Archive | 2021
Evaluation of nested PCR for diagnosis of Cyclospora cayetanensis in a sample of immunosuppressed and diarrheic patients in Turkey
Abstract
Background: Cyclospora cayetanensis is a food-borne coccidian parasite that causes cyclosporiasis in humans and possibly in other animals. It presents with watery diarrhea and other related symptoms. Since detection of oocysts may be difficult with histological stains, a negative result should not exclude the possibility of C. cayetanensis. PCR methods can achieve more sensitive detection of the parasite. Objective: The presence of C. cayetanensis was investigated in an immunosuppressed patient group, diarrhea patient group, and in both immunosuppressed and diarrhea patient group using the modified acid-fast staining and nested polymerase chain reaction (nPCR) methods. Subjects and Methods: Included in the study were 80 patients with immune suppression, 50 patients with diarrhea, and 70 patients with both immune suppression and diarrhea. The clinical findings of these patients were recorded, stool samples were collected and examined using both the modified acid-fast (AF) staining and nPCR methods. Results: The overall detection rate of C. cayetanensis was 8% and 12% using the modified AF and nPCR, respectively. C. cayetanensis was detected in 5% of immunosuppressed patients, 12%, in patients with diarrhea and 20% in patients with both immune suppression and diarrhea. Statistically significant relationships were identified between the frequency of C. cayetanensis and abdominal pain (P<0.01), nausea (P<0.01), fatigue (P<0.01), diarrhea (P<0.05), and weight loss (P<0.01). Conclusion: nPCR gave a higher rate of cyclosporiasis, and it is more appropriate especially in cases with recurrent prolonged symptoms. PARASITOLOGISTS UNITED JOURNAL 142 disease and that the rates of cyclosporiasis determined in Turkey do not reflect reality. The aim of our study was to investigate the presence of C. cayetanensis in an immunosuppressed non-diarrheic patient group, in diarrheic patient group, and in both immunosuppressed and diarrheic patient group using nPCR and modified AF methods. SUBJECTS AND METHODS This cross-sectional study was conducted in the Parasitology Laboratory of the Dursun Odabas Medical Center of the Van Yuzuncu Yil University, between January 2018 and May 2019. Sample and patient groups: Included in the study were 80 patients with immune suppression, 50 patients with diarrhea, and 70 patients with both immune suppression and diarrhea. Stool samples were collected from these patient groups. Sex, age, patient status, and clinical findings were recorded for each patient. Microscopic stool examination: For identification of C. cayetanensis oocysts fecal suspensions of the formol-ether concentration technique[5] were stained with modified AF staining[6]. The slides were examined under a Leica DM500 microscope (Leica Microsystems, Wetzlar, Germany) at a magnification of X1000. DNA extraction: DNA extraction was performed as described in the GeneMATRIX Stool DNA Purification Kit (Gdańsk, Poland) manual from whole stool samples. The Lyticase enzyme from Arthrobacter Luteus (L2524; Sigma-Aldrich, St. Louis, MO, USA) was used to weaken or break down the oocyst wall before extraction. Enzymes were added to the samples and incubated at 25°C for 15 min. The samples were then incubated at 95°C for 30 min in a dry block heater and vortexed at five-min intervals during the incubation period. All other procedures were carried out according to the kit’s procedure instructions. PCR and electrophoresis: nPCR was performed using the methods and primers specified by Orlandi et al.[7]. In the first stage of the PCR, F1E 5′-TACCCAATGAAAACAGTTT-3′ and R2B 5′-CAGGAGAAGCCAAGGTAGG-3′ were the primers used to amplify the ~636 bp region of the 18S rRNA gene region of Cyclospora and Eimeria species. The reaction was adjusted to a total volume of 50 μL, containing 25 μL of Tag 2x Master Mix (12.5 mM MgCl2), 0.5 mM MgCl2 and 0.2μM of each primer, and 1 μL of sample DNA. Next, 1 μL of the amplicon obtained for the second stage of the nPCR was used. In the second stage of the nPCR, the primers were used to amplify the region of ~298 bp from the 18S rRNA gene region of Cycylospora species. The second nPCR reaction was carried out under the conditions specified in the previous step. Reactions were performed on the Applied Biosystems SimpliAmp Thermal Cycler (Thermo Fisher Scientific Inc., Waltham, MA, USA). The first PCR was programmed for a total of 35 cycles, each at 94°C for 30 s, then 53°C for 30 s, and 72°C for 90 s. nPCR was adjusted for a total of 25 cycles, at 94°C for 15 s and 66°C for 15 s. Since the primary binding temperature in the nPCR phase was close to the activity temperature of the Tag polymerase, no extension temperature (72°C) was required. In both PCR procedures, an additional administration was done at 95°C for 15 min before the first cycle for the denaturation step. Following the last cycle, an extension step at 72°C (66°C for nPCR) for 10 min was performed. To display the results of the nPCR procedure, 15 μL of PCR reaction products was run on agarose gel (1%) electrophoresis and visualized in a UVP Gel documentation system (Ultra-Violet Products Ltd., Upland, CA, USA). Statistical analysis: MINITAB (ver: 14) statistics package program was used for data statistical analysis. The frequency of parasite prevalence was expressed as number and percentage according to the relevant categorical variables. Significance was calculated using the Chi-square (X2) test to compare between quantitative data, and Z-ratio test to compare the rates of noise. Odds values were calculated for the risk of occurrence of parasites. The sensitivity and specificity values of the methods used (e.g., PCR) were calculated to determine its diagnostic efficacy. Statistical significance level was taken as 5% (P<0.05) in calculations and SPSS (IBM SPSS for Windows, Ver. 21) statistics package program was used for calculations. Ethical considerations: Ethical approval was obtained from The Van Yuzuncu Yil University Ethics Committee approved the study (No: 18). Informed consent form was obtained from the patients included in the study.