First molecular characterization of Escherichia coli O157:H7 isolates from clinical samples in Paraguay using whole-genome sequencing Molecular characterization of Escherichia coli O157 isolates from Paraguay / Primera caracterización molecular de aislamientos de Escherichia coli O157:H7 provenientes de muestras clínicas en Paraguay utilizando secuenciación de genoma completo

Abstract Escherichia coli O157:H7 is a foodborne pathogen implicated in numerous outbreaks worldwide that has the ability to cause extra-intestinal complications in humans. The Enteropathogens Division of the Central Public Health Laboratory (CPHL) in Paraguay is working to improve the genomic characterization of Shiga toxin-producing E. coli (STEC) to enhance laboratory-based surveillance and investigation of foodborne disease outbreaks. Whole genome sequencing (WGS) is proposed worldwide to be used in the routine laboratory as a high-resolution tool that allows to have all the results in a single workflow. This study aimed to carry out for the first time, the genomic characterization by WGS of nine STEC O157:H7 strains isolated from human samples in Paraguay. We were able to identify virulence and resistance mechanisms, MLST subtype, and even establish the phylogenetic relationships between isolates. Furthermore, we detected the presence of strains belonging to hypervirulent clade 8 in most of the isolates studied.

Resumen Escherichia coli O157:H7 es un patógeno transmitido por alimentos implicado en numerosos brotes en todo el mundo y es capaz de causar complicaciones extraintestinales en humanos. La sección de «Enteropatógenos¼ del Laboratorio Central de Salud Pública trabaja en mejorar la caracterización genómica de STEC, de modo de potenciar la vigilancia laboratorial y la investigación de brotes de enfermedades transmitidas por alimentos. La secuenciación de genoma completo (WGS, por sus siglas en inglés) se propone a nivel mundial como una herramienta de alta resolución para ser utilizada en el laboratorio de rutina, ya que permite obtener todos los resultados en un único proceso. El objetivo de este trabajo fue llevar a cabo, por primera vez, la caracterización genómica por WGS de nueve cepas STEC O157:H7 aisladas en Paraguay a partir de muestras de origen humano. Pudimos identificar los factores de virulencia, los mecanismos de resistencia, el subtipo MLST, e incluso pudimos establecer la relación filogenética entre los aislamientos. Además, detectamos que la mayoría de las cepas pertenecían al clado hipervirulento 8.

Human papillomavirus genotyping using next generation sequencing (NGS) in cervical lesions: Genotypes by histologic grade and their relative proportion in multiple infections.

Sensitive and specific genotyping of human papillomaviruses (HPVs) is critical for the surveillance and monitoring of the vaccine effectiveness. Here, HPV genotypes were identified in 137 cervical samples with different histology (79 ≤CIN1 and 58 CIN3+) using Nested-PCR followed by Next-Generation sequencing (NGS) and relative proportions for each genotype in multiple infections were computed. All samples had been previously genotyped by PCR-Reverse Blotting Hybridization (PCR-RBH) thus allowing for a concordance analysis between both techniques. Multiple infections were present in 85% of ≤CIN1 cases compared to only 41% in CIN3+ cases (p<0.001). Among ≤CIN1 cases a towering genotypic diversity was observed, considering both low (LR-) and high risk (HR-) HPV genotypes; while among CIN3+, diversity was lower, HR-HPVs prevailing in most cases, especially HPV16. Furthermore, the predominance of HR-HPV genotypes in the proportions identified in each sample was higher in CIN3+ cases [(HPV16 (62.5%), followed by HPV31 and HPV58 (8.3% each)], than in ≤CIN1 cases [(HPV16 (17.7%), followed by HPV52 (14.7%) and HPV31 (10.3%)]. Agreement between PCR-RBH and NGS was higher than 90% for all genotypes (with an overall Kappa of 0.7), even though NGS identified eighty-nine positive results for HPV genotypes that had not been detected by PCR-RBH, evidencing its greater sensitivity. These results suggest that a reduction in genotypic diversity and/or an increase in the relative proportion of HR-HPVs in multiple infections can be considered as a biomarker for the potential risk of malignant progression.

OXA-48 Carbapenemase in Klebsiella pneumoniae Sequence Type 307 in Ecuador.

Antibiotic resistance is on the rise, leading to an increase in morbidity and mortality due to infectious diseases. Klebsiella pneumoniae is a Gram-negative bacterium that causes bronchopneumonia, abscesses, urinary tract infection, osteomyelitis, and a wide variety of infections. The ubiquity of this microorganism confounds with the great increase in antibiotic resistance and have bred great concern worldwide. K. pneumoniae sequence type (ST) 307 is a widespread emerging clone associated with hospital-acquired infections, although sporadic community infections have also been reported. The aim of our study is to describe the first case of Klebsiella pneumoniae (ST) 307 harboring the blaOXA-48-like gene in Ecuador. We characterized a new plasmid that carry OXA-48 and could be the source of future outbreaks. The strain was recovered from a patient with cancer previously admitted in a Ukrainian hospital, suggesting that this mechanism of resistance could be imported. These findings highlight the importance of programs based on active molecular surveillance for the intercontinental spread of multidrug-resistant microorganisms with emergent carbapenemases.

Diagnóstico de un brote de Salmonella Typhimurium en Chinchillas (Chinchilla lanigera) mediante la electroforesis en gel de campo pulsado / Diagnosis of an autbreak of Salmonella Typhimurium in chinchillas (Chinchilla lanigera) by pulsed-fiel gel electrophoresis

Empleando estudios anatomopatológicos y microbiológicos se examinó a un grupo de chinchillas (Chinchilla lanigera) adultas que murieron súbitamente en 2012 en una granja de la ciudad de La Plata (Buenos Aires, Argentina). Se aisló Salmonella enterica serovar Typhimurium (S. Typhimurium) del hígado, el bazo, el corazón, los pulmones, los riñones y los intestinos de los cinco animales evaluados. Los cinco aislamientos estudiados (uno por animal) fueron sensibles a ampicilina, cefalotina, cefotaxima, ácido nalidíxico, gentamicina, estreptomicina, cloranfenicol, fosfomicina, nitrofurantoína y trimetoprima-sulfametoxazol, y resistentes a tetraciclina. El análisis de dichos aislamientos por electroforesis en gel de campo pulsado [pulsed-field gel electrophoresis (PFGE)] con XbaI mostró un perfil electroforético idéntico con 15 bandas, idéntico a su vez al patrón ARJPXX01.0220 del banco nacional argentino de datos de PulseNet, que cuenta con patrones de PFGE de Salmonella. El presente trabajo describe por primera vez el diagnóstico postmortem de un brote de salmonelosis en chinchillas usando un método molecular, como la electroforesis en gel en campo pulsado

Adult chinchillas (Chinchilla lanigera) that had suddenly died in a commercial farm located in La Plata City, Buenos Aires Province, Argentina, in July 2012 were macroscopically, histopathologically, and microbiologically examined. Salmonella enterica serovar Typhimurium (S. Typhimurium) was isolated from the liver, spleen, heart, lungs, kidneys and intestines from each of the five animals evaluated. The five strains were susceptible to ampicillin, cephalotin, cefotaxime, nalidixic acid, gentamicin, streptomycin, chloramphenicol, fosfomycin, nitrofurantoin and trimethoprimsulfamethoxazole, and resistant to tetracycline. Each of the five S. Typhimurium isolates was analyzed by XbaI- pulsed-field gel electrophoresis (PFGE), showing an identical electrophoretic profile with 15 defined bands, which was found to be identical to pattern ARJPXX01.0220 of the PulseNet Argentine National database of Salmonella PFGE patterns. This is the first work describing the postmortem diagnosis of an outbreak of salmonellosis in chinchillas by using molecular methods such as PFGE

Experiencias en la vigilancia epidemiológica de agentes patógenos transmitidos por alimentos a través de electroforesis en campo pulsado (PFGE) en el Perú / Experiences in the epidemiological surveillance of foodborne pathogens by pulsed field gel electrophoresis (PFGE) in Peru

Las enfermedades transmitidas por alimentos (ETA) y otras enfermedades entéricas infecciosas ocurren a menudo como brotes y son causa de morbilidad y mortalidad en todo el mundo. En el Perú, son un importante problema de salud pública y son causados por una gran variedad de agentes infecciosos. Para la investigación epidemiológica se utiliza una variedad de métodos de tipificación. Una de las herramientas más importantes en la subtipificación molecular de patógenos bacterianos es la técnica de la electroforesis en campo pulsado (PFGE), que es un método altamente resolutivo que permite la discriminación entre diferentes aislamientos bacterianos epidemiológicamente relacionados. El Instituto Nacional de Salud (INS) del Perú integra las redes WHO Global Foodborne Infections Network y la Red PulseNet América Latina y Caribe, con quienes comparte los perfiles genéticos de las cepas patógenas aisladas, permitiendo comparar los genotipos de cepas semejantes halladas en diferentes países y reconocer la ocurrencia de brotes epidémicos en la región, fortaleciendo el sistema de vigilancia epidemiológica regional y generando una rápida respuesta conjunta entre países. Se presenta la experiencia de los dos últimos años sobre los avances en la utilización de estas herramientas estratégicas que nos ha permitido caracterizar patrones de genotipo de principales patógenos implicados en ETA a partir de aislamientos recuperados de la red de laboratorios del Perú.

Foodborne diseases and other enteric infections often occur as outbreaks and cause morbidity and mortality all over the world. In Perú, they represent a serious public health problem, and are caused by a great variety of infectious agents. For epidemiological research, a wide array of typification methods are used. One of the most important tools for the molecular subtyping of bacterial pathogens is the Pulsed Field Gel Electrophoresis (PFGE), which is a highly precise method that allows the discrimination between different bacterial isolates which are epidemiologically related. The Instituto Nacional de Salud del Perú (INS) is part of the WHO Global Foodborne Infections Network (WHO-GFN) and of the PulseNet Latin American and Caribbean Net (PN-AL & C), with whom it shares the genetic profiles of the isolated pathogenic strains, so that it is possible to compare de genotypes of similar strains found in different countries and to identify the occurrence of epidemic outbreaks in the region, strengthening the regional system of epidemiological surveillance and generating a rapid, coordinated response between the countries. We present the two last years´ experience including the advances in the use of these strategic tools that have allowed us to characterize genotype patterns implicated in foodborne diseases from isolates recovered in the laboratory network of Peru.

A polymorphism at the 3'-UTR region of the aromatase gene defines a subgroup of postmenopausal breast cancer patients with poor response to neoadjuvant letrozole.

BACKGROUND: Aromatase (CYP19A1) regulates estrogen biosynthesis. Polymorphisms in CYP19A1 have been related to the pathogenesis of breast cancer (BC). Inhibition of aromatase with letrozole constitutes the best option for treating estrogen-dependent BC in postmenopausal women. We evaluate a series of polymorphisms of CYP19A1 and their effect on response to neoadjuvant letrozole in early BC. METHODS: We analyzed 95 consecutive postmenopausal women with stage II-III ER/PgR [+] BC treated with neoadjuvant letrozole. Response to treatment was measured by radiology at 4th month by World Health Organization (WHO) criteria. Three polymorphisms of CYP19A1, one in exon 7 (rs700519) and two in the 3'-UTR region (rs10046 and rs4646) were evaluated on DNA obtained from peripheral blood. RESULTS: Thirty-five women (36.8%) achieved a radiological response to letrozole. The histopathological and immunohistochemical parameters, including hormonal receptor status, were not associated with the response to letrozole. Only the genetic variants (AC/AA) of the rs4646 polymorphism were associated with poor response to letrozole (p = 0.03). Eighteen patients (18.9%) reported a progression of the disease. Those patients carrying the genetic variants (AC/AA) of rs4646 presented a lower progression-free survival than the patients homozygous for the reference variant (p = 0.0686). This effect was especially significant in the group of elderly patients not operated after letrozole induction (p = 0.009). CONCLUSIONS: Our study reveals that the rs4646 polymorphism identifies a subgroup of stage II-III ER/PgR [+] BC patients with poor response to neoadjuvant letrozole and poor prognosis. Testing for the rs4646 polymorphism could be a useful tool in order to orientate the treatment in elderly BC patients.