Real-time Microscopy Research Articles

Characteristics of the monkeypox virus isolate obtained from the first patient in Russia and its sensitivity to 7-[N-(4-trifluoromethylbenzoyl)-hydrazinocarbonyl]-tricyclo-[3.2.2.0^2,4]non-8-en-6-carboxylic acid

Introduction. Since early May 2022, more than 90,000 cases of monkeypox virus infection have been reported in more than 70 countries around the World. This is the largest outbreak of monkeypox ever recorded outside of Africa. The aim of the study is to confirm the first case of monkeypox in Russia, to isolate and sequence a new strain of monkeypox virus (MPXV), and to assess its sensitivity to the 7-[N-(4-trifluoromethylbenzoyl)-hydrazinocarbonyl]-tricyclo-[3.2.2.0^2,4]non-8-en-6-carboxylic acid (NIOCH-14) antipox drug. Materials and methods. The biological materials obtained from the affected area of the skin (contents of vesicles), a nasopharyngeal smear, sputum and venous blood from a patient with suspected monkeypox were used. The disease was confirmed by PCR followed by determination of the nucleotide sequence of viral DNA by sequencing. Isolation of the new MPXV strain from clinical samples was carried out in Vero E6 cells. The antiviral effectiveness of NIOCH-14 against the new MPXV strain was assessed using an adapted spectrophotometric method. Results. A diagnostic study of the biological samples of a patient who returned from a tourist trip to European countries with complaints of skin rashes all over the body revealed MPXV DNA. A new strain of MPXV was isolated from vesicles in Vero E6 cells, and the genomic sequence MPXV-pustule S45 was assembled using high-throughput parallel sequencing (NGS). Discussion. The effectiveness of the finished dosage form of NIOCH-14 against the new strain of MPXV based on the results of determining the 50% virus inhibitory concentration (IC50) was 0.02 μg/mL, and the selectivity index (SI) was 15,000. Conclusion. In this study, the pathogen of monkeypox was detected and identified using real-time PCR, NGS and electron microscopy, and the first imported case of this disease in Russia was confirmed. It has been proven that the drug NIOCH-14 exhibits high antiviral activity in vitro against the new MPXV strain.

Effects of Temperature Fluctuations on Surface Mobility of Atomic Steps and Oxidation Dynamics in High-Temperature Alloys.

In contrast to the traditional perspective that thermal fluctuations are insignificant in surface dynamics, here we report their influence on surface reaction dynamics. Using real-time low-energy electron microscopy imaging of NiAl(100) under both vacuum and O2 atmospheres, we demonstrate that transient temperature variations substantially alter the direction of atom diffusion between the surface and bulk, leading to markedly different oxidation outcomes. During heating, substantial outward diffusion of atoms from the bulk to the surface results in step growth. Conversely, cooling induces considerable inward diffusion of adatoms, producing a distinct oxide morphology. In both scenarios, initially formed oxide islands impede local atomic step mobility, thereby increasing step length due to mass transfer between the surface and bulk, with atomic steps acting as adatom sinks during heating and sources during cooling. Furthermore, we show that this pinning effect on atomic step mobility can be mitigated by applying persistent temperature fluctuations. Understanding these nuances is vital for accurately predicting and dynamically manipulating the performance of active materials in various chemical processes under transient thermal conditions.

Testosterone deficiency aggravates diet-induced non-alcoholic fatty liver disease by inducing hepatocyte ferroptosis via targeting BMAL1 in mice.

Testosterone deficiency is linked to an increased prevalence of non-alcoholic fatty liver disease (NAFLD), although the mechanisms underlying this association are not fully understood. Ferroptosis, a regulated cell death pathway driven by iron-dependent lipid peroxidation, has been suggested to play a role in NAFLD pathogenesis. Since testosterone deficiency is associated with lipid disorders and iron deposition, we hepothesize that ferroptosis may be involved in the pathogenesis of diet-induced NAFLD exacerbated by testosterone deficiency. Apolipoprotein E (APOE-/-) mice were subjected to sham surgery or bilateral castration and subsequently fed a high-fat diet for 16weeks. Liver gene expression was analyzed using RNA sequencing. Additional assessments included blood analysis, histological staining, measurement of iron and antioxidant enzyme levels, quantitative real-time PCR, Western blotting, and electron microscopy. The effects of testosterone on ferroptosis induced by free fatty acids (FFAs) and Erastin were further investigated in HepG2 cells in vitro. Testosterone deficiency resulted in increased hepatic lipid accumulation and macrovesicular steatosis in high-fat diet-fed APOE-/- mice, accompanied by hepatic inflammation, fibrosis, and elevated liver enzyme levels. Transcriptomic analysis revealed that testosterone deficiency affects ferroptosis and circadian rhythm-related signaling pathways. Castrated APOE-/- mice exhibited significantly higher hepatic iron deposition, lipid peroxidation, and expression of key ferroptosis-related proteins, along with decreased Brain and muscle ARNT-like gene 1 (BMAL1) protein expression. In vitro, testosterone treatment reduced lipid and iron accumulation and lipid peroxidation in HepG2 cells subjected to FFAs and Erastin. Moreover, BMAL1 knockdown negated the protective effects of testosterone against ferroptosis in hepatocytes. Our study demonstrated that testosterone deficiency exacerbates NAFLD induced by a high-fat diet by promoting hepatocyte ferroptosis through modulation of the circadian protein BMAL1.

Microfluidics to Follow Spatiotemporal Dynamics at the Nucleo-Cytoplasmic Interface During Plant Root Growth.

Nuclear dynamics refers to global/local changes in the molecular and spatial organization of genomic DNA that can occur during development or in response to environmental stress signals and eventually impact genomic functions. In plants, nuclear dynamics relies notably on the connection of the nucleus with the cytoskeleton during development. It orchestrates genomic functions in response to developmental and environmental cues. This is particularly true in the plant root system, which is constantly exposed to a wide range of internal and external stimuli. Currently, studying nuclear dynamics in a growing root is challenging due to limitations regarding real-time imaging for quantitative analyses under controlled conditions. Microfluidic systems for plant cell studies are valuable analytical tools that provide precise control of culture conditions together with live-imaging capabilities at high temporal and spatial resolutions. Herein, we describe a microfluidic platform to unravel dynamically and noninvasively nuclear organization in the seedling root system exposed to various treatments. As exemplified here, our microfluidic platform can be conveniently used for real-time microscopy imaging and quantitative analysis of fine nuclear morphological changes upon modifying cytoskeleton dynamics. Importantly, our system can be applied to a wide variety of microscopic means including high-resolution microscopy to investigate diverse subcellular compartments or nuclear domains in Arabidopsis thaliana roots.

A Study on the Development of Real-Time Chamber Contamination Diagnosis Sensors.

Plasma processes are critical for achieving precise device fabrication in semiconductor manufacturing. However, polymer accumulation during processes like plasma etching can cause chamber contamination, adversely affecting plasma characteristics and process stability. This study focused on developing a real-time sensor system for diagnosing chamber contamination by quantitatively monitoring polymer accumulation. A quartz crystal sensor integrated with flexible printed circuit boards was designed to measure the frequency shifts corresponding to polymer thickness changes. An impedance probe was also employed to monitor variations in the plasma discharge characteristics. The sensor demonstrated high reliability with a measurement scatter of 2.5% despite repeated plasma exposure. The experimental results revealed that polymer accumulation significantly influenced the plasma impedance, and this correlation was validated through real-time monitoring and scanning electron microscopy (SEM). The study further showed that the sensor could detect the transition point of the plasma state changes under varying process gas conditions, enabling the early detection of potential process anomalies. These findings suggest that the developed sensor system can be crucial for diagnosing plasma and chamber conditions, providing valuable data for optimizing preventive maintenance schedules. This advancement offers a pathway for improving process reliability and extending the operational lifetime of semiconductor manufacturing equipment.

Optical diffraction tomography using a self-reference module

Optical diffraction tomography enables label-free, 3D refractive index (RI) imaging of biological samples. We present a novel, cost-effective approach to ODT that employs a modular design incorporating a self-reference holographic capture module. This two-part system consists of an illumination module and a capture module that can be seamlessly integrated with any life-science microscope using an automated alignment protocol. The illumination module employs a galvo-scanner system, providing precise control over the angular illumination, while the capture module utilises the principle of self-reference off-axis holography. The design has a compact form factor, simple alignment, and reduced cost. Furthermore, our system offers the capability to switch between two imaging modalities, ODT and real-time synthetic aperture digital holographic microscopy (SA-DHM), a unique feature not found in other setups. Experimental results are provided using a kidney cancer cell line. Experimental results are provided using a kidney cancer cell line.

Interplay between reactive oxygen species and ERK activation in cervical cancer cells.

Among the types of cancer affecting women, cervical cancer (CC) is a public health problem with high global incidence and mortality rates. It is currently classified into three main histological types: squamous cell carcinoma (SCC), adenocarcinoma (AC), and adenosquamous (ASC) carcinoma. All of them lack a targeted therapy. The primary risk factor for CC is Human Papilloma Virus (HPV) infection, which is known to increase reactive oxygen species (ROS), contributing to malignant transformation and tumor progression. At basal levels, ROS can function as second messengers in signaling pathways, and elevated concentrations have been linked to their overactivation. One of these, the ERK pathway, is implicated in both cell proliferation and differentiation and is often dysregulated in cancer, promoting malignant transformation. Several studies have proposed antioxidant supplementation or ERK inhibitors as potential therapies. In vitro studies were performed using CC cell lines. ROS levels were evaluated by flow cytometry; cellular proliferation, death and migration were evaluated using real-time microscopy; cell viability was evaluated with crystal violet staining, and phosphorylated ERK levels were evaluated by Western Blot. A bioinformatic analysis was done in a cervical cancer database. We elucidate part of the complex interplay between ROS and ERK pathway in CC pro-tumorigenic characteristics. Through bioinformatic analysis, we found distinct ROS and ERK activation patterns across CC tumor samples from different histological types. However, in vitro, ROS regulated migration and viability in CC, with no discernible variance based on histological classification. ERK activation, however, differed according to the histological type with SCC displaying increased ERK activation compared to AC and regulating cellular migration in SCC cells. Our study identifies a potential synergistic interaction between ROS and ERK inhibitors, highlighting the therapeutic promise of combinatorial targeting for CC treatment. These findings underscore the importance of personalized approaches aimed at improving the outcomes of CC patients.

Chronic Strongyloides stercoralis infection in Fijian migrants to the UK.

Introduction. Strongyloides stercoralis, the human threadworm, is a parasitic nematode with global distribution, estimated to infect over 600 million people. Chronic infection is often asymptomatic, but hyperinfection and dissemination syndromes can occur in the immunosuppressed with high case fatality rates. Whilst strongyloidiasis is endemic in Fiji, its prevalence in Fijian migrant groups in the UK is unknown.Gap Statement. No previous studies have been conducted on the prevalence of Strongyloides and other gastrointestinal parasites (GIPs) in Fijian migrants to the UK.Aim. We conducted a cross-sectional study of the prevalence of GIPs in a Fijian migrant population.Methodology. Participants completed a questionnaire on residence, travel and clinical symptoms and were asked to provide a serum sample for S. stercoralis IgG ELISA, venous blood samples for eosinophil count and a faecal sample for charcoal culture, multiplex real-time PCR (rtPCR) and microscopy after formalin-ethyl acetate concentration. Sequencing was performed on pooled Strongyloides larvae for nuclear 18S rRNA hyper-variable regions (HVRs) I and IV.Results. A total of 250 participants (94% male) with median (range) age 37 (20-51) years entered the study, 15 (1-24) years since leaving Fiji. S. stercoralis IgG ELISA was positive in 87/248 (35.1 %) and 14/74 (18.9 %) had a GIP detected in faeces. This included 7/74 (9.5 %) with Strongyloides and 5/74 (6.8 %) with hookworms. Dermatological symptoms were more common in those with Strongyloides, and eosinophilia (>0.5×109 cells per litre) was present in 55.6% of those with positive S. stercoralis IgG. rtPCR was the most sensitive faecal diagnostic test for Strongyloides and hookworms in faeces. Sequences of nuclear 18S rRNA for HVRs I and IV confirmed the presence of S. stercoralis.Conclusion. This first cross-sectional study in Fijian migrants found a high rate of chronic infection with GIPs, particularly S. stercoralis. Faecal microscopy was insensitive compared to charcoal culture, rtPCR or serology, demonstrating the importance of specialist parasitological tests when investigating people with a suspected chronic infection. Our study highlights an overlooked burden of strongyloidiasis in the UK and has implications for screening and treatment programmes in Fiji and for migrants from Fiji.

Early and late phases of liver sinusoidal endothelial cell (LSEC) defenestration in mouse model of systemic inflammation

BackgroundLiver sinusoidal endothelial cells (LSECs) have transcellular pores, called fenestrations, participating in the bidirectional transport between the vascular system and liver parenchyma. Fenestrated LSECs indicate a healthy phenotype of liver while loss of fenestrations (defenestration) in LSECs is associated with liver pathologies.MethodsWe introduce a unique model of systemic inflammation triggered by the deletion of Mcpip1 in myeloid leukocytes (Mcpip1fl/flLysMCre) characterised by progressive alterations in LSEC phenotype. We implement multiparametric characterisation of LSECs by using novel real-time atomic force microscopy supported with scanning electron microscopy and quantitative fluorescence microscopy. In addition, we provide genetic profiling, searching for characteristic genes encoding proteins that might be connected with the structure of fenestrations.ResultsWe demonstrate that LSECs in Mcpip1fl/flLysMCre display two phases of defenestration: the early phase, with modest defenestration that was fully reversible using cytochalasin B and the late phase, with severe defenestration that is mostly irreversible. By thorough analysis of LSEC porosity, elastic modulus and actin abundance in Mcpip1fl/flLysMCre and in response to cytochalasin B, we demonstrate that proteins other than actin must be additionally responsible for inducing open fenestrations. We highlight several genes that were severely affected in the late but not in the early phase of LSEC defenestration shedding a light on complex structure of individual fenestrations.ConclusionsThe presented model of LSEC derived from Mcpip1fl/flLysMCre provides a valuable reference for developing novel strategies for LSEC refenestration in the early and late phases of liver pathology.

Mitochondrial Viscosity Probes: Iridium(III) Complexes Induce Apoptosis in HeLa Cells.

Mitochondrial viscosity has emerged as a promising biomarker for diseases such as cancer and neurodegenerative disorders, yet accurately measuring viscosity at the subcellular level remains a significant challenge. In this study, we synthesized and characterized three cyclometalated iridium(III) complexes (Ir1-Ir3) containing 5-fluorouracil derivatives as ligands. Among these, Ir1 selectively induced apoptosis in HeLa cells by increasing mitochondrial production of reactive oxygen species (ROS), which triggered a cascade of events leading to mitochondrial dysfunction. Additionally, the fluorescence lifetime of Ir1 demonstrated high sensitivity to intracellular viscosity changes, enabling real-time fluorescence lifetime imaging microscopy (FLIM) of cellular micro-viscosity during apoptosis. These findings underscore the potential of cyclometalated Ir(III) complexes for both therapeutic and diagnostic applications at the subcellular level.

Real-time four-dimensional scanning transmission electron microscopy through sparse sampling

Abstract Four-dimensional scanning transmission electron microscopy (4-D STEM) is a state-of-the-art image acquisition mode used to reveal high and low mass elements at atomic resolution. The acquisition of the electron momenta at each real space probe location allows for various analyses to be performed from a single dataset, including virtual imaging, electric field analysis, as well as analytical or iterative extraction of the object induced phase shift. However, the limiting factor in 4-D STEM is the speed of acquisition which is bottlenecked by the read-out speed of the camera, which must capture a convergent beam electron diffraction (CBED) pattern at each probe position in the scan. Recent developments in sparse sampling and image inpainting (a branch of compressive sensing) for STEM have allowed for real-time recovery of sparsely acquired data from fixed monolithic detectors, Further developments in compressive sensing for 4-D STEM have also demonstrated that acquisition speeds can be increased, i.e., live video rate 4-D imaging is now possible. In this work, we demonstrate the first practical implementations of compressive 4-D STEM for real-time inference on two different scanning transmission electron microscopes.

Real-Time, High-Throughput Microscopic Quantification of Human Neutrophil Extracellular Trap Release and Assessing the Pharmacology of Antagonists.

Neutrophils play an important role in innate immune defense by using several strategies, including the release of neutrophil extracellular traps (NETs) in a process referred to as NETosis. However, in the past two decades, it has become clear that the accumulation of NETs in tissues contributes to the pathophysiology of multiple inflammatory and autoimmune diseases. Therefore, interest in the development of NETosis antagonists has risen. Variable and non-standardized methods to detect and analyze NETosis were developed concomitantly, each with its own advantages and limitations. Here, we describe a real-time microscopy method for the quantification of human NET release, allowing to study NETosis as well as NET inhibition in a high-throughput manner. The surface area-based semi-automated analysis recognizes NETs and distinguishes them from non-netting activated neutrophils. We demonstrate that the non-physiological NETosis inducers, calcium ionophore and phorbol-12-myristate-13-acetate (PMA), trigger the release of NETs with different characteristics and kinetics. Furthermore, we show that this approach allows studying NET release in response to disease-relevant stimuli, including immune complexes, N-Formylmethionine-leucyl-phenylalanine (fMLF), monosodium urate crystals, and calcium pyrophosphate crystals. To exemplify the utility of this method to study NETosis antagonists, we used CIT-013, a first-in-class monoclonal antibody inhibitor of NET release. CIT-013 targets citrullinated histone H2A and H4 and efficiently inhibits NET release with an IC50 of 4.6 nM. Other anti-histone antibodies tested lacked this NETosis-inhibitory capacity. Altogether, we demonstrate that this protocol enables specific, reliable, and reproducible high-throughput quantification of NETs, enhancing the study of NET release characteristics, kinetics, and pharmacology of NETosis antagonists.

Real-time high-resolution microscopy reveals how single-cell lysis shapes biofilm matrix morphogenesis.

During development, multiscale patterning requires that cells organize their behavior in space and time. Bacteria in biofilms must similarly dynamically pattern their behavior with a simpler toolkit. Like in eukaryotes, morphogenesis of the extracellular matrix is essential for biofilm development, but how it is patterned has remained unclear. Here, we explain how the architecture of eDNA, a key matrix component, is controlled by single cell lysis events during Pseudomonas aeruginosa biofilm development. We extend single-cell imaging methods to capture complete biofilm development, characterizing the stages of biofilm development and visualizing eDNA matrix morphogenesis. Mapping the spatiotemporal distribution of single cell lysis events reveals that cell lysis is restricted to a specific biofilm zone. Simulations indicate that this patterning couples cell lysis to growth, more uniformly distributing eDNA throughout the biofilm. Finally, we find that patterning of cell lysis is organized by nutrient gradients that act as positioning cues.

Direct method for microscale manipulation at liquid-liquid interfaces in ionic liquid media with real-time electron microscopy observation

A deep understanding of the processes in soft matter systems with liquid-liquid phase boundaries is of particular importance for materials science, chemistry and life sciences. A vast variety of physicochemical techniques have been proposed for the study of interfacial properties of liquid systems. Among them, electron microscopy methods occupy an important place due to the possibility of direct observation of the sample areas of interest with high spatial resolution; however, the harsh conditions of the electron microscope chamber impose significant restrictions on the possibilities of observing and manipulating unprotected liquids and related soft systems. To overcome these difficulties, in this work, we developed a methodology for direct probing of liquid-liquid interfaces with simultaneous control of the process using electron microscopy. Practically relevant liquid mixtures based on vacuum-compatible ionic liquid (IL) with water additives were probed with micrometer accuracy in real time inside an electron microscope chamber, which made it possible to reveal the role of specific ions aggregation and electrostatic phenomena in the stabilization of liquid microdomains. To test the versatility of the proposed approach, the morphology of a typical IL/water mixture was examined using a series of electron microscopes of various configurations, and it was shown that the best level of contrast between two chemically related liquid phases can be obtained in the case of a cold field emission electron source in combination with an in-lens secondary electron detector, regardless of the specific instrument manufacturer.

Real-time segmentation and classification of whole-slide images for tumor biomarker scoring

Histopathology image segmentation and classification are essential for diagnosing and treating breast cancer. This study introduced a highly accurate segmentation and classification for histopathology images using a single architecture. We utilized the famous segmentation architectures, SegNet and U-Net, and modified the decoder to attach ResNet, VGG and DenseNet to perform classification tasks. These hybrid models are integrated with Stardist as the backbone, and implemented in a real-time pathologist workflow with a graphical user interface. These models were trained and tested offline using the ER-IHC-stained private and H&E-stained public datasets (MoNuSeg). For real-time evaluation, the proposed model was evaluated using PR-IHC-stained glass slides. It achieved the highest segmentation pixel-based F1-score of 0.902 and 0.903 for private and public datasets respectively, and a classification-based F1-score of 0.833 for private dataset. The experiment shows the robustness of our method where a model trained on ER-IHC dataset able to perform well on real-time microscopy of PR-IHC slides on both 20x and 40x magnification. This will help the pathologists with a quick decision-making process.

Microfluidic Laser-Induced Nucleation of Iron (II,III) Oxide Nanoparticle-Doped Supersaturated Aqueous KCl Solutions.

A capillary-based microfluidic system designed for nonphotochemical laser-induced nucleation (NPLIN) studies coupled with real-time microscopy was used to study NPLIN of iron (II,III) oxide doped aqueous KCl solutions. Supersaturation was achieved by lowering the solution temperature using thermoelectric cooling, and heating was used for the dissolution of crystals downstream to prevent clogging during the flow. The effect of nanoparticle concentration, supersaturation, laser intensity, and filtration was studied. We report laser-induced nucleation using laser intensities as low as 1 MW/cm2 with nanoparticle number densities of ∼109 particles per mL of solution at KCl supersaturations from 1.06 to 1.08. The number of crystals increased with increasing laser intensity, supersaturation, and nanoparticle concentration. We discuss our results with respect to the colloidal impurity-heating mechanism hypothesis and propose a semiempirical model based on the nanoparticle heating and bubble formation due to the absorption of laser energy.

SGLT2 inhibitors protect against diabetic cardiomyopathy and atrial fibrillation through a CaMKII independent mechanism.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been implicated as an important mediator of the increasingly evident cardioprotective benefits exerted by sodium-glucose transport protein 2 channel inhibitors (SGLT2i). However, the exact nature of the relationship between CaMKII and SGLT2i remains unclear. Here, we find that empagliflozin but not dapagliflozin attenuated susceptibility to atrial fibrillation (AF) in a type 2 diabetic (T2D) mouse model. However, both empagliflozin and dapagliflozin protected from diabetic cardiomyopathy in T2D mice. We then used real-time microscopy of neonatal rat ventricular cardiomyocytes (NRVMs) with the CaMKII biosensor - CaMKAR to demonstrate that direct inhibition of CaMKII is not essential for the effects of SGLT2i in these cells. Therefore, we conclude that the benefits of SGLT2i in heart disease likely occur through indirect modulation of CaMKII activity, or possibly through an alternative pathway altogether.

Real-time PCR for malaria diagnosis and identification of Plasmodium species in febrile patients in Cubal, Angola

BackgroundMalaria is the parasitic disease with the highest morbimortality worldwide. The World Health Organization (WHO) estimates that there were approximately 249 million cases in 2022, of which 3.4% were in Angola. Diagnosis is based on parasite identification by microscopy examination, antigen detection, and/or molecular tests, such as polymerase chain reaction (PCR). This study aimed to evaluate the usefulness of real-time PCR as a diagnostic method for malaria in an endemic area (Cubal, Angola).MethodsA cross-sectional study was carried out at the Hospital Nossa Senhora da Paz in Cubal, Angola, including 200 patients who consulted for febrile syndrome between May and July 2022. From each patient, a capillary blood sample was obtained by finger prick for malaria field diagnosis [microscopy and rapid diagnostic test (RDT)] and venous blood sample for real-time PCR performed at the Hospital Universitario Vall d’Hebron in Barcelona, Spain. Any participant with a positive result from at least one of these three methods was diagnosed with malaria.ResultsOf the 200 participants included, 54% were female and the median age was 7 years. Malaria was diagnosed by at least one of the three techniques (microscopy, RDT, and/or real-time PCR) in 58% of the participants, with RDT having the highest percentage of positivity (49%), followed by real-time PCR (39.5%) and microscopy (33.5%). Of the 61 discordant samples, 4 were only positive by microscopy, 13 by real-time PCR, and 26 by RDT. Plasmodium falciparum was the most frequent species detected (90.63%), followed by P. malariae (17.19%) and P. ovale (9.38%). Coinfections were detected in ten participants (15.63%): six (60%) were caused by P. falciparum and P. malariae, three (30%) by P. falciparum and P. ovale, and one (10%) triple infection with these three species. In addition, it was observed that P. falciparum and P. malariae coinfection significantly increased the parasite density of the latter.ConclusionsRDT was the technique with the highest positivity rate, followed by real-time PCR and microscopy. The results of the real-time PCR may have been underestimated due to suboptimal storage conditions during the transportation of the DNA eluates. However, real-time PCR techniques have an important role in the surveillance of circulating Plasmodium species, given the epidemiological importance of the increase in non-falciparum species in the country, and can provide an estimate of the intensity of infection.Graphical abstract

Establishment of a reverse genetics system for virulent systemic feline calicivirus using circular polymerase extension reaction

SummaryFeline caliciviruses can cause oral and upper respiratory tract infections in cats. However, a virulent and systemic feline calicivirus (VS-FCV) variant implicated in multisystem lesions and death in cats has emerged recently. To date, the mechanism underlying virulence variations in VS-FCV remains unclear. The aim of the present study was to provide a tool for exploring genetic variation in VS-FCV, by constructing an infectious clone of VS-FCV SH/2014. First, a full-length cDNA molecular clone of VS-FCV SH/2014 strain, which contains an Xba I recognition site generated by mutating one base (A→T) as a genetic marker, was constructed using the circular polymerase extension reaction (CPER) method. Second, the full-length cDNA clone was introduced into Crandell-Rees feline kidney cells using liposomes to rescue recombinant VS-FCV SH/2014 (rVS-FCV SH/2014). Third, the rescued viruses were identified by real-time PCR, immunofluorescence assay, western blotting, and electron microscopy. The full-length cDNA molecular clone of the VS-FCV SH/2014 strain was successfully constructed and that rVS-FCV SH/2014 could be rescued efficiently. rVS-FCV SH/2014 had the expected genetic markers and morphology and growth characteristics similar to those of the parental virus. The reverse genetics system provides a research platform for future studies on VS-FCV genetic variation and pathogenesis.

Comparative evaluation of real-time polymerase chain reaction, conventional PCR and microscopy for detection of Babesia bigemina in bovines of Punjab (India).

Babesia bigemina is a major cause of bovine babesiosis, an economically devastating tick-borne disease that needs timely diagnosis for precise treatment. In present investigation, the detection efficacy of real-time PCR (qPCR) in comparison to conventional PCR and microscopy targeting 18S ribosomal gene was evaluated on 95 bovines (70 cattle and 25 buffaloes) suspected for babesiosis. Real-time PCR was standardized with the 10-fold serial dilutions in duplication of the given positive control (2 × 106 copy number) ranging from 106 to 100 copy number/µL and mean Ct value of each dilution was taken to extrapolate the curve. The samples with Ct value 36.92 of 10 copy number/µL were considered as positive. Out of 95 samples, 5 (5.26%), 21 (22.10%) and 49 (51.58%) positive by microscopy, conventional PCR and real-time PCR were in corresponding range of > 106-104, 104-103, and 103-<10 copy number/µL, respectively. The concordance of real-time PCR with conventional PCR and microscopy was moderate (Kappa = 0.523) and slight (Kappa = 0.09), respectively. The cows were at four times risk than the buffaloes for B. bigemina infection (Odds ratio:3.85, CI:1.4255-10.4370). This pioneer report from Punjab state (India) of application of real-time PCR to detect B. bigemina in bovines was found to be more sensitive than conventional PCR and microscopy that needs further investigations on a greater number of random samples.