Abstract
Battling infection is a major healthcare objective. Untreated infections can rapidly evolve toward the condition of sepsis in which the body begins to fail and resuscitation becomes critical and tenuous. Identification of infection followed by rapid antimicrobial treatment are primary goals of medical care, but precise identification of offending organisms by current methods is slow and broad spectrum empirical therapy is employed to cover most potential pathogens. Current methods for identification of bacterial pathogens in a clinical setting typically require days of time, or a 4- to 8-h growth phase followed by DNA extraction, purification and PCR-based amplification. We demonstrate rapid (70–120 min) genetic diagnostics methods utilizing loop-mediated isothermal amplification (LAMP) to test for 15 common infection pathogen targets, called the Infection Diagnosis Panel (In-Dx). The method utilizes filtration to rapidly concentrate bacteria in sample matrices with lower bacterial loads and direct LAMP amplification without DNA purification from clinical blood, urine, wound, sputum and stool samples. The In-Dx panel was tested using two methods of detection: (1) real-time thermocycler fluorescent detection of LAMP amplification and (2) visual discrimination of color change in the presence of Eriochrome Black T (EBT) dye following amplification. In total, 239 duplicate samples were collected (31 blood, 122 urine, 73 mucocutaneous wound/swab, 11 sputum and two stool) from 229 prospectively enrolled hospital patients with suspected clinical infection and analyzed both at the hospital and by In-Dx. Sensitivity (Se) of the In-Dx panel targets pathogens from urine samples by In-Dx was 91.1% and specificity (Sp) was 97.3%, with a positive predictive value (PPV) of 53.7% and a negative predictive value (NPV) of 99.7% as compared to clinical microbial detection methods. Sensitivity of detection of the In-Dx panel from mucocutaneous swab samples was 65.5% with a Sp of 99.3%, and a PPV of 84% and NPV of 98% as compared to clinical microbial detection methods. Results indicate the LAMP-based In-Dx panel allows rapid and precise diagnosis of clinical infections by targeted pathogens across multiple culture types for point-of-care utilization.
Highlights
Rapid and accurate diagnoses, paired with appropriate and effective interventions, are important to stemming the disease process, maintaining economically feasible care and reducing long-term morbidity of infected patients
Primers were designed to be exclusive to one organism of interest except for mecA which is a generalized antibiotic resistance gene target with known associations to a number of clinical pathogens including S. aureus and S. epidermidis, a coagulase-negative Staphylococcus species which has been clinically recognized as a commensal skin contaminant, an opportunistic pathogen, and potential microbial gene-transfer reservoir (Becker et al, 2014; Rossi et al, 2017)
Detection thresholds for primer sets using purified DNA ranged from 5 pg at 27 min to 50 fg at 20 min (Proteus mirabilis) by LightCycler analysis (Supplementary Figure 1)
Summary
Rapid and accurate diagnoses, paired with appropriate and effective interventions, are important to stemming the disease process, maintaining economically feasible care and reducing long-term morbidity of infected patients. Systematic approaches to early sepsis identification and intervention including timely broad-spectrum antibiotic administration and adequate fluid volume resuscitation have yielded definite improvements in patient outcomes and health care resource utilization. In the period it takes for final culture results, administration of broad-spectrum antibiotics is provided ensure organism eradication. Improving upon this method of nonspecific antimicrobial administration and lengthy identification period is achieved with fast and precise species identification as well as antibiotic resistance gene identification. Longterm medical problems include increased health care costs due to difficulty in pathogen eradication, as microbial populations gain strength against future drug administration
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