Nt A and 100 ml of Binding Reagent B were added to the extraction tube. The bound DNA was washed once with 1 ml of Wash Buffer A and 5 times with 1 ml of Wash Buffer B. 100 ml of elution buffer was added to the tube followed by incubation at 90uC for 5 minutes. Elute was used directly in PCR reactions. 2. Real-time PCR on ABI 7500. PCR reactions were run using the DNA extracted using the Trueprep-MAG protocol. 4 ml of extracted DNA was mixed with 6 ml of the Truenat MTB mastermix and real-time PCR was performed on ABI 7500 (Applied Biosystems) under the following cycling conditions: 1 min at 95uC and 45 cycles of 10 s at 95uC and 34 s at 58uC.3. Real-time PCR on chip. 5 ml of DNA extracted added to the Truenat MTB microchip (Fig. 3) and the real-time PCR was done using a pre-programmed profile on the device. Results were observed on the screen and compared to the results obtained on the ABI 7500 using the same mastermix. 3. Buffers, reagents and mastermixes. All buffers and reagents used for nucleic acid extraction and all mastermixes used for PCR are proprietary components of the Truenat MTB kit.Table 2. Performance of PCR tests in various patient groups.Smear Truenat MTB + 2 In-house nested PCR + 2 117 3 59 47 + 119 1 2 55Culture + 132 9 2 42S+C+ (n = 112)S2C2 (n = 77)111341354111135doi:10.1371/journal.pone.0051121.tTruenat MTB MedChemExpress 370-86-5 DiagnosisTable 3. Comparison of Truenat MTB results with in-house nested PCR results.Nested PCR Truenat MTB + 2 doi:10.1371/journal.pone.0051121.t003 + 160 16 2 14Statistical AnalysisEvaluation of the Truenat MTB test was performed done in comparison to the other molecular methods for detection of Mycobacterium tuberculosis DNA from sputum, following the STARD recommendations [9]. Sensitivity, Specificity, Positive Predictive Value, Negative Predictive Value, Positive Likelihood Ratio, Negative Likelihood Ratio were calculated by using Bayesian sensitivity/specificity calculator and ROC curve and forest plot were calculated using Meta disc (version 1.4).Among the S2C+ specimens, 75.86 (22/29) were Truenat MTB positive and 82.76 (24/29) were positive by the IS6110 nested PCR protocol. The Truenat MTB results were largely concordant with the inhouse nested PCR results, 196 of 226 get ML-281 specimens showed the same result by either PCR test (Table 3). 1317923 Of the 30 discordant results, 16 specimens were MTB positive by nested PCR but not by Truenat. Of this group, 3 specimens were CRS2 and treatment naive but consequently false positives. On the other hand, 14 of the 30 were MTB positive by Truenat but not nested by PCR. Of this group, all 14 were CRS+ and on antitubercle treatment indicating no false positives Performance estimates of all tests using the CRS as a reference standard are presented in Table 4 As can be seen, the PCR tests have higher sensitivity than smear and culture tests. The IS6110 nested PCR protocol had a PCR inhibition rate of 8.4 (19/226) where the PCR reaction had to be repeated after the DNA was diluted as 1:1 with sterile water. Liquid culture had an average time to positivity (TTP) of 25 days, in-house nested PCR had a TTP of 7 days (additional 7 days if PCR was inhibited) and the Truenat MTB test had a TTP of approximately 1 hour.ResultsAs shown in fig. 4, outcome of study out of total 230 specimens screened, 4 were detected as nontuberculous mycobacteria (NTM) by phenotypic MGIT and hence were excluded from this study. Of the remaining 226 sputum specimens, 141 were MTB culture positive(C+) and 8.Nt A and 100 ml of Binding Reagent B were added to the extraction tube. The bound DNA was washed once with 1 ml of Wash Buffer A and 5 times with 1 ml of Wash Buffer B. 100 ml of elution buffer was added to the tube followed by incubation at 90uC for 5 minutes. Elute was used directly in PCR reactions. 2. Real-time PCR on ABI 7500. PCR reactions were run using the DNA extracted using the Trueprep-MAG protocol. 4 ml of extracted DNA was mixed with 6 ml of the Truenat MTB mastermix and real-time PCR was performed on ABI 7500 (Applied Biosystems) under the following cycling conditions: 1 min at 95uC and 45 cycles of 10 s at 95uC and 34 s at 58uC.3. Real-time PCR on chip. 5 ml of DNA extracted added to the Truenat MTB microchip (Fig. 3) and the real-time PCR was done using a pre-programmed profile on the device. Results were observed on the screen and compared to the results obtained on the ABI 7500 using the same mastermix. 3. Buffers, reagents and mastermixes. All buffers and reagents used for nucleic acid extraction and all mastermixes used for PCR are proprietary components of the Truenat MTB kit.Table 2. Performance of PCR tests in various patient groups.Smear Truenat MTB + 2 In-house nested PCR + 2 117 3 59 47 + 119 1 2 55Culture + 132 9 2 42S+C+ (n = 112)S2C2 (n = 77)111341354111135doi:10.1371/journal.pone.0051121.tTruenat MTB DiagnosisTable 3. Comparison of Truenat MTB results with in-house nested PCR results.Nested PCR Truenat MTB + 2 doi:10.1371/journal.pone.0051121.t003 + 160 16 2 14Statistical AnalysisEvaluation of the Truenat MTB test was performed done in comparison to the other molecular methods for detection of Mycobacterium tuberculosis DNA from sputum, following the STARD recommendations [9]. Sensitivity, Specificity, Positive Predictive Value, Negative Predictive Value, Positive Likelihood Ratio, Negative Likelihood Ratio were calculated by using Bayesian sensitivity/specificity calculator and ROC curve and forest plot were calculated using Meta disc (version 1.4).Among the S2C+ specimens, 75.86 (22/29) were Truenat MTB positive and 82.76 (24/29) were positive by the IS6110 nested PCR protocol. The Truenat MTB results were largely concordant with the inhouse nested PCR results, 196 of 226 specimens showed the same result by either PCR test (Table 3). 1317923 Of the 30 discordant results, 16 specimens were MTB positive by nested PCR but not by Truenat. Of this group, 3 specimens were CRS2 and treatment naive but consequently false positives. On the other hand, 14 of the 30 were MTB positive by Truenat but not nested by PCR. Of this group, all 14 were CRS+ and on antitubercle treatment indicating no false positives Performance estimates of all tests using the CRS as a reference standard are presented in Table 4 As can be seen, the PCR tests have higher sensitivity than smear and culture tests. The IS6110 nested PCR protocol had a PCR inhibition rate of 8.4 (19/226) where the PCR reaction had to be repeated after the DNA was diluted as 1:1 with sterile water. Liquid culture had an average time to positivity (TTP) of 25 days, in-house nested PCR had a TTP of 7 days (additional 7 days if PCR was inhibited) and the Truenat MTB test had a TTP of approximately 1 hour.ResultsAs shown in fig. 4, outcome of study out of total 230 specimens screened, 4 were detected as nontuberculous mycobacteria (NTM) by phenotypic MGIT and hence were excluded from this study. Of the remaining 226 sputum specimens, 141 were MTB culture positive(C+) and 8.
