Pneumococcal Serotype Identification by Capsular Sequence Typing (CST): A Modified Novel Approach for Serotyping Directly in Clinical Samples

As almost 60–70% of Invasive Pneumococcal Disease (IPD) is identified by nonculture methods in Greece, serotyping is of high importance for the better monitoring of pneumococcal serotypes due to the availability of conjugate vaccines. The aim of the study was the modification and direct application of the Capsular Sequence Typing (CST) assay in clinical samples in order to serotype Streptococcus pneumoniae culture-negative, Polymerase Chain Reaction (PCR_-positive samples, followed by CST group specific single-tube PCR assays. A two-step PCR modified assay was applied on a total of 306 samples (such as CSF, blood, pleural and middle ear fluids, isolates) obtained from 283 patients with IPD. The overall performance permits a rapid, accurate and cost-effective method for nonculture pneumococcal serotyping. As the management of IPD is closely related to the continuous monitoring of pneumococcal serotypes, the proposed approach proved to be a valuable tool for the typing and epidemiological monitoring of S. pneumoniae, for the evaluation of the overall impact of vaccination programs in the era of pneumococcal conjugate vaccines, in order to initiate the appropriate vaccination strategy.

Isolation and Molecular Identification of Foot and Mouth Disease Virus Circulating Around Central Ethiopia

Background: Foot and mount disease (FMD) is a highly contagious, economically and politically significant transboundary animal disease which specifically affects all cloven-hoofed animals; cattle, pig, goat, sheep and many wild artiodactyls. Five of the seven (O, A, C, SAT1, SAT2, SAT3 and Asia 1) serotypes of FMD virus (O, A, C, SAT1, SAT2) are endemic in Ethiopia; however, limited information on the current FMDV status and the circulating serotype is available in the country. Therefore, this study was conduct to isolate and molecularly identify the FMD viruses using a panel of virological detection assays. Methods: An outbreak-based cross-sectional study was conducted in Addis Ababa and Bishoftu during 2013 and 2014 to isolate and to molecularly identify the circulating serotype of FMDV. A total of 20 samples were collected from clinically infected cattle and pigs during the outbreak and virus isolation and molecular serotype identification was carried out at the National Veterinary Institute (NVI), Bishoftu Ethiopia. Cells were monitored for cytopathic effects (CPE) daily and frozen when CPE were developed. Serotyping of FMD viruses were made by applying classical PCR from cell cultures showing CPE.Results: All tested samples showed cytopathic effect (CPE) on BHK-21 cell culture and serotype O was identified using PCR. The DNA bands were visualized to the expected sizes.Conclusions: Based on these result, a continuous understanding of the molecular epidemiology of the disease along with the proper vaccination matching for the circulating serotype; O is critical for implementation effective control and prevention programs eventually for the eradication of the disease.

Serotype Distribution and Characteristics of the Minimum Inhibitory Concentrations of Streptococcus pneumoniae Isolated from Pediatric Patients in Kunming, China

Streptococcus pneumoniae (S. pneumoniae) is the main conditional pathogen of acute respiratory infection in infants, children, and older adults worldwide. It was great significant to identify the epidemic characteristics of serotypes and antibiotic susceptibility for the prevention and treatment of S. pneumoniae diseases. This research assessed the serotype distribution and the minimum inhibitory concentrations (MICs) of S. pneumoniae isolated from pediatric patients to provide information on the epidemiology and antibiotic resistance of S. pneumoniae in Kunming, China. A total of 140 S. pneumoniae isolates were collected from pediatric patients at Kunming Children’s Hospital from January 2016 to October 2017. Serotype identification was done by Quellung reaction and multiplex polymerase chain reaction. MICs were determined by E-test. 140 isolates distributed in 13 types of serotypes. The top-three prevalent serotypes were 19F, 19A, and 6B. The immunization coverage rate of 13-valent pneumococcal conjugate vaccine (PCV) was relatively higher and should be introduced into the vaccination program in the region. MIC50 of penicillin, ceftriaxone, and levofloxacin was 1 μg/mL. MIC50 for meropenem and vancomycin was 0.38 μg/mL. MIC90 of penicillin, ceftriaxone, and levofloxacin was 1.5 μg/mL and that of meropenem and vancomycin was 0.5 μg/mL. The MIC90 of erythromycin was > 256 μg/mL. In summary, S. pneumoniae had low resistance rates to penicillin, ceftriaxone, levofloxacin, vancomycin, and meropenem, and these antibiotics could be the first-line agents for children with pneumococcal infections in Kunming.

Risk factors of Dengue fever in urban areas of district Rawalpindi, Pakistan- 2017 (Preprint)

BACKGROUND During August 2017, an increased number of suspected dengue fever cases were reported in the hospitals of district Rawalpindi. A case control study was conducted to measure the extent of the outbreak, to determine the risk factors, and recommend preventive measures. OBJECTIVE To determine the risk factors and recommend control measures. METHODS A case was defined as an acute febrile illness with one or more of the following; retro-orbital pain, headache, rash, myalgia, arthralgia, and hemorrhage, confirmed with ELISA among residents of Rawalpindi district from 30th Aug- 30th Oct. 2017. All ELISA confirmed cases were recruited from the hospital. Age and sex matched controls were selected from the same community. Frequencies, univariate and multivariate analysis was performed using epi Info 7. RESULTS Total 373 cases were recruited. The mean age was 36±2.9 years (range:10-69yeras) and 75% were male. The most affected age group was 21-30 years (AR 40%) followed by 31-40 years (AR 23%). Two deaths were reported (CFR 0.53%). The most frequent signs/symptoms were; fever (100%), myalgia (86%) headache (86%), and retro-orbital pain (73%). Serotype identification carried out in 322 cases and DEN-2 was dominant (34%, n=126). Contact with a confirmed dengue case (OR 4.27, CI: 3.14-5.81, P<0.0001) and stored water in open containers at home (OR 2.04, CI: 1.53-2.73, P <0.0001) and travel to a dengue outbreak area (OR 2.88, CI: 2.12-3.92, P<0.0001) had higher odds while the use of mosquito repellents (OR 0.12, CI 0.09-0.18, P < 0.0001) and regular water supply at home (OR 0.03, CI: 0.02-0.04, P <0.0001) showed a protective effect. Geographical distribution was limited to densely populated areas and all the water samples were tested positive for dengue larvae. CONCLUSIONS Stored water in containers inside houses and subsequent mosquito breeding was the most probable cause of this outbreak. The study led to a recommendation to undertake activities to improve the use of repellents and remove sources of breeding (indoor uncovered stored water).

A Targeted Sequencing Assay for Serotyping Escherichia coli Using AgriSeq Technology

The gold standard method for serotyping Escherichia coli has relied on antisera-based typing of the O- and H-antigens, which is labor intensive and often unreliable. In the post-genomic era, sequence-based assays are potentially faster to provide results, could combine O-serogrouping and H-typing in a single test, and could simultaneously screen for the presence of other genetic markers of interest such as virulence factors. Whole genome sequencing is one approach; however, this method has limited multiplexing capabilities, and only a small fraction of the sequence is informative for subtyping or identifying virulence potential. A targeted, sequence-based assay and accompanying software for data analysis would be a great improvement over the currently available methods for serotyping. The purpose of this study was to develop a high-throughput, molecular method for serotyping E. coli by sequencing the genes that are required for production of O- and H-antigens, as well as to develop software for data analysis and serotype identification. To expand the utility of the assay, targets for the virulence factors, Shiga toxins (stx1, and stx2) and intimin (eae) were included. To validate the assay, genomic DNA was extracted from O-serogroup and H-type standard strains and from Shiga toxin-producing E. coli, the targeted regions were amplified, and then sequencing libraries were prepared from the amplified products followed by sequencing of the libraries on the Ion S5™ sequencer. The resulting sequence files were analyzed via the SeroType Caller™ software for identification of O-serogroup, H-type, and presence of stx1, stx2, and eae. We successfully identified 169 O-serogroups and 41 H-types. The assay also routinely detected the presence of stx1a,c,d (3 of 3 strains), stx2c−e,g (8 of 8 strains), stx2f (1 strain), and eae (6 of 6 strains). Taken together, the high-throughput, sequence-based method presented here is a reliable alternative to antisera-based serotyping methods for E. coli.

Determining the serotype composition of mixed samples of pneumococcus using whole-genome sequencing

Serotyping of Streptococcus pneumoniae is a critical tool in the surveillance of the pathogen and in the development and evaluation of vaccines. Whole-genome DNA sequencing and analysis is becoming increasingly common and is an effective method for pneumococcal serotype identification of pure isolates. However, because of the complexities of the pneumococcal capsular loci, current analysis software requires samples to be pure (or nearly pure) and only contain a single pneumococcal serotype. We introduce a new software tool called SeroCall, which can identify and quantitate the serotypes present in samples, even when several serotypes are present. The sample preparation, library preparation and sequencing follow standard laboratory protocols. The software runs as fast as or faster than existing identification tools on typical computing servers and is freely available under an open source licence at https://github.com/knightjimr/serocall. Using samples with known concentrations of different serotypes as well as blinded samples, we were able to accurately quantify the abundance of different serotypes of pneumococcus in mixed cultures, with 100 % accuracy for detecting the major serotype and up to 86 % accuracy for detecting minor serotypes. We were also able to track changes in serotype frequency over time in an experimental setting. This approach could be applied in both epidemiological field studies of pneumococcal colonization and experimental laboratory studies, and could provide a cheaper and more efficient method for serotyping than alternative approaches.

Evaluation of Molecular Serotyping Assays for Shigella flexneri Directly on Stool Samples

Shigella flexneri is prevalent worldwide and is the most common Shigella species in many countries. At least 19 S. flexneri serotypes exist, and serotype information is important for epidemiologic and vaccine development purposes. We evaluated the performance of real time PCR (PCR) assays for O-antigen modification genes to identify the major serotypes on isolates and direct stool samples. The assays were formulated into two multiplex panels: one panel included gtrII, gtrV, gtrX, oac, and wzx6 to identify S. flexneri serotype 2a, 2b, 3a, 5a, 5b, 6, and X, the other panel included ipaH, gtrI, gtrIc, gtrIV, to confirm Shigella detection and further identify S. flexneri serotype 1a, 1b, 1d, 3b, 4a, 4b, 7a, and 7b. We first evaluated 283 Shigella isolates and PCR serotyping demonstrated 97.0% (95% confidence interval 93.0% - 99.0%) sensitivity and 99.9% (99.9%-100%) specificity compared to conventional serotyping. The assays were then utilized on direct stool specimens. A quantitative detection algorithm was developed with a validation set of 174 Shigella culture positive stool samples, and further tested with a derivation set of 164 samples. The PCR serotyping on stool achieved 93% (89%-96%) sensitivity and 99% (99%-100%) specificity compared to serotyping. Most discrepancies were genotypic-phenotypic discordance, not genotypic failure. These real-time PCR assays provide an efficient and novel tool for S. flexneri serotype identification.

Determining the serotype composition of mixed samples of pneumococcus using whole genome sequencing

ABSTRACTSerotyping of Streptococcus pneumoniae is a critical tool in the surveillance of the pathogen and development and evaluation of vaccines. Whole-genome DNA sequencing and analysis is becoming increasingly common and is an effective method for pneumococcal serotype identification of pure isolates. However, because of the complexities of the pneumococcal capsular loci, current analysis software requires samples to be pure (or nearly pure) and only contain a single pneumococcal serotype. We introduce a new software tool called SeroCall, which can identify and quantitate the serotypes present in samples, even when several serotypes are present. The sample preparation, library preparation and sequencing follow standard laboratory protocols. The software runs as fast or faster than existing identification tools on typical computing servers and is freely available under an open source license at https://github.com/knightjimr/serocall. Using samples with known concentrations of different serotypes as well as blinded samples, we were able to accurately quantify the abundance of different serotypes of pneumococcus in mixed cultures, with 100% accuracy for detecting the major serotype and up to 86% accuracy for detecting minor serotypes. We were also able to track changes in serotype frequency over time in an experimental setting. This approach could be applied in both epidemiologic field studies of pneumococcal colonization as well as in experimental lab studies and could provide a cheaper and more efficient method for serotyping than alternative approaches.