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GLOBVAC-Global helse- og vaksin.forskn

Improved diagnosis of cryptosporidiosis: Evaluation of a low-cost point-of-care cryptosporidiosis test in children in Ethiopia

Alternative title: Improved diagnosis of cryptosporidiosis: Evaluation of a low-cost point-of-care cryptosporidiosis test in children in Ethiopia

Awarded: NOK 3.9 mill.

CRYPTO-POC is a University of Bergen-led PhD project that includes partners at Vestfold Hospital Trust (Norway), Norwegian University of Life Sciences (Norway), Jimma University (Ethiopia) and Copenhagen university (Denmark). The protozoan parasite Cryptosporidium is a major cause of childhood diarrhoea and death. There is no vaccine and only one approved drug, however new and better drugs are in the development pipeline. A test-and-treat strategy will require a reliable and affordable point-of-care test that is appropriate and accurate for use in resource-poor settings. Accuracy and operational feasibility of currently available or newly developed tests will however need to be established on the ground, in the intended clinical place of use. Durable low-power light-emitting diode fluorescence microscopes and auramine-phenol stains (LED-AP) are now commonly available as they have been rolled out for tuberculosis testing in poor countries. The motivating idea behind CRYPTO-POC is that the LED-AP method might serve the dual purpose of both tuberculosis and point-of-care diagnostic testing for cryptosporidiosis. Our primary objective was therefore to estimate the accuracy and operational performance of LED-AP for the diagnosis of cryptosporidiosis in children with diarrhoea. The population under study was children under 5 in an area in Ethiopia with high rates of diarrhoea and malnutrition and is likely to be representative of many globally important high-impact settings. A ?paper dipstick? rapid test with the novelty of not requiring storage in a fridge was also evaluated. From December 2016 to July 2018, 1384 children with diarrhoea were enrolled from Jimma Medical Centre - a regional university hospital - and from Serbo Health Center, in a rural area 22 km east of Jimma. We also enrolled 946 community controls, with no diarrhoea, from two geographically defined catchment areas around both enrolment sites. The core study on diagnostic accuracy of LED-AP and the paper dipstick concluded with a sensitivity close to 90 percent for both methods, while maintaining high specificity. The LED-AP method could easily be integrated with existing tuberculosis diagnostic laboratory infrastructure in low- and middle-income countries and is a much better alternative than the widely used Ziehl-Neelsen method. A publication showing these results is now in press. The project also collected data on local risk factors for diarrhoea and cryptosporidiosis and conducted a 2-month follow-up study to assess the duration of symptoms and parasite excretion in children with cryptosporidiosis. The case-control substudy and the follow-up study will further investigate the relationship between disease severity and malnutrition and other underlying health conditions. Laboratory data collected from the case-control study will also be used for future studies on what other microbes that cause diarrhoea in Ethiopia. An assessment of the prevalence of prolonged and persistent diarrhea in the study population and their cooccurrence with acute malnutrition is already published. It showed that three out of four children with prolonged diarrhea did not have acute malnutrition, but children with prolonged diarrhea present with a different symptomatology than children with acute diarrhea. Further statistical analysis and write-up of the risk factor study and follow-up study is currently ongoing. The CRYPTO-POC-project has so far formed the basis of two other, locally grounded, PhD projects and several smaller projects at the MSc level.

The CRYPTO-POC study was a evaluation of LED-microscopy and a rapid test as diagnostic methods for cryptosporidiosis. This was a field study in an Ethiopian hospital and a rural health centre. These tests can be considered useful in clinical trials of new Cryptosporidium therapeutics and as a diagnostic test in comparable low-income settings where nitazoxanide, the only currently approved drug, is available. Any health facility that has an operational LED microscope and where auramine-phenol stain is available for TB diagnosis should consider establishing standard operating procedures for AP staining and microscopy for Cryptosporidium oocysts. We found a cryptosporidiosis prevalence of 9% by LED-AP in diarrhoea cases, and we believe this is a representative setting for Ethiopia. Detection of Cryptosporidium by LED-AP was strongly associated with diarrhoea. This affordable point-of-care diagnostic method could enable treatment trials for anti-parasitic compounds in Ethiopia.

The protozoan parasite Cryptosporidium is a major cause of childhood diarrhoea and death. There is no vaccine and only one approved drug; however new drugs are under development. A test-and-treat strategy will require a reliable and affordable point-of-care test that is not available at present. We propose that light-emitting diode fluorescence microscopes and auramine-phenol stains (LED-AP) that are currently being rolled out in poor countries can serve the dual purpose of tuberculosis and Cryptosporidium point-of-care diagnostic testing. Our primary objective is therefore to estimate the accuracy and operational performance of LED-AP for the diagnosis of Cryptosporidium in children with diarrhoea in Ethiopia. Inclusion sites are Jimma University Teaching Hospital and a rural Health Centre 55km away in a university-run Health and Demographics Surveillance Site. This setup is ideal for building capacity in diarrhoeal research as there is a long-standing collaboration in malnutrition research between JUTH and University of Copenhagen. CRYPTO-POC is a University of Bergen-led PhD project that includes partners from these established research groups. We will include all children under 5 y that present to the hospital or health center with diarrhoea, aiming to include 1500 children over a 12-month period. Questionnaires (risk factors), clinical data and a stool sample will be collected and examined by both LED-AP and the gold standard (immunofluorescent antibody test) by different examiners in a blinded manner, allowing estimation of diagnostic accuracy. Operational performance will also be assessed. We hypothesise that the sensitivity of LED-AP is at least 70%. A sub-study to assess the longitudinal pattern of Cryptosporidium oocyst shedding will include Cryptosporidium-positive children with and without severe acute malnutrition and is expected to provide valuable insights on the association between oocyst shedding, symptom severity, malnutrition and HIV.

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GLOBVAC-Global helse- og vaksin.forskn