ABSTRACT
The use of genotypic assays for determining drug resistance in human immunodeficiency virus (HIV) type 1 (HIV-1)-infected patients is increasing. These tests lack standardization and validation. The aim of this study was to evaluate several tests used for the determination of HIV-1 drug resistance. Two genotypic tests, the Visible Genetics TruGene HIV-1 Genotyping Kit and the Applied Biosystems HIV Genotyping System, were compared using 22 clinical samples. Genotyping results were also obtained from an independent reference laboratory. The Visible Genetics and Applied Biosystems genotyping tests identified similar mutations when differences in the drug databases and reference strains were taken into account, and 19 of 21 samples were equivalent. The concordance between the two assays was 99% (249 of 252 mutation sites). Mutations identified by the reference laboratory varied the most among those identified by the three genotypic tests, possibly because of differences in the databases. The concordance of the reference laboratory results with the results of the other two assays was 80% (201 of 252). Samples with 500 to 750 HIV RNA copies/ml could be sequenced by the Visible Genetics and Applied Biosystems assays using 1 ml of input. The Visible Genetics and Applied Biosystems assays both generated an accurate sequence. However, the throughput of the Visible Genetics assay is more limited and may require additional instruments. The two assays differ technically but are similar in overall complexity. Data analysis in the two assays is straightforward, but only the reports provided by Visible Genetics contain information relating mutations to drug resistance. HIV drug resistance genotyping by sequencing is a complex technology which presents a challenge for analysis, interpretation, and reporting.
Subject(s)
Anti-HIV Agents/pharmacology , HIV Protease/genetics , HIV Reverse Transcriptase/genetics , HIV-1/drug effects , Sequence Analysis, DNA/methods , Drug Resistance, Microbial/genetics , Genotype , HIV Infections/virology , HIV-1/classification , HIV-1/genetics , Humans , Microbial Sensitivity Tests , Mutation , RNA, Viral/blood , Reagent Kits, Diagnostic , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
We evaluated the performance characteristics of the COBAS AMPLICOR Hepatitis C Virus (HCV) MONITOR Test, version 2.0. Dilution studies using patient specimens demonstrated a lower limit of detection of 1,000 copies per milliliter. The assay was linear from 1,000 to 1 million HCV RNA copies per milliliter. Within-run precision and between-run precision were acceptable (approximately 0.100 and 0.14 SD for log10 [copies per milliliter]). A comparison of this version of the test (y), with the manual AMPLICOR HCV MONITOR Test, version 1.0 (x), yielded the following Deming regression equation: y = 1.004(+/- 0.04)x + 0.654(+/- 0.22); Sy/x¿D = 0.336; n = 92; r2 = 0.846; r = 0.920. Further comparison of the COBAS version 2.0 assay (x) with the QUANTIPLEX HCV bDNA Test (y) yielded the following Deming regression equation: y = 0.943 (+/- 0.130)x + 0.473 (+/- 0.717); Sy/x¿D = 0.194; n = 26; r2 = 0.600; r = 0.774. Version 2.0 detected the spectrum of HCV genotypes better than version 1.0.
Subject(s)
Hepacivirus/genetics , Hepatitis C/diagnosis , RNA, Viral/analysis , Reagent Kits, Diagnostic/standards , Evaluation Studies as Topic , Genotype , Hepacivirus/classification , Humans , Reproducibility of Results , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Legionella spp. are a common cause of community-acquired respiratory tract infections and an occasional cause of nosocomial pneumonia. A PCR method for the detection of legionellae in respiratory samples was evaluated and was compared to culture. The procedure can be performed in 6 to 8 h with a commercially available DNA extraction kit (Qiagen, Valencia, Calif.) and by PCR with gel detection. PCR is performed with primers previously determined to amplify a 386-bp product within the 16S rRNA gene of Legionella pneumophila. We can specifically detect the clinically significant Legionella species including L. pneumophila, L. micdadei, L. longbeachae, L. bozemanii, L. feeleii, and L. dumoffii. The assay detects 10 fg (approximately two organisms) of legionella DNA in each PCR. Of 212 clinical specimens examined by culture, 100% of the culture-positive samples (31 of 31) were positive by this assay. By gel detection of amplification products, 12 of 181 culture-negative samples were positive for Legionella species by PCR, resulting in 93% specificity. Four of the 12 samples with discrepant results (culture negative, PCR positive) were confirmed to be positive for Legionella species by sequencing of the amplicons. The legionella-specific PCR assay that is described demonstrates high sensitivity and high specificity for routine detection of legionellae in respiratory samples.
Subject(s)
Legionella pneumophila/isolation & purification , Legionella/isolation & purification , Legionellosis/diagnosis , Base Sequence , Bronchoalveolar Lavage Fluid/microbiology , DNA Primers , Genetic Variation , Humans , Legionella/classification , Legionella/genetics , Legionella pneumophila/classification , Legionella pneumophila/genetics , Legionnaires' Disease/diagnosis , Lung/microbiology , Molecular Sequence Data , Pleural Effusion/microbiology , Polymerase Chain Reaction/methods , RNA, Ribosomal, 16S/genetics , Reproducibility of Results , Sensitivity and Specificity , Sequence Alignment , Sequence Homology, Nucleic Acid , Sputum/microbiologyABSTRACT
The ultrasensitive Amplicor HIV-1 Monitor test (Roche Diagnostic Systems) was evaluated for precision, linearity, and sensitivity and was compared to the standard Amplicor assay. The ultrasensitive assay reliably quantified samples in the range from 50 to 50,000 human immunodeficiency virus type 1 RNA copies/ml with acceptable correlation with the standard Amplicor test.
Subject(s)
Acquired Immunodeficiency Syndrome/blood , HIV-1/isolation & purification , RNA, Viral/blood , Viral Load , Humans , Regression Analysis , Reproducibility of Results , Sensitivity and Specificity , Virology/methodsABSTRACT
An ELISA for measuring thyroglobulin (Tg) in serum was developed with polyclonal antibodies to Tg on the solid phase and two monoclonal antibodies to Tg as the second antibodies. The assay has a detection limit of 1 microgram/L, a within-run imprecision (CV) of <7%, and a between-run CV of < 10%. Parallelism of the assay was shown in dilution studies, in which the percent observed/expected values for n = 5 autoantibody-containing samples gave a mean of 99% (SD 13.1 %); for n = 5 samples with undetectable autoantibody concentrations, the mean was 103% (SD 11.8%). The correlation of the ELISA with an RIA for Tg in 46 normal samples was ELISA = 1.11(RIA) + 0.52, S y/x = 2.23, SD intercept = 0.54, SD slope = 0.03, range = 0 to 53 micrograms/L, r = 0.980. Comparison of the ELISA with a reference laboratory RIA for 29 clinical samples gave a correlation of: ELISA = 1.53(RIA) - 0.48, S y/x = 9.00, SD intercept = 2.19, SD slope = 0.10, range = 0 to 98 micrograms/L, r = 0.950. To provide additional information concerning the reliability of the Tg measurement in samples containing autoantibodies to Tg, we developed a procedure for determining the percent recovery. A percent recovery greater than or equal to 80% indicates minimal interference by autoantibodies in this assay. The assay is straightforward to perform, results can be posted within 8 h, and the routinely good recovery of Tg in the presence of Tg autoantibodies indicates minimal autoantibody interference in this assay.
Subject(s)
Autoantibodies/blood , Enzyme-Linked Immunosorbent Assay/methods , Thyroglobulin/blood , Thyroglobulin/immunology , Adult , Enzyme-Linked Immunosorbent Assay/statistics & numerical data , Female , Humans , Male , Middle Aged , Quality Control , Radioimmunoassay , Reference Values , Sensitivity and SpecificityABSTRACT
By taking advantage of a newly available microplate counter for radioactivity and the organic solvent-resistant, pigmented microplates, we have successfully established radioimmunoassays (RIA) for both CA-GI and CA-BR on microplate for routine clinical use. In the process of assay development, we found that both pigmented PicoPlate, made of acrylonitrile, and polystyrene Microlite 2 can be coated with antialpha fetoprotein (AFP) and antinerve growth factor (NGF) and used for setting up immunoassays for AFP and nerve growth factors. There were no problems following a test format of either competitive binding or sandwich design. Microlites 2 is recommended over PicoPlate because Microlites 2 is made of polystyrene, which is less expensive and separable into 8-well strips or even single wells. Single-well separation allows for the use of regular gamma counters in case Topcount is unavailable. We also found that the sensitivity of these tests was not significantly affected even though Topcount counts the weaker beta emissions. Similar dose-response curves could also be generated between original Biomira tube assays and assays using PicoPlate or Microlite 2 coated with protein antigens CA-Br and CA-GI. Excellent correlations were also obtained between the microplate assays and the Biomira tube assays for CA-GI and CA-Br using groups of serum specimens from cancer patients. We recommend the development of various RIAs on the microplate: it requires less reagents and less sample handling by the technologists and it can be essentially automated.
