By: 1 March 2010

Laboratory detection of Human papillomavirus (HPV), Chlamydia trachomatis and Neisseria gonorrhoeae in liquid-based cervicovaginal cytology specimens is now based on identification of the DNA sequences unique to these infectious agents. However, current commercial test kits rely on nucleotide probe hybridization to determine DNA sequences, which may lead to diagnostic errors due to cross-reactivity. The aim of this study was to find a practical approach to perform automated Sanger DNA sequencing in clinical laboratories for validation of the DNA tests for these three infectious agents.

A crude proteinase K digestate of 5% of the cells collected in a liquid-based cervicovaginal cytology specimen was used for the detection of DNA molecules specific for HPV, C trachomatis and N gonorrhoeae, and for preparation of materials suitable for direct automated DNA sequencing. Several sets of commercially available polymerase chain reaction (PCR) primers were used to prepare nested PCR amplicons for direct DNA sequencing.

Some variants of HPV-16 and HPV-31 were found to share an at least 34-base long sequence homology downstream of the GP5+ binding site, and all HPV-6 and HPV-11 variants shared an upstream 34-base sequence including part of the GP5+ primer. Accurate HPV genotyping frequently required more than 34-bases for sequence alignments to distinguish some of the HPV genotype variants with closely related sequences in this L1 gene hypervariable region. Using the automated Sanger DNA sequencing method for parallel comparative studies on split samples and to retest the residues of samples previously tested positive for C trachomatis and/or for N gonorrhoeae, we also found false-negative and false-positive results as reported by two commercial nucleic acid test kits.

Identification of a signature DNA sequence by the automated Sanger method is useful for validation of HPV genotyping and for molecular testing of C trachomatis and N gonorrhoeae in liquid-based cervicovaginal Papanicolaou (Pap) cytology specimens for clinical laboratories with experience in molecular biology to increase the specificity of these DNA-based tests. However, even a highly specific test for high-risk HPV genotyping may have unacceptably low positive predictive values for precancer lesion in populations with a low cervical cancer prevalence rate.

Human papillomavirus (HPV), Chlamydia trachomatis and Neisseria gonorrhoeae are the causative agents for the three most common sexually transmitted infections in women. Newly introduced laboratory diagnostic procedures for these infectious agents are mostly nucleic acid-based, relying on detection and identification of a DNA sequence specific for the infectious agent with or without DNA replication (amplification) by polymerase chain reaction (PCR). The tests incorporating PCR amplification in the procedure, often referred to as nucleic acid amplification (NAA) tests, are extremely sensitive, capable of detecting a single copy of target DNA. The result of a DNA-based test may be validated by identifying a signature sequence of the target DNA and used for accurate genotyping if HPV DNA is the target1-8.

The commercial NAA tests for C trachomatis and N gonorrhoeae, all relying on probe hybridization for DNA sequence determination, are claimed to have a sensitivity of 85% and a specificity of 97-99%, depending on the evaluating method chosen for comparison9. These tests tend to generate unacceptably low positive predictive value (PPV)10 in low prevalence populations. As a result, the Centers for Disease Control and Prevention (CDC) has issued recommendations that an additional test with different method or with the same method be performed to confirm a positive NAA test for C trachomatis and N gonorrhoeae to reduce the possibility of false-positive results that may have adverse medical, social and psychological impacts on the patient, but also cautions that such a supplementary test result might itself be falsely negative9. Clinical laboratories often find these tests are associated with 5.3% of initially C trachomatis-positive results and 10.7% of initially N gonorrhoeae-positive results that cannot be confirmed by repeated testing of the original sample11.

In the past few years the cost of performing automated DNA sequencing has decreased considerably. This article gives a brief summary on the experience of applying this research tool in HPV genotyping and in the identification of C trachomatis and N gonorrhoeae DNA in liquid-based Pap cytology specimens to serve the physicians who want unequivocal evidence to validate each test result.

Clinical Materials
The clinical samples used for the HPV study were 2,020 alcohol-preserved liquid-based cervicovaginal Pap cytology specimens (ThinPrep or Surepath) routinely submitted by 2 private gynecologists who provided general obstetric and gynecologic care to local residents in Milford, Connecticut. Milford has a rural and suburban population of 50,000+, in which 93.6% of the residents are non-Hispanic whites according to the latest available census12. The incidence of cervical cancer in the 2,020 patients selected for this study was considered to be representative of or below that in the state of Connecticut in which the cervical cancer rate is 6.8 per 100,000 women13. Publication of the laboratory data with blinded patient identities was approved by the Milford Hospital IRB.

The cervicovaginal specimens were collected from women below age 30 who had a Pap cytology finding of atypical squamous cells of undetermined significance (ASCUS) or more severe, and from women 30 years and older in conjunction with Pap cytology screening when the clinical specimens were submitted for HPV testing with or without concomitant request for C trachomatis or N gonorrhoeae testing (Figure 1). The Pap cytology slides were screened by a commercial cytology laboratory and all abnormal slides reviewed by the 2 author pathologists for final classification.

Figure 1. Age distribution of 2,020 private patients tested for HPV in Milford, CT.

The cases classified as “ASCUS favoring a reactive process” under the Bethesda system14 were grouped with the normal cases under less than ASCUS (<ASCUS) for the purpose of analysis in this report. Samples submitted in the liquid-based Pap cytology vial for C trachomatis and/or N gonorrhoeae testing were generally from “asymptomatic” women 15 to 65 years old regardless of Pap cytology findings. A total of 510 liquid-based Pap cytology specimens were tested for C trachomatis DNA and a total of 507 tested for N gonorrhoeae DNA.

Primary and Nested PCR
The cellular debris derived from 5% of a liquid-based Pap cytology collection was pelleted for primary and nested PCR amplification of HPV, C trachomatis or N gonorrhoeae DNA without DNA extraction or purification, as previously described15-17.

The residues of 26 endocervical samples which had been tested positive by the BD Probe Tec™ ET Chlamydia trachomatis and Neisseria gonorrhoeae Amplified DNA Assays (Becton, Dickinson, Sparks, MD) were retested by DNA sequencing for comparison16.

All positive nested PCR products were validated by on-line Basic Local Alignment Search Tool (BLAST) algorithms for final validation of the signature sequence.

The numbers of cases analyzed did not include 38 cases which were considered inadequate for failed PCR amplification of a human