The present and future research agenda of sporotrichosis on the silver anniversary of zoonotic sporotrichosis in Rio de Janeiro, Brazil.

Twenty-five years have passed since the initial observation of endemic zoonotic sporotrichosis in Rio de Janeiro, Brazil. Since then, this disease has spread throughout South America. Accompanying the emergence of this mycosis, some progress has been made, including the expansion of a research network in this field and higher visibility of sporotrichosis within government authorities and funding agencies. However, there are still some challenges to curbing the expansion of this disease in the coming years. These include the development of rapid and accurate diagnostic tests, new antifungal drugs, particularly for the treatment of extracutaneous manifestations of sporotrichosis, and more comprehensive care for cats with sporotrichosis. Including these actions in the sporotrichosis research agenda is required so as to change the development of this disease in the years to come.

Development and validation of a new quantitative reverse transcription PCR assay for the diagnosis of human sporotrichosis.

Sporotrichosis is an emergent public health problem. The mycological diagnosis of this infection is based on culture, which is fastidious and may represent a biohazard for technicians. Although not widely implemented in routine diagnosis, molecular methodologies are fast, have good accuracy, and can be easily standardized, aiding in the early diagnosis of neglected mycoses. This study aimed at implementing a new pan-Sporothrix quantitative reverse transcription PCR (RT-qPCR) assay, and then validating it on clinical samples from confirmed human sporotrichosis cases. A total of 68 human samples with culture-confirmed diagnosis of sporotrichosis were collected from 64 patients followed at a Brazilian reference center for endemic mycoses. These samples were submitted to whole nucleic acid extraction, followed by an RT-qPCR protocol. The limit of detection was 244 fg, the efficiency was 2.0 (100%), and the assay could amplify the genetic material of the three major clinically relevant species of the genus Sporothrix. Among the 68 samples analyzed, 62 were positive in RT-qPCR, showing an overall sensitivity of 91.18%, which variated according to the type of biological sample: 96.72% in skin samples (n = 61) and 100% in respiratory samples (n = 3), whereas all cerebrospinal fluid specimens (n = 4) were negative. The specificity was 100% when tested in 25 samples from patients with other mycoses and tuberculosis. In addition, DNA from 93 fungal species did not yield positive results, confirming the high specificity of this test. Our RT-qPCR presented high sensitivity and specificity, representing an excellent tool for a fast and reliable diagnosis of human sporotrichosis.

Sporotrichosis is a deep mycosis with limited laboratorial techniques for fast diagnosis. We developed an assay able to detect the genetic material of fungal agents of sporotrichosis, and validated it in human specimens from patients with this disease, obtaining high positivity and specificity.

In vitro activity of Schinus terebinthifolius extract and fractions against Sporothrix brasiliensis.

BACKGROUND: Sporothrix brasiliensis is the causative agent of zoonotic cases of sporotrichosis in Brazil and is associated with atypical and severe presentations in cats, dogs, and humans. Sporotrichosis treatment is usually time- and cost-consuming, sometimes with poor response and host toxicity. Schinus terebinthifolius has proven efficacy against bacteria and fungi of clinical interest. OBJECTIVE: To determine the in vitro activity of S. terebinthifolius against S. brasiliensis. METHODS: Five S. brasiliensis isolates and three reference strains were subjected to a hydroethanol extract derived from the leaves of S. terebinthifolius and its fractions. The minimal inhibitory concentration (MIC) was determined using the broth microdilution method according to the M38-A2 CLSI guidelines. Also, the fungicidal/fungistatic activity of the extract and fractions was studied. FINDINGS: The crude extract of S. terebinthifolius inhibited the growth of S. brasiliensis (MIC: 0.5-1.0 µg/mL), while the partitioned extracts dichloromethane, ethyl acetate, and butanol demonstrated growth inhibition at 8 µg/mL due to a fungistatic activity. MAIN CONCLUSIONS: Due to its in vitro efficacy against S. brasiliensis and its known pharmacological safety, S. terebinthifolius is a candidate to be tested using in vivo models of sporotrichosis.

Sporotrichosis After Tattooing Caused by Sporothrix brasiliensis.

Sporotrichosis is a subcutaneous mycosis caused by pathogenic species of the genus Sporothrix. Since 1998, the number of cases of sporotrichosis due to Sporothrix brasiliensis has grown significantly in Rio de Janeiro, Brazil. Nearly all cases are related to cats as the main source of fungal infection. We report two cases of sporotrichosis following tattoos, a transmission form of S. brasiliensis not yet reported. The first patient, a 22-year-old female, had cutaneous sporotrichosis, fixed form, over a tattoo in her lumbar region. The lesion appeared 12 weeks after she was tattooed. The second patient, a 27-year-old female, had a lymphocutaneous sporotrichosis over a forearm tattoo. The lesion appeared two weeks after she was tattooed. In both cases there was no history of contact with cats or other plausible source of infection. The present study highlights that other non-zoonotic forms of transmission of S. brasiliensis may occur in endemic areas.

Zoonotic sporotrichosis with greater severity in Rio de Janeiro, Brazil: 118 hospitalizations and 11 deaths in the last 2 decades in a reference institution.

Since 1998, there has been an increase in the number of cat-transmitted cases of human sporotrichosis in Rio de Janeiro state, and severe forms are observed especially when associated with human immunodeficiency virus (HIV). A retrospective search of the INI/ Fiocruz database was conducted to identify sporotrichosis cases, hospitalized and deceased patients, between 1999 and 2015. There were 3917 adult patients diagnosed, 75 of them hospitalized, and 11 died. We conclude there is still a progression toward hyperendemic levels and greater severity has been demonstrated mainly in a socially excluded population.

A case of sporotrichosis caused by different Sporothrix brasiliensis strains: mycological, molecular, and virulence analyses.

BACKGROUND: Sporotrichosis is a subcutaneous mycosis caused by dimorphic pathogenic fungi belonging to the Sporothrix genus. Pathogenic Sporothrix species typically produce melanin, which is known to be a virulence factor. OBJECTIVES: The aim of this study was to perform phenotypic, genotypic, and virulence analyses of two distinct Sporothrix brasiliensis strains isolated from the same lesion on a patient from Rio de Janeiro. METHODS AND FINDINGS: Genotypic analyses by partial sequencing of the calmodulin, ß-tubulin, and chitin synthase genes, as well as polymerase chain reaction (PCR)-fingerprinting by T3B, M13, and GACA, showed that the isolates were very similar but not identical. Both isolates had similar phenotypic characteristics and effectively produced melanin in their yeast forms, accounting for their ability of causing disease in a murine sporotrichosis model. Remarkably, isolate B was albino in its environmental form but caused more severe disease than the pigmented A isolate. CONCLUSIONS: These findings indicate that the patient was infected by two genetically and biologically distinct S. brasiliensis that vary in their production of melanin in their environmental forms. The results underscore the importance of characterizing phenotypically different isolates found in the same clinical specimen or patient.

Cutaneous tuberculosis and HIV infection at a referral centre in Rio de Janeiro, Brazil.

BACKGROUND: Cutaneous tuberculosis (CTB) is a rare extrapulmonary form of tuberculosis (TB). Despite the increase in the number of cases of TB and HIV, few cases of CTB have been reported. OBJECTIVE: To describe CTB cases among patients with HIV infection from a cohort with tuberculosis. METHODS: We describe a series of 15 CTB and HIV cases, based on secondary data from 2000 to 2016. Diagnosis was based on isolation of Mycobacterium tuberculosis in culture or clinical response to anti-tuberculous treatment associated with positive smear or histopathologic findings from affected skin or an adjacent lymph node. FINDINGS: Scrofuloderma was present in 12 (80%) patients and solitary gumma in three (20%) patients. One case of scrofuloderma was associated with papulonecrotic tuberculid. Seven (46.6%) patients had pulmonary TB. Diagnosis was based on culture in nine patients (60%). The median CD4 cell count was 262 cells/µL. All patients were cured at the end of treatment (median time 6 months). Three patients presented with immune reconstitution inflammatory syndrome. CONCLUSIONS: In this study, CTB associated with HIV infection presented as localised forms or in association with pulmonary TB. In patients with HIV who have subacute and chronic skin lesions, CTB should be considered in differential diagnosis, which may represent a good opportunity for early diagnosis of active TB.

Refractory sporotrichosis due to Sporothrix brasiliensis in humans appears to be unrelated to in vivo resistance.

Sporotrichosis is a subacute to chronic infection caused by members of the Sporothrix schenckii complex. Itraconazole is the first choice antifungal drug for treating this infection, with terbinafine and potassium iodide as alternatives and amphotericin B used in cases of severe infections. Correlation of antifungal susceptibility data with the clinical outcome of the patients is scarce. The aim of this study was to correlate clinical and mycological data in patients with refractory sporotrichosis. In this work, antifungal susceptibilities, determined according to the reference M38-A2 CLSI protocol, of 25 Sporothrix strains, isolated from seven human cases of sporotrichosis with adversities in the treatment, are presented. Tested drugs included itraconazole, ketoconazole, posaconazole, voriconazole, terbinafine, and amphotericin B. Fungi were identified using the T3B PCR fingerprinting. This method identified all strains as Sporothrix brasiliensis and also demonstrated a high degree of similarity between the strains. In general, voriconazole was ineffective against all strains, and elevated minimal inhibitory concentrations (MICs) were observed for amphotericin B. High itraconazole and terbinafine MICs were not observed in S. brasiliensis isolates from patients of this study. Moreover, a significant increase in itraconazole and terbinafine MIC values from strains isolated from the same patient in different periods was not observed. The results suggest that the antifungal susceptibility to terbinafine and itraconazole determined by the reference method does not play an important role in therapeutic failure of sporotrichosis and that acquisition of resistance during prolonged antifungal treatment is not likely to occur in S. brasiliensis.

Hepatic Disease with Portal Hypertension and Acute Juvenile Paracoccidioidomycosis: A Report of Two Cases and Literature Review.

Paracoccidioidomycosis (PCM) is a neglected systemic mycosis endemic to Latin America caused by dimorphic fungi of the genus Paracoccidioides. The acute juvenile PCM is a severe type of presentation that usually affects young vulnerable patients and rarely progresses to portal hypertension. Here, two cases of liver disease and portal hypertension as complications of acute juvenile PCM are reported. Diagnosis of PCM was performed by isolation of the fungus and molecular identification of the strains provided through partial sequencing of two protein encoding genes, arf and gp43. Genotypic analysis revealed that Paracoccidioides brasiliensis S1 was the phylogenic species involved in both cases. Patients presented a good clinical response to amphotericin B and sulfamethoxazole-trimethoprim. These results highlight the importance of the interdisciplinary approach in patients with severe forms of PCM to avoid and treat complications, and the necessity of further investigations focusing on host-pathogen interaction in order to explain the broad clinical spectrum in PCM as well as the severity and poor outcome in some clinical cases.

Acute dacryocystitis: another clinical manifestation of sporotrichosis.

Sporotrichosis associated with exposure to domestic cats is hyperendemic in Rio de Janeiro, Brazil. A review of the clinical records at our institute revealed four patients with clinical signs of dacryocystitis and a positive conjunctival culture for Sporothrix who were diagnosed with Sporothrix dacryocystitis. Three patients were children (< 13 years of age) and one patient was an adult. Two patients reported contact with a cat that had sporotrichosis. Dacryocystitis was associated with nodular, ulcerated lesions on the face of one patient and with granulomatous conjunctivitis in two patients; however, this condition manifested as an isolated disease in another patient. All of the patients were cured of the fungal infections, but three patients had chronic dacryocystitis and one patient developed a cutaneous fistula. Sporotrichosis is usually a benign disease, but may cause severe complications when the eye and the adnexa are affected. Physicians, especially ophthalmologists in endemic areas, should be aware of the ophthalmological manifestations and complications of sporotrichosis.

Sporothrix brasiliensis Causing Atypical Sporotrichosis in Brazil: A Systematic Review.

Zoonotic sporotrichosis, a subcutaneous mycosis caused by Sporothrix brasiliensis, has become hyperendemic and a serious public health issue in Brazil and an emerging disease throughout the world. Typical sporotrichosis is defined as fixed or lymphocutaneous lesion development, however, reports of atypical presentations have been described in hyperendemic areas, which may result in a worse prognosis. Thus, considering an increase in atypical cases and in more severe extracutaneous cases and hospitalizations reported in Brazil, we aimed to perform a systematic review to search for hypersensitivity reactions (HRs) and extracutaneous presentations associated with zoonotic sporotrichosis. A systematic review was performed, following the PRISMA guidelines to search for atypical/extracutaneous cases (mucosal, osteoarthritis, HRs, pulmonary, meningeal) of zoonotic sporotrichosis. A total of 791 published cases over 26 years (1998-2023) in eleven Brazilian states were reviewed. Most cases corresponded to a HR (47%; n = 370), followed by mucosal (32%; n = 256), multifocal (8%; n = 60), osteoarthritis (7%; n = 59), meningeal (4%; n = 32), and pulmonary (2%; n = 14) infections. When available (n = 607), the outcome was death in 7% (n = 43) of cases. Here, we show a frequent and worrisome scenario of zoonotic sporotrichosis in Brazil, with a high and dispersed incidence of atypical/extracutaneous cases throughout the Brazilian territory. Therefore, educational measures are necessary to make health professionals and the overall population aware of this fungal pathogen in Brazil as well as in other countries in the Americas.

Trends in the Epidemiological and Clinical Profile of Paracoccidioidomycosis in the Endemic Area of Rio de Janeiro, Brazil.

Paracoccidioidomycosis (PCM) is a neglected endemic mycosis in Latin America. Most cases occur in Brazil. It is classified as PCM infection and PCM disease and is subdivided into chronic (adult type) or acute (juvenile type) disease, with the latter being less frequent and more severe. In 2016, we reported an increase in the numbers of patients diagnosed with acute PCM after a highway's construction. We conducted a study at INI-Fiocruz, a reference center for infectious diseases, including endemic mycoses, in Rio de Janeiro, Brazil, aiming to deepen the analysis of this new clinical and epidemiological profile of PCM. The authors developed a retrospective study including 170 patients diagnosed with PCM between 2010 and 2019. There was an increase in the number of atypical and severe forms, starting in 2014. In subsequent years, we detected a higher incidence of adverse outcomes with patients requiring more hospitalizations and an increased mortality rate. We estimate that PCM has become more severe throughout the Rio de Janeiro state, affecting a greater number of young individuals and leading to a greater number of and longer hospitalizations. Surveillance measures and close monitoring of future notification data in the state, with emphasis on children, adolescents, and young adults are necessary for a better understanding of the perpetuation of this public health challenge.

The State of the Art in Transcriptomics and Proteomics of Clinically Relevant Sporothrix Species.

Proteomics provide a robust approach to profile and quantify proteins within cells, organs, or tissues, providing comprehensive insights about the dynamics of cellular processes, modifications, and interactions. Similarly, understanding the transcriptome is essential to decipher functional elements of the genome, unraveling the mechanisms of disease development and the molecular constituents of cells and tissues. Some thermodimorphic fungi of the genus Sporothrix cause sporotrichosis, a subcutaneous mycosis of worldwide relevance. The transcriptome and proteome of the main Sporothrix species of clinical interest can elucidate the mechanisms underlying pathogenesis and host interactions. Studies of these techniques can contribute to the advancement of novel diagnostic and therapeutic strategies. A literature review was carried out, addressing all articles based on proteomics using mass spectrometry and transcriptomics of Sporothrix spp. Twenty-one studies were eligible for this review. The main findings include proteins and genes involved in dimorphism, cell differentiation, thermotolerance, virulence, immune evasion, metabolism, cell adhesion, cell transport, and biosynthesis. With the spread and emergence of sporotrichosis in different countries, ongoing research efforts and new discoveries are welcome to advance knowledge about this mycosis and its agents.

Sporotrichosis in Older Adults: A Cohort Study of 911 Patients from a Hyperendemic Area of Zoonotic Transmission in Rio de Janeiro, Brazil.

Generally, older people tend to suffer from more severe infections than younger adults. In addition, there are accumulations of comorbidities and immune senescence in some cases. This cohort study evaluated the clinical and epidemiological characteristics of older adults (≥60 years old) with sporotrichosis. The cohort consisted of 911 patients with a median age of 67 years, most of whom were female (72.6%), white (62.1%), and afflicted with comorbidities (64.5%). The lymphocutaneous form occurred in 62% of the patients, followed by the fixed form (25.7%), cutaneous disseminated form (8.9%), and extracutaneous/disseminated forms (3.3%). In this study, we draw attention to the frequency of osteoarticular involvement (2.1%) secondary to skin lesions such as osteomyelitis and/or tenosynovitis. A clinical cure was achieved in 87.3% of cases. Itraconazole was used in 81.1% of cases, while terbinafine was used in 22.7% of cases, usually in low doses. Survival analysis showed that the median treatment time was 119 days, and the multiple Cox model demonstrated that the presentation of a black coloration and diabetes was associated with a longer treatment time required to establish a cure. Therefore, these subgroups should be monitored more closely to reduce possible difficulties during treatment. It would be interesting to conduct more studies analyzing older adults with sporotrichosis from different geographic areas to better comprehend the disease in this group.

Sporotrichosis in the nasal mucosa: A single-center retrospective study of 37 cases from 1998 to 2020.

BACKGROUND: Sporotrichosis is a subcutaneous or implantation mycosis caused by some species of the genus Sporothrix. Rio de Janeiro state, Brazil, experiences hyperendemic levels of zoonotic sporotrichosis, with increasing cases of disseminated disease, especially in people living with HIV (PLHIV). Involvement of the nasal mucosa is rare and occurs isolated or in disseminated cases, with a delayed resolution. METHODOLOGY/PRINCIPAL FINDINGS: This study aimed to describe the epidemiological, clinical, and therapeutic profiles of 37 cases of sporotrichosis with involvement of the nasal mucosa treated at the ear, nose, and throat (ENT) outpatient clinic of the Instituto Nacional de Infectologia Evandro Chagas, Fundação Oswaldo Cruz, from 1998 to 2020. Data were reviewed from the medical records and stored in a database. The Mann-Whitney test was used to compare the means of quantitative variables, and Pearson chi-square and Fisher's exact tests were used to verify the association between qualitative variables (p<0.05). Most patients were males, students or retirees, with a median age of 38 years, residents in the municipality of Rio de Janeiro, and infected through zoonotic transmission. Disseminated sporotrichosis forms in patients with comorbidities (mostly PLHIV) were more common than the isolated involvement of the mucosa. The main characteristics of lesions in the nasal mucosa were the presence/elimination of crusts, involvement of various structures, mixed appearance, and severe intensity. Due to therapeutic difficulty, itraconazole was combined with amphotericin B and/or terbinafine in most cases. Of the 37 patients, 24 (64.9%) healed, with a median of 61 weeks of treatment, 9 lost follow-up, 2 were still treating and 2 died. CONCLUSIONS: Immunosuppression was determinant to the outcome, with worse prognosis and lower probability of cure. Notably in this group, the systematization of the ENT examination for early identification of lesions is recommended to optimize the treatment and outcome of the disease.

Osteoarticular Sporotrichosis of the Knee Caused by Sporothrix brasiliensis: Two Similar Cases with Different Outcomes.

Sporotrichosis is the most frequent subcutaneous or implantation mycosis in Latin America, and its transmission occurs as a result of traumatic inoculation into the skin by organic matter containing the thermodimorphic fungi of the genus Sporothrix. Although cutaneous forms are more common, another important site is the osteoarticular system, whose hematogenous involvement is commonly associated with disseminated forms, especially in people who have an immunosuppressive condition, such as HIV/AIDS, chronic steroid use, and alcohol abuse. We present two cases of osteoarticular sporotrichosis of the knee caused by Sporothrix brasiliensis and followed up at our institution, with different outcomes. In the cases presented here, aging, anatomical sites, comorbidities, subtherapeutic serum levels, low adherence to treatment, and late diagnosis for different reasons may explain the observed outcomes. Early diagnosis of Sporothrix infection is critical in preventing complications, including death. We also highlight the importance of multidisciplinary follow-up and adherence to treatment for a favorable outcome.

Paracoccidioidomycosis and pregnancy: A 40-year single-center cohort study in the endemic area of Rio de Janeiro, Brazil.

The occurrence of acute paracoccidioidomycosis (PCM) in urban areas of the Rio de Janeiro state, Brazil, has emerged in recent years. Therefore, young populations, including pregnant women, are at a higher risk of infection. Furthermore, young women undergoing itraconazole treatment for PCM have increased chances to get pregnant because this medication may reduce the effectiveness of contraceptives. Acute PCM is invasive, reaching abdominal organs, posing a maternal-fetal risk. PCM treatment in pregnant women is also challenging due to the teratogenicity associated with the currently available oral drugs. There are scarce studies on PCM and pregnancy, mainly consisting of case reports and experimental murine models that highlight the severity of this association. We conducted a database research at a PCM reference center in Rio de Janeiro state from 1980 to 2020. We included patients diagnosed with PCM who were pregnant shortly before, at admission, or at any moment of their PCM follow-up care. Data related to pregnancy, childbirth, and the newborn were obtained from the Brazilian official public databases. We also reviewed the epidemiological and clinical features of these patients. During the study period, we identified 18 pregnant patients, with a median age of 26 years (range: 16-38). Among these cases, six (33.3%) were detected in the last 5 years, and 14 (77.8%) presented acute PCM, supporting the recent shift in the epidemiological profile towards acute PCM. Most pregnancies occurred during PCM treatment (n = 11, 61.1%), which led to challenges in the therapeutic management. Maternal-fetal complications occurred in some of these cases, including vaginal bleeding (n = 1), preeclampsia (n = 1), prematurity (n = 2), low birth weight (n = 4), and fetal deaths (n = 2). PCM during pregnancy presents a significant public health concern in the context of the emergence of acute PCM in urban areas.

Evaluation of Five Non-Culture-Based Methods for the Diagnosis of Meningeal Sporotrichosis.

Sporotrichosis is the main subcutaneous mycosis worldwide. Several complications, including meningeal forms, can be observed in immunocompromised individuals. The sporotrichosis diagnosis is time-consuming due to the culture's limitations. The low fungal burden in cerebrospinal fluid (CSF) samples is another important drawback in the diagnosis of meningeal sporotrichosis. Molecular and immunological tests can improve the detection of Sporothrix spp. in clinical specimens. Therefore, the following five non-culture-based methods were evaluated for the detection of Sporothrix spp. in 30 CSF samples: (i) species-specific polymerase chain reaction (PCR); (ii) nested PCR; (iii) quantitative PCR; (iv) enzyme-linked immunosorbent assay (ELISA) for IgG detection; and (v) ELISA for IgM detection. The species-specific PCR was unsuccessful in the diagnosis of the meningeal sporotrichosis. The other four methods presented substantial levels of sensitivity (78.6% to 92.9%) and specificity (75% to 100%) for the indirect detection of Sporothrix spp. Both DNA-based methods presented similar accuracy (84.6%). Both ELISA methods were concomitantly positive only for patients with sporotrichosis and clinical signs of meningitis. We suggest that these methods should be implemented in clinical practice to detect Sporothrix spp. in CSF early, which may optimize treatment, augment the chances of a cure, and improve the prognosis of affected individuals.

Sporotrichosis in HIV-infected patients: report of 21 cases of endemic sporotrichosis in Rio de Janeiro, Brazil.

Sporotrichosis is endemic in Rio de Janeiro, Brazil, and cases have been reported to be associated with HIV. This article describes the clinical manifestations and evolution of sporotrichosis in HIV-positive patients and constitutes the largest case series reported to date. There were 21 HIV-positive patients with sporotrichosis diagnosed by the recovery of the etiologic agent from 1999-2009. Sixteen patients (76.2%) were men and five (23.8%) were women, with a mean age of 41.2 years. Seven of these individuals were previously unaware of their HIV infection. Mean CD4 count was 346.4 cells/µl. The most frequent clinical presentations of sporotrichosis in these patients were the lymphocutaneous and disseminated form (seven patients each, 33.3%), followed by the widespread cutaneous form in five (23.8%), and fixed form in the remaining two (9.5%). In patients with the disseminated forms, clinical manifestations involved the skin in six, mucosa (nasal, oral, or conjunctival) in four, bone in two, and meninges in two. Eleven (52.4%) patients received itraconazole and eight (38.1%) amphotericin B contributing to an overall cure rate of 81%. Spontaneous cure was observed in one patient. The clinical forms of sporotrichosis varied according to the patients' immune status. The results demonstrate the importance of sporotrichosis as an opportunistic infection associated with AIDS in countries where the mycosis occurs.