Andrology: The screening for cytomegalovirus antibody in semen donors and recipients within a donor insemination programme

A total of 67 semen donors (62 Caucasian and five non-Caucasian)were tested for the presence of immunoglobulin G antibodiesto cytomegalovirus (CMV). Ten (16%) of the Caucasian donorstested positive for CMV, while four (80%) of the non-Caucasiandonors were positive. A total of 131 women receiving donor insemination(114 Caucasian and 17 non-Caucasian) were tested. Of these,43 (38%) of the Caucasian recipients tested positive for CMV,while 16 (94%) of the non–Caucasian recipients were positive.There was a significant association between the incidence ofpositive tests and the age of the Caucasian recipients.     

Detection of herpes simplex virus type 1 DNA in bilateral human trigeminal ganglia and optic nerves by polymerase chain reaction

Herpes simplex virus type 1 (HSV-1) DNA was extracted from human trigeminal ganglia of 121 cadavers aged between 3 months and 94 years, and its PCR amplification was performed for the RL2 HSV-1 sequence using two pairs of primers. The HSV-1 DNA was detected in 74 of 121 patients (61%); 70/74 bilaterally, 3/74 only on the left side, and 1/74 only on the right side. Although the PCR-positive rate was significantly higher in advanced age, the correlation between the PCR-positive rate and gender was unclear. Additionally, HSV-1 DNA was not detected in any of the 50 optic nerve samples.     

Absence of human herpes virus 8 in semen from healthy Danish donors.

Epidemiological data indicate a sexual route of transmission of acquired immune deficiency syndrome (AIDS) associated Kaposi's sarcoma. Recently human herpes virus 8 (HHV-8) has been proposed as the aetiological agent for development of Kaposi's sarcoma. Further the virus has been reported in semen obtained from healthy men. In Denmark strict biochemical and microbiological criteria are used in combination with an intensive interview to select semen donors. Despite these strict criteria, HHV-8 may be transmitted to a recipient and even the child by the use of donor semen. We used four different polymerase chain reaction (PCR) and one nested PCR to test semen from 100 Danish donors for the presence of HHV-8 DNA. All 100 samples were consistently negative for HHV-8 DNA, while only one sample (1%) was positive for cytomegalovirus DNA. As HHV-8 was not demonstrated in any of the semen samples, we conclude that the frequency of HHV-8 in semen from Danish donors is very low.     

Determination of hepatitis C virus genotypes by melting-curve analysis of quantitative polymerase chain reaction products

Hepatitis C virus (HCV) is the major causative agent of non-A and non-B viral hepatitis. Factors associated with disease progression following HCV infection include the viral genotype, the patient's alcohol consumption and viral load. In this study, the COBAS AMPLICOR HCV MONITOR test, a commercially available quantitative assay for HCV RNA, was used for HCV genotyping analysis. Amplification products obtained from 100 HCV-positive cases were subjected to real-time polymerase chain reaction (PCR) typing using a single pair of fluorescence resonance energy transfer (FRET) probes and melting-curve analysis. Of 100 samples tested, two inhibited the PCR, two samples yielded discrepancies between our results and the reference laboratory results and the remaining samples provided correct typing. The present report suggests that HCV genotypes can be determined rapidly with FRET probes directly from COBAS AMPLICOR MONITOR test PCR products.     

An analytical method for R5 repeated structure in varicella-zoster virus DNA by polymerase chain reaction

The tandem direct reiteration, R5, in varicella-zoster virus (VZV) genome consists of 88-bp and 24-bp elements. The R5 structure was examined by the polymerase chain reaction to differentiate between various strains. A sense primer was designed at the 88-bp element and an antisense primer at the site separated from R5, in which we expected to detect multiple bands corresponding to repeating numbers of 88-bp elements. Thirty-four wild strains and a vaccine strain were analyzed using this method. The 34 wild strains showed two to four bands corresponding to numbers of 88-bp elements, in which the strain showing three bands was found to be predominant. The vaccine strain also revealed three bands.     

Detection of HPV DNA in trichilemmomas by polymerase chain reaction

Paraffin sections of 11 formalin-fixed trichilemmomas were investigated for the presence of human papillomavirus (HPV) DNA by the polymerase chain reaction (PCR) with the degenerated consensus primer pairs. PCRs were conducted with different annealing temperatures. When the annealing temperature was reduced from 55°C to 50°C, amplification products of the expected size were obtained for all 11 cases investigated. Determination of the HPV type was performed by cloning and sequencing of the amplification products. The sequence analysis of the eleven cloned amplicons gave the following data: based on sequence comparison with published amino acid sequences, the best homology was found to epidermodysplasia verruciformis (EV)-associated HPVs (supergroup B). In four specimens an HPV type 23 related type was found; five specimens contained HPV sequences which did not match with one of the known HPV types, but had the closest homology to HPV types 15, 17, and 37. Three of the HPV variants which had not been characterised, displayed identical sequences. Two additional HPV amplification fragments displayed 100% homology to HPV-6b. These results demonstrate, for the first time, the presence of HPV DNA in trichilemmomas. The sequence data suggest that HPV variants or types in trichilemmoma are members of the EV-associated HPV supergroup B. J Med Virol 51:119–125, 1997. © 1997 Wiley-Liss, Inc.     

Identification and typing of molluscum contagiosum virus in clinical specimens by polymerase chain reaction

A polymerase chain reaction (PCR) which enables the detection of molluscum contagiosum virus (MCV) genomes in either fresh or formalin-fixed clinical specimens is described. The primers used were designed to amplify a 167 bp region of the 3.8kbp HindIII fragment K of the MCV 1 genome. The ability of this PCR to detect three common MCV types (1, 1v and 2) in clini-cal specimens was confirmed using frozen extracts from 75 molluscum lesions, and digests of single sections of 11 formalin-fixed, paraf-fin-embedded lesions; all of which had been previously typed by Southern hybridisation. In addition, 2 specimens previously negative by hybridisation were shown to be positive for MCV DNA by PCR. Confirmation of the identity of the PCR products and distinction between the two major MCV types (MCV 1/1v versus MCV 2) was achieved by comparison of the results of cleavage with the restriction endonucleases HhaI and SacI. Sequencing of the PCR products revealed complete homology between MCV 1 and 1v, but minor nucleotide variations between MCV 1/1v and MCV 2 were identified. As well as providing a highly sensitive means of diagnosis, the technique may also prove useful for investigations into the pathogenesis, epidemiology and natural history of molluscum contagiosum infection. J. Med. Virol. 53:205–211, 1997. © 1997 Wiley-Liss, Inc.     

Failure to detect hepatitis C virus (HCV) genome by polymerase chain reaction in human anti-HCV-positive intravenous immunoglobulins

The prevalence of HCV antibodies was determined by a second-generation ELISA and a four-antigen rccombinant immunoblot assay in nine intravenous immunoglobulin (IVIG) preparations commercially available in Italy. In addition, the clinical safety of six of them was ascertained by polymerase chain reaction (PCR) of HCV RNA and a prospective study in 14 patients with immunodeficiency disorders. Results indicated that all IVIG preparations were anti-HCV-positive. However, there were substantial variations in their anti-HCV antibody titres. The preparations retained IgG subelass reactivilies to HCV-associated structural (C223) and non-structural (C33c, C100-3) proteins. Our sensitive and specific PCR assay was unable to detect HCV RNA in the six preparations tested. Clinical surveillance of IVIG-trcated patients prospectivcly evaluated over a mean period of 83 months failed to delect clinical and/or biochemical evidence of hepatitis.     

应用原位PCR技术检测阴茎癌组织中人乳头瘤病毒DNA

目的：研究人乳头瘤病毒（ＨＰＶ）与阴茎癌的关系。方法：应用原位ＰＣＲ技术对４６例阴茎癌组织中的ＨＰＶ１６和ＨＰＶ１８ＤＮＡ进行检测。结果：３３例阴茎癌中检测到了ＨＰＶＤＮＡ（７１．７％），其中２９例ＨＰＶ１６ＤＮＡ阳性（６３．０％），６例ＨＰＶ１８ＤＮＡ阳性（１３．０％），２例ＨＰＶ１６和ＨＰＶ１８ＤＮＡ均阳性，４例ＨＰＶ１８ＤＮＡ阳性而ＨＰＶ１６阴性；２例淋巴结转移癌，３例癌旁不典型增生组织和１例癌旁增生组织ＨＰＶ１６ＤＮＡ阳性。结论：阴茎癌与ＨＰＶ１６、ＨＰＶ１８感染有密切关系，原位ＰＣＲ技术是一项敏感性高、特异性强的技术。     

Indeterminate western blot patterns in a cohort of individuals at high risk for human immunodeficiency virus (HIV-1) exposure

Our objective was to map serial patterns of Western blot reactivity over time of a cohort of initially ELISA-negative, Western blot-indeterminate individuals from a high-risk group and to determine if these individuals were at increased risk of harboring occult HIV-1 infection. A 2-year prospective study used serial ELISA, two types of Western blot, immunologic profiles, HIV-1 culture, and analysis by polymerase chain reaction. Subjects were 20 ELISA-negative, Western blot indeterminate homosexual volunteers and 20 matched seronegative controls. Results showed that 19 of 20 study subjects completed a mean of 17.0 months of clinical and laboratory follow-up. Reactivities with p24 and/or with p55 were the two most commonly observed Western blot patterns, occurring in 70% of individuals. Specific Western blot reactivity was dependent upon the particular immunoblot preparation being used and varied considerably on a longitudinal basis. No individual pattern appeared predictive of an increased likelihood of subsequent seroconversion to HIV-1 relative to controls. By all other criteria including polymerase chain reaction analysis, samples from 17 of 19 individuals remained negative for HIV-1 at each time point. Two individuals evolved from an indeterminate to a positive Western blot and, simultaneously, from a negative to a positive polymerase chain reaction analysis, during follow-up. Our conclusions were as follows. ELISA-negative, Western blot-indeterminate individuals from a high-risk group show marked variability in immunoblot findings over time, and these patterns do not appear predictive of an increased likelihood of infection. Polymerase chain reaction analysis is clearly of value in providing confirmation of the low probability of infection in this group, although in patients who do become infected, detection by this test may not always precede diagnosis by serologic methods.     

Detection of cytomegalovirus in human placental cells by polymerase chain reaction

Human cytomegalovirus (HCMV) is the most common cause of viral intrauterine infection. Progress in rapid, specific, and dependable detection of HCMV has recently been achieved by the use of DNA hybridization techniques and other molecular methods. We examined 21 placentas after delivery for the presence of HCMV DNA by polymerase chain reaction (PCR). To test the reliability of the PCR for the detection of HCMV DNA in clinical specimens, two simple PCR assays and a real-time quantitative PCR were used. PCR analysis of villous and decidual cells showed that HCMV DNA was present in 16 placentas (76.2%). Transmission of HCMV infection to chorionic villi was confirmed in 11 organs (52.4%), and congenital infections in newborns were detected in 9 cases (42.8%). These results suggest that HCMV genome detection in placentas at later gestational ages is common. Our results demonstrated that detection of HCMV DNA in placental tissues by DNA amplification provides a specific and sensitive method for diagnosis of intrauterine HCMV infection.     

Evaluation of the polymerase chain reaction for the detection of human papillomavirus from urine

The ability to detect the presence of human pap-illomavirus (HPV)-DNA sequences in urine was evaluated using polymerase chain reaction (PCR). DMA was purified and extracted from urine samples, and subjected to 40 cycles of amplification using the consensus primer pair MY11 and MY09. Coamplification using the β-globin primers, GH20 and PC04, was performed as an internal reaction control. Following assay optimization, urine samples from 22 women undergoing examination for cervical dysplasia were tested for the presence of HPV-DNA. PCR assay results were correlated with cytologic and histo-logic findings as well as Vira Type(tm) assay results. Overall, HPV was detected by PCR in 16 (76%) of the interpretable samples. HPV sequences were detected in 13 (87%) of the 15 specimens from women showing evidence of condylomata, dysplasia, or invasive carcinoma. HPV was detected in 3 (50%) of the women whose cytologic or his-tologic results were either negative or showed benign atypia. Although the sample size in this study is small, our results show that HPV can be detected by PCR in a majority of individuals showing evidence of HPV infection. The method described provides a means for the clinical laboratory to detect a broad range of HPV types from using a sample obtained by noninvasive techniques. The ability to easily obtain urine would allow for increased numbers of individuals to be tested, and thus, aid in our understanding of HPV. © 1995 Wiley-Liss, Inc.     

Detection of human immunodeficiency virus-1 by polymerase chain reaction and virus cultivation

Peripheral blood of 57 patients with antibodies to human immunodeficiency virus 1 (HIV-1) and of five HIV-1 seronegative subjects at risk for HIV-1 infection were analysed by polymerase chain reaction (PCR) and virus isolation. The virus was recovered from peripheral blood cells in 89% and from plasma in 75% of the HIV-1 seropositive cases. In contrast, proviral HIV-1 DNA was detected in all HIV-1 seropositive patients by dot blot hybridization of the amplified fragments. The intensities of the dot blot reactions were less pronounced in asymptomatic HIV-1 seropositive individuals than in patients with acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC), suggesting an increase in proviral DNA with advancing disease. Three of five seronegative patients with signs or symptoms suggesting HIV-1 infection, but none of the controls, were positive for HIV-1 DNA by one or two primer pairs. These results show a high sensitivity of the PCR for detecting HIV-1 DNA in patients of all stages of HIV-1 infection. Proviral DNA can also be detected in some individuals without detectable antibodies to the virus. The virus load in peripheral blood, as determined by virus cultivation and PCR, seems to increase with progression of the infection.     

Demonstration of coxsackie virus RNA in formalin-fixed tissue sections from childhood myocarditis cases by in situ hybridization and the polymerase chain reaction

This is a combined study using in situ hybridization and the polymerase chain reaction to investigate the presence of Coxsackie virus RNA in formalin-fixed tissue from cases of childhood myocarditis. Of the ten cases studied, two were positive by both methods. The virus RNA was predominantly located in areas showing an inflammatory cell infiltrate and myofibre necrosis. These findings suggest that direct lytic infection of myocytes by virus is responsible for myocaiditis in these cases, rather than an autoimmune process, which has been suggested previously. The findings in one case, where the virus showed a marked sub-endocardial distribution, may have implications for the aetiology of endocardial fibroelastosis by confirming a viral tropism for this location. The techniques used in this study are easily repeatable and can be directly applied to look for viruses in a number of other diseases where a viral aetiology is suspected.     

Large-scale screening for human parvovirus B19 DNA in clinical specimens by dot blot hybridization and polymerase chain reaction

Large-scale screening for human parvovirus B19 (619) DNA in serum samples was carried out by both dot blot hybridization and the polymerase chain reaction (PCR). Dot blot hybridization was undertaken with a digoxigenin-labeled DNA probe. Serum samples from four patients were pooled and tested by a dot blot hybridization assay. When a dot was positive, each of the four samples was tested separately to identify the positive sample. The PCR template was the DNA extracted from mixed serum samples from 10 patients. When B19 DNA was positive by PCR, each of the ten samples was tested separately. A total of 7, 969 serum samples were tested by dot blot hybridization and 15 samples (11 patients) were positive for B19 DNA; 7, 038 serum samples were tested by PCR and 71 samples (50 patients) were positive. Large-scale screening for B19 DNA by PCR suggested a broader spectrum of clinical manifestations associated with B19 infection. © Wiley-Liss, Inc.     

Short communication: previous semen donors and their views regarding the sharing of information with offspring

BACKGROUND: The UK government has decided to introduce, from 2005, rules that will allow donor-conceived persons to have access to identifying information concerning their donor. This has led to many concerns regarding future gamete donor recruitment. METHODS: Semen donors who had been recruited between 1988 and 2002 were invited to take part in a telephone interview. The interview sought these previous donors' views on issues associated with recruitment, attitudes regarding information sharing and views concerning the offspring. Responses regarding information sharing were compared with their views recorded at the time of recruitment. RESULTS: All 32 donors were recruited altruistically. Eighteen (56%) held the same views concerning the provision of identifying information as they did at the time of recruitment. Of those who had changed their views, eight (25%) expressed a willingness to be more open and four (12%) now wished to be anonymous having previously been unsure. Half of the donors would still have donated if they had been required to be identified to offspring, one-quarter would not have and one-quarter were undecided, although the majority of these said they may have donated under an open system. CONCLUSION: The study shows that it is possible to recruit identifiable donors at this clinic and this suggests that it may be possible for other clinics to do likewise.     

Detection of human herpesviruses 6 and 7 in heart transplant recipients by a multiplex polymerase chain reaction method

In order to evaluate the possible reactivation of human herpesviruses 6 (HHV-6) and 7 (HHV-7) after heart transplantation, buffy-coat and plasma specimens from 21 transplant patients and 56 healthy blood donors were examined for HHV-6 and HHV-7 DNA by polymerase chain reaction. Human herpesvirus 6 and HHV-7 infection or reactivation has been suggested to play a role in cytomegalovirus disease progression in renal transplant recipients. In the present study, however, no significant difference in the prevalence of HHV-6 and HHV-7 was found between the immunosuppressed and the healthy population; moreover, no viral reactivation was found in the heart transplant recipients.