From Human Papillomavirus (HPV) Detection to Cervical Cancer Prevention in Clinical Practice.

The newly gained knowledge of the viral etiology in cervical carcinogenesis has prompted industrial interests in developing virology-based tools for cervical cancer prevention. Due to the long incubation period from viral infection to developing an invasive cancer, a process whose outcome is influenced by numerous life-style and genetic factors, the true efficacy of the genotype-specific human papillomavirus (HPV) vaccines in cervical cancer prevention cannot be determined for another 30 years. Most HPV DNA test kits designed to replace the traditional Papanicolaou (Pap) smears for precancer detection lack the analytical sensitivity and specificity to comprehensively detect all potentially carcinogenic HPVs and to perform reliable genotyping. The authors implemented the classic nested PCR and Sanger DNA-sequencing technology for routine HPV testing. The results showed a true negative HPV PCR invariably indicates the absence of precancerous cells in the cytology samples. However, 80.5% of single positive HPV-16 tests and 97.3% of single positive HPV-18 tests were associated with a negative or a largely self-reversible Pap cytology. Routine sensitive and reliable HPV type-specific or perhaps even variant-specific methods are needed to address the issues of persistence of HPV infection if a virology-based primary cervical screen is used to replace the Pap cytology screening paradigm.

DNA sequencing diagnosis of off-season spirochetemia with low bacterial density in Borrelia burgdorferi and Borrelia miyamotoi infections.

A highly conserved 357-bp segment of the 16S ribosomal RNA gene (16S rDNA) of Borrelia burgdorferi sensu lato and the correspondent 358-bp segment of the Borrelia miyamotoi gene were amplified by a single pair of nested polymerase chain reaction (PCR) primers for detection, and the amplicons were used as the templates for direct Sanger DNA sequencing. Reliable molecular diagnosis of these borreliae was confirmed by sequence alignment analysis of the hypervariable regions of the PCR amplicon, using the Basic Local Alignment Search Tool (BLAST) provided by the GenBank. This methodology can detect and confirm B. burgdorferi and B. miyamotoi in blood samples of patients with off-season spirochetemia of low bacterial density. We found four B. miyamotoi infections among 14 patients with spirochetemia, including one patient co-infected by both B. miyamotoi and B. burgdorferi in a winter month when human exposure to tick bites is very limited in the Northeast of the U.S.A. We conclude that sensitive and reliable tests for these two Borrelia species should be implemented in the microbiology laboratory of hospitals located in the disease-endemic areas, for timely diagnosis and appropriate treatment of the patients at an early stage of the infection to prevent potential tissue damages.

Detection of borreliae in archived sera from patients with clinically suspect Lyme disease.

The diagnoses of Lyme disease based on clinical manifestations, serological findings and detection of infectious agents often contradict each other. We tested 52 blind-coded serum samples, including 20 pre-treatment and 12 post-treatment sera from clinically suspect Lyme disease patients, for the presence of residual Lyme disease infectious agents, using nested PCR amplification of a signature segment of the borrelial 16S ribosomal RNA gene for detection and direct DNA sequencing of the PCR amplicon for molecular validation. These archived sera were split from the samples drawn for the 2-tier serology tests performed by a CDC-approved laboratory, and are used as reference materials for evaluating new diagnostic reagents. Of the 12 post-treatment serum samples, we found DNA evidence of a novel borrelia of uncertain significance in one, which was also positive for the 2-tier serology test. The rest of the post-treatment sera and all 20 control sera were PCR-negative. Of the 20 pre-treatment sera from clinically suspect early Lyme disease patients, we found Borrelia miyamotoi in one which was 2-tier serology-negative, and a Borrelia burgdorferi in two-one negative and one positive for 2-tier serology. We conclude that a sensitive and reliable DNA-based test is needed to support the diagnosis of Lyme disease and Lyme disease-like borreliosis.

Early Lyme disease with spirochetemia - diagnosed by DNA sequencing.

BACKGROUND: A sensitive and analytically specific nucleic acid amplification test (NAAT) is valuable in confirming the diagnosis of early Lyme disease at the stage of spirochetemia. FINDINGS: Venous blood drawn from patients with clinical presentations of Lyme disease was tested for the standard 2-tier screen and Western Blot serology assay for Lyme disease, and also by a nested polymerase chain reaction (PCR) for B. burgdorferi sensu lato 16S ribosomal DNA. The PCR amplicon was sequenced for B. burgdorferi genomic DNA validation. A total of 130 patients visiting emergency room (ER) or Walk-in clinic (WALKIN), and 333 patients referred through the private physicians' offices were studied. While 5.4% of the ER/WALKIN patients showed DNA evidence of spirochetemia, none (0%) of the patients referred from private physicians' offices were DNA-positive. In contrast, while 8.4% of the patients referred from private physicians' offices were positive for the 2-tier Lyme serology assay, only 1.5% of the ER/WALKIN patients were positive for this antibody test. The 2-tier serology assay missed 85.7% of the cases of early Lyme disease with spirochetemia. The latter diagnosis was confirmed by DNA sequencing. CONCLUSION: Nested PCR followed by automated DNA sequencing is a valuable supplement to the standard 2-tier antibody assay in the diagnosis of early Lyme disease with spirochetemia. The best time to test for Lyme spirochetemia is when the patients living in the Lyme disease endemic areas develop unexplained symptoms or clinical manifestations that are consistent with Lyme disease early in the course of their illness.

Increased sensitivity and specificity of Borrelia burgdorferi 16S ribosomal DNA detection.

The DNA of Borrelia burgdorferi spirochetes extracted by ammonium hydroxide was used as the template for nested polymerase chain reaction (PCR) amplification of the species-specific 16S ribosomal DNA (rDNA). The primers were those well known to be specific for signature sequence amplification of the B burgdorferi sensu lato 16S ribosomal RNA gene. The positive 293-base-pair nested PCR amplicon was subjected to routine direct automated Sanger sequencing. A 50-base sequence excised randomly from the sequencing electrophoretogram between the 2 nested PCR primer binding sites was sufficient for the Basic Local Alignment Search Tool (BLAST) analysis to validate the B burgdorferi sensu lato 16S rDNA without a reasonable doubt. Nested PCR increased the sensitivity of DNA detection by 100- to 1,000-fold. DNA sequence validation based on BLAST algorithms using the GenBank database practically eliminates any possibility of false-positive results due to molecular misidentification. This technology may be a valuable supplement to the current serologic tests for Lyme disease.

Signature sequence validation of human papillomavirus type 16 (HPV-16) in clinical specimens.

AIMS: Persistent infection indicated by detection of human papillomavirus 16 (HPV-16) on repeat testing over a period of time poses the greatest cervical cancer risk. However, variants of HPV-16, HPV-31 and HPV-33 may share several short sequence homologies in the hypervariable L1 gene commonly targeted for HPV genotyping. The purpose of this study was to introduce a robust laboratory procedure to validate HPV-16 detected in clinical specimens, using the GenBank sequence database as the standard reference for genotyping. METHODS: A nested PCR with two pairs of consensus primers was used to amplify the HPV DNA released in crude proteinase K digest of the cervicovaginal cells in liquid-based Papanicolaou cytology specimens. The positive nested PCR products were used for direct automated DNA sequencing. RESULTS: A 48-base sequence downstream of the GP5+ priming site, or a 34-base sequence upstream thereof, was needed for unequivocal validation of an HPV-16 isolate. Selection of a 45-base, or shorter, sequence immediately downstream of the GP5+ site for Basic Local Alignment Search Tool sequence analysis invariably led to ambiguous genotyping results. CONCLUSIONS: DNA sequence analysis may be used for differential genotyping of HPV-16, HPV-31 and HPV-33 in clinical specimens. However, selection of the signature sequence for Basic Local Alignment Search Tool algorithms is crucial to distinguish certain HPV-16 variants from other closely related HPV genotypes.

Validation of human papillomavirus genotyping by signature DNA sequence analysis.

BACKGROUND: Screening with combined cytologic and HPV testing has led to the highest number of excessive colposcopic referrals due to high false positive rates of the current HPV testing in the USA. How best to capitalize on the enhanced sensitivity of HPV DNA testing while minimizing false-positive results from its lower specificity is an important task for the clinical pathologists. METHODS: The HPV L1 gene DNA in liquid-based Pap cytology specimens was initially amplified by the degenerate MY09/MY11 PCR primers and then re-amplified by the nested GP5+/GP6+ primers, or the heminested GP6/MY11, heminested GP5/MY09 primers or their modified equivalent without sample purification or DNA extraction. The nested PCR products were used for direct automated DNA sequencing. A 34- to 50-base sequence including the GP5+ priming site was selected as the signature sequence for routine genotyping by online BLAST sequence alignment algorithms. RESULTS: Of 3,222 specimens, 352 were found to contain HPV DNA, with 92% of the positive samples infected by only 1 of the 35 HPV genotypes detected and 8% by more than 1 HPV genotype. The most common genotype was HPV-16 (68 isolates), followed by HPV-52 (25 isolates). More than half (53.7%) of the total number of HPV isolates relied on a nested PCR for detection although the majority of HPV-16, -18, -31, -33 -35 and -58 isolates were detected by a single MY09/MY11 PCR. Alignment of a 34-base sequence downstream of the GP5+ site failed to distinguish some isolates of HPV-16, -31 and -33. Novel variants of HPV with less than "100% identities" signature sequence match with those stored in the Genbank database were also detected by signature DNA sequencing in this rural and suburban population of the United States. CONCLUSION: Laboratory staff must be familiar with the limitations of the consensus PCR primers, the locations of the signature sequence in the L1 gene for some HPV genotypes, and HPV genotype sequence variants in order to perform accurate HPV genotyping.

Molecular tests for human papillomavirus (HPV), Chlamydia trachomatis and Neisseria gonorrhoeae in liquid-based cytology specimen.

BACKGROUND: 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. METHODS: A crude proteinase K digest 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. RESULTS: 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. CONCLUSION: 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.

HPV infection among women in a representative rural and suburban population of the USA.

OBJECTIVE: To study the prevalence of low-risk and high-risk HPV genotypes in a largely suburban, non-Hispanic, white female population of the USA, and to determine the positive predictive value of one-occasion HPV detection and genotyping using high-grade squamous intraepithelial lesion (HSIL) cytology as the endpoint for clinical evaluation. METHODS: HPV DNA present in liquid-based cytology specimens collected by gynecologists in private practice was amplified using nested polymerase chain reaction. HPV DNA was validated by signature DNA sequencing for accurate genotyping. RESULTS: Of 2633 specimens, 278 were positive for HPV DNA of any genotype. Among 255 single HPV infections, the most prevalent genotype was HPV-16 (n=50; 19.6%), followed by HPV-52 (n=24; 9.4%). Only 10 specimens, all positive for a high-risk HPV, were associated with an HSIL cytology result. Among them were 6 of the 50 specimens (12%) tested positive for HPV-16. One novel HPV-39 variant was detected in repeat testing in a patient with persistent HPV infection. CONCLUSION: DNA sequencing is a useful method for increasing the specificity of HPV genotyping as an aid to follow persistent high-risk HPV infections to reduce excessive colposcopies in populations with low cancer prevalence.

DNA sequencing validation of Chlamydia trachomatis and Neisseria gonorrhoeae nucleic acid tests.

DNA sequencing was used to confirm Chlamydia trachomatis and Neisseria gonorrhoeae nucleic acids in endocervical swab samples. DNA in residues of the samples with positive results by 2 commercial kits was subjected to nested polymerase chain reaction (PCR) amplification. The nested PCR amplicons were used as templates for direct automated DNA sequencing. A 40-base signature sequence was sufficient to achieve unequivocal validation of C trachomatis cryptic plasmid and gonococcal opa gene DNA. DNA with a signature sequence specific for C trachomatis was identified in all 14 samples and for N gonorrhoeae in all 13 samples with positive results by the commercial kits for these 2 microbes. In a low-prevalence population, PCR retesting of 289 samples with initial negative results by a non-nucleic acid amplification assay revealed 3 samples positive for C trachomatis and 2 samples positive for N gonorrhoeae that were missed by the commercial kit. DNA sequencing is a useful tool in validating molecular diagnostics.

Routine human papillomavirus genotyping by DNA sequencing in community hospital laboratories.

BACKGROUND: Human papillomavirus (HPV) genotyping is important for following up patients with persistent HPV infection and for evaluation of prevention strategy for the individual patients to be immunized with type-specific HPV vaccines. The aim of this study was to optimize a robust "low-temperature" (LoTemp) PCR system to streamline the research protocols for HPV DNA nested PCR-amplification followed by genotyping with direct DNA sequencing. The protocol optimization facilitates transferring this molecular technology into clinical laboratory practice. In particular, lowering the temperature by 10 degrees C at each step of thermocycling during in vitro DNA amplification yields more homogeneous PCR products. With this protocol, template purification before enzymatic cycle primer extensions is no longer necessary. RESULTS: The HPV genomic DNA extracted from liquid-based alcohol-preserved cervicovaginal cells was first amplified by the consensus MY09/MY11 primer pair followed by nested PCR with GP5+/GP6+ primers. The 150 bp nested PCR products were subjected to direct DNA sequencing. The hypervariable 34-50 bp DNA sequence downstream of the GP5+ primer site was compared to the known HPV DNA sequences stored in the GenBank using on-line BLAST for genotyping. The LoTemp ready-to-use PCR polymerase reagents proved to be stable at room temperature for at least 6 weeks. Nested PCR detected 107 isolates of HPV in 513 cervicovaginal clinical samples, all validated by DNA sequencing. HPV-16 was the most prevalent genotype constituting 29 of 107 positive cases (27.2%), followed by HPV-56 (8.5%). For comparison, Digene HC2 test detected 62.6% of the 107 HPV isolates and returned 11 (37.9%) of the 29 HPV-16 positive cases as "positive for high-risk HPV". CONCLUSION: The LoTemp ready-to-use PCR polymerase system which allows thermocycling at 85 degrees C for denaturing, 40 degrees C for annealing and 65 degrees C for primer extension can be adapted for target HPV DNA amplification by nested PCR and for preparation of clinical materials for genotyping by direct DNA sequencing. HPV genotyping is performed by on-line BLAST algorithm of a hypervariable L1 region. The DNA sequence is included in each report to the physician for comparison in following up patients with persistent HPV infection, a recognized tumor promoter in cancer induction.