Evolutionary trends in antifungal resistance: a meta-analysis.

The present paper includes a meta-analysis of literature data on 318 species of fungi belonging to 34 orders in their response to 8 antifungal agents (amphotericin B, caspofungin, fluconazole, itraconazole, ketoconazole, posaconazole, terbinafine, and voriconazole). Main trends of MIC results at the ordinal level were visualized. European Committee on Antimicrobial Susceptibility Testing and Clinical & Laboratory Standards Institute (CLSI) clinical breakpoints were used as the staff gauge to evaluate MIC values ranging from resistance to susceptibility, which were subsequently compared with a phylogenetic tree of the fungal kingdom. Several orders (Hypocreales, Microascales, and Mucorales) invariably showed resistance. Also the basidiomycetous orders Agaricales, Polyporales, Sporidiales, Tremellales, and Trichosporonales showed relatively high degrees of azole multi-resistance, while elsewhere in the fungal kingdom, including orders with numerous pathogenic and opportunistic species, that is, Onygenales, Chaetothyiales, Sordariales, and Malasseziales, in general were susceptible to azoles. In most cases, resistance vs susceptibility was consistently associated with phylogenetic distance, members of the same order showing similar behavior. IMPORTANCE: A kingdom-wide the largest set of published wild-type antifungal data comparison were analyzed. Trends in resistance in taxonomic groups (monophyletic clades) can be compared with the phylogeny of the fungal kingdom, eventual relationships between fungus-drug interaction and evolution can be described.

Usefulness of Sona Aspergillus Galactomannan LFA with digital readout as diagnostic and as screening tool of COVID-19 associated pulmonary aspergillosis in critically ill patients. Data from a multicenter prospective study performed in Argentina.

COVID-19-associated pulmonary aspergillosis (CAPA) incidence varies depending on the country. Serum galactomannan quantification is a promising diagnostic tool since samples are easy to obtain with low biosafety issues. A multicenter prospective study was performed to evaluate the CAPA incidence in Argentina and to assess the performance of the lateral flow assay with digital readout (Sona Aspergillus LFA) as a CAPA diagnostic and screening tool. The correlation between the values obtained with Sona Aspergillus LFA and Platelia® EIA was evaluated. In total, 578 serum samples were obtained from 185 critically ill COVID patients. CAPA screening was done weekly starting from the first week of ICU stay. Probable CAPA incidence in critically ill patients was 10.27% (19/185 patients when LFA was used as mycological criteria) and 9% (9/100 patients when EIA was used as mycological criteria). We found a very good correlation between the two evaluated galactomannan quantification methods (overall agreement of 92.16% with a Kappa statistic value of 0.721). CAPA diagnosis (>0.5 readouts in LFA) were done during the first week of ICU stay in 94.7% of the probable CAPA patients. The overall mortality was 36.21%. CAPA patients' mortality and length of ICU stay were not statistically different from for COVID (non-CAPA) patients (42.11 vs 33.13% and 29 vs 24 days, respectively). These indicators were lower than in other reports. LFA-IMMY with digital readout is a reliable tool for early diagnosis of CAPA using serum samples in critically ill COVID patients. It has a good agreement with Platelia® EIA. LAY SUMMARY: The incidence of COVID-associated pulmonary aspergillosis (CAPA) in critically-ill Argentinian patients was established (10.27%). Serum galactomannan quantification was useful as a screening tool for this mycosis. A good agreement between Platelia® EIA and Sona Aspergillus LFA is reported.

Role of Antifungal Combinations in Difficult to Treat Candida Infections.

Candida infections are varied and, depending on the immune status of the patient, a life-threatening form may develop. C. albicans is the most prevalent species isolated, however, a significant shift towards other Candida species has been noted. Monotherapy is frequently indicated, but the patient's evolution is not always favorable. Drug combinations are a suitable option in specific situations. The aim of this review is to address this problem and to discuss the role of drug combinations in difficult to treat Candida infections. A search for eligible studies in PubMed and Google Scholar databases was performed. An analysis of the data was carried out to define in which cases a combination therapy is the most appropriate. Combination therapy may be used for refractory candidiasis, endocarditis, meningitis, eye infections and osteomyelitis, among others. The role of the drug combination would be to increase efficacy, reduce toxicity and improve the prognosis of the patient in infections that are difficult to treat. More clinical studies and reporting of cases in which drug combinations are used are needed in order to have more data that support the use of this therapeutic strategy.

Recent developments in less known and multi-resistant fungal opportunists.

Fungal infections have increased in recent years due to host factors, such as oncohaematological and transplant-related disorders, immunosuppressive therapy, and AIDS. Additionally, molecular and proteomic facilities have become available to identify previously unrecognizable opportunists. For these reasons, reports on less-known and recalcitrant mycoses, such as those caused by black fungi, hyaline filamentous fungi, coelomycetes, Mucorales, and non-Candida yeasts have emerged. In this review, novel taxonomy in these groups, which often are multi-resistant to one or several classes of antifungals, is discussed. Clinical presentations, diagnosis and current treatment of some major groups are summarised.

Alternative treatment of fungal infections: Synergy with non-antifungal agents.

Fungal infections are responsible for high mortality rates in immunocompromised and high-risk surgical patients. Therapy failures during the last decades due to increasing multidrug resistance demand innovative strategies for novel and effective antifungal drugs. Synergistic combinations of antifungals with non-antifungal agents highlight a pragmatic strategy to reduce the development of drug resistance and potentially repurpose known compounds with other functions to bypass costly and time-consuming novel drug development.

In Vitro and In Vivo Evaluation of Voriconazole-Containing Antifungal Combinations against Mucorales Using a Galleria mellonella Model of Mucormycosis.

Mucorales are resistant to most antifungals. Mucormycosis associated mortality is unacceptable and new treatment approaches are needed. The objectives of this work were (i) to evaluate the nature and intensity of the in vitro effect of three drugs combinations which included voriconazole (plus amphotericin B, posaconazole and caspofungin) against 25 strains of six different Mucorales species; (ii) to evaluate a Galleria mellonella mucormycosis model; and (iii) to establish if any in vitro⁻in vivo correlation exists. As expected, amphotericin B and posaconazole were the most active drugs when tested alone. However, species-specific differences were found. The ΣFICs varied according to the used combination. Only five strains showed synergism when voriconazole was combined with posaconazole and three strains when combined with amphotericin B. Microscopic hyphae alteration were observed for some isolates when confronted against drugs combinations. Using a Galleria mellonella mucormycosis model, better survival was seen in voriconazole plus amphotericin B and plus caspofungin combined treatments when compared with AMB alone for R. microsporus. These survival improvements were obtained using a 32-fold lower amphotericin B doses when combined with VRC than when treated with the polyene alone. These lower antifungal doses emulate the antifungal concentrations where the microscopic hyphae alterations were seen.

Differential distribution patterns of Fonsecaea agents of chromoblastomycosis, exemplified by the first case due to F. monophora from Argentina.

Chromoblastomycosis is a mutilating infection of the skin and subcutaneous tissues caused by melanized fungi belonging to the order Chaetothyriales. Proven cases of the main agent, Fonsecaea pedrosoi are mainly limited to (sub)tropical, humid climates of Latin and Central America and the Caribbean. Fonsecaea monophora has a global distribution along the equator. Cases outside the (sub)tropics have thus far mostly been considered to have been imported, but here we report the first endemic case by F. monophora from Argentina. Patient was a 82-year-old rural female worker from Corrientes, a province with a dry continental climate.

Multiplex PCR designed to differentiate species within the Candida glabrata complex / PCR multiplex diseñada para diferenciar las especies del complejo Candida glabrata

Background. No phenotypic methods are available to unequivocally differentiate species within the Candida glabrata complex. Aims. To develop a new multiplex PCR method to differentiate between the three species of the C. glabrata species complex, as well as using it to study a C. glabrata collection to discover strains of the newly described species. Methods. The method was developed based on the Internal Transcribed Spacer (ITS) sequence differences between the species. It was validated by using a blinded collection of strains and, finally, the new molecular method was used to study a collection of 192 C. glabrata species complex strains. The obtained results were compared with ITS sequencing. Results. The proposed method showed 100% concordance with ITS sequencing and proved to be effective for clinical and epidemiological applications. Two Candida bracarensis and three Candida nivariensis were found out of the 192 studied strains (0.93% and 1.40% prevalence, respectively). Conclusions. A fast, inexpensive, robust and highly reproducible multiplex PCR method is presented. Its usefulness is demonstrated by studying a large collection of C. glabrata sensu lato strains (AU)

Antecedentes. No hay métodos fenotípicos disponibles para diferenciar las especies del complejo Candida glabrata. Objetivos. Diseñar un método de PCR multiplex para diferenciar las tres especies del complejo C. glabrata y usarlo para estudiar una colección de cepas identificadas anteriormente como C. glabrata. Métodos. El método fue desarrollado con base en las diferencias de la secuencia internal transcribed spacer (ITS) entre las especies. El método se validó mediante el uso de una colección de cepas incógnitas y se utilizó posteriormente para estudiar una colección de 192 cepas. Los resultados se compararon con las secuencias ITS. Resultados. El método propuesto mostró 100% de concordancia con la secuenciación de las regiones ITS y demostró ser eficaz clínica y epidemiológicamente. Se identificaron dos aislamientos de Candida bracarensis y tres de Candida nivariensis dentro de las 192 cepas identificadas fenotípicamente como C. glabrata (prevalencia de 0,93% y 1,40%, respectivamente). Conclusiones. Presentamos un método de PCR múltiplex rápido, económico y fiable. La utilidad de la metodología queda demostrada con el estudio de una gran colección de cepas de C. glabrata sensu lato (AU)

Acute pulmonary involvement by paracoccidiodomycosis disease immediately after kidney transplantation: Case report and literature review.

Paracoccidioides brasiliensis is the cause of paracoccidioidomycosis, one of the most important systemic mycoses in Latin America. Human disease has been observed in a limited geographic and ecological niche, and it is attributed to exposure to the fungus in soil. Most primary infections are subclinical, as the infection is contained by the host mainly through cell-mediated immune response. However, as the fungus has the ability to survive in a dormant state for long periods, an impairment of the immune response may lead to reactivation and clinical disease. Surprisingly, paracoccidioidomycosis has rarely been reported in transplanted patients. The aim of this communication is to report a case occurring in a kidney recipient in an acute clinical form immediately after transplantation, and to review the available information on previously reported cases.

Multiplex PCR designed to differentiate species within the Candida glabrata complex.

BACKGROUND: No phenotypic methods are available to unequivocally differentiate species within the Candida glabrata complex. AIMS: To develop a new multiplex PCR method to differentiate between the three species of the C. glabrata species complex, as well as using it to study a C. glabrata collection to discover strains of the newly described species. METHODS: The method was developed based on the Internal Transcribed Spacer (ITS) sequence differences between the species. It was validated by using a blinded collection of strains and, finally, the new molecular method was used to study a collection of 192 C. glabrata species complex strains. The obtained results were compared with ITS sequencing. RESULTS: The proposed method showed 100% concordance with ITS sequencing and proved to be effective for clinical and epidemiological applications. Two Candida bracarensis and three Candida nivariensis were found out of the 192 studied strains (0.93% and 1.40% prevalence, respectively). CONCLUSIONS: A fast, inexpensive, robust and highly reproducible multiplex PCR method is presented. Its usefulness is demonstrated by studying a large collection of C. glabrata sensu lato strains.

Name changes in medically important fungi and their implications for clinical practice.

Recent changes in the Fungal Code of Nomenclature and developments in molecular phylogeny are about to lead to dramatic changes in the naming of medically important molds and yeasts. In this article, we present a widely supported and simple proposal to prevent unnecessary nomenclatural instability.

Set of classical PCRs for detection of mutations in Candida glabrata FKS genes linked with echinocandin resistance.

Clinical echinocandin resistance among Candida glabrata strains is increasing, especially in the United States. Antifungal susceptibility testing is considered mandatory to guide therapeutic decisions. However, these methodologies are not routinely performed in the hospital setting due to their complexity and the time needed to obtain reliable results. Echinocandin failure in C. glabrata is linked exclusively to Fks1p and Fks2p amino acid substitutions, and detection of such substitutions would serve as a surrogate marker to identify resistant isolates. In this work, we report an inexpensive, simple, and quick classical PCR set able to objectively detect the most common mechanisms of echinocandin resistance in C. glabrata within 4 h. The usefulness of this assay was assessed using a blind collection of 50 C. glabrata strains, including 16 FKS1 and/or FKS2 mutants.

Influence of capsule size on the in vitro activity of antifungal agents against clinical Cryptococcus neoformans var. grubii strains.

Cryptococcosis causes disseminated disease in AIDS patients. In contrast to what occurs in laboratory conditions, a large capsule is produced by Cryptococcus neoformans in vivo during infection. The aim of this study was to compare the in vitro activity of different antifungal agents against 34 clinical isolates of C. neoformans var. grubii without or with capsule induction (CLSI, CLSI-C, respectively), following the CLSI M27A3 document. Capsule induction was obtained by addition of NaHCO(3) and incubation with CO(2). The geometric means of the MICs, in µg ml(-1), for CLSI and CLSI-C cultures, respectively, were 1.9 and 9.8 for fluconazole; 0.04 and 0.08 for itraconazole; 0.04 and 0.05 for voriconazole; 0.16 and 0.38 for amphotericin B; and 1.6 and 5.6 for 5-flucytosine. Thus fluconazole showed the highest MICs after capsule induction. Determination of antifungal activity after capsule induction may be clinically relevant and could be used to evaluate the correlation between in vitro results and clinical outcome.

Antifungal susceptibility and phylogeny of opportunistic members of the order mucorales.

The in vitro susceptibilities of 66 molecularly identified strains of the Mucorales to eight antifungals (amphotericin B, terbinafine, itraconazole, posaconazole, voriconazole, caspofungin, micafungin, and 5-fluorocytosine) were tested. Molecular phylogeny was reconstructed based on the nuclear ribosomal large subunit to reveal taxon-specific susceptibility profiles. The impressive phylogenetic diversity of the Mucorales was reflected in susceptibilities differing at family, genus, and species levels. Amphotericin B was the most active drug, though somewhat less against Rhizopus and Cunninghamella species. Posaconazole was the second most effective antifungal agent but showed reduced activity in Mucor and Cunninghamella strains, while voriconazole lacked in vitro activity for most strains. Genera attributed to the Mucoraceae exhibited a wide range of MICs for posaconazole, itraconazole, and terbinafine and included resistant strains. Cunninghamella also comprised strains resistant to all azoles tested but was fully susceptible to terbinafine. In contrast, the Lichtheimiaceae completely lacked strains with reduced susceptibility for these antifungals. Syncephalastrum species exhibited susceptibility profiles similar to those of the Lichtheimiaceae. Mucor species were more resistant to azoles than Rhizopus species. Species-specific responses were obtained for terbinafine where only Rhizopus arrhizus and Mucor circinelloides were resistant. Complete or vast resistance was observed for 5-fluorocytosine, caspofungin, and micafungin. Intraspecific variability of in vitro susceptibility was found in all genera tested but was especially high in Mucor and Rhizopus for azoles and terbinafine. Accurate molecular identification of etiologic agents is compulsory to predict therapy outcome. For species of critical genera such as Mucor and Rhizopus, exhibiting high intraspecific variation, susceptibility testing before the onset of therapy is recommended.

Evaluation of the in vitro activity of amphotericin B by time-kill curve methodology against large and small capsulate C. neoformans isolates.

We have evaluated and compared the activity of amphotericin B (AMB) by time-kill curve methodology against 20 clinical Cryptococcus neoformans isolates in which capsule induction in vitro was performed. Overall, large capsulated isolates were more resistant to killing by AMB over time when compared with those small capsulate ones.

Synthesis and antifungal activity of some substituted phenothiazines and related compounds.

Several phenothiazines and related compounds were synthesized and their antifungal activity was evaluated in vitro. The results observed for α-chloro-N-acetyl phenothiazine led us to choose this compound as a lead in the search of antifungal agents.

Chromoblastomycosis: an overview of clinical manifestations, diagnosis and treatment.

Chromoblastomycosis is one of the most frequent infections caused by melanized fungi. It is a subcutaneous fungal infection, usually an occupational related disease, mainly affecting individuals in tropical and temperate regions. Although several species are etiologic agents, Fonsecaea pedrosoi and Cladophialophora carrionii are prevalent in the endemic areas. Chromoblastomycosis lesions are polymorphic and must be differentiated from those associated with many clinical conditions. Diagnosis is confirmed by the observation of muriform cells in tissue and the isolation and the identification of the causal agent in culture. Chromoblastomycosis still is a therapeutic challenge for clinicians due to the recalcitrant nature of the disease, especially in the severe clinical forms. There are three treatment modalities, i.e., physical treatment, chemotherapy and combination therapy but their success is related to the causative agent, the clinical form and severity of the chromoblastomycosis lesions. There is no treatment of choice for this neglected mycosis, but rather several treatment options. Most of the patients can be treated with itraconazole, terbinafine or a combination of both. It is also important to evaluate the patient's individual tolerance of the drugs and whether the antifungal will be provided for free or purchased, since antifungal therapy must be maintained in long-term regimens. In general, treatment should be guided according to clinical, mycological and histopathological criteria.

In vitro interactions of antifungal agents against clinical isolates of Fusarium spp.

The in vitro activities of amphotericin B (AMB), itraconazole (ITC), voriconazole (VCZ) and terbinafine (TBF) alone and in the combinations AMB+VCZ, TBF+ITC and TBF+VCZ were evaluated against 29 clinical isolates of Fusarium spp. (15 Fusarium solani, 7 Fusarium oxysporum, 2 Fusarium decemcellulare, 2 Fusarium dimerum and 3 other Fusarium spp.). Minimum inhibitory concentrations were determined using the method of the Clinical and Laboratory Standards Institute and the interaction activity was calculated using the fractional inhibitory concentration index. The four antifungal drugs tested alone showed very limited activity against most of the isolates. In contrast, the combination TBF+VCZ showed synergy for 21 isolates. The combination AMB+VCZ showed synergism for only five strains. No interaction or antagonism was observed among the remaining strains. TBF+ITC showed no interaction for 18 strains. The in vitro antifungal activity of the drugs alone and in combination varied for different species. These results corroborate previous in vitro studies in which the combination TBF+VCZ showed synergy against Fusarium spp., although further studies are needed to elucidate its potential usefulness for therapy.

Activity and post antifungal effect of chlorpromazine and trifluopherazine against Aspergillus, Scedosporium and zygomycetes.

The phenothiazine compounds chlorpromazine and trifluopherazine are antipsychotic agents that exhibit antimicrobial activity against bacteria, some protozoa and yeasts. Data of activity against filamentous fungi are lacking. The in vitro activity and postantifungal effect (PAFE) of chlorpromazine and trifluopherazine was determined against Aspergillus species, zygomycetes and Scedosporium species. In vitro susceptibility testing was performed with CLSI M38A and the PAFE was determined with previously established methods. Both drugs inhibited the growth of all fungi tested at concentrations of 16 to 64 microg ml(-1). For Aspergillus species the mean PAFE was 3.7 and 4.7 h; for zygomycetes, 3.1 and 3.4 h; for Scedosporium, 4.3 and 5.3 h for chlorpromazine and trifluoroperazine respectively. These are the first drugs shown to induce PAFE against Scedosporium. We show that phenothiazine compounds have in vitro antifungal activity and exhibit PAFE against a broad range of filamentous fungal pathogens. Although the exact mechanism of action is unknown, further studies are needed to explore the clinical usefulness of phenothiazine compounds.