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  . According to the World Health Organization (WHO), approximately 20% - 25% of the world population has some kind of cutaneous fungal infection    .
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    . 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  . 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   .
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  . With the advent of molecular biology, conventional morphological taxonomy was influenced by data obtained DNA sequencing which improved the taxonomic system  . 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   . 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  .
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.
2. Population and Methods
This study was carried out at the Infectious Diseases Outpatient Unit in a Brazilian Teaching Hospital from August 2014 to December 2015. A total of 398 adult patients of both genders were randomicaly recruited and divided into three groups: I composed by 153 HIV-infected individuals with skin, hair or nail lesions, II composed by 153 HIV-infected without apparent clinical lesions in these sites and III composed by 92 non-HIV-patients with clinical lesions similar to group I seen at the Dermatology Outpatient Clinic of the same hospital. The inclusion criteria were: both genders, age over 18 years, that was receiving antiretroviral therapy and agree to participate of the study. Those who referred antifungal use within 30 days prior to the recruitment or disagreed to participate were excluded.
Epidemiological and clinical data of the HIV-infected patients with dermatomycosis were registered and a clinical exam was performed. Nail and cutaneous samples were obtained by scraping while hair fragments were obtainied by removing a small amount of hair containing their bulb. Concomitantly, nail scrapings samples of the 153 HIV-infected without clinical lesions were obtained.
These clinical samples were examined by direct microscopic using potassium hydroxide 20% (KOH) in order to identify fungal structures. Then, they were seeded in Sabouraud Dextrose Agar added with 0.05 mg∙mL−1 chloramphenicol and Mycosel Agar, incubated at 28˚C ± 2˚C and observed for up to 30 days. Identification of the yielded filamentous fungi was carried out using the microculture technique in Potato Dextrose Agar for seven days at room temperature.
2.2. Molecular Identification of Isolates
The DNA extraction was performed by the phenol-chloroform method  . 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  . Briefly, each reaction contained a final volume of 50 μL with 50 ng of template DNA, 5 μL of the 1X PCR buffer (10 mM Tris-HCl pH 8.3, 50 mM KCl, 0.1%, 1.5 mM MgCl2), 0.25 mM dNTP’s (dATP, dCTP, dGTPe dTTP), 1.25 U Taq DNA polymerase (Invitrogen, Brazil) and 70 pmol of each primer. The PCR was performed under the following conditions: initial denaturation at 94˚C (3 min) followed by 29 cycles with denaturation at 94˚C (30 s), association at 57˚C (30 s), extension at 72˚C (45 s) and one cycle 10 minutes at 72˚C.
Next, the DNA was digested using the restriction enzyme MvaI Fast Digest (Thermo Scientific, USA) according to the manufacturer’s instructions. The fragments were detected by electrophoresis in 2% agarose gel at 80 V for 2 hours. The profiles were visually identified by comparison with the restriction patterns already described   . 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 alignments were performed in the Clustal W software  . 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  . The Bootstrap analysis with 1000 replicates was used to support the tree nodes.
2.3. Statistical Analysis
Data analysis was performed using Epi Info software (Statistical Package for the Social Sciences, version 7.0, USA) and GraphPad Prism (version 5.0, USA). The verification of normal distribution of the quantitative variables was done by the Shapiro-Wilk test. Continuous variables with normal distribution were expressed as mean ± standard deviation and those with ab-normal distribution were expressed in median, with maximum, minimum and percentiles values. Mann-Whitney and Fisher exact tests were used to evaluate the differences and frequencies among groups. Values of p < 0.05 were considered statistically significant.
The project was approved by the Research Ethics Board of the Triângulo Mineiro Federal University in Uberaba, Minas Gerais State, Brazil under protocol number: 1716.
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.
Table 1. Main demographic, epidemiological and clinical data of the HIV-infected and Non-HIV patient groups.
*Three patients from the HIV-infected group were excluded due to unavailable data; **SD: standard deviation.
Table 2. Mycological diagnosis according with the presence or absence of skin, hair or nail lesions in HIV-infected patients.
*KOH: potassium hydroxide 20%.
Table 3. Epidemiological and clinical data of patients with positive culture for dermatophytes.
NA: not available.
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.
3.2. Sequencing of the ITS Region and Phylogenetic Analysis
Ten (31.25%) of isolates were randomly selected for ITS sequencing. The generated ITS fragments presented approximately 630 bp. Of these, seven (70%) were adequately confirmed being five T. rubrum and two T. interdigitale. The sequences presented 97% to 100% of identity in relation to clinical isolates from USA, Australia, India, China, Russia and Japan.
The comparison of these sequences with the 29 reference sequences from GenBank, demonstrated that the T. rubrum isolates herein identified appear to have a greater phylogenetic homegeneity in comparison to the CBS 392.58 reference
Figure 1. Agarose gel (2%) representative of the band pattern after digestion with Mva I. ATCC: MYA4439 Trichophyton interdigitale; D04, D15, D16, D32, D34, D35, D40, D41: Trichophyton rubrum; D22, D07: not digest samples; D20, D39: excluded isolates. MM: LowRanger 100 bp DNA Ladder (NorGen Biotek, Canada). Color: Ethidium Bromide (0.5 μg/mL).
strain. Otherwise, the isolates D33 and D38 were grouped together with the anthropophilic strains of T. mentagrophytes complex (Figure 2).
The remaining three isolates formerly identified as Trichophyton sp. by their phenotypic aspects were identified within Ascomycetes: Lecanicillium aphanocladii (MH346472), Preussia sp. (MH346502) and Montagnulaceae sp. (MH348207).
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   . 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   .
The relationship between dermatophytosis and HIV infection remains poorly understood, regarding their frequency, causative species and therapeutical outcome   . Of 306 HIV-infected patients herein evaluated, 50% presented cutaneous, hair or nail lesions, often with atypical and heterogeneous appearance. Despite fungal ethiology had been suspected, other causes should be excluded and for this direct exam, culture and molecular methods are pivotal to improve the diagnosis accuracy and therapeutical decision  .
In the present report, 39.87% and 47.06% of HIV-infected patients with skin, hair or nail lesions presented direct exam and positive fungal culture respectively. Whereas in non-HIV individuals, 46.74% and 52.17% of cases presented direct exam and culture positive respectively. These figures were similar with those
Figure 2. The evolutionary history of the phylogenetic tree was inferred by using the Maximum Likelihood method based on the Tamura-Nei model. The tree with the highest log likelihood (−1415.8245) is shown. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.3745)). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. All positions containing gaps and missing data were eliminated. There were a total of 437 positions in the final dataset and 36 isolates were tested. The isolates are described according species of Trichophyton, strain ID, GenBank number, and the host from where they were recovered (e.g. human, horse, or chicken). The isolates described in bold are described in this study. The reference strain Epidermophyton floccosum CBS 358.93 was used as outgroup. The bootstrap values above 80% are described next to the branches.
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  .
The frequency of dermatophytes isolation between HIV-infected patients and non-HIV individuals was similar, a finding in line with another report where 105 HIV-infected patients and 63 non-HIV individuals were evaluated. According to the authors, there was no correlation between the T CD4+ cell count and the prevalence of dermatophytosis in HIV patients  .
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  . 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  . In contrast, another study from the same country described a high frequency (70%) of Tinea corporis in 20 HIV-infected patients  .
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   . 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   .
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   .
The therapy to superficial mycoses in HIV/AIDS patients is usually started with topical antifungals, whereas the use of systemic drugs is recommended in cases where there is no improvement or when lesions are extensive, deep or widespread. Several reports have pointed out that oral medicaments such as terbinafine, intraconazole or fluconazole are the most appropriate therapeutic options for these individuals. According with other authors, they must be prescribed from two to six weeks except those who present oniocomycosis and require more time of treatment   . Patients with dermatomycosis herein described received topical or oral itraconazole with clinical remission of their lesions except those who presented nail involvement and exhibited a poor response.
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  . 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   . Recently, a whole-genome analysis showed a clonal population profile of this species, which may explain the genetic homogeneity of the complex  .
In accordance to the current taxonomy of dermatophytes, the anthropophilic isolates are separated from the zoophilic ones constituting the T. mentagrophytes species complex  . 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  .
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  .
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.
This work was supported by Fundação de Amparo a Pesquisa de Minas Gerais (FAPEMIG)-grant #APQ-02633-16. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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.
T.B.F. and M.L.S.V. conceived the study; T.B.F., L.S.L.J. and B.S.P. collected the data; T.B.F. and L.B.S. performed experiments; T.B.F., L.B.S., L.E.A.S., K.F.P. and M.L.S.V analyzed data; F.A.A.S. and L.R.G. contributed new methods; T.B.F., D.J.M., K.F.P and M.L.S.V. led the writing.
Table S1. List of the of the patients and isolates identified in the study containing the anatomical site, HIV status, CD4+ count, direct microscopy, phenotypic identification, ITS-RFLP and ITS sequencing.
NA: Not available.