MEDICAL DIAGNOSTIC LABORATORIES, L.L.C. Leukorrhea Panel Chlamydia trachomatis, ,

The for C. trachomatis is 6-14 days. In Chlamydia trachomatis women, the primary site of infection is the ; specifically, EPIDEMIOLOGY columnar cells in the transitional zone and the endocervix. For this reason, it will not cause infections of the ectocervix Chlamydia is the most or vaginal walls. Cervical ectopy, or abnormal positioning common bacterial sexually of the cervix, can predispose one to chlamydial infection transmitted disease in the by exposing susceptible columnar epithelial cells. It is world with an estimated 90 estimated that 60% to 80% of sexually active adolescent million new cases annually females have cervical ectopy (1). This phenomenon may (1). It is the number one also account for the association between oral contraceptive reported disease in the use and chlamydia since women taking oral contraceptives United States (5). An estimated 3 million new cases occur are prone to cervical ectopy (1). each year in the U.S. alone (4). Rates of infection in the U.S. have steadily increased in the last 15 years. 1998- 2002 saw a higher incidence in the southern portion of the CLINICAL SIGNIFICANCE U.S. than in other regions (5). The highest incidence was in It is estimated that 80% of all infected individuals are adolescents and young adults, 15 to 24 years of age (4). In asymptomatic (3). Due to the lack of signs or symptoms 2004, a national study conducted from the University of North in some patients, it is believed that of all infected Carolina at Chapel Hill determined that 4.9% of young adults individuals, 25% of men and 40% of infected women in the U.S. were infected with chlamydia (15). Risk factors will go undiagnosed (6). Women who are symptomatic for infection include low socioeconomic status, young age, may experience abnormal or a burning non-white race, unmarried, multiple sex partners, new sex sensation when urinating. If infection progresses into the partner, history of infection, and lack of barrier upper genital tract, they may experience lower abdominal contraceptive devices. pain, lower back pain, painful intercourse, nausea, fever, or bleeding between menstrual cycles. The appearance of PATHOGENESIS an infected cervix can vary greatly, from completely normal upon examination, to the presence of severe hypertrophic Chlamydia trachomatis, the cause of the sexually transmitted erosions and mucopurulent endocervical discharge. disease chlamydia, is an obligate intracellular parasite. This organism is unique, in that it is similar to both and In women, the most common manifestation of infection viruses. It is similar to bacteria because it contains RNA and is mucopurulent . It is estimated that 20% of DNA and has a rigid cell wall. It is similar to viruses because women with mucopurulent cervicitis will harbor Chlamydia it is an obligate intracellular parasite that depends on the trachomatis (14). Lack of proper treatment of infection hosts cells for nutrients and energy. Much of the fibrosis and of the lower genital tract can progress to upper genital scarring associated with C. trachomatis infection is thought to tract infection and pelvic inflammatory disease (PID), be due, in large part, by a cell-mediated immune response. as well as other very serious complications, such as, Chlamydial heat-shock proteins (HSPS) share antigens with tubal factor , ectopic , chronic pelvic other bacterial and human HSPS. Therefore, the immune pain, lymphogranuloma venereum (LGV), , response cannot only target chlamydia, but host cells and , and urethritis. Chlamydia infection can also tissues as well. The cytokine production associated with C. increase the risk for acquisition of HIV (2). trachomatis is one that is delayed and prolonged, as opposed to the quick short production associated with other pathogens. In pregnant women, chlamydia can have very serious Both of these characteristics, in combination with the often consequences including preterm delivery, low birth persistent infections of chlamydia, may account for many of weight, premature rupture of the membranes (PROM), the severe complications, including (1). and postpartum endometritis. There is a 60% to 70% risk of transmission of chlamydia to a neonate if it passes through the birth canal of an infected mother (1). It is antibody is used to stain the system for the evaluation estimated that 25% to 50% of exposed infants will develop of characteristic intracytoplasmic inclusions containing inclusion within two weeks of birth and 10% to elementary and reticulate bodies of C. trachomatis. There 20% will develop within 3 to 4 months of birth are certain disadvantages to this technique. Specimen (1). Chlamydia trachomatis is implicated in 20% to 60% viability is greatly affected by collection techniques and of all during the first six months of life (14). transport conditions. Results may take anywhere from 3 to Transmission is not known to occur in-utero. Therefore, 7 days depending on growth. Although specificity is nearly neonates delivered by C-section are not at risk unless there 100%, reported sensitivity is as low as 70% to 85% (3). is premature rupture of the membranes. Therefore, culture is not a good screening test.

In men, symptoms include difficulty urinating, pain or burning Point-of-care tests have recently been introduced. They can upon urination, penile discharge, testicular tenderness, and typically be performed within 30 minutes in the physician’s redness or swelling of the urethral opening at the tip of office. In some settings, such as in clinics, it may be useful the penis. Complications can include prostatitis, proctitis, to have an immediate answer if it is anticipated that the epididymitis, sterility, or narrowing (stricture) of the urethra patient may not return. These tests use antibodies that that may require surgery to correct. target lipopolysaccharides (LPS) that are found in all chlamydia species as well as other microorganisms that Some complications can occur in both men and women. could produce false-positive results (4). Point-of-care Lymphogranuloma venereum is a manifestation of tests have the lowest reported sensitivity and specificity Chlamydia trachomatis that primarily infects the lymph of any test available, 52% to 85% and 95% respectively system. Although cases do occur sporadically in the (3). These tests are generally not recommended due to U.S., it is usually associated with recent travel to endemic the lack of quality control as well as poor performance countries. Reiter’s syndrome consists of a classic triad of characteristics. urethritis, conjunctivitis, and reactive arthritis. Patients may also exhibit characteristic mucocutaneous lesions. Other Table 1. Sensitivities of Chlamydia trachomatis detection tests. sites of infection can include the throat (pharyngitis), rectum Method Sensitivity (proctatitis), and eye (conjunctivitis). Gram Stain 20% DIAGNOSIS Cell Culture 70% - 85% Laboratory diagnosis of chlamydia is crucial for definitive Point-of-Care Testing 52% - 85% diagnosis. Gram stain is not possible due to the lack of Enzyme immunoassay (EIA) 60% - 80% peptidoglycan in the cell wall. Cytological techniques consist Direct Fluorescent assay (DFA) 60% - 80% of Giemsa-stained cell scrapings that are visually inspected Pace-2 GenProbe 75% for cells with inclusion bodies. This technique is only useful in diagnosing inclusion conjunctivitis in newborns. Studies Hybrid Capture-II (HCII) Digene 75% Transcription Mediated Amplification show that only 20% of cervical infections are detected in this 89% manner (1). (TMA) GenProbe APTIMA 2 Strand Displacement Technology (SDA) 84% Serological methods have a very limited value when Becton Dickinson BDProbeTec investigating uncomplicated genital tract infection (4). Ligase Chain Reaction (LCR) 86% They are often inconclusive due to the long-lasting Abbott LCx immune response that may be present from a previous Real-Time PCR Technology 99% infection and the cross-reactivity with other bacteria including other chlamydia species. Therefore, serological Enzyme immunoassays (EIA) use immunohistochemical assays, such as the complement fixation test and the reactions to detect chlamydial antigens. However, microimmunofluorescence test are not appropriate specimen viability can be affected by collection techniques screening methods. and transport conditions. The reported sensitivity of this assay is approximately 60% to 80% (10, 11). Many of these Due to the fact that Chlamydia trachomatis is an obligate assays also target lipopolysaccharides (LPS) that are found intracellular parasite, it cannot be cultured on artificial in all chlamydia species as well as other microorganisms media. Cell culture techniques are commonly utilized. that could produce false-positive results (4). Therefore The specimen is introduced into a susceptible cell line in a specificity is low as well. They require either blocking tissue culture system. A fluorescein-conjugated monoclonal assays or confirmatory assays for all positive specimens. Direct fluorescent antibody (DFA) techniques use with chemiluminescent DNA probes are used to visualize the fluoroscein-labeled specific antibodies to detect chlamydial rRNA amplification products spectrophotometrically. Due antigens. The specimen is applied and fixed to a glass slide to the fact that this test is based upon a multiplex format, which is stained. The stained slide is reviewed using a simultaneous detection of both Neisseria gonorrhoeae and fluorescent microscope for stained elementary bodies. DFA Chlamydia trachomatis, competition inhibition can occur. requires a highly skilled and experienced microscopist for A phenomenon occurs when a high concentration of one reliable results. Sensitivity may be even lower when there target masks a low concentration of another target resulting is a very low bacterial load in the specimen. Specimen in false-negatives. The reported sensitivity of this assay is viability can be affected by collection techniques and as low as 89.3% in some studies (4). transport conditions. Many of these assays also target lipopolysaccharides (LPS) that are found in all chlamydia The Becton Dickinson BDProbeTec assay utilizes strand species as well as other microorganisms that could produce displacement (SDA) technology which uses amplification false-positive results (4). Therefore specificity is low as well. primers and a fluorescent detector probe for amplification The reported sensitivity of this assay is approximately 50% and detection of target DNA. A restriction endonuclease to 70% (12). DFA is not typically utilized as a screening test produces site-specific nicks within a double-stranded DNA or for primary diagnostic purposes. It is generally used for target. A new single-stranded DNA displaces the nicked confirmatory purposes only. strand. As the SDA process continues, a fluorescent signal is released and detected via fluorescent energy transfer. Nucleic acid hybridization (nucleic acid probe) tests such Specimens must be stored and transported at a refrigerated as the Pace-2 Gen-Probe and Digene Hybrid Capture temperature, a requirement which may be a serious limitation II are also available. The Pace-2 Gen-Probe utilizes a for some facilities. The reported sensitivity of this assay is chemiluminescently-labeled, single-stranded DNA probe 83.7% (4). to detect ribosomal RNA of C. trachomatis. Positive test results, as well as those that fall within a “grey-zone”, must The Abbott LCx assay utilizes Ligase Chain Reaction (LCR) be confirmed by a second method. This assay is limited technology. In LCR, specimens are denatured through in that target RNA is less stable than DNA and specimen heating to release DNA. Two pairs of complementary integrity may therefore be an issue depending upon oligonucleotide probes hybridize to a specific region of the collection and storage conditions. There is no amplification target DNA. The probes are ligated via DNA-ligase to form step, therefore, sensitivity of the assay is as low as 75% (9). a copy of the target DNA which is available for continued The Digene Hybrid Capture II test is a nucleic acid probe that amplification. The product is then detected via microparticle utilizes signal amplification. This test uses a single-stranded enzyme immunoassay (MEIA). A limitation of the procedure RNA probe to hybridize with specific DNA sequences of C. is the fact that grossly bloody specimens are inappropriate trachomatis. These RNA/DNA hybrids are captured by for testing. Sensitivity of this assay is as low as 85.5% (4). antibody, which is immobilized on microplate wells. The hybrids are detected with a secondary antibody conjugated The Roche Amplicor assay utilizes Polymerase to alkaline phosphatase, which is a chemiluminescent tag. Chain Reaction (PCR) technology. In this assay, two Emitted signals are measured by a luminometer. The use oligonucleotide primers and DNA polymerase react with the of multiple antibodies, each conjugated to an enzyme that target DNA in the specimen through cycles of denaturation, binds to the captured hybrid, results in signal amplification. annealing, and extension, resulting in amplification of This assay requires that positive tests, and those that specific nucleic acid sequences. The resulting amplicon fall within a “grey-zone”, must be confirmed by a second hybridizes to a probe which is bound to microparticles. method. The reported sensitivity of this assay is 95% (8). The product is detected via a biotinavidin reaction which results in colorimetric formation. The Roche Amplicor Nucleic acid amplification tests are all designed to amplify test was developed to detect both Neisseria gonorrhoeae specific nucleic acid sequences for a particular organism. and Chlamydia trachomatis simultaneously. As was Although the theory behind the technique is similar, the described for the Genprobe APTIMA 2 Combo Assay, tests themselves vary greatly in amplification methods tests prepared in multiplex formats can be affected and target sequences. This variation in test design by competition inhibition and result in false negatives. produces differences in sensitivities and specificities of each test system. The Gen-Probe APTIMA 2 Combo The recent advent of Real-Time PCR technology allows Assay utilizes transcription-mediated amplification (TMA) for the detection of PCR amplification while the reaction technology. Target ribosomal RNA (rRNA) molecules from is proceeding. Conventional PCR methods only allow the the specimen are captured and separated. Hybridization visualization of product at the end of the reaction or end- Specificity was determined theoretically by blast analysis of point analysis. In addition to the highly specific primers that the primers and probe sequences against the GenBank ‘nr’ are used in a PCR reaction, Real-Time PCR utilizes a probe database and experimentally by attempting amplification of to enhance the sensitivity and specificity of the assay. The DNA extracted from 39 known human pathogens and other validation studies performed by MDL, prior to offering testing organisms purchased from ATCC and mixed in various for a specific pathogen by PCR, are used to establish the cocktails. None of the organisms listed in Table 2 were efficacy of the application (sensitivity and specificity) as well amplified. as interference studies. In interference studies, the reaction Table 2. Validated organisms purchased from the ATCC for testing is spiked with potential amplification interfering substances specificity for Chlamydia trachomatis. (i.e., blood, mucus, genomic DNA) and perform C. Adenovirus Gardnerella vaginalis trachomatis amplification assays. The studies performed Anaplasma phagocytophilum by MDL establish the ability of the PCR method to detect Aspergillus fumigatus HHV-6 specific genetic sequences of a target pathogen within a Babesia microti HHV-8 given clinical specimen. Bacteroides fragilis HPV-16 bacilliformis HSV-1 Sensitivity was determined using serial dilutions of DNA HSV-2 extracted from known validated organisms purchased Bartonella quintana HTLV-1 from the American Type Cultures Collection (ATCC) and Borrelia burgdorferi Mobiluncus curtisii subcloned plasmid standards. A range of 108 to 100 copies Brucella abortis Mobiluncus mulieris Candida albicans Mycoplasma fermentans can be detected with an r2 value of 0.99899, (see Figure 1). Candida glabrata Mycoplasma genitalium Candida parapsilosis Mycoplasma hominus Candida tropicalis Mycoplasma pneumoniae Chlamydia pneumoniae Mycoplasma salivarium Chlamydia psittaci Neiserria gonorrhoeae CMV Trichomonas vaginalis Coxsackie Virus Trichosporon Cryptococcus neoformans Ureaplasma urealyticum EBV

RECOMMENDATIONS Figure1. pCtrachMF plasmid dilutions: 108, 107,106,105,104,103, and102 copies/reaction. In 2001, the U.S. Preventive Services Task Force published their recommendations regarding screening for Interference from other substances inherent to the specimen chlamydia in the American Journal of Preventive Medicine. type was determined by adding 500 ng of DNA extracted from They strongly recommend that all sexually active women a clinical sample that had tested negative for the pathogens aged 25 and younger, and other asymptomatic women at in question to a dilution of plasmid standards. The addition increased risk for infection, be screened for chlamydia of the extracted sample DNA did not significantly alter the 7 3 (13). They also recommend that clinicians routinely screen detection of the target DNA. A range of 10 to 10 copies can asymptomatic pregnant women aged 25 and younger 2 be detected with an r value of 0.99606, (see Figure2). and others at increased risk for infection for chlamydial infection (13). Recommendations for the treatment and management of patients can be found in the May 10, 2002 edition of Morbidity and Mortality Weekly Report (MMWR), Vol. 51: No. RR-6, pages 32-36.

Figure 2. pCtrachMF plasmid dilutions. Red lines represent plasmid alone. Blue lines represent plasmid with DNA. From left to right: 107, 106, 105,104, and 103 copies/reaction. for this infection is typically 3-5 days. Transmission more Neisseria gonorrhoeae frequently occurs from male to female (17). Some risks factors for infection include low socioeconomic status, EPIDEMIOLOGY early onset of sexual activity, unmarried status, a history Gonorrhea is one of the of previous gonorrhea infection, illicit drug abuse, and oldest diseases known to prostitution. A population with steadily increasing risk of man. References date contracting a gonorrhea infection is women 15–19 years back as far as the Old of age. Testament and ancient Chinese writings. Today, CLINICAL SIGNIFICANCE gonorrhea is the second most commonly reported disease in the U.S. (20). The The clinical manifestation in men is usually acute incidence of gonorrhea declined in the mid 1940’s with symptomatic urethritis. However, pharyngeal, anorectal, the introduction of penicillin. Although initially there was a and disseminated infections are also possible. In women, steady decline, 1975 saw a peak occurrence with 1 million infections are often asymptomatic. When manifested, cases in the U.S. (16). In 2002, 351,852 cases were symptoms may include vaginal discharge, dysuria, reported (20). Consistent with previous years, 2002 saw , menorrhagia, pelvic discomfort, a higher incidence in the southern portion of the U.S. than infection of the periurethral glands, Bartholin glands, and in other regions (20). Several important epidemiological anorectum. In women with endocervical infections, 35% to changes have occurred regarding gonorrhea infection 50% of women also have anal infections, and 10% to 20% in recent years. Between 1987 and 1996, the reported have coexistent pharyngeal infections (16, 17). In women cases originating from STD clinics as compared to private with lower genital tract infections, 15% to 20% also have providers shifted from twice as many to slightly more than an upper genital tract infection such as pelvic inflammatory one-half (16). Also, from 1966 to 1996 the male to female disease (PID) (16). The cervix can appear healthy or may ratio went from 3:1 to 1:1 (16). have an inflamed canal with ectopy and mucopurulent exudates.

PATHOGENESIS Due to the fact that gonorrhea can have serious The gram-negative diplococci bacteria, Neisseria consequences for both the mother and the neonate, it is gonorrhoeae, is the causative agent of gonorrhea. Humans crucial to screen pregnant women for infection. Studies are the only known natural host. Due to its affinity for report that the incidence of gonorrhea during pregnancy can columnar or pseudostratified epithelium, it is most commonly vary from rare to approximately 10% (18). Complications detected in the genital tract. The primary site of involvement that can occur during pregnancy include amniotic is usually the endocervical canal and the transition zone of infection syndrome, premature rupture of the membranes, the cervix. Vaginal infection is rare except in children, post- chorioamnionitis, premature birth, intrauterine growth menopausal women, and in women who acquire infection retardation, neonatal sepsis, and postpartum endometritis. during pregnancy. N. gonorrhoeae’s unique ability to alter Another major concern of infection during pregnancy is the surface structures allows increased pathogenicity, facilitates risk of gonorrheal ophthalmia neonatorum. 30% to 35% epithelial surface attachment, and enables evasion of of exposed neonates will acquire Neisseria gonorrhoeae the host’s immune response (16). N. gonorrhoeae also from an infected mother during a vaginal delivery (16). If produces lipopolysaccharides which act as endotoxins. left untreated, it can progress to corneal ulceration, corneal These substances have cytotoxic effects on the host scarring, and blindness. epithelium, which can produce systemic symptoms such as fever and toxicity. By the mere action of replication, N. Nongenital infections include conjunctivitis through gonorrhoeae alters the local microbiologic environment. autoinoculation or disseminated gonococcal infection This action creates an environment conducive to select (DGI). DGI occurs in about 0.1% to 0.3% of those infected. components of the vaginal flora and inhibitory to others However, some studies report an incidence as high as 3% in (29). high-risk groups (17).

Transmission of N. gonorrhoeae is almost entirely through DIAGNOSIS sexual contact. It can also be transmitted via the passage Diagnosis of gonorrhea infections has traditionally relied of a neonate through an infected mother’s birth canal or upon Gram stain, culture, and immunochemical techniques. via autoinoculation from the hands of an infected person Gram stain techniques, which consist of microscopic spreading a genital infection to their eye. Incubation time visualization of the organism on stained slides, have a reported sensitivity which varies from 40% to 70% (8). hybrids are detected with a secondary antibody conjugated Culture techniques have a reported sensitivity as low to alkaline phosphatase, which is a chemiluminescent tag. as 50% (23). The accuracies of these tests are greatly Emitted signals are measured by a luminometer. The use impacted by the adequacy of the clinical specimen and of multiple antibodies, each conjugated to an enzyme that transport conditions, particularly when transporting to off- binds to the captured hybrid, results in signal amplification. site facilities. Some assays have inherent growth-related This assay requires that positive tests, as well as those that issues that can delay results by as much as three days fall within a “grey-zone”, must be confirmed be confirmed by or more. Immunochemical detection methods, such as a second method. The reported sensitivity of this assay is the Gonozyme antigen detection test, have a reported 90.8% (22). sensitivity as low as 60% in women (24). Although this type of test seems to have good sensitivity and specificity in men, The second generation of molecular diagnostic tests are it is not recommended for women as a screening test due to nucleic acid amplification tests (NAAT). The Gen-Probe its low sensitivity. APTIMA 2 test utilizes transcription-mediated amplification (TMA) technology. Target ribosomal RNA (rRNA) molecules Table 3. Sensitivities of Neisseria gonorrhoeae detection tests. from the specimen are captured and separated. Hybridization Method Sensitivity with chemiluminescent DNA probes are used to visualize the rRNA amplification products spectrophotometrically. Due Gram Stain 40% - 70% to the fact that this test is based upon a multiplex format, Culture 50% simultaneous detection of both Neisseria gonorrhoeae and Immunochemical (Gonozyme, Abbott 60% Chlamydia trachomatis, competition inhibition can occur. Laboratories A phenomenon occurs when a high concentration of one Gen Probe Pace-2 97% target masks a low concentration of another target resulting in false-negatives. The reported sensitivity of this assay is Hybrid Capture-II (HCII) Digene) 91% 89.3% (24). Transcription Mediated Amplification 89% (TMA) GenProbe APTIMA 2 The Becton Dickinson BDProbeTec assay utilizes strand Strand Displacement Technology (SDA) 84% displacement (SDA) technology which uses amplification Becton Dickinson BDProbeTec primers and a fluorescent detector probe for amplification Ligase Chain Reaction (LCR) Abbott 92% and detection of target DNA. A restriction endonuclease LCx produces site-specific nicks within a double-stranded DNA Polymerase Chain Reaction (PCR) 96.8% target. A new single-stranded DNA displaces the nicked Roche Amplicor strand. As the SDA process continues, a fluorescent signal is released and detected via fluorescent energy transfer. Real-Time PCR Technology 99% Specimens must be stored and transported at a refrigerated temperature, a requirement which may be a serious limitation for some facilities. The reported sensitivity of this Advances in molecular techniques have resulted in assay is 83.7% (24). the introduction of several new diagnostic tests for N. gonorrhoeae. Initial assays incorporated nonamplified The Abbott LCx assay utilizes Ligase Chain Reaction (LCR) DNA probes, such as the Pace 2 system by Gen-Probe. technology. In LCR, specimens are denatured through This assay utilizes a chemiluminescently-labeled, single- heating to release DNA. Two pairs of complementary stranded DNA probe to detect ribosomal RNA of N. oligonucleotide probes hybridize to a specific region of the gonorrhoeae. Positive test results, as well as those that target DNA. The probes are ligated via DNA ligase to form fall within a “grey-zone”, must be confirmed by a second a copy of the target DNA which is available for continued method. This assay is limited in that target RNA is less amplification. The product is then detected via microparticle stable than DNA and specimen integrity may therefore be enzyme immunoassay (MEIA). A limitation of the procedure an issue depending upon collection and storage conditions. is the fact that grossly bloody specimens are inappropriate The reported sensitivity of this assay is 97.1% (21). for testing. The reported sensitivity of this assay is 92.1% (25). The Digene Hybrid Capture II test is a nucleic acid probe that utilizes signal amplification. This test uses a single-stranded The Roche Amplicor assay utilizes Polymerase RNA probe to hybridize with specific DNA sequences of N. Chain Reaction (PCR) technology. In this assay, two gonorrhoeae. These RNA/DNA hybrids are captured by oligonucleotide primers and DNA polymerase react with the antibody, which is immobilized on microplate wells. The annealing, and extension resulting in amplification of specific nucleic acid sequences. The resulting amplicon hybridizes to a probe which is bound to microparticles. The product is detected via a biotinavidin reaction which results in colorimetric formation. The Roche Amplicor test was developed to detect both Neisseria gonorrhoeae and Chlamydia trachomatis simultaneously. As was described for the Genprobe APTIMA 2 Combo Assay, tests prepared in multiplex formats can be affected by competition inhibition and result in false negatives. The reported sensitivity of this assay is 96.8% (25).

Figure 3. pNgonRao plasmid dilutions, 109, 108, 107, 106, 105, SDA techniques such as the BDProbeTec (Becton 4 3 2 Dickinson) as well as the Amplicor PCR (Roche) method, 10 , 10 , and 10 copies/reaction. have documented issues with specificity. Both assays can produce false positive results due to cross-reactivity with other Neisseria species which may be found as normal flora, including N. lactamica, N. cinerea, N. sicca, and N. subflava (26).

The recent advent of Real-Time PCR technology allows for the detection of PCR amplification while the reaction is proceeding. Conventional PCR methods only allow the visualization of product at the end of the reaction or end- point analysis. In addition to the highly specific primers that are used in a PCR reaction, Real-Time PCR utilizes a Figure 4. pNgonRao plasmid dilutions. Red lines represent probe to enhance the sensitivity and specificity of the assay. plasmid alone. Blue lines represent plasmid with DNA. From left The gene targeted in this assay is the omp85 gene which is to right: 107, 106, 105, 104, and 103 copies/reaction. highly conserved and specific to N. gonorrhoeae. Specificity was determined theoretically by blast analysis of the primers and probe sequences against the GenBank ‘nr’ The validation studies performed by MDL, prior to offering database and experimentally by attempting amplification of testing for a specific pathogen by PCR, are used to establish DNA extracted from 42 known human pathogens and other the efficacy of the application (sensitivity and specificity) as organisms purchased from ATCC and mixed in various well as interference studies. In interference studies, the cocktails. None of the organisms listed in Table 4 were reaction is spiked with potential amplification interfering amplified. substances (i.e., blood, mucus, genomic DNA) and perform N. gonorrhoeae amplification assays. The studies Table 4. Validated organisms purchased from the ATCC for performed by MDL establish the ability of the PCR method testing specificity for Neisseria gonorrhoeae. to detect specific genetic sequences of a target pathogen Adenovirus Gardnerella vaginalis Anaplasma phagocytophilum Helicobacter pylori within a given clinical specimen. Sensitivity was determined Aspergillus fumigatus HHV-6 using serial dilutions of DNA extracted from known validated Babesia microti HHV-8 organisms purchased from the American Type Cultures Bacteroides fragilis HPV-16 HSV-1 Collection (ATCC) and subcloned plasmid standards. A Bartonella henselae HSV-2 range of 109 to 100 copies per reaction can be reproducibly Bartonella quintana HTLV-1 detected with an r2 value of 0.99754 (See Figure 3). Borrelia burgdorferi Mobiluncus curtisii Brucella abortis Mobiluncus mulieris Candida albicans Mycoplasma fermentans Interference from other substances inherent to the Candida glabrata Mycoplasma genitalium specimen type was determined by adding 500 ng of DNA Candida parapsilosis Mycoplasma hominus Candida tropicalis Mycoplasma pneumoniae extracted from a clinical sample that had tested negative Chlamydia pneumoniae Mycoplasma salivarium for the pathogens in question to a dilution of plasmid Chlamydia psittaci Neisseria lactamica standards. The addition of the extracted sample DNA did Chlamydia trachomatis Neiserria mucosa CMV not significantly alter the detection of the target DNA. A Coxsackie Virus Neisseria perflava range of 5x106 to 5x102 copies can be detected with an r2 Cryptococcus neoformans Trichosporon value of 0.80308 (See Figure 4). EBV Ureaplasma urealyticum RECOMMENDATIONS Surface proteins on Trichomonads appear to damage genital epithelium by direct contact (31). This direct injury Current recommendations for Neisseria gonorrhoeae results in the development of micro ulcerations. Although include routine screening for asymptomatic women at high local and systemic humoral responses, as well as delayed risk of infection. Also during pregnancy all high risk women hypersensitivity type reactions do occur, the specific role should be screened (27). Bright Futures, the American of T. vaginalis virulence factors is not yet completely Medical Association Guidelines for Adolescent Preventive understood. Services (GAPS), and the American Academy of Pediatrics (AAP) recommend annual screening of sexually active CLINICAL SIGNIFICANCE adolescents (28). Due to the high incidence of , screening for Chlamydia trachomatis should routinely be The incubation period for infection is 5-28 days (31). It has performed on adults with gonorrhea or treatment given to been documented that up to 50% of infected women are eradicate possible co-infection (28). Recommendations for asymptomatic (30). Symptoms may often begin or worsen the treatment and management of patients can be found in during . They can include increased vaginal the May 10, 2002 edition of Morbidity and Mortality Weekly discharge which can have a foul smell, be frothy, or yellow- Report (MMWR), Vol. 51: No. RR-6, pages 36-42. green to gray in color. Some women may also experience dysuria, vulvovaginal soreness or irritation, itching, and painful intercourse or urination. Infection is self limited in 20% of infected women (31). It is primarily transmitted Trichomonas vaginalis through sexual contact. However, due to the ability of T. EPIDEMIOLOGY vaginalis to survive for hours under moist conditions, it is possible for non-sexual transmission to occur (31, 32). Although Trichomonas Prevalence studies report two very different groups with vaginalis was first the highest incidences of infection; young sexually active described in 1836, it was women and in older women who have no evidence for not recognized as a true sexually transmitted infection (37). pathogen of the lower genital tract until 1947. Although T. vaginalis can be detected from the endocervix More than 180 million in 90% of infected women, it does not cause endocervical cases of Trichomoniasis occur worldwide each year (37). disease (31). Infection typically manifests as There are an estimated 3 million new cases annually (31). of the vaginal walls or exocervix. Upon visualization of In the U.S. between 5% to 74% of women and 5% to 29% the cervix or during colposcopy, characteristic punctate of men are infected (33, 36). It is believed that prevalence mucosal hemorrhages of the cervix may be seen. This in men is described less due to asymptomatic infection. phenomenon is often referred to as strawberry or - Overall, it is believed that the actual occurrence of T. bitten cervix. Complications of infection can include vaginalis infection is underestimated due to many health care emphysematosa, premature labor, low birth practitioners rely on insensitive wet-mount preparations for weight, postabortal infections, and premature rupture of detection (33). The highest incidence occurs in women with the membranes. Neonates may acquire Trichomonas as multiple sexual partners and groups with other increased they pass through the birth canal of an infected mother. It rates of sexually transmitted diseases. Other risk factors has been documented that 2% to 17% of female newborns include race, older age, previous sexually transmitted will acquire infection (2). This may manifest as pharyngeal infections (STI), prostitution, pregnancy, and drug use. or respiratory infection. Due to the inflammatory response associated with T. vaginalis infection, it is believed to PATHOGENESIS facilitate the acquisition and transmission of HIV (31, 33). Trichomoniasis is caused by the protozoan parasite Trichomonas vaginalis. Trichomonas, of the family In men, infection is mainly asymptomatic. However, it has Trichomonadida, consists of five species. Trichomonas been isolated in 5% to 15% of men with non-gonococcal vaginalis infects the , urethra, and paraurethral urethritis. Symptoms can include a very mild, often glands. This protozoan has four interiorly placed flagella unrecognized discharge and slight burning after urination and an undulating membrane which produce a characteristic or ejaculation. In men it may cause urethritis, epididymitis, tumbling motility. Changes in the levels of endogenous superficial penile ulcerations, and may involve the prostate vaginal microflora, such as a reduction of Lactobacillus gland. species facilitates establishment of other infections, for example, Trichomonas. or denatured, through elevations in temperature. As the DIAGNOSIS temperature cools, primers anneal to the target strand and Due to the nonspecific symptoms which are shared between allow nucleotides to bind, forming a complementary region. many sexually transmitted diseases, diagnosis cannot occur The temperature elevates slightly to allow completion of based on clinical presentation alone (31). Traditionally, the synthesis of a new strand. At the end of one cycle the physicians would perform a wet mount by mixing collected amount of DNA is doubled. Through 20 to 40 repetitive vaginal secretions with saline solution on a glass slide cycles, exponential amplification of the DNA is achieved. and visualizing it under a microscope. Identification would occur by characteristic movements of flagella or an The recent advent of Real-Time PCR technology allows undulating membrane. False negatives may result due for the detection of PCR amplification while the reaction to low parasitemia levels and the length of time between is proceeding. Conventional PCR methods only allow the specimen collection and analysis. Sensitivity of this assay visualization of product at the end of the reaction or end- varies greatly with the experience and thoroughness of the point analysis. In addition to the highly specific primers that clinician. It is as low as 52% in some studies (35). are used in a PCR reaction, Real-Time PCR utilizes a probe to enhance the sensitivity and specificity of the assay. Culture can be performed by using special media such as modified Diamond’s or Trichosel media. However, this The validation studies performed by MDL, prior to offering procedure is very time consuming and requires the growth testing for a specific pathogen by PCR, are used to of motile trichomonads. This method is not used often establish the efficacy of the application (sensitivity and due to the fact that results can take as long as 7 days. specificity) as well as interference studies. In interference This procedure has a sensitivity as low as 78% (35). It is studies, the reaction is spiked with potential amplification possible to identify T. vaginalis during a Pap smear analysis, interfering substances (i.e., blood, mucus, genomic DNA) but the sensitivity is only 52% to 67% (30). There is also a and perform T. vaginalis amplification assays. The studies low specificity, even when utilizing a special stain such as performed by MDL establish the ability of the PCR method acridine orange. Sensitivity with this special stain is only to detect specific genetic sequences of a target pathogen 66% (31). within a given clinical specimen. Sensitivity was determined using serial dilutions of DNA extracted from known validated Enzyme-linked immunosorbent assay (ELISA) is available organisms purchased from the American Type Cultures for the detection of T. vaginalis antigens in vaginal secretions. Collection (ATCC) and subcloned plasmid standards. A In this assay, a sandwich technique uses biotinylated anti-T. range of 5x108 to 50 copies can be detected with an r2 value vaginalis antibodies which bind to antigens in the specimen. of 0.99832 (See Figure 5). Horseradish peroxidase-conjugated streptavidin and ABTS are used to produce a colorimetric reaction for product detection. This test has a sensitivity of 82% and a specificity of 73% (34).

Direct fluorescent antibody (DFA) techniques use fluoroscein-labeled specific antibodies to detect T. vaginalis antigens. The specimen is applied and fixed to a glass slide which is stained. The stained slide is reviewed using a fluorescent microscope for stained membrane, cytoplasm, and nuclei of Trichomonas organisms. DFA requires a highly skilled and experienced microscopist for reliable results. Sensitivity may be even lower when there is a very low bacterial load in the specimen. Specimen viability can be affected by collection techniques and transport conditions. This test has a sensitivity of 85% and a specificity of 99% (34). Figure 5. pTvagMF plasmid dilutions, 5x108, 5x107, 5x106, 5x105, 5x104, 5x103, 5x102, and 50 copies/reactions. The polymerase chain reaction (PCR) method is a nucleic acid amplification test. In the PCR method, double stranded DNA from the specimen is separated into single strands, Interference from other substances inherent to the RECOMMENDATIONS specimen type was determined by adding 500 ng of DNA Due to the fact that Trichomoniasis often occurs with other extracted from a clinical sample that had tested negative STDs such as chlamydia and gonorrhea with rates of 15% to for the pathogens in question to a dilution of plasmid 28% and 10%, respectively (33), current recommendations standards. The addition of the extracted sample DNA did include screening of concurrent STDs in both men and not significantly alter the detection of the target DNA. A 6 2 2 women. Sexual partners should be treated simultaneously. range of 5x10 to 5x10 copies can be detected with an r Recommendations for the treatment and management value of 0.99606 (See Figure 6). of patients can be found in the May 10, 2002 edition of Morbidity and Mortality Weekly Report (MMWR), Vol. 51: No. RR-6 pages, 44-45.

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