A Randomized, Controlled, Noninferiority, Multicenter Trial of Systemic vs Intralesional Treatment With Meglumine Antimoniate for Cutaneous Leishmaniasis in Brazil.

BACKGROUND: Meglumine antimoniate (MA) remains the main treatment for cutaneous leishmaniasis (CL). Uncontrolled studies suggest that intralesional MA (IL-MA) may be noninferior and safer than systemic MA (S-MA). METHODS: Multicenter, randomized, controlled, open-label, phase 3 clinical trial to evaluate the efficacy and toxicity of IL-MA in 3 infiltrations at 14-day intervals compared with S-MA (10-20 mg Sb5+/kg/day, 20 days) for CL, with noninferiority margin of 20%. Primary and secondary outcomes were definitive cure at day 180 and epithelialization rate at day 90 of treatment, respectively. A 2-year follow-up was performed to assess relapses and emergence of mucosal lesions. Adverse events (AEs) were monitored according to the Division of AIDS AE grading system. RESULTS: We evaluated 135 patients. The cure rates (95% confidence interval) for IL-MA and S-MA treatment were, respectively, 82.8% (70.5-91.4) and 67.8% (53.3-78.3) per protocol (PP) and 70.6% (58.3-81.0) and 59.7% (47.0-71.5) per intention to treat (ITT). The epithelialization rates of the IL-MA and S-MA treatment were, respectively, 79.3% (66.6-88 + 8) and 71.2% (57.9-82.2) PP and 69.1% (55.2-78.5) and 64.2% (50.0-74.2) ITT. AEs in the IL-MA and S-MA groups were, respectively, clinical, 45.6% and 80.6%; laboratory, 26.5% and 73.1%; and electrocardiogram, 8.8% and 25.4%. Ten participants in the S-MA group and 1 in the IL-MA group were discontinued due to severe or persistent AEs. CONCLUSIONS: IL-MA provides a similar cure rate and results in less toxicity compared with S-MA and may be used as first-line therapy for CL patients. CLINICAL TRIALS REGISTRATION: REBEC: RBR-6mk5n4.

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.

Clinical and laboratory profiles of patients with early spontaneous healing in cutaneous localized leishmaniasis: a historical cohort study.

BACKGROUND: Skin ulcers in American cutaneous leishmaniasis (ACL) may heal spontaneously after months/years. However, few cases may present quick heal even during diagnosis procedure (early spontaneous healing- ESH). The main objective of this study was to compare ESH patients with cases requiring specific treatment [non-ESH (NESH)]. METHODS: A historical cohort study of ACL patients (n = 445) were divided into 2 groups: ESH - spontaneously healed patients (n = 13; 2.90%), and NESH- treated patients (n = 432; 97.10%). We compared clinical and laboratorial findings at diagnosis, including the lesion healing process. RESULTS: ESH patients had a higher percentage of single lesions (p = 0.027), epithelialized lesion on initial examination (p = 0.001), lesions located in the dorsal trunk (p = 0.017), besides earlier healing (p < 0.001). NESH presents higher frequency of ulcerated lesions (p = 0.002), amastigotes identified in histopathology exams (p = 0.005), positive cultures (p = 0.001), and higher positivity in ≥3 parasitological exams (p = 0.030). All ESH cases were positive in only a single exam, especially in PCR. CONCLUSIONS: ESH group apparently presented a lower parasitic load evidenced by the difficulty of parasitological confirmation and its positivity only by PCR method. The absence or deficiency of specific treatment is commonly identified as predisposing factors for recurrence and metastasis in ACL. However, due to the drugs toxicity, the treatment of cases which progress to early spontaneous healing is controversial. ESH patients were followed for up to 5 years after cure, with no evidence of recrudescence, therefore suggesting that not treating these patients is justifiable, but periodic dermatological and otorhinolaryngological examinations are advisable to detect a possible relapse.

Effect of secondary infection on epithelialisation and total healing of cutaneous leishmaniasis lesions.

BACKGROUND: Cutaneous leishmaniasis (CL) generally presents with a single or several localised cutaneous ulcers without involvement of mucous membranes. Ulcerated lesions are susceptible to secondary contamination that may slow the healing process. OBJECTIVE: This study verified the influence of non-parasitic wound infection on wound closure (epithelialisation) and total healing. METHODS: Twenty-five patients with a confirmed diagnosis of CL and ulcerated lesions underwent biopsy of ulcer borders. One direct microbial parameter (germ identification in cultures) and four indirect clinical parameters (secretion, pain, burning sensation, pruritus) were analysed. FINDINGS Biopsies of ten lesions showed secondary infection by one or two microorganisms (Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis, Streptococcus pyogenes and Candida parapsilosis). "Secretion" and "burning sensation" influenced epithelialisation time but not total healing time. Positive detection of germs in the ulcer border and "pain" and "pruritus" revealed no influence on wound closure. CONCLUSIONS: Our borderline proof of clinical CL ulcer infection inhibiting CL wound healing supports the need to follow antimicrobial stewardship in CL ulcer management, which was recently proposed for all chronic wounds.

Low dose systemic or intralesional meglumine antimoniate treatment for American tegumentary leishmaniasis results in low lethality, low incidence of relapse, and low late mucosal involvement in a referral centre in Rio de Janeiro, Brazil (2001-2013).

BACKGROUND: American tegumentary leishmaniasis (ATL) is a non-lethal parasitic disease that presents with cutaneous (CL) and mucosal (ML) clinical forms. ATL treatment aims at healing the lesions and preventing the development of the late mucosal form. Systemic meglumine antimoniate (MA) therapy with 10-20 mg Sb5+/kg/day is the first choice of treatment. However, alternative therapies using 5 mg Sb5+/kg/day or intralesional (IL) MA are the usual regimens at the National Institute of Infectious Diseases (NIID), Rio de Janeiro, Brazil. OBJECTIVES: To evaluate lethality and the incidence of relapse and development of late ML in CL patients treated at NIID from 2001 until 2013. METHODS: Data were recovered from records of all ATL patients diagnosed during that period. FINDINGS: Out of 777 patients, 753 were treated with MA (96.9%). Of those, 89.1% received alternative therapy of 9.9% IL and 79.2% systemic 5 mg Sb5+/kg/day. Some patients required 1-3 additional courses of treatment, thus making a total of 997 courses; 85.2% of them were subjected to alternative therapies. Lethality was 0.1%, relapse incidence 5.8%, and late ML incidence 0.25%. As a final outcome for the 777 patients, 95.9% were cured, 0.1% died and 4.0% were not able to follow-up. MAIN CONCLUSIONS: Alternative MA schedules resulted in low lethality without increase of relapse or late ML incidence.

Apoptosis and frequency of total and effector CD8+ T lymphocytes from cutaneous leishmaniasis patients during antimonial therapy.

BACKGROUND: Leishmaniasis is an important parasitic disease affecting millions worldwide. Human cutaneous leishmaniasis (CL) is endemic in Rio de Janeiro, Brazil, where is caused by Leishmania braziliensis. The adaptive immune response is accountable for the healing of CL and despite of key role of CD8+ T cells in this immune response little is known about the CD8+ T lymphocytes frequencies, apoptosis and antigen-responsive CD8+ T lymphocytes of CL patients during antimonial therapy. METHODS: Using flow cytometry, we examined total and effector CD8+ T cells from CL patients before (PBT), during (PDT) and after (PAT) treatment for apoptosis and frequencies upon isolation and after in vitro L. braziliensis antigens (LbAg)-stimulation culture. Besides, a correlation study between immunological findings and lesion size was done. RESULTS: PDT showed lower frequencies of total CD8+ T lymphocytes and higher levels of apoptosis of these cells, which were also observed following LbAg-stimulation culture. Regarding effector CD8+ T cells, high frequencies were observed in PDT, while lower frequencies were observed in PAT. Interestingly, PDT showed higher frequencies of apoptotic-effector CD8+ T lymphocytes. Similar results were seen after in vitro antigenic-stimulation assays. Correlation analysis showed that the greater the size of lesion, the smaller the frequency of effector CD8+ T lymphocytes in PDT and PAT, as well as a positive correlation between apoptotic-effector CD8+ T cells frequency and lesion size of PDT. CONCLUSIONS: Changes in effector CD8+ T-lymphocyte frequencies, during and after treatment, seem to represent a critical stage to generate an efficient immune response and suggest that these cells would be evolved in the triggering or in the resolution of lesion, under the influence of therapy. This hypothesis opens new perspectives to clarify controversial statements about the protective or deleterious role of CD8+ T cells in the cure or aggravation of CL and the new approach of evaluating patients during treatment proved to be of utmost importance for understanding the immune response in the healing process of human CL.

T-cell receptor Vß repertoire of CD8+ T-lymphocyte subpopulations in cutaneous leishmaniasis patients from the state of Rio de Janeiro, Brazil.

In human cutaneous leishmaniasis (CL), the immune response is mainly mediated by T-cells. The role of CD8+ T-lymphocytes, which are related to healing or deleterious functions, in affecting clinical outcome is controversial. The aim of this study was to evaluate T-cell receptor diversity in late-differentiated effector (LDE) and memory CD8+ T-cell subsets in order to create a profile of specific clones engaged in deleterious or protective CL immune responses. Healthy subjects, patients with active disease (PAD) and clinically cured patients were enrolled in the study. Total CD8+ T-lymphocytes showed a disturbance in the expression of the Vß2, Vß9, Vß13.2, Vß18 and Vß23 families. The analyses of CD8+T-lymphocyte subsets showed high frequencies of LDE CD8+T-lymphocytes expressing Vß12 and Vß22 in PAD, as well as effector-memory CD8+ T-cells expressing Vß22. We also observed low frequencies of effector and central-memory CD8+ T-cells expressing Vß2 in PAD, which correlated with a greater lesion size. Particular Vß expansions point to CD8+ T-cell clones that are selected during CL immune responses, suggesting that CD8+ T-lymphocytes expressing Vß12 or Vß22 are involved in a LDE response and that Vß2 contractions in memory CD8+T-cells are associated with larger lesions.

Self-reporting of hearing loss and tinnitus in the diagnosis of ototoxicity by meglumine antimoniate in patients treated for American tegumentary Leishmaniasis.

INTRODUCTION: American Tegumentary Leishmaniasis (ATL) treatment is based on pentavalent antimonials (Sb5+), but these drugs have been associated to several adverse effects. Hearing loss and tinnitus during treatment with meglumine antimoniate (MA) have already been reported. This study aimed to describe the usefulness of self-reporting of hearing loss and tinnitus in diagnosing MA-induced ototoxicity. METHODS: A prospective longitudinal study was conducted with 102 patients with parasitological diagnosis of ATL, treated with different MA schemes. The presence of clinical auditory toxicity was defined as the emergence or worsening of self-reporting hearing loss and/or tinnitus during monitoring. Measures of sensitivity, specificity, and the positive and negative predictive value of the patient's self-reporting of hearing loss and tinnitus in relation to the result of the audiometric test (considered the gold standard) were calculated. RESULTS: The age of the evaluated patients ranged from 15 to 81 years, with a median of 41 years, and most were male (73.5%). Seventy-five patients (73.5%) had cutaneous leishmaniasis and 27 (26.5%) mucosal leishmaniasis. Eighty-six patients (84.3%) received intramuscular (IM) treatment and 16 (15.7%) were treated with intralesional MA. During treatment, 18 (17,6%) had tinnitus and 7 (6,9%) had complaint of hearing loss. 53 (52%) patients had cochlear toxicity confirmed by tone threshold audiometry and high frequency audiometry, from which 60% received a dose of 20 mg Sb5+/kg/day (p = 0.015) and 96.2% were treated with IM MA (p = 0.001). Tinnitus has greater specificity and positive predictive value than hearing loss, with a low number of false positives, but with a high false negative value. CONCLUSION: Although the large number of false negatives suggests that self-report of hearing loss or tinnitus cannot be considered a good screening test for referring the patient to an audiometry, the low number of false positives suggests the need to value the patient's complaint for referral. Otherwise, this study reinforces the importance of audiological monitoring during treatment with MA, especially in those patients with self-reporting of hearing loss or tinnitus when treated with 20 mg Sb5+/kg/day via IM.

Sporothrix schenckii and Sporotrichosis.

Sporotrichosis, which is caused by the dimorphic fungus Sporothrix schenckii, is currently distributed throughout the world, especially in tropical and subtropical zones. Infection generally occurs by traumatic inoculation of soil, plants, and organic matter contaminated with the fungus. Certain leisure and occupational activities, such as floriculture, agriculture, mining, and wood exploitation, are traditionally associated with the mycosis. Zoonotic transmission has been described in isolated cases or in small outbreaks. Since the end of the 1990s there has been an epidemic of sporotrichosis associated with transmission by cats in Rio de Janeiro, Brazil. More than 2,000 human cases and 3,000 animal cases have been reported. In humans, the lesions are usually restricted to the skin, subcutaneous cellular tissue, and adjacent lymphatic vessels. In cats, the disease can evolve with severe clinical manifestations and frequent systemic involvement. The gold standard for sporotrichosis diagnosis is culture. However, serological, histopathological, and molecular approaches have been recently adopted as auxiliary tools for the diagnosis of this mycotic infection. The first-choice treatment for both humans and cats is itraconazole.

Is There Any Difference in the In Situ Immune Response in Active Localized Cutaneous Leishmaniasis That Respond Well or Poorly to Meglumine Antimoniate Treatment or Spontaneously Heal?

Localized cutaneous leishmaniasis caused by Leishmania braziliensis can either respond well or poorly to the treatment or heal spontaneously; It seems to be dependent on the parasite and/or host factors, but the mechanisms are not fully understood. We evaluated the in situ immune response in eighty-two active lesions from fifty-eight patients prior to treatment classified as early spontaneous regression (SRL-n = 14); treatment responders (GRL-n = 20); and non-responders (before first treatment/relapse, PRL1/PRL2-n = 24 each). Immunohistochemistry was used to identify cell/functional markers which were correlated with the clinical characteristics. PRL showed significant differences in lesion number/size, clinical evolution, and positive parasitological examinations when compared with the other groups. SRL presented a more efficient immune response than GRL and PRL, with higher IFN-γ/NOS2 and a lower percentage of macrophages, neutrophils, NK, B cells, and Ki-67+ cells. Compared to SRL, PRL had fewer CD4+ Tcells and more CD163+ macrophages. PRL1 had more CD68+ macrophages and Ki-67+ cells but less IFN-γ than GRL. PRL present a less efficient immune profile, which could explain the poor treatment response, while SRL had a more balanced immune response profile for lesion healing. Altogether, these evaluations suggest a differentiated profile of the organization of the inflammatory process for lesions of different tegumentary leishmaniasis evolution.

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.

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.

Comparison between Colorimetric In Situ Hybridization, Histopathology, and Immunohistochemistry for the Diagnosis of New World Cutaneous Leishmaniasis in Human Skin Samples.

New world cutaneous leishmaniasis (NWCL) is an anthropozoonosis caused by different species of the protozoan Leishmania. Colorimetric in situ hybridization (CISH) was shown to satisfactorily detect amastigote forms of Leishmania spp. in animal tissues, yet it was not tested for the diagnosis of human NWCL. The aim of this study was to compare CISH, histopathology (HP), and immunohistochemistry (IHC) techniques to diagnose NWCL in human cutaneous lesions. The sample comprised fifty formalin-fixed, paraffin-embedded skin biopsy specimens from patients with NWCL caused by L. (V.) braziliensis. These specimens were analyzed by CISH, using a generic probe for Leishmania, IHC, and HP to assess the sensitivity of these methods by using a parasitological culture as a standard reference. Additional specimens from three patients diagnosed with cutaneous mycoses were also included to evaluate cross-reactions between CISH and IHC. The sensitivities of IHC, CISH, and HP for detecting amastigotes was 66%, 54%, and 50%, respectively. IHC, unlike CISH, cross-reacted with different species of fungi. Together, these results demonstrate that CISH may be a complementary assay for the detection of amastigote in the laboratorial diagnosis routine of human NWCL caused by L. (V.) braziliensis.

Parasite species variation and impact of spatial displacement of the population on cutaneous leishmaniasis in Rio de Janeiro, Brazil.

BACKGROUND: Cutaneous leishmaniasis results from complex interactions between human beings, vectors and the environment. Parasitic species differ in epidemiological and geographical contexts. METHODS: We studied a retrospective cohort of 696 patients with cutaneous leishmaniasis treated at a reference centre in the state of Rio de Janeiro, Brazil, between 2000 and 2015. We analysed displacements due to work, leisure and migrations with identification of Leishmania species. RESULTS: The geographic distribution of autochthonous cases showed that >95% of infections occurred in urban areas. In the state of Rio de Janeiro, most cases were concentrated in the cities surrounding forest parks and nature conservation areas. The same applies to the city of Rio de Janeiro, where these infections occurred in the neighbourhoods surrounding some mountain and forest areas. The non-displacement group included 575 (82.6%) patients and the displacement group included 121 (17.4%) patients. Leishmania (Viannia) braziliensis predominated in both groups. Other species were found in the displacement group. CONCLUSIONS: The disordered urbanization of the state of Rio de Janeiro in recent decades has created conditions for the emergence of urban foci of transmission close to forest areas. Changes in the environment, movement of infected individuals and adaptation of sandflies may have contributed to this.

Treatment of cutaneous sporotrichosis with itraconazole--study of 645 patients.

BACKGROUND: Itraconazole has become the first choice for treatment of cutaneous sporotrichosis. However, this recommendation is based on case reports and small series. The safety and efficacy of itraconazole were evaluated in 645 patients who received a diagnosis on the basis of isolation of Sporothrix schenckii in Rio de Janeiro, Brazil. METHODS: A standard regimen of itraconazole (100 mg/day orally) was used. Clinical and laboratory adverse events were assessed a grades 1-4. A multivariate Cox model was used to analyze the response to treatment. RESULTS: The median age was 43 years. Lymphocutaneous form occurred in 68.1% and fixed form in 23.1%. Six hundred ten patients (94.6%) were cured with itraconazole (50-400 mg/day): 547 with 100 mg/day, 59 with 200-400 mg/day, and 4 children with 50 mg/day. Three patients switched to potassium iodide, 2 to terbinafine, and 4 to thermotherapy. Twenty-six were lost to follow-up. Clinical adverse events occurred in 18.1% of patients using 100 mg/day and 21.9% of those using 200-400 mg/day. The most frequent clinical adverse events were nausea and epigastric pain. Laboratory adverse events occurred in 24.1%; the most common was hypercholesterolemia, followed by hypertriglyceridemia. Four hundred sixty-two patients (71.6%) completed clinical follow-up, and all remained cured. Only 2 variables were significant in explaining the cure: patients with erythema nodosum healed faster, and lymphocutaneous form took longer to cure. CONCLUSIONS: In the current series, the therapeutic response was excellent with the minimum dose of itraconazole, and there was a low incidence of adverse events and treatment failure.

The in situ inflammatory profile of lymphocutaneous and fixed forms of human sporotrichosis.

The most common clinical presentations of sporotrichosis are the lymphocutaneous (LC) and fixed cutaneous (F) forms, but little is known about the immunopathologic differences between them. The aim of this study was to evaluate through immunohistochemistry the composition of the in situ inflammatory reaction so as to correlate the results with the clinical presentation of the disease. The following two groups of patients were involved in the studies, i.e., LC (n=19) and F (n=11) patients. Those with the LC form, in contrast to F patients, were found to have a larger number of lesions (P=0.001), of longer duration (P=0.026) and require a more extended course of treatment (P=0.049). LC patients also presented a greater fungal burden (LC:0-6.5; F:0-1.5; P=0.021), a higher percentage of neutrophils (median LC:24.7%; F:6.7%, P=0.002), CD4(+) cells (median LC:40.9%; F:30.0%, P=0.0024), CD22(+) cells (median LC:15.3%; F:2.9%, P=0.048), and higher intensity of NOS2 expression (P=0.009). Thus, our data identified differences in cell profile and inflammatory activity in lesions of LC and F forms of human sporotrichosis.

Histopathology of cutaneous sporotrichosis in Rio de Janeiro: a series of 119 consecutive cases.

BACKGROUND: Sporotrichosis is the most common subcutaneous mycosis in Rio de Janeiro. Histopathological examination reveals diffuse granulomatous and suppurative dermatitis, and the fungus is rarely identifiable in tissue. We describe the histopathological features of cutaneous sporotrichosis, and investigate the association between them and the lack of visualization of the fungus. METHODS: A total of 119 samples of confirmed sporotrichosis cases were studied. The characteristics of the inflammatory infiltrate, the presence of epidermal changes, necrosis and fibrosis, and the parasite burden were analyzed. The association between histopathological changes and the lack of visualization of the fungus was evaluated using prevalence ratios. RESULTS: Granulomas were observed in all samples, suppurative granulomas in 100 (84%) and diffuse dermatitis in 114 (95.8%). Liquefaction and caseous necrosis were present in 78 (65.5%) and 52 (43.7%) samples, respectively. The fungus was not seen in 77 (64.7%) samples. Epithelioid, tuberculoid or foreign-body-type granulomas, caseous, fibrinoid or absent necrosis, predominance of lymphocytes among nonphagocytic cells and fibrosis were associated with the lack of visualization of the fungus. CONCLUSION: The histopathological characteristics of sporotrichosis were variable when the causative fungus was not seen. In a proper context, the diagnosis of sporotrichosis remains a potential consideration even in the absence of demonstrable yeast.

Comparison of the sensitivity of imprint and scraping techniques in the diagnosis of American tegumentary leishmaniasis in a referral centre in Rio de Janeiro, Brazil.

American tegumentary leishmaniasis (ATL) is an infectious disease that presents a wide spectrum of clinical manifestations making parasitological tests important for its diagnosis. Direct examination, although considered of low sensitivity is still employed mainly in areas with poor laboratory infrastructure. The aim of this study was to standardize the method of collecting and reading the scraping procedure and to then compare sensitivity of this procedure on two sites of the lesion (outer edge-OE and inner edge-IE) and of the imprint against the reference method (isolation in culture) in a group of 110 patients treated at a Referral Center in Rio de Janeiro, Brazil. ATL diagnosis was confirmed in 40 patients (36.4%), 39 cases were caused by L. braziliensis and 1 by L. amazonensis. Imprint was positive in 28 patients and scraping in OE in 17 and in IE in 25 patients, resulting in sensitivity of 70%, 42.5%, and 62.5% respectively. When the three direct examinations were combined, sensitivity value attained 77.5%. Aspects related to ease and quality of the collected material, pain intensity and frequency of bleeding in the scraping procedure were also broached and discussed in this study. The parameters of accuracy presented indicate that the direct methods can be safely used in ATL diagnosis, principally in IE scraping, as it is easy to produce and the examination is not costly, which allows the procedure to be repeated at different moments which, in turn, increases the possibility of finding the parasite. Despite that the direct methods are technically widespread, they are not standardized and the parameters of accuracy are unknown. If we consider the high incidence of leishmaniasis in low-income areas, the implantation of standardized and selective methods would provide advances in the diagnosis of leishmaniasis.

Serine proteases profiles of Leishmania (Viannia) braziliensis clinical isolates with distinct susceptibilities to antimony.

Glucantime (SbV) is the first-line treatment against American Tegumentary Leishmaniasis. Resistance cases to this drug have been reported and related to host characteristics and parasite phenotypes. In this study, 12 Leishmania (Viannia) braziliensis isolates from patients that presented clinical cure (Responders-R) and relapse or therapeutic failure (Non-responders-NR) after treatment with antimony, were analyzed. These parasites were assessed by in vitro susceptibility to SbIII and SbV, serine proteases activity measured with substrate (z-FR-AMC) and specific inhibitors (TLCK, AEBSF and PMSF). In vitro susceptibility of axenic amastigotes to SbIII showed a significant difference between R and NR groups. The protease assays showed that TLCK inhibited almost 100% of activity in both axenic amastigotes and promastigotes while AEBSF inhibited around 70%, and PMSF showed lower inhibition of some isolates. Principal component and clustering analysis performed with these data yielded one homogeneous cluster with only NR isolates and three heterogeneous clusters with R and NR isolates. Additionally, differential expression of subtilisins (LbrM.13.0860 and LbrM.28.2570) and TXNPx (LbrM.15.1080) was evaluated in promastigotes and axenic amastigotes from both groups. The results showed a higher expression of LbrM.13.0860 and LbrM.15.1080 genes in axenic amastigotes, while LbrM.28.2570 gene had the lowest expression in all isolates, regardless of the parasite form. The data presented here show a phenotypic heterogeneity among the parasites, suggesting that exploration of in vitro phenotypes based on SbIII and serine proteases profiles can aid in the characterization of L. (V.) braziliensis clinical isolates.

An old drug and different ways to treat cutaneous leishmaniasis: Intralesional and intramuscular meglumine antimoniate in a reference center, Rio de Janeiro, Brazil.

BACKGROUND: Treatment of cutaneous leishmaniasis (CL) remains challenging since the drugs currently used are quite toxic, thus contributing to lethality unrelated to the disease itself but to adverse events (AE). The main objective was to evaluate different treatment regimens with meglumine antimoniate (MA), in a reference center in Rio de Janeiro, Brazil. METHODOLOGY: A historical cohort of 592 patients that underwent physical and laboratory examination were enrolled between 2000 and 2017. The outcome measures of effectiveness were epithelialization and complete healing of cutaneous lesions. AE were graded using a standardized scale. Three groups were evaluated: Standard regimen (SR): intramuscular (IM) MA 10-20 mg Sb5+/kg/day during 20 days (n = 46); Alternative regimen (AR): IM MA 5 mg Sb5+/kg/day during 30 days (n = 456); Intralesional route (IL): MA infiltration in the lesion(s) through subcutaneous injections (n = 90). Statistical analysis was performed through Fisher exact and Pearson Chi-square tests, Kruskal-Wallis, Kaplan-Meier and log-rank tests. RESULTS: SR, AR and IL showed efficacy of 95.3%, 84.3% and 75.9%, with abandonment rate of 6.5%, 2.4% and 3.4%, respectively. IL patients had more comorbidities (58.9%; p = 0.001), were mostly over 50 years of age (55.6%), and had an evolution time longer than 2 months (65.6%; p = 0.02). Time for epithelialization and complete healing were similar in IL and IM MA groups (p = 0.9 and p = 0.5; respectively). Total AE and moderate to severe AE that frequently led to treatment interruption were more common in SR group, while AR and IL showed less toxicity. CONCLUSIONS/SIGNIFICANCE: AR and IL showed less toxicity and may be good options especially in CL cases with comorbidities, although SR treatment was more effective. IL treatment was an effective and safe strategy, and it may be used as first therapy option as well as a rescue scheme in patients initially treated with other drugs.