Identificación directa de microorganismos a partir de hemocultivos positivos utilizando espectrometría de masas (MALDI-TOF MS) / Direct identification of microorganisms in positive blood culture bottles by mass spectrometry (MALDI-TOF MS)

Resumen La espectrometría de masas (MALDI-TOF MS) permite la identificación de microorganismos directamente de las colonias en pocos minutos. En este estudio se ha desarrollado y evaluado un protocolo reducido para identificar microorganismos directamente de las botellas de hemocultivos positivos en 30 minutos con una alta sensibilidad y especificidad, utilizando MALDITOF. Un total de 2535 hemocultivos positivos fueron estudiados por el método directo de MALDI-TOF MS, a partir de una alícuota de sangre de las botellas y el método de colonia, utilizando los cultivos desarrollados en medios sólidos. Del total de hemocultivos positivos incluidos en este estudio, 2381 (93,9%) fueron monomicrobianos y 146 (5,8%) polimicrobianos. Mil trescientos treinta (55,9%) de los aislamientos correspondieron a cocos gram positivos, 922 (38,7%) a bacilos gram negativos, 60 (2,5%) a anaerobios, 36 (1,5%) a bacilos gram positivos y 13 a levaduras. La concordancia global entre ambos métodos fue del 81,7% a nivel de especie (90,0% para bacilos gram negativos, 76,7% para cocos gram positivos y 33,3% para bacilos gram positivos). Se identificó al menos un germen en el 88% de las botellas positivas con desarrollo polimicrobiano. Los resultados del presente estudio demostraron que el protocolo basado en MALDI-TOF MS permite la identificación microbiana directamente de hemocultivos positivos en un tiempo corto, con una alta precisión, con excepción de los bacilos gram positivos.

Abstract Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) enables the identification of microorganisms directly from colonies within minutes. In this study this technology was adapted and tested for use with blood culture bottles, thus allowing identification in 30 minutes once the blood culture is detected as positive by the automate. A total of 2535 blood culture bottles reported as positive were tested by MALDI-TOF MS directly from positive blood culture bottles and colonies. A total of 2381 (93.9%) and 146 (5.8%) of the positive blood cultures were monomicrobial and polymicrobial, respectively. And 1330 (55.9%), 922 (38.7%), 60 (2.5%), 36 (1.5%) and 13 of the isolates were gram-positive cocci (GPC), gram-negative bacilli (GNB), anaerobic bacteria, gram-positive bacilli (GPB) and yeast respectively. Concordance between both methods was 81.7% (76.7% of GPC, 90% of GNB, 74.2% of anaerobic bacteria and 33.3% of GPB) in monomicrobial cultures. Eighty eight per cent of the polymicrobial cultures were identified correctly in at least one of the two bacteria. The results of the present study show that this fast, MALDI-TOF MS based method allows microbial identification directly from positive blood culture in a short time, with a high accuracy, with the exception of gram-positive bacilli.

Resumo A espectrometria de massa (MALDI-TOF MS) permite a identificação de microorganismos diretamente das colônias em minutos. Nesse estudo, foi desenvolvido um protocolo reduzido para identificar microrganismos diretamente das garrafas de hemoculturas positivas em 30 minutos com alta sensibilidade e especificidade, utilizando MALDI-TOF. Um total de 2535 hemoculturas positivas foram relatadas -o método direto de MALDI-TOF MS, a partir de uma alíquota de sangue dos vidros e o método de colônia, a partir das culturas desenvolvidas em meios sólidos. Do total de hemoculturas positivas incluídas neste estudo, 2.381 (93,9%) eram monomicrobianas e 146 (5,8%) eram polimicrobianas. Mil trezentos e trinta (55,9%) dos isolados corresponderam a cocos gram-positivos, 922 (38,7%) bacilos gram-negativos, 60 (2,5%) anaeróbios, 36 (1,5%) bacilos gram-positivos e 13 leveduras. A concordância geral entre os dois métodos foi de 81,7% em nivel de especie (90,0% para bacilos gram-negativos, 76,7% para cocos gram-positivos e 33,3% para bacilos gram-positivos). Pelo menos um germe foi identificado em 88% dos vidros positivos com desenvolvimento polimicrobiano. Os resultados do presente estudo demonstraram que o protocolo baseado em MALDI-TOF MS permite a identificação microbiana diretamente de hemoculturas positivas em um curto espaço de tempo, com alta precisão, com exceção de bacilos gram-positivos.

Direct Identification and Antimicrobial Susceptibility Testing of Bacteria From Positive Blood Culture Bottles by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry and the Vitek 2 System

BACKGROUND: We evaluated the reliability and accuracy of the combined use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) bacterial identification and Vitek 2 antimicrobial susceptibility testing (AST) for bacteria from positive blood culture bottles. METHODS: Direct identification and AST were performed in parallel to the standard methods in monomicrobial positive blood culture bottles. In total, 254 isolates grown on aerobic and/or anaerobic bottles were identified with MALDI-TOF Vitek MS (bioMerieux, France), and 1,978 microorganism/antimicrobial agent combinations were assessed. For isolates from anaerobic bottles, an aliquot of the culture broth was centrifuged, washed, and filtered through a nylon mesh. For isolates from aerobic/pediatric bottles, a lysis step using 9.26% ammonium chloride solution and 2% saponin solution was included. RESULTS: The overall correct identification rate was 81.8% (208/254) and that for gram-positive/gram-negative isolates was 73.9%/92.6%, respectively, and it was 81.8%, 87.6%, and 57.9% for isolates from aerobic, anaerobic, and pediatric bottles, respectively. Identification was not possible in 45 cases, and most of these isolates were streptococci (N=14) and coagulase-negative staphylococci (N=11). Misidentification occurred only in one case. Compared with standard methods, direct AST showed 97.9% (1,936/1,978) agreement with very major error of 0.25%, major error of 0.05%, and minor error of 1.8%. CONCLUSIONS: This simple and cost-effective sample preparation method gives reliable results for the direct identification and AST of bacteria. For the identification of streptococci and coagulase-negative staphylococci, the method should be further improved.

Comparison of four methods for rapid identification of Staphylococcus aureus directly from BACTEC 9240 blood culture system.

Purpose: Differentiation of Staphylococcus aureus (S. aureus) from coagulase-negative staphylococci is very important in blood stream infections. Identification of S. aureus and coagulase-negative staphylococci (CoNS) from blood cultures takes generally 18-24 h after positive signaling on continuously monitored automated blood culture system. In this study, we evaluated the performance of tube coagulase test (TCT), slide agglutination test (Dry Spot Staphytect Plus), conventional polymerase chain reaction (PCR) and LightCycler Staphylococcus MGrade kit directly from blood culture bottles to achieve rapid identification of S. aureus by using the BACTEC 9240 blood culture system. Materials and Methods: A total of 129 BACTEC 9240 bottles growing gram-positive cocci suggesting Staphylococci were tested directly from blood culture broths (BCBs) with TCT, Dry Spot Staphytect Plus, conventional PCR and LightCycler Staphylococcus MGrade kit for rapid identification of S. aureus. Results: The sensitivities of the tests were 99, 68, 99 and 100%, respectively. Conclusion: Our results suggested that 2 h TCT was found to be simple and inexpensive method for the rapid identification of S. aureus directly from positive blood cultures.