Traditional Blood Culture Research Articles

Combination of a Rapid Diagnostic Assay and Antimicrobial Stewardship Intervention for Gram-Negative Bacteremia.

Traditional blood cultures for gram-negative bacteremia can take up to 72 hours or more to return results, prolonging the duration of empiric broad-spectrum intravenous antibiotics. The Accelerate Pheno system provides rapid identification and susceptibilities for blood cultures in gram-negative bacteremia. Current data on its clinical utility are mixed overall, so the system requires further research. A multicenter, retrospective quasi-experimental study was conducted comparing the Accelerate Pheno rapid diagnostic system with antimicrobial stewardship intervention and traditional blood cultures alone. A total of 264 patients with blood cultures with gram-negative bacteria growth were included in the final analysis (102 pre-intervention, 162 post-intervention). The antimicrobial stewardship team made 364 recommendations in 152/162 (93.8%) patients in the post group. Duration of intravenous therapy was shorter (P < .001) for the post-intervention group (median, 4.0 days) compared with the pre-intervention group (median, 7.5 days). Hospital length of stay was also shorter (P < .001) for the post-intervention group (median, 5.1 days) compared with the pre-intervention group (median, 7.0 days). Readmission rates within 30 days were reduced (P = .042) post-intervention (13.0%) compared with pre-intervention (22.6%). In the post-intervention group, a larger proportion of patients were transitioned to oral therapy at any point (126/162, 77.8%) compared with pre-intervention (62/102, 60.8%; P < .001). These results suggest that the Accelerate Pheno system, with active review and intervention by a multidisciplinary antimicrobial stewardship team, is a useful tool in improving both patient-centric and antimicrobial stewardship outcomes.

Microbial cell-free DNA-sequencing as an addition to conventional diagnostics in neonatal sepsis.

Bloodstream infections remain a challenge for neonatologists, as traditional culture-based methods are time-consuming and rely on adequate blood volume. Next-generation sequencing (NGS) offers an alternative, as it can identify microbial cell-free DNA (mcfDNA) in a small blood sample, providing rapid pathogen detection. This study aimed to assess the diagnostic performance of DISQVER®-NGS compared to blood cultures in neonatal patients with suspected sepsis. In neonates with suspected sepsis, blood cultures and samples for NGS were prospectively collected. Patients were divided into four categories: 1) sepsis, blood culture positive, 2) clinical sepsis, culture negative, 3) suspected sepsis, 4) validation cohort. NGS detected bacterial, viral or fungal mcfDNA in 24 of 82 samples. Blood cultures were collected in 46 of 84 patients (15/46 positive). DISQVER® correctly identified pathogens in 9/15 patients with a positive blood culture, two with intrinsic resistance to their antibiotic regimen. In seven samples NGS reported the mcfDNA of bacteria that could have theoretically grown in culture but did not. NGS may enhance sensitivity in sepsis diagnostics by detecting mcfDNA in neonates with suspected sepsis. Interpreting NGS results requires correlation with clinical data, laboratory values, and routine microbiological tests for a comprehensive understanding of the patient's condition. Conventional blood culture methods have limitations in accuracy and turnaround time. The study aimed to investigate the diagnostic performance of the Next-Generation Sequencing method DISQVER® compared to traditional blood cultures in neonatal patients with suspected sepsis. Our findings suggest that NGS has the potential to augment the precision of conventional diagnostic techniques, can lead to improved detection of pathogens and targeted treatment approaches in neonatal sepsis. It is emphasized that further validation and integration with clinical and microbiological data are required to ensure optimal clinical utility.

Blood culture-free ultra-rapid antimicrobial susceptibility testing.

Treatment assessment and patient outcome for sepsis depend predominantly on the timely administration of appropriate antibiotics1-3. However, the clinical protocols used to stratify and select patient-specific optimal therapy are extremely slow4. In particular, the major hurdle in performing rapid antimicrobial susceptibility testing (AST) remains in the lengthy blood culture procedure, which has long been considered unavoidable due to the limited number of pathogens present in the patient's blood. Here we describe an ultra-rapid AST method that bypasses the need for traditional blood culture, thereby demonstrating potential to reduce the turnaround time of reporting drug susceptibility profiles by more than 40-60 h compared with hospital AST workflows. Introducing a synthetic beta-2-glycoproteinI peptide, a broad range of microbial pathogens are selectively recovered from whole blood, subjected to species identification or instantly proliferated and phenotypically evaluated for various drug conditions using a low-inoculum AST chip. The platform was clinically evaluated by the enrolment of 190 hospitalized patients suspected of having infection, achieving 100% match in species identification. Among the eight positive cases, six clinical isolates were retrospectively tested for AST showing an overall categorical agreement of 94.90% with an average theoretical turnaround time of 13 ± 2.53 h starting from initial blood processing.

Development of two multiplex PCR assays for rapid detection of eleven Gram-negative bacteria in children with septicemia

AimThis study aimed to develop a multiplex PCR assay for simultaneous detection of major Gram-negative etiologies of septicemia and evaluate its performance.MethodsMultiplex PCR (mPCR) assays were developed targeting 11 bacterial strains. Species-specific primers were confirmed using known clinical isolates and standard strains. Gradient PCR was performed on each primer against its target bacterial gene to determine its optimal amplification condition. The minimum detectable DNA concentration of the two assays was evaluated by adjusting bacterial DNA concentration to 100 ng/μL and, tenfold serially diluting it up to 10 pg/μL with DNAse-free water. The diagnostic accuracy of mPCR assays was established by subjecting the assays to 60 clinical blood samples.ResultsTwo mPCR assays were developed. Optimal primer annealing temperature of 55 °C was established and utilized in the final amplification conditions. The assays detected all targeted bacteria, with a 100 pg minimum detectable DNA concentration. Pathogens were not detected directly from whole blood, but after 4 h and 8 h of incubation, 41% (5/12) and 100% (12/12) of the bacteria were detected in culture fluids, respectively. The assays also identified Salmonella spp. and Klebsiella pneumoniae co-infections and extra pathogens (1 E. coli and 2 K. pneumoniae) compared with culture. The sensitivity and specificity of the mPCR were 100.0% (71.7–100.0) and 98.0% (90.7–99.0), respectively. The area under the ROC curve was 1.00 (1.00–1.00).ConclusionsThe mPCR assays demonstrated substantial potential as a rapid tool for septicemia diagnosis alongside the traditional blood culture method. Notably, it was able to identify additional isolates, detect co-infections, and efficiently detect low bacterial DNA loads with high sensitivity, implying its value in enhancing efficiency of diagnosis of septicemia.

Short turnaround time of seven to nine hours from sample collection until informed decision for sepsis treatment using nanopore sequencing

Bloodstream infections (BSIs) and sepsis are major health problems, annually claiming millions of lives. Traditional blood culture techniques, employed to identify sepsis-causing pathogens and assess antibiotic susceptibility, usually take 2–4 days. Early and accurate antibiotic prescription is vital in sepsis to mitigate mortality and antibiotic resistance. This study aimed to reduce the wait time for sepsis diagnosis by employing shorter blood culture incubation times for BD BACTEC™ bottles using standard laboratory incubators, followed by real-time nanopore sequencing and data analysis. The method was tested on nine blood samples spiked with clinical isolates from the six most prevalent sepsis-causing pathogens. The results showed that pathogen identification was possible at as low as 102–104 CFU/mL, achieved after just 2 h of incubation and within 40 min of nanopore sequencing. Moreover, all the antimicrobial resistance genes were identified at 103–107 CFU/mL, achieved after incubation for 5 h and only 10 min to 3 h of sequencing. Therefore, the total turnaround time from sample collection to the information required for an informed decision on the right antibiotic treatment was between 7 and 9 h. These results hold significant promise for better clinical management of sepsis compared with current culture-based methods.

Utilization of Platelet-Rich Plasma (PRP) in the Performance of Blood Cultures

Sepsis is a leading cause of death and morbidity. A precise microbiologic diagnosis in these patients reduces mortality, morbidity, hospital length of stay and costs. Current blood culture technology is woefully inadequate in the detection of circulating bacteria, with detection rates in the range of only 32 to 71%. Platelets play a key role in innate immunity. Platelets interact directly with innate immune cells, exert important immunomodulatory effects, and promote leucocyte migration to sites of infection. During active bacterial infection, platelets release platelet factor -4 which binds onto a variety of bacterial surfaces, thus rendering bacteria more susceptible to opsonization by leucocytes. Platelet surface Toll-like receptors play a key role in regulating bacterial phagocytosis by leucocytes. FcgammaRllA mediates immune complex mediated platelet activation for killing of opsonized bacteria. Platelets also have been observed to “corral” circulating bacteria by forming hollow spheres around them. For these reasons, we performed a prospective pilot study involving 100 patients with suspected sepsis testing the hypothesis that BC performed with PRP is superior to traditional BC performed with whole blood in detecting circulating bacteria. PRP was prepared via centrifugation of blood contained in 3 cc CBC tubes containing EDTA obtained upon admission. PRP and whole blood were injected into separate and identical culture media bottles and cultures were incubated identically in our state-of-the art microbiology laboratory. PRP culture technique detected two patients with circulating bacteria in whom traditional blood cultures were negative: Enterobacter faecalis in an elderly woman with bilateral pneumonia and Burkolderia capecia in an immunocompromised elderly man with pneumonia. In two patients with urosepsis culture results were concordant with identification of E. coli in both techniques. In eight patients, traditional BC technique detected circulating bacteria, wherein PRP-generated cultures were negative: three with Klebsiella pneumoniae, two with Enterococcus faecalis, two with E. Coli, and one with anaerobic Bacteroides Theta-iota group. Three positive cultures generated by traditional methodology were deemed contaminants by I.D. consultation. No contaminants or false positives were detected by the PRP-generated technique. Thus, in this pilot study, evaluating the utility of blood cultures prepared from platelet-rich-plasma, two patients were detected to harbor pathogenic circulating bacteria which was undetected by standard state-of-the-art blood culture techniques. Two distinct disadvantages of BC prepared from PRP relative to traditional BC technique in this study were the time delay in preparing PRP from the patient's CBC tube: these had been stored in the lab for typically 4 to 48 hours before centrifugation and subsequent inoculation, and the dramatic difference in volume represented by PRP vs. traditional BC: typically .5 to 1.1 cc vs. 32 to 40 cc, respectively. An additional disadvantage was the presence of EDTA in CBC tubes, which is confirmed to exert antibacterial properties. The results of this pilot study are intriguing. Further studies comparing PRP-generated BC and traditional BC in the detection of circulating bacteria with emerging identification techniques such as metrogenomic analysis in detecting bacterial DNA, RNA or peptide signatures would be interesting.

Clinical Application of a Multiplex Droplet Digital PCR in the Rapid Diagnosis of Children with Suspected Bloodstream Infections.

Droplet digital PCR (ddPCR) recently has been shown to be a potential diagnostic tool for adults with bloodstream infections (BSIs); however, its application in children remains obscure. In this study, 76 blood samples of children with suspected BSIs were synchronously detected by traditional blood cultures (BCs) and ddPCRs. Our team validated the diagnostic performance of ddPCR including sensitivity, specificity, and positive and negative predictive values. The 76 pediatric patients from the hematology department (67.1%), the pediatric intensive care unit (PICU, 27.6%), and other departments (5.2%) were enrolled. The positive rate of ddPCR results was 47.9%, whereas that for BC was 6.6%. In addition, the time consumption of ddPCR was shorter, only for 4.7 ± 0.9 h, in comparison with the detection timing of BC (76.7 ± 10.4 h, p < 0.01). The levels of agreement and disagreement between BC and ddPCR were 96.1% and 4.2%, and the negative agreement reached 95.6%. The sensitivity of ddPCR was 100%, with corresponding specificities ranging from 95.3 to 100.0%. In addition, a total of nine viruses were identified by ddPCR. In China, the multiplexed ddPCR first could be a tool for the rapid and accurate diagnosis of children with suspected BSIs and can be an early indicator of the possibility of viraemia in children with immunosuppression.

Updates in Laboratory Identification of Invasive Fungal Infection in Neonates.

Invasive fungal infection (IFI) in immunocompromised neonates is significantly associated with high morbidity and mortality and has become the third most common infection in Neonatal Intensive Care Units. The early diagnosis of IFI for neonatal patients is difficult because of the lack of specific symptoms. The traditional blood culture remains the gold standard in clinical diagnosis for neonatal patients but it requires a long duration, which delays treatment initiation. Detections of fungal cell-wall components are developed for early diagnosis but the diagnostic accuracy in neonates needs to be improved. PCR-based laboratory methods, such as real-time PCR, droplet digital PCR, and the cationic conjugated polymer fluorescence resonance energy transfer (CCP-FRET) system, distinguish the infected fungal species by their specific nucleic acids and show a high sensitivity and specificity. Particularly, the CCP-FRET system, which contains a cationic conjugated polymer (CCP) fluorescent probe and pathogen-specific DNA labeled with fluorescent dyes, could identify multiple infections simultaneously. In the CCP-FRET system, the CCP and fungal DNA fragments can self-assemble into a complex with an electrostatic interaction and the CCP triggers the FRET effect under ultraviolet light to make the infection visible. Here, we summarize the recent laboratory methods for neonatal IFI identification and provide a new perspective for early clinical fungal diagnosis.

Clinical Diagnosis Application of Metagenomic Next-Generation Sequencing of Plasma in Suspected Sepsis.

We analyzed the clinical concordance of mNGS test results from blood samples and improved the clinical efficiency of mNGS in the diagnosis of suspected sepsis pathogens. In this study, 99 samples of suspected blood flow infection were included for plasma mNGS, and the correlation between mNGS results and blood culture results, serum inflammatory indices, clinical symptoms and antibiotic treatment was analyzed, as well as the comparison with the detection rate of BALF pathogens, as well as the classification of different pathogens in the mNGS results were analyzed. The mNGS pathogen detection rate was higher than that of traditional blood culture (83.02% vs 35.82%). The rate of the mNGS results being consistent with the clinical diagnosis was also higher than that of traditional blood culture (58.49% vs 20.75%). This study shows that bacteria and fungi are the main pathogens in sepsis, and viral sepsis is very rare. In this study, 32% of sepsis patients were secondary to pneumonia. Compared with the pathogen detection rate using alveolar lavage fluid, the detection rate from plasma mNGS was 62.5%. Samples were also easy to sample, noninvasive, and more convenient for clinical application. This study shows that compared with blood culture, the detection rate of mNGS pathogen that meets the diagnosis of sepsis is higher. We need a combination of multiple indicators to monitor the early diagnosis and treatment of sepsis.

885. Integrated Blood Culture and Molecular Diagnostic Device Reduces Time-to-Result to a Quarter of Traditional Methods

Abstract Background Prolonged antibiotic exposure in infants with negative blood cultures is associated with adverse outcomes, disruption of the developing microbiome, and increased carriage of antibiotic resistance genes. However, early administration of antibiotics significantly reduces risk of infant morbidity and mortality caused by infections, and therefore faster culture-to-identification is highly desirable. We combined blood culture (BC) and PCR-based pathogen identification into a single digital microfluidic (DMF) device to speed up BC results. Methods Our DMF BC+PCR device has a custom-built BC bottle incubator, which is connected to a disposable cartridge that performs sample preparation and PCR. Standard BC bottles were inoculated with K. pneumoniae, then incubated in either the DMF BC device or a traditional BC device (BD BACTEC 9050). Samples were drawn from the DMF BC bottle at 3, 4 and 5 hours and loaded in the cartridge for automated sample processing and PCR. Time-to-positivity for the DMF BC+PCR device was determined using cycle threshold and compared with the traditional method, which uses fluorescence from BC bottle followed by PCR on a separate device. Results Bacterial presence could be detected as quickly as 3 hours post inoculation (Ct values of 34, 31 and 28 at 3, 4, and 5 hrs) with five replicates of K. pneumoniae samples on the DMF BC+PCR device (growing 10x every hr), compared to &amp;gt; 12 hrs for the traditional method (10.5 hr culture and 2 hr PCR identification). Additionally, for 5 common pathogens that cause neonatal sepsis (K. pneumoniae, E. coli, S. aureus, S. agalactiae, or S. epidermidis), we also demonstrated that the DMF BC bottle incubator and the traditional BC device show similar growth curves using optical methods to determine growth. Conclusion The DMF BC system yields bacterial growth rates equivalent to traditional methods, but can drastically reduce time to pathogen identification, from 12 hrs to 3 hrs by leveraging PCR sensitivity to determine growth rather than fluorescence in the BC bottles. To our knowledge, this is the first demonstration of using an integrated microfluidic PCR platform to speed up culture and identification. Further work is ongoing to include antibiotic susceptibility testing and expand the panel of pathogens detected on the same cartridge. Disclosures Laura Huning, PhD, Baebies, Inc.: Stocks/Bonds Devika Varma, PhD, Baebies, Inc.: Stocks/Bonds Abbey Jackson, PhD, Baebies, Inc.: Stocks/Bonds Peter Bohlen, n/a, Baebies, Inc.: Stocks/Bonds Rainer Ng, PhD, Baebies, Inc.: Stocks/Bonds Vamsee Pamula, PhD, Baebies, Inc.: Stocks/Bonds.

Primary exploring the value of metagenomic next-generation sequencing in detecting pathogenic bacteria of cholangitis with biliary atresia after Kasai operation.

To evaluate the value of metagenomic next-generation sequencing (mNGS) in detecting pathogenic bacteria of cholangitis for patients with biliary atresia after Kasai operation. This study retrospectively analyzed patients of biliary atresia with cholangitis after Kasai operation who were admitted to Xi'an Children's Hospital from July 2019 to December 2021. Both blood culture and mNGS were carried out in all of these patients. We compared the detection rate of pathogenic bacteria, pathogenic bacteria spectrum, test time, inflammatory indicators and liver function. All the patients were followed up for 0.5-3years to evaluate the onset of cholangitis and the survival status of autologous liver. This study included total of 30 cholangitis occurred in 25 patients. There were significant differences in the detection rate of pathogenic bacteria [23.3 vs.73.3%, P < 0.05] and the test time [120 (114.5-120) vs.16 (16-21) h, P < 0.001] between the blood culture and mNGS. These two methods showed significant statistical differences in comparing inflammatory indicators (CRP, PCT) and liver function (TB, DB, GGT) before and after anti-infection. Four kinds of bacteria were detected by blood cultures and ten kinds of bacteria were detected by mNGS. Cholangitis occurred 3times in one case (4%) and twice in three cases (12%). Autologous liver survived in 17 cases (68%). Comparing with traditional blood culture, mNGS is more efficient, convenient and accurate in the detection of pathogens. It provides a new method for accurately detecting pathogenic bacteria of cholangitis after Kasai operation.

Clinical application value of peripheral blood metagenomic next-generation sequencing test for patients with hematological diseases accompanied by fever

Objective: To investigate the clinical application value of peripheral blood metagenomic next-generation sequencing (mNGS) test for patients with hematological diseases accompanied by fever. Methods: The blood mNGS results and clinical data of inpatients with hematological diseases accompanied by fever treated in the Hematology Department of Tianjin Medical University General Hospital in March 2020 to June 2021were retrospectively analyzed. A total of 90 patients with 98 cases of specimens were included. The pathogen distribution characteristics and mNGS test performance were analyzed. Results: The positive rate of peripheral blood mNGS was significantly higher than that of traditional examination (68.37% vs 37.76%, P<0.001) and blood culture (68.37% vs 9.18%, P<0.001) . Viral, bacterial, and fungal infections accounted for 38.81%, 14.93%, and 2.99% in patients with single-pathogen infections, respectively. Polymicrobial infections accounted for 43.28%, in which viral and bacterial coinfections were the most common type (25.37%) . There were 55 virus-positive cases (82.09%) , 30 bacteria-positive cases (44.78%) , and 14 fungus-positive cases (20.90%) . The clinical approval rate of peripheral blood mNGS was 64.63% (63/98) . The sensitivity, specificity, positive predictive value, and negative predictive value (NPV) of peripheral blood mNGS were 75.68%, 36.07%, 41.79%, and 70.97%, respectively, and the overall consistency rate with traditional examination was 51.02%. Of the 22 pulmonary infection cases with no detectable pathogens by conventional tests, the pathogens were identified by peripheral blood mNGS in 14 cases, 10 of which were clinically approved. Conclusion: The positive rate of peripheral blood mNGS was significantly higher than that of blood culture and traditional laboratory examination. Peripheral blood mNGS had a high clinical recognition rate, sensitivity, and NPV in the detection of pathogens in patients with hematological diseases accompanied by fever.

Efficacy of VERIGENE® Blood Culture Assays in Accurately and Rapidly Detecting Gram-Negative and Gram-Positive Pathogens.

Rapid administration of appropriately indicated antibiotics is crucial in septic patients. Sepsis data supports that there is a higher risk of mortality for each hour delay from triage to antibiotic therapy, as well as for inappropriate antibiotic selection. There are a variety of rapid microbial detection systems, such as VERIGENE®, used in acute care facilities to rapidly detect bacteremia and identify resistance markers. Our study investigates the usefulness of VERIGENE® assays in accurately detecting Gram-positive and Gram-negative pathogens when compared to traditional blood culture analysis systems, such as VITEK®. 819 Gram-positive and 373 Gram-negative blood samples were collected and tested using both VERIGENE® and VITEK®. Statistical tests were two-tailed and observations were defined as statistically significant if P ≤ 0.05. VERIGENE® detected a pathogen in 816/819 (99.6%) samples of the Gram-positive blood cultures and 367/373 (98.3%) samples of the Gram-negatives compared to 805/819 (98.3%) and 367/373 (98.4%), respectively, using VITEK®. Gram-positive cultures had a sensitivity of 99.5% and a specificity of 27.3% (PPV 99.0%, NPV 42.9%, 98.7% accuracy) with VERIGENE analysis. Gramnegatives had a sensitivity of 99.2% and a specificity of 20.0% (PPV 98.9%, NPV 25.0%, 98.4% accuracy). Although statistically insignificant (P = 0.25), VERIGENE® was 1.3% more likely to identify Gram-positive bacteria when compared to conventional methods. Overall, we concluded that VERIGENE® assays are valuable in their ability to rapidly detect microorganisms and resistance markers, given their high sensitivities. This allows for select targeted therapy in patients with sepsis and can ultimately reduce mortality rates.

Clinical Application and Influencing Factor Analysis of Metagenomic Next-Generation Sequencing (mNGS) in ICU Patients With Sepsis

ObjectiveTo analyze the clinical application and related influencing factors of metagenomic next-generation sequencing (mNGS) in patients with sepsis in intensive care unit (ICU).MethodsThe study included 124 patients with severe sepsis admitted to the ICU in the First Affiliated Hospital of Zhengzhou University from June 2020 to September 2021. Two experienced clinicians took blood mNGS and routine blood cultures of patients meeting the sepsis diagnostic criteria within 24 hours after sepsis was considered, and collection the general clinical data.ResultsmNGS positive rate was higher than traditional blood culture (67.74% vs. 19.35%). APACHE II score [odds ratio (OR)=1.096], immune-related diseases (OR=6.544), and hypertension (OR=2.819) were considered as positive independent factors for mNGS or culture-positive. The sequence number of microorganisms and pathogen detection (mNGS) type had no effect on prognosis. Age (OR=1.016), female (OR=5.963), myoglobin (OR=1.005), and positive virus result (OR=8.531) were independent risk factors of sepsis mortality. Adjusting antibiotics according to mNGS results, there was no statistical difference in the prognosis of patients with sepsis.ConclusionmNGS has the advantages of rapid and high positive rate in the detection of pathogens in patients with severe sepsis. Patients with high APACHE II score, immune-related diseases, and hypertension are more likely to obtain positive mNGS results. The effect of adjusting antibiotics according to mNGS results on the prognosis of sepsis needs to be further evaluated.

Comparing multiplex and multiplex real-time polymerase chain reaction with traditional blood culture in bacterial detection among patients with septicemia

Comparing multiplex and multiplex real-time polymerase chain reaction with traditional blood culture in bacterial detection among patients with septicemia

Validation of an Isothermal Amplification Platform for Microbial Identification and Antimicrobial Resistance Detection in Blood: A Prospective Study.

Background: Recent advances in nucleic acid amplification technique (NAAT)-based identification of pathogens in blood stream infections (BSI) have revolutionized molecular diagnostics in comparison to traditional clinical microbiology practice of blood culture. Rapid pathogen detection with point-of-care diagnostic-applicable platform is prerequisite for efficient patient management. The aim of the study is to evaluate an in-house developed, lyophilized OmiX-AMP pathogen test for the detection of top six BSI-causing bacteria along with two major antimicrobial resistance (AMR) markers of carbapenem and compare it to the traditional blood culture-based detection.Materials and methods: One hundred forty-three patients admitted to the Medical Intensive Care Unit, Narayana Hrudayalaya, Bangalore, with either suspected or proven sepsis, of either gender, of age ≥18 years were enrolled for the study. Pathogen DNA extracted from blood culture sample using OmiX pReP method was amplified at isothermal conditions and analyzed in real time using OmiX Analysis software.Results: Among the processed 143 samples, 54 were true negative, 83 were true positive, 3 were false negative, and 2 were false positive as analyzed by OmiX READ software. Gram-negative bacteria (91.3%) and gram-positive bacteria (75%) were detected with 100% specificity and 95.6% sensitivity along with the AMR marker pattern with a turnaround time of 4 hours from sample collection to results.Conclusion: OmiX-AMP pathogen test detected pathogens with 96.5% concordance in comparison to traditional blood culture. Henceforth, OmiX-AMP pathogen test could be used as a readily deployable diagnostic kit even in low-resource settings.How to cite this article: Maheshwarappa HM, Guru P, Mundre RS, Lawrence N, Majumder S, Sigamani A, et al. Validation of an Isothermal Amplification Platform for Microbial Identification and Antimicrobial Resistance Detection in Blood: A Prospective Study. Indian J Crit Care Med 2021;25(3):299–304.

Microfluidic Chips for Sepsis Diagnosis.

This chapter discusses two microfluidic-based approaches for early sepsis detection that achieve a higher accuracy than traditional blood culture analysis. Patient blood samples were included in this work to validate the performance of our chips in diagnosing sepsis. The single-parameter chip demonstrated the increased accuracy if using CD64 as a biomarker for sepsis detection compared with C-reactive protein (CRP) and procalcitonin (PCT) when applied alone. In addition, a multiparameter chip measuring a combined panel of CD25, CD64, and CD69, and achieved a high accuracy with an Area Under the Receiver Operating Characteristic Curve (AUROC) of 0.978. The combined panel was also able to detect culture-negative patients and provided a faster diagnosis. Besides, microfluidics has advantages of less time consuming, easier to manufacture, less sample loading, less complex, and portable. Therefore, our approach is of great potential to become a bedside sepsis detection method.

306. Rapid Detection Of Bloodstream Infections, Including Molecular Characterization, From Whole Blood

BackgroundDiagnosis of bloodstream infections (BSI) and treatment with appropriate antimicrobials is dependent upon fast and accurate information about the microorganism(s). The long time taken for a blood culture result, microbial identification and antimicrobial susceptibility testing (AST), can lead to poor antimicrobial stewardship. Many antimicrobial change decisions are based on the results of a Gram stain, with this being the first result available. Having results from a rapid test, direct from blood, which can confirm BSI and characterize the causative pathogen(s) would provide an improvement in antimicrobial stewardship and patient care.MethodsSepsiSTAT® is a rapid molecular test, developed by Momentum Bioscience Ltd, for the detection of BSI, with a time-to-result of < 4 hours. It uses whole blood to detect viable microorganisms whilst also providing molecular characterization. Microorganisms are extracted from the sample using a proprietary process involving capture on magnetic microbeads. This is followed by Enzymatic Template Generation and Amplification (ETGA®) for ultra-sensitive, universal detection of viable bacterial and fungal species, based on detecting microbial DNA polymerase activity. Simultaneously, molecular characterization also provides genus/species identification. The detection limits of SepsiSTAT® were evaluated for a broad panel of microorganisms, representing 80% of BSI reported to Public Health England (2018 report).ResultsThese results show a median detection limit (n=5) of < 10 cfu/mL in blood for microorganisms representing 77.4% of reported BSI, including key organisms such as E. coli, S. epidermidis and C. albicans. Notably, S. aureus, P. aeruginosa, E. faecalis, P. mirabilis and S. marcescens were all detected at < 1 cfu/mL.ConclusionSepsiSTAT® can detect microbes in low numbers, with a turnaround time substantially faster than traditional blood culture. Future development will aim to shorten time-to-results to < 3 hours, further benefiting patient outcomes and antimicrobial stewardship. Future studies in a clinical setting will seek to further demonstrate the efficacy and rapid turnaround time of the SepsiSTAT® test.Disclosures All Authors: No reported disclosures

Biomarkers for sepsis – past, present and future

Sepsis is, in many patients, very difficult to recognise, especially early on and in the elderly and those with multiple comorbidities1. This difficulty leads to delayed treatment in some, and over-treatment in others in whom bacterial infection does not exist. One large study of 2579 patients admitted to critical care for presumed sepsis showed that 13% had a post-hoc infection likelihood of “none” and an additional 30% of only “possible”2. With increasing recognition of the many detrimental yet usually covert effects of antibiotics, such agents can only cause harm when given unnecessarily. There is a pressing need for reliable, early, sensitive, and specific biomarkers to (i) indicate the presence of infection, (ii) to indicate the likelihood that these infected patients will go on to develop organ dysfunction (sepsis), and (iii) to identify which specific treatments (e.g. immunomodulatory) should be administered to which patient in terms of timing, dosing, and duration.Infection diagnostics have traditionally relied upon Gram stain and culture; the yield is low and often several days elapse before an organism is identified, speciated, and its antibiotic resistance pattern determined. Newer molecular diagnostics are arriving at an impressive pace and offer the opportunity for point-of-care testing at the bedside to identify micro-organisms (at least, the commonest pathogens), and some indication of antibiotic sensitivity, within minutes of sampling. Remarkably, bacteria within the lung can also be imaged in real time. As an example of the power of molecular diagnostics, one study involving 529 patients in nine European ICUs demonstrated that from 616 blood culture samples, polymerase chain reaction/electrospray ionization-mass spectrometry identified a pathogen in 228 cases (37%) whereas traditional blood cultures were positive in just 68 (11%)3.For sepsis, current biomarkers such as C-reactive protein and procalcitonin are generally fairly sensitive but are too non-specific to accurately diagnose infection as the cause of inflammation, nor to identify which infected/inflamed patients will proceed to organ failure. Many patients will thus be unnecessarily treated with antibiotics while a smaller number may be inappropriately not treated. It is unlikely that a single biomarker will yield all the necessary information so technologies that can measure multiple markers will probably be more useful. Such devices are being developed, often for point-of-care testing, and include PCR (polymerase chain reaction), lateral flow, microfluidics, and nanotechnology4,5. These promise to deliver results in 30-75 minutes at the bedside with no need for involvement of the main hospital laboratory. The challenge now is to find the best biomarkers.Finally, for treatment selection, it has become clear that sepsis is an umbrella syndrome with many patient subsets within. Inflammatory and hyperinflammatory phenotypes have been described from a combination of clinical and biological markers. At least from retrospective studies, it appears that these subsets respond differently to fluid, PEEP (positive end-expiratory pressure), oxygen, and corticosteroids. So targeted treatment may become a reality in the not-too-distant future though prospective validation is first needed.

Rapid Diagnosis of Blood Stream Infections in ICU: Recent Advances

Blood stream infections and sepsis are major causes of hospitalization in most intensive care units around the globe, especially in developing countries like Nepal. Although the sepsis guidelines emphasize the role of early institution of appropriate antibiotics, it is practically challenging due to delayed turnover time of currently available diagnostic tests. Modifications in traditional blood culture methods, use of molecular techniques and availability of biomarkers have raised hope in rapid detection of blood stream infections.