الفهرس 
LITERATURE REVIEWOnly 14 pages are availabe for public view
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Abstract
Monoplex and multiplex PCR-based assays were developed and compared with standard conventional methods for rapid detection of specified indicator pathogens, Pseudomonas aeruginosa, Staphylococcus aureus, E. coli, Salmonella spp. and Candida albicans, in nonsterile pharmaceutical preparations. A total of 280 non-sterile pharmaceutical preparations obtained either from different pharmaceutical companies or purchased randomly from various retail pharmacies in Egypt were subjected to microbial limit testing using standard conventional techniques. These samples comprised 165 preparations for oral use and 115 preparations for topical use. Method suitability testing was conducted and any antimicrobial property present in the product was neutralized before routine testing. Attempts were also made to identify other contaminants.
Microbial contaminants were recovered from 27.6 % of the tested products with oral preparations showing a higher incidence of contamination (33.75%) in comparison to topical preparations (19.1%). The level of fungal contamination was less than that of bacteria (11.3% and 20% respectively). For bacterial counts, six products (3 syrups, 2 tablets and 1 cream) were found to exceed the United States Pharmacopeia (USP) specified limits whilst for fungal counts, three syrups exceeded the USP specified limits. Regarding the isolates, 8 were USP indicator pathogens (15%); five S. aureus isolates (5.5%) were recovered from a cream, a gel, an ointment, a tablet and a syrup; one Pseudomonas aeruginosa isolate (1.1%) was recovered from a capsule; one E. coli isolate (1.1%) and one Candida albicans isolate (1.1%) were recovered from syrups. No Salmonella spp. was recovered from any tested preparation. These eight isolates were identified by molecular techniques through the application of PCR using species specific primers for each suspected pathogen. Specific primers were selected for
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amplification of nuc gene of S. aureus, invA gene of Salmonella spp., 16S RNA gene of E. coli, oprL gene of Pseudomonas aeruginosa and 25S rRNA gene of Candida albicans. Each isolate together with the relevant positive control, produced detectable DNA bands with similar migration on the gel and of the expected molecular size. Specificity of the PCR assays was investigated and demonstrated where amplicons of the expected sizes were produced only in the presence of the respective DNA templates. Sensitivity of the assays was evaluated by the use of extracted pure genomic DNA, whole cells, and pharmaceutical preparations artificially inoculated with different dilutions of the indicator pathogens.
The detection limit of monoplex PCR assays for the topical microbial targets was estimated at 100 fg purified DNA for Pseudomonas aeruginosa and 1 fg purified DNA for both Staphylococcus aureus and Candida albicans and 10¹ CFU/ml for Pseudomonas aeruginosa and 10⁰ CFU/ml for both Staphylococcus aureus and Candida albicans, in pure cultures and artificially inoculated topical pharmaceutical preparation. The detection limits of multiplex PCR assays for the simultaneous detection of the three pathogens were estimated at 10¹ CFU/ml for Pseudomonas aeruginosa and 10⁰ CFU/ml for Staphylococcus aureus and Candida albicans, in mixed cultures and artificially inoculated pharmaceutical preparations, after two successive rounds of PCR.
Similarly, the detection limit of monoplex PCR assays for the oral microbial targets was estimated at 10 fg purified DNA for E. coli and 1 fg purified DNA for Salmonella spp. and 10⁰ CFU/ml for both pathogens in pure cultures and artificially inoculated pharmaceutical preparations. The detection limit of multiplex PCR assays, for the simultaneous detection of the two oral pathogens, was estimated at 10⁰ CFU/ml for both pathogens, in mixed cultures and artificially inoculated pharmaceutical preparations.
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Thus, there was a 100% correlation between conventional detection methods and the PCR assays however, the standard methods required 5–6 days, whereas PCR detection and identification was completed in 6-8 h. Accordingly, we can conclude that PCR assays provide specific, reliable results and allow for cost-effective, rapid detection of indicator pathogens in nonsterile pharmaceutical products.