ELISA with recombinant antigen Lb6H validated for the diagnosis of American tegumentary leishmaniasis.

American tegumentary leishmaniasis (ATL) diagnosis is an open question, and the search for a solution is urgent. The available tests that detect the etiological agent of the infection are specific for ATL diagnosis. However, they present disadvantages, such as low sensitivity and the need for invasive procedures to obtain the samples. Immunological methods (leishmanin skin test and search for anti-Leishmania antibodies) are good alternatives to the etiological diagnosis of ATL. Presently, we face problems with disease confirmation due to the discontinuity in the production of leishmanin skin test antigen, particularly in resource-poor settings. Aiming to diagnose ATL, we validated rLb6H-ELISA for IgG antibodies using 1,091 samples from leishmaniasis patients and healthy controls, divided into four panels, living in 19 Brazilian endemic and non-endemic states. The rLb6H-ELISA showed a sensitivity of 98.6% and a specificity of 100.0%, with the reference panel comprising 70 ATL patient samples and 70 healthy controls. The reproducibility evaluation showed a coefficient of variation of positive samples ≤ 8.20% for repeatability, ≤ 17,97% for reproducibility, and ≤ 8.12% for homogeneity. The plates sensitized with rLb6H were stable at 4°C and -20°C for 180 days and 37°C for seven days, indicating 12 months of validity. In samples of ATL patients from five research and healthcare centers in endemic and non-endemic areas, rLb6H-ELISA showed a sensitivity of 84.0%; no significant statistical difference was observed among the five centers (chi-square test, p = 0.13). In samples of healthy controls from four areas with different endemicity, a specificity of 92.4% was obtained; lower specificity was obtained in a visceral leishmaniasis high endemicity locality (chi-square test, p<0.001). Cross-reactivity was assessed in 166 other disease samples with a positivity of 13.9%. Based on the good diagnostic performance and the reproducibility and stability of the antigen, we suggest using ELISA-rLb6H to diagnose ATL.

Detection of Internal Transcribed Spacer 1 and hsp70 Genetic Markers Using Restriction Fragment Length Polymorphisms and Sequencing in Identification of Leishmania Species Causing Tegumentary Leishmaniasis in Brazil.

The identification of Leishmania species that cause tegumentary leishmaniasis (TL) is important for taxonomic and prognostic purposes. Molecular analysis using different Leishmania genomic targets is the most useful method for identifying Leishmania species. Therefore, we evaluated the performance of ribosomal RNA internal transcribed spacer 1 (ITS1) and heat shock protein (hsp70) genetic markers by polymerase chain reaction (PCR), followed by restriction fragment length polymorphism analysis (RFLP) and sequencing, for identification of Leishmania species. Samples from 84 Brazilian patients were amplified. Internal transcribed spacer 1 PCR followed by RFLP (HaeIII) [ITS1-RFLP (HaeIII)] identified 46.4% (39/84) of the samples as compatible with the Viannia subgenus. Internal transcribed spacer 1 PCR followed by sequencing (ITS1-sequencing) identified Leishmania (Viannia) braziliensis in 91.7% (77/84) of the TL samples, Leishmania (Leishmania) amazonensis in 3.6% (3/84), L. (V.) guyanensis in 2.4% (2/84), and L. (L.) infantum in 1.2% (1/84). One of the samples showed the same proportion of similarity with L. (V.) guyanensis and L. (V.) panamensis. hsp70 nested PCR followed by RFLP (HaeIII) [nested hsp70-RFLP (HaeIII)] identified 91.7% (77/84) of the samples as compatible with L. (V.) braziliensis/L. (V.) naiffi, 3.6% (3/84) with L. (L.) amazonensis, 1.2% (1/84) with L. (L.) infantum, and 3.6% (3/84) with L. (V.) guyanensis. hsp70 PCR followed by sequencing (hsp70-sequencing) identified L. (V.) braziliensis in 91.7% (77/84) of the TL samples, L. (L.) amazonensis in 3.6% (3/84), L. (V.) guyanensis in 3.6% (3/84), and L. (L.) infantum in 1.2% (1/84). Our findings clearly showed that nested hsp70-RFLP (HaeIII) is better than ITS1-RFLP (HaeIII) and that ITS1 or hsp70 PCR followed by sequencing was adequate for identifying Leishmania species. We also found that Leishmania (Viannia) braziliensis is the most common species causing TL in Brazil. Therefore, sequencing multiple target genes such as ITS1 and hsp 70 is more accurate than RFLP for identifying Leishmania species.

Seasonality of sporotrichosis in Brazil: A modelled analysis of the epidemic in São Paulo, 2011-2020.

BACKGROUND: Sporotrichosis is an endemic subcutaneous mycosis classically caused by the Sporothrix schenckii species complex. Recently, sporotrichosis has emerged in Brazil as a cat-transmitted epidemic caused by a new species, Sporothrix brasiliensis. OBJECTIVES: To survey the clinical-epidemiological profile of all sporotrichosis cases diagnosed between 2011 and 2020 at a reference hospital in São Paulo metropolitan area and evaluate the annual distribution of cases in relation to seasonality. METHODS: Patients' demographic and clinical-epidemiological data were surveyed. A generalized linear model was fitted to relate the quarterly number of sporotrichosis cases detected between 2015 and 2019 with precipitation and temperature series. Prediction of the number of cases from 2011 to 2014 was attempted based on the fitted model without the trend component that appears from 2015. RESULTS: Among 271 suspected cases admitted during 2011-2020, 254 were confirmed by fungal isolation and/or clinical-epidemiological criteria. We observed that 2015 onwards the number of cases regularly increased during Autumn and Winter, the driest and coldest stations of the year. We verified that temperature series affected the number of cases (p = .005) because an increase of 1°C in the temperature series was associated with a 14.24% decrease in the average cases number, with the average number of cases increasing by 10.96% (p < .0001) every quarter, corresponding to an annual increase of 52%. Between 2011 and 2014, the predicted number of sporotrichosis cases averaged 10-12 per year, with 33%-38% occurring in the winter. CONCLUSION: We hypothesize that sporotrichosis seasonality is associated with the felines' oestrus cycle, which may provide alternative, cat-directed approaches to the sporotrichosis epidemic control.

A neglected disease. Human sporotrichosis in a densely populated urban area in São Paulo, Brazil: clinical-epidemiological and therapeutic aspects.

Human sporotrichosis is caused by different Sporothrix species; however, Sporothrix brasiliensis is the main species, usually related to cat transmission in urban areas. A retrospective descriptive study was conducted at the Institute of Infectology Emílio Ribas from 2010 to 2018. Demography, clinical, diagnostic, and therapeutic data were obtained from medical records. Polymerase chain reaction of the calmodulin gene was performed to identify Sporothrix species. In addition, to evaluate the spread of the disease across São Paulo metropolitan region, TerraView version 4.2.2 software was used for geocoding cases according to residence addresses. Kernell's maps using QGIS software version 2.16.3 were constructed to determine the concentration of cases. Results: 260 cases of sporotrichosis were diagnosed between 2010 and 2018. We observed a 700% increment in the number of human cases in the 2016-2018 triennium compared with the 2013-2015 triennium. Female adults with a median age of 46 years old were the predominant infected group associated with cats' exposition at home care, although the age range of all patients was 01 to 86 years old. The main epidemiological risk of acquiring sporotrichosis was contact with cats, reported by 96.5% of the patients. Molecular identification showed that most of the tested isolates were Sporothrix brasiliensis. Lymphocutaneous form was observed in 59.2% and fixed cutaneous form in 37.5% of the patients. Regarding treatment, itraconazole was the main drug used (94.2%) with a cure rate of 98.8%. We observed an important spread of human sporotrichosis involving cat transmission caused by Sporothrix brasiliensis in a densely populated area of São Paulo state. These results are important to alert clinicians and dermatologists about the occurrence and progression of a neglected tropical disease in an urban area and the urgent necessity to include sporotrichosis as a differential diagnosis in the clinical investigation routine.