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.

Detection of RNA complementary to herpes simplex virus DNA in human cervical squamous cell neoplasms.

Nonneoplastic and neoplastic cervical biopsy specimens were examined by in situ hybridization to 125I-labeled DNA of herpes simplex virus (HSV), adenovirus, and bacteriophage lambda DNA's, and quantitative hybridization data were obtained using a Video Image Analyser. HSV-specific RNA was detected in 72% of cervical intraepithelial neoplasia, 60% of squamous cervical carcinomas, 2% of nonneoplastic cervices, and 9% of primary adenocarcinomas of the cervix. None of the tissues gave positive hybridization with adenovirus or lambda DNA probes. In paired biopsies of cervical intraepithelial neoplasia and nonneoplastic epithelium from 29 individuals, HSV-specific RNA was detected only in the epithelium of the neoplastic sample and not in the nonneoplastic control. Infectious HSV-2 was isolated from a low proportion (2%) of both ectocervical swabs and cell-free tissue extracts of patients examined, suggesting that the HSV-specific RNA detected in squamous cell neoplasms was not due to overt infections.

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.

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.

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.

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.

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.

An outbreak of Q fever affecting postal workers in Oxfordshire.

Twenty-five people in Oxfordshire were found to have had clinical illness due to Q fever in the 3 months from April to June 1983. Twelve cases were diagnosed through the routine laboratory diagnostic service. Five of these were postmen, four of whom worked in a sorting office where an outbreak of illness similar to influenza had been noted by the Occupational Health Nurse. Thirteen cases were diagnosed by active case-finding in this sorting office but investigation failed to define the source of the outbreak. Nine of the 18 postal workers were found to have antibodies to phase I Coxiella burnetii antigen. The significance of these antibodies is discussed. Surveillance for over 2 years has not revealed anyone with symptoms or signs suggestive of chronic Q fever. An outbreak of Q fever among postal workers has not previously been described. We recommend continued surveillance for this enigmatic condition.

Murine pneumonia virus: seroepidemiological evidence of widespread human infection.

More than 75% of a random sample of adult human sera exhibited moderate to high murine pneumonia virus (PVM)-neutralizing activity. There was no correlation between PVM-neutralizing activity and respiratory syncytial virus or parainfluenza type 3 virus-neutralizing activities of the same sera. In children the proportion of sera with moderate to high titres increased with age, indicating early exposure to infection. Seroconversion (i.e. greater than fourfold increase in titre) was observed in four of 108 paired samples of previously undiagnosed respiratory infections. These observations suggest that the human population is frequently exposed to infection with PVM or an antigenically related virus. The sera of patients suffering from Paget's disease of bone tended to exhibit higher than normal PVM-neutralizing titres in comparison with the sera of patients with other bone diseases. Thus, PVM (or an antigenically related virus) resembles some other parainfluenza viruses in being circumstantially associated with Paget's disease of bone.

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.

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.

Ultraviolet irradiation of herpes simplex virus (type 1): delayed transcription and comparative sensitivites of virus functions.

The delay in the replication of herpes simplex virus surviving u.v. irradiation occurs after the uncoating of virus, as judged by sensitivity to DNase. It occurs before translation, judged by the kinetics of appearance of various virus-specific proteins, and before transcription, judged by the detection of virus-specific RNA by in situ hybridization. Since the delays in both transcription and translation are reversed by photoreactivation, the simplest hypothesis is that pyrimidine dimers directly obstruct transcription;unless these are broken by photoreactivating enzymes, there will be transcriptional delay until reactivating processes have repaired the lesion. The u.v. sensitivities of the abilities to induce various enzymes (thymidine kinase, DNase and DNA polymerase) were only about four times less than that of infectivity. The The ability to induce the three enzymes was three times less sensitive than that of the structural antigen (Band II).

Detection of RNA complementary to herpes-simplex virus in mononuclear cells from patients with Behçet's syndrome and recurrent oral ulcers.

Viral DNA probes were used in in-situ hybridisation to detect the complementary RNA in mononuclear cells from the blood of normal subjects and patients with Behçet's syndrome and recurrent oral ulcers. Hybridisation of 125I-labelled DNA probes was detected by means of autoradiography and quantitated with a video image-analysis technique. Hybridisation between herpes-simplex virus type 1 (HSV-1) DNA and the complementary RNA in mononuclear cells was significantly greater in 10 of 20 patients with Behçet's syndrome than in controls. Consistently, mononuclear cells from fewer patients showed significant hybridisation with the HSV-2 probe, and the grain counts were also lower. Control DNA probes from adenovirus 2, bacteriophage lambda, and Micrococcus lysodeikticus did not show significant hybridisation. Further separation of the patients into the four types of Behçet's syndrome revealed that mononuclear cells in 8 of 10 patients with the ocular or arthritic types showed significant hybridisation of RNA to the HSV-1 DNA probe, compared with 2 of 10 patients with the mucocutaneous or neurological types. Mononuclear cells from 4 of 8 patients with minor but not major aphthous ulcers also showed significant RNA hybridisation to HSV-1 DNA. The results suggest that at least part of the HSV genome is present and transcribed in peripheral-blood mononuclear cells--probably lymphocytes--of patients with the ocular and arthritic types of Behçet's syndrome and minor aphthous ulcers. The immunopathogenesis of these diseases might therefore be associated with HSV-1.

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.

African Kaposi's sarcoma and AIDS.

16 Zambian patients with Kaposi's sarcoma (KS) were studied to determine whether they had evidence of lymphopenia with decreased T helper/T suppressor (Th/Ts) ratios or previous infection with opportunistic pathogens. Serological tests for viruses commonly associated with the acquired immunodeficiency syndrome (AIDS) were also carried out. 12 patients had a decreased Th/Ts and 2 of these were also lymphopenic. Serological evidence for infection with Toxoplasma and with Pneumocystis was present but this was not significantly more common in KS patients than in controls. All 16 patients had antibodies to cytomegalovirus (CMV), 15 had antibodies to Epstein-Barr virus and 13 to human T leukaemia virus (HTLV) infected cells. 5 patients had evidence of previous infection with hepatitis B virus. African patients with KS seem to have an immunological and virological profile similar to that seen in American patients with AIDS.

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.

Detection of RNA complementary to herpes simplex virus in human oral squamous cell carcinoma.

Biopsy specimens from patients with oral squamous cell carcinoma were examined by in situ hybridisation for evidence of RNA complementary to herpes simplex virus (HSV) type 1, HSV type 2, and adenovirus type 2. RNA complementary to HSV was found in 66% of carcinomas and 33% of non-malignant lesions from other patients. In a further study with internally paired controls, RNA complementary to HSV was found in 53% of carcinoma biopsy specimens but in no biopsy specimens of normal oral mucosa from the same patients. RNA complementary to HSV was found in over 50% of oral squamous cell carcinomas.

Abnormal tubular structures associated with the granular endoplasmic reticulum of neocortical neurons in a biopsy from a patient with Alzheimer's disease.

The electron microscope has been used to examine a diagnostic biopsy of frontal neocortex which showed the light microscopic features of Alzheimer's disease. In addition to plaques and tangles, the biopsy showed some neurons which contained abnormal tubular profiles specifically associated with their granular endoplasmic reticulum. In transverse section the profiles consisted of two concentric layers of trilaminar unit membrane with an overall diameter of approximately 75 nm and they appeared to lie within the cisternae of granular endoplasmic reticulum. When cut longitudinally, the structures appeared as elongated tubes lying within the endoplasmic reticulum, but a number of ends were found where the inner membrane formed a closed tube and the outer membrane folded back and was continuous with the membrane forming the outer layer of the sac of endoplasmic reticulum. These profiles do not appear to have been described previously although similarities between their ultrastructure and that of the coat of certain unusual forms of Rhabdovirus are noted.

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.