PCR detection of human papillomavirus: comparison between MY09/MY11 and GP5+/GP6+ primer systems

Human papillomavirus (HPV) is an etiologic agent of cervical cancer and is the most common sexually transmitted disease in women. PCR amplification of HPV genomes is the most sensitive method for the detection of cervicovaginal HPV. We have compared the two most commonly used PCR primer sets, MY09/MY11 (MY-PCR) and GP5+/GP6+ (GP+-PCR), for the detection of HPV DNA in cervicovaginal lavage samples from 208 women. Oligonucleotide probes for 39 different HPV types were used. Both primer sets amplified a wide spectrum of HPV genotypes and detected similar overall prevalences of 45% (94 of 208) and 43% (89 of 208), respectively. The MY-PCR system detected 27 of 30 (90%) samples with multiple HPV types, whereas the GP+-PCR system detected 14 of 30 (47%) samples with multiple HPV types. Differences in the detection of HPV types 35, 53, and 61 were noted between the two primer systems. Serial dilution of plasmid templates indicated a 3-log decrease in the amplification of HPV type 35 by MY-PCR and HPV types 53 and 61 by GP+-PCR. These results indicate that although the MY-PCR and GP+-PCR identified nearly equivalent prevalences of HPV in a set of clinical samples, differences in the detection of specific types and infections with multiple types were found. Differences in the sensitivities and characteristics of the PCR systems for the detection of HPV within clinical samples should be considered when comparing data between studies and/or in designing new studies or clinical trials.     

Group-specific differentiation between high- and low-risk human papillomavirus genotypes by general primer-mediated PCR and two cocktails of oligonucleotide probes

In recent years, general primer-mediated PCR assays have been developed to detect a broad spectrum of human papillomavirus (HPV) genotypes. In this study, a procedure enabling a simple group-specific differentiation of high-risk (HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -54, -56, and -58) and low risk (HPV-6, -11, -34, -40, -42, -43, and 44) HPVs following an HPV general primer-mediated (GP5+/GP6+) PCR is presented. By computer-assisted sequence analysis, oligonucleotides (30-mers) specific for 19 different HPV genotypes were selected from the internal part of the 150-bp GP5+/GP6(+)-amplified region. These oligo probes were tested for specificity in a Southern blot analysis of PCR products derived from the same panel of HPV types. No cross-hybridizations were found. The sensitivities of the oligo probes varied from the femtogram level for the well-amplified HPV types like HPV-16 and -18 to the picogram level for the less-well amplified HPV types like HPV-39 and -51. These sensitivities were reached when the oligo probes were applied both individually and in a cocktail. On the basis of these results, two cocktail oligo probes that enabled a specific and sensitive differentiation between low- and high-risk HPV types were composed.     

Comprehensive study of several general and type-specific primer pairs for detection of human papillomavirus DNA by PCR in paraffin-embedded cervical carcinomas

We have compared the efficacies of three general primer pairs for the detection of human papillomavirus (HPV) DNA in formaldehyde-fixed paraffin-embedded carcinomas. The use of these primer pairs leads to underestimates of the HPV prevalence (GP5/6, 61.1%; CPI/IIG, 57.4%; MY09/11, 46.9%; combined, 72.8%). The efficacy of each primer pair seemed to be inversely correlated to the length of the amplimer produced. By using newly developed type-specific primer pairs (amplimer length, approximately 100 bp), an increase in HPV DNA detection (87.6%) was found.     

Detection of human papillomavirus DNA with in situ hybridisation in oral squamous carcinoma in a rural black population

Intra-oral carcinoma is the third most common malignancy among men in developing countries, and carries a high mortality rate, particularly in Africa, where patients often present initially with lesions at an advanced stage. The present study was undertaken to determine the prevalence of human papillomavirus (HPV) DNA in oral squamous carcinoma in the west of the Northern Transvaal, an area where a large number of new cases has been diagnosed over the past few years. Paraffin blocks from 66 cases (51 men, 15 women; mean age 58.7 years) of oral squamous carcinoma were randomly selected. Blocks contained samples of both tumour and adjacent normal epithelium. The presence of HPV antigen was established by means of immunocytochemistry and HPV DNA by in situ hybridisation with radiolabelled probes for HPV-6, 11, 16 and 18. Immunocytochemistry for viral antigen was negative in all the specimens. HPV-18 was detected in normal epithelium adjacent to the tumour in one case only. It appears from our study that HPV is of limited importance in oral squamous cell carcinogenesis in the population studied.     

Seroprevalence of human papillomavirus types 6 and 16 capsid antibodies in homosexual men

Human papillomavirus (HPV) has been implicated in the pathogenesis of anal carcinoma, which is increased in homosexual men. Little is known about the serologic response to HPV in normal or immunosuppressed men; therefore, HIV-infected and -uninfected homosexual men were screened for HPV-6 and -16 capsid antibodies. HIV-infected men had increased HPV DNA detection but did not significantly differ in the prevalence of serum HPV antibodies. HPV-6 DNA detection and the presence of anal warts were significantly correlated with serum antibody overall and in the HIV-infected subgroup. HPV-16 DNA detection was not significantly correlated with serum antibody overall or in either subgroup; however, HIV-infected men with high-grade anal squamous intraepithelial lesions were significantly more likely to have HPV-16 antibodies. HIV-infected men are able to generate an antibody response to HPV, and a lack of serum HPV antibodies cannot explain the increased HPV-associated disease seen in HIV-infected men.     

The L1 major capsid protein of human papillomavirus type 16 variants affects yield of virus-like particles produced in an insect cell expression system

The L1 major capsid proteins of six human papillomavirus type 16 (HPV-16) strains were expressed in insect cells by using recombinant baculoviruses. Virus-like particles (VLPs) which appeared similar to empty virions were identified by electron microscopy for all HPV strains investigated. However, the yield of VLPs produced varied in a range from 1 to 79 depending on the HPV-16 strain. The L1 proteins of these strains differed by up to 15 amino acids from the L1 protein of the prototype HPV-16 strain. Mutations in the amino acid region from residues 83 to 97 seemed to affect the level of expression of the L1 protein. These results are important when considering the development of HPV vaccines and serological tests. They indicate that strains inducing high levels of VLP production must be selected for the development of vaccines. Moreover, the L1 proteins of all strains investigated were able to bind with DNA. We also investigated the seroreactivities of VLPs derived from three different HPV-16 strains from Algeria, Senegal, and the Philippines by testing sera from women from 11 countries in immunoglobulin G-specific enzyme-linked immunosorbent assays. We observed a strong correlation between the reactivities of the three different VLP variants, independent of the geographical origin of the sera investigated. These results indicate that the three strains investigated are serologically cross-reactive despite the fact that their L1 proteins differ in 14 amino acids and suggest that VLPs derived from only one HPV-16 strain could be sufficient for the development of an HPV-16 vaccine and anti-HPV-16 tests.     

Human papillomavirus type 11 (HPV-11) neutralizing antibodies in the serum and genital mucosal secretions of African green monkeys immunized with HPV-11 virus-like particles expressed in yeast

It has been shown previously that immunization of animals with recombinant virus-like particles (VLPs) consisting of the viral capsid proteins L1 or L1 plus L2 protected animals against experimental viral challenge. However, none of these experimental models addresses the issue of whether systemic immunization with VLPs elicits a neutralizing antibody response in the genital mucosa. Such a response may be necessary to protect the uterine cervix against infection with genital human papillomavirus (HPV) types. African green monkeys systemically immunized with HPV-11 VLPs expressed in Saccharomyces cerevisiae and formulated on aluminum adjuvant elicited high-titered HPV-11 VLP-specific serum antibody responses. Sera from these immunized monkeys neutralized HPV-11 in the athymic mouse xenograft system. Significant levels of HPV-11-neutralizing antibodies also were observed in cervicovaginal secretions. These findings suggest that protection against HPV infection of the uterine cervix may be possible through systemic immunization with HPV VLPs.     

Detection of human papillomavirus in routinely processed biopsy specimens from laryngeal papillomas: evaluation of reproducibility of polymerase chain reaction and DNA in situ hybridization procedures

The frequency of human papillomavirus (HPV) in laryngeal papillomas varies largely among different studies. DNA in situ hybridization (ISH) has been the most widely used method for detection of HPV. The aim of this study was to compare the reproducibility and sensitivity of ISH with polymerase chain reaction (PCR) in 35 specimens of laryngeal papillomas routinely fixed in buffered or unbuffered formalin. Out of 12 specimens fixed in buffered formalin, 10 were positive for HPV 6/11 using ISH. The procedure was repeated three times and three specimens were positive only twice. Nine biopsies were positive for HPV using PCR with consensus primers (My 09/11) on dewaxed tissue without extracting DNA. In three repeated PCRs, the results were inconsistent in three samples. After DNA extraction, all 12 samples were positive with PCR. Of the 23 specimens fixed in unbuffered formalin, 14 were HPV-positive with ISH, while only one was positive with PCR. We concluded that PCR with My 09/11 consensus primers is a highly sensitive method for detection of HPV in laryngeal papillomas fixed in buffered formalin, but useless for samples fixed in unbuffered formalin. When DNA was extracted from the former type of fixed tissue, the results were highly reproducible. In contrast to PCR, ISH appeared to be less influenced by fixation procedure, but it was not as reproducible and sensitive as PCR. Negative results did not necessarily mean absence of HPV.     

Surface conformational and linear epitopes on HPV-16 and HPV-18 L1 virus-like particles as defined by monoclonal antibodies

A panel of 24 monoclonal antibodies (MAbs) was generated against human papillomavirus (HPV) types 16 and 18 L1 virus-like particles (VLPs). The MAbs were screened for reactivity to a variety of VLPs prepared from HPV-6, -11, -16, -18, -31, -33, -35, and -45, cottontail rabbit papillomavirus, bovine papillomavirus type 1, and a set of 35 overlapping 20-amino-acid peptides spanning the entire HPV-16 L1 gene. Type-specific linear and conformational surface epitopes were detected as well as several cross-reactive linear epitopes that showed various levels of cross-reactivity between different genital HPV and animal papillomavirus L1s. Most of the linear epitopes were mapped using synthetic peptides, and the epitopes were identified as being either surface or buried within the VLP as defined by the pattern of reactivity in ELISA using intact and disrupted VLP antigen. These MAbs may be useful reagents to help define neutralizing epitopes of HPV-16 and -18 when infectivity assays become available, and to define the regions of L1 that are exposed on the surface or buried within the assembled capsid.     

Generation of type-specific probes for the detection of single-copy human papillomavirus by a novel in situ hybridization method

The integration of human papillomavirus (HPV) DNA is associated with the pathogenesis of HPV-associated malignancies. The ability, however, of standard in situ hybridization (ISH) to detect low-copy integrated HPV DNA is limited. We describe the generation of HPV type-specific biotin-labeled DNA probes and a novel ISH method that uses the catalyzed reporter deposition (CARD) system for the detection of single-copy target HPV DNA in formalin-fixed, paraffin-embedded tissue. Consensus primers flanking the noncoding region of HPVs were used to generate biotin-labeled HPV-6b, -11, -16 and -18 probes by polymerase chain reaction (PCR). The probes were used for ISH with the novel technique of CARD to increase the sensitivity of the assay. Tissue blocks were prepared from CaSki (500-600 copies of HPV-16), SiHa (1-2 copies of HPV-16), and HeLa (10-50 copies of HPV-18) cell lines, as well as from an HPV-negative cell line, C33A, and then tested to demonstrate the sensitivity and specificity of the probes. Surgical specimens were used to show the clinical applicability of this technique. We successfully detected HPV-16 DNA in CaSki and SiHa cells but not in HeLa or C33A cells. HPV-18 DNA was detected in HeLa cells but not in CaSki, SiHa, or C33A cells. Sensitivity was increased when ISH was performed using probes with more biotin incorporation or when more cycles of signal amplification were employed, but significant nonspecific background was observed after more than two cycles of signal amplification. The probes generated in this study detected specific types of HPV in surgical specimens with much higher sensitivity than did conventional ISH. We concluded that our new method was highly sensitive and could be applied to formalin-fixed, paraffin-embedded clinical material for the detection of HPV.     

Genotyping of 27 human papillomavirus types by using L1 consensus PCR products by a single-hybridization, reverse line blot detection method

Amplification of human papillomavirus (HPV) DNA by L1 consensus primer systems (e.g., MY09/11 or GP5(+)/6(+)) can detect as few as 10 to 100 molecules of HPV targets from a genital sample. However, genotype determination by dot blot hybridization is laborious and requires at least 27 separate hybridizations for substantive HPV-type discrimination. A reverse blot method was developed which employs a biotin-labeled PCR product hybridized to an array of immobilized oligonucleotide probes. By the reverse blot strip analysis, genotype discrimination of multiple HPV types can be accomplished in a single hybridization and wash cycle. Twenty-seven HPV probe mixes, two control probe concentrations, and a single reference line were immobilized to 75- by 6-mm nylon strips. Each individual probe line contained a mixture of two bovine serum albumin-conjugated oligonucleotide probes specific to a unique HPV genotype. The genotype spectrum discriminated on this strip includes the high-risk, or cancer-associated, HPV genotypes 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 55, 56, 58, 59, 68 (ME180), MM4 (W13B), MM7 (P291), and MM9 (P238A) and the low-risk, or non-cancer-associated, genotypes 6, 11, 40, 42, 53, 54, 57, 66, and MM8 (P155). In addition, two concentrations of beta-globin probes allowed for assessment of individual specimen adequacy following amplification. We have evaluated the performance of the strip method relative to that of a previously reported dot blot format (H. M. Bauer et al., p. 132-152, in C. S. Herrington and J. O. D. McGee (ed.), Diagnostic Molecular Pathology: a Practical Approach, (1992), by testing 328 cervical swab samples collected in Digene specimen transport medium (Digene Diagnostics, Silver Spring, Md.). We show excellent agreement between the two detection formats, with 92% concordance for HPV positivity (kappa = 0.78, P < 0.001). Nearly all of the discrepant HPV-positive samples resulted from weak signals and can be attributed to sampling error from specimens with low concentrations (<1 copy/microliter) of HPV DNA. The primary advantage of the strip-based detection system is the ability to rapidly genotype HPVs present in genital samples with high sensitivity and specificity, minimizing the likelihood of misclassification.     

Human papillomavirus type 16 DNA detected by the polymerase chain reaction in non-cancer tissues of the head and neck

Cancer-free tissues from various anatomical subsites in the head and neck were examined by the polymerase chain reaction (PCR) for the incidence of human papillomavirus (HPV) types 16 and 18. We detected HPV-16 DNA in 9 of 103 samples (8.7%), including specimens from the paranasal sinuses, tonsil, hypopharynx and larynx. However, no HPV-16/18 DNA was detected by Southern hybridization in these 9 samples. The significance of the presence of HPV-16 DNA in non-cancer tissues is still unknown, but PCR detection only of high-risk HPV DNA in head and neck cancer should be evaluated cautiously because of its ubiquity in this region.     

Association of the antibodies against human papillomavirus 16 E4 and E7 proteins with cervical cancer positive for human papillomavirus DNA

Occurrence of the antibodies against human papillomavirus (HPV) 16 proteins E4 and E7 is specifically but independently associated with cervical cancer. To correlate HPV DNA and antibody data, we examined the biopsy specimens and sera, by polymerase chain reaction (PCR) and by ELISA, respectively, from 51 patients with cervical cancer (including 3 recurrent cases) and 22 with cervical intra-epithelial neoplasia. Consensus primers for the L1 region were used for PCR and bacterially expressed, purified fusion protein HPV-16 E4 and non-fusion protein HPV-16 E7 were used for ELISA. HPV-16 DNA and other HPV types were detected in 17 and 25, respectively, out of 51 cases of cervical cancer. Ten out of the 17 HPV-16-DNA-positives were positive either for anti-E4 or for anti-E7: positivities for anti-E4, for anti-E7, and for both were 6/17, 5/17 and 1/17 respectively. Three anti-E7-positives consisted of those for HPV-33, -52 and -58 DNA, suggesting that limited cross-reaction occurred between the HPV types. Among the HPV-16-DNA-positive cases of cancer, lymph-node or distant metastasis was recorded more frequently in the seropositives than in the seronegatives. Our results show that the HPV-16 anti-E4 or anti-E7 occurs in some, but not in all, of the HPV-16-DNA-positive cases, and support the hypothesis that the presence of the HPV-16 antibodies can be used as a marker for possible metastasis.     

Pediatric respiratory papillomatosis: prognostic role of viral typing and cofactors

Children with recurrent respiratory papillomatosis vary greatly in their clinical disease course. Many have mild disease with eventual remission while others present with an early aggressive airway obstructive course. This study consisted of 24 pediatric patients whose specimens underwent polymerase chain reaction analysis for cytomegalovirus (CMV), herpes simplex virus (HSV), and human papillomavirus (HPV) type. Nineteen of 24 specimens contained enough DNA for this study. None of the specimens were found to contain DNA from HPV-16, -18, -31, -33; CMV; or HSV, which contrasts with our previous findings in adults. Ten patients were infected by HPV-11 and seven of these underwent tracheotomy because of an aggressive tumorigenic clinical course. Nine patients were infected by HPV-6 alone of whom only two required a tracheotomy (P = 0.05, Fisher's Exact Test). The early airway obstructive course associated with HPV-11, however, had no bearing on achieving eventual disease remission, with decannulation achieved in eight of nine children.     

Detection of human papillomavirus DNA in laryngeal squamous cell carcinoma by polymerase chain reaction

Although human papillomaviruses (HPVs) have been found in many, but not all, tumours of the oral cavity, nose, pharynx and larynx, the true role of HPV in malignant tumours of the head and neck is still unclear. The presence of HPV DNA was investigated in 45 fresh squamous cell carcinoma (SCC) specimens and in 29 normal mucosa specimens collected from 45 primary laryngeal SCC patients. HPV DNA was detected using the polymerase chain reaction (PCR) with consensus primers that detect HPV types 6, 11, 16 and 18.9 of the 45 patients (20%) were HPV positive; the presence of HPV was also detected in the corresponding normal laryngeal mucosa of four of the 29 specimens (14%). No statistically significant differences were found between the presence of HPV DNA in normal specimens and in neoplastic mucosa specimens. No correlation was found between HPV DNA positive tumours and size, T classification, lymph node involvement and histological grading. This study adds further evidence suggesting a possible role of HPV DNA infection in laryngeal carcinogenesis.     

Human papillomavirus type 11 recombinant L1 capsomeres induce virus-neutralizing antibodies

The human papillomavirus type 11 (HPV-11) L1 major capsid protein can be trypsinized to generate recombinant capsomeres that retain HPV genotype-restricted capsid antigenicity (M. Li, T. P. Cripe, P. A. Estes, M. K. Lyon, R. C. Rose, and R. L. Garcea, J. Virol. 71:2988-2995, 1997). In the present study, HPV-11 virion-neutralizing monoclonal antibodies H11.F1 and H11.H3, previously characterized as recognizing two distinct HPV-11 capsid-neutralizing antigenic domains (S. W. Ludmerer, D. Benincasa, and G. E. Mark III, J. Virol. 70:4791-4794, 1996), were each found to be highly immunoreactive with trypsin-generated capsomeres in an enzyme-linked immunosorbent assay (ELISA). Capsomeres were used to generate high-titer polyclonal immune sera that demonstrated HPV genotype-restricted reactivity by ELISA. The capsomere antisera were then tested in an in vitro infectivity assay and found to neutralize HPV-11 virions. In this assay, HPV-11 capsomere polyclonal antisera exhibited neutralization titers (10(-5) to 10(-6)) comparable to those obtained with a virion-neutralizing antiserum raised previously against intact HPV-11 VLPs (R. C. Rose, R. C. Reichman, and W. Bonnez, J. Gen. Virol. 75:2075-2079, 1994). These results indicate that highly immunogenic, genotype-restricted HPV capsid-neutralizing antigenic domains are contained entirely within capsomeres. Thus, capsomeres may be viable vaccine candidates for the prevention of HPV disease.     

Detection of human papillomavirus DNA sequences in oral squamous cell carcinomas and their relation to p53 and proliferating cell nuclear antigen expression

BACKGROUND: The etiology of oral squamous cell carcinoma (SCC) is still obscure. Since human papillomavirus (HPV) DNAs are associated with carcinoma of the uterine cervix, carcinomas of the oral cavity were investigated to ascertain if these viruses are present in squamous carcinomas of this anatomic site. METHODS: Seventy-seven oral mucosal SCCs were examined for the presence of HPV DNAs by polymerase chain reaction and dot blot hybridization. Immunohistochemical detection of proliferating cell nuclear antigen (PCNA) and p53 was performed and single strand conformation polymorphism analysis for p53 was undertaken. In situ hybridization detection of HPV-16 DNA also was performed. RESULTS: Human papillomavirus-16 DNA was detected in 23 cases of oral SCC and both HPV-16 and HPV-18 DNA were detected in one case of tongue SCC. Human papillomavirus DNAs were detected of 11 of 33 tongue, 4 of 15 gingival, 2 of 4 palate, 2 of 5 buccal mucosa, 3 of 7 maxillary sinus, and 2 of 11 the floor of the mouth SCCs. None were detected in SCCs of the retromolar region (0/2). Immunohistochemical examination for p53 was performed in 26 cases of oral SCC and the accumulation of p53 protein was observed in 6 cases (i.e., in 4 of 17 HPV DNA-negative cases and in 2 of 9 HPV DNA-positive cases). Single strand conformation polymorphism analysis confirmed gene mutations in all 6 cases. Human papillomavirus-16 DNA was predominantly identified in cancer cells that showed a morphologic resemblance to basal cells and its hybridized signal in keratinized cells was reduced by in situ hybridization detection. Immunohistochemical detection of PCNA revealed its cooccurrence with HPV-16 DNA in cancer cells. CONCLUSIONS: These results suggest that HPV-16 DNA sequences may have the capability to maintain the proliferative state of epithelial cells, and may contribute to the production of malignant phenotypes.     

Epidural anaesthesia for caesarean section in an achondroplastic dwarf

Telomerase is a ribonucleoprotein that synthesizes telomeric DNA on chromosomal ends. Telomerase activation has been seen in many immortal cell lines and cancers. Telomerase activity was analyzed in prostate carcinoma; in coexistent prostatic intraepithelial neoplasia (PIN), benign prostatic hyperplasia (BPH), atrophy and normal tissue; and in benign prostate glands. Telomerase activity was detected in 80 of 87 (92%) prostate cancers. Forty-one matched samples (from a total of 32 cases) were available for comparative analysis. The presence of telomerase activity in adjacent PIN, BPH, and normal tissue was correlated with telomerase activity in the malignant epithelium. In these adjacent tissues, telomerase activity was found in 11 of 15 (73%) PINs, 13 of 26 (50%) BPHs, and 1 of 6 (16%) atrophy and 4 of 11 (36%) normal tissues. In contrast to the BPH tissue from cancer-bearing glands, all 16 BPH specimens from patients only diagnosed with BPH were telomerase activity negative. In cancer samples, there was no correlation between telomerase activity and Gleason grade or preoperation prostate-specific antigen level. Our data indicate that telomerase activity is present in most prostate cancers. The high rate of telomerase activity in the benign-appearing areas of these glands may be attributed either to the presence of occult cancer cells or to early molecular alterations of cancer that were histologically inapparent.     

Comparison of PCR- and hybrid capture-based human papillomavirus detection systems using multiple cervical specimen collection strategies

This study compared the performances of three human papillomavirus (HPV) detection tests with specimens collected by three alternative procedures. The HPV tests included the Hybrid Capture Tube test (HCT), the microplate-based Hybrid Capture II test (HC II), and the MY09-MY11 L1 consensus primer PCR-based assay. Initial cervical specimens were collected from study subjects with a broom device, and after Papanicolaou smears were made, residual specimens were placed into PreservCyt (PC), a liquid cytology medium. A second specimen was collected from each subject and placed into Digene Specimen Transport Medium (STM). The device for collection of the second specimen alternated with consecutive subjects between a conical cytology brush and a Dacron swab. At the 1.0-pg/ml cutoff, the results of the HC II agreed well with those of the PCR. Specifically, when PCR data were restricted to the types found by the HC II (HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68), there was greater than 90% agreement between the HC II and PCR results with both STM and PC. At a lower cutoff (0.2 pg/ml), HC II-positive results increased further, especially when the test was applied to the PC specimens. However, false-positive HC II results were more often observed at the 0.2-pg/ml cutoff. HC II yielded the highest HPV positivity with specimens placed into PC, followed by specimens collected with a conical brush and placed into STM and, last, by those collected with a Dacron swab and placed into STM. Our results demonstrate the utility of both the STM and PC specimen collection methods and show good agreement between the HC II and PCR.