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.

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.

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.

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.