Rapid bacterial identification by MALDI-TOF MS directly from blood cultures and rapid susceptibility testing: A simple approach to reduce the turnaround time of blood cultures

Abstract Antimicrobial treatment of patients with bloodstream infections (BSI) is time-sensitive. In an era of increasing antimicrobial resistance, rapid detection and identification of bacteria with antimicrobial susceptibility are critical for targeted therapy early in the disease course. This study describes the performance of a rapid method for identifying and testing antimicrobial susceptibility of Gram-negative bacteria performed directly from blood culture bottles in a routine microbiology laboratory. A total of 284, 120, and 24 samples were analyzed by rapid identification (Rid), rapid susceptibility testing (RAST), and rapid broth microdilution for polymyxin B (rMIC), respectively, and compared with standard methods. Our protocol was able to identify 93% of isolates at the species level using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We obtained 100% agreement for RAST compared to the standard method and 96% agreement for rMIC. Our protocol has proven to be an excellent tool for rapid identification of Gram-negative bacilli causing BSIs. It can also be used in microbiology laboratory routine along with RAST and faster polymyxin microdilution, especially for carbapenemase-producing bacteria, allowing for rapid, simple, accurate, and cost-effective diagnosis.

Vancomycin resistant Enterococcus spp (VRE): follow up during 9 years in a tertiary teaching hospital in southern Brazil

Introduction: Infection with vancomycin-resistant Enterococcus spp (VRE) has been a worldwide problem since mid 1980’s and, in Brazil, since 1996. This study was conducted to evaluate the experience with VRE in our institution. Methods: A prospective cohort study from 2000 to 2009 was conducted at Hospital São Lucas da PUCRS. All hospitalized patients with VRE positive culture were included and followed from their diagnosis until they were negative for VRE or their discharge. Only the first admission for each VRE positive patient was included. Pulsed field gel electrophoresis (PFGE) was performed to determine how VRE had spread. Results: A total of 315 cases of VRE were identified, 224 of which were isolated from rectal swabs. Vancomycin-resistant/ampicilin susceptible Enterococcus faecalis were identified in 312 isolates. PFGE was performed in 47 VRE isolates that presented an indistinguishable migratory profile. The median length of hospital stay and length of stay before VRE isolation were 46 days and 21 days, respectively; 52% of the patients were aged 60 and above. The annual distribution of the new VRE cases showed a clear decrease from 2000 to 2009. Discussion: This study shows a substantial VRE colonization (71%) with a homogenous pattern that emphasizes its transversal spread. Predominance of E. faecalis differs from the literature which largely describes a higher prevalence of vancomycin-resistant Enterococcus faecium. The follow up of VRE during 9 years in our institution highlighted the importance of continuous surveillance to prevent outbreaks in our hospital.

Bacteriologia da fibrose cística / Cystic fibrosis bacteriology

O exame bacteriológico é um dos principais parâmetros que auxiliam o diagnóstico e manuseio da infecção respiratória dos pacientes com Fibrose Cística (FC). Os microrganismos que colonizam e infectam o paciente fibrocístico determinam o tratamento, a qualidade de vida, as perspectivas para o transplante e a sua sobrevida global. A identificação precisa de patógenos respiratórios é essencial para o tratamento da infecção, seja como guia para o uso adequado de antibióticos por longos períodos para os pacientes com infecção bacteriana crônica ou para a aplicação adequada de medidas de controle de infecção. Embora exista um espectro limitado de patógenos respiratórios classicamente associados à doença respiratóriana FC, um número crescente de microrganismos vem sendo reconhecido como potenciais agentes patogênicos. O espectro de patógenos em FC varia com a idade do paciente, mas, de uma forma geral, é bem estabelecido na literatura que existem quatro bactérias “clássicas”: Staphylococcus aureus, Haemophilus influenzae, Pseudomonas aeruginosa e o complexo B. cepacia (CBC)...

The bacteriological culture is one of the main parameters that support the diagnosis and management of the respiratoryinfection in patients with cystic fibrosis (CF). The microorganisms that colonize and infect CF patients determine the treatment,quality of life, the lung transplant possibility and their overall survival. The accurate identification of respiratory pathogensis essential for the treatment of infection, either to guide the appropriate use of antibiotics for long periods to patientswith chronic bacterial infection or to the proper implementation of infection control measures. Although there is a limitedspectrum of respiratory pathogens classically associated with the respiratory disease in CF, an increasing number of microorganismshas been recognized as potential pathogens. The spectrum of pathogens in CF varies with the patients age but,in general, it is well established in the literature that there are four "classic" pathogens: Staphylococcus aureus, Haemophilusinfluenzae, Pseudomonas aeruginosa and the B. cepacia complex (Bcc)...

Molecular identification of Burkholderia cepacia complex and species distribution among cystic fibrosis patients seen at the reference center in southern Brazil / Identificação molecular do complexo Burkholderia cepacia e distribuição por espécie entre pacientes com fibrose cística em um centro de referência no sul do Brasil

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.

Identification, antimicrobial resistance and genotypic characterization of Enterococcus spp. isolated in Porto Alegre, Brazil / Identificação, resistência antimicrobiana e caracterização genotípica de Enterococcus spp. isolados em Porto Alegre, Brasil

In the past two decades the members of the genus Enterococcus have emerged as important nosocomial pathogens worldwide. In the present study, we evaluated the antimicrobial resistance and genotypic characteristics of 203 Enterococcus spp. recovered from different clinical sources from two hospitals in Porto Alegre, Rio Grande do Sul, Brazil. The species were identified by conventional biochemical tests and by an automated system. The genetic diversity of E. faecalis presenting high-level aminoglycoside resistance (HLAR) was assessed by pulsed-field gel electrophoresis of chromosomal DNA after SmaI digestion. The E. faecalis was the most frequent specie (93.6 percent), followed by E. faecium (4.4 percent). The antimicrobial resistance profile was: 2.5 percent to ampicillin, 0.5 percent to vancomycin, 0.5 percent teicoplanin, 33 percent to chloramphenicol, 2 percent to nitrofurantoin, 66.1 percent to erythromycin, 66.5 percent to tetracycline, 24.6 percent to rifampicin, 30 percent to ciprofloxacin and 87.2 percent to quinupristin-dalfopristin. A total of 10.3 percent of the isolates proved to be HLAR to both gentamicin and streptomycin (HLRST/GE), with 23.6 percent resistant only to gentamicin (HLR-GE) and 37.4 percent only to streptomycin (HLRST). One predominant clonal group was found among E. faecalis HLR-GE/ST. The prevalence of resistance among beta-lactam antibiotics and glycopeptides was very low. However, in this study there was an increased number of HLR Enterococcus which may be spreading intra and inter-hospital.

Nas últimas duas décadas os membros do gênero Enterococcus emergiram como importantes patógenos nosocomiais ao redor do mundo. No presente estudo, nós avaliamos a resistência antimicrobiana e as características genotípicas de 203 Enterococcus spp. obtidos de diferentes fontes clínicas em dois hospitais de Porto Alegre, Rio Grande do Sul, Brasil. As espécies foram identificadas por testes bioquímicos convencionais e por um sistema automatizado. A diversidade genética de E. faecalis demonstrando resistência à altos níveis de aminoglicosídeos (HLAR) foi avaliada através da análise do DNA cromossômico após digestão com a enzima SmaI, seguido por eletroforese em campo pulsado. O E. faecalis foi a espécie mais freqüente (93,6 por cento), seguido por E. faecium (4,4 por cento). O perfil de resistência antimicrobiana foi: 2,5 por cento para ampicilina, 0,5 por cento para vancomicina, 0,5 por cento para teicoplanina, 33 por cento para cloranfenicol, 2 por cento para nitrofurantoína 66,1 por cento para eritromicina, 66,5 por cento para tetraciclina, 24,6 por cento para rifampicina, 30 por cento para ciprofloxacino e 87,2 por cento para quinupristina-dalfopristina. Um total de 10,3 por cento dos isolados apresentaram HLAR para ambos gentamicina e estreptomicina (HLR-ST/GE), sendo 23,6 por cento resistentes somente a gentamicina (HLR-GE) e 37,4 por cento somente a estreptomicina (HLR-ST). Um grupo clonal predominante foi encontrado em E. faecalis HLR-GE/ST. A prevalência de resistência a antibióticos ²-lactâmicos, e em particular aos glicopeptídeos, foi muito baixa. Entretanto, neste estudo, houve um número crescente de Enterococcus HLAR que podem estar se disseminando intra e interhospitais.

Polymerase chain reaction as a useful and simple tool for rapid diagnosis of tuberculous meningitis in a Brazilian tertiary care hospital

Meningitis is a severe and potentially fatal form of tuberculosis. The diagnostic workup involves detection of acid-fast bacilli (AFB) in the cerebrospinal fluid (CSF) by microscopy or culture, however, the difficulty in detecting the organism poses a challenge to diagnosis. The use of the polymerase chain reaction (PCR) in the diagnostic approach to Mycobacterium tuberculosis (MTB) meningitis has been reported as a fast and accurate method, with several commercial kits available. As an alternative, some institutions have been developing inexpensive in house assays. In our institution, we use an in house PCR for tuberculosis. We analyzed the performance of our PCR for the diagnosis of MTB meningitis in 148 consecutive patients, using MTB culture as gold standard. The sensitivity and specificity of CSF PCR for the diagnosis of MTB meningitis was 50 percent and 98.6 percent respectively with a concordance with CSF mycobacterial culture of 96 percent (Kappa=0.52). In contrast to CSF cultures for MTB, our PCR test is a fast, simple and inexpensive tool to diagnose tuberculous meningitis with a performance similar to that obtained with the available commercial kits.

Susceptibility of Staphylococcus aureus isolates to vancomycin at a university hospital in southern Brazil

The in vitro susceptibilty of Staphylococcus aureus to vancomycin was evaluated from impatients at a Brazilian University Hospital by the Etest and a screening method. No vancomycin intermediate (VISA) or vancomycin resistant (VRSA) S. aureus isolate was identified. Three patients presented as heteroresistant VISA (h-VISA) isolates but none of them received vancomycin previously.

A suscetibilidade in vitro de Staphylococcus aureus à vancomicina foi avaliada em pacientes internados em um Hospital Universitário Brasileiro pelo Etest e por um método de triagem. Nenhuma amostra de S. aureus resistente (VRSA) ou com resistência intermediária (VISA) à vancomicina foi isolada. Três pacientes tiveram amostras heteroresistentes VISA (h-VISA), mas nenhum destes recebeu vancomicina previamente.

Clinical failure of vancomycin treatment of Staphylococcus aureus infection in a tertiary care hospital in southern Brazil

We describe a case of clinical failure of vancomycin treatment of Staphylococcus aureus infection and the laboratory characteristics of the organism in a tertiary referral university hospital in southern Brazil. An 11-month-old male patient presented with pneumonia and S. aureus was isolated from his respiratory tract. Initial treatment with oxacillin and gentamicin was ineffective. Vancomycin was added to the regimen as the patient worsened, but after the 30th day of vancomycin treatment S. aureus was isolated from the blood. This isolate had a minimum inhibitory concentration (MIC) for vancomycin of 4 æg/mL. After pre-incubation with vancomycin the isolate displayed an increase in the expression of vancomycin resistance and colonies grew in the presence of up to 12 æg/mL vancomycin. Based on these results, and considering that the patient had not responded to vancomycin, the isolate was considered to be S. aureus heteroresistant to vancomycin (SAHV). The SAHV proved to be similar, based on DNA macrorestriction analysis, to methicillin resistant S. aureus (MRSA) isolates from other patients in the hospital who had responded to vancomycin treatment. Our findings underline the need to improve methods in the clinical laboratory to detect the emergence of S. aureus clinically resistant to vancomycin . The fact that the isolate emerged in the blood 30 days after vancomycin treatment was initiated suggests that the organism was originally an MRSA that had acquired the ability to circumvent the mechanism of action of vancomycin