Smartphone-based pathogen diagnosis in urinary sepsis patients.

BackgroundThere is an urgent need for rapid, sensitive, and affordable diagnostics for microbial infections at the point-of-care. Although a number of innovative systems have been reported that transform mobile phones into potential diagnostic tools, the translational challenge to clinical diagnostics remains a significant hurdle to overcome.MethodsA smartphone-based real-time loop-mediated isothermal amplification (smaRT-LAMP) system was developed for pathogen ID in urinary sepsis patients. The free, custom-built mobile phone app allows the phone to serve as a stand-alone device for quantitative diagnostics, allowing the determination of genome copy-number of bacterial pathogens in real time.FindingsA head-to-head comparative bacterial analysis of urine from sepsis patients revealed that the performance of smaRT-LAMP matched that of clinical diagnostics at the admitting hospital in a fraction of the time (~1 h vs. 18-28 h). Among patients with bacteremic complications of their urinary sepsis, pathogen ID from the urine matched that from the blood - potentially allowing pathogen diagnosis shortly after hospital admission. Additionally, smaRT-LAMP did not exhibit false positives in sepsis patients with clinically negative urine cultures.InterpretationThe smaRT-LAMP system is effective against diverse Gram-negative and -positive pathogens and biological specimens, costs less than $100 US to fabricate (in addition to the smartphone), and is configurable for the simultaneous detection of multiple pathogens. SmaRT-LAMP thus offers the potential to deliver rapid diagnosis and treatment of urinary tract infections and urinary sepsis with a simple test that can be performed at low cost at the point-of-care. FUND: National Institutes of Health, Chan-Zuckerberg Biohub, Bill and Melinda Gates Foundation

LcrV Synthesis Is Altered by DNA Adenine Methylase Overproduction in Yersinia pseudotuberculosis and Is Required To Confer Immunity in Vaccinated Hosts

Yersinia pseudotuberculosis mutants that overproduce the DNA adenine methylase (Dam(OP) Yersinia) are attenuated, confer robust protective immune responses, and synthesize or secrete several Yersinia outer proteins (Yops) under conditions that are nonpermissive for synthesis and secretion in wild-type strains. To understand the molecular basis of immunity elicited by Dam(OP) Yersinia, we investigated the effects of Dam overproduction on the synthesis and localization of a principal Yersinia immunogen, LcrV, a low-calcium-responsive virulence factor involved in Yop synthesis, localization, and suppression of host inflammatory activities. Dam overproduction relaxed the stringent temperature and calcium regulation of LcrV synthesis. Moreover, the LcrV-dependent synthesis and localization of the actin cytotoxin, YopE, were shown to be relaxed in Dam(OP) cells, suggesting that the synthesis and localization of Yops can occur via both LcrV-dependent and -independent mechanisms. Last, the immunity conferred by Dam(OP) Yersinia was strictly dependent on the presence of LcrV, which may result from its role (i) as an immunogen, (ii) as an immunomodulator of host anti-inflammatory activities, or (iii) in the altered synthesis and localization of Yops that could contribute to immunogen repertoire expansion

Rise of the microbes.

Infectious diseases continue to plague the modern world. In the evolutionary arms race of pathogen emergence, the rules of engagement appear to have suddenly changed. Human activities have collided with nature to hasten the emergence of more potent pathogens from natural microbial populations. This is evident in recent infectious disease outbreaks, the events that led to their origin, and lessons learned: influenza (2009), meningitis (Africa, 2009), cholera (Haiti, 2010), E. coli (Germany, 2011) and Salmonella (USA, 2012). Developing a comprehensive control plan requires an understanding of the genetics, epidemiology and evolution of emergent pathogens for which humans have little or no pre-existing immunity. As we plot our next move, nature's genetic lottery continues, providing the fuel to transform the most unlikely infectious disease scenarios into reality

Altered Levels of Salmonella DNA Adenine Methylase Are Associated with Defects in Gene Expression, Motility, Flagellar Synthesis, and Bile Resistance in the Pathogenic Strain 14028 but Not in the Laboratory Strain LT2

Comparative genomic analysis has revealed limited strain diversity between Salmonella pathogenic and nonpathogenic isolates. Thus, some of the relative virulence and host-immune response disparities may be credited to differential gene regulation rather than gross differences in genomic content. Here we show that altered levels of Salmonella DNA adenine methylase (Dam) resulted in acute defects in virulence-associated gene expression, motility, flagellin synthesis, and bile resistance in the Salmonella pathogenic strain 14028 but not in avirulent laboratory strain LT2. The defects in motility exhibited by 14028 in response to altered Dam levels was not dependent on the presence of the regulatory protein, RpoS. The transitioning between flagellar types (phase variation) was also differentially regulated in 14028 versus LT2 in response to dam levels, resulting in distinct differences in flagellin expression states. These data suggest that differential gene regulation may contribute to the relative virulence disparities observed between Salmonella serovars that are closely related at the DNA level