Th The prevalence of Cryptosporidium Parvum infection in adults presenting with acute diarrhea to diagnostic medical laboratories in Jahrom in 2014

Kargar Jahromi Zahra1, Davami Mohamad Hasan*1, Solhjoo Kavous1, Khajei Mohammad2, Kargar Jahromi Hossein1

Received:8/24/2015 Revised:10/25/2015 Accepted: 20/12/2015

1. Zoonoses Research Center, Jahrom University of Medical Sciences, Jahrom, 2. Student Research Committee, Jahrom University of Medical Sciences, Jahrom, Iran

Pars Journal of Medical Sciences, Vol. 13, No.4, Winter 2016 Par J Med Sci 2016;13(4):7-13

Abstract Introduction: Cryptosporidium parvum is an Apicomplexan protozoan that can cause acute diarrhea in humans and animals. The present study was conducted to determine the prevalence of this infection in adults with acute diarrhea presenting to diagnostic medical laboratories in Jahrom, Iran, in 2014. Materials & Methods: The present descriptive study was conducted on 117 adults with acute diarrhea presenting to diagnostic medical laboratories in Jahrom. The patients’ epidemiological data were collected using a questionnaire. After they were condensed with Formalin-Ether and stained with modified Ziehl- Neelsen, the patients' stool samples were examined for cryptosporidium parvum oocysts both under the microscope and using the Nested-PCR method. The data obtained were analyzed in SPSS-16. Results: Of the total of 117 adults presenting with acute diarrhea, only one (0.9%) was found to be Cryptosporidium parvum positive both through the Ziehl-Neelsen staining and the Nested-PCR method. Conclusion: The results of the examinations with microscopic and molecular methods showed a very low percentage of cryptosporidium infection in adults. Given the higher cost of the molecular method compared to the microscopic method, the modified acid-fast staining method can be used as an alternative, provided that it is performed by technicians who are experienced in detecting this parasite.

Keywords: Cryptosporidium Parvum, Adult, Prevalence

Introduction Cryptosporidiosis is a disease caused by micron - obligate intracellular protozoan protozoa of the genus cryptosporidium that mainly infects gastrointestinal belonging to the apicomplexa- group, and epithelium and –sometimes the respiratory clinically appears as a gastroenteritis-like system in vertebrates (2). This parasite was syndrome (1). Cryptosporidium is a 2-6 first identified in the gastric epithelium of * Corresponding author, Address: Zoonoses Research Center, Jahrom University of Medical Sciences, Jahrom, Iran Tel: 07154340406 Email: [email protected] 7 The prevalence of Cryptosporidium Parvum infection Kargar Jahromi Z et al mice by Ernest Edward Tyzzer in 1907, hospitalized adults with diarrhea, five who named it Morris Cryptosporidium (3). cases of cryptosporidium oocytes were At least eight of the 29 known species of identified and the prevalence was reported this parasite cause diseases (4). Due to the as 3.5% (16). – There is an increase in the similarity - - between human and bovine prevalence of this parasite in developing cryptosporidium and - their cross countries - like Iran. Due to the specialized transmission-, parvum –has been detection method, the contamination introduced ----- as the main cause of the assessment - is not usually - possible in disease in - immunodeficient - patients. medical laboratories. The stool sample- Accordingly, it is essential to know how assessment in terms of contamination with this protozoan is transmitted to humans. cryptosporidium is not routinely Thus, human cryptosporidium that often performed in – the laboratories across the infects people is named cryptosporidium country, and it is only performed - if parvum or genotype 1 (5), and bovine deemed necessary by the doctor. Specific cryptosporidium parvum that not only methods are required for the detection of infects humans, but also other mammals, is cryptosporidium oocytes in stool such as named genotype 2. Because of the Ziehl-Neelsen staining-, or other methods difference in sequence and also in such as auramine-rhodamine staining-, - electrophoretic pattern after the digestion monocolonal antibodies, or molecular – of the gene by restriction enzymes, these techniques such as PCR (17). The majority two species are considered as two distinct of studies - conducted so far have been genetic species (6). This parasite spreads concerned with frequency and clinical universally and infects humans through symptoms of contamination with this drinking water and eating food parasite in children and -immunodeficient contaminated with spored oocytes (1). patients, and very few studies have been This disease can – range from - moderate conducted on adults. Thus, the present to severe depending on the severity of study was conducted with the aim to symptoms, patient’s immune system, investigate the frequency of infection with infected site in the body, and one's cryptosporidium parvum in adults nutritional status (4). In an presenting with diarrhea to medical immunocompetent host, cryptosporidium laboratories in Jahroom. infection usually appears as spontaneously limiting intestinal symptoms (7). In - Materials and Methods immunodeficient patients, this infection In the present descriptive study, sample may be intestinal or - extraintestinal. size was calculated as 117, which were Regardless of the patient's immune system, collected by convenient sampling from the most common clinical symptom is 117 adults presenting with acute diarrhea diarrhea (8). The prevalence of to medical laboratories in Jahroom from cryptosporidium varies according to mid-June 2014 to late March 2015. The climate- and - the minimum of 5% to samples wer transferred to the laboratory 12.7% has been reported in developing within an hour. The samples belonged to countries (8). Studies conducted in Iran - people from Jahroom and its six show the prevalence of infection with this surrounding villages (Heidarabad, parasite as 1.5% in adults with HIV and Chahrtaqi, Mobarakabad, Hakan, 7% in children (9-14). In different parts of qotbabad, and simakan). Data including Iran, the prevalence varies from 2.2% in age, gender, drinking water conditions, the south, 4.1% in the west, and 7% in contact with livestock, and place of southeast to 7.7% in northwest (15). In a residence (urban, rural) were collected study conducted by Khalili et al. in 2008, from their parents in the course of in Shahrkord, on 146 stool samples from sampling. Patients who had taken anti-

Pars Journal of Medical Sciences Vol. 13, No.4, Winter 2016 8 The prevalence of Cryptosporidium Parvum infection Kargar Jahromi Z et al parasitic medication or antibiotics were 3), and in the second stage, 434bp piece excluded from the study. Samples were was proliferated using primer pairs (PF: 5- condensed using formalin-ether technique, AAGCTCGTAGTTGGATTTCTG-3) and in which one gram of stool sample was (PR: 5- mixed with 10 ml of formalin 10% and TAAGGTGCTGAAGGAGTAAGG-3) filtered into the centrifuge tube through (18). Then, the PCR product underwent two layers of gauze. Three milliliters of electrophoresis and was observed using the diethyl-ether was added to each tube and Gel Document (Gel Doc) device. then vigorously stirred until fully mixed. The mixture obtained was then Results centrifuged. Contents of each tube were Of 117 adults of mean age 45.83±19.72 emptied and the sediment was spread onto years, 64 (54.7%) were male and 53 a glass slide and - air-dried (19). Smears (45.3%) were female, of whom, 95 were stained using the modified acid-fast - (81.2%) lived in the city and 22 (18.8%) in staining method. Alkaline fuchsin solution villages. Modified acid-fast staining was poured on slides and they were gently technique showed positive results in terms heated over the Bunsen burner such that of cryptosporidium contamination in a 28- they do not boil. Slides were then year-old urban man (0.9%). Of all incubated at room temperature for 5 participants, 104 (88.9%) had no contact minutes, rinsed with tap water and de- with livestock, 4 (3.4%) had constant stained with sulfuric acid 2% for 1-2 contact and 9 (7.7%) had little contact with minutes, and rinsed again with tap water livestock. The person infected had little and dried at room temperature. They were contact with livestock. City pipe water then stained with methylene blue for one was available to 108 (92.3%) participants, minute, rinsed with tap water, and dried at and rural pipe- water to 9 (7.7%). room temperature. Then using emersion Cryptosporidium infected person had oil, they were examined for access to city pipe water. The – nestedPCR cryptosporidium under an optical assessment of all participants produced the microscope (magnification 100×) (20). same results as those of acid-fast staining Figure 1 shows 4-6 micron technique. Out of 117 participants, the cryptosporidium oocytes in light red in a same infected person in staining and blue background. To perform molecular microscopic assessment was also found tests, the DNA was first extracted using infected with cryptosporidium parvum in Qiagen stool DNA extraction kit the molecular method. Figure 1 shows the (Germany) using the manufacturer’s one positive sample and a number of instructions, and – then the nested PCR negative electrophoresed samples. In the test was performed on all samples. In the present study, the frequency and the first stage, 1056bp piece was proliferated frequency distribution of infection with using primer pairs (PF: 5- cryptosporidium were determined based CCACATCTAAGGAAGGCAGC-3) and on the study variables. (PR: 5-ATGGATGCATCAGTGTAGCG-

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Figure 1: Detection of cryptosporidium parvum in Nested PCR technique Column 1: Positive control of cryptosporidium parvum Column 2: Negative control Column 3: Sample from the patient infected with cryptosporidium parvum Column 4-5: Samples from patients not infected with cryptosporidium parvum

Discussion cryptosporidium in children under 5 years The present study results - confirmed the of age attending -Hajar hospital in infection with cryptosporidium parvum in Shahrkord was reported as 2% (19). In a adults with acute diarrhea in the city of study by Dabirzadeh et al. - in Zahedan, – Jahrom for the first time. Cryptosporidium in children under five years of age infection was observed in 0.9% out of 117 admitted to the pediatric hospital, the patients with gastroenteritis studied using infection with this parasite was 4.7% (24). modified acid-fast staining and Nested In a study conducted by Postchian et al. in PCR techniques. In a study conducted by Isfahan on all age groups, the prevalence Khalili et al. in Chaharmahal-Bakhtiari, of this infection was reported as 3.9% (25). 3.5% of adults were infected with In a- study conducted in Tehran on cryptosporidium (3). In - a study, children under 10,- the prevalence of this conducted by Dalimi et al, on 1128 stool infection was 2.4% (26). This was reported samples from patients attending Aliasghar, 4.7% in Khoramabad (27), 4.04% in Mofid, and Imam Khomeini hospitals in Ardebil (28), and 3.26% in Semnan (29). Tehran, cryptosporidium parvum was The contradictory results -- related to the detected in 10 samples (0.8%), which is prevalence of cryptosporidium across the very close to the present study results (21). world may be due to various factors such Similarly, in a study - conducted, in as one's lifestyle, cultural and economic Jahrom by Kargar et al. (2014), on status, -living in urban or rural areas, children with acute diarrhea using the geographical climate and -their health staining technique, the infection rate of knowledge. In the present study, the 1.9% with this parasite was reported - frequency of infection with this protozoon which is also close to the present study in male participants was 0.9%, which was results (22). In a- study conducted in different from 0% in the female Qazvin, Ghoreishi et al. reported the participants- and disagrees with similar prevalence of this infection in children domestic (29-31) and foreign (32-35) with gastroenteritis to be 0.3% (23). In studies with similar odds for infection. another study by Khalili et al., the These results are similar to those obtained prevalence of the infection with in studies conducted in Korea and

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Slovenia that showed a higher prevalence cases in modified acid-fast method, which among male samples - (36-37). In a study may have been due to the difference in test conducted by Dabirzadeh et al. in method (condensing), modified acid-fast Zahedan, the infection level was higher in staining and magnification ×1000 that boys than in girls, but not statistically eliminated false positive cases and more significantly, which agrees with the accurately detected actual cryptosporidium present study (38). In a study conducted in oocytes. In Germany, Bylak et al. Shahrekord on 504 diarrhea samples, 2% considered the specificity of infection with cryptosporidium parasite - immunological methods sufficient for was detected in 12 samples, which is - screening stool samples for close to the present study figures, but – cryptosporidium, and reported that the higher in initial sample size (39). Given PCR method does not enhance the the above studies, since the immune accuracy of detection of oocytes (29), system improves with aging, the infection which agrees with the present study that with this parasite decreases. In the present showed no greater accuracy with study, the person found infected was in molecular method (without considering the contact with livestock. In the study staining method). conducted in Semnan, the infection was For the early treatment of this disease and observed in 3.26% of people that were in prevention of its spread, its detection is contact with livestock, and this essential, especially in infection specialist relationship was statistically significant pediatric hospitals, where oocytes of (29). In the present study, the molecular cryptosporidium parvum should be technique was also used to detect infection differentiated from larger yeast cyclospora with cryptosporidium, which produced the oocytes (8-10 microns) using ocular same result as that of modified acid-fast micrometer. Hence, the correct detection method (0.9%). In their study conducted in of this parasite requires experience and 1998 in Australia, Morgan et al. compared morphological knowledge of PCR and staining methods used for cryptosporidium parvum oocytes (41). detection of cryptosporidium in diarrheic Given the high cost and unavailability of human stool samples. Of 511 stool molecular tests in conventional samples, 36 samples were found positive laboratories, it is recommended that using the PCR technique. - Only 29 smears obtained from sedimentation in samples could be diagnosed using formalin-ether method be replaced by PCR modified acid-fast staining method, for detecting cryptosporidium oocytes in equivalent of 7 false-negative samples. stool samples by experts. Previous studies Moreover, positive samples in PCR have proposed PCR as a useful method for showed 5 false positive results in modified differentiation of pathogenic genotypes of acid-fast method (40). In a study this protozoan (40). Given the negligible conducted in Tehran by Salehi et al. on percentage of infection cases found in the 2500 samples using both methods, 30 present study, it is recommended that positive samples were detected in modified larger sample sizes -be used for the acid-fast staining and no false positive determination of the relationship between cases, and all were reported positive in the percentage of this infection and other PCR technique. Of negative cases, –two variables affecting this clinical disease. positive ones - were detected using the PCR technique, which suggested false Conclusion negative caused by the staining method. The present study results showed a very The results obtained by Morgan et al. low percentage of infection with disagree with those of the present study cryptosporidium in adults using that showed no false positive or negative microscopic and molecular methods.

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Given that percentage of infection was the University of Medical Sciences, for his same in both methods and also the high cooperation and the Research Deputy of cost of molecular tests for detecting Jahrom University of Medical Sciences for cryptosporidium, modified acid-fast funding this research. This article is part of method on the condition that it is the professional doctorate thesis – of performed in the presence of an expert for Jahrom University of Medical Sciences. microscopic detection of this parasite can be beneficial. Conflicts of Interest Acknowledgments The authors did not express any conflicts We thank Dr. Agholi, the assistant of interest. professor of parasitology at Fasa

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