Tion. The sensitivity and specificity for detecting ethambutol was 56.19 and 81 which is very low as compared to FQ and second line injectables, but is in concordance to other studies [13,17]. The most common mutation detected by the assay was M306V 74.32 (55/74) followed by M306I 25.67 (9/74) which is high in case of M306V as compared to previous studies. Other mutations are required to be targeted [23] by the assay to increase its sensitivity and specificity. It has been observed that genotypic analysis identified high rate of mutations (91.4 ) at codon 306 of the embB gene in comparison to phenotypic analysis, where phenotypic test failed to identify EMB resistance [24]. But 40 (46/115) of the EMB resistance cases were not detected by the assay, which is same as reported in the previous study. In the current scenario, detection of EMB resistance by targeting mutations at 306 codon has a mixed opinion by different authors which is due to poorer inter-laboratory performance for EMB than some other drugs, and it has been suggested that discrepancies between genotypic and phenotypic 301-00-8 testing may be due to difficulties with phenotypic testing [23,25,26]. FQ and second line injectables are resorted to be used in treatment of cases that are treatment failures, relapses or MDR/ XDR-TB suspects. Detection of resistance to FQ and second line injectables by conventional method (a two step process) takes approximately 15?0 days to report DST results, due to slow growing nature of M. tuberculosis [13,27]. The time required to detect resistance by MTBDRsl is 1? days after receiving the sample. Table 4. Genotypic emb 1480666 pattern obtained by MTBDRsl assay on 170 clinical sediments.Phenotypic DST RCodon mutation No of Isolates M306I M306V 19 56 85 10 5.88 (10/170) 25.67 (19/74) 75.6 (56/74)SWT1 Indeterminatedoi:10.1371/journal.pone.0049433.tEvaluation of Genotype MTBDRsl AssayTable 5. Statistical summary of GenoTypeMTBDRsl assay.FQ Sensitivity Specificity Positive predict Value (PPV) Negative predict Value (NPV) Prevalence Diagnostic Accuracy 91 98 99 88 62 94SLD 100 100 1 1 14.67 100EMB 56.19 81 88.06 43.21 61 63.51doi:10.1371/journal.pone.0049433.tAlthough, there are numerous molecular based test like Multiplex allele specific (MAS-PCR) [28], Reverse Line Blot Hybridization (RLBH) [29], Hetero duplex analysis [30] that targets gyrA, rrs, embB to detect resistance, but to confirm the amplified product might require sequencing facility as reference standard to detect the mutation in coordination to abovementioned technique. Unfortunately, not all laboratories are equipped with sequencing facility as it is Chebulagic acid site expensive and requires modern expertise. In this study 21 strains were XDR, of which MTBDRsl was able to detect 95.23 (20/21) of cases but a single XDR strain was detected as FQ-sensitive, but resistant to Second line injectable and EMB by the assay, as the resistant to this strain might be due to other resistant mechanism [5] as there was no mutation detected by sequencing of the hot spot region. The limitation of the study is it included smear positive sputum samples only. In conclusion, to break the chain of ongoing transmission a rapid molecular method, like MTBDRsl, would limit or decrease the rate of transmission by early detection of the resistance. 1662274 Although the assay does not replace the phenotypic DST it is helpful in rapid detection of drug resistance among resistant suspects.Author ContributionsConceived and designed the.Tion. The sensitivity and specificity for detecting ethambutol was 56.19 and 81 which is very low as compared to FQ and second line injectables, but is in concordance to other studies [13,17]. The most common mutation detected by the assay was M306V 74.32 (55/74) followed by M306I 25.67 (9/74) which is high in case of M306V as compared to previous studies. Other mutations are required to be targeted [23] by the assay to increase its sensitivity and specificity. It has been observed that genotypic analysis identified high rate of mutations (91.4 ) at codon 306 of the embB gene in comparison to phenotypic analysis, where phenotypic test failed to identify EMB resistance [24]. But 40 (46/115) of the EMB resistance cases were not detected by the assay, which is same as reported in the previous study. In the current scenario, detection of EMB resistance by targeting mutations at 306 codon has a mixed opinion by different authors which is due to poorer inter-laboratory performance for EMB than some other drugs, and it has been suggested that discrepancies between genotypic and phenotypic testing may be due to difficulties with phenotypic testing [23,25,26]. FQ and second line injectables are resorted to be used in treatment of cases that are treatment failures, relapses or MDR/ XDR-TB suspects. Detection of resistance to FQ and second line injectables by conventional method (a two step process) takes approximately 15?0 days to report DST results, due to slow growing nature of M. tuberculosis [13,27]. The time required to detect resistance by MTBDRsl is 1? days after receiving the sample. Table 4. Genotypic emb 1480666 pattern obtained by MTBDRsl assay on 170 clinical sediments.Phenotypic DST RCodon mutation No of Isolates M306I M306V 19 56 85 10 5.88 (10/170) 25.67 (19/74) 75.6 (56/74)SWT1 Indeterminatedoi:10.1371/journal.pone.0049433.tEvaluation of Genotype MTBDRsl AssayTable 5. Statistical summary of GenoTypeMTBDRsl assay.FQ Sensitivity Specificity Positive predict Value (PPV) Negative predict Value (NPV) Prevalence Diagnostic Accuracy 91 98 99 88 62 94SLD 100 100 1 1 14.67 100EMB 56.19 81 88.06 43.21 61 63.51doi:10.1371/journal.pone.0049433.tAlthough, there are numerous molecular based test like Multiplex allele specific (MAS-PCR) [28], Reverse Line Blot Hybridization (RLBH) [29], Hetero duplex analysis [30] that targets gyrA, rrs, embB to detect resistance, but to confirm the amplified product might require sequencing facility as reference standard to detect the mutation in coordination to abovementioned technique. Unfortunately, not all laboratories are equipped with sequencing facility as it is expensive and requires modern expertise. In this study 21 strains were XDR, of which MTBDRsl was able to detect 95.23 (20/21) of cases but a single XDR strain was detected as FQ-sensitive, but resistant to Second line injectable and EMB by the assay, as the resistant to this strain might be due to other resistant mechanism [5] as there was no mutation detected by sequencing of the hot spot region. The limitation of the study is it included smear positive sputum samples only. In conclusion, to break the chain of ongoing transmission a rapid molecular method, like MTBDRsl, would limit or decrease the rate of transmission by early detection of the resistance. 1662274 Although the assay does not replace the phenotypic DST it is helpful in rapid detection of drug resistance among resistant suspects.Author ContributionsConceived and designed the.
