diagnosis of Chlamydia trachomatis infections. J. Clin. Microbiol. 26:1735â. 1737. 4. Gaydos, C. A., L. Bobo, L. Welsh, E. W. Hook III, R. Viscidi, and T. C.. Quinn.
JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1997, p. 2679–2680 0095-1137/97/$04.0010 Copyright © 1997, American Society for Microbiology
Vol. 35, No. 10
Failure To Detect Chlamydia trachomatis in Cell Culture by Using a Monoclonal Antibody Directed against the Major Outer Membrane Protein ¨ RAN WADELL,1 JENS BOMAN,1* CHARLOTTE GAYDOS,2 PER JUTO,1 GO
AND
THOMAS C. QUINN2
Department of Clinical Virology, The University Hospital of Umeå, Umeå University, Umeå, Sweden,1 and Department of Medicine, The Johns Hopkins University, Baltimore, Maryland2 Received 31 March 1997/Returned for modification 10 June 1997/Accepted 7 July 1997
Two commercially available monoclonal antibodies for cell culture confirmation of Chlamydia trachomatis were compared in two prospective studies and one large retrospective study. In total, more than 33,000 genital specimens were cultured in parallel and stained with both antibodies, one of which was directed against the major outer membrane protein (MOMP) and one of which was directed against the lipopolysaccharide (LPS). We found the anti-LPS-based assay to be more sensitive and as specific as the anti-MOMP-based assay for C. trachomatis cell culture confirmation of genital specimens. tember 1995 for C. trachomatis analysis were included. For positive samples the fluorescence was graded from 6 to 3 (6, organisms just visible; 1, light-green staining; 2, moderate fluorescent-green staining; and 3, intense fluorescent-green staining) according to Montalban et al. (6). A total of 969 samples were analyzed, 58 of which were toxic to the cell culture and, therefore, were not included in this study. Of the remaining 911 samples, 32 (3.5%) were culture positive by the anti-LPS antibody, whereas 28 (3.1%) were positive by the anti-MOMP antibody. All anti-MOMP-positive samples were positive by the anti-LPS antibody. The fluorescence intensity was equal for 22 samples; however, in 6 samples (21%) the fluorescence was stronger with more numerous and larger inclusion bodies by the anti-LPS assay (Table 1). In order to investigate whether the Swedish results could be confirmed by another laboratory, and to establish whether LPS-positive–MOMP-negative specimens were true C. trachomatis culture positives, all genital samples cultured at the Johns Hopkins University Chlamydia Research Laboratory from January 1991 through December 1996 were reviewed. A total of 31,266 genital specimens were cultured for C. trachomatis in cycloheximide-treated McCoy cells in duplicate wells in 96-well microtiter plates. After 72 h of incubation, each of the two wells was fixed and stained with the above-described commercially available monoclonal antibodies; for all specimens the first well was stained with the anti-LPS monoclonal antibody (Kallestad) and the second well was stained with the anti-MOMP monoclonal antibody (Syva). There were 277 (0.89%) specimens toxic to cell culture, and these were not included as part of the study. Of the remaining 30,989 cultures, 2,519 were culture positive (prevalence, 8.13%). A total of 2,484 cultures were positive by both monoclonal antibodies, whereas 35 were positive only by the anti-LPS monoclonal antibody. None of these isolates were positive for Chlamydia pneumoniae by using a species-specific monoclonal antibody. Thus, 1.4% of the C. trachomatis-positive samples were negative by the anti-MOMP antibody (Table 2). In addition, 301 (12.1%) of the culture-positive samples stained lightly (6, approximately 60%; 1, 40%) with the anti-MOMP monoclonal antibody and had normal staining (2 and 3) with the anti-LPS monoclonal antibody. Of the LPS-positive–MOMP-negative cultures, a subset was typed with serovar-specific monoclonal antibodies or by PCR (1, 4). The majority (85%) were sero-
Chlamydia trachomatis is one of the most common sexually transmitted bacterial infections in the world with an estimated number of 50 million new cases each year (11). Since genital chlamydial infections can cause severe clinical diseases, especially in women (2), correct diagnosis and treatment are imperative. Several diagnostic methods are used including cell culture, enzyme immunoassay, and direct fluorescent assay, and recently, different nucleic acid amplification methods have been introduced. Agreement between different methods is not always very good (7, 9). Chlamydia sp. in cell culture is usually identified by means of monoclonal antibodies that recognize the major outer membrane protein (MOMP) or the lipopolysaccharide (LPS). In order to investigate whether the Chlamydia antigen to be detected is of importance for identifying C. trachomatis in cell culture, we conducted comparative studies using monoclonal antibodies recognizing the MOMP of C. trachomatis (MicroTrak Chlamydia trachomatis Culture Confirmation test; Behring Diagnostica/Syva Company, San Jose, Calif.) and the LPS of Chlamydia (Pathfinder Chlamydia Culture Confirmation Assay, Sanofi Diagnostics Pasteur/Kallestad, Chaska, Minn.). In a preliminary study performed in February 1995, 1,456 genital samples were cultured for 48 h in parallel in cycloheximide-treated McCoy cells in duplicate wells in 96-well microtiter plates and Chlamydia inclusions were confirmed by the two antibodies in one of the wells for each antibody. In order to create conditions as identical as possible for the two evaluated antibodies, the choice between the wells was standardized so that the first and the second well were used equally often for each antibody. Fifty-three C. trachomatis-positive-samples were detected by the anti-LPS antibody compared to only 43 such samples (81% of the number of positive samples detected by the anti-LPS antibody) by the anti-MOMP antibody. Due to the discrepancy between the results of the two tests we decided to repeat the study with a protocol that was accepted by both companies. Therefore, all genital samples sent to the Department of Clinical Virology, University Hospital of Northern Sweden, Umeå, Sweden, from 28 August to 29 Sep* Corresponding author. Mailing address: Department of Clinical Virology, The University Hospital of Umeå, S-901 85 Umeå, Sweden. Phone: 46 90 7851304. Fax: 46 90 129905. E-mail: jens.boman@climi .umu.se. 2679
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TABLE 1. Grading of fluorescence intensity in 6 of 28 positive samples in which the intensities were different between the anti-LPS and anti-MOMP antibodies Intensity of fluorescencea Sample
Anti-LPS antibody
Anti-MOMP antibody
1 2 3 4 5 6
2 1 1 2 1 1
1 6 6 6 6 6
a
Shown as 6, 1, and 2 as defined in the text.
typed as C. trachomatis serovar J. In addition, the light-staining positive cultures were predominantly serovar J (63%) but also included serovars D (17%), E (15%), and F (5%). The clinical impact of not diagnosing the J serovar and the others (D, E, and F) is dependent on their frequency. To answer such a question, van de Laar et al. (10) compiled data on the distribution of serovars of genital infections due to C. trachomatis from 14 published studies (6 from the United States and 8 from Europe) and found that the J, D, E, and F serovars are rather prevalent in both the United States and Europe. In total, 5,189 isolates (2,795 from the United States and 2,394 from Europe) were included in the compilation. The average percentages of C. trachomatis serotypes J, D, E, and F in the United States were 6.5, 22.7, 23.7, and 18.7%, respectively, and in Europe they were 6.2, 13.2, 38.8, and 19.3%, respectively. Since all patients in our studies of whom the results of the two tests differed had signs and symptoms compatible with a genital chlamydial infection, and there was nothing to suggest another diagnosis, the results thus indicate that the broadly reacting anti-LPS antibody is more sensitive than the antiMOMP antibody. It may be argued that the anti-LPS antibody, due to its broader range and ability to detect chlamydial spe-
TABLE 2. Summary of two Swedish prospective and one U.S. retrospective study comparing the sensitivities of anti-LPSa and anti-MOMPb antibodies for culture confirmation of C. trachomatis in genital specimens No. of positive samples (%) Type of study (location)
No. of samples
Prospective (Sweden) Prospective (Sweden) Retrospective (United States) Total
1,456 911 30,989 33,356
Both antibodies
Anti-LPS antibody only
43 (81) 28 (88) 2,484 (98.6) 2,555 (98.1)
10 (19) 4 (12) 35 (1.4) 49 (1.9)
a Pathfinder Chlamydia Culture Confirmation Assay, Sanofi Diagnostics Pasteur/Kallestad. b MicroTrak Chlamydia trachomatis Culture Confirmation test, Behring Diagnostica/Syva Company.
cies other than C. trachomatis (6), yields a lower specificity in the detection of C. trachomatis. Nonetheless, from a clinical point of view this is not a problem since all chlamydial species infecting the genital tract should be treated. Furthermore, there is very little evidence of genital infections in humans with other species of Chlamydia. For direct fluorescent assay staining of elementary bodies, Cles et al. (3) have shown in in vitro experiments that monoclonal antibodies against the MOMP of C. trachomatis may produce a brighter and more consistent fluorescence than monoclonal antibodies against the LPS. However, in the study by Cles et al. (3), the anti-LPS antibody from Kallestad was not used. The explanation as to why the anti-LPS assay had a higher sensitivity in culture confirmation in our studies might be due to a more pronounced capability of detecting both forms of the organism (elementary bodies and reticulate bodies) (8) or to the presence of new MOMP variants of C. trachomatis, which have been shown to be able to escape neutralization by both monoclonal antibodies and human immune sera (5). In conclusion, we found the anti-LPS antibody to be more sensitive than the anti-MOMP antibody and as specific as the antiMOMP-based assay for C. trachomatis cell culture confirmation of genital specimens. REFERENCES 1. Barnes, R. C., S. P. Wang, C. C. Kuo, and W. E. Stamm. 1985. Rapid immunotyping of Chlamydia trachomatis with monoclonal antibodies in a solid-phase enzyme immunoassay. J. Clin. Microbiol. 22:609–613. 2. Centers for Disease Control and Prevention. 1991. Pelvic inflammatory disease: guidelines for prevention and management. Morbid. Mortal. Weekly Rep. 40:1–25. 3. Cles, L. D., K. Bruch, and W. E. Stamm. 1988. Staining characteristics of six commercially available monoclonal immunofluorescence reagents for direct diagnosis of Chlamydia trachomatis infections. J. Clin. Microbiol. 26:1735– 1737. 4. Gaydos, C. A., L. Bobo, L. Welsh, E. W. Hook III, R. Viscidi, and T. C. Quinn. 1992. Gene typing of Chlamydia trachomatis by polymerase chain reaction and restriction endonuclease digestion. Sex. Transm. Dis. 19:303– 308. 5. Lampe, M. F., K. G. Wong, L. M. Kuehl, and W. E. Stamm. 1997. Chlamydia trachomatis major outer membrane protein variants escape neutralization by both monoclonal antibodies and human immune sera. Infect. Immun. 65: 317–319. 6. Montalban, G. S., P. M. Roblin, and M. R. Hammerschlag. 1994. Performance of three commercially available monoclonal reagents for confirmation of Chlamydia pneumoniae in cell culture. J. Clin. Microbiol. 32:1406–1407. 7. Quinn, T. C., C. Gaydos, M. Shepherd, L. Bobo, E. W. Hook III, R. Viscidi, and A. Rompalo. 1996. Epidemiologic and microbiologic correlates of Chlamydia trachomatis infection in sexual partnerships. JAMA 276:1737–1742. 8. Stephens, R. S., M. R. Tam, C. C. Kuo, and R. C. Nowinski. 1982. Monoclonal antibodies to Chlamydia trachomatis: antibody specificities and antigen characterization. J. Immunol. 128:1083–1089. 9. Tong, C. Y. W., and I. A. Tait. 1996. Under-diagnosis of female genital Chlamydia trachomatis infection. Genitourin. Med. 72:144–145. 10. van der Laar, M. J. W., J. Lan, Y. T. H. P. van Duynhoven, J. S. A. Fennema, J. M. Ossewaarde, A. J. C. van den Brule, G. J. J. van Doornum, R. A. Coutinho, and J. A. R. van den Hoek. 1996. Differences in clinical manifestations of genital chlamydial infections related to serovars. Genitourin. Med. 72:261–265. 11. World Health Organization. 1995. Global programme on AIDS. An overview of selected curable sexually transmitted diseases. World Health Organization, Geneva, Switzerland.
