Molecular Tools Research Articles

Methods used in sea turtle genetic and molecular studies

Sea turtles have a tropical and subtropical distribution and can be found in nearly all seas and oceans. They have been the subject of considerable genetic research. However, it does not yet appear that the molecular techniques used for these genetic studies follow a consensus or universal set of tools to be followed for subsequent studies. This is not desirable since it may preclude data exchange and use among studies. Thus, the aim of this review was to survey the main genetic and molecular methods used for sea turtle research worldwide. To achieve this goal, a total of 95 scientific papers were compiled from online databases. We considered articles that used molecular tools for genetic analysis and provided detailed locality data. The following aspects were assessed: species studied, local of sample collection, type of tissue used for molecular studies and type of genetic material used. The seven known sea turtle species have different distribution patterns, with some overlapping of occurrence. Chelonia mydas has been studied genetically along the coasts of all continents. Skin is the most common type of tissue used for molecular analyses. From genetic studies on sea turtles, it is possible to verify the occurrence of hybridism. This phenomenon is relevant to the conservation of the species and was reported in six articles on this topic. Therefore, it is considered that genetic and molecular assays in sea turtles are important tools for biological evaluation and protection. The aim was to survey the main methods used in genetic and molecular research on marine turtles. Keywords: DNA, Chelonian, Testudines, marine turtles

Evaluating culture-free targeted next-generation sequencing for diagnosing drug-resistant tuberculosis: a multicentre clinical study of two end-to-end commercial workflows.

Drug-resistant tuberculosis remains a major obstacle in ending the global tuberculosis epidemic. Deployment of molecular tools for comprehensive drug resistance profiling is imperative for successful detection and characterisation of tuberculosis drug resistance. We aimed to assess the diagnostic accuracy of a new class of molecular diagnostics for drug-resistant tuberculosis. We conducted a prospective, cross-sectional, multicentre clinical evaluation of the performance of two targeted next-generation sequencing (tNGS) assays for drug-resistant tuberculosis at reference laboratories in three countries (Georgia, India, and South Africa) to assess diagnostic accuracy and index test failure rates. Eligible participants were aged 18 years or older, with molecularly confirmed pulmonary tuberculosis, and at risk for rifampicin-resistant tuberculosis. Sensitivity and specificity for both tNGS index tests (GenoScreen Deeplex Myc-TB and Oxford Nanopore Technologies [ONT] Tuberculosis Drug Resistance Test) were calculated for rifampicin, isoniazid, fluoroquinolones (moxifloxacin, levofloxacin), second line-injectables (amikacin, kanamycin, capreomycin), pyrazinamide, bedaquiline, linezolid, clofazimine, ethambutol, and streptomycin against a composite reference standard of phenotypic drug susceptibility testing and whole-genome sequencing. Between April 1, 2021, and June 30, 2022, 832 individuals were invited to participate in the study, of whom 720 were included in the final analysis (212, 376, and 132 participants in Georgia, India, and South Africa, respectively). Of 720 clinical sediment samples evaluated, 658 (91%) and 684 (95%) produced complete or partial results on the GenoScreen and ONT tNGS workflows, respectively, with 593 (96%) and 603 (98%) of 616 smear-positive samples producing tNGS sequence data. Both workflows had sensitivities and specificities of more than 95% for rifampicin and isoniazid, and high accuracy for fluoroquinolones (sensitivity approximately ≥94%) and second line-injectables (sensitivity 80%) compared with the composite reference standard. Importantly, these assays also detected mutations associated with resistance to critical new and repurposed drugs (bedaquiline, linezolid) not currently detectable by any other WHO-recommended rapid diagnostics on the market. We note that the current format of assays have low sensitivity (≤50%) for linezolid and more work on mutations associated with drug resistance is needed. This multicentre evaluation demonstrates that culture-free tNGS can provide accurate sequencing results for detection and characterisation of drug resistance from Mycobacterium tuberculosis clinical sediment samples for timely, comprehensive profiling of drug-resistant tuberculosis. Unitaid.

Molecular tool for efficient breeding of DOMINANT Greenshell laying hens and significant refinement of phenotypic selection focused on eggshell color

In recent decades, interest in non-traditionally colored eggs has increased. For breeders, this market interest means breeding lines of laying hens that lay eggs of varied colors, such as the blue-green eggshells (Dominant Greenshell) in this study. This study presents the results of genotyping the polymorphism of the O locus responsible for shell coloration and photometric measurement of eggshell color based on the CIELAb system, which was carried out on the unique Czech breeding population Dominant Greenshell. The aim was to use a combination of phenotyping using the CIELab System method and genotyping of the O locus using the end-point PCR approach with the main focus on the accuracy of distinguishing shell color genotypes, streamlining the selection of dominant homozygotes in the O locus, optimizing this technology for the most efficient and cost-effective selection procedure in practical hen breeding. The optometric method was able to reliably distinguish only dominant and recessive phenotypes and eliminate from the population only undesirable recessive homozygotes with a white colored shell. The parameter a* (redness/greenness) from the CIELab color space turned out to be absolutely key for distinguishing dominant and recessive phenotypes. Using the CART methodology, a classification tree built on discriminating optometric characteristics a-blunt was obtained, however, for the group of desirable O/O homozygotes, the selection approach would result in incorrect genotyping of 31% of individuals. Therefore, a combined approach based on rapid and simple elimination of recessive homozygotes using phenotyping (CIELab photometric measurement) and molecular identification of the EAV-HP insertion in the SLCO1B3 gene in dominant phenotypes, regardless of color intensity affected by laying time/order, and allowing reliable elimination, has proven to be the most effective method to distinguish heterozygotes from the breeding population. The combination of optometric and molecular selection methods then leads to more efficient selection, reduction of overall selection costs. This process led to the stabilization of the breeding population within one generation and the achievement of a pure homozygous line with regard to eggshell color.

Isoniazid resistance pattern among pulmonary tuberculosis patients in Bangladesh: An exploratory study

ObjectivesIn high tuberculosis (TB) burden countries such as Bangladesh, research and policy tend to focus on rifampicin (RIF)-resistant TB patients, leaving RIF-sensitive but isoniazid (INH)-resistant (Hr-TB) patients undiagnosed. Our study aims to determine the prevalence of INH resistance among pulmonary TB patients in selected health care facilities in Bangladesh. MethodsThis study was conducted across nine TB Screening and Treatment Centres situated in Bangladesh. Sputum samples from 1084 Xpert-positive pulmonary TB patients were collected between April 2021 and December 2022 and cultured for drug susceptibility testing. Demographic and clinical characteristics of Hr-TB and drug-susceptible TB patients were compared. ResultsAmong available drug susceptibility testing results of 998 culture-positive isolates, the resistance rate of any INH regardless of RIF susceptibility was 6.4% (64/998, 95% CI: 4.9–8.2). The rate was significantly higher in previously treated (21.1%, 16/76, 95% CI: 12.0–34.2) compared with newly diagnosed TB patients (5.2%, 48/922, 95% CI: 3.8–6.9) (p < 0.001). The rate of Hr-TB was 4.5% (45/998, 95% CI: 3.3–6.0), which was also higher among previously treated patients (6.6%, 5/76, 95% CI: 1.4–13.5) compared with newly diagnosed TB patients (4.3%; 40/922, 95% CI: 3.1–5.9) (p 0.350). Most importantly, the rate of Hr-TB was more than double compared with MDR-TB (4.5%, 45/998, vs. 1.9%, 19/998) found in the current study. DiscussionThis study reveals a high prevalence of Hr-TB, surpassing even that of the multi-drug-resistant TB in Bangladesh. This emphasizes the urgent need to adopt WHO-recommended molecular tools at the national level for rapid detection of INH resistance so that patients receive timely and appropriate treatment.

Palladium–iridium nanocubes modified with a high-affinity DNA aptamer as paired viral diagnostic and therapeutic tools

The COVID-19 pandemic emphasizes the need for the development of molecular tools that can be used as effective diagnostic and therapeutic agents. Herein we investigate the potential of aptamer-dressed nanomaterials both as diagnostics and therapeutics using SARS-CoV-2 as a model. The nanomaterials are based on the palladium-iridium (Pd–Ir) nanocubes modified with monomeric, dimeric or trimeric aptamers that exhibit varying affinities for the spike protein of SARS-CoV-2. These nanomaterials were first examined for diagnostic potential through the creation of a nanozyme-linked aptamer assay (NLAA) that takes advantage of the peroxidase-mimicking activity of Pd–Ir nanocubes. The trimeric aptamer-based NLAA demonstrated a limit of detection (LOD) of 9.3 ​× ​103 cp/mL for pseudoviruses expressing the spike protein of SARS-CoV-2, 172- and 12.9-fold lower than that of the monomeric and dimeric aptamer-based NLAAs, respectively. Upon testing with 60 clinical saliva samples, the trimeric aptamer-based NLAA achieved a specificity of 100 ​% and a sensitivity of 86.7 ​%. The same nanomaterials were also examined for the ability to block viral entry to host cells. The trimeric aptamer-conjugated nanocubes exhibited a superior neutralizing ability, with an IC50 value of 6.4 pM, 2.7- and 10.1-fold lower than that of the dimeric and monomeric aptamer nanocubes. Moreover, the trimeric aptamer-conjugated nanocubes exhibited excellent biostability and biocompatibility. Overall, our study provides a framework for combating future viral pandemics through the development of a paired biosensor and neutralizing agent made of the same aptamer-modified nanomaterial that recognizes an important viral surface protein like the spike protein of SARS-CoV-2.

Exploring the Genetic Diversity of Mycoplasma hyopneumoniae in Pigs with Pneumonia and Pleurisy at Slaughter.

Mycoplasma (M.) hyopneumoniae is the key pathogen of the porcine respiratory disease complex (PRDC) and contributes to pleurisy in pigs. Due to its limited metabolism and laborious cultivation, molecular tools are useful for diagnosis. This study investigated the genetic diversity of M. hyopneumoniae in slaughter pigs with pneumonia and pleurisy, and it assessed co-infections by Pasteurella multocida type A (PM), Actinobacillus pleuropneumoniae (APP), and swine influenza virus A (sIVA). Lungs (n = 70) with different pleurisy scores and lesions compatible with M. hyopneumoniae infection were collected for convenience. Macroscopic and microscopic evaluations were performed. M. hyopneumoniae was detected using qPCR, and MLST was used for genetic characterization. Co-infections with PM and APP were also evaluated by qPCR, while the immunohistochemistry assessed sIVA infection. All lungs were positive for M. hyopneumoniae. Histopathology confirmed M. hyopneumoniae-associated lesions. MLST characterization was possible in 25 lungs and revealed 10 distinct allelic profiles, with none matching known sequence types in the public database. Co-infections were detected in 40% of the samples with APP and 32% with PM, with 12% showing both pathogens and 52% of the samples presenting microscopic lesions compatible with sIVA infection. The diverse genetic profiles found underscore the need for research on isolation and potential pathogenic variations.

Structure and functional studies of Avt1, a novel peptide from the sea anemone Aulactinia veratra

Sea anemones are a rich source of peptide toxins spanning a diverse range of biological activities, typically targeting proteins such as ion channels, receptors and transporters. These peptide toxins and their analogues are usually highly stable and selective for their molecular targets, rendering them of interest as molecular tools, insecticides and therapeutics. Recent transcriptomic and proteomic analyses of the sea anemone Aulactinia veratra identified a novel 28-residue peptide, designated Avt1. Avt1 was produced using solid-phase peptide synthesis, followed by oxidative folding and purification of the folded peptide using reversed-phase high-performance liquid chromatography. The liquid chromatography-mass spectrometry profile of synthetic Avt1 showed a pure peak with molecular mass 6 Da less than that of the reduced form of the peptide, indicating the successful formation of three disulfide bonds. The solution structure determined by NMR revealed that Avt1 adopts an inhibitor cystine knot (ICK) fold, in which a ring is formed by two disulfide bonds with a third disulfide penetrating the ring to create the pseudo-knot. This structure provides ICK peptides with high structural, thermal and proteolytic stability. Consistent with its ICK structure, Avt1 was resistant to proteolysis by trypsin, chymotrypsin and pepsin, although it was not a trypsin inhibitor. Avt1 at 100 nM showed no activity in patch–clamp electrophysiological assays against several mammalian voltage-gated ion channels, but has structural features similar to toxins targeting insect sodium ion channels. Although sequence homologues of Avt1 are found in a number of sea anemones, this is the first representative of this family to be characterised structurally and functionally.

Molecular Genetic Insights into the Stress Responses and Cultivation Management of Zoysiagrass: Illuminating the Pathways for Turf Improvement

Zoysiagrass (Zoysia spp.) and its hybrids are known for their low maintenance requirements and are widely utilized as warm-season turfgrass, which offers considerable ecological, environmental, and economic benefits in various environments. Molecular genetic approaches, including the identification and genetic engineering of valuable gene resources, present a promising opportunity to enhance the quality and performance of zoysiagrass. This review surveys the recent molecular genetic discoveries in zoysiagrass species, with a focus on elucidating plant responses to various abiotic and biotic stresses. Furthermore, this review explores the notable advancements in gene function exploration to reduce the maintenance demands of zoysiagrass cultivation. In addition, we discuss the achievements and potential of contemporary molecular and genetic tools, such as omics approaches and gene editing technologies, in developing zoysiagrass cultivars with desirable traits. Overall, this comprehensive review highlights future strategies that may leverage current molecular insights to accelerate zoysiagrass improvement and further promote sustainable turf management practices.

Epidemiology of Hepatitis C virus among thalassemia patients in Al-Muthanna Province

One of the main causes of blood transfusion problem including thalassemia, is the hepatitis C virus (HCV). In total, 1125 patients with thalassemia (635 men and 490 women, ages 2-23 years) took part in the trial from January 1, 2019, to December 31, 2021. Prior to PCR assay, the clinical symptoms, complete blood count (CBC) and particular hemoglobin tests were used to diagnose the thalassemia patients. Three blood transfusions every month were on an average. 1125 patients in total underwent ELISA testing to identify anti-HCV antibody (IgG) in serum. There were 111 (9.8%) Hepatitis C virus infections. In comparison to 2019 and 2021, the year 2020 had the highest numbers of thalassemia patients 400 (36%) and hepatitis C virus infections 41 (36.9%). Hepatitis C virus infected male individuals with thalassemia are more prevalent than female individuals. Compared to married, widowed and divorced states, the single status had the highest rate of hepatitis C virus infection. In comparison to urban residence, the number and percentage of Hepatitis C virus infections were highest in rural areas. To lessen the spread of the virus and eradicate it, we recommend using routine laboratory testing of blood donors and Thalassemia patients and the use of molecular biology tools like the PCR assay in the early diagnosis of the virus.

Potential Performance of Two New RT-PCR and RT-qPCR Methods for Multiplex Detection of Dengue Virus Serotypes 1-4 and Chikungunya Virus in Mosquitoes.

Mosquitoes of the genus Aedes are the most important arthropod disease vector. Dengue virus (DENV) and Chikungunya virus (CHIKV) are the main arboviruses distributed throughout the world. Based on entomo-virological surveillance, appropriate public health strategies can be adopted to contain cases and control outbreaks. This study aims to show the potential performance of two new molecular methods for detecting DENV serotypes and CHIKV in mosquitoes. Mosquitoes were collected in urban and sylvatic areas of Bobo-Dioulasso, Burkina Faso, between July and August 2023. DENV and CHIKV were screened using new multiplex RT-PCR and RT-qPCR methods. A total of 2150 mosquitoes were trapped, consisting of 976 Aedes (959 Ae. aegypti, 6 Ae. furcifer, and 11 Ae. vittatus) and 1174 Culex sp. These were grouped into 39 pools, with each pool containing a maximum of 30 mosquitoes. Molecular screening revealed that 7.7% (3/39) of the pools were positive for DENV. Specifically, DENV-1 was detected in one pool (1/3), and DENV-3 was found in two pools (2/3). All pools tested negative for CHIKV. The overall minimum infection rate (MIR) of DENV in this study was 3.07 (95% CI: 2.24-19.86). This study shows the usefulness of our new molecular tools for the surveillance of DENV serotypes and CHIKV.

Identification and phylogenetic analysis of carbapenemase genes from clinical strains of Klebsiella pneumoniae.

Klebsiella pneumoniae is an encapsulated Gram-negative bacterium that is responsible for numerous infections in healthcare facilities worldwide and is frequently isolated. The World Health Organization has listed K. pneumoniaeas as a critical antibiotic resistant bacterial pathogen for which new antibiotics are urgently needed. This study aimed to use molecular tools to identify and examine antibiotic resistance in clinical strains of K. pneumoniae. A total of 15 unduplicated K. pneumoniae strains isolated from patient samples with multidrug-resistant (MDR) profiles were subjected to polymerase chain reaction (PCR) to amplify the most common carbapenem resistance genes. (GTG)5 PCR and phylogenetic analysis were performed to identify the genetic relationship between the strains. All strains yielded a (GTG)5-PCR profile, and this allowed us to group these strains into 8 groups according to the size and number of characteristic bands. Phylogenetic analysis was done using the free software UPGMA and a single bacterial clone with a correlation coefficient of over 97% was identified. New Delhi metallo-beta-lactamase NDM-like (blaNDM) carbapenem resistance genes were detected in three strains of K. pneumoniae, which represented a resistance rate of 20%. However, carbapenemases type A [Klebsiella pneumoniae carbapenemase (KPC) and imipenem-hydrolysing beta-lactamase (IMI), type D [oxacillinase-48 (OXA-48)], and other metallo-β-lactamase [Verona integron-encoded metallo-beta-lactamase (VIM), and enzyme active on imipenem (IMP)] were not detected. We identified and grouped the blaNDM resistance genes of Klebsiella pneumonia strains.

Identification and Differentiation of the Fusarium graminearum NX-2 Chemotype Using High-Resolution Melting (HRM).

Fusarium head blight causes significant yield losses in wheat and other cereals and contaminates grain products with trichothecene mycotoxins. F. graminearum isolates are classified into different chemotypes depending on the type of mycotoxin produced, including the type B trichothecenes 3-acetyl deoxynivalenol (3-ADON), 15-acetyl deoxynivalenol (15-ADON), nivalenol (NIV), and the recently identified type A trichothecene NX-2. Molecular tools to differentiate NX-2 producers from other chemotypes have remained relatively laborious and time consuming. In this study, we developed and validated a high-resolution melting (HRM) assay that can identify NX-2 producers quickly and cost-effectively. By analyzing TRI1 coding sequences from 183 geographically diverse isolates representing all four F. graminearum chemotypes, we selected a 75-base pair region containing four non-synonymous single nucleotide polymorphisms (SNPs) that are specific to the NX-2 genotypes. The amplicon generated two HRM profiles, one of which was specific for only NX-2. We confirmed that the assay is robust across qPCR platforms and unambiguously differentiates NX-2 from other chemotypes using a panel of 72 diverse isolates previously collected from North America. The HRM assay was also successful in identifying NX-2 producers directly from DNA extracted from infected wheat spikes with varying levels of disease severity and fungal DNA. The assay can detect as little as 0.01 ng of fungal DNA in a background of 50 ng of plant DNA. This new diagnostic assay can be used for high-throughput molecular detection of the NX-2 chemotype of F. graminearum from infected plant samples and culture collections, thus making it a valuable tool for surveys of contemporary and historical FHB pathogen populations.

Advances in the Clinical Study of Nuclear Overhauser Enhancement.

Nuclear overhauser enhancement is a confounding factor arising from the in vivo application of a chemical exchange saturation transfer technique in which two nuclei in close proximity undergo dipole cross-relaxation. Several studies have shown applicability and efficacy of nuclear overhauser enhancement in observing tumors and other lesions in vivo. Thus, this effect could become an emerging molecular imaging research tool for many diseases. Moreover, nuclear overhauser enhancement has the advantages of simplicity, noninvasiveness, and high resolution and has become a major focus of current research. In this review, we summarize the principles and applications of nuclear overhauser enhancement. LEVEL OF EVIDENCE: 2 TECHNICAL EFFICACY: Stage 1.

Distinct Communities of Bacteria and Unicellular Eukaryotes in the Different Water Masses of Cretan Passage Water Column (Eastern Mediterranean Sea).

Elucidating marine microbiota diversity and dynamics holds significant importance due to their role in maintaining vital ecosystem functions and services including climate regulation. This work aims to contribute in the understanding of microbial ecology and networking in one of the world's most understudied marine regions, the Eastern Mediterranean Sea. High-throughput 16S and 18S rRNA gene sequencing analysis was applied to study the diversity of bacteria and unicellular eukaryotes in the different water masses of the Cretan Passage during two seasonally-different sampling expeditions. We assumed that microbial associations differ between the surface and deepwater masses and created co-occurrence networks to evaluate this hypothesis. Our results unveiled vertical variations in both bacterial and unicellular eukaryotic diversity with species fluctuations indicative of seasonality being recorded in the surface water mass. Heterotrophic taxa and grazers related to organic matter degradation and nutrient cycling were enriched in the deepest water layers. Moreover, surface waters presented a higher number of microbial associations indicating abundant ecological niches compared to the deepest layer, possibly related to the lack of bottom-up resources in the oligotrophic deep ocean. Overall, our data provide insight in a heavily stressed, yet underexplored, marine area that requires further research to unravel the ecological roles of marine microbes. To our knowledge, this is the first study that combines molecular biology tools to provide data on both planktic prokaryotes and unicellular eukaryotes across the different water masses in this marine region of the Eastern Mediterranean basin.

A novel nucleic acid extraction system integrating a switching valve-assisted microfluidic cartridge and a constant pressure pump

BackgroundNucleic acid extraction (NAE) is essential in molecular diagnostics, genetic engineering, and DNA sequencing. While advances in switching valve-assisted, self-contained microfluidic (S2M) cartridges have improved the NAE processes, challenges persist in streamlining workflows, reducing processing times, and enhancing multi-target detection. Achieving these objectives requires precise control of on-chip fluid dynamics and efficient transfer of nucleic acid solutions (NAS) to detection areas. However, research on novel S2M cartridge designs and compatible fluid control systems is still limited. ResultThis study presented an innovative NAE system that integrated an S2M cartridge, a constant pressure (CP) pump, a metal heating bath, a rotating lever, and a magnet. The S2M cartridge contained the reagents for both NAE and detection, while the CP pump provided stable, precise, and rapid fluid delivery, outperforming traditional peristaltic and injection pumps, especially for handling small volumes and reducing pulsation. A custom rotating lever connects reagent chambers, facilitating mixing, cleaning, elution, and NAS transfer. This system showed advantages over manual methods, achieving all processes in 20 min. The CP pump-assisted S2M cartridge enabled rapid MBs re-suspension with a recovery rate of 80 %. The system's sensitivity, repeatability, and ease of use were also confirmed. The NAE system can extract Escherichia coli DNA (50 CFU mL^-1) from food and serum samples and viral DNA (HBV, HCV, HIV) at 200 copies mL^-1 from serum samples. SignificanceThis study highlighted the need for an efficient fluid system in the S2M cartridge for stable NAE. By streamlining NAE and facilitating on-chip reagent preparation, the system enhanced point-of-care diagnostics. Although manual steps remain, the CP pump effectively managed fluid processes, paving the way for automated pathogen detection. Moreover, when combined with visual detection reagents, the NAE system showed promise as a direct molecular diagnostic tool.

Prefrontal synaptic regulation of homeostatic sleep pressure revealed through synaptic chemogenetics.

Sleep is regulated by homeostatic processes, yet the biological basis of sleep pressure that accumulates during wakefulness, triggers sleep, and dissipates during sleep remains elusive. We explored a causal relationship between cellular synaptic strength and electroencephalography delta power indicating macro-level sleep pressure by developing a theoretical framework and a molecular tool to manipulate synaptic strength. The mathematical model predicted that increased synaptic strength promotes the neuronal "down state" and raises the delta power. Our molecular tool (synapse-targeted chemically induced translocation of Kalirin-7, SYNCit-K), which induces dendritic spine enlargement and synaptic potentiation through chemically induced translocation of protein Kalirin-7, demonstrated that synaptic potentiation of excitatory neurons in the prefrontal cortex (PFC) increases nonrapid eye movement sleep amounts and delta power. Thus, synaptic strength of PFC excitatory neurons dictates sleep pressure in mammals.

PCR-based RFLP and ERIC-PCR patterns of Helicobacter pylori strains linked to multidrug resistance in Egypt.

H. pylori infects approximately 50% of the world's population that causes chronic gastritis, and may lead to peptic ulcer disease (PUD). H. pylori-induced chronic infections are associated with gastric adenocarcinoma and low-grade gastric lymphoma. In Egypt, H. pylori strains are widespread and became resistant to antimicrobial agents, thus advanced typing methods are needed to differentiate infectious strains that are resistant to antibiotics, and therefore earlier prognosis and infection control. The main objectives were (i) to determine susceptibility of infectious H. pylori strains to some antimicrobial agents that are currently used in eradication therapy in Egypt; (ii) to identify diverse strains commonly detected in the gastrointestinal (GIT) endoscopy units in Egypt through phenotypic and genotypic analyses. In this observational study we isolated 167 isolates from 232 gastric biopsies (antrum and corpus) of patients who were admitted to the upper GIT endoscopy units in five governmental Egyptian hospitals. Antimicrobial susceptibility patterns were investigated using Kirby Bauer disc diffusion and agar dilution Minimum Inhibitory Concentrations (MICs) methods. Phenotypic characterization was based on biotyping and antibiogram typing techniques. Genotypic characterization was carried out using PCR-based Restriction Fragment Length Polymorphism (RFLP) and Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR analyses. H. pylori isolates were highly resistant to diverse antimicrobial agents including Metronidazole, Fluoroquinolones, Macrolides, Amoxycillin, Tetracycline and Gentamicin. Two factors contributed to the increased resistance of H. pylori to the conventional therapy seen in Egypt: (i) Metronidazole and Amoxycillin are inexpensive and available drugs being abused by patients; (ii) the regional prescribing practice of Macrolids commonly used to treat upper respiratory and urinary tract infections. Five different biotypes were identified depending on the ability of the isolates to synthesize different enzymes. Nine antibiogram types were identified. PCR-RFLP analysis revealed fifteen different fingerprints while ERIC-PCR revealed 22 fingerprints. Biotyping alone or in combination with antibiogram typing are highly useful molecular tools in the prognosis of strain relatedness. PCR-RFLP and ERIC-PCR acquired good discriminatory power for identifying H. pylori infectious sub-types.

Monocentric evaluation of the Novaplex dermatophyte multiplex qPCR assay in the diagnosis of dermatophytoses.

Superficial fungal infections caused by dermatophytes are a prevalent global health concern. Rapid and accurate diagnosis of these pathogens through molecular tools would offer a substantial advantage for early detection and effective treatment. The conventional fungal culture presents inherent limitations, including extended result delivery delay and variable sensitivity. This study aimed to evaluate the performance of the multiplex real-time PCR Novaplex dermatophyte assay (Seegene) in comparison to traditional mycological methods including direct examination and culture. A total of 312 nail, skin, and scalp samples collected from patients with suspected superficial fungal infections for mycological diagnosis were retrospectively subjected to the Novaplex dermatophyte assay. Overall, 170 (54.6%) and 186 (59.6%) samples tested positive for dermatophyte culture and dermatophyte PCR, respectively. The concordance between PCR and culture for dermatophyte detection was 87.2%. There were 158 culture-positive/PCR-positive samples, 12 culture-positive/PCR-negative samples, and 28 culture-negative/PCR-positive samples. The sensitivity of PCR against culture varied according to the dermatophyte target, ranging from 90.5% (Trichophyton mentagrophytes/interdigitale/benhamiae), 91.2% (Trichophyton rubrum), to 100% (Microsporum spp. and Trichophyton tonsurans). When considering the final diagnosis using composite criteria, the sensitivity and specificity for the diagnosis of dermatophytosis were 92.9% and 96.6% for PCR, 86.7% and 100% for culture, and 95.4% and 92.2% for direct examination and culture combined, respectively. The Seegene Novaplex dermatophyte assay is an easy-to-use automated one-step extraction-PCR system that offers satisfactory performance for routine diagnosis of dermatophytoses in clinical laboratories, particularly in non-specialized centers. However, it cannot fully replace conventional mycology due to its inability to detect mold infections and to identify dermatophytes at the species level.

Lymph Node Histology and Metagenomic Sequencing in a child with Cat Scratch Disease.

A 4-year-old boy presented with fever and lymphadenopathy. Despite antibiotic treatment for suspected lymphadenitis, there was minimal improvement. A lymph node biopsy revealed granulomas, raising suspicion of cat scratch disease (CSD). The boy had been scratched by a kitten a month prior. Metagenomic testing of the tissue confirmed the presence of Bartonella henselae, the causative agent of CSD. It is important to incorporate molecular diagnostic tools into clinical practice for precise and timely diagnosis of infectious diseases.

Multi-spectral image transformer descriptor classification combined with molecular tools for early detection of tomato grey mould

Accurate early detection of plant diseases is a critical milestone in the development of self-navigating robots for precision agriculture and still remains one of the most significant challenges. Plant pathogens, like Botrytis cinerea causing grey mould disease, pose significant threats to agriculture and food safety. This study focuses on the early detection of B. cinerea in tomato crops using Artificial Intelligence. Specifically, the Deep Learning (DL) YOLOv8 architecture was employed to apply single class segmentation on plant captures to extract the tomato leaves locational information. Each plant capture includes five images from hyperspectral wavelengths (at 460, 540, 640, 775 and 875 nm) and one RGB images. The leaf segmentation achieved 81.7 % Mean Average Precision (mAP) at Intersection over Union (IoU) threshold 0.5 (mAP50). Then the for each leaf segment, and array of descriptors is extracted through various Transformer models. Finally, the descriptors are classified through a KNN or an LSTM solution and the results of all the descriptors for each leaf from each of the Transformer models, for each of the six images of the capture, are ensembled to yield the class of each leaf. The best ensemble results were produced by only accumulating results from some of the Transformer and specifically the MaxViT-L, the ViT-B (P:16 × 16 – C), the VOLO (D:5) and the XCIT-L (L:24 – P:16 × 16), by only using the results provided by the images at the hyperspectral wavelengths at 540, 640 nm and the RGB image, and by applying the LSTM solution. The process achieved a 79.41 % classification accuracy and 71.12 % F1-Score for all classes. Additionally, early, rapid and accurate detection of grey mould at the early stages of the infection or at latent infections when symptoms are not visible was assessed by comparative qPCR-based methods (qRT-PCR) for fungal biomass estimation in plant tissues and the rest of this study's methodology. The findings of this study represent a significant advancement in crop disease detection and management, and highlight the potential for integrating these methods into on-site digital systems (robots, mobile apps, etc.) under real-world settings. The results, demonstrate the effectiveness of combining segmentation and transformer-based classification models for early and accurate detection of grey mould disease.