Hyper-prevalence of submicroscopic Plasmodium falciparum infections in a rural area of western Kenya with declining malaria cases

Background The gold standard for diagnosing Plasmodium falciparum infection is microscopic examination of Giemsa-stained peripheral blood smears. The effectiveness of this procedure for infection surveillance and malaria control may be limited by a relatively high parasitaemia detection threshold. Persons with microscopically undetectable infections may go untreated, contributing to ongoing transmission to mosquito vectors. The purpose of this study was to determine the magnitude and determinants of undiagnosed submicroscopic P. falciparum infections in a rural area of western Kenya. Methods A health facility-based survey was conducted, and 367 patients seeking treatment for symptoms consistent with uncomplicated malaria in Homa Bay County were enrolled. The frequency of submicroscopic P. falciparum infection was measured by comparing the prevalence of infection based on light microscopic inspection of thick blood smears versus real-time polymerase chain reaction (RT-PCR) targeting P. falciparum 18S rRNA gene. Long-lasting insecticidal net (LLIN) use, participation in nocturnal outdoor activities, and gender were considered as potential determinants of submicroscopic infections. Results Microscopic inspection of blood smears was positive for asexual P. falciparum parasites in 14.7% (54/367) of cases. All of these samples were confirmed by RT-PCR. 35.8% (112/313) of blood smear negative cases were positive by RT-PCR, i.e., submicroscopic infection, resulting in an overall prevalence by RT-PCR alone of 45.2% compared to 14.7% for blood smear alone. Females had a higher prevalence of submicroscopic infections (35.6% or 72 out of 202 individuals, 95% CI 28.9–42.3) compared to males (24.2%, 40 of 165 individuals, 95% CI 17.6–30.8). The risk of submicroscopic infections in LLIN users was about half that of non-LLIN users (OR = 0.59). There was no difference in the prevalence of submicroscopic infections of study participants who were active in nocturnal outdoor activities versus those who were not active (OR = 0.91). Patients who participated in nocturnal outdoor activities and use LLINs while indoors had a slightly higher risk of submicroscopic infection than those who did not use LLINs (OR = 1.48). Conclusion Microscopic inspection of blood smears from persons with malaria symptoms for asexual stage P. falciparum should be supplemented by more sensitive diagnostic tests in order to reduce ongoing transmission of P. falciparum parasites to local mosquito vectors.

White Blood Cell Image Classification Using Deep Learning

The microscopic inspection of blood smears provides diagnostic information concerning patients’ health status. For example, the presence of infections, leukemia, and some particular kinds of cancers can be diagnosed based on the results of the classification and the count of white blood cells. The traditional method for the differential blood count is performed by experienced operators. They use a microscope and count the percentage of the occurrence of each type of cell counted within an area of interest in smears. Obviously, this manual counting process is very tedious and slow. In addition, the cell classification and counting accuracy may depend on the capabilities and experiences of the operators. Therefore, the necessity of an automated differential counting system becomes inevitable. In this paper, CNN models are used. In order to achieve good performance from deep learning methods, the network needs to be trained with large amounts of data during the training phase. We take the images of the white blood cells for the training phase and train our model on them. With this method we achieved good accuracy than traditional methods. And we can generate the results within the seconds also.

Multi-Scale Input Strategies for Medulloblastoma Tumor Classification using Deep Transfer Learning

Medulloblastoma (MB) is a primary central nervous system tumor and the most common malignant brain cancer among children. Neuropathologists perform microscopic inspection of histopathological tissue slides under a microscope to assess the severity of the tumor. This is a timeconsuming task and often infused with observer variability. Recently, pre-trained convolutional neural networks (CNN) have shown promising results for MB subtype classification. Typically, high-resolution images are divided into smaller tiles for classification, while the size of the tiles has not been systematically evaluated. We study the impact of tile size and input strategy and classify the two major histopathological subtypes-Classic and Desmoplastic/Nodular. To this end, we use recently proposed EfficientNets and evaluate tiles with increasing size combined with various downsampling scales. Our results demonstrate using large input tiles pixels followed by intermediate downsampling and patch cropping significantly improves MB classification performance. Our top-performing method achieves the AUC-ROC value of 90.90% compared to 84.53% using the previous approach with smaller input tiles.

Applications of Digital Microscopy and Densely Connected Convolutional Neural Networks for Automated Quantitation of Babesia-Infected Erythrocytes

BackgroundClinical babesiosis is diagnosed, and parasite burden is determined, by microscopic inspection of a thick or thin Giemsa-stained peripheral blood smear. However, quantitative analysis by manual microscopy is subject to observer bias, slide distribution errors, statistical sampling error, recording errors, and is inherently burdensome from time management and workflow efficiency standpoints. As such, methods for the automated measurement of percent parasitemia in digital microscopic images of peripheral blood smears could improve clinical accuracy, relative to the predicate method.MethodsIndividual erythrocyte images (shape: 70×70×3) were manually labeled as “parasite” or “normal” and were used to train a model for binary image classification. The best model was then used to calculate percent parasitemia from a clinical validation dataset, and values were compared to a clinical reference value. Lastly, model interpretability was examined using an integrated gradient to identify pixels most likely to influence classification decisions.ResultsThe precision and recall of the model during development testing were 0.92 and 1.00, respectively. In clinical validation, the model returned increasing positive signal with increasing mean reference value. However, there were two highly erroneous false positive values returned by the model. Lastly, the model incorrectly assessed three cases well above the clinical threshold of 10%. The integrated gradient suggested potential sources of false positives including rouleaux formations, cell boundaries, and precipitate as deterministic factors in negative erythrocyte images.ConclusionsWhile the model demonstrated highly accurate single cell classification and correctly assessed most slides, several false positives were highly incorrect. This project highlights the need for integrated testing of ML-based models, even when models in the development phase perform well.

A Digital Microscopic Inspection of Dentinal Defects after Using Endodontic Retreatment Files

Aim. The study aimed at evaluating the incidence of dentinal defects after root canal retreatment with ProTaper Universal retreatment (PTUR) and XP-endo Shaper and Finisher R (XP). Materials and Methods. Sixty extracted single-rooted human premolars were selected and divided into 4 groups of 15 teeth each. In the negative control group, the teeth were left unprepared. In the positive control group, the teeth were prepared with ProTaper Next and obturated with no further retreatment. In the PTUR and XP groups, the teeth were prepared and obturated followed by removal of the filling material at body temperature using PTUR and XP instruments, respectively. The roots were then sectioned at 3, 6, and 9 mm from the apex and observed under a digital microscope to detect defects. Results. PTUR group showed significantly higher ( p value <0.05) incidence of defects than the other groups. Comparison of no defects versus defects between groups in different areas of root canals demonstrated significant difference among the groups in the apical and cervical regions. Conclusion. Within the limitations of the present study, PTUR files created significantly more dentinal defects than XP files, with most of those defects at the cervical and apical areas of the root canals.

Environmental Effect on Fatigue Behavior of Epoxy Coating Used for Water Tanks

The fatigue limit and lifetime of epoxy-based coatings may be affected by various factors, especially the environmental effects. This paper evaluates the impact of air, potable water media, and pollution gases (CO2, H2S, and SO2) on the fatigue performance of two types of epoxy-based coatings (polyamine and polyamide epoxy-based coatings) used as lining for potable water storage tanks. The fatigue test apparatus is assembled in the laboratory and utilized for testing. Different factors are discussed, including absorption, adsorption, and the reaction of environmental gasses with polyamine and polyamide coating surfaces. The influence of porosity on the epoxy-based coatings is experimentally determined, and its effects on fatigue limit and fatigue life are discussed in detail. As a result, the coatings were applied to improve the fatigue resistance of stainless steel. The fatigue limits of both types of coatings tested in potable water are lower than the value obtained when tested in air or gas environments. The fatigue limit of polyamine coating is greater than the polyamide coating. The microscopic inspection indicated a different mechanism for initiating fatigue crack, and the test environments are affected by the nature of the fracture surface.

1265. AT-527, an Oral Purine Nucleotide Prodrug Exhibiting Potent In Vitro Antiviral Activity Against Human Coronaviruses, Including SARS-CoV-2

Background Coronaviruses (CoVs) are the causative pathogens of several human diseases, including seasonal respiratory infections (HCoV-229E and HCoV-OC43), Middle East respiratory syndrome (MERS-CoV), severe acute respiratory syndrome (SARS-CoV-1) and the novel CoV recently identified as the virus responsible for the current COVID-19 pandemic, SARS-CoV-2. AT-527 is currently in Phase 2 clinical trials and has demonstrated potent activity and a well-tolerated safety profile in HCV-infected subjects. Here we report the in vitro activity of AT-511, the free base form of AT-527, against SARS-CoV-2 and other CoVs. Methods BHK-21, Huh-7, RD and differentiated normal human bronchial epithelial (dNHBE) cell cultures were exposed to virus and serial dilutions of test compounds. Independent assessments of antiviral activity were obtained by determining effective concentrations of test compounds required to 1) prevent half-maximal (EC50) virus-induced cytopathic effect (CPE) using MTT or neutral red staining and 2) produce virus yield reductions (VYR) by 90% (EC90) using standard endpoint dilution CCID50 assays in Vero 76 cells. Half maximal cytotoxicity of test compounds was determined by dye (MTT or neutral red) staining in the absence of added virus or by microscopic inspection (dNHBE cells only). Results Table 1 presents the in vitro activities of AT-511 against several coronaviruses. Also included in these assays are the antiviral activities of potential COVID-19 oral treatments, including chloroquine, hydroxychloroquine and N4-hydroxycytidine. Table 1. In Vitro Activity of AT-511 Against Various Human Coronaviruses Conclusion The data demonstrate the potent in vitro activity of AT-511 against several CoVs, with individual EC90 values ranging from 0.34 to 1.2 µM against HCoV-229E, HCoV-OC43, SARS-CoV-1 and SARS-CoV-2 and less activity against MERS-CoV (average EC90 = 36 µM). The potent in vitro antiviral activity of AT-511 against SARS-CoV-2 (EC90 = 0.55 µM), associated with the AT-527 safety profile in treated HCV patients, support the ongoing clinical evaluation of the safety and efficacy of AT-527 in COVID-19 patients. Disclosures Steven S. Good, MS, Atea Pharmaceuticals, Inc. (Employee) Adel Moussa, PhD, Atea Pharmaceuticals, Inc. (Employee) Xiao-Jian Zhou, PhD, Atea Pharmaceuticals, Inc. (Employee) Keith Pietropaolo, B.A., Atea Pharmaceuticals, Inc. (Employee) Jean-Pierre Sommadossi, PhD, Atea Pharmaceuticals, Inc. (Board Member)

Label-free imaging and classification of live P. falciparum

Manual microscopic inspection of fixed and stained blood smears has remained the gold standard for Plasmodium parasitemia analysis for over a century. Unfortunately, smear preparation consumes time and reagents, while manual microscopy is skill-dependent and labor-intensive. Here, we demonstrate that label-free microscopy combined with deep learning enables both life stage classification and accurate parasitemia quantification. Using a custom-built microscope, we find that deep-ultraviolet light enhances image contrast and resolution, achieving four-category classification of Plasmodium falciparum blood stages at an overall accuracy greater than 99%. To increase accessibility, we extended our method to a commercial brightfield microscope using near-ultraviolet and visible light. Both systems were tested extrinsically by parasitemia titration, revealing superior performance over manually-scored Giemsa-stained smears, and a limit of detection below 0.1%. Our results suggest that label-free microscopy combined with deep learning could eliminate the need for conventional blood smear analysis.

AgNO3 Sterilizes Grains of Barley (Hordeum vulgare) without Inhibiting Germination—A Necessary Tool for Plant–Microbiome Research

To understand and manipulate the interactions between plants and microorganisms, sterile seeds are a necessity. The seed microbiome (inside and surface microorganisms) is unknown for most plant species and seed-borne microorganisms can persist and transfer to the seedling and rhizosphere, thereby obscuring the effects that purposely introduced microorganisms have on plants. This necessitates that these unidentified, seed-borne microorganisms are removed before seeds are used for studies on plant–microbiome interactions. Unfortunately, there is no single, standardized protocol for seed sterilization, hampering progress in experimental plant growth promotion and our study shows that commonly applied sterilization protocols for barley grains using H2O2, NaClO, and AgNO3 yielded insufficient sterilization. We therefore developed a sterilization protocol with AgNO3 by testing several concentrations of AgNO3 and added two additional steps: Soaking the grains in water before the sterilization and rinsing with salt water (1% (w/w) NaCl) after the sterilization. The most efficient sterilization protocol was to soak the grains, sterilize with 10% (w/w) AgNO3, and to rinse with salt water. By following those three steps, 97% of the grains had no culturable, viable microorganism after 21 days based on microscopic inspection. The protocol left small quantities of AgNO3 residue on the grain, maintained germination percentage similar to unsterilized grains, and plant biomass was unaltered. Hence, our protocol using AgNO3 can be used successfully for experiments on plant–microbiome interactions.

A safety evaluation of mixed human milk oligosaccharides in neonatal farm piglets

Human Milk Oligosaccharides (HMOs) are the third most abundant, solid component of human milk after lactose and fat. As novel processes are developed to cost-effectively produce commercial volumes of these oligosaccharides, they are becoming more common components of infant formulas worldwide. The study evaluated the safety of a novel mixture of HMOs in a neonatal piglet model with the objective of identifying potential effects during the sensitive, preweaning developmental stage of life. The mixture of HMOs (HMO MIX 1) was composed of 2′-fucosyllactose (2′-FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT), 3′-sialyllactose (3′-SL), and 6′-sialyllactose (6′-SL), and was administered to 2-day old piglets at either 5.75 or 8.0 g/L for a period of 21 days. Piglets in the 5.75 and 8.0 g/L HMO MIX 1 dosing groups did not exhibit differences in body weight, food consumption, or feed efficiency. Analysis of clinical chemistry parameters on Study Day 7 and Study Day 21 did not demonstrate any effects that could be attributed to HMO MIX 1, nor were there any findings in organ weight, macroscopic, or microscopic inspection of tissues that could be attributed to this oligosaccharide blend. Therefore, since administration of HMO MIX 1 in a liquid diet up to 8.0 g/L resulted in no toxicologically-relevant effects in comparison with animals fed a control diet, this study supports the safety of this ingredient for addition to infant formula products.