Evaluating Pharmacy Students' Teamwork Attitudes in Virtual COVID-19 Emergency Department Simulations: A Pilot Study.

OBJECTIVE: This study explores the impact of virtual simulation training on the transformation of teamwork attitudes among pharmacy students in a simulated severe COVID-19 pneumonia scenario in the emergency department. METHODS: From July 2022 to January 2023, 16 pharmacy students, along with other health care students, participated in interprofessional simulation rounds. Each pharmacy student was assigned specific days for participation, using either a 3-dimensional computer or a virtual reality headset to manage a patient with severe COVID-19 pneumonia in the virtual emergency department. The TeamSTEPPS Teamwork Attitudes Questionnaire (T-TAQ) was used for pre- and post-training assessments. RESULTS: The mean baseline T-TAQ score was 119.44 ± 10.63, showing a significant post-training improvement to a mean score of 130.88 ± 8.98 (Hedges' g = 1.52). Stratification by academic year and device type revealed no significant impact on the learning experience. Remarkable enhancements in teamwork attitudes were observed after training, specifically in team structure, situation monitoring, mutual support, and communication domains. CONCLUSION: These findings indicate that virtual simulation training in scenarios such as severe COVID-19 effectively augments teamwork attitudes among pharmacy students, preparing them for collaborative practice in high-stakes emergency medicine settings.

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.

Targeted sequencing analysis pipeline for species identification of human pathogenic fungi using long-read nanopore sequencing.

Among molecular-based techniques for fungal identification, Sanger sequencing of the primary universal fungal DNA barcode, the internal transcribed spacer (ITS) region (ITS1, 5.8S, ITS2), is commonly used in clinical routine laboratories due to its simplicity, universality, efficacy, and affordability for fungal species identification. However, Sanger sequencing fails to identify mixed ITS sequences in the case of mixed infections. To overcome this limitation, different high-throughput sequencing technologies have been explored. The nanopore-based technology is now one of the most promising long-read sequencing technologies on the market as it has the potential to sequence the full-length ITS region in a single read. In this study, we established a workflow for species identification using the sequences of the entire ITS region generated by nanopore sequencing of both pure yeast isolates and mocked mixed species reads generated with different scenarios. The species used in this study included Candida albicans (n = 2), Candida tropicalis (n = 1), Nakaseomyces glabratus (formerly Candida glabrata) (n = 1), Trichosporon asahii (n = 2), Pichia kudriavzevii (formerly Candida krusei) (n = 1), and Cryptococcus neoformans (n = 1). Comparing various methods to generate the consensus sequence for fungal species identification, the results from this study indicate that read clustering using a modified version of the NanoCLUST pipeline is more sensitive than Canu or VSEARCH, as it classified species accurately with a lower abundance cluster of reads (3% abundance compared to 10% with VSEARCH). The modified NanoCLUST also reduced the number of classified clusters compared to VSEARCH, making the subsequent BLAST+ analysis faster. Subsampling of the datasets, which reduces the size of the datasets by approximately tenfold, did not significantly affect the identification results in terms of the identified species name, percent identity, query coverage, percentage of reads in the classified cluster, and the number of clusters. The ability of the method to distinguish mixed species within sub-populations of large datasets has the potential to aid computer analysis by reducing the required processing power. The herein presented new sequence analysis pipeline will facilitate better interpretation of fungal sequence data for species identification.

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.

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.

Neutrophil extracellular traps and phagocytosis in Pythium insidiosum.

Neutrophils are innate immune cells that play crucial roles in response to extracellular pathogens, including bacteria and fungi. Pythium insidiosum (P insidiosum) is a fungus-like pathogen that causes "pythiosis" in mammals. This study investigated in vitro function of human neutrophils against P. insidiosum. We demonstrated the killing mechanism of neutrophils when incubated with P. insidiosum zoospores (infective stage), such as phagocytosis and neutrophil extracellular traps (NETs). Healthy human neutrophils significantly reduced six strains of live zoospores isolated from different sources compared to the condition without neutrophils (p < 0.001), observed by colony count and trypan blue staining. As our results showed the killing ability of neutrophils, we further investigated the neutrophil killing mechanism when incubating with zoospores. Our study found that only two strains of heat-killed zoospores significantly induced phagocytosis (p < 0.01). Co-culture of heat-killed zoospores and neutrophils demonstrated NET formation, which was detected by immunofluorescence staining using DAPI, anti-myeloperoxidase, and anti-neutrophil elastase and quantitated under the fluorescence microscope. In addition, the level of cell-free DNA released from neutrophils (as a marker of NET production) after incubation with zoospores showed significantly increased levels when compared with unstimulated neutrophils (p < 0.001). Our findings demonstrate that neutrophils revealed the NET formation in response to P. insidiosum zoospores. This study is the first observation of the neutrophil mechanism against P. insidiosum, which could provide a better understanding of some parts of the innate immune response during pythiosis.

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.

A Review: Antimicrobial Therapy for Human Pythiosis.

Human pythiosis is associated with poor prognosis with significant mortality caused by Pythium insidiosum. Antimicrobials' in vitro and in vivo results against P. insidiosum are inconsistent. Although antimicrobials are clinically useful, they are not likely to achieve therapeutic success alone without surgery and immunotherapy. New therapeutic options are therefore needed. This non-exhaustive review discusses the rationale antimicrobial therapy, minimum inhibitory concentrations, and efficacy of antibacterial and antifungal agents against P. insidiosum. This review further provides insight into the immunomodulating effects of antimicrobials that can enhance the immune response to infections. Current data support using antimicrobial combination therapy for the pharmacotherapeutic management of human pythiosis. Also, the success or failure of antimicrobial treatment in human pythiosis might depend on the immunomodulatory effects of drugs. The repurposing of existing drugs is a safe strategy for anti-P. insidiosum drug discovery. To improve patient outcomes in pythiosis, we suggest further research and a deeper understanding of P. insidiosum virulence factors, host immune response, and host immune system modification by antimicrobials.

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.

The association of cytomegalovirus infection and cytomegalovirus serostatus with invasive fungal infections in allogeneic haematopoietic stem cell transplant recipients: a systematic review and meta-analysis.

BACKGROUND: In allogeneic haematopoietic stem cell transplant (allo-HSCT) recipients, the inter-relationship between post-transplant cytomegalovirus (CMV) and subsequent invasive fungal infections (IFIs) is conflicting and the association of CMV serostatus with IFIs has not been evaluated. OBJECTIVES: To determine the relationship between CMV infection/serostatus and IFIs in allo-HSCT populations. DATA SOURCES: A systematic literature search was conducted from existence until 11 July 2021 using Medline, Embase and ISI Web of Science databases. STUDY ELIGIBILITY CRITERIA: Cross-sectional, prospective cohort, retrospective cohort and case-control studies that reported allo-HSCT recipients with CMV and without CMV who developed or did not develop IFIs after CMV infection. PARTICIPANTS: Allo-HSCT recipients. INTERVENTIONS: Not applicable. METHODS: A systematic search, screening, data extracting and assessing study quality were independently conducted by two reviewers. The Newcastle-Ottawa scale was used to assess risk of bias. data were analysed using the pooled effect estimates of a random-effects model. RESULTS: A total of 18 and 12 studies were included for systematic review and meta-analysis, respectively. Post-transplant CMV infection significantly increased the risk of IFIs with a pooled hazard ratio (pHR) of 2.58 (1.78, 3.74), I2 = 75%. Further subgroup analyses by timing of IFIs, CMV definitions, study continents, study design and adjustment of effect estimates showed that post-transplant CMV infection consistently increased the risk of subsequent IFIs. High-risk CMV serostatus (D-/R+) increased the risk of IFIs with a pooled odds ratio (OR) of 1.33 (1.04, 1.71), I2 = 0%, but low-risk CMV serostatus (D-/R-) decreased the risk of IFIs with a pOR of 0.69 (0.55, 0.87), I2 = 0%. CONCLUSIONS: Post-transplant CMV infection and high-risk CMV serostatus increased the risk of IFIs, but low-risk CMV serostatus decreased risk of IFIs among allo-HSCT recipients. Further studies are needed to identify at-risk allo-HSCT recipients as well as to focus on fungal diagnostics and prophylaxis to prevent this fungal-after-viral phenomenon.

Vascular pythiosis caused by Pythium aphanidermatum: the first case report in Asia.

BACKGROUND: Pythium, soil-borne plant pathogens, are in the class Oomycetes. They are not true fungi, but are related to diatom and algae. There are two human pathogens including P. insidiosum and P. aphanidermatum. To date, only one case of pythiosis caused by P. aphanidermatum has been reported. We present herein the first case of P. aphanidermatum vascular pythiosis in Asia. CASE PRESENTATION: A 47-year-old Thai woman, living in North Thailand, with ß thalassemia/hemoglobin E presented with acute recurrent arterial insufficiency of both legs. Emergent embolectomy with clot removal was performed. The pathology of the clot exhibited noncaseous granulomatous inflammation with many fungal hyphal elements. PCR identified P. aphanidermatum with 100% identity. Final diagnosis is vascular pythiosis. Unfortunately, the patient eventually expired after treatment with itraconazole, terbinafine, azithromycin, and doxycycline. CONCLUSIONS: To date, only one case of pythiosis caused by P. aphanidermatum has been reported. We present herein the first case of P. aphanidermatum vascular pythiosis in Asia.

Clinical Characteristics, Histopathology, and Treatment Outcomes of Pythium Keratitis: A Retrospective Cohort Study.

PURPOSE: To identify factors associated with the prognosis in Pythium keratitis. PATIENTS AND METHODS: The medical records of 25 patients (26 eyes) diagnosed with Pythium keratitis at a referral institution were reviewed. The demographic and clinical characteristics, treatment, microbiological diagnosis, histopathological features, and outcomes were recorded. The histopathological specimens were reviewed. The patients were divided into a globe removal group and a globe salvage group. Univariate analysis was used to identify factors associated with poor outcomes. RESULTS: Fifteen eyes (57.7%) were removed. Patients in the globe removal group were on average 16.4 years older (95% CI 6.98 to 25.88) than those in the globe salvage group, received the first medication (either topical antifungals or antibiotics) later than one day after the onset of symptoms (RR = 2.75, 95% CI 1.18 to 6.42), and had a maximal diameter of the infiltration area ≥6 mm (RR = 3.14, 95% CI 1.17 to 8.45). The globe removal group showed satellite, multifocal, or total corneal infiltration patterns (RR = 2.82, 95% CI 1.03 to 7.74) and a hypopyon (RR = 3.43, 95% CI 1.26 to 9.35) as risk factors. The histopathological examination showed a higher density of Pythium in the globe removal group than the globe salvage group (median 376 (interquartile range 323, 620) versus 107 (interquartile range 16, 260) hyphae per high power field; P = 0.035). CONCLUSION: The risk of globe removal in patients with Pythium keratitis increased with age, delayed initial topical antifungal or antibiotic treatment, advanced disease at presentation, and dense Pythium hyphae infiltration of the cornea. Early recognition and treatment are critical to successfully eradicate the infection.

CryptoType - Public Datasets for MALDI-TOF-MS Based Differentiation of Cryptococcus neoformans/gattii Complexes.

Yeasts of the Cryptococcus neoformans/gattii species complexes are human pathogens mostly in immune compromised individuals, and can cause infections from dermal lesions to fungal meningitis. Differences in virulence and antifungal drug susceptibility of species in these complexes indicate the value of full differentiation to species level in diagnostic procedures. MALDI-TOF MS has been reported to sufficiently discriminate these species. Here, we sought to re-evaluate sample pre-processing procedures and create a set of publicly available references for use with the MALDI Biotyper system. Peak content using four different pre-processing protocols was assessed, and database entries for 13 reference strains created. These were evaluated against a collection of 153 clinical isolates, typed by conventional means. The use of decapsulating protocols or mechanical disruption did not sufficiently increase the information content to justify the extra hands-on-time. Using the set of 13 reference entries created with the standard formic acid extraction, we were able to correctly classify 143/153 (93.5%) of our test isolates. The majority of the remaining ten isolates still gave correct top matches; only two isolates did not give reproducible identifications. This indicates that the log score cut-off can be lowered also in this context. Ease to identify cryptococcal isolates to the species level is improved by the workflow evaluated here. The database references are freely available from https://github.com/oliverbader/BioTyper-libraries for incorporation into local diagnostic systems.

Does Post-Transplant Cytomegalovirus Increase the Risk of Invasive Aspergillosis in Solid Organ Transplant Recipients? A Systematic Review and Meta-Analysis.

BACKGROUND: Cytomegalovirus (CMV) and invasive aspergillosis (IA) cause high morbidity and mortality in solid organ transplant (SOT) recipients. There are conflicting data with respect to the impact of CMV on IA development in SOT recipients. METHODS: A literature search was conducted from existence through to 2 April 2021 using MEDLINE, Embase, and ISI Web of Science databases. This review contained observational studies including cross-sectional, prospective cohort, retrospective cohort, and case-control studies that reported SOT recipients with post-transplant CMV (exposure) and without post-transplant CMV (non-exposure) who developed or did not develop subsequent IA. A random-effects model was used to calculate the pooled effect estimate. RESULTS: A total of 16 studies were included for systematic review and meta-analysis. There were 5437 SOT patients included in the study, with 449 SOT recipients developing post-transplant IA. Post-transplant CMV significantly increased the risk of subsequent IA with pORs of 3.31 (2.34, 4.69), I2 = 30%. Subgroup analyses showed that CMV increased the risk of IA development regardless of the study period (before and after 2003), types of organ transplantation (intra-thoracic and intra-abdominal transplantation), and timing after transplant (early vs. late IA development). Further analyses by CMV definitions showed CMV disease/syndrome increased the risk of IA development, but asymptomatic CMV viremia/infection did not increase the risk of IA. Conclusions: Post-transplant CMV, particularly CMV disease/syndrome, significantly increased the risks of IA, which highlights the importance of CMV prevention strategies in SOT recipients. Further studies are needed to understand the impact of programmatic fungal surveillance or antifungal prophylaxis to prevent this fungal-after-viral phenomenon.

Clinical Outcomes of Radical Surgery and Antimicrobial Agents in Vascular Pythiosis: A Multicenter Prospective Study.

Vascular pythiosis is a rare, neglected, life-threatening disease with mortality of 100% in patients with incomplete surgical resection or patients with persistently elevated serum ß-d-glucan (BDG). The study was conducted to understand the clinical outcomes of new treatment protocols and potential use of erythrocyte sedimentation rate (ESR) and c-reactive protein (CRP) as alternative monitoring tools, given recent favorable minimum inhibitory concentrations (MICs) of antibacterial agents and prohibitive cost of serum BDG in Thailand. A prospective cohort study of patients with vascular pythiosis was conducted between February 2019 and August 2020. After diagnosis, patients were followed at 0.5, 1, 1.5, 3, and 6 months. Descriptive statistics, Spearman's correlation coefficient, and general linear model for longitudinal data were used. Amongst the cohort of ten vascular pythiosis patients, four had residual disease after surgery. Among four with residual disease, one developed disseminated disease and died, one developed relapse disease requiring surgery, and two were successfully managed with antimicrobial agents. The spearman's correlation coefficients between BDG and ESR, and between BDG and CRP in patients without relapse or disseminated disease were 0.65 and 0.60, respectively. Tetracyclines and macrolides had most favorable minimum inhibitory concentrations and synergistic effects were observed in combinations of these two antibiotic classes. Adjunctive use of azithromycin and doxycycline preliminarily improved survival in vascular pythiosis patients with residual disease. Further studies are needed to understand the trends of ESR and CRP in this population.

Drug-drug interaction between itraconazole capsule and efavirenz in adults with HIV for talaromycosis treatment.

OBJECTIVES: To assess the pharmacokinetic of itraconazole capsule formulation and its active metabolite, hydroxyitraconazole, in adults with HIV diagnosed with talaromycosis in an endemic area, and to evaluate the drug-drug interaction between itraconazole/hydroxyitraconazole (ITC/OH-ITC) and efavirenz. METHODS: Open-label, single arm, sequential pharmacokinetic study. Eligible subjects were adults with HIV, ≥18 years old, with confirmed talaromycosis, initiating itraconazole capsule as part of standard talaromycosis treatment, in whom efavirenz-based ART was anticipated. Steady-state pharmacokinetic assessments (pre-dose and at 1, 3, 4, 5, 6, 8 and 12 h post dose) were performed for itraconazole/hydroxyitraconazole without and with efavirenz use. Mid-dose efavirenz concentrations were also assessed. Pharmacokinetics parameters were calculated using non-compartmental analysis. RESULTS: Ten subjects (70% male) were enrolled. At entry, median (range) age was 29.5 years (22-64), and CD4 cell count was 18.0 (1-39) cells/mm3. Geometric mean (95% CI) of itraconazole and hydroxyitraconazole AUC0-12 without efavirenz were 9097 (6761-12 239) and 11 705 (8586-15 959) ng·h/mL, respectively, with a median metabolic ratio of OH-ITC : ITC of 1.3 (95% CI 0.9-1.9). Intra-subject comparison revealed that both itraconazole and hydroxyitraconazole exposures were significantly reduced with concomitant efavirenz use, with the mean AUC0-12 of itraconazole and hydroxyitraconazole being 86% (71%-94%) and 84% (64%-97%) lower, respectively. With efavirenz, itraconazole trough concentrations were also below the recommended therapeutic level (0.5 µg/mL). All subjects had mid-dose efavirenz concentrations >1000 ng/mL. CONCLUSIONS: Concomitant administration of itraconazole capsule with efavirenz significantly reduced itraconazole and hydroxyitraconazole exposures. The clinical impact of this drug-drug interaction on talaromycosis treatment or prophylaxis in the era of potent ART needs further evaluation.

Endotoxemia and circulating bacteriome in severe COVID-19 patients.

BACKGROUND: When severe, COVID-19 shares many clinical features with bacterial sepsis. Yet, secondary bacterial infection is uncommon. However, as epithelium is injured and barrier function is lost, bacterial products entering the circulation might contribute to the pathophysiology of COVID-19. METHODS: We studied 19 adults, severely ill patients with COVID-19 infection, who were admitted to King Chulalongkorn Memorial Hospital, Bangkok, Thailand, between 13th March and 17th April 2020. Blood samples on days 1, 3, and 7 of enrollment were analyzed for endotoxin activity assay (EAA), (1 → 3)-ß-D-glucan (BG), and 16S rRNA gene sequencing to determine the circulating bacteriome. RESULTS: Of the 19 patients, 13 were in intensive care and 10 patients received mechanical ventilation. We found 8 patients with high EAA (≥ 0.6) and about half of the patients had high serum BG levels which tended to be higher in later in the illness. Although only 1 patient had a positive blood culture, 18 of 19 patients were positive for 16S rRNA gene amplification. Proteobacteria was the most abundant phylum. The diversity of bacterial genera was decreased overtime. CONCLUSIONS: Bacterial DNA and toxins were discovered in virtually all severely ill COVID-19 pneumonia patients. This raises a previously unrecognized concern for significant contribution of bacterial products in the pathogenesis of this disease.

In Vitro Susceptibility of Thai Pythium insidiosum Isolates to Antibacterial Agents.

Human pythiosis is a life-threatening human disease caused by Pythium insidiosum In Thailand, vascular pythiosis is the most common form and carries a mortality rate of 10 to 40%, despite aggressive treatment with radical surgery, antifungal agents, and immunotherapy. Itraconazole and terbinafine have been the mainstay of treatment, until recently, based on case report data showing potential synergistic effects against Brazilian P. insidiosum isolates. However, the synergistic effects of itraconazole and terbinafine against Thai P. insidiosum isolates were not observed. This study tested the in vitro susceptibilities of 27 Thai human P. insidiosum isolates (clade II, n = 17; clade IV, n = 10), 12 Thai environmental P. insidiosum isolates (clade II, n = 4; clade IV, n = 8), and 11 non-Thai animal P. insidiosum isolates (clade I, n = 9; clade II, n = 2) to antibiotics in eight antibacterial classes to evaluate alternative effective treatments. Tetracycline and macrolide antibiotics demonstrated in vitro activity against Thai P. insidiosum isolates, with doxycycline MICs (1 to 16 µg/ml), minocycline MICs (1 to 4 µg/ml), tigecycline MICs (1 to 4 µg/ml), azithromycin MICs (1 to 16 µg/ml), and clarithromycin MICs (0.125 to 8 µg/ml) being the lowest, on average. Synergistic effects of tetracyclines and macrolides were also observed.