Daniel P. Kolk

Highly Sensitive Multiplex Assay for Detection of Human Immunodeficiency Virus Type 1 and Hepatitis C Virus RNA

ABSTRACT Various nucleic acid assays have been developed and implemented for diagnostics and therapeutic monitoring of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) infections. The high-throughput, semiautomated assays described here were developed to provide a method suitable for screening plasma specimens for the presence of HIV-1 and HCV RNAs. Three assays were developed: a multiplex HIV-1/HCV assay for simultaneous detection of HIV-1 and HCV, and discriminatory assays for specific detection of HIV-1 and HCV. The assay systems utilize three proprietary technologies: (i) target capture-based sample preparation, (ii) transcription-mediated amplification (TMA), and (iii) hybridization protection assay (HPA). An internal control is incorporated into each reaction to control for every step of the assay and identify random false-negative reactions. The assays demonstrated a sensitivity of at least 100 copies/ml for each target, and they detected with similar sensitivity all major variants of HCV and HIV-1, including HIV-1 group O strains. Assay sensitivity for one virus was not affected by the presence of the other. The specificity of these TMA-driven assays was ≥99.5% in both normal donor specimens and plasma containing potentially interfering substances or other blood-borne pathogens. Statistical receiver operating characteristic plots of 1 − specificity versus sensitivity data determined very wide analyte cutoff values for each assay at the point at which the assay specificity and sensitivity were both ≥99.5%. The sensitivity, specificity, and throughput capability predict that these assays will be valuable for large-volume plasma screening, either in a blood bank setting or in other diagnostic applications.

Dengue viremia in blood donors from Honduras, Brazil, and Australia.

BACKGROUND: Dengue fever and hemorrhagic disease are caused by four dengue virus (DENV) serotypes (DENV‐1 to ‐4), mosquito‐borne flaviviruses with increasing incidence, and expanding global distributions. Documented transfusion transmission of West Nile virus raised concern regarding transfusion‐transmitted DENV.

A cross-sectional study of a prototype carcinogenic human papillomavirus E6/E7 messenger RNA assay for detection of cervical precancer and cancer.

Purpose: To evaluate carcinogenic human papillomavirus (HPV) mRNA for E6 and E7 mRNA detection on clinical specimens to identify women with cervical precancer and cancer. Experimental Design: We evaluated a prototype assay that collectively detects oncogenes E6/E7 mRNA for 14 carcinogenic HPV genotypes on a sample of liquid cytology specimens (n = 531), masked to clinical data and to the presence of HPV genotypes detected by PGMY09/11 L1 consensus primer PCR assay. Results: We found an increasing likelihood of testing positive for carcinogenic HPV E6/E7 mRNA with increasing severity of cytology (PTrend < 0.0001) and histology (PTrend < 0.0001), with 94% of cervical intraepithelial neoplasia grade 3 (CIN3) histology cases (46 of 49) and all five cancer cases testing positive for carcinogenic HPV E6/E7 mRNA. Overall, fewer specimens tested positive for carcinogenic HPV E6/E7 mRNA than for carcinogenic HPV DNA (P < 0.0001, McNemars χ2 test), especially in women with <CIN1 (P < 0.0001). We also found that using a higher positive cutpoint for detection of carcinogenic HPV E6/E7 mRNA improved the association of positive test results with cervical precancer and cancer by reducing the number of test positives in women without precancer without reducing clinical sensitivity for cervical precancer and cancer compared with detection of carcinogenic HPV E6/E7 mRNA using a lower positive cutpoint by the same assay and with detection of carcinogenic HPV DNA. Conclusions: We found that carcinogenic HPV E6/E7 mRNA is a potentially useful biomarker for detection of cervical precancer and cancer and warrants further evaluation.

Significant Closure of the Human Immunodeficiency Virus Type 1 and Hepatitis C Virus Preseroconversion Detection Windows with a Transcription-Mediated-Amplification-Driven Assay

ABSTRACT While the present generation of serology-based assays has significantly decreased the number of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) infections acquired by transfusion, the possibility of infected donations escaping detection still exists. The average seronegative viremic window duration during which immunological assays are unable to detect the virus is estimated to be between 16 and 22 days for HIV-1 and approximately 70 days for HCV. Significant reduction of detection window duration was demonstrated using a nucleic acid amplification assay, the Procleix HIV-1/HCV Assay, which utilizes transcription-mediated amplification technology to simultaneously detect HIV-1 and HCV RNAs. For 26 commercially available HIV-1 seroconversion panels tested, specimens were reactive in the HIV-1/HCV assay at the same time as or earlier than in serological assays. Overall, the HIV-1/HCV assay was able to reduce the detection window duration by an average of 14 days and 6 days compared to tests relying on recognition of HIV-1 antibody and p24 antigen, respectively. For 24 commercially available HCV seroconversion panels tested, the specimens were reactive in the HIV-1/HCV assay at an earlier blood sampling date than in serological assays, reducing the detection window duration by an average of 26 days. Similar results were obtained in testing the HIV-1 and HCV seroconversion panels in the virus-specific HIV-1- and HCV-discriminatory assays, respectively. In conclusion, the HIV-1/HCV assay and corresponding discriminatory assays significantly reduced detection window durations compared to immunoassays.

High Throughput Assay for the Simultaneous or Separate Detection of Human Immunodeficiency Virus (HIV) and Hepatitis Type C Virus (HCV)

© 1998 S. Karger GmbH, Freiburg Fax (07 61) 4 52 07 14 www.karger.com Received: December 2, 1997 Accepted: March 9, 1998 S. H. McDonough Gen-Probe Incorporated 10210 Genetic Center Drive San Diego, CA 92121 (USA) Fax +1 619 41 088 71 This article is also accessible online at: http://BioMedNet.com/karger High Throughput Assay for the Simultaneous or Separate Detection of Human Immunodeficiency Virus (HIV) and Hepatitis Type C Virus (HCV) S. H. McDonough C. Giachetti Y. Yang D. P. Kolk E. Billyard L. Mimms

Sensitive detection of genetic variants of HIV-1 and HCV with an HIV-1/HCV assay based on transcription-mediated amplification

This paper describes a comprehensive study of hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) genotype sensitivity of the transcription-mediated amplification (TMA)-based HIV-1/HCV assay, developed and manufactured by Gen-Probe Incorporated (San Diego, CA) for screening human plasma specimens in blood bank settings. The TMA HIV-1/HCV assay is a qualitative, in vitro nucleic acid testing system used for initial screening. HIV-1 and HCV discriminatory assays are used to distinguish between HIV-1 and HCV infection or co-infection. In this study, multiple unique specimens representing HCV genotypes 1-6 were tested at various dilutions. The results show that the TMA HIV-1/HCV assay and the TMA HCV discriminatory assay have similar HCV genotype sensitivity, as both assays detected all six genotypes at 100 copies/ml and nearly all replicates tested at 30 copies/ml. Similarly, numerous unique specimens representing HIV-1 group M subtypes (A-G), HIV-1 group N, and group O specimens were tested at various dilutions. The TMA HIV-1/HCV assay and the TMA HIV-1 discriminatory assay were found to have similar HIV-1 subtype sensitivity; all variants at 100 copies/ml and nearly all at 30 copies/ml were detected. These results indicate that the TMA assays meet the sensitivity requirements for blood screening in blood banks worldwide.