Dermatophytes Species Isolated of HIV-Infected Patients Identified by ITS-RFLP and ITS Region Sequencing from Triângulo Mineiro, Minas Gerais State of Brazil

Dermatomycoses are a group of diseases worldwide distributed and affect skin, hair and nails. Several fungal species can be envolved and keratinophilic fungi belonging to the genera Trichophyton, Microsporum and Epidermophyton are very common. These infections are globally distributed and their incidence has progressively increased. Despite their high incidence, few studies have been carried out in HIV-infected patients, regarding frequency, clinical presentations, and species identification. This study aimed to evaluate some epidemiological and clinical aspects of dermatophytosis in HIV-infected patients and to attempt phenotypical and molecular characteristics of their agents. Of 398 patients included, 306 were HIV-infected. Clinical data and samples of skin, hair and nail lesions were simultaneously obtained. The dermatophytes yielded in culture were phenotypically identified and evaluated by ITS-RFLP and ITS (Internal Transcribed Space) sequencing. Dermatophytes frequency in HIV-infected patients with cutaneous lesions was 11.76% similar to the 15.22% observed in non-HIV individuals. Trichophyton rubrum was the most common isolated species in both groups. Throught ITS-RFLP, 29/32 (90.63%) of dermatophytes isolates were identified at the species level. From the 10 isolates randomly selected for ITS sequencing, seven confirmed the ITS-RFLP results while three Trichophyton sp. were not identified as dermatophytes. The identification of these species in HIV-infected patients is highly desirable in order to improve the knowledge of their freHow to cite this paper: Bragine-Ferreira, T., de Lima-Júnior, L.S., Silva, L.B., Andrade-Silva, L.E., Mora, D.J., Prudente, B.S., Ferreira-Paim, K., Santos, F.A.A., Goulart, L.R. and Silva-Vergara, M.L. (2019) Dermatophytes Species Isolated of HIV-Infected Patients Identified by ITS-RFLP and ITS Region Sequencing from Triângulo Mineiro, Minas Gerais State of Brazil. Advances in Microbiology, 9, 790-803. https://doi.org/10.4236/aim.2019.99048 Received: July 25, 2019 Accepted: September 2, 2019 Published: September 5, 2019 Copyright © 2019 by author(s) and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access T. Bragine-Ferreira et al. DOI: 10.4236/aim.2019.99048 791 Advances in Microbiology quency, geographical distribution and relation to clinical presentation.


Introduction
Dermatomycoses are common cutaneous infections of keratinized tissues, caused by several fungal species which invade the corneous layer of skin, hair and nails. Among these, keratinophilic fungi belonging to the genera Trichophyton, Microsporum and Epidermophyton are very common. These infections are globally distributed and their incidence has progressively increased [1]. According to the World Health Organization (WHO), approximately 20% -25% of the world population has some kind of cutaneous fungal infection [1] [2] [3].
These infections can have acute or chronic clinical presentation, depending on the degree of inflammation and it is hypothesized that these mycoses trend to be more frequent and severe in immunocompromised individuals [4] [5] [6]. The HIV infection is one of the most important risk factors to the occurrence of fungal infections and due to the scarcity of data, the prevalence, clinical features, diagnosis and outcome of the dermatomycosis in HIV-infected individuals are still unclear [7]. Moreover, cellular immunity is pivotal in the control of these infections and Th1 and Th2 responses balance can determinate the progression of the infection in such patients [8] [9].
The taxonomy of dermatophytes remained confused for a long time, until the discovery of the sexual reproduction in keratinophilic species, which led to the introduction of the biological concept of species [10]. With the advent of molecular biology, conventional morphological taxonomy was influenced by data obtained DNA sequencing which improved the taxonomic system [11]. One of the sequencing targets is the Internal Transcribed Spacer (ITS) region of ribosomal DNA, which is considered the main barcode of the fungal kingdom [12] [13]. Furthermore, the sequencing of different genes, specially using the genealogical concordance phylogenetic species recognition (GCPSR) can be useful to improve fungal diagnosis, biodiversity range, species conservation and taxonomy accuracy [11]. The present report aimed to evaluate some epidemiological and clinical aspects of dermatophytosis in HIV-infected patients and to attempt characterizes phenotipic and molecular aspects of their etiological agents in a teaching hospital from Brazil.

Population
This study was carried out at the Infectious Diseases Outpatient Unit in a Bra-

Molecular Identification of Isolates
The DNA extraction was performed by the phenol-chloroform method [14]. Then, a PCR reaction of the ITS region of ribosomal DNA was performed using ITS-1 and ITS-4 primers as previously described with modifications [15].  [17]. The strain ATCC MYA4439 of Trichophyton interdigitale was used as a positive control.
The amplified DNA of ten randomly selected isolates was submitted to the sequencing reaction using the BigDye terminator 3.1 reagent kit (Applied Biosystems, Foster City, CA, USA). The sequences obtained were analyzed in the software Chromas-pro 1.5 and then compared with the available data in the National Center of Biotechnological Information (NCBI-Nacional Center for Biotechnolgy Information), including sequences of reference strains. Multiple align-  [18]. The best model used in the phylogenetic tree was chosen from the software JModel test using the Bayesian Information Criterion (BIC). The Tamura Nei substitution model with Gamma distribution was the best model for the aligned dataset and used to construct the phylogenetic tree. The phylogenetic analysis was performed in the MEGA 7.0 software with the Maximum likelihood method [19]. The Bootstrap analysis with 1000 replicates was used to support the tree nodes.

Results
Of 306 HIV-infected patients, 178 (58.17%) were male (mean age: 43.86 ± 12.24 years) ( Table 1). Of 153 HIV patients with cutaneous, hair or nails lesions, in 61 (39.87%) at least one fungal structure was observed by direct examination whereas in 72 (47.06%) of cases a positive fungal culture was obtained. The presence of skin, hair or nail lesions was significantly associated with the chance to obtain a positive fungal direct exam or culture (p < 0.001) ( Table 2). Of 92 non-HIV patients evaluated, 55 (59.78%) were female (mean age: 52.64 ± 13.63 years). Of these, 43 (46.74%) and 48 (52.17%) presented a fungal positive direct exam and culture, respectively ( Table 2).
Among the recovered fungal isolates, 32 were phenotypically identified as dermatophytes, of which 18 (56.25%) were obtained from HIV-infected patients while the remaining 14 (43.75%) were from non-HIV patients (Table S1).
The frequency of dermatophytes in HIV-infected patients with clinical lesions (11.76%) was similar to that observed in non-HIV patients (15.22%) (p = 0.4406). Trichophyton rubrum was predominant followed by Trichophyton interdigitale in both groups (Table S1).
Of the 18 HIV-infected patients with dermatophytes isolation, 14 (77.78%) were males (mean age: 48.22 years) while the 9 (64.29%) of the 14 non-HIV patients with isolated dermathophytes were female (mean age: 50.93 years). Most individuals were from urban areas and had a low scholar level. Other epidemiological and clinical data are described in Table 3 and Table S1.

ITS-RFLP
Of 32 isolated dermatophytes, 29 (90.63%) were identified at the species level by the ITS-RFLP profiles. All 18 isolates phenotypically characterized as T. rubrum were confirmed by ITS-RFLP. Of the six isolates previously identified as T. interdigitale, four presented the typical profile of this species while two were reclassified as T. rubrum (Figure 1). Two isolates previously identified as Microsporum canis and T. verrucosum were confirmed as well (data not shown).
From the six isolates (D04, D05, D07, D22, D30 and D32) previously identified as Trichophyton sp., three (D04, D30 and D32) were confirmed as T. rubrum by the ITS-RFLP (Figure 1). The remaining isolates did not present any pattern of digestion by this technique.

Discussion
Dermatophytes comprise a group of pathogenic filamentous fungi that cause cutaneous mycoses and due to their ubiquity in nature and high transmissibility, the infections caused by these agents are often considered a neglected problem [20] [21]. Currently, HIV infection is considered one of the major global public health problems and a wide gamut of diseases with cutaneous involvement in HIV-infected patients has been already reported. Due to the immunosuppresion, atypical skin lesions can lead to clinical misdiagnosis and treatment difficulties  reported from India, where the positivity rate of direct microscopy and culture were respectively 49% and 51% in 100 non-HIV patients with clinical suspicious of dermatophytosis [26].  [7].

The frequency of dermatophytes isolation between HIV-infected patients and
Although the relation of dermatophytosis and HIV-infected patients is still unclear, some authors pointed out an inverse relationship between T CD4 + cells count and the frequency and extension of cutaneous lesions in these individuals [9]. Most patients herein evaluated exhibited high T CD4 + cell counts and undetectable viral load values, which reinforce that they presented adequate adhesion to antiretroviral therapy and partially could explain the low frequency of dermatophytes recovered despite 306 patients were evaluated. Herein and as described for other mycosis, the HIV infection can change the clinical presentation without alters the rate incidence. Maybe, the stratification of CD4 + values and the inclusion of patients with advanced immunodeficiency could identify different risk categories for dermatophytes infection.
Regarding the anatomic site, the toenails and feet were the sites where dermatophytes were more frequently isolated. The severity of the nail involvement with chronic and distrophic changes and a poor antifungal response is a common clinical observation in these patients. This finding agrees with literature data and confirms the results from a recent Brazilian survey where onicomycosis and Tinea pedis were the most common clinical pictures in 84 HIV-infected patients evaluated [27]. In contrast, another study from the same country described a high frequency (70%) of Tinea corporis in 20 HIV-infected patients [28].
Most dermatophytes species herein identified belong to the Trichophyton genus which is in line with other reports that point out 90% of the dermatophytosis around the world are caused by species of this genus [29] [30]. Although T. rubrum is found as the main agent of dermatophytosis in HIV-infected patients, it presents global distribution and also is the most common agent recovered from clinical specimens [7] [27]. The T. verrucosum isolate was confirmed by ITS-RFLP and recovered from a patient who referred frequent contact with domestic animals. Despite this zoophilic species is an uncommon agent in humans, its isolation could be clinically relevant since it was recovered from an HIV-infected individual. This species has been commonly associated with farm activities and cattle are known as the main infection source. However, accidental transmission can also occur by contaminated objects [31] [32].
The Despite, culture continues to be one of the most used methods for mycological diagnosis, the ITS-RFLP allowed the identification of 90.63% of the isolated dermatophytes at the species level. The obtained band patterns were variable in size among the different species, which allowed the visual differentiation among T. rubrum, T. interdigitale, M. canis and T. verrucosum [17]. The T. rubrum complex includes species with different phenotypic characteristics, however this variability has not been homogeneously observed in molecular studies. In these cases, the restriction pattern from other DNA regions or the use of additional enzymes could increase the accuracy of the method [16] [33]. Recently, a wholegenome analysis showed a clonal population profile of this species, which may explain the genetic homogeneity of the complex [34].
In accordance to the current taxonomy of dermatophytes, the anthropophilic isolates are separated from the zoophilic ones constituting the T. mentagrophytes species complex [11]. The T. interdigitale isolates herein identified presented the same restriction pattern on ITS-RFLP and were grouped together with the anthropophilic ones in the phylogenetic analysis. A recent report with 60 clinical isolates of dermatophytes showed that the most of them belonged to the anthropophilic variant of T. mentagrophytes complex [35].
Despite, the DNA sequencing is a reliable technique for fungal identification species and the ITS region is relatively conserved in dermatophytes species, the combination of different genes sequencing of these species by MLST (Multilocus Sequence Typing) undoubtedly is pivotal to improve their identification [11].
Despite the low number of isolates evaluated, the results herein showed that identification based on morphological characteristics of dermatophytes species presents high level of complexity and accuracy limitations, although it is still one of the most used method in the context of clinical mycological diagnosis. Otherwise the ITS-RFLP appears to be useful and reliable tool to identify and to differentiate dermatophytes at the species level. In addition, this technique is still cheaper and simpler than any sequencing method, especially when taking into account limited-resource settings.
Moreover, the sequencing is considered the ideal method to identify the dermatophytes with more precision at the species level and to observe their intraspecific variability. However, logistical and economical issues around the world remain as the main obstacle for its wide implementation as well.

Ethical Approval
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study.

Conflicts of Interest
There are no conflicts of interest to report.