Detection of adenoviruses in stool specimens by nucleic acid spot hybridization

Nucleic acid hybridization was used for the detection of adenovirus DNA in stool specimens, and the results were compared with those obtained by a radioimmunoassay (RIA) for adenovirus hexon antigen. DNA from 40 specimens, 18 of which were positive by RIA, were spotted onto nitrocellulose filters and analyzed by hybridization using radioactively labeled adenovirus-2 DNA or a cloned DNA fragment from enteric adenovirus-41 as probes. With the adenovirus-2 DNA probe, 15 of the 18 RIA-positive specimens were also positive in the hybridization assay, and one of the RIA negative specimens was also scored as positive. The cloned adenovirus-41 fragment gave a positive signal with five specimens, all of which were also detected with the adenovirus-2 DNA probe. The results show that hybridization is an alternative method for detection of adenovirus in stool specimens. The sensitivity of the assay is comparable to that of the RIA.     

Detection of specific types of human papillomavirus in cervical scrapes, anal scrapes, and anogenital biopsies by DNA hybridization

Specific varieties of human papillomavirus (HPV) infecting the anogenital region were detected in clinical samples by use of a filter hybridization technique suitable for rapid screening of cervical and anal scrapes. In this way possibly benign types (HPV6 and HPV11) could be differentiated from types thought to be capable of malignant transformation (HPV 16 and HPV 18). Cervical or anal canal cells were applied directly to nylon filters and fixed by u.v. irradiation before hybridization with mixed viral DNA probes under both low- and high-stringency conditions. In addition, probe for the human Alu-repeated DNA sequence was used to assess the relative amount of total nucleic acids in each sample applied to the filter. HPV DNA was detected in 3 of 19 cervical scrapes from patients with no past or present history of wart virus infection or cervical dysplasia. Within a positive study group totalling 71 patients, HPV (6/11 or 16/18) was detected in cervical scrapes from 24% of 41 patients who did not have visible genital dysplasia, 30% of 27 patients with visible genital dysplasia or cervical intraepithelial neoplasia (CIN) I, and in 1 of 3 patients with past CIN II/III. In addition, HPV6/11 or 16/18 DNA was detected in anal scrapes from 3 of 6 male patients and in 85% of genital biopsies. A notably high proportion (4/6) of vaginal condylomata were positive with both the HPV6/11 and the HPV16/18 mixed viral DNA probes. Of the biopsies prepared for histopathology and positive for HPV DNA, the HPV group-specific antigen could be detected in only 60%.     

Detection of human papillomavirus deoxyribonucleic acid by filter in situ hybridization during pregnancy

Samples taken from 101 healthy pregnant women (49 over and 52 under the 20-week gestational period) and 108 healthy nonpregnant women were tested for human papillomavirus (HPV) types. Using 6, 11, 16, and 18 HPV DNA probes, 3-5 x 10(5) exfoliated cells scraped from the cervix were tested by filter in situ hybridization (FISH). Thirty-five of the pregnant women (34.6%) had evidence of the presence of HPV DNA: with 11.8% (12/101) HPV 6; 7.9% (8/101) HPV 11; 8.9% (9/101) HPV 16; and 5.9% (6/101) HPV 18 positivity. HPV DNA was detected in 20.4% (22/108) of the non-pregnant women. Compared with the healthy, nonpregnant group, the higher level of asymptomatic cervical HPV infection was mainly due to the accumulation of HPV 16 and 18 nucleic acids during the gestational period: with detection of HPV 16 in 8/49 cases (16.3%) and of HPV 18 DNA sequences in 4/49 (7.6%) cases. Screening 6-8 weeks after delivery indicated a decline of HPV positivity. Of the 4/12 HPV type 16 positive mothers, only one retained the presence of HPV 16 DNA, whereas neither of the 2/12 type 18 positive women reacted after birth with the type 18 radioactive probe.     

用核酸分子杂交技术产前诊断人巨细胞病毒感染

用ELISA法筛选早孕妇女和中孕引产妇女积压人巨细胞病毒IgM(HCMV-IgM)，早孕妇女阳性者在旱12．20周(中孕引产妇女阳性者在引产时)留羊水，用生物素标记的HCMV-DNA探针斑点杂吏检测其羊水中的HCMV-DNA。对胎儿巨细胞病毒惑染作出产前诊断。结果：早孕妇女384例．血HCMV-IgM阳性26例，此26例羊水HCMV-DNA阳性9例，其中7例出生时留脐血进一步检测HCMV-IgM和HCMV-DNA。证实有HCMV感染。中孕引产妇女125例。血HCMV-IgM阳性10例。此10例羊水HCMV-DNA阳性4例。     

Detection of human papillomavirus (HPV) type 16 and 52b in cervical cancer tissues by Southern blot hybridization and polymerase chain reaction (PCR)

DNA samples from cancer tissues diagnosed histologically as squamous cell carcinoma of the uterine cervix were examined for the presence of human papillomavirus (HPV) genome DNA by Southern blot hybridization. Of 63 specimens, 32 were found to hybridize with HPV DNA probes; 23 specimens (35%) with HPV type 16 (HPV16), one (2%) with HPV type 18 (HPV18), four (7%) with HPV type 52b (HPV52b), and four others (7%) weakly with HPV52b. Specimens negative for HPV DNA with Southern blot hybridization were subjected to polymerase chain reaction (PCR) to determine the presence of HPV DNA under more sensitive conditions. After PCR using one set of primers specific for HPV16 and HPV52b, 7 out of 31 specimens were found to have HPV16 DNA. None was positive for HPV52b DNA. Our results indicate that HPV52b, as well as HPV16 and HPV18, is associated with squamous cell carcinoma of uterine cervix, and more sensitive determination of HPV infection can be made by amplification of the viral genome by PCR.     

Detection by PCR of human papillomavirus genotypes in cervical lesions of Senegalese women

In order to analyse human papillomavirus (HPV) infection in the Senegalese population, HPV DNA was sought in 65 women with evidence of cervical cytological abnormality and in 72 pregnant women. Ninety-four percent of the patients were positive for HPV DNA as compared to 24% of pregnant women. HPV 16 was detected in cervical smears in 42% of cases, HPV 18 in 39%, HPV 6 in 26%, HPV 11 in 15%, HPV 45 in 10%, HPV 52 in 3%, and HPV 31, HPV 33 and HPV 68 in 1.5%. HPV 16 and HPV 18 were detected in 16% and 7% respectively of pregnant women. HPV DNA of unknown type was detected in 6% of cases, and multiple HPV infections were observed in 28% of cases. Low risk genital HPVs (6/11) were detected in smaller proportions (17%) among high grade squamous intraepithelial lesions (SILs) than the low grade SILs (43%). High risk HPVs (16/18) were detected in high proportions both in low and high grade SIL lesions, though the highest frequency (70%) was observed among patients with high grade lesions. In conclusion, the results confirm that HPV infections are frequent in Senegal and that HPV 18 and 45 are detected in a high proportion of patients in Africa. © 1996 Wiley-Liss, Inc.     

Detection of human papillomavirus in urine and cervical swabs from patients with invasive cervical cancer

Despite the high prevalence of both human papillomavirus (HPV) infections and cervical cancer among Zimbabwean women, the ability to test for HPV infection of the uterine cervix is limited by a lack of an easy sample collection method that does not require gynecological examination. The presence of HPVs in urine and cervical swab samples collected from 43 women who presented with invasive cervical cancer was investigated. HPV detection was done by means of degenerate primers in a nested polymerase chain reaction (PCR). Typing of HPVs was done using restriction fragment length polymorphism (RFLP) analysis. HPV was identified and typed in 98% (42/43) of cervical swabs and 72% (31/43) of paired urine samples. HPV type 16 was the most common (25/42, 59%), followed by types: 33 (13/42, 31%), 18 (6/42, 14%), and 31 (1/42, 2%). Type-specific concordance between cervical and urine samples was high (22/28, 79%). Therefore, the HPV types identified in urine samples in most cases represent the same HPV type infecting the cervical epithelium. The results suggest that urine may be a practical sample for testing of HPV urogenital infection. Further research is required before the detection of HPV in urine can be applied in the routine cervical screening programs.     

Human papillomavirus integration: detection by in situ hybridization and potential clinical application

Human papillomaviruses (HPVs) are accepted as a necessary cause of cervical neoplasia. However, the benefits of testing simply for high-risk HPV types are limited because of their high prevalence in intraepithelial lesions of all grades, the majority of which regress if left untreated. One factor considered to be of key importance for the progression of intraepithelial lesions to invasive disease is integration of HPV into the host cell genome. Although questions remain about the prevalence of integration amongst pre-invasive lesions, sensitive in situ hybridization techniques utilizing tyramide reagents may aid determination of the significance of HPV infection by enabling routine detection of both high-risk HPV and its physical status. This will provide important data relevant not only to our understanding of the biology of HPV-associated neoplasia, but also potentially to clinical testing for HPV.     

Detection of human papillomavirus in reprocessed routine Papanicolaou smears by DNA hybridization

DNA dot blot hybridization was used for the detection of human papillomavirus (HPV) types 6, 11, 16, and 18 in reprocessed routinely collected Papanicolaou smears. DNA was extracted from the smears with alkaline lysis and applied onto a nitrocellulose filter. The specificity and sensitivity of the dot blot hybridization on reprocessed Papanicolaou smears were confirmed by Southern blot analysis of selected samples, using CaSki, SiHa cell lines as positive controls and HF 32 as negative controls. From 42 normal smears and 44 abnormal smears with koilocytosis present, 9 (21%) and 43 (98%), respectively, were positive for HPV 6/11/16 or 18. These results show that reprocessed Papanicolaou smears in combination with DNA hybridization have potential application in retrospective studies on the prevalence and distribution of HPV infection.     

Detection of high-risk human papillomavirus subtypes in cervical glandular neoplasia by in situ hybridization

In situ hybridization (ISH) was performed on paraffin-embedded tissues to detect multiple high-risk human papillomavirus (HPV) subtypes in 27 cases of cervical adenocarcinoma in situ (AIS) and adenocarcinoma (CA) specimens. These results were compared with those of HPV detection by HPV-PCR genotyping and p16 immunohistochemistry in the same specimens. Of the 27 cases, 17 (63%) showed HPV-DNA by HPV-ISH, including 3 metastatic lesions. HPV-DNA was detected in 18 cases (67%) by PCR. The concordance rate between HPV-ISH and HPV-PCR genotyping was 74% with moderate agreement (Kappa value, 0.41). HPV-16 was identified in 5 cases, HPV-18 in 2 cases, and HPV-45 in 1 case. Combining the results of HPV-ISH and HPV-PCR/genotyping, 22 cases (81.5%) were considered HPV positive. Immunohistochemical staining of p16 indicated that 25 (93%) cases were positive; however, 4 of these cases were HPV-negative by both PCR and ISH. Combining HPV-ISH and HPV-PCR/genotyping techniques demonstrated a high sensitivity of HPV detection in FFPE tissues from cervical glandular neoplasias. In contrast, p16 immunohistochemistry seemed to have a low specificity for determining HPV status in cervical glandular neoplasia. HPV-ISH is useful for recognizing the distribution of HPV in AIS and CA tissues and visualizing signal patterns, and may be a useful tool to confirm the cervical origin of neoplasias and metastatic lesions.     

Detection of human papillomavirus DNA in cervical lesions by in-situ DNA hybridization

Authors examined paraffin sections of 50 cervical specimens from 34 cases for the presence of human papillomavirus (HPV) type 6b, 11, 16, 18, 31 and 33 by in-situ hybridization using 35S-labelled HPV DNA probes. Specimens were classified according to the degree of dysplasia after histological examination. Viral nucleic acids were detected in 30 of 50 tissues (60%) in which 15 specimens had single, 10 double, 4 triple and 1 quadruple viral infections. In some cases, different viral nucleic acids were detected at separate sites in the same patient. Overall, no great variation in the frequency of each HPV was detected, but a pattern became apparent when the frequencies were compared with the grade of dysplasia. CIN II/III lesions contained one or more of the HPV types 16, 18, 31, 33 which are frequently associated with cervical carcinoma. In-situ hybridization offers sensitive means of investigating viral infection, gene expression and neoplastic transformation.     

Genotyping of human papillomavirus in paraffin embedded cervical tissue samples from women in ethiopia and the Sudan

Cervical cancer is the most frequent female malignancy in most developing countries. Previous studies have demonstrated a strong association of human papillomavirus (HPV) infection with dysplasia and carcinoma of the uterine cervix. The objective of this study was to identify the prevailing HPV genotypes responsible for the development of cervical cancer among women in Ethiopia and the Sudan. A molecular characterization of HPV was done on 245 paraffin embedded cervical biopsy samples collected from the two countries. Amplification of HPV and subsequent genotyping was done using SPF10 primers and Line probe assay. Of samples collected from Ethiopian patients, 93% (149/160) and 13% (21/160) had high risk and low risk HPV genotypes, respectively. Among samples collected from the Sudan, 94% (80/85) harbored high risk and 11.7% (10/85) low risk HPV genotypes. Human papillomavirus 16 was the most frequent genotype identified in samples from Ethiopia (91%, 136/149) and the Sudan (82.5%, 66/80). HPV 52, 58, and 18 were the second, third and fourth common genotypes identified in Ethiopia, whereas HPV 18, 45, and 52 were the second, third, and fourth genotypes identified in samples collected from the Sudan. Thus, individuals living in different geographical localities should receive vaccines based on the specific genotypes circulating in the area and a vaccine targeting HPV 16, 18, 45, 52, and 58 may be optimal for the control of cervical cancer in the two countries. J. Med. Virol. 85:282–287, 2013. © 2012 Wiley Periodicals, Inc.     

A high frequency of human papillomavirus DNA sequences in cervical carcinomas of Indian women as revealed by Southern blot hybridization and polymerase chain reaction.

Ninety-six colposcopically directed biopsies from squamous epithelial carcinoma of the uterine cervix and 22 age-matched normal control biopsy specimens were examined by both Southern blot hybridization and polymerase chain reaction (PCR) for the presence of different human papillomavirus (HPV) DNA types. Cancer of the uterine cervix, which is the most common malignant disease in Indian women, showed a high frequency (98%) of HPV as compared to those reported from other parts of the world. HPV type 16 was found to be the dominant (64%) type while the frequency of HPV type 18 was very low (3%). On individual typing of HPV, no biopsy was found to contain any other known HPV types under stringent conditions of hybridization except a single case of HPV type 11. Only one case of double infection with HPV types 16 and 18 was recorded. Under low stringency conditions of hybridization with a mixed probe of HPV types 16 and 18, 29 additional biopsies were found to be positive. Southern blot hybridization alone detected HPV DNA in 92% of the cases but none in the controls. By PCR, six (6.25%) more cases and four (18.18%) healthy women were found to be positive for HPVs. Analysis of the physical state of HPV 16 indicated integration in about 70% of carcinoma cases while 30% of them were in episomal form. The findings suggest that infection with HPV is an important etiologic factor for the development of cervical cancer, that a number of such tumours may arise without HPV infection, and that integration of the viral DNA into host genome is not always essential for malignant progression.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nucleic acid spot hybridization with nonradioactive labeled probes in screening for human papillomavirus DNA sequences

We examined 161 human tissue samples using the spot hybridization technique with nonradioactive labeled DNA probes of human papillomavirus (HPV). Whole cells were spotted on nitrocellulose filters; DNA of the cells was denatured and fixed to the filter. Then the DNA spots were hybridized to nonradioactive labeled DNA and monitored by a sandwich immunoenzymatic reaction. This technique is simple, sensitive, specific, requires no special equipment, and can be used in clinical settings. HPV DNA was found in 92% of samples in which, on the basis of histologic and colposcopic criteria, its presence was suspected, as well as in 31 samples where it was not suspected.     

Variability of polymerase chain reaction-based detection of human papillomavirus DNA is associated with the composition of vaginal microbial flora

The results of repeated human papillomavirus (HPV) DNA testing were compared to changes in cervical pathology and the composition of vaginal microorganisms. A cohort of 19 women with HPV cervical infections in the absence of cervical intraepithelial neoplasia at enrollment was reexamined on average at 7.3-month intervals over a 2-year period. At each follow-up visit, cytological and colposcopic examinations were done and vaginal microorganisms were assessed quantitatively by Gram staining of secretions, and anaerobic and aerobic culture. HPV genotypes 6, 11, 16, and 18 were detected by polymerase chain reaction analysis using DNA isolated from exfoliated cervical cells. The detection of HPV DNA was significantly associated with carriage of Grade II flora (P < 0.001), isolation of Gardner ella vagina/is (P = 0.03), Ureaplasma urealyti cum (P = 0.04), Candida albicans (P = 0.01), Bac teroides species (P = 0.01), and overgrowth by anaerobes (P = 0.004). Normal vaginal flora, characterised by the predominance of Lactoba cillus species, was significantly associated (P < 0.001) with a negative HPV test. The detection of HPV DNA is associated with the composition of microorganisms present in the vagina at the time of testing. © 1994 Wiley-Liss, Inc.     

应用量子点荧光原位杂交技术检测人宫颈鳞癌中人乳头状瘤病毒16/18的感染

目的 探讨荧光标志物量子点在荧光原位杂交技术检测人宫颈癌组织中人乳头状瘤病毒16/18(HPV16/18)感染中的应用.方法 以量子点荧光原位杂交(QD-FISH)和显色原位杂交方法(CISH)分别检测80例宫颈鳞癌活检组织中HPV16/18的感染情况,对其结果进行统计学分析.结果 QD-FISH检测宫颈鳞癌活检组织中HPV16/18阳性率为88.8%(71/80),高于CISH检出的阳性率(80%,64/80),但差异无统计学意义(P=0.127),并且HPV16/18感染的阳性率随着宫颈癌级别的上升而上升.结论 QD-FISH检测HPV感染的灵敏性和特异性均高于CISH,可作为筛查宫颈癌的一种方法.     

Detection of multiple human papillomavirus types in the lower genital tract correlates with cervical dysplasia

Some human papillomavirus (HPV) types, such as HPV 16, are clearly associated with cervical dysplasia; however, the role played by other HPV types occasionally found in dysplasia is less certain. In addition, most methods used to detect HPV in clinical specimens cannot easily distinguish among more than two or three HPV types in a single specimen. Therefore, the significance of infection with multiple HPV types is not known. To address this question, we analyzed cervicovaginal lavage specimens from three cohorts of women for HPV DNA using a PCR/reverse blot assay system that permits the detection and partial quantitation of 26 genital HPV types. As expected, 94.1% of women who had dysplasia (n = 34) and 71.4% of women who had atypical squamous cells of uncertain significance (ASCUS) (n = 21) on cytology had HPV DNA detected compared to 54.5% of age matched women with normal cytology. HPV 16 DNA was detected in 35% of dysplasia patients compared to 9% of cytologic normals (P = 0.0044). Dysplasia patients had a mean of 3.29 (range 0-10) different HPV types detected compared to 1.04 (range 0-7) HPV types among those with normal cytology (P < 0.0001). These data support a possible role for multiple HPV types in the development or progression of cervical dysplasia.     

Molecular detection and genotyping of human papillomavirus in cervical carcinoma biopsies in an area of high incidence of cancer from Moroccan women

Cervical cancer is a leading cause of cancer‐related deaths in developing countries, and the human papillomavirus (HPV) is linked etiologically to cervical cancer. Eighty nine cervical carcinoma biopsies collected from women visiting the Oncologic Center in Casablanca (Centre Hospitalier Universitaire Ibn Rochd, Morocco) for cervical cancer symptoms, were screened for HPV DNA by polymerase chain reaction amplification with subsequent typing by hybridization with specific oligonucleotides for HPV types 16, 18, 31, 33, 45, and 59. Using very high stringency hybridization the HPV types could be easily distinguished. After preliminary clinical sorting, 92% (82/89) of the samples were found to be HPV‐positive. Among the samples infected by a single HPV, type 16 was the most frequent 36.6% (30/82) of the positive samples, followed by HPV 18; 19.5% (16/82). Double or even multiple infections by the different HPV types were also detected (35.5% of the positive samples); dual infections were the more frequent, with the following combinations of HPVs: HPV16/HPV18 (21% of the positives samples) and HPV16/HPV45 (8.5%). J. Med. Virol. 81:678–684, 2009 © 2009 Wiley‐Liss, Inc.     

Detection of rare and possibly carcinogenic human papillomavirus genotypes as single infections in invasive cervical cancer

The contribution of carcinogenic human papillomavirus (HPV) types to the burden of cervical cancer has been well established. However, the role and contribution of phylogenetically related HPV genotypes and rare variants remains uncertain. In a recent global study of 8977 HPV‐positive invasive cervical carcinomas (ICCs), the genotype remained unidentified in 3.7% by the HPV SPF₁₀ PCR–DEIA–LiPA₂₅ (version 1) algorithm. The 331 ICC specimens with unknown genotype were analysed by a novel sequence methodology, using multiple selected short regions in L1. This demonstrated HPV genotypes that have infrequently or never been detected in ICC, ie HPV26, 30, 61, 67, 68, 69, 73 and 82, and rare variants of HPV16, 18, 26, 30, 34, 39, 56, 67, 68, 69, 82 and 91. These are not identified individually by LiPA₂₅ and only to some extent by other HPV genotyping assays. Most identified genotypes have a close phylogenetic relationship with established carcinogenic HPVs and have been classified as possibly carcinogenic by IARC. Except for HPV85, all genotypes in α‐species 5, 6, 7, 9 and 11 were encountered as single infections in ICCs. These species of established and possibly carcinogenic HPV types form an evolutionary clade. We have shown that the possibly carcinogenic types were detected only in squamous cell carcinomas, which were often keratinizing and diagnosed at a relatively higher mean age (55.3 years) than those associated with established carcinogenic types (50.9 years). The individual frequency of the possibly carcinogenic types in ICCs is low, but together they are associated with 2.25% of the 8338 included ICCs with a single HPV type. This fraction is greater than seven of the established carcinogenic types individually. This study provides evidence that possibly carcinogenic HPV types occur as single infections in invasive cervical cancer, strengthening the circumstantial evidence of a carcinogenic role. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Detection and genotyping of human papillomavirus in cervical samples from Italian patients

Human papillomaviruses (HPVs) are etiological agents of cervical cancer. In order to assess the epidemiological incidence and frequency of different HPV types, we applied a polymerase chain reaction (PCR)-direct sequencing approach based on the use of MY09/MY11 primers as compared to Hybrid Capture assay. Cervical samples were taken from 1,500 women, both with normal and abnormal cytological smears, and we found an incidence of 6.6% of HPV infection in Brescia. Overall, 97 samples tested HPV-positive, yielding 18 HPV types. The four most frequent HPV types were: HPV 16, -31, -6, and -58. This approach could be used in ordinary laboratory settings for quick and reliable typing of known and novel HPVs from clinical specimens and it could also be applied to anti-cancer vaccine development.