Juan C Román

Porin alterations present in non-carbapenemase-producing Enterobacteriaceae with high and intermediate levels of carbapenem resistance in Chile

The main goal of this work was to identify the mechanisms responsible for carbapenem resistance in 61 Chilean clinical isolates of Enterobacteriaceae (Enterobacter spp., Serratia marcescens, Morganella morganii, Escherichia coli and Klebsiella pneumoniae) with reduced susceptibility to at least one carbapenem (ertapenem, imipenem or meropenem). All of the isolates were analysed for the presence of carbapenemases, extended spectrum β-lactamases (ESBLs), AmpC enzymes and outer-membrane proteins. None of the isolates exhibited carbapenemase activity nor did they have any of the carbapenemase genes that were screened for. Most of the 61 strains produced at least one ESBL and/or one AmpC enzyme and either lost their porins or had altered porins according to sequence analysis. The distribution of ESBLs and AmpC enzymes was different among the species studied. Resistance in K. pneumoniae and E. coli isolates was associated with ESBLs; in M. morganii isolates, resistance was attributed to overexpression of an AmpC enzyme; and in Enterobacter spp. isolates, resistance was associated with both types of enzymes. In K. pneumoniae isolates, porin integrity was more a determinant of carbapenem resistance than the presence of ESBLs, whereas in isolates of Enterobacter spp., M. morganii and S. marcescens, the presence of an overexpressed AmpC enzyme was associated with higher imipenem and meropenem MIC values. Therefore, carbapenem resistance in Chilean isolates is not due to true carbapenemases but rather to a combination of porin loss/alteration and β-lactamase activity. The fact that carbapenemases were not detected in this study is unique, given that many countries in the region have already reported the presence of these enzymes.

M-protein gene-type distribution and hyaluronic acid capsule in group A Streptococcus clinical isolates in Chile: association of emm gene markers with csrR alleles.

Streptococcus pyogenes causes a variety of infections because of virulence factors such as capsular hyaluronic acid and M protein. The aim of this study was to determine emm types and capsule phenotype in 110 isolates of S. pyogenes from patients with invasive (sterile sites) and non-invasive (mainly pharyngitis) infections in Chile, and the relationship between both virulence factors. The most abundant types found were emm12, emm1, emm4 and emm28 and their distribution was similar to that seen in Latin America and developed countries, but very different from that in Asia and Pacific Island countries. Ten of 16 emm types identified in pharyngeal isolates were found in sterile-site isolates, and three of nine emm types of sterile-site isolates occurred in pharyngeal isolates; three emm subtypes were novel. The amount of hyaluronic acid was significantly higher in sterile-site isolates but did not differ substantially among emm types. Only three isolates were markedly capsulate and two of them had mutations in the csrR gene that codes for a repressor of capsule synthesis genes. We found a non-random association between emm types and csrR gene alleles suggesting that horizontal gene transfer is not freely occurring in the population.

Análisis de los fenotipos y genotipos de resistencia a eritromicina y clindamicina en cepas de Streptococcus pyogenes aisladas en Chile en un período de 10 años

Background : Macrolide and lincosamide resistance in Streptococcus pyogenes is due to the acquisition of mef, ermB and ermA genes, which confer different resis- tance phenotypes, namely M, MLSBconstitut ive and MLSBinducible respectively. The last report of resistance in Chile was done in the period 1990-1998, in which resistance to macrolides was 5.4%, with M phenotype as the predominant one. Aim : To characterize the evolution of erythromycin and clindamycin resistance and their associated genes in S. pyogenes strains iso lated from patients with invasive and non- invasive infections in the period 1996 to 2005. Material and Methods: Resistance to erythromycin and clindamycin was determined in 1,282 clinical isolates using the disk diffusion test. Resistant isolates were analyzed by polymerase chain reaction (PCR) for the presence of the above mentioned resistance genes. Results : Global resistance to erythromycin and clindamycin was 3.5 and 0.7% respectively. Eighty percent of the resistant strains possessed the M. phenotype. Conclusions : Resistance levels of S. pyogenes have decreased in Chile in the last years. Most resistant strains have M phenotype in contrast to many countries in which the MLSB constitutive phenotype is the predominant one

Presencia de metalo-ß-lactamasas en Pseudomonas aeruginosa resistente a imipenem

Background: Metallo-s-lactamases (MBL) confer high resistance to carbapenems in Pseudomonas aeruginosa (Psae). They are encoded in mobile elements of different genes (VIM, IMP, SMP, GIM), along with other resistance genes. Aim: To detect the presence of MBL in imipenem resistant Psae strains. Material and methods: Fifty-nine imipenem resistant Psae strains isolated from January 2004 to August 2005 in a University Clinical Hospital, were included. The presence of MBL was studied by Etest (phenotypic) and genotypic polymerase chain reaction (PCR) methods. To rule out a nosocomial outbreak, MBL positive strains, were studied by pulse field gel electrophoresis. Results: The presente of MBL was detected in eleven strains. AH were type VIM and were not clonally related. There was no concordance between phenotypic and genotypic MBL detecting methods. AH the strains were also multiresistant. Conclusions: The presence of MBL was detected in 19% of imipenem resistant Psae strains

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.

Comparación de adenosina deaminasa y detección de anticuerpos anti-antígeno A60 para el diagnóstico de meningitis tuberculosa

BACKGROUND Diagnosis of tuberculous meningitis (TBM) is hampered by the lack of rapid and accurate diagnostic tools. We evaluated the immunological response to Mycobacterium tuberculosis anti-A60 antibodies in cerebrospinal fluid (CSF) in comparison to adenosine deaminase (ADA) determination, for the diagnosis of TBM. METHODS A total of 63 CSF samples were analyzed by indirect ELISA for the detection of anti- A60 IgG, IgM and IgA. These include samples from 17 patients with confirmed TBM and 46 control patients with other infections. RESULTS The mean individual anti-A60 IgM, IgG and IgA CSF antibody titers were significantly higher in TBM in comparison with control groups (p < 0.01). The best discriminatory CSF antibody for confirming TBM diagnosis was IgM, with an area under the receiver operating characteristic curve of 0.928 (95%CI 0.834-0.978), compared to 0.863 (95% CI: 0.752-0.936) for ADA testing (p = NS). The sensitivity of anti- A60 IgM CSF antibody titers (cutoff > 0.06 U/ml) was 94.1% compared to 88.2% for ADA (cutoff > 6.2 U/ml), p = NS. Both anti A60 IgM and ADA showed the same moderate specificity (80.4%). Two cases of TBM were correctly identified by anti-A60 IgM but missed by ADA. CONCLUSION The ELISA test for anti-antigen A60 antibodies (IgM) is a rapid and sensitive tool for the rapid diagnosis of TBM that can be a complement to ALDA determination. The specificity of both tests is still a limitation in TBM diagnosis.

Evaluación de un sistema automatizado de siembra de orinas para urocultivos

Resumen Introduccion : Los sistemas automatizados han facili-tado el flujo de trabajo, mejorado la estandarizacion, la trazabilidad, disminuido el error humano y la carga de tra-bajo en los laboratorios. A pesar de que la microbiologia ha permanecido poco automatizada, en los ultimos anos han aparecido nuevas herramientas para la automatizacion de la etapa pre analitica. Objetivos: Evaluar el desempeno de un sistema automatizado de siembra de urocultivos y la concordancia con la siembra manual convencional en el recuento semicuantitativo de colonias. Materiales y Metodos: 495 muestras de orinas fueron sembradas segun nuestro protocolo habitual y comparadas con las placas de CPS® obtenidas con PREVI™ Isola en cuanto a positividad/negatividad, muestras polimicrobianas, especies de bacterias aisladas, recuentos y necesidad de resembrar. Resultados: Hubo concordancia en 98,97% de los positivos y negativos, en 99,39% de las muestras polimicrobianas, en 99,76% de las especies aisladas y en 98,56% de los recuentos. La necesidad de resiembra disminuyo de 12,1% a un 1,1% usando este sistema au-tomatizado.

Vigilancia de enterobacterias productoras de carbapenemasas en cultivos rectales en un hospital universitario de Santiago, Chile

Introduccion: Los cultivos clinicos detectan solo un tercio de los pacientes colonizados con enterobacterias resistentes a carbapenemicos, su identificacion temprana permitiria implementar precauciones de contacto y asi interrumpir su transmision. En nuestro hospital no hay descritas infecciones causadas por enterobacterias productoras de carbapenemesas Objetivo: Realizar vigilancia de colonizacion intestinal por enterobacterias productoras de carbapenemasas en pacientes ingresados a nuestro hospital. MaterialyMetodos: Se realizaron cultivos de vigilancia rectal en pacientes con cinco o mas dias de hospitalizacion en una unidad de paciente critico y cuidados especiales de nuestro hospital, en forma mensual, desde julio a diciembre del ano 2011. Posteriormente se realizaron tests fenotipicos (test de Hodge modificado y test de acido fenilboronico) para detectar enterobacterias productoras de carbapenemasas y busqueda confirmatoria de seis carbapenemasas del grupo A y B de la clasificacion de Ambler mediante reaccion de polimerasa en cadena (RPC). Resultados: Durante este periodo, se llevaron a cabo 241 vigilancias rectales; de estas, 38 enterobacterias resultaron resistentes o con susceptibilidad intermedia a carbapenemicos por metodo de dilucion en agar, las que correspondieron a 30 pacientes estudiados. Todas las RPC fueron negativas. Conclusion: A pesar del significativo numero de cepas resistentes a carbapenemicos, en los pacientes analizados no se encontraron enterobacterias productoras de carbapenemasas. Destacamos la importancia de la realizacion de cultivos de vigilancia antes de tener casos clinicos sintomaticos detectados.

Evaluación de la técnica Xpert® MTB/RIF para la detección de Mycobacterium tuberculosis complex en muestras extra-pulmonares

Extra-pulmonary tuberculosis (TB) represents the 26.2% of total TB cases in Chile. Culture is the gold standard method, but the process is extremely slow. Xpert®MTB/RIF technique detects Mycobacterium tuberculosis complex (MTBc) through real time PCR in less than 3 h. However, it has been validated only for respiratory specimens. We aimed to determine the performance of Xpert®MTB/RIF test in detecting MTBc in extra-respiratory specimens compared with a combined gold standard consisting in a positive (liquid and solid) mycobacterial culture and/or a positive validated molecular method (q-RPC, Cobas®TaqMan®-MTB). Fifty extra-respiratory specimens were analyzed, from which 25 were positive and 25 negative for MTBc based on the combined gold standard. The 25 positive specimens had a positive result by Xpert®MTB/RIF; from the 25 negative specimens, 24 had a negative result and one had a positive result. We obtained an overall concordance of 98% between Xpert®MTB/RIF and the combined gold standard. Xpert®MTB/RIF test was able to detect 12 smear-negative specimens and 3 culture-negative specimens, all of them corresponding to extra-pulmonary TB cases. Xpert®MTB/RIF showed similar sensitivity to q-RPC in detecting MTBc in extra-respiratory specimens. This procedure allowed a substantial reduction in the time of diagnosis.

Impacto de una técnica automatizada rápida en la identificación de SAMR/ERV en pacientes trasladados desde otros centros hospitalarios

INTRODUCTION Identification of patients with methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococci (VRE) is essential to limit the spread of these agents, through the use of isolation and contact precautions. Traditional microbiology has a long turn around time (3-5 days) extending the time of isolation, increasing complexity and cost of these patients. OBJECTIVES To implement a new real time polymerase chain reaction (PCR) GeneXpert R for SAMR and VRE detection. To compare costs and turn around time of PCR versus traditional cultures. METHODS Two periods were compared, in the first, traditional microbiology (standard group) was used, and in the second, only PCR was used (PCR group). RESULTS MRSA or VRE were identified in 29.9% of patients in the PCR group and in 9.6% in the standard group. Turn around time was 15 ± 9 hours in PCR group and 53 ± 23 hours in standard group. PCR group had a net cost of USD 245 per patient and standard group USD 530 per patient. DISCUSSION PCR technique GeneXpert R for MRSA and VRE had a positive impact in the management of these patients and justifies its inclusion.