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Literature reviews > Articles for review > Berg et al. Reliability of the Amplicor internal ... |
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A positive internal control signal but inhibition of the C. trachomatis signal was obtained in urine specimens with low numbers of C. trachomatis, leading to false negative results using the Amplicor assay. Reliability of the Amplicor internal control to disclose false-negative Chlamydia trachomatis PCR results. Question Is the internal control for the Amplicor C. trachomatis PCR assay preferentially amplified in samples with inhibitory substances, thereby affecting the reliability of the internal control for identifying false negative results? Design Urine specimens that were negative for both the internal control and C. trachomatis by the Amplicor PCR assay were used to examine the effects of inhibitory substances on the amplification of C. trachomatis and the internal control. Participants Five hundred fourteen men less than 35 years old attending the Oslo municipal STI clinic were tested. Description of Tests and Diagnostic Standard First-void urine specimens were tested using the Amplicor C. trachomatis PCR assay (F. Hoffmann-La Roche), the LCx C. trachomatis LCR assay (Abbott Laboratories), and the Amplified C. trachomatis TMA assay (Gen-Probe).Specimens were considered inhibitory if the Amplicor internal control signal was <2.0 and the C. trachomatis signal was <0.8 (n = 4) or if the PCR result was negative and either of the other assay results was positive for C. trachomatis (n = 0).New aliquots of the 4 inhibitory specimens were processed with and without spiking with approximately 50 C. trachomatis elementary bodies per mL by centrifugation and removal of the supernatant.The spiked and non-spiked urine pellets were split and 0, 25, 50, or 100 μl/mL of urine supernatant was added back. The 8 aliquots from each specimen were analyzed by the Amplicor C. trachomatis PCR assay. Inhibitory samples were prepared artificially by adding 0.1 to 1% urea, NaCl, FeCl3, CaCl2, hemoglobin, hemin, or bilirubin dissolved in water to Amplicor PCR reaction mixtures containing approximately two copies of C. trachomatis cryptic plasmid DNA. Main Outcome Measures The effect on amplification of the internal control and of C. trachomatis of different amounts of inhibitory urine supernatant added to urine cell pellets and of known inhibitory substances added to Amplicor PCR reaction mixtures were determined. Main Results The internal control amplified in 3 of 4 of the non-spiked urine cell pellets without added supernatant.All were negative for C. trachomatis.The internal control failed to amplify in two specimens when 50 μl/mL of urine supernatant was added and in one of the specimens when 100 μl/mL of urine supernatant was added.The internal control and C. trachomatis amplified in the same 3 of 4 spiked urine cell pellets without added supernatant.The signals for both the internal control and C. trachomatis were reduced with increasing amount of urine supernatant added to the spiked pellets.However, the inhibition did not affect the amplification of the internal control as much as the C. trachomatis.In the experiments using artificial inhibitory specimens, the inclusion of NaCl gave a selective inhibition of amplification of the C. trachomatis compared to the internal control. Authors' Conclusions Selective inhibition of the C. trachomatis target and the preferential amplification of the internal control were observed when clinical and experimental urine specimens containing inhibitory substances were analyzed using the Amplicor C. trachomatis PCR assay. The positive internal control signal may be misleading for the detection of possible false-negative results. Source of funding:Abbott laboratories, Gen-Probe, and Roche Diagnostic Systems supplied reagents and equipment. For correspondence:Einar S. Berg, SMAB, Division for Infectious Disease Control, Norwegian Institute of Public Health, PO Box 4404 Nydalen, N-0403 Oslo, Norway.E-mail address:esbe@fhi.no |
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