FR2648150A1 - Process for the detection of Endamoeba histolytica in fecal materials - Google Patents

Abstract

The present invention relates to tropical medicine, specifically to the branch of parasitology. The aim of the invention is to detect infection by the parasite Endamoeba histolytica by means of analysis of fecal materials. This process makes it possible to detect, with high sensitivity and high precision, amoebiases. The object of the invention is the addition of a sufficient quantity of fecal material, diluted in buffer solution, to a solid surface which has been previously sensitised with an antibody against histolysin. After removal of the unbound biological material, the bound histolysin is visualised directly and it is reacted with a specific chromogenic or fluorogenic substrate. The change in absorbance or fluorescence of the reaction liquid is indicative of infection. The process is applicable to parasitology, epidemiology, epidemiological monitoring and taxonomy.

Description

 The present invention relates to biochemistry, tropical medicine and parasitology, especially to a highly sensitive and precise method for the immunochemical detection of Endamoeba histolytica in faeces.

About 10% of the world's population is infected with Endamoeba histolytica. Amoebiasis is responsible for 40,000 to 100,000 deaths each year worldwide (Amoebiasis and the fight against antimicrobials. Bull. WHO 63 (5): 821-831 (1985)). In some countries, for example in Mexico, amoebiasis is one of the ten main causes of death from parasitic disease and globally is only preceded by malaria and schistosomiasis (Walsh. JA (1986)
Problems in recognition and diagnosis of amebiasis: Estimation of the global magnitude cf morbidity and mortality. Rev.

Infect. Say. 8 (2): 228-238. The microscopic examination of my fecal sorters is quite boring, difficult, expensive and depends on the skill and experience of the technician. Even the most experienced technicians confuse E. trophozoldes. histolytica and the main phages having ingested erythrocytes (alsh, 1986).

 Furthermore, due to the intermittent excretion of cysts, three or more samples should be examined over several days to detect 80% to 90% of infections. Examination of a single sample per person only detects 40% to 50% of genuinely infected cases (Mathur.TN and J. Kaur (1973). The frequency of excretion of cyst of Endamoeba histolytica in known cases of non- dysenteric amoebic colitis based on 21 stool examinations. Indian J. Med. Res. 61: 330-333).

 Despite the use of several different methods for examining feces, we manage to obtain many false positives resulting from confusion with macrophages or non-pathogenic protozoa.

 There are two types of current methods for detecting this parasite: direct microscopic assessment of the morphology of the parasite's cysts or trophozold in faeces, and immunological tests for the detection of antigens in faeces.

 Microscopic observation is the procedure usually used in the clinic. This process consists in dispersing a sample of fresh or preserved faeces in an appropriate preservative as soon as it is removed. This dispersion can be colored with suitable dyes. We then observe under an optical microscope. The appearance of Endamoeba histolytica cysts (or rarely trophozoids) is the criterion for considering a positive case. The process can be combined with a precise concentration technique for protozoan cysts.

 Observation under the microscope has many disadvantages.

In the first place, the trophozoide is fragile and generally does not appear in its integrity in the feces.

In addition, infected people do not excrete cysts in all their evacuations. Besides, usually the cysts are confused with those of other non-pathogenic protozoa, or even with host cells or food particles. (Healy. G.R. (1972) Laboratory Diagnosis of a amoebiasis. Clin. Trop. Med. Volume II, Ed. Kevin, M.

Cahill University Press: 150-165: Harrys. J. (1982). Amoebiasis: A review J. Roy; Soc. Med. 75: 190-197).

 On the other hand, several immunological tests have been described for detecting antigens in feces.

Grundy (1987) describes a multi-phase Elisa (Grundy, M.S.

A. Voller; D. Warhurst (1987 An Enzyme-Linked Immunosorbent
Assay for the detection of Endamoeba histolytica. Antigens in Faecal material. Trans. Roy. Soc. Too much. Med. Hyg. 81 627-632).

The method consists in sensitizing a microtiter plate with specific sheep antibodies directed against antigens of E. trophozoides. histolytica. A suspension of faeces is then added and the mixture is incubated for 2.5 h at 4 C. The plate is washed and a solution of rabbit antibodies specific for E. antigens is added. hlstolvtica and incubate for another 2.5 h. The plate is washed again and the conjugate solution (sheep antibodies specific for rabbit immunoglobulins conjugated with the peroxidase enzyme) added. The plate is again washed and cn then added the substrate of the peroxidase (o-phenylenediamine). The reaction is stopped and the optical density of the products is measured at 492 rm.

 This process has a low sensitivity, which is why cases carrying E. cysts. histolytica are not reported as actual. In addition, the feces of patients with other parasites give higher results than those of healthy people.

 A detection kit has started to be marketed by Millipore Co. It is based on a "sandwich-ELISA" test developed by Root, D. M. et al. (Root, D.M.; F.X. Cole: J.

Williamson, (1978). The Development and Standardization of an E'ISA Method for the Detection of Endamoeba histolvtica Antigens in Fecal Samples, Arch, Invest. Med. Mex. 9, 203-210).

The method consists in sensitizing discs of 6 mm this diameter of immunoabsorbent material with specific antibodies against the antigens of E. histolytica, to place these discs on a strip which is immersed for 16 hours in the sample of diluted feces, to wash the strip, to add a second antibody conjugated with peroxidase at room temperature for two hours, posteriorly the substrate and finally, to stop the reaction. The process was withdrawn from the market due to its low sensitivity. In addition, there are interferences with the substances that are present in the faeces, such as lipids, biopigments, blood, some drugs and food, as well as the substances that contain humes and peroxidases.
ne rut of the junior process of the present application is the sensitive and precise detection of Endamoeba histolytlca in the faeces.

 This method is new in that, unlike the previous methods, the antibodies with which the solid surface is sensitized are specific for histolysin, the protease dependent on E. cysteine. histolytica.

Another new element is that, to obtain the result, the enzymatic activity of histolysine intervenes on a specific substrate. Then there is no need for the addition of another enzyme (conjugate). This fact increases the specificity of the process, because histolysin has so far only been found in E. histolytica.

The object of the process is to sensitize a solid surface of polystyrene, polyvinyl chloride or other synthetic surface or Staphylococcus aureus with a solution (0.05-2 μm / ml) of specific antibodies against histolysin. This solid surface is then brought into contact for 1 to 24 hours with a dilution of faeces (5% to 50%). The solid surface is washed five times with PBS-T20 (phosphate buffered saline, 0.05% TWEEN 20). Add a pH 6-9.5 buffer, which contains cysteine or 2-2 dithiothreitol 0.5-2 nM, and a concentration between 0.05 and 10 nM of the substrate having the formula
Benzyloxycarbonyle-XYC where
X = Arginyl or phenylalanyl
Y = Arginine
C = Beta-naphthylamide or amino methyl coumarin
or NPhNO2
Incubation is carried out for 1 to 72 hours at 25 to 400 ° C. and the colored reaction products are determined visually or spectrophotometrically and the fluorescent products by visual assessment of the fluorescence under ultraviolet light or by a fluorometer.

 The determination of the alterative products can be carried out natively after having retained the reaction by adding a retarding solution which contains a histolysin inhibitor, or by varying the pH or denaturalization of the histolysine. This retarding solution may contain 4-diazo-2 ', 3-dimethylazobenzene (22.5 µg / ml) or 100 nM monochlorinated sodium acetate.

Example of implementation of the invention
Example No. 1
A polyvinyl chloride plate is sensitized with a solution of specific antibodies against histolysin (0.05 μg / ml). So we put on the plate a 50% dilution of feces of patients with
E. histolytica and healthy people for 24 hours. The surface of the plate is washed five times with
PBS-T20. Add the substrate (TRIS 20 mM pH 7.5, 2 nM, 2.2 dithiothreitoi, benzyloxycarbonyl-arginyl-arginineamino methyl coumarin 2 mM) and incubate at 400C for 1 hour. Acetic acid is pierced until pH 3 is obtained and the result obtained is observed by illuminating the plate with ultraviolet rays (360 nm). Fluorescence is observed in the parts of the plate where the samples from the infected patients have been added.

Example N02
A polystyrene plate with specific antibodies is sensitized against Xistolysine (2 ug / ml). A 5% dilution of positive and negative faeces is placed on the plate for one hour at 40C. The surface of the plate is washed five times with PBS-T20. The substrate buffer is added (Carbonate-bicabonate 200 nM,
Ph 9.5, 0.5 nM Cysteine, benzyloxycarbonyl-phenylalanylearginine-amino-methyl coumarin, 0.05 nM) and incubated at 25 ° C for 48 hours. The reaction mixture is read in a fluorimeter. The device is set to 100% fluorescence with a 0.0625 nM solution of amino methyl coumarin, excitation wavelength 360 rsn and emission wavelength 460 nm. Values greater than 1 are obtained for positive samples.

Example # 3.

 A suspension of S. aureus is sensitized with a solution of specific antibodies against histolysin (0.0; ug / ml). The suspension is brought into contact with a dilution (20%) of faeces from persons positive and negative for E. histolytica for 3 hours. The staphylococci are washed three times by centrifugation and resuspension.

Buffer pH 7.5, benzyloxycarbonyl-arginylearginine-4-methoxy-beta-naphthylamide 10 nM is added. Incubate for 1 hour at 40 ° C. The reaction is stopped with ethylene diamino tetra acetic acid 0.9%, TWEEN 20 1%, 4-diazo-2 ', 3-dimethylazobenzene (22.5 ug / ml Centrifuge and read the optical density at 520 nm in the supernatant. Cases positive for E. histolytica show a major optical density at 0.300.

 In order to show the effectiveness of the proposed process, an analysis of 150 volunteers was carried out. We collected faeces which correspond to 3 to 6 different donations from these volunteers. All the samples were analyzed by microscopy technicians experienced by the microscopic convolutional process (with the concentration technique.

 At the same time, the determination was carried out by the method proposed in the present patent application.

 In Table 1 are reported the results obtained from the analysis made by the two methods.

Results of the analysis by the pro
assigned proposed in this request (+)
Analysis results (+) 28 4 microscopic (-) 0 118
Among the positive cases by microscopy, 8 cases were taken to make a comparison analysis of sensitivity: 4 were positive under microscopy in the first examination, 2 after 3 examinations and 2 after 6 examinations, which confirms the best sensitivity. of the present invention, which is comparable to the analysis by microscopy of 6 samples of faeces of different donations. There are elements which suggest that the 4 cases negative by the present method and positive by microscopy are errors in microscopic detection. First, the above cases were positive, only in the first series of six microscopic determinations.

 Secondly, the faeces of these people were cultivated in a Robinson medium with the aim of putting E. histolytica aside, and from which the organism could not be isolated.

 The faeces of patients with other intestinal parasites who were not E carriers were also analyzed. his tolytica. The results are shown in Table 2.

Table 2
Analysis of cross-reactions with other intestinal parasites.

Parasite Result by the proposed process observed .-) (+)
T. TRICHIURA 33 0
G. LAMBLIA 10 0
E. NANA 34 o
E. COLI 12 0
B. HOMINIS 30 0
S. STERCORALIS 3 0
A. LUMBROCOIDS 5 0
E. VERMICULARIS ~ 12 0
Cross-reactives with other parasites have not been observed. Positive cases received anti-amoebic treatment (diloxanide 500 mg / 8h for 10 days). The faeces of 8 of them were collected 10 days after finishing the treatment and analyzed. All cases were negative.

 A group of five positive samples and five negative samples was analyzed immediately after evacuation and after being stored at 250C for one week.

No significant variations in the results were observed.

Advantages of the proposed solution
The present method has higher specificity, because the specificity of the antibody-antigen interaction is potentiated by the enzyme-substrate specificity. This is why it is possible to adjust the process conditions in order to obtain a higher sensitivity. This allows reliable detection of intestinal amoebiasis by a single sample of faeces (compared to 6 or more necessary for diagnosis by microscopy). Given that the object of the method is the determination of the enzymatic activity, there is no need for the presence of the intact parasite (either in a form of cyst or trophozoite) in the sample analyzed.

 Samples can be kept frozen without a specific deadline, or at 250C for at least one week.

 In the process, the appreciation of the morphological characteristics of the parasite does not intervene, this is why the error inherent in the address of the observer is avoided.

 Due to its superior efficiency and low cost, this method can be used for screening in the population.

Claims (2)

 1. Method for the detection of Endamoeba hlstolvtica in faeces, using a solid surface of polystyrene, polyvinyl chloride, microorganisms such as for example Stathvlococcus aureus, or other synthetic or natural material sensitized with antibodies on which takes place a chromogenic or fluorogenic enzymatic reaction, characterized in that the solid surface is sensitized with a solution of 0.05 to 2 mg / ml of specific antibodies against histoiysine, said solid surface being brought into contact with a dilution of materials faeces (5-50%) from 1 hour to 24 hours and being washed five times with DBS-T20 (phosphate buffered saline, 0.05% TWEEN 20), add the substrate buffer and incubate from 1 hour to 72 hours from 250C to 400C.  2. Method according to claim 1, characterized in that the substrate buffer, pH 6-9.5 contains cysteine or 2-2 ', dithiothreitol 0.5-2 mM and a concentration of 0.05 to 10 mM of the substrate having the formula  Benzyloxycarbonyle-X-Y-C where: X = Argiryl or phenylalanyl Y = Arginine C = Beta-naphthylamide or  4-metn'oxy- 2-napntylamide cu go amino methyl coumarine or  N? HNO