The first human case report of molecularly confirmed co-infection of Brucella melitensis and Coxiella burnetii: A case report.

Co-infection with Brucella melitensis and Coxiella burnetii has been rarely reported. To date, there are only two co-infection case reports from Croatia and China which diagnosed the infections mainly through the use of serological tests. In this report, we present the first case of molecularly confirmed B. melitensis bacteremia and C. burnetii spondylodiscitis co-infection in a goat dairy farmer who presented with lumbosacral spondylodiscitis and bilateral psoas abscesses. From the blood culture, B. melitensis was identified by using 16S rRNA gene sequencing and specific PCR. Lumbar bone tissue was found to be positive for C. burnetii using multiplex real-time PCR and was confirmed with a positive result from conventional PCR which detected the infection through the identification of the IS1111 gene. The patient's condition improved after decompressive laminectomy was performed and administration of antibiotics regimen: intravenous gentamicin, oral rifampicin, and oral doxycycline. From our case, it is important to raise awareness of this underreported co-infection with multiple zoonotic diseases, especially Q fever and brucellosis, which share the same exposure risk. Moreover, we also emphasize the use of advanced molecular techniques to improve the diagnostic efficiency and reduce the use of time-consuming procedures among patients who are continuously exposed to such risk factors in areas with high seroprevalence of these zoonotic diseases.

Needs assessment of a pythiosis continuing professional development program.

BACKGROUND: Pythiosis is a rare disease with high mortality, with over 94% of cases reported from Thailand and India. Prompt diagnosis and surgery improves patient outcomes. Therefore, continuing professional development (CPD) is essential for early recognition. However, a needs assessment related to a pythiosis CPD program has not been performed. OBJECTIVES: We conducted a needs assessment to develop a pythiosis CPD program. PATIENTS/METHODS: We conducted a survey study with 267 King Chulalongkorn Memorial Hospital residents (141 internal medicine (IM) residents and 126 surgery residents). A 30-item survey consisting of a knowledge assessment, demographic section, and an attitudes portion was distributed both electronically and via paper. The data was analyzed with descriptive and inferential statistics. RESULTS: Sixty-seven percent completed the survey (110/141 IM residents, 70/126 surgery residents). The mean score [95% confidence interval] on the knowledge assessment was 41.67% [39.64%-43.69%] across all objectives. The three domains with the highest scores were pythiosis risk factors (67.22% correct), microbiologic characteristics (50.83%), and radiographic interpretation (50.56%). The three domains with the lowest scores were laboratory investigation (15.00%), epidemiology (29.17%), and symptomatology (30.83%). Most participants noted that the program should be online with both synchronous and asynchronous sessions, with a preferred length of 60-90 minutes per session. CONCLUSION: The pythiosis CPD program should emphasize education regarding symptomatology, laboratory investigation, and epidemiology, all of which are critical for the early detection of pythiosis to decrease mortality from this devastating disease. Most respondents felt this program was necessary and should be implemented in a virtual blended format.

Ex vivo observation of Pythium insidiosum-antigen treated neutrophils on three Pythium insidiosum strains isolated from vascular pythiosis patients.

The mechanisms of Pythium insidiosum-antigen (PIA) immunotherapy activating a patient's immune system are unknown. We evaluated the interleukin-8 (IL-8) serum levels during P. insidiosum infection and after vaccination with PIA in vascular pythiosis cases. Furthermore, we studied the anti-P. insidiosum activity of neutrophils stimulated with various concentrations of PIA ex vivo in 3 strains of P. insidiosum isolated from vascular pythiosis patients. IL-8 serum levels were evaluated using the ELISA technique. We assessed the effect of PIA-stimulated neutrophils on the viability of zoospores using MTT assay, visualized neutrophil extracellular trap (NET) formation via microscopy, and measured the levels of double-stranded DNA (dsDNA) using PicoGreen dsDNA quantitation assay in 3 strains of P. insidiosum isolated from vascular pythiosis patients. Serum levels of IL-8 gradually lowered from the early to the end phases of vaccination with PIA among the surviving group of vascular pythiosis cases. Neutrophils stimulated with 0.01 µg/ml PIA reduced zoospore viability significantly compared to PIA-unstimulated neutrophils for strain 1 and strain 3 (p < .05). Neutrophils stimulated with 0.01, 0.1, 1, and 10 µg/ml PIA exhibited significantly lower zoospore viability than PIA-unstimulated neutrophils for strain 2 (p < .05). IL-8 can be used as a biomarker for monitoring vascular pythiosis cases treated with the PIA vaccine. Also, anti-P. insidiosum activity of PIA-stimulated neutrophils was probably due to the disruption of cellular activity in zoospores rather than the mechanisms based on the formation of NETs.

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.

Gas chromatography-flame ionization detector for sweat based COVID-19 screening.

Simple approach for rapid screening of corona virus disease 2019 (COVID-19) has been developed. This applied gas chromatography-flame ionization detector (GC-FID) analyzing the potential compound marker in sweat samples obtained from COVID-19 positive and negative volunteers in Bangkok, Thailand. The samples were collected by using cotton rods for 15 min, heated at 90 °C for 5 min, and the volatile compounds in the headspace (HS) were injected (5.00 mL) at 150 °C and separated within 13.7 min. The marker peak was tentatively identified as p-cymene by the authentic standard injection and comparison with the GC-mass spectrometry (GC-MS) and comprehensive two-dimensional GC (GC × GC)-MS analysis. Possible mechanisms for the presence of p-cymene were proposed. The marker peak area thresholds were then varied and optimized via construction of the receiver operating characteristic (ROC) curve. With the optimum threshold, the established method offered the accuracy, sensitivity and specificity of 96 %. This method was insignificantly affected (p-value >0.05) by genders, body mass indices, ages, and use of deodorants as well as the p-cymene containing food. However, the performance could be affected by the population with personal hygiene or experiencing the microbiomes producing p-cymene.

Attenuated immunogenicity of SARS-CoV-2 vaccines and risk factors in stem cell transplant recipients: a meta-analysis.

Immunogenicity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination is diminished in hematopoietic stem cell transplant (HSCT) recipients. To summarize current evidence and identify risk factors for attenuated responses, 5 electronic databases were searched since database inceptions through 12 January 2023 for studies reporting humoral and/or cellular immunogenicity of SARS-CoV-2 vaccination in the HSCT population. Using descriptive statistics and random-effects models, extracted numbers of responders and pooled odds ratios (pORs) with 95% confidence intervals (CIs) for risk factors of negative immune responses were analyzed (PROSPERO: CRD42021277109). From 61 studies with 5906 HSCT recipients, after 1, 2, and 3 doses of messenger RNA (mRNA) SARS-CoV-2 vaccines, the mean antispike antibody seropositivity rates (95% CI) were 38% (19-62), 81% (77-84), and 80% (75-84); neutralizing antibody seropositivity rates were 52% (40-64), 71% (54-83), and 78% (61-89); and cellular immune response rates were 52% (39-64), 66% (51-79), and 72% (52-86). After 2 vaccine doses, risk factors (pOR; 95% CI) associated with antispike seronegativity were male recipients (0.63; 0.49-0.83), recent rituximab exposure (0.09; 0.03-0.21), haploidentical allografts (0.46; 0.22-0.95), <24 months from HSCT (0.25; 0.07-0.89), lymphopenia (0.18; 0.13-0.24), hypogammaglobulinemia (0.23; 0.10-0.55), concomitant chemotherapy (0.48; 0.29-0.78) and immunosuppression (0.18; 0.13-0.25). Complete remission of underlying hematologic malignancy (2.55; 1.05-6.17) and myeloablative conditioning (1.72; 1.30-2.28) compared with reduced-intensity conditioning were associated with antispike seropositivity. Ongoing immunosuppression (0.31; 0.10-0.99) was associated with poor cellular immunogenicity. In conclusion, attenuated humoral and cellular immune responses to mRNA SARS-CoV-2 vaccination are associated with several risk factors among HSCT recipients. Optimizing individualized vaccination and developing alternative COVID-19 prevention strategies are warranted.

A fluorescence-based sweat test sensor in a proof-of-concept clinical study for COVID-19 screening diagnosis.

During the corona virus disease 2019 (COVID-19) pandemic period, rapid screening of covid-19 patients has been of great interest by developing a fluorescent sensor for complexation with nonanal, which is a marker for Covid-19 detection in sweat. Solid phase micro-extraction gas chromatography-mass spectrometry (SPME GC-MS) was initially used to quantify nonanal in armpit sweat samples based on an external calibration curve. A sample containing a nonanal content above the threshold of 1.04 µL is expected to be COVID-19 positive with a sensitivity and specificity of 87% and 89%, respectively, validated by comparison with RT-PCR results. For more practical applications, helicene dye-encapsulated ethyl cellulose, namely EC@dyeNH, was applied to screen 140 sweat samples collected from the foreheads of volunteers. The mixed sensor and sweat solution droplets were then visualized and imaged under blacklight. The COVID-19 positive droplets exhibited yellow fluorescence emission, the brightness of which could be measured by using ImageJ in the grey scale. With the optimum color intensity of >73 for positive results, the screening performance was observed with a sensitivity and specificity of 96% and 93%, respectively. The overall test time of this method is approximately less than 15 min. This alternative method offers a promising practical screening approach for the diagnosis of COVID-19 in sweat.

Self-enhancement lateral flow immunoassay for COVID-19 diagnosis.

Equipment-free colorimetric-based lateral flow immunoassay (LFIA) is the most convenient and popular tool for various applications, including diagnostic tools requiring high sensitivity for the detection of pathogens. Thus, improvements and developments of LFIA are constantly being reported. Herein, we enriched the sensitivity of LFIA using the gold enhancement principle, emphasizing needlessly complicated apparatus, only one step for the strip test operation, and typical time incubation (15 min) process. Self-enhanced LFIA was then executed for subsequent flows by overlapping the additionally enhanced pad composed of gold ions and reducing agent on the conjugate pad and the sample pad. Self-enhanced LFIA was performed to detect SARS-CoV-2 antigens in saliva. The obtained result depicted that the achieved sensitivity was up to tenfold compared with that of conventional LFIA by visual measurements. The detection limits of self-enhanced LFIA detecting nucleocapsid protein antigens in the saliva sample was 0.50 and 0.10 ng/mL employed by naked eye detection and calibration curve-based calculation, respectively. When the proposed device was applied to 207 human saliva samples, the diagnostic performance presented a 96.10 % sensitivity and 99.23 % specificity. This self-enhanced LFIA could be implemented in large-scale production and demonstrates higher sensitivity with effortless use, which meets the requirements for point-of-care testing and on-field mass screening.

Ribavirin treatment for respiratory syncytial virus infection in patients with haematologic malignancy and haematopoietic stem cell transplant recipients: a systematic review and meta-analysis.

BACKGROUND: Ribavirin use for respiratory syncytial virus (RSV) infection in patients with haematologic malignancies (HM) and haematopoietic stem cell transplant (HSCT) recipients remains controversial. OBJECTIVES: To summarize the current evidence of ribavirin treatment in association with mortality and progression to lower respiratory tract infection (LRTI) among patients with HM/HSCT with RSV infection. DATA SOURCES: MEDLINE, Embase, and the Institute for Scientific Information Web of Science. STUDY ELIGIBILITY CRITERIA: Randomized controlled trials and observational studies investigating the effects of ribavirin, compared with treatment without ribavirin, for RSV infection. PARTICIPANTS: Patients with HM/HSCT. INTERVENTIONS: Ribavirin versus no ribavirin. ASSESSMENT OF RISK OF BIAS: The risk of bias in non-randomized studies of exposure (ROBIN-E). METHODS OF DATA SYNTHESIS: The random-effects model was used to calculate the pooled OR (pOR) with 95% CI for the pooled effect estimates of ribavirin benefits. Grading of recommendation assessment, development, and evaluation was used to evaluate the certainty of evidence. RESULTS: One randomized controlled trial and 14 observational studies were included, representing 1125 patients with HM/HSCT. Ribavirin use was not associated with lower all-cause or RSV-associated mortality with pORs [95% CI] of 0.81 [0.40, 1.66], I2 = 55% (low certainty of evidence) and 0.48 [0.11, 2.15], I2 = 64% (very low certainty of evidence), respectively. In subgroup analyses, ribavirin use was associated with lower mortality in patients with HM/HSCT with LRTI with pOR [95% CI] of 0.19 [0.07, 0.51], I2 = 0% (moderate certainty of evidence). In subgroup analyses among studies providing adjusted OR, ribavirin use was associated with lower all-cause mortality with pOR of 0.41 [0.23, 0.74], I2 = 0% (moderate certainty of evidence). In addition, aerosolized ribavirin was associated with lower progression to LRTI with pOR [95% CI] of 0.27 [0.09, 0.80], I2 = 71% (low certainty of evidence). CONCLUSIONS: Ribavirin may be a reasonable option to treat RSV in patients with HM/HSCT in the absence of other effective antiviral agents.

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.

Prevalence of Ocular Candidiasis and Candida Endophthalmitis in Patients With Candidemia: A Systematic Review and Meta-Analysis.

BACKGROUND: Infectious diseases and ophthalmology professional societies have disagreed regarding ocular screening in patients with candidemia. We aimed to summarize the current evidence on the prevalence of ocular candidiasis (OC) and Candida endophthalmitis (CE) according to the standardized definitions. METHODS: A literature search was conducted from the inception date through 16 October 2022 using PubMed, Embase, and SCOPUS. Pooled prevalence of ocular complications was derived from generalized linear mixed models (PROSPERO CRD42022326610). RESULTS: A total of 70 and 35 studies were included in the meta-analysis for OC and concordant CE (chorioretinitis with vitreous involvement), respectively. This study represented 8599 patients with candidemia who underwent ophthalmologic examination. Pooled prevalences (95% CI) of OC, overall CE, concordant CE, and discordant CE were 10.7% (8.4-13.5%), 3.1% (2.1-4.5%), 1.8% (1.3-2.6%), and 7.4% (4.5-12%) of patients screened, respectively. Studies from Asian countries had significantly higher concordant CE prevalence (95% CI) of patients screened (3.6%; 2.9-4.6%) compared with studies from European countries (1.4%; .4-5%) and American countries (1.4%; .9-2.2%) (P <.01). Presence of total parenteral nutrition and Candida albicans was associated with CE, with pooled odds ratios (95% CI) of 6.92 (3.58-13.36) and 3.02 (1.67-5.46), respectively. CONCLUSIONS: Prevalence of concordant CE overall and among Asian countries was 2 and 4 times higher than the prevalence previously reported by the American Academy of Ophthalmology (AAO) of <0.9%, respectively. There is an urgent need to study optimal screening protocols and to establish joint recommendations by the Infectious Diseases Society of America and AAO.

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 hours. However, the spike gene sequencing or NGS retains the advantage of detecting newly emerged variants.

The In Vitro Efficacy of Activated Charcoal in Fecal Ceftriaxone Adsorption among Patients Who Received Intravenous Ceftriaxone.

Broad-spectrum antibiotics can kill both pathogens and gut microbiota. Reducing exposure to excess intestinal antibiotics could theoretically protect gut microbiota homeostasis. Recently, engineered charcoals, gel microparticles, and resin beads have demonstrated efficacy in intestinal antibiotic adsorption in animal studies. We report the first in vitro study evaluating human fecal antibiotic adsorption efficacy of conventional activated charcoal (AC). We collected fecal samples from eight patients who received intravenous (IV) ceftriaxone after admission to King Chulalongkorn Memorial Hospital, Thailand, during January−March 2020. Fecal ceftriaxone was measured by indirect competitive enzyme-linked immunoassays. Three different doses of AC were mixed with fecal samples under a specified protocol. The geometric mean reduction in fecal ceftriaxone concentration when mixed with AC 30 mg/g feces was 0.53 (95% CI 0.33−0.85, p-value < 0.001), meaning 47% adsorption efficacy. Increased adsorption was found with higher doses, 71% and 87% for AC 150 and 500 mg/g feces, respectively. In conclusion, the usual food-poisoning-care dose of conventional AC, 30 mg/g feces, demonstrated dose-dependent and significant fecal ceftriaxone adsorption. Conventional oral AC might be a pragmatic and inexpensive option for the protection of gut microbiota in patients receiving IV ceftriaxone. However, in vivo studies and microbiome analysis are needed for further evidence.

First case series and literature review of coronavirus disease 2019 (COVID-19) associated pulmonary tuberculosis in Southeast Asia: Challenges and opportunities.

BACKGROUND: Subclinical tuberculosis (TB) is accidentally detected by radiologic and microbiologic findings. Transmission by those with subclinical TB could delay prevention effort. However, our study demonstrated positive aspect of COVID-19 outbreak as it could allow subclinical TB to be detected faster through a chest X-Ray (CXR). METHODS: This cross-sectional cohort study aimed to report demographics, comorbidities, and outcomes related to early detection of TB among COVID-19 patients, and to elaborate the association between SARS-CoV-2 and pulmonary TB. Data of patients with SARS-CoV-2 co-infection with Mycobacterium tuberculosis (MTB) diagnosed between March 2020 - March 2022 was collected. RESULTS: Out of 12,275 COVID-19 patients, 26 were definitively diagnosed with MTB infection (mean age 48.16 ± 20.17 years). All cases that had suspicious CXR that were not typical for COVID-19, were tested for MTB. On average, pulmonary TB was diagnosed after admission 5(3-10) days, the treatment initiation period was 3(1-5) days from the TB diagnosis. CONCLUSIONS: This suggests an early detection of tuberculosis among COVID-19 patients by quicker screening CXR and sputum comparing to previous symptom guided screening. Thereby reducing the chance of TB transmission demonstrated during COVID-19 pandemic. So, clinicians should be aware of pulmonary tuberculosis in COVID-19 patients with atypical radiologic findings.

Immunogenicity of SARS-CoV-2 vaccines in patients with multiple myeloma: a systematic review and meta-analysis.

Patients with multiple myeloma (MM) have a diminished immune response to coronavirus disease 2019 (COVID-19) vaccines. Risk factors for an impaired immune response are yet to be determined. We aimed to summarize the COVID-19 vaccine immunogenicity and to identify factors that influence the humoral immune response in patients with MM. Two reviewers independently conducted a literature search in MEDLINE, Embase, ISI Web of Science, Cochrane library, and Clinicaltrials.gov from existence until 24 May 24 2022. (PROSPERO: CRD42021277005). A total of 15 studies were included in the systematic review and 5 were included in the meta-analysis. The average rate (range) of positive functional T-lymphocyte response was 44.2% (34.2%-48.5%) after 2 doses of messenger RNA (mRNA) COVID-19 vaccines. The average antispike antibody response rates (range) were 42.7% (20.8%-88.5%) and 78.2% (55.8%-94.2%) after 1 and 2 doses of mRNA COVID-19 vaccines, respectively. The average neutralizing antibody response rates (range) were 25% (1 study) and 62.7% (53.3%-68.6%) after 1 and 2 doses of mRNA COVID-19 vaccines, respectively. Patients with high-risk cytogenetics or receiving anti-CD38 therapy were less likely to have a humoral immune response with pooled odds ratios of 0.36 (95% confidence interval [95% CI], 0.18, 0.69), I2 = 0% and 0.42 (95% CI, 0.22, 0.79), I2 = 14%, respectively. Patients who were not on active MM treatment were more likely to respond with pooled odds ratio of 2.42 (95% CI, 1.10, 5.33), I2 = 7%. Patients with MM had low rates of humoral and cellular immune responses to the mRNA COVID-19 vaccines. Further studies are needed to determine the optimal doses of vaccines and evaluate the use of monoclonal antibodies for pre-exposure prophylaxis in this population.

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.

Anti-Inflammatory Effects and Decreased Formation of Neutrophil Extracellular Traps by Enoxaparin in COVID-19 Patients.

Neutrophil Extracellular Traps (NETs) are a contributing factor of vascular thrombosis and alveolar damage in COVID-19 patients. As enoxaparin is currently used to inhibit vascular thrombosis, this study aimed to investigate whether enoxaparin also reduced inflammation and NETs in COVID-19 patients. Patients with COVID-19 infection were classified into three groups: mild, moderate, and severe (n = 10 for all groups). Plasma was collected from patients and healthy donors (n = 10). Neutrophils isolated from healthy controls were incubated with COVID-19 or healthy plasma, and with or without enoxaparin pretreatment in vitro. Neutrophils and plasma isolated from patients treated with enoxaparin were also investigated. The levels of inflammatory cytokines and NET products such as dsDNA, NE, MPO−DNA and Histone−DNA complexes in plasma and supernatants were measured using immunofluorescence staining and ELISA kits. The expression of inflammatory signaling genes by neutrophils (RELA, SYK, ERK and PKC) was measured using real-time qPCR. The levels of NET products were elevated in the plasma of COVID-19 patients, particularly in the severe group (p < 0.01). Moreover, plasma from the severe group enhanced NET formation (p < 0.01) from neutrophils in vitro. Enoxaparin pretreatment in vitro decreased plasma-induced NETs in a dose-dependent manner and down-regulated the expression of inflammatory genes (p < 0.05). Patients treated with prophylactic enoxaparin showed lower inflammatory cytokine levels and expression of inflammatory genes (p < 0.05). Increased NETs were associated with the severity of COVID-19 infection, particularly in patients with severe pneumonia, and could be used as biomarkers to assess disease severity. Enoxaparin pretreatment inhibited NETs and reduced the expression of inflammatory cytokines, and these effects mostly persisted in patients treated with prophylactic enoxaparin.

Immunogenicity and Risk Factors Associated With Poor Humoral Immune Response of SARS-CoV-2 Vaccines in Recipients of Solid Organ Transplant: A Systematic Review and Meta-Analysis.

Importance: Recipients of solid organ transplant (SOT) experience decreased immunogenicity after COVID-19 vaccination. Objective: To summarize current evidence on vaccine responses and identify risk factors for diminished humoral immune response in recipients of SOT. Data Sources: A literature search was conducted from existence of database through December 15, 2021, using MEDLINE, Embase, Web of Science, Cochrane Library, and ClinicalTrials.gov. Study Selection: Studies reporting humoral immune response of the COVID-19 vaccines in recipients of SOT were reviewed. Data Extraction and Synthesis: Two reviewers independently extracted data from each eligible study. Descriptive statistics and a random-effects model were used. This report was prepared following the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) reporting guideline. Data were analyzed from December 2021 to February 2022. Main Outcomes and Measures: The total numbers of positive immune responses and percentage across each vaccine platform were recorded. Pooled odds ratios (pORs) with 95% CIs were used to calculate the pooled effect estimates of risk factors for poor antibody response. Results: A total of 83 studies were included for the systematic review, and 29 studies were included in the meta-analysis, representing 11 713 recipients of SOT. The weighted mean (range) of total positive humoral response for antispike antibodies after receipt of mRNA COVID-19 vaccine was 10.4% (0%-37.9%) for 1 dose, 44.9% (0%-79.1%) for 2 doses, and 63.1% (49.1%-69.1%) for 3 doses. In 2 studies, 50% of recipients of SOT with no or minimal antibody response after 3 doses of mRNA COVID-19 vaccine mounted an antibody response after a fourth dose. Among the factors associated with poor antibody response were older age (mean [SE] age difference between responders and nonresponders, 3.94 [1.1] years), deceased donor status (pOR, 0.66 [95% CI, 0.53-0.83]; I2 = 0%), antimetabolite use (pOR, 0.21 [95% CI, 0.14-0.29]; I2 = 70%), recent rituximab exposure (pOR, 0.21 [95% CI, 0.07-0.61]; I2 = 0%), and recent antithymocyte globulin exposure (pOR, 0.32 [95% CI, 0.15-0.71]; I2 = 0%). Conclusions and Relevance: In this systematic review and meta-analysis, the rates of positive antibody response in solid organ transplant recipients remained low despite multiple doses of mRNA vaccines. These findings suggest that more efforts are needed to modulate the risk factors associated with reduced humoral responses and to study monoclonal antibody prophylaxis among recipients of SOT who are at high risk of diminished humoral response.

Neutrophil Extracellular Traps in Severe SARS-CoV-2 Infection: A Possible Impact of LPS and (1→3)-ß-D-glucan in Blood from Gut Translocation.

Due to limited data on the link between gut barrier defects (leaky gut) and neutrophil extracellular traps (NETs) in coronavirus disease 2019 (COVID-19), blood samples of COVID-19 cases-mild (upper respiratory tract symptoms without pneumonia; n = 27), moderate (pneumonia without hypoxia; n = 28), and severe (pneumonia with hypoxia; n = 20)-versus healthy control (n = 15) were evaluated, together with in vitro experiments. Accordingly, neutrophil counts, serum cytokines (IL-6 and IL-8), lipopolysaccharide (LPS), bacteria-free DNA, and NETs parameters (fluorescent-stained nuclear morphology, dsDNA, neutrophil elastase, histone-DNA complex, and myeloperoxidase-DNA complex) were found to differentiate COVID-19 severity, whereas serum (1→3)-ß-D-glucan (BG) was different between the control and COVID-19 cases. Despite non-detectable bacteria-free DNA in the blood of healthy volunteers, using blood bacteriome analysis, proteobacterial DNA was similarly predominant in both control and COVID-19 cases (all severities). In parallel, only COVID-19 samples from moderate and severe cases, but not mild cases, were activated in vitro NETs, as determined by supernatant dsDNA, Peptidyl Arginine Deiminase 4, and nuclear morphology. With neutrophil experiments, LPS plus BG (LPS + BG) more prominently induced NETs, cytokines, NFκB, and reactive oxygen species, when compared with the activation by each molecule alone. In conclusion, pathogen molecules (LPS and BG) from gut translocation along with neutrophilia and cytokinemia in COVID-19-activated, NETs-induced hyperinflammation.