Hepatitis E virus infection in Thai blood donors.

BACKGROUND: Hepatitis E virus (HEV) infection in several industrialized and developing countries is associated with the consumption of pork and other meat products, an exposure risk among the majority of blood donors. We aimed to evaluate the prevalence of HEV in plasma from healthy blood donors in Thailand. STUDY DESIGN AND METHODS: We screened blood samples collected between October and December 2015, from 30,115 individual blood donors in 5020 pools of six, for HEV RNA using in-house real-time reverse-transcription polymerase chain reaction (RT-PCR). Thrice-reactive samples were subjected to a commercial real-time RT-PCR (cobas HEV test) and evaluated for anti-HEV immunoglobulin M and immunoglobulin G antibodies. Genotyping using nested RT-PCR, nucleotide sequencing, and phylogenetic analysis was performed. RESULTS: Twenty-six donors were positive for HEV RNA by the in-house assay, nine of whom were also positive by cobas test. None of the latter were reactive for anti-HEV immunoglobulin M or immunoglobulin G antibodies. Six samples were successfully genotyped and found to be HEV genotype 3. Thus, the frequency of HEV infection among healthy Thai blood donors is 1 in 1158. CONCLUSION: The presence of HEV RNA in the Thai blood supply was comparable to the rates reported in western European countries, but higher than in North America and Australia.

A molecular epidemiological study of the hepatitis B virus in Thailand after 22 years of universal immunization.

Hepatitis B virus (HBV) infection affects an estimated two billion people worldwide. Since 1992, Thailand implemented universal HBV vaccination as part of the expanded program on immunization (EPI) for newborns. This study aims to compare genotypes and characterize HBV by assessing pre-S/S and basic core promoter (BCP)/precore (PC) mutations in populations born before and after EPI implementation. A nationwide serosurvey conducted in 2014 assessed the impact of universal HBV vaccination in Thailand. Two cohort groups were established based on whether they were born before or after 1992. HBV DNA was amplified from HBsAg positive samples by PCR and sequenced. HBV genotypes, pre-S/S regions, and BCP/PC mutations were characterized. From a total of 5,964 subjects, there were 2,805 (47.0%) and 3,159 (53.0%) individuals who were born before and after EPI implementation, respectively. The overall prevalence of HBsAg was 2.2%. The prevalence of HBsAg was significantly higher in the before EPI group (4.3%) than in the after EPI group (0.3%) (P < 0.001). HBV DNA was detected in 119 samples; 111 HBV-positive samples (93%) were genotype C (subgenotype C1). The "a" determinant mutation was only detected in the "before EPI" group. Twenty-two years after implementation of the EPI program, the HBV carrier rate is significantly reduced. The most prevalent genotype for the remaining HBV was C1. The "vaccine escape" mutant, especially the "a" determinant, was not detected after the launch of the EPI program, and the current HBV vaccine remains highly effective.

Lineage-specific detection of influenza B virus using real-time polymerase chain reaction with melting curve analysis.

Influenza B viruses comprise two lineages, Victoria (B/Vic) and Yamagata (B/Yam), which co-circulate globally. The surveillance data on influenza B virus lineages in many countries often underestimate the true prevalence due to the lack of a rapid, accurate, and cost-effective method for virus detection. We have developed a real-time PCR with melting curve analysis for lineage-specific differential detection of influenza B virus. By amplifying a region of the hemagglutinin gene using real-time PCR with SYBR Green I dye, B/Vic and B/Yam could be differentiated based on their melting temperature peaks. This method was efficient (B/Vic = 93.2 %; B/Yam 97.7 %), sensitive (B/Vic, 94.6 %; B/Yam, 96.3 %), and specific (B/Vic, 97.7 %; B/Yam, 97.1 %). The lower detection limit was 10(2) copies per microliter. The assay was evaluated using 756 respiratory specimens that were positive for influenza B virus, obtained between 2010 and 2015. The incidence of influenza B virus was approximately 18.9 % of all influenza cases, and the percentage was highest among children aged 6-17 years (7.57 %). The overall percentage of mismatched influenza B vaccine was 21.1 %. Our findings suggest that real-time PCR with melting curve analysis can provide a rapid, simple, and sensitive lineage-specific influenza B virus screening method to facilitate influenza surveillance.

Genetic and antigenic characterization of hemagglutinin of influenza A/H3N2 virus from the 2015 season in Thailand.

Antigenic changes in the HA1 domain of the influenza A/H3N2 hemagglutinin (HA) present a challenge in the design of the annual influenza vaccine. We examined the genetic variability in the nucleotide and amino acid of encoding HA1 sequences of the influenza A/H3N2 virus during the 2015 influenza season in Thailand. Toward this, the HA genes of 45 influenza A/H3N2 strains were amplified and sequenced. Although a clade 3C.3a strain (A/Switzerland/9715293/2013) was chosen for the 2015 vaccine, phylogenetic analysis demonstrated that strains belonging to clade 3C.2a (96 %) instead of clade 3C.3a (4 %) were circulating that year. Sequence analysis showed that seven codons were under positive selection, five of which were located inside the antigenic epitopes. The percentages of the perfect match vaccine efficacy (VE) estimated by the P epitope model against circulating strains suggested antigenic drift of the dominant epitopes A and B, which contributed to reduced VE of the 2015 vaccine. However, the 2016 vaccine strain (A/Hong Kong/4801/2014) was closely related and well matched against the circulating strain (mean of VE = 79.3 %). These findings provide data on the antigenic drift of the influenza A/H3N2 virus circulating in Thailand and further support continual monitoring and surveillance of the antigenic changes on HA1.

The correlation between hepatitis C core antigen and hepatitis C virus RNA levels with respect to human immunodeficiency virus status, hepatitis C virus genotype and interferon-lambda-4 polymorphism.

OBJECTIVES: Serum hepatitis C virus (HCV) core antigen (HCVcAg) concentrations correlate with HCV RNA levels in HCV monoinfected patients. Data in HCV/HIV coinfected patients are still limited. We aim to compare the use of HCVcAg measurement with respect to HIV status, HCV genotypes, interferon-lambda-4 (IFNL4) polymorphism and clinical parameters. METHODS: We analyzed an untreated cohort of 104 patients with HCV monoinfection and 85 patients with HCV/HIV coinfection. Serum HCVcAg was measured by a commercial chemiluminescent microparticle immunoassay. The presence of IFNL4 polymorphism ss469415590 was identified by real-time PCR. RESULTS: log10 HCVcAg levels were significantly correlated with corresponding log10 HCV RNA levels (r = 0.889, p < 0.001), but not with ALT levels and liver stiffness. The correlation between HCV RNA and HCVcAg was particularly high in coinfected patients and those with high viremia. Mean log10 HCVcAg concentration was significantly higher in coinfected patients than in monoinfected patients. Patients harboring the TT/TT genotype of ss469415590 had significantly higher levels of log10 HCVcAg than those with the non-TT/TT genotype. HCVcAg levels were similar across HCV genotypes. CONCLUSIONS: HCVcAg concentrations had an excellent correlation with HCV RNA levels, particularly in HCV/HIV-coinfected individuals and might be associated with IFNL4 polymorphism. HCVcAg testing could be used as an alternative to HCV RNA assays in resource-limited settings.

Bufavirus in fecal specimens of patients with and without diarrhea in Thailand.

Bufavirus (BuV) was initially discovered in fecal samples from children with acute diarrhea. In this study, we determined the prevalence, distribution, and genotype(s) of BuV in Thailand. A total of 1,495 diarrheal and 741 non-diarrheal stool specimens were collected and analyzed. A portion of the NS1 gene of BuV was amplified by nested RT-PCR. Phylogenetic analysis was performed to classify the BuV strains found. We detected bufavirus (BuV) in diarrheal (4/1495; 0.27%) but not in non-diarrheal specimens (0/726). All four strains belonged to BuV genotype 1. BuV could be detected in adults and children, but its role in causing acute diarrhea remains unclear.

Diagnostic accuracy of urine heparin binding protein for pediatric acute pyelonephritis.

UNLABELLED: Timely antibiotic initiation for acute pyelonephritis (APN) can prevent renal complications. We investigated whether urine heparin binding protein (UHBP), a cytokine released from activated neutrophils, was a useful diagnostic tool for APN. Febrile children with presumed APN were prospectively enrolled between January and September 2013, and divided into two groups based on urine cultures. UHBP levels were measured at enrollment in all children and 1 month after antibiotic treatment in children with APN. UHBP levels in children with APN at baseline and 1 month versus controls were 47.0 ± 8.4 and 16.6 ± 3.8 vs. 15.0 ± 2.9 ng/mL, respectively (p < 0.001). Test performance characteristics were calculated against a gold standard of positive urine cultures and compared with leukocyte esterase (LE) and nitrite measured by dipsticks and pyuria by microscopy. The sensitivity and specificity for UHBP levels ≥34 ng/mL were 100 and 100 %. Spearman's rank coefficient was used to assess the associations between routine laboratory tests and UHBP levels. Significant positive correlations were found with pyuria grade (Spearman's rho = 0.62; p < 0.001), neutrophil count (rho = 0.38; p = 0.03), and platelet count (rho = 0.39; p = 0.03). CONCLUSIONS: UHBP is a valid adjunctive diagnostic tool for aiding clinicians in making rapid treatment decisions for APN.

NOROVIRUS OUTBREAK AT A DAYCARE CENTER IN BANGKOK, 2014.

Norovirus is a leading cause of acute non-bacterial gastroenteritis worldwide, affecting developing and developed countries, both children and adults. This study describes an outbreak of acute gastroenteritis at a daycare center of a tertiary level hospital in Bangkok, Thailand during October 2014. Although none of the staff became symptomatic, 8 of 11 children attending the center and 4 of their household contacts developed acute gastroenteritis. No pathogenic bacteria or rotavirus were detected in their evaluation; however, 3 out of 7 stool samples from the cases were positive for norovirus GII.17. Reverse transcriptase polymerase chain reaction analysis with sequence and phylogenetic analysis revealed the viral strain was the same strain reported from Taiwan in 2013. Because norovirus is a frequent cause of outbreaks in crowded conditions, early detection and preventive measures are important to control outbreaks.

Molecular characterization of human respiratory syncytial virus, 2010-2011: identification of genotype ON1 and a new subgroup B genotype in Thailand.

This study reports the molecular epidemiology and genetic characterization of human respiratory syncytial virus (RSV) samples collected in Thailand from January 2010 to December 2011. In total, 1,315 clinical samples were collected from Bangkok and Khon Kaen provinces and were screened by semi-nested PCR for RSV infection. We found 74 samples (27.7 %) and 71 samples (6.8 %) to be RSV positive for Bangkok and Khon Kaen, respectively, and we sequenced 122 of these samples. Phylogenetic analysis revealed that 100 of the RSV-A-positive samples clustered into either genotype NA1 or the recently discovered genotype ON1 strain, which has a 72-nucleotide duplication in the second variable region of its G protein. Moreover, 22 of the RSV-B-positive samples clustered into four genotypes; BA4, BA9, BA10 and genotype THB, first described here. The NA1 genotype was found to be the predominant strain in 2010 and 2011. The ON1 strain detected in this study first emerged in 2011 and is genetically similar to ON1 strains characterized in other counties. We also describe the THB genotype, which was first identified in 2005 and is genetically similar to the GB2 genotype. In conclusion, this study indicates the importance of molecular epidemiology and characterization of RSV in Thailand in order to better understand this virus. Further studies should be conducted to bolster the development of antiviral agents and a vaccine.

Evaluation of the rapid test for human rotavirus A in Thai children with acute gastroenteritis.

BACKGROUND: Rapid tests are widely used to detect rotavirus A; however, pediatricians are concerned whether the rapid test can still accurately detect the virus. Therefore, this study evaluated the performance of the rotavirus rapid test by comparing it to the one-step RT-PCR method. METHODS: Seven hundred fifty-five stool samples were collected from children with acute diarrhea. All samples were processed immediately after arrival with the SD BIOLINE rota rapid test and one-step RT-PCR method. RESULTS: The detection rates of rotavirus A were 40.79% and 41.91% for the rapid test and one-step RT-PCR, respectively. The rapid test had 93.57% sensitivity and 96.17% specificity. Most of the different genotypes of rotavirus A were detected with the SD rapid test. CONCLUSIONS: Although the rapid test is able to quickly give results, we found that it has high false positive and negative rates. Thus, other highly sensitive methods such as one-step RT-PCR are still required for true diagnosis.

Diphtheria outbreak in Thailand, 2012; seroprevalence of diphtheria antibodies among Thai adults and its implications for immunization programs.

An age distribution shift in diphtheria cases during a 2012 outbreak in northeastern of Thailand suggests adults are increasingly at risk for infection in Thailand. Data regarding immunity against diphtheria among the adult Thai population is limited. We review a 2012 diphtheria outbreak in Thailand and conducted a nationwide seroepidemiological survey to determine the prevalence of diphtheria antibodies among Thai adults in order to inform immunization programs. A total of 41 confirmed cases, 6 probable cases and 101 carriers of diphtheria were reported from northeastern and upper southern Thailand. The diphtheria outbreak in northeastern Thailand occurred among adults aged > or =15 years; sporadic cases occurred among children from upper southern Thailand. We conducted a seroepidemiological survey of 890 Thai adults from 4 age groups (20-29, 30-39, 40-49 and 50-59 years) in 7 different geographical areas of Thailand (Chiang Mai, Ratchaburi, Chon Buri, Nakhon Si Thammarat, Phitsanulok, Khon Kaen and Songkhla). Diptheria toxin antibody levels were measured with a commercially available ELISA test. The seroprotection rate ranged from 83% to 99%, with the highest in eastern Thailand (Chon Buri, 99%) and the lowest in northern Thailand (Chiang Mai, 83%). Diphtheria antibodies declined with increasing age. We recommend one doseof diphtheria-tetanus toxoid (dT) vaccine once after 20 years of age in order to boost the antibody and revaccinations every 10 years to prevent future outbreaks.

Human parainfluenza virus infection in Thai children with lower respiratory tract infection from 2010 to 2013.

Human parainfluenza virus (HPIV) is a common cause of upper and lower respiratory illness in infants and young children. In order to classify the HPIV isolates circulating in the central part of Thailand, 650 samples obtained from the lower respiratory tract of patients from two hospital pediatric wards during 2010 to 2013, were analyzed for the presence and types of HPIVs by multiplex semi-nested PCR of hemagglutinin-neuraminidase (HN) gene. The results showed that 4.8% of the samples were positive for HPIV, among which 0.5%, 2.5% and 1.5% were positive for HPIV-1, HPIV-3, and HPIV-4, respectively, and none were positive for HPIV-2. A phylogenetic tree constructed from 31 HPIV HN gene sequences compared to those in GenBank showed greater than 80% identity to other reference strains. Prevalence of HPIV infection and phylogenetic characteristics of the circulating HPIVs may help explain the impact of HPIVs infection in Thai children.

Prevalence and genetic characterization of human coronaviruses in southern Thailand from July 2009 to January 2011.

Emergence of viruses belonging to the coronavirus family has been widespread in the past, causing respiratory infections in humans, such as severe acute respiratory syndrome (SARS). This study investigated the prevalence of human coronavirus (HCoV) and characterized the molecular viral genetics. We collected 1,254 samples from patients diagnosed with respiratory infection in southern Thailand from July 2009 to January 2011 and screened for HCoV by RT-PCR and genotyped by BLAST analysis of nsp12 gene. Phylogenetic analysis was performed based on S gene sequences. Thirty-five of 1,254 samples were positive for HCoV. Viral genotyping revealed 4 genotypes with HCoV-OC43 being the predominant genotype. Viral prevalence and genotype distribution were not in accordance with seasonal distribution. Phylogenetic analysis and deduced amino acid sequences of the S gene showed amino acid variations in each genotype. The S gene sequence of HCoV-OC43 genotype indicated that it resulted from recombination between subgenotypes B and C. Viral genetics analysis disclosed genetic variations of HCoV and additionally, it can provide information suitable for monitoring and prevention of the emergence and re-emergence of various types of coronavirus.

Hand, foot, and mouth disease caused by coxsackievirus A6, Thailand, 2012.

In Thailand, hand, foot, and mouth disease (HFMD) is usually caused by enterovirus 71 or coxsackievirus A16. To determine the cause of a large outbreak of HFMD in Thailand during June-August 2012, we examined patient specimens. Coxsackievirus A6 was the causative agent. To improve prevention and control, causes of HFMD should be monitored.

Molecular characterization of human adenovirus infection in Thailand, 2009-2012.

BACKGROUND: Human adenovirus (HAdV) can cause a wide spectrum of human diseases worldwide. METHODS: Using PCR and sequence analysis, we investigated HAdV infection prevalence in the Thai population for four years from January 2009 to December 2012. We collected Nasopharyngeal swab/aspirate (NP) specimens from patients in Bangkok, Khon Kaen, and Nakhon Si Thammarat province and fecal specimens only from Bangkok and Khon Kaen province. RESULTS: We observed HAdV infection in 1.04% (82/7,921) of NP samples and in 5.84% (76/1,301) of fecal specimens. HAdV-B3 (32%) and HAdV-C1 (31%) were the genotypes most commonly associated with NP specimens followed by HAdV-C2 (13%) and HAdV-C5 (12%). In fecal specimens, we found that 25% harbored HAdV-F41 followed by HAdV-C1 (18%), HAdV-C2 (16%), and HAdV-B3 (13%). Out of all population subsets, children below the age of 3 years were the most likely to be HAdV positive (63.29%). In addition, HAdV infection occurred throughout the year without a seasonal distribution pattern, although HAdV infection of NP samples peaked from January-April while HAdV infection peaked from January to March and then again from May to July in fecal samples. CONCLUSIONS: This study has for the first time reported the HAdV infection rate in Thai NP and fecal specimens from 2009-2012. We observed that HAdV-B3 and HAdV-C1 were commonly found in NP specimens, and that HAdV-F41 was the most prevalence in fecal specimens in Thailand during the study period.

Hepatitis E virus genotype 3f sequences from pigs in Thailand, 2011-2012.

Phylogenetic analysis of partial ORF1 and ORF2 genes of Hepatitis E virus (HEV) strains from pigs in Thailand during 2011-2012 was performed. The result indicated that the current Thai strains belonged to the genotype 3 subgroup 3f, which were similar to the previous HEVs circulating in humans in Thailand.

Molecular analysis of hepatitis B virus associated with vaccine failure in infants and mothers: a case-control study in Thailand.

Perinatal transmission of hepatitis B virus (HBV) has been controlled incompletely despite adequate immunoprophylaxis in infants. The aim of this study was to characterize virological factors of HBV associated with vaccine failure in Thailand. Sera of 14 infected infants (13 HBeAg-positive and one HBeAg-negative) with vaccine failure and their respective mothers (group M1) were tested quantitatively for HBV DNA by real-time PCR, HBV genotypes and mutations were characterized by direct sequencing. Sera collected from 15 HBeAg-positive (group M2) and 15 HBeAg-negative (group M3) mothers whose infants had been vaccinated successfully served as controls. The results showed that group M1 and group M2 mothers had equal titers of HBV DNA but higher titers than group M3. All infected infants and their respective mothers had the same HBeAg status and HBV genotypes. DNA analysis in a pair of HBeAg-negative infant and mother revealed that both were infected with an HBV precore mutant (G1896A). Escape mutants in the "a" determinant region (residues 144 and 145) were detected in two (14%) infected infants. The prevalence of BCP mutations/deletions in groups M2 and M3 was higher significantly than in group M1 (P = 0.022 and P < 0.001, respectively). In conclusion, instead of the HBeAg status, a high titer of HBV DNA in mothers was the major contributor to perinatal transmission of HBV. Escape mutants might be associated with vaccine failure in some infants. BCP mutations/deletions in mothers might contribute to the prevention of mother-to-infant transmission of HBV.

Complete coding sequence and molecular analysis of hepatitis A virus from a chimpanzee with fulminant hepatitis.

BACKGROUND: Hepatitis A virus (HAV) infects both humans and non-human primates, in experimentally infected chimpanzees is typically milder than in humans. In 1982, Abe and Shikata reported a first case of a chimpanzee with fulminant hepatitis caused by spontaneous HAV infection, and the underlying mechanisms of the disease remain unknown. METHODS: To characterize denoted CFH-HAV, we conducted cloning and near full-length sequence analysis. RESULTS: Phylogenetic analyses of VP1-2A and complete sequence comparison between various genotypes and the sample sequence showed clustering in genotype IB. Based on BLAST analysis, the sequence was most closely related to the wild-type (HM175/WT) isolate. Amino acid and nucleic acid similarities were 99.8% and 94.41%, respectively. CONCLUSIONS: The chimpanzee may have been infected with human HAV genotype IB. The substitutions in VP2, VP4, 2B, 2C, and 3D, which may enhance virus proliferation, contributed to disease severity culminating in fulminant hepatic failure.

Evaluation of rapid influenza virus tests in patients with influenza-like illness in Thailand.

BACKGROUND: The influenza virus is responsible for causing major respiratory tract symptoms. A fast, accurate diagnosis is essential for efficient treatment, especially in patients with complications. The Rapid Influenza Diagnostic Tests (RIDTs) for influenza detection have been developed to subtype the influenza virus. The re-evaluation of the rapid test is needed in terms of specificity, sensitivity, and accuracy. METHODS: From August 13, 2010 to September 22, 2011, 1,076 nasal aspirates were obtained from patients, ages ranging from 15 days to 98 years, with symptoms of influenza-like illness (ILI) and evaluated by 2 types of RIDTs, Standard Diagnosis (SD) and QuickVue (QV) Rapid tests followed by real-time RT-PCR. The results from the rapid test diagnoses were compared to those from real-time RT-PCR. RESULTS: During 2010 and 2011, the estimated sensitivity of the SD rapid test for seasonal H3, human pandemic H1N1, and influenza B infection was 49.4%, specificity was 84.1%, positive predictive value was 47.6%, and negative predictive value was 85% while those of the QV rapid test were 63.4%, 96.7%, 94.8%, and 80.3%, respectively. Infant patients (< or = 5 years) yielded less false negatives while adolescents and adults (older than 5 years) showed more false negatives 8.8% and 15.2%, respectively. Using rapid test diagnosis, H3N2 influenza virus was found with more false negative results (11.1%) than the other viruses (1.1 - 3.5%). The SD rapid test appeared to be more sensitive than the QV test during high season activity while the QV test was more sensitive during the period of low influenza virus activity. CONCLUSIONS: Due to persistent genetic drift of the influenza virus, the available RIDTs should be re-evaluated each year. During 2010 - 2011, the QV rapid test showed more reliable results than the SD rapid test. However, the false negative results of H3N2 influenza virus detection during its peak should be cause for concern. Some of the results, e.g. patients with complications, should be compared with real-time RT-PCR as the gold standard method for detecting influenza virus infection.

Polyomavirus reactivation in pediatric patients with systemic lupus erythematosus.

Polyomavirus (PyV) infection usually persists without any symptoms in normal individuals. In immunocompromised patients including patients with systemic lupus erythematosus (SLE), PyV reactivation occurs with a high prevalence and can cause severe clinical complications. In this study, reactivation of six PyV [JC, BK, WU, KI, merkel cell (MC) and trichodysplasia spinulosa (TS)] was investigated in terms of prevalence, clinical implications and correlation with urine transforming growth factor (TGF)-ß1 expression in 50 SLE patients aged less than 18 years. Clinical characteristics were obtained from medical record review. PyV viruria was assessed by nested polymerase chain reaction. Urine TGF-ß1 was measured with ELISA. The mean age was 13 ± 2.8 years. The prevalence of JC and BK viruria was 16% and 32%, respectively. WU, KI, MC and TS were not isolated from any urine specimens. Co-reactivation of 2 PyV was not detected. Urine TGF-ß1 levels in patients with JC viruria, with BK viruria and without PyV viruria were 0.27 ± 0.09, 0.10 ± 0.05 and 0.13 ± 0.09 ng/mg of urine creatinine, respectively. Cumulative doses of cyclophosphamide per body weight and urine TGF-ß1 levels were higher in JC viruria than in other groups (p < 0.05). The prevalence of JC and BK reactivation was higher in pediatric patients with SLE than in the normal population. JC reactivation in pediatric patients with SLE was correlated with the administration of high-dose cyclophosphamide and increased urine TGF-ß1 levels. Surveillance of JC reactivation is recommended in pediatric patients with chronic and severe SLE receiving high-dose cyclophosphamide.