An unusual diarrheal outbreak in the community in Eastern Thailand caused by Norovirus GII.3[P25].

BACKGROUND: Sentinel laboratory surveillance for diarrheal disease determined norovirus to be the most common cause of non-bacterial gastroenteritis in people during the COVID-19 pandemic in Thailand. An increase in patients presenting with diarrhea and vomiting in hospitals across Chanthaburi province between December 2021 and January 2022 led to the need for the identification of viral pathogens that may be responsible for the outbreak. METHODS: Fecal samples (rectal swabs or stool) from 93 patients, of which 65 patients were collected during the December 2021 to January 2022 outbreak, were collected and screened for viral infection by real-time RT-PCR. Positive samples for norovirus GII were then genotyped by targeted amplification and sequencing of partial polymerase and capsid genes. Full genome sequencing was performed from the predominant strain, GII.3[P25]. RESULTS: Norovirus was the most common virus detected in human fecal samples in this study. 39 of 65 outbreak samples (60%) and 3 of 28 (10%) non-outbreak samples were positive for norovirus genogroup II. One was positive for rotavirus, and one indicated co-infection with rotavirus and norovirus genogroups I and II. Nucleotide sequences of VP1 and RdRp gene were successfully obtained from 28 of 39 positive norovirus GII and used for dual-typing; 25/28 (89.3%) were GII.3, and 24/28 (85.7) were GII.P25, respectively. Norovirus GII.3[P25] was the predominant strain responsible for this outbreak. The full genome sequence of norovirus GII.3[P25] from our study is the first reported in Thailand and has 98.62% and 98.57% similarity to norovirus found in China in 2021 and the USA in 2022, respectively. We further demonstrate the presence of multiple co-circulating norovirus genotypes, including GII.21[P21], GII.17[P17], GII.3[P12] and GII.4[P31] in our study. CONCLUSIONS: An unusual diarrhea outbreak was found in December 2021 in eastern Thailand. Norovirus strain GII.3[P25] was the cause of the outbreak and was first detected in Thailand. The positive rate during GII.3[P25] outbreak was six times higher than sporadic cases (GII.4), and, atypically, adults were the primary infected population rather than children.

High Transmission Rates of Early Omicron Subvariant BA.2 in Bangkok, Thailand.

The emergence of Omicron as the fifth variant of concern within the SARS-CoV-2 pandemic in late 2021, characterized by its rapid transmission and distinct spike gene mutations, underscored the pressing need for cost-effective and efficient methods to detect viral variants, especially given their evolving nature. This study sought to address this need by assessing the effectiveness of two SARS-CoV-2 variant classification platforms based on RT-PCR and mass spectrometry. The primary aim was to differentiate between Delta, Omicron BA.1, and Omicron BA.2 variants using 618 COVID-19-positive samples collected from Bangkok patients between November 2011 and March 2022. The analysis revealed that both BA.1 and BA.2 variants exhibited significantly higher transmission rates, up to 2-3 times, when compared to the Delta variant. This research presents a cost-efficient approach to virus surveillance, enabling a quantitative evaluation of variant-specific public health implications, crucial for informing and adapting public health strategies.

Clinical characterization of COVID-19 breakthrough infections, Philippines.

We clinically characterized PCR detected breakthrough infections among partially/fully vaccinated cases with majority given an inactivated vaccine, CoronaVac. From 1 March to 15 July 2021, we detected 182 SARS-CoV-2 infections among vaccinated cases with 129 classified as breakthrough infections. Majority were male, 30-39 y.o., and were asymptomatic or mildly symptomatic with few severe cases. Alpha, Beta and Delta VOCs were detected from sequenced breakthrough infections. Healthcare workers had significantly lower Ct values(higher viral loads) versus non-HCWs. Our results underscore the importance of regular PCR screening for HCWs due to the risk of SARS-CoV-2 transmission from asymptomatic breakthrough infections and provide evidence supporting administration of a booster dose especially to HCWs.

Coding-Complete Genome Sequences of Alpha and Delta SARS-CoV-2 Variants from Kamphaeng Phet Province, Thailand, from May to July 2021.

We report coding-complete genome sequences of 44 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains of the alpha and delta variants identified from patients in Kamphaeng Phet, Thailand. Two nonsense mutations in open reading frame 3a (ORF3a) (G254*) and ORF8 (K68*) were found in the alpha variant sequences. Two lineages of the delta variant, B.1.617.2 and AY.30, were found.

Coding-Complete Genome Sequences of 11 SARS-CoV-2 B.1.1.7 and B.1.351 Variants from Metro Manila, Philippines.

Here, we report the complete genome sequences of 11 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants from the Philippines. Lineage analysis showed 3 B.1.1.7 and 8 B.1.351 sequences. One B.1.1.7 sequence contained two additional mutations, F318N and V320F, with V320F located in the receptor binding domain of the S1 subunit.

SARS-CoV-2 Among Military and Civilian Patients, Metro Manila, Philippines.

INTRODUCTION: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) belonging to the family Coronaviridae and genus Betacoronavirus is the causative agent of COVID-19 disease and was first identified in Wuhan, China. SARS-CoV-2 spread globally with >28 million cases and 911,000 deaths recorded worldwide as of September 12, 2020. The Philippines reported the first case of community transmission on March 5, 2020, and despite the government imposing one of the longest and strictest lockdowns in Southeast Asia, the number of confirmed COVID-19 cases still surged with >250,000 cases and 4,000 deaths reported as of September 12, 2020. It is important to estimate the burden and impact of SARS-CoV-2 on the military population since this can affect the military readiness. MATERIALS AND METHODS: Nasopharyngeal and oropharyngeal swabs were collected and SARS-CoV-2 real-time RT-PCR testing was performed on the samples. Phylogenetic analysis was performed using sequences from 23 SARS-CoV-2-positive specimens from this study and sequences retrieved from GenBank and GISAID databases. RESULTS: From April 14 to August 15, 2020, a total of 12,432 specimens were tested with 763 (6%) unique individuals testing positive for SARS-CoV-2 by rRT-PCR. In the military population, majority of the patients who were tested (80%) and those who tested positive for SARS-CoV-2 (86%) were male. Military and civilian status was available for 7,672 patients with 515/5,042 (10%) positive among military patients and 248/2,630 (9%) positive among civilian patients. Both military and civilian populations had the highest case counts of SARS-CoV-2-positive cases in the 21- to 30- and 31- to 40-year-old age groups, while the 71- to 80-year-old age group had the highest proportion (18%) of SARS-CoV-2-positive cases. Sequencing analysis showed 19 different variants in the 23 genomes. Twenty of the 23 genomes were classified under clade GR/B1.1, 2 genomes were classified under clade GR/B1.1.28, and 1 genome was classified under Clade O/B.6. Twenty-two of the 23 sequences collected after June 25, 2020, contained the D614G mutation. CONCLUSION: We describe here the results of SARS-CoV-2 testing for military and civilian patients and personnel. The 21- to 30- and 31- to 40-year-old age groups had the highest case counts of SARS-CoV-2-positive cases. Sequencing results showed the presence of the D614G mutation in the spike protein in a majority of specimens collected from the end of June to July 2020.

Genomic surveillance of SARS-CoV-2 in Thailand reveals mixed imported populations, a local lineage expansion and a virus with truncated ORF7a.

Coronavirus Disease 2019 (COVID-19) is a global public health threat. Genomic surveillance of SARS-CoV-2 was implemented in March of 2020 at a major diagnostic hub in Bangkok, Thailand. Several virus lineages supposedly originated in many countries were found, and a Thai-specific lineage, designated A/Thai-1, has expanded to be predominant in Thailand. A virus sample in the SARS-CoV-2 A/Thai-1 lineage contains a frame-shift deletion at ORF7a, encoding a putative host antagonizing factor of the virus.

Coding-Complete Genome Sequences of 23 SARS-CoV-2 Samples from the Philippines.

Here, we report the coding-complete genome sequences of 23 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) samples from the Philippines. Sequences were obtained from nasopharyngeal and oropharyngeal swabs from coronavirus disease 2019 (COVID-19)-positive patients. Mutation analysis showed the presence of the D614G mutation in the spike protein in 22 of 23 genomes.

Complete Coding Sequences of 22 East/Central/South African Genotype Chikungunya Virus Isolates from Thailand (2018 to 2019).

The coding-complete genome sequences of 22 chikungunya virus strains collected from the 2018-2019 outbreak in Thailand are reported. All sequences belong to the East/Central/South African (ECSA) genotype and contain two mutations, E1:K211E and E2:V264A, which were previously shown to be associated with increased viral infectivity, dissemination, and transmission in Aedes aegypti.

Molecular epidemiology of dengue fever outbreaks in Bhutan, 2016-2017.

Dengue continues to pose a significant public health problem in tropical and subtropical countries. In Bhutan, first outbreak of dengue fever (DF) was reported in 2004 in a southern border town, followed by sporadic cases over the years. In this study, we analysed DF outbreaks that occurred in 3 different places during the years 2016 and 2017. A total of 533 cases in 2016 and 163 in 2017 were suspected of having of DF, where young adults were mostly affected. A total of 240 acute serum specimens collected and analyzed for serotype by nested RT-PCR revealed predominance of serotypes 1 and 2 (DENV-1 and 2). Phylogenetic analysis using envelope gene for both the serotypes demonstrated cosmopolitan genotype which were closely related to strains from India, indicating that they were probably imported from the neighboring country over the past few years.

Retrospective use of next-generation sequencing reveals the presence of Enteroviruses in acute influenza-like illness respiratory samples collected in South/South-East Asia during 2010-2013.

BACKGROUND: Emerging and re-emerging respiratory pathogens represent an increasing threat to public health. Etiological determination during outbreaks generally relies on clinical information, occasionally accompanied by traditional laboratory molecular or serological testing. Often, this limited testing leads to inconclusive findings. The Armed Forces Research Institute of Medical Sciences (AFRIMS) collected 12,865 nasopharyngeal specimens from acute influenza-like illness (ILI) patients in five countries in South/South East Asia during 2010-2013. Three hundred and twenty-four samples which were found to be negative for influenza virus after screening with real-time RT-PCR and cell-based culture techniques demonstrated the potential for viral infection with evident cytopathic effect (CPE) in several cell lines. OBJECTIVE: To assess whether whole genome next-generation sequencing (WG-NGS) together with conventional molecular assays can be used to reveal the etiology of influenza negative, but CPE positive specimens. STUDY DESIGN: The supernatant of these CPE positive cell cultures were grouped in 32 pools containing 2-26 supernatants per pool. Three WG-NGS runs were performed on these supernatant pools. Sequence reads were used to identify positive pools containing viral pathogens. Individual samples in the positive pools were confirmed by qRT-PCR, RT-PCR, PCR and Sanger sequencing from the CPE culture and original clinical specimens. RESULTS: WG-NGS was an effective way to expand pathogen identification in surveillance studies. This enabled the identification of a viral agent in 71.3% (231/324) of unidentified surveillance samples, including common respiratory pathogens (100/324; 30.9%): enterovirus (16/100; 16.0%), coxsackievirus (31/100; 31.0%), echovirus (22/100; 22.0%), human rhinovirus (3/100; 3%), enterovirus genus (2/100; 2.0%), influenza A (9/100; 9.0%), influenza B, (5/100; 5.0%), human parainfluenza (4/100; 4.0%), human adenovirus (3/100; 3.0%), human coronavirus (1/100; 1.0%), human metapneumovirus (2/100; 2.0%), and mumps virus (2/100; 2.0%), in addition to the non-respiratory pathogen herpes simplex virus type 1 (HSV-1) (172/324; 53.1%) and HSV-1 co-infection with respiratory viruses (41/324; 12.7%).

Human Sentinel Surveillance of Influenza and Other Respiratory Viral Pathogens in Border Areas of Western Cambodia.

Little is known about circulation of influenza and other respiratory viruses in remote populations along the Thai-Cambodia border in western Cambodia. We screened 586 outpatients (median age 5, range 1-77) presenting with influenza-like-illness (ILI) at 4 sentinel sites in western Cambodia between May 2010 and December 2012. Real-time reverse transcriptase (rRT) PCR for influenza was performed on combined nasal and throat specimens followed by viral culture, antigenic analysis, antiviral susceptibility testing and full genome sequencing for phylogenetic analysis. ILI-specimens negative for influenza were cultured, followed by rRT-PCR for enterovirus and rhinovirus (EV/RV) and EV71. Influenza was found in 168 cases (29%) and occurred almost exclusively in the rainy season from June to November. Isolated influenza strains had close antigenic and phylogenetic relationships, matching vaccine and circulating strains found elsewhere in Cambodia. Influenza vaccination coverage was low (<20%). Western Cambodian H1N1(2009) isolate genomes were more closely related to 10 earlier Cambodia isolates (94.4% genome conservation) than to 13 Thai isolates (75.9% genome conservation), despite sharing the majority of the amino acid changes with the Thai references. Most genes showed signatures of purifying selection. Viral culture detected only adenovirus (5.7%) and parainfluenza virus (3.8%), while non-polio enteroviruses (10.3%) were detected among 164 culture-negative samples including coxsackievirus A4, A6, A8, A9, A12, B3, B4 and echovirus E6 and E9 using nested RT-PCR methods. A single specimen of EV71 was found. Despite proximity to Thailand, influenza epidemiology of these western Cambodian isolates followed patterns observed elsewhere in Cambodia, continuing to support current vaccine and treatment recommendations from the Cambodian National Influenza Center. Amino acid mutations at non-epitope sites, particularly hemagglutinin genes, require further investigation in light of an increasingly important role of permissive mutations in influenza virus evolution. Further research about the burden of adenovirus and non-polio enteroviruses as etiologic agents in acute respiratory infections in Cambodia is also needed.

Detection of Zika Virus Infection in Thailand, 2012-2014.

Zika virus (ZIKV) is an emerging mosquito-borne pathogen with reported cases in Africa, Asia, and large outbreaks in the Pacific. No autochthonous ZIKV infections have been confirmed in Thailand. However, there have been several cases reported in travelers returning from Thailand. Here we report seven cases of acute ZIKV infection in Thai residents across the country confirmed by molecular or serological testing including sequence data. These endemic cases, combined with previous reports in travelers, provide evidence that ZIKV is widespread throughout Thailand.

Monitoring and improving the sensitivity of dengue nested RT-PCR used in longitudinal surveillance in Thailand.

BACKGROUND: AFRIMS longitudinal dengue surveillance in Thailand depends on the nested RT-PCR and the dengue IgM/IgG ELISA. OBJECTIVE: To examine and improve the sensitivity of the nested RT-PCR using a panel of archived samples collected during dengue surveillance. STUDY DESIGN: A retrospective analysis of 16,454 dengue IgM/IgG ELISA positive cases collected between 2000 and 2013 was done to investigate the sensitivity of the nested RT-PCR. From these cases, 318 acute serum specimens or extracted RNA, previously found to be negative by the nested RT-PCR, were tested using TaqMan real-time RT-PCR (TaqMan rRT-PCR). To improve the sensitivity of nested RT-PCR, we designed a new primer based on nucleotide sequences from contemporary strains found to be positive by the TaqMan rRT-PCR. Sensitivity of the new nested PCR was calculated using a panel of 87 samples collected during 2011-2013. RESULTS AND CONCLUSION: The percentage of dengue IgM/IgG ELISA positive cases that were negative by the nested RT-PCR varied from 17% to 42% for all serotypes depending on the year. Using TaqMan rRT-PCR, dengue RNA was detected in 194 (61%) of the 318 acute sera or extracted RNA previously found to be negative by the nested RT-PCR. The newly designed DENV-1 specific primer increased the sensitivity of DENV-1 detection by the nested RT-PCR from 48% to 88%, and of all 4 serotypes from 73% to 87%. These findings demonstrate the impact of genetic diversity and signal erosion on the sensitivity of PCR-based methods.

The impact of primer and probe-template mismatches on the sensitivity of pandemic influenza A/H1N1/2009 virus detection by real-time RT-PCR.

BACKGROUND: In response to the 2009 H1N1 pandemic the US CDC and WHO rapidly developed and distributed a real-time RT-PCR kit to detect this strain in clinical samples. The results from the WHO swH1 primer and probe set exhibited diverse sensitivities for the 2009 influenza A/H1N1 strains in Southeast Asia (SEA). OBJECTIVE: Investigate the primer and probe-template mismatches among the 2009 influenza A/H1N1 strains in SEA that reduced the real-time RT-PCR sensitivity. STUDY DESIGN: Thirty-seven swH1 positive samples categorized into sensitive and insensitive groups based on real-time RT-PCR results were selected for hemagglutinin (HA) gene sequencing. The sequence in swH1 primer and probe binding regions of the viruses was examined for mismatches. Phylogenetic analysis was performed to investigate the diversity among these viruses. Primers and probe were redesigned to match each of our sequences and tested to determine the impact on sensitivity. RESULTS: HA sequencing of the viruses isolated from patients with high and low sensitivities revealed that a single mismatch at the 3rd base of the probe reduced sensitivity in 23/37 viruses. Homologous primers and probes increased the sensitivity (mean difference 4.66Ct P<0.0001). Phylogenetic tree revealed that the viruses in this study clustered into two groups, coinciding with RT-PCR sensitivity. CONCLUSION: Results obtained indicate that at least two variants of the novel H1N1 transmitting in SEA and the mutations in HA gene have a direct effect on the detection by using WHO swH1 primer and probe set.