Evaluation of enzyme-linked immunosorbent assay using crude Leishmania and recombinant antigens as a diagnostic marker for canine visceral leishmaniasis

The performances of ELISA assays with different antigen preparations, such as Leishmania amazonensis or L. chagasi lysates and the recombinant antigens rK-39 and rK-26, were compared using sera or eluates from dried blood collected on filter paper to detect anti-Leishmania antibodies in dogs from a visceral leishmaniasis-endemic area in Brazil. Of 115 IFAT-reactive dogs at 1:40 titre, 106 (92.2%) were positive in parasitological exams (skin and/or spleen). These animals were compared to healthy animals (n = 25), negative for IFAT at a titre of 1:40 and parasitological exams. The sensitivities of crude and recombinant antigens were similar and remarkably high for both sera and eluates (97-100%). Specificity was higher than 96% for sera and eluates for different antigens, except for L. chagasi antigen using eluates (88%). Concordance values among the tests were higher either for sera or eluates (J = 0.95-1.00). High concordances were observed between sera and eluates tested with different antigens (kappa = 0.93-0.97). Crude and recombinant antigens identified different clinical phases of canine leishmaniasis. These results show that eluates could be used in canine surveys to identify L. chagasi infection. Recombinant antigens added little when compared to crude antigen in identifying positive dogs. Cross-reactivity with other diseases whose distribution often overlaps VL-endemic areas is a limitation of crude antigen use however.

Participation of cytokines in the necrotic-inflammatory lesions in the heart and skeletal muscles of Calomys callosus infected with Trypanosoma cruzi

Calomys callosus, a sylvatic reservoir of Trypanosoma cruzi, when infected with the Colombian strain (Biodeme Type III, T. cruzi I ) develops necrotic-inflammatory lesions and intense early fibrogenesis in the heart and skeletal muscles, that spontaneously regress. Participation of pro-inflammatory and pro-fibrogenic cytokines, such as tumor necrosis factor-alpha (TNF-alpha), gamma interferon (IFN-gamma) , and tumor growth factor-beta (TGF-beta), in the pathogenesis of the lesions is herein studied. Eighty C. callosus weighing 20 to 30 g were used. Seventy of them were inoculated with the Colombian strain (10(5) blood forms) and 10 were maintained as intact non-infected controls. After infection, C. callosus were sacrificed at different time-points from 15 to 70 days. The heart and skeletal muscle were processed for histopathology and cryopreserved for immunohistochemistry. Early necrotic lesions of parasitized skeletal muscle and myocardium with intense inflammatory lesions were present. Search for the in situ presence of TNF-alpha and IFN-gamma, was performed using rat-IgG anti-mouse antibodies against these cytokines. For the in situ search of TGF-beta, rabbit IgG anti-mouse antibodies were used. Immunolabeling of the cytokines in tissues of infected C. callosus was successful. The cytokines TNF-alpha, IFN-gamma , and TGF-beta were detected in the cytoplasm of macrophages and in the necrotic material from 15 to 45 days post-infection, decreasing their intensity until complete disappearance by the 65th day, which correlated with subsiding histopathological lesions. These findings suggest the participation of these cytokines in the control of parasite multiplication, in the development of an early fibrogenesis and in the regression of fibrotic-inflammatory lesions observed in C. callosus.

Related factors to human toxocariasis in a rural community of Argentina

The objective of this study was to evaluate the relationship between toxocariasis frequency and demographic, environmental, sanitary variables, eosinophylia, and other intestinal parasites in a rural population of Argentina. Serological examination of 100 individuals was carried out by using ELISA technique for the detection of anti-toxocara antibodies. Eosinophiles in peripheral blood, presence of intestinal parasites, and demographic, environmental, and socio-cultural data were evaluated. Eighty-one feces samples of dogs belonging to the studied people were analyzed to detect eggs of Toxocara canis. Thirty of them were from 30 dogs and 51 were pools from dog feces. Samples of dirt from around the homes (n: 47) and from public park (n: 4) were taken. To determine the associations, the c² and Fisher tests were used. The seroprevalence was 23%. Eosinophilia in peripheral blood was detected in 86.95% seropositive individuals and in 37.66% seronegative individuals (p < 0.001, OR = 11.03). Of the 23 people with positive serology, 69.56% had at least one intestinal parasite. All individuals with positive serology had dogs in their homes. Among the dog owners there was a significant association between the presence of anti-toxocara antibodies and home flooding. Eggs of T. canis were detected in the feces of 5/81 dogs and three of these dogs belonged to individuals with positive serology. Eggs of Toxocara spp. were found in 41.17% of the dirt samples, eight of which came from the area surrounding the homes of individuals with positive serology (p = 0.032; OR = 4.36). Taking into account all the variables influencing the frequency of toxocariasis in this population, the implementation of Public Health programs specifically focused on anti-parasitic treatment of dogs is recommended.

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.

Protection against hepatitis B by the Butang® recombinant vaccine in newborn children in South Brazil

The prevention of hepatitis B by vaccination is one of the most efficient tools to avoid the transmission of the virus. This study evaluated the immunogenicity of the national vaccine Butang® in children born in Campo Mourão City, state of Paraná, Brazil, aged 7 to 12 months, by determining the anti-HBsAg antibodies levels after completion of the National Immunization Program Protocol for hepatitis B. All 70 children evaluated by the MEIA method (immune-enzymatic micro particles) showed seroconversion to the Butang® vaccine. Nine children (12.9%) presented a low response, with anti-HBs titers between 11 and 100 mUI/ml; 39 children (55.7%) showed a good response to the vaccine, with titers between 101 and 1000 mUI/ml; and 22 children (31.4%) showed antibodies titers higher than 1000 mUI/ml. The mean titer of the anti-HBs antibody titers was 1408.1 ± 2870.26 mUI/ml (15.7 to 19560.0 mUI/ml). The levels of antibodies produced by the prematurely-born children were not statistically different from those found in the newborns. Fifty-five children were also evaluated through the ELFA method (ELISA with a final detection in fluorescence), which presented similar results. The results obtained in our study corroborated the effectiveness of the national vaccine Butang® in newborn children of Campo Mourão City, Paraná, even if they were premature.

Seroprevalence of hepatitis B and C virus markers among blood donors in Rio de Janeiro, Brazil, 1998-2005

The prevalence of infection by hepatitis B (HBV) and C (HCV) viruses varies among geographical regions. In order to determine the prevalence of HBV and HCV infection in voluntary blood donors we evaluated the prevalence of HBsAg, anti-HBc, and anti-HCV markers of 128,497 blood donor samples collected from 1998 to 2005 in the state of Rio de Janeiro. These markers were analyzed by immunoenzymatic tests, as determined by the Ministry of Health. Data were obtained from the Sorology Laboratory of the Hemoterapy Service of the Instituto Nacional de Câncer, Rio de Janeiro. Overall prevalence estimates were: 0.27% for HBsAg, 3.68% for anti-HBc, and 0.90% for anti-HCV. There was a significant decrease in the overall prevalence of HBsAg (from 0.36 to 0.14%) and anti-HBc (from 6.12 to 2.05%) in the period encompassed between 1998-2005. Similarly, there was a decline in anti-HCV prevalence rates in Brazilian blood donors, from 1.04% in 1998 to 0.79% in 2004, with an increase of HCV prevalence to 1.09% in 2005. These prevalence estimates were higher than those found in other countries, indicating high rates of infection by HBV and HCV and a persistent risk of HBV and HCV transmission by transfusion.

Plasmodium vivax thrombospondin related adhesion protein: immunogenicity and protective efficacy in rodents and Aotus monkeys

The thrombospondin related adhesion protein (TRAP) is a malaria pre-erythrocytic antigen currently pursued as malaria vaccine candidate to Plasmodium falciparum. In this study, a long synthetic peptide (LSP) representing a P. vivax TRAP fragment involved in hepatocyte invasion was formulated in both Freund and Montanide ISA 720 adjutants and administered by IM and subcutaneous routes to BALB/c mice and Aotus monkeys. We measured specific humoral immune responses in both animal species and performed a sporozoite challenge in Aotus monkeys to assess the protective efficacy of the vaccine. After immunization both mice and Aotus seroconverted as shown by ELISA, and the specific anti-peptide antibodies cross reacted with the parasite in IFAT assays. Only two out of six immunized animals became infected after P. vivax sporozoite challenge as compared with four out of six animals from the control group. These results suggest that this TRAP fragment has protective potential against P. vivax malaria and deserves further studies as vaccine candidate.

Abnormal humoral immune response to influenza vaccination in pediatric type-1 human immunodeficiency virus infected patients receiving highly active antiretroviral therapy

Given that highly active antiretroviral therapy (HAART) has been demonstrated useful to restore immune competence in type-1 human immunodeficiency virus (HIV-1)-infected subjects, we evaluated the specific antibody response to influenza vaccine in a cohort of HIV-1-infected children on HAART so as to analyze the quality of this immune response in patients under antiretroviral therapy. Sixteen HIV-1-infected children and 10 HIV-1 seronegative controls were immunized with a commercially available trivalent inactivated influenza vaccine containing the strains A/H1N1, A/H3N2, and B. Serum hemagglutinin inhibition (HI) antibody titers were determined for the three viral strains at the time of vaccination and 1 month later. Immunization induced a significantly increased humoral response against the three influenza virus strains in controls, and only against A/H3N2 in HIV-1-infected children. The comparison of post-vaccination HI titers between HIV-1+ patients and HIV-1 negative controls showed significantly higher HI titers against the three strains in controls. In addition, post vaccination protective HI titers (defined as equal to or higher than 1:40) against the strains A/H3N2 and B were observed in a lower proportion of HIV-1+ children than in controls, while a similar proportion of individuals from each group achieved protective HI titers against the A/H1N1 strain. The CD4+ T cell count, CD4/CD8 T cells ratio, and serum viral load were not affected by influenza virus vaccination when pre- vs post-vaccination values were compared. These findings suggest that despite the fact that HAART is efficient in controlling HIV-1 replication and in increasing CD4+ T cell count in HIV-1-infected children, restoration of immune competence and response to cognate antigens remain incomplete, indicating that additional therapeutic strategies are required to achieve a full reconstitution of immune functions.

The role of triple infection with hepatitis B virus, hepatitis C virus, and human immunodeficiency virus (HIV) type-1 on CD4+ lymphocyte levels in the highly HIV infected population of North-Central Nigeria

We set out to determine the seroprevalence of hepatitis B and C among human immunodeficiency virus type-1 (HIV-1) infected individuals in North-Central Nigeria to define the influence of these infections on CD4+ lymphocytes cells among our patients as access to antiretroviral therapy improves across the Nigerian nation. The CD4+ values of 180 confirmed HIV-1 infected individuals were enumerated using a superior fluorescence-activated cell sorter system. These patients were tested for the presence of hepatitis B surface antigen and anti-hepatitis C virus (HCV) using third generation enzyme-linked immunosorbent assays. Fifty (27.8%) patients had active hepatitis B virus (HBV) infection while 33 (18.3%) tested positive for anti-HCV antibody. Of these infections, 110 (61.1%), 37 (20.6%), and 20 (11.1%) had HIV only, HBV/HIV-only, and HCV/HIV-only respectively. A HBV/HCV/HIV coinfection prevalence of 7.2% (13 patients) was recorded. Patients coinfected with HIV/HBV/HCV appeared to have lower CD4+ counts (mean = 107 cells/µl; AIDS defining) when compared to HBV/HIV-only (mean = 377 cells/µl), HCV/HIV-only (mean = 373 cells/µl) and patients with mono HIV infection (mean = 478 cells/µl). Coinfection with HBV or HCV is relatively common among HIV-infected patients in Nigeria and should be a big consideration in the initiation and choice of therapy.

Prevalence, genotypes and risk factors associated with hepatitis C virus infection in hemodialysis patients in Campo Grande, MS, Brazil

A survey was conducted among the hemodialysis units of the city of Campo Grande, located in the state of Mato Grosso do Sul in the Mid-west region of Brazil, with the aim of investigating the prevalence, risk factors, and genotypes of hepatitis C virus (HCV) infection. A total of 163 patients were interviewed in five dialysis units. Serum samples were screened for anti-HCV. Positive samples were tested for HCV RNA and genotyped. The prevalence of anti-HCV was 11% (95% CI: 6.8-17.1). A history of transfusion with blood that was not screened for anti-HCV and length of time on hemodialysis were associated with HCV infection. HCV RNA was detected in 12 samples: ten were of genotype 1, subtypes 1a (75%) and 1b (8.3%), and two were of genotype 3, subtype 3a (16.7%).

Improving the production of the denatured recombinant N-terminal domain of rhoptry-associated protein 2 from a Plasmodium falciparum target in the pathology of anemia in falciparum malaria

Rhoptry-associated protein 2 (RAP2) is known to be discharged from rhoptry onto the membrane surface of infected and uninfected erythrocytes (UEs) ex vivo and in vitro and this information provides new insights into the understanding of the pathology of severe anemia in falciparum malaria. In this study, a hexahistidine-tagged recombinant protein corresponding to residues 5-190 of the N-terminal of Plasmodium falciparum RAP2 (rN-RAP2) was produced using a new method of solubilization and purification. Expression was induced with D-lactose, a less expensive alternative inducer to the more common isopropyl-²-D-thio-galactopyranosidase. The recombinant protein was purified using two types of commercially-available affinity columns, iminodiacetic and nitrilotriacetic. rN-RAP2 had immunogenic potential, since it induced high titers of anti-RAP2 antibodies in mice. These antibodies recognized full-length RAP2 prepared from Triton X-100 extracts from two strains of P. falciparum. In fact, the antibody recognized a 29-kDa product of RAP2 cleavage as well as 82 and 70-kDa products of RAP1 cleavage. These results indicate that the two antigens share sequence epitopes. Our expressed protein fragment was shown to contain a functional epitope that is also present in rhoptry-derived ring surface protein 2 which attaches to the surface of both infected and UEs and erythroid precursor cells in the bone marrow of malaria patients. Serum from malaria patients who developed anemia during infection recognized rN-RAP2, suggesting that this protein fragment may be important for epidemiological studies investigating whether immune responses to RAP2 exacerbate hemolysis in falciparum malaria patients.

Effectiveness and safety of isoniazid chemoprophylaxis for HIV-1 infected patients from Rio de Janeiro

The clinical and epidemiological characteristics, adverse events, treatment adherence and effectiveness of isoniazid chemoprophylaxis were analyzed in a cohort of 138 tuberculosis/HIV-coinfected patients. An open, non-randomized, pragmatic prophylactic trial was conducted on adult patients with a normal chest X-ray and positive tuberculin skin test (> 5 mm) who received isoniazid chemoprophylaxis (300 mg/day) for six months. The mean of follow up was 2.8 years (SD 1.3). Adherence to chemoprophylaxis was 87.7% (121/138). Only one patient presented tuberculosis after the end of chemoprophylaxis, corresponding to 0.3 cases per 100 persons per year. The relative risk of some adverse effects was 4.6 times higher (95% CI: 1.9-11.5) in patients with positive anti-HCV serology (4/9, 44.4%) compared to those with negative serology (12/129, 9.6%) (p = 0.002). This study provides evidence regarding the effectiveness and safety of a short and self-administered isoniazid regimen. We recommend the implementation of this routine by health service practitioners.

Toxoplasma gondii: further studies on the subpellicular network

The association of the pellicle with cytoskeletal elements in Toxoplasma gondii allows this parasite to maintain its mechanical integrity and makes possible its gliding motility and cell invasion. The inner membrane complex (IMC) resembles the flattened membrane sacs observed in free-living protozoa and these sacs have been found to associate with cytoskeletal proteins such as articulins. We used immunofluorescence microscopy to characterise the presence and distribution of plateins, a sub-family of articulins, in T. gondii tachyzoites. A dispersed labelling of the whole protozoan body was observed. Electron microscopy of detergent-extracted cells revealed the presence of a network of 10 nm filaments distributed throughout the parasite. These filaments were labelled with anti-platein antibodies. Screening the sequenced T. gondii genome, we obtained the sequence of an IMC predicted protein with 25% identity and 42% similarity to the platein isoform alpha 1 present in Euplotes aediculatus, but with 42% identity and 55% similarity to that found in Euglena gracilis, suggesting strong resemblance to articulins.

Prevalence and genotypes of hepatitis C virus among injecting drug users from Salvador-BA, Brazil

Hepatitis C virus (HCV) is the major infectious disease agent among injecting drug users (IDUs), with seroprevalence ranging from 50-90%. In this paper, serological and virological parameters were investigated among 194 IDUs, 94 ex-IDUs and 95 non-IDUs that were sampled by the "snowball" technique in three localities renowned for both intense drug use and trafficking activities in Salvador, Brazil. The majority of the participants were male, but sex and mean age differed significantly between IDUs/ex-IDUs and non-IDUs (p < 0.05). Anti-HCV screening revealed that 35.6%, 29.8% and 5.3% of samples from IDUs, ex-IDUs and non-IDUs, respectively, were seropositive. HCV-RNA detection confirmed that the prevalence of infection was 29.4%, 21.3% and 5.3% for IDUs, ex-IDUs and non-IDUs, respectively. Genotyping analysis among IDUs/ex-IDUs determined that 76.9% were infected with genotype 1, 18.5% with genotype 3 and 4.6% with a mixed genotype; this result differed significantly from non-IDUs, where genotype 3 was the most frequent (60%), followed by genotype 1 (20%) and a mixed genotype (20%). We report a significantly higher prevalence of HCV infection in IDUs/ex-IDUs compared to the control group (p < 0.001). Although the sample size of our study was small, the differences in HCV genotype distribution reported herein for IDUs/ex-IDUs and non-IDUs warrant further investigation.

Aseptic meningitis caused by Leptospira spp diagnosed by polymerase chain reaction

Leptospirosis is a zoonotic disease caused by the pathogenic Leptospira spp. The clinical presentations are diverse, ranging from undifferentiated fever to fulminant disease including meningeal forms. The neurological leptospirosis forms are usually neglected. The aim of this study was to investigate leptospirosis as the cause of aseptic meningitis using different diagnostic techniques including the polymerase chain reaction (PCR). Thirty-nine cerebrospinal fluid (CSF) samples from patients presenting with meningeal abnormalities, predominance of lymphocytes and negative results by traditional microbiological tests were processed by leptospiral culture, anti-leptospiral antibody response and PCR. Leptospira spp DNA was detected in 23 (58.97%) of the CSF samples. Anti-leptospiral antibodies were found in 13 (33.33%) CSF samples. Twelve CSF samples were positive by PCR assay and negative by microscopic agglutination test (MAT) assay. Two CSF samples were positive by MAT and negative by PCR. The positive and negative agreement between both tests was 11 and 14, respectively. CSF samples from six cases of unknown diagnosis were positive by PCR assay. Eight cases showed positive results using PCR and MAT. Leptospirosis could be detected by PCR assay from the 3rd-26th day after illness onset. The sensitivity of the PCR was assessed with confirmed cases of leptospirosis (by MAT) and found to be 89.5%. All CSFs were negative by culture. PCR was found to be a powerful tool for diagnosing meningitis cases of leptospirosis. We recommend that it may be used as a supplementary diagnostic tool, especially in the early stages of the disease, when other diagnostic techniques such as serology are not sensitive.