Genome characterization and mutation analysis of human influenza A virus in Thailand

The influenza A viruses have high mutation rates and cause a serious health problem worldwide. Therefore, this study focused on genome characterization of the viruses isolated from Thai patients based on the next-generation sequencing technology. The nasal swabs were collected from patients with influenza-like illness in Thailand during 2017-2018. Then, the influenza A viruses were detected by reverse transcription-quantitative polymerase chain reaction and isolated by MDCK cells. The viral genomes were amplified and sequenced by Illumina MiSeq platform. Whole genome sequences were used for characterization, phylogenetic construction, mutation analysis and nucleotide diversity of the viruses. The result revealed that 90 samples were positive for the viruses including 44 of A/ H1N1 and 46 of A/H3N2. Among these, 43 samples were successfully isolated and then the viral genomes of 25 samples were completely amplified. Finally, 17 whole genomes of the viruses (A/H1N1, n=12 and A/H3N2, n=5) were successfully sequenced with an average of 232,578 mapped reads and 1,720 genome coverage per sample. Phylogenetic analysis demonstrated that the A/H1N1 viruses were distinguishable from the recommended vaccine strains. However, the A/H3N2 viruses from this study were closely related to the recommended vaccine strains. The nonsynonymous mutations were found in all genes of both viruses, especially in hemagglutinin (HA) and neuraminidase (NA) genes. The nucleotide diversity analysis revealed negative selection in the PB1, PA, HA, and NA genes of the A/H1N1 viruses. High-throughput data in this study allow for genetic characterization of circulating influenza viruses which would be crucial for preparation against pandemic and epidemic outbreaks in the future.

Metagenomic analysis of viral genes integrated in whole genome sequencing data of Thai patients with Brugada syndrome

Brugada syndrome (BS) is an autosomal dominant inheritance cardiac arrhythmia disorder associated with sudden death in young adults. Thailand has the highest prevalence of BS worldwide, and over 60% of patients with BS still have unclear disease etiology. Here, we performeda new viral metagenome analysis pipeline called VIRIN and validated it with whole genome sequencing (WGS) data of HeLa cell lines and hepatocellular carcinoma. Then the VIRIN pipelinewas applied to identify viral integration positions from unmapped WGS data of Thai males, including 100 BS patients (case) and 100 controls. Even though the sample preparation had noviral enrichment step, we can identify several virus genes from our analysis pipeline. The predominance of human endogenous retrovirus K (HERV-K) viruses was found in both cases andcontrols by blastn and blastx analysis. This study is the first report on the full-length HERV-Kassembled genomes in the Thai population. Furthermore, the HERV-K integration breakpointpositions were validated and compared between the case and control datasets. Interestingly,Brugada cases contained HERV-K integration breakpoints at promoters five times more oftenthan controls. Overall, the highlight of this study is the BS-specific HERV-K breakpoint positionsthat were found at the gene coding region "NBPF11" (n = 9), "NBPF12" (n = 8) and longnon-coding RNA (lncRNA) "PCAT14" (n = 4) region. The genes and the lncRNA have been reported to be associated with congenital heart and arterial diseases. These findings provide another aspect of the BS etiology associated with viral genome integrations within the humangenome.

Comparison of Four Human Papillomavirus Genotyping Methods: Next-generation Sequencing, INNO-LiPA, Electrochemical DNA Chip, and Nested-PCR

BACKGROUND: Human papillomavirus (HPV) infection causes cervical cancer, thus necessitating early detection by screening. Rapid and accurate HPV genotyping is crucial both for the assessment of patients with HPV infection and for surveillance studies. METHODS: Fifty-eight cervicovaginal samples were tested for HPV genotypes using four methods in parallel: nested-PCR followed by conventional sequencing, INNO-LiPA, electrochemical DNA chip, and next-generation sequencing (NGS). RESULTS: Seven HPV genotypes (16, 18, 31, 33, 45, 56, and 58) were identified by all four methods. Nineteen HPV genotypes were detected by NGS, but not by nested-PCR, INNO-LiPA, or electrochemical DNA chip. CONCLUSIONS: Although NGS is relatively expensive and complex, it may serve as a sensitive HPV genotyping method. Because of its highly sensitive detection of multiple HPV genotypes, NGS may serve as an alternative for diagnostic HPV genotyping in certain situations.

Interpretation of microscopic agglutination test for leptospirosis diagnosis and seroprevalence

Determination of antibody titer by microscopic agglutination test (MAT) has been used as a tool for leptospirosis diagnosis. Four fold or greater rise in antibody titers between acute and convalescent sera suggests recent Leptospira infection. In addition, results obtained by MAT have been used to predict infecting serovars. However, cross reactivity among various Leptospira serovars have been reported when patient sera were tested with a battery of Leptospira serovars. This study demonstrates cross- reactivity among several Leptospira serovars when MAT was performed on leptospirosis sera. The data support a role of MAT as a tool for diagnosis. However, for information on infecting serovars, Leptospira isolation and molecular identification should be performed.

Interpretation of microscopic agglutination test for leptospirosis diagnosis and seroprevalence

Determination of antibody titer by microscopic agglutination test (MAT) has been used as a tool for leptospirosis diagnosis. Four fold or greater rise in antibody titers between acute and convalescent sera suggests recent Leptospira infection. In addition, results obtained by MAT have been used to predict infecting serovars. However, cross reactivity among various Leptospira serovars have been reported when patient sera were tested with a battery of Leptospira serovars. This study demonstrates cross- reactivity among several Leptospira serovars when MAT was performed on leptospirosis sera. The data support a role of MAT as a tool for diagnosis. However, for information on infecting serovars, Leptospira isolation and molecular identification should be performed.

Cytokine levels in patients with chikungunya virus infection

OBJECTIVE@#To investigate cytokine profile in patients with chikungunya virus (CHIKV) infection.@*METHODS@#Twenty eight pairs of serum samples collected from CHIKV infected patients during the outbreak of chikungunya fever in South Thailand in 2008 were obtained. A multiple cytokine assay for detection of 17 cytokines was performed.@*RESULTS@#In the acute stage of CHIKV infection, the patients had significantly higher levels of interleukin-6, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, monocyte chemotactic protein 1 and tumor necrosis factor alpha than the control (P<0.001, P=0.023, P=0.015, P<0.001 and P=0.024, respectively). When the disease developed to the recovery stage, the patients had significantly lower levels of interleukin-6, granulocyte-macrophage colony-stimulating factor, monocyte chemotactic protein 1 and macrophage inflammatory protein beta than in the acute stage (P<0.001).@*CONCLUSIONS@#This study provides additional information that these cytokines could play roles in pathogenesis of CHIKV infection and could be used as disease biomarkers or drug targets.

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

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 assay's 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.

Erythrocyte Binding Preference of Human Pandemic Influenza Virus A and Its Effect on Antibody Response Detection

BACKGROUND: Validation of hemagglutination inhibition (HI) assays is important for evaluating antibody responses to influenza virus, and selection of erythrocytes for use in these assays is important. This study aimed to determine the correlation between receptor binding specificity and effectiveness of the HI assay for detecting antibody response to pandemic influenza H1N1 (pH1N1) virus. METHODS: Hemagglutination (HA) tests were performed using erythrocytes from 6 species. Subsequently, 8 hemagglutinating units of pH1N1 from each species were titrated by real-time reverse transcription-PCR. To investigate the effect of erythrocyte binding preference on HI antibody titers, comparisons of HI with microneutralization (MN) assays were performed. RESULTS: Goose erythrocytes showed most specific binding with pH1N1, while HA titers using human erythrocytes were comparable to those using turkey erythrocytes. The erythrocyte binding efficiency was shown to have an impact on antibody detection. Comparing MN titers, HI titers using turkey erythrocytes yielded the most accurate results, while those using goose erythrocytes produced the highest geometric mean titer. Human blood group O erythrocytes lacking a specific antibody yielded results most comparable to those obtained using turkey erythrocytes. Further, pre-existing antibody to pH1N1 and different erythrocyte species can distort HI assay results. CONCLUSIONS: HI assay, using turkey and human erythrocytes, yielded the most comparable and applicable results for pH1N1 than those by MN assay, and using goose erythrocytes may lead to overestimated titers. Selection of appropriate erythrocyte species for HI assay allows construction of a more reliable database, which is essential for further investigations and control of virus epidemics.

Molecular genome tracking of East, Central and South African genotype of Chikungunya virus in South-east Asia between 2006 and 2009

OBJECTIVE@#To understand the epidemiology of the East, Central and South African (ECSA) genotype of Chikungunya virus (CHIKV) in terms of emerging and re-emerging infections, this study has been aimed at investigating the evolutionary parameters, genomic signatures and molecular tracking of the CHIKV ECSA genotype in South-east Asia and coastal areas of the Indian Ocean between 2006 and 2009 by using phylogenetic analysis and the Bayesian Markov Chain Monte Carlo (BMCMC) evolutionary estimation.@*METHODS@#Nearly complete genome sequences of 53 CHIKV isolates from all genotypes were subjected to phylogenetic analysis and evolutionary parameter estimation. The amino acids of 67 of ECSA genotype during 2006 to 2009 were compared for finding molecular signature tracking. The ECSA genotype signatures were visualized to find the possible transmission root was projected onto a geographic map.@*RESULTS@#Phylogenetic analysis showed the ECSA genotype was divided into 2 groups. The first group comprises viruses from India and Southeast Asian countries. The second group consists of strains typically circulating in Sri Lanka in 2008. The evolutionary parameters of these groups depicted the time of the most recent common ancestor at approximately 7.5 years ago. The genomic signatures revealed the positions of amino acid variation in each group.@*CONCLUSIONS@#The molecular evolution projected onto a geographical map showed the routes of CHIKV transmission from 2006 to 2009. Molecular tracking will assist in understanding transmission routes, epidemiology and molecular evolution of CHIKV.

Overwhelming strongyloidiasis

Background: Overwhelming strongyloidiasis defined by multi-organ dissemination causes severe morbidity and high mortality.Objective: To report a case of disseminated strongyloidiasis presenting with unusual gastrointestinal manifestations in an immunocompromised host.Methods: A Thai girl with myasthenia gravis treated by chronic administration of corticosteroids presented with disseminated strongyloidiasis. Pulmonary and gastrointestinal symptoms were the clinical manifestations of hyperinfection or disseminated strongyloidiasis.Results: Strongyloid larvae were found in her sputum, stool, and peritoneal fluid. They were present in all layers of the intestinal wall. She did not respond to oral antihelminthic drugs (albendazole). Subcutaneous ivermectin was administered. She succumbed to unresponsive cardiac arrest that was unresponsive to standard resuscitation protocols due to severe septicemia. Pulmonary and gastrointestinal symptoms were the clinical manifestations of hyperinfection or disseminated strongyloidiasis.Conclusion: Serial stool examination should be performed prior to the onset and during immunosuppressive treatment.

Novel H1N1 2009 influenza virus infection in Bangkok, Thailand:effects of school closures

Background: Influenza virus is the major cause of respiratory illness, especially in young and older age groups. Since 1918, many subtypes, defined by hemagglutinine (HA) and neuraminidase (NA), have caused global infections or pandemics. The recently isolated swine origin influenza virus (S-OIV) subtype H1N1 has been defined by the World Health Organization (WHO) as the cause of the present influenza pandemic. Objective: To describe and attempt to predict the epidemiology of the novel H1N1 2009 in Bangkok and to evaluate the effects of school closures during the outbreaks. Materials and methods: The first two human cases infected by this S-OIV subtype H1N1 or H1N1 2009 in Thailand have been reported in May 12, 2009 by the Ministry of Public Health. Between May 12 and July 30, 2009, 1212 nasopharyngeal (NP) swabs from four private hospitals and Chulalongkorn Hospital, Bangkok have been sent to a laboratory for Influenza virus diagnosis. The diagnosis was based on real time RT-PCR for seasonal influenza (H1, H3) and H1N1 2009. Results: One thousand two hundreds and twelve specimens of patients with influenza like illness were tested using real time RT-PCR detection. Between mid June and early July, the number of H1N1 2009 increased rapidly with a high prevalence among the 6- to 20-year olds. School closure policy, long public holiday, and additional preventive measures have led to a rapid reduction in the number of H1N1 2009 positive patients. Conclusion: Preventive measures including school closures are important to slow down the outbreak and thus enable health care centers to cope with the large number of patients. Everyone should play a role in delaying the spread of this pandemic.

Platelet count in predicting bleeding complication after elective endoscopy in children with portal hypertension and thrombocytopenia

Background: Thrombocytopenia is a frequent and challenging clinical disorder in patients with portal hypertension. It can increase the risk of bleeding associated with invasive procedures. Standard treatment for thrombocytopenia usually consists of platelet transfusions, which may cause transfusion-related complications including viral or bacterial infection, allo-immunization and febrile non-hemolytic reactions. Uncertainty still exists as to the platelet level at which transfusion is indicated in conjunction with invasive procedures such as endoscopy.Objective: To determine the predictive value of platelet level for post elective endoscopy bleeding in children with portal hypertension and thrombocytopenia.Patients and methods: Children with portal hypertension and thrombocytopenia (platelet count \< 100,000 per μL) were enrolled. Those who had active gastrointestinal (GI) bleeding at the time of admission were excluded. All patients underwent elective upper GI endoscopy for esophagogastric varices surveillance and tested for complete blood count.Results: There were 41 children (male:female = 20:21) with portal hypertension enrolled in this study. Age ranged from 2-16 years (mean±SD = 8.42±4.32 years). The etiology of portal hypertension was biliary atresia in 19 and extra-hepatic portal vein obstruction in 22. Thirty of 41 experienced previous upper GI bleeding. Endoscopic finding was 32 esophageal varices (EVs), two EVs plus gastric varices, and seven without varices. Twenty-one patients underwent endoscopy without therapeutic procedure and 20 went through endoscopic variceal ligation. Two patients developed GI bleeding post endoscopy. Platelet count of children with and without post endoscopy bleeding was not significantly different (68,500±40,305 vs. 73,025±24,030/μL, respectively; p=0.8). By applying receiver operating characteristic (ROC) curves, a platelet count cut-off value of 41,500/μL was obtained, which gave positive and negative predictive values of 12.5% and 96.9%, respectively. The accuracy of this cut-off value as evaluated by applying ROC curves was 80.5%.Conclusion: Patients with platelet count of more than 40,000/μL were almost certainly safe to undergo elective endoscopy without prophylactic platelet transfusions and hence minimize the cost and complications of transfusion.

Crigler-Najjar syndrome: a case report

Background: Crigler-Najjar syndrome (CN) clinically manifests as intense unconjugated hyperbilirubinemia without evidence of hemolysis. At present, over 90 genetic variations such as mutations, insertions, or deletions have been described in the five exons of the UDP-glucuronosyltransferase (UGT1A1) gene responsible for defect of bilirubin conjugation.Objective: To report a case of a female CN type I child who presented with unconjugated hyperbilirubinemia, normal liver function tests, and normal ultrasonographic images of the liver.Results: Peak total bilirubin in this patient was 27.6 mg/dL. She developed kernicterus despite prolonged daily home phototherapy. Exons 1-5 of the UGT1A1 gene were amplified by polymerase chain reaction (PCR) and the UGT1A1 gene was screened for mutations by direct DNA sequencing. Molecular genetic analysis showed that this patient was homozygous for a nonsense mutation at nucleotide number 715 (715C→T) in exon 1 resulting in the replacement of glutamine (CAG, amino acid 239) by a stop codon (TAG).Conclusion: Detection of this genetic defect is essential for gene therapy and can be used as a prenatal screening test to identify the affected offspring.

LipL32 mRNA expression in kidneys, livers and lungs of hamsters infected with pathogenic Leptospira

Background: LipL32 is an outer membrane protein of pathogenic Leptospira. Humans and animals infected with Leptospira elicite strong immune responses to this protein. LipL32 induced cytokine expression in mouse proximal tubule cells which suggests its involvement in kidney immunopathogenesis. In addition to kidneys, livers and lungs are others organs frequently affected in leptospirosis. Objective: To demonstrate LipL32 expression in kidney, liver and lung tissues of hamsters infected with pathogenic Leptospira. Methods: RNA was extracted from kidney, liver and lung tissues of hamsters infected with pathogenic Leptospira. LipL32 expression in tissues was investigated using reverse transcription and nested PCR. Results: LipL32 mRNA expression was detected in kidney, liver and lung tissues of hamsters infected with pathogenic Leptospira. No PCR product was detected when tissues from non-infected hamsters were investigated. Conclusion: The data demonstrate that LipL32 is expressed in kidney, liver and lung tissues of animals infected with Leptospira. Further study is needed to demonstrate whether its expression is involved in pathogenesis in those organs.