Abstract Background Eugenol shows both antibacterial and antiparasitic activities, suggesting that it might be evaluated as an option for the treatment of praziquantel-resistant schistosome. Methods The in vitro activities of three eugenol derivatives (FB1, FB4 and FB9) on adult worms from Schistosoma mansoni were examined by fluorescence and scanning electron microscopy to analyze effects on the excretory system and integument damage, respectively. Biochemical tests with verapamil (a calcium channel antagonist) and ouabain (a Na+/K+-ATPase pump inhibitor) were used to characterize eugenol derivative interactions with calcium channels and the Na+/K+-ATPase, while in silico analysis identified potential Na+/K+-ATPase binding sites. Results The compounds showed effective doses (ED50) of 0.324 mM (FB1), 0.167 mM (FB4), and 0.340 mM (FB9). In addition, FB4 (0.322 mM), which showed the lowest ED50, ED90 and ED100 (p < 0.05), caused the most damage to the excretory system and integument, according to both fluorescence and scanning electron microscopy analysis. The death of adult worms was delayed by ouabain treatment plus FB1 (192 versus 72 hours) and FB9 (192 versus 168 hours), but the response to FB4 was the same in the presence or absence of ouabain. Besides, no changes were noted when all of the eugenol derivatives were combined with verapamil. Moreover, FB1 and FB9 inhibited Na+/K+-ATPase activity according to in silico analysis but FB4 did not show a time-dependent relationship and may act on targets other than the parasite Na+/K+-ATPase. Conclusion Eugenol derivatives, mainly FB4 when compared to FB1 and FB9, seem to act more effectively on the integument of adult S. mansoni worms.

Abstract INTRODUCTION: In most Strongyloides stercoralis infected individuals, nematoidosis occurs asymptomatically, but in immunocompromised patients, it can cause hyperinfection. Serological techniques seem to be a good alternative for detecting this parasite. METHODS The frequency of seropositivity for strongyloidiasis in Alfenas, MG, was estimated using the enzyme linked immunosorbent assay on blood samples, between May and August of 2015. RESULTS: Out of 258 samples tested, 53.9% were positive, and the frequency of seropositive individuals was higher in the peripheral districts of the municipality. CONCLUSIONS: The results indicate high seropositivity rates for strongyloidiasis among the residents of Alfenas city.

SUMMARY Visceral leishmaniasis is a systemic and chronic disease and dogs are the main reservoir of the etiologic agent, Leishmania infantum (syn L. chagasi). A serological and molecular investigation of canine visceral leishmaniasis (CVL) was performed in the municipality of Alfenas, located in the southern region of Minas Gerais, where the disease is not endemic. Samples from 87 dogs were submitted to serological tests including the Dual Path Platform (DPP (r) ) CVL Bio-Manguinhos rapid test, an in-house enzyme-linked immunosorbent assay (ELISA) and an immunofluorescence antibody test (IFAT), as well as molecular techniques such as a conventional polymerase chain reaction (PCR) with the RV1/RV2 primers and a quantitative PCR (qPCR) with the LinJ31, Ldon and DNApol primers. Of the 87 serum samples, eight (9.2%) were positive for Leishmania using the DPP rapid test, but only four (4.6%) were confirmed by ELISA and two (2.3%) by IFAT. In these two serologically confirmed cases, spleen and liver samples were positive by all the employed molecular and parasitological procedures performed on spleen samples. When whole blood samples were used in the molecular assays, two samples (2.3%) were positive only by qPCR. DNA extracted and amplified from the spleens of seropositive dogs was sequenced, showing 100% of similarity with the Leishmania infantum (syn L. chagasi) sequence. Thus, the first cases of CVL have been confirmed in the Alfenas region, suggesting the importance of canine surveys in non-endemic municipalities for CVL to monitor disease progression and to prevent outbreaks.

No Brasil, cães domésticos são considerados como os principais reservatórios da leishmaniose visceral zoonótica, devido à clara correlação positiva existente entre as curvas de infecção humana e canina. Este estudo objetivou a realização de um inquérito sorológico da leishmaniose visceral canina (LVC) em cães abrigados em um canil público de Juiz de Fora, Minas Gerais, Brasil, através do teste rápido imunocromatográfico TR DPP®. Adicionalmente, a PCR convencional e/ou em tempo real foi usada para detectar/confirmar a infecção por L. infantum apenas nos animais DPP positivos. Dos 400 cães estudados, a maioria não apresentou sinais clínicos para a LVC (p < 0,05) e quinze (3,8%) foram sororreativos ao DPP. Não houve diferença estatisticamente significativa entre os cães com DPP positivo e os sinais clínicos para a doença (p > 0,05). PCR convencional e em tempo real confirmaram a infecção por L. infantum em nove (75,0%) dos doze animais DPP positivos que permaneceram vivos durante o estudo. Este é o primeiro estudo soroepidemiológico sobre LVC realizado no município de Juiz de Fora, e os resultados reforçam a idéia de que esta doença está em processo de expansão e urbanização no Brasil. Além disto, este estudo destaca o uso do DPP como uma alternativa para o diagnóstico da LVC em cidades de médio e grande porte, devido à facilidade de execução.

In Brazil, domestic dogs are branded as the primary reservoir for zoonotic visceral leishmaniasis, due to the clear positive correlation observed between human and canine infection rates. This study aimed to carry out a serological survey of canine visceral leishmaniasis (CVL) in dogs housed at a public kennel in the municipality of Juiz de Fora, Minas Gerais State, Brazil, using the immunochromatographic TR DPP® CVL rapid test. Additionally, conventional and/or real time PCR assay was used to detect and confirm L. infantum infection in the DPP positive dogs only. Of the 400 dogs studied, most did not present clinical signs for CVL (p < 0.05), and fifteen (3.8%) were seropositive in the DPP test. There was no statistically significant difference between the DPP seropositive dogs and the clinical signs of the disease (p > 0.05). Both conventional and real time PCR tests confirmed L. infantum infection in nine (75.0%) of the twelve DPP seropositive dogs that remained alive during the follow-up period. This study is the first seroepidemiologic survey of CVL held in the city of Juiz de Fora, and the results reinforce the idea that this disease is currently in a process of expansion and urbanization in Brazil. Furthermore, this study highlights the use of the DPP test as an alternative for diagnosing CVL in large and mid-sized cities, due to its ease of implementation.

INTRODUÇÃO: Analisou-se a positividade da reação em cadeia da polimerase (PCR) na detecção de DNA de Leishmania em pacientes. MÉTODOS: DNA extraído foi submetido a L150/L152, 13Y/13Z e PCR seminested (snPCR). RESULTADOS: Resultados foram evidenciados por bandas de aproximadamente 120; 720 e 670pb para L150/L152, 13Y/13Z e snPCR, respectivamente. Positividades para L150/L152, 13Y/13Z e snPCR foram 76,9; 56,4 e 69,2 (p > 0,05), para suspeitos; e 93,7; 68,7 e 84,4 (p < 0,05) para confirmados, respectivamente. CONCLUSÕES: Resultados preliminares mostraram que os ensaios, principalmente L150/L152 e snPCR, podem detectar DNA de Leishmania e têm potencial para diagnóstico laboratorial das leishmanioses.

INTRODUCTION: The study analyzed positivity of polymerase chain reaction (PCR) on detection of DNA from Leishmania in patients' samples. METHODS: Extracted DNA was submitted to L150/L152, 13Y/13Z, and seminested PCR (snPCR). RESULTS: Results were evidenced by bands of approximately 120, 720, and 670 bp for L150/L152, 13Y/13Z, and snPCR, respectively. L150/L152, 13Y/13Z, and snPCR positivity indexes were 76.9, 56.4, and 9.2 (p>0.05), respectively, for suspected and 93.7, 68.7, and 84.4 (p<0.05), respectively, for confirmed. CONCLUSIONS: Preliminary results showed that these assays, mainly L150/L152 and snPCR, can detect Leishmania DNA and carry potential on laboratory diagnosis of leishmaniasis.

Neste trabalho, foi avaliado o perfil de isotipos de imunoglobulinas anti-Paracoccidioides brasiliensis em soros de pacientes com formas crônica e aguda de paracoccidiodomicoses usando antígeno total e tratado com meta-periodato. Todos os tipos de imunoglobulinas presentes nos soros de pacientes com formas aguda e crônica apresentaram alta reatividade ao antígeno total quando comparado ao tratado com meta-periodato (P < 0,05). Houve maior reatividade de IgG e IgM anti-antígeno tratado com meta-periodato em soros de pacientes com forma aguda da doença (P < 0,05), enquanto IgA foi mais reativa em soros da forma crônica (P < 0,05). Houve maior reatividade de IgG1 e IgG2 com antígeno total e tratado com meta-periodato em soros de pacientes comparados aos com outras parasitoses (P < 0,05). Além disso, IgG1 de pacientes com a forma aguda reconhecem antígenos de 19kDa, 27kDa e 31kDa por western blot. Assim, os resultados sugerem que alterações nos epitopos de antígenos de Paracoccidioides brasiliensis podem auxiliar no aprimoramento do imunodiagnóstico da paracoccidioidomicose.

In this study, we evaluated the profile of anti-Paracoccidioides brasiliensis immunoglobulin isotypes in serum from patients with the acute and chronic forms of paracoccidioidomycosis, using the whole Paracoccidioides brasiliensis antigen and the antigen treated with sodium metaperiodate. All the immunoglobulin isotypes present in the serum from patients with the acute and chronic forms of paracoccidioidomycosis presented higher reactivity towards the whole antigen than to the antigen treated with metaperiodate (P < 0.05). The reactivity of IgG and IgM to the antigen treated with metaperiodate was greater in serum from patients with the acute form of the disease (P < 0.05), while IgA was more reactive in serum from patients with the chronic form (P < 0.05). There was greater reactivity of IgG1 and IgG2 to the whole antigen and the antigen treated with metaperiodate in the serum from patients with paracoccidioidomycosis than there was in serum from patients with other parasitic infections (P < 0.05). Furthermore, IgG1 from patients with the acute form recognized the 19kDa, 27kDa and 31kDa antigens in the western blot test. Thus, the results suggest that modifications to the epitopes of Paracoccidioides brasiliensis antigens may help to improve the immunodiagnosis of paracoccidioidomycosis.

O presente trabalho descreve inicialmente um cão com sintomas característicos de leishmaniose visceral. Amostra de soro desse cão foi positiva por imunofluorescência indireta (IFI) conduzida no IgG total anti-Leishmania em 1999. Além disso, tecidos desse cão foram positivos por reação em cadeia pela polimerase (PCR) conduzida em 2004, identificando DNA de Leishmania no cerebelo, fígado, rim e intestino. Esta é a primeira vez que um cão com leishmaniose visceral autóctone foi descrito no Município de Bom Sucesso, Minas Gerais, Brasil. O achado desse cão reagente à IFI levou a uma investigação epidemiológica nesse município. Essa investigação foi conduzida de março de 1999 a dezembro de 2005. Vinte e dois de um total de 734 (3%) cães examinados foram reagentes à IFI. Entre os 22 cães IFI reagentes, seis apresentaram sintomas característicos de leishmaniose visceral canina. Os resultados desta investigação epidemiológica foram enviados às autoridades locais e estaduais de saúde pública requerendo medidas preventivas e de controle para leishmaniose visceral de forma a interromper a transmissão da doença e evitar a ocorrência de casos humanos.

The article begins by describing a dog with characteristic symptoms of visceral leishmaniasis. A serum sample from this animal was positive by indirect immunofluorescence (IIF) performed in anti-Leishmania total IgG in 1999. Tissues from the same dog were also positive by polymerase chain reaction (PCR) in 2004, identifying Leishmania DNA in the cerebellum, liver, kidney, and intestine. This is the first report of a dog with autochthonous visceral leishmaniasis in the county of Bom Sucesso, Minas Gerais State, Brazil. The finding of this IIF-positive dog led to a canine visceral leishmaniasis epidemiological investigation in the county. The investigation was conducted from March 1999 to December 2005. IIF was positive for Leishmania in 22 (3%) of 734 examined dogs. Among the 22 IIF-positive dogs, six presented characteristic symptoms of canine visceral leishmaniasis. The results of this epidemiological investigation were sent to local and State public health authorities, requesting visceral leishmaniasis control and preventive measures to interrupt transmission of the disease and avoid the occurrence of human cases.

O presente trabalho descreve inicialmente um cão com sintomas característicos de leishmaniose visceral. Amostra de soro desse cão foi positiva por imunofluorescência indireta (IFI) conduzida no IgG total anti-Leishmania em 1999. Além disso, tecidos desse cão foram positivos por reação em cadeia pela polimerase (PCR) conduzida em 2004, identificando DNA de Leishmania no cerebelo, fígado, rim e intestino. Esta é a primeira vez que um cão com leishmaniose visceral autóctone foi descrito no Município de Bom Sucesso, Minas Gerais, Brasil. O achado desse cão reagente à IFI levou a uma investigação epidemiológica nesse município. Essa investigação foi conduzida de março de 1999 a dezembro de 2005. Vinte e dois de um total de 734 (3%) cães examinados foram reagentes à IFI. Entre os 22 cães IFI reagentes, seis apresentaram sintomas característicos de leishmaniose visceral canina. Os resultados desta investigação epidemiológica foram enviados às autoridades locais e estaduais de saúde pública requerendo medidas preventivas e de controle para leishmaniose visceral de forma a interromper a transmissão da doença e evitar a ocorrência de casos humanos.

The article begins by describing a dog with characteristic symptoms of visceral leishmaniasis. A serum sample from this animal was positive by indirect immunofluorescence (IIF) performed in anti-Leishmania total IgG in 1999. Tissues from the same dog were also positive by polymerase chain reaction (PCR) in 2004, identifying Leishmania DNA in the cerebellum, liver, kidney, and intestine. This is the first report of a dog with autochthonous visceral leishmaniasis in the county of Bom Sucesso, Minas Gerais State, Brazil. The finding of this IIF-positive dog led to a canine visceral leishmaniasis epidemiological investigation in the county. The investigation was conducted from March 1999 to December 2005. IIF was positive for Leishmania in 22 (3%) of 734 examined dogs. Among the 22 IIF-positive dogs, six presented characteristic symptoms of canine visceral leishmaniasis. The results of this epidemiological investigation were sent to local and State public health authorities, requesting visceral leishmaniasis control and preventive measures to interrupt transmission of the disease and avoid the occurrence of human cases.

Neste estudo, descrevemos etapas iniciais de padronização de uma nova metodologia para detecção de anticorpos antipromastigotas vivas de Leishmania (Viannia) braziliensis, pela citometria de fluxo e a análise de sua aplicabilidade para estudos clínicos. Foram avaliados 39 indivíduos com sorologia convencional (RIFI) positiva para leishmaniose, classificados quanto à ausência/presença de lesão (L- e L+). Os resultados foram expressos sob a forma de percentual de parasitas fluorescentes positivos (PPFP). A análise dos dados, na diluição 1:1.024, permitiu distinguir 95% dos pacientes L+ como um grupo de alta reatividade (PPFP>50%) e 72% dos indivíduos L- como um grupo de baixa reatividade (PPFP<50%). A análise dos títulos da reação de imunofluorescência indireta não demonstrou nenhuma relação com a ausência/presença de lesão. Em conjunto, nossos dados sugerem a aplicabilidade da citometria de fluxo na identificação dos casos de infecção ativa, o que não tem sido possível através das reações sorológicas convencionais.

In the current study we described initial standardization steps of a new methodology to detect anti-live Leishmania (Viannia) braziliensis promastigote antibodies by flow cytometry, followed by analysis of its applicability to clinical studies. We have studied 39 individuals with positive conventional serology to leishmaniasis, classified according to the absence/presence of cutaneous lesions (L- and L+). The results were expressed as percentage of positive fluorescent parasites (PPFP). Data analysis at dilution of 1:1,024, allowed the distinction of 95% of L+ patients as a group of high reactivity (PPFP>50%) and 72% of L- individuals as a group of low reactivity (PPFP<50%). The analysis of immunofluorescence assay titers did not show any relationship with the absence/presence of lesion. Together, our data support the applicability of flow cytometry to identify cases of active infection, which has not been possible through conventional serological reactions.