Diagnosis of septic shock by serum measurement of human neutrophil lipocalin by a rapid homogeneous assay.

BACKGROUND: Human neutrophil lipocalin (HNL) is a marker of neutrophil activation and has a high efficacy in diagnosing bacterial infections. In this study, we applied the AlphaLISA technique to measure the serum level of HNL, evaluate HNL's efficacy in diagnosing septic shock, and identify any association between HNL level and septic patients' prognosis. METHODS: We collected 146 serum samples from the Fifth Medical Center of Chinese PLA General Hospital. HNL was measured by AlphaLISA and results were compared with commercial ELISA kits. We studied 78 patients admitted to the ICU with sepsis and data on their clinical and physiological characteristics were recorded. Blood levels of HNL, procalcitonin (PCT), high-sensitivity C-reactive protein (hs-CRP), and lactate were measured. A receiver operating characteristic (ROC) curve was used to evaluate the performance of each marker. RESULTS: The AlphaLISA assay for serum HNL had a detection range from 1.5 ng/mL to 1000 ng/mL, with a detection limit of 1 ng/mL and a detection time of approximately 25 min. The AlphaLISA assay's results were in high agreement with ELISA results (R2 = 0.9413). HNL levels were analyzed in sepsis patients, and HNL was significantly higher in sepsis patients with shock compared to sepsis patients without shock (median 356.47 ng/mL vs 158.93 ng/mL, P < 0.0001) and in the 28-day non-survivor group compared to the 28-day survivor group (median 331.83 ng/mL vs 175.17 ng/mL, P < 0.0001). ROC curve analysis was performed for the biomarkers. In differentiating the diagnosis of septic shock from sepsis patients, HNL was the most effective marker (AUC = 0.857), followed by PCT (AUC = 0.754) and hs-CRP (AUC = 0.627). In predicting the prognosis of septic patients, lactate had the best effect (AUC = 0.805), followed by HNL (AUC = 0.784), PCT (AUC = 0.721), and hs-CRP (AUC = 0.583). CONCLUSIONS: As an assessment tool, we found that our AlphaLISA had good consistency with an ELISA and had several other advantages, including requiring a shorter processing time and detecting a wider range of serum HNL concentrations. Monitoring serum HNL levels of patients admitted to the ICU might be useful in distinguishing sepsis patients who have septic shock from other sepsis patients, indicating its value in the prediction of sepsis patient prognosis.

Development of an AlphaLISA assay for sensitive and accurate detection of influenza B virus.

Objective: Influenza B virus (IBV) is highly contagious, spreads rapidly, and causes seasonal epidemic respiratory disease in the human population, especially in immunocompromised people and young children. Clinical manifestations in this high-risk population are often more severe than in immunocompetent hosts and sometimes atypical. Therefore, rapid, and accurate detection of IBV is important. Methods: An amplified luminescent proximity homogeneous assay linked immunosorbent assay (AlphaLISA) was developed for detection of IBV by optimizing the ratio of IBV antibody-labeled receptor beads, streptavidin-conjugated donor beads and biotinylated IBV antibody, as well as the optimal temperature and time conditions for incubation. Assay sensitivity, specificity and reproducibility were evaluated. A total of 228 throat swab samples and inactivated influenza B virus were tested by AlphaLISA and lateral flow colloidal gold-based immunoassay (LFIA). Results: AlphaLISA produced the best results for detection of inactivated influenza B virus when IBV antibody-labeled acceptor beads were 50 µg/ mL, streptavidin-conjugated donor beads were 40 µg/mL, and biotinylated IBV antibody was 0.5 µg/mL at 37°C for 15-10 min. Under these conditions, AlphaLISA had a limit of detection of 0.24 ng/mL for the detection of influenza B nucleoprotein, did not cross react with other common respiratory viruses, and showed good reproducibility with inter-assay coefficient of variation (CV) and intra-assay CV < 5%. The results of 228 clinical throat swab samples showed good agreement between AlphaLISA and LFIA (Kappa = 0.982), and AlphaLISA showed better sensitivity than LFIA for detecting inactivated influenza B virus. Conclusion: AlphaLISA showed higher sensitivity and throughput in the detection of IBV and can be used for IBV diagnosis and epidemic control.

Detection of Pathogens and Antimicrobial Resistance Genes in Ventilator-Associated Pneumonia by Metagenomic Next-Generation Sequencing Approach.

Background: The early identification of pathogens and their antibiotic resistance are essential for the management and treatment of patients affected by ventilator-associated pneumonia (VAP). However, microbiological culture may be time-consuming and has a limited culturability of many potential pathogens. In this study, we developed a rapid nanopore-based metagenomic next-generation sequencing (mNGS) diagnostic assay for detection of VAP pathogens and antimicrobial resistance genes (ARGs). Patients and Methods: Endotracheal aspirate (ETA) samples from 63 patients with suspected VAP were collected between November 2021 and July 2022. Receiver operating characteristic (ROC) curves were established to compare the pathogen identification performance of the target pathogen reads, reads percent of microbes (RPM) and relative abundance (RA). The evaluation of the accuracy of mNGS was performed comparing with the gold standard and the composite standard, respectively. Then, the ARGs were analyzed by mNGS. Results: ROC curves showed that RA has the highest diagnostic value and the corresponding threshold was 9.93%. The sensitivity and specificity of mNGS test were 91.3% and 78.3%, respectively, based on the gold standard, while the sensitivity and specificity of mNGS test were 97.4% and 100%, respectively, based on the composite standard. A total of 13 patients were virus-positive based on mNGS results, while the coinfection rate increased from 27% to 46% compared to the rate obtained based on clinical findings. The mNGS test also performed well at predicting antimicrobial resistance phenotypes. Patients with a late-onset VAP had a significantly greater proportion of ARGs in their respiratory microbiome compared to those with early-onset VAP (P = 0.041). Moreover, the median turnaround time of mNGS was 4.43 h, while routine culture was 72.00 h. Conclusion: In this study, we developed a workflow that can accurately detect VAP pathogens and enable prediction of antimicrobial resistance phenotypes within 5 h of sample receipt by mNGS.

Development of a rapid homogeneous immunoassay for detection of rotavirus in stool samples.

Rotavirus is the main pathogen causing acute viral gastroenteritis. Accurate and rapid diagnosis of rotavirus infection is important to determine appropriate treatment, prevention of unnecessary antibiotics use and control of infection spread. In this study, we established a rapid, accurate, and sensitive amplified luminescent proximity homogeneous assay linked immunosorbent assay (AlphaLISA) for detecting rotavirus and evaluated its efficacy in human stool samples. Our results demonstrated that the sensitivity of AlphaLISA (5-8) significantly exceeded that of the immunochromatographic assay (ICA, 5-4) for rotavirus antigen detection. The intra-assay and inter-assay coefficients of variation were 2.99-3.85% and 5.27-6.51%, respectively. Furthermore, AlphaLISA was specific for rotavirus and did not cross-react with other common diarrhea viruses. AlphaLISA and real-time reverse transcription polymerase chain reaction (RT-qPCR, which is considered a gold standard for detecting diarrhea viruses) tests showed consistent results on 235 stool samples, with an overall consistency rate of 97.87% and a kappa value of 0.894 (P < 0.001). The overall consistency rate of ICA compared with RT-qPCR was 95.74%. AlphaLISA showed better consistency with RT-qPCR than the routinely used ICA for rotavirus detection in stool samples. The AlphaLISA method can be used in clinical practice for the rapid, accurate, and sensitive detection of rotavirus infection.