Characteristics and evolution of hemagglutinin and neuraminidase genes of Influenza A(H3N2) viruses in Thailand during 2015 to 2018.

Background: Influenza A(H3N2) virus evolves continuously. Its hemagglutinin (HA) and neuraminidase (NA) genes have high genetic variation due to the antigenic drift. This study aimed to investigate the characteristics and evolution of HA and NA genes of the influenza A(H3N2) virus in Thailand. Methods: Influenza A positive respiratory samples from 2015 to 2018 were subtyped by multiplex real-time RT-PCR. Full-length HA and NA genes from the positive samples of influenza A(H3N2) were amplified and sequenced. Phylogenetic analysis with the maximum likelihood method was used to investigate the evolution of the virus compared with the WHO-recommended influenza vaccine strain. Homology modeling and N-glycosylation site prediction were also performed. Results: Out of 443 samples, 147 (33.18%) were A(H1N1)pdm09 and 296 (66.82%) were A(H3N2). The A(H3N2) viruses circulating in 2015 were clade 3C.2a whereas sub-clade 3C.2a1 and 3C.2a2 dominated in 2016-2017 and 2018, respectively. Amino acid substitutions were found in all antigenic sites A, B, C, D, and E of HA but the majority of the substitutions were located at antigenic sites A and B. The S245N and N329S substitutions in the NA gene affect the N-glycosylation. None of the mutations associated with resistance to NA inhibitors were observed. Mean evolutionary rates of the HA and NA genes were 3.47 × 10 -3 and 2.98 × 10-3 substitutions per site per year. Conclusion: The influenza A(H3N2) virus is very genetically diverse and is always evolving to evade host defenses. The HA and NA gene features including the evolutionary rate of the influenza A(H3N2) viruses that were circulating in Thailand between 2015 and 2018 are described. This information is useful for monitoring the genetic characteristics and evolution in HA and NA genes of influenza A(H3N2) virus in Thailand which is crucial for predicting the influenza vaccine strains resulting in high vaccine effectiveness.

Inhibitory efficiency of Andrographis paniculata extract on viral multiplication and nitric oxide production.

Andrographis paniculata (Burm. F.) Nees is a medicinal plant previously reported with broad-spectrum antivirals but the mode of inhibition remains elusive. The objective of this study was to identify the most active fraction from A. paniculata ethanol extract (APE, APE-2A, APE-2B and APE-2C) and dry powder extract (APSP) against influenza A (H3N2), representing RNA viruses, and herpes simplex virus-1 (HSV-1), representing DNA viruses. The results showed that the fractions APSP, APE, APE-2B, and APE-2C directly neutralized the HSV-1 and influenza A (H3N2) when incubated at room temperature for 60 min before infecting the cells. The results also showed that the additional APE-2A fraction also directly neutralized the influenza A (H3N2), but not the HSV-1. The APE, APE-2B and APE-2C inhibited the HSV-1 by more than 0.5 log when the fractions were introduced after infection. Similarly, the APSP and APE inhibited the influenza A (H3N2) more than 0.5 log after infection. Only 50 µg/mL APE-2C inhibited the viruses greater than 0.5 log. In addition, A. paniculata extracts were also evaluated for their interfering capacities against nitric oxide (NO) production in LPS-activated RAW 264.7 macrophages. As well, APE-2C potently inhibited NO production at the IC50 of 6.08 µg/mL. HPLC and LC-MS analysis indicated that the most actively antiviral fractions did not contain any andrographolide derivatives, whereas the andrographolide-rich fractions showed moderate activity.

A performance comparison between fluorescent immunoassay and immunochromatography for rapid dengue detection in clinical specimens.

Dengue virus (DENV 1-4) infection has been a global health threat where no specific treatment is currently available. Therefore, a rapid and accurate diagnosis is critical for an appropriate management as it could reduce the burden of severe clinical manifestation. Currently, dengue immunochromatography (IC) is commonly used to primarily differentiate acute febrile illnesses. Fluorescent immunoassay (FIA) utilized a highly sensitive detection system and claimed 70-100% sensitivity and 83.5-91.7% specificity for dengue infection in a preliminary report. This report recruited samples with acute febrile illnesses sent for dengue screening and tested IC and FIA in parallel. The performance of both tests was verified by a definitive diagnosis retrieved from combinatorial reverse transcription-quantitative polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA) for IgM and IgG confirmation tests. Results showed that the viral nonstructural protein (NS1) performance of FIA was slightly higher than IC with the sensitivity, specificity, PPV, NPV, agreement, kappa, and its standard error at 79.11, 92.28, 86.81, 87.31, 352 (87.13%), 0.725 ± 0.035, respectively; whereas those of the IC were at 76.58, 92.28, 86.43, 85.98, 348 (86.14%), 0.703 ± 0.037, respectively. Moreover, the IgM and IgG performance of FIA had higher specificity, PPV, and agreement than the IgM IC performance, suggesting that the FIA was more specific but less sensitive for antibody detection. No correlation was observed in IgM and IgG levels of ELISA and FIA assays. In conclusion, the FIA and IC were highly sensitive, specific, and substantially agreed in NS1 detection but moderately agreed in IgM and IgG detection.

Low Measles Seropositivity Rate among Thai Adolescents in the Thai National Immunization Program.

To achieve the goal of measles elimination, herd immunity with 95% seroprotection in the community is required. This study aimed to describe the measles seropositivity rate among Thai children and adolescents. A cross-sectional study was conducted among children aged 3−18 years in Bangkok and its suburbs. Measles IgG antibodies were measured using a EUROIMMUN enzyme-linked immunosorbent assay kit. Seropositivity is defined as a measles IgG titer of ≥200 IU/L, due to a correlation with a >85% positive rate with a plaque reduction neutralizing titer of >120. Factors associated with seropositivity were analyzed using logistic regression analysis. From May to July 2020, 570 children with a median (IQR) age of 11.7 (9.4−14.8) years were enrolled. The geometric mean titer (GMT) of anti-measles IgG was 281 IU/L (95% CI; 257−306). The proportion of children with seropositivity was inversely correlated with age; 3−5 years 85.3%, 6−9 years 72.5%, 10−14 years 50.7%, and 15−18 years 56.3%. Adolescents aged 10−18 years had a lower measles seropositivity rate compared with young children; aOR 0.29 (95% CI 0.17−0.48). Only half of the adolescents who received two doses of measles-containing vaccine maintained measles IgG above the seropositive level. A measles booster dose for young adults may be needed to achieve the measles elimination goal.

Sensitivity and specificity of anti-double-stranded RNA immunofluorescence for universal detection of viral infection in respiratory specimens.

BACKGROUND: Emerging viruses could be detected before reaching pandemic level if universal viral detection screening was routinely used. Double-stranded RNA (dsRNA) is the only common antigen across most viral families. Anti-dsRNA immunofluorescence has shown promising results in vitro; however, its diagnostic value in respiratory specimens has not been evaluated. METHODS: Consecutive inpatient cases of suspected respiratory viral infections were prospectively enrolled. Respiratory samples were collected and divided for anti-dsRNA immunofluorescence (index test) and 19-subtypes respiratory virus microarray (reference standard). Using fluorescence microscopy, positive or negative anti-dsRNA IF results were determined independently by two raters. RESULTS: By microarray, 108 and 87 samples were positive and negative for viruses, respectively. The anti-dsRNA IF sensitivity was 83.3% (95%CI 76.1%-90.2%), while specificity was 87.4% (95%CI 80.8%-93.7%). CONCLUSIONS: Anti-dsRNA IF is simple to perform, with acceptable accuracy, and suitable for point-of-care respiratory virus screening. Unlike most molecular techniques, known viral genome sequences are not required.

Antibody responses to SARS-CoV-2 in patients with differing severities of coronavirus disease 2019.

BACKGROUND: A greater understanding of the antibody response to SARS-CoV-2 in an infected population is important for the development of a vaccination. AIM: To investigate SARS-CoV-2 IgA and IgG antibodies in Thai patients with differing severities of COVID-19. METHODS: Plasma from the following patient groups was examined: 118 adult patients with confirmed SARS-CoV-2 infections, 49 patients under investigation (without confirmed infections), 20 patients with other respiratory infections, and 102 healthy control patients. Anti-SARS-CoV-2 enzyme-linked immunosorbent assay (ELISA) from EUROIMMUN was performed to assess for IgA and IgG antibodies. The optical density (OD) ratio cutoff for a positive result was 1.1 for IgA and 0.8 for IgG. Additionally, the association of the antibody response with both the severity of disease and the date after onset of symptoms was analyzed. RESULTS: A total of 289 participants were enrolled and 384 samples analyzed from March 10 to May 31, 2020. Patients were categorized, based on their clinical manifestations, as mild (n = 59), moderate (n = 27), or severe (n = 32). The overall sensitivity of IgA and IgG from the samples collected after day 7 of the symptoms was 87.9% (95% CI: 79.8-93.6) and 84.8% (95% CI: 76.2-91.3), respectively. Compared to the mild group, the severe group had significantly higher levels of spike 1 (S1) antigen-specific IgA and IgG. All patients in the moderate and severe groups had S1-specific IgG, while 20% of the patients in the mild group did not have any IgG detected after two weeks after the onset of symptoms. Interestingly, in the severe group, the SARS-CoV-2 IgG level was significantly higher in males than females (p = 0.003). CONCLUSION: The serological test for SARS-CoV-2 has a high sensitivity more than two weeks after the onset of illness. Additionally, the serological response differs among patients based on sex as well as the severity of infection.

Prevalence and Characteristics of Pediatric Healthcare Workers without Immunity to Varicella zoster Virus.

This study aimed to determine the proportion of varicella non-immune pediatric healthcare workers (HCWs) of the Pediatrics Department of King Chulalongkorn Memorial Hospital and to determine cost-effective strategies for identifying non-immune personnel. A cross-sectional study using a self-administered questionnaire to determine HCWs' histories of chickenpox or 2-dose varicella vaccination was conducted. From a total of 699 HCWs, 653 HCWs (93%), including 145 physicians (22%), 297 nurses (46%), and 211 administrative staff (32%), responded to questionnaires. There were 475 HCWs (73%) who had a history of chickenpox, 58 (9%) who had completed the 2-dose varicella vaccine schedule, and 120 (18%) whose varicella-zoster virus (VZV) immunity status was uncertain. In total, 107 HCWs (89%) were tested for VZV IgG, 90 of whom had immunity, and 17 were determined to be non-immune. After combining history and VZV IgG test results, the prevalence of non-immune HCWs was 2.6% (95%CI 1.4-3.8), with those ≤40 years of age at higher risk of non-immunity. Implementing a strategy that involves testing of only those with an unknown VZV status and vaccination for only those determined to be non-immune costs 1,801 United States dollar (USD), less than the total cost (4,601 USD) for vaccinating all HCWs with uncertain status.

The immunogenicity and safety of live attenuated varicella-zoster virus vaccine in human immunodeficiency virus-infected children.

BACKGROUND: The live attenuated varicella vaccine is recommended for HIV-infected children who are not severely immunosuppressed. This study aimed to assess the immunogenicity and safety of varicella vaccination among HIV-infected children who had severe immunosuppression before receiving antiretroviral therapy. METHODS: Sixty HIV-infected children with no history of chickenpox or herpes zoster infection with CD4 T lymphocyte counts ≥ 15% or ≥ 200 cell/mm were enrolled. Two doses of varicella vaccine were administered at the time of enrollment and at 3 months. Varicella zoster virus (VZV) antibody was tested at baseline and 3 months after each dose by the enzyme-linked immunosorbent assay technique. An antibody titer >20 HU/mL was regarded as protective. RESULTS: The median (interquartile range) of age, CD4 nadir, and current CD4 percentage were 11.2 (8.5-12.8) years, 9.5% (3-14), and 28% (22-32), respectively. Fifty-seven children (95%) received antiretroviral therapy for a median of 27 months. Among 34 children (57%) who were VZV seronegative at baseline, 11.8% (95% CI, 3.3%-27.5%) and 79.4% (95% CI, 62.1%-91.3%) were VZV seroconverted after first and second dose of vaccine, respectively. Children who had VZV seroconversion were more likely to have HIV RNA <1.7 copies/mL (92.6% vs. 71.4%, P = 0.18). Among 26 children who were seropositive at baseline, the geometric mean titers were increased from 56.7 to 107.9 and 134.6 unit/mL, respectively. Local and systemic reactions of grade 1 and 2 were reported in 13% and 4% of children, respectively. There was a trend toward better response among children with younger age, high CD4, and viral suppression. CONCLUSIONS: Administration of the 2 doses of varicella vaccine resulted in high seroconversion rates without serious adverse reactions. Varicella vaccination for HIV-infected children should be encouraged.

Molecular and demographic analysis of respiratory syncytial virus infection in patients admitted to King Chulalongkorn Memorial Hospital, Thailand, 2007.

OBJECTIVES: To preliminary preview the molecular character and its possible clinical correlation of RSV subgroups in Thailand. DESIGN: Cross-sectional analytic design. SETTING: Admitted acute lower respiratory tract infection patients of King Chulalongkorn Memorial Hospital, Bangkok, during Jun-Dec, 2007 were recruited. SAMPLE: Nasopharyngeal aspirations were collected. MAIN OUTCOME MEASURES: All samples were analyzed for the presence of RSV glycoprotein G gene by reverse transcription PCR. Molecular character of each subgroup was determined by sequencing. Admission records were also analyzed for clinical correlations. RESULTS: Equal infectivity and severity of both RSV subgroups to the patients was shown. Mixed infection was shown to be as common as each single infection, higher than previously reported. GA2 of subgroup A and BA-IV of subgroup B were the most widespread genotypes and showed their monophyletic origins. From admission records, either type of infection did not show significantly preference in demographic record or clinical severity. Comorbidity, however, was statistically significant that more congenital heart disease was found in negative RSV cases, while more chronic pulmonary disease was in positive cases. Nevertheless, the clinical severity was insignificantly different suggesting that only patients with chronic pulmonary underlying were prone to be infected with RSV. CONCLUSIONS: This preliminary RSV study showed prevalence of subgroups, types of infection, and common genotypes in an epidemic, uncorrelated to demography or clinical severity.

Randomized control trial of live Lactobacillus acidophilus plus Bifidobacterium infantis in treatment of infantile acute watery diarrhea.

OBJECTIVE: To evaluate the effectiveness of live Lactobacillus acidophilus plus Bifidobacterium infantis in the treatment of acute watery diarrhea. DESIGN: Open, randomized control trial. SETTING: King Chulalongkorn Memorial Hospital, Bangkok, Thailand. MATERIAL AND METHOD: Seventy-one infants (aged 1-24 months) with acute watery diarrhea that presented at Department of Pediatrics, King Chulalongkorn Memorial Hospital, Bangkok were enrolled after parental signed informed consent. They were randomized into 2 groups. The Study group (n = 35) received live Lactobacillus plus Bifidobacterium (3 x 10(9) CFU) bid and ORS and the Control group (n = 36) received ORS only. All infants received lactose free milk. Case record forms were completed daily for 5 days by the parents. RESULTS: All 71 infants completed the present study. There was no difference of the patients'characteristics and baseline clinical symptoms between the study group and the control group. Live Lactobacillus plus Bifidobacterium shortened the diarrhea duration (1.6 +/- 0.7 days vs 2.9 +/- 1.7 days, p < 0.01) compared to controls. However the stool frequency and duration of hospitalization were not significantly different (p > 0.05, study group vs control group). CONCLUSION: Live Lactobacillus acidophilus plus Bifidobacterium infantis may be an effective treatment for acute watery diarrhea in infants. The 2-day course treatment can significantly shorten the duration of diarrhea.

The prevalence of varicella-zoster virus infection in normal healthy individuals aged above 6 months.

The prevalence of varicella-zoster virus (VZV) infection was studied by determining the presence of IgG antibody to VZV (anti-VZV IgG) using ELISA method. Three hundred and fifty sera collected from Thai healthy individuals aged above 6 months (mean age +/- standard deviation = 14.9 +/- 11.4) were tested, the prevalence of VZV infection was 64.6% (225/350). All samples were randomly sampling from healthy children and blood donors who visited the hospital and clssified into 7 groups, 50 samples each, according to their age, i.e., group 1; 6 months-3 years, group 2; 4-6 years, group 3; 7-9 years, group 4; 10-14 years, group 5; 15-19 years, group 6; 20-24 years and group 7; above 25 years. The prevalence of VZV infection were 12%, 42%, 64%, 70%, 78%, 84% and 100% respectively. The mean amount of anti-VZV IgG among groups of positive VZV infection (225 samples) was 86.8 +/- 29.7 unit/ml. The mean amount of anti-VZV IgG was highest in 6 months-3 years age group (113.6 +/- 39.2 unit/ml). Significant difference of the mean amount of anti- VZV IgG was found between group 1, 3, 6 and other groups (p-value < 0.05). There was a correlation between history of varicella and the presence of anti-VZV IgG in the serum. 95.3% of individuals with positive history has already had the antibody. The important associated factors that might involve VZV infection were age, number of members in family and place of exposure to VZV infection. Other factors, such as sex and income did not show any association to VZV infection.

Prevalence of parvovirus B19 infection in Thai young adults.

The prevalence of antibody to human parvovirus B19 in 128 Thai healthy young adults was measured. Antibodies of the immunoglobulin G (IgG) class were investigated in serum samples of 51 males and 77 females aged 18-24 years (mean 19.83; SD 1.07) by a commercial enzyme-linked immunosorbent assay (ELISA) using high specific recombinant parvovirus B 19 antigen. Only 14 out of 128 (10.94%) sera were found positive, including 6 males and 8 females. No sex preponderance was observed. The amount of antibody calculated as antibody index was not statistically significant difference between genders.