Rhizopus Arrhizus Research Articles

First Report of Rhizopus arrhizus (syn. R. oryzae) Causing Garlic Bulb Soft Rot in Hebei Province, China.

Rhizopus soft rot occurs on the succulent tissues of vegetables, fruits, and ornamental plants throughout the world (Cui et al. 2019). When the garlic is in the seedling stage in the fields (Fig. S1) in November 2021, a disease outbreak on garlic bulbs suspected as Rhizopus soft rot occurred in Daming County, Handan City, Hebei Province of China (N 36°17', E 115° 13'). This disease symptom was first found in the garlic seedling stage in China. Disease incidence was 10% to 30% in cultivated garlic bulbs. There were soft water-soaked lesions on the surface of diseased garlic bulbs and the interiors were brown and soft. In the disease severe field, white to gray mycelia were observed on the diseased garlic bulbs. Infected garlic bulbs were sampled to isolate and determine the identity of the disease-causing organism. Symptomatic bulbs were surface sterilized with 1% NaClO for 2 min, dipped in 75% ethanol for 3 min and rinsed three times with autoclaved distilled water. Small pieces of the inner decayed tissue were removed and cultured on potato dextrose agar (PDA) at 28°C for 2 to 3 days. Five white colonies grew on PDA and then they became brownish gray to blackish-gray mycelium. The fungal strains were purified by hyphal-tip isolation method. To determine the identity of the five isolated fungi, we analyzed their internal transcribed spacer (ITS) region sequences (Jung et al. 2012). BLAST analysis of the ITS sequences from DSF-0-2 (accession no. ON706022), DSF-0-3 (accession no. ON706021), DSF-0-4 (accession no. ON706020), DSF-0-5 (accession no. ON706019) and DSF-0-6 (accession no. ON706018) were all 100% identical with Rhizopus arrhizus (syn. Rhizopus oryzae). Phylogenetic trees were constructed using the neighbor-joining method of MEGA11 based on the sequences of ITS rRNA gene (Walther et al. 2013). Phylogenetic trees indicated that isolates were most likely Rhizopus arrhizus (syn. Rhizopus oryzae) (Fig. S2). We selected one isolated strain, DSF-0-2, for characterize the morphology and test its ability to cause garlic bulb soft rot. Under the microscope, nonseptate rhizoids, sporangia, and sporangiospores were observed (Fig. S1). Sporangiospores were unequal, subglobose, numerous irregular, or oval, and 9.7 (6.2 - 12.5) × 6.5 (4.1 - 8.5) μm (n = 50) in diameter. The sporangia were globose, black, 121.5 (65 - 198) μm (n = 50) in diameter. Based on the rDNA-ITS sequencing and the morphological characteristics, the DSF-0-2 isolate was identified as Rhizopus arrhizus (syn. Rhizopus oryzae) (Zheng et al. 2007; Abeywickrama et al. 2020). To complete Koch's postulates, surface-sterilized healthy garlic bulbs were inoculated with R. arrhizus isolate DSF-0-2. A 1.0-ml sterile syringe was used to inject 50 μl of a 106 conidia/ml suspension into each of five healthy bulbs. As a control, garlic bulbs were treated with sterile distilled water. The inoculated and control bulbs were incubated at 28°C for 7 days. The bulbs inoculated with R. arrhizus DSF-0-2 showed symptoms of water soaking, and the tissues were brown and soft throughout the bulb at 7 days (Fig. S1). Results of the three trials were the same. No symptoms were observed in the control group. R. arrhizus was reisolated from the symptomatic garlic bulb and confirmed as such based-on colony and sporangia morphology and ITS sequence. There were some reports that R. arrhizus infects cassava tubers and potato tubers (Amadioha and Markson 2007; Cui et al. 2019). To our knowledge, this is the first report of R. arrhizus (syn. Rhizopus oryzae) associated with soft rot on garlic bulb in the seedling stage in China. This disease has posed a potential threat during garlic seedling stage in the field. Management measures should be considered before this disease outbreaks widely. Garlic bulbs died in the seedling stage, which caused production reduction, serious economic loss and soil pollution. This finding may help to take effective control measures for this disease.

Microbiology Profile of COVID-19-Associated Rhino-Orbital Mucormycosis Pathogens in South India.

This study describes the microbiological and histopathological features of patients with COVID-19-associated rhino-orbital mucormycosis (ROM) seen at the L V Prasad Eye Institute between May and August 2021. Diagnosed clinically and radiologically, 24 patients with ROM were included in the study. Deep nasal swabs or endoscopically collected nasal swabs or orbital tissues were submitted for microbiological evaluation and invitro susceptibility testing by microbroth dilution for natamycin, amphotericin B, caspofungin, posaconazole, ketoconazole, and voriconazole. Cultures were processed by 28S ribosomal DNA polymerase chain reaction and molecular sequencing. A portion of orbital tissues was also sent for histopathological evaluation. The age of the patients ranged from 27 to 75 (mean 48.58 ± 14.09) years and the majority (79%) were male. Nineteen patients were known to be diabetic prior to developing ROM and 18 patients had recovered from active COVID-19 infection. Thirteen patients had a history of hospitalization during COVID-19 infection and eight received steroids. Of the 24 samples, microbiological evaluation identified Rhizopus arrhizus in 12, Rhizopus microsporus in 9, Lichtheimia ramosa in 2, and Rhizopus delemar in 1. Twelve isolates were tested for antifungal susceptibility and all were susceptible to natamycin and amphotericin B. The susceptibility to posaconazole was high, with minimum inhibitory concentration (MIC) < 2 µg/mL for 10/12 (84%) isolates, whereas the MIC of other drugs varied. Histopathological examination of tissues showed acute fulminant disease, granuloma formation, and vascular invasion by the fungal pathogens in these specimens. Rhizopus arrhizus was predominantly associated with ROM and most isolates were susceptible to amphotericin B and posaconazole. Further studies are needed to corroborate the findings and explain possible underlying links.

Fungal Biomarkers in Traditional Starter Determine the Chemical Characteristics of Turbid Rice Wine from the Rim of the Sichuan Basin, China.

The fungal community in Qu plays a key role in the formation of turbid rice wine (TRW) style. The Sichuan Basin and its surrounding areas have become one of the main TRW production regions in China; however, the fungal community in Qu and how they affect the characteristics of TRW remain unknown. Therefore, this study provided insight into the fungal biomarkers in Qu from Guang'an (GQ), Dazhou (DQ), Aba (AQ), and Liangshan (LQ), as well as their relationships with compounds in TRW. The main biomarkers in GQ were Rhizopus arrhizus, Candida glabrata, Rhizomucor pusillus, Thermomyces lanuginosus and Wallemia sebi. However, they changed to Saccharomycopsis fibuligera and Mucor indicus in DQ, Lichtheimia ramose in AQ, and Rhizopus microsporus and Saccharomyces cerevisiae in LQ. As a response to fungal biomarkers, the reducing sugar, ethanol, organic acids, and volatile compounds were also changed markedly in TRWs. Among important volatile compounds (VIP > 1.00), phenethyl alcohol (14.1-29.4%) was dominant in TRWs. Meanwhile, 3-methyl-1-butanol (20.6-56.5%) was dominant in all TRWs except that fermented by GQ (GW). Acetic acid (29.4%) and ethyl palmitate (10.1%) were dominant in GW and LW, respectively. Moreover, GQ biomarkers were positively correlated with acetic acid and all unique important volatile compounds in GW. DQ biomarkers had positive correlations with unique compounds of acetoin and ethyl 5-chloro-1,3,4-thiadiazole-2-carboxylate in DW. Meanwhile, the AQ biomarkers were positively correlated with all AW unique, important, and volatile compounds. Although there were not any unique volatile compounds in LW, 16 important volatile compounds in LW were positively related to LQ biomarkers. Obviously, biomarkers in different geographic Qu played vital roles in the formation of important volatile compounds, which could contribute specific flavor to TRWs. This study provided a scientific understanding for future efforts to promote the excellent characteristics of TRW by regulating beneficial fungal communities.

Hyalohyphomycete or zygomycete? Challenges with microscopic diagnosis

Background: Invasive mould infections are associated with profound morbidity. Direct microscopic examination of specimens is a useful tool for laboratories to advise clinicians on the presumptive mould identity. Nonetheless, pitfalls abound. Here, we highlight how microscopy may not always be straightforward. Case description: A 69-year-old male with T cell lymphoma was admitted for chemotherapy. Shortly after conditioning, he developed hand cellulitis which progressed to a 10 cm necrotic ulcer despite vancomycin and piperacillin-tazobactam. Skin biopsy was performed. Direct Gram stain and calcofluor white stain revealed narrow hyphae with suggestion of septa and 45 degree branching. Given the clinical syndrome (eschar in an immunocompromised host), we communicated our suspicion of Fusarium to the clinician, who started voriconazole. Overnight, a cottony mould grew. Lactophenol cotton blue stain revealed irregular, broad, ribbon-like hyphae with sporangia. The clinician was informed of the possibility of Mucorales. Antifungals were switched to liposomal amphotericin B. Internal transcribed spacer sequencing subsequently confirmed the mould as Rhizopus arrhizus. Antifungal susceptibility testing revealed MICs of 1 μg/ml and >8 μg/ml to amphotericin B and voriconazole respectively. Discussion: Direct microscopy findings should always be correlated with colonial morphology. Clinicians should be promptly informed of critical findings so appropriate treatment can be instituted.

Complement, but Not Platelets, Plays a Pivotal Role in the Outcome of Mucormycosis In Vivo

Mucormycetes, a heterogeneous group of fungi, induce a life-threatening disease called mucormycosis. Immune deficiencies represent a major risk factor; hence, we wanted to illuminate the role of complement and platelets in the defense against mucormycetes. Rhizopus arrhizus (Ra), Rhizopus microsporus (Rm), Lichtheimia ramosa (Lr), Lichtheimia corymbifera (Lc), Rhizomucor pusillus (Rmp), and Mucor circinelloides (Mc) spores were opsonized with human and mouse serum, and C1q, C3c, and terminal complement complex (C5b-9) deposition was measured. Additionally, thrombocytopenic, C3-deficient, or C6-deficient mice were intravenously infected with selected isolates. Survival and immunological parameters were monitored, and fungal burden was determined and compared to that of immunocompetent and neutropenic mice. In vitro experiments showed significant differences in complement deposition between mucormycetes. Mc isolates bound up to threefold more human C5b-9 than other mucormycetes. Lr, Lc, and Mc bound high levels of murine C3c, whereas human C3c deposition was reduced on Mc compared to Lr and Lc. Murine C3c deposition negatively correlated with virulence. Complement deficiencies and neutropenia, but not thrombocytopenia, were shown to be a risk factor for a lethal outcome. Complement deposition varies between mucormycetes. Additionally, we demonstrated that complement and neutrophilic granulocytes, but not platelets, play an important role in a murine model of disseminated mucormycosis.

Processing Mechanism of Massa Medicata Fermentata Based on the Correlation Analysis of Strains, Chemical Compositions and Anti-Inflammatory Activity.

The traditional Chinese medicine of fermented medicine may be under the involvement of multiple strains and the interaction between these microorganisms. Liu Shenqu (Massa Medicata Fermentata, MMF) is one of the most widely used fermented medicines, whose potential processing mechanism is still unclear. In this work, UPLC/MS and GNPS methods were employed to rapidly predict chemical compositions in MMF. Moreover, the dynamic changes of strains, chemical compositions and anti-inflammatory activity of MMF during fermentation process were investigated, and subsequently strains-chemical compositions-efficacy interactions were revealed by Pearson correlation analysis and partial least squares regression (PLSR) analysis. As a result, 24 components were identified, and the potential strains including Bacillus, Burkholderia_Caballeronia_Paraburkholderia, Enterobacter, Aspergillus heterocaryoticus, Rhizopus arrhizus, Kazachstania bulderi, which related to the production of anti-inflammatory active ingredients were exposed. These results demonstrated chemical compositions-strains-efficacy interactions during fermentation of MMF, and provide reference for the exploration of the processing mechanism of MMF.

COVID-19 associated Mucormycosis (CAM): Implications for perioperative physicians - A narrative review.

Mucormycosis once considered a rare disease with an incidence of 0.005 to 1.7 per million, has become one of the greatest menaces during the coronavirus disease (COVID-19) pandemic. India alone has contributed to nearly 70% of the global caseload of COVID-associated mucormycosis (CAM) and it had even been declared as a notifiable disease. Second wave of COVID-19 pandemic saw a steep rise in the incidence of mucormycosis and these patients have been presenting to anesthesiologists for various surgical procedures due to its primary or secondary sequelae. Rhino-orbito-cerebral mucormycosis (ROCM) is the commonest manifestation and is caused by Rhizopus arrhizus. Injudicious use of corticosteroids in vulnerable patients could have been a major contributing factor to the sudden rise in ROCM during the pandemic. Concerns related to anesthetic management include COVID-19 infection and post COVID sequalae, common presence of uncontrolled diabetes mellitus, possibility of difficult mask-ventilation and/or intubation, various drug therapy-associated adverse effects, and interaction of these drugs with anesthetic agents. Thorough preoperative optimization, multidisciplinary involvement, perioperative care, and vigilance go a long way in improving overall outcomes in these patients.

Safety evaluation of the food enzyme triacylglycerol lipase from the non-genetically modified Rhizopus arrhizus strain AE-N.

The food enzyme triacylglycerol lipase (EC 3.1.1.3) is produced with the non-genetically modified Rhizopus arrhizus strain AE-N by Amano Enzyme Inc. It is considered free from viable cells of the production organism. The food enzyme is intended to be used in an immobilised form in the modification of fats and oils by interesterification. Since residual amounts of total organic solids (TOS) are removed during refinement of the fats and oils, dietary exposure was not calculated. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panelidentified a no observed adverse effect level of 1,806mg TOS/kg body weight (bw) per day, the highest dose tested. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panelconsidered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood for this to occur is low. Based on the data provided, the Panelconcluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Safety evaluation of the food enzyme glucan 1,4-α-glucosidase from the non-genetically modified Rhizopus arrhizus strain AE-G.

The food enzyme glucan 1,4-α-glucosidase (4-α-d-glucan glucohydrolase; EC 3.2.1.3) is produced with the non-genetically modified Rhizopus arrhizus strain AE-G by Amano Enzyme Inc. The food enzyme is free from viable cells of the production organism. The applicant proposed the use of the food enzyme in baking processes, coffee processing and manufacture of enzymatically modified dairy ingredients (EMDI). The Panelconsidered only the baking processes as the relevant intended use of this food enzyme. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.94mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not raise a safety concern. The systemic toxicity was assessed by a repeated dose 90-day oral toxicity study in rats. The Panelidentified a no observed adverse effect level of 1,868mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 1,987. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and one match was found. The Panelconsidered that under the intended conditions of use the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panelconcluded that this food enzyme does not give rise to safety concerns when used in baking processes under the intended conditions of use.

Modelling the Kinetics of Tartrazine Sorption by the Rhizopus arrhizus Biomass

Rhizopus arrhizus is utilized in many biotechnological applications such as in the manufacturing of enzymes, including pectinases, amylases, proteases, cellulases, and phytases, and metabolites such as lactic acid, ethanol and fumaric acids. Its spent biomass is useful in the food and feed industry. Its usage as a biosorption agent especially dye sorption is an emerging and important application. In this study we explore 16 adsorption kinetics model of tartrazine by R. arrhizus using nonlinear regression. Based on the statistical indicators especially penalty-based error functions such as adjusted coefficient of determination (R2), Root-Mean-Square Error (RMSE), corrected Akaike Information Criterion (AICc), Bayesian Information Criterion (BIC), Hannan-Quinn Information Criterion (HQC) and Marquardt's percentage standard deviation (MPSD) shows that the pseudo-2nd order (PSO) was the best model followed by pseudo-nth order and Fractal-like Pseudo-2nd Order. The parameter of the PSO model gave a value of equilibrium adsorption capacity, qe of 9.367 mg g-1 (95% confidence interval (C.I.), 9.250 to 9.485) and k2 (g/(mg.min)) of 0.037 (95%, C.I., 0.032 to 0.041). The nonlinear regression exercise allows the uncertainty determination of the parameters to be carried out.

353. Evaluation of the in vitro activity of amphotericin B used in the liposomal formulation AmBisome against contemporary human fungal pathogens

Abstract Background Amphotericin B is an antifungal drug whose main limitation is toxicity. To reduce these side effects, liposomal formulations have been developed, being AmBisome (L-AmB, Gilead Science Inc) the most commonly used. We describe the in vitro antifungal activity of L-AmB component used in the AmBisome formulation Methods We performed a multicenter study to determine the susceptibility to L-AmB using CLSI and EUCAST reference methods. Contemporary human fungal pathogenic species (14 yeast spp and 23 mould spp from different geographical regions were included. Other antifungals (triazoles and echinocandins) were also evaluated. Results L-AmB showed activity against yeast species using both EUCAST and CLSI protocols. The strongest activity was shown against Candida albicans (GM-EUCAST 0.24 mg/L (n=85), CLSI 0.23 mg/L (n=111)), C. parapsilosis (GM-EUCAST 0.32 mg/L (n=82), CLSI 0.32 mg/L (n=124)), C. glabrata (GM-EUCAST 0.32 mg/L (n=84), CLSI 0.38 mg/L (n=145)), C. auris (GM-EUCAST 0.46 mg/L (n=20), CLSI 0.31 mg/L (n=22)). In filamentous fungi, lowest MICs were found for Aspergillus fumigatus (GM-EUCAST 0.62 mg/L (n=77), CLSI 0.65 mg/L (n=124)), and A. niger (GM-EUCAST 0.25 mg/L (n=72) , CLSI 0.32 mg/L (n=85)), while for other Aspergillus species, there was a higher variability in the MICs (A. flavus, GM-EUCAST 1.2 mg/L (n=71), CLSI 1.75 mg/L (n=76); A. terreus GM-EUCAST 1.7 mg/L (n=65), CLSI 1.04 mg/L (n=71); A. nidulans GM-EUCAST 1.38 mg/L (n=39), CLSI 0.73 mg/L (n=39)). For other genera from filamentous fungi, L-AmB had a strong activity (GM around 0.2-0.5 mg/L for both methods, including Talaromyces marneffei, Rhizopus arrhizus and R. microscoporus, Lichtheimia corymbifera, Mucor circinelloides). The less susceptible species corresponded to multi-resistant (MDR) species, such as Lomentospora prolificans and Cunninghamella bertholletiae. Conclusion The L-AmB component of AmBisome showed activity against most contemporary human fungal pathogens and only showed limited activity against some MDR mould species. Disclosures Oscar Zaragoza, Principal Investigator, Gilead: Grant/Research Support Teresa Merino-Amador, n/a, Gilead: Grant/Research Support Cristina de Armentia, n/a, Gilead: Grant/Research Support Cornelia Lass-Flörl, PhD, Gilead: Grant/Research Support Jochem B. Buil, PhD, Gilead: Grant/Research Support Paul E. Verweij, PhD, Gilead: Grant/Research Support Patrick C. Y. Woo, PhD, Gilead: Grant/Research Support Chi-Ching Tsang, PhD, Gilead: Grant/Research Support P. Lewis White, PhD, Associates of Cape Cod: Honoraria|F2G: Advisor/Consultant|Gilead: Grant/Research Support|IMMY: Honoraria|Pfizer: Advisor/Consultant|Pfizer: Honoraria Jessica Price, PhD, Gilead: Grant/Research Support Carolyn Grimes, PhD, Gilead: Grant/Research Support Alessandro C. Pasqualotto, PhD, Gilead: Grant/Research Support Manuel Cuenca-Estrella, PhD, Gilead: Grant/Research Support.

Blood Mucorales PCR to track down Aspergillus and Mucorales co-infections in at-risk hematology patients: A case-control study.

Serum Mucorales PCR can precede the final diagnosis of invasive mucormycosis by several days or weeks and could therefore be useful as a non-invasive screening tool. We assessed the performance of a commercial Mucorales PCR assay (MucorGenius®, PathoNostics, Maastricht, The Netherlands) on prospectively collected banked sera from hematology patients at risk for invasive mould infections. We evaluated if there is an underestimated incidence of missed Mucorales co-infections in patients with invasive aspergillosis (IA). We tested Mucorales PCR on the sera of all patients with a diagnosis of at least possible IA (EORTC-MSGERC consensus criteria) before the start of any antifungal therapy, and in a control group of similar high-risk hematology patients without IA (in a 1:4 ratio). When a positive Mucorales PCR was observed, at least 5 serum samples taken before and after the positive one were selected. Mucorales PCR was performed in 46 diagnostic serum samples of cases and in 184 controls. Serum Mucorales PCR was positive in 4 cases of IA (8.7%; 12.9% of probable cases) and in 1 control case (0.5%) (p=0.0061, OR=17.43 (1.90-159.96). Post-mortem cultures of the positive control became positive for Rhizopus arrhizus. Mortality of IA cases with and without a positive Mucorales PCR was not significantly different. Only in the PCR positive control case, serial serum samples before and after the diagnostic sample were also positive. It is not entirely clear what a positive Mucorales PCR in these cases implies since the 4 Mucorales PCR positive cases were treated with antifungals with activity against Mucorales. In addition, PCR was positive only once. This study does not provide enough evidence to implement Mucorales PCR screening. However, our findings emphasize once more the importance of considering the possibility of dual mould infections, even in patients with a positive galactomannan detection.

Appraisal of selected ethnomedicinal plants as alternative therapies against onychomycosis: Evaluation of synergy and time-kill kinetics.

Introduction: This study aims at the biological profiling of Allium sativum, Zingiber officinale, Nigella sativa, Curcuma longa, Mentha piperita, Withania somnifera, Azadirachta indica, and Lawsonia inermis as alternatives against onychomycosis to combat the treatment challenges. Methods: An extract library of aqueous (DW), ethyl acetate (EA), and methanol (M) extracts was subjected to phytochemical and antioxidant colorimetric assays to gauge the ameliorating role of extracts against oxidative stress. RP-HPLC quantified therapeutically significant polyphenols. Antifungal potential (disc diffusion and broth dilution) against filamentous (dermatophytes and non-dermatophytes) and non-filamentous fungi (yeasts; Candida albicans), synergistic interactions (checkerboard method) with terbinafine and amphotericin-B against resistant clinical isolates of dermatophytes (Trichophyton rubrum and Trichophyton tonsurans) and non-dermatophytes (Aspergillus spp., Fusarium dimerum, and Rhizopus arrhizus), time-kill kinetics, and protein estimation (Bradford method) were performed to evaluate the potential of extracts against onychomycosis. Results: The highest total phenolic and flavonoid content along with noteworthy antioxidant capacity, reducing power, and a substantial radical scavenging activity was recorded for the extracts of Z. officinale. Significant polyphenolics quantified by RP-HPLC included rutin (35.71 ± 0.23µg/mgE), gallic acid (50.17 ± 0.22µg/mgE), catechin (93.04 ± 0.43µg/mgE), syringic acid (55.63 ± 0.35µg/mgE), emodin (246.32 ± 0.44µg/mgE), luteolin (78.43 ± 0.18µg/mgE), myricetin (29.44 ± 0.13µg/mgE), and quercetin (97.45 ± 0.22µg/mgE). Extracts presented prominent antifungal activity against dermatophytes and non-dermatophytes (MIC-31.25μg/ml). The checkerboard method showed synergism with 4- and 8-fold reductions in the MICs of A. sativum, Z. officinale, M. piperita, L. inermis, and C. longa extracts and doses of amphotericin-B (Amp-B) and terbinafine (against non-dermatophytes and dermatophytes, respectively). Furthermore, the synergistic therapy showed a time-dependent decrease in fungal growth even after 9 and 12h of treatment. The inhibition of fungal proteins was also observed to be higher with the treatment of synergistic combinations than with the extracts alone, along with the cell membrane damage caused by terbinafine and amp-B, thus making the resistant fungi incapable of subsisting. Conclusion: The extracts of A. sativum, Z. officinale, M. piperita, L. inermis, and C. longa have proven to be promising alternatives to combat oxidative stress, resistance, and other treatment challenges of onychomycosis.

Dynamic full-field optical coherence tomography as complementary tool in fungal diagnostics

Histopathology and microscopic examination of infected tissue are the gold standards to prove the diagnosis of invasive fungal infection (IFI). Yet, they suffer from essential limitations that hamper rapid diagnosis and require the future development of new imaging tools dedicated to fungal diagnostics. To this end, the present work introduces the first use of dynamic full-field optical coherence tomography (D-FF-OCT) for the visualization of microscopic filamentous fungi. Data collected from the observation of three different fungal species (Nannizzia gypsea, Aspergillus fumigatus and Rhizopus arrhizus) confirm the ability of D-FF-OCT to visualize not only the main structures of all selected fungal species (hyphae, spores, conidia, sporulating structures), but also the metabolic activity of the organisms, which could provide additional help in the future to better characterize the signature of each fungal structure. These results demonstrate how D-FF-OCT could serve as potential complementary tool for rapid diagnosis of IFI in both intensive and non-intensive care units.

Aspergillus Was the Dominant Genus Found during Diversity Tracking of Potentially Pathogenic Indoor Fungal Isolates.

Viable airborne pathogenic fungi represent a potential health hazard when exposing vulnerable persons in quantities exceeding their resilience. In this study, 284 indoor fungal isolates from a strain collection of indoor fungi were screened for pathogenic potential through the ability to grow in neutral pH at 37 °C and 30 °C. The isolates were collected from 20 locations including 14 problematic and 6 non-problematic ordinary buildings. Out of the screened isolates, 170 isolates were unable to grow at 37 °C, whereas 67 isolates growing at pH 7.2 at 37 °C were considered as potential opportunistic pathogens. Forty-seven isolates growing at 30 °C but not at 37 °C were considered as less likely pathogens. Out of these categories, 33 and 33 strains, respectively, were identified to the species level. The problematic buildings included known opportunistic pathogens: Aspergillus calidoustus, Trichoderma longibrachiatum, Rhizopus arrhizus and Paecilomyces variotii, as well as less likely pathogens: Aspergillus versicolor, Chaetomium cochliodes, Chaetomium globosum and Chaetomium rectangulare. Opportunistic pathogens such as Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger and Aspergillus tubingensis and less likely pathogens such as Aspergillus westerdijkiae, Chaetomium globosum and Dichotomopilus finlandicus were isolated both from ordinary and from problematic buildings. Aspergillus was the dominant, most diverse genus found during screening for potentially pathogenic isolates in the indoor strain collection. Studies on Aspergillus niger and Aspergillus calidodoustus revealed that tolerance to cleaning chemicals may contribute to the adaptation of Aspergillus species to indoor environments.

Violacein and its antifungal activity: comments and potentialities.

Violacein is an important natural antimicrobial pigment that is mainly produced by Chromobacterium violaceum and Janthinobacterium lividum. It presents a significant range of effects against phytopathogenic and human fungi, besides being featured as having low toxicity, and by its important ecological role in protecting amphibian species and applications in dyed medical fabric. The hypothesis about violacein's action mechanisms against mucormycosis (Rhizopus arrhizus) and candidiasis (Candida auris) is herein discussed based on data available in the scientific literature.

P327 Prevalence of orofacial Mycoses in COVID-19patients: experience from a tertiary care center in Northern India

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM ObjectiveDue to coronavirus disease (COVID-19) a new group of patients at risk emerged with COVID-19-associated mycoses (CAM). The studies, evaluating the prevalence of CAM are missing in India.MethodTo assess CAM prevalence in a tertiary care hospital in India, we applied direct microscopy, fungal culture, and histopathology on respiratory specimens of 285 critically ill COVID-19 patients admitted between September 2020 and March 2022.ResultAmong the 285 patients, 187 were male, and 98 were female. A total of 34.03% had mucor (33.01% Rhizopus arrhizus;1.02% R. microsporus), 59.51% had Aspergillus (50% Aspergillus flavus; 41.17% A. fumigatus; 2.94% A. terreus; 2.35% A. niger), 3.04% had both (Rhizopus arrhizus + Aspergillus flavus), and 3.01% (Schizophyllum sps 5; Fusarium 1, Paecilomyces variotii 1) had other types of mycosis on fungal smear and culture.ConclusionConsistent with others, our findings underline the importance of microbiological/pathological assessment in patients with predispositions for COVID-19-associated mycoses but due to the low prevalence, a routine screening seems not to be indicated currently. However, multicenter studies are desirable for substantiation of findings. A high index of clinical suspicion, diagnosis at an early stage, and use of antifungal agents are essential for a successful outcome.

P135 Immunological correlation with clinicomycological aspects of fungal rhinosinusitis during COVID-19 pandemic

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM Background and ObjectivesTo determine the relation between disease spectrum, etiology, and immunological markers of fungal rhinosinusitis.Material and MethodsDiagnostic nasal endoscopy was used for the collection of sinus and nasal tissue samples which were processed as per standard mycological procedure for microscopy, culture and histopathological examination (HPE). Enzyme-linked immunosorbent assay was used for total IgE, serum IL-6, IL-10, TNF-α levels detection.ResultsOut of 148 samples, 70% were diagnosed as FRS. COVID-19 infection 46.9% was the most common comorbid condition. Use of steroid therapy 46.9% was the most common predisposing factor. Nasal obstruction 62.2% was the most common clinical feature but headache (P-value .002) and eye discharge were statistically significant (P-value .005). KOH mount revealed findings in 84.69% patients in this study. Combination of KOH, HPE, and fungal culture revealed findings in 62.2% cases. Rhizopus arrhizus 27.6% was the most common isolate Aspergillus flavus and Rhizopus arrhizus were statistically significant (P-value .003, .026 respectively). IL-6 59.2% was the most common serological marker raised in this study. PNS imaging revealed that 100% of the patients had single or multiple sinus involvement and maxillary sinus (P-value .038) was significantly associated. Acute invasive fungal rhinosinusitis 75.5% was the most common type of FRS. Mortality was 17.34% in this study.ConclusionFRS is an important emerging public health problem and a cause of mortality and morbidity, particularly in immunocompromised patients. Furthermore, in the current scenario of COVID-19 pandemic, FRS has become more fatal and an entity of immediate and grave concern than ever before.

P086 Clinical outcome and in vitro antifungal susceptibility of clinical isolates of rhino-orbito-cerebral mycosis associated with post COVID 19 from North India

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM ObjectivesThis study aimed primarily to determine the etiology, clinical features, and comorbidities of patients with rhino-orbito-cerebral mycosis. Secondly, antifungal susceptibility pattern of the isolates and lineage by ITS-sequencing was also studied.MethodsThe study was conducted from May to December 2021 on all suspected cases of rhino-orbito-cerebral mycosis in post-COVID-19 patients at a tertiary care center. Data pertaining to demographics, recent COVID-19 infection, clinical features, comorbidities, laboratory, radiological investigations, management, and outcomes were collected after obtaining informed consent from all patients. Staging of ROCM was done using the proposed code Mucor and diagnosis of COVID-19 was done on basis of real-time polymerase chain reaction (RT-PCR) test. KOH Mount examination, fungal culture, and histopathological examination was performed on samples collected endoscopically or post-debridement. Mucormycosis was proven based on fungal culture or specific histological features from biopsy specimens. In vitro susceptibility profiles for antifungal drugs as per CLSI microbroth dilution method (M38-A2) was studied by HiMIC™ plate (HiMedia) for amphotericin B, voriconazole, posaconazole, itraconazole, and isavuconazole. MIC ranges and the drug concentrations required to inhibit 50% (MIC50) or 90% of isolates (MIC90) were determined. ITS Sequencing was also performed on representative isolates.ResultsA total of 70 patients were diagnosed with mucormycosis. Rhino-orbital and rhino-orbito-cerebral forms were observed in 35.7% of cases each. Diabetes mellitus (DM) was present in 95.7% patients while 78.5% of the patients were treated with corticosteroids in recent past, and 25.7% presented with active COVID-19 pneumonia. Most cases showed onset of symptoms of mucormycosis between 29 ± 17 days from diagnosis of COVID-19. On imaging, orbit was involved in 52.8% and cranial involvement is seen in 35.7% of patients. Diagnosis of mucormycosis was established on KOH direct microscopy 68.6%, culture 47.14%, histopathology 55.7%. Isolates obtained were Rhizopus arrhizus (42.4%), Apophysomyces variabilis (3.03%), and Aspergillus spp (69.7%) while mixed infection was seen in 42.4%. The MIC50 and MIC90 of amphotericin B for R. arrhizus strains were 0.25 and 4 μg/ml; and MIC50 and MIC90 results for itraconazole, posaconazole, and isavuconazole were 8 and 8, 2 and 2, and 2 and 8 μg/ml respectively. Aspergillus spp was susceptible to amphotericin B (38.8%), itraconazole (50%), voriconazole (50%), posaconazole (11.1%), and isavuconazole (44.4%). Overall treatment included intravenous amphotericin B along with functional endoscopic sinus surgery (FESS)/paranasal sinus (PNS) debridement in 68.2%, orbital exenteration in 4.2%, orbital decompression in 11.4% patients and partial maxillectomy in 22.8% cases. Intraorbital injection of amphotericin B was administered in 15.7%. At final follow-up, mortality was 19.7%. In vitro MICs showed that amphotericin B was the most active compound against most species.ConclusionHigh index of suspicion, early diagnosis, and appropriate management of mucormycosis can improve survival. Rational use of steroids and strict glycemic control in diabetic patients can prevent occurrence of mucormycosis. Use of standard methods for antifungal susceptibility testing to guide antifungal treatment may be clinically useful in cases of treatment failure.

P356 An aeromycological study of diagnostic microbiology laboratory at a tertiary care center

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMObjectivesTo evaluate the aeromycoflora of diagnostic microbiology laboratory at a tertiary care center.MethodsThis is an ecological prospective analytical study. This pilot study was conducted at a diagnostic microbiology laboratory in which the Bacteriology section and Mycology section were the study areas. One indoor and one outdoor community area were taken as community controls and histopathology lab was taken as Laboratory control. The sampling was done every fortnight for a period of 1 year from March 2021 to February 2022.Environmental air samples were collected using the passive settle plate method based on sedimentation on 90 mm Sabouraud Dextrose Agar plates. The plates were exposed for 30 mins at a height of 1 m and a distance of 1m from the wall as per standard references.After sample collection, plates were incubated at 22°C for a maximum of 3 weeks or till the time the colonies were countable. The colonies were counted and the total CFU/m3 was calculated using the Omeliansky Formula. The identification of the isolates was done using standard mycological protocols. Statistical analysis using paired student t-test was also performed.ResultsA total of 13 types of fungi were isolated in the study belonging to the following genera- Aspergillus, Alternaria, Curvularia, Bipolaris, Cladosporium, Fonseacea, Fusarium, Penicillium, and Rhizopus. In the Bacteriology section, the highest frequency was of A. niger (63%) followed by A. flavus (50%) while in the mycology section the highest frequency of A. fumigatus (50%) followed by A. flavus (46%) was observed.Certain fungal species known to cause serious infections like A. flavus, Rhizopus arrhizus, Fusarium spp., Penicillium spp., Curvularia spp., were isolated only in the laboratory environment and were absent in the Community Controls.The total CFU/m3 ranged from 26.11 to 576.64 in mycology section and 26.11 to 419.37 in Bacteriology section while in the indoor community control the range was between 26.11 to 235.89 CFU/m3 which was found to be statistically significantly lesser using T-test when compared to the laboratory environment. A seasonal variation with higher counts during the autumn months (September-October) was observed in both the labs. Seasonal variation in the distribution of fungi was observed especially in the case of Rhizopus.ConclusionHealthcare workers spending a significant time in this environment need to be made aware of the quality of air in laboratories and initiation of appropriate intervention to make the laboratory environment safer to work as in this study it was found that more pathogenic fungi were grown in a laboratory environment which is clearly due to the processing of clinical samples in labs as compared to the community environment.The use of standard aseptic precautions, biosafety cabinets, fumigation of laboratories, and regular housekeeping activities would help to decrease the aerosols generated in the labs.The results from this study will be useful to spread awareness and help in formulating guidelines for the air quality of laboratories. However, aeromycology data from more such studies over a larger number of labs from different demographic areas are needed to enable a better understanding of the role of the formulation of standards for a safer laboratory environment.