Detection of PCR inhibitors in cervical specimens by using the AMPLICOR Chlamydia trachomatis assay

Abstract

To determine the 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؇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 specimens 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 >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.

Use of Ligase Chain Reaction with Urine versus Cervical Culture for Detection of Chlamydia trachomatis in an Asymptomatic Military Population of Pregnant and Nonpregnant Females Attending Papanicolaou Smear Clinics

“…This value is of the same order of magnitude as that reported by others for inhibition in urine specimens (2.6 and 1.8%) for amplified testing (3,15). Most investigators now believe that inhibitors to amplification exist for both urine and cervical specimens (3,15,35). A combination of heat treatment (95°C for 10 min) and 10-fold dilution of the processed specimen reduced inhibition of PCR from 19 to 4% in one study (35).…”

“…It is possible that freezing and thawing of these processed urine specimens reduced or destroyed some LCR inhibitors. Freezing and thawing reduced the inhibition from 19 to 16% in one study (35). Additionally, the experiment which tested the archived urine of 65 pregnant women demonstrated only three (4.6%) inhibited specimens.…”

“…Most investigators now believe that inhibitors to amplification exist for both urine and cervical specimens (3,15,35). A combination of heat treatment (95°C for 10 min) and 10-fold dilution of the processed specimen reduced inhibition of PCR from 19 to 4% in one study (35). The pH of the cervical mucosa was partly correlated with inhibitors (35).…”

Detection of cutaneous herpes simplex virus infections by immunofluorescence vs. PCR

“…Also the correct amplification procedure should be proven for every individual PCR tube as PCR inhibition can occur. 16,17 This can easily be accomplished with internal controls. Internal controls, or mimics, contain the same primer sequences as the target, but a different intermediate sequence and can thus be differentiated from the target.…”

Performance of a commercial polymerase chain reaction test for endocervical Chlamydia trachomatis infection in a university hospital population

“…Inhibitors of the PCR process have been described in cervical specimens at a frequency of 19% by Verkooyen et al, 28 using the method of detection we used. Using more sophisticated methods, Bass et al 20 found inhibitors in 4% of their specimens.…”