-genome sequencing (WGS) for its predictive accuracy, use in TB drug-resistance surveillance and ability to quantify the effects of resistance-associated mutations on MICs of anti-TB drugs.MethodsWe used WGS to measure the susceptibility of 4880 isolates to ten anti-TB drugs; for pyrazinamide, we used BACTEC MGIT 960. We determined the accuracy of WGS by comparing the prevalence of drug resistance, measured by WGS, with the true prevalence, determined by phenotypic susceptibility testing. We used the Student–Newman–Keuls test to confirm MIC differences of mutations.ResultsResistance to isoniazid, rifampin and ethambutol was highly accurately predicted with at least 92.92% (95% confidence interval [CI], 88.19–97.65) sensitivity, resistance to pyrazinamide with 50.52% (95% CI, 40.57–60.47) sensitivity, and resistance to six second-line drugs with 85.05% (95% CI, 80.27–89.83) to 96.01% (95% CI, 93.89–98.13) sensitivity. The rpoB S450L, katG S315T and gyrA D94G mutations always confer high-level resistance, while rpoB L430P, rpoB L452P, fabG1 C-15T and embB G406S often confer low-level resistance or sub-epidemiological cutoff (ECOFF) MIC elevation.ConclusionWGS can predict phenotypic susceptibility with high accuracy and could be a valuable tool for drug-resistance surveillance and allow the detection of drug-resistance level; It can be an important approach in TB drug-resistance surveillance and for determining therapeutic schemes.     

Pyrazinamide resistance among South African multidrug-resistant Mycobacterium tuberculosis isolates

Pyrazinamide is important in tuberculosis treatment, as it is bactericidal to semidormant mycobacteria not killed by other antituberculosis drugs. Pyrazinamide is also one of the cornerstone drugs retained in the treatment of multidrug-resistant tuberculosis (MDR-TB). However, due to technical difficulties, routine drug susceptibility testing of Mycobacterium tuberculosis for pyrazinamide is, in many laboratories, not performed. The objective of our study was to generate information on pyrazinamide susceptibility among South African MDR and susceptible M. tuberculosis isolates from pulmonary tuberculosis patients. Seventy-one MDR and 59 fully susceptible M. tuberculosis isolates collected during the national surveillance study (2001 to 2002, by the Medical Research Council, South Africa) were examined for pyrazinamide susceptibility by the radiometric Bactec 460 TB system, pyrazinamidase activity (by Wayne's assay), and sequencing of the pncA gene. The frequency of pyrazinamide resistance (by the Bactec system) among the MDR M. tuberculosis isolates was 37 of 71 (52.1%) and 6 of 59 (10.2%) among fully sensitive isolates. A total of 25 unique mutations in the pncA gene were detected. The majority of these were point mutations that resulted in amino acid substitutions. Twenty-eight isolates had identical mutations in the pncA gene, but could be differentiated from each other by a combination of the spoligotype patterns and 12 mycobacterial interspersed repetitive-unit loci. A high proportion of South African MDR M. tuberculosis isolates were resistant to pyrazinamide, suggesting an evaluation of its role in patients treated previously for tuberculosis as well as its role in the treatment of MDR-TB.     

Detection of Mycobacterium tuberculosis resistance mutations to rifampin and isoniazid by real-time PCR

Objective: The objective of our study was to evaluate the use of a real-time polymerase chain reaction (PCR)-based technique for the prediction of phenotypic resistance of Mycobacterium tuberculosis. Materials and Methods: We tested 67 M tuberculosis strains (26 drug resistant and 41 drug susceptible) using a method recommended for the LightCycler platform. The susceptibility testing was performed by the absolute concentration method. For rifampin resistance, two regions of the rpoB gene were targeted, while for identification of isoniazid resistance, we searched for mutations in katG and inhA genes. Results: The sensitivity and specificity of this method for rapid detection of mutations for isoniazid resistance were 96% (95% CI: 88% to 100%) and 95% (95% CI: 89% to 100%), respectively. For detection of rifampin resistance, the sensitivity and specificity were 92% (95% CI: 81% to 100%) and 74% (95% CI: 61% to 87%), respectively. The main isoniazid resistance mechanism identified in our isolates is related to changes in the katG gene that encodes catalase. We found that for rifampin resistance the concordance between the predicted and observed phenotype was less than satisfactory. Conclusions: Using this method, the best accuracy for genotyping compared with phenotypic resistance testing was obtained for detecting isoniazid resistance mutations. Although real-time PCR assay may be a valuable diagnostic tool, it is not yet completely satisfactory for detection of drug resistance mutations in M tuberculosis.