SARS-CoV-2 Variants Detection Strategies in Wastewater Samples Collected in the Bangkok Metropolitan Region.

Wastewater surveillance is considered a promising approach for COVID-19 surveillance in communities. In this study, we collected wastewater samples between November 2020 and February 2022 from twenty-three sites in the Bangkok Metropolitan Region to detect the presence of SARS-CoV-2 and its variants for comparison to standard clinical sampling. A total of 215 wastewater samples were collected and tested for SARS-CoV-2 RNA by real-time PCR with three targeted genes (N, E, and ORF1ab); 102 samples were positive (42.5%). The SARS-CoV-2 variants were determined by a multiplex PCR MassARRAY assay to distinguish four SARS-CoV-2 variants, including Alpha, Beta, Delta, and Omicron. Multiple variants of Alpha-Delta and Delta-Omicron were detected in the wastewater samples in July 2021 and January 2022, respectively. These wastewater variant results mirrored the country data from clinical specimens deposited in GISAID. Our results demonstrated that wastewater surveillance using multiple signature mutation sites for SARS-CoV-2 variant detection is an appropriate strategy to monitor the presence of SARS-CoV-2 variants in the community at a low cost and with rapid turn-around time. However, it is essential to note that sequencing surveillance of wastewater samples should be considered complementary to whole genome sequencing of clinical samples to detect novel variants.

Telepharmacy during home isolation: drug-related problems and pharmaceutical care in COVID-19 patients receiving antiviral therapy in Thailand.

BACKGROUND: Home isolation has been proposed for coronavirus disease 2019 (COVID-19) patients with mild symptoms to avoid hospital overcrowding. This study aimed to describe the drug-related problems (DRPs) and the pharmaceutical care of home-isolating COVID-19 patients in Thailand. METHODS: Our cross-sectional study was undertaken from July 1 to September 30, 2021, at the King Chulalongkorn Memorial Hospital, Thailand. Patients who were ≥ 18 years old, were diagnosed with mild COVID-19 by real-time polymerase chain reaction (RT-PCR), and were able to isolate at home while receiving an antiviral agent and standard symptomatic treatment were enrolled. Infectious disease pharmacists provided a telepharmacy service on days 1 and 3 after the COVID-19 diagnosis. RESULTS: A total of 197 patients met the study criteria. Their median age was 45 years, and their most common underlying disease was hypertension (44.29%). All patients exhibited excellent anti-COVID-19 drug adherence. We identified 125 DRPs, including adverse reactions (68%), and the unnecessary use of products (62.40%). Moreover, 91 patients (46.19%) reported the use of supplements or herbs, with vitamin C being the main supplement (37.36%). Pharmacists provided 36 recommendations and received 33 questions from COVID-19 patients. CONCLUSIONS: Our study demonstrates that telepharmacy is an essential service for detecting and preventing DRPs in home-isolating COVID-19 patients.

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.

Impact of SARS-CoV-2 infection on the profiles and responses of innate immune cells after recovery.

BACKGROUNDS: SARS-CoV-2 infection results in a broad spectrum of clinical outcomes, ranging from asymptomatic to severe symptoms and death. Most COVID-19 pathogenesis is associated with hyperinflammatory conditions driven primarily by myeloid cell lineages. The long-term effects of SARS-CoV-2 infection post recovery include various symptoms. METHODS: We performed a longitudinal study of the innate immune profiles 1 and 3 months after recovery in the Thai cohort by comparing patients with mild, moderate, and severe clinical symptoms using peripheral blood mononuclear cells (n = 62). RESULTS: Significant increases in the frequencies of monocytes compared to controls and NK cells compared to mild and moderate patients were observed in severe patients 1-3 months post recovery. Increased polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) were observed in all recovered patients, even after 3 months. Increased IL-6 and TNFα levels in monocytes were observed 1 month after recovery in response to lipopolysaccharide (LPS) stimulation, while decreased CD86 and HLA-DR levels were observed regardless of stimulation. A multiplex analysis of serum cytokines performed at 1 month revealed that most innate cytokines, except for TNFα, IL4/IL-13 (Th2) and IFNγ (Th1), were elevated in recovered patients in a severity-dependent manner. Finally, the myelopoiesis cytokines G-CSF and GM-CSF were higher in all patient groups. Increased monocytes and IL-6- and TNFα-producing cells were significantly associated with long COVID-19 symptoms. CONCLUSIONS: These results reveal that COVID-19 infection influences the frequencies and functions of innate immune cells for up to 3 months after recovery, which may potentially lead to some of the long COVID symptoms.

SARS-CoV-2 Omicron variant emerged under immune selection.

The SARS-CoV-2 Omicron variant (B.1.1.529 lineage) escapes antibodies that neutralize the ancestral virus. We tested human serum panels from participants with differing infection and vaccination status using a multiplex surrogate virus neutralization assay targeting 20 sarbecoviruses. We found that bat and pangolin sarbecoviruses showed significantly less neutralization escape than the Omicron variant. We propose that SARS-CoV-2 variants have emerged under immune selection pressure and are evolving differently from animal sarbecoviruses.

Impact of SARS-CoV2 Infection on the Profiles and Responses of Innate Immune Cells After Recovery

Backgrounds SARS-CoV-2 infection results in a broad spectrum of clinical outcomes, ranging from asymptomatic to severe symptoms and death. Most COVID-19 pathogenesis is associated with hyperinflammatory conditions driven primarily by myeloid cell lineages. The long-term effects of SARS-CoV-2 infection post recovery include various symptoms. Methods We performed a longitudinal study of the innate immune profiles 1 and 3 months after recovery in the Thai cohort by comparing patients with mild, moderate, and severe clinical symptoms using peripheral blood mononuclear cells (n=62). Results Significant increases in the frequencies of monocytes compared to controls and NK cells compared to mild and moderate patients were observed in severe patients 1-3 months post recovery. Increased polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) were observed in all recovered patients, even after 3 months. Increased IL-6 and TNFα levels in monocytes were observed 1 month after recovery in response to lipopolysaccharide (LPS) stimulation, while decreased CD86 and HLA-DR levels were observed regardless of stimulation. A multiplex analysis of serum cytokines performed at 1 month revealed that most innate cytokines, except for TNFα, IL4/IL-13 (Th2) and IFNγ (Th1), were elevated in recovered patients in a severity-dependent manner. Finally, the myelopoiesis cytokines G-CSF and GM-CSF were higher in all patient groups. Increased monocytes and IL-6- and TNFα-producing cells were significantly associated with long COVID-19 symptoms. Conclusions These results reveal that COVID-19 infection influences the frequencies and functions of innate immune cells for up to 3 months after recovery, which may potentially lead to some of the long COVID symptoms.

Homologous or Heterologous COVID-19 Booster Regimens Significantly Impact Sero-Neutralization of SARS-CoV-2 Virus and Its Variants.

We determined the levels of neutralizing antibodies against the SARS-CoV-2 ancestral strain, Delta and Omicron variants of concern (VOCs), in 125 healthcare workers who received CoronaVac as their primary vaccination and later received either a single ChAdOx1 or a combi-nation of two consecutive boosters using either two ChAdOx1 doses or a ChAdOx1 or BNT162b2 as the primary and second boosters, respectively, or two doses of BNT162b2. The titers 12 weeks after primary vaccination were inadequate to neutralize all strains. After a single ChAdOx1 booster, the levels of neutralization at Day 30 varied significantly, with only a small proportion of participants developing neutralizing titers against Omicron at Day 7 and 30. The two doses of ChAdOx1 as the booster induced the lowest activity. A combination ChAdOx1 and BNT162b2 induced greater neutralization than by two doses of ChAdOx1. Two doses of BNT162b2 as the booster had the maximal activity against Omicron VOC.

Cold agglutinin disease following SARS-CoV-2 and Mycoplasma pneumoniae co-infections.

SARS-CoV-2 and other respiratory co-infections may occur. As Mycoplasma pneumoniae and various viruses can cause cold agglutinin disease (CAD), the presence of CAD in COVID-19 patients should indicate the need of investigations for those pathogens.

Behavioral–biological surveillance of emerging infectious diseases among a dynamic cohort in Thailand

Background Interactions between humans and animals are the key elements of zoonotic spillover leading to zoonotic disease emergence. Research to understand the high-risk behaviors associated with disease transmission at the human-animal interface is limited, and few consider regional and local contexts. Objective This study employed an integrated behavioral–biological surveillance approach for the early detection of novel and known zoonotic viruses in potentially high-risk populations, in an effort to identify risk factors for spillover and to determine potential foci for risk-mitigation measures. Method Participants were enrolled at two community-based sites (n = 472) in eastern and western Thailand and two hospital (clinical) sites (n = 206) in northeastern and central Thailand. A behavioral questionnaire was administered to understand participants’ demographics, living conditions, health history, and animal-contact behaviors and attitudes. Biological specimens were tested for coronaviruses, filoviruses, flaviviruses, influenza viruses, and paramyxoviruses using pan (consensus) RNA Virus assays. Results Overall 61/678 (9%) of participants tested positive for the viral families screened which included influenza viruses (75%), paramyxoviruses (15%), human coronaviruses (3%), flaviviruses (3%), and enteroviruses (3%). The most salient predictors of reporting unusual symptoms (i.e., any illness or sickness that is not known or recognized in the community or diagnosed by medical providers) in the past year were having other household members who had unusual symptoms and being scratched or bitten by animals in the same year. Many participants reported raising and handling poultry (10.3% and 24.2%), swine (2%, 14.6%), and cattle (4.9%, 7.8%) and several participants also reported eating raw or undercooked meat of these animals (2.2%, 5.5%, 10.3% respectively). Twenty four participants (3.5%) reported handling bats or having bats in the house roof. Gender, age, and livelihood activities were shown to be significantly associated with participants’ interactions with animals. Participants’ knowledge of risks influenced their health-seeking behavior. Conclusion The results suggest that there is a high level of interaction between humans, livestock, and wild animals in communities at sites we investigated in Thailand. This study highlights important differences among demographic and occupational risk factors as they relate to animal contact and zoonotic disease risk, which can be used by policymakers and local public health programs to build more effective surveillance strategies and behavior-focused interventions.

Behavioral-biological surveillance of emerging infectious diseases among a dynamic cohort in Thailand.

BACKGROUND: Interactions between humans and animals are the key elements of zoonotic spillover leading to zoonotic disease emergence. Research to understand the high-risk behaviors associated with disease transmission at the human-animal interface is limited, and few consider regional and local contexts. OBJECTIVE: This study employed an integrated behavioral-biological surveillance approach for the early detection of novel and known zoonotic viruses in potentially high-risk populations, in an effort to identify risk factors for spillover and to determine potential foci for risk-mitigation measures. METHOD: Participants were enrolled at two community-based sites (n = 472) in eastern and western Thailand and two hospital (clinical) sites (n = 206) in northeastern and central Thailand. A behavioral questionnaire was administered to understand participants' demographics, living conditions, health history, and animal-contact behaviors and attitudes. Biological specimens were tested for coronaviruses, filoviruses, flaviviruses, influenza viruses, and paramyxoviruses using pan (consensus) RNA Virus assays. RESULTS: Overall 61/678 (9%) of participants tested positive for the viral families screened which included influenza viruses (75%), paramyxoviruses (15%), human coronaviruses (3%), flaviviruses (3%), and enteroviruses (3%). The most salient predictors of reporting unusual symptoms (i.e., any illness or sickness that is not known or recognized in the community or diagnosed by medical providers) in the past year were having other household members who had unusual symptoms and being scratched or bitten by animals in the same year. Many participants reported raising and handling poultry (10.3% and 24.2%), swine (2%, 14.6%), and cattle (4.9%, 7.8%) and several participants also reported eating raw or undercooked meat of these animals (2.2%, 5.5%, 10.3% respectively). Twenty four participants (3.5%) reported handling bats or having bats in the house roof. Gender, age, and livelihood activities were shown to be significantly associated with participants' interactions with animals. Participants' knowledge of risks influenced their health-seeking behavior. CONCLUSION: The results suggest that there is a high level of interaction between humans, livestock, and wild animals in communities at sites we investigated in Thailand. This study highlights important differences among demographic and occupational risk factors as they relate to animal contact and zoonotic disease risk, which can be used by policymakers and local public health programs to build more effective surveillance strategies and behavior-focused interventions.

Use of qRT-PCR for SARS-CoV-2 sgRNA leader for the therapeutic plan: a preliminary report on 10 patients.

INTRODUCTION: Duration of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) shedding is important for infection control. The presence of SARS-CoV-2 subgenomic RNA (sgRNA) leader indicates that the virus is replicative. This study examined the shedding duration of SARS-CoV-2 sgRNA leader and genomic RNA (gRNA) in diverse respiratory specimens. METHODOLOGY: One hundred and eleven respiratory specimens collected sequentially from 10 COVID-19 patients with real-time RT-PCR SARS-CoV-2 orf1ab gene confirmed positive admitted to King Chulalongkorn Memorial Hospital were examined for SARS-CoV-2 E sgRNA leader and E gRNA by using Real-time reverse transcription PCR (qRT-PCR). These specimens included nasopharyngeal swab and throat swabs, nasal swab and throat swabs, sputum, and endotracheal aspirate, and were collected from the first day of admission until the time of orf1ab real-time RT-PCR negative of at least 2-4 consecutive days. RESULTS: E sgRNA leader could only be detectable in specimens with ≥ 1E+05 virus E gene copies per ml within the first 15 days after hospitalization. SARS-CoV-2 sgRNA leader was undetectable from one to 15 days earlier than that of gRNA in all patients. Re-shedding of sgRNA was evident in 2 cases, both on a single occasion after being undetectable for 3-10 days. CONCLUSIONS: Assessment of the presence of sgRNA leader may be useful for therapeutic planning.

Dynamics of Neutralizing Antibody and T-Cell Responses to SARS-CoV-2 and Variants of Concern after Primary Immunization with CoronaVac and Booster with BNT162b2 or ChAdOx1 in Health Care Workers.

Inactivated SARS-CoV-2 vaccine (CoronaVac) is commonly used in national immunization programs. However, the immune response significantly declines within a few months. Our study assessed the immune response against SARS-CoV-2 after receiving booster shots of BNT162b2 or ChAdOx1 among health care workers who previously received CoronaVac as their primary immunization. Fifty-six participants who received ChAdOx1 and forty-two participants who received BNT162b2 were enrolled into this study, which evaluated immune responses, including anti-SARS-CoV-2 spike total antibodies (Elecsys®), surrogated viral neutralization test (sVNT) to ancestral strain (cPass™; GenScript), five variants of concern (Alpha, Beta, Gamma, Delta, and Omicron) (Luminex; multiplex sVNT) and the ELISpot with spike (S1 and S2) peptide pool against the ancestral SARS-CoV-2 strain. The samples were analyzed at baseline, 4, and 12 weeks after primary immunization, as well as 4 and 12 weeks after receiving the booster. This study showed a significant increase in anti-SARS-CoV-2 spike total antibodies, sVNT, and T-cell immune response after the booster, including against the Omicron variant. Immune responses rapidly decreased in the booster group at 12 weeks after booster but were still higher than post-primary vaccination. A fourth dose or a second booster should be recommended, particularly in health care workers.

Host genetic factors of COVID-19 susceptibility and disease severity in a Thai population.

Host genetic factors have been shown to play a role in SARs-CoV-2 infection in diverse populations. However, the genetic landscape differs among various ethnicities; therefore, we explored the host genetic factors associated with COVID-19 disease susceptivity and disease severity in a Thai population. We recruited and genotyped 212 unrelated COVID-19 Thai patients and 36 controls using AxiomTM Human Genotyping SARs-COV-2 array, including 847,384 single nucleotide polymorphisms related to SARs-COV-2 pathogenesis, immune response, and related comorbidity No SNPs passed the genome-wide significance threshold of p value <1 × 10-8. However, with a threshold of p value <1 × 10-5, a locus on chromosome 5q32 was found to have a suggestive association with COVID-19 disease susceptibility (p value 6.9 × 10-6; Q-Q plot λ = 0.805, odds ratio 0.02). Notably, IL17B is a gene located in this linkage disequilibrium block and is previously shown to play a part in inflammation and pneumonia. Additionally, a suggestive locus on chromosome 12q22, harboring EEA1 and LOC643339, was associated with COVID-19 disease severity (p value 1.3 × 10-6 - 4.4 × 10-6, Q-Q plot λ = 0.997, odds ratio 0.28-0.31). EEA1 is involved in viral entry into cells, while LOC643339 is a long non-coding RNA. In summary, our study suggested loci on chromosomes 5q32 and 12q22 to be linked to COVID-19 disease susceptibility and disease severity, respectively. The small sample size of this study may lessen the likelihood that the association found is real, but it could still be true. Further study with a larger cohort is required to confirm these findings.

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.

Short-term immune response after inactivated SARS-CoV-2 (CoronaVac®, Sinovac) and ChAdOx1 nCoV-19 (Vaxzevria®, Oxford-AstraZeneca) vaccinations in health care workers.

BACKGROUND: Inactivated SARS-CoV-2 (CoronaVac®, Sinovac, or SV) and ChAdOx1 nCoV-19 (Vaxzevria®, Oxford-Astra Zeneca, or AZ) vaccines have been administered to the health care workers (HCWs). OBJECTIVE: To determine the short-term immune response after the SV and AZ vaccinations in HCWs. METHODS: In this prospective cohort study, HCWs who completed a 2-dose regimen of the SV or AZ were included. Immune response was evaluated by surrogate viral neutralization test (sVNT) and anti-SARS-CoV-2 total antibody. Blood samples were analyzed at 4 and 12 weeks after the complete vaccination. The primary outcome was the seroconversion rate at 4-weeks after complete immunization. RESULTS: Overall, 185 HCWs with a median (IQR) age of 40.5 (30.3-55.8) years (94 HCWs in the SV group and 91 in the AZ group) were included. At 4 weeks after completing the SV vaccination, 60.6% (95%CI: 50.0-70.6%) had seroconversion evaluated by sVNT (≥ 68% inhibition), comparable to the patients recovered from mild COVID-19 infection (69.0%), with a rapid reduction to 12.2% (95%CI: 6.3-20.8) at 12 weeks. In contrast, 85.7% (95%CI: 76.8-92.2%) HCWs who completed two doses of the AZ for 4 weeks had seroconversion, comparable to the COVID-19 pneumonia patients (92.5%), with a reduction to 39.2% (95%CI: 28.4-50.9%) at 12 weeks. When using the anti-SARS-CoV-2 total antibody level (≥ 132 U/ml) criteria, only 71.3% HCWs in the SV group had seroconversion, compared to 100% in the AZ group at 4 weeks. CONCLUSIONS: A rapid decline of short-term immune response in the HCWs after the SV vaccination indicates the need for a vaccine booster, particularly during the ongoing spreading of the SARS-CoV-2 variants of concern.

COVID-19 and HIV infection co-pandemics and their impact: a review of the literature.

Coronavirus disease 2019 (COVID-19) was first detected in December 2019. In March 2020, the World Health Organization declared COVID-19 a pandemic. People with underlying medical conditions may be at greater risk of infection and experience complications from COVID-19. COVID-19 has the potential to affect People living with HIV (PLWH) in various ways, including be increased risk of COVID-19 acquisition and interruptions of HIV treatment and care. The purpose of this review article is to evaluate the impact of COVID-19 among PLWH. The contents focus on 4 topics: (1) the pathophysiology and host immune response of people infected with both SARS-CoV-2 and HIV, (2) present the clinical manifestations and treatment outcomes of persons with co-infection, (3) assess the impact of antiretroviral HIV drugs among PLWH infected with COVID-19 and (4) evaluate the impact of the COVID-19 pandemic on HIV services.

Endotoxin Adsorbent Therapy in Severe COVID-19 Pneumonia.

INTRODUCTION: Uncontrolled systemic inflammation may occur in severe coronavirus disease 19 (COVID-19). We have previously shown that endotoxemia, presumably from the gut, may complicate COVID-19. However, the role of endotoxin adsorbent (EA) therapy to mitigate organ dysfunction in COVID-19 has not been explored. METHODS: We conducted a retrospective observational study in COVID-19 patients who received EA therapy at the King Chulalongkorn Memorial Hospital, Bangkok, Thailand, between March 13 and April 17, 2020. Relevant clinical and laboratory data were collected by inpatient chart review. RESULTS: Among 147 hospitalized COVID-19 patients, 6 patients received EA therapy. All of the 6 patients had severe COVID-19 infection with acute respiratory distress syndrome (ARDS). Among these, 5 of them were mechanically ventilated and 4 had complications of secondary bacterial infection. The endotoxin activity assay (EAA) results of pre-EA therapy ranged from 0.47 to 2.79. The choices of EA therapy were at the discretion of attending physicians. One patient was treated with oXiris® along with continuous renal replacement therapy, and the others received polymyxin B hemoperfusion sessions. All patients have survived and were finally free from the mechanical ventilation as well as had improvement in PaO2/FiO2 ratio and decreased EAA level after EA therapy. CONCLUSIONS: We demonstrated the clinical improvement of severe COVID-19 patients with elevated EAA level upon receiving EA therapy. However, the benefit of EA therapy in COVID-19 ARDS is still unclear and needs to be elucidated with randomized controlled study.

Multiple clades of SARS-CoV-2 were introduced to Thailand during the first quarter of 2020.

In early January 2020, Thailand became the first country where a coronavirus disease 2019 (COVID-19) patient was identified outside China. In this study, 23 whole genomes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from patients who were hospitalized from January to March 2020 were analyzed, along with their travel histories. Six lineages were identified including A, A.6, B, B.1, B.1.8, and B.58, among which lineage A.6 was dominant. Seven patients were from China who traveled to Thailand in January and early February. Five of them were infected with the B lineage virus, and the other two cases were infected with different lineages including A and A.6. These findings present clear evidence of the early introduction of diverse SARS-CoV-2 clades in Thailand.