[Show abstract][Hide abstract] ABSTRACT: The emergence of a novel coronavirus (CoV) as the cause of severe acute respiratory syndrome (SARS) catalyzed the development of rapid diagnostic tests. Stool samples have been shown to be appropriate for diagnostic testing for SARS CoV, although it has been recognized to be a heterogeneous and difficult sample that contains amplification inhibitors. Limited information on the efficiency of extraction methods for the purification and concentration of SARS CoV RNA from stool samples is available. Our study objectives were to determine the optimal extraction method for SARS CoV RNA detection and to examine the effect of increased specimen volume for the detection of SARS CoV RNA in stool specimens. We conducted a multicenter evaluation of four automated and four manual extraction methods using dilutions of viral lysate in replicate mock stool samples, followed by quantitation of SARS CoV RNA using real-time reverse transcriptase PCR. The sensitivities of the manual methods ranged from 50% to 100%, with the Cortex Biochem Magazorb method, a magnetic bead isolation method, allowing detection of all 12 positive samples. The sensitivities of the automated methods ranged from 75% to 100%. The bioMérieux NucliSens automated extractor and miniMag extraction methods each had a sensitivity of 100%. Examination of the copy numbers detected and the generation of 10-fold dilutions of the extracted material indicated that a number of extraction methods retained inhibitory substances that prevented optimal amplification. Increasing the volume of sample input did improve detection. This information could be useful for the extraction of other RNA viruses from stool samples and demonstrates the need to evaluate extraction methods for different specimen types.
Journal of Clinical Microbiology 09/2006; 44(8):2681-8. · 4.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background: Self-collected vaginal swabs [VS] tested by nucleic acid amplification tests, are candidates for C. trachomatis screening programs to identify and treat infected asymptomatic women. The objectives were to evaluate the performance of the Aptima Combo 2 [AC2] (Gen-Probe Inc.) and Amplicor CT [AMP] (Roche) tests on self-collected VS to detect lower genital tract CT-infections in women.
Methods: We collected a cervical swab [CS], VS and first void urine [FVU] from 300 women. Each sample was processed within 48 hours into the AC2 and AMP assays using published protocols. A VS protocol was established for AMP following their CS method. An infected patient was determined to be positive in more than one specimen or when a single specimen was positive in both assays.
Results: There were 67 [22.3%] women infected with CT. AC2 detected 100% and AMP identified 67.2% by VS. The results for CS were 95.5% by AC2 and 89.4% by AMP. For VS, FVU and CS the AC2 assay confirmed 98.1, 94.3 and 90.6% of the AMP-positives but the AMP confirmed only 52.3, 46.5 and 47.7% of the AC2-positives. Using the AMP amplification control, 33 CS [11%], 27 FVU [9%] and 7 VS [2.3%] were recorded inhibitory. Only 4 samples were found inhibitory in AC2. Seven patients with inhibitory AMP samples were CT-infected in another sample. Equivocal readings were encountered in 18 specimens by AMP and 5 by AC2. Both inhibitory and equivocal samples required retesting.
Conclusions: Significantly more CT-infected women were identified by AC2 testing of any of the three specimen types, than by AMP testing [p<0.001]. VS testing by AC2 identified the greatest number of infections. Using AMP to confirm AC2 results was inadequate. Inhibition of AMP appeared to play a role in its clinical sensitivity.
Infectious Diseases Society of America 2005 Annual Meeting; 10/2005