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Screening Tests To Detect Chlamydia trachomatis and Neisseria gonorrhoeae Infections - 2002

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• Collecting, Transporting, and Storing Specimens
• Laboratory Implementation of NAATS
• Test of Cure, Treatment Failure, and Antimicrobial Resistance
• Sexual Assault and Sexual Abuse

Collecting, Transporting, and Storing Specimens

Correct specimen collection and handling techniques are critical for all methods used to identify C. trachomatis and N. gonorrhoeae. Even diagnostic tests with the highest performance characteristics cannot produce accurate results when specimens submitted to the laboratory are incorrectly collected. Recommendations for transporting and storing specimens are summarized in this report (Appendix D).

Clinicians require training and periodic assessment to maintain correct technique for specimen collection (125--127). The presence of columnar epithelial cells has been associated with increased sensitivity in the majority of studies that evaluated collection of endocervical specimens for C. trachomatis screening with NAATs as well as other types of tests (125--132). How these results apply to other types of specimens is unclear. Obtaining columnar cells is less critical for detecting N. gonorrhoeae than for detecting C. trachomatis.

Collecting and Transporting Specimens for Screening

Specimen Collection Recommendations Applicable to Culture and Nonculture Tests

Although the requirement for columnar endocervical cells applies less to N. gonorrhoeae than to C. trachomatis, guidelines included in this report are appropriate for both organisms (Box 9).

BOX 9. Specimen-collection guidelines

* Source: Sellors J, Chernesky M, Pickard L, et al. Effect of time elapsed since previous voiding on the detection of Chlamydia trachomatis antigens in urine. Eur J Clin Microbiol Infect Dis 1993;12:285–9.

Specific Requirements for C. trachomatis Culture

Swabs with plastic or wire shafts can be used to obtain specimens for cell culture (133--135). Swab tips can be made of cotton, rayon, or Dacron,^® but should not be made of calcium alginate ( 133--136). Swabs with wooden shafts should not be used because the wood might contain substances that are toxic to C. trachomatis or tissue culture cells (133--135). As part of routine quality control, samples of each lot of swabs that are used to collect specimens for C. trachomatis isolation should be screened for possible inhibition of C. trachomatis growth and toxicity to tissue culture cells.

The substitution of an endocervical brush for a swab might increase the sensitivity of culture for endocervical specimens from nonpregnant women (137). However, using an endocervical brush might induce bleeding. Although such bleeding does not interfere with the isolation of C. trachomatis, patients should be advised regarding possible spotting.

When culture isolation of C. trachomatis from women is to be performed, processing a specimen from the urethra as well as the endocervix can increase sensitivity by 23% (138). Placing the two specimens in the same transport container is acceptable. The viability of C. trachomatis organisms must be maintained during transport to the laboratory (28).

• When the elapsed time between specimen collection and inoculation is ≤24 hours, specimens should be stored at 4^ºC and inoculated in cell culture as quickly as possible.
• When specimens cannot be inoculated in ≤24 hours, the specimens should be maintained at ≤--70^ºC.
• Specimens for culture should never be stored at --20^ºC or in frost-free freezers.

Specific Requirements for N. gonorrhoeae Culture

Swabs with plastic or wire shafts can be used to obtain specimens for culture. Swab tips can be made of rayon, Dacron, or calcium alginate. Obtaining a second cervical specimen for N. gonorrhoeae culture is associated with an increase in sensitivity (139). Inoculating the two specimens on the same culture plate is acceptable.

The viability of N. gonorrhoeae organisms must be maintained during transport to the laboratory; therefore, the following recommendations are made for transporting specimens:

• Specimens should be inoculated directly onto a selective or nonselective (if the specimen is from a sterile site) nutritive medium (e.g., Martin-Lewis or chocolate agar containing IsoVitaleX,^® respectively).
• Specimens should be incubated at 35^ºC--36.5^ºC in a CO₂-enriched atmosphere immediately after collection.
• For transport to a local laboratory, placement of the inoculated plates in a CO₂-enriched atmosphere is more critical than incubation at 35^ºC--36.5^ºC (140--142). Inoculated media can be held at room temperature in a CO₂-enriched atmosphere for ≤5 hours without a loss of viability.
• For transport to a remote laboratory, specimens should be inoculated onto commercial transport media (e.g., JEMBEC^® or Transgrow media). Inoculated media should be incubated for 18--24 hours before being transported, and the specimen should arrive ≤48 hours after collection from the patient. Specimens should not be transported in Stuart's, Amies', or other such media; specimens are diluted and organisms might lose viability if delays in shipping occur. If specimens must be transported in extreme hot or cold conditions, the specimens should be placed in an insulated Styrofoam^® container. Detailed directions for collection of specimens for culture of N. gonorrhoeae have been described elsewhere (12 ,143 ,144).

Adequacy of Endocervical Specimens

Without endocervical specimen quality assurance, ≥10% of specimens collected for C. trachomatis testing are probably unsatisfactory because they contain secretions or exudate, but lack endocervical cells (126 ,128 ,145). A substantially reduced likelihood exists of detecting C. trachomatis in inadequate specimens by all tests, including NAATs (125--132).

Assessing Endocervical Specimen Quality.
Health-care providers and laboratorians are encouraged to evaluate the effect on endocervical C. trachomatis test positivity rates of monitoring the endocervical columnar cell content of specimens and then providing feedback and, as indicated, training to clinicians. Until proven unnecessary, routine or periodic assessment of endocervical specimen quality is recommended for all types of C. trachomatis tests. A report presenting alternative methods of measuring specimen quality has been developed by the National Chlamydia Laboratory Committee for the CDC-supported C. trachomatis screening program and distributed to constituent screening programs (additional information is available at http://www.aphl.org/chlamydia_lab.cfm). The majority of published studies have categorized specimens dichotomously as either containing the appropriate endocervical cells or not. In one study, a dose-response association between quantity of the appropriate endocervical cells and the C. trachomatis positivity rate was demonstrated by using a multiple-category scale for quantity of appropriate cells (132). This study also prescribed a systematic process for reviewing slides. Diff-Quick stain has been described as efficient and inexpensive (125).

The principal purpose of assessing the quality of endocervical specimens is to determine whether the sensitivity of C. trachomatis tests would be enhanced by providing feedback and training to clinicians. No evaluations have been published of the cost-effectiveness of alternative approaches to measuring the quality of endocervical cell specimens and improving specimen-collection practices, and no studies have correlated measures of specimen adequacy with test positivity for specimens collected for N. gonorrhoeae testing.

Reporting and Follow-Up of Inadequate Specimens.
An inadequate specimen should be reported as inadequate. Whenever possible, a second specimen should be collected for repeat testing.

Collecting Specimens for Indications Other Than Screening

Anatomic site-specific recommendations for application of C. trachomatis and N. gonorrhoeae tests, including for medicolegal applications, are summarized in this report (Appendices A and B). Multiple sources provide directions for collecting specimens for N. gonorrhoeae testing (12 ,143 ,144).

Laboratory Implementation of NAATS

NAATs require more attention to procedural details and development and maintenance of quality control systems than other nonculture screening tests (e.g., EIAs and nonamplified nucleic acid hybridization assays). An increase in such requirements results from the susceptibility of NAAT amplification enzymes to inhibition and the potential of NAATs to generate cross-contaminating target and to detect limited quantities of target present as a contaminant. Despite close attention to quality control, concerns regarding consistency of test performance and reproducibility persist, as indicated by reports of heterogeneity of results in clinical trials (78 ,80) and varying rates of reproducing positive results (146--151).

As laboratories and health-care providers transition to amplification tests, certain critical concerns should be addressed, including

• clinician training whenever a change occurs in the testing method. Training should address
  • indications for test use (e.g., appropriate types of specimens);
  • general instruction in obtaining adequate specimens from any site and specific instruction in obtaining a proper endocervical specimen (i.e., one that contains endocervical cells rather than ectocervical cells or vaginal material);
  • requirements for storage and transport; and
  • interpretation of test results.
• monitoring of specimen collection and transport and periodic reinforcement of staff training.
• development of standard laboratory operating procedures and quality-assurance protocols based on package inserts and any supplementary manufacturer instructions. Procedures and protocols should address  
  
  • adoption of prescribed work areas and specimen handling procedures to avoid cross-contamination, which is of heightened importance because of the inherent sensitivity of amplification tests;
  • use of positive and negative controls, including a positive control from culture stock or known positive clinical specimens in addition to the control provided in the commercial kit;
  • trade-offs of using amplification controls to identify inhibitors (e.g., reducing false-negative results but decreasing throughput); and
  • creation of a data system that alerts laboratorians when a run includes an unusual number of positive specimens or when positive specimens are clustered within a run.
• manufacturer-based training of laboratory staff with periodic retraining.
• CLIA requirements for verifying or establishing test performance characteristics. If a laboratory is adopting an FDA-cleared test that is classified under CLIA as a high-complexity test, CLIA requires conducting a study to verify that the test performs according to the manufacturer's package insert claims (24). If the laboratory is adopting a test that has not been cleared by FDA or is adopting a modification of an FDA-cleared test, CLIA requires a more extensive study to establish performance specifications, because FDA-cleared package insert specifications are lacking (24) (Appendix C).
• participation in a proficiency testing program.

Test of Cure, Treatment Failure, and Antimicrobial Resistance

Test-of-cure is not recommended as a routine procedure after therapy for C. trachomatis or N. gonorrhoeae infection with first-line CDC-recommended treatment regimens, except after C. trachomatis therapy during pregnancy (152). Nonculture tests that are performed <3 weeks after completion of antimicrobial therapy might be falsely positive because of the presence of nonviable organisms; this applies in particular to NAATs (153--160).

CDC recommends that clinicians contact their local or state health department for guidance and to arrange for antimicrobial susceptibility testing of isolates from patients apparently failing CDC-recommended therapy for C. trachomatis infection or CDC-recommended or FDA-approved therapy for N. gonorrhoeae infection. For this purpose, a patient's infection is considered to have failed therapy if the patient is laboratory-test--positive for C. trachomatis or N. gonorrhoeae after treatment and the patient provides a history of having complied with the prescribed therapy and denies posttreatment sexual exposure to an untreated or new sex partner.

Because knowledge is limited regarding the ability of C. trachomatis to develop antimicrobial resistance, CDC encourages health departments to inform CDC of treatment failures and, if possible, arrange for shipment of a swab specimen suitable for tissue culture for test confirmation. To report apparent C. trachomatis treatment failure, contact the following:

Surveillance and Special Studies Section, Mail Stop E-02
Epidemiology and Surveillance Branch
Division of STD Prevention
Centers for Disease Control and Prevention
1600 Clifton Rd., N.E.
Atlanta, GA 30333
Phone: 404-639-8371
Attention: Susan Wang, M.D.
or Hillard Weinstock, M.D.

To submit specimens for C. trachomatis testing, contact the following:

Chlamydia Laboratory
Syphilis and Chlamydia Branch, Mail Stop D-13
Centers for Disease Control and Prevention
1600 Clifton Rd., NE
Atlanta, GA 30333
Phone: 404-639-3785
Attention: John Papp, Ph.D.

CDC encourages health departments to inform CDC of N. gonorrhoeae treatment failures and, if possible, arrange for N. gonorrhoeae culture and testing of any isolate for susceptibility to CDC-recommended regimens used to treat patients. If susceptibility testing cannot be performed at a local or state laboratory, isolates should be submitted to CDC as should any isolate that is resistant to a CDC-recommended therapy. Antimicrobial susceptibility testing should be performed according to recommendations of the National Committee for Clinical Laboratory Standards (NCCLS), and results should be interpreted according to criteria designated by NCCLS. If NCCLS has not provided criteria for resistance for a CDC-recommended therapy (e.g., cephalosporins), an isolate is considered to be resistant if the isolate fails to meet the NCCLS criteria for susceptibility. NCCLS might not have designated criteria for definition of a susceptible category for antimicrobial agents that are used for gonorrhea treatment. When no NCCLS criterion is available, consult http://www.cdc.gov/std/gonorrhea/lab/diskdiff.htm  for additional information or contact the Neisseria Reference Laboratory for further guidance. To report apparent N. gonorrhoeae treatment failure, contact the following:

Surveillance and Special Studies Section, Mail Stop E-02
Epidemiology and Surveillance Branch
Division of STD Prevention
Centers for Disease Control and Prevention
1600 Clifton Rd., N.E.
Atlanta, GA 30333
Phone: 404-639-8371
Attention: Susan Wang, M.D.
or Hillard Weinstock, M.D.

To submit specimens for N. gonorrhoeae testing, contact the following:

Neisseria Reference Laboratory, Unit 31
Gonorrhea Research Branch, Mail Stop C-13
Bldg. 1 South/Room B260
Centers for Disease Control and Prevention
1600 Clifton Rd., N.E.
Atlanta, GA 30333
Attention: David Trees, Ph.D., 404-639-2134
or Joan S. Knapp, Ph.D., 404-639-3470

In addition, statewide programs should be maintained to routinely isolate gonococcal strains and monitor antimicrobial susceptibilities to CDC-recommended therapies and to other FDA-cleared therapies with established usage in the state.

Sexual Assault and Sexual Abuse

Detailed information concerning evaluation and treatment of suspected victims of sexual assault or abuse can be obtained from the 2002 STD treatment guidelines (152). Presented here are general guidelines pertaining only to C. trachomatis and N. gonorrhoeae infections (Box 10). 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. Because of the health and legal implications of test results, the additional time, labor, and cost of performing tests that are sensitive and highly specific are justified. Using highly specific tests is critical with preadolescent children for whom the diagnosis of a sexually transmitted infection might lead to initiation of an investigation for child abuse. Local legal requirements and guidance should be sought for maintaining and documenting a chain of custody for specimens and results that might be used in a legal investigation.

BOX 10. General guidelines for testing specimens related to possible sexual assault or abuse

 

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