Multiplex PCR to Streptococcus pneumoniae serotype identification directly in cerebrospinal fluid samples.

Streptococcus pneumoniae causes invasive diseases of significant public health concern, such as meningitis. The culture of cerebrospinal fluid (CSF) samples, the standard technique for meningitis diagnoses, is not always positive. Consequently, meaningful information about the etiological agent is lost, which can compromise effective epidemiological surveillance and the improvement of immunization policies. This study aims to standardize a method to genotype pneumococcus in the CSF samples which could mitigate the absence of isolated strains, and also evaluate the prediction of this assay. We applied eight multiplex PCR (mPCR) assays to CSF samples paired with the Quellung reaction applied to the isolated strains. We also compared different master mix kits in the mPCR. Moreover, a retrospective study was conducted with CSF samples considered pneumococcus positive due to the presence of the lytA gene. Results showed that genotyping by the mPCR correlated 100% with the Quellung reaction, and genotyping was dependent on the master mix applied. In the retrospective study (2014-2020), 73.4% were successfully genotyped. The analyses of the receiver operating characteristic curve showed that the cycle threshold (Ct value) around 30 for the lytA gene had a 75% positive chance of successful genotyping, whereas with a Ct value > 35, the chance was 12.5%. Finally, we observed that genotype 19A was prevalent in the period (12%), information unknown until now due to the lack of isolated strains. Therefore, the mPCR of CSF samples can efficiently predict S. pneumoniae serotypes, especially in the absence of isolated strains, which can be a great tool for pneumococcal serotype surveillance.

Historical, current, and emerging tools for identification and serotyping of Shigella.

The Shigella genus includes serious foodborne disease etiologic agents, with 4 species and 54 serotypes. Identification at species and serotype levels is a crucial task in microbiological laboratories. Nevertheless, the genetic similarity between Shigella spp. and Escherichia coli challenges the correct identification and serotyping of Shigella spp., with subsequent negative repercussions on surveillance, epidemiological investigations, and selection of appropriate treatments. For this purpose, multiple techniques have been developed historically ranging from phenotype-based methods and single or multilocus molecular techniques to whole-genome sequencing (WGS). To facilitate the selection of the most relevant method, we herein provide a global overview of historical and emerging identification and serotyping techniques with a particular focus on the WGS-based approaches. This review highlights the excellent discriminatory power of WGS to more accurately elucidate the epidemiology of Shigella spp., disclose novel promising genomic targets for surveillance methods, and validate previous well-established methods.

Epidemiology of dengue fever in Guatemala.

Dengue fever occurs worldwide and about 1% of cases progress to severe haemorrhage and shock. Dengue is endemic in Guatemala and its surveillance system could document long term trends. We analysed 17 years of country-wide dengue surveillance data in Guatemala to describe epidemiological trends from 2000 to 2016.Data from the national dengue surveillance database were analysed to describe dengue serotype frequency, seasonality, and outbreaks. We used Poisson regression models to compare the number of cases each year with subsequent years and to estimate incidence ratios within serotype adjusted by age and gender. 91,554 samples were tested. Dengue was confirmed by RT-qPCR, culture or NS1-ELISA in 7097 (7.8%) cases and was IgM ELISA-positive in 19,290 (21.1%) cases. DENV1, DENV2, DENV3, and DENV4 were detected in 2218 (39.5%), 2580 (45.9%), 591 (10.5%), and 230 (4.1%) cases. DENV1 and DENV2 were the predominant serotypes, but all serotypes caused epidemics. The largest outbreak occurred in 2010 with 1080 DENV2 cases reported. The incidence was higher among adults during epidemic years, with significant increases in 2005, 2007, and 2013 DENV1 outbreaks, the 2010 DENV2 and 2003 DENV3 outbreaks. Adults had a lower incidence immediately after epidemics, which is likely linked to increased immunity.

Testing New Peptides From Toxoplasma gondii SAG1, GRA6, and GRA7 for Serotyping: Better Definition Using GRA6 in Mother/Newborns Pairs With Risk of Congenital Transmission in Mexico.

Toxoplasma gondii variant influences clinical profile in human congenital and ocular toxoplasmosis. Parasite genotyping represents a challenge due to insufficient amount of genetic material of the protozoan in the host samples, and isolates are hard to obtain, especially from pediatric patients. An alternative is serotyping, which is based on the presence of specific antibodies against polymorphic proteins related to virulence; the more widely used are GRA6 and GRA7, but most works report cross reactions among the classical strains (I, II, and III). We designed new peptides of GRA6, GRA7, and SAG1 proteins, with more SNPs among the three clonal strains than those previously designed. This was done by identifying BcR and polymorphic epitopes by means of bioinformatics; then we designed peptides with linkers joining the specific regions and predicted their 3D structure. With the commercial molecules synthesized on the basis of these designs, we tested 86 serum samples from 42 mother/newborn pairs and two congenitally infected newborns, by indirect ELISA. We implemented a strategy to determine the serotype based on scatter plots and a mathematical formula, using ratios among reactivity indexes to peptides. We found low frequency of samples reactive to GRA7 and SAG1, and cross reactions between GRA6 serotypes I and III; we modified these later peptides and largely improved distinction among the three clonal strains. The chronicity of the infection negatively affected the reactivity index against the peptides. Serotyping both members of the mother/child pair improves the test, i.e., among 26% of them only one member was positive. Serotype I was the most frequent (38%), which was congruent with previous genotyping results in animals and humans of the same area. This serotype was significantly more frequent among mothers who transmitted the infection to their offspring than among those who did not (53 vs. 8%, p = 0.04) and related to disease dissemination in congenitally infected children, although non-significantly. In conclusion, serotyping using the improved GRA6 peptide triad is useful to serotype T. gondii in humans and could be implemented for clinical management and epidemiological studies, to provide information on the parasite type in specific areas.

Development of a pooled antigen for use in the macroscopic slide agglutination test (MSAT) to detect Sejroe serogroup exposure in cattle.

This study aimed to develop and evaluate a pooled antigen for use in the macroscopic slide agglutination test (MSAT) to detect cattle positive for the Sejroe serogroup. To this end, 193 bovine serum samples from different Pará State regions (Amazonia) were subjected to a reference microscopic agglutination test (MAT) for the serological diagnosis of leptospirosis using 11 serovars representing the Sejroe serogroup: Hardjo-prajitno; Hardjo-bovis; Sejroe; Wolffi; Guaricura (Bov.G.); Guaricura (M4/98); Ricardi; Gorgas; Recreo; Polonica and Medanensis. The three most prevalent serovars in the MAT were selected for the development of a pooled antigen for use in MSAT; subsequently, the 193 serum were assessed with the macroscopic slide agglutination test (MSAT) containing the developed antigen. The Kappa test was used to determine the general agreement between the MAT and MSAT results. As a result, of the 193 serum samples, 155 (80.3%) were reactive, and 38 (19.7%) were non-reactive in the MAT; Hardjo-prajitno, Wolffi and Medanensis were the three most prevalent serovars. Of the 193 serum samples tested in the MSAT using the developed pooled antigen, 114 were reactive (59.0%), and 79 (41.0%) were non-reactive; the Kappa coefficient was 0.52 (CI 95%, 0.40-0.63), indicating moderate agreement between the two tests. The MSAT with the pooled antigen including the most prevalent serovars detected bovines with the Sejroe serogroup exposure, mainly in animals with high titters in the MAT, and could be used to screen herds suspected of acute infection by this serogroup in Pará State.

Comparison of six commercial systems for the detection of non-O157 STEC in meat and vegetables.

Shiga toxin-producing Escherichia coli (STEC) are important pathogens transmitted by food that may cause severe illness in human beings. Thus, systems for STEC detection in food should have increasingly higher sensitivity and specificity. Here we compared six commercial systems for non-O157 STEC detection in meat and vegetables and determined their sensitivity, specificity and repeatability. A total of 46 samples (meat n = 23; chard n = 23) were experimentally contaminated with strains O26:H11, O45:H-, O103:H2, O111:NM, O121:H19 and O145:NM isolated in Argentina. Strain detection was confirmed by isolation according to ISO 13136:2012. Detection of the stx and eae genes in meat samples was highly satisfactory with all commercial kits, but only five had 100% sensitivity and specificity in chard. Of four kits evaluated for serogroup detection, three had 100% sensitivity and specificity, and one had 93.7% sensitivity and 100% specificity. All kits were adequate to analyze meat but not vegetable samples, and were not therefore validated for the latter matrix. The challenge for microbiology laboratories is to identify the advantages and disadvantages of the available kits for STEC detection in food based on a clear knowledge of the particular needs of each laboratory.

Genomic analysis of human invasive Salmonella enterica serovar Typhimurium ST313 isolate B3589 from India.

Salmonella Typhimurium ST313 is known to cause invasive disease in sub Saharan African (sSA) countries while the same sequence type is often associated with gastro-intestinal infections in the UK and Brazil. Although S. Typhimurium has been frequently isolated from human samples in India, the prevalence and invasive nature of infection of ST313 is currently unknown. The present study elucidates the phenotypic and genotypic characteristics of S. Typhimurium strain B3589 that belongs to ST313. The isolate was subjected to serotyping and antimicrobial susceptibility test to understand its phenotypical characteristics. Whole genome sequencing and comparative genomic analysis was carried out to provide an insight into S. Typhimurium ST313 lineage in India. The results suggests antibiotic resistance against aminoglycoside was associated with the presence of aminoglycoside modifying enzymes aac(6')-Ia in the genome. Phylogenetic analysis revealed the India-ST313 isolates are genotypically distinct from the known African, UK and Brazilian ST313 lineages. The isolate possess the characteristic prophage gene repertoire except BTP-5. The presence of BTP-1 and more importantly bstA virulence gene has been the distinguishable feature of strain B3589 among other non-African isolates. In addition the genome degradation of African ST313 lineage-2 was not conserved in the Indian ST313 isolates. Fewer genome degradation events as well as the absence of plasmid mediated MDR locus suggest the Indian ST313 isolates are of low risk. The identification of ST313 isolates in India reveals the previously unknown characteristics of ST313 S. Typhimurium isolated from India.

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

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.

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.

Maternal Immunization with Pneumococcal Polysaccharide Vaccine: Persistence of Maternal Antibodies in Infants.

OBJECTIVE: To evaluate the level and the persistence of maternal antibodies in infants after maternal immunization with pneumococcal polysaccharide vaccine (Pn23V). METHODS: Pregnant women were assigned to two groups, during routine low-risk pre-natal visits. The first Group (VAC) received the Pn23V vaccine shortly after enrolment at 28 weeks or later, and the second Group (NO_VAC) received no vaccine. To investigate the antibody persistence, we collected blood samples from the mothers after 1 month of delivery and from the infants at 1 and 6 months of age. RESULTS: Antibody titers were measured for serotypes 1, 6B and 14. Geometric mean antibody concentrations of specific immunoglobulin G were significantly higher in the vaccinated group compared with unvaccinated controls for all three serotypes tested. CONCLUSION: Despite the antibody level's decline, at 6 months of age, proportions >0.35 µg/ml remained higher in the infants of vaccinated mothers than controls for all three serotypes.

Serotipos de Streptococcus pneumoniae responsables de enfermedad invasiva en niños cubanos / Streptococcus pneumoniae serotypes responsible for invasive disease affecting Cuban children

Introducción: Streptococcus pneumoniae es causa importante de morbilidad y mortalidad a nivel mundial, fundamentalmente en niños < 5 años. En Cuba aún no se introdujo la vacunación antineumocócica, pero desde 2014, con el propósito de sentar las bases para la evaluación de su impacto, se lleva a cabo un protocolo de vigilancia centinela de la enfermedad neumocócica invasiva en niños ≤ 5 años. Objetivos: notificar los serotipos de S. pneumoniae responsables de enfermedad neumocócica invasiva en la población pediátrica cubana, y valorar la contribución de ese protocolo a la vigilancia. Métodos: se determinaron los serotipos de todos los aislamientos pediátricos invasivos y los recuperados de otitis media aguda, recibidos en el Laboratorio Nacional de Referencia de Neumococo, del Instituto de Medicina Tropical Pedro Kourí, entre 2013-2015. Se utilizó el método de hinchazón capsular empleando el juego de reactivos Pneumotest. Resultados: se notificaron 141 aislamientos invasivos en edad pediátrica. Predominaron los responsables de neumonías (76 vs. 49 aislamientos meníngeos) y la mayoría de estos fueron aportados por los hospitales involucrados en la vigilancia centinela (75 por ciento; 57/76). El 85,8 por ciento de los aislamientos quedaron contenidos en siete serotipos, que por orden de frecuencia fueron: 14, 19A, 6A, 19F, 6B, 3 y 23F. La cobertura serotípica de las diferentes vacunas neumocócicas multivalentes con posibilidades de ser empleadas se estimó entre 54 y 90 por ciento. Conclusiones: tras la introducción de la vacunación cabría esperar una reducción de la enfermedad neumocócica invasiva debida a los serotipos contenidos en las vacunas conjugadas disponibles, pero se insiste en la necesidad de fortalecer la vigilancia clínico-epidemiológica que se hace hoy de esta entidad en el país(AU)

Introduction: Streptococcus pneumoniae is a significant cause of morbidity and mortality worldwide mainly in children younger than 5 years. The pneumococcal vaccination has not been yet put into practice in Cuba; however, since 2014 a protocol of sentinel surveillance of the invasive pneumococcal disease in children aged 5 years or less is being implemented to lay the foundations for the evaluation of the impact of this vaccine. Objectives: to report on the S. pneumoniae serotypes responsible for the invasive pneumococcal disease in the Cuban pediatric population and to assess the contribution of this protocol to surveillance. Methods: the serotypes of all the invasive pediatric isolates and the recovered ones of acute otitis media were determined by the national laboratory of pneumococcal reference of Pedro Kourí Institute of Tropical Medicine from 2013 to 2015. The capsular swelling method was used with the Penumotest reagent set. Results: one hundred and forty one invasive isolates were reported at pediatric ages. The isolates causing pneumonia predominated (76 vs. 49 meningeal isolates) and most of them were provided by hospitals involved in the sentinel surveillance project (57 out of 76; 75 percent). In this regard, 85.8 percent of isolates belonged to seven serotypes that were in order of frequency the following: 14, 19A, 6A, 19F, 6B, 3 and 23F. The serotype coverage of the various multivalent pneumococcal vaccines of possible use was estimated at 54-90 percent. Conclusions: after the introduction of the vaccinations, one might expect that a reduction of the invasive pneumococcal disease occurs due to the serotypes included in the available conjugate vaccines, but emphasis is made on the need of strengthening at present the clinical and epidemiological surveillance system for this disease nationwide(AU)

A Luminex-based single DNA fragment amplification assay as a practical tool for detecting and serotyping dengue virus.

Dengue is a mosquito-borne viral infection that can evolve from subclinical to severe forms of disease. Early recognition during initial primary and secondary infections correlates with a reduced case-fatality rate in susceptible groups. The aim of this study was to standardize a DNA hybridization assay based on the Luminex technology for detecting and serotyping dengue virus (DENV). Reference DENVs representing the four different serotypes were used as controls to standardize the test. For validation, 16 DENV isolates obtained from a reference laboratory were analyzed in a double-blind manner to validate the test. Sixty blood samples from patients suspected of having dengue fever were used to evaluate the methodology after the validation step, and the results were compared with the reference semi-nested RT-PCR. Additionally, five human samples of each Zika and Chikungunya confirmed patients were used for specificity analysis. The Luminex-based assay correctly identified all 16 DENV isolates. In the evaluation step, the results of the RT-PCR/Luminex assay showed a concordance of 86.7% with those of the semi-nested RT-PCR. None of other virus infection samples was amplified. This is the first description of a hybridization assay that can discriminate the four DENV serotypes using probes against a single DENV sequence. The results indicated that the RT-PCR/Luminex DENV assay designed and evaluated in this study is a valuable additional tool for the early and rapid detection and serotyping of DENV, which could, in the future, be applied to new targets such as the Zika and Chikungunya viruses.

The Relevance of a Novel Quantitative Assay to Detect up to 40 Major Streptococcus pneumoniae Serotypes Directly in Clinical Nasopharyngeal and Blood Specimens.

For epidemiological and surveillance purposes, it is relevant to monitor the distribution and dynamics of Streptococcus pneumoniae serotypes. Conventional serotyping methods do not provide rapid or quantitative information on serotype loads. Quantitative serotyping may enable prediction of the invasiveness of a specific serotype compared to other serotypes carried. Here, we describe a novel, rapid multiplex real-time PCR assay for identification and quantification of the 40 most prevalent pneumococcal serotypes and the assay impacts in pneumonia specimens from emerging and developing countries. Eleven multiplex PCR to detect 40 serotypes or serogroups were optimized. Quantification was enabled by reference to standard dilutions of known bacterial load. Performance of the assay was evaluated to specifically type and quantify S. pneumoniae in nasopharyngeal and blood samples from adult and pediatric patients hospitalized with pneumonia (n = 664) from five different countries. Serogroup 6 was widely represented in nasopharyngeal specimens from all five cohorts. The most frequent serotypes in the French, South African, and Brazilian cohorts were 1 and 7A/F, 3 and 19F, and 14, respectively. When both samples were available, the serotype in blood was always present as carriage with other serotypes in the nasopharynx. Moreover, the ability of a serotype to invade the bloodstream may be linked to its nasopharyngeal load. The mean nasopharyngeal concentration of the serotypes that moved to the blood was 3 log-fold higher than the ones only found in the nasopharynx. This novel, rapid, quantitative assay may potentially predict some of the S. pneumoniae serotypes invasiveness and assessment of pneumococcal serotype distribution.

Complete genome of a dengue virus serotype 4 strain from Amazonas, Brazil.

Dengue virus (DENV) infections represent a significant concern for public health worldwide, being considered as the most prevalent arthropod-borne virus regarding the number of reported cases. In this study, we report the complete genome sequencing of a DENV serotype 4 isolate, genotype II, obtained in the city of Manaus, directly from the serum sample, applying Ion Torrent sequencing technology. The use of a massive sequencing technology allowed the detection of two variable sites, one in the coding region for the viral envelope protein and the other in the nonstructural 1 coding region within viral populations.

Molecular Serogrouping of Listeria monocytogenes from Brazil Using PCR.

We assessed the serotype distribution of Listeria monocytogenes isolates from clinical, beef, and environment samples using two PCR-based protocols for serogrouping. A panel of 134 isolates (22 clinical samples, 79 samples of beef cuts, and 33 samples from the beef processing environment) were subjected to conventional serology and identified as serotypes 1/2a (n = 12), 1/2b (n = 21), 1/2c (n = 71), and 4b (n = 30). Isolates from clinical samples were predominantly serotype 4b, and the most prevalent serotype among the beef cut and environment samples was 1/2c. The protocol described by M. Doumith, C. Buchrieser, P. Glaser, C. Jacquet, and P. Martin (J. Clin. Microbiol. 42:3819-3822, 2004) produced contradictory results for seven 1/2a isolates, which were positive for lmo1118 and had the profile IIc (serotypes 1/2c and 3c). Fifteen serotype 4b isolates amplified the target lmo0737, with the atypical profile IVb variant 1. The results obtained with the protocol described by M. K. Borucki and D. R. Call (J. Clin. Microbiol. 41:5537-5540, 2003) were in full agreement with those of the conventional serology. We recommend using this multiplex PCR approach by adding one pair of the reported primers to the panel to reduce total effort by one PCR while maintaining specificity. We present additional recommendations to improve the efficiency and reproducibility of this serogrouping assay.

Twenty Years of Listeria in Brazil: Occurrence of Listeria Species and Listeria monocytogenes Serovars in Food Samples in Brazil between 1990 and 2012.

Listeria spp. isolated from different food products and collected from 12 Brazilian states were sent to the Laboratory of Bacterial Zoonoses (Oswaldo Cruz Institute, Brazil) for identification. The aims of this study were to characterize these isolates, from 1990 to 2012, by using biochemical, morphological, and serotyping tests, and to analyze the distribution of L. monocytogenes serotypes on different food products and geographical locations. Serotyping was performed using polyclonal somatic and flagellar antisera. Of 5953 isolates, 5770 were identified as Listeria spp., from which 3429 (59.4%) were L. innocua, 2248 (38.9%) were L. monocytogenes, and 93 (1.6%) were other Listeria spp. L. innocua was predominantly isolated from 1990 to 2000, while L. monocytogenes was from 2001 to 2012. Regarding the serotype distribution in the foods, serotypes 1/2a and 4b were most common in processed meat and ready-to-eat products, respectively; serotypes 1/2a, 1/2b, and 4b were the most common in nonprocessed meat. The results above confirm the presence of the main serotypes of L. monocytogenes in different parts of the food chain from three regions of the country and emphasize the importance of improving the control measures, as tolerance zero policy and microbiological surveillance in Brazil.

Dengue in the State of Rio Grande do Norte, Brazil, 2010-2012.

OBJECTIVE: To present results of virological surveillance and epidemiological aspects of dengue in the State of Rio Grande do Norte, Brazil. METHODS: A total of 1581 cases, reported from 2010 to 2012 at various health centres in the state, were analysed by viral isolation and/or RT-PCR for viral detection and typing. To identify whether different genotypes were circulating in the state during this period, sequencing of the complete E gene for DENV (1485 bp in length) was performed directly from patient serum samples. RESULTS: All four serotypes of dengue virus circulated in Rio Grande do Norte, with the introduction of DENV-4 in the state in 2011. In 2012, DENV-4 represented 100% of positive confirmed cases. 53.97% of cases occurred in Natal. Case numbers peaked in April (21%) and May (23%). Genetic characterisation of circulating strains confirmed the circulation of genotypes V, south-east Asian/American and II, respectively, for DENV-1, DENV-2 and DENV-4. CONCLUSIONS: This work furthers a better understanding of dengue viruses in the State of Rio Grande do Norte. Strengthening control efforts in the region is important considering the impact of dengue.

Characterization of Carriage Isolates of Neisseria meningitides in the Adolescents and Young Adults Population of Bogota (Colombia).

BACKGROUND: Meningococcal carriage studies are important to improve our understanding of the epidemiology of meningococcal disease. The aim of this study was to determine the prevalence of meningococcal carriage and the phenotypic and genotypic characteristics of isolates collected from a sample of students in the city of Bogotá, Colombia. MATERIALS AND METHODS: A total of 1459 oropharyngeal samples were collected from students aged 15-21 years attending secondary schools and universities. Swabs were plated on a Thayer Martin agar and N. meningitidis was identified by standard microbiology methods and PCR. RESULTS: The overall carriage prevalence was 6.85%. Carriage was associated with cohabitation with smokers, and oral sex practices. Non-groupable and serogroup Y isolates were the most common capsule types found. Isolates presented a high genetic diversity, and circulation of the hypervirulent clonal complexes ST-23, ST-32 and ST-41/44 were detected. CONCLUSION: The meningococcal carriage rate was lower than those reported in Europe and Africa, but higher than in other Latin American countries. Our data also revealed antigenic and genetic diversity of the isolates and the circulation of strains belonging to clonal complexes commonly associated with meningococcal disease.

Revisiting molecular serotyping of Streptococcus pneumoniae.

BACKGROUND: Ninety-two Streptococcus pneumoniae serotypes have been described so far, but the pneumococcal conjugate vaccine introduced in the Brazilian basic vaccination schedule in 2010 covers only the ten most prevalent in the country. Pneumococcal serotype-shifting after massive immunization is a major concern and monitoring this phenomenon requires efficient and accessible serotyping methods. Pneumococcal serotyping based on antisera produced in animals is laborious and restricted to a few reference laboratories. Alternatively, molecular serotyping methods assess polymorphisms in the cps gene cluster, which encodes key enzymes for capsular polysaccharides synthesis in pneumococci. In one such approach, cps-RFLP, the PCR amplified cps loci are digested with an endonuclease, generating serotype-specific fingerprints on agarose gel electrophoresis. METHODS: In this work, in silico and in vitro approaches were combined to demonstrate that XhoII is the most discriminating endonuclease for cps-RFLP, and to build a database of serotype-specific fingerprints that accommodates the genetic diversity within the cps locus of 92 known pneumococci serotypes. RESULTS: The expected specificity of cps-RFLP using XhoII was 76% for serotyping and 100% for serogrouping. The database of cps-RFLP fingerprints was integrated to Molecular Serotyping Tool (MST), a previously published web-based software for molecular serotyping. In addition, 43 isolates representing 29 serotypes prevalent in the state of Minas Gerais, Brazil, from 2007 to 2013, were examined in vitro; 11 serotypes (nine serogroups) matched the respective in silico patterns calculated for reference strains. The remaining experimental patterns, despite their resemblance to their expected in silico patterns, did not reach the threshold of similarity score to be considered a match and were then added to the database. CONCLUSION: The cps-RFLP method with XhoII outperformed the antisera-based and other molecular serotyping methods in regard of the expected specificity. In order to accommodate the genetic variability of the pneumococci cps loci, the database of cps-RFLP patterns will be progressively expanded to include new variant in vitro patterns. The cps-RFLP method with endonuclease XhoII coupled with MST for computer-assisted interpretation of results may represent a relevant contribution to the real time detection of changes in regional pneumococci population diversity in response to mass immunization programs.

Serogroup-specific bacterial engineered glycoproteins as novel antigenic targets for diagnosis of shiga toxin-producing-escherichia coli-associated hemolytic-uremic syndrome.

Human infection with Shiga toxin-producing Escherichia coli (STEC) is a major cause of postdiarrheal hemolytic-uremic syndrome (HUS), a life-threatening condition characterized by hemolytic anemia, thrombocytopenia, and acute renal failure. E. coli O157:H7 is the dominant STEC serotype associated with HUS worldwide, although non-O157 STEC serogroups can cause a similar disease. The detection of anti-O157 E. coli lipopolysaccharide (LPS) antibodies in combination with stool culture and detection of free fecal Shiga toxin considerably improves the diagnosis of STEC infections. In the present study, we exploited a bacterial glycoengineering technology to develop recombinant glycoproteins consisting of the O157, O145, or O121 polysaccharide attached to a carrier protein as serogroup-specific antigens for the serological diagnosis of STEC-associated HUS. Our results demonstrate that using these antigens in indirect ELISAs (glyco-iELISAs), it is possible to clearly discriminate between STEC O157-, O145-, and O121-infected patients and healthy children, as well as to confirm the diagnosis in HUS patients for whom the classical diagnostic procedures failed. Interestingly, a specific IgM response was detected in almost all the analyzed samples, indicating that it is possible to detect the infection in the early stages of the disease. Additionally, in all the culture-positive HUS patients, the serotype identified by glyco-iELISAs was in accordance with the serotype of the isolated strain, indicating that these antigens are valuable not only for diagnosing HUS caused by the O157, O145, and O121 serogroups but also for serotyping and guiding the subsequent steps to confirm diagnosis.