Prevalence of hepatitis C virus infection in quilombo remnant communities in Central Brazil

In order to determine the prevalence of hepatitis C virus (HCV) infection in quilombo remnant communities in Central Brazil, 1,007 subjects were interviewed in all 12 communities existing in Mato Grosso do Sul State, Central Brazil. Blood samples were collected and sera were tested for anti-HCV by enzyme-linked immunosorbent assay. Positive samples were retested for confirmation using a line immunoassay and were also subjected to HCV RNA detection. The prevalence of HCV infection was 0.2%. This finding shows a low prevalence of HCV infection in quilombo remnant communities in Central Brazil.

Confirming the presence of HTLV-1 infection and the absence of HTLV-2 in blood donors from Arequipa, Peru

Epidemiological studies conducted in Peru disclosed HTLV-1 to be prevalent in different ethnic groups, and found HTLV-2 in some Amazonian Indians and in men who have sex with men. No data concerning HTLV-1/2 infection in blood donors from Arequipa, a highlands region in southern Peru, is available. We searched for the presence of HTLV-1 and HTLV-2 antibodies in 2,732 serum samples obtained from blood donors from this geographic area. HTLV-1/2-specific antibodies were detected using an enzyme-linked immunosorbent assay (ELISA) and were confirmed by Western blot (WB). Reactive sera had their blood bags discarded from donation, and the demographic characteristics of the donors were analyzed. Thirty-five sera (1.2%) were HTLV seroreactive by ELISA, and 25 were confirmed HTLV-1-positive by WB. One serum disclosed HTLV-positivity, and the remaining nine serum samples showed indeterminate results by WB; three of which had an HTLV-1 indeterminate Gag profile. The median age of HTLV-positive individuals was 34.6 years; 27 were male and eight were female. All individuals were from southern Peru: 27 from Arequipa, five from Puno, and three from Cuzco. HTLV co-positivity with hepatitis B (five sera) and syphilis (one serum) were detected. Previous transfusion and tattooing were observed in two and one individuals, respectively. No serum was positive for HTLV/HIV co-infection. This study confirmed, for the first time, HTLV-1 infection and the absence of HTLV-2 infection in blood donors from Arequipa, Peru and suggests vertical transmission as the major route of HTLV-1 transmission and acquisition in this geographic region.

A serological study of cysticercosis in patients with HIV

Neurocysticercosis (NCC) has attained the importance of one of the most common cause of focal brain lesions in patients infected with HIV (human immunodeficiency virus). Adequate data regarding the rate of this co-infection is lacking. Therefore, the present study was carried out to determine the prevalence of cysticercosis among HIV patients residing in Puducherry or its neighboring districts of Tamil Nadu State, India. A total of one hundred blood samples were collected from HIV seropositive cases visiting JIPMER hospital, Puducherry, between June 2007 and May 2008. Enzyme immunotransfer blot (EITB) and enzyme linked immunosorbent assay (ELISA) were used to demonstrate anti- T. solium larval stage antibodies and Co-agglutination (Co-A) test was used to detect T. solium larval stage antigens in sera. Two HIV seropositive cases were found positive for anti-T. solium larval stage antibody by EITB and four were positive by ELISA. Only one sample was positive by both EITB and ELISA. No serum sample was found positive for T. solium larval stage antigen by Co-A test. The overall seropositivity detected by all the methods was 5% in this study group. The accurate clinical diagnosis of NCC in HIV is difficult due to deranged immunological parameters in the HIV infected patients. The results of this study provides important data on the prevalence of cysticercosis in HIV positive patients in Puducherry and neighboring areas which was previously unknown. This study will also increase awareness among physicians and public health agencies about T. solium cysticercosis in the selected group.

Circulation of antibodies against yellow fever virus in a simian population in the area of Porto Primavera Hydroelectric Plant, São Paulo, Brazil

Yellow fever (YF) is an acute viral infectious disease transmitted by mosquitoes which occurs in two distinct epidemiological cycles: sylvatic and urban. In the sylvatic cycle, the virus is maintained by monkey's infection and transovarian transmission in vectors. Surveillance of non-human primates is required for the detection of viral circulation during epizootics, and for the identification of unaffected or transition areas. An ELISA (enzyme-linked immunosorbent assay) was standardized for estimation of the prevalence of IgG antibodies against yellow fever virus in monkey sera (Alouatta caraya) from the reservoir area of Porto Primavera Hydroelectric Plant, in the state of São Paulo, Brazil. A total of 570 monkey sera samples were tested and none was reactive to antibodies against yellow fever virus. The results corroborate the epidemiology of yellow fever in the area. Even though it is considered a transition area, there were no reports to date of epizootics or yellow fever outbreaks in humans. Also, entomological investigations did not detect the presence of vectors of this arbovirus infection. ELISA proved to be fast, sensitive, an adequate assay, and an instrument for active search in the epidemiological surveillance of yellow fever allowing the implementation of prevention actions, even before the occurrence of epizootics.

Discrepancies and consequences of indirect hemagglutination, indirect immunofluorescence and Elisa tests for the diagnosis of Chagas disease

Using the indirect hemagglutination (IH), indirect immunofluorescence (IIF) and enzyme linked immunosorbent assay (ELISA) tests for the diagnosis of Chagas disease, 4000 serum samples were examined. This study was conducted with different purposes: clinical interest, research support and parasitological monitoring of those patients with Chagas disease who were treated with heart transplantations. The tests occurred without patient selection and in accordance with the medical requests. The results showed discrepancies and brought about several questions, considering the different results that all three methods showed when considered together. What was found brought about concerns and we suggest the adoption of different measures, aiming to avoid these mismatches in the context of this disease.

THE SCHISTOSOMULA TEGUMENT ANTIGEN AS A POTENTIAL CANDIDATE FOR THE EARLY SEROLOGICAL DIAGNOSIS OF SCHISTOSOMIASIS MANSONI

If Schistosoma mansoni infection could be detected in its early stages, especially before the egg deposition in the host tissues, the development of severe pathologic lesions could be efficiently prevented. We therefore developed an indirect enzyme-linked immunosorbent assay based on the detection of specific IgG against schistosomula antigens (ELISA-SmTeg). The assay was applied in sera samples from non-infected and infected mice collected seven and 15 days post-infection. The results were compared to the number of adult worms obtained by perfusion of the murine hepatic system 50 days post-infection. The sensitivity and specificity of the ELISA-SmTeg were 100% (p = 0.0032 and 0.0048 respectively for seven and 15 days of infection) with a cutoff value of 0.15 (p = 0.0002). Our findings show a novel low-cost serological assay using antigens which are easy to obtain, which was able to detect all the infected mice as early as seven days post-infection.

Toxocariasis in children attending a Public Health Service Pneumology Unit in Paraná State, Brazil

The enzyme-linked immunosorbent assay (ELISA) is the most widely used tool to detect anti-Toxocara IgG antibodies for both serodiagnostic and seroepidemiological surveys on human toxocariasis. In the last eight years a high prevalence of toxocariasis (32.2-56.0%) has been reported in children attending public health units from municipalities in the state of Paraná, Brazil. Therefore, the aim of this work was to compare the frequency found among the general child population with that of children attending a public pneumology service in Maringá, Paraná, Brazil and describe the laboratorial, clinical and epidemiological findings. The research was conducted at the Consórcio Público Intermunicipal de Saúde do Setentrião Paranaense (CISAMUSEP) from July 2009 to July 2010 among children aged between one and 15 years. From a total of 167 children studied, only 4.2% (7/167) tested positive for anti-Toxocara spp. IgG antibodies and presented mild eosinophilia (2/7), increased serum IgE levels (6/7) and a positive allergy test for mites (5/7). The presence of pets (dogs or cats) at home did not correlate with the seroprevalence. In conclusion, cases of toxocariasis involving the respiratory tract are rare in children attending a public health pneumology unit in the northwestern region of Paraná State, despite the high prevalence of this type of toxocariasis among the infantile population attending Basic Health Units in the same geographical area.

High occurrence of Entamoeba histolytica in the municipalities of Ariquemes and Monte Negro, State of Rondônia, Western Amazonia, Brazil

Introduction: Entamoeba histolytica infections were investigated in residents of the Ariquemes and Monte Negro municipalities in Rondônia State, Brazil. Methods: Stool samples of 216 individuals were processed by the spontaneous sedimentation method and analyzed by microscopy for detection of the E. histolytica/E. dispar complex, followed by the immunoassay method using an enzyme-linked immunosorbent assay-based kit for the E. histolytica stool antigen. Results: E. histolytica/E. dispar cysts were present in 61% (50/82) and 44% (59/134) of the samples from Ariquemes and Monte Negro respectively, with a significant difference in the occurrence of infection between the two populations [p < 0.05; χ2 = 5.2; odds ratio = 2.0 (1.1 - 3.6)]. The E. histolytica antigen detection rate was 36.6% (30/82) for stool samples from Ariquemes, and 19.4% (26/134) for stool taken from the residents of Monte Negro. The rate of the occurrence of amoebiasis was significantly higher in the population from Ariquemes [p < 0.05; χ2 = 7.8; odds ratio = 2.4 (1.2 - 4.7)]. Discussion: Due to the high occurrence of E. histolytica infected residents diagnosed in the region and the unavailability in local clinics of a test to distinguish between the two Entamoeba species, physicians should consider treating E. histolytica/E.dispar infections. Conclusion: The results indicate that E. histolytica infection is highly endemic in the studied areas.

FATAL OUTCOME OF INFECTION BY DENGUE 4 IN A PATIENT WITH THROMBOCYTOPENIC PURPURA AS A COMORBID CONDITION IN BRAZIL

Dengue is currently a major public-health problem. Dengue virus (DENV) is classified into four distinct serotypes, DENV 1-4. After 28 years of absence, DENV-4 was again detected in Brazil in 2010 in Roraima State, and one year later, the virus was identified in the northern Brazilian states of Amazonas and Pará, followed by Rio de Janeiro and São Paulo. In Minas Gerais, the first confirmed case of DENV-4 occurred in the municipality of Frutal in 2011 and has now been isolated from a growing number of patients. Although DENV-2 is associated with the highest risk of severe forms of the disease and death due to the infection, DENV-4 has also been associated with severe forms of the disease and an increasing risk of hemorrhagic manifestations. Herein, the first fatal case of confirmed DENV-4 in Brazil is reported. The patient was an 11-year-old girl from the municipality of Montes Claros in northern Minas Gerais State, Brazil. She had idiopathic thrombocytopenic purpura as a comorbid condition and presented with a fulminant course of infection, leading to death due to hemorrhagic complications. Diagnosis was confirmed by detection of Dengue-specific antibodies using IgM capture enzyme-linked immunosorbent assay and semi-nested RT-PCR. Primary care physicians and other health-care providers should bear in mind that DENV-4 can also result in severe forms of the disease and lead to hemorrhagic complications and death, mainly when dengue infection is associated with coexisting conditions.

PARTICIPATION OF TICKS IN THE INFECTIOUS CYCLE OF CANINE VISCERAL LEISHMANIASIS, IN TERESINA, PIAUÍ, BRAZIL

In this study, we detected Leishmania spp. infection in R. sanguineus collected from dogs that were naturally infected with L. (L.) infantum. We examined 35 dogs of both sexes and unknown ages. The infected dogs were serologically positive by the immunofluorescence antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA), and Quick Test-DPP (Dual Path Platform), as well as parasitological examination of a positive skin biopsy or sternal bone marrow aspiration. Ten negative dogs were included as controls. The ticks that infested these dogs were collected in pools of 10 adult females per animal. The PCR was performed with specific primers for Leishmania spp., which amplified a 720-bp fragment. Of the 35 analyzed samples, a product was observed in eight samples (8/35; 22.9%). We conclude that the presence of parasite DNA suggests that ticks participate in the zoonotic cycle of canine visceral leishmaniasis, in the city of Teresina, Piauí.

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.

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.

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.

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.