Effects of myofibril-palatinose conjugate as a phosphate substitute on meat emulsion quality.

The objective of this study was to investigate a replacement for phosphate in meat products. Protein structural modification was employed in this study, and grafted myofibrillar protein (MP) with palatinose was added to meat emulsion without phosphate. Here, 0.15% of sodium polyphosphate (SPP) was replaced by the same (0.15%) concentration and double (0.3%) the concentration of grafted MP. Although the thermal stability was decreased, the addition of transglutaminase could increase stability. The rheological properties and pH also increased with the addition of grafted MP and transglutaminase. The addition of grafted protein could be perceived by the naked eye by observing a color difference before cooking, but it was not easy to detect after cooking. The cooking loss, emulsion stability, water holding capacity, lipid oxidation, and textural properties improved with the addition of grafted MP. However, the excessive addition of grafted MP and transglutaminase was not recommended to produce a high quality of phosphate replaced meat emulsion, and 0.15% was identified as a suitable addition ratio of grafted MP.

Exploring the vaccine-induced immunity against severe acute respiratory syndrome coronavirus 2 in healthcare workers.

We aimed to analyze the kinetics of T-cell-mediated and B-cell-mediated humoral immune responses against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) before and after booster vaccination, as well as the impacts of the in vitro test results the type of vaccination on the prediction of SARS-CoV-2 infection. A total of 240 healthcare workers vaccinated twice were serially tested using an interferon gamma release assay (IGRA) and a neutralizing antibody (nAb). At the end of the study, we investigated the history of SARS-CoV-2 infection of all the enrolled participants to analyze the effects of the test results and the type of vaccination on SARS-CoV-2 infection. Overall, the positive rates were 52.3% and 80.0% for IGRA and 84.6% and 100% for the nAb test before and after booster vaccination, respectively. However, the positive rates were 52.8% for IGRA and 100% for nAb 3 months after booster vaccination. The in vitro test results and the type of vaccination were not associated with SARS-CoV-2 infection. The antibody response caused by the SARS-CoV-2 vaccination lasted more than 6 months, although the response of the T-cells disappeared rapidly after 3 months. However, these in vitro results and the type of vaccination cannot be used for predicting the risk of SARS-CoV-2 infection.

Performance evaluation of newly developed surrogate virus neutralization tests for detecting neutralizing antibodies against SARS-CoV-2.

We evaluated newly developed surrogate virus neutralization tests (sVNT) for detecting neutralizing antibodies (NAbs) against the receptor binding domain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). VERI-Q SARS-CoV-2 Neutralizing Antibody Detection ELISA Kit (MiCo BioMed, Gyeonggi-do, Republic of Korea, hereafter, "eCoV-CN") is an enzyme-linked immunosorbent assay-based sVNT, and VERI-Q SARS-CoV-2 Neutralizing Antibody Rapid Test Kit (MiCo BioMed, hereafter, "rCoV-RN") is a point-of-care lateral-flow immunochromatography test with auto-scanner. A total of 411 serum samples were evaluated. Both evaluations used a 50% plaque reduction neutralization test (PRNT50) as the gold standard. Compared with PRNT50, the eCoV-CN showed 98.7% positive percent agreement (PPA), 96.8% negative percent agreement (NPA), 97.4% total percent agreement (TPA), with kappa values of 0.942. The rCoV-RN showed 98.7% PPA, 97.4% NPA, 97.8% TPA, and kappa values of 0.951, comparing to PRNT50. Neither assay indicated cross-reactivity for other pathogens, and the signal indexes were statistically significantly correlated to the PRNT50 titer. The two evaluated sVNTs show comparable performances to the PRNT50 with the advantages of technical simplicity, speed, and do not require cell culture facilities.

Evaluation of ichroma™ COVID-19 interferon gamma release assay for detection of vaccine-induced immunity in healthcare workers.

OBJECTIVES: We compared the performance of a new interferon gamma release assay (IGRA) format assay, the ichroma™ COVID-19 IGRA (IGRA-SARS), with that of the widely used QuantiFERON SARS-CoV-2 ELISA kit (QFN-SARS) in vaccinated healthcare workers (HCWs). Additionally, we analyzed the long-term changes in IGRA results after the final vaccine dose. METHODS: A total of 383 specimens from 281 HCWs were enrolled in this study, and the results of SARS-IGRA and QFN-SARS assays were compared. In addition, we performed the receive operator curve analysis to estimate the optimal cut-off value for IGRA-SARS. RESULTS: For all specimens, IGRA-SARS and QFN-SARS showed 75.7% and 64.2% of the positive results, respectively. The absolute agreement between IGRA-SARS and QFN-SARS was 80.0%, and the Fleiss' κ value was 0.525, indicating moderate agreement. ROC curve analysis of the IGRA-SARS results showed a cut-off value of >0.254 IU/mL, which was consistent with the manufacturer's specifications. The positive rates of both IGRA assays decreased significantly after a postvaccination period of 6 months. CONCLUSIONS: IGRA-SARS showed acceptable performance in the detection of vaccine-induced immunity against COVID-19; however, harmonization of IGRA assays has not yet been achieved. Additionally, the significant decline of positive rates of IGRA after the last vaccination would support the necessity of booster vaccination after a postvaccination period of 6 months.

Performance Evaluation of Bruker Biotyper, ASTA MicroIDSys, and VITEK-MS and Three Extraction Methods for Filamentous Fungal Identification in Clinical Laboratories.

Filamentous fungi are a major cause of life-threatening infections in immunocompromised patients; thus, rapid and accurate identification is critical. Filamentous fungal identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been demonstrated with high sensitivity and reproducibility; however, its wider application has been limited in clinical laboratories because of practical challenges such as database availability or lack of standardization. In this study, we compared the performance of the Bruker Biotyper, ASTA MicroIDSys, and Vitek MS for 84 clinical filamentous fungal isolates. Moreover, the sensitivity of three independent sample preparation methods (direct, on plate, in tube) was compared. Bruker Biotyper identified 71.43% (60/84) of isolates correctly (species, genus, or complex/group level). ASTA MicroIDSys and Vitek MS showed accuracy rates of 70.24% (59/84) and 55.95% (47/84), respectively. We found that any difference in sensitivity may be attributed to the database of the systems. In addition, the "in tube" method showed the highest sensitivity among the three methods; however, there was no statistical difference among them. For the broader application of MALDI-TOF MS for filamentous fungal identification, further studies from multiple perspectives are required.

Comparative Analysis of Cutaneous Fungi in Atopic Dermatitis Patients and Healthy Individuals.

Background: Atopic dermatitis (AD) is a chronic relapsing inflammatory skin disease triggered by diverse factors. Microbes are one of the crucial risk factors for AD development or exacerbation. However, the effect of a fungal burden on AD has been overlooked compared to bacteria. Objective: This study aimed to comparatively analyze cutaneous fungal distribution between AD patients and healthy individuals by polymerase chain reaction (PCR)-based analysis. Methods: Skin samples of AD outpatients and healthy individuals collected at the Chung-Ang University were analyzed. Representative AD-associated fungal genera, Candida, dermatophytes, and Malassezia , were analyzed using specific primer and amplification methods. Amplicons were sequenced, and the fungal distribution of both groups were compared. Results: Totally, 211 patients and 23 healthy individuals were studied. Of the 211 patients, 10.90% (23/211) had Candida species, whereas 0% (0/23) healthy individuals showed its presence. The most frequently detected species in patients was Candida albicans (5.21%) followed by Candida parapsilosis (3.79%). For dermatophytes, 1.42% (3/211) of patients showed positive results, whereas 0% (0/23) healthy individuals showed positive results. Malassezia species were identified in 20.85% (44/211) and 8.70% (2/23) in patients and healthy individuals, respectively. Malassezia restricta was the most frequently identified species in the AD patient group, and the only species found in the healthy control group. Conclusion: The distribution of Candida spp., dermatophytes, and Malassezia spp. are altered with AD development.

Oxidative Stress Causes Vacuolar Fragmentation in the Human Fungal Pathogen Cryptococcus neoformans.

Vacuoles are dynamic cellular organelles, and their morphology is altered by various stimuli or stresses. Vacuoles play an important role in the physiology and virulence of many fungal pathogens. For example, a Cryptococcus neoformans mutant deficient in vacuolar functions showed significantly reduced expression of virulence factors such as capsule and melanin synthesis and was avirulent in a mouse model of cryptococcosis. In the current study, we found significantly increased vacuolar fragmentation in the C. neoformans mutants lacking SOD1 or SOD2, which respectively encode Zn, Cu-superoxide dismutase and Mn-superoxide dismutase. The sod2 mutant showed a greater level of vacuole fragmentation than the sod1 mutant. We also observed that the vacuoles were highly fragmented when wild-type cells were grown in a medium containing high concentrations of iron, copper, or zinc. Moreover, elevated temperature and treatment with the antifungal drug fluconazole caused increased vacuolar fragmentation. These conditions also commonly cause an increase in the levels of intracellular reactive oxygen species in the fungus, suggesting that vacuoles are fragmented in response to oxidative stress. Furthermore, we observed that Sod2 is not only localized in mitochondria but also in the cytoplasm within phagocytosed C. neoformans cells, possibly due to copper or iron limitation.

Evaluation of rapid SARS-CoV-2 antigen tests, AFIAS COVID-19 Ag and ichroma COVID-19 Ag, with serial nasopharyngeal specimens from COVID-19 patients.

We evaluated the diagnostic accuracy of two newly developed, point-of-care, rapid antigen tests (RATs) for detecting SARS-CoV-2, the AFIAS COVID-19 Ag and the ichromaTM COVID-19 Ag, and investigated antigen kinetics. A total of 200 serially collected nasopharyngeal (NP) specimens from 38 COVID-19 patients and 122 specimens from negative controls were analyzed. Diagnostic sensitivity and specificity were assessed in comparison to molecular test results and subdivided according to targeted genes (E, RdRP, and N) and days post-symptom onset (PSO). For the kinetics evaluation, cut-off-indices from serial NP specimens were used according to the number of days PSO. Both RATs showed sensitivity of 91.3‒100% for specimens with cycle threshold (Ct) < 25. The specificity of AFIAS was 98.7‒98.9% and that of ichromaTM was 100.0%. The kappa values of AFIAS and ichromaTM for the molecular testing of specimens with Ct < 25 (RdRP) were 0.97 and 1.00, respectively. The sensitivity of AFIAS and ichromaTM for all genes was lower for specimens collected at 8‒14 PSO than for those collected before 7-days PSO. The kinetics profiles showed that antigen levels gradually decreased from ≤ 7-days PSO to > 22-days PSO. Both RATs showed excellent specificity and acceptable sensitivity for NP specimens with higher viral loads and for specimens collected within 7-days PSO. Hence, they have the potential to become useful tools for the early detection of SARS-CoV-2. However, because of concerns about false negativity, RATs should be used in conjunction with molecular tests.

Involvement of Mrs3/4 in Mitochondrial Iron Transport and Metabolism in Cryptococcus neoformans.

Mitochondria play a vital role in iron uptake and metabolism in pathogenic fungi, and also influence virulence and drug tolerance. However, the regulation of iron transport within the mitochondria of Cryptococcus neoformans, a causative agent of fungal meningoencephalitis in immunocompromised individuals, remains largely uncharacterized. In this study, we identified and functionally characterized Mrs3/4, a homolog of the Saccharomyces cerevisiae mitochondrial iron transporter, in C. neoformans var. grubii. A strain expressing an Mrs3/4-GFP fusion protein was generated, and the mitochondrial localization of the fusion protein was confirmed. Moreover, a mutant lacking the MRS3/4 gene was constructed; this mutant displayed significantly reduced mitochondrial iron and cellular heme accumulation. In addition, impaired mitochondrial iron-sulfur cluster metabolism and altered expression of genes required for iron uptake at the plasma membrane were observed in the mrs3/4 mutant, suggesting that Mrs3/4 is involved in iron import and metabolism in the mitochondria of C. neoformans. Using a murine model of cryptococcosis, we demonstrated that an mrs3/4 mutant is defective in survival and virulence. Taken together, our study suggests that Mrs3/4 is responsible for iron import in mitochondria and reveals a link between mitochondrial iron metabolism and the virulence of C. neoformans.

Genomic Tandem Quadruplication is Associated with Ketoconazole Resistance in Malassezia pachydermatis.

Malassezia pachydermatis is a commensal yeast found on the skin of dogs. However, M. pachydermatis is also considered an opportunistic pathogen and is associated with various canine skin diseases including otitis externa and atopic dermatitis, which usually require treatment using an azole antifungal drug, such as ketoconazole. In this study, we isolated a ketoconazole-resistant strain of M. pachydermatis, designated "KCTC 27587," from the external ear canal of a dog with otitis externa and analyzed its resistance mechanism. To understand the mechanism underlying ketoconazole resistance of the clinical isolate M. pachydermatis KCTC 27587, the whole genome of the yeast was sequenced using the PacBio platform and was compared with M. pachydermatis type strain CBS 1879. We found that a ~84-kb region in chromosome 4 of M. pachydermatis KCTC 27587 was tandemly quadruplicated. The quadruplicated region contains 52 protein coding genes, including the homologs of ERG4 and ERG11, whose overexpression is known to be associated with azole resistance. Our data suggest that the quadruplication of the ~84-kb region may be the cause of the ketoconazole resistance in M. pachydermatis KCTC 27587.