Endolysin, a Promising Solution against Antimicrobial Resistance

Antimicrobial resistance (AMR) is a global crisis for human public health which threatens the effective prevention and control of ever-increasing infectious diseases. The advent of pandrug-resistant bacteria makes most, if not all, available antibiotics invalid. Meanwhile, the pipeline of novel antibiotics development stagnates, which prompts scientists and pharmacists to develop unconventional antimicrobials. Bacteriophage-derived endolysins are cell wall hydrolases which could hydrolyze the peptidoglycan layer from within and outside of bacterial pathogens. With high specificity, rapid action, high efficiency, and low risk of resistance development, endolysins are believed to be among the best alternative therapeutic agents to treat multidrug resistant (MDR) bacteria. As of now, endolysins have been applied to diverse aspects. In this review, we comprehensively introduce the structures and activities of endolysins and summarize the latest application progress of recombinant endolysins in the fields of medical treatment, pathogen diagnosis, food safety, and agriculture.

Recent Improvements in CRISPR-Based Amplification-Free Pathogen Detection

Molecular diagnostic (MDx) methods directly detect target nucleic acid sequences and are therefore an important approach for precise diagnosis of pathogen infection. In comparison with traditional MDx techniques such as PCR, the recently developed CRISPR-based diagnostic technologies, which employ the single-stranded nucleic acid trans-cleavage activities of either Cas12 or Cas13, show merits in both sensitivity and specificity and therefore have great potential in both pathogen detection and beyond. With more and more efforts in improving both the CRISPR trans-cleavage efficiencies and the signal detection sensitivities, CRISPR-based direct detection of target nucleic acids without preamplification can be a possibility. Here in this mini-review, we summarize recent research progresses of amplification-free CRISPR-Dx systems and explore the potential changes they will lead to pathogen diagnosis. In addition, discussion of the challenges for both detection sensitivity and cost of the amplification-free systems will also be covered.

Self-Cognizant Bionic Liquid Sensor for Pathogen Diagnosis

As observed in the outbreaks of SARS and swine flu, as well as many other infectious diseases, the huge volume of human traffic across numerous enclosed public venues has posed immense challenges to preventing the spread of communicable diseases. There is an urgent need for effective disease surveillance management in public areas under pandemic outbreaks. The physicochemical properties associated with ionic liquids make them particularly suited for molecular communications in sensing networks where low throughput is quite adequate for pathogen detection. This paper presents a self-cognizant system for rapid diagnosis of infectious disease using a bionic sensor such that testing can be supported without collecting a fluid sample from a subject through any invasive methods. The system is implemented for testing the performance of the proposed bionic liquid sensing network.

Case Report: Diagnosis of Klebsiella pneumoniae Invasive Liver Abscess Syndrome With Purulent Meningitis in a Patient From Pathogen to Lesions

As a determinant human pathogen, Klebsiella pneumoniae is known to cause rare K. pneumoniae liver abscess syndrome (KLAS) which was more common in Asia in early-stage and reported increasingly outside Asia now. Patients with KLAS who have septic metastatic ocular or central nervous system (CNS) lesions are associated with high morbidity and mortality. Relatively infrequent adult community-acquired K. pneumoniae meningitis have been documented and most were with poor prognosis. In this paper, we reported a case of KLAS presenting purulent meningitis as disease onset. While negative results were obtained in the bacterial culture of CSF, blood, or liver pus, metagenomic next-generation sequencing (mNGS) of CSF, and blood samples which were synchronously performed demonstrated Klebsiella pneumoniae as the pathogenic microorganism (13,470 and 5,318 unique reads, respectively). The ultimately cured patient benefited from rapid pathogen diagnosis, early percutaneous drainage of the abscess, and prompt appropriate antibiotic administration. Our case highlights the importance of clinicians using mNGS for early pathogen diagnosis of this disease.

Design of Inverted Nano-Cone Arrayed SERS Substrate for Rapid Detection of Pathogens

Rapid detection of bacteria is a very critical and important part of infectious disease treatment. Sepsis kills more than 25 percent of its victims, resulting in as many as half of all deaths in hospitals before identifying the pathogen for patients to get the right treatment. Raman spectroscopy is a promising candidate in pathogen diagnosis given its fast and label-free nature, only if the concentration of the pathogen is high enough to provide reasonable sensitivity. This work reports a new design of surface-enhanced Raman spectroscopy (SERS) substrate which will provide high enough sensitivity and fast and close contact of the target structure to the optical hot spots for immunomagnetic capturing-based bacteria-concentrating technique. The substrate uses inverted nanocone structure arrays made of transparent PDMS (Polydimethylsiloxane) to funnel the light from the bottom to the top of the cones where plasmonic gold nanorods are located. A high reflective and low loss layer is deposited on the outer surface of the cone. Given the geometry of cones, photons are multi-reflected by the outer layer and thus the number density of photons at hotspots increases by an order of magnitude, which could be high enough to detect immunomagnetically densified bacteria.

#35: Rapid, Non-invasive Detection and Serial Monitoring of Invasive Fungal Infections in Immunocompromised Children Using the Karius Test (a Plasma-based Microbial Cell-free DNA Sequencing Test)

Background A diverse spectrum of invasive molds and fungi cause serious opportunistic infections in immunocompromised (IC) children. Their overlap in clinical presentation can make it challenging to differentiate among etiologies and optimally tailor antifungal therapy. Current methods to identify these pathogens lack sensitivity, are limited by long turnaround times and require an array of individual tests on invasively obtained specimens. The delay or lack of a pathogen diagnosis in combination with the reliance on invasive procedures leads to a dependence on broad empiric therapy, the development of antimicrobial resistance and increases in morbidity and mortality. Rapid, non-invasive diagnosis of invasive fungal infections through next-generation sequencing (NGS) of plasma microbial cell-free DNA (mcfDNA) offers a means to overcome these limitations. Methods Karius® Test (KT) results were reviewed for detections of Aspergillus, non-Aspergillus molds and Pneumocystis jirovecii (PJP) in children. KT, developed and validated in Karius’ CLIA certified/CAP accredited lab, detects microbial cell-free DNA (mcfDNA) can assist with the diagnosis of invasive infections. McfDNA is extracted, NGS is performed, human sequences removed and remaining sequences aligned to a curated pathogen database of >1400 organisms. Organisms present above a statistical threshold are reported and quantified. For > 85% of tests the time to result reporting is 24 hours from sample receipt. Clinical information was included from data submitted with the requisition or obtained at the time of reporting from clinical consultations with the provider. Results KT detected 7 different species of Aspergillus in 61 patients (74% IC, 40% with a pulmonary focus). KT detected 15 different non-Aspergillus molds in 51 patients (80% IC, 36% with a pulmonary focus). KT detected PJP in 37 patients (73% IC, 76% with a pulmonary focus, 54% with a DNA virus co-detection and 32% with a herpesvirus co-detections). There were 31 subjects with serial monitoring (97% IC, 70% with a pulmonary focus) including 48% with Aspergillus, 39% with non-Aspergillus molds and 12% with PJP (Figure 1). 71% of subjects demonstrated a decline in the quantitative mcfDNA signal over time; the duration of a positive mcfDNA signal ranged from 3–92 days (median 16 days, SD 22.4). Conclusions Plasma mcfDNA NGS offers a rapid, non-invasive means of detecting a broad diversity of invasive pathogens that overlap in their clinical presentations and are difficult to identify in immunocompromised children. The rapid turnaround time, non-invasive sampling and 1-sample-1000+test-solution may lead to a faster time to pathogen diagnosis, faster time to targeted therapy and obviate the need for invasive diagnostic procedures. The ability with a single test to concomitantly diagnose co-pathogens including reactivating herpesviruses that modulate the progression of principal infecting fungal pathogens (i.e. cytomegalovirus modulation of PJP) can help optimize care. Additionally, this convenient non-invasive means of serial testing of invasive fungal infections may serve as an indicator of burden of infection, provide insight into treatment efficacy and ultimately help define the length and mode (medical/surgical) of therapy required to improve outcomes. Additional studies correlating the mcfDNA signal with individual patient clinical and radiographic parameters will be important to further define the utility of serial mcfDNA monitoring.

Multicenter prospective surveillance study of viral agents causing meningoencephalitis

The frequency of bacterial factors causing central nervous system infections has decreased as a result of the development of our national immunization program. In this study, it is aimed to obtain the data of our local surveillance by defining the viral etiology in cases diagnosed with meningoencephalitis for 1 year. Previously healhty 186 children, who applied with findings suggesting viral meningoencephalitis to 8 different tertiary health centers between August 2018 and August 2019, in Istanbul, were included. The cerebrospinal fluid (CSF) sample was evaluated by polymerase chain reaction. The M:F ratio was 1.24 in the patient group, whose age ranged from 1 to 216 months (mean 40.2 ± 48.7). Viral factor was detected in 26.8%. Enterovirus was the most common agent (24%) and followed by Adenovirus (22%) and HHV type 6 (22%). In the rest of the samples revealed HHV type 7 (10%), EBV (6%), CMV (6%), HSV type 1 (6%), Parvovirus (4%) and VZV (2%). The most common symptoms were fever (79%) and convulsions (45.7%). Antibiotherapy and antiviral therapy was started 48.6% and 4% respectively. Mortality and sequela rate resulted 0.53% and 3.7%, respectively. This highlights the importance of monitoring trends in encephalitis in Turkey with aview to improving pathogen diagnosis for encephalitis and rapidly identifying novel emerging encephalitis-causing pathogens that demand public health action especially in national immunisation programme.

Clinical utility of metagenomic next-generation sequencing in suspected central nervous system infectious pediatric patients with empirical treatment: a cohort study

Background: Metagenomic next generation sequencing (mNGS) is becoming an increasingly available diagnostic method used to identify a broad range of pathogens. However, the optimal role of mNGS in clinical diagnostic schema remains uncertain, especially in pediatric patients suspected central nervous system (CNS) infection and treated with empirical antibiotic. The purpose of this study was to investigate the usefulness of cerebrospinal fluid (CSF) mNGS in the pediatric patients.Methods: We performed a retrospective review of suspected CNS infection patients who had CSF mNGS test from April 2019 to December 2020. Results and clinical impact of mNGS test were collected. We investigated the usefulness of CSF mNGS in clinical impact and diagnosis. Results: We enrolled 57 pediatric patients with empirical treatment. A total of 39 CNS infection patients were diagnosed, and 27 patients were identified by mNGS that only 2 of 27 were co-identified by CSF culture. In all of the patients, 75.4% (45 cases) had changed (addition or de-escalation) in antimicrobial therapy according to the results of the mNGS test. By each CSF mNGS test as a whole, the positive and negative percent agreement were 69.2% (95% CI: 54.1-84.4%) and 61.1% (95% CI: 36.2-86.1%), respectively, and true negatives of negative mNGS tests were 50% (95% CI: 27.3 – 72.7%).Conclusion: In this study, CSF mNGS test improved the diagnosis of neurologic infections and adjusted antibiotic therapy in the vast majority of cases. Consequently, for patients with empirical treatment, CSF mNGS should be used more in pathogen diagnosis and clinical therapy.