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Different Salmonella serovars generally display different antigenic formulae , but there are some exceptions. For instance, the same antigenic formula , 6,7:c:1,5, is shared by Salmonella enterica serovar, Paratyphi C, Typhisuis, and Choleraesuis. Moreover, three biotypes have been described within the S . Choleraesuis serovar. A distinction among such biotypes can only be based on biochemical behaviors (biotyping) posing serious concerns when rapid characterization is required. The study of an outbreak of severe epizootic salmonellosis in wild boars occurred in Italy between 2012 and 2014 and the typing of the isolates recovered from the outbreak were used to test different approaches for serovar identification. A number of 30 S . Choleraesuis var. Kunzendorf isolates from the outbreak were typed by means of four different methods to derive serovar and biotype: (i) slide agglutination method followed by biochemical tests, (ii) suspension array xMAP® Salmonella Serotyping Assay (SSA), (iii) whole genome sequencing (WGS) and data analysis using SeqSero tool, and (iv) WGS and data analysis using Salmonella TypeFinder tool. Slide agglutination, xMAP® SSA and WGS, followed by SeqSero analysis, are methods that infer the serovars according to the White-Kauffmann-Le Minor (WKL) scheme, based exclusively on antigens. Using these methods, isolates with incomplete antigenic formulae could be misleadingly excluded from an outbreak. On the contrary, WGS followed by Salmonella TypeFinder data analysis, which predicts the serotype on the basis of Multilocus sequence typing (MLST), might be able to cluster together isolates belonging to the same outbreak irrespective of the antigenic formula . Results suggest the benefit of routine use of a combination of in silico MLST and antigenic formula analysis to solve specific ambiguous case studies for outbreak investigation purposes.

Multiple serotypes and topotypes of foot-and-mouth disease virus (FMDV) circulate in endemic areas, posing considerable impacts locally. In addition, introductions into new areas are of great concern. Indeed, in recent years, multiple FMDV outbreaks, caused by topotypes that have escaped from their original areas, have been recorded in various parts of the world. In both cases, rapid and accurate diagnosis, including the identification of the serotype and topotype causing the given outbreaks, plays an important role in the implementation of the most effective and appropriate measures to control the spread of the disease. In the present study, we describe the performance of a range of diagnostic and typing tools for FMDV on a panel of vesicular samples collected in northern Tanzania (East Africa, EA) during 2012–2018. Specifically, we tested these samples with a real-time RT-PCR targeting 3D sequence for pan-FMDV detection; an FMDV monoclonal antibody-based antigen (Ag) detection and serotyping ELISA kit; virus isolation (VI) on LFBKαVβ6 cell line; and a panel of four topotype-specific real-time RT-PCRs, specifically tailored for circulating strains in EA. The 3D real-time RT-PCR showed the highest diagnostic sensitivity, but it lacked typing capacity. Ag-ELISA detected and typed FMDV in 71% of sample homogenates, while VI combined with Ag-ELISA for typing showed an efficiency of 82%. The panel of topotype-specific real-time RT-PCRs identified and typed FMDV in 93% of samples. However, the SAT1 real-time RT-PCR had the highest (20%) failure rate. Briefly, topotype-specific real-time RT-PCRs had the highest serotyping capacity for EA FMDVs, although four assays were required, while the Ag-ELISA, which was less sensitive, was the most user-friendly, hence suitable for any laboratory level. In conclusion, when the four compared tests were used in combination, both the diagnostic and serotyping performances approached 100%.

A One Health approach to the epidemiology, management, surveillance, and control of leptospirosis relies on accessible and accurate diagnostics that can be applied to humans and companion animals and livestock. Diagnosis should be multifaceted and take into account exposure risk, clinical presentation, and multiple direct and/or indirect diagnostic approaches. Methods of direct detection of Leptospira spp. include culture, histopathology and immunostaining of tissues or clinical specimens, and nucleic acid amplification tests (NAATs). Indirect serologic methods to detect leptospiral antibodies include the microscopic agglutination test (MAT), the enzyme-linked immunosorbent assay (ELISA), and lateral flow methods. Rapid diagnostics that can be applied at the point-of-care; NAAT and lateral flow serologic tests are essential for management of acute infection and control of outbreaks. Culture is essential to an understanding of regional knowledge of circulating strains, and we discuss recent improvements in methods for cultivation, genomic sequencing, and serotyping. We review the limitations of NAATs, MAT, and other diagnostic approaches in the context of our expanding understanding of the diversity of pathogenic Leptospira spp. Novel approaches are needed, such as loop mediated isothermal amplification (LAMP) and clustered regularly interspaced short palindromic repeats (CRISPR)-based approaches to leptospiral nucleic acid detection.

Streptococcus suis is a significant porcine pathogen and zoonotic agent responsible for infectious diseases in humans worldwide. It is classified into 29 serotypes, each with varying geographical prevalence and pathogenicity. Hence, serotyping of S. suis is crucial for active surveillance, outbreak monitoring, and infection control. This study developed a novel MassARRAY-based single assay to simultaneously identify S. suis species and differentiate all 29 serotypes. The assay targeted glutamate dehydrogenase ( gdh ) and recombination/repair protein ( recN ) for species identification, and capsular polysaccharide ( cps ) genes for serotyping. Based on single nucleotide polymorphisms (SNPs) at position 483 of cpsK gene, the assay accurately distinguished between two pairs of serotypes: ½ and 2, as well as 1 and 14. The assay, validated with genomic DNA from 105 whole-genome sequencing (WGS)-confirmed isolates, demonstrated 100% specificity and sensitivity for both species identification and serotyping. In the evaluation with 143 field isolates, the results demonstrated perfect agreement between the MassARRAY-based assay and WGS for species identification, with 100% sensitivity and specificity, and for serotyping, with 99.15% sensitivity and 100% specificity (κ-value = 0.98). The agreement between WGS-based serotyping and multiplex PCR serotyping was substantial (κ = 0.65). Moreover, the results showed that the limit of detection for both species and serotypes ranged from 1 to 10 pg of gDNA per reaction.

Dengue is the most prevalent mosquito-borne viral disease and a major public health problem worldwide. Most primary infections with the four dengue virus serotypes (DENV1–4) are inapparent; nonetheless, whether the distribution of symptomatic versus inapparent infections by serotype varies remains unknown. Here, we present (1) the evaluation of a DENV1–4 envelope domain III multiplex microsphere-based assay (EDIII-MMBA) to serotype inapparent primary infections and (2) its application leveraging 17 years of prospective sample collection from the Nicaraguan Pediatric Dengue Cohort Study (PDCS). We analysed primary DENV infections in the PDCS from 2004 to 2022 detected by inhibition ELISA (iELISA) or RT-PCR. First, we evaluated the performance of the EDIII-MMBA for serotyping with samples characterised by RT-PCR or focus reduction neutralisation test. Next, we analysed a subset of inapparent primary DENV infections in the PDCS with the EDIII-MMBA to evaluate the epidemiology of inapparent infections. Remaining infections were inferred using stochastic imputation, taking year and neighbourhood into account. Infection incidence and percentage of inapparent, symptomatic, and severe infections were analysed by serotype. Between Aug 30, 2004, and March 10, 2022, a total of 5931 DENV-naive participants were followed in the PDCS. There were 1626 primary infections (382 symptomatic, 1244 inapparent) detected by iELISA or RT-PCR over the study period. The EDIII-MMBA demonstrated excellent overall accuracy (100%, 95% CI 95·8–100) for serotyping inapparent primary DENV infections when evaluated against gold-standard serotyping methods. Of the 1244 inapparent infections, we analysed 574 (46%) using the EDIII-MMBA. We found that the majority of primary infections were inapparent, with DENV3 exhibiting the highest likelihood of symptomatic (pooled odds ratio compared with DENV1: 2·13, 95% CI 1·28–3·56) and severe (6·75, 2·01–22·62) primary infections, whereas DENV2 was similar to DENV1 in both analyses. Considerable within-year and between-year variation in serotype distribution between symptomatic and inapparent infections and circulation of serotypes undetected in symptomatic cases were observed in multiple years. Our study indicates that case surveillance skews the perceived epidemiological footprint of DENV. We reveal a more complex and intricate pattern of serotype distribution in inapparent infections. The substantial differences in infection outcomes by serotype emphasises the need for vaccines with balanced immunogenicity and efficacy across serotypes. National Institute of Allergy and Infectious Diseases (National Institutes of Health) and Bill & Melinda Gates Foundation. For the Spanish translation of the abstract see Supplementary Materials section.

Roughly half the world's population live in dengue-endemic countries, but no vaccine is licensed. We investigated the efficacy of a recombinant, live, attenuated tetravalent dengue vaccine. In this observer-masked, randomised, controlled, monocentre, phase 2b, proof-of-concept trial, healthy Thai schoolchildren aged 4–11 years were randomly assigned (2:1) to receive three injections of dengue vaccine or control (rabies vaccine or placebo) at months 0, 6, and 12. Randomisation was by computer-generated permuted blocks of six and participants were assigned with an interactive response system. Participants were actively followed up until month 25. All acute febrile illnesses were investigated. Dengue viraemia was confirmed by serotype-specific RT-PCR and non-structural protein 1 ELISA. The primary objective was to assess protective efficacy against virologically confirmed, symptomatic dengue, irrespective of severity or serotype, occurring 1 month or longer after the third injection (per-protocol analysis). This trial is registered at ClinicalTrials.gov, NCT00842530. 4002 participants were assigned to vaccine (n=2669) or control (n=1333). 3673 were included in the primary analysis (2452 vaccine, 1221 control). 134 cases of virologically confirmed dengue occurred during the study. Efficacy was 30·2% (95% CI −13·4 to 56·6), and differed by serotype. Dengue vaccine was well tolerated, with no safety signals after 2 years of follow-up after the first dose. These data show for the first time that a safe vaccine against dengue is possible. Ongoing large-scale phase 3 studies in various epidemiological settings will provide pivotal data for the CYD dengue vaccine candidate. Sanofi Pasteur.

Foot-and-mouth disease (FMD) is a devastating infectious viral disease of cloven-hoofed animals. Differentiating FMD from other vesicular diseases is difficult based on only clinical symptoms, requiring an appropriate laboratory diagnostic test. The double-antibody sandwich (DAS)-ELISA is a reliable diagnostic technique for antigen detection and serotyping of FMDV. However, classical DAS-ELISAs use polyclonal antibodies (pAbs), which are inconsistent in yields and limited in large-scale applications compared to hybridoma cell-secreted laboratory-made monoclonal antibodies (mAbs). Therefore, this study aimed to develop simplified and sensitive FMD serotype-specific DAS-ELISAs using HRP-conjugated mAbs and a TMB substrate. Six FMDV serotype-specific mAb-DAS-ELISAs were developed. All assays were optimized using BEI-inactivated FMD antigens. Real-time reverse-transcriptase PCR (RRT-PCR) was also used to verify the detection efficiency of all assays. Known negative and positive 10% tissue suspensions of different animal origins were examined to calculate the diagnostic specificity (DSp) and sensitivity (DSe). Serotype-specific mAb-DAS-ELISAs demonstrated 100%, 97%, 97%, 99%, 99%, and 94% DSp and 100%, 95%, 90%, 95%, 100%, and 100% DSe for serotypes O, A, Asia-1, SAT-1, SAT-2, and SAT-3, respectively. The detection efficiency of mAb-DAS-ELISAs was better than that of classical DAS-ELISAs. Also, all assays demonstrated minimal cross-reactivity and optimal reproducibility. Therefore, the mAb-DAS-ELISAs developed in this study could be useful for detecting and serotyping FMDV and ultimately replacing the classical DAS-ELISA.

Glaesserella parasuis (GPS) is an important bacterial pathogen of swine. Serotype identification has presented a bottleneck in GPS research since it was first identified as the pathogen causing Glässer’s disease in pigs in 1910. This paper presents a systematic review of the history of the development and application of gel immunodiffusion (GID), indirect hemagglutination assay (IHA), and polymerase chain reaction (PCR) typing methods for GPS, and the discovery of their shared antigenic basis. It provides a systematic theoretical overview of the immunology and principles underlying the three typing methods and offers new ideas for research into the prevention and control of Glässer’s disease. In 1992, GPS was first classified into serotypes 1–15 using GID based on GPS heat-stable antigens, but about 25% of the strains were found to be non-typeable, and the composition of their antigens for serotyping was unclear. In 2003, the IHA method was established based on saline-extracted antigens of GPS, whose sensitivity and typing rate were higher than for GID, although about 15% of strains were still found to be non-typeable. The results of IHA and GID typing are roughly consistent, since they share the same GPS surface polysaccharide serotyping antigens, although whether these are capsular polysaccharides, lipopolysaccharides, or other polysaccharides, remains to be determined. In 2013, the Capsular polysaccharide (CPS) synthetic gene clusters from GPS serotypes 1–15 were successfully analyzed, confirming that CPS is essential for the formation of antigens for serotyping. In 2015, primers were designed based on the specific target genes of GPS capsules to establish a PCR typing method (H-PCR) for GPS, which, however, could not identify serotypes 5 and 12. In 2017, a new PCR typing method (J-PCR) was established based on the specific target genes of GPS capsules, which could identify serotypes 5 and 12. A combination of the two PCR typing methods enables the typing of almost all GPS strains, and the consistency with GID and IHA was verified using molecular biological methods. The antigenic basis of the three typing methods was shown to involve the GPS capsule. PCR typing methods are characterized by simple operation, fast speed, and low cost, and can successfully solve many problems in GID and IHA serotyping, and so have become widely adopted.

Background Non-typhoid Salmonella Typhimurium ( S. Typhimurium) accounts for a high number of registered salmonellosis cases, and O-serotyping is one important tool for monitoring epidemiology and spread of the disease. Moreover, variations in glucosylated O-antigens are related to immunogenicity and spread in the host. However, classical autoagglutination tests combined with the analysis of specific genetic markers cannot always reliably register phase variable glucose modifications expressed on Salmonella O-antigens and additional tools to monitor O-antigen glucosylation phenotypes of S. Typhimurium would be desirable. Results We developed a test for the phase variable O-antigen glucosylation state of S. Typhimurium using the tailspike proteins (TSP) of Salmonella phages 9NA and P22. We used this ELISA like tailspike adsorption (ELITA) assay to analyze a library of 44 Salmonella strains. ELITA was successful in discriminating strains that carried glucose 1-6 linked to the galactose of O-polysaccharide backbone (serotype O1) from non-glucosylated strains. This was shown by O-antigen compositional analyses of the respective strains with mass spectrometry and capillary electrophoresis. The ELITA test worked rapidly in a microtiter plate format and was highly O-antigen specific. Moreover, TSP as probes could also detect glucosylated strains in flow cytometry and distinguish multiphasic cultures differing in their glucosylation state. Conclusions Tailspike proteins contain large binding sites with precisely defined specificities and are therefore promising tools to be included in serotyping procedures as rapid serotyping agents in addition to antibodies. In this study, 9NA and P22TSP as probes could specifically distinguish glucosylation phenotypes of Salmonella on microtiter plate assays and in flow cytometry. This opens the possibility for flow sorting of cell populations for subsequent genetic analyses or for monitoring phase variations during large scale O-antigen preparations necessary for vaccine production.

Dengue is the most prevalent arboviral disease of humans. The host and virus variables associated with dengue virus (DENV) transmission from symptomatic dengue cases (n = 208) to Aedes aegypti mosquitoes during 407 independent exposure events was defined. The 50% mosquito infectious dose for each of DENV-1-4 ranged from 6.29 to 7.52 log10 RNA copies/mL of plasma. Increasing day of illness, declining viremia, and rising antibody titers were independently associated with reduced risk of DENV transmission. High early DENV plasma viremia levels in patients were a marker of the duration of human infectiousness, and blood meals containing high concentrations of DENV were positively associated with the prevalence of infectious mosquitoes 14 d after blood feeding. Ambulatory dengue cases had lower viremia levels compared with hospitalized dengue cases but nonetheless at levels predicted to be infectious to mosquitoes. These data define serotype-specific viremia levels that vaccines or drugs must inhibit to prevent DENV transmission.