Ded by WHO. [8] Among the SLD kanamycin was tested at a critical concentration of 5 mgm/ml [11]. The drug powders were procured from Sigma laboratories except for moxifloxacin and ethambutol which were procured from Cipla and Becton Dickinson respectively. For Quality Control on a routine weekly basis a known genotypic confirmed resistant Desoxyepothilone B strain was put up for DST in MGIT 960 and H37Rv as the pan susceptible strain. Biannual proficiency testing is performed as per College of American Pathologists (CAP) guidelines by sequencing five resistant and susceptible isolates for the hot spot regions for each of the SLD.SequencingEqual number of representative discrepant sediments of phenotypic resistance and genotypic susceptible as well as phenotypic susceptible and genotypic resistance would be sequenced.Stastical AnalysisSensitivity, specificity, positive predicative value (PPV), Negative predicative Value (NPV) was calculated in comparison to MGIT phenotypic DST (gold standard). Metadisc software (Ver1.4) was used to calculate the Sensitivity, specificity, positive predicative value (PPV), Negative predicative Value (NPV).Results Phenotypic DSTOf the 170 sediments analyzed 77.64 (132/170) are MDR (INH+RIF) R of these 15.9 (21/132) are XDR, 5.88 (10/170) were monoresistant to isoniazid and susceptible to rifampicin, ofloxacin, moxifloxacin and second line aminoglycosides tested (kanamycin, AG-221 site amikacin and capreomycin). Three (1.7 ) were resistant to only ofloxacin and moxifloxacin but susceptible to the other drugs tested, a single isolate was resistant to isoniazid as well as fluoroquinolones but susceptible to other drugs. 12.9 (22/ 170) were susceptible to all drugs tested. Phenotypic FQ-DST. Of the total 170 sample sediments 99 isolates were resistant to ofloxacin and moxifloxacin, 4 isolatesGenotype MTBDR sl AssayThe procedure was divided into 3 steps:N NDNA extraction: 500 microlitre of decontaminated sediment is centrifuged at 14,000 rpm for 15minutes; this is resuspended in 100 microlitre of sterile distilled water. This was further processed with heat lysis and sonication. Mulitplex polymerase chain reaction (PCR) amplification with biotinylated primers: PCR was performed in Eppendorff thermal cyclers with the following cycling conditions, Denaturation 95uC/15 24272870 min, initial Denaturation 950 C/30 sec, Annealing 58uC/2 min,(10 cycles) Denaturation 95uC/25 sec,Evaluation of Genotype MTBDRsl Assaywere resistant to ofloxacin alone. Three phenotypic sensitive strains detected as resistant by the Genotype MTBDRsl assay; of these one sample was revived from 1407003 contaminated specimen, which on repeat DST continued to be susceptible. The other two specimens were repeated for phenotypic DST and it showed resistance correlating with the assay. Thus after the resolution of the discrepancies, there were 101 strains resistant to both ofloxacin and moxifloxacin and 65 strains were susceptible to both. [Table 1]. Phenotypic second line aminoglycosides DST. Of the total 170 sediments, 148 isolates were susceptible to KAN, AM, CAP; 16 isolates were resistant to KAN, AM, CAP and 6 were resistant to KAN and AM but sensitive to CAP. [Table 1]. Phenotypic DST-EMB. Out of 170 sediments 114 isolates were resistance to EMB and 56 susceptible. [Table 1].Genotype MTBDRsl Assay Second Line Injectables Results16 sediments resistant to second line injectables were correctly identified by MTBDRsl assay as resistant; by hybridization to one of the mutant rrs probe MUT1.Ded by WHO. [8] Among the SLD kanamycin was tested at a critical concentration of 5 mgm/ml [11]. The drug powders were procured from Sigma laboratories except for moxifloxacin and ethambutol which were procured from Cipla and Becton Dickinson respectively. For Quality Control on a routine weekly basis a known genotypic confirmed resistant strain was put up for DST in MGIT 960 and H37Rv as the pan susceptible strain. Biannual proficiency testing is performed as per College of American Pathologists (CAP) guidelines by sequencing five resistant and susceptible isolates for the hot spot regions for each of the SLD.SequencingEqual number of representative discrepant sediments of phenotypic resistance and genotypic susceptible as well as phenotypic susceptible and genotypic resistance would be sequenced.Stastical AnalysisSensitivity, specificity, positive predicative value (PPV), Negative predicative Value (NPV) was calculated in comparison to MGIT phenotypic DST (gold standard). Metadisc software (Ver1.4) was used to calculate the Sensitivity, specificity, positive predicative value (PPV), Negative predicative Value (NPV).Results Phenotypic DSTOf the 170 sediments analyzed 77.64 (132/170) are MDR (INH+RIF) R of these 15.9 (21/132) are XDR, 5.88 (10/170) were monoresistant to isoniazid and susceptible to rifampicin, ofloxacin, moxifloxacin and second line aminoglycosides tested (kanamycin, amikacin and capreomycin). Three (1.7 ) were resistant to only ofloxacin and moxifloxacin but susceptible to the other drugs tested, a single isolate was resistant to isoniazid as well as fluoroquinolones but susceptible to other drugs. 12.9 (22/ 170) were susceptible to all drugs tested. Phenotypic FQ-DST. Of the total 170 sample sediments 99 isolates were resistant to ofloxacin and moxifloxacin, 4 isolatesGenotype MTBDR sl AssayThe procedure was divided into 3 steps:N NDNA extraction: 500 microlitre of decontaminated sediment is centrifuged at 14,000 rpm for 15minutes; this is resuspended in 100 microlitre of sterile distilled water. This was further processed with heat lysis and sonication. Mulitplex polymerase chain reaction (PCR) amplification with biotinylated primers: PCR was performed in Eppendorff thermal cyclers with the following cycling conditions, Denaturation 95uC/15 24272870 min, initial Denaturation 950 C/30 sec, Annealing 58uC/2 min,(10 cycles) Denaturation 95uC/25 sec,Evaluation of Genotype MTBDRsl Assaywere resistant to ofloxacin alone. Three phenotypic sensitive strains detected as resistant by the Genotype MTBDRsl assay; of these one sample was revived from 1407003 contaminated specimen, which on repeat DST continued to be susceptible. The other two specimens were repeated for phenotypic DST and it showed resistance correlating with the assay. Thus after the resolution of the discrepancies, there were 101 strains resistant to both ofloxacin and moxifloxacin and 65 strains were susceptible to both. [Table 1]. Phenotypic second line aminoglycosides DST. Of the total 170 sediments, 148 isolates were susceptible to KAN, AM, CAP; 16 isolates were resistant to KAN, AM, CAP and 6 were resistant to KAN and AM but sensitive to CAP. [Table 1]. Phenotypic DST-EMB. Out of 170 sediments 114 isolates were resistance to EMB and 56 susceptible. [Table 1].Genotype MTBDRsl Assay Second Line Injectables Results16 sediments resistant to second line injectables were correctly identified by MTBDRsl assay as resistant; by hybridization to one of the mutant rrs probe MUT1.
