cotH Genes Are Necessary for Normal Spore Formation and Virulence in Mucor lusitanicus.

Mucormycosis is an invasive fungal infection caused by certain members of the fungal order of Mucorales. The species most frequently identified as the etiological agents of mucormycosis belong to the genera Rhizopus, Lichtheimia, and Mucor. The frequency of systemic mucormycosis has been increasing, mainly because of increasing numbers of susceptible patients. Furthermore, Mucorales display intrinsic resistance to the majority of routinely used antifungal agents (e.g., echinocandins and short-tailed azoles), which limits the number of possible therapeutic options. All the above-mentioned issues urge the improvement of molecular identification methods and the discovery of new antifungal targets and strategies. Spore coat proteins (CotH) constitute a kinase family present in many pathogenic bacteria and fungi and participate in the spore formation in these organisms. Moreover, some of them can act as virulence factors being receptors of the human GRP78 protein during Rhizopus delemar-induced mucormycosis. We identified 17 cotH-like genes in the Mucor lusitanicus genome database. Successful disruption of five cotH genes in Mucor was performed using the CRISPR-Cas9 system. The CotH3 and CotH4 proteins play a role in adaptation to different temperatures as well as in developing the cell wall structure. We also show CotH4 protein is involved in spore wall formation by affecting the total chitin content and, thus, the composition of the spore wall. The role of CotH3 and CotH4 proteins in virulence was confirmed in two invertebrate models and a diabetic ketoacidosis (DKA) mouse model. IMPORTANCE Current treatment options for mucormycosis are inadequate, resulting in high mortality rates, especially among immunosuppressed patients. The development of novel therapies for mucormycosis has been hampered by lack of understanding of the pathogenetic mechanisms. The importance of the cell surface CotH proteins in the pathogenesis of Rhizopus-mediated mucormycosis has been recently described. However, the contribution of this family of proteins to the virulence of other mucoralean fungi and their functionality in vital processes remain undefined. Through the use of the CRISPR-Case9 gene disruption system, we demonstrate the importance of several of the CotH proteins to the virulence of Mucor lusitanicus by using three infection models. We also report on the importance of one of these proteins, CotH4, to spore wall formation by affecting chitin content. Therefore, our studies extend the importance of CotH proteins to Mucor and identify the mechanism by which one of the CotH proteins contributes to the development of a normal fungal cell wall, thereby indicating that this family of proteins can be targeted for future development of novel therapeutic strategies of mucormycosis.

Molecular identification, phylogeny and antifungal susceptibilities of dematiaceous fungi isolated from human keratomycosis.

PURPOSE: To investigate the dematiaceous fungal profile of patients with ocular mycoses attending a tertiary eye care hospital in Coimbatore, India METHODS: The identification of dematiaceous fungus based on their morphology, their genotypes, and the measurement of the minimum inhibitory concentrations (MICs) using microdilution method of routinely used antifungal drugs were all compared. RESULTS: A total of 148 dematiaceous fungi were isolated during a study period of 27 months. Isolates were confirmed as Curvularia spp. (n = 98), Exserohilum spp. (n = 32), Alternaria spp. (n = 14), Exophiala spp. (n = 2), Cladosporium sp. (n = 1) and Aureobasidium sp. (n = 1). Out of 50 well grown isolates characterized genotypically based on the amplification and sequencing of the ITS region of the ribosomal RNA gene cluster and subsequent BLAST analysis, Curvularia lunata (n = 24), C. aeria (n = 1), C. spicifera (n = 8), C. hawaiiensis (n = 1), C. maydis (n = 2), C. papendorfii (n = 2), C. geniculata (n = 3), C. tetramera (n = 2) and Exs. rostratum (n = 7) were identified. In vitro antifungal susceptibilities of the most tested dematiaceous isolates showed that voriconazole had a MIC50 of 0.25 µg ml-1, while amphotericin B had a MIC50 of 0.25 µg ml-1 for Curvularia spp. and Alternaria spp. CONCLUSION: Voriconazole proved to be the most effective drug against the pigmented filamentous fungi, followed by amphotericin B, itraconazole and econazole.

Systematic review and network meta-analysis (NMA) for cladribine tablets in achieving sustained disability improvement (SDI) in multiple sclerosis.

INTRODUCTION: This study was performed to compare probabilities of SDI on the Expanded Disability Status Scale (EDSS) in patients with relapsing-remitting multiple sclerosis (RRMS), treated with cladribine tablets (CT) or fingolimod (FTY), natalizumab (NAT), alemtuzumab (ALE) and ocrelizumab (OCR). CLINICAL RATIONALE FOR THE STUDY: Progression of neurological disability as measured by the EDSS has been a common endpoint in multiple sclerosis (MS) trials. Novel therapies can not only slow this process, but in some patients even reverse it. This effect can be measured by the sustained disability improvement (SDI) - an endpoint that seems to continuously gain importance in clinical practice. Despite that, SDI has rarely been explored as an outcome in MS clinical studies, mostly as post-hoc analyses from randomised trials or as retrospective analyses based on patient registry records. MATERIAL AND METHODS: A systematic review was conducted in Medline, Embase and Cochrane to identify clinical trials (RCT or non-RCT) evaluating 6-month SDI. An indirect comparison via network meta-analysis (NMA) was performed. Bayesian inference with Markov chains Monte Carlo methods were applied. RESULTS: Eight trials presenting SDI results and applicable for NMA were included: six non-RCTs, with control groups selected by propensity score matching, and two RCTs. NMA results revealed that probability of achieving 6-month SDI with CT was significantly higher compared to all other high efficacy disease-modifying drugs with available data - HR (95% Crl - Bayesian Credibility Interval) vs. FTY: 4.98 (2.11-11.79); vs. NAT: 3.12 (1.31-7.27); vs. ALE: 9.29 (3.40-25.21). The main results were confirmed in the sensitivity analyses. CONCLUSIONS: Of all considered therapies, treatment with cladribine tablets was associated with a higher probability of sustained disability improvement in RRMS patients. As this conclusion is based on available clinical data of limited quality, future studies, as well as real-world data, would be valuable to provide further evidence regarding the comparative effectiveness of RRMS therapies.

Differential Gene Expression of Mucor lusitanicus under Aerobic and Anaerobic Conditions.

Mucor lusitanicus and some other members of the fungal order Mucorales display the phenomenon of morphological dimorphism. This means that these fungi aerobically produce filamentous hyphae, developing a coenocytic mycelium, but they grow in a multipolar yeast-like form under anaerobiosis. Revealing the molecular mechanism of the reversible yeast-hyphal transition can be interesting for both the biotechnological application and in the understanding of the pathomechanism of mucormycosis. In the present study, transcriptomic analyses were carried out after cultivating the fungus either aerobically or anaerobically revealing significant changes in gene expression under the two conditions. In total, 539 differentially expressed genes (FDR < 0.05, |log2FC| ≥ 3) were identified, including 190 upregulated and 349 downregulated transcripts. Within the metabolism-related genes, carbohydrate metabolism was proven to be especially affected. Anaerobiosis also affected the transcription of transporters: among the 14 up- and 42 downregulated transporters, several putative sugar transporters were detected. Moreover, a considerable number of transcripts related to amino acid transport and metabolism, lipid transport and metabolism, and energy production and conversion were proven to be downregulated when the culture had been transferred into an anaerobic atmosphere.

Characterization of Three Pleiotropic Drug Resistance Transporter Genes and Their Participation in the Azole Resistance of Mucor circinelloides.

Mucormycosis is a life-threatening opportunistic infection caused by certain members of the fungal order Mucorales. This infection is associated with high mortality rate, which can reach nearly 100% depending on the underlying condition of the patient. Treatment of mucormycosis is challenging because these fungi are intrinsically resistant to most of the routinely used antifungal agents, such as most of the azoles. One possible mechanism of azole resistance is the drug efflux catalyzed by members of the ATP binding cassette (ABC) transporter superfamily. The pleiotropic drug resistance (PDR) transporter subfamily of ABC transporters is the most closely associated to drug resistance. The genome of Mucor circinelloides encodes eight putative PDR-type transporters. In this study, transcription of the eight pdr genes has been analyzed after azole treatment. Only the pdr1 showed increased transcript level in response to all tested azoles. Deletion of this gene caused increased susceptibility to posaconazole, ravuconazole and isavuconazole and altered growth ability of the mutant. In the pdr1 deletion mutant, transcript level of pdr2 and pdr6 significantly increased. Deletion of pdr2 and pdr6 was also done to create single and double knock out mutants for the three genes. After deletion of pdr2 and pdr6, growth ability of the mutant strains decreased, while deletion of pdr2 resulted in increased sensitivity against posaconazole, ravuconazole and isavuconazole. Our result suggests that the regulation of the eight pdr genes is interconnected and pdr1 and pdr2 participates in the resistance of the fungus to posaconazole, ravuconazole and isavuconazole.

CRISPR-Cas9-Based Mutagenesis of the Mucormycosis-Causing Fungus Lichtheimia corymbifera.

Lichtheimia corymbifera is considered as one of the most frequent agents of mucormycosis. The lack of efficient genetic manipulation tools hampers the characterization of the pathomechanisms and virulence factors of this opportunistic pathogenic fungus. Although such techniques have been described for certain species, the performance of targeted mutagenesis and the construction of stable transformants have remained a great challenge in Mucorales fungi. In the present study, a plasmid-free CRISPR-Cas9 system was applied to carry out a targeted gene disruption in L. corymbifera. The described method is based on the non-homologous end-joining repair of the double-strand break caused by the Cas9 enzyme. Using this method, short, one-to-five nucleotide long-targeted deletions could be induced in the orotidine 5'-phosphate decarboxylase gene (pyrG) and, as a result, uracil auxotrophic strains were constructed. These strains are applicable as recipient strains in future gene manipulation studies. As we know, this is the first genetic modification of this clinically relevant fungus.

Response of Human Neutrophil Granulocytes to the Hyphae of the Emerging Fungal Pathogen Curvularia lunata.

Curvularia lunata is an ascomycete filamentous fungus causing local and invasive phaeohyphomycoses in both immunocompromised and immunocompetent patients. Neutrophils are crucial participants of the first line host defense against fungal infections. They migrate to the infected site and eliminate the infectious agents by various mechanisms including phagocytoses, oxidative damage, or formation of neutrophil extracellular trap (NET). Neutropenia may be a risk factor for phaeohyphomycoses, and restoration of the neutrophil function can improve the outcome of the infection. In the present study, interaction of primary human neutrophil granulocytes with the hyphae C. lunata was examined and compared to that with the well characterized filamentous fungal pathogen Aspergillus fumigatus. Neutrophils could recognize the serum opsonized hyphae of C. lunata and attach to them. Myeloperoxidase release was also activated by a soluble factor present in the culture supernatant of the fungus. Induction of the oxidative burst was found to depend on serum opsonization of the hyphae. Although extracellular hydrogen peroxide production was induced, the fungus efficiently blocked the oxidative burst by acidifying the reaction environment. This blockage also affected the NET forming ability of the neutrophils.

Agricultural systems as potential sources of emerging human mycoses caused by Trichoderma: a successful, common phylotype of Trichoderma longibrachiatum in the frontline.

Trichoderma species are abundant in different agricultural habitats, but some representatives of this genus, mainly clade Longibrachiatum members are also emerging as causative agents of various human diseases with even fatal outcome. Strains of these species frequently show resistance to commonly used azole antifungals. Based on previous data it is hypothesized that Trichoderma isolates identified in human infections derive from environmental-including agricultural-origins. We examined Trichoderma longibrachiatum Rifai and Trichoderma bissettii Sandoval-Denis & Guarro strains recovered from four novel cases of human mycoses, along with isolates from previous case reports and different agricultural habitats, using multilocus phylogenetic analysis, BIOLOG Phenotype Microarrays and Etest. Strains attributed to T. bissettii were more abundant in both clinical and agricultural specimens compared to T. longibrachiatum. The majority of the isolates of both taxa could tolerate >256, >32 and >32 µg/ml fluconazole, itraconazole and posaconazole, respectively. None of the obtained results revealed characteristic differences between strains of clinical and agricultural origin, nor between the two taxa, supporting that agricultural environments may be significant sources of infections caused by these emerging human fungal pathogens. Furthermore, based on our findings we propose the re-classification of T. bissettii as T. longibrachiatum f. sp. bissettii.

New Species of the Genus Curvularia: C. tamilnaduensis and C. coimbatorensis from Fungal Keratitis Cases in South India.

Members of the genus Curvularia are melanin-producing dematiaceous fungi of increasing clinical importance as causal agents of both local and invasive infections. This study contributes to the taxonomical and clinical knowledge of this genus by describing two new Curvularia species based on isolates from corneal scrapings of South Indian fungal keratitis patients. The phylogeny of the genus was updated based on three phylogenetic markers: the internal transcribed spacer (ITS) region of the ribosomal RNA gene cluster as well as fragments of the glyceraldehyde-3-phosphate dehydrogenase (gpdh) and translation elongation factor 1-α (tef1α) genes. The maximum likelihood phylogenetic tree constructed from the alignment of the three concatenated loci revealed that the examined isolates are representing two new, yet undescribed, Curvularia species. Examination of colony and microscopic morphology revealed differences between the two species as well as between the new species and their close relatives. The new species were formally described as Curvularia tamilnaduensis N. Kiss & S. Kocsubé sp. nov. and Curvularia coimbatorensis N. Kiss & S. Kocsubé sp. nov. Antifungal susceptibility testing by the broth microdilution method of CLSI (Clinical & Laboratory Standards Institute) revealed that the type strain of C. coimbatorensis is less susceptible to a series of antifungals than the C. tamilnaduensis strains.

Characterization of Aspergillus tamarii Strains From Human Keratomycoses: Molecular Identification, Antifungal Susceptibility Patterns and Cyclopiazonic Acid Producing Abilities.

Aspergillus tamarii appears to be an emerging aetiological agent of human keratomycoses in South India. The investigated strains were isolated from six suspected fungal keratitis patients attending a tertiary care eye hospital in Coimbatore (Tamil Nadu, India), and were initially identified by the microscopic examinations of the scrapings and the cultures. Our data suggest that A. tamarii could be easily overlooked when identification is carried out based on morphological characteristics alone, while the sequence analysis of the calmodulin gene can be used successfully to recognize this species accurately. According to the collected clinical data, ocular trauma is a common risk factor for the infection that gradually developed from mild to severe ulcers and could be healed with an appropriate combined antifungal therapy. Antifungal susceptibility testing revealed that A. tamarii strains are susceptible to the most commonly used topical or systemic antifungal agents (i.e., econazole, itraconazole and ketoconazole) except for natamycin. Moreover, natamycin proved to be similarly less effective than the azoles against A. tamarii in our drug interaction tests, as the predominance of indifferent interactions was revealed between natamycin and econazole and between natamycin and itraconazole as well. Four and five isolates of A. tamarii were confirmed to produce cyclopiazonic acid (CPA) in RPMI-1640 - which is designed to mimic the composition of human extracellular fluids - and in yeast extract sucrose (YES) medium, respectively, which is a widely used culture medium for testing mycotoxin production. Although a ten times lower mycelial biomass was recorded in RPMI-1640 than in YES medium, the toxin contents of the samples were of the same order of magnitude in both types of media. There might be a relationship between the outcome of infections and the toxigenic properties of the infecting fungal strains. However, this remains to be investigated in the future.

CRISPR-Cas9-mediated disruption of the HMG-CoA reductase genes of Mucor circinelloides and subcellular localization of the encoded enzymes.

Terpenoid compounds, such as sterols, carotenoids or the prenyl groups of various proteins are synthesized via the mevalonate pathway. A rate-limiting step of this pathway is the conversion of 3-methylglutaryl-CoA (HMG-CoA) to mevalonic acid catalyzed by the HMG-CoA reductase. Activity of this enzyme may affect several biological processes, from the synthesis of terpenoid metabolites to the adaptation to various environmental conditions. In this study, the three HMG-CoA reductase genes (i.e. hmgR1, hmgR2 and hmgR3) of the ß-carotene producing filamentous fungus, Mucor circinelloides were disrupted individually and simultaneously by a recently developed in vitro plasmid-free CRISPR-Cas9 method. Examination of the mutants revealed that the function of hmgR2 and hmgR3 are partially overlapping and involved in the general terpenoid biosynthesis. Moreover, hmgR2 seemed to have a special role in the ergosterol biosynthesis. Disruption of all three genes affected the germination ability of the spores and the sensitivity to hydrogen peroxide. Disruption of the hmgR1 gene had no effect on the ergosterol production and the sensitivity to statins but caused a reduced growth at lower temperatures. By confocal fluorescence microscopy using strains expressing GFP-tagged HmgR proteins, all three HMG-CoA reductases were localized in the endoplasmic reticulum.

In vitro Interactions of Pseudomonas aeruginosa With Scedosporium Species Frequently Associated With Cystic Fibrosis.

Members of the Scedosporium apiospermum species complex are the second most frequently isolated pathogens after Aspergillus fumigatus from cystic fibrosis (CF) patients with fungal pulmonary infections. Even so, the main risk factors for the infection are unrevealed. According to previous studies, bacterial infections might reduce the risk of a fungal infection, but an antibacterial therapy may contribute to the airway colonization by several fungal pathogens. Furthermore, corticosteroids, which are often used to reduce lung inflammation in children and adults with CF, are also proved to enhance the growth of A. fumigatus in vitro. Considering all the above discussed points, we aimed to test how Pseudomonas aeruginosa influences the growth of scedosporia and to investigate the potential effect of commonly applied antibacterial agents and corticosteroids on Scedosporium species. Direct interactions between fungal and bacterial strains were tested using the disk inhibition method. Indirect interactions via volatile compounds were investigated by the plate-in-plate method, while the effect of bacterial media-soluble molecules was tested using a modified cellophane assay and also in liquid culture media conditioned by P. aeruginosa. To test the effect of bacterial signal molecules, antibacterial agents and corticosteroids on the fungal growth, the broth microdilution method was used. We also investigated the germination ability of Scedosporium conidia in the presence of pyocyanin and diffusible signal factor by microscopy. According to our results, P. aeruginosa either inhibited or enhanced the growth of scedosporia depending on the culture conditions and the mode of interactions. When the two pathogens were cultured physically separately from each other in the plate-in-plate tests, the presence of the bacteria was able to stimulate the growth of several fungal isolates. While in direct physical contact, bacterial strains inhibited the fungal growth. This effect might be attributed to bacterial signal molecules, which also proved to inhibit the germination and growth of scedosporia. In addition, antibacterial agents showed growth-promoting, while corticosteroids exhibited growth inhibitory effect on several Scedosporium isolates. These data raise the possibility that a P. aeruginosa infection or a previously administered antibacterial therapy might be able to increase the chance of a Scedosporium colonization in a CF lung.

South Indian Isolates of the Fusarium solani Species Complex From Clinical and Environmental Samples: Identification, Antifungal Susceptibilities, and Virulence.

Members of the Fusarium solani species complex (FSSC) are the most frequently isolated fusaria from soil. Moreover, this complex solely affects more than 100 plant genera, and is also one of the major opportunistic human pathogenic filamentous fungi, being responsible for approximately two-third of fusariosis cases. Mycotic keratitis due to Fusarium species is among the leading causes of visual impairment and blindness in South India, but its management is still challenging due to the poor susceptibility of the isolates to conventional antifungal drugs. Aims of the present study were to isolate South Indian clinical and environmental FSSC strains and identify them to species level, to determine the actual trends in their susceptibilities to antifungal therapeutic drugs and to compare the virulence of clinical and environmental FSSC members. Based on the partial sequences of the translation elongation factor 1α gene, the majority of the isolates-both from keratomycosis and environment-were confirmed as F. falciforme, followed by F. keratoplasticum and F. solani sensu stricto. In vitro antifungal susceptibilities to commonly used azole, allylamine and polyene antifungals were determined by the CLSI M38-A2 broth microdilution method. The first generation triazoles, fluconazole and itraconazole proved to be ineffective against all isolates tested. This phenomenon has already been described before, as fusaria are intrinsically resistant to them. However, our results indicated that despite the intensive agricultural use of azole compounds, fusaria have not developed resistance against the imidazole class of antifungals. In order to compare the virulence of different FSSC species from clinical and environmental sources, a Drosophila melanogaster model was used. MyD88 mutant flies having impaired immune responses were highly susceptible to all the examined fusaria. In wild-type flies, one F. falciforme and two F. keratoplasticum strains also reduced the survival significantly. Pathogenicity seemed to be independent from the origin of the isolates.

Exophiala dermatitidis Endophthalmitis: Case Report and Literature Review.

We report a case of a 59-year-old male patient with a postoperative fungal infection of the left eye. A dark-pigmented yeast, Exophiala dermatitidis (previously known as Wangiella dermatitidis), was identified from the culture of the biopsy taken from the posterior capsule. The infection was successfully eradicated by a combination of surgical and medical (i.e., voriconazole and fluconazole) treatment. This is the first report of successfully treated E. dermatitidis endophthalmitis, which demonstrates that a prompt and aggressive antifungal therapy combined with surgical intervention is necessary to prevent vision loss in cases of endophthalmitis due to Exophiala species. Beside the case description, we also aim to provide a literature review of previously reported eye infections caused by Exophiala species in order to help the future diagnosis and management of the disease.

Interaction of THP-1 Monocytes with Conidia and Hyphae of Different Curvularia Strains.

Interaction of the human monocytic cell line, THP-1 with clinical isolates of three Curvularia species were examined. Members of this filamentous fungal genus can cause deep mycoses emerging in both immunocompromised and immunocompetent patients. It was found that monocytes reacted only to the hyphal form of Curvularia lunata. Cells attached to the germ tubes and hyphae and production of elevated levels of interleukin (IL)-8 and IL-10 and a low level of TNF-α were measured. At the same time, monocytes failed to produce IL-6. This monocytic response, especially with the induction of the anti-inflammatory IL-10, correlates well to the observation that C. lunata frequently cause chronic infections even in immunocompetent persons. Despite the attachment to the hyphae, monocytes could not reduce the viability of the fungus and the significant decrease in the relative transcript level of HLA-DRA assumes the lack of antigen presentation of the fungus by this cell type. C. spicifera and C. hawaiiensis failed to induce the gathering of the cells or the production of any analyzed cytokines. Monocytes did not recognize conidia of Curvularia species, even when melanin was lacking in their cell wall.

In vitro activity of calcium channel blockers in combination with conventional antifungal agents against clinically important filamentous fungi.

Despite the current therapeutic options, filamentous fungal infections are associated with high mortality rate especially in immunocompromised patients. In order to find a new potential therapeutic approach, the in vitro inhibitory effect of two antiarrhythmic agents, diltiazem and verapamil hydrochloride were tested against different clinical isolates of ascomycetous and mucoralean filamentous fungi. The in vitro combinations of these non-antifungal drugs with azole and polyene antifungal agents were also examined. Susceptibility tests were carried out using the broth microdilution method according to the instructions of the Clinical and Laboratory Standards Institute document M38-A2. Checkerboard microdilution assay was used to assess the interactions between antifungal and non-antifungal drugs. Compared to antifungal agents, diltiazem and verapamil hydrochloride exerted a relatively low antifungal activity with high minimal inhibitory concentration values (853-2731 µg/ml). Although in combination they could increase the antifungal activity of amphotericin B, itraconazole and voriconazole. Indifferent and synergistic interactions were registered in 33 and 17 cases, respectively. Antagonistic interactions were not revealed between the investigated compounds. However, the observed high MICs suggest that these agents could not be considered as alternative systemic antifungal agents.

Recurrent Scedosporium apiospermum mycetoma successfully treated by surgical excision and terbinafine treatment: a case report and review of the literature.

BACKGROUND: Scedosporium apiospermum is an emerging opportunistic filamentous fungus, which is notorious for its high levels of antifungal-resistance. It is able to cause localized cutaneous or subcutaneous infections in both immunocompromised and immunocompetent persons, pulmonary infections in patients with predisposing pulmonary diseases and invasive mycoses in immunocompromised patients. Subcutaneous infections caused by this fungus frequently show chronic mycetomatous manifestation. CASE REPORT: We report the case of a 70-year-old immunocompromised man, who developed a fungal mycetomatous infection on his right leg. There was no history of trauma; the aetiological agent was identified by microscopic examination and ITS sequencing. This is the second reported case of S. apiospermum subcutaneous infections in Hungary, which was successfully treated by surgical excision and terbinafine treatment. After 7 months, the patient remained asymptomatic. Considering the antifungal susceptibility and increasing incidence of the fungus, Scedosporium related subcutaneous infections reported in the past quarter of century in European countries were also reviewed. CONCLUSIONS: Corticosteroid treatment represents a serious risk factor of S. apiospermum infections, especially if the patient get in touch with manure-enriched or polluted soil or water. Such infections have emerged several times in European countries in the past decades. The presented data suggest that besides the commonly applied voriconazole, terbinafine may be an alternative for the therapy of mycetomatous Scedosporium infections.

In vitro susceptibility of Scedosporium isolates to N-acetyl-L-cysteine alone and in combination with conventional antifungal agents.

In recent years, Scedosporium species have been more commonly recognized from severe, difficult-to-treat human infections, such as upper respiratory tract and pulmonary infections. To select an appropriate therapeutic approach for these infections is challenging, because of the commonly observed resistance of the causative agents to several antifungal drugs. Therefore, to find a novel strategy for the treatment of pulmonary Scedosporium infections the in vitro antifungal effect of a mucolytic agent, N-acetyl-L-cysteine and its in vitro combinations with conventional antifungals were investigated. Synergistic and indifferent interactions were registered in 23 and 13 cases, respectively. Antagonism was not revealed between the compounds.

Antifungal Susceptibility and Phylogeny of Opportunistic Members of the Genus Fusarium Causing Human Keratomycosis in South India.

Fusarium species are reported frequently as the most common causative agents of fungal keratitis in tropical countries such as India. Sixty-five fusaria isolated from patients were subjected to multilocus DNA sequencing to characterize the spectrum of the species associated with keratitis infections in India. Susceptibilities of these fusaria to ten antifungals were determined in vitro by the broth microdilution method. An impressive phylogenetic diversity of fusaria was reflected in susceptibilities differing at species level. Typing results revealed that the isolates were distributed among species in the species complexes (SCs) of F. solani (FSSC; n = 54), F. oxysporum (FOSC; n = 1), F. fujikuroi (FFSC; n = 3), and F. dimerum (FDSC; n = 7). Amphotericin B, voriconazole, and clotrimazole proved to be the most effective drugs, followed by econazole.

Antifungal Effect of Essential Oils against Fusarium Keratitis Isolates.

The present study was carried out to investigate the antifungal effects of Cinnamomum zeylanicum, Citrus limon, Juniperus communis, Eucalyptus citriodora, Gaultheria procumbens, Melaleuca alternifolia, Origanum majorana, Salvia sclarea, and Thymus vulgaris essential oils against Fusarium species, the most common etiologic agents of filamentous fungal keratitis in South India. C. zeylanicum essential oil showed strong anti-Fusarium activity, whereas all the other tested essential oils proved to be less effective. The main component of C. zeylanicum essential oil, trans-cinnamaldehyde, was also tested and showed a similar effect as the oil. The in vitro interaction between trans-cinnamaldehyde and natamycin, the first-line therapeutic agent of Fusarium keratitis, was also investigated; an enhanced fungal growth inhibition was observed when these agents were applied in combination. Light and fluorescent microscopic observations revealed that C. zeylanicum essential oil/trans-cinnamaldehyde reduces the cellular metabolism and inhibits the conidia germination. Furthermore, necrotic events were significantly more frequent in the presence of these two compounds. According to our results, C. zeylanicum essential oil/trans-cinnamaldehyde provides a promising basis to develop a novel strategy for the treatment of Fusarium keratitis.