Nucleic acid detection as a diagnostic tool in polyomavirus JC induced progressive multifocal leukoencephalopathy

Procedures involved in the diagnosis of JC virus central nervous system infection range from detection of virus specific products in biopsy material to demonstration of viral DNA in cerebrospinal fluid by PCR. Despite the fact that PCR is the most sensitive method for the detection of virus in clinical specimens, diagnostic evaluation is increasingly difficult in view of the possible subclinical activation of persistent JCV infection in the central nervous system of high risk patients. Therefore, PML diagnosis by molecular detection of JCV DNA in biopsy material was compared with diagnosis by PCR on CSF of patients with and without PML. Evaluation of the diagnostic techniques revealed that stereotactic biopsy based PCR diagnosis at present combines speed and sensitivity with the highest specificity available. Although the non invasive technique of JCV detection in CSF by PCR is even more sensitive leading to detection of about 20 genome equivalents per 1 microl of CSF, the specificity of the method is limited by subclinical presence of JCV DNA in CSF of neurologically asymptomatic HIV infected patients. Additionally, autopsy proven PML cases remaining JCV negative in PCR on CSF become a common finding. Therefore, in cases where biopsy is not performed, diagnosis of PML can only be achieved in combination with neurological and radiological diagnosis.     

A comparative study of nucleolar organizer region, proliferating cell nuclear antigen and epidermal growth factor receptor staining in colon tumours

The prevalence of hepatitis A virus (HAV) antibody in people has decreased from year to year in Japan. A sequential outbreak occurred in an institution for the mentally handicapped people in Chiba City in the summer of 1995. Eight people were infected including 7 residents and one staff member. We tested to detect antigen in fecal samples by ELISA and PCR for early diagnosis for hepatitis A infection. Four sera and 5 feces were obtained from 5 patients between 2 and 8 days after the onset of symptoms. The anti-HAV IgM was found to be positive in 4 sera examined. The HAV antigen was detected in 3 out of 5 feces using ELISA. An existence of inhibitor in 2 negative specimens against the ELISA was suggested by the recovery test of added antigen. HAV RNA was extracted by CTAB method from feces and detected in 4 our of 5 specimens in PCR amplification and in all of 5 specimens in nested PCR amplification. The sequence of PCR products in the P1/P2 junction of the HAV genome revealed that the virus associated with the outbreak belongs to HAV subgenotype IA. HAV RNA was detected in ELISA negative specimens and in the specimen from a patient 2 days after the onset of symptoms using PCR amplification by CTAB method. These results indicate that PCR amplification was useful for the early diagnosis of hepatitis A infection.     

Human immunodeficiency virus infection among high-risk seronegative prostitutes in Nairobi

To determine the frequency and duration of antibody-negative human immunodeficiency virus (HIV) infection among heterosexually exposed African women, 56 HIV-seronegative female prostitutes in Nairobi were studied. Polymerase chain reaction (PCR) was used to detect HIV DNA in peripheral blood at enrollment, and women were followed prospectively with serologic testing to determine HIV seroincidence. Six women (11%) were infected with HIV by PCR criteria at enrollment. Seroconversion occurred in 5 of these subjects within 1-12 months, while the sixth remained seronegative when last evaluated at 5 months. The cumulative annual seroconversion rate in the entire cohort was 38%. Using maximum likelihood analysis, the mean interval between HIV infection and seroconversion was estimated to be between 3 and 4 months, similar to that described for homosexual men and blood product recipients in the United States. Prolonged HIV infection in the absence of antibodies appears to be uncommon in this setting.     

Possibility for detecting HIV-1 in bone transplant by PCR using the HIV-1 microtiter plate assay

It is known that HIV can be transmitted by allogenous bone transplantation. Hitherto neither chemical nor physical methods have existed to allow reliable disinfection and sterilization of bone specimens without reducing osteogenetic potency. Only demonstration or exclusion of the presence of HIV-1 in a bone specimen guarantees that infection will not occur. The method now presented for HIV detection is based on a polymerase chain reaction (PCR). This HIV microtiter-plate assay combines amplification of DNA molecules with a staining reaction. In cultures containing HIV-infected cells definite detection of viruses was possible when 50-100 cells per specimen were infected. Examination of 137 HIV-negative and 25 HIV-positive bone specimens showed sensitivity of 96% and specificity of 97.8% for the test. In subsequent studies, after drying on filter paper viral DNA was again demonstrable by the PCR. This means safe handling and uncomplicated transportation of non-infectious specimens to a central analysis laboratory are possible. This HIV test offers the possibility of quick and safe demonstration that specimens are free of HIV and is therefore likely to enhance the safety of bone transplantation considerably.     

Blood pH in the umbilical artery at birth: an analysis of data from patients delivered in Hesse between 1986 and 1989

A nonradioactive in situ hybridization method is described for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) in cell cultures and in formalin-fixed paraffin-embedded tissue sections originating from experimentally infected pigs and from 1 field case. A 174 bp cDNA probe targeting the viral RNA encoding the nucleocapsid protein of a Canadian PRRSV isolate was generated by polymerase chain reaction. The cDNA probe was labeled by random priming with digoxigenin-dUTP using a commercially available kit. The ability of the digoxigenin-labeled probe to specifically detect PRRSV RNA was tested on cultured cells infected with 6 Canadian PRRSV isolates, a US PRRSV isolate and the European Lelystad isolate. The probe detected all Canadian PRRSV isolates tested as well as the US PRRSV isolate but did not detect the Lelystad isolate. In addition, when tested on formalin-fixed paraffin-embedded tissue sections from pigs experimentally infected with several Canadian isolates and from a field case, a strong signal without background staining was obtained. Our results indicate that nonradioactive in situ hybridization could represent a useful tool for the detection of PRRSV in routinely fixed and processed tissues. In situ hybridization could also be used to differentiate infection by North American and European Lelystad-like PRRSV isolates.     

Comparative investigations of practice-oriented methods for the detection of viruses in food with Aujeszky infection in swine as an example

In order to detect contamination in foodstuffs originating from animals, three different diagnostic methods were tested in comparison: cultivation in permissive cell cultures, microparticle antigen capture system per FACS (MAS) and polymerase chain reaction (PCR). Assessment implied relevance for health, sensitivity and specificity. Aujeszky infection in swine served as a model. The blood and organs of healthy, but persistently infected as well as specifically diseased animals were examined. In addition, artificially Aujeszky-contaminated milk, black pudding and minced meat were included in the comparison of methods. Basically, all three methods of detecting contamination in raw foodstuffs originating from animals were useful. The virus detection in cell cultures as well as the efficacy of MAS were inhibited by meat products according to their preparation (e.g., virus protein denaturation). PCR turned out to be the only reliable method to confirm the contamination in foodstuffs. Using PCR, viral contamination in foodstuffs originating from healthy but persistently infected animals could be detected. Each of the three virus detection systems has various advantages and disadvantages. They are listed and discussed in detail. With regard to the relevance of health, virus detection in raw meat via cell culture remains the preferred method. Besides the detection of an individual virus, routine examinations of foodstuffs for unknown zoonotic virus contamination, sets based on permissive cell cultures, primer sets for the PCR as well as sets based on various monoclonal antibodies for MAS have to be developed and made available at the diagnostic laboratories.     

Mother to child transmission of hepatitis C virus: prospective study of risk factors and timing of infection in children born to women seronegative for HIV-1. Tuscany Study Group on Hepatitis C Virus Infection

OBJECTIVE: To determine the risk factors for and timing of vertical transmission of hepatitis C virus in women who are not infected with HIV-1. DESIGN: Follow up for a median of 28 (range 24-38) months of babies born to women with antibodies to hepatitis C virus but not HIV-1. SUBJECTS: 442 mothers and babies, of whom 403 completed the study. MAIN OUTCOME MEASURES: Presence of antibodies to hepatitis C virus and viral RNA and alanine aminotransferase activity in babies. Presence of viral RNA, method of infection with hepatitis C, method of delivery, and type of infant feeding in mothers. RESULTS: 13 of the 403 children had acquired hepatitis C virus infection at the end of follow up. All these children were born to women positive for hepatitis C virus RNA; none of the 128 RNA negative mothers passed on the infection (difference 5%, 95% confidence interval 2% to 7%). 6 children had viral RNA immediately after birth. 111 women had used intravenous drugs and 20 had received blood transfusions. 11 of the infected children were born to these women compared with 2 to the 144 with no known risk factor (difference 7%, 2% to 12%). CONCLUSIONS: This study suggests that in women not infected with HIV only those with hepatitis C virus RNA are at risk of infecting their babies. Transmission does seem to occur in utero, and the rate of transmission is higher in women who have had blood transfusions or used intravenous drugs than in women with no known risk factor for infection.     

Human immunodeficiency virus infection of the human thymus and disruption of the thymic microenvironment in the SCID-hu mouse

Infection with the human immunodeficiency virus (HIV) results in immunosuppression and depletion of circulating CD4+ T cells. Since the thymus is the primary organ in which T cells mature it is of interest to examine the effects of HIV infection in this tissue. HIV infection has been demonstrated in the thymuses of infected individuals and thymocytes have been previously demonstrated to be susceptible to HIV infection both in vivo, using the SCID-hu mouse, and in vitro. The present study sought to determine which subsets of thymocytes were infected in the SCID-hu mouse model and to evaluate HIV-related alterations in the thymic microenvironment. Using two different primary HIV isolates, infection was found in CD4+/CD8+ double positive thymocytes as well as in both the CD4+ and CD8+ single positive subsets of thymocytes. The kinetics of infection and resulting viral burden differed among the three thymocyte subsets and depended on which HIV isolate was used for infection. Thymic epithelial (TE) cells were also shown to endocytose virus and to often contain copious amounts of viral RNA in the cytoplasm by in situ hybridization, although productive infection of these cells could not be definitively shown. Furthermore, degenerating TE cells were observed even without detection of HIV in the degenerating cells. Two striking morphologic patterns of infection were seen, involving either predominantly thymocyte infection and depletion, or TE cell involvement with detectable cytoplasmic viral RNA and/or TE cell toxicity. Thus, a variety of cells in the human thymus is susceptible to HIV infection, and infection with HIV results in a marked disruption of the thymic microenvironment leading to depletion of thymocytes and degeneration of TE cells.     

Long-term lipid-based total parenteral nutrition activates mononuclear cells and modulates membrane lipid composition in pigs

This study aims to assess the possible strain-dependent variations in detection of Toxoplasma antigens and antibodies. The virulent RH strain or avirulent Beverley strain of T. gondii were injected into mice, intraperitoneally, and their antigens, antibodies and parasites were identified from the blood or tissues; liver, brain and spleen by ELISA, Western blot and PCR. In mice infected with RH strain, circulating antigens and parasitemia were first detected from 2 days after infection, and Toxoplasma DNA were found in the blood, liver, brain and spleen from 3 days after infection. It was impossible to detect specific IgM and IgG antibodies to T. gondii, and any specific band was not found by Western blot. In mice infected with Beverley strain, circulating antigens were detected between day 10 and day 35. The Toxoplasma DNA was found in the blood and liver from day 15 until day 60, and in the brain from day 20. But Toxoplasma DNA in the spleen were mainly detected between day 10 and day 30. The IgM antibodies were first appeared on day 10 post-infection, and were noted obviously increased between day 15 and 25. The IgG antibodies were first detected on day 15, and showed progressively increased titers. The antibody binding bands were specific according to infection period. Sera from mice infected with Beverley strain reacted mainly with the antigen of 27.5-kDa and 32.5-kDa. In conclusion, mice infected with RH strain revealed Toxoplasma antigens strongly, but not antibodies. However, mice infected with Beverley strain revealed both the Toxoplasma antigens and antibodies. The present results showed that immune responses are different between avirulent and virulent T. gondii.     

Detection of HIV-1 nucleic acid and HIV-1 antibodies in needles and syringes used for non-intravenous injection

BACKGROUND: HIV antibodies and HIV DNA have been detected in needles and syringes that have been used for intravenous injections in HIV-infected persons. During intravenous injection, blood is typically aspirated into the lumen of the syringe. During intramuscular or subcutaneous injection, however, blood is not usually introduced into the syringe. OBJECTIVES: To investigate the presence of HIV antibodies, HIV proviral DNA, HIV RNA, and human DNA in needles and syringes that had been used for intramuscular or subcutaneous injection in persons known to have HIV infection. METHODS: Discarded disposable needles and syringes used by health-care personnel for medically indicated intramuscular or subcutaneous injections of HIV-infected patients were collected. Residual material was extracted from the syringes. The extracts were analyzed by enzyme immunoassay for the presence of HIV antibodies. PCR was conducted to detect HIV and human DNA, as well as HIV RNA. RESULTS: HIV antibodies were detected in 16 (6.2%) out of 260 syringes. Human DNA or HIV-specific DNA were not detected. A second set of 80 syringes was collected to examine the presence of HIV RNA. HIV RNA was detected in three (3.8%) out of 80 syringes. CONCLUSION: This analysis demonstrates that the risk of transmitting HIV from syringes that have been used for intramuscular or subcutaneous injection may be low, but is not zero.     

Apparent enhancement of perinatal transmission of human immunodeficiency virus type 1 by high maternal anti-gp160 antibody titer

The presence of antibodies able to enhance infection in vitro in sera from human immunodeficiency virus (HIV)-1-infected patients raises the possibility that antibodies exert a deleterious activity during natural infection. The anti-HIV-1 humoral response and plasma HIV-1 RNA were measured in a cohort of 98 infected mothers, included in the French Prospective Study on Pediatric HIV Infection, 49 of whom transmitted HIV to their children. Transmission from mother to child was associated with antibody responses to the envelope gp160 (P = .009 for serum dilution of 1/400) and to a highly conserved domain of the transmembrane glycoprotein (P = .055 for serum dilution of 1/400) and with plasma HIV-1 RNA levels (P < .0001). Multivariate logistic regression indicated that a high anti-gp160 response and a high plasma virus load are independent risk factors for perinatal transmission of HIV-1 (odds ratio, 3.4; 95% confidence interval, 1.1-9.9 for anti-gp160; odds ratio, 2.8; 95% confidence interval, 1.6-5.0 for virus load).     

Vasectomy and human immunodeficiency virus type 1 in semen

PURPOSE: Human immunodeficiency virus type 1 (HIV) is cultured more often from seminal cells than seminal plasma. Because vasectomy causes dramatic reductions in seminal cells and also eliminates secretions from proximal sites in the male reproductive tract, vasectomy may change the potential infectiousness of semen. MATERIALS AND METHODS: We used polymerase chain reaction (PCR) assays to measure HIV ribonucleic acid (RNA) in seminal plasma and HIV deoxyribonucleic acid (DNA) in seminal cells from 46 asymptomatic, seropositive men before and after vasectomy. RESULTS: HIV RNA levels in semen correlated only weakly with blood levels (r = 0.22, p = 0.03). Of 183 semen specimens assayed for cell-free HIV RNA and proviral DNA 37 (20%) were positive for HIV RNA only, 41 (22%) were positive for HIV DNA only, and 18 (10%) were positive for RNA and DNA. Thus, detection of HIV RNA in seminal plasma was not associated with detection of HIV DNA in seminal cells. HIV RNA was present in 23 of 82 specimens (28%) (mean 2.87 log copies/ml.) before vasectomy and in 38 of 121 specimens (31%) after vasectomy (mean 2.81 log copies/ml.). CONCLUSIONS: These findings suggest that direct measurement of HIV levels in semen is necessary to assess the potential for sexual transmission, most cell-free HIV in seminal plasma arises distal to the vas deferens, and vasectomy may have minimal impact on the infectiousness of HIV seropositive men on sexual partners.     

Enzyme-linked immunosorbent assay for serological survey of equine arteritis virus in racehorses

To examine antibodies against equine arteritis virus (EAV), an enzyme-linked immunosorbent assay (ELISA) using purified virus antigen was developed. The results of ELISA were compared with those of serum neutralization (SN) tests. The ELISA absorbance values and the SN titers in sera collected weekly from EAV-infected horses showed a similar pattern. The ELISA could detect antibody to EAV in horses experimentally infected with not only a homologous virus strain, which was used as the ELISA antigen, but also a heterologous strain. Using the ELISA, serum samples collected in 1996 from racehorses in three prefectures (Hokkaido, Ibaraki, and Shiga) were examined and there was no evidence of recent EAV infection among these racehorse populations in Japan. The ELISA should be a simple and highly specific method for rapid screening of EAV infection in racehorses.     

Plants transformed with a mutant alfalfa mosaic virus coat protein gene are resistant to the mutant but not to wild-type virus

Transgenic tobacco plants expressing the wild-type (wt) coat protein (CP) gene of alfalfa mosaic virus (AIMV) have been shown to be resistant to infection with viral particles and RNAs or to infection with viral particles only. The difference in resistance of these plants to RNA inocula was found to correlate with a difference in the expression level of the transgene. Plants expressing a mutant AIMV CP with the N-terminal serine residue changed to glycine have been shown to be susceptible to infection with wt viral particles or RNAs. By site-directed mutagenesis of AIMV cDNA a viable mutant virus encoding CP with the same N-terminal mutation was obtained. Plants expressing wt or mutant CP were resistant to the mutant virus, demonstrating that a single amino acid substitution in CP did not permit the virus to overcome CP-mediated resistance. Although the mutant CP did not confer resistance to wt virus when expressed in transgenic plants, it was still effective in classical cross-protection: plants infected with the mutant virus were resistant to severe strain of AIMV. A model to explain the data is discussed.     

Viral serology and detection

Viral detection is an important part of clinical hepatology. For many years practical clinical tests have been serological but recently newer molecular techniques have become available for virus detection, although these have yet to become routine and some, such as PCR of viral nucleic acid in blood or tissue are not yet consistently reliable. Serology remains the mainstay at present for routine diagnosis. Hepatitis A testing in clinical practice is entirely serological, the IgM response representing acute infection and the IgG response immunity, although more sophisticated molecular techniques have been applied experimentally. A second agent of epidemic enteral hepatitis, the hepatitis E virus, has recently been cloned and sequenced and serological tests for this virus are available, although experience in their use is necessarily limited and a commercial IgM assay has yet to be produced. Serological tests for the hepatitis B virus are well developed. The IgM anticore response differentiates acute infection from chronic, the latter being characterized by the persistence of hepatitis B surface antigen for over six months. Chronic carriers are at risk of liver damage and this risk is best assessed by the amount of viral DNA circulating, which can be determined using a hybridization assay. More sensitive techniques such as the branched chain DNA assay or PCR can detect lower levels of viral DNA but their clinical relevance remains to be established. The hepatitis D virus is defective and relies on hepatitis B to replicate. Serology for antibody and antigen is well established although PCR for circulating viral genome may come to supplant hepatic viral antigen as a test for hepatitis D replication. For hepatitis C serology is feasible only for antibodies, not antigens; although early tests were prone both to false positives and false negatives, current versions are more reliable. PCR has been much used for detection of hepatitis C RNA in blood and tissues and a bDNA assay is now commercially available. Cytomegalovirus detection is confounded by the problem of distinguishing asymptomatic viral replication from disease. Serology is helpful, especially in primary infections, but viral culture is a widely used method. PCR (especially quantitative modifications) or the pp65 antigenaemia assay are experimental approaches which may prove specific enough for general use.     

Heat-stabilized albumin microspheres as a sustained drug delivery system for the antimetabolite, 5-fluorouracil

A nested polymerase chain reaction (PCR) test was developed to examine infection with the bovine lentivirus, bovine immunodeficiency-like virus (BIV), in cattle. Primers were designed to amplify 2 separate regions of the pol and env segments of the BIV genome. Two calves were experimentally infected with an isolate derived from the original strain of BIV, R29, or with a recent field isolate, FL491. Serial blood samples were collected and examined by virus isolation, protein immunoblot, and nested PCR. The nested PCR test detected BIV infection by 3 days after inoculation, earlier than the other 2 methods, and continued to identify infected cattle 9 to 15.5 months after inoculation, even when results from virus isolation and serology became negative. Nested PCR also detected multiple-size env products in samples obtained later in the infection from the calf that received FL491, giving evidence that viral quasispecies were selected during in vivo replication of the virus. Results indicated that the nested PCR test is more sensitive than virus isolation or serology for the detection of BIV infection in cattle.     

False-positive HIV-1 test results in a low-risk screening setting of voluntary blood donation. Retrovirus Epidemiology Donor Study

CONTEXT: Persons at risk of human immunodeficiency virus 1 (HIV-1) infection, have been classified incorrectly as HIV infected because of Western blot results, but the frequency of false-positive Western blot results is unknown. OBJECTIVES: To determine the frequency of false-positive HIV-1 Western blot results in US blood donors and to make projections to other screened populations. Secondarily, to validate an algorithm for evaluating possible false-positive cases. DESIGN: A retrospective cohort study of HIV-1 enzyme immunoassay (EIA) and Western blot results from large blood donor screening programs in which donors with suspected false-positive Western blot results underwent HIV-1 RNA polymerase chain reaction (PCR) testing and follow-up HIV-1 serology. SETTING: Five US blood centers participating in the Retrovirus Epidemiology Donor Study. PARTICIPANTS: More than 5 million allogeneic and autologous blood donors who successfully donated blood at 1 of the 5 participating centers from 1991 through 1995. MAIN OUTCOME MEASURES: Rate of false positivity by Western blot and true HIV-1 infection status as determined by HIV-1 RNA PCR and by serologic follow-up of blood donors more than 5 weeks after donation. RESULTS: Of 421 donors who were positive for HIV-1 by Western blot, 39 (9.3%) met the criteria of possible false positivity because they lacked reactivity to p31. Of these, 20 (51.3%) were proven by PCR not to be infected with HIV-1. The false-positive prevalence was 4.8% of Western blot-positive donors and 0.0004% (1 in 251000) of all donors (95% confidence interval, 1 in 173000 to 1 in 379000 donors). CONCLUSIONS: A false diagnosis of HIV-1 infection can result from the combination of EIA and Western blot testing in blood donor and other HIV-1 screening programs. Individuals with a positive Western blot result lacking the p31 band should be counseled that, although they may be HIV infected, there is uncertainty about this conclusion. These individuals should be further evaluated by RNA PCR testing (if feasible) and HIV serologic analysis on a follow-up sample.     

In vivo and in vitro detection of canine distemper virus nucleoprotein gene with digoxigenin-labelled RNA, double-stranded DNA probes and oligonucleotides by in situ hybridization

A single-stranded RNA, two double-stranded (ds) DNA probes and 10 oligonucleotides labelled with digoxigenin were comparatively evaluated for their usefulness to detect canine distemper virus (CDV) nucleoprotein RNA in in vitro infected Vero cells and in tissues of dogs with spontaneous CDV infection by in situ hybridization (ISH). In addition, results were compared to CDV nucleoprotein antigen distribution as demonstrated by immunohistochemistry. The RNA probe was derived from the virulent A75/17 strain, the DNA and oligonucleotide probes from the avirulent Onderstepoort strain of CDV. The two DNA probes were 287 and 126 base pairs long. For ISH, various factors including fixatives, proteolytic digestion, probe concentration, hybridization conditions and detection systems were compared. All probes were suitable for demonstration of CDV RNA in in vitro infected cells, regardless of the CDV strain employed. In vivo CDV nucleic acid was detected by RNA and the dsDNA probes. However, the probes varied substantially with respect to sensitivity and specificity. The CDV RNA probe was far superior in sensitivity when compared to the DNA probes. Furthermore, the shorter DNA probe displayed a higher sensitivity, indicating that length of the probe is an important parameter when selecting probes. Oligonucleotides displayed only rarely a positive signal and caused frequently hybridization signals in the nucleus, which where considered not specific for CDV. Summarized, the present study reveals that RNA probes are currently the most sensitive tool for detection of CDV RNA in tissues.