Background: The rapid identification (ID) of microorganisms from blood specimens allows a prompt and adequate patient’s management. Blood cultures (BCs) are the gold standard for diagnosing bloodstream infections but suffer from long time-to-result. Over the years, Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight mass spectrometry (MALDI-TOF-MS) has proved to be a valid support for the direct identification of microorganisms cultured on solid media and many different in-house pre-treatment protocols have been developed to identify the microorganisms in blood cultures. In this study, the MALDI-TOF Sepsityper workflow was evaluated. Methods: A total of 58 positive BCs was selected to implement the sample preparation procedure according to the manufacturers’ instructions. One ml of each BC was purified to obtain a pellet by a quick-to-perform purification protocol standardized to allow a direct ID (dID). In case of uncertain or invalid outcome an additional protein extraction step was needed for the identification (eID). In parallel, further aliquots of each BC were used for both microscopic examination with Gram staining technique and subculture onto solid media. Results: As a result of microscopic examination, Gram-positive (GP) and Gram-negative (GN) bacteria were contained in 38 (65.5%; 38/58) and 20 (34.5%; 20/58) positive BCs, respectively. For 68.4% (26/38) of BCs-containing-GP bacteria, an identification at species level was obtained by performing a dID; for the remaining 12 ones an eID was needed, and an identification at species level was obtained in 83.3% (10/12). On the other hand, for 75% (15/20) of BCs-containing-GN bacteria the dID allows the identification at species level and for the remaining 25% (5/20) an eID has been necessary. The subculture results showed 96.6% agreement with the Sepsityper workflow results. Conclusions: The Sepsityper workflow demonstrated to be a helpful tool in identifying the microorganisms from blood cultures. This method showed a high identification capability for both GP and GN bacteria, either performing a dID or an eID. In particular, the additional eID allowed the identification of 25.9% cases with uncertain or invalid outcome obtained by dID. In addition to its significant performance, Sepsityper workflow relies on a cheap, easy- and rapid-to-perform protocol highly standardized and therefore it demonstrated sensitivity to pre-analytic and analytic variables.

Evaluation of the MALDI-TOF mass spectrometry Sepsityper workflow for the identification of microorganisms in positive blood cultures / Calderaro, Adriana; Buttrini, Mirko; Martinelli, Monica; Covan, Silvia; Di Maio, Alan; Montecchini, Sara; Farina, Benedetta; Arcangeletti, Maria Cristina; Chezzi, Carlo; De Conto, Flora. - (2022).

Evaluation of the MALDI-TOF mass spectrometry Sepsityper workflow for the identification of microorganisms in positive blood cultures

Adriana Calderaro;Mirko Buttrini;Monica Martinelli;Silvia Covan;Sara Montecchini;Maria Cristina Arcangeletti;Carlo Chezzi;Flora De Conto
2022

Abstract

Background: The rapid identification (ID) of microorganisms from blood specimens allows a prompt and adequate patient’s management. Blood cultures (BCs) are the gold standard for diagnosing bloodstream infections but suffer from long time-to-result. Over the years, Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight mass spectrometry (MALDI-TOF-MS) has proved to be a valid support for the direct identification of microorganisms cultured on solid media and many different in-house pre-treatment protocols have been developed to identify the microorganisms in blood cultures. In this study, the MALDI-TOF Sepsityper workflow was evaluated. Methods: A total of 58 positive BCs was selected to implement the sample preparation procedure according to the manufacturers’ instructions. One ml of each BC was purified to obtain a pellet by a quick-to-perform purification protocol standardized to allow a direct ID (dID). In case of uncertain or invalid outcome an additional protein extraction step was needed for the identification (eID). In parallel, further aliquots of each BC were used for both microscopic examination with Gram staining technique and subculture onto solid media. Results: As a result of microscopic examination, Gram-positive (GP) and Gram-negative (GN) bacteria were contained in 38 (65.5%; 38/58) and 20 (34.5%; 20/58) positive BCs, respectively. For 68.4% (26/38) of BCs-containing-GP bacteria, an identification at species level was obtained by performing a dID; for the remaining 12 ones an eID was needed, and an identification at species level was obtained in 83.3% (10/12). On the other hand, for 75% (15/20) of BCs-containing-GN bacteria the dID allows the identification at species level and for the remaining 25% (5/20) an eID has been necessary. The subculture results showed 96.6% agreement with the Sepsityper workflow results. Conclusions: The Sepsityper workflow demonstrated to be a helpful tool in identifying the microorganisms from blood cultures. This method showed a high identification capability for both GP and GN bacteria, either performing a dID or an eID. In particular, the additional eID allowed the identification of 25.9% cases with uncertain or invalid outcome obtained by dID. In addition to its significant performance, Sepsityper workflow relies on a cheap, easy- and rapid-to-perform protocol highly standardized and therefore it demonstrated sensitivity to pre-analytic and analytic variables.
Evaluation of the MALDI-TOF mass spectrometry Sepsityper workflow for the identification of microorganisms in positive blood cultures / Calderaro, Adriana; Buttrini, Mirko; Martinelli, Monica; Covan, Silvia; Di Maio, Alan; Montecchini, Sara; Farina, Benedetta; Arcangeletti, Maria Cristina; Chezzi, Carlo; De Conto, Flora. - (2022).
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11381/2919542
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