Clinical and diagnostic virology最新文献

Background: The laboratory diagnosis of herpes simplex infection may require rapid (direct) tests, as well as cell cultures, for detection of the virus in clinical samples. The quantity of virus present in clinical samples is variable and this may depend on the period from onset of rash. In addition, not all patients may show obvious symptoms with this infection. The successful culture of herpes simplex virus requires prompt transportation after collection of the specimen as the virus is easily inactived. Hence, rapid and culture tests would enable detection of non-viable and viable viruses.

Study Design: We describe the rapid detection of HSV by EIA directly in various clinical samples using commercially available polyclonal sera. In addition, specimens were inoculated in microwell cell cultures and 4 days post inoculation the culture fluids were tested for HSV and subtyped by a similar EIA (culture amplified EIA).

Results: The direct EIA showed an endpoint detection of 100 TCID₅₀/ml, sensitivity of 92% (all specimen types) and specificity of 100%. The direct EIA sensitivity was 97% in non-genital specimens and 88% in genital specimens. The culture amplified EIA showed a sensitivity of 95% compared to all confirmed HSV positive samples.

Conclusions: The results of the HSV rapid tests were available within 24 h from receipt of specimens. Specimens which were culture negative/direct EIA positive were confirmed by blocking antisera. Culture positive specimens which were direct EIA negative were confirmed by subtyping of the virus.

Background: The quantitation of viral nucleic acids in biological fluids has become increasingly desirable over the past several years. To this end, a number of quantitative molecular procedures have been developed. Objectives: The objective was to review the current literature on the molecular techniques used in the quantitation of viral nucleic acids and to assess the appropriateness of these methods for clinical use. Results: Assays involving both target and signal amplification are now available for the accurate and precise quantitation of viral burden in infected patients. These methods include quantitative polymerase chain reaction (PCR), branched chain signal amplification (bDNA), nucleic acid sequence-based amplification (NASBA) and the SHARP signal and hybrid capture systems. Our understanding of the natural history and pathogenesis of viruses such as the human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), cytomegalovirus (CMV) and Epstein–Barr virus (EBV) may be greatly facilitated by accurate determinations of viral and infected cell burden. Quantitation of viral load in infected individuals may also be useful to assess disease progression, monitor the efficacy of therapy and to predict treatment failure and the emergence of drug-resistant viruses. Conclusion: Precise, accurate and reproducible quantitation of viral load is now feasible. Molecular assays for viral quantitation should have a considerable impact on medical research and clinical care.

Background: The second generation Hybrid Capture microplate-based human papillomavirus (HPV) test (HC II) was examined to determine its sensitivity for identification of cervical intraepithelial neoplasia (CIN) by two different cervical specimen collection methods.

Objectives: A cohort of 115 women with a mean age of 34.6 years (SD 9.1), referred to colposcopy with a history of abnormal cytology, was studied to compare HPV prevalence and viral load in low grade CIN vs. high grade CIN.

Study design: Prior to the application of acetic acid, cervical specimens were obtained by either method 1 or 2, as follows: method 1: A cotton-tipped swab was applied to the ectocervix and endocervix for a Papanicolaou (Pap) smear. Next, a special cone-shaped cervical brush was applied to the endocervix, the ectocervix, and to the posterior vaginal vault and suspended in 1.0 ml of transport medium for HPV testing. Method 2: a Pap smear was taken with a cyto standard cylindrical cytology brush from the endocervix, and ectocervix, and the remaining cells were suspended in 3 ml phosphate-buffered saline (PBS) for HPV testing. Next, a Dacron-tipped swab was used to take a specimen from the ectocervix and posterior fornix and suspended in the same PBS solution.

Results and conclusions: Histological evaluation of cervical punch biopsies or conization specimens showed CIN I in 39 cases, CIN II in 32 cases, and CIN III in 44 cases. High risk (HR) HPVs were detected with collection method 1 vs. collection method 2 in 70 (14/20) vs. 74% (14/19) of CIN I; 100 (17/17) vs. 87% (13/15) of CIN II; and 96 (25/26) vs. 94% (17/18) of CIN III (all differences not statistically significant). The estimated viral load of each collection method was significantly higher in patients with CIN II or III compared with CIN I, independent of the collection method used (P≤0.03). Collection method 1 gave higher relative HPV viral load estimates than method 2 for CIN II/III, but this difference was not statistically significant. We conclude that HC II has a high clinical sensitivity for CIN II/III (over 90%) and that high grade disease (CIN II/III) has a greater relative HPV viral load than low grade disease (CIN I). Method 1 appears to be better than method 2 for collecting cervical specimens, especially if viral load is considered a useful risk marker for high grade CIN or in cases of low-level HPV infection.

Background: Rhinoviruses have long been associated with mild upper respiratory illness in both adults and children. However, the role of rhinoviruses as lower respiratory tract pathogens has not been fully characterized. Previous data suggests that rhinoviruses may cause severe lower respiratory illness in young children or infants.

Objectives: The present study describes the clinical presentations, severity of illness and outcomes for a large cohort of pediatric patients with documented rhinovirus infections.

Subjects and methods: A retrospective chart review was done on 93 pediatric patients from whom 101 nasopharyngeal or endotracheal specimens were positive by viral culture for a rhinovirus. All patients were hospitalized or seen in the pediatric emergency department at The Children’s Hospital of Philadelphia between 1 January, 1990 and 31 May, 1996.Results: Of the 93 patients, 52 were male and 41 female. The age range was 0 days to 18 years with 25 (27%) less than 3 months, 42 (45%) between 3 and 12 months and 26 (28%) over the age of 12 months. Clinical presentations on evaluation in the emergency department or admission included 78 (84%) patients with acute respiratory illness, 13 (17%) with fever and suspected sepsis and 11 (12%) with other complaints. Reported physical findings on examination included one or more lower respiratory symptoms or signs of acute distress and fever greater than or equal to 38.1°C. A total of 64 (69%) children were noted to have significant past medical histories, including 28 (44%) with prematurity or complicated neonatal courses, 11 (17%) with prior reactive airways, 8 (12%) with congenital cardiac disease and 7 (11%) with neurologic disorders. Of the patients, 29 (31%) were considered to be otherwise healthy children with no underlying dysfunctions. The mean duration of hospitalization for 69 patients admitted with respiratory illness who did not develop subsequent unrelated complications was 3.7 days. No significant bacterial or fungal pathogens were identified in 91% of the cases.

Conclusions: This study shows that rhinoviruses were associated with severe lower respiratory illness and hospitalization in a large pediatric population and that rhinovirus infection was a complicating factor in those patients with underlying or predisposing conditions.

Background: The family of picornaviridae has been studied extensively: the structure of the virion and its replication strategy are known in molecular detail. Nevertheless, infections with the multitude of enteroviruses still cause widespread epidemics, serious disease and a diversity of clinical syndromes ranging from central nervous system involvement to light febrile illness. Infections with more than 100 human rhinovirus types are an important economic factor.

Objective: In order to treat and control picornavirus infections with their great diversity of manifestations the pathogenesis of diseases must be better understood, e.g. concerning virus spread in the organism or molecular detail of the disease processes, such as cell tropism of the virus or cytokine and immunologic actions. Elucidation of mechanisms of virus transmission in the population is also needed.

Study design: This review discusses aspects of our present knowledge of the pathogenesis, transmission, prophylaxis and treatment of picornavirus infections.

Conclusions: The need for further development of selective antiviral substances, safe vaccines and basic research in picornavirology is stressed.

Background: Several lines of evidence suggest that enterovirus infections may be involved in the etiology of the insulin-dependent diabetes mellitus (IDDM). Often in the literature, a reference is given to specifically diabetogenic strains of enterovirus but there is no systematic assessment about the generation of such strains in the course of evolution or about their abundance among the 64 enterovirus serotypes pathogenic to man. If enteroviruses truly are involved in the etiology of IDDM, a possibility to prevent the disease with enterovirus vaccines might become feasible. In such a situation it would be important to know which serotypes and strains are the most important ones, and whether there would be differences between the strains as regards the pathogenetic mechanisms involved.

Objective: To present a brief summary of the basic biology of enteroviruses, on existing data of genetic variation of enteroviruses, and on molecular epidemiology of human enteroviruses with special reference to the different epidemiological modes of their putative involvement in the pathogenesis of IDDM.

Conclusions: Like RNA viruses in general, enteroviruses exist as a quasispecies, a mixture of genetic microvariants with a vast potential to adapt to new environments. This means that specifically beta cell-tropic and potentially diabetogenic variants could, in theory, emerge sporadically during systemic infection of any individual. The patterns of genetic diversification of enteroviruses, cocirculation of separate genetic lineages in the human populations, and the assumed geographical restrictions of endemic transmission of the lineages, allow one to hypothesize that populations with a high persisting IDDM incidence might be endemically infected by some specific strains of enteroviruses. However, so far, there is no systematically collected data supporting this hypothesis.

Background: Observations in humans and the results of experiments on laboratory animals have provided evidence that coxsackieviruses of group B (CVB) are major etiologic agents of acute and chronic enterovirus myocarditis and various other virus-induced diseases.

Objective: This minireview briefly summarizes the investigations to elucidate various molecular mechanisms for the induction and maintenance of persistent CVB infections. With regard to the recent findings that CVB may use several different receptor proteins, this article focuses on virus-host cell interactions and the potential impact of these interactions for enteroviral replication.

Study design: The interaction of CVB with specific cell surface proteins was analyzed in cultured cell lines and murine tissues at the level of virus attachment and virus internalization. As example for the interaction of CVB with intracellular proteins, the state of p21^rasGTPase-activating protein (RasGAP) was investigated in mock-infected and CVB3-infected HeLa cells.

Results and conclusions: The experiments to elucidate the virus receptor interactions revealed the necessity to differentiate between CVB attachment proteins and proteins involved in virus internalization. Since more than one protein may be required to initiate the uptake of CVB into permissive host cells, a model of the putative interaction of these proteins within a multimeric receptor complex is proposed. It is further tempting to speculate that the presence of multiple attachment proteins may influence the tissue tropism of CVB as well as pathogenicity.

Background: Insulin-dependent diabetes mellitus or type 1 diabetes is a disease with a diverse aetiology. Epidemiological studies examining newly diagnosed, recent onset IDDM patients have suggested a role for viruses in the aetiology of IDDM (Yoon, 1995, Diabetes/Metabolism Reviews 11, 83–107). Important candidates are the enteroviruses, in particular coxsackieviruses B3 and B4. The latter can cause diabetes in animals (Clements et al., 1995, Lancet 346, 221–223).

Objectives: We have developed a quantitative PCR method for the detection of enterovirus genomes in biological samples. The quantitative PCR will be used to screen for enteroviruses in blood of diabetes patients and their relatives by testing a Blood Diabetes Register.

Study design: A substantial amount of data has been collected on enterovirus induced IDDM, our study is original in so far as it will be: (1) a quantitative study, not only the presence of viral genome sequences in blood will be determined, but also their concentrations (viral load); and (2) a longitudinal study, samples are and will be collected as a function of time. Positive PCR samples will be quantified using the standard addition method.

Results: The test is specific for enteroviruses, since all enteroviruses were detected with equal sensitivity. Viruses belonging to other picornavirus genera scored negative (even up to 3×10⁶ genome copies). An equal detection limit of 10 genome copies was found for all enteroviruses.

Conclusions: The developed method will permit us to generate quantitative and longitudinal data of enterovirus genomes in blood of diabetes patients and their relatives, which might help in the elucidation of the relationship between enteroviruses and IDDM.