Laboratory-Based Prospective Surveillance for Community Outbreaks of Shigella spp. in Argentina

Background: To implement effective control measures, timely outbreak detection is essential. Shigella is the most common cause of bacterial diarrhea in Argentina. Highly resistant clones of Shigella have emerged, and outbreaks have been recognized in closed settings and in whole communities. We hereby report our experience with an evolving, integrated, laboratory-based, near real-time surveillance system operating in six contiguous provinces of Argentina during April 2009 to March 2012. Methodology To detect localized shigellosis outbreaks timely, we used the prospective space-time permutation scan statistic algorithm of SaTScan, embedded in WHONET software. Twenty three laboratories sent updated Shigella data on a weekly basis to the National Reference Laboratory. Cluster detection analysis was performed at several taxonomic levels: for all Shigella spp., for serotypes within species and for antimicrobial resistance phenotypes within species. Shigella isolates associated with statistically significant signals (clusters in time/space with recurrence interval ≥365 days) were subtyped by pulsed field gel electrophoresis (PFGE) using PulseNet protocols. Principal Findings In three years of active surveillance, our system detected 32 statistically significant events, 26 of them identified before hospital staff was aware of any unexpected increase in the number of Shigella isolates. Twenty-six signals were investigated by PFGE, which confirmed a close relationship among the isolates for 22 events (84.6%). Seven events were investigated epidemiologically, which revealed links among the patients. Seventeen events were found at the resistance profile level. The system detected events of public health importance: infrequent resistance profiles, long-lasting and/or re-emergent clusters and events important for their duration or size, which were reported to local public health authorities. Conclusions/Significance: The WHONET-SaTScan system may serve as a model for surveillance and can be applied to other pathogens, implemented by other networks, and scaled up to national and international levels for early detection and control of outbreaks

Standardization and international multicenter validation of a PulseNet pulsed-field gel electrophoresis protocol for subtyping Shigella flexneri isolates

Fil: Pichel, Mariana. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas; Argentina.Fil: Brengi, Silvina P. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas; Argentina.Fil: Cooper, Kara L. F. Centers for Disease Control and Prevention; Estados Unidos.Fil: Ribot, Efrain M. Centers for Disease Control and Prevention; Georgia.Fil: Al-Busaidy, Suleiman. Central Public Health Laboratory; Omán.Fil: Araya, Pamela. Instituto de Salud Pública de Chile; Chile.Fil: Fernández, Jorge. Instituto de Salud Pública de Chile; Chile.Fil: Vaz, Tania Ibelli. Instituto Adolfo Lutz; Brazil.Fil: Kam, Kai Man. Public Health Laboratory Centre; Japón.Fil: Morcos, Myriam. Regional Center at the U.S. Naval Medical Research Unit #3 (NAMRU-3). Global Disease Detection (GDD); Egipto.Fil: Nielsen, Eva M. Statens Serum Institut; Dinamarca.Fil: Nadon, Celine. National Microbiology Laboratory; Canadá.Fil: Pimentel, Guillermo. Regional Center at the U.S. Naval Medical Research Unit #3 (NAMRU-3). Global Disease Detection (GDD); Egipto.Fil: Pérez-Gutiérrez, Enrique. PAHO/WHO. Health Surveillance; Panamá.Fil: Gerner-Smidt, Peter. Centers for Disease Control and Prevention; Georgia.Fil: Binsztein, Norma. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas; Argentina.Shigella flexneri is one of the agents most frequently linked to diarrheal illness in developing countries and often causes outbreaks in settings with poor hygiene or sanitary conditions. Travel is one of the means by which S. flexneri can be imported into developed countries, where this pathogen is not commonly seen. A robust and discriminatory subtyping method is needed for the surveillance of S. flexneri locally and regionally, and to aid in the detection and investigation of outbreaks. The PulseNet International network utilizes standardized pulsedfield gel electrophoresis (PFGE) protocols to carry out laboratory-based surveillance of foodborne pathogens in combination with epidemiologic data. A multicenter validation was carried out in nine PulseNet laboratories located in North and South America, Europe, and Asia, and it demonstrated that a new protocol is highly robust and reproducible for subtyping of S. flexneri. This protocol, already approved for PulseNet laboratories, applies NotI and XbaI as primary and secondary restriction enzymes, respectively, under electrophoresis conditions of initial switch time of 5 s to final switch time of 35 s, at 6 volts/cm

Characterization and subtyping of Cronobacter spp. from imported powdered infant formulae in Argentina

Fil: Terragno, R. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas; Argentina.Fil: Salve, Angela. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas; Argentina.Fil: Pichel, Mariana. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas; Argentina.Fil: Epszteyn, Sergio. Gobierno de la Ciudad de Buenos Aires. Dirección General de Higiene y Seguridad Alimentaria; Argentina.Fil: Brengi, Silvina P. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas; Argentina.Fil: Binsztein, Norma. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas; Argentina.Cronobacter spp. (Enterobacter sakazakii), have been associated with severe foodborne infections in neonates and immunocompromised infants. In Argentina, we have isolated Cronobacter spp. from three different brands of imported powdered infant formulae (PIF). The objectives of this work were to characterize the recovered isolates phenotypically and to evaluate the use of a Pulsed-Field Gel Electrophoresis (PFGE) protocol for Cronobacter spp. subtyping. Out of 23 isolates studied from three brands of PIF (20 of brand A, 1 of brand B and 2 of brand C), 22 were identified as C. sakazakii and 1 as C. malonaticus. All isolates were susceptible to twelve antimicrobial agents assayed. The 19 C. sakazakii isolates of brand A showed five XbaI-PFGE patterns and the genetic clusters revealed by XbaI were confirmed with a second restriction enzyme, SpeI. The isolate from brand B showed the same XbaI and SpeI patterns as those of a group of isolates of brand A, suggesting a possible common source of contamination. The C. sakazakii isolates of brand C exhibited two unique XbaI-PFGE patterns, unrelated to the rest. Different genetic subtypes were found among isolates of a single batch of PIF from brand A and the single C. malonaticus strain also showed a distinct XbaI-PFGE pattern

Successful management with fosfomycin + ceftazidime of an infection caused by multiple highly-related subtypes of multidrug-resistant and extensively drug-resistant KPC-producing Serratia marcescens

Fil: Rodriguez, Cecilia. Centro de Referencia para Lactobacilos-Consejo Nacional de Investigaciones Científicas y Técnicas (CERELA-CONICET), San Miguel de Tucumán, Tucumán; Argentina.Fil: Brengi, Silvina P. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio Enterobacterias; Argentina.Fil: Cáceres, Mariel Agustina. Laboratorio de Bacteriología del Hospital 'Ángel C. Padilla', San Miguel de Tucumán, Tucumán; Argentina.Fil: Mochi, Silvana. Laboratorio de Bacteriología del Hospital 'Ángel C. Padilla', San Miguel de Tucumán, Tucumán; Argentina.Fil: Viñas, María R. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio Enterobacterias; Argentina.Fil: Rizza, Constanza Antich. Laboratorio de Microbiología del Hospital del Niño Jesús, San Miguel de Tucumán, Tucumán; Argentina.Fil: Merletti, G. Laboratorio de Microbiología del Hospital del Niño Jesús, San Miguel de Tucumán, Tucumán; Argentina.Fil: Bru, Elena. Centro de Referencia para Lactobacilos-Consejo Nacional de Investigaciones Científicas y Técnicas (CERELA-CONICET), San Miguel de Tucumán, Tucumán; Argentina.Fil: Assa, José Daniel. Laboratorio de Microbiología del Hospital del Niño Jesús, San Miguel de Tucumán, Tucumán; Argentina.Fil: Raya, Raúl Ricardo. Centro de Referencia para Lactobacilos-Consejo Nacional de Investigaciones Científicas y Técnicas (CERELA-CONICET), San Miguel de Tucumán, Tucumán; Argentina.Fil: Centrón, Daniela. Instituto de Microbiología y Parasitología Médica, Facultad de Medicina, Universidad de Buenos Aires (IMPaM, UBA-CONICET), Buenos Aires, Argentina

Development and Validation of a PulseNet Standardized Protocol for Subtyping Isolates of Cronobacter Species

Cronobacter (formerly known as Enterobacter sakazakii) is a genus comprising seven species regarded as opportunistic pathogens that can be found in a wide variety of environments and foods, including powdered infant formula (PIF). Cronobacter sakazakii, the major species of this genus, has been epidemiologically linked to cases of bacteremia, meningitis in neonates, and necrotizing enterocolitis, and contaminated PIF has been identified as an important source of infection. Robust and reproducible subtyping methods are required to aid in the detection and investigation, of foodborne outbreaks. In this study, a pulsed-field gel electrophoresis (PFGE) protocol was developed and validated for subtyping Cronobacter species. It was derived from an existing modified PulseNet protocol, wherein XbaI and SpeI were the primary and secondary restriction enzymes used, generating an average of 14.7 and 20.3 bands, respectively. The PFGE method developed was both reproducible and discriminatory for subtyping Cronobacter species

PCR-Based Method for Shigella flexneri Serotyping: International Multicenter Validation

Fil: Brengi, Silvina P. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio Enterobacterias; Argentina.Fil: Sun, Qiangzheng. Chinese Center for Disease Control and Prevention. National Institute for Communicable Diseases Control and Prevention. State Key Laboratory for Infectious Disease Prevention and Control, Changping, Beijing; China.Fil: Bolaños, Hilda. Centro Nacional de Referencia de Bacteriología, Inciensa, Cartago; Costa Rica.Fil: Duarte, Francisco. Centro Nacional de Referencia de Bacteriología, Inciensa, Cartago; Costa Rica.Fil: Jenkins, Claire. Public Health England. Gastrointestinal Bacteria Reference Unit, Londres, Inglaterra.Fil: Pichel, Mariana. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio Enterobacterias; Argentina.Fil: Shahnaij, Mohammad. International Center for Diarrheal Disease Research. Laboratory Sciences and Services Division. Enteric and Food Microbiology Laboratory Dhaka; Bangladés.Fil: Sowers, Evangeline G. Centers for Disease Control and Prevention. National Institute for Escherichia and Shigella Reference Unit, Atlanta, Georgia, Estados Unidos.Fil: Strockbine, Nancy. Centers for Disease Control and Prevention. National Institute for Escherichia and Shigella Reference Unit, Atlanta, Georgia, Estados Unidos.Fil: Talukder, Kaisar A. International Center for Diarrheal Disease Research. Laboratory Sciences and Services Division. Enteric and Food Microbiology Laboratory Dhaka; Bangladés.Fil: Derado, Gordana. Centers for Disease Control and Prevention. Biostatistics and Information Management Office. Waterborne and Environmental Diseases. Division of Foodborne, Atlanta, Georgia, Estados Unidos.Fil: Viñas, María R. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio Enterobacterias; Argentina.Fil: Kam, Kai Man. The Chinese University of Hong Kong. Faculty of Medicine. School of Public Health and Primary Care. Stanley Ho Centre for Emerging Infectious Diseases, Hong Kong; China.Fil: Xu, Jianguo. Chinese Center for Disease Control and Prevention. National Institute for Communicable Diseases Control and Prevention. State Key Laboratory for Infectious Disease Prevention and Control, Changping, Beijing; China.Shigella spp. are a leading cause of human diarrheal disease worldwide, with Shigella flexneri being the most frequently isolated species in developing countries. This serogroup is presently classified into 19 serotypes worldwide. We report here a multicenter validation of a multiplex-PCR-based strategy previously developed by Q. Sun, R. Lan, Y. Wang, A. Zhao, et al. (J Clin Microbiol 49:3766-3770, 2011) for molecular serotyping of S. flexneri This study was performed by seven international laboratories, with a panel of 71 strains (researchers were blind to their identity) as well as 279 strains collected from each laboratory's own local culture collections. This collaborative work found a high extent of agreement among laboratories, calculated through interrater reliability (IRR) measures for the PCR test that proved its robustness. Agreement with the traditional method (serology) was also observed in all laboratories for 14 serotypes studied, while specific genetic events could be responsible for the discrepancies among methodologies in the other 5 serotypes, as determined by PCR product sequencing in most of the cases. This work provided an empirical framework that allowed the use of this molecular method to serotype S. flexneri and showed several advantages over the traditional method of serological typing. These advantages included overcoming the problem of availability of suitable antisera in testing laboratories as well as facilitating the analysis of multiple samples at the same time. The method is also less time-consuming for completion and easier to implement in routine laboratories. We recommend that this PCR be adopted, as it is a reliable diagnostic and characterization methodology that can be used globally for laboratory-based shigella surveillance

Dendrogram showing the genetic relatedness of isolates of <i>S. flexneri</i> 2 included in Event 1 and 6.

<p>Isolates recovered in Santa Rosa, La Pampa (Event 1 and 6), and selected isolates for comparison, including one from an outbreak in San Luis and three from sporadic cases in 2009. The rectangle highlights isolates of <i>S. flexneri</i> 2 recovered in March–April 2009 (Event 1) and October–November 2009 (Event 6).</p

Detection of events of <i>Shigella</i> by the SaTScan software, January 2012–March 2012 period.

<p>MFP: (the most frequent pattern).</p>a<p>: % indistinguishable isolates with the first enzyme and confirmed with the second enzyme.</p>b<p>: bands of difference (bd) to the MFP.</p

Events with epidemiologic evidence.

<p>Prov/Lab: Province/Laboratory.</p><p>LA: Latin America.</p

Dendrogram showing the genetic relatedness of <i>S. sonnei</i> SXT resistant isolates included in the Event 7.

<p>Isolates recovered in Santa Rosa, La Pampa in January – February 2010. The rectangle highlights the 7 <i>S. sonnei</i> isolates with an indistinguishable PFGE pattern which had not previously been recorded in the National Data Base (NDB). The remaining 7 isolates identified statistically and epidemiologically as part of Event 7, exhibited high genetic relatedness (from 91.4 to 97.4% similarity, 1 to 3 bands of difference) to the most frequent pattern within the event, confirming the relation of the cases.</p