Clinical and diagnostic virology最新文献

Background: The quantitative cytomegalovirus-antigenemia (CMV-Ag) assay is an important technology in the regimen of tests utilized in the care and management of acquired immunodeficiency syndrome (AIDS) and other immunocompromised patient groups. Performance of this assay is contingent upon the appropriate processing of the polymorphonuclear leukocyte (PMNL) compartment of the peripheral blood. However, a cell input standard in the performance of the CMV-Ag assay, has not been established. Interpretive differences between laboratories utilizing the CMV-Ag assay may reflect this lack of test uniformity.

Objectives: To determine the effect of different PMNL concentrations on the quantitation of CMV in peripheral blood. The leukocyte concentration resulting in optimal rates of viral detection, will be compared with the shell vial assay-indirect immunofluorescent assay (SVA-IFA), and conventional tube culture (TC-CPE).

Study design: A total of 74 freshly collected blood specimens were tested by the CMV-Ag assay, using cytospin preparations consisting of 2×10⁵, 4×10⁵ and in preliminary experiments, 8×10⁵ PMNLs/slide. Data obtained from these studies were compared to SVA-IFA and TC-CPE. Viral load was monitored among 11 symptomatic patients through sequential testing of these patients at the start of ganciclovir (GCV), foscarnet (PFA), or combination drug therapy.

Results: Among 74 blood specimens tested by the CMV-Ag assay, cytospin preparations consisting of 4×10⁵ compared with 2×10⁵ PMNLs/slide, affected a mean positive cell increase of 215% (P=0.03). PMNL slide preparations consisting 8×10⁵ cells produced background levels which prevented accurate reading of slides. The CMV-Ag assay was more sensitive than the SVA-IFA, but equivalent to TC-CPE. Among 11 patients started on drug therapy, viral load was markedly reduced in 8 within 2–3 weeks; three patients (2 deceased within 3 weeks after receiving therapy), showed no decrease in viral load. One patient was identified as harboring a PFA resistant strain.

Conclusions: A PMNL concentration of 4×10⁵ cells facilitated the reading of CMV-Ag assay slide preparations. The modified CMV-Ag assay furthermore, is applicable in the monitoring of viral load for the tracking of susceptible or resistant CMV strains.

Background: Hantaviruses cause two serious human diseases: hantavirus pulmonary syndrome and hemorrhagic fever with renal syndrome. At least nine hantaviruses are known to be pathogenic for humans and numerous others, with unknown disease potential, have been detected in rodents. Assays to quickly identify specific hantaviruses would be useful both for clinical diagnosis and in risk assessment studies.

Objectives: The goal of our study was to develop and test a specific and sensitive PCR-based assay for identification and differentiation of hantaviruses.

Study design: We developed an assay that combined RNA-PCR amplification and colorimetric enzymatic detection to identify representative European, Asian, and north American hantaviruses. RNAs from 18 hantavirus strains of nine species were amplified in the presence of digoxigenin-dUTP by using a single pair of oligonucleotide primers and polymerase chain reaction (PCR) performed by using rTth DNA polymerase. Digoxigenin-labeled PCR products were hybridized in solution to virus type-specific biotinilated probes, captured onto streptavidin-coated microtiter plates and detected by horseradish peroxidase-labeled anti-digoxigenin antibodies and a chromogenic substrate.

Results and conclusions: The assay correctly identified each homologous virus type tested. The detection limit of the assay was approximately 15 PFU or at least 50 copies of the viral genome. The assay is simple and strain-specific and is adaptable for automation, making it more practical than other available techniques for accurate and reliable diagnosis and typing of hantaviruses.

Background: Herpesvirus infections of the central nervous system are often severe but are fortunately rare. The incidence of these infections has however, increased in recent years as a consequence of an increase in the number of immune-compromised individuals. New diagnostic procedures have improved our ability to diagnose these infections and herpesviruses may yet be implicated as the cause of further neurological diseases with no known aetiology. Methodological standards for selection and evaluation of patient materials are essential to the provision of reliable diagnosis, yet few studies have addressed this important issue.

Objectives: To describe and define methodological standards and reference methodology for diagnosis of herpesvirus infections of the CNS.

Study design: Information gathered by literature review.

Results: Only for herpes simplex encephalitis is there sufficient data to allow the definition of reference methodology. Good methodological standards exist but few studies have adhered to these standards. As methods for the detection of specific intrathecal antibody synthesis are well established yet under-used in diagnostic virology, the principle of these measurements is reviewed in some detail.

Conclusions: Herpesvirus infections of the CNS are of increasing importance. High quality, multi-centre studies are needed to establish the value of the new diagnostic test procedures if further improvement in the diagnostic sensitivity and specificity of these procedures is to be achieved.

Background: Human papillomaviruses (HPV) are now considered etiologic agents of cancer of the uterine cervix. Adjunctive diagnostic procedures for the detection of HPV infection could increase the sensitivity of primary and secondary screening of cervical cancer. HPV testing could also improve the specificity of screening programs resulting in avoidance of overtreatment and saving of costs for confirmatory procedures.

Objectives: To review the rationale of HPV testing in genital diseases and the potential applications of HPV DNA detection methods for clinical and epidemiological purposes.

Results: Progression of HPV infection is associated with the persistence of HPV infection, involvement of high-risk HPV types, high HPV viral load in specimens, integration of viral DNA and possibly the presence of cofactors. The design of HPV diagnostic tests will need to take into account these parameters of disease progression. HPV DNA detection techniques based on signal-amplification are standardized, commercially available and detect several high-risk HPV types. They increase the sensitivity of screening for high-grade and low-grade lesions. Although they may yield false-negative results in the presence of significant HPV-related disease, new test formats could resolve this weakness. Amplification techniques are ideal instruments for epidemiologic purposes since they minimize misclassification of HPV infection status and allow for the detection of low viral burden infections. They are currently not readily applicable to diagnostic laboratories.

Conclusions: Before recommending HPV testing, prospective trials of untreated LSIL with HPV testing as well as the determination of the efficacy and cost-effectiveness of novel HPV tests, need to be completed.

Background: PCR assays for detection of herpes simplex virus DNA sequences in clinical specimens are more sensitive than cell culture.

Objective: A non-isotopic PCR assay (glycoprotein B HSV-2 assay, Roche Molecular Systems) for detection of HSV-2 DNA sequences was evaluated on 234 clinical specimens.

Study design: A total of 125 patients (73 women, 40 men, 12 unknown) provided 162 samples contained in viral transport medium. Samples were first inoculated in cell culture, centrifuged at 12 000 × g, lyzed and kept frozen. A total of 77 women provided 42 cervicovaginal lavages and 30 vaginal tampons that were lyzed, tested with two isotopic HSV-2 PCR assays and kept frozen. All these samples were subsequently thawed and amplified with the glycoprotein B HSV-2 assay using generic primers for HSV glycoprotein B gene. Amplicons were captured on microplates with a HSV-2-specific probe and were detected with avidin-peroxidase and substrate.

Results: Of the 162 samples submitted to viral culture, HSV-2 was isolated from 73 while 89 did not contain HSV-2. All the 73 specimens with culture-proven HSV-2 infections tested positive with glycoprotein B HSV-2 assay (sensitivity of 100%). Herpesviruses other than HSV-2 were isolated from 34 samples that were negative with glycoprotein B HSV-2 assay. Two culture-negative samples tested positive in the glycoprotein B HSV-2 assay (specificity of 98.7%). The latter samples could not be retested in confirmatory isotopic HSV-2 PCR tests. HSV-2 DNA sequences could also be detected directly in cervical lavages or vaginal tampons from 13 women with the glycoprotein B HSV-2 assay.

Conclusion: Detection and typing of HSV-2 in clinical samples, including those collected in viral transport medium, can be accomplished with PCR assays using the AMPLICOR format.

Background: It is not known whether the measurement of serum hepatitis C virus (HCV) RNA by reverse transcription polymerase chain reaction (RT-PCR) could improve the management of patients with chronic hepatitis C being treated with interferon.

Objectives: We analysed, in a pilot study, the relations between the variations of HCV-RNA and alanine aminotransferase (ALT) serum levels in 18 anti-HCV positive patients treated with interferon.

Study design: Serum HCV-RNA was measured, using a non competitive coamplification assay (Amplicor HCV Monitor™), before (at 3, 2 and 1 months and baseline), during (first, third and sixth month) and after treatment for at least 8 months (range 8–17 months). HCV-RNA levels fluctuations were correlated with those of ALT and treatment outcome. According to the ALT pattern, four patients were non responders, seven partial responders, four relapsers and two long term responders.

Results: The median and mean baseline HCV-RNA levels were significantly different in patients infected by HCV type 1, 2 and 3, being 248 449, 235 506; 4170, 17 866 and 22 315, 79 273 molecules per ml, respectively (P < 0.0001). We did not find any significant difference between median and mean baseline viremia of responders and non responders. After 1 month of treatment viremia was below the sensitivity levels of the assay in 77.7% ($\text{14}\text{18}$) of the patients who normalized ALT, at least temporarily. On the contrary, HCV-RNA remained detectable in non responders.

Conclusions: Our data suggest that HCV-RNA detection using Amplicor Monitor™ at the first month of treatment can be useful to identify non responders, avoiding three additional months of treatment as would be required by ALT monitoring alone. During the post-treatment follow-up, persistence of undetectable HCV-RNA and normal ALT levels helps to identify long term responders from patients with the risk of relapse in spite of biochemical remission.

Background: Immunofluorescence assay (IFA) of viral antigens in nasal aspirates is largely used for the diagnosis of respiratory syncytial virus (RSV), parainfluenzavirus (PIV) type 3 and adenovirus (AdV) infections, whilst rhinovirus (RV) are detected by virus isolation technique (VIT) only. Using the two techniques, IFA and VIT, a significant number of specimens remain negative in spite of clinical and epidemiological presumptions of viral infection.

Objectives and study design: The polymerase chain reaction (PCR) should improve the sensitivity of viral detection in clinical specimens. From October 1995 to March 1996, 277 nasal aspirates from hospitalized infants were tested simultaneously by IFA, VIT, polymerase chain reaction and hybridization with a DNA enzyme immunoassay (PCR-EIA) for RSV, PIV-3, AdV and RV.

Results: RSV were detected in 177 (64%) samples, PIV-3 in 23 (8%), RV in 40 (14%), and AdV in 30 (10%). PCR-EIA detected RSV in more samples 173 (62%) than $\text{IFA}\text{VIT}$: 109 (39%) (P < 10⁻⁷). In most cases (79%), RSV-infected infants had lower respiratory tract disease, and routine and PCR techniques were positive. Out of the 23 PIV-3 infections, 12 were $\text{IFA}\text{VIT}\text{-}$ and PCR-EIA-positive, and 11 $\text{IFA}\text{VIT}\text{-}\text{negative}$ and PCR-EIA-positive. For RV, 35 (87%) specimens were PCR EIA-positive and 11 (27%) culture-positive; for AdV 30 samples were PCR-EIA-positive and four were culture-positive. Simultaneous viral infections were revealed in a significantly higher proportion than in conventional techniques: 18% ($\text{50}\text{277}$) versus 2.5% ($\text{7}\text{277}$); P < 10⁻⁷. One RSV infection in four was associated with the presence of another virus, mainly PIV-3 (16 cases) and AdV (13 cases).

Conclusions: PCR-EIA detects more positive-specimens than $\text{IFA}\text{VIT}$, 1.5 times more for RSV, 1.9 for PIV-3, 4 for RV and 10 for AdV, respectively. This increased sensitivity of viral detection by PCR-EIA compared to the $\text{IFA}\text{VIT}$ could suggest that samples containing low levels of virus are missed by routine methods $\text{IFA}\text{VIT}$, and consequently, RSV or PIV-3, and above all RV or AdV are overlooked as agents of respiratory diseases. However, apart from the fact that the economic and convenient aspects of virus diagnostic cannot be missed, it is difficult to answer the following questions: what is the meaning of the detection of a viral sequences in nasal aspirates of infants. or may PCR