Phenotypic Methods Research Articles

A Skeleton-Based Method of Root System 3D Reconstruction and Phenotypic Parameter Measurement from Multi-View Image Sequence

The phenotypic parameters of root systems are vital in reflecting the influence of genes and the environment on plants, and three-dimensional (3D) reconstruction is an important method for obtaining phenotypic parameters. Based on the characteristics of root systems, being featureless, thin structures, this study proposed a skeleton-based 3D reconstruction and phenotypic parameter measurement method for root systems using multi-view images. An image acquisition system was designed to collect multi-view images for root system. The input images were binarized by the proposed OTSU-based adaptive threshold segmentation method. Vid2Curve was adopted to realize the 3D reconstruction of root systems and calibration objects, which was divided into four steps: skeleton curve extraction, initialization, skeleton curve estimation, and surface reconstruction. Then, to extract phenotypic parameters, a scale alignment method based on the skeleton was realized using DBSCAN and RANSAC. Furthermore, a small-sized root system point completion algorithm was proposed to achieve more complete root system 3D models. Based on the above-mentioned methods, a total of 30 root samples of three species were tested. The results showed that the proposed method achieved a skeleton projection error of 0.570 pixels and a surface projection error of 0.468 pixels. Root number measurement achieved a precision of 0.97 and a recall of 0.96, and root length measurement achieved an MAE of 1.06 cm, an MAPE of 2.37%, an RMSE of 1.35 cm, and an R2 of 0.99. The whole process of reconstruction in the experiment was very fast, taking a maximum of 4.07 min. With high accuracy and high speed, the proposed methods make it possible to obtain the root phenotypic parameters quickly and accurately and promote the study of root phenotyping.

Discordance in genotypic and phenotypic drug susceptibility results: time to reconsider critical concentration of rifampicin.

Tuberculosis (TB) remains a leading cause of morbidity and mortality worldwide, killing millions every year. The emergence of multidrug-resistant and extensively drug-resistant TB forms poses a challenge to the TB control programs. In the past few decades, several molecular tests for rapid detection and drug resistance determination have been developed. But these can miss the genetic mutations that confer low-level resistance to rifampicin (RIF), a critical constituent for treating drug-susceptible TB. On the other hand, for the phenotypic methods, a cutoff value is fixed, known as critical concentration (CC). The current WHO-endorsed CC for rifampicin is 1.0 μg/mL in liquid culture for confirmation of drug resistance; because of that in this system too, low-level RIF resistance may not be correctly identified. Therefore, it is important that either the CC for phenotypic methods is lowered or the specific mutations are included in the molecular tests. This study provides important insights in that direction.

Enhancing lipid production in plant cells through automated high-throughput genome editing and phenotyping

Abstract Plant bioengineering is a time-consuming and labor-intensive process with no guarantee of achieving desired traits. Here, we present a fast, automated, scalable, high-throughput pipeline for plant bioengineering (FAST-PB) in maize (Zea mays) and Nicotiana benthamiana. FAST-PB enables genome editing and product characterization by integrating automated biofoundry engineering of callus and protoplast cells with single-cell matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). We first demonstrated that FAST-PB could streamline Golden Gate cloning, with the capacity to construct 96 vectors in parallel. Using FAST-PB in protoplasts, we found that PEG2050 increased transfection efficiency by over 45%. For proof-of-concept, we established a reporter-gene-free method for CRISPR editing and phenotyping via mutation of high chlorophyl fluorescence 136 (HCF136). We show that diverse lipids were enhanced up to sixfold using CRISPR activation of lipid controlling genes. In callus cells, an automated transformation platform was employed to regenerate plants with enhanced lipid traits through introducing multi-gene cassettes. Lastly, FAST-PB enabled high-throughput single-cell lipid profiling by integrating MALDI-MS with the biofoundry, protoplast, and callus cells, differentiating engineered and unengineered cells using single-cell lipidomics. These innovations massively increase the throughput of synthetic biology, genome editing, and metabolic engineering and change what is possible using single-cell metabolomics in plants.

Anti-biofilm activity of carvacrol-thymoquinone nanocarriers on vulvovaginal candidiasis isolates.

Given the recurrent nature of vulvovaginal candidiasis (VVC), the restricted availability of effective antifungal agents, and the recent rise in drug resistance, this study sought to assess the antifungal efficacy of carvacrol-thymoquinone delivered via a nanocarrier on Candida isolates obtained from patients with VVC. Isolates were identified using phenotypic and genotypic methods. Nanocarriers were synthesized using the thin-film hydration method. The antifungal activity of carvacrol-thymoquinone was evaluated using the broth microdilution method (CLSIM27-A3). The impact of nanocarriers on the biofilm formation capabilities of Candida isolates was assessed using the MTT assay. Data were analyzed using the Mann-Whitney U test. The nanocarrier exhibited a spherical morphology with a diameter measuring 50 nm. The nano-formulated drug combination could inhibit biofilm formation in C. albicans at half the minimum inhibitory concentration and in C. glabrata at the minimum inhibitory concentration. Our results suggest that the carvacrol-thymoquinone nanocarrier can be studied further in vivo for potential use in the treatment of recurrent VVC.

Study of Phenotypic and Molecular Approaches of Potyvirus plumpoxi Resistance Evaluation in New Czech Apricot Hybrids

This study investigates the resistance of 17 apricot hybrids to Potyvirus plumpoxi (PPV) with the use of phenotyping, serological assays [double-antibody-sandwich enzyme-linked immunosorbent assay (DAS-ELISA)], and molecular marker analyses. The expression of symptoms expression was evaluated on four occasions throughout the course of the experiment, while the presence of the virus in the plant was diagnosed by DAS-ELISA. In addition, the genotyping with simple sequence repeat (SSR) markers (PGS1.21, PGS1.23) and ParPMC2 allele-specific polymerase chain reaction (PCR) for the purpose of evaluating PPV resistance. A total of five new apricot hybrids were evaluated as resistant to PPV, while five hybrids exhibited resistance despite the presence of some PPV symptoms. In addition, three hybrids demonstrated genotype-phenotype incongruences (GPIs). The statistical analysis revealed a significant relation between PPV-positive plants and the absence of resistance-associated alleles, confirming that allelic composition is strongly associated with phenotypic resistance. The average value of symptoms evaluation was 0.3 ± 0.1 for resistant hybrids and 1.7 ± 0.3 and 1.5 ± 0.4 for susceptible hybrids evaluated by PGS1.21 and PGS1.23, respectively. The Tukey’s honestly significant difference (HSD) test showed the significant difference between genotypes evaluated by ParPMC2 marker as “RR” and “RS” (resistant) from “SS” (susceptible). However, discrepancies in hybrid resistance evaluation highlight the complexity of inheritance and the potential errors in genotyping. These results indicate that the most reliable method for evaluating PPV resistance is the time-consuming phenotyping method. Conversely, the use of SSR markers greatly improves the effectiveness of breeding.

Annotated image dataset with different stages of European pear rust for UAV-based automated symptom detection in orchards.

The evaluation of fruit genetic resources regarding a resistance to pathogens is an essential basis for subsequent selection in fruit breeding. Both genetic analysis and phenotyping of defined traits are important tools and provide decision data in the evaluation process. However, the phenotyping of plants is often carried out 'by hand' and remains the bottleneck in fruit breeding and fruit growing. The development of a digital and UAV (unmanned aerial vehicle)-based phenotyping method for the assessment of genotype-specific susceptibility or resistance against diseases in orchards would significantly increase the efficiency of plant breeding. In this framework, a workflow for drone-based monitoring of pathogens in orchards was developed using the European pear rust (Gymnosporangium sabinae) as model pathogen. Pear rust is widespread in orchards and causes conspicuous, clearly visible, yellow to orange-colored disease symptoms. In this paper, we provide a dataset with expert-annotated high-resolution RGB images with pear rust symptoms. For data collection, ten UAV-flight campaigns were realized between 2021 and 2023 under various weather conditions and with different flight parameters in the experimental orchard of the Julius Kühn-Institute for Breeding Research on Fruit Crops in Dresden-Pillnitz (Germany). 1394 images were captured of different pear genotypes, including varieties, wild species and progeny from breeding. The dataset contains manually labelled images with a size of 768 × 768 pixels of leaves infected with pear rust at different stages of development, labelled as class GYMNSA, as well as background images without symptoms. Each leaf with pear rust symptoms was annotated with the drawing method by two points (bounding boxes) using the Computer Vision Annotation Tool (CVAT, v1.1.0) [1] and presented in YOLO 1.1 file format (.txt files). A total of 584 annotated images and 162 background images, organized into a training and validation set, are included in the GYMNSA dataset. This GYMNSA dataset can be used as a resource for researchers and developers working on drone-based plant disease monitoring systems.

P-816. Diagnostic Performance of Molecular Sample-to-Answer Tests for Bloodstream Infections in the Clinical Setting: a Systematic Literature Review and Meta-Analysis

Abstract Background Rapid identification of bloodstream pathogens and antimicrobial resistance (AMR) genes by molecular tests from positive blood cultures (PBCs) have the potential to improve patient management. A systematic review and meta-analysis was conducted to evaluate diagnostic test accuracy (DTA) of molecular tests compared to phenotypic methods for detecting pathogens and AMR genes in the clinical setting.Figure 1.Summary sensitivity and specificity for total gram-negative bacteria and gram-positive bacteria, with subgroup by multiple and single test panel coverage. Methods MEDLINE, EMBASE, and Cochrane databases were searched from inception to October 25, 2023. Search of conference proceedings and forward and backward searching were also done. Studies evaluating DTA of commercially available molecular sample-to-answer tests versus phenotypic methods (reference standard) in adults and children with PBCs were eligible. Risk of bias (RoB) was assessed using QUADAS-2. Summary DTA outcomes were estimated using bivariate random-effects model for gram-negative bacteria (GNB), gram-positive bacteria (GPB), yeast, gram-negative (GN) AMR, gram-positive (GP) AMR, and targets within these groups when reported by ≥ 2 studies (PROSPERO CRD42023488057).Figure 2.Summary sensitivity and specificity for antimicrobial resistance detected in gram-negative bacteria Results Seventy-four studies including 24,634 samples were analyzed, most of which showed low RoB or applicability concerns. Compared with phenotypic methods, molecular tests demonstrated high DTA across all target groups, with sensitivity ≥ 92% and PPV ≥ 99% for total GNB (N=43 studies), GPB (N=38), yeast (N=24), GN AMR (N=35), and GP AMR (N=39). Specificity and NPV were 100% and 98% for GNB and 100% and 97% for GPB, respectively, among molecular tests targeting both groups (not estimable for molecular tests targeting GNB only or GPB only as true negatives are not applicable). Specificity and NPV were ≥ 99% for other target groups. High DTA was also observed for individual pathogen targets (≥ 93% sensitivity, 100% specificity, ≥ 96% PPV, and ≥ 99% NPV). Five of 7 AMR genes had high sensitivity (91%-99%) and specificity (≥ 99%). Sensitivity was lower for 2 carbapenemase genes IMP (N=4; 69%, 95% CI 15%–96%) and VIM (N=4; 70%, 95% CI 38%–90%), of which detection was missed in P. aeruginosa.Figure 3.Summary sensitivity and specificity for antimicrobial resistance detected in gram-positive bacteria Conclusion Results show excellent DTA of molecular sample-to-answer tests compared to traditional culture in identifying a broad panel of pathogens and detecting AMR in GNB and GPB.Table 1.Summary of diagnostic test accuracy outcomes for targets evaluated across studies Disclosures Joan-Miquel Balada-Llasat, PharmD/PhD, bioMerieux: Advisor/Consultant|bioMerieux: Grant/Research Support Tammy C. Bleak, PharmD, MSc, bioMerieux, Inc: employee Sarah Jiudice, MPH, bioMerieux, Inc: employee Tristan T. Timbrook, PharmD, bioMérieux: Stocks/Bonds (Public Company)

ФЕНОТИПИЧЕСКИЕ И МОЛЕКУЛЯРНО-ГЕНЕТИЧЕСКИЕ МЕТОДЫ ОПРЕДЕЛЕНИЯ АНТИБИОТИКОРЕЗИСТЕНТНОСТИ МИКРООРГАНИЗМОВ В ВЕТЕРИНАРИИ

The paper presents scientific approaches to solving a global problem, namely the increasing antibiotic resistance among opportunistic and weakly pathogenic microorganisms, which pose a significant risk both in human medicine and in animal husbandry. In this regard, phenotypic, analytical and molecular methodo-logical methods for determining and studying the antibiotic resistance of microorganisms are presented. Of the phenotypic methods, the method of standard disks and the method of serial dilutions, which are based on the diffusion of antibacterial drugs from the carrier onto a solid nutrient medium and inhibition of the growth of the studied culture of the microorganism, have received wide application. Among the analytical methods for detecting antibiotic resistance, electrophoretic, immunochemical (immunochromatography), UV and mass spectrometry are distinguished. The most promising and effective is mass spectrometry, in particular the MALDI-TOF test, which is the most optimal for routine determination of the sensitivity of a number of cultures of microorganisms. The molecular approach, namely the use of PCR using molecular beacons and DNA genes, expands the possibility of monitoring the resistance of microorganisms, and makes it possible to determine the mechanism of formation of resistance to antibacterial drugs by the presence of resistance genes. The analysis of methodological approaches made it possible to identify the following determinants of resistance: carbapenemase in Enterobacterales spp, P. aeruginosa and Acineto-bacter; Extended-spectrum B-lactamases in Enterobacterales; mcr-mediated encoded resistance to poly-myxins in gram-negative microorganisms; mecA/mecC, mediated resistance to beta-lactams in S.aureus; and vanA/vanB, mediated resistance to glycopeptides in E. faecium and E. faecalis. An analysis of the data of available scientific publications allows us to state that different methodological approaches provide different accuracy in indicating variants of resistance determinants. It should be noted that the presence of several databases of antibiotic resistance genes makes it difficult to use them to determine the phenotype of drug sensitivity. However, the use of genomic data to determine antibiotic resistance is crucial for the development of clinical metagenomics.

P-882. Comparison of Epidemiology, Risk factors, Antimicrobial susceptibility and Outcome of Anaerobic Bacteremia: 2011-2013 and 2020- 2022

Abstract Background Anaerobes are again entering the spotlight as patients become more complex and antimicrobial resistances continue to mount as a result of their widespread use. The aim of our study was to compare the incidence, risk factors and susceptibility patterns of anaerobic bacteremia during 1st Jan 2011-31st Dec 2013 (period 1) and 1st Jan 2020-31st Dec 2022 (period 2) in community hospitals and acute care centres in Edmonton and the North Zone in Alberta. The percentages of anaerobic organisms isolated in 2011-2013 and 2020-2022 Methods • Microbiologic data extracted from laboratory information system • Demographic and clinical data extracted from the EMR system • Blood cultures were incubated on the BD BACTEC 9240™ system or the BD BACTEC™ FX Blood Culture System • Organism identification was performed by Vitek® 2 ANC ID card, traditional phenotypic methods, Vitek® MALDI-TOF and, failing these, genomic sequencing • Susceptibility testing by E-test; interpretation as per CLSI or EUCAST guidelines The percentages of susceptible Bacteroides species to different antimicrobials in 2011-2013 and 2020-2022 Results Period 1: 319 anaerobic bacteremia were identified. 247 were included in the study after exclusion of repeat positives (within the first 2 weeks of the first positive culture) and contamination. Total number of isolates were 279 and susceptibilities were available on 252 during this period. Period 2: 830 anaerobic bacteremia were identified. 467 anaerobic bacteremia were included in the study after exclusion of repeat positives and contamination. The total number of isolates were 539 and susceptibilities were available on 417 isolates during this study period. The total number of anaerobic bacteremia almost doubled from 82 cases per year to 156 cases per year during the study decade. The majority were monomicrobial bacteremia (71% and 85%) with the gastrointestinal tract identified as the commonest source. The most common isolated anaerobe was Bacteroides species. Metronidazole susceptibilities for all anaerobes were above 90%. The crude 30-day mortality rates were 22.7% and 25.8% in period 1 and 2 respectively. Conclusion The total number of anaerobic bacteremia increased significantly in the 9-year interval. No significant changes in metronidazole susceptibility were observed between both periods of time. Disclosures All Authors: No reported disclosures

High-throughput phenotyping of wheat root angle and coleoptile length at different temperatures using 3D-printed equipment

BackgroundInnovation in crop establishment is crucial for wheat productivity in drought-prone climates. Seedling establishment, the first stage of crop productivity, relies heavily on root and coleoptile system architecture for effective soil water and nutrient acquisition, particularly in regions practicing deep planting. Root phenotyping methods that quickly determine coleoptile lengths are vital for breeding studies. Traditionally, direct selection for root and coleoptile traits has been limited by the lack of suitable phenotyping methods, genetic and phenotypic complexity, and poor repeatability in sampling. In this study, we innovated a method utilizing 3D printing technology to measure root angle and coleoptile length in wheat seedlings. We evaluated seedlings from eight different wheat genotypes across varying temperatures and validated our findings through image processing techniques.ResultsThe analysis of variance in root architecture revealed significant differences among genotypes for root angle. Temperature treatments also significantly influenced shoot length, number of roots and total root length. The Tosunbey genotypes exhibited the highest root angle and the lowest root angle was observed in Altindane genotypes. Additionally, we observed that increasing the temperature led to an increase in seedling root length. Similarly, the coleoptile architecture analysis showed significant differences among genotypes in coleoptile length, leaf length, number of roots, and total root length. Temperature treatments and deep sowing applications significantly affected these traits as well. The Tosunbey and Müfitbey genotypes exhibited the longest coleoptile length, whereas the Nevzatbey genotype showed the shortest.ConclusionSelecting for a narrow root angle and a high number of seminal roots can result in deeper, more branched root systems. Furthermore, developing wheat genotypes with longer coleoptiles can enhance plant production and early vigor, particularly with deep sowing. Our method, using the eqiupments producing by 3D printing technology enables high-throughput phenotyping of wheat roots and coleoptiles, offering new insights into root and coleoptile system regulation at different temperature conditions. This method can be seamlessly integrated into breeding programs to enhance drought tolerance, rapidly phenotyping populations for root and coleoptile characteristics.

Advances in digital camera-based phenotyping of Botrytis disease development.

Botrytis cinerea is an important generalist fungal plant pathogen that causes great economic losses. Conventional detection methods to identify B. cinerea infections rely on visual assessments, which are error prone, subjective, labor intensive, hard to quantify, and unsuitable for artificial intelligence (AI) and machine learning (ML) applications. New, often camera-based, techniques provide objective digital data by remote and proximal sensing. We detail the B. cinerea infection process and link this with conventional and novel detection methods. We evaluate the effectiveness of current digital phenotyping methods to detect, quantify, and classify disease symptoms for disease management and breeding for resistance. Finally, we discuss the needs, prospects, and challenges of digital camera-based phenotyping.

Investigating the validity of mCIM and sCIM phenotypic methods in screening Pseudomonas aeruginosa isolates producing IMP, VIM, and NDM metallo-beta-lactamases isolated from burn wounds.

Metallo-beta-lactamase-producing Pseudomonas aeruginosa (P. aeruginosa) is a major pathogen in burn wounds, often exhibiting high levels of antibiotic resistance, which complicates treatment strategies. This study deals with the validity of the modified Carbapenem Inactivation Method (mCIM) and the simplified Carbapenem Inactivation Method (sCIM) phenotypic tests for screening metallo-beta-lactamase (MBL) production by P. aeruginosa isolates from a referral burn center in Iran. Forty isolates were obtained between January and June 2021 and identified using conventional biochemical methods. Antimicrobial susceptibility testing was conducted following Clinical and Laboratory Standards Institute (CLSI) 2021 guidelines. mCIM based on CLSI 2023 guidelines was used to detect carbapenemase production. sCIM was also used based on previously developed protocols. PCR was performed to detect blaIMP, blaVIM, and blaNDM genes. The results were analyzed using SPSS and MedCalc. We observed a 90% resistance rate to imipenem and high resistance to other antibiotics, with multidrug-resistant (MDR) strains constituting 95% of the isolates. The mCIM test demonstrated high sensitivity (87.50%) and high negative predictive value (89.47%) and moderate specificity (70.83%) and moderate positive predictive value (66.67%) for detecting MBLs. In contrast, the sCIM test was unreliable, indicating a need for more standardized testing protocols. This study underscores the importance of accurate and timely detection of carbapenemase production to guide effective treatment.

METHOD FOR PHENOTYPIC IDENTIFICATION OF ACINETOBACTER SEIFERTII BACTERIA

Abstract. The aim of the study is to increase the accuracy of Acinetobacter seifertii species identification by the phenotypic method. Study objects: 5 strains of A. seifertii (3 isolated in the microbiological laboratory at the Military Medical Academy, 2 strains obtained from the Pasteur Institute (St. Petersburg)). Clinical strains of the A. baumannii group (Ab group) isolated at the Military Medical Academy in 2023-2024 were also studied: A. baumannii (n=152), of which 37 strains of the tryptophandestruens biovar, A. nosocomialis (n=12), A. pittii (n=6), 3 strains of A. calcoaceticus from the Neva River. The type of such strains was determined by MALDI-TOF mass spectrometry. The belonging of the strains to the Ab group was established by taxonomic tests. The biovar A. baumannii bv. tryptophandestruens was determined by a chromogenic reaction on sodium benzoate-containing nutrient medium. Urease of rapid activity was detected by a micro-volumetric method assessed 3 hours later. Utilization of D-xylose as the only carbon source was determined on a nutrient medium containing (g/l): D-xylose 2.0; NH4CI 5.0; NH4NO3 1.0; Na2SO4 2.0; K2HPO4 3.0; KH2PO4 1.0; MgSO4 0.1; bromothymol blue 1.6% aqueous solution 4 ml; bacteriological agar 15.0; distilled water 1 l; pH 7.2 ± 0.2; sterilization at 112oC 20 min. A pure bacterial culture was studied, in which signs of the Ab group and growth on a sodium acetate-containing nutrient medium were previously detected (control of no auxotrophy). The agar bacteria culture was suspended in 0.1 ml of 0.85% sodium chloride solution, one loop of bacterial suspension was sown with a stroke on a nutrient medium with D-xylose and a medium without D-xylose (control), incubated aerobically at 30oC 24-48 hours. The absence of bacterial growth on a medium with D-xylose and a medium without D-xylose in the presence of their growth on a nutrient medium with sodium acetate indicates belonging to the species A. seifertii. Studies showed that all A. seifertii strains do not utilize D-xylose, and all strains of other species of the Ab group utilize D-xylose. A. seifertii bacteria do not utilize L-arabinose, however, 29.6±3,7% of A. baumannii strains do not utilize L-arabinose, including all 37 strains of the tryptophandestruens biovar, which determines the unreliability of this test for the identification of A. seifertii. Most strains of A. seifertii (4 out of 5) have a urease of rapid activity, which suggests their proximity to the species A. nosocomialis. A method has been developed for identification of A. seifertii bacteria based on a set of phenotypic features of Ab group bacteria with a test for D-xylose utilization. It has been established that the use of the D-xylose utilization test in conjunction with the detection of rapid urease activity allows to accurately identify the bacteria A. seifertii and A. nosocomialis among other species of the Ab group.

Isolation and Molecular Detection of Antimicrobial Resistance Genes in Escherichia coli and Staphylococcus Species from Joint Ill Cases of Calves in Puducherry

Background: Joint ill is a septicemic polyarthritis condition due to the localization of pathogenic bacteria within the joints of young calves. The emergence of antibiotic resistant pathogen in such cases may lead to further complication in treatment. Thus, the present study was aimed to isolate and identify the bacterial pathogens and its antimicrobial resistant genes from joint ill cases in calves. Methods: A total of 31 pus swabs were collected from joint ill cases from calves and subjected for bacterial isolation and identification by phenotypic and genotypic method followed by determination of its antimicrobial resistant genes (tet and mecA gene) and antibiogram. Result: Based on colony characters, microscopic observation, biochemical tests, 17/31 (54.83%) Escherichia coli and 14/31 (45.16%) Staphylococcus aureus were isolated and identified phenotypically. All the isolates were further confirmed by polymerase chain reaction using E. coli and S. aureus species specific primers. The antimicrobial resistant genes carrying E. coli and S. aureus isolates were also detected, in which 4/17 (23.5%) isolates were positive for tet gene and 4/14 (28.57%) were positive for the mecA gene. The antibiotic susceptibility test showed that the isolates were highly sensitive to Enrofloxacin (100%) and Gentamicin (77%), but were resistant to Amoxyclav (70%) and tetracycline (50%). On conclusion, E. coli and S. aureus are the most common bacterial pathogens identified from this study. The presence of antimicrobial resistant genes (tet and mecA) and antibiogram pattern of the bacterial isolates indicating the possible treatment failure and serious public health risks in future.

Neuroendocrine Tumors: Germline Genetics and Hereditary Syndromes.

The vast majority of neuroendocrine 'neoplasms (NENs) are sporadic, although recent evidence has indicated that a subset of these cancers may also originate as a result of genetic germline mutations. To date, 10% of these cancers can be linked to an inherited genetic syndrome. Genetic diagnosis is crucial for patients with a suspected hereditary NEN syndrome, as it recognizes patients carrying germline mutations and allows for personalized clinical follow-up, considering the higher risk of developing other tumours. The potential for early genetic detection has significant implications for the treatment of patients with hereditary NEN syndrome, as it may facilitate the delivery of precision therapy that differs from that typically provided to other patients. Thus, the integration of genotypic and phenotypic diagnostic methods help clinicians to provide more informed treatment and to extend appropriate prevention to family members.

A Comparison and Assessment of Different Phenotypic Methods for Biofilm Identification and Antimicrobial Correlation from Clinical Isolates of Acinetobacter

A Comparison and Assessment of Different Phenotypic Methods for Biofilm Identification and Antimicrobial Correlation from Clinical Isolates of Acinetobacter

Assessment of typing methods, virulence genes profile and antimicrobial susceptibility for clinical isolates of Proteus mirabilis

Proteus mirabilis (P. mirabilis) is one of the most important causative pathogens associated with complicated urinary tract infections with a 20% incidence. For epidemiological determinations, several phenotypic and molecular typing methods have been implicated. Sixty P. mirabilis isolated undergo antibiotic susceptibility test by standard Kirby Bauer method. They showed high resistance to nitrofurantoin and trimethoprim/sulfamethoxazole that appear mainly in 3rd age group. The 2nd age group comprised most of the resistant isolates to the tested antibiotics. A total of 73.33% of isolates were classified as multi drug resistance (MDR) and 78.3% of isolates were distributed in several antibiotypes with MAR index over 0.2. Twenty-one isolates were strong biofilm-producers and they were significantly related to MDR. Different virulence factors as protease, urease and hemolysin production are detected. Detection of several virulence genes by PCR; zapA and ureC were harbored by all isolates, followed by rsbA (95%), ureA and flaA (93%), hpmA (91.7%) and mrpA (73.3%). Determination of genetic diversity between isolates was performed by different methods (RAPD, ISSR, ERIC, BOX-AIR and REP-PCR) by using several parameters as typeability and discriminatory power indicating that ERIC-PCR was the best method followed by REP-PCR 1R. Rand’s & Wallace coefficients were used for calculating the congruence among typing methods. Conclusions: The results obtained from both conventional and molecular typing methods indicated that molecular methods are superior to conventional methods in the discrimination of isolates. ERIC-PCR and Rep-PCR provide high discrimination ability among P. mirabilis clinical isolates contributing to epidemiological studies.

Automated Phenotypic Analysis of Mature Soybean Using Multi-View Stereo 3D Reconstruction and Point Cloud Segmentation

Phenotypic analysis of mature soybeans is a critical aspect of soybean breeding. However, manually obtaining phenotypic parameters not only is time-consuming and labor intensive but also lacks objectivity. Therefore, there is an urgent need for a rapid, accurate, and efficient method to collect the phenotypic parameters of soybeans. This study develops a novel pipeline for acquiring the phenotypic traits of mature soybeans based on three-dimensional (3D) point clouds. First, soybean point clouds are obtained using a multi-view stereo 3D reconstruction method, followed by preprocessing to construct a dataset. Second, a deep learning-based network, PVSegNet (Point Voxel Segmentation Network), is proposed specifically for segmenting soybean pods and stems. This network enhances feature extraction capabilities through the integration of point cloud and voxel convolution, as well as an orientation-encoding (OE) module. Finally, phenotypic parameters such as stem diameter, pod length, and pod width are extracted and validated against manual measurements. Experimental results demonstrate that the average Intersection over Union (IoU) for semantic segmentation is 92.10%, with a precision of 96.38%, recall of 95.41%, and F1-score of 95.87%. For instance segmentation, the network achieves an average precision (AP@50) of 83.47% and an average recall (AR@50) of 87.07%. These results indicate the feasibility of the network for the instance segmentation of pods and stems. In the extraction of plant parameters, the predicted values of pod width, pod length, and stem diameter obtained through the phenotypic extraction method exhibit coefficients of determination (R2) of 0.9489, 0.9182, and 0.9209, respectively, with manual measurements. This demonstrates that our method can significantly improve efficiency and accuracy, contributing to the application of automated 3D point cloud analysis technology in soybean breeding.

Epidemiological Analysis of a K. pneumoniae NDM Outbreak in a Temporary Ward for Patients with Primary COVID-19 Infection

The COVID-19 pandemic made the medical community realize how large a problem it would face. The epidemiological situation forced the opening of additional wards, the so-called “COVID wards”, where an increase in the rate of coexisting bacterial infections was observed. We report a hospital outbreak due to New Delhi carbapenemases producing K. pneumoniae clones. Twenty-eight K. pneumoniae strains were analyzed from patients with primary COVID-19 infection. The drug susceptibility of the strains was determined by the diffusion–circulation method and E-test. Phenotypic and PCR methods confirmed the production of carbapenemases. The phylogenetic similarity of the obtained strains was examined using pulsed-field electrophoresis. Most strains were isolated from bronchoalveolar lavage. All isolates obtained were resistant to β-lactams and fluoroquinolones. All strains produced New Delhi carbapenemases and were classified into two genetic clusters, A and B. Eight risk factors for secondary bacterial infection were analyzed. Following an intervention involving hand hygiene, strict contact prevention, and cleaning of the hospital environment and medical devices, this outbreak was successfully brought under control.

Advances in microfluidic platforms for tumor cell phenotyping: from bench to bedside.

Heterogeneities among tumor cells significantly contribute towards cancer progression and therapeutic inefficiency. Hence, understanding the nature of cancer through liquid biopsies and isolation of circulating tumor cells (CTCs) has gained considerable interest over the years. Microfluidics has emerged as one of the most popular platforms for performing liquid biopsy applications. Various label-free and labeling techniques using microfluidic platforms have been developed, the majority of which focus on CTC isolation from normal blood cells. However, sorting and profiling of various cell phenotypes present amongst those CTCs is equally important for prognostics and development of personalized therapies. In this review, firstly, we discuss the biophysical and biochemical heterogeneities associated with tumor cells and CTCs which contribute to cancer progression. Moreover, we discuss the recently developed microfluidic platforms for sorting and profiling of tumor cells and CTCs. These techniques are broadly classified into biophysical and biochemical phenotyping methods. Biophysical methods are further classified into mechanical and electrical phenotyping. While biochemical techniques have been categorized into surface antigen expressions, metabolism, and chemotaxis-based phenotyping methods. We also shed light on clinical studies performed with these platforms over the years and conclude with an outlook for the future development in this field.