and specificity were 62.3% (95% CI, 57.9% to 66.6%) and 98.2% (CI, 97.5% to 98.7%), respectively. The positive and negative likelihood ratios were 34.5 (CI, 23.8 to 45.2) and 0.38 (CI, 0.34 to 0.43), respectively. Sensitivity estimates were highly heterogeneous, which was partially explained by lower sensitivity in adults (53.9% [CI, 47.9% to 59.8%]) than in children (66.6% [CI, 61.6% to 71.7%]) and a higher sensitivity for influenza A (64.6% [CI, 59.0% to 70.1%) than for influenza B (52.2% [CI, 45.0% to 59.3%). Limitation: Incomplete reporting limited the ability to assess the effect of important factors, such as specimen type and duration of influenza symptoms, on diagnostic accuracy. Conclusion: Influenza can be ruled in but not ruled out through the use of RIDTs. Sensitivity varies across populations, but it is higher in children than in adults and for influenza A than for influenza B. Primary Funding Source: Canadian Institutes of Health Research.

Accuracy of Rapid Influenza Diagnostic Tests

SUMMARY Extraintestinal pathogenic Escherichia coli (ExPEC) strains are responsible for a majority of human extraintestinal infections globally, resulting in enormous direct medical and social costs. ExPEC strains are comprised of many lineages, but only a subset is responsible for the vast majority of infections. Few systematic surveillance systems exist for ExPEC. To address this gap, we systematically reviewed and meta-analyzed 217 studies (1995 to 2018) that performed multilocus sequence typing or whole-genome sequencing to genotype E. coli recovered from extraintestinal infections or the gut. Twenty major ExPEC sequence types (STs) accounted for 85% of E. coli isolates from the included studies. ST131 was the most common ST from 2000 onwards, covering all geographic regions. Antimicrobial resistance-based isolate study inclusion criteria likely led to an overestimation and underestimation of some lineages. European and North American studies showed similar distributions of ExPEC STs, but Asian and African studies diverged. Epidemiology and population dynamics of ExPEC are complex; summary proportion for some STs varied over time (e.g., ST95), while other STs were constant (e.g., ST10). Persistence, adaptation, and predominance in the intestinal reservoir may drive ExPEC success. Systematic, unbiased tracking of predominant ExPEC lineages will direct research toward better treatment and prevention strategies for extraintestinal infections.

Global Extraintestinal Pathogenic Escherichia coli (ExPEC) Lineages.

The human microbiome includes trillions of bacteria, many of which play a vital role in host physiology. Numerous studies have now detected bacterial DNA in first-pass meconium and amniotic fluid samples, suggesting that the human microbiome may commence in utero. However, these data have remained contentious due to underlying contamination issues. Here, we have used a previously described method for reducing contamination in microbiome workflows to determine if there is a fetal bacterial microbiome beyond the level of background contamination. We recruited 50 women undergoing non-emergency cesarean section deliveries with no evidence of intra-uterine infection and collected first-pass meconium and amniotic fluid samples. Full-length 16S rRNA gene sequencing was performed using PacBio SMRT cell technology, to allow high resolution profiling of the fetal gut and amniotic fluid bacterial microbiomes. Levels of inflammatory cytokines were measured in amniotic fluid, and levels of immunomodulatory short chain fatty acids (SCFAs) were quantified in meconium. All meconium samples and most amniotic fluid samples (36/43) contained bacterial DNA. The meconium microbiome was dominated by reads that mapped to Pelomonas puraquae. Aside from this species, the meconium microbiome was remarkably heterogeneous between patients. The amniotic fluid microbiome was more diverse and contained mainly reads that mapped to typical skin commensals, including Propionibacterium acnes and Staphylococcus spp. All meconium samples contained acetate and propionate, at ratios similar to those previously reported in infants. P. puraquae reads were inversely correlated with meconium propionate levels. Amniotic fluid cytokine levels were associated with the amniotic fluid microbiome. Our results demonstrate that bacterial DNA and SCFAs are present in utero, and have the potential to influence the developing fetal immune system.

/pdf/the-not-so-sterile-womb-evidence-that-the-human-fetus-is-4jkt7radr5.pdf

The Not-so-Sterile Womb: Evidence That the Human Fetus Is Exposed to Bacteria Prior to Birth

Fusarium ranks as one of the world’s most economically destructive and species-rich groups of mycotoxigenic plant pathogens (Aoki et al. 2014). These ubiquitous molds produce a plethora of toxic secondary metabolites, such as trichothecenes, zearalenone, fumonisins, and enniatins, which pose a significant threat to agricultural biosecurity, food safety, and plant, human and animal health (Marasas et al.1984). Fusarial-induced diseases of virtually every economically important plant cost the global agricultural economy multi-billion euro losses annually. Moreover, phylogenetically diverse fusaria, including plant pathogens (Short et al. 2011), cause infections in humans, with those involving the cornea and nails being the most common (Chang et al.2006 and references therein). Because fusaria are broadly resistant to the spectrum of antifungals currently available, disseminated infections in patients who are artificially immunosuppressed or immunocompromised and severely neutropenic are typically fatal (Balajee et al.2009). The likely reservoir of nosocomial fusarioses is the plumbing system, which has been shown to harbor the most common human opportunistic fusaria (Kuchar 1996; Short et al.2011). Accurate identification of the etiological and/or toxigenic agent is central to disease management and infection control (Wingfield et al. 2012). Thus, the primary focus of this mini-review is to provide a contemporary guide to the following three web-accessible resources for DNA sequence-based identification of Fusarium: FUSARIUM-ID (http://isolate.fusariumdb.org/; Geiser et al.2004; Park et al. 2010), Fusarium MLST (http://www.cbs.knaw.nl/fusarium/; O’Donnell et al.2010), and NCBI GenBank (http://www.ncbi.nlm.nih.gov/). The following brief overview of Fusarium phylogenetic diversity is provided as background information for the sections on DNA sequence-based identification.

/pdf/dna-sequence-based-identification-of-fusarium-current-status-191wairrla.pdf

DNA sequence-based identification of Fusarium : Current status and future directions

The purpose of this review is to present the most common and emerging DNA-based methods used to generate data for biodiversity and biomonitoring studies. As environmental assessment and monitoring programmes may require biodiversity information at multiple levels, we pay particular attention to the DNA metabarcoding method and discuss a number of bioinformatic tools and considerations for producing DNA-based indicators using operational taxonomic units (OTUs), taxa at a variety of ranks and community composition. By developing the capacity to harness the advantages provided by the newest technologies, investigators can "scale up" by increasing the number of samples and replicates processed, the frequency of sampling over time and space, and even the depth of sampling such as by sequencing more reads per sample or more markers per sample. The ability to scale up is made possible by the reduced hands-on time and cost per sample provided by the newest kits, platforms and software tools. Results gleaned from broad-scale monitoring will provide opportunities to address key scientific questions linked to biodiversity and its dynamics across time and space as well as being more relevant for policymakers, enabling science-based decision-making, and provide a greater socio-economic impact. As genomic approaches are continually evolving, we provide this guide to methods used in biodiversity genomics.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/mec.14478

Scaling up: A guide to high-throughput genomic approaches for biodiversity analysis.

Recent advancements in next-generation sequencing (NGS) have provided the foundation for modern studies into the composition of microbial communities. The use of these NGS methods allows for the detection and identification of (‘difficult-to-culture’) microorganisms using a culture-independent strategy. In the field of routine clinical diagnostics however, the application of NGS is currently limited to microbial strain typing for epidemiological purposes only, even though the implementation of NGS for microbial community analysis may yield clinically important information. This lack of NGS implementation is due to many different factors, including issues relating to NGS method standardization and result reproducibility. In this review article, the authors provide a general introduction to the most widely used NGS methods currently available (i.e., targeted amplicon sequencing and shotgun metagenomics) and the strengths and weaknesses of each method is discussed. The focus of the publication then shifts toward 16S rRNA gene NGS methods, which are currently the most cost-effective and widely used NGS methods for research purposes, and are therefore more likely to be successfully implemented into routine clinical diagnostics in the short term. In this respect, the experimental pitfalls and biases created at each step of the 16S rRNA gene NGS workflow are explained, as well as their potential solutions. Finally, a novel diagnostic microbiota profiling platform (‘MYcrobiota’) is introduced, which was developed by the authors by taking into consideration the pitfalls, biases, and solutions explained in this article. The development of the MYcrobiota, and future NGS methodologies, will help pave the way toward the successful implementation of NGS methodologies into routine clinical diagnostics.

/pdf/understanding-and-overcoming-the-pitfalls-and-biases-of-next-1o2x9it3rc.pdf

Understanding and overcoming the pitfalls and biases of next-generation sequencing (NGS) methods for use in the routine clinical microbiological diagnostic laboratory.

Multilocus sequence typing (MLST) is a widely used system for typing microorganisms by sequence analysis of housekeeping genes. The main advantage of MLST in comparison to other typing techniques is the unambiguity and transferability of sequence data. However, a main disadvantage is the high cost of DNA sequencing. Here we introduce a high-throughput MLST (HiMLST) method that employs next-generation sequencing (NGS) technology (Roche 454), to generate large quantities of high-quality MLST data at low costs. The HiMLST protocol consists of two steps. In the first step MLST target genes are amplified by PCR in multi-well plates. During this PCR the amplicons of each bacterial isolate are provided with a unique DNA barcode, the multiplex identifier (MID). In the second step all amplicons are pooled and sequenced in a single NGS-run. The MLST profile of each individual isolate can be retrieved easily using its unique MID. With HiMLST we have profiled 575 isolates of Legionella pneumophila, Staphylococcus aureus, Pseudomonas aeruginosa and Streptococcus pneumoniae in mixed species HiMLST experiments. In conclusion, the introduction of HiMLST paves the way for a broad employment of the MLST as a high-quality and cost-effective method for typing microbial species.

/pdf/high-throughput-multilocus-sequence-typing-bringing-4nuaymuif3.pdf

High-throughput multilocus sequence typing: bringing molecular typing to the next level.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1469-0691.2006.01562.x

Legionnaires’ disease and gardening

https://www.clinicalmicrobiologyandinfection.com/article/S1198-743X(14)60939-3/pdf

Ruud Jansen

Papers

Understanding and overcoming the pitfalls and biases of next-generation sequencing (NGS) methods for use in the routine clinical microbiological diagnostic laboratory.

High-throughput multilocus sequence typing: bringing molecular typing to the next level.

Legionnaires’ disease and gardening

Genotypic comparison of clinical Legionella isolates and patient-related environmental isolates in The Netherlands, 2002-2006

Outbreak detection and secondary prevention of Legionnaires' disease: a national approach.