Prevalence of saliva immunoglobulin A antibodies reactive with severe acute respiratory syndrome coronavirus 2 among Japanese people unexposed to the virus.

While the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a threat to public health as the number of cases and COVID-19-related deaths are increasing worldwide, the incidence of the virus infection is extremely low in Japan compared with many other countries. To explain this uncommon phenomenon, we investigated the prevalence of naturally occurring ("natural") antibodies, focusing on those of the secretory immunoglobulin A (sIgA) form, reactive with SARS-CoV-2 among Japanese people. One hundred and eighty healthy Japanese volunteers of a wide range of age who had been considered to be unexposed to SARS-CoV-2 participated in this study. Saliva samples and blood samples were collected from all of the 180 participants and 139 adults (aged ≥ 20 years) included therein, respectively. The determination of saliva IgA antibodies, mostly comprising sIgA antibodies, as well as serum IgA and immunoglobulin G antibodies, reactive with the receptor binding domain of the SARS-CoV-2 spike-1 subunit proteins was conducted using an enzyme-linked immunosorbent assay. The major findings were that 52.78% (95% confidence interval, 45.21%-60.25%) of the individuals who had not been exposed to SARS-CoV-2 were positive for saliva IgA antibodies with a wide range of levels between 0.002 and 3.272 ng/mL, and that there may be a negative trend in positivity for the antibodies according to age. As we had expected, a frequent occurrence of assumable "natural" sIgA antibodies reactive with SARS-CoV-2 among the studied Japanese participant population was observed.

The Molecular Identification and Antifungal Susceptibilities of Aspergillus Species Causing Otomycosis in Tochigi, Japan.

Aspergillus species are the most common pathogenic fungi involved in otomycosis, an infection of the outer ear canal. In this study, we examined the incidence of Aspergillus infections and the antifungal susceptibilities of 30 Aspergillus species isolates from patients with otomycosis who visited Saiseikai Utsunomiya Hospital between August 2013 and July 2016. Based on the morphological test results, the strains were identified as Aspergillus niger sensu lato (20 strains), A. terreus sensu lato (7 strains), and A. fumigatus sensu lato (3 strains). In contrast, the molecular identifications based on analyzing the isolates' partial ß-tubulin gene sequences revealed them to be A. niger sensu stricto (12 strains), A. tubingensis (8 strains), A. terreus sensu stricto (7 strains), and A. fumigatus sensu stricto (3 strains). The antifungal susceptibility test results indicated that strains of A. tubingensis and A. niger sensu stricto displayed lower susceptibilities to ravuconazole, compared with the other isolates. The Aspergillus strains from this study showed low minimum inhibitory concentrations toward the azole-based drugs efinaconazole, lanoconazole, and luliconazole. Therefore, these topical therapeutic agents may be effective for the treatment of otomycosis.

How histopathology can contribute to an understanding of defense mechanisms against cryptococci.

Invasive fungal infections, particularly those considered opportunistic, have become a common and significant complication of procedures performed in advanced contemporary medicine. Among such infections, cryptococcosis, which is usually caused by infection with Cryptococcus neoformans and Cryptococcus gattii, is particularly problematic because this fungal infection occurs in immunocompromised and apparently immunocompetent individuals. It has been largely accepted that Cryptococcus species are recognized by cellular receptors and that Th1-type immune responses play an important role in defense mechanisms against the yeast. However, the interaction between the yeast and host tissue varies depending on the characteristics of the yeast and the immune status of the host. To gain a better understanding of the pathophysiology of cryptococcosis, we wish to emphasize the usefulness of histopathological examinations, because it allowed more detailed information of an extremely complex interaction between the causative yeasts and tissue response. In the present review, we describe the pathophysiology of cryptococcosis as largely revealed in our previous histopathological investigations of the experimental infection.

Short-term therapy with luliconazole, a novel topical antifungal imidazole, in guinea pig models of tinea corporis and tinea pedis.

Luliconazole is a novel topical antifungal imidazole with broad-spectrum and potent antifungal activity. The drug is under clinical development in the United States for management of dermatophytosis with a short-term treatment regimen. The present study was undertaken to investigate the clinical benefit of short-term therapy with luliconazole cream in guinea pig models of tinea corporis and tinea pedis induced with Trichophyton mentagrophytes. The dose-dependent therapeutic efficacy of topical luliconazole cream (0.02 to 1%), measured by macroscopic improvement of skin lesions and by fungal eradication as determined by a culture assay, was demonstrated using a tinea corporis model. The improvement in skin lesions seen with luliconazole cream was observed even at a concentration of 0.02%, and its efficacy at 0.1% was equal to that of 1% bifonazole cream. The efficacy of short-term therapy with 1% luliconazole cream, which is used for clinical management, was investigated using the tinea corporis model (4- and 8-day treatment regimens) and the tinea pedis model (7- and 14-day treatment regimens). The 1% luliconazole cream completely eradicated the fungus in half or less of the treatment time required for 1% terbinafine cream and 1% bifonazole cream, as determined by a culture assay for both models. These results clearly indicate that 1% luliconazole cream is sufficiently potent for short-term treatment for dermatophytosis compared to existing drugs. Luliconazole is expected to be useful in the clinical management of dermatophytosis.

Effect of a dietary supplement containing glucosamine hydrochloride, chondroitin sulfate and quercetin glycosides on symptomatic knee osteoarthritis: a randomized, double-blind, placebo-controlled study.

BACKGROUND: Oral glucosamine and chondroitin sulfate, alone and in combination, have been used worldwide for the treatment of osteoarthritis (OA), but their efficacy is controversial. This clinical study was aimed at investigating the potential of a dietary supplement containing glucosamine and chondroitin sulfate in combination with derivatives of quercetin, a naturally occurring flavonoid, (GCQ supplement) for knee OA care. RESULTS: A randomized, double-blind, placebo-controlled study was conducted in 40 Japanese subjects with symptomatic knee OA. Subjects were randomly assigned to GCQ supplement (1200 mg glucosamine hydrochloride, 60 mg chondroitin sulfate and 45 mg quercetin glycosides per day) or placebo and the treatment and follow-up were continued for 16 weeks. The results of symptomatic efficacy assessment based on Japanese Orthopaedic Association criteria showed that scores for two of the four symptom/function subscales, as well as the aggregate scores, were significantly improved at week 16 or earlier in the GCQ group compared to the placebo group. Moreover, analyses of cartilage metabolism biomarkers showed a trend of improvement in type II collagen synthesis/degradation balance in the GCQ group during follow-up. CONCLUSION: GCQ supplement was thought to be more effective than placebo in decreasing the intensity of knee OA-associated clinical symptoms.

Correlation of in vitro activity and in vivo efficacy of itraconazole intravenous and oral solubilized formulations by testing Candida strains with various itraconazole susceptibilities in a murine invasive infection.

OBJECTIVES: To examine whether in vitro antifungal susceptibility test results correlate with in vivo efficacy of two cyclodextrin-solubilized itraconazole formulations (intravenous and oral) against Candida in a murine model of invasive infection. METHODS: A selected set of 12 Candida spp. strains with various itraconazole susceptibilities were tested. We studied the efficacy of intravenous and oral itraconazole administered once daily at dosages of 0.63, 2.5, 10 and 40 mg/kg body weight in mice lethally infected with each tested strain. Survival of mice in each treated group was monitored daily until the death of all control mice and compared between groups. RESULTS: Survival of mice infected with 9 of 12 Candida strains with itraconazole MICs of ≤0.016-2.0 mg/L was significantly prolonged by treatment with intravenous itraconazole at dosages of 2.5 or 10 mg/kg and above. In contrast, the other three strains resistant to 8 mg/L itraconazole in vitro were refractory to the therapy, even at the highest itraconazole dosage (40 mg/kg). Closely similar in vivo data were obtained with the oral itraconazole therapy. The effective doses of the two itraconazole formulations increased with increasing itraconazole MICs for the infecting strains. CONCLUSIONS: The in vivo efficacy of intravenous and oral itraconazole correlated with the in vitro susceptibility data.

[Drug properties of fosravuconazole L-lysine ethanolate (NAILIN® Capsules 100†mg), a new oral azole therapeutic for onychomycosis: an analysis based on non-clinical and clinical trial data].

Ravuconazole is a fourth generation azole exerting strong antifungal activity, with low drug-drug interaction and hepatic dysfunction risks. Fosravuconazole l-lysine ethanolate (fosravuconazole; NAILIN® Capsules 100 mg) was developed as a ravuconazole prodrug. Ravuconazole exerts strong antifungal activity against various pathogenic fungi including dermatophytes and Candida. Through prodrug formation, pharmacokinetic improvement was achieved, and bioavailability after oral administration reached 100%. The plasma ravuconazole concentration became 10-35 times higher than with current oral anti-onychomycosis drugs, and showed good skin and nail tissue transition plus tissue retention. This improvement obtained with fosravuconazole reflects its superior pharmacokinetic properties. We conducted a clinical trial with fosravuconazole orally administered once a day (100 mg ravuconazole) for 12 weeks in Japanese onychomycosis patients. The ravuconazole concentration in nail tissues exceeded the MIC90 against dermatophytes, even after treatment completion. Furthermore, the placebo-controlled, double-blind, comparative trial showed significantly superior effects (at 48 weeks after starting treatment, with a complete cure rate of 59.4%, a marked clinical improvement rate of 83.1%, and a mycological cure rate by direct microscopy of 82.0%). The major adverse reactions were laboratory abnormalities and gastrointestinal disorders with no severe symptoms, suggesting good tolerability. Fosravuconazole has fewer drug-drug interactions, is not affected by food, and is also expected to improve medication adherence since the administration period is only 12 weeks and there is no drug-free period as required with pulse therapy. Thus, fosravuconazole has many favorable pharmacological properties and can reasonably be expected to become a new oral treatment option for onychomycosis.

Efficacy of Luliconazole Against Broad-Range Filamentous Fungi Including Fusarium solani Species Complex Causing Fungal Keratitis.

PURPOSE: Fungal keratitis can be difficult to medically treat. Topical antifungals are usually applied empirically as the initial option in treating fungal keratitis. Natamycin (NAT) and/or voriconazole (VRCZ) have been widely used in the treatment of fungal keratitis. However, Fusarium solani species complex (FSSC), which are the dominant species of fungal keratitis, are resistant to VRCZ. This study investigated in vitro efficacy of luliconazole (LLCZ), a new imidazole antifungal, against FSSC and other filamentous fungi. METHODS: A total of 18 Fusarium isolates and 7 others were grown on potato dextrose agar at 30 and 37°C. For Fusarium, species identification and phylogenetic tree analysis were performed based on elongation factor-1α (EF-1α) DNA sequencing. The broth microdilution method was used for antifungal susceptibility testing of 11 antifungal drugs including LLCZ. RESULTS: The 18 identified Fusarium isolates belonged to FSSC (n = 13), Fusarium oxysporum species complex (FOSC; n = 2), Fusarium chlamydosporum species complex (FCSC; n = 1), Fusarium incarnatum-equiseti species complex (FIESC; n = 1), and Fusarium fujikuroi species complex (FFSC; n = 1). We further divided 13 FSSC isolates into 3 clades, FSSC5 (n = 8), FSSC3 + 4 (n = 4), and FSSC9-a (n = 1), with 8 FSSC strains growing at 37°C. LLCZ showed lowest minimum inhibitory concentrations (MICs) against all tested filamentous fungi, with a MIC90 against the Fusarium species of 0.06 µg/mL, whereas MIC90 for NAT and VRCZ were 4 and 8 µg/mL, respectively. CONCLUSIONS: LLCZ has the strongest in vitro antifungal activity among all drugs used against broad-range filamentous fungi including FSSC. LLCZ may potentially be a new medical treatment option for fungal keratitis.

Histopathological study on experimental endophthalmitis induced by bloodstream infection with Candida albicans.

To investigate the details of the pathophysiology of endogenous fungal endophthalmitis (EFE), we performed sequential histological and ophthalmoscopic examination on a rabbit model comparing immunocompromised EFE developed using a steroid with an immunocompetent one intravenously inoculated with Candida albicans. The ophthalmoscopic examination and histological analysis of the retina in both groups demonstrated that lesions appear on the equator of the eyeball and then spread toward the posterior pole. It has been speculated that, because of the unique innate vasculature system of the equator, there is a sudden, decrease of shear stress in rheologically, resulting in adhesion of yeast cells to the endothelial cells. Histological examination revealed that the degree of polymorphonuclear leukocyte (PMN) infiltration was equivalent in the two groups. However, the appearance of PMN was delayed and the number of fungi was higher in the state of hyphae and/or pseudohyphae in the steroid-treated group. Furthermore, the eyeball was found to be the second earliest organ involved in candidemia. Our results indicate that ophthalmic examination is useful to monitor the development and systemic involvement of endophthalmitis in patients with candidemia.

Oral intake of soy isoflavone aglycone improves the aged skin of adult women.

A double-blind, placebo-controlled trial was conducted to clinically evaluate the effect of soy isoflavone aglycone on the aged skin of middle-aged women. Twenty-six women volunteers in their late 30s and early 40s were randomly assigned to receive either a test food (13 women, the test food group) or a placebo food (13 women, the control group). These groups were given the test food (40 mg of soy isoflavone aglycone per day) and placebo, respectively, for 12 wk. The extent of linear and fine wrinkles at the lateral angle of the eyes was selected as the major evaluation criterion to assess the effects of foods, and the wrinkles' area ratio was used as the evaluation parameter. The extent of skin microrelief at the lateral angle of eyes and that of malar skin elasticity were used as secondary evaluation criteria, and the skin microrelief's area ratio and recovery of skin elasticity were used as the respective evaluation parameters. These parameters were assessed 4, 8, and 12 wk after the start of the test food or placebo intake. The test food group showed a statistically significant improvement of fine wrinkles at week 12 (p<0.05) and of malar skin elasticity at week 8 (p<0.05), compared with the control group. Although there was no significant difference between the test food group and the control group regarding effects on skin microrelief at lateral angle of the eyes, a significant intragroup improvement was observed at week 8 in the test food group (p<0.05). As for the test food safety, none of the subjects presented adverse symptoms during the study period or discontinued the intake of the test food. These data indicate that the oral intake of 40 mg soy isoflavone aglycones per day improves the aged skin of middle-aged women.

Combined effect of heat, essential oils and salt on fungicidal activity against Trichophyton mentagrophytes in a foot bath.

This work was originally undertaken to determine the effective conditions of essential oils against Trichophyton mentagrophytes in vitro for the treatment of tinea pedis in a foot bath. Agar blocks implanted with T. mentagrophytes were immersed in 0.1% aqueous agar containing two-fold dilutions of essential oils with or without sodium chloride at 27 degrees C, 37 degrees C and 42 degrees C for 10 and 20 min. The number of surviving mycelia on the agar blocks was determined from the standard curves of the colony diameter and original inocula of the conidia. At the same time, the thermal effect on the cellular morphology was examined using SEM. Most fungal mycelia (99.7%) were killed after treatment at 42 degrees C for 20 min without essential oil. The fungicidal activity of essential oils was markedly enhanced by treating at 42 degrees C for 20 min as compared with that at 27 degrees C, showing 1/4 - 1/32-fold reduction of minimum fungicidal concentration (MFC to kill 99.99%). The order of the fungicidal activity of 11 essential oils was oregano, thyme thymol, cinnamon bark > lemongrass > clove, palmarose, peppermint, lavender > geranium Bourbon, tea tree > thyme geraniol oils. MFCs were further reduced to 1/2 - 1/8 by the addition of 10% sodium chloride. The salt effect was explained, at least partly, by an increase in mycelial adsorption of antifungal constituents in the presence of sodium chloride. Considerable hyphal damage was done at 27 degrees C by the essential oils, but no further alteration in morphology of the hyphae treated at 42 degrees C with or without oil was observed by SEM. The inhibitory effect of heat and oils was also observed against mycelia of T. rubrum and conidia of T. mentagrophytes. Thermotherapy combined with essential oils and salt would be promising to treat tinea pedis in a foot bath.

Rapid identification of Trichophyton tonsurans by specific PCR based on DNA sequences of nuclear ribosomal internal transcribed spacer (ITS) 1 region.

BACKGROUND: Trichophyton tonsurans, a dermatophyte implicated in an international epidemic of tinea capitis, was also found to be responsible for infecting wrestling and Judo athletes nationwide in Japan since 2001. OBJECTIVE: A rapid and highly accurate means of identifying this pathogen has been required to control the infection. We have developed a T. tonsurans-specific PCR method based on the DNA sequences of the nuclear ribosomal internal transcribed spacer 1 region. SUBJECTS: Eighteen species of six genera of standard strains and 75 strains of clinically isolated Trichophyton species were used in this study. METHODS: A T. tonsurans-specific PCR primer pair (tonsF1 and tonsR1) was designed on the nuclear ribosomal internal transcribed spacer 1 region, located between 18S and 5.8S rDNA. Fungal DNA was extracted from the colonies grown on culture plates, and the specificity of the PCR primers was tested. RESULTS: The specific PCR product was amplified from the standard strain of T. tonsurans and from five strains isolated from black dot ringworms, but there was no band from the 70 clinical isolates of other Trichophyton species. This T. tonsurans-specific PCR method was able to detect 10 pg of T. tonsurans genomic DNA with ethidium bromide staining. CONCLUSIONS: A PCR identification system specific for T. tonsurans is rapid, sensitive, and specific.

[Studies on the correlation of the method currently used by the Japanese Society for Medical Mycology and its modified method with the NCCLS M27-A2 microdilution method for azole susceptibility testing of Candida species].

To evaluate the currently used Japanese Society for Medical Mycology (JSMM) method for testing the azole susceptibility of yeasts, the activities of fluconazole and itraconazole were tested against recently collected clinical isolates of Candida spp. (n=946) and compared with the National Committee for Clinical Laboratory Standards (NCCLS) M27-A2 microdilution reference method. Favorable correlation with the M27-A2 method was not seen for isolates of C. albicans, C. tropicalis or other Candida spp., particularly their trailing-growth isolates. However, the degree of correlation and agreement of MIC values were markedly improved when testing was performed by the modified JSMM method in which the end-point to be read was changed from IC80 (for the current JSMM method) to IC50. These results suggest that there is an urgent need to revise the current JSMM method.

A one-enzyme PCR-RFLP assay for identification of six medically important Candida species.

Early identification of Candida isolates to the species level is necessary for effective antifungal therapy, and can also facilitate control of hospital infections. Phenotype-based methods for identifying Candida species are often difficult and time-consuming. Molecular biological techniques provide a useful alternative approach. In the present study, the ITS1-5.8S-ITS2 regions of fungal rRNA genes were amplified with universal primers in 20 standard strains. Digestion of the PCR products with one restriction enzyme, MspI, allowed discrimination of medically important Candida species, including C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. krusei, and C. guilliermondii. Using this method, we successfully identified 137 clinical isolates of Candida. Among them, C. albicans was identified as the most common species, followed by C. parapsilosis, C. tropicalis, C. glabrata, C. krusei, and C. guilliermondii. This method is a simple, rapid, and cost-effective method for differentiation between species that is applicable in clinical laboratories.

Development of safety culture tube for molds and proposed procedure for collecting conidia or fixing strains to control fungal infection and allergy.

The conidia of filamentous fungi can be easily blown into the air and tend to be contaminants in the laboratory environment. We developed a new "safety culture tube for fungi" to prevent biohazards and a procedure for collecting conidia for passage or fixing strains was proposed.

A novel method to estimate the contribution of the vapor activity of essential oils in agar diffusion assay.

By the combined use of agar diffusion, agar vapor and agar vapor-inhibitory assays, contribution of the vapor activity of essential oils was quantitatively estimated. The test organisms were Trichophyton mentagrophytes and Aspergillus fumigatus. Agar vapor assay was used to confirm the vapor activity of the oils. The parameter delta defined as a contribution index of the vapor activity was calculated by (1 - b-c/a-c) x 100, where a is inhibitory diameter in the diffusion assay, b is inhibitory diameter in the vapor-inhibitory assay and c is diameter of the sealed ring in the vapor-inhibitory assay (21 mm). Many of the essential oils examined showed a delta value near 100, thus providing the major contribution of the vapor activity to the inhibitory diameter. Essential oils containing aldehyde as major constituent showed low delta value, indicating the major inhibition was due to agar diffusion. Major essential oil components behaved similarly; the delta value was increased in the following order: aldehyde < phenol < alcohol < ester, oxide, hydrocarbon, indicating the enhanced contribution of the vapor activity in that order.

Potential of Ravuconazole and its Prodrugs as the New OralTherapeutics for Onychomycosis.

Onychomycosis is a fungal infection of the nail apparatus caused by dermatophytes, Candida and non-dermatophytic molds. It is highly prevalent in the general population worldwide and also responsible for significant morbidity and complications and does not usually cure itself. Thus, the condition needs to be treated in view of physical and psychological problems produced. Currently, oral medications using terbinafine are the most effective therapy, but it has relatively limited therapeutic success, particularly for long-term management. Such existing oral therapies are associated with high recurrence rates and treatment failure, as well as with potential adverse events and drug-drug interactions. In the light of these issues, development of more efficacious and safer alternatives for the treatment of onychomycosis is warranted.Ravuconazole and its prodrugs are promising new drug candidates for oral therapy of onychomycosis, among which a water-soluble prodrug, mono-lysine phosphoester derivative (E1224 or BFE1224) is in the most advanced stage of clinical development; a Phase II dose-finding study has been successfully completed and Phase III comparative studies are in progress in Japan.This review aims to summarize our current status of knowledge and information on ravuconazole and its prodrugs, particularly BFE1224, as the potential oral treatment option for onychomycosis. It also summarize the clinical features of onychomycosis with particular stress on its etiology, epidemiology, and current therapeutic options and their limitations. Given its clinical usefulness, BFE1224 may become a valuable addition to the current armamentarium for the treatment of onychomycosis.

A simple PCR-RFLP method for identification and differentiation of 11 Malassezia species.

A PCR-RFLP method targeted toward 26S rDNA and with 2 restriction enzymes, CfoI and BstF51, was developed to identify 11 Malassezia species. Not only type and standard strains but also 13 clinical isolates were identified successfully in this study. The results of identifications were confirmed by DNA sequencing.

Metalloprotease gene of Arthroderma gypseum.

The full-length cDNA sequence for metalloprotease (MEP) of Arthroderma gypseum (one of the teleomorphs of the Microsporum gypseum complex) was determined by the 5'-rapid amplification of cDNA ends (RACE) and 3'-RACE methods using cDNA as a template. The full-length cDNA sequence of the MEP (2,670 bp) gene was proved to encode 677 amino acids. The amino acid sequence of the A. gypseum MEP gene shared about 89 and 66% sequence similarity with the conserved region of the Microsporum canis MEP gene and Aspergillus fumigatus, respectively. Southern hybridization analysis of genomic DNA with an MEP probe gave many distinct bands in BamHI, EcoRI and HindIII digests of genomic DNA from A. gypseum. Reverse transcriptase-PCR analysis suggested that keratin might stimulate the expression of MEP mRNA in A. gypseum.

Differentiation of Candida albicans and Candida dubliniensis using a single-enzyme PCR-RFLP method.

Candida dubliniensis is a novel Candida spp. that is similar to Candida albicans with respect to several phenotypic characteristics. However, they differ from each other with respect to epidemiology, pathogenesis, and the rapid development of resistance to fluconazole. In the present study, we used a single-enzyme PCR-restriction fragment length polymorphism (RFLP) technique to differentiate C. dubliniensis from C. albicans. The amplified ITS region of C. dubliniensis was digested once using the enzyme B1nI, whereas that of C. albicans remained intact. All standard strains tested were identified successfully by this method. None of 140 clinical isolates identified morphologically as C. albicans were recognized as C. dubliniensis based on their PCR-RFLP pattern. Our PCR-RFLP method easily differentiated C. dubliniensis from C. albicans, and this result was also demonstrated with standard strains.