Passive case detection for canine visceral leishmaniasis control in urban Brazil: Determinants of population uptake.

BACKGROUND: In Brazil, the transmission of Leishmania infantum in urban settings is closely related to infection among dogs, with occasional transmission to humans. Serological screening of dogs for Leishmania spp. infection on requests of their owners (passive case detection) represents a frequent, but little studied, practice within the scope of Brazilian public health. This study identified factors associated with canine visceral leishmaniasis (CVL) diagnosis-seeking behavior of dog owners in Rondonópolis (236,000 inhabitants), a municipality in Central-Western Brazil where VL is endemic. Also, we evaluated the profile of dog owners and their animals screened on free demand. METHODOLOGY/PRINCIPAL FINDINGS: Using mixed effects negative binomial regression, we modelled the number of dogs screened for Leishmania infection on free demand per neighborhood from 2011 to 2016 as a function of time-dependent predictors (current or recent canine seropositivity and human VL incidence), distance to the screening site, and demographic variables. We assessed potential delays in the effect of time-dependent predictors on the outcome. Among 12,536 dogs screened for Leishmania infection, 64.2% were tested during serosurveys and 35.8% were tested on free demand. Of these, 63.9% were positive. Uptake of screening under free demand was strongly associated with higher levels of canine seropositivity in the neighborhood (current or recent) and decreasing distance to the screening site. A subsample of dog owners (n = 93) who sought CVL screening between 2016 and 2017 were interviewed in more detail. Owners with better socioeconomic status and dogs with apparent CVL clinical manifestations prevailed among them. CONCLUSIONS/SIGNIFICANCE: To support timely CVL management, passive case detection along with awareness activities aimed at dog owners should be encouraged in endemic areas. Screening sites should be prioritized in accessible zones, as well as in socio-economically disadvantage areas. In parallel, CVL active case detection should be continued as a surveillance tool to guide control actions.

Human visceral leishmaniasis in Central-Western Brazil: Spatial patterns and its correlation with socioeconomic aspects, environmental indices and canine infection.

In this ecological study, we investigated spatial patterns of human visceral leishmaniasis (VL) incidence, its correlation with socioeconomic aspects, environmental indices (obtained through remote sensing) and canine VL during 2011-2016 in the municipality of Rondonópolis, a relevant endemic area for VL in Central-Western Brazil. Human VL cases were georeferenced and point patterns were analyzed by univariate Ripley's K function and Kernel density estimation (KDE). Poisson-based scan statistics were used to investigate spatial and spatiotemporal clusters of human VL incidence at the neighborhood level. Socioeconomic and environmental characteristics were compared between neighborhoods within and outside spatial human VL clusters. Also, we assessed the correlation between smoothed human VL incidence and canine VL seropositivity rates within and between neighborhoods. Human VL cases were clustered up to 2000 m; four hotspots were identified by KDE in peripheral areas. Spatial and spatiotemporal low-risk clusters for human VL were identified in central and southern areas. Neighborhoods within spatial low-risk cluster presented higher mean income, literacy rate, sanitary sewage service coverage and lower altitude, compared to the rest of the municipality. A positive correlation was found between the occurrence of human and canine VL. On the northern outskirts, high human VL incidence was spatially correlated with high canine VL seropositivity in surrounding neighborhoods. In conclusion, human VL demonstrated a heterogeneous, aggregated and peripheral spatial pattern. This distribution was correlated with intra-urban socioeconomic differences and canine VL seropositivity at the neighborhood level.

Factors associated with Leishmania spp. infection in domestic dogs from an emerging area of high endemicity for visceral leishmaniasis in Central-Western Brazil.

The factors associated with Leishmania spp. infection in dogs are still poorly understood. This study aimed to identify such factors among domestic dogs from the Brazilian municipality of Rondonópolis, which recently emerged as an endemic area with intense transmission of human visceral leishmaniasis (VL). It was a household-based cross-sectional study conducted between 2016 and 2017. Interviews were conducted with 405 dog owners considering their socioeconomic characteristics, and environmental aspects of the household and its adjacent areas. In addition, 600 dogs were evaluated regarding physical characteristics, behavior, and care provided by the owner. Seropositive animals were those that showed reactivity in a rapid immunochromatographic test and enzyme-linked immunosorbent assay, which are currently recommended for canine visceral leishmaniasis (CVL) diagnosis in Brazil. A multivariable logistic regression analysis was employed to determine the associated factors. Low social class [OR = 2.0; 95%CI = (1.2-3.2)], presence of acerola tree (Malpighia emarginata) in the yard [OR = 2.2; 95%CI = (1.2-4.1)], presence of more than one dog in the household [OR = 2.0; 95%CI = (1.3-3.3)], horse and/or cattle breeding [OR = 12.0; 95%CI = (1.9-73.6)], existence of houses with yard adjacent to the home [OR = 4.0; 95%CI = (1.3-12.2)], presence of apparent signs consistent with CVL [OR = 10.6; 95%CI = (5.8-19.4)], dog staying mostly in the yard during the day [(OR = 4.8; 95%CI = (1.1-21.4)], and lack of cleaning of the dog's shelter [(OR = 1.9; 95%CI = (1.1-3.2)] were identified as the factors associated with Leishmania spp. infection. These results support the importance of socioeconomic and environmental aspects in the occurrence of Leishmania spp. infection. In addition, they may be useful in guiding control strategies in areas where zoonotic VL is endemic.

Therapeutic Response in Adult Patients with Nonsevere Chronic Paracoccidioidomycosis Treated with Sulfamethoxazole-Trimethoprim: A Retrospective Study.

According to the Brazilian Consensus on Paracoccidioidomycosis (PCM), itraconazole is the drug of choice for treatment. However, the combination of sulfamethoxazole and trimethoprim (SMX-TMP) is most commonly used in clinical practice because of its higher availability in the public health services. The aims of this study were to evaluate the therapeutic response of patients with nonsevere chronic PCM to SMX-TMP and highlight the factors related to treatment failure. An adequate therapeutic response was defined as completely improved disease signs and symptoms after medication use for a minimum of 6 months, followed by normalized hematological and biochemical changes, radiological improvements, and negative mycological examination findings. Medical records were analyzed for 244 patients with nonsevere chronic PCM who were treated between 1998 and 2014. In total, 41.9% of the patients had PCM for ≥ 8 months. Seven (2.9%) patients were coinfected with human immunodeficiency virus (HIV). The median (25%, 75% percentiles) treatment duration was 21 (10, 25) months. Adequate treatment adherence was reported by 68.3% of patients. In addition, 73.6% of patients exhibited an adequate therapeutic response. The majority (82.6%) of patients who were treated with SMX-TMP for > 24 months displayed an adequate therapeutic response, and the frequency of adequate therapeutic response gradually decreased as the duration of treatment decreased. Treatment nonadherence (P < 0.001) and PCM-HIV coinfection (P = 0.019) were factors associated with therapeutic failure. The study results support the good efficacy of SMX-TMP. Attention should be given to PCM-HIV coinfection, emphasizing the concern of a higher risk of PCM therapeutic failure in these patients.

Allele-specific antibodies to Plasmodium vivax merozoite surface protein-1: prevalence and inverse relationship to haemoglobin levels during infection.

BACKGROUND: Antigenic polymorphisms are considered as one of the main strategies employed by malaria parasites to escape from the host immune responses after infections. Merozoite surface protein-1 (MSP-1) of Plasmodium vivax, a promising vaccine candidate, is a highly polymorphic protein whose immune recognition is not well understood. METHODS AND RESULTS: The IgG responses to conserved (MSP-119) and polymorphic (block 2 and block 10) epitopes of PvMSP-1 were evaluated in 141 P. vivax infected patients. Ten recombinant proteins corresponding to block 2 (variants BR07, BP29, BP39, BP30, BEL) and block 10 (BR07, BP29, BP39, BP01, BP13) often observed in Brazilian P. vivax isolates were assessed by ELISA in order to determine levels of specific antibodies and their respective seroprevalence. The magnitude and the frequency of variant-specific responses were very low, except for BR07 variant (>40%), which was the predominant haplotype as revealed by block 10 PvMSP-1 gene sequencing. By contrast, 89% of patients had IgG against the C-terminal conserved domain (PvMSP-119), confirming the high antigenicity of this protein. Using multiple linear and logistic regression models, there was evidence for a negative association between levels of haemoglobin and several IgG antibodies against block 2 variant antigens, with the strongest association being observed for BP39 allelic version. This variant was also found to increase the odds of anaemia in these patients. CONCLUSIONS: These findings may have implications for vaccine development and represent an important step towards a better understanding of the polymorphic PvMSP-1 domain as potential targets of vaccine development. These data highlight the importance of extending the study of these polymorphic epitopes of PvMSP-1 to different epidemiological settings.

Changes in serum lipid profile in the acute and convalescent Plasmodium vivax malaria: A cohort study.

Although serum lipids are known to be altered in Plasmodium falciparum-induced malaria, little is known about such changes due to Plasmodium vivax infection. This cohort study assessed serum concentrations of triglycerides, total cholesterol, low-density lipoprotein (LDL), and high-density lipoprotein (HDL) in 164 patients in the acute phase of malaria caused by P. vivax and characterized these changes in the convalescent phase after treatment with chloroquine and primaquine. Compared to reference values, serum total cholesterol, LDL, and HDL levels were lower and triglyceride levels were higher in the acute phase. Moreover, the parasite density was negatively correlated with LDL (r=-0,189; p=0.027) and HDL (r=-0,256; p=0.001) serum levels. Eighty patients returned for clinical and laboratory revaluation 7-12days after treatment initiation. All patients showed parasite clearance and the absence of symptoms during the convalescent phase. Analysis of the serum lipids of these 80 patients showed significant increases in the serum levels of total cholesterol (p<0.0001), LDL (p<0.0001), and HDL (p<0.0001) as well as a significant reduction in triglycerides (p=0.004), indicating a trend towards a return to normal levels. This transient change in lipid profile between the acute and convalescent stages may be useful for the clinical monitoring of patients treated for vivax malaria.

Assumed white blood cell count of 8,000 cells/µL overestimates malaria parasite density in the Brazilian Amazon.

Quantification of parasite density is an important component in the diagnosis of malaria infection. The accuracy of this estimation varies according to the method used. The aim of this study was to assess the agreement between the parasite density values obtained with the assumed value of 8,000 cells/µL and the automated WBC count. Moreover, the same comparative analysis was carried out for other assumed values of WBCs. The study was carried out in Brazil with 403 malaria patients who were infected in different endemic areas of the Brazilian Amazon. The use of a fixed WBC count of 8,000 cells/µL to quantify parasite density in malaria patients led to overestimated parasitemia and resulted in low reliability when compared to the automated WBC count. Assumed values ranging between 5,000 and 6,000 cells/µL, and 5,500 cells/µL in particular, showed higher reliability and more similar values of parasite density when compared between the 2 methods. The findings show that assumed WBC count of 5,500 cells/µL could lead to a more accurate estimation of parasite density for malaria patients in this endemic region.

Sensitivity of nested-PCR for plasmodium detection in pooled whole blood samples and its usefulness to blood donor screening in endemic areas.

Transfusion-transmitted malaria is a severe disease with high fatality rate. Most Brazilian blood banks in the Amazon region perform malaria screening using microscopic examination (thick smears). Since low parasite concentrations are expected in asymptomatic blood donors a high sensitivity test should be used for donor screening. This study determined the sensitivity of a nested-PCR for plasmodium detection in pooled samples. We performed a one-stage criterion validation study with 21 positive samples pooled with samples from ten negative volunteer until three different concentrations were reached (0.33; 0.25; 0.20 parasites/µL - p/µL). Nested PCR was performed as described by Snounou et al. (1993). Sensitivities (and confidence intervals) were determined by stratum of final parasite concentration on the pooled samples. All samples with parasitemia values of 0.33 and 0.25 p/µL had 100% sensitivity (95%CI=86.3-100). One negative result was obtained from a sample with 0.20 p/µL sensitivity=95.2% (95%CI=76.2-99.9). Compared to parasitemia detectable under ideal conditions of thick smear, this nested-PCR in pooled sample was able to detect 40 times more parasites per microliter. Nested-PCR in pooled samples should be considered as a high sensitive alternative to thick smear for donor screening in blood banks at endemic regions. Local authorities need to assess cost:benefit advantages of this method compared to alternatives.

Multiple-clone activation of hypnozoites is the leading cause of relapse in Plasmodium vivax infection.

BACKGROUND: Plasmodium vivax infection is characterized by a dormant hepatic stage, the hypnozoite that is activated at varying periods of time after clearance of the primary acute blood-stage, resulting in relapse. Differentiation between treatment failure and new infections requires characterization of initial infections, relapses, and clone multiplicity in vivax malaria infections. METHODOLOGY/PRINCIPAL FINDINGS: Parasite DNA obtained from primary/relapse paired blood samples of 30 patients with P. vivax infection in Brazil was analyzed using 10 molecular markers (8 microsatellites and MSP-1 blocks 2 and 10). Cloning of PCR products and genotyping was used to identify low-frequency clones of parasites. We demonstrated a high frequency of multiple-clone infections in both primary and relapse infections. Few alleles were identified per locus, but the combination of these alleles produced many haplotypes. Consequently, the majority of parasites involved in relapse showed haplotypes that were distinct from those of primary infections. Plasmodium vivax relapse was characterized by temporal variations in the predominant parasite clones. CONCLUSIONS/SIGNIFICANCE: The high rate of low frequency alleles observed in both primary and relapse infections, along with temporal variation in the predominant alleles, might be the source of reported heterologous hypnozoite activation. Our findings complicate the concept of heterologous activation, suggesting the involvement of undetermined mechanisms based on host or environmental factors in the simultaneous activation of multiple clones of hypnozoites.

Analysis of genetic variability of Plasmodium vivax isolates from different Brazilian Amazon areas using tandem repeats.

Few genetic markers have been described to analyze populations of Plasmodium vivax. The genetic variability of P. vivax has been analyzed mainly among isolates taken from areas ranging from hyper- to holoendemic areas. These studies of genetic variability have neglected many areas with different epidemiologic profiles. The purpose of this study was to analyze the genetic variability of P. vivax isolates from four different Brazilian Amazon areas. We chose to study the five most polymorphic tandem repeats (TRs) identified so far. All TRs studied were polymorphic in at least one studied population, with a modal allele at nearly all loci. Expected heterozygosity ranged from 0.462 to 0.666 and did not correlate with the repeat array length. The genetic distances among the populations varied from 0.027 to 0.241, and did not correlate with their geographic separation. Tandem repeats identified in P. vivax isolates failed to allow geographic clustering.

Polymorphism at the apical membrane antigen 1 locus reflects the world population history of Plasmodium vivax.

BACKGROUND: In malaria parasites (genus Plasmodium), ama-1 is a highly polymorphic locus encoding the Apical Membrane Protein-1, and there is evidence that the polymorphism at this locus is selectively maintained. We tested the hypothesis that polymorphism at the ama-1 locus reflects population history in Plasmodium vivax, which is believed to have originated in Southeast Asia and is widely geographically distributed. In particular, we tested for a signature of the introduction of P. vivax into the New World at the time of the European conquest and African slave trade and subsequent population expansion. RESULTS: One hundred and five ama-1 sequences were generated and analyzed from samples from six different Brazilian states and compared with database sequences from the Old World. Old World populations of P. vivax showed substantial evidence of population substructure, with high sequence divergence among localities at both synonymous and nonsynonymous sites, while Brazilian isolates showed reduced diversity and little population substructure. CONCLUSION: These results show that genetic diversity in P. vivax AMA-1 reflects population history, with population substructure characterizing long-established Old World populations, whereas Brazilian populations show evidence of loss of diversity and recent population expansion. NOTE: Nucleotide sequence data reported is this paper are available in the GenBanktrade mark database under the accession numbers EF031154 - EF031216 and EF057446 - EF057487.

Púrpura em paciente com estrongiloidíase disseminada / Purpura in patient with disseminated strongiloidiasis

A infecção pelo Strongyloides stercoralis em associação com imunosupressão pode manifestar-se com lesões em múltiplos órgãos e sistemas, caracterizando a forma disseminada da doença. Lesões cutâneas não são freqüentemente relatadas e, se presentes, manifestam-se como rash e petéquias. Púrpuras bem definidas são pouco descritas. No presente trabalho é descrito um caso de estrongiloidíase disseminada, com acometimento cutâneo em forma de púrpura, que se desenvolveu em um paciente timectomizado e usuário crônico de corticosteróide devido à miastenia gravis.

[Purpura in patient with disseminated strongiloidiasis]. / Púrpura em paciente com estrongiloidíase disseminada.

The association of systemic corticosteroid therapy and dissemination of Strongyloides stercoralis has been increasingly documented in the literature. Skin involvement in disseminated strongyloidiasis has been reported and the most commonly described cutaneous manifestations are rash and petechial eruptions. We present a case of an immunosuppressed man that developed disseminated strongyloidiasis with extensive purpura.

[Entomophthoramycosis (zygomycosis) caused by Conidiobolus coronatus in Mato Grosso (Brazil): case report]. / Entomoftoromicose (zigomicose) causada por Conidiobolus coronatus em Mato Grosso (Brasil): relato de caso.

Zygomycosis is a subcutaneous mycosis caused by soil fungi, such as Conidiobolus coronatus. In general, the main clinical manifestation is a chronic rhinofacial tumor. We report the first case of zygomycosis (entomophthoramycosis) caused by Conidiobolus coronatus, occurring in Mato Grosso, West Brazil.

Entomoftoromicose (zigomicose) causada por Conidiobolus coronatus em Mato Grosso (Brasil): relato de caso / Entomophthoramycosis (zygomycosis) caused by Conidiobolus coronatus in Mato Grosso (Brazil): case report

Zigomicoses são micoses subcutâneas causadas por fungos do solo, que geralmente manifestam-se como uma infiltração granulomatosa crônica da submucosa nasal, estendendo-se para o tecido subcutâneo e pele da face. Descreve-se aqui o primeiro caso de zigomicose nasofacial causada pelo Conidiobolus coronatus, ocorrendo em Mato Grosso, Brasil.

IgG isotype to C-terminal 19 kDa of Plasmodium vivax merozoite surface protein 1 among subjects with different levels of exposure to malaria in Brazil.

Subclasses of antibodies to the C-terminal 19 kDa fragment of the Plasmodium vivax merozoite surface protein 1 (PvMSP-1(19)) were assessed among subjects with distinct degrees of malaria exposure in the Brazilian endemic area. The PvMSP-1(19) specific IgG1and IgG3 levels were low among subjects with long-term exposure (approximately 19 years) when compared to subjects less and sporadically exposed (<1 year). No statistically difference was observed in IgG subclass distribution of antibodies from symptomatic Plasmodium-infected patients, asymptomatic parasite carriers and non-infected subjects living in a same mesoendemic area. Subjects briefly exposed to a P. vivax outbreak living in a rural community outside the endemic area were also evaluated to measure the persistence of specific antibodies. IgG anti-PvMSP-1(19) antibodies persisted in 40% of the subjects who had had malarial symptoms 8 months before and decreased after 7 years (28%). Specific IgG1 were the predominant isotype. Our study emphasizes the highly immunogenicity of the PvMSP-1(19) and points toward its possible use as a potential malaria vaccine.

Increased polyclonal immunoglobulin reactivity toward human and bacterial proteins is associated with clinical protection in human Plasmodium infection.

BACKGROUND: Polyclonal B-cell activation is well known to occur in Plasmodium infections, but its role in pathogenesis or protection remains unclear. However, protective properties of natural antibodies have previously been demonstrated in other contexts. METHODS: Sera from asymptomatic and symptomatic Plasmodium-infected subjects locally detected in a survey study in the Brazilian Amazon, and from unexposed and exposed but presently uninfected control subjects, were assayed by a standardized quantitative immunoblot method allowing simultaneous detection of IgG or IgM reactivity to a large number of parasite-unrelated proteins. RESULTS: In subjects free of coinfection with hepatitis B virus, IgG reactivity to human brain antigens and Escherichia coli proteins was strikingly enhanced in asymptomatic Plasmodium-infected individuals when compared to such with clinical malaria symptoms, or to uninfected control subjects. This difference was most characteristic for limited exposure times (less than ten years locally, or 20 years in endemic areas). It was more significant than a similar trend found for IgG to Plasmodium falciparum antigens, and unrelated to parasitaemia levels. Asymptomatic subjects with comparatively short exposure characteristically showed relatively elevated IgG versus IgM reactivity. Polyclonal IgG reactivity appears triggered by previous P. falciparum but not Plasmodium vivax malaria. CONCLUSION: The observed difference in polyclonal antibody production seems related to intrinsic activation states of infected individuals, rather than to parasite-antigen specific immune responses. However, it appears influenced by preceding stimuli. This supports the idea that acquired clinical immunity may not exclusively depend on antigen-specific responses, but also on the individual polyclonal reaction.

Association of the IgG response to Plasmodium falciparum merozoite protein (C-terminal 19 kD) with clinical immunity to malaria in the Brazilian Amazon region.

The antibody response to the C-terminal 19-kD fragment of Plasmodium falciparum merozoite surface protein-1 (PfMSP1-19) was investigated in groups of subjects living in areas of Brazil with different levels of malaria transmission. The prevalence and the levels of IgG to PfMSP1-19 increased with the time of exposure and were positively correlated with the absence of clinical symptoms in parasitemic patients. The frequency of positive response and the mean level of IgG were higher in areas where malaria prevalence was more intense, especially among asymptomatic patients. The serum absorbance values of the IgG1 isotype were significantly higher among subjects with long-term exposure and in asymptomatic infections. These data suggest a protective role of IgG1 in naturally acquired immunity in spite of the unstable transmission levels in the Brazilian Amazon.

Diagnóstico de paracoccidioidomicose disseminada grave em aspirado de medula óssea: relato de caso / Severe disseminated paracoccidioidomycosis diagnosis in bone marrow aspirate: a case report

Acute or chronic disseminated paracoccidioidomycosis can be associated with changes in blood and bone marrow cell counts, mainly in the severe forms of this disease. However, there are few reports about the microbiological confirmation of the mycosis in bone marrow tissue. The present report describes a case of an adult patient with severe chronic multifocal paracoccidioidomycosis, whose etiological diagnosis has been done by the microscopical exam and culture fo the bone marrow aspirate. The authors emphasize the importance of these exams as an alternative way for the diagnosis of suspected cases of severe paracoccidioidomycosis.