Outbreak of influenza and SARS-CoV-2 at the Armed Forces of the Philippines Health Service Education and Training Center, September 25-October 10, 2023.

In the last week of September 2023, a surge of influenza-like illness was observed among students of the Armed Forces of the Philippines (AFP) Health Service Education and Training Center, where 48 (27 males and 21 females; age in years: mean 33, range 27-41) of 247 military students at the Center presented with respiratory symptoms. Between September 25 and October 10, 2023, all 48 symptomatic students were evaluated with real-time reverse transcription polymerase chain reaction and sequencing for both influenza and SARS-CoV-2. Thirteen (27%) students were found positive for influenza A/H3 only, 6 (13%) for SARS-CoV-2 only, and 4 (8%) were co-infected with influenza A/H3 and SARS-CoV-2. Seventeen influenza A/ H3N2 viruses belonged to the same clade, 3C.2a1b.2a.2a.3a, and 4 SARSCoV-2 sequences belonged to the JE1.1 lineage, indicating a common source outbreak for both. The influenza A/H3N2 circulating virus belonged to a different clade than the vaccine strain for 2023 (3C.2a1b.2a.2a). Only 4 students had received the influenza vaccine for 2023. In response, the AFP Surgeon General issued a memorandum to all military health institutions on October 19, 2023 that mandated influenza vaccination as a prerequisite for enrollment of students at all education and training centers, along with implementation of non-pharmaceutical interventions and early notification and testing of students exhibiting influenza-like-illness.

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.

Simultaneous detection of omicron and other SARS-CoV-2 variants by multiplex PCR MassARRAY technology.

The rapid emergence of SARS-CoV-2 variants with high severity and transmutability adds further urgency for rapid and multiplex molecular testing to identify the variants. A nucleotide matrix-assisted laser-desorption-ionization time-of-flight mass spectrophotometry (MALDI-TOF MS)-based assay was developed (called point mutation array, PMA) to identify four major SARS-CoV-2 variants of concern (VOCs) including Alpha, Beta, Delta, and Omicron (namely PMA-ABDO) and differentiate Omicron subvariant (namely PMA-Omicron). PMA-ABDO and PMA-Omicron consist of 24 and 28 mutation sites of the spike gene. Both PMA panels specifically identified VOCs with as low as 10 viral copies/µl. The panel has shown a 100% concordant with the Next Generation Sequencing (NGS) results testing on 256 clinical specimens with real-time PCR cycle threshold (Ct) values less than 26. It showed a higher sensitivity over NGS; 25/28 samples were positive by PMA but not NGS in the clinical samples with PCR Ct higher than 26. Due to the mass of nucleotide used to differentiate between wild-type and mutation strains, the co-infection or recombination of multiple variants can be determined by the PMA method. This method is flexible in adding a new primer set to identify a new emerging mutation site among the current circulating VOCs and the turnaround time is less than 8 h. However, the spike gene sequencing or NGS retains the advantage of detecting newly emerged variants.

Simultaneous Detection of Omicron and Other SARS-CoV-2 Variants by Multiplex PCR MassARRAY Technology.

The rapid emergence of SARS-CoV-2 variants with high severity and transmutability adds further urgency for rapid and multiplex molecular testing to identify the variants. A nucleotide matrix-assisted laser-desorption-ionization time-of-flight mass spectrophotometry (MALDI-TOF MS)-based assay was developed (called point mutation array, PMA) to identify four major SARS-CoV-2 variants of concern (VOCs) including Alpha, Beta, Delta, and Omicron (namely PMA-ABDO) and differentiate Omicron subvariant (namely PMA-Omicron). PMA-ABDO and PMA-Omicron consist of 24 and 28 mutation sites of the spike gene. Both PMA panels specifically identified VOCs with as low as 10 viral copies/ µl. The panel has shown a 100% concordant with the Next Generation Sequencing (NGS) results testing on 256 clinical specimens with real-time PCR cycle threshold (Ct) values less than 26. It showed a higher sensitivity over NGS; 25/28 samples were positive by PMA but not NGS in the clinical samples with PCR Ct higher than 26. Due to the mass of nucleotide used to differentiate between wild-type and mutation strains, the co-infection or recombination of multiple variants can be determined by the PMA method. This method is flexible in adding a new primer set to identify a new emerging mutation site among the current circulating VOCs and the turnaround time is less than 8 hours. However, the spike gene sequencing or NGS retains the advantage of detecting newly emerged variants.

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.

Limited and Short-Lasting Virus Neutralizing Titers Induced by Inactivated SARS-CoV-2 Vaccine.

In vitro determination of severe acute respiratory syndrome coronavirus 2 neutralizing antibodies induced in serum samples from recipients of the CoronaVac vaccine showed a short protection period against the original virus strain and limited protection against variants of concern. These data provide support for vaccine boosters, especially variants of concern circulate.

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.

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.

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.

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%).

Epidemiological and Molecular Characterization of Dengue Virus Circulating in Bhutan, 2013-2014.

Dengue is one of the most significant public health problems in tropical and subtropical countries, and is increasingly being detected in traditionally non-endemic areas. In Bhutan, dengue virus (DENV) has only recently been detected and limited information is available. In this study, we analyzed the epidemiological and molecular characteristics of DENV in two southern districts in Bhutan from 2013-2014. During this period, 379 patients were clinically diagnosed with suspected dengue, of whom 119 (31.4%) were positive for DENV infection by NS1 ELISA and/or nested RT-PCR. DENV serotypes 1, 2 and 3 were detected with DENV-1 being predominant. Phylogenetic analysis of DENV-1 using envelope gene demonstrated genotype V, closely related to strains from northern India.

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.

Evidence of West Nile virus infection in Nepal.

BACKGROUND: Acute febrile illness is common among those seeking medical care and is frequently treated empirically with the underlying illness remaining undiagnosed in resource-poor countries. A febrile illness study was conducted 2009-2010 to identify known and unknown pathogens circulating in Nepal. METHOD: Study methods included diagnostic testing and preliminary ELISA screening of acute and convalescent samples for diseases both known and unknown to be circulating in Nepal, including West Nile virus (WNV). The molecular assays including Polymerase Chain Reaction (PCR), Sanger sequencing and ultra deep sequencing on MiSeq Illumina Platform were conducted to further confirm the presence of WNV. RESULTS: The study enrolled 2,046 patients presenting undifferentiated febrile illness with unknown etiology. Sera from 14 out of 2,046 patients were tested positive for west nile virus (WNV) by nested Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Only two out of 14 cases were confirmed for the presence of WNV by sequencing and identified as WNV lineage 1 phylogentically. The two patients were adult males with fever and no neurological symptoms from Kathmandu and Bharatpur, Nepal. CONCLUSION: Two out of 2,046 serum samples contained fragments of WNV genome resembling WNV lineage 1, which is evidence of the continued spread of WNV which should be considered a possible illness cause in Nepal.

Demographic, clinical and laboratory findings among adult and pediatric patients hospitalized with dengue in the Philippines.

We evaluated the differences in demographic, clinical, and laboratory findings between adult and pediatric patients hospitalized with dengue fever. Ninety patients with dengue infection admitted at San Lazaro Hospital (SLH), Manila from September 2005 to January 2006 were included in the study. The cases were laboratory-confirmed to have dengue infection. The majority of dengue cases (92%) had secondary dengue infection (median age = 18, age range: 2-37) while the remainder (8%) had a primary dengue infection (median age = 12, age range: 7-22). Nearly all the patients (99%) had dengue hemorrhagic fever (DHF). Sixty-five of the cases (72%) had serotype data: 2 (3%) were dengue virus serotype 1 (DENV-1) (median age = 17), 12 (18%) had DENV-2 (median age = 17.5), 38 (59%) had DENV-3 (median age = 16) and 13 (20%) had DENV-4 (median age = 18). The initial signs, symptoms and laboratory results except hematocrit (p = 0.02) and hemoglobin (p = 0.02) did not differ significantly between adults and children. During the study period, half the cases were adults (218 years; n = 45) and half were children (<18 years; n = 45). The ages of cases ranged from 2 to 37 years (median = 17 years) and the peak incidence was 15-19 years. Dengue is often considered as a pediatric disease. Additional studies are needed to determine if an age shift is occurring and where.

Kinetics of chikungunya infections during an outbreak in Southern Thailand, 2008-2009.

The Indian Ocean chikungunya epidemic re-emerged in Thailand in August 2008. Forty-five adults with laboratory-confirmed chikungunya in Songkhla province, Thailand were clinically assessed and serially bled throughout the acute and convalescent phase of the disease. Patient symptoms, antibody responses, and viral kinetics were evaluated using observational assessments, polymerase chain reaction (PCR), and serological assays. All subjects experienced joint pain with 42 (93%) involving multiple joints; the interphalangeal most commonly affected in 91% of the subjects. The mean duration of joint pain was 5.8 days, 11 (25%) experiencing discomfort through the duration of the study. Rash was observed in 37 (82%) subjects a mean 3.5 days post onset of symptoms. Patents were positive by PCR for a mean of 5.9 days with sustained peak viral load through Day 5. The IgM antibodies appeared on Day 4 and peaked at Day 7 and IgG antibodies first appeared at Day 5 and rose steadily through Day 24.