Nonisotopic detection and typing of human papillomavirus DNA in genital samples by the line blot assay. The Canadian Women's HIV study group.

The line blot assay, a gene amplification method that combines PCR with nonisotopic detection of amplified DNA, was evaluated for its ability to detect human papillomavirus (HPV) DNA in genital specimens. Processed samples were amplified with biotin-labeled primers for HPV detection (primers MY09, MY11, and HMB01) and for beta-globin detection (primers PC03 and PC04). Amplified DNA products were hybridized by a reverse blot method with oligonucleotide probe mixtures fixed on a strip that allowed the identification of 27 HPV genotypes. The line blot assay was compared to a standard consensus PCR test in which HPV amplicons were detected with radiolabeled probes in a dot blot assay. Two hundred fifty-five cervicovaginal lavage specimens and cervical scrapings were tested in parallel by both PCR tests. The line blot assay consistently detected 25 copies of HPV type 18 per run. The overall positivity for the DNA of HPV types detectable by both methods was 37.7% (96 of 255 samples) by the line blot assay, whereas it was 43. 5% (111 of 255 samples) by the standard consensus PCR assay. The sensitivity and specificity of the line blot assay reached 84.7% (94 of 111 samples) and 98.6% (142 of 144 samples), respectively. The agreement for HPV typing between the two PCR assays reached 83.9% (214 of 255 samples). Of the 37 samples with discrepant results, 33 (89%) were resolved by avoiding coamplification of beta-globin and modifying the amplification parameters. With these modifications, the line blot assay compared favorably to an assay that used radiolabeled probes. Its convenience allows the faster analysis of samples for large-scale epidemiological studies. Also, the increased probe spectrum in this single hybridization assay permits more complete type discrimination.

Mycoplasma fermentans DNA is infrequently detected in urine specimens from renal transplant recipients.

Mycoplasma fermentans is a likely causative agent of HIV-associated nephropathy. In a pilot study, M. fermentans DNA was detected with polymerase chain reaction (PCR) in urine samples from renal allograft recipients; nine (39.1%) out of 23 renal allograft recipients (most of whom had chronic allograft rejection) and none of the 20 controls, were infected with M. fermentans. A cross-sectional study was conducted to investigate the prevalence of M. fermentans in urine samples from renal allograft recipients. Midstream urine samples were centrifuged at 13,000 x g, purified with QIAamp and tested with PCR using RW004/RW005 and an internal control to screen for the presence of inhibitors. Of the 264 participants recruited, 263 completed the questionnaire (172 men, 92 women); 53 had chronic renal allograft rejection, 106 had chronic renal dysfunction without rejection, 69 had a normal renal allograft for more than 3 months and 35 had a renal allograft for less than 3 months. All urine samples yielded positive results for the internal control. Mycoplasma fermentans DNA was detected once i prospectively collected urine samples. The only individual infected with M. fermentans was also seropositive for HIV-1. This study demonstrates that M. fermentans can be at most sporadically detected in urine from patients living with a renal allograft but is not implicated in chronic rejection of allograft.

Detection of human papillomavirus DNA in cervical lavage specimens by a nonisotopic consensus PCR assay.

A gene amplification method that combines PCR with an enzyme immunoassay (PCR-EIA) for quantitation of amplified DNA was developed for the detection of human papillomavirus (HPV). Samples were amplified with consensus primers MY09 and MY11. Amplified DNA products were reacted in solution with type-specific nested RNA probes labelled with digoxigenin-11-UTP. Hybrids were captured on a microtiter plate coated with an antidigoxigenin antibody. Bound DNA-RNA hybrids were quantitated by the addition of an alkaline phosphatase-labelled monoclonal antibody directed against DNA-RNA hybrids and a fluorogenic substrate. The detection limit of PCR-EIA was six copies of HPV type 18 DNA in the original specimen. The assay was used to assess HPV infection of the uterine cervixes of 65 women referred to a colposcopy clinic. In 66 cervicovaginal lavage specimens, all 23 HPV strains detected by a standard isotopic PCR assay were also detected by the PCR-EIA (sensitivity, 100%; 95% confidence interval, 85.2 to 100%). Forty-two of the 43 samples that did not contain HPV types 6/11, 16, 18, 31, 33, 35, and 45 were also negative by PCR-EIA, for a specificity of 97.7%. Low-level cross-reactivity was encountered between HPV types 18 and 45 as well as between types 33 and 58. PCR-EIA provides a convenient means of objectively measuring PCR-amplified HPV DNA from common genital HPV types.

Clinical evaluation of Amplicor HIV-1 test for detection of human immunodeficiency virus type 1 proviral DNA in peripheral blood mononuclear cells.

We report here the clinical evaluation of Amplicor polymerase chain reaction (PCR) assay for the detection of the human immunodeficiency virus type 1 (HIV-1) in peripheral blood mononuclear cells (PBMCs). Results obtained with Amplicor HIV-1 test were compared to serological status and a standard PCR assay using SK38/SK39 and oligomer hybridization with SK19. A panel of 208 well-characterized specimens was analyzed, including PBMC lysates from 47 antibody-negative high-risk individuals, eight antibody-negative low-risk subjects, two subjects with acute retroviral disease, 35 asymptomatic seropositive subjects (59 samples) with CD4 counts > 400/mm3, 31 patients (46 samples) with AIDS-related complex (ARC), 30 patients (40 specimens) with AIDS, and six seropositive patients with unknown clinical status. Amplicor demonstrated a specificity of 100% and a sensitivity of 98.7%. Of the two false-negative samples with Amplicor, one was negative for beta-globin amplification, whereas a dilution of the other sample turned positive for HIV-1. Inhibitors of Taq polymerase were thus believed to be responsible for the negative results. This study demonstrates that commercialized nonisotopic PCR assays reach adequate levels of sensitivity and specificity for diagnosis of HIV-1 infection and could be considered in clinical situations in which serology is not helpful.

Absence of prolonged immunosilent infection with human immunodeficiency virus in individuals with high-risk behaviors.

PURPOSE: The presence in some individuals of a prolonged phase of infection with human immunodeficiency virus type 1 (HIV-1) before seroconversion remains controversial. This study was undertaken to determine with a sensitive in vitro amplification technique, the polymerase chain reaction (PCR), whether seronegative individuals with high-risk behaviors could harbor HIV-1 sequences in their peripheral blood mononuclear cells (PBMCs) and remain seronegative for more than 6 months. PATIENTS AND METHODS: Seronegative individuals who engaged in unprotected anogenital intercourse with HIV-1-infected partners or with more than 10 individuals per year, and seronegative individuals who shared needles with seropositive partners, were recruited prospectively over 18 months. HIV-1 DNA and RNA sequences were detected in PBMCs of these individuals with three PCR assays using SK38/SK39, SK145/SK431, and SK68/SK69. Seronegative but PCR-positive patients were also evaluated with p24 antigen capture assay, radioimmunoprecipitation assay, and Western blot. The latter patients were followed prospectively to reproduce PCR-positive results and monitor serologic responses. RESULTS: Sixty-one men and 18 women, with an average age of 34.1 +/- 7.6 years, were recruited: 56 were homosexual men, 18 were heterosexual women, and 5 were heterosexual men. Amplification reactions for HIV-1 of 104 PBMC specimens from 79 patients with negative or indeterminate serologies revealed that 4 patients (5.1%) were positive with PCR for HIV-1 DNA and RNA at the time of enrollment. Positive amplification reactions could not be reproduced in prospective samples for one patient. The analysis of a variable human genomic locus in this patient's PBMCs demonstrated that the first PCR-positive sample and following PCR-negative samples originated from different patients, suggesting a specimen mix-up. Two of the three PCR-positive seronegative patients had symptoms suggestive of acute retroviral disease. Sera from all three patients contained p24 antigen. Two patients seroconverted within 1 month whereas one patient could not be followed prospectively. CONCLUSION: Prolonged infection with HIV-1 without seroconversion was not found in our population of patients at very high risk for HIV-1 infection. All PCR-positive patients seroconverted in less than 1 month.

Detection of polymerase chain reaction-amplified human immunodeficiency virus type 1 proviral DNA with a digoxigenin-labeled RNA probe and an enzyme-linked immunoassay.

An enzyme-linked immunoassay (EIA) combined with a solution hybridization (SH) reaction was devised to detect human immunodeficiency virus type 1 (HIV-1) provirus amplified by the polymerase chain reaction (PCR). In this nonisotopic PCR assay, designated PCR-EIASH, a fragment of the HIV-1 gag gene from peripheral blood mononuclear cells (PBMCs) was first amplified with biotinylated primers. The biotinylated amplified DNA segment was reacted in solution with an internal RNA probe labeled with digoxigenin-11-UTP. Hybrids were captured in a microtiter plate coated with streptavidin. Specific bound hybrids were quantitated by the addition of an enzyme-labeled antibody against digoxigenin and a fluorogenic substrate. The hybridization, immunological, and amplification parameters of PCR-EIASH were optimized as follows: 12.5 pmol of each primer was used in the PCR; the reannealing reaction of amplified products with the RNA probe, which was used at 0.30 microgram/ml, was completed in 30 min at 70 degrees C in 2x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate). Five copies of HIV-1 DNA diluted in a lysate of 100,000 PBMCs from a seronegative control could be detected by PCR-EIASH with a signal of 41 +/- 3 fluorescent units above a background noise of 13 +/- 2 fluorescent units. A total of 91 PBMC lysates from 91 seropositive patients sampled once and 20 PBMC lysates from 10 seropositive patients sampled twice were tested in duplicate in the PCR-EIASH; 107 samples were positive in duplicate tests, 1 sample was indeterminate, and 3 samples were negative. Of the latter three samples, one became positive by diluting the cell lysate, suggesting the presence of an inhibitor of Taq polymerase. The three samples negative for HIV-1 by PCR-EIASH were also negative when amplified with SK145-SK39 and detected with 32P-labeled SK102.

Evaluation of infection with human immunodeficiency virus type 1 by using nonisotopic solution hybridization for detection of polymerase chain reaction-amplified proviral DNA.

A convenient assay combining solution hybridization and enzyme immunoassay for DNA-RNA hybrids (polymerase chain reaction-enzyme immunoassay [PCR-EIA]) was developed to detect human immunodeficiency virus type 1 (HIV-1) provirus amplified by the PCR and was compared with oligomer hybridization with 32P-labeled SK19. In PCR-EIA, a fragment of the HIV-1 gag gene from peripheral blood mononuclear cells was first amplified with primer pair SK38/SK39 or O1/O2. PCR-amplified material was reacted in solution with a biotinylated RNA probe. Biotinylated hybrids were measured in a microtiter-plate EIA with antibiotin antibody and a beta-D-galactosidase-conjugated monoclonal antibody to DNA-RNA hybrids. Ten copies of HIV-1 DNA could be detected by PCR-EIA by using two different sets of primers. HIV-1 DNA was detected in 104 of 108 peripheral blood mononuclear cell samples by using SK38/39 and oligomer hybridization, in 104 of 108 samples by using SK38/SK39 and PCR-EIA, and in 104 of 108 samples by using O1/O2 and PCR-EIA. HIV-1 provirus was detected in 107 of 108 samples by using a combination of two sets of primers. One sample from a seropositive patient was negative in all three PCR assays, and six samples gave discordant results between primer pairs. Six of the latter samples scored negative in a PCR for beta-globin but became positive when the sample was diluted before amplification. When applied to clinical samples, PCR-EIA generated results similar to those of an isotopic assay for detection of amplified DNA.