Serological diagnosis of Chagas disease: evaluation and characterisation of a low cost antigen with high sensitivity and specificity

In spite of evident progress in the serology of Chagas disease, the requirement for new diagnostic antigens persists. We have evaluated different antigens obtained from Trypanosoma cruzi grown in medium rich in nutrients or under nutrient stress, autoclaved or sonicated and fractionated by differential centrifugation. The resulting antigens were evaluated for diagnosis of Chagas disease using ELISA. Immunofluorescence of the parasites demonstrated that nutrient stress induced changes in the distribution and density of antigens recognised by a pool of sera from experimentally infected mice. When evaluated using ELISA, it was evident that most fractions had good sensitivity but poor specificity. Surprisingly, the best specificity and sensitivity was observed with parasites cultured under nutrient stress and autoclaved. Furthermore this antigen had low cross reactivity with sera from other parasitic diseases, Leishmaniasis in particular. Western blot analysis demonstrated that autoclaving seems to non-specifically eliminate cross-reactive antigens. In conclusion, autoclaving epimastigotes of T. cruzi, after nutrient stress, allowed us to obtain an antigen that could be used in the serological diagnosis of Chagas disease.

Distribution and habitat preferences of the genus Biomphalaria (Gastropoda: Planorbidae) in Cuba

A study was carried out to determine the distribution and habitat preferences of several species of the genus Biomphalaria. Samples were taken at 350 freshwater locations in Cuba. Three species of Biomphalaria (Biomphalaria havanensis, Biomphalaria helophila and Biomphalaria pallida) were recorded based on their distribution. Of the three species, B. havanensis has the weakest distribution because it was identified in only one locality. The other species, B. helophila and B. pallida, are abundant in rivers and dams and have large populations in Cuba. However, the only species that appears to occur in ecosystems shared with thiarids is B. pallida, possibly due to recent introduction of thiarids, but always in fewer numbers. Here we discuss the possibility of these species to act as intermediary hosts of Schistosoma mansoni in Cuba over the basis of occurrence in natural and anthropic habitats.

Classification of immature mosquito species according to characteristics of the larval habitat in the subtropical province of Chaco, Argentina

To classify mosquito species based on common features of their habitats, samples were obtained fortnightly between June 2001-October 2003 in the subtropical province of Chaco, Argentina. Data on the type of larval habitat, nature of the habitat (artificial or natural), size, depth, location related to sunlight, distance to the neighbouring houses, type of substrate, organic material, vegetation and algae type and their presence were collected. Data on the permanence, temperature, pH, turbidity, colour, odour and movement of the larval habitat's water were also collected. From the cluster analysis, three groups of species associated by their degree of habitat similarity were obtained and are listed below. Group 1 consisted of Aedes aegypti. Group 2 consisted of Culex imitator, Culex davisi, Wyeomyia muehlensi and Toxorhynchites haemorrhoidalis separatus. Within group 3, two subgroups are distinguished: A (Psorophora ferox, Psorophora cyanescens, Psorophora varinervis, Psorophora confinnis, Psorophora cingulata, Ochlerotatus hastatus-oligopistus, Ochlerotatus serratus, Ochlerotatus scapularis, Culex intrincatus, Culex quinquefasciatus, Culex pilosus, Ochlerotatus albifasciatus, Culex bidens) and B (Culex maxi, Culex eduardoi, Culex chidesteri, Uranotaenia lowii, Uranotaenia pulcherrima, Anopheles neomaculipalpus, Anopheles triannulatus, Anopheles albitarsis, Uranotaenia apicalis, Mansonia humeralis and Aedeomyia squamipennis). Principal component analysis indicates that the size of the larval habitats and the presence of aquatic vegetation are the main characteristics that explain the variation among different species. In contrast, water permanence is second in importance. Water temperature, pH and the type of larval habitat are less important in explaining the clustering of species.

Specificity of immunoblotting analyses in eosinophilic meningitis

Angiostrongylus cantonensis and Gnathostoma spinigerum are the two most common causative parasites of eosinophilic meningitis (EOM). Serological tests are helpful tools for confirming the identity of the pathogen. Recent reports determined the specificity of such tests by using normal healthy controls. There have been limited studies done to rule out the cross-reactivity between these two causative parasites of EOM. This study aims to assess the specificity of the serological test in EOM by using each condition as a control for the other. Thirty-three patients with a diagnosis of EOM were enrolled. Sera from 22 patients with a positive 29-kDa antigenic diagnostic band of A. cantonensis were tested for the 21 and 24-kDa antigenic bands of G. spinigerum. Similarly, sera of 11 gnathostomiasis patients were tested for the 29-kDa diagnostic band for A. cantonensis. Only one patient in the angiostrongyliasis group had a positive result for the 21 and 24-kDa antigenic bands of G. spinigerum, while no gnathostomiasis patients showed a positive result for the 29-kDa antigenic band of A. cantonensis. The specificity of the 21 and 24-kDa antigenic bands for gnathostomiasis and the 29-kDa antigenic band for A. cantonensis was 95.5% and 100%, respectively. The antigenic bands for the diagnosis of gnathostomiasis and angiostrongyliasis in EOM were highly specific.

Habitat suitability of Anopheles vector species and association with human malaria in the Atlantic Forest in south-eastern Brazil

Every year, autochthonous cases of Plasmodium vivax malaria occur in low-endemicity areas of Vale do Ribeira in the south-eastern part of the Atlantic Forest, state of São Paulo, where Anopheles cruzii and Anopheles bellator are considered the primary vectors. However, other species in the subgenus Nyssorhynchus of Anopheles (e.g., Anopheles marajoara) are abundant and may participate in the dynamics of malarial transmission in that region. The objectives of the present study were to assess the spatial distribution of An. cruzii, An. bellator and An. marajoara and to associate the presence of these species with malaria cases in the municipalities of the Vale do Ribeira. Potential habitat suitability modelling was applied to determine both the spatial distribution of An. cruzii, An. bellator and An. marajoara and to establish the density of each species. Poisson regression was utilized to associate malaria cases with estimated vector densities. As a result, An. cruzii was correlated with the forested slopes of the Serra do Mar, An. bellator with the coastal plain and An. marajoara with the deforested areas. Moreover, both An. marajoara and An. cruzii were positively associated with malaria cases. Considering that An. marajoara was demonstrated to be a primary vector of human Plasmodium in the rural areas of the state of Amapá, more attention should be given to the species in the deforested areas of the Atlantic Forest, where it might be a secondary vector.

Appraisal of Phlebotomus argentipes habitat suitability using a remotely sensed index in the kala-azar endemic focus of Bihar, India

Visceral leishmaniasis, or kala-azar, is recognised as a serious emerging public health problem in India. In this study, environmental parameters, such as land surface temperature (LST) and renormalised difference vegetation indices (RDVI), were used to delineate the association between environmental variables and Phlebotomus argentipes abundance in a representative endemic region of Bihar, India. The adult P. argentipes were collected between September 2009-February 2010 using the hand-held aspirator technique. The distribution of P. argentipes was analysed with the LST and RDVI of the peak and lean seasons. The association between environmental covariates and P. argentipes density was analysed a multivariate linear regression model. The sandfly density at its maximum in September, whereas the minimum density was recorded in January. The regression model indicated that the season, minimum LST, mean LST and mean RDVI were the best environmental covariates for the P. argentipes distribution. The final model indicated that nearly 74% of the variance of sandfly density could be explained by these environmental covariates. This approach might be useful for mapping and predicting the distribution of P. argentipes, which may help the health agencies that are involved in the kala-azar control programme focus on high-risk areas.

Specificity of the rapid rK39 antigen-based immunochromatographic test Kalazar Detect(r) in patients with cutaneous leishmaniasis in Brazil

The aim of this study was to evaluate the specificity of a rapid immunochromatographic test that was developed to detect antibodies against the rK39 antigen for the diagnosis of visceral leishmaniasis (VL). This evaluation was performed using sera from patients with a confirmed diagnosis of active cutaneous leishmaniasis. The sera from 272 patients with a confirmed diagnosis of localised cutaneous leishmaniasis (CL) who resided in an area endemic for Leishmania braziliensis in Brazil were obtained before the initiation of antileishmanial treatment. Kalazar Detect(r)(InBios, Seattle, WA) recombinant K39 antigen-based immunochromatographic strips were used according to the manufacturer's instructions. The test results were evaluated independently by two examiners in sequential order. The positive controls for the test included five serum samples from five patients with parasitologically confirmed diagnosis of VL caused by Leishmania infantum in Brazil. Overall, 100% of the samples obtained from patients with CL were negative, confirming the absence of a serological cross-reaction for individuals with cutaneous disease when these patients were evaluated using the rapid test. The lack of a cross-reaction in patients who were infected by parasites of the same genus highlights the specificity of the rK39 antigen for the diagnosis of VL in areas with the sympatric circulation of L. braziliensis and L. infantum.

Sensitivity and specificity of parallel or serial serological testing for detection of canine Leishmania infection

In Brazil, human and canine visceral leishmaniasis (CVL) caused byLeishmania infantum has undergone urbanisation since 1980, constituting a public health problem, and serological tests are tools of choice for identifying infected dogs. Until recently, the Brazilian zoonoses control program recommended enzyme-linked immunosorbent assays (ELISA) and indirect immunofluorescence assays (IFA) as the screening and confirmatory methods, respectively, for the detection of canine infection. The purpose of this study was to estimate the accuracy of ELISA and IFA in parallel or serial combinations. The reference standard comprised the results of direct visualisation of parasites in histological sections, immunohistochemical test, or isolation of the parasite in culture. Samples from 98 cases and 1,327 noncases were included. Individually, both tests presented sensitivity of 91.8% and 90.8%, and specificity of 83.4 and 53.4%, for the ELISA and IFA, respectively. When tests were used in parallel combination, sensitivity attained 99.2%, while specificity dropped to 44.8%. When used in serial combination (ELISA followed by IFA), decreased sensitivity (83.3%) and increased specificity (92.5%) were observed. Serial testing approach improved specificity with moderate loss in sensitivity. This strategy could partially fulfill the needs of public health and dog owners for a more accurate diagnosis of CVL.

New host, geographic records, and histopathologic studies ofAngiostrongylus spp (Nematoda: Angiostrongylidae) in rodents from Argentina with updated summary of records from rodent hosts and host specificity assessment

To date, 21 species of the genus Angiostrongylus (Nematoda: Angiostrongylidae) have been reported around the world, 15 of which are parasites of rodents. In this study, new host, geographic records, and histopathologic studies of Angiostrongylus spp in sigmodontine rodents from Argentina, with an updated summary of records from rodent hosts and host specificity assessment, are provided. Records of Angiostrongylus costaricensis from Akodon montensis andAngiostrongylus morerai from six new hosts and geographical localities in Argentina are reported. The gross and histopathologic changes in the lungs of the host species due to angiostrongylosis are described. Published records of the genus Angiostrongylus from rodents and patterns of host specificity are presented. Individual Angiostrongylusspecies parasitise between one-19 different host species. The most frequent values of the specificity index (STD) were between 1-5.97. The elevated number of host species (n = 7) of A. morerai with a STD = 1.86 is a reflection of multiple systematic studies of parasites from sigmodontine rodents in the area of Cuenca del Plata, Argentina, showing that an increase in sampling effort can result in new findings. The combination of low host specificity and a wide geographic distribution of Angiostrongylus spp indicates a troubling epidemiological scenario although, as yet, no human cases have been reported.

Ovipositing behaviour of Compsobracon mirabilis (Szépligeti) (Hymenoptera, Braconidae) in a cerrado habitat, southeastern Brazil

The oviposition behaviour of the braconid parasitoid, Compsobracon mirabilis ( Szépligeti, 1901) is described. Observations were conducted in a cerrado region located in Três Marias, Minas Gerais, Brazil. The oviposition occurred in a branch of Alibertia concolor (Cham.) K. Schum. 1889 (Rubiaceae), inside of which there were thirteen larvae of an unidentified species of Lepidoptera.

Ocupação diferencial do habitat por vespas do gênero Trypoxylon (Trypargilum) Latreille (Hymenoptera, Crabronidae)

Vespas do gênero Trypoxylon apresentam comportamento solitário e aprovisionam seus ninhos de forma massiva com aranhas paralisadas. Algumas espécies utilizam cavidades preexistentes para nidificação, o que facilita sua amostragem e estudo. Neste trabalho, dados sobre a biologia de nidificação de quatro espécies de Trypoxylon (Trypargilum) - T. rogenhoferi, T. lactitarse, T. aurifrons e T. nitidum - são apresentados. As espécies foram amostradas por meio de ninhos-armadilha durante três anos em Araras e dois anos em Rifaina e São Carlos (São Paulo). As localidades de estudo foram subdivididas em sítios de amostragem. Foram obtidos 2.698 ninhos de himenópteros solitários, dos quais 2.268 foram fundados por espécies de Trypoxylon. Intensa atividade de nidificação foi observada principalmente na estação quente e chuvosa (outubro-março). Os ninhos-armadilha utilizados para nidificação pelas diferentes espécies apresentaram dimensões significativamente diferentes. A família de aranhas mais utilizada para aprovisionamento foi Araneidae; no entanto, as espécies de vespas diferiram quanto às espécies forrageadas. O principal parasitóide das quatro espécies foi Melittobia, mas indivíduos das famílias Chrysididae, Ichneumonidae, Chalcididae e Sarcophagidae também foram verificados. Observou-se que as espécies de Trypoxylon coexistem temporalmente e que nas três localidades cada espécie nidificou com maior freqüência em um sítio particular, sugerindo uma ocupação diferencial do habitat. A partição do habitat aparentemente homogêneo pode ser resultado de "competição aparente" mediada por inimigos naturais comuns.

Efeito da estrutura de habitat sobre a abundância de parasitóides Pseudacteon Coquillett (Diptera, Phoridae) em ninhos de Solenopsis invicta Buren (Hymenoptera, Formicidae)

A influência de características do habitat na abundância dos parasitóides Pseudacteon Coquillett (Diptera, Phoridae) de Solenopsis invicta Buren (Hymenoptera, Formicidae) foi estudada em um agroecossistema orgânico diversificado no sudeste do Brasil. Os parasitóides foram coletados durante 30 minutos após a perturbação de ninhos da formiga lava-pé em áreas com culturas anuais ou perenes. Foram coletados no total 228 parasitóides de quatro espécies diferentes em 61,90% dos 84 ninhos da formiga lava-pé perturbados. Pseudacteon obtusus Borgmeier foi a espécie mais abundante (70 fêmeas), seguida por Pseudacteon litoralis Borgmeier (37 fêmeas), Pseudacteon tricuspis Borgmeier (23 fêmeas) e Pseudacteon solenopsidis Schmitz (1 fêmea). Pseudacteon litoralis foi mais freqüente em ninhos presentes nas culturas perenes que anuais enquanto que P. obtusus e P. tricuspis tiveram um padrão oposto. Correlações significativamente negativas foram encontradas para as abundâncias de P. litoralis e P. obtusus com a temperatura do ar. Houve correlação significativamente positiva entre a abundância dos parasitóides e o tamanho dos ninhos da formiga lava-pé. Considerando que os forídeos são importantes inimigos naturais de S. invicta, esse estudo fornece informações para o manejo de S. invicta em agroecossistemas tropicais diversificados.

What is the importance of open habitat in a predominantly closed forest area to the dung beetle (Coleoptera, Scarabaeinae) assemblage?

What is the importance of open habitat in a predominantly closed forest to the dung beetle assemblage? The Atlantic Forest in Brazil is one of the most highly disturbed ecosystems and is mainly represented by fragmented areas. However, in places where human disturbances have ceased, certain areas are showing a natural regeneration pattern. The aim of the present study was to determine how the dung beetle assemblage responds to distinct habitat structures in a fragment of Atlantic Forest. For such, open and closed forest areas were sampled in a fragment of the Atlantic Forest in the northeastern region of Brazil. Pitfall traps baited with excrement and carrion were used to collect the beetles. A total of 7,267 individuals belonging to 35 species were captured. Canthon chalybaeus and C. mutabilis were restricted to open areas. Nearly 90% of the individuals of C. aff. simulans and Deltochilum aff. irroratum were identified in these areas. A higher percentage (> 50%) of Canthon staigi, Dichotomius aff. depressicolis and D. aff. sericeus occurred in closed areas. Abundance differed between areas, with higher values in closed areas. Richness was not influenced by the habitat structure. NMDS ordination exhibited the segregation of areas and ANOSIM confirmed that this variable explained the assemblage of dung beetle species. The findings of the present study validate that open areas are associated to more restrictive conditions, limiting a higher abundance of dung beetle. Although situated near preserved fragments, the studied open areas increase the heterogeneity of the general landscape.

Mutants of common bean alpha-amylase inhibitor-2 as an approach to investigate binding specificity to alpha-amylases

Despite the presence of a family of defense proteins, Phaseolus vulgaris can be attacked by bruchid insects resulting in serious damage to stored grains. The two distinct active forms of a-amylase inhibitors, a-AI1 and a-AI2, in P. vulgaris show different specificity toward a-amylases. Zabrotes subfasciatus a-amylase is inhibited by a-AI2 but not by a-AI1. In contrast, porcine a-amylase is inhibited by a-AI1 but not by a-AI2. The objective of this work was to understand the molecular basis of the specificity of two inhibitors in P. vulgaris (a-AI1 and a-AI2) in relation to a-amylases. Mutants of a-AI2 were made and expressed in tobacco plants. The results showed that all the a-AI2 mutant inhibitors lost their activity against the insect a-amylases but none exhibited activity toward the mammalian a-amylase. The replacement of His33 of a-AI2 with the a-AI1-like sequence Ser-Tyr-Asn abolished inhibition of Z. subfasciatus a-amylase. From structural modeling, the conclusion is that the size and complexity of the amylase-inhibitor interface explain why mutation of the N-terminal loop and resultant abolition of Z. subfasciatus a-amylase inhibition are not accompanied by gain of inhibitory activity against porcine a-amylase.

Specificity and selectivity improvement in doping analysis using comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry

Comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry was used for the identification of forty doping agents. The improvement in the specificity was remarkable, allowing the resolution of analytes that could not be done by one-dimensional chromatographic systems. The sensitivity observed for different classes of prohibited substances was clearly below the value required by the World Anti-Doping Agency. In addition time-of-flight mass spectrometry gives full spectrum for all analytes without any interference from the matrix, resulting in selectivity improvements. These results could support the implementation of an exhaustive monitoring approach for hundreds of doping agents in a single injection.