Direct determination of pyrazinamide susceptibility by sputum MODS culture at neutral pH: the MODS-PZA assay.

Pyrazinamide (PZA) is considered the pivot drug in all tuberculosis treatment regimens, due to its particular action on the persistent forms of Mycobacterium tuberculosis. However, no drug susceptibility test (DST) is considered sufficiently reliable for routine application. Although molecular tests are endorsed, their application is limited to known PZA resistance associated mutations. Microbiological DSTs for PZA have been restricted by technical limitations, especially the necessity for an acidic pH. Here, for the first time, MODS culture at neutral pH was evaluated using high PZA concentrations (400 and 800 μg/mL) to determine PZA susceptibility directly from sputum samples. Sputum samples were cultured with PZA for up to 21 days at 37°C. Plate reading was performed at two timepoints: R1 (mean 10 days) and R2 (mean 13 days) for each PZA concentration. A consensus reference test (CRT), composed of MGIT-PZA, pncA sequencing, and the classic Wayne test, was used. One hundred and eighty-two samples were evaluated. The sensitivity and specificity for 400 μg/mL ranged from 76.9% - 89.7% and 93.0% - 97.9%, respectively, and for 800 μg/mL ranged from 71.8% - 82.1% and 95.8% - 98.6%, respectively. Compared to MGIT- PZA, our test showed a similar turnaround time (median 10 and 12 days for PZA sensitive and resistant isolates, respectively). In conclusion, MODS-PZA is presented as a fast, simple, and low-cost DST that could complement the MODS assay to evaluate resistance to the principal first-line anti-tuberculosis drugs. Further optimization of test conditions would be useful, in order to increase its performance.

Mutations in pncA, the gene encoding PZAse, have been reported as the primary cause of PZA resistance (7,14). It has been estimated that between 40 and 80% of multidrugresistant TB (MDR-TB) and 100% of extensively drug-resistant TB (XDR-TB) strains have also been reported as resistant to PZA (5,(15)(16)(17). Even though PZA is considered the only first-line drug to be preserved in all future treatment regimens (18)(19)(20)(21), PZA DST is not routinely performed and is seldom used to plan a personalized treatment regimen (9).
The development of molecular diagnostic tests to detect PZA resistance have been limited due to the large number of mutations along the pncA gene (14), the lack of a mutation hot spot, and the fact that not all mutations cause PZAse impairment or have been associated with a specific PZA susceptibility phenotype (15,(22)(23)(24)(25). However, recently some genotypic DST have been reported with considerable sensitivity and specificity, such the Genoscholar PZA-TB II LPA (Nipro Corporation, Japan), which is a line probe assay (LPA), and a high-resolution melting assay. Previous studies have reported an agreement of 97.6% between the PZA-TB II LPA and a composite reference (pncA sequencing plus phenotypic DST) when heteroresistance is excluded (26), and a sensitivity of 93.2% and a specificity of 91.2% when the test was compared to only pncA sequencing (27). On the other hand, the high-resolution melting assay has been described as having an agreement of 94 and 84% with pncA sequencing and MGIT-PZA, respectively (28). Nonetheless, one limitation for the immediate application of these tests is that the genetic diversity of pncA mutations can vary appreciably between different geographical regions, affecting the sensitivity and specificity of the test (26,27). As a matter of fact, the correlation between pncA sequencing and detection of mutations with PZA resistance varies among countries low and high endemicities, with reported estimates of 41% in Taiwan, 67% in South Africa, 72 to 84% in Brazil, 91% in China, and 97% in China and Japan (7,18).
There is currently no WHO-endorsed phenotypic drug susceptibility test (DST) for detecting PZA resistance due to various technical limitations affecting the utility of currently available assays. These limitations include long turnaround times, poor reproducibility, requirements for an acidic pH, and medium alkalization due to inoculum size (9,15,29,30). Performing direct phenotypic DST for PZA has been hampered by the difficulty in demonstrating anti-TB activity of the drug when analyzed under conventional culture conditions. The widely accepted explanation for this is the requirement of an acidic environment for PZA to exert anti-TB activity versus the pH close to neutral of the culture medium (9,10,18,(31)(32)(33). At present, the Bactec MGIT 960 PZA (MGIT-PZA) assay is the accepted standardized phenotypic DST (34). However, several publications have reported a high rate of false PZA resistance and poor reproducibility, both drawbacks related to pH variation produced by the inoculum size used in the assay (35)(36)(37)(38)(39).
The microscopic observation drug susceptibility (MODS) assay is a relatively rapid and inexpensive direct drug susceptibility test used in the diagnosis of TB and MDR-TB (40)(41)(42) and XDR-TB (43)(44)(45). Previous studies have evaluated the use of MODS culture for analysis of PZA resistance using PZA concentrations ranging between 6.25 and 3,200 g/ml (46)(47)(48). These studies have compared MODS for PZA susceptibility determination against the MGIT-PZA (46), the proportion method (47), and the absolute concentration method (48), reporting sensitivity and specificity ranges of 95.5 to 100% and 93.3 to 100%, respectively. In all cases, a high concordance between MODS and the selected reference method was observed, although all tests were performed with cultured M. tuberculosis strains and not directly from clinical sputum samples.
In the present study, for the first time, we evaluated the use of MODS to determine PZA resistance directly from sputum samples. MODS-PZA is a test that was conceived as a complement for the traditional MODS test to determine resistance to all the first-line antituberculosis drugs. MODS-PZA has been standardized based on the fact that several studies have reported that stress factors can strengthen PZA activity even in nonacidic media (6,9,31,(49)(50)(51). These stress factors must affect the metabolism of M. tuberculosis in order to predispose the mycobacteria to show a PZA-sensitive For pncA sequencing, one full loop of a 21-day M. tuberculosis culture was suspended in 100 mM Tris-EDTA buffer (pH 8.0). The suspension was inactivated by heating to 80°C for 30 min, and genomic DNA was extracted after a modified proteinase K-chloroform protocol (58). Briefly, 100 l of 10 mg/ml of lysozyme was added, and the sample was incubated overnight at 37°C in agitation. The following day for sample digestion, 20 mg/ml of proteinase K and 10% sodium dodecyl sulfate was added, and the sample was incubated at 65°C for 3 h. After that, 5 M NaCl-10% CTAB (cetyltrimethylammonium bromide) was added, and the sample was incubated at 65°C for another 15 min. Then, a solution of phenol-chloroformisoamilic alcohol (25:24:1) was added in equal volumes, and this was mixed gently. The sample was centrifuged at 10,000 rpm at 4°C for 10 min. The upper layer was transferred to a new tube, and  phenol-chloroform was added (1:1). The sample was centrifuged at 10,000 rpm at 4°C for 10 min. This step was repeated. To precipitate the DNA, 95% ethanol was added, and the sample was incubated at -80°C for 30 min. The sample was centrifuged at 10,000 rpm at 4°C for 10 min, and a wash step was performed with 70% ethanol; the pellet was then dried at room temperature. For resuspension, 50 l of Tris-EDTA buffer (pH 8.0) was added. PCR was performed using the primers P1 (5=-GTCGGTCATGTTCGC ATCG-3=; from Ϫ105 bp upstream of pncA) and P6 (5=-GCTTTGCGGCGAGCGCTCCCA-3=; from 60 bp downstream of pncA) (59). The PCR cycling parameters were as follows: one cycle of 94°C for 4 min; followed by 30 cycles of 94°C for 45 s, 58°C for 60 s, and 72°C for 60 s; with a final step of 94°C for 5 min. The PCR product length was 720 bp. Sequencing was performed using the same pair of primers. The M. tuberculosis H37Rv reference strain (NC_000962.3) was used as a reference for the pairwise sequence alignment in order to detect mutations in the pncA gene and its putative promoter. PZA susceptibility for the mutations detected was assigned according to the Tuberculosis Drug Resistance Mutation Database (60).
The classic Wayne test was performed as previously described (61). Briefly, three full loops of M. tuberculosis 21-day culture were inoculated in tubes containing Dubos medium, followed by incubation at 37°C for 7 days. Positive (H37Rv) and negative (DM097) controls were included. At day 7, 1 ml of 1% ferrous ammonium sulfate (SAF; Sigma-Aldrich) was added. After 5 min of incubation at room temperature, each tube was read. A positive result (i.e., a PZA-susceptible strain) was reported when a pink band was observed at the top of the media. In contrast, a negative result (i.e., a PZA-resistant strain) was reported when there was an absence of the pink band.
Data analysis. The sensitivity and specificity (95% CI) of MODS-PZA was calculated by comparison with the CRT in a 2 ϫ 2 contingency table for each PZA concentration and plate reading day. McNemar's test was used to determine significant differences between the sensitivities and specificities calculated. The Fisher exact test was used to determine the association between growth level in culture and the determined PZA susceptibility. Agreement between the dichotomous MODS-PZA, the CRT, and MGIT-PZA was estimated by using the kappa statistic. All statistical tests were reported with 5% significance using STATA v14.0.

RESULTS
Study population. A total of 183 sputum samples were collected. One was excluded from the analysis due to inconsistent results. Of the remaining 182 samples, 51.1% (93/182) were reported as RIF and INH susceptible, 30.22% (55/182) were MDR-TB (i.e., resistant to both RIF and INH), 12.09% (22/182) were monoresistant to INH, and 6.59% (12/182) were monoresistant to RIF. Data regarding patient age and sex, site of sample collection, tuberculosis treatment, and sputum acid-fast microscopy results are presented in Table 1.
According to MGIT-PZA, 75.27% (137/182) of the samples were reported as PZA susceptible, and 24.73% (45/182) as PZA resistant. Six samples did not agree with the CRT. Four of these discordant samples had the K48T and F81S pncA mutations, which have been reported as showing a PZA-resistant phenotype by MGIT-PZA when this method was performed using 100 g/ml of PZA. However, a higher critical concentration of PZA (300 g/ml) classified these isolates as PZA susceptible (data not shown) in concordance with the classical Wayne test and pncA sequencing. The other two presented a wild-type genotype.
Finally, by pncA sequencing, 26.92% (49/182) of the samples reported a nonsynonymous pncA mutation (i.e., in its putative promoter or in the gene itself), and 73.08% (133/182) displayed the wild-type genotype. Q10R was the most frequently observed mutation (28.57%), followed by H51R (18.37%), and K48T (16.33%). All other reported mutations were found in fewer than five samples, including one sample with the A-11C (nucleotide) mutation in the pncA promoter ( Table 3). The P62S, K48T, and F81S mutations (i.e., observed in this study) were linked to a PZA-susceptible phenotype according to the Tuberculosis Drug Resistance Mutation Database (54). Overall, 79.12% (144/182) and 20.88% (38/182) of samples were reported as PZA susceptible and resistant, respectively. Just one sample was discordant with the CRT, in which pncA sequencing did not report any mutations, but both MGIT-PZA and the classic Wayne test were reported as showing PZA resistance (see Table S1 in the supplemental material).
Growth level in culture. M. tuberculosis culture growth was classified into four growth levels: high, regular, low, and microcolonies only. Ninety-three percent of the samples reported at least a low level of growth in culture, including 94.4% of the PZA-sensitive isolates (determined by the CRT) and 89.74% of the PZA-resistant isolates ( Table 4). No significant association (P ϭ 0.107) was found between the level of growth on culture and PZA susceptibility, as determined by CRT.
MODS-PZA performance. A similar percentage of PZA-susceptible and -resistant samples was observed in both PZA wells (400 and 800 g/ml) at the first plate reading (R1). Twelve samples showed a conversion to PZA resistance at 400 g/ml PZA for the second plate reading (R2), and eight samples showed a conversion to PZA resistance at 800 g/ml. For R1, three samples showed discordant results at between 400 and 800 g/ml PZA. One of these samples presented a wild-type genotype and was a All samples were selected according to the following criteria: a minimum sample volume of 2 ml and a positive sputum acid-fast smear microscopy result (health center results). b Paucibacillary, 1 to 9 bacilli in 100 fields; 1ϩ, 10 to 99 bacilli in 100 fields; 2ϩ, 1 to 10 bacilli per field in 50 fields; 3ϩ, 10 bacilli per field in 20 fields. determined to be PZA susceptible by the CRT. The remaining two samples were PZA resistant, with nucleotide deletions in the pncA gene. Likewise, for R2 seven samples showed discordant results. Three samples were determined to be PZA resistant by the CRT, and all of these had the Q10R mutation and a deletion in the pncA gene. The other four samples were determined to be PZA susceptible according to the CRT, and just one had the F81S mutation. No significant association was observed between PZA susceptibility determined in R1 and the level of growth using either 400 g/ml (P ϭ 0.069) or 800 g/ml (P ϭ 0.064) PZA. However, a significant association was reported for PZA susceptibility in R2 at both 400 g/ml and 800 g/ml PZA (P ϭ 0.045 and P ϭ 0.021, respectively). Although not significant, an association was observed for R1 at both concentrations, with a tendency toward an increase (approximately 40 to 50%) for a high growth level for PZA-resistant isolates from R1 to R2 in both concentrations. This event did not occur for PZA-susceptible isolates, for which the high growth level percentage was around 50% at both R1 and R2. The percentages for the other growth levels did not show any changes from R1 to R2 (data not shown). Accuracy measures for MODS-PZA. Results obtained at each PZA concentration and reading day were compared independently to the CRT in order to evaluate sensitivity and specificity. For both drug concentrations, the sensitivity and specificity were Ͼ70 and Ͼ90%, respectively (Table 5). When using a PZA concentration of 400 g/ml, a significant difference was observed between R1 and R2 in specificity (P ϭ 0.016) but not in their sensitivity (P ϭ 0.063). On the other hand, no significant  a Four levels of growth were determined according to microscopic evaluation: "Microcolonies" indicates fewer than 20 microcolonies in the well, "low" refers to a barely covered well, "regular" indicates half of the well was covered, and "high" indicates a completely covered well. No significant association (P Ͼ 0.05) was found between PZA susceptibility and the level of growth. b As determined by CRT.
difference in sensitivity or specificity was observed when using PZA concentrations of 800 g/ml between R1 and R2 (both P ϭ 0.125). In addition, no significant difference was observed in sensitivity and specificity between 400 and 800 g/ml of PZA in R1 (P ϭ 0.5 and P ϭ 1.0, respectively) and in R2 (P ϭ 0.25 and P ϭ 0.125, respectively). A kappa index and agreement percentage of 0.79 and 93.41%, respectively, were reported for a PZA concentration of 400 g/ml, and 0.77 and 92.86%, respectively, for a PZA concentration of 800 g/ml; both in R1. Similarly, in R2 a kappa index and agreement percentage of 0.78 and 92.31%, respectively, were reported for 400 g/ml PZA, and 0.79 and 92.86%, respectively, for 800 g/ml PZA. From the samples that had discordant results for MODS-PZA at 400 g/ml PZA and the CRT, those that were reported as PZA resistant by MODS-PZA but PZA susceptible by the CRT showed either a wild-type genotype or the P62S or F81S mutations in the pncA gene. However, these mutations have been previously reported in the PZAresistant phenotype associated with mutations represented in a M. tuberculosis mutant pncA library, where the PZA susceptibility was detected by MGIT 960 and bioinformatics prediction (62). On the other hand, samples that were reported to be PZA susceptible by MODS-PZA but PZA resistant by CRT had Q10R, Q10P, H51R, H71R, and A-11C mutations detected in the pncA gene and its putative promoter. All of these mutations have been reported to confer PZA resistance with high confidence (60,63). No differences between bacillary load (i.e., by sputum acid-fast smear or level of growth in culture) were observed (data not shown). The same results were observed for isolates with discordancy between MODS-PZA performed at 800 g/ml PZA and the CRT, including samples in which pncA deletions (nucleotide deletions Δ375-389 and Δ456 -466) were detected and yet were reported as PZA susceptible in 800 g/ml of PZA.
MODS-PZA was compared to MGIT-PZA since both tests are based on the principle that culture in the presence of the drug would inhibit bacterial growth. A kappa index of 0.74 and an agreement percentage of 91.21% were found for 400 g/ml at R1, a kappa index of 0.72 and agreement of 90.66% were found for 800 g/ml at R1, a kappa index of 0.79 and agreement of 92.31% for 400 g/ml were found at R2, and finally a kappa index of 0.77 and an agreement of 91.76% for 800 g/ml were found at R2. The median turnaround time for MODS-PZA was 10 days (range, 5 to 24 days) for R1, and the R2 was performed a median of 3.1 days after R1.

DISCUSSION
This study reports, for the first time, the application of MODS culture to determine PZA resistance directly from sputum culture through the evaluation of PZA activity at neutral pH (MODS-PZA). Unlike other studies that have evaluated the application of MODS for PZA resistance determination in M. tuberculosis strains (46)(47)(48), our findings have demonstrated the possibility of directly evaluating sputum samples with high PZA concentration (400 and 800 g/ml) without the need for additional processing except for a decontamination step. Including PZA in the spectrum of M. tuberculosis drugs that are currently tested in MODS (RIF, INH, and second-line drugs) (41)(42)(43)64) will give a complete drug susceptibility test that could be applied routinely in low-income countries.
The principal limitation for traditional phenotypic DST for PZA susceptibility deter- mination has been the requirement of an acidic pH in order to allow PZA to show antituberculosis activity (15,29,30). In vivo, a concentration peak in blood between 20 and 60 g/ml (1 to 2 h postdose) is active against M. tuberculosis (65). These concentrations are active during the early phase of infection because the physiological pH is reduced due to ongoing inflammatory processes. According to the proposed mechanism of action for PZA, when POA is released in an acidic medium (pH 5.5), some molecules are protonated (due to its pKa of 2.9) and reenter the cytoplasm, showing its bactericidal effect (66). In vitro, a similar condition can be obtained when the culture is performed at pH 5.5 (using an MIC of 50 g/ml). However, an acidic pH has been reported to inhibit M. tuberculosis growth (30,31). Theoretically, higher concentrations of PZA can show antituberculosis activity at a higher pH. Therefore, an active PZA concentration can be estimated for a particular pH using the Henderson-Hasselbach equation (67). One disadvantage of this methodology is that susceptibility results of DST using higher drug concentrations cannot be directly related to pharmacokinetic parameters determined in vivo. However, it should be considered that the objective of any DST is the discrimination between susceptible and resistant strains. For this study, PZA concentrations of 400 and 800 g/ml were selected to evaluate PZA susceptibility based on the expected active PZA concentration for a pH of 6.8 (estimated concentration, 992 g/ml) and some past studies that reported a PZA activity of 300 to 400 g/ml in 7H12 broth at pH 6.0 to 6.2 (68)(69)(70). Even though no significant differences were reported between the sensitivities of the two reading time points (R1 and R2) in this study (P Ͼ 0.05), reported that sensitivities were lower than those shown in other studies. All other studies (46)(47)(48) have reported a sensitivity close to 95% but used 100 g/ml PZA as the critical concentration and at a lower pH (6.0); although these studies evaluated higher concentrations of PZA, ranging from 6.25 to 3,200 g/ml, 100 g/ml was used as the cutoff for sensitivity determination. However, this concentration has been shown to lead to the false reporting of isolates as resistant (36,39,68) since it is lower than the expected PZA concentration for pH 6.0. It is important to bear in mind that none of those studies used a CRT as a gold standard: they compared MODS for PZA against MGIT-PZA (46), the broth microdilution method (47), and the proportion method (48).
Another important difference that may account for the observed sensitivity variation is that those studies used a McFarland dilution of M. tuberculosis strains previously isolated in Lowenstein-Jensen medium as the inoculum, rather than direct inoculation of sputum samples. This could therefore indicate that a larger number of viable mycobacteria were evaluated than would be found in clinical samples. The specificity, which ranged from 93.0 to 98.6%, was similar to that reported in other studies close to 95% (46,48); Ghiraldi et al. (47) reported a sensitivity of 100%, but the number of PZA-resistant strains included in that study was small (n ϭ 8).
Discordant samples that were reported as PZA sensitive by MODS-PZA but PZA resistant by CRT showed mutations in the pncA gene or the putative promoter, independently of the concentration of PZA. The mutations observed in these isolates were H51R, H71R, Q10P, Q10R, A-11C, Δ375-389, and Δ456 -466. All of these mutations have been strongly associated with PZA-resistant phenotypes (11,22,25,63). These mutations affect the catalytic site or the metal-binding site of PZAse, eliminating its enzymatic activity (11,22,63). Almost all of these isolates were MDR-TB or monoresistant to RIF (88.89% for 400 g/ml and 90.91% for 800 g/ml PZA in R1 and 100% for both 400 and 800 g/ml PZA in R2) and demonstrated at least a low level of growth in control wells. On the other hand, discordant samples that were reported as PZA resistant by MODS-PZA but PZA sensitive by CRT showed a wild-type genotype or carried the F81S or P62S mutations, which are reported as being associated with PZA-sensitive phenotypes in the Tuberculosis Drug Resistance Mutation Database (60). These isolates were MDR-TB, monoresistant to INH, or non-MDR-TB. The growth level in culture did not show the same tendency as the previously described discordant samples: these samples were reported as either microcolonies only or having a high growth level. Although no plausible explanation was found for these observations, some similar discordances have been reported elsewhere (48). PZA-sensitive strains with a wild-type genotype that were reported as PZA resistant by MODS showed a PZA-sensitive phenotype by the proportion method in Lowenstein-Jensen medium, with an MIC ranging from 600 to 1,200 g/ml. Likewise, a PZA-sensitive strain with the L35P mutation (associated with a PZA-sensitive phenotype) was reported as PZA resistant in MODS with an MIC of 1,200 g/ml PZA.
Upon comparison to MGIT-PZA, since both assays have the same rationale, the test showed a sensitivities ranging from 68.9 to 84.4% for R1 and from 64.4 to 75.6% for R2 for 400 and 800 g/ml PZA, respectively. The specificity fell within a narrower range of 94.9 to 98.7%, with an agreement close to 91.0%. However, it must be noted that MGIT-PZA is performed with M. tuberculosis cultures. A previous study (71) that evaluated the performance of MGIT-PZA directly from sputum samples reported uninterpretable results for 163 samples, varying from 23 to 66% among all of the participating laboratories. Moreover, the percentage of successful MGIT-PZA procedures performed directly from sputum samples compared to traditional MGIT-PZA was 59% (range, 34 to 77%). Therefore, MODS-PZA could be comparable to the "direct MGIT-PZA" method. Median turnaround time of MODS-PZA were 10 and 12 days for PZA-sensitive isolates (range, 5 to 24 days) and PZA-resistant isolates (range, 5 to 21 days), respectively; the traditional MGIT-PZA reports a similar turnaround time ranging from 4 to 21 days (71).
It is important to mention that it remains vital to define the exact mechanism of action of PZA in order to develop the most suitable susceptibility test for this drug, which is considered the pivot in most current and future treatments. More data are needed to increase our understanding of the synergy between different factors that can enhance or reduce PZA activity under controlled in vitro conditions. This is the first time that MODS culture has been adapted to evaluate PZA susceptibility directly from sputum samples, obtaining a relatively inexpensive, simple, and fast phenotypic DST. Currently, the implementation of any MODS based DSTs (such as MODS-PZA) will have limitations related to laboratory infrastructure (MODS requires biosafety level 2 facilities) (72), equipment acquisition (inverted microscope), and personnel training in the interpretation of results. However, some advantages of the MODS test should be highlighted. Compared to other commercial cultures or LPAbased assays, MODS is less expensive (approximately U.S. $4 to $5 compared to U.S. $15 to $56, not considering any additional equipment) (73,74). Although there is no considerable cost difference compared to gene sequencing (US $8 to $12), in limitedresource countries this procedure is usually outsourced, which implies an additional and expensive shipping service (28). Regarding the infrastructure and equipment implementation, every phenotypic DST has similar minimum requirements with the exception of automatized tests (i.e., MGIT-PZA) which increase the total cost of the assay due to the acquisition of the specific instrument. Although molecular tests (such as LPA-based assays) may not require additional expensive instrumentation, they face an important downside since not all pncA mutations have been reported and associated with PZA resistance (26,27). Consequently, growth-based tests are still considered the standard test (or at least a complementary test) for PZA susceptibility determination (27,39).
Recently, our group have reported an adaptation of the classic Wayne test on MODS culture directly from sputum samples (75). This test permits an indirect determination of PZA resistance by the detection of POA produced at pH 7.0 (i.e., with a sensitivity and specificity of 92.7 and 99.3%, respectively) without the necessity for a primary culture. However, one critical point of this test is the addition of PZA and ferrous ammonium sulfate. For this, PZA is added after M. tuberculosis growth for an average of 10 days and then, after three more incubation days, ferrous ammonium sulfate is added in order to determine PZA susceptibility phenotype. As a result, the culture plate must be opened twice when M. tuberculosis has just shown growth. This means that the risk of cross contamination or culture spillover could increase if technicians do not work very carefully. Thus, MODS-PZA is an alternative direct phenotypic DST which does not require the addition of any reagent or the manipulation of the plate, other than for microscopic evaluation (i.e., the culture plate is never opened after the sputum sample inoculation).
In spite of the sensitivity and specificity values achieved in this study, more work is needed to evaluate the effect of other stress factors on MODS-PZA, since adjusting for these may enable a further increase in its accuracy. The final goal is the development of a complete, reliable, fast, and low-cost PZA susceptibility assay that could reduce the rate of nondiagnosed PZA resistance cases, especially in low-setting countries where TB is still an endemic disease.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. SUPPLEMENTAL FILE 1, XLSX file, 0.02 MB.