Detection of bacterial contamination in platelet concentrates from Brazilian donors by molecular amplification of the ribosomal 16S gene.

OBJECTIVE: The aim of our work was to establish a semi-automated high-throughput DNA amplification method for the universal screening of bacteria in platelet concentrates (PCs). BACKGROUND: Among cases of transfusion transmission of infectious agents, bacterial contamination ranks first in the number of events, morbidity and mortality. Transmission occurs mainly by transfused PCs. Automated culture is adopted by some blood banks for screening of bacterial contamination, but this procedure is expensive and has a relatively long turnaround time. METHODS: PCs were spiked with suspensions of five different bacterial species in a final concentration of 1 and 10 colony-forming units (CFU) per millilitre. After incubation, the presence of bacteria was investigated by real-time polymerase chain reaction (PCR) and by the Enhanced Bacterial Detection System (eBDS, Pall) assay as a reference method. Real-time PCR amplification was performed with a set of universal primers and probes targeting the 16S rRNA gene. Co-amplification of human mitochondrial DNA served as an internal control. RESULTS: Using the real-time PCR method, it was possible to detect the presence of all bacterial species tested with an initial concentration of 10 CFU mL-1 24 h after contamination, except for Staphylococcus hominis. The PCR assay also detected, at 24 h, the presence of Serratia marcescens and Enterobacter cloacae with an initial concentration of 1 CFU mL-1 . CONCLUSIONS: The real-time PCR assay may be a reliable alternative to conventional culture methods in the screening of bacterial contamination of PCs, enabling bacterial detection even with a low initial concentration of microorganisms.

State of the art: Vest Nile Virus circulation surveillance in Italy and transfusion risk early prevention methods.

After the Chikungunya outbreak in 2007 in Italy, a national Plan for the surveillance of human vector-borne diseases has been implemented and annually updated on the basis of the epidemiological changes based-evidences. The transfusion Authorities cooperates, since 2008, with public health services and veterinary (entomological and ornithological) surveillance systems. In some Italian regions, a common protocol for exchanging data is in place to identify the West Nile Virus (WNV) circulation in birds and mosquitoes: the goal is to anticipate the introduction of WNV-NAT screening in blood donors and, on the other hand, to limit testing only in geographic areas where the virus circulation is actual. The integration of surveillance activities and a multidisciplinary approach made it possible to introduce efficient and preventive measures for reducing the risk of of transmission of WNV trough blood, tissues and organ donation.

Prevalência e risco transfusional para doenças infectocontagiosas em doadores de sangue / Prevalence and transfusional risc for infect diseases among blood donors

Objetivos: Conhecer a prevalência e incidência para doenças infecto-contagiosas entre doadores de sangue em hemocentro regional do sul do país e estabelecer o risco transfusional. Materiais e Métodos: Foram analisadas as doações com marcadores sorológicos positivos entre os anos de 2003 a 2005. Resultados: Para o ano de 2003 observou-se uma prevalência de 2,76% para hepatite B, 0,013% para a hepatite C e 0,067% para a sífilis. O risco transfusional estimado no ano para a hepatite B foi de 0,17 casos/milhão de doações. Para o ano de 2004 observou-se uma prevalência de 2,75% para a hepatite B, 0,013% para HIV/AIDS, 0,026% para o HTLV, 0,013% para a doença de Chagas e 0,13% para a sífilis. No ano, o risco transfusional estimado para a hepatite B foi de 0,32 casos/milhão de doações, para o HTLV foi de 0,02 casos/ milhão de doações e sífilis com 0,09 casos/milhão de doações. Em 2005 observou-se prevalência de 2,3% para a hepatite B, 0,012% para o HTLV, 0,012% para a doença de Chagas, 0,15% para a sífilis e 0,012% para a hepatite C. No ano o risco transfusional estimado para a hepatite B teve taxa de 0,16 casos/milhão de doações e sífilis com taxa de 0,03 casos/milhão de doações. Conclusões: hepatite B e sífilis são as condições mais prevalentes e incidentes e trazem um maior risco de transmissão via transfusão sanguínea.

Replacement of HIV p24 Ag test by a multiplex RT-PCR method for primary screening of blood donors

Nucleic Acid Testing (NAT) as a tool for primary screening of blood donors became a reality in the end of the 1990 decade. We report here the development of an "in-house" RT-PCR method that allows the simultaneous (multiplex) detection of HCV and HIV-RNA in addition to an artificial RNA employed as an external control. This method detects all HIV group M subtypes, plus group N and O, with a detection threshold of 500 IU/mL. After validation, the method replaced p24 Ag testing, in use for blood donation screening since 1996 at our services. From July 2001 to February 2006, 102,469 donations were tested and 41 (0.04 percent) were found HIV-RNA reactive. One NAT-only reactive donation (antibody non-reactive) was observed, with subsequent seroconversion of the implied donor, giving a yield of 1:102,469. This rate is in contrast to the international experience that reports a detection of approximately 1:600,000 - 1:3,100,000 of isolated HIV-RNA donations.

O uso de testes de ácidos nucleicos (NAT) na rotina de triagem de doadores de sangue tornou-se uma realidade ao final da década de 1990. Descreve-se aqui uma metodologia de RT-PCR multiplex "in-house" que permite a detecção simultânea dos RNAs dos vírus HIV e HCV além de uma molécula artificial de RNA usada como controle externo. O método detecta todos os subtipos de HIV do grupo M e também do grupo N e O, com uma sensibilidade de 500 UI/mL. Após validação, este teste substituiu o do antígeno p24, até então na rotina de triagem em nosso laboratório, desde 1996. De julho de 2001 a fevereiro de 2006 foram testadas 102.469 doações e 41 (0.04 por cento) foram NAT reativas. Uma doação NAT isoladamente reativa (anticorpo não-reativa) foi detectada com soroconversão subseqüente do doador, portanto, o rendimento do NAT nesta população até o presente momento é de 1:102.469. Este número contrasta com a experiência obtida internacionalmente, onde taxas de 1:600.000 - 1:3.100.000 foram descritas.

Primary screening of blood donors by nat testing for HCV-RNA: development of an "in-house" method and results

An "in-house" RT-PCR method was developed that allows the simultaneous detection of the RNA of the Hepatitis C Virus (HCV) and an artificial RNA employed as an external control. Samples were analyzed in pools of 6-12 donations, each donation included in two pools, one horizontal and one vertical, permitting the immediate identification of a reactive donation, obviating the need for pool dismembering. The whole process took 6-8 hours per day and results were issued in parallel to serology. The method was shown to detect all six HCV genotypes and a sensitivity of 500 IU/mL was achieved (95 percent hit rate). Until July 2005, 139,678 donations were tested and 315 (0.23 percent) were found reactive for HCV-RNA. Except for five false-positives, all 310 presented the corresponding antibody as well, so the yield of NAT-only donations was zero, presenting a specificity of 99.83 percent. Detection of a window period donation, in the population studied, will probably demand testing of a larger number of donations. International experience is showing a rate of 1:200,000 - 1:500,000 of isolated HCV-RNA reactive donations.

Desenvolveu-se uma metodologia própria ("in-house") baseada em RT-PCR, que permite detectar simultaneamente o RNA do vírus HCV e de um RNA artificial empregado como controle externo. As amostras são analisadas em pools de 6-12 doações, cada doação sendo incluída em dois pools diferentes, um horizontal e um vertical, permitindo a identificação imediata de uma doação reativa, sem a necessidade de desmembrar-se um pool reativo. O processo todo consumiu de 6-8 horas diárias e os resultados foram emitidos em paralelo à sorologia. O método detectou os seis genótipos de HCV, com um limite de sensibilidade de 500 UI/mL (95 por cento hit rate). Até julho de 2005 haviam sido testadas 139.678 doações com a detecção de 315 (0,23 por cento) doações reativas para HCV-RNA. Exceto cinco falso-positivas, todas estas doações também apresentavam o respectivo anticorpo, portanto não se detectou nenhuma doação em janela imunológica. A especificidade foi de 99,83 por cento. A detecção de amostra em janela imunológica, nesta população de doadores, provavelmente demandará a análise de um número maior de doações, espelhando-se na experiência internacional que tem mostrado a detecção de amostras HCV-RNA isoladas em 1:200.000 - 1:500.000 doações.

La seguridad de las reservas sanguíneas en la República Mexicana durante los años 1999 a 2003 / Safety of the blood supply in Mexico from 1999 to 2003

Introduction. The incidence of the infection by the viruses of the human immunodeficiency (HIV), hepatitis B (HBV) and hepatitis C (HCV) has diminished enormously in developed countries during the last 20 years; nevertheless, in our country we do not know such an incidence and, therefore, the safety of our blood supply. Material and methods. We performed a retrospective analysis at the Centro Nacional de la Transfusión Sanguínea (CNTS) assessing 17,176,298 serologic tests including HIV, HCV and HBV carried on 5,725,432 blood units collected and informed to the CNTS from January 1999 to December 2003 by all the Mexican blood banks. Prevalence, incidence and residual risk of each one of the aforementioned serologic markers were calculated. Results. The five years mean prevalence for HIV, HBV and HCV has remained steady. The residual risk (RR) when hemagglutination test was employed was 1:977 for HCV; 1:1,564 for HBV and 1:1,262 for HIV. Whereas the RR when ELISA was performed decreased to 1:2,781 for HCV; 1:3,185 for HBV and 1:9,969 for HIV. If nucleic acid amplification test were employed, RR would be 1:8,170 for HBV; 1:9,915 for HCV and 1:19,939 for HIV. Conclusions. The theoretical risk for transfusion-transmitted diseases in our country is still worrisome.

Introducción. La transmisión del virus de la inmunodeficiencia humana (VIH), de la hepatitis C (VHC) y de la hepatitis B (VHB) por transfusión sanguínea ha disminuido de manera significativa en los países industrializados durante los últimos 20 años; sin embargo, en nuestro país aún no conocemos dicha incidencia y consecuentemente la seguridad de nuestras reservas sanguíneas. Material y métodos. Se realizó un estudio retrospectivo en el Centro Nacional de la Transfusión Sanguínea (CNTS) analizando 17,176,298 pruebas serológicas incluyendo VIH, VHC y VHB realizadas a 5,725,432 unidades de sangre captadas e informadas al CNTS de enero de 1999 a diciembre de 2003 por todos los bancos de la República Mexicana. Se calcularon la prevalencia, la incidencia y el riesgo residual para cada uno de los marcadores serológicos mencionados. Resultados. La prevalencia en los cinco años para el VIH, VHB y VHC se ha mantenido estable entre los donantes. El riesgo residual encontrado con la prueba de hemaglutinación fue de 1:977 para el VHC; de 1:1,564 para el VHB y de 1:1,262 para el VIH. Con la prueba de ELISA el riesgo descendió a 1:2,781 para el VHC; 1:3,185 para el VHB y 1:9,969 para el VIH. Si se empleara la prueba de amplificación de ácidos nucleicos, el riesgo disminuiría a 1:8,170 para el VHB; 1:9,915 para el VHC y a 1:19,939 para el VIH. Conclusiones. El riesgo de transmitir infecciones por transfusión sanguínea en nuestro país es todavía preocupante.