Early infection and asymptomatic spread of hepatitis A virus in a public child care center in Rio de Janeiro, Brazil: should attending children under two years of age be vaccinated?

A cross-sectional study was conducted in order to identify hepatitis A virus (HAV) serological markers in 418 individuals (mean age, 16.4 years; range, 1 month-80 years) at a public child care center in Rio de Janeiro, Brazil, as well as to analyze risk factors and determine circulating genotypes. Serum samples were tested using an enzyme immunoassay. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect and characterize HAV RNA, and sequencing was performed. Anti-HAV antibodies and IgM anti-HAV antibodies were detected, respectively, in 89.5% (374/418) and 10.5% (44/418) of the individuals tested. Acute HAV infection in children was independently correlated with crawling (p < 0.05). In 56.8% (25/44) of the IgM anti-HAV-positive individuals and in 33.3% (5/15) of the IgM anti-HAV-negative individuals presenting clinical symptoms, HAV RNA was detected. Phylogenetic analysis revealed co-circulation of subgenotypes IA and IB in 93.3% (28/30) of the amplified samples. In present study, we verify that 79% (30/38) of children IgM anti-HAV-positive were asymptomatic. In child care centers, this asymptomatic spread is a more serious problem, promoting the infection of young children, who rarely show signs of infection. Therefore, vaccinating children below the age of two might prevent the asymptomatic spread of hepatitis A.

The effect of early infection with Echinostoma paraensei on the interaction of Schistosoma mansoni with Biomphalaria glabrata and Biomphalaria tenagophila

Infection caused by the trematode Echinostoma paraensei has been shown to interfere in the natural resistance to infection by Schistosoma mansoni. Biomphalaria glabrata is susceptible to infection, while Taim isolate Biomphalaria tenagophila is resistant to infection by S. mansoni. These two snail species were assessed for infection with E. paraensei two days after exposure to S. mansoni miracidia. The number of B. tenagophila and B. glabrata infected with E. paraensei was lower in co-infected group, suggesting an antagonistic relationship. B. glabrata showed an increase in its susceptibility to S. mansoni, whereas B. tenagophila maintained its refractoriness to S. mansoni infection. Weekly comparisons made between the E. paraensei cercariae released from B. tenagophila and B. glabrata mono-infected snails revealed no quantitative differences. In contrast, S. mansoni cercariae released were higher in the B. glabrata co-infected group. Mortality rates were significantly greater in both species pertaining to co-infected group and unexpected mortalities were also observed in B. tenagophila exposed only to S. mansoni miracidia. Our study revealed that the B. tenagophila Taim isolate is susceptible to E. paraensei infection, although infection did not alter its resistance to S. mansoni infection.

Vaccination with Trypanosoma rangeli modulates the profiles of immunoglobulins and IL-6 at local and systemic levels in the early phase of Trypanosoma cruzi experimental infection

In America, there are two species of Trypanosoma that can infect humans: Trypanosoma cruzi, which is responsible for Chagas disease and Trypanosoma rangeli, which is not pathogenic. We have developed a model of vaccination in mice with T. rangeli epimastigotes that protects against T. cruzi infection. The goal of this work was to study the pattern of specific immunoglobulins in the peritoneum (the site of infection) and in the sera of mice immunized with T. rangeli before and after challenge with T. cruzi. Additionally, we studied the effects triggered by antigen-antibodies binding and the levels of key cytokines involved in the humoral response, such as IL-4, IL-5 and IL-6. The immunization triggered the production of antibodies reactive with T. cruzi in peritoneal fluid (PF) and in serum, mainly IgG1 and, to a lesser magnitude, IgG2. Only immunized mice developed specific IgG3 antibodies in their peritoneal cavities. Antibodies were able to bind to the surface of the parasites and agglutinate them. Among the cytokines studied, IL-6 was elevated in PF during early infection, with higher levels in non-immunized-infected mice. The results indicate that T. rangeli vaccination against T. cruzi infection triggers a high production of specific IgG isotypes in PF and sera before infection and modulates the levels of IL-6 in PF in the early periods of infection.

A contribution to the diagnosis of Capillaria hepatica infection by indirect immunofluorescence test

A highly specific pattern of immunofluorescence was noted when sera from Capillaria hepatica-infected rats were tested against the homologous worms and eggs present either in paraffin or cryostat sections from mouse liver. The pattern was represented by a combined apple green fluorescence of the internal contents of worms and eggs, which persisted in serum-dilutions of 1:400 up to 1:1600. Unequivocal fluorescent pattern was observed from 15 days up to 3 months following inoculation of rats with embryonated C. hepatica eggs and such result was confirmed by the ELISA. After the 4th month of infection, the indirect immunofluorescence test turned negative, probably revealing the extinction of parasitism, however the ELISA was contradictory, disclosing high levels of antibodies in this period . The IIF was also negative when control normal rat sera and sera from rats administered by gavage with immature C. hepatica eggs (spurious infection), or for reactions made against Schistosoma mansoni eggs, although a weakly positive pattern occurred with Fasciola hepatica eggs. The indirect immunofluorescence test may be recommended for use with human sera to detect early C. hepatica infection in special clinical instances and in epidemiological surveys, since it is a simple, inexpensive, and reliable test, presenting excellent sensitivity and specificity. Although the diagnosis is positive only during early infection, this is the period when the symptoms are usually more severe and the need for differential diagnosis is greater.

Analysis of the expression of toll-like receptors 2 and 4 and cytokine production during experimental Leishmania chagasi infection

Toll-like receptors (TLRs) recognise pathogen-derived molecules and influence immunity to control parasite infections. This study aimed to evaluate the mRNA expression of TLRs 2 and 4, the expression and production of the cytokines interleukin (IL)-12, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, IL-17, IL-10 and transforming growth factor (TGF)-β and the production of nitric oxide (NO) in the spleen of mice infected with Leishmania chagasi. It also aimed to evaluate any correlations between mRNA expression TLR2 and 4 and cytokines and NO production. Infection resulted in increased TLR2-4, IL-17, TNF-α and TGF-β mRNA expression during early infection, with decreased expression during late infection correlating with parasite load. IFN-γ and IL-12 mRNA expression decreased at the peak of parasitism. IL-10 mRNA expression increased throughout the entire time period analysed. Although TGF-β, TNF-α and IL-17 were highly produced during the initial phase of infection, IFN-γ and IL-12 exhibited high production during the final phase of infection. IL-10 and NO showed increased production throughout the evaluated time period. In the acute phase of infection, there was a positive correlation between TLR2-4, TNF-α, IL-17, NO, IL-10 and TGF-β expression and parasite load. During the chronic phase of infection, there was a positive correlation between TLR2-4, TNF-α, IL-17 and TGF-β expression and parasite load. Our data suggest that infection by L. chagasi resulted in modulation of TLRs 2 and 4 and cytokines.

Rapid Characterization of S. mansoni Expression Library Clones of Potential Interest

A S. mansoni adult worm cDNA expression library was screened with sera from baboons in a early phase after infection. The clones that were positive with the early infection sera were examined for reactivity with pre-infection sera and heterologous infection sera. In order to discriminate a positive antibody reaction from the reactivity due to residual anti-E. coli antibodies, an unrelated cDNA clone was plated with the positive clone. The unrelated clone provided the negative background and the contrast necessary to discern a positive antibody reaction. In this way, we were able to eliminate selected clones that were positive with the pre-infection sera or heterologous infection sera. This characterization of the expression library clones enabled us to quickly target only clones with the desired pattern of antibody reactivity for sequencing, subcloning, and expressing

Kinetics of Trypanosoma cruzi destruction in the mouse spleen

Massive destruction of parasitized splenic macrophages was histologically observed at the height of a virulent infection caused by Trypanosoma cruzi (Y strain) in the mouse. This was coincident with a sudden drop in parasitemic curve. Most of the animals died at this point, probably due to the liberation of toxic products, such as TNF, following the massive destruction of parasitized cells. However, parasitized-cell destruction indicated the transition from susceptibility to resistance. Although it has been extensively studied in vitro, this study contributes with the morphological counterpart observed in vivo by optical and electron microscopy. When infected animals were specifically treated during early infection transition to chronic phase was immediately observed without splenic parasitism. Animals that apparently recovered from massive cell-destruction in the spleen showed evidences of a rapid restoration of splenic architecture.

Integrate Study of a Bolivian Population Infected by Trypanosoma cruzi, the Agent of Chagas Disease

A cross section of a human population (501 individuals) selected at random, and living in a Bolivian community, highly endemic for Chagas disease, was investigated combining together clinical, parasitological and molecular approaches. Conventional serology and polymerase chain reaction (PCR) indicated an active transmission of the infection, a high seroprevalence (43.3%) ranging from around 12% in < 5 years to 94.7% in > 45 years, and a high sensitivity (83.8%) and specificity of PCR. Abnormal ECG tracing was predominant in chagasic patients and was already present among individuals younger than 13 years. SAPA (shed acute phase antigen) recombinant protein and the synthetic peptide R-13 were used as antigens in ELISA tests. The reactivity of SAPA was strongly associated to Trypanosoma cruzi infection and independent of the age of the patients but was not suitable neither for universal serodiagnosis nor for discrimination of specific phases of Chagas infection. Anti-R-13 response was observed in 27.5% only in chagasic patients. Moreover, anti-R13 reactivity was associated with early infection and not to cardiac pathology. This result questioned previous studies, which considered the anti-R-13 response as a marker of chronic Chagas heart disease. The major clonets 20 and 39 (belonging to Trypanosoma cruzi I and T. cruzi II respectively) which circulate in equal proportions in vectors of the studied area, were identified in patients' blood by PCR. Clonet 39 was selected over clonet 20 in the circulation whatever the age of the patient. The only factor related to strain detected in patients' blood, was the anti-R-13 reactivity: 37% of the patients infected by clonet 39 (94 cases) had anti-R13 antibodies contrasting with only 6% of the patients without clonet 39 (16 cases).

Capillaria hepatica in rats: focal parasitic hepatic lesions and septal fibrosis run independent courses

Capillaria hepatica causes two main lesions in the liver of rats: multifocal chronic inflammation, directly related to the presence of disintegrating parasites and their eggs, and a process of systematized septal fibrosis. The comparative behavior of these two lesions was investigated in rats experimentally infected with 600 embryonated eggs, following either corticosteroid treatment or specific antigenic stimulation, in an attempt to understand the relationship between these two lesions, and the pathogenesis of septal fibrosis. The two treatments differently modified the morphological aspects of the focal parasitic-related lesions, but did not interfere with the presentation of diffuse septal fibrosis, although a mild decrease in the degree of fibrosis occurred in corticoid-treated animals. These findings indicate that although the two lesions are C. hepatica induced, they are under different pathogenetic control, the induction of septal fibrosis being triggered during early infection to follow an independent pathway.

Hepatic stereology of schistosomiasis mansoni infected-mice fed a high-fat diet

High-fat diets induce weight gain and fatty liver in wild-type mice. Schistosomiasis mansoni infection also promotes hepatic injury. This study was designed to quantify hepatic alterations in schistosomiasis mansoni-infected mice fed a high fat-rich chow compared to mice fed a standard rodent chow, using stereology. Female SW mice fed each either high-fat diet (29% lipids) or standard chow (12% lipids) over 8 months, and then were infected with Schistosoma mansoni cercariae. Four experimental groups were studied: infected mice fed a high-fat diet (IHFC) or standard chow (ISC), uninfected mice fed a high-fat diet (HFC) or standard chow (SC). Mice were sacrificed during early infection (9 weeks from exposure). The following hepatic biometry and the stereology parameters were determined: volume density (hepatocytes [h], sinusoids [s], steatosis [st] and hepatic fibrosis [hf]); numerical density (hepatocyte nuclei - Nv[h]); absolute number of total hepatocyte N[h], normal hepatocyte N[nh], and binucleated hepatocyte N[bh], percentage of normal hepatocyte P[nh] and binucleated hepatocyte P[bh]. IHFC and HFC groups exhibited TC, HDL-C, LDL-C, and body mass significantly greater (p < 0.05) than control group. No significant differences were found regards liver volume (p = 0.07). Significant differences were observed regards P[nh] (p = 0.0045), P[bh] (p = 0.0045), Nv[h] (p = 0.0006), N[h] (p = 0.0125), N[bh] (p = 0.0164) and N[nh] (p = 0.0078). IHFC mice group presented 29% of binucleated hepatocytes compared to HFC group (19%), ISC group (17%) and SC (6%). Volume density was significantly different between groups: Vv[h] (p = 0.0052), Vv[s] (p = 0.0025), Vv[st] (p = 0.0004), and Vv[hf] (p = 0.0007). In conclusion, schistosomiasis mansoni infection with concurrent high-fat diet promotes intensive quantitative changes in hepatic structure, contributing to an increasing on hepatic regeneration.

EXPERIMENTAL INFECTION WITH Toxocara cati IN PIGS: MIGRATORY PATTERN AND PATHOLOGICAL RESPONSE IN EARLY PHASE

Experimental inoculations of approximately 100,000 infective Toxocara cati larval eggs were done in twelve pigs. The T. cati eggs used for inoculation were collected from cat's feces. Another group of three pigs served as an uninfected control. Groups of infected pigs were euthanized at seven, 14, 21, and 28 days post-inoculation (dpi). Tissue samples were taken for digestion and histopathology changes in early phase. The number of larvae recovered from the lungs peaked at seven and 14 dpi and were also present at 21, and 28 dpi. Larvae of T. cati were present in the lymph nodes of the small and large intestine at seven, 14, and 28 dpi and at seven, 14, 21, and 28 dpi respectively. In other studied tissues, no larvae or less than one larva per gram was detected. The pathological response observed in the liver and lungs at seven and 14 dpi, showed white spots on the liver surface and areas of consolidation were observed in the lungs. The lungs showed an inflammatory reaction with larvae in center at 28 dpi. In the liver we observed periportal and perilobular hepatitis. The lymph nodes of the intestines displayed eosinophil lymphadenitis with reactive centers containing parasitic forms in some of them. The granulomatous reaction was not observed in any tissues. The role of the other examined tissues had less significance. The relevance of this parasite as an etiological agent that leads to disease in paratenic hosts is evident.

Interferon-gamma is required for the late but not early control of Leishmania amazonensis infection in C57Bl/6 mice

The critical role of interferon-gamma (IFN-g) in the resistance of C57Bl/6 mice to Leishmania major is widely established but its role in the relative resistance of these animals to L. amazonensis infection is still not clear. In this work we use C57Bl/6 mice congenitally deficient in the IFN-g gene (IFN-g KO) to address this issue. We found that IFN-g KO mice were as resistant as their wild-type (WT) counterparts at least during the first two months of infection. Afterwards, whereas WT mice maintained lesion growth under control, IFN-g KO mice developed devastating lesions. At day 97 of infection, their lesions were 9-fold larger than WT controls, concomitant with an increased parasite burden. At this stage, lesion-draining cells from IFN-g KO mice had impaired capacity to produce interleukin-12 (IL-12) and tumour necrosis factor-a in response to parasite antigens whereas IL-4 was slightly increased in comparison to infected WT mice. Together, these results show that IFN-g is not critical for the initial control of L. amazonensis infection in C57Bl/6 mice, but is essencial for the developmente of a protective Th1 type immune response in the later stages.

Schistogram changes after administration of antischistosomal drugs in mice at the early phase of Schistosoma mansoni infection

Mice infected with Schistosoma mansoni were treated with oxamniquine, praziquantel, artesunate at the pre-patent phase, aiming at observing schistogram alterations. Half of the animals were perfused five days post-treatment for counting and classification of immature worms, based on pre-established morphological criteria (schistogram); the remaining animals were evaluated 42 or 100 days after infection and perfusion of the portal-system was performed for collection and counting of adult worms and oogram. It was observed that oxamniquine and artesunate treatment administered at the pre-postural phase causes significant reduction in the number of immature and adult worms. However, there was little reduction with praziquantel when used at the dose of 400 mg/kg for treatments administered 14, 15, 21 or 23 days post-infection. Artesunate was responsible for significant alterations in development of young worms, as well as for a higher number of worms presenting intestinal damages. Immature adult worms were detected in mice treated with artesunate or oxamniquine at the pre-patent phase of infection and recovered by perfusion 100 days after infection. Schistogram proved to be a very useful tool for experimental evaluation of the activity of antischistosomal drugs and a good model to identify the most sensitive stages to drugs.

Induction of cell-mediated immunity during early stages of infection with intracellular protozoa

Toxoplasma gondii and Trypanosoma cruzi are intracellular parasites which, as part of their life cycle, induce a potent cell-mediated immunity (CMI) maintained by Th1 lymphocytes and IFN-g. In both cases, induction of a strong CMI is thought to protect the host against rapid parasite multiplication and consequent pathology and lethality during the acute phase of infection. However, the parasitic infection is not eliminated by the immune system and the vertebrate host serves as a parasite reservoir. In contrast, Leishmania sp, which is a slow growing parasite, appears to evade induction of CMI during early stages of infection as a strategy for surviving in a hostile environment (i.e., inside the macrophages which are their obligatory niche in the vertebrate host). Recent reports show that the initiation of IL-12 synthesis by macrophages during these parasitic infections is a key event in regulating CMI and disease outcome. The studies reviewed here indicate that activation/inhibition of distinct signaling pathways and certain macrophage functions by intracellular protozoa are important events in inducing/modulating the immune response of their vertebrate hosts, allowing parasite and host survival and therefore maintaining parasite life cycles.

The role of natural killer cells in the early period of infection in murine cutaneous leishmaniasis

In order to study the role of natural killer (NK) cells during the early period of Leishmania infection, BALB/c mice were selectively and permanently depleted of NK cells by injection with 90Sr and subsequently infected with Leishmania (Leishmania) amazonensis (HSJD-1 strain). 90Sr is known to selectively deplete NK cells, leaving an intact T- and B-cell compartment and preserving the ability to produce both interferon alpha and IL-2. This method of depletion has advantages when compared with depletion using anti-NK cell monoclonal antibodies because the effect is permanent and neither activates complement nor provokes massive cell death. In the present study, after one month of treatment with 90Sr, the depletion of NK cells was shown by a more than ten-fold reduction in the cytotoxic activity of these cells: 2 x 106 spleen cells from NK-depleted animals were required to reach the same specific lysis of target cells effected by 0.15 x 106 spleen cells from normal control animals. The histopathology of the skin lesion at 7 days after Leishmania infection showed more parasites in the NK cell-depleted group. This observation further strengthens a direct role of NK cells during the early period of Leishmania infection.