Comparison of different phenotypic methods of detection of methicillin resistance in staphylococcus aureus with the molecular detection of mec-a gene

OBJECTIVE: To evaluate accuracy, cost-effectiveness and ease to perform different phenotypic methods i.e. Cefoxitin 30 microg disc, Oxacillin 1microg disc and Oxacillin agar screening plate (6microg/ml ) for early and accurate identification of MRSA by comparing with the detection of mec-A gene in our clinical isolates. DESIGN: A comparative study. PLACE AND DURATION OF STUDY: Clinical samples submitted in the Clinical Microbiology Laboratory at Aga Khan University Hospital, Karachi, from 1st August to 31st October 2006. MATERIAL AND METHODS: Out of 200 clinical samples, conventional Polymerase chain reaction (PCR) was done on 62 pure biochemically identified S. aureus isolates for mec-A gene detection. Phenotypic methods for detecting methicillin sensitivity (Cefoxitin 30 microg disc, Oxacillin 1 microg disc and Oxacillin agar screening plate) were also used according to the recommended incubation time, duration and temperature on the same isolates. RESULTS: Out of 62 isolates of S. aureus, mec-A gene were detected (MRSA) in 32, whereas 30 were mec-A gene negative (MSSA). Cefoxitin disc and agar screening plate correctly identify all MRSA isolates with the sensitivity and specificity of 100%. Single isolate was false, positively detected as sensitive with Oxacillin 1microg disc, due to which, the sensitivity and negative predictive value of this method were reduced to 96.9% and 96.8% respectively, while positive predictive value and specificity remained 100%. CONCLUSION: Comparing different phenotypic methods for MRSA screening in routine microbiology laboratory, Cefoxitin disc and Oxacillin agar screening has better sensitivity and specificity comparative to Oxacillin disc. However, Cefoxitin disc can be preferred especially for small laboratories because it is easy to perform, do not require special technique and media preparation is consequently more cost-effective

P450 Oxidoreductase deficiency: Analysis of mutations and polymorphisms.

Cytochrome P450 oxidoreductase (POR) is required for metabolic reactions of steroid and drug metabolizing cytochrome P450 proteins located in endoplasmic reticulum. Mutations in POR cause a complex set of disorders resembling combined deficiencies of multiple steroid metabolizing enzymes. The P450 oxidoreductase deficiency (PORD) was first reported in patients with symptoms of defects in steroidogenic cytochrome P450 enzymes and ambiguous genitalia, and bone malformation features resembling Antley-Bixler syndrome. POR is now classified as a separate and rare form of congenital adrenal hyperplasia (CAH), which may cause disorder of sexual development (DSD). Since the initial description of PORD in 2004, a large number of POR mutations and polymorphisms have been described. In this report we have performed computational analysis of mutations and polymorphisms in POR linked to metabolism of steroids and xenobiotics and pathology of PORD from the reported cases. The mutations in POR that were identified in patients with disruption of steroidogenesis also have severe effects on cytochrome P450 proteins involved in metabolism of drugs. Different variations in POR show a range of diverse effects on different partner proteins that are often linked to the location of the particular variants. The variations in POR that cause defective binding of co-factors always have damaging effects on all partner proteins, while the mutations causing subtle structural changes may lead to altered interaction with partner proteins and the overall effect may be different for each individual partner. Computational analysis of available sequencing data and mutation analysis shows that Japanese (R457H), Caucasian (A287P) and Turkish (399-401) populations can be linked to unique founder mutations. Other mutations identified so far were identified as rare alleles or in single isolated reports. The common polymorphism of POR is the variant A503V which can be found in about 27% of alleles in general population but there are remarkable differences among different sub populations