Background and Rationale:

Chikungunya is a viral disease transmitted by Aedes mosquitoes. Chikungunya virus (CHIKV) is an arbovirus that belongs to the family of Togaviridae and genus Alphavirus. The virus was first discovered in 1952 and used to cause sporadic outbreaks.

In recent years, there have been several reports of large scale outbreaks of Chikungunya virus infection in several parts of Southern India. Over, 2000 cases of Chikungunya fever have also been reported from Malegaon town in Nasik district, Maharashtra state, India between February-March 2006. In October 2009, 2–3 months after an outbreak of a febrile disease with joint pain on the eastern coast of Madagascar, serologic markers for chikungunya virus (CHIKV) in n= 1,244 pregnant women were assessed and IgG sero-prevalence against CHIKV was 45%. The recent outbreaks of this disease have been associated with fatalities and chronic and persistent disability particularly the persistence of joint pain. Chikungunya is thus considered as an important re-emerging public health problem in both tropical and temperate countries, where the distribution of the Aedes mosquito vectors continues to expand.

Given this backdrop, several pharmaceutical companies are in the process of developing vaccines to combat the disease. One such vaccine has been developed by Bharat Biotech and is undergoing clinical development at the KEM Hospital. A Phase I study [a study in normal, healthy participants is currently underway at this institute after regulatory and Ethics Committee approval- EC- PHARMA-10/16].

While screening normal, healthy participants for inclusion into this study the study team found 13/53 [25%] participants to be positive for anti- Chikungunya antibodies by an ELISA test. When questioned, these 13 participants did not give any past history of Chikungunya infection indicating that they suffered from asymptomatic infection. When the team did a literature review of the prevalence of anti-Chikungunya antibodies in the general population, there were no studies. Only three studies on antibody prevalence have been done in the country and these are largely confined to patients who have been hospitalized with a diagnosis of Chikungunya.

A disease like Chikungunya that has seen a re-emergence would benefit from a biomarker that facilitates its diagnosis and helps assess prognosis. Biomarker studies being with an assessment of plasma proteins that are up or down regulated during and after the infection – an analysis that can be done by proteomics- a study of an entire set of proteins that are modified by an organism or system.

Thus, the present study is planned with the objective of carrying out a quantitative proteomic analysis of patients who are positive for anti-Chikungunya antibodies including those from the above-mentioned study (EC/PHARMA-10/16) as well as those who have active infection and analyse proteins that are upregulated or downregulated in them.

Objective:

To assess via a quantitative proteomic analysis the differential expression of proteins in Chikungunya antibody positive patients (symptomatic and asymptomatic) versus age and sex matched healthy controls.

Methods:

Ethics: The study will be submitted to the Institutional Ethics Committee for approval. Written, informed consent will be taken from all participants.  A copy of the submission form will be submitted to the Research Society

Study design: Cross sectional

Sample Size calculation: No formal sample size calculation

Adults: The department is currently doing a study EC/PHARMA-10/2016 where a new Chikungunya vaccine is being tested in normal, healthy participants. A total of n = 25 participants who are asymptomatic but tested positive (>11 standard units), n = 25 who satisfy the confirmed case definition of Chikungunya (NVBDCP) and n = 50 controls who have tested negative (<11 standard units) will be enrolled in this cross-sectional study.

Paediatrics: A total of n = 10 participants who are asymptomatic but tested positive (>11 standard units), n = 10 who satisfy the confirmed case definition of Chikungunya (NVBDCP) and n = 20 controls who have tested negative (<11 standard units) will be enrolled in this cross-sectional study.

Screening: All participants will be screened (via medical history and physical examination only) to establish the eligibility criteria as outlined below. This includes negative antibodies, CHIKV IgG, by ELISA method.

Selection criteria:

Adults: All asymptomatic participants from the Chikungunya vaccine trial (EC/PHARMA-10/16), aged ≥18 and ≤50 years and who are anti-CHIKV antibody positive (>11 standard units) will be enrolled as asymptomatic adult cases. These are the participants who have been excluded from the vaccine study due to antibody positivity. Symptomatic confirmed adult cases (NVBDCP) will be enrolled from the Department of Medicine. Age and sex matched adult controls will be taken from those relatives who accompany patients to the Therapeutic Drug Monitoring OPD run by the Department on Fridays and Saturdays. All participants will be enrolled after written, informed consent.

Paediatrics: All asymptomatic paediatric participants from who are CHIKV IgG antibody positive without clinical manifestation will be enrolled as asymptomatic cases. Symptomatic confirmed paediatric cases (NVBDCP) will be enrolled from the Department of Paediatric. Age and sex matched paediatric controls will be taken from those relatives who accompany patients to the Therapeutic Drug Monitoring OPD run by the Department on Fridays and Saturdays. All participants will be enrolled after written, assent and informed consent from the LAR.

Study procedure: A total of 5 ml of blood will be collected from all study participants for assessing the CHIKV antibody. This will only be a qualitative estimation [yes/no]. There will be two study visits (Visit 1 - [Screening] + visit-2 [collection of blood for proteomic analysis]). If a participant who is asymptomatic from a potential control group [since at the point of screening we will not know whether a “control” will tests positive or negative for the antibody] and tests positive (>11 standard units), he/she will be enrolled as an asymptomatic case and participants who fulfil the confirmed case definition (NVBDCP) will be enrolled as symptomatic cases.  A total of 5 ml of blood will be collected for proteomic analysis will be processed in centrifuge at 3000 rpm and plasma will be separated. Plasma will be stored under-20 degree celsius till shipment to central laboratory (IIT Bombay). Details of the proteomics analysis are given in Appendix 1.

Statistical Analysis:

Descriptive statistics (mean and SD, median and range and percentages) will be used to present the quantitative and qualitative data. Odds ratio will be used as a measure of association between the proteins seen in cases versus controls. Chi square or Fisher exact test will be used for categorical data to establish the strength of the association. A p value of less than 5% will be considered as statistically significant. Graph Pad statistical software version 3 will be used for statistical analysis.

Anticipated outcomes:

Chikungunya is a viral disease transmitted by Aedes mosquitoes. Chikungunya virus (CHIKV) is an arbovirus that belongs to the family of Togaviridae and genus Alphavirus. The virus was first discovered in 1952 and used to cause sporadic outbreaks.

In recent years, there have been several reports of large scale outbreaks of Chikungunya virus infection in several parts of Southern India. Over, 2000 cases of Chikungunya fever have also been reported from Malegaon town in Nasik district, Maharashtra state, India between February-March 2006. In October 2009, 2–3 months after an outbreak of a febrile disease with joint pain on the eastern coast of Madagascar, serologic markers for chikungunya virus (CHIKV) in n= 1,244 pregnant women were assessed and IgG sero-prevalence against CHIKV was 45%. The recent outbreaks of this disease have been associated with fatalities and chronic and persistent disability particularly the persistence of joint pain. Chikungunya is thus considered as an important re-emerging public health problem in both tropical and temperate countries, where the distribution of the Aedes mosquito vectors continues to expand.

Given this backdrop, several pharmaceutical companies are in the process of developing vaccines to combat the disease. One such vaccine has been developed by Bharat Biotech and is undergoing clinical development at the KEM Hospital. A Phase I study [a study in normal, healthy participants is currently underway at this institute after regulatory and Ethics Committee approval- EC- PHARMA-10/16].

While screening normal, healthy participants for inclusion into this study the study team found 13/53 [25%] participants to be positive for anti- Chikungunya antibodies by an ELISA test. When questioned, these 13 participants did not give any past history of Chikungunya infection indicating that they suffered from asymptomatic infection. When the team did a literature review of the prevalence of anti-Chikungunya antibodies in the general population, there were no studies. Only three studies on antibody prevalence have been done in the country and these are largely confined to patients who have been hospitalized with a diagnosis of Chikungunya.

A disease like Chikungunya that has seen a re-emergence would benefit from a biomarker that facilitates its diagnosis and helps assess prognosis. Biomarker studies being with an assessment of plasma proteins that are up or down regulated during and after the infection – an analysis that can be done by proteomics- a study of an entire set of proteins that are modified by an organism or system.

Thus, the present study is planned with the objective of carrying out a quantitative proteomic analysis of patients who are positive for anti-Chikungunya antibodies including those from the above-mentioned study (EC/PHARMA-10/16) as well as those who have active infection and analyse proteins that are upregulated or downregulated in them.

Objective:

To assess via a quantitative proteomic analysis the differential expression of proteins in Chikungunya antibody positive patients (symptomatic and asymptomatic) versus age and sex matched healthy controls.

Methods:

Ethics: The study will be submitted to the Institutional Ethics Committee for approval. Written, informed consent will be taken from all participants.  A copy of the submission form will be submitted to the Research Society

Study design: Cross sectional

Sample Size calculation: No formal sample size calculation

Adults: The department is currently doing a study EC/PHARMA-10/2016 where a new Chikungunya vaccine is being tested in normal, healthy participants. A total of n = 25 participants who are asymptomatic but tested positive (>11 standard units), n = 25 who satisfy the confirmed case definition of Chikungunya (NVBDCP) and n = 50 controls who have tested negative (<11 standard units) will be enrolled in this cross-sectional study.

Paediatrics: A total of n = 10 participants who are asymptomatic but tested positive (>11 standard units), n = 10 who satisfy the confirmed case definition of Chikungunya (NVBDCP) and n = 20 controls who have tested negative (<11 standard units) will be enrolled in this cross-sectional study.

Screening: All participants will be screened (via medical history and physical examination only) to establish the eligibility criteria as outlined below. This includes negative antibodies, CHIKV IgG, by ELISA method.

Selection criteria:

Adults: All asymptomatic participants from the Chikungunya vaccine trial (EC/PHARMA-10/16), aged ≥18 and ≤50 years and who are anti-CHIKV antibody positive (>11 standard units) will be enrolled as asymptomatic adult cases. These are the participants who have been excluded from the vaccine study due to antibody positivity. Symptomatic confirmed adult cases (NVBDCP) will be enrolled from the Department of Medicine. Age and sex matched adult controls will be taken from those relatives who accompany patients to the Therapeutic Drug Monitoring OPD run by the Department on Fridays and Saturdays. All participants will be enrolled after written, informed consent.

Paediatrics: All asymptomatic paediatric participants from who are CHIKV IgG antibody positive without clinical manifestation will be enrolled as asymptomatic cases. Symptomatic confirmed paediatric cases (NVBDCP) will be enrolled from the Department of Paediatric. Age and sex matched paediatric controls will be taken from those relatives who accompany patients to the Therapeutic Drug Monitoring OPD run by the Department on Fridays and Saturdays. All participants will be enrolled after written, assent and informed consent from the LAR.

Study procedure: A total of 5 ml of blood will be collected from all study participants for assessing the CHIKV antibody. This will only be a qualitative estimation [yes/no]. There will be two study visits (Visit 1 - [Screening] + visit-2 [collection of blood for proteomic analysis]). If a participant who is asymptomatic from a potential control group [since at the point of screening we will not know whether a “control” will tests positive or negative for the antibody] and tests positive (>11 standard units), he/she will be enrolled as an asymptomatic case and participants who fulfil the confirmed case definition (NVBDCP) will be enrolled as symptomatic cases.  A total of 5 ml of blood will be collected for proteomic analysis will be processed in centrifuge at 3000 rpm and plasma will be separated. Plasma will be stored under-20 degree celsius till shipment to central laboratory (IIT Bombay). Details of the proteomics analysis are given in Appendix 1.

Statistical Analysis:

Descriptive statistics (mean and SD, median and range and percentages) will be used to present the quantitative and qualitative data. Odds ratio will be used as a measure of association between the proteins seen in cases versus controls. Chi square or Fisher exact test will be used for categorical data to establish the strength of the association. A p value of less than 5% will be considered as statistically significant. Graph Pad statistical software version 3 will be used for statistical analysis.

Anticipated outcomes: