Descriptive Project Title:

Genetic variation in seasonal Cryptosporidial infections within a city in Northeastern Brazil.

Country of Interest:

Brazil

Proposed Dates for project:

May 27, 2007 to August 4, 2007

Describe why you are seeking this global health experience, including ties to personal goals, professional goals and your academic career at UVa. :

I am seeking this global health experience so that I can be introduced to international fieldwork. Academically, there is only so much learning that can be done in the classroom; practical fieldwork experience will allow me to perform real science that will be far more educating and useful to me in the long term. I am a third year student completing a degree in biochemistry, and I have no concrete plans for what I want to do with my degree. This grant presents an excellent opportunity to bring more definition to my post-graduation plans. I cannot decide between attending medical school and attending graduate school to study epidemiology and public health. Additionally, if this international fieldwork experience proves to be fulfilling, it may convince me to take two years off afterwards to join the Peace Corps, something I have long considered doing. In short, I hope that it will change me in some way so that I can make more informed decisions about my post-graduate future. While I believe performing global health research in any setting would be useful, I selected this project principally for the strong academic and research ties between the University of Virginia and the Federal University of Cearà. Their research collaboration has existed for more than 25 years and the laboratory in Fortaleza is well equipped. It also has a support network for potentially confused undergraduates such as me. I would not be a lonely student, set loose with a spiral notebook and a pen on the edge of the Serengeti. This collaboration provides structure and resources that makes completion of a quantitative research project both feasible and minimally stressful.

Host Organization name and supervisor name:

Institute of Biomedecine, Federal University of Cearà, Dr. Reinaldo Oria

Host organization purpose / mission:

The research collaboration between the University of Virginia and the Federal University of Cearà (UFC), is jointly run by Drs. Richard Guerrant and Aldo Lima. The laboratory where the majority of research will be preformed is under the auspices of the Institute of Biomedicine at UFC and the Center for Global Health at UVA. The collaboration's research focus is on enteric diseases, with an emphasis on the long term effects of diarrhea on child development. Current projects range from the testing of potential probiotics in mice to the long term assessment of physical and cognitive ability of children.

Your job description:

I will work within the context of the ongoing study as a laboratory worker under Dr. Reinaldo Oria. The majority of the time will be spent genotyping crypotosporidia samples, though data interpretation and analysis will also be performed. By adding to the knowledge base of the ongoing studies, we hope to either elucidate current questions or inspire new ones. While the majority of my time will be spent in the laboratory, I also intend to assist with some of the projects involving the collection of field data. This way I could spend some time within the community, and not just in a laboratory. I must also emphasize that there is a high likelihood that if granted funding, the actual research performed will not resemble this proposal. If my principle intent is to add to the knowledge base of the ongoing study, then there is no need for strict adherence to this research plan. I am flexible and exist to assist with whichever research questions are deemed most important, and while this proposal may be appropriate and feasible now, it is impossible to know what the needs of the ongoing research projects will be six months from now. Given that this proposal is being submitted in conjunction with another biochemistry student it is our hope that in the succeeding six months the Fortaleza lab can help us identify a research question that we could both research. This way, we could assist each other in the laboratory to answer this question and in the process, actually perform work and collect data that would be useful to the ongoing study. While our current proposals are both feasible, we also want to ensure that our work is of maximal benefit to the ongoing study, and this requires us to wait until we are closer to the summer term.

Description of the target population:

The target population is the pediatric population of Fortaleza Brazil, and indirectly, the developing world. We hope that the performed experiments will improve the understanding of enteric diseases and how they are related to developmental deficiencies in children.

What are the targeted learning objectives associated with your project?:

I intend to quantify the genetic variability (i.e. species and genotype) of Cryptosporidial infections within a collection of frozen pediatric stool samples. This will be accomplished through seven to eight 40 hour weeks of laboratory work in Fortaleza. The remaining two to three weeks will be used to analyze the collected data. By the end of the summer term, the preliminary results will be reported to the ongoing study and a final report will be written the following fall semester

Background or rationale for your learning objective(s):

Diarrheal diseases, which account for ~2 million deaths and millions more disability adjusted life years (DALYs), disproportionately affect the peoples of the developing world where access to sanitation and drinking water is limited. The highest burden of disease is on the weakest member of a community: the old, the immuno-compromised, and most importantly, young children, who account for up to 90% of diarrhea related deaths . The magnitude of diarrhea's burden, though, is not simply defined by the great number of deaths it causes, but also in the long term health outcomes of children who suffer from high rates of diarrhea. These repeated incidences of diarrhea in the first two years of life can result in severe malnutrition and micronutrient deficiencies, which have well documented negative effects on long term physical and mental development , . The magnitude and severity of this long term morbidity has also been underestimated; recent data suggests that the DALYs associated with early childhood diarrhea (ECD) may be two to four times higher than previously thought, resulting in a burden that is greater than that attributed to the number of yearly diarrhea deaths . Additionally, the ongoing University of Virginia-Federal University of Cearà study reported in 2006 that repeated incidents of ECD predict impaired school performance years later . Since the amount of schooling received correlates directly to productivity and socioeconomic success later in life , it is of great importance that in the quest to reduce long term poverty levels, adequate resources be allocated towards reducing the severity and number of ECD events. Clearly, expanded research is needed to help reduce both the short term morbidity of severe diarrhea and also to determine the long term developmental effects of diarrheal disease. Protozoa of the genus Cryptosporidium are one of the main causative agents of ECD, especially in tropical climates such as Brazil. Cryptosporidia generally causes an acute, short term case of diarrhea, but in the young, the cases can linger for weeks, resulting in the aforementioned long term detrimental effects . Cryptosporidiosis is spread by oocysts through the fecal-oral route and infection generally results from fecal ingestion of contaminated food and water, though person to person transmission is also possible. These oocysts are resistant to common chlorine disinfectants and can also survive outside of hosts for months. The genus Cryptosporidium contains many different species and genotype strains that can cause cryptosporidiosis in humans, many of which are non-specific to humans. This means that there can be extensive transmission between humans and other mammalian hosts, particularly from livestock, but also household pets and wildlife . Some of the most common species and genotype strains that cause cryptosporidiosis in humans are C. parvum bovine genotype, C. parvum human genotype, C. hominis, C. meleagridis, C. felis, and C. canis. In Fortaleza, Cryptosporidium infection rates peak during the rainy season (defined as January through May), with approximately 90% of all cryptosporidiosis cases seen in the first six months of the year; this is confirmed by a longitudinal pediatric study affiliated with the ongoing collaboration. In the remainder of the year, months can pass without a single diagnosed case. What no study in Fortaleza has expressly set out to determine, though, is which species and strains are represented in this seasonal spike.

Your hypothesis or question, associated with your objective(s):

Hypothesis: There will be significant variation in speciation and genotypes of Cryptosporidium within the seasonal spike of cases. Independent variable: Date of sample collection. Dependent variables: Species/genotype of Cryptosporidia represented and frequency of cases.

Methodology for data collection / analysis:

Following a postpositivistic approach, one first screens frozen samples for Cryptosporidium oocysts microscopically using either an immunofluorescent assay or the rapid dimethyl sulfoxide-modified acid-fast stain. Cryptosporidial DNA can then be extracted directly from frozen fecal specimens (0.5 g/sample) by a phenol-chloroform extraction. This is followed by nested PCR amplification of the 18S rRNA strand and restriction fragment length polymorphism . Speciation and genotyping can then be determined from the band patterns on the gel electrophoresis plate. Species and frequency data will then be recorded and subjected to statistical analysis.

Benefits: What benefits can reasonably be expected from your project (Benefits may be to the participants, to the knowledge base of the area:

Given that this proposal relies on a limited collection of frozen samples, it will likely function as a prospective study that may inspire future work. The sample size and time available for research are insufficient to provide statistically significant conclusions, which would instead require a longitudinal study. Also, since the majority of Cryptosporidia infections are asymptomatic, testing only diarrheal samples does not provide an accurate picture of infection rates. That said, some inferences could still be made. Simply knowing which species and genotypes are represented would be useful to the study. Since many of the species and strands that cause Cryptosporidium in humans are not human-specific, knowing which species are responsible for the cases could help elucidate the origin of an outbreak or cluster of cases. Strains the reappear yearly would be considered endemic in the community, while cases of rarer zoonotic strains could suggest isolated cases. Additionally, many of the nonhuman strains were originally identified in infections of HIV(+) and other immuno-compromised patients. With increasing frequency though, infections of HIV(-) individuals by these nonhuman strains is being seen. If multiple strains are represented in the frozen pediatric samples, it could inspire a new study to see what the current distribution of cases are and if there have been any significant increases in certain strain infections since these samples were collected (average age of 5 years). In conclusion, my hope is that gaining a better understanding of the genetic variation of Cryptosporidial infections will inspire further investigations.

Risks: What risks can reasonably be expected from your project? Are there any possible physical, psychological, professional or personal risks and/or hazards for the participants? (Please be sensitive regarding potential risks for participants, partic:

In most studies such as this, the safety and privacy of the children would be the chief concern of the researcher. Fortunately, though, these concerns were already addressed when the samples were first collected and frozen. Additionally, patient identities will be masked for the study. For the proposed research there are no foreseeable risks to the children. Risks to personal safety, though, abound. Within the laboratory, there is the risk of infection, along with other chemical safety concerns. These can be easily addressed by strict adherence to lab protocol. Also, Fortaleza is a large city of almost 2.5 million people. My health and physical safety are potentially at risk, particularly since I do not speak Portuguese. These risks can be mitigated by never traveling alone, avoiding dangerous areas of the city, and trying not to stand out, though this is likely more difficult.

What will be done with what you've learning during your project? How will this information be shared with the people in the location where you conduct your project? Do you expect to present, publish or disseminate your findings in a public forum (othe:

Any data collected will be presented to the ongoing study; the hope is that our data may inspire future studies and that the data could eventually be integrated into a publication. Apart from CGH's reporting requirements, I will also be presenting my project to Dr. Demas's introductory chemical research seminar. If it is possible, though, my ultimate intent is to continue my research or related research here at UVA so that I may complete a distinguished major's thesis my fourth year.

If animals are involved, then (online training and following questions):

No animals

Estimated Budget including vaccines, health insurance, evacuation insurance, requisite visas, international airfare, meals and lodging, project supplies and the total that you are requesting from CGH. :

Budget Round Trip Ticket $ 1,600.00 Brazilian Visa $ 180.00 Room and Board (25$/night for 10 weeks) $ 1,800.00 In-country Transportation $ 250.00 Vaccinations/Prophylactic Medicines $ 200.00 $ 4,030.00 Request from CGH $ 3000.00