Rapid phenotypic methods to improve the diagnosis of bacterial bloodstream infections: meeting the challenge to reduce the time to result (original) (raw)
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Scientific Reports, 2018
The current culture-based approach for the diagnosis of bloodstreams infection is incommensurate with timely treatment and curbing the prevalence of multi-drug resistant organisms (MDROs) due to its long time-to-result. Bloodstream infections typically involve extremely low (e.g., <10 colonyforming unit (CFU)/mL) bacterial concentrations that require a labor-intensive process and as much as 72 hours to yield a diagnosis. Here, we demonstrate a culture-free approach to achieve rapid diagnosis of bloodstream infections. An immuno-detection platform with intrinsic signal current amplification was developed for the ultrasensitive, rapid detection, identification (ID) and antibiotic susceptibility testing (AST) of infections. With its capability of monitoring short-term (1-2 hours) bacterial growth in blood, the platform is able to provide 84-minute simultaneous detection and ID in blood samples below the 10 CFU/mL level and 204-minute AST. The susceptible-intermediate-resistant AST capacity was demonstrated. The current standard for diagnosis of bloodstream infections, including device associated infections (e.g., central line-associated bloodstream infections, prosthetic valve endocarditis), requires sequential bacterial detection, identification (ID), and antibiotic susceptibility testing (AST) via traditional blood cultures. This culture-based three-step diagnostic approach is not optimal with profound clinical implications. First, the time to result for blood cultures typically ranges from 24 to more than 48 hours 1,2. Typical AST requires an additional 16-24 hours of culture of the isolated pathogen 3. Understanding that early antimicrobial therapy reduces mortality in bloodstream infections, patients are given empiric, broad-spectrum antibiotics pending culture results. This one-size-fits-all use of antibiotics results in opportunistic infections, drug-related toxicities, and antibiotic resistance. The prevalence of multi-drug resistant organisms (MDROs) is poised to be one of the greatest threats to global public health as new MDROs emerge over time 4. Each year, in the United States, over 2 million people acquire serious infections with bacteria that are resistant to one or more of the antibiotics designed to treat those infections 5. At least 23,000 people die each year as a direct result of these antibiotic-resistant infections 5. Many more die from other conditions that are complicated by an antibiotic-resistant infection. Second, blood cultures have low sensitivity. When bacteria in the blood are in low numbers (<10 CFU/mL) growth is sufficiently slow to produce a negative result 6-8. Worse still, certain bacteria do not grow at all under standard culture conditions 9. Up to 30% of prosthetic valve endocarditis is initially culture negative 10. Third, cultures are frequently contaminated by normal skin flora 11 , which can grow rapidly and out-compete certain pathogens in the culture media. Repeating cultures to confirm contamination versus infection further extend the time to diagnosis. A rapid diagnostic for bacterial detection, ID, and more importantly, AST would reduce exposure time to broad-spectrum antibiotics and allow for rapid de-escalation to pathogen targeted therapy. Non-culture, molecular diagnostic methods are rapidly being incorporated into standard medical microbiology laboratories. Most techniques are based on nucleic acid detection and/or amplification (e.g., polymerase chain reaction). However,
Official Journal of the Association of Medical Microbiology and Infectious Disease Canada, 2020
Background: Our laboratory uses matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI) and the VITEK 2 system (DV2) directly from positive blood cultures (BC) for organism identification (ID) and antimicrobial susceptibility testing (AST). Our objective was to compare direct MALDI–DV2 with a commercial BC ID–AST platform, the Accelerate Pheno system (AXDX), in the ID–AST of clinical and seeded BC positive for gram-negative bacilli (GNB). Methods: BC positive for GNB were collected over a 3-mo period and tested using AXDX and direct MALDI–DV2 and compared with conventional methods. A subset of sterile BC were seeded with multi-drug-resistant GNB. Results: Twenty-nine clinical samples and 35 seeded samples were analyzed. Direct MALDI had a higher ID failure rate (31.0%) than AXDX (3.4%; p < 0.001). Time to ID–AST was 1.5–6.9 h, 5.8–16.5 h, and 21.6–33.0 h for AXDX, direct MALDI–DV2, and conventional methods, respectively ( p < 0.001). For clinical s...
Rapid identification of bacteria from positive blood cultures
2005
Bacteremia results in significant morbidity and mortality, especially among patient populations that are immunocompromised. Broad-spectrum antibiotics are administered to patients suspected to have bloodstream infections that are awaiting diagnosis that depends on blood culture analysis. Significant delays in identification of pathogens can result, primarily due to the dependence on growth-based identification systems. To address these limitations, we took advantage of terminal restriction fragment (TRF) length polymorphisms (T-RFLP) due to 16S ribosomal DNA (rDNA) sequence diversity to rapidly identify bacterial pathogens directly from positive blood culture. TRF profiles for each organism were determined by sizing fragments from restriction digests of PCR products derived from two sets of 16S rDNA-specific fluorescent dye-labeled primers. In addition, we created a TRF profile database (TRFPD) with 5,899 predicted TRF profiles from sequence information representing 2,860 different bacterial species. TRF profiles were experimentally determined for 69 reference organisms and 32 clinical isolates and then compared against the predicted profiles in the TRFPD. The predictive value of the profiles was found to be accurate to the species level with most organisms tested. In addition, identification of 10 different genera was possible with profiles comprising two or three TRFs. Although it was possible to identify Enterobacteriaceae by using a profile of three TRFs, the similarity of the TRF profiles of these organisms makes differentiation of species less reliable with the current method. The ability to rapidly (i.e., within ϳ8 h) identify bacteria from blood cultures has potential for reducing unnecessary use of broad-spectrum antibiotics and promoting more timely prescription of appropriate antibiotics.
Rapid diagnosis of bloodstream infections using a culture-free phenotypic platform
Communications medicine, 2024
Background Bloodstream infections (BSIs) are a life-threatening acute medical condition and current diagnostics for BSIs suffer from long turnaround time (TAT). Here we show the validation of a rapid detection-analysis platform (RDAP) for the diagnosis of BSIs performed on clinical blood samples Methods The validation was performed on a cohort of 59 clinical blood samples, including positive culture samples, which indicated confirmed bloodstream infections, and negative culture samples. The bacteria in the positive culture samples included Gram-positive and Gram-negative pathogenic species. RDAP is based on an electrochemical sandwich immunoassay with voltage-controlled signal amplification, which provides an ultra-low limit of detection (4 CFU/mL), allowing the platform to detect and identify bacteria without requiring culture and perform phenotypic antibiotic susceptibility testing (AST) with only 1-2 h of antibiotic exposure. The preliminary diagnostic performance of RDAP was compared with that of standard commercial diagnostic technologies. Results Using a typical clinical microbiology laboratory diagnostic workflow that involved sample culture, agar plating, bacteria identification using matrix-assisted laser desorption ionization time-of-flight (MALDI TOF) mass spectrometry, and AST using MicroScan as a clinical diagnostic reference, RDAP showed diagnostic accuracy of 93.3% and 95.4% for detection-identification and AST, respectively. However, RDAP provided results at least 15 h faster. Conclusions This study shows the preliminary feasibility of using RDAP to rapidly diagnose BSIs, including AST. Limitations and potential mitigation strategies for clinical translation of the present RDAP prototype are discussed. The results of this clinical feasibility study indicate an approach to provide near real-time diagnostic information for clinicians to significantly enhance the treatment outcome of BSIs.
Journal of Clinical Microbiology, 2013
Conventional blood culturing using automated instrumentation with phenotypic identification requires a significant amount of time to generate results. This study investigated the speed and accuracy of results generated using PCR and pyrosequencing compared to the time required to obtain Gram stain results and final culture identification for cases of culture-confirmed bloodstream infections. Research and physician-ordered blood cultures were drawn concurrently. Aliquots of the incubating research blood culture fluid were removed hourly between 5 and 8 h, at 24 h, and again at 5 days. DNA was extracted from these 6 time point aliquots and analyzed by PCR and pyrosequencing for bacterial rRNA gene targets. These results were then compared to those of the physician-ordered blood culture. PCR and pyrosequencing accurately identified 92% of all culture-confirmed cases after a mean enrichment time of 5.8 ± 2.9 h. When the time needed to complete sample processing was included for PCR and ...
Clinical Microbiology and Infection, 2013
For septic patients, delaying the initiation of antimicrobial therapy or choosing an inappropriate antibiotic can considerably worsen their prognosis. This study evaluated the impact of rapid microbial identification (RMI) from positive blood cultures on the management of patients with suspected sepsis. During a 6-month period, RMI by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was performed for all new episodes of bacteraemia. For each patient, the infectious disease specialist was contacted and questioned about his therapeutic decisions made based on the Gram staining and the RMI. This information was collected to evaluate the number of RMIs that led to a therapeutic change or to a modification of the patient's general management (e.g. fast removal of infected catheters). During the study period, 277 new episodes of bacteraemia were recorded. In 71.12% of the cases, MALDI-TOF MS resulted in a successful RMI (197/277). For adult and paediatric patients, 13.38% (21/157) and 2.50% (1/40) of the RMIs, respectively, resulted in modification of the treatment regimen, according to the survey. In many other cases, the MALDI-TOF MS was a helpful tool for infectious disease specialists because it confirmed suspected cases of contamination, especially in the paediatric population (15/40 RMIs, 37.50%), or suggested complementary diagnostic testing. This study emphasizes the benefits of RMI from positive blood cultures. Although the use of this technique represents an extra cost for the laboratory, RMI using MALDI-TOF MS has been implemented in our daily practice.
Clinical Laboratory, 2013
Usually, 18-48 h are needed for the identification of microbial pathogens causing urinary tract infections (UTIs) by urine culture. Moreover, antimicrobial susceptibility testing (AST) takes an additional 18-24 h. Rapid identification and AST of the pathogens allow fast and precise treatment. The objective of this study was to shorten the time of diagnosis of UTIs by combining pathogen screening through flow cytometry, microbial identification by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS), and AST using the VITEK 2 system for the direct analysis of urine samples. We analyzed 1,638 urine samples from patients with suspected UTIs submitted to the microbiology laboratory for culture. Each urine sample had an approximate volume of 30 mL and was divided into three aliquots. Urine processing included differential centrifugation and two washes to enrich the bacterial fraction for direct MALDI-TOF MS and direct AST. From a total of 1,638 urine samples, 307 were found to be positive through UF-1000i screening. Among them, 265 had significant growth of a singlemicroorganism. Direct identification was obtained in 229 (86.42%) out of these 265 samples, and no pathogens were misidentified. Moreover, species-level identification was obtained in 163 (88.59%) out of the 184 samples with Gram-negative bacteria, and 27 (38.03%) out of the 71 samples with Gram-positive bacteria. VITEK 2 AST was performed for 117 samples with a single-microorganism. Enterobacteriaceae data showed an agreement rate of antimicrobial categories of 94.83% (1,229/1,296), with minor, major, and very major error rates of 4.17% (54/1,296), 0.92% (12/1,296), and 0.08% (1/1,296), respectively. For Enterococcus spp., the overall categorical agreement was 92.94% (158/170), with a minor error rate of 2.94% (5/170) and major error rate of 4.12% (7/170). The turnaround time of this combined protocol to diagnose UTIs was 1 h for pathogen identification and 6-24 h for AST; noteworthily, only 6-8 h are needed