Rotavirus infection in children in Southeast Asia 2008–2018: disease burden, genotype distribution, seasonality, and vaccination

Background Rotaviruses (RVs) are recognized as a major cause of acute gastroenteritis (AGE) in infants and young children worldwide. Here we summarize the virology, disease burden, prevalence, distribution of genotypes and seasonality of RVs, and the current status of RV vaccination in Southeast Asia (Cambodia, Indonesia, Lao People’s Democratic Republic, Malaysia, Myanmar, Philippines, Singapore, Thailand, and Vietnam) from 2008 to 2018. Methods Rotavirus infection in Children in Southeast Asia countries was assessed using data from Pubmed and Google Scholars. Most countries in Southeast Asia have not yet introduced national RV vaccination programs. We exclude Brunei Darussalam, and Timor Leste because there were no eligible studies identified during that time. Results According to the 2008–2018 RV surveillance data for Southeast Asia, 40.78% of all diarrheal disease in children were caused by RV infection, which is still a major cause of morbidity and mortality in children under 5 years old in Southeast Asia. Mortality was inversely related to socioeconomic status. The most predominant genotype distribution of RV changed from G1P[8] and G2P[4] into the rare and unusual genotypes G3P[8], G8P[8], and G9P[8]. Although the predominat strain has changed, but the seasonality of RV infection remains unchanged. One of the best strategies for decreasing the global burden of the disease is the development and implementation of effective vaccines. Conclusions The most predominant genotype distribution of RV was changed time by time. Rotavirus vaccine is highly cost effective in Southeast Asian countries because the ratio between cost per disability-adjusted life years (DALY) averted and gross domestic product (GDP) per capita is less than one. These data are important for healthcare practitioners and officials to make appropriate policies and recommendations about RV vaccination.


Rotavirus (RV) history
RV was first identified in cattle in 1969 [1]. The virus appeared similar to those that cause diarrhea in mice [2], calves [3], and a virus identified from a rectal swab of a healthy monkey [4]. In May 1973, Bishop, Davidson, Holmes, and Ruck examined ultrathin sections of duodenal mucosa from children with acute gastroenteritis (AGE) by electron microscopy (EM), and found abundant viral particles in the epithelial cell linings of the upper villous surface which were similar in appearance to the RVs discovered in animals before [5]. EM also revealed 70-nm particles in negatively stained fecal extracts [6,7]. The viral particle was initially identified by several names including reovirus-like, orbivirus-like, duovirus, infantile gastroenteritis virus, or a "new" virus.
The wheel-like structure observed on EM eventually led to the naming concensus of Rotavirus (rota is Latin for wheel) [8]. RVs have now been shown to be a cause of diarrhea in the young of many mammalian and avian species [9].

RV morphology
RVs are 70-nm, non-enveloped RNA viruses belonging to the family Reoviridae. The RV genome consists of 11 segments of double-stranded RNA (dsRNA) surrounded by a triple-layered capsid. Each genomic fragment encodes protein of different function. The outer layer proteins (viral protein [VP] 4 and VP7) mediate attachment and penetration; the inner layer is composed of VP2 protein and encloses the viral genome and the minor protein VP1, the viral RNA-dependent RNA polymerase, and VP3, the viral capping enzyme. The middle layer is composed of VP6 which interacts with and stabilized the inner and outer layer [9]. VP6 defines species/group and subgroup specificities [10][11][12]. All RNA segments, except for segment 11, are monocistronic, encoding either structural viral proteins (VP1 to VP4, VP6, and VP7) or non-structural proteins (NSP1 to NSP5). Genome segment 11 codes for two proteins: NSP5 and NSP6 [9]. RVs can be differentiated by a dual classification system, based on the two outer capsid proteins, VP7 and VP4, that determine the G (VP7, glycoprotein) and P (VP4, protease-sensitive) genotypes [13]. At least 36 G types and 51 P types have so far been identified in humans and animals [14].
Indirect immunofluorescence techniques targeting VP6 are used to differentiate RV species. RVs are currently differentiated into at least nine species, designated A to I and a tentative tenth species, J. RVA infects in birds and mammals; RVB, RVC, RVE, RVH, and RVI have been detected in one or more mammalian hosts; RVD, RVF, and RVG have been detected only in birds; RVJ infects bats [18][19][20]. Table 1 shows rotavirus groups and its host.

RV infection burden
RVs were recognized as a major cause of AGE in infants and young children in 1973 [2,7]. RV is the leading cause of diarrhea-associated mortality among children younger than 5 years, although the burden of RV has decreased during the past decade. RV infections were responsible for approximately 128,515 deaths annually among children younger than 5 years. RV constitutes 1 of the 13 diarrhea etiologic agents measured in the 2016 Global Burden of Disease Study [37]. It is the most prevalent agent causing severe diarrhea in both developed and developing countries [38,39]. After RV vaccine introduction in developed countries, norovirus become the predominant viral pathogen that caused AGE in children. Norovirus prevalence remained stable or increased, whereas rotavirus activity dramatically decreased [40][41][42]. Nevertheles, from 2000 to 2013 in Southeast Asia,  [25,32] approximately 50.7% (n = 10,765) of total diarrhea mortality was associated with RV disease [43]. Figure 1 shows the prevalence and death caused by diarrheal disease and RV in children under 5 years old from 1990 to 2017 in Southeast Asia. The prevalence of diarrheal diseases in Southeast Asia countries varies, but the mortality trend associated with diarrhea and especially RV infection has been decreasing in recent years. Lao People's Democratic Republic [PDR] reports one of the highest death rates in this period. However, improvement in hygiene and sanitation combined with the introduction of the rotavirus vaccine has contributed to decreasing RV infection [46].
We collected RV surveillance data from 9 countries in Southeast Asia (Cambodia, Indonesia, Lao PDR, Malaysia, Myanmar, Philippines, Singapore, Thailand, and Vietnam) for the years 2008-2018 to estimate the proportion of RV gastroenteritis (RVGE) ( Table 2). A total of 52,579 stool samples were collected of which 21, 444 (40.78%) were RV positive. Acute diarrheal disease caused by RV is still a major cause of morbidity and mortality in children under 5 years old in developing countries, which may be attributed to the regions' lower standards of living and hygiene conditions [46]. However, our study revealed that as the gross domestic product (GDP) per capita increases, and the economic status of Southeast Asian countries improves, the RV mortality rate steadily declines (Fig. 2). Higher socioeconomic status (SES) can improve sanitation, hygiene practices, and healthcare facilities to support better living conditions and decrease the RV mortality in children.

RV genotype distribution
Among the data from Southeast Asia countries examined, the most predominant genotype distribution of RV has changed except in Lao PDR and Malaysia. In 2009-2013, G1P [8] and G2P [4] were the most predominant genotypes, but starting 2014, it changed into the rare and unusual genotypes G3P [8], G8P [8], and G9P [8]. Several uncommon RV genotypes such as G2P [8], G8P [6], G5P [19], G9P [4], G9P [6], and G1P7 [5] were identified in the surveillance data. The presence of such diversity among RV isolates provides insight into the evolution of these strains, which can arise due to point mutations, genetic rearrangements, reassortment events, and interspecies transmission [13,80,81]. Circulating RV strain appears diverse despite RV vaccination, which may enable the increase in the prevalence of non-vaccine strains. Thus, the circulation of strains in which vaccines have lower efficacy eventually impairs vaccine effectiveness [82]. In the Philippines, where the Rotarix® vaccine was introduced in July 2012, the frequency of RVGE cases caused by G1P [8] decreased while the circulation of G9P [8] increased significantly [83]. The genotype distribution of RV in Southeast Asia is shown in Table 3.

RV seasonality
Meteorological conditions have an indirect yet important impact on the epidemiology of human rotavirus infection. Weather-related low indoor relative humidity and indoor crowding may be key factors in the epidemiology of rotavirus disease. Hospitalizations for rotavirus gastroenteritis tended to be more common after a cold or dry month than after a warm or wet corresponding calendar month [86].
The temporal trend of Cambodian RV infection shows substantial year-round transmission with prominent peaks during colder, dry months. Peaks typically occurred between November and May [47]. In Indonesia, RV infection was present throughout the year and did not demonstrate clear annual seasonality [55]. Conversely, infection generally peaks during the rainy season in Singapore and Malaysia. An outbreak of RV infection was observed from January to March [65,87]. Positive RV cases increased in number and proportion during the dry season (January-April) each year in Lao PDR [88]. In Myanmar and the Philippines, RV infection has a strong seasonal peak in colder, drier months, as seen in other Asian countries. The highest rate of RV infection occurred in January and February [61,63]. RV cases in Thailand were most prevalent during the cooler months, specifically from January to March, but RV was detected every month in the northern part of Thailand, where the weather is relatively cooler compared to the rest of the country [68]. In Vietnam, RV was detected every month, but most RV gastroenteritis (GE) cases occurred between December and May [78]. Figure 3 shows the RV seasonality in Southeast Asian countries.
The seasonal pattern in RV varies by climatic region and is also associated with local weather. A reduction in RV rates was associated with increased temperature and precipitation [89]. There is a significant association  between increased numbers of estimated positive RV cases and lower humidity, rain, and temperature. In children younger than 2 years old, RV was the pathogen most frequently identified in the winter, dry, or cool/dry seasons [90]. In tropical climates, the higher temperature was associated with a greater decrease in RV than in humid mid-latitude climates [91]. It had been suggested that the seasonal pattern may be driven by airborne transmission of the disease [92].

RV vaccination
One of the best strategies for decreasing the global burden of disease is the development and implementation of effective vaccines [43]. RV is the most common cause of vaccine-preventable severe diarrhea [93]. The World Health Organization (WHO) recommended that RV vaccines be included in immunization programs in the European region and the Americas in 2006. In 2009, following efficacy studies in low-income countries (LICs) and lower-middle-income countries (LMICs) in Africa and Asia, the WHO recommended the use of RV vaccines in all National Immunization Programs (NIPs) [94]. RV vaccines had been introduced in 101 countries by the end of 2018, and global coverage was estimated to be at 35% [95]. Most countries in Southeast Asia have not yet introduced national RV vaccination programs. Among Asian countries, only the Philippines, and recently Thailand have introduced the vaccines on a limited basis [96]. The Global Alliance for Vaccines and Immunizations (GAVI), also known as the Vaccine Alliance, actively supports RV vaccination by subsidizing the cost in eligible countries. In LMIC, the budget impact is an important criterion for funding new interventions, particularly for large public health investments such as new vaccines. By the end of 2018, GAVI had funded RV vaccine introductions in 45 countries [97].

RV vaccine history
Research to develop a safe, effective RV vaccine began in the mid-1970s when investigators demonstrated that previous infection with animal RV strains protected laboratory animals from experimental infection with human RVs [98]. The first multivalent live oral reassortant vaccine developed was RotaShield® (a rhesus RV tetravalent [RRV-TV] vaccine) in the late 1980s [99]. The RRV-TV vaccine was licensed in August 1998 for routine use in children in the United States at 2, 4, and 6 months of age due to its proven efficacy [100]. In 1999, RotaShield® was voluntarily withdrawn from the U.S. market due to an increased risk of intussusception within 3 to 14 days after the first dose in infants < 3 months of age [101].
Due to the past association of intussusception and the earlier RV vaccine, large safety studies were performed both for RV1 and RV5 before market authorization [102]. Rotarix® vaccine were highly efficacious in protecting infants against severe rotavirus gastroenteritis [66,103] and were not associated with an increased risk of intussusception [104][105][106]. The pentavalent rotavirus vaccine (RotaTeq®) was highly efficacious against severe rotavirus gastroenteritis and provided substantial protection against rotavirus gastroenteritis of any severity. A significantly increased risk of intussusception in vaccine recipients was not detected [107]. In February 2014, WHO reviewed global intussusception data and found that the risk of intussusception following current rotavirus vaccines remains small compared to the benefits of preventing the impact of severe diarrhea [108]. At the end of 2018 there are four globally available WHO-prequalified oral vaccines (Rotarix® and RotaTeq®, Rotavac® and Rotasiil®) [109], one rotavirus vaccine licensed in China (Lanzhou lamb RV vaccine), one in Vietnam (Rotavin-M1), and there are several candidates in development [110].
Two RV vaccines, Rotarix® and RotaTeq®, have been developed by Glaxo Smith Kline and Merck, respectively. Rotarix® is a live attenuated monovalent vaccine derived from the most common human RV strain, G1P [8]. Rota-Teq® is a live attenuated pentavalent vaccine containing mono-reassortant strains with genes encoding the human G1, G2, G3, G4, and P [8] protein in the genetic background of a bovine RV strains [39,100]. These vaccines are highly effective for the global prevention of severe diarrhea and are included in the NIPs or phased subnational introductions in 101 countries by the end of 2018 [95,111].
Rotavac® (Bharat Biotech International Limited) is a monovalent human-bovine RV vaccine. The vaccine consists of the 116E RV strain, which is a naturally occurring reassortant strain G9P [11], containing 1 bovine RV gene P [11] and 10 human RV genes [112]. Rotavac® is the first to be introduced into a public vaccination program as of April 2016 when it was introduced in four states in India [113]. Rotasiil® is a live attenuated human-bovine reassortant pentavalent RV vaccine that contains genes encoding the VP7 of serotypes G1, G2, G3, G4, and G9. In March and September 2018 Rotavac® and Rotasiil®, respectively achieved WHO prequalification. Rotavac® has a vaccine efficacy of 53.6% for severe RV diarrhea in India [112], while Rotasiil® has efficacies of 60.5 to 66.7% in India [114] and Niger respectively [115]. Rotasiil® can safely be delivered with decreased dependence on the availability of a cold chain [116].
The Lanzhou Institute of Biological Products manufactures the Lanzhou Lamb RV vaccine (LLR). It is a monovalent lamb vaccine strain G10P [12], attenuated by cell passage [117], and was licensed in China in 2000. When given to children between 9 and 35 months old, one dose of the LLR vaccine conferred partial protection [118]. Vaccine effectiveness in children under 5 years of age was recently estimated at 35% (13 to 52%) against RV diarrhea and 53% (15 to 75%) against moderate-tosevere RV diarrhea based on a large case-controlled study [119].
Rotavin-M1 vaccine is manufactured by the Center for Research and Production of Vaccines and Biologicals and was licensed for use in Vietnam in 2012. The vaccine was derived from an attenuated strain, G1P [8], isolated from a Vietnamese child. A clinical trial found the vaccine to be safe and immunogenic in Vietnamese infants [120].
Another candidate RV vaccine, RV3-BB, was developed from a neonatal strain G3P [6] identified in Australia, with ongoing early clinical studies conducted in New Zealand and now underway in Indonesia. It was also successfully implemented for vaccination of neonates [121]. Table 4 describes the comparison of all oral RV vaccines developed and used so far.
Intussusception, neutralizing antibodies present in breast milk, as well as the lower vaccine effectiveness in less developed settings has stimulated interest in an alternative, parenteral approach to immunization [123][124][125][126]. The inactivated rotavirus particles, protein sub-units or virus-like particles (VLPs, structurally-similar to live virus) are being investigated as rotavirus vaccine candidates [125,127,128].

Vaccine efficacy
RV vaccination does not completely protect young children against infection, but it reduce the severity of RVGE [135]. RV vaccines are highly effective in preventing severe gastroenteritis in young children during the first 5 years of their life, particularly in developed countries [136]. The SES of a country seems to influence RV vaccine effectiveness [137]. Vaccination was predicted to prevent 93, 86, and 51% of severe RVGE in high, middle, and low SES, respectively [126]. Analysis of the data for the Asia region found median vaccine effectiveness of 94% in low child mortality countries, 64% in medium child mortality countries, and 49% in high child mortality countries [138]. Factors that might contribute to this phenomenon including gut microbiota, genetic factors, transplacental antibodies, enteric pathogens, and environmental enteropathy [139,140]. Evidence suggests that vaccine efficacy may vary by setting, due to regional differences in circulating RV vaccine strains and reduced efficacy of oral vaccines in settings with a high prevalence of malnutrition and gastrointestinal infections [141]. Pooled efficacy estimate of Rotarix® and RotaTeq® against severe RVGE in industrialized countries is 88% during the first year of age and 83% during the second year. However, RV vaccine efficacy is much lower in countries where the mortality rate for children under 5 years of age is high [142]. The efficacy of Rotarix® and RotaTeq® in the U.S. depends on the level of exposure during the RV season [143]. It can be concluded that vaccine efficacy is affected by individual factors such as nutritional level, gut microbiota, genetic factor, transplacental antibody and environmental enteropathy and external factors including SES, circulating vaccine strain, childhood mortality rate, and RV season in each country. Additionally, RV vaccination confers herd protection among infants and children under 5 years old who had not been vaccinated [144][145][146]. In developing countries with lower RV vaccine efficacy and coverage, indirect protection gain from herd immunity is more significant than in industrialized countries where vaccine efficacy and coverage exceed 90% [142].

Vaccine introduction in Southeast Asia
Many countries in Southeast Asia have not implemented national RV vaccination programs including Cambodia, Indonesia, Lao PDR, Malaysia, Myanmar, Singapore, Thailand and Vietnam. One reason is because of uncertainties regarding the cost-effectiveness of incorporating RV vaccination into the NIP. In addition, the vaccine's decreased efficacy in LIC settings has discouraged its introduction. Prevention of diarrhea in these countries has focused on patient treatment and the management of water quality, sanitation, and hygiene [84].
In Indonesia, the RV vaccine has been commercially available since 2011. Indonesia's national vaccine manufacturer, PT. Bio Farma, Bandung, is developing an RV vaccine using G3P [6] strain in collaboration with the Murdoch Children's Research Institute in Melbourne, Australia [125]. Bio Farma is currently driving clinical development, intending to introduce the vaccine into the Indonesian NIP by 2021, and eventually develop a product for the global market [128].
Currently Rotarix® and RotaTeq® are commercially available in Malaysia through private health providers [147]. However, RV vaccine is not included in Malaysia NIP because it is not considered permissible under Islamic shariah law (halal) [84]. The current oral rotavirus vaccines use porcine trypsin in the manufacturing process [148]. There are also concerns about competing public health priorities and price [84]. The Health Ministry recently said the RV vaccine would be included in NIP if the associated mortality rate for children aged 5 and below exceeded 10%. However, the childhood mortality rates in Malaysia was 0.5% in 2014 and 2.9% in 2015 [149].
In Myanmar and Lao PDR where individual incomes are relatively low, international assistance will support for the RV vaccine introduction in 2020. The total amount of GAVI support for Myanmar is $4,088,000 [150]. Lao PDR is also planning RV vaccine introductions into the NIP in near future and assistance will be provided the Asian Development Bank and GAVI through an accelerated transition program [84].
In July 2012, the Philippines became the first Asian country to introduce RV vaccines into its NIP. The Philippines has initially focus on immunizing children living in the poorest communities, which have the highest child morbidity and mortality rates from the diarrheal disease [151]. The target population was identified by the Department of Social Welfare and Development, but there were challenges with nationwide vaccine distribution. In 2014, vaccine introduction was limited to the Caraga region, where it was co-administered with oral polio vaccine and the pentavalent vaccine. By 2015, vaccine coverage was close to 90% in the province of Agusan del Sur within this region but subsequently decreased due to a supply shortage [84]. In Agusan del Sur, the RV vaccine became available to the poorest quintile in September 2012; in January 2013, availability was expanded to all age-eligible children in two municipalities, San Francisco and Prosperidad; it was available to the entire province in July 2014. RV vaccine introduction was associated with a substantial decline in diarrheal hospitalizations and outpatient consultations for diarrhea in Agusan del Sur, Philippines [152].
Two live-attenuated, orally administrable RV vaccines Rotarix® and RotaTeq® were licensed in Singapore in October 2005 and July 2007, respectively [106]. To date, RV vaccination is optional in Singapore [65].
The National Vaccine Committee of Thailand considered the introduction of an RV vaccine in 2010 in Sukhothai and Petchabun. Sukhothai province began a routine immunization program with an RV vaccine in October 2011. Evaluation of the first introduction was completed in 2017 and concluded that RV vaccine was highly effective in preventing diarrheal hospitalizations and conferring herd protection among older children who had not been vaccinated [144]. Therefore, universal RV vaccination for infants has been implemented in Thailand since January 2020.
Vietnam is located in a region of high RV infection incidence and eligible for financial support to introduce vaccines into the expanded program of immunization (EPI) from the GAVI [153]. In 2012, the local vaccine manufacturer Polyvac licensed Rotavin-M1, which is based on an attenuated G1P [8] strain. Rotavin-M1 will be offered to children less than 1 year through a two-dose schedule, vaccinating infants at 2 and 4 months [120].
Rotarix® and RotaTeq® are also licensed in Vietnam and are available in the private sector, with approximately 590, 000 doses imported since 2017. That same year, the Government approved the introduction of RV vaccination into Vietnam's NIP by September 2019 with GAVI support. In 2021, the national government will pay for 80% of the vaccine cost, while GAVI will cover the remaining 20% and all operational costs. By 2022, all costs will be covered by the government [84].

Health and economic impacts of RV vaccination
In LMICs, understanding the short-and long-term impact of intervention adoption on national budgets is critical for ensuring program sustainability [154]. Both budget impact and cost effectiveness are key criteria, among others, for policy makers deciding how to allocate limited resources [155].. Vaccination would be considered a worthwhile investment for improving general childhood development and health levels in most LIC. The highest reduction in burden would be achieved in countries with a high disease burden (≥200 RV deaths per 100,000 children under 5 years old), but a similar reduction would be achieved in countries with a medium burden (100-200 RV deaths per 100,000 children under 5 years old) because disease burden reduction also depends heavily on population size and countryspecific vaccine efficacy adjusted for local RV serotype distributions [156].
For GAVI non-eligible countries, the price for Rotarix® is $2.49-7.27, and for RotaTeq® is $3.65-5.09 [157]. For GAVI-eligible countries, the price per dose will depend on the country's gross national income per capita averaged over the previous 3 years. As such Indonesia, Malaysia, the Philippines, Singapore, and Thailand are fully not eligible for GAVI vaccine prices and will have to rely on self-financing [158]. For LIC in Asia, introducing vaccines would halve RV-related deaths and medical visits, leading to significant cost reductions [159].
WHO-CHOICE (CHOosing Interventions that are Cost-Effective) uses the GDP as an indicator to develop the following widely referenced categories of cost-effectiveness. Disability-adjusted life years (DALYs) averted is a widely used indicator that allows easy comparison with a 'no vaccination' strategy and with others public health interventions. The ratio between costs per DALY averted and GDP per capita less than one is defined as highly cost effective. The ratio between 1 to < 3 and ≥ 3 are defined as cost effective and not cost effective, respectively [160]. The lowest and highest GDP values were in Myanmar with $1326 and Malaysia with $11,239, respectively [79]. Figure 4 shows the comparison between costs per DALY averted with each country's GDP in 2018. We excluded the Philippines because the RV vaccine already included in the country's NIP and Singapore because Singapore is a high-income country. According to categories of cost-effectiveness, RV vaccine introduction into Southeast Asia countries is highly cost-effective because the ratio between costs per DALY averted and GDP per capita it is less than one.

Conclusions
According to 2008-2018 RV surveillance data for Southeast Asia, 40.78% of all diarrheal cases were causes by RV. Acute diarrheal disease caused by RV is still a major cause of morbidity and mortality in children under 5 years old in Southeast Asia. Among all assessed Fig. 4 Comparison between cost per DALY averted and GDP per capita in Southeast Asia. Introducing the rotavirus vaccine in Southeast Asia is highly cost-effective because the ratio is less than one [79,107,137,161] countries, the most predominant genotype distribution of RV changed from G1P [8] and G2P [4] into the rare and unusual genotypes G3P [8], G8P [8], and G9P [8]. Although the predominant RV strain has been changed, but the seasonality of RV infection remains unchanged. Continuous surveillance is necessary to determine whether they are regional genotype differences. Epidemiological data on RV prevalence will greatly facilitate vaccine development. In the mean time, the development of new vaccines will be needed if RVs are able to evade current vaccine immunity. More effective vaccines may also further decrease RV infection in children in LIC and LMIC, where currently available vaccines provide moderate efficacy. RV vaccine efficacy is affected by individual factors and external factors. Although most countries in Southeast Asia have not yet introduced national RV vaccination programs, such introduction is projected to be highly cost-effective because the ratio between costs per DALY averted and GDP per capita it is less than one. Authors' contributions FBL drafted the manuscript. FBL and YP wrote the article and prepared the figures. SV, NW, and YP revised and finished the final version of the manuscript. All authors read and approved the final manuscript.

Authors' information
Fajar Budi Lestari is a doctoral student at the Inter-Department of Biomedical Science, Faculty of Graduate School, at Chulalongkorn University, in Bangkok. Her primary research interests are zoonotic diseases and molecular epidemiology and evolution of rotavirus in humans and animals. Availability of data and materials Not applicable.
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.