ELISA TEST FOR DETECTION OF BLASTOCYSTIS SPP. IN HUMAN FAECES: COMPARISON OF THREE METHODS

Transcription

1 ELISA TEST FOR DETECTION OF BLASTOCYSTIS SPP. IN HUMAN FAECES: COMPARISON OF THREE METHODS István Kucsera, Mónika Molnár, Eszter Gályász, József Danka, Erika Orosz National Center for Epidemiology, Department of Parasitology, Budapest, Hungary National Center for Epidemiology Department of Parasitology 1097 Budapest, Gyáli út 2-6 Hungary Phone/Fax: Mob: kucsera.istvan@oek.antsz.hu ikucsera@gmail.com

2 Introduction: Blastocystis is a common intestinal parasitic micro-eukaryote with a worldwide distribution and is often the most frequently detected parasite found in both humans and non-human hosts and known to be genetically very diverse. It has been divided into numerous subtypes nine of which have been identified in humans to date. It was found that ST3 is the predominant subtype in human population. The taxonomic classification of Blastocystis hominis is mired in controversy. It has been previously considered as yeasts, fungi, or ameboid, flagellated, or sporozoan protozoa. Recently, based on molecular studies, has been placed within an informal group, the stramenopiles (Silberman et al. 1996). Cavalier-Smith (1998) considers stramenopiles to be identical to his infrakingdom Heterokonta under the kingdom Chromista. Therefore, according to Cavalier-Smith, B. hominis is a heterokontid chromista. Knowledge of the life cycle and transmission is still under investigation, therefore this is a proposed life cycle for B. hominis. Transmission is through the spread of cysts via the faecal-oral route by ingestion of contaminated water or food. Some debate exists as to whether B. hominis is pathogenic in immunocompetent individuals. A variety of clinical symptoms: nausea, anorexia, abdominal pain, flatulence and acute or chronic diarrhoea have been described, although Bh has been frequently found in asymptomatic individuals, too. Generally, the prevalence in developing countries is higher than in developed countries, which has been linked to standards of hygiene, waste disposal, exposure to animals, and consumption of contaminated food or water.

3 a b b/b9/four_common_forms_of_blastocystis_homi nis_valzn.jpg d c B. hominis: a) vakuolar form, culture, 500x b) phase contrast microscopy, 500x c) Indian ink, 1250x d) cyst form, 500x e) cyst form, 1250x (Dr. István Kucsera, NCE) e

4 Advantages and disadvantages of diagnostic methods: Detection of Blastocystis is routinely performed by microscopy, culture, and sedimentation concentration technique. These methods are time consuming, laborious and require special skilled personnel. Microscopy is difficult since Blastocystis has several morphological forms (vacuolar, cyst, amoeboid, granular, multivacuolar, and avacuolar). Concentration technics destroy some of the forms during stool processing, therefore are unreliable. Culture requires 2-4 days for diagnosis and may allow preferential growth of specific strains while eliminating others. ELISA-based test for detection of Blastocystis antigens in stool is fast, sensitive, specific and could be a proper alternative to currently used methods, especially the microscopy. In the other hand, it could detect only one pathogen, Blastocystits. Objectives: This work compares results of stool examination by microscopy, cultivation and antigen detection test.

5 Material and Methods: 78 stool samples routinely sent to the laboratory have been tested microscopically and by CoproELISA Blastocystis. 67 samples were tested with cultivation. For microscopic examination direct wet mount and Modified Merthiolate-Iodine -Formalin (MIF) preparation, for cultivation modified Boeck and Drbohlav s diphasic medium and for Blastocystis antigen detection CoproELISA Blastocystis Enzyme-Linked Immunosorbent Assay (ELISA) for the detection of Blastocystis spp. antigens in human faeces (Savyon Diagnostics, ISRAEL)

6 Principle of the Test Plates are coated with specific polyclonal antibodies directed against Blastocystis antigens. The absorbance is proportional to the number of Blastocystis cells present in the sample. Stool Collection 2. Preserved stool: The test is compatible with specimens that were fixed in 10% formalin or in Sodium Acetate Formalin (SAF). Preserved samples can be stored at room temperature for up to 24 months. The test is not compatible with stool specimens fixed in Polyvinyl Alcohol (PVA). Test Procedure for manual use Test Procedure for automation use

7 RESULTS Direct wet mount, Modified Merthiolate-Iodine Formalin (MIF) preparation and cultivation were performed continuously according to routine diagnostic procedure. CoproELISA Blastocystis was performed in 4 steps: , , ,

8 Sample number Microscopic Cultivation CoproELISA Bh Performed COV =OD nc +0,15=0,125+0,15=0, Positive control 1,637 positive 0,1250 negative Negative control 0,0530 negative Giardia intestinalis, Blastocystis Blastocystis 1,9200 positive Giardia intestinalis Blastocystis 1,8480 positive Giardia intestinalis, Blastocystis Entamoeba histolytica/dispar, Endolimax nana, Entamoeba coli Blastocystis 1,7580 positive Blastocystis, Endolimax nana 0,1840 negative negative negative 0,0530 negative negative negative 0,1000 negative negative Blastocystis 1,7600 positive negative Blastocystis 0,3900 positive negative negative 2,2150 positive negative Blastocystis 1,8700 positive negative Blastocystis 3,3200 positive negative Not performed 0,0530 negative negative negative 0,0460 negative negative negative 0,0500 negative negative Not performed 0,7270 positive Blastocystis Blastocystis 1,4750 positive negative negative 0,0900 negative negative Not performed 0,1020 negative 19 / negative Blastocystis 1,0580 positive 20 / negative Blastocystis 1,1580 positive 21 / Blastocystis Blastocystis 1,2090 positive 22 / Blastocystis Blastocystis 1,0810 positive

9 Sample number Microscopic Cultivation CoproELISA Bh Performed COV =OD nc +0,15=0,076+0,15=0,226 Positive control 1,638 positive Negative contro 0,076 negative negative negative 0,0660 negative negative negative 0,0660 negative Entamoeba coli negative 0,0630 negative negative negative 0,0350 negative negative negative 0,0520 negative Entamoeba coli, Entamoeba histolytica/dispar, Blastocystis Blastocystis 1,2890 positive negative negative 0,0700 negative negative negative 0,0420 negative negative negative 0,0660 negative negative negative 0,0330 negative Blastocystis Blastocystis 0,9290 positive negative negative 0,0400 negative 35 VA negative negative 0,0640 negative negative negative 0,0520 negative Blastocystis Blastocystis 0,2770 positive Blastocystis Blastocystis 3,4160 positive Negative Not performed 0,0730 negative Negative Not performed 0,0660 negative 41 / Blastocystis Blastocystis 1,1140 positive 42 / Blastocystis Blastocystis 1,2140 positive

10 Sample number Microscopic Cultivation CoproELISA Bh COV =OD nc +0,15=0,072+0,15=0,222 Positive control 1,435 positive Negative contro 0,072 negative negative negative 0,0970 negative Entamoeba coli, Entamoeba histolytica/dispar negative 0,3580 positive Entamoeba coli, Endolimax nana, Trichuris trichiura negative 2,8650 positive negative negative 0,0430 negative Entamoeba coli, Enterobius vermicularis Blastocystis 2,4700 positive negative negative 0,0360 negative Blastocystis Blastocystis 1,6340 positive negative negative 0,1340 negative negative negative 0,0560 negative negative negative 0,0360 negative Blastocystis Blastocystis 2,5520 positive negative negative 0,0500 negative negative negative 0,0460 negative negative negative 0,0310 negative negative negative 0,0290 negative negative negative 0,0770 negative negative negative 0,0810 negative Blastocystis Blastocystis 1,8520 positive negative negative 0,0660 negative Blastocystis Blastocystis 1,8660 positive negative Not performed 0,0460 negative negative negative 0,0470 negative Blastocystis Blastocystis 2,3610 positive negative negative 0,1780 negative negative negative 0,0380 negative 68 CSA negative negative 0,0950 negative 69 SZD1 negative Not performed 0,3280 positive Trichuris trichiura negative 0,1060 negative Giardia intestinalis Not performed 0,0710 negative negative Not performed 0,1450 negative

11 Sample number Microscopic Cultivation CoproELISA Bh Performed COV =OD nc +0,15=0,086+0,15=0,236 Positive control 1,3940 positive Negative contro 0,0860 negative negative negative 0,1020 negative negative negative 0,5270 positive 75 SZD2 negative negative 0,0450 negative negative Blastocystis 0,3490 positive negative Not performed 0,0240 negative 78 XY negative Not performed 0,0830 negative

12 ELISA 67 samples were tested with both cultivation and CoproELISA Blastocystis ELISA positive 25 Cultivation positive: 26 ELISA negative 1 Cultivation positive negative positive 25 4 Cultivated samples 67 Cultivation negative 41 ELISA positive 4 ELISA negative 37 negative 1 37 Sensitivity: 96,2% Specificity: 90.2% Efficiency of the Copro ELISA Blastocystis hominis test

13 SUMMARIZING THE RESULTS: microscopically in 20,5% (16/78) of samples Blastocystis was detected, by cultivation in 38,8% (26/67) and by ELISA 39,7% (31/78). Comparing cultivation and ELISA: 26 by cultivation positive samples were positive in 96.2% (25/26) by ELISA. Testing 41 culture-negative samples by ELISA further 4 positives have been detected (9.7%, 4/41). Based on this comparison the calculated parameters of the ELISA test are: Sensitivity: 96.2% and Specificity: 90.2%. Number of specimens with other microscopy detected parasites Parasite No samples E. histolytica/dispar+endolimax nana+entamoeba coli 1 Entamoeba coli 1 Entamoeba coli+endolimax nana+trichuris trichiura 1 Entamoeba coli+entamoeba histolytica/dispar 1 Entamoeba coli+enterobius vermicularis 1 Trichuris trichiura 1 Giardia intestinalis 2 Ʃ 8

14 In 16 microscopic Blastocystis positive samples Blastocystis was only parasite in 13 cases, in 2 Giardia intestinalis and in 1 Entamoeba coli and Entamoeba histolytica /dispar was present in addition to Blastocystis. From 78 samples only 11 had anamnestic data included, 6 patients of them had abdominal discomfort or diarrhoea. Conclusions: The ELISA results showed high correlation with results obtained by cultivation, as a standard method. Some differences in results of cultivation and antigen detection could be perhaps explained by presents of different subtypes of Bh. Antigen ELISA is expeditious in providing reliable results. Considering this, the antigen ELISA is expected to be the method of choice for diagnosis of Blastocystis in the common laboratory.

15 Thank you for your attention