Scielo RSS <![CDATA[Memórias do Instituto Oswaldo Cruz]]> vol. 111 num. 8 lang. es <![CDATA[SciELO Logo]]> <![CDATA[Experimental infection of <em>Phlebotomus perniciosus</em> by bioluminescent <em>Leishmania infantum</em> using murine model and artificial feeder]]> Leishmaniasis is a vector-borne disease that is transmitted by sandflies and caused by obligate intracellular protozoa of the genus Leishmania. In the present study, we carried out a screening on the experimental infection of Phlebotomus pernioucus by bioluminescent Leishmania infantum using murine model and artificial feeder. We developed a real-time polymerase chain reaction (RT-PCR)-based method to determine individually the number of Leishmania promastigotes fed by infected flies. Among 1840 new emerged female sand flies, 428 were fed on the infected mice. After their death, they were analysed individually by RT-PCR. Our results demonstrated just a single Leishmania positive female at sixth day post meal. A total of 1070 female sand flies were exposed in contact with artificial feeder containing the human blood with two different quantities of Leishmania parasites: 2.106/mL and 1.107/mL. A blood meal including 1.107/mL LUC-promastigotes was proposed to 270 females and 75 (28%) flies were engorged. Among them, 44 (59%) were positive by RT-PCR analysis, with a relative average of 50551 Leishmania parasites. In case of blood feeding of females with 2.106/mL promastigotes, 57 out of 800 (7%) females succeed to feed from artificial feeder which 22 (39%) were positive with a relative average of 6487 parasites. <![CDATA[Calculation of the ELISA’s cut-off based on the change-point analysis method for detection of <em>Trypanosoma cruzi</em> infection in Bolivian dogs in the absence of controls]]> In ELISAs, sera of individuals infected by Trypanosoma cruzi show absorbance values above a cut-off value. The cut-off is generally computed by means of formulas that need absorbance readings of negative (and sometimes positive) controls, which are included in the titer plates amongst the unknown samples. When no controls are available, other techniques should be employed such as change-point analysis. The method was applied to Bolivian dog sera processed by ELISA to diagnose T. cruzi infection. In each titer plate, the change-point analysis estimated a step point which correctly discriminated among known positive and known negative sera, unlike some of the six usual cut-off formulas tested. To analyse the ELISAs results, the change-point method was as good as the usual cut-off formula of the form “mean + 3 standard deviation of negative controls”. Change-point analysis is therefore an efficient alternative method to analyse ELISA absorbance values when no controls are available. <![CDATA[Human and canine visceral leishmaniasis in an emerging focus in Araçuaí, Minas Gerais: spatial distribution and socio-environmental factors]]> This study aimed to analyse the spatial distribution of human (2007-2013) and canine (2013) visceral leishmaniasis (VL) in the city of Araçuaí, Minas Gerais, Brazil, and identify the socio-environmental factors related to their occurrence. The spatial distribution of human and canine cases was analysed by kernel density estimation (KDE) and the K function. The KDE values were analysed for correlation between human and canine LV and for normalised difference vegetation index (NDVI). Socio-environmental aspects of household structures and surroundings were evaluated. The spatial distribution of human and canine VL cases exhibited a significant aggregated pattern in distances greater than 350 and 75 m, respectively. The higher occurrence of human and canine infection occurred in the central area of the city. A positive correlation between the densities of human and canine cases was observed, as well as a negative correlation between NDVI and densities of human and canine cases. Socio-environmental analysis revealed that the large amount of animals, organic material from trees and deficiencies in environmental sanitation are possibly contributing to the continuation of the transmission cycle of Leishmania infantum in Araçuaí. These results can contribute to the planning by competent agencies to reduce the incidence of infection in the city. <![CDATA[Intralesional meglumine antimoniate for the treatment of localised cutaneous leishmaniasis: a retrospective review of a Brazilian referral centre]]> Although intralesional meglumine antimoniate (MA) infiltration is considered an option for cutaneous leishmaniasis (CL) therapy and is widely used in the Old World, there have been few studies supporting this therapeutic approach in the Americas. This study aims to describe outcomes and adverse events associated with intralesional therapy for CL. This retrospective study reviewed the experience of a Brazilian leishmaniasis reference centre using intralesional MA to treat 31 patients over five years (2008 and 2013). The median age was 63 years (22-86) and the median duration time of the lesions up to treatment was 16 weeks. In 22 patients (71%), intralesional therapy was indicated due to the presence of contraindications or previous serious adverse events with systemic MA. Other indications were failure of systemic therapy or ease of administration. Intralesional treatment consisted of one-six infiltrations (median three) for a period of up to 12 weeks. The initial (three months) and definitive (six months) cure rates were 70.9% and 67.7%, respectively. Most patients reported mild discomfort during infiltration and no serious adverse events were observed. In conclusion, these results show that the intralesional MA efficacy rate was very similar to that of systemic MA treatment, and reinforce the need for further studies with adequate design to establish the efficacy and safety of this therapeutic approach. <![CDATA[Comparison of optical microscopy and quantitative polymerase chain reaction for estimating parasitaemia in patients with kala-azar and modelling infectiousness to the vector <em>Lutzomyia longipalpis</em>]]> Currently, the only method for identifying infective hosts with Leishmania infantum to the vector Lutzomyia longipalpis is xenodiagnosis. More recently, quantitative polymerase chain reaction (qPCR) has been used to model human reservoir competence by assuming that detection of parasite DNA indicates the presence of viable parasites for infecting vectors. Since this assumption has not been proven, this study aimed to verify this hypothesis. The concentration of amastigotes in the peripheral blood of 30 patients with kala-azar was microscopically verified by leukoconcentration and was compared to qPCR estimates. Parasites were identified in 4.8 mL of peripheral blood from 67% of the patients, at a very low concentration (average 0.3 parasites/mL). However, qPCR showed 93% sensitivity and the estimated parasitaemia was over a thousand times greater, both in blood and plasma, with higher levels in plasma than in blood. Furthermore, the microscopic count of circulating parasites and the qPCR parasitaemia estimates were not mathematically compatible with the published proportions of infected sandflies in xenodiagnostic studies. These findings suggest that qPCR does not measure the concentration of circulating parasites, but rather measures DNA from other sites, and that blood might not be the main source of infection for vectors. <![CDATA[Adamantylidene-substituted alkylphosphocholine TCAN26 is more active against <em>Sporothrix schenckii</em> than miltefosine]]> Sporotrichosis is the most frequent subcutaneous mycosis in the world and its increasing incidence has led to the search for new therapeutic options for its treatment. In this study, we demonstrated that three structural analogues of miltefosine (TCAN26, TC19, and TC70) showed inhibitory activity against Sporothrix schenckii sensu stricto and that TCAN26 was more active in vitro than miltefosine against several isolates. Scanning electron microscopy showed that S. schenckii exposure to TCAN26 resulted in cells that were slightly more elongated than untreated cells. Transmission electron microscopy showed that TCAN26 treatment induced loss of the regular cytoplasmic electron-density and altered the cell envelope (disruption of the cell membrane and cell wall, and increased cell wall thickness). Additionally, TCAN26 concentrations required to kill S. schenckii cells were lower than concentrations that were cytotoxic in mammalian cells, and TCAN26 was more selective than miltefosine. Thus, the adamantylidene-substituted alkylphosphocholine TCAN26 is a promising molecule for the development of novel antifungal compounds, although further investigations are required to elucidate the mode of action of TCAN26 in S. schenckii cells. <![CDATA[Comparative growth of spotted fever group <em>Rickettsia</em> spp. strains in Vero cells]]> In Brazil, the spotted fever group (SFG) Rickettsia rickettsii and Rickettsia parkeri related species are the etiological agents of spotted fever rickettsiosis. However, the SFG, Rickettsia rhipicephali, that infects humans, has never been reported. The study of growth dynamics can be useful for understanding the infective and invasive capacity of these pathogens. Here, the growth rates of the Brazilian isolates R. rickettsii str. Taiaçu, R. parkeri str. At#24, and R. rhipicephali HJ#5, were evaluated in Vero cells by quantitative polymerase chain reaction. R. rhipicephali showed different kinetic growth compared to R. rickettsii and R. parkeri. <![CDATA[Ultrastructure of Zika virus particles in cell cultures]]> Zika virus (ZIKV) has infected thousands of Brazilian people and spread to other American countries since 2015. The introduction of ZIKV brought a strong impact to public health in Brazil. It is of utmost importance to identify a susceptible cell line that will enable the isolation and identification of the virus from patient samples, viral mass production, and testing of drug and vaccine candidates. Besides real-time reverse transcriptase polymerase chain reaction diagnosis for detecting the viral genome, virus isolation in cell lines was useful in order to study the structure of the viral particle and its behaviour inside cells. Analysis of ZIKV infected cell lines was achieved using transmission electron microscopy (TEM). Blood was obtained from a Brazilian patient during the first days after presenting with signs of the disease, and ZIKV from the patient’s blood was isolated in the C6/36 mosquito cell line. Afterwards, Vero cells were inoculated with the viral suspension, fixed six days after inoculation, embedded in polymers, and ultra-thin cut. Like dengue viruses, this flavivirus showed numerous virus particles present inside cellular vesicles thereby confirming the susceptibility of the Vero cell line to ZIKV replication. TEM is a unique technique available to make the virus visible. <![CDATA[Solubility as a limiting factor for expression of hepatitis A virus proteins in insect cell-baculovirus system]]> The use of recombinant proteins may represent an alternative model to inactivated vaccines against hepatitis A virus (HAV). The present study aimed to express the VP1 protein of HAV in baculovirus expression vector system (BEVS). The VP1 was expressed intracellularly with molecular mass of 35 kDa. The VP1 was detected both in the soluble fraction and in the insoluble fraction of the lysate. The extracellular expression of VP1 was also attempted, but the protein remained inside the cell. To verify if hydrophobic characteristics would also be present in the HAV structural polyprotein, the expression of P1-2A protein was evaluated. The P1-2A polyprotein remained insoluble in the cellular extract, even in the early infection stages. These results suggest that HAV structural proteins are prone to form insoluble aggregates. The low solubility represents a drawback for production of large amounts of HAV proteins in BEVS. <![CDATA[Comparative genomic analysis of Brazilian <em>Leptospira kirschneri</em> serogroup Pomona serovar Mozdok]]> Leptospira kirschneri is one of the pathogenic species of the Leptospira genus. Human and animal infection from L. kirschneri gained further attention over the last few decades. Here we present the isolation and characterisation of Brazilian L. kirschneri serogroup Pomona serovar Mozdok strain M36/05 and the comparative genomic analysis with Brazilian human strain 61H. The M36/05 strain caused pulmonary hemorrhagic lesions in the hamster model, showing high virulence. The studied genomes presented high symmetrical identity and the in silico multilocus sequence typing analysis resulted in a new allelic profile (ST101) that so far has only been associated with the Brazilian L. kirschneri serogroup Pomona serovar Mozdok strains. Considering the environmental conditions and high genomic similarity observed between strains, we suggest the existence of a Brazilian L. kirschneri serogroup Pomona serovar Mozdok lineage that could represent a high public health risk; further studies are necessary to confirm the lineage significance and distribution.