Epidemiological Investigation of COVID-19 in Laos Using a One-Health Approach in Human and Animals

1. INTRODUCTION

This project is mainly based on two major questions that remain unsolved. The first one
is the origin of the outbreak and the events that led to the transmission of SARS-CoV-2
to humans. The second is the striking epidemiological situation in Laos, in which only a
few cases have been observed despite similar biogeographical and socio-ecological
characteristics shared with South-Western China.The diversity of wild fauna (notably in
southern China and in South-East Asia, where bat diversity peaks to over 100 species),
associated with the wide circulation of coronaviruses, especially in Laos PDR, and their
evolutionary and zoonotic potential, indicate that (past or future) emergences in Laos
can be expected. In addition, given the massive population movement between Laos and
China, it was expected a significant spread of COVID-19 from China to Laos. In order to
respond to these two main questions, the investigators formulate the following working
hypotheses to address in this project.

Assumption 1: COVID-19 may massively spread to the Laos population in the near future
through the arrival in the country of infected individuals.

Assumption 2: SARS-CoV-2 has been already circulating in Laos before the appearance of
COVID-19 outbreak in China.

Assumption 3: SARS-CoV-2 may jump from wildlife and emerge into the Lao human population
in the future Assumption 4: Some genetic closely related viruses (SARS-CoV-2-like
viruses) have already circulated in Laos.

2. OBJECTIVES

The general objective of the project is to elucidate the natural history of COVID-19 by
investigating the circulation of the virus in Lao PDR following a ONE-HEALTH approach
before (retrospective approach) and after (prospective approach) the initial appearance
of the outbreak in China.

In particular, the investigators will investigate the progressions of the outbreak and
the reasons behind the relative low number of COVID-19 positive patients observed to
date in the country, despite sharing biogeographical and socio-ecological features and
trans-border with South-Western China.

Under this general heading, the secondary objectives are:

In humans:

To determine COVID-19 circulation within the cohort before the appearance of the first
positive cases in December 2019 in Wuhan, China To determine COVID-19 progression, over
a one-year period from the beginning of the project To evaluate the SARS-CoV-2
asymptomatic infections To assess past SARS-CoV-2 circulation

In animals:

To investigate SARS-CoV-2 transmission to pets and domestic animals

3. METHODOLOGY

In humans:

The project will be conducted within a community-based cohort of 1092 households,
including 5400 study participants, followed-up between March 2015 and February 2019 for
influenza-like illness investigation and causative agents detection (LACORIS project,
Respiratory Infectious Disease among Cohorts in Vientiane Capital, Lao PDR), located in
the Vientiane metropolitan area in Lao PDR (reference 1 and internal reports).

- Retrospective approach: To determine COVID-19 circulation within the cohort before
the appearance of the first positive cases in December 2019 in Wuhan, China, by
retrospective SARS-CoV-2 RNA detection in samples collected from a cohort of 1092
households, including 5400 study participants, followed-up between March 2015 and
February 2019 (LACORIS project) (1 and internal reports).

- Prospective approach: To determine COVID-19 progression, over a one-year period
from the beginning of the project, by SARS-CoV-2 RNA detection in individuals
expressing an influenza-like illness.

- For this, intra-familial and intra-community transmission rate and context will be
evaluated by SARS-CoV-2 RNA detection in contacts of COVID-19 positive cases
detected during the prospective phase of the project.

- To assess past SARS-CoV-2 circulation by screening specific antibodies from saliva
swabs in a randomized group of the cohort study.

In animals:

- To investigate SARS-CoV-2 transmission to pets and domestic animals by RNA detection
in animals living with or near positive cases detected during the prospective phase of
the project.

4. STUDY SITES AND POPULATION

The study will cover 1092 households, including 5400 study participants living in 25
villages within the administrative boundaries of the Vientiane metropolitan area. A
household was defined as a group of individuals living on the same plot of land (one or
more houses) or in the same building (apartment) and sharing their meals All study
co-investigators will be trained before the beginning and during the study on the
specific aspects of the study.

5. RESEARCH FLOWCHART

5.1 Flowchart of the participant with ILI symptoms

All family members more than 6 months living with SARS-CoV-2 positive individuals will have
non-invasive exam for SARS-CoV-2 detection (nasal, throat, swab, saliva) and a questionnaire.

Participation in the study

Information: All participants will be handed a written information form to participate in the
research. This form will provide information on the scope and intent of the study as well as
the obligations of the study investigators and expectations from cohort members.

Collection of consent: Before the beginning of the study, an appointment with the
investigative surveillance team at the household will be scheduled to explain the details of
the project. These details include taking respiratory swab samples in case of influenza-like
disease for the patient and family members. The team will hand in the information form and
will collect the written consent form from each willing household member, previously signed
by three people (Interviewer, parent or guardian, witness/family member)

Cohort participation:

The project study will extend over a year period and will begin as soon as the ethical
clearance will be obtained. Households will be selected from household registries and will be
enrolled into the cohort via door-to-door sampling, according to the database already set up
at CILM. A household is defined as a group of individuals living on the same plot of land
(one or more houses) or in the same building (apartment) and sharing meals. Over a year,
active case finding for influenza-like illness (ILI) will be conducted among the cohort
through weekly phone calls (N=500) to each household by staff at CILM. To increase the
likelihood of pathogen identification, study participants will be encouraged to self-report
occurrence of symptoms to allow early collection of specimens. As soon as a case meets the
ILI-WHO criteria, a free of charge visit will be organised on the same day by the
field-trained healthcare staff (the investigative surveillance team) to interview and verify
the disease eligibility criteria, to complete a disease investigation questionnaire, where
details about the symptomatology will be collected.

Diagnosis results and ILI Follow-up:

The results of COVID 19 analysis (positive or negative) will be transmitted to the NCLE
(National Center for Laboratory and Epidemiology).

If the result is COVID19 positive, the NCLE will send it to the "National Taskforce Committee
for prevention, control and response COVID-19 pandemic". The COVID-19 positive patient will
be taken care by this national Committee and followed up according to the official
procedures.

According to the Committee recommendations, all family members living with the COVID-19
patient will be sampled in order to look for asymptomatic infections. In all other cases
(detection of another respiratory virus or negative results), the result will be returned on
site, in written form, directly to the participant (adult or children between 12 and 18 years
old) or to the parent or the guardian (children under 12 years old or adult with psychiatric
disorders) by the interviewer. The participants will be referred to medical doctors or to the
local healthcare structures to be treated. There are four general hospitals in Vientiane and
several local healthcare structures in each of the 25 villages included in our study. When a
patient will develop ILI, the doctor in our team will advise him on any treatment to be
taken, on the preventive measures to be taken to avoid contamination of other members of his
family and will direct him to the closest healthcare structure. Except in exceptional cases,
no logistical (transport) or medical support is recommended in the project.

Patients or their family will be attended 60 days later to determine the overall health
status, including hospitalisation or death.

5.2 End of research

At the end of the study, a final report will be communicated to each household and the
results will be explained by the interviewer.

Each week, the head of the household will be contacted by phone to ask if one member of his
family has developed signs of illness. If there is no news from a participant for 3
consecutive weeks, an investigator will go on site to conduct more precise investigations.

5.3 Questionnaires

Overall, the study includes two kind of questionnaires:

A household enrolment questionnaire, including sociodemographic details. Census questions
asked upon enrollment to build a database of participating villages for purposes of weekly
home visits, disease investigation, and follow-up investigation. This questionnaire will be
filled in by the interviewer for each household, at the time of the enrolment, when the
information form will be handed in and the signature of the informed consent will be
obtained.

A disease investigation questionnaire, including details of the ILI disease. Questions asked
will provide information on the person reporting with illness and include basic clinical
observations and other information relevant to understanding the source of the illness. This
questionnaire will be filled in by the interviewer at the time of the sample collection.

Specimens to be collected using standard protocols include:

- Upper Respiratory swabs (saliva, throat and nasal swabs): samples will be collected on
transport medium according to the recommendations of the "National Taskforce Committee
for prevention, control and response COVID-19 pandemic". Each sample will be collected
by trained field staff using sterile Nylon swabs with plastic shafts and inserted in the
sterile cryovial containing universal transport media (UTM).

- The samples will be placed and transported in refrigerated coolers to CILM for testing.

- The patient identification number (see below) will be linked by a relational database
and pre-printed labels placed on the questionnaire and on any specimen collected

- Specific qPCR for SARS-CoV2 (targeting RdRp, N and E genes) as well as Pan-coronavirus
targeting RdRp will be performed on RNA extracted from retrospective and prospective
nasal swabs, throat swabs, and sputum specimens. The analyses will be performed at CILM
(Vientiane, Laos). Each positive sample will be sequenced and compared to reference
sequences.

- Specific IgG testing will be performed by luminex and ELISA technologies from saliva
swabs.

- After testing, the samples will be stored at -80°C at CILM until further analysis upon
request for at least 15 years. The samples could be destroyed after at least 15 years
upon request from the authorities of Lao PDR and the staff manager of CILM.

- Upon request from the coordinating investigators of the study (Phimpha Paboriboune and
Eric Leroy) and agreement of the scientific committee, genetic materials from positive
samples will be sent to MIVEGEC UMR IRD 224-CNRS 5290-Université de Montpellier - IRD
(911, avenue Agropolis, 34394 Montpellier France) for further analyses that cannot be
performed at CILM or for whole genome sequencing.