Molecular Epidemiological Study of Human Adenovirus in Children with Acute Flaccid Paralysis and Gastroenteritis in Pakistan

Abstract

Infectious diseases are among the leading cause of disability and mortality in Pakistan. Epidemics of new and old infectious agents appear from time to time, having a negative impact on people and magnifying the burden on the economy. In Pakistan, among major causes of mortality in children less than five years, diarrhea contributes 11% to the total mortality rates. Before reaching age five, one in every eleven children in Pakistan dies. Post neonatal deaths are due to diarrhea, pneumonia, meningitis and sepsis. WHO collaborated rotavirus surveillance program screen the diarrheal samples for rotavirus but no system presents in country for other diarrhea causing viruses. In addition to this, Pakistan also harbors the highest numbers of poliomyelitis cases in the world, threatening global eradication goals. Surveillance for poliomyelitis relies on investigation and reporting of children who develop acute flaccid paralysis (AFP). However, the country making tremendous efforts for the eradication of polio with the help of its extensive AFP surveillance activities. But a recent report indicates the increased in number of non-polio AFP cases from 2018 (14 per 100,000 children aged < 15 years) to 2019 (16.2 per 100,000 children aged < 15 years) which highlighted the role of other infectious and non-infectious agents that may be responsible in causing AFP. Pakistan is still struggling to cope with AFP cases, which are caused by agents other than poliovirus, but among these agent’s adenovirus is ignored. In our country, there is no system present for the screening of non-polio AFP causing adenoviruses while there is data present from other countries that focused on the human adenoviruses responsible for causing AFP. However, the infectious morbidity and mortality is huge in country, Pakistan had no formal training programs in infectious diseases, and a tremendous shortage of individuals with the requisite skills to conduct independent research in infectious diseases affecting Pakistani children. In light of above mentioned facts, investigation of adenovirus prevalence among children with AFP as well as in diarrheal samples was then conducted with the aim of testing its association with the disease. Acute flaccid paralysis (AFP) is an infrequent clinical syndrome includes a sudden onset of flaccid paralysis in one or more limbs in children less than 15 years old. Infectious and non-infectious agents, (e.g. metabolic disorders), trauma and metal toxicity as well as post-infectious autoimmune conditions (e.g. Guillain-Barre Syndrome) may be involved in the establishment of this syndrome in humans. The recognized viral etiologies of AFP including poliovirus, nonpolio enteroviruses, flaviviruses such as West Nile, St Louis, and Japanese encephalitis viruses, herpesviruses, adenoviruses, and others. AFP includes the participation of spinal cord or anterior horn cell (“non poliovirus” anterior myelitis) which is considered to happen rarely. For the study of AFP, 172 stool samples were collected from all over Pakistan under the framework of AFP surveillance activities for polio eradication program during 2017. All of these samples were polio and enteroviruses negative. So these samples were screened for the presence of human adenoviruses. Several genotypes of human adenoviruses were isolated from stool samples. The results of current analysis showed the presence of HAdV in a substantial portion of AFP cases, but then again more research is required to elucidate the function of adenovirus, however, conduction of such studies provide the basis for future comprehensive analysis in order to provide health authorities about the causes and patterns of AFP along with the statistics needed to avoid contact to or evade risk factors comprised in AFP cases and prevalence. According to results, total 89/172 (51.74%) samples were confirmed for adenoviruses. Mostly children with age below 5 years (n= 83/ 89, 93.25%) were affected with the infection. In the study the detection rate of adenoviruses in AFP cases were found throughout year with male (n= 52/89, 58.42%), more affected then females (n= 37/89, 41.57%). The prevalence rate of adenoviruses for different provinces as Punjab (54.11%), Sindh (52.94%), KPK (50%), Baluchistan (50%), FATA (57.14%), GB (25%) and AJK (33.33%). The results of our study showed that the adenovirus genotypes were present with the occurrence rate as HAdV-A31= 24.24%; HAdV-A18 = 3.03%; HAdV-B3 = 9.09%; HAdVB7 = 12.12%; HAdV-C1 = 6.06%; HAdV-D19 = 6.06%, and HAdVF41= 39.39%. The phylogenetic analysis of adenovirus strain showed viral distinct lineages close genetic relation with the global strains. To the best of our knowledge, this is the first work that provide the comprehensive outline on prevalence, genetic diversity and molecular epidemiology of human adenoviruses strains present in stool samples of AFP patients. Second part of the study includes gastroenteritis and diarrhea infection which are a major public health problem resulting in significant morbidity and mortality each year with substantial economic losses. Internationally, the principal pathogens of pediatric diarrhea have been the enteric viruses, particularly rotavirus. Few other childhood diarrheas related enteric viruses includes norovirus, human adenovirus (HAdV), human astrovirus, and sapovirus. Understanding of the pathogens that are responsible in producing diarrhea is precarious for prevention and management of the diarrheal diseases. Pakistan is at high risk of epidemics because of overcrowded cities, unsafe drinking water, inadequate sanitation, poor socioeconomic conditions, low health awareness and inadequate vaccination coverage. The diarrheal diseases contribute to the major disease burden under five along with pneumonia in Pakistan. Unlike vaccine preventable diseases like Polio, measles and maternal & neonatal tetanus, Pakistan does not have any formal responsive surveillance system to monitor the burden of diarrheal diseases and to respond to the outbreaks. In order to evaluate the association of adenovirus infections in the acute gastroenteritis cases, this study was carried out during 2017-2018 at five main hospitals of the country; Benazir Bhutto Hospital, Rawalpindi (BBH), Mayo Hospital, Lahore (MHL), The Children's Hospital, Lahore, Kharadar General Hospital (KGH), Karachi and National Institute of Child Health (NICH), Karachi among hospitalized patients having age less than five years with acute gastroenteritis. In these areas to our information, no research study highlighting the association of adenovirus in causing gastroenteritis had been conducted. Diarrheal ailments aided to a substantial source of illness and death particularly in the developing countries like Pakistan however, the standard data regarding the presence of adenoviruses related with gastro-enteric infections is not presently obtainable for Pakistan. The objective of this study is to help in better understanding of the clinical and molecular epidemiology of enteric adenoviruses related with acute gastroenteritis in hospitalized children in Pakistan.

Total 204/1,118 (18.24%) samples were found positive for human adenovirus. The prevalence of adenovirus in BBH was 13.90% with females (64.51%) were more affected then males (35.48%). The higher detection (45.16%) was observed in children with age ≤ 9 months without any seasonal peculiarity. In MHL, 18.84% infection was found with increased rate (42.30%) in children having age ≤ 9 months. Mostly males (53.84%) were affected than females (46.15%). The virus was found mostly in summer months. In CHL the detection percentage for adenovirus was 32.07%). Mostly children (68.62%) having age ≤ 12 months were affected with higher rate of detection found in winter. Males were more (70.58%) affected as compared to females (29.41%). In case of KGH, 12.08% were found positive for adenoviruses with females (54.54%) more infected than males (45.45%). Increased detection (81.81%) was observed in children with age ≤ 18 months without any seasonal peculiarity. In NICH, the prevalence rate for adenovirus was 17.8%. Increased detection (60.8%) was found in children with ≤9 months of age. Males (63.5%) were more infected with virus then females (36.5%). The detection of virus was found more in winter. Similarly, the co-infection rate of adenovirus with rotavirus was also studied which was found as 26.76%. In this study, the incidence of viral co-infection was more in male cases comparative to females without any statistical difference present among them. According to our results, children with co-infection did not that much differ from those with mono-infection with regard to clinical parameters. The molecular analysis of adenovirus positive cases revealed the presence of several genotypes in the diarrheal cases. Genetic characterization of adenoviruses indicated a huge diversity in Pakistani population. The prevalence of these diverse genotypes pose the great impact of the variants to cause disease severity in pediatrics population. The detected genotypes include HAdV-B3, B7, C2, D28, F40, and F41. Among these types, HAdV-F40 was isolated with increased frequency followed by HAdV-F41. The phylogenetic analysis of adenoviruses showed viral lineages with close genetic relation with the global strains. Our findings confirm the earlier findings that Pakistan is among one the five countries where diarrheal morbidities is considerably high and advocate implementing robust surveillance system for gastro-enteric infections in Pakistan with the immediate introduction sound preventive measures. The presence of adenovirus in diarrheal samples other than rotavirus increases the importance of other viruses to be associated with diarrheal infection, which needs to be consider seriously on government level. Similarly, presence of adenoviruses in AFP cases which are poliovirus and other enterovirus negative must be taken into consideration and furthermore explore its presence in the samples with the association to the disease. In conclusion, our results may describe the association of adenoviruses in AFP as well as diarrheal infections in Pakistan. The results of this study provide the epidemiological picture of adenoviruses in cases of AFP and gastroenteritis. These outcomes are constant with the reports of other studies that signifies the prevalence of adenoviruses in AFP and gastroenteritis. However, additional molecular and clinical analysis is needed.