Human papillomavirus infection and squamous cell carcinoma of the conjunctiva.

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.

Detection and typing of human papillomavirus DNA in penile carcinoma: evidence for multiple independent pathways of penile carcinogenesis.

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.

Using a new HPV detection system in epidemiological research: change of views on cervical dyskaryosis?

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.

Comparative analysis of human papillomavirus infections in cervical scrapes and biopsy specimens by general SPF(10) PCR and HPV genotyping.

Human papillomavirus (HPV) can be detected by DNA amplification from clinical samples. The aim of the present study was to compare the HPV status in both cervical scrape and biopsy specimens obtained from 174 patients, using the recently developed broad spectrum SPF(10) PCR-LiPA method. The detection rate of HPV in these materials was determined and the spectrum of HPV genotypes was compared. Cervical scrapes and biopsy specimens were obtained, either on the same day (group I), or with an interval of up to almost 2 years (group II, mean interval 97 days, range 1-469 days). HPV DNA was amplified by SPF(10) PCR and detected in a microtitre plate hybridization assay. Of the HPV-positive cases, the genotype was determined by reverse hybridization of the same SPF(10) amplimer on a line probe assay (LiPA), discriminating between HPV genotypes 6, 11, 16, 18, 31, 33-35, 39, 40, 42-45, 51-54, 56, 58, 59, 66, 68, 70, and 74. The results showed that the detection rate and the spectrum of HPV genotypes in cervical scrapes and the corresponding biopsy specimens were highly comparable in both patient groups, even when multiple genotypes were present. In both groups, multiple HPV genotypes were more frequently detected in cervical scrapes than in the corresponding biopsy specimens. In conclusion, HPV infection can be diagnosed in cervical scrapes and biopsy specimens using the SPF(10) PCR-LiPA system. Analysis of cervical scrapes accurately reflects the spectrum of HPV genotypes in the patient's cervical region, even with a sampling interval between the cervical scrape and the biopsy specimen.

Molecular detection and genotyping of human papillomavirus.

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.

Prevalence of human papillomavirus DNA in different histological subtypes of cervical adenocarcinoma.

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.

Development and clinical evaluation of a highly sensitive PCR-reverse hybridization line probe assay for detection and identification of anogenital human papillomavirus.

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.

Detection and typing of human papillomavirus in cervical carcinomas in Russian women: a prognostic study.

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.

Novel short-fragment PCR assay for highly sensitive broad-spectrum detection of anogenital human papillomaviruses.

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.

Efficacy of interferon dose and prediction of response in chronic hepatitis C: Benelux study in 336 patients.

BACKGROUND/AIMS: In an attempt to improve the limited efficacy of treatment of chronic hepatitis C with interferon-alpha 3 MU tiw, we studied the effects of double-dose therapy followed by downward titration, and analyzed the pre- and pertreatment factors associated with response or non-response. METHODS: Three hundred and fifty-four consecutive patients in 19 centers were randomized to interferon-alpha 3 MU tiw for 6 months or 6 MU tiw for 8 weeks followed by down-titration (3,1 MU tiw) till alanine aminotransferase remained normal and plasma HCV RNA was repeatedly undetectable. The primary outcome measure was sustained alanine aminotransferase and HCV RNA response 6 months after treatment. RESULTS: Three hundred and thirty-six patients received treatment. The sustained response rate for patients receiving 3 MU tiw for 6 months was 14% (9-21%,) and for patients receiving double dose tiw for 8 weeks and thereafter titrated therapy 15% (10-21%) (p=0.8). Pretreatment factors associated with a sustained alanine aminotransferase plus HCV RNA response were the absence of cirrhosis, presence of genotype 2 or 3, a low viral load and, in addition, a low alanine aminotransferase/aspartate aminotransferase ratio; a model was developed to allow estimation of the chance of response for the individual patient. The most powerful predictor of sustained response, however, was plasma HCV RNA at week 4; a positive test virtually precluded a sustained response (1.7%, 0.4-5.0%). If week 4 HCV RNA was not detectable, the chance of a sustained response was 21% (12-34%) for genotype 1 versus 40% (28-54%) for the others (p=0.02). Six MU tiw led to a significantly higher week 4 HCV RNA response (47% not detectable) than 3 MU (37%) (p=0.02). During down-titration this difference in viral on-treatment response was lost. CONCLUSIONS: In the treatment of hepatitis C, an early HCV RNA response is a prerequisite for long-term efficacy. Doubling the initial interferon dose increases this early response, but subsequent downward titration negates this effect, especially in genotype 1.

Hepatitis C virus genotypes: epidemiological and clinical associations. Benelux Study Group on Treatment of Chronic Hepatitis C.

In a cohort of 292 chronic hepatitis C patients living in the Benelux countries the relationship between viral genotype and geographical origin, route of transmission, clinical characteristics and severity of liver disease was analyzed. HCV-RNA isolates could be classified by the Line Probe Assay (LiPA) as 1a, 1b, 2, 3, 4 or 5 in 286 (98%) cases. Patients of European origin were predominantly infected with HCV subtype 1b (164/254, 65%, CI 58-70%), as were patients of Asian origin (7/13, 54%). Patients originating from Surinam (South America) had predominantly type 2 (9/10, 90%), whereas Africans were mainly infected with type 4 (7/9, 77%). Blood transfusion was the mode of transmission in 142 (50%) patients, intravenous drug abuse (IVDA) in 40 (14%), occupational needle accident or tattoo in 11 (4%); no obvious source of infection was found in 93 (33%). In patients infected by blood transfusion, subtype 1b was predominant (70%, CI 61-77%), whereas subtypes la and 3 were predominant in those infected by IVDA (25% and 45%, respectively, p<0.001). Cirrhosis was observed in 68 (24%) patients; in multivariate analysis, factors independently related to cirrhosis were: the duration of infection, age and prior hepatitis B. No significant relationship was found between the severity of fibrosis or liver inflammation and the HCV (sub)types. In summary, in this large cohort of patients in the Benelux countries the hepatitis C virus (sub)type present was clearly related to the country of origin and the route of transmission, but not to the severity of liver disease.

Hepatitis G virus: prevalence and sequence analysis in blood donors of São Paulo, Brazil.

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.

Analysis of hepatitis C virus isolates by serotyping and genotyping.

Hepatitis C virus (HCV)-positive sera from 106 chronically infected patients which had previously been genotyped were characterized by serotyping. Genotypes were determined by a first-generation line probe assay (INNO-LiPA HCV) and by sequence analyses of the core, core-E1, and NS5B regions. HCV serotypes were determined by measuring type-specific antibodies to NS4-derived peptide antigens (Murex HCV serotyping 1-6 assay). Of 106 serum samples, serotype-specific antibodies were detected in 88 (sensitivity, 83.0%), and 77 (specificity, 87.5%) of these serotypeable samples revealed a corresponding serotype (total concordance, 72.6%). Eleven samples revealed discrepant results as follows. (i) Five serum samples in which only a single genotype was detected contained an additional serotype. (ii) In one sample with two genotypes only one serotype was detected. (iii) In five isolates the serotype (all serotype 1) was completely different from the genotype. Double infections, as determined by genotyping, were confirmed by serotyping in two of four cases. Of 11 serum samples from chronically infected hemodialysis patients, 7 (64%) were reactive in the serotyping assay. In conclusion, genotyping allows discrimination between (sub)types but requires the relatively complex reverse transcriptase PCR. The novel serotyping assay offers an alternative method to distinguish the major types of HCV, although the sensitivity of the assay may be limited by the immunocompetence of the infected host.

Hepatitis C virus genotyping by means of 5'-UR/core line probe assays and molecular analysis of untypeable samples.

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.

Analysis of hepatitis C virus genotypes by a line probe assay and correlation with antibody profiles.

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.

Rapid detection of hepatitis C virus RNA by direct capture from blood.

A new diagnostic assay for hepatitis C virus RNA detection is described. HCV genomic RNA is captured onto streptavidin-coated magnetic beads by solution hybridization with biotinylated complementary oligonucleotides. The specificity of the capture assay is confirmed using different capture oligonucleotides as well as sera representing different types of HCV. Sensitivity was determined by testing serial dilutions of a HCV infected plasma. A panel of 50 sera was tested for anti-HCV by a Line Immunoassay and for HCV-RNA by both a conventional guanidinium extraction method and the new capture assay. The specificity of the capture assay was 95.8% and the sensitivity was 92.3% compared to the standard protocol. This method provides a rapid and simple alternative for HCV-RNA detection in blood samples.

Detection of the human papilloma virus type 16 mRNA-transcripts in cytological abnormal scrapings.

The RNA-polymerase chain reaction (PCR) was carried out on cervical scrapings to detect and analyze transcripts from the E6-E7 open reading frames (ORF) of human papilloma virus type 16 (HPV16). The method, described previously for cervical squamous carcinomas and cervical intraepithelial neoplasias, was adapted to cervical scrapings. A primer set and two different probes specific for the E6-E7 ORFs were selected. One of the probes was able to detect the amplification products from the full length, the major, and the minor transcripts whereas the other was specific for the major transcript only. To check the quality of the mRNA in the cervical scrapings, a primer set and a probe specific for the human keratin mRNA were selected. A group of 17 abnormal cytological cervical scrapes, which were positive for HPV16 DNA, was analyzed. In this group the human papilloma virus was not always transcriptionally active, as HPV16 mRNA transcripts were detected only in about one-half (8/17) of the samples. These findings suggest that the RNA-PCR method on cervical scrapings may be very useful for epidemiological studies on the role of transcriptionally active/inactive HPV16 genes in the pathogenesis of an HPV16 infected lesion.

Fibrinogen and albumin synthesis are regulated at the transcriptional level during the acute phase response.

During acute inflammation or after administration of monocytic products, an enhanced transcription of the fibrinogen polypeptide genes and a reduced transcription of the albumin gene were observed. The changes in the fibrinogen polypeptide transcriptional rate were found to precede the change in albumin gene transcription. These findings indicate that the altered synthesis of fibrinogen and albumin during inflammation are regulated at the transcriptional level and are most probably mediated by monocytic products (including interleukin-1).