Hybrid Capture Technology

The hybrid capture 2 (HC2) technology is the platform for signal-amplified, nucleic acid tests (for review, seeLorincz and Anthony, 2001). HC2 systems areavail-able to detect human papillomavirus (HPV), cytomegalovirus (CMV), Chlamydia trachomatis (CT), Neisseria gonorrhoeae (GC), and hepatitis B virus (HBV). An assay for herpes simplex virus (HSV) is in development.

Cervical cancer is one of the few malignancies for which the cause has been identified: the human papillomavirus, a small DNA tumor virus that belongs to the family Papovaviridae and is sexually transmitted (Schiffman, 2000; Munoz, 2003). There are more than 100 types of HPV. Low-risk types of HPV may cause genital warts. High-risk types have been shown to cause most cases of cervical cancer. The HC2 HPV test uses two RNA probe cocktails to differentiate between carcinogenic and low-risk HPV types. Thirteen types are implicated in the pathogenesis of High-Grade squamous intraepithelial lesion (HSIL) and invasive cancer: 16,18, 31, 33,35,39,45,51,52,56,58,59, and 68. Five probes detect low-risk viral types associated with Low-Grade squamous intraepithelial lesion (LSIL): 6, 11,42,43, and 44. This test is used to screen patients with ASCUS (atypical squamous cells of undermined significance). Pap smear results determine the need for referral to col-poscopy. HC2 HR HPV DNA test was initially approved for follow-up evaluation in women with inconclusive Pap-test results. It has a proven 99% negative predictive value (NPV) (Manos, 1999; Solomon, 2001). The negative predictive value of HPV DNA testing would be of particular significance in excluding HPV-associated dysplasias in postmenopausal women diagnosed with ASCUS (Manos, 1999).

The test was approved in 2003 by the U.S. Food and Drug Administration (FDA) for cervical cancer screening, in conjunction with a Pap test, in women age 30 and older. The HC2 HR HPV DNA test (marketed as the DNAwithPAP) became Digene's flagship product. Specimens containing the target DNA hybridize with a specific HPV RNA probe cocktail. An RLU measurement equal to or greater than the cutoff value (CO) indicates the presence of HR HPV DNA sequences in the specimen. A study (Kulmala, 2004) comparing performance of the hybrid capture 2 assay and polymerase chain reaction (PCR) in screening HPV did not find much difference between the two. The results of PCR and the HC2 assay were concordant for 85% of samples, resulting in substantial reproducibility. Hybrid capture 2 has been shown to have similar analytic sensitivity to some PCR methods for HPV DNA detection (Clavel, 1998; Peyton, 1998).

Rapidly emerging as a standard of practice in cervical cancer screening, the HPV test helps clinical diagnosis of women who are most at risk of having or developing cervical cancer. In addition, one sample collected and rinsed in Cytyc's ThinPrep Pap Test vial Cytyo Corp., Marlborough, MA can be used for both the Pap test and the HPV test. Another liquid-based method, AutoCyte PREP TriPath Imaging, Inc., Burlington, NC, is expected to be used for the same purpose. HPV, chlamydia, and gonorrhea testing can be performed using one sample. Cervical specimens are collected with a broom collection device and rinsed in the ThinPrep System PreservCyt solution with the Digene Cervical Sampler. The Digene Sample Conversion Kit is used to allow the HPV DNA test to be performed on the same specimen that the ThinPrep Pap Test is performed on. In addition, cervical biopsies are collected in Digene Specimen Transport Medium.

Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC) are the most common bacterial infections of the lower genital tract. The CT/GC Probe Cocktail contains a probe mixture specifically chosen to eliminate or minimize cross-reactivity with DNA sequences from human cells, other bacterial species, Chlamydia species other than CT, or GC. The CT/GC Probe Cocktail supplied with the hc2 CT/GC DNA test is complementary to approximately 4% of the CT genomic DNA (1 x 106 bp) and 7500 bp or 100% of the cryptic plasmid; and 0.5% of the GC genomic DNA. A specimen positive by the HC2 CT/GC DNA test must be tested by HC2 CT-ID DNA test or HC2 GC-ID DNA test or another method to verify organism detection. The Digene CT/GC, CT-ID, and GC-ID tests are designed for the detection of CT and GC from cervical specimens collected using the Digene Cervical Sampler or from urine specimens processed using the Digene Urine Prep Kit for male specimens. The Digene Urine Preparation Kit for male specimens is required to process male urine specimens for use with any of the Digene CT/GC tests (Girdner, 1999; Schachter, 1999; Dawin, 2002). It is recommended that positive results be confirmed by another method if the likelihood of N. gonorrhoeae or C. trachomatis infection is uncertain or questioned. Analytical sensitivity of the HC2 CT/GC DNA test to detect Chlamydia ranges from 50 to 2500 CFUs/assay (1000 to 50,000 CFUs/mL). The lower limit of detection for GC isolates ranges from 25 to 5000 CFUs/assay (500 to 100,000 CFUs/mL).

HC2 CMV DNA test is the first molecular diagnostic test to be FDA cleared for the qualitative detection of human cytomegalovirus DNA in peripheral white blood cells isolated from whole blood (Mazzulli, 1999). Active CMV infection in immunosuppressed and immunocompromised patients, such as solid organ transplant, bone marrow transplant, and HIV-positive/AIDS patients, can be detected more accurately. Analytical studies using cloned HPV plasmid DNA demonstrated that the assay using high-risk probe could detect these types at levels ranging from 0.62 pg/mL to 1.39 pg/mL. Analytic sensitivity for CMV is

0.48 pg/mL. The RNA probe for CMV is about 40,000 bp, about 17% of the CMV genome.

The HBV DNA test is a signal amplification test to quantify hepatitis B viral DNA in human serum. The test detects HBV ad and ay subtypes. Standard test dynamic range is 1.42 x 105 to 1.7 x 109 copies/mL (0.5 to 6000 pg/mL) using a sample volume of 30 mL. Ultrasensitive dynamic range is 4.7 x 103 to 5.6 x 107 copies/mL (0.017 to 200 pg/mL) using a sample volume of 1 mL, which is concentrated by centrifugation. The Digene HC2 assay and the PCR assay had similar intra-assay and inter-assay variabilities. For the patients with HBV DNA levels detectable by the HC2 assay, the HBV DNA levels obtained by the HC2 assay and by the PCR assay showed an excellent correlation. The PCR assay was more sensitive than the HC2 assay and more suitable for monitoring low levels of HBV viremia (Yuan, 2004; Konnick, 2005).

Rapid capture system (RCS), a semiautomated pipetting and dilution system, provides high-volume labs with the ability to run high-risk HPV, CT and GC, and HBV HC2 tests. Automation of RCS does not include sample denaturation, as well as the chemiluminescent signal detection and result reporting that are performed using the microplate luminometer system (DML 2000 Instrument, Digene, Gaithersburg, MD). This system handles up to 352 specimens (4 microplates) in 8 h. Thus, the HC2 system offers multiple testing using a single platform. A single sample can be tested for HPV, CT and GC, and, in the future, HIV-1 and HSV, a test currently under development (Cullen, 1997). This system provides a comprehensive risk-screening approach based on one patient visit.

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