Detection of PCR inhibitors in cervical specimens by using the AMPLICOR Chlamydia trachomatis assay
To determine that susceptibility of AMPLICOR Chlamydia trachomatis PCR to inhibitory factors possibly present in cervical specimens, we obtained cervical specimens from 200 gynecology patients attending our outpatient clinic. The prevalence of C. trachomatis infection was 4.1%, as determined by cell culture. All AMPLICOR specimens were tested in one procedure as described by the manufacturer, and after the specimen was spiked with C. trachomatis, several other pretreatment protocols were used. Complete inhibition of the PCR was observed in 38 (19%) cervical specimens. Heat treatment at 95 degrees C, freeze-thawing, or 10-fold dilution of the samples reduced the initial inhibition to 9, 16, or 9%, respectively. A combination of heat treatment and 10-fold dilution reduced the inhibition to 4% of the samples. A second specimen type (swabs inoculated in 0.2 M sucrose phosphate buffer [2SP]) was also evaluated. A 10-fold dilution of the spiked 2SP specimen resulted in an inhibition rate of 6%, which was comparable to that obtained by centrifugation of the 2SP specimen prior to processing. Furthermore, it was shown that the inhibition was not correlated with blood contamination. Processing the specimens on the day of collection or the day after resulted in a higher inhibition rate than did delayed processing (27.6 versus 15.5%, respectively). An inverse correlation was found between the concentration of C. trachomatis added to the sample and the rate of inhibition observed. The inhibition was partly correlated with the pH of the cervical mucosa. Decreased inhibition was found at pH values of > or = 7.5. The effects of blood, pH, and delay in processing were all evaluated by using the AMPLICOR specimen. We conclude that the susceptibility of AMPLICOR C. trachomatis PCR to inhibiting factors in cervical specimens can be significantly reduced if the pretreatment procedure includes heat treatment or the use of 2SP transport medium. Also, a 10-fold dilution of the clinical specimen followed by heat treatment will largely prevent the inhibition of this PCR.
|Keywords||Cervix Uteri/*microbiology, Chlamydia Infections/diagnosis/microbiology, Chlamydia trachomatis/*genetics/*isolation & purification, DNA, Bacterial/analysis/genetics, Evaluation Studies, Female, Heat, Humans, Polymerase Chain Reaction/*methods/statistics & numerical data, Sensitivity and Specificity, Uterine Cervical Diseases/diagnosis/microbiology|
Verkooyen, R.P.A.J., Luijendijk, A., Huisman, W.M., Goessens, W.H.F., Kluytmans, J.A.J.W., van Rijsoort-Vos, J.H., & Verbrugh, H.A.. (1996). Detection of PCR inhibitors in cervical specimens by using the AMPLICOR Chlamydia trachomatis assay. Journal of Clinical Microbiology. Retrieved from http://hdl.handle.net/1765/8636