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Salivary antibodies as markers of recent acute cryptosporidiosis in children under the age of 5 in Zambia

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Michelo Simuyandi
Michelo Simuyandi

This document outlines a study to evaluate salivary antibodies as markers for recent Cryptosporidium infection in children under 5 years old in Zambia. It proposes a case-control study comparing saliva samples to standard diagnostic methods using serum and stool samples. The study aims to assess the sensitivity and specificity of salivary antibodies for detecting recent Cryptosporidium infection up to 6 months prior. It also proposes a retrospective study using existing saliva samples from a cohort study to examine antibody profiles over time and associations with other factors.

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Salivary antibodies as markers of recent acute cryptosporidiosis in children under the age of 5 in Zambia upgrading presentation by MICHELO SIMUYANDI 15th February, 2013 Department of Disease Control, Faculty of Infectious & Tropical Diseases SUPERVISOR: JOE BROWN
Outline for presentation Introduction Diarrhoeal disease and Cryptosporidium Emerging results from GEMS Current methods and practices used in diagnostics and surveillance Suggested alternative methods Saliva Literature review Previous uses of salivary assays Problem statement Research questions
Outline contd Part 1: Case-control study Aims and objectives Covariates Sample size calculation Collection, processing, and analytical Plan for data analysis Retrospective study Description and plan of analysis Work to date, logistics
Introduction: burden of disease An estimated 10% of deaths in children in 2010 were attributed to diarrhoea Liu et al. (2012) most preventable by WASH Highest mortality among malnourished and HIV+ in Zambia 67.3% (261/388) of children with SAM had diarrhoea with an OR 2.5 of mortality over those with adequate nutrition (Zambia, Irena et al. 2011) In peri urban Lusaka, 35% of the children have HIV and severe opportunistic infections with 76% if them classified as stunted (unpublished data personal communication)
Liu et al. 2012
Introduction: Crypto Global Enteric Multi-Center Survey (GEMS): Cryptosporidium spp. among top aetiologic agents of diarrhoea in children Associated with mortality in children, particularly <2 Much higher priority than previously thought Call for simple rapid diagnostics Limitations of current methods In Zambia: prevalence of 6% - 30.7%, highest in children and HIV+ children with severest outcomes (Amadi et al (2001), Nchito et al. (1998), Siwila et al (2010) and Siwila et al. (2011)
Salivary antibodies as markers of recent acute cryptosporidiosis in children under the age of 5 in Zambia
Next 3 slides from the GEMS study http://www.slideshare.net/PGPR/the-global-enteric-multicenter-study-gems- etiology-burden-of-moderate-severe-diarrheal-disease-in-africa-asia
Salivary antibodies as markers of recent acute cryptosporidiosis in children under the age of 5 in Zambia
Salivary antibodies as markers of recent acute cryptosporidiosis in children under the age of 5 in Zambia
Salivary antibodies as markers of recent acute cryptosporidiosis in children under the age of 5 in Zambia
Current methods: good for diagnostics, bad for field surveillance/monitoring Stool: low compliance Serum: multiple needle sticks usually not possible Saliva: promising alternative Other outcome measures used in surveillance Self-report: highly subject to bias WAZ: may not be specific to diarrhoea
Current methods Sample Methods Strengths Limitations Stool Microscopy Cheap easy to Less sensitive use in field can not distinguish C.parvum from setting C.hominis Affected by intermittent shedding Labour intensive Highly specialised analyst Low compliance in community settings Stool Immuno- Very sensitive Cost, labour intensive, highly labelling specialised analyst Low compliance in community settings Stool PCR/RT-PCR Very sensitive Cost, standardisation, variability of results due to choice of DNA/RNA extraction methods, choice of primers and detection probes, needs validation Low compliance in community settings
Current methods Sample Methods Strengths Limitations Blood ELISA Very sensitive Invasiveness of sample collection (serum) Blood PCR/RT-PCR Very sensitive Invasiveness of sample collection, Cost, (serum) standardisation, variability of results due to choice of DNA/RNA extraction methods, choice of primers and detection probes, needs other methods for validation Reported Interviews, Cheap and easy Potential for bias, observer induced diarrhoea diaries behaviour modification, captures limited information on cause
Alternative: saliva Sample Methods Strengths Questions/Limitations Saliva ELISA Sensitive, specific Low conc. of protein of interest Easy to collect Variability due to physiological sample changes in body Lack of consensus on collection and post collection processing and storage Do markers stay elevated for long enough for this to be used in surveillance?
Knowns All the three classes of immunoglobulin (IgA, IgM and IgG) have been measured in saliva Total recoverable dependent on many factors Variety of pathogens: protozoa, viruses, bacteria, fungi Collection, processing, and storage conditions reported (though little consensus) Has been piloted in proof-of-concept studies in the USA in multi-plex format (on adults only) Giardia and non-WASH related pathogens have been tested in clinical diagnostic applications in lower-income countries
From Griffin et al 2011 (USEPA) Griffin et al. 2011
Problem statement Reliable, objective, persistent biomarker needed for tracking diarrhoeal diseases Surveillance and intervention studies Salivary antibody measures present potential advantages over available alternatives Ease of collection and processing No studies of salivary antibodies for tracking diarrhoeal diseases have been conducted in a lower-income setting Immune response may be very different Focus on incident cases in children
Approach: two studies A case-control study comparing saliva with standard methods (serum antibodies, stool samples) in children over 3-60months(study 1) Cases: confirmed Crypto infection, symptomatic Controls: confirmed seronegative for Crypto A retrospective study of saliva samples from a longitudinal cohort study (study 2) Existing stored samples of saliva from 482 persons Associated data: stool parasites, self-report, WAZ, and extensive WSH exposure-related data
Case-control study
Research questions Primary Do salivary antibodies (sIgA and IgG) correlate with serum antibody measures specific to Cryptosporidium spp. in acute cryptosporidiosis patients? Can salivary antibody measures indicate cryptosporidiosis in the recent past (up to 6 months following clinical presentation)?
Aims Primary To assess the utility of salivary antibody measures in indicating recent Cryptosporidium spp. infection up to 6 months following clinical presentation in children under 5 Secondary To assess growth measures (IGF-1, WAZ) in acute cryptosporidiosis patients less than 5 years up to 6 months post clinical presentation
Objectives To determine the sensitivity and specificity of salivary antibody measures in Cryptosporidium spp. diagnosis To examine the associations between salivary immunological response and the following co- factors: age, sex, HIV status, major co-infections, malnutrition, breastfeeding, and anthropometric data and IGF-1 in study participants To asses salivary antibody profiles in children less than 5 years up to 6 months post clinical presentation with Cryptosporidium spp. infection PERSISTANCE OF THE ANTIBODY RESPONSE
Objectives contd To estimate the prevalence of Cryptosporidium spp. infection in children under 5 years presenting at the University Teaching Hospital in Lusaka Data collected during recruitment To compare the effects of storage temperature, time and post collection processing on amount of recoverable total antibody Methods development (before recruitment) To propose a method for saliva collection, processing and storage for field studies in Zambia Production of simple, brief guidelines for use and further development of assays
Overview of case-control study CASES: 200 patients with cryptosporidiosis (confirmed by stool samples and symptomatic) under 5 years of age CONTROLS: 200 matched controls (seronegative, no Cryptosporidium spp. infection based on stools), matching based on HIV status, age within 1 year Cases and controls from paediatric out-patient admission centre of UTH and followed up from their respective homes or hospital if admitted MEASURING: stool, saliva, serum, WAZ, IGF-1, all covariates SAMPLE POINTS: t = 0 (enrolment), monthly up to t=6 months
Matching on HIV status & age HIV & associated factors Cryptosporidium spp. infection Antibody levels Age & associated factors
Covariates Justification Reference Reduction in the total antibody (Skott et al. 1999) HIV infection production (Brandtzaeg 2007) Variation of sIgA from 0 to 3months (Gleeson et al. 1995) Age of age and reported consistent up to 4 years, age related immunity has been reported Inflammation caused by any gut (Hieshima et al. 2003) Co-infections (Giardia infection will stimulate production of ,Ascaris, Malaria, Shistosoma total sIgA and IgG mansoni, Roravirus, Salmonella) Vitamin A has been known to (Ross 2012) Nutritional status mediate immune response including mediation of inflammation which has an effect on mucosal immunity
Covariates Justification Reference Presence of Cryptosporidium spp. (Korpe et al. 2013) Breastfeeding Specific IgA antibodies in breast milk is protective against infection Depending on type, altered natural Parent administered flora can affect the levels of medication antibodies and response Nitazoxanide is not effective against (Beatrice Amadi et al. Treatment given by cryptosporidium in immune 2009b; Beatrice Amadi health centre compromised patients but effective et al. 2002) in immune competent Inflammation or colonization may (Lycke et al. 2013) Recent vaccination cause affect the expression levels of antibodies
Sample size calculation Covariate % Sample size 50 HIV 10 200 45 Number of children enrolled in Breastfeeding 40 25 40 35 30 Recent 50 15 each group vaccination 25 Power 20 70% Nutritional 50 15 status 15 80% 10 90% Co-infection 20 50 5 0 25% 50% 75% Detectable difference in means alpha = 0.05, SD = mean, assume antibody responses are normally distributed
Work Pilot and validation of methods Pilot Recruitment , screening ,consent Cross section and enrolment study Controls(matched by age and Cases sex) Receive standard care Standard care Monthly Follow Paired samples of Monthly Follow Controls that sero-convert or have +ve saliva & stool will be moved to cases blood, stool up up Anthropometrics Case-control study ELISA and microscopy screening for parasites on Outcome monthly samples measures Total controls at end of study Total cases at end of study Data analysis
Stool screening http://www.mayomedicallaboratories.com/articles/hottopics/transcripts/2009/2009-3a-intestinal/2009-3a-intestinal.html
Serum For HIV testing if the child has not been tested CD4+ count and other differentials (at first visit only) Malaria For serum for Cryptosporidium spp. specific antibodies ELISA tests
ELISA Cryptosporidium antigen Crypto spp. IGF-1
Data analysis Still in development Taking SME Antibody response: comparison of means Stratified by major co-variates Regression analysis to identify influence of co- variates on salivary antibody response Persistence of marker At what point is the signal no longer there Also stratified by major co-variates
Retrospective study of salivary samples from existing longitudinal cohort study
Overview Our team carried out a longitudinal cohort study from September 2011 to October 2012 (with planned follow up in May 2013) in an urban community in Lusaka, Zambia Primary purpose of this study is to evaluate the use and effectiveness of a novel water quality intervention in an HIV- impacted population We therefore have access to a wide range of water, sanitation, and hygiene exposure data as well as key health outcome data (stool, self- report, anthropometrics) from 2,364 individuals
Archived saliva samples Saliva samples from 482 people over the course of the study collected 123 by expectoration, placed on ice for transportation and stored at -80oC 259 samples collected using an oral swab, placed on ice for transportation and stored at- 80oC We plan to collect more in May 2012
Self-report data The primary self-report health outcome measure we used was Highly Credible Gastrointestinal Illness (HCGI) (Payment et al. 1997; Hellard et al. 2001; Colford et al. 2002; Colford et al. 2005) Cases are defined as any of the following: (i) vomiting, (ii) watery diarrhoea, (iii) soft diarrhea and abdominal cramps, or (iv) nausea and abdominal cramps. 7 day, 48 hour, 24 hour recall at multiple time points
Clinic-based surveillance Nurse practitioner in clinic assigned to study cohort Free clinic within 30 minutes walk from all households Cases encouraged to report for diagnosis and treatment on a voluntary basis throughout surveillance period
Anthropometrics and stool All children under 5 years of age were measured for height and weight to calculate weight-for-age (WAZ) and height-for-age (HAZ) z-scores to classify wasting, stunting, and underweight, respectively. Stool samples taken from volunteers for analysis Multiple time points
Primary question Do adults and children with confirmed Cryptosporidium infection show significantly higher Cryptosporidium-specific salivary antibody titres compared with stool-negative controls? Secondary question Is measured salivary IGF-1 in children significantly reduced at low WAZ or reported/clinically confirmed diarrhoea prevalence?
Data analysis Plan still in development Primary question: comparison of means in antibody response between those shedding oocysts and others Secondary question: correlation in IGF-1 (outcome measure) with anthropometrics, self-reported diarrhoea, and associated WSH exposure variables through regression analysis
Work to date Collection of samples for retrospective study Existing Ethics covers analysis of these samples Systematic literature review on methods and existing knowledge of these metrics Secured office space at the UTH, laboratory space and all equipment (ELISA plate reader and washer, FACS machine and a microscope) that we need for the study has been offered to us the Tropical Gastroenterology and Nutrition Group (TORPGAN) Secured funding for the study
Filling knowledge and skills gaps I am currently taking DL courses at LSHTM (Analysis and Design of Research Studies and Statistical Methods in Epidemiology) Author aid scientific writers workshop Online Ethics course by NIH Assembled my advisory committee and local advisory team
Acknowledgements NIH for the funding LSHTM Environmental Health Group TROPGAN for laboratory and office space Advisory committee Name of member Affiliated Institutions 1 Dr.Paul Kelly Barts and London and TROPGAN 2 Prof. Ian Sanderson Barts and London 3 Dr. Beatrice Amadi University Teaching Hospital and TROPGAN 4 Mellissa Kapulu University of Zambia, and Biological Sciences Department, Jenner Institute University of Oxford
Thank you all for listening
No consensus on the best collection method for maximum antibody yield
Evaluation of saliva sample collection and processing (pilot methods development work) t0=2hrs t1=24hrs t2=48hrs t3=7days t4=14days Test for total immunoglobulin at the 5 time points Samples from healthy adult and compared amongst the different volunteers stored in four aliquots at different time points temperatures(28oC, 4oC, -4oC and - 80oC)
Other data Height and weight to calculate weight-for-age (WAZ), height- for-age (HAZ), and weight-for-height (WHZ) z-scores to classify wasting, stunting, and underweight Major co-infections identified at enrolment Some may be identified later through stool sampling (e.g., parasites) Other relevant household and individual characteristics will be recorded during home visitation Breastfeeding Other major covariates Factors affecting saliva production (time since eating, etc) Self-report health data collected from caregiver at each visit
IGF-1 Linear growth IGF-1 Diarrhoea Zinc
Stool screening data 25 20 15 10 5 0

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Salivary antibodies as markers of recent acute cryptosporidiosis in children under the age of 5 in Zambia

  • 1. Salivary antibodies as markers of recent acute cryptosporidiosis in children under the age of 5 in Zambia upgrading presentation by MICHELO SIMUYANDI 15th February, 2013 Department of Disease Control, Faculty of Infectious & Tropical Diseases SUPERVISOR: JOE BROWN
  • 2. Outline for presentation Introduction Diarrhoeal disease and Cryptosporidium Emerging results from GEMS Current methods and practices used in diagnostics and surveillance Suggested alternative methods Saliva Literature review Previous uses of salivary assays Problem statement Research questions
  • 3. Outline contd Part 1: Case-control study Aims and objectives Covariates Sample size calculation Collection, processing, and analytical Plan for data analysis Retrospective study Description and plan of analysis Work to date, logistics
  • 4. Introduction: burden of disease An estimated 10% of deaths in children in 2010 were attributed to diarrhoea Liu et al. (2012) most preventable by WASH Highest mortality among malnourished and HIV+ in Zambia 67.3% (261/388) of children with SAM had diarrhoea with an OR 2.5 of mortality over those with adequate nutrition (Zambia, Irena et al. 2011) In peri urban Lusaka, 35% of the children have HIV and severe opportunistic infections with 76% if them classified as stunted (unpublished data personal communication)
  • 5. Liu et al. 2012
  • 6. Introduction: Crypto Global Enteric Multi-Center Survey (GEMS): Cryptosporidium spp. among top aetiologic agents of diarrhoea in children Associated with mortality in children, particularly <2 Much higher priority than previously thought Call for simple rapid diagnostics Limitations of current methods In Zambia: prevalence of 6% - 30.7%, highest in children and HIV+ children with severest outcomes (Amadi et al (2001), Nchito et al. (1998), Siwila et al (2010) and Siwila et al. (2011)
  • 8. Next 3 slides from the GEMS study http://www.slideshare.net/PGPR/the-global-enteric-multicenter-study-gems- etiology-burden-of-moderate-severe-diarrheal-disease-in-africa-asia
  • 12. Current methods: good for diagnostics, bad for field surveillance/monitoring Stool: low compliance Serum: multiple needle sticks usually not possible Saliva: promising alternative Other outcome measures used in surveillance Self-report: highly subject to bias WAZ: may not be specific to diarrhoea
  • 13. Current methods Sample Methods Strengths Limitations Stool Microscopy Cheap easy to Less sensitive use in field can not distinguish C.parvum from setting C.hominis Affected by intermittent shedding Labour intensive Highly specialised analyst Low compliance in community settings Stool Immuno- Very sensitive Cost, labour intensive, highly labelling specialised analyst Low compliance in community settings Stool PCR/RT-PCR Very sensitive Cost, standardisation, variability of results due to choice of DNA/RNA extraction methods, choice of primers and detection probes, needs validation Low compliance in community settings
  • 14. Current methods Sample Methods Strengths Limitations Blood ELISA Very sensitive Invasiveness of sample collection (serum) Blood PCR/RT-PCR Very sensitive Invasiveness of sample collection, Cost, (serum) standardisation, variability of results due to choice of DNA/RNA extraction methods, choice of primers and detection probes, needs other methods for validation Reported Interviews, Cheap and easy Potential for bias, observer induced diarrhoea diaries behaviour modification, captures limited information on cause
  • 15. Alternative: saliva Sample Methods Strengths Questions/Limitations Saliva ELISA Sensitive, specific Low conc. of protein of interest Easy to collect Variability due to physiological sample changes in body Lack of consensus on collection and post collection processing and storage Do markers stay elevated for long enough for this to be used in surveillance?
  • 16. Knowns All the three classes of immunoglobulin (IgA, IgM and IgG) have been measured in saliva Total recoverable dependent on many factors Variety of pathogens: protozoa, viruses, bacteria, fungi Collection, processing, and storage conditions reported (though little consensus) Has been piloted in proof-of-concept studies in the USA in multi-plex format (on adults only) Giardia and non-WASH related pathogens have been tested in clinical diagnostic applications in lower-income countries
  • 17. From Griffin et al 2011 (USEPA) Griffin et al. 2011
  • 18. Problem statement Reliable, objective, persistent biomarker needed for tracking diarrhoeal diseases Surveillance and intervention studies Salivary antibody measures present potential advantages over available alternatives Ease of collection and processing No studies of salivary antibodies for tracking diarrhoeal diseases have been conducted in a lower-income setting Immune response may be very different Focus on incident cases in children
  • 19. Approach: two studies A case-control study comparing saliva with standard methods (serum antibodies, stool samples) in children over 3-60months(study 1) Cases: confirmed Crypto infection, symptomatic Controls: confirmed seronegative for Crypto A retrospective study of saliva samples from a longitudinal cohort study (study 2) Existing stored samples of saliva from 482 persons Associated data: stool parasites, self-report, WAZ, and extensive WSH exposure-related data
  • 21. Research questions Primary Do salivary antibodies (sIgA and IgG) correlate with serum antibody measures specific to Cryptosporidium spp. in acute cryptosporidiosis patients? Can salivary antibody measures indicate cryptosporidiosis in the recent past (up to 6 months following clinical presentation)?
  • 22. Aims Primary To assess the utility of salivary antibody measures in indicating recent Cryptosporidium spp. infection up to 6 months following clinical presentation in children under 5 Secondary To assess growth measures (IGF-1, WAZ) in acute cryptosporidiosis patients less than 5 years up to 6 months post clinical presentation
  • 23. Objectives To determine the sensitivity and specificity of salivary antibody measures in Cryptosporidium spp. diagnosis To examine the associations between salivary immunological response and the following co- factors: age, sex, HIV status, major co-infections, malnutrition, breastfeeding, and anthropometric data and IGF-1 in study participants To asses salivary antibody profiles in children less than 5 years up to 6 months post clinical presentation with Cryptosporidium spp. infection PERSISTANCE OF THE ANTIBODY RESPONSE
  • 24. Objectives contd To estimate the prevalence of Cryptosporidium spp. infection in children under 5 years presenting at the University Teaching Hospital in Lusaka Data collected during recruitment To compare the effects of storage temperature, time and post collection processing on amount of recoverable total antibody Methods development (before recruitment) To propose a method for saliva collection, processing and storage for field studies in Zambia Production of simple, brief guidelines for use and further development of assays
  • 25. Overview of case-control study CASES: 200 patients with cryptosporidiosis (confirmed by stool samples and symptomatic) under 5 years of age CONTROLS: 200 matched controls (seronegative, no Cryptosporidium spp. infection based on stools), matching based on HIV status, age within 1 year Cases and controls from paediatric out-patient admission centre of UTH and followed up from their respective homes or hospital if admitted MEASURING: stool, saliva, serum, WAZ, IGF-1, all covariates SAMPLE POINTS: t = 0 (enrolment), monthly up to t=6 months
  • 26. Matching on HIV status & age HIV & associated factors Cryptosporidium spp. infection Antibody levels Age & associated factors
  • 27. Covariates Justification Reference Reduction in the total antibody (Skott et al. 1999) HIV infection production (Brandtzaeg 2007) Variation of sIgA from 0 to 3months (Gleeson et al. 1995) Age of age and reported consistent up to 4 years, age related immunity has been reported Inflammation caused by any gut (Hieshima et al. 2003) Co-infections (Giardia infection will stimulate production of ,Ascaris, Malaria, Shistosoma total sIgA and IgG mansoni, Roravirus, Salmonella) Vitamin A has been known to (Ross 2012) Nutritional status mediate immune response including mediation of inflammation which has an effect on mucosal immunity
  • 28. Covariates Justification Reference Presence of Cryptosporidium spp. (Korpe et al. 2013) Breastfeeding Specific IgA antibodies in breast milk is protective against infection Depending on type, altered natural Parent administered flora can affect the levels of medication antibodies and response Nitazoxanide is not effective against (Beatrice Amadi et al. Treatment given by cryptosporidium in immune 2009b; Beatrice Amadi health centre compromised patients but effective et al. 2002) in immune competent Inflammation or colonization may (Lycke et al. 2013) Recent vaccination cause affect the expression levels of antibodies
  • 29. Sample size calculation Covariate % Sample size 50 HIV 10 200 45 Number of children enrolled in Breastfeeding 40 25 40 35 30 Recent 50 15 each group vaccination 25 Power 20 70% Nutritional 50 15 status 15 80% 10 90% Co-infection 20 50 5 0 25% 50% 75% Detectable difference in means alpha = 0.05, SD = mean, assume antibody responses are normally distributed
  • 30. Work Pilot and validation of methods Pilot Recruitment , screening ,consent Cross section and enrolment study Controls(matched by age and Cases sex) Receive standard care Standard care Monthly Follow Paired samples of Monthly Follow Controls that sero-convert or have +ve saliva & stool will be moved to cases blood, stool up up Anthropometrics Case-control study ELISA and microscopy screening for parasites on Outcome monthly samples measures Total controls at end of study Total cases at end of study Data analysis
  • 32. Serum For HIV testing if the child has not been tested CD4+ count and other differentials (at first visit only) Malaria For serum for Cryptosporidium spp. specific antibodies ELISA tests
  • 33. ELISA Cryptosporidium antigen Crypto spp. IGF-1
  • 34. Data analysis Still in development Taking SME Antibody response: comparison of means Stratified by major co-variates Regression analysis to identify influence of co- variates on salivary antibody response Persistence of marker At what point is the signal no longer there Also stratified by major co-variates
  • 35. Retrospective study of salivary samples from existing longitudinal cohort study
  • 36. Overview Our team carried out a longitudinal cohort study from September 2011 to October 2012 (with planned follow up in May 2013) in an urban community in Lusaka, Zambia Primary purpose of this study is to evaluate the use and effectiveness of a novel water quality intervention in an HIV- impacted population We therefore have access to a wide range of water, sanitation, and hygiene exposure data as well as key health outcome data (stool, self- report, anthropometrics) from 2,364 individuals
  • 37. Archived saliva samples Saliva samples from 482 people over the course of the study collected 123 by expectoration, placed on ice for transportation and stored at -80oC 259 samples collected using an oral swab, placed on ice for transportation and stored at- 80oC We plan to collect more in May 2012
  • 38. Self-report data The primary self-report health outcome measure we used was Highly Credible Gastrointestinal Illness (HCGI) (Payment et al. 1997; Hellard et al. 2001; Colford et al. 2002; Colford et al. 2005) Cases are defined as any of the following: (i) vomiting, (ii) watery diarrhoea, (iii) soft diarrhea and abdominal cramps, or (iv) nausea and abdominal cramps. 7 day, 48 hour, 24 hour recall at multiple time points
  • 39. Clinic-based surveillance Nurse practitioner in clinic assigned to study cohort Free clinic within 30 minutes walk from all households Cases encouraged to report for diagnosis and treatment on a voluntary basis throughout surveillance period
  • 40. Anthropometrics and stool All children under 5 years of age were measured for height and weight to calculate weight-for-age (WAZ) and height-for-age (HAZ) z-scores to classify wasting, stunting, and underweight, respectively. Stool samples taken from volunteers for analysis Multiple time points
  • 41. Primary question Do adults and children with confirmed Cryptosporidium infection show significantly higher Cryptosporidium-specific salivary antibody titres compared with stool-negative controls? Secondary question Is measured salivary IGF-1 in children significantly reduced at low WAZ or reported/clinically confirmed diarrhoea prevalence?
  • 42. Data analysis Plan still in development Primary question: comparison of means in antibody response between those shedding oocysts and others Secondary question: correlation in IGF-1 (outcome measure) with anthropometrics, self-reported diarrhoea, and associated WSH exposure variables through regression analysis
  • 43. Work to date Collection of samples for retrospective study Existing Ethics covers analysis of these samples Systematic literature review on methods and existing knowledge of these metrics Secured office space at the UTH, laboratory space and all equipment (ELISA plate reader and washer, FACS machine and a microscope) that we need for the study has been offered to us the Tropical Gastroenterology and Nutrition Group (TORPGAN) Secured funding for the study
  • 44. Filling knowledge and skills gaps I am currently taking DL courses at LSHTM (Analysis and Design of Research Studies and Statistical Methods in Epidemiology) Author aid scientific writers workshop Online Ethics course by NIH Assembled my advisory committee and local advisory team
  • 45. Acknowledgements NIH for the funding LSHTM Environmental Health Group TROPGAN for laboratory and office space Advisory committee Name of member Affiliated Institutions 1 Dr.Paul Kelly Barts and London and TROPGAN 2 Prof. Ian Sanderson Barts and London 3 Dr. Beatrice Amadi University Teaching Hospital and TROPGAN 4 Mellissa Kapulu University of Zambia, and Biological Sciences Department, Jenner Institute University of Oxford
  • 46. Thank you all for listening
  • 47. No consensus on the best collection method for maximum antibody yield
  • 48. Evaluation of saliva sample collection and processing (pilot methods development work) t0=2hrs t1=24hrs t2=48hrs t3=7days t4=14days Test for total immunoglobulin at the 5 time points Samples from healthy adult and compared amongst the different volunteers stored in four aliquots at different time points temperatures(28oC, 4oC, -4oC and - 80oC)
  • 49. Other data Height and weight to calculate weight-for-age (WAZ), height- for-age (HAZ), and weight-for-height (WHZ) z-scores to classify wasting, stunting, and underweight Major co-infections identified at enrolment Some may be identified later through stool sampling (e.g., parasites) Other relevant household and individual characteristics will be recorded during home visitation Breastfeeding Other major covariates Factors affecting saliva production (time since eating, etc) Self-report health data collected from caregiver at each visit
  • 50. IGF-1 Linear growth IGF-1 Diarrhoea Zinc

Editor's Notes

  • #23: Secondary aim will be justified later and clarified in the discussion