Medical Device Link.

Originally Published IVD Technology November/December 2004

The Rapid Capture system by Digene Corp. (Gaithersburg, MD) is an automated platform that has been approved by FDA for hybrid capture testing.

More than 4.5 million women between the ages of 18 and 50 report at least one chronic gynecological condition each year.¹ Human immunodeficiency virus (HIV), human papillomavirus (HPV), Chlamydia trachomatis, Neisseria gonorrhoeae, and herpes simplex virus (HSV) are among the most common sexually transmitted disease (STD) pathogens. Although most STD pathogens can be detected using traditional methods such as culture or serology, nucleic acid tests offer improved accuracy in diagnosing active infections. This article will focus on noninvasive molecular diagnostic methods for detecting important bacterial and viral diseases in the female lower genital tract.

Some early nucleic acid methods include Southern blotting, in situ hybridization (ISH), and solution hybridization. While such tests demonstrated considerable progress when they were first introduced, they were time consuming and lacked adequate clinical sensitivity. Nonisotopic nucleic acid tests developed in the late 1970s were even less sensitive than the isotopic assays.

A significant breakthrough in nucleic acid testing was the development of target and signal amplification methods that improved sensitivity and specificity. Tests based on enzymatic target amplification, such as polymerase chain reaction (PCR), increase target molecules to levels that can be easily detected using various reporter systems. Signal amplification methods enhance the signal without changing the number of target molecules. Such methods can involve amplified immunostaining detection systems using labeled antibodies or other affinity pairs, special labeled branched probes, or antihybrid alkaline phosphatase labeled antibodies (see Table I).

Clinical Utility and Test Validation

Table I. Target and signal amplification nucleic acid test features (click to enlarge).

For women and their families to benefit from nucleic acid tests, medical professionals, laboratories, and IVD manufacturers must ensure that tests are validated as safe, clinically effective, and reproducible. Health professionals should especially consider and be aware of the difference between the analytical and clinical performance characteristics of nucleic acid tests, which are often misinterpreted.

For example, the most analytically sensitive tests are required for detecting many pathogens such as C. trachomatis and N. gonorrhoeae. Due to the asymptomatic nature of such infections and their easily treatable nature, detecting any organism has been presumed to equal the presence of disease. Such considerations have driven testing requirements for C. trachomatis and N. gonorrhoeae to ultrasensitive levels, while the relatively low prevalence of these pathogens in the general population (<5%) necessitates greater test specificity.

In contrast, the natural history of HPV infection is complex, and only a small subpopulation of women infected by carcinogenic types of HPV progress to cancer. HPV treatments are costly and invasive, making it critical to avoid overtreatment. Certain nucleic acid tests with very high analytical sensitivity for HPV DNA, such as most PCR tests, are unsuitable or suboptimal for clinical use because they detect many transient HPV infections. Cervical cancer screening requires a careful balance of HPV test sensitivity and specificity that is correlated to disease and not merely detection of virus. Validating clinical sensitivity and specificity of each new HPV nucleic acid test requires exfoliated cervical cell specimens from women with at least 150 corresponding biopsies with confirmed high-grade cervical intraepithelial neoplasia (CIN) and cancer from a representative population of several thousand women.

Another important factor in test validation is determining the appropriateness of the probes used for detecting the target pathogens. With more than 100 HPV types and a large number of C. trachomatis and N. gonorrhoeae serotypes, auxotypes, and variants, the probes selected must demonstrate generalizable sensitivity and specificity for disease in the intended use populations. Such proper probe selection is essential since ethnic and demographic differences can influence the prevalence of variants and disease effects. The possibility that common genetic alterations, such as deletions or mutations, could negatively affect nucleic acid tests must also be considered. For example, deletion of the HPV L1 region is a common occurrence in high-grade CIN lesions and cervical cancers, and can lead to false-negative results for amplified primer systems directed to this region.

Figure 1. Human papillomavirus structure.

IVD manufacturers are legally and ethically responsible for validating the performance of their nucleic acid tests. Manufacturers must conduct extensive studies to confirm analytical sensitivity, specificity, reproducibility, and stability, as well as carefully designed multicenter prospective studies to demonstrate safety and clinical effectiveness. In addition, manufacturers must establish procedures that assure high-quality production and long-term reproducibility of test reagents.

Clinical testing laboratories are also responsible for validating the tests, particularly when implementing home-brew methods, using analyte specific reagents (ASRs), or deviating from an IVD manufacturer’s recommended procedures. Laboratories must maintain rigorous training and quality control procedures, clear documentation, and monitoring systems to ensure high-quality results.

Moreover, IVD manufacturers must educate physicians about correctly interpreting molecular test results within the context of the stated performance characteristics. A recent article discusses the negative impact of overinterpreting nucleic acid amplification test results without considering test limitations.² In one clinical setting, five false-positive N. gonorrhoeae results were reported based on PCR tests that were not confirmed using another nucleic acid amplification test or culture. The manufacturer made such recommendations due to the poor positive predictive value in low-prevalence populations.

Human Papillomavirus

Figure 2. Papanicolaou smear.

High-risk human papillomaviruses are the causal agents for CIN and cervical carcinoma (see Figure 1). Since the recognition of cancerous cells in cervical scrapes preceded the discovery of carcinogenic types of HPV by more than 40 years, cytological screening for cancer using the Papanicolaou (Pap) technique has been the standard of care in developed countries (see Figure 2).³ However, the Pap smear test is limited by poor sensitivity and subjectivity of interpretation. As a result, 20–50% of invasive cervical cancers remain undetected in women who have been adequately screened.⁴

Because no method for growing HPV in culture exists, and immunological methods are inadequate, clinical detection of HPV is based exclusively on nucleic acid techniques. The current methods are ISH, PCR, and hybrid capture.

Commercially available ISH kits have been developed by Ventana Medical Systems Inc. (Tucson, AZ), Kreatech Biotechnology (Amsterdam, The Netherlands), and DakoCytomation (Glostrup, Denmark). ISH methods are applied to cervical biopsies or Pap smear samples and use labeled DNA probes with various detection systems. The results are evaluated using light microscopy and are useful for observing the presence of HPV DNA and cellular dysplastic features in the same preparation. However, the main disadvantage of ISH is poor sensitivity (60–75%) for high-grade CIN and cervical cancer.⁵ FDA has not validated and approved any ISH tests for clinical use.

The hybrid capture 2 (hc2) test by Digene Corp. (Gaithersburg, MD), a signal amplification technology, detects the 13 most common carcinogenic types of HPV. In several international clinical studies, hc2 demonstrated a 95% or better clinical sensitivity for biopsy-proven high-grade CIN and cancer.^6-7 Comparisons between the sensitivity of cytology alone and cytology with hc2 are shown (see Figure 3). The hc2 test is the only FDA-approved HPV test for triage of atypical squamous cells of undetermined significance cytology results and as a primary screening test in conjunction with Pap smears for women who are 30 years of age and older. An automated platform, the Rapid Capture system, has been developed and has been approved by FDA for hc2 testing. This system allows screening of up to 352 samples in a single laboratory shift.

PCR methods for HPV detection, genotyping, and quantitation of viral load employ consensus or type-specific primers and various signaling systems. HPV genotyping with PCR offers researchers a useful tool for epidemiological studies and evaluating HPV vaccines. Real-time PCR provides viral load results from reaction curves generated by monitoring PCR reaction kinetics in real time. Some recent studies comparing PCR and hybrid capture for detecting HPV showed good agreement. However, PCR may produce false-positives if the detection limit is set too low. PCR may also miss true infections if specimens contain inhibitors or the L1 region of the virus is deleted. PCR methods for detecting HPV are currently for research use only, and their clinical performance as a cervical cancer screening application has not been validated.

With the availability of sensitive and specific HPV detection methods, new research has focused on triage assays that can provide accurate prognostic information for patients who are HPV positive. For example, multiple ongoing studies are evaluating protein biomarkers that are overexpressed as a result of HPV infection, such as cell cycle regulatory or proliferative proteins (i.e., p16, Ki-67).⁸

Chlamydia trachomatis and Neisseria gonorrhoeae

The Centers for Disease Control and Prevention (Atlanta) recommend screening for C. trachomatis and N. gonorrhoeae for sexually active women less than 25 years old and for all women with risk factors such as new or multiple sexual partners.⁹ Traditional detection methods are based on culture in McCoy cells for C. trachomatis and selective agar media for N. gonorrhoeae. While such methods provide good sensitivity and specificity, C. trachomatis culture in particular is complex and time consuming.

Table II. Commercially available nucleic acid tests for detecting C. trachomatis and N. gonorrhoeae (click to enlarge).

Several molecular diagnostic tests are used for the rapid and accurate detection of C. trachomatis and N. gonorrhoeae (see Table II). Such tests can be divided into three categories: non-amplified probe hybridization tests, signal amplification tests, and nucleic acid amplification tests.

The Pace 2 system by Gen-Probe Inc. (San Diego) is one type of probe hybridization test that relies on chemiluminescent detection of ribosomal RNA through the use of labeled DNA probes. This test is used for testing endocervical specimens. In general, non-amplified probe hybridization tests are easy to use and are quite robust. However, they lack the sensitivity of nucleic acid amplification tests and appear to be even less sensitive than traditional culture, which precludes their use with certain specimens such as urine.¹⁰ The non-amplified probe tests are gradually being replaced by the more sensitive tests.

Digene’s hc2 test for detecting C. trachomatis and N. gonorrhoeae employs the same signal amplification-based technology used for the hc2 HPV test. The hc2 test has been validated on the Rapid Capture system instrument for detecting C. trachomatis and N. gonorrhoeae in endocervical specimens, but not in urine.

Each of the three primary nucleic acid amplification tests for C. trachomatis and N. gonorrhoeae detection utilize a slightly different technology platform. The Cobas Amplicor system by Roche Molecular Diagnostics (Pleasanton, CA) relies on PCR to amplify specific DNA regions of the target sequence. The products of this reaction are then hybridized with oligonucleotide probes that can be detected colorimetrically. The Aptima Combo 2 by Gen-Probe utilizes transcription mediated amplification to replicate specific regions of ribosomal RNA found in C. trachomatis and N. gonorrhoeae organisms. Such amplified products are then hybridized with oligonucleotide DNA probes and detected through a chemiluminescent reaction. The BD ProbeTec ET system by BD Diagnostics (Sparks, MD) uses strand displacement amplification, followed by a homogeneous assay format involving fluorescent energy transfer to detect the amplified products.

The analytical and clinical sensitivity of nucleic acid amplification tests is excellent. They can be used with a variety of clinical specimens, including female urine, and they provide a high degree of flexibility in obtaining noninvasive samples. However, nucleic acid amplification tests do have some drawbacks that must be considered. Such tests are more expensive than other technologies, are technically demanding, and require specialized areas in a laboratory to limit cross-contamination. The Cobas Amplicor system has been shown to cross-react with nonpathogenic isolates, such as Neisseria subflava and Neisseria cinerea, leading to a reduced positive predictive value in low-prevalence populations. Although nucleic acid amplification tests are also subject to false- negative results, many IVD manufacturers now provide an internal control to identify inhibitory specimens.

Herpes Simplex Virus

Early, accurate, and sensitive detection of HSV infection or recurrence is important, especially for pregnant women and immunosuppressed patients. This allows for optimal patient management, including antiviral therapy and counseling targeted to changing sexual practices.

Figure 3. Comparison of the sensitivity of cytology alone and cytology with hybrid capture for high-grade cervical disease and cancer (click to enlarge).

The most commonly used tests for HSV are based on serological methods. Serological tests such as the HerpeSelect ELISA and HerpeSelect Immunoblot by Focus Technologies (Herndon, VA), and the Cobas Core HSV-2 IgG EIA and Cobas Core Anti-HSV-I/II by Roche Molecular Diagnostics detect antibodies specific to either HSV-1 or HSV-2. Such assays are very reliable tests for the detection of primary infection. However, they cannot indicate when or where on the body the primary infection took place. Because it takes approximately three months to develop antibodies against either type of HSV, such tests could generate false-negative results if performed too soon after primary infection.¹¹

During the past few years, several target amplification methods have been developed for the detection and typing of HSV. Such methods employ duplex LightCycler PCR or real-time PCR and provide higher sensitivity compared with traditional tests.

Conclusion

Even as new and more-sophisticated nucleic acid technologies become available, many challenges remain. While matters of sexuality and reproductive health are less of a taboo than in the past, it is still too often the case that women are not aware of the range of diagnostic and treatment options. Women’s health organizations, medical associations, and the IVD industry should shoulder the educational burden to provide women with information, facts, and statistics. A recent U.S. national survey by the Kaiser Family Foundation showed that most adolescents and young adults learn about sexual health from friends and media, which are not reliable sources of such information. Open discussion of sexual health issues can help to alleviate the stigma associated with STDs.

Physician awareness of medical, technological, and socioeconomic aspects of women’s reproductive health should also be promoted in order to improve the overall quality of healthcare for women. In addition, all women, regardless of their economic situation, should have access to new diagnostic tools. This is especially important considering the high prevalence of STDs in poorer populations with limited access to comprehensive healthcare (see Figure 4).

Figure 4. The prevalence of chlamydia infection in different ethnic groups in young adults between 16 and 26 years old (Source: Kaiser Family Foundation Daily Reproductive Health Report, May 2004) (click to enlarge).

STDs have so many negative implications that even when people feel comfortable talking about sex, they often avoid this subject. Unfortunately, lack of communication and adequate knowledge may lead to otherwise avoidable tragedies. The problem does not concern only women’s health—it also affects their families, friends, and their social and professional environments. Knowledge, technology, and adequate resources are the solutions for improving the health and quality of life of all those at risk.

References

1. “Women’s Health in the U.S.,” National Institute of Allergy and Infectious Diseases Web site (Bethesda, MD: NIAID, 2004 [accessed 29 September 2004]); available from Internet: www.niaid.nih.gov/publications/ womenshealth/womenshealth.pdf.

2. LA Shrier et al., “Limitations of Screening Tests for the Detection of Chlamydia trachomatis in Asymptomatic Adolescent and Young Adult Women,” American Journal of Obstetrics and Gynecology 3, vol. 190 (2004): 654–662.

3. AT Lorincz and RM Richart, “Human Papillomavirus DNA Testing as an Adjunct to Cytology in Cervical Screening Programs,” Archives of Pathology & Laboratory Medicine 8, vol. 127 (2003): 959–968.

4. J Salmeron et al., “Comparison of HPV-Based Assays with Papanicolaou Smears for Cervical Cancer Screening in Morelos State, Mexico,” Cancer Causes & Control 6, vol. 14 (2003): 505.

5. AT Hesselink et al., “Comparison of Hybrid Capture 2 with In Situ Hybridization for the Detection of High-Risk Human Papillomavirus in Liquid-Based Cervical Samples,” Cancer 1, vol. 102 (2004): 11–18.

6. J Cuzick et al., “Management of Women Who Test Positive for High-Risk Types of Human Papillomavirus: The HART Study,” The Lancet 9399, vol. 362 (2003): 1871–1876.

7. L Kuhn et al., “Human Papillomavirus DNA Testing for Cervical Cancer Screening in Low-Resource Settings,” Journal of the National Cancer Institute 10, vol. 92 (2000): 818–825.
8. N Murphy et al., “p16INK4A as a Marker for Cervical Dyskaryosis: CIN and cGIN in Cervical Biopsies and ThinPrep Smears,” Journal of Clinical Pathology 1, vol. 56 (2003): 56–63.

9. “CDC Sexually Transmitted Diseases Treatment Guidelines,” Morbidity and Mortality Weekly Report RR-6, vol. 51 (2002).

10. B Van Der Pol et al., “Multicenter Evaluation of the BD ProbeTec ET System for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae in Urine Specimens, Female Endocervical Swabs, and Male Urethral Swabs,” Journal of Clinical Microbiology 3, vol. 39 (2001): 1008–1016.

11. A Wald and R Ashley-Morrow, “Serological Testing for Herpes Simplex Virus (HSV)-1 and HSV-2 Infection,” Clinical Infectious Diseases supp. 2, vol. 35 (2002): S173–S182.

Iwona Mielzynska, PhD, was formerly director of product development at Digene Corp. (Gaithersburg, MD) and is currently an independent consultant. Allison Cullen is vice president of product development, Kevin J. Modarress, PhD, is manager of product development, and Attila T. Lorincz, PhD, is senior vice president of research and development and chief scientific officer at Digene Corp. The authors can be reached at mielzynska@aol.com, allison.cullen @digene.com, kevin.modarress@ digene.com, and attila.lorincz@ digene.com, respectively.

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