Epidemic outbreak of acute haemorrhagic conjunctivitis caused by coxsackievirus A24 in Thailand, 2014.

Acute haemorrhagic conjunctivitis outbreaks are often attributed to viral infection. In 2014, an unprecedented nationwide outbreak of infectious conjunctivitis occurred in Thailand, which affected >300 000 individuals over 3 months. To identify and characterize the virus responsible for the epidemic, eye swab specimens from 119 patients were randomly collected from five different provinces. Conserved regions in the enteroviral 5'-UTR and adenovirus hexon gene were analysed. Enterovirus was identified in 71·43% (85/119) of the samples, while no adenovirus was detected. From enterovirus-positive samples, the coxsackievirus A24 variant (70·59%, 84/119) and echovirus (0·84%, 1/119) were identified. Additional sequencing of full-length VP1 and 3C genes and subsequent phylogenetic analysis revealed that these clinical isolates form a new lineage cluster related to genotype IV-C5. In summary, the coxsackievirus A24 variant was identified as an aetiological agent for the recent acute haemorrhagic conjunctivitis outbreak in Thailand.

Complete genotype constellation of human rotavirus group A circulating in Thailand, 2008-2011.

This study has identified diverse and re-assorted group A rotavirus (RVA) strains by sequence and phylogenetic analysis of the 11 genomic segments. The 22 cases investigated in this study were collected from children with diarrhea between 2008 and 2011. The RVA genomic constellations identified in this study were identified as G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 22.7% (5/22); G2-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2 27.3% (6/22); G3-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 18.2% (4/22); G3-P[9]-I3-R3-C3-M3-A3-N3-T3-E3-H6 4.6% (1/22); G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 9.1% (2/22); G12-P[6]-I1-R1-C1-M1-A1-N1-T1-E1-H1 4.6% (1/22) and G12-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 13.6% (3/22). Two RVA strains, possessing a complete AU-1-like genomic backbone, showed re-assortment for genes 3 and 11, revealing possible zoonotic re-assortment events between human and canine strains. In addition, one of the analyzed strains revealed a G12 specificity for VP7 in combination with a porcine-like P[6] VP4 and a complete Wa-like constellation. Continuous surveillance of rotavirus strains and their evolution may be useful for understanding the emergence of novel strains through interspecies genome re-assortment between human and animal viruses.

Molecular epidemiology and genetic history of hepatitis C virus subtype 3a infection in Thailand.

OBJECTIVE: Among all hepatitis C virus (HCV) infections, subtype 3a is the most common genotype in Thailand. This study investigates the molecular epidemiology and epidemic history of HCV subtype 3a in Thailand. METHODS: Three hundred and fifty-six serum samples were collected from HCV-infected Thai patients. The virus was isolated, after which the core and NS5B regions were sequenced. Subsequently, the HCV genotype was classified by phylogenetic analysis based on the core and NS5B regions. Molecular evolution analysis of HCV subtype 3a was estimated using BEAST (Bayesian Evolutionary Analysis by Sampling Trees) v.1.5.4. RESULTS: Based on our phylogenetic analyses, subtype 3a (38.5%) was the most prevalent, followed by 1a (21%), 1b (13.8%), genotype 6 (19.9%) [comprised of subtypes 6e (0.3%), 6f (11%), 6i (1.9%), 6j (1.9%) and 6n (4.8%)] and 3b (5.6%). Our phylogenetic tree indicates the existence of a specific group of HCV subtype 3a strains in the Thai population. Molecular evolutionary analysis dated the most recent common ancestor of the Thai HCV subtype 3a strains as existing approximately 200 ago, and a Bayesian skyline plot showed that this particular strain spread to Thailand during the mid-1970s and early 1980s. This period overlaps with the Vietnam War (1955-1975) and the widespread use of injection stimulants introduced by the US Army during this time. CONCLUSION: The estimated history of HCV subtype 3a infection in Thailand may help to predict the future burden of HCV-related diseases and facilitate better public health control and surveillance.

Elevation of serum galectin-3 and liver stiffness measured by transient elastography in biliary atresia.

BACKGROUND AND AIM: Biliary atresia (BA) is an intractable neonatal liver disease characterized by progressive fibrosclerotic obliteration of the extrahepatic biliary tree. The aim of this study was to evaluate serum galectin-3 in postoperative BA patients and the association between galectin-3, clinical outcome and liver stiffness score. METHODS: 58 BA patients post Kasai operation and 20 controls were enrolled. None of the patients had undergone liver transplantation. BA patients were classified into 2 groups according to their serum total bilirubin (TB) levels (TB<2 mg/dL, no jaundice vs. TB≥2 mg/dL, persistent jaundice) and alanine aminotransferase (ALT) levels (ALT<45 IU/L, normal ALT vs. ALT≥45 IU/L, elevated ALT). Serum galectin-3 levels were determined by enzyme-linked immunosorbent assay. Liver stiffness scores were measured by transient elastography (FibroScan). RESULTS: BA patients had higher serum galectin-3 levels (5.1±0.3 vs. 3.8±0.4 ng/mL, p=0.01) and greater liver stiffness values than healthy controls (29.7±3.0 vs. 5.1±0.5 kPa, p<0.001). Serum galectin-3 levels were markedly elevated in BA patients with jaundice compared to those without jaundice (6.4±0.5 vs. 4.4±0.3 ng/mL, p=0.001). Furthermore, BA patients with elevated ALT displayed significantly higher levels of serum galectin-3 than those with normal ALT (5.9±0.4 vs. 3.8±0.3 ng/mL, p=0.001). Additionally, BA patients with portal hypertension had considerably higher serum galectin-3 levels than those without portal hypertension (6.1±0.4 vs. 3.7±0.3 ng/mL, p<0.001). CONCLUSIONS: Increased serum galectin-3 is associated with a poor outcome in postoperative BA patients. Serum galectin-3 could be used as a biochemical parameter reflecting the deterioration of liver function and the severity of liver fibrosis in postoperative BA.

Evidence of protection against clinical and chronic hepatitis B infection 20 years after infant vaccination in a high endemicity region.

Vaccination against hepatitis B virus (HBV) immediately after birth prevents neonatal infection by vertical transmission from HBV carrier mothers. There is an ongoing debate whether infant vaccination is sufficient to protect against infection when exposed to HBV later in life. We studied 222 Thai infants born to HBsAg -/+ and HBeAg -/+ mothers who were vaccinated with recombinant hepatitis B vaccine at 0-1-2-12 months of age. A subset of 100 subjects received a booster dose at age 5 years. Blood samples collected yearly for 20 years were examined for anti-HBs antibodies and serological markers of hepatitis B infection (anti-HBc, HBsAg, and in selected cases HBeAg, anti-HBe, HBV DNA). During the 20-year follow-up, no subject acquired new chronic HBV infection or clinical hepatitis B disease. During the first decade, possible subclinical breakthrough HBV infection (anti-HBc seroconversion) was only observed in subjects born to HBsAg +/HBeAg + mothers (6/49 [12.2%]). During the second decade, breakthrough HBV infections were detected in all groups (18/140 [12.8%]). Increases in anti-HBs concentrations that were unrelated to additional HBV vaccination or infection were detected in approximately 10% of subjects in each decade. Primary infant vaccination with a recombinant hepatitis B vaccine confers long-term protection against clinical disease and new chronic hepatitis B infection despite confirmed hepatitis B exposure.

Entire genome characterization of Chikungunya virus from the 2008-2009 outbreaks in Thailand.

The resurgence of Chikungunya virus (CHIKV) in the southern, northeastern and northern parts of Thailand, inflicting approximately 46,000 reported cases since October 2008 until December 2009, has raised public health concerns. In the present study, we characterized nearly complete genome sequences of four CHIKV isolates obtained from 2008 to 2009 outbreaks in Thailand. Phylogenetic analysis was performed to determine the relationships of the study viruses with previously reported isolates. Results showed that 2008-2009 Thailand isolates belonged to the East, Central and South African genotype and were most closely related to isolates detected in Malaysia and Singapore in 2008. This was in contrast to isolates from all previous outbreaks in Thailand which were caused by an Asian genotype. We describe several novel mutations in Thailand isolates that warrants further investigation on characterization of CHIKV from different parts of the country to better understand the molecular epidemiology of Chikungunya fever outbreaks in Thailand.

Increased level of hepatocyte growth factor in children with dengue virus infection.

BACKGROUND: Evidence of hepatocellular damage is common in dengue-infected individuals. Hepatocyte growth factor (HGF), a key cytokine responsible for liver regeneration, may play a prognostic role in dengue virus infection. AIM: To determine the relationship between serum HGF level and disease severity in patients with dengue virus infection. METHODS: Serum samples from 27 children [17 dengue fever (DF), ten dengue haemorrhagic fever (DHF)] with serologically confirmed dengue virus infection during the febrile, toxic stages and at follow-up were analysed for HGF. Serum samples obtained from nine healthy children served as the control group. RESULTS: In dengue-infected patients, serum HGF was significantly higher at the febrile and toxic stages than at follow-up (p<0.05). In comparison with DF, patients with DHF had a greater level of HGF at the febrile stage (p<0.05). A cut-off HGF level of 1220 pg/mL obtained during the febrile stage showed a sensitivity of 90% and a specificity of 53% for predicting clinical progression to DHF (area under the ROC curve 0.75). CONCLUSION: Serum HGF level at the early stage of dengue virus infection is elevated and may be a useful predictor for clinical progression to DHF.

Correlation of circulating endoglin with clinical outcome in biliary atresia.

BACKGROUND AND AIM: Biliary atresia (BA) is a chronic progressive inflammatory disorder of the extrahepatic and intrahepatic biliary system in children. The aim of the present study was to investigate circulating endoglin levels in BA patients compared with healthy controls and to determine the relationship between plasma endoglin levels and outcome parameters of BA patients after Kasai operation. METHODS: Fifty-five postoperative BA patients and 14 healthy controls were recruited. The patients were divided into two groups based on their serum total bilirubin levels (TB<34.2, no jaundice vs. TB>or=34.2 micromol/L, persistent jaundice) and serum alanine aminotransferase (ALT<45, normal ALT vs. ALT>or=45 IU/L, high ALT). Circulating endoglin levels were analyzed by enzyme-linked immunosorbent assay. RESULTS: Average levels of plasma endoglin were significantly higher in BA patients compared to healthy controls (7.8+/-0.4 vs. 6.5+/-0.4 ng/mL; P=0.02). BA patients with persistent jaundice had higher plasma endoglin levels than those without jaundice (9.2+/-0.8 vs. 6.9+/-0.3 ng/mL; P=0.006). Furthermore, the concentrations of plasma endoglin in BA patients with high ALT were significantly higher compared to those with normal ALT (8.5+/-0.5 vs. 6.3+/-0.5 ng/mL, P=0.003). In addition, BA patients with portal hypertension had more elevated plasma endoglin levels than those without portal hypertension (8.8+/-0.6 vs. 6.1+/-0.3 ng/mL, P=0.001). Plasma endoglin was positively correlated with serum ALT (r=0.36, P=0.007) and serum GGT (r=0.44, P=0.001). CONCLUSION: High circulating endoglin correlated with a poor outcome for BA. Plasma endoglin can be utilized as a potential biomarker reflecting the severity of ongoing liver injury and biliary obstruction in BA patients after Kasai procedure.

Molecular characterization of Hepatitis A virus causing an outbreak among Thai navy recruits.

Hepatitis A virus (HAV) infection is a communicable disease, typically transmitted by faecal-oral contamination. HAV outbreaks usually occur in endemic areas. We report an outbreak of HAV from June to July, 2008 among Thai navy recruits who had received training at the Sattahip Navy Base, Chonburi province, Thailand. Upon conclusion of the training, the recruits were deployed to serve at several navy bases across the country. Secondary cases of HAV infection were reported among military personnel from these navy bases. To elucidate origin and distribution of these outbreaks, we characterized the genome and genotype of HAV isolated from the different navy bases. Sera and stool from the subjects were tested for antiHAV IgM, antiHAV IgG and HAV RNA. Subsequently, molecular characterization of HAV was performed by nucleotide sequencing of the VP1-P2A region, BLAST/FASTA and phylogenetic analysis. HAV RNA was detected in specimens obtained from different areas. All isolated strains clustered in the same lineage and belonged to genotype 1A. They shared nearly 100% genome homology indicating a single point source of this outbreak. This study provides essential baseline data as a reference for genetic analysis of HAV strains causing future outbreaks. Early detection of HAV infection and identification of the source by using molecular characterization and prompt preventive measures will hopefully prevent further outbreaks.

Serum transforming growth factor-beta1 and epidermal growth factor in biliary atresia.

BACKGROUND AND AIM: Biliary atresia (BA) is a serious liver disease in children. Since transforming growth factor-beta1 (TGF-beta1) and epidermal growth factor (EGF) are involved in the hepatic reparative process, our objective was to investigate whether serum TGF-beta1 and serum EGF levels were associated with therapeutic outcomes in BA. METHODS: Serum levels of TGF-beta1 and EGF were determined with the ELISA method in 67 postoperative BA patients with a median age of 7 years and in 10 age-comparable healthy children. The BA patients were then divided into two groups depending on their therapeutic outcome: good outcome (jaundice-free) and poor outcome (persistent jaundice). Clinical data, serum TGF-beta1 and serum EGF levels were compared between the two groups of BA patients. Correlation analysis of serum TGF-beta1 with serum EGF was carried out. Data are expressed as mean +/- SD. RESULTS: Serum TGF-beta1 levels of BA patients were higher than those of controls (86.6 +/- 15.7 vs. 75.7 +/- 8.8 ng/ml, p = 0.0362). However, there was no difference in serum EGF between BA patients and controls (133.1 +/- 66.6 vs. 125.4 +/- 88.9 pg/ml, p = 0.744). Further subgroup analysis showed that patients with good outcomes (n = 40) had higher serum TGF-beta1 and serum EGF levels than patients with poor outcomes (TGF-beta1: 91.2 +/- 16.5 vs. 79.6 +/- 11.7 ng/ml, p = 0.002; EGF: 148.5 +/- 65.0 vs. 110.3 +/- 63.4 pg/ml, p = 0.02). In addition, serum TGF-beta1 was positively correlated with serum EGF (Pearson's r = 0.3418, p = 0.0046). CONCLUSION: Elevated serum TGF-beta1 and serum EGF levels were associated with a good outcome in BA patients. There was a positive correlation between serum TGF-beta1 and serum EGF. This suggests that the resultant TGF-beta1 and EGF pathways may be involved in the pathophysiological process in postoperative BA.

Elevated serum soluble E-selectin is associated with poor outcome and correlated with serum ALT in biliary atresia.

BACKGROUND AND AIM: Biliary atresia (BA) is a serious liver disease. Our objective was to investigate possible roles of serum soluble E-selectin (sE-selectin) in BA. METHODS: During their annual follow-up, the serum levels of sE-selectin were determined by ELISA in 53 postoperative BA patients and 10 healthy children. The patients were categorized into two groups according to their jaundice status. Comparisons of demographic data and serum sE-selectin levels between jaundice-free patients and jaundice patients were performed. Correlation analysis was carried out of serum E-selectin with serum ALT and serum GGT. Data are expressed as mean and SD (ng/mL). RESULTS: The serum sE-selectin of BA patients was higher than that of controls (114.1 +/- 44.0 vs. 88.7 +/- 22.2; p = 0.01). Further subgroup analysis showed that there was an increase in serum sE-selectin levels of BA patients with jaundice (n = 21) compared to those without jaundice (n = 32) (129.7 +/- 48.6 vs. 103.9 +/- 38.1; p = 0.035). Also, serum E-selectin was positively correlated with serum ALT, a marker for liver injury (Pearson r = 0.355, p = 0.009), but not with serum GGT (Pearson r = 0.223, p = 0.12). CONCLUSION: Elevated serum sE-selectin was associated with a poor outcome of BA. There was a positive correlation between serum sE-selectin and serum ALT. E-selectin probably plays a role in the pathophysiology of liver injury in postoperative BA.

Molecular characterisation of the hepatitis A virus circulating in the 2001-2005 outbreaks in Thailand.

In a recent study of hepatitis A virus (HAV) in Thailand, viral isolates recovered during several outbreaks of infection that occurred between 2001 and 2005 were genotyped and subjected to phylogenetic analysis. Anti-HAV IgM was detected, by ELISA, in many of the 283 serum samples that were collected from the provinces of Suphanburi, Songkhla, Chiangrai and Lampang: 40 (48.2% of those investigated), 38 (47.5%), 25 (41.0%) and 32 (54.2%), respectively. The HAV RNA in the positive samples was reverse transcribed and amplified, using a nested PCR focussed on the VP1-2A region, before the nucleotides of the VP1-2A region of each HAV-RNA-positive sample were sequenced. All the isolates investigated clustered in subgenotype IA and, hence, are closely related to the strains previously investigated in Thailand. When the genome of one sample from an outbreak in Lampang (LP014) was fully sequenced, the results of genome comparison and phylogenetic analysis again indicated subgenotype 1A, which appears to be the predominant form of HAV circulating throughout Thailand.

Rapid detection and strain identification of porcine reproductive and respiratory syndrome virus (PRRSV) by real-time RT-PCR.

AIMS: To develop and validate assays based on real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for rapid detection and strain identification (European and North American strains) of porcine reproductive and respiratory syndrome virus (PRRSV) by using SYBR Green I and TaqMan probe chemistries. METHODS AND RESULTS: This study describes two alternative assays based on real-time RT-PCR for rapid detection and strain identification of PRRSV in comparison with conventional RT-PCR. The first assay utilized SYBR Green I with melting curve analysis; another assay was performed using strain-specific TaqMan probes. Primers were selected from the conserved regions within ORF7 (N) of both strains whereas two TaqMan probes labelled with different fluorescent dyes were specifically designed for each strain. The result of strain identification was confirmed by direct sequencing. Both assays can be used for rapid detection and strain identification of PRRSV with a sensitivity of 10(4) and 10(3) copies microl(-1) for SYBR Green and TaqMan probe, respectively. CONCLUSIONS: Real-time RT-PCR is a powerful method combining rapidity, specificity and efficiency for large-scale screening and strain identification of PRRSV. SIGNIFICANCE AND IMPACT OF THE STUDY: The data indicate that the methods developed are invaluable for detecting low levels of PRRSV infection in swine.

Genetic analysis of influenza A virus (H5N1) derived from domestic cat and dog in Thailand.

Complete genome sequences of H5N1 viruses derived from a domestic cat "A/Cat/Thailand/KU-02/04" and dog "A/Dog/Thailand/KU-08/04" were comprehensively analyzed and compared with H5N1 isolates obtained during the 2004 and 2005 outbreaks. Phylogenetic analysis of both cat and dog viruses revealed that they are closely related to the H5N1 viruses recovered from avian influenza outbreaks of the same period. Genetic analysis of 8 viral gene segments showed some evidence of virulence in mammalian species. In summary, the H5N1 viruses that infected a domestic cat and dog are highly pathogenic avian influenza viruses that are virulent in mammalian species, potentially indicating transmission of H5N1 viruses from domestic animals to humans.

Declining trend in the seroprevalence of infection with hepatitis A virus in Thailand.

Since the mid 1970s, infection with hepatitis A virus (HAV) in Thailand has shifted from hyper-endemic to mesoendemic. In 2004, to explore this trend in prevalence further, 3997 subjects from four geographically distinct provinces of Thailand were tested, in a commercial ELISA, for antibodies to HAV. The results indicate that the seroprevalence of HAV continues to fall, almost certainly because the profound socio-economic development that has occurred over the last few decades in Thailand has brought with it significant improvements in sanitation and personal hygiene. As exposure to HAV declines, however, the risks of symptomatic and potentially severe infection in adulthood (rather than asymptomatic infection during childhood) and of epidemics of such infection, which would lead to profound economic loss, increases. Improvements in hygiene and sanitation to reduce exposure to the virus and measures to reduce the incidence of symptomatic disease in those infected, such as vaccination (which may only be cost-effective when targeted at high-risk groups), need to be carefully considered.

The incidence of rotavirus a isolates of G genotype in Thailand in 2002-2004.

Rotaviruses are the leading cause of severe gastroenteritis among infants and young children worldwide. Between November 2002 and March 2004, 36 stool specimens of 108 children with acute diarrhea in Bangkok, Thailand were found positive for Rotavirus A (RV-A) by RT-PCR. The 36 isolates were subjected to genotyping by RFLP analysis and direct sequencing of a part of the gene for major outer capsid glycoprotein VP7. The sequences obtained were subjected to phylogenetic analysis. Among the isolates the genotypes G1 (5.6%), G2 (69.4 %) and G9 (25.0 %) were found. Comparison of these results with those of previous studies covering the period of 1982-1999 revealed a changing pattern of RV-A G genotypes and thus contributed to the understanding of RV-A epidemiology in Thailand. Any vaccine to be developed against this virus should target the G9 genotype as one of common global genotypes.

Nucleotide sequence analysis of nucleocapsid protein gene of canine distemper virus isolates in Thailand.

The C-terminal part of the nucleocapsid protein gene of 13 canine distemper virus (CDV) isolates from Thailand, were analyzed. The nucleotide sequences were assigned to two clusters; cluster A exhibited a high degree of homology with the vaccine strain Onderstepoort, 99.10 and 97.61%, respectively, in the two isolates examined. Cluster B appeared closely related to virulent strains registered in the GeneBank database and to the virulent reference strain (A75/17); a total of 11 samples were analyzed, with 94.63-99.10% homology at the same position. The deduced amino acid sequences correlated with the two-nucleotide sequence clusters. However, there was no association among the CDV groups with histories of vaccination, sex, ages, clinical findings and evidence of viral antigen in tissues.