[Evaluation of Real-Time Polymerase Chain Reaction in Pneumocystis jirovecii Laboratory Diagnosis]
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[Evaluation of Real-Time Polymerase Chain Reaction in Pneumocystis jirovecii Laboratory Diagnosis]
Pneumocystis jirovecii is a human species-specific and cause fatal infections such as P.jirovecii pneumonia (PCP) in immunocompromised people. Although direct microscopy is the gold standard in the diagnosis of microorganisms, molecular methods such as polymerase chain reaction (PCR), which is required in patients with immunosuppressive virus non-human immunodeficiency (HIV) with low P.jirovecii load. In this study, we aimed to evaluate the value of real-time PCR (RT-PCR) in laboratory diagnosis P.jirovecii. Bronchoalveolar lavage (BAL) specimens from 658 patients sent to Dokuz Eylul University Medical Center Hospital Parasitology Laboratory on suspicion of PCP were included in this study.
BAL fluids were evaluated for identification P.jirovecii mitochondrial genes coding ribosomal large subunit (mtLSUrRNA) using RT-PCR. In addition, staining and Gomori’s methenamine silver (GMG) staining tests that are applied to all samples and nested PCR (n-PCR) assay was applied to the positive samples detected by real time PCR. Ninety-two (14.3%) of the samples were positive by RT-PCR. Of the 92 patients, 85 (92.4%) with n-PCR positive. Only seven of the specimens have P.jirovecii cysts and trophozoites with microscopic examination.
Mean threshold cycle (CT) values Rt-PCR positive patients was 29.7 (18.17 ≤ CT ≤ 37.96). P.jirovecii load in these patients was calculated as 2.6 x 101 to 6.15 x 107 copies / ml. The difference between the average value of CT positive and negative results of n-PCR was statistically significant (p <0.01). Rt-PCR CT values of samples with positive microscopy; 18.2, 20.9, 22.2, 24.3, 24.7, 26.5, 29.7. The difference between CT means of samples with positive and negative microscopy was statistically significant (p <0.05). When positive patients are grouped according to their diagnosis; Lowest average CT values (CTmean = 24.8) was found in HIV-positive patients.
On the other hand, the values of CT was found to be significantly lower in patients with organ transplants (CTmean = 26.15) and in the group of patients with collagen-vascular-inflammatory (CTmean = 27.8). This study shows that RT-PCR is an effective method in the diagnosis P.jirovecii in the laboratory. N-conventional PCR method was found to be more successful than the RT-PCR in the presence of a very low density of organisms; Direct microscopy is generally found to be positive in a sample with a higher load than P.jirovecii.
[Evaluation of Real-Time Polymerase Chain Reaction in Pneumocystis jirovecii Laboratory Diagnosis]
Droplet Digital Validation Polymerase Chain Reaction for Salmonella spp. count
Salmonellosis is a foodborne illness caused by Salmonella spp. Although cell culture is the gold standard for the identification, molecular methods were validated to be an alternative, because of their speed, selectivity, and specificity. A trickle of simplex and duplex digital polymerase chain reaction (ddPCR) based method for the identification and quantification of Salmonella using the TTR, the gene sequences INVA, Hila, spaQ, and SIIA passed.
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This method has a high specificity, working hose between 8 and 8000 cp / uL reaction ddPCR, limit of detection of 0.5 copies / uL, and precision ranges between 5 and 10% measured as the standard deviation of repeatability. Relative standard uncertainty of measurement is between 2 and 12%. This tool will improve food safety in products of national consumption and will improve the competitiveness in the trade of agricultural products.
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