A Laboratory Perspective on an Epidemiological Pattern of Infectious Gastroenteritis: A Five-year Surveillance between 2016 to 2020 from Established Private Healthcare Centers within Klang Valley in Malaysia

Infectious gastroenteritis is endemic globally and caused by bacteria, viruses and parasites. The study determined the epidemiological pattern of infectious gastroenteritis within selected urban areas in Malaysia. Analysis of 745 laboratory requests was conducted based on FilmArray gastrointestinal assay and socio-demographic details from suspected cases in KPJ hospitals in Klang Valley, Malaysia, between 2016 to 2020. Descriptive analysis and Fisher-Freeman-Halton Exact testing were performed to ascertain the relationship between socio-demographics with the type of infections. Of 745 requests, 288 (38.7%) were caused by one etiological agent (mono-infection), while the remaining 334 (44.8%) were due to more than one agent (co-infection). Mono-infection was significantly higher among adolescents (n=9; 47.4%) and young adults (n=37; 40.2%) in comparison to adults (n=18; 35.3%). Whereas co-infection was significantly higher in infants (n=216; 49.2%). Mono-infection was mainly caused by bacteria (n=194/288; 67.4%) with Salmonella spp. was prevalent (16.6%), followed by EPEC (12.5%), Campylobacter spp. (11.9%) and Norovirus (10.5%). The co-infections (n=334) were identified with a combination of EPEC and the following bacteria or viruses (Norovirus=38, Campylobacter spp.=30, Salmonella spp.=26, EAEC=22, Rotavirus=19, Adenovirus=10). The month of August to October was recognised as the peak season for infectious gastroenteritis. The present findings may indicate contamination of infectious agents to the food supply and food processing chain as well as reduced hygiene in food sanitation leading to foodborne health issues, particularly among the children in Klang Valley, Malaysia.


INTRODUCTION
Infectious gastroenteritis is a common disease in children and adults globally caused by enteric pathogens such as bacteria, viruses and parasites. 1,2The typical clinical manifestations are vomiting and watery diarrhoea due to the secretion of enterotoxin, with or without stomach, small and large intestines inflammation. 3,4Infectious gastroenteritis originates from contaminated food, water or person-to-person contact. 5,6Despite firm regulations of food safety and sanitation, the diarrheal disease remains a significant cause of mortality and morbidity in children and adults, mainly from lower-middle-income countries. 7iarrheal diseases accounted for one in nine child deaths worldwide and became the second leading cause of death among children under five. 8The percentage of deaths caused by diarrhoea in children under five years of age in 2019 was reported to be highest in Nigeria (18%), 10 to 15% in most African countries, 8.0% in Myanmar, 7.5% in Cambodia, 6.5% in India, and 4.8% in Indonesia. 7While in Malaysia, the reported mortality rate due to diarrhoea in children under five years was 0.8% in 2019. 7In contrast, recent data revealed that the positivity rate of diarrheal diseases among children under five was 4.4%. 9ruses are the primary etiological agents responsible for almost 70% of children's acute gastroenteritis (AGE) cases. 1,10The common enteric viruses detected globally are Rotavirus, Norovirus, Adenovirus and Sapovirus. 1,2,10,11otavirus is the most frequent causative agent of AGE in children less than five years old. 12eanwhile, Norovirus is a leading etiological agent of infectious gastroenteritis across all age groups globally. 2,12,13Bacteria including Salmonella spp.and Campylobacter 1 become the second most common etiological agent of gastroenteritis, with Salmonella spp.being the most common enteric pathogen reported in countries such as Africa, 14,15 the USA, Canada 16 and Korea. 2,6he etiologic diagnosis of infectious diarrhoea can be challenging because almost all types of enteric pathogens produce similar clinical manifestations, occurrence of multiple infectious agents causing AGE, and the source of contamination, whether from the food, water, environment or animal exposure.Thus, these limit an appropriate and timely epidemiological surveillance report.
Detection of concomitant or multiple enteric pathogens using multiplex molecular assay provides a significant platform to assist the physicians in prescribing pathogen-specific treatment, provision related infection control and public health measures in the event of an outbreak.Hence, this study aimed to determine the epidemiological pattern of infectious gastroenteritis by incorporating the sociodemographic characteristics, etiological agents, type of infections and seasonal peaks based on the private laboratory data within the Klang Valley, Malaysia.The present study's findings may improve our national surveillance data on infectious gastroenteritis.These will provide insight and aid relevant authorities in formulating the appropriate health policy concerning foodborne illness.

Ethics statement
The ethical approvals were obtained from Universiti Teknologi MARA (UiTM) Research Ethics Committee (REC/654/19) and KPJ Clinical and Research Ethics Review Committee (CRERC/15092020) for a duration of seven years from 2016 until 2022.

Study design and population
A single-centre, retrospective study on identifying the etiological agents causing gastroenteritis from 2016 to 2020 was conducted at the Molecular Diagnostic Laboratory (MDL), KPJ Lablink Central Laboratory, Kuala Lumpur, Malaysia.The MDL provides molecular diagnostic services for infectious diseases for all the KPJ hospitals within the Klang Valley.The data on the etiological agents were retrieved from the FilmArray gastrointestinal panel assay (FAGP) (BioFire Diagnostics-bioMerieux, Italy).The Klang Valley is located in the central of the west coast of Peninsular Malaysia, with an estimated eight million population (Figure 1). 17It is Malaysia's most urbanised population, a centre for industry and commerce hub.Klang Valley consists of several cities, including Kuala Lumpur, Ampang Jaya, Petaling Jaya, Subang Jaya, Shah Alam, Klang, and Rawang (Figure 1), with seven KPJ hospitals across these cities.

Data collection and definition
A total of 745 laboratory requests for FAGP at KPJ Lablink Medical Laboratory, Kuala Lumpur, were analyzed between January 1 st , 2016 to December 31 st , 2020.The laboratory requests were from seven KPJ hospitals (Table 1) and the Central Laboratory in Klang Valley, Malaysia (Table 1).Only laboratory requests with complete information of socio-demographic details and result of type of gastrointestinal (GI) infections (mono-infection/co-infection) were included in this study.Mono-infection was defined as gastroenteritis caused by a single agent, virus, bacteria or parasite.While co-infection is characterised by gastroenteritis caused by more than one agent, which involves any combination between virus, bacteria and parasite.The age was categorized as an infant (< 2-year-old), children (2 to 10-year-old), adolescent (11 to 17-yearold), young adult (18 to 40-year-old), adult (41 to 65-year-old), and elderly (> 66-year-old) based on WHO's classification. 19The laboratory result was grouped into non-infectious gastroenteritis, mono-infection and co-infection accordingly.

Statistical analysis
Descriptive analysis incorporating the frequency and percentage for the age and gender of the patients with laboratory variables were performed using SPSS Statistics software (SPSS, IBM New York USA; version 21).Statistical differences from the data were assessed using the Fisher-Freeman-Halton Exact Test for categorical variables.A p-value < 0.05 was considered statistically significant.The minimum sample size required to represent the urban area of Klang Valley in the present study is 65, as determined using a formula by Pourhoseingholi et al. 20 Therefore, 745 laboratory requests were sufficient to provide significant statistical power.

Distribution of enteric pathogens
Table 3 summarises the distribution of pathogens according to the types of infection, gender, age groups and location of the private hospitals.Bacteria (n=194/288) was the main pathogen for mono-infection across the gender and age groups (p<0.001).In comparison, there was no data on parasites causing infectious gastroenteritis.In co-infection, a combined     A total of 334 different co-infection combinations were detected from 622 confirmed cases, as shown in Table 4.The most common combinations were EPEC with virus and EPEC with bacteria (Norovirus GI/ GII, n=38; Campylobacter, n=30; Salmonella spp., n=26; EAEC, n=22; Rotavirus A, n=19; Adenovirus F 40/41, n=10), as well as EAEC in association with Norovirus GI/GII (n=15) (Table 5).

Seasonality of the enteric pathogens
The rate of confirmed infectious gastroenteritis appeared lowest in the year 2016 (n=19/28; 67.9%) and 2020 (n=45/65; 70.3%) as compared to 2017 (n=127/148; 85.8%), 2018 (n=178/210; 84.8%) and 2019 (n=245/295; 85.8), however, the trend was not statistically significant (p>0.05).Figure 2 shows a monthly distribution of confirmed infectious gastroenteritis (Figure 2A) and the most common pathogens recorded (Figure 2B to 2H) for five consecutive years.The peak season for confirmed infectious gastroenteritis was from August until October throughout the five years (2016 to 2020) (Figure 2A).The Salmonella spp. was continuously detected throughout the year with three seasonal peaks: Jan, April, June and October (Figure 2B).The EPEC demonstrated a peak season from May until November (Figure 2C).While the detection rate of Campylobacter spp. was increased above the median positive rate between March and July (Figure 2D).The detection rate for Norovirus GI/ GII was raised between July and December (Figure 2E).While C. difficile toxin A/B displayed two peak seasons: April and May, September and November (Figure 2F).Rotavirus had three peak seasons: January, March and April, and August until October (Figure 2G).In contrast, EAEC was detected highest in February, June, August and October (Figure 2H).

DISCUSSION
We report the five years of data on the epidemiology of infectious gastroenteritis from 745 cases across all age groups involving stool testing using a multiplex molecular array.The multiplex molecular array such as FAGP assists physicians in differentiating bacteria, viruses and parasites as responsible etiological agents.Despite high sensitivity, multiple molecular arrays might detect nucleic acids from non-viable microorganisms unrelated to infectious diarrhoea.However, it provides a prompt diagnosis by detecting 13 bacteria, four parasites and five different viruses simultaneously.Thus, enabling precise patient treatment and reducing the need of antibiotic prescription and health burden due to unnecessary hospitalisation.
In the studied populations, 83.5% (monoinfection, n=288/745; co-infection, n=334/745) of the samples were positive, which was higher than the previously reported study in Malaysia (4.4%) 9 and a recent study in Italy with only 56.2%. 10 The higher positive rate could be attributed to the utilisation of multiplex molecular arrays from 2016 to 2020 in KPJ hospitals within the Klang Valley, Malaysia.It was noted that there were increased of laboratory requests for FAGP from KPJ Rawang Specialist Hospital, Selangor, owing to the escalation of suspected infectious gastroenteritis cases in Rawang, Selangor, and the availability of the test offered by the laboratory which reflects the advantages of the multiplex molecular detection of enteric pathogens and strain identification.Rawang is a semi-urban area within Klang Valley, with the main economy contributed by agriculture and industrial activities.The intensification of infectious gastroenteritis cases could be attributed to the contamination of infectious agents into the water and food supply and reduced hygiene and sanitation.However, these could not be confirmed as there is no specific epidemiological study on infectious gastroenteritis has been carried out in Rawang.
A higher prevalence of mono-infection in males and among adolescents, young adults and adult patients was observed in contrast to females and infants.The higher rates among adolescents and adults were contributed by the bacteria such as Salmonella spp., EPEC and Campylobacter spp.This probably owing to that Salmonella spp.and Campylobacter spp.were often associated with travelling. 20,21Whereas EPEC has been associated with contaminated food, water, or person-toperson contact. 5,6The median age of the studied population is two years old, which supports infectious gastroenteritis commonly occurring in children below five years of age. 8Besides that, the co-infection cases were significantly higher than mono-infection in the present study, particularly among the infant age group.This could be due to children frequently being exposed to various environmental pathogens at home, daycare, pets, and playgrounds.][24][25] The ability of multiple molecular arrays to distinguish enteric pathogens in positive samples provides insightful and improved epidemiological data for infectious gastroenteritis.Our analysis found that mono-infection is caused mainly by bacteria (67.4%; n=194/288) with Salmonella spp., EPEC and Campylobacter.Our findings are consistent with other previously reported studies in Africa, 14,15 the USA and Canada, 16 Korea, 2,6 Malaysia, 26 and Italy. 10almonella spp., Campylobacter and Norovirus have been known as common enteric pathogens detected in Africa, 14,15 the USA and Canada, 16 and Korea. 2,6Interestingly, we observed that there is an emergence of EPEC as one of the prevalent enteric pathogens.For EPEC, an inference is difficult to make as several studies reported an unclear aetiology role of EPEC in infectious diarrhoea. 10,27Recently, EPEC was associated with environmental enteric dysfunction (EED) and affected child growth, thus highlighting the need to formulate a vaccine to reduce the burden and combat childhood malnutrition. 27Furthermore, the National Health and Morbidity Survey 2016 conducted by the Ministry of Health Malaysia on 15,188 children below five years old found that the risk of diarrhoea was higher in children who consumed untreated water and associated with less privileged socio-demographic. 9Nevertheless, our study detected the emergence of EPEC in Malaysia, particularly within the urban area, which notified its clinical importance in Malaysia and required further investigation.We noted that mixed infection between bacteria and virus is prevalent, with 334 coinfection combinations observed.Our report on the frequent mixed infections between viruses and bacteria was in tandem with other epidemiological reports. 10,26,28Distinctly, we found that the prevalence of co-infection is between EPEC with 38 Norovirus, 30 Campylobacter, 26 Salmonella spp., 22 EAEC, 19 Rotavirus and ten Adenovirus strains (Table 4).Thus, the co-infection between EPEC with several viruses and bacteria species highlights the emergence of EPEC as one of the prevalent enteric pathogens in Malaysia.Hence, detailed epidemiological studies should be performed, and public health measures should be revised as EPEC may cause severe gastroenteritis with a substantial hospitalization rate in the near future.
Enteropathogenic E.coli (EPEC) has been identified as a common pathogen for infectious gastroenteritis in children besides rotavirus. 25,29A comparative study carried out by Ayedun et al. 29 revealed that the prevalence of EPEC infection among children in Nigeria, Korea, Sweden and Tanzania was correlated with the risk of limited access to clean water, poor hygiene and sanitation.Thus, there is likelihood for EPEC to spread and contaminate the food supply and food processing chain. 30,31Furthermore, the prevalence of EPEC has been associated with developing nations, as reviewed by Alharbi and colleagues. 30Among the reported possible reasons for high EPEC infection in developing nations such as Malaysia are the consumption of insufficient pasteurized milk, direct contact with animal manure and poor handwashing practice in day care or school for children, exposure to the contaminated swimming pools or fecally contaminated rivers or lake. 30ur time series analysis over the five years detected that the peak season for infectious gastroenteritis was between August until October (Figure 2A).The rise of confirmed infectious gastroenteritis during the peak season was contributed by the higher detection rate of Salmonella spp.(Figure 2B), EPEC (Figure 2C), Norovirus (Figure 2E), Rotavirus (Figure 2G) and EAEC (Figure 2H).The rate of confirmed infectious gastroenteritis increased from 2016 until 2019, showing higher utilisation of multiplex molecular assay in detecting enteric pathogens in KPJ hospitals.Conversely, as observed in Italy, lower testing and detection rates were observed in 2020 due to the reduced referral cases and hospitalisation during the SARS-CoV-2 pandemic. 10t would be tempting to speculate that movement restriction order and social distancing modulated the circulation of enteric pathogens.Therefore, it would be interesting to determine the circulation pattern of the enteric pathogens from 2021 onwards.
Each of the prevalent enteric pathogens reported here has shown a specific seasonality trend where Salmonella spp. was continuously detected throughout the year, EPEC (May until November), Campylobacter spp.(March until July), Norovirus GI/GII (July until December), C. difficile toxin A/B and Rotavirus showed cyclical patterns, respectively.C. difficile toxin A/B had two seasons (April and May, September until November), whereas Rotavirus had three seasons (January, March and April, August until October).Whereas EAEC has shown more dispersal peak seasons in February, June, August and October.Overall, within the peak season of infectious gastroenteritis between August until October over the five years, the highest positive cases were observed in September and October.Salmonella spp., EPEC, Norovirus GI/GII, C. difficile toxin A/B, Rotavirus and EAEC were predominant during these two months.
The highest occurrence of infectious gastroenteritis in the tropical region is reportedly occur during rainy season, while in temperate regions the highest cases were observed during winter. 30There is a possibility to have a correlation between the rainy season in September and October with the peak season of positive infectious gastroenteritis cases in Klang Valley, as gathered in the present study.However, the correlation analysis between the rising cases and rainfall could not be confirmed due to the unavailability of meteorology data between the periods, which is worth exploring.Nevertheless, the spatiotemporal of infectious gastroenteritis in Klang Valley, Malaysia, over five years from 2016 to 2020 has been established.

COCLUSION
The present study describes temporal c i rc u l at i n g e nte r i c p at h o ge n s ca u s i n g

Figure 2 .
Figure 2. Monthly distribution of infectious gastroenteritis and the most prevalent pathogens detected in 622 confirmed cases by multiplex RT-PCR assay for five years from 2016 until 2020.A: total confirmed infectious gastroenteritis in the study period; B: Salmonella spp.; C: EPEC; D: Campylobacter; E: Norovirus GI/GII; F: C. difficile toxin A/B; G: Rotavirus; H: EAEC

Table 1 .
The list of KPJ hospitals in Klang Valley with the number of laboratory requests Location of the Klang Valley within the borders of the state of Selangor and the Federal Territory of Kuala Lumpur in the central part of the west coast of Peninsular Malaysia, where the focus area of study for the data catchment area of KPJ hospital.Reprinted from Map of Peninsular Malaysia.

Table 3 .
Distribution of enteric pathogens identified in mono and co-infection of infectious gastroenteritis by multiplex RT-PCR assay according to the gender, age and location of KPJ hospitals within Klang Valley, Malaysia, from 2016 to 2020

Table 5 .
Distribution of co-infection in infectious gastroenteritis consisting of 334 different combinations detected by multiplex RT-PCR assay in KPJ hospitals within Klang Valley, Malaysia, from 2016 to 2020