Immunochromatographic Test for Detection of Cryptosporidium in Malignant Children Before and During Chemotherapy in Basra

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1 International Journal of Innovative Biochemistry & Microbiology Research 4(1):1-9, Jan.-Mar SEAHI PUBLICATIONS, ISSN: Immunochromatographic Test for Detection of Cryptosporidium in Malignant Children Before and During Chemotherapy in Basra Sabeeha M. Abdul-Hussein¹, Abdulmohsin H. Jassim², Jinan G. Hassan³ Department of Medical Microbiology, College of Medicine, Basra University, Basra, Iraq. ABSTRACT Immunochromatographic (IC) tests may play an important role in the future diagnosis of parasitic diseases because of their speed and simplicity of use. A recently developed test to detect Cryptosporidium. Microscopy the gold standard reference techniques and the results of this IC test were compared with those obtained with ELISA and IC single test for the parasite. One hundred stool samples were assayed from malignant children aged 2 month to 14 years before and during (6 to 8 weeks) chemotherapy. Using microscopy,9 sample before chemotherapy and 15 during chemotherapy were diagnosed as positive for Cryptosporidium. Results of IC tests show 13 positive sample before chemotherapy and 33 during chemotherapy sensitivities of 100% and specificities were of 95.9 % before chemotherapy, when,results of IC tests show sensitivities of 100% and specificities were of 80.2 % during chemotherapy when compare with staining method. Keywords: Cryptosporidiosis, Immunochromatographic test (IC), Cryptosporidium, ELISA, INTRODUCTION Cryptosporidiosis is a parasitic disease caused by an apicomplexan protozoa of the genus Cryptosporidium ; Cryptosporidium parvum is the specific infective agent in human (Ikechukwu et al.,2011).cryptosporidium are waterborne, obligate intracellular protozoan parasite,(mosallanejad et al.,2010). Ernest Edward Tyzzer in 1907 was the first person successively to identify the oocyst of Cryptosporidium parasite (Fayer,2004). The first case of human cryptosporidiosis was reported in 1976(Nime et al., 1976). Cryptosporidiosis, long considered to be a veterinary disease, has emerged as a serious human health problem, being the most frequent secondary diagnosis in people with AIDS and significantly contributing to their mortality. Human Cryptosporidium parvum associated disease is the result of zoonotic or anthroponotic transmission of the parasite s infectious stages, the oocysts. The parasite is transmitted via a fecal - oral route and very frequently via contaminated water and food ( Graczyk T.K., et al., 2003 ). Diarrheal caused by Cryptosporidium was estimated in immunocompetent individuals between 1%-10% of diarrheal disease worldwide. It is also found in 6% of all patients with acquired immunodeficiency syndrome (AIDS) and in 21% of AIDS patients with diarrhea (Ayalew,2006). The life cycle of Cryptosporidium is complex(casemore,1990). In this study determining the most sensitive and specific method for diagnosing the parasite to be a guidative for diagnostic laboratories. SUBJECTS AND METHODS A total of 100 fecal specimens were collected from malignant children aged 2 month to 14 years before and during (6 to 8 weeks) chemotherapy in Pediatric Oncology Center at Basrah children s specialty Hospital in Basrah Province during May to November 2015; 100 stool specimen from symptomatic and asymptomatic hospitalized children (including males and females).stool samples were collected and examined using Formalin-Ethyl Acetate Sedimentation method, a modified acid fast staining method,immunochromatographic test (IC) and ELISA. Stool samples must be collected in clean containers 1

2 without any additives and stored at 2 8 C. If stored for more than 3 days, the sample must be frozen at 20 C. In this case, the sample must be completely thawed out and brought to room temperature before testing begins. 1. Concentration of Fecal Samples :- Stool samples were concentrated using the Formalin-Ethyl Acetate Sedimentation technique to achieve a better result in the a modified acid fast staining method( Garcia and Bruckner,1993). 2. Staining Method:- Two thin smears were prepared from concentrated materials, dried in room air and fixed, then stained with Kinyoun s Acid-Fast and Modified Ziehl-Neelsen acid fast staining method,then examined under a microscope using the high-power, using oil immersion(garcia and Bruckner,1993 ). The oocysts of Cryptosporidium spp. were described pink-red Oocysts are 4 to 6 µm in diameter, and four sporozoites may be present internally. The background should stain uniformly blue(garcia and Bruckner,1993). 3.Immunochromatographic test (IC) :- The RIDA Quick Cryptosporidium test (R-Biopharm AG, Darmstadt, Germany) was used. This test uses monoclonal antibodies directed against specific membrane proteins of Cryptosporidium. The method is a one-step lateral flow immunochromatographic assay containing specific antibodies labelled to coloured latex particles (blue for Cryptosporidium) to generate a visual signal. Latex labelled specific antibodies are applied into a porous filter affixed to a membrane. Capture specific antibodies are also applied at different levels on the membrane. Capture specific antibodies are also applied at different levels on the membrane. To perform the test, a faecal specimen is suspended in antigen extraction buffer and added to the porous filter containing latex labelled antibodies. If the target antigens are present in the specimens, these antibodies specifically bind to the antigens. The resulting latex labelled antigen/antibody complexes flow by capillarity action from the filter through the membrane. Here they are bound and immobilized by the redeposited capture antibodies, which are specific against the same protozoan antigens or against the latex labelled antibodies (as an internal control). This allows colour particles to concentrate and form a visible line. Indeed, after binding, immune complexes become visible and permit visual inspection of positive reactions. Change of colour on the control line is necessary to validate the test and its non-appearance, with or without colour changes on the test lines, invalidates the test. For the accomplishment of the method, reagents were stored at room temperature (20-25 C) until use. The tests were conducted at room temperature following the recommended manufacturer s instructions. Briefly, 1 ml of extraction buffer was placed into a test tube. Approximately 50 mg of stool sample were added, homogenized by a vortex mixer, and allowed to settle for 3 minutes. Then, four drops of the clear supernatant were placed into the round inlet opening of the test cassette. After 5 minutes, results were visually read. A negative result was represented by the appearance of a single blue band in the control area of the cassette. A positive result was indicated by the presence of a red(cryptosporidium positive ) band in the test areas of the cassette. 3. Enzyme Linked Immunosorbent Assay (ELISA) :- Enzyme Linked Immunoassay (ELISA) for the detection of C. parvum in faecal samples. RIDIASCREEN test (C 1201). (Art. No.: C GmbH, Darmstadt, Germany) The test was used for in vitro diagnosis of Cryptosporidium. This test is enzyme immunoassay for the qualitative determination of C. parvum in faecal samples and performed according to instructions of the kits. Microwell plate and reagents were brought to room temperature (20-25 ºC). The washing buffer was diluted with distilled water 1:10, followed by dilution the faecal sample with the sample dilution buffer diluent 1:11. The required micro well strips were placed in the frame followed by addition of 2 drops or 100 µl positive control (control +), negative control (diluent), or sample. After that 2 drops or 100 µl conjugate were added. Incubation was at room temperature (20-25ºC) for 60 min followed by Wash for five times with 300 µl diluted wash buffer followed by addition of 2 drops or 100 µl substrate. The faecal sample was incubated again at room temperature (20-25ºC) in the dark for 15 min followed by adding 1 drop or 50 µl stop reagent. Finally, photometric measurement was carried out at 450 nm (optional reference wave length > 600 nm). As indicated by the manufacturer instruction s, samples were considered positive when their extinction was more than 10% above the calculated cut-off, equivocal when their extinctions were within ± 10% of the cut-off and negative when their extinctions were 10% below the calculated cut-off. 2

3 Statistical methods: Statistical package of social science (SPSS) version 20 was used to analyze data, Chi-square (ᵡ²) test was used to assess the significance of difference between groups and variable, P-value less than 0.05 was considered to be statistically significant. RESULTS The results showed that the total infectivity rate of Cryptosporidium spp. in children was (31.1%) during chemotherapy in comparison with(12.3%) before chemotherapy ( Table 1). The number of positive samples were 33 out of 106 patients during chemotherapy and 13 out of 106 before chemotherapy. A total of 106 stool samples were microscopically examined for the presence of Cryptosporidium,9 were positive for Cryptosporidium before chemotherapy and 15 were positive for Cryptosporidium during chemotherapy, but were examined by immunochromatographic test (IC) and ELISA shows 13 positive for Cryptosporidium before chemotherapy and 33 positive for Cryptosporidium during chemotherapy. In( Table 2 )show Results of IC tests 13positive sample before chemotherapy and 33 during chemotherapy sensitivities of 100% and specificities were of 96.7 % before chemotherapy, when,results of IC tests show sensitivities of 100% and specificities were of 81.2 % during chemotherapy when compare with staining method. Table (1) Cryptosporidium by staining method,ic test and ELISA before and during chemotherapy Patients group Staining method ᵡ² P value Positive Negative No. % No. % Before chemotherapy During chemotherapy Patients group IC test &ELISA ᵡ² P value Positive Negative No. % No. % Before chemotherapy During chemotherapy

4 Figure (1) Cryptosporidium by staining method before and during chemotherapy Figure (2) Cryptosporidium by IC&ELISA before and during chemotherapy 4

5 Table(2) Diagnosis of Cryptosporidium by Immunochromatographic test (IC) and ELISA in comparison with staining method ( standard method) before and during chemotherapy Before chemotherapy Test Result Staining Method No. Patients Total Positive Negative Ic Test Positive (100) Negative (95.9) ELISA Positive (100) Negative 0 93 (95.9) 93 During chemotherapy Test Result Staining Method No. Patients Total Positive Negative IC Test Positive (100) Negative (80.2) ELISA Positive (100) Negative 0 73 (80.2) 73 5

6 Figure (3) Comparison between staining method, Immunochromatographic test (IC) and ELISA before and during chemotherapy In Table (2) A total of 106 stool samples were microscopically examined for the presence of Cryptosporidium,9 were positive for Cryptosporidium before chemotherapy and 15 were positive for Cryptosporidium during chemotherapy, but were examined by Immunochromatographic test (IC) and ELISA shows 13 positive for Cryptosporidium before chemotherapy and 33 positive for Cryptosporidium during chemotherapy. Also in Table (2) show Results of Immunochromatographic test (IC) and ELISA (13)positive sample before chemotherapy and (33) during chemotherapy sensitivities of (100% )and specificities were of (95.9 %) before chemotherapy, when,results of Immunochromatographic test (IC) and ELISA show sensitivities of (100%) and specificities were of (80.2 % )during chemotherapy when compare with staining method. In table (3) From among hundred and six patients with malignant diseases before chemotherapy 13 (12.26 %) were found to excrete Cryptosporidium oocyst in their stool compared to 33(31.13%) of patients with malignant diseases during chemotherapy,while among hundred and six patients with malignant diseases before chemotherapy 66( 62.3 % ) were found to be positive for various species of intestinal parasites to 40 ( 37.7 %) of patients with malignant diseases during chemotherapy (Table 4-2). Statistically, the difference was very highly significant ( ᵡ² = , df =2, p <0.05). Table (3) Parasitic and Cryptosporidium infections among patients with malignant disease before and during chemotherapy Patients group No. examined Parasitic infections Cryptosporidium infections +ve % +ve % Before chemotherapy During chemotherapy X 2 =15.598, df =2, p value=

7 DISCUSSION Comparison of the results of the new test with Staining Method for detection of Cryptosporidium infection in all samples from children, as well as detection of Immunochromatographic Test and ELISA Specificity & sensitivity were summarized in Table(1 and 2). Immunochromatographic Test allows the reliable detection of oocysts in one step does not require any special skill and is simple to use. Among several authors who performed Antigen detection by immunoassays, Agnamey et al. (2011) concluded that these methods have become a well-established aid to microscopic examination for the diagnosis of cryptosporidiosis. Good sensitivities and specificities have been reported for some of these tests in their comparative study. Cryptosporidium was found in (12.3%) in different types of malignancy in patients before chemotherapy and (31.1%) in different types of malignancy in patients during chemotherapy. These results are differently to some studies, as in in Basra city which found cryptosporidiosis among cancer patients to be (8%)( Mahdi et al.,2002),also in Basra by Mahdi (9.9%)( Mahdi et al.,1998) and in Baghdad (14.78%) by Al-Warid et al.(2012 ).In Kuwait found Cryptosporidium about (3.4%) ( Iqbal et al.,2011). In Egypt, cryptosporidiosis in immunocompromised children about (4.8%)( El-Mahallawy et al.,2014 ). In Iran found in children suffered from hematopoietic malignancy about (4.2%)( Dehkordy et al., 2010). In Ethiopia about (10.4%) ( Adamu,2010), in German, cryptosporidium was found in (12.6% )of patients with colorectal cancer (Sulżyc- Bielicka et al., 2012). In India found Cryptosporidium in (3.8%) (Sharma et al., 2013). explanation the differences and disparities in rates of Cryptosporidium infection among children of cancer patients because the patients have a lack of interaction between nutrition and immunity of the patient due to exposure to chemotherapy and malnutrition one recurring infected reasons, also patients who are exposed to chemotherapy weakens the cells and spread the parasite to the weakness of the patient's immunity and exposure and contact with infected animals.( Rudrapantna et al.,1997; Berenji et al.,2007 ) But did not report in previous studies to know the preparation of Cryptosporidium infection during chemotherapy.while was found intestinal parasite in (62.3%) different types of malignancy in children before chemotherapy and (37.7 %) in different types of malignancy in children during chemotherapy, this significant reflect two major changing reduction in the all-percent per dent and increasing in Cryptosporidium infection. These results are differently to some studies, as in Basra was found (49.5%) in different types of malignancy(mahdi et al.,1998),in Baghdad was found parasitic infections about (71.4%)(Hussein et al., 2011). In Egypt found parasitic intestinal about (12.9%)( Rivera et al., 1989), Botero et al. in 2003 reported about (32.9%) of intestinal parasites found in leukemia patients(botero et al., 2003).In Iran Zabolinejad et al. found Intestinal Parasites in Children with Lymphohematopoietic Malignancy in (35.9% )( Zabolinejad et al.,2013). In this study we addressed several diagnosed Cryptosporidium ways and comparison between them and the standard method, the results were the emergence of positive cases in the examination IC and ELISA and negative in the staining method for the same patient. This result is due to the number of oocysts, which take during the examination where in the way staining method need oocysts about 500,000 oocysts per gram of stool(weber B et al.,1991),but in ELISA needed about 500 oocysts per gram in stool (Chappell C L et al., 1999) and in IC needed about 1000 oocysts per gram in stool (Llorente T M et al., 2002). So observed proportion of positive cases in Immunochromatographic test and ELISA more than in staining method because in staining method needed high number of oocysts,also it needs to be versed examiner to detect oocysts due to the small size of the oocysts by microscopic examination((mchardy I H et al.,2014). CONCLUSION We can conclude that Immunochromatographic Test is a specific, sensitive and easy to use test for diagnostic of Cryptosporidium oocysts in stools. To our knowledge, this is the first study in IRAQ that uses both RIDASCREEN Cryptosporidium ELISA coproantigenic test and RIDA Quick Cryptosporidium test to detect Cryptosporidium infection in humans and compare their sensitivity and specificity in malignant children before and during chemotherapy. 7

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