Invasive group A streptococcal (GAS) infections associated with the sudden onset of shock, multi-organ failure, necrotizing fasciitis/myonecrosis and death emerged worldwide in the mid-1980s. Despite modern antibiotic regimens and intensive care measures, morbidity remains high and mortality ranges from 30 - 60%. Nearly half of all patients with GAS necrotizing fasciitis/myonecrosis develop infection at the site of minor, nonpenetrating trauma or muscle strain. In the absence of an obvious portal of entry and with a history of minor trauma, patients typically receive an incorrect diagnosis and are sent home, only to return to the ER 24-48 hours later in florid shock and organ failure. In these rapidly progressing """"""""cryptic"""""""" infections, mortality is 70- 85%. Survivors undergo extensive surgical debridement including amputations and prolonged hospitalization. The single most important factor contributing to the exceedingly high morbidity and mortality in these cryptic infections is a delay in diagnosis. The principal reason patients with cryptic GAS infections seek medical attention is pain;however, the absence of other clinical clues at this stage of infection makes diagnosis extremely difficult. Even if an astute physician were to suspect a cryptic infection, there is no available test to confirm this suspicion. To remedy this diagnostic shortfall and improve clinical outcomes, we will combine, for the first time, 1) the well-known ability of GAS exotoxins to modify host proteins plus 2) the knowledge that injured muscles express unique proteins during regeneration, as the basis for a novel approach to diagnosis. Specifically, we hypothesize that an exotoxin-modified form of a human injury-associated protein circulates in patients during the very earliest stages of cryptic GAS myonecrosis and that this pathogen-modified host protein (PmHP) could be exploited as a biomarker to detect infection well before life-threatening complications develop. Seeking PmHPs as evidence of early infection constitutes an entirely new approach to infection detection and could provide a new paradigm for infectious disease diagnosis. Studies proposed will utilize our existing animal model of injury-induced cryptic GAS infection in combination with state-of-the-art mass spectrometry (MS) analyses to seek PmHPs suitable for development of a rapid, sensitive and specific diagnostic test for sub-clinical crypti GAS infection. Two complimentary approaches will be taken. The first is a vimentin-focused approach that extends our previously published findings supporting a role for vimentin/GAS interaction in these cryptic infections. For this, we will apply classical immunologic protein isolation methods followed by MS analysis to determine whether an exotoxin-modified form of vimentin (a muscle injury-associated protein) can be detected in circulation of experimental animals with cryptic GAS infection. The second is an unbiased proteomics approach. In collaboration with scientists at the Pacific Northwest National Laboratory, an unbiased LC-MS-based systems proteomics approach will be used to screen the murine tibialis anterior (TA) muscle proteome for candidate biomarkers that discriminate between 1) normal muscle, 2) muscle injury alone and 3) injury complicated by GAS infection in animals treated with and without NSAIDs. Here, muscle digests will be subjected to initial 2D-LC-MS/MS analyses to generate a comprehensive accurate mass and time (AMT) tag database for use in subsequent quantitative LC-MS analyses using high resolution MS (e.g., Orbitrap) and the AMT tag approach. The high resolution and mass measurement accuracy (at ppm levels) of this technique will allow us to determine GAS toxin-induced modifications to host peptide/proteins in the highly complex muscle homogenate samples based upon fragmentation patterns. The application of this robust technique to biomarker discovery for early-stage cryptic infection is th first of its kind and will yield a solid foundation for new diagnostic test development. Bringing such a new diagnostic tool to the clinic would profoundly affect management decisions and dramatically improve patient outcomes for such an insidious but rapidly progressing and devastating disease.

Public Health Relevance

Cryptic group A streptococcal infections develop at sites of minor injury that do not break the skin. Without an obvious portal of bacterial entry, the correct diagnosis is often delayed until after shock and multi-organ failure develop. By this time, patients require emergent surgery, including amputation, and intensive care measures. Mortality is 60-85% and survivors undergo prolonged hospitalization. Even if an astute physician suspected early developing infection, there are no diagnostic tests to confirm this suspicion. To remedy this shortfall, this project takes a totally new approach to diagnosis, seeking for the firs time pathogen-modified host proteins as biomarkers that discern between injury alone and injury complicated by infection. The goal is to develop a highly specific and sensitive diagnostic test that could be applied well before life-threatening complications develop. Early diagnosis would ensure earlier therapeutic intervention, reduce the need for amputation, decrease the incidence of multi-organ failure, lessen costs and markedly improve clinical outcomes.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Exploratory/Developmental Grants (R21)
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Special Emphasis Panel (ZRG1-IDM-A (80))
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GU, Xin-Xing
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Idaho Veterans Research / Education Fdn
United States
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