PCR-based identification of Burkholderia pseudomallei.

DNA amplification techniques are being used increasingly in clinical laboratories to confirm the identity of medically important bacteria. A PCR-based identification method has been in use in our centre for 10 years for Burkholderia pseudomallei and was used to confirm the identity of bacteria isolated from cases of melioidosis in Ceará since 2003. This particular method has been used as a reference standard for less discriminatory methods. In this study we evaluated three PCR-based methods of B. pseudomallei identification and used DNA sequencing to resolve discrepancies between PCR-based results and phenotypic identification methods. The established semi-nested PCR protocol for B. pseudomallei 16-23s spacer region produced a consistent negative result for one of our 100 test isolates (BCC #99), but correctly identified all 71 other B. pseudomallei isolates tested. Anomalous sequence variation was detected at the inner, reverse primer binding site for this method. PCR methods were developed for detection of two other B. pseudomallei bacterial metabolic genes. The conventional lpxO PCR protocol had a sensitivity of 0.89 and a specificity of 1.00, while a real-time lpxO protocol performed even better with sensitivity and specificity of 1.00, and 1.00. This method identified all B. pseudomallei isolates including the PCR-negative discrepant isolate. The phaC PCR protocol detected the gene in all B. pseudomallei and all but three B. cepacia isolates, making this method unsuitable for PCR-based identification of B. pseudomallei. This experience with PCR-based B. pseudomallei identification methods indicates that single PCR targets should be used with caution for identification of these bacteria, and need to be interpreted alongside phenotypic and alternative molecular methods such as gene sequencing.

PCR-based identification of Burkholderia pseudomallei / Identificação de Burkholderia pseudomallei baseada em PCR

DNA amplification techniques are being used increasingly in clinical laboratories to confirm the identity of medically important bacteria. A PCR-based identification method has been in use in our centre for 10 years for Burkholderia pseudomallei and was used to confirm the identity of bacteria isolated from cases of melioidosis in Ceará since 2003. This particular method has been used as a reference standard for less discriminatory methods. In this study we evaluated three PCR-based methods of B. pseudomallei identification and used DNA sequencing to resolve discrepancies between PCR-based results and phenotypic identification methods. The established semi-nested PCR protocol for B. pseudomallei 16-23s spacer region produced a consistent negative result for one of our 100 test isolates (BCC #99), but correctly identified all 71 other B. pseudomallei isolates tested. Anomalous sequence variation was detected at the inner, reverse primer binding site for this method. PCR methods were developed for detection of two other B. pseudomallei bacterial metabolic genes. The conventional lpxO PCR protocol had a sensitivity of 0.89 and a specificity of 1.00, while a real-time lpxO protocol performed even better with sensitivity and specificity of 1.00, and 1.00. This method identified all B. pseudomallei isolates including the PCR-negative discrepant isolate. The phaC PCR protocol detected the gene in all B. pseudomallei and all but three B. cepacia isolates, making this method unsuitable for PCR-based identification of B. pseudomallei. This experience with PCR-based B. pseudomallei identification methods indicates that single PCR targets should be used with caution for identification of these bacteria, and need to be interpreted alongside phenotypic and alternative molecular methods such as gene sequencing.

As técnicas de amplificação de DNA estão sendo cada vez mais utilizadas em laboratórios clínicos para a confirmação da identificação de bactérias que têm importância médica. Um método de identificação de Burkholderia pseudomallei baseado em PCR tem sido usado em nosso centro há 10 anos e foi utilizado para confirmar a identificação de bactérias isoladas de casos de melioidose no Ceará desde 2003. Este método particular tem sido usado como padrão ouro para métodos menos discriminatórios. Nesse estudo, avaliamos três métodos de identificação de B. pseudomallei baseados em PCR e usamos seqüenciamento de DNA para solucionar discrepâncias entre os resultados baseados em PCR e os métodos de identificação fenotípica. O estabelecido protocolo de PCR semi-nested para a região espacial 16-23s da B. pseudomallei produziu um consistente resultado negativo para um de nossos 100 isolados testados (BCC#99), mas identificou corretamente todos os outros 71 isolados de B. pseudomallei. Uma variação anômala da seqüência foi detectada na região interna do sítio de ligação do primer reverso para este método. Métodos de PCR foram desenvolvidos para a detecção de outros dois genes bacterianos metabólicos de B. pseudomallei. O protocolo de PCR IpxO convencional teve sensibilidade de 0,89 e especificidade de 1,0, enquanto que o PCR em tempo real mostrou-se ainda melhor, com sensibilidade de 1,0 e especificidade de 1,0. Este método identificou todos os isolados de B. pseudomallei, incluindo o isolado discrepante que teve o PCR negativo. O protocolo de PCR phaC detectou o gene de todos os B. pseudomallei e em todos exceto três isolados de B. cepacia, tornando este método de identificação de B. pseudomallei baseado em PCR inadequado. Esta experiência com métodos de identificação de B. pseudomallei baseados em PCR indica que devemos ter precaução quando estes forem utilizados sozinhos para identificação dessa bactéria e que eles necessitam ser interpretados em conjunto com métodos fenotípicos e moleculares alternativos, tais como seqüenciamento genético.

Melioidosis, northeastern Brazil.

Melioidosis was first recognized in northeastern Brazil in 2003. Confirmation of additional cases from the 2003 cluster in Ceará, more recent cases in other districts, environmental isolation of Burkholderia pseudomallei, molecular confirmation and typing results, and positive serosurveillance specimens indicate that melioidosis is more widespread in northeastern Brazil than previously thought.