44 Nucleic Acid Amplification Tests for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae AMY L. LEBER, GERRI S. HALL, AND WILLIAM D. LEBAR COORDINATING EDITOR
SUSAN E. SHARP
Cumitech CUMULATIVE TECHNIQUES AND PROCEDURES IN CLINICAL MICROBIOLOGY
Cumitech 30A
Selection and Use of Laboratory Procedures for Diagnosis of Parasitic Infections of the Gastrointestinal Tract (2003)
Cumitech 31
Verification and Validation of Procedures in the Clinical Microbiology Laboratory (1997)
Cumitech 32
Laboratory Diagnosis of Zoonotic Infections: Viral, Rickettsial, and Parasitic Agents Obtained from Food Animals and Wildlife (1999)
Cumitech 33
Laboratory Diagnosis of the Mycobacterioses (1994)
Laboratory Safety, Management, and Diagnosis of Biological Agents Associated with Bioterrorism (2000)
Cumitech 34
Cumitech 18A
Laboratory Diagnosis of Hepatitis Viruses (1998)
Laboratory Diagnosis of Mycoplasmal Infections (2001)
Cumitech 35
Postmortem Microbiology (2001)
Cumitech 19A
Laboratory Diagnosis of Chlamydia trachomatis Infections (1999)
Cumitech 36
Biosafety Considerations for Large-Scale Production of Microorganisms (2002)
Cumitech 21
Laboratory Diagnosis of Viral Respiratory Disease (1986)
Cumitech 37
Cumitech 23
Infections of the Skin and Subcutaneous Tissues (1988)
Laboratory Diagnosis of Bacterial and Fungal Infections Common to Humans, Livestock, and Wildlife (2003)
Cumitech 24
Rapid Detection of Viruses by Immunofluorescence (1988)
Cumitech 38
Human Cytomegalovirus (2003)
Cumitech 39
Cumitech 26
Laboratory Diagnosis of Viral Infections Producing Enteritis (1989)
Competency Assessment in the Clinical Microbiology Laboratory (2003)
Cumitech 40
Cumitech 27
Laboratory Diagnosis of Zoonotic Infections: Bacterial Infections Obtained from Companion and Laboratory Animals (1996)
Packing and Shipping of Diagnostic Specimens and Infectious Substances (2004)
Cumitech 41
Detection and Prevention of Clinical Microbiology Laboratory-Associated Errors (2004)
Cumitech 42
Infections in Hemopoietic Stem Cell Transplant Recipients (2005)
Cumitech 43
Cystic Fibrosis Microbiology (2006)
Cumitech 44
Nucleic Acid Amplification Tests for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae (2006)
Cumitech 1C Cumitech 2B
Blood Cultures IV (2005)
Cumitech 3B
Quality Systems in the Clinical Microbiology Laboratory (2005)
Cumitech 7B Cumitech 10A
Lower Respiratory Tract Infections (2004)
Cumitech 12A
Laboratory Diagnosis of Bacterial Diarrhea (1992)
Cumitech 13A
Laboratory Diagnosis of Ocular Infections (1994)
Cumitech 16A
Cumitech 28
Cumitech 29
Laboratory Diagnosis of Urinary Tract Infections (1998)
Laboratory Diagnosis of Upper Respiratory Tract Infections (2006)
Laboratory Diagnosis of Zoonotic Infections: Chlamydial, Fungal, Viral, and Parasitic Infections Obtained from Companion and Laboratory Animals (1996) Laboratory Safety in Clinical Microbiology (1996)
Cumitechs should be cited as follows, e.g.: Leber, A. L., G. S. Hall, and W. D. LeBar. 2006. Cumitech 44, Nucleic Acid Amplification Tests for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae. Coordinating ed., S. E. Sharp. ASM Press, Washington, D.C. Editorial Board for ASM Cumitechs: Alice S. Weissfeld, Chair; Maria D. Appleman, Vickie Baselski, B. Kay Buchanan, Mitchell l. Burken, Roberta Carey, Linda Cook, Lynne Garcia, Susan L. Mottice, Michael Saubolle, David L. Sewell, Daniel Shapiro, Susan E. Sharp, James W. Snyder, Allan Truant. Effective as of January 2000, the purpose of the Cumitech series is to provide consensus recommendations regarding the judicious use of clinical microbiology and immunology laboratories and their role in patient care. Each Cumitech is written by a team of clinicians, laboratorians, and other interested stakeholders to provide a broad overview of various aspects of infectious disease testing. These aspects include a discussion of relevant clinical considerations; collection, transport, processing, and interpretive guidelines; the clinical utility of culture-based and non-culture-based methods and emerging technologies; and issues surrounding coding, medical necessity, frequency limits, and reimbursement. The recommendations in Cumitechs do not represent the official views or policies of any third-party payer. Copyright © 2006 ASM Press American Society for Microbiology 1752 N St. NW Washington, DC 20036-2904 All Rights Reserved 10 9 8 7 6 5 4 3 2 1
Address editorial correspondence to ASM Press, 1752 N St. NW, Washington, DC 20036-2904, USA E-mail:
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Nucleic Acid Amplification Tests for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae Amy L. Leber Quest Diagnostics, Nichols Institute, 33608 Ortega Highway, San Juan Capistrano, CA 92690
Gerri S. Hall Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195
William D. LeBar Hospital Consolidated Laboratories-Providence Hospital, 23775 Northwestern Highway, Southfield, MI 48075
COORDINATING EDITOR: Susan E. Sharp Kaiser Permanente, 13705 Airport Way, Portland, OR 97230
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Biology of the Organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Chlamydia trachomatis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Neisseria gonorrhoeae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Epidemiology and Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Chlamydia and Gonorrhea Infections in Women . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Chlamydia and Gonorrhea Infections in Men . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Other Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Review of Testing Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Culture . . . . . . . . . . . . Staining . . . . . . . . . . . . Enzyme Immunoassay Probe . . . . . . . . . . . . . NAATs . . . . . . . . . . . . . Serology . . . . . . . . . . .
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5 5 7 7 7 7
Specimen Types and Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Endocervical Specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Inhibition with Endocervical Specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Urethral Specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Noninvasive Specimen Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Urine Specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Liquid-Based Cytology Specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Vaginal Specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Nonurogenital Sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Specimen Pooling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Review of Current NAATs for C. trachomatis and N. gonorrhoeae Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Ligase Chain Reaction: LCx Probe System (Abbott Laboratories, Abbott Park, Ill.) . . . . . . . 10 PCR: COBAS AMPLICOR CT/NG Test (Roche Diagnostics Corporation, Indianapolis, Ind.) . 11 Transcription-Mediated Amplification: APTIMA (Gen-Probe Inc., San Diego, Calif.) . . . . . . 16 Strand Displacement Amplification: BD ProbeTecET (Becton Dickinson and Company, Sparks, Md.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Hybrid Capture: Hybrid Capture 2 CT/GC DNA Tests (Digene Corporation, Gaithersburg, Md.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 New Technologies and Laboratory-Developed Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Interpretation and Reporting of Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 Test of Cure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Children and Sexual Abuse or Assault Cases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
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CUMITECH 44 Reporting Comments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Kit Controls . . . . . . . . . . External Controls . . . . . . Positivity Rate . . . . . . . . Proficiency Testing . . . . . Contamination Concerns
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31 33 33 33 33
Considerations for Laboratories Performing NAATs . . . . . . . . . . . . . . . . . . . 33 Verification of a Test Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Guidelines for Screening and Testing for Infection . . . . . . . . . . . . . . . . . . . . 37 Symptomatic Testing . . . . . . . . . . . . . . . . Asymptomatic Screening . . . . . . . . . . . . . Laboratory Guidelines for Screening Tests Effectiveness of Screening . . . . . . . . . . .
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37 37 37 38
Coding and Reimbursement Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
INTRODUCTION All the world loves loving, but it entails the bothersome inconveniences of pregnancy and disease. Richard Gordon, The Alarming History of Medicine (St. Martin’s Press, New York, N.Y., p. 143, 1993)
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exually transmitted diseases impact millions of people worldwide, and Chlamydia trachomatis and Neisseria gonorrhoeae represent two of the most common of these infections. Laboratory diagnosis has evolved from culture, to direct detection by staining and enzyme immunoassay, and then to use of nucleic acid probes to detect both organisms. With the advent of nucleic acid amplification tests (NAATs) for the detection of C. trachomatis and N. gonorrhoeae, the quality of testing for both organisms has improved greatly, and these tests have arguably become the standard of care for diagnosis. Amplified techniques may present new challenges to laboratorians. The exquisite sensitivity of these methods is both a blessing and a curse. Changes to the work practices and physical facility may be necessary, along with a vigilant quality control program. This Cumitech is intended as a guide for those interested in either bringing C. trachomatis and N. gonorrhoeae amplified testing into their laboratories for the first time or improving the testing processes already in place. All aspects of the testing process are covered in this document, including a review of those assays that are commercially available. The commercial tests tend to change rapidly, but it is hoped that much of the information provided will be applicable for laboratories regardless of the particular NAAT used.
BIOLOGY OF THE ORGANISMS Chlamydia trachomatis Organisms in the genus Chlamydia are gram-negative, nonmotile, obligate intracellular parasites of eukaryotic cells. The chlamydiae do not carry out energy metabolism and are entirely dependent on the host cell to supply them with ATP. C. trachomatis is classified in the order Chlamydiales and family Chlamydiaceae, which has recently been divided into two genera, Chlamydia and Chlamydophila (71). Four species are recognized, C. trachomatis, Chlamydia muridarum, Chlamydia suis, and Chlamydophila pneumoniae. The chlamydiae have a distinctive dimorphic life cycle that takes place within the cytoplasm of the host cell. Stages in this cycle consist of a nonreplicating infectious particle called the elementary body and a replicative form called the reticulate body found within the cytoplasm of infected cells. The chlamydial developmental cycle is initiated by the ingestion of the infective elementary body by the host cell. The elementary body is a metabolically inert, electrondense particle 0.2 to 0.6 m in diameter that contains the genome and a cryptic plasmid (164). RNA polymerase, ribosomes, and ribosomal subunits are also present in the elementary body (10). Following attachment and endocytosis, the elementary body differentiates into a noninfectious reticulate body within a cytoplasmic vacuole in the infected host cell. Transcription, protein synthesis, and DNA replication all take place within this structure. The reticulate body divides by binary fission, forming particles that develop into the infectious elementary bodies. After multiple division cycles, the newly developed infectious elementary bodies are released by rupture
CUMITECH 44
Nucleic Acid Amplification Tests for Detection of C. trachomatis and N. gonorrhoeae
of the host cell. The chlamydial development cycle is completed within 48 to 72 h (170). All members of the Chlamydiaceae express a familyspecific lipolysaccharide epitope (25), and structural integrity is maintained by three proteins, including a 40-kDa major outer membrane protein, a cysteinerich 60-kDa protein, and a low-molecular-weight cysteine-rich lipoprotein (97, 174). It is the major outer membrane protein complex that is responsible for antigenic differences between species and subspecies. Based on antigenic differences, C. trachomatis is divided into 15 main serovars comprising two human biovars, the lymphogranuloma venereum (LGV) biovar (L1, L2, L3) and the trachoma biovar (A–K, B, Ba) (220). The Chlamydiae, in general, have a small genome. In addition to chromosomal DNA, most strains of C. trachomatis contain a 7,500-base pair plasmid common to all serovars (164). This plasmid is the target used in many of the DNA-based amplified molecular assays. Ribosomal RNA, specifically 23S rRNA, is also used as a target for amplified nucleic acid hybridization procedures. All 15 of the C. trachomatis serovars may be detected by commercially available DNA or RNA amplification methods (64, 125). Neisseria gonorrhoeae N. gonorrhoeae, the agent of gonorrhea, was first described by Albert Neisser in 1879 (158). Members of the genus Neisseria are gram-negative coccal organisms that may occur in pairs or short chains. The gonococcus has a tendency to occur in pairs, with adjacent sides flattened. All members of the genus are aerobic, produce cytochrome oxidase (except Neisseria elongata), grow best at 35 to 37°C and do not form spores (108). Capneic incubation and increased humidity may stimulate growth, whereas some gonococcal strains have an obligate requirement for CO2. Gonococci infect nonciliated columnar epithelial cells. Once attached, they enter the cell by endocytosis and multiply on the basement membrane. Gonococcal adherence is mediated by the antigenic characteristics of surface proteins and pili. Pili are hairlike appendages that extend from the surface of the cell and are composed of protein subunits called pilin (98). These structures are important in the initial attachment of the organism to the host cell and also impart increased resistance to phagocytosis. Pili can undergo both phase and antigenic variation, which assists in the evasion of the immune system. Once attached, other surface proteins including Opa (Protein II, P.II) assist in the adhesion of gonococci to mucosal surfaces (193). Protein I (P.I, Por) is a porin protein located in the outer membrane of the gonococcus and is involved in the penetration of the host cell. Organisms express
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one of two forms of P.I—IA or IB—which differ antigenically from strain to strain. Antibodies to P.I acquired during infection are bactercidal and prevent repeated attacks of salpingitis with strains of the same P.I type (17, 18, 175, 193). All strains of N. gonorrhoeae contain an outer membrane P.III (Rmp) found in complex with P.I and bacterial lipooligosaccharide (LOS) (18). This LOS antigen is released by autolysis of cells and mediates the inflammatory response associated with the symptoms of gonorrhea (89). Gonococcal LOS is responsible for mucosal damage in fallopian tube organ cultures through local release of tumor necrosis factor alpha. In strains that cause systemic infection, LOS binds serum sialic acid, preventing complement activation and blocking antibodies from binding to surface proteins (17, 175). Antibodies also may be produced against P.III that may react on the gonococcal surface by blocking bactericidal antibodies directed against LOS and P.I. Iron acquisition is necessary to support bacterial growth and invasion. Gonococci obtain host iron complexes through the expression of transferrin (Tbp1, Tbp2) and lactoferrin (Lbp) receptors in their outer membrane. These receptors are induced under low iron conditions and are able to extract iron from transferrin, lactoferrin, heme, and hemoglobin. The transferrin receptor has been shown to be required for experimental urethral infection in male volunteers (187). N. gonorrhoeae may also produce immunoglobulin A (IgA) proteases (110). These proteases cleave the heavy chain of the IgA1 at specific points within the hinge region. Once cleaved, the IgA loses its activity. Although products of IgA1 cleavage have been found in genital secretions of females with gonorrhea, it has been shown that N. gonorrhoeae does not require IgA1 protease activity to produce experimental urethritis in males.
EPIDEMIOLOGY AND DISEASE Chlamydia and gonorrhea are the two most commonly reported notifiable diseases in the United States, with an estimated 3 million new C. trachomatis and 1 million new N. gonorrhoeae infections annually (34, 70). In 2003, 877,478 chlamydial infections and 335,104 cases of gonorrhea were reported to the Centers for Disease Control and Prevention (CDC) from all 50 states and the District of Columbia. The case count corresponds to a rate of 304.3 cases of C. trachomatis infection per 100,000 population, a 5.1% increase over 2002. Increases have been seen for greater than 10 years in reported C. trachomatis infections in women and are likely attributable to expanded screening programs and the
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use of more sensitive testing such as NAATs. The case rate for N. gonorrhoeae in 2003 was 116.2 cases per 100,000 population, the lowest ever reported and a 10% decrease since 1999. The rate of coinfection is less clear, but patients infected with N. gonorrhoeae often are coinfected with C. trachomatis. Miller et al. (149) found a prevalence of 0.3% coinfection in a representative population of young adults in the United States. Among persons with N. gonorrhoeae infection, the prevalence of C. trachomatis infection was very high at 70%, and those infected with C. trachomatis had a prevalence of N. gonorrhoeae of 7.3%. These data support the recommendation that patients treated for gonococcal infection should also be treated routinely with a regimen effective against uncomplicated genital C. trachomatis infection (35). Infections with C. trachomatis and N. gonorrhoeae are probably significantly underreported because both infections are often asymptomatic (201). Also, most data are collected from select populations that may not reflect all susceptible individuals (34). In a recent study of a nationally representative sample of 14,322 young adults aged 18 to 26 years in the United States, the overall prevalence of C. trachomatis infection was 4.19%, and for N. gonorrhoeae infections, prevalence was 0.43% (149). The authors state that these rates are higher than those proposed by the CDC. Risk factors for infection with these organisms include lower socioeconomic status, early sexual activity, multiple sexual partners, non-white race, illicit drug use, prostitution, and younger age (15 to 25 years) (149). Within a given population there may be reservoirs of infection called “core group transmitters” who are more likely to become infected and transmit infection (173). Treatment focused on such individuals would be a useful strategy for effective disease control. Transmission of C. trachomatis and N. gonorrhoeae is both horizontal and vertical. Sexual contact, including vaginal, anal, and oral contact, is the most common mode of transmission; infection passed from mother to infant during birth also occurs. C. trachomatis infections of the genital tract are caused by biovars D–K. The organism targets the squamocolumnar epithelial cells of the endocervix and upper genital tract in women. The bacteria also infect the conjunctiva, urethra, and rectum in men and women. Infants are commonly infected in the epithelial cells of the respiratory track, leading to pneumonia. The squamocolumnar epithelial cells in mucous membranes are the site of gonoccocal infections in adults. Mothers can infect infants during delivery; the most common manifestation is gonococcal conjunctivitis (ophthalmia neonatorum), but infection also can lead to arthritis and septicemia.
CUMITECH 44
Chlamydia and Gonorrhea Infections in Women As stated previously, urogenital infection is often asymptomatic or minimally symptomatic in women infected with C. trachomatis or N. gonorrhoeae. Symptomatic infection may result in abnormal vaginal discharge, bleeding, abdominal pain, and dsyuria. Symptoms usually appear within 1 to 3 weeks after exposure for C. trachomatis and 10 days for N. gonorrhoeae. Infections can extend to the endometrium and fallopian tubes, resulting in abdominal pain, low back pain, nausea, fever, abnormal bleeding, and pain during intercourse. Women generally suffer more serious complications than men, including pelvic inflammatory disease, infertility, and ectopic pregnancy. Up to 40% of women with untreated urogenital chlamydia infection develop pelvic inflammatory disease (PID). Among women with PID, 18% will suffer from chronic pain, 20% will be left infertile, and 9% will have tubal pregnancies (223). Perihepatitis (FitzHugh–Curtis syndrome) can occur with both organisms. It is more commonly associated with C. trachomatis than N. gonorrhoeae, and evidence of concomitant salpingitis may or may not be present. In one study, a prior cervical C. trachomatis infection was associated with an increased risk for development of invasive cervical cancer (219). More data are needed to make a conclusive link between these two conditions. Chlamydia and Gonorrhea Infections in Men Urethral infection with C. trachomatis and N. gonorrhoeae can result in asymptomatic or symptomatic infection in males. Chlamydial urethritis is more likely to be asymptomatic than gonococcal urethritis: nongonococcal urethritis and postgonococcal urethritis. N. gonorrhoeae infections in men are often symptomatic, with a shorter incubation period (average 4 days) compared to that of C. trachomatis (7 to 14 days). Men with symptomatic infection with either organism may present with dysuria and urethral discharge. Pain and swelling in the testicles are uncommon. Complications of untreated infection include epidimitis, prostatitis and proctitis, and systemic disease. An additional concern is the finding that human immunodeficiency virus transmission is facilitated by infection with C. trachomatis and N. gonorrhoeae (51, 75). Other Diseases Sexual exposure to C. trachomatis or N. gonorrhoeae can result in rectal infections in both men and women. Women may have anal infections by extension from the cervix. Rectal infection in both men and women may be symptomatic with discharge, anal itching, soreness, bleeding, or tenesmus. Rectal infection may also cause no symptoms. Oropharyn-
CUMITECH 44
Nucleic Acid Amplification Tests for Detection of C. trachomatis and N. gonorrhoeae
geal infections occur after N. gonorrhoeae exposure and may result in sore throat but are most often asymptomatic and resolve without intervention, and transmission to another individual is uncommon (187). Pharyngitis caused by C. trachomatis has been reported and correlates with recent sexual contact (113). Studies using serology testing to link C. trachomatis infection to nonstreptococcal communityacquired pharyngitis were most likely detecting crossreacting C. pneumoniae antibodies (189). Conjunctivitis in adults occurs and is linked with autoinoculation of the eye with genital secretions. For C. trachomatis infection, the disease is very similar to milder trachoma. It is seen in approximately 1% of cases with proven genital infections (171). Gonococcal conjunctivitis tends to be more severe and necessitates prompt therapy (187). Rarely, genital chlamydial infection can cause disseminated disease. In men and women, acute aseptic arthritis can result from untreated urogenital infection. In men, this reactive arthritis in combination with skin lesions, conjunctivitis, and urethritis is called Reiter’s syndrome. Approximately 1% of men presenting with chlamydial urethritis have the arthritis syndrome, and one-third of these have Reiter’s syndrome (117). Disseminated gonococcal infections occur in 0.5 to 3% of infected individuals; the most common manifestations are septic arthritis and dermatitis (101). Among sexually active young adults, disseminated gonococcal infection is a common cause of infective arthritis. Birth of an infant to a mother who is infected with C. trachomatis or N. gonorrhoeae can result in infection in the baby, with organisms being detectable from the conjunctiva, anus, oropharynx, urethra, and vagina. As noted above, C. trachomatis infection most commonly manifests as pneumonia, but it is also a common cause of neonatal conjunctivitis. Neonatal infection with N. gonorrhoeae can result in conjunctivitis and other less common but serious diseases. Trachoma and Lymphogranuloma Venereum Two additional diseases caused by C. trachomatis are trachoma and LGV. These diseases have been reviewed extensively elsewhere and are covered briefly in this document (137, 138, 186, 222). Trachoma is the leading cause of preventable blindness in the world and is caused by C. trachomatis biovars A, B, Ba, and C. Infection with serovars L1, L2, and L3 causes LGV, a sexually transmitted disease that is endemic in Africa, Asia, and South America and sporadic elsewhere. Both LGV and trachoma are uncommon in the United States; however, a recent outbreak of LGV occurred in The Netherlands. This finding, along with cases in Europe, suggests that there may be an increase in LGV cases in the United States, especially among men who have sex with men (32).
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REVIEW OF TESTING METHODS Methods for the laboratory diagnosis of C. trachomatis and N. gonorrhoeae include culture and nonculture techniques. Below are brief descriptions of each of the available methods that have been or continue to be used. Summaries of the various assays and their advantages and disadvantages for the detection of C. trachomatis and N. gonorrhoeae infections are listed in Tables 1 and 2. Culture Culture is available for both organisms and is still considered the recommended method in certain situations. These situations include testing specimen types other than urogenital; testing urogenital specimens from infants and children or in cases of possible sexual abuse; and “test of cure” (in rare instances when that is indicated). Culture for C. trachomatis requires that well-collected endocervical or urethral specimens be centrifuged onto a monolayer of tissue culture cells, such as McCoy fibroblasts or human foreskin cells. After cells are incubated for at least 2 days, specific fluorescent antibody stains are used to detect the inclusions characteristic of cells infected by C. trachomatis. In many laboratories, if the first stain is negative, a blind passage of the tissue culture is made into a new monolayer and the test is repeated. This is done to enhance recovery of C. trachomatis. This can be labor-intensive and, compared to the NAATs that are discussed in this Cumitech, such cultures can be as much as 30% less sensitive than amplification (15). Culture for the recovery of N. gonorrhoeae remains a very sensitive method for the detection of this organism; however, the problems with transportation inherent in culture methods often make nonculture methods more convenient, efficient, and attractive. One significant advantage of N. gonorrhoeae cultures is the ability to perform antimicrobial susceptibility testing, which is not possible with the nonculture techniques. Specificity of culture for either C. trachomatis or N. gonorrhoeae is considered to be 100%. Staining Staining directly for the presence of C. trachomatis and N. gonorrhoeae is possible. Cytopathology can be performed on Papanicolaou’s (Pap) smears or other tissue specimens and examined for the presence of inclusions typical of C. trachomatis; however, the sensitivity of this method is less than that of other available diagnostic methods. The sensitivity and specificity of cytology for C. trachomatis in 159 women in a study from Ontario, Canada, were 13.3% and 90.3%, respectively, although the negative predictive value was 90.9% (215). Another study
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Table 1. Advantages and disadvantages of assays for detection of C. trachomatis Method
Advantages
Culture
Specificity; any specimen type can be cultured
Cytopathology
Performed on Pap smears
Direct fluorescent antibody stain
Specificity; can be used to determine suitability of specimen; can be used in discrepancy testing when two assays are compared Batching of large volumes of specimens, especially for screening; some success with urine samples
Enzyme immunoassay
DNA probe
NAAT
Easy to perform and rapid turnaround time; can use conjunctival specimens as well as endocervical and urethral specimens Sensitive and specific assays; batching of specimens possible; automation possible
Disadvantages Up to 30% less sensitive than NAAT; labor intensive; slow turnaround time; cell lines need to be purchased or maintained in the laboratory; experienced technologist required for setup and reading of cell cultures Low sensitivity; can be low specificity; labor intensive; need experienced individuals to read adequately Reduced sensitivity as compared to NAAT; timeconsuming since each must be read individually; only genital specimens Sensitivity can be low; specificity can be less than needed, unless blocking antibody tests are done for confirmation; availability of reagents may be a problem Less sensitive than NAAT by as much as 30%
Concern about contamination by amplicon; can be costly; in low-prevalence populations, need to consider confirmation of positives; manual processing of specimens can be time-consuming; cannot be used in cases of sexual abuse and/or in children; cannot be used for nongenitourinary specimens Positive results could indicate past or present infection or infection with other species of Chlamydia
Serology
Only useful in the diagnosis of LGV infections
Table 2.
Advantages and disadvantages of assays for detection of N. gonorrhoeae
Method Culture
Gram stain
Direct fluorescent antibody stain Enzyme immunoassay DNA probe NAAT
Serology a
NA, not available.
Advantages
Disadvantages
Ease of performance in most laboratories; can be 100% sensitive if transport can be controlled; specificity great; any specimen can be cultured; viable organism available for susceptibility testing if needed Easy to perform; can be performed rapidly on most specimens; good sensitivity and specificity in male urethral specimens NAa
If transportation of cultures cannot be optimized, organisms may be nonviable and limit recovery; requires overnight incubation or longer for detection
NA Easy to perform and rapid turnaround time; can use endocervical and urethral specimens Sensitive and specific assays; batching of specimens possible; automation possible
NA
Specificity not acceptable for making diagnosis in female endocervical specimens due to possible presence of other diplococci Not readily available NA May be slightly less sensitive than culture Concern about contamination by amplicon; can be costly; in low-prevalence populations, need to consider confirmation of positives; manual processing of specimens can be time-consuming; cannot be used in cases of sexual abuse and/or in children; cannot be used for nongenitourinary specimens NA
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Nucleic Acid Amplification Tests for Detection of C. trachomatis and N. gonorrhoeae
demonstrated 100% sensitivity for the Pap smear versus PCR and an enzyme immunoassay in 125 pregnant females; however, the positive predictive value for the smear in this study was only 75% (9). In the same study, the sensitivity, specificity, and positive and negative predictive values were 100% for PCR. Use of the Gram stain for the detection of N. gonorrhoeae is relatively easy, and the finding of intracellular gram-negative diplococci, consistent with N. gonorrhoeae, can be used to make the diagnosis of gonorrhea from a male urethral specimen. The sensitivity of the Gram stain has been reported at 89 to 95% versus culture and as high as 99.6% versus the PACE 2 probe assay in symptomatic male patients; the specificity is 94% in this same population (114). Because of problems with specificity in the female endocervical specimens, Gram stains must be used in conjunction with culture or other means of confirmation for diagnosis. Direct fluorescent antibody (DFA) staining is available for C. trachomatis and N. gonorrhoeae, although rarely used for the latter. DFA is the only available method that allows assessment of the quality of the specimens that have been submitted, but it can be a time-consuming, subjective, and labor-intensive procedure. Sensitivity of the C. trachomatis DFA assay in most studies is considered less than or equivalent to that of culture (107, 129). Specificity is usually shown to be 97% (129). The primary use of the DFA today is for discrepancy testing in studies in which NAATs or other nonculture methods are being compared to culture or to each other. Enzyme Immunoassay Enzyme immunoassays (EIAs) have been available for C. trachomatis and N. gonorrhoeae and afford the capability of batch testing for a less laborconsuming method of detection. EIA for N. gonorrhoeae is no longer commercially available and is not covered further. The Wampole MicroTrak (Wampole Laboratories, Princeton, N.J.) for C. trachomatis detection has been reported most widely in the literature. The sensitivity ranges from 61 to 98%, with specificities from 96 to 100% (48). Wampole has recently discontinued distribution of the MicroTrak products; Abbott’s Chlamydiazyme (Abbott Laboratories, Abbott Park, Ill.) is no longer available in the United States. The Bio-Rad Pathfinder Chlamydia EIA (Bio-Rad Laboratories, Hercules, Calif.) for the detection of C. trachomatis is available in the United States, but references in the literature are limited. Specificity of the EIA can be enhanced if one uses a competitive assay with blocking antibody, for example, to confirm a positive result. EIA is not performed in many laboratories today since the advent of NAATs. In a study comparing NAATs versus EIA for
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the detection of C. trachomatis, Forward reported a 46% increase in positive results with a $18 price savings (Canadian dollars) per test when PCR was utilized as compared to EIA (78). Probe The DNA probe (PACE 2; Gen-Probe Inc., San Diego, Calif.) assay became the procedure used in many laboratories for the detection of C. trachomatis and N. gonorrhoeae prior to the advent of NAAT. Sensitivity and specificity for the probes have been shown to be slightly less than those of culture (78% sensitive versus culture), but turnaround times are much better for C. trachomatis in particular (15, 127). For laboratories where transport of specimens for culture is problematic, the probe assay can be a good alternative to culture for N. gonorrhoeae. Laboratories today could combine a NAAT for C. trachomatis, to enhance recovery, and a probe for N. gonorrhoeae, to maximize sensitivity, in a cost-effective manner (40, 45). NAATs The commercially available NAATs are displayed in Table 3. Many studies using NAATs have demonstrated a sensitivity approaching 100%, with some variation in specificity, depending on the population being studied. One of the striking advantages of a NAAT is the ability to use urine as a specimen. Although there are limited studies in the literature that demonstrate use of urine with EIAs, this specimen type can only be reliably tested if NAAT is employed. In analyzing NAATs, or any assays for the detection of C. trachomatis or N. gonorrhoeae, one must be careful to note several factors such as the prevalence of the analyte in the population studied, the specimen type, whether endocervical or urethral swabs or urine is used, and the gender of the patients. All of these variables will affect the overall results and their analysis. Likewise, to which assay the “new” or test assay is being compared will strongly influence comparative results. In most studies today, an “expanded gold standard” is being used as comparators for NAATs, since no one test can be considered as the sole “correct” answer. A recent publication by Katz et al. cautions about the validity of N. gonorrhoeae test results when using a NAAT without taking into consideration disease prevalence and the predictive positive value of a positive test in the population being tested (116). Serology Serologic assays for C. trachomatis are available in the form of a complement fixation assay, a microimmunofluorescence assay, and EIA; however, their utility for the diagnosis of acute infection is very limited.
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Table 3.
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Chlamydia trachomatis and N. gonorrhoeae NAATs available and FDA cleared in the United States (2005)
NAAT Product
Principle of assay
Amplicor, COBAS AMPLICOR CT/NG assays
PCR
BD ProbeTec ET CT/GC and CT assays APTIMA Combo 2, CT, and GC assays
Strand displacement amplification (SDA) Transcription-mediated amplification (TMA)
Hybrid Capture 2 CT/NG, CT, and GC assays
Hybrid Capture (HC)
Manufacturer
FDA-cleared sample types
Roche Diagnostics, Corp., Indianapolis, Ind.
Female, cervical swab, FCU,a,b PreservCyt: cMale, urethral swab, FCU Becton Dickinson, and Company, Female, cervical swab, FCU: Sparks, Md. Male, urethral swab, FCU Gen-Probe Inc., San Female, cervical swab, Diego, Calif. vaginal swab, PreservCyt, FCU: Male, urethral swab, FCU Digene Corporation, Female, cervical brush or Gaithersburg, Md. swab
a
FCU, first-catch urine. Roche PCR not cleared for N. gonorrhoeae testing on female urine. PreservCyt liquid Pap medium for collection of cervical cells. Clearance granted to Cytyc Corporation, Boxborough, Mass., not Roche Diagnostics.
b c
Serologic cross-reactivity between Chlamydia species has been well documented (15, 126). In addition, antibodies are long-lived, and a positive result cannot distinguish past or present exposure to the organism. If one obtains an acute and convalescent titer and a fourfold rise is present, this may indicate acute, active infection with C. trachomatis, but it would require 1 month for the rise to occur. This time delay renders serology inappropriate for the diagnosis of acute infection and administration of appropriate antimicrobials (46). The presence of IgM, although rarely positive, is not helpful since it could actually represent exposure to other species of Chlamydia, specifically C. pneumoniae (15). Serology is the method of choice, however, for diagnosis of LGV, a specific infection with the L biovars of C. trachomatis. Culture and other nonculture assays are not available for the specific diagnosis of LGV. Chlamydia serological assays have also been used in the evaluation of infertility and other gynecologic problems, but their accuracy is still under investigation (111, 151, 199).
SPECIMEN TYPES AND COLLECTION Proper specimen collection is critical for all methods used to detect C. trachomatis and N. gonorrhoeae infections. Specimens for testing by the commercially available NAATs must be obtained as directed by the manufacturer in the package insert. Each of these assays is approved for specific specimen types and has its own assay-specific collection and transport device (Table 3). Other specimen types or transport systems must be validated by the user (50). The performance characteristics of NAATs have been shown to be related to specimen adequacy. The presence of columnar epithelial cells has been associated with superior assay performance for NAAT and other methods. Studies have demonstrated that the sensitivity of commercially available assays is af-
fected by the quality of the endocervical specimen; variations in sensitivities range up to 10% for specimens containing endocervical cells compared to inadequate specimens (12, 118, 119, 136, 221). The need to obtain columnar cells to detect N. gonorrhoeae is less critical than to detect C. trachomatis (33). The CDC recommends routine or periodic assessment of endocervical specimen quality (33). Endocervical Specimens The procedure for collecting female endocervical swabs is well known. Briefly, a cleaning swab should be used to remove secretions and mucus from the cervical os. An endocervical swab or brush supplied by the manufacturer should be inserted 1 to 2 cm into the cervical canal and rotated for 15 to 30 s (33). The swab should then be placed in the medium supplied for transport. Inhibition with Endocervical Specimens The increased sensitivity of NAATs over culture is well documented; however, early studies revealed problems with inhibitors for certain types of amplification reactions. Bauwens et al. (11) reported reduced sensitivity of PCR with endocervical specimens due to the presence of inhibitors. These inhibitors were considered labile as many of the repeat tests from falsely negative PCR reactions became positive after storage. Verkooyen et al. (211) described factors that were associated with inhibition of PCR, including a higher cervical pH (cervical mucosa pH of 7.5) and specimen processing on the day of collection. Unlike some other publications, these authors noted that blood did not affect PCR amplification. Many techniques have been used to facilitate removal of inhibitory substances. Transport of swabs dry rather than in PCR transport medium resulted in fewer false-negative specimens (120). Reduction or removal of inhibitors has been accomplished by dilution of specimens 1:10, heat treatment (95°C for 10
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Nucleic Acid Amplification Tests for Detection of C. trachomatis and N. gonorrhoeae
min), a combination of heat treatment and 10-fold dilution, freeze-thawing, and holding specimens at 4°C (211). These same techniques have been applied to urine as well (42, 140). Inhibition of amplification in specimens can be detected by the use of internal amplification controls. Currently such controls are available only for PCR and strand displacement amplification (SDA) assays. The use of these controls is optional and up to the discretion of each laboratory. When introducing NAAT with either of the above methods, it is prudent to include the amplification control until such time an inhibitory rate with common specimen types can be computed. Unpublished data from one of the authors’ own laboratories showed an inhibition rate of 0.37% in over 52,000 endocervical and urethral specimens tested by PCR (W. D. LeBar, unpublished data). The use of an amplification control was found to be of limited value for use with vaginal swab specimens (56). Because of difficulties with urine specimen preparation, amplification controls are recommended when available. Urethral Specimens For collection of a male urethral swab, the patient should not have urinated within the previous 1 h. If the patient has a discharge, the exudates may be collected and examined by Gram stain for organisms typical of N. gonorrhoeae. A thin-wired swab is introduced 2 to 4 cm into the urethra, rotated, withdrawn, and placed into transport medium for testing. Noninvasive Specimen Types NAATs have proved to be more sensitive (with equivalent specificity) than other nonculture technologies used for the diagnosis of C. trachomatis and N. gonorrhoeae infections. This increased sensitivity has extended the ability to test a variety of noninvasively collected sample types such as first-catch urine (FCU) or vaginal swabs from females. Noninvasive samples are useful in screening at-risk populations and for epidemiological studies. Urine Specimens The use of FCU as a specimen for chlamydial diagnosis was first reported by Caul et al. (30), using an EIA compared to direct fluorescent monoclonal antibody. Since that time, FCU from both males and females has been found to be an appropriate specimen for NAAT. The ease of collection of this sample type has permitted the expansion of screening programs beyond the traditional clinic setting (3, 74, 81, 128, 144, 179, 205, 207). In males, NAATs using FCU have been found to be sensitive and specific in both symptomatic and asymptomatic patients and may be
9
considered the test of choice for the diagnosis of urethral chlamydial infection (74, 109, 205, 207). For females, urine NAATs can be affected by a variety of factors. The presence of phosphates, nitrates, crystals, beta-human chorionic gonodotropin, and hemoglobin is associated with inhibition of amplification (41, 42, 140, 161). This inhibition is transient and can be reduced by removing residual urine from the pellet after processing and by storing the specimen overnight in the freezer or refrigerator (41). The volume of urine processed has also been shown to have an effect on NAAT sensitivity. Each NAAT has specific FCU volume requirements for testing. Moncada et al. (152) recently demonstrated that the sensitivity of NAAT varied drastically when specimen volumes ranging from 20 to 90 ml of urine were collected and analyzed. Verkooyen et al. (212) reported the results of a study designed to assess the abilities of laboratories to detect C. trachomatis in a panel of urine samples by various NAATs. Although the specificity for this group of specimens was high, sensitivity problems occurred frequently, underscoring the need to adhere to all manufacturers’ stated protocols. A recent review has summarized the English language literature describing chlamydial and gonococcal testing with self-collected urine or vaginal specimens outside of clinic settings (77). Liquid-Based Cytology Specimens The introduction of liquid-based cytology has made possible the screening of residual materials for chlamydial and gonococcal infection. Using cervical scrapings collected for cervical cytology, Lentrichia et al. (130) demonstrated the feasibility of detecting chlamydia from a fluid-based sample. Liquid-based cytology samples collected in Autocyte preservative fluid from women at high risk for chlamydia infection and tested by a modified LCx were compared to the standard Abbott LCx method (Abbott Laboratories, Abbott Park, Ill.). There was agreement for 588 of 590 specimens (6). The collection of endocervical scrapings into PreservCyt (Cytyc Corporation, Boxborough, Mass.) has been shown to be an acceptable process for sample collection for different NAATs (14, 230). Chlamydial DNA was found to be stable in PreservCyt for 6 weeks at room temperature (14). Koumans et al. (124) examined the agreement between specimens collected in PreservCyt and multiple NAATs for the detection of C. trachomatis and N. gonorrhoeae. In this study, test performances were similar for LCx-PreservCyt, LCx-cervix, and LCxurine, with sensitivities from 93 to 99% for C. trachomatis and 81 to 83% for N. gonorrhoeae and specificities from 95.5 to 99% for C. trachomatis and 99.1 to 99.6% for N. gonorrhoeae using an infected patient PCR-based standard. At present, collection of
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liquid-based cytology samples in PreservCyt fluid is approved by the U.S. Food and Drug Administration (FDA) for testing for chlamydia and gonococcal infection on the COBAS AMPLICOR (Roche Diagnostics, Indianapolis, Ind.) and APTIMA Combo 2 (Gen-Probe Inc., San Diego, Calif.). Vaginal Specimens A question could be posed as to what is the best specimen to use to detect C. trachomatis and N. gonorrhoeae infection in women. After reports of the successful application of urine for testing, there were a number of reports that vaginal or vulvar swabs may be useful as noninvasively collected samples for clinical and epidemiological studies (194, 196, 226). Numerous studies have demonstrated that self-collected vaginal swabs, introital swabs, and tampons are appropriate for diagnosis of genital tract infection, perform as well as physician-collected endocervical swabs, and are suitable for routine screening of asymptomatic women (4, 23, 28, 38, 56, 88, 178, 192, 227). These noninvasive specimen types have been shown to be preferable to patients (105, 169, 185, 210). In January 2004, the Gen-Probe APTIMA vaginal swab specimen collection kit received FDA clearance. This collection device allows sample collection by the physician or by the patient, under medical supervision, for C. trachomatis and N. gonorrhoeae testing. Nonurogenital Sites Collection of samples from ocular, pharyngeal, and rectal sites follows standard protocols. Currently, no NAAT is FDA cleared for performance with specimens from any of the above sites. Conjunctival swabs may be indicated for conjunctivitis in adults and in newborns or infants with neonatal conjunctivitis or pneumonia consistent with C. trachomatis infection (33). Pharyngeal swabs may be indicated from patients exposed during oral sex, neonates with conjunctivitis or pneumonia consistent with C. trachomatis infection, or in cases of possible sexual abuse (33). Rectal swabs may be indicated in patients with a history of receptive anal intercourse or proctitis or in cases of possible sexual abuse (33). PCR has performed comparably to culture for detecting C. trachomatis in conjunctival and pharyngeal specimens from infants with conjunctivitis (69, 96, 197). NAATs have been evaluated for diagnosis of chlamydial and gonococcal infection in rectal and pharyngeal specimens. In general, these tests have performed with comparable sensitivity to culture; however, there have been few published studies (85, 132, 133, 162, 190, 229). These uses are not FDA cleared, and consideration needs to be given to potential cross-reacting species of Neisseria that may produce false-positive results.
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Specimen Pooling Pooling of specimens has been investigated in public health laboratories and in facilities processing large numbers of specimens. Depending on the prevalence of infection, considerable reagent cost-savings and technologist time-savings may be achieved by the pooling when compared to testing individual samples. This strategy employs screening samples in groups. If any group is positive, individual specimens within the group are then retested to find the positive sample. Most of the chlamydia pooling studies have been performed with urine specimens; however, recent studies have been extended to vaginal and endocervical samples as well (47, 59, 115, 155, 166). Pooling has been shown to be a sensitive and specific strategy to reduce the number of tests performed without sacrificing accuracy. It should be noted that pooling of specimens has not been FDA cleared for any nucleic acid amplification method; therefore, verification must be performed by any laboratory implementing this strategy (50).
REVIEW OF CURRENT NAATS FOR C. TRACHOMATIS AND N. GONORRHOEAE DETECTION Ligase Chain Reaction: LCx Probe System (Abbott Laboratories, Abbott Park, Ill.) The Abbott LCx C. trachomatis and N. gonorrhoeae assays were removed from the market in 2003. Much of the literature about other commercial NAATs used these assays as a comparator; therefore, LCx will be covered briefly. The LCx assays used ligase chain reaction (LCR) amplification technology (Abbott Laboratories, package insert, LCx Probe System Chlamydia trachomatis Assay, List No. 9B11-91, 2000; Abbott Laboratories, package insert, LCx Probe System Neisseria gonorrhoeae Assay, List No. 8A48-82; 2001). The principle of LCR involves the release of singlestranded DNA from the specimen in transport buffer by using heat. Separate assays for C. trachomatis and N. gonorrhoeae meant that individual reactions were required to detect the two analytes. The prepared sample is added to the LCR mixture consisting of oligonucleotide probes, thermostable ligase, polymerase enzymes, and individual oligonucleotides. The four probes are designed in pairs that hybridize to single-stranded DNA target sequences in the exposed single-stranded DNA in the sample preparation. The C. trachomatis assay targets a conserved DNA sequence on a cryptic plasmid, and the N. gonorrhoeae assay targets a DNA sequence of the opa gene. The polymerase enzyme then fills in the gap between the hybridized probe pairs. The ligase
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Nucleic Acid Amplification Tests for Detection of C. trachomatis and N. gonorrhoeae
enzyme covalently joins the pairs of probes to form an amplification product complementary to the target sequence. After multiple rounds of replication in the thermocycler, the amplified product is detected on the LCx analyzer by means of the immunoreactive labeling of the probes in the amplicon detected by microparticle enzyme immunoassay. LCx C. trachomatis and N. gonorrhoeae assays were FDA cleared for testing of female endocervical and male urethral swabs and female and male FCU specimens from symptomatic and asymptomatic individuals. The LCx probe system consisted of an LCx analyzer for product detection, a thermocycler for product amplification, centrifuges, and a dry bath for specimen processing. Contamination control was achieved by unidirectional workflow, surface decontamination with 10% bleach, and chemical inactivation of amplicons with hydrogen peroxide plus a metal chelator, resulting in a 107-fold reduction in product. The LCx probe system contained no internal inhibition control for individual samples. A negative result may have been due to lack of target or target level below the sensitivity of the assay (true negative) or inhibitors of amplification present in the specimen (false negative). Studies on urine specimens for the detection of C. trachomatis demonstrated that nitrates and phosphate may inhibit LCR (41, 140, 198). Inhibition could be removed by overnight storage of samples at 4 or 70°C and by dilution (140, 161, 198). In one study of C. trachomatis detection in female urines, inhibition of LCR amplification was seen in approximately 3% of samples. The authors concluded that most inhibition would disappear during transport and that additional steps to remove inhibition for every urine were not warranted (161). Future Development Abbott Laboratories currently has no commercial product available in the United States for the amplified detection of C. trachomatis and N. gonorrhoeae. In collaboration with another company, artus GmbH, they are marketing a commercial real-time PCR test for detection of C. trachomatis. Known as the RealArt C. trachomatis PCR, this test is CE-marked and available throughout the world, except the United States, Canada, and Japan. Abbott does not intend to sell this product in the United States. Abbott also has a fully automated sample preparation/real-time PCR system in development. It has agreements with Tecan of Zurich, Switzerland, for the development and commercialization of two automated sample preparation systems, the m1000 and m2000sp. Introduction of C. trachomatis/N. gonorrhoeae duplex and C. trachomatis-only NAATs in the United States is planned using the totally automated
11
m2000 sample preparation system linked to real-time homogeneous PCR technology. PCR amplification will be detected homogeneously with random-coiled oligonucleotide probes using a real-time thermal cycler/detection instrument codeveloped with Applied Biosystems. (J. Yu, Abbott Laboratories, personal communication). PCR: COBAS AMPLICOR CT/NG Test (Roche Diagnostics Corporation, Indianapolis, Ind.) Principle of Procedure Roche Molecular Systems first marketed a manual assay (Roche AMPLICOR CT/NG Test) utilizing PCR for the simultaneous detection of C. trachomatis (CT) and N. gonorrhoeae (NG) in microwell plate format. In the early 1990s, a semiautomated format, the Roche COBAS AMPLICOR CT/NG test, was introduced, and this has become one of the three most widely used systems for the simultaneous detection of these two pathogens. Because the majority of Roche users are performing the COBAS AMPLICOR method, further discussion of the manual AMPLICOR is limited. The method of amplification for the COBAS is PCR (Roche Diagnostics, package insert, COBAS AMPLICOR CT/NG Test for Chlamydia trachomatis, Revision 1.0, 1999; Roche Diagnostics, package insert, COBAS AMPLICOR CT/NG Test for Neisseria gonorrhoeae, Revision 3, 1999). The COBAS test detects both C. trachomatis and N. gonorrhoeae simultaneously and includes an internal control to detect the presence of any inhibitors in the specimen. There are four major steps in the procedure: specimen processing; PCR of the target DNA; hybridization of the amplified DNA to oligonucleotide probes; and detection of the hybridized probe and amplicon. For sample processing, specimens are treated with a detergent solution to release the organism’s DNA. A second detergent solution is then added to prepare the lysed specimen for amplification. The processed specimens are added to the amplification tubes containing a reaction mixture with biotinylated primers, thermostable Thermus aquaticus DNA polymerase (Taq Pol), and excess deoxynucleotide triphosphates (dNTPs). The reaction mixture is heated to denature the double-stranded DNA, exposing the specific primer target sequences allowing primers to bind, and extending the target region by the action of Taq Pol. The COBAS AMPLICOR analyzer automatically repeats this process (thermocycling), thus doubling the amount of amplicon DNA for each of 35 cycles. The target for C. trachomatis primers is a region of 270 nucleotides within the cryptic plasmid DNA and for N. gonorrhoeae, a region of 201 nucleotides within the cryptic plasmid in the M.Ngo P11 gene. Following PCR amplification, the amplicons
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Leber et al.
are chemically denatured to form single-stranded DNA. The denatured specimen is transferred to detection cups along with a suspension of magnetic particles coated with an oligonucleotide probe specific for the target region (C. trachomatis, N. gonorrhoeae, or internal control). The magnetic particles are washed to remove unbound material, avidinhorseradish peroxidase conjugate is added, and the particles are washed again. The substrate solution containing hydrogen peroxide and 3,3,5,5-tetramethylbenzidine is added to each reaction. In the presence of hydrogen peroxide, the particle-bound horseradish peroxidase catalyzes the oxidation of 3,3,5,5-tetramethylbenzidine to form a colored complex. The absorbance is then measured by the analyzer at a wavelength of 660 nm. Instrumentation Performance of the COBAS assay requires a vortex mixer, microcentrifuge, heating block, the COBAS AMPLICOR analyzer, and printer. Specimen processing occurs manually. The COBAS analyzer, a benchtop system, automates the amplification and detection steps of the PCR testing process. It combines five instruments into one (thermal cycler, automatic pipettor, incubator, washer, and reader). The instrument allows barcode data entry and has a 48-sample capacity per run. Sample Collection and Transport The COBAS AMPLICOR CT test is FDA cleared for testing female endocervical and male urethral swabs from symptomatic and asymptomatic patients. The COBAS AMPLICOR NG test is FDA cleared for testing endocervical specimens, male urethral specimens from symptomatic patients, and male urine specimens. The COBAS AMPLICOR NG test is not cleared by the FDA for testing on female urine or urethral swabs from asymptomatic males. Swabs are collected and transported in Chlamydia culture transport medium such as 2SP, SPG, Bartels ChlamTrans (Bartels, Inc.), or M4 Culture Transport Medium (Microtest, Inc.). Various laboratories have reported on the use of M4 and M4RT media for transport (7). M4RT is more convenient for use than M4 because it does not require refrigeration before inoculation. Media lots must be qualified for use in each individual laboratory (Roche Diagnostics, package insert, COBAS AMPLICOR CT/NG Test for Chlamydia trachomatis, Revision 1.0, 1999; Roche Diagnostics, package insert, COBAS AMPLICOR CT/NG Test for Neisseria gonorrhoeae, Revision 3, 1999). Swabs may be transported at 18 to 25°C provided that the total time of storage and transport is less than 1 h. Specimens should be refrigerated if testing is delayed for more than 1 h. If testing is delayed for longer times, swabs may be stored refrigerated
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for 7 days or frozen at 20°C or colder for up to 30 days. For urine specimens, the patient must not have urinated during the previous 2 h. FCU (first 10 to 50 ml) is collected in a clean polypropylene container. Urine specimens are stable for up to 24 h at 18 to 25°C. If urine will not be processed within 24 h, it can be held refrigerated for 7 days or frozen at 20°C or colder for up to 2 months. The presence of inhibitor substances in specimens may prevent PCR amplification. Swabs for the PCR assay are collected from endocervix or urethra after the external surface is cleansed to remove excess mucus and debris. If much of the mucus remains in the specimen that is being submitted to the laboratory or the specimen is very bloody, PCR may be inhibited, causing a falsenegative result (200). Urine specimens have also been shown to contain inhibitory substances that could result in false-negative results (2, 140, 200). As with other amplification assays, testing of specimens outside the urogenital tract with COBAS CT/NG PCR is not FDA cleared. There are some publications in which specimen types, such as rectal and pharyngeal, have performed adequately with COBAS (85, 133). Vaginal samples, including those that have been self-collected, have been reported to provide results comparable to results with wellcollected endocervical samples (80, 178). Liquid cytology transport media, such as ThinPrep and SurePath, have been used to transport samples for processing of Pap smears, human papilloma-virus detection, and C. trachomatis and N. gonorrhoeae PCR testing, all from the same sample (14, 124). In 2002, Cytyc Corporation announced that FDA clearance was granted for C. trachomatis and N. gonorrhoeae testing directly from the ThinPrep Pap Test collection vial using COBAS AMPLICOR PCR. Approval was issued to Cytyc, not Roche Diagnostics. Instructions for testing are not provided in the AMPLICOR product insert but must be obtained from Cytyc. Inhibition and Contamination Controls There is an optional C. trachomatis and N. gonorrhoeae internal control (IC), run simultaneously with each specimen that provides a means of identifying any substances in the specimens that could inhibit PCR amplification. The IC is a recombinant plasmid DNA containing primer regions identical to those of the C. trachomatis target sequence, a randomized internal sequence of similar length and base composition as the N. gonorrhoeae and C. trachomatis target sequences, and a unique probe-binding region that differentiates it from the target amplicon. The IC is introduced into each amplification reaction to be coamplified with target DNA from the clinical specimen. The amount of the IC added is set at 20 copies
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Nucleic Acid Amplification Tests for Detection of C. trachomatis and N. gonorrhoeae
per reaction; this acts to monitor for adequate amplification of the lower level of sensitivity of the assay to prevent the reporting of a false-negative result. If the IC is negative, the negative results of the clinical samples should be questioned; repeat testing of the sample after a freeze-thaw or dilution procedure may eliminate the inhibition and allow positive IC and correct results with the clinical samples. As mentioned above, use of the IC is optional. The control is amplified in each reaction, but the user decides whether the product is detected and reported. In the presence of large amounts of target DNA, the PCR reactants in the COBAS CT/NG test may be consumed, leading to failure of the IC to amplify. A single set of primer oligonucleotides is used to amplify both the IC and target C. trachomatis DNA. All three targets (C. trachomatis, N. gonorrhoeae, and IC) also draw from a common pool of dNTPs and polymerase. Thus, IC amplification may be suppressed due to competition in the samples containing large amounts of target C. trachomatis DNA. One group of investigators was able to demonstrate this suppression of the IC signal by testing samples of known increasing amounts of target C. trachomatis DNA with IC included. About 5,000 to 50,000 copies per milliliter of sample were needed to decrease the IC response; a concentration of 2.5 105 had to be present to bring the IC down to background levels. Compared to the IC, C. trachomatis and N. gonorrhoeae target DNAs were less sensitive to competition. Approximately 2.5 105 copies per test sample of C. trachomatis or N. gonorrhoeae DNA were required to produce a small decrease in the signal generated by 25 copies of N. gonorrhoeae or C. trachomatis DNA (172). A second advantage of use of the IC in the COBAS AMPLICOR system is to monitor for potential competition between the C. trachomatis and N. gonorrhoeae target DNA if both are present. If only one of the pathogens is determined to be positive in a sample in which the IC is negative, results for the second analyte may not be truly negative. Rather, there may have been a competition between the two targets. This competition has been shown to occur only when the target of one is 10fold more than that of the second (172). To avoid contamination of specimens by amplicons, AmpErase and deoxyuridine triphosphates (dUTPs) are added to each reaction vessel in the COBAS procedure. AmpErase is an enzyme, uracil-N-glycosylase, that recognizes and catalyzes the destruction of DNA strands containing deoxyuridine, but not DNA containing thymidine. Deoxyuridine is not contained in naturally occurring DNA, so when it is added as one of the dNTPs in the Master Mix reagent, amplicons that contain deoxyuridine triphosphate will be destroyed before amplification of target DNA. Amp-
13
Erase is inactive above 55°C, and hence cannot destroy target amplicon during the thermal cycling steps. Following amplification, any residual enzyme is denatured by the addition of denaturation solution by the COBAS analyzer. Unidirectional workflow and environmental monitoring for amplicon contamination are also recommended, as for any NAAT. Result Interpretation Results of the COBAS AMPLICOR are read colorimetrically at 660 nm, and results are displayed as optical density (OD) readings; a result 3.5 is considered positive and 2.0 is negative. The assay is qualitative and no correlation can be made between the magnitude of a positive absorbance signal and the number of cells infected with C. trachomatis or the number of CFU of N. gonorrhoeae. Results between 2.0 and 3.5 are in what is called an equivocal zone, and the manufacturer recommends that with N. gonorrhoeae results in the equivocal zone, the assay should be repeated two more times and the results of the two of three or three of three same answers should be reported. On the repeat samples, a positive 2.0 is accepted as positive. For C. trachomatis, the manufacturer’s recommendation is to ask for a new specimen if results are in the equivocal zone. If the results are found to be inhibitory (i.e., if the IC does not amplify), and results of the C. trachomatis and/or N. gonorrhoeae assay are negative, a new specimen should be requested or repeat testing of specimens can be attempted. Elimination of inhibition has been demonstrated after repeat testing subsequent to one of these three methods: dilution of the samples, freezing of the sample at 70°C, or refrigeration for at least 24 h (140). If the inhibition is no longer seen, results can be reported as determined without inhibition; if inhibition is still present after repeat testing with one of the suggested methods, then equivocal results should be reported and repeat specimens requested. Performance Characteristics Sensitivity of the COBAS CT/NG test is excellent as compared to that of other NAATs. There are variances between males and females and between genital swabs and urine samples, but overall a good sensitivity has been seen in most studies. The prevalence of C. trachomatis and N. gonorrhoeae in the population studies accounts for differences between studies, but again, that is true for all of the amplification assays. Tables 4 and 5 summarize the performance characteristics of the COBAS CT/NG tests. In literature reviews comparing PCR to culture, EIA, or probe assays, enhanced sensitivity and specificity for urogenital samples, especially for the detection of C. trachomatis, have been demonstrated. Black (15) reviewed over 11 published reports on the
CX CX FCU FCU UR UR FCU FCU UR CX FCU UR FCU CX FCU UR FCU
1,098, F (asymptomatic)
1,138, F (symptomatic)
1,098, F (asymptomatic) 1,138, F (symptomatic) 710, M (asymptomatic)
1,230, M (symptomatic)
710, M (asymptomatic)
1,230, M (symptomatic)
442, F
447, F
442, F
565, M
565, M
2,010, F
1,248, F
371, M
254, M
7.2
7.2a
2.4
2.4
8.8
7.8
6.3
6
11.3
20.6
12.5
19.0
8.1 9.2 11.7
9.1
7.7
9.2
Prevalence (%)
Patient infected statusb (culture, alternate target PCR on swab and urine) Patient infected statusb (culture, alternate target PCR on swab and urine) Patient infected statusb (culture, alternate target PCR on swab and urine) Patient infected statusb (culture, alternate target PCR on swab and urine)
Culture or LCR
Culture or LCR, alternate target PCR
Culture or alternate target PCR, DFA
Culture or alternate target PCR, DFA
Culture or alternate target PCR, DFA
Culture or alternate target PCR, DFA Culture or alternate target PCR, DFA Culture or alternate target PCR, DFA
Culture or alternate target PCR, DFA
Culture or alternate target PCR, DFA
Culture and PCR (manual)
Reference methoda
b
F, female; M, male, CX, endocervical swab; UR, urethral swab. True positives are defined as two or more positive results with reference tests in any combination of method and sample type.
CX
Specimen types
654, Fa
No. of subjects, sex
94.4
100
95.1
96.5
95.9
92.2
96.4
78.6
94
92.5
92.1
97.4
93.3 93.3 98.8
96.1
97.6
93.3
Sensitivity (%)
100
98.5
99.8
99.4
99.4
99.1
99.8
98.8
99.2
98.5
98.4
98.7
99.6 98.5 98.7
99.2
99.5
99.7
Specificity (%)
100
89.1
92.9
87.4
94
89.9
96.4
81.5
92
94.0
89.1
94.6
95.4 86.0 91.1
92.5
94.3
99.3
Positive predictive value (%)
99.6
100
99.8
99.8
99.6
99.4
99.8
98.6
99.2
98.1
98.9
99.4
99.4 99.3 99.8
99.6
99.8
96.4
Negative predictive value (%)
Leber et al.
a
Livengood et al., 2001 (135) Van Der Pol et al., 2000 (207) Van Der Pol et al., 2000 (207) Van Der Pol et al., 2000 (207) Van Der Pol et al., 2000 (207) Van Der Pol et al., 2000 (207) Van Der Pol et al., 2000 (207) Van Der Pol et al., 2000 (207) Pasternack et al., 1997 (165) Puolakkainen et al., 1998 (168) Puolakkainen et al., 1998 (168) Puolakkainen et al., 1998 (168) Puolakkainen et al., 1998 (168) Vincelette et al., 1999 (214) Vincelette et al., 1999 (214) Vincelette et al., 1999 (214) Vincelette et al., 1999 (214)
Reference
Table 4. Recent studies evaluating the Roche COBAS AMPLICOR assay for detection of C. trachomatis
14 CUMITECH 44
and PCR (manual) or alternate target or alternate target or alternate target or alternate target or alternate target or alternate target F, female; M, male; CX, endocervical swab; UR, urethral swab; NA, not available. a
CX CX FCU UR UR FCU FCU 618, Fa 2,192, F 2,192, F 714, M (asymptomatic) 1,267, M (symptomatic) 714, M (asymptomatic) 1,267, M (symptomatic) Livengood, 2001 (135) Martin, 2000 (145) Martin, 2000 (145) Martin, 2000 (145) Martin, 2000 (145) Martin, 2000 (145) Martin, 2000 (145)
4.4 6.3 6.2 2.9 29 2.0 29
Specimen types No. of subjects, sex
Prevalence (%)
Culture Culture Culture Culture Culture Culture Culture
Comparison methoda
PCR PCR PCR PCR PCR PCR
96.3 96.4 79.6 90.5 99.5 78.6 96
100 99.5 99.8 99.0 99.0 99.9 99.9
99.8 NA NA NA NA NA NA
100 NA NA NA NA NA NA
Nucleic Acid Amplification Tests for Detection of C. trachomatis and N. gonorrhoeae
Reference
Table 5. Recent studies evaluating the Roche COBAS AMPLICOR assay for detection of N. gonorrhoeae
Sensitivity (%)
Specificity (%)
Positive predictive value (%)
Negative predictive value (%)
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15
use of the manual AMPLICOR PCR for the detection of C. trachomatis. She demonstrated that sensitivity was 64 to 96% and specificity was 96% for PCR, as compared to culture and other amplification assays or combinations of the two. There are minimal reported data on the use of the manual AMPLICOR NG test. In the early 1990s, an automated format, the Roche COBAS AMPLICOR C. trachomatis/ N. gonorrhoeae PCR test was introduced, and today it is more widely used than the AMPLICOR CT/NG test. Equivalence in performance was found when COBAS was compared to the manual AMPLICOR PCR, with a much easier throughput and workflow than the manual AMPLICOR CT/NG assay (Tables 4 and 5). Puolakkainen et al. (168) compared COBAS to LCx for the detection of C. trachomatis and found that in 97% of the samples tested, results were identical between the two “automated” methods. Specificity for C. trachomatis in the PCR assays is 99%; specificity for N. gonorrhoeae is good; however, there have been reported problems with crossreactions with species of Neisseria other than N. gonorrhoeae (72, 73, 131). The AMPLICOR assay targets a sequence in the cytosine DNA methyltransferase gene of N. gonorrhoeae. Similar sequences are present in some strains of “normal flora” Neisseria species, such as N. sicca, N. subflava, N. lactamica, N. flavescens, and N. cinerea. Although the latter organisms are usually present in the oropharyngeal cavity, they may be present in the genital tract and may result in false-positive results with the N. gonorrhoeae AMPLICOR assay. In a study that correlated specificity of the assay with positive predictive values (PPV) based on the type of specimen assayed, male urine samples achieved as high as 83% PPV as compared to a low of 13.3% PPV with female endocervical samples (63). In that same study, a correlation was shown between the absorbance values (OD) of the assay (the measurement of positive detection in the assay) and false-positive results. If the OD reading was in the positive range, i.e., 3.5, the likelihood that a positive N. gonorrhoeae result was true was 65% as compared to a 10% likelihood if the OD was in the range of 2.5 to 3.5. Results of a positive N. gonorrhoeae result in the latter range (2.5 to 3.499), or what some people would call the “gray zone,” should be repeated twice, as per the manufacturer’s package insert instructions. Further confirmation of any positive N. gonorrhoeae result before reporting should be considered if NAAT is the method used for screening (33). Leslie et al. (131) found a confirmation rate of 86.2% for urethral swabs in a population of male patients with a 7% prevalence of N. gonorrhoeae. The confirmation rate in cervicovaginal specimens was only 5.7%, but this represented only 3 of 1,256 patients in a population with 0.24%
16
Leber et al.
prevalence. When a real-time PCR assay (Roche LightCycler), targeting different genes than the COBAS assay, was used to confirm N. gonorrhoeae results of 172 endocervical samples from women, 59.8% of the COBAS positive samples confirmed as positive on the LightCycler assay (195). Future Directions Improvements in the sensitivity for detection of C. trachomatis in the COBAS AMPLICOR system continue to appear in the literature. Pretreatment of clinical samples with an erythrocyte lysis buffer and additional centrifugation steps aided in detecting what were considered to be low-level infections missed with the routine procedure of sample preparation (160). For the future, Roche is planning to automate the sample processing by means of the MagnaPure for extraction of DNA or use of the more automated Tecan systems for many of the pipetting steps of the procedures for setting up the A-rings. Roche’s TaqMan instrumentation, utilizing real-time PCR technology, may also become a format for amplification assays for C. trachomatis and N. gonorrhoeae, either in addition to or as a replacement of the COBAS. Transcription-Mediated Amplification: APTIMA (Gen-Probe Inc., San Diego, Calif.) Principles of Procedure The APTIMA assays are second-generation target amplification nucleic acid tests for the qualitative detection and differentiation of rRNA from C. trachomatis and/or N. gonorrhoeae. The APTIMA assays combine the technologies of target capture, transcription-mediated amplification (TMA), and the dual kinetics assay (DKA) (Gen-Probe Inc., package insert, APTIMA Combo 2 Assay, IN0097-01 Rev. A, 2003). APTIMA Combo 2 (AC2), which detects C. trachomatis and N. gonorrhoeae, received FDA clearance in August 2001. APTIMA CT (ACT) for detection of C. trachomatis and APTIMA GC (AGC) for detection of N. gonorrhoeae use the same technology as AC2. These two tests were FDA cleared in 2005. The first-generation test, Amp CT, utilizes TMA for the detection of C. trachomatis only; it does not utilize target capture or DKA. The performance characteristics of Amp CT served as the basis for development of the second-generation assay and have been published elsewhere (57, 74, 84, 156). In the APTIMA assays, the target rRNA molecules are released in the specimen transport solution and then isolated from the samples by the use of capture oligomers in a method called target capture. This method differs from other NAATs that use crude lysis for isolation of nucleic acid. By isolating the rRNA
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away from other components in the sample, inhibitor substances are removed. The capture oligomers contain sequences complementary to specific regions of the target molecules as well as a string of deoxyadenosine residues. A separate capture oligomer is used for each target. During the hybridization step, the sequence-specific regions of the capture oligomers bind to specific regions of the target molecules. The capture oligomer-target complex is then captured out of solution by decreasing the temperature of the reaction to room temperature. This temperature reduction allows hybridization to occur between the deoxyadenosine region on the capture oligomer and the polydeoxythymidine molecules that are covalently attached to the magnetic particles. The microparticles, including the captured target molecules bound to them, are pulled to the side of the reaction vessel by magnets and the supernatant is aspirated. The particles are washed to remove residual specimen matrix that may contain amplification reaction inhibitors. After the target capture steps are completed, the specimens are ready for amplification. The AC2 assay replicates a specific region of the 23S rRNA from C. trachomatis and a specific region of the 16S rRNA from N. gonorrhoeae via DNA intermediates. For the ACT and AGC assays, the rRNA targets are different from those used in AC2; the C. trachomatis assay replicates a specific region of the 16S rRNA, and the N. gonorrhoeae target is a 16S rRNA target different from that in AC2. A unique set of primers is used for each target molecule. The rRNA amplification product sequences (amplicon) are detected by nucleic acid hybridization. Singlestranded chemiluminescent DNA probes, which are complementary to a region of each target amplicon, are labeled with different acridinium ester molecules. The labeled DNA probes combine with amplicon to form stable RNA:DNA hybrids. The selection reagent differentiates hybridized from unhybridized probe, eliminating the generation of signal from unhybridized probe. During the detection step, light emitted from the labeled RNA:DNA hybrids is measured as photon signals in a luminometer and is reported as relative light units (RLU). In DKA, differences in the kinetic profiles of the C. trachomatis and N. gonorrhoeae labeled probes allow the differentiation of signal; kinetic profiles are derived from measurements of photon output during the detection read time. The chemiluminescent detection reaction for C. trachomatis signal has very rapid kinetics and has the “flasher” kinetic type. The chemiluminescent detection reaction for N. gonorrhoeae signal is relatively slower and has the “glower” kinetic type. Assay results are determined by a cutoff based on the total RLU and the kinetic curve type.
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Nucleic Acid Amplification Tests for Detection of C. trachomatis and N. gonorrhoeae
Instrumentation The AC2 assay can be performed with two platforms, the direct tube sampling (DTS) systems and the TIGRIS DTS system. For the single-analyte tests (ACT and AGC), the DTS systems are the approved platform. DTS systems include DTS 400, DTS 800, and DTS 1600. These systems are designed for low-, medium-, and high-volume laboratories yielding up to 400, 800, or 1,600 results, respectively, in an 8-h shift. Equipment for the DTS systems includes three water baths, a multitube vortexer, a luminometer for measurement of chemiluminescent emission, and the target capture system (TCS). More recently a dry bath-vortexer has been developed to replace the water baths and vortexer (S. Yamagata, M. Kennedy, F. Li, K. Dickey, E. Brown, and T. Calderoni, Abstr. 104th Gen. Meet. Am. Soc. Microbiol., abstr. C-282, p. 175, 2004). The DNA extraction is performed on the TCS platform: for the DTS 800 and DTS 1600, this involves a modified Tecan workstation. The TIGRIS DTS system received FDA clearance in December 2003 and is the first fully automated molecular testing system for C. trachomatis and N. gonorrhoeae. Currently only AC2 can be used on this platform. The TIGRIS system offers direct tube sampling utilizing the specimen collection devices with piercible caps; both swab and urine collection devices are directly loaded on the instrument along with reagents. No other manual manipulation of specimens is required. All steps of specimen processing, amplification, detection, and result analysis are carried out by the instrumentation. There is positive sample identification throughout the process, and results are automatically linked to reagent lot numbers and can be uploaded to a laboratory information system. The throughput as reported by the manufacturer is approximately 500 samples in an 8 h shift and up to 1,000 samples in approximately 13 h. The TIGRIS DTS analyzer is the size of a common chemistry analyzer, measuring 69 36 72 inches and weighing 1,300 pounds. In a study performed by Novak et al., performance of the TIGRIS versus the DTS 1600 system with the AC2 assay was evaluated (S. M. NovakWeekly and A. M. Vannier, Abstr. 104th Gen. Meet. Am. Soc. Microbiol., abstr. C-281, p. 175, 2004). Using over 10,000 specimens, the authors found excellent agreement for C. trachomatis and N. gonorrhoeae in swabs and urines, with 98% or greater concordance between results on both platforms. Similar agreement between TIGRIS and DTS results was seen in a multicenter study with 1,061 urine and swab specimens (E. W. Hook, P. Fine, M. Cohen, A. Weissfeld, D. Willis, Y. Wang, and B. Fugikawa, Abstr. 104th Gen. Meet. Am. Soc. Microbiol., abstr. C-280,
17
p. 174, 2004). These authors also concluded that use of the TIGRIS reduced operator-related error that could result in the reporting of incorrect results. Sample Collection and Transport The AC2, ACT, and AGC assays are FDA cleared for testing endocervical, vaginal, and male urethral swab specimens and female and male urine specimens. The assays are approved for testing in both symptomatic and asymptomatic individuals. Rectal, oropharyngeal, conjunctival, and female urethral specimens are not approved for testing. The APTIMA collection devices contain specimen buffer and have a penetrable top. Once inoculated, the tube is not opened for sample processing and is pierced directly to remove the sample. This feature minimizes the risk of cross-contamination in the laboratory. Swab specimens are transported at 2 to 30°C and are stable for 60 days. Urine specimens can be transported at 2 to 30°C in either the primary collection container or in the urine specimen transport tube. Urine specimens must be transferred to the manufacturer’s transport tube within 24 h and before assay. After transfer, urine specimens are stable for up to 30 days at 2 to 30°C. Both swab and urine specimens in transport tubes can be stored frozen at 20 to 70°C for up to 90 days. The APTIMA vaginal swab specimen collection kit received FDA clearance in January 2004. It is the first kit that enables patients to self-collect vaginal swab specimens, under the direction of medical professionals, for C. trachomatis and N. gonorrhoeae detection with the APTIMA assays. The swab is inserted into the vagina about 2 inches past the introitus and gently rotated for 10 to 30 s. The swab is then withdrawn with care not to touch skin and placed in the transport tube. The PACE collection device, used for Gen-Probe nonamplified C. trachomatis and N. gonorrhoeae probes, can also be used to test male urethral and endocervical swab samples with the AC2 assay. These samples require use of the APTIMA adaptor kit for dilution of the PACE medium. Liquid cytology media such as PreservCyt (Cytyc Corporation, Marlborough, Mass.) and SurePath (TriPath Imaging Inc., Burlington, N.C.) can be tested; however, only PreservCyt has been FDA cleared (August 2005). Several researchers have demonstrated success testing both media in AC2 for the detection of C. trachomatis and N. gonorrhoeae. (124; B. Weinbaum, R. Pilla, M. Alvarez, M. Shih, C. Eng, and R. Johnson, Abstr. 104th Gen. Meet. Am. Soc. Microbiol., abstr. C-285, p. 175, 2004; R. Johnson, B. Weinbaum, R. Pilla, M. Alvarez, M. Shih, and C. Eng, Abstr. 104th Gen. Meet. Am. Soc. Microbiol., abstr. C-283, p. 175, 2004).
18
Leber et al.
Inhibition and Contamination Control There is no inhibition control for individual samples in the APTIMA assays. With the use of target capture for isolation of rRNA, the effect of inhibitors in the sample is minimized. In spiking studies conducted by Chong et al. (44), urine from 415 pregnant and nonpregnant females was spiked with 12 C. trachomatis elementary bodies and assayed on APTIMA and LCx. False-negative rates of 0.48% and 13% were detected with the APTIMA and LCx, respectively. Repeat testing after overnight storage reduced the false-negative rate to 0% for APTIMA and 5.4% with LCx. Additional experiments suggested that the APTIMA assay has a higher sensitivity and better nucleic acid extraction or fewer problems with inhibition than LCx (44). Contamination control is accomplished by unidirectional workflow and amplicon deactivation. The laboratory should be designed so that work proceeds from reagent preparation through DKA. The manufacturer strongly suggests a separate and dedicated area for the DKA. After detection has been completed, the samples are removed from the luminometer and placed in a buffer bleach solution to deactivate amplicons. Work surfaces and equipment are decontaminated with a 1:1 solution of bleach and water. Regular monitoring of the environment is recommended to check for contamination. Swabs of surfaces and water in water baths are tested in the APTIMA assays, and any positive results indicate that additional cleaning is needed. Result Interpretation AC2 test results are automatically interpreted by the manufacturer’s software and are presented as individual C. trachomatis and N. gonorrhoeae results. The final patient results are calculated by using an algorithm that incorporates several elements, including the kinetic output (measured in RLUs) and kinetic type/curve shape of the light-off reaction. A test result may be negative, equivocal, positive, or invalid for C. trachomatis and N. gonorrhoeae. Along with the qualitative results, the RLU values for each sample are printed on the sample report. The ACT and AGC assays have result interpretation and reporting similar to that of AC2; the result is negative, equivocal, positive, or invalid and the result is based on RLU and kinetic type/curve shape. Individual testing for C. trachomatis or N. gonorrhoeae is also possible with the AC2. The software suppresses the result for the analyte that is not ordered. With this mode of testing, the RLU for the reaction, which includes both analytes, is still reported and may indicate the presence of the unordered analyte. For example, high RLUs in a C. trachomatis negative specimen may indicate that N. gonorrhoeae is
CUMITECH 44
present in the sample. However, because the algorithm is based on RLU and curve kinetics, the presence of N. gonorrhoeae cannot be automatically inferred. Performance Characteristics As the newest of the commercially available NAATs for the detection of C. trachomatis and N. gonorrhoeae, there are relatively few peer-reviewed publications concerning the performance characteristics of AC2 and the ACT and AGC reagents. Tables 6 and 7 summarize data from publications and abstracts about the three tests. Analytical performance has been assessed in studies, and it appears that the APTIMA technology may have greater sensitivity than other NAATs. In a study using urine spiked with C. trachomatis elementary bodies (EB), AC2 was found to have a limit of detection of 0.01 EB versus 12 EB for the LCR assay (44). Another spiking study comparing AC2 to AMPLICOR PCR showed an analytical sensitivity of 0.008 EB versus 0.5, respectively (106). In clinical studies, APTIMA Combo 2 has demonstrated comparable sensitivity and specificity when compared to other NAATs. Gaydos et al. (82) compared AC2, BD ProbeTec, and LCR for the detection of C. trachomatis in 506 FCU specimens from a highrisk population. They found no statistically significant differences in the sensitivity and specificity of the three NAATs using a rotating gold standard. Another evaluation by Gaydos et al. (81) found AC2 performance comparable to or higher than that of other NAATs for the detection of C. trachomatis and N. gonorrhoeae in female swab and urine specimens. Unlike reports for other NAATs, they demonstrated that the sensitivity and specificity were comparable for the swab and FCU. LCR, PCR, and SDA have lower sensitivity for urines, probably due to inhibitors present in this sample type. Using other NAATs as referee methods, researchers have found positive results by AC2 that are negative by the other methods (152, 177). These results may be due to a lower specificity or, conversely, a high sensitivity that cannot be confirmed by the less sensitive NAAT (imperfect gold standard). The use of rRNA in TMA assays increases the number of target molecules per cell. This increase in target increases the sensitivity of the test. Also, TKA may remove inhibitor that can lead to false negatives by other methods. When the APTIMA reagents for individual analytes are used for confirmation of such discrepant samples, many are confirmed as positive. In a study comparing the performance of ACT and AGC assays to AC2, LCx, culture, and DFA, the ACT and AGC reagents were found to be suitable for confirmation of positive tests in urine and swab specimens (24). There was 100% concordance with the
FCU
506, M and F
1,389, F
1,391, F
2,457 total
Gaydos et al., 2003 (81)
Gaydos et al., 2003 (81)
Martin et al., 2001d
NA
FCU
Patient infected status (LCx, SDA on swab and urine) Patient infected status (LCx, SDA on swab and urine)
Patient infected statusb (LCR, PCR, and TMA on swab and urine) Specimenc (LCR and PCR) LCR and SDA Patient infected status (LCR, PCR on swab and urine) Patient infected status (LCR, PCR on swab and urine) Patient infected status (LCR, PCR on swab and urine) Patient infected status (LCR, PCR on swab and urine) Patient infected status (LCR, PCR on swab and urine) Patient infected status (LCR, PCR on swab and urine) Patient infected status (LCR, PCR on swab and urine) Patient infected status (LCx, PCR on swab and urine)
Comparison methoda
94.9
94.7 91.5
97.9
95.3
94.2
97.9
96.9
94.7
99.4 100 94.2
92.1
Sensitivity (%)
99.0
98.9 94.6
98.9
98.9
97.6
98.5
97.5
98.9
97.4 98.8 97.6
97.7
Specificity (%)
b
F, female; M, male; CX, endocervical swab; UR, urethral swab; NA, not available. True positives are defined as two or more positive results with reference tests in any combination of method and sample type. c True positives are defined as two or more positive results by test methods only for a single sample type. d D. H. Martin, E. W. Hook, D. Ferrero, D. Willis, J. Schachter, A. Weissfeld, C. Gaydos, and T. Quinn, Abstr. Int. Soc. Sex. Transm. Dis. Res. p. 97, 2001. e D. V. Ferrero, C. A. Gaydos, T. C. Quinn, E. W. Hook, D. H. Martin, J. Schachter, A. Weissfeld, and D. Willis, Abstr. Int. Soc. Sex. Transm. Dis. Res. p. 97, 2001. f D. H. Martin, C. L. Cammarata, and B. Smith, Abstr. 102nd Gen. Meet. Am. Soc. Microbiol., abstr. C-174, p. 131, 2002.
a
15.0 NA
FCU CX
1,391, F 271, F
25.8
Martin et al., 2002f
FCU
F FCU
CX
F
1,095, M
FCU
M
NA
15.0
15.0
14.8
13.3
Prevalence (%)
Ferrero et al., 2001e
UR
M
FCU
CX
CX
Specimen types
1,411, Fa
No. of subjects, sex
Moncado et al., 2004 (152) Gaydos et al., 2004 (82)
Reference(s)
Table 6. Recent studies evaluating Gen-Probe APTIMA assay for detection of C. trachomatis
NA
93.8 NA
95.8
NA
93.8
93.8 87.4
NA
Positive predictive value (%)
NA
99.1 NA
99.3
NA
99.1
100 99.0
NA
Negative predictive value (%)
CUMITECH 44 Nucleic Acid Amplification Tests for Detection of C. trachomatis and N. gonorrhoeae 19
FCU
CX
FCU
1,391, F
269, F
FCU
FCU
F
1,095, M
CX
F
NA
8.6
29.0
NA
8.6
8.6
8.7
Prevalence (%) Patient infected statusb (LCx, PCR on swab and urine) Specimenc (LCR and PCR) Patient infected status (LCR, PCR on swab and urine) Patient infected status (LCR, PCR on swab and urine) Patient infected status (culture or LCR and PCR on swab and urine) Patient infected status (culture or LCR and PCR on swab and urine) Patient infected status (culture or LCR and PCR on swab and urine) Patient infected status (culture or LCR and PCR on swab and urine) Patient infected status (culture or LCR and PCR on swab and urine) Patient infected status (LCR and culture on swab and urine) Patient infected status (LCR and culture on swab and urine) Patient infected status (LCR, SDA on swab and urine) Patient infected status (LCR, SDA on swab and urine)
Comparison method
99.0
100
91.3
98.5
91.1
99.2
98.1
99.2
100
100
99.3
99.6
99.3
98.7
99.6
97.9
99.3
98.6 98.7
99.2 99.2 91.3
98.7
Specificity (%)
98.5
Sensitivity (%)
b
F, female; M, male; CX, endocervical swab; UR, urethral swab; NA, not available. True positives are defined as two or more positive results with reference tests in any combination of method and sample type. c True positives are defined as two or more positive results by test methods only for single sample type. d D. H. Martin, E. W. Hook, D. Ferrero, D. Willis, J. Schachter, A. Weissfeld, C. Gaydos, and T. Quinn, Abstr. Int. Soc. Sex. Transm. Dis. Res. p. 97, 2001. e D. V. Ferrero, C. A. Gaydos, T. C. Quinn, E. W. Hook, D. H. Martin, J. Schachter, A. Weissfeld, and D. Willis, Abstr. Int. Soc. Sex. Transm. Dis. Res. p. 97, 2001. f D. H. Martin, C. L. Cammarata, and B. Smith, Abstr. 102nd Gen. Meet. Am. Soc. Microbiol., abstr. C-174, p. 131, 2002.
a
Martin et al., 2002f
Ferrero et al., 2001e
FCU
2,546 total
Martin et al., 2001d
FCU
M
1,484, F
Gaydos et al., 2003 (81)
CX
UR
1,479, F
Gaydos et al., 2003 (81)
CX
Specimen types
M
1,489, Fa
No. of subjects, sex
Moncado et al., 2004 (152)
Reference
Table 7. Recent studies evaluating Gen-Probe APTIMA assay for detection of N. gonorrhoeae
NA
92.1
99.1
NA
92.1
NA 88.1
NA
Positive predictive value (%)
NA
99.2
99.4
NA
99.2
NA 99.9
NA
Negative predictive value (%)
20 Leber et al. CUMITECH 44
CUMITECH 44
Nucleic Acid Amplification Tests for Detection of C. trachomatis and N. gonorrhoeae
Combo 2 assay in both sample types. All of the APTIMA assays (AC2, ACT, and AGC) detected more positives than culture, DFA, or LCx. Use of other NAATs for confirmation of positive samples with AC2 and AC and AG assays may not be advisable due to differences in sensitivity. In a study by Schachter et al. (177), separate swab and urine samples were collected from each patient for testing with AC2, ACT, and BD ProbeTec C. trachomatis assay. The authors found that the use of BD to confirm AC2 and AC positive C. trachomatis samples resulted in incorrectly reporting approximately 15% of the AC and AC2 positive samples as negative. Conversely, use of the AC2 or AC assays for confirmation of C. trachomatis positive BD samples resulted in confirmation of 96.9% of specimens. Such findings are important in light of the recommendation to confirm positive screening tests in low-prevalence populations. Confirmation must be done with a test that is at least as sensitive as the initial assay. Future Directions With the first fully automated instrument on the market, Gen-Probe will hopefully develop other NAATs for testing on the TIGRIS platform. This may make it more economically feasible for laboratories to justify the cost of the instrumentation. Gen-Probe has stated its intent to standardize its assays on the TIGRIS platform. Analytes that have been mentioned include human papillomavirus, Trichomonas, human immunodeficiency virus, hepatitis C virus, and PCA3 (a prostate cancer marker under investigation) (M. Bott, personal communication). Strand Displacement Amplification: BD ProbeTecET (Becton Dickinson and Company, Sparks, Md.) Principles of Procedure The BD ProbeTecET (BDP) C. trachomatis and N. gonorrhoeae amplified DNA assays are a secondgeneration NAAT system for the detection of chlamydial and gonococcal infection from endocervical, urethral, or urine specimens. The BDP C. trachomatis and N. gonorrhoeae assays are based on the simultaneous amplification and detection of organisms’ target DNA by isothermal SDA and real-time fluorescence detection using a fluorescent detector probe (134, 218). Targets for amplification are the C. trachomatis cryptic plasmid and the pilin gene inverting protein sequence of N. gonorrhoeae (134). SDA is an isothermal process that utilizes a series of primers, DNA polymerase, and a restriction enzyme to exponentially amplify the unique nucleic acid target sequences. SDA consists of two processes, target generation and exponential target amplification
21
(134, 218). Throughout the process of target generation, double-stranded DNA is heat denatured, creating two single-stranded copies. A series of amplification primers for copying the base sequence and bumper primers for displacing the newly created strands combine with DNA polymerase to form altered targets capable of exponential amplification. The exponential amplification process begins with single-stranded DNA strands with restricted enzyme recognition sites from the target generation phase. Amplification primers bind to each strand at its complementary DNA sequence. DNA polymerase uses the primer to identify a location to extend the primer from its 3 end, using the altered target as a template for adding individual nucleotides. The extended primer forms a double-stranded DNA segment containing a complete restriction enzyme recognition site at each end. The restriction enzyme binds to the double-stranded DNA segment at its recognition site. The restriction enzyme dissociates from the recognition site after having cleaved only one strand of the double-sided segment, forming a nick. DNA polymerase recognizes the nick and extends the strand from the site, displacing the previously created strand. The recognition site is repeatedly nicked and restored by the restriction enzyme and DNA polymerase with continuous displacement of DNA strands containing the target segment. Each displaced strand is then available to anneal with amplification primers, and the process continues with repeated nicking, extension, and displacement of new DNA strands, resulting in exponential amplification of the original DNA target (134, 218). Single-stranded DNA probes containing fluorescein and rhodamine labels are present in the reaction mixture. A stem-loop structure occupies the space between the two fluorochromes. Before target amplification, the fluorochromes are located close enough to each other so that any excitation of the fluorescein results in the transfer of the emitted energy to the rhodamine molecule. This results in very little emission from the excited fluorescein molecule being detected. After the SDA reaction, the probe is converted to a double-stranded molecule that is cleaved by a restriction enzyme. This separates the two fluorochromes to the extent that no energy transfer from the excited fluorescein to rhodamine can occur. The fluorescence detected from the excited fluorescein label indicates amplification of the specific target sequence (134). Instrumentation The BDP system instrumentation components consist of an expandable, programmable pipettor, lysing heater and sample rack, priming and warming heater, and the BD ProbeTecET reader (134). This system
22
Leber et al.
requires manual processing of samples and pipetting of reagents throughout the testing. The BD VIPER (VIPER) sample processor has been developed to minimize pipetting and reduce handson time that is associated with the BDP C. trachomatis and N. gonorrhoeae assays. For manual users, samples are processed, lysed, and then manually transferred from the sample tubes to the priming and amplification wells. The VIPER automates the pipetting of samples from sample tubes to the priming wells and from priming to amplification wells. The instrument also replaces the current priming and warming heaters. In the first comparison of the VIPER to manual methods, negative cervical and urine pools were spiked with varying levels of chlamydial elementary bodies and gonococcal particles. The analytical sensitivities for C. trachomatis and N. gonorrhoeae from endocervical swabs were not significantly different from those seen with the manual procedure. For urine samples, the limits of detection were significantly lower for both C. trachomatis and N. gonorrhoeae using the automated procedure (T. Schlitzer, T. Fort, T. Hansen, and P. Johnson, Abstr. 102nd Gen. Meet. Am. Soc. Microbiol., abstr. C-161, p. 128, 2002). The automated assay has been validated by analyzing specimens previously run with the manual assay in duplicate for between-run variability and found to be concordant (D. E. Anamani., L. E. Burgess, S. Vicki, L. Chaffee, and G. J. Tsongalis, Abstr. 19th Annu. Meet. Clin. Virol. Symp., abstr. S48, 2003). In addition, the authors compared VIPER automation to the Abbott LCx system and commented on superior throughput, ease of operation, and maintenance with the BD system. Blackwell et al. found the sensitivity and specificity of VIPER automation for C. trachomatis to be comparable to the manual assay using pools of four endocervical swabs (G. Blackwell, F. Jamieson, G. Riley, and M. Gorus, Abstr. 19th Annu. Meet. Clin. Virol. Symp., abstr. TM19, 2004). By pooling, this study noted a cost savings of 30 to 40%, which was attributed to reduced reagent and consumable usage. Approximately 15 linear feet are required to accommodate the VIPER and two ProbeTec instruments. The manufacturer’s estimated throughput on a daily basis (swab specimens per 8-h shift) is 360 specimens for C. trachomatis/N. gonorrhoeae with amplification control and 552 specimens for C. trachomatis/N. gonorrhoeae without amplification control. Sample Collection and Transport The BDP is approved for the direct detection of C. trachomatis and N. gonorrhoeae from endocervical swabs, male urethral swabs, and female and male
CUMITECH 44
urine specimens from asymptomatic and symptomatic individuals. To collect female endocervical samples, a cleaning swab is used first to remove mucus. Endocervical specimens are then collected with a polyurethanetipped swab (CULTURETTE DIRECT; Becton Dickinson). Male urethral specimens are collected using the MiniTip CULTURETTE DIRECT. Swab specimen types can be transported without preservatives to the testing laboratory at 2 to 27°C and are stable for 4 to 6 days. At the testing site swabs are expressed into sample diluent tube and the swab is discarded. The diluent tubes are recapped and vortexed for 5 s. The expressed sample is placed into the lysing heater (114°C) and cooled for 15 min at room temperature before the assay is performed. Urine specimens are collected in sterile, plastic, preservative-free containers. Collection of the first 15 to 20 ml of voided urine is optimal, with a maximum volume of 60 ml. The patient should not have voided for 1 h prior to collection. A urine-processing pouch is added to the sample cup, and the cup is then capped and transported to the testing laboratory. Samples may be held for 2 days at 15 to 27°C or 4 to 6 days at 2 to 8°C. The urine-processing pouch must be in contact with the specimen for a minimum of 2 h before to processing. Urine specimens are mixed by swirling, and 4 ml is pipetted into an empty sample tube. The tubes are capped and centrifuged for 30 min. The supernatant is decanted and 2 ml of sample diluent is added to each tube. The samples are placed into the lysing heater (114°C) and cooled for 15 min at room temperature before the assay is performed. Inhibition and Contamination Control An amplification control for inhibition testing is available in the CT/GC/amplification control reagent pack for use with the manual method. The control is included for each specimen and should identify samples containing amplification inhibitors that could prevent detection of target DNA. For result interpretation, different MOTA (method other than acceleration) values are used whether or not the amplification control is included. The use of the amplification control is optional and, depending on the type of specimens tested, may not have a noticeable impact on the test performance characteristics (56). It is currently not available for use on the VIPER instrument. According to the manufacturer, dedicated work areas are not required because of the assay’s design (Becton Dickinson, BD ProbeTec ET C. trachomatis and N. gonorrhoeae Amplified DNA Assays, package insert L000203, 1999). Contamination is prevented through standard laboratory precautions such as changing gloves and decontaminating work surfaces
CUMITECH 44
Nucleic Acid Amplification Tests for Detection of C. trachomatis and N. gonorrhoeae
with a 1% solution of bleach in distilled/deionized water. Plates containing amplification microwells are sealed before they are moved from the priming and warming heater to the ProbeTec instrument. The use of sealed plastic bags is suggested for disposal of amplification microwells to prevent environmental contamination with amplicons. After the assay is completed, all countertops and instrument surfaces should be disinfected as above. Wipe tests of work areas and equipment surfaces, including the lysing heater, rack, priming and warming heater, microwell carriers, pipettor handles, and ProbeTec instrument, should be performed at least monthly to monitor for the presence of DNA contamination. Although separate work areas are not mandatory, they are advisable for sample processing and amplification/detection to prevent amplicon contamination. Of particular note, the amplification control generates amplicon that is indistinguishable from the amplicon generated by N. gonorrhoeae, thus increasing the risk of false-positive N. gonorrhoeae results. Result Interpretation The presence or absence of C. trachomatis and N. gonorrhoeae is determined by relating MOTA values for the sample to predetermined cutoff values (134). The MOTA is used to determine the magnitude of the signal generated in the reaction; however, this does not correlate with the number of organisms in the specimen. When testing without the amplification control, MOTA scores of 10,000 are considered positive for C. trachomatis and/or N. gonorrhoeae DNA. When testing is performed with the amplification control, MOTA scores of 2,000 are considered positive for C. trachomatis and/or N. gonorrhoeae DNA. The package insert refers to samples with MOTA values between 2,000 and 10,000 as “low positive” samples and states that there is a decreased likelihood of the results being a true positive as compared to results with MOTA values greater than 10,000. CDC guidelines suggest that consideration be given to additional testing for persons with positive screening results for C. trachomatis and N. gonorrhoeae in low-prevalence populations, which results in a lower PPV (33). All nucleic acid amplification assays may be prone to problems with reproducibility of positive test results (90, 167). A recent article described a repeat test algorithm for samples with MOTA scores greater than or equal to the manufacturer’s cutoff (58). Poor reproducibility (21 of 26 C. trachomatis and 4 of 12 N. gonorrhoeae) was noted with MOTA scores between 2,000 and 9,999 for both analytes. The reproducibility of both analytes with initial MOTA scores of 10,000 increased to 96.7%. It is also noteworthy that Neisseria cinerea
23
and other Neisseria species may cause false positives in the BDP N. gonorrhoeae assay (163). Confirmatory testing in low-prevalence populations or in patients with history or clinical signs inconsistent with gonococcal infection is suggested. Performance Characteristics A number of published studies have evaluated the performance of the assays. Tables 8 and 9 summarize the performance characteristics of the BDP C. trachomatis and N. gonorrhoeae assays. The performance of the BDP has been compared to other nucleic acid amplification systems and found to be equivalent. In the first published study using this technology, the BDP was 100% sensitive and specific when compared to the LCx (134). The BDP performs comparably to other amplification technologies for the detection of C. trachomatis with male urine and female endocervical specimens (26, 37, 147, 205, 209); however, in a study of a relatively high-prevalence population the sensitivity of the assay using female urine specimens was notably reduced (147). In the largest study comparing the BDP to culture and LCx, the assays were comparable for the detection of C. trachomatis and N. gonorrhoeae, but indeterminate assays occurred more often with the BDP assay and urine specimens (205). Akduman et al. (3) demonstrated greater sensitivity using the BDP for detecting N. gonorrhoeae in urine samples when compared to female endocervical and male urethral specimens. Future Directions Becton Dickinson is developing a second-generation SDA, the C. trachomatis Diplex, which incorporates an internal amplification control to monitor the presence of amplification inhibitors and to verify the existence of proper conditions for amplification (R. A. McMillian, T. L. Fort, T. L. Hellyer, and R. Moore, Abstr. 104th Gen. Meet. Am. Soc. Microbiol., abstr. C-275, p. 173, 2004). The performance characteristics of the diplex assay were comparable to those of the currently available assay with both urine and endocervical swabs. Addressing automation, an extraction control based on the binding of DNA and RNA to iron oxide particles has been developed for use with a prototype BD VIPER equipped for nucleic acid extraction (J. M. Harris, T. Brink, and C. Keys, Abstr. 104th Gen. Meet. Am. Soc. Microbiol., abstr. C-293, p. 177, 2004). The extraction control functions as a process control and verifies that conditions exist for binding, washing, and recovering nucleic acids from vaginal swabs and urine.
272, F
11.8
LCR, PCR on CX or FCU LCR, PCR on CX or FCU LCR Two or more positives between PCR, LCR, SDA, culture Two or more positives between PCR, LCR, SDA, culture
LCR PCR Patient infected statusb (culture, or LCR and DFA on swab or urine) Culture or two positive tests PCR, SDA, LCR Culture (CX, UR) LCR, PCR LCR, PCR LCR, PCR Culture or LCR and SDA positive Culture or LCR and SDA positive LCR LCR Patient infected statusb (culture, or LCR on CX or FCU) Patient infected statusb (culture, or LCR on CX or FCU) Patient infected statusb (culture, or LCR on CX or FCU)
Expanded standard methoda
78.1
95.5 98 96 100 90.6
100
90.6
96 95.5 77.3 90.9 100 100 96.6 94.1 96.9
94.7
100 95.3 90.0
Sensitivity (%)
NA
96.1 100 100 100 NA
96.7
98.4
100 100 100 100 99.3 98.6 97.3 95.1 96.6
100
100 99.3 97.3
Specificity (%)
NA
77.7 100 100 100 NA
81.4
87.8
100 100 100 100 89.2 82 83.5 85.6 77.5
100
100 95.9 NA
Positive predictive value (%)
b
97.1
99.3 99.6 99.4 100 98.8
100
98.8
99.4 99.5 97.3 97.3 100 100 99.5 98.1 99.6
99.4
100 99.2 NA
Negative predictive value (%)
F, female; M, male; CX, endocervical swab; UR, urethral swab; NA, not applicable. True positives are defined as two or more positive results with reference tests in any combination of method and sample type. c D. V. Ferrero, L. Buck-Barrington, H. Meyers, G. S. Hall, M. Tuohy, D. Wilson, J. Schachter, J. Moncada, and F. Pang, Abstr. 99th Gen. Meet. Am. Soc. Microbiol., abstr. C-120, p. 129, 1999. d L. S. Leister, K. A. Crotchfelt, B. VanderPol, R. B. Jones, K. Smith, C. Lenderman, E. W. Hook, T. C. Quinn, and C. A. Gaydos, Abstr. 99th Gen. Meet. Am. Soc. Microbiol., abstr. C-115, p. 127, 1999. e J. C. Lovchick, L. M. Peralta, C. M. Brown, D. M. Hilligoss, Abstr. 99th Gen. Meet. Am. Soc. Microbiol., abstr. C-119, p. 128, 1999. f D. Fuller, T. Davis, P. Lineback, M. Milish, and L. Jasper, Abstr. 100th Gen. Meet. Am. Soc. Microbiol., abstr. C-376, p. 220, 2000. g L. Chandler, B. Reisner, J. Fraser, P. Fulcher, D. Fahlen, T. Gateley, and G. Woods, Abstr. 100th Gen. Meet. Am. Soc. Microbiol., abstr. C-375, p. 219, 2000. h K. Homick, J. B. Myers, L. A. Garringer, D. Amsterdam, and S. J. Zimmerman, Abstr. 100th Gen. Meet. Am. Soc. Microbiol., abstr. C-377, p. 220, 2000.
a
FCU
293, F 272, F
Chandler et al., 2000g Homick et al., 2000h
12.3 12.7 12.7 10.2 11.8
FCU CX FCU CX, FCU CX
12.9
CX
377, F
170, F
10.9
Fuller et al., 2000f
Lovechik et al., 1999e
Leister et al., 1999d
10.2
FCU
419, M and F 715, M 291, F 291, F 657, F 588, F 468, F 658, M 294, F
Browning et al., 2001 (26) McCartney et al., 2001 (147)
Ferrero et al., 1999c
733, F
Van Dyck et al., 2001 (209)
8.2 13.8 13.0
11.7 9.2 9.1 9.1 5.0 5.0 12.4 23.1 10.9
CX, FCU FCU CX, FCU, and UR CX
122, Fa 1,224, M and F 2,109, M and F
Little et al., (134) Chan et al., (37) Van Der Pol et al., 2001 (205)
Prevalence (%)
FCU FCU FCU CX CX FCU CX FCU CX
Specimen types
No. of subjects, sex
Reference
Table 8. Recent studies evaluating the BD ProbeTec assay for detection of C. trachomatis
24 Leber et al. CUMITECH 44
3,544, M and F 644, F 578, F F, 471 463, F 663, M 294, F
Akduman et al., 2002 (3) Ferrero et al., 1999c
377, F 377, F 293, F 272, F
6.1 6.1 6.5 8.8
10.6
U CX FCU CX, FCU CX
11.7
7.1
FCU CX
3.6 1.5 1.5 12.1 11.0 29.5 7.1
FCU CX FCU CX FCU FCU CX
1
0.8 2.6
Prevalence (%)
Patient infected statusb (culture, or LCR on CX or FCU) Patient infected statusb (culture, or LCR on CX or FCU) Patient infected statusb (culture, or LCR on CX or FCU) Patient infected statusb (culture, or LCR on CX or FCU) Consensus LCR, PCR on CX or FCU Consensus LCR, PCR on CX or FCU LCR Two or more positives between PCR, LCR, SDA, culture
LCR PCR Patient infected statusb (culture, or LCR on swab or urine) Culture or two positive tests PCR, SDA, LCR Culture Culture or LCR and SDA positive Culture or LCR and SDA positive LCR
Reference method
98 100 100 100
88.9
100
84.2
99.2 100 90 96.6 84.4 94.1 100
93.9
100 100 95.9
Sensitivity (%)
100 99 99.6 NA
97.5
98.3
98.8
99.3 99.7 99.6 97.3 96.9 95.1 99.6
100
100 99.7 98.6
Specificity (%)
100 95.6 95 NA
80
86.9
84.2
84.9 83.3 81.8 93.3 86.2 92.7 95.4
100
100 88.2 NA
Positive predictive value (%)
b
99.7 100 100 100
98.7
100
98.8
99.9 100 99.8 99.8 98.2 98.6 100
99.4
100 100 NA
Negative predictive value (%)
F, female; M, male; CX, endocervical swab; UR, urethral swab. True positives are defined as two or more positive results with reference tests in any combination of method and sample type. c D. V. Ferrero, L. Buck-Barrington, H. Meyers, G. S. Hall, M. Tuohy, D. Wilson, J. Schachter, J. Moncada, and F. Pang, Abstr. 99th Gen. Meet. Am. Soc. Microbiol., abstr. C-120, p. 129, 1999. d L. S. Leister, K. A. Crotchfelt, B. VanderPol, R. B. Jones, K. Smith, C. Lenderman, E. W. Hook, T. C. Quinn, and C. A. Gaydos, Abstr. 99th Gen. Meet. Am. Soc. Microbiol., abstr. C-115, p. 127, 1999. e J. C. Lovchick, L. M. Peralta, C. M. Brown, and D. M. Hilligoss, Abstr. 99th Gen. Meet. Am. Soc. Microbiol., abstr. C-119, p. 128, 1999. f D. Fuller,T. Davis, P. Lineback, M. Milish, and L. Jasper, Abstr. 100th Gen. Meet. Am. Soc. Microbiol., abstr. C-376, p. 220, 2000. g L. Chandler, B. Reisner, J. Fraser, P. Fulcher, D. Fahlen, T. Gateley, and G. Woods, Abstr. 100th Gen. Meet. Am. Soc. Microbiol., abstr. C-375, p. 219, 2000. h K. Homick, J. B. Myers, L. A. Garringer, D. Amsterdam, and S. J. Zimmerman, Abstr. 100th Gen. Meet. Am. Soc. Microbiol., abstr. C-377, p. 220, 2000.
a
Chandler et al., 2000g Homick et al., 2000h
Fuller et al., 2000f
Lovechik et al., 1999e
170, F
733, F
Van Dyck et al., 2001 (209)
Leister et al., 1999d
CX, FCU FCU CX, FCU, UR
122, Fa 1,224, M and F 2,109, M and F
Little et al., 1999 (134) Chan et al., 2000 (37) Van Der Pol et al., 2001 (205) CX
Specimen types
No. of subjects, sex
Reference
Table 9. Recent studies evaluating BD ProbeTec assays for detection of N. gonorrhoeae
CUMITECH 44 Nucleic Acid Amplification Tests for Detection of C. trachomatis and N. gonorrhoeae 25
26
Leber et al.
Hybrid Capture: Hybrid Capture 2 CT/GC DNA Tests (Digene Corporation, Gaithersburg, Md.) Principles of Procedure The Hybrid Capture 2 (HC2) assays developed by Digene are nucleic acid hybridization assays with signal amplification that utilize microplate chemiluminescent detection of C. trachomatis and N. gonorrhoeae DNA from endocervical specimens. Three testing formats are available: CT/GC DNA for the simultaneous detection of C. trachomatis and N. gonorrhoeae, CT-ID DNA for detection of C. trachomatis, and GC-ID for detection of N. gonorrhoeae (Digene Corporation, package insert, Hybrid Capture 2 CT/GC DNA Test Version 2.0, catalog no. 5130-1220, 2001; Digene Corporation, package insert, Hybrid Capture 2 CT-ID DNA Test Version 2.0, catalog no. 5135-1220, 2001; Digene Corporation, package insert, Hybrid Capture 2 GC-ID DNA Test Version 2.0, catalog no. 5140-1220, 2001). Unlike the other NAATs discussed above, HC involves a signal amplification rather than target amplification. This chemistry, like the other NAAT assays, achieves improved sensitivity over probe and culture (150). Specimens containing the target nucleic acid are denatured to release single-stranded DNA and then hybridized with RNA probes in solution. The probe cocktails contain a mixture of complementary RNA sequences to the genomic and plasmid DNAs. The CT probes are complementary to approximately 39,300 bp (4%) of the C. trachomatis genomic DNA and 7,500 bp (100%) of the cryptic plasmid; the NG probes are complementary to 9,700 bp (0.5%) of the N. gonorrhoeae genomic DNA and 4,200 bp (100%) of the cryptic plasmid. There are about 10 copies of the cryptic plasmid in each C. trachomatis organism, and 25 per genome in most N. gonorrhoeae isolates, respectively, hence a natural amplification of signal in each cell. The DNA:RNA hybrids are captured onto the surface of a microplate well coated with antibodies specific for RNA:DNA hybrids. Immobilized hybrids are then reacted with alkaline phosphatase conjugated antibodies specific for RNA:DNA hybrids and detected with a chemiluminescent substrate. Several alkaline phosphate molecules are conjugated to each antibody. Multiple conjugated antibodies bind to each captured hybrid, resulting in substantial signal amplification. As the substrate is cleaved by the bound alkaline phosphatase, light is emitted that is measured as RLUs on a luminometer. The intensity of the light emitted denotes the presence or absence of target DNA in the specimen. Instrumentation The HC2 CT/GC, CT-ID, and GC-ID assays can be run in two formats. The manual method involves the
CUMITECH 44
use of a water bath, shaker, plate heater, and luminometer. For partial automation, the Rapid Capture System (RCS) is the newest addition to the HC assays. RCS is a programmable 96-well microplate processor that integrates liquid handling, shaking, and washing with software specific to testing HC2 CT/GC. The sample processing is still performed manually, and after processing on RCS, the microwell plates must be manually placed in the luminometer for reading. Each run of the RCS has the capacity to test up to four 96-well plates, with 88 wells each for samples plus 8 controls per plate. However, the flexible format allows any number of samples to be run from 1 to 352, although maximal efficiency would be obtained with larger batches. Sample Collection and Transport The Digene HC2 CT/GC, CT-ID, and GC-ID DNA tests are FDA cleared for testing with female cervical samples collected with the cervical sampler (cervical brush with specimen transport medium) and the female swab specimen collection kit (Dacron swab and specimen transport medium). No other sample types have received FDA clearance for testing. Specimens are collected after the endocervix is cleaned of mucus and debris as per other nonculture systems. Samples may be held up to 2 weeks at room temperature and shipped without refrigeration to the laboratory. At the testing site, the samples may be held at 4°C for an additional 7 days or frozen up to 3 months before the assay is performed. Inhibition and Contamination Control Because HC2 uses signal amplification, no amplicons are generated as in target amplification techniques such as PCR, TMA, and SDA. Therefore, the risk of contamination from amplicons is not present. Care should be taken, however, to prevent crosscontamination from sample to sample by utilizing good laboratory technique and unidirectional workflow. Inhibition, as seen with target amplification, is not a factor in HC2. A specific control for inhibition of the signal amplification reaction is not included in the test. Result Interpretation The Digene HC2 CT/GC assay is used to screen a sample for the presence of either or both of the pathogens. If positive, the individual HC2 assays, CT-ID and GC-ID, can be performed to determine whether C. trachomatis, N. gonorrhoeae, or both are present in the specimen. The results for each specimen are generated by the Digene qualitative software based on the ratio of RLUs for the sample divided by the cutoff value (RLU/CO). The cutoff value for the test is the mean number of RLUs of three replicates of a positive control that is tested in each run. Results are
99.7 98 99.8 F, female; M, male; CX, endocervical swab; UR, urethral swab; PACE 2, Gen-Probe PACE DNA probe. Two sites were used in the study with different prevalence rates. c Rapid Capture System. b
a
Darwin, 2002 (61)
Van Der Pol, 2002 (208)
27
97 PACE 2; discrepants with TMA and PCR 8.4 UR
99 100 100 93.3 Culture or PCR 13.6
4.7; 3.7b 9.6 13.6 CX CX CX
1,746, F 1,370, F 330, F All samples 330, F C. trachomatis only N. gonorrhoeae 1,202, M Modarress, 1999 (150) Schachter, 1999 (176) Van Der Pol, 2002c (208)
CX
NA NA 99.3 NA NA 100 99.8 98.2 100 100 97.7 95.6
93.3 99.2 97.2
Culture; discrepants, with DFA and PCR PACE 2; discrepants with PCR Culture; discrepants with DFA Culture or PCR 12.6; 7.8b CX 669, Fa Darwin, 2002 (60)
Positive predictive value (%) Specificity (%) Sensitivity (%) Comparison method Prevalence (%) Specimen types No. of subjects, sex, sample site
Recent studies evaluating Digene Hybrid Capture 2 Assays for detection of C. trachomatis Table 10.
Performance Characteristics There have been several published studies examining the performance of HC2 for the detection of C. trachomatis and N. gonorrhoeae. These studies are summarized in Tables 10 and 11. Girdner et al. (83) compared HC2 CT-ID to culture and PCR for the detection of C. trachomatis from 587 endocervical samples. A specimen was considered positive if culture was positive for C. trachomatis or if two of these tests were positive: HC2 CT-ID, AMPLICOR PCR, or DFA staining. The latter was only performed on discrepant samples that were culture negative but positive by HC2 and/or PCR. The Digene CT-ID assay demonstrated a sensitivity of 95%, a specificity of 99%, and positive and negative predictive values of 92.5% and 99.4%, respectively. Although much of the processing of the CT-ID in this format was manual, it was well adapted for a high throughput; 90 clinical samples could be processed per microplate in 5 h. In a study comparing the manual HC2 versus the RCS instrument, 352 samples plus 32 controls were run in a single shift; 3.5 h of that time required no hands-on involvement from a technologist. The performance of HC2 RCS was equivalent to that of the manual HC2. In the same study, the RCS showed excellent performance on 330 endocervical samples when compared to PCR and culture for the detection of C. trachomatis and N. gonorrhoeae. For the detection of chlamydial infection, HC2-RCS had sensitivity and specificity similar to those of PCR and an improved sensitivity compared to that of culture (see Tables 10 and 11). For the identification of gonococcal infections, all assays performed similarly (208). In a study by Darwin et al. (60), the HC2 CT/GC combination assay was found to have 94.8% sensitivity and 99.8% specificity when compared to HC2 CT-ID and GC-ID or culture. Tables 10 and 11 give the results of using the Digene HC2 CT/GC to detect the specific pathogens in endocervical specimens. For the detection of C. trachomatis, in relatively highprevalence populations (4 to 14%), sensitivities 93% and specificities of 98% were found in a variety of studies. For N. gonorrhoeae, in populations with 1% to 15% prevalence, sensitivities 92% and specificities 98% were found. Excellent predictive values were also found for both pathogens. However, some of these studies compared HC2 to culture or DNA probe (PACE 2), and not to other NAATs directly. Although discrepancies were adjudicated with other NAATs, results for sensitivity may be lower if direct comparisons are made to available amplification assays by testing on all specimens with NAATs.
Negative predictive value (%)
either positive (RLU/CO 1.0) or negative (RLU/CO 1.0). There are no equivocal or indeterminant results for HC assays.
99.7
Nucleic Acid Amplification Tests for Detection of C. trachomatis and N. gonorrhoeae
Reference
CUMITECH 44
99.9
99.4
99.8
The Digene HC2 CT/GC assays are FDA approved for use on female endocervical samples but not for male urethral samples or urine samples from males or females. However, one study by Darwin et al. (61) examined their use with 1,202 male urethral specimens. The authors found that HC2 had excellent performance characteristics comparable to results with female genital samples. The Digene HC2 CT/GC assay might be considered for use in a laboratory that receives predominantly female endocervical samples in a relatively low-prevalence population, in which there would be few positives that would have to be confirmed by additional assays. In a population with an overall prevalence of C. trachomatis and N. gonorrhoeae less than 3 to 4%, few GC-ID or CT-ID assays or other specific tests would have to be performed. On the other hand, one could process the CT-ID and GCID assays individually if the volume of samples was low, but prevalence higher.
b
a
F, female; M, male; CX, endocervical swab; UR, urethral swab; PACE 2, Gen-Probe PACE DNA probe. Two sites were used in the study with different prevalence rates. c Rapid Capture System.
98.9 14.6 Darwin, 2002 (61)
UR
PACE 2; discrepants with TMA or PCR
100 99.4 6.4 CX Van Der Pol, 2002 (208)
2.8; 0.8 6.9 6.4 CX CX CX
1,746, F 1357, F 330, F All samples 330, F; only C. trachomatis/ N. gonorrhoeae 1,202; M Modarress, 1999 (150) Schachter, 1999 (176) Van Der Pol, 2002c (208)
Culture or PCR
100
91.3
100 87.5 99.7 98.5 99 100 92.6 100
98.8 99.8 92.2 CX 669, Fa Darwin, 2002 (60)
3.8; 6.3b
Culture; discrepants, with DFA and PCR PACE 2; discrepants with PCR Culture; discrepants with DFA Culture or PCR
Specificity (%)
Positive predictive value (%)
CUMITECH 44
Sensitivity (%) Comparison method Prevalence (%) Specimen types No. of subjects, sex, sample site Reference
Table 11. Recent studies evaluating Digene Hybrid Capture 2 assays for detection of N. gonorrhoeae
98.8
Leber et al. Negative predictive value (%)
28
Future Directions It would be most efficient to be able to perform multiple tests from a single specimen in addition to C. trachomatis and N. gonorrhoeae. A study reported in 2000 compared cervical specimens collected in Cytyc Thin Prep PreservCyt solution to those collected using Digene transport medium (K. J. Modarress, H. E. Richter, J. R. Schwebke, M. Venglarik, B. Cotton, D. Ball, A. P. Cullen, A. T. Lorincz, and W. J. Payne, Abstr. 100th Gen. Meet. Am. Soc. Microbiol., abstr. C-371, p. 218, 2000). Both samples were tested with the HC2 CT/GC DNA test. Agreement between the two specimen types was 98.6% for C. trachomatis and 98.0% for N. gonorrhoeae. Use of the liquid Pap medium would permit testing for human papillomavirus, C. trachomatis, and N. gonorrhoeae from the same sample used for Pap smear preparation. FDA clearance of PreservCyt or other liquid Pap for testing with the HC2 assays is still awaited along with FDA clearance for male urethral and male and female urine specimens. New Technologies and Laboratory-Developed Assays As with other areas of laboratory medicine, nucleic acid-based techniques have had a major impact on and will continue to change the way testing is done. The most significant developments over the past decade for the detection of chlamydia and gonorrhea infections have involved NAATs. More work is needed to improve their performance, validate novel specimen types, and increase automation of testing and standardization of sample processing. However, there are numerous publications on new innovations and technologies for the detection of these organisms.
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Nucleic Acid Amplification Tests for Detection of C. trachomatis and N. gonorrhoeae
Some may be used in development of next-generation commercial assays and some may be used in laboratory-developed tests. Most clinical laboratories in the United States use one of the FDA-cleared NAATs; however, if a laboratory chooses to develop its own assay, the analytical performance characteristics must be fully verified. Real-Time PCR Real-time PCR involves the amplification and detection of nucleic acid sequences in a closed system (49, 139). Amplicons are generated and detected during the reaction process, as opposed to postamplification end-point detection used in conventional techniques. Real-time PCR assays generally utilize fluorescent reporter molecules, and the amount of reporter signal detected increases proportionally with the number of amplicons generated. Multiple detection chemistries exist, including hybridization probes (LightCycler probes; Roche Molecular Systems, Indianapolis, Ind.), hydrolysis probes (TaqMan probes; Applied Biosystems, Foster City, Calif.), molecular beacon probes, minor groove-binding probes, and doublestranded DNA-binding dyes such as SYBR green. There are also several instrumentation platforms produced by companies such as Roche Diagnostics (LightCycler and COBAS TaqMan); Cepheid, Sunnyvale, Calif. (SmartCycler); Applied Biosystems, Foster City, Calif. (7000, 7500, 7700, and 7900HT); Corbett Research, Sydney, Australia (Rotor-gene); Bio-Rad Laboratories, Hercules, Calif. (i-Cycler); and others. Several publications have reported the use of realtime PCR for the detection of C. trachomatis and N. gonorrhoeae (22, 66, 123, 141, 195, 224). Koenig et al. (123) compared the BD ProbeTecET C. trachomatis and N. gonorrhoeae assay (Becton Dickinson) to a laboratory-developed real-time PCR assay targeting the portion of the ccp gene present on a cryptic plasmid of N. gonorrhoeae and the 7.5-kb cryptic plasmid of C. trachomatis. In specimens initially positive for N. gonorrhoeae by BD ProbeTec, the overall agreement between the two systems for N. gonorrhoeae detection from 155 clinical specimens was only 77.4%, with agreement particularly low (24.1%) when the BD ProbeTec MOTA scores ranged from 2,000 to 19,999. Results for C. trachomatis showed a 91.2% agreement when 114 clinical specimens were tested. The agreement between the two systems improved to 96% when only MOTA scores of 30,000 were retested by the laboratorydeveloped assays. The authors proposed an algorithm for selective repeat testing with the real-time PCR assay for samples giving equivocal BDP C. trachomatis results and N. gonorrhoeae results with MOTA scores of 2,000 to 9,999.
29
Boel et al. (22) compared the use of two laboratorydeveloped real-time PCR (LightCycler) assays and two EIA-based PCR assays for the confirmation of positive COBAS N. gonorrhoeae results. Of 765 male and female urogenital and nasopharyngeal specimens positive for N. gonorrhoeae in the COBAS assay, only 229 (30%) were confirmed positive. Some of the samples were determined to contain Neisseria species other than N. gonorrhoeae. Of the two target regions examined (16S rRNA and ccp cryptic plasmid), only the 16S rRNA target was reliable. With the cppB assays, the authors found that 5.7% of the N. gonorrhoeae positive samples lacked the gene, giving a false-negative results. Both studies described above demonstrate that real-time PCR assays may be useful for confirming positive results obtained with one of the FDA-cleared tests. NASBA Nucleic acid sequence-based amplification (NASBA) (bioMérieux, Inc., Durham, N.C.) is a transcriptionbased amplification method that amplifies RNA from either an RNA or DNA target (36, 121). It is an isothermal process, similar to TMA, that relies on the simultaneous activity of three enzymes: avian myoblastosis virus-reverse transcriptase, RNase H, and T7 polymerase. Real-time detection can be performed with molecular beacon probes, or electrochemiluminescence technology can be used in a postamplification detection step. Several publications have reported the use of NASBA for the detection of C. trachomatis. Morre et al. (153), using two different primer sets in NASBA, found it was more sensitive than conventional PCR methods. Using both the cryptic plasmid and omp1 targets of C. trachomatis, NASBA gave a limit of detection (LOD) of 1 inclusion-forming unit (IFU), whereas the 16S rRNA target gave even better sensitivity with a LOD of 103 IFU. This compared to the LOD of 102 IFU for the most sensitive PCR with primers to the plasmid DNA. In another study, Morre et al. (154) compared the use of 16S rRNA NASBA and cryptic plasmid-based PCR for their ability to monitor the effect of antibiotic treatment in women with genital C. trachomatis infection. The RNA target was found up to 2 weeks following antimicrobial therapy in cervical and urine samples compared to DNA, which was found in some samples for up to 3 weeks. NASBA was more effective in monitoring test of cure than DNA PCR, which can be present in nonviable organisms and detectable 3 to 4 weeks after successful treatment has ended (216). Mahony et al. (142) compared NASBA for C. trachomatis and N. gonorrhoeae 16S rRNA to C. trachomatis PCR and N. gonorrhoeae culture and showed that the test has excellent analytical sensitivity and
30
Leber et al.
clinical performance characteristics. The authors found an LOD of 1 IFU for C. trachomatis and 1 CFU for N. gonorrhoeae with the NASBA assays on bacterial stock solutions and 100 copies for 16S rRNA transcript constructs for both C. trachomatis and N. gonorrhoeae. When testing clinical specimens, NASBA resulted in sensitivity and specificity of 100% and 98.6%, respectively, for C. trachomatis; for N. gonorrhoeae, the NASBA had sensitivity and specificity of 97.9% and 98.7%. Although NASBA looks to be a promising technology for the detection of C. trachomatis and N. gonorrhoeae, there is currently no FDA-cleared assay available, and the development of such an assay does not appear to be on the near horizon. A NucliSens Basic Kit is available that contains all qualitycontrolled reagents and standardized procedures for RNA release, isolation, amplification, and detection. Users can develop their own assays by designing primers and probes for targets of interest such as C. trachomatis and N. gonorrhoeae.
Multiplex Testing and Microarrays Testing for multiple organisms from a single sample type, or multiplexing, is growing in popularity in clinical laboratories. Detection of C. trachomatis and N. gonorrhoeae from a single sample makes sense because the organisms share a mode of transmission, have similar risk profiles, and infect similar body sites. These facts have led to NAATs that combine testing for both organisms. Additional sexually transmitted disease agents may be detectable from these same samples, although care must be taken to ensure that the infecting organism’s nucleic acid can be found in the same specimen collection site. For example, testing of endocervical swabs for Trichomonas vaginalis may not be appropriate since the organism infects primarily the vagina and ectocervix. One sample may not fit all situations. Through innovations such as microarray technology, multiplexing is achievable for the simultaneous detection of numerous microorganisms (21, 27, 29, 62, 191, 213). A microarray is a solid support, such as a glass slide, that is used as a platform for immobilization of nucleotide sequences. These nucleotides act as probes for complementary nucleic acid sequences in the specimen. Many hundreds of probes can be deposited on one slide with great precision with proper instrumentation. Further development is needed before microarray technology is useful in the routine clinical laboratory for diagnosis of disease. This so-called “lab on a chip” technology may dramatically change the way clinical microbiology is performed by allowing a rapid and comprehensive screening approach to testing for infectious diseases.
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INTERPRETATION AND REPORTING OF RESULTS Current commercially manufactured amplified methods for detecting C. trachomatis and N. gonorrhoeae nucleic acid generate qualitative results; the result is positive, negative, and, in some cases, equivocal. The package inserts for these tests contain instructions for the interpretation of results and serve as the guidelines for reporting. Laboratories should consider reporting results as “detected” or “not detected” for the organism’s nucleic acid instead of positive or negative (i.e., C. trachomatis DNA detected) (157) along with indicating, either in the test name or in the result fields, that a NAAT was used to generate results. This emphasizes that a negative result does not rule out infection and may be due to limitations in assay sensitivity or the presence of inhibitors in the sample that led to a falsely negative result. Also, with a report of “detected,” the presence of the organism’s nucleic acid versus viable organisms is emphasized and must be correlated with the clinical picture to determine whether active disease or infection is present. For assays with an internal amplification control, the presence of inhibitors can be detected and thus prevents reporting of false-negative results. Inclusion of additional information on the report is also useful to aid clinicians in interpreting tests. See “Reporting Comments” below. When they are used as a screening test, all positive tests for C. trachomatis and N. gonorrhoeae NAATs should be considered presumptive evidence of infection (33). Because positive results can have significant adverse consequences and psychological effects, consideration must be given to the specificity of the methodology and the prevalence of disease in the population. The PPV of the result for the individual patient is influenced by both factors. As is discussed later in this Cumitech, in low-prevalence populations a positive result for C. trachomatis and/or N. gonorrhoeae may not reflect infection. For N. gonorrhoeae cross-reactivity between the pathogenic and nonpathogenic Neisseria species has been demonstrated for some NAATs that can affect specificity (145, 206) (Becton Dickinson, BD ProbeTecET C. trachomatis and N. gonorrhoeae amplified DNA assays, package insert L000203, 1999). However, these nonpathogenic species are rarely isolated from urogenital sites. Cross-reactivity among chlamydia species in NAATs has not been noted. Because of concerns about specificity and PPV, additional testing after a positive screening test may be necessary in certain populations to ensure the reliability of a positive result (33). Equivocal results mean that the presence or absence of the organism’s nucleic acid cannot be determined. Testing of a second sample is recommended.
CUMITECH 44
Nucleic Acid Amplification Tests for Detection of C. trachomatis and N. gonorrhoeae
Test of Cure After initial diagnosis of chlamydial and gonococcal infections, test of cure is not recommended for most patients treated with recommended first-line antimicrobials. Exceptions include cases of suspected treatment failure, noncompliance or reinfection, and pregnant women treated for C. trachomatis infection (35). If NAAT is done for test of cure for C. trachomatis infection, specimens should be collected 3 weeks following the end of treatment. NAAT done before 3 weeks may be falsely positive due to continued shedding of nonviable organisms (79, 154, 225). Culture should be used for test of cure following treatment of N. gonorrhoeae infections (35). This practice allows susceptibility testing in cases where agents with known resistance are used to treat gonorrhea. Children and Sexual Abuse or Assault Cases The populations used to evaluate NAATs for C. trachomatis and N. gonorrhoeae for FDA clearance include mostly sexually active adolescents and adults (13 years of age). These tests are not approved for rectogenital testing in children, and their performance is not clearly established in this population (94). Because the prevalence of infections in children is expected to be lower than that in adults, the predictive value of positive results may be very low. To date, culture of C. trachomatis and N. gonorrhoeae remains necessary for medicolegal purposes. With specificities of nearly 100%, culture is still considered the gold standard for issues such as child sexual abuse and rape (94, 95). Protocols need to be written and distributed to personnel where specimens from cases of sexual abuse may be collected. Use of nonamplified methods may miss some cases of sexual abuse; however, the implications of false-positive reports are paramount. In some instances, a NAAT may be requested in addition to culture to ensure maximal sensitivity. Further studies are needed to establish the utility of the amplified tests in the setting of child sexual abuse. Reporting Comments Suggested reporting comments for C. trachomatis and N. gonorrhoeae testing are given in Table 12. These comments are designed to communicate reasons for the rejection of specimens and special consideration for unusual circumstances. In most situations, specimens not collected in the manner described in the manufacturer’s package insert should be rejected unless these sample types have been validated by the testing laboratory. This also applies to sample types not included in the package insert. Federal regulations require all clinical testing to be performed only after the performance characteristics of the test have been validated, either by a kit manufacturer (and ver-
31
ified by the clinical laboratory) or by the clinical laboratory itself in the case of a user-developed assay or a modification of a marketed assay (50). QUALITY CONTROL Quality control is important for all laboratory tests, but especially for NAATs due to their exquisite sensitivity and the risk of contamination. Because of the possible social and psychological ramifications of reporting a positive N. gonorrhoeae or C. trachomatis result, and the requirement to report results to state health departments and partners of infected individuals, care needs to be taken to ensure the accuracy of results at all times. A study of five laboratories performing C. trachomatis NAATs on urine samples from asymptomatic men has shown variance within and between laboratories in the performance of similar assays. The authors recommend that strict adherence to quality control, proper training, and continuous monitoring of test performance characteristics are essential (112). Several key points about quality control for C. trachomatis and N. gonorrhoeae NAATs are discussed below. The reader is referred to the recent National Committee for Clinical Laboratory Standards (now named Clinical and Laboratory Standards Institute, Wayne, Pa.) document MM3-A for a useful and complete review of guidelines for quality control in molecular infectious disease diagnostics (157). Kit Controls The manufacturer’s directions in the package insert should be followed for the number and frequency of controls necessary for each of the NAAT assays. In general, positive and negative controls are included with each batch of specimens that is being tested for each of the two pathogens. All controls must perform as expected for the results to be acceptable for reporting. Roche COBAS AMPLICOR: a positive and negative control is run with each A-ring. The C. trachomatis and N. gonorrhoeae positive control materials are noninfectious plasmid DNA containing sequences specific for each organism. The C. trachomatispositive control serves as the N. gonorrhoeae-negative control; the N. gonorrhoeae-positive control serves as the C. trachomatis-negative control. An internal inhibition control is provided, which is plasmid DNA containing the C. trachomatis primer-binding sequences and a unique probe-binding region. BD ProbeTec ET: a positive and negative control is included with each test run. The positive control contains C. trachomatis and N. gonorrhoeae linearized plasmid DNA; the negative control contains salmon testes DNA. An amplification control is provided,
32
Leber et al.
Table 12.
CUMITECH 44
Suggested reporting comments for C. trachomatis and N. gonorrhoeae NAAT results
Sample type/situation
Action
C. trachomatis or N. gonorrhoeae nucleic acid detected
Report result: detected
C. trachomatis or N. gonorrhoeae nucleic acid not detected C. trachomatis or N. gonorrhoeae nucleic acid equivocal Inhibition present
Report result: not detected
Report comment Additional testing is recommended after an initial positive screening test if a low positive predictive value can be expected, such as in low-prevalence populations, or if a false-positive result would have serious psychosocial or legal consequences. A result of not detected does not eliminate the possibility of infection.
Report result: equivocal
Results are inconclusive. Please submit a new specimen for repeat or alternate testing if clinically indicated.
Report result: inhibition noted
A valid result cannot be determined. Inhibition may be due to lubricants, mucus, blood, or other substances. Please submit a new specimen for repeat or alternate testing if clinically indicated.
Sample not collected according to manufacturer’s directions
Reject sample
Sample type other than those included in manufacturer’s package insert Vaginal sample from hysterectomized patient
Reject sample
Testing not performed. Specimen was not collected properly. (Additional description of inappropriate collection may be added such as “Large cleaning swab submitted.”) Testing not performed. Sample type is not acceptable for testing. (Recommend alternate testing, such as culture, for the specimen if appropriate.) Interpret results with caution. This sample type has not been validated by the manufacturer or the laboratory.
Sexual assault or abuse
Patient age less than 13 years or prepubescent/ menarche
Reject sample or consult with clinician before testing Reject sample or consult with counsel or clinician before testing Reject sample
Culture is the recommended method for detecting C. trachomatis and N. gonorrhoeae in urogenital, pharyngeal, and rectal specimens in cases of sexual abuse or assault. Nucleic acid amplification testing for C. trachomatis (or N. gonorrhoeae) has not been validated for testing and is not recommended for prepubertal children. Culture is the recommended test for this population.
which functions as the inhibition control, and consists of the same linearized plasmid used for the N. gonorrhoeae-positive control. Gen-Probe APTIMA: a positive and negative control is included with each test run. The C. trachomatis- and N. gonorrhoeae-positive control materials are noninfectious RNA containing sequences specific for each organism. The C. trachomatis-positive control serves as the N. gonorrhoeae-negative control; the N. gonorrhoeae-positive control serves as the C.
References 33
Roche Diagnostics, package insert, COBAS AMPLICOR CT/NG Test for Chlamydia trachomatis, Revision 1.0, 1999; Roche Diagnostics, package insert, COBAS AMPLICOR CT/NG Test for Neisseria gonorrhoeae, Revision 3, 1999; Becton Dickinson, BD ProbeTec ET Chlamydia trachomatis and Neisseria gonorrhoeae amplified DNA assays, package insert L000203, 1999 Alternate specimen collection procedures must be verified by laboratory if not included in FDA-cleared package insert (50). Sample types must be validated by laboratory if not included in FDA-cleared package insert (50). Women with hysterectomies are capable of acquiring and transmitting STDs (39, 122). Data, experience, and court cases are insufficient to assess the applicability of NAATs to detect C. trachomatis or N. gonorrhoeae in investigating sexual assault and abuse (33). 33, 94
trachomatis-negative control. No inhibition control is provided due to use of target capture for sample processing. Digene Hybrid Capture 2: a negative control and positive control is included in each test run. The C. trachomatis and N. gonorrhoeae controls are cloned C. trachomatis and N. gonorrhoeae DNA targets, composed of the same plasmid construct for each individual organism. The negative control contains carrier DNA. No inhibition control is provided.
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Nucleic Acid Amplification Tests for Detection of C. trachomatis and N. gonorrhoeae
The IC, if provided, should be employed as directed. If the IC is negative (i.e., suggesting that inhibitory substances are present in the sample), and the sample is negative for one or both analytes, the negative results should not be reported. Rather, the specimen should be rerun after being frozen, refrigerated, or diluted. If, after the second run, the amplification control is positive, indicating no inhibition, then results can be reported as positive or negative for the test sample and the analyte in question. If there is noted inhibition and the analytes are positive, the positive sample results can be reported without repeat testing. Consideration can be given to omitting the IC if the laboratory demonstrates an acceptably low rate of inhibition in a given sample type (157). External Controls For all NAATs, it is advisable to test external control material to monitor assay performance. The controls provided in commercial kits are nucleic acid sequences and do not represent whole organisms (see above). Therefore, they do not represent specimen matrices, such as urine or cervical cells, containing infectious organisms. To adequately control for all steps of the NAAT, including specimen processing and extraction, an additional whole organism control in a suitable matrix is advisable. These controls should be taken through all steps of the procedure (157). A number of companies manufacture whole organism controls for use as positive external controls. Such controls should be at concentrations in the low range of assay to challenge assay sensitivity and not serve as a source of contamination (157). Positivity Rate To avoid false-positive results due to contamination, it is advisable to set up a system in the laboratory whereby data are collected to determine the prevalence of each pathogen and hence the number of positive results to be expected per day, per run, or by some other appropriate parameter. Once the expected positivity rate is determined, results should be checked each day to determine if that limit has been exceeded. If it has, steps should be taken to investigate and correct any problems that are uncovered. For example, if there are more male genital samples on a particular day, one might expect a higher number of positive results due to the higher rate of symptomatic infections in this population. Also, the tally system should exclude results for repeat testing of positives. Such results are included for counting only in the initial run. If an obvious explanation is not found for an elevated positivity rate, other methods of tracing could involve repeat testing, making physician calls to determine the likelihood of a positive result, and within the laboratory, checking on where
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the positives were found in relationship to each other and to the positive control on the testing run. Returning to the initial specimen and reprocessing would be appropriate, but the possibility of contamination of the original specimen must be considered. Requesting additional samples from the patient is always an option, although in many cases, this is not possible or causes hardships on both patient and physician. If any repeat testing is required because of possible laboratory error, the patients should not be charged for the additional assay(s). Proficiency Testing Proficiency testing is required for all laboratories and determines what testing can be performed (50). Proficiency programs should be designed to make certain reliable and reproducible results are obtained and to evaluate the skill of the personnel performing testing. Comparison to other laboratories also participating in proficiency programs allows the credibility of results to be determined. External proficiency testing is available via agencies such as the College of American Pathology (CAP, Northfield, Ill.). A CAP survey is available for C. trachomatis and N. gonorrhoeae amplified testing, and all laboratories performing this testing should participate. For further details, see “Considerations for Laboratories.” Additionally, an internal blind testing program may be considered in addition to external programs. Blind retesting of specimens allows assessment of competency for individual technicians as well as the analytical performance of the test. Contamination Concerns Use of any of the NAATs demands strict control procedures to prevent contamination of specimens (see “Considerations for Laboratories Performing NAATs” below). This can include the use of various chemical or physical controls. An example of chemical control is uracil-N-glycosylase (AmpErase) in the reaction as is found in the Roche AMPLICOR products, and closed amplification systems such as realtime amplification represent physical control measures. In addition, designated areas for specimen processing, amplification setup, and amplification/ detection and strict adherence to unidirectional workflow are mandatory (148).
CONSIDERATIONS FOR LABORATORIES PERFORMING NAATs A variety of technical and procedural issues must be considered before implementing nucleic acid amplification testing for C. trachomatis and N. gonorrhoeae (Table 13). These issues include the physical limitations of laboratory space, availability of equipment
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Table 13. Considerations for laboratories performing nucleic acid amplification testing for C. trachomatis and N. gonorrhoeae Patient and client Population of patients Type of populations to be tested High-risk/symptomatic Screening/asymptomatic Prevalence Acceptable specimen sources Ease of sample collection Invasive versus noninvasive collection procedures Client education Sample collection and transport Result interpretation Turnaround time requirements Laboratory Physical facilities Space requirements Electrical and other requirements Location within the laboratory Air flow Storage of reagents Storage of tested specimens Waste disposable Equipment Manufacturer provided equipment Additional equipment and material requirements Pipetters Barrier pipette tips Incubators Refrigerator/freezers Preventive maintenance Instrument throughput Interface between instrument and lab information system Personnel Level of expertise Training Competency assessment
Laboratory (continued) Contamination control Unidirectional workflow Separation of pre- and postamplification area Decontamination of equipment and materials Contamination monitoring Result interpretation and reporting Acceptance/rejection criteria Improper collection Testing in children Sexual abuse Nonvalidated sample types Result and rejection messages Confirmatory testing for positive samples Quality control Verification and validation of assay performance Tracking of positivity rates Monitoring for contamination (wipe testing) External control material Proficiency testing External programs (CAP) Internal programs Financial considerations Cost per test Labor costs Reimbursement FDA-cleared kit Laboratory-developed (home brew) assay Research use only Investigational use only reagents Payer mix Cost-versus-benefit analysis
for assay performance, training and maintaining staff competence, selection of an assay platform(s), and client-related issues. Education of clients concerning proper specimen selection, collection, handling, and transport is important. Choice of a particular NAAT may also be influenced by patient population; clinical performance is influenced by prevalence of disease. In addition, ease of collection and patient preference as to acceptable specimen types should be considered. The sensitivity of NAATs dictates that contamination control be a top priority when designing or restructuring existing laboratory space. The performance of NAATs can be separated into four steps consisting of reagent preparation, sample preparation, amplification, and detection. In a perfect setting, these steps would be carried out in separate rooms, but in laboratories with limited space, configuration of these areas as individual stations within one or two rooms is practical. Each laboratory area should be labeled, and each step of assay performance from specimen
handling to personnel movement should provide for unidirectional workflow from preamplification to detection areas. Physical separation of preamplification and postamplification areas and control of airflow are primary mechanisms of protecting against product carryover from aerosol contamination (148). Other methods to prevent contamination include environmental monitoring for amplicon and regular decontamination of work surfaces. Decontamination can be accomplished with bleach solutions, enzymatic inactivation of amplified target by uracil-Nglycosylase, or surface irradiation with UV light (148). Each NAAT approved for C. trachomatis and N. gonorrhoeae testing has its own specific instrumentation provided by the manufacturer. Instrumentation unique to each assay must have regular preventive maintenance and a quality control program established to ensure proper instrument function. All instrument activities involved in assay performance must be monitored as prescribed by the manufacturer. Temperature-dependent equipment should be moni-
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Nucleic Acid Amplification Tests for Detection of C. trachomatis and N. gonorrhoeae
tored and values recorded daily. Pipettors should be calibrated at specified intervals to ensure precision and accuracy. Use of aerosol barrier pipette tips is mandatory to prevent cross-contamination between specimens. Biological safety cabinets must be certified and calibrated at least annually. Cabinets as well as dead air boxes should be decontaminated at the start and end of each working day or immediately when a spill occurs. To prevent amplicon carryover, dedicated equipment should be available for each work area (148). Examples include water baths, incubators, heating blocks, and pipettors. Equipment or materials that can be moved should be clearly marked so that if they do get moved, it is obvious and appropriate decontamination can be performed. Items such as pens, pencils, and radios are often overlooked but should also be properly handled to prevent contamination. Although NAATs for the diagnosis of C. trachomatis and N. gonorrhoeae have been available for some time, they remain largely manual procedures. The skill of the individual performing the testing is critical to the quality of the results. Personnel chosen to perform this type of testing should have good organizational skills, be detail oriented in their approach to work, and be well versed in aseptic and molecular technique. It is the laboratory director’s responsibility to ensure that all personnel have the appropriate training, experience, and education for the type and complexity of testing performed (50). Because NAATs are classified as highly complex procedures, the competency of individuals performing these tests must be verified semiannually for the first year and documented annually thereafter (50, 68). The CAP also requires that laboratories have an adequate training program for new technologists and should have a continuing medical laboratory education program for the technical staff. These requirements are listed in the CAP molecular pathology checklist and outlined elsewhere (13, 52, 146, 203, 217, 228). All laboratories should participate in a formal proficiency test program. The CAP program provides challenges for both C. trachomatis and N. gonorrhoeae NAATs. The survey, HC6, contains five challenges per shipment and is issued three times per year. Samples representing both swab and urine samples are included. Internal proficiency testing with previously characterized positive and negative patient samples is useful in demonstrating consistency of results among laboratory staff members. Internal and external proficiency testing should be rotated among all staff members and be handled in the same manner as clinical specimens. New-hire training, proficiency testing, annual competency assessment, and ongoing continuing education programs ensure that quality results are consistently obtained.
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Once the decision has been made to implement NAATs for the diagnosis of C. trachomatis and N. gonorrhoeae, the laboratory must choose a platform. Laboratories must acknowledge that the cost of NAATs will be greater than that of culture or nonculture tests for either of these organisms. Depending on the payer mix, reimbursement may offset the higher cost per test. NAAT manufacturers have different requirements for laboratory space, separation of work areas, and other equipment. The type of testing performed (e.g., screening asymptomatic individuals, confirmatory testing, use in epidemiologic studies, or strictly diagnostic purposes) should be considered before selection (33). The sensitivity and specificity of all of the NAATs described are comparable; however, their performance with different specimen types may vary. A laboratory must also consider the predominant specimen types to be tested to adjust specimen preparation time and workflow accordingly. Throughput of the various NAATs will differ from system to system based on the instrumentation’s use of amplification controls. Laboratories must also consider client turnaround time requirements, technician time, and maintenance. All clients must be instructed in proper specimen collection and transport techniques. Laboratories should supply clients with informational materials concerning specimen selection, specimen collection, the use of specific transport devices for a particular assay, and the appropriate transport conditions. Clients must be informed that strict adherence to these requirements will ensure optimal assay performance.
VERIFICATION OF A TEST METHOD When a laboratory has selected a test to replace an existing method, or implements a new method, for diagnosing C. trachomatis and N. gonorrhoeae infections, it must demonstrate that it can meet the analytic system requirements described by Centers for Medicare and Medicaid Services (50). Before reporting patient results with an FDA-cleared NAAT, the laboratory must demonstrate that it can obtain results comparable to those described in the manufacturer’s package insert. The performance characteristics compared include accuracy, precision, and reportable range (50). Results should be compared to the manufacturer’s package insert claims and evaluated accordingly (180). These guidelines do not apply to any test system used in a laboratory before 24 April 2003. Recommendations for conducting studies to evaluate C. trachomatis and N. gonorrhoeae tests have been reviewed previously (8, 33). If a laboratory chooses to modify an FDA-cleared test system or uses a system for which performance
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specifications are not provided by the manufacturer, before reporting results they must establish and maintain documentation of the performance characteristics for the following: accuracy, precision, analytical sensitivity, analytical specificity to include interfering substances, reportable range, reference intervals, and any performance characteristic required in test performance (50). The traditional reference test for determining C. trachomatis and N. gonorrhoeae infection is culture. There are many inherent variables in the performance of culture for these agents. The necessity for maintaining viability, transport issues, and lack of standardization affect culture results for each of these organisms. Unless a culture has been contaminated, it is assumed that culture is 100% specific. Demonstration of chlamydial inclusions stained with monoclonal anti-C. trachomatis antibodies or isolation of N. gonorrhoeae from an infected site with confirmation by biochemical testing is a fairly clear demonstration of infection. Culture for these agents is less than a perfect standard as the sensitivity is generally less than 100% because of the variables mentioned above. The specificity of many of the early nonculture tests was underestimated as these methods were compared against less sensitive culture techniques. Likewise, increased sensitivity of nonculture methods may be expected if the new procedure is more likely to detect culture-positive rather than culture-negative specimens (16). There are many evaluations of NAAT performance characteristics in the literature. Many of these use a sample that determines whether a subject is infected at a single anatomic site rather than true infection status. The use of highly sensitive methods allows the laboratory to more accurately define infection status by testing specimens from different anatomic sites, e.g., endocervical and urine. Estimates of test sensitivity are probably overestimated when the standard for infection is based on a positive test from a single specimen type rather than multiple sites because use of multiple samples identifies more infections (33, 179, 205, 207). The infected patient is a more appropriate standard for evaluation of the performance of NAATs rather than a specimen-based standard. It is expected that NAAT sensitivity will exceed that of culture or other nonamplified nonculture procedures. These apparent false-positive NAATs may be subjected to supplemental testing to determine their true status. This discrepant analysis is applied to identify true positive infections that were missed by a less sensitive standard. Discrepant analysis has been used to arbitrate chlamydia laboratory tests for many years (181, 182). Proponents of discrepancy testing argue that estimates of sensitivity and specificity based on discrepant analysis are less biased
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than those based on traditional gold standards. However, use of discrepant analysis has been criticized as biased in favor of new tests (91–93). Cumitech 31 describes the protocol for verification of tests that are performed as described in the manufacturer’s package insert (67). The Clinical Laboratory Improvement Amendments mandate 30 comparisons in order to validate a new test methodology. It is suggested that the minimum number of paired specimens that should be run in parallel with the existing test or reference method is that number that would give at least 20 positives and 50 negatives (67). For endocervical specimens, a swab should be obtained for each test and placed in the transport medium approved by the manufacturer. For urine, the appropriate number of specimens from patients of each gender should be obtained for at least 10 positives from each gender. It may be appropriate to obtain fewer positives if the test has been verified for other specimen types (50). For test reproducibility, it is suggested that at a minimum 10 to 20 specimens should be repeated. The tests selected should represent all possible types of results, both positive and negative, and include specimens near the assay cutoff. A laboratory’s comparison of commercially available NAATs may be complicated by the necessity to collect multiple patient samples, which might require approval from an internal review board and informed consent. In addition, each of these assays has its own approved transport. Currently, the user of any amplification system is locked into using the manufacturer’s specific transport device. Use of a transport device specific for a particular NAAT in another test system represents an off-label use and must be subject to the verification procedure described above. Once the transport is validated, ongoing quality control should be performed. Each new lot of the new transport type should be tested with positive and negative controls to ensure the consistency of the procedure. The use of a universal type of nucleic acid transport device would be more appropriate as laboratories, particularly those serving a large diverse client base, may receive more than one transport device for the particular NAAT that is offered in-house. Transport media such as M4 and M4RT (Remel, Lenexa, Kans.) and 2-SP and FlexTrans (Bartels) wet and dry transports have been validated with different amplification systems for C. trachomatis and N. gonorrhoeae (7, 65, 76, 80, 120, 150; J. Harrison, R. Sautter, and W. LeBar, Abstr. 98th Gen. Meet. Am. Soc. Microbiol., abstr. C-48, p. 139, 1998; R. Kendrick, R. Sautter, W. LeBar, and T. Rudolph, Abstr. 15th PASCV Meet., abstr. S34, 1999; J. Harrison, M. Garassi, S. Wierzbicki, and W. LeBar, Abstr. 104th Gen. Meet. Am. Soc. Microbiol., abstr. C-286, p. 176, 2004; J. Harrison, M. Garassi, S. Wierzbicki, and W.
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Nucleic Acid Amplification Tests for Detection of C. trachomatis and N. gonorrhoeae
LeBar, Abstr. 20th PASCV Meet., abstr. TM25, 2004; G. S. Hall, J. Tuohy, T. Katanik, D. Wilson, and G. W. Procop, Abstr. 102nd Gen. Meet., Am. Soc. Microbiol., abstr. C-162, p. 129, 2002). Recently, a device called the Nucleic Acid Transport from Medical Packaging Corporation (Camarillo, Calif.) was approved for collection and transport of samples for testing for C. trachomatis and N. gonorrhoeae in endocervical and urethral samples with the BD ProbeTec ET, Roche COBAS AMPLICOR, and Gen-Probe PACE 2.
GUIDELINES FOR SCREENING AND TESTING FOR INFECTION Symptomatic Testing Current recommendations for patient management include testing for C. trachomatis and N. gonorrhoeae in all symptomatic individuals with the most sensitive and specific test available (35). For C. trachomatis infections, NAATs are preferred for testing urogenital specimens because of the high sensitivity relative to other tests. The preferred test for N. gonorrhoeae is culture for female endocervical specimens if collection and transport of the inoculated media are adequate to maintain viability. Culture allows monitoring for antimicrobial resistance if the patient fails therapy. A Gram-stained smear for males with urethral discharge with culture backup gives adequate performance for the detection of N. gonorrhoeae in most instances (33). Asymptomatic Screening Annual screening of all sexually active women under the age of 26 for C. trachomatis infection has been recommended by the CDC, the U.S. Preventive Services Task Force, and a variety of medical organizations (35, 53, 99, 159). Screening is also indicated for all pregnant women and older women with risk factors, such as sexual contact with an untreated partner or sex with a new or multiple partners. Followup screening, at 3 to 4 months after antimicrobial treatment, is recommended because of the high rate of reinfection in women who have had a C. trachomatis infection in the preceding several months (35). This rescreening is distinct from test of cure, a practice that is not routinely recommended. Screening for N. gonorrhoeae infection is recommended in women at high risk of sexually transmitted diseases and in pregnant women with high risk or living in areas with a high N. gonorrhoeae prevalence (35). The U.S. Preventive Services Task Force recommends screening all sexually active women, including those who are pregnant, for gonorrhea infection if they are at increased risk for infection. At-risk popu-
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lations include the young (less than 25 years of age) and those with individual or population risk factors (202). Presently there are no recommendations for screening for C. trachomatis or N. gonorrhoeae in men. As many infections with these organisms are asymptomatic in males, this population represents a major reservoir for transmission of new infections and reinfection of treated partners. Further research is needed to determine if screening young sexually active males would be of benefit in reducing prevalence and preventing infection in women (54). Currently, through partner notification, some of the infected males are identified and may receive treatment, but research indicates that this is not an effective method to reduce prevalence rates (86). Expedited partner therapy, a process by which patients are given antimicrobials to give to their sex partners, is an alternative approach that is gaining recognition. Several studies have shown this approach to be effective in preventing reinfection with C. trachomatis and N. gonorrhoeae (87, 183). Laboratory Guidelines for Screening Tests In 2002, the CDC issued recommendations for the selection, performance, and interpretation of screening tests for C. trachomatis and N. gonorrhoeae infections (33). This document is a useful reference for all laboratories performing this testing, particularly those using NAATs. A key point in this document is that all screening tests should be considered presumptive evidence of infection. Additional testing should be considered for persons with a positive screening test result if a false-positive result would have a serious adverse consequence. Routine additional testing should be considered when the prevalence of either C. trachomatis or N. gonorrhoeae infection is low, resulting in a low PPV (e.g., 90%). Additional testing increases the specificity and can include retesting of the specimen or collection and testing of a new specimen (Table 14). A two-tiered testing scheme may be directed by the ordering physician for individual patients or by the laboratory for all positive specimens or for samples from select groups of patients with low prevalence. Either way, the interpretation of the combined results must be clear. A positive result with the screening and an additional test is more likely to be a true positive and represent infection. A positive screening test and a negative additional test may mean the initial result was falsely positive and the patient is not infected. Alternately, the additional test may be falsely negative because of factors such as administration of therapy based on the positive screening test.
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Table 14. Considerations for additional testing after a positive screening NAAT for C. trachomatis or N. gonorrhoeaea C. trachomatis Only another NAAT test is appropriate for confirmation of a positive screening test. N. gonorrhoeae Culture with confirmation is the preferred test after a positive NAAT, providing the culture specimen integrity can be maintained. Use of a NAAT after positive screen is acceptable but has had limited evaluation Samples for additional testing (in order of preference) Test second specimen with a different test that uses a different target or format. Test the original specimen with a different test that uses a different target or format. Repeat the original test on the original specimen. a
Adapted from reference 33.
Effectiveness of Screening The goal of screening is to detect infection in asymptomatic individuals and prevent subsequent adverse health outcomes and the associated costs to society. The economic impact of C. trachomatis and N. gonorrhoeae infections in young adults in the United States is significant; the estimated direct medical cost exceeds $325 billion annually (43). Numerous publications have examined the effectiveness of C. trachomatis screening programs (1, 100, 102–104, 143, 184). These programs have been shown to reduce the prevalence of infection and to reduce the number of cases of PID and other sequelae, and they are cost-effective. In a study by Scholes et al. (184) screening for C. trachomatis infection among high-risk sexually active women reduced the incidence of PID by 56%. This study used culture and EIA for the detection of C. trachomatis. Other studies supporting the effectiveness of C. trachomatis screening in sexually active women found that NAATs were also cost-effective despite the increased cost of this type of testing compared to nonamplified methods. The NAATs are more sensitive and allow detection of more infected individuals (103, 104, 128). Despite the recommendations from multiple groups and increasing coverage for testing by insurance providers, the rate of C. trachomatis screening in the United States remains low (54, 55, 188). In a study by the CDC, data from the Health Plan Employer Data and Information Set during 1999 to 2001 were examined to determine the rate of screening in commercial and Medicaid insurance plans (31). Among sexually active females aged 16 to 26 years, screening increased over the 3-year period, but by 2001 only 26% of these women were screened for C. trachomatis in the commercial plans and 38% in
CUMITECH 44
Medicaid plans. Clearly screening needs to increase in order to improve the quality of care and reduce the costs associated with C. trachomatis infections among young sexually active youth. The advent of noninvasive sample types such as urine and vaginal swabs for NAATs is hoped to increase the acceptance of screening. Novel approaches such as NAATs through pharmacy-based screening and mail-in testing also warrant further study if the goal of sustained and widespread reduction in C. trachomatis infection is to be realized (19, 20, 204).
CODING AND REIMBURSEMENT ISSUES The Current Procedural Terminology (CPT) codes for amplified probe testing of C. trachomatis and N. gonorrhoeae are 87491 and 87591, respectively (5). Note that both target amplification (e.g., PCR, TMA, and SDA) and signal amplification techniques (e.g., Hybrid Capture) are accepted methodologies for these codes. These codes are both method and analyte specific so little interpretation is required for accurate coding. Codes for molecular diagnostic procedures (83890–83901, 83912) should not be included because the amplified probe technique is a comprehensive procedure, which provides the relevant information that is available through other techniques such as molecular diagnostics. Therefore, procedures 83890–83901 and 83912 should not be submitted for separate reimbursement when submitted with procedures 87491 and 87591. In most states, justification of medical necessity is not required for these two CPT codes; no limited coverage codes from the International Classification of Diseases 9th Revision, Clinical Modification (ICD-9-CM) are required for reimbursement in the outpatient or inpatient settings. At the time of writing, reimbursement for 87491 and 87591 ranges from approximately $22 for Medicaid to a range of $40 to $70 for third-party payers. Each laboratory must research reimbursement rates to determine if internalization of C. trachomatis and N. gonorrhoeae NAAT will be financially feasible. REFERENCES 1. Addiss, D. G., M. L. Vaughn, D. Ludka, J. Pfister, and J. P. Davis. 1993. Decreased prevalence of Chlamydia trachomatis infection associated with a selective screening program in family planning clinics in Wisconsin. Sex. Transm. Dis. 20:28–35. 2. Airell, A., L. Ottosson, S. M. Bygdeman, H. Carlberg, P. Lidbrink, A. K. Ruden, and K. Elfgren. 2000. Chlamydia trachomatis PCR (Cobas Amplicor) in women: endocervical specimen transported in a specimen of urine versus endocervical and urethral speci-
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mens in 2-SP medium versus urine specimen only. Int. J. STD AIDS 11:651–658. 3. Akduman, D., J. M. Ehret, K. Messina, S. Ragsdale, and F. N. Judson. 2002. Evaluation of a strand displacement amplification assay (BD ProbeTec-SDA) for detection of Neisseria gonorrhoeae in urine specimens. J. Clin. Microbiol. 40:281–283. 4. Alary, M., C. Poulin, C. Bouchard, M. Fortier, G. Murray, S. Gingras, M. Aube, and C. Morin. 2001. Evaluation of a modified sanitary napkin as a sample self-collection device for the detection of genital chlamydial infection in women. J. Clin. Microbiol. 39:2508–2512. 5. American Medical Association. 2003. Current Procedural Terminology. AMA Press, Chicago, Ill. 6. Anguenot, J. L., F. de Marval, P. Vassilakos, R. Auckenthaler, V. Ibecheole, and A. Campana. 2001. Combined screening for Chlamydia trachomatis and squamous intra-epithelial lesions using a single liquidbased cervical sample. Hum. Reprod. 16:2206–2210. 7. Aslanzadeh, J., and M. Jones. 2002. Comparison of M4 and M4RT media for transporting cervical swab samples for PCR detection of Chlamydia trachomatis and Neisseria gonorrhoeae. Ann. Clin. Lab. Sci. 32: 61–64.
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15. Black, C. M. 1997. Current methods of laboratory diagnosis of Chlamydia trachomatis infections. Clin. Microbiol. Rev. 10:160–184. 16. Black, C. M., J. Marrazzo, R. E. Johnson, E. W. Hook III, R. B. Jones, T. A. Green, J. Schachter, W. E. Stamm, G. Bolan, M. E. St. Louis, and D. H. Martin. 2002. Head-to-head multicenter comparison of DNA probe and nucleic acid amplification tests for Chlamydia trachomatis infection in women performed with an improved reference standard. J. Clin. Microbiol. 40:3757–3763. 17. Blake, M. S., and E. C. Gotschlich. 1987. Functional and immunologic properties of pathogenic Neisseria surface proteins, p. 377–400. In M. Inouye (ed.), Bacterial Outer Membranes as Model Systems. Wiley, New York, N.Y. 18. Blake, M. S., L. M. Wetzler, E. C. Gotschlich, and P. A. Rice. 1989. Protein III: structure, function, and genetics. Clin. Microbiol. Rev. (2 Suppl):S60–S63. 19. Bloomfield, P. J., C. Kent, D. Campbell, L. Hanbrook, and J. D. Klausner. 2002. Community-based chlamydia and gonorrhea screening through the United States mail, San Francisco. Sex. Transm. Dis. 29:294–297. 20. Bloomfield, P. J., K. C. Steiner, C. K. Kent, and J. D. Klausner. 2003. Repeat chlamydia screening by mail, San Francisco. Sex. Transm. Infect. 79:28–30.
8. Association of Public Health Laboratories. 2001. Practical Guidelines for Chlamydia Test Verification, p. 1–5. National Chlamydia Laboratory Committee, Washington, D.C.
21. Bodrossy, L., and A. Sessitsch. 2004. Oligonucleotide microarrays in microbial diagnostics. Curr. Opin. Microbiol. 7:245–254.
9. Banuelos Panuco, C. A., I. Deleon Rodriguez, J. T. Hernandez Mendez, L. A. Martinez Guzman, D. Akle Fierro, J. Miranda Murillo, and E. Reyes Maldonado. 2000. Detection of Chlamydia trachomatis in pregnant women by the Papanicolaou technique, enzyme immunoassay and polymerase chain reaction. Acta Cytol. 44:114–123.
22. Boel, C. H., C. M. van Herk, P. J. Berretty, G. H. Onland, and A. J. van den Brule. 2005. Evaluation of conventional and real-time PCR assays using two targets for confirmation of results of the COBAS AMPLICOR Chlamydia trachomatis/Neisseria gonorrhoeae test for detection of Neisseria gonorrhoeae in clinical samples. J. Clin. Microbiol. 43:2231–2235.
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