The efficacy of modified Ziehl-Neelsen (MZN) and antigen detection enzyme linked immunosorbent assay (ELISA) for detection of cryptosporidia in the stool of gastrointestinal cancer Sudanese patients.

The efficacy of modified Ziehl-Neelsen (MZN) and antigen detection enzyme linked immunosorbent assay (ELISA) for detection of cryptosporidia in the stool of gastrointestinal cancer Sudanese patients. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The efficacy of modified Ziehl-Neelsen (MZN) and antigen detection enzyme linked immunosorbent assay (ELISA) for detection of cryptosporidia in the stool of gastrointestinal cancer Sudanese patients. Dafalla Sidig Mohamed Ali Abdullah, Qutoof Hashim Taha, Tayebalasma Mohamed Ahmed Taher, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3856151/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Cryptosporidiosis diagnosis based on demonstration of the parasite microscopically in the stool through modified Ziehl-Neelsen (MZN) or auramine phenol staining techniques. The primary goal of this study was to compare between the efficacy of cryptospridia antigen detection potential in the fecal specimens using ELISA and Oocyst detection by MZN staining methods for the diagnosis of human cryptosporidiosis among some Sudanese patients with gastrointestinal cancer. One hundred and eighty-one patients suspected of having gasterointestinal cancer based on their clinical presentation were enrolled in this study. Cryptosporidiosis was diagnosed using polymerase chain reaction (PCR). MZN staining was conducted for fresh stool samples to detect Cryptosporidium Oocyst. Results: Of 181 cancer suspected patients, 55 (30.5%) were confirmed as having gastrointestinal cancer. Of the 55 cancer patients 41(74.5%) were confirmed as having colorectal cancer, 7 (12.7%) small intestine cancer, 4(7.3%) liver cancer, 3(5.5%) esophageal cancer. Of the confirmed cancer patients MZN identified 24 (43.6%) and ELISA identified 31(56.4%) as having cryptospridia Oocyst and antigens in their fecal samples, respectively. Based on PCR outcome, the infection rates was significantly higher in colorectal cancer patients than in other cancer patients (P = 0.02). Depending on the criteria of ‘true positive’ samples, sensitivity of MZN and ELISA was 81.06% and 94.25% respectively. Conclusions: Fecal antigen ELISA detection test has the advantage of accurate diagnosis of Cryptosporidiosis over MZN staining technique especially in immunocompromised patients. Cryptosporidiosis Cryptosporidium Gastrointestinal cancer Antigen detection Enzyme linked immunosorbent assay Ziehl-Neelsen staining Background Cryptosporidium is enteric protozoan parasite which infect the gastrointestinal tract of humans, animals, and birds [ 1 , 2 ]. The mechanisms by which these parasites influence the gastrointestinal tract of their host is unclear. Gastrointestinal malignancies mortality, accounting for nearly one-third of all cancer incidences worldwide [ 3 – 5 ]. Numerous cancers of the digestive tract have been linked to genetic or epigenetic alterations. Though, infectious organisms are responsible for one-fifth of the incidences of cancer worldwide. As a result, a variety of gastrointestinal malignancies have been linked to a wide spectrum of infectious agents, including some viruses, bacteria, and parasites [ 6 , 7 ]. Globally, Cryptosporidium emerged a significant contributor to diarrheal sickness, particularly in infants and immunocompromised individuals [ 8 , 9 ]. Depending on the host's nutritional and immune status, as well as the infecting species, the range of intestinal infections varies from an asymptomatic carrier state to severe diarrhoea 7 . Self-limiting diarrhoea and transitory gastroenteritis lasting up to 2 weeks are common in immunocompetent individuals and they often recover without medication, indicating a successful host-antiparasite immune response. However, it is frequently accompanied by chronic or cholera-like diarrhoea in immunocompromised people, such as cancer patients who receive immunosuppressive medications, and can be fatal if neglected [ 8 , 9 ]. The conventional method for detection of Cryptosporidium in faeces using either modified Ziehl–Neelson (MZN) or auramine phenol Staining techniques [ 10 ]. However, the sensitivity of these approaches is poor for detecting Cryptosporidium spp [ 10 ]. Polymerase chain reaction (PCR) is regarded as the most reliable technique for differentiating between Cryptosporidium spp. is the polymerase chain reaction (PCR) method [ 11 ]. It is often challenging to detect these protozoa readily using traditional MZN technique, making misdiagnosis unavoidable. This is due to the presence of several acid-fast microorganisms in faeces and other faecal structures that resemble cryptosporidium in size, such as yeast, pollen, and other protozoa like Cyclospora [ 12 ]. Due to this method's drawbacks, reliable detection requires the presence of a high concentration of oocysts per gram of stool and well-trained technician. In Sudan, (MZN) is used widely by the medical practitioners and laboratory personnel. Faecal Ag-capture enzyme linked immunosorbent assay (ELISA) and molecular diagnosis are scarcely used. To our knowledge, there are no studies evaluated the efficacy of cryptosporidium antigen detection in faecal specimens using MZN technique particularly in Sudanese patients with gastrointestinal cancer. Therefore, this study was carried out to (1) detect cryptosporidium oocyst and its specific antigens using MZN and ELISA, respectively, (2) determine the prevalence of Cryptosporidiosis among gastrointestinal cancer patients admitted to Oncology hospital in Khartoum - Sudan, (3) assess the efficacy of ELISA antigen detection test in comparison to MZN stain. In this study molecular identification was used as reference method for diagnosis of cryptosporidiosis. Methods Study design In this clinical cross-sectional study, 181 stool samples from suspected cancer patients referred to Khartoum oncology hospital, included gastrointestinal cancer suspected patients during the study period. The stool specimens used for further investigations. Each stool sample was divided into three parts: one immediately was subjected for wet mount preparation, part of the sample kept at -20˚C for Ag-capture ELISA and still other concentrated before it was subjected for MZN staining and molecular detection. The stool samples were immediately examined using direct preparation to detect cyst, protozoan trophozoites. Wet mount with iodine for glycogen and protozoan cyst nuclei. Concentration of Fecal Samples To achieve a better staining outcome when using the MZN staining approach. The concentration technique was carried out as follows: One gram of feces was emulsified in seven milliliters of 10% formalin, and then three milliliters of ether were added. The mixture was violently shaken after the addition of the ether. In preparation for MZN staining, the sediments were smeared on microscope slides. Modified Zeil-Neelson staining (MZN) technique Stool after being concentrated, stained with modified Zeil-Neelson staining techniques. The stained slides were examined under a light microscope using (40X,100X). Cryptosporidium Fecal Ag-Detection ELISA To detect the presence of cryptosporidium fecal antigen in the stools. DNA extraction and molecular identification With some modification to remove DNA inhibitors as described previously and the manufacturer’s guidelines were precisely followed [ 13 ]. DNA amplification With some modification as described previously and the manufacturer’s guidelines were precisely followed [ 14 ]. Statistical analysis Data analysis were performed using SPSS (version 20.0, IBM, Armonk, NY, USA) and GraphPad prism six software’s. The differences between different groups were assessed by Pearson chi-square (χ2) or Fisher’s exact test where indicated. P < 0.05 was considered statistically significant. Results One hundred and sixty thee (163) cancer suspected patients were included in this study, 55 (30.5%) were confirmed as having gastrointestinal cancer. Of the 55 identified patients 41(74.5%) were confirmed as having colorectal cancer, 7 (12.7%) small intestine cancer, 4(7.3%) liver cancer, 3(5.5%) esophageal cancer. Modified Zeil-Neelson staining (MZN) Twenty-four (14.7%) out of 163 samples were tested positive in the MZN staining technique. Of the 55 confirmed cancer patients, MZN identified cryptosporidium oocyst in the stool of 21 (38.1%) of colorectal cancer, 2(28.6%) of small intestine cancer, and none with liver cancer. Cryptosporidium Fecal Ag-Detection ELISA Of 55 confirmed cancer patients, ELISA identified 31(56.4%), of these, 29 with colorectal cancer and 2 with small intestine cancer) as having cryptospridia Oocyst and antigens in their fecal samples (Table 2 ). Molecular identification (PCR) Patients with colorectal cancer (n = 32; 58.7.3%), followed by individuals with small intestine cancer (n = 3; 5.4%) and liver cancer (n = 1; 1.8%) (Table 1 ). Based on PCR outcome, the infection rates was significantly higher in colorectal cancer patients than in other cancer patients (χ2 = 9.7, df = 1, P = 0.002). Table 1 Occurrences of Cryptosporidium spp. in gastrointestinal cancer patients and controls with different test methods Group Participants n(163) PCR MZN ELISA Χ2/df/P-value + - +( %) + - +(%) + - +(%) Controls 108 3 105 2.7 1 107 0.9 2 106 1.8 GIT cancers Colorectal 41 32 9 78 21 20 51.2 29 12 70.7 9.7/1/0.002 Small Intestine 7 3 3 42.8 2 5 28.6 2 5 42.8 24.0/1/0.003 Liver cancer 4 1 3 25 0 3 0 0 0 0 10.0/1/0.002 Total 143 39 120 24 135 33 123 Comparison between MZN staining and Ag-detection ELISA techniques performance To determine the efficiency of the diagnostic tests under evaluation, PCR was regarded as gold standard because many researchers have reported that its performance was excellent (table 3). There was no significant variation between MZN staining and Ag-detection ELISA test in terms of specificity the predictive values. However, they showed significant differences in terms of sensitivity. In this study, Ag-detection ELISA recorded a higher prevalence of cryptosporidiosis (20.25%) compared to that obtained by MZN (15.9%). Table 2 Diagnostic accuracy of MZN and ELISA for cryptosporidiosis Variables MZN (95% CI) ELISA (95% CI) Prevalence 15.9% 20.25% Sensitivity 78.9%(62.7–90.4) 93.9% (79.8–99.3) Specificity 99.1%(95.6–99.9) 99.2% (95.8–99.9) Positive predictive value 96.8%(80.9–99.5) 96.9%(81.5–99.6) Negative predictive value 93.9%(89.2–96.6) 98.5%(94.4–99.6) Positive likelihood ratio 97.9 (13.8-694.3) 122.7 (17.3–862.1) Negative likelihood ratio 0.21 (0.11–0.39) 0.06(0.02–0.23) Discussion The parasitic protozoa cryptosporidium spp . is the significant cause of extremely dangerous diarrhea particularly in immunocompromised hosts. In Sudan, there is a lack of reports evaluating and comparing diagnostic tests used for diagnosing the intestinal cryptosporidiosis in the community. This may be in part due to the fact that cryptosporidium infection is largely neglected by health care professionals or simply to the inconvenient laboratory management of cryptosporidium detection. Most laboratories use the conventional methods that have low sensitivity such as MZN. Because Gastro Intestinal Tract (GIT) malignancy is best indicator for immunocomprimization associated with the prevalence of cryptosporidiosis, we investigated cryptosporidiosis in GIT cancer patients with chronic diarrhea admitted to the oncology hospital. Participants diagnosed with cancer were classified based on their clinical and test outcomes into those with: colorectal cancer, small intestine cancer, liver cancer. Cancer suspected individuals and who have diarrhea were also included as controls to determine the prevalence of cryptosporidiosis and the effectiveness of each investigation method under study. In this study, molecular identification revealed a significant association between cryptosporidium infection and GIT cancer, with prevalence rate reaching 16.9% among all participants. Our study identified high occurrence of cryptosporidiosis in 32(58.2%) colorectal cancer patients, 3 (42.8%) small intestine cancer cases, and 1 (25%) liver cancer case (p = 0.002) . This is consistent with results reported by several researchers whom found that individuals with colorectal cancers are more likely to have cryptosporidiosis than patients with other GIT malignancies. However, because our study included more individuals with colorectal cancer, the positivity rate was higher in this cohort of patients [ 15 – 19 ]. To evaluate the efficiency of MZN and Ag-detection ELISA and compare their performance in cryptosporidium detection, PCR was regarded as gold standard because many researchers have reported that its performance was excellent [ 20 ]. Microscopic identification of cryptosporidium oocyst showed a lower prevalence rate when compared to molecular identification in the same population. Considering the cancer type, the infection rate was significantly higher in individuals with colorectal cancer (21; 38.2%) than in participants with small intestinal cancer cases (2; 3.6%). The most likely reason is that individual with colorectal cancer execrate more parasites as they pass through short digestive canal. Overall, MZN was found to be 78.9% sensitive with a specificity of 99.1%, which is consistent with findings from earlier studies. The poor sensitivity exhibited by MZN staining is likely due to the presence of a number of acid-fast bacteria in feces and other fecal structures that are similar in size to Cryptosporidium, including yeast, pollen and other protozoa like cyclospora. Another reason is that poor stain absorption by Oocyst occasionally [ 21 – 23 ]. Immunodetection of cryptosporidium antigen revealed a lower prevalence rate in comparison with the molecular identification in the same population. Nevertheless, it was superior to the microscopic identification. Considering the cancer type, the infection rate showed by Ag-detection ELISA was significantly higher in individuals with colorectal cancer (29; 70.7%) than in participants with small intestinal cancer cases (2; 42.8%). Ag-detection ELISA test consistently outperformed the MZN test in terms of sensitivity, but not on terms of specificity, and positive and negative predictive values [ 24 , 25 ]. Conclusions This study concludes that the prevalence of cryptosporidium infection in Sudanese patients is relatively high particularly in individuals whose impaired immune system and presented with chronic diarrhea and, consequently, physicians and laboratory staff need to lend an opening eye for this infection and include it into their daily routine. Abbreviations ELISA Enzyme linked immunosorbent assay GIT Gastro Intestinal Tract MZN Modified Ziehl-Neelsen PCR Polymerase chain reaction SPSS Statistical Packages for Social Sciences Declarations Ethics approval and consent to participate Ethical approval was obtained from the from Ministry of Health Ethical Research Committee in accordance with the Declaration of Helsinki Principles, and the agreement was taken from Dentistry hospital administration before sample and data collection. The aims and objectives of the study along with its procedure, methods, risks and benefits of this study were explained to each participant in easily understandable local language , and written informed consent was taken from each patient. The patient’s information was highly secured and not used for other purposes than scientific inquiry. Ethical clearance code number: MH-RES/17-022-08 Date: 3/11/2022 Consent for publication All authors have reviewed the manuscript and consent to its publication. Availability of data and materials The data sets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not- for- profit sectors. Author’s contribution DSM and QHT and TMA conceived the design and carried out the experiments. ERO and THA and SSA obtained, analysed and interpreted the data. SM and AAI wrote and revised the manuscript. AAI provides financial support for all experiments. All authors have read and approved the manuscript. Acknowledgements Thanks for all participants involved in this research. References Zhao Z, Li X, Zhang N, Li J, Zhao N, Gao M et al (2023) Multiple Regulations of Parasitic Protozoan Viruses: A Double-Edged Sword for Protozoa. mBio 14(1):e0264222 Deng S, He W, Gong AY, Li M, Wang Y, Xia Z et al (2023) Cryptosporidium uses CSpV1 to activate host type I interferon and attenuate antiparasitic defenses. Nat Commun 14(1):1456 Ferlay J, Colombet M, Soerjomataram I et al (2019) Estimating the global cancer incidence and mortality in 2018: GLOBOCAN sources and methods. Int Cancer 144:1941–1953 Abd El-Latif NF, Kandil NS, Shamseya M, Elwany YN, Ibrahim HS (2023) Role of Cryptosporidium spp in Development of Colorectal Cancer. Asian Pac J Cancer Prev 24(2):667–674 Ali MI, Wahab WMAE, Hassan A, Ryan U, Xiao L, Arafa WM, Hamdy DA (2023) Detection of unusual Cryptosporidium parvum subtype in patients with gastrointestinal cancer in Egypt. Parasitol Res 122(2):597–606 Huang Q, Huang S, Kuang W, Yi J, Xiao S, Zhao F, Xiao G (2023) Health risks of Cryptosporidium and Giardia in the application of surface water and septic tank effluent in Chinese agriculture: Impact on cancer patients identified by quantitative microbial risk assessment. Food Microbiol 111:104213 Bauer J, Kaske M, Oehm A, Schnyder M (2023) Dynamics of protozoal excretion in the faeces of calves during the first 28 days after arrival at the fattening farm indicate infection before regrouping and show poor temporal correlation with diarrhoea. Parasit Vectors 16(1):338 Petri WAO, Hossain B, Kabir M, So HH, Moreau GB, Nayak U, Ma JZ, Noor Z, Faruque A, Alum M, Haque R, Petri WA Jr, Gilchrist CA Children develop Immunity to cryptosporidiosis in a high transmission intensity area. medRxiv [Preprint]. 2023 Aug 9:2023.06.28.23292000. Korpe P, Ni Z, Kabir M, Alam M, Ferdous T, Ara R, Munday RM, Haque R, Duggal P (2023) Prospective Cohort Study of Cryptosporidium Infection and Shedding in Infants and Their Households. Clin Infect Dis 76(12):2178–2186 Ahmed SAA, Quattrocchi A, Elzagawy SM, Karanis P, Gad SEM (2023) Diagnostic Performance of Toluidine Blue Stain for Direct Wet Mount Detection of Cryptosporidium Oocysts: Qualitative and Quantitative Comparison to the Modified Ziehl-Neelsen Stain. Diagnostics (Basel) 13(15):2557 Jingyi J, Ping Y, Jian X, Jia C, Xujian M, Qiong L, Bowen T, Fengming W (2023) Efficacy of a membrane concentration method combined with real-time PCR for detection of Giardia and Cryptosporidium in drinking water. Lett Appl Microbiol 76(10):ovad121 Sharma K, Srivastava S, Tak V (2023) Cryptosporidiosis in India and the World: A Review. Infect Disord Drug Targets 23(5):e030423215404 Robinson G, Chalmers RM (2020) Preparation of Cryptosporidium DNA for Whole Genome Sequencing. Methods Mol Biol . ; 2052:129–138 Moreira NC, Cabrine-Santos M, de Oliveira-Silva MB (2020) Evaluation of Different Oocyst DNA Extraction Methods for Cryptosporidium spp. Research in Environmental Samples. Acta Parasitol 65(4):995–998 Sawant M, Baydoun M, Creusy C, Chabé M, Viscogliosi E, Certad G, Benamrouz-Vanneste S (2020) Cryptosporidium and Colon Cancer: Cause or Consequence? Microorganisms 8(11):1665 Certad G (2022) Is Cryptosporidium a hijacker able to drive cancer cell proliferation? Food Waterborne Parasitol 27:e00153 Sulżyc-Bielicka V, Kołodziejczyk L, Jaczewska S, Bielicki D, Safranow K, Bielicki P, Kładny J, Rogowski W (2018) Colorectal cancer and Cryptosporidium spp. infection. PLoS ONE 13(4):e0195834 Taghipour A, Rayatdoost E, Bairami A, Bahadory S, Abdoli A (2022) Are Blastocystis hominis and Cryptosporidium spp. playing a positive role in colorectal cancer risk? A systematic review and meta-analysis. Infect Agent Cancer 17(1):32 Kalantari N, Gorgani-Firouzjaee T, Ghaffari S, Bayani M, Ghaffari T, Chehrazi M (2020) Association between Cryptosporidium infection and cancer: A systematic review and meta-analysis. Parasitol Int 74:101979 Allam AF, El-Taweel HA, Farag HF, Mohamed AFI, Khalil SS, Shehab AY (2021) Performance of formalin ethyl acetate, percoll sedimentation and ELISA for the detection of Cryptosporidium infection in asymptomatic children. J Parasit Dis 45(2):319–323 Ibrahim HS, Shehab AY, Allam AF, Mohamed MA, Farag HF, Tolba MM (2021) Detection and Molecular Identification of Cryptosporidium Species Among Children with Malignancies. Acta Parasitol 66(2):377–383 Aghamolaie S, Rostami A, Fallahi Sh, Tahvildar Biderouni F, Haghighi A, Salehi N (2016) Evaluation of modified Ziehl-Neelsen, direct fluorescent-antibody and PCR assay for detection of Cryptosporidium spp. in children faecal specimens. J Parasit Dis 40(3):958–963 Mohammad SM, Ali M, Abdel-Rahman SA, Moustafa RA, Salama MA (2021) Molecular prevalence of Cryptosporidium isolates among Egyptian children with cancer. J Parasit Dis 45(3):746–753 Abdou NMI, AlAzemi MS, Al-Sayegh MT, Majeed QAH (2022) Performance of diagnostic assays used to detect Cryptosporidium oocysts in faecal samples of cattle in Kuwait and genotyping of Cryptosporidium species. BMC Vet Res 18(1):336 Abdel Gawad SS, Ismail MAM, Imam NFA, Eassa AHA, Abu-Sarea EY (2018) Detection of Cryptosporidium spp. in Diarrheic Immunocompetent Patients in Beni-Suef, Egypt: Insight into Epidemiology and Diagnosis. Korean J Parasitol 56(2):113–119 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3856151","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":267013262,"identity":"6c612ef3-897f-4b25-b743-fa31fd8f09dc","order_by":0,"name":"Dafalla Sidig Mohamed Ali Abdullah","email":"","orcid":"","institution":"National University - Sudan","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Dafalla","middleName":"Sidig Mohamed Ali","lastName":"Abdullah","suffix":""},{"id":267013263,"identity":"ebcd0a9f-228b-423e-bf27-d241c0528e7d","order_by":1,"name":"Qutoof Hashim Taha","email":"","orcid":"","institution":"National University - Sudan","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qutoof","middleName":"Hashim","lastName":"Taha","suffix":""},{"id":267013264,"identity":"173e6f09-f8b2-4219-bb72-21d1ee0d1152","order_by":2,"name":"Tayebalasma Mohamed Ahmed Taher","email":"","orcid":"","institution":"National University - Sudan","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tayebalasma","middleName":"Mohamed Ahmed","lastName":"Taher","suffix":""},{"id":267013265,"identity":"09fff45c-da28-4bc4-9af9-248111c06a64","order_by":3,"name":"Esra Rodwan Omer","email":"","orcid":"","institution":"National University - Sudan","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Esra","middleName":"Rodwan","lastName":"Omer","suffix":""},{"id":267013266,"identity":"b0ec8a53-496e-4014-a476-d10ed9787b15","order_by":4,"name":"Tagwa Hafiz Abdelkabeer Alrasheed","email":"","orcid":"","institution":"National University - Sudan","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tagwa","middleName":"Hafiz Abdelkabeer","lastName":"Alrasheed","suffix":""},{"id":267013267,"identity":"0d69bb81-86e2-477a-93a6-044d79fb41a9","order_by":5,"name":"Sababil Salih Abdalla","email":"","orcid":"","institution":"National University - Sudan","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sababil","middleName":"Salih","lastName":"Abdalla","suffix":""},{"id":267013268,"identity":"7be4e3e4-dd4f-4a3d-ba50-f5df42691e6d","order_by":6,"name":"Salah Marjan","email":"","orcid":"","institution":"National University - Sudan","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Salah","middleName":"","lastName":"Marjan","suffix":""},{"id":267013269,"identity":"14588df4-709b-49fb-8238-18b7cec606f9","order_by":7,"name":"Alkhair Abd Almahmoud Idris","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA50lEQVRIie3Pv+rCMBDA8ZPAZQl2banoK1wpOOqrVASnIIKTODgInXwAfQunzpaAXequCOKfVQc3Bwejjr8fjW4O+W4H+XA5AJvtF+OIAARV5JPXLMpGwt4kdEQOsNAEPyMArdlUvggYiTPGzv7W25Xmm0t6uspGBYEdjusC4irMggn1GW27bUqTtv4YhqEsWqN47AqKkLay7qYJ00SgX0Rqmnh3igRt8icZmQkpXPp6i+tNxZMoMwkUdvwKReQIGdIqyQQywy3VLK5753s0inke7AfJsOnw8eFUeP7f2HfPbTabzfZPD7znQPYZ6O2ZAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-9278-5591","institution":"Ahfad University for Women","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Alkhair","middleName":"Abd Almahmoud","lastName":"Idris","suffix":""}],"badges":[],"createdAt":"2024-01-12 07:53:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3856151/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3856151/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50373514,"identity":"66a24a28-0a55-497e-a879-7a5009152516","added_by":"auto","created_at":"2024-01-30 14:10:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":477139,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3856151/v1/afb3113e-68de-46ed-b415-7306498f9239.pdf"}],"financialInterests":"","formattedTitle":"The efficacy of modified Ziehl-Neelsen (MZN) and antigen detection enzyme linked immunosorbent assay (ELISA) for detection of cryptosporidia in the stool of gastrointestinal cancer Sudanese patients.","fulltext":[{"header":"Background","content":"\u003cp\u003eCryptosporidium is enteric protozoan parasite which infect the gastrointestinal tract of humans, animals, and birds [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The mechanisms by which these parasites influence the gastrointestinal tract of their host is unclear. Gastrointestinal malignancies mortality, accounting for nearly one-third of all cancer incidences worldwide [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNumerous cancers of the digestive tract have been linked to genetic or epigenetic alterations. Though, infectious organisms are responsible for one-fifth of the incidences of cancer worldwide. As a result, a variety of gastrointestinal malignancies have been linked to a wide spectrum of infectious agents, including some viruses, bacteria, and parasites [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Globally, Cryptosporidium emerged a significant contributor to diarrheal sickness, particularly in infants and immunocompromised individuals [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Depending on the host's nutritional and immune status, as well as the infecting species, the range of intestinal infections varies from an asymptomatic carrier state to severe diarrhoea\u003csup\u003e\u003cb\u003e7\u003c/b\u003e\u003c/sup\u003e. Self-limiting diarrhoea and transitory gastroenteritis lasting up to 2 weeks are common in immunocompetent individuals and they often recover without medication, indicating a successful host-antiparasite immune response. However, it is frequently accompanied by chronic or cholera-like diarrhoea in immunocompromised people, such as cancer patients who receive immunosuppressive medications, and can be fatal if neglected [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The conventional method for detection of Cryptosporidium in faeces using either modified Ziehl\u0026ndash;Neelson (MZN) or auramine phenol Staining techniques [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, the sensitivity of these approaches is poor for detecting Cryptosporidium spp [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Polymerase chain reaction (PCR) is regarded as the most reliable technique for differentiating between Cryptosporidium spp. is the polymerase chain reaction (PCR) method [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. It is often challenging to detect these protozoa readily using traditional MZN technique, making misdiagnosis unavoidable. This is due to the presence of several acid-fast microorganisms in faeces and other faecal structures that resemble cryptosporidium in size, such as yeast, pollen, and other protozoa like Cyclospora [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Due to this method's drawbacks, reliable detection requires the presence of a high concentration of oocysts per gram of stool and well-trained technician.\u003c/p\u003e \u003cp\u003eIn Sudan, (MZN) is used widely by the medical practitioners and laboratory personnel. Faecal Ag-capture enzyme linked immunosorbent assay (ELISA) and molecular diagnosis are scarcely used. To our knowledge, there are no studies evaluated the efficacy of cryptosporidium antigen detection in faecal specimens using MZN technique particularly in Sudanese patients with gastrointestinal cancer. Therefore, this study was carried out to (1) detect cryptosporidium oocyst and its specific antigens using MZN and ELISA, respectively, (2) determine the prevalence of Cryptosporidiosis among gastrointestinal cancer patients admitted to Oncology hospital in Khartoum - Sudan, (3) assess the efficacy of ELISA antigen detection test in comparison to MZN stain. In this study molecular identification was used as reference method for diagnosis of cryptosporidiosis.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design\u003c/h2\u003e \u003cp\u003eIn this clinical cross-sectional study, 181 stool samples from suspected cancer patients referred to Khartoum oncology hospital, included gastrointestinal cancer suspected patients during the study period. The stool specimens used for further investigations. Each stool sample was divided into three parts: one immediately was subjected for wet mount preparation, part of the sample kept at -20˚C for Ag-capture ELISA and still other concentrated before it was subjected for MZN staining and molecular detection.\u003c/p\u003e \u003cp\u003eThe stool samples were immediately examined using direct preparation to detect cyst, protozoan trophozoites. Wet mount with iodine for glycogen and protozoan cyst nuclei.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eConcentration of Fecal Samples\u003c/h2\u003e \u003cp\u003eTo achieve a better staining outcome when using the MZN staining approach. The concentration technique was carried out as follows: One gram of feces was emulsified in seven milliliters of 10% formalin, and then three milliliters of ether were added. The mixture was violently shaken after the addition of the ether. In preparation for MZN staining, the sediments were smeared on microscope slides.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eModified Zeil-Neelson staining (MZN) technique\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eStool after being concentrated, stained with modified Zeil-Neelson staining techniques. The stained slides were examined under a light microscope using (40X,100X).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCryptosporidium Fecal Ag-Detection ELISA\u003c/h2\u003e \u003cp\u003eTo detect the presence of cryptosporidium fecal antigen in the stools.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eDNA extraction and molecular identification\u003c/h2\u003e \u003cp\u003eWith some modification to remove DNA inhibitors as described previously and the manufacturer\u0026rsquo;s guidelines were precisely followed [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDNA amplification\u003c/h2\u003e \u003cp\u003eWith some modification as described previously and the manufacturer\u0026rsquo;s guidelines were precisely followed [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData analysis were performed using SPSS (version 20.0, IBM, Armonk, NY, USA) and GraphPad prism six software\u0026rsquo;s. The differences between different groups were assessed by Pearson chi-square (χ2) or Fisher\u0026rsquo;s exact test where indicated. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eOne hundred and sixty thee (163) cancer suspected patients were included in this study, 55 (30.5%) were confirmed as having gastrointestinal cancer. Of the 55 identified patients 41(74.5%) were confirmed as having colorectal cancer, 7 (12.7%) small intestine cancer, 4(7.3%) liver cancer, 3(5.5%) esophageal cancer.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eModified Zeil-Neelson staining (MZN)\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eTwenty-four (14.7%) out of 163 samples were tested positive in the MZN staining technique. Of the 55 confirmed cancer patients, MZN identified cryptosporidium oocyst in the stool of 21 (38.1%) of colorectal cancer, 2(28.6%) of small intestine cancer, and none with liver cancer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCryptosporidium Fecal Ag-Detection ELISA\u003c/h2\u003e \u003cp\u003eOf 55 confirmed cancer patients, ELISA identified 31(56.4%), of these, 29 with colorectal cancer and 2 with small intestine cancer) as having cryptospridia Oocyst and antigens in their fecal samples (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMolecular identification (PCR)\u003c/h2\u003e \u003cp\u003ePatients with colorectal cancer (n\u0026thinsp;=\u0026thinsp;32; 58.7.3%), followed by individuals with small intestine cancer (n\u0026thinsp;=\u0026thinsp;3; 5.4%) and liver cancer (n\u0026thinsp;=\u0026thinsp;1; 1.8%) (Table\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Based on PCR outcome, the infection rates was significantly higher in colorectal cancer patients than in other cancer patients (χ2\u0026thinsp;=\u0026thinsp;9.7, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eOccurrences of \u003cem\u003eCryptosporidium\u003c/em\u003e spp. in gastrointestinal cancer patients and controls with different test methods\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c3\" namest=\"c2\" rowspan=\"2\"\u003e \u003cp\u003eParticipants n(163)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePCR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMZN\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eELISA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eΧ2/df/P-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"1\" nameend=\"c9\" namest=\"c9\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+ - +( %)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+ - +(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+ - +(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"1\" nameend=\"c9\" namest=\"c9\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControls\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e108\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e3 105 2.7\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1 107 0.9\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2 106 1.8\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGIT cancers\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c9\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eColorectal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e41\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e\u003cb\u003e32 9 78\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e21 20 51.2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e29 12 70.7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e9.7/1/0.002\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c9\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSmall Intestine\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e\u003cb\u003e3 3 42.8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e2 5 28.6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e2 5 42.8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e24.0/1/0.003\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c9\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLiver cancer\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e\u003cb\u003e1 3 25\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0 3 0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0 0 0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e10.0/1/0.002\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c9\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e143\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e\u003cb\u003e39 120\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e24 135\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e33 123\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c9\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eComparison between MZN staining and Ag-detection ELISA techniques performance\u003c/h2\u003e \u003cp\u003eTo determine the efficiency of the diagnostic tests under evaluation, PCR was regarded as gold standard because many researchers have reported that its performance was excellent (table 3). There was no significant variation between MZN staining and Ag-detection ELISA test in terms of specificity the predictive values. However, they showed significant differences in terms of sensitivity. In this study, Ag-detection ELISA recorded a higher prevalence of cryptosporidiosis (20.25%) compared to that obtained by MZN (15.9%).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDiagnostic accuracy of MZN and ELISA for cryptosporidiosis\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariables\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMZN (95% CI)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eELISA (95% CI)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePrevalence\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15.9%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20.25%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSensitivity\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e78.9%(62.7\u0026ndash;90.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e93.9% (79.8\u0026ndash;99.3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSpecificity\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e99.1%(95.6\u0026ndash;99.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e99.2% (95.8\u0026ndash;99.9)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePositive predictive value\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e96.8%(80.9\u0026ndash;99.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e96.9%(81.5\u0026ndash;99.6)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNegative predictive value\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e93.9%(89.2\u0026ndash;96.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e98.5%(94.4\u0026ndash;99.6)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePositive likelihood ratio\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e97.9 (13.8-694.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e122.7 (17.3\u0026ndash;862.1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNegative likelihood ratio\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.21 (0.11\u0026ndash;0.39)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.06(0.02\u0026ndash;0.23)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe parasitic protozoa \u003cem\u003ecryptosporidium spp\u003c/em\u003e. is the significant cause of extremely dangerous diarrhea particularly in immunocompromised hosts. In Sudan, there is a lack of reports evaluating and comparing diagnostic tests used for diagnosing the intestinal cryptosporidiosis in the community. This may be in part due to the fact that cryptosporidium infection is largely neglected by health care professionals or simply to the inconvenient laboratory management of cryptosporidium detection. Most laboratories use the conventional methods that have low sensitivity such as MZN.\u003c/p\u003e \u003cp\u003eBecause Gastro Intestinal Tract (GIT) malignancy is best indicator for immunocomprimization associated with the prevalence of cryptosporidiosis, we investigated cryptosporidiosis in GIT cancer patients with chronic diarrhea admitted to the oncology hospital. Participants diagnosed with cancer were classified based on their clinical and test outcomes into those with: colorectal cancer, small intestine cancer, liver cancer. Cancer suspected individuals and who have diarrhea were also included as controls to determine the prevalence of cryptosporidiosis and the effectiveness of each investigation method under study.\u003c/p\u003e \u003cp\u003eIn this study, molecular identification revealed a significant association between cryptosporidium infection and GIT cancer, with prevalence rate reaching 16.9% among all participants. Our study identified high occurrence of cryptosporidiosis in 32(58.2%) colorectal cancer patients, 3 (42.8%) small intestine cancer cases, and 1 (25%) liver cancer case (p\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.002)\u003c/b\u003e. This is consistent with results reported by several researchers whom found that individuals with colorectal cancers are more likely to have cryptosporidiosis than patients with other GIT malignancies. However, because our study included more individuals with colorectal cancer, the positivity rate was higher in this cohort of patients [\u003cspan additionalcitationids=\"CR16 CR17 CR18\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo evaluate the efficiency of MZN and Ag-detection ELISA and compare their performance in cryptosporidium detection, PCR was regarded as gold standard because many researchers have reported that its performance was excellent [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Microscopic identification of cryptosporidium oocyst showed a lower prevalence rate when compared to molecular identification in the same population. Considering the cancer type, the infection rate was significantly higher in individuals with colorectal cancer (21; 38.2%) than in participants with small intestinal cancer cases (2; 3.6%). The most likely reason is that individual with colorectal cancer execrate more parasites as they pass through short digestive canal. Overall, MZN was found to be 78.9% sensitive with a specificity of 99.1%, which is consistent with findings from earlier studies.\u003c/p\u003e \u003cp\u003eThe poor sensitivity exhibited by MZN staining is likely due to the presence of a number of acid-fast bacteria in feces and other fecal structures that are similar in size to Cryptosporidium, including yeast, pollen and other protozoa like cyclospora. Another reason is that poor stain absorption by Oocyst occasionally [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eImmunodetection of cryptosporidium antigen revealed a lower prevalence rate in comparison with the molecular identification in the same population. Nevertheless, it was superior to the microscopic identification. Considering the cancer type, the infection rate showed by Ag-detection ELISA was significantly higher in individuals with colorectal cancer (29; 70.7%) than in participants with small intestinal cancer cases (2; 42.8%). Ag-detection ELISA test consistently outperformed the MZN test in terms of sensitivity, but not on terms of specificity, and positive and negative predictive values [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study concludes that the prevalence of cryptosporidium infection in Sudanese patients is relatively high particularly in individuals whose impaired immune system and presented with chronic diarrhea and, consequently, physicians and laboratory staff need to lend an opening eye for this infection and include it into their daily routine.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eELISA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEnzyme linked immunosorbent assay\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eGIT\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGastro Intestinal Tract\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eMZN\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eModified Ziehl-Neelsen\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ePCR\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePolymerase chain reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eSPSS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eStatistical Packages for Social Sciences\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval was obtained from the from Ministry of Health Ethical Research Committee in accordance with the Declaration of Helsinki Principles, and the agreement was taken from Dentistry hospital administration before sample and data collection.\u003c/p\u003e\n\u003cp\u003eThe aims and objectives of the study along with its procedure, methods, risks and benefits of this study were explained to each participant in easily understandable local language\u003cspan dir=\"RTL\"\u003e,\u003c/span\u003e and written informed consent was taken from each patient. The patient\u0026rsquo;s information was highly secured and not used for other purposes than scientific inquiry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical clearance code number:\u003c/strong\u003e MH-RES/17-022-08\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDate:\u003c/strong\u003e 3/11/2022\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have reviewed the manuscript and consent to its publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data sets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not- for- profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDSM and QHT and TMA conceived the design and carried out the experiments. ERO and THA and SSA obtained, analysed and interpreted the data. SM and AAI wrote and revised the manuscript. AAI provides financial support for all experiments. All authors have read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThanks for all participants involved in this research.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhao Z, Li X, Zhang N, Li J, Zhao N, Gao M et al (2023) Multiple Regulations of Parasitic Protozoan Viruses: A Double-Edged Sword for Protozoa. mBio 14(1):e0264222\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeng S, He W, Gong AY, Li M, Wang Y, Xia Z et al (2023) Cryptosporidium uses CSpV1 to activate host type I interferon and attenuate antiparasitic defenses. Nat Commun 14(1):1456\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFerlay J, Colombet M, Soerjomataram I et al (2019) Estimating the global cancer incidence and mortality in 2018: GLOBOCAN sources and methods. Int Cancer 144:1941\u0026ndash;1953\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbd El-Latif NF, Kandil NS, Shamseya M, Elwany YN, Ibrahim HS (2023) Role of Cryptosporidium spp in Development of Colorectal Cancer. Asian Pac J Cancer Prev 24(2):667\u0026ndash;674\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAli MI, Wahab WMAE, Hassan A, Ryan U, Xiao L, Arafa WM, Hamdy DA (2023) Detection of unusual Cryptosporidium parvum subtype in patients with gastrointestinal cancer in Egypt. Parasitol Res 122(2):597\u0026ndash;606\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang Q, Huang S, Kuang W, Yi J, Xiao S, Zhao F, Xiao G (2023) Health risks of Cryptosporidium and Giardia in the application of surface water and septic tank effluent in Chinese agriculture: Impact on cancer patients identified by quantitative microbial risk assessment. Food Microbiol 111:104213\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBauer J, Kaske M, Oehm A, Schnyder M (2023) Dynamics of protozoal excretion in the faeces of calves during the first 28 days after arrival at the fattening farm indicate infection before regrouping and show poor temporal correlation with diarrhoea. Parasit Vectors 16(1):338\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePetri WAO, Hossain B, Kabir M, So HH, Moreau GB, Nayak U, Ma JZ, Noor Z, Faruque A, Alum M, Haque R, Petri WA Jr, Gilchrist CA Children develop Immunity to cryptosporidiosis in a high transmission intensity area. medRxiv [Preprint]. 2023 Aug 9:2023.06.28.23292000.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKorpe P, Ni Z, Kabir M, Alam M, Ferdous T, Ara R, Munday RM, Haque R, Duggal P (2023) Prospective Cohort Study of Cryptosporidium Infection and Shedding in Infants and Their Households. Clin Infect Dis 76(12):2178\u0026ndash;2186\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhmed SAA, Quattrocchi A, Elzagawy SM, Karanis P, Gad SEM (2023) Diagnostic Performance of Toluidine Blue Stain for Direct Wet Mount Detection of Cryptosporidium Oocysts: Qualitative and Quantitative Comparison to the Modified Ziehl-Neelsen Stain. Diagnostics (Basel) 13(15):2557\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJingyi J, Ping Y, Jian X, Jia C, Xujian M, Qiong L, Bowen T, Fengming W (2023) Efficacy of a membrane concentration method combined with real-time PCR for detection of Giardia and Cryptosporidium in drinking water. Lett Appl Microbiol 76(10):ovad121\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharma K, Srivastava S, Tak V (2023) Cryptosporidiosis in India and the World: A Review. Infect Disord Drug Targets 23(5):e030423215404\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRobinson G, Chalmers RM (2020) Preparation of Cryptosporidium DNA for Whole Genome Sequencing. \u003cem\u003eMethods Mol Biol\u003c/em\u003e. ; 2052:129\u0026ndash;138\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoreira NC, Cabrine-Santos M, de Oliveira-Silva MB (2020) Evaluation of Different Oocyst DNA Extraction Methods for Cryptosporidium spp. Research in Environmental Samples. Acta Parasitol 65(4):995\u0026ndash;998\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSawant M, Baydoun M, Creusy C, Chab\u0026eacute; M, Viscogliosi E, Certad G, Benamrouz-Vanneste S (2020) Cryptosporidium and Colon Cancer: Cause or Consequence? Microorganisms 8(11):1665\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCertad G (2022) Is Cryptosporidium a hijacker able to drive cancer cell proliferation? Food Waterborne Parasitol 27:e00153\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSulżyc-Bielicka V, Kołodziejczyk L, Jaczewska S, Bielicki D, Safranow K, Bielicki P, Kładny J, Rogowski W (2018) Colorectal cancer and Cryptosporidium spp. infection. PLoS ONE 13(4):e0195834\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTaghipour A, Rayatdoost E, Bairami A, Bahadory S, Abdoli A (2022) Are Blastocystis hominis and Cryptosporidium spp. playing a positive role in colorectal cancer risk? A systematic review and meta-analysis. Infect Agent Cancer 17(1):32\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKalantari N, Gorgani-Firouzjaee T, Ghaffari S, Bayani M, Ghaffari T, Chehrazi M (2020) Association between Cryptosporidium infection and cancer: A systematic review and meta-analysis. Parasitol Int 74:101979\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAllam AF, El-Taweel HA, Farag HF, Mohamed AFI, Khalil SS, Shehab AY (2021) Performance of formalin ethyl acetate, percoll sedimentation and ELISA for the detection of Cryptosporidium infection in asymptomatic children. J Parasit Dis 45(2):319\u0026ndash;323\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIbrahim HS, Shehab AY, Allam AF, Mohamed MA, Farag HF, Tolba MM (2021) Detection and Molecular Identification of Cryptosporidium Species Among Children with Malignancies. Acta Parasitol 66(2):377\u0026ndash;383\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAghamolaie S, Rostami A, Fallahi Sh, Tahvildar Biderouni F, Haghighi A, Salehi N (2016) Evaluation of modified Ziehl-Neelsen, direct fluorescent-antibody and PCR assay for detection of Cryptosporidium spp. in children faecal specimens. J Parasit Dis 40(3):958\u0026ndash;963\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMohammad SM, Ali M, Abdel-Rahman SA, Moustafa RA, Salama MA (2021) Molecular prevalence of Cryptosporidium isolates among Egyptian children with cancer. J Parasit Dis 45(3):746\u0026ndash;753\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbdou NMI, AlAzemi MS, Al-Sayegh MT, Majeed QAH (2022) Performance of diagnostic assays used to detect Cryptosporidium oocysts in faecal samples of cattle in Kuwait and genotyping of Cryptosporidium species. BMC Vet Res 18(1):336\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbdel Gawad SS, Ismail MAM, Imam NFA, Eassa AHA, Abu-Sarea EY (2018) Detection of Cryptosporidium spp. in Diarrheic Immunocompetent Patients in Beni-Suef, Egypt: Insight into Epidemiology and Diagnosis. Korean J Parasitol 56(2):113\u0026ndash;119\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cryptosporidiosis, Cryptosporidium, Gastrointestinal cancer, Antigen detection, Enzyme linked immunosorbent assay, Ziehl-Neelsen staining","lastPublishedDoi":"10.21203/rs.3.rs-3856151/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3856151/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground:\u003c/h2\u003e \u003cp\u003eCryptosporidiosis diagnosis based on demonstration of the parasite microscopically in the stool through modified Ziehl-Neelsen (MZN) or auramine phenol staining techniques. The primary goal of this study was to compare between the efficacy of cryptospridia antigen detection potential in the fecal specimens using ELISA and Oocyst detection by MZN staining methods for the diagnosis of human cryptosporidiosis among some Sudanese patients with gastrointestinal cancer. One hundred and eighty-one patients suspected of having gasterointestinal cancer based on their clinical presentation were enrolled in this study. Cryptosporidiosis was diagnosed using polymerase chain reaction (PCR). MZN staining was conducted for fresh stool samples to detect Cryptosporidium Oocyst.\u003c/p\u003e\u003ch2\u003eResults:\u003c/h2\u003e \u003cp\u003eOf 181 cancer suspected patients, 55 (30.5%) were confirmed as having gastrointestinal cancer. Of the 55 cancer patients 41(74.5%) were confirmed as having colorectal cancer, 7 (12.7%) small intestine cancer, 4(7.3%) liver cancer, 3(5.5%) esophageal cancer. Of the confirmed cancer patients MZN identified 24 (43.6%) and ELISA identified 31(56.4%) as having cryptospridia Oocyst and antigens in their fecal samples, respectively. Based on PCR outcome, the infection rates was significantly higher in colorectal cancer patients than in other cancer patients (P\u0026thinsp;=\u0026thinsp;0.02). Depending on the criteria of \u0026lsquo;true positive\u0026rsquo; samples, sensitivity of MZN and ELISA was 81.06% and 94.25% respectively.\u003c/p\u003e\u003ch2\u003eConclusions:\u003c/h2\u003e \u003cp\u003eFecal antigen ELISA detection test has the advantage of accurate diagnosis of Cryptosporidiosis over MZN staining technique especially in immunocompromised patients.\u003c/p\u003e","manuscriptTitle":"The efficacy of modified Ziehl-Neelsen (MZN) and antigen detection enzyme linked immunosorbent assay (ELISA) for detection of cryptosporidia in the stool of gastrointestinal cancer Sudanese patients.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-16 03:48:50","doi":"10.21203/rs.3.rs-3856151/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"da54b84f-3ef0-4587-a34e-7117bc3f17de","owner":[],"postedDate":"January 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-01-30T14:02:17+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-16 03:48:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3856151","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3856151","identity":"rs-3856151","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}