Evaluation of pathogen from the FilmArray meningitis/encephalitis panel and recommendations on atypical findings.

BACKGROUND: Infectious meningoencephalitis is a potentially fatal clinical condition that causes inflammation of the central nervous system secondary to the installation of different microorganisms. The FilmArray meningitis/encephalitis panel allows the simultaneous detection of 14 pathogens with results in about one hour. OBJECTIVE: This study is based on retrospectively evaluating the implementation of the FilmArray meningitis/encephalitis panel in a hospital environment, highlighting the general results and, especially, analyzing the consistency of the test results against the clinical and laboratory conditions of the patients. METHODS: Data were collected through the results reported by the BioFire FilmArray system software from the meningitis/encephalitis panel. The correlated laboratory tests used in our analysis, when available, included biochemical, cytological, direct and indirect microbiological tests. RESULTS: In the analyzed period, there were 496 samples with released results. Of the total of 496 samples analyzed, 88 (17.75%) were considered positive, and 90 pathogens were detected, and in 2 of these (2.27%) there was co-detection of pathogens. Viruses were the agents most frequently found within the total number of pathogens detected. Of the 496 proven samples, 20 (4.03%) were repeated, 5 of which were repeated due to invalid results, 6 due to the detection of multiple pathogens and 9 due to disagreement between the panel results and the other laboratory tests and/or divergence of the clinical-epidemiological picture. Of these 20 repeated samples, only 4 of them (20%) maintained the original result after repeating the test, with 16 (80%) being non-reproducible. The main factor related to the disagreement of these 16 samples during retesting was the detection of bacterial agents without any relationship with other laboratory tests or with the patients' clinical condition. CONCLUSION: In our study, simply reproducing tests with atypical results from the FilmArray meningitis/encephalitis panel proved, in most cases, effective and sufficient for interpreting these results.

ANTECEDENTES: A meningoencefalite infecciosa é uma condição clínica potencialmente fatal que causa inflamação do sistema nervoso central secundária à instalação de diversos microrganismos. O painel de meningite/encefalite FilmArray permite a detecção simultânea de 14 patógenos, com resultados em cerca de uma hora. OBJETIVO: Este estudo baseia-se em avaliar retrospectivamente a implementação do painel de meningite/encefalite FilmArray em ambiente hospitalar, destacando os resultados gerais e, principalmente, analisando a consistência dos resultados do teste frente às condições clínicas e laboratoriais dos pacientes. MéTODOS: Os dados foram coletados por meio dos resultados relatados pelo software do sistema BioFire FilmArray do painel de meningite/encefalite. Os exames laboratoriais correlacionados utilizados em nossa análise, quando disponíveis, incluíram exames bioquímicos, citológicos, microbiológicos diretos e indiretos. RESULTADOS: No período analisado, foram 496 amostras com resultados divulgados. Do total de 496 amostras analisadas, 88 (17,75%) foram consideradas positivas e 90 patógenos foram detectados, sendo que em duas destas (2,27%) houve codetecção de patógenos. Os vírus foram os agentes mais frequentemente encontrados dentro do total de patógenos detectados. Das 496 amostras analisadas, 20 (4,03%) foram repetidas, sendo 5 repetidas por resultado inválido, 6 pela detecção de múltiplos patógenos e 9 por discordância dos resultados do painel com os demais exames laboratoriais e/ou divergência do quadro clínico-epidemiológico. Destas 20 amostras repetidas, apenas 4 delas (20%) mantiveram o resultado original após a repetição do teste, sendo 16 (80%) não reprodutíveis. O principal fator relacionado à discordância destas 16 amostras na retestagem foi a detecção de agentes bacterianos sem qualquer relação com os demais exames laboratoriais ou com o quadro clínico dos pacientes. CONCLUSãO: Em nosso estudo, a simples repetição dos testes com resultados atípicos do painel de meningite/encefalite FilmArray mostrou-se, na maior dos casos, efetiva e suficiente para a interpretação destes achados.

Evaluation of pathogen from the FilmArray meningitis/encephalitis panel and recommendations on atypical findings / Avaliação do patógeno do painel de meningite/encefalite FilmArray e recomendações sobre achados atípicos

Abstract Background Infectious meningoencephalitis is a potentially fatal clinical condition that causes inflammation of the central nervous system secondary to the installation of different microorganisms. The FilmArray meningitis/encephalitis panel allows the simultaneous detection of 14 pathogens with results in about one hour. Objective This study is based on retrospectively evaluating the implementation of the FilmArray meningitis/encephalitis panel in a hospital environment, highlighting the general results and, especially, analyzing the consistency of the test results against the clinical and laboratory conditions of the patients. Methods Data were collected through the results reported by the BioFire FilmArray system software from the meningitis/encephalitis panel. The correlated laboratory tests used in our analysis, when available, included biochemical, cytological, direct and indirect microbiological tests. Results In the analyzed period, there were 496 samples with released results. Of the total of 496 samples analyzed, 88 (17.75%) were considered positive, and 90 pathogens were detected, and in 2 of these (2.27%) there was co-detection of pathogens. Viruses were the agents most frequently found within the total number of pathogens detected. Of the 496 proven samples, 20 (4.03%) were repeated, 5 of which were repeated due to invalid results, 6 due to the detection of multiple pathogens and 9 due to disagreement between the panel results and the other laboratory tests and/or divergence of the clinical-epidemiological picture. Of these 20 repeated samples, only 4 of them (20%) maintained the original result after repeating the test, with 16 (80%) being non-reproducible. The main factor related to the disagreement of these 16 samples during retesting was the detection of bacterial agents without any relationship with other laboratory tests or with the patients' clinical condition. Conclusion In our study, simply reproducing tests with atypical results from the FilmArray meningitis/encephalitis panel proved, in most cases, effective and sufficient for interpreting these results.

Resumo Antecedentes A meningoencefalite infecciosa é uma condição clínica potencialmente fatal que causa inflamação do sistema nervoso central secundária à instalação de diversos microrganismos. O painel de meningite/encefalite FilmArray permite a detecção simultânea de 14 patógenos, com resultados em cerca de uma hora. Objetivo Este estudo baseia-se em avaliar retrospectivamente a implementação do painel de meningite/encefalite FilmArray em ambiente hospitalar, destacando os resultados gerais e, principalmente, analisando a consistência dos resultados do teste frente às condições clínicas e laboratoriais dos pacientes. Métodos Os dados foram coletados por meio dos resultados relatados pelo software do sistema BioFire FilmArray do painel de meningite/encefalite. Os exames laboratoriais correlacionados utilizados em nossa análise, quando disponíveis, incluíram exames bioquímicos, citológicos, microbiológicos diretos e indiretos. Resultados No período analisado, foram 496 amostras com resultados divulgados. Do total de 496 amostras analisadas, 88 (17,75%) foram consideradas positivas e 90 patógenos foram detectados, sendo que em duas destas (2,27%) houve codetecção de patógenos. Os vírus foram os agentes mais frequentemente encontrados dentro do total de patógenos detectados. Das 496 amostras analisadas, 20 (4,03%) foram repetidas, sendo 5 repetidas por resultado inválido, 6 pela detecção de múltiplos patógenos e 9 por discordância dos resultados do painel com os demais exames laboratoriais e/ou divergência do quadro clínico-epidemiológico. Destas 20 amostras repetidas, apenas 4 delas (20%) mantiveram o resultado original após a repetição do teste, sendo 16 (80%) não reprodutíveis. O principal fator relacionado à discordância destas 16 amostras na retestagem foi a detecção de agentes bacterianos sem qualquer relação com os demais exames laboratoriais ou com o quadro clínico dos pacientes. Conclusão Em nosso estudo, a simples repetição dos testes com resultados atípicos do painel de meningite/encefalite FilmArray mostrou-se, na maior dos casos, efetiva e suficiente para a interpretação destes achados.

The Gastrointestinal Load of Carbapenem-Resistant Enterobacteriacea Is Associated With the Transition From Colonization to Infection by Klebsiella pneumoniae Isolates Harboring the blaKPC Gene.

Background: Healthcare-associated infections by carbapenem-resistant Klebsiella pneumoniae are difficult to control. Virulence and antibiotic resistance genes contribute to infection, but the mechanisms associated with the transition from colonization to infection remain unclear. Objective: We investigated the transition from carriage to infection by K. pneumoniae isolates carrying the K. pneumoniae carbapenemase-encoding gene bla KPC (KpKPC). Methods: KpKPC isolates detected within a 10-year period in a single tertiary-care hospital were characterized by pulsed-field gel electrophoresis (PFGE), multilocus sequencing typing, capsular lipopolysaccharide and polysaccharide typing, antimicrobial susceptibility profiles, and the presence of virulence genes. The gastrointestinal load of carbapenem-resistant Enterobacteriaceae and of bla KPC-carrying bacteria was estimated by relative quantification in rectal swabs. Results were evaluated as contributors to the progression from carriage to infection. Results: No PGFE type; ST-, K-, or O-serotypes; antimicrobial susceptibility profiles; or the presence of virulence markers, such yersiniabactin and colibactin, were associated with carriage or infection, with ST437 and ST11 being the most prevalent clones. Admission to intensive and semi-intensive care units was a risk factor for the development of infections (OR 2.79, 95% CI 1.375 to 5.687, P=0.005), but higher intestinal loads of carbapenem-resistant Enterobacteriaceae or of bla KPC-carrying bacteria were the only factors associated with the transition from colonization to infection in this cohort (OR 8.601, 95% CI 2.44 to 30.352, P<0.001). Conclusion: The presence of resistance and virulence mechanisms were not associated with progression from colonization to infection, while intestinal colonization by carbapenem-resistant Enterobacteriacea and, more specifically, the load of gastrointestinal carriage emerged as an important determinant of infection.

Clostridioides difficile laboratory diagnostic techniques: a comparative approach of rapid and molecular methods.

Clostridioides difficile infection is a public health problem because of it is easily spread; with harmful consequences, it is essential to reduce hospital costs and prevent its dissemination by having a precise diagnosis. The gold standard for its diagnosis is polymerase chain reaction (PCR); however, the technique is not available for all laboratories due to the high cost. New approaches using non-molecular tests to detect C. difficile and toxin A/B production has been proposed to improve cost benefits. The objective of this study is to compare molecular methods (PCR) and rapid methods (immunochromatographic test and enzymatic immunoassay). A series of tests comprising these diagnostic techniques was performed with 50 patients with a clinical diagnosis for Clostridioides difficile on GeneXpert® devices test; a calculation of the sensitivity was executed, followed by a comparison of the efficiency of all techniques. Greater sensitivity was observed in the PCR-based methods (BD MAX™ and BioFire FilmArray®) and the GDH-based assays (RIDASCREEN® and Alere Techlab®). The proposed algorithm represents minor monetary disadvantages but a significant temporal optimization of 10%. Future studies concerning both positive and negative results could be advantageous because of the possibility of calculating more method concordance indexes, such as the specificity and Kappa index, in addition to being able to indicate a monetary profit if the proposed algorithm was applied due to the nonproceeding PCR cases.

Mutagenesis Induced by Sub-Lethal Doses of Ciprofloxacin: Genotypic and Phenotypic Differences Between the Pseudomonas aeruginosa Strain PA14 and Clinical Isolates.

Bacterial resistance is a severe threat to global public health. Exposure to sub-lethal concentrations has been considered a major driver of mutagenesis leading to antibiotic resistance in clinical settings. Ciprofloxacin is broadly used to treat infections caused by Pseudomonas aeruginosa, whereas increased mutagenesis induced by sub-lethal concentrations of ciprofloxacin has been reported for the reference strain, PAO1, in vitro. In this study we report increased mutagenesis induced by sub-lethal concentrations of ciprofloxacin for another reference strain, PA14-UCBPP, and lower mutagenesis for clinical isolates when compared to the reference strain. This unexpected result may be associated with missense mutations in imuB and recX, involved in adaptive responses, and the presence of Pyocin S2, which were found in all clinical isolates but not in the reference strain genome. The genetic differences between clinical isolates of P. aeruginosa and the reference PA14-UCBPP, often used to study P. aeruginosa phenotypes in vitro, may be involved in reduced mutagenesis under sub-lethal concentrations of CIP, a scenario that should be further explored for the understanding of bacterial fitness in hospital environments. Moreover, we highlight the presence of a complete umuDC operon in a P. aeruginosa clinical isolate. Even though the presence of umuDC did not contribute to a significant increase in mutagenesis, it highlights the dynamic exchange of genetic material between bacterial species in the hospital environment.

Comparative Genomics of Nonoutbreak Pseudomonas aeruginosa Strains Underlines Genome Plasticity and Geographic Relatedness of the Global Clone ST235.

Pseudomonas aeruginosa is an important opportunistic pathogen in hospitals, responsible for various infections that are difficult to treat due to intrinsic and acquired antibiotic resistance. Here, 20 epidemiologically unrelated strains isolated from patients in a general hospital over a time period of two decades were analyzed using whole genome sequencing. The genomes were compared in order to assess the presence of a predominant clone or sequence type (ST). No clonal structure was identified, but core genome-based single nucleotide polymorphism (SNP) analysis distinguished two major, previously identified phylogenetic groups. Interestingly, most of the older strains isolated between 1994 and 1998 harbored exoU, encoding a cytotoxic phospholipase. In contrast, most strains isolated between 2011 and 2016 were exoU-negative and phylogenetically very distinct from the older strains, suggesting a population shift of nosocomial P. aeruginosa over time. Three out of 20 strains were ST235 strains, a global high-risk clonal lineage; these carried several additional resistance determinants including aac(6')Ib-cr encoding an aminoglycoside N-acetyltransferase that confers resistance to fluoroquinolones. Core genome comparison with ST235 strains from other parts of the world showed that the three strains clustered together with other Brazilian/Argentinean isolates. Despite this regional relatedness, the individuality of each of the three ST235 strains was revealed by core genome-based SNPs and the presence of genomic islands in the accessory genome. Similarly, strain-specific characteristics were detected for the remaining strains, indicative of individual evolutionary histories and elevated genome plasticity.

Evaluation of two commercial methods for the susceptibility testing of Candida species: Vitek 2® and Sensititre YeastOne®.

BACKGROUND: An increased incidence of fungal infections caused by Candida species, especially Candida glabrata and Candida krusei, which are less susceptible to azoles, has been observed. Standardized susceptibility testing is essential for clinical management and for monitoring the epidemiology of resistance. AIMS: We evaluated the performance of two different susceptibility testing commercial methods, Vitek 2® and Sensititre YeastOne®, and compared them with the standard broth microdilution method (CLSI). METHODS: A total of 80 isolates of several Candida species (Candida albicans, Candida parapsilosis complex, Candida tropicalis, C. glabrata and C. krusei) were selected for this study. RESULTS: We analyzed the categorical agreement (CA) between the methods, stratifying the disagreements. The average CA between the methods was 96.3% for Vitek 2® and 84% for Sensititre YeastOne®. No very major errors were observed. Major errors and minor errors were found for all the isolates tested. With the azoles, both Vitek 2® and Sensititre YeastOne® had good and similar performance levels, except for C. tropicalis and C. krusei (Sensititre YeastOne® showed low CA, 56.2%). With the echinocandins, both methods showed good performance for C. albicans, C. parapsilosis and C. tropicalis. However, we observed important discrepancies for C. krusei with caspofungin: Vitek 2® had 100% CA while Sensititre YeastOne® had only 25%. With amphotericin B, both Vitek 2® and Sensititre YeastOne® had good performance with high CA. CONCLUSIONS: Despite the limited isolates tested, we concluded that both methods have good performance and are reliable for antifungal susceptibility testing. However, caspofungin activity against C. krusei and C. glabrata should be interpreted carefully when using Sensititre YeastOne® because we observed a low CA.

Evaluation of a new rapid test for carbapenemase detection in carbapenem resistant Enterobacteriaceae.

We evaluated a new phenotypic test for carbapenemase detection. A total of 100 Enterobacteriaceae isolates were selected. The test was compared with conventional PCR for bla(KPC) and bla(NDM) detection. We found 100% sensitivity and specificity, suggesting that this test may be a feasible alternative for rapid carbapenemase detection.

Polyclonal anti-intimin antibody: immunological characterization and its use in EPEC and EHEC diagnosis

EPEC e EHEC constituem um risco significativo para a saúde pública em diferentes partes do mundo. Ambas colonizam a mucosa intestinal e subvertem as funções celulares do epitélio intestinal ao produzirem uma lesão histopatológica característica, conhecida por lesão A/E (attaching-and-effacing), na qual a intimina é uma das proteínas envolvidas. A família das intiminas apresenta também uma região conservada, que compreende os aminoácidos de 388 a 667 (Int 388-667). O objetivo do presente trabalho foi a obtenção de um anticorpo policlonal contra a região conservada de intimina. A caracterização fenotípica das amostras de EPEC e EHEC utilizando este anticorpo permitiu observar-se a maneira variável que ele reconhece os diversos subtipos de intimina e sugere que ele seja uma ferramenta para detecção destes patógenos, sendo o ensaio de immuno-dot o método de captura de escolha.

Capture immunoassay for LT detection produced by enterotoxigenic Escherichia coli in bacterial isolates

O objetivo do presente trabalho foi a padronização de um imunoensaio de captura para detecção de amostras de E. coli produtoras da toxina LT-I. Este ensaio de captura foi desenvolvido utilizando-se a fração enriquecida em IgG do anticorpo policlonal anti-LT e um anticorpo monoclonal caracterizado como IgG2b. Através deste método verificou-se uma clara distinção entre cepas de E. coli produtoras e não produtoras da toxina (p< 0,0001), sendo a sensibilidade do método de 78%, a especificidade de 94% e a eficiência de 92%. Assim, o imunoensaio de captura mostrou-se como uma ferramenta sensível para a detecção de amostras de E. coli que produzem a enterotoxina termo-lábil, podendo ser aplicado em laboratórios clínicos e inquéritos epidemiológicos em paises em desenvolvimento.

Polyclonal anti-intimin antibody: immunological characterization and its use in EPEC and EHEC diagnosis

Intimins are outer membrane proteins expressed by enteric bacterial pathogens capable of inducing intestinal attachment-and-effacement lesion (A/E). Through immunoblotting, immunofluorescence, flow citometry and immunogold we observed that the obtained polyclonal antibody against conserved intimin region recognizes the different intimin subtypes and suggests that it can be used as a tool for EPEC and EHEC detection. Besides, immuno-dot assay seems to be a possible alternative as a capture method.

EPEC e EHEC constituem um risco significativo para a saúde pública em diferentes partes do mundo. Ambas colonizam a mucosa intestinal e subvertem as funções celulares do epitélio intestinal ao produzirem uma lesão histopatológica característica, conhecida por lesão A/E (attaching-and-effacing), na qual a intimina é uma das proteínas envolvidas. A família das intiminas apresenta também uma região conservada, que compreende os aminoácidos de 388 a 667 (Int 388-667). O objetivo do presente trabalho foi a obtenção de um anticorpo policlonal contra a região conservada de intimina. A caracterização fenotípica das amostras de EPEC e EHEC utilizando este anticorpo permitiu observar-se a maneira variável que ele reconhece os diversos subtipos de intimina e sugere que ele seja uma ferramenta para detecção destes patógenos, sendo o ensaio de immuno-dot o método de captura de escolha.

Capture immunoassay for LT detection produced by enterotoxigenic Escherichia coli in bacterial isolates

A capture enzyme-linked immunosorbent assay (ELISA), which detects LT-I toxin produced by enterotoxigenic Escherichia coli strains, has beendeveloped. This capture assay was performed using the IgG enriched fraction of anti-LT-I antiserum and IgG2b anti-LT-I monoclonal antibody and allowed a clear distinction between E. coli LT-I - producing and non-producing strains. The estimated accuracy of the assay is 78% for sensitivity, 94% for specificity and 92% for efficiency. Thus, the capture immunoassayis a sensitive tool for detection of E. coli, which produces heat-labile enterotoxin, and is suitable for use in clinical laboratories and epidemiological surveys in developing world.

O objetivo do presente trabalho foi a padronização de um imunoensaio de captura para detecção de amostras de E. coli produtoras da toxina LT-I. Este ensaio de captura foi desenvolvido utilizando-se a fração enriquecida em IgG do anticorpo policlonal anti-LT e um anticorpo monoclonal caracterizado como IgG2b. Através deste método verificou-se uma clara distinção entre cepas de E. coli produtoras e não produtoras da toxina (p 0,0001), sendo a sensibilidade do método de 78%, a especificidade de 94% e a eficiência de 92%. Assim, o imunoensaio de captura mostrou-se como uma ferramenta sensível para a detecção de amostras de E. coli que produzem a enterotoxina termo-lábil, podendo ser aplicado em laboratórios clínicos e inquéritos epidemiológicos em paises em desenvolvimento.