Molecular diagnostics is impacting nearly every discipline in the laboratory. Molecular infectious disease testing has revolutionized the diagnosis of viral infections, sexually transmitted infections and emerging pathogens. This process has been felt in a wide variety of patient populations, including HIV-infected individuals, pediatrics, transplant recipients and women. This article focuses on the impact of molecular diagnostics on women's health, including public health indicators, molecular testing for human papillomavirus, Neisseria gonorrhoeae and Chlamydia trachomatis, bacterial vaginosis, and evolving tools to identify highrisk patients.
Sexually transmitted infections (STIs) are an unfortunate fact of life. While the numbers of certain organisms, particularly NG and CT temporarily dipped due to increased use of condoms at the onset of the AIDS epidemic, they continue to maintain a strong presence. With more than 3 million cases of CT and 700,000-plus cases of NG annually, CT and NG are now the most common reported STIs worldwide. Now included in the plethora of infections gaining more laboratory attention are Trichomonas vaginalis, Ureaplasma urealyticum, Mycoplasma genitalium and Mycoplasma hominis.
Traditional Diagnostic Approaches
On the women's health timeline, the "molecular diagnoses" of NG and CT have been around the longest. Traditional microbial identification methods typically relied on phenotypes, such as morphologic features, growth variables and biochemical utilization of organic substrates. Under reported and not easily detected, mucopurulent cervicitis (leukorrhoea) was a frequent but nonspecific, clinical presentation. Phenotypic identification by Gram stain and cultures for NG became routine. Because of morphologic overlap, Gram stains for NG on endocervical discharge were contraindicated as phenotypic constructs could be confused with that of NG, resulting in false-positive results.
Early in the 1980s the surprising prevalence of CT in the sexually active population of the United States became apparent. Not only was CT as prevalent as NG, but it also could lead to pelvic inflammatory disease, infant pneumonia, conjunctivitis, ectopic pregnancy, urethritis/urinary tract infections and infertility. While morphology was a traditional approach for NG, CT was more problematic as it is an obligate intracellular pathogen. With the understanding of these potentially serious outcomes, particularly in women for whom the disease was often asymptomatic, the wider utilization of diagnostic tests gained a new urgency.
The introduction of nucleic acid detection methods revolutionized diagnosis of both CT and NG. The GenProbe (now Hologic Inc.) PACE System included non-amplified DNA probe assays for CT and GC. First introduced in 1988, the PACE was fairly easy to implement in routine clinical laboratories. Designed for endocervical, male urethral and conjunctival swab specimens, this molecular method involved a hybridization step and subsequent signal amplification developed to selectively detect ribosomal nucleic acid (rRNA), present in more copies per cell. The assay was capable of slightly higher sensitivity than detection of a single chromosomal target. While the original sensitivity was relatively low compared to a reference standard (i.e., culture), this technology played an important role in establishing molecular methods as a more rapid alternative to culture.
There are now several commercially available direct detection and culture identification nucleic probes that have been cleared by the U.S. Food and Drug Administration. The procedures for the use of DNA probes are now well standardized, and the advent of synthetic short oligonucleotide DNA probes has shortened the time required for probe assay.
Despite the ease of use and popularity of the DNA probe approach, nucleic acid analysis without amplification often has the disadvantage of low sensitivity (high detection limits). Nucleic acid amplification techniques increase sensitivity dramatically while retaining a high specificity. Polymerase chain reaction (PCR) is the best-developed and most widely used method of nucleic acid amplification. PCR is based on the ability of DNA polymerase to copy a strand of DNA by elongation of complementary strands initiated from a pair of closely spaced chemically synthesized oligonucleotide primers.
The first benchtop system to fully automate amplification and detection for PCR testing, the COBAS AMPLICOR CT/NG Test (Roche) incorporated PCR in a multiplex assay that allowed the simultaneous amplification using biotinylated primer pairs specific for NG and CT, capture of the bound amplification products using magnetic particles labeled with specific oligonucleotide probes, and detecting the bound products colorimetrically. The first commercial PCR assay approved by the FDA, implementation of the technology into clinical laboratories was rapid.
Testing methodologies and automation have improved testing for CT/NG. Sensitivity and specificity have been improved while cross contamination has decreased. The batch-based testing approach of most modern formats creates a real risk of internal or specimen-handling based contamination, leading to false positive results. Reporting a false-positive result for any sexually-transmitted infection can have ruinous consequences, both for the patient and for the patient's contacts. Alternately, other confounding factors, such as inhibition (due to blood, mucin and feminine hygiene products), can result in false-negative results. With the incorporation of internal process controls, manufacturers are moving toward correcting this problem (ProbeTec Qx Amplified DNA assay, BD, GenProbe Aptima assay). This has helped these become what many feel is the standard of the industry today.
Laboratories now have choices for testing (BD ProbeTec [Becton Dickinson], APTIMA COMBO 2 Assay [Hologic GenProbe], Infiniti CT/NG [Autogenomics], Abbott RealTime CT/NG, cobas 4900 CT/NG [Roche]). Next generation testing with specific, sensitive and rapid detection using novel stable genetic targets for CT/NG allows laboratories to incorporate testing with ease and confidence. Adopting these approaches, in addition to on demand testing versus batch and minimal manipulation is of paramount importance (Xpert CT/NG, Cepheid).
CT/NG testing is only part of the sexually transmitted disease problem. Half of all non-gonococcal venereal diseases are caused by Ureaplasma (U. urealyticum) and Mycoplasma (M. genitalium, M. hominis). These pathogens can be found in 70% of sexually active humans. These are often not detected in plate culture and are difficult to identify under microscope. These are acute infections in women causing diffuse, malodorous, yellow-green vaginal discharge with vulvar irritation, pelvic inflammatory disease (PID), urethritis (NGU), cervicitis, endometriosis and pyelonephritis. Of concern are post-infection complications, which include infertility, premature labor, low birth weight, chorioamnionitis, pre-rupture of the membranes (PROM), and post-partum complications.
Another issue, vaginitis, accounts for more outpatient visits for women than any other reason. Vaginitis, in?ammation of the vaginal wall caused by one or more of these infections presents with a spectrum of vulvovaginal symptoms including itching, burning, irritation and abnormal discharge. Organisms causing vaginitis include vulvovaginal candidiasis (yeast) (Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis), bacterial vaginosis (Bacteroides fragilis, Gardenerella vaginalis, Mobiluncus mulieris, Mobiluncus curtisii, Atopobium vaginae, Prevotella bivia), and Trichomoniasis vaginalis. While traditional testing for causation is either performed in physician laboratories, or not at all in some instances, the infections result in reduced quality of life, primary care resources, HIV transmission, infertility, early pregnancy loss, premature labor and delivery and neonatal intensive care. Office-based molecular testing for vaginitis include a molecular probe assay (Affirm VPIII [BD]) to test for G. vaginalis, T. vaginalis and C. albicans. Newer amplification assays that incorporate all of the benefits of molecular testing are gaining popularity for these causative pathogens (i.e., Infiniti STD 6-Quad, Autogenomics).
Ongoing HPV Concerns
Viral infections, such as the human papillomavirus (HPV), are also mainstream in the clinical laboratory. HPV is a common cause of dysplasia, intraepithelial neoplasia and carcinoma in the female genital tract. Cervical cancer is second only to breast cancer as most frequent female malignancy and cause of cancer death. Certain HPV types, such as types 16 and 18, have been regarded as high-risk cancer-associated HPVs, whereas types 6 and 11 are regarded as low-risk HPVs. Use of DNA hybridization assays in cervical swabs or fresh cervical biopsy specimens to determine HPV infection and viral types has provided helpful information for clinical assessment and treatment of patients. Guidelines for HPV testing have been established. Recently, the U.S. Preventive Services Task Force (USPSTF) recommended that the HPV test is appropriate for some women as part of routine cervical cancer screening.1 This study determined that HPV testing can reliably detect cervical cancer better than the Pap smear. The identification of HPV type and persistence of infection are key determinants in progression monitoring. Laboratory testing for HPV is routine. Several manufacturers have kept up with the increasing need for differentiating between high risk and in some cases, genotyping (cobas 4800 [Roche], Cervista HPV HR, Gen-Probe APTIMA-HPV Assay [Hologic] and digene HC2 HPV DNA Test (Qiagen) ). The FDA has approved the majority of these HPV assays for clinical laboratory diagnostics.
More problematic, of course, are women who test positive for a high-risk genotype by molecular methods, yet are negative by Pap smear. Are these women on the path to developing cervical cancer? Newer laboratory assays have shown that a gene named TERC may help physicians recognize molecular changes to cervical cells that increase the probability of cervical cancer in women. Molecular based methods are able to target TERC and identify changes that would predict malignancy (Figure 1). Information gained will enable physicians to detect women who are more likely to progress to a cancerous state without treatment.. Women with high risk results may benefit from additional cervical biopsies at colposcopy and more aggressive observation and treatment, while women with low-risk results may undergo lesser follow-up procedures.
All in all, molecular testing is here to stay. Infectious disease testing makes up more than half of overall molecular diagnostic market revenues. Outside of CT/NG and HPV, laboratories have been slow to expand for women's health testing. The rapidly changing and increasingly competitive landscape of diagnostics continues to stimulate demand for molecular testing. Women's health will ultimately benefit.
Dr. Lowery-Nordberg is director of molecular diagnostics at Delta Pathology in Shreveport, La., and professor of medicine and pediatrics at Louisiana State University Health in Shreveport.
- Ann Intern Med. 19 June 2012;156(12):880-891