Taxonomy of Candida parapsilosis complex isolated from neonates and the role of Hsp90 inhibitors to enhanced the antifungal activity of micafungin.

Species from Candida parapsilosis complex are frequently found in neonatal candidemia. The antifungal agents to treat this infection are limited and the occurrence of low in vitro susceptibility to echinocandins such as micafungin has been observed. In this context, the chaperone Hsp90 could be a target to reduce resistance. Thus, the objective of this research was to identify isolates from the C. parapsilosis complex and verify the action of Hsp90 inhibitors associated with micafungin. The fungal identification was based on genetic sequencing and mass spectrometry. Minimal inhibitory concentrations were determined by broth microdilution method according to Clinical Laboratory and Standards Institute. The evaluation of the interaction between micafungin with Hsp90 inhibitors was realized using the checkerboard methodology. According to the polyphasic taxonomy, C. parapsilosis sensu stricto was the most frequently identified, followed by C. orthopsilosis and C. metapsilosis, and one isolate of Lodderomyces elongisporus was identified by genetic sequencing. The Hsp90 inhibitor geladanamycin associated with micafungin showed a synergic effect in 31.25% of the isolates, a better result was observed with radicicol, which shows synergic effect in 56.25% tested yeasts. The results obtained demonstrate that blocking Hsp90 could be effective to reduce antifungal resistance to echinocandins.

Caspofungin alone or combined with polymyxin B are effective against mixed biofilm of Aspergillus fumigatus and carbapenem-resistant Pseudomonas aeruginosa.

Aspergillus fumigatus and Pseudomonas aeruginosa biofilms are associated to the recalcitrant and persistent infections due to resistance to antimicrobials. Here, we evaluated the effect of antimicrobials on single and mixed biofilms of A. fumigatus and P. aeruginosa (carbapenem-resistant and susceptible strains) determining total biomass by crystal violet, cell viability by colony forming unit count, and microscopy. Polymyxin B (PMB) had the best action on P. aeruginosa biofilms inhibiting the biomass (2-4 µg/mL) and it was efficient reducing the viable bacterial cells. Amphotericin B (AMB) and caspofungin (CAS) were the best antifungal at inhibiting A. fumigatus biofilms and reducing fungal viability at concentration ≥1 and ≥ 16 µg/mL, respectively. In addition, CAS was able to significantly reduce P. aeruginosa viability in mixed biofilms. CAS combined with PMB also significantly reduced the mixed biofilm biomass and fungal and bacterial viability mainly against carbapenem-resistant bacterium. The light and fluorescence microscopy showed alterations on hyphae morphology and confirmed the increase of fungal and bacterial death cells after combined therapy of mixed biofilms. Taken together, our work showed that CAS alone and its combination with PMB showed better potential in reducing mixed biofilm biomass and fungal and bacterial viability, even for the carbapenem-resistant P. aeruginosa strain.

The spread of cat-transmitted sporotrichosis due to Sporothrix brasiliensis in Brazil towards the Northeast region.

BACKGROUND: Sporotrichosis is a worldwide subcutaneous mycosis caused by Sporothrix spp. In the past, this infection was associated with armadillo hunting, horticulturists, miners, and gardeners, being considered an implantation mycosis acquired by plant debris injury. Nevertheless, since the late nineties, it has been considered a zoonotic disease in Brazil. Here we report a case series of 121 patients with cat-transmitted sporotrichosis seen in Northeast Brazil. METHODOLOGY/PRINCIPAL FINDINGS: Patient's demographic, clinical data, and length of treatment were recorded. In addition, a mycological examination and further PCR confirmation of species identification were performed. One hundred and twenty two patients were diagnosed with subcutaneous sporotrichosis from October 2016 to December 2019, while PCR revealed that 71 of them were due to S. brasiliensis. The majority of the individuals were female (n = 86; 70.5%). Patient's age ranged from 5 to 87 years old. The clinical forms found were lymphocutaneous (58.2%) and fixed cutaneous (39.4%). Interestingly, 115 patients reported previous contact with cats diagnosed with sporotrichosis. Patients were successfully treated with itraconazole and potassium iodide. CONCLUSIONS/SIGNIFICANCE: Our study adds important contributions for the investigation of the spread of cat-transmitted subcutaneous sporotrichosis in Brazil, specifically towards the Northeast region of a continental-size country. It will also help clinicians to be aware of the existence and importance to accurately diagnose sporotrichosis and treat patients with this infectious disease in the lowest income region of Brazil.

Oral colonization by Candida spp. in liver transplant patients: Molecular identification and antifungal susceptibilityOral colonization by Candida spp. in liver transplant.

Candida species are commensal to normal oral microbiota; however, they can cause infections if immune functions are reduced. The aim of this study was to investigate oral colonization, identify species, and test the susceptibility profile to antifungals. A descriptive study included 97 liver transplant patients who attended the transplant center of a referral hospital in southern Brazil. Two oral swab collections were performed, with a 6-month gap between collections. The samples were identified by sequencing the internal transcribed spacer ITS region of the ribosomal DNA. The sensitivity test was performed with fluconazole, amphotericin B, and micafungin using a broth microdilution method recommended by Clinical and Laboratory Standards Institute document M27-A4. Eighty-two patients were investigated and 15 were excluded for presenting clinical infection. The identification of yeasts showed colonization in 66% and 61.9% in collections A and B, respectively. Candida albicans was the most prevalent species in both collections (n = 29/50 and n = 27/49, respectively). In 31 (62%) patients, the yeast species remained the same for 6 months, and in 19 (38%) the colonizing species was substituted. Thirty-two isolates from collection A were sensitive (S) to Fluconazole, 13 sensitive dose-dependent (SDD), and five resistant (R). In collection B, 32 were S, 12 SDD, and 5 R. For amphotericin B and micafungin, all isolates were sensitive. With knowledge of the species and identification of strains resistant to fluconazole, useful information can be alerts about the emergence of antifungal resistance strains. LAY SUMMARY: Study of great importance because it is the first investigation that identifies Candida in the oral cavity of liver transplant patients, allowing an understanding of epidemiology and contributing to the knowledge about strains resistant to fluconazole.

Efficacy of voriconazole in vitro and in invertebrate model of cryptococcosis.

Cryptococcosis is a common opportunistic infection in patients with advanced HIV infection and may also affect immunocompetent patients. The available antifungal agents are few and other options are needed for the cryptococcosis treatment. In this work, we first analyzed the virulence of twelve C. neoformans and C. gattii strains assessing capsule thickness, biofilms formation, and survival and morbidity in the invertebrate model of Galleria mellonella and then we evaluated the antifungal activity of voriconazole (VRC) in vitro and in vivo also using G. mellonella. Our results showed that all Cryptococcus spp. isolates were able to produce capsule and biofilms, and were virulent using G. mellonella model. The VRC has inhibitory activity on planktonic cells with MIC values ranging from 0.03 to 0.25 µg/mL on Cryptococcus spp.; and these isolates were more tolerant to fluconazole (ranging from 0.25 to 16 µg/mL), the triazol agent often recommended alone or in combination with amphotericin B in the cryptococcosis therapy. In contrast, mature biofilms were less susceptible to the VRC treatment. The VRC (10 or 20 mg/kg) treatment of infected G. mellonella larvae significantly increased the larval survival when compared to the untreated group for the both Cryptococcus species and significantly decreased the fungal burden and dissemination in the larval tissue. Our findings corroborate with the literature data, supporting the potential use of VRC as an alternative for cryptococcosis treatment. Here, we emphasize the use of G. mellonella larval model as an alternative animal model for studies of antifungal efficacy on mycosis, including cryptococcosis.

Candida tropicalis geographic population structure maintenance and dispersion in the coastal environment may be influenced by the climatic season and anthropogenic action.

Candida tropicalis is a pathogenic yeast with worldwide recognition as the second or third more frequently isolated species in Latin America, for both superficial and systemic infections. Because of its high prevalence, and growing clinical interest, it is essential to understand genetic variability patterns of this important Candida species in the tropics. Besides belonging to the human normal microbiota, C. tropicalis may be found in other warm blood animals and in the environment, including water and sand of beaches. The aims of the present study were to evaluate genotypic and phenotypic variability of 62 isolates of C. tropicalis obtained from the coastal environment in Northeast Brazil using microsatellite and MALDI-TOF/MS comparisons. There was a relatively low correspondence between these typing techniques employed. Therefore, further studies are needed to consolidate the use of MALDI-TOF/MS as a yeast typing tool. Nevertheless, the two methods employed demonstrated the heterogeneity of C. tropicalis in a coastal environment. We also found relative maintenance of the population structure within the same season, which may reinforce the idea that this species presents the potential to remain in the environment for a long period of time. In addition, highly related strains were found within different geographic points of collection, demonstrating that this strain may be dispersed at long distances, probably influenced by anthropogenic actions and driven by the sea tides and wind.

Trichosporon inkin meningitis in Northeast Brazil: first case report and review of the literature.

BACKGROUND: Trichosporon species may colonize the skin, respiratory tract and gastrointestinal tract of human beings. The yeast is recognized as etiological agent of white piedra, a superficial mycosis. Nevertheless, immunocompromised hosts may develop invasive Trichosporonosis. Central nervous system trichosporonosis is a very rare clinical manifestation. In fact, only a few cases have been published in the literature and none of them was caused by Trichosporon inkin. CASE PRESENTATION: Here we report the first clinical case of meningoencephalitis due to this species in a female previously healthy patient under corticosteroids and antibiotics therapy for several months. She was submitted to an invasive procedure to remove a left sided acoustic neuroma and further developed a cerebrospinal fistula. After some days of the procedure, she presented a predominantly and intensive occipital holocranial headache, followed by vomiting, hyporexia, weight loss, asthenia, irritability, difficulty to concentrate and rotator vertigo. The patient further developed a cerebrospinal fistula in the occipital region and was submitted to a surgical correction. After several months of clinical interventions, she was diagnosed with CNS Trichosporonosis, after Magnetic Resonance Imaging and positive microbiological cultures obtained within two different occasions (2 weeks apart). Despite the antifungal therapy with Amphotericin B and Voriconazole, the patient did not survive. CONCLUSIONS: Despite CNS Fungal infections are mostly due to Cryptococcus spp., other emergent yeasts, such as T. inkin may be considered as a likely etiological agent. This is the first case report of CNS Trichosporonosis, where species identification was performed with rDNA sequencing.

Genetic diversity of medically important and emerging Candida species causing invasive infection.

BACKGROUND: Genetic variation in the ribosomal DNA (rDNA) internal transcribed spacer (ITS) region has been studied among fungi. However, the numbers of ITS sequence polymorphisms in the various Candida species and their associations with sources of invasive fungal infections remain poorly investigated. Here, we characterized the intraspecific and interspecific ITS diversity of Candida spp. strains collected from patients with bloodstream or oroesophageal candidiasis. METHODS: We selected cultures of representative medically important species of Candida as well as some rare and emerging pathogens. Identification was performed by micromorphology and by biochemical testing using an ID32C system, as well as by the sequencing of rDNA ITS. The presence of intraspecific ITS polymorphisms was characterized based on haplotype networks, and interspecific diversity was characterized based on Bayesian phylogenetic analysis. RESULTS: Among 300 Candida strains, we identified 76 C. albicans, 14 C. dubliniensis, 40 C. tropicalis, 47 C. glabrata, 34 C. parapsilosis (sensu stricto), 31 C. orthopsilosis, 3 C. metapsilosis, 21 Meyerozyma guilliermondii (C. guilliermondii), 12 Pichia kudriavzevii (C. krusei), 6 Clavispora lusitaniae (C. lusitaniae), 3 C. intermedia, 6 Wickerhamomyces anomalus (C. pelliculosa), and 2 C. haemulonii strains, and 1 C. duobushaemulonii, 1 Kluyveromyces marxianus (C. kefyr), 1 Meyerozyma caribbica (C. fermentati), 1 Pichia norvegensis (C. norvegensis), and 1 Lodderomyces elongisporus strain. Out of a total of seven isolates with inconsistent ID32C profiles, ITS sequencing identified one C. lusitaniae strain, three C. intermedia strains, two C. haemulonii strains and one C. duobushaemulonii strain. Analysis of ITS variability revealed a greater number of haplotypes among C. albicans, C. tropicalis, C. glabrata and C. lusitaniae, which are predominantly related to endogenous sources of acquisition. Bayesian analysis confirmed the major phylogenetic relationships among the isolates and the molecular identification of the different Candida spp. CONCLUSIONS: Molecular studies based on ITS sequencing are necessary to identify closely related and emerging species. Polymorphism analysis of the ITS rDNA region demonstrated its utility as a genetic marker for species identification and phylogenetic relationships as well as for drawing inferences concerning the natural history of hematogenous infections caused by medically important and emerging Candida species.

International Society of Human and Animal Mycology (ISHAM)-ITS reference DNA barcoding database--the quality controlled standard tool for routine identification of human and animal pathogenic fungi.

Human and animal fungal pathogens are a growing threat worldwide leading to emerging infections and creating new risks for established ones. There is a growing need for a rapid and accurate identification of pathogens to enable early diagnosis and targeted antifungal therapy. Morphological and biochemical identification methods are time-consuming and require trained experts. Alternatively, molecular methods, such as DNA barcoding, a powerful and easy tool for rapid monophasic identification, offer a practical approach for species identification and less demanding in terms of taxonomical expertise. However, its wide-spread use is still limited by a lack of quality-controlled reference databases and the evolving recognition and definition of new fungal species/complexes. An international consortium of medical mycology laboratories was formed aiming to establish a quality controlled ITS database under the umbrella of the ISHAM working group on "DNA barcoding of human and animal pathogenic fungi." A new database, containing 2800 ITS sequences representing 421 fungal species, providing the medical community with a freely accessible tool at http://www.isham.org/ and http://its.mycologylab.org/ to rapidly and reliably identify most agents of mycoses, was established. The generated sequences included in the new database were used to evaluate the variation and overall utility of the ITS region for the identification of pathogenic fungi at intra-and interspecies level. The average intraspecies variation ranged from 0 to 2.25%. This highlighted selected pathogenic fungal species, such as the dermatophytes and emerging yeast, for which additional molecular methods/genetic markers are required for their reliable identification from clinical and veterinary specimens.

Trichosporon inkin: an uncommon agent of scalp white piedra. Report of four cases in Brazilian children.

We report four cases of scalp white piedra (SWP) in Brazilian female children. Morphological and physiological approaches gave inconsistent results for identifying Trichosporon to species level, while the sequencing of the intergenic spacer 1 region of ribosomal DNA accurately identified the agent of SWP as T. inkin. These cases emphasize the occurrence of this species causing this type of infection. The molecular identification of the suspected agent is needed for appropriate epidemiological surveillance of superficial mycoses caused by Trichosporon species.