Sensitivities of radioimmunoprecipitation assay and PCR for detection of human T-lymphotropic type II infection.

Five hundred forty-eight uncoagulated blood specimens from intravenous drug users infected with human T-lymphotropic virus type II (HTLV-II) were used to evaluate the sensitivities of the radioimmunoprecipitation assay (RIPA) and PCR for detecting HTLV-II-infected people. The sensitivities of both RIPA and PCR were found to be dependent on the HTLV-II antibody titer, as determined by the immunofluorescence assay. Neither of these recommended confirmatory methods was as sensitive for detecting weakly reactive HTLV-II specimens as the immunofluorescence assay, Western blotting (immunoblotting), or a modified licensed enzyme immunoassay. Use of RIPA and PCR to determine the reliabilities of other tests may sometimes give erroneous results.

Cloning the Cryptococcus neoformans TRP1 gene by complementation in Saccharomyces cerevisiae.

We have cloned the phosphoribosyl anthranilate isomerase (PRAI)-encoding gene (TRP1) of Cryptococcus neoformans by genetic complementation in Saccharomyces cerevisiae. Sequence analysis of this gene revealed it to be 939 bp in length, and without known promoter or termination sequences. Unlike some of the filamentous fungi, where PRAI enzymatic activity is controlled by a trifunctional gene product, the C. neoformans PRAI appears to be unifunctional. PRAI of C. neoformans exhibits 39% amino acid (aa) sequence identity compared to the S. cerevisiae counterpart. The TRP1 gene of C. neoformans maps to different size chromosomes in strains with different serotypes. The cloning of this gene for vector constructions, and the demonstration that S. cerevisiae can be used as a surrogate for C. neoformans gene expression, should help with the molecular studies of this significant fungal pathogen in our increasing immunocompromised population.

Detection and differentiation of antibodies to human T-cell lymphotropic virus types I and II by the immunofluorescence method.

We compared the sensitivities of the prototype human T-cell lymphotropic virus type I (HTLV-I)- and HTLV-II-transformed cell lines, MT2 and Mo-T, with that of an HTLV-II-infected cell line, clone 19, established in our laboratory, in the immunofluorescence (IF) test for detection of antibody to HTLV-I and HTLV-II. In addition, IF antibody titers with the three antigens were determined, and the results were compared with HTLV-I and HTLV-II typing by polymerase chain reaction (PCR). The MT2 cell line was more sensitive than the two HTLV-II cell lines for detecting HTLV-I antibody by IF, and clone 19 was more sensitive than Mo-T or MT2 for measuring HTLV-II antibody. In the titration study, the antigen that gave the highest titer correlated completely with the HTLV type determined by PCR, indicating that the relatively simple IF titration method can be used for differentiating HTLV-I and HTLV-II antibody in sera and plasmas.

Comparison of immunofluorescence, enzyme immunoassay, and Western blot (immunoblot) methods for detection of antibody to human T-cell leukemia virus type I.

A total of 3,349 serum samples were screened by the immunofluorescence (IF) method for antibody to human T-cell leukemia virus type I (HTLV-I). Only 9 of 2,409 specimens from selected individuals, blood bank donors, patients with encephalitis-meningitis, and human immunodeficiency virus antibody-positive homosexual or bisexual men were reactive by IF. In addition, 940 serum samples from intravenous drug abusers were tested by IF and also by an HTLV-I enzyme immunoassay (EIA) method. Of these, 222 (24%) were positive for both HTLV-I and HTLV-II antigens by IF, and 191 of these 222 were also reactive in the HTLV-I EIA. Of the 31 IF-positive, EIA-negative serum samples, 20 exhibited optical density readings greater than or equal to 70% of the positive cutoff in the EIA, and 29 samples reacted with 1 or more bands in the Western blot (immunoblot) test. An additional 10 specimens that were EIA negative reacted only with HTLV-I by IF. Differences in staining morphology and in reactions on HTLV-I and HTLV-II antigens before and after absorption of the serum specimens with HTLV-I and HTLV-II-infected cell pellets revealed six distinct serological patterns by IF. These results indicate that infections by HTLV-I or by another closely related retrovirus(es) occur in California. Further studies utilizing statistically valid sampling methods are needed to estimate true prevalence rates among various groups. IF and Western blot tests should supplement the EIA method to maximize sensitivity and specificity of test procedures.