Clinical and diagnostic virology最新文献

Background: Rapid laboratory methods for the early detection of cytomegalovirus (CMV) are needed for the prevention of CMV disease in transplant recipients. These methods should not only be able to detect the virus but also be highly predictive for CMV disease.

Objective: The clinical value of a simple and rapid nested plasma polymerase chain reaction (PCR) was evaluated by comparing the results with CMV pp65 antigen detection in leukocytes (CMV antigenemia assay), virus isolation from leukocytes, CMV IgG and IgM antibody response and clinical data.

Study design: A total of 471 EDTA blood samples were collected from 85 kidney transplant patients during a 3–4 month period after transplantation. CMV DNA was amplified directly from 10 μl of plasma while 150 000 separated leukocytes were stained for CMV pp65 antigen by each of two monoclonal antibodies. A total of one million leukocytes were used for virus isolation. The PCR protocol used in the present study involves a simple alkaline lysis technique for isolating DNA directly from plasma which is easy and rapid to perform.

Results: Twenty-eight patients developed symptomatic CMV infection while asymptomatic infection occurred in 29 patients. CMV pp65 antigen detection had a 75% sensitivity and a 57% positive predictive value for CMV disease development, compared with 64% and 79% sensitivity and 49% and 46% positive predictive value for CMV DNA and viremia, respectively. The median time until detection of CMV in patients with symptomatic CMV infection was 26 days after transplantation, compared with 49 days in asymptomatic patients by any of the methods used. Early appearance (within 8 weeks) of CMV pp65 antigen and CMV DNA had high predictive values for symptomatic infection; repeated detection of pp65 antigen and CMV DNA were more common in symptomatic patients.

Conclusions: CMV antigenemia assay and plasma PCR can be used for pre-symptomatic diagnosis of CMV infection. Virus isolation and CMV serology in most cases provide a post-symptomatic diagnosis. The best marker for monitoring kidney transplant patients might be the quantitative CMV antigenemia assay.

Background: When virologic and molecular diagnostic techniques are unavailable, the diagnosis of varicella zoster virus (VZV) infection depends on clinical criteria and histologic evaluation of skin biopsy specimens or Tzank preparations. These methods can misdiagnose chickenpox and zoster, particularly when the clinical manifestations are atypical.

Objective: To improve diagnosis in these settings, we developed an in situ hybridization technique for the detection of VZV utilizing a fluorescein-labeled oligonucleotide probe visualized with anti-fluorescein alkaline phosphatase-conjugated antibody.

Study design: We retrospectively examined 26 paraffin-embedded skin biopsy specimens with histologic features consistent with VZV or herpes simplex virus (HSV) infection and 11 control cases by in situ hybridization. In situ hybridization for VZV and HSV-1 was compared with polymerase chain reaction (PCR) for VZV and HSV-1 and clinical and histologic examination.

Results: Thirteen of the 26 study cases and two of the 11 control cases were positive for VZV by in situ hybridization. When compared with PCR, in situ hybridization was 92% sensitive and 88% specific. When compared with clinical diagnosis, in situ hybridization was 86% sensitive and 87% specific. All cases of chickenpox had VZV-positive inflammatory cells in the dermis but this finding was less frequent among the cases of zoster.

Conclusions: This in situ hybridization technique is a sensitive and specific method for the diagnosis of VZV in skin lesions that is applicable to most histopathology laboratory settings. In addition, in situ hybridization reveals individual infected cells and may provide insight into the pathogenesis of VZV skin infection.

Background: The traditional methods used in the diagnosis of dengue infection do not lend themselves to field application. As such, clinical specimens have to be sent to a central laboratory for processing which invariably leads to delay. This affects patient management and disease control. The development of the dengue IgM dot enzyme immunoassay has opened up the possibility of carrying out the test in peripheral health settings.

Objectives: This multicentre study was conducted to evaluate a new, commercial nitrocellulose membrane based IgM capture enzyme immunoassay.

Study design: The sensitivity and specificity of the test were compared with in-house dengue IgM enzyme-linked immunoassays routinely performed by each of the selected centres. Known positive and negative dengue specimens, as well as specimens from non-dengue cases, were included in the evaluation.

Results: Based on 402 specimens tested by the six centres, the sensitivity was 92.1% and specificity 88.1%, with an overall agreement of 92.8% when compared with IgM EIA assays performed on microplates.

Conclusions: The results suggest that this commercial kit has a role to play in the diagnosis of dengue infection, especially in peripheral health settings.

Background: A number of strategies, such as subtractive cDNA libraries and high through-put sequencing, have been devised to assess differential gene expression. Most of these approaches, however, are cumbersome and/or require tremendous technological power. In this paper, we describe a method, RNA fingerprinting using arbitrarily primed polymerase chain reaction (RAP-PCR), that is rapid, less cumbersome and can differentiate low levels of mRNA expression.

Objectives: To identify genes that are differentially expressed following human immunodeficiency virus type 1 (HIV-1) infection in different cell types by RAP-PCR.

Study design: RNA was extracted from both HIV-1-infected and uninfected HUT78 cells and peripheral blood mononuclear cells (PBMCs), reverse transcribed, and RAP-PCR amplified using numerous primer sets.

Results: Three genes, γ-actin, the HIV-1 nef and an unknown sequence, were identified as being differentially expressed in HUT78 cells. The level of γ-actin mRNA expression is increased after HIV infection and, as expected, the nef gene was solely expressed in HIV-infected cells. In contrast, the unknown mRNA is down-regulated by HIV. Northern blot analysis and/or specific PCR confirmed the differential expression of these three genes. RNA fingerprinting using phytohemagglutinin (PHA)-activated PBMCs infected by HIV in vitro, revealed that γ-actin is still up-regulated by HIV, whereas the unknown product no longer shows down-regulation.

Conclusion: These results illustrate the usefulness of the RAP-PCR method for isolating and identifying differentially expressed genes during HIV-1 infection of primary lymphocytes.

Background: Human herpesvirus type 6 (HHV-6), an ubiquitous virus, is the causative agent for exanthem subitum. The virus is frequently associated with lymphoproliferative disorders and other diseases. Recently, we have reported the frequent presence of HHV-6 in oral carcinoma and the present study extends the observation to cervical carcinoma.

Objective: To examine the presence of HHV-6 in cervical carcinoma.

Study design: Formalin-fixed, paraffin-embedded cervical carcinoma tissues were examined for the presence of HHV-6 by immunohistochemistry using two monoclonal antibodies that react to HHV-6-encoded p41/38 and gp 116/64/54. In situ hybridization with variant-specific probes were used to type the HHV-6 DNA sequences present.

Results: A total of 14/26 (53.9%) carcinoma tissue specimens and 5/8 (62.5%) normal tissue specimens were positive for viral antigens. In situ hybridization studies revealed the presence of HHV-6 DNA sequences in 10/26 (38.5%) carcinoma tissue specimens and 1/8 (12.5%) normal tissue specimens. In the normal tissue, the HHV-6 was present in the endocervical ciliated columnar-epithelial cells and some cells in the subepithelial mucosa but in the carcinoma, the transformed cells were positive for the virus.

Conclusion: HHV-6 viral proteins and DNA were found in more than one third of the cervical tissue examined suggesting possible viral expression in these tumours. The significance of the distribution and role of the HHV-6 in cervical tissue remains unclear. Since HHV-6 has an oncogenic potential, the virus may cooperate with other transforming agents for the progression of the disease.

Background: Variability in the profile of antigen-reactive bands in Western blot for serodiagnosis of human immunodeficiency virus (HIV) infection may result in disagreement regarding interpretation of positive result, due to lack of consensus in the interpretive criteria laid down by various organisations.

Objectives: The objectives of this study were (i) to find out the extent of disagreement over various criteria regarding interpretation of positivity in Western blot and (ii) to review the discordance by retesting the discordant specimens using recombinant antigens as well as by performing repeat Western blot in follow-up specimens.

Study design: A total of 467 specimens from high-risk groups, diagnosed positive for HIV type-1 (HIV-1) infection by the criteria of at least one of the five organisations, viz. Association of State and Public Health Laboratories Directors (ASTPHLD), Consortium for Retrovirus Serology (CRSS), American Red Cross (ARC) and World Health Organisation (WHO), were analysed to find out the extent of discordance between various criteria for interpretation of Western blot positivity. The discordant specimens were subjected to line immunoassay (LIA) using recombinant antigens. Also, follow-up Western blots were performed in case of discordant specimens at 6, 12 and 24 weeks intervals.

Results: We observed that criteria laid down by ASTPHLD, CDC and CRSS scored all the specimens as positive while ARC and WHO criteria scored 13 (2.8%) and 18 (3.8%) of specimens, respectively, as negatives which were detected as positives by other criteria (discordant specimens). The gp41 reactive band was the most frequently missing band, being undetectable in 11.6% of specimens while bands reactive to p24, p31, gp120 and gp160 could not be recorded in 1.9%, 9.4% and 3.2% and 1.5% of specimens, respectively. Testing of the discordant specimens with recombinant antigen preparation and with repeat Western blot in follow-up specimens collected at 6, 12 and 24 weeks demonstrated all bands undetectable in initial Western blot, except 25% of gp41 reactive bands.

Conclusions: It is felt that before selecting any criterion for Western blot positivity, it should be evaluated in the local population at risk for HIV-1 infection with additional or follow-up tests.

Background: Determination of the immune status against measles in young adults requires careful evaluation of the laboratory methods because of waning immunity. The hemagglutination inhibition (HI) test and enzyme-linked immunosorbent assay (ELISA) may lack the sensitivity required to detect very low levels of antibodies. In addition, the correlation between ELISA-IgG assays and the degree of protection from measles is not well defined.

Objectives: (a) Evaluation of a commonly used measles ELISA-IgG test kit in comparison with the hemagglutination inhibition (HI) test which corresponds strongly to virus neutralization; (b) determination of false negative rates of the ELISA-IgG and the HI tests; (c) evaluation of the ELISA-IgG test kit as a quantitative assay.

Study design: One hundred and eighty serum samples collected from 60 vaccinated young adults immediately before vaccination and 14 and 28 days postvaccination, were tested comparatively by HI and by a commercial ELISA-IgG kit. For evaluation of false negative rates, postvaccination sera of a cohort of 48 vaccinees with negative HI or ELISA-IgG prevaccination sera were tested for IgM. Sixty-three of the samples were also titrated by the ELISA-IgG kit using serial dilutions, for comparison with HI titers.

Results: Using the HI test as a reference method, the ELISA-IgG kit was found to have overall accuracy of 81%, sensitivity of 80% and specificity of 84%. The false negative and the false positive rates were 20% and 16%, respectively. In contrast, when we used postvaccination IgM test to distinguish between true and false prevaccination negatives in both the HI and ELISA-IgG tests, we found that the false negative rates were 75.6% by ELISA and 72.5% by HI, and the false positive rates were 2.4% and 0%, respectively. Serum titers determined by the ELISA-IgG test were generally 5–10-fold higher than the corresponding HI titers, but without a consistent correlation.

Conclusions: Both the ELISA-IgG and the HI tests frequently failed to detect residual immunity. The two tests also did not correlate well with each other suggesting that different antigenic determinants of the virus are involved in each assay and therefore the HI test should not be used as a reference method for evaluation of the sensitivity of ELISA IgG kits.

Background: Novel commercial kits based on antibody reactivity to purified heterophile antigens have recently been introduced for the diagnosis of Epstein-Barr (EB) virus-associated infectious mononucleosis (IM). It is important to determine possible improvements in the performance and reliability of such tests for the diagnosis of IM.

Objective: To evaluate the reliability of six commercially available kits for the rapid diagnosis of IM in comparison to EB-virus-specific serology.

Study design: In total, 100 sera, 53 from patients with serologically verified primary EB virus infection and 47 from EB-virus-immune or -susceptible patients, were used to evaluate the six rapid test kits: Monolatex, Mono-Latex, Mono-Lex (latex agglutination-based kits), Mono-Plus, IM-Check and Clearview IM (solid-phase-based kits). EB-virus-specific serologies including detection of viral capsid antigen IgM and IgG and EB nuclear antigen-1 IgG, were used as reference methods.

Results: Compared with the reference methods, the sensitivities and specificities of the heterophile antibody test kits were 70–92% and 96–100%, respectively. IM-Check had a low sensitivity and was difficult to read. The remaining kits performed well.

Conclusion: Monolatex, Mono-Latex, Mono-Lex, Mono-Plus and Clearview IM can be recommended for the confirmation of EB-virus-associated infectious mononucleosis. Clearview IM combined a high sensitivity and specificity with a very simple one-step solid-phase-based procedure.

Objective: We present a prospective study of the correlation between the human cytomegalovirus (HCMV) quantitative antigenemia with monoclonal antibody to p72 protein (immediate-early antigen) and the number of infected cell foci detected in the shell-vial culture. A comparative study was made of the value of quantitative antigenemia (pp65 and p72) in 14 patients.

Results: The average value of the pp65 antigenemia was 195 pp65-positive PMNLs per 10⁵ PMNLs (range 10–1000) and that of the p72, 21 p72-positive PMNLs per 10⁵ PMNLs (range 0–120) (P < 0.001). The p72 antigenemia value represented 10.7% of the pp65 value (range 4.4–70%). A statistical correlation was observed between the total number of infected cell foci detected in the shell-vial culture and the total number of p72-positive PMNLs (P < 0.001), but not with the number of pp65-positive PMNLs (P = 0.4). A study of the number of infected cell foci detected in the shell-vial per 100 000 PMNLs inoculated showed a statistical correlation with the value of the p72 antigenemia (P < 0.001).

Conclusions: According to results, there seems to be a general population of PMNLs carrying viral particles which are detected by means of the pp65 monoclonal antibody, and a subpopulation carrying active and replicative viral particles which is detected with the p72 antibody. This last subpopulation would be responsible for the formation of infected cell foci in the shell-vial culture. However due to the technical difficulties presented by the routine performance of p72 antigenemia, we recommend the routine application of the quantitative shell-vial culture and the use of the number of infected cell foci × 100 000 PMNLs inoculated as a parameter of replicative viral load for the diagnosis of infection and disease caused by HCMV.

Genotyping of hepatitis C virus (HCV) is important because of its clinical and epidemiological implications. We report here the distribution of HCV genotypes in various patient groups in Finland using a rapid and reliable HCV typing method based on restriction fragment length polymorphism (RFLP) of the amplified DNA from the 5′ non-coding region of the genome. The reaction product from the primary, diagnostic PCR (nested, one tube system) was used in genotyping. From 264 Finnish sera we identified HCV genotypes 1a (14%), 1b (24%), 2b (20%), 3a (41%) and 1a + 1b (1%). Only one patient with genotype 2a was identified. From four Egyptian blood donors, types 1b and 4 were found. Genotype 3a was more often associated with i.v. drug abuse and younger age profile (less than 30 years).