Clinical and diagnostic virology最新文献

Background: Two biotypes of pestiviruses, cytopathogenic (cp) and non-cytopathogenic (noncp) viruses, are distinguished by their effects on tissue culture cells. In contrast to the bovine viral diarrhoea virus (BVDV) system, only a few cp border disease virus (BDV) and cp classical swine fever virus (CSFV) strains have been described. Antigenically closely related noncp and cp BVDV can be isolated from cattle with fatal mucosal disease (MD) and are called a virus pair. The generation of cp BVDV in an animal persistently infected with noncp BVDV is regarded as causative for the development of MD.

Objectives: To analyse viral pairs of BVDV at the molecular level and thereby identify differences between the viruses of each pair.

Study design: BVDV pairs were isolated from several animals coming down with MD; the genomes of the respective BVD viruses were sequenced on cDNA level. Studies concerning the polyprotein processing of each strain were carried out.

Results: Molecular analysis of BVDV pairs demonstrated a linkage between RNA recombination, generation of NS3 and the onset of fatal MD.

Conclusion: The molecular analysis of BVDV pairs revealed that the respective cp strains arise by RNA recombination from noncp viruses.

Background: Millions of individuals are estimated to become infected with dengue virus each year, particularly in tropical and subtropical regions. Mortality is low but infection can lead to a severe form of dengue, characterised by haemorrhage and shock. A safe and effective vaccine against dengue is still not available.

Objective: To use the successful construction of dengue type 4 virus (DEN4) cDNA, which yields infectious RNA transcripts, to provide a new approach to the development of safe and effective dengue vaccines.

Study design: The 3′ and 5′ noncoding (NC) regions of the genome were targeted to construct DEN4 deletion mutants, because the sequences in these regions are thought to play an important role in the regulation of viral replication. DEN4 cDNA was also employed to construct a viable chimeric virus with dengue type 1, 2 or 3 antigenicity, by substitution of heterotypic structural protein genes.

Results: Most viable mutants, recovered from the cDNA constructs, were partially restricted for growth in simian cells as analysed by plaque morphology assay and viral yield analysis. Several 3′ NC deletion mutants which exhibited a range of growth restriction in cell culture were further evaluated for infectivity and immunogenicity in rhesus monkeys. Occurrence and duration of viraemia were reduced for these deletion mutants, compared to the wild type DEN4. Analysis of antibody response to infection in rhesus monkeys also indicated that some of these mutants were attenuated. These DEN4 deletion mutants represent promising live dengue vaccine candidates that merit further clinical evaluation. Chimera DEN1/DEN4 or DEN2/DEN4 which expresses DEN1 or DEN2 antigenicity were also used to infect monkeys. Most monkeys immunised with these chimeric viruses, singly or in combination, developed high titres of neutralising antibodies and were protected against homotypic wild type DEN1 or DEN2 challenge.

Conclusions: DEN4 and its derived chimeric viruses of other three dengue serotype specificity, that contain appropriate attenuating mutations, have a potential use in a tetravalent live vaccine against dengue.

Background: Natural hepatitis C virus (HCV) infection elicits poor immunity. Although HCV proteins elicit immune responses in virtually all cases of infection, the great majority of HCV infections become chronic. Currently, no vaccine is available for HCV despite an estimated incidence of approximately 50 000 new cases per year in the USA alone.

Objectives: To discuss how the problems associated with developing a vaccine against HCV infection may be overcome and describe recent progress made towards overcoming these problems and developing a vaccine.

Study design: A cytofluorimetric assay that can assess the ability of a serum sample to neutralise the binding of the HCV-envelope glycoprotein E2 to human cells (neutralisation of binding or NOB assay) was developed. The assay was used to assess the levels of antibodies capable of neutralising E2 binding in the sera of vaccinated and carrier chimpanzees.

Results: Low titres of NOB antibodies were found in the majority of chimpanzees challenged with HCV infection. Chimpanzees immunised with the E1/E2 heterodimer developed NOB antibodies and high levels of neutralising antibodies. These chimpanzees were not protected from challenge with heterologous virus but were protected from subsequent chronic infection.

Conclusions: A subunit vaccine composed of recombinant HCV proteins may protect from infection or chronic infection by different HCV genotypes.

Background: Dengue virus infection may be asymptomatic or lead to undifferentiated febrile illness or dengue haemorrhagic fever and dengue shock syndrome (DHF/DSS). The major clinical manifestations of DHF/DSS are high fever, haemorrhage, hepatomegaly and circulatory failure.

Objectives: The relatively high level of viraemia only a few days after infection may reflect a large number of replication sites. However, the degree of cell injury in fatal cases of DHF/DSS is not sufficient to explain death and suggests metabolic disturbance rather than tissue destruction. This theory was investigated in this study.

Results: We demonstrated that replication of dengue virus in infected cells induces stress leading to apoptotic cell death in vitro and in vivo.

Conclusions: The elimination of apoptotic bodies by phagocytic cells is a previously unsuspected pathway of dengue virus clearance from infected tissues. However, the mechanisms of host defence involving apoptosis and phagocytic cell activation may cause local tissue injury or transient homeostasis imbalance and may trigger further deleterious events.

Background and objectives: Rubella virus (RV) produces a subtle and slow-developing cytopathic effect in Vero cells that is difficult to recognize, especially at low multiplicities of infection. In order to facilitate the detection of RV in cell culture, we standardized a low-pH virus-mediated cell-fusion assay.

Study design: The incubation periods, temperatures, pH and multiplicity of infection were established. The specificity of the method was tested by immunofluorescence assay and cell-fusion inhibition by specific sera.

Results: Six days post infection, Vero cells were treated for 5 min with fusion medium. After that, monolayers were incubated with medium at neutral pH for 16 h and then stained. Gigantic cells with multiple nuclei were observed.

Conclusions: The method allowed the observation of unequivocal images that are easier to recognize than the cytopathic effect caused by RV in the same cell line. At the same time, the method is simple, accessible and shown to be specific to demonstrate the replication of several strains and isolates of RV in Vero cells.

Background: Acute lower respiratory infection (ALRI) is one of the main causes of morbidity and mortality in small children. Objective: The aim of this study was to determine the frequency, seasonality and association with clinical entities of respiratory syncytial virus (RSV) and adenoviruses in children with ALRI. Study design: During 2 consecutive years (1991–1992), 168 children under 2 years of age hospitalized due to ALRI in a public pediatric hospital of Buenos Aires, Argentina, were studied. RSV and adenoviruses were investigated on nasopharyngeal aspirates (NPA) by indirect immunofluorescence (IIF). HEp-2 cells were used for adenovirus isolation. Results: RSV was detected in 36.3% and adenoviruses in 14.3% of the cases (P<0.0001). All adenoviruses detected by IIF were also isolated in culture. Out of 61 RSV cases, 57% corresponded to bronchiolitis and 43% to pneumonia. Ninety-two per cent of children with RSV were less than 1 year old and 70% were less than 5 months. The highest number of RSV cases were observed during winter, with a clear peak in July. Seventy-one per cent of adenovirus cases were associated with pneumonia and only 24% with bronchiolitis (P<0.02), and predominated in children older than 5 months of age (P<0.0001). Adenoviruses were detected in almost all months of the year with a small peak at the end of winter and beginning of spring. No significant differences in clinical features at admission, breast feeding or malnutrition were observed among children with RSV or adenovirus diagnosis versus those with no viral etiology. The overall fatality rate was 2.4%. In all fatal cases adenovirus was detected in NPA. Thus, fatality rate among patients with adenoviruses reached 16.7%. Conclusions: Our findings show the importance of RSV and adenoviruses associated with ALRI in hospitalized children under 2 years of age and the different epidemiological patterns of the two viruses in Buenos Aires, Argentina.

Background: Puumala virus (PUU), a member of Hantavirus genus, is the causative agent of nephropathia epidemica (NE), a milder form of hemorrhagic fever with renal syndrome (HFRS). Rapid diagnosis is essential for clinical management of NE.

Objectives: To evaluate the usefulness of recombinant protein-based IgM (direct- and μ-capture) and IgG (direct- and antigen (Ag)-capture) enzyme immunoassays (EIA) in early diagnosis of NE in comparison to IgG immunofluorescence assay (IF), and to find out the time limit for PUU-specific antibody seroconversion.

Study design: The specific IgM and IgG antibody responses in serum were analyzed in 109 patients (235 serial sera) and 114 patients (233 serial sera), respectively. The serum panel used was selected from a larger material according to the availability of information concerning the date after onset of symptoms, the panel also containing NE patients who had been IgG-IF negative in their first (early) samples to find out the possible differences between sensitivities of the EIAs and IF.

Results: All NE patients tested became IgM-positive at the latest on the 6th (μ-capture EIA) or 7th (direct-IgM EIA) day after onset of symptoms. Out of a panel of very early NE-patient sera (n=38) that could not be detected by IgG-IF, 66% were already positive with both direct-IgM EIA and μ-capture EIA. When comparing IgG EIAs and IgG-IF, 98% of IF-positive sera from NE patients were also positive with direct-IgG EIA, and 99% with Ag-capture IgG EIA. Out of a panel of very early NE-patient sera (n=37) that could not be detected by IgG-IF, 57% were positive with direct-IgG EIA, and 27% with Ag-capture IgG EIA.

Conclusions: The baculovirus-expressed PUU-N-based IgG and IgM EIAs were found most suitable for NE diagnosis, giving the opportunity in some cases for earlier diagnosis as compared with PUU-IgG IF.

Background: Rapid diagnosis of dengue infection is essential to patient management and disease control. The development of a rapid (5 min) immunochromatographic test and a 2 h commercial capture enzyme linked immunosorbent assay (ELISA) for anti-dengue IgM and IgG antibodies may lead to more rapid and accurate testing in peripheral health settings and diagnostic laboratories.

Objectives: Evaluate two new commercial tests for dengue serology (Dengue Rapid test and Dengue Duo ELISA; PanBio, Brisbane, Australia).

Study design: The sensitivity and specificity of the tests were compared with in-house dengue IgM ELISA and hemagglutination-inhibition (HAI) assays using known positive and negative dengue specimens, as well as specimens from non-dengue cases.

Results: Both assays showed excellent sensitivity in the diagnosis of both primary and secondary dengue infection (100%). In both assays, IgG levels showed excellent correlation with hemagglutination-inhibition (HAI) assay, and these could be used to distinguish between primary and secondary dengue infections (92 and 97% of patients correctly classified in the rapid test and Duo ELISA, respectively). Specificity in both assays was 89% when sera from patients, with no apparent dengue infection, typhoid, leptospirosis and malaria, were tested.

Conclusions: These tests should be a useful aid in confirming the clinical diagnosis of dengue infection. The rapid test will be particularly valuable in peripheral health settings, while the ELISA has a place in central testing laboratories.

Background: The role of the cytomegalovirus (CMV) in the respiratory morbidity and mortality of HIV-infected patients remains unclear. This is due in part to difficulties in making an accurate and rapid diagnosis. There has been a limited number of studies, often with few or no AIDS patients, on the use of DNA–DNA in situ hybridization (ISH) and polymerase chain reaction to diagnose CMV respiratory infection directly on bronchoalveolar fluid samples.

Objectives: To compare the centrifugation culture (CC), ISH, and nested-primer polymerase chain reaction (npPCR) techniques on bronchoalveolar fluid for the diagnosis of respiratory CMV infection.

Study design: Samples were obtained prospectively from a group of 35 HIV-infected homosexual men evaluated for pneumonia at a university hospital. Sensitivity, specificity, and predictive values of the three techniques were measured and compared, using the conventional roller tube cell culture (CRTC) as the gold standard.

Results: Sensitivity, specificity, positive and negative predictive values were as follows: 86%, 86%, 90%, and 80% for the CC; 5%, 100%, 100%, and 41% for ISH; and 86%, 57%, 75%, and 73% for npPCR. Of the six false positive samples by npPCR, two were positive by CC (none by ISH). If the latter were considered true positives, the specificity and positive predictive values of npPCR would increase to 67% and 83%, respectively.

Conclusions: CC appeared to be the best of the three techniques compared in this study for diagnosis of respiratory CMV infection in HIV-infected patients. The sensitivity and predictive values of DNA–DNA ISH were very poor. Results with npPCR were acceptable, and this technique may be considered in situations when rapid diagnosis of CMV infection is necessary.