Development and Evaluation of Multiplex PCR for the Detection of Carbapenemase-Producing Enterobacteriaceae / 대한임상미생물학회지

BACKGROUND: The isolation of carbapenemase-producing Enterobacteriaceae (CPE) has become increasingly common. Continuous surveillance for these organisms is essential because their infections are closely related to outbreaks of illness and are associated with high mortality rates. The aim of this study was to develop and evaluate multiplex PCR as a means of detecting several important CPE genes simultaneously. METHODS: We aimed to develop a multiplex PCR that could detect seven CPE genes simultaneously. The multiplex PCR was composed of seven primer sets for the detection of KPC, IMP, VIM, NDM-1, GES, OXA-23, and OXA-48. We designed different PCR product sizes of at least 100 bp. We evaluated the performance of this new test using 69 CPE-positive clinical isolates. Also, we confirmed the specificity to rule out false-positive reactions by using 71 carbapenem-susceptible clinical strains. RESULTS: A total of 69 CPE clinical isolates showed positive results and were correctly identified as KPC (N=14), IMP (N=13), OXA-23 (N=12), OXA-48 (N=11), VIM (N=9), GES (N=5), and NDM (N=5) by the multiplex PCR. All 71 carbapenem-susceptible clinical isolates, including Enterococcus faecalis , Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa, showed negative results. CONCLUSION: This multiplex PCR can detect seven CPE genes at a time and will be useful in clinical laboratories.

Identificación de genes de resistencia a carbapenémicos en enterobacterias de hospitales de Perú, 2013-2017 / Identification of carbapenem-resistant genes in enterobacteria from peruvian hospitals, 2013-2017

RESUMEN La diseminación global de carbapenemasas es de importancia en la salud pública. El objetivo del estudio es describir la presencia de genes de resistencia a carbapenémicos tipo KPC y metalobetalactamasas en enterobacterias aisladas de 12 hospitales y remitidos al Laboratorio de Referencia Nacional de Infecciones Intrahospitalarias del Instituto Nacional de Salud de Perú durante los años 2013 al 2017. Las cepas fueron identificadas por métodos convencionales, la resistencia antimicrobiana fue determinada por métodos fenotípicos, bioquímicos y la presencia de genes de resistencia, se detectaron por PCR convencional. Se identificaron 83 cepas con carbapenemasas: 26 (31,3 %) portando el gen blaKPC, 56 (67,5 %) el gen blaNDM y una (1,2 %) cepa con el gen blaIMP. Es el primer reporte que da a conocer los genes de carbapenemasas circulantes en hospitales de Perú, por lo que se requiere mejorar la vigilancia para tener un mejor conocimiento de la situación en Perú.

ABSTRACT The global spread of carbapenemases is a significant public health concern. The aim of this report is to describe the presence of KPC-type carbapenem-resistant genes and enterobacteria isolated in 12 hospitals and forwarded to the Peruvian National Institute of Health's National Infection Reference Laboratory during the period between 2013 and 2017. The strains were identified by conventional methods; antimicrobial resistance was determined by phenotypic and biochemical methods. The presence of resistant genes was detected by conventional PCR. Eighty-three (83) strains harboring carbapenemases were identified: 26 (31.3%) carrying the blaKPC gene, 56 (67.5%) the blaNDM gene, and one strain (1.2%) with the blaIMP gene. This is the first report that shows the circulating carbapenemases genes in Hospitals in Peru of cases submitted for their confirmation to the National Reference Laboratory, so it is necessary to improve the surveillance to better understand their situation in our country.

Surveillance Culture of Carbapenemase-Producing Enterobacteriaceae in a Tertiary-Care Hospital / 대한임상미생물학회지

BACKGROUND: Carbapenem-resistant Enterobacteriaceae (CRE) are increasingly being reported throughout the world, which is a significant problem for patient treatment and infection control. Carbapenem-resistance in Enterobacteriaceae is mainly due to carbapenem-hydrolyzing β-lactamase, which tends to spread through genetic mobile elements. Therefore, the detection of carbapenemase-producing Enterobacteriaceae (CPE) carriers is particularly important for the prevention and epidemiological monitoring of these infections. In this study, we performed surveillance cultures for CPE in patients admitted to the hospital and evaluated the prevalence of CPE. METHODS: Stool cultures were obtained from a total of 228 patients at our tertiary-care hospital between March and May 2017. Stool specimens were inoculated on ChromID CARBA agar (bioMérieux, France) and incubated for 18-24 hours. Suspicious colonies with pink or bluish-green color were screened for CPE by the modified Hodge test (MHT) and carbapenemase inhibition test (CIT). We performed PCR to detect five carbapenemase genes, bla(KPC), bla(IMP), bla(VIM), bla(NDM), and bla(OXA-48). RESULTS: Among 228 isolates, seven were suspicious for CPE: four Klebsiella pneumoniae, one Escherichia coli, one Enterobacter aerogenes, and one Serratia marcescens. Two K. pneumoniae isolates showed positive reactions in both the modified Hodge test and inhibition test with phenylboronic acid. By PCR, bla(KPC) was identified in these two K. pneumoniae isolates. CONCLUSION: Our results showed a very low prevalence (2/228, 0.9%) of CPE in our tertiary-care hospital based on surveillance culture in a recent three month period.

Bacteriemia por enterobacterias resistentes a carbapenems: Un estudio transversal / Bacteremia due to carbapenem-resistant Enterobacteriaceae: A cross-sectional study

Objetivos: Describir las características clínicas, los esquemas de antibiótico empleados y el pronóstico en términos de mortalidad intrahospitalaria y efectos adversos en pacientes con bacteriemia por enterobacterias con prueba fenotípica para carbapenemasas positiva. Material y métodos: Estudio de corte trasversal en un hospital de tercer nivel (Medellín, Colombia), en pacientes con bacteriemia por enterobacterias resistentes a carbapenems (CRE) detectados entre enero del 2010 y diciembre del 2013. Se presentan las variables continuas con medianas y rangos intercuartiles (RIQ) y las categóricas con porcentajes. Resultados: Se incluyeron 64 casos con un promedio de edad de 62 ± 14 años, 66% (n = 42) hombres. El 60% (n = 38) se encontraban en la UCI, y la mediana de APACHE-II fue de 17 (RIQ: 12-22), con alta comorbilidad (puntaje Charlson de 3; RIQ: 2-5). La mediana de estancia previa a la bacteriemia fue de 21 días (RIQ: 13-39). El 64% correspondieron a Klebsiella pneumoniae , el 20% a Serratia marcescens y el 11% a Enterobacter spp. El 45% tenían tamización positiva previa a la bacteriemia. La mortalidad a los 28 días fue del 51,6% (n = 33) y ocurrió con una mediana de 5 días luego de detectada la bacteriemia (RIQ: 2-17). El tratamiento definitivo fue combinado en el 76,6% de los casos, pero no hubo un esquema de combinación prevalente. Se reportaron efectos adversos en uno de cada 3 pacientes, y la mediana de estancia hospitalaria fue de 46 días (RIQ: 26-76). La mortalidad a 28 días de pacientes tratados con carbapenems (n = 27), colistina (n = 27) o tigeciclina (n = 18), solos o en cualquier combinación, fue del 40,7, del 55,2 y del 55,7%, respectivamente. Discusión: Los pacientes incluidos tenían altos índices de comorbilidad y exposición al ambiente nosocomial, como en estudios previamente publicados. La mortalidad a 28 días fue comparable a la reportada en otros estudios. Se encontró menor mortalidad en pacientes tratados con terapias combinadas que incluían carbapenems, similar a lo reportado en un estudio clínico reciente en pacientes con bacteriemia por Klebsiella pneumoniae productora de carbapenemasas. Conclusiones: La bacteriemia por CRE afecta pacientes muy enfermos y se acompaña de elevadamortalidad. Se detecta colonización en casi la mitad de los pacientes antes del desarrollo deinfección. Hay heterogeneidad en el manejo antimicrobiano, pero la inclusión de carbapenemsen el esquema de tratamiento combinado podría asociarse con menor mortalidad.

Objectives: To describe the clinical features, antibiotic regimes and prognosis in terms of inpatient mortality and adverse effects in patients with Enterobacteriaceae bacteremia anda positive carbapenemase-detecting phenotypic test. Materials and methods: A cross-sectional study was conducted at a tertiary hospital (Medellín,Colombia). Patients with blood stream infections by carbapenems-resistant Enterobacteriaceae(CRE) diagnosed from January, 2010 to December, 2013 were included. Continuous variables are presented as medians and interquartile ranges (IQR), and categorical variables are presentedas percentages. Results: Sixty-four cases were included, with a mean age of 62 ± 14; 66% were male (n = 42).A total of 60% (n = 38) were admitted to the ICU and the median APACHE-II score was 17 (IQR:12-22), with high comorbidity (Charlson score = 3, IQR: 2-5). The median hospital stay prior to the diagnosis of bacteremia was 21 days (IQR: 13-39). Klebsiella pneumoniae was isolated in 64%, Serratia marcescens in 20% and Enterobacter spp. in 11% of the cases. Some 45% had apositive screening before the diagnosis of bacteremia. Mortality at 28 days was 51.6% (n = 33)and occurred in a median of 5 days (IQR: 2-17) after bloodstream infection was detected. Definitive treatment was a combination of antibiotics for 76.6%, but no combination scheme was prevalent. Adverse effects were observed in one of 3 patients and the median hospital stay was46 days (IQR: 26-76). Mortality at 28 days was 40.7% when patients were treated with a combination that included carbapenems agents (n = 27), compared with 55.2% for colistin (n = 27) and 55,7% for tigecycline (n = 18). Discussion: A high comorbidity index and nosocomial environment exposure were observed,as in previously published studies. The 28-day mortality was comparable to that reported inother studies. There was less mortality in patients treated with a combination that includeda carbapenem agent, as was reported in a recent clinical study on patients with bacteremia Klebsiella pneumoniae carbapenemase. Conclusions: CRE bacteremia is seen in very ill patients and is associated with high mortality. Bacterial colonization was detected in nearly half the patients prior to development of infection. The current antimicrobial therapy is heterogeneous, but the inclusion of a carbapenems agent in combination therapy may be associated with lower mortality.

Detection of Carbapenemases in Clinical Enterobacteriaceae Isolates Using the VITEK AST-N202 Card / 감염과화학요법

BACKGROUND: The rapid and accurate detection of carbapenemase-producing Enterobacteriaceae (CPE) in clinical microbiology laboratories is essential for the treatment and control of infections caused by these microorganisms. This study was performed to evaluate the ability of the VITEK AST-N202 card to detect CPE isolates. MATERIALS AND METHODS: A total of 43 (Klebsiella pneumoniae, n = 37; Escherichia coli, n = 3; and Enterobacter cloacae, n = 3) CPE isolates and 79 carbapenemase-non-producing Enterobacteriaceae (CNE) isolates were included in this study. The CPE isolates harbored KPC-2 (n = 11), KPC-3 (n = 20), GES-5 (n = 5), VIM-2 (n = 2), IMP-1 (n = 1), NDM-1 (n = 2), or OXA-232 (n = 2). Of the 79 CNE isolates, eight K. pneumoniae isolates were resistant to ertapenem, imipenem, and meropenem, while the remaining 71 isolates were susceptible to the carbapenems. Antimicrobial susceptibilities were tested using the VITEK AST-N202 card, and the results were interpreted as positive when the isolates showed resistant or intermediate results. Modified-Hodge tests (MHTs) were performed using ertapenem or meropenem disks for the screening of carbapenemase production. Polymerase chain reaction (PCR) and direct sequencing were used to identify beta-lactamase genes. RESULTS: Sensitivity of MHT with ertapenem and meropenem disks for the detection of carbapenemase was 81.4% (35/43) and 81.4% (35/43), respectively, and a combination with both antibiotic disks increased the sensitivity to 88.4% (38/43). Specificity of the MHT was 100% (79/79) for the CNE isolates. Sensitivity of ertapenem, imipenem, and meropenem as assessed by the VITEK AST-N202 card was 100% (43/43), 93% (40/43), and 95.3% (41/43), respectively. Specificity (89.8%, 71/79) of the test with each carbapenem was improved to 100% (71/71) when eight carbapenem-resistant CNE isolates were excluded from the testing. CONCLUSION: The VITEK AST-N202 card showed high sensitivity for the detection of carbapenemases in Enterobacteriaceae strains. PCR and sequencing experiments for the detection of carbapenemases are recommended when clinical Enterobacteriaceae isolates show non-susceptibility to carbapenems.