ABSTRACT

Objective

We aimed to investigate the frequency of Cryptosporidium spp. in immunosuppressed patients who were admitted with diarrhea by microscopic, serological, and molecular methods and to evaluate the results in comparison with the direct fluorescent antibody (DFA) method.

Methods

We analyzed 90 stool samples from immunosuppressed patients with diarrhea. All stool samples were examined using modified acid-fast staining, polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) tests for the detection of Cryptosporidium spp. samples examined with the DFA method were selected randomly, including samples found positive in other diagnostic tests. Stool samples that were positive in any of these diagnostic tests were evaluated by immunochromatographic card test.

Results

Our study included 90 samples, of which 44 (48.8%) and 46 (51.2%) were from male and female patients, respectively. The mean age of the patients was 37.8±27.5. Thirty-one (34.4%) of the samples were from pediatric patients and their mean age was 5.3±4.34. The methods used did not show a statistically significant difference between the positivity status and patient age and gender (p>0.05). Cryptosporidium spp. positivity was detected in five samples (5.6%), two samples (2.2%), and one sample (1.1%) by using the modified acid-fast staining, PCR, and ELISA methods, respectively. Six of 51 samples to which the DFA method was applied were detected positive. In 4 of the 7 stool samples that were found positive by any of these methods, Cryptosporidium spp. was detected positive by the immunochromatographic card test.

Conclusion

When the DFA method is accepted as the gold standard method in the diagnosis of Cryptosporidium spp., the modified acid-fast method can be used in routine diagnosis with high sensitivity and specificity. Besides, it immunochromatographic diagnostic tests thought to be useful in laboratories with intensive workflow due to their practical use and rapid results.

Keywords:

Cryptosporidium, diarrhea, direct fluorescent antibody, immunosuppressive

INTRODUCTION

The severity and duration of infections caused by Cryptosporidium spp. vary depending on the type, immunity, and host age (1). Cryptosporidiosis involves the jejunum and ileum in patients, making diarrhea the most typical clinical finding of the infection (2). Other less frequently detected clinical findings are abdominal pain, nausea, and vomiting (3). Extraintestinal system involvement, such as cholecystitis, pancreatitis, and cholangitis can also be observed in immunosuppressive patient groups, such as acquired immunodeficiency syndrome (AIDS) (1).

Respiratory cryptosporidiosis commonly occurs in children, and although the presentation is generally asymptomatic, pulmonary infiltration and respiratory distress can be detected (4). Cough is the main symptom when the lungs or trachea is involved(1). In addition, cryptosporidiosis persisting for a long time in childhood may lead to growth and developmental retardation (5).

Additionally, asymptomatic infections have also been reported(3). Anorexia, depression, myalgia, headache, and fatigue have been reported in asymptomatic cases. Asymptomatic patients are more common than diagnosed patients in developed or developing countries (6-8).

Although clinical symptoms vary individually, the excreted amount of oocyst is closely related to the immune status and host age. The infection, which generally has an asymptomatic course in healthy people or heals spontaneously within 2-3 weeks causes more severe clinical symptoms, particularly in children, those with insufficient oral intake, and immunosuppressed and elderly people (9). Persistent diarrhea lasting for a long time can become chronic and lead to resulting in death (10).

Infections caused by intestinal parasites are commonly seen in immunosuppressed patients. Parasitic agents can cause diarrhea varying in severity and may result in mortality (11). In developing countries, the frequency of diarrhea due to parasitic agents was >90% in the immunosuppressed patient population. Cryptosporidium is one of the most frequently detected pathogens in these patients (12).

Cases of this agent may not be adequately detected due to symptoms of cryptosporidiosis being similar to other gastroenteritis symptoms, stool samples of patients complaining of diarrhea not being routinely evaluated for Cryptosporidium, and not using advanced diagnostic techniques in many cases (13).

Cryptosporidiosis can be diagnosed using histological and molecular methods, microscopic examination, and serological techniques, such as direct fluorescent antibody (DFA) and enzyme-linked immunosorbent assay (ELISA)(14, 15). The development of microscopical techniques revolutionized the morphological sciences, progressively providing new levels of magnification and resolution for exploring biological and non-biological samples. Furthermore, the high resolution of microscopic techniques also allowed the detection and visualization of different types of microorganisms, by exploring in a deep way the causative agent of different pathologies (16).

Microscopic examination is the most commonly preferred method. However, evaluating Cryptosporidium oocysts using direct microscopic examination is challenging due to their small size which is very similar to yeasts. The chance of diagnosing Cryptosporidium spp. can be increased with concentration methods (17, 18). Microscopic examination after acid-fast staining is preferable because it is cheap, allows the internal structure of oocysts to be evaluated, and preparations can be stored for a long time thanks to permanent staining. However, due to disadvantages such as the need for a long time and experience for evaluation and low reproducibility, different diagnostic methods are also needed (17).

Molecular techniques are diagnostic methods that provide rapid results with high specificity and sensitivity based on the amplification of the appropriate gene region. Nowadays, it has started to replace conventional diagnostic techniques based on the identification of phenotypic characteristics and even became the gold standard for the detection of some microorganisms. It can distinguish between closely species or subtypes. It is also of great importance in the diagnosis of microorganisms that have lost their viability during the transport phase (19).

The DFA method, which detects surface antigens, technique offers the highest combination of sensitivity and specificity and is considered the gold standard by many laboratories (20).

We aimed to investigate the frequency of Cryptosporidium spp. in immunosuppressed patients who were admitted with diarrhea by microscopic, serological, and molecular methods and to evaluate the results in comparison with the DFA method.

METHODS

This prepared study was evaluated and approved by Necmettin Erbakan University Faculty of Medicine Hospital Non-Drug and Non-Medical Device Research Ethics Committee on 15.04.2022 with decision number 2022/3755. Since our study was conducted on stool samples routinely sent to the microbiology laboratory, patient consent was not obtained. This study, with project number 221518022, was supported by Necmettin Erbakan University Scientific Research Project Coordination Office.

Gender, age, clinical and diagnostic information of the patients were obtained from our hospital’s laboratory information system. The study included patients receiving immunosuppressive therapy after transplantation, patients receiving chemotherapy due to malignant solid tumors or hematological malignancies, patients immunosuppressed due to hematological malignancies, patients receiving corticosteroid therapy for more than two months, and patients diagnosed with primary immunodeficiency.

A simple sedimentation technique was applied to the samples for fecal concentration. Subsequently, direct microscopic examination was performed with physiological saline (0.9% NaCl) on the slides. For stained microscopic examination, a smear was prepared from the stool samples, and modified acid-resistant Ehrlich-Ziehl-Neelsen (EZN) was performed. We also recorded other parasitic agents detected during direct microscopic examination or stained (modified acid-resistant staining) microscopic examination was recorded.

The remaining samples were divided into three for ELISA, DFA, and polymerase chain reaction (PCR) analyses without adding any preservative solution, divided into 1.5 mL eppendorf tubes, and kept at -80 °C to be stored until analyses.

In the ELISA method, the Cryptosporidium RIDASCREENR, C 1201 r-Biopharm commercial kit was used to detect Cryptosporidium parvum and Cryptosporidium hominis antigens.

Before the molecular study, the QIAamp DNA Stool Mini Kit (QIAGEN GmbH, Hilden, Germany) was used for DNA extraction of stool samples. Subsequently, probe-based real-time PCR analysis of the extracted samples was performed using the Roche real-time PCR device (Roche, Switzerland 2004). The probe and primer sequences defined in the PCR study in the literature were used for Cryptosporidium (21). These sequences are shown in Table 1.

PCR primers and probes (Table 1) were designed using the BioEdit software program, version 7.0.9.0 (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) to create Clustal W (23) alignments of representative Cryptosporidium sequences from GenBank [National Center for Biotechnology Information (NCBI)]; http://www.ncbi.nlm.nih.gov/GenBank/). The Primer Express software program (Applied Biosystems, Warrington, United Kingdom) was used to calculate melting temperatures and check for undesirable inter and intramolecular binding. Primer and probe sequences were then checked for cross-reactions with nontarget sequences on the GenBank database using the basic local alignment search tool (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi).

Cryptosporidium and Giardia antigens were investigated using the DFA method with the Merifluor Cryptosporidium/Giardia Direct Immunofluorescence Assay (DFA; Meridian Diagnostics, Inc., Cincinnati, Ohio, USA) commercial kit. Samples examined with the DFA method were selected randomly by including samples found positive in other diagnostic tests. The samples detected as positive by any of the diagnostic methods were evaluated with an immunochromatographic card test as a using the commercial Entamoeba/Giardia/Crypto Combo Rapid Cassette test (Microcult, Biotech, China). ELISA, DFA, PCR and card tests were used following the manufacturer’s recommendations.

Using the DFA method as the reference method, the positive and negative predictive values, specificity, sensitivity and accuracy rates of serological, molecular and microscopic methods were determined. The accuracy value was found by dividing the sum of the true positive and true negative values by the total data.

Statistical Analysis

Statistical analysis of the data was analyzed with SPSS (Statistical Package for Social Sciences) ver.23 statistical package program. Continuous variables are expressed as mean ± standard deviation, minimum and maximum values, and categorical variables are expressed as numbers and percentages. Differences between categorical variables were examined with Pearson chi-square and Fisher-Freeman-Halton Exact tests. The significance level was determined as p<0.05.

RESULTS

The frequency of Cryptosporidium spp. was investigated in 90 immunosuppressed patients with diarrhea, of whom 31 were children, between May 2022-December 2022.

The ages of the patients ranged between 0 and 81 years, and the mean age was 37.8±27.5 years. Moreover, 31 (34.4%) samples were from pediatric patients, of which 18 (58%) and 13 (42%) were from men and women, respectively, and their mean age was 5.3±4.34 years. Of the 59 adult patients, 26 (44%) and 33 (56%) were men and women, respectively. Of the 90 included samples, 44 (48.8%) and 46 (51.2%) were from men and women patients, respectively. The methods used did not show a statistically significant difference between the positivity status and patient age (p=0.688) or between the positivity status and sex (p=0.261).

The study patients frequently consist of patients in pediatrics, oncology and hematology clinics. The positivity rates of patients coming from these departments are 11.1%, 11.7% and 8.3% respectively. In this study, there were 4 patients from the immunology and allergic diseases clinics, and the positivity rate in this patient group was 25%. Table 2 shows the distribution and rates of patients included in the study according to clinics.

Among the study participants, 54.4%, 14.4%, 11.1%, 8.8%, and 7.7% were diagnosed with a malignant solid tumor, lymphoma, primary immunodeficiency, multiple myeloma, and leukemia, respectively.

In the microscopic examination using acid-fast modified EZN staining, Cryptosporidium spp. oocysts were detected in five samples (Figure 1). Cyclospora spp. oocyst was seen in one sample. In microscopic examination, Giardia spp. and Blastocystis spp. were detected in 2 and 26 samples, respectively.

Cryptosporidium positivity was detected in one and two samples using ELISA and PCR methods, respectively. In this study, in which Cryptosporidium and Giardia antigens were investigated by DFA in 51 stool samples, including those positive in any of the microscopic, ELISA or PCR evaluations, Cryptosporidium spp. was found positive in six samples and Giardia spp. was found positive in three samples (Figure 2).

Because the DFA is considered the gold standard method, the DFA results were considered as the main results in the comparative evaluation of the methods in statistical analysis. Based on the DFA method, the modified acid-fast staining method had a sensitivity, specificity, positive predictive value, negative predictive value, and accuracy rate of 83.33%, 100%, 100%, 97.82%, and 98.03%, respectively. Based on the DFA method, PCR had a sensitivity, specificity, positive predictive value, negative predictive value, and accuracy rate of 33.3%, 100%, 100%, 91.83%, and 92.15%, respectively. Based on the DFA method, ELISA had a sensitivity, specificity, positive predictive value, negative predictive value, and accuracy rate of 0%, 97.77%, 0%, 88%, and 86.27%, respectively. The specificity and sensitivity, positive and negative predictive values and accuracy rates of serological, molecular and microscopic methods compared to the gold standard method the DFA method, are shown in Table 3. The stool samples of seven patients found to be positive with any of the other diagnostic tests were re-examined with the immunochromatographic card test, resulting in four samples being positive. The characteristics of patients with Cryptosporidium positivity are shown in Table 4.

DISCUSSION

Cryptosporidium spp., one of the factors causing infectious diarrhea, is an opportunistic agent that inhabits most commonly in the jejunum and less frequently in organs, such as the lungs, gallbladder, and liver. This parasite can cause mortality in older people and children, in the immunosuppressive patient group, and malnourished individuals (20). Initially, it causes watery diarrhea in immunosuppressive patients. Furthermore, it also causes gastroenteritis with a more benign course in immunocompetent individuals (24).

After evaluating the literature, sex was not associated with the incidence of Cryptosporidium (25). In our study, sex did not affect the positivity of Cryptosporidium. Cryptosporidium is an important cause of diarrhea in childhood, and the parasite is more symptomatic in children (4). In this study, the positivity rate was higher in children than in the adult age group, but the difference was not statistically significant.

This parasite is found worldwide and may be the underlying cause of diseases with diarrhea as the main symptom (26). Cryptosporidium, which is associated with premature deaths and growth and development retardation in childhood, caused a waterborne epidemic affecting 400,000 people in Milwaukee, USA, in the 1990s. Currently, it can be found almost everywhere, except in Antarctica. Although Cryptosporidium is detected in 1% of immunocompetent people in developed countries, it is 5-10% in countries with low socioeconomic levels. This rate varies between 10% and 25% in children who consult with complaints of diarrhea (4).

Because this pathogen is more commonly detected in immunocompromised patients, epidemiological studies were mostly conducted on immunosuppressed patients with diarrhea (27). Therefore, the data were highly inconsistent, which may be due to the stage of the infection, characteristics and number of the evaluated patient population, sensitivity of the diagnostic methods, and the climatic conditions (28).

Cryptosporidium spp. positivity was found in 6.3% of 427 immunocompromised patients with diarrhea in a study conducted in Egypt, 12.6% of 87 patients with colorectal malignancy and diarrhea in a study conducted in Poland (29, 30), 12% of 137 HIV patients with diarrhea, and 12.4% of 84 immunocompromised patients with diarrhea (31, 32).

In the studies evaluating Cryptosporidium spp. incidence in the immunocompromised patient population in Türkiye , Cryptosporidium spp. positivity was found in 6.4% of 47 patients diagnosed with chronic renal failure (33),5.3% of 94 immunosuppressive patients with malignant solid tumors receiving chemotherapy and complaining of diarrhea for >5 days, and 8.3% in 72 stool samples of immunosuppressive patients with diarrhea (34, 35).

In our study, Cryptosporidium spp. was detected in 7 (7.8%) of 90 patients with complaints of immunosuppressive diarrhea, by at least one of the methods used.

The DFA method is used as the gold standard in reference laboratories in the USA and Europe (4). Therefore, in our study, based on DFA, in the modified acid-fast EZN staining method, the sensitivity and specificity were 83.33% and 100%, respectively. The high cost of DFA tests is the most important problem that limits their use in routine laboratories. Modified EZN staining, which has high sensitivity and specificity, can be used to detect Cryptosporidium spp. as a more cost-effective method in experienced laboratories, particularly in sensitive groups, such as immunosuppressed and pediatric patients.

Depending on the commercial kit used, the efficacy of the ELISA method varies. In our study, ELISA was unsuccessful in detecting Cryptosporidium spp. In another study, PCR was considered 10³-10⁴times more sensitive than the ELISA method (36). Moreover, false-positive results can be due to cross-reactions against common antigens in many pathogens, particularly apicomplexan parasites (27). The high cost of this method is another disadvantage, as well as sample collection before the examination (34).

A previous study evaluated 80 stool samples of patients presenting with gastrointestinal system complaints, and Cryptosporidium spp. positivity was found in three (3.75%) and five samples (6.25%) by using acid-fast staining and ELISA, respectively. Three positive samples detected using acid-fast staining were also positive with the ELISA technique (37).In another study, the ELISA method detected Cryptosporidium spp. in 2.8% of 723 stool samples in the patient group presenting with the same complaints (27).In our study, the ELISA method detected positivity in only one patient, which could not be confirmed by other diagnostic methods. The low sensitivity of the ELISA method may be related to the kit used or the Cryptosporidium spp. oocysts may not have been distributed homogeneously in the stool of the positive samples.

Recently, molecular methods have been preferred because they can perform genotyping based on nucleic acid analysis, and the opportunity to easily evaluate many samples, and guide the treatment process (38). In order to apply molecular biology techniques in studies with genetic material, DNA and RNA molecules must be obtained in pure form before PCR, taking into account that they will also contain inhibitors. Otherwise, results may be masked by taq polymerase enzyme inhibitors in the sample. The basic components used in PCR are target DNA or RNA (template), taq DNA polymerase enzyme, primers, deoxynucleotides, buffer liquid, pH, Mg+2 ions. Temperature parameters (especially annealing temperature) and Mg+2 concentration that are not in the appropriate range, and the use of poor quality polymerase enzyme may lead to erroneous PCR results. Additionally, the design of oligonucleotide primers is only possible with known strains of microorganisms and known sequences of these strains. False negatives may occur in inappropriate primer selections. Another factor that may cause problems in the functions of PCR is unexpected mutations that occur in microbial genomes (19). In conditions where parasite oocysts are not homogeneously distributed throughout the stool, molecular techniques detect fewer positive samples than microscopic examination (39). Furthermore, polysaccharides, bile salts, or bilirubin in the stool can inhibit the PCR study (34). If oocyst damage occurs before DNA isolation PCR can not detect Cryptosporidium spp., resulting in discrepant data between microscopic evaluation data and PCR results (39, 40).The inclusion of molecular techniques in routine diagnosis will only be possible when these problems are solved and ensure a certain standardization (40).

In our study, only two patients were positive using PCR, and based on the DFA test, the sensitivity and specificity were 33.3% and 100%, respectively. False negative results in PCR studies are thought to be caused by inhibitors in the stool sample, problems in the DNA extraction stage, or primer probe optimization. Some studies in the literature also showed that PCR had lower sensitivity than microscopic evaluation (38, 41).

In a study conducted in our country on 36 immunosuppressed patients with diarrhea, the Cryptosporidium was investigated using microscopic examination with modified acid-fast staining, molecular techniques, and DFA testing. Based on the data, one sample and three samples were positive with acid-fast staining and DFA method, respectively; however, no positive sample could be detected with the molecular technique used (41). Another study evaluating 80 immunosuppressed patients with diarrhea, three, five, and four positive samples were found by microscopic examination, ELISA, and DFA, respectively, whereas PCR did not detect positivity (38).

In the stool samples of 90 immunosuppressive patients in our study, positivity was detected in five, one, and two samples by microscopic examination, ELISA, and PCR, respectively. All the positive samples were positive with DFA, except for the positive sample detected by ELISA, and DFA positivity was detected in 6 of the 51 samples studied. An immunochromatographic rapid diagnostic test was used in seven positive samples by any of the methods, and four samples were positive with the rapid diagnostic test. Rapid diagnostic tests will be useful in rapid diagnosis in laboratories with intensive sample studies, particularly in sensitive groups with chronic diarrhea. In the study, Cyclospora spp., another opportunistic pathogen, were detected in one patient, and Giardia spp., an important causative agent of diarrhea, were detected in three patients.

The inadequate effectiveness of the commercial kit used in the ELISA test and the problems experienced in the DNA extraction phase and/or primer probe optimization in PCR caused limitations in our study. In addition, DFA and immunochromatographic rapid diagnostic tests could not be applied to all samples. The small number of positive samples does not allow for a fully accurate assessment of the usefulness of these methods. Studies in which all diagnostic techniques are applied on more samples are needed.

CONCLUSION

Our study especially showed that the probability of detection of Cryptosporidium spp. in stool samples from immunosuppressed patients with diarrhea should not be underestimated. Considering that the number of immunosuppressive patients increases daily, rapid and reliable detection of this agent is extremely important in the routine work of laboratories, particularly for the immunosuppressive patient group. Therefore, diagnostic algorithms should be created for the diagnosis of Cryptosporidium spp. in secondary and tertiary care hospitals with more immunosuppressive patient follow-up.

Although diagnostic methods used to detect Cryptosporidium oocysts have advantages, each has its disadvantages. Based on the gold standard method DFA, modified acid-fast staining has high sensitivity and specificity, but it requires labor and technical experience. Performing modified acid-fast staining and evaluating all stool samples received in laboratories with intensive sample flow may not be possible. However, in high-risk patient groups, evaluation of samples using modified acid-fast staining as a minimum diagnostic method will be useful for the diagnosis of Cryptosporidium. Besides, it immunochromatographic diagnostic tests will be useful in laboratories with intensive workflow due to their practical use and rapid results.

Ethics

Ethics Committee Approval: Non-Drug and Non-Medical Device Research Ethics Committee on 15.04.2022 with decision number 2022/3755.

Informed Consent: Since our study was conducted on stool samples routinely sent to the microbiology laboratory, patient consent was not obtained.

Authorship Contributions

Surgical and Medical Practices: D.B., Concept: F.E.T., Design: F.E.T., Data Collection or Processing: D.B., Analysis or Interpretation: F.E.T., Literature Search: D.B., Writing: D.B.

Conflict of Interest: No conflict of interest was declared by the authors.

Financial Disclosure: This study, with project number 221518022, was supported by Necmettin Erbakan University Scientific Research Project Coordination Office.

References

Sen ZS, Tasar MA, Kaya FD, Gunes A, Vezir E, Gulsen HH. A rare complication of acute diarrhae caused byCryptosporidium: possible hepatobiliary system involvement in a child without immunodeficiency. Mikrobiyol Bul. 2019; 53: 464-71.

Yalçın S, Doğan NY, Mor N. Determination ofCryptosporidium spp. by molecular methods in different water resources of Erzincan province. Erzincan University Journal of Science and Technology. 2019; 12: 1-13.

Ekici A, Unlu A, Aydemir S, Barlik F, Yilmaz H. Subtyping ofCryptosporidium parvum obtained from humans and calves in Van, Turkey. Iran J Parasitol. 2022; 17: 366-74.

Miman O, Saygi G. Temel Tibbi Parazitoloji. Istanbul: Istanbul Yayınevi; 2018: 241-248.

Hossain MJ, Powell H, Sow SO, Omore R, Roose A, Jones JCM, et al. Clinical and epidemiologic features ofCryptosporidium-associated diarrheal disease among young children living in sub-Saharan Africa: the vaccine impact on diarrhea in Africa (VIDA) study. Clin Infect Dis. 2023; 76: S97-S105.

Robinson P, Okhuysen PC, Chappell CL, Lewis DE, Shahab I, Janecki A, et al. Expression of tumor necrosis factor alpha and interleukin 1 beta in jejuna of volunteers after experimental challenge withCryptosporidium parvum correlates with exposure but not with symptoms. Infect Immun. 2001; 69: 1172-4.

Dann SM, Wang HC, Gambarin KJ, Actor JK, Robinson P, Lewis DE, et al. Interleukin-15 activates human natural killer cells to clear the intestinal protozoan cryptosporidium. J Infect Dis. 2005; 192: 1294-302.

Huang DB, White AC. An updated review onCryptosporidium and Giardia. Gastroenterol Clin North Am. 2006; 35: 291-314.

Zhao W, Ren G, Jiang W, Wang L, Wang J, Yuan Z, et al. Genetic characterizations ofCryptosporidium spp. from children with or without diarrhea in Wenzhou, China: high probability of zoonotic transmission. BMC Microbiol. 2024: 24: 113.

Aydemir S, Durmaz H, Aydemir ME, Kılıc S, Demir A, Halidi AG, et al. Investigation of the effect of pasteurization on the viability ofCryptosporidium parvum in cow’s milk by propidium monoazide qPCR. Mikrobiyol Bul. 2023; 57: 660-6.

Atasever M, Mazlum H, Cevik B, Uzum H. Investigating the quality of public fountain water in Gümüşhane province and the presence ofCryptosporidium spp. using the PCR method. Turkish Journal of Agriculture Food Science and Technology. 2024; 12: 1530-8.

Zorbozan O, Quliyeva G, Tunalı V, Özbilgin A, Turgay N, Gökengin AD. Intestinal protozoa in HIV-infected patients: A retrospective analysis. Turk J Parazitol. 2018; 42: 187-90.

Khalil SB, Mirdha BR, Paul J, Panda A, Makharia G, Chaudhry R, et al. Development and evaluation of molecular methods for detection ofCryptosporidium spp. in human clinical samples. Exp Parasitol. 2016; 170: 207-13.

Sears CL, Kirkpatrick BD. Cryptosporidiosis and isosporiosis. In: Principles and Practice of Clinical Parasitology; 2001; 139-164.

Çuhadar V, Sengül M, Mete E. Investigation ofCryptosporidium parvum in water resources in Mardin province. Turk Mikrobiyol Cemiy Derg. 2023; 53: 156-62.

Torge D, Bernardi S, Ciciarelli G, Macchiarelli G, Bianchi S. Dedicated protocol for ultrastructural analysis of farmed rainbow trout (Oncorhynchus mykiss) tissues with red Mark syndrome: The Skin-Part One. Methods and Protocols. 2024; 7: 37.

Usluca S, Babur C, Kilic S. Current status in intestinal parasitic İnfections:a reference laboratory results. Klimik J. 2020; 33: 307-14.

Centers for Disease Control and Prevention (CDC). Laboratory Identification of Parasites of Public Health Concern. Cryptosporidiosis. June 3, 2024. Available from: https://www.cdc.gov/dpdx/cryptosporidiosis/index.html (accessed: March 7, 2025).

Kahya S, Büyükcangaz E, Carlı KT. Polimeraz zincir reaksiyonu (PCR) optimizasyonu. Uludag Univ J Fac Vet Med. 2013; 32: 31-8.

Centers for Disease Control and Prevention (CDC). Diagnostic Procedures. September 24, 2024. Available from: https://www.cdc.gov/dpdx/diagnosticprocedures/index.html (accessed: March 7, 2025).

Hadfield SJ, Robinson G, Elwin K, Chalmers RM. Detection and differentiation ofCryptosporidium spp. in human clinical samples by use of real-time PCR. J Clin Microbiol. 2011; 49: 918-24.

Morgan UM, Constantine CC, Forbes DA, Thompson RC. Differentiation between human and animal isolates ofCryptosporidium parvum using rDNA sequencing and direct PCR analysis. J Parasitol. 1997; 83: 825-30.

Thompson JD, Higgins DG, Gibson TJ. Clustal W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994; 22: 4673-80.

Şahin S, Ağaoğlu S, Alemdar S.Cryptosporidium ve Cryptosporidiosis. Doğruer Y, editör. Gıda kaynaklı paraziter hastalıklar. Ankara: Türkiye Klinikleri; 2018; 35-41. (Turkish)

Ekici A, Gunay C, Sahin M, Aydemir S, Yılmaz H. Spread of intestinal parasites in patients presenting with gastrointestinal complaints. Turk J Parazitol. 2023; 47: 224-8.

Leder K, Weller PF. Epidemiology, clinical manifestations, and diagnosis of cryptosporidiosis.2017. Available from: https://www.uptodate.com/contents/epidemiology-clinical-manifestations-and-diagnosis-of-cryptosporidiosis.

Beyhan YE, Yılmaz H. Investigation ofCryptosporidium spp. Antigen by ELISA in stool specimens: nine year evaluation. Turk J Parazitol. 2020; 44: 68-71.

Brooks G, Carroll KC, Butel JS, Morse SA, Mietzner TA. Jawetz, Melnick, Adelberg Tıbbi Mikrobiyoloji. Yenen Ş, editör. Ankara: 2014.

Abaza SM, Makhlouf LM, El-Shewy KA, El-Moamly AA. Intestinal opportunistic parasites among different groups of immunocompromised hosts. J Egypt Soc Parasitol. 1995; 25: 713-27.

Sulżyc-Bielicka V, Kołodziejczyk L, Jaczewska S, Bielicki D, Kładny J, Safranow K. Prevalence ofCryptosporidium spp. in patients with colorectal cancer. Pol Przegl Chir. 2012; 84: 348-51.

Sane SS, Thakar MR, Mehendale SM. Opportunistic parasitic infections in HIV/AIDS patients presenting with diarrhoea by the level of immunesuppression. Indian J Med Res. 2009; 130: 63-6.

Jayalakshmi JB, Appalaraju B, Mahadevan K. Evaluation of an enzyme-linked immunoassay for the detection ofCryptosporidium antigen in fecal specimens of HIV/AIDS patients. Indian J Pathol Microbiol. 2008; 51: 137-8.

Sari C, Sari K, Ertug S. Investigation of the frequency ofCryptosporidium spp. and Blastocystis hominis in patients with chronic renal failure. Turk J Parazitol. 2003; 27: 187-90.

Karabey M, Can H, Öner TÖ, Döşkaya M, Alak SE, Döşkaya AD, et al.Cryptosporidium spp. during chemotherapy: a cross-sectional study of 94 patients with malignant solid tumor. Ann Saudi Med. 2021; 41: 293-8.

Yıldız M, Çöplü N, Kılıç S, Babür C, Öncül Ö, Esen B. Investigation ofCryptosporidium spp. in solid tumor cases with diarrhea. Turk J Parazitol. 2001; 25: 1-8.

Leng X, Mosier DA, Oberst RD. Simplified method for recovery and PCR detection ofCryptosporidium DNA from bovine feces. Appl Environ Microbiol. 1996; 62: 643-47.

Tamer GS, Gülenç S. Investigation ofCryptosporidium spp. antigens in stool by ELISA. Turk J Parazitol. 2008; 32: 198-201.

Yılmaz, A. The investigation ofCryptosporidium spp. in patients with diarrhea by microscopic and ELISA methods. Selcuk Med J. 2016; 32: 61-4.

Magi B, Canocchi V, Tordini G, Cellesi C, Barberi A.Cryptosporidium infection: diagnostic techniques. Parasitol Res. 2006; 98: 150-2.

Amar CFL, Dear PH, McLauchlin J. Detection and identification by real time PCR/RFLP analyses ofCryptosporidium species from human faeces. Lett Appl Microbiol. 2004; 38: 217-22.

Ulçay A, Görenek L, Coşkun O, Araz E, Acar A, Eyigün CP. Diagnosis of intestinal-protozoa in patients with immune deficiency. Turk J Parazitol. 2008; 32: 328-33.

Investigation of the Frequency of Cryptosporidium spp. in Immunosuppressed Patients with Diarrhea