RESUMO
Introduction. The alarming rise in urinary tract infection (UTI) antimicrobial resistance has resulted from a combination of high prevalence, low specificity and the lack of a rapid, point-of-care (POC) antibiotic susceptibility test (AST), which has led to the overuse/inappropriate use of antibiotics.Aim. This study aimed to evaluate the performance of a rapid POC phenotypic AST device in reporting susceptibility information within 2 h.Methodology. Instrument calibration was performed with model bacteria and fluorescent microbeads to determine the dynamic range and limit of detection for quantifying concentrations of bacteria and demonstrate the ability to rapidly differentiate susceptible and resistant model bacteria. We then evaluated 30 presumptive UTI-positive patient urine samples in a clinical pilot study using a panel of 5 common UTI antibiotics plus a growth control and compared our results to the hospital standard of care AST.Results. Our device was able to robustly detect and quantify bacteria concentrations from 50 to 105 colony-forming units (c.f.u.) ml-1. The high sensitivity of this measurement technique enabled the device to differentiate between susceptible and resistant model bacteria with 100 % specificity over a 2 h growth period. In the clinical pilot study, an overall categorical agreement (CA) of 90.7 % was observed (sensitivity=91.4 %, specificity=88.9 %, n=97) with performance for individual drugs ranging from 85 % CA (ceftazidime) to 100 % (nitrofurantoin).Conclusions. By reducing the typical timeframe for susceptibility testing from 2-3 days to 2 h, our POC phenotypic AST can provide critical information to clinicians prior to the administration of antibiotic therapy.
Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Testes de Sensibilidade Microbiana/métodos , Sistemas Automatizados de Assistência Junto ao Leito , Infecções Urinárias/microbiologia , Bactérias/isolamento & purificação , Humanos , Projetos Piloto , Sensibilidade e Especificidade , Fatores de Tempo , Urina/microbiologiaRESUMO
Sepsis due to antimicrobial resistant pathogens is a major health problem worldwide. The inability to rapidly detect and thus treat bacteria with appropriate agents in the early stages of infections leads to excess morbidity, mortality, and healthcare costs. Here we report a rapid diagnostic platform that integrates a novel one-step blood droplet digital PCR assay and a high throughput 3D particle counter system with potential to perform bacterial identification and antibiotic susceptibility profiling directly from whole blood specimens, without requiring culture and sample processing steps. Using CTX-M-9 family ESBLs as a model system, we demonstrated that our technology can simultaneously achieve unprecedented high sensitivity (10 CFU per ml) and rapid sample-to-answer assay time (one hour). In head-to-head studies, by contrast, real time PCR and BioRad ddPCR only exhibited a limit of detection of 1000 CFU per ml and 50-100 CFU per ml, respectively. In a blinded test inoculating clinical isolates into whole blood, we demonstrated 100% sensitivity and specificity in identifying pathogens carrying a particular resistance gene. We further demonstrated that our technology can be broadly applicable for targeted detection of a wide range of antibiotic resistant genes found in both Gram-positive (vanA, nuc, and mecA) and Gram-negative bacteria, including ESBLs (blaCTX-M-1 and blaCTX-M-2 families) and CREs (blaOXA-48 and blaKPC), as well as bacterial speciation (E. coli and Klebsiella spp.) and pan-bacterial detection, without requiring blood culture or sample processing. Our rapid diagnostic technology holds great potential in directing early, appropriate therapy and improved antibiotic stewardship in combating bloodstream infections and antibiotic resistance.
Assuntos
Antibacterianos/farmacologia , Enterobacteriaceae/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Reação em Cadeia da Polimerase , Enterococos Resistentes à Vancomicina/efeitos dos fármacos , Enterobacteriaceae/isolamento & purificação , Humanos , Dispositivos Lab-On-A-Chip , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Técnicas Analíticas Microfluídicas/instrumentação , Tamanho da Partícula , Propriedades de Superfície , Enterococos Resistentes à Vancomicina/isolamento & purificaçãoRESUMO
Current cancer detection systems lack the required sensitivity to reliably detect minimal residual disease (MRD) and recurrence at the earliest stages when treatment would be most effective. To address this issue, we present a novel liquid biopsy approach that utilizes an integrated comprehensive droplet digital detection (IC3D) digital PCR system which combines microfluidic droplet partitioning, fluorescent multiplex PCR chemistry, and our rapid 3D, large-volume droplet counting technology. The IC3D ddPCR assay can detect cancer-specific, ultra-rare genomic targets due to large sample input and high degree of partitioning. We first demonstrate our droplet digital PCR assay can robustly detect common cancer mutants including KRAS G12D spiked in wild-type genomic background or isolated from patient samples with 100% specificity. We then demonstrate that the IC3D ddPCR system can detect oncogenic KRAS G12D mutant alleles against a background of wild-type genomes at a sensitivity of 0.00125-0.005% with a false positive rate of 0% which is 50 to 1000× more sensitive than existing commercial liquid biopsy ddPCR and qPCR platforms, respectively. In addition, our technology can uniquely enable detection of circulating tumor cells using their genetic markers without a pre-enrichment step, and analysis of total tumor DNA isolated from blood samples, which will increase clinical sensitivity and specificity, and minimize inter-assay variability. Therefore, our technology holds the potential to provide clinicians with a powerful decision-making tool to monitor and treat MRD with unprecedented sensitivity for earlier stage intervention.