Title
Epidemiology and serological evaluation of flavivirus infection in Thailand
Study sites
Department of Disease Control, Thailand Ministry of Public Health (MoPH), Nonthaburi, Thailand
Thailand MoPH-US Centers for Disease Control and Prevention collaboration (TUC), Nonthaburi, Thailand
Oregon Health and Sciences University, Portland, Oregon, USA
Funded By
Oregon Health and Sciences University
Thai Ministry of public health/US CDC collaboration
Principal Investigator
Dr. Chuleeporn Jiraphongsa, Office of the Senior Expert Committee, Department of Disease Control, Ministry of Public Health, Thailand. Email: jiraphongsa@gmail.com; Tel: 662 590 3221 ; Fax 662 590 3221
Research Team Lead
Dr. Apinya Niramitsantipong, Division of Vector Borne Diseases, Department of Disease Control,
Ministry of Public Health, Thailand. Email: yayamedrec@gmail.com; Tel: 662 591 8422 ; Fax 662 590 3141
Co-Investigators
Dr. Chantana Padungtod, Director, Division of Vector Borne Diseases, Department of Disease Control, Ministry of Public Health, Thailand. Email: cpadungt@gmail.com; Tel: 662 591 8422 ; Fax 662 590 3141
Dr. Piraporn Utachee, Division of Vector Borne Diseases, Department of Disease Control,
Ministry of Public Health, Thailand. Email: pirapornaom@gmail.com; Tel: 662 591 8422 ; Fax 662 590 3141
Dr. Marcel Curlin, Division of Infectious Diseases, Oregon Health and Sciences University
Email curlin@ohsu.edu; Tel (503) 418 0174; Fax: 502 494 4264
Dr. William Messer, Division of Infectious Diseases, Oregon Health and Sciences University,
Email messer@ohsu.edu; Tel: (503) 494 2185; Fax: 502 494 4264
Dr. Andrew Hickey, Thailand MOPH-US CDC collaboration
Email: ANH9@cdc.gov; Tel: 662 591 5444 ; Fax 662 580 0696
Ms. Wanna Leelawiwat, Thailand MOPH-US CDC collaboration
Email: hpx0@cdc.gov; Tel: 662 5915444 ; Fax 662 580 0696
Mx. Hiro Ross, MD candidate, School of Medicine, Oregon Health and Sciences University
Email: rosshi@ohsu.edu; Tel: 541 880 6862; Fax: none
Ms. Francie Goodstein, MD candidate, School of Medicine, Oregon Health and Sciences University OHSU
Email goodstef@ohsu.edu; Tel: 503) 418 0174: Fax: none
Rationale
An accurate understanding of geographic and temporal seroprevalence trends is vital to reduce the public health impact of common flavivirus infections, including zika virus, dengue virus and Japanese encephalitis virus. Laboratory assays currently in use to examine the seroprevalence of these pathogens suffer from cross-reactivity and may mis-represent true seroprevalence. For technical and other reasons, our knowledge of flavivirus seroepidemiology in Thailand remains incomplete. This study offers an opportunity to examine historical and current flavivirus serodistribution, and will contribute to laboratory capacity within the Department of Disease Control, Ministry of Public Health.
Study Objectives:
Objective 1: Describe flavivirus seroprevalence in a representative historical sample population in Thailand
Objective 2: Evaluate seroprevalences of other regionally important flaviviruses (dengue, Japanese encephalitis virus) in a representative historical sample population in Thailand.
Objective 3: Optimize and operationalize accurate neutralizing antibody assays at the Ministry of Public Health, Department of Disease Control, Division of Vector-borne Diseases
Study Timeline
The total study period is projected to be 15 months. The anticipated timeline is as follows:
Target Period Projected Milestone
Month 0-1 Retrieve specimens from repository
Month 2-3 Optimize the neutralizing antibody assay
Month 4-12 Perform neutralizing antibody assay on all specimens
Month 13-15 Data analysis and final report preparation
Study Design:
Retrospective cross-sectional seroprevalence study, using archived serum specimens obtained and maintained by the TUC collected between 1992 and now.
Population: Deidentified serum and plasma samples are selected from a collection of more than 350,000 specimens taken from patients enrolled in various studies between 1992 and the present.
Sample Size Justification:
Sample size is based on availability of a limited number of archived specimens for this project.
Evaluation of Objectives:
Seroprevalence values will be summarized using standard descriptive statistics including percentages, proportions, and percent change per year.
LIST OF ABBREVIATIONS
CDC US Centers for Disease Control and Prevention
CO2 Carbon Dioxide
DENV Dengue Virus
EDTA Ethylenediaminetetraacetic Acid
ERC Ethics Review Committee
HIV Human Immunodeficiency Virus
IC50 50% Inhibitory Concentration
IRB Institutional Review Board
MEM Minimal Essential Media
MOPH Thai Ministry of Public Health
Nab Neutralizing Antibody
NT Neutralizing Titer
OHSU Oregon Health and Sciences University
PBMC Peripheral Blood Mononuclear Cells
PI Principal Investigator
PRNT Plaque reduction neutralization test
SE-Asia Southeast Asia
TUC Thai Ministry of Public Health / US CDC Collaboration
ZIKV Zika Virus
PROTOCOL SUMMARY
Title:
Epidemiology and serological evaluation of flavivirus infection in Thailand
Objectives:
Objective 1: Describe flavivirus seroprevalence in a representative historical sample population in Thailand
Objective 2: Evaluate seroprevalences of other regionally important flaviviruses (dengue, Japanese encephalitis virus) in a representative historical sample population in Thailand.
Objective 3: Optimize and operationalize accurate neutralizing antibody assays at the Ministry of Public Health, Department of Disease Control, Division of Vector-borne Diseases
Hypotheses:
ZIKV has been transmitted to humans in Thailand at low levels in the late 1990s, and increased in prevalence between 1998 and 2015.
Study Design:
Retrospective cross-sectional seroprevalence study, using archived serum specimens obtained and maintained by the TUC collected between 1992 and now.
Population: Deidentified serum and plasma samples are selected from a collection of more than 350,000 specimens taken from patients enrolled in various studies between 1992 and the present.
Description of study:
A convenience sample of archived plasma maintained by the TUC will be retrieved for study. Samples are anonymized, and all identifying data including name, date of birth, place of residence, contact information have been de-linked from the participant and/or specimen identification. Information on year of sampling, gender and age in years will be retained. Samples will be subjected to serological neutralizing antibody assays to determine the seroprevalence of flavivirus (ZIKV, dengue, Japanese encephalitis virus) neutralizing antibodies in these samples, with appropriate controls. Neutralizing antibody titer data will be tabulated for analysis. Titers above a cutoff threshold will be considered positive. Data will be presented as percent positive by year. Following study completion, any remaining samples will be returned to the TUC specimen repository.
BACKGROUND:
Zika virus (ZIKV) was first isolated from rhesus macaques from the Zika forest region of Uganda in the 1940s. Between the 1950s and 2007 ZIKV is thought to have silently spread to several African and Asian countries. Prior to 2007, ZIKV infection was considered to be clinically insignificant. Between 2007 and the present, 3 major ZIKV epidemic outbreaks have been noted, in the Yap islands (2007), Oceania (2013–2014), and South and Central America (2015–2016). The 2015 epidemic centered on the Americas was notable for its association with several significant clinical manifestations including Guillain-Barré syndrome, and neurological disorders in infants born to infected mothers. Because of the rapid spread through South and Latin America and the potentially serious health consequences observed during this epidemic, the WHO declared a public health emergency of international concern in February 2016. By 2017, the number of new ZIKV cases in the Americans had declined substantially.
The epidemiology of ZIKV associated with these recognized epidemics has been intensively studied. However, while Aedes aegypti is endemic in Southeast Asia, our knowledge of the epidemiology of ZIKV infection in this region is incomplete. ZIKV infection has been less frequently reported in Southeast Asia than it was in prior epidemics. However, current information is largely based on older studies [1-3] and a relatively small number of more recent reports [4-8]. Presently, we know little of the extent and trajectory ZIKV infection in Southeast Asia, and there is a poor understanding of its potential to cause large-scale outbreaks and associated congenital malformations [7, 8]. For reasons that are currently unknown, a rise in congenital malformations such as microcephaly has not been reported in association with ZIKV infection in Thailand.
Estimating the burden of ZIKV in Thailand is complicated by unreported asymptomatic infections, the clinical similarity between of ZIKV dengue infection, and the high degree of serological cross-reactivity between ZIKV and other circulating flaviviruses using standard assays [9]. The use of plaque reduction neutralization tests (PRNT) is currently the “gold standard” for assessing serologic evidence of exposure to flaviviruses [10], but is hampered by cross-reactivity between flavivirus antibodies. This creates uncertainty in the interpretation of assays in places such as Thailand where dengue and other flaviviruses co-circulate at high prevalence [11-13]. A modified PRNT assay capable of addressing this issue will be used to detect viral antibodies using recombinant ZIKV, Dengue virus, and possibly other relevant flaviviruses as reference strains [14]. Together with the availability of archived specimens, there is now a unique opportunity to conduct a population-representative serosurvey to study the historical epidemiology of ZV seroreactivity and seroreactivity to other common flaviviruses in Thailand. We expect this work to contribute expanded diagnostic capacity and investigator capacity to the Thai Ministry of Public Health by adding new diagnostic tools to perform viral seroepidemiology that will clearly define national seroepidemiological survey data and trends and expert advance virology laboratory training of young investigators.
OBJECTIVES:
Objective 1: Describe flavivirus seroprevalence in a representative historical sample population in Thailand
Objective 2: Evaluate seroprevalences of other regionally important flaviviruses (dengue, Japanese encephalitis virus) in a representative historical sample population in Thailand.
Objective 3: Optimize and operationalize accurate neutralizing antibody assays at the Ministry of Public Health, Department of Disease Control, Division of Vector-borne Diseases
HYPOTHESES:
ZIKV has been transmitted to humans in Thailand at low levels in the late 1990s, and increased in prevalence between 1998 and 2015.
METHODS:
A. Study design: Retrospective cross-sectional seroprevalence study, using archived serum specimens obtained and maintained by the TUC
B. Number of study participants: Total N: approximately 1800
C. Population: Serum and plasma samples are selected from a collection of more than 350,000 specimens, taken from patients enrolled in various studies between the years of 1990 and the present. Stored samples available from study listed below are to be used:
D. Selection Criteria: A computer algorithm will be implemented to maintain balanced selection by age and gender. if sample volume and/or condition is inadequate.
F. Sample type: Frozen serum or plasma, estimated volume 0.5 ml
G. Sample data: As available: Participant gender and age at sampling; Participant residence region; Year of sample taken from participant
H. Specimen requirements and handling: MoPH ethics review committee approval, additional OHSU IRB approval, CDC reliance.
I. Laboratory methods: Assays will be performed at the Thai Ministry of Public Health by established methods. Modified plaque reduction neutralization assay will be performed on collected samples to establish neutralizing antibody titer (NT) to ZIKV, dengue serotypes 1-4 and JEV. Briefly, Vero cells will be grown in 96-well culture plates in minimum essential medium supplemented with 2% fetal bovine serum. Reference virus stocks for dengue serotypes 1-4 and ZIKV will be grown with human-derived cell lines. Test serum will be heat-inactivated at 56 degrees Celsius to remove complement. Serial dilutions of heat-inactivated serum samples will be performed. Dilution aliquots will be incubated with each reference virus at predetermined titers in buffered growth medium at 37 degrees C. Virus-antibody mixture from each sample will be applied to confluent monolayers of Vero cells in 96-well plates. Each well will be overlaid with 0.9% low-melting-point agarose, MEM + 10% heat-inactivated fetal bovine serum with 0.5% vitamin, L-glutamine, 2.5% of 7.5% sodium bicarbonate solution, and 1000 units of neomycin-streptomycin, or another appropriate overlay solution. Plates will be incubated at 35 degrees C in 5% CO2 for 4 to 5 days or until cytopathic effect is observed. After completion of incubation, wells will be stained with labeled antibodies, and plaques, representing foci of infection, will be counted manually following completion of incubation. A reduction in plaque count of 50% will be used as the neutralization endpoint. All assays will be performed in triplicate with appropriate positive and negative controls.
J. Data Analysis: Stored demographic data associated with each sample will be collected at the time of sample selection. The neutralizing antibody titer will be collected at the time of assay, and demographic and related data will be entered manually into a spreadsheet format. These data fields are (if available): ZIKV neutralizing titer, participant age, participant gender, year the sample was taken, and the postal code of participant residence Entries will be verified independently by a second person to avoid error. The prevalence of ZIKV will be predicted based on the proportion of seropositivity of the samples. ZIKV problems will be categorized by year of specimen collection.
k. Data sharing: Data from this study will be made available to all listed study investigators for analysis and publication. Public sharing of data will be performed at a time and to the extent required by MoPH and CDC data sharing policies.
l. Study location: All laboratory work for this project will be performed within the laboratory of Dr. Piraporn Utachee, Thai Ministry of Public Health, Dept. of Disease Control, Div. of Vector Borne Diseases, Nonthaburi, Thailand.
PROJECT COORDINATION
This project concept has been developed in collaboration between the Oregon Health and Sciences University, the Thai Ministry of public health /US CDC collaboration. Samples for this project will be selected from the specimen repository managed by the TUC, and will be transferred to the laboratory of MOPH, Department of disease control, Division of Vector Borne Diseases. All samples will be processed in the division of vector borne diseases. Trainees from OHSU will collaborate with DVBD laboratory staff to perform the required assays. Any remaining samples will be returned to the TUC specimen repository at TUC request. Data derived the project will be shared among all study investigators. Data from this project will be co-authored and co-published in collaboration with all appropriate Thai MOPH, TUC and OHSU team members.
TIMELINE
Concept development – Up to May 2021
ERC review – June-September 2021
Specimen retrieving – October 2021
Laboratory processing – November 2021-July 2022
Data analysis – August 2022 to October 2022
ETHICAL CONSIDERATIONS
Risk to participants: This study is a noninterventional retrospective serological survey of anonymized stored specimens. Only samples de-linked from identifying information other than year of sampling, age and gender will be used in this project. No communication will take place with any study subjects, no study procedures will be performed on study subjects, and no identifying information will be used during the course of the study. There are therefore no foreseeable risks to study subjects, either through direct injury or through loss of privacy.
Benefits to the Thai Ministry of Public Health: The immediate goal of this project is to share recent methodological developments in serological diagnosis with colleagues and collaborators at the Thai Ministry of Public Health. If successful, at the completion of this project, the primary laboratory within the Department of Disease Control will possess the necessary reagents, equipment and expertise to perform a highly sensitive, highly accurate neutralization assay for the detection of flavivirus infections. Together, the Thai team and guests from OHSU will apply this method to a set of samples that will shed light on the historical and current epidemiology of flavivirus infections in a sample population in Thailand. These methods and information will benefit current and future epidemiological work in this area by the Thai Ministry of Public Health
Association and Benefits to Thai society: Efforts to reduce the impact of endemic and epidemic flavivirus infections such as Dengue, Zika and Japanese Encephalitis virus depend on accurate measures of local and national prevalence and incidence. Without such measures, it is impossible to understand the magnitude of the infection, track changes over time, and assess the impact of control measures and public health policies. Unfortunately, current methods are insensitive and suffer from extensive cross-reactivity, and therefore limiting the usefulness of data collected by standard methods. The end result of this study will be to establish enhanced methodology for performing serological studies of flaviviral infections that is sensitive, highly specific in discriminating between related viral infections, and scalable for high-throughput use. Studies using these enhanced methods will provide a basis to inform public health policy priorities, design control programs, and assess the efficacy of public health measures over time. Ultimately better control of flaviviral infections will lead to improved health outcomes for the people of Thailand.
BUDGET
The study is funded by Oregon Health and Sciences University, OR, USA (approximately 20,000 USD).
CONFLICT OF INTEREST
The investigators of the study do not have any commercial interests or other financial conflicts of interest related to the conduct of this study
REFERENCES
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