The present invention is concerned with a method for the isolation of chlamydia for diagnosis of chlamydial infection, in which method a clinical sample is inoculated in the cells of a culture cell line, incubated, and the inclusions formed are detected most appropriately photo icroscopically after colouring.

Chlamydia trachomatis is a prokaryote orga- nism which infects eukaryote cells as an obligatory cell-internal parasite. Chlamydia trachomatis causes several diseases in man, and awareness of its signifi¬ cance for the public health is rapidly increasing (Paavonen, J.,1979, "Chlamydial infections. Micro- biological, diagnostic and clinical aspects", Med.Biol. 54:135-151; Paavonen, J., P. Saikku, E.Vesterinen, B.Meyer, E.Vartiainen, and E.Saksela, 1978, "Genital chlamydial infections in patients attending a gynaeco¬ logical outpatient clinic", Br. J.Vener. Pis. 54:257-261; Schachter, J., 1978, "Chlamydial infections", N. Engl. Med. 298:428-435, 490-495, 540-549; Taylor-Robinson, D. and B.J. Thomas, 1980, "The role of Chlamydia tracho¬ matis in genital-tract and associated diseases", J. Clin. Pathol. 33:205-235.). A break-through proper of the laboratory diagnoses of chlamydial infections was caused by the development of the tissue culture method for the isolation of the organism in irradiated McCoy cells (Gordon, F.B., H.R. Dressier, A.L. Quan, .T. McQuilkin, and J.I. Thomas, 1972, "Effect of ionizing radiation on susceptibility of McCoy cell cultures to Chlamydia trachomatis", Appl. Microbiol. 23:123-129; Gordon, F.B. and A.L. Quan, 1965, "Isolation of the trachoma agent in cell culture", Proc. Soc■ Biol. Med. 118:354-359). The McCoy cell line originally used is closely related to the fibroblastic cancer cell line L929 of mice (Blyth, W.A., and J. Taverene, 1972, "Cultivation of TRIC agents: a comparison between the

use of BHK-21 and irradiated McCoy cells", J.Hyg. 72: 121-128) . Besides the McCoy cell line, it has been established that many other cell lines are also suscep¬ tible to chlamydia (Croy, T.R. , C.C. Kuo, and S.P. Wang, 1975, "Comparative susceptibility of eleven mammalian cell lines to infection with trachoma organ¬ isms", J.Clin. Microbiol. 1:434-439; Rota, T.R., 1977, "Chlamydia trachomatis in cell culture. Susceptibility of seven established mammalian cell types in vitro. Adaption of trachoma organisms to McCoy and BHK-21 cells". In Vitro 13:280-292) , but only few of them are sufficiently susceptible to be used for the isolation of the organism from clinical samples.

In addition to the clinical sample being centrifuged onto the cell layer, the cells are usually pre-treated or after-treated so as to prevent their division and to increase the cell-internal division of chlamydia. The practical problems related to irradiation have resulted in the use of alternative methods in the . treatment of the cells.

It has been reported that irradiation or treatment with IUdR, cytocalasine B or with cyclo- heximide increases the susceptibility of McCoy cells to chlamydia. Some scientists have alleged that untreated McCoy cells are equally susceptible as irradiated cells for the isolation of chlamydia (Hobson, D., F.W.A. Johnson, E. Rees, and I.A. Taita, 1974, "Simplified method for diagnosis of genital and occular infection with chlamydia". Lancet ii:555) , but some others have again established that they are less susceptible and that owing to the small size of the inclusions it is difficult to ascertain and count the inclusions (Evans, R.T. and D. Taylor-Robinson, 1979, "Comparison of various McCoy cell treatment procedures used for detection of Chlamydia trachomatis", J. Clin. Microbiol. 10:198-201). Many laboratories have found that irradiation is a difficult operation, and attempt to avoid the acquisition of a

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cobalt source for the purpose of irradiation. Likewise, all compounds that inhibit the metabolism of cells and all cytotoxic compounds may have some effects of health risk on the laboratory personnel. The employment of different cell lines and treatments will establish that the best technique for the isolation of chlamydia has not been discovered as yet.

Even though the cycloheximide treatment of cells is widely considered to be the most sensitive method, irradiation is still used in many laboratories to increase the susceptibility of cells to infection.

The method in accordance with the present invention is mainly characterized in that the sample is inoculated in the cells of a cultured cell line grown out of epithelial cells of an embryo of the mouse species Mus musculus castaneous.

An epithelial cell is the primary object of a chlamydial infection in man. Since it is difficult to produce non-transformed, pure epithelial cell cul- tures, experimental infection of epithelial cells has been difficult to produce. Successful culture of a stable epithelial cell line isolated from the embryo of the mouse species Mus musculus castaneous, MMC-E (Rapp, U.R., J. Keski-Oja, and U.I. Heine, 1979, "Establishment and characterization of the epithelial mouse embryo cell line MMC-E", Cancer Res. 39:4111-4118) and character¬ ization of the cell line have permitted a study in vitro of various aspects of such diseases as usually produce al¬ terations in the epithelium. The MMC-E cell line was originally isolated from an embryo of Mus musculus castaneous and was classified as epithelial on the basis of its morphology. In a MMC-E culture, dense joints and desmosomic struc¬ tures are seen when examined as highly enlarged. In immύnofluorescence, little quantities of the fibronec- tine of the cell surface are seen and, moreover, out of the cultures, by means of virus transformation, REA

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poorly differentiated carcinomas are obtained in the "nude-mice". The cells also contain keratin filaments, which are a specific feature of the epithelial cells (Keski-Oja, J., Lehto, V.-P., and Virtanen I., 1981, "Keratin filaments of cultured mouse epithelial cells are rapidly affected by epidermal growth factor", J. Cell Biol. 90:531-541). The MMC-E cells are not tumorigenic in "nude-mice", and therein large quantities of receptors of "epidermal growth factor" (EGF) appear. The EGF may stimulate the cells to divide themselves, and produces morphological alterations resembling differen¬ tiation (Keski-Oja, J., I.U. Heine, U.R. Rapp, arid B. Wetzel, 1980, "Epidermal growth factor-induced alterations in proliferating mouse epithelial cells", Exp. Cell Res. 128:279-290. In the culture situation, the "murine sarcoma" growth factors producea transformed phenotype in these cells (Keski-Oja, J. , J.E. DeLarco, U.R. Rapp, and G.J. Todaro, 1980, "Murine sarcoma growth factors affect the growth and morphology of cultured mouse epithelial cells", J. Cell Physiol. 104:41-46).

In this connection, the susceptibility of these mouse embryo epithelial cells to Chlamydia tracho¬ matis was studied. It was found out that MMC-E cell cultures were susceptible to Chlamydia trachomatis without any treatment if dense, old cultures are used, whereat the growth of the cells, but not the growth of chlamydia, is inhibited.

The untreated embryo epithelial cell line MMC-E of Mus musculus castaneous is equally susceptible to infection with Chlamydia trachomatis as the standard McCoy method in which irradiated cells are used, and it possesses remarkable advantages both in clinical routine diagnosis and in_ vitro when studying an experi¬ mental chlamydial infection of epithelial cells. MMC-E cells are susceptible to infection with

Chlamydia trachomatis without any pre- or after-treat¬ ment, and they are comparable with irradiated McCoy

cells, which are usually used for the isolation. MMC-E cells treated with cycloheximide permit routine dia¬ gnoses of chlamydia infections in ever more and more laboratories and are useful in studies in vitro of the mechanisms related to chlamydial infections of epi¬ thelial cells.

The use of untreated MMC-E cells for the isolation of chlamydia is useful for routine diagnoses of chlamydial infections, at least in such laboratories in which the pre-treatments of McCoy cells are too difficult to perform. The use of MMC-E cells thereby permits the performance of chlamydia isolation tests in ever more and more laboratories.

Below, an exemplifying embodiment of the invention will be described in more detail. Cell culture

The MMC-E cells were grown in plastic flasks treated for cell culture (Tissue culture plastic petri dish) in BHK-liquid to which 10 % of calf embryo serum and 50 μg/mg of gentamycin had been added. The origi¬ nal cells were detached for passaging with trypsin-EDTA on a weekly basis in the ratio of 1:5. About 3 x 10^ cells were grown in 1.0 ml of nutrient liquid in a plane-bottom plastic tube, which contained a round, 13 mm diameter sterilized covering glass to which the cultured cells adhered. The tubes were incubated at 35°C under 5 % carbon-dioxide areation, and they were ready for inoculation of the sample when the bottom was filled with cells 1 to 2 days later. Irradiated McCoy cell - cultures were prepared in similar tubes (see above) in a conventional way (Terho, P., 1978, "Isolation techniques of Chlamydia trachomatis from patients with nonspecific urethritis", Dermatol. Monatsschr. 164:515-520). C. trachomatis control strain

The strain used was ___ trachomatis serotype L2 (434Bu) , which had been originally obtained from an

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ophtalmological clinic in London. It was passaged in McCoy cells irradiated in a laboratory in the way described (Saikku, P., and J. Paavonen, 1978, "Single- antigen immunofluorescence test for chlamydial anti- bodies", -J___.-_--C_-l_--i--h---_._-__M_-_i-_c___r--o---b___io—— l--.— 8:119-121). The infecti- vity of this control strain was 5 x 10 inclusion-forming units per ml as defined in irradiated McCoy cell tubes. The 10-fold series dilutions of the control strain in a 0.2 M sucrose - 0.02 M phosphate solution (2SP) (Gordon, F.B., and A.L. Quan, 1965, "Isolation of the trachoma agent in cell culture"-, Proc. Soc. Biol. Med. 118:354-359) , ^■ to which 3 % of calf embryo serum, 50 μg/ml of gentamycin and 25 U/ml of nystatin had been added, had been prepared right before being used. Clinical samples

As study materialwere used 110 samples from the urethra and from the neck of the womb taken in the outpatient clinic of dermatological and venereal diseases of the University of Helsinki in May 1980 from patients who had non-gonococcal uteritis or cervicitis. Titration of inoculum and of infectivity

All the inoculi were made as two parallels both out of the MMC-E cell line and out of the McCoy cell line. 0.1 ml of the control strains of both cell lines was applied to the tubes, which contained con¬ fluent, i.e. fully grown cultures of untreated MMC-E cells or irradiated McCoy cells. Cells inoculated with a 0.2 M sucrose-0.02 M phosphate buffer were used as negative controls. The clinical samples were melted, if they had been frozen, and homogenized by stirring over 20 seconds. The sample was divided into two portions of equal size (0.5 ml each) and inoculated into the cell cultures. The sample was applied by centrifuging at 3000 x g over 1 hour at 35°C. The cells were incubated for two days at 37°C. For iodine colouring, the nutrient liquid was removed from the tube and 1 ml of

methanol was added, and the mix was allowed to stand for 10 minutes at the room temperature. The methanol was removed and 1 ml of colouring liquid sold under the trade mark Lugol was added (manufacturer: Merck, Darmstadt) , and the mix was allowed to stand for 20 minutes at the room temperature. The colouring liquid was removed and the covering glass was removed out of the tube. The counting of the iodine-coloured inclu¬ sions was performed by means of the standard technique under microscope (Paavonen, J. , M. Kousa, P. Saikku, E. Vesterinen, E. Jansson, and A. Lassus, 1978, "Examination of men with nongonococcal urethritis and their sexual partners for Chlamydia trachomatis and Ureaplasma Urealyticum", Sex. Transm. Pis. 5:93-96) . The inclusions in the untreated MMC-E cells were of a size corresponding to those in the McCoy cells and easy to detect. Such isolation tubes containing untreated MMC-E cells can be prepared on the daily basis, Thereby the problem is avoided that is invol- ved in having to calculate when to irradiate the cells and to grow the cells on the covering glass for several days before obtaining the sample. The suggested method is forthwith ready for use for routine culture of chlamydia. Iodine colouring proved suitable for the screening of large quantities of samples.

In stead of untreated MMC-E cells, it is also possible to use, e.g., MMC-E cells treated with cyclo- heximide. The treatment then takes place in the same way as the treatment of McCoy cells.