Evaluation of a rapid immunochromatographic test for detection of KPC in clinical isolates of Enterobacteriaceae and Pseudomonas species.

The KPC K-SeT immunochromatographic test (Coris BioConcept®, Gembloux, Belgium) has been widely used for detection of KPC in Enterobacteriaceae with reported sensitivities and specificities of 100%. However, to our knowledge, there are no reports of its use in KPC-positive Pseudomonas species. We evaluated the KPC K-SeT test in 36 clinical isolates of Enterobacteriaceae (21 KPC-positive and 15 KPC-negative) and 20 Pseudomonas species (5 KPC-positive and 15 KPC-negative) using conventional PCR for carbapenemase genes as the reference method. The KPC K-SeT test detected 25 out of 26 KPC-positive isolates (96.1%). The undetected isolate was 1 P. aeruginosa bearing the mutation D179Y in the omega loop region of KPC-2 carbapenemase. This mutation was already reported in Enterobacteriaceae as conferring resistance to ceftazidime-avibactam. To our knowledge, this is the first report of evaluation of KPC K-SeT test in KPC-positive P. aeruginosa isolates.

[Test evaluation and strategy proposal to detect and to characterize carbapenemase-producing gram negative bacilli]. / Evaluación de test rápidos y diseño de una estrategia para la detección y caracterización de carbapenemasas en cepas de bacilos gramnegativos.

INTRODUCTION: The detection of carbapenemase-producing gram negative bacilli is complicated, because there are available multiple options of test. The confirmation of the enzyme by molecular characterization is not available in all laboratories in our country. OBJECTIVE: To propose a fast, efficient and simple strategy to detect and confirm CPB. MATERIALS AND METHODS: 39 CPB isolates and 8 non-producing were used to evaluate the phenotypic test Carba NP, CarbAcineto NP and Blue-Carba, validating the test Xpert® Carba-R, to be used directly with bacterial colonies with conventional PCR. RESULTS: The sensitivity of Carba NP, CarbAcineto NP and Blue-Carba was 79,5; 87,2 y 84,6%, respectively; and specificity was 79.5; 87.2 and 84.6%, respectively. The limit of detection of Xpert® Carba-R was different for each carbapenemasa: 40.8 ufc/reaction to KPC and NDM and 30.6 ufc/reaction to VIM. DISCUSSION: On isolates with decreased susceptibility to carbapenems we propose to use as screening the test CarbAcineto NP, follow by Xpert®Carba-R to characterize the carbapenemase and adopt specific infection control measures.

Evaluación de test rápidos y diseño de una estrategia para la detección y caracterización de carbapenemasas en cepas de bacilos gramnegativos / Test evaluation and strategy proposal to detect and to characterize carbapenemase-producing gram negative bacilli

Resumen Introducción: La detección de bacilos gramnegativos productores de carbapenemasas es compleja, existiendo actualmente varios test disponibles. La confirmación mediante la caracterización molecular de la enzima no está disponible en todos los laboratorios del país. Objetivo: Plantear una estrategia rápida, eficiente y sencilla para la detección y confirmación de carbapenemasas en cepas de bacilos gramnegativos. Material y Métodos: Se utilizaron 39 aislados productores y ocho no productores de carbapenemasas para evaluar los test fenotípicos Carba NP, CarbAcineto NP, Blue-Carba y validar el test molecular Xpert® Carba-R directo de la colonia en comparación con RPC convencional. Resultados: La sensibilidad para Carba NP, CarbAcineto NP y Blue-Carba fue de 79,5; 87,2 y 84,6%, respectivamente; mientras que la especificidad fue de 100; 100 y 87,5%, respectivamente. La concordancia entre RPC convencional y Xpert® Carba-R fue de 100%. El límite de detección para Xpert® Carba-R fue diferente según el tipo de carbapenemasa: 40,8 ufc/reacción par KPC y NDM y 30,6 ufc/reacción para VIM. Discusión: En aislados con susceptibilidad disminuida a carbapenémicos se propone realizar un tamizaje con CarbAcineto NP, para luego caracterizar la carbapenemasa con Xpert® Carba-R y adoptar las medidas de contención específica: para cada caso.

Introduction: The detection of carbapenemase-producing gram negative bacilli is complicated, because there are available multiple options of test. The confirmation of the enzyme by molecular characterization is not available in all laboratories in our country. Objective: To propose a fast, efficient and simple strategy to detect and confirm CPB. Materials and Methods: 39 CPB isolates and 8 non-producing were used to evaluate the phenotypic test Carba NP, CarbAcineto NP and Blue-Carba, validating the test Xpert® Carba-R, to be used directly with bacterial colonies with conventional PCR. Results: The sensitivity of Carba NP, CarbAcineto NP and Blue-Carba was 79,5; 87,2 y 84,6%, respectively; and specificity was 79.5; 87.2 and 84.6%, respectively. The limit of detection of Xpert® Carba-R was different for each carbapenemasa: 40.8 ufc/reaction to KPC and NDM and 30.6 ufc/reaction to VIM. Discussion: On isolates with decreased susceptibility to carbapenems we propose to use as screening the test CarbAcineto NP, follow by Xpert®Carba-R to characterize the carbapenemase and adopt specific infection control measures.

[Presence of Bordetella holmesii in an outbreak of pertussis in Chile]. / Presencia de Bordetella holmesii en brote epidémico de coqueluche en Chile.

The incidence of whooping cough in Chile ranges from 4.1 and 7.5 per hundred thousand inhabitants. B. pertussis detection is performed by Real Time PCR (Q-PCR) directed to the insertion sequence IS481. However, this sequence is also found in the genome of B. bronchiseptica and B. holmesii. The latter is also a respiratory pathogen whose clinical features are similar to B. pertussis. However, it is important to differentiate between these species because in immunosuppressed patients B. holmesii is more likely to cause bacteremia and is less susceptible to erythromycin. The goal of this work is to measure prospectively and retrospectively the presence of B. holmesii in samples reported positive for B. pertussis in the period 2010-2011. During this period, 1994 nasopharyngeal specimens entered the laboratory for Bordetella sp. PCR, of which 224 were positive. The analysis by Q-PCR directed to the recA gene of B. holmesii of all 224 positive samples determined a prevalence of B. holmesii of 0.6% (12/1994). Because of its more aggressive behavior in immunosupressed patients and its different resistance pattern, routine screening of B. pertussis and B. holmesii is currently performed for all samples in which Bordetella sp PCR is initially detected.

Presencia de Bordetella holmesii en brote epidémico de coqueluche en Chile / Presence of Bordetella holmesii in an outbreak of pertussis in Chile

The incidence of whooping cough in Chile ranges from 4.1 and 7.5 per hundred thousand inhabitants. B. pertussis detection is performed by Real Time PCR (Q-PCR) directed to the insertion sequence IS481. However, this sequence is also found in the genome of B. bronchiseptica and B. holmesii. The latter is also a respiratory pathogen whose clinical features are similar to B. pertussis. However, it is important to differentiate between these species because in immunosuppressed patients B. holmesii is more likely to cause bacteremia and is less susceptible to erythromycin. The goal of this work is to measure prospectively and retrospectively the presence of B. holmesii in samples reported positive for B. pertussis in the period 2010-2011. During this period, 1994 nasopharyngeal specimens entered the laboratory for Bordetella sp. PCR, of which 224 were positive. The analysis by Q-PCR directed to the recA gene of B. holmesii of all 224 positive samples determined a prevalence of B. holmesii of 0.6% (12/1994). Because of its more aggressive behavior in immunosupressed patients and its different resistance pattern, routine screening of B. pertussis and B. holmesii is currently performed for all samples in which Bordetella sp PCR is initially detected.

La incidencia de coqueluche en Chile varía entre 4,1 y 7,5 por 100.000 habitantes. La detección de Bordetella pertussis se realiza por RPC-tiempo real (Q-RPC) dirigida a la secuencia de inserción IS481. Sin embargo, esta secuencia se encuentra también en el genoma de B. bronchiseptica y B. holmesii. Este último es también un patógeno respiratorio que produce un cuadro similar a B. pertussis. Sin embargo, es importante diferenciar entre estas especies porque en pacientes inmunosuprimidos B. holmesii tiene mayor tendencia a causar bacteriemia y además es menos susceptible a eritromicina. El objetivo de este trabajo es determinar, prospectiva y retrospectivamente, la presencia de B. holmesii en muestras informadas positivas para B. pertussis en el período 2010-2011. Durante ese período ingresaron al laboratorio 1. 994 muestras de hisopado nasofaríngeo para RPC de Bordetella sp., de las cuales 224 fueron positivas. El análisis por Q-RPC dirigido al gen recA de B. holmesii de las 224 muestras positivas determinó una prevalencia de B. holmesii de 0,6% (12/1994). Debido al comportamiento más agresivo en inmunosuprimidos y al patrón de resistencia de B. holmesi, se decide incorporar la detección de rutina de B. pertussis y B. holmesii en todas las muestras en que se detecta inicialmente la presencia de Bordetella sp.

[Impact of mass spectrometry by MALDI-TOF MS for the rapid identification of aerobic and anaerobic bacteria of clinical importance]. / Impacto de la espectrometría de masas por MALDI-TOF MS en la identificación rápida de bacterias aeróbicas y anaeróbicas de importancia clínica.

BACKGROUND: MALDI-TOF MS (Matrix Assisted Laser Desorption Ionization -Time of Flight Mass Spectrometry) technology, recently introduced in the microbiology laboratory has proven to be a precise and rapid method for bacterial identification. OBJECTIVE: To evaluate the performance, costs associated and turnaround time of MALDI-TOF in a routine laboratory. MATERIAL AND METHOD: Five hundred and sixty one clinical isolates (281 aerobes and 280 anaerobes) previously identified by conventional methods were evaluated. Discordances were resolved by means of 16S rRNA sequencing. RESULTS: MALDI-TOF identified 95, 7% of the aerobes isolates and 86, 4% of the anaerobes. The groups with better performance were the enterobacteriacea and Bacteroides spp with 95% and 100% identification at the species level. The error rate of MALDI-TOF and conventional methods compared to sequencing was 0, 39% and 9, 4% respectively. The costs associated were 8 times lower with a turnaround time of 6 hours. CONCLUSION: MALDI-TOF proved to be simple, precise and less expensive technology compared to the traditional methods.

Impacto de la espectrometría de masas por MALDI-TOF MS en la identificación rápida de bacterias aeróbicas y anaeróbicas de importancia clínica / Impact of mass spectrometry by MALDI-TOF MS for the rapid identification of aerobic and anaerobic bacteria of clinical importance

Background: MALDI-TOF MS (Matrix Assisted Laser Desorption Ionization -Time of Flight Mass Spectrometry) technology, recently introduced in the microbiology laboratory has proven to be a precise and rapid method for bacterial identification. Objective: To evaluate the performance, costs associated and turnaround time of MALDI-TOF in a routine laboratory. Material and Method: Five hundred and sixty one clinical isolates (281 aerobes and 280 anaerobes) previously identified by conventional methods were evaluated. Discordances were resolved by means of 16S rRNA sequencing. Results: MALDI-TOF identified 95, 7% of the aerobes isolates and 86, 4% of the anaerobes. The groups with better performance were the enterobacteriacea and Bacteroides spp with 95% and 100% identification at the species level. The error rate of MALDI-TOF and conventional methods compared to sequencing was 0, 39% and 9, 4% respectively. The costs associated were 8 times lower with a turnaround time of 6 hours. Conclusion: MALDI-TOF proved to be simple, precise and less expensive technology compared to the traditional methods.

Introducción: La tecnología MALDI-TOF MS (Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry) incorporada recientemente en el laboratorio de microbiología ha demostrando ser un método rápido y preciso para la identificación bacteriana. Objetivo: Evaluar el desempeño de MALDI-TOF para la identificación de aislados clínicos, comparar los costos asociados y el tiempo en la entrega de resultados en un laboratorio de rutina. Material y Método: Se evaluaron un total de 561 aislados de pacientes (281 aeróbicos y 280 anaeróbicos estrictos) identificados previamente por métodos convencionales, los que fueron identificados por MALDI-TOF. Las discordancias fueron resueltas mediante secuenciación del 16S ARNr. Resultados: MALDI-TOF identificó adecuadamente a 95,7% de los aislados aeróbi-cos y 86,4% de los anaeróbicos estrictos, observándose el mayor porcentajes de identificación a nivel de especie en los grupos de enterobacterias y Bacteroides spp (95 y 100% respectivamente). La tasa de error de MALDI-TOF y métodos convencionales vs secuenciación fue de 0,39 y 9,4%, respectivamente. El costo asociado por identificación fue ocho veces menor que el de los métodos tradicionales con una demora promedio de seis horas en la entrega de resultados. Conclusión: MALDI-TOF mostró ser una tecnología simple, precisa y de menor costo que los métodos tradicionales.