Study Title:

A Prospective Cohort Study To Compare The Burden Of Medically Attended and Community RSV,
hMPV, SARS-CoV-2 and influenza LRTI In Infants And Children Under 5 Years Of Age and
adults (16-60 years) In Rural India

Institution Name MAHAN Trust

Investigator Contact Information:

Dr. Ashish Satav Mahatma Gandhi Tribal Hospital, Karmgram, Utavali. Tahsil Dharni,
District: Amaravati, Maharashtra. India pin: 444702 Phone : +919423118877 Email:
drashish@mahantrust.org

Protocol ID MAHAN RSV 2025 1

Section #2- Core Protocol

2.1 Objectives & Hypotheses

2.1 Main objectives : Primary: To determine the population-based epidemiology of
respiratory syncytial virus (RSV) lower respiratory tract illness (LRTI) in infants and
children less than 5 years of age and adults, elderly people in rural India.

Secondary: To determine the population-based epidemiology of human metapneumovirus
(hMPV), SARS-CoV-2 and influenza LRTI in infants and children less than 5 years of age
and adults, elderly people in rural India, in relation to RSV.

2.1.1 Clinical hypotheses. This is primarily a descriptive observational study. However,
investigators have proposed null hypotheses in the statistical methods, addressing each
of the objectives and sub objectives

2.2 Background & Rationale, Significance of Selected Topic & Preliminary Data Respiratory
syncytial virus (RSV) is the most common cause of infection of the upper and lower
respiratory tract in infants and young children worldwide and is a major public health
burden.1 2 It has been estimated that globally in 2019 there were 33.0 million episodes
of RSV acute lower respiratory tract infections (LRTI) in children less than 5 years of
age resulting in about 3.6 million hospitalizations, with 6.6 million episodes occurring
in infants < 6 months of age and 1.4 million RSV associated ALRI hospitalizations 3. It
was estimated that India bears a fifth of the global burden of illness (6.2 million
episodes of RSV LRTI).3 Preliminary Data from our study the burden of RSV as a cause of
mortality in rural infants and children under 2 years of age.

Results of Virology testing: In the 12 134 subjects, there were 5308 episodes of
non-severe LRTI 2481 episodes of severe LRTIs and 1808 of very severe LRTIs. 4842 NP
swabs were collected and tested by Real time RT-PCR for 16 respiratory viruses including
RSV subtyping. During 2021 and 2022 additionally the samples were tested for SARS CoV 2.
Out of 4842 almost 55% samples i.e., 2637 were positive for one or more respiratory
viruses. RSV was detected in 740 cases (360 RSV A and 380 RSV B) followed by rhinovirus
in 722 cases, PIV was detected in 345, Influenza 332, hMPV in 251, Adenovirus detected in
136. SARS CoV 2 was detected in 19 cases. RSV was detected in alternate year outbreaks
with a dominance of RSV A or RSV B subtypes. In 2016 RSV A was predominantly in
circulation where as in 2017 and 2018 RSV B was exclusively in circulation. In 2021 RSV A
predominated. (Figure 1) RSV and hMPV alternate and Influenza occurs annually.

RSV Mortality: Fifteen of 16(94%) children with RSV died of LRTI, 14 in the community and
1 in the hospital. The case fatality ratios for severe RSV LRI in the first 6 months of
life were 3/52(7.1%) and 1/36(2.8%) in the community and hospital respectively. Of those
with very severe LRTI in the community, 17.6% died. There were no very severe RSV LRTI
hospital deaths. The adjusted RSV LRTI mortality rates ranged from 1.0 - 3.0/1000 child
years (CY) overall, and 2.0 - 6.1/1000 CY, accounting for 20% of the LRTI deaths and 10%
of the post neonatal infant mortality.4

2.3 Study Design Melghat is a hilly, forested area spread over >4000 km2 in Maharashtra
state, Central India, with population of 2,80,000 scattered over 320 villages,
distributed in 2 blocks. The government primary health care delivery is through
sub-center with paramedical workers for 2-5 villages, a Primary Health Center (PHC) with
a doctor and nurses for every 30 villages, a rural sub-district hospital (RH) and 2
district hospitals 1 for mother and children and the other for adults. MAHAN hospital,
where the study is based is a charitable trust hospital that provides free or low-cost
care to the community.

Investigators propose an active surveillance observational study in Melghat of a cohort
of all newborns, infants and children under 5 years, and adults in age group of 16-60
years, for 36 months, of LRTI detected at home, primary health centers or hospitals in
the catchment area of 30 villages under surveillance. Active surveillance for LRTI cases
in the community will be performed through weekly home visits by village health workers
(VHWs) The VHWs are local women living in and accepted by the community. They have been
trained using standard WHO materials, translated into Hindi with a refresher course every
6 months. A nasopharyngeal swab will be obtained from children and adults (16-60 years)
with non-severe, severe or very severe LRTI and those who die. A counselor is posted at
every hospital and PHC who will capture every hospitalization of children under 5 and
adults from the 30 villages, will identify any children from the 30 villages who visit
the OPD with an LRTI and collect a swab from them. Children/adult with any lower
respiratory illness will have a swab collected at admission and from any child/adult who
dies. Samples (2000) will be tested for a panel of respiratory viruses first with a
screening PCR(RSV, hMPV, SARS CoV 2 and Influenza) followed by subtyping when positive
(RSV A and RSV B; hMPV A and hMPV B; Influenza A H1N1 pdm2009, H3N2 and Influenza B and
SARS CoV 2 ) at the National Institute of Virology, Pune.

2.7 Study Procedures Study Population: At the start of the study All children under the
age of five years and adults (16-60 years) will be recruited for prospective observation
for LRTI, after obtaining informed consent from the parent/caretaker. Subsequently using
pregnancy monitoring, all parents of live-born neonates will be approached for consent to
follow-up their children for the next 3 years. Subjects will be followed until they reach
the age of 5 years or 60 years, migrate permanently out of the 30 villages, withdrew
consent, or expire.

Health worker training:

All VHWs, and their supervisors and health center/hospital based counselors will be
re-trained using a modified World Health Organization (WHO) methodology, in the
recognition of WHO defined Pneumonia5, and its recording on paper case report forms as
well as how to collect nasopharyngeal (NP) swabs in infants and children, and adults
(16-60 years).

Subject monitoring in the community:

Our experience is that VHWs develop a keen rapport with the families who they follow with
weekly home visits, specifically following the respiratory health of the subjects. Any
child or adult with an LRTI, will have a NP swab collected for pathogen detection.
Treatment will follow Govt. of Maharashtra guidelines that permits VHW's to prescribe
amoxicillin for pneumonia/ within 24 hours of the assessment, supervisors will visit the
subject and conduct a respiratory assessment as well. All subjects with LRTI who are
hospitalized, will be followed up at home one and two weeks later. (Follow up form) VHWs
are also informed of community deaths and, after grief counseling is offered to the
family, either by a supervisor or local trained traditional healers or nurses, with
informed consent, NP swabs will be obtained from the deceased child whenever feasible.
(the community is used to this)

Subject monitoring in the health centers and hospitals:

Counselors working in the 14 government facilities will monitor the outpatient and
conduct a daily census of all new admissions, and after obtaining informed consent, (from
children and adults from the 30 villages) will administer a short questionnaire and
collect a NP sample from children (0-5 years) and adults (16-60 years) with LRTI or
sepsis, as well as all who die. (if they have not been already collected by the VHW in
the village) for an acute illness visit . Total samples to be collected 2000.

Nasopharyngeal swab collection and sample processing:

NP swabs will be collected using flocked swabs and transported in viral transport medium
to MAHAN hospital where they will be stored at 4-80C. Batches are transported to the ICMR
-National Institute of Virology Pune for pathogen testing. The samples will be tested in
a 2-step manner (screening and subtyping) as outlined previously by real time PCR using
standardized protocols6,7.

Verbal Autopsy:

A supervisor and a VHW from the community will conduct a verbal autopsy (VA) within 2
weeks of the death. The VA uses standardized Indian government protocols modified for use
in Maharashtra incorporating local terminology8,9. The narratives are read by two
independent trained physicians, and where there is discordance, a third Senior
pediatrician adjudicates the difference.

All pathogen testing is done at India's premier virology Institute, National Institute of
Virology, Pune.

2.8 Study duration and study timelines

The study will proceed in four major phases over a 4-year implementation period:

2.10Statistical Analysis and Sample Size Justification Statistical Analysis Plan All data
will be entered into a RedCap database and exported for analysis. All statistical
analyses will be done by the Biostatistician Dr Raje, who is part of the study team,

Statistical methods The demographic and socio-economic status of each subject included in
the cohort will be summarized according to scale of measurement. Continuous variables
with be expressed in terms of mean and standard deviation, while categorical variables
will be summarized in terms of frequencies and percentages.

Primary objective

1. To prospectively determine the burden (clinical features and mortality) of RSV LRTI
in a cohort of infants and children under 5 and adults (16-60 years) in 30 villages,
over 3 years, and all newborns entering the cohort during the 3 years of the study.

2. To determine the incidence (per 1000 child years/adult years of observation) of the
following classifications of RSV LRTI, using the WHO classification of disease:
non-severe pneumonia, severe pneumonia, very severe pneumonia, RSV hospitalization,
RSV mortality.

3. To evaluate the RSV subgroup specific (RSV A and RSV B) burden of illness and to
compare severity based on subgroups.

Power/Sample Size:

Investigators plan to recruit a cohort of 22,121 individuals, comprising:

- 2,726 children aged 0-5 years (U5C)

- 19,395 adults aged 16-60 years, These sample sizes are powered to detect
statistically significant differences (α = 0.05, power = 80%) in virus-specific
respiratory disease incidence between subgroups (e.g., age, nutrition status, tribal
subpopulation) and to analyse long-term sequelae patterns in RSV-positive vs.
RSV-negative individuals using lung function tests.

2.16 Plan for Policy Translation Proactive dissemination and policy translation plan will
be implemented.

- Continuous Stakeholder Engagement:

- Evidence-Based Advocacy:

- Targeted Dissemination of Findings:

- Policy Briefs:

- Academic Publications:

- Conferences and Meetings:

- Public interest litigation to courts.

- Public and Community-Level Communication:

2.17 Local Stakeholder Engagement Strategy for Engaging Local Stakeholders To maximize
the study's relevance, impact, and long-term sustainability, this plan outlines a
dedicated strategy for the early and continuous engagement of local health authorities
and policymakers.

1. Initial Partnership Building and Alignment

2. Formation of a Stakeholder Advisory Group

3. Regular and Transparent Communication

4. Integration with Existing Health Systems o