Chlamydia trachomatis Infections - Journal of Clinical Microbiology

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Mar 22, 1988 - Oriel, J. D., A. L. Johnson, D. Barlow, B. J. Thomas, K. Nayyar, and P. Reeve. 1978. Infection of the uterine cervix with Chla- mydia trachomatis.
Vol. 26, No. 7

JOURNAL OF CLINICAL MICROBIOLOGY, JUIY 1988, P. 1428-1430 0095-1137/88/071428-03$02.00/0 Copyright C 1988, American Society for Microbiology

Simplified Culture Procedure for Large-Scale Screening for Chlamydia trachomatis Infections MAXWELL I. LEES,* DIANE M. NEWNAN, AND SUZANNE M. GARLAND

Microbiology Section, Department of Pathology, Royal Women's Hospital, Melbourne 3053, Victoria, Australia Received 24 August 1987/Accepted 22 March 1988

A method that uses a 48-well tissue culture cluster tray system for the isolation of Chlamydia trachomatis is described. The cluster tray system was as sensitive (100%) as and more time efficient than the conventional cover slip method, thereby being considerably cost saving. With both culture methods, the prevalence rates of genital carriage of C. trachomatis in women attending clinics for legal abortion and for cervical dysplasia were 5% (31 of 641 patients) and 2% (3 of 148 patients), respectively.

Chlamydia trachomatis is now recognized as the most common sexually transmitted agent in developed countries, being isolated from 20 to 30% of unselected women attending sexually-transmissible-disease clinics (9). In the female genital tract it is a causal agent of mucopurulent cervicitis, endometritis, salpingitis, and Fitz-Hugh-Curtis syndrome, and there have been reports of chlamydial genital tract infections preceding infertility (6, 13). In pregnant patients carrying chlamydia, apart from postpartum endometritis and salpingitis, transmission from mother to infant can occur, resulting in neonatal inclusion conjunctivitis, pneumonitis, bronchiolitis, or otitis media (1). Of concern is that in up to two-thirds of women, cervical carriage is symptom free (9). Although rapid diagnostic tests are available, cell culturing is still recognized as the most sensitive method for the detection of genital tract chlamydial infections. This is particularly the case when the expected rate of infection is lower than that in a typical sexually-transmissible-disease clinic setting (12). We report a more time-efficient technique for the isolation of C. trachomatis that uses 48-well tissue culture cluster trays without cover slips. This technique was used concurrently with the conventional method of glass cover slips in plastic vials to screen for C. trachomatis in two different patient groups attending the Royal Women's Hospital, Melbourne, Victoria, Australia. The study population consisted of 647 patients (age, 12 to 46 years; average age, 25.4 years; ages unknown for 11 patients) attending the Pregnancy Advisory Service (PAS) Clinic and requesting a legal termination of pregnancy plus 149 patients (age, 14 to 63 years; average age, 30.0 years; age unknown for 1 patient) attending the Dysplasia Clinic for the first time from April 1986 to May 1987, inclusively, at the Royal Women's Hospital, Melbourne, Victoria, Australia. Endocervical swabs were taken from each patient with cotton-tipped aluminum E.N.T. swabs (Medical Wire and Equipment Co., Wiltshire, England) after cervical mucus or pus was wiped away. Each swab was placed into transport medium, transported on ice to the laboratory, and either cultured the same day or stored at -70°C until processed. HeLa 229 cells (Commonwealth Serum Laboratories, Parkville, Victoria, Australia) were passed twice weekly in GM, which contained Eagle minimum essential medium with nonessential amino acids, 2 mM L-glutamine, Earle salts *

(GIBCO Laboratories, Grand Island, New York) supplemented with 8% fetal bovine serum (Flow Laboratories, Herts, England), 0.022 M sodium bicarbonate, 6 g of HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) per liter (Sigma Chemical Co., St. Louis, Mo.), and 20 ,ug of gentamicin per ml. The medium for the growth of C. trachomatis in HeLa 229 cells (MM) differed from GM by the reduction of fetal bovine serum from 8 to 1% and by the addition of 0.056 M glucose, 20 ,ug of amphotericin B per ml, and 150 ,ug of vancomycin per liter. Transport medium for specimen collection consisted of sucrose-phosphate-glutamate (2) modified by the addition of antibiotics as described for MM. A stock culture of C. trachomatis UW31 (serovar K) (kindly provided by D. Graham, Department of Microbiology, University of Melbourne, Parkville, Victoria, Australia) was passaged in HeLa 229 cells, divided into aliquots containing 5 x 108 inclusion-forming units per ml in sucrose-phosphate-glutamate, and stored at -70°C. Cover slip cultures were prepared by adding 0.5 ml of a suspension of HeLa 229 cells in GM at a concentration of 2.75 x 105 cells per ml to a sterile 5-ml white-capped plastic vial (Johns Division, James Hardie Health Care, Melbourne, Victoria, Australia) containing a 12-mm-diameter glass cover slip (Mediglass, Bondi Beach, New South Wales, Australia) and were incubated at 37°C. Cultures were also prepared in 48-well tissue culture cluster trays (Costar, Badhoevedorp, The Netherlands) by adding 0.4 ml of a suspension of HeLa 229 cells in GM at a concentration of 3.5 x 105 cells per ml to each well of the trays and were incubated at 37°C. In both cases, confluent growth was obtained within 24 h. The GM for both cover slip and cluster tray culture procedures was removed and replaced with 1 and 0.6 ml of MM, respectively. Clinical specimens were thawed and vortexed for 30 s each, and 200 ,ul of each was inoculated concurrently into each of the cover slip and tray cell cultures. Both types of cultures were centrifuged at 1,800 x g for 1 h at 37°C in a model J6-B centrifuge (Beckman Instruments, Inc., Fullerton, Calif.) and incubated for 48 h at 37°C. All cultures were then fixed in methanol and stained with 25 ,ul of fluoresceinconjugated antichlamydial monoclonal antibody (Syva Co., Palo Alto, Calif.). After mounting was done, cover slips were scanned for inclusions at x 100 on a Laborlux 12 fluorescence microscope (Leitz/Opto-Metric Div. of E. Leitz Inc., Rockleigh, N.J.) with the morphology of inclusions confirmed at x 400, whereas trays were scanned at x 100 on a Fluovert microscope (Leitz) with the morphology of inclusions confirmed at x200.

Corresponding author. 1428

VOL. 26, 1988

NOTES

TABLE 1. Titration of C. trachomatis UW31 (serovar K) in cluster trays and glass cover slips Dilution

1 X 5x 2.5x 1.3x 6x 3 x

10-5 10-6 10-6 10-6

10-7

10-7

1.5x10-7 8 x 10-8

Cluster trays

Cover slips

3,564 ± 501 1,229 ± 99 675 ± 95 324 ± 74 127 ± 21 58 ± 8 47±16 5± 5

34±8 15

9

To ascertain the comparative sensitivity of the two systems, we inoculated doubling dilutions of a 10-5 solution of

the laboratory stock strain of C. trachomatis in triplicate as 200-,ul volumes into cover slip and tray cultures concurrently. Otherwise, all procedures were identical to those already described for clinical specimens. Inclusions were counted in 30 fields at x 100, with the exception of counts of less than one inclusion per field, in which case whole wells or cover slips were used for counting. Results expressed in inclusion-forming units per milliliter are shown in Table 1 and indicate that the limits of detection of the two culture systems were comparable and that both systems were capable of detecting low-titer samples. A potential concern with the cluster tray system is the possibility of cross-contamination because of the closeness of adjacent wells and the sharing of a common lid between different specimens. To test for this, we alternately inoculated wells with 200 pul of a 10-5 dilution of the stock strain of C. trachomatis and 200 pul of sterile MM. The inoculation pattern resulted in diagonally aligned rows of positive and negative wells with parallel adjacent wells being alternately positive and negative. All wells were then incubated, the contents were fixed and stained, and inclusions were counted by the procedure already described. There was no evidence of cross-contamination in cluster trays alternately inoculated with positive and negative samples. All of the positive wells contained similar numbers of inclusions, 3,224 + 323 inclusion-forming units per ml (mean + standard deviation), and negative wells contained no inclusions. Of the 796 patient specimens processed, 7 were inadequate because of toxic changes in the cell cultures (6 of 647 PAS Clinic specimens and 1 of 149 Dysplasia Clinic specimens). The results for the remaining 789 specimens cultured are shown in Table 2. The rates of isolation of C. trachomatis in the PAS Clinic and the Dysplasia Clinic were found to be 5 and 2%, respectively. Apart from one specimen which was positive in the cluster tray system and negative in the cover slip-vial system, all results were identical. ConseTABLE 2. Rate of isolation of C. trachomatis from PAS Clinic and Dysplasia Clinic patients by cell culturing with glass cover slips and cluster trays Patient category

TABLE 3. Prevalence of C. trachomatis according to age group in PAS Clinic and Dysplasia Clinic patients

Inclusion-forming units/mI (mean ± SD) in:

3,171 + 246 1,640 66 758 22 373 23 130 15 94 3

No. of patients tested by both methods

No.

(%) of patients positive for:

Cover slips

Trays

PAS Clinic Dysplasia Clinic

641 148

31

(4.8)

3

(2.0)

32 (5.0) 3 (2.0)

Totals

789

34 (4.3)

35 (4.4)

1429

No. of patients positive/no. tested in the following category:

Age (yr)

PAS Clinic

12/134 15/198 1/148 4/150

'19

20-24 25-29

.30

(9.0) (7.6) (0.7) (2.7)

32/630 (5.1)

Totals

(%)

Dysplasia Clinic 0/8 (0) 3/37 (8.1) 0/42 (0) 0/60 (0) 3/147 (2.0)

excluded from the PAS Clinic category, and one patients patient was excluded from the Dysplasia Clinic category because their ages were unknown. a

Eleven

were

quently, the sensitivity of the cluster tray system as compared with the cover slip-vial system was 100%. The use of 24-well and 96-well microdilution trays for the

tissue culture isolation of C. trachomatis has been reported previously (3, 5, 11, 15). Comparisons of trays and conventional cover slips and vials have been made with 96-well microdilution trays, and while significant gains in the efficiency of handling specimens have been reported, the sensitivity has been reduced, attributed largely to the necessary reduction in inoculum volume for each specimen (5). With a 48-well cluster tray the difference in receptacle size between the two methods is minimal (11.25-mm-diameter well in the tray and 12-mm-diameter cover slip in a 13-mm-diameter vial); consequently, the inoculum volumes are the same. Our laboratory can now handle 48 specimens in cluster trays in the time it previously took to handle 20 specimens in the conventional way, representing a considerable savings in time and therefore cost per test. Enzyme immunoassay and direct fluorescent-antibody tests for chlamydial antigen still offer advantages over our simplified tissue culture isolation procedure, particularly for less-specialized laboratories. However, the clinical settings in which these rapid antigen tests are suitable for use are limited. Lipkin et al. (4) have pointed out that when the prevalence rate of C. trachomatis for a patient population falls to a level of 5%, as in our study population, the positive predictive value for the direct fluorescent-antibody test with a sensitivity of 70% and a specificity of 94% as compared with that of tissue culturing becomes 38%, a value which is unacceptably low. Consequently, we believe that when the prevalence of C. trachomatis is likely to be lower than that in a typical sexually-transmissible-disease clinic setting, tissue culturing must still be the method of choice for reliably detecting C. trachomatis. Our results for the prevalence of C. trachomatis in the two populations studied are the first reported from this country. The 2% chlamydia isolation rate for our Dysplasia Clinic patients is lower than those in other reports, which varied from 4.1% (14) to 16% (10). This discrepancy could be explained by the fact that our groups were older (average age, 30 years [Table 3]). The 5% carriage rate found at our institution for women attending clinics for legal abortion is similar to those reported by Moller et al. (8) for a Danish group (5.3%) and a Swedish group (4.9%) and by Mardh et al. (7) for a Swedish group (6.1%). Moller et al. (8) reported that pelvic inflammatory disease occurred in 20% of cases following therapeutic abortion in cervical carriers of chlamydiae. Morbidity, suggestive of upper genital tract involvement, following termination of pregnancy occurred in 19% of our patients who were carriers of chlamydia (S. Garland,

1430

J. CLIN. MICROBIOL.

NOTES

unpublished observation). Consequently, it is imperative that pelvic inflammatory disease and the associated risks of infertility, chronic abdominal pain, and tubal pregnancy are prevented. Our study highlights that C. trachomatis predominantly occurs in young (84% of our patients were less than 25 years old [Table 3]), single (77%), and nulliparous (77%) women. Therefore, chlamydia screening and appropriate treatment of all PAS Clinic patients, together with concurrent treatment of their sexual partners, are routine in our hospital. We believe that this is a cost-effective exercise, given the cost to the community of the treatment of pelvic inflammatory disease and its complications and the expense of in vitro fertilization programs, for which these patients may become candidates. The technical assistance and critical assessment of S. Murphy and the secretarial assistance of D. Rouch are gratefully acknowledged. The medical and nursing staff of the PAS Clinic and Dysplasia Clinic are also sincerely thanked for their participation. LITERATURE CITED 1. Alexander, E. R., and H. R. Harrison. 1983. Role of Chlamydia trachomatis in perinatal infection. Rev. Infect. Dis. 5:713-719. 2. Bovarnick, M. R., J. C. Miller, and J. C. Snyder. 1950. The influence of certain salts, amino acids, sugars, and proteins on the stability of rickettsiae. J. Bacteriol. 59:509-522. 3. Coudron, P. E., D. P. Fedorko, M. S. Dawson, L. G. Kaplowitz, R. R. Brookman, H. P. Dalton, and B. A. Davis. 1986. Detection of Chlamydia trachomatis in genital specimens by the Microtrak direct specimen test. Am. J. Clin. Pathol. 85:89-92. 4. Lipkin, E. S., J. V. Moncada, M.-A. Shafer, T. E. Wilson, and J. Schachter. 1986. Comparison of monoclonal antibody staining and culture in diagnosing cervical chlamydial infection. J. Clin. Microbiol. 23:114-117. 5. Mallinson, H., S. Sikotra, and O. P. Arya. 1981. Cultural method for large-scale screening for Chlamydia trachomatis genital

infection. J. Clin. Pathol. 34:712-718. 6. Mardh, P.-A. 1986. Ascending chlamydial infection in the female genital tract, p. 173-184. In J. D. Oriel, G. Ridgway, J. Schachter, D. Taylor-Robinson, and M. Ward (ed.), Chlamydial infections. Cambridge University Press, Cambridge. 7. Mardh, P.-A., I. Helin, S. Bobeck, J. Laurin, and T. Nillson. 1980. Colonization of pregnant and puerperal women and neonates with Chlamydia trachomatis. Br. J. Vener. Dis. 56:96100. 8. Moller, B. R., S. Ahrons, J. Laurin, and P.-A. Mardh. 1982. Pelvic infection after elective abortion associated with Chlamydia trachomatis. Obstet. Gynecol. 59:210-213. 9. Oriel, J. D., A. L. Johnson, D. Barlow, B. J. Thomas, K. Nayyar, and P. Reeve. 1978. Infection of the uterine cervix with Chlamydia trachomatis. J. Infect. Dis. 137:443-451. 10. Paavonen, J., E. Vesterinen, B. Meyer, P. Saikku, J. Suni, E. Purola, and E. Saksela. 1979. Genital Chlamydia trachomatis infections in patients with cervical atypia. Obstet. Gynecol.

54:289-291. 11. Pothier, P., and A. Kazilerczak. 1986. Comparison of cell culture with two direct chlamydia tests using immunoperoxidase or enzyme-linked immunosorbent assay. Eur. J. Clin. Microbiol. 5:569-572. 12. Ridgway, G. L. 1986. The laboratory diagnosis of chlamydial infection, p. 539-549. In J. D. Oriel, G. Ridgway, J. Schachter, D. Taylor-Robinson, and M. Ward (ed.), Chlamydial infections. Cambridge University Press, Cambridge. 13. Schachter, J. 1978. Chlamydial infection (second of three parts). N. Engl. J. Med. 298:490-495. 14. Schachter, J., E. C. Hill, E. B. King, V. R. Coleman, P. Jones, and K. F. Meyer. 1975. Chlamydial infection in women with cervical dysplasia. Am. J. Obstet. Gynecol. 123:753-757. 15. Smith, J. W., R. E. Rogers, B. P. Katz, J. F. Brickler, P. L. Lineback, B. Van Der Pol, and R. B. Jones. 1987. Diagnosis of chlamydial infection in women attending antenatal and gynecologic clinics. J. Clin. Microbiol. 25:868-872.