Ana Lúcia Ribeiro Gonçalves

Hydrophobic fractions from Strongyloides venezuelensis for use in the human immunodiagnosis of strongyloidiasis

The objective of the present research was to evaluate detergent and aqueous phases of total saline (TS) and alkaline extracts of Strongyloides venezuelensis for human strongyloidiasis immunodiagnosis. Total extracts and detergent and aqueous antigenic fractions were separated using Triton X-114 and were examined by enzyme-linked immunosorbent assay (ELISA) and immunoblotting (IB) tests to detect immunoglobulin G (IgG). Serum samples were obtained from 120 individuals: 40 strongyloidiasis patients (group I), 40 patients with other parasitic diseases (group II), and 40 apparently healthy individuals (group III). Each extract provided a different profile of antigenic components as recognized by IgG in IB. The detergent fraction of the TS extract demonstrated the highest sensitivity and specificity for ELISA and IB. The results indicated that the detergent saline fraction, purified from S. venezuelensis, furnished the most valid results for the strongyloidiasis immunodiagnosis and could be employed as an alternative antigen and as a useful source of specific polypeptides.

Prevalence of intestinal parasites in preschool children in the region of Uberlândia, State of Minas Gerais, Brazil

INTRODUCTION Children are an important high-risk group for helminth and protozoa infections. Daycare centers are environments where children have proven to be more susceptible to acquiring intestinal parasites. Thus, the purpose of this study was to verify the prevalence of intestinal parasites in children who attended the two daycare centers maintained by the local government of Uberlândia, State of Minas Gerais, Brazil. METHODS Fecal samples were collected from 133 children (73 children at the Public Preschool for Early Childhood Education, PPECE A, and 60 at the PPECE B) following identification according to sex and age and agreement to participate by parents or guardians who signed the free, informed consent form. The samples were examined by the Lutz method. RESULTS Coproparasitological tests performed on 133 children showed that 29.3% of them were parasitized for enteroparasites or commensals, 6.7% of the children presented polyparasitism. Among the protozoa, Giardia lamblia were the most prevalent and Hymenolepis nana were the most frequent among the helminths. CONCLUSIONS Thus, analysis of the results showed that intestinal parasites still represent a public health problem, especially among children and in areas where the socioeconomic and educational conditions are less favorable.

Specific IgG and IgA to larvae, parthenogenetic females, and eggs of Strongyloides venezuelensis in the immunodiagnosis of human strongyloidiasis.

The aim of this study was to detect levels of IgG and IgA by enzyme-linked immunosorbent assay (ELISA) using alkaline extracts of larvae, adult female worms, and eggs of Strongyloides venezuelensis as antigen. One hundred twenty serum samples divided into 3 groups were analysed: group I (40 strongyloidiasis patients), group II (40 patients with other parasitic infections), and group III (40 healthy subjects). Statistical variations were analyzed using analysis of variance. There was a significant statistical difference (P < 0.001) in the detection of antibodies in group I between larvae and female antigens and between larvae and egg antigens, with higher positivity using larvae antigen. The larvae antigen showed the highest values for sensitivity, specificity, and diagnostic efficiency in ELISA. This study is the first that examines the use of adult female worm and egg antigens to detect antibodies for human strongyloidiasis diagnosis compared with the larval extract. By comparing all 3 extracts, larval antigens demonstrated better diagnostic parameters.

A glance at Listeria and Salmonella cell invasion: Different strategies to promote host actin polymerization

The facultative intracellular bacterial pathogens Listeria monocytogenes and Salmonella enterica have evolved multiple strategies to invade a large panel of mammalian cells. These pathogens use the host cell actin system for invasion and became a paradigm for the study of host-pathogen interactions and bacterial adaptation to mammalian hosts. The key signaling component that these pathogens use to orchestrate actin remodeling is the Arp2/3 complex, which is related to polymerization of actin filaments. These bacterial pathogens are able to trigger distinct invasion mechanisms. On the one hand, L. monocytogenes invade a host cell in a way dependent on the specific interactions between bacterial and host cell proteins, which in turn activate the host cell actin polymerizing machinery that culminates with bacterial internalization. Also, Listeria escapes from the newly formed parasitophorous vacuole and moves among adjacent cells by triggering actin polymerization. On the other hand, Salmonella invades a host cell by delivering into the cytoplasm virulence factors which directly interact with host regulators of actin polymerization which leads to bacterial uptake. Moreover, Salmonella avoids vacuole lyses and modulates the early and late endosomal markers presented in the vacuole membrane. This mini-review focuses on the different pathways that L. monocytogenes and S. enterica activate to modulate the actin cytoskeleton in order to invade, to form the parasitophorous vacuole, and to migrate inside host cells.

Major histocompatibility complex (MHC) class II but not MHC class I molecules are required for efficient control of Strongyloides venezuelensis infection in mice

Strongyloides stercoralis is an intestinal nematode capable of chronic, persistent infection and hyperinfection of the host; this can lead to dissemination, mainly in immunosuppressive states, in which the infection can become severe and result in the death of the host. In this study, we investigated the immune response against Strongyloides venezuelensis infection in major histocompatibility complex (MHC) class I or class II deficient mice. We found that MHC II−/− animals were more susceptible to S. venezuelensis infection as a result of the presence of an elevated number of eggs in the faeces and a delay in the elimination of adult worms compared with wild‐type (WT) and MHC I−/− mice. Histopathological analysis revealed that MHC II−/− mice had a mild inflammatory infiltration in the small intestine with a reduction in tissue eosinophilia. These mice also presented a significantly lower frequency of eosinophils and mononuclear cells in the blood, together with reduced T helper type 2 (Th2) cytokines in small intestine homogenates and sera compared with WT and MHC I−/− animals. Additionally, levels of parasite‐specific immunoglobulin M (IgM), IgA, IgE, total IgG and IgG1 were also significantly reduced in the sera of MHC II−/− infected mice, while a non‐significant increase in the level of IgG2a was found in comparison to WT or MHC I−/− infected mice. Together, these data demonstrate that expression of MHC class II but not class I molecules is required to induce a predominantly Th2 response and to achieve efficient control of S. venezuelensis infection in mice.

A new faecal antigen detection system for Strongyloides venezuelensis diagnosis in immunosuppressed rats.

This study was performed with the objective of developing innovative procedures for the diagnosis of strongyloidiasis. Enzyme-linked immunosorbent assay (ELISA) was conducted to detect coproantigen in the faecal samples of normal and of immunosuppressed rats using an anti-L3 polyclonal antibody produced in rabbits. Analysis revealed the kinetics of egg shedding in the non-immunosuppressed and immunosuppressed rats infected with S. venezuelensis. Further analysis verified the ability of the immune serum to detect L3 antigens in faecal samples from infected animals. The number of eggs recovered in the faeces at 8 days p.i was significantly higher for both groups. Immunosuppressed animals eliminated increased quantities of eggs. The immune serum was able to detect 0.39 microg/ml of L3 antigens. The antigen recognition in the immunosuppressed group was anticipated on the 8th day p.i. In conclusion, these results may represent a first step in the development of a rapid coproantigen detection kit for strongyloidiasis.

Increased susceptibility to Strongyloides venezuelensis infection is related to the parasite load and absence of major histocompatibility complex (MHC) class II molecules.

In human and murine models strongyloidiasis induce a Th2 type response. In the current study we investigated the role of different loads of Strongyloides venezuelensis in the immune response raised against the parasite and the participation of the major histocompatibility complex (MHC) class II molecule in the disease outcome in face of the different parasite burden. The C57BL/6 wild type (WT) and MHC II(-/-) mice were individually inoculated by subcutaneous injection with 500 or 3000 S. venezuelensis L3. The MHC II(-/-) mice infected with 3000L3 were more susceptible to S. venezuelensis infection when compared with WT groups, in which the parasite was completely eliminated. The production of Th2 cytokines and specific IgG1 or IgE antibodies against parasite were significantly lowered in MHC II(-/-) infected mice with different larvae inoculums. The infection of MHC II(-/-) mice with S. venezuelensis induced slight inflammatory alterations in the small intestine, and these lesions were lower when compared with WT mice, irrespective of the parasite load utilized to infect animals. Finally, we concluded that MHC class II molecules are essential in the immune response against S. venezuelensis mainly when infection occurs with high parasite inoculum.

Detection of parasite-specific IgG and IgA in paired serum and saliva samples for diagnosis of human strongyloidiasis in northern Paraná state, Brazil

Human strongyloidiasis is an infection caused by the helminth Strongyloides stercoralis that can be fatal, especially in immunosuppressed patients. The aim of this study is to evaluate parasite-specific IgG and IgA levels using S. venezuelensis third-stage (L3) infective larvae alkaline extract as a heterologous antigen by ELISA in paired serum and saliva samples with improved sensitivity and specificity. Individuals from northern Paraná state, Brazil were divided into three groups: 30 patients copropositive for S. stercoralis (Group I); 30 clinically healthy individuals (Group II); and 30 patients copropositive for other parasites (Group III). The area under ROC curve (AUC), an overall index of diagnostic accuracy, and Kappa index were calculated. Data were analyzed using analysis of variance (ANOVA) followed by a Kruskal-Wallis test. Probability (p) values of <0.05 were regarded as significant. In Group I, IgG was detected in 96.7% serum and in 6.7% saliva samples. IgG was not detected in Group II. In Group III, cross-reactivity was observed for serum IgG in 26.7% and in 6.7% for saliva samples. In Group I, IgA was detected in 76.7% serum and 56.7% saliva samples. In Group II, 3.3% were positive for IgA in serum, whereas IgA was not detected in any saliva samples. Group III showed 6.7% serum and 26.7% saliva-positive samples. The sensitivity values for detection of IgG and IgA in serum samples were 96.7% and 76.7%, respectively. In saliva samples, the sensitivity values for detection of IgG and IgA were 6.7% and 56.7%, respectively. The specificity value was 100% for the detection of IgG in serum and for detection of IgG and IgA in saliva, and 96.7% for detection of IgA in serum samples. The proper choice of immunological diagnosis to supplement parasitological methods is essential to estimate the true prevalence of the parasite, and will permit analysis of population immune response profiles, particularly in northern Paraná state, where there are no previous reports.

Antigen, antibody and immune complex detection in serum samples from rats experimentally infected with Strongyloides venezuelensis

In order to establish an antigen, antibody and immune complex detection by enzyme-linked immunosorbent assay (ELISA) in serum samples, normal or immunocompromised Wistar rats experimentally infected with Strongyloides venezuelensis were used. The microtitre plates were coated with IgG anti-S. venezuelensis for antigen and immune complex detection and with alkaline parasite extract for antibody detection. Analysis revealed at least 12.5 μg/mL of S. venezuelensis specific antigens in serum samples. Assay for antigen detection was not a good approach for evaluating infection in normal or immunocompromised rats. In normal rats IgG specific for S. venezuelensis was preferentially detected during the first 13 days post-infection (p.i.) and immune complex detection was significantly reduced in 21 p.i. day. On the other hand, in immunocompromised rats, IgG and immune complex were detected during the entire kinetic (5, 8, 13 and 21 p.i). These results suggest that immune complex screening seems to be an alternative for early strongyloidiasis diagnosis in immunocompromised individuals.

A novel approach based on antigen, antibody and immune complex detection in bronchoalveolar lavage fluid samples from rats experimentally infected with Strongyloides venezuelensis.

This study was performed in order to develop a novel approach based on antigen, antibody and immune complex detection by enzyme-linked immunosorbent assay (ELISA) in bronchoalveolar lavage fluid (BALF) samples. For that purpose Wistar rats immunosuppressed or not were experimentally infected with Strongyloides venezuelensis. The microtiter plates were coated with alkaline parasite extract for antibody detection and with IgG anti-S. venezuelensis for antigen and immune complex detection. The immune serum was able to detect 1.56 μg/mL of L3 antigens in BALF samples. ELISA sensitivity was 96.6%, 71.6% and 91.6% for antigen, antibody and immune complex, respectively, and the specificity was 100% for all methods. Antigen detection in BALF samples showed to be a good approach for evaluating the kinetics of infection in non immunosuppressed or immunosuppressed rats. IgG was detected in non immunosuppressed rats from day 8 p.i. and in immunosuppressed rats from day 2 p.i. Moreover, immune complex was detected during the entire kinetic for both groups. In conclusion, association of antigen, antibody and immune complex detection in BALF samples seems to be an alternative approach for early strongyloidiasis diagnosis particularly in immunosuppressed individuals.