FI104218B - Diagnostic method - Google Patents

Description

104218

Diagnostic method

The present invention relates to a method for detecting the presence of Chlamydia trachomatis in Papanicolaou-stained cytologic brush specimens and to a reagent useful in the method.

Chlamydia trachomatis is the most important etiologic factor in pelvic inflammatory disease (PID). PID often causes infertility and ectopic pregnancies in women of childbearing age: after a single PID caused by chlamydia, infertility occurs in 10 to 25% of patients [Schachter, J., New England Journal of Medicine 298 (428-435) 1978; Svensson, L. O. et al. Acta Obstetrica et Gynecologica Scandinavia 70 (587-590) 1991].

Clinical specimens are usually taken from patients with symptoms of the disease. However, asymptomatic carriers are common, especially in the young age group 15 (20-25 years) and may lead to long-term complications, which are expensive to treat. The incidence of genital chlamydia infections in asymptomatic women may be as high as 5.4-15%, and chlamydial antibodies have been reported in 19.3% of women treated in infertility clinics [Edet, E.E., British Journal of Clinical Practice 47 (1993) 21-22; Driscoll, G.E. et al., 20 Medical Journal of Australia 160: 46 (1994)]. Therefore, patients with or suffering from Chlamydia trachomatis infection should be identified and treated despite possible asymptoms in order to avoid cumbersome, personal problems and costly consequences for society related to chlamydia infections and in particular infantile infections.

Chlamydia trachomatis is a small intracellular bacterium with an adhesive form of only about 0.3 micrometres in diameter, the so-called elemental-tare body. This infectious element is highly adherent to the cell surface from which it is phagocyted into the cell. In in-30 clusters within the host cell, Chlamydia multiplies into a reproductive form. Thousands of new infectious elemental fragments that are released from cells may be produced in such chlamyria dialysis. Chlamydia infection can be treated, for example, with tetracycline, erythromycin or clindamycin, but treatment failures have been described [Jones, R. B., J. Infect. Dis. 162: 1309-1315 (1990); 35 Munday, P. E., et al., Genitour. Med. 71: 24-26 (1995)]. This may result in a latent asymptomatic infection.

• 2 104218

Currently, several methods are available for the detection of Chlamydia trachomatis, such as culture, ELISA, immunofluorescence and DNA techniques. In recent years, RNA-DNA hybridization, which is almost invariably performed on a urine sample of 5 symptomatic patients, sometimes including a vaginal or cervical mucosa, has become a routine method for detecting chlamydia infection. Extensive screening programs have not been implemented, even if the need for such screening is obvious. However, large-scale mass screening programs using Papanicolaou staining of a bulk cell sample are performed to detect cervical cancers in many countries around the world. However, hitherto, cytological staining has not been able to reliably diagnose Chlamydia trachomatis, although cellular staining often indicates infection. The reagents and conditions used in Papanicolaou staining alter the antigenicity of any chlamydial bacterium present in the sample, and thus the bacterium has not previously been detected with, for example, antibody reagents. In order to detect chlamydia, it has always been necessary to take a sample.

It is obvious that detection of asymptomatic chlamydial infection would be socially desirable and desirable. In addition, it would be highly advantageous that this could be combined with an existing screening method, such as the cellular assay used for screening for, for example, cervical cancer.

A method has now been developed to unexpectedly restore the antigenic properties of Chlamydia trachomatis and to detect the bacterium from the same Papanicolaou-stained cell specimen widely used, for example, in cervical cancer screening, even if the specimen can be stained again with Papanicolaou. according to need. It is possible to restore the antigenic properties even months after Papanicolaou staining, thus allowing a retrospective detection of a possible chlamydial infection.

This method can substantially facilitate the detection of chlamydia infections in a population undergoing cytologic profiling. The invention provides a method for detecting the presence of Chlamydia trachomatis in Papanicolaou-stained cytological specimens without altering the cell morphology of the original specimen.

The invention relates to a method for detecting Chlamydia trachomatis in Papanicolaou-stained cytological specimens. The method comprises the steps of: a) decolorizing a Papanicolaou-stained specimen on a slide removed by incubation in a decolorization solution *, an acidic buffer solution having an acidic pH, for 5-30 minutes, and washing the decolorization solution out of the sample for example, a buffer solution free of oxidant 5; b) incubating the sample in a reflux reagent solution containing the alkaline earth metal salt component (s), the sugar component, and the amino acid component in an acidic pH buffer for 30 minutes to 12 hours, and washing the reflux reagent with, for example, buffered saline; c) the sample is treated with a solution containing a labeled identification reagent specific for Chlamydia trachomatis for 20 min -12 hours; and d) detecting the presence of any Chlamydia trachomatis bacterium by appropriate means.

The invention also relates to a reagent containing an alkaline earth metal salt component (s), a sugar component and an amino acid component in a buffer solution having a pH in the acidic range.

The invention further relates to a method for restoring the antigenicity of Chlamydia trachomatis -20 in a Papanicolaou-stained cytological specimen by treating the specimen with a reconstitution reagent of the invention.

Fig. 1 is a fluorescence microscope image showing elemental fragments of Chlamydia trachomatis as bright spots in Buffalo Green 25 Monkey cell culture.

Figure 2 is a fluorescence microscope image showing elemental fragments of Chlamydia trachomatis as bright spots on a specimen of a culture positive patient.

The 30 oxidant buffer solution used as the decolorization solution in the process of the invention may be, for example, 0.3-1.8% perhalogen acid solution in a suitable buffer having a clearly acidic pH (pH below 6.0, for example 3.5-5). 5) such as 0.01-0.2 M mol / L in phosphate buffer, citrate buffer or Tris-HCl buffer. Preferably, periodic acid and its salts are used as a 0.5 to 1% solution in a 0.05 to 0.1 mol / L phosphate pouch 35 pH 4.0 to 5.5. A particularly preferred decolorizing reagent is 1% periodic acid in 0.1 M phosphate buffer, pH 5.0.

• * 4 104218

The recovery reagent solution contains calcium magnesium chloride and / or other divalent ions in the concentration of 10-40 mmol / l as alkaline earth metal salts. A preferred recovery reagent solution contains 20 mmol / l calcium chloride and 20 mmol / l magnesium chloride. The sugar component is glucose 5 or sucrose in a concentration of 5 to 40 mmol / l. A preferred reconstituting reagent solution contains 10-20 mmol / L, most preferably 15 mmol / L glucose. The amino acid component of the reagent is a basic or hydrophilic amino acid, for example lysine, in an amount of 10-40 mg / ml. A preferred reconstituting reagent solution contains 25 to 35 mg / ml, most preferably 30 mg / ml lysine.

The pH of the reconstitution reagent is in the acidic range, for example, 3.5 to 5.5. The reagent buffer may be any suitable buffer, such as 0.01 to 0.2 mol / L phosphate, citrate or Tris-HCl. Preferably, a 0.1 mol / L phosphate buffer having a pH of 3.5 to 5.5, most preferably 5.0 is used.

The recognition reagent used in the method of the invention may be any labeled poly- or monoclonal antibody or genetic probe as long as it is specific for Chlamydia trachomatis and recognizes all known serovars thereof and does not cross-react with Chlamydia psittaci pneumoniae, Chlamydia psittaci or other organisms known to readily cross-react with at least anti-chlamydia-20 antibodies. The detection reagent may be labeled with any detectable label, such as fluorescent, phosphorescent, chemiluminescent, or color labeled, or other detectable labeling method, such as biotin-avidin method, radioactive or enzymatic label such as analytical phosphatase. Preferred identification reagent genes are antibodies labeled with a fluorescent agent such as fluorescein.

The presence of Chlamydia trachomatis is detected depending on the labeled detection reagent used in a known manner. Such methods are well known to those skilled in the art. For example, if a fluorescent labeled antibody is used, detection may be; perform fluorescence microscopically. When using a radioactive label, the operation can be performed by autoradiography. Enzyme labeling is usually detected by color reaction.

For simplicity and sensitivity of use, the preferred labeled detection reagent is a fluorescent labeled antibody that can be detected by fluorescence microscopy at 104218 * 5. In a fluorescence microscope, the tracers are visualized by exposure to UV light filtered with a suitable excitation filter for the tracer. The light emitted by the tracer is filtered by their specific emission filter, whereby their characteristic color can be observed under the microscope. Opposite dyes, which are intended to improve the separation of cell morphology (eg Evans Blue), must not impair the fluorescence of the tracer.

The incubation times used in the method can range from 5 minutes to overnight incubation. Preferably, incubation times suitable for the practical implementation of method 10 are selected, for example, so that the method can be conveniently performed within one or a working day. The incubation time in the recovery and detection reagent may thus be, for example, from 30 minutes to 2-4 hours or overnight.

The cytological specimen may be any Papanicolaou-stained bulk 15 cell sample for the presence of Chlamydia trachomatis, for example, a cervical mucosa, vaginal mucosa, eye, or urethral specimen. The sample may be a fresh Papanicolaou-stained specimen or, for example, an archived specimen for subsequent investigation for the presence of Chlamydia trachomatis. Respectively, stain specimens may be re-stained with Papanicolaou staining for archiving or other purposes, since the method of the invention does not cause any change in cell morphology.

Papanicolaou-stained slides are removed in a manner known per se, for example by incubation in xylene if the coverslips are fixed with xylene-based fixing reagent.

The method of the present invention for detecting the presence of Chlamydia trachomatis is particularly suitable for combination with screening assays where it is possible that individuals with chlamydia infection may be present. For example, using the method of the invention, it is possible to investigate chlamydial infection from Papanicolaou-stained smear specimens taken in cervical cancer screening studies and to restore the smear specimens to their original state.

The present invention will be described in detail by the following examples. These examples are given merely to illustrate the invention further and should not be construed as limiting the invention in any way.

• · 6 104218

Example 1

Commercially available Papanicolaou-stained slides (15) containing Chlamydia trachomatis elemental slides in Buffalo Green Monkey cell culture (Cellabs Pty Ltd., Brookvale, Australia) were first incubated in xylene until the slide was removed, followed by washing with slides. minutes. The slides were then washed twice for five minutes with absolute ethanol and then decolorized by incubating the slides in a solution of 1% periodic acid in 0.1 M phosphate buffer (pH 5.0) for 15 minutes. After decolorization, the ob-10 slide was washed with 0.1 M phosphate buffer (pH 5.0) at least twice for two minutes. The slides were then incubated for 30 minutes in Recovery Solution containing 20 mM calcium chloride, 20 mM magnesium chloride, 15 mM glucose, 30 mg / ml lysine, 10% normal mouse serum and 0.05% sodium azide, in a humid chamber at room temperature. The slides were then washed with 0.1 M phosphate-buffered saline (PBS) twice for two minutes. The slides were dried for five minutes so that the next fluorescein-labeled antibody to be added remained in the selected area as a drop and the rest of the slide was allowed to dry completely. Next, a droplet (25-30 microliters) of a solution (Cellabs Pty 20 Ltd., Brookvale, Australia) containing a fluorescein conjugated monoclonal antibody (Cellabs clone 512F) specific for the major outer membrane of Chlamydia trachomatis and Evipr Blue dye and the slide were incubated for 60 minutes in a humidified chamber at 37 ° C. Finally, the slide was rinsed with PBS and the cover slide was fixed for immunofluorescence microscopic observation.

Corresponding Papanicolaou non-stained control slides (15) (Cellabs Pty Ltd., Brookvale, Australia) were stained directly with fluorescein-conjugated monoclonal antibody combined with Evans Blue.

In microscopic examination, the areas of interest selected for cytological examination on the slides were marked with a diamond pen on the backside of the lenses. The areas with a diameter of 8-10 mm corresponded to the areas of normal m fluorescence antibody staining. Fluorescence microscopy used epifluorescence, a fluorescein strip filter, and 40, 60 or 100x 35 oil immersion lenses.

• «7 104218

When samples were examined under fluorescence microscopy, all Papanicoiaou-stained slide glasses containing chlamydial elementary tare showed bright fluorescent eemeral pieces (Fig. 1). The elemental specimens did not differ from the elemental specimens that were present on the corresponding Papanicolaou non-stained slide glasses.

Example 2

Chlamydia trachomatis cervical lymphadenoplasty samples (12), of which seven were from patients with Chlamydia trachomatis positive culture and five were from patients with 10 Chlamydia trachomatis positive, negative, methanol, ethanol and Carbowax (Mekalasi Co., Helsinki), or 90% ethanol. Fluorescence antibody staining control slides (Cellabs), initially fixed with absolute methanol, were reattached in the same manner as patient samples. After attachment, the slides 15 were stained with conventional Papanicolaou staining and covered with a slide glass using a xylene solution. The samples were then treated and stained on Papanicolaou-stained slides as described in Example 1. When examining the samples treated according to the invention under fluorescence microscopy, different amounts of Chlamydia trachoma-20 tis eemental specimens were detected in all culture positive samples (Fig. 2).

Example 3

Urine samples from ten young abortion patients were tested for Chlamydia trachomatis using the Base 2 RNA-DNA Hybridization Kit (Gen-Probe Inc., San Diego, California, USA). Three of the samples were chlamydial positive-25. Papanicolaou-stained cervical specimens from the same patients were processed and stained as described in Example 1 for Papanicolaou-stained slides, and the results were interpreted blindly unaware of the results of the hybridization method. The method of the invention identified the same three klamy diapositive samples as the hybridization method.

Example 4 -: From the 64 routine screening samples, areas suggestive of Chlamydia infection with cellular changes were selected and marked with a diamond pen on the backside of the glasses. This made it possible to reduce the amount of detection reagent and the costs involved. The samples were then processed and stained on Papanico-35 laou-stained slides as in Example 1. Of the examined specimens, 39 were • <8 104218 normal in nature and 25 had evidence of changes suggestive of cervical inflammation. The results are shown in Table 1.

Six (27%) of the 22 specimens showing changes suggestive of cervical inflammation were positive for fluorescence staining.

In one sample (2.4%) of the 42 samples with normal cytology, 12 elemental samples were detected by fluorescence staining, which was interpreted as positive (Table 1).

table 1

Comparison of Direct Fluorescence Antibody Staining to Cytological Examination in 64 Cervical Screening Specimens

Cytology Fluorescence Microscopy __ (number of cases) _

Inflammation_Atypia_Positive_Negative_

Lievä_jts__8_

Moderate ns_2_3_

Vaikea_jts_3__

Slight ASCUS__2_

Kohtalainen_ASCUS__1__

Difficult_ ASCUS__J_

Slight_ LSIL__1_

Moderate_LSIL__J_ ns_ASCUS__1_. ns LSIL 2 ns_ [ns_ [1_38_ ASCUS = untyped Finnish atypical cells 15 LSIL = low grade epithelial injury ns = normal cytology

Claims (13)

A method for detecting the presence of the Chlamydia trachoma-f / s bacterium in Papanicolaou-stained cytologic cell samples, characterized in that it comprises steps in which a) a Papanicolaou-stained sample is on a slide and from which the cover glass has been removed, decolorized by incubation in a decolorization solution which is a buffer solution containing a reducing agent and whose pH is in the range, for 5-30 minutes, and the decolorization solution is washed away from the sample; b) the sample is incubated in an ethereal reagent solution containing an alkaline earth metal salt component (s), a sugar component and an amino acid component in a buffer solution whose pH is in acidic range, for 30 minutes to 12 hours, and the ethereal reagent solution is washed away; C) treating the sample with a solution containing an identification reagent specific to the Chlamydia trachomatis bacterium for 20 minutes -12 hours; and d) the presence of any Chlamydia trachomatis bacterium is detected in a suitable manner. 20 Process according to claim 1, characterized in that the ethereal reagents contain as alkaline earth metal salt component calcium and magnesium chloride and / or optionally other divalent ions in a concentration of 10 - 40 mmol / l, advantageously 20 mmol / l calcium and magnesium chloride, as sugar. glucose or sucrose at a concentration of 5 - 40: 25 mmol / l, advantageously at a concentration of 10 - 20 mmol / l, and as amino acid component lysine at a concentration of 10 - 40 mg / ml, advantageously 25 - 35 mg / l. ml, in a 0.01 - 0.1 mol / l phosphate, citrate or Tris-HCl buffer whose pH is 3.5-5.5. Process according to Claim 1, characterized in that the etherification reagents contain as alkaline earth metal salt component 20 mmol / l calcium chloride and 20 mmol / l magnesium chloride, as sugar component 15 mmol / l glucose and as amino acid component 30 mg / ml lysine in a 0.1 mol / l phosphate buffer whose pH is 5.0. Process according to claim 1, characterized in that the decolorizing reagent is a 0.3 - 1.8% by weight perhalogenic acid solution in a suitable buffer whose pH is below 6.0, advantageously in a 0.01-0.0. 1 mol / l phosphate buffer, citrate buffer or Tris-HCl buffer whose pH is 3.5 - 5.5. Process according to claim 1, characterized in that the decolorising reagent is a periodic acid or a salt thereof as a 0.5 - 1%% solution in a 0.05 - 0.1 mol / l phosphate buffer whose pH is 4.0 - 5.5. 6. A process according to claim 1, characterized in that the decolorizing reagent is 1% by weight of periodic acid in a 0.1 mol / l phosphate buffer whose pH is 5.0. 7. A method according to claim 1, characterized by the identification reagent being a stamped poly or monoclonal antibody or genetic engineering probe specific to the Chlamydia trachomatis bacterium and identifying all of its known serovars and not cross-reacting with the Chlamydia ps / tfac / p bacterium, Chlamydia the bacterium or other organisms known to readily cross-react with at least anti-chlamydia antibodies. 15 Method according to claim 7, characterized in that the identifying reagents are stamped with any detectable stamp, such as a fluorescent, phosphorous, chemiluminescent or dye stamp or by another detectable stamping method, such as the biotin-avidin method, a radioactive or enzymatic stamp, alkaline phosphatase or peroxidase. Method according to claim 7, characterized in that the identification reagent is an antibody which has been tuned with a fluorescent agent, such as fluorescein. 10. Reagent reagent, characterized in that it contains an alkaline earth metal salt component (s), a sugar component and an amino acid component in a buffer solution the pH of which is in the acidic region, wherein the alkaline earth metal salt component is calcium and magnesium chloride and / or magnesium chloride. a concentration of 10-40 mmol / l, the sugar component is glucose or sucrose at a concentration of 5 - 40 mmol / l, advantageous glucose at a concentration of 10-20 mmol / l, and the amino acid component is a basic or hydrophilic amino acid, advantageous lysine, at a concentration of 10 - 40 mg / ml, advantageously 25 - 35 mg / ml, and the buffer is a 0.01-0.2 mol / l phosphate, citrate or Tris-HCl buffer or a corresponding buffer whose pH is 3.5 - 5.5. 104218 11. Reagent reagent according to claim 10, characterized in that it contains 20 mmol / l calcium chloride, 20 mmol / l magnesium chloride, 15 mmol / l glucose and 30 mg / ml lysine in a 0.1 mol / l phosphate buffer whose pH is 5.0 . 12. Reagent reagent according to claim 10, characterized in that it further contains an agent or agent which stabilizes the reagents and promotes its shelf life. A method for recovering the antigen of the Chlamydia tracoma / bacterial bacterium in a Papanicolaou-stained cytological sample, characterized in that the sample is treated with a recirculating reagent according to any of claims 10-12.