Cellular and humoral immune responses against the Plasmodium vivax MSP-119 malaria vaccine candidate in individuals living in an endemic area in north-eastern Amazon region of Brazil

Abstract Background Plasmodium vivax merozoite surface protein-1 (MSP-1) is an antigen considered to be one of the leading malaria vaccine candidates. PvMSP-1 is highly immunogenic and evidences suggest that it is target for protective immunity against asexual blood stages of malaria parasites. Thus, this study aims to evaluate the acquired cellular and antibody immune responses against PvMSP-1 in individuals naturally exposed to malaria infections in a malaria-endemic area in the north-eastern Amazon region of Brazil. Methods The study was carried out in Paragominas, Pará State, in the Brazilian Amazon. Blood samples were collected from 35 individuals with uncomplicated malaria. Peripheral blood mononuclear cells were isolated and the cellular proliferation and activation was analysed in presence of 19 kDa fragment of MSP-1 (PvMSP-119) and Plasmodium falciparum PSS1 crude antigen. Antibodies IgE, IgM, IgG and IgG subclass and the levels of TNF, IFN-γ and IL-10 were measured by enzyme-linked immunosorbent assay. Results The prevalence of activated CD4+ was greater than CD8+ T cells, in both ex-vivo and in 96 h culture in presence of PvMSP-119 and PSS1 antigen. A low proliferative response against PvMSP-119 and PSS1 crude antigen after 96 h culture was observed. High plasmatic levels of IFN-γ and IL-10 as well as lower TNF levels were also detected in malaria patients. However, in the 96 h supernatant culture, the dynamics of cytokine responses differed from those depicted on plasma assays; in presence of PvMSP-119 stimulus, higher levels of TNF were noted in supernatant 96 h culture of malaria patient’s cells while low levels of IFN-γ and IL-10 were verified. High frequency of malaria patients presenting antibodies against PvMSP-119 was evidenced, regardless class or IgG subclass.PvMSP-119-induced antibodies were predominantly on non-cytophilic subclasses. Conclusions The results presented here shows that PvMSP-119 was able to induce a high cellular activation, leading to production of TNF and emphasizes the high immunogenicity of PvMSP-119 in naturally exposed individuals and, therefore, its potential as a malaria vaccine candidate.

Space-time cluster analysis of American visceral leishmaniasis in Bauru, São Paulo State, Brazil

A space-time analysis of American visceral leishmaniasis (AVL) in humans in the city of Bauru, São Paulo State, Brazil was carried out based on 239 cases diagnosed between June 2003 and October 2008. Spatial analysis of the disease showed that cases occurred especially in the city's urban areas. AVL annual incidence rates were calculated, demonstrating that the highest rate occurred in 2006 (19.55/100,000 inhabitants). This finding was confirmed by the time series analysis, which also showed a positive tendency over the period analyzed. The present study allows us to conclude that the disease was clustered in the Southwest side of the city in 2006, suggesting that this area may require special attention with regard to control and prevention measures.

Anti-Leishmania infantum IgG Antibody Avidity in Visceral Leishmaniasis

IgG avidity tests are used to discriminate acute from chronic infections. There are few reports on the IgG avidity profile of patients with visceral leishmaniasis (VL). This study investigated the anti-Leishmania IgG avidity in patients with classic VL (n = 10), patients showing clinical cure after treatment (n = 18), and asymptomatic subjects with at least one positive Leishmania test (n = 20). All subjects were from areas in Brazil where VL is endemic. Serum samples were collected from each subject on two different occasions. IgG avidity was evaluated by Western blotting. The proportion of high-avidity antibodies was higher in all samples from patients with classic VL. In contrast, low-avidity antibodies predominated in subjects with a history of VL, including 13 cases (72.2%) in the first assessment and 14 (77.8%) in the second. Fifteen (75%) of the asymptomatic subjects presented a predominance of low-avidity antibodies in the first assessment, and the frequency of high-avidity antibodies increased over time in seven subjects (35%) of this group. Antibodies against the 14- and/or 16-kDa antigen fraction were detected in the first assessment in all patients with classic VL, in 10 (55.5%) treated patients, and in 10 (50%) asymptomatic subjects. These were high-avidity antibodies in most cases. In the asymptomatic group, an increase in IgG avidity against the 14- and/or 16-kDa antigen fraction was observed in three cases (15%). The results indicate distinct responses in infected and asymptomatic subjects, probably associated with the length of time after infection. In this respect, IgG avidity tests represent a new approach to better characterize asymptomatic VL.

Resposta imune à saliva de flebotomíneos (diptera: psychodidae) em indivíduos residentes em áreas urbana e periurbana no nordeste do brasil

Visceral leishmaniasis (VL) has a wide geographical distribution in tropical and subtropical areas of the planet, which is a protozoan parasite of the genus Leishmania. This pathogen is transmitted to the host through the sandflies bite, with its saliva, the immune response that leads to both. In the state of Rio Grande do Norte, 85% of the sand flies captured is Lutzomyia longipalpis, but the second most abundant, Lutzomyia evandroi, it deserves emphasis because its wide distribution and eclectic behavior. The exposure of people living in endemic areas for the insect vector VL greatly increases the chances of infection. This study aimed to evaluate aspects of the epidemiological profile of VL in endemic areas of human and nonendemic in the metropolitan area of Natal, as well as verify the abundance and seasonal fluctuations of sandflies species in two counties endemic for VL. Were collected in the municipalities of Nísia Floresta, Parnamirim, São Gonçalo do Amarante and Macaíba, of which groups of females were separated for further dissection of the salivary glands and identification of species. The blood samples used were from individuals of two Natal s districts where it has never been reported cases of VL and neighborhoods of Parnamirim applicants who present cases of VL. In the municipality of Nísia Floresta, the most abundant species was L. evandroi with 38.39%, followed by L. longipalpis with 36.22%, L. walkeri 19.67% L. lenti 3.81%, L. wellcomei 1.39% and L. whitmani 0.52%. Already in Parnamirim the proportions were L. walkeri with 73.15%, L. evandroi with 10.55%, L. wellcomei 7.63%, L. longipalpis 6.37%, L. whitmani 1.46%, L. sordellii 0.52%, L. intermedia 0.21 and L. shanonni 0.1%. In both municipalities was observed higher abundance of species distributed in the initial months of the year, as February and March. The study showed that no difference in exposure to the vector of VL among individuals from endemic and non endemic area for this disease. But there are differences in exposure between individuals of L. longipalpis and L. evandroi, confirming the great powers of the first vector. It was also characterized as predominant phenotype in the population of endemic areas who had negative serologic responses to antigens of Leishmania and result in negative Montenegro skin test (DTH), indicating that much of the population hasn t been bitten by infected insects

The conundrum of human visceral leishmaniasis in Emilia-Romagna, Italy: are wild and peridomestic animals potential reservoirs?

Leishmaniasis is a complex parasitic disease caused by intracellular protozoans of the genus Leishmania mainly transmitted by the bite of sand flies. In Italy, leishmaniasis is caused by Leishmania infantum, responsible for the human visceral and canine leishmaniases (HVL and CanL, respectively). Within Emilia-Romagna region, Italy, recent molecular studies indicated that L. infantum strains circulating in dogs and humans are different. This suggests that an animal reservoir other than dog should be evaluated in the epidemiology of HVL in Emilia-Romagna. Therefore, the main aim of this PhD project was to investigate the role of wild and peridomestic mammals as potential animal reservoirs of L. infantum in the regional zones where HVL foci are still active, also evaluating the possible role of arthropod vectors other than phlebotomine sandflies as vectors of Leishmania spp. in the sylvatic cycle of the protozoa. Overall, 206 specimens of different animal species (roe deer, rats, mice, badgers, hares, polecats, foxes, beech martens, bank voles, hedgehogs, and shrews), collected in Emilia-Romagna were screened for Leishmania with a real-time PCR, revealing a prevalence of 33% for roe deer (first report in this species). Positivity was also found in brown rats (10.6%), black rats (13.1%), mice (10%), badgers (25%), hedgehogs (80%) and bank voles (11%). To distinguish the two strains of L. infantum circulating in Emilia-Romagna, a nested PCR protocol optimized for animal tissues was developed, demonstrating that over 90% of L. infantum infections in roe deer were due to the strain isolated from humans and suggesting their possible role as reservoirs in the study area. Furthermore, the presence of Leishmania kDNA was detected in unfed larvae, nymphs and males of questing Ixodes ricinus ticks collected in regional parks of Emilia-Romagna suggesting their possible role in the transmission of L. infantum in a sylvatic or rural cycle.

Phlebotomines (Diptera, Psychodidae) in the Speleological Province of the Ribeira Valley: 3. Serra district - area of hostels for tourists who visit the Parque Estadual do Alto Ribeira (PETAR), state of São Paulo, Brazil

Phlebotomines (Diptera, Psychodidae) in the Speleological Province of the Ribeira Valley: 3. Area of hostels for tourists who visit the Parque Estadual do Alto Ribeira (PETAR), state of São Paulo, Brazil. The study characterizes some ecological aspects of the phlebotomine fauna in an endemic area of cutaneous leishmaniasis (CL) situated in the Serra district, Iporanga municipality where the hostels for tourists visiting the PETAR are located. Captures were undertaken on a smallholding and a small farm situated near the hostels, monthly between January/2001 and December/2003 with automatic light traps (ALT) in pigsty, hen-house and veranda of a domicile at the two sites, and in peridomicile of the small farm also with black/white Shannon traps. With the ALT a total of 87,224 phlebotomines representing 19 species and also two hybrids of Nyssomyia intermedia (Lutz & Neiva) and Nyssomyia neivai (Pinto) and two anomalous specimens were captured. The standardized index species abundance was for Ny. intermedia = 1.0 and Ny. neivai = 0.935. The highest frequencies of the smallholding occurred in the pigsty, the Williams' mean/capture for Ny. intermedia being 63.7 specimens and for Ny. neivai 29.2, and on the small farm, in the hen-house, Ny. intermedia 402.6 and Ny. neivai 116.2. A total of 863 phlebotomines (Ny. intermedia: 75.4%; Ny. neivai: 24.3%) were captured with black/white Shannon traps; females of both species being predominant in the white trap. The high frequencies of Ny. intermedia and Ny. neivai, both implicated in CL transmission, indicate the areas presenting risk of the disease.

Occurrence of anti-Neospora caninum and anti-Toxoplasma gondii antibodies in dogs with visceral leishmaniasis

Uninfected dogs and those naturally infected with Leishmania chagasi exhibiting different clinical forms of disease were evaluated for the presence of anti-Neospora caninum and anti-Toxoplasma gondii antibodies. Blood samples were collected from 110 mongrel dogs. Sera were tested using the indirect fluorescent antibody test (IFAT), and the animals with visceral leishmaniasis (VL) (n=60) were classified clinically. Out of the 110 sera investigated, 5 (4.5%) were positive for N. caninum (IFAT≥50) and 36 (32.7%) for T. gondii (IFAT≥16). Anti-L. chagasi antibody titers in asymptomatic dogs (n=10) were found to be significantly lower (P<0.05) than those in oligosymptomatic ones (n=22), which were in turn significantly lower (P<0.05) than those in symptomatic ones (n=28). No association between Leishmania and N. caninum infections was observed. Among dogs infected with L. chagasi, a tendency (P=0.053) towards an association between the infection with T. gondii and the appearance of VL symptoms was observed, suggesting that the clinical manifestation of VL in dogs may enhance their susceptibility to T. gondii. The possible influence of the immunosuppressive status of canine leishmaniasis in the different clinical forms of the disease is discussed.

New Epidemiological Data on Brazilian Spotted Fever in an Endemic Area of the State of Sao Paulo, Brazil

The present work evaluated rickettsial infection in dogs and their ticks in an area endemic for Brazilian spotted fever (BSF) in the metropolitan area of Sao Paulo, Brazil, where the tick Amblyomma aureolatum was presumed to be the vector of the disease. Ticks were collected on dogs from 185 houses, encompassing single infestations by Rhipicephalus sanguineus, Amblyomma aureolatum, Amblyomma longirostre, or Amblyomma sp. in dogs from 60 (32.4%), 77 (41.6%), 2 (1.1%), and 25 (13.5%) houses, respectively; 19 (10.3%) houses had dogs with mixed infestations by R. sanguineus and A. aureolatum; 1 (0.5%) house had dogs with infestations by A. aureolatum and A. longirostre; and 1 (0.5%) house had dogs with infestations by R. sanguineus and Amblyomma sp. Overall, A. aureolatum was present in dogs from 97 (52.4%) houses, and R. sanguineus in dogs from 80 (43.2%) houses. A total of 287 ticks (130 A. aureolatum and 157 R. sanguineus) infesting dogs from 98 houses were selected for testing by polymerase chain reaction (PCR) targeting rickettsial genes. Overall, 3.1% of the A. aureolatum ticks were infected by Rickettsia bellii, and 1.3% of the R. sanguineus were infected by Ricketttsii rickettsii. For serology, we selected 23 dogs living in and in the vicinity of the house where the R. rickettsii-infected ticks were collected. The indirect fluorescent antibody (IFA) test detected antibodies reactive with R. rickettsii in sera from 16 (69.6%) dogs, with titers ranging from 256 to 32,768. It is established that Amblyomma aureolatum is a vector of R. rickettsii in the Sao Paulo metropolitan area, but our results highlight for the first time in Brazil, a possible role of R. sanguineus in the epidemiology of R. rickettsii, corroborating previous findings in Mexico and the United States, where R. sanguineus has been implicated in the transmission of R. rickettsii to humans.

Cost-effectiveness analysis of humanitarian relief interventions: visceral leishmaniasis treatment in the Sudan

Spending by aid agencies on emergencies has quadrupled over the last decade, to over US$ 6 billion. To date, cost-effectiveness has seldom been considered in the prioritization and evaluation of emergency interventions. The sheer volume of resources spent on humanitarian aid and the chronicity of many humanitarian interventions call for more attention to be paid to the issue of 'value for money'. In this paper we present data from a major humanitarian crisis, an epidemic of visceral leishmaniasis (VL) in war-torn Sudan. The special circumstances provided us, in retrospect, with unusually accurate data on excess mortality, costs of the intervention and its effects, thus allowing us to express cost-effectiveness as the cost per Disability Adjusted Life Year (DALY) averted. The cost-effectiveness ratio, of US$ 18.40 per DALY (uncertainty range between US$ 13.53 and US$ 27.63), places the treatment of VL in Sudan among health interventions considered 'very flood value for money' (interventions of less than US$ 25 per DALY). We discuss the usefulness of this analysis to the internal management of the VL programme, the procurement of funds for the programme, and more generally, to priority setting in humanitarian relief interventions. We feel that in evaluations of emergency interventions attempts could be made more often to perform cost-effectiveness analyses, including the use of DALYs, provided that the outcomes of these analyses are seen in the broad context of the emergency situation and its consequences on the affected population. This paper provides a first contribution to what is hoped to become an international database of cost-effectiveness studies of health outcome such as the DALY.

Climate and acute/subacute paracoccidioidomycosis in a hyper-endemic area in Brazil

Methods Stepwise regression of annual data was applied to model incidence, calculated based on 91 cases, from lagged variables: antecedent precipitation, air temperature, soil water storage, absolute and relative air humidity, and Southern Oscillation Index (SOI). Results Multiple regression analyses resulted in a model, which explains 49% of the incidence variance, taking into account the absolute air humidity in the year of exposure, soil water storage and SOI of the previous 2 years. Conclusions The correlations may reflect enhanced fungal growth after increase in soil water storage in the longer term and greater spore release with increase in absolute air humidity in the short term.

In situ immune responses to interstitial pneumonitis in human visceral leishmaniasis

Lung disease during active human visceral leishmaniasis is frequently reported. As such, studies have associated pulmonary symptoms to interstitial pneumonitis with a mononuclear infiltrate. However, the immune response in this condition has never been described before. The aim of this study was to determine the immunophenotypic pattern and cytokine profile of lung involvement (IPL) in human visceral leishmaniasis. Quantitative methods of analysis were performed using immunohistochemistry, and were compared with a control group of normal lung. Interstitial macrophages and cd8 cells were increased in IPL, and IL-4 as well as TNF-alpha displayed increased expression when compared to the control group. This inflammatory process with a Th2 pattern, as suggested by increased IL-4 and low IFN-gamma expression, is consistent with the immune response in other organs of visceral leishmaniasis. The microenvironment of the immune response in this condition is associated with lung disease in patients with interstitial pneumonitis related to visceral leishmaniasis, increasing the chance of bacterial infection.

Decrease in Mycobacterium tuberculosis specific immune responses in patients with untreated psoriasis living in a tuberculosis endemic area

Tuberculosis has emerged as a major concern in patients with immuno-mediated diseases, including psoriasis, undergoing treatment with biologicals. However, it is not known whether the chronically activated immune system of psoriasis patients interferes with their Mycobacterium tuberculosis (Mtb)-specific immunity, especially in tuberculosis-endemic areas like Brazil. We evaluated T-cell responses to a Mtb lysate and to the recombinant Mtb proteins ESAT-6 and Ag85B of tuberculin skin test (TST) positive and TST negative patients with severe or mild/moderate, untreated psoriasis in three different assays: lymphocyte proliferation, enzyme immunoassay for interferon (IFN)-gamma and interleukin (IL)-10 production by peripheral blood mononuclear cells and overnight enzyme immunospot (ELISpot) for enumerating IFN-gamma-secreting cells. In our cohort, a low proportion (29%) of the severe psoriasis patients tested were TST-positive. IFN-gamma and IL-10 secretion and T-cell proliferation to Mtb antigens were reduced in TST-negative but not in TST-positive patients with severe psoriasis when compared to healthy controls with the same TST status. Similarly, severe psoriasis patients had decreased cytokine secretion and proliferative response to phytohemagglutinin. However, most psoriasis patients and healthy controls showed detectable numbers of IFN-gamma-secreting effector-memory T-cells in response to Mtb antigens by ELISpot. TST-negative, mild/moderate psoriasis patients had responses that were mostly intermediary between TST-negative controls and severe psoriasis patients. Thus, patients with severe psoriasis possess decreased anti-Mtb central memory T-cell responses, which may lead to false-negative results in the diagnosis of TB infection, but retain T-cell memory-effector activity against Mtb antigens. We hypothesize that the latter may confer some protection against tuberculosis reactivation.

TGF-beta and mesenchymal hepatic involvement after visceral leishmaniasis

The liver involvement in the human visceral leishmaniasis (VL) has been related to parasitism and activated Kupffer cells with further occasional fibrotic alterations, especially after long-term disease without treatment. However, fibrotic alterations have been reported after therapy, whose clinical finding is the persistence of hepatomegaly. Fibrotic involvement of the liver after therapy was never well understood, and the aim of this study was to evaluate this finding through ultrastructural and morphometric analysis. A case-control study was performed with 20 patients (15 cases and five controls). Cases included patients with persistent hepatomegaly (residual) after treatment of VL submitted to liver biopsy to exclude other causes of liver enlargement, including serum tests of viral hepatitis. The material was evaluated by electron microcopy allowing ultrastructural with morphometric analysis of medium portion of hepatic lobule. Narrow sinusoidal lumen and prominent Kupffer cells were found with insignificant alterations of hepatocytes, pit, and endothelial cells. On ultrastructural analysis, the enlargement of the space of Disse was due to fibrous collagen, increase of number of Ito cells, and nonfibrous extracellular matrix that were associated with Kupffer cells enlargement. Immunohistochemistry showed an intense expression of TGF-beta in patients with VL. These findings suggest a production of TGF-beta by Kupffer cells that resulted in the characteristic fibrotic involvement of the liver. Residual hepatomegaly in visceral leishmaniasis could result from sustained Kupffer cell activation with perihepatocytic fibrosis.

The risk of chikungunya fever in a dengue-endemic area

Background. Chikungunya, an alphavirus of the Togaviridae family, causes a febrile disease transmitted to humans by the bite of infected Aedes mosquitoes. This infection is reaching endemic levels in many Southeast Asian countries. Symptoms include sudden onset of fever, chills, headache, nausea, vomiting, joint pain with or without swelling, low back pain, and rash. According to the World Health Organization, there are 2 billion people living in Aedes-infested areas. In addition, traveling to these areas is popular, making the potential risk of infections transmitted by the bite of infected Aedes mosquitoes very high. Methods. We proposed a mathematical model to estimate the risk of acquiring chikungunya fever in an Aedes-infested area by taking the prevalence of dengue fever into account. The basic reproduction number for chikungunya fever R-0chik can be written as a function of the basic reproduction number of dengue R-0dengue by calculating the ratio R-0chik/R-0dengue. From R-0chik, we estimated the force of infection and the risk of acquiring the disease both for local residents of a dengue-endemic area and for travelers to this area. Results. We calculated that R-0chik is 64.4% that of R-0dengue. The model was applied to a hypothetical situation, namely, estimating the individual risk of acquiring chikungunya fever in a dengue-endemic area, both for local inhabitants (22% in steady state) and for visiting travelers (from 0.31% to 1.23% depending on the time spent in the area). Conclusions. The method proposed based on the output of a dynamical model is innovative and provided an estimation of the risk of infection, both for local inhabitants and for visiting travelers.

Therapeutic evaluation of free and liposome-loaded furazolidone in experimental visceral leishmaniasis

Drug delivery systems are promising pharmaceutical formulations used to improve the therapeutic index of drugs. In this study, we developed a liposomal formulation of furazolidone that targets Leishmania (Leishmania) chagasi amastigotes in a hamster model. Using laser scanning confocal microscopy, it was demonstrated that the liposomal drug co-localised with L. (L.) chagasi amastigotes within macrophages. Liposomal furazolidone administered intraperitoneally at 0.5 mg/kg for 12 consecutive days reduced spleen (74%) and liver (32%) parasite burden at a 100-fold lower dose than the free drug. Free furazolidone (50 mg/kg) also effectively reduced spleen (82.5%) and liver (85%) parasites; its in vitro activity against promastigotes and intracellular amastigotes demonstrated a high degree of parasite selectivity. Thus, furazolidone, both in the free and liposome-loaded formulation, is an effective inhibitor of L. (L.) chagasi, representing a possible cost-effective drug candidate for the treatment of visceral leishmaniasis. (C) 2010 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.