Correlating rrs and eis promoter mutations in clinical isolates of Mycobacterium tuberculosis with phenotypic susceptibility levels to the second-line injectables

Objective/background: The in vitro drug-susceptibility testing of Mycobacterium tuberculosis reports isolates as resistant or susceptible on the basis of single critical concentrations. It is evident that drug resistance in M. tuberculosis is quite heterogeneous, and involves low level, moderate level, and high level of drug-resistant phenotypes. Thus, the aim of our study was to correlate rrs [X52917] and eis [AF144099] promoter mutations, found in M. tuberculosis isolates, with corresponding minimum inhibitory concentrations of amikacin, kanamycin, and capreomycin

Methods: Ninety M. tuberculosis clinical isolates were analyzed in this study. The minimum inhibitory concentrations were determined by MGIT 960 for 59 isolates with resistance-associated mutations in the rrs and eis promoter gene regions, and 31 isolates with wild-type sequences, as determined by the GenoType MTBDRsI [version 1] assay

Results: The rrs A1401G mutation was identified in 48 isolates resistant to the second- line injectables. The eis promoter mutations C-14T [n = 3], G-10C [n = 3], G-10A [n = 3], and C-12T [n = 2] were found within 11 isolates with various resistance profiles to the second-line injectables. Thirty-one isolates had wild-type sequences for the rrs and eis promoter gene regions of interest, one of which was amikacin, kanamycin, and capreomycin resistant. The isolates with the rrs A1401G mutation had amikacin, kanamycin, and capreomycin minimum inhibitory concentrations of >40 mg/L, >20 mg/L, and 515 mg/L, respectively. The isolates with eis promoter mutations had amikacin, kanamycm, and capreomycin minimum inhibitory concentrations of 0.25-1.0 mg/L, 0.625-10 mg/ L, and 0.625-2.5 mg/L, respectively

Micronucleus assay- an early diagnostic tool to assess genotoxic changes in tobacco and related habits

Objective: Micronuclei are induced in cells by a variety of substances, like UV radiation, infrared rays, X-radiations, and chemicals. Among them tobacco- specific nitrosamines have been reported to be potent mutagenic agents which are thought to be responsible for the induction of chromosomal aberrations resulting in production of micronuclei. The main aim of our study is to compare MN frequency among subjects, chewing tobacco only, chewing and smoking tobacco only, and chewing, smoking with alcohol, and to co-relate with control subjects

Methods: Healthy subjects are included in the study and divided into four groups having 20 subjects in each group. Group-I is chewing only, group-II chewing and smoking, group-III chewing and smoking with alcohol, group-IV control. Smears were made from buccal exfoliated cells and stained with DNA specific stain Acridine orange. Frequency on MNC per 100 cells was assessed with, one way ANOVA and Tukey HSD Multiple Comparisons test with p<0.05

Results: The mean number of MN was 2.3, 2.4, 3.6 in the group of chewing only, chewing and smoking, chewing, smoking and alcohol respectively. While assessing MN in the controls, out of 20 cases, 19 showed no MN among the cells examined while 1 patient showed 1 MN each per 100 cells examined

Conclusion: The present study concludes that MN is a better surrogate biomarker to predict genotoxicity for tobacco related habits

Is a composite reference standard [CRS] an alternative to culture in assessment and validation of a single tube nested in-house PCR for TB diagnosis?

Introduction: Delay in diagnosis of paucibacillary extra pulmonary tuberculosis [TB] and of smear negative TB has hampered the efforts taken by Control Programs to curb its spread. Better efforts to control spread of TB require more accurate and rapid diagnostic test

Aims: To facilitate early diagnosis of TB directly from clinical specimens, we have standardized and validated the use of a single tube in-house nested PCR in comparison against culture and composite reference standard [CRS]

Methods: Single tube nested PCR was performed using primers targeting Insertion Sequence [IS] 6110 of Mycobacterium tuberculosis complex. Microbiological techniques includes AFB smear microscopy, and cultivation on solid egg-based medium [Lowenstein-Jensen [LJ]] and on liquid culture medium using BACTEC MGIT 960 system, BD Microbiology Systems

Results: The sensitivity and specificity of PCR against culture was observed to be 89.7% [95% CI: 84.1-93.5] and 73.1% [95% CI: 67.4-78.1] respectively and that against CRS criteria was 80.2% [95% CI: 75.1-84.5] and 97.1% [95% CI: 92.9-98.9] respectively. PCR showed 100% [111/111, 95% CI: 97-100] sensitivity for smear positive specimens. For smear negative specimens sensitivity and specificity of PCR against culture was observed to be 78.4% [69/88, 95% CI: 68.4-86.5] and 77.3% [204/264, 95% CI: 71.7-82.2] respectively and that against CRS was 68.1% [124/182, 95% CI: 60.8-74.8] and 97.1% [165/170, 95% CI: 93.3-99] respectively

Conclusion: CRS criteria were observed to be better than culture for assessing the diagnostic accuracy of PCR test