Serological, clinical and epidemiological evaluation of toxocariasis in urban areas of south Brazil

Toxocariasis is a worldwide public-health problem that poses major risks to children who may accidentally ingest embryonated eggs of Toxocara. The objectives of this study were to investigate the occurrence of anti-Toxocara spp. antibodies in children and adolescents and the variables that may be involved, as well as environmental contamination by Toxocara spp. eggs, in urban recreation areas of north central mesoregion, Paraná State, Brazil. From June 2005 to March 2007. a total of 376 blood samples were collected by the Public Health Service from children and adolescents one to 12 years old, of both genders. Samples were analyzed by the indirect ELISA method for detection of anti-Toxocara antibodies. Serum samples were previously absorbed with Ascaris suum antigens, and considered positive with a reagent reactivity index >1. Soil samples from all of the public squares and schools located in the four evaluated municipalities that had sand surfaces (n = 19) or lawns (n = 15) were analyzed. Of the 376 serum samples, 194 (51.6%) were positive. The seroprevalence rate was substantially higher among children aging one to five years (p = 0.001) and six to eight years (p = 0.022). The clinical signs and symptoms investigated did not show a statistical difference between seropositive and seronegative individuals (p > 0.05). In 76.5% of the investigated recreation places, eggs of Toxocara were detected in at least one of the five collected samples. Recreation areas from public schools were 2.8 times more contaminated than from public squares. It is important to institute educational programs to inform families and educators, as well as to improve sanitary control of animals and cleaning of the areas intended for recreation in order to prevent toxocariasis.

Canine visceral leishmaniasis: study of methods for the detection of IgG in serum and eluate samples

The Brazilian Ministry of Health recommends the culling and euthanasia of dogs with a positive serological test for canine visceral leishmaniasis (CVL). In the Municipality of Rio de Janeiro, the technique used for the diagnosis of CVL is the indirect fluorescent antibody test (IFAT), using blood samples eluted on filter paper (eluate). A dog survey was conducted over a period of one year in the region of Carapiá, in order to evaluate the diagnosis of CVL in this region. All animals underwent clinical examination, and blood samples (serum and eluate) were collected for analysis by enzyme immunoassay (ELISA) and IFAT. A skin biopsy was obtained for parasitological examination (culture). A total of 305 animals were studied and Leishmania chagasi was isolated from nine animals. Sensitivity and specificity were 100% and 96.6% for ELISA, respectively, 100% and 65.5% for IFAT (cut-off at a 1:40 dilution), 100% and 83.4% for IFAT (cut-off at a 1:80 dilution), and 22.2% and 97.0% for eluate IFAT. In conclusion, ELISA was the best tool for the diagnosis of CVL among the serological techniques tested. The present results suggest the need for a better evaluation of filter paper IFAT as the only diagnostic method for CVL in the Municipality of Rio de Janeiro.

Peritubular Capillaries C4d Deposits in Renal Allograft Biopsies and Anti HLA I/II Alloantibodies Screening. Incidence and Clinical Importance

Aim: To characterise clinically the patients with C4d in peritubular capillaries deposits (C4dPTCD) and/or circulating anti-HLA class I/II alloantibodies. To determine the correlation between positive C4dPTCD and circulating anti-HLA class I/II alloantibodies during episodes of graft dysfunction. Subjects and Methods: C4d staining was performed in biopsies with available frozen tissue obtained between January 2004 and December 2006. The study was prospective from March 2005, when a serum sample was obtained at the time of biopsy to detect circulating anti-HLA class I/II alloantibodies. Results: We studied 109 biopsies in 86 cadaver renal transplant patients. Sixteen of these (14.7%) presented diffuse positive C4dPTCD. There was a 13.5% rate of +C4dPTCD incidence within the first six months of transplantation and 16% after six months (p>0.05). Half of the +C4dPTCD in the first six months was associated with acute humoral rejection. After six months, the majority of +C4dPTCD (n=7/8) was present in biopsies with evidence of interstitial fibrosis/tubular atrophy and/or transplant glomerulopathy. The C4dPTCD was more frequent in patients with positive anti-HCV antibodies(p<0.0001), a previous renal transplant (p=0.007), and with a panel reactivity antibody (PRA) ≥ 50%(p=0.0098). The anti-HCV+ patients had longer time on dialysis (p=0.0019) and higher PRA(p=0.005). Circulating anti-HLA I/II alloantibodies were screened in 46 serum samples. They were positive in 10.9% of samples, all obtained after six months post transplant. Circulating alloantibodies were absent in 92.5% of the C4d negative biopsies. Conclusion: We found an association between the presence of C4dPTCD and 2nd transplant recipients,higher PRA and the presence of anti-HCV antibodies. The presence of HCV antibodies is not a risk factor for C4dPTCD per se, but appears to reflect longer time on dialysis and presensitisation. In renal dysfunction a negative alloantibody screening is associated with a reduced risk of C4dPTCD (<10%).

Ankylosing spondylitis: genomic and functional characterization of candidate genes and their repercussion in clinical practice

RESUMO: Introdução: A espondilite anquilosante (EA) é uma doença inflamatória crónica caracterizada pela inflamação das articulações sacroilíacas e da coluna. A anquilose progressiva motiva uma deterioração gradual da função física e da qualidade de vida. O diagnóstico e o tratamento precoces podem contribuir para um melhor prognóstico. Neste contexto, a identificação de biomarcadores, assume-se como sendo muito útil para a prática clínica e representa hoje um grande desafio para a comunidade científica. Objetivos: Este estudo teve como objetivos: 1 - caracterizar a EA em Portugal; 2 - investigar possíveis associações entre genes, MHC e não-MHC, com a suscetibilidade e as características fenotípicas da EA; 3 - identificar genes candidatos associados a EA através da tecnologia de microarray. Material e Métodos: Foram recrutados doentes com EA, de acordo com os critérios modificados de Nova Iorque, nas consultas de Reumatologia dos diferentes hospitais participantes. Colecionaram-se dados demográficos, clínicos e radiológicos e colhidas amostras de sangue periférico. Selecionaram-se de forma aleatória, doentes HLA-B27 positivos, os quais foram tipados em termos de HLA classe I e II por PCR-rSSOP. Os haplótipos HLA estendidos foram estimados pelo algoritmo Expectation Maximization com recurso ao software Arlequin v3.11. As variantes alélicas dos genes IL23R, ERAP1 e ANKH foram estudadas através de ensaios de discriminação alélica TaqMan. A análise de associação foi realizada utilizando testes da Cochrane-Armitage e de regressão linear, tal como implementado pelo PLINK, para variáveis qualitativas e quantitativas, respetivamente. O estudo de expressão génica foi realizado por Illumina HT-12 Whole-Genome Expression BeadChips. Os genes candidatos foram validados usando qPCR-based TaqMan Low Density Arrays (TLDAs). Resultados: Foram incluídos 369 doentes (62,3% do sexo masculino, com idade média de 45,4 ± 13,2 anos, duração média da doença de 11,4 ± 10,5 anos). No momento da avaliação, 49,9% tinham doença axial, 2,4% periférica, 40,9% mista e 7,1% entesopática. A uveíte anterior aguda (33,6%) foi a manifestação extra-articular mais comum. Foram positivos para o HLA-B27, 80,3% dos doentes. Os haplótipo A*02/B*27/Cw*02/DRB1*01/DQB1*05 parece conferir suscetibilidade para a EA, e o A*02/B*27/Cw*01/DRB1*08/DQB1*04 parece conferir proteção em termos de atividade, repercussão funcional e radiológica da doença. Três variantes (2 para IL23R e 1 para ERAP1) mostraram significativa associação com a doença, confirmando a associação destes genes com a EA na população Portuguesa. O mesmo não se verificou com as variantes estudadas do ANKH. Não se verificou associação entre as variantes génicas não-MHC e as manifestações clínicas da EA. Foi identificado um perfil de expressão génica para a EA, tendo sido validados catorze genes - alguns têm um papel bem documentado em termos de inflamação, outros no metabolismo da cartilagem e do osso. Conclusões: Foi estabelecido um perfil demográfico e clínico dos doentes com EA em Portugal. A identificação de variantes génicas e de um perfil de expressão contribuem para uma melhor compreensão da sua fisiopatologia e podem ser úteis para estabelecer modelos com relevância em termos de diagnóstico, prognóstico e orientação terapêutica dos doentes. -----------ABSTRACT: Background: Ankylosing Spondylitis (AS) is a chronic inflammatory disorder characterized by inflammation in the spine and sacroiliac joints leading to progressive joint ankylosis and in progressive deterioration of physical function and quality of life. An early diagnosis and early therapy may contribute to a better prognosis. The identification of biomarkers would be helpful and represents a great challenge for the scientific community. Objectives: The present study had the following aims: 1- to characterize the pattern of AS in Portuguese patients; 2- to investigate MHC and non-MHC gene associations with susceptibility and phenotypic features of AS and; 3- to identify candidate genes associated with AS by means of whole-genome microarray. Material and Methods: AS was defined in accordance to the modified New York criteria and AS cases were recruited from hospital outcares patient clinics. Demographic and clinical data were recorded and blood samples collected. A random group of HLA-B27 positive patients and controls were selected and typed for HLA class I and II by PCR-rSSOP. The extended HLA haplotypes were estimated by Expectation Maximization Algorithm using Arlequin v3.11 software. Genotyping of IL23R, ERAP1 and ANKH allelic variants was carried out with TaqMan allelic discrimination assays. Association analysis was performed using the Cochrane-Armitage and linear regression tests as implemented in PLINK, for dichotomous and quantitative variables, respectively. Gene expression profile was carried out using Illumina HT-12 Whole-Genome Expression BeadChips and candidate genes were validated using qPCR-based TaqMan Low Density Arrays (TLDAs). Results: A total of 369 patients (62.3% male; mean age 45.4±13.2 years; mean disease duration 11.4±10.5 years), were included. Regarding clinical disease pattern, at the time of assessment, 49.9% had axial disease, 2.4% peripheral disease, 40.9% mixed disease and 7.1% isolated enthesopathic disease. Acute anterior uveitis (33.6%) was the most common extra-articular manifestation. 80.3% of AS patients were HLA-B27 positive. The haplotype A*02/B*27/Cw*02/DRB1*01/DQB1*05 seems to confer susceptibility to AS, whereas A*02/B*27/Cw*01/DRB1*08/DQB1*04 seems to provide protection in terms of disease activity, functional and radiological repercussion. Three markers (two for IL23R and one for ERAP1) showed significant single-locus disease associations. Association of these genes with AS in the Portuguese population was confirmed, whereas ANKH markers studied did not show an association with AS. No association was seen between non-MHC genes and clinical manifestations of AS. A gene expression signature for AS was established; among the fourteen validated genes, a number of them have a well-documented inflammatory role or in modulation of cartilage and bone metabolism. Conclusions: A demographic and clinical profile of patients with AS in Portugal was established. Identification of genetic variants of target genes as well as gene expression signatures could provide a better understanding of AS pathophysiology and could be useful to establish models with relevance in terms of susceptibility, prognosis, and potential therapeutic guidance.

ISOLATION OF Cryptococcus neoformans FROM ENVIRONMENTAL SAMPLES COLLECTED IN SOUTHEASTERN NIGERIA

SUMMARY Cryptococcosis caused by Cryptococcus neoformans is the second most common fungal opportunistic pathogen and a lifethreatening infection with serious clinical manifestations especially in HIV/AIDS and other immunocompromised patients. In Nigeria, HIV/AIDS infection has reached an alarming level. Despite this, information on the presence of this fungus in clinical and environmental samples is very scanty in Nigeria and many other parts of Africa. We set out to evaluate the presence of Cryptococcus neoformans or C. gattii in pigeon droppings obtained from Southeastern Nigeria. One hundred and seventy-seven samples of pigeon droppings from six sample types were collected. The area covered comprised of ten cities and other locations spanning across five States in Nigeria. Using established techniques, Cryptococcus neoformans was isolated from 39 of the 177 (22.0%) samples overall. No C. gattiiwas isolated. Most of the isolates (32.4%) were recovered from dovecotes (11 of 34) followed closely by samples taken from markets (31.8%; seven of 22) and least from the church (4.0%; one of 25). The highest isolation rate (38.9%) was found in samples from Enugu-Ezike(seven of 23) while the least came from Afikpoand the other locations each with 9.1% isolation rate. This is the first large-scale screening of Cryptococcus neoformans from pigeon droppings in Nigeria. The ecological and epidemiological significance of these findings are discussed.

Bronchial Asthma in Children: Clinical and Epidemiologic Approach in Different Portuguese Speaking Countries

BACKGROUND: Geographical differences in asthma prevalence are currently accepted, but evidence is sparse due to the lack of multicentre studies using the same protocol. OBJECTIVES: To compare the prevalence of asthma and atopy among schoolchildren from Portuguese speaking countries (ISAAC and Portuguese Study) and evaluate some environmental variables, such as house dust mite exposure. MATERIAL AND METHODS: Significant random samples of schoolchildren studied with standard validated methods--questionnaires, skin prick tests, methacholine bronchial challenge tests; dust bed sampling for analysis of mite antigens. RESULTS: In the ISAAC study, in the 13-14 year-old age group, statistical significant differences were found, with higher wheezing prevalence in Brazil than in Portugal (two-fold). In the Portuguese Study, atopy prevalence ranged between 6.0 and 11.9% in Sal and S. Vicente (Cape Verde), up to 48.6 and 54.1% in Macau and Madeira. Active asthma had the higher values in Madeira (14.6%), and the lower in Macau (1.3%). Cape Verde had intermediate asthma prevalence (10.6 and 7.0%). The bronchial challenge test was positive in 25, 66 and 70% of asthmatic children from Sal, S. Vicente and Madeira respectively. Significant HDM antigen concentrations (Der p1) were found in Cape Verde and Madeira. CONCLUSIONS: There are significant variations in asthma and atopy prevalence between these pediatric populations. The reasons remain under discussion, but genetics linked to race, seem to play a central role, modulated by environmental and lifestyle variables.

HTLV-I associated myelopathy in the northern region of Brazil (Belém-Pará): serological and clinical features of three cases

Three patients (males, black, ages 37, 40 and 57) attended a university clinic with a progressive paraparesis of obscure origin. One patient who referred disease duration of more than 16 years, showed diminished deep reflexes, bilateral Babinski's sign, diminished sensation of vibration, abnormal bladder function and back pain. The other two patients (with one and six years of disease duration) complained of weakness in one leg, increased deep reflexes and back pain. Babinski's sign and bladder disturbance were also present in the patient with six years of disease. Blood samples tested by an enzyme immune assay and a discriminatory Western blot were positive for HTLV-I. The familial analysis of one patient showed a possible pattern of sexual and vertical transmission of the virus. To the best of our knowledge, these are the first cases of a proven association between HTLV-I and TSP/HAM in Belem, Para, and emphasize the need to actively look for cases of neurological disease associated to the virus.

Longitudinal clinical and serological survey of abdominal angiostrongyliasis in Guaporé, southern Brazil, from 1995 to 1999

Abdominal angiostrongyliasis is a zoonotic infection caused by Angiostrongylus costaricensis, a nematode with an intra-vascular location in the mesentery. Our objective was to address several aspects of the natural history of this parasitosis, in a longitudinal clinical and seroepidemiological study. A total of 179 individuals living in a rural area with active transmission in southern Brazil were followed for five years (1995-1999) resulting in yearly prevalence of 28.2%, 4.2%, 10%, 20.2% and 2.8% and incidences of 0%, 5.9%, 8% and 1.5%, respectively. Both men and woman were affected with higher frequencies at age 30-49 years. In 32 individuals serum samples were collected at all time points and IgG antibody reactivity detected by ELISA was variable and usually persisting not longer than one year. Some individual antibody patterns were suggestive of re-infection. There was no association with occurrence of abdominal pain or of other enteroparasites and there was no individual with a confirmed (histopathologic) diagnosis. Mollusks were found with infective third-stage larvae in some houses with an overall prevalence of 16% and a low parasitic burden. In conclusion, abdominal angiostrongyliasis in southern Brazil may be a frequent infection with low morbidity and a gradually decreasing serological reactivity.

Retrospective study of the occurrence of Cyclospora cayetanensis at Clinical Hospital of the University of São Paulo Medical School, SP

Cyclospora cayetanensis causes watery diarrhea in tropical countries, among travelers and after ingestion of contaminated water and food. Very little is known about its epidemiology, pathogenic aspects and reservoirs. In Brazil, its prevalence is unknown and to date there have been reports of three outbreaks. We report here a retrospective study of 5,015 stool samples from 4,869 patients attended at Clinical Hospital of the University of São Paulo Medical School, SP, Brazil between April 1996 and January 2002, with 14 cases of Cyclospora cayetanensis being detected there was a prevalence of 0.3%. Of the 14 infected patients, the mean age was 38 years and 71.4% were female. Ten patients presented symptoms; six presented levels of immunological markers and five patients were immunodeficient.

Clinical, epidemiological and laboratory aspects of patients with American cutaneous leishmaniasis in the State of Pernambuco

The diagnosis for American cutaneous leishmaniasis is based on an association of clinical, epidemiological and laboratory characteristics. The present study identified the circulating species of Leishmania in the State of Pernambuco, described its clinical-epidemiological characteristics and diagnosed the disease. Nineteen patients presenting active lesions who had been diagnosed through clinical evaluation and laboratory tests were selected. The tests included direct investigation, in vitro culturing, Montenegro skin test, indirect immunofluorescence and polymerase chain reaction. The Montenegro Skin Test showed positive results in 89% of the patients; indirect immunofluorescence, in 79%; direct investigation, in 58%; and polymerase chain reaction in 75%. Seven Leishmania (Viannia) braziliensis samples were isolated from these patients and were characterized by means of specific monoclonal antibodies. These data confirm that a combination of different diagnosis techniques is needed in order to obtain efficient results and that, so far, Leishmania (Viannia) braziliensis is the only species responsible for American cutaneous leishmaniasis infection in Pernambuco. Thus, it is essential to identify the parasite species involved in cases of human disease in an endemic area in order to determine the clinical and epidemiological characteristics, especially with regard to diagnosis, therapy development and disease prognosis.

Novel Cryopreservation Strategies for Cell‐Therapies and Pre‐Clinical Research

The expanding need for complex biologics for therapeutic applications, in‐vitro pharmacology and toxicology studies and fundamental research demands the production of banks of well‐characterized and safety‐tested stocks of a large number of cell/tissue samples. This implies the development of effective cryopreservation methodologies that can cope with process scalability and automation and must reflect the biological and physical properties of the cells as these can be significantly altered by the process.

High molecular mass fraction in clinical isolates of Paracoccidioides brasiliensis

INTRODUCTION: Different serum levels of the IgG/IgE for Paracoccidioides brasiliensis high mass molecular (hMM) fraction (~366kDa) in the acute and chronic forms of the disease have been reported. Considering the nonexistence of hMM fraction investigation involving clinical isolates of P. brasiliensis, the present study aimed to investigate the presence of the hMM fraction (~366kDa) in cell free antigens (CFA) from P. brasiliensis clinical isolates. METHODS: CFA from 10 clinical isolates and a reference strain (Pb18) were submitted to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by gel image capturing and densitometer analysis. Additionally, CFA from 20 isolates and Pb18 were analyzed by capture ELISA (cELISA) using polyclonal (polAb) or monoclonal (mAb) antibodies to the hMM fraction. RESULTS: The presence of the hMM component was observed in CFA of all samples analyzed by SDS-PAGE/densitometry and by cELISA. In addition, Pearson's correlation test demonstrated stronger coefficients between hMM fraction levels using pAb and mAb (R = 0.853) in cELISA. CONCLUSIONS: The soluble hMM fraction was present in all the P. brasiliensis clinical isolates analyzed and the reference strain Pb18, which could be used as a source of this antigen. The work also introduces for first time, the cELISA method for P. brasiliensis hMM fraction detection. Analysis also suggests that detection is viable using polAb or mAb and this methodology may be useful for future investigation of the soluble hMM fraction (~366kDa) in sera from PCM patients.

Cocirculation of two dengue virus serotypes in individual and pooled samples of Aedes aegypti and Aedes albopictus larvae

INTRODUCTION: To detect dengue virus, eggs of Aedes sp were collected in the city of Belo Horizonte, Brazil, in 2007. METHODS: Egg samples were subsequently hatched and the larvae were tested for the presence of dengue virus RNA by RT-PCR. RESULTS: Among the Aedes aegypti larvae samples, 163 (37.4%) out of 435 were positive, including 32 (10.9%) of 293 individual larvae samples concomitantly positive for two serotypes. CONCLUSIONS: Virological surveillance detecting coinfected vectors in the field could represent an important strategy for understanding the numerous factors involved in the transmission and clinical presentation of dengue.

Species distribution and in vitro fluconazole susceptibility of clinical Candida isolates in a Brazilian tertiary-care hospital over a 3-year period

INTRODUCTION: In this study, we aimed at identifying Candida isolates obtained from blood, urine, tracheal secretion, and nail/skin lesions from cases attended at the Hospital Universitário de Londrina over a 3-year period and at evaluating fluconazole susceptibilities of the isolates. METHODS: Candida isolates were identified by polymerase chain reaction (PCR) using species-specific forward primers. The in vitro fluconazole susceptibility test was performed according to EUCAST-AFST reference procedure. RESULTS: Isolates were obtained from urine (53.4%), blood cultures (19.2%), tracheal secretion (17.8%), and nail/skin lesions (9.6%). When urine samples were considered, prevalence was similar in women (45.5%) and in men (54.5%) and was high in the age group >61 years than that in younger ones. For blood samples, prevalence was high in neonates (35%) and advanced ages (22.5%). For nail and skin samples, prevalence was higher in women (71.4%) than in men (28.6%). Candida albicans was the most frequently isolated in the hospital, but Candida species other than C. albicans accounted for 64% of isolates, including predominantly Candida tropicalis (33.2%) and Candida parapsilosis (19.2%). The trend for non-albicans Candida as the predominant species was noted from all clinical specimens, except from urine samples. All Candida isolates were considered susceptible in vitro to fluconazole with the exception of isolates belonging to the intrinsically less-susceptible species C. glabrata. CONCLUSIONS: Non-albicans Candida species were more frequently isolated in the hospital. Fluconazole resistance was a rare finding in our study.

Clinical and laboratory alterations in dogs naturally infected by Leishmania chagasi

INTRODUCTION: Canine visceral leishmaniasis (CVL) is a zoonotic disease with different clinical manifestations. Parasitism often occurs in bone marrow, but changes have been observed in peripheral blood and serum biochemical parameters. The aim of this study was to evaluate the hematological and biochemical parameters in dogs naturally infected by Leishmania chagasi. METHODS: Eighty-five adult dogs of both sexes and various weights and ages from the Zoonosis Control Center of Fortaleza (CCZ) were used, selected by immunofluorescence assay (IFA) and considered positive with IFA titers greater than 1:40 and by visualizing amastigotes of Leishmania chagasi in smears obtained by bone marrow aspiration. The dogs (n = 85) were grouped according to clinical signs: negative (CN = 7), subclinical (CS = 10), and clinical (CC = 68). Blood samples were collected for determination of hematological and biochemical serum values. The experimental protocol was approved by the CEUA/UECE. RESULTS: The most frequent clinical signs were cachexia (77.9%), keratitis (61.8%), and lymphadenopathy (55.9%), and 86.8% of the animals showed more than one clinical sign characteristic of CVL. In CC were observed reductions in red blood cells (63%), hematocrit (72%), and hemoglobin (62%), as well as leukocytosis (33%), neutropenia (28%), thrombocytopenia (50%), uremia (45%), hyperproteinemia (53%, p<0.05), hypergammaglobulinemia (62%, p<0.01), and hypoalbuminemia (58%). CONCLUSIONS: Animals with the clinical form of the disease demonstrate hematological and biochemical changes consistent with anemia, uremia, hyperproteinemia, and hyperglobulinemia, which present themselves as strong clinical markers of visceral leishmaniasis associated with the signs previously reported.