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Background: Squamous cell carcinoma of the conjunctiva (SCCC) is associated with HIV-related immunosuppression, but human papillomavirus virus (HPV) is also suspected to have a role. We carried out a case-control study to assess the role of cutaneous and mucosal HPV types in SCCC, conjunctival dysplasia, and their combination (SCCC/dysplasia) in Uganda.Methods: We compared HPV prevalence in frozen biopsies from 94 SCCC cases (79 of whom were found to be HIV-positive), 39 dysplasia cases (34 HIV-positive), and 285 hospital controls (128 HIV-positive) having other eye conditions that required surgery. Highly sensitive PCR assays that detect 75 HPV types were used. Odds ratios (ORs) and 95% confidence intervals (CIs) were computed, adjusting for, or stratifying by age, sex, and HIV status.Results: Cutaneous HPV types were detected in 45% of SCCC cases, 41% of dysplasia cases and 11% of controls. Human papillomavirus virus 5 and 8 were the most common types in SCCC, and most often occurred in combination with other types. Associations were observed between SCCC/dysplasia and detection of both single (OR=2.3; 1.2-4.4) and multiple (OR=18.3; 6.2-54.4) cutaneous HPV types, and were chiefly based on findings in HIV-positive patients. Cutaneous HPV infections were rarely observed among HIV-negative patients and the association with SCCC/dysplasia was not significant (OR=2.4; 0.6-9.6) among them. Squamous cell carcinoma of the conjunctiva/dysplasia risk and mucosal HPV types were not associated in either HIV-positive or HIV-negative patients.Conclusions: We detected cutaneous HPV types in nearly half of SCCC/dysplasia cases and often multiple types (HPV5 and 8 being most common). The role of HIV (confounder or strong enhancer of cutaneous HPV carcinogenicity) is still uncertain. [ABSTRACT FROM AUTHOR]

Background: Identification of human papillomavirus (HPV) DNA in cervical tissue is important for understanding cervical carcinogenesis and for evaluating cervical cancer prevention approaches. However, HPV genotyping using formalin-fixed, paraffin-embedded (FFPE) tissues is technically challenging. We evaluated the performance of four commonly used genotyping methods on FFPE cervical specimens conducted in different laboratories and compared to genotyping results from cytological samples., Methods: We included 60 pairs of exfoliated-cell and FFPE specimens from women with histologically confirmed cervical intraepithelial lesions grade 2 or 3. Cytology specimens were genotyped using the Linear Array assay. Four expert laboratories processed tissue specimens using different preparation methods and then genotyped the resultant sample preparations using four different HPV genotyping methods: SPF10-PCR DEIA LiPA25 (version 1), Inno-LiPA, Linear Array and the Onclarity assay. Percentage agreement, kappa statistics and McNemar's chi-square were calculated for each comparison of different methods and specimen types., Results: Overall agreement with respect to carcinogenic HPV status for FFPE samples between different methods was: 81.7, 86.7 and 91.7% for Onclarity versus Inno-LiPA, Linear Array and SPF-LiPA25, respectively; 81.7 and 85.0% for Linear Array versus Inno-LiPA and SPF-LiPA25, respectively; and 86.7% for SPF-LiPA25 versus Inno-LiPA. Type-specific agreement was >88.3% for all pair-wise comparisons. Comparisons with cytology specimens resulted in overall agreements from 80 to 95% depending on the method and type-specific agreement was >90% for most comparisons., Conclusions: Our data demonstrate that the four genotyping methods run by expert laboratories reliably detect HPV DNA in FFPE specimens with some variation in genotype-specific detection.

Two commercial HPV tests target the same 65 bp fragment of the human papillomavirus genome (designated SPF10): the original HPV SPF10 PCR-DEIA-LiPA25 system, version 1, (LiPA25) and the INNO-LiPA HPV Genotyping Extra (INNO-LiPA). The original SPF10 LiPA25 system was designed to have high analytical sensitivity and applied in HPV vaccine and epidemiology studies worldwide. But due to apparent similarities, this test can be easily confused with INNO-LiPA, a more recent assay of which the intended use, i.e., epidemiological or clinical, is currently unclear. The aim was to compare the analytical sensitivity of SPF10 LiPA25 to that of INNO-LiPA on the level of general HPV detection and genotyping. HPV testing by both assays was performed on the same DNA isolated from cervical swab (n = 365) and biopsy (n = 42) specimens. In cervical swabs, SPF10 LiPA25 and INNO-LiPA identified 35.3% and 29.3% multiple infections, 52.6% and 51.5% single infections, and no HPV type in 12.1% and 19.2%, respectively. Genotyping results were 64.7% identical, 26.0% compatible and 9.3% discordant between both methods. SPF10 LiPA25 detected significantly more genotypes (p < 0.001). The higher analytical sensitivity of SPF10 LiPA25 was confirmed by the MPTS123 genotyping assay. HPV positivity by the general probes in SPF10 DEIA was significantly higher (87.9%) than by those on INNO-LiPA (77.0%) (kappa = 0.592, p < 0.001). In cervical biopsies, SPF10 LiPA25 and INNO-LiPA identified 21.4% and 9.5% multiple types, 76.2% and 81.0% single types, and no type in 2.4% and 9.5%, respectively. Between both tests, the identification of genotypes was 76.3% identical, 14.3% compatible and 9.5% discordant. Overall, significantly more genotypes were detected by SPF10 LiPA25 (kappa = 0.853, p = 0.022). HPV positivity was higher by the SPF10 DEIA (97.6%) than by the INNO-LiPA strip (92.9%). These results demonstrate that SPF10 LiPA25 is more suitable for HPV genotyping in epidemiologic and vaccine-related studies, due to its higher analytical sensitivity., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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HPV16 variants correlate with geographic origin and ethnicity. The association between infection with a specific variant and the cervical disease risk remains unclear. We studied the prevalence, persistence and association with cervical intraepithelial neoplasia (CIN) of different HPV16 variants, using cervical swabs and whole tissue sections (WTS) of biopsies from 548 women in the placebo group of a HPV16/18 vaccine trial. In HPV16-positive samples, HPV16 variants were identified by a reverse hybridization assay (RHA). Laser-capture micro-dissection (LCM) was performed for localized detection of HPV. HPV16 variants were determined in 47 women. Frequency of mixed HPV16 variant infections was lower (8.5%) than for multiple HPV genotypes (39.1%). Among 35 women having consecutive HPV16 variant-positive swabs, 32 (91.4%) had the same variant while in three (8.6%) women a change in variant(s) was observed. HPV16-positive WTS were obtained from 12 women having consecutive HPV16 variant-positive swabs. The same variant was present in WTS of 10 women, while two were negative. WTS of five women were histologically normal. A single HPV16 variant was detected in four women having CIN1-3, while additional HPV genotypes were found in three other women having CIN2 and CIN3. In the WTS of one woman with mixed genotypes, the HPV16 variant was assigned to a CIN2 lesion by LCM. HPV16 variant infections can be effectively studied in cervical swabs and tissue specimens by the HPV16 variant RHA. Multiple HPV16 variants in one woman are rare. The HPV16 genotype consistently detected in follow-up samples usually involves a persistent infection with the same variant.

Human papillomavirus (HPV) epidemiological and vaccine studies require highly sensitive HPV detection and genotyping systems. To improve HPV detection by PCR, the broad-spectrum L1-based SPF10 PCR DNA enzyme immunoassay (DEIA) LiPA system and a novel E6-based multiplex type-specific system (MPTS123) that uses Luminex xMAP technology were combined into a new testing algorithm. To evaluate this algorithm, cervical swabs (n = 860) and cervical biopsy specimens (n = 355) were tested, with a focus on HPV types detected by the MPTS123 assay (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 6, and 11). Among the HPV-positive samples, identifications of individual HPV genotypes were compared. When all MPTS123 targeted genotypes were considered together, good overall agreement was found (κ = 0.801, 95% confidence interval [CI], 0.784 to 0.818) with identification by SPF10 LiPA, but significantly more genotypes (P < 0.0001) were identified by the MPTS123 PCR Luminex assay, especially for HPV types 16, 35, 39, 45, 58, and 59. An alternative type-specific assay was evaluated that is based on detection of a limited number of HPV genotypes by type-specific PCR and a reverse hybridization assay (MPTS12 RHA). This assay showed results similar to those of the expanded MPTS123 Luminex assay. These results confirm the fact that broad-spectrum PCRs are hampered by type competition when multiple HPV genotypes are present in the same sample. Therefore, a testing algorithm combining the broad-spectrum PCR and a range of type-specific PCRs can offer a highly accurate method for the analysis of HPV infections and diminish the rate of false-negative results and may be particularly useful for epidemiological and vaccine studies.

Background: Isolates of HPV16 comprise six variants: European (Eu), Asian (As), Asian-American (AA), North American (NA), African-1 (AF1), and African-2 (AF2) with different carcinogenic potentials. Highly reliable automatable techniques for HPV variant genotyping would be helpful to confirm the role of variants in cervical cancer in large epidemiological studies., Objective: To validate the performance of a novel assay for identification of HPV16 variants., Study Design: The test is a multiplex PCR amplifying four small fragments from the E6 open reading frame (ORF). Variants are identified in a reverse hybridization assay with variant specific probes. The novel assay was compared to sequence analysis of the E6 ORF in 68 clinical samples. In addition, HPV16 variant distribution was studied in 218 cervical samples from women with normal cytology, squamous cell carcinoma (SCC) and adenocarcinoma of countries in Africa, Asia and South-America., Results: There was 95.6% agreement between the test and sequencing. Analysis of the clinical panel including 218 positive samples revealed worldwide distribution patterns of HPV16 variants. Finally, a threefold increased risk for SCC with grouped Eu and As variants in South-American countries as compared to controls was found, although the association was not statistically significant., Conclusions: The novel assay is a reliable and simple technique, distribution patterns of HPV16 variants in different world regions and disease associations could be established and it may be useful in further epidemiological studies investigating the role of HPV16 variants in cervical carcinogenesis., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: While the association of human papillomavirus (HPV) with cervical cancer is well established, the influence of HIV on the risk of this disease in sub-Saharan Africa remains unclear. To assess the risk of invasive cervical carcinoma (ICC) associated with HIV and HPV types, a hospital-based case-control study was performed between September 2004 and December 2006 in Kampala, Uganda. Incident cases of histologically-confirmed ICC (N=316) and control women (N=314), who were visitors or care-takers of ICC cases in the hospital, were recruited. Blood samples were obtained for HIV serology and CD4 count, as well as cervical samples for HPV testing. HPV DNA detection and genotyping was performed using the SPF10/DEIA/LiPA25 technique which detects all mucosal HPV types by DEIA and identifies 25 HPV genotypes by LiPA version 1. Samples that tested positive but could not be genotyped were designated HPVX. Odds ratios (OR) and 95% confidence intervals (CI) were calculated by logistic regression, adjusting for possible confounding factors., Results: For both squamous cell carcinoma (SCC) and adenocarcinoma of the cervix, statistically significantly increased ORs were found among women infected with HPV, in particular single HPV infections, infections with HPV16-related types and high-risk HPV types, in particular HPV16, 18 and 45. For other HPV types the ORs for both SCC and adenocarcinoma were not statistically significantly elevated. HIV infection and CD4 count were not associated with SCC or adenocarcinoma risk in our study population. Among women infected with high-risk HPV types, no association between HIV and SCC emerged. However, an inverse association with adenocarcinoma was observed, while decrease in CD4 count was not associated with ICC risk., Conclusions: The ORs for SCC and adenocarcinoma were increased in women infected with HPV, in particular single HPV infections, infections with HPV16- and 18-related types, and high-risk HPV types, specifically HPV16, 18 and 45. HIV infection and CD4 count were not associated with SCC or adenocarcinoma risk, but among women infected with high-risk HPV types there was an inverse association between HIV infection and adenocarcinoma risk. These results suggest that HIV and CD4 count may have no role in the progression of cervical cancer.

Background: Human Papillomavirus (HPV) detection results comparing paraffin embedded cervical tissue and other cervical specimens have been done with varying degrees of agreement. However, studies comparing freshly frozen specimens and paraffin embedded specimens of invasive cervical carcinomas are lacking. The aim of the study was to compare HPV detection using SPF10 broad-spectrum primers PCR followed by DEIA and genotyping by LiPA25 (version 1) between freshly frozen cervical tissue samples and paraffin embedded blocks of cervical tissue from the same patient. There were 171 pairs of paraffin embedded and freshly frozen samples analyzed from cervical carcinoma cases from Kampala, Uganda., Results: 88.9% (95% CI: 83.2%-93.2%) of paraffin embedded samples were HPV positive compared with 90.1% (95% CI: 84.6%-94.1%) of freshly frozen samples, giving an overall agreement in HPV detection between fresh tissue and paraffin embedded tissue at 86.0% (95% CI: 79.8%-90.8%). Although the proportion of HPV positive cases in freshly frozen tissue was higher than those in paraffin blocks, the difference was not statistically significant (p > 0.05). In both types of tissues, single HPV infections were predominant, with HPV16 accounting for 47% of positive cases. Comparison in the overall agreement, taking into accounts not only positivity in general, but also HPV types, showed a 65% agreement (complete agreement of 59.7%, partial agreement of 5.3%) and complete disagreement of 35.0%. HPV detection in squamous cell carcinomas (SCC) and adenocarcinomas (ADC) was similar in fresh tissue or paraffin blocks (p ≥ 0.05).p16 immunostaining in samples that had at least one HPV negative results showed that 24 out of 25 cases had an over-expressed pattern., Conclusions: HPV DNA detection was lower among ADC as compared to SCC. However, such differences were minimized when additional p16 testing was added, suggesting that the technical issues may largely explain the HPV negative cases.

A large number of human papillomavirus (HPV) types, distributed over five papillomavirus genera, are detectable in the skin. HPV types belonging to the alpha, gamma, and mu genera have been detected in cutaneous warts. A state-of-the-art HPV genotyping assay for these cutaneous wart-associated HPV types does not exist although warts constitute a highly prevalent skin condition, especially in children (33%) and organ transplant recipients (45%). Cutaneous warts are again the focus of attention as their clinical relevance rises with the increasing number of chronically immunosuppressed patients. The objective of this study was to develop and evaluate a DNA-based genotyping system for all known cutaneous wart-related HPV types using PCR and Luminex xMAP technology. The broad-spectrum PCR amplified DNA of all known wart-associated HPV types from the genera alpha (HPVs 2, 3, 7, 10, 27, 28, 29, 40, 43, 57, 77, 91, and 94), gamma (HPVs 4, 65, 95, 48, 50, 60, and 88), mu (HPVs 1 and 63), and nu (HPV41). The probes were evaluated using plasmid HPV DNA and a panel of 45 previously characterized cutaneous wart biopsy specimens showing high specificity. HPV was also identified in 96% of 100 swabs from nongenital cutaneous warts. HPV types 1, 2, 27, and 57 were the most prevalent HPV types detected in 89% of the swabs. In conclusion, this Luminex-based genotyping system identifies all known cutaneous wart HPV types including phylogenetically related types, is highly HPV type specific, and is suitable for large-scale epidemiological studies.

Background: While infections with human papillomavirus (HPV) are highly prevalent among sexually active young women in Uganda, information on incidence, clearance and their associated risk factors is sparse. To estimate the incidence, prevalence and determinants of HPV infections, we conducted a prospective follow-up study among 1,275 women aged 12-24 years at the time of recruitment. Women answered a questionnaire and underwent a pelvic examination at each visit to collect exfoliated cervical cells. The presence of 42 HPV types was evaluated in exfoliated cervical cells by a polymerase chain based (PCR) assay (SPF10-DEIA LiPA)., Results: Three hundred and eighty (380) of 1,275 (29.8%) women were followed up for a median time of 18.5 months (inter-quartile range 9.7-26.6). Sixty-nine (69) women had incident HPV infections during 226 person-years of follow-up reflecting an incidence rate of 30.5 per 100 person-years. Incident HPV infections were marginally associated with HIV positivity (RR = 2.8, 95% CI: 0.9 - 8.3). Clearance for HPV type-specific infections was frequent ranging between 42.3% and 100.0% for high- and 50% and 100% for low-risk types. Only 31.2% of women cleared all their infections. Clearance was associated with HIV negativity (Adjusted clearance = 0.2, 95% CI: 0.1 - 0.7) but not with age at study entry, lifetime number of sexual partners and multiplicity of infections. The prevalence of low-grade squamous intraepithelial lesions (LSILs) was 53/365 (14.5%). None of the women had a high-grade cervical lesion (HSIL) or cancer. Twenty-two (22) of 150 (14.7%) HPV negative women at baseline developed incident LSIL during follow-up. The risk for LSIL appeared to be elevated among women with HPV 18-related types compared to women not infected with those types (RR = 3.5, 95% CI: 1.0 - 11.8)., Conclusions: Incident HPV infections and type-specific HPV clearance were frequent among our study population of young women. These results underscore the need to vaccinate pre-adolescent girls before initiation of sexual activity.

Rotavirus infections can be diagnosed in stool samples by serological and molecular methods. We developed a novel reverse transcriptase PCR (RT-PCR) method for the amplification of rotavirus RNA and a reverse hybridization assay on a strip to detect amplimers and identify the specific G and P genotypes present in human stool specimens. An additional aim was to permit specific identification of the rotavirus G1P[8] strain, used in the Rotarix vaccine. Novel broad-spectrum PCR primers were developed for both VP4 and VP7, permitting the amplification of a wide range of rotavirus genotypes. Primer sets comprise mixtures of defined primer sequences. For the identification of G and P genotypes, two reverse hybridization strip assays were developed. Both the VP4 and the VP7 strip contain universal probes for the detection of VP4 and VP7 sequences, irrespective of the G or P genotype. The VP4 strip contains type-specific probes for P[4], P[6], P[8], P[9], and P[10]. The VP7 strip contains type-specific probes for G1, G2, G3, G4, G5, G6, G8, and G9. In addition, probes to distinguish between wild-type G1 and G1 vaccine strain sequences were present. Testing by analysis of multiple reference strains confirmed that both RT-PCR methods allowed the detection of a broad spectrum of genotypes. RT-PCR for VP7 was more sensitive than RT-PCR for VP4, but all samples identified as positive for rotavirus antigen by an enzyme-linked immunosorbent assay (ELISA) were also positive for both VP4 and VP7. The high specificity of the reverse hybridization method was confirmed by sequence analysis as well as by type-specific PCR, and the vaccine strain could also be specifically identified. The reverse hybridization method permits accurate identification of mixed infections with different genotypes. Rotavirus genotypes for which no type-specific probes were present on the strip were adequately identified by the universal detection probes. The assay was formally validated by analyses of specificity, sensitivity, precision, accuracy, and robustness. In a panel of 149 ELISA-positive stool samples, comparison with conventional type-specific RT-PCR methods revealed the superiority of the novel method, mainly in cases of mixed rotavirus infections. This novel method permits highly accurate detection and identification of human rotavirus infections in stool samples. This validated assay could be useful for large-scale epidemiological and clinical trials.

The proportion of women who have already been exposed to human papillomavirus (HPV) infection by the time they first become pregnant, and the influence of pregnancy and delivery on the course of HPV infection are unclear. In Kampala, Uganda, 987 young primiparous pregnant women aged <25 years had gynaecological examination and liquid-based cytology. In the follow-up, women acted as their own controls, i.e., 1st/2nd versus 3rd trimesters (105 women), and during pregnancy versus after delivery (289 women). HPV was assessed using highly sensitive PCR assays. Prevalence of HPV and HIV infections at baseline were 60.0% and 7.3%, respectively. HPV16 and 18 were detected in 8.4% and 5.8%, respectively, i.e., less frequently than HPV51 (8.7%) and 52 (12.1%). At follow-up new HPV infections were detected in 42.9% of women between the 1st/2nd and 3rd trimesters, and 38.1% between pregnancy and delivery, but 50.4% and 71.8% of HPV infections, respectively, cleared, leaving HPV prevalence unchanged in the different periods. Prevalence of cytological abnormalities diminished after delivery (from 21.2% to 12.4%). Presence of genital warts and sexually transmitted infections other than HPV were the strongest risk factors for prevalent or incident HPV infection. Clearance was lower among HIV-positive women. In conclusion, HPV prevalence was high in primiparous women in Uganda, but pregnancy did not seem to be a period of special vulnerability to the infection., ((c) 2008 Wiley-Liss, Inc.)

Knowledge about the burden of Human Papillomavirus (HPV) infections in Sub-Saharan Africa is very limited. We collected cervical samples from 262 women from the general population and 241 tumor samples from women with invasive cervical cancer in Mozambique and tested them for HPV genotyping by the SPF(10)-LiPA(25) PCR system. Among the 195 women without cervical abnormalities by cytology HPV prevalence was 75.9%. In this group of women, the most frequently identified HPV types among HPV-positive women were in descending order of frequency: HPV51 (23.6%), HPV35 (19.6%), HPV18 (14.2%), HPV31 (13.5%) and HPV52 (12.8%). In women with cervical cancer HPV DNA detection was 100%. The type-specific distribution of the most frequent types in descending order of frequency was: HPV16 (47.0%), HPV18 (31.3%), HPV51 (14.8%), HPV52 (14.3%), HPV45 (12.6%), HPV35 (10.4%), HPV33 (4.8%) and HPV31 (2.6%). HPVs 16/18 and HPVs 16/18/31/45 were detected in 71.7% and 80.9% of cervical cancer tissue, respectively. While HPVs 51 and 35 were the two most common types in cytologically normal women in Mozambique, HPVs 16 and 18 remained the two most frequently identified types in cervical cancer. The introduction of an efficacious HPV 16/18 vaccine could potentially prevent the occurrence of 72% of cervical cancer cases and up to 81% of the cases if full cross-protection against HPVs 31 and 45 is assumed.

Chlamydia trachomatis (Ct) comprises distinct serogroups and serovars. The present study evaluates a novel Ct amplification, detection, and genotyping method (Ct-DT assay). The Ct-DT amplification step is a multiplex broad-spectrum PCR for the cryptic plasmid and the VD2-region of ompl. The Ct-DT detection step involves a DNA enzyme immunoassay (DEIA) using probes for serogroups (group B, C, and intermediate) and the cryptic plasmid, permitting sensitive detection of 19 Ct serovars (A, B/Ba, C, D/Da, E, F, G/Ga, H, I/Ia, J, K, L1, L2/L2a, and L3) without any cross-reactivity with other Chlamydia species and pathogenic bacteria or commensal organisms of the genital tract. Ct-positive samples are analyzed by a nitrocellulose-based reverse hybridization assay (RHA) containing probes for the 19 different serovars and for the cryptic plasmid. The sensitivity of the PCR-DEIA on clinical specimen is equivalent to that of the Cobas TaqMan assay [kappa = 0.95 (95% confidence interval = 0.92 to 0.99)]. Using the RHA, 98% of the Ct-DT detection step-positive samples could be typed. Analysis of cervical swabs from Uganda and The Netherlands revealed that the most common serovars in Uganda are G/Ga (45%), E (21%), K (13%), and F (8%), and in The Netherlands serovars E (38%), F (23%), G/Ga (11%), and D/Da (7%) were most common. Thus, multiplex broad-spectrum PCR in combination with DEIA and RHA permits highly sensitive and specific detection and identification of Ct serovars.

Infections with human papillomaviruses (HPVs) belonging to the genus Betapapillomavirus have been linked to the development of non-melanoma skin cancer. Although persistence is expected, systematic investigation of this aspect of betapapillomavirus (beta-PV) infection has not been conducted. This study investigated the prevalence and persistence of 25 known beta-PV types in the skin of immunocompetent individuals. Over a 2 year period, eight consecutive plucked eyebrow hair samples taken from 23 healthy individuals were analysed for the presence of beta-PV DNA. Using a recently published general beta-PV PCR and genotyping method, 61% of the individuals were beta-PV DNA positive for one or more types at intake, whereas during follow-up this percentage rose to 96%. HPV23 was the most frequently detected beta-PV type. Type-specific beta-PV DNA was detected over 6 months or longer in 74% of the individuals. In 57% of the individuals, DNA from multiple beta-PV types was detected simultaneously for 6 months or longer. When the detection intervals of all beta-PV type-specific infections in the study population were considered, a substantial proportion, 48%, lasted at least half a year. The consistent beta-PV patterns found over time in most individuals strongly suggested that beta-PV DNA detection in plucked eyebrow hairs reveals true beta-PV infection. If the minimum interval of detection was set at 6 months, persistent beta-PV infections were found in the majority of the study population (74%).

The use of a single broad-spectrum human papillomavirus (HPV) DNA-based PCR test may fail to detect lower concentrations of HPV DNA due to competition between different genotypes in mixed infections. To improve HPV detection by PCR, broad-spectrum and type-specific (TS) PCRs were combined, with a focus on HPV-16 and HPV-18. Cervical and cervicovaginal cell samples were obtained from 1,113 healthy women (age range, 15 to 25 years) participating in an HPV-16/HPV-18 candidate vaccine efficacy trial. These samples were tested by a broad-spectrum SPF(10) PCR-DNA enzyme immunoassay, followed by a primer SPF(10)-based line probe assay (SPF(10) LiPA), and HPV-16- and HPV-18-TS PCRs. The results for the majority of the HPV-16/18 SPF(10) LiPA-positive samples were confirmed by TS-PCR (kappa values, 0.775 for HPV-16 and 0.785 for HPV-18). However, TS PCR revealed additional positive samples among those that contained other HPV genotypes due to competition. Conversely, SPF(10) LiPA identified HPV-16 or -18 in samples that remained negative by TS PCR as a result of sampling variation. Analysis of follow-up samples from more than 1,000 women confirmed that the combination of SPF(10)-LiPA with additional HPV-16- and HPV-18-TS PCR diminishes the rate of false-negative diagnosis. The combination of broad-spectrum and TS PCRs resulted in a novel testing algorithm. This combination of assays is more accurate than either method alone, and the novel algorithm offers a highly accurate and effective method for the analysis of HPV infections.

Human papillomavirus can be detected by amplification of viral DNA. A novel one-step PCR (PM-PCR) was evaluated for amplification of a 117-bp fragment from the E1 region. It permitted ultrasensitive detection of all 25 known human papillomavirus genotypes from the beta-papillomavirus genus. The intra- and intertypic sequence variations of the 77-bp interprimer region were studied. Genotype-specific probes as well as general probes were selected for the 25 established beta-papillomavirus types, and a reverse hybridization assay (RHA) was developed (PM-PCR RHA method). The analytical sensitivity of the PM-PCR RHA method was 10 to 100 viral genomes. The one-step PM-PCR turned out to be more sensitive than the previously described nested MaHa-PCR for beta-papillomavirus detection. The PM-PCR RHA method was able to detect and identify beta-papillomavirus types in frozen patient material as well as in poorly amplifiable material such as formalin-fixed, paraffin-embedded skin biopsy specimens. Inter- and intralaboratory variability experiments showed that the reproducibility of the assay was very high. In conclusion, the one-step PM-PCR together with the RHA allows extremely sensitive, specific, and reproducible detection of beta-papillomavirus DNA as well as reliable identification of beta-papillomavirus genotypes in both fresh and paraffin-embedded patient material.

Infection with human immunodeficiency virus (HIV) and the resulting immunosuppression are associated with an increased risk for human papillomavirus (HPV) persistence and related malignancies. In the present study we investigated the prevalence of HPV in urine samples from 104 HIV-infected men with low CD4+ cell counts (<100 per mm(3)) and 115 urine samples from HIV-negative men. A high prevalence of HPV DNA (39.4%) was found in the HIV patients. Most of the HPV types were high risk (81.4%), with HPV 52 as the most prevalent type (12.5%), followed by HPV 18 (6.7%), HPV 35 (5.8%), and HPV 70 (4.8%). Multiple HPV genotypes were observed in 17 (41%) of the 41 HPV- and HIV-positive men. In contrast, only 11 (9.6%) HPV DNA-positive cases were observed among the 115 HIV-uninfected men, and 3 (27.3%) contained multiple genotypes. Quantitative analyses indicated that the HPV viral load, as measured in urine samples, is significantly higher in HIV-positive men compared to HIV-negative men. In the present study we show that urine samples are useful for detecting HPV DNA, there is a high prevalence of HPV in HIV-positive men, and the HPV viral load is substantially higher in HIV-positive than in HIV-negative men. More studies are needed to evaluate the risk and natural development of HPV-related malignancies in HIV-positive men.

Background: HPV infection in young women is common. However only a certain number of HPV genotypes are oncogenic. It is necessary for high risk HPV infection to persist at the cervix for a considerable time before oncogenesis occurs., Objectives: To look for persistence of high risk HPV in women attending a colposcopy clinic. Two DNA detection methods were used and the results compared to determine the rates of persistent, resolved and acquired infections over a 6-month period. HPV genotyping was used to determine type specific persistence., Study Design: One hundred and thirty-eight women were tested for HPV infection when attending the colposcopy clinic at UCLH and then tested again at a subsequent visit approximately 6 months later. HPV DNA was detected by the Digene HC II assay using the high risk probes only and by PCR with the SPF10 primer set. All SPF10 PCR-positive samples were then specifically genotyped by a Line Probe Assay (LiPA) [Kleter et al. 1999. J. Clin. Microbiol. 1999;37:2508]., Results: At entry of the study high risk HPV was detected in 43% of the samples by Digene HC II and in 60% of the samples by SPF10/LiPA. Thirty-eight (28%) of the women had a true persistent infection with the same high risk HPV genotype over a median period of 6.3 months. Nine (7%) women resolved one HR HPV infection after their first colposcopy visit, but obtained a different high risk HPV infection by the time they were tested at their second visit as identified by LiPA. Thirty-seven (27%) of the 138 women had mixed HPV infections, representing 45% of all those infected., Conclusions: The SPF10/LiPA assay detected more high risk infections than the Digene HC II assay. The Digene HC II assay was unable to distinguish between persistent infections with the same high risk genotype and those where the genotype had changed between visits.

Human papillomaviruses (HPVs) comprise more than 100 genotypes. The mucosal types can be divided into high-risk and low-risk (LR) types depending on the associated disease risk. HPV infection is mainly diagnosed by molecular methods, since reliable serological tools are not available and culture of the virus is not possible. Accurate molecular diagnostic techniques that can be used to inform patient management and follow-up after treatment are now available for detection and identification of HPV. The diagnosis of HPV infections in patients at risk of disease in a clinical setting requires a different approach from that used for epidemiological studies, vaccination trials and natural history studies. This review describes the different molecular methods available for HPV detection and genotyping and their possible clinical utility.

Background: In sub-Saharan Africa, cervical cancer is the leading cancer among women. The causative role of different human papillomavirus (HPV) types in cervical cancer is established, but the distribution of HPV types within this region is largely unknown., Goal: The goal was to study the distribution of HPV among family planning clinic attendees in Nairobi, Kenya., Study Design: This was a cross-sectional study of persons attending a family planning center in Nairobi, Kenya., Results: HPV data of 429 women were analyzed; 7.0% had low-grade intraepithelial lesions, 6.8% had high-grade intraepithelial lesions, and 0.23% had invasive cancer. One hundred ninety samples (44.3%) were HPV-positive (28.4% were positive for multiple types). The most common HPV types were HPV 52 (17.9% of positive samples), HPV 16 (14.7%), HPV 35 (11.6%), and HPV 66 (9.0%). The risk of high-grade squamous intraepithelial lesions (HSIL) was 88.5 times higher (95% CI, 8.5-1.4 x 10 ) in HPV 16-positive women than in HPV-negative women. Relative risks were 54.3 (95% CI, 4.0-1.4 x 10 ) for HPV 35, 49.2 (95% CI, 3.6-9.5 x 10 ) for HPV 52, and 21.7 (95% CI, 0.0-1.9 x 10 ) for HPV 18. The prevalence of HSIL was not increased in association with HIV-positivity, yet HIV-1 was significantly associated with high-risk HPV types ( P< 0.00001)., Conclusion: The pattern of HPV distribution in this population was different from that in other regions in the world, which has important consequences for HPV vaccine development.

A group of 208 human immunodeficiency virus (HIV)-infected women in Brazil were studied for the presence of human papillomavirus with the general SPF(10) PCR primer set. Virtually all (98%) women were found positive for human papillomavirus (HPV) DNA. Genotyping by the reverse hybridization line probe assay (HPV-LiPA) revealed a high prevalence of multiple genotypes (78.9% of the cases), with an average of 3.1 genotypes per patient (range, 1 to 10 genotypes). HPV 6 was the most prevalent genotype and was observed in 80 (39.2%) patients, followed by types 51 (31.9%), 11 (26.0%), 18 (24.0%), and 16 (22.5%). Of the genotypes detected, 40.9% were low-risk genotypes. Twenty-two (10.5%) patients showed normal (Pap I) cytology, 149 (71.6%) patients had inflammation (Pap II), and 28 patients (13.4%) had a Pap III score. The prevalence of high-risk genotypes increased with the cytological classification. There were no significant associations between the number of HPV genotypes detected and the cytological classification, HIV viral load, and CD4 count in these patients. In conclusion, the highly sensitive SPF(10) LiPA system shows that a very high proportion of HIV-infected women in Brazil are infected with HPV and often carry multiple HPV genotypes.

Human papillomavirus (HPV) is the main etiological agent of cervical cancer. There is a large number of HPV genotypes and therefore a need to distinguish the high risk HPV genotypes associated with invasive cancer from the low risk. Because persistence of high risk HPV infection is necessary for progression of a pre-invasive cervical change one needs to identify the individual genotype to see if it persists. PCR amplification of HPV DNA is described using two consensus primer systems from cervical cells. Amplified HPV DNA was genotyped using a reverse hybridization line probe assay (LiPA). HPV DNA was amplified from 42% of samples by MY09/11 and from 80% by SPF10. In 42 samples HPV DNA was detected by both primer sets and in 38 samples only the SPF10 primers detected HPV DNA. The LiPA detected 21 different HPV genotypes (13 high risk) in this cohort of samples. Forty-three percent contained a single HPV genotype and 24% contained multiple infections (2-5 genotypes). Overall, high risk HPV genotypes were detected in 48% of the cervical samples, the most frequent types were 16, 18, 31, and 51. The proportion of high risk HPV genotypes increased with more severe cytological abnormalities. This study demonstrates that the SPF10 primer set is more sensitive than the MY09/11 primer set and that genotyping by LiPA tells us if the HPV infection is caused by a high risk type and if the infection is mixed. Additionally LiPA provides information about the individual genotype when looking for persistence of infection. HPV DNA detection and genotyping is therefore a useful tool in the colposcopy clinic, used in conjunction with cytology., (Copyright 2002 Wiley-Liss, Inc.)

A comparison of two PCR-based human papillomavirus (HPV) DNA detection and genotyping systems (PGMY LBA and SPF(10) LiPA) was conducted in two laboratories. Both systems are based on broad-spectrum PCR for the detection of HPV DNA, followed by reverse hybridization with type-specific probes. A total of 400 selected cervical scrape specimens in PreservCyt solution (55% normal cytology, 18% atypical squamous cells of unknown significance, 14.8% low-grade squamous intraepithelial lesions [SIL], and 12.5% high-grade SIL) were tested for the presence of HPV DNA. In this selected group of specimens, the overall agreement between the two methods for the detection of any HPV DNA was high (kappa = 0.859). When the 20 common HPV genotypes identified by both methods were considered (HPV types 6, 11, 16, 18, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 56, 58, 59, 66, and 68), compatible genotype-specific results were observed in 96.5% of the samples, even when multiple HPV genotypes were present. However, for some specific HPV genotypes, there were significant differences in HPV detection by the two methods. PGMY LBA detected more HPV type 42 (P = 0.002), HPV type 56 (P = 0.039), and HPV type 59 (P < 0.001), whereas SPF(10) LiPA detected more HPV type 31 (P < 0.001) and HPV type 52 (P = 0.031). For the remaining genotypes, including HPV types 16 and 18, the results obtained by the two methods were not significantly different. In general, both genotyping methods are highly suitable for clinical and epidemiological studies.

Recent studies have indicated that the use of the MIB-1 immunostaining may be useful in distinguishing endocervical neoplasia from benign nonneoplastic lesions. We sought to investigate this finding further with a specific emphasis on the common benign processes that may result in a nonspecific increase of MIB-1 staining. In this study we quantified the MIB-1 immunostaining in the mucinous endocervical epithelium (n=45) and in tubal metaplasia (n=28) during the proliferative and secretory phases (hormonal influence), in the mucinous endocervical epithelium in cases of cervicitis (inflammation) (n=10), in cases with a history of a recent biopsy (regeneration) (n=15), endocervical polyps (benign growth) (n=8), in the endocervical glands adjacent to a squamous intraepithelial lesion (human papilloma virus [HPV] infection) (n=63), and in in situ and invasive cervical adenocarcinomas (n=30). All cases with increased MIB-1 staining were subsequently tested for the presence of HPV DNA. The range of MIB-1 staining in the benign endocervical epithelium was from 0% to 48% and in the neoplastic epithelium from 25% to 84%. MIB-1 staining below 10% always reflected a benign process and MIB-1 staining higher than 50% was always associated with a neoplasia. Rare benign cases (tubal metaplasia during the proliferative phase, glands adjacent to squamous intraepithelial lesions, and cases with a history of a recent biopsy) had increased MIB-1 index, which overlapped with the neoplastic cases. In conclusion, MIB-1 is a useful marker of endocervical neoplasia, although in rare cases an overlap between benign and neoplastic cases may exist.

Human papillomavirus infections are associated with the development of cervical neoplasia. Human papillomavirus is a group of heterogeneous viruses, comprising many genotypes, which can be divided into high-risk and low-risk types, depending on their association with disease. Therefore, accurate molecular diagnostic tools are required for detection and identification of human papillomavirus. Monitoring of human papillomavirus infection is necessary for adequate patient management and follow-up during treatment. This review describes the different molecular methods available for human papillomavirus detection and identification of genotypes.

To clarify the role of human papillomavirus (HPV) in penile cancer we evaluated the prevalence of HPV DNA in different histological subtypes of penile carcinoma, dysplasia, and condyloma using a novel, sensitive SPF10 HPV polymerase chain reaction assay and a novel genotyping line probe assay, allowing simultaneous identification of 25 different HPV types. Formalin-fixed, paraffin-embedded tissue samples were collected from the United States and Paraguay. HPV DNA was detected in 42% cases of penile carcinoma, 90% cases of dysplasia, and 100% cases of condyloma. There were significant differences in HPV prevalence in different histological cancer subtypes. Although keratinizing squamous cell carcinoma and verrucous carcinoma were positive for HPV DNA in only 34.9 and 33.3% of cases, respectively, HPV DNA was detected in 80% of basaloid and 100% of warty tumor subtypes. There was no significant difference in HPV prevalence between cases from Paraguay and the United States. In conclusion, the overall prevalence of HPV DNA in penile carcinoma (42%) is lower than that in cervical carcinoma (approximately 100%) and similar to vulvar carcinoma (approximately 50%). In addition, specific histological subtypes of penile cancer--basaloid and warty--are consistently associated with HPV, however, only a subset of keratinizing and verrucous penile carcinomas is positive for HPV DNA, and thus these two tumor groups seem to develop along different pathogenetic pathways.

Unlabelled: The prevalence of human papillomavirus (HPV) rises with increasing histological severity of neoplasia, more cigarettes smoked per day and higher lifetime number of sexual partners in women with cervical dyskaryosis. Recently, the highly sensitive SPF10 primers and Inno-LiPA (line probe assay) HPV prototype research assay became available for the detection and typing of HPV., Background: using this system, we challenged the previously reported findings., Study Design: the study group comprised 304 women referred because of abnormal pap smears in whom a histological diagnosis was made. Data on the lifetime number of sexual partners and smoking behaviour were obtained by questionnaire. HPV analysis was performed on cervical scrapes obtained at the enrollment visit., Results: oncogenic HPV was found in 288 (95%) women. A total of 86 (30%) out of these 288 women disclosed multiple types. HPV 16 occurred significantly less often in multiple infections than was expected on the basis of chance alone. The grade of neoplasia was significantly associated with the presence of oncogenic HPV, and this association depended on the presence of HPV type 16. No association was found between grade of neoplasia and the presence of multiple HPV types. Neither the lifetime number of sexual partners nor smoking were associated with oncogenic HPV, the five most frequent HPV types separately or the presence of multiple types., Conclusion: we conclude that the association between the detection of HPV and the epidemiological risk factors, as found with the GP5/6 PCR in the past, could not be confirmed when using SPF10 PCR primers and LiPA HPV genotyping. We suggest that the number of sexual partners and smoking may be determinants of high HPV viral load rather than determinants of the presence of HPV per se.

The prevalence of human papilloma virus (HPV) DNA in different histological subtypes of cervical adenocarcinoma and related tumors was examined using formalin-fixed, paraffin-embedded tissue samples from 105 primary cervical adenocarcinomas and adenosquamous carcinomas. Broad-spectrum HPV DNA amplification and genotyping was performed with the SPF10 primer set and line probe assay (LiPA), respectively. HPV DNA was detected in 82 of 90 (91%) mucinous adenocarcinomas, encompassing endocervical, intestinal, and endometrioid histological subtypes, and in nine of nine adenosquamous tumors (100%). HPV DNA was not detected in any nonmucinous adenocarcinomas including clear cell, serous, and mesonephric carcinomas (0/6). The most common viral types detected in adenocarcinoma were HPV 16 (50%) and HPV 18 (40%), followed by HPV 45 (10%), HPV52 (2%), and HPV 35 (1%). Multiple HPV types were detected in 9.7% of the cases. In conclusion, mucinous adenocarcinomas and adenosquamous carcinomas of the cervix demonstrate a very high prevalence of HPV DNA, similar to that reported for cervical squamous cell carcinoma. Only rare histological variants of cervical adenocarcinoma seem unrelated to HPV infection.

Human papillomavirus (HPV) can be detected by amplification of viral DNA. A novel PCR primer set generating a short PCR fragment (SPF PCR) was used for amplification of a fragment of only 65 bp from the L1 region and permitted ultrasensitive detection of a broad spectrum of HPV genotypes. The intra- and intertypic sequence variations of the 22-bp interprimer region of this amplimer were studied. Among 238 HPV sequences from GenBank and clinical specimens, HPV genotypes were correctly identified based on the 22-bp sequence in 232 cases (97.2%). Genotype-specific probes for HPV genotypes 6, 11, 16, 18, 31, 33 to 35, 39, 40, 42 to 45, 51 to 54, 56, 58, 59, 66, 68, 70, and 74 were selected, and a reverse hybridization line probe assay (LiPA) (the INNO-LiPA HPV prototype research assay) was developed. This LiPA permits the use of amplimers generated by the SPF as well as the MY 09/11 primers. The assay was evaluated with a total of 1, 354 clinical specimens, comprising cervical scrapes (classifications ranging from normal cytology to severe dyskaryosis) and formalin-fixed, paraffin-embedded cervical carcinoma samples. LiPA results were highly concordant with sequence analysis of the SPF amplimer, genotype-specific PCR, and sequence analysis of amplimers generated by MY 09/11 primers. The sensitivity of the SPF primers was higher than that of the GP5(+)/6(+) primers over a broad range of HPV types, especially when multiple HPV genotypes were present. In conclusion, the SPF LiPA method allows extremely sensitive detection of HPV DNA as well as reliable identification of HPV genotypes in both cervical smears and paraffin-embedded materials.

Background: The correlation between human papillomavirus (HPV) infection and tumor prognosis in 159 Russian women with cervical carcinoma was investigated. The presence of various HPV types was correlated with the histologic parameters of the carcinomas and with their immunoreactivity with antibodies to p53, Ki-67-Ag, and bcl-2., Methods: Formalin fixed, paraffin embedded tissue specimens representing 159 cases of International Federation of Gynecology and Obstetrics Stage I and II were used. HPV DNA was detected by polymerase chain reaction (PCR) using a general primer set that targets the L1 region and synthesizes a product of only 65 base pairs. The HPV types were determined by direct sequencing and compared with known HPV types., Results: All 159 carcinomas were positive for HPV. HPV 16 (64.8%) was most frequently found, followed by HPV 18 (10.7%) and HPV 45 (8.2%). In 6 patients (3.8%), HPV types could not been further classified, and these cases were therefore categorized as HPV X. Although a trend was noted toward poorer prognosis for women with carcinomas harboring HPV types 16, 18, and 45 than for patients with carcinomas harboring HPV types 31, 33, 35, 52, 56, 58, and 68, the differences were not statistically significant. The prevalence of adenocarcinoma and adenosquamous carcinoma was higher among HPV 18 positive patients than among patients with the other known HPV types (P=0.0002)., Conclusions: The rate of HPV positivity in these 159 cervical carcinomas was 100%. These findings challenge the assumption that HPV negative cervical carcinomas exist. This high rate might be attributed to the use of a new broad-spectrum HPV PCR test. HPV typing in cervical carcinoma was not significantly related to clinical outcome. HPV 18 was significantly more frequently found in adenocarcinoma and adenosquamous carcinoma. The possibility of classifying HPV 45 as an oncogenic high risk type should be considered.

A novel set of polymerase chain reaction (PCR) primers, designated SPF1 and SPF2 and located in the L1 region, was developed for universal detection of human papillomavirus (HPV). A short PCR fragment (SPF) of only 65 pb was synthesized. SPF amplimers were detected in a microtiter-based hybridization system, using a mixture of oligonucleotide probes. The SPF system allowed detection of at least 43 different HPV genotypes. The clinical performance of the novel SPF system was assessed in three different patient groups. 1) Analysis of 534 cervical scrapes, obtained from treated patients, showed that the detection rate in 447 (83.7%) scrapes with normal cytology was significantly higher using the SPF system as compared with the universal primer set GP5+/6+ (P < 0.001). 2) The SPF assay detected HPV DNA in 299 (98.4%) of 304 scrapes with cytological dyskaryosis. 3) The SPF system detected HPV DNA in 100% of 184 formalin-fixed, paraffin-embedded cervical carcinoma specimens. In conclusion, the novel SPF system permitted universal and highly sensitive detection of HPV DNA in diverse clinical materials and may improve the molecular diagnosis and epidemiology of this important virus.

Background and Objectives: Hepatitis G virus (HGV) is a recently discovered viral agent transmitted by blood, which was firstly identified in patients with acute or chronic liver disease. HGV prevalence in US blood donors was recently found to average 1-2%. We report a much higher HGV frequency among blood donors of São Paulo, Brazil., Materials and Methods: 200 serum samples were submitted to RT-PCR using primers directed to the 5' untranslated region and nonstructural 5A (NS5A) region. PCR products were analyzed by gel electrophoresis and Southern blot hybridization., Results: Of the 200 specimens, 18 (9%; 95% CI 5.4-13.8%) were positive by both sets of primers. Sequence analysis of the NS5A PCR products revealed a homology of 96.3%. Of the 18 HGV-positive samples, only one was positive for anti-HBc and all were anti-HCV- and HCV-RNA-negative., Conclusion: Such a high prevalence of HGV in a nonsymptomatic population suggests that this is a benign agent.

To test the theoretical possibility of 5'-UR mistyping between hepatitis C virus subtypes 1a and 1b, we combined a 5'-UR/Core line probe assay (LiPA) with a nested PCR system and retested 183 sera, previously genotyped as type 1a or 1b and originating mainly from Western Europe. Eight percent of these were found to be wrongly subtyped. Based on this method, 3 additional subtypes of type 1 were discovered (1d-1f). Randomly selected European type 2 sera (n = 18) were tested with a similar type 2 5'-UR/Core LiPA. They were unexpectedly found to belong to subtype 2c in the majority of cases. Among serum samples originating from South-East Asia, several additional genotypes (7a, 7c, 7d, and 9a) were detected which had 5'-UR sequence motifs indistinguishable from genotype 1. Based on 13,203 pairwise comparisons in the 340-bp NS5B region, classification into types, subtypes, and isolates was obtained in 99.8% of all cases by using the phylogenetic border value of 0.328 for subtypes/types and 0.127 for isolates/subtypes; and evidence for a 10th major type of HCV was provided. Combination of all available HCV sequence data from the 447-bp Core/E1 region and the NS5B 340-bp and 222-bp regions provided evidence for the existence of 10 types, including 50 subtypes. Previously, extensive studies involving genotypes 1a, 1b, 2a, and 2b indicated the importance of HCV subtyping in interferon treatment and progression of chronic liver disease. The herein described expansion in the number of HCV types and subtypes should help improve diagnosis, treatment and possibly prophylaxis of hepatitis C liver disease.

The 5' untranslated regions derived from 54 patients with a chronic hepatitis C virus infection were analyzed to determine the (sub)type of hepatitis C virus. Labelled polymerase chain reaction products from 5' untranslated region were used as probes for reverse hybridization in a line probe assay (Inno-LiPA) and results were validated by comparison with direct sequencing data. Five different genotypes could be distinguished based on 5' untranslated region sequence diversity. Results of typing by line probe assay and direct sequencing were similar. Antibody responses against core, NS-3, NS-4 and NS-5 epitopes were detected by RIBA-4 and Inno-LIA HCVAb II confirmatory assays. There was no consistent correlation between genotype and anti-HCV responses, although types 2, 3 and 4 hepatitis C virus isolates show poor reactivity with NS-4 ep!%"pes.