Dimitrov, B. | Search bioRxiv

bioRxiv2018

FLASH: A next-generation CRISPR diagnostic for multiplexed detection of antimicrobial resistance sequences

The growing prevalence of deadly microbes with resistance to previously life-saving drug therapies is a dire threat to human health. Detection of low abundance pathogen sequences remains a challenge for metagenomic Next Generation Sequencing (NGS). We introduce FLASH (Finding Low Abundance Sequences by Hybridization), a next-generation CRISPR/Cas9 diagnostic method that takes advantage of the efficiency, specificity and flexibility of Cas9 to enrich for a programmed set of sequences. FLASH-NGS achieves up to 5 orders of magnitude of enrichment and sub-attomolar gene detection with minimal background. We provide an open-source software tool (FLASHit) for guide RNA design. Here we applied it to detection of antimicrobial resistance genes in respiratory fluid and dried blood spots, but FLASH-NGS is applicable to all areas that rely on multiplex PCR.

microbiology↗

bioRxiv2018

Etiology of fever in Ugandan children: identification of microbial pathogens using metagenomic next-generation sequencing and IDseq, a platform for unbiased metagenomic analysis

BackgroundFebrile illness is a major burden in African children, and non-malarial causes of fever are uncertain. We built and employed IDseq, a cloud-based, open access, bioinformatics platform and service to identify microbes from metagenomic next-generation sequencing of tissue samples. In this pilot study, we evaluated blood, nasopharyngeal, and stool specimens from 94 children (aged 2-54 months) with febrile illness admitted to Tororo District Hospital, Uganda.\n\nResultsThe most common pathogens identified were Plasmodium falciparum (51.1% of samples) and parvovirus B19 (4.4%) from blood; human rhinoviruses A and C (40%), respiratory syncytial virus (10%), and human herpesvirus 5 (10%) from nasopharyngeal swabs; and rotavirus A (50% of those with diarrhea) from stool. Among other potential pathogens, we identified one novel orthobunyavirus, tentatively named Nyangole virus, from the blood of a child diagnosed with malaria and pneumonia, and Bwamba orthobunyavirus in the nasopharynx of a child with rash and sepsis. We also identified two novel human rhinovirus C species.\n\nConclusionsThis exploratory pilot study demonstrates the utility of mNGS and the IDseq platform for defining the molecular landscape of febrile infectious diseases in resource limited areas. These methods, supported by a robust data analysis and sharing platform, offer a new tool for the surveillance, diagnosis, and ultimately treatment and prevention of infectious diseases.

genomics↗