Serotyping Assay Research Articles

Detection and characterization of HIV-1 group O and HIV-2 in the Central African Republic.

The Central African Republic (CAR) is characterized by widespread HIV epidemic with notable prevalence and genetic diversity. We herein analysed the genetic diversity of atypical non-M HIV-1 strains. In-house serotyping assays for variants of HIV-1 (M, N, O, P) and HIV-2 were used to test a biological collection of 6092 HIV-seropositive blood samples collected between 2003 and 2014 at the Institut Pasteur de Bangui. Samples indicative of recombinant M/O groups, HIV-2, or those that yield doubtful/negative results underwent further PCR tests and sequencing. We found six atypical HIV strains: specifically, three (0.05%) HIV-1 group O strains (subtype H) detected in samples from 2005, 2008 and 2009, alongside three (0.05%) HIV-2 strains (two group A and one group B) identified in samples from 2007 and 2009. HIV-1/O strains showed a genetic link to Cameroon and Gabon strains. This study highlights the dominance of HIV-1/M in the CAR's HIV epidemic over time and underscores the infrequent occurrence of HIV-1 group O and HIV-2 strains. These findings validate the efficacy of WHO-recommended HIV testing protocols and emphasize the need for adaptive surveillance and management strategies to confront the complexities introduced by the genetic diversity of HIV strains.

Molecular serotyping of diarrheagenic Escherichia coli with a MeltArray assay reveals distinct correlation between serotype and pathotype

ABSTRACT Diarrheagenic Escherichia coli serotypes are associated with various clinical syndromes, yet the precise correlation between serotype and pathotype remains unclear. A major barrier to such studies is the reliance on antisera-based serotyping, which is culture-dependent, low-throughput, and cost-ineffective. We have established a highly multiplex PCR-based serotyping assay, termed the MeltArray E. coli serotyping (EST) assay, capable of identifying 163 O-antigen-encoding genes and 53 H-antigen-encoding genes of E. coli. The assay successfully identified serotypes directly from both simulated and real fecal samples, as demonstrated through spike-in validation experiments and a retrospective study. In a multi-province study involving 637 E. coli strains, it revealed that the five major diarrheagenic pathotypes have distinct serotype compositions. Notably, it differentiated 257 Shigella isolates into four major Shigella species, distinguishing them from enteroinvasive E. coli based on their distinct serotype profiles. The assay’s universality was further corroborated by in silico analysis of whole-genome sequences from the EnteroBase. We conclude that the MeltArray EST assay represents a paradigm-shifting tool for molecular serotyping of E. coli, with potential routine applications for comprehensive serotype analysis, disease diagnosis, and outbreak detection.

Establishment and Modification of Ninety-seven Pneumococcal Serotyping Assays Based on Quantitative Real-time Polymerase Chain Reaction

ObjectiveTo establish and modify quantitative real-time polymerase chain reaction (qPCR)-based serotyping assays to distinguish 97 pneumococcal serotypes. MethodsA database of capsular polysaccharide (cps) loci sequences was generated, covering 97 pneumococcal serotypes. Bioinformatics analyses were performed to identify the cps loci structure and target genes related to different pneumococcal serotypes with specific SNPs. A total of 27 novel qPCR serotyping assay primers and probes were established based on qPCR, while 27 recombinant plasmids containing serotype-specific DNA sequence fragments were constructed as reference target sequences to examine the specificity and sensitivity of the qPCR assay. A panel of pneumococcal reference strains was employed to evaluate the capability of pneumococcal serotyping. ResultsA total of 97 pneumococcal serotyping assays based on qPCR were established and modified, which included 64 serotypes previously reported as well as an additional 33 serotypes. Twenty-seven novel qPCR serotyping target sequences were implemented in the pneumococcal qPCR serotyping system. A total of 97 pneumococcal serotypes, which included 52 individual serotypes and 45 serotypes belonging to 20 serogroups, could not be identified as individual serotypes. The sensitivity of qPCR assays based on 27 target sequences was 1–100 copies/μL. The specificity of the qPCR assays was 100%, which were tested by a panel of 90 serotypes of the pneumococcal reference strains. ConclusionA total of 27 novel qPCR assays were established and modified to analyze 97 pneumococcal serotypes.

Evaluation of Legionella pneumophila SGUT Serotypes Isolated from Bath Water Using a Multiplex-PCR Serotyping Assay.

Legionella pneumophila, the primary causative agent of Legionnaires' disease, is classified into at least 15 serogroups (SGs). Before genotyping, serotyping is first performed to limit the sources of L. pneumophila infections that caused an outbreak. In addition to conventional assays using monoclonal or polyclonal antisera, serotyping using multiplex polymerase chain reaction (M-PCR) was recently developed for L. pneumophila. In this study, we applied the M-PCR system to 41 strains that remained to be SGUT (untypable) by slide agglutination tests among the 220 L. pneumophila strains isolated from bath water in Kobe City during 2016-2020, to determine SG-genotypes (SGg) by PCR amplification of the specific target gene of the SGs. Among the 41 SGUT strains, SGg4/10/14 was the most predominant (24/41, 58.5%), followed by SGg1 (7/41, 17.1%). Seven strains, except for the strains determined as SGg1, were identified as belonging to a single SGg by M-PCR serotyping (SGg5 [3/41, 7.3%], SGg8 [3/41, 7.3%], and SGg7 [1/41, 2.4%]). Furthermore, we found that the seven strains identified as SGg1 harbored particular genotypes. In conclusion, the M-PCR serotyping assay will be helpful for investigating the distribution of L. pneumophila in environmental and clinical settings.

Enhanced detection and serotyping of foot-and-mouth disease virus serotype O, A, and Asia1 using a novel multiplex real-time RT-PCR.

Rapid and accurate detection and serotyping of foot-and-mouth disease (FMD) virus (FMDV) is essential for implementing control policies against emergent FMD outbreaks. Current serotyping assays, such as VP1 reverse transcription-polymerase chain reaction (RT-PCR)/sequencing (VP1 RT-PCR/sequencing) and antigen detection enzyme-linked immunosorbent assay (ELISA), have problems with increasing serotyping failure of FMDVs from FMD outbreaks. This study was conducted to develop a multiplex real-time RT-PCR for specific detection and differential serotyping of FMDV serotype O, A, and Asia 1 directly from field clinical samples. Primers and probes were designed based on 571 VP1 coding region sequences originated from seven pools. Multiplex real-time RT-PCR using these primers and probes demonstrated serotype-specific detection with enhanced sensitivity compared to VP1 RT-PCR/sequencing for reference FMDV (n = 24). Complete serotyping conformity between the developed multiplex real-time RT-PCR and previous VP1 RT-PCR/sequencing was demonstrated using FMDV field viruses (n = 113) prepared in cell culture. For FMDV field clinical samples (n = 55), the serotyping rates of multiplex real-time RT-PCR and VP1 RT-PCR/sequencing were 92.7% (51/55) and 72.7% (40/55), respectively. Moreover, the developed multiplex real-time RT-PCR demonstrated improved FMDV detection (up to 33.3%) and serotyping (up to 67.7%) capabilities for saliva samples when compared with 3D real-time RT-PCR and VP1 RT-PCR/sequencing, during 10 days of challenge infection with FMDV serotype O, A, and Asia 1. Collectively, this study suggests that the newly developed multiplex real-time RT-PCR assay may be useful for the detection and differential serotyping of FMDV serotype O, A, and Asia 1 in the field.

Phenotypic and genomic characterization for Salmonella isolates recovered from meat products in Beijing wholesale markets, 2014-2015

Objective: To analyze the phenotypic and genomic characteristics of Salmonella isolates recovered from meat products in Beijing wholesale markets. Methods: A total of 336 Salmonella strains from meat products collected from wholesale markets in Beijing were tested for antimicrobial resistance to 25 antimicrobial compounds by micro-broth dilution method; whole genome data were sequenced, followed by the serotype and ST type prediction by Seqsero2 and SISTR software, and the drug resistance genes and virulence factors were also predicted with CARD and VFDB databases of Abricate software; Salmonella serotyping assay kit and serum agglutination method were used for serotype confirmation of some isolates with different genome prediction results. Results: The resistance rates to Nalidixic acid and Ampicillin were 62.5% (210/336) and 55.1% (185/336), respectively, and all isolates were susceptible to Tigecyclin, Cefoxitin and Carbapenem antimicrobial compounds; 207 isolates (61.6%, 207/336) were multi-drug resistant, some could even be resistant to ten categories of drugs at the same time, and the most common antimicrobial resistance spectrum was NAL-AMP-SAM. A total of 24 serotypes were detected with predominant serotypes of Enteritidis (34.5%, 116/336), Derby (17.3%, 58/336) and Indiana (10.4%, 35/336). A total of 27 ST types were detected, the dominant type was ST11; ST types were in good consistency with serotypes; The detection rates of resistant genes referred to aminoglycosides, fluoroquinolones, β-lactams, sulfonamides and tetracyclines are more than 48%, and the first two reached 100%. The prediction of drug resistance genes was consistent with the results of antimicrobial resistance phenotype. A total of 122 virulence genes were predicted, 74 of which existing among all isolates. Conclusion: Salmonella in meat from the wholesale markets of Beijing has a high proportion of multiple drug resistance, a complex drug resistance spectrum, a variety of serotypes and ST types, and a high carrying rate of drug resistance gene and virulence gene; drug resistance phenotype and genotype are relatively consistent.

Direct Real-Time PCR for the Detection and Serotyping of Haemophilus influenzae without DNA Extraction.

To monitor the burden and changes in Haemophilus influenzae (Hi) disease, direct real-time PCR (drt-PCR) assays have been developed for Hi detection in monoplex form and its six serotypes in triplex form, directly from cerebrospinal fluid (CSF) specimens. These assays target the phoB gene for the species detection (Hi-phoB) and serotype-specific genes in region II of the capsule biosynthesis locus (Hi-abf and Hi-cde), identified through comparative analysis of Hi and non-Hi whole-genome sequences. The lower limit of detection (LLD) is 293 CFU/mL for the Hi-phoB assay and ranged from 11 to 130 CFU/mL for the triplex serotyping assays. Using culture as a reference method, the sensitivity and specificity of Hi-phoB, Hi-abf, and Hi-cde were 100%. Triplex serotyping assays also showed 100% agreement for each serotype compared to their corresponding monoplex serotyping assay. These highly sensitive and specific drt-PCR assays do not require DNA extraction and thereby reduce the time, cost, and handling required to process CSF specimens. Furthermore, triplex drt-PCR assays combine the detection of three serotypes in a single reaction, further improving testing efficiency, which is critical for laboratories that process high volumes of Hi specimens for surveillance and diagnostic purposes.

Improved Genomic Identification, Clustering, and Serotyping of Shiga Toxin-Producing Escherichia coli Using Cluster/Serotype-Specific Gene Markers.

Shiga toxin-producing Escherichia coli (STEC) have more than 470 serotypes. The well-known STEC O157:H7 serotype is a leading cause of STEC infections in humans. However, the incidence of non-O157:H7 STEC serotypes associated with foodborne outbreaks and human infections has increased in recent years. Current detection and serotyping assays are focusing on O157 and top six (“Big six”) non-O157 STEC serogroups. In this study, we performed phylogenetic analysis of nearly 41,000 publicly available STEC genomes representing 460 different STEC serotypes and identified 19 major and 229 minor STEC clusters. STEC cluster-specific gene markers were then identified through comparative genomic analysis. We further identified serotype-specific gene markers for the top 10 most frequent non-O157:H7 STEC serotypes. The cluster or serotype specific gene markers had 99.54% accuracy and more than 97.25% specificity when tested using 38,534 STEC and 14,216 non-STEC E. coli genomes, respectively. In addition, we developed a freely available in silico serotyping pipeline named STECFinder that combined these robust gene markers with established E. coli serotype specific O and H antigen genes and stx genes for accurate identification, cluster determination and serotyping of STEC. STECFinder can assign 99.85% and 99.83% of 38,534 STEC isolates to STEC clusters using assembled genomes and Illumina reads respectively and can simultaneously predict stx subtypes and STEC serotypes. Using shotgun metagenomic sequencing reads of STEC spiked food samples from a published study, we demonstrated that STECFinder can detect the spiked STEC serotypes, accurately. The cluster/serotype-specific gene markers could also be adapted for culture independent typing, facilitating rapid STEC typing. STECFinder is available as an installable package (https://github.com/LanLab/STECFinder) and will be useful for in silico STEC cluster identification and serotyping using genome data.

VPsero: Rapid Serotyping of Vibrio parahaemolyticus Using Serogroup-Specific Genes Based on Whole-Genome Sequencing Data.

Vibrio parahaemolyticus has emerged as a significant enteropathogen in human and marine habitats worldwide, notably in regions where aquaculture products constitute a major nutritional source. It is a growing cause of diseases including gastroenteritis, wound infections, and septicemia. Serotyping assays use commercially available antisera to identify V. parahaemolyticus strains, but this approach is limited by high costs, complicated procedures, cross-immunoreactivity, and often subjective interpretation. By leveraging high-throughput sequencing technologies, we developed an in silico method based on comparison of gene clusters for lipopolysaccharide (LPSgc) and capsular polysaccharide (CPSgc) by firstly using the unique-gene strategy. The algorithm, VPsero, which exploits serogroup-specific genes as markers, covers 43 K and all 12 O serogroups in serotyping assays. VPsero is capable of predicting serotypes from assembled draft genomes, outputting LPSgc/CPSgc sequences, and recognizing possible novel serogroups or populations. Our tool displays high specificity and sensitivity in prediction toward V. parahaemolyticus strains, with an average sensitivity in serogroup prediction of 0.910 for O and 0.961 for K serogroups and a corresponding average specificity of 0.990 for O and 0.998 for K serogroups.

Development of a Multiplex-PCR Serotyping Assay for Characterizing Legionella pneumophila Serogroups Based on the Diversity of Lipopolysaccharide Biosynthetic Loci.

Legionella pneumophila, which is the main cause of Legionnaires' disease, comprises at least 15 serogroups (SGs). We show here the diversity of lipopolysaccharide biosynthetic loci among serogroups and describe the development of a PCR serotyping assay for 15 SGs based on the sequences of LPS biosynthetic loci. Using this multiplex-PCR (M-PCR) system, serogroups were detected using primers that specifically amplify the sequences of SG1, SG2, SG5, SG7, SG8, SG9, SG11, SG13, SG3/15, and SG6/12. When PCR products of the expected sizes were not detected, we used primers that identified SG4/10/14. The PCR serotyping system specifically amplified the sequences corresponding to SGs of 238 L. pneumophila strains. This method will be very useful for conducting epidemiological studies and investigating outbreak of Legionnaires' disease.

The Distribution of Several Genomic Virulence Determinants Does Not Corroborate the Established Serotyping Classification of Bacillus thuringiensis.

Bacillus thuringiensis, commonly referred to as Bt, is an object of the lasting interest of microbiologists due to its highly effective insecticidal properties, which make Bt a prominent source of biologicals. To categorize the exuberance of Bt strains discovered, serotyping assays are utilized in which flagellin serves as a primary seroreactive molecule. Despite its convenience, this approach is not indicative of Bt strains’ phenotypes, neither it reflects actual phylogenetic relationships within the species. In this respect, comparative genomic and proteomic techniques appear more informative, but their use in Bt strain classification remains limited. In the present work, we used a bottom-up proteomic approach based on fluorescent two-dimensional difference gel electrophoresis (2D-DIGE) coupled with liquid chromatography/tandem mass spectrometry(LC-MS/MS) protein identification to assess which stage of Bt culture, vegetative or spore, would be more informative for strain characterization. To this end, the proteomic differences for the israelensis-attributed strains were assessed to compare sporulating cultures of the virulent derivative to the avirulent one as well as to the vegetative stage virulent bacteria. Using the same approach, virulent spores of the israelensis strain were also compared to the spores of strains belonging to two other major Bt serovars, namely darmstadiensis and thuringiensis. The identified proteins were analyzed regarding the presence of the respective genes in the 104 Bt genome assemblies available at open access with serovar attributions specified. Of 21 proteins identified, 15 were found to be encoded in all the present assemblies at 67% identity threshold, including several virulence factors. Notable, individual phylogenies of these core genes conferred neither the serotyping nor the flagellin-based phylogeny but corroborated the reconstruction based on phylogenomics approaches in terms of tree topology similarity. In its turn, the distribution of accessory protein genes was not confined to the existing serovars. The obtained results indicate that neither gene presence nor the core gene sequence may serve as distinctive bases for the serovar attribution, undermining the notion that the serotyping system reflects strains’ phenotypic or genetic similarity. We also provide a set of loci, which fit in with the phylogenomics data plausibly and thus may serve for draft phylogeny estimation of the novel strains.

Development of a Luminex microbead-based serotyping assay for Glaesserella parasuis

Glaesserella parasuis consists of 15 serovars with some of them highly virulent and some of them avirulent. As killed vaccines do not provide crossprotection across serovars, serotyping is of importance. Serotyping, previously done by gel diffusion, is now done by multiplex PCR followed by electrophoresis. Accurately differentiating 15 serovars by electrophoresis is problematic. To overcome this problem, a Luminex microbead-based multiplex assay was used to differentiate the serovars. The assay consisted of a multiplex PCR assay followed by hybridisation to microbeads which were then analysed on a Luminex machine. The newly developed assay was compared to the multiplex serotyping PCR and the gel diffusion/indirect haemagglutination assay (GD/IHA). The microbead-based assay worked very well for the 15 reference strains but when used on the 74 Australian field strains displayed some problems. The main problems were with the eight out of nine serovar 4 field isolates and the five serovar 7 and three serovar 14 field isolates. While the microbead-based assay could differentiate between the serovar 5 and 12 reference strains, which the serovar multiplex PCR could not, all four field isolates identified by GD/IHA as serovar 12 were identified as serovar 5 by the microbead-based assay. Serovar 4 has been noted to have a high diversity especially among strains from different countries. Our work clearly shows that the diversity of strains at both the national and the international level has to be taken into account when developing diagnostic assays.

PCR-based detection and serovar identification of Salmonella in retail meat collected from wet markets in Metro Manila, Philippines.

This study aimed to detect Salmonella from retail meat collected from nine wet markets in Metro Manila, and identify the subtypes of Salmonella isolates using molecular serotyping assays from previously developed primers. Of the 720 collected meat samples, 57.64% were found to be Salmonella-contaminated. The most predominant serogroup was Salmonella O:3, and Salmonella serogroups O:4, O:6,7, O:8, O:9, and undetermined serogroups were also found. Most frequently detected isolates in bovine meat were S. 3:e,h:1,6 (putative identity: S. Anatum) and S: 4:e,h:1,2 (putative identity: S. Saintpaul), in porcine meat was S. 3:e,h:1,6 (putative identity: S. Anatum), and S. 8:i:z6 (putative identity: S. Kentucky) was common in poultry products. This study also demonstrated retail meat samples were contaminated with multiple Salmonella serogroups and serovars. This is the first Philippine study that utilized PCR-based assays to characterize Salmonella isolates down to a serovar level and provides baseline information regarding Salmonella prevalence and serovar distribution in retail meat. Molecular serotyping performed in this study can be used as an alternative approach to traditional serotyping in surveillance of Salmonella in the Philippines since the latter is expensive, time-consuming, and requires skilled technicians.

Production of foot-and-mouth disease virus SAT2 VP1 protein

The seven serotypes of foot-and-mouth disease virus (FMDV) differ on the surface exposed regions on the VP1, 2 and 3 proteins. Amongst the three, the VP1 protein has been produced the most for use in serotyping assays for some of the Euro-Asian serotypes. In this study the VP1 protein of the FMDV SAT2/ZIM/7/83 was expressed in Escherichia coli BL21 cells in Luria broth and EnPresso® B media in shake flasks. Production was further developed and the VP1 protein was produced at 2.15 g L−1 in fed-batch fermentations at 2 L scale. The protein formed insoluble inclusion bodies that were isolated, denatured and refolded. When tested in ELISA, the protein was found to be highly reactive with serum from a SAT2 vaccinated guinea pig, and not reactive to SAT1 and SAT3 antisera. These results open avenues to evaluate recombinantly expressed VP1 proteins for differentiation of the three Southern African Territories serotypes of FMDV that co-occur in Southern and East Africa. In addition, this could mitigate the need for employing virus as reagent, or having to raise reagent antibodies.

Development for Clinical Use of a Multiplexed Immunoassay Using Sputum Samples for Streptococcus pneumoniae: a Non-Culture-Based Approach for Serotype-Specific Detection.

The multiplexed immunoassay (MIA) is an automated, monoclonal antibody-based serotyping assay that uses culture lysates of Streptococcus pneumoniae This study describes the development and validation of applying MIA directly to sputum samples for the serotype-specific detection of S. pneumoniae Sputum optimization involved liquefaction and fractionation. The subjects included 173 adult patients from whom both pneumococcal isolates cultured from sputum samples and the corresponding sputum samples were available at the Korea University Hospital from March 2012 to June 2015. Pneumococcal lysates and the sputum fraction were separately evaluated by MIA with a set A reaction to identify 27 serotypes (24 vaccine serotypes and serotypes 6C, 6D, and 11E). MIA results were validated by multiplex PCR (mPCR). Among the 173 patients analyzed, the pneumococcal isolate MIA detected a single set A serotype in 104 patients, and the corresponding sputum MIA showed concordant results with additional multiple serotypes in 21 patients. For the remaining 69 patients whose pneumococcal isolates were not determined to be set A serotypes by the pneumococcal isolate MIA, the corresponding sputum MIA identified additional set A serotypes (single serotypes, n = 17; multiple serotypes, n = 4). Serotypes 3 and 11A/D/F were the most commonly detected serotypes in both the pneumococcal isolate and sputum MIA analyses. However, serotype 8 was the most prevalent serotype detected only by the sputum MIA. The results of mPCR, performed for validation, showed a high concordance with the results of the sputum MIA. In conclusion, MIA using sputum samples enables the accurate, rapid, direct, and serotype-specific detection of S. pneumoniae, which may improve postvaccination serotype surveillance.

An alarmingly high nasal carriage rate of Streptococcus pneumoniae serotype 19F non-susceptible to multiple beta-lactam antimicrobials among Vietnamese children

BackgroundUnderstanding the relationship between serotype epidemiology and antimicrobial susceptibility of Streptococcus pneumoniae is essential for the effective introduction of pneumococcal conjugate vaccines (PCVs) and control of antimicrobial-resistant pneumococci.MethodsWe conducted a community-based study in Nha Trang, central Vietnam, to clarify the serotype distribution and pattern of S. pneumoniae antimicrobial susceptibility in children under 5 years of age and to identify risk factors for carrying antimicrobial-resistant strains. Nasopharyngeal swabs collected from children with acute respiratory infections (ARIs) hospitalized between April 7, 2008, and March 30, 2009, and from healthy children randomly selected in July 2008 were subjected to bacterial culture. Minimum inhibitory concentrations (MICs) against S. pneumoniae were determined, and multiplex-polymerase chain reaction (PCR) serotyping assays were performed. Logistic regression was applied to identify risk factors.ResultsWe collected 883 samples from 331 healthy children and 552 ARI cases; S. pneumoniae was isolated from 95 (28.7%) healthy children and 202 (36.6%) ARI cases. Age and daycare attendance were significantly associated with pneumococcal carriage. In total, 18.0, 25.8 and 75.6% of the isolates had high MICs for penicillin (≥4 μg/ml), cefotaxime (≥2 μg/ml) and meropenem (≥0.5 μg/ml), respectively. The presence of pneumococci non-susceptible to multiple beta-lactams was significantly associated with serotype 19F (Odds Ratio: 4.23) and daycare attendance (Odds Ratio: 2.56) but not ARIs, age or prior antimicrobial use. The majority of isolates non-susceptible to multiple beta-lactams (90%) were PCV13 vaccine serotypes.ConclusionsS. pneumoniae serotype 19F isolates non-susceptible to multiple beta-lactams are widely prevalent among Vietnamese children. Vaccine introduction is expected to significantly increase drug susceptibility.

Source and Relevance of the BK Polyomavirus Genotype for Infection After Kidney Transplantation.

BackgroundBK polyomavirus (BKPyV)–associated nephropathy (BKPyVAN) is a major threat for kidney transplant recipients (KTRs). The role of specific BKPyV genotypes/serotypes in development of BKPyVAN is poorly understood. Pretransplantation serotyping of kidney donors and recipients and posttransplantation genotyping of viremic recipients, could reveal the clinical relevance of specific BKPyV variants.MethodsA retrospective cohort of 386 living kidney donor-recipient pairs was serotyped before transplantation against BKPyV genotype I–IV viral capsid protein 1 antigen, using a novel BKPyV serotyping assay. Replicating BKPyV isolates in viremic KTRs after transplantation were genotyped using real-time polymerase chain reaction and confirmed by means of sequencing. BKPyV serotype and genotype data were used to determine the source of infection and analyze the risk of viremia and BKPyVAN.ResultsDonor and recipient BKPyV genotype and serotype distribution was dominated by genotype I (>80%), especially Ib, over II, III and IV. Donor serotype was significantly correlated with the replicating genotype in viremic KTRs (P < .001). Individual donor and recipient serotype, serotype (mis)matching and the recipient replicating BKPyV genotype were not associated with development of viremia or BKPyVAN after transplantation.ConclusionsBKPyV donor and recipient serotyping and genotyping indicates the donor origin of replicating BKPyV in viremic KTRs but provides no evidence for BKPyV genotype–specific virulence.

Streptococcus infantis, Streptococcus mitis, and Streptococcus oralis Strains With Highly Similar cps5 Loci and Antigenic Relatedness to Serotype 5 Pneumococci.

Streptococcus pneumoniae is a highly impactful bacterial pathogen on a global scale. The principal pneumococcal virulence factor and target of effective vaccines is its polysaccharide capsule, of which there are many structurally distinct forms. Here, we describe four distinct strains of three Mitis group commensal species (Streptococcus infantis, Streptococcus mitis, and Streptococcus oralis) recovered from upper respiratory tract specimens from adults in Kenya and the United States that were PCR-positive for the pneumococcal serotype 5 specific gene, wzy5. For each of the four strains, the 15 genes comprising the capsular polysaccharide biosynthetic gene cluster (cps5) shared the same order found in serotype 5 pneumococci, and each of the serotype 5-specific genes from the serotype 5 pneumococcal reference strain shared 76–99% sequence identity with the non-pneumococcal counterparts. Double-diffusion experiments demonstrated specific reactivity of the non-pneumococcal strains with pneumococcal serotype 5 typing sera. Antiserum raised against S. mitis strain KE67013 specifically reacted with serotype 5 pneumococci for a positive Quellung reaction and stimulated serotype 5 specific opsonophagocytic killing of pneumococci. Four additional commensal strains, identified using PCR serotyping assays on pharyngeal specimens, revealed loci highly homologous to those of pneumococci of serotypes 12F, 15A, 18C, and 33F. These data, in particular the species and strain diversity shown for serotype 5, highlight the existence of a broad non-pneumococcal species reservoir in the upper respiratory tract for the expression of capsular polysaccharides that are structurally related or identical to those corresponding to epidemiologically significant serotypes. Very little is known about the genetic and antigenic capsular diversity among the vast array of commensal streptococcal strains that represent multiple diverse species. The discovery of serotype 5 strains within three different commensal species suggests that extensive capsular serologic overlap exists between pneumococci and other members of the diverse Mitis group. These findings may have implications for our current understanding of naturally acquired immunity to S. pneumoniae and pneumococcal serotype distributions in different global regions. Further characterization of commensal strains carrying homologs of serotype-specific genes previously thought to be specific for pneumococci of known serotypes may shed light on the evolution of these important loci.

Identification and characterization of epizootic hemorrhagic disease virus serotype 6 in cattle co-infected with bluetongue virus in Trinidad, West Indies

Epizootic hemorrhagic disease virus (EHDV) is an economically important virus that can cause severe clinical disease in deer and to a lesser extent cattle. This study set out to determine and characterize which EHDV serotypes were circulating in Trinidad. Serum and whole blood samples were collected monthly for six months from a cohort of cattle imported to Trinidad from the USA. Results revealed that all the cattle seroconverted to EHDV within six months of their arrival, with EHDV RNA being detected in the samples just prior to antibodies, as expected. Serotyping assays revealed that a single serotype (EHDV-6) was circulating in the cattle. Sequencing of the surface viral protein (VP2) of EHDV-6, followed by phylogenetic analysis, revealed that the Trinidad EHDV-6 strain was closely related to EHDV-6 viruses found in Guadeloupe (2010), Martinique (2010) and USA (2006), with 96–97.2% nucleotide identity. The Trinidad EHDV-6 VP-2 shared 97.2% identity with the Australian EHDV-6 prototype strain, classifying it within the eastern topotype clade. Bayesian coalescent analysis support Australia as the most probable source for the EHDV-6 VP2 sequences in the Americas and Caribbean region and suggests that the they diverged from the Australian prototype strain around 1966 (95% HPD 1941–1979).

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

BackgroundThe 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. ObjectivesTo develop and evaluate a genotype-specific IgG antibody-based BKPyV serotyping assay, in order to classify kidney transplant donors and recipients accordingly. Study designVP1 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. ResultsSeroreactivity 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. ConclusionsDespite 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.