A combination of GRA3, GRA6 and GRA7 peptides offer a useful tool for serotyping type II and III Toxoplasma gondii infections in sheep and pigs.

The clinical consequences of toxoplasmosis are greatly dependent on the Toxoplasma gondii strain causing the infection. To better understand its epidemiology and design appropriate control strategies, it is important to determine the strain present in infected animals. Serotyping methods are based on the detection of antibodies that react against segments of antigenic proteins presenting strain-specific polymorphic variations, offering a cost-effective, sensitive, and non-invasive alternative to genotyping techniques. Herein, we evaluated the applicability of a panel of peptides previously characterized in mice and humans to serotype sheep and pigs. To this end, we used 51 serum samples from experimentally infected ewes (32 type II and 19 type III), 20 sheep samples from naturally infected sheep where the causative strain was genotyped (18 type II and 2 type III), and 40 serum samples from experimentally infected pigs (22 type II and 18 type III). Our ELISA test results showed that a combination of GRA peptide homologous pairs can discriminate infections caused by type II and III strains of T. gondii in sheep and pigs. Namely, the GRA3-I/III-43 vs. GRA3-II-43, GRA6-I/III-213 vs. GRA6-II-214 and GRA6-III-44 vs. GRA6-II-44 ratios showed a statistically significant predominance of the respective strain-type peptide in sheep, while in pigs, in addition to these three peptide pairs, GRA7-II-224 vs. GRA7-III-224 also showed promising results. Notably, the GRA6-44 pair, which was previously deemed inefficient in mice and humans, showed a high prediction capacity, especially in sheep. By contrast, GRA5-38 peptides failed to correctly predict the strain type in most sheep and pig samples, underpinning the notion that individual standardization is needed for each animal species. Finally, we recommend analyzing for each animal at least 2 samples taken at different time points to confirm the obtained results.

Comparing serotyping with whole-genome sequencing for subtyping of non-typhoidal Salmonella enterica: a large-scale analysis of 37 serotypes with a public health impact in the USA.

Serotyping has traditionally been used for subtyping of non-typhoidal Salmonella (NTS) isolates. However, its discriminatory power is limited, which impairs its use for epidemiological investigations of source attribution. Whole-genome sequencing (WGS) analysis allows more accurate subtyping of strains. However, because of the relative newness and cost of routine WGS, large-scale studies involving NTS WGS are still rare. We aimed to revisit the big picture of subtyping NTS with a public health impact by using traditional serotyping (i.e. reaction between antisera and surface antigens) and comparing the results with those obtained using WGS. For this purpose, we analysed 18 282 sequences of isolates belonging to 37 serotypes with a public health impact that were recovered in the USA between 2006 and 2017 from multiple sources, and were available at the National Center for Biotechnology Information (NCBI). Phylogenetic trees were reconstructed for each serotype using the core genome for the identification of genetic subpopulations. We demonstrated that WGS-based subtyping allows better identification of sources potentially linked with human infection and emerging subpopulations, along with providing information on the risk of dissemination of plasmids and acquired antimicrobial resistance genes (AARGs). In addition, by reconstructing a phylogenetic tree with representative isolates from all serotypes (n=370), we demonstrated genetic variability within and between serotypes, which formed monophyletic, polyphyletic and paraphyletic clades. Moreover, we found (in the entire data set) an increased detection rate for AARGs linked to key antimicrobials (such as quinolones and extended-spectrum cephalosporins) over time. The outputs of this large-scale analysis reveal new insights into the genetic diversity within and between serotypes; the polyphyly and paraphyly of certain serotypes may suggest that the subtyping of NTS to serotypes may not be sufficient. Moreover, the results and the methods presented here, leading to differentiation between genetic subpopulations based on their potential risk to public health, as well as narrowing down the possible sources of these infections, may be used as a baseline for subtyping of future NTS infections and help efforts to mitigate and prevent infections in the USA and globally.

Application of Toxoplasma gondii GRA15 peptides in diagnosis and serotyping.

Previous studies showed that three clonal strain types (I, II, and III) of Toxoplasma gondii can be distinguished using serotyping based on a series of polymorphic proteins. However, to establish a systematic serotyping method with higher resolution even being equal to that of genotyping, more specific peptide markers are needed. The objective of the present study was to determine the possibility of the polymorphic dense granule protein 15 (GRA15) for diagnosis and serotyping of T. gondii infection. Three different T. gondii GT1 strain GRA15 gene fragments encoding a 584-residue peptide, a 199-residue peptide and a 84-residue peptide were amplified, expressed and purified, respectively. Anti-T. gondii GT1 strain antibodies, anti-T. gondii RH strain antibodies and anti-T. gondii PRU strain antibodies were used for immunoblotting analysis of the three peptides. Western blotting analysis showed that the 584-residue peptide of GT1 strain GRA15 was a potential candidate for serological diagnosis of T. gondii infection. RH strain from GT1 strain could be distinguished by serotyping based on the GRA15199 or GRA1584, and T. gondii GT1 strain could be distinguished from PRU strain by using serotyping based on the GRA1584. These findings reveal, for the first time, a novel potential role of GRA15-derived peptides in diagnosis and serological differentiation of T. gondii infection.

Serotyping of Toxoplasma gondii Infection Using Peptide Membrane Arrays.

The intracellular parasite Toxoplasma gondii can cause chronic infections in most warm-blooded animals, including humans. In the USA, strains belonging to four different Toxoplasma clonal lineages (types 1, 2, 3, and 12) are commonly isolated, whereas strains not belonging to these lineages are predominant in other continents such as South America. Strain type plays a pivotal role in determining the severity of Toxoplasma infection. Therefore, it is epidemiologically relevant to develop a non-invasive and inexpensive method for determining the strain type in Toxoplasma infections and to correlate the genotype with disease outcome. Serological typing is based on the fact that many host antibodies are raised against immunodominant parasite proteins that are highly polymorphic between strains. However, current serological assays can only reliably distinguish type 2 from non-type 2 infections. To improve these assays, mouse, rabbit, and human infection serum were reacted against 950 peptides from 62 different polymorphic Toxoplasma proteins by using cellulose membrane peptide arrays. This allowed us to identify the most antigenic peptides and to pinpoint the most relevant polymorphisms that determine strain specificity. Our results confirm the utility of previously described peptides and identify novel peptides that improve and increase the specificity of the assay. In addition, a large number of novel proteins showed potential to be used for Toxoplasma diagnosis. Among these, peptides derived from several rhoptry, dense granule, and surface proteins represented promising candidates that may be used in future experiments to improve Toxoplasma serotyping. Moreover, a redesigned version of the published GRA7 typing peptide performed better and specifically distinguished type 3 from non-type 3 infections in sera from mice, rabbits, and humans.

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.

Identification of Salmonella Taxon-Specific Peptide Markers to the Serovar Level by Mass Spectrometry.

We present an LC-MS/MS pipeline to identify taxon-specific tryptic peptide markers for the identification of Salmonella at the genus, species, subspecies, and serovar levels of specificity. Salmonella enterica subsp. enterica serovars Typhimurium and its four closest relatives, Saintpaul, Heidelberg, Paratyphi B, and Muenchen, were evaluated. A decision-tree approach was used to identify peptides common to the five Salmonella proteomes for evaluation as genus-, species-, and subspecies-specific markers. Peptides identified for two or fewer Salmonella strains were evaluated as potential serovar markers. Currently, there are approximately 140 000 assembled bacterial genomes publicly available, more than 8500 of which are for Salmonella. Consequently, the specificity of each candidate peptide marker was confirmed across all publicly available protein sequences in the NCBI nonredundant (nr) database. The performance of a subset of candidate taxon-specific peptide markers was evaluated in a targeted mass-spectrometry method. The presented workflow offers a marked improvement in specificity over existing MALDI-TOF-based bacterial identification platforms for the identification of closely related Salmonella serovars.

Development and evaluation of a BK polyomavirus serotyping assay using Luminex technology.

BACKGROUND: The BK polyomavirus (BKPyV) is subdivided into four genotypes. The consequences of each genotype and of donor-recipient genotype (mis)match for BKPyV-associated nephropathy (BKPyVAN) in kidney transplant recipients (KTRs) are unknown. OBJECTIVES: To develop and evaluate a genotype-specific IgG antibody-based BKPyV serotyping assay, in order to classify kidney transplant donors and recipients accordingly. STUDY DESIGN: VP1 antigens of six BKPyV variants (Ib1, Ib2, Ic, II, III and IV) were expressed as recombinant glutathione-s-transferase-fusion proteins and coupled to fluorescent Luminex beads. Sera from 87 healthy blood donors and 39 KTRs were used to analyze seroreactivity and serospecificity against the different BKPyV genotypes. Six sera with marked BKPyV serotype profiles were analyzed further for genotype-specific BKPyV pseudovirus neutralizing capacity. RESULTS: Seroreactivity was observed against all genotypes, with seropositivity rates above 77% comparable for KTRs and blood donors. Strong cross-reactivity (r > 0.8) was observed among genotype I subtypes, and among genotypes II, III and IV. Seroresponses against genotypes I and IV seemed genuine, while those against II and III could be out(cross)competed. GMT (Luminex) and IC50 (neutralization assay) values showed good agreement in determining the genotype with the strongest seroresponse within an individual. CONCLUSIONS: Despite some degree of cross-reactivity, this serotyping assay seems a useful tool to identify the main infecting BKPyV genotype within a given individual. This information, which cannot be obtained otherwise from nonviremic/nonviruric individuals, could provide valuable information regarding the prevalent BKPyV genotype in kidney donors and recipients and warrants further study.

Fiber-based fluorescent microsphere immunoassay (FMIA) as a novel multiplex serodiagnostic tool for simultaneous detection and differentiation of all clinically relevant fowl adenovirus (FAdV) serotypes.

The recent emergence of fowl aviadenovirus (FAdV) induced disease outbreaks in chicken flocks worldwide, with distinct aetiologies confined to particular FAdV species and serotypes, is increasingly urging the need for specific and mass-applicable antibody screening systems. Despite this exigency, there are to date no available serological procedures which satisfactorily combine the criteria for sensitive detection of antibodies against FAdVs, diagnostic reliability in face of cross-reactions and requirements for a rapid and large-scale application. In order to address this gap, a multiplexed fluorescent microsphere immunoassay (FMIA) based on recombinant FAdV fiber proteins from six different serotypes FAdV-1, -2, -4, -8a, -8b and -11 was developed, which enabled simultaneous detection of antibodies against all clinically relevant serotypes in a single reaction within a high throughput setting. Based on a panel of >300 monospecific antisera raised against each of the 12 FAdV serotypes, 100% serotype-specificity was demonstrated for FAdV-1 (FAdV-A) and FAdV-4 (FAdV-C) fiber-based analytes. Analytes based on serotypes affiliated to FAdV-D and FAdV-E exhibited moderately lower specificities of 91.2-95.7%. This was attributed almost exclusively to mutual recognition between FAdV-2 and -11 field strains and to a much lesser extent to reference strains, supporting earlier proposals to merge them into a single serotype. Similarly, extensive cross-reactions between FAdV-8a and -8b were noted. Altogether intraspecies cross-reactions can be attributed to viruses with a close etiological intersection. Antisera against other important avian viruses remained negative by the FMIA, further validating its specificity. Compared to the virus-neutralization (VN) test, FMIA and individual fiber-based enzyme-linked immunosorbent assays (ELISAs) were equally sensitive in the detection of sera against FAdV-2 and -11, as well as FAdV-8a and -8b field strains, while they were even superior to VN test in detection of FAdV-1 and FAdV-4 responses, likely attributed to a relative abundance of fiber antibodies early upon infection. Moreover, application of the FMIA on field samples comprising a diversified response against all 12 FAdV serotypes further consolidated its specificity and agreement with VN test.

Enhancing the sensitivity of Dengue virus serotype detection by RT-PCR among infected children in India.

Dengue surveillance relies on reverse transcription-polymerase chain reaction (RT-PCR), for confirmation of dengue virus (DENV) serotypes. We compared efficacies of published and modified primer sets targeting envelope (Env) and capsid-premembrane (C-prM) genes for detection of circulating DENV serotypes in southern India. Acute samples from children with clinically-diagnosed dengue were used for RT-PCR testing. All samples were also subjected to dengue serology (NS1 antigen and anti-dengue-IgM/IgG rapid immunochromatographic assay). Nested RT-PCR was performed on viral RNA using three methods targeting 654bp C-prM, 511bp C-prM and 641bp Env regions, respectively. RT-PCR-positive samples were validated by population sequencing. Among 171 children with suspected dengue, 121 were dengue serology-positive and 50 were dengue serology-negative. Among 121 serology-positives, RT-PCR detected 91 (75.2%) by CprM654, 72 (59.5%) by CprM511, and 74 (61.1%) by Env641. Among 50 serology-negatives, 10 (20.0%) were detected by CprM654, 12 (24.0%) by CprM511, and 11 (22.0%) by Env641. Overall detection rate using three methods sequentially was 82.6% (100/121) among serology-positive and 40.0% (20/50) among serology-negative samples; 6.6% (8/120) had co-infection with multiple DENV serotypes. We conclude that detection of acute dengue was enhanced by a modified RT-PCR method targeting the 654bp C-prM region, and further improved by using all three methods sequentially.

Unusual severe case of hemolytic uremic syndrome due to Shiga toxin 2d-producing E. coli O80:H2.

BACKGROUND: Hemolytic uremic syndrome (HUS) is one of the most common causes of acute renal failure in children, with the majority of cases caused by an infection with Shiga toxin-producing Escherichia coli (STEC). Whereas O157 is still the predominant STEC serotype, non-O157 serotypes are increasingly associated with STEC-HUS. However, little is known about this emerging and highly diverse group of non-O157 serotypes. With supportive therapy, STEC-HUS is often self-limiting, with occurrence of chronic sequelae in just a small proportion of patients. CASE DIAGNOSIS/TREATMENT: In this case report, we describe a 16-month-old boy with a highly severe and atypical presentation of STEC-HUS. Despite the presentation with multi-organ failure and extensive involvement of central nervous system due to extensive thrombotic microangiopathy (suggestive of atypical HUS), fecal diagnostics revealed an infection with the rare serotype: shiga toxin 2d-producing STEC O80:H2. CONCLUSIONS: This report underlines the importance of STEC diagnostic tests in all children with HUS, including those with an atypical presentation, and emphasizes the importance of molecular and serotyping assays to estimate the virulence of an STEC strain.

Discrepancy between Hepatitis C Virus Genotypes and NS4-Based Serotypes: Association with Their Subgenomic Sequences.

Determination of hepatitis C virus (HCV) genotypes plays an important role in the direct-acting agent era. Discrepancies between HCV genotyping and serotyping assays are occasionally observed. Eighteen samples with discrepant results between genotyping and serotyping methods were analyzed. HCV serotyping and genotyping were based on the HCV nonstructural 4 (NS4) region and 5'-untranslated region (5'-UTR), respectively. HCV core and NS4 regions were chosen to be sequenced and were compared with the genotyping and serotyping results. Deep sequencing was also performed for the corresponding HCV NS4 regions. Seventeen out of 18 discrepant samples could be sequenced by the Sanger method. Both HCV core and NS4 sequences were concordant with that of genotyping in the 5'-UTR in all 17 samples. In cloning analysis of the HCV NS4 region, there were several amino acid variations, but each sequence was much closer to the peptide with the same genotype. Deep sequencing revealed that minor clones with different subgenotypes existed in two of the 17 samples. Genotyping by genome amplification showed high consistency, while several false reactions were detected by serotyping. The deep sequencing method also provides accurate genotyping results and may be useful for analyzing discrepant cases. HCV genotyping should be correctly determined before antiviral treatment.

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)

[Two Outbreaks of Yersinia enterocolitica O:8 Infections in Tokyo and the Characterization of Isolates].

Although the number of outbreaks caused by Yersinia enterocolitica has been very small in Japan, 4 outbreaks were occurred during the 2 years between 2012 and 2013. We describe herein 2 outbreaks which were examined in Tokyo in the present study. Outbreak 1: A total of 39 people (37 high school students and 2 staff) stayed at a hotel in mountain area in Japan had experienced abdominal pain, diarrhea and fever in August, 2012. The Y. enterocolitica serogroup O:8 was isolated from 18 (64.3%) out of 28 fecal specimens of 28 patients. The infection roots could not be revealed because Y. enterocolitica was not detected from any meals at the hotel or its environment. Outbreak 2: A total of 52 students at a dormitory had diarrhea and fever in April, 2013. The results of the bacteriological and virological examinations of fecal specimens of patients showed that the Y. enterocolitica serogroup O:8 was isolated from 24 fecal specimens of 21 patients and 3 kitchen staff. We performed bacteriological and virological examination of the stored and preserved foods at the kitchen of the dormitory to reveal the suspect food. For the detection of Y. enterocolitica, food samples. together with phosphate buffered saline (PBS) were incubated at 4 degrees C for 21 days. Then, a screening test for Y. enterocolitica using realtime-PCR targeting the ail gene was performed against the PBS culture. One sample (fresh vegetable salad) tested was positive on realtime-PCR. No Y. enterocolitica was isolated on CIN agar from the PBS culture because many bacteria colonies other than Y. enterocolitica appeared on the CIN agar. After the alkaline-treatments of the culture broth or the immunomagnetic beads concentration method using anti-Y. enterocolitica O:8 antibodies, Y. enterocolitica O:8 which was the same serogroup as the patients' isolates was successfully isolated from the PBS culture. The fresh vegetable salad was confirmed as the incrimination food of this outbreak.

Simultaneous Rapid Detection and Serotyping of Cronobacter sakazakii Serotypes O1, O2, and O3 by Using Specific Monoclonal Antibodies.

Cronobacter sakazakii is a foodborne pathogen associated with rare but often lethal infections in neonates. Powdered infant formula (PIF) represents the most frequent source of infection. Out of the identified serotypes (O1 to O7), O1, O2, and O3 are often isolated from clinical and PIF samples. Serotype-specific monoclonal antibodies (MAbs) suitable for application in enzyme immunoassays (EIAs) for the rapid detection of C. sakazakii have not yet been developed. In this study, we created specific MAbs with the ability to bind toC. sakazakii of serotypes O1, O2, and O3. Characterization by indirect EIAs, immunofluorescence, motility assays, and immunoblotting identified lipopolysaccharide (LPS) and exopolysaccharide (EPS) as the antigenic determinants of the MAbs. The established sandwich EIAs were highly sensitive and were able to detect between 2 × 10(3)and 9 × 10(6)CFU/ml. Inclusivity tests confirmed that 93% of serotype O1 strains, 100% of O2 strains, and 87% of O3 strains were detected at low cell counts. No cross-reactivity with >100 strains of Cronobacter spp. and other Enterobacter iaceae was observed, except for that with C. sakazakii serotype O3 and Cronobacter muytjensii serotype O1. Moreover, the sandwich EIAs detected C. sakazakii in PIF samples artificially contaminated with 1 to 10 bacterial cells per 10 g of sample after 15 h of preenrichment. The use of these serotype-specific MAbs not only allows the reliable detection of C. sakazakii strains but also enables simultaneous serotyping in a simple sandwich EIA method.

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.

Epidemic Pseudomonas aeruginosa infection in patients with cystic fibrosis is not a risk factor for poor clinical Outcomes following lung transplantation.

BACKGROUND: Epidemic strains of Pseudomonas aeruginosa (ePA) causing infection in cystic fibrosis (CF) have been commonly identified from clinics around the world. ePA disproportionally impacts CF patient pre-transplant outcomes manifesting in increased exacerbation frequency, worsened treatment burden and increased rate of lung function decline, and disproportionally leads to death and/or transplantation. As other CF factors such as pre-transplant infection with multi-resistant organisms, and isolation of P. aeruginosa in the post transplant graft, may impact post-transplant outcomes, we sought to determine if infection with ePA similarly adversely impact post-transplant outcomes. METHODS: Between 1991-2014, 53 CF patients from our center received lung transplants. Bacterial strain typing was performed retrospectively on isolates collected prior to transplantation. Comprehensive chart reviews were performed to obtain baseline patient characteristics and post-transplant outcomes. RESULTS: Of the 53 transplanted patients, 57% of patients were infected with ePA prior to transplant; the other 43% of patients had unique strains of P. aeruginosa. Mean age at transplant was 29.0years for ePA and 33.3years for unique (p=0.04). There were no differences in overall survival (HR=0.75, 95% CI 0.31-1.79), bronchiolitis obliterans syndrome (BOS) free survival (HR 1.43, 95% CI 0.54-4.84) or all other assessed outcomes including exacerbation frequency, chronic renal failure, acute cellular rejections, Aspergillus infection, airway stenosis, and post-transplant lymphoproliferative disorder. CONCLUSION: Unlike pre-transplant outcomes, CF patients infected with ePA do not experience worse post-transplant outcomes than those infected with unique strains. Therefore, lung transplantation should be considered for all patients with P. aeruginosa infection and end stage lung disease, irrespective of infection with ePA.

Streptococcus pneumoniae oropharyngeal colonization in children and adolescents with cystic fibrosis.

BACKGROUND: This study was designed to evaluate Streptococcus pneumoniae (S. pneumoniae) carriage rates in patients with cystic fibrosis (CF). METHODS: An oropharyngeal swab was obtained from 212 CF children and adolescents enrolled during routine clinical visits. DNA from swabs was analyzed by real-time polymerase chain reaction. RESULTS: A total of 42 (19.8%) CF patients (mean age±standard deviation [SD], 12.0±3.3years) were colonized by S. pneumoniae. Carriage was more common in younger patients and tended to decline with age. Administration of systemic and/or inhaled antibiotics in the last 3months significantly correlated with a reduced carrier state [odds ratio (OR) 0.23, 95% confidence interval (CI) 0.07-0.69, and OR 0.26, 95% CI 0.08-0.77, respectively]. Vitamin D serum levels ≥30ng/mL were less common in carriers than that in non-carriers (OR 0.35; 95% CI 0.08-1.49). In both the vaccinated and unvaccinated subjects, serotypes 19F, 5, 4, and 9V were the most commonly carried serotypes. CONCLUSIONS: S. pneumoniae carrier state of school-age children and adolescents with CF is more prevalent than previously thought, and pneumococcal conjugate vaccination administered in the first year of life does not reduce the risk of re-colonization in later childhood and adolescence.

Assessing the public health risk of Shiga toxin-producing Escherichia coli by use of a rapid diagnostic screening algorithm.

Shiga toxin-producing Escherichia coli (STEC) is an enteropathogen of public health concern because of its ability to cause serious illness and outbreaks. In this prospective study, a diagnostic screening algorithm to categorize STEC infections into risk groups was evaluated. The algorithm consists of prescreening stool specimens with real-time PCR (qPCR) for the presence of stx genes. The qPCR-positive stool samples were cultured in enrichment broth and again screened for stx genes and additional virulence factors (escV, aggR, aat, bfpA) and O serogroups (O26, O103, O104, O111, O121, O145, O157). Also, PCR-guided culture was performed with sorbitol MacConkey agar (SMAC) and CHROMagar STEC medium. The presence of virulence factors and O serogroups was used for presumptive pathotype (PT) categorization in four PT groups. The potential risk for severe disease was categorized from high risk for PT group I to low risk for PT group III, whereas PT group IV consists of unconfirmed stx qPCR-positive samples. In total, 5,022 stool samples of patients with gastrointestinal symptoms were included. The qPCR detected stx genes in 1.8% of samples. Extensive screening for virulence factors and O serogroups was performed on 73 samples. After enrichment, the presence of stx genes was confirmed in 65 samples (89%). By culture on selective media, STEC was isolated in 36% (26/73 samples). Threshold cycle (CT) values for stx genes were significantly lower after enrichment compared to direct qPCR (P < 0.001). In total, 11 (15%), 19 (26%), 35 (48%), and 8 (11%) samples were categorized into PT groups I, II, III, and IV, respectively. Several virulence factors (stx2, stx2a, stx2f, toxB, eae, efa1, cif, espA, tccP, espP, nleA and/or nleB, tir cluster) were associated with PT groups I and II, while others (stx1, eaaA, mch cluster, ireA) were associated with PT group III. Furthermore, the number of virulence factors differed between PT groups (analysis of variance, P < 0.0001). In conclusion, a diagnostic algorithm enables fast discrimination of STEC infections associated with a high to moderate risk for severe disease (PT groups I and II) from less-virulent STEC (PT group III).

Molecular methods for serovar determination of Salmonella.

Salmonella is a diverse foodborne pathogen, which has more than 2600 recognized serovars. Classification of Salmonella isolates into serovars is essential for surveillance and epidemiological investigations; however, determination of Salmonella serovars, by traditional serotyping, has some important limitations (e.g. labor intensive, time consuming). To overcome these limitations, multiple methods have been investigated to develop molecular serotyping schemes. Currently, molecular methods to predict Salmonella serovars include (i) molecular subtyping methods (e.g. PFGE, MLST), (ii) classification using serovar-specific genomic markers and (iii) direct methods, which identify genes encoding antigens or biosynthesis of antigens used for serotyping. Here, we reviewed reported methodologies for Salmonella molecular serotyping and determined the "serovar-prediction accuracy", as the percentage of isolates for which the serovar was correctly classified by a given method. Serovar-prediction accuracy ranged from 0 to 100%, 51 to 100% and 33 to 100% for molecular subtyping, serovar-specific genomic markers and direct methods, respectively. Major limitations of available schemes are errors in predicting closely related serovars (e.g. Typhimurium and 4,5,12:i:-), and polyphyletic serovars (e.g. Newport, Saintpaul). The high diversity of Salmonella serovars represents a considerable challenge for molecular serotyping approaches. With the recent improvement in sequencing technologies, full genome sequencing could be developed into a promising molecular approach to serotype Salmonella.