The influence of intestinal parasites on Plasmodium vivax-specific antibody responses to MSP-119 and AMA-1 in rural populations of the Brazilian Amazon.

BACKGROUND: Polyparasitism is a common condition in humans but its impact on the host immune system and clinical diseases is still poorly understood. There are few studies of the prevalence and the effect of malaria-intestinal parasite co-infections in the immune response to malaria vaccine candidates. The present study determines whether the presence of malaria and intestinal parasites co-infection is associated with impaired IgG responses to Plasmodium vivax AMA-1 and MSP-119 in a rural population of the Brazilian Amazon. METHODS: A cross-sectional survey was performed in a rural area of Rondonia State and 279 individuals were included in the present study. At recruitment, whole blood was collected and Plasmodium and intestinal parasites were detected by microscopy and molecular tests. Blood cell count and haemoglobin were also tested and antibody response specific to P. vivax AMA-1 and MSP-119 was measured in plasma by ELISA. The participants were grouped according to their infection status: singly infected with Plasmodium (M); co-infected with Plasmodium and intestinal parasites (CI); singly infected with intestinal parasites (IP) and negative (N) for both malaria and intestinal parasites. RESULTS: The prevalence of intestinal parasites was significantly higher in individuals with malaria and protozoan infections were more prevalent. IgG antibodies to PvAMA-1 and/or PvMSP-119 were detected in 74 % of the population. The prevalence of specific IgG was similar for both proteins in all four groups and among the groups the lowest prevalence was in IP group. The cytophilic sub-classes IgG1 and IgG3 were predominant in all groups for PvAMA-1 and IgG1, IgG3 and IgG4 for PvMSP-119. In the case of non-cytophilic antibodies to PvAMA-1, IgG2 was significantly higher in IP and N group when compared to M and CI while IgG4 was higher in IP group. CONCLUSIONS: The presence of intestinal parasites, mainly protozoans, in malaria co-infected individuals does not seem to alter the antibody immune responses to P. vivax AMA-1 and MSP-119. However, IgG response to both AMA1 and MSP1 were lower in individuals with intestinal parasites.

IL10A genotypic association with decreased IL-10 circulating levels in malaria infected individuals from endemic area of the Brazilian Amazon.

BACKGROUND: Cytokines play an important role in human immune responses to malaria and variation in their production may influence the course of infection and determine the outcome of the disease. The differential production of cytokines has been linked to single nucleotide polymorphisms in gene promoter regions, signal sequences, and gene introns. Although some polymorphisms play significant roles in susceptibility to malaria, gene polymorphism studies in Brazil are scarce. METHODS: A population of 267 individuals from Brazilian Amazon exposed to malaria was genotyped for five single nucleotide polymorphisms (SNPs), IFNG + 874 T/A, IL10A-1082G/A, IL10A-592A/C, IL10A-819 T/C and NOS2A-954G/C. Specific DNA fragments were amplified by polymerase chain reaction, allowing the detection of the polymorphism genotypes. The polymorphisms IL10A-592A/C and IL10A-819 T/C were estimated by a single analysis due to the complete linkage disequilibrium between the two SNPs with D' = 0.99. Plasma was used to measure the levels of IFN-γ and IL-10 cytokines by Luminex and nitrogen radicals by Griess reaction. RESULTS: No differences were observed in genotype and allelic frequency of IFNG + 874 T/A and NOS2A-954G/C between positive and negative subjects for malaria infection. Interesting, the genotype NOS2A-954C/C was not identified in the study population. Significant differences were found in IL10A-592A/C and IL10A-819 T/C genotypes distribution, carriers of IL10A -592A/-819 T alleles (genotypes AA/TT + AC/TC) were more frequent among subjects with malaria than in negative subjects that presented a higher frequency of the variant C allele (p < 0.0001). The presence of the allele C was associated with low producer of IL-10 and low parasitaemia. In addition, the GTA haplotypes formed from combinations of investigated polymorphisms in IL10A were significantly associated with malaria (+) and the CCA haplotype with malaria (-) groups. The IL10A-1082G/A polymorphism showed high frequency of heterozygous AG genotype in the population, but it was not possible to infer any association of the polymorphism because their distribution was not in Hardy Weinberg equilibrium. CONCLUSION: This study shows that the IL10A-592A/C and IL10A-819 T/C polymorphisms were associated with malaria and decreased IL-10 levels and low parasite density suggesting that this polymorphism influence IL-10 levels and may influence in the susceptibility to clinical malaria.

Evaluating seroprevalence to circumsporozoite protein to estimate exposure to three species of Plasmodium in the Brazilian Amazon.

BACKGROUND: Brazil has seen a great decline in malaria and the country is moving towards elimination. However, for eventual elimination, the control program needs efficient tools in order to monitor malaria exposure and transmission. In this study, we aimed to evaluate whether seroprevalence to the circumsporozoite protein (CSP) is a good tool for monitoring the exposure to and/or evaluating the burden and distribution of Plasmodium species in the Brazilian Amazon. METHODS: Cross-sectional surveys were conducted in a rural area of Porto Velho, Rondônia state. Parasite infection was detected by microscopy and polymerase chain reaction. Antibodies to the sporozoite CSP repeats of Plasmodium vivax, P. falciparum, and P. malariae (PvCS, PfCS, and PmCS) were detected using the enzyme-linked immunosorbent assay technique. Human leukocyte antigen (HLA)-DRB1 and DQB1 genes were typed using Luminex® xMAP® technology. RESULTS: The prevalence of immunoglobulin G against P. vivax CSP peptide (62%) was higher than P. falciparum (49%) and P. malariae (46%) CSP peptide. Most of the studied individuals had antibodies to at least one of the three peptides (72%), 34% had antibodies to all three peptides and 28% were non-responders. Although the majority of the population was not infected at the time of the survey, 74.3% of parasite-negative individuals had antibodies to at least one of the CSPs. Importantly, among individuals carrying the haplotypes DRB1*04~DQB1*03, there was a significantly higher frequency of PfCS responders, and DRB1*16~DQB1*03 haplotype for PvCS and PfCS responders. In contrast, HLA-DRB1*01 and HLA-DQB1*05 allelic groups were associated with a lack of antibodies to P. vivax and P. falciparum CSP repeats, and the haplotype DRB1*01~DQB1*05 was also associated with non-responders, including non-responders to P. malariae. CONCLUSIONS: Our results show that in low transmission settings, naturally acquired antibody responses against the CSP repeats of P. vivax, P. falciparum, and P. malariae in a single cross-sectional study may not represent a valuable marker for monitoring recent malaria exposure, especially in an area with a high prevalence of P. vivax. Furthermore, HLA class II molecules play an important role in antibody response and require further study with a larger sample size. It will be of interest to consider HLA analysis when using serosurveillance to monitor malaria exposure among genetically diverse populations.

Field trial of efficacy of the Leish-tec® vaccine against canine leishmaniasis caused by Leishmania infantum in an endemic area with high transmission rates.

BACKGROUND: Because domestic dogs are reservoir hosts for visceral leishmaniasis (VL) in Brazil, one of the approaches used to reduce human disease incidence is to cull infected dogs. However, the results of controlled intervention trials based on serological screening of dogs and killing of seropositive animals are equivocal. A prophylactic vaccine to protect dogs from being infectious to the sand fly vector could be an effective strategy to provide sustained control. Here, we investigated whether a currently licensed commercial subunit rA2 protein-saponin vaccine (Leish-tec®) had an additional effect to dog culling on reducing the canine infectious populations. METHODOLOGY/PRINCIPAL FINDINGS: This prospective study was conducted in an L. infantum highly endemic area of southeast Brazil. At the onset of the intervention, all of the eligible dogs received through subcutaneous route a three-dose vaccine course at 21-day intervals and a booster on month 12. For the purpose of comparison, newly recruited healthy dogs were included as the exposed control group. To ascertain vaccine-induced protection, dogs were screened on clinical and serological criteria every 6 months for a 2-year follow-up period. Antibody-based tests and histopathological examination of post-mortem tissue specimens from euthanized animals were used as a marker of infection. The standardized vaccine regime, apart from being safe, was immunogenic as immunized animals responded with a pronounced production of anti-A2-specific IgG antibodies. It should be noted the mean seroconversion time for infection obtained among immunized exposed dogs (~ 18 months), which was twice as high as that for unvaccinated ones (~ 9 months). After two transmission cycles completed, the cumulative incidence of infection did differ significantly (P = 0.016) between the vaccinated (27%) and unvaccinated (42%) dogs. However, the expected efficacy for the vaccine in inducing clinical protection was not evident since 43% of vaccine recipients developed disease over time. Our estimates also indicated that immunoprophylaxis by Leish-tec® vaccine in addition to dog culling might not have an impact on bringing down the incidence of canine infection with L. infantum in areas of high transmission rates. CONCLUSIONS/SIGNIFICANCE: Leish-tec® as a prophylactic vaccine showed promise but needs to be further optimized to be effective in dogs under field conditions, and thereby positively impacts human incidence.

Plasmodium vivax Cell-Traversal Protein for Ookinetes and Sporozoites: Naturally Acquired Humoral Immune Response and B-Cell Epitope Mapping in Brazilian Amazon Inhabitants.

The cell-traversal protein for ookinetes and sporozoites (CelTOS), a highly conserved antigen involved in sporozoite motility, plays an important role in the traversal of host cells during the preerythrocytic stage of Plasmodium species. Recently, it has been considered an alternative target when designing novel antimalarial vaccines against Plasmodium falciparum. However, the potential of Plasmodium vivax CelTOS as a vaccine target is yet to be explored. This study evaluated the naturally acquired immune response against a recombinant P. vivax CelTOS (PvCelTOS) (IgG and IgG subclass) in 528 individuals from Brazilian Amazon, as well as the screening of B-cell epitopes in silico and peptide assays to associate the breadth of antibody responses of those individuals with exposition and/or protection correlates. We show that PvCelTOS is naturally immunogenic in Amazon inhabitants with 94 individuals (17.8%) showing specific IgG antibodies against the recombinant protein. Among responders, the IgG reactivity indexes (RIs) presented a direct correlation with the number of previous malaria episodes (p = 0.003; r = 0.315) and inverse correlation with the time elapsed from the last malaria episode (p = 0.031; r = -0.258). Interestingly, high responders to PvCelTOS (RI > 2) presented higher number of previous malaria episodes, frequency of recent malaria episodes, and ratio of cytophilic/non-cytophilic antibodies than low responders (RI < 2) and non-responders (RI < 1). Moreover, a high prevalence of the cytophilic antibody IgG1 over all other IgG subclasses (p < 0.0001) was observed. B-cell epitope mapping revealed five immunogenic regions in PvCelTOS, but no associations between the specific IgG response to peptides and exposure/protection parameters were found. However, the epitope (PvCelTOSI136-E143) was validated as a main linear B-cell epitope, as 92% of IgG responders to PvCelTOS were also responders to this peptide sequence. This study describes for the first time the natural immunogenicity of PvCelTOS in Amazon individuals and identifies immunogenic regions in a full-length protein. The IgG magnitude was mainly composed of cytophilic antibodies (IgG1) and associated with recent malaria episodes. The data presented in this paper add further evidence to consider PvCelTOS as a vaccine candidate.

Seroprevalence of infection with Toxoplasma gondii in indigenous Brazilian populations.

The prevalence of Toxoplasma gondii in indigenous Brazilian tribes with different degrees of acculturation was studied in the Enawenê-Nawê, an isolated tribe, in the state of Mato Grosso, the Waiãpi, with intermittent non-Indian contacts, in the state of Amapa, and the Tiriyó, with constant non-Indian contacts, in the state of Para. An IgG-enzyme-linked immunosorbent assay (IgG-ELISA) or an IgG/IgM-indirect immunofluorescence antibody (IFA) assay were performed for the detection of antibodies to T. gondii in 2000-2001. Both assays showed that the Tiriyó had the lowest crude seroprevalence (55.6%), the Enawené-Nawé the highest crude seroprevalence (80.4%), and the Waiãpi an intermediate crude seroprevalence (59.6%). The age-adjusted prevalence (95% confidence intervals) values for the Tiriyó, Enawenê-Nawê, and Waiãpi were 57.3% (53.4, 61.1%), 78.8% (72.2, 85.7%), and 57.7% (52.5, 62.9%), respectively. Contact with non-Indians probably did not influence the prevalence of the infection. However, differential contact with soil-harboring oocysts from wild felines may be responsible for the various seroprevalences in the different tribes.

Human retroviruses (HIV and HTLV) in Brazilian Indians: seroepidemiological study and molecular epidemiology of HTLV type 2 isolates.

To investigate serological, epidemiological, and molecular aspects of HTLV-1, HTLV-2, and HIV-1 infections in Amerindian populations in Brazil, we tested 683 and 321 sera from Tiriyo and Waiampi Indians, respectively. Both HIV-1 and HTLV-2 infections were detected at low prevalence among the Tiriyos whereas only HTLV-1 was present among the Waiampis, also at low prevalence. Analysis of the nucleotide sequence of the 631 bp of the env gene obtained from the three HTLV-2 isolates detected among the Tiriyos demonstrated by restriction fragment length polymorphism that these viruses belong to subtype IIa. Phylogenetic analysis of this same fragment showed that these sequences cluster closer to HTLV-2 isolates from intravenous drug users living in urban areas of southern Brazil than to the same gene sequence studied in another Brazilian tribe, the Kayapos. Our results confirm the distribution of Brazilian HTLV-2 sequences in a unique cluster I and cluster IIa and suggest that there is a considerable degree of diversity within this cluster. We also report for the first time HIV-1 infection among Brazilian Amerindians.

Cross-immunity experiments between different species or strains of Leishmania in rhesus macaques (Macaca mulatta).

This study evaluates cross-immunity in rhesus monkeys (Macaca mulatta) previously infected with one species of Leishmania and have had self-cured disease or were cured by antimony-based therapy upon development of full-blown disease. We found that a self-healing cutaneous leishmaniasis (CL) following experimental infection with Leishmania (Leishmania) major induces significant protection for L. (L.) amazonensis and L. (Viannia) guyanensis, and was dependent on time of re-challenge by L (L.) amazonensis after animals had recovered from primary lesions, but lacked protection against L. (V.) braziliensis. In contrast, monkeys that recovered from L. (V.) braziliensis CL or L. (L.) chagasi visceral leishmaniasis following chemotherapeutic intervention were protected by challenge with L. (V.) braziliensis and L (L.) amazonensis. These findings indicate the relative variability in protection after self-cure or drug-cured experimental leishmaniasis to challenge by heterologous leishmanial parasites. Further studying the immune response may provide information regarding relevant factors influencing cross-protective immunity.

[Serological survey of Toxoplasma gondii infection in isolated Amerindians, Mato Grosso]. / Inquérito sorológico para a infecção por Toxoplasma gondii em ameríndios isolados, Mato Grosso.

This study determined the occurrence of the antibodies anti-Toxoplasma gondii among the Enawenê-Nawê, an indigenous population of Mato Grosso. These inhabit a vast wild area, with rare contacts with non-Indians. They do not keep domestic animals, including cats. Their diet is based on insects, cassava, corn, honey and mushrooms, they do not consume meat, except fish. Based on the above, serologic tests ELISA-IgG and indirect fluorescent antibody test for IgG/IgM were performed. From 148 samples, 80.4% positive for IgG by ELISA or indirect fluorescent antibody test. No IgM reagent cases were detected. In that group the seropositivity rates increased significantly with age from 50% to 95%. Having analyzed their customs and habits, together with the high seropositivity found, it is suggested that the presence of wild felines in the vicinity of the village and areas where water collects could play an important role as an infection source, contaminating soil and consequently insects and mushrooms consumed by the Indians.

Clinical and parasitological protection in a Leishmania infantum-macaque model vaccinated with adenovirus and the recombinant A2 antigen.

BACKGROUND: Visceral leishmaniasis (VL) is a severe vector-born disease of humans and dogs caused by Leishmania donovani complex parasites. Approximately 0.2 to 0.4 million new human VL cases occur annually worldwide. In the new world, these alarming numbers are primarily due to the impracticality of current control methods based on vector reduction and dog euthanasia. Thus, a prophylactic vaccine appears to be essential for VL control. The current efforts to develop an efficacious vaccine include the use of animal models that are as close to human VL. We have previously reported a L. infantum-macaque infection model that is reliable to determine which vaccine candidates are most worthy for further development. Among the few amastigote antigens tested so far, one of specific interest is the recombinant A2 (rA2) protein that protects against experimental L. infantum infections in mice and dogs. METHODOLOGY/PRINCIPAL FINDINGS: Primates were vaccinated using three rA2-based prime-boost immunization regimes: three doses of rA2 plus recombinant human interleukin-12 (rhIL-12) adsorbed in alum (rA2/rhIL-12/alum); two doses of non-replicative adenovirus recombinant vector encoding A2 (Ad5-A2) followed by two boosts with rA2/rhIL-12/alum (Ad5-A2+rA2/rhIL12/alum); and plasmid DNA encoding A2 gene (DNA-A2) boosted with two doses of Ad5-A2 (DNA-A2+Ad5-A2). Primates received a subsequent infectious challenge with L. infantum. Vaccines, apart from being safe, were immunogenic as animals responded with increased pre-challenge production of anti-A2-specific IgG antibodies, though with some variability in the response, depending on the vaccine formulation/protocol. The relative parasite load in the liver was significantly lower in immunized macaques as compared to controls. Protection correlated with hepatic granuloma resolution, and reduction of clinical symptoms, particularly when primates were vaccinated with the Ad5-A2+rA2/rhIL12/alum protocol. CONCLUSIONS/SIGNIFICANCE: The remarkable clinical protection induced by A2 in an animal model that is evolutionary close to humans qualifies this antigen as a suitable vaccine candidate against human VL.

The effect of removing potentially infectious dogs on the numbers of canine Leishmania infantum infections in an endemic area with high transmission rates.

To assess the effect of the rapid removal of potentially infectious dogs on the prevalence and incidence of canine infections, a prospective study was undertaken in an area endemic for Leishmania infantum. We used serological testing based on the rapid DPP rK28 fusion protein chromatographic immunoassay for this dog screening-and-culling intervention trial. The outcome was evaluated by measuring seropositivity and sero-conversion/-reversion rates for canine infection. Our estimates indicated that concomitant detection and elimination of seropositive dogs with active disease may affect the numbers of canine infections and disease burden temporarily, although it is insufficient as a measure to interrupt the zoonotic L. infantum transmission. However, most of the asymptomatic, seropositive dogs continuously exhibit low levels of antibodies and/or reverted, remaining seronegative thereafter. In the process of waiting for an effective vaccine, one option for canine reservoir control may be to identify these possibly genetically resistant animals and promote their expansion in the population.

Evaluation of a novel chromatographic immunoassay based on Dual-Path Platform technology (DPP® CVL rapid test) for the serodiagnosis of canine visceral leishmaniasis.

Canine visceral leishmaniasis (CVL) is the major source of human visceral leishmaniasis (VL) and is transmitted from dogs to sand flies to humans. To control the spread of this disease, early and accurate detection of infected dogs is critical but challenging. Here we demonstrate the potential of the Dual-Path Platform (DPP(®)) CVL rapid test for detecting K26/K39-reactive antibodies in sera from clinically symptomatic (n=60) and asymptomatic (n=60) Leishmania infantum-infected dogs. For the specificity evaluation, assays were performed using known negative diagnostic serum samples (n=59) and cross-reaction control sera (n=11) from animals born in a VL-free area of Brazil. The diagnostic kit displayed high specificity (96%) but low sensitivity (47%) in identifying parasite-positive dogs without signs of CVL. However, the test sensitivity was significantly higher (98%) in diseased cases, indicating that this convenient test may be useful to identify the most infectious dogs. Efforts should be pursued to obtain a more sensitive DPP-multiplexed test parameter (i.e. based on simultaneous yet separate antibody detection of carefully selected multiple antigens of diagnostic utility) for effective serodiagnosis of early-infected dogs, as this will likely allow more efficient canine removal regimens than those used in practice by public health services.

In situ characterization of the granulomatous immune response with time in nonhealing lesional skin of Leishmania braziliensis-infected rhesus macaques (Macaca mulatta).

We have recently introduced a macaque (Macaca mulatta) model of Leishmania braziliensis-induced self-healing cutaneous leishmaniasis in which the T cell-mediated inflammatory response effectively promotes parasite clearance and granuloma resolution. Here we show that macaques infected with a highly pathogenic L. braziliensis strain displayed longstanding granulomatous lesions which lasted until the end of the observation period (52 weeks). Immunoperoxidase staining of representative tissue sections indicated that distinct cell populations (CD3, CD4, CD8, CD20, Foxp3, CD20, CD68, HLA-DR, CCL2, and CXCL-10) change uniformly during infection, suggesting that the same components of the local immune response are working in unison. This model also confirmed that granuloma formation is orchestrated by diverse inflammatory mediators that are important for T helper type 1 (Th1) cell development and macrophage effector functions. Cytometry analysis of ex vivo granuloma-derived leukocytes revealed accumulation of distinct functional subsets of effector and regulatory T cells into the inflamed skin. We provide evidence that local interleukin (IL)-10 production by both Foxp3(+) and Foxp3(-) CD4(+) T subsets is likely important in promoting lesional granuloma maintenance. Further studying the immune suppression mechanisms that induces granulomas in L. braziliensis-infected macaques may reveal new opportunities for therapeutic control of this important human disease.

Cross-sectional and longitudinal epidemiologic surveys of human and canine Leishmania infantum visceral infections in an endemic rural area of southeast Brazil (Pancas, Espirito Santo).

In an endemic rural area of southeast Brazil, surveys confirmed that dogs serve as peridomestic reservoirs of Leishmania infantum. It is likely that the lack of efficient control is because presently used diagnostic tests miss positive dogs. Overall, 57% of the dogs had specific antibodies, but the canine infection was not uniformly fatal and many seropositive dogs remained asymptomatic or even spontaneously recovered. Furthermore, 42% of the human residents became leishmanin-positive reactors and 47% had positive serology at the initial survey, but our estimates also point at a high recovery rate among the infected population with time. The delayed-type hypersensitivity (DTH) reaction to Leishmania was a good indicator of resistance to infection in this particular epidemiologic situation. The lack of any significant differences in infection rates by gender or age indicate that all of the population was at an equal risk of infection and most people were infected in the peridomestic setting.

Impact of 3 years ivermectin treatment on onchocerciasis in Yanomami communities in the Brazilian Amazon.

In the current study, it was assessed, for the first time, the effect of ivermectin treatment administered twice a year on the prevalence and morbidity of onchocerciasis in the hyperendemic Yanomami communities of the Roraima State (Brazil). Physical and parasitological examinations were carried out every 6 months until six drug rounds of treatment were completed. The coverage during the six rounds of ivermectin treatment ranged from 89% to 92% of the eligible Yanomami population. Overall, comparison of results at pre-treatment with results after six rounds of treatment, the prevalence of infection had declined from 87% to 42% (P<0.0001, CI 95%=0.05-0.22); the community microfilarial load (CMFL) fell from 1.17 to 0.53Mf/mg of skin; and the crude intensity of infection (MFL-Total) decreased from 18.95 to 1.96Mf/mg of skin during the same period (P<0.0001, for both microfilarial loads). Although no significant difference was observed between microfilarial densities in skin snips from iliac crest and scapula after the 6th round of ivermectin treatment it was observed that the prevalence of positive skin snips was significantly higher when skin snips were taken from iliac crest (42%) than from scapula (8%) (P=0.001, CI 95%=3.41-22.67). After six rounds of ivermectin treatments, no significant differences were observed in the prevalences of palpable nodules and of onchodermatitis in relation to pre-treatment prevalences, from 45% to 41% and from 17% to 20% (P>0.05, for both). These findings suggest that mass population treatment should continue without interruption and achieve higher levels of drug coverage in order to alleviate disease manifestations and interrupt infection transmission to hasten the elimination of onchocerciasis in Yanomami communities. In addition, the sensitivity of iliac crest snips for parasitological assessment in epidemiological surveillance of Yanomami communities may increase the acceptance of the population in biopsy sampling and seems to be a good choice for assessing the success of control programs.

Comparative evaluation of enzyme-linked immunosorbent assays based on crude and recombinant leishmanial antigens for serodiagnosis of symptomatic and asymptomatic Leishmania infantum visceral infections in dogs.

The diagnosis of visceral leishmaniasis remains difficult in rural areas where the disease is endemic, and serologic methods still need assessment, as they are not very sensitive for the detection of asymptomatic infectious dogs. Here we present data on the development of enzyme-linked immunosorbent assay (ELISA)-based methods for the detection of antibodies against recombinant leishmanial antigens (namely, the recombinant K26 [rK26] and rK39 antigens from Leishmania infantum and the rA2 protein from Leishmania donovani) in comparison to ELISAs employing crude soluble antigen (CSA). The assays utilized sera from known negative controls (n=25) and clinically asymptomatic (n=50) and symptomatic (n=50) dogs with confirmed L. infantum infections. Additional studies were also done using sera from animals harboring other infections (n=14) for the evaluation of cross-reactivity. Our study indicated that rK26 and rK39 used in ELISAs provided very high sensitivities for the detection of symptomatic dogs (94% and 100%, respectively), followed by CSA (88%) and rA2 (70%). Conversely, rA2 was more sensitive for asymptomatic dogs (88%) than rK39 and rK26 (both 66%) and CSA (30%). Some cross-reactivity in sera from dogs with other infections (Leishmania braziliensis and Leptospira interrogans) was identified, but the rA2 protein provided the greatest specificity (98%). Data further indicate that all three recombinant proteins must be used in parallel to detect essentially all infected dogs. Efforts should be made to develop a cheap and reliable serologic test based on epitope selection from these diagnostic markers for the sensitive detection of L. infantum-infected dogs.

Inquérito sorológico para a infecção por Toxoplasma gondii em ameríndios isolados, Mato Grosso / Serological survey of Toxoplasma gondii infection in isolated Amerindians, Mato Grosso

Este estudo determinou a ocorrência de anticorpos anti-Toxoplasma gondii em população indígena do Mato Grosso, os Enawenê-Nawê. Estes habitam uma vasta região selvagem, com raros contatos com não-índios. Não apresentam animais domésticos, inclusive gatos. A dieta é baseada em insetos, mandioca, milho, mel e fungos e não se alimentam de carne, exceto de peixe. Com base no exposto, desenvolveu-se análise sorológica, por meio de ELISA - IgG e IFI - IgG e IgM. De 148 soros, 80,4 por cento foram ELISA ou IFI- IgG positivos. Não foram detectados casos de IgM reagentes. Nesse grupo as taxas de soropositividade aumentaram significativamente com a idade, de 50 por cento a 95 por cento. Analisando-se os hábitos e costumes, aliados à alta soropositividade encontrada, sugere-se que a presença de felinos silvestres nas imediações da aldeia e coleções de água poderia ter papel importante como fonte de infecção, contaminando o solo e, conseqüentemente, os insetos e fungos consumidos pelos índios.

A biologia da Leishmania (Viannia) braziliensis no modelo experimental primata Macaca mulatta / The biology of the Leishmania (Viannia) braziliensis in the experimental model primate Macaca mulatta

Infecções naturais com o protozoário parasita Leishmania (Viannia) braziliensis Vianna 1911 (Kinetoplastida: Trypanosomatidae) causam doença humana em várias áreas (sub )tropicais no continente Americano (em pelo menos 15 países), constituindo um importante problema de saúde pública. A leishmaniose cutânea (LC) pela L. (V.) braziliensis distingue-se de outras formas da doença pela maior cronicidade, latência e morbidade da infecção, com tendência para ocorrer lesões metastáticas na pele e/ou mucosa nasofaríngea. Estudos taxonômicos têm revelado o polimorfismo genético em populações naturais de L. (V.) braziliensis, porém os fatores de risco envolvidos na expressão clínica diferenciada da doença são ainda obscuros. Nossos estudos sobre infecções com L. (V.) braziliensis em macacos rhesus (Macaca mulatta) foram feitos para caracterizar a evolução de diferentes cepas do patógeno e as respostas imunes adaftativas neste modelo experimental. Um inóculo padronizado de promastigotas (10 ) foi injetado, via intradérmica, na arcada superciliar ou no antebraço de cada macaco. As infecções de longa duração (25-33 meses) reproduziram as úlceras cutâneas auto-resolutivas ou persistentes, assim como a LC crônica recidivante devido à reativação da infecção latente. O decurso da lesão primária foi muito variável, não somente para os isolados mostrando diferenças moleculares, mas também para animais individuais em grupos infectados com a mesma cepa do parasito.

Entretanto, 80% dos símios infectados com o isolado de lesão disseminada desenvolveram lesões metastáticas persistentes na pele e/ou mucosa. Digno de nota, o comprometimento granulomatoso da mucosa nasal foi observado em 2 de 33 (6%) dos rhesus infectados. A persistência do parasito em lesões ativas ou cicatrizadas foi documentada na maioria dos primatas. Foram caracterizados genótipos específicos representando parasitos isolados de humanos e macacos (contudo, sem ter havido qualquer relação aparente com fenótipos clínicos específicos), confirmando assim a estrutura genética multiclonal de L. (V.) braziliensis. Respostas imunes adaptativas específicas (Le., anticorpos anti-Leishmania e reações in vivo e in vitro de linfócitos T específicos) foram induzidas no decurso das infecções. A resistência clínica à L. (V.) braziliensis foi aparentemente associada com a resposta T H1 específica, e os animais curados da infecção primária adquiriram imunidade protetora sólida contra re-infecção homóloga (Parasitology 2003, 127:437-447). Em outro estudo (Am J Trop Med Hyg 2004, 71 :297-305), demonstramos a variabilidade relativa em ensaios de proteção cruzada entre diferentes espécies ou cepas variantes de Leishmania em macacos rhesus.

A investigação mais detalhada da resposta imune neste sistema primata poderá esclarecer fatores relevantes influenciando a proteção heteróloga. O modelo macaco rhesus-L. (V.) braziliensis foi também explorado para avaliar a eficácia leishmanicida do antimoniato de Nmetillglucamina (J Parasitol 2005, 91:976-978). As respostas terapêuticas nos animais experimentais usando doses reduzida e convencional (5 e 20 mg/kg/dia por 28 dias, respectivamente) de Sbv foram similares aos de pacientes com LC. Os primatas curaram suas lesões após o tratamento, porém com parasitismo críptico e/ou recidiva. Concluindo, como a resposta granulomatosa à L. (V.) braziliensis em M. mulatta reproduz a patologia humana, este modelo torna-se útil para estudar a fisiopatologia/eventos imunoregulatórios associados com a evolução da doença, assim como em testes pré-clínicos de novas drogas e vacinas candidatas.