Targeted Next-Generation Sequencing Assay for Direct Detection and Serotyping of Salmonella from Enrichment.

In this study, an automated, targeted next-generation sequencing (tNGS) assay to detect and serotype Salmonella from sample enrichments was evaluated. The assay generates millions of reads to detect multiple Salmonella-specific genes and serotype-specific alleles, detecting all Salmonella spp. tested to date, and serotyping 62 common Salmonella serotypes. Accuracy was tested on 291 pure reference cultures (251 Salmonella, 40 non-Salmonella), 21 artificially contaminated poultry carcass rinse samples, and 363 naturally contaminated poultry environmental samples. Among the 291 pure reference cultures, the automated tNGS assay resulted in 100% detection accuracy, 100% serotyping accuracy for the claimed serotypes, and 0% false positives. The limit of detection was estimated at 5 × 104 CFU/mL by testing enumerated cultures of strains representative of six serotypes. In cocontamination studies with mixtures of two serotypes (Enteritidis, Typhimurium, Kentucky, Infantis, and Newport) at a 1:1 ratio, tNGS detected both serotypes with 100% accuracy. The assay demonstrated 100% accuracy in artificially contaminated poultry carcass rinse sample enrichments. Targeted NGS was highly effective in detecting Salmonella in samples collected from poultry production facilities. Results demonstrated that tNGS could detect Salmonella and provide accurate serotyping information consistent with conventional serology. These findings highlight the reliable and efficient performance of a fully automated tNGS Salmonella assay in detecting and identifying Salmonella strains in complex matrices, reducing the time to results from 4 to 5 days required by the traditional isolation and serotyping to 10-12 h for tNGS after primary enrichment.

Growth assessment of Salmonella enterica multi-serovar populations in poultry rinsates with commonly used enrichment and plating media.

Isolation of Salmonella from enrichment cultures of food or environmental samples is a complicated process. Numerous factors including fitness in various selective enrichment media, relative starting concentrations in pre-enrichment, and competition among multi-serovar populations and associated natural microflora, come together to determine which serovars are identified from a given sample. A recently developed approach for assessing the relative abundance (RA) of multi-serovar Salmonella populations (CRISPR-SeroSeq or Deep Serotyping, DST) is providing new insight into how these factors impact the serovars observed, especially when different selective enrichment methods are used to identify Salmonella from a primary enrichment sample. To illustrate this, we examined Salmonella-positive poultry pre-enrichment samples through the selective enrichment process in Tetrathionate (TT) and Rappaport Vassiliadis (RVS) broths and assessed recovery of serovars with each medium. We observed the RA of serovars detected post selective enrichment varied depending on the medium used, initial concentration, and competitive fitness factors, all which could result in minority serovars in pre-enrichment becoming dominant serovars post selective enrichment. The data presented provide a greater understanding of culture biases and lays the groundwork for investigations into robust enrichment and plating media combinations for detecting Salmonella serovars of greater concern for human health.

Identification of pneumococcal serotypes with individual recognition of vaccine types by a highly multiplexed real-time PCR-based MeltArray approach.

BACKGROUND: Pneumococcus serotyping is important for monitoring serotype epidemiology, vaccine-induced serotypes replacement and emerging pathogenic serotypes. However, the lack of high-resolution serotyping tools has hindered its widespread implementation. METHODS: We devised a single-step, multiplex real-time polymerase chain reaction (PCR)-based MeltArray approach termed PneumoSero that can identify 92 serotypes with individual recognition of 54 serotypes, including all 24 currently available vaccine types. The limit of detection (LOD) and the ability to coexisting serotypes were studied, followed by analytical evaluation using 92 reference pneumococcal strains and 125 non-pneumococcal strains, and clinical evaluation using 471 pneumococcus isolates and 46 pneumococcus-positive clinical samples. RESULTS: The LODs varied with serotypes from 50 to 100 copies per reaction and 10 % of the minor serotypes were detectable in samples containing two mixed serotypes. Analytical evaluation presented 100 % accuracy in both 92 reference pneumococcal strains and 125 non-pneumococcal strains. Clinical evaluation of 471 pneumococcus isolates displayed full concordance with Sanger sequencing results. The 46 clinical specimens yielded 45 typeable results and one untypeable result. Of the 45 typeable samples, 41 were of a single serotype and four were of mixed serotypes, all of which were confirmed by Sanger sequencing or separate PCR assays. CONCLUSION: We conclude that the PneumoSero assay can be implemented as a routine tool for pneumococcal serotyping in standard microbiology laboratories and even in clinical settings.

Combined usage of serodiagnosis and O antigen typing to isolate Shiga toxin-producing Escherichia coli O76:H7 from a hemolytic uremic syndrome case and genomic insights from the isolate.

IMPORTANCE: Hemolytic uremic syndrome (HUS) is a life-threatening disease caused by Shiga toxin-producing Escherichia coli (STEC) infection. The treatment approaches for STEC-mediated typical HUS and atypical HUS differ, underscoring the importance of rapid and accurate diagnosis. However, specific detection methods for STECs other than major serogroups, such as O157, O26, and O111, are limited. This study focuses on the utility of PCR-based O-serotyping, serum agglutination tests utilizing antibodies against the identified Og type, and isolation techniques employing antibody-conjugated immunomagnetic beads for STEC isolation. By employing these methods, we successfully isolated a STEC strain of a minor serotype, O76:H7, from a HUS patient.

Molecular identification and characterisation of Mannheimia haemolytica.

Mannheimia haemolytica is known as one of the major bacterial contributors to Bovine Respiratory Disease (BRD) syndrome. This study sought to establish a novel species-specific PCR to aid in identification of this key pathogen. As well, an existing multiplex PCR was used to determine the prevalence of serovars 1, 2 or 6 in Australia. Most of the 65 studied isolates originated from cattle with a total of 11 isolates from small ruminants. All problematic field isolates in the identification or serotyping PCRs were subjected to whole genome sequencing and bioinformatic analysis. The field isolates were also subjected to rep-PCR fingerprinting. A total of 59 out of the 65 tested isolates were conformed as M. haemolytica by the new species-specific PCR which is based on the rpoB gene. The confirmed M. haemolytica field isolates were assigned to serovars 1 (24 isolates), 2 (seven isolates) and 6 (26 isolates) while two of the isolates were negative in the serotyping PCR. The two non-typeable isolates were assigned to serovar 7 and 14 following whole genome sequencing and bioinformatic analysis. The rep-PCR typing resulted in five major clusters with serovars 1 and 6 often within the same cluster. The M. haemolytica-specific PCR developed in this work was species specific and should be a valuable support for frontline diagnostic laboratories. The serotyping results support the relative importance of serovars 1 and 6 in bovine respiratory disease.

Poultry Processing Interventions Reduce Salmonella Serovar Complexity on Postchill Young Chicken Carcasses as Determined by Deep Serotyping.

Nearly 20% of salmonellosis cases are attributed to broilers, with renewed efforts to reduce Salmonella during broiler production and processing. A limitation to Salmonella culture is that often a single colony is picked for characterization, favoring isolation of the most abundant serovar found in a sample, while low abundance serovars can remain undetected. We used a deep serotyping approach, CRISPR-SeroSeq (serotyping by sequencing the clustered regularly interspaced palindromic repeats), to assess Salmonella serovar complexity during broiler processing and to determine the impact of antimicrobial interventions upon serovar population dynamics. Paired hot rehang and postchill young chicken carcasses were collected from establishments across the United States from August to November 2022. CRISPR-SeroSeq was performed on Salmonella culture-positive hot rehang (n = 153) and postchill (n = 38) samples, including 31 paired hot rehang and postchill samples. Multiple serovars were detected in 48.4% (74/153) and 7.9% (3/38) of hot rehang and postchill samples, respectively. On average, hot rehang carcasses contained 1.6 serovars, compared to 1.1 serovars at postchill (Mann Whitney U, p = 0.00018). Nineteen serovars were identified with serovar Kentucky the most common at hot rehang (72.5%; 111/153) and postchill (73.7%; 28/38). Serovar Infantis prevalence was higher at hot rehang (39.9%; 61/153) than in postchill (7.9%; 3/38). At hot rehang, serovar Enteritidis was outnumbered by other serovars 81.3% (13/16) of the time but was always the single or most abundant serovar detected when it was present at postchill (n = 5). We observed 98.4% (188/191) concordance between traditional isolation with serotyping and CRISPR-SeroSeq. Deep serotyping was able to explain serovar discrepancies between paired hot rehang and postchill samples when only traditional isolation and serotyping methods were used. These data demonstrate that processing interventions are effective in reducing Salmonella serovar complexity.

Comparison of next generation technologies and bioinformatics pipelines for capsular typing of Streptococcus pneumoniae.

Whole genome sequencing (WGS)-based approaches for pneumococcal capsular typing have become an alternative to serological methods. In silico serotyping from WGS has not yet been applied to long-read sequences produced by third-generation technologies. The objective of the study was to determine the capsular types of pneumococci causing invasive disease in Catalonia (Spain) using serological typing and WGS and to compare the performance of different bioinformatics pipelines using short- and long-read data from WGS. All invasive pneumococcal pediatric isolates collected in Hospital Sant Joan de Déu (Barcelona) from 2013 to 2019 were included. Isolates were assigned a capsular type by serological testing based on anticapsular antisera and by different WGS-based pipelines: Illumina sequencing followed by serotyping with PneumoCaT, SeroBA, and Pathogenwatch vs MinION-ONT sequencing coupled with serotyping by Pathogenwatch from pneumococcal assembled genomes. A total of 119 out of 121 pneumococcal isolates were available for sequencing. Twenty-nine different serotypes were identified by serological typing, with 24F (n = 17; 14.3%), 14 (n = 10; 8.4%), and 15B/C (n = 8; 6.7%) being the most common serotypes. WGS-based pipelines showed initial concordance with serological typing (>91% of accuracy). The main discrepant results were found at the serotype level within a serogroup: 6A/B, 6C/D, 9A/V, 11A/D, and 18B/C. Only one discrepancy at the serogroup level was observed: serotype 29 by serological testing and serotype 35B/D by all WGS-based pipelines. Thus, bioinformatics WGS-based pipelines, including those using third-generation sequencing, are useful for pneumococcal capsular assignment. Possible discrepancies between serological typing and WGS-based approaches should be considered in pneumococcal capsular-type surveillance studies.

A TaqMan-based RT-qPCR assay for serotyping of Southern African territories (SAT) 2 strains of Foot-and-Mouth disease virus (FMDV) in Southern Africa.

OBJECTIVE: Determining the serotype of circulating virus strains is important in implementing effective vaccination. In this study, Foot-and-Mouth Disease (FMD) Southern African territory 2 (SAT2) specific primers and TaqMan probe were designed towards rapid SAT2 detection and serotyping. The primers were tested by endpoint reverse transcription (RT) polymerase chain reaction (PCR) and quantitative PCR (RT-qPCR) using the vaccine strain SAT2035. The SAT2 serotype-specific RT-qPCR assay was compared with currently used ELISA and VP1 sequencing using Cohen's kappa statistics. RESULTS: The primers yielded amplicons of band size 190 bp during endpoint RT-PCR. When coupled with the probe, the primers reaction efficiency was determined to be 99% with an r2 value of 0.994. The results show that the SAT2 assay has comparable performance to VP1 sequencing (k = 1) and a moderate degree of agreement with ELISA (k = 0.571). The data shows that the newly designed assay could be considered for serotyping of SAT2 strains. However, for this assay to be complete there is a need to design effective SAT1 and SAT3 primers and probes that can be multiplexed to target other serotypes that co-circulate within relevant FMD endemic pools. For future implementation of the assay there is also a need to increase the number of field samples towards validation of the assay.

PfaSTer: a machine learning-powered serotype caller for Streptococcus pneumoniae genomes.

Streptococcus pneumoniae (pneumococcus) is a leading cause of morbidity and mortality worldwide. Although multi-valent pneumococcal vaccines have curbed the incidence of disease, their introduction has resulted in shifted serotype distributions that must be monitored. Whole genome sequence (WGS) data provide a powerful surveillance tool for tracking isolate serotypes, which can be determined from nucleotide sequence of the capsular polysaccharide biosynthetic operon (cps). Although software exists to predict serotypes from WGS data, most are constrained by requiring high-coverage next-generation sequencing reads. This can present a challenge in respect of accessibility and data sharing. Here we present PfaSTer, a machine learning-based method to identify 65 prevalent serotypes from assembled S. pneumoniae genome sequences. PfaSTer combines dimensionality reduction from k-mer analysis with a Random Forest classifier for rapid serotype prediction. By leveraging the model's built-in statistical framework, PfaSTer determines confidence in its predictions without the need for coverage-based assessments. We then demonstrate the robustness of this method, returning >97 % concordance when compared to biochemical results and other in silico serotyping tools. PfaSTer is open source and available at: https://github.com/pfizer-opensource/pfaster.

Is it time to move on to gene-based Salmonella typing: Evidence and implications.

Non-typhoidal Salmonella (NTS) is the major cause of foodborne infections globally, with considerable morbidity and mortality. The accurate identification of Salmonella serovars is important in disease management and public health surveillance. However, traditional serotyping methods are laborious, time-consuming and may produce ambiguous results. In this study, we evaluated traditional serotyping and seven gene-based multilocus sequence typing (MLST) methods to determine the serogroups of Salmonella strains. This study analysis suggests that MLST based serotyping is accurate in serogroup identification and discrimination of Salmonella serovars compared to the traditional serotyping method and can be implemented in routine clinical practice.

SSP: An In Silico Tool for Salmonella Species Serotyping Using the Sequences of O-Antigen Biosynthesis Proteins and H-Antigen Filament Proteins.

Over 2500 Salmonella species (alternatively, serovars) encompassing different combinations of O-, H1- and H2-antigens are present in nature and cause millions of deaths worldwide every year. Since conventional serotyping is time-consuming, a user-friendly Salmonellaspecies serotyping (SSP) web tool (https://project.iith.ac.in/SSP/) is developed here to predict the serotypes using Salmonella protein(s) or whole proteome sequences. Prior to SSP implementation, a detailed analysis of protein sequences involved in O-antigen biosynthesis and H-antigen formation is carried out to assess their serotype specificity. Intriguingly, the results indicate that the initializing transferases WbaP, WecA and GNE can efficiently distinguish the O-antigens, which have Gal, GlcNAc and GalNAc as initial sugars respectively. Rigorous analysis shows that Wzx and Wzy are sufficient to distinguish the O-types. Exceptionally, some situations warrant additional proteins. Thus, 150 additional transferases, RfbE for O2, O9 and O9,46 types, Orf17.4 for O3,10 and O1,3,19 types, WecB, WbbE and WbbF for O54 and, Wzm and Wzt for O67 are utilized in serotyping. An in-depth analysis of 302 reference datasets representing 56 H1- and 20 H2-types leads to the identification and utilization of 61 unique sequence patterns of FliC and FljB in H-typing. A test dataset of 2136 whole proteome sequences covering 740 Salmonella serovars, including 13 new species are successfully predicted with 99.72% accuracy. Prior to this, all the O-, H1- and H2-antigens are predicted accurately when tested independently. Indeed, SSP also identifies wrongly annotated Salmonella species; hence, it can easily identify new species that emerge with any combination of O-, H1- and H2-antigens. Thus, SSP can act as a valuable tool in the surveillance of Salmonella species.

A novel immuno-molecular strategy for the detection of Streptococcus pneumoniae serotypes in human cerebrospinal and middle ear fluids.

Streptococcus pneumoniae is one of the most common microorganisms causing acute otitis media (AOM) in children. While bacterial culture of middle ear fluid (MEF) is the gold standard to detect the etiological organisms, several host and pathogen factors impact the survival of the organisms resulting in false negatives. To overcome this limitation, we have developed and validated an innovative multiplex immuno-molecular assay to screen and detect the S. pneumoniae 15-valent pneumococcal conjugate vaccine (PCV15; STs 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F) vaccine serotypes in MEF. This novel in vitro approach involves two-step testing. First, the MEF specimens were tested for highly conserved pneumococcal genes, autolysin, lytA, and pneumolysin, ply using direct PCR to identify pneumococcus positive specimens. The pneumococcus positive specimens were screened for the presence of vaccine serotype specific pneumococcal polysaccharides using a 15-plex Pneumococcal Antigen Detection (PAD) assay, with specific capture and detection monoclonal antibodies. Due to the lack of availability of MEF samples, cerebrospinal fluid (CSF) was used as the surrogate matrix for the development and validation of the PCR-PAD assays. The PCR and PAD assays were separately evaluated for sensitivity and specificity. Subsequently, the PCR-PAD assays were cross-validated with human MEF samples (n = 39) which were culture confirmed to contain relevant bacterial strains. The combined PCR-PAD assays demonstrated high rate of agreement 94.9% (95% CI; 82.7, 99.4%) with historical Quellung serotype data of these MEF samples. This novel PCR-PAD assay demonstrates the feasibility of combining molecular and immunological assays to screen and identify PCV15 pneumococcal vaccine serotypes in AOM clinical samples.

ShigaPass: an in silico tool predicting Shigella serotypes from whole-genome sequencing assemblies.

Shigella is one of the commonest causes of diarrhoea worldwide and a major public health problem. Shigella serotyping is based on a standardized scheme that splits Shigella strains into four serogroups and 60 serotypes on the basis of biochemical tests and O-antigen structures. This conventional serotyping method is laborious, time-consuming, impossible to automate, and requires a high level of expertise. Whole-genome sequencing (WGS) is becoming more affordable and is now used for routine surveillance, opening up possibilities for the development of much-needed accurate rapid typing methods. Here, we describe ShigaPass, a new in silico tool for predicting Shigella serotypes from WGS assemblies on the basis of rfb gene cluster DNA sequences, phage and plasmid-encoded O-antigen modification genes, seven housekeeping genes (EnteroBase's MLST scheme), fliC alleles and clustered regularly interspaced short palindromic repeats (CRISPR) spacers. Using 4879 genomes, including 4716 reference strains and clinical isolates of Shigella characterized with a panel of biochemical tests and serotyped by slide agglutination, we show here that ShigaPass outperforms all existing in silico tools, particularly for the identification of Shigella boydii and Shigella dysenteriae serotypes, with a correct serotype assignment rate of 98.5 % and a sensitivity rate (i.e. ability to make any prediction) of 100 %.

Optimization of a high-throughput nanofluidic real-time PCR to detect and quantify of 15 bacterial species and 92 Streptococcus pneumoniae serotypes.

Sensitive tools for detecting concurrent colonizing pneumococcal serotypes are needed for detailed evaluation of the direct and indirect impact of routine pneumococcal conjugate vaccine (PCV) immunization. A high-throughput quantitative nanofluidic real-time PCR (Standard BioTools 'Fluidigm') reaction-set was developed to detect and quantify 92 pneumococcal serotypes in archived clinical samples. Nasopharyngeal swabs collected in 2009-2011 from South African children ≤ 5 years-old, previously serotyped with standard culture-based methods were used for comparison. The reaction-set within the 'Fluidigm' effectively amplified all targets with high efficiency (90-110%), reproducibility (R2 ≥ 0.98), and at low limit-of-detection (< 102 CFU/ml). A blind analysis of 1 973 nasopharyngeal swab samples showed diagnostic sensitivity > 80% and specificity > 95% compared with the referent standard, culture based Quellung method. The qPCR method was able to serotype pneumococcal types with good discrimination compared with Quellung (ROC-AUC: > 0.73). The high-throughput nanofluidic real-time PCR method simultaneously detects 57 individual serotypes, and 35 serotypes within 16 serogroups in 96 samples (including controls), within a single qPCR run. This method can be used to evaluate the impact of current PCV formulations on vaccine-serotype and non-vaccine-serotype colonization, including detection of multiple concurrently colonizing serotypes. Our qPCR method can allow for monitoring of serotype-specific bacterial load, as well as emergence or ongoing transmission of minor or co-colonizing serotypes that may have invasive disease potential.

Characterization of Shigella flexneri Serotype 6 Strains Isolated from Bangladesh and Identification of a New Phylogenetic Cluster.

A significant cause of shigellosis in Bangladesh and other developing countries is Shigella flexneri serotype 6. This serotype has been subtyped, on the basis of the absence or presence of a group-specific antigen, E1037, into S. flexneri 6a and 6b, respectively. Here, we provided rationales for the subclassification, using several phenotypic and molecular tools. A set of S. flexneri 6a and 6b strains isolated between 1997 and 2015 were characterized by analyzing their biochemical properties, plasmid profiles, virulence markers, pulsed-field gel electrophoresis (PFGE) results, and ribotype. Additionally, the genomic relatedness of these subserotypes was investigated with global isolates of serotype 6 using publicly available genomes. Both subserotypes of S. flexneri 6 agglutinated with monoclonal antiserum against S. flexneri (MASF) B and type VI-specific antiserum (MASF VI) and were PCR positive for O-antigen flippase-specific genes and virulence markers (ipaH, ial, sen, and sigA). Unlike S. flexneri 6a strains, S. flexneri 6b strains seroagglutinated with anti-E1037 antibodies, MASF IV-I. Notably, these two antigenically distinct subserotypes were clonally diverse, showing two distinct PFGE patterns following the digestion of chromosomal DNA with either XbaI or IceuI. In addition, hybridization of a 16S rRNA gene probe with HindIII-digested genomic DNA yielded two distinguishing ribotypes. Genomic comparison of S. flexneri subserotype 6a and 6b strains from Bangladesh indicated that, although these strains were in genomic synteny, the majority of them formed a unique phylogroup (PG-4) that was missing for the global isolates. This study supports the subserotyping and emphasizes the need for global monitoring of the S. flexneri subserotypes 6a and 6b. IMPORTANCE Shigella flexneri serotype 6 is one of the predominant serotypes among shigellosis cases in Bangladesh. Characterization of a novel subserotype of S. flexneri 6 (VI:E1037), agglutinated with type 6-specific antibody and anti-E1037, indicates a unique evolutionary ancestry. PFGE genotyping supports the finding that these two antigenically distinct subserotypes are clonally diverse. A phylogenetic study based on single-nucleotide polymorphism (SNP) data revealed that these two subserotypes were in genomic synteny, although their genomes were reduced. Interestingly, a majority of the S. flexneri 6 strains isolated from Bangladesh form a novel phylogenetic cluster. Therefore, this report underpins the global monitoring and tracking of the novel subserotype.

Combined Quantification and Deep Serotyping for Salmonella Risk Profiling in Broiler Flocks.

Despite a reduction of Salmonella contamination on final poultry products, the level of human salmonellosis cases attributed to poultry has remained unchanged over the last few years. There needs to be improved effort to target serovars which may survive antimicrobial interventions and cause illness, as well as to focus on lessening the amount of contamination entering the processing plant. Advances in molecular enumeration approaches allow for the rapid detection and quantification of Salmonella in pre- and postharvest samples, which can be combined with deep serotyping to properly assess the risk affiliated with a poultry flock. In this study, we collected a total of 160 boot sock samples from 20 broiler farms across four different integrators with different antibiotic management programs. Overall, Salmonella was found in 85% (68/80) of the houses, with each farm having at least one Salmonella-positive house. The average Salmonella quantity across all four complexes was 3.6 log10 CFU/sample. Eleven different serovars were identified through deep serotyping, including all three key performance indicators (KPIs; serovars Enteritidis, Infantis, and Typhimurium) defined by the U.S. Department of Agriculture-Food Safety and Inspection Service (USDA-FSIS). There were eight multidrug resistant isolates identified in this study, and seven which were serovar Infantis. We generated risk scores for each flock based on the presence or absence of KPIs, the relative abundance of each serovar as calculated with CRISPR-SeroSeq (serotyping by sequencing the clustered regularly interspaced palindromic repeats), and the quantity of Salmonella organisms detected. The work presented here provides a framework to develop directed processing approaches and highlights the limitations of conventional Salmonella sampling and culturing methods. IMPORTANCE Nearly one in five foodborne Salmonella illnesses are derived from chicken, making it the largest single food category to cause salmonellosis and indicating a need for effective pathogen mitigation. Although industry has successfully reduced Salmonella incidence in poultry products, there has not been a concurrent reduction in human salmonellosis linked to chicken consumption. New efforts are focused on improved control at preharvest, which requires improved Salmonella surveillance. Here, we present a high-resolution surveillance approach that combines quantity and identity of Salmonella in broiler flocks prior to processing which will further support improved Salmonella controls in poultry. We developed a framework for this approach, indicating that it is possible and important to harness deep serotyping and molecular enumeration to inform on-farm management practices and to minimize risk of cross-contamination between flocks at processing. Additionally, this framework could be adapted to Salmonella surveillance in other food animal production systems.

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.

[Serotyping methods of Streptococcus pneumonia].

More than 100 serotypes of Streptococcus pneumonia have been identified, which has been one bottleneck problem for pneumococcal disease diagnosis, surveillance, development of pneumococcal vaccine and effectiveness evaluation of pneumococcal vaccines. Three categories of approaches for pneumococcal serotyping will be discussed including phenotyping based on anti-serum, biochemical typing based on pneumococcal capsular characteristics and genotyping based on pneumococcal capsular locus sequences. We reviewed the development and applications of different serotyping of pneumococcus to provide guidance for pneumococcal disease prevention and control.

Variants of Streptococcus pneumoniae Serotype 14 from Papua New Guinea with the Potential to Be Mistyped and Escape Vaccine-Induced Protection.

Streptococcus pneumoniae (the pneumococcus) is a human pathogen of global importance, classified into serotypes based on the type of capsular polysaccharide produced. Serotyping of pneumococci is essential for disease surveillance and vaccine impact measurement. However, the accuracy of serotyping methods can be affected by previously undiscovered variants. Previous studies have identified variants of serotype 14, a highly invasive serotype included in all licensed vaccine formulations. However, the potential of these variants to influence serotyping accuracy and evade vaccine-induced protection has not been investigated. In this study, we screened 1,386 nasopharyngeal swabs from children hospitalized with acute respiratory infection in Papua New Guinea for pneumococci. Swabs containing pneumococci (n = 1,226) were serotyped by microarray to identify pneumococci with a divergent serotype 14 capsule locus. Three serotype 14 variants ('14-like') were isolated and characterized further. The serotyping results of these isolates using molecular methods varied depending on the method, with 3/3 typing as nontypeable (PneumoCaT), 3/3 typing as serotype 14 (seroBA), and 2/3 typing as serotype 14 (SeroCall and quantitative PCR). All three isolates were nontypeable by phenotypic methods (Quellung and latex agglutination), indicating the absence of capsule. Illumina and nanopore sequencing were employed to examine their capsule loci and revealed unique mutations. Lastly, when incubated with sera from vaccinated individuals, the 14-like isolates evaded serotype-specific opsonophagocytic killing. Our study highlights the need for phenotypic testing to validate serotyping data derived from molecular methods. The convergent evolution of capsule loss underscores the importance of studying pneumococcal population biology to monitor the emergence of pneumococci capable of vaccine escape, globally. IMPORTANCE Pneumococcus is a pathogen of major public health importance. Current vaccines have limited valency, targeting a subset (up to 20) of the more than 100 capsule types (serotypes). Precise serotyping methods are therefore essential to avoid mistyping, which can reduce the accuracy of data used to inform decisions around vaccine introduction and/or maintenance of national vaccination programs. In this study, we examine a variant of serotype 14 (14-like), a virulent serotype present in all currently licensed vaccine formulations. Although these 14-like pneumococci no longer produce a serotype 14 capsule, widely used molecular methods can mistype them as serotype 14. Importantly, we show that 14-like pneumococci can evade opsonophagocytic killing mediated by vaccination. Despite the high accuracy of molecular methods for serotyping, our study reemphasizes their limitations. This is particularly relevant in situations where nonvaccine type pneumococci (e.g., the 14-likes in this study) could potentially be misidentified as a vaccine type (e.g., serotype 14).

Validation of Fourier Transform Infrared Spectroscopy for Serotyping of Streptococcus pneumoniae.

Fourier transform infrared (FT-IR) spectroscopy (IR Biotyper; Bruker) allows highly discriminatory fingerprinting of closely related bacterial strains. In this study, FT-IR spectroscopy-based capsular typing of Streptococcus pneumoniae was validated as a rapid, cost-effective, and medium-throughput alternative to the classical phenotypic techniques. A training set of 233 strains was defined, comprising 34 different serotypes and including all 24 vaccine types (VTs) and 10 non-vaccine types (NVTs). The acquired spectra were used to (i) create a dendrogram where strains clustered together according to their serotypes and (ii) train an artificial neural network (ANN) model to predict unknown pneumococcal serotypes. During validation using 153 additional strains, we reached 98.0% accuracy for determining serotypes represented in the training set. Next, the performance of the IR Biotyper was assessed using 124 strains representing 59 non-training set serotypes. In this setting, 42 of 59 serotypes (71.1%) could be accurately categorized as being non-training set serotypes. Furthermore, it was observed that comparability of spectra was affected by the source of the Columbia medium used to grow the pneumococci and that this complicated the robustness and standardization potential of FT-IR spectroscopy. A rigorous laboratory workflow in combination with specific ANN models that account for environmental noise parameters can be applied to overcome this issue in the near future. The IR Biotyper has the potential to be used as a fast, cost-effective, and accurate phenotypic serotyping tool for S. pneumoniae.