Laishram Chandreshwor Singh

Expression of genes related to multiple drug resistance and apoptosis in acute leukemia: response to induction chemotherapy

Resistance to chemotherapy is a major impediment to the successful treatment of acute leukemia (AL). Expression of genes involved in drug resistance and apoptosis may be responsible for this. This study aimed to investigate the expression of drug resistance (MDR1, MRP1, LRP, BCRP, GSTP1, DHFR) and apoptotic genes (p53, BCL-2, Survivin) in adult acute leukemias and compare them with clinical and hematological findings and response to induction chemotherapy. Eighty-five patients with AL [45 with acute myeloid leukemia (AML) and 40 with acute lymphoblastic leukemia (ALL)] were used as a study group. Real-time PCR results showed that expression level of MDR1 was significantly higher in AML whereas expression of DHFR, BCRP and Survivin was significantly higher in ALL patients. In AML, significant correlation was observed between LRP and MRP1 (r(s)=0.44, p=0.016), LRP and DHFR (r(s)=0.41, p=0.02), MDR1 and BCL-2 (r(s)=0.38, p=0.03). Expression of GSTP1 and LRP correlated with high white blood count (p=0.03 and p=0.03) and BCL-2 with high peripheral blast count (p=0.009). MDR1 expression was significantly associated with the expression of immature stem cell marker CD34 (p=0.002). In ALL, significant association was found between LRP gene and female sex (p<0.0001), LRP and B-ALL patients (p=0.04) and LRP and BCR/ABL positive patients (p=0.004). High expression of MDR1 and BCL-2 in AML and MRP1 gene in ALL was associated with response to induction chemotherapy (p=0.001, p=0.02 and p=0.007 respectively). These results showed the potential clinical relevance of MDR1, MRP1 and BCL-2 in adult patients with acute leukemia in the context of induction chemotherapy.

Community acquisition of β-lactamase producing Enterobacteriaceae in neonatal gut

BackgroundCommensal flora constitutes a reservoir of antibiotic resistance. The increasing variety of β-lactamases and the emergence of Carbapenem resistant Enterobacteriaceae (CRE) in community, raise concerns regarding efficacy of β-lactams. It is important to know the exact load of antibiotic resistance in the absence of any antibiotic selection pressure including via food and water.In the present study gut colonization in neonates with no direct antibiotic pressure was used as a model to evaluate β-lactam resistance in the community.ResultsIn this prospective study, 75 healthy, vaginally delivered, antibiotic naive, breast fed neonates were studied for gut colonization by Extended spectrum β-lactamases (ESBL), AmpC β-lactamases hyperproducing Enterobacteriaceae and CRE on day 0, 21 and 60. Total 267 Enterobacteriaceae were isolated and E.coli was the predominant flora. ESBL, AmpC and coproduction was seen in 20.6%, 19.9% and 11.2% isolates respectively. ESBL carriage increased threefold from day 1 to 60 showing predominance of CTX-M group 15 (82.5%), ampC genes were heterogeneous. Colonization with CRE was rare, only one baby harboured Enterobacter sp positive for kpc-2. The reservoirs for these genes are likely to be mother and the environment.ConclusionsData strongly suggests that in absence of any antibiotic pressure there is tremendous load of antibiotic resistance to β-lactam drugs. Wide spread presence of ESBL and AmpC can drive rapid emergence and dissemination of CRE. This is the first report from India which depicts the smaller picture of true antibiotic pressure present in the Indian community.

Association of interleukin-1β -511 C/T polymorphism with tobacco-associated cancer in northeast India: a study on oral and gastric cancer.

The IL-1β -511 C/T polymorphism is associated with increased IL-1 production and with increased risk of developing cancers. In this study, 251 patients (125 with gastric cancer [GC] and 126 with oral cancer [OC]) and 207 normal controls from northeast (NE) India were genotyped for the IL-1β -511 C/T polymorphism by PCR-restriction fragment length polymorphism (RFLP) and sequencing. Analysis of results showed betel-quid chewing to be a major risk factor (OR = 2.01, 95% CI = 1.05-3.87; P = 0.035) for OC. Inheritance of the IL-1β -511 CT or TT resulted in a 2.6- to 3.05-fold increase in the risk of developing OC relative to that of participants who possessed the reference genotype (OR = 2.57, 95% CI = 1.06-6.22; P = 0.036 and OR = 3.05, 95% CI = 1.22-7.63; P = 0.017), after adjusting for potential confounders. The dominant genetic model also confirmed the presence of the T allele as a significant risk factor for OC (OR = 2.72, 95% CI = 1.15-6.42; P = 0.02). In GC, interaction of the CT genotype with tobacco and betel-quid chewing habits conferred a significant 78% and 89% reduced risk of cancer, respectively. In conclusion, for the NE Indian population, the IL-1β -511 CC and CT genotypes were significantly associated with increased risk of OC. However, the interaction of the CT genotype with risk habits may play a preventive role for GC but not for OC.

Association of androgen receptor, prostate-specific antigen, and CYP19 gene polymorphisms with prostate carcinoma and benign prostatic hyperplasia in a north Indian population

The genes involved in androgen pathway and metabolism have been reported to contribute considerably to prostate carcinoma (CaP) risk. The present study investigated the association of androgen receptor (AR), prostate-specific antigen (PSA or KLK3), and cytochrome P450 (CYP19) gene polymorphisms in CaP (n=105) and benign prostatic hyperplasia (BPH) (n=120) in comparison to normal healthy controls (n=106) in an Indian population. We also evaluated the functional consequences of these gene variants on AR and PSA mRNA expression. Significant association of short AR CAG repeats (≤24) with risk of CaP (odds ratios [OR]=2.98, p<0.001) and BPH (OR=1.96, p=0.01) was observed; however, CYP19 gene polymorphism was not found to be associated with disease phenotype (p>0.05). PSA G-158A SNP was found to be significantly associated with risk of CaP (AA: OR=2.68, p=0.016 and GA: OR=2.07, p=0.018) p-trend 0.031 and BPH (AA: OR=3.46, p<0.001 and GA: OR=2.47, p=0.03) p-trend 0.009, respectively. PSA G-158A genotype independently increased the risk of developing BPH (OR=16.37, p<0.001), irrespective of AR CAG repeat length. Using quantitative real-time polymerase chain reaction, we found a significant upregulation of AR and PSA mRNA expression in CaP comparison to BPH. While short AR CAG (≤24) repeats were associated with higher AR mRNA expression in CaP (p=0.002), the PSA SNP did not correlate with its mRNA expression. Interestingly, significantly higher risk estimates for CaP were observed for the combined analysis of short AR CAG and CYP19 genotypes (A2A2) (OR=7.18, p<0.001) or A2A3 (OR=7.60, p=0.004). Our results suggest significant association of androgen signaling gene polymorphisms with risk of CaP and BPH and provide evidence for a putative functional role of AR CAG repeat in regulating its mRNA expression and warrant the need of larger studies in the Indian population to confirm our results.

Characterization of non-classical quinolone resistance in Salmonella enterica serovar Typhi: Report of a novel mutation in gyrB gene and diagnostic challenges

Objective To establish the relative importance of Salmonella enterica serovar Typhi with non-classical quinolone resistance. Methods Eight hundred and ninety-one isolates of S. Typhi, isolated between 2004 and 2011, were tested for antibiotic susceptibility determination using disc diffusion and E-test. The mechanisms of fluoroquinolone resistance were studied in a sub-set of the NALS (nalidixic acid susceptible) isolates by wave nucleic acid fragment analysis of PCR products from gyrA, gyrB, parC and parE and from the plasmid borne determinants: qnrA,B,S; aac(6′)-Ib-cr and qepA. To assess genetic relatedness multi-locus variable number tandem repeat analysis was carried out using five loci. Results Eighty isolates with a nalidixic acid MIC of <32 mg/L (NALS) and a ciprofloxacin MIC of >0.064 mg/L CIPI (ciprofloxacin reduced susceptibility) were found. In 36 NALS CIPI isolates two distinct genotypes were identified when compared with 16 susceptible controls: Group B (n = 34), mutation in gyrB at codon 464, NAL MIC of 3–12 mg/L and CIP MIC of 0.064–0.5 mg/L.; and Group C, mutation in gyrA at codon 83 (n = 2) NAL MIC of 16 mg/L and CIP MIC of 0.25–0.38 mg/L. Group B isolates were found in different strain backgrounds as defined by MLVA. Conclusion The use of nalidixic acid to screen for reduced susceptibility to fluoroquinolones in S. Typhi misses CIPI-NALS isolates, an established phenotype in India.

Detection of mutations in gyrB using denaturing high performance liquid chromatography (DHPLC) among Salmonella enterica serovar Typhi and Paratyphi A

Background Fluoroquinolone resistance is mediated by mutations in the quinolone-resistance determining region (QRDR) of the topoisomerase genes. Denaturing high performance liquid chromatography (DHPLC) was evaluated for detection of clinically important mutations in gyrB among Salmonella. Methods Salmonella Typhi and S. Paratyphi A characterised for mutation in QRDR of gyrA, parC and parE were studied for mutation in gyrB by DHPLC and validated by sequencing. Results The DHPLC analysis was able to resolve the test mutant from isolates with wild type gyrB and distinguished mutants from other mutant by peak profile and shift in retention time. Three sequence variants were detected at codon 464, and a novel mutation Ser→Thr was also detected. gyrB mutation was associated with non classical quinolone resistance (NALS-CIPDS) in 34 isolates of S. Typhi only and was distinct from classical quinolone resistance associated with gyrA mutations (NALR-CIPDS). Conclusions DHPLC is effective for the detection of mutation and can reduce the need for sequencing to detect clinically significant gyrB mutations. GenBank accession nos KF993966, KF993965 and KF993964.