Neutrophil Extracellular Traps in Severe SARS-CoV-2 Infection: A Possible Impact of LPS and (1→3)-β-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)-β-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

Survey of laboratory practices for diagnosis of fungal infection in seven Asian countries: An Asia Fungal Working Group (AFWG) initiative

An online survey of mycology laboratories in seven Asian countries was conducted to assess the status, competence, and services available. Country representatives from the Asia Fungal Working Group (AFWG) contacted as many laboratories performing mycology diagnosis as possible in their respective countries, requesting that the laboratory heads complete the online survey. In total, 241 laboratories responded, including 71 in China, 104 in India, 11 in Indonesia, 26 in the Philippines, four in Singapore, 18 in Taiwan, and seven in Thailand. Overall, 129/241 (53.5%) surveyed mycology laboratories operate as separate designated mycology laboratories, 75/241 (31.1%) conduct regular formal staff training, 103/241 (42.7%) are accredited, and 88/157 (56.1%) participate in external quality assurance scheme (EQAS) programs. Microscopy and culture methods are available in nearly all laboratories, although few perform DNA sequencing (37/219; 16.9%) or use matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS) (27/219; 12.3%) for isolate identification. Antifungal susceptibility testing is performed in 142/241 (58.9%) laboratories, mainly for yeasts. The most commonly performed nonculture diagnostic is cryptococcal antigen testing (66 laboratories), followed by galactomannan testing (55), polymerase chain reaction (PCR) diagnosis (37), and beta-D-glucan testing (24). Therapeutic drug monitoring is conducted in 21 laboratories. There is almost no access to advanced diagnostic tests, like galactomannan, β-D-glucan, and PCR, in the surveyed laboratories in Indonesia, the Philippines, and Thailand. These results highlight the need for development of quality laboratories, accreditation and training of manpower in existing laboratories, and access to advanced non–culture-based diagnostic tests to facilitate the diagnosis of fungal infections in Asia

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

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

<i>Helicobacter pylori</i>, Protected from Antibiotics and Stresses Inside <i>Candida albicans</i> Vacuoles, Cause Gastritis in Mice

Due to (i) the simultaneous presence of Helicobacter pylori (ulcer-induced bacteria) and Candida albicans in the stomach and (ii) the possibility of prokaryotic–eukaryotic endosymbiosis (intravacuolar H. pylori in the yeast cells) under stresses, we tested this symbiosis in vitro and in vivo. To that end, intravacuolar H. pylori were induced by the co-incubation of C. albicans with H. pylori under several stresses (acidic pH, non-H. pylori-enrichment media, and aerobic environments); the results were detectable by direct microscopy (wet mount) and real-time polymerase chain reaction (PCR). Indeed, intravacuolar H. pylori were predominant under all stresses, especially the lower pH level (pH 2–3). Interestingly, the H. pylori (an amoxicillin-sensitive strain) inside C. albicans were protected from the antibiotic (amoxicillin), while extracellular H. pylori were neutralizable, as indicated by the culture. In parallel, the oral administration of intravacuolar H. pylori in mice caused H. pylori colonization in the stomach resulting in gastritis, as indicated by gastric histopathology and tissue cytokines, similar to the administration of free H. pylori (extra-Candida bacteria). In conclusion, Candida protected H. pylori from stresses and antibiotics, and the intravacuolar H. pylori were able to be released from the yeast cells, causing gastric inflammation with neutrophil accumulations

Clinicopathological features and outcomes of pythiosis

Objectives: Vascular pythiosis is a life-threatening infection caused by the oomycete Pythium insidiosum. This article reports the clinical presentation, serodiagnosis, pathology, and outcomes seen at the authors’ institution. Methods: The cases of patients with proven vascular pythiosis at Ramathibodi Hospital, Mahidol University, Bangkok, Thailand from January 2006 to December 2016 were analyzed retrospectively. Results: Thirteen patients were analyzed, eight of whom had underlying thalassemias. Of the remaining five patients, one had aplastic anemia, one had myelodysplasia, one had acute leukemia, one had cirrhosis, and one had alcoholism. Neutropenic patients showed a rapid clinical deterioration. Atypical presentations including carotid arteritis, aneurysm, brain abscess, and stroke occurred in the non-thalassemic patients. Serology yielded positive results in all cases, with a rapid turnaround time. Serology has the advantage of providing a presurgical diagnosis, which allows prompt surgery and clinical cure to be achieved. Pathology revealed a neutrophilic response in the acute phase and a later shift to granuloma. Immunotherapy in combination with itraconazole and terbinafine was given. The amputation rate was 77%, and disease-free surgical margins were achieved in five cases (38%). The mortality rate was 31%. Conclusions: This study highlights new aspects of pythiosis, such as the unusual host, clinical presentation, serology as a marker for rapid diagnosis, histopathology, and outcomes. Early recognition of the disease with prompt multimodality treatment may improve survival

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

Cutaneous blastomycosis and dermatophytic pseudomycetoma in a Persian cat from Bangkok, Thailand

This is a case report of concurrent of blastomycosis and pseudomycetoma in a 3 year-old Persian cat from Bangkok, Thailand. Histopathology from antemortem and postmortem samples revealed blastomycosis and dermatophyte pseudomycetoma. The PCR analysis of the formalin-embedded tissue of antemortem sample confirmed that blastomycosis was caused by Blastomyces dermatitidis. Dermatophyte infection was caused by Microsporum canis. According to the author's knowledge, this is the first case of Blastomyces dermatitidis and dermatophyte pseudomycetoma in South-East Asia

Antibodies in the Sera of Host Species with Pythiosis Recognize a Variety of Unique Immunogens in Geographically Divergent Pythium insidiosum Strains▿

Studies by Western blot analyses have shown that antibodies in the sera of host species infected by Pythium insidiosum recognized several prominent proteins expressed by this fungus-like pathogen. Although these studies have utilized sera from infected patients and relevant local strains of P. insidiosum, the results are difficult to compare because of the lack of method standardization. In an effort to resolve this issue, we have utilized standardized methodologies to evaluate six P. insidiosum strains from Asia and the Americas and 15 serum samples from cattle, cats, dogs, horses, and humans with pythiosis from the same geographical regions. Our data show that the antibodies present in these sera recognize a wide variety of unique P. insidiosum immunogenic proteins. Although some of the prominent proteins in this study have been previously reported, several others have yet to be described. For instance, a ∼28-kDa-molecular-mass antigen was detected by the antibodies in all serum samples evaluated. However, this antigen was strongly expressed by only one of the strains evaluated. A diffuse ∼51-kDa protein was not detected by the antibodies in the human sera; but it was recognized by the antibodies in the sera of cattle, cats, dogs, and horses. This antigen was expressed by only two of the strains investigated. Several other similar examples were also observed. The variation of the P. insidiosum protein profile identified by the antibodies in the sera evaluated indicates that some geographically diverged P. insidosum strains expressed some unique immunogens in vitro and that during natural infection (in vivo) P. insidiosum might express a broader number of antigens variably detected by individuals within the same species but especially across species