Rapid Classification of Serum from Patients with Paracoccidioidomycosis Using Infrared Spectroscopy, Univariate Statistics, and Linear Discriminant Analysis (LDA).

Paracoccidioidomycosis (PCM) is a systemic mycosis that is diagnosed by visualizing the fungus in clinical samples or by other methods, like serological techniques. However, all PCM diagnostic methods have limitations. The aim of this study was to develop a diagnostic tool for PCM based on Fourier transform infrared (FTIR) spectroscopy. A total of 224 serum samples were included: 132 from PCM patients and 92 constituting the control group (50 from healthy blood donors and 42 from patients with other systemic mycoses). Samples were analyzed by attenuated total reflection (ATR) and a t-test was performed to find differences in the spectra of the two groups. The wavenumbers that had p < 0.05 had their diagnostic potential evaluated using receiver operating characteristic (ROC) curves. The spectral region with the lowest p value was used for variable selection through principal component analysis (PCA). The selected variables were used in a linear discriminant analysis (LDA). In univariate analysis, the ROC curves with the best performance were obtained in the region 1551-1095 cm-1. The wavenumber that had the highest AUC value was 1264 cm-1, achieving a sensitivity of 97.73%, specificity of 76.01%, and accuracy of 94.22%. The total separation of groups was obtained in the PCA performed with a spectral range of 1551-1095 cm-1. LDA performed with the eight wavenumbers with the greatest weight from the group discrimination in the PCA obtained 100% accuracy. The methodology proposed here is simple, fast, and highly accurate, proving its potential to be applied in the diagnosis of PCM. The proposed method is more accurate than the currently known diagnostic methods, which is particularly relevant for a neglected tropical mycosis such as paracoccidioidomycosis.

Prediction of melanin content of Fonsecaea pedrosoi using Fourier transform infrared spectroscopy (FTIR) and chemometrics.

Fungal melanin contributes to the survival and virulence of pathogenic fungi, such as Fonsecaea pedrosoi, which is responsible for causing chromoblastomycosis. The objective of this study was to employ Fourier transform infrared spectroscopy (FTIR) to predict the melanin content of F. pedrosoi. The melanin content, in percentage, was previously determined using gravimetry for twenty-six clinical isolates. Quintuplicate spectra of each isolate were obtained using attenuated total reflection (ATR) within the range of 4000 to 650 cm-1. To predict the melanin content, modeling was performed using partial least squares regression (PLS) in the region 1800 - 750 cm-1. Two models were tested: PLS and successive projections algorithms for interval selection in partial least squares (iSPA-PLS). The best modeling results were achieved using iSPA-PLS with one factor. The calibration set exhibited a determination coefficient (R2) of 0.9745 and a root mean square error of cross-validation (RMSECV) of 0.0977. In the prediction set, the R2 value was 0.9711, and the root mean square error of prediction (RMSEP) was 0.0999. Modeling with FTIR and multivariate calibration provides a valuable means of predicting fungal melanin content, which is simpler and more robust, thereby contributing to the advancement of this field of study.

Epidemiological characteristics of leprosy from 2000 to 2019 in a state with low endemicity in southern Brazil.

BACKGROUND: Leprosy is an infectious and contagious disease caused by Mycobacterium leprae and is mainly characterized by lesions in the skin and peripheral nerves. In Brazil, it is a public health problem due to its high endemicity. However, the state of Rio Grande do Sul presents low endemicity of this disease. OBJECTIVE: To characterize the epidemiological profile of leprosy in the state of Rio Grande do Sul from 2000 to 2019. METHODS: This was a retrospective observational study. Epidemiological data were collected from the Notifiable Diseases Information System (SINAN, Sistema de Informação de Agravos de Notificação). RESULTS: Among the 497 municipalities in the state, 357 (71.8%) registered cases of leprosy in the assessed period, with an average of 212 (81.5%) new cases per year. The average detection rate was 1.61 new cases per 100,000 inhabitants. The male sex was predominant (51.9%) and the mean age was 50.4 years. Regarding the epidemiological clinical profile; 79.0% of the patients were multibacillary; 37.5% presented the borderline clinical form; 16% had grade 2 physical disability at diagnosis and bacilloscopy was positive in 35.4% of cases. As for treatment, 73.8% of the cases were treated with the standard multibacillary therapeutic regimen. STUDY LIMITATIONS: There were missing/inconsistent data in the database available. CONCLUSIONS: The findings observed in this study indicate that the state presents a low endemicity profile of the disease and these results can support adequate health policies relevant to the reality of Rio Grande do Sul, inserted in a national scenario of highly endemic leprosy.

Epidemiological characteristics of leprosy from 2000 to 2019 in a state with low endemicity in southern Brazil

Abstract Background Leprosy is an infectious and contagious disease caused by Mycobacterium leprae and is mainly characterized by lesions in the skin and peripheral nerves. In Brazil, it is a public health problem due to its high endemicity. However, the state of Rio Grande do Sul presents low endemicity of this disease. Objective To characterize the epidemiological profile of leprosy in the state of Rio Grande do Sul from 2000 to 2019. Methods This was a retrospective observational study. Epidemiological data were collected from the Notifiable Diseases Information System (SINAN, Sistema de Informação de Agravos de Notificação). Results Among the 497 municipalities in the state, 357 (71.8%) registered cases of leprosy in the assessed period, with an average of 212 (81.5%) new cases per year. The average detection rate was 1.61 new cases per 100,000 inhabitants. The male sex was predominant (51.9%) and the mean age was 50.4 years. Regarding the epidemiological clinical profile; 79.0% of the patients were multibacillary; 37.5% presented the borderline clinical form; 16% had grade 2 physical disability at diagnosis and bacilloscopy was positive in 35.4% of cases. As for treatment, 73.8% of the cases were treated with the standard multibacillary therapeutic regimen. Study limitations There were missing/inconsistent data in the database available. Conclusions The findings observed in this study indicate that the state presents a low endemicity profile of the disease and these results can support adequate health policies relevant to the reality of Rio Grande do Sul, inserted in a national scenario of highly endemic leprosy.

Using infrared spectroscopy of serum and chemometrics for diagnosis of paracoccidioidomycosis.

Paracoccidioidomycosis (PCM) is a systemic granulomatous mycosis endemic to Latin America, whose etiologic agents are fungi of the genus Paracoccidioides. PCM is usually diagnosed by microscopic observation of the fungus in biological samples, combined or not with other techniques such as serological methods. However, all currently used diagnostic methods have limitations. The objective of this study was to develop a method based on Fourier transform infrared spectroscopy (FTIR) and chemometric analysis for PCM diagnosis. We included 224 serum samples: 132 PCM sera, 24 aspergillosis sera, 10 cryptococcosis sera, 8 histoplasmosis sera, and 50 sera from healthy blood donors. Samples were analyzed by attenuated total reflection (ATR), and chemometric analyses including exploratory analysis through principal component analysis (PCA) and a classification method (PCM and non-PCM) through orthogonal partial least squares discriminant analysis (OPLS-DA). The spectra were similar, with the main bands up to approximately 1652 cm-1 and 1543 cm-1 (amide I and amide II bands). This same region was mainly responsible for the partial separation of the samples in PCA. The OPLS-DA model correctly classified all serum samples with only one latent variable, with a determination coefficient (R²) higher than 0.999 for both the calibration set and prediction set. Sensitivity and specificity were 100% for both sets, showing better performance than the reference diagnostic methods. Therefore, the use of FTIR/ATR together with OPLS-DA modeling proved to be a promising method for PCM diagnosis.

Antifungal susceptibility of the endophytic fungus Rhinocladiella similis (URM 7800) isolated from the Caatinga dry forest in Brazil.

The present study reports a new occurrence of Rhinocladiella similis isolated as an endophytic fungus in the Caatinga dry tropical forest in Brazil and describes its antifungal susceptibility. The isolate R. similis URM 7800 was obtained from leaves of the medicinal plant Myracrodruon urundeuva. Its morphological characterization was performed on potato dextrose agar medium and molecular analysis using the ITS rDNA sequence. The antifungal susceptibility profile was defined using the Clinical and Laboratory Standards Institute (CLSI) protocol M38-A2. The colony of isolate URM 7800 showed slow growth, with an olivaceous-gray color and powdery mycelium; in microculture, it showed the typical features of R. similis. In the antifungal susceptibility test, isolate URM 7800 showed high minimal inhibitory concentration (MIC) values for amphotericin B (>16 µg/mL), voriconazole (16 µg/mL), terbinafine (>0.5 µg/mL), and caspofungin (>8 µg/mL), among other antifungal drugs. Pathogenic melanized fungi are frequently isolated in environments where humans may be exposed, and these data show that it is essential to know if these isolates possess antifungal resistance.

Head and neck manifestations of paracoccidioidomycosis: A retrospective study of histopathologically diagnosed cases in two medical centers in southern Brazil.

BACKGROUND: Paracoccidoidomycosis (PCM) is a granulomatous systemic mycosis endemic in southern Brazil. OBJECTIVES: Analyze the clinical and epidemiological characteristics of cases of PCM with head and neck manifestations in southern Brazil. MATERIAL AND METHODS: Retrospective study of histopathologically diagnosed cases of PCM with head and neck manifestations referred to two medical centers in the municipality of Santa Cruz do Sul, state of Rio Grande do Sul, Brazil, during a 10-year period (2011-2020). RESULTS: Twenty-eight patients were selected. The number of cases usually ranged from one to four per year, although 11 cases were diagnosed in 2019. Most patients were between 40 and 59 years old. In total, 21 patients were men and seven were women (male:female ratio 3:1). Most (92%) were Caucasian and 46% were smokers. Patients were from 12 municipalities in the central-eastern region of the state. The most frequent site of the lesions was the larynx. Associated HIV and squamous cell carcinoma were both present in three cases. CONCLUSIONS: This is the first study to analyze PCM cases from central-eastern Rio Grande do Sul. PCM is endemic in southern Brazil, and more epidemiological data are needed, considering that its incidence may be higher than currently estimated, as demonstrated by our results.

Epidemiological characteristics and trends of leprosy in children and adolescents under 15 years old in a low-endemic State in Southern Brazil.

Leprosy is an infectious and contagious disease affecting skin and nerves. The number of cases in individuals under 15 years old is one of the parameters used in Brazil as an indicator of endemic permanence of the disease and its continuous transmission. Rio Grande do Sul State, in Southern Brazil, is low-endemic to leprosy. However, the disease remains a public health problem. This is a retrospective, observational and analytical study of a historical series of new cases of leprosy in children under 15 years old diagnosed in the period from 2000 to 2019, in all health units in Rio Grande do Sul State. Seventy-seven new cases were notified. The male gender was predominant in 53.2% of the cases (n=41). The average age was 10.4 years (standard deviation of 2.9), with predominance of the age group between 10 and 15 incomplete years old. The most frequent operational classification was multibacillary, in 62.3% of cases (n=48), and the most common clinical form was borderline, in 38.9% of cases (n=28). The predominant disability degree in the sample was grade zero, in 80.0% of the cases (n=60), but in 4.0% (n=3) the grade assessed was 2. In 54.0% of cases (n=27), bacilloscopy was performed, with positive results in 36.0% (n=9) of the exams. Multibacillary cases, with physical disability and/or positive bacilloscopy, draws attention that that the diagnosis is frequently not made in early stages.

Molecular identification and antifungal susceptibility of 75 clinical isolates of Trichophyton spp. from southern Brazil.

BACKGROUND: Dermatophytoses affect more than 20% of the world's population and is caused by filamentous fungi, mainly of the genus Trichophyton. The species identification through microscopic direct examination and culture methods is challenging, with molecular presenting high sensitivity and specificity. Although there are several therapy options for dermatophyte infections, treatment failures and antifungal resistance are growing concerns. OBJECTIVE: This study aimed to identify clinical isolates of Trichophyton spp. from southern Brazil using molecular methods and determine their in-vitro antifungal susceptibility. MATERIAL AND METHODS: Seventy-five isolates were identified through sequencing of the ITS region. The exposure to seven antifungals drugs was performed according to protocol M28-A2 of the Clinical and Laboratory Standards Institute (CLSI). RESULTS: Sixty-one isolates (81%) were identified as T. interdigitale, which differs from the epidemiological data present in the literature. Thirteen isolates were identified as T. rubrum and one as T. tonsurans. Terbinafine was the most effective antifungal, followed by itraconazole and voriconazole, which is in accordance with the results reported in previous studies. CONCLUSIONS: The use of molecular methods to identify Trichophyton spp. clinical isolates and the performance of susceptibility tests are relevant to epidemiological data, identification of the emergence of antifungal resistance, and to help to translate the in-vitro antifungal susceptibility results into clinical practice.

Effect of Melanin Biosynthesis Inhibition on the Antifungal Susceptibility of Chromoblastomycosis Agents.

Chromoblastomycosis (CBM) is a chronic subcutaneous infection caused by genera of melanized fungi: Fonsecaea, Cladophialophora, Phialophora, Exophiala, and Rhinocladiella. Melanin is a virulence factor known to influence antifungal susceptibility. A specific inhibitor of melanin biosynthesis is tricyclazole. The aim of this study was to evaluate the effect of melanin inhibition on antifungal susceptibility of chromoblastomycosis agents and describe the susceptibility profiles of some unusual CBM agents. Seventy-six clinical isolates, representing 13 species of the five main genera of CBM agents, were studied. The antifungal susceptibility testing was performed according to the M38-A2 protocol of CLSI (Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi, 3rd ed., CLSI Standard M38, 2017). In the melanin inhibition test, 16 mg/liter of tricyclazole was added to the medium used in the inoculum preparation and the susceptibility assay. CBM agents were less susceptible to amphotericin B than azoles and terbinafine. The unusual species showed similar susceptibility profiles to those of other species of the same genera. With tricyclazole exposure, MICs of terbinafine, posaconazole, and itraconazole for Fonsecaea spp. significantly decreased (P < 0.05). For Phialophora spp., this reduction was significant for posaconazole and itraconazole. For the other genera, there was a reduction in MICs of terbinafine and itraconazole; however, the statistical tests were not significant. Melanin inhibition can increase the antifungal susceptibility of most CBM agents to itraconazole and terbinafine, the main drugs used in the disease treatment. This increased susceptibility may open up new possibilities for therapy in refractory cases of CBM and/or cases caused by resistant fungal strains. Further studies are needed to confirm the same results in vivo.

Rapid classification of chromoblastomycosis agents genera by infrared spectroscopy and chemometrics supervised by sequencing of rDNA regions.

Chromoblastomycosis (CBM) is a skin and subcutaneous infection caused by species of seven fungal genera. Identification of CBM species is performed by DNA sequencing of one or more genes, which becomes a time-consuming work. Fourier Transform Infrared Spectroscopy (FTIR) has been used for the identification of other microorganisms, however, only one CBM genus was evaluated by FTIR analysis to date. Therefore, the study is aimed to differentiate the CBM agents for identification at genera level using FTIR supervised by Internal Transcribed Spacer (ITS) rDNA region. Seventy-seven isolates of the main five CBM genera were prepared for Attenuated Total Reflection FTIR (ATR-FTIR) with a new methodology using slices of dry fungus in glass fixing-modeling proposed in this study. The algorithm Hierarchical Cluster Analysis (HCA) was used to analyze the differences and similarities between species through the spectra. Orthogonal Partial Least Square Discriminant Analysis (OPLS-DA) allowed to correctly classify all samples of five CBM genera. The ATR-FTIR/OPLS-DA models highlighted important contributions of regions attributed to NH and OH stretching, amide I of proteins, polysaccharides bands and fingerprint region for the complete differentiation of the genera investigated. Thus, FTIR can be a fast and inexpensive alternative for identification of CBM agents.

Melanin and chromoblastomycosis agents: Characterization, functions, and relation with antifungals.

Melanins are a diverse group of dark pigments with similar properties. In fungi, the most studied is the dihydroxynaphtalene (DHN)-melanin, present in several species including all the chromoblastomycosis agents, a chronic, disabling, and recalcitrant subcutaneous mycosis. It is synthesized in a pathway known as the pentaketide pathway, which has the agrochemical tricyclazole as an inhibitor, widely used in in vitro studies because it does not prevent the growth of fungi. There are different methodologies for qualitative and quantitative analyses of DHN-melanin, which made it possible to discover its important structural and antioxidant functions, with melanin acting as a protective factor against the host's immune system. Also, it can interact with some of the main antifungals of medical interest, reducing its activity and the susceptibility of fungi to these agents. This review aims to discuss the aspects of DHN-melanin, focusing on chromoblastomycosis, bringing the main findings of the published scientific studies, and highlighting the need for further research to understand this important fungal pathogenicity and a virulence factor.

Prediction of itraconazole minimum inhibitory concentration for Fonsecaea pedrosoi using Fourier Transform Infrared Spectroscopy (FTIR) and chemometrics.

Fonsecaea pedrosoi is one of the main agents of chromoblastomycosis, a chronic subcutaneous mycosis. Itraconazole (ITC) is the most used antifungal in its treatment, however, in vitro antifungal susceptibility tests are important to define the best therapy. These tests are standardized by the Clinical and Laboratory Standards Institute (CLSI), but these protocols have limitations such as the high complexity, cost and time to conduct. An alternative to in vitro susceptibility test, which overcomes these limitations, is FTIR. This study determined the minimum inhibitory concentration (MIC) of itraconazole for F. pedrosoi, using FTIR and chemometrics. The susceptibility to ITC of 36 strains of F. pedrosoi was determined according to CLSI and with the addition of tricyclazole (TCZ), to inhibit 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis. Strains were grown in Sabouraud agar and prepared for Attenuated Total Reflection (ATR)/FTIR. Partial least squares (PLS) regression was performed using leave-one-out cross-validation (by steps of quintuplicates), then tested on an external validation set. A coefficient of determination (R²) higher than 0.99 was obtained for both the MIC-ITC and MIC-ITC+TCZ ATR/PLS models, confirming a high correlation of the reference values with the ones predicted using the FTIR spectra. This is the first study to propose the use of FTIR and chemometric analyses according to the M38-A2 CLSI protocol to predict ITC MICs of F. pedrosoi. Considering the limitations of the conventional methods to test in vitro susceptibility, this is a promising methodology to be used for other microorganisms and drugs.