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Background: Elderly individuals are considered an at-risk population, susceptible to enteric infections; and Cryptosporidium spp. is an apicomplexan protozoan considered to be one of the most common protozoa causing diarrhea. Cryptosporidiosis causes elevation of many pro-inflammatory cytokines like tumor necrosis factor-alpha (TNF-α) which may play a role in pathogenesis of the disease. Objectives: This study was designed for detection and genotyping of Cryptosporidium spp. in elderly patients and the relationship of infection with copro TNF-α. Diagnosis was by evaluation of permanent acid-fast cold Kinyoun's (AF) staining, immunochromatographic detection (ICT), and ELISA in comparison to molecular diagnosis as gold standard diagnostic method. Subjects and Methods: Stool samples were collected from 270 elderly patients aged above 60 years old attending outpatient clinics of Internal Medicine Hospital, Cairo University. Samples were examined microscopically by direct wet mount, and AF staining, and then subjected to ICT, ELISA, and nested PCR (nPCR) assays. Positive samples by nPCR were then subjected to Restriction fragment length polymorphism (RFLP) to detect Cryptosporidium genotypes. Copro-levels of TNF-α were measured to assess their relationship with cryptosporidiosis. Results: Cryptosporidiosis detection rates of 3.7%, 6.3%, 6.7%, 3.7% were determined by microscopic examination after AF staining, ICT, ELISA and nPCR, respectively. When RFLP was performed on nPCR positive samples, eight and two samples were assigned as genotype 1 and 2, respectively. Moreover, TNF-α was significantly correlated with cryptosporidiosis. Conclusion: The elderly are highly vulnerable to cryptosporidiosis. Immunodiagnosis and molecular techniques are fundamental for the diagnosis of cryptosporidiosis. Cryptosporidiosis significantly affects copro TNF-α. PARASITOLOGISTS UNITED JOURNAL 78 cases of cryptosporidiosis are mainly caused by two species: C. parvum and C. hominis[16]. However, other species as C. felis, C. canis, C. meleagridis, C. muris, and C. suiscan also infect humans[17]. It was observed that cryptosporidiosis correlates with the elevation of many pro-inflammatory cytokines such as TNF-α and interleukin (IL)-1β; these cytokines are key stimulators of prostaglandin synthesis[18]. In the intestinal wall, prostaglandins are expressed in response to these pro-inflammatory cytokines and may contribute to diarrhea by altering chloride secretion. Thus, TNF-α, IL-1β and prostaglandins have been assumed to have a role in the pathogenesis of cryptosporidiosis[19]. The aim of our study is to relate cryptosporidiosis in elderly patients to copro TNF-α. Simultaneously, we compared the diagnostic sensitivity and specificity of the AF stain, and the commercially available coproantigen tests; Cryptosporidium ELISA, and RIDA® QUICK-ICT against PCR as the reference method[20,21]. SUBJECTS AND METHODS This descriptive analytical study was conducted at Medical Parasitology, and Clinical Pathology departments, Cairo University, Faculty of Medicine during the period from May 2019 to June 2020. Subjects: The study included 270 elderly diarrheic patients who were selected from Internal Medicine Hospital, outpatients' clinic, Cairo University. Inclusion criteria include both sexes, aged above 60 years, with diarrhea. The patient was considered diarrheic when complaining of abnormal increase in stool liquidity and frequency of bowel motions. Patients were subjected to thorough history taking including type of drinking water, level of education, residence, vomiting, history of hepatitis C, and abdominal pain, as well as clinical examination. Stool samples collection: A single fecal sample from each patient was collected in a dry, clean, leak-proof plastic container. The macroscopic examination of the fecal samples was recorded before processing. Each sample was divided into 2 parts; fresh and frozen at -20 ̊C. I. Detection rate of cryptosporidiosis 1. Microscopic examination: The stool samples were first examined by direct wet mount after the addition of a drop of iodine, and by formalin-ethyl acetate sedimentation techniques to aid visualization of protozoa cysts, helminthes eggs and larvae. 2. Permanent cold AF Kinyoun's staining: Thin stool smears were prepared from the sediment obtained by the formalin ethyl acetate sedimentation procedure and stained with cold AF stain for detection of oocysts. A commercially available readily prepared AF stain was used (Kinyoun Kit cat no 25765-1, Polysciences, Germany). Oval/round bodies, about 4-5 μm, of Cryptosporidium oocysts stained pink to red to deep purple, often with darker staining around the periphery, were identified against a blue background. In some of the oocysts, four sporozoites were visible[22]. 3. Immunochromatographic test (ICT): Fresh samples were also processed using RIDA® QUICK Cryptosporidium test (Biopharm, Germany) for the qualitative determination of C. parvum in stool samples. This test is a fast single-step ICT in which the sample was positive if the red and blue bands are visible, the sample was negative if only the blue band is visible and not valid if there were no visible bands. Likewise, changes in band color that appear after 10 minutes or later were also without any diagnostic value and not used for evaluation. 4. Enzyme linked immunosorbent assay (ELISA): Part of each frozen fecal specimens was processed for detection of Cryptosporidium copro-antigen using RIDASCREEN® Cryptosporidium ELISA (Cryptosporidium-C1201, Germany) according to the manufacturer’s instructions[23]. In order to establish the cut-off point, extinction units (0.15) were added to the measured extinction for the negative control. The specimen was considered positive if the extinction rate was 10% higher than the calculated cut-off value, while samples with extinctions 10% less than calculated cut-off were considered negative. 5. Nested PCR (nPCR)[24,25]: In this study, nPCR was considered the gold standard method for diagnosis of cryptosporidiosis[20,21]. Genomic DNA extraction from stool samples was done using FavorPrepTM Table 1. The thermocycler program for nPCR reactions. Steps Process Primary reaction Secondary reaction Cycle number Temp. Time Temp. Time 1 2 3 4 5 6 Initial denaturation Denaturation Annealing Extension Final extension Infinite 95°C 94°C 63°C 72°C 72°C 4°C 4 min 1 min 1 min 1 min 10 min ∞ 95°C 94°C 54°C 72°C 72°C 4°C 4 min 50 sec 30 sec 50 sec 10 min ∞ 1 35 35 35 1 -Cryptosporidiosis in elderly patients Amin et al., 79 stool DNA isolation Mini Kit (Cat. No. FASTI 001, Favorgen Biotech corporation Ping-Tung, Taiwan). Extracted copro-DNA was amplified by nPCR (Table 1) targeting the gene encoding Cryptosporidium oocyst wall protien (COWP). Primers used to amplify 796 bp fragment were BCOWPF (5́-ACC GCT TCT CAA CAA CCA TCT TGT CCT C-3̀) and BCOWPR (5́CGC ACC TGT TCC CAC TCA ATG TAA ACC C-3̀)[24]. Nested primers to amplify 553 bp fragment were Cry-15 (5́-GTA GAT AAT GGA AGA GAT TGT G-3̀) and Cry-9 (5-GGA CTG AAA TAC AGG CAT TAT CTT G-3̀)[25]. The volume of reaction in the 1ry and 2ry reactions was 25 μl including 12.5 μl PCR master mix, 0.1 μl Taq polymerase, forward and reverse primer 1 μl each, 2 μl templet DNA and distilled water up to 25 μl. Reagents used in the 1ry and 2ry reactions were similar, except for the templet DNA. The amplified amplicons of 553 bp generated from the secondary reaction were subjected to 1.5% agarose gel electrophoresis and visualized by a UV trans-illuminator after being stained with ethidium bromide[24,25]. II. Genotyping: Positive samples by nPCR were then subjected to RFLP with restriction enzyme cleavage Rsa I to determine Cryptosporidium genotypes. The reaction was performed in a volume of 30 μl. Included reagents were 10 μl PCR product (Target DNA), 17 μl Nuclease-free water, 2 μl Green buffer and 1 μl Rsa I Enzyme. Genotype 1 was considered if Rsa I digestion resulted in 4 bands: 34 bp, 106 bp, 125 bp and 285 bp, and genotype 2 if Rsa I digestion resulted in 3 bands: 34 bp, 106 bp and 410 bp. III. Measurement of copro-level of TNF-α: This was measured by an ELISA method (Eagle Biosciences, KR9610, USA) using fecal samples according to the manufacturer's instructions[26]. Statistical analysis: Data were coded and entered using the statistical package SPSS version 25. Data were processed using mean, standard deviation, median, minimum, and maximum in quantitative data and using frequency (count) and relative frequency (percentage) for categorical data. Comparisons between quantitative variables were done using the non-parametric MannWhitney test[27]. For comparing categorical data, Chi square (X2) test was performed. Fisher exact test was used instead when the expected frequency was less than five[28]. Standard diagnostic indices including sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and diagnostic efficacy were calculated as described elsewhere[29]. Statistical significance was considered when P value was lower than 0.05. Ethical consideration: Participants in this study were informed of the aim of this study and a consent was taken from each. The study followed the Ethical Guidelines of Faculty of Medicine; Cairo University Institutional which agreed with the 1964 Helsinki Declaration. Cryptosporidium spp. infected cases were informed and treated.

parasitologists united journal

Detection rate and genotyping of Cryptosporidium spp. and its relation to copro TNF-α in elderly Egyptians attending outpatient clinics of Cairo University Hospitals