Microfluidics have become an enabling technology for point-of-care and personalized diagnostics. Desirable capabilities of microfluidics-based diagnostic devices include simplicity, portability, low cost and the performance of multiplexed and quantitative measurements, ideally in a high-throughput format. Here we present the multiplexed volumetric bar-chart chip (V-Chip), which integrates all these capabilities in one device. A key feature of the V-Chip is that quantitative results are displayed as bar charts directly on the device—without the need for optical instruments or any data processing or plotting steps. This is achieved by directly linking oxygen production by catalase, which is proportional to the concentration of the analyte, with the displacement of ink along channels on the device. We demonstrate the rapid quantification of protein biomarkers in diverse clinical samples with the V-Chip. The development of the V-Chip thus opens up the possibility of greatly simplified point-of-care and personalized diagnostics.

The sequences reported in this paper have been deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (see URLs) with the Bioproject accession number PRJNA434389 and the NCBI Gene Expression Omnibus (GEO) under accession number GSE113058. ETHICS STATEMENTAll mouse studies were performed in accordance with a protocol (AUP-0318-0016) approved by the Institutional Animal Care and Use Committee at Houston Methodist Research Institute. All studies with human blood and blood components were performed in accordance with a protocol (01-I-N055) approved by the Institutional Review Board for human subjects, National Institute of Allergy and Infectious Diseases. All study volunteers gave written informed consent.

dTo obtain new information about Streptococcus pyogenes intrahost genetic variation during invasive infection, we sequenced the genomes of 2,954 serotype M1 strains recovered from a nonhuman primate experimental model of necrotizing fasciitis. A total of 644 strains (21.8%) acquired polymorphisms relative to the input parental strain. The fabT gene, encoding a transcriptional regulator of fatty acid biosynthesis genes, contained 54.5% of these changes. The great majority of polymorphisms were predicted to deleteriously alter FabT function. Transcriptome-sequencing (RNA-seq) analysis of a wild-type strain and an isogenic fabT deletion mutant strain found that between 3.7 and 28.5% of the S. pyogenes transcripts were differentially expressed, depending on the growth temperature (35°C or 40°C) and growth phase (mid-exponential or stationary phase). Genes implicated in fatty acid synthesis and lipid metabolism were significantly upregulated in the fabT deletion mutant strain. FabT also directly or indirectly regulated central carbon metabolism genes, including pyruvate hub enzymes and fermentation pathways and virulence genes. Deletion of fabT decreased virulence in a nonhuman primate model of necrotizing fasciitis. In addition, the fabT deletion strain had significantly decreased survival in human whole blood and during phagocytic interaction with polymorphonuclear leukocytes ex vivo. We conclude that FabT mutant progeny arise during infection, constitute a metabolically distinct subpopulation, and are less virulent in the experimental models used here. Streptococcus pyogenes is a Gram-positive bacterium that causes a range of diseases in humans, including pharyngitis, superficial and deep skin infections, acute rheumatic fever, poststreptococcal glomerulonephritis, bacteremia, and necrotizing fasciitis ("flesh-eating disease") (1, 2). Globally, there are over 700 million group A streptococcus (GAS) infections annually (3). It has been estimated that 9,000 to 11,500 cases of invasive GAS disease occur each year in the United States, resulting in 1,000 to 1,800 deaths annually (4; http://www.cdc.gov). Rheumatic fever, necrotizing fasciitis, and toxic shock syndrome are responsible for the high morbidity and mortality rates due to GAS infections (5).Advances in DNA sequencing and related technologies have facilitated genome-wide dissection of genetic events involved in colonization (6), immune evasion (7), virulence (8-10), and the evolution and spread of highly virulent S. pyogenes clones (11-13). To advance our understanding of S. pyogenes molecular-pathogenesis events occurring during invasive infection, we performed whole-genome sequencing of 2,954 isolates recovered from a nonhuman primate model of necrotizing fasciitis. The resulting genome sequence data stimulated us to construct an isogenic fabT deletion mutant strain and to compare its global transcriptome and virulence attributes with those of the wild-type parental strain. MATERIALS AND METHODSBacterial strains and growth conditions. Escherichia coli strai...

Serotype M28 group A streptococcus (GAS) is a common cause of infections such as pharyngitis ("strep throat") and necrotizing fasciitis ("flesh-eating" disease). Relatively little is known about the molecular mechanisms underpinning M28 GAS pathogenesis. Whole-genome sequencing studies of M28 GAS strains recovered from patients with invasive infections found an unexpectedly high number of missense (amino acid-changing) and nonsense (protein-truncating) polymorphisms in (egulator fov), leading us to hypothesize that altered RocA activity contributes to M28 GAS molecular pathogenesis. To test this hypothesis, an isogenic deletion mutant strain was created. Transcriptome sequencing (RNA-seq) analysis revealed that RocA inactivation significantly alters the level of transcripts for 427 and 323 genes at mid-exponential and early stationary growth phases, respectively, including genes for 41 transcription regulators and 21 virulence factors. In contrast, RocA transcriptomes from other GAS M protein serotypes are much smaller and include fewer transcription regulators. The mutant strain had significantly increased secreted activity of multiple virulence factors and grew to significantly higher colony counts under acid stress RocA inactivation also significantly increased GAS virulence in a mouse model of necrotizing myositis. Our results demonstrate that RocA is an important regulator of transcription regulators and virulence factors in M28 GAS and raise the possibility that naturally occurring polymorphisms in in some fashion contribute to human invasive infections caused by M28 GAS strains.