Clinical and diagnostic virology最新文献

Background: Large numbers of measles virus (MV) specimens are processed in our laboratory each year as part of a molecular epidemiological study of MV in South Africa. The development of a sensitive, rapid virus isolation system is needed to cope with the number of specimens processed.

Objectives: A comparison was made of centrifugation-enhanced shell vial culture and standard tissue culture using B95a cells for the isolation of MV from throat swabs and urine.

Study design: The rapid method was initially evaluated using Schwarz vaccine virus and then compared to standard culture using throat swab specimens.

Results: The shell vial assay proved to be ten times more sensitive than standard culture in the initial evaluation. Of 43 throat swab specimens, 37 (86%) were positive and 6 (14%) negative in standard culture using B95a cells. The specimens were removed after adsorption in standard culture, frozen and then used in the shell vial assay. It was found that $\text{16}\text{27}$ were positive in the shell vial assay (24 of these 27 being positive in standard culture,) and 8 negative and 8 specimens gave an indeterminate result. For the 45 urine specimens used in the shell vial assay, 71% were positive, 11% negative and 18% gave an indeterminate result, due to too few cells being present for antigen determination by indirect fluorescent antibody assay. Results were obtained in 4 days, as opposed to the average of 14 days for confirmed isolation in standard culture.

Conclusion: Rapid culture substantially reduced total test time, was less labour-intensive and was as sensitive as standard culture for the isolation of measles virus from clinical specimens.

Background: Serological markers such as HBsAg and anti-HIV may be present in serum at very high concentrations and this may give rise to erroneous diagnoses due to cross-contamination.

Objectives: To investigate poor equipment maintenance and use, including contamination with human serum, as a potential source of erroneous assay results.

Study design: The potential of microtitre plate washers and micropipettors to transfer material between microplate wells and between specimens was examined. For the study of micropipettors we recruited 19 UK diagnostic laboratories.

Results: Four out of seven plate washers in use, until adjusted, had the potential to cause false positive HBsAg reactions. The centering of the probes that delivered the wash fluid, delivery pressure, wash volume and the use of a pre-programmed card to direct the washing procedure were important variables. We investigated soiling of tip cones of micropipettors. In every laboratory human IgG could be detected in at least a third of eluates from micropipettor tip cones; only 31 (14%) of 222 showed no evidence of contamination with human serum. Only one laboratory submitted eluates devoid of specific antibodies. Anti-HAV was the marker most commonly found (n = 68), followed by HBsAg (n = 27) and anti-HIV (n = 20). Seven micropipettor eluates from two laboratories were radioactively contaminated.

Conclusions: Recommended precautions are regular checking, cleaning and servicing of equipment, care in interpreting weak reactions, reference back to serum left on the clot of the original specimen and testing of a follow up specimen. Poorly maintained immunoassay equipment can readily generate false positive results due to low-level cross-contamination, particularly with the current highly sensitive HBsAg and anti-HIV assays.

Background: Comparative field utility of selected HIV-1 assays using homologous collections of serum, urine and oral mucosal transudate (OMT) was determined in adult populations from a tuberculosis hospital and STD clinic in Djibouti, East Africa.

Study design: Enzyme immunoassay with confirmatory Western blot was performed on all serum specimens for comparison with rapid, instrument-free assays (SUDS HIV-1, Murex; TestPack Abbott; and COMBAIDS HIV 1 + 2, SPAN Diagnostics) using various specimen sources. Delayed (48 h post-collection) testing was also performed on urine. Sensitivity and specificity for the rapid assays, in descending order, were as follows: serum SUDS HIV-1 assay (100%, 98.3%), serum COMBAIDS assay (98.4%, 99.6%), and OMT SUDS HIV-1 assay (98.4%, 94.5%).

Results: The OMT EIA optical density cutoff value was modified resulting in an improved specificity from 89.1 to 99.6%; however, sensitivity decreased from 100 to 98.5%. Urine EIA and rapid assays demonstrated unacceptable test performance for use as a screening test.

Background: Hepatitis C virus (HCV) serotyping has been proposed as an alternative assay to determine the respective genotype as it is more rapid, simple and less expensive than polymerase chain reaction (PCR) based typing methods.

Objectives: A serotyping assay was compared with a genotyping assay to determine the infecting hepatitis C virus type in chronically HCV infected patients eligible for interferon therapy.

Study design: An enzyme immunoassay (HC01, Murex^™) was tested to identify HCV types 1, 2 and 3 specific antibodies in 134 PCR-positive sera from chronically infected patients which had been previously genotyped by a reverse hybridization assay (INNO-LiPA HCV I, Innogenetics). Respectively nine and seven sera were from HIV-seropositive and hemodialysis patients. Unreactive sera and those with discrepant results were retested by a new version (HC02) extended to types 4, 5 and 6.

Results: The distribution frequency of HCV genotypes was subtype 1a, 16.4%; subtype 1b, 46.3%; subtype 2a, 7.5%; subtype 3a, 20.9%; type 4, 4.5%; type 5, 0.7%; and co-infections, 3.7%. Among all the patients, 95% were of type 1, 2 or 3. The antibody reactivities of hemodialysis ($\text{1}\text{7}$; P < 0.05) and HIV-seropositive patients ($\text{4}\text{9}$; P = 0.06) were lower than for the patients seen at the hepatology unit ($\text{87}\text{118}$). For these latter patients, the serotyping assay was interpretable in 71% and concordant in 64% of the samples with the genotyping assay. Out of the 84 samples with interpretable results, 75 sera were correctly serotyped (89% specificity). The two mixed results obtained by serotyping did not correspond to genotype coinfections (n = 3) and reciprocally. Six discrepancies were ruled out by the new assay, but the 2 untypeable sera remained unsolved, and four out of six sera with genotype 4 were serotyped as type 5.

Conclusions: Serotyping could be an attractive approach if the reactivity was improved and the subtyping possible.

Background: Mutations in the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) gene confer resistance to antiviral drugs acting on RT. Current methods employed to detect such resistance require time-consuming culture techniques during which selective pressures may affect the outcome of the test.

Objectives: We sought to determine whether drug-susceptible and drug-resistant HIV-1 derived from clinical specimens could be distinguished by the effects of the active form of the drug on the enzyme activity in a quantitative, cell-free RT assay.

Study design: Polyethylene glycol (PEG)-precipitated virus was obtained from 7-day culture supernatants. RT activity in the lysed viral extracts was measured in the presence of increasing concentrations of the active form of the drug being tested. IC₅₀ (50% inhibitory concentration) values were determined by application of the median effect equation.

Results: Assays from nine post-nevirapine therapy isolates gave IC₅₀ values at least 2 logs greater than pre-nevirapine isolates. The method also correctly distinguished between isolates sensitive and resistant to 2′,3′-dideoxyinosine (ddI), but not between the ZDV-sensitive and ZDV-resistant isolates tested. The results agreed with data obtained by sequencing and by culture-based susceptibility assays.

Conclusions: This technique appears to offer a simple, rapid method for determining the resistance of HIV-1 isolates to nevirapine, ddI and possibly other RT-inhibiting drugs. The method is not useful for identifying resistance to ZDV.

Background: Influenza virus infections are a major cause of morbidity and the identification of the type or subtype of a clinical isolate has important clinical and epidemiological implications.

Objectives: To evaluate the ability of a reverse transcription-polymerase chain reaction (RT-PCR) assay to type and subtype clinical human isolates of influenza virus.

Study design: Reference strains of influenza $\text{A}\text{H1N1}$, $\text{A}\text{H3N2}$, and B viruses and human clinical isolates of influenza virus representing antigenic variants from the last 15 years were evaluated using an RT-PCR assay.

Results: Amplicons of 325, 198 and 365 base pairs in length were obtained from RNA extracted from influenza $\text{A}\text{H1N1}$, $\text{A}\text{H3N2}$ and B viruses, respectively. All human-derived $\text{A}\text{H1N1}$, $\text{A}\text{H3N2}$, and B reference strains and antigenic variants tested were correctly identified.

Conclusions: RT-PCR is an effective alternative to traditional methods for typing and subtyping influenza viruses.

Background: Multiplex polymerase chain reaction (PCR) has been established as a general technique for the simultaneous amplification of different target sequences. Uses of multiplex include pathogens identification, linkage analysis and genetic disease diagnosis. The high sensitivity of PCR may produce false-positive results due to contamination with previously amplified material.

Objectives: To develop a multiplex PCR technique that can simultaneously detect and discriminate human immunodeficiency virus types 1 and 2 ($\text{HIV}\text{-1}\text{2}$) and human T-lymphotropic virus types 1 and 2 ($\text{HTLV-I}\text{II}$) proviral sequences. Such a method should incorporate a system that prevents the occurrence of false-positive results.

Study design: Combinations of four primer pairs, one for each retrovirus, were assayed in order to determine the combination of oligonucleotides as well as the PCR conditions that yield the most specific and sensitive coamplification of proviral sequences. To prevent contamination with DNA from previous PCR amplifications, the uracil N-glycosylase (UNG) system was incorporated into the coamplification format.

Results: A combination of primer pairs from the gag region of HIV-1, env of HIV-2, pol of HTLV-I and tax of HTLV-II yielded specific and sensitive coamplification of proviral sequences. The UNG system was incorporated and shown to be efficient in the degradation of contaminating DNA. In the evaluation of a serologically well established panel of singly and dually infected individuals, the assay detected $\text{20}\text{22}\text{HIV-1}$, $\text{8}\text{10}\text{HIV-2}$, $\text{8}\text{8}\text{HTLV-I}$ and $\text{8}\text{8}\text{HTLV-II}$ infections.

Conclusions: A multiplex PCR method for the detection and discrimination of $\text{HIV}\text{-1}\text{2}$ and $\text{HTLV-I}\text{II}$ has been developed. Under standardized conditions, all four proviral sequences were detected in a specific and sensitive manner. The evaluation of a panel of clinical specimens from infected individuals by one or more retroviruses showed that the technique detected most of the infected individuals. A low viral load may explain cases where multiplex PCR failed to detect target sequences.