STUDY OF THE PREVALENCE OF Capillaria hepatica IN HUMANS AND RODENTS IN AN URBAN AREA OF THE CITY OF PORTO VELHO, RONDÔNIA, BRAZIL

Introduction: Hepatic capillariosis, caused by Capillaria hepatica (Calodium hepaticum) (Bancroft, 1893), Travassos, 1915 (Nematoda, Trichinelloidea, Capillariidae), is a common zoonosis in rodents but is rare in humans. Seventy-two cases in humans have been reported worldwide since the first case was described by MACARTHUR in 192417,27. This study aimed to determine the prevalence of Capillaria hepatica in humans and rodents in an urban area of Porto Velho, the capital of Rondônia, in Brazil. Methods: After conducting a census of the area, 490 residents were randomly selected, and, after signing a term of consent, provided blood samples that were screened for anti-Capillaria hepatica antibodies. Simultaneously, rats were captured to assess the prevalence of this parasite in rodents by histopathological examination in liver sections. Results: A prevalence of 1.8% was found among residents who had specific antibodies at a dilution of 1:150, indicating exposure to parasite eggs; 0.8% of the subjects also had positive titers at a dilution of 1:400, indicating true infection. The prevalence in rats was 2%. Conclusions: The prevalence of infection with this parasite among humans and rats was low. While the prevalence encountered among humans was within the limits reported in the literature, the prevalence among rodents was much lower.

MEMBRANE FRACTIONS FROM Strongyloides venezuelensis IN THE IMMUNODIAGNOSIS OF HUMAN STRONGYLOIDIASIS

Strongyloides venezuelensis is a parasitic nematode of rodents frequently used to obtain heterologous antigens for the immunological diagnosis of human strongyloidiasis. The aim of this study was to evaluate membrane fractions from S. venezuelensis for human strongyloidiasis immunodiagnosis. Soluble and membrane fractions were obtained in phosphate saline (SS and SM) and Tris-HCl (TS and TM) from filariform larvae of S. venezuelensis. Ninety-two serum samples (n = 92) were obtained from 20 strongyloidiasis patients (Group I), 32 from patients with other parasitic diseases (Group II), and 40 from healthy individuals (Group III), and were analyzed by enzyme-linked immunosorbent assay (ELISA). Soluble fractions (SS and TS) showed 90.0% sensitivity and 88.9% specificity, whereas the membrane fractions (SM and TM) showed 95.0% sensitivity and 94.4% specificity. The present results suggest the possible use of membrane fractions of S. venezuelensis as an alternative antigen for human strongyloidiasis immunodiagnosis.

ROTAVIRUS GENOTYPES CIRCULATING IN BRAZIL, 2007-2012: IMPLICATIONS FOR THE VACCINE PROGRAM

SUMMARY Regarding public health in Brazil, a new scenario emerged with the establishment of universal rotavirus (RV) vaccination programs. Herein, the data from the five years of surveillance (2007-2012) of G- and P-type RV strains isolated from individuals with acute gastroenteritis in Brazil are reported. A total of 6,196 fecal specimens were investigated by ELISA and RT-PCR. RVs were detected in 19.1% (1,181/6,196). The peak of RV incidence moved from June-August to September. RV was detected less frequently (19.5%) among children ≤ 5 years than in older children and adolescents (6-18 years) (40.6%). Genotype distribution showed a different profile for each year: G2P[4] strains were most prevalent during 2007-2010, G9P[8] in 2011, and G12P[8] in 2012. Mixed infections (G1+G2P[4], G2+G3P[4]+P[8], G2+G12P[8]), unusual combinations (G1P[4], G2P[6]), and rare strains (G3P[3]) were also identified throughout the study period. Widespread vaccination may alter the RV seasonal pattern. The finding of RV disease affecting older children and adolescents after vaccine implementation has been reported worldwide. G2P[4] emergence most likely follows a global trend seemingly unrelated to vaccination, and G12, apparently, is emerging in the Brazilian population. The rapidly changing RV genotype patterns detected during this study illustrate a dynamic population of co-circulating wildtype RVs in Brazil.

IDENTIFICATION OF SANDFLIES (Diptera: Psychodidae: Phlebotominae) BLOOD MEALS IN AN ENDEMIC LEISHMANIASIS AREA IN BRAZIL

SUMMARY The aim of this study was to identify blood meals of female sandflies captured in the municipality of Governador Valadares, an endemic area of visceral and cutaneous leishmaniasis, in the State of Minas Gerais, Brazil. From May 2011 to January 2012, captures were performed using HP light traps in four districts. There were 2,614 specimens (2,090 males and 524 females) captured; 97 engorged females were identified belonging to the species Lutzomyia longipalpis (82.1%) and Lutzomyia cortelezzii (17.9%). Considering simple and mixed feeding, the enzyme-linked immunosorbent assay revealed a predominance of chicken blood (43.6%) in Lutzomyia longipalpis, showing the important role that chickens exert around the residential areas of Governador Valadares. This finding increases the chances of sandflies contact with other vertebrates and consequently the risk of leishmaniasis transmission.

CANINE VISCERAL LEISHMANIASIS CASE INVESTIGATION IN THE JACARE REGION OF NITEROI, RIO DE JANEIRO, BRAZIL

SUMMARY American visceral leishmaniasis is a vector-borne zoonosis in expansion in Brazil. Dogs are the main urban reservoir. Departing from a case of canine visceral leishmaniasis (CVL) in Jacaré, Niterói, Rio de Janeiro State, an epidemiological canine and entomological study was performed to assess the extension of the disease at the location. Sample was collected around the case and the dogs identified by serological tests (rapid double platform immunochromatographic exams, immunoenzymatic assay/ELISA, indirect immunofluorescence/IFAT). The parasitological diagnosis was performed in animals positive in at least one of these tests. The entomological study was carried out by using light traps and manual collection. The associations between canine variables and outcome (ELISA and IFAT reagents) were assessed by the chi-square test and adjusted by multivariate logistic regression for those associations with p < 0.1 in the bivariate analysis. Seventeen cases of CVL were detected among 110 evaluated dogs (prevalence of 15.5%). Presence of ectoparasites (OR 6.5; 95% CI 1.1-37.4), animals with clinical signs (OR 9.5; 95% CI 1.2-76.6), and previous cases of CVL in the same house (OR 17.9; 95% CI 2.2-147.1) were associated with the outcome. Lutzomyia longipalpiswas not detected. Our results are indicative of an ongoing transmission in the area.

EFFICACY OF NITAZOXANIDE AGAINST Toxocara canis: LARVAL RECOVERY AND HUMORAL IMMUNE RESPONSE IN EXPERIMENTALLY INFECTED MICE

SUMMARY The efficacy of nitazoxanide (NTZ) against toxocariasis was investigated in an experimental murine model and results were compared to those obtained using mebendazole. Sixty male BALB/c mice, aged six to eight weeks-old, were divided into groups of 10 each; fifty were orally infected with 300 larvaed eggs of T. canisand grouped as follows, G I: infected untreated mice; G II: infected mice treated with MBZ (15 mg/kg/day) 10 days postinfection (dpi); G III: infected mice treated with NTZ (20 mg/kg/day) 10 dpi; G IV: infected mice treated with MBZ 60 dpi; G V: infected mice treated with NTZ 60 dpi; GVI: control group comprising uninfected mice. Mice were bled via retro-orbital plexus on four occasions between 30 and 120 dpi. Sera were processed using the ELISA technique to detect IgG anti- Toxocaraantibodies. At 120 dpi, mice were sacrificed for larval recovery in the CNS, liver, lungs, kidneys, eyes and carcass. Results showed similar levels of anti- ToxocaraIgG antibodies among mice infected but not submitted to treatment and groups treated with MBZ or NTZ, 10 and 60 dpi. Larval recovery showed similar values in groups treated with NTZ and MBZ 10 dpi. MBZ showed better efficacy 60 dpi, with a 72.6% reduction in the parasite load compared with NTZ, which showed only 46.5% reduction. We conclude that administration of these anthelmintics did not modify the humoral response in experimental infection by T. canis. No parasitological cure was observed with either drug; however, a greater reduction in parasite load was achieved following treatment with MBZ.

Evaluation of a new vaccine based on pDNA and recombinant protein against Helicobacter pylori

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

ADENOSINE DEAMINASE ACTIVITY AND SERUM C-REACTIVE PROTEIN AS PROGNOSTIC MARKERS OF CHAGAS DISEASE SEVERITY

SUMMARY Chagas disease is a public health problem worldwide. The availability of diagnostic tools to predict the development of chronic Chagas cardiomyopathy is crucial to reduce morbidity and mortality. Here we analyze the prognostic value of adenosine deaminase serum activity (ADA) and C-reactive protein serum levels (CRP) in chagasic individuals. One hundred and ten individuals, 28 healthy and 82 chagasic patients were divided according to disease severity in phase I (n = 35), II (n = 29), and III (n = 18). A complete medical history, 12-lead electrocardiogram, chest X-ray, and M-mode echocardiogram were performed on each individual. Diagnosis of Chagas disease was confirmed by ELISA and MABA using recombinant antigens; ADA was determined spectrophotometrically and CRP by ELISA. The results have shown that CRP and ADA increased linearly in relation to disease phase, CRP being significantly higher in phase III and ADA at all phases. Also, CRP and ADA were positively correlated with echocardiographic parameters of cardiac remodeling and with electrocardiographic abnormalities, and negatively with ejection fraction. CRP and ADA were higher in patients with cardiothoracic index ≥ 50%, while ADA was higher in patients with ventricular repolarization disturbances. Finally, CRP was positively correlated with ADA. In conclusion, ADA and CRP are prognostic markers of cardiac dysfunction and remodeling in Chagas disease.

IMMUNODIAGNOSIS OF HUMAN STRONGYLOIDIASIS: USE OF SIX DIFFERENT ANTIGENIC FRACTIONS FROM Strongyloides venezuelensis PARASITIC FEMALES

SUMMARY The aim of this study was to evaluate six different antigenic fractions from Strongyloides venezuelensis parasitic females for the immunodiagnosis of human strongyloidiasis. Soluble and membrane fractions from S. venezuelensis parasitic females were prepared in phosphate-buffered saline (SSF and SMF, respectively), Tris-HCl (TSF and TMF, respectively), and an alkaline buffer (ASF and AMF, respectively). Serum samples obtained from patients with strongyloidiasis or, other parasitic diseases, and healthy individuals were analyzed by enzyme-linked immunosorbent assay (ELISA). Soluble fractions SSF, TSF, and ASF showed 85.0%, 75.0%, and 80.0% sensitivity and 93.1%, 93.1%, and 87.5% specificity, respectively. Membrane fractions SMF, TMF, and AMF showed 80.0%, 75.0%, and 85.0% sensitivity, and 95.8%, 90.3%, and 91.7% specificity, respectively. In conclusion, the present results suggest that the fractions obtained from parasitic females, especially the SSF and SMF, could be used as alternative antigen sources in the serodiagnosis of human strongyloidiasis.

Toxoplasma-SPECIFIC IgG SUBCLASS ANTIBODY RESPONSE IN CEREBROSPINAL FLUID SAMPLES FROM PATIENTS WITH CEREBRAL TOXOPLASMOSIS

SUMMARY Cerebral toxoplasmosis can be highly debilitating and occasionally fatal in persons with immune system deficiencies. In this study, we evaluated the Toxoplasma gondii-specific IgG subclass antibody response in 19 cerebrospinal fluid (CSF) samples from patients with cerebral toxoplasmosis who had a positive IgG anti-T. gondii ELISA standardized with a cyst antigen preparation. There were no significant differences between the rates of positivity and the antibody concentrations (arithmetic means of the ELISA absorbances, MEA) for IgG1 and IgG2, but the rates of positivity and MEA values for these two IgG subclasses were significantly higher than those for IgG3 and IgG4. The marked IgG2 response in CSF from patients with cerebral toxoplasmosis merits further investigation.

EXPERIMENTAL PARACOCCIDIOIDOMYCOSIS IN PREGNANT RATS

Paracoccidioidomycosis (PCM), caused by the dimorphic fungus Paracoccidioides brasiliensis (Pb), is the most prevalent systemic mycosis in Latin America. There are few reports in the literature about the disease damages during pregnancy and the consequences to the fetuses and breeding. This study evaluated the implications of PCM during pregnancy on offspring and mothers in Wistar rats. Groups of rats were submitted to systemic Pb infection, by intraperitoneal infusion, and mated 30 days after the infection date. Immediately after birth, rats and neonates were sacrificed to obtain organs for standard histological examination, morphometric analysis, fungi recovery by plating (CFU) and dosing of anti-Pb antibodies by ELISA. There were no stillbirths or miscarriages, however, the fetuses from infected pregnant rats had lower body and organ weight but the fertility rate was 100%. The largest number of CFU was recovered from the organ of pregnant rats, the pathological examination revealed more severe infection in the same group, further on the largest number of granulomas and fungal field. It can be concluded that the PCM was more severe in the group of pregnant rats, with implications to the weight of offspring.

DISSEMINATED FUNGAL INFECTION WITH ADRENAL INVOLVEMENT: REPORT OF TWO HIV NEGATIVE BRAZILIAN PATIENTS

Paracoccidioidomycosis and histoplasmosis are systemic fungal infections endemic in Brazil. Disseminated clinical forms are uncommon in immunocompetent individuals. We describe two HIV-negative patients with disseminated fungal infections, paracoccidioidomycosis and histoplasmosis, who were diagnosed by biopsies of suprarenal lesions. Both were treated for a prolonged period with oral antifungal agents, and both showed favorable outcomes.

IDENTIFICATION OF CANINE VISCERAL LEISHMANIASIS IN A PREVIOUSLY UNAFFECTED AREA BY CONVENTIONAL DIAGNOSTIC TECHNIQUES AND CELL-BLOCK FIXATION

After the report of a second case of canine visceral leishmaniasis (CVL) in São Bento da Lagoa, Itaipuaçu, in the municipality of Maricá, Rio de Janeiro State, an epidemiological survey was carried out, through active search, totaling 145 dogs. Indirect immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA), and rapid chromatographic immunoassay based on dual-path platform (DPP(r)) were used to perform the serological examinations. The parasitological diagnosis of cutaneous fragments was performed by parasitological culture, histopathology, and immunohistochemistry. In the serological assessment, 21 dogs were seropositive by IFA, 17 by ELISA, and 11 by DPP(r), with sensitivity of 66.7%, 66.7% and 50%, and specificity of 87.2%, 90.2% and 94%, respectively for each technique. The immunohistochemistry of bone marrow using the cell-block technique presented the best results, with six positive dogs found, three of which tested negative by the other parasitological techniques. Leishmania sp. was isolated by parasitological culture in three dogs. The detection of autochthonous Leishmania infantum in Itaipuaçu, and the high prevalence of seropositive dogs confirm the circulation of this parasite in the study area and alert for the risk of expansion in the State of Rio de Janeiro.

Homogneous time resolved fluorescence: a new technique for detection and quantifications of infliximab in autoimmune patients

RESUMO:As terapias biológicas revolucionaram o tratamento das doenças autoimunes nos últimos anos. Tipicamente têm como alvos mediadores importantes no mecanismo das doenças. Os antagonistas do fator de necrose tumoral-α (TNF-α) são um grupo de agentes biológicos muito prescrito, pois estão indicados no tratamento de doenças imuno-mediadas comuns, tais como artrite reumatoide, artrite idiopática juvenil, artrite psoriática, espondilite anquilosante, doença de Crohn e colite ulcerosa. Com o uso frequente de inibidores do TNF-α, tem-se tornado evidente que estes agentes têm um potencial imunogénico importante, que pode comprometer o prognóstico a longo prazo dos doentes cronicamente tratados. A produção de anticorpos anti-fármaco parece causar falência terapêutica secundária em muitos doentes. Um dos efeitos dos anticorpos anti-fármaco é o aumento da eliminação do fármaco. A eliminação do fármaco, por sua vez, varia entre indivíduos, refletindo diferentes perfis farmacocinéticos. A determinação dos níveis séricos mínimos do agente anti-TNF-α é assim muito informativa e pode auxiliar nas decisões terapêuticas. Contudo, os testes imunológicos para determinar as concentrações séricas do fármaco não estão facilmente disponíveis na prática clínica. De forma a investigar uma nova técnica potencialmente fidedigna e prática para a deteção e quantificação dos agentes biológicos anti-TNF-α, foi testada a técnica por HTRF (homogeneous time-resolved fluorescence resonance energy transfer) para a determinação de concentrações séricas de infliximab. Apesar de apresentar algumas limitações relacionadas com as condições de leitura da fluorescência, esta técnica provou obter resultados próximos das concentrações obtidas por ELISA (enzyme-linked immunosorbent assay) bridging. Adicionalmente, tem a vantagem de ser de execução muito mais fácil e rápida. Deste modo, a técnica por HTRF poderá ser otimizada e tornar-se uma valiosa ferramenta laboratorial para orientar as decisões terapêuticas em doentes autoimunes com falência da terapêutica anti-TNF-α.--------- ABSTRACT: Biologic therapies revolutionized the treatment of autoimmune diseases in the last years. Typically, they target important disease mediators. Tumor necrosis factor-alpha (TNF-α) antagonists constitute a very prescribed group of biologic agents as they are indicated for the treatment of common immune-mediated diseases, such as rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn’s disease and ulcerative colitis. With the increasing use of TNF-α inhibitors it has been noticed that they have an important immunogenic potential that can compromise long-term outcomes in chronically treated patients. The production of anti-drug antibodies seems to cause secondary therapeutic failure in many patients. One of the effects of anti-drug antibodies is the enhancement of drug clearance. Drug clearance, in turn, varies among individuals, reflecting different pharmacokinetic profiles. Determination of serum anti-TNF-α drug trough levels is though very informative and could support treatment decisions. However, immunologic assays to determine drug serum concentrations are not readily available in clinical practice. In order to investigate a potentially reliable and practical new technique for detection and quantification of anti-TNF-α biologic agents, homogeneous time-resolved fluorescence resonance energy transfer (HTRF) technique was tested for determination of serum infliximab concentrations. Although presenting some limitations related with fluorescence reading conditions, this technique proved to give results close to the concentrations obtained by the widely used bridging enzyme-linked immunosorbent assay (ELISA). In addition, it has the advantage of being much easier and faster to perform. Thus, HTRF technique can be optimized and become a valuable laboratorial tool to guide treatment decisions in autoimmune patients with anti-TNF-α therapy failure.

Anti-Phospholipase A2 Receptor Antibodies in the Diagnosis of Idiopathic Membranous Nephropathy

Circulating anti-phospholipase A2 receptor antibodies (anti-PLA2R) have been described in 70% to 80% of the patients with idiopathic membranous nephropathy (iMN), but not in patients with secondary membranous nephropathy or other glomerular diseases. The goal of this study was to evaluate the sensitivity and specificity of the assay for anti-PLA2R in the diagnosis of iMN. Anti-PLA2R IgG, Elisa and immunofluorescence tests were used to detect circulating anti-PLA2R. These tests were applied in 53 patients who had a kidney biopsy. Of these, 38 had histological diagnosis of membranous nephropathy (MN) and the remaining had other glomerular diseases. The MN was classified as idiopathic in 33 patients after clinical exclusion of secondary causes. Anti-PLA2R were positive in 57.6% of the patients with iMN. All patients with secondary membranous nephropathy or other glomerular diseases did not show circulating anti-PLA2R. The sensitivity was 57.6% (CI 39.2-74.5) and specificity 100% (CI 47.8-100), AUC 0.788; p < 0.0001 for the detection of iMN. 71.4% of the iMN patients that tested negative for anti-PLA2R were in partial or complete remission. The detection of anti-PLA2R in the studied population had a specificity of 100% for the iMN diagnosis. Prior treatments seem to make the test negative and contribute to a lower sensitivity.