Recent Biotechnological Applications of Polyhydroxyalkanoates (PHA) in the Biomedical Sector-A Review.

Petroleum-derived plastics are materials of great importance for the contemporary lifestyle, and are widely used commercially because they are low cost, resistant, malleable, and weightless, in addition to their hydrophobic character. However, some factors that confer the qualities of these materials also cause problems, mainly environmental, associated with their use. The COVID-19 pandemic aggravated these impacts due to the high demand for personal protective equipment and the packaging sector. In this scenario, bioplastics are environmentally positive alternatives to these plastics due to their applicability in several areas ranging from packaging, to biomedicine, to agriculture. Polyhydroxyalkanoates (PHAs) are biodegradable biopolymers usually produced by microorganisms as an energy reserve. Their structural variability provides a wide range of applications, making them a viable option to replace polluting materials. PHAs can be applied in various biotechnology sectors, such as producing drug carriers and scaffolds for tissue engineering. This review aimed to survey works published in the last five years on the study and biotechnological application of PHAs in the biomedical sector, exploring the versatility and advantages of their use and helping to understand how to enhance their application.

Cyanobacteria: A Promising Source of Antifungal Metabolites.

Cyanobacteria are a rich source of secondary metabolites, and they have received a great deal of attention due to their applicability in different industrial sectors. Some of these substances are known for their notorious ability to inhibit fungal growth. Such metabolites are very chemically and biologically diverse. They can belong to different chemical classes, including peptides, fatty acids, alkaloids, polyketides, and macrolides. Moreover, they can also target different cell components. Filamentous cyanobacteria have been the main source of these compounds. This review aims to identify the key features of these antifungal agents, as well as the sources from which they are obtained, their major targets, and the environmental factors involved when they are being produced. For the preparation of this work, a total of 642 documents dating from 1980 to 2022 were consulted, including patents, original research, review articles, and theses.

In Silico Analysis of a GH3 ß-Glucosidase from Microcystis aeruginosa CACIAM 03.

Cyanobacteria are rich sources of secondary metabolites and have the potential to be excellent industrial enzyme producers. ß-glucosidases are extensively employed in processing biomass degradation as they mediate the most crucial step of bioconversion of cellobiose (CBI), hence controlling the efficiency and global rate of biomass hydrolysis. However, the production and availability of these enzymes derived from cyanobacteria remains limited. In this study, we evaluated the ß-glucosidase from Microcystis aeruginosa CACIAM 03 (MaBgl3) and its potential for bioconversion of cellulosic biomass by analyzing primary/secondary structures, predicting physicochemical properties, homology modeling, molecular docking, and simulations of molecular dynamics (MD). The results showed that MaBgl3 derives from an N-terminal domain folded as a distorted ß-barrel, which contains the conserved His-Asp catalytic dyad often found in glycosylases of the GH3 family. The molecular docking results showed relevant interactions with Asp81, Ala271 and Arg444 residues that contribute to the binding process during MD simulation. Moreover, the MD simulation of the MaBgl3 was stable, shown by analyzing the root mean square deviation (RMSD) values and observing favorable binding free energy in both complexes. In addition, experimental data suggest that MaBgl3 could be a potential enzyme for cellobiose-hydrolyzing degradation.

Análise dos prazos de oferta dos medicamentos incorporados no Componente Especializado da Assistência Farmacêutica, entre 2012 e 2016 / Analysis of supply deadlines for drugs incorporated in the Specialized Component of Pharmaceutical Assistance between 2012 and 2016

Introdução: O acesso a medicamentos no Sistema Único de Saúde (SUS) é preconizado pelas políticas de assistência farmacêutica e de incorporação de tecnologias. Desde 2011, com a implantação da Comissão Nacional de Incorporação de Tecnologias no SUS (Conitec), o prazo máximo para oferta das tecnologias deve ser de até 180 dias, a partir da data de publicação da portaria de incorporação. Objetivo: analisar prazo da oferta dos medicamentos incorporados e alocados no Componente Especializado da Assistência Farmacêutica (CEAF) entre 2012 e 2016, abrangendo cinco anos de início da norma de incorporação. Método: foi realizado estudo analítico retrospectivo, utilizando-se bases de dados da APAC/SIA-SUS relativas às dispensações que ocorreram entre 2012 e 2017. O prazo de oferta foi definido como a data da primeira dispensação. Resultados: 1.198 registros de primeira dispensação foram computados entre 2012 a 2017, sendo que apenas 12% (n = 144/1.198) atenderam ao prazo estabelecido e a média de atraso daqueles que não cumpriram o prazo foi de 14 meses. Conclusão: O prazo médio ficou fora do limite estabelecido, evidenciando a necessidade de aprimoramento dos processos relacionados à oferta dos medicamentos incorporados, bem como reavaliação da regra estabelecida tendo em vista perfil apresentado e trâmites necessários relacionados aos processos de aquisição e dispensação.

Ultra-high-performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight mass spectrometry and molecular networking analysis to investigate the chemodiversity of bioactive extracts of Annona jahnii Saff. fungi from the Brazilian Amazon.

RATIONALE: Annona species are of interest for the isolation of bioactive molecules; however, studies of Annona jahnii Saff. are limited. The exploration of bioactive metabolites of endophytes isolated from this species is unprecedented and allows the preservation of the host plant, in addition to enabling the discovery of compounds with promising biological activities. METHODS: Ethyl acetate extracts from the cultured media of five fungi were obtained. The antioxidant capacity of the extracts was measured using the 1,1-diphenyl-2-picrylhydrazyl free radical method. Antimicrobial activity was determined using the microdilution method in broth in 96-well plates. The exploration of the metabolic profile of the extracts and dereplication of the compounds were performed using ultra-high-performance liquid chromatography/electrospray ionization/tandem mass spectrometry (UHPLC/ESI-MS/MS) combined with analysis using molecular networking (MN). RESULTS: A total of 1818 MS features were detected in the five selected extracts, of which 39 compounds were putatively identified. The secondary metabolites with the highest abundance were alkaloids, naphthopyrons, and cytochalasins. Other secondary metabolites include fumonisins, coumarin, and a meroterpenoid. Most of these compounds are related to specific biological properties such as antioxidant, anti-inflammatory, antimicrobial, antiviral, and antitumor activities. Extracts F398 and F403 showed inhibitory activity of the four pathogens tested. Extracts F475 and F506 did not inhibit the growth of Staphylococcus aureus, and F407 did not inhibit the growth of Escherichia coli in addition to having potent antioxidant activity, with IC50 values of 10 µg/mL or less. CONCLUSIONS: The use of UHPLC/ESI-MS/MS data combined with MN proved useful in the dereplication of bioactive molecules of complex extracts that are still unexplored. These initial investigations should significantly assist in further research and increase the efficiency and speed in the discovery of new sources of secondary metabolites and new natural products.

Identification of potential inhibitors of Schistosoma mansoni purine nucleoside phosphorylase from neolignan compounds using molecular modelling approaches.

Schistosomiasis is a parasitic disease that is part of the neglected tropical diseases (NTDs), which cause significant levels of morbidity and mortality in millions of people throughout the world. The enzyme purine nucleoside phosphorylase from Schistosoma mansoni (SmPNP) represents a potential target for discovering new agents, and neolignans stand out as an important class of compounds. In this work, molecular modeling studies and biological assays of a set of neolignans were conducted against the PNP enzymes of the parasite and the human homologue (HssPNP). The results of the molecular docking described that the neolignans showed good complementarity by the active site of SmPNP. Molecular dynamics (MD) studies revealed that both complexes (Sm/HssPNP - neolignan compounds) were stable by analyzing the root mean square deviation (RMSD) values, and the binding free energy values suggest that the selected structures can interact and inhibit the catalytic activity of the SmPNP. Finally, the biological assay indicated that the selected neolignans presented a better molecular profile of inhibition compared to the human enzyme, as these ligands did not have the capacity to inhibit enzymatic activity, indicating that these compounds are promising candidates and that they can be used in future research in chemotherapy for schistosomiasis.Communicated by Ramaswamy H. Sarma.

Anabaenopeptins: What We Know So Far.

Cyanobacteria are microorganisms with photosynthetic mechanisms capable of colonizing several distinct environments worldwide. They can produce a vast spectrum of bioactive compounds with different properties, resulting in an improved adaptative capacity. Their richness in secondary metabolites is related to their unique and diverse metabolic apparatus, such as Non-Ribosomal Peptide Synthetases (NRPSs). One important class of peptides produced by the non-ribosomal pathway is anabaenopeptins. These cyclic hexapeptides demonstrated inhibitory activity towards phosphatases and proteases, which could be related to their toxicity and adaptiveness against zooplankters and crustaceans. Thus, this review aims to identify key features related to anabaenopeptins, including the diversity of their structure, occurrence, the biosynthetic steps for their production, ecological roles, and biotechnological applications.

Glucosidase Inhibitors Screening in Microalgae and Cyanobacteria Isolated from the Amazon and Proteomic Analysis of Inhibitor Producing Synechococcus sp. GFB01.

Microalgae and cyanobacteria are good sources for prospecting metabolites of biotechnological interest, including glucosidase inhibitors. These inhibitors act on enzymes related to various biochemical processes; they are involved in metabolic diseases, such as diabetes and Gaucher disease, tumors and viral infections, thus, they are interesting hubs for the development of new drugs and therapies. In this work, the screening of 63 environmental samples collected in the Brazilian Amazon found activity against ß-glucosidase, of at least 60 min, in 13.85% of the tested extracts, with Synechococcus sp. GFB01 showing inhibitory activity of 90.2% for α-glucosidase and 96.9% against ß-glucosidase. It was found that the nutritional limitation due to a reduction in the concentration of sodium nitrate, despite not being sufficient to cause changes in cell growth and photosynthetic apparatus, resulted in reduced production of α and ß-glucosidase inhibitors and differential protein expression. The proteomic analysis of cyanobacteria isolated from the Amazon is unprecedented, with this being the first work to evaluate the protein expression of Synechococcus sp. GFB01 subjected to nutritional stress. This evaluation helps to better understand the metabolic responses of this organism, especially related to the production of inhibitors, adding knowledge to the industrial potential of these cyanobacterial compounds.

Culture of Mycobacterium smegmatis in Different Carbon Sources to Induce In Vitro Cholesterol Consumption Leads to Alterations in the Host Cells after Infection: A Macrophage Proteomics Analysis.

During tuberculosis, Mycobacterium uses host macrophage cholesterol as a carbon and energy source. To mimic these conditions, Mycobacterium smegmatis can be cultured in minimal medium (MM) to induce cholesterol consumption in vitro. During cultivation, M. smegmatis consumes MM cholesterol and changes the accumulation of cell wall compounds, such as PIMs, LM, and LAM, which plays an important role in its pathogenicity. These changes lead to cell surface hydrophobicity modifications and H2O2 susceptibility. Furthermore, when M. smegmatis infects J774A.1 macrophages, it induces granuloma-like structure formation. The present study aims to assess macrophage molecular disturbances caused by M. smegmatis after cholesterol consumption, using proteomics analyses. Proteins that showed changes in expression levels were analyzed in silico using OmicsBox and String analysis to investigate the canonical pathways and functional networks involved in infection. Our results demonstrate that, after cholesterol consumption, M. smegmatis can induce deregulation of protein expression in macrophages. Many of these proteins are related to cytoskeleton remodeling, immune response, the ubiquitination pathway, mRNA processing, and immunometabolism. The identification of these proteins sheds light on the biochemical pathways involved in the mechanisms of action of mycobacteria infection, and may suggest novel protein targets for the development of new and improved treatments.

Characterization and Biotechnological Potential of Intracellular Polyhydroxybutyrate by Stigeoclonium sp. B23 Using Cassava Peel as Carbon Source.

The possibility of utilizing lignocellulosic agro-industrial waste products such as cassava peel hydrolysate (CPH) as carbon sources for polyhydroxybutyrate (PHB) biosynthesis and characterization by Amazonian microalga Stigeoclonium sp. B23. was investigated. Cassava peel was hydrolyzed to reducing sugars to obtain increased glucose content with 2.56 ± 0.07 mmol/L. Prior to obtaining PHB, Stigeoclonium sp. B23 was grown in BG-11 for characterization and Z8 media for evaluation of PHB nanoparticles' cytotoxicity in zebrafish embryos. As results, microalga produced the highest amount of dry weight of PHB with 12.16 ± 1.28 (%) in modified Z8 medium, and PHB nanoparticles exerted some toxicity on zebrafish embryos at concentrations of 6.25-100 µg/mL, increased mortality (<35%) and lethality indicators as lack of somite formation (<25%), non-detachment of tail, and lack of heartbeat (both <15%). Characterization of PHB by scanning electron microscopy (SEM), X-ray diffraction (XRD), differential scanning calorimeter (DSC), and thermogravimetry (TGA) analysis revealed the polymer obtained from CPH cultivation to be morphologically, thermally, physically, and biologically acceptable and promising for its use as a biomaterial and confirmed the structure of the polymer as PHB. The findings revealed that microalgal PHB from Stigeoclonium sp. B23 was a promising and biologically feasible new option with high commercial value, potential for biomaterial applications, and also suggested the use of cassava peel as an alternative renewable resource of carbon for PHB biosynthesis and the non-use of agro-industrial waste and dumping concerns.

Influence on Secondary Metabolism of Piper nigrum L. by Co-Inoculation with Arbuscular Mycorrhizal Fungi and Fusarium solani f. sp. piperis.

To elucidate defense mechanisms of Piper nigrum against fusariosis, an experiment based on co-inoculation of arbuscular mycorrhizal fungi (AMF) and Fusarium solani f. sp. piperis was performed. Variations in secondary metabolism in plants infected with F. solani f. sp. piperis (FUS) and co-inoculated with AMFs and F. solani (AMF + FUS) were monitored at 7- and 21-days post inoculations (dpi). The pathogen induced a decrease in oxygenated sesquiterpenes (82.0-77.4%), and changes in the concentrations of the main compounds, α-muurolene, α-muurolol, and 2E-hexenal in the leaves. It was observed that the concentration of 2E-hexenal decreased at 7 dpi, α-muurolene decreased at 21 dpi, and α-muurolol increased at 21 dpi. There was a prevalence of sesquiterpene and monoterpene hydrocarbons in the roots, such as ß-caryophyllene, δ-elemene, and limonene. The infection and co-inoculation induced greater production of phenolics in the roots at 7 dpi. The enzymatic activity of phenylalanine ammonia-lyase decreased in the leaves at 21 dpi and in the roots on both days, while the lipoxygenase activity decreased only in the roots at 21 dpi. The results demonstrated that co-inoculation with AMFs and F. solani induces changes in the defense metabolism of P. nigrum, but it is not efficient in the biocontrol of fusariosis during the evaluated period.

Current Knowledge on Microviridin from Cyanobacteria.

Cyanobacteria are a rich source of secondary metabolites with a vast biotechnological potential. These compounds have intrigued the scientific community due their uniqueness and diversity, which is guaranteed by a rich enzymatic apparatus. The ribosomally synthesized and post-translationally modified peptides (RiPPs) are among the most promising metabolite groups derived from cyanobacteria. They are interested in numerous biological and ecological processes, many of which are entirely unknown. Microviridins are among the most recognized class of ribosomal peptides formed by cyanobacteria. These oligopeptides are potent inhibitors of protease; thus, they can be used for drug development and the control of mosquitoes. They also play a key ecological role in the defense of cyanobacteria against microcrustaceans. The purpose of this review is to systematically identify the key characteristics of microviridins, including its chemical structure and biosynthesis, as well as its biotechnological and ecological significance.

Optimization of Polyhydroxybutyrate Production by Amazonian Microalga Stigeoclonium sp. B23.

The present work established the optimization and production of biodegradable thermoplastic polyhydroxybutyrate (PHB) from Amazonian microalga Stigeoclonium sp. B23. The optimization was performed in eight different growth media conditions of Stigeoclonium sp. B23, supplemented with sodium acetate and sodium bicarbonate and total deprivation of sodium nitrate. B23 was stained with Nile Red, and PHB was extracted and quantified by correlating the amount of fluorescence and biopolymer concentration through spectrofluorimetry and spectrophotometry, respectively. Our results detected the production of PHB in Stigeoclonium sp. B23 and in all modified media. Treatment with increased acetate and bicarbonate and without nitrate gave the highest concentration of PHB, while the treatment with only acetate gave the lowest among supplemented media. Our results showed a great potential of Stigeoclonium sp. B23, the first Amazonian microalga reported on PHB production. The microalga was isolated from a poorly explored and investigated region and proved to be productive when compared to other cyanobacterial and bacterial species. Additionally, microalga biomass changes due to the nutritional conditions and, reversely, biopolymer is well-synthetized. This great potential could lead to the pursuit of new Amazonian microalgae species in the search for alternative polyesters.

Determination of Volatile Organic Compounds and Antibacterial Activity of the Amazonian Cyanobacterium Synechococcus sp. Strain GFB01.

Cyanobacteria exhibit great biotechnological potential due to their capacity to produce compounds with various applicability. Volatile organic compounds (VOCs) possess low molecular weight and high vapor pressure. Many volatiles produced by microorganisms have biotechnological potential, including antimicrobial activity. This study aimed to investigate the VOCs synthesized by cyanobacterium Synechococcus sp. strain GFB01, and the influence of nitrate and phosphate on its antibacterial potential. The strain was isolated from the surface of the freshwater lagoon Lagoa dos Índios, Amapá state, in Northern Brazil. After cultivation, the VOCs were extracted by a simultaneous distillation-extraction process, using a Likens-Nickerson apparatus (2 h), and then identified by GC-MS. The extracts did not display inhibitory activity against the Gram-positive bacteria tested by the disk-diffusion agar method. However, the anti-Salmonella property in both extracts (methanol and aqueous) was detected. The main VOCs identified were heptadecane (81.32%) and octadecyl acetate (11.71%). To the best of our knowledge, this is the first study of VOCs emitted by a cyanobacterium from the Amazon that reports the occurrence of 6-pentadecanol and octadecyl acetate in cyanobacteria.

Cyanobacterial Polyhydroxyalkanoates: A Sustainable Alternative in Circular Economy.

Conventional petrochemical plastics have become a serious environmental problem. Its unbridled use, especially in non-durable goods, has generated an accumulation of waste that is difficult to measure, threatening aquatic and terrestrial ecosystems. The replacement of these plastics with cleaner alternatives, such as polyhydroxyalkanoates (PHA), can only be achieved by cost reductions in the production of microbial bioplastics, in order to compete with the very low costs of fossil fuel plastics. The biggest costs are carbon sources and nutrients, which can be appeased with the use of photosynthetic organisms, such as cyanobacteria, that have a minimum requirement for nutrients, and also using agro-industrial waste, such as the livestock industry, which in turn benefits from the by-products of PHA biotechnological production, for example pigments and nutrients. Circular economy can help solve the current problems in the search for a sustainable production of bioplastic: reducing production costs, reusing waste, mitigating CO2, promoting bioremediation and making better use of cyanobacteria metabolites in different industries.

Arbuscular Mycorrhizal Fungi Colonization Promotes Changes in the Volatile Compounds and Enzymatic Activity of Lipoxygenase and Phenylalanine Ammonia Lyase in Piper nigrum L. 'Bragantina'.

Arbuscular mycorrhizal fungi (AMF) have been used to promote numerous benefits to plants. In this study, we evaluated the symbiosis between AMF species (Rhizophagus clarus, Claroideoglomus etunicatum) and Piper nigrum L. 'Bragantina'. Volatile compounds, lipoxygenase (LOX) and phenylalanine ammonia-lyase (PAL) activities, and total phenolic content were monitored from 1 to 60 days post-inoculation (dpi). Hyphae, arbuscles, and vesicles were observed during the root colonization. In the leaves, AMF induced an increase of sesquiterpene hydrocarbons (54.0%-79.0%) and a decrease of oxygenated sesquiterpenes (41.3%-14.5%) at 7 dpi and 60 dpi (41.8%-21.5%), respectively. Cubenol, the main volatile compound of leaves, showed a significant decrease at 7 dpi (21.5%-0.28%) and 45 dpi (20.4%-18.42%). ß-caryophyllene, the major volatile compound of the roots, displayed a significant reduction at 45 dpi (30.0%-20.0%). LOX increased in the roots at 21, 30, and 60 dpi. PAL was higher in leaves during all periods, except at 60 dpi, and increased at 21 and 45 dpi in the roots. The total phenolic content showed a significant increase only in the roots at 30 dpi. The results suggested that AMF provided changes in the secondary metabolism of P. nigrum, inducing its resistance.

Optimization of Light Intensity and NaNO3 Concentration in Amazon Cyanobacteria Cultivation to Produce Biodiesel.

The objective of this study, for the first time, was to optimize Amazonian cyanobacterial culture conditions for improving cell productivity and lipid content, by analyzing the effect of light intensity and nitrogen concentration, for empirically evaluating biodiesel quality parameters. The strains Synechocystis sp. CACIAM05, Microcystis aeruginosa CACIAM08, Pantanalinema rosaneae CACIAM18, and Limnothrix sp. CACIAM25, were previously identified by morphological and molecular analysis (16S rRNA) and were selected based on their production of chlorophyll a and dry cell weight. Then, factorial planning (22) with central points was applied, with light intensity and NaNO3 concentration as independent variables. As response variables, cell productivity and lipid content were determined. Statistical analysis indicated that for all strains, the independent variables were statistically significant for cell productivity. Analysis of the fatty acid composition demonstrated diversity in the composition of the fatty acid profile from the experimental planning assays of each strain. The Biodiesel Analyzer software predicted the biodiesel quality parameters. CACIAM05 and CACIAM25 obtained better parameters with low levels of light intensity and NaNO3 concentration, whereas CACIAM08 and CACIAM18 obtained better parameters with low NaNO3 concentrations and high luminous intensity.

Effects of inoculation by arbuscular mycorrhizal fungi on the composition of the essential oil, plant growth, and lipoxygenase activity of Piper aduncum L.

The aim of this study was to evaluate the changes in the production of secondary metabolites Piper aduncum seedlings were inoculated by spores of the arbuscular mycorrhizal fungi (AMF) Rhizophagus clarus and Claroideoglomus etunicatum. P. aduncum seedlings were inoculated by spores of R. clarus and C. etunicatum and then, development parameters, root colonization, lipoxygenase (LOX) activity, and essential oil (OE) chemical composition were monitored at 30, 60 and 90 days' post-inoculation (dpi). The inoculation had influenced the plant height and root length at 30 and 90 dpi and microscopic analysis of roots showed the presence of hyphae, arbuscules and vesicles in the inoculated plants. Phenylpropanoids and sesquiterpene hydrocarbons were the main compounds in the EO. In the leaves, the concentration of phenylpropanoids showed a decrease, mainly at 60 dpi, with increased sesquiterpene hydrocarbon production. The main compounds were dillapiole, myristicin, and germacrene D; the dillapiole concentration decreased in all treatments. LOX activity had an increase in the leaves and roots at 90 dpi. These results suggest that alterations in the secondary metabolites of P. aduncum can be induced by its mechanisms of resistance during AMF interaction.

Insights Into Limnothrix sp. Metabolism Based on Comparative Genomics.

Currently only four genome sequences for Limnothrix spp. are publicly available, and information on the genetic properties of cyanobacteria belonging to this genus is limited. In this study, we report the draft genome of Limnothrix sp. CACIAM 69d, isolated from the reservoir of a hydroelectric dam located in the Amazon ecosystem, from where cyanobacterial genomic data are still scarce. Comparative genomic analysis of Limnothrix revealed the presence of key enzymes in the cyanobacterial central carbon metabolism and how it is well equipped for environmental sulfur and nitrogen acquisition. Additionally, this work covered the analysis of Limnothrix CRISPR-Cas systems, pathways related to biosynthesis of secondary metabolites and assembly of extracellular polymeric substances and their exportation. A trans-AT PKS gene cluster was identified in two strains, possibly related to the novel toxin Limnothrixin biosynthesis. Overall, the draft genome of Limnothrix sp. CACIAM 69d adds new data to the small Limnothrix genome library and contributes to a growing representativeness of cyanobacterial genomes from the Amazon region. The comparative genomic analysis of Limnothrix made it possible to highlight unique genes for each strain and understand the overall features of their metabolism.

A focussed single-view hand-held echocardiography protocol for the detection of rheumatic heart disease.

BACKGROUND: Echocardiographic screening represents an opportunity for reduction in the global burden of rheumatic heart disease. A focussed single-view screening protocol could allow for the rapid training of healthcare providers and screening of patients. OBJECTIVE: The aim of this study was to determine the sensitivity and specificity of a focussed single-view hand-held echocardiographic protocol for the diagnosis of rheumatic heart disease in children. METHODS: A total of nine readers were divided into three reading groups; each interpreted 200 hand-held echocardiography studies retrospectively as screen-positive, if mitral regurgitation ⩾1.5 cm and/or any aortic insufficiency were observed, or screen-negative from a pooled study library. The performance of experts receiving focussed hand-held protocols, non-experts receiving focussed hand-held protocols, and experts receiving complete hand-held protocols were determined in comparison with consensus interpretations on fully functional echocardiography machines. RESULTS: In all, 587 studies including 76 on definite rheumatic heart disease, 122 on borderline rheumatic heart disease, and 389 on normal cases were available for analysis. The focussed single-view protocol had a sensitivity of 81.1%, specificity of 75.5%, negative predictive value of 88.5%, and a positive predictive value of 63.2%; expert readers had higher specificity (86.1 versus 64.8%, p<0.01) but equal sensitivity. Sensitivity - experts, 96% and non-experts, 95% - and negative predictive value - experts, 99% and non-experts, 98% - were better for definite rheumatic heart disease. False-positive screening studies resulting from erroneous identification of mitral regurgitation and aortic insufficiency colour jets increased with shortened protocols and less experience (p<0.01). CONCLUSION: Our data support a focussed screening protocol limited to parasternal long-axis images. This holds promise in making echocardiographic screening more practical in regions where rheumatic heart disease remains endemic.