Sample pooling methods for efficient pathogen screening: Practical implications.

Due to the large number of negative tests, individually screening large populations for rare pathogens can be wasteful and expensive. Sample pooling methods improve the efficiency of large-scale pathogen screening campaigns by reducing the number of tests and reagents required to accurately categorize positive and negative individuals. Such methods rely on group testing theory which mainly focuses on minimizing the total number of tests; however, many other practical concerns and tradeoffs must be considered when choosing an appropriate method for a given set of circumstances. Here we use computational simulations to determine how several theoretical approaches compare in terms of (a) the number of tests, to minimize costs and save reagents, (b) the number of sequential steps, to reduce the time it takes to complete the assay, (c) the number of samples per pool, to avoid the limits of detection, (d) simplicity, to reduce the risk of human error, and (e) robustness, to poor estimates of the number of positive samples. We found that established methods often perform very well in one area but very poorly in others. Therefore, we introduce and validate a new method which performs fairly well across each of the above criteria making it a good general use approach.

Variant site strain typer (VaST): efficient strain typing using a minimal number of variant genomic sites.

BACKGROUND: Targeted PCR amplicon sequencing (TAS) techniques provide a sensitive, scalable, and cost-effective way to query and identify closely related bacterial species and strains. Typically, this is accomplished by targeting housekeeping genes that provide resolution down to the family, genera, and sometimes species level. Unfortunately, this level of resolution is not sufficient in many applications where strain-level identification of bacteria is required (biodefense, forensics, clinical diagnostics, and outbreak investigations). Adding more genomic targets will increase the resolution, but the challenge is identifying the appropriate targets. VaST was developed to address this challenge by finding the minimum number of targets that, in combination, achieve maximum strain-level resolution for any strain complex. The final combination of target regions identified by the algorithm produce a unique haplotype for each strain which can be used as a fingerprint for identifying unknown samples in a TAS assay. VaST ensures that the targets have conserved primer regions so that the targets can be amplified in all of the known strains and it also favors the inclusion of targets with basal variants which makes the set more robust when identifying previously unseen strains. RESULTS: We analyzed VaST's performance using a number of different pathogenic species that are relevant to human disease outbreaks and biodefense. The number of targets required to achieve full resolution ranged from 20 to 88% fewer sites than what would be required in the worst case and most of the resolution is achieved within the first 20 targets. We computationally and experimentally validated one of the VaST panels and found that the targets led to accurate phylogenetic placement of strains, even when the strains were not a part of the original panel design. CONCLUSIONS: VaST is an open source software that, when provided a set of variant sites, can find the minimum number of sites that will provide maximum resolution of a strain complex, and it has many different run-time options that can accommodate a wide range of applications. VaST can be an effective tool in the design of strain identification panels that, when combined with TAS technologies, offer an efficient and inexpensive strain typing protocol.

Differentiating Botulinum Neurotoxin-Producing Clostridia with a Simple, Multiplex PCR Assay.

Diverse members of the genus Clostridium produce botulinum neurotoxins (BoNTs), which cause a flaccid paralysis known as botulism. While multiple species of clostridia produce BoNTs, the majority of human botulism cases have been attributed to Clostridium botulinum groups I and II. Recent comparative genomic studies have demonstrated the genomic diversity within these BoNT-producing species. This report introduces a multiplex PCR assay for differentiating members of C. botulinum group I, C. sporogenes, and two major subgroups within C. botulinum group II. Coding region sequences unique to each of the four species/subgroups were identified by in silico analyses of thousands of genome assemblies, and PCR primers were designed to amplify each marker. The resulting multiplex PCR assay correctly assigned 41 tested isolates to the appropriate species or subgroup. A separate PCR assay to determine the presence of the ntnh gene (a gene associated with the botulinum neurotoxin gene cluster) was developed and validated. The ntnh gene PCR assay provides information about the presence or absence of the botulinum neurotoxin gene cluster and the type of gene cluster present (ha positive [ha+] or orfX+). The increased availability of whole-genome sequence data and comparative genomic tools enabled the design of these assays, which provide valuable information for characterizing BoNT-producing clostridia. The PCR assays are rapid, inexpensive tests that can be applied to a variety of sample types to assign isolates to species/subgroups and to detect clostridia with botulinum neurotoxin gene (bont) clusters.IMPORTANCE Diverse clostridia produce the botulinum neurotoxin, one of the most potent known neurotoxins. In this study, a multiplex PCR assay was developed to differentiate clostridia that are most commonly isolated in connection with human botulism cases: C. botulinum group I, C. sporogenes, and two major subgroups within C. botulinum group II. Since BoNT-producing and nontoxigenic isolates can be found in each species, a PCR assay to determine the presence of the ntnh gene, which is a universally present component of bont gene clusters, and to provide information about the type (ha+ or orfX+) of bont gene cluster present in a sample was also developed. The PCR assays provide simple, rapid, and inexpensive tools for screening uncharacterized isolates from clinical or environmental samples. The information provided by these assays can inform epidemiological studies, aid with identifying mixtures of isolates and unknown isolates in culture collections, and confirm the presence of bacteria of interest.