J.C. Booth

Prevalence and distribution of BK virus subtypes in healthy people and immunocompromised patients detected by PCR-restriction enzyme analysis.

BACKGROUND Four antigenic subtypes of BK virus (BKV) have recently been characterised by both genomic subtyping and serological reactivity. OBJECTIVES To study the prevalence and distribution of subtypes of BKV in different groups of patients. STUDY DESIGN Urine specimens were collected from 33 bone marrow transplant (BMT) recipients, from 101 HIV-infected patients, from 15 children aged 2-5 and from 40 pregnant women were tested for BKV DNA by polymerase chain reaction (PCR) and subtyped using a PCR-sequencing (PCR-S) and a modified PCR-restriction enzyme analysis (PCR-RE) methods. RESULTS BKV DNA was detected in 12/18 (67%) of BMT patients with haematuria and 5/15 (33%) without. Overall BKV DNA was detected in 45% of HIV-infected patients, the prevalence of BKV DNA increased with greater immunosuppression as defined by CD4 cell counts. BKV DNA was detected in urine samples from 27% of children and 47% of pregnant women. Four stable BKV subtypes were detected in these patient groups. Dual infections with more than one subtype were identified in urine samples from HIV-infected patients, children and pregnant women but not in the samples from bone marrow recipients. CONCLUSION This study has confirmed the high prevalence of BKV infection in immunocompromised patients and suggests that stable BKV subtypes with conserved sequences are circulating in the human population. The techniques of PCR-S and PCR-RE described in this study are sufficiently sensitive for subtyping BKV direct from clinical specimens.

MICROBIAL CAUSES OF MENTAL RETARDATION THE ROLE OF PRENATAL INFECTIONS WITH CYTOMEGALOVIRUS, RUBELLA VIRUS, AND TOXOPLASMA

Abstract Mentally retarded children, all under 6 years of age and living at home with their parents, were studied for evidence of congenital infection with cytomegalovirus, rubella virus, and toxoplasma. They were compared with normal children of the same ages. A significant association was found between cytomegalovirus infection and microcephalic mental deficiency. This virus may account for about 10% of such cases. Rubella and toxoplasmosis together probably were responsible for about 2-3% of all the cases of mental deficiency.

A polymerase chain reaction to detect a spliced late transcript of human cytomegalovirus in the blood of bone marrow transplant recipients

A reverse transcription (RT) nested polymerase chain reaction (PCR) procedure is described for detecting RNA to a spliced late gene (SLG) of human cytomegalovirus (CMV), the product of which (175 bp) is easily differentiated in agarose gels from the product when the target is unspliced viral RNA or DNA (258 bp). The SLG-RT-PCR has been compared against a semi-quantitative PCR for CMV DNA in buffy-coat specimens collected weekly after bone marrow transplantation from 3 patients and against the results of culturing these specimens for CMV both by conventional virus isolation, based on the detection of cytopathic effect, and by the early detection of infected cells by staining with virus-specific monoclonal antibodies. The detection of CMV RNA by SLG-RT-PCR correlated well with the detection of infective virus but only when the results of both culture methods were combined, in that neither culture method alone was as sensitive as the SLG-RT-PCR. The presence of SLG RNA in the circulation is of value as a marker of active CMV infection.

Quantification of cytomegalovirus DNA in blood specimens from bone marrow transplant recipients by the polymerase chain reaction

A nested PCR system for cytomegalovirus (CMV) DNA in blood specimens from bone marrow transplant recipients is described, in which the biotinylated tritium-labelled product from the second round of PCR is quantified using streptavidin-coated fluorometric Scintillation Proximity Assay (SPA) beads (Amersham, UK). This assay has been compared with a PCR procedure based on limiting-dilution, in which the end-point is determined visually following electrophoresis in agarose gel. The two systems were shown to be equivalent in sensitivity and specificity on testing stored serial blood samples from six CMV antibody-positive allogeneic bone marrow transplant patients who developed viraemia as detected by conventional methods of virus isolation in tissue culture.

Rapid detection of mumps virus by the polymerase chain reaction

A procedure for detecting mumps virus in under 48 h was developed using the PCR. The sensitivity of the PCR amplification reaction and of the detection of the PCR product was significantly improved by: (i) enriching for viral template RNAs by overnight culture of the virus in Vero cells and (ii) substitution of polyacrylamide gel analysis for agarose gel electrophoresis. The technique was capable of detecting 1-20 infectious units of virus or an equivalent of 1-10 pg of mumps virus-specific plasmid DNA.

A simple immunoalkaline phosphatase method for the rapid diagnosis of cytomegalovirus (CMV) infection

Abstract Eight hundred and eighty-four specimens, comprising mainly buffy coats, urines, throat swabs and other respiratory tract specimens, were examined for the presence of cytomegalovirus (CMV) by conventional virus isolation in tissue culture and by a rapid alkaline phosphatase technique. The rapid method has an overall sensitivity of 81·2%, compared with conventional virus isolation, and a specificity of 95·8%. The sensitivity for buffy coat specimens, however, was only 53·3%.

Early detection of cytomegalovirus (CMV) infection in bone marrow transplant patients by reverse transcription-PCR for CMV spliced late gene UL21.5: a two site evaluation

BACKGROUND Bone marrow transplant (BMT) patients at risk of developing cytomegalovirus (CMV) pneumonitis are identified routinely by the early detection of virus in blood. For early diagnosis of CMV infection, the RNA-based approach demonstrates advantages when compared with the current CMV antigen and DNA detection methods. OBJECTIVES We have evaluated our previously developed reverse transcription-polymerase chain reaction (RT-PCR) to a spliced late CMV gene (SLG; J. Virol. Methods 56 (1996), 139) to monitor CMV infection in BMT patients at two clinical sites. The diagnostic value of the SLG RT-PCR was compared with the routine CMV antigen and DNA detection methods. STUDY DESIGN Weekly blood samples from BMT patients were tested for CMV during the first 3 months post-transplant. The qualitative SLG RT-PCR, semiquantitative DNA PCR, and viral antigen tests were compared. The RNA and DNA PCR results were analysed in terms of their temporal relationship and consistency of CMV detection and compared with CMV infection diagnosed by viral antigen tests. RESULTS Of the 101 BMT recipients studied, 25 developed CMV antigenemia and/or DNAemia resulting in symptomatic infection in two patients. All CMV PCR-positive patients were either CMV seropositive pretransplant or received marrow from seropositive donor. The highest incidence of CMV infection was seen in seropositive recipients (R+) irrespective of the donors status. Detection of CMV infection by SLG RNA preceded CMV DNA detection by 0-2 weeks (median 1 week) and CMV antigen detection by 0-8 weeks (median 3 weeks). Once detected, the SLG RNA remained consistently positive before antiviral treatment was commenced. Both the SLG RNA and CMV DNA detection methods had the same clinical sensitivity, specificity, positive and negative predictive values of 100, 94, 80 and 100%, respectively. CONCLUSIONS The RT-PCR for SLG RNA proved to be the earliest indicator of CMV infection in BMT patients demonstrating a sustained pattern of CMV detection during the 3 months post-transplant period. Although very similar in its diagnostic performance to CMV DNA PCR the SLG RNA RT-PCR does not require quantitation and provides an efficient and ongoing indication of active CMV infection.

Sequence variation within the carboxyl terminus of the nucleoprotein gene of mumps virus strains.

BACKGROUND On rare occasions, clinically apparent mumps virus infection and meningitis can be caused by vaccine virus as well as wild type strains. This has been shown by nucleotide sequencing of short regions of the viral genome. OBJECTIVES We wished to confirm and extended these findings by investigating for strain differences within the carboxyl terminus of the nucleoprotein (NP) gene, which is known to be highly variable amongst paramyxoviruses in general. STUDY DESIGN A 576 bp carboxyterminal fragment of NP was amplified by reverse transcriptase (RT) PCR from several different strains and was sequenced directly. RESULTS Three Urabe vaccine variants and five clinical isolates previously classified as Urabe strains revealed the same carboxyterminal pattern for NP. Three other isolates and the Leningrad-3 vaccine strain showed characteristic differences of up to 5% compared to the Urabe strains, with U/C substitutions being predominant. CONCLUSIONS The carboxyterminal sequence of NP varies significantly between mumps virus strains such that it can be used for differentiating between vaccine and wild-type isolates.

Enteroviral pharyngitis diagnosed by reverse transcriptase-polymerase chain reaction.

The role of enteroviruses in childhood pharyngitis was investigated using enteroviral specific reverse transcriptase-polymerase chain reaction (RT-PCR). Viral/bacterial throat swabs were taken from 50 children with acute pharyngitis and 26 controls. A positive culture was identified in only 26% of children with pharyngitis (adenovirus 10%, group A streptococci 2%), and none of the controls. Enteroviral RT-PCR was positive in 8% of the pharyngitis group and none of the controls. Enteroviruses are an important cause of pharyngitis in childhood.

Cytomegalovirus viraemia in HIV infection : association with intercurrent infection

The aim of this retrospective study was to investigate the clinical significance of cytomegalovirus (CMV) viraemia in HIV-infected subjects (with or without AIDS) who had attended this hospital during a 45 month period. They were reviewed regularly and, when clinically indicated, tested for CMV viraemia. The blood of 105 subjects was cultured for CMV and 34 had at least one episode of CMV viraemia during the review period. The viraemia was present during CMV disease in nine of the 34 positive patients and was the only detectable infection in another two. In the remaining 23 patients, CMV viraemia occurred in association with intercurrent opportunistic infection. Among these 23 patients, the viraemia resolved in 12 after treatment (or natural resolution) of the intercurrent infection and only one of these 12 developed CMV disease (mean review period: 8 months). In another seven patients, CMV viraemia persisted despite treatment (or natural resolution) of the intercurrent infection and four subsequently developed CMV disease (mean review period: 4 months) (P = 0.08, Fishers exact test). From the remaining four patients, no specimens for CMV culture were obtained after treatment of the intercurrent infection. The CD4 count was higher in the 12 patients in whom there was resolution of the viraemia [mean CD4 60 x 10(6)/l] compared with the seven in whom the viraemia persisted [mean CD4 45 x 10(6)/l]. These findings suggest that in some HIV-positive patients, CMV viraemia was potentiated by intercurrent infection with another micro-organism and that its treatment was sufficient to mitigate the CMV disease.