Improvement in the performance of HIV screening kits.

SUMMARY: The aim of this study was to assess the performance of HIV screening kits introduced over a 12-year period. HIV kits used by the National Blood Service (NBS) were assessed in the context of other HIV kits employed by diagnostic and reference laboratories. Thirty-three HIV screening kits were assessed and 13 had the potential to be used by the NBS. Specimens applied to NBS evaluations included 2000 HIV-negative specimens collected from blood donors, 200 HIV-positive specimens and 21 seroconversion panels, with larger numbers applied to the latter two categories prior to implementation of Communauté Européennes (CE) marking. The 33 HIV kits gave repeat reactive rates, based on HIV-negative specimens, of between 0% and 0.8% (and between 0% and 0.2% for kits relevant to the NBS). When examined for diagnostic sensitivity, the 33 kits gave sensitivities between 99.78% and 100%. Kits relevant to NBS gave sensitivities of 100% except one kit, which failed to detect one anti-HIV-2-positive specimen. Twenty-six kits were compared for detection of primary HIV infection. Of these, the 10 combined HIV antigen/antibody kits examined were more sensitive than other formats and have been exclusively adopted by NBS where operational considerations allow. Their added seroconversion sensitivity makes them the screening method of choice for populations at increased risk, e.g. in sexually transmitted infection (STI) clinics. The regular review of evaluation results has demonstrated a continuing improvement over time in the performance of HIV screening kits and contributed to advances in blood safety.

A study of infectious intestinal disease in England: microbiological findings in cases and controls.

A study was undertaken to identify the microorganisms and toxins in stool specimens associated with infectious intestinal disease (IID) among cases in the community and presenting to general practitioners (GPs) and in asymptomatic controls. Population based cohorts were recruited from practice lists in 70 practices and followed for 26 weeks (cohort component). Seven hundred and sixty-one cases of IID identified from the cohorts, 2893 cases who presented to GPs in 34 of the practices (GP component), and age/sex matched control subjects (555 and 2264, respectively) submitted stool specimens by post for comprehensive microbiological examination. Campylobacter spp (12.2% of stools tested), rotavirus group A (7.7%), and small round structured virus (SRSV) (6.5%) were the organisms most commonly detected in the GP component. SRSV was identified in 7.0% of cases in the community cohort. No target microorganisms or toxins were identified in 45.1% and 63.1% of cases in the two components. Aeromonas spp, Yersinia spp, and some enterovirulent groups of Escherichia coli were detected as frequently in controls as in cases. The higher frequency of detection of campylobacter, salmonella, and rotavirus among cases who presented to GPs than among those in the community suggests that those pathogens cause more severe illness. No enteropathogens were detected from a large proportion of cases although comprehensive standard methods were used to seek them.

Pathogenesis of pneumovirus infections in mice: detection of pneumonia virus of mice and human respiratory syncytial virus mRNA in lungs of infected mice by in situ hybridization.

The pathogenesis of pneumonia virus of mice (PVM) and human respiratory syncytial virus (HRSV) in BALB/c mice were investigated by using in situ hybridization to detect virus mRNA in fixed lung sections. Following intranasal inoculation with 120 p.f.u. PVM the pattern of hybridization showed that virus mRNA was initially detected within 2 days in alveolar cells. As the infection progressed the number of hybridizing alveolar cells increased and signal was also detected in cells lining the terminal bronchioles. By days 4 to 5 post-infection areas of morphological abnormality could be seen, particularly in the strongly hybridizing regions of the lung, and this correlated with the appearance of clinical signs of infection. In animals which survived the infection virus-specific mRNA could not be detected 10 days post-infection. Mice infected with 1500 p.f.u. HRSV showed significant differences in the distribution of virus-specific mRNA when compared to the pattern seen with PVM. HRSV mRNA was detected over large areas, but predominantly in peribronchiolar and perivascular regions of the lungs 5 days post-infection. The yield of PVM from infected mouse lungs was considerably higher than that of HRSV. The possible implications of these results for the use of the mouse model for pneumovirus infections are discussed.

Low detection rate and maternal provenance of hepatitis B virus S gene mutants in cases of failed postnatal immunoprophylaxis in England and Wales.

Hepatitis B virus (HBV) infection occurred despite full passive-active immunoprophylaxis in 20 of 321 infants born to mothers seropositive for hepatitis B e antigen. In 2 (12%) of 17 infected infants, mother-infant DNA sequence mismatches were found in a segment of the HBV S gene coding for antigenic determinants of the HBV surface antigen (HBsAg) amplified from sera by polymerase chain reaction (PCR). Point substitutions occurred in codons 120, 134, and 144 of the HBsAg polypeptide in the variant sequence of 1 infant and in codon 126 in the other; all were missense mutations. Mutant sequences could not be recovered from maternal sera by PCR cloning but were selectively generated using an amplification refractory mutation system. The frequency of potential vaccine escape mutants is therefore low, and these preexist maternally as minor variants.

Herpes simplex virus encephalitis in a mouse model: PCR evidence for CNS latency following acute infection.

We have used a mouse model of herpes simplex encephalitis produced by intranasal inoculation of virus to study the expression of viral immediate early, early and late genes and latency associated transcript (LAT) in trigeminal ganglia and brain at various times after inoculation. A PCR technique was used to detect the viral gene transcripts. All viral genes were expressed between post-inoculation days 1 and 13. On post-inoculation day 42 when the acute infection had subsided only the LAT could be detected, most commonly (70%) in the trigeminal ganglion but also, in 50% of mice, in the brain stem, in 40% in olfactory bulbs and in 20% in cerebrum and cerebellum. These findings suggest that latent infection by HSV-1 may be relatively readily established in the CNS as well as in sensory ganglia. The frequency of establishment of latency appears to be related to the neuroanatomical accessibility of each brain region to the site of entry of the virus.

Use of anti-GOR testing in the screening of blood donors for hepatitis C virus infection.

Sera from 12/1,155 (1%) anti-HCV-negative (Elisa) UK blood donors were found to be anti-GOR positive. None out of 12 of those sera were positive for HCV RNA by the reverse transcriptase/polymerase chain reaction (RT/PCR). In a cohort of 316 anti-HCV Elisa-positive sera, 27/57 RIBA-positive, and 1/188 RIBA-negative sera were anti-GOR positive, resulting in a sensitivity of 47% and a specificity of 99.5% for anti-GOR as a marker for RIBA-confirmed HCV infection. Four out of 71 (6%) of RIBA-indeterminate sera were anti-GOR positive. Donors with anti-GOR reactivity were more likely to have antibodies against each of the individual HCV antigens represented in the RIBA, and those antibodies were of greater intensity, when compared to the anti-GOR negative cohort. Twenty out of 36 (55.6%) of RT/PCR-positive sera were anti-GOR positive, compared to 1/7 (14.3%) of RT/PCR-negative sera (p = 0.09). The usefulness of anti-GOR testing of UK blood donors is discussed in the light of these results.

3' terminal nucleotide sequence of human astrovirus type 1 and routine detection of astrovirus nucleic acid and antigens.

Human astrovirus type 1 was purified by caesium chloride density-gradient centrifugation and the virus was located using an immunodot blot technique with polyclonal rabbit serum, which reacted with all five serotypes. The virus banded with a density of 1.33 g/ml. RNA was extracted from the purified virus, converted into double-stranded cDNA, using an oligo(dT) primer, and cloned into plasmid and M13 vectors. The sequence of the 3' end of astrovirus RNA adjacent to the poly(A) tract was determined. This sequence showed no significant homology with the equivalent region of other positive-sense RNA viruses. Synthetic oligonucleotide primers were designed to amplify specifically astrovirus type 1 RNA in a polymerase chain reaction.

Chicken pox infection (varicella zoster virus) and acute monoarthritis: evidence against a direct viral mechanism.

A 9 year old boy developed acute monoarthritis of the left knee concurrent with the appearance of a varicella zoster virus (VZV) rash. Repeated VZV DNA hybridisation of the cells within the synovial fluid and synovial membrane failed to show any evidence of intracellular virus. Virus was isolated from synovial fluid 24 hours after the start of clinical infection but not later. These findings suggest that the mechanism of the arthritis is not due to viral replication inside the swollen joint.

Failure to detect cytomegalovirus DNA in pancreas in type 2 diabetes.

We have attempted to confirm the finding of cytomegalovirus (CMV) nucleic acid sequences in pancreas sections from patients with type 2 diabetes in a large population since this finding has major implications for the pathogenesis of the disorder. A highly sensitive nested polymerase chain reaction method was developed to detect the immediate-early CMV gene in DNA extracted from wax-embedded tissue sections. We could not confirm the previous findings; CMV DNA was not detected in pancreas sections from 43 type 2 diabetic patients or from 38 non-diabetic age-matched subjects, although the method detected CMV DNA, even in very low concentrations, in positive controls.

Coxsackie virus B4 infection of the mouse pancreas: I. Detection of virus-specific RNA in the pancreas by in situ hybridisation.

The pathology of Coxsackie virus B4 (CVB4) infection in a murine model was investigated by in situ hybridisation using a biotinylated cDNA probe derived from CVB4. During the acute phase of infection virus RNA sequences were detected in the exocrine pancreas of 60% of mice infected with a pancreotropic variant of CVB4. A positive hybridisation signal was observed in other organs in some animals including the heart and liver of 1 mouse 28 days after infection. The cellular distribution of virus RNA sequences corresponded well with the histological findings in most tissues. Possible causes for failure of hybridisation in some infected pancreases are discussed in conjunction with potential application of the technique in human pancreas biopsy samples.

Epidemiology of parainfluenza virus type 3 in England and Wales over a ten-year period.

We have analysed data on respiratory syncytial (RS) and parainfluenza type 3 (PF3) viruses reported to the Communicable Disease Surveillance Centre, London, over the period 1978-87. These confirm the annual winter epidemic of RS virus and show that, in England and Wales, PF3 is a summer infection with regular yearly epidemics.

Use of betapropiolactone to disinfect fresh tissue without impairing antigenicity: method applicable to human immunodeficiency virus (HIV) positive tissue.

A method for inactivating viruses in tissues is reported that does not impair the antigenicity of the Coxsackie virus or of some common tissue antigens, a common problem with standard tissue fixation methods. Tissues can be placed briefly in Betapropiolactone before cryostat sectioning without any adverse effect on preservation or antigen expression. It is suggested that use of Betapropiolactone is applicable to tissues harbouring or exposed to the human immunodeficiency virus (HIV). As betapropiolactone has been reported to be carcinogenic in rodents any potential danger can be avoided by basic simple precautions.

Experimental murine herpes simplex encephalitis: immunohistochemical detection of virus antigens.

Inbred Balb/c female mice were intranasally inoculated with 1 microliter of a suspension of herpes simplex virus type 1 adjusted to contain the required infectivity. Doses of virus inoculated ranged from 1 X 10(2) plaque forming units (p.f.u.) to 1 X 10(6) p.f.u. Humoral and cellular immunological response of the animals was studied for the acute phase of the disease, defined as being up to 14 days post infection. Localization of virus-antigen positive cells, detected by immunoperoxidase staining, was monitored daily for the acute phase. Immune responses directly reflected virus input, with the highest response to 1 X 10(6) p.f.u. of herpes simplex virus. The neuro-anatomical localization of virus-antigen positive cells was found mainly in the hippocampus and entorhinal cortex for inoculum doses of 1 X 10(6) p.f.u. and mainly in the brain stem for inoculum doses 5 X 10(2) p.f.u. The lower mortality rate of animals inoculated with 5 X 10(2) p.f.u. has identified this dose as suitable for use in the study of sites of latency within the central nervous system and in the investigation of reactivation of herpes simplex virus type 1 in causing encephalitis.

The detection of coxsackievirus RNA in cardiac tissue by in situ hybridization.

A cloned cDNA probe derived from coxsackie B4 virus-infected cell RNA was shown to hybridize to the RNA of a number of different enteroviruses including coxsackie A and B viruses, echoviruses and poliovirus. The probe was used to detect virus-specific RNA sequences in cardiac tissue obtained from patients diagnosed as having a coxsackievirus infection. Virus RNA was detected using the technique of in situ hybridization in 46% (6/13) cases, but none was found in normal, control, cardiac samples. Two distinct patterns of infection were observed. The significance of these differences and the possible uses of the technique are discussed.

HTLV-1, HIV-1, hepatitis B and hepatitis delta in the Pacific and South-East Asia: a serological survey.

Blood samples from 13 locations in the Pacific and South-East Asia were tested for evidence of infection with human T-cell lymphotropic virus type-1 (HTLV-1), human immunodeficiency virus (HIV-1), hepatitis B virus (HBV) and hepatitis delta virus (HDV). No samples were positive for antibody to HIV-1. Antibodies to HTLV-1 were found in samples from five locations, the maximum prevalence being 19%, in Vanuatu. Serological markers of HBV infection were found in all locations, the maximal prevalence being 88%, in Majuro, Micronesia. Antibodies to HDV in HBsAg positive sera were found in six locations with a maximum prevalence of 81% in Kiribati.

PIP: In a serological survey of samples taken throughout the Pacific and South-East Asia for HILV-1, HIV-1, HBV, and HDV infection, 9 of the samples tested positive for antibodies to HIV-1. HTLV-1 antibodies were found in 2 groups. Other findings showed 13 or 81% of HBsAG positive sera when tested for anti-HDV. Of all the locations surveyed, serological markers of HBV infection were noted. The maximum prevalence (88%) of HBV infection was found in Majuro, Micronesia. The greatest evidence of antibodies to HDV in HBsAG positive sera of 6 locations was found in kiribati (84% prevalence). The survey studied haemoglobinopathies from samples taken between May 1985 and February 1986 from New Guinea, Philippines, Vanuatu, French Polynesia, Palau and the Federated States of Micronesia. The survey volunteers were adult blood donors attending ante-natal clinic. Samples were also taken from a Tuvaluan community of immigrant workers in Nauru and from people from Kapingamarangi who have been resettled onto Ponape.

Routine full blood counts as indicators of acute viral infections.

Over a period of three weeks about 9000 full blood counts were analysed on the Technicon H6000 automated haematology machine. From these, 62 patients were identified who had abnormally high numbers of large unstained white cells; these patients were followed up for evidence of viral infection. Seventeen were either lost to follow up or in chronic renal failure; of the remaining 45 patients, 40 had viral infections, 26 of which were due to Epstein-Barr virus. In the presence of a raised number of large unstained white cells, an IgM test for Epstein-Barr virus is recommended, followed by routine serology when necessary.