Microbiological testing of pharmaceuticals and cosmetics in Egypt.

BACKGROUND: Microbial contamination of pharmaceuticals poses a great problem to the pharmaceutical manufacturing process, especially from a medical as well as an economic point of view. Depending upon the product and its intended use, the identification of isolates should not merely be limited to the United States Pharmacopeia (USP) indicator organisms. RESULTS: Eighty-five pre-used non-sterile pharmaceuticals collected from random consumers in Egypt were examined for the eventual presence of bacterial contaminants. Forty-one bacterial contaminants were isolated from 31 of the tested preparations. These isolates were subjected to biochemical identification by both conventional tests as well as API kits, which were sufficient for the accurate identification of only 11 out of the 41 bacterial contaminants (26.8%) to the species level. The remaining isolates were inconclusively identified or showed contradictory results after using both biochemical methods. Using molecular methods, 24 isolates (58.5%) were successfully identified to the species level. Moreover, polymerase chain reaction (PCR) assays were compared to standard biochemical methods in the detection of pharmacopoeial bacterial indicators in artificially-contaminated pharmaceutical samples. CONCLUSION: PCR-based methods proved to be superior regarding speed, cost-effectiveness and sensitivity. Therefore, pharmaceutical manufacturers would be advised to adopt PCR-based methods in the microbiological quality testing of pharmaceuticals in the future.

Recombinant expression of the alternate reading frame protein (ARFP) of hepatitis C virus genotype 4a (HCV-4a) and detection of ARFP and anti-ARFP antibodies in HCV-infected patients.

HCV is a single-stranded RNA virus with a single open reading frame (ORF) that is translated into a polyprotein that is then processed to form 10 viral proteins. An additional eleventh viral protein, the alternative reading frame protein (ARFP), was discovered relatively recently. This protein results from a translational frameshift in the core region during the expression of the viral proteins. Recombinant expression of different forms of ARFP was previously done for HCV genotypes 1 and 2, and more recently, genotype 3. However, none of the previous studies addressed the expression of ARFP of HCV genotype 4a, which is responsible for 80 % of HCV infections in the Middle East and Africa. Moreover, the direct detection of the ARFP antigen in HCV-infected patients was never studied before for any HCV genotype. In the present study, recombinant ARFP derived from HCV genotype 4a was successfully expressed in E. coli and purified using metal affinity chromatography. The recombinant ARFP protein and anti-ARFP antibodies were used for detection of ARFP antigen in patients' sera, employing competitive enzyme-linked immunosorbent assay (ELISA) procedures. Furthermore, the recombinant antigen was also used to detect and quantify anti-ARFP antibodies in HCV-infected Egyptian patients at different stages of pegylated interferon/ribavirin therapy, using an ELISA assay. The ARFP antigen was detectable in 69.4 % of RNA-positive sera, indicating that ARFP antigen is produced during the natural course of HCV infection. In addition, significant levels of anti-ARFP antibodies were present in 41 % of the serum samples tested. The important diagnostic value of the recombinant ARFP antigen was also demonstrated.

Potential Co-prevalence of Plasmid-Mediated Quinolone Resistance Determinant qepA and 16S rRNA Methylase rmtB among E.coli Clinical Isolates from Alexandria- Egypt.

BACKGROUND: Fluoroquinolone-resistant Gram-negative pathogens have been increasingly reported from most regions of the world over the last decade. A new plasmid-mediated fluoroquinolone efflux pump gene (qepA) is known to be associated with the 16S rRNA methylase gene (rmtB) that confers resistance to aminoglycosides. AIM: To investigate the potential co-prevalence of qepA and rmtB genes in Escherichia coli (E. coli) clinical isolates collected from Egyptian medical facilities. MATERIAL AND METHODS: A total of 225 non-duplicate E. coli isolates were collected between 2008 and 2009 from two hospitals in Alexandria. Isolates were initially tested for their antibiotic susceptibility by disc diffusion method. Isolates exhibited quinolone and aminoglycosides co-resistance profile were screened for the presence of qepA and rmtB genes. The effect of efflux pump inhibitor, phenylarginine-beta-naphthylamide (PAßN) on the minimum inhibitory concentration (MIC) of ciprofloxacin, levofloxacin and gentamicin against these strains was tested and log activity index was calculated. Using checkerboard titration method, the combinations of gentamicin with ciprofloxacin against the strains harboring qepA and rmtB genes were tested and the fractional inhibitory concentrations (FIC) were calculated. RESULTS: Forty-five E. coli isolates exhibited quinolone and aminoglycosides co-resistance profile. Of them, two E. coli isolates were positive for qepA, and three harbored rmtB genes. No association between both genes was detected. The calculated log activity index revealed a reduction in MIC of the fluoroquinolones with PAßN but not of gentamicin. FIC calculated here for gentamicin/ciprofloxacin combinations reflected either antagonism or indifference against the strains harboring qepA and rmtB genes. CONCLUSION AND RECOMMENDATIONS: qepA as well as rmtB genes-carrying E. coli strains could become a greater nosocomial infection problem with appropriate foci of selective pressure. Therefore, public health support for active surveillance for plasmid mediated fluoroquinolones, aminoglycosides resistance determinants among clinical E. coli isolates should be encouraged. In addition, the effect of efflux pumps needs to be considered in the design of future antibiotics as their synergistic role may pave the way to novel combination therapies that could be used against these strains.