Tests to Detect C. trachomatis and N. gonorrhoeae

Isolation and identification of Chlamydia trachomatis. Specimen collection swabs for C. trachomatis culture must have a plastic or wire shaft and either rayon, dacron, or cytobrush tip. Other materials might inhibit isolation. Specimen collection for C. trachomatis culture is invasive requiring insertion of a swab 2–3 cm into the male urethral or 1–2 cm into the endocervical canal followed by two or three rotations to collect sufficient columnar or cuboidal epithelial cells. Following the collection, culture samples should be stored in an appropriate transport media such as sucrose phosphate glutamate buffer or M4 media (Thermal Scientific, Lenexa, Kansas) and transported at ≤4°C to the laboratory within 24 hours of collection to maximize recovery of viable organisms. If transport is delayed >24 hours, the transport media containing the specimen should be stored at -70°C. The specimen is inoculated by centrifugation onto a confluent monolayer of McCoy, HeLa 229, or Buffalo green monkey kidney cells that support growth of C. trachomatis (42–46). Once the specimen has been inoculated, 2μg/ml of cycloheximide should be added to the growth medium to suppress protein synthesis by the host eukaryotic cell (47). Inoculated cells are harvested after 48–72 hours of growth; infected cells develop characteristic intracytoplasmic inclusions that contain substantial numbers of C. trachomatis elementary and reticulate bodies.

The cell monolayers are reacted with either genus specific or species-specific fluorescein-conjugated monoclonal antibodies to allow specific visualization of the chlamydial inclusions with an epifluorescent microscope. Cell culture detection of C. trachomatis is highly specific if a C. trachomatis major outer membrane protein (MOMP)–specific stain is used. Monoclonal antibodies directed against the family-specific lipopolysaccharide (LPS) of Chlamydiaceae cost less but might stain bacteria that share LPS antigens. LPS stains might be suitable for routine use, but a species-specific (MOMP) stain is recommended in situations requiring increased specificity (48–50). Less specific inclusion-detection methods using iodine or Giemsa stain are not recommended (48–50).

Cell culture methods vary among laboratories, resulting in substantial interlaboratory variation in performance (51). The shell vial method of culture uses a larger inoculum with a reduced risk for crosscontamination and therefore provides better accuracy than the 96-well microtiter plate method (42,43). In certain laboratories, higher sensitivities are obtained by performing a blind pass in which an inoculated cell monolayer is allowed to incubate for 48–72 hours, after which the monolayer is disrupted and used to inoculate a fresh monolayer that is stained after 48–72 hours of incubation to allow for another cycle of growth (49).

Despite the technical difficulties, cell culture, when performed by an experienced analyst, was the most sensitive diagnostic test for chlamydial infection until the introduction of NAATs (28,52). The relatively low sensitivity, extended testing turnaround time, difficulties in standardization, labor intensity, technical complexity, stringent specimen collection and transport requirements, and relatively high cost are disadvantages of cell culture isolation of C. trachomatis. Recommended procedures for C. trachomatis isolation and culture detection using a species specific stain must be followed when using this test in cases of suspected child sexual assault in boys and extragenital infections in girls.

Isolation and identification of N. gonorrhoeae. Because of its high specificity (>99%) and sensitivity (>95%), a Gram stain of a male urethral specimen that demonstrates polymorphonuclear leukocytes with intracellular Gram-negative diplococci can be considered diagnostic for infection with N. gonorrhoeae in symptomatic men. However, because of lower sensitivity, a negative Gram stain should not be considered sufficient for ruling out infection in asymptomatic men. In addition, Gram stains of endocervical specimens, pharyngeal, or rectal specimens also are not sufficient to detect infection and therefore are not recommended. Specific testing for N. gonorrhoeae is recommended because of the increased utility and availability of highly sensitive and specific testing methods and because a specific diagnosis might enhance partner notification.

If multiple specimens are being collected from an anatomic site, N. gonorrhoeae culture specimens should be obtained first; this sequence maximizes the load collected, which increases the likelihood of a successful culture (39). Specimens collected for gonorrhea culture should be obtained by using swabs with plastic or wire shafts and rayon, Dacron, or calcium alginate tips. Other swab material such as wood shafts and cotton tips might be inhibitory or toxic to the organism and should be avoided. Although collection of epithelial cells is less important for culture detection of N. gonorrhoeae, swabs should be inserted 2–3 cm in the male urethra or 1–2 cm into the endocervical canal followed by two or three rotations. In cases of urethritis, collection of the exudate is sufficient for N. gonorrhoeae culture.

Several nonnutritive swab transport systems are available, and some studies suggest that these transport systems might maintain gonococcal viability for up to 48 hours in ambient temperatures (53–55). However, environmental conditions might vary by location and season, which could affect the viability of gonorrhea in these transport systems; thus, additional local validation of transport conditions might be needed. Culture medium transport systems are preferred because there are some advantages over swab transport systems (e.g., extended shelf life and better recovery because cultivated isolates are being transported rather than a clinical specimen) (39). Culture medium is inoculated with the swab specimen and then placed immediately into a CO2- enriched atmosphere for transportation to the laboratory. Because N. gonorrhoeae has demanding nutritional and environmental growth requirements, optimal recovery rates are achieved when specimens are inoculated directly and when the growth medium is incubated in an increased CO2 environment as soon as possible.

Methods of gonococcal culture have been described elsewhere (39,56). Specimens from normally nonsterile sites are streaked on a selective (e.g., Thayer-Martin or Martin-Lewis) medium and specimens from sterile sites are streaked on nonselective (e.g., chocolate agar) medium. Culture media for N. gonorrhoeae isolation include a base medium supplemented with chocolatized (heated) equine or bovine blood to support the growth of the gonococcus. Commercially prepared chocolate agar containing synthetic hemin and growth factors for N. gonorrhoeae are available from various vendors. Selective media differ from routine culture media in that they contain antimicrobial agents (i.e., vancomycin, colistin, and nystatin or another antifungal agent) that inhibit the growth of other bacteria and fungi. Using selective media might improve isolation if the anatomic source of the specimen normally contains other bacterial species although some strains of N. gonorrhoeae have been demonstrated to be inhibited on selective media (57). Inoculated media are incubated at 35°C–36.5°C in an atmosphere supplemented with 5% CO2 and examined at 24 and 48 hours postcollection. Supplemental CO2 can be supplied by a CO2 incubator, candle-extinction jar using unscented candles (e.g., votive candles) or CO2-generating tablets.

Isolates recovered from a genital specimen on selective medium that are Gram-negative diplococcus- and oxidasepositive might be presumptively identified as N. gonorrhoeae (39). A presumptive identification indicates only that a Gramnegative, oxidase-positive diplococcus (e.g., any Neisseria species or Branhamella catarrhalis) might be isolated from such specimens. Certain coccobacilli, including Kingella denitrificans, might appear to be Gram-negative diplococci in Gram-stained smears. A confirmed laboratory diagnosis of N. gonorrhoeae requires additional biochemical tests (Table 1). A presumptive test result is sufficient to initiate antimicrobial therapy, but additional tests must be performed to confirm the identity of an isolate as N. gonorrhoeae (39).

Culture for N. gonorrhoeae is inexpensive to perform from genital sites and is specific and sensitive if the specimen is collected and transported properly to the laboratory. However, it is less than ideal for routine diagnostics because of stringent collection and transport requirements, and confirmation might take several days from time of specimen collection. The primary advantage of isolating N. gonorrhoeae by culture is the ability to characterize the isolate further by antimicrobial susceptibly testing and genetic analysis if necessary. Cephalosporins are the sole class of antibiotics recommended for the treatment of N. gonorrhoeae infections in CDC’s 2010 STD treatment guidelines (19), and the availability of gonoccocal culture capacity at the local level is an important consideration if a patient fails therapy (58).

TABLE 1. Characteristics of gram-negative, oxidase-positive Neisseria and related species of human origin

Antibiotic susceptibility testing. Gonorrhea treatment is complicated by the ability of N. gonorrhoeae to develop resistance to antimicrobial therapies. Genetic mutations and/or acquisition of genetic material from closely related bacteria species might result in antibiotic-resistant N. gonorrhoeae. Plasmid mediated resistance to penicillin can be conferred by extrachromosomal genes encoding for β-lactamase that destroys penicillin (59,60). Resistance to tetracycline also might occur when the organism acquires an extrachromosomal gene from streptococcus, the tetM gene that allows for ribosomal protein synthesis that is normally impaired by tetracycline (61). Testing for these plasmid genes provides limited information because genetic changes in the chromosome also might confer resistance to penicillin and tetracycline in addition to spectinomycin and fluoroquinolones (62,63). Testing specimens for genetic alterations in the chromosome requires a complete understanding of the complex and multiple mechanisms associated with resistance. For example, chromosomal-mediated resistance to penicillin can alter penicillin binding, penetration, or efflux (64). Resistance to fluoroquinlones results from mutations in DNA gyrase (gyrA) or topoisomerase (parC) resulting in decreased drug penetration and increased efflux (65,66). Penicillin-, tetracycline-, and fluoroquinolone-resistant N. gonorrhoeae isolates now are disseminated widely throughout the United States and globally (67). These antimicrobial agents no longer are recommended regimens for N. gonorrhoeae treatment, and thus susceptibility testing is not needed to make recommendations for clinical management. Laboratory capacity for N. gonorrhoeae culture and antibiotic susceptibility testing is critical to monitor for emerging resistance. Updated information regarding N. gonorrhoeae antibiotic susceptibility testing is available from CDC at

Assessing N. gonorrhoeae isolates for antibiotic susceptibility requires viable isolates because accurate genetic markers of antibiotic resistance to recommended therapies have not been documented. Agar plate dilution testing that provides minimum inhibitory concentration values of tested antibiotics is the preferred method for testing the susceptibility of N. gonorrhoeae but might be too difficult to perform in laboratories with limited capacity and low testing volumes. Disk diffusion and E-test are simpler methods for determining susceptibilities of gonococcal isolates, although cefixime E-test strips are not FDA-cleared for use in the United States. Isolates that appear to be sess susceptible than the current Clinical and Laboratory Standards Institute (CLSI) interpretive criteria for susceptible organisms (68) (available at should be submitted to CDC for reference testing using the agar plate dilution method because there are no CLSI interpretive criteria for resistance to CDC-recommended therapeutic agents. Procedures for agar dilution and disk diffusion testing are available at Clinicians who diagnose N. gonorrhoeae infection in a patient with suspected treatment failure should contact their local or state public health laboratory or local clinical laboratory for guidance on submitting specimens for culture and susceptibility testing. Local and state public health laboratory directors are encouraged to maintain culture and antimicrobial susceptibility testing capabilities for N. gonorrhoeae or identify public health or private laboratories in their area with such capacity if they do not perform such testing.

No standard procedures exist to assess in vitro susceptibility of C. trachomatis to antibiotics (69). Further research is required to determine the relationship between in vitro data and outcome of treatment.

Nucleic acid amplification tests (NAATs). As of May 2013, five manufacturers had commercially available and FDA-cleared NAAT assay platforms for the detection of C. trachomatis and N. gonorrhoeae in the United States. NAAT assays are recommended for detection of urogenital infections caused by C. trachomatis and N. gonorrhoeae infections in women and men with and without symptoms. These tests have been shown to be cost-effective in preventing sequelae due to these infections (70–72). A list of FDA-cleared specimen types and transport and storage requirements is provided (Table 2). These include Abbott RealTime m2000 CT/NG (Abbott Molecular Inc. Des Plaines, Illinois), Amplicor and cobas CT/NG test (Roche Molecular Diagnostics, Branchburg, New Jersey); Aptima, (Hologic/Gen-Probe, San Diego, California); BD ProbeTec ET and Qx (Becton Dickinson, Sparks, Maryland), and Xpert CT/ NG Assay (Cepheid, Sunnyvale, California) (Table 2). NAATs are designed to amplify and detect nucleic acid sequences that are specific for the organism being detected. Similar to other nonculture tests, NAATs do not require viable organisms. The increased sensitivity of NAATs is attributable to their theoretic ability to produce a positive signal from as little as a single copy of the target DNA or RNA. This high sensitivity has allowed the use of less invasively collected specimens such as first catch urines and vaginal swabs to detect shed organisms. Use of such specimens greatly facilitates screening.

Commercial tests differ in their amplification methods and their target nucleic acid sequences (Table 2). The two Roche tests and the Abbott RealTime CT/NG use polymerase chain reaction (PCR) and both Becton Dickinson tests use strand displacement amplification (SDA) to amplify C. trachomatis DNA sequences in the cryptic plasmid that is found in >99% of strains of C. trachomatis. The Hologic/Gen-Probe Aptima Combo 2 assay for C. trachomatis uses transcription-mediated amplification (TMA) to detect a specific 23S ribosomal RNA target. The Roche cobas CT/NG test, Abbott, Becton Dickinson, and Hologic/Gen-Probe tests detect the new variant of C. trachomatis (nvCT) strain. These nucleic acid amplification methods also are used to detect N. gonorrhoeae, and each manufacturer has marketed a duplex assay that allows for simultaneous detection of both organisms. The nucleic acid primers used by commercial NAATs for C. trachomatis are not known to detect DNA from other bacteria found in humans. However, the primers employed by the Becton Dickinson N. gonorrhoeae NAATs might detect nongonococcal Neisseria species (73–76) (Table 3). Most commercial NAATs have been cleared by FDA to detect C. trachomatis and N. gonorrhoeae in vaginal and endocervical swabs from women, urethral swabs from men, and first catch urine from both men and women (Table 2).

Because NAATs are so sensitive, efforts are warranted to prevent contamination of specimens in the clinic or spread of environmental amplicon in the laboratory. Laboratories should follow standard molecular method techniques, clean workspaces and equipment frequently, include multiple negative controls in each run, and monitor the rate of indeterminate and positive results as a change in monthly trends might indicate a need to investigate the accuracy of the results. Environmental monitoring might be required as recommended by the manufacturer. If environmental amplicons are found, robust cleaning of the laboratory is needed until negative results are obtained. Steps to prevent cross-contamination include proper testing of laboratory workflow design and strict adherence to testing and quality assurance protocols.

Performance of Tests to Detect C. trachomatis and N. gonorrhoeae

Studies assessing the performance of NAATs might include test algorithms that use multiple NAATs, nonculture and culture tests as reference standards. Regardless of the analytic study design, the performance characteristics are relative to the standards used at the time of evaluation. When less sensitive methods are used as the reference standard, the specificity of the test under evaluation is likely to be underestimated. Conversely, the sensitivity of older assays was likely overestimated because of the relative poor performance of the assays used as standards at the time.

Because no gold standard exists, researchers compared two versions of the patient-infected-status algorithm (PISA) to assess the performance of NAATs. Using simulations with latent-class models, these researchers concluded that PISAbased methods can produce biased estimates of sensitivity and specificity that changed markedly as the true prevalence changes (77). However, there is no consensus on the optimal approach to evaluating the performance of NAATs, and better methods are needed (78). Until better methods become available, these recommendations support continuing reliance on NAATS based on their approval by FDA for indicated clinical use.

Simply quoting sensitivity and specificity data from package inserts or published studies is not useful because the numbers are estimates and are valid only within the context of the particular evaluation. Variables that can impact on these numbers include what comparison tests were used, in which population the evaluation was performed, and whether calculations were made on the basis of an infected patient standard or a direct comparison of specimens.

Nevertheless, despite the absence of a criterion standard, valid generalizations can be made. All diagnostic tests including NAATs can generate inaccurate results, and it is important for laboratorians and clinicians to understand test limitations. Certain false positives and false negatives can occur as a consequence of specimen collection, test operation, and laboratory environment. However, NAATs are far superior in overall performance compared with other C. trachomatis and N. gonorrhoeae culture and nonculture diagnostic methods. NAATs offer greatly expanded sensitivities of detection, usually well above 90%, while maintaining very high specificity, usually ≥99%. NAATs typically detect 20%–50% more chlamydial infections than could be detected by culture or earlier nonculture tests (20). The increment for detection of gonococcal infections is somewhat less.

TABLE 2. Food and Drug Administration–cleared* specimen types and requirements for the transport and storage of specimens for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae by nucleic acid amplification test (NAAT) type

TABLE 3. Chlamydia trachomatis and Neisseria gonorrhoeae Nucleic Acid Amplification Test (NAAT) target sequences and possible false reactions, by test type