problem for civilian and military populations alike, and in the Warfighter, these .... Wound Infections Department, Walter Reed Army Institute of Research, ...
Development of Rapid, Multiplex PCR-Based Diagnostics to Improve the Treatment of Traumatic Wound Infections in the Injured Warfighter Janos Luka1, Yuanzheng Si1, Daniel V. Zurawski1, Michael G. Stockelman2, and Stuart D. Tyner1 1 Wound Infections Department, Walter Reed Army Institute of Research, Bacterial Diseases Branch, Silver Spring, MD . 2Wound Infection Department, Naval Medical Research Center, Silver Spring, MD ABSTRACT Despite a wide array of available antimicrobial agents, wound infections continue to pose a major problem for the Wounded Warrior, especially as pathogens develop antimicrobial resistance. Most combat-related wound infections are polymicrobial and contain not only diverse types of bacteria, but also fungi. Conventional identification of bacterial or fungal pathogens does not exist in many Role 3 (Combat Support Hospital) environments and certainly not in Roles 1 or 2. Classical microbiology takes considerable time (days to weeks) to identify any potential wound pathogens. These delays and limitations ensure that empiric antimicrobial therapy, uninformed by lab results in Roles 1 through 3, remains standard. The purpose of this study was to develop a rapid multiplex real-time laboratory diagnostic assay (less than 2 hours) for concurrent quantitation and identification of bacterial and fungal wound pathogens at Role 3. This method includes detection of most common carbapenemase resistance genes, which potentially enables earlier and more appropriate antimicrobial therapy early in the recovery process.
BACKGROUND The emergence of Multidrug Resistant (MDR) skin and soft tissue infections has become a global problem for civilian and military populations alike, and in the Warfighter, these infections are especially problematic when associated with traumatic/blast injuries. Up to 90% of the isolates from patients with traumatic wounds are MDRs. Most notably, the MDR infections of combat-injured personnel returning from service include the “ESKAPE” pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) (1,2). The management of these infections may be complex, requiring prolonged courses of combination broad-spectrum antibiotics in conjunction with surgical debridement. During the course of prolonged antibiotic therapy, soldiers are potentially exposed to multi-organ toxic effects from these antibiotics which in turn can lead to increased morbidity and mortality in patients. Surgical debridement is also used to further remove tissue harboring recalcitrant infections. Increasing the tissue loss in a severely wounded soldier can debilitate well beyond the damage of the original injury. In addition to the toll wound infections take on individual patients, there is a massive financial burden associated with the care of these patients. The total resources for inpatient treatment and multiple re-hospitalizations associated with infection puts a significant additional burden on both medical personnel and the patient. Therefore, there is an urgent need to develop effective strategies to improve the quality and effectiveness of treatment (3). To improve patient care and outcomes, resulting in lower costs and an improved quality of life for the recovering Warfighter, requires not only new therapeutics but vastly improved diagnostics. Improved rapid diagnostic tests could allow clinicians to make more informed decisions regarding therapy options, or when to close a wound, etc. Primer design: Broad range primers for detection and quantitation of all bacteria and fungi were identified by in silico analysis from the 16S and 18S regions, respectively. Specific primers for multiplexing of ESKAPE pathogens and twelve most common carbapenemase genes were also selected based on in silico analysis. The specificities of the primer sets were successfully validated using a battery of bacteria, fungi and yeast DNA purification: A commercial kit for rapid sample processing with adequate yield of all pathogen DNAs (bacterial and fungal species) was identified from MoBios Laboratories, which is compatible with tissue, exudates, and other complex sample matrices. DNA amplification: A multiplexing assay, based on High resolution Melting Analysis (HRM) using the selected primers, was developed for quantitation and identification of the most frequently detected wound infection associated microbes and their antibiotic resistance status. The performance of the assay was confirmed in a blinded study using 16 bacterial and 10 fungal samples most frequently detected in wound infections. For Sensitivity and Limit of Detection determination, multiple pathogens were mixed into tissue samples. PCR assays were run on Roche Lightcycler 480 with High Resolution Melting Analysis software. The authors would like to acknowledge funding via the Defense Medical Research and Development Program (DMRDP) grant # D_MIDCTA_I_11_J2_490
METHODS
FUNDING:
DISCLAIMER: Material has been reviewed by the Walter Reed Army Institute of Research. views of the Department of the Army or the Department of Defense.
Melting temperatures associated with resistance genes
Pathogens and genes selected for assay development TABLE 1 Pathogens and antibiotic resistance genes identified by Multiplex PCR Bacteria Fungi, Yeast Antibiotic Resistance genes Staph. aureus Aspergillus sp KPC Staph. capitis Penicillium sp VIM Staph. haemolyticus Chaetomium sp NDM-1 Klebsiella pneumoniae Aureobasidium TEM Escherichia coli Symbiotaphrina kochii OXA-48 Serratia marcescens Cladosporium IMP Acinetobacter baumannii Coniochaeta velutina FOX Pseudomonas aeruginosa Coniosporium sp MOX Pseudomonas stutzeri Malassezia DHA Enterobacter cloacae Candida CMY-2 Enterococcus durans ACT Enterococcus faecalis ACC Enterococcus faecium Moganella morganii Citrobacter freundii Shigella flexneri
Table 1 shows the pathogens and antibiotic resistance genes which were selected for assay development. These pathogens have been identified by Trauma Infectious Disease Outcome Study (TIDOS) to be most frequently associated with wound infections.
Melting temperature values of amplified products of the multiplex PCR assay for six carbapenem resistance genes (KPC, TEM, NDM, OXA, VIM, IMP) and ampC β-lactamase genes blaMOX, blaFOX, blaCMY-2, blaDHA, blaACC, and blaACT. Identification of resistance genes is based on melting temperatures (Tm) associated with each amplification product.
RESULTS
Sensitivity - Limit of Detection TABLE 2 Pathogens and antibiotic resistance genes identified by Multiplex PCR Bacteria LoD # detected % detected Staph. aureus 400 CFU/ml 15/15 100% Staph. capitis 500 CFU/ml 15/15 100% Staph. haemolyticus 800 CFU/ml 15/15 100% Klebsiella pneumoniae 400 CFU/ml 15/15 100% Escherichia coli 600 CFU/ml 15/15 100% Serratia marcescens 800 CFU/ml 15/15 100% Acinetobacter baumannii 600 CFU/ml 15/15 100% Pseudomonas aeruginosa 900 CFU/ml 15/15 100% Pseudomonas stutzeri 800 CFU/ml 15/15 100% Enterobacter cloacae 1000 CFU/ml 15/15 100% Enterococcus durans 1200 CFU/ml 15/15 100% Enterococcus faecalis 900 CFU/ml 15/15 100% Enterococcus faecium 1400 CFU/ml 15/15 100% Moganella morganii 1300 CFU/ml 15/15 100% Citrobacter freundii 2000 CFU/ml 15/15 100% Shigella flexneri 700 CFU/ml 15/15 100% Fungi, Yeast Aspergillus sp 600 Ge/ml 15/15 100% Penicillium sp 800 Ge/ml 15/15 100% Chaetomium sp 1200 Ge/ml 15/15 100% Aureobasidium 2500 Ge/ml 15/15 100% 15/15 Symbiotaphrina kochii 1700 Ge/ml 100% Cladosporium 1600 Ge/ml 15/15 100% Coniochaeta velutina 2500 Ge/ml 15/15 100% Coniosporium sp 3000 Ge/ml 15/15 100% Malassezia 2200 Ge/ml 15/15 100% Candida 1800 Ge/ml 15/15 100%
TABLE 3 Multiplex PCR for antibiotic resistance genes Resistance gene Tm KPC 90.6 NDM-1 84.3 OXA-48 81.9 VIM 88.4 IMP 80.2 TEM 83.4 ampC β-lactamase genes MOX 85.8 FOX 86.7 CMY-2 84.2 DHA 85.0 ACC 82.1 ACT 83.1
Sensitivity - Limit of Detection A sensitivity study was performed to determine the lowest concentration of pathogen that can be consistently detected (at least 95% detection or LoD95) in spiked specimens • Serial 2-fold dilutions of individual organisms were tested and used to obtain an initial estimate of LoD95. • LoD95 was confirmed by testing 15 samples. If less than 14/15 positive results were obtained, a new LoD concentration was selected, tested, and confirmed (Table 2). • Multiple pathogens were combined in a single specimen (multi-spike), each at its LoD concentration, to confirm equivalent sensitivity in samples containing one or more pathogens (i.e. coinfection) Figure 1
HRM signals associated with selected pathogens
Figure 1 shows the unique HRM signals associated with selected pathogens in a multiplex assay. PCR assay was run three times and signals from each run was over layered to show reproducibility of the signals.
• In this study we identified a commercially available microbial DNA purification kit (UltraClean® Microbial DNA Isolation Kit, MoBio Laboratoires) which efficiently isolate both bacterial and fungal DNA from biological samples in less than 15 minutes. • Primers for multiplex quantitative PCR were developed based on in silico analysis and were utilized for the assay development. • Pathogens for assay development were selected from the Trauma Infectious Disease Outcome Study (TIDOS) as to be most frequently associated with wound infections. (Table 1) • Using the developed primers and the HRM based quantitative PCR method, we have successfully quantitated and identified all microbial agents in spiked samples containing multiple bacterial and fungal agents at various quantities and proportions. • The sensitivity and Limit of Detection (LoD) has been determined using spiked tissue samples (Table 2) and was similar or better than commercially available PCR kits detecting only single microbial agent (4). • A multiplex PCR assay was also developed based on melting temperature differences (Tm) as shown in Table 3. • The was validated for detection of six carbapenem resistance genes (KPC, TEM, NDM, OXA, VIM, IMP) and detection of ampC β-lactamase genes blaMOX, blaFOX, blaCMY-2, blaDHA, blaACC, and blaACT • The developed assay will be validated using over 400 archived wound samples in the future.
CONCLUSIONS • A commercially available DNA purification kit has been identified which can simultaneously purify both bacterial and fungal DNA, especially suitable for purification of DNA from notoriously difficult fungus like Aspergillus. • A real-time multiplex diagnostic assay was developed for simultaneous identification of bacterial and fungal agents present in wound infections. • Sensitivity and Limit of Detection was determined and was similar or better than commercially available PCR kits detecting only single microbial agent. • A multiplex PCR was also developed for detection of most frequent antibiotic resistance genes. • Sample processing, PCR and HRM analysis can be done in less than two hours, allowing results to be rapidly communicated to the physicians.
References: 1. 2. 3. 4.
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