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Recommendations for the Laboratory-Based Detection of *Chlamydia trachomatis* and *Neisseria gonorrhoeae* — 2014








Recommendations Continued						Print
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			Detection of Genitourinary *C. trachomatis* and *N. gonorrhoeae* Infections in WomenDetection of Genitourinary *C. trachomatis* and *N. gonorrhoeae* Infections in MenDetection of Extragenital *C. trachomatis* and *N. gonorrhoeae* Infections in Men and WomenDetection of Genitourinary and Extragenital *C. trachomatis* and *N. gonorrhoeae* Infections in Cases of Sexual AssaultDetection of lymphogranuloma venereum InfectionsAdditional ConsiderationsTests Not Recommended for Routine Use
		
Detection of Genitourinary *C. trachomatis* and *N. gonorrhoeae* Infections in Women
Screening programs have been demonstrated to reduce both the prevalence of *C. trachomatis* infection and rates of PID in women (79,80). Sexually active women aged ≤25 years and women aged >25 years with risk factors (e.g., those who have a new sex partner or multiple partners) should be screened annually for chlamydial infections (81).
The prevalence of gonorrhea varies widely among communities and populations; health-care providers should consider local gonorrhea epidemiology when making screening decisions. Although widespread screening is not recommended, targeted screening of young women (i.e., those aged ≤25 years) at increased risk for infection is a primary component of gonorrhea control in the United States because gonococcal infections among women are frequently asymptomatic. For sexually active women, including those who are pregnant, the U.S. Preventive Services Task Force (USPSTF) recommends that clinicians provide gonorrhea screening only to those at increased risk for infection (e.g., women with previous gonorrhea infection, other STDs, new or multiple sex partners, and inconsistent condom use; those who engage in commercial sex work and drug use; women in certain demographic groups; and those living in communities with a high prevalence of disease). USPSTF does not recommend screening for gonorrhea in women who are at low risk for infection (81).
For female screening, specimens obtained with a vaginal swab are the preferred specimen type. Vaginal swab specimens are as sensitive as cervical swab specimens, and there is no difference in specificity (82–87). Self-collected vaginal swabs are equivalent in sensitivity and specificity to those collected by a clinician (83,88). Cervical samples are acceptable when pelvic examinations are done, but vaginal swab specimens are an appropriate sample type, even when a full pelvic exam is being performed. Cervical specimens collected into a liquid cytology medium for Pap screening are acceptable for NAATs that have been cleared by FDA for such specimen types (Table 2). However, following Pap screening, there should be a clinical indication for reflex additional testing of liquid cytology specimens for chlamydia and gonorrhea since these specimen types are more widely used in older populations at low risk for infection. First catch urine from women, while acceptable for screening, might detect up to 10% fewer infections when compared with vaginal and endocervical swab samples (82,87,89) (Box 2).
BOX 2. *Chlamydia trachomatis* and *Neisseria gonorrhoeae* testing in women
Nucleic acid amplification tests (NAATs) are the recommended test method.
A self- or clinician-collected vaginal swab is the recommended sample type. Self-collected vaginal swab specimens are an option for screening women when a pelvic exam is not otherwise indicated.
An endocervical swab is acceptable when a pelvic examination is indicated.
A first catch urine specimen is acceptable but might detect up to 10% fewer infections when compared with vaginal and endocervical swab samples.
An endocervical swab specimen for *N. gonorrhoeae* culture should be obtained and evaluated for antibiotic susceptibility in patients that have received CDC recommended antimicrobial regimen as treatment, and subsequently had a positive *N. gonorrhoeae* test result (positive NAAT ≥7 days after treatment), and did not engage in sexual activity after treatment.
Detection of Genitourinary *C. trachomatis* and *N. gonorrhoeae* Infections in Men
*C. trachomatis* and N. *gonorrhoeae* control efforts in men differ substantially from those recommended for women. Although chlamydia prevalence data have provided a basis for setting age guidelines for routine annual screening and behavioral guidelines for targeted screening in women (11), no such consensus has been reached regarding control program definitions in men who have sex with women (12). Although there are no recommendations to screen heterosexual men, it USPSTF suggests testing to test sexually active heterosexual men in clinical settings with a high prevalence of *C. trachomatis* (e.g., STD clinics, adolescent clinics, and detention and correctional facilities) and among persons entering the Armed Forces or the National Job Training Program (81).
The prevalence of *N. gonorrhoeae* varies widely among communities and populations; health-care providers should consider the local gonorrhea epidemiology when making screening recommendations. There is insufficient evidence for or against routine screening for gonorrhea in sexually active heterosexual men at increased risk for infection (81). However, it is not recommended to screen for gonorrhea infections in men at low risk for infection (81).
Overwhelming evidence described the performance of male first catch urine samples as equivalent to, and in some situations superior to, urethral swabs (23,90). Use of urine samples is highly acceptable and might improve the likelihood of uptake of routine screening in men (Box 3).
BOX 3. *Chlamydia trachomatis* and *Neisseria gonorrhoeae* testing in men
Nucleic acid amplification tests (NAATs) are the recommended test method.
A first catch urine is the recommended sample type and is equivalent to a urethral swab in detecting infection.
A urethral swab specimen for *N. gonorrhoeae* culture should be obtained and evaluated for antibiotic susceptibility in patients who have received CDC recommended an antimicrobial regimen as treatment, and subsequently had a positive *N. gonorrhoeae* test result (positive NAAT ≥7 days after treatment), and did not engage in sexual activity after treatment.
Detection of Extragenital *C. trachomatis* and *N. gonorrhoeae* Infections in Men and Women
Infections with *C. trachomatis* and *N. gonorrhoeae* are common in extragenital sites in certain populations such as MSM. Because extragenital infections are common in MSM, and most infections are asymptomatic (91), routine annual screening of extragenital sites in MSM is recommended. No recommendations exist regarding routine extragenital screening in women because studies have focused on genitourinary screening, but rectal and oropharyngeal infections are not uncommon.
A 2003 study that assessed NAATs for diagnosing *C. trachomatis* and *N. gonorrhoeae* infections in multiple anatomic sites in MSMs (91) used Becton Dickinson’s ProbeTec NAAT, which had been validated previously for such use. Among 6,434 MSM attending an STD clinic or a gay men’s clinic, the study found that the prevalence by site for *C. trachomatis* was 7.9% for the rectum, 5.2% urethral, and 1.4% pharyngeal; and prevalence by site for *N. gonorrhoeae* was 6.9% for the rectum, 6% urethral, and 9.2% pharyngeal. The great majority (84%) of the gonococcal and chlamydial rectal infections were asymptomatic. More than half (53%) of *C. trachomatis* and 64% of *N. gonorrhoeae* infections were at nonurethral sites and would have been missed if the traditional approach to screening of men by testing only urethral specimens had been used.
The scope of the problem of extragenital infection in MSM is not known at the national level. In 2007, CDC coordinated an evaluation of MSM attending several community-based organizations and public or STD clinics and found that of approximately 30,000 tests performed, 353 (5.4%) MSM were positive for rectal infection with *N. gonorrhoeae*, and 468 (8.9%) were positive for rectal *C. trachomatis*. Pharyngeal *N. gonorrhoeae* tests were positive for 759 MSM (5.3%), and 54 (1.6%) were positive for *C. trachomatis* (92).
In the United Kingdom, some studies on screening MSMs have been performed using NAATs (93,94), and in one study of 3,076 MSM attending an STD clinic, there was an 8.2% prevalence of infection with *C. trachomatis* in the rectum and 5.4% in the urethra. The majority (69%) of the men with *C. trachomatis* were asymptomatic, stressing the need for screening (94).
A study that compared culture to two NAATs (Hologic/ Gen-Probe’s Aptima Combo2 [AC2]) and Becton Dickinson’s ProbeTec) for the detection of *C. trachomatis* and *N. gonorrhoeae* in pharyngeal and rectal specimens collected from 1,110 MSM being seen in an STD clinic confirmed all NAAT positive results when either the original test or a test using alternate primers was positive (95). For oropharyngeal *N. gonorrhoeae*, sensitivities were 41% for culture, 72% for SDA, and 84% for AC2; and for rectal *N. gonorrhoeae*, sensitivities were 43% for culture, 78% for SDA, and 93% for AC2. For oropharyngeal infections with *C. trachomatis* (for which only nine infections were detected), sensitivities were 44% for culture, 67% for SDA, and 100% for AC2; for rectal *C. trachomatis*, sensitivities were 27% for culture, 63% for SDA, and 93% for AC2. Specificities were >99.4% for all specimens, tests, and anatomic sites. The number of infections detected was more than doubled when a more sensitive NAAT was used as compared with the use of standard culture. Other researchers also have demonstrated the superiority of NAATs as compared with culture for diagnosing *C. trachomatis* and *N. gonorrhoeae* in rectal and oropharyngeal sites (36,75,76).
Although commercially available NAATs are recommended for testing genital tract specimens, they have not been cleared by FDA for the detection of *C. trachomatis* and *N. gonorrhoeae* infections of the rectum and oropharynx (Box 4). Results from commercially available NAATs can be used for patient management if the laboratory has established specifications for the performance characteristics according to CLIA regulations (96). If a moderate complexity test such as the GeneXpert is modified in any manner, the test defaults to high complexity and the laboratory must meet all high complexity CLIA requirements, including those for personnel. Certain NAATs that have been demonstrated to detect commensal *Neisseria* species in urogenital specimens might have comparable low specificity when testing oropharyngeal specimens for *N. gonorrhoeae*. Thus, a *N. gonorrhoeae* NAAT that does not react with nongonococcal commensal *Neisseria* species is recommended when testing oropharyngeal specimens (Table 3).
BOX 4. Detection of *Chlamydia trachomatis* and *Neisseria gonorrhoeae* infections in the rectum and oropharynx
Nucleic acid amplification tests (NAATs) are the recommended test method for rectal and oropharyngeal specimens.
Laboratories must be in compliance with CLIA for test modifications since these tests have not been cleared by the FDA for these specimen types.
Commensal *Neisseria* species commonly found in the oropharynx might cause false positive reactions in some NAATs, and further testing might be required for accuracy.
A rectal or oropharyngeal swab specimen for *N. gonorrhoeae* culture should be obtained and evaluated for antibiotic susceptibility in patients who have received CDC recommended antimicrobial regimen as treatment, had a subsequent positive *N. gonorrhoeae* test result (positive NAAT ≥7 days after treatment), and did not engage in sexual activity after treatment.
Detection of Genitourinary and Extragenital *C. trachomatis* and *N. gonorrhoeae* Infections in Cases of Sexual Assault
Detailed information about evaluation and treatment of suspected victims of sexual assault can be obtained from the 2010 STD treatment guidelines (19). General recommendations pertaining only to *C. trachomatis* and *N. gonorrhoeae* testing are presented here. Examination of victims is required for two purposes: 1) to determine if an infection is present so that it can be successfully treated and 2) to acquire evidence for potential use in a legal investigation. Testing to satisfy the first purpose requires a method that is highly sensitive, whereas satisfying the second purpose requires a method that is highly specific. Although NAATs meet these criteria, acceptance of any test results is determined by local legal authorities. Local legal requirements and guidance also should be sought for maintaining and documenting a chain of custody for specimens and results that might be used in a legal investigation and for which test results are accepted as evidence.
NAATs for *C. trachomatis* and *N. gonorrhoeae* are preferred for the diagnostic evaluation of adult sexual assault victims, from any sites of penetration or attempted penetration (97,98). Data on use of NAATs for detection of *N. gonorrhoeae* in children are limited. Consultation with an expert is necessary before use of NAATs for this indication in children to minimize the possibility of positive reactions with nongonococcal *Neisseria* species and other commensals. NAATs can be used as alternative to culture with vaginal specimens or urine specimens from girls. Culture remains the preferred method for urethral specimens from boys and extragenital specimens (pharynx and rectum) in boys and girls.
Using highly specific tests is critical with prepubescent children for whom the diagnosis of a sexually transmitted infection might lead to initiation of an investigation for child abuse. Specimen collection for culture for *N. gonorrhoeae* includes the pharynx and rectum in boys and girls, the vagina in girls, and the urethra in boys. Cervical specimens are not recommended for prepubertal girls. For boys with a urethral discharge, a meatal specimen discharge is an adequate substitute for an intra-urethral swab specimen. Standard culture procedures must be followed. Gram stains are inadequate to evaluate prepubertal children for *N. gonorrhoeae* and should not be used to diagnose or exclude *N. gonorrhoeae*. Specimens from the vagina, urethra, pharynx, or rectum should be streaked onto selective media for isolation of *N. gonorrhoeae*, and all presumptive isolates of *N. gonorrhoea*e should be identified definitively by at least two tests that involve different principles (e.g., biochemical, enzyme substrate, or serologic). Isolates should be preserved to enable additional or repeated testing.
Cultures for *C. trachomatis* can be collected from the rectum in both boys and girls and from the vagina in girls. The likelihood of recovering *C. trachomatis* from the urethra of prepubertal boys is too low to justify the trauma involved in obtaining an intraurethral specimen. However a meatal specimen should be obtained if urethral discharge is present. Pharyngeal specimens for *C. trachomatis* are not recommended for children of either sex because the yield is low, perinatally acquired infection might persist beyond infancy, and culture systems in some laboratories use antibody stains that do not distinguish between *C. trachomatis* and *C. pneumoniae*. All specimens must be retained for additional testing, if necessary, regardless of a positive or negative test result.
Only standard culture systems for the isolation of *C. trachomatis* should be used. The isolation of *C. trachomatis* should be confirmed by microscopic identification of inclusions by staining with fluorescein-conjugated monoclonal antibody MOMP specific for *C. trachomatis*; stains using monoclonal antibodies directed against LPS should not be used. EIAs are not acceptable confirmatory methods. Isolates should be preserved. Nonculture tests for *C. trachomatis* such as EIAs and DFA are not sufficiently specific for use in circumstances involving possible child abuse or sexual assault. NAATs can be used for detection of *C. trachomatis* in vaginal specimens or urine from girls. No data exist on the use of nucleic acid amplification tests in boys and extragenital specimens (rectum) in boys and girls. Culture remains the preferred method for extragenital sites in these circumstances.
Detection of lymphogranuloma venereum Infections
Serologic testing for LGV is not widely available in the United States. The chlamydial complement fixation test (CFT), which measures antibody against group-specific lipopolysaccharide antigen, has been used as an aid in the diagnosis of LGV. A CFT titer ≥1:64 typically can be measured in the serum of patients with bubonic LGV (99). The microimmunofluorescence (MIF) test was initially developed for serotyping strains of *C. trachomatis* isolated from the eye and genital tract but was soon adapted to measure antibody responses in patients with chlamydial infections. Although the original MIF method was complicated, involving the titration of sera against numerous antigens, it was found to have many advantages when compared with the CFT (99). The MIF test can be used detect type-specific antibody and different immunoglobulin classes. The MIF test is more sensitive than the CFT with a larger proportion of patients developing an antibody response and at higher titer. Patients with LGV tend to have broadly crossreactive MIF titers that are often greater than 1:256 (99). Microtiter plate format enzyme immunoassays have been developed but comparative performance data are lacking. Serologic test interpretation for LGV is not standardized, tests have not been validated for clinical proctitis presentations, and *C. trachomatis* serovarspecific serologic tests are not widely available. More detailed information concerning the diagnosis and treatment of LGV has been published (19).
Genital and lymph node specimens (i.e., lesion swab or bubo aspirate) can be tested for *C. trachomatis* by culture, direct immunofluorescence, or nucleic acid detection. Commercially available NAATs for *C. trachomatis* detect both LGV and non-LGV *C. trachomatis* but cannot distinguish between them. Additional molecular procedures (e.g., PCR-based genotyping) can be used to differentiate LGV from non-LGV *C. trachomatis*, but these are not widely available (100–104).
For patients presenting with proctitis, *C. trachomatis* NAAT testing of a rectal specimen is recommended. While a positive result is not definitive diagnosis of LGV, the result might aid in a presumptive clinical diagnosis of LGV proctitis.
Additional Considerations
**Supplemental testing of NAAT-positive specimens.** In 2002, CDC recommended that consideration be given to performing an additional test routinely after a positive NAAT screening test for *C. trachomatis* and *N. gonorrhoeae* (20). This approach was advised to improve the positive predictive value (PPV) of a NAAT screening test. This was particularly important when the test was used in a population with a low prevalence of infection. However, studies since 2002 addressing the utility of routine repeat testing of positive specimens demonstrated >90% concurrence with the initial test for either *C. trachomatis* or *N. gonorrhoeae* (105–107). Therefore, routine additional testing following a positive NAAT screening test for *C. trachomatis* no longer is recommended by CDC unless otherwise indicated in the product insert. Some NAATs might detect nongonococcal *Neisseria* species (Table 3). When these NAATs are used, consideration should be given to retest these specimens with an alternate target assay if the anatomic site from which the specimen was collected is typically colonized with these commensal organisms, e.g., oropharyngeal specimens. As with any diagnostic test, if there is a clinical or laboratory reason to question a test result, a repeat test should be considered.
**Test interpretation.** The laboratory should interpret and report results according to the manufacturer’s package insert instructions In the event of discordant results from multiple tests, the report should indicate the results of both the initial and any additional tests. An interpretation of “inconclusive,” “equivocal,” or “indeterminate” would be most appropriate. A new specimen should be requested for testing in these situations. All test results should be interpreted by clinicians within the context of the patient-specific information to determine appropriate patient management.
**Test of cure.** Culture is the only method that can be used to properly assess the efficacy of antibiotic therapy because commercial NAATs are not FDA-cleared for use as a test of cure. Residual nucleic acid from bacteria rendered noninfective by antibiotics might still give a positive *C. trachomatis* NAAT up to 3 weeks after therapy (108,109). Detection of *N. gonorrhoeae* nucleic acid has been observed for up to 2 weeks following therapy although the vast majority of patients who were treated effectively for gonorrhea had a negative NAAT 1 week after treatment (110). However, data from these studies were derived from older NAATs and should be repeated with current NAATs.
**Pooling of specimens.** The superior performance characteristics of NAATs for detection of *C. trachomatis* and *N. gonorrhoeae* have led some researchers to pool urine specimens in an attempt to reduce the higher material costs associated with their use (111–113). Samples of individual specimens are first combined into a pool, which is then tested by a NAAT. If the pool is negative, all specimens forming the pool are reported as negative. If the pool is positive, a second aliquot of each specimen that contributed to the pool is tested individually. The potential cost-savings with pooling increases with decreasing prevalence of infection, because more specimens can be included in a pool without increasing the probability of a pool testing positive. The number of specimens pooled to achieve the greatest cost savings for a particular prevalence can be calculated (111). Available evidence indicates that pooled aliquots from up to 10 urine specimens can be a cost-effective alternative to testing individual specimens without any loss of sensitivity or specificity (111). Savings from reduced reagent costs have ranged from 40% to 60% (111). However, the increased complexity of the pooling protocol might require more personnel time to deconstruct positive pools for individual specimen testing. The use of pooled specimens for testing is not cleared by FDA and, therefore, the CLIA requirements applicable to modifying a test procedure must be met before implementation and reporting results intended to guide patient care. Laboratories must be aware that the process of pooling specimens requires extensive handling of samples which increases the potential for cross-contamination. Studies for pooling clinical specimens other than urine are required before extending this recommendation.
Tests Not Recommended for Routine Use
**Direct fluorescent antibody (DFA) tests.** This assay should not be used for routine testing of genital tract specimens. Rather, DFA tests are the only FDA-cleared tests for ocular *C. trachomatis* infections. Depending on the commercial product used, the antigen that is detected by the antibody in the *C. trachomatis* DFA procedure is either the MOMP or LPS molecule. Specimen material is obtained with a swab or endocervical brush, which is then rolled over the specimen well of a slide. After the slide has dried and the fixative applied, the slide can be stored or shipped at ambient temperature. The laboratory should process the slide <7 days after the specimen has been obtained. Staining consists of flooding the smear with fluorescein-labeled monoclonal antibody that binds to *C. trachomatis* elementary bodies. Stained elementary bodies are then identified by fluorescence microscopy. Only *C. trachomatis* organisms will stain with the anti-MOMP antibodies used in commercial kits. The anti-LPS monoclonal antibodies will react with family-specific epitopes found within the LPS of *Chlamydiaceae* and might cross-react with LPS of other bacteria. The procedure requires an experienced microscopist and is labor-intensive and time-consuming. No DFA tests exist for the direct detection of *N. gonorrhoeae* in clinical specimens.
**Nucleic acid hybridization/probe tests.** Two nucleic acid hybridization assays are FDA-cleared to detect *C. trachomatis* or *N. gonorrhoeae*: the Hologic/Gen-Probe PACE 2 and the Digene Hybrid Capture II assays. Both the PACE and Hybrid Capture assays can detect *C. trachomatis* or *N. gonorrhoeae* in a single specimen. The Hybrid Capture assay is not widely available and the PACE 2C test was discontinued December 31, 2012.
**Nucleic acid genetic transformation tests.** The Gonostat test (Sierra Diagnostics, Incorporated, Sonora, California) uses a gonococcal mutant that grows when genetically altered by DNA extracted from a swab specimen containing *N. gonorrhoeae*. *N. meningitidis* causes false-positive results (114). The test has received limited evaluation in published studies (115–118), which included an evaluation of its use with mailed specimens (117). Amplified and hybridization tests that detect *N. gonorrhoeae* nucleic acid have better performance characteristics than the Gonostat test. The gonorrhea nucleic acid genetic transformation test might have some utility in settings that lack the stringency for gonorrhea culture. However, it is not recommended as an alternative test to *N. gonorrhoeae* NAATs. A genetic transformation test is not available for detection of *C. trachomatis* infection.
**Enzyme immunoassay (EIA) tests.** A substantial number of EIA tests have been marketed for detecting *C. trachomatis* infection. The performance and cost characteristics of EIA tests for *N. gonorrhoeae* infection have not made them competitive with other available tests (56). *C. trachomatis* EIA tests detect chlamydial LPS, and there is the potential for false-positive results caused by crossreaction with LPS of other microorganisms. Manufacturers have developed blocking assays that verify positive EIA test results to improve specificity. None of the EIAs are as sensitive or specific as NAATs, and their use is discouraged.
**Serology tests.** Serology has little, if any, value in testing for uncomplicated genital *C. trachomatis* infection. It should not be used for screening because previous chlamydial infection might or might not elicit a systemic antibody response. Infections caused by LGV serovars of *C. trachomatis* tend to invade to the draining lymph nodes resulting in a greater likelihood of detectable systemic antibody response and might aid in diagnosis of inguinal (but not rectal) disease (99). The complement fixation test was classically used for this purpose but has been replaced by the more sensitive species-specific microimmunofluorescence test. A serologic screening or diagnostic assay is not available for *N. gonorrhoeae*.
							
					
							Last Reviewed:  March 31, 2017						Source: 	Division of STD Prevention, National Center for HIV, Viral Hepatitis, STD, and TB Prevention, Centers for Disease Control and Prevention
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Introduction
Methods
Recommendations
Recommendations Continued
Conclusion
Appendix


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