Epidemiological profile and antifungal susceptibility pattern of Trichosporon species in a tertiary care hospital in Chandigarh, India

Background and Purpose: Trichosporon species are ubiquitous in nature which are associated with fatal opportunistic invasive infections, especially in immunocompromised patients. The present study aimed to evaluate the epidemiological and clinical details, as well as the antifungal susceptibility pattern of the patients with Trichosporon infections. Materials and Methods: In total, 50 clinical isolates of Trichosporon species from various samples were included in this study. The samples were isolated over a period of 18 months from patients in a tertiary hospital in North India. The isolates were characterised phenotypically with Vitek MS (bioMérieux, France). Trichosporon spp. were isolated from urine (30%), nail (30%), tissue (16%), pleural fluid (14%), and sputum (5%). In total, majority of the isolates were of Trichosporon asahii (92%), followed by Trichosporon mucoides (6%), and Trichosporon ovoides (2%). It is noteworthy that most of the reported cases were from intensive care unit (34%). Results: Intravenous catheters, antibiotics, and antifungal uptake were significantly associated risk factors with Trichosporon infection. All invasive isolates were observed to be resistant in vitro to caspofungin and exhibited high minimum inhibitory concentration (MIC) values against amphotericin B, fluconazole, and 5-flucytosine. The MICs for voriconazole and posaconazole were low. Conclusion: Trichosporonosis is being increasingly reported all around the world, including India. The results of this study highlighted the importance of early detection and treatment for this emerging yeast and also added to the ongoing surveillance for the antifungal susuceptibility pattern for this fungus.


Introduction
richosporon species was first discovered in 1865 as a benign infection from a patient with white Piedra. Currently, Trichosporon species have been increasingly recognized as opportunistic pathogens capable of causing invasive diseases, especially in immunosuppressed patients [1]. Multiple species of the genus Trichosporon were used to be called collectively T. beigelii, while currently, the genus Trichosporon comprises 50 species with 16 human pathogens [2,3].
In the past three decades, Trichosporon species have been implicated in invasive infections in the immunocompromised hosts. Moreover, there have been rare reports of trichosporonosis in immunocompetent patients [4]. Most cases have been reported among neutropenic patients with hematological or solid organ malignancies, and bone marrow or solid organ transplantation. Other patients at risk for invasive disease include patients with AIDS, extensive burns, intravascular catheters; patients who receive corticosteroids or undergo heart valve surgery and liver transplantation; and patients on dialysis [4].
Trichosporon species exhibit variable MICs against amphotericin B and moderate susceptibility to fluconazole and itraconazole [5]. This fungus exhibits intrinsic resistance to echinocandins, as evidenced by the high MICs reported until now, and the reports of breakthrough infections among patients receiving these antifungals [6]. The clinical picture of trichosporonosis resembles that of invasive candidiasis, a neutropenic patient with an acute febrile illness not responding to empirical broad-spectrum antibiotics or even empirical antifungal agents. The patient may rapidly develop multiorgan failure and become septic. According to the literature review, the prognosis is dismal as the case fatality rate is as high as 77% [7]. Lack of background knowledge impairs the proper diagnosis and treatment of Trichosporon species. The majority of the studies on this disease in India and other countries around the world are either retrospective or case reports. Therefore, there is a need for prospective studies, especially in developing countries, like India, due to the increase in the number of cases as well as the presence of diverse risk factors in both immunocompetent and immunocompromised patients [4]. Such studies can facilitate the timely diagnosis, analysis, and documentation of various risk factors and improve the treatment of the patient based on the reported MICs of the isolates. The present study aimed to investigate the epidemiology of this disease, including the clinical presentation with associated risk factors and antifungal susceptibility.

Materials and Methods
This prospective observational study was conducted on all the samples received in the Mycology Laboratory wherein Trichosporon species had been isolated for 18 months (from January 2016 to June 2017). The samples included blood, urine, pus, sputum, peritoneal fluid, hair, nail, skin scrapings, or any other body fluid collected from both admitted and outpatients that were suspected of any fungal infection. A detailed pro forma was filled with various information about the demographic characteristics of the patients, fungal isolates, antifungal susceptibilities, and the final outcome of the patients. On the basis of sample site and presentation, we broadly categorized our cases into invasive and superficial trichosporonosis, similar to Colombo et al. [4].
In total, 50 patients of all age ranges from whom Trichosporon was isolated on culture were included in the study. Slide and tube KOH mounts were prepared with 10-20% KOH and examined for the presence of fungal elements, such as arthroconidia, blastoconidia, hyphae, or pseudohyphae [8]. Samples were inoculated for culture on Sabouraud dextrose agar (SDA) (HiMedia, India) tube slants with chloramphenicol and gentamicin both with and without cycloheximide . Each medium was inoculated in duplicate and incubated at 25°C and 37°C. The subcultures from blood culture bottles were performed on respective SDAs [8,9].
The SDA slants were observed for fungal growth daily for one week and twice a week for the next three weeks. The fungal growth was identified and assessed conventionally by standard mycological methods on the basis of macroscopic morphological features, a microscopic examination by lactophenol cotton blue mount, cornmeal morphology, and urease and sugar assimilation tests [10]. Confirmatory detection was performed with Vitek MS (bioMérieux, France), a commercially available MALDI-TOF MS platform [11].
Antifungal susceptibility testing was performed by micro-broth dilution technique for amphotericin B, 5flucytosine, fluconazole, itraconazole, voriconazole, posaconazole, and caspofungin according to the Referencence Method for Broth Dilution Antifungal Susceptibility Testing of Yeast by the Clinical Laboratory Standard Institute (CLSI) (M27-A3). [12]. Moreover, the C. parapsilosis ATCC 22019 and C. krusei ATCC 6258 were used as quality strains. The tested antifungals were provided in the form of powders by a commercial source (Sigma-Aldrich, USA). The MICs were read after 24 h of incubation at 37°C.
The association between categorical variables was determined using Fisher's exact test. A two-sided pvalue of less than 0.05 was considered statistically significant. The study was conducted after obtaining ethical approval from the Ethical Clearance Committee of the Government Medical College and Hospital, Chandigarh, India. The present research was conducted based on the ethical guidelines for biomedical research on human subjects According to the Central Ethics Committee on Human Research of Indian Council of Medical Research, New Delhi, India in 2006 [13] and the Declaration of Helsinki of 2008 [14].

Discussion
Trichosporonosis is an emerging cause of morbidity and mortality in both developing as well as developed countries [4]. Trichosporon species have been increasingly recognized as opportunistic pathogens capable of causing invasive disease, especially in immunosuppressed patients [8]. Population of immunocompromised patients is on the rise which means that the fraction of at-risk population is increasing making more people prone to the development of this disease [6]. This is consistent with an increasing incidence rate of disseminated Trichosporon infections in humans alongside other invasive fungal infections [8].
On the basis of presentation and sample site, we broadly categorized our cases into invasive and superficial trichosporonosis, similar to Colombo et al. [4]. Based on the results, 35 (70%) out of the 50 Trichosporon isolates included in this study were of invasive trichosporonosis, most of which were isolated from urine (n=15, 30%). Furthermore, 30% (n=15) of the isolates were superficial trichosporonosis, all of which were isolated from the nail. In 2015, Montoyo et al. also observed a similar sample distribution, wherein 26 (66%) out of the total 39 cases were from urine and only five of them (12%) were from nail samples [15]. It is noteworthy that urine was the commonest sample in other studies as well [16,17]. India has a hot humid climate which could be accounted for the high number of onychomycosis cases in this study since it facilitates the growth of this fungi.
Although the male to female ratio of the total subjects was 1:1, most of the cases of invasive trichosporonosis were male (60%). Nevertheless, this ratio was opposite regarding superficial trichosporonosis since females accounted for 73% of cases. The male preponderance in invasive trichosporonosis was reported by most studies, while Colombo et al. in their study reported female preponderance in superficial trichosporonosis [4,18,19]. In the present study, the mean age of the participants was 49.5±11.32 years. Maximum number of cases (n=17, 34%) were observed in the age range of 41-50 years both in invasive and superficial trichosporonosis. Yang et al. and Wei sun et al. reported the maximum number of invasive and superficial trichosporonosis cases in the age ranges of ≥ 66 years and 70 years, respectively [16,19]. Besides, in the present study, trichosporonosis was mostly found in the younger age group which clearly indicates the growing severity of the disease.
Maximum reported cases (34%) in this study and some previous studies were from ICU [18,20]. Based on the findings, trichosporonosis had a significant relationship with antibiotic and antifungal intake and intravenous catheters (P≤0.05). Moreover, it was found that invasive trichosporonosis had a significant association with malignancy, surgery, bronchial asthma with COPD, urinary catheter, antibiotic and immunosuppressant intake, and ICU stay. In addition, antifungal intake (itraconazole) was significantly associated with superficial trichosporonosis.
Based on risk factor analysis, the history of Curr Med Mycol, 2021, 7(1): 19-24 23 antibiotic intake was observed in 100% and 82.2 % of patients in other studies [18,19]. In the present study, 70% of subjects had a history of antibiotic intake. Moreover, corticosteroid intake history was observed in 26% of cases in this research and 34.1% and 53% of patients in other studies [18,20]. In addition, immunosuppression history was observed in 31.7% of participants in a study performed by Almeida junior et al. while in this study 10% of subjects had a history of immunosuppression [18]. In a study carried out by Wei sun et al., 60.8% of cases used a urinary catheter, while in the present study 44% of patients had catheterization [19]. Furthermore, in the aforementioned study, a history of diabetes was seen in 34.7% of cases which was the same in the present study [19].
It can be said that T. asahii, T. asteroides, T. cutaneum, T. inkin, T. mucoides, and T. ovoides are common human pathogens causing superficial and disseminated infections [21]. More specifically, T. asahii and T. mucoides appear to be much more common in cases of systemic mycosis in immunocompromised patients. Moreover, T. inkin and T. ovoides are associated with pubic white piedra and white piedra of the head, respectively, while T. asteroids and T. cutaneum are associated with superficial skin lesions [4,21].
It must be mentioned that T. asahii was the only species isolated from all the cases of invasive trichosporonosis in this study. Moreover, it was the most common species in superficial trichosporonosis, accounting for 92% of cases. Other isolated species were T. mucoides (n=3, 6%) and T. ovoides (n=1, 2%). Similar results were found by Almeida et al. who also reported T. asahii as the most common species in urine samples of hospitalized patients [18]. Taverna [16]. Furthermore, Kalkanci et al. in their study found that 81.3% of cases were infected by T. asahii [17]. Based on previous studies, there is consistency in clinical dominance of T. asahii in Brazil, China, Japan, Spain, Taiwan, Thailand, and Turkey 118 [16].
Until now, there have been no recommendations on MIC breakpoints for Trichosporon from the two main consortia, CLSI and EUCAST [9]. The MICs for T. asahii have been discussed here as it was the main isolated species in this study. Distribution of MICs against amphotericin B is quite heterogeneous. Higher  [15]. Besides, even the present study produced similar results with MIC values of 0.125-16 µg/ml. The MIC50 and MIC90 had wide ranges in various studies such as 0.5-16 µg/ml and 2-16 µg/ml, respectively [16,24].
The strains in this study had very high MIC50 (16 µg/ml) and MIC90 (16µg/ml) values. The MIC values for T. mucoides (8-≥16 µg/ml) and T. ovoides (≥16 µg/ml) were also high. It must be noted that 5flucytosine is not usually a preferred medication for Trichosporon. The MIC values of 5-flucytosine were high in this study (0.125-≥16 µg/ml), similar to those in the study performed by Kalkanci [22].
The MIC50 of fluconazole in this research was 4 µg/ml while in other studies it was within the range of 0.5-8 µg/ml [5,19]. The MIC90 in this study was 8 µg/ml which has been reported to be as high as 64 µg/ml in one of the previous studies [23]. The T. mucoides (0.125-4 µg/ml) and T. ovoides had lower MICs (≤0.125 µg/ml) in this study. Iitraconazole had lower MICs (0.125-0.25 µg/ml) which has also been observed by others [17,19,22], while few studies have reported it to have higher MICs [23][24][25].
It must be noted that the results of other studies in this regard are similar to those of the present research [15,22]. Trichosporon has been considered inherently resistant to caspofungin with constantly elevated MICs [4,18]. It showed higher MICs in this research similar to other studies [15,18].

Conclusion
Results of the present study emphasize the dynamic nature of Trichosporon spp. in the immunocom-

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Curr Med Mycol, 2021, 7(1): 19-24 promised and immunocompetent hosts. Their frequency raises the concern of the increase of Trichosporon. This study will add to the ongoing surveillance of antifungal susceptibility patterns. Interval surveillance of this type is an essential component in the development of institutional guidelines for prophylaxis, empiric, or pre-emptive therapy for such life-threatening infections.