A population genetics analysis in clinical isolates of Sporothrix schenckii based on calmodulin and calcium/calmodulin‐dependent kinase partial gene sequences

Sporotrichosis is a subcutaneous mycosis that is caused by diverse species of Sporothrix . High levels of genetic diversity in Sporothrix isolates have been reported, but few population genetics analyses have been documented. To analyse the genetic variability and population genetics relations of Sporothrix schenckii Mexican clinical isolates and to compare them with other reported isolates. We studied the partial sequences of calmodulin and calcium/calmodulin‐dependent kinase genes in 24 isolates; 22 from Mexico, one from Colombia, and one ATCC ^®6331?; the latter was used as a positive control. In total, 24 isolates were analysed. Phylogenetic, haplotype and population genetic analyses were performed with 24 sequences obtained by us and 345 sequences obtained from GenBank. The frequency of S. schenckii sensu stricto was 81% in the 22 Mexican isolates, while the remaining 19% were Sporothrix globosa . Mexican S. schenckii sensu stricto had high genetic diversity and was related to isolates from South America. In contrast, S. globosa showed one haplotype related to isolates from Asia, Brazil, Spain and the USA . In S. schenckii sensu stricto, S. brasiliensis and S. globosa, haplotype polymorphism (θ) values were higher than the nucleotide diversity data (π). In addition, Tajima's D plus Fu and Li's tests analyses displayed negative values, suggesting directional selection and arguing against the model of neutral evolution in these populations. In addition, analyses showed that calcium/calmodulin‐dependent kinase was a suitable genetic marker to discriminate between common Sporothrix species.     

Molecular typing of Sporothrix schenckii isolates from cats in Malaysia

Epidemiological data on the aetiologic agents of feline sporotrichosis in Malaysia have not been reported, though human sporotrichosis in Malaysia is reported to be transmitted primarily via cat scratch. To the best of our knowledge, the present report is the first study of the molecular epidemiology of Sporothrix schenckii isolates from cats with sporotrichosis in Malaysia. In the present work, we characterised 18 clinical isolates from cats in Malaysia based on molecular properties, including sequence analyses of the calmodulin gene and the rDNA ITS region and selective PCR of mating type (MAT) loci. In this study, isolates from feline sporotrichosis were identified as a S. schenckii sensu stricto by sequence analyses of the calmodulin gene and the internal transcribed spacer (ITS) region. Notably, phylogenetic analysis of the ITS confirmed assignment to clinical clade D (and not C) of S. schenckii sensu stricto. Therefore, clinical clade D of S. schenckii sensu stricto appeared to be the prevailing source of feline sporotrichosis in Malaysia. The ratio of MAT1‐1‐1:MAT1‐2‐1 in these Malaysian isolates was found to be 1 : 0. This result suggested that a clonal strain of S. schenckii is the prevailing causative agent of feline sporotrichosis in Malaysia.     

Seventeen years of subcutaneous infection by Aspergillus flavus; eumycetoma confirmed by immunohistochemistry

Chronic subcutaneous infections caused by Aspergillus species are considered to be extremely rare. Because these fungi are among the most common laboratory contaminants, their role as eumycetoma causative agents is difficult to ascertain. Here, we report the first case of A. flavus eumycetoma confirmed by isolation, molecular identification and immunohistochemical analysis. Patient was a 55‐year‐old male from Sudan suffering from eumycetoma on his left foot for a period of 17 years. He developed swelling, sinuses and white grain discharge was observed. He has been operated nine times and was treated with several regimens of ketoconazole and itraconazole without improvement. Initial diagnosis based on histology and radiology was Scedosporium eumycetoma. However, examination of the biopsy revealed A. flavus, which was identified by molecular analysis and MALDI‐TOF MS. Immunohistochemistry using antibody directed against Aspergillus species was positive. Because of the earlier treatment failures with ketoconazole and itraconazole, therapy with voriconazole was initiated. However, in vitro susceptibility testing yielded a lower Minimum Inhibitory Concentration (MIC) value for itraconazole (0.25 μg ml^?1) than for voriconazole (1 μg ml^?1). Based on the presented results, A. flavus can be considered as one of the agents of white‐grain eumycetoma.     

Trichosporon asahii infection presenting as chronic meningo‐ventriculitis and intra ventricular fungal ball: a case report and literature review

Central nervous system trichosporonosis is a rare clinical entity and so far only six cases including three each of brain abscess and meningitis has been on record. We report a rare case of chronic meningo‐ventriculitis and intraventricular fungal ball due to Trichosporon asahii in an 18‐year‐old immunocompetent male from Burundi, east Africa. Neuroendoscopy showed multiple nodules and a fungal ball within the ventricle, which on culture grew T. asahii. He was initially empirically treated with liposomal amphotericin B. However, the antifungal susceptibility testing of T. asahii isolate revealed high minimum inhibitory concentration for amphotericin B (2 μg ml^?1), flucytosine (16 μg ml^?1) and caspofungin (2 μg ml^?1) but exhibited potent activity for voriconazole, posaconazole, itraconazole and fluconazole. The patient rapidly succumbed to cardiac arrest before antifungal therapy could be changed. Although disseminated trichosporonosis has been increasingly reported the diagnosis represents a challenge especially in rare clinical settings such as intraventricular fungal ball in the present case, which has not been described previously.     

Modest efficacy of voriconazole against murine infections by Sporothrix schenckii and lack of efficacy against Sporothrix brasiliensis

The efficacy of voriconazole (VRC) was evaluated against two strains of each of the two most common species causing sporotrichosis, Sporothrix schenckii sensu stricto and Sporothrix brasiliensis, using a murine model of disseminated infection. Voriconazole was administered at doses of 20 or 40 mg kg^?1 per day by gavage. The drug showed some efficacy, especially at 40 mg kg^?1 per day, in prolonging the survival and reducing fungal load in spleen and liver in mice infected with S. schenckii, whereas in animals infected with S. brasiliensis the drug did not work.     

Mating genotypes and susceptibility profiles of clinical isolates of Candida glabrata from Turkey

The sexual cycle of Candida glabrata is not known; however, genomic evidence is indicative of recombination among subpopulations and the genome harbours genes necessary for undergoing mating and meiosis, which may increase fitness. The relationship between specific mating type‐like (MTL) loci and antifungal susceptibility is not well understood in C. glabrata. We investigated different combinations of clinical C. glabrata isolate mating types and their antifungal susceptibility profiles. Allele profiles of the mating genes of 103 clinical C. glabrata isolates were identified, and their antifungal susceptibility to azoles, echinocandins and amphotericin B were compared. The majority (88.3%) of screened isolates harboured the a allele in the locus. The MTL1, MTL2 and MTL3 loci harboured a (88.3%), a (95.1%), and α (71.8%) alleles, respectively. The C. glabrata isolates were susceptible to echinocandins but displayed high minimal inhibitory concentrations (MICs) for azoles. The MIC ranges and MIC90 values of all isolates were 1.0 to ≥64 and 8.0 μg mL^?1 for fluconazole, 0.06 to ≥16.0 and 0.5 μg mL^?1 for voriconazole, 0.06 to ≥16.0 and 1.0 μg mL^?1 for posaconazole, ≤0.015 to 0.06, and 0.03 μg mL^?1 for caspofungin, ≤0.015 to 0.06 and 0.015 μg mL^?1 for anidulafungin and 0.5‐2 and 2.0 μg mL^?1 for amphotericin B, respectively. The mating gene alleles of the clinical C. glabrata isolates were not associated with differences in the MICs of the tested antifungals, except for the MTL3 α‐allele and echinocandins. The mating genotypes of the clinical C. glabrata isolates had no recognisable common effect on antifungal susceptibility.     

An anti‐Aspergillus protein from Escherichia coli DH5α: Putative inhibitor of siderophore biosynthesis in Aspergillus fumigatus

An antifungal protein designated as anti‐Aspergillus protein (AAP), produced by Escherichia coli DH5α, was purified and characterised. It exhibited a molecular weight of 60 kDa on Sodium dodecyl sulphate–polyacrylamide gel electrophoresis analysis and depicted 99% purity on ultra performance liquid chromatography. The purified protein manifested antimycotic potential against pathogenic isolates of Aspergillus spp., depicting a minimum inhibitory concentration in the range 15.62–31.25 μg ml^?1 and 5.0–10.0 μg per disc, using microbroth dilution, spore germination inhibition and disc diffusion assays respectively. In vitro toxicity tests demonstrated that it showed no toxicity against human erythrocytes at doses up to 1000 μg ml^?1. Matrix‐assisted laser desorption ionisation–Time‐of‐flight analysis of trypsin‐digested peptides of purified protein and subsequent Mascot search revealed that several peptides of AAP have identity with bacterial siderophore biosynthetic protein, i.e. non‐ribosomal peptide synthetase enzyme, involved in critical step of fungal siderophore biosynthesis. Siderophore‐based inhibition was further corroborated by Chrome azurol S assay. Hence, the antagonistic effect might be the result of impediment in siderophore‐mediated iron uptake and transport process which may cause critical consequences on Aspergillus growth and virulence.     

Serum posaconazole levels among haematological cancer patients taking extended release tablets is affected by body weight and diarrhoea: single centre retrospective analysis

The posaconazole extended release tablet formulation was developed to improve bioavailability relative to the oral suspension. Therapeutic drug monitoring has been used to optimise posaconazole dosing to achieve a target trough level ≥0.7 μg ml^?1. We retrospectively evaluated 28 patients with haematological malignancies who received posaconazole tablets for antifungal prophylaxis. Posaconazole serum trough levels were obtained 5 days after initiation of therapy. Mean trough level was 1.19 ± 0.63 μg ml^?1, and 71% achieved a trough level ≥0.7 μg ml^?1. Diarrhoea was associated with lower mean trough levels (0.65 ± 0.08 μg ml^?1 vs. 1.31 ± 0.13 μg ml^?1), P = 0.002. Mean trough levels were lower in patients ≥90 kg (0.74 ± 0.09 μg ml^?1) vs. <90 kg (1.32 ± 0.14 μg ml^?1), P = 0.002 and in patients with body mass index (BMI) ≥30 (0.89 ± 0.13 μg ml^?1) vs. BMI <30 (1.29 ± 0.14 μg ml^?1), P = 0.05. Posaconazole delayed release tablets attain appropriate trough levels in most patients, but patients with a higher weight and those experiencing diarrhoea are more likely to have lower levels.     

Susceptibility profile and epidemiological cut‐off values of Cryptococcus neoformans species complex from Argentina

Epidemiological cut‐off values (ECVs) based on minimal inhibitory concentration (MIC) distribution have been recently proposed for some antifungal drug/Cryptococcus neoformans combinations. However, these ECVs vary according to the species studied, being serotypes and the geographical origin of strains, variables to be considered. The aims were to define the wild‐type (WT) population of the C. neoformans species complex (C. neoformans) isolated from patients living in Argentina, and to propose ECVs for six antifungal drugs. A total of 707 unique C. neoformans isolates obtained from HIV patients suffering cryptococcal meningitis were studied. The MIC of amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole and posaconazole was determined according to the EDef 7.2 (EUCAST) reference document. The MIC distribution, MIC₅₀, MIC₉₀ and ECV for each of these drugs were calculated. The highest ECV, which included ≥95% of the WT population modelled, was observed for flucytosine and fluconazole (32 μg ml^?1 each). For amphotericin B, itraconazole, voriconazole and posaconazole, the ECVs were: 0.5, 0.5, 0.5 and 0.06 μg ml^?1 respectively. The ECVs determined in this study may aid in identifying the C. neoformans strains circulating in Argentina with decreased susceptibility to the antifungal drugs tested.     

Comparative in vitro fungicidal activity of echinocandins against Candida albicans in peritoneal dialysis fluids

The peritoneal dialysis (PD)‐associated peritonitis caused by fungi is a relatively rare, but very serious disease. PD fluids (PDFs) affect inhibitory efficacy on the microorganisms' growth, which may compromise the affectivity of some antimicrobials. The purpose of this study was to investigate in vitro the fungicidal effectiveness of echinocandins in diverse PDFs. The fungicidal efficacy of caspofungin (CAS), anidulafungin (ANA), micafungin (MYC) against five clinical isolates of Candida albicans was studied in the different PDFs using time‐kill curves. As control substance amphotericin B was used. Echinocandins showed slower and reduced killing of C. albicans in PDFs when compared with the time‐kill curves in control bouillon. At concentration of 8 × minimal inhibitory concentration (MIC) the greatest reduction in the growth of C. albicans was seen by ANA in lactate‐buffered Nutrineal PD4^® with 1.1% amino acid (2.33 ± 0.52 log₁₀ CFU ml^?1), and by CAS and MYC in lactate‐buffered Dianeal PD4^® with 1.36% glucose (2.36 ± 0.89 log₁₀ CFU ml^?1 and 2.36 ± 0.99 log₁₀ CFU ml^?1 respectively). Using high concentration of 128 × MIC echinocandins achieved fungicidal effect in all PDFs. PDFs may significantly impair the activities of echinocandins, but fungicidal activity of drugs can be achieved at high concentration of 128 × MIC.     

Molecular epidemiology and in vitro antifungal susceptibility testing of 108 clinical Cryptococcus neoformans sensu lato and Cryptococcus gattii sensu lato isolates from Denmark

Cryptococcosis is mainly caused by members of the Cryptococcus gattii/Cryptococcus neoformans species complexes. Here, we report the molecular characterisation and in vitro antifungal susceptibility of Danish clinical cryptococcal isolates. Species, genotype, serotype and mating type were determined by amplified fragment length polymorphism (AFLP) fingerprinting and qPCR. EUCAST E.Def 7.2 MICs were determined for amphotericin B, flucytosine, fluconazole, voriconazole and isavuconazole. Most isolates were C. neoformans (serotype A; n = 66) and belonged to genotype AFLP1/VNI (n = 61) or AFLP1B/VNII (n = 5) followed by Cryptococcus deneoformans (serotype D; genotype AFLP2, n = 20), C. neoformans × C. deneoformans hybrids (serotype AD; genotype AFLP3, n = 13) and Cryptococcus curvatus (n = 2). Six isolates were C. gattii sensu lato, and one isolate was a C. deneoformans × C. gattii hybrid (genotype AFLP8). All isolates were amphotericin B susceptible. Flucytosine susceptibility was uniform MIC₅₀ of 4–8 mg l^?1 except for C. curvatus (MICs >32 mg l^?1). Cryptococcus gattii sensu lato isolates were somewhat less susceptible to the azoles. MICs of fluconazole (>32 mg l^?1), voriconazole (≥0.5 mg l^?1) and isavuconazole (0.06 and 0.25 mg l^?1 respectively) were elevated compared to the wild‐type population for 1/19 C. deneoformans and 1/2 C. curvatus isolates. Flucytosine MIC was elevated for 1/61 C. neoformans (>32 mg l^?1). Antifungal susceptibility revealed species‐specific differential susceptibility, but suggested acquired resistance was an infrequent phenomenon.     

Invasive infections due to Saprochaete and Geotrichum species: Report of 23 cases from the FungiScope Registry

Saprochaete and Geotrichum spp. are rare emerging fungi causing invasive fungal diseases in immunosuppressed patients and scarce evidence is available for treatment decisions. Among 505 cases of rare IFD from the FungiScope^? registry, we identified 23 cases of invasive infections caused by these fungi reported from 10 countries over a 12‐year period. All cases were adults and previous chemotherapy with associated neutropenia was the most common co‐morbidity. Fungaemia was confirmed in 14 (61%) cases and deep organ involvement included lungs, liver, spleen, central nervous system and kidneys. Fungi were S. capitata (n=14), S. clavata (n=5), G. candidum (n=2) and Geotrichum spp. (n=2). Susceptibility was tested in 16 (70%) isolates. All S. capitata and S. clavata isolates with the exception of one S. capitata (MIC 4 mg/L) isolate had MICs>32 mg/L for caspofungin. For micafungin and anidulafungin, MICs varied between 0.25 and >32 mg/L. One case was diagnosed postmortem, 22 patients received targeted treatment, with voriconazole as the most frequent first line drug. Overall mortality was 65% (n=15). Initial echinocandin treatment was associated with worse outcome at day 30 when compared to treatment with other antifungals (amphotericin B ± flucytosine, voriconazole, fluconazole and itraconazole) (P=.036). Echinocandins are not an option for these infections.     

Inibitory effects of linalool on fungal pathogenicity of clinical isolates of Microsporum canis and Microsporum gypseum

In humans worldwide, Microsporum sp. is a frequent agent of dermatophytsosis. When considering the emergence of resistant fungi and the clinical relevance of dermatophytosis, terpene antifungal activity is of great interest. Linalool is a monoterpene alcohol with pharmacological properties. In this study, antifungal in vitro activity of linalool and ketoconazole (as a positive control) were evaluated against clinical isolates of M. canis and M. gypseum. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of each drug were determined by broth microdilution. The effects of the drugs (1/2MIC, MIC, 2xMIC) on radial mycelial growth, conidial production and germination were analysed. The effect on the fungal cell membrane (release of intracellular material) was also investigated. Linalool (MIC: 128 μg/mL) and ketoconazole (MIC: 64 μg/mL) were effective in inhibiting all dermatophytes studied. The MFC values of linalool ranged between 128 and 256 μg/mL, whereas ketoconazole showed MFC values of from 64 to 256 μg/mL. Linalool (at MIC and 2xMIC) and ketoconazole (at 1/2MIC, MIC, 2xMIC) inhibited mycelial growth (P < 0.05). The drugs (1/2MIC, MIC, 2xMIC) were also active on conidiogenesis and conidia germination, causing complete inhibition (P < 0.05). Linalool caused leakage of intracellular material. Our study supports the use of linalool as a potential antifungal agent against M. canis and M. gypseum.     

In vitro activity of colistin as single agent and in combination with antifungals against filamentous fungi occurring in patients with cystic fibrosis

Because published reports indicate that the antibiotic colistin (COL) has antifungal properties, this study investigated the antifungal in vitro activity of COL as single agent and in combination with the antifungal compounds voriconazole (VRC), caspofungin (CAS) and amphotericin B (AMB) against Scedosporium/Pseudallescheria spp., Exophiala dermatitidis and Geosmithia argillacea. In total, susceptibility was determined for 77 Scedosporium/Pseudallescheria spp., 82 E. dermatitidis and 17 G. argillacea isolates. The minimal inhibitory concentrations (MICs) of COL and the antifungals as single compound and in combination were determined with MIC test strips. Drug interactions were detected by crossing the MIC test strips at a 90º angle. The fractional inhibitory concentration index was used to categorise the drugs’ interaction. The MIC₅₀ value of COL was 12 μg ml^?1 for S. prolificans, 16 μg ml^?1 for P. apiosperma, 16 μg ml^?1 for P. boydii, 12 μg ml^?1 for E. dermatiditis and 6 μg ml^?1 for G. argillacea. VRC was the most active drug in combination without any antagonism with the exception of few P. boydii isolates. COL as single agent and in most combinations with antifungals exhibits in vitro antifungal activity against filamentous ascomycetes occurring in cystic fibrosis patients and may offer a novel therapeutic option, especially for multidrug‐resistant S. prolificans.     

Susceptibility of Malassezia pachydermatis to aminoglycosides

Previous studies have evaluated the action of gentamicin against Malassezia pachydermatis. The aim of this study was to evaluate in vitro susceptibility of M. pachydermatis to the aminoglycosides— gentamicin, tobramycin, netilmicin and framycetin. The minimum inhibitory concentration (MIC) of gentamicin was determined following methods M27‐A3 microdilution and Etest^®. The Etest^® was used to determine the minimum inhibitory concentration (MIC) of the tobramycin and netilmicin. The Kirby‐Bauer test was used to determine the antibiotic susceptibility to the framycetin. The MIC50 and MIC90 were 8.12 μg/mL and 32.5 μg/mL by microdilution method for gentamicin. The MIC50, determined by the Etest^®, was 8 μg/mL for gentamicin and netilmicin and 64 μg/mL for tobramycin. The MIC90 was 16 and 32 μg/mL for gentamicin and netilmicin respectively. The MIC90 was outside of the detectable limits for tobramycin. To framycetin, 28 strains (40%) of the 70 M. pachydermatis isolates tested showed a diameter of 22 mm, 22 strains (31.42%) showed a diameter of 20 mm, 16 strains showed a diameter of ≤ 18 mm, and only 5.71% of the isolates showed a diameter of ≥ 22 mm. This study provides evidence of high in vitro activity of the aminoglycosides—gentamicin, tobramycin, netilmicin and framycetin against M. pachydermatis. For gentamicin Etest^® showed similar values of MIC50 y MIC90 that the obtained by microdilution method. We considered Etest^® method could be a good method for these calculations with aminoglycosides.     

Glutathione as a promising anti‐hydrophobicity agent against Malassezia spp.

The genus Malassezia has recently attracted wide attention in medical microbiology and dermatology as a pathogen. They are lipophilic yeasts possessing high level of cell surface hydrophobicity (CSH). l ‐glutathione (GSH) is a ubiquitous antioxidant which offers protection against microbial infections. This study is intended to investigate the role of GSH as a potential anti‐hydrophobicity agent against Malazessia spp. Microbial adherence to hydrocarbon assay was performed to assess the anti‐hydrophobicity activity (AHA) of GSH against four Malassezia spp. The assay revealed that GSH at 400 μg ml^?1 concentration inhibited CSH, ranging from 84% to 95% in M. furfur, M. globosa, M. restricta and M. sympodialis without killing the cells. The AHA of GSH was corroborated by auto‐aggregation assay and zeta‐potential measurement, through which delayed cell aggregation was observed due to reduction in CSH level and not by modification in cell surface charge. In addition, colony‐forming unit assay was performed in which 62–93% of CSH reduction was observed in Malassezia spp. tested. Furthermore, GSH treatment enhanced the sensitivity of Malassezia spp. towards human blood at the rate of 64–72%. The AHA was further confirmed through Fourier transform infrared analysis. Thus, this study portrays GSH as a prospective therapeutic alternative for Malassezia‐mediated infections.     

The role of drug efflux pumps in Malassezia pachydermatis and Malassezia furfur defence against azoles

This study aims to evaluate the effect of efflux pump modulators (EPMs) on the minimal inhibitory concentration (MIC) of fluconazole (FLZ) and voriconazole (VOR) in Malassezia furfur and Malassezia pachydermatis. The in vitro efficacy of azoles, in combination with EPMs (ie haloperidol‐HAL, promethazine‐PTZ and cyclosporine A‐CYS), against 21 M. furfur from bloodstream infection patients and 14 M. pachydermatis from the skin of dogs with dermatitis, was assessed using a broth microdilution chequerboard analysis. Data were analysed using the model‐fractional inhibitory concentration index (FICI) method. The MIC of FLZ and VOR of Malassezia spp. decreased in the presence of sub‐inhibitory concentrations of HAL and/or PTZ. The synergic effect was observed only in strains with FLZ MIC≥128 μg/mL for M. furfur, FLZ MIC≥64 μg/mL for M. pachydermatis and VOR MIC≥4 μg/mL in both Malassezia spp. These results suggest that the drug efflux pumps are involved as defence mechanisms to azole drugs in Malassezia yeast. The synergism might be related to an increased expression of efflux pump genes, eventually resulting in azole resistance phenomena. Finally, the above FLZ and VOR MIC values might be considered the cut‐off to discriminate susceptible and resistant strains.     

Evolution of procalcitonin,C‐reactive protein and fibrinogen levels in neutropenic leukaemia patients with invasive pulmonary aspergillosis or mucormycosis

Unlike bacterial infections, the value of procalcitonin (PCT) in detecting fungal infections in leukaemia patients is not clear. To determine whether the monitoring of PCT coupled with C‐reactive protein (CRP) and fibrinogen (Fib) could be helpful in the management of pulmonary aspergillosis (IPA) or mucormycosis (PM), we retrospectively analysed the evolution of PCT, CRP and Fib levels in 94 leukaemia patients with proven/probable IPA (n = 77) or PM (n = 17) from D?12 to D12 relative to IFI onset defined as D0. Overall, 2140 assays were performed. From D?12 to D0, 12%, 5% and 1.4% of patients had PCT >0.5, 1 and 1.5 μg l^?1, respectively, while CRP was >50, 75 and 100 mg l^?1 in 84%, 70% and 57% and Fib was >4, 5 and 6 g l^?1 in 96%, 80% and 61% of cases respectively (P < 10^?7). The same trends were observed from D1 to D12. Overall, between D?12 and D12, only 6.4% of patients had PCT >1.5 μg l^?1, while CRP >100 mg l^?1 and Fib >6 g l^?1 were observed in 80% and 75% of cases respectively (P < 10^?7). In leukaemia patients, IPA or PM was accompanied by a significant increase in CRP and Fib while PCT remained low.     

Photodynamic fungicidal efficacy of hypericin and dimethyl methylene blue against azole‐resistant Candida albicans strains

Antimicrobial photodynamic therapy (aPDT) is an emerging alternative to treat infections based on the use of photosensitisers (PSs) and visible light. To investigate the fungicidal effect of PDT against azole‐resistant Candida albicans strains using two PSs with a different mechanism of action, hypericin (HYP) and 1,9‐dimethyl methylene blue (DMMB), comparing their efficacy and the reactive oxygen species (ROS) species involved in their cytotoxicity. Azole‐resistant and the azole‐susceptible C. albicans strains were used. Solutions of 0.5 and 4 McFarland inoculum of each Candida strain were treated with different concentrations of each PS, and exposed to two light‐emitting diode light fluences (18 and 37 J cm^?2). Mechanistic insight was gained using several ROS quenchers. The minimal fungicidal concentration of HYP for ≥3 log₁₀ CFU reduction (0.5 McFarland) was 0.62 μmol l^?1 for most strains, whereas for DMMB it ranged between 1.25 and 2.5 μmol l^?1. Increasing the fluence to 37 J cm^?2 allowed to reduce the DMMB concentration. Higher concentrations of both PSs were required to reach a 6 log₁₀ reduction (4 McFarland). H₂O₂ was the main phototoxic species involved in the fungicidal effect of HYP‐aPDT whereas ¹O₂ was more important for DMMB‐based treatments. aPDT with either HYP or DMMB is effective in killing of C. albicans strains independent of their azole resistance pattern. HYP was more efficient at low fungal concentration and DMMB at higher concentrations.     

Evaluation of improvement of onychomycosis in HIV‐infected patients after initiation of combined antiretroviral therapy without antifungal treatment

Onychomycosis in HIV‐infected patients has a prevalence of 20–44% and is more frequently seen with CD4⁺ T cell counts ≤450 cel μl^?1. There are case reports of improvement in onychomycosis after initiation of combined antiretroviral therapy (cART), but there are no prospective studies that prove the existence and frequency of this phenomenon. The aim of this study was to evaluate if HIV‐infected patients with onychomycosis who begin cART improve and/or cure without antifungal treatment. We included HIV‐infected patients with onychomycosis who had not started cART and nor received antifungal therapy during 6 months prior to the study. We evaluated affected the nails with the Onychomycosis Severity Index (OSI); nail scrapings were collected and direct microscopy with potassium hydroxide (KOH) as well as mycological culture were performed. We repeated these procedures at 3 and 6 months to assess changes. CD4 T cell counts and HIV viral load were obtained. A total of 16 patients were included, with male gender predominance (68.7%); distal and lateral subungual onychomycosis (DLSO) was the most common form (31.3%). Trichophyton rubrum was the most frequently isolated microorganism. OSI decreased 21.5% at 3 months and 40% at 6 months after initiation of antiretrovirals (P = 0.05). We found a non‐significant tendency towards improvement with higher CD4⁺ T cell counts and with viral loads <100 000 copies ml^?1. This could be due to the increase in CD4⁺ T cells, decreased percentage of Treg (CD4⁺CD25⁺) among CD4⁺ Tcells and/or a decreased viral load; further studies are necessary to prove these hypothesis.