Clinical and diagnostic virology最新文献

Background: Influenza A infections are an important cause of morbidity and mortality in the elderly and patients affected by chronic diseases or immunodeficiencies. Treatment and prevention of infection in hospitals and nursing homes often involve the use of amantadine, but resistant viruses may arise.

Objectives: To assess the effectiveness of specific and sensitive methods for rapid screening and sequence confirmation of amantadine resistance, and the occurrence of amantadine resistance in recent influenza A virus isolates in Canada.

Study design: A chicken antiserum-based enzyme linked immunoassay (ELISA) was developed and used to screen fifty influenza A isolates for amantadine resistance. Drug sensitivity was expressed as a percentage of virus growth inhibition. The efficiency of the assay was compared to that of a monoclonal antibody (mab)-based ELISA using influenza A strains from 1968 to 1994. Specific PCR primers, generated to amplify the M2 gene region where amantadine resistance mutations occur, were tested over a wide range of strains. Direct sequencing of the PCR fragments was performed to confirm the presence of resistance mutations.

Results: The polyclonal antiserum-based ELISA detected antigens from all recent H1N1 strains and H3N2 strains tested at an inoculum dilution ten-fold lower than the mab-based ELISA. Primers for the detection of amantadine resistance mutations consistently amplified a wide range of strains. The direct sequencing of the RT-PCR amplicons generated, detected resistance mutations in reassortant and control viruses, the only strains found resistant by ELISA. All influenza A isolates (H3N2, H1N1) tested, except resistant controls and two reassortant viruses, were amantadine-sensitive as indicated by greater than 50% virus growth inhibition.

Conclusions: Influenza A virus susceptibility to amantadine could be detected by using an antiserum-based ELISA, offering a simple and more sensitive alternative to the mab-based assay. Coupled with direct sequencing of the M2 gene, it provides a reliable way to detect and confirm resistance in influenza isolates. However, no resistant clinical isolates were detected in the sample.

Background: The reference method for detecting specific Epstein-Barr virus (EBV) antibodies is indirect immunofluorescence (IF) with EBV-infected cells. The availability of protein purified from infected cells and more recently of recombinant polypeptides designed to contain immunodominant epitopes, has enabled the development of commercial enzyme-linked immunosorbent assays (ELISA) for the specific serodiagnosis of EBV infection.

Objective: Evaluation of ELISA-based EBV serodiagnosis in comparison with indirect immunofluorescence.

Study design: We have first compared three commercial ELISA test systems with our in house indirect immunofluorescence assay for classifying correctly a set of serum samples into clinical categories (acute infection, past infection, interfering non-EBV infection, persistent infection). Additionally a prospective analysis with the best performing ELISA test (Enzygnost) was then carried out by running the ELISA test in parallel with the indirect immunofluorescence assay on 324 consecutive clinical samples sent to our laboratory for EBV serodiagnosis.

Results: For the serodiagnosis of past EBV infection and acute EBV infection all three commercial ELISAs performed well in comparison with indirect immunofluorescence. When testing samples positive for cytomegalovirus (CMV), Toxoplasma or herpes simplex IgM, interference in the IgM tests was observed with the three ELISAs. In some instances we could demonstrate that the positive IgM results were due to EBV reactivation. The observed discrepancies between ELISA and IF for the serodiagnosis of chronic EBV infection or EBV reactivation, point to the difficulty for the serodiagnosis of persistent EBV infection on single serum samples. According to our prospective study the EBV IgG determination was accurate. A positive IgM result was not always indicative of an acute infection. Positive IgM results due to EBV reactivation were observed. A positive EBV nuclear antigen (EBNA) IgG result in those samples precluded acute infection.

Conclusions: 90–95% of samples could be classified correctly into clinical categories by a two parameter ELISA system detecting IgG and IgM against a standardized mixture of EBV antigens, allowing standardization and automation of EBV-specific serology. The absence of EBNA IgG was useful as a second line confirmatory assay for acute EBV infection.

Background: It is important to investigate a possible cross-reaction of anti-rubella IgM in the IDEIA^™ Parvovirus B19 IgM test because many B19 infections are either asymptomatic or have clinical symptoms similar to those of rubella virus infections. Epstein-Barr virus (EBV) IgM, cytomegalovirus (CMV) IgM, measles IgM and rheumatoid factor (RF) IgM cross-reactions were also studied.

Objectives: In the period from February to September 1994 (including a parvovirus B19 epidemic) more than 10 000 serum samples were examined for parvovirus B19 IgM in Denmark. This gave an opportunity to evaluate the commercial IDEIA^™ Parvovirus B19 ELISA kit (DAKO A/S, Glostrup, Denmark), which was used routinely at Statens Serum Institut from the beginning of 1994 and onwards.

Study design: A total of 123 parvovirus B19 IgM positive sera were tested for reactivity in rubella IgM EIA. A total of 78 rubella IgM positive sera, 60 EBV VCA-IgM positive sera, 30 CMV IgM positive sera and 24 measles virus IgM positive sera were tested for reaction in IDEIA^™ Parvovirus B19 IgM test. Finally, 25 parvovirus IgM positive sera were tested for specific IgM against measles virus, EBV (VCA), CMV and for RF.

Results: One anti-B19 IgM positive serum sample reacted positively in the rubella IgM test. Of rubella IgM positive serum samples 4% cross-reacted in IDEIA^™ Parvovirus B19 IgM test, as did 17 and 20% of EBV VCA-IgM and CMV IgM positive serum samples respectively. None of measles virus IgM positive serum samples cross-reacted in the IDEIA^™ Parvovirus B19 IgM test. Of 25 initially parvovirus B19 IgM positive sera 20% cross-reacted in EBV VCA IgM test and 8% in the CMV IgM test. None reacted positively in measles virus IgM test; 28% showed weak reactivity in RF IgM test.

Conclusions: Precautions must be taken when results of IgM assays are interpreted. Epidemiological and clinical observations must be considered.

Background: The Argene Biosoft 1C3 and the Biotest C10,C11 monoclonal antibodies are two of the most commonly used commercially available antibody reagents for the cytomegalovirus (CMV) pp65 antigenemia assay.

Objectives: The sensitivities of these two reagents were compared in peripheral blood specimens received for CMV antigenemia testing.

Study design: A total of 1149 peripheral blood specimens were processed for CMV antigenemia testing. Duplicate slides were stained with the Biosoft 1C3 and Biotest C10,C11 monoclonal antibodies.

Results: A total of 158 specimens gave a positive result by one or both antibodies. One hundred and forty five were positive by the Biosoft antibody and 130 were positive by the Biotest antibody. Positive cell counts were significantly higher on cell preparations stained by the Biosoft antibody (Wilcoxon signed rank, P < 0.001) and the Biosoft antibody detected twice as many low-level positive specimens as the Biotest.

Conclusions: The Biosoft antibody reagent was superior to the Biotest reagent for the detection of CMV antigenemia. This is an important factor since early detection is essential for appropriate initiation of preemptive antiviral therapy, particularly in transplant recipients at high risk of CMV disease.

Background: Saliva is increasingly being investigated as an alternative to serum for diagnostic and epidemiological testing even though antibody levels are substantially lower in buccal cavity fluids. However, there has been little study on whether buccal cavity activity and/or the timing of saliva sampling affects the diagnostic outcome, particularly in seropositive subjects. The absence of influence by these factors may be critical to the use of saliva for pre-vaccination screening for example.

Objectives: The effects of eating, brushing of teeth and circadian rhythm on the measureable salivary immune status of 42 healthy individuals known to be serum and saliva anti-HAV positive were examined.

Study design: A total of 141 saliva samples obtained from the 42 healthy subjects, before and after meals, before and after brushing of teeth and at various timepoints throughout the day, were assayed for total anti-HAV using an in-house saliva based enzyme-immunoassay, previously shown to have a 100% correlation in terms of sensitivity and specificity with a serum based assay.

Results: The results indicated that total anti-HAV titres varied according to the time of day and that eating had no significant effect on the total anti-HAV titre, but brushing of teeth did. Titres never varied to the extent that a result was falsely negative at any timepoint.

Conclusion: These results confirm the usefulness of saliva as a diagnostic sample for the detection of hepatitis A antibody, regardless of sampling times, eating or tooth-brushing.

Background: The COBAS AMPLICOR^™ (CA) instrument for the amplification and detection steps of the AMPLICOR^™ molecular diagnostic assays has recently been introduced. The system contains a single thermal cycler with two independently controlled heating/cooling blocks, a pipettor, a magnetic particle washer, a photometer and an incubator.

Objective: The performance of the CA instrument was evaluated in a routine diagnostic laboratory for the detection of hepatitis C virus (HCV) RNA. The new system was compared with the corresponding microwell plate assay (AMPLICOR HCV Test).

Study design: Routine clinical sera (350) from hemodialysis patients and patients with chronic HCV infection and interferon therapy were studied. If discrepant results were obtained, both assays were repeated (specimen preparation, amplification and detection); in addition, the HCV copy number was determined with the AMPLICOR HCV MONITOR^™ Test.

Results: There was a correlation between the CA HCV Test and the AMPLICOR HCV Test in 341 of 350 specimens (97%). After resolution of 9 discrepant results, the CA HCV Test gave a sensitivity of 97.8% and a specificity of 99.4%. The most common reason for discrepant results was a low HCV RNA copy number.

Conclusion: The CA system was found to be a labor-saving, fast and reliable instrument for the amplification and detection steps of a RT-PCR molecular assay for detection of HCV RNA.

Background: Previous studies have shown the diagnostic utility of qualitative detection of herpes simplex virus (HSV) DNA by the polymerase chain reaction (PCR) in cerebrospinal fluid samples (CSF) from patients with herpes simplex encephalitis (HSE).

Objectives: To determine whether quantitation of HSV DNA in CSF could be useful for monitoring efficacy of antiviral therapy and provide prognostic indications.

Study design: A quantitative PCR assay using an internal control for evaluation of PCR efficiency and detection of PCR inhibitors was developed and used for retrospective testing of 98 CSF samples from 26 patients with serologically diagnosed HSE during the period 1980–1995.

Results: HSV DNA was detected in 36 CSF samples from 23 patients. PCR positivity was 100% for CSF samples collected within 10 days after onset, and 30.4 and 18.7% for samples collected 11–20 and 21–40 days later, respectively. The 3 PCR-negative patients had their first CSF collected 14, 16, and 28 days after onset, respectively. Three of 98 (3.1%) CSF samples were completely or partially inhibitory to PCR. Initial DNA levels were not significantly different in patients with HSE due to either primary or recurrent HSV infection. In addition, they were not related to severity of clinical symptoms nor were predictive of the outcome. A progressive decrease in viral DNA levels was observed both in patients who received acyclovir therapy and in a small number of untreated patients.

Conclusions: This study: (i) confirms the high sensitivity of PCR for the diagnosis of HSE; (ii) emphasizes the need for an internal control of amplification to achieve maximal sensitivity and perform reliable quantitation of viral DNA; and (iii) suggests that CSF might not be the best specimen to investigate in studies of the natural history of HSE.

Background and objective: Systemic interferon α-2b treatment reduces relapses of genital human papillomavirus (HPV) lesions in some but not all females. The aim of the present study was to investigate possible predictive pretreatment factors for the outcome of therapy.

Material and methods: HPV DNA status and HPV antibody response were evaluated in 100 randomized patients treated with laser ablation and systemic interferon α-2b or placebo, and followed up to 6 months.

Results: Overall, adjuvant therapy with systemic interferon-α did not differ from placebo. However, detectable diagnostic phase levels of serum antibodies to e.g. HPV16 open reading frame (ORF) E2 derived peptide ¹⁴¹EEASVTVVEGQVDYY¹⁵⁵ predicted 10-fold difference in the risk of recurrence of HPV infection following adjuvant interferon α-2b therapy as compared with placebo (odds ratio, OR, 0.5, 95% confidence interval (CI), 0.1–2.3; OR, 4.6, 95% CI 0.5–41, respectively). This trend was statistically significant in the whole study population (2P < 0.05), and in patients with high viral load (2P < 0.01).

Conclusions: Evaluation of the E2 antibody responses may help to identify women with genital HPV lesions who respond to systemic interferon α-2b treatment.