Highly sensitive extraction-free saliva-based molecular assay for rapid diagnosis of SARS-CoV-2.

The COVID-19 pandemic highlighted the necessity of fast, sensitive, and efficient methods to test large populations for respiratory viruses. The "gold standard" molecular assays for detecting respiratory viruses, such as quantitative polymerase chain reaction (qPCR) and reverse transcription qPCR (RT-qPCR), rely on invasive swab samples and require time-consuming and labor-intensive extraction processes. Moreover, the turnaround time for RT-qPCR-based assays is too lengthy for rapid screening. Extraction-free saliva-based methods provide a non-invasive sampling process with a fast turnaround time and are suitable for high-throughput applications. However, when used with a standard RT-qPCR system, the absence of extraction significantly reduces the assays' sensitivity. Here, using a novel optical modulation biosensing (OMB) platform, we developed a rapid and highly sensitive extraction-free saliva-based molecular assay. We blindly tested 364 paired nasopharyngeal swabs and saliva samples from suspected SARS-CoV-2 cases in Israel. Compared with the gold standard swab-based RT-qPCR assay, the sensitivity of the extraction-free saliva-based OMB assay is 90.7%, much higher than the sensitivity of extraction-free saliva-based RT-qPCR assay (77.8%) with similar specificity (95.3% and 97.6%, respectively). Moreover, out of 12 samples identified by the OMB-based assay as positive, 8 samples were collected from hospitalized patients in a COVID-19 ward and were verified to be SARS-CoV-2-positive upon admission, indicating that the actual clinical sensitivity and specificity of the OMB assay are higher. Considering its user-friendly saliva-based protocol, short and cost-effective extraction-free process, and high clinical accuracy, the OMB-based molecular assay is very suitable for high-throughput testing of large populations for respiratory viruses. IMPORTANCE: Three years after the SARS-CoV-2 outbreak, there are no molecular tests that combine low-cost and straightforward sample preparation, effective sample handling, minimal reagent and disposable requirements, high sensitivity, and high throughput required for mass screening. Existing rapid molecular techniques typically sacrifice certain requirements to meet others. Yet, localized outbreaks of novel viral diseases happen daily in different parts of the world. In this context, respiratory diseases are of specific importance, as they are frequently airborne and highly contagious, with the potential for a rapid global spread. The widely accepted opinion is that another pandemic is just a question of time. To ensure that the containment efforts for the upcoming "disease X" are successful, introducing rapid, high-throughput, and highly sensitive diagnostic methods for detecting and identifying pathogens is critical. A few months into the pandemic, saliva was suggested as a diagnostic matrix for SARS-CoV-2 detection. The collection of saliva does not require swabs and is minimally invasive. In particular, extraction-free saliva-based assays require fewer reagents and disposables, and therefore are faster and cheaper, offering an appealing alternative for low-income countries. Unfortunately, current extraction-free saliva-based detection methods, such as direct RT-qPCR or isothermal amplification, have either low sensitivity or low throughput. Therefore, we believe that the presented highly sensitive ht-OMBi platform and the extraction-free saliva-based molecular assay can become an essential tool in the infectious disease monitoring toolbox.

Clinical isolate characteristics and demographics of patients with C.jejuni and C.coli infections in Northern Israel, 2015-2021.

C.coli is a significant cause of foodborne gastroenteritis worldwide, with the majority of cases attributed to C.jejuni. Although most clinical laboratories do not typically conduct antimicrobial susceptibility testing for C.coli, the rise in resistant strains has underscored the necessity for such testing and epidemiological surveillance. The current study presents clinical isolate characteristics and demographics of 221 patients with C.coli (coli and jejuni) infections in Northern Israel, between 2015 and 2021. Clinical and demographic data were collected from patient medical records. Susceptibility to erythromycin, tetracycline, ciprofloxacin, and gentamicin was assessed using the standard E-test. No significant correlations were found between bacterial species and patient ethnicity, patient gender, or duration of hospitalization. In contrast, significant differences were found between infecting species and patient age and age subgroup (P < 0.001). Furthermore, erythromycin resistance was observed in only 0.5% of the study population, while resistance to ciprofloxacin, tetracycline, and gentamicin was observed in 95%, 93%, and 2.3% of the population, respectively. The presented study underscores the need for routine surveillance of C.coli antibiotic resistance.

Nucleic acid hybridization-based detection of pathogenic RNA using microscale thermophoresis.

Infectious diseases are one of the world's leading causes of morbidity. Their rapid spread emphasizes the need for accurate and fast diagnostic methods for large-scale screening. Here, we describe a robust method for the detection of pathogens based on microscale thermophoresis (MST). The method involves the hybridization of a fluorescently labeled DNA probe to a target RNA and the assessment of thermophoretic migration of the resulting complex in solution within a 2 to 30-time window. We found that the thermophoretic migration of the nucleic acid-based probes is primarily determined by the fluorescent molecule used, rather than the nucleic acid sequence of the probe. Furthermore, a panel of uniformly labeled probes that bind to the same target RNA yields a more responsive detection pattern than a single probe, and moreover, can be used for the detection of specific pathogen variants. In addition, intercalating agents (ICA) can be used to alter migration directionality to improve detection sensitivity and resolving power by several orders of magnitude. We show that this approach can rapidly diagnose viral SARS-CoV2, influenza H1N1, artificial pathogen targets, and bacterial infections. Furthermore, it can be used for anti-microbial resistance testing within 2 h, demonstrating its diagnostic potential for early pathogen detection.

The Impact of a Positive Biofire® FilmArray® Gastrointestinal Panel Result on Clinical Management and Outcomes.

The gold standard diagnostic method for gastrointestinal infections is stool culture, which has limited sensitivity and long turnaround time. Infection diagnosis recently shifted to syndrome-based panel assays. This study employed the FilmArray® Gastrointestinal Panel, which detects 22 pathogens simultaneously, to investigate gastrointestinal infection and pathogen distribution in 91 stool samples of patients hospitalized at the Tzafon Medical Center, Israel, during 2020, and to compare the clinical and demographic data of negative vs. positive samples. Among the 61 positive samples (67%), the most common pathogen was Campylobacter (34.4%). Positive test results were associated with a slightly younger patient age (p = 0.012), significantly higher post-diagnosis use of antibiotics (63.9% vs. 36.7%; p = 0.014), and shorter length of stay and time to discharge (p = 0.035, p = 0.003, respectively) than negative test results. To conclude, the FilmArray® Gastrointestinal Panel enabled the early identification of causative infectious agents and enhanced clinical management and outcomes.

Impact of Helicobacter pylori virulence markers on clinical outcomes in adult populations.

BACKGROUND: In recent years, associations between specific virulence markers of Helicobacter pylori (H. pylori) and gastrointestinal disorders have been suggested. AIM: To investigate the presence of virulence factors including vacuolating cytotoxin A genotypes (s1m1, s1m2, s2m1, and s2m2), cytotoxin-associated gene A (CagA), and urease activity in H. pylori strains isolated from Arab and Jewish populations in northern Israel and to assess associations between these factors and patients' demographics and clinical outcomes. METHODS: Patients (n = 108) who underwent gastroscopy at the Baruch Padeh Medical Center, Poriya due to symptomatic gastroduodenal pathologies as part of H. pylori diagnosis were enrolled in the study. Gastric biopsy specimens were collected from the antrum of the stomach. Clinical condition was assessed by clinical pathology tests. Bacteria were isolated on modified BD Helicobacter Agar (BD Diagnostics, Sparks, MD, United States). Bacterial DNA was extracted, and PCR was performed to detect CagA and vacuolating cytotoxin A genes. Urease activity was assessed using a rapid urease test. RESULTS: A significant correlation was found between disease severity and patient ethnicity (P = 0.002). A significant correlation was found between CagA presence and the s1m1 genotype (P = 0.02), which is considered the most virulent genotype. Further, a higher level of urease activity was associated with isolates originating from the Jewish population. Moreover, higher urease activity levels were measured among CagA-/s1m1 and CagA-/s2m2 isolates. CONCLUSION: Our study highlights the importance of incorporating molecular methods for detection of virulence markers of H. pylori in order to tailor optimal treatments for each patient. Further investigation should be performed regarding associations between H. pylori virulence factors and ethnicity.

Characterization of community-acquired Clostridioides difficile strains in Israel, 2020-2022.

Background: The prevalence of community-acquired Clostridioides difficile infection (CA-CDI) has been rising, due to changes in antibiotics prescribing practices, emergence of hypervirulent strains and improved diagnostics. This study explored CA-CDI epidemiology by examining strain diversity and virulence factors of CA-CDI isolates collected across several geographical regions in Israel. Methods: Stool samples of 126 CA-CDI patients were subjected to PCR and an immunoassay to identify toxin genes and proteins, respectively. Toxin loci PaLoc and PaCdt were detected by whole-genome sequencing (WGS). Biofilm production was assessed by crystal violet-based assay. Minimum inhibitory concentration was determined using the Etest technique or agar dilution. WGS and multi-locus sequence typing (MLST) were used to classify strains and investigate genetic diversity. Results: Sequence types (ST) 2 (17, 13.5%), ST42 (13, 10.3%), ST104 (10, 8%) and ST11 (9, 7.1%) were the most common. All (117, 92.8%) but ST11 belonged to Clade 1. No associations were found between ST and gender, geographic area or antibiotic susceptibility. Although all strains harbored toxins genes, 34 (27%) produced toxin A only, and 54 (42.9%) strains produced toxin B only; 38 (30.2%) produced both toxins. Most isolates were biofilm-producers (118, 93.6%), primarily weak producers (83/118, 70.3%). ST was significantly associated with both biofilm and toxin production. Conclusion: C. difficile isolates in Israel community exhibit high ST diversity, with no dominant strain. Other factors may influence the clinical outcomes of CDI such as toxin production, antibiotic resistance and biofilm production. Further studies are needed to better understand the dynamics and influence of these factors on CA-CDI.

Antibiotic resistance patterns of Helicobacter pylori in North Israel - A six-year study.

BACKGROUND: One main challenge in Helicobacter pylori (H. pylori) eradication is its increasing antibiotic resistance. Additionally, resistance rates vary between geographic areas and periods. However, data are limited since susceptibility testing is not routinely performed. Thus, it is valuable to gather data regarding H. pylori's resistance rates in Israel that would aid in better adjustment of treatment. MATERIALS AND METHODS: The study included 540 H. pylori isolates, recovered from gastric biopsy samples of patients who had undergone endoscopy, during 2015-2020, at the Padeh Poriya Medical Center. Antibiotic susceptibility testing to amoxicillin, clarithromycin, metronidazole, levofloxacin, rifampicin, and tetracycline was performed using the Etest technique. Data regarding participants' sex, age, and ethnic group were collected. For every antibiotic and for multi-resistance, generalized linear models were used to estimate crude and adjusted estimated differences in mean MIC and odds ratios (ORs) for every year, compared with the reference year 2015. RESULTS: The highest resistance rates were for clarithromycin and metronidazole (46.3% and 16.3%, respectively). Patients above 18 had higher resistance rate to rifampicin and multi-resistance (3.3% and 14.8%), compared with patients under 18 (0.5% and 8.4%, respectively). Resistance rates for levofloxacin, rifampicin, and multi-resistance were significantly higher among Arab patients, compared with Jewish patients. During the 6-year surveillance, a significant annual trend in MIC for metronidazole and in ORs for metronidazole, levofloxacin, and multi-resistance were observed (after adjustment). During 2020 compared with 2015, significant increased ORs were observed for levofloxacin and metronidazole [5.72 (1.03-31.84); 4.28 (1.30-14.14), respectively]. CONCLUSIONS: In light of the remarkable changes in antibiotic resistance of H. pylori during the study's period and the increasing resistance rates to various antibiotics, it is very important to continuously monitor H. pylori antibiotic susceptibly. In order to increase eradication rates of this bacterium, therapy regimes must be based on an updated antibiotic resistance data.

Rapid SARS-CoV-2 Detection Using the Lucira™ Check It COVID-19 Test Kit.

The need for the early identification of SARS-CoV-2 has let to a quest for reliable tests that meet the standards of polymerase chain reaction (PCR) tests, on the one hand, and are low-cost, easy-to-use, and fast, on the other hand. One such test is the Lucira™ Check It COVID-19 Test kit ("Lucira") (Lucira Health, Inc., Emeryville, CA, USA), which utilizes real-time loop-mediated isothermal amplification technology, developed for at-home use. This study evaluated the clinical sensitivity and specificity of Lucira in identifying the virus in 190 nasopharyngeal samples collected between January and October 2021. Each sample was also subjected to RT-PCR. All negative RT-PCR results were paralleled by a negative Lucira result. Out of 90 participants who had a positive RT-PCR result, 82 (91.1%) tested positive by Lucira. Among the 72 symptomatic participants, 67 (93%) tested positive by Lucira. All samples with a positive RT-PCR result with a threshold cycle (Ct) > 36, yielded a negative Lucira result. In addition, a significant positive correlation was found between Ct and time-to-positivity with Lucira (R = 0.8612, p < 0.0001). The implementation of such a portable and affordable assay may aid in breaking the COVID-19 transmission chain.

High throughput optical modulation biosensing for highly sensitive and rapid detection of biomarkers.

Rapid, highly sensitive, and high-throughput detection of biomarkers at low concentrations is invaluable for early diagnosis of various diseases. In many highly sensitive immunoassays, magnetic beads are used to capture fluorescently labeled target molecules. The target molecules are then quantified by detecting the fluorescent signal from individual beads, which is time consuming and requires a complicated and expensive detection system. Here, we demonstrate a high-throughput optical modulation biosensing (ht-OMB) system, which uses a small permanent magnet to aggregate the beads into a small detection volume and eliminates background noise by steering a laser beam in and out of the cluster of beads. Shortening the aggregation, acquisition, and well-to-well scanning transition times enables reading a 96-well plate within 10 min. Using the ht-OMB system to detect human Interleukin-8, we demonstrated a limit of detection of 0.14 ng/L and a 4-log dynamic range. Testing 94 RNA extracts from 36 confirmed RT-qPCR SARS-CoV-2-positive patients (Ct≤40) and 58 confirmed RT-qPCR SARS-CoV-2-negative individuals resulted in 100% sensitivity and 100% specificity.

Characterization of Clostridioides difficile Strains, the Disease Severity, and the Microbial Changes They Induce.

BACKGROUND: Clostridioides difficile infection (CDI) is a major nosocomial disease. The characteristics of different strains, the disease severity they cause, their susceptibility to antibiotics, and the changes they inflict on gut microbiome, have not been comprehensively studied in Israel. METHODS: A severity score was calculated for 70 patients. Stool samples were tested for toxins presence using a special kit. Bacteria were isolated, identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) and antibiotic susceptibility tests were performed for several antibiotics. Strains were classified by Multi-locus sequence typing (MLST), and changes in gut microbiome were tested. RESULTS: ST04 (22.5%) and ST37 (12.7%) were the most frequent strains. Clade (phylogenetic lineage) 1 was the most (81.4%) prevalent. We found significant associations between ST and age (p = 0.024) and between ST and moxifloxacin susceptibility (p = 0.001). At the clade level, we found significant associations with binary toxin gene occurrence (p = 0.002), and with susceptibility to both metronidazole and vancomycin (p = 0.024, 0.035, respectively). Differences in intestine microbiome were affected by age, clades' distribution and STs. CONCLUSIONS: By defining the characteristics of the different strains and clades, clinicians can choose medical interventions based on the predicted response or disease severity associated with each strain, enabling new advances in the field of personalized medicine.

MicroRNA-423 may regulate diabetic vasculopathy.

To test the hypothesis that microRNAs may play a role in diabetic retinopathy, we measured the levels of different markers [microRNAs, vascular endothelial growth factor (VEGF), nitric oxide (NO), and total antioxidant capacity (TAO)] in patients with type 2 diabetes mellitus (T2DM) and microvascular complications. Sixty-nine patients were recruited: 22 healthy subjects, ten T2DM patients without retinopathy, 22 with nonproliferative diabetic retinopathy, and 15 with proliferative diabetic retinopathy (PDR). Serum levels of NO, VEGF, TAO and 16 candidate microRNAs were measured. Additionally, the mRNA levels of endothelial nitric oxide synthase (eNOS), induced NOS (iNOS), C reactive protein (CRP), VEGF, tumor necrosis factor α (TNFα), PON2, p22, and SOD2 were measured in human vascular endothelial cells cultured in the presence of pooled sera from the subject groups. Plasma miR-423 levels showed a significant ~ twofold decrease in patients with PDR compared to controls. P lasma NO levels were significantly higher in retinopathy, VEGF levels were significantly lower, and TAO was significantly decreased. eNOS mRNA levels were lower in the cells of T2DM patients without retinopathy, but higher in PDR. PON2, p22, and SOD2 mRNA levels were all significantly lower in PDR. CRP, TNFα, iNOS, and VEGF mRNA levels showed no significant association with disease status. Lowered miR-423 levels in diabetic patients showed a correlation with VEGF and an inverse correlation between NO and eNOS expression. Our findings suggest a cross talk between miR-423 and VEGF signaling, affecting eNOS function. miR-423 may be involved in the regulation of diabetic vascular retinal proliferation.