Geographical origin of chronic Chagas disease patients in Brazil impacts the performance of commercial tests for anti-T. cruzi IgG

BACKGROUND Chagas disease, caused by Trypanosoma cruzi, affects nearly six million people worldwide. Various serological tests have been developed for its diagnosis. OBJECTIVE Examine the performance of a set of commercial immunological assays in relation to the geographical origin of the patient sample comparing four states of Brazil: Amazonas (AM), Mato Grosso do Sul (MS), Minas Gerais (MG) and Piauí (PI). METHODS Seven immunoassays were employed to detect anti-T. cruzi IgG antibodies in 379 patient samples that had been previously diagnosed using the two-step protocol required by the Brazilian Ministry of Health. FINDINGS A significant variation in the percent reactive was calculated for the samples from AM and MS, while the PI and MG showed a significant variation in the percent non-reactive. The average reactivity index was significantly higher for samples from the states of PI and MG states than AM and MS. MAIN CONCLUSIONS All tests presented a satisfactory performance overall. Yet, variations were observed that were associated to the region of origin of the samples. Our analyses suggest that future evaluations of immunoassays should include a sampling of sera from regions where the test will be applied in addition to the available International Biological Reference Standards.

Application of WHO International Biological Reference Standards to evaluate commercial serological tests for chronic Chagas disease.

BACKGROUND Chagas disease, resulting from Trypanosoma cruzi infections, continues to be a health concern mainly in Latin American countries where the parasite is endemic. The laboratory diagnosis of a chronic infection is determined through serological assays for antibodies against T. cruzi and several tests are available that differ in key components, formats and methodologies. To date, no single test meets the criteria of a gold standard. The situation is further complicated by the difficulties associated with performance comparisons between different immunoassays or methodologies executed at different times and geographical areas. OBJECTIVE To improve the diagnosis of Chagas disease, the WHO coordinated the development of two International Biological Reference Standards for antibodies against anti-T. cruzi: NIBSC 09/186 and NIBSC 09/188 that respectively represent geographical regions with the highest prevalence of TcII and TcI lineages of the parasite. METHODS The principle goal of this study was to verify the behavior of these standards when assayed by several commercially available serological tests that employ different methods to capture and detect human anti-T. cruzi antibodies. FINDINGS AND MAIN CONCLUSIONS The results reinforce the recommendation that these standards be considered for performance evaluations of commercialised immunoassays and should be an integral step in the development of new test components or assay paradigms.

Application of WHO International Biological Reference Standards to evaluate commercial serological tests for chronic Chagas disease

BACKGROUND Chagas disease, resulting from Trypanosoma cruzi infections, continues to be a health concern mainly in Latin American countries where the parasite is endemic. The laboratory diagnosis of a chronic infection is determined through serological assays for antibodies against T. cruzi and several tests are available that differ in key components, formats and methodologies. To date, no single test meets the criteria of a gold standard. The situation is further complicated by the difficulties associated with performance comparisons between different immunoassays or methodologies executed at different times and geographical areas. OBJECTIVE To improve the diagnosis of Chagas disease, the WHO coordinated the development of two International Biological Reference Standards for antibodies against anti-T. cruzi: NIBSC 09/186 and NIBSC 09/188 that respectively represent geographical regions with the highest prevalence of TcII and TcI lineages of the parasite. METHODS The principle goal of this study was to verify the behavior of these standards when assayed by several commercially available serological tests that employ different methods to capture and detect human anti-T. cruzi antibodies. FINDINGS AND MAIN CONCLUSIONS The results reinforce the recommendation that these standards be considered for performance evaluations of commercialised immunoassays and should be an integral step in the development of new test components or assay paradigms.

Inmunoserología y métodos moleculares para el diagnóstico de Chagas: revisión sistemática rápida / Immunoserology and molecular techniques for diagnosis of Chagas´ disease: rapid systematic review / Imunoserologia e métodos moleculares para o diagnóstico de Chagas: revisão sistemática rápida

Con el objetivo de incrementar la precisión diagnóstica, la Organización Mundial de la Salud recomienda la realización de dos o más pruebas inmunoserológicas para el diagnóstico de la enfermedad de Chagas en etapa crónica. El objetivo de este trabajo fue realizar una revisión sistemática rápida acerca del desempeño de las técnicas inmunoserológicas y métodos moleculares en la población general. Se identificaron 178 estudios de los cuales fueron incluidos nueve. Las técnicas de ELISA mostraron la mayor sensibilidad (82-98%) y especificidad (96-100%). Los métodos rápidos mostraron valores de sensibilidad entre 88-93% y especificidad 97- 100%, mientras que los métodos moleculares (PCR) presentaron niveles muy variables de sensibilidad (22-92%) y especificidad (70-100%). Estos resultados muestran que las técnicas de ELISA cuentan con una sensibilidad y especificidad adecuadas. La PCR, al igual que los métodos rápidos, mostró una gran variabilidad en los resultados, debido principalmente a la heterogeneidad de la técnicas y profusión de métodos elaborados de manera in house.

In order to increase diagnostic accuracy, the World Health Organization recommends performing two or more immunoserological tests for the diagnosis of Chagas disease in chronic stage. The aim of this work was to make a rapid systematic review of the performance of immunoserological techniques and molecular methods in the general population. A total of 178 studies were identified, nine ofwhich were included. ELISA techniques showed the highest sensitivity (82-98%) and specificity (96-100%). Rapid methods presented values of sensitivity between 88-93% and 97-100% of specificity, while the molecular methods (PCR) showed highly variable levels of sensitivity (22-92%) and specificity (70-100%). These results indicate that ELISA techniques have adequate sensitivity and specificity. PCR, as well as rapid methods, showed great variability in the results, mainly due to the heterogeneity of the techniques and abundance of "in-house" methods.

Visando a aumentar a precisão do diagnóstico, a Organização Mundial da Saúde recomenda executar dois ou mais testes imunoserológicos para o diagnóstico na fase crônica da doença de Chagas. O objetivo deste trabalho foi fazer uma revisão sistemática rápida sobre o desempenho de técnicas imunoserológicas e métodos moleculares na população em geral. Foram identificados 178 estudos dos quais se incluíram nove. Técnicas de ELISA mostraram a maior sensibilidade (82-98%) e especificidade (96-100%). Métodos rápidos apresentaram valores de sensibilidade e especificidade de 88-93% e 97-100%, respectivamente enquanto que os métodos moleculares (PCR) tinham níveis extremamente variáveis de sensibilidade (22-92%) e especificidade (70-100%). Estes resultados mostram que as técnicas de ELISA possuem sensibilidade e especificidade adequadas. A PCR, bem como os métodos rápidos, mostrou grande variabilidade nos resultados, principalmente devido à heterogeneidade das técnicas e profusão de métodos desenvolvidos de modo in house.

Quality Control in Screening for Infectious Diseases at Blood Banks. Rationale and Methodology.

Quality control procedures are indispensable to ensure the reliability of the results provided by laboratories responsible for serological screening in blood banks. International recommendations on systems of quality management classify as a top component the inclusion of two types of control: (a) internal quality control (IQC) and (b) external quality control (EQC). In EQC it is essential to have, at least, a monthly frequency of laboratory assessment. On the other hand, IQC involves the daily use of low-reactivity control sera, which should be systematically added in all run, carried out in the laboratory for each parameter. Through the IQC analysis some variations in the criteria of run acceptance and rejection may be revealed, but it is of paramount importance to ensure the previous definition of these criteria and even more importantly, the adherence to them; and that corresponds to the validation of analytical runs of each test. Since 2010 this has been, for instance, the experience of the PNCQ*, developing external quality control programmes on serology for blood banks. These programmes use samples of lyophilized sera well-characterized for the reactivity related to the parameters used for the serological screening of blood donors. The programmes have used blind panels of six samples for monthly assessments. In the last 50 assessments, which involved 68 blood banks in Brazil, a significant number of instances of non-compliance were observed in all monthly assessments. These results provide strong support to the recommendation of systematic monthly assessments. (*) National Quality Control Programme (PNCQ).

Comparative evaluation of 11 commercialized rapid diagnostic tests for detecting Trypanosoma cruzi antibodies in serum banks in areas of endemicity and nonendemicity.

Chagas disease is one of the main public health issues in Latin America. Increasingly during the past few decades, Trypanosoma cruzi infection has been detected in North America, Europe, and the Western Pacific, mainly as a result of population movement. The limited availability of rapid serological diagnostic tests hinders rapid diagnosis and early treatment in areas of endemicity and nonendemicity. In collaboration with 11 national reference laboratories (NRLs) from different geographical areas, we evaluated the performances of commercialized serological rapid diagnostic tests (RDT) for T. cruzi infection. Eleven commercialized T. cruzi infection RDTs were evaluated on a total of 474 samples extensively tested with at least three different techniques for Chagas disease, maintained at controlled low temperatures, and stored in the serum banks of the 11 NRLs. We measured the sensitivity, specificity, and concordance of each RDT and provided an additional questionnaire to evaluate its ease of use. The selected RDTs in this study were performed under controlled laboratory conditions. Out of the 11 RDTs, we found 8 of them to be useful, with the cassette format favored over the strip. We did not observe significant differences in RDT performances in the different regions. Overall, the performance results were lower than those disclosed by the manufacturers. The results of this evaluation validate the possibility of using RDTs to diagnose Chagas disease, thereby decreasing the time to treatment at a primary health care facility for patients who are willing to be treated. Further studies should be conducted in the laboratory and in the field to confirm these data, expressly to evaluate reproducibility in resource-limited settings, or using whole blood in clinical settings in areas of endemicity and nonendemicity.

Desempenho de testes sorológicos para sífilis, treponêmicos (ELISA) e não treponêmicos (VDRL e RPR), na triagem sorológica para doadores de sangue- confirmação dos resultados por meio de três testes treponêmicos (FTAABS, WB e TPHA) / Evaluation of serological tests for syphilis, treponemal (ELISA) and non-treponemal (VDRL and RPR), in blood donors serological screening: confirmation of results by three treponemal tests (FTAABS, WB and TPHA)

Este estudo constitui uma análise do desempenho do teste sorológico treponêmico ELISA Recombinante Wiener lab. e de seis testes não treponêmicos (Laborclin: VDRL Bras. RPR Bras e RPR TRUST: Wama: VDRL e RPR; Wiener:USR) na triagem sorológica de doadores de sangue. Os resultados repetidamente reagentes (RR) foram confirmados por meio de três testes treponêmicos: FTAabs (Wama), WB (in house) e TPHA (bioMérieux). Foram analisadas 2990 amostras de soro. O teste VDRL (USR), utilizado inicialmente na triagem sorológica, apresentou reatividade em 11 (0,4por cento) amostras, com títulos variando de 1/1 até 1/32. Nos demais testes não treponêmicos, observou-se reatividade em um número de amostras que variou de 4 a 14, num total de 20 (0,7por cento) amostras das 2990 analisadas. O teste treponêmico ELISA apresentou resultados RR em 42 (1,4por cento) das amostras analisadas, entre as quais 8 (0,3por cento) se mostraram reagentes a, pelo menos, um teste não treponêmico. Das 42 amostras RR pelo teste ELISA submetidas aos testes confirmatórios de FTA-abs, TPHA e WB, 9 (21,4por cento) foram negativas para os três testes, 8 (19,1por cento) foram positivas para WB e TPHA e negativas para o FTA-abs e 25 (59,5por cento) foram positivas para os três testes. A especificidade do teste ELISA atingiu 99,7por cento, considerando-se o WB e o TPHA como confirmatórios. As 12 amostras que apresentaram resultados não reagentes no teste ELISA, mas mostraram algum tipo de reatividade para os testes não treponêmicos, tiveram resultado negativo nos testes confirmatórios utilizados. Mesmo que o descarte de bolsas seja maior quando se utiliza teste treponêmico, essa conduta parece ser a mais segura na triagem sorológica de doadores de sangue.

Evaluation of serological tests to identify Trypanosoma cruzi infection in humans and determine cross-reactivity with Trypanosoma rangeli and Leishmania spp.

Five commercially available enzyme-linked immunosorbent assays (ELISAs), one in-house ELISA, and two hemagglutination assays were evaluated to determine their diagnostic accuracy for Chagas' disease in two studies. In study 1, ELISA kits showed 100% sensitivity, but specificities ranged from 82.84% to 100% when leishmaniasis cases were included and from 95.57% to 100% when leishmaniasis cases were excluded. Kits using recombinant antigens or synthetic peptides are more specific than those using crude extracts from Trypanosoma cruzi epimastigote forms. Kits evaluated in Panama, in study 2, showed 75% to 100% sensitivity and 97.12% to 100% specificity. These data were obtained by using a Western blot assay with T. cruzi trypomastigote excreted-secreted antigens as a reference test to confirm T. cruzi infection.

[External serology quality control programs developed in Latin America with the support of PAHO from 1997 through 2000]. / Programas de control externo de calidad en serología desarrollados en América Latina con el apoyo de la OPS entre 1997 y 2000.

OBJECTIVE: To evaluate the quality of serological screening of blood donors in five groups of blood banks in Latin America that participated over the 1997-2000 period in an external serology control project developed with support from the Pan American Health Organization (PAHO). METHODS: With assistance from PAHO, the Serology Authority of the Pro-Blood Foundation/Blood Center of São Paulo (Fundação Pró-Sangue/Hemocentro de São Paulo), of São Paulo, Brazil, carried out the external quality control project and served as its "organizing center" (OC). The OC developed five external serology quality control "programs" (ESQCPs), or external evaluation activities, for the respective groups of participating blood banks. There was one ESQCP each in 1997, 1999, and 2000, and there were two in 1998. In these five programs, the number of participating blood banks ranged from 13 to 21, and the number of countries ranged from 11 to 16. In each program, the OC used a set of 24 blinded sera samples with different reactivities for the various infectious agents for which screening is obligatory in Brazil. Each participating institution in each program received a sera set, to be processed using that institution's standard screening procedures. After returning its results to the OC, each participant received an answer key for the sera set, to be used in evaluating its own performance. All the individual results were kept strictly confidential. At the end of each program, the OC prepared and sent to all the participants a final report that contained information on the overall results from that program. RESULTS: An analysis of the five programs showed that there was a lack of homogeneity among the countries with respect to the strategies and the parameters used in screening blood donors. Few laboratories screened for human T-cell-lymphotropic virus (beginning with the 1997 program, the respective rates were 17%, 27%, 35%, 39%, and 45%). Rates of screening were also low for antibodies to the hepatitis B core antigen (again, beginning with the 1997 program, the rates were 42%, 27%, 39%, 50%, and 60%). There were also important differences with respect to which tests and which combinations of tests were used, making it hard to compare the types of screening done. In the five programs, with the various tests used, the overall rate of false positive results fluctuated around 2%. The highest false positive rate for any of the tests, 4.6%, was for antibodies against the hepatitis C virus. The lowest false positive rate, 0.4%, was for antibodies against Trypanosoma cruzi. CONCLUSIONS: These results show the need for PAHO to continue using these external quality control programs as well as other activities in order to strengthen the procedures for serological screening blood banks in Latin America, until there is more uniformity in the procedures that the countries use.

Programas de control externo de la calidad en serología desarrollados en América Latina con el apoyo de la OPS entre 1997 y 2000 / External serology quality control programs developed in Latin America with the support of PAHO from 1997 through 2000

OBJETIVOS: Con el apoyo de la Organización Panamericana de la Salud (OPS), desarrollamos entre 1997 y 2000 cinco programas de control externo de la calidad en serología (PCECS) en los que participaron entre 13 y 21 bancos de sangre de 11 a 16 países de América Latina. El objetivo fue evaluar el desempeño de los bancos de sangre con respecto al tamizaje serológico realizado en donantes de sangre. MÉTODOS: Como herramienta de trabajo utilizamos conjuntos de 24 muestras de sueros anónimos con reactividades variables para los parámetros de uso obligatorio en el tamizaje serológico de donantes de sangre en Brasil. En cada PCECS enviamos un multipanel a cada institución participante para que lo procesara en las mismas condiciones de su rutina de tamizaje. Cada participante recibió la clave del multipanel para autoevaluación, después de haber devuelto los resultados obtenidos en su laboratorio. Se mantuvo siempre la más estricta confidencialidad sobre los resultados obtenidos individualmente. Al terminar de cada programa, el Centro Organizador (Superintendencia de Serología de la Fundaçäo Pró-Sangue/Hemocentro de Säo Paulo) elaboró un informe final que contenía toda la información obtenida en el programa y que fue enviado a los participantes. RESULTADOS: En el análisis de los cinco PCECS se observó falta de homogeneidad entre los países con respecto a las estrategias y a los parámetros utilizados en el tamizaje de donantes de sangre. Pocos laboratorios practicaron el tamizaje de los virus linfotrópico de células T humanas (HTLV) (17 por ciento, 27 por ciento, 35 por ciento, 39 por ciento y 45 por ciento, respectivamente y en orden creciente para los cinco PCECS) y de anticuerpos contra el antígeno nuclear del virus de la hepatitis B (anti-HBc) (42 por ciento, 27 por ciento, 39 por ciento, 50 por ciento y 60 por ciento). También se observaron diferencias importantes en cuanto a las pruebas o combinaciones de pruebas utilizadas, lo cual puede dificultar el estudio comparativo de los tipos de tamizaje. El número total de resultados positivos falsos osciló alrededor del 2 por ciento, correspondiendo el mayor valor al tamizaje de anticuerpos contra el virus de la hepatatis C (anti-VHC) (4,6 por ciento) y el menor a anti-Trypanosoma cruzi (0,4 por ciento). CONCLUSIONES: Los resultados obtenidos en este trabajo demuestran la necesidad de continuar las acciones de la OPS en América Latina para reforzar los procedimientos de...

Resultados de un programa de control de calidad externo del tamizaje serológico de anticuerpos contra Trypanosoma cruzi en donantes de sangre de Brasil / The results of an external quality control program for serological screening for antibodies against Trypanosoma cruzi in blood donors in Brazil

OBJETIVOS: A partir de 1995 en Brasil se iniciaron programas de control de calidad externo de laboratorios de serología (PCCES) abiertos a la participación de laboratorios de bancos de sangre públicos y privados. Estos programas se han puesto en práctica tres veces al año desde 1995 y cuentan con la participación de más de 100 entidades. El objetivo de este trabajo es analizar los resultados de los ocho últimos programas de evaluación del tamizaje serológico para la enfermedad de Chagas, que se realizaron entre abril de 1999 y agosto de 2001. MÉTODOS: Participaron en los ocho programas laboratorios de serología de instituciones públicas y privadas. El número de laboratorios participantes que entregó los resultados en cada uno de los ocho PCCES fue de 94, 90, 85, 94, 100, 103, 102 y 116, respectivamente. Al inicio de cada PCCES se envió un panel enmascarado a cada institución participante, con un plazo de 60 días para informar los resultados del procesamiento de las muestras. Se utilizaron pruebas de inmunoadsorción enzimática (ELISA), inmunofluorescencia indirecta (IFI) y hemaglutinación indirecta (HAI). Posteriormente, cada institución recibió del centro organizador (PANEL) la clave con los resultados correctos para su autoevaluación. Los paneles estaban compuestos por 24 muestras de sueros con diferentes reactividades a los marcadores obligatorios en el tamizaje serológico de donantes de sangre en Brasil, incluido el de muestras negativas. RESULTADOS: La técnica de ELISA fue la más utilizada en el tamizaje (92 por ciento-98 por ciento). La estrategia de tamizaje más usada por los laboratorios participantes fue la combinación de una prueba de ELISA y una de HAI (58 por ciento-83 por ciento). La mayoría de los laboratorios participantes obtuvieron resultados correctos en los diferentes programas sin resultados negativos falsos (83,6 por ciento-98,1 por ciento). De las 5 406 muestras de suero positivas a anti-Trypanosoma cruzi que hubo en los ocho programas, 85 (1,6 por ciento) fueron notificadas de negativas por 58 laboratorios, pero solamente 14 de ellos dejaron de identificar una o más muestras positivas por no conseguir detectarlas por medio de ninguna de las pruebas de tamizaje utilizadas. No se observó ninguna diferencia significativa entre bancos en los ocho programas (P = 0,5936). No se observó asociación entre los resultados negativos falsos y una muestra o sistema diagnóstico en particular...

Erros e anomalias mais comuns em laboratórios de sorologia / The most frequent errors and abnormalities in serological laboratories

Erros e anormalidades ocorrem frequentemente em laboratórios desorologia. Com o objetivo de se evitar erros é importante a adoção de medidas preventivas de controle para sua redução e eliminação da possibilidade dos mesmos. Os erros mais comumente observados são: a) variação de resultados em metodologias dqerentes; h) contaminação gerando resultados falso positivos; c) erros durante a transcrição de resultados; d)perfomance de kits e equipamentos inadequados; e) alterações no comportamento das reações entre os vários lotes dos kits.É recomendado que determinações que são consideradas de grande importância adotem as seguintes medidas: 1) qualificação e treinamento contínuo do pessoal técnico envolvido; 2) incorporação de programas de qualidade interna; 3) participação em programasde qualidade externo; 4) escolha de kits após prévia avaliação; 5) escolha dos equipamentos envolvidos nos testes baseados na existência de manutenção prévia e assistência técnica qualificada.

Errors and abnormalities occur frequently in serological laboratories. In order to avoid such errors, it is important to adopt preventative and control measures, the former reduces the occurrence of errors and the latter allows for the detection of errors, eliminating the possibility that they go undetected, and taking the necessary steps to correct them. The more common observed errors are: a) Varyingresults between different methodologies. b) Problems with contamination, generating FalsePositive results. c) Errors made duringtranscription of results, giving either False Positive or False Negative results. d) False Positive or False Negative results, for other reasons, including thepeformance of the kits and equipment used. e) Alterations in the behavior of the reactions between kits from different lot numbers. It is recommended that some measures, which are considered of great importance, be adopted andmaintained: 1) Qualification and continualtraining of the technical personnel. 2)Incorporation of internal Qualiy Control Program. 3) Participation in external Quality Control programs (Quality Assessment). 4) The choosing of kits based on prior evaluation. 5) The choosing of equipment based on the existence of preventive maintenance and qualified technical assistance.

Anti-HBc testing for blood donations in areas with intermediate hepatitis B endemicity

En otros estudios se ha evaluado el menor riesgo de infecciones transmitidas por transfusión sanguínea que acarrea el tamizaje de anticuerpos contra los antígenos nucleares de la hepatitis B (anti-HBc), sumado a la prueba detectora de antígenos de superficie (HbsAg). No obstante, la mayoría de estos estudios se basaron en datos procedentes de zonas con baja endemicidad por hepatitis B o de bancos de sangre de países desarrollados, donde la mayoría de la sangre proviene de personas que han donado previamente. A fin de examinar los pros y los contras del tamizaje de anticuerpos anti-HBc en el Brasil, los autores describen los antecedentes y la interpretación de las pruebas de tamizaje para prevenir la hepatitis postransfusión. También presentan los datos sobre el tamizaje de la hepatitis B en todas las regiones del Brasil. No hay pruebas suficientes para suspender el tamizaje de los anticuerpos anti-HBc en el país. Debe darse alta prioridad a comparar el costo-efectividad de un marcador basado en la biología molecular con el de la actual prueba detectora de anticuerpos anti-HBc aplicada comúnmente, a fin de mejorar las medidas de tamizaje que garantizan la inocuidad de la sangre

Chronic hepatitis C virus infections in brazilian patients: association with genotypes, clinical parameters and response to long term alpha interferon therapy

No presente estudo, avaliamos a importancia clinico-epidemiologica da genotipagem do VHC em 130 pacientes com diagnostico histologico de hepatite cronica C e sua influencia na resposta terapeutica sustentada. Nao se observou associacao entre os genotipos e os aspectos clinico-epidemiologicos como sexo, idade, vias de trasmissao, presenca ou nao de cirrose e tempo de infeccao. Dos 130 pacientes, 113 foram submetidos a tratamento com...