Michelle M. Stempien

Rapid and precise quantification of HIV-1 RNA in plasma using a branched DNA signal amplification assay.

The level of human immunodeficiency virus type 1 (HIV-1) RNA in human plasma has been quantitated directly with use of a solid-phase nucleic acid hybridization assay, based on branched DNA (bDNA) signal amplification technology with chemiluminescent detection. Signal amplification is accomplished by the incorporation of sites for 1,755 alkaline phosphatase-labeled probes per genome of HIV-1, after successive hybridization of target-specific oligonucleotides and bDNA amplifier molecules. The assay is performed in microwells, much like an immunoassay, and is amenable to routine laboratory use. Reproducibility and specificity studies indicated that the bDNA method was precise and showed no reactivity with seronegative donors. HIV-1 RNA levels were quantitated for 348 seropositive specimens, with a detection rate of 83% for those specimens from patients with < 500 CD4+ T-cell counts. Plasma RNA levels were found to change with disease stage, and in response to antiviral therapy. Quantitation of HIV-1 RNA in the plasma of HIV-1-infected patients, with use of the bDNA assay, may be a useful method for monitoring HIV-1 disease progression and therapeutic response.

Short and long‐term effects of interferon on serum markers of hepatitis C virus replication

Abstract Hepatitis C virus RNA (HCV‐RNA) and serological markers of HCV infection were measured in 30 patients with chronic hepatitis C who had been treated with interferon (IFN). Patients were classified into four groups according to serum alanine aminotransferase (ALT) levels after treatment. These were: as complete responders (CR); partial responders (PR); transient responders (TR); and non‐responders (NR). In all 11 patients in the CR group, HCV‐RNA disappeared from serum for at least 24 months and anti‐c100‐3 decreased progressively during this time. In the PR group, four of five patients were positive for HCV‐RNA in spite of the improvement of ALT levels and decline of anti‐c100‐3. In the TR and NR groups, HCV‐RNA disappeared transiently or remained persistently positive. The results indicate that IFN‐mediated improvement of ALT and decrease of anti‐HCV (anti‐c100‐3) were not always related to the disappearance of HCV‐RNA from serum. On the other hand, sustained disappearance of HCV‐RNA from serum was demonstrated in the patients who did not have post‐treatment ALT relapse. This indicates that IFN can eradicate HCV from serum in some patients and provide a clinical remission of chronic hepatitis C.

Comparison of selective polymerase chain reaction primers and differential probe hybridization of polymerase chain reaction products for determination of relative amounts of codon 215 mutant and wild-type HIV-1 populations.

A mutation at codon 215 of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) gene results in decreased sensitivity to zidovudine (ZDV). In order to follow changes in codon 215 mutant (MUT) and wild-type (WT) populations in the plasma of patients during therapy, two polymerase chain reaction (PCR) procedures were investigated. The first was a nested, selective PCR, wherein a first round with viral-specific primers was followed by a second round with allele-specific primers. Although the procedure is relatively sensitive, some samples in the first round of PCR could not be amplified. In mixing experiments, mispriming of the MUT primer made relative determination of quantities subjective and difficult. Differential hybridization of PCR product with probes specific for codon 215 MUT or WT sequences was also investigated. A probe directed to a highly conserved region of the RT gene in the amplified PCR product was used to determine the total amount of PCR product analyzed. Differential hybridization was linear and reproducible over several logs of MUT:WT ratios, and determination of a 1:100 ratio of MUT:WT was readily achieved. When applied to longitudinal samples from three patients, dramatic changes in each population were readily apparent. These changes were evaluated with regard to viral load.

The specificity of pilin DNA sequences for the detection of pathogenic Neisseria

A nucleic acid hybridization assay for the detection of the pilin gene of Neisseria gonorrhoeae has been devised. The method involves solution hybridization of pilin specific synthetic oligonucleotide probes to genomic DNA in crude cell lysates. This is followed by capture of the probe-target complex onto a microtitre dish well, signal amplification and labelling based on horseradish peroxidase conjugated to oligonucleotides. Detection is achieved with a chemiluminescent enzyme substrate. With a detection limit of about 20,000 cells, the 4-h assay is as sensitive as a radioactive dot-blot method. Over 150 strains of Neisseria gonorrhoeae collected from a variety of sources were detected with the assay. Several N. meningitidis serogroups were also found to react positively. No reactivity was observed with non-pathogenic Neisseria spp. or with other known pathogenic or normal microbial inhabitants of the human urogenital tract.