Factors influencing PCR detection of viruses in cerebrospinal fluid of patients with suspected CNS infections.

BACKGROUND: Polymerase chain reaction (PCR) is used to detect viruses in the cerebrospinal fluid (CSF) of patients with neurological disease. However, data to assist its use or interpretation are limited. OBJECTIVE: We investigated factors possibly influencing viral detection in CSF by PCR, which will also help clinicians interpret positive and negative results. METHODS: CSF from patients with was tested for human herpesviruses types 1-6, JC virus, enteroviruses, and Toxoplasma gondii. The likelihood of central nervous system (CNS) infection was classified as likely, possible, or unlikely. PCR findings in these categories were compared using single variable and logistic regression analysis. RESULTS: Of 787 samples tested, 97 (12%) were PCR positive for one or more viruses. Of episodes likely to be CNS viral infections, 30% were PCR positive compared to 5% categorised as unlikely. The most frequent positive findings were Epstein Barr virus (EBV), enteroviruses, and herpes simplex virus (HSV). Enteroviruses and HSV were found predominantly in the likely CNS viral infection group, whereas EBV was found mainly in the unlikely group. Positive PCR results were more likely when there were 3-14 days between symptom onset and lumbar puncture, and when CSF white cell count was abnormal, although a normal CSF did not exclude a viral infection. CONCLUSIONS: The diagnostic yield of PCR can be maximised by using sensitive assays to detect a range of pathogens in appropriately timed CSF samples. PCR results, in particular EBV, should be interpreted cautiously when symptoms cannot readily be attributed to the virus detected.

Rubella.

Maternal rubella is now rare in many developed countries that have rubella vaccination programmes. However, in many developing countries congenital rubella syndrome (CRS) remains a major cause of developmental anomalies, particularly blindness and deafness. WHO have provided recommendations for prevention of CRS, and, encouragingly, the number of countries introducing rubella vaccination programmes has risen. However, declining uptake rates due to concerns about the measles-mumps-rubella vaccine in the UK, and increasing numbers of cases in some European countries coupled with poor uptake rates might jeopardise this progress. Surveillance of postnatally and congenitally acquired infection is an essential component of CRS prevention since rubella is difficult to diagnose on clinical grounds alone. Laboratory differentiation of rubella from other rash-causing infections, such as measles, parvovirus B19, human herpesvirus 6, and enteroviruses in developed countries, and various endemic arboviruses is essential. Reverse transcriptase PCR and sequencing for diagnosis and molecular epidemiological investigation and detection of rubella-specific IgG and IgM salivary antibody responses in oral fluid are now available.

Hepatitis A booster vaccination: is there a need?

Hepatitis A is one of the most common vaccine-preventable infectious diseases in the world. Effective vaccines against hepatitis A have been available since 1992, and they provide long-term immunity against the infection. However, there is no worldwide consensus on how long protection will last or whether there will be a need for hepatitis A virus (HAV) booster vaccinations in the future. In most countries, booster-vaccination policy is guided by manufacturers' recommendations, national authorities, or both. In June, 2002, a panel of international experts met to review the long-term immunogenicity and protection conferred by HAV vaccine in different population groups. Data have shown that after a full primary vaccination course, protective antibody amounts persist beyond 10 years in healthy individuals, and underlying immune memory provides protection far beyond the duration of anti-HAV antibodies. The group concluded that there is no evidence to lend support to HAV booster vaccination after a full primary vaccination course in a healthy individual. However, further investigations are needed before deciding if boosters can be omitted in special patient-groups.

Hepatitis B vaccine -- do we need boosters?

This review analyses the cumulated data from a number of long-term follow-up studies among infants, children and adults vaccinated against hepatitis B in industrialised and developing countries. Despite low or undetectable antibody responses years after vaccination, the development of HBsAg was a rarity and, if present, only transient. Some vaccinees developed anti-HBc responses but none developed an HB carrier state or clinical manifestations of disease. Studies demonstrating anamnestic responses among those with low or undetectable anti-HBs levels following challenge with HB vaccine, together with the production of anti-HBs in circulating B-cells by spot ELISA, confirmed the presence of immune memory among vaccinees. Anamnestic anti-HBs responses all correlate close in kinetics and magnitude with proliferative T-cell responses. The accumulated data from studies assessed in this Review indicate that protection is dependent on immune memory, rather than declining anti-HBs responses and add additional weight to the European Consensus recommendations (12) that following a complete course of vaccination, booster doses are unnecessary in immunocompetent persons. If implemented, this recommendation will have considerable cost benefits world-wide.

Lifelong protection against hepatitis B: the role of vaccine immunogenicity in immune memory.

Long-term protection against hepatitis B (HB) disease is dependent on persistence of a strong immune memory. This paper presents and discusses new knowledge that allows a better understanding of the mechanisms of long-term protection following hepatitis B vaccination. Studies have revealed links between specific lymphoproliferation, the in vivo humoral response and immune memory. The strength of immune memory and of a subsequent secondary immune response can therefore be predicted by the antibody response following primary vaccination. Vaccine antigen dose and structure have been identified as important influences in the primary antibody response and development of immune memory. The data and considerations presented support the use of highly immunogenic HB vaccines in order to provide long-lasting protection against HB disease.

Cervical lesions are associated with human papillomavirus type 16 intratypic variants that have high transcriptional activity and increased usage of common mammalian codons.

Human papillomavirus type 16 (HPV-16) is a major cause of cervical neoplasia, but only a minority of HPV-16 infections result in cancer. Whether particular HPV-16 variants are associated with cervical disease has not yet been clearly established. An investigation of whether cervical neoplasia is associated with infection with HPV-16 intratypic variants was undertaken by using RFLP analyses in a study of 100 HPV-16 DNA-positive women with or without neoplasia. RFLP variant 2 was positively associated [odds ratio (OR)=2.57] and variant 5 was negatively associated with disease (OR=0.2). Variant 1, which resembles the reference isolate of HPV-16, was found at a similar prevalence among those with and without neoplasia. Variants 1 and 2 were also more likely to be associated with detectable viral mRNA than variant 5 (respectively P=0.03 and P=0.00). When HPV-16 E5 ORFs in 50 clones from 36 clinical samples were sequenced, 19 variant HPV-16 E5 DNA sequences were identified. Twelve of these DNA sequences encoded variant E5 amino acid sequences, 10 of which were novel. Whilst the associations between HPV-16 E5 RFLP variants and neoplasia could not be attributed to differences in amino acid sequences, correlation was observed in codon usage. DNA sequences of RFLP variant 2 (associated with greatest OR for neoplasia) had a significantly greater usage of common mammalian codons compared with RFLP pattern 1 variants.

Problems in the interpretation of HIV-1 viral load assays using commercial reagents.

During routine monitoring of human immunodeficiency virus (HIV) viral load, two problems arose. First, a number of patients, the majority being African, were found to have low viral loads by the Chiron branched-chain DNA assay in conjunction with low CD4(+) cell numbers. In order to determine whether this was due to failure of the branched-chain DNA assay to detect non-B subtypes of HIV, selected samples were subtyped and HIV RNA quantified by branched-chain DNA, NASBA, and the Roche Monitor RT-PCR assay. Twenty-eight (97%) of 29 Africans were infected with a non-B subtype of HIV and 15 (93.7%) of 16 non-Africans with subtype B. Twenty-three samples had a low viral load by branched-chain DNA, which was confirmed by the NASBA and RT-PCR assays. All three assays detected B and non-B subtypes with similar efficiency; NASBA failed to detect HIV RNA in a small number of non-B samples. Discrepancies between viral load and CD4(+) cell numbers did not appear therefore to be related to subtype. Second, while quantification of HIV RNA was being conducted using version 2 of the branched-chain DNA assay (lower detection limit 500 HIV RNA copies/ml) the manufacturers had developed a more sensitive assay and a comparative evaluation was therefore conducted. In approximately 30% of samples the viral load was up to 10 times higher with the more sensitive assay. These experiences emphasise the importance of close collaboration between the clinic and the laboratory.

A phase I trial in HIV negative healthy volunteers evaluating the effect of potent adjuvants on immunogenicity of a recombinant gp120W61D derived from dual tropic R5X4 HIV-1ACH320.

Thirty healthy HIV negative volunteers were randomised to receive 200 micrograms of rgp120W61D in either: 3D-MPL and QS21, with an oil and water emulsion (SBAS-2) (13); or 3D-MPL and QS21 (SBAS-1) (11); or alum (six). Immunizations were given at 0, 4 and 28 weeks and 23 (77%) participants completed the schedule. Adverse events were more frequent (P < 0.001) and more severe (P < 0.001) in the SBAS-2 group. Binding antibodies to the homologous rgp120W61D were detected after the first immunisation only in those receiving SBAS-1 and SBAS-2, were maximal after the third immunization in all three groups, and persisted to week 84 only in the novel adjuvant groups. These differences were significant (p = 0.02). Neutralising antibodies to TCLA-strains of HIV-1 were observed after the second immunization in all three groups, were maximal after the third immunization, but did not neutralise homologous or heterologous PBMC derived primary HIV-1 isolates. Proliferative T-cell responses to rgp120W61D were maximal after the second immunization and reached very high values in the SBAS-2 group. HIV-1 specific CD8+ MHC Class I restricted cytotoxic T-lymphocytes were not seen in a subset of participants tested at a single timepoint. SBAS-2 with rgp120W61D induced antibody titres as high as those seen in HIV infection, but the quality of the antibodies remained different in that there was no evidence of primary isolate neutralisation. Although cell-mediated immunity was enhanced by SBAS-2 in terms of lymphoproliferative responses, HIV-1 specific CD8+ cytotoxicity was not demonstrated.

Identification of hepatitis C virus seroconversion resulting from nosocomial transmission on a haemodialysis unit: implications for infection control and laboratory screening.

Hepatitis C virus (HCV) seroconversion was detected by routine screening in a haemodialysis patient, Patient 1. Serological investigations were undertaken over the following 3 months to determine if further transmission to other patients on the unit had occurred. No additional cases were identified. Twenty-two haemodialysis patients known to have HCV infection were investigated using molecular epidemiological methods to determine if transmission between patients had occurred. HCV viraemia was demonstrated by polymerase chain reaction in 19 of 22 patients (86%). Genotyping showed that eight patients were infected with genotype 1, three with genotype 3 and eight, including Patient 1, with genotype 2. Phylogenetic analysis of viral sequences from the eight patients with genotype 2 revealed three, including Patient 1,with a novel subtype of HCV type 2, and revealed close similarity between viral sequences from patient 1 and those from one other patient, suggesting transmission. This was consistent with haemodialysis histories. Among other patients with genotype 2, there were two with subtype 2a and three others with three separate novel subtypes, as yet undesignated. With the exception of patient 1, all patients infected with novel subtypes were of Afro-Caribbean origin. The HCV prevalence among patients on the haemodialysis unit was high (14%), which may reflect the ethnicity of our haemodialysis population. This case emphasises the risk of nosocomial transmission and the importance of infection control procedures on haemodialysis units, and highlights the usefulness of molecular epidemiological techniques for the investigation of outbreaks of HCV infection.

High risk genital papillomavirus infections are spread vertically.

It is well recognised that high-risk human papillomaviruses (HPVs) are spread by sexual activity, but the possibility of non-sexual transmission remains controversial. We present evidence for vertical transmission from at least 30% HPV positive mothers to their infants, resulting in persistent infection in children. That the mother is the source of infant infection has been confirmed by DNA sequencing. We also discuss the evidence for oral HPV-16 infection in children. In our own studies, HPV-16 DNA was detected in buccal cells from 48% children, aged 3-11 and transcriptionally active infection was confirmed in some children. Other studies have reported prevalences of 19%-27% among children less than 11 years of age. Studies that have failed to detect high-risk HPVs in children have used techniques which were insufficiently sensitive to detect the low levels of virus present. Serological studies also suggest that < or = 45% prepubertal children have acquired HPV-16. Thus, convincing evidence is now available for vertical transmission of high risk HPVs, which probably results in widespread infection among children. The consequences of such infections remain to be elucidated.

Medical students' risk of infection with bloodborne viruses at home and abroad: questionnaire survey.

OBJECTIVE: To determine risks of exposure to and prevention of bloodborne virus infections among medical students during their elective period. DESIGN: Questionnaire study of students returning from their electives in 1997-8. SETTING: Urban teaching hospital. SUBJECTS: 220 final year medical students. RESULTS: 148 students (67%) returned questionnaires; all had been vaccinated against hepatitis B. 65 respondents (44%) had visited areas of relatively high endemicity for HIV, although 27 (42%) of these, all of whom had visited areas other than sub-Saharan Africa, were unaware of this. All but one had discussed their elective with advisers. Four students experienced percutaneous or mucosal exposure to potentially infectious body fluids, three in areas with a high prevalence of HIV infection. 44 respondents (30%) had experienced at least one such exposure during their clinical training; 75% of these exposures were unreported. 34% (13/38) students who visited areas known to have a high prevalence of HIV infection took with them a starter pack of zidovudine for post-exposure prophylaxis; 53% (20) took latex gloves and 63% (24) a medi-kit. None of the 27 students who were unaware that the areas they visited had a relatively high prevalence of HIV infection took zidovudine; only 15% (4) took gloves and 30% (8) a medi-kit. CONCLUSIONS: Medical schools should produce, regularly update, and implement guidelines regarding protection from bloodborne viruses during clinical studies, including electives. Education and training in infection control should start at the earliest opportunity.

Development and evaluation of quantitative-competitive PCR for quantitation of coxsackievirus B3 RNA in experimentally infected murine tissues.

A method is described for quantitation of enterovirus RNA in experimentally infected murine tissues. Viral RNA was extracted from tissue samples and amplified by reverse transcriptase PCR in the presence of an internal standard RNA. The ratio of PCR product derived from viral RNA and internal standard RNA was then determined using specific probes in a post-PCR electrochemiluminescent hybridization assay. This provided an estimate of the viral RNA copy number in the original sample, and detection of PCR product derived from internal standard RNA validated sample processing and amplification procedures. RNA copy number correlated with viral infectivity of cell culture-derived virus, and one tissue culture infective dose was found to contain approximately 10(3) genome equivalents. The ratio of RNA copy number to infectivity in myocardial tissue taken from mice during the acute phase of coxsackievirus B3 myocarditis was more variable ranging from 10(4)-10(7), and was dependent on the stage of infection, reflecting differential rates of clearance for viral RNA and viral infectivity. The assay is rapid, and could facilitate investigations which currently rely upon enterovirus quantitation by titration in cell culture. This would be useful for experimental studies of viral pathogenesis, prophylaxis and antiviral therapy.

Rubella virus and chronic joint disease: is there an association?

Synovial fluid samples and/or biopsies from 79 patients with various chronic inflammatory joint diseases or traumatic joint injury were tested for rubella virus (RV) in order to confirm or refute results from other studies that suggested RV as a cause of chronic inflammatory joint disease. Sixty-eight of the 72 patients tested had RV antibodies. RV RNA was detected by reverse transcription-PCR in the synovial fluid cells from two patients. RV was also isolated by cell culture from the synovial fluid of one of these two patients. This patient was a 42-year-old female with common variable immune deficiency and Mycoplasma hominis arthritis, while the other was a 68-year-old female with rheumatoid arthritis. While these results fail to confirm that RV is associated with chronic inflammatory joint disease, they suggest that RV may persist within a joint and be reactivated when cell-mediated immunity is suppressed.

Feasibility of named antenatal HIV screening in an inner city population.

The object of this study was to assess the resource use, feasibility, uptake and consumers' perspective of introducing routine named human immunodeficiency virus (HIV) testing into an inner city hospital antenatal clinic (St Thomas Hospital, part of Guy's and St Thomas Hospital Trust). Following the introduction of a new service offering routine named HIV testing at booking appointments, the length of the appointments were recorded over a three-month period and compared with appointment lengths in the previously existing service. Women being offered routine named HIV testing were asked to complete a questionnaire before and after their appointment to assess knowledge, attitudes and acceptability of HIV testing. Subjects were three-hundred-and-eighty-one pregnant women attending the antenatal clinic for their booking appointment. There was no significant difference in the length of the booking appointments following the introduction of offering routine named HIV testing. One-hundred-and-fifty-eight women (42%) consented to testing with no positive results detected. There was a significant rise in the uptake of the test over the three-month period. No other variables (demographics, perceived risk, having read the leaflet) were predictive of uptake. Anxiety scores were significantly less after the booking appointment. Routine named HIV testing was feasibly introduced into a hospital antenatal clinic with minimum resource implications. Further study is needed to monitor the ongoing uptake rate and identification of HIV-positive women before a decision can be made as to the long-term benefit of this new service. Women considered it acceptable to be offered the HIV test and were less anxious after their appointments.