Identification of Two New Sequences of Flagellin-Encoding Gene in Escherichia coli Using PCR and Sequencing-Based Methods.

Escherichia coli has traditionally been serotyped using antisera against the O and H antigens. However, a proportion of E. coli isolates are nonmotile and, in addition, some isolates do not react with the currently available H-typing sera. Alternative molecular methods have been developed based on the detection of genes encoding for H antigens. In this study, we studied 13 serologically nontypable H antigen E. coli strains using polymerase chain reaction (PCR) and sequencing-based methods. We found two new sequences of flagellin-encoding gene, for each of which a specific antiserum was produced to confirm their expression. Sequencing of the flagellin gene offers a rapid determination of E. coli H antigens and could be used to detect potential novel flagellar antigens.

Production of a latex agglutination reagent for the rapid diagnosis of cryptococcal meningitis / Producción de un reactivo de látex para el diagnóstico rápido de criptococosismeníngea

Abstract Cryptococcosis is a fungal disease affecting more than one million people per yearworldwide. Its main etiological agents are Cryptococcus neoformans species complex and Cryp-tococcus gattii species complex. Cryptococcal meningitis (CM) is considered an AIDS-definingcondition. Rapid diagnosis by cryptococcal antigen assays, either the latex agglutination test(LA) or the lateral flow assay, is key to decreasing mortality due to cryptococcal disease. Theaim of the study was to develop a latex agglutination reagent (LA-ANLIS) for the rapid and reliable diagnosis of cryptococcosis in Argentina. This reagent will be produced in order to supplythe NMLN (National Mycology Laboratory Network). The evaluation of LA-ANLIS performanceand its comparison with the Cryptococcus Antigen Latex Agglutination Test System (LA-IMMY)(Immuno-Mycologics, Inc., USA) were conducted in 94 samples of cerebrospinal fluid. LA-ANLISand LA-IMMY compared exhibited 100% positive agreement and 97% negative agreement. LA-ANLIS showed 94% sensitivity and 97% specificity with the positive and negative predictivevalues of 94% and 97%, respectively. The LA-ANLIS is a reliable, reproducible and cost-effectivereagent, especially useful in countries where the commercial kit is not generally available andmust be obtained at a high cost. National production of reagents is the best choice for a reliableaccess to the rapid diagnosis of CM in Argentina.

Resumen La criptococosis es una enfermedad fúngica que afecta a más de un millón de personas por año en todo el mundo. Los principales agentes etiológicos pertenecen a los complejos de especies Cryptococcus neoformans y Cryptococcus gattii. La criptococosis meníngea (CM) se considera una enfermedad marcadora de sida. El diagnóstico rápido de esta enfermedad a través de la detección del antígeno de Cryptococcus, ya sea por aglutinación en partículas de látex o por inmunocromatografía, es clave para disminuir la mortalidad. El objetivo del presente estudio fue desarrollar un reactivo de aglutinación en partículas de látex para el diagnóstico rápido y certero de la CM en Argentina. Este reactivo (denominado en adelante LA-ANLIS) será producido para abastecer a la Red Nacional de Laboratorios de Micología. Se evaluó el desempeno del reactivo LA-ANLIS, y se realizó una comparación con el reactivo comercial Immuno-Mycologics, Inc. (en adelante, LA-IMMY) utilizando 94 muestras de líquido cefalorraquídeo. Hubo un 100% de acuerdo positivo y un 97% de acuerdo negativo entre los resultados obtenidos con los reactivos LA-ANLIS y LA-IMMY. El reactivo LA-ANLIS mostró una sensibilidad del 94% y una especificidad del 97%; los valores predictivos positivo y negativo fueron del 94 y del 97%, respectivamente. Se concluye que el LA-ANLIS es un reactivo confiable y rentable, que arroja resultados reproducibles, por lo que es especialmente útil en países donde los reactivos comerciales generalmente no están disponibles o sus costos son elevados. La producción nacional de reactivos es la mejor opción para asegurar el acceso de todos los hospitales al diagnóstico rápido de la CM en Argentina.

Production of a latex agglutination reagent for the rapid diagnosis of cryptococcal meningitis.

Cryptococcosis is a fungal disease affecting more than one million people per year worldwide. Its main etiological agents are Cryptococcus neoformans species complex and Cryptococcus gattii species complex. Cryptococcal meningitis (CM) is considered an AIDS-defining condition. Rapid diagnosis by cryptococcal antigen assays, either the latex agglutination test (LA) or the lateral flow assay, is key to decreasing mortality due to cryptococcal disease. The aim of the study was to develop a latex agglutination reagent (LA-ANLIS) for the rapid and reliable diagnosis of cryptococcosis in Argentina. This reagent will be produced in order to supply the NMLN (National Mycology Laboratory Network). The evaluation of LA-ANLIS performance and its comparison with the Cryptococcus Antigen Latex Agglutination Test System (LA-IMMY) (Immuno-Mycologics, Inc., USA) were conducted in 94 samples of cerebrospinal fluid. LA-ANLIS and LA-IMMY compared exhibited 100% positive agreement and 97% negative agreement. LA-ANLIS showed 94% sensitivity and 97% specificity with the positive and negative predictive values of 94% and 97%, respectively. The LA-ANLIS is a reliable, reproducible and cost-effective reagent, especially useful in countries where the commercial kit is not generally available and must be obtained at a high cost. National production of reagents is the best choice for a reliable access to the rapid diagnosis of CM in Argentina.

Prevalence, antimicrobial resistance profile and comparison of methods for the isolation of salmonella in chicken liver from Argentina.

This study was conducted to estimate the apparent prevalence of Salmonella spp. in chicken livers obtained from markets in Entre Ríos, Argentina, using two culture methods (preenrichment and direct selective agar plating). We also determined the antimicrobial resistance of Salmonella isolated strains and evaluated the performance of the two culture methods and selective-differential plating media used for Salmonella isolation. Of 666 chicken livers studied, 32 organs (4.8%) related to 4 poultry slaughterhouse companies were positive for Salmonella sp. using one or two culture methods. Fifty Salmonella strains were isolated from the positive liver samples and were typed into 3 serovars: S. ser. Schwarzengrund (78%), S. ser. Enteritidis (18%), and S. ser. Typhimurium 4(%). More than one Salmonella serovar was found in livers belonging to two chicken slaughterhouse companies. All strains were susceptible to all antibiotics tested, with the exception of erythromycin (100% resistant) and streptomycin (22% intermediate sensitivity). Overall, 32 (4.80%) and 3 (0.45%) of the chicken liver samples were positive for Salmonella sp. in preenrichment method and direct selective agar plating method, respectively; these percentages were significantly different (P=0.0001; kappa=0.16). There was also a statistical difference in relative accuracy, sensitivity and negative predictive value between the preenrichment method and the direct selective agar plating method; the first had greater values for these parameters than the direct selective agar plating method. These parameters were statistically different between MacConkey agar (MCA) and modified lysine iron (MLIA) in the two culture methods; the second had greater values than MCA for both culture methods. This study shows that even though serovars that are important for public health were isolated, the prevalence of Salmonella sp. is low in chicken livers from Entre Rios, Argentina. The isolated strains do not have multi-resistance patterns. Furthermore, the preenrichment method and MLIA are superior to the direct selective agar plating method and MCA for Salmonella sp. isolation from chicken liver samples, respectively.

Design of Two Multiplex PCR Assays for Serotyping Shigella flexneri.

Shigella flexneri is a major health problem in developing countries. There are 19 serotypes recognized based on O-antigen structure and its typing is important for epidemiological purposes. However, the diversity of serotypes and the difficulties presented by phenotypic serotyping, for example, unavailable antisera for less common antigens, require the implementation of molecular techniques. In this study, we developed two multiplex PCR assays targeting the O-antigen synthesis genes and the O-antigen modification genes, for the rapid identification of S. flexneri serotypes 1/7, 2, 4, 5, and 6 (PCR A) and serotype 7 and group antigenic factors (3,4; 6; 7,8; E1037) (PCR B). A total of 73 S. flexneri strains representing 18 serotypes, except serotype 1d, were used in the study. Specific amplification patterns were obtained for each of the different serotypes. All strains tested had concordant results with phenotypic and genotypic serotyping; therefore, its implementation in the microbiology clinical laboratory will significantly improve S. flexneri serotyping.

AA479 antiserum: new reagent for the serotype characterization of atypical variants of Shigella flexneri.

Shigella flexneri is divided into 13 serotypes based on the combination of antigenic determinants present in the O-antigen. A new O-antigen modification with phosphoethanolamine has been identified. The presence of this antigenic determinant (called E1037) is recognized by monoclonal antibody MASF IV-1. Given the increasing incidence of these new variants and the difficulty in supplying the monoclonal antibody to our country, we produced a polyclonal antiserum (AA479) through immunization with a S. flexneri Xv strain. The antiserum specificity was assessed by slide agglutination against isolates from clinical cases and a culture collection representing all Shigella serotypes. The results obtained demonstrated a 100% correlation between AA479 absorbed antiserum and monoclonal antibody MASF IV-1. The availability of AA479 antiserum in every public hospital in Argentina will allow us to identify atypical S. flexneri isolates in order to strengthen Shigella surveillance in our country and to compare with global epidemiological data.

Antisuero AA479: nuevo reactivo para la serotipificación de variantes atípicas se Shigella flexneri / AA479 antiserum: new reagent for the serotype characterization of atypical variants of Shigella flexneri

Shigella flexneri is divided into 13 serotypes based on the combination of antigenic determinants present in the O-antigen. A new O-antigen modification with phosphoethanolamine has been identified. The presence of this antigenic determinant (called E1037) is recognized by monoclonal antibody MASF IV-1. Given the increasing incidence of these new variants and the difficulty in supplying the monoclonal antibody to our country, we produced a polyclonal antiserum (AA479) through immunization with a S. flexneri Xv strain. The antiserum specificity was assessed by slide agglutination against isolates from clinical cases and a culture collection representing all Shigella serotypes. The results obtained demonstrated a 100% correlation between AA479 absorbed antiserum and monoclonal antibody MASF IV-1. The availability of AA479 antiserum in every public hospital in Argentina will allow us to identify atypical S. flexneri isolates in order to strengthen Shigella surveillance in our country and to compare with global epidemiological dat

Shigella flexneri se divide en al menos 13 serotipos sobre la base de la combinación de determinantes antigénicos presentes en el antígeno O. Se identificó una nueva modificación del antígeno O con fosfoetanolamina. La presencia de este determinante antigénico (denominado E1037) es reconocida por el anticuerpo monoclonal MASF IV-1. Teniendo en cuenta la incidencia creciente de estas nuevas variantes y la dificultad en la provisión del anticuerpo monoclonal para nuestro país, se elaboró un antisuero de tipo policlonal (AA479) mediante la inmunización con un cultivo de S. flexneri Xv. La especificidad del antisuero se evaluó por aglutinación en lámina con aislamientos clínicos y cultivos de colección, con lo que quedaron representados todos los serotipos de Shigella. Los resultados obtenidos demostraron una correlación del 100% entre el antisuero AA479 absorbido y el anticuerpo monoclonal MASF IV-1. La disponibilidad del antisuero AA479 en todos los hospitales públicos de Argentina permitirá identificar los aislamientos atípicos de S. flexneri; de esta forma se podrá fortalecer la vigilancia de Shigella en nuestro país y comparar con los datos epidemiológicos a nivel global

AA479 antiserum: new reagent for the serotype characterization of atypical variants of Shigella flexneri. / AA479 antiserum: new reagent for the serotype characterization of atypical variants of Shigella flexneri.

Shigella flexneri is divided into 13 serotypes based on the combination of antigenic determinants present in the O-antigen. A new O-antigen modification with phosphoethanolamine has been identified. The presence of this antigenic determinant (called E1037) is recognized by monoclonal antibody MASF IV-1. Given the increasing incidence of these new variants and the difficulty in supplying the monoclonal antibody to our country, we produced a polyclonal antiserum (AA479) through immunization with a S. flexneri Xv strain. The antiserum specificity was assessed by slide agglutination against isolates from clinical cases and a culture collection representing all Shigella serotypes. The results obtained demonstrated a 100

correlation between AA479 absorbed antiserum and monoclonal antibody MASF IV-1. The availability of AA479 antiserum in every public hospital in Argentina will allow us to identify atypical S. flexneri isolates in order to strengthen Shigella surveillance in our country and to compare with global epidemiological data.

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.

Characterization of lipid A profiles from Shigella flexneri variant X lipopolysaccharide.

RATIONALE: In developing countries, Shigella flexneri (Sf) is the major causative agent of the endemic shigellosis (bacillary dysentery) responsible annually for one million fatalities mostly among infants. Lipopolysaccharides (LPSs) are characteristic components of the outer membrane of the overwhelming majority of Gram-negative bacteria. Since lipid A is essential for the viability of the Gram-negative bacteria, it is subject to extensive chemical studies with new analytical techniques. METHODS: Lipid A was released by mild acid hydrolysis from the lipopolysaccharide which was obtained via the phenol/water extraction, purified and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and matrix-assisted laser desorption/ionization laser-induced dissociation tandem mass spectrometry (MALDI-LID-MS/MS). RESULTS: A detailed structural study of the whole lipid A obtained from S. flexneri variant X was carried out for the first time. Thus, we have shown that lipid A is a heterogeneous mixture having different numbers of acylated and phosphoethanolamine groups attached to the diglucosamine backbone. Furthermore, we found in the phenol phase an unusual hepta-acylated lipid A species, although the abundance was very low. CONCLUSIONS: MALDI-TOF-MS allowed us to unravel the lipid A heterogeneity, which was not previously reported in Sf LPS. It is well known that slight variations of the chemical structure of lipid A may change its biological activity. Thus, the knowledge of the detailed chemical structure represents an essential step for further development of new preventive or therapeutically active compounds.

Identification and characterization of serine proteinase inhibitors from Neospora caninum.

Two cDNA clones obtained from the Neospora caninum Expressed Sequence Tag project were selected by their homology with the Toxoplasma gondii serine proteinase inhibitor (serpin) gene, TgPI-1 and TgPI-2. One of them, named NcPI-H, showed several premature stop codons. The other cDNA, named NcPI-S, encoded a 79 amino acid protein containing a putative signal peptide and only one non-classical Kazal domain. Two other N. caninum EST sequences (NcEST1 and NcEST2) and one from Eimeria tenella (EtPI-S) were retrieved from the database. Amino acid sequence analysis suggested that NcEST1 and NcEST2 might be the N. caninum counterparts of TgPI-1 and TgPI-2, respectively. EtEST-S, as NcPI-S, is a single domain serpin. The open reading frame encoding the mature version of NcPI-S was expressed as recombinant protein, fused to a 6 histidine tag in Escherichia coli. Specific rabbit antiserum generated against the recombinant NcPI-S was used in immunoblot assays. Bands of 20, 30, 40, and 66-kDa were detected by SDS-PAGE of whole parasite homogenate. In addition, when an anti-TgPI-1 serum was used, bands of 25 and 35-kDa were detected indicating that there is no cross-reactivity between both serpins, and showing as well, the presence of another putative serpin in N. caninum. The recombinant protein NcPI-S, inhibited bacterial subtilisin completely, and showed lower inhibitory capacity on human neutrophil elastase, animal trypsin, and chymotrypsin, suggesting differences in effectiveness.