Diagnosis of Multisystem Inflammatory Syndrome in Children by a Whole-Blood Transcriptional Signature.

BACKGROUND: To identify a diagnostic blood transcriptomic signature that distinguishes multisystem inflammatory syndrome in children (MIS-C) from Kawasaki disease (KD), bacterial infections, and viral infections. METHODS: Children presenting with MIS-C to participating hospitals in the United Kingdom and the European Union between April 2020 and April 2021 were prospectively recruited. Whole-blood RNA Sequencing was performed, contrasting the transcriptomes of children with MIS-C (n = 38) to those from children with KD (n = 136), definite bacterial (DB; n = 188) and viral infections (DV; n = 138). Genes significantly differentially expressed (SDE) between MIS-C and comparator groups were identified. Feature selection was used to identify genes that optimally distinguish MIS-C from other diseases, which were subsequently translated into RT-qPCR assays and evaluated in an independent validation set comprising MIS-C (n = 37), KD (n = 19), DB (n = 56), DV (n = 43), and COVID-19 (n = 39). RESULTS: In the discovery set, 5696 genes were SDE between MIS-C and combined comparator disease groups. Five genes were identified as potential MIS-C diagnostic biomarkers (HSPBAP1, VPS37C, TGFB1, MX2, and TRBV11-2), achieving an AUC of 96.8% (95% CI: 94.6%-98.9%) in the discovery set, and were translated into RT-qPCR assays. The RT-qPCR 5-gene signature achieved an AUC of 93.2% (95% CI: 88.3%-97.7%) in the independent validation set when distinguishing MIS-C from KD, DB, and DV. CONCLUSIONS: MIS-C can be distinguished from KD, DB, and DV groups using a 5-gene blood RNA expression signature. The small number of genes in the signature and good performance in both discovery and validation sets should enable the development of a diagnostic test for MIS-C.

Clinical Diagnostics of Bacterial Infections and Their Resistance to Antibiotics-Current State and Whole Genome Sequencing Implementation Perspectives.

Antimicrobial resistance (AMR), defined as the ability of microorganisms to withstand antimicrobial treatment, is responsible for millions of deaths annually. The rapid spread of AMR across continents warrants systematic changes in healthcare routines and protocols. One of the fundamental issues with AMR spread is the lack of rapid diagnostic tools for pathogen identification and AMR detection. Resistance profile identification often depends on pathogen culturing and thus may last up to several days. This contributes to the misuse of antibiotics for viral infection, the use of inappropriate antibiotics, the overuse of broad-spectrum antibiotics, or delayed infection treatment. Current DNA sequencing technologies offer the potential to develop rapid infection and AMR diagnostic tools that can provide information in a few hours rather than days. However, these techniques commonly require advanced bioinformatics knowledge and, at present, are not suited for routine lab use. In this review, we give an overview of the AMR burden on healthcare, describe current pathogen identification and AMR screening methods, and provide perspectives on how DNA sequencing may be used for rapid diagnostics. Additionally, we discuss the common steps used for DNA data analysis, currently available pipelines, and tools for analysis. Direct, culture-independent sequencing has the potential to complement current culture-based methods in routine clinical settings. However, there is a need for a minimum set of standards in terms of evaluating the results generated. Additionally, we discuss the use of machine learning algorithms regarding pathogen phenotype detection (resistance/susceptibility to an antibiotic).

A Portable RT-LAMP/CRISPR Machine for Rapid COVID-19 Screening.

The COVID-19 pandemic has changed people's lives and has brought society to a sudden standstill, with lockdowns and social distancing as the preferred preventative measures. To lift these measurements and reduce society's burden, developing an easy-to-use, rapid, and portable system to detect SARS-CoV-2 is mandatory. To this end, we developed a portable and semi-automated device for SARS-CoV-2 detection based on reverse transcription loop-mediated isothermal amplification followed by a CRISPR/Cas12a reaction. The device contains a heater element mounted on a printed circuit board, a cooler fan, a proportional integral derivative controller to control the temperature, and designated areas for 0.2 mL Eppendorf® PCR tubes. Our system has a limit of detection of 35 copies of the virus per microliter, which is significant and has the capability of being used in crisis centers, mobile laboratories, remote locations, or airports to diagnose individuals infected with SARS-CoV-2. We believe the current methodology that we have implemented in this article is beneficial for the early screening of infectious diseases, in which fast screening with high accuracy is necessary.

Evaluation of the ID NOW among symptomatic individuals during the Omicron wave.

Introduction. Starting in December, 2020, the ID NOW was implemented throughout the province of Alberta, Canada (population 4.4 million) in various settings.Gap statement. ID NOW's test performance with SARS-CoV-2 Omicron variant BA.1 is unknown.Aim. To assess the ID NOW performance among symptomatic individuals during the BA.1 Omicron wave and compare it to previous SARS-CoV-2 variant waves.Methodology. The ID NOW was assessed in two locations among symptomatic individuals: rural hospitals and community assessment centres (AC) during the period 5-18 January 2022. Starting 5 January, Omicron represented >95 % of variants detected in our population. For every individual tested, two swabs were collected: one for ID NOW testing and the other for either reverse-transcriptase polymerase chain reaction (RT-PCR) confirmation of negative ID NOW results or for variant testing of positive ID NOW results.Results. A total of 3041 paired samples were analysed (1139 RT-PCR positive). From this, 1873 samples were from 42 COVID-19 AC and 1168 from 69 rural hospitals. ID NOW sensitivity for symptomatic individuals presenting to community AC and rural hospitals was 96.0 % [95 % confidence interval (CI) 94.5-97.3 %, n=830 RT-PCR positive], and 91.6 % (95 % CI 87.9-94.4 %, n=309 RT-PCR positive), respectively. SARS-CoV-2 positivity rate was very high for both populations (44.3 % at AC, 26.5 % in hospital).Conclusions. Sensitivity of ID NOW SARS-CoV-2, compared to RT-PCR, is very high during the BA.1 Omicron wave, and is significantly higher when compared to previous SARS-CoV-2 variant waves.

Prospective population-level validation of the Abbott ID NOW severe acute respiratory syndrome coronavirus 2 device implemented in multiple settings for testing asymptomatic and symptomatic individuals.

OBJECTIVE: Diagnostic evaluation of the ID NOW coronavirus disease 2019 (COVID-19) assay in various real-world settings among symptomatic and asymptomatic individuals. METHODS: Depending on the setting, the ID NOW testing was performed using oropharyngeal swabs (OPSs) taken from patients with symptoms suggestive of COVID-19, asymptomatic close contacts, or asymptomatic individuals as part of outbreak point prevalence screening. From January to April 2021, a select number of sites switched from using OPS to combined oropharyngeal and nasal swab (O + NS) for ID NOW testing. For every individual tested, two swabs were collected by a health care worker: one swab (OPS or O + NS) for ID NOW testing and a separate swab (OPS or nasopharyngeal swab) for RT-PCR. RESULTS: A total of 129 112 paired samples were analysed (16 061 RT-PCR positive). Of these, 81 697 samples were from 42 COVID-19 community collection sites, 16 924 samples were from 69 rural hospitals, 1927 samples were from nine emergency shelters and addiction treatment facilities, 23 802 samples were from six mobile units that responded to 356 community outbreaks, and 4762 O + NS swabs were collected from three community collection sites and one emergency shelter. The ID NOW assay sensitivity was the highest among symptomatic individuals presenting to community collection sites (92.5%; 95% CI, 92.0-93.0%) and the lowest for asymptomatic individuals associated with community outbreaks (73.9%; 95% CI, 69.8-77.7%). Specificity was >99% in all populations tested. DISCUSSION: The sensitivity of ID NOW severe acute respiratory syndrome coronavirus 2 testing is the highest when used in symptomatic community populations not seeking medical care. Sensitivity and positive predictive value drop by approximately 10% when tested on asymptomatic populations. Using combined oropharyngeal and nasal swabs did not improve the performance of ID NOW assay.

Self-directed molecular diagnostics (SdMDx) system for COVID-19 via one-pot processing.

Rapid, sensitive, and specific detection of the severe acute respiratory syndrome coronavirus (SARS-CoV)- 2 during early infection is pivotal in controlling the spread and pathological progression of Coronavirus Disease 2019 (COVID-19). Thus, highly accurate, affordable, and scalable point-of-care (POC) diagnostic technologies are necessary. Herein, we developed a rapid and efficient self-directed molecular diagnostic (SdMDx) system for SARS-CoV-2. This system combines the sample preparation step, including virus enrichment and extraction processes, which involve dimethyl suberimidate dihydrochloride and diatomaceous earth functionalized with 3-aminopropyl(diethoxy)methylsilane, and the detection step using loop-mediated isothermal amplification-lateral flow assay (LAMP-LFA). Using the SdMDx system, SARS-CoV-2 could be detected within 47 min by hand without the need for any larger instruments. The SdMDx system enabled detection as low as 0.05 PFU in the culture fluid of SARS-CoV-2-infected VeroE6 cells. We validated the accuracy of the SdMDx system on 38 clinical nasopharyngeal specimens. The clinical utility of the SdMDx system for targeting the S gene of SARS-CoV-2 showed 94.4% sensitivity and 100% specificity. This system is more sensitive than antigen and antibody assays, and it minimizes the use of complicated processes and reduces contamination risks. Accordingly, we demonstrated that the SdMDx system enables a rapid, accurate, simple, efficient, and inexpensive detection of SARS-CoV-2 at home, in emergency facilities, and in low-resource sites as a pre-screening platform and POC testing through self-operation and self-diagnosis.

Counter-propagating Gaussian beam enhanced Raman spectroscopy for rapid reagentless detection of respiratory pathogens in nasal swab samples

Co-circulation of respiratory viruses compounded by similarities in clinical presentation and mode of transmission underscores the need for broad range pathogen detection. Accurate identification and diagnosis at the point-of-need is critical to limiting disease spread. A novel point-of-need Raman spectroscopy-based platform is described for rapid detection of multiple respiratory pathogens in nasal swab samples with high sensitivity and specificity. The system takes advantage of a counter-propagating Gaussian beam focused within the sample chamber that augments the Raman signal of pathogens. Combined with multiclass machine learning spectral analysis via Gradient Boosting Machine, accurate identification of SARS-CoV-2, human coronaviruses OC43, NL63, 229E, Influenza A (H1N1), respiratory syncytial virus, and Streptococcus pyogenes in spiked clinical nasal swab samples was demonstrated at 99% sensitivity and 93% specificity. The limit of detection was assessed using binary class Support Vector Machine with SARS-CoV-2 in nasal swab samples against negative control at 2.2 × 104 virions/swab. The spectrometer can be operated by minimally trained personnel with software-generated diagnostic yes/no results in 2 min or less, making it well suited for point-of-need applications. Furthermore, adaptive algorithms can detect and differentiate new and emerging variants using a Raman spectral database.

ADAPTABLE ENGINEERING OF CELLULOSE-BASED VERTICAL FLOW ASSAYS FOR RAPID DIAGNOSTICS - THE CASE OF COVID-19

Paper microfluidics has had a rich history in medical diagnostics owing to their portability, low-cost and capacity for mass manufacture. While nitrocellulose has widespread use in commercial paper-based assays, shortages can become a bottleneck for deployment. Here, we seek to overcome this limitation by enabling swift and efficient production of cellulose-based paper assays with minimal substrate processing via protein engineering. We demonstrate good clinical and lab-based performance for both serological and antigen rapid tests and their compatibility with roll-to-roll mass manufacturing, which validates our proposed workflow for commercialization. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.

Limitations of Molecular and Antigen Test Performance for SARS-CoV-2 in Symptomatic and Asymptomatic COVID-19 Contacts.

COVID-19 has brought unprecedented attention to the crucial role of diagnostics in pandemic control. We compared severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) test performance by sample type and modality in close contacts of SARS-CoV-2 cases. Close contacts of SARS-CoV-2-positive individuals were enrolled after informed consent. Clinician-collected nasopharyngeal (NP) swabs in viral transport media (VTM) were tested with a routine clinical reference nucleic acid test (NAT) and PerkinElmer real-time reverse transcription-PCR (RT-PCR) assay; positive samples were tested for infectivity using a VeroE6TMPRSS2 cell culture model. Self-collected passive drool was also tested using the PerkinElmer RT-PCR assay. For the first 4 months of study, midturbinate swabs were tested using the BD Veritor rapid antigen test. Between 17 November 2020 and 1 October 2021, 235 close contacts of SARS-CoV-2 cases were recruited, including 95 with symptoms (82% symptomatic for ≤5 days) and 140 asymptomatic individuals. Reference NATs were positive for 53 (22.6%) participants; 24/50 (48%) were culture positive. PerkinElmer testing of NP and saliva samples identified an additional 28 (11.9%) SARS-CoV-2 cases who tested negative by reference NAT. Antigen tests performed for 99 close contacts showed 83% positive percent agreement (PPA) with reference NAT among early symptomatic persons, but 18% PPA in others; antigen tests in 8 of 11 (72.7%) culture-positive participants were positive. Contacts of SARS-CoV-2 cases may be falsely negative early after contact, but more sensitive platforms may identify these cases. Repeat or serial SARS-CoV-2 testing with both antigen and molecular assays may be warranted for individuals with high pretest probability for infection.

Field Evaluation of the New Rapid NG-Test® SARS-CoV-2 Ag for Diagnosis of COVID-19 in the Emergency Department of an Academic Referral Hospital.

Background: As the COVID-19 pandemic resurges affecting large numbers of patients, rapid, and accurate diagnosis using point-of-care tests is very important. Objectives: To evaluate the NG-Test® SARS-CoV-2 Ag (NG-Test) immunoassay for qualitative detection of SARS-CoV-2 antigen in nasopharyngeal (NP) and oropharyngeal (OP) samples compared with RT-PCR, in patients attending the Emergencies of an academic referral hospital. Methods: All adult ambulatory patients presenting to the Emergencies of "Attikon" University hospital (Athens, Greece) within three consecutive hours per day between December 2020 and March 2021 and for whom SARS-CoV-2 PCR testing was requested were included. Two NP and one OP samples obtained from each participant were analyzed to determine the diagnostic performance [sensitivity, specificity, positive/negative predictive values (PPV/NPV)] of the NG-Test (NP/OP swabs) in comparison to the reference RT-PCR (NP swab). Results: Overall, 134/263 (51%) patients tested were RT-PCR positive, whereof 108 (overall sensitivity 81%, 95% CI 73-87%) were NP NG-Test positive (PPV 99%, NPV 83%) and 68 (overall sensitivity 51%, 95% CI 42-59%) were OP NG-Test positive (PPV 100%, NPV 66%). The test's specificity (95% CI) was 99% (95-100%) and 100% (96-100%) for NP and OP swabs, respectively. The assay's sensitivity (95% CI) for high viral load (Ct ≤25) was 99% (92-100%) and 71% (60-81%) for NP and OP swabs, respectively. Conclusions: NG-Test using NP swabs detected almost all patients with high viral loads, showing satisfactory performance as a point-of-care test for NP samples obtained from patients with acute infection.

Dealing with a Pandemic: Emerging Tools, Solutions, and Challenges.

As a result of the COVID-19 pandemic, nations across the globe have responded by attempting to understand how the virus was spreading in their communities, in order to isolate cases, reduce morbidity and mortality, and avoid overwhelming healthcare facilities. In this article, we describe the global response to tracking the virus and discuss new technological advances in molecular testing that have been deployed and developed to track and mitigate COVID-19. We also discuss how the successes and failures observed in the COVID-19 pandemic can be extrapolated to improve our ability to respond to the next pandemic.

High Sensitivity, Rapid Detection of Virus in High Traffic Environments.

The global pandemic caused by the SARS-CoV-2 virus has underscored the need for rapid, simple, scalable, and high-throughput multiplex diagnostics in non-laboratory settings. Here we demonstrate a multiplex reverse-transcription loop-mediated isothermal amplification (RT-LAMP) coupled with a gold nanoparticle-based lateral flow immunoassay (LFIA) capable of detecting up to three unique viral gene targets in 15 min. RT-LAMP primers associated with three separate gene targets from the SARS-CoV-2 virus (Orf1ab, Envelope, and Nucleocapsid) were added to a one-pot mix. A colorimetric change from red to yellow occurs in the presence of a positive sample. Positive samples are run through a LFIA to achieve specificity on a multiplex three-test line paper assay. Positive results are indicated by a characteristic crimson line. The device is almost fully automated and is deployable in any community setting with a power source.

One Swab Fits All: Performance of a Rapid, Antigen-Based SARS-CoV-2 Test Using a Nasal Swab, Nasopharyngeal Swab for Nasal Collection, and RT-PCR Confirmation from Residual Extraction Buffer.

BACKGROUND: Point-of-care SARS-CoV-2 antigen tests have great potential to help combat the COVID-19 pandemic. In the performance of a rapid, antigen-based SARS-CoV-2 test (RAT), our study had 3 main objectives: to determine the accuracy of nasal swabs, the accuracy of using nasopharyngeal swabs for nasal collection (nasalNP), and the effectiveness of using residual extraction buffer for real-time reverse-transcriptase PCR (RT-PCR) confirmation of positive RAT (rPan). METHODS: Symptomatic adults recently diagnosed with COVID-19 in the community were recruited into the study. Nasal samples were collected using either a nasalNP or nasal swab and tested immediately with the RAT in the individual's home by a health care provider. 500 µL of universal transport media was added to the residual extraction buffer after testing and sent to the laboratory for SARS-CoV-2 testing using RT-PCR. Parallel throat swabs tested with RT-PCR were used as the reference comparators. RESULTS: One hundred and fifty-five individuals were included in the study (99 nasal swabs, 56 nasalNP). Sensitivities of nasal samples tested on the RAT using either nasal or nasalNP were 89.0% [95% confidence interval (CI) 80.7%-94.6%] and 90.2% (95% CI 78.6%-96.7%), respectively. rPan positivity agreement compared to throat RT-PCR was 96.2%. CONCLUSIONS: RAT reliably detect SARS-CoV-2 from symptomatic adults in the community presenting within 7 days of symptom onset using nasal swabs or nasalNP. High agreement with rPan can avoid the need for collecting a second swab for RT-PCR confirmation or testing of variants of concern from positive RAT in this population.

Assessing Differential Binding of Aggregation-Induced Emission-Based Luminogens to Host Interacting Surface Proteins of SARS-CoV-2 and Influenza Virus-An in silico Approach.

Early detection of asymptomatic cases through mass screening is essential to constrain the coronavirus disease 2019 (COVID-19) transmission. However, the existing diagnostic strategies are either resource-intensive, time-consuming, or less sensitive, which limits their use in the development of rapid mass screening strategies. There is a clear pressing need for simple, fast, sensitive, and economical diagnostic strategy for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) screening even in resource-limited settings. In the current work, we assessed the in silico feasibility of directly labeling virus surface proteins using fluorogenic molecules with aggregation-induced emission (AIE) property. Here, we present the results for binding of two such AIE probes, phosphonic acid derivative of tetraphenyl ethylene (TPE-P) and sulfonic acid derivative of tetraphenyl ethylene (TPE-S), to SARS-CoV-2 spike protein based on in silico docking studies. Our results show that both TPE-P and TPE-S bind to angiotensin converting enzyme 2 (ACE2)-binding, and N-terminal domains of SARS-CoV-2 spike protein. Molecular dynamic simulations have revealed specific nature of these interactions. We also show that TPE-P and TPE-S bind to hemagglutinin protein of influenza virus, but the interaction strength was found to be different. This difference in interaction strength may affect the emission spectrum of aforementioned AIE probes. Together, these results form a basis for the development of AIE-based diagnostics for differential detection of SARS-CoV-2 and influenza viruses. We believe that these in silico predictions certainly aid in differentially labeling of the both viruses toward the development of rapid detection by AIE probes.

Development of a Novel Loop Mediated Isothermal Amplification Assay (LAMP) for the Rapid Detection of Epizootic Haemorrhagic Disease Virus.

Epizootic haemorragic disease (EHD) is an important disease of white-tailed deer and can cause a bluetongue-like illness in cattle. A definitive diagnosis of EHD relies on molecular assays such as real-time RT-qPCR or conventional PCR. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a cost-effective, specific, and sensitive technique that provides an alternative to RT-qPCR. We designed two sets of specific primers targeting segment-9 of the EHD virus genome to enable the detection of western and eastern topotypes, and evaluated their performance in singleplex and multiplex formats using cell culture isolates (n = 43), field specimens (n = 20), and a proficiency panel (n = 10). The limit of detection of the eastern and western RT-LAMP assays was estimated as ~24.36 CT and as ~29.37 CT in relation to real-time RT-qPCR, respectively, indicating a greater sensitivity of the western topotype singleplex RT-LAMP. The sensitivity of the western topotype RT-LAMP assay, relative to the RT-qPCR assay, was 72.2%, indicating that it could be theoretically used to detect viraemic cervines and bovines. For the first time, an RT-LAMP assay was developed for the rapid detection of the EHD virus that could be used as either a field test or high throughput screening tool in established laboratories to control the spread of EHD.

Rapid COVID-19 Molecular Diagnostic System Using Virus Enrichment Platform.

The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus (SARS-CoV)-2, is rapidly spreading and severely straining the capacities of public health communities and systems around the world. Therefore, accurate, rapid, and robust diagnostic tests for COVID-19 are crucial to prevent further spread of the infection, alleviate the burden on healthcare and diagnostic facilities, and ensure timely therapeutic intervention. To date, several detection methods based on nucleic acid amplification have been developed for the rapid and accurate detection of SARS-CoV-2. Despite the myriad of advancements in the detection methods for SARS-CoV-2, rapid sample preparation methods for RNA extraction from viruses have rarely been explored. Here, we report a rapid COVID-19 molecular diagnostic system that combines a self-powered sample preparation assay and loop-mediated isothermal amplification (LAMP) based naked-eye detection method for the rapid and sensitive detection of SARS-CoV-2. The self-powered sample preparation assay with a hydrophilic polyvinylidene fluoride filter and dimethyl pimelimidate can be operated by hand, without the use of any sophisticated instrumentation, similar to the reverse transcription (RT)-LAMP-based lateral flow assay for the naked-eye detection of SARS-CoV-2. The COVID-19 molecular diagnostic system enriches the virus population, extracts and amplifies the target RNA, and detects SARS-CoV-2 within 60 min. We validated the accuracy of the system by using 23 clinical nasopharyngeal specimens. We envision that this proposed system will enable simple, facile, efficient, and inexpensive diagnosis of COVID-19 at home and the clinic as a pre-screening platform to reduce the burden on the medical staff in this pandemic era.

Glycan-Based Flow-Through Device for the Detection of SARS-COV-2.

The COVID-19 pandemic, and future pandemics, require diagnostic tools to track disease spread and guide the isolation of (a)symptomatic individuals. Lateral-flow diagnostics (LFDs) are rapid and of lower cost than molecular (genetic) tests, with current LFDs using antibodies as their recognition units. Herein, we develop a prototype flow-through device (related, but distinct to LFDs), utilizing N-acetyl neuraminic acid-functionalized, polymer-coated, gold nanoparticles as the detection/capture unit for SARS-COV-2, by targeting the sialic acid-binding site of the spike protein. The prototype device can give rapid results, with higher viral loads being faster than lower viral loads. The prototype's effectiveness is demonstrated using spike protein, lentiviral models, and a panel of heat-inactivated primary patient nasal swabs. The device was also shown to retain detection capability toward recombinant spike proteins from several variants (mutants) of concern. This study provides the proof of principle that glyco-lateral-flow devices could be developed to be used in the tracking monitoring of infectious agents, to complement, or as alternatives to antibody-based systems.

Rapid identification of bacteria from respiratory samples of patients hospitalized in intensive care units, with FilmArray Pneumonia Panel Plus.

OBJECTIVES: This study aimed to evaluate the performance of FilmArray Pneumonia Panel Plus (FA-PP) for the detection of typical bacterial pathogens in respiratory samples from patients hospitalized in intensive care units (ICUs). METHODS: FA-PP was implemented for clinical use in the microbiology laboratory in March 2020. A retrospective analysis on a consecutive cohort of adult patients hospitalized in ICUs between March 2020 and May 2020 was undertaken. The respiratory samples included sputum, blind bronchoalveolar lavage (BBAL) and protected specimen brush (PSB). Conventional culture and FA-PP were performed in parallel. RESULTS: In total, 147 samples from 92 patients were analysed; 88% had coronavirus disease 2019 (COVID-19). At least one pathogen was detected in 46% (68/147) of samples by FA-PP and 39% (57/147) of samples by culture. The overall percentage agreement between FA-PP and culture results was 98% (93-100%). Bacteria with semi-quantitative FA-PP results ≥105 copies/mL for PSB samples, ≥106 copies/mL for BBAL samples and ≥107 copies/mL for sputum samples reached clinically significant thresholds for growth in 90%, 100% and 91% of cultures, respectively. FA-PP detected resistance markers, including mecA/C, blaCTX-M and blaVIM. The median turnaround time was significantly shorter for FA-PP than for culture. CONCLUSIONS: FA-PP may constitute a faster approach to the diagnosis of bacterial pneumonia in patients hospitalized in ICUs.