ABSTRACT
La espectrometría de masas (EM) (matrix assisted laser desorption ionization-time of flight) MALDI-TOF demostró ser una herramienta robusta para la identificación de numerosos grupos taxonómicos. No obstante, presenta limitaciones. Una ventaja clave de la técnica es la flexibilidad para la incorporación de espectros proteicos de microorganismos ausentes en la base de datos comercial. Dada la prevalencia de Burkholderia contaminans en los pacientes fibroquísticos en Argentina, y a que en ellos es crucial el diagnóstico microbiológico rápido y confiable, la EM MALDI-TOF surge como una herramienta estratégica. El objetivo del trabajo fue desarrollar una base de datos adicional con espectros peptídicos de aislamientos de referencia de B. contaminans. La misma demostró ser exitosa para la identificación del 97% de los aislamientos analizados. Por lo cual la EM MALDI-TOF con la base de datos extendida resultó ser una herramienta útil para la identificación y diferenciación de otras especies relacionadas a B. contaminans.
MALDI-TOF (matrix assisted laser desorption ionization-time of flight) mass spectrometry (MS) proved to be a robust tool for the identification of numerous taxonomic groups. However, it has limitations. A key advantage of this technique is the flexibility for the incorporation of protein profiles of microorganisms not included in the commercial database. Due to the prevalence of Burkholderia contaminans in fibrocystic patients in Argentina and the fact that rapid and reliable microbiological diagnosis is crucial in them, MALDI-TOF MS emerges as a strategic tool. The aim of this work was to develop an additional database with peptide spectra of reference isolates of B. contaminans. This database demonstrated to be successful for the identification of 97% of the isolates analyzed. Therefore, MALDI-TOF MS with the extended database was a useful tool for the identification and differentiation of other related species to B. contaminans.
Subject(s)
Humans , Databases, Factual , Bacteriological Techniques , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Burkholderia/isolation & purification , Species Specificity , Bacterial Proteins/analysis , Algorithms , Reproducibility of Results , Burkholderia Infections/complications , Burkholderia Infections/microbiology , Burkholderia/classification , Burkholderia/chemistry , Cystic Fibrosis/complications , Cystic Fibrosis/microbiologyABSTRACT
Aim: The objective was to describe an outbreak of bloodstream infections by Burkholderia cepacia complex (Bcc) in bone marrow transplant and hematology outpatients. Methods: On February 15, 2008 a Bcc outbreak was suspected. 24 cases were identified. Demographic and clinical data were evaluated. Environment and healthcare workers' (HCW) hands were cultured. Species were determined and typed. Reinforcement of hand hygiene, central venous catheter (CVC) care, infusion therapy, and maintenance of laminar flow cabinet were undertaken. 16 different HCWs had cared for the CVCs. Multi-dose heparin and saline were prepared on counter common to both units. Findings: 14 patients had B. multivorans (one patient had also B. cenopacia), six non-multivorans Bcc and one did not belong to Bcc. Clone A B. multivorans occurred in 12 patients (from Hematology); in 10 their CVC had been used on February 11/12. Environmental and HCW cultures were negative. All patients were treated with meropenem, and ceftazidime lock-therapy. Eight patients (30%) were hospitalized. No deaths occurred. After control measures (multidose vial for single patient; CVC lock with ceftazidime; cleaning of laminar flow cabinet; hand hygiene improvement; use of cabinet to store prepared medication), no new cases occurred. Conclusions: This polyclonal outbreak may be explained by a common source containing multiple species of Bcc, maybe the laminar flow cabinet common to both units. There may have been contamination by B. multivorans (clone A) of multi-dose vials.
O objetivo foi descrever um surto de infecções da corrente sanguínea por complexo B. cepacia (Bcc) nos ambulatórios de hematologia e transplante de medula óssea. Métodos: Em 15/02/2008, um surto de Bcc foi suspeitado. 24 casos foram identificados. Os dados demográficos e clínicos foram avaliados. Mãos de profissionais da saúde e ambiente foram cultivadas. Espécies foram determinadas e tipadas. Reforço da higiene das mãos, cuidados com cateteres, terapia de infusão e manutenção da câmara de fluxo laminar foram realizadas. 16 profissionais de saúde (PS) diferentes manipularam os cateteres. Heparina multidoses e soro eram preparadas em um balcão comum a ambas as unidades. Resultados: 14 pacientes tiveram B. multivorans (um paciente teve também B. cenopacia), 6 Bcc não-multivorans e um teve um agente não pertencente a Bcc. Clone A de B. multivorans ocorreu em 12 pacientes (da Hematologia), em 10 o cateter havia sido utilizado nos dias 11 ou 12 de fevereiro. Culturas ambientais e de PS foram negativos. Todos os pacientes foram tratados com meropenem e selo de ceftazidima. Oito pacientes (30%) foram hospitalizados. Não ocorreram mortes. Após as medidas de controle, nenhum novo caso ocorreu. Conclusões: Este surto policlonal pode ser explicado por uma fonte comum contendo várias espécies de Bcc, talvez a câmara de fluxo laminar comum a ambas as unidades. Pode ter havido contaminação por B. multivorans (clone A) de frascos multi-dose.
Subject(s)
Adolescent , Adult , Child , Child, Preschool , Female , Humans , Male , Middle Aged , Young Adult , Bacteremia/microbiology , Burkholderia Infections/microbiology , Burkholderia cepacia complex/isolation & purification , Catheter-Related Infections/microbiology , Disease Outbreaks , Bone Marrow Transplantation , Bacteremia/epidemiology , Burkholderia Infections/epidemiology , Catheter-Related Infections/epidemiology , Hematologic DiseasesABSTRACT
Las especies del complejo Burkholderia cepacia (CBC) son capaces de causar infecciones crónicas del tracto respiratorio en pacientes con fibrosis quística y en otros individuos inmunocomprometidos. La mayoría de estas especies exhiben alta resistencia a la terapia antibiótica, lo que genera la necesidad de una detección rápida y precisa para poder implementar estrategias de control adecuadas. En este trabajo se utilizó la técnica de reacción en cadena de la polimerasa (PCR) para amplificar el gen recA (PCR-recA), con el fin de identificar microorganismos pertenecientes al CBC. Con este método molecular como referencia, se evaluó la sensibilidad (S) y la especificidad (E) de dos sistemas de identificación comerciales automatizados, VITEK 2 y API 20NE (bioMérieux®), así como también el valor de las pruebas bioquímicas manuales más representativas para la identificación de estos microorganismos. El método VITEK 2 presentó una S del 71,1 % y una E del 100 %; para el método API 20NE, estos valores fueron 69,7 % y 90,2 %, respectivamente. En cuanto a las pruebas fenotípicas manuales, los resultados obtenidos fueron más heterogéneos, lo que posiblemente se deba a que estas bacterias podrían sufrir presión selectiva para sobrevivir en pacientes crónicos y perder factores fenotípicos característicos. La técnica de PCR-recA resultó de fácil implementación, por lo que cabe considerar a esta técnica de identificación como una opción viable, aun en laboratorios de diagnóstico clínico de mediana complejidad.
Species belonging to the Burkholderia cepacia complex (BCC) are capable of causing chronic respiratory tract infections in patients suffering from cystic fibrosis as wel as in immunocompromised individuals. Most of these species are highly resistant to antibiotic therapy, generating the need for their rapid and accurate detection for the proper treatment and clinical management of these patients. In this wok, the polymerase chain reaction (PCR) technique based on the amplification of the recA gene (PCR-recA) was applied for an accurate identification of bacteria belonging to the BCC. Sensitivity (S) and specificity (E) of two biochemically-based commercial automated systems, API 20NE and VITEK 2 (bioMérieux®), and of the most representative biochemical manual tests for the identification of the Burkholderia cepacia complex were herein evaluated. The commercial systems VITEK 2 and API 20NE showed the following sensitivity and specificity vaues for identification to the species level, S: 71.1 %, E: 100 %, S: 69.7 %, E: 90.2 %, respectively. More complex results were observed for phenotypic manual tests, since BCC bacteria can undergo selective pressure to survive in chronic patients causing the loss of their typical phenotypic characteristics. The PCR-recA technique was easy to implement even in medium-complexity clinical diagnostic laboratories.
Subject(s)
Humans , Bacterial Typing Techniques/methods , Burkholderia Infections/microbiology , Burkholderia cepacia complex/isolation & purification , Reagent Kits, Diagnostic , Respiratory Tract Infections/microbiology , Automation , Bacterial Proteins/genetics , Burkholderia Infections/diagnosis , Burkholderia Infections/etiology , Colorimetry/methods , Cystic Fibrosis/complications , Disease Susceptibility , DNA, Bacterial/genetics , Genes, Bacterial , Genotype , Polymerase Chain Reaction/methods , Reference Standards , Reproducibility of Results , Rec A Recombinases/genetics , Respiratory Tract Infections/diagnosis , Respiratory Tract Infections/etiology , Sensitivity and Specificity , SoftwareABSTRACT
Background: Burkholderia complex (Bcc) infections in cystic fibrosis (CF) patients are associated with decline in lung function and reduced survival. The potential transmissibility of Bcc among CF patients has been reported, indicating that strict segregation of CF patients with Bcc is crucial. Aims: To standardize the PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) assay in order to identify Bcc species and to establish the prevalence of Bcc species and their susceptibility profile among CF patients seen at the Hospital de Clínicas de Porto Alegre (HCPA). Methods: The classification of the clinical isolates recovered from respiratory tract specimens of CF patients as Bcc was achieved using the API-20NE® phenotypic commercial system. The identification of the Bcc species was performed using PCR-RFLP. The antimicrobial disc diffusion susceptibility testing was performed according to the CLSI (2006). Results: API-20NE® was able to identify Bcc isolates (244 specimens), such as B. cepacia, indicating that it was not able to distinguish among the Bcc species. The PCR-RFLP molecular method discriminated the eight reference Bcc species, thus validating the method for clinical isolates. Bcc prevalence determined by PCR-RFLP was 10.6% (26/244). The molecular analysis identified B. cenocepacia in 53.8% (14/26) of infected patients, B. multivorans in 15.4% (4/26), and B. vietnamiensis and B. ambifaria in 7.7% (2/26). The antibiotic resistance profile was variable among Bcc species. Conclusions: The PCR-RFLP method was validated for the identification of Bcc species. B. cenocepacia proved to be the most prevalent species among the CF patients seen at the HCPA.
Introdução: Infecções por bactérias do complexo Burkholderia cepacia (CBC) em pacientes com fibrose cística (FC) estão associadas a declínio da função pulmonar e diminuição da sobrevida. O potencial de transmissibilidade de CBC entre pacientes com FC é uma realidade, tornando-se importante a estrita segregação dos pacientes infectados.Objetivos: Padronizar a técnica de PCR-RFLP (reação em cadeia da polimerase seguida de clivagem com enzimas derestrição) para diferenciação das espécies de CBC e estabelecer a prevalência dessas espécies e seus perfis de sensibilidade em pacientes com FC atendidos no Hospital de Clínicas de Porto Alegre (HCPA). Métodos: A identificação dos isolados clínicos do trato respiratório de pacientes com FC como CBC foi feita pelo sistema deidentificação fenotípica comercial API-20NE®. A diferenciação das espécies de CBC foi realizada por PCR-RFLP, e o teste de suscetibilidade aos antimicrobianos por disco-difusão foi realizado de acordo com o CLSI (2006).Resultados: O sistema API-20NE® identificou todos os isolados do CBC (244 amostras) como B. cepacia, indicando claramente que não distingue as espécies do complexo. O método molecular de PCR-RFLP discriminou as oito espécies de referência de CBC, validando o método para isolados clínicos. A prevalência de CBC por PCR-RFLP foi de 10,6% (26/244).A análise molecular apontou B. cenocepacia colonizando em 53,8% (14/26) dos pacientes infectados, B. multivorans em 15,4% (4/26) e B. vietnamiensis e B. ambifaria em 7,7% (2/26). O perfil de resistência entre as espécies de CBC para os antibióticos testados foi variado. Conclusão: Foi validada a aplicação do método molecular PCR-RFLP para identificar espécies de CBC, e B. cenocepaciafoi a espécie mais prevalente entre os pacientes fibrocísticos atendidos no HCPA.