Epidemiologic Notes and Reports False-Positive Results with the Use of Chlamydia Tests in the Evaluation of Suspected Sexual Abuse -- Ohio, 1990

On June 21, 1990, a commercial laboratory reported to a private residential-care facility for profoundly retarded persons in Ohio that rectal cultures from 10 residents tested positive for Chlamydia trachomatis. This report summarizes the epidemiologic and laboratory investigation by public health officials in Ohio and at CDC, which concluded that the C. trachomatis results were false-positive.

On June 6, a female resident of the facility (index resident) who had undergone a hysterectomy several years earlier was evaluated in a hospital emergency room for vaginal bleeding. Because sexual abuse was suspected, vaginal specimens were obtained for culture of Neisseria gonorrhoeae and nonculture (i.e., enzyme immuno assay (EIA)) detection of C. trachomatis and sent to the local health department. On June 12, the facility was notified that the chlamydia test was positive, and a 10-day course of doxycycline was initiated.

On June 18, the facility's medical staff collected multiple specimens for chlamydia and gonorrhea testing with swabs from 25 of the other 26 residents (one recently admitted resident was not tested) of the same unit as the index resident and sent the specimens to a commercial laboratory for analysis. Specimens included rectal swabs from all residents for culture of C. trachomatis and N. gonorrhoeae, urethral swabs from males and cervical swabs from females for nucleic acid probe assays to detect C. trachomatis and N. gonorrhoeae, and urethral swabs from males for chlamydia culture. On June 21, the laboratory reported that rectal cultures were positive for chlamydia in 10 residents (six female and four male; age range: 10-25 years); all other specimens were negative. On June 23, pharyngeal swabs for chlamydia culture were obtained from the 10 residents with positive rectal cultures, and doxycycline therapy was initiated for these 10 residents. On June 25, four of the 10 pharyngeal cultures were reported by the commercial laboratory as positive for C. trachomatis.

From June 25 through June 28, rectal, pharyngeal, urethral (males), and cervical (females) swabs for chlamydia culture and rectal swabs for gonorrhea culture were obtained from the 75 residents of the remaining three units of the facility and from the 15 residents of the first unit who initially tested negative. All specimens were sent to the commercial laboratory that had tested the specimens obtained on June 18 and June 23; chlamydia cultures were positive in three residents (in two patients, rectal only, and one patient, rectal and pharyngeal). On June 29, all male staff of the facility and female staff of the index resident's unit were asked to volunteer to be cultured for C. trachomatis; rectal, pharyngeal, and urethral swabs were obtained from males, and rectal and pharyngeal swabs from females. All specimens from staff members for chlamydia culture were sent to the local health department; none were positive.

On June 22 and June 25, the commercial laboratory reported that it had used immunofluorescence (IF) staining to identify chlamydial inclusions in cell culture; this report implied the true presence of chlamydia in the rectal specimens obtained June 18. On July 2, however, the laboratory indicated that 9 months previously it had changed from the IF method to a new EIA confirmation method for detecting chlamydial antigen in cell culture and that only the EIA method had been used to identify the 10 rectal and four pharyngeal specimens as positive, as well as to identify as positive the specimens from the three residents of the facility obtained during the week of June 25.

On July 6, to compare the EIA culture confirmation and standard IF culture confirmation methods, duplicate rectal and pharyngeal specimens were obtained from the three residents identified as infected during the week of June 25 and from three residents who had tested negative; these specimens were tested by both the commercial laboratory (using both IF and EIA culture confirmation) and the chlamydia laboratory of CDC's Division of Sexually Transmitted Diseases Laboratory Research, Center for Infectious Diseases (IF culture confirmation only). None of the specimens from these six residents were positive by IF culture confirmation at either laboratory. However, five rectal and two pharyngeal specimens were positive by EIA culture confirmation at the commercial laboratory.

The CDC chlamydia laboratory also performed standard IF culture confirmation on residual transport media from the 10 rectal and four pharyngeal specimens initially reported as positive by EIA culture confirmation. No chlamydia were detected in any of these specimens. However, because these 14 transport media had not been stored optimally before transport to CDC, the viability of any chlamydial organisms present would be reduced. Therefore, CDC also analyzed these specimens (and those obtained from the six residents on July 6) by polymerase chain reaction (PCR) using C. trachomatis-specific primers to amplify a portion of the 16s RNA gene; PCR results were also negative. Finally, CDC tested serum specimens from the 16 residents who had positive chlamydia cultures by EIA confirmation and the index resident for antibodies to C. trachomatis and C. pneumoniae (formerly C. psittaci TWAR) using the microimmunofluorescence test and immunoblotting; no IgG or IgM antibodies to C. trachomatis were detected. IgG antibody to C. pneumoniae, consistent with a past infection, was detected in one resident.

Based on the failure to detect C. trachomatis (or C. pneumoniae) in any specimens by conventional culture techniques, negative PCR results, and the absence of serologic evidence of infection, the investigators concluded that the initial reports of positive chlamydia cultures represented false-positive results. Reported by: KJ Dorian, MS, F Holtzhauer, PhD, WC Myers, MS, Columbus Health Dept; JM Moser, MD, TJ Halpin, MD, State Epidemiologist, Ohio Dept of Health. Clinical Research Br, Div of STD/HIV Prevention, Center for Prevention Svcs; Molecular Epidemiology and Pathogenesis Br, Div of Sexually Transmitted Diseases Laboratory Research, Center for Infectious Diseases, CDC.

Editorial Note

Editorial Note: Both culture and nonculture methods for the detection of C. trachomatis are widely used in the United States. When performed by experienced technologists, cell culture isolation of C. trachomatis is the most sensitive and specific test. The three types of nonculture methods commercially available for detecting C. trachomatis directly in clinical specimens are EIA, direct immunofluorescence staining of smears (DIF), and nucleic acid probe tests.

In this report, the index resident was tested for chlamydia because of suspicion of sexual abuse, and a nonculture (EIA) chlamydia test performed on a vaginal specimen (a site for which this test is not approved) was positive. However, because the physical findings were not consistent with sexual abuse and serologic evidence of infection was not present, the nonculture results appear to have been false-positive. Based on the evaluation of this resident and subsequent investigation, three important issues concerning the use of laboratory tests to identify C. trachomatis should be emphasized. First, chlamydia tests should be used only on specimens for which they are approved. Each antigen detection and nucleic acid probe test for C. trachomatis is approved for use only on specimens from certain anatomic sites. When used on specimens from sites for which they are not approved, the likelihood of false-positive results is higher. False-positive results have been reported with the use of EIA on vaginal specimens from children (1-3) and rectal specimens from adults and children (4-6) and with DIF staining of rectal smears from adults and children (1,5). EIA and DIF false-positives may result from cross-reactivity with other bacteria commonly present in the anogenital area, including strains of Acinetobacter calcoaceticus, Escherichia coli, Gardnerella vaginalis, group A and group B streptococci, N. gonorrhoeae, Proteus vulgaris, and Staphylococcus aureus (2-4,7-9).

Second, only standard chlamydia cultures should be used in the evaluation of suspected sexual abuse (10) or other situations in which the possibility of a false-positive result is unacceptable. The two components necessary to culture C. trachomatis are a cell-culture system to amplify the number of organisms from a clinical specimen and an inclusion detection method (11). IF staining is the most sensitive detection method available. False-positive results should not occur with IF staining since the chlamydial inclusions have a characteristic morphology and unique staining pattern (12). Nonculture tests for chlamydia are not recommended and should not be used in the evaluation of suspected sexual abuse because of the possibility of false-positive results.

The method used by the commercial laboratory involved in this report uses the standard cell culture system but with EIA detection of chlamydial antigens rather than an inclusion staining method. EIA involves an automated optical density endpoint reading that is proportional to the amount of antigen present. EIA detection has the potential to generate false-positive results because EIA detects solubilized chlamydial antigens that would be derived from the inoculated cell culture, as well as cross-reacting antigens from other organisms present in clinical specimens. Those organisms may be present more commonly in rectal and pharyngeal specimens than in cervical and urethral specimens and may be amplified in the cell culture system if they are resistant to the antimicrobials usually added to the transport medium and cell culture to suppress microbial contamination.

Finally, the term chlamydia "culture" should imply the use of visual identification of characteristic chlamydial intracellular inclusions in cell culture, because this method is specific. Any method that detects solubilized chlamydial macromolecules (e.g., proteins, lipopolysaccharide, DNA, and RNA) after inoculation and incubation of a specimen in cell culture is more likely to yield false-positive results than visual identification of inclusions and therefore may be less specific. Thus, laboratories that claim to be performing chlamydia "culture" should use only standard methods to grow and detect chlamydia. The decision to use EIA (or other methods that do not require visual identification of characteristic inclusions by a trained technician) to identify the presence of chlamydia in cell culture requires full understanding of the advantages and limitations of these methods.

References

1. Hammerschlag MR, Rettig PJ, Shields ME. False positive

results with the use of chlamydial antigen detection tests in the evaluation of suspected sexual abuse in children. Pediatr Infect Dis J 1988;7:11-4.

2. Porder K, Sanchez N, Roblin PM, McHugh M, Hammerschlag MR. Lack of specificity of Chlamydiazyme for detection of vaginal chlamydial infection in prepubertal girls. Pediatr Infect Dis J 1989;8:358-60.

3. Goudswaard F, Sabbe L, van Belzen C. Interference by gram-negative bacteria in the enzyme immunoassay for detecting Chlamydia trachomatis. J Infect 1989;18:94-6.

4. Riordan T, Ellis DA, Matthews PI, Ratcliffe SF. False positive results with an ELISA for detection of chlamydia antigen. J Clin Pathol 1986;39:1276-7.

5. Pratt BC, Tait IA, Anyaegbunam WI. Rectal carriage of Chlamydia trachomatis in women. J Clin Pathol 1989;42:1309-10.

6. Rothburn MM, Mallinson H, Mutton KJ. False-positive ELISA for Chlamydia trachomatis recognised by atypical morphology on fluorescent staining. Lancet 1986;2:982-3.

7. Krech T, Gerhard-Fsadni D, Hofmann N, Miller SM. Interference of Staphylococcus aureus in the detection of Chlamydia trachomatis by monoclonal antibodies. Lancet 1985;1:1161-2.

8. Saikku P, Puolakkainen M, Leinonen M, et al. Cross-reactivity between Chlamydiazyme and Acinetobacter strains. N Engl J Med 1986;314:922.

9. Taylor-Robinson D, Thomas BJ, Osborn MF. Evaluation of enzyme immunoassay (Chlamydiazyme) for detecting Chlamydia trachomatis in genital tract specimens. J Clin Pathol 1987;40:194-9. 10. CDC. 1989 Sexually transmitted diseases treatment guidelines. MMWR 1989;38(no. S-8): 41-3. 11. CDC. Chlamydia trachomatis infections: policy guidelines for prevention and control. MMWR 1985;34(no. 3S):57S. 12. Barnes RC. Laboratory diagnosis of human chlamydial infections. Clin Microbiol Rev 1989;2:119-36.

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