Kesmanee Praianantathavorn

Geographic distribution of hepatitis C virus genotype 6 subtypes in Thailand

The nucleotide sequence of hepatitis C virus (HCV) genotype 6 found mostly in south China and south‐east Asia, displays profound genetic diversity. The aim of this study to determine the genetic variability of HCV genotype 6 (HCV‐6) in Thailand and locate the subtype distribution of genotype 6 in various geographic areas. Four hundred nineteen anti‐HCV positive serum samples were collected from patients residing in ‐ the central part of the country. HCV RNA positive samples based on reverse transcriptase‐ polymerase chain reaction (RT‐PCR) of the 5′UTR were amplified with primers specific for the core and NS5B regions. Nucleotide sequences of both regions were analyzed for the genotype by phylogenetic analysis. To determine geographic distribution of HCV‐6 subtypes, a search of the international database on subtype distribution in the respective countries was conducted. Among 375 HCV RNA positive samples, 71 had HCV‐6 based on phylogenetic analysis of partial core and NS5B regions. The subtype distribution in order of predominance was 6f (56%), 6n (22%), 6i (11%), 6j (10%), and 6e (1%). Among the 13 countries with different subtypes of HCV‐6, most sequences have been reported from Vietnam. Subtype 6f was found exclusively in Thailand where five distinct HCV‐6 subtypes are circulating. HCV‐6, which is endemic in south China and south‐east Asia, displays profound genetic diversity and may have evolved over a considerable period of time. J. Med. Virol. 82:257–262, 2010.

Long-term humoral and cellular immune response to hepatitis B vaccine in high-risk children 18-20 years after neonatal immunization.

Eighty-seven high-risk individuals in Thailand who had received a complete course of recombinant HBV vaccine 18-20 y ago were investigated with regard to their immunological memory. To evaluate humoral immunity, anti-HBs antibody titers were measured. Cellular immunity was determined by ELISPOT to detect HBV-specific IFN-gamma-producing cells. Overall 83.9% of participants developed circulating anti-HBs (titer > or = 1 mIU/mL) and 58.6% were seroprotected (titer > or = 10 mIU/mL). As for cellular immunity, 50.6% were positive on ELISPOT. Moreover, there was no correlation between the level of anti-HBs and positive ELISPOT results. However, the majority of participants (81.8%) who were positive for IFN-gamma-producing cells were seropositive, but only 50% of seropositive participants were ELISPOT-positive. Thus, 18-20 y after immunization, it appears that a second booster dose should be considered, especially in high-risk groups.

Multiplex real-time RT-PCR for detecting chikungunya virus and dengue virus

Abstract Objective To develop diagnostic test for detection chikungunya virus (CHIKV and Dengue virus (DENV) infection. Methods We have performed a rapid, accurate laboratory confirmative method to simultaneously detect, quantify and differentiate CHIKV and DENV infection by single-step multiplex real-time RT-PCR. Results The assays sensitivity was 97.65%, specificity was 92.59% and accuracy was 95.82% when compared to conventional RT-PCR. Additionally, there was no cross-reaction between CHIKV, DENV, Japanese encephalitis virus, hepatitis C, hepatitis A or hepatitis E virus. Conclusions This rapid and reliable assay provides a means for simultaneous early diagnosis of CHIKV and DENV in a single-step reaction.

Seroprevalence and Genotype of Hepatitis C Virus among Immigrant Workers from Cambodia and Myanmar in Thailand

Objective: There is a large number of immigrant workers from Cambodia and Myanmar in Thailand. The aim of our study was to determine seroprevalence and genotypes of hepatitis C virus (HCV) in this group. Methods: Immigrants aged between 15 and 60 years (1,431 Cambodians and 1,594 Myanmarese) were recruited into this study. Each sample was screened for anti-HCV by ELISA. RNA was extracted from seropositive samples and RT-PCR was performed in order to amplify the HCV core region. Each sample was subsequently sequenced, and the genotype was determined by phylogenetic analysis. Results: The prevalence of HCV infection in immigrant workers from Cambodia and Myanmar was 33 (2.3%) and 27 (1.69%) samples, respectively. Of the anti-HCV-positive individuals, 25 (75.8%) from Cambodia and 15 (55.6%) from Myanmar harbored viral RNA. Phylogenetic analysis showed that the predominant HCV genotypes in this group were 1a, 1b, 3a, 3b and 6 (6e, 6f, 6m, 6p and 6r). Most HCV isolates can be found in Thailand, though some subtypes of HCV-6 are uncommon. Conclusions: This study shows the HCV seroprevalence and genotypes among immigrant Cambodians and Myanmarese which may reflect the prevalence in each country and closely relate to the prevalence in the guest country.

Serum adiponectin and transient elastography as non-invasive markers for postoperative biliary atresia

BackgroundBiliary atresia (BA) is a progressive inflammatory disorder of the extrahepatic bile ducts leading to the obliteration of bile flow. The purpose of this study was to determine serum adiponectin in BA patients and to investigate the relationship of adiponectin with clinical parameters and liver stiffness scores.MethodsSixty BA patients post Kasai operation and 20 controls were enrolled. The mean age of BA patients and controls was 9.6 ± 0.7 and 10.1 ± 0.7 years, respectively. BA patients were classified into two groups according to their serum total bilirubin (TB) levels (non-jaundice, TB < 2 mg/dl vs. jaundice, TB ≥ 2 mg/dl) and liver stiffness (insignificant fibrosis, liver stiffness < 7 kPa vs. significant fibrosis, liver stiffness ≥ 7 kPa). Serum adiponectin levels were analyzed by enzyme-linked immunosorbent assay. Liver stiffness scores were examined by transient elastography (FibroScan).ResultsBA patients had markedly higher serum adiponectin levels (15.5 ± 1.1 vs. 11.1 ± 1.1 μg/ml, P = 0.03) and liver stiffness than controls (30.1 ± 3.0 vs. 5.1 ± 0.5 kPa, P < 0.001). Serum adiponectin levels were significantly elevated in BA patients with jaundice compared with those without jaundice (24.4 ± 1.4 vs. 11.0 ± 0.7 μg/ml, P < 0.001). In addition, BA patients with significant liver fibrosis had remarkably greater serum adiponectin than insignificant fibrosis counterparts (17.7 ± 1.2 vs. 9.4 ± 1.1 μg/ml, P < 0.001). Subsequent analysis revealed that serum adiponectin was positively correlated with total bilirubin, hyaluronic acid, and liver stiffness (r = 0.58, r = 0.46, and r = 0.60, P < 0.001, respectively).ConclusionsSerum adiponectin and liver stiffness values were higher in BA patients compared with normal participants. The elevated serum adiponectin levels also positively correlated with the degree of hepatic dysfunction and liver fibrosis. Accordingly, serum adiponectin and transient elastography could serve as the useful non-invasive biomarkers for monitoring the severity and progression in postoperative BA.

Molecular Evolution of Human H1N1 and H3N2 Influenza A Virus in Thailand, 2006–2009

Background Annual seasonal influenza outbreaks are associated with high morbidity and mortality. Objective To index and document evolutionary changes among influenza A H1N1 and H3N2 viruses isolated from Thailand during 2006–2009, using complete genome sequences. Methods Nasopharyngeal aspirates were collected from patients diagnosed with respiratory illness in Thailand during 2006–2009. All samples were screened for Influenza A virus. A total of 13 H1N1 and 21 H3N2 were confirmed and whole genome sequenced for the evolutionary analysis using standard phylogenetic approaches. Results Phylogenetic analysis of HA revealed a clear diversification of seasonal from vaccine strain lineages. H3N2 seasonal clusters were closely related to the WHO recommended vaccine strains in each season. Most H1N1 isolates could be differentiated into 3 lineages. The A/Brisbane/59/2007 lineage, a vaccine strain for H1N1 since 2008, is closely related with the H1N1 subtypes circulating in 2009. HA sequences were conserved at the receptor-binding site. Amino acid variations in the antigenic site resulted in a possible N-linked glycosylation motif. Recent H3N2 isolates had higher genetic variations compared to H1N1 isolates. Most substitutions in the NP protein were clustered in the T-cell recognition domains. Conclusion In this study we performed evolutionary genetic analysis of influenza A viruses in Thailand between 2006–2009. Although the current vaccine strain is efficient for controlling the circulating outbreak subtypes, surveillance is necessary to provide unambiguous information on emergent viruses. In summary, the findings of this study contribute the understanding of evolution in influenza A viruses in humans and is useful for routine surveillance and vaccine strain selection.

Human microRNAs profiling in response to influenza A viruses (subtypes pH1N1, H3N2, and H5N1).

MicroRNAs (miRNAs) play an important role in regulation of gene silencing and are involved in many cellular processes including inhibition of infected viral replication. This study investigated cellular miRNA expression profiles operating in response to influenza virus in early stage of infection which might be useful for understanding and control of viral infection. A549 cells were infected with different subtypes of influenza virus (pH1N1, H3N2 and H5N1). After 24 h post-infection, miRNAs were extracted and then used for DNA library construction. All DNA libraries with different indexes were pooled together with equal concentration, followed by high-throughput sequencing based on MiSeq platform. The miRNAs were identified and counted from sequencing data by using MiSeq reporter software. The miRNAs expressions were classified into up and downregulated miRNAs compared to those found in non-infected cells. Mostly, each subtype of influenza A virus triggered the upregulated responses in miRNA expression profiles. Hsa-miR-101, hsa-miR-193b, hsa-miR-23b, and hsa-miR-30e* were upregulated when infected with all three subtypes of influenza A virus. Target prediction results showed that virus infection can trigger genes in cellular process, metabolic process, developmental process and biological regulation. This study provided some insights into the cellular miRNA profiling in response to various subtypes of influenza A viruses in circulation and which have caused outbreaks in human population. The regulated miRNAs might be involved in virus–host interaction or host defense mechanism, which should be investigated for effective antiviral therapeutic interventions.

Kinetics of serum HBsAg and intrahepatic cccDNA during pegylated interferon therapy in patients with HBeAg-positive and HBeAg-negative chronic hepatitis B.

This study was aimed at comparing clinical applicability of serum HBsAg quantification in relation to intrahepatic covalently closed‐circular DNA (cccDNA) in patients with HBeAg‐positive and HBeAg‐negative chronic hepatitis B (CHB) treated with pegylated interferon (PEG‐IFN) monotherapy for 48 weeks. Overall, 32 and 36 patients with HBeAg‐positive and HBeAg‐negative CHB, respectively were recruited. Paired liver biopsies at baseline and end of therapy were analyzed for cccDNA. Virological response (VR) at 48 weeks post‐treatment was defined as HBeAg clearance (for HBeAg‐positive CHB) and HBV DNA <2,000 IU/ml (for both groups). The results demonstrated that baseline levels of all viral markers were higher in the HBeAg‐positive group than the HBeAg‐negative group. Baseline HBsAg correlated with cccDNA in the HBeAg‐positive group (r = 0.452, P = 0.009) but not in the HBeAg‐negative group (r = 0.018, P = 0.919). However, the magnitude of cccDNA and HBsAg decline at end of treatment was not different between groups. The reduction of HBsAg showed a positive correlation with cccDNA decline in HBeAg‐positive and HBeAg‐negative CHB (r = 0.544, P = 0.001 and r = 0.364, P = 0.029, respectively). Overall, responders had more decline in cccDNA and HBsAg levels compared with non‐responders. Patients with serum HBsAg decline of >1.0 log10 IU/ml during treatment archived VR and HBsAg clearance of 80% and 30%, respectively. In conclusion, serum HBsAg represented a better surrogate marker of intrahepatic cccDNA in patients with HBeAg‐positive CHB compared to those with HBeAg‐negative CHB. On‐treatment, HBsAg reduction of 1.0 log10 IU/mL was associated with a high probability of subsequent VR and HBsAg clearance in patients receiving PEG‐IFN therapy. J. Med. Virol. 89:130–138, 2017.

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.

Original Research: Analysis of hepatic microRNA alterations in response to hepatitis B virus infection and pegylated interferon alpha-2a treatment.

Interferons play important roles in defense mechanisms against viral infection, and thus interferon therapy has been a standard treatment in chronic hepatitis B patients. Interferons signaling pathways promote interferon-inducible genes including microRNAs. In this research, we aimed to determine microRNAs expression profiles in vitro and in vivo. For in vitro model, Huh7 cells were transfected with or without hepatitis B virus plasmid for 6 h, and then treated with 100 ng of pegylated-interferon alpha-2a for 24 h. In vivo, we defined microRNAs expression profiles in pair-liver tissues of chronic hepatitis B patients in comparison between before and after treatment of pegylated-interferon alpha-2a for 48 weeks. Cellular small RNAs were extracted followed by library preparation. To determine microRNAs expression profiles, the next-generation sequencing was carried out on MiSeq platform (Illumina®). In vitro analysis demonstrated that microRNAs can be classified into up-regulated and down-regulated microRNAs in response to hepatitis B virus, interferon, and combination of hepatitis B virus and interferon. Moreover, in vivo analysis revealed microRNAs profiles in non-responders, responders without hepatitis B surface antigen clearance, and responders with hepatitis B surface antigen clearance. The target genes of the candidate microRNAs were determined in terms of roles in cellular pathways and immune response, which might be related to treatment in chronic hepatitis B patients. Results revealed that two down-regulated microRNAs including miR-185-5p and miR-186-5p were correlated in both in vitro and in vivo studies. These two microRNAs might be represented as specific hepatic microRNAs responding to hepatitis B virus and pegylated-interferon alpha-2a treatment, which may remarkable and attractive for further study involving in the association of their target genes and prediction of pegylated-interferon alpha-2a response. Interestingly, microRNAs expression patterns might be useful for understanding the response mechanism and serve as biomarkers for prediction of pegylated-interferon alpha-2a treatment response in patients with chronic hepatitis B.