Antigenic typing of brazilian rabies virus samples isolated from animals and humans, 1989-2000

Amostras de vírus rábico isoladas de animais e humanos no período de 1989 a 2000 foram tipificadas antigenicamente com a utilização de um painel de anticorpos monoclonais contra a nucleoproteína viral, pré-estabelecido para o estudo da epidemiologia molecular do vírus rábico isolado nas Américas. As amostras testadas foram isoladas no laboratório de diagnóstico do Instituto Pasteur e outros centros de diagnóstico de raiva no Brasil. Além das cepas de vírus rábico fixo CVS-31/96-IP, mantida em cérebro de camundongos e a PV-BHK/97, mantida em cultura de células, cepas de vírus rábico isoladas de cães, gatos, bovinos, eqüinos, morcegos, ovinos, caprino, suínos, raposa, sagüí, coatí, guaxinim e humanos, totalizaram 330 amostras. Seis variantes antigênicas foram definidas, compatíveis com perfís observados no painel de anticorpos monoclonais pré-estabelecido utilizado, as de número 2 (cão), 3 (Desmodus rotundus), 4 (Tadarida brasiliensis), 5 (Vampiro da Venezuela), 6 (Lasiurus cinereus) e Lab (reagente a todos os anticorpos utilizados), além de outros seis perfís desconhecidos, não compatíveis com aqueles observados no painel utilizado. A maior variabilidade foi observada entre as amostras isoladas de morcegos insetívoros e a variante mais comum isolada entre as espécies foi a variante 3 (Desmodus rotundus). Estes fatos podem representar a existência de múltiplos ciclos de transmissão independentes, envolvendo diferentes espécies de morcegos.

Potential force of infection of human rabies transmitted by vampire bats in the Amazonian region of Brazil

Human rabies tansmitted by bats has acquired greater epidemiologic relevance in various Latin American countries, just when cases transmitted by dogs have decreased. Concern has been heightened by reports of increased rates of bats biting humans in villlages in the Amazonian region of Brazil. The aim of the present work was to estimate the potential force of infection (per capita rate at which susceptible individuals acquire infection) of human rabies transmitted by the common vampire bat if the rabies virus were to be introduced to a colony of bats close to a village with a high rate of human bites. The potential force of infection could be then used to anticipate the size of a rabies outbreak in control programs. We present an estimator of potential incidence, adapted from models for malaria. To obtain some of the parameters for the equation, a cross-sectional survey was conducted in Mina Nova, a village of gold prospectors in the Amazonian region of Brazil with high rates of bates biting humans. Bats were captured near dwellings and sent to the Rabies Diagnostic Laboratory at the Center for Control of Zoonoses (São Paulo, Brazil) to be examined. To estimate the force of infection, a hypothetical rabies outbreak among bats was simulated using the actual data obtained in the study area. of 129 people interviewed, 23.33% had been attacked by a vampire bat during the year prior to the study, with an average of 2.8 bites per attacked person. Males (29.41%) were attacked more often than females (11.36%); also, adults (29.35%) were attacked more often than children (8.33%). None of the 12 bats captured in Mina Nova tested positive for rabies, but the force of infection for a hypothetical outbreak was estimated to be 0.0096 per person per year. This risk represents 0.96 cases per 100 area residents, giving an incidence of 1.54 cases of bat-transimtted buman rabies per year in the village of Mina Nova (160 inhabitants). The estimated risk is comparable with what has been observed in similar Brazilian villages.

Detection and quantification of antibodies to Newcastle disease virus in ostrich and rhea sera using a liquid phase blocking enzyme-linked immunosorbent assay

A liquid phase blocking ELISA (LPB-ELISA) was adapted for the detection and quantification of antibodies to Newcastle disease virus. Sera from vaccinated and unvaccinated commercial flocks of ostriches (Struthio camelus) and rheas (Rhea americana) were tested. The purified and nonpurified virus used as the antigen and the capture and detector antibodies were prepared and standardized for this purpose. The hemagglutination-inhibition (HI) test was regarded as the reference method, the cutoff point for the LPB-ELISA was determined by a two-graph receiver operating characteristic analysis. The LPB-ELISA titers regressed significantly (P < 0.0001) on the HI titers with a high correlation coefficient (r = 0.875). The two tests showed good agreement ( = 0.82; P < 0.0001), relative sensitivity (90.91%) and specificity (91.18%), and accuracy (91.02%), suggesting that they are interchangeable.

Use of lytic bacteriophage for control of experimental Escherichia coli septicemia and meningitis in chickens and calves

A lytic bacteriophage, which was previously isolated from sewage and which attaches to the K1 capsular antigen, has been used to prevent septicemia and a meningitis-like infection in chickens caused by a K1+ bacteremic strain of Escherichia coli. Protection was obtained even when administration of the phage was delayed until signs of disease appeared. The phage was able to multiply in the blood. In newly borne colostrum-deprived calves given the E. coli orally, intramuscular inoculation of phage delayed appearance of the bacterium in the blood and lengthened life span. With some provisos there is considerable potential for this approach to bacterial-disease therapy.

Evaluation of IFA, MAT, ELISAs and immunoblotting for the detection of anti-Toxoplasma gondii antibodies in paired serum and aqueous humour samples from experimentally infected pigs

The study evaluated the efficiency of diagnostic laboratory methods to detect anti-Toxoplasma gondii antibodies in paired serum and aqueous humour samples from experimentally infected pigs. 18-mixed breed pigs were used during the experiment; these were divided into two groups, G1 (infected group, n = 10) and G2 (uninfected group, n = 8). Infection was performed with 4 × 10 4 VEG strain oocysts at day 0 by the oral route in G1 animals. All pigs were euthanized at day 60, when retina, aqueous humour, and blood samples were collected. Anti-T. gondii antibody levels were assessed in serum (s) and aqueous humour (ah) by indirect immunofluorescence assay (IFA), modified agglutination test (MAT), m-ELISA (using crude membranes from T. gondii tachyzoites as antigen) and r-ELISA (using rhoptries from T. gondii tachyzoites as antigen). Polymerase chain reactions (PCR) of samples from the retina were performed by using Tox4 and Tox5 primers. Antibody titers of G1 animals ranged from 128 to 1024 and from 16 to 256 in serum and aqueous humour, respectively. There were differences in the correlation coefficients between IFA(s) × IFA (ah) (r = 0.62, P = 0.05), MAT(s) × MAT (ah) (r = 0.97, P < 0.0001); however, there was no significant difference between r-ELISA(s) × r-ELISA (ah) (r = 0.14, P = 0.7). Antibodies present in serum and aqueous humour recognized similar antigens. Samples of retina were positive by PCR in 30% (3/10) of infected pigs. G2 animals remained without antibody levels and were PCR negative throughout the experiment. These results suggest that the use of a combination of tests and immunoblotting for paired aqueous humour and serum samples could improve the sensitivity and specificity for the diagnosis of ocular toxoplasmosis. © 2007 Elsevier Ltd. All rights reserved.

Epidemiologia da brucelose bovina nos municípios de Altinópolis e Santo Antônio da Alegria, Estado de São Paulo: prevalência, fatores de risco e métodos de diagnóstico

Pós-graduação em Medicina Veterinária - FCAV

Contribution to the laboratory diagnosis of human cryptosporidiosis

A criptosporidiose humana é uma infecção causada pelo Cryptosporidium sp, um protozoário coccídio de patogenicidade emergente e responsável por severa e prostrante diarréia aquosa em humanos, principalmente em indivíduos imunodeprimidos. O diagnóstico, feito através da utilização de esfregaços de fezes submetidos a técnicas de concentração e coloração específica pela fucsina-carbólica tem oferecido bons resultados em nosso laboratório. Tendo em vista o longo tempo despendido para a observação dos esfregaços considerando-se a pequenez das formas e o contraste da coloração, realizamos modificações no procedimento técnico da coloração ácido-resistente que resultaram em sensível melhoria das preparações: a fucsina-carbólica passou a ser deixada sobre o esfregaço por período de 3 minutos (LENNETTE et al., 1985) e procedeu-se a substituição da solução de álcool-ácido sulfúrico a 5% (HENRIKSEN & POHLENZ, 1981) por solução de ácido clorídrico a 0,5% em álcool etílico 70%, por cerca de 2 minutos (contribuição original). Estas alterações promoveram melhor remoção do excesso de fucsina-carbólica, aumentando a eficiência da etapa de descoloração e consequentemente otimizando o contraste do processo de coloração. Nestas condições, as lâminas examinadas em microscópio óptico em aumentos de 250x e 1000x tiveram a visualização dos oocistos do protozoário facilitada, sendo os mesmos observados em contraste destacado com pigmentação intensa de cor rosa-avermelhada contra coloração de fundo azulada. Vale destacar que estas modificações oferecem vantagens de rápido processamento do material e facilidade de visualização do protozoário, diminuindo o tempo de microscopia, tornando a análise das lâminas mais rápida e menos cansativa, agilizando o diagnóstico.

Frequency of Blastocystis hominis and other intestinal parasites in stool samples examined at the Parasitology Laboratory of the School of Pharmaceutical Sciences at the São Paulo State University, Araraquara

Blastocystis hominis é um protozoário, causador de infecção intestinal denominada blastocistose humana, cujo diagnóstico é realizado pelo exame coproparasitológico e por meio de técnicas de coloração permanente. Este estudo foi desenvolvido para avaliar a freqüência da infecção por Blastocystis hominis em habitantes da região de Araraquara/SP, bem como comparar diferentes métodos para a pesquisa desse protozoário em amostras de fezes. Foram estudadas 503 amostras de fezes submetidas ao exame direto a fresco, às técnicas de Faust e cols, Lutz e de Rugai e cols, além das colorações pela hematoxilina férrica, tricrômio e de Kinyoun modificada. Entre as 503 amostras examinadas, 174 (34,6%) apresentaram-se positivas para a presença de parasitas intestinais. O protozoário e o helminto mais freqüentes foram Entamoeba coli (14,6%) e Strongyloides stercoralis (6,7%), respectivamente. Blastocystis hominis foi observado em 23 (4,6%) amostras fecais com consistência predominantemente pastosa, não caracterizando quadro diarréico. Apesar da baixa freqüência de Blastocystis hominis encontrada na região de Araraquara, comparativamente a outras regiões brasileiras, é importante a realização do diagnóstico laboratorial desse protozoário. O encontro de Blastocystis hominis em material fecal é indicativo de contaminação de alimentos e água de consumo, desde que se admita a rota de transmissão oral-fecal desse parasita, o que implica na orientação da população sobre as medidas de saneamento básico e higiene como meio para se controlar problemas de saúde ocasionados pelos enteroparasitas.

A Novel Diagnostic Target in the Hepatitis C Virus Genome

BackgroundDetection and quantification of hepatitis C virus (HCV) RNA is integral to diagnostic and therapeutic regimens. All molecular assays target the viral 5'-noncoding region (59-NCR), and all show genotype-dependent variation of sensitivities and viral load results. Non-western HCV genotypes have been under-represented in evaluation studies. An alternative diagnostic target region within the HCV genome could facilitate a new generation of assays.Methods and FindingsIn this study we determined by de novo sequencing that the 3'-X-tail element, characterized significantly later than the rest of the genome, is highly conserved across genotypes. To prove its clinical utility as a molecular diagnostic target, a prototype qualitative and quantitative test was developed and evaluated multicentrically on a large and complete panel of 725 clinical plasma samples, covering HCV genotypes 1-6, from four continents (Germany, UK, Brazil, South Africa, Singapore). To our knowledge, this is the most diversified and comprehensive panel of clinical and genotype specimens used in HCV nucleic acid testing (NAT) validation to date. The lower limit of detection (LOD) was 18.4 IU/ml (95% confidence interval, 15.3-24.1 IU/ml), suggesting applicability in donor blood screening. The upper LOD exceeded 10(-9) IU/ml, facilitating viral load monitoring within a wide dynamic range. In 598 genotyped samples, quantified by Bayer VERSANT 3.0 branched DNA (bDNA), X-tail-based viral loads were highly concordant with bDNA for all genotypes. Correlation coefficients between bDNA and X-tail NAT, for genotypes 1-6, were: 0.92, 0.85, 0.95, 0.91, 0.95, and 0.96, respectively; X-tail-based viral loads deviated by more than 0.5 log10 from 5'-NCR-based viral loads in only 12% of samples (maximum deviation, 0.85 log10). The successful introduction of X-tail NAT in a Brazilian laboratory confirmed the practical stability and robustness of the X-tail-based protocol. The assay was implemented at low reaction costs (US$8.70 per sample), short turnover times (2.5 h for up to 96 samples), and without technical difficulties.ConclusionThis study indicates a way to fundamentally improve HCV viral load monitoring and infection screening. Our prototype assay can serve as a template for a new generation of viral load assays. Additionally, to our knowledge this study provides the first open protocol to permit industry-grade HCV detection and quantification in resource-limited settings.

Accuracy of routine diagnostic tests used in paracoccidioidomycosis patients at a university hospital

The identification of appropriate laboratory measures to confirm clinical hypotheses is important in routine paracoccidioidomycosis medical care. The clinical records and laboratory reports of 401 paracoccidioidomycosis patients attended at the Tropical Diseases Area, Faculdade de Medicina de Botucatu, from 1974 to 2008 were reviewed. Direct mycological (DM), cell block (CB), histopathological (HP), and double immunodiffusion (DID) tests were evaluated before treatment. Typical Paracoccidioides brasiliensis yeast forms were observed in clinical specimens of 86% of the patients, but 14% were detected only by serological test. DM of 51 different tissue specimens produced 74.5% sensitivity, and 62.5% sensitivity was observed in 112 sputum samples. CB in 483 sputum samples generated 55.3% sensitivity. HP performed in 239 samples from different tissues revealed 96.7% sensitivity. Serology carried out in 351 patients and 200 healthy controls provided 90.0% sensitivity, 100.0% specificity, 100.0% positive predictive value, 85.1% negative predictive value and 93.6% accuracy. Comparisons of laboratory measurements performed in the same patient showed that sensitivity decreases from HP to DID to CB and DM, with the last two assays providing similar sensitivities. This study demonstrated that P. brasiliensis identification by HP, CB, and/or DM associated with DID is sufficient to establish the laboratorial diagnosis of paracoccidioidomycosis in practically all cases. (C) 2011 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

Postlarval stages and growth patterns of the spider crab Pyromaia tuberculata (Brachyura, Majidae) from laboratory-reared material

The spider crab Pyromaia tuberculata was introduced into southeastern Brazil; ovigerous material was collected and reared in the laboratory. Morphologic changes and growth patterns of post-larval development are reported. Results show that within-stage size variation is lowest in mature stages, especially in the case of females in which there is an apparent size threshold for the last juvenile stages to undergo the puberty molt. A prepuberty molt taking place at the fourth crab stage is indicated by analyzing the allometric growth of the abdomen in females. In contrast, the same procedure using the allometric growth of chelae failed in detecting both the prepuberty and puberty molts in males. Conversely to females, which develop a complex brood chamber at the puberty molt, the enlargement of chelae was not consistent in all postpuberty males. The short instar sequence of this species, in no case exceeding nine stages, is marked by conspicuous morphologic alterations achieved at each molt. Almost all stages can be identified by examining diagnostic features of rostrum, abdomen, sternum, and pleopods.

Tuberculosis diagnosis after bleach processing for early stage tuberculosis laboratory capacity building

The diagnosis of tuberculosis is seriously hampered in the absence of standard biosafety laboratory facilities for specimen concentration and Mycobacterium tuberculosis culture. Within a laboratory twinning arrangement, heat-fixed direct smear and sediment from 74 bleach-processed and 20 non-processed specimens from Cumura Hospital, Guinea-Bissau, were sent to Lisbon for molecular evaluation of rifampicin resistance. Sequence analysis of a 369 base-pair ppoB locus detected 3.2% (3/94) resistant specimens. To our knowledge, this represents the first report on the molecular analysis of M. tuberculosis from bleach-processed sputum, an alternative to current diagnostic practice in low-resource settings.

The diagnostic value of Gram stain for initial identification of the etiologic agent of peritonitis in CAPD patients

Objective: To evaluate the effectiveness of the Gram stain in the initial diagnosis of the etiologic agent of peritonitis in continuous ambulatory peritoneal dialysis (CAPD). Design: Retrospective study analyzing the sensitivity (S), specificity (SS), positive predictive value (+PV), and negative predictive value (-PV) of the Gram stain relating to the results of cultures in 149 episodes of peritonitis in CAPD. The data were analyzed in two studies. In the first, only the cases with detection of a single agent by Gram stain were taken (Study 1). In the second, only the cases with two agents in Gram stain were evaluated (Study 2). Setting: Dialysis Unit and Laboratory of Microbiology of a tertiary medical center. Patients: Sixty-three patients on regular CAPD who presented one or more episodes of peritonitis from May 1992 to May 1995. Results: The positivity of Gram stain was 93.2% and the sensitivity was 95.7%. The values of S, SS, +PV, and -PV were respectively: 94.9%, 53.5%, 68.3%, and 90.9% for gram-positive cocci and 83.3%, 98.8%, 95.2%, and 95.6% for gram-negative bacilli. The association of gram-positive cocci plus gram-negative bacilli were predictive of growth of both in 6.8%, growth of gram-positive cocci in 13.7%, and growth of gram-negative bacilli in 72.5%. Conclusions: The Gram stain is a method of great value in the initial diagnosis of the etiologic agent of peritonitis in CAPD, especially for gram-negative bacilli.

Diagnostic accuracy of semi-quantitative and quantitative culture techniques for the diagnosis of catheter-related infections in newborns and molecular typing of isolated microorganisms

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)