Authors Camilo J. Acosta (corresponding author), Claudia M. Galindo, Mohammad Ali, R. Leon Ochiai, M. Carolina Danovaro-Holliday, Anne-Laure Page, Jin Kyung Park, Hyejon Lee, Mahesh K. Puri, Bernard Ivanoff, John D. Clemens and Zhi-Yi Xu, International Vaccine Institute, Research Park, San 4-8 Bongcheon-7-Dong, Kwanak-gu, Seoul, South Korea 151-818. Tel: +1-610-7873112; Fax: 1-610-7877057; E-mail: firstname.lastname@example.org Remon Abu Elyazeed, Epidemiology Unit, Enteric Disease Research Program, US NAMRU 3, Cairo, Egypt. Vu Dinh Thiem, Do Gia Canh, Dang Duc Anh and Dang Duc Trach, National Institute of Hygiene and Epidemiology, Hanoi, Vietnam. Yang Jin, Yang Honghui and Dong Bai-qing, Guangxi Center for Disease Prevention and Control, Nanning, Guangxi, China. Magdarina D Agtin, Rooswanti Soeharno and Cyrus H. Simanjuntak, National Institute of Health Research and Development, Ministry of Health Republic of Indonesia, Indonesia. Narain H. Punjabi, NAMRU-2, Jakarta, Indonesia. Dipika Sur, Byomkesh Manna and Sujit Kumar Bhattacharya, National Institute of Cholera and Enteric Diseases, Kolkata, India. Qamaruddin Nizami and Zulfikar Bhutta, Aga Khan University, Karachi, Pakistan. Tikki Pang, Research Policy and Cooperation, World Health Organization, Geneva, Switzerland. Allan Donner, University of Western Ontario, Ont., Canada.
Phase-III vaccine efficacy trials typically employ individually randomized designs intended to ensure that measurements of vaccine protective efficacy reflect only direct vaccine effects. As a result, decisions about introducing newly licensed vaccines into public health programmes often fail to consider the substantially greater protection that may occur when a vaccine is deployed in public health programmes, due to the combination of direct plus indirect vaccine protective effects. Vaccine total protection can be better evaluated with cluster randomized trials. Such a design was considered to generate policy relevant data to accelerate the rationale introduction of the licensed typhoid fever Vi polysaccharide (PS) vaccine in Asia by the Diseases of the Most Impoverished (DOMI) typhoid fever programme. The DOMI's programme multi-country study is one of the largest cluster randomized vaccine trials ever mounted in Asia, which includes approximately 200 000 individuals. Its main objective is to determine the effectiveness of a licensed Vi PS vaccine. The rationale and design of this study are discussed. Preliminary results are presented that determined the final planning of the trial before immunization. Important methodological and practical issues regarding vaccine cluster randomized designs are illustrated.
A multi-disciplinary programme (DOMI*) to accelerate the introduction of typhoid fever vaccines in partner Asian developing countries has been underway since the year 2001. Here we describe the rationale and design behind the decision to conduct one of the core projects of DOMI's typhoid fever research agenda: a multi-country effectiveness trial to evaluate the typhoid fever Vi polysaccharide (PS) vaccine and present preliminary results of the pilot phase that guided decisions in the final study design and planning of the trial.
The typhoid fever global prevalence has been recently estimated to be between 21 and 22 million cases and approximately 200 000 deaths for the year 2000 (Crump et al. 2004). In Asia, annual incidence rates have been placed at 198 per 100 000 in Vietnam (Mekong Delta) (Lin et al. 2000) and 980 per 100 000 in India (Delhi) (Sinha et al. 1999). The burden has been aggravated by the emergence of multi-drug-resistant strains that limit treatment options (Wain & Kidgell 2004).
In the absence of an affordable programme to assure safe water and better sanitation conditions in developing countries, vaccination of high-risk populations is considered the most promising strategy for the control of typhoid fever (WHO 2003). Two new-generation licensed typhoid fever vaccines are moderately efficacious and lack significant side effects: the live, attenuated oral vaccine, Ty21a (Germanier & Fuer 1975) and the injectable subunit vaccine, Vi PS (Robbins & Robbins 1984). Phase III licensing and randomized controlled trials in several countries have documented the vaccines’ safety and moderate efficacy but neither has been adopted in public health programmes in developing countries. These vaccines are registered for populations over 2 years of age and therefore cannot be deployed through currently available channels such as the expanded programme of immunization in developing countries, which covers basically under-ones. Such constraint is amplified by the little enthusiasm from public health authorities of introducing vaccines of moderate efficacy. These limitations could be mitigated by generating effectiveness (the net balance of benefits and adverse effects observed when a vaccine is applied routinely in practice) (Clemens et al. 1996) and cost-effectiveness data to support informed policy decisions.
The DOMI typhoid fever programme specific goals are to demonstrate the effectiveness and feasibility of providing Vi PS vaccine under the actual programmatic conditions of impoverished Asian countries and to enable an unambiguous and realistic evaluation of Vi PS vaccine within a public health setting. It includes studies in epidemiology (Acosta et al. 2004), socio-behaviour (Kaljee et al. 2004), economics, and policy (DeRoeck et al. 2005) and to date, large-scale vaccination campaigns have been launched since 2001 in five areas: Hechi, China; Karachi, Pakistan; Hue, Vietnam; North Jakarta, Indonesia; and Kolkata, India.†
The choice of the subunit Vi PS vaccine was based on: consistent efficacy results (55–72%) (Acharya et al. 1987; Klugman et al. 1996; Wang et al. 1997; Yang et al. 2001), even in areas of high typhoid fever incidence; a single-dose regimen; lack of patent protection; no strict cold chain requirements and currently manufactured by local vaccine producers.
Researchers in participating countries and from the International Vaccine Institute (IVI) agreed to evaluate the vaccine by use of a multi-country cluster randomized, evaluation-blinded, controlled trial. The primary objective of this core study is to determine the protective effectiveness of the Vi PS vaccine following mass campaign immunizations administration in a single-dose schedule. Other objectives were to determine the vaccine's cost-effectiveness, safety, and immunogenicity. The study is also evaluating logistic feasibility and factors that influence how the vaccine can be added to immunization programmes. Table 1 provides detailed information on study design, objectives, outcome measurements, and current status in each study site.
Table 1. Diseases of the most impoverished typhoid Vi PS vaccine projects
Study location; collaborating institutions
Study design and subjects
Sample and cluster size (80% power to detect a 50% vaccine protection at a 5% level; loss to follow up, 10%)
Status (updated until first quarter of 2004)
Hechi city (urban), Guangxi province, China
Open cluster-randomized control effectiveness trial
Sample size: 96 468
Pilot phase: October 2001–February 2003
Expected incidence of typhoid fever in placebo: 0.5/1000
Fever episode with S. typhi isolated from blood culture
Second census completed: January 2003
Registered in project census
Total cluster size: 120
Fever episode in which S. Typhi isolated from blood culture or fever ≥3 days and positive serology-proven typhoid fever
Vaccination: April–May 2003
Age: 5–60 years
Type of cluster geographic
Guangxi Health and Anti-Epidemic Centre
Target population: 100 000
Local and systemic events within 3 days after immunization
Expected incidence of typhoid fever in placebo: 0.5/1,000
Second census completed: September 2003
National Institute of Hygiene and Epidemiology (NIHE)
Total cluster size: 68
Vaccination: December 2003–January 2004
Registered in project census
Type of cluster: school-based
Age: 5–18 years
Target population: 70 000
Fever (>37.5°C, axillary)
Pilot phase: May 2003–April 2004
Wards 29 and 30 (slums), Kolkata, West Bengal, India
Evaluation-blinded cluster- randomized control effectiveness trial
Sample size: 38,721
Expected incidence of typhoid fever in placebo: 2/1,000
Census 2: April 2004
National Institute of Cholera and Enteric Diseases (NICED)
Total cluster size: 80
Vaccination: September 2004
Registered in project census
Type of cluster: geographic (premises)
Age: ≥2 years
Target population: 70 000
Fever (>37.5 °C, axillary)
Tanjung Priok and Koja subdistricts (urban), North Jakarta, Indonesia
Logistic and feasibility study
5000 school children in random subgroup of primary schools in selected sub-districts.
Vaccine logistics and feasibility
Pilot phase: August 2001–June 2003
Ministry of Health; National Institute of Health Research and Development (NIHRD)
Resources (human and other)
Vaccination: February 2004
Attending selected schools
Vaccine (procurement, storage, transport, delivery, storage and handling procedures)
Age: 6–11 years
Safe vaccination practices (e.g. vaccine quality, administration, and disposal of sharps)
Detailed costs of vaccine delivery (project and vaccination)
Fever (>37.5 °C, axillary)
Private vaccination costs
Figure 1 explains the different study phases of the trial. Three censuses are to be conducted throughout the complete study period (a minimum of 3 years) to generate: the sampling frame for vaccine trial participation; denominators for incidence rates, baseline population characteristics and migration rates. Prior to the vaccination phase, a 1-year pilot phase will take place to test feasibility of the trial and obtain information on background typhoid fever incidence. During vaccination, teams will immunize by clusters after assessing eligibility of the consenting study population. Typhoid fever cases are to be documented by the study health facilities for 2 years after immunization. Pilot phase and mass immunization campaigns have been completed in all the five study sites and disease and mortality surveillance is underway (Table 1).
A randomized controlled study with an already licensed vaccine was considered relevant. Other designs such as pre- and post-evaluations or case-control studies are known to be prone to bias. Information on vaccine performance with the proposed design in the target population under ordinary public health programmes is to yield objective and realistic results.
A cluster-randomized design allows mimicking the way the Vi PS vaccine would be delivered under public health programme conditions (Hall & Aaby 1990; Clemens 2003). Under this design, the unit of allocation is clusters of individuals. The boundaries demarcating these clusters resemble a unit to be targeted under ordinary conditions (geographic areas or schools). The estimation of the magnitude of total protective effect (direct and indirect) conferred when vaccines are administered to groups (as will usually occur in practice) and evaluation of the impact of vaccination on practical outcomes of public health importance (cost-benefit profile) will be generated.
Eligibility for participation corresponded to the anticipated eligibility of a national immunization programme: school-aged children for Vietnam and Indonesia; all population above age 5 years for China and those above 2 years for India and Pakistan. The vaccine formulation, mode of vaccine delivery, safety measures, constitution of vaccine teams and choice of venue for vaccinating simulated programmatic conditions. In each country, the vaccine campaign lasted 4–8 weeks and was launched in a way that it would not interfere with regular national immunization programmes.
The use of a vaccine in the control arm raised several issues. The moderate efficacy of the Vi PS was known and local investigators requested a vaccine that would benefit the control arm. Hepatitis A vaccine was selected for the majority of the study areas. Like the Vi PS vaccine, it is formulated in individual pre-filled syringes, although the content is not fully identical (solution vs. suspension). Several measures were introduced to ensure ‘blindness’. All vaccines were coded with one of the two letter codes. Furthermore the study staffs responsible (laboratory personnel) for the ascertainment of the outcome were blind to the trial vaccine assignment (evaluation-blinding). Because review board members argued that in most of the study areas the sero-prevalence of hepatitis A is high in early childhood that the benefit in the control arm would be low or moderate, it was agreed to offer Vi PS to the control arm at the end of the study. In China, where the producers of the Vi PS vaccine did not have an adequate blind control, the lyophilized meningococcal A PS vaccine was employed as the active non-masked control.
A community-based passive surveillance system relied on febrile patients, participating in the study, attending the existent health facilities (both private and public). Medical personnel interviewed, examined, and obtained a venous blood specimen for laboratory investigation from all patients (targeted age-group) living in the study area with fever of ≥3 days of duration (prolonged fever). At each study site all relevant clinical information was recorded by use of standard procedures and forms. Printed census booklets and/or computer-based search were used to identify study subjects and their unique identification number during presentation to any project health outpost.
Blood samples were inoculated in sterile Bactec PlusTM (Becton-Dickinson, NJ, USA) bottles and processed to identify Salmonella enterica subspecies enterica serovar Typhi (Salmonella typhi) Serological typhoid fever tests used were: Widal (Biorad, CA, USA), TyphidotTM (Malaysian Bio-Diagnostics Research, Selangor Darul Ehsan, Malaysia), and IDL-TubexTM (IDL Biotech, Sollentuna, Sweden). Bacteriologic and serologic tests were prepared at a central laboratory at each site. All Salmonella isolates were sent to the Centre for Tropical Diseases (Oxford Research Unit, Ho Chi Minh City, Vietnam) for re-confirmation of the strain.
Throughout the study local treatment guidelines were used and S. typhi resistance patterns were determined. All culture-proven subjects or those with positive serology test results were visited at home on days 7, 14, 30 and 90 after onset of illness to confirm the patient's identification and to assess clinical progress and typhoid fever-related disability.
Deaths are documented through project health facilities, censuses and national death reports. Verbal autopsy (VA) forms are completed for all trial participant deaths, regardless of site of death. A shorter, infectious disease-focused and all ages-targeted VA questionnaire was designed to detect cause of death (AMMP 1997).
Adverse events (AEs) following immunization
The date, time of onset, outcome and intensity, and relationship to vaccination were documented for AE through three mechanisms: (i) observation for 30 min after vaccination of all vaccine recipients to detect immediate serious AEs; (ii) solicited AEs were sought for 3 days after vaccination in a randomly selected sub-sample of participants and (iii) unsolicited AEs detected at the project health facilities were recorded throughout the total study period. Trained study staff treated immediate serious AEs and vaccination centres were outfitted with basic emergency equipment. Transportation to a hospital was assured in the event of an immediate serious AE.
Vi immune responses are being monitored in a random sample (200–300 per study site) of both Vi PS and control vaccine recipients. Samples are collected at three points: before immunization, 6 weeks after immunization, and at the end of the 2-year follow up. All quantitative serum Vi titres (IgG) will be measured by enzyme immunosorbent assay (ELISA), and samples will be tested pair-wise. The sample size is based on the comparison between expected seroconversion rates in each study arm.
Data collection and storage
A generic database management system (DBMS) was designed for all sites. The system includes: dual data entry, reporting of errors, editing and logical consistency. All steps are documented and back-up provisions are in place. The DBMS team is responsible for archiving and storing data (electronic and hard copies) at local sites and at the IVI (Seoul). This system assures safety, security and confidentiality of the data.
Feasibility and safety of the immunization campaign
To assess the logistic feasibility and safety of the campaigns, the study teams recorded information on resources required (human and other), vaccine (procurement, storage, transport, delivery, storage and handling procedures), safe vaccination practices (e.g. vaccine quality, administration and disposal of sharps) and detailed costs of vaccine delivery (project and vaccination level and private vaccination costs).
Geographic information system (GIS) mapping of the study areas is used to demarcate the clusters and map the distribution of typhoid fever cases in relation to spatial variables that could be considered confounders (such as distance to the nearest health facilities) (Ali et al. 2004). Buffer zones were not used; hence in the study area all clusters were randomized either to Vi PS or the active control vaccine.
Clusters were randomly assigned to the Vi PS vaccine group or to the control vaccine group to one of two codes, by an independent statistician. In order to ensure balance among clusters, we used a stratified randomization scheme and the principal stratification factors considered were: cluster size, type of cluster (urban vs. rural; elementary vs. middle vs. high school; slum location, etc.), cluster-based typhoid fever incidence, cluster-based blood culture incidence and distance to health facility.
Sample sizes (Table 1) were calculated based on the cluster design (Hayes & Bennett 1999) for two endpoints: effectiveness and immunogenicity. A sensitivity analysis was performed and a conservative estimate of sample size was selected for each site based on an 80% power to detect a 50% vaccine protection at a 5% level of significance (two-sided). This sample size calculation assumed an annual minimum typhoid fever incidence observed during the 1-year pilot phase.
Statistical analysis of the endpoints will take into account the cluster randomization (Donner & Klar 2000; Hayes et al. 2000) and will be based on the generalized estimating equations. Vi vaccine effectiveness will be estimated by calculating risk differences of typhoid fever between those who received an active control and those who received Vi vaccine over a 2-year period. The incidence of typhoid fever will be defined as fever together with a positive blood culture. Analysis of the surveillance data will be based on a fixed cohort approach. Should an imbalance occur at the time of the analysis, the adequate logistic model will be fitted to control for it.
The analytical plan was developed at an initial stage of the study. A meta-analysis is planned and the design effect (variation between the designs of each pooled study) will be taken into account (Donner et al. 2001).
The principles that govern biomedical research involving human subjects apply to this project and all researchers followed the Declaration of Helsinki (WMA 2002) and the International Conference on Harmonization's Good Clinical Practice Guidelines (EU, MHLW & FDA 1997). The application of these principles in developing countries poses complex ethical and practical challenges. An independent data and safety monitoring board with clinical, statistical, bioethical, and typhoid fever expertise monitors the trials. Full protocols were developed jointly with local investigators and submitted, before initiation of the projects, to national review boards, the World Health Organization Ethics Review Committee, and the IVI institutional review board. The consent process has included appropriate information dissemination and assured both individual/parental and community consent. The documentation of this process has been adapted to the cultural characteristics of the study site and to the large-scale dimensions of the study.
Below are some preliminary data obtained during the pilot phase of the trial that caused us to reconsider and adjust the study design and milestone indicators.
Demographic data derived from the first census (Table 2) provided the potential target population in each site. Some sites had to be expanded to include an adjacent area to increase the study power. Data on willingness to participate (Table 2) in the census also gave us insight on participation coverage. After providing general information on the aim of the project, 24% of the households interviewed in Hue (Vietnam) answered positively to the question on willingness to participate; in other country study sites the proportion was over 87%. In Vietnam the trial information strategy was planned well in advance to the immunization campaigns and included local health and education authorities and the parents.
Table 2. Selected results of census 1 in the multi-country Vi PS vaccine study
North Jakarta (Indonesia)
Hue city (Vietnam)
* In the denominator the total population interviewed.
18 700 (12)
25 992 (9)
10 161 (17)
40 254 (25)
79 777 (28)
33 838 (22)
20 198 (35)
77 969 (49)
118 367 (42)
73 408 (48)
22 567 (39)
18 091 (11)
32 695 (12)
23 726 (16)
24 957 (9)
11 386 (7)
160 262 (100)
281 788 (100)
152 141 (100)
58 197 (100)
Population willing to participate in project*
147 380/160 196 (92)
67 629/281 788 (24)
100 171/115 139 (87)
13 519/15 021 (90)
Prevalence (point), 3 ≥ days of fever in past month*
12 737/160 262 (8)
989/85 152 (1)
657/132 450 (1)
780/24 404 (3)
Because a cluster trial design is prone to disease detection bias and complete surveillance systems are desirable, we derived the point prevalence data of febrile cases from the initial census (Table 2). This allowed us to estimate the maximum expected numbers of prolonged fever cases at the community level. Hence we had an estimate of the degree of coverage of the disease surveillance systems during the lead-in phase.
Other data from the initial census showed that health-seeking behaviour for prolonged fever varied by country (Figure 2) whereas in North Jakarta (Indonesia) and Karachi (Pakistan) most patients attend public health facilities; in Hue (Vietnam) persons are more likely to seek health care from private practitioners and pharmacies. The Hechi (China) febrile population attends hospitals. Initially efforts were made to have all available health facilities (including private) in the surveillance network; however, in North Jakarta (Indonesia), the private sector support was not feasible. Thus, the initial design was deemed not possible in Jakarta and it was decided to evaluate logistics and feasibility without a control arm.
Classically, efficacy determinations of typhoid fever vaccine trials have relied on positive S. typhi blood culture as the main end point. Initial results of the pilot phase surveillance period, showed unexpectedly low positive S. typhi blood culture incidences and several fold higher serologic-based incidences (data not shown). Therefore, we developed an additional case definition to enable a more comprehensive picture of the burden of the disease and to evaluate the ‘full’ protective impact of the vaccine. This new end point includes febrile subjects with negative blood cultures who are positive for a pre-specified combination of serological results. Sensitivity will be maintained at its highest level while keeping specificity at 100%.
The planning and design of this multi-country trial illustrate several challenges beginning with the decision to carry out such a trial, given the tremendous investment entailed. Nevertheless if the results of the DOMI typhoid fever effectiveness trial proved promising, they would strongly support decisions by policy-makers with regard to introduction of a typhoid fever vaccine into national immunization programmes, which in turn might encourage other endemic countries to use the vaccine.
Another significant hurdle was the justification for utilizing a controlled cluster-randomized evaluation with an already licensed vaccine. However the scarcity of resources for delivering public health interventions in the DOMI study sites makes the need for this sort of objective evidence compelling.
Third was the difficulty in applying standardized clinical and microbiological protocols to yield comparable results between countries. This required bridging technical gaps in remote settings that usually lack human, economic, and technical resources while introducing international research standards.
Our experience above with cluster randomized trials (CRTs) brings to light certain points that despite the proliferation of literature dealing with methodological challenges are still not being appreciated by trial investigators when planning such large scale CRT with a vaccine (Donner & Klar 2002). First, because of less statistical efficiency, in comparison with individually randomized trials, CRTs should be avoided. Strong reasons to conduct CRT include: interventions applied at the cluster level (schools, geographically determined health areas) and to simulate public health practice and hence generate effectiveness results. Nevertheless, total protective effects if found in such a practical design are less likely to be extrapolated to other settings because that would depend on local conditions such as vaccine coverage, logistics (e.g. cold chain, injection techniques, potency for the vaccine, etc.) in each country.
Second, there is an ongoing debate regarding informed consent in CRT. In the typhoid fever trials written individual informed consent was deemed necessary by the IRBs even when this procedure distracts the design from its original practical approach and may have impact in vaccine intake.
Third, distance to the health facilities has been described as a potential confounding variable in vaccine trials (Alonso et al. 1994). Hence cluster-baseline data on health seeking behaviour ought to be supported by base-line cluster specific GIS generated maps and analysis. The incidence rate of other similar infectious diseases (disease indicator) should be measured in the vaccine and control groups to test for similarity between the two groups with respect to health seeking behaviour.
Fourth, an important potential source of bias in estimating the incidence rates of the disease is under-ascertainment due to the failure of study participants with relevant symptoms to seek care from healthcare providers and facilities associated with the surveillance system, and the use of antibiotics prior to collection of specimens for culture yielding a lower than expected culture-proven incidence rate. In our study efforts were made to include all health facilities, both private and public. Additionally the wide used of antibiotics in this area prompted us to use typhoid fever serology tests to capture negative S. typhi culture typhoid fever cases. Such disease surveillance limitations may call to have harder end-points as primary objectives, for example overall mortality when planning large vaccine CRT.
Large-scale vaccination campaigns have been launched in China, Pakistan, Vietnam, Indonesia and India among over 200 000 individuals. Some results on feasibility, logistics, vaccine coverage and safety are available (Yang et al. 2005). Effectiveness results will be available by the year 2006. These results shall help to further enlighten the value of cluster randomized vaccine trials.
The disease of the most impoverished (DOMI) programme coordinated by the International Vaccine Institute (IVI) and supported the Bill and Melinda Gates Foundation has as main goal to accelerate the development and introduction of vaccines against cholera, typhoid fever and shigellosis.
In collaboration with the Guangxi Center for Disease Prevention and Control, China; the National Institute of Health and Development, Ministry of Heath and NAMRU-2, Indonesia; the National Institute of Hygiene and Epidemiology, Ministry of Health, Vietnam; The Aga Khan University and Hospital, Karachi, Pakistan and The National Institute of Cholera and Enteric Diseases, Kolkata, India.
This work was supported by the Diseases of the Most Impoverished Programme, funded by the Bill and Melinda Gates Foundation. We are indebted to the late Dang Duc Trach for his contribution and encouragement to bring vaccines for those in need. We thank the following persons: Shousun Szu, Wang Bingrui, Jeremy Farrar, John M. Albert, Varaprasad Reddy, Nirmal Kumar Ganguly, Lalit Kant, Afia Zafar, Rumina Hasan, Yanning Gao, Hasbullah DVM, Lorenz von Seidlein, Jacqueline Deen, Luis Jodar, Moshaddeque Hussein, Paul Kilgore, Kok-hai Ong, T. Afifah Ibrahim, Michael Goon, Eun Young Kim, Sue Kyoung Jo, John Wain, Amanda Walsh, Hans Bock, Didier Leboulleux, and more than 500 staff working for the following institutions: Guangxi Center for Prevention and Disease Control (CDC), China; Jiangsu CDC, China; South East University, Nanjing China; Lanzhou Institute, China; National Institute of Hygiene and Epidemiology, Vietnam; Thua Thien Hue Preventive Medicine Center, Vietnam; Oxford University – Wellcome Trust, Tropical Unit HCM city, Vietnam; India Council Medical Research, India; National Institute of Cholera and Enteric Disease, India; Aga Khan University, Pakistan; NIHRD and US-NAMRU2, Indonesia; GlaxoSmithKline; Shanta Biotechnics, India; University of North Carolina, USA; University of Western Ontario, Canada; WHO, Switzerland; NIH-US and The IVI, Korea.