upgrading presentation by

MICHELO SIMUYANDI15th February, 2013

Department of Disease Control, Faculty of Infectious & Tropical DiseasesSUPERVISOR: JOE BROWN

Salivary antibodies as markers of recent acute

cryptosporidiosis in children under the age of 5 in Zambia

Introduction• Diarrhoeal disease and Cryptosporidium

• Emerging results from GEMS• Current methods and practices used in

diagnostics and surveillance• Suggested alternative methods

• SalivaLiterature review

• Previous uses of salivary assaysProblem statement Research questions

Outline for presentation

Outline cont’dPart 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urban Lusaka, 35% of the children have HIV and severe opportunistic infections with 76% if them classified as stunted (unpublished data personal communication)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)

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

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

Sample Methods Strengths Limitations

Stool Microscopy Cheap easy to use in field setting

Less sensitivecan not distinguish C.parvum from C.hominisAffected by intermittent shedding Labour intensiveHighly specialised analystLow compliance in community settings

Stool Immuno-labelling

Very sensitive Cost, labour intensive, highly specialised analystLow 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 validationLow compliance in community settings

Current methods

Sample Methods Strengths Limitations

Blood(serum)

ELISA Very sensitive Invasiveness of sample collection

Blood(serum)

PCR/RT-PCR Very sensitive Invasiveness of sample collection, Cost, standardisation, variability of results due to choice of DNA/RNA extraction methods, choice of primers and detection probes, needs other methods for validation

Reported diarrhoea

Interviews, diaries

Cheap and easy Potential for bias, observer induced behaviour modification, captures limited information on cause

Current methods

Alternative: salivaSample Methods Strengths Questions/Limitations

Saliva ELISA Sensitive, specificEasy to collect sample

Low conc. of protein of interestVariability due to physiological changes in bodyLack of consensus on collection and post collection processing and storageDo 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 cont’d

• 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 & ageHIV &

associated factors

Antibody levelsCryptosporidium

spp. infection

Age & associated

factors

Covariates Justification Reference

HIV infection Reduction in the total antibody production

(Skott et al. 1999) (Brandtzaeg 2007)

Age Variation of sIgA from 0 to 3months of age and reported consistent up to 4 years, age related immunity has been reported

(Gleeson et al. 1995)

Co-infections

(Giardia ,Ascaris, Malaria, Shistosoma mansoni, Roravirus, Salmonella)

Inflammation caused by any gut infection will stimulate production of total sIgA and IgG

(Hieshima et al. 2003)

Nutritional status Vitamin A has been known to mediate immune response including mediation of inflammation which has an effect on mucosal immunity

(Ross 2012)

Covariates Justification Reference

Breastfeeding Presence of Cryptosporidium spp. Specific IgA antibodies in breast milk is protective against infection

(Korpe et al. 2013)

Parent administered medication

Depending on type, altered natural flora can affect the levels of antibodies and response

Treatment given by health centre

Nitazoxanide is not effective against cryptosporidium in immune compromised patients but effective in immune competent

(Beatrice Amadi et al. 2009b; Beatrice Amadi et al. 2002)

Recent vaccination Inflammation or colonization may cause affect the expression levels of antibodies

(Lycke et al. 2013)

Sample size calculation

Covariate % Sample size

HIV 10 200

Breastfeeding 40 25

Recent vaccination

50 15

Nutritional status 50 15

Co-infection 20 50

25% 50% 75%0

5

10

15

20

25

30

35

40

45

50

70%80%90%

Detectable difference in means alpha = 0.05, SD = mean, assume antibody re-

sponses are normally distributed

Num

ber o

f chi

ldre

n en

rolle

d in

ea

ch g

roup Power

Pilot and validation of methods

Monthly Follow

up

Recruitment , screening ,consent and enrolment

Controls(matched by age and sex)

Standard care

CasesReceive standard care

Total controls at end of study Total cases at end of study

Data analysis

Controls that sero-convert or have +ve stool will be moved to cases

Monthly Follow

up

Case-control studyCross section

study Pilot W

ork

• Paired samples of saliva &

blood, stool• Anthropometric

s• Outcome

measures

ELISA and microscopy screening for parasites on

monthly samples

http://www.mayomedicallaboratories.com/articles/hottopics/transcripts/2009/2009-3a-intestinal/2009-3a-intestinal.html

Stool screening

Serum

For HIV testing if the child has not been testedCD4+ count and other differentials (at first visit only)MalariaFor serum for Cryptosporidium spp. specific antibodies ELISA tests

ELISACrypto spp.IGF-1

Cryptosporidium antigen

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 writer’s 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 t2=48hrs t3=7days t4=14dayst1=24hrs

Samples from healthy adult volunteers stored in four aliquots at different temperatures(28oC,

4oC, -4oC and -80oC)

Test for total immunoglobulin at the 5 time points and compared amongst the different time points

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

DiarrhoeaIGF-1

Zinc

Stool screening data

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