Insight into the antifungals used to address human infection due to Trichosporon spp.: a scoping review

Trichosporonosis infections have been increasing worldwide. Providing adequate treatment for these infections remains a challenge. This scoping review contains information about potential antifungals to treat this pathology. Using online databases, we found 76 articles published between 2010 and 2020 related to this topic. Classic antifungals, molecules and biomolecules, repositioned drugs and natural products have been tested against species of Trichosporon. Experimental research has lacked depth or was limited to in vitro and in vivo tests, so there are no promising new candidates for the clinical treatment of patients with trichosporonosis. Furthermore, most studies did not present appropriate scientific criteria for drug tests, compromising their quality.


Identifying the research question
The research question was as follows: what types of antifungals involving classic drugs, new molecules and biomolecules, and repositioned drugs have been evaluated against Trichosporon spp.?
Identifying relevant studies Using PubMed and Web of Science, an electronic database search was conducted in groups to find original articles published between 1 January 2010 and 31 May 2020. Two groups were established. The first group was for general search and used the keywords 'Trichosporon and treatment or antifungal'. The second group was a more specific search and used the keywords 'Trichosporon' and 'treatment or antifungal' and 'biofilm'. Only full papers, letters and short communications were considered. All material was published in the English language.

Study selection
Each article was independently assessed for inclusion and quality. One author performed the initial review of publications using the following inclusion criteria: studies needed to have a focus on Trichosporon spp. antifungal susceptibility. In addition, only studies that came from full papers, letters or short communications were considered. The authors separated the articles in groups according to the treatment used (classic antifungals, molecules and biomolecules, repositioned drugs, natural products) using spreadsheets in Google Drive. Characteristics of the articles were recorded in the spreadsheets, such as the article title, author, scientific magazine and publication.
Scientific criteria were set for the exclusion of articles. Articles that did not include Trichosporon spp. or did not present clear minimal inhibitory concentration (MIC) results as well as case reports and reviews were excluded. Studies that determined the MIC by guidelines different from the Clinical & Laboratory Standards Institute were also excluded due the scarcity data. The authors filtered abstracts or full articles using the eligibility criteria. When there was uncertainty about inclusion or exclusion, they evaluated the study and made a decision together.

Article characterization
Articles included in this study were reviewed in detail by all the authors. They were selected to summarize the main data. The following descriptors were used: article aim; classic drugs, molecules and biomolecules, repositioned drugs or natural products tested; study type (in vitro or in vivo); main results; characterization of clinical isolates of Trichosporon spp. (number, species and material in which it was isolated); whether it had been tested on biofilm; protocol and susceptibility method used; and treatment effectiveness determined by MIC determination.

Results & discussion
Trichosporon spp. isolates Using the previously described method, 76 articles were identified involving several Trichosporon spp. (Figure 1); the most studied were T. asahii, T. inkin, T. faecale and T. asteroides [14][15][16]. The majority of clinical isolates were from human biological samples (70 of 76; 92.1%); urine and blood were the most frequent. Fungal infections caused by Trichosporon spp. on nails, skin or hair and in fluids, secretions or biopsy materials were also identified. Thus, these fungi do not have a specific human infection site and can be present in any situation according to patient vulnerability.
Classic drugs evaluated against Trichosporon spp. Various therapies for Trichosporon spp. have been studied over the years. Guidelines have considered that azoles are the primary drug class in therapies, as several species are resistant in vitro to amphotericin B (MIC ≥2 μg/ml), flucytosine (MIC 4-128 μg/ml) and echinocandins (MIC >16 μg/ml) [26]. However, different clinical isolates of the same species have great variability in antifungal susceptibility tests [9,10].  There is great variability in the response of this genus to antifungals. Also, comparing different studies is difficult because of the methodologies used, which are often based on the protocols of Candida spp. Generally, the observed studies presented results showing MIC variations. In this review, 11 of the observed articles provided individual MIC values for the tested samples ( Figure 2) [9][10][11][12][27][28][29][30][31][32][33]. Although there is this great variability in the MIC of FLC, VRC and AMB, concentrations <10 μg/ml are the most frequently used in antifungal susceptibility tests against Trichosporon spp. and were thus used to build the box plot in Figure 2.

Review
Results from several of the observed studies were obtained from the in vitro antifungal susceptibility of the fungi in only planktonic cells. However, infections by Trichosporon spp. are generally associated with biofilm formation, particularly on invasive medical devices [28]. Interestingly, all articles showed that classic drugs alone do not have a positive effect against Trichosporon biofilms [15,22,31,34,35], demonstrating the need for new therapeutic options for this fungal infection. Regarding Trichosporon spp., few studies correlate the in vitro susceptibility profile with the clinical response of the patient. Tsai et al. [28] reported positive outcomes in patients who provided positive cultures for T. asahii and had their clinical characteristics and outcome compared. For five of the patients in this study, MIC values for FLC were low (0.25-2 μg/ml) and they were treated with low doses of FLC (100-400 mg/day); all five were discharged. However, this dosage was not standardized, and more clinical studies are needed to establish guidelines for treatment (e.g., in the management of candidiasis) [36]. Thus, studies similar to those performed by Pfaller et al. [37][38][39] with Candida spp. are currently needed for Trichosporon spp. because their susceptibility cutoff points are not sufficiently clear to guide patient therapy.
Because of several limitations, such as off-target toxicity and drug resistance, there is a need for new, safe and more effective antifungal agents. In addition, Trichosporon spp. often present intrinsic resistance or reduced susceptibility to some drugs, hindering research for potential agents [5].
Biomolecules & synthetic molecules against Trichosporon spp. Current research mostly focuses on obtaining or synthesizing molecules that are potentially active against Trichosporon spp. and also improving antifungals that are already available [1]. Some studies examined the potential of molecules that act on the Trischosporon cell and its virulence factors, such as filamentation, adhesion and biofilm formation ( Figure 3). In addition, the syntheses of compounds with specific antifungal action on planktonic and biofilm cells are also being investigated.
The similarities between fungal and mammalian cells impede the development of novel antifungals with ideal characteristics, such as broad-spectrum effectiveness, enhanced bioavailability, minimal toxicity and minimal side effects. Consequently, drug development against invasive fungal pathogens has been slow.

Drug repositioning as an alternative to new therapeutic options
Drug repositioning is an alternative strategy to new antifungal therapeutic options against Trichosporon spp. Previously approved compounds used for other purposes or drugs that were shelved after failure in clinical trials could be quickly and inexpensively brought into clinical practice [51,52]. Some previously approved compounds have been evaluated regarding antifungal properties.
Sertraline, an antidepressant and anxiolytic from selective serotonin reuptake inhibitor class, exhibited synergic effects in vitro against T. asahii with AMB, CAS and FLC in planktonic cells and with AMB in biofilm forms [53]. The antifungal activity of this antidepressant was first observed in three patients with premenstrual dysphoric disorder and recurrent vulvovaginal candidiasis (CVVR). Clinical symptoms of CVVR disappeared in these patients after treatment with sertraline during therapy. However, they returned when the treatment was interrupted [54]. Other in vitro studies with sertraline showed its antifungal activity against Aspergillus [55], Candida [56,57] and Cryptococcus [58,59]. Sertraline has also shown in vivo anticryptococcal activity [60], but randomized clinical studies for cryptococcal meningitis treatment with sertraline have not shown positive results [61,62].
Tacrolimus is an immunosuppressant widely used for the prevention of transplant rejection, blocking the immune system through inhibition of calcineurin. It was tested against T. asahii. In vitro results showed a synergic effect with AMB and CAS on azole-sensitive isolates but not against resistant strains [63].
Nonsteroidal antiinflammatory drugs, such as aspirin, ibuprofen and diclofenac sodium, have shown potential antifungal activity against pathogenic fungi under planktonic and biofilm forms [64]. One study indicated the potential effects of AMB/ibuprofen and CAS/ibuprofen combinations against T. asahii isolated from patients with trichosporonosis [65].
Ritonavir, an HIV aspartyl protease inhibitor, was tested against T. asahii and T. inkin. This drug inhibited Trichosporon growth, reduced protease activity, decreased cell adhesion and biofilm formation and also altered the structure and matrix composition of the biofilm. Synergism was not observed between ritonavir and antifungals [66].
Diphenyl diselenide and ebselen, which mimic the antioxidant effect of the glutathione peroxidase, were tested against Trichosporon strains. Both compounds showed synergism with some antifungals, mainly with AMB and some other tested antifungals. However, they demonstrated antagonism when combined with FLC [67].
Sodium butyrate, a histone deacetylase inhibitor, reduced the adhesion, biofilm metabolic activity and biomass of T. asahii and T. inkin [68]. In addition, this compound inhibited planktonic growth, interfered with the filamentation of these species, affecting adhesion, development and maturation of the biofilm.
Although drug repositioning is an interesting alternative, it could be applied more appropriately to finding efficient antifungal activity addressing Trichoporon spp. The selection of compounds for experimental investigation should be based on in-depth studies on specific fungal targets, such as the use of high-throughput screening that integrates automation and computational advances with biological knowledge. This allows for evaluation of thousands or even millions of molecules and increases the chances of finding drug candidates [51]. High-throughput screening can be performed using in silico methods [69] or in vitro experiments [70]. Comparative genomics should also be used when selecting novel compounds, which allows for safe molecule targeting when acting against certain fungal targets [71].
Antifungal studies of natural products Natural products have historically been a source of antifungal drugs, such as the polyenes nystatin and AMB, which are potent antifungal antibiotics against a large number of fungi and were initially isolated from strains of Streptomycetes [72]. In the context of the current review, we found 15 published articles involving natural products evaluated against species from the Trichosporon genus. Details regarding 13 of these articles are shown in Table 1. However, the likelihood of discovery of new natural products for antifungal treatments is remote.
A variety of natural products have been explored for their antifungal activity, and most come from plants [20,23,[73][74][75][76][77][78]. Studies have examined the antifungal activity of compounds produced by bees (propolis) [79], synthesized from natural products [80][81][82] and extracted from snake venom [83]. However, no promising candidates for drugs from natural products were found. As far as we know, no in vivo studies have been performed with natural products that targeted Trichosporon spp., including in experimental animals.
In vitro tests of antifungal activity from natural products have been used in only a few small studies. One study tested a compound in vitro against a Trichosporon spp. biofilm [80]. According these authors, resveratrol and Regrapex-R-forte (a dietary supplement that contains the extract of Vitis vinifera grape and extract of Polygonum cuspidatum root) showed antibiofilm effects. They both inhibited biofilm formation and eradicated mature biofilm. 10 The in vitro results with this compound were comparable to AMB, the most efficient antimycotic agent [72]. Such antifungal action could be added to a nutritional effect, recomposing the endogenous intestinal microbiota, and thereby avoiding the dysbiosis, a relevant point today [84]. However, the use of various methods to assess the antifungal properties of natural products hampers study comparison. In addition, several studies only reported data from agar disk diffusion, which provides information from a simple screening. The MIC and minimal fungicidal concentration (MFC) provided by dilution tests are more contributory and allow for the observance of greater variability. In general, MIC values ranged from 0 to 200 μg/ml among the compounds studied. All of them showed significant in vitro activity according to preestablished criteria [85], as MIC values were between 100 and 625 μg/ml. Despite these promising preliminary results, these compounds have a long road ahead before they will be eligible for clinical trials. Some of these compounds should be evaluated for in vitro and in vivo toxicity and their pharmacological potential.

Conclusion & future perspective
In the past 10 years, new potential antifungals for trichosporonosis treatment have not been found, and the need for effective drugs remains. Several compounds have been tested, but none have shown promising results. In general, most studies did not present appropriate scientific criteria for drug tests, compromising their quality.
Thus, new studies presenting well-defined scientific criteria are essential. For example, the selection of compounds for experimental investigation needs to be suitable and based on in-depth studies on fungal cell targets. In addition, new studies need to address Trichosporon biofilms (an important drug-resistance factor) and correlate susceptibility in vitro with patient clinical response. Another strategy would be to pursue further tests on in vitro and in vivo toxicity of drugs that are already under study and also evaluate their pharmacological potential.

Executive summary
Background • Trichosporonosis has been increasing worldwide, and there are no effective treatments for it. Aim • To collect information about potential antifungals tested against Trichosporon spp. Methods • A search in PubMed and Web of Science for relevant articles regarding potential antifungals for Trichosporon spp.

Results & discussion
• Related articles were divided into three topics according to the origin of the compound, such as biomolecules and molecules, repositioned drugs and natural products.

Conclusion
• Several compounds have been tested but without promising results.
• Most studies did not present appropriate scientific criteria for drug tests thus compromising the quality of the research.  Table 1, assisted in the correction of the manuscript and helped to review.