Combination Effect of Diurnal Exposure to Sucrose and Nocturnal Exposure to Lactose on Enamel Demineralization.

We have hypothesized that the association between human milk and caries in breastfeeding children could be explained by the combination of a diurnal cariogenic diet with the nocturnal lactose fermentation, conditions simulated in this experimental study. Cariogenic biofilm was formed on bovine enamel slabs, which were exposed 8x/day for 3 min to a 10% sucrose solution, simulating a highly cariogenic diurnal diet, or 50 mM NaCl solution (control). Simulating the nocturnal retention of milk in mouth, biofilms were transferred to culture medium containing 0.7% lactose for 2 h, or only to culture medium (control). Four groups were designed (n = 12): Ctrl, no exposure to diurnal sucrose or nocturnal lactose; Lac, only nocturnal exposure to lactose (2 h); Suc, only diurnal exposure to sucrose (8x/day); and Suc→Lac, diurnal exposure to sucrose (8x/day) followed by nocturnal exposure to lactose (2 h). The medium was changed 3x/day, at the beginning of the day and after diurnal and nocturnal exposures. Calcium in the medium was determined as a chemical indicator of partial demineralizations occurred during the diurnal and the nocturnal treatments; the medium pH was also determined. After 96 h of growth, biofilms were harvested to evaluate CFU, biomass, and extracellular polysaccharides, soluble and insoluble. The percentage of enamel surface hardness loss (%SHL) was evaluated as cumulative demineralization. Data were analyzed by one-way ANOVA and Tukey's test (α = 5%). Highest %SHL (p < 0.05) was found for the Suc→Lac (40.6%) group when compared to Suc (32.1%), Lac (6.6%), and Ctrl (2.4%) groups. Calcium released during the diurnal and nocturnal treatments was, respectively, Suc→Lac = Suc > Lac = Ctrl and Suc→Lac > Lac > Suc = Ctrl (p < 0.05). Regarding the Ctrl group, calcium released from nocturnal lactose fermentation by the Suc→Lac group was 4-fold greater than that provoked by the Lac group. The findings were supported by the pH of the media. The data suggest that the biofilm formed under diurnal exposure to sucrose enhances the cariogenicity of nocturnal exposure to lactose.

Dystrophic calcinosis cutis in chronic venous disease of the lower limbs.

Lower limb ulcers secondary to chronic venous disease (CVD) are a significant public health problem in Brazil and account for about 70% of these ulcers. Despite recent technological advances and the various therapeutic options for treatment of these chronic injuries, several factors may be involved in resistance to treatment. Dystrophic calcinosis cutis (DCC) is a rare and often underdiagnosed condition that, when in conjunction with CVD, may be associated with a refractory healing process. In this article, we report a case of DCC in a patient with CVD and discuss its etiology, pathophysiology and possible treatment options.

Mitochondria-Rich Fraction Isolated From Mesenchymal Stromal Cells Reduces Lung and Distal Organ Injury in Experimental Sepsis.

OBJECTIVES: To ascertain whether systemic administration of mitochondria-rich fraction isolated from mesenchymal stromal cells would reduce lung, kidney, and liver injury in experimental sepsis. DESIGN: Animal study. SETTING: Laboratory investigation. SUBJECTS: Sixty C57BL/6 male mice. INTERVENTIONS: Sepsis was induced by cecal ligation and puncture; sham-operated animals were used as control. At 24 hours after surgery, cecal ligation and puncture and Sham animals were further randomized to receive saline or mitochondria-rich fraction isolated from mesenchymal stromal cells (3 × 106) IV. At 48 hours, survival, peritoneal bacterial load, lung, kidney, and liver injury were analyzed. Furthermore, the effects of mitochondria on oxygen consumption rate and reactive oxygen species production of lung epithelial and endothelial cells were evaluated in vitro. MEASUREMENTS AND MAIN RESULTS: In vitro exposure of lung epithelial and endothelial cells from cecal ligation and puncture animals to mitochondria-rich fraction isolated from mesenchymal stromal cells restored oxygen consumption rate and reduced total reactive oxygen species production. Infusion of exogenous mitochondria-rich fraction from mesenchymal stromal cells (mitotherapy) reduced peritoneal bacterial load, improved lung mechanics and histology, and decreased the expression of interleukin-1ß, keratinocyte chemoattractant, indoleamine 2,3-dioxygenase-2, and programmed cell death protein 1 in lung tissue, while increasing keratinocyte growth factor expression and survival rate in cecal ligation and puncture-induced sepsis. Mitotherapy also reduced kidney and liver injury, plasma creatinine levels, and messenger RNA expressions of interleukin-18 in kidney, interleukin-6, indoleamine 2,3-dioxygenase-2, and programmed cell death protein 1 in liver, while increasing nuclear factor erythroid 2-related factor-2 and superoxide dismutase-2 in kidney and interleukin-10 in liver. CONCLUSIONS: Mitotherapy decreased lung, liver, and kidney injury and increased survival rate in cecal ligation and puncture-induced sepsis.

Alopecia in anticoagulated patients.

Alopecia is a common complication of anticoagulant therapy that may have important psychological repercussions for patients, especially female patients, and can interfere with the decision to extend anticoagulation. This review aims to describe the mechanisms potentially involved in the genesis of alopecia during anticoagulant therapy, since these are not yet fully understood, and discusses the existing therapies for the most appropriate management.

The PARP inhibitor olaparib exerts beneficial effects in mice subjected to cecal ligature and puncture and in cells subjected to oxidative stress without impairing DNA integrity: A potential opportunity for repurposing a clinically used oncological drug for the experimental therapy of sepsis.

Poly(ADP-ribose) polymerase (PARP) is involved in the pathogenesis of cell dysfunction, inflammation and organ failure during septic shock. The goal of the current study was to investigate the efficacy and safety of the clinically approved PARP inhibitor olaparib in experimental models of oxidative stress in vitro and in sepsis in vivo. In mice subjected to cecal ligation and puncture (CLP) organ injury markers, circulating and splenic immune cell distributions, circulating mediators, DNA integrity and survival was measured. In U937 cells subjected to oxidative stress, cellular bioenergetics, viability and DNA integrity were measured. Olaparib was used to inhibit PARP. The results show that in adult male mice subjected to CLP, olaparib (1-10 mg/kg i.p.) improved multiorgan dysfunction. Olaparib treatment reduced the degree of bacterial CFUs. Olaparib attenuated the increases in the levels of several circulating mediators in the plasma. In the spleen, the number of CD4+ and CD8+ lymphocytes were reduced in response to CLP; this reduction was inhibited by olaparib treatment. Treg but not Th17 lymphocytes increased in response to CLP; these cell populations were reduced in sepsis when the animals received olaparib. The Th17/Treg ratio was lower in CLP-olaparib group than in the CLP control group. Analysis of miRNA expression identified a multitude of changes in spleen and circulating white blood cell miRNA levels after CLP; olaparib treatment selectively modulated these responses. Olaparib extended the survival rate of mice subjected to CLP. In contrast to males, in female mice olaparib did not have significant protective effects in CLP. In aged mice olaparib exerted beneficial effects that were less pronounced than the effects obtained in young adult males. In in vitro experiments in U937 cells subjected to oxidative stress, olaparib (1-100 µM) inhibited PARP activity, protected against the loss of cell viability, preserved NAD+ levels and improved cellular bioenergetics. In none of the in vivo or in vitro experiments did we observe any adverse effects of olaparib on nuclear or mitochondrial DNA integrity. In conclusion, olaparib improves organ function and extends survival in septic shock. Repurposing and eventual clinical introduction of this clinically approved PARP inhibitor may be warranted for the experimental therapy of septic shock.

Secondary lymphedema of limbs and chikungunya fever.

Certain systemic viral infections can be related to development of vascular complications, such as deep venous thrombosis and lymphedema of lower and upper limbs. These links have been well-established in patients with human immunodeficiency virus (HIV), hepatitis C, or influenza. Recently introduced into the American continent (2013), chikungunya virus is an arbovirus transmitted by mosquitoes of the Aedes genus and is the etiologic agent of chikungunya fever (CF), but its relationship to these vascular complications has not yet been consolidated. However, the CF outbreak that occurred during 2015 and 2016 resulted in the first cases described in the medical literature of acute and chronic vascular complications secondary to infection by this arbovirus. In this report, we describe the case of a patient who developed lymphedema of upper and lower limbs after an episode of CF.

Cold-adapted enzymes produced by fungi from terrestrial and marine Antarctic environments.

Antarctica is the coldest, windiest, and driest continent on Earth. In this sense, microorganisms that inhabit Antarctica environments have to be adapted to harsh conditions. Fungal strains affiliated with Ascomycota and Basidiomycota phyla have been recovered from terrestrial and marine Antarctic samples. They have been used for the bioprospecting of molecules, such as enzymes. Many reports have shown that these microorganisms produce cold-adapted enzymes at low or mild temperatures, including hydrolases (e.g. α-amylase, cellulase, chitinase, glucosidase, invertase, lipase, pectinase, phytase, protease, subtilase, tannase, and xylanase) and oxidoreductases (laccase and superoxide dismutase). Most of these enzymes are extracellular and their production in the laboratory has been carried out mainly under submerged culture conditions. Several studies showed that the cold-adapted enzymes exhibit a wide range in optimal pH (1.0-9.0) and temperature (10.0-70.0 °C). A myriad of methods have been applied for cold-adapted enzyme purification, resulting in purification factors and yields ranging from 1.70 to 1568.00-fold and 0.60 to 86.20%, respectively. Additionally, some fungal cold-adapted enzymes have been cloned and expressed in host organisms. Considering the enzyme-producing ability of microorganisms and the properties of cold-adapted enzymes, fungi recovered from Antarctic environments could be a prolific genetic resource for biotechnological processes (industrial and environmental) carried out at low or mild temperatures.

Defining the clinical relevance of red blood cell autoantibodies by Monocyte Monolayer Assay.

BACKGROUND: The Monocyte Monolayer Assay (MMA) is an in vitro simulation of red blood cell (RBC) alloantibody behavior. It has been classically applied to predict the risks of post-transfusion hemolytic reactions when transfusing incompatible RBC units. Quantifying erythrophagocytosis by MMA may be an interesting option for situations where there is doubt whether a RBC autoantibody is mediating significant hemolysis. Here, we present three situations involving RBC autoantibodies in which the MMA was decisive for clarifying the diagnosis and choosing the best clinical treatment. CASE REPORT: Case 1. Pregnant patient with severely anemic fetus exhibited warm autoantibody without signs of hemolysis. MMA revealed 30% of monocyte index (MI) highlighting that fetal hemolysis was caused by maternal autoantibody. Prednisone was prescribed with fetal clinical improvement. Cases 2 and 3. Two patients with the diagnosis of mixed auto-immune hemolytic anemia and poor response to corticosteroids were evaluated using MMA. The resulting MI was less than 10% in both cases, suggesting that the cold-agglutinin rather than the warm auto-IgG was responsible for overt hemolysis. Treatment with rituximab was begun, with good clinical response. CONCLUSION: MMA can be used to evaluate the ability of RBC autoantibodies to mediate overt hemolysis. It can be especially useful to determine the role played by cold and warm auto-antibodies in mixed auto-immune hemolytic disease, helping to define the best treatment option.

Venous thromboembolism prophylaxis on flights.

Civil aviation has seen a steady increase in the number of scheduled flights over the last ten years and, as a result, more passengers are traveling by air. This has been associated with an increase in flight-related diseases, especially on long-haul flights. One of the most feared complications during flights is venous thromboembolism (VTE), but its true incidence is difficult to measure because of a lack of consensus on elements such as the definition of how long after landing a VTE can be considered to be related to a flight and even how long a flight must last to be considered of long duration. There has been much discussion of the pathophysiological mechanisms of flight-related VTE, of which passengers are at greatest risk, and of what prophylactic measures can be adopted safely and effectively. The purpose of this review is to clarify these points and describe current consensual conduct.

RHD and RHCE genotyping by next-generation sequencing is an effective strategy to identify molecular variants within sickle cell disease patients.

BACKGROUND: The complexity of Rh genetic variation among sickle cell disease (SCD) patients is high. Conventional molecular assays cannot identify all genetic variants already described for the RH locus as well as foresee novel alleles. Sequencing RHD and RHCE is indicated to broaden the search for Rh genetic variants. AIMS: To standardize the Next Generation Sequencing (NGS) strategy to assertively identify Rh genetic variants among SCD patients with serologic suspicion of Rh variants and evaluate if it can improve the transfusion support. METHODS: Thirty-five SCD patients with unexplained Rh antibodies were enrolled. A NGS-based strategy was developed to genotype RHD and RHCE using gene-specific primers. Genotype and serological data were compared. RESULTS: Data obtained from the NGS-based assay were gene-specific. Ten and 25 variant RHD and RHCE alleles were identified, respectively. Among all cases of unexplained Rh antibodies, 62% had been inaccurately classified by serological analysis and, of these, 73.1% were considered as relevant, as were associated with increased risk of hemolytic reactions and shortage of units suitable for transfusion. CONCLUSION: The NGS assay designed to genotype RH coding regions was effective and accurate in identifying variants. The proposed strategy clarified the Rh phenotype of most patients, improving transfusion support.

Use of Low-Dose Alendronate Improves Cranial Bone Repair and Is Associated With an Increase of Osteocalcin: An Experimental Study.

PURPOSE: The objective of the present study was to evaluate cranial bone repair and remodeling after systemic application of alendronate (ALN), using histologic analysis, histometric analysis, and immunohistochemistry (osteocalcin). MATERIALS AND METHODS: Female rabbits (n = 28) were randomly divided into 2 groups: control (C) and ALN treated (A). Group A received 3 systemic intraperitoneal injections of ALN weekly for 4 weeks, at a dose of 0.2 mg/kg. Group C received intraperitoneal injections of physiologic saline for the same period. After 4 weeks, all the rabbits underwent surgery to create 2 noncritical defects on the calvaria (5 mm in diameter). The groups were divided into 2 subgroups for sacrificing, at 15 and 60 postoperative days. After death, the analyses were performed. The data were also analyzed statistically. RESULTS: Histologic analysis of group C revealed healing in the dense connective tissue and trabecular bone, and the presence of compact bone with osteoblastic activity was noted in both 15- and 60-day subgroups. In group A, at 15 days, the presence of conjunctive tissue with osteoblastic activity and intense, compact, newly formed bone was observed. At 60 days, the created bone defect in group A showed a large amount of neoformed compact bone with a surface of dense modeled connective tissue and the presence of adipocytes and trabecular bone. The histometric analysis confirmed that a statistically significant difference was present between groups C and A when comparing the measured bone area and the area of connective tissue. Group A presented with a statistically significant larger amount of bone area in the 60-day subgroup than in the 15-day subgroup. The immunohistochemical analysis showed stronger immunostaining for osteocalcin in group A. CONCLUSIONS: Within the limits of the present study, our results have shown that the systemic application of ALN, at a dose of 0.2 mg/kg, increased the repair and remodeling of cranial bone.

Would Be Prophylactic Administrations of Low Concentration of Alendronate an Alternative for Improving the Craniofacial Bone Repair? A Preliminary Study Focused in the Period of Cellular Differentiation and Tissue Organization.

BACKGROUND: Alendronate (ALN) is a nitrogen-bisphosphonate that may induce an anabolic effect on craniofacial bone repair when administrated in low doses. Based on this premise, this study analyzed the influence of prophylactic low doses of ALN on bone healing in defects created in rabbit mandible. METHODS: A 5 × 2-mm diameter deep defect was created in the calvaria of 28 rabbits. Fourteen of these rabbits received previously 50 µg/kg of 1% sodium ALN for 4 weeks, while the other rabbits received only 0.9% physiological saline solution (control). Animals were euthanized at 15 and 60 days postsurgery (n = 7), and the data were analyzed using histomorphometry and immunohistochemistry using the anti-CD34, bone morphogenetic protein -2 (BMP-2), and transforming growth factor (TGF)-ß1 antibodies. RESULTS: On the 15th day postsurgery, the specimens that received previous treatment with ALN demonstrated large vascular lumen and intense positivity to CD34 either concentrated in endothelium or cells spread among the reparative tissue. These results coincided with intense positivity for BMP-2+ cells and TGF-ß1 that was concentrated in both cells and perivascular area. In contrast, the control group revealed scarce cells that exhibited CD34, BMP-2+, and the TGF-ß1 was restricted for perivascular area on well-formed granulation tissue. These patterns of immunohistochemical result, especially found on the 15th day of analysis, seem to be responsible for the development of larger quantities of bone matrix in the specimens that receive ALN on the 60th day postsurgery. CONCLUSION: These preliminary results showed that the prophylactic administration of low doses of ALN might be an alternative to craniofacial bone craniofacial bone repair because it increases the immunopositivity for TGF-ß1 and consequently improves the CD34+ and BMP-2+ cells on reparative sites.

Antibody-enhanced dengue disease generates a marked CNS inflammatory response in the black-tufted marmoset Callithrix penicillata.

Severe dengue disease is often associated with long-term neurological impairments, but it is unclear what mechanisms are associated with neurological sequelae. Previously, we demonstrated antibody-enhanced dengue disease (ADE) dengue in an immunocompetent mouse model with a dengue virus 2 (DENV2) antibody injection followed by DENV3 virus infection. Here we migrated this ADE model to Callithrix penicillata. To mimic human multiple infections of endemic zones where abundant vectors and multiple serotypes co-exist, three animals received weekly subcutaneous injections of DENV3 (genotype III)-infected supernatant of C6/36 cell cultures, followed 24 h later by anti-DENV2 antibody for 12 weeks. There were six control animals, two of which received weekly anti-DENV2 antibodies, and four further animals received no injections. After multiple infections, brain, liver, and spleen samples were collected and tissue was immunolabeled for DENV3 antigens, ionized calcium binding adapter molecule 1, Ki-67, TNFα. There were marked morphological changes in the microglial population of ADE monkeys characterized by more highly ramified microglial processes, higher numbers of trees and larger surface areas. These changes were associated with intense TNFα-positive immunolabeling. It is unclear why ADE should generate such microglial activation given that IgG does not cross the blood-brain barrier, but this study reveals that in ADE dengue therapy targeting the CNS host response is likely to be important.

Pulpal calcifications in orthodontically moved teeth: Scoping review.

Background: The application of forces during orthodontic treatment can induce pulpal calcifications, characterized by the deposition of mineralized tissue in the pulp cavity space, there may be repercussions on dental procedures, especially endodontic treatment. The objective of this article is to map the scientific evidence and any gaps in knowledge regarding the relationship between orthodontics and dental pulp calcifications. Material and Methods: The study comprised a scoping review whose guiding question was: "What is the scientific evidence of the association between pulpal calcifications and orthodontic treatment?" Two independent reviewers searched the PubMed, Scopus, and Web of Science databases and the grey literature. Original articles and observational and clinical trials, which addressed the repercussions on the pulp cavity of teeth submitted to orthodontic treatment, were included. There was no language restriction or limitation of the year of publication until October 2022. Results: After critical reading and applying the eligibility criteria, 11 studies were included in this scoping review: six observational and five experimental studies. A statistically significant association of orthodontic treatment with changes in pulp cavity volume and increased incidence of pulp stones was observed among the studies findings. Conclusions: The orthodontic force can promote changes in the dental pulp that may cause direct implications in other dental treatments, especially endodontic treatment. Key words:Dental pulp calcifications, pulp node, pulp obliteration, orthodontic treatment, orthodontics.

Disentangling a Neotropical pest species complex: genetic diversity and population structure of the native rice stink bug Oebalus poecilus and the invasive O. ypsilongriseus.

BACKGROUND: A first step in any pest management initiative is recognizing the existing problem - identifying the pest species and its abundance and dispersal capacities. This is not simple and even more challenging when insidious (invasive) species are involved constituting a pest complex. Understanding a species' population diversity and structure can provide a better understanding of its adaptation and relative pest potential. Such is the need for the native rice stink bug Oebalus poecilus and the invasive O. ypsilongriseus in low and high flatlands of South America. RESULTS: The genetic structure differed between both rice stink bug species (FST  = 0.157, P = 0.001), where 84% of the overall genetic variability takes place within species and three genetic groups were recognized through Bayesian approach (K = 3). Oebalus poecilus exhibited slightly higher genetic diversity (HE  = 0.253) and structuring (FST  = 0.050, P = 0.001) than the invasive O. ypsilongriseus (HE  = 0.211; FST  = 0.038, P = 0.013). Nonetheless, only the former exhibited significant correlation between genetic and geographic distances (r = 0.48, P = 0.013). CONCLUSION: Despite the pointed peculiarities, the obtained results indicate overlap in both species' occurrence and similar genetic structure allowing for a compound problem to be dealt with as the complex requires managing without, as yet, a prevailing species or a niche specialization. © 2022 Society of Chemical Industry.

Development of nintedanib nanosuspension for inhaled treatment of experimental silicosis.

Silicosis is an irreversible and progressive fibrotic lung disease caused by massive inhalation of crystalline silica dust at workplaces, affecting millions of industrial workers worldwide. A tyrosine kinase inhibitor, nintedanib (NTB), has emerged as a potential silicosis treatment due to its inhibitory effects on key signaling pathways that promote silica-induced pulmonary fibrosis. However, chronic and frequent use of the oral NTB formulation clinically approved for treating other fibrotic lung diseases often results in significant side effects. To this end, we engineered a nanocrystal-based suspension formulation of NTB (NTB-NS) possessing specific physicochemical properties to enhance drug retention in the lung for localized treatment of silicosis via inhalation. Our NTB-NS formulation was prepared using a wet-milling procedure in presence of Pluronic F127 to endow the formulation with nonadhesive surface coatings to minimize interactions with therapy-inactivating delivery barriers in the lung. We found that NTB-NS, following intratracheal administration, provided robust anti-fibrotic effects and mechanical lung function recovery in a mouse model of silicosis, whereas a 100-fold greater oral NTB dose given with a triple dosing frequency failed to do so. Importantly, several key pathological phenotypes were fully normalized by NTB-NS without displaying notable local or systemic adverse effects. Overall, NTB-NS may open a new avenue for localized treatment of silicosis and potentially other fibrotic lung diseases.

The Complex Role of the Microbiome in Non-Small Cell Lung Cancer Development and Progression.

In recent years, there has been a growing interest in the relationship between microorganisms in the surrounding environment and cancer cells. While the tumor microenvironment predominantly comprises cancer cells, stromal cells, and immune cells, emerging research highlights the significant contributions of microbial cells to tumor development and progression. Although the impact of the gut microbiome on treatment response in lung cancer is well established, recent investigations indicate complex roles of lung microbiota in lung cancer. This article focuses on recent findings on the human lung microbiome and its impacts in cancer development and progression. We delve into the characteristics of the lung microbiome and its influence on lung cancer development. Additionally, we explore the characteristics of the intratumoral microbiome, the metabolic interactions between lung tumor cells, and how microorganism-produced metabolites can contribute to cancer progression. Furthermore, we provide a comprehensive review of the current literature on the lung microbiome and its implications for the metastatic potential of tumor cells. Additionally, this review discusses the potential for therapeutic modulation of the microbiome to establish lung cancer prevention strategies and optimize lung cancer treatment.

Development and international validation of custom-engineered and code-free deep-learning models for detection of plus disease in retinopathy of prematurity: a retrospective study.

BACKGROUND: Retinopathy of prematurity (ROP), a leading cause of childhood blindness, is diagnosed through interval screening by paediatric ophthalmologists. However, improved survival of premature neonates coupled with a scarcity of available experts has raised concerns about the sustainability of this approach. We aimed to develop bespoke and code-free deep learning-based classifiers for plus disease, a hallmark of ROP, in an ethnically diverse population in London, UK, and externally validate them in ethnically, geographically, and socioeconomically diverse populations in four countries and three continents. Code-free deep learning is not reliant on the availability of expertly trained data scientists, thus being of particular potential benefit for low resource health-care settings. METHODS: This retrospective cohort study used retinal images from 1370 neonates admitted to a neonatal unit at Homerton University Hospital NHS Foundation Trust, London, UK, between 2008 and 2018. Images were acquired using a Retcam Version 2 device (Natus Medical, Pleasanton, CA, USA) on all babies who were either born at less than 32 weeks gestational age or had a birthweight of less than 1501 g. Each images was graded by two junior ophthalmologists with disagreements adjudicated by a senior paediatric ophthalmologist. Bespoke and code-free deep learning models (CFDL) were developed for the discrimination of healthy, pre-plus disease, and plus disease. Performance was assessed internally on 200 images with the majority vote of three senior paediatric ophthalmologists as the reference standard. External validation was on 338 retinal images from four separate datasets from the USA, Brazil, and Egypt with images derived from Retcam and the 3nethra neo device (Forus Health, Bengaluru, India). FINDINGS: Of the 7414 retinal images in the original dataset, 6141 images were used in the final development dataset. For the discrimination of healthy versus pre-plus or plus disease, the bespoke model had an area under the curve (AUC) of 0·986 (95% CI 0·973-0·996) and the CFDL model had an AUC of 0·989 (0·979-0·997) on the internal test set. Both models generalised well to external validation test sets acquired using the Retcam for discriminating healthy from pre-plus or plus disease (bespoke range was 0·975-1·000 and CFDL range was 0·969-0·995). The CFDL model was inferior to the bespoke model on discriminating pre-plus disease from healthy or plus disease in the USA dataset (CFDL 0·808 [95% CI 0·671-0·909, bespoke 0·942 [0·892-0·982]], p=0·0070). Performance also reduced when tested on the 3nethra neo imaging device (CFDL 0·865 [0·742-0·965] and bespoke 0·891 [0·783-0·977]). INTERPRETATION: Both bespoke and CFDL models conferred similar performance to senior paediatric ophthalmologists for discriminating healthy retinal images from ones with features of pre-plus or plus disease; however, CFDL models might generalise less well when considering minority classes. Care should be taken when testing on data acquired using alternative imaging devices from that used for the development dataset. Our study justifies further validation of plus disease classifiers in ROP screening and supports a potential role for code-free approaches to help prevent blindness in vulnerable neonates. FUNDING: National Institute for Health Research Biomedical Research Centre based at Moorfields Eye Hospital NHS Foundation Trust and the University College London Institute of Ophthalmology. TRANSLATIONS: For the Portuguese and Arabic translations of the abstract see Supplementary Materials section.