Utility of itraconazole and terbinafine in mucormycosis: a proof-of-concept analysis.

An epidemic of mucormycosis followed the second wave of COVID 19 in the state of Uttar Pradesh, India in May 2021. This epidemic, however, had additional challenges to offer in the form of acute shortage of all forms of amphotericin B, posaconazole and isavuconazole. It was, therefore, planned to assess the trends in minimum inhibitory concentration (MIC) of antifungal agents, viz itraconazole and terbinafine, and provide a template for personalized therapy to see whether the results could be translated clinically. This is an observational, single-center study. Samples comprising nasal swab, nasal and paranasal sinus tissue, brain tissue, brain abscess and orbital content, derived from 322 patients from northern India with mucormycosis, of whom 215 were male and 107 were female, were used for analysis. Cultures were identified both by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and conventional methods of identification. Antifungal susceptibility was done for amphotericin B, posaconazole, isavuconazole, itraconazole and terbinafine as per Clinical Laboratory Standard Institute M38-A2. The outcome was identification of the species of mucormycosis and susceptibility to itraconazole and terbinafine besides other primary antifungal agents. Patients or the public were not involved in the design, or conduct, or reporting or in the dissemination plans of our research. Of 322 patients, 203 were culture-positive, of whom 173 were positive by both MALDI-TOF and conventional methods of identification. Final antifungal susceptibility testing was available for 150 patients. The most common Mucorales found to cause this epidemic was Rhizopus oryzae, followed by R. microsporus Amphotericin B, posaconazole and isavuconazole had low MIC values in 98.8% of all Mucorales identified. The MIC of itraconazole was species-dependent. 97.7% of Roryzae had MIC ≤2 µg/mL. However, only 36.5% of Rmicrosporus had MIC ≤2 µg/mL. For terbinafine, 85.2% of R. microsporus had MIC ≤2 µg/mL. We conclude that identification at the species level is required as antifungal susceptibilities seem to be species-dependent. Assessment of the efficacy of itraconazole and terbinafine warrants further studies with clinical assessment and therapeutic drug monitoring as they seem to be potential candidates especially when the primary agents are not available.

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 in vitro 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.

Indications that the Antimycotic Drug Amphotericin B Enhances the Impact of Platelets on Aspergillus.

Platelets are currently thought to harbor antimicrobial functions and might therefore play a crucial role in infections, e.g., those caused by Aspergillus or mucormycetes. The incidence of invasive fungal infections is increasing, particularly during the coronavirus disease 2019 (COVID-19) pandemic, and such infections continue to be life-threatening in immunocompromised patients. For this reason, the interaction of antimycotics with platelets is a key issue to evaluate modern therapeutic regimens. Amphotericin B (AmB) is widely used for the therapy of invasive fungal infections either as deoxycholate (AmB-D) or as a liposomal formulation (L-AmB). We showed that AmB strongly activates platelets within a few minutes. AmB concentrations commonly measured in the blood of patients were sufficient to stimulate platelets, indicating that this effect is highly relevant in vivo. The stimulating effect was corroborated by a broad spectrum of platelet activation parameters, including degranulation, aggregation, budding of microparticles, morphological changes, and enhanced adherence to fungal hyphae. Comparison between the deoxycholate and the liposomal formulation excluded the possibility that the liposomal part of L-Amb is responsible for these effects, as no difference was visible. The induction of platelet activation and alteration by L-AmB resulted in the activation of other parts of innate immunity, such as stimulation of the complement cascade and interaction with granulocytes. These mechanisms might substantially fuel the antifungal immune reaction in invasive mycoses. On the other hand, thrombosis and excessive inflammatory processes might occur via these mechanisms. Furthermore, the viability of L-AmB-activated platelets was consequently decreased, a process that might contribute to thrombocytopenia in patients.

Epidemiology and Antifungal Susceptibilities of Mucoralean Fungi in Clinical Samples from the United States.

The global incidence of mucormycosis has increased in recent years owing to higher numbers of individuals at risk for these infections. The diagnosis and treatment of this aggressive fungal infection are of clinical concern due to differences in species distribution in different geographic areas and susceptibility profiles between different species that are capable of causing highly aggressive infections. The purpose of this study was to evaluate the epidemiology and susceptibility profiles of Mucorales isolates in the United States over a 52-month period. Species identification was performed by combined phenotypic characteristics and DNA sequence analysis, and antifungal susceptibility testing was performed by CLSI M38 broth microdilution for amphotericin B, isavuconazole, itraconazole, and posaconazole. During this time frame, 854 isolates were included, representing 11 different genera and over 26 species, of which Rhizopus (58.6%) was the predominant genus, followed by Mucor (19.6%). The majority of isolates were cultured from the upper and lower respiratory tracts (55%). Amphotericin B demonstrated the most potent in vitro activity, with geometric mean (GM) MICs of ≤0.25 µg/ml against all genera with the exception of Cunninghamella species (GM MIC of 1.30 µg/ml). In head-to-head comparisons, the most active azole was posaconazole, followed by isavuconazole. Differences in azole and amphotericin B susceptibility patterns were observed between the genera with the greatest variability observed with isavuconazole. Awareness of the epidemiology of Mucorales isolates and differences in antifungal susceptibility patterns in the United States may aide clinicians in choosing antifungal treatment regimens. Further studies are warranted to correlate these findings with clinical outcomes.

Improving the Cellular Selectivity of a Membrane-Disrupting Antimicrobial Agent by Monomer Control and by Taming.

Antimicrobial resistance represents a significant world-wide health threat that is looming. To meet this challenge, new classes of antimicrobial agents and the redesign of existing ones will be required. This review summarizes some of the studies that have been carried out in my own laboratories involving membrane-disrupting agents. A major discovery that we made, using a Triton X-100 as a prototypical membrane-disrupting molecule and cholesterol-rich liposomes as model systems, was that membrane disruption can occur by two distinct processes, depending on the state of aggregation of the attacking agent. Specifically, we found that monomers induced leakage, while attack by aggregates resulted in a catastrophic rupture of the membrane. This discovery led us to design of a series of derivatives of the clinically important antifungal agent, Amphotericin B, where we demonstrated the feasibility of separating antifungal from hemolytic activity by decreasing the molecule's tendency to aggregate, i.e., by controlling its monomer concentration. Using an entirely different approach (i.e., a "taming" strategy), we found that by covalently attaching one or more facial amphiphiles ("floats") to Amphotericin B, its aggregate forms were much less active in lysing red blood cells while maintaining high antifungal activity. The possibility of applying such "monomer control" and "taming" strategies to other membrane-disrupting antimicrobial agents is briefly discussed.

LY6E Restricts Entry of Human Coronaviruses, Including Currently Pandemic SARS-CoV-2.

C3A is a subclone of the human hepatoblastoma HepG2 cell line with strong contact inhibition of growth. We fortuitously found that C3A was more susceptible to human coronavirus HCoV-OC43 infection than HepG2, which was attributed to the increased efficiency of virus entry into C3A cells. In an effort to search for the host cellular protein(s) mediating the differential susceptibility of the two cell lines to HCoV-OC43 infection, we found that ArfGAP with dual pleckstrin homology (PH) domains 2 (ADAP2), gamma-interferon-inducible lysosome/endosome-localized thiolreductase (GILT), and lymphocyte antigen 6 family member E (LY6E), the three cellular proteins identified to function in interference with virus entry, were expressed at significantly higher levels in HepG2 cells. Functional analyses revealed that ectopic expression of LY6E, but not GILT or ADAP2, in HEK 293 cells inhibited the entry of HCoV-O43. While overexpression of LY6E in C3A and A549 cells efficiently inhibited the infection of HCoV-OC43, knockdown of LY6E expression in HepG2 significantly increased its susceptibility to HCoV-OC43 infection. Moreover, we found that LY6E also efficiently restricted the entry mediated by the envelope spike proteins of other human coronaviruses, including the currently pandemic SARS-CoV-2. Interestingly, overexpression of serine protease TMPRSS2 or amphotericin treatment significantly neutralized the IFN-inducible transmembrane 3 (IFITM3) restriction of human coronavirus (CoV) entry, but did not compromise the effect of LY6E on the entry of human coronaviruses. The work reported herein thus demonstrates that LY6E is a critical antiviral immune effector that controls CoV infection and pathogenesis via a mechanism distinct from other factors that modulate CoV entry.IMPORTANCE Virus entry into host cells is one of the key determinants of host range and cell tropism and is subjected to the control of host innate and adaptive immune responses. In the last decade, several interferon-inducible cellular proteins, including IFITMs, GILT, ADAP2, 25CH, and LY6E, had been identified to modulate the infectious entry of a variety of viruses. Particularly, LY6E was recently identified as a host factor that facilitates the entry of several human-pathogenic viruses, including human immunodeficiency virus, influenza A virus, and yellow fever virus. Identification of LY6E as a potent restriction factor of coronaviruses expands the biological function of LY6E and sheds new light on the immunopathogenesis of human coronavirus infection.