Complete genome analyses of G12P[8] rotavirus strains from hospitalized children in Surabaya, Indonesia, 2017-2018.

Rotavirus A (RVA) is a major viral cause of acute gastroenteritis (AGE) worldwide. G12 RVA strains have emerged globally since 2007. There has been no report of the whole genome sequences of G12 RVAs in Indonesia. We performed the complete genome analysis by the next-generation sequencing of five G12 strains from hospitalized children with AGE in Surabaya from 2017 to 2018. All five G12 strains were Wa-like strains (G12-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1) and were clustered into lineage-III of VP7 gene phylogenetic tree. STM430 sample was observed as a mixed-infection between G12 and G1 strains: G12/G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1. A phylogenetic tree analysis revealed that all five Indonesian G12 strains (SOEP379, STM371, STM413, STM430, and STM433) were genetically close to each other in all 11 genome segments with 98.0%-100% nucleotide identities, except VP3 and NSP4 of STM430, suggesting that these strains have originated from a similar ancestral G12 RVA. The VP3 and NSP4 genome segments of STM430-G12P[8] were separated phylogenetically from those of the other four G12 strains, probably due to intra-genotype reassortment between the G12 and G1 Wa-like strains. The change from G12P[6] lineage-II in 2007 to G12P[8] lineage-III 2017-2018 suggests the evolution and diversity of G12 RVAs in Indonesia over the past approximately 10 years.

Molecular epidemiology and genetic diversity of norovirus infection in children hospitalized with acute gastroenteritis in East Java, Indonesia in 2015-2019.

Noroviruses are recognized as a leading cause of outbreaks and sporadic cases of acute gastroenteritis (AGE) among individuals of all ages worldwide, especially in children <5 years old. We investigated the epidemiology of noroviruses among hospitalized children at two hospitals in East Java, Indonesia. Stool samples were collected from 966 children with AGE during September 2015-July 2019. All samples were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) for the amplification of both the RNA-dependent RNA polymerase (RdRp) and the capsid genes of noroviruses. The genotypes were determined by phylogenetic analyses. In 2015-2019, noroviruses were detected in 12.3% (119/966) of the samples. Children <2 years old showed a significantly higher prevalence than those ≥2 years old (P = 0.01). NoV infections were observed throughout the year, with the highest prevalence in December. Based on our genetic analyses of RdRp, GII.[P31] (43.7%, 31/71) was the most prevalent RdRp genotype, followed by GII.[P16] (36.6%, 26/71). GII.[P31] was a dominant genotype in 2016 and 2018, whereas GII.[P16] was a dominant genotype in 2015 and 2017. Among the capsid genotypes, the most predominant norovirus genotype from 2015 to 2018 was GII.4 Sydney_2012 (33.6%, 40/119). The most prevalent genotype in each year was GII.13 in 2015, GII.4 Sydney_2012 in 2016 and 2018, and GII.3 in 2017. Based on the genetic analyses of RdRp and capsid sequences, the strains were clustered into 13 RdRp/capsid genotypes; 12 of them were discordant, e.g., GII.4 Sydney[P31], GII.3[P16], and GII.13[P16]. The predominant genotype in each year was GII.13[P16] in 2015, GII.4 Sydney[P31] in 2016, GII.3[P16] in 2017, and GII.4 Sydney[P31] in 2018. Our results demonstrate high detection rates and genetic diversity of norovirus GII genotypes in pediatric AGE samples from Indonesia. These findings strengthen the importance of the continuous molecular surveillance of emerging norovirus strains.

Molecular detection of a new pathotype enteroaggregative haemorrhagic Escherichia coli (EAHEC) in Indonesia, 2015.

Enteroaggregative haemorrhagic Escherichia coli (E. Coli, EAHEC) has been identified as the agent responsible for one of the largest outbreaks of gastroenteritis and Haemolytic-uremic syndrome (HUS) that is transmitted through food in Germany in 2011. The hypervirulent pathotype has a unique combination of two pathogens namely enterohemorrhagic E.coli strain (EHEC) which produces shiga/verotoxin and enteroaggregative E.coli toxins (EAEC) which produces toxins similar to ST and hemolysin. The toxin produced by the EAHEC strain is a hybrid pathotype that combines the virulence potential of the EAEC and EHEC strains that will damage the microcirculation, cause vasculitis and other toxic effects. The purpose of this study was to determine the percentage of samples infected with enteroaggregative hemorrhagic E. coli bacteria (EAHEC) in pediatric diarrhea patients at DR. Soetomo Hospital, Surabaya, Indonesia, 2015. This study used PCR (Polymerase Chain Reaction) method to detect enteroaggregative E. coli strains (CVD432 and aaic genes) and enterohemorrhagic E.coli (eae gene).The results showed that 33 out of 40 (82,5%) stool samples examined were detected enteroaggregative E. coli (EAEC), 4 out of 40 (10%) enterohemorrhagic E. coli (EHEC) and 3 out of 40 (7,5%) enteroaggregative haemorrhagic E. coli bacteria (EAHEC), which caused diarrhea in pediatric diarrhea patients at Dr. Soetomo General Hospital. The unique combination of genomic features of the Surabaya outbreak strain, containing characteristics from pathotypes EAEC and EHEC, suggested that it represents a new pathotype enteroaggregative haemorrhagic E. coli (EAHEC). It is expected that development of specific primer design and sequencing are needed to continue in this research.

Effects of probiotics on the enhancement of the innate mucosal immune response against pathogenic bacteria.

BACKGROUND AND OBJECTIVES: Probiotics have been widely used for host immune system enhancement but with limited knowledge regarding the immunomodulation mechanisms by which they assist the mucosal innate immune response. We investigated the effects of probiotics on the modulation of the innate mucosal immune response particularly in association with Toll-like receptor (TLR)-2, TLR-4 and nuclear factor-kappa B (NF-κB) p65 and p105. MATERIALS AND METHODS: We randomized 24 male BALB/c mice into four groups. Two groups were administered probiotics for 21 consecutive days; one of these groups was challenged with Lipopolysaccharide (LPS) on day 15. The third group was challenged with only LPS. The fourth group remained untreated. All mice were sacrificed after 21 days. An immunohistochemistry procedure on the ileum was performed and monoclonal antibodies specific for TLR-2, TLR-4 and NF-κB p65 and p105 were used for the analysis of innate lymphoid cells. RESULTS: In the LPS-only treated group, there was a significant decrease in p105, indicating an alternative transcription pathway for the process of pro-inflammatory cytokine production. In the probiotics-only treated group there was significant enhancement of TLR-2 and TLR-4 and NF-κB p65 and p105. When mice treated with probiotics were exposed to LPS, there was a significant decrease in NF-κB p65 and p105, indicating employment of the classical pathway for pro-inflammatory cytokine production. CONCLUSION: Probiotics can enhance the innate mucosal immune response in healthy mice and can maintain the homeostasis of the gut mucosal immune response against LPS through the activation of the classical NF-κB pathway.

Effects of a multispecies synbiotic on intestinal mucosa immune responses.

BACKGROUND AND OBJECTIVES: Probiotics and prebiotics are known to regulate immune responses. A synbiotic is a product that combines probiotics and prebiotics in a single dosage form. In this study, we attempt to present the effects of a multispecies synbiotic on intestinal mucosa immune responses after exposure to Escherichia coli O55:B5 lipopolysaccharide (LPS). MATERIALS AND METHODS: Totally 21 male Balb/c mice were randomly classified into two groups. The K-I group received LPS and a synbiotic, and the K-II group received LPS alone. The synbiotic was administered for 21 consecutive days, whereas LPS was administered once on the 15th day. Specifically, a synbiotic containing 1 × 109 colony forming units (CFUs) of the probiotic combination of Lactobacillus acidophilus PXN 35, L. casei subsp. casei PXN 37, L. rhamnosus PXN 54, L. bulgaricus PXN 39, Bifidobacterium breve PXN 25, B. infantis PXN 27 and Streptococcus thermophilus PXN 66 and the prebiotic fructo-oligosaccharide was administered through an orogastric tube. Immunohistochemistry was performed to measure immunoglobulin A (IgA) levels for humoral immune responses and CD4+ and CD8+ levels for cellular immune responses. RESULTS: An independent-samples t-test revealed significant increases of the numbers of IgA- (p = 0.027) and CD4-expressing cells (p = 0.009) but not the number of CD8-expressing cells in the K-I group compared with those in the K-II group. CONCLUSION: The multispecies synbiotic had immunoregulatory effects on IgA and CD4 expression in LPS-exposed mice.

Comparison of Helicobacter pylori stool antigen, salivary IgG, serum IgG, and serum IgM as diagnostic markers of H. pylori infection in children.

BACKGROUND AND OBJECTIVES: Various non-invasive diagnostic tests are available for the detection of Helicobacter pylori infection. The aim of this study was to compare the sensitivity and specificity of HpSA, salivary IgG, serum IgG, and serum IgM to those of endoscopic-biopsy as the gold standard for the diagnosis of H. pylori infection. MATERIALS AND METHODS: This is a cross-sectional study performed among pediatric patients at Dr. Soetomo General Hospital (Surabaya, Indonesia). Fecal, blood, and saliva samples were collected from all subjects. The results of the HpSA, salivary IgG, serum IgG, and serum IgM tests were compared to the results of endoscopic-biopsy as the gold standard. RESULTS: Of the 37 study participants, H. pylori infection was confirmed in 5 (13.33%) with serum IgG, 23 (63.33%) with serum IgM, 15 (40%) with HpSA, and 26 (70.97%) with salivary IgG. The salivary IgG enzyme-linked immunosorbent assay (ELISA) was the only diagnostic test with significantly different results, as compared to biopsy (p = 0.017). CONCLUSION: The results of this study showed that HpSA, salivary IgG, and serum IgG and IgM were not sufficient to replace endoscopic-biopsy as the gold standard for the diagnosis of H. pylori infection.

Effect of Lactobacillus plantarum IS-10506 on blood lipopolysaccharide level and immune response in HIV-infected children.

BACKGROUND AND OBJECTIVES: HIV enteropathy may cause disruption of the intestinal barrier, leading to a loss of CD4+ T cells, increased intestinal permeability, and microbial translocation. Lactobacillus plantarum IS-10506 has the ability to improve gut barrier function. This study investigated the effect of L. plantarum IS-10506 on a number of biomarkers of enteropathy-related damage in HIV-infected paediatric patients undergoing antiretroviral therapy (ARV). MATERIALS AND METHODS: A randomized, double-blind, placebo-controlled study was conducted on 2-18 year-old children, diagnosed as HIV infected according to the WHO 2007 criteria who had received ARV for ≥ 6 months. Subjects were excluded if ARV therapy was discontinued or the patients took probiotics ≥ 2 weeks prior to the study or during the study period. Subjects were randomized into a probiotic group and placebo group. The probiotic group received L. plantarum IS-10506 2.86 × 1010 cfu/day for 6 days. Blood lipopolysaccharide (LPS) level, serum CD4+ T cell count, serum CD8+ T cell count, CD4+/CD8+ T cell ratio, and faecal sIgA level were assessed as biomarkers. RESULTS: Twenty-one subjects completed this study. The blood LPS level decreased significantly in the probiotic group (p = 0.001). There was no significant difference in absolute CD4+ T cell count, percent CD4+ cells, absolute CD8+ T cell count, CD4+/CD8+ T cell ratio, or faecal sIgA. No serious adverse events were reported. CONCLUSION: The probiotic L. plantarum IS-10506 reduced the blood LPS level but showed no effect on the humoral mucosa and systemic immune response in HIV-infected children undergoing ARV therapy.

Effect of the probiotic Lactobacillus plantarum IS-10506 on BDNF and 5HT stimulation: role of intestinal microbiota on the gut-brain axis.

BACKGROUND AND OBJECTIVES: Microbial communities residing in the gut play a major role in the communication between the gut and the brain through neural, immune, and hormonal routes. Changes in abundance of beneficial intestinal bacteria can affect health of individuals. Conversely, drugs, disease, diet and other factors can alter the gut microbiome. However, there is limited information on the effect of exogenous factors on gut microbiota. In this study, we investigated whether a beneficial bacterium, the probiotic Lactobacillus plantarum IS-10506, can stimulate the gut-brain axis using Wistar rats. MATERIALS AND METHODS: The animals were divided into two groups: one received L. plantarum IS strain 10506 supplementation, while the control group received no treatment. Activation of the gut-brain axis was evaluated by immunohistochemical analysis of intestinal and brain serotonin (5-HT) and brain neurotrophin (NT), serotonin transporter (5-HTT), and brain-derived neurotrophic factor (BDNF) levels. RESULTS: The results showed that BDNF (p< 0.000), NT (p< 0.000007), and 5-HTT (p< 0.000007) expression was upregulated in the brain along with intestinal 5-HT (p< 0.000) level in rats treated with L. plantarum strain IS-10506 relative to the control group. CONCLUSION: The probiotic L. plantarum IS-10506 stimulates the gut-brain axis and can potentially promote brain development and function.

Molecular Epidemiology and Clinical Features of Rotavirus Infection Among Pediatric Patients in East Java, Indonesia During 2015-2018: Dynamic Changes in Rotavirus Genotypes From Equine-Like G3 to Typical Human G1/G3.

Group A rotavirus (RVA) is the most important cause of severe gastroenteritis among children worldwide, and effective RVA vaccines have been introduced in many countries. Here we performed a molecular epidemiological analysis of RVA infection among pediatric patients in East Java, Indonesia, during 2015-2018. A total of 432 stool samples were collected from hospitalized pediatric patients with acute gastroenteritis. None of the patients in this cohort had been immunized with an RVA vaccine. The overall prevalence of RVA infection was 31.7% (137/432), and RVA infection was significantly more prevalent in the 6- to 11-month age group than in the other age groups (P < 0.05). Multiplex reverse transcription-PCR (RT-PCR) revealed that the most common G-P combination was equine-like G3P[8] (70.8%), followed by equine-like G3P[6] (12.4%), human G1P[8] (8.8%), G3P[6] (1.5%), and G1P[6] (0.7%). Interestingly, the equine-like strains were exclusively detected until May 2017, but in July 2017 they were completely replaced by a typical human genotype (G1 and G3), suggesting that the dynamic changes in RVA genotypes from equine-like G3 to typical human G1/G3 in Indonesia can occur even in the country with low RVA vaccine coverage rate. The mechanism of the dynamic changes in RVA genotypes needs to be explored. Infants and children with RVA-associated gastroenteritis presented more frequently with some dehydration, vomiting, and watery diarrhea, indicating a greater severity of RVA infection compared to those with non-RVA gastroenteritis. In conclusion, a dynamic change was found in the RVA genotype from equine-like G3 to a typical human genotype. Since severe cases of RVA infection were prevalent, especially in children aged 6 to 11 months or more generally in those less than 2 years old, RVA vaccination should be included in Indonesia's national immunization program.

Genotyping and clinical factors in pediatric diarrhea caused by rotaviruses: one-year surveillance in Surabaya, Indonesia.

BACKGROUND: Rotavirus infections are a major cause of diarrhea in children in both developed and developing countries. Rotavirus genetics, patient immunity, and environmental factors are thought to be related to the severity of acute diarrhea due to rotavirus in infants and young children. The objective of this study was to provide a correlation between rotavirus genotypes, clinical factors and degree of severity of acute diarrhea in children under 5 years old in Surabaya, Indonesia. METHODS: A cross-sectional study was conducted in children aged 1-60 months with acute diarrhea hospitalized in Soetomo Hospital, Surabaya, Indonesia from April to December 2013. Rotavirus in stool specimens was identified by ELISA and genotyping (G-type and P-type) using multiplex reverse transcription PCR. Severity was measured using the Ruuska and Vesikari scoring system. The clinical factors were investigated included patient's age (months), hydration, antibiotic administration, nutritional state, co-bacterial infection and co-viral infection. RESULTS: A total of 88 children met the criteria; 80.7% were aged 6-24 months, watery diarrhea was the most common type (77.3%) and 73.6% of the subjects were co-infected with bacteria, of which pathogenic Escherichia coli was the most common (42.5%). The predominant VP7 genotyping (G-type) was G2 (31.8%) and that of VP4 genotyping (P-type) was P[4] (31.8%). The predominant rotavirus genotype was G2P[4] (19.3%); G1P[4] and G9P[4] were uncommon with a prevalence of 4.5%. There were significant differences between the common genotype and uncommon genotype with respect to the total severity score of diarrhea (p <0.05). G3, G4 and G9 were significantly correlated with severe diarrhea (p = 0.009) in multivariate analyses and with frequency of diarrhea (>10 times a day) (p = 0.045) in univariate analyses, but there was no significant correlation between P typing and severity of diarrhea. For combination genotyping of G and P, G2P[4] was significantly correlated with severe diarrhea in multivariate analyses (p = 0.029). CONCLUSIONS: There is a correlation between rotavirus genotype and severity of acute diarrhea in children. Genotype G2P[4] has the highest prevalence. G3, G4, G9 and G2P[4] combination genotype were found to be associated with severe diarrhea.