Clinical evaluation of a highly multiplexed CRISPR-based diagnostic assay for diagnosing lower respiratory tract infection: a prospective cohort study.

OBJECTIVE: Accurate and rapid identification of causative pathogens is essential to guide the clinical management of lower respiratory tract infections (LRTIs). Here we conducted a single-centre prospective study in 284 patients suspected of lower respiratory tract infections to evaluate the utility of a nucleic acid test based on highly multiplexed polymerase chain reaction (PCR) and CRISPR-Cas12a. METHODS: We determined the analytical and diagnostic performance of the CRISPR assay using a combination of reference standards, including conventional microbiological tests (CMTs), metagenomic Next-Generation Sequencing (mNGS), and clinical adjudication by a panel of experts on infectious diseases and microbiology. RESULTS: The CRISPR assay showed a higher detection rate (63.0%) than conventional microbiological tests (38.4%) and was lower than metagenomic Next-Generation Sequencing (72.9%). In detecting polymicrobial infections, the positivity rate of the CRISPR assay (19.4%) was higher than conventional microbiological tests (3.5%) and lower than metagenomic Next-Generation Sequencing (28.9%). The overall diagnostic sensitivity of the CRISPR assay (67.8%) was higher than conventional microbiological tests (41.8%), and lower than metagenomic Next-Generation Sequencing (93.2%). CONCLUSIONS: Considering the low cost, ease of operation, short turnaround time, and broad range of pathogens detected in a single test, the CRISPR assay has the potential to be implemented as a screening tool for the aetiological diagnosis of lower respiratory tract infections patients, especially in cases where atypical bacteria or coinfections are suspected.

Clinical diagnostic value of metagenomic next-generation sequencing in patients with acute infection in emergency department.

Objective: To explore the value of metagenomic next-generation sequencing (mNGS) and culture in microbial diagnosis of patients with acute infection. Methods: We retrospectively analyzed 206 specimens from 163 patients who were admitted to the emergency department of The First Affiliated Hospital of Sun Yat-sen University between July 2020, and July 2021. We evaluated the diagnostic efficacy of mNGS and in-hospital traditional culture. Results: The total positive rate of mNGS was significantly higher than that culture methods (71.4 % vs 40.8 %, p < 0.001), while the sensitivity and accuracy of mNGS were found to be 92.9 % and 88.2 % respectively. However, culture exhibited superior specificity with a value of 92.6 % compared to 75.9 % for mNGS. The detection efficiency of mNGS and culture for fungi was comparable, but mNGS showed superior performance for bacterial detection. In the analysis of sepsis samples, mNGS outperformed traditional culture methods in diagnosing various types of samples, especially for sputum and bronchoalveolar lavage fluid. Among the identified infections, bacterial infections were the most common single infection (37.5 %). Additionally, bacterial-fungal infections represented the most prevalent form of mixed infection (77.3 %). Candida albicans and Staphylococcus aureus were identified as the predominant pathogens in the survival and death groups, respectively. No significant differences in microbial diversity were observed. Conclusion: Compared to culture methods, mNGS demonstrates superior positive rates, sensitivity, and accuracy in the rapid detection of acute infections, particularly in critically ill patients such as those with sepsis. This capability establishes a foundation for the swift and precise identification of pathogens, allowing for the analysis of clinical indicators and patient prognosis based on the extensive data generated from mNGS.

Getting Up to Speed: Rapid Pathogen and Antimicrobial Resistance Diagnostics in Sepsis.

Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. Time to receive effective therapy is a primary determinant of mortality in patients with sepsis. Blood culture is the reference standard for the microbiological diagnosis of bloodstream infections, despite its low sensitivity and prolonged time to receive a pathogen detection. In recent years, rapid tests for pathogen identification, antimicrobial susceptibility, and sepsis identification have emerged, both culture-based and culture-independent methods. This rapid narrative review presents currently commercially available approved diagnostic molecular technologies in bloodstream infections, including their clinical performance and impact on patient outcome, when available. Peer-reviewed publications relevant to the topic were searched through PubMed, and manufacturer websites of commercially available assays identified were also consulted as further sources of information. We have reviewed data about the following technologies for pathogen identification: fluorescence in situ hybridization with peptide nucleic acid probes (Accelerate PhenoTM), microarray-based assay (Verigene®), multiplex polymerase chain reaction (cobas® eplex, BioFire® FilmArray®, Molecular Mouse, Unyvero BCU SystemTM), matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (Rapid MBT Sepsityper®), T2 magnetic resonance (T2Bacteria Panel), and metagenomics-based assays (Karius©, DISQVER®, Day Zero Diagnostics). Technologies for antimicrobial susceptibility testing included the following: Alfed 60 ASTTM, VITEK® REVEALTM, dRASTTM, ASTar®, Fastinov®, QuickMIC®, ResistellTM, and LifeScale. Characteristics, microbiological performance, and issues of each method are described, as well as their clinical performance, when available.

Nanopore Sequencing Technology: A Reliable Method for Pathogen Diagnosis in Elderly Patients with Community-Acquired Pneumonia.

Purpose: Next-generation sequencing of the metagenome (mNGS) is gaining traction as a valuable tool for diagnosing infectious diseases. Compared to mNGS, pathogen detection based on Oxford Nanopore Technology further shortens the detection time. This study seeks to assess the efficacy of Nanopore sequencing in identifying pathogens associated with community-acquired pneumonia (CAP) among elderly individuals in China. Patients and Methods: From January 2023 to June 2023, elderly patients with CAP were prospectively recruited from Hangzhou First People's Hospital. A comprehensive set of clinical data was gathered, and bronchoalveolar lavage (BAL) fluid samples were collected. Concurrently, pathogen identification was performed using conventional microbiological diagnostic methods, Illumina sequencing, and Nanopore sequencing, and the diagnostic efficacy of pathogen detection was compared. Results: The study included a total of 29 patients. The diagnostic positivity rates of traditional microbiological detection, Illumina sequencing, and Nanopore sequencing were 24.1%, 51.7%, and 48.3%, respectively. Their diagnostic specificities were 91.7%, 50%, and 75%, respectively. Compared to traditional microbiological detection, both Nanopore and Illumina sequencing showed significantly increased sensitivity. However, Nanopore sequencing exhibited relatively better consistency with the final clinical comprehensive diagnosis, with a Kappa value of 0.574. This outperformed traditional microbiological detection and Illumina sequencing, which had a Kappa value of 0.296 and 0.402, respectively. In addition, Nanopore sequencing required the shortest turnaround time. Conclusion: Nanopore sequencing technology demonstrates as a reliable and rapid method for detecting pathogens in elderly patients with CAP.

Minute level ultra-rapid and thousand copies level high-sensitive pathogen nucleic acid identification based on contactless impedance detection microsensor.

Early screening for pathogens is crucial during pandemic outbreaks. Nucleic acid testing (NAT) is a valuable method for keeping pathogens from spreading. However, the long detection time and large size of the instruments involved significantly limited the efficiency of detection. This work described an integrated NAT microsensor that facilitated rapid and extremely sensitive detection based on nucleic acid amplification (NAA) on a chip. The biochip consisted of two layers incorporating a heater, a thermometer, an interdigital electrode (IDE) and a reaction chamber. The Pt electrode based heater and thermometer were utilized to maintain a specific temperature for the sample in the chamber. The thermometer exhibited a good linear correlation with a sensitivity of 9.36 Ω/°C and the heater achieved a heating efficiency of approximately 6.5 °C/s. Multiple ions were released during NAA, resulting in a decrease in the impedance of the amplification system solution. A large signal of impedance was generated by the released ions due to its linear correlation with the logarithm of the ion concentration. With this detection principle, IDE was employed for real-time monitoring of the in-chip reaction system impedance and NAA process. Specific nucleic acids from two pathogens (SARS-CoV-2, Vibrio vulnificus) were detected with this microsensor. The samples were qualitatively analyzed on microchip within 3 min, with a limit of detection (LOD) of 103 copies/µL. The proposed sensor presented several advantages, including reduced NAT time and increased sensitivity. Consequently, it has shown significant potential in rapid and high-quality nucleic acid testing for the field of epidemic prevention.

Application value of metagenomic next-generation sequencing in hematological patients with high-risk febrile neutropenia.

Background: Metagenomic next-generation sequencing (mNGS) is a novel non-invasive and comprehensive technique for etiological diagnosis of infectious diseases. However, its practical significance has been seldom reported in the context of hematological patients with high-risk febrile neutropenia, a unique patient group characterized by neutropenia and compromised immune responses. Methods: This retrospective study evaluated the results of plasma cfDNA sequencing in 164 hematological patients with high-risk febrile neutropenia. We assessed the diagnostic efficacy and clinical impact of mNGS, comparing it with conventional microbiological tests. Results: mNGS identified 68 different pathogens in 111 patients, whereas conventional methods detected only 17 pathogen types in 36 patients. mNGS exhibited a significantly higher positive detection rate than conventional methods (67.7% vs. 22.0%, P < 0.001). This improvement was consistent across bacterial (30.5% vs. 9.1%), fungal (19.5% vs. 4.3%), and viral (37.2% vs. 9.1%) infections (P < 0.001 for all comparisons). The anti-infective treatment strategies were adjusted for 51.2% (84/164) of the patients based on the mNGS results. Conclusions: mNGS of plasma cfDNA offers substantial promise for the early detection of pathogens and the timely optimization of anti-infective therapies in hematological patients with high-risk febrile neutropenia.

From Shadows to Spotlight: Enhancing Bacterial DNA Detection in Blood Samples through Cutting-Edge Molecular Pre-Amplification.

One of the greatest challenges to the use of molecular methods for diagnostic purposes is the detection of target DNA that is present only in low concentrations. One major factor that negatively impacts accuracy, diagnostic sensitivity, and specificity is the sample matrix, which hinders the attainment of the required detection limit due to the presence of residual background DNA. To address this issue, various methods have been developed to enhance sensitivity through targeted pre-amplification of marker sequences. Diagnostic sensitivity to the single molecular level is critical, particularly when identifying bloodstream infections. In cases of clinically manifest sepsis, the concentration of bacteria in the blood may reach as low as one bacterial cell/CFU per mL of blood. Therefore, it is crucial to achieve the highest level of sensitivity for accurate detection. In the present study, we have established a method that fills the analytical gap between low concentrations of molecular markers and the minimum requirements for molecular testing. For this purpose, a sample preparation of whole blood samples with a directly downstream pre-amplification was developed, which amplifies specific species and resistance markers in a multiplex procedure. When applying pre-amplification techniques, the sensitivity of the pathogen detection in whole blood samples was up to 100 times higher than in non-pre-amplified samples. The method was tested with blood samples that were spiked with several Gram-positive and Gram-negative bacterial pathogens. By applying this method to artificial spiked blood samples, it was possible to demonstrate a sensitivity of 1 colony-forming unit (CFU) per millilitre of blood for S. aureus and E. faecium. A detection limit of 28 and 383 CFU per ml of blood was achieved for E. coli and K. pneumoniae, respectively. If the sensitivity is also confirmed for real clinical blood samples from septic patients, the novel technique can be used for pathogen detection without cultivation, which might help to accelerate diagnostics and, thus, to decrease sepsis mortality rates.

Clinical metagenomic sequencing of plasma microbial cell-free DNA for febrile neutropenia in patients with acute leukaemia.

OBJECTIVES: To evaluate the diagnostic performance and clinical impact of metagenomic next-generation sequencing (mNGS) of plasma microbial cell-free DNA (mcfDNA) in febrile neutropenia (FN). METHODS: In a 1-year, multicentre, prospective study, we enrolled 442 adult patients with acute leukaemia with FN and investigated the usefulness of mNGS of plasma mcfDNA for identification of infectious pathogens. The results of mNGS were available to clinicians in real time. The performance of mNGS testing was evaluated in comparison with blood culture (BC) and a composite standard that incorporated standard microbiological testing and clinical adjudication. RESULTS: In comparison with BC, the positive and negative agreements of mNGS were 81.91% (77 of 94) and 60.92% (212 of 348), respectively. By clinical adjudication, mNGS results were categorized by infectious diseases specialists as definite (n = 76), probable (n = 116), possible (n = 26), unlikely (n = 7), and false negative (n = 5). In 225 mNGS-positive cases, 81 patients (36%) underwent antimicrobials adjustment, resulting in positive impact on 79 patients and negative impact on two patients (antibiotics overuse). Further analysis indicated that mNGS was less affected by prior antibiotics exposure than BC. DISCUSSION: Our results indicate that mNGS of plasma mcfDNA increased the detection of clinically significant pathogens and enabled early optimization of antimicrobial therapy in patients with acute leukaemia with FN.

Clinical Application and Evaluation of Metagenomic Next-Generation Sequencing for Lower Respiratory Tract Infections and Human Tumor Screening.

Background: To evaluate the clinical value of metagenomic next-generation sequencing (mNGS) in screening of lower respiratory tract infections (LRTIs) and human tumors. Methods: Human samples included bronchoalveolar lavage fluid (BALF), sputum, lung biopsy tissue, and peripheral blood from 188 patients who were admitted to our hospital between January 2020 and September 2022 were analyzed using mNGS for simultaneous pathogen and chromosome copy number variation (CNV) detection. Traditional microbial culture and comprehensive microbial test (CMT) were also conducted. The diagnostic efficiencies of the three methods (mNGS, traditional culture, and CMT groups) were compared. Results: Among the 188 patients, 149 (79.3%) were in the LRTIs group and 39 (20.7%) were in the non-LRTIs group. The diagnostic sensitivity and accuracy of the mNGS group were higher than those of the traditional culture and CMT groups (P < 0.001; P < 0.001; P < 0.001; P < 0.001), and the specificity was higher than that of the CMT group (P = 0.039) but lower than that of the traditional culture group (P = 0.006). The positive predictive values of the mNGS and traditional culture groups were higher than that of the CMT group (P = 0.004; P = 0.011). The negative predictive value of the mNGS group was higher than that of the CMT group (P = 0.003). In addition, all samples were subjected to simultaneous chromosome CNV detection, and 8% (15/188) were positive for CNV. Of the 15 patients, 10 were initially misdiagnosed as non-neoplastic diseases, with a misdiagnosis rate of 66.7% (10/15). The BALF CNV test was performed on 13 patients diagnosed with primary or metastatic lung cancer, with a positivity rate of 38.5%. Conclusion: The sensitivity and accuracy of pathogen diagnosis using mNGS were better than those of traditional culture and CMT. CNV detection is an important auxiliary diagnostic tool for cancer, particularly for screening occult tumors.

Clinical evaluation of metagenomic next-generation sequencing in unbiased pathogen diagnosis of urinary tract infection.

BACKGROUND: Early availability of pathogen identification in urinary tract infections (UTIs) has critical importance in disease management. Metagenomic next-generation sequencing (mNGS) has the potential to transform how acute and serious infections are diagnosed by offering unbiased and culture-free pathogen detection. However, clinical experience with application of the mNGS test is relatively limited. METHODS: We therefore established a MinION-based mNGS pathogens diagnostic platform and evaluated its potential for clinical implementation in UTIs with clinical samples. 213 urine samples from patients with suspected UTIs were included and subjected to mNGS testing using the MinION platform. mNGS results were compared to the gold standard of clinical culture and composite standard of combining clinical testing, confirmatory qPCR testing, and clinical adjudication by doctors. RESULTS: The mNGS exhibited a sensitivity of 81.4% and a specificity of 92.3%, along with a positive predictive value of 96.6%, a negative predictive value of 64.9%, and an overall accuracy of 84.4%, all of which were determined based on the gold standard of routine culture results. When assessed against the composite standard, the sensitivity and specificity both increased to 89.9% and 100%, respectively, while the accuracy rose to 92.4%. Notably, the positive predictive value and negative predictive value also saw improvements, reaching 100% and 76.8%, respectively. Moreover, this diagnostic platform successfully identified dsDNA viruses. Among the 65 culture-negative samples, the viral detection rate reached 33.8% (22/65) and was subsequently validated through qPCR. Furthermore, the automatic bioinformatics pipeline we developed enabled one-click analysis from data to results, leading to a significant reduction in diagnosis time. CONCLUSION: These results demonstrate that the pathogen detection performance of mNGS is sufficient for diagnostic testing in clinical settings. As the method is generally unbiased, it can improve diagnostic testing of UTIs and other microbial infections.

Case report: Intraabdominal infection of Mycobacterium syngnathidarum in an immunocompetent patient confirmed by whole-genome sequencing.

Background: The taxonomic group of non-tuberculous mycobacteria (NTM) encompasses more than 190 species and subspecies, some of which can cause pulmonary and extrapulmonary diseases across various age groups in humans. However, different subspecies exhibit differential drug sensitivities, and traditional detection techniques struggle to accurately classify NTM. Therefore, clinicians need more effective detection methods to identify NTM subtypes, thus providing personalized medication for patients. Case presentation: We present the case of a 47-year-old female patient diagnosed with an intraabdominal infection caused by Mycobacterium syngnathidarum. Despite computed tomography of the chest suggesting potential tuberculosis, tuberculosis infection was ruled out due to negative TB-DNA results for ascites fluid and sputum and limited improvement of lung lesions after treatment. Additionally, acid-fast staining and Lowenstein-Jensen culture results revealed the presence of mycobacterium in ascites fluid. Subsequent whole-genome sequencing (WGS) confirmed the DNA sequences of Mycobacterium syngnathidarum in colonies isolated from the ascites fluid, which was further corroborated by polymerase chain reaction and Sanger sequencing. Ultimately, the patient achieved a complete recovery following the treatment regimen targeting Mycobacterium syngnathidarum, which involved clarithromycin, ethambutol hydrochloride, pyrazinamide, rifampicin, and isoniazid. Conclusion: This is the first reported case of Mycobacterium syngnathidarum infection in humans. Mycobacterium syngnathidarum was detected by WGS in this case, suggesting that WGS may serve as a high-resolution assay for the diagnosis of different subtypes of mycobacterium infection.

Metagenomic next-generation sequencing of bronchoalveolar lavage fluid assists in the diagnosis of pathogens associated with lower respiratory tract infections in children.

Worldwide, lower respiratory tract infections (LRTI) are an important cause of hospitalization in children. Due to the relative limitations of traditional pathogen detection methods, new detection methods are needed. The purpose of this study was to evaluate the value of metagenomic next-generation sequencing (mNGS) of bronchoalveolar lavage fluid (BALF) samples for diagnosing children with LRTI based on the interpretation of sequencing results. A total of 211 children with LRTI admitted to the First Affiliated Hospital of Guangzhou Medical University from May 2019 to December 2020 were enrolled. The diagnostic performance of mNGS versus traditional methods for detecting pathogens was compared. The positive rate for the BALF mNGS analysis reached 95.48% (95% confidence interval [CI] 92.39% to 98.57%), which was superior to the culture method (44.07%, 95% CI 36.68% to 51.45%). For the detection of specific pathogens, mNGS showed similar diagnostic performance to PCR and antigen detection, except for Streptococcus pneumoniae, for which mNGS performed better than antigen detection. S. pneumoniae, cytomegalovirus and Candida albicans were the most common bacterial, viral and fungal pathogens. Common infections in children with LRTI were bacterial, viral and mixed bacterial-viral infections. Immunocompromised children with LRTI were highly susceptible to mixed and fungal infections. The initial diagnosis was modified based on mNGS in 29.6% (37/125) of patients. Receiver operating characteristic (ROC) curve analysis was performed to predict the relationship between inflammation indicators and the type of pathogen infection. BALF mNGS improves the sensitivity of pathogen detection and provides guidance in clinical practice for diagnosing LRTI in children.

Argonaute protein-based nucleic acid detection technology.

It is vital to diagnose pathogens quickly and effectively in the research and treatment of disease. Argonaute (Ago) proteins are recently discovered nucleases with nucleic acid shearing activity that exhibit specific recognition properties beyond CRISPR-Cas nucleases, which are highly researched but restricted PAM sequence recognition. Therefore, research on Ago protein-mediated nucleic acid detection technology has attracted significant attention from researchers in recent years. Using Ago proteins in developing nucleic acid detection platforms can enable efficient, convenient, and rapid nucleic acid detection and pathogen diagnosis, which is of great importance for human life and health and technological development. In this article, we introduce the structure and function of Argonaute proteins and discuss the latest advances in their use in nucleic acid detection.

The application of metagenomic next-generation sequencing in pathogen diagnosis: a bibliometric analysis based on Web of Science.

Background: Infectious disease is a large burden on public health globally. Metagenomic next-generation sequencing (mNGS) has become popular as a new tool for pathogen diagnosis with numerous advantages compared to conventional methods. Recently, research on mNGS increases yearly. However, no bibliometric analysis has systematically presented the full spectrum of this research field. Therefore, we reviewed all the publications associated with this topic and performed this study to analyze the comprehensive status and future hotspots of mNGS for infectious disease diagnosis. Methods: The literature was searched in the Web of Science Core Collection and screened without year or language restrictions, and the characteristics of the studies were also identified. The outcomes included publication years, study types, journals, countries, authorship, institutions, frontiers, and hotspots with trends. Statistical analysis and visualization were conducted using VOSviewer (version 1.6.16) and CiteSpace (version 6.1. R3). Results: In total, 325 studies were included in the analysis after screening. Studies were published between 2009 and 2022 with a significantly increasing number from 1 to 118. Most of the studies were original articles and case reports. Frontiers in Cellular and Infection Microbiology and Clinical Infectious Disease were the most commonly cited and co-cited journals. Institutions and researchers from China contributed the most to this field, followed by those from the USA. The hotspots and frontiers of these studies are pneumonia, tuberculosis, and central nervous system infections. Conclusion: This study determined that mNGS is a hot topic in the diagnosis of infectious diseases with development trends and provides insights into researchers, institutions, hotspots and frontiers in mNGS, which can offer references to related researchers and future research.

CRISPR/Cas-Based Diagnostics in Agricultural Applications.

Pests and disease-causing pathogens frequently impede agricultural production. An early and efficient diagnostic tool is crucial for effective disease management. Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated protein (Cas) have recently been harnessed to develop diagnostic tools. The CRISPR/Cas system, composed of the Cas endonuclease and guide RNA, enables precise identification and cleavage of the target nucleic acids. The inherent sensitivity, high specificity, and rapid assay time of the CRISPR/Cas system make it an effective alternative for diagnosing plant pathogens and identifying genetically modified crops. Furthermore, its potential for multiplexing and suitability for point-of-care testing at the field level provide advantages over traditional diagnostic systems such as RT-PCR, LAMP, and NGS. In this review, we discuss the recent developments in CRISPR/Cas based diagnostics and their implications in various agricultural applications. We have also emphasized the major challenges with possible solutions and provided insights into future perspectives and potential applications of the CRISPR/Cas system in agriculture.

Comparison of Third-Generation Sequencing Technology and Traditional Microbiological Detection in Pathogen Diagnosis of Lower Respiratory Tract Infection.

BACKGROUND: It is common to obtain a low detection rate and unsatisfactory detection results in complex infection or rare pathogen detection. This retrospective study aimed to illustrate the application value and prospect of the third-generation sequencing technology in lower respiratory tract infection disease. METHODS: This study recruited 70 patients with lower respiratory tract infection (LRTI). Pathogen detection of bronchoalveolar lavage fluid (BALF) from all patients was performed using nanopore metagenomic sequencing technology and traditional culture. BALF culture combined with quantitiative PCR (qPCR) was used as a reference standard to analyze the sensitivity and specificity of nanopore sequencing technology. The current study also collected the examination results of enrolled samples using technical methods sputum culture, tuberculosis DNA (TB-DNA), and Xpert MTB/RIF and analyzed the detection efficiency of nanopore sequencing for Mycobacterium tuberculosis. RESULTS: The positive rates of pathogens in 70 BALF samples detected by conventional culture and nanopore sequencing were 25.71% and 84.29%, respectively. Among the 59 positive BALF cases using nanopore sequencing, a total of 31 pathogens were identified, of which the proportions of bacteria, fungi, viruses, and other pathogens were 50%, 17%, 32%, and 1%, respectively. Using the results combined with culture and qPCR detection methods as the standard, the pathogen detection of BALF using nanopore sequencing had a sensitivity of 70% and a specificity of 91.7%. Additionally, the positive rate of the detection of M. tuberculosis using nanopore sequencing was 33.3% (6/18). The clinical medication plans of 74.3% (52/70) of the patients were referred to the nanopore sequencing results, of which 31 cases changed their treatment strategy, 21 supported the previous treatment plans, and 90% (47/52) of the patients finally had clinical improvement. CONCLUSIONS: BALF detection using nanopore sequencing technology improves the process of detecting pathogens in patients with LRTI, especially for M. tuberculosis, fungi, and viruses, by reducing the report time from three days to six hours. The clinical application prospect of nanopore sequencing technology is promising in the pathogen diagnosis of LRTI.

Detection, Diagnosis, and Preventive Management of the Bacterial Plant Pathogen Pseudomonas syringae.

Plant diseases caused by the pathogen Pseudomonas syringae are serious problems for various plant species worldwide. Accurate detection and diagnosis of P. syringae infections are critical for the effective management of these plant diseases. In this review, we summarize the current methods for the detection and diagnosis of P. syringae, including traditional techniques such as culture isolation and microscopy, and relatively newer techniques such as PCR and ELISA. It should be noted that each method has its advantages and disadvantages, and the choice of each method depends on the specific requirements, resources of each laboratory, and field settings. We also discuss the future trends in this field, such as the need for more sensitive and specific methods to detect the pathogens at low concentrations and the methods that can be used to diagnose P. syringae infections that are co-existing with other pathogens. Modern technologies such as genomics and proteomics could lead to the development of new methods of highly accurate detection and diagnosis based on the analysis of genetic and protein markers of the pathogens. Furthermore, using machine learning algorithms to analyze large data sets could yield new insights into the biology of P. syringae and novel diagnostic strategies. This review could enhance our understanding of P. syringae and help foster the development of more effective management techniques of the diseases caused by related pathogens.

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.

Metagenomic next-generation sequencing clinches the diagnosis of Legionella pneumonia in a patient with acute myeloid leukemia: A case report and literature review.

Legionella pneumonia caused by Legionella pneumophila is a multi-system disease that is a life-threatening, acute, and severe form of pneumonia. L. pneumophila is widespread and the clinical manifestations of Legionella pneumonia are similar to those of typical and atypical pneumonia. Current diagnostic scores and radiologic evidence have limited diagnostic value. Thus, it is likely that many cases of Legionella pneumonia remain unreported. We describe a woman with a medical history of acute myeloid leukemia who suffered from repeated fever, and no relief following initial empirical antibiotic treatment. Ultimately, she was diagnosed with Legionella pneumonia based on metagenomic next-generation sequencing (mNGS). We also performed a systematic review of the literature and identified 5 other patients who were diagnosed with Legionella pneumonia using mNGS, and reviewed their clinical characteristics, biological characteristics, epidemiological features, laboratory results, clinical findings, and treatments. This literature review showed that accurate etiological diagnosis is becoming increasingly essential for a definitive diagnosis and treatment strategies. The clinical manifestations of Legionella pneumonia are non-specific, and many routine laboratory diagnostic tests cannot identify Legionella. mNGS, an indispensable approach for identifying microorganisms, can provide a promising tool for the rapid and accurate etiological diagnosis methods contributing to early diagnosis, early treatment, and improved prognosis, especially for uncommon species such as L. pneumophila.