FI63778C - FOERFARANDE FOER ISOLERING AV KLAMYDIA - Google Patents

Description

63778

The present invention relates to a method for isolating chlamydia for the diagnosis of chlamydial infection, in which a clinical sample is inoculated into cells of a cultured cell line, incubated and the resulting inclusions are preferably detected by light microscopy after staining.

Chlamydia trachomatis is a prokaryotic organism that infects eukaryotic cells as an absolute intracellular parasite. Chlamydia trachomatis causes several diseases in humans, and awareness of its importance for public health is growing rapidly (Paavonen, J. 1979. Chlamydial infections. Microbiological, diagnostic, and clinical aspects. Med. Biol. 54: 135-151; Paavonen, J. , P.Saikku, E.Vesterinen, B.Meyer, E.Vartiainen, and E.Saksela 1978.

15 Genital chlamydial infections in patients attending a gynecological outpatient Clinic. Br. J.Vener. Dis. 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 trachomatis 20 in genital tract and associated diseases.J. Clin. Pathol.

33: 205-235.) The actual breakthrough in laboratory diagnoses of chlamydial infections was caused by the development of a tissue culture method for isolating the organism in irradiated McCoy cells (Gordon, F.B., H.R. Dressier, A.L. Quan, W.T.

25 McQuilkin, and J.I. Thomas. 1972. Effect of ionizing radiation on the susceptibility of McCoy cell cultures to Chlamydia trachomatis.Appi.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 mouse fibroblast cancer cell line L929 (Blyth, WA, 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).

In addition to the McCoy cell line, many other cell lines have been shown to be susceptible to chlamydia (Croy, T.R., C.C. Kuo, and S.P. Wang. 1975- Comparative suspectibility of Eleven mammalian cell lines to infection with trachoma organisms.

2 63778 J.Clin. Microbiol. 1: 434-439; Rota, T.R. 1977. Chlamydia trachomatis in cell culture. Susceptibility of seven established mammalian cell types in vitro. Adaptation of; trachoma organisms to McCoy and BHK-21 cells. In vitro; 5 13: 280-292.), But few are sensitive enough to be used to isolate the organism from clinical specimens.

In addition to centrifuging the clinical specimen over the cell layer, the cells are usually pretreated or post-treated to prevent their proliferation and to increase the intracellular proliferation of chlamydia. Practical problems with radiation have led to the use of alternative methods in cell processing.

Irradiation or treatment with IUdR, cytocalazine,! 15 B or cycloheximide has been reported to increase I |

McCoy cell susceptibility to chlamydia. Some scientists are j! v argued that untreated McCoy cells are as j, sensitive as irradiated cells to isolate chlamydia j; (Hobson, D., FWA Johnson, E. Rees, and IA Taita.1974. 1; 20 Simplified method for diagnosis of genital and occular infections with Chlamydia. Lancet ii: 555), but some have shown that they are less sensitive and due to the small size of the inclusions, it is difficult to detect and count the inclusions (Evans, RT and D. Taylor-Robinson. 1979.;! 25 Comparison of various McCoy cell treatment procedures used (for detection of Chlamydia trachomatis. J. Clin. Microbiol .! j

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10; 198-201). Radiation has been found by many laboratories to be a difficult procedure and attempts to avoid obtaining a source of cobalt for irradiation. not yet found.

55 Although cycloheximide treatment of cells is widely considered to be the most sensitive method, irradiation is still used in many laboratories to increase the sensitivity of cells to 3 63778 hours.

The method of the present invention is mainly characterized in that the sample is inoculated from fetal epithelial cells of Mus musculus castaneous into cells of a cultured 5 cell line.

The epithelial cell is the primary target of human chlamydial infection. Because untransformed, pure epithelial cell cultures are difficult to obtain, experimental infection of epithelial cells has been difficult to perform. Successful culturing of a permanent epithelial cell line, MMC-E, isolated from the fetus of Musculus 10 castaneous mice (Rapp, UR, J. Central Oja, and UI Heine 1979- Establishment and characterization of the epithelial mouse embryo cell line MMC-E. Cancer Res. 39: ^ 111- ^ 113) and the characterization of the cell line character-15 have made it possible to study various aspects of such diseases in vitro, which usually cause changes in the epithelium.

The MMC-E cell line was originally isolated from the fetus of Mus musculus castaneous and classified as epithelial based on its morphology. In MMC-E culture, tight junctions and desmosome-like structures are seen when viewed at high magnification. Immunofluorescence shows small amounts of cell surface fibronectin, and in addition, cultures produce poorly differentiated carcinomas by viral transformation in “nude mice.” Cells also contain keratin strands, a special feature of epithelial cells (Keski-Oja, J., Lehto, V.-P. 1981. Keratin filaments of cultured mouse epithelial cells are rapidly affected by epidermal qrowth factor J. Cell Biol.

30 90: 531-5 ^ 1). MMC-E cells are non-tumorigenic in "nude mice" and express high levels of epidermal growth factorin1 (EGP) receptors. EGF can stimulate cell division and induce differentiation-like morphological changes (Keski-Oja, J., IU Heine , UR

35 Rapp, and B. Wetzel. 19Ö0. Epidermal growth factor-induced Alterations in proliferating mouse epithelial cells.Exp.

Cell Res. 128: 279-290). "Murine sarcoma" growth factors produce a phenotype transformed in these cells in culture (Keski-Oja, J., JE DeLarco, UR Rapp, and GJ Todaro. 1980. Murine sarcoma growth factors affect the growth and morphology of cultured mouse epithelial cells .5 J. Cell Physiol. 104: 41-46).

In this context, the susceptibility of these mouse fetal epithelial cells to Chlamydia trachomatis was studied. It was found that MMC-E cell cultures were susceptible to Chlamydia trachomatis infection without any treatment if dense, old cultures were used, thereby inhibiting cell growth but not chlamydia growth.

The untreated fetal Musculus castaneous fetal epithelial cell line MMC-E is as susceptible to Chlamydia trachomatis infection as the standard McCoy method using 15 irradiated cells and has significant advantages in both routine clinical diagnosis and in vitro epithelialization; when examining experimental chlamydial infection of cells.

MMC-E cells are susceptible to Chlamydia trachomatis infection without any pre- or post-treatment and are comparable to irradiated McCoy cells commonly used in isolation. Cycloheximide-treated MMC-E cells allow for the routine diagnosis of chlamydial infections in an increasing number of laboratories and are useful in.

in vitro studies of the mechanisms involved in chlamydial infections of epithelial cells. i Use of untreated MMC-E cells for chlamydia.

isolation is useful in routine diagnoses of chlamydial infections, at least in laboratories where pretreatments of McCoy cells are too difficult to perform.

The use of MMC-E cells thus allows chlamydia isolation tests to be performed in an increasing number of laboratories.

An embodiment of the invention will be described in more detail below. f

Colon culture 35 MMC-E cells were grown in Tissue culture plastic Petri dish 5 63778 in BHK supplemented with 10% fetal calf serum and 50 pg / mg gentamicin. Stem cells were detached for weekly 1: 5 tryosin-EDTA for oasis staining. Approximately 3 x 10 cells were cultured in 1.0 ml of 5 medium in a flat-bottomed plastic tube containing a round, 13 mm diameter sterilized coverslip to which the cultured cells adhered. . The tubes were incubated at 35 ° C, 5% carbon dioxide aeration, and were ready for inoculation of the sample, with the bottom filling the cells 10 1-2 days later.

Irradiated McCoy cell cultures were prepared in similar tubes (see above) conventionally (Terho, P. 1978. Isolation techniques of Chlamydia trachomatis from patients with nonspecific urethritis. Dermatol. Monatsschr.

15 164: 515-520).

C. trachomatis control strain The strain used was C. trachomatis serotype L2 (434Bu), originally obtained from an ophthalmology clinic in London. It was passaged in laboratory irradiated McCoy cells as described (Saikku, P., and J. Paavonen. 1978. Single-antigen immunofluorescence test for chlamydial antibodies. J. Clin. Microbiol. 8: 119-121). The infectivity of this control strain was 5 x 10 6 inclusion units / ml as determined in irradiated McCoy 25 cell tubes. Serial 10-fold dilutions of control strain in 0.2 M sucrose-0.02 M phosphate solution (2SP) (Gordon, PB, and AL Quan. 1965 · Isolation of the trachoma agent in cell culture. Proc.Soc.Biol.Med. 118 : 354-359) supplemented with 3% fetal calf serum, 50 μg / ml gentamicin-30 and 25 U / ml nystatin were prepared just prior to use.

Clinical specimens

A total of 110 urethral and cervical samples from patients with non-gonococcal urethritis or cervicitis taken in May 1980 at the Outpatient Clinic of Dermatology and Venereology of the University of Helsinki were used as research material.

63778 6

Inoculation and titration of infectivity

All inoculations were performed in duplicate from both the MMC-E and McCoy cell lines. 0.1 ml of control strains from both cell lines were applied to tubes containing confluent-5, i.e. cultures filling the bottom of the culture vessel, untreated MMC-E cells or irradiated McCoy cells. Cells inoculated with 0.2 M sucrose-0.02 M phosphate buffer were used as negative controls.

Clinical samples were thawed if frozen and homogenized by stirring for 20 seconds.

The sample was divided into two equal sized portions (0.5 ml each) and inoculated into cell cultures.

The sample was applied by centrifugation at 3000 xg for 1 hour at 35 ° C. The cells were incubated for two days at 37 ° C. For iodine-15 staining, the medium was removed from the tube and 1 ml of methanol was added and allowed to stand for 10 minutes at room temperature. The methanol was removed and 1 ml of Lugol dye (manufactured by Merck, Darmstadt) was added and allowed to stand for 20 minutes at room temperature. The staining liquid was removed and the coverslip was taken from the tube. Calculation of iodine-stained inclusions was performed according to standard techniques under a microscope (Paavonen, J., M. Kousa, P. Saikku, E. Vesterinen, E. Jansson, and A. Lassus.1978. Examination of men with nongonococcal 25 urethritis and their sexual partners for Chlamydia trachomatis and Ureaplasma Urealyticum.Sex. Transm.Dis. 5: 93-96).

Inclusions in untreated MMC-E cells were similar in size to McCoy cells and easy to detect. Insulation tubes containing such untreated MMC-E cells can be prepared daily.

This avoids the problem of calculating when to irradiate the cells and culturing them on a coverslip for many days before receiving the sample. The presented method is immediately ready for use in routine chlamydia culture. Iodine staining proved to be suitable for screening large volumes of samples.

Instead of untreated MMC-E cells, it is also possible to use e.g. MMC-E treated with cycloheximide.