Altered indirect hemagglutination method for easy serotyping of Haemophilus parasuis / Método de Hemaglutinação Indireta alterado para facilitar a sorotipificação de Haemophilus parasuis

Glässer's disease is an emergent bacterial disease that affects swine husbandries worldwide causing important economic losses. The aetiological agent, Haemophilus parasuis, is currently divided in fifteen serovars but an increasing number of non-typeable serovars have been reported. Indirect hemagglutination (IHA) is indicated as a serotyping method for H. parasuis. In the present study, we describe an additional step that aims to work around a possible obstacle in the original protocol that may compromise the outcome of this assay. We observed that the choice of anticoagulant for blood collection influences and/or impairs spontaneous adsorption of H. parasuis antigens on sheep red blood cells (SRBCs). However, regardless of the anticoagulant used, chemical treatment of SRBCs with tannic acid induces a stable antigen adsorption (sensitization step). The addition of 1% BSA to SRBCs washing buffer and to antisera dilution augments IHA specificity. Tannic acid treated SRBCs combined with thermo-resistant H. parasuis antigens increases the assay resolution. Thus, our results demonstrate an improvement in the technique of H. parasuis serotyping that will prove valuable to understand Glässer's disease epidemiology and to better characterize serovars involved in outbreaks.(AU)

A Doença de Glässer é uma doença bacteriana emergente que afeta a produção de suínos em todo o mundo e causa importantes perdas econômicas. O agente etiológico, Haemophilus parasuis, é atualmente dividido em quinze sorovares; no entanto, um número crescente de cepas não tipificáveis tem sido relatado. O teste de hemaglutinação indireta (IHA) tem sido utilizado para a sorotipificação de H. parasuis. Neste estudo, descrevemos uma alteração no protocolo original de IHA e que supera uma limitação específica que pode comprometer o uso geral deste ensaio. Descobrimos que o tipo de anticoagulante utilizado para coletar os eritrócitos ovinos (SRBCs) pode comprometer a adsorção espontânea dos antígenos do H. parasuis. Por outro lado, o tratamento químico dos SRBCs com ácido tânico promove uma adsorção antigênica estável (passo de sensibilização) e independente do anticoagulante utilizado. O uso de 1% de BSA durante as lavagens dos SRBCs e na diluição dos antissoros incrementa a especificidade da IHA e, a combinação dos SRBCs tratados quimicamente com antígenos de H. parasuis termo-resistentes aumentam a resolução da IHA. Nossos resultados destacam uma melhoria na principal técnica de sorotipificação de H. parasuis, que auxiliará diretamente no entendimento da epidemiologia da Doença de Glässer e na caracterização dos sorovares envolvidos em surtos da doença.(AU)

Frecuencia de antígenos del sistema sanguíneo Rh y del sistema Kell en donantes de sangre / Frequency of antigens in Rh and Kell blood system in blood donors

Introducción: los estudios inmunohematológicos que se realizan a los donantes de sangre se orientan a proporcionar al receptor una terapia transfusional compatible con el sistema sanguíneo ABO y antígeno D del sistema Rh. Sin embargo, como una forma de incrementar la seguridad transfusional, surge el interés de ampliar la gama de antígenos a determinar y por ende, a compatibilizar previo a una transfusión sanguínea. Objetivo: determinar la frecuencia de los cinco antígenos mayores del sistema Rh y los antígenos K1 y K2 del sistema Kell en donantes voluntarios de sangre. Métodos: Estudio descriptivo transversal que incluyó 200 donantes voluntarios de sangre del Centro Productivo Regional de Sangre del Maule (CPRSM) seleccionados al azar. Se realizó fenotipificación de los cinco antígenos mayores del sistema Rh. y el antígeno K1 y K2 del sistema Kell. Se utilizó la técnica de hemaglutinación en tubo, con sueros monoespecíficos y DG Gel® Coombs. Se calculó la frecuencia fenotípica de los antígenos D, C, c, E y e del sistema Rh., y K1 y K2 del sistema Kell, en porcentajes. A partir de la frecuencia de los fenotipos Rh. se determinó la frecuencia del genotipo más probable de dicho sistema. Para el Kell se estimó el genotipo en base al fenotipo. Resultados: sistema Rh: 96 por ciento de las muestras estudiadas presentaba el antígeno D, 97,5 por ciento el antígeno e; 35,5 por ciento el antígeno E; 79 por ciento el antígeno C y 65,5 por ciento el antígeno c. El genotipo más frecuente fue CDe/CDe. Sistema Kell: se encontró una frecuencia del 4 por ciento para el antígeno K1, mientras que el antígeno K2 presenta una frecuencia del 99,5 por ciento. Al nivel de frecuencia genotípica se detectó que el 96 por ciento de la población tiene un genotipo homocigoto para K2 (kk). Conclusiones: La frecuencias de los siete antígenos estudiados es similar a la descrita en otras poblaciones(AU)

Introduction: Immunologic studies performed on blood donors are directed to provide a transfusion therapy compatible with ABO blood group system and Rh system D antigen in the recipient. However, as a way to increase transfusion safety, the interest of expanding the range of antigens to determine and therefore to be tested for compatibility prior to a blood transfusion, arises. Aim: To determine the frequency of the five major antigens of Rh. system and K1 and K2 antigens of the Kell system in blood donors. Methods: Cross-sectional study including 200 randomly selected voluntary blood donors from Centro Productivo Regional de Sangre del Maule (CPRSM). Phenotyping of five major antigens of Rh. system and K1 and K2 Kell system antigens was carried out. The tube hemagglutination technique with monospecific Coombs sera and DG Gel ® was used. The Rh. system D, C, c, E, e antigens and Kell system K1 and K2 antigen phenotypic frequencies were calculated in percentages. The most likely Rh.genotype was determined from the phenotype frequency of this system. Similarly, in Kell system the genotype frequency was determined based on phenotype. Results: In the Rh.system, 96 percent of the samples studied had D antigen; 97.5 percent had the e antigen, and 35.5 percent the E antigen. Antigen C was present in 79 percent and c in 65.5 percent. The most frequent Rh. genotype was CDe/CDe. In Kell system, K1 antigen presented a frequency of 4 percent, while antigen K2 presented a 99.5 percent. Regarding genotypic frequency, a 96 percent of the population showed a K2 (kk) homozygous genotype(AU) Conclusion: The frequency of the seven antigens studied is similar to that described in other populations(AU)

Comparison of seven diagnostic tests to detect Trypanosoma cruzi infection in patients in chronic phase of Chagas disease / Comparación de siete pruebas diagnósticas para detectar infección por Trypanosoma cruzi en pacientes en fase crónica de la enfermedad de Chagas

Objective: To compare the diagnostic performance of seven methods to determine Trypanosoma cruzi infection in patients with chronic Chagas disease. Methods: Analytical study, using the case-control design, which included 205 people (patients with Chagasic cardiomyopathy, n= 100; control group, n= 105). Three enzyme linked immunosorbent assays, one indirect hemagglutination assay and one immunochromatographic test were assessed. Additionally, DNA amplification was performed via the PCR method using kinetoplast and nuclear DNA as target sequences. For the comparative analysis of diagnostic tests, the parameters used were sensitivity, specificity, positive and negative predictive values, Receiver Operator Characteristic (ROC), positive and negative likelihood ratio, as well as κ quality analysis. Results: The commercial Bioelisa Chagas test showed the highest sensitivity (98%), specificity (100%), and positive and negative predictive values; additionally it had the highest discriminatory power. Otherwise, the amplification of T. cruzi DNA in blood samples showed low values of sensitivity (kinetoplast DNA= 51%, nuclear DNA= 22%), but high values of specificity (100%), and moderate to low discriminatory ability. Conclusion: The comparative analysis among the different methods suggests that the diagnostic strategy of T. cruzi infection in patients with chronic Chagas disease can be performed using ELISA assays based on recombinant proteins and/or synthetic peptides, which show higher diagnosis performance and can confirm and exclude the diagnosis of T. cruzi infection. The molecular methods show poor performance when used in the diagnosis of patients with chronic Chagas disease.

Objetivo: Comparar la capacidad diagnóstica de siete métodos para determinar infección por Trypanosoma cruzi, en pacientes con enfermedad de Chagas crónica. Métodos: Estudio analítico de casos y controles, que incluyó 205 personas (pacientes con miocardiopatía chagásica, n= 100; grupo control, n= 105). Se evaluaron tres inmunoensayos enzimáticos, una hemaglutinación indirecta y una inmunocromatografia. Adicionalmente, se realizó amplificación de ADN de T. cruzi por reacción en cadena de la polimerasa utilizando como secuencias diana ADN de kinetoplasto y nuclear. Para el análisis comparativo de las pruebas diagnósticas, los parámetros utilizados fueron sensibilidad, especificidad, valores predictivo positivo y negativo, análisis ROC, razón de verosimilitud positiva y negativa, así como análisis de calidad κ. Resultados: La prueba Bioelisa para Chagas mostró la mayor sensibilidad (98%), especificidad (100%) y valores predictivos positivo y negativo; además ésta tuvo el mayor poder discriminatorio. En contraste, los ensayos de amplificación de ADN de T. cruzi mostraron baja sensibilidad (ADN de kinetoplasto= 51%, ADN nuclear= 22%), alta especificidad (100%) y de moderada a baja capacidad discriminatoria. Conclusión: El análisis comparativo entre los métodos sugiere utilizar como estrategia diagnóstica en pacientes crónicos con enfermedad de Chagas, los ensayos de ELISA con proteínas recombinantes y/o péptidos sintéticos por mostrar un rendimiento diagnóstico superior y tener la capacidad de confirmar y descartar el diagnóstico de infección por T. cruzi. Los métodos moleculares muestran pobre rendimiento para ser utilizados en el diagnóstico de pacientes en fase crónica con enfermedad de Chagas.

Avaliação sorológica de Toxoplasma gondii, em ovinos criados em dois municípios do nordeste paraense. Considerações relativas ao consumo humano de produtos e subprodutos dessa espécie / Evaluation serological of Toxoplasma gondii in sheep reared in two municipalities of northeastern Pará. Considerations for human consumption of products

No intuito de realizar uma investigação sorológica da toxoplasmose natural em ovinos, amostras de 350 soros de ovinos foram submetidas à reação de hemaglutinação indireta (HAI) para detecção de anticorpos anti-Toxoplasma gondii. Desta forma, objetivou-se nesta pesquisa realizar uma investigação sorológica inicial sobre a ocorrência da infecção pelo T.gondii em ovinos de dois municípios do Estado do Pará e a correlação da enfermidade em humanos por consumo de produto e subprodutos dessa espécie.

Identification of the diagnostic and epidemiological potential of camel strain G6 in human cystic echinococcosis

Recent molecular studies detected the presence of camel G6 genotype in human samples in different countries including Egypt. However, none of them studied the diagnostic and epidemiological role of camel G6 genotype in patients' sera with cystic echinococcosis [CE]. Detection of camel G6 strain in patients' sera by polymerase chain reaction [PCR] and determination of changes in the genotype profile that might be influenced by the predominant transmission cycle during a certain time. Eighty subjects were divided into 2 main groups with 3 subgroups in each: Group I [31 CE cases with positive G6 PCR in parasite material obtained from their cysts] subdivided into Group IA [21 hepatic CE], Group IB [5 pulmonary CE], Group IC [5 multiple organ CE], Group II [49 control subjects] including Group IIA [29 patients with other parasitic diseases], Group IIB [10 patients with space occupying lesions] and Group IIC [10 healthy individuals]. DNA was extracted from CE patients' sera for amplification and sequencing. Hot-start specific G6 PCR for patients' sera [PCRs] revealed that all CE cases [100%] were of G6 genotype, with 100% sensitivity and 100% specificity and a specific band at 254 bp. Indirect hemagglutination test [IHAT] showed 61.29% sensitivity and 95.92% specificity. DNA sequencing of the amplified DNA fragments of patient's sera showed 100% homology with extracts from parasite materials taken from their own cysts [Gen Bank under the accession no. from GQ476732 to GQ476735], as well as with that of an Argentinean reference strain [provided from WHO reference laboratory]

Estudo comparativo entre os testes adotados pelo PNCEBT para o diagnóstico sorológico da brucelose em bovinos / Comparative study of serological tests for bovine brucellosis diagnosis adopted by the Brazilian Program for animal brucellosis and tuberculosis control and eradication

Avaliaram-se comparativamente as provas do antígeno acidificado tamponado (AAT), a combinação das provas de soroaglutinação lenta e 2-mercaptoetanol (2-ME) e a reação de fixação de complemento (RFC), provas preconizadas pelo Programa Nacional de Controle e Erradicação de Brucelose Tuberculose (PNCEBT). Para tanto, foram alisadas 1.061 amostras de soros bovinos. Os dados foram analisados pelo indicador kappa, adotando-se como ponto de corte o título 25 no 2-ME e 4 na RFC. Constatou-se sensibilidade relativa de 99,6%, 98,8% e 91,1%, respectivamente, para o AAT, a 2-ME e a RFC, e especificidade relativa de 83,9%, 96,2% e 100,0%. A comparação entre os testes adotados pelo programa apontou concordância boa entre o teste de triagem (AAT) e os testes confirmatórios (kappa: 2-ME = 0,80; e RFC = 0,73) e concordância ótima quando os testes confirmatórios foram comparados entre si (kappa = 0,86). No entanto foram encontrados soros com título elevado em um dos testes confirmatórios e resultado negativo no outro, o que reforça a ideia de que o diagnóstico sorológico da brucelose é mais confiável quando obtido por meio dos resultados de vários testes.

Serum samples from 1,061 bovine were analyzed by serological diagnostic techniques adopted by the Brazilian Program for Animal Brucellosis and Tuberculosis Control and Eradication in order to compare, as a screening test, the rose Bengal plate test (RBPT), and as confirmatory tests, the 2-mercaptoethanol plus standard tube agglutination test (2-ME), and the complement fixation test (CFT). Relative sensitivity of 99.6%, 98.8% and 91.1% were observed, respectively, for RBPT, 2-ME and CFT, and relative specificity of 83.9%, 96.2% and 100.0%. The agreement between tests was analyzed by the kappa statistic (2-ME cut-off = titer 25, and CFT cutoff = titer 4). RBPT showed a substantial agreement (kappa: 2-ME = 0.80; and RFC = 0.73) with the confirmatory tests, and excellent agreement between the confirmatory tests (kappa = 0.86) was observed. However sera with a negative result in one of the confirmatory tests and a high titer in the other were observed, reinforcing that the serological diagnosis of bovine brucellosis is more reliable when based on the results of several tests.

Evaluation of ELISA and IHAT in serological diagnosis of proven cases of human hydatidosis

Cystic hydatid disease [Hydatidosis] is the most serious tape-worm infection prevalent in the cattle and sheep raising area of the world. Hydatidosis in man [as an accidental host] is caused by infection with the ova containing larval stage of Echinococcus granulosus. In the last decade, different techniques have been employed for sero-diagnosis of hydatidosis; as IHA, IFA, ELISA, CCLE [Counter Current Immuno-electrophoresis]. This paper evaluated the validity of ELISA and IHA. Since whole hydatid cyst fluid was used as a source of antigen for serodiagnosis. Thirty surgical and pathological hydatidosis proven patients were examined. The sensitivity and specificity of ELISA were 96.7% and 97.5% respectively, and that of IRA were 86.7%, and 95% respectively

Surveillance on bacteria associated with avian influenza virus outbreaks in commercial broiler flocks in Egypt

Avian influenza [AI] [H5N1] outbreaks with very high mortality in twenty four [24] broiler flocks in Egypt provinces [El-Behira, El-Garbia, Kafer El-Sheikh, and Alexandria] were subjected to laboratory investigations in order to detect Enterobacteria associated with Avian influenza virus [AI] outbreaks in broiler flocks. Samples were collected from heart blood and liver for bacteriological examination. The samples for viral isolation were inoculated into specific pathogen free [SPF] embryonated chicken eggs [9-11 days old]. The allantoic fluids [AF] were harvested 36 hours post incubation and were tested by using Haemagglutination [HA] test, Haemagglutination-inhibition [HI] test, Agar gel precipitation test [AGPT] and Reverse Transcription Polymerase Chain Reaction [RTPCR]. Escherichia coli and Salmonella were determined by using a specific media for isolation and identification of both enterobacteria isolates. Twenty isolates out of twenty four [20 / 24] flocks which represent 83.33% of examined outbreaks having haemagglutination-inhibition titer by using avian influenza [AI] common type A antibodies ranging from 24 to28. The same isolates were also confirmed by Agar gel precipitation test [AGPT] and Reverse Transcription Polymerase Chain Reaction [RT-PCR]. A total of 16 samples out of 20 [16 / 20] [represented 80%] examined samples from broiler flocks associated with Avian influenza virus outbreaks proved to be E.coli while a total of 4 samples[4 / 20] [represented 20%] proved to be Salmonella enteritidis. The results of bacterial isolates were confirmed by biochemical test and serological identification

Evaluation of Two ELISA and Two Indirect Hemagglutination Tests for Serodiagnosis of Pulmonary Hydatid Disease

To establish a definite diagnosis for pulmonary hydatid disease, combination of radiology and serology is useful. In this study, 19 preoperative sera from patients with surgically confirmed pulmonary hydatidosis, 40 sera from patients with other parasitosis and pulmonary diseases, and 20 sera from healthy donors were evaluated using 4 different serological tests, i.e., the commercial ELISA (ELISA-kit) test, the ELISA (ELISA-lab) test prepared in our laboratory, the commercial indirect hemagglutination assay kit (IHA-kit) test, and the IHA test using sensitized sheep red blood cells with tannic acid (IHA-TA). The ELISA-kit was the most sensitive (84.2%) and the most specific test (100.0%). The ELISA-kit also demonstrated the highest positive (100.0%) and negative (95.2%) predictive values. The sensitivity of the ELISA-lab test, that we prepared, was found to be 73.6%, whereas the IHA-kit test and the IHA-TA test were found to be 73.6% and 68.4%, respectively. The specificity of these tests was 96.6%, 98.3%, and 83.3%, respectively. When all 4 tests were assessed together, it was found that the sensitivity had risen to 94.7%. When the ELISA-kit was assessed with the IHA-kit and IHA-TA together, it was found that the sensitivity was 89.5% and 84.2%, respectively. Likewise, the combination of the ELISA-lab and IHA-kit or IHA-TA allowed us to achieve a sensitivity of 84.2% in cases of pulmonary echinococcosis. In conclusion, the diagnosis would be imminent if least 2 tests were applied together.

Screening and Identification of Unexpected Red Cell Antibodies by Simultaneous LISS/Coombs and NaCl/Enzyme Gel Methods

We evaluated the clinical usefulness of simultaneous LISS/Coombs and NaCl/Enzyme testing using the gel method for screening and identification of unexpected antibodies in 15,014 samples. When unexpected antibodies were detected by either screening test, those antibodies were identified using both the LISS/Coombs and the NaCl/Enzyme gel test. The positive screening rates of the LISS/Coombs, NaCl/Enzyme, and combined tests (excluding 25 autoantibody cases) were 0.48%, 1.29%, and 1.39%, respectively. Among the 57 samples positive by both screening methods, the antibodies in 19.3% could be identified only by the NaCl/Enzyme method. Among the 137 samples positive only by NaCl/Enzyme screening, 74.5% showed positive results in antibody identification only by the NaCl/Enzyme test, although 7.3% were also positive in the LISS/Coombs test. The NaCl/Enzyme method thus showed about threefold higher detection rates than the LISS/Coombs method, especially in screening for Rh antibodies, and higher exact identification rates and discriminatory power for identifying mixed antibodies. Addition of the NaCl/Enzyme method to routine laboratory procedures may detect and identify considerable numbers of significant antibodies that might be missed if only the LISS/Coombs method is used.

Evaluation of Automated Architect Syphilis TP as a Diagnostic Laboratory Screening Test for Syphilis / 대한진단검사의학회지

BACKGROUND: The aim of the study was to establish a new syphilis test algorithm using Architect Syphilis TP (Abbott Japan, Japan: AST), a fully automated treponemal antibody test, as a screening test in a university hospital laboratory. We evaluated performance characteristics of AST in various patient groups. METHODS: A total of 1,357 serum samples obtained from patients at a university hospital from June to August, 2008 were categorized into checkup, preoperative, other diseases, diagnosis (clinically suspected of syphilis), and follow up groups. We compared the results of AST with those of RPR (N=1,276) or Treponema pallidum hemagglutination assay (TPHA, N=81). Samples with discrepant results between RPR or TPHA and AST were retested by fluorescent treponemal antibody absorption test (FTA-ABS) and all patients' clinical records were thoroughly reviewed. RESULTS: The positive rate of AST was significantly higher than that of RPR in preoperative and other diseases groups and was the same as that of RPR in diagnosis group. There were no significant differences in check up and follow up groups. The results of AST showed 97.4% (1,243/1,276) and 97.5% (79/81) concordance rates with those of RPR and TPHA, respectively. Among 26 RPR-AST discrepant and FTA-ABS confirmed cases, there were 20 RPR false-negatives, 4 RPR false-positives, 1 AST false-negative, and 1 AST false-positive. CONCLUSIONS: Based on the results and literature review, we established a new syphilis test algorithm using AST as a screening test, which would be helpful for detection of more syphilis patients including latent infections.

Ascaris lumbricoides: epitopes del Sistema P por método de cinética de la hemaglutinación / Ascaris lumbricoides: P system epithopes by haemogglutination kinetics method

Experiencias previas demostraron epitopes P1 en Ascaris lumbricoides por inhibición de la aglutinación. La cinética de la hemaglutinación aplica la extinción óptica relativa producida por un haz de luz trasmitido a través de una suspensión de pequeñas partículas, y permite obtener una estimación paramétrica de la tasa de hemaglutinación. El objetivo de este trabajo fue utilizar este método para demostrar la presencia de epitopes del Sistema P en extractos de A. lumbricoides. La técnica de inhibición de la aglutinación permitió seleccionar 10 extractos: 3 con y 7 sin epitopes P1. Se registró la cinética de la reacción Control: anti- P1- eritrocitos - P1 y la cinética de la misma reacción, previo contacto del anticuerpo con el extracto. Se calcularon y se compararon los valores de ³ EOR % (variación en el porcentaje de extinción óptica relativa) para ambas reacciones. Los resultados evidenciaron la presencia de epitopes P1, que no habían sido detectados por inhibición de la aglutinación, en 3 extractos. Para los extractos restantes, hubo coincidencia entre los resultados obtenidos por los dos métodos. La cinética de la hemaglutinación es sencilla, rápida y fácilmente adaptable para las experiencias en que se necesite evaluar una reacción antígeno-anticuerpo a través de la hemaglutinación.

Summary Previous experiences have demonstrated P1 epithopes in Ascaris lumbricoides by inhibition agglutination. Haemogglutination kinetics applies the relative optical extinction produced on a light beam transmitted through a suspension of small particles, giving a parametric estimate of the haemagglutination rate.The aim was to use this method for the demonstration of P System epithopes presence in A. lumbricoides extracts. inhibition agglutination test enabled the selection of 10 extracts: 3 with and 7 without P1 epithopes. Kinetics of anti- P1 - P1 erythrocyte control reaction and kinectics of the same reaction with a previous contact between antibody and extract were registered. ³ EOR % values (relative optical extinction variation) for both reactions were calculated and compared between themselves. The results proved P1 epithopes presence in 3 extracts, which had not been detected by inhibition agglutination. The results coincided for the remaining extracts by both methods. Haemogglutination kinetics is simple and fast and it is easily adapted for experiences in which the investigator needs to assess the antibody-antigen reaction by haemogglutination.

Reverse passive haemagglutination (RPHA) test for detection of mycobacterial antigen in the cerebrospinal fluid for diagnosis of tubercular meningitis.

BACKGROUND: Various serological techniques have been developed to detect antibodies and antigens in the cerebrospinal fluid (CSF) for diagnosis of tubercular meningitis. Most of the serological assays are ELISA based. Attempts have been made to use much simpler antigen detection techniques like the reverse passive haemagglutination (RPHA)which is simple and cost-effective. AIMS: To evaluate the reverse passive haemagglutination (RPHA) test for detection of mycobacterial antigens in the CSF for diagnosis of tubercular meningitis. METHODS: In the present study, we have made the use of polyclonal antiserum against heat killed whole Mycobacterium tuberculosis bacilli to sensitize the RBCs in RPHA to detect antigens in clinically suspected cases. A total of 46 cases (clinically suspected TBM 24, culture proven TBM 2, non- TBM cases 20) were included in the present study for detecting M. tuberculosis antigen in the CSF specimens. RESULTS: Of the 26 test CSF specimens, 13 CSF specimens were positive by RPHA while 4 of the 20 control CSF specimens were also reactive. Two culture positive specimens included in the study were positive by RPHA. Of the 4 control CSF specimens positive by RPHA, 3 were culture proven cases of pneumococcal meningitis and 1 was a case of cryptococcal meningitis. The RPHA is found to be 50% sensitive and 80% specific; and showed a 76.4 % positive predictive value and a 55.2 % negative predictive value. CONCLUSION: The RPHA is a simple test that could be used as an adjunct in diagnosing TBM. It does not require any special equipment or technically trained or skilled manpower. It is economical and can be afforded for use in community where TBM is more prevalent. Even though the present study showed a poor sensitivity and specificity, further identification, characterization and evaluation of better immuno-dominant and specific antigens or epitopes, and the usage of antibodies developed against such mycobacterial antigens might improve the sensitivity and specificity of this test.

Prevalence of echinococcosis in street dogs in tripole district, Libya

Cystic echinococcosis due to E. granulosus is a serious public health and livestock economy problem in Libya. Kato thick smear examination of 50 street dogs stools showed that they had Echinococcus granulosus [58%], Taenia spp. [14%], Diplydium caninum [16%], Toxocara canis [121%] and 20% were parasite-free. The stool examination using Kato thick smear was more sensitive and more specific that the indirect haemaggltination test. The results were discussed with general review of the disease in Libya

Risk factors contributing to fascioliasis endemicity in a focus in Dakahlia governorate 1- human host

Zoonotic fascioliasis is a problem not only in Dakahlia Governorate, but also in other Egyptian Governorates. Two hundreds and twenty patients randomly selected with suggesting manifestations were examined for fascioliasis. A total of 23 [10.4%] were positive by Kato thick smears, of which 21 were from Kafr El-Hessah and two from Oweish El-Hager. The proven human fascioliasis was examined for anti -Fasciola antibodies by IHAT and ELISA [Fhes], haemoglobin level, eosinophils percent, serum bilirubin and liver function tests. IHAT gave 82.61% positive results [19/23], and ELISA gave 100% positive results [23/23]. The clinical signs ranged between splenomegaly and ascitis in 4.34% for each up to hepatomegaly in 73.91% and mild fever in 78.26% but 2 cases were asymptomatic. Mild eosinophilia and moderate anaemia were recorded with means of 11 +/- 5.8 and 10 +/- 1.3 respectively. Mean serum bilirubin was not elevated [0.91 +/- 0.51 gm/dl]. Liver function tests [AST within normal range in all cases; <40 unit /ml but one patient had ALT above normal; >45 unit/ml]

Levantamento soro-epidemiológico da infecção pelo vírus da Anemia Infecciosa Eqüina, da Influenza Eqüina-2 e do Herpesvírus Eqüino-1 em rebanhos do sul do Estado do Pará, Brasil / Seroepidemiological survey of infection of equine infectious anemia, equine influenza type 2 and equine herpesvirus type 1 in herds of south Pará State, Brazil

Os vírus da anemia infecciosa eqüina (EIAV), da influenza eqüina tipo 2 (EIV-2) e o herpesvírus eqüino tipo 1 (EHV-1) são agentes causadores de enfermidades que podem causar graves prejuízos econômicos. O objetivo deste presente estudo foi estimar a freqüência de anticorpos contra o EIAV, EIV-2 e o EHV-1 em rebanhos do sul do Estado do Pará, Brasil. Os anticorpos contra EIAV, EIV-2 e EHV- 1 foram detectados pelo teste de IDGA, pelo método de inibição da hemaglutinação e pela técnica de soroneutralização (TCID50 =100), respectivamente. Amostras de sangue de 672, 514 e de 506 equídeos saudáveis e sem histórico de vacinação contra nenhum dos três vírus foram testadas, respectivamente, para EIAV, EIV-2, EHV-1. A seguinte freqüência de soro reativos foi observada: 1,34% para o EIAV; 35,79% para o EIV-2; 45,45% para o EHV-1. Estes resultados indicam que estes agentes estão presentes no rebanho paraense e a adoção de medidas de manejo e profilaxia devem ser priorizadas, garantindo deste modo, a prosperidade da eqüideocultura brasileira.

Equine infectious anemia virus (EIA V), equine influenza virus type 2 (EIV-2) and equine herpesvirus type 1 (EHV-1) are the causal agents of diseases that may bring economical Iosses. The aim of this present study was to estimate the frequency of antibodies against EIAV, EIV-2 and EHV-1 in herds of south Pará State, Brazil. Antibodies against EIAV, EIV-2 and EHV-1 were detected by AGID, hemagglutination inhibition method and serum neutralization technique (TCID50 =100), respectively. Blood samples of 572, 514, and 506 healthy equine unvaccinated against any of the three viruses were tested, respectively, for EIAV, EIV-2 and EHV-1. The following frequencies of serum reactors animals were observed: EIAV,1,34%; EIV-2, 35,79%; EHV-1, 45,45%. These results show that the agents are present in herds from Pará herds and the adoption of measures of management and prophylaxis should be prioritized, ensuring, thereby, the prosperity of brazilian's breeding equine.

Anticuerpos anti-Trypanosoma cruzi en donantes de sangre / Serological screening of antibodies to Trypanosoma cruzi

La vía transfusional es la segunda ruta más común de infección de la enfermedad de Chagas en regiones endémicas. Para el tamizaje de donantes de sangre, la OMS recomienda el uso de al menos dos ensayos. Las pruebas convencionales, Enzimoinmunoensayo (ELISA) y hemo-aglutinación indirecta (HAI), no son suficientemente sensibles y específicas. El objetivo de este trabajo fue estudiar la prevalencia de anti-T.cruzi en nuestra población de donantes de sangre, y comparar las sensibilidades y especificidades relativas de diferentes ensayos. Evaluamos 12479 donantes consecutivos con HAI y ELISA y de ellos, seleccionamos un panel de 138 muestras para estudiar sensibilidad y 784 para especificidad. La seroprevalencia fue 1,89 por ciento (ES: 0,122; IC: 1,65-2,14; p<0,05) y 1,10 por ciento (ES: 0,094; IC: 0,91-1,29; p<0,05) se confirmaron positivos. De los ELISAs, el recombinante presentó la mayor especificidad relativa y se observó una muy buena performance en cuanto a la sensibilidad, con valores de densidad óptica más elevados. Este ensayo es útil para tamizaje o como un tercer método para confirmar resultados discordantes en donantes de sangre.

Optimización y validación de un método de Elisa en tubo para la determinación de la potencia de preparados de inmunoglobulina Anti-D / ELISA in tube to determine potency of anti-D immunoglobuline

La cuantificación de la potencia en preparaciones de inmunoglobulina anti-D (Ig anti-D) requiere de metodologías costosas y de difícil ejecución, como lo es el método de hemaglutinación automatizado. Ante la necesidad de implementar métodos alternativos, se optimizó y validó un enzimoinmunoanálisis (ELISA) competitivo, utilizando tubos de poliestireno como fase sólida. El método consistió en: a) adsorción de glóbulos rojos grupo 0 Rh positivo (fenotipo R1R1) a la fase sólida; b) reacción competitiva de, cantidades variables de Ig anti-D no marcada (muestra de anti-D o preparación de referencia) y de una cantidad constante del Ac monoclonal biotinilado (Brad-5 biotinilado), por los sitios de unión D de los góbulos rojos; c) revelado de la unión del Brad-5 mediante el conjugado extravidina/fosfatasa alcalina; d) punto final con OHNa 3M; e) lectura de absorbancia a 405nm. La potencia relativa de la muestra problema respecto del estándar se determinó mediante el modelo estadístico de líneas paralelas. Resultados: la validación arrojó los siguientes resultados: rango lineal entre 0.094 y 1.5 ug/ml con un coeficiente de correlación (R²) de 0.99, precisión intermedia entre 5.7 y 28.1 (CV por ciento), repetibilidad entre 1.6 y 15.8 (CV por ciento) y una recuperación del 100 por ciento (entre 90 y 110 por ciento). Los controles de especificidad fueron satisfactorios. Según los resultados obtenidos, el método de ELISA descripto puede ser utilizado para la determinación de la potencia en preparaciones de Ig anti-D como así también en pooles de plasma y semielaborados. Por su robustez y fácil implementación es un método aplicable en laboratorios de baja complejidad.

Nuevo uso de un medio de preservación de eritrocitos para la prueba inversa / New use of preservation medium for erythrocytes to perfom inverse test

Fundamento: los medios actuales de preservación de eritrocitos humanos (MPE) artesanales son eficaces por cortos períodos. Objetivo: obtener un MPE útil por un período prolongado, para efectuar la prueba inversa del grupo sanguíneo ABO en donantes de sangre y receptores transfusionales. Material y método: la técnica se basó en utilizar el MPE de la OMS para controles de calidad de contadores hematológicos (glutaraldehído 0,37 por ciento, formaldehído 2,7 por ciento, citrato trisódico 26 por ciento, y antibióticos). Se prepararon distintos lotes al 3 y 5 por ciento de glóbulos preservados a partir de sangre de bolsa de donantes con serología no reactiva. Se ensayaron 1.080 muestras. Se evaluaron reacciones en platina y en tubo. Se comparó la reactividad de los glóbulos en MPE y en solución salina. Se evaluó el grado de discrepancia con respecto a la prueba directa ABO dentro de los 40 días de preparados los reactivos. Resultados: los resultados obtenidos en platina dentro de las 2 semanas mostraron una discrepancia total del 2,3 por ciento, siendo en todos los casos, aglutinación débil. Las discrepancias fueron resueltas al pasar a soporte tubo. De las 1030 muestras evaluadas en soporte tubo con MPE se obtuvo una discrepancia del 0,7 por ciento dentro de los 40 días de preparación. El grado de discrepancia en función del tiempo de preparación reactivo se incrementó del 0,2 al 0,7 por ciento entre la tercera y la cuarta semanas. Estos porcentajes no difieren con los descriptos en la bibliografía con otros reactivos. Fijando el límite de discrepancia en 0,2 por ciento y en soporte tubo, el reactivo es estable hasta los 21 días. Conclusiones: el presente trabajo se considera un aporte a la sección de inmunohematología para evitar la preparación diaria de suspensiones y economizar en la compra de reactivo comercial. A futuro también podría utilizarse como MPE en los paneles para anticuerpos irregulares.

Diagnóstico serológico de sífilis en pacientes cubanos con VIH/SIDA / Serological diagnosis of syphilis in HIV/AIDS Cuban

Se utilizó la prueba no treponémica de detección rápida de reaginas plasmáticas y la prueba treponémica de hemaglutinación de Treponema pallidum, en la detección de la infección por T. pallidum en 60 hombres que presentaban la infección VIH/SIDA con diagnóstico clínico-epidemiológico de sífilis. Se confirmó que 30 por ciento presentaba sífilis reciente adquirida sintomática o latente y que 10 por ciento poseía marcadores de infección pasada tratada o de sífilis tardía adquirida latente, mientras que en 60 por ciento restante no se detectó reactividad serológica. Se realizó, además, un estudio de seroprevalencia de anticuerpos reagínicos por detección rápida de reaginas plasmáticas en 59 mujeres con VIH/SIDA, utilizando como control 67 mujeres negativas a este virus, todas sin síntomas compatibles con la infección sifilítica. Se obtuvo que 20,3 y 11,9 por ciento, respectivamente, mostraban reactividad, lo que estableció un diagnóstico probable de sífilis o una serorresitencia a una sífilis anterior. Estos resultados muestran una fuerte asociación entre sífilis y VIH/SIDA y que ambas enfermedades pueden coexistir en un mismo paciente