Low-Cost Methods for Molecular Characterization of Mutant Plants: Tissue Desiccation, DNA Extraction and Mutation Discovery: Protocols

Plant Breeding/Biotechnology; Biological Techniques; Nucleic Acid Chemistr

Low-Cost Methods for Molecular Characterization of Mutant Plants: Tissue Desiccation, DNA Extraction and Mutation Discovery: Protocols

Plant Breeding/Biotechnology; Biological Techniques; Nucleic Acid Chemistr

Emergence of Undetectable Malaria Parasites: A Threat under the Radar amid the COVID-19 Pandemic?

Rapid diagnostic tests (RDTs) play a critical role in malaria diagnosis and control. The emergence of Plasmodium falciparum parasites that can evade detection by RDTs threatens control and elimination efforts. These parasites lack or have altered genes encoding histidine-rich proteins (HRPs) 2 and 3, the antigens recognized by HRP2-based RDTs. Surveillance of such parasites is dependent on identifying false-negative RDT results among suspected malaria cases, a task made more challenging during the current pandemic because of the overlap of symptoms between malaria and COVID-19, particularly in areas of low malaria transmission. Here, we share our perspective on the emergence of P. falciparum parasites lacking HRP2 and HRP3, and the surveillance needed to identify them amid the COVID-19 pandemic

Snapshot PCR surveillance for SARS-CoV-2 in hospital staff in England

Background: Significant nosocomial transmission of SARS-CoV-2 has been demonstrated. Understanding the prevalence of SARS-CoV-2 carriage amongst HCWs at work is necessary to inform the development of HCW screening programmes to control nosocomial spread. / Methods: Cross-sectional ‘snapshot’ survey from April-May 2020; HCWs recruited from six UK hospitals. Participants self-completed a health questionnaire and underwent a combined viral nose and throat swab, tested by Polymerase Chain Reaction (PCR) for SARS-CoV-2 with viral culture on majority of positive samples. / Findings: Point prevalence of SARS-CoV-2 carriage across the sites was 2.0% (23/1152 participants), median cycle threshold value 35.70 (IQR:32.42–37.57). 17 were previously symptomatic, two currently symptomatic (isolated anosmia and sore throat); the remainder declared no prior or current symptoms. Symptoms in the past month were associated with threefold increased odds of testing positive (aOR 3.46, 95%CI 1.38–8.67; p = 0.008). SARS-CoV-2 virus was isolated from only one (5%) of nineteen cultured samples. A large proportion (39%) of participants reported symptoms in the past month. / Interpretation: The point-prevalence is similar to previous estimates for HCWs in April 2020, though a magnitude higher than in the general population. Based upon interpretation of symptom history and testing results including viral culture, the majority of those testing positive were unlikely to be infectious at time of sampling. Development of screening programmes must balance the potential to identify additional cases based upon likely prevalence, expanding the symptoms list to encourage HCW testing, with resource implications and risks of excluding those unlikely to be infectious with positive tests. / Funding: Public Health England

Drug-resistant genotypes and multi-clonality in Plasmodium falciparum analysed by direct genome sequencing from peripheral blood of malaria patients.

Naturally acquired blood-stage infections of the malaria parasite Plasmodium falciparum typically harbour multiple haploid clones. The apparent number of clones observed in any single infection depends on the diversity of the polymorphic markers used for the analysis, and the relative abundance of rare clones, which frequently fail to be detected among PCR products derived from numerically dominant clones. However, minority clones are of clinical interest as they may harbour genes conferring drug resistance, leading to enhanced survival after treatment and the possibility of subsequent therapeutic failure. We deployed new generation sequencing to derive genome data for five non-propagated parasite isolates taken directly from 4 different patients treated for clinical malaria in a UK hospital. Analysis of depth of coverage and length of sequence intervals between paired reads identified both previously described and novel gene deletions and amplifications. Full-length sequence data was extracted for 6 loci considered to be under selection by antimalarial drugs, and both known and previously unknown amino acid substitutions were identified. Full mitochondrial genomes were extracted from the sequencing data for each isolate, and these are compared against a panel of polymorphic sites derived from published or unpublished but publicly available data. Finally, genome-wide analysis of clone multiplicity was performed, and the number of infecting parasite clones estimated for each isolate. Each patient harboured at least 3 clones of P. falciparum by this analysis, consistent with results obtained with conventional PCR analysis of polymorphic merozoite antigen loci. We conclude that genome sequencing of peripheral blood P. falciparum taken directly from malaria patients provides high quality data useful for drug resistance studies, genomic structural analyses and population genetics, and also robustly represents clonal multiplicity

Persistence of Plasmodium falciparum parasitemia after artemisinin combination therapy: evidence from a randomized trial in Uganda

Artemisinin resistance is rapidly spreading in Southeast Asia. The efficacy of artemisinin-combination therapy (ACT) continues to be excellent across Africa. We performed parasite transcriptional profiling and genotyping on samples from an antimalarial treatment trial in Uganda. We used qRT-PCR and genotyping to characterize residual circulating parasite populations after treatment with either ACT or ACT-primaquine. Transcripts suggestive of circulating ring stage parasites were present after treatment at a prevalence of >25% until at least 14 days post initiation of treatment. Greater than 98% of all ring stage parasites were cleared within the first 3 days, but subsequently persisted at low concentrations until day 14 after treatment. Genotyping demonstrated a significant decrease in multiplicity of infection within the first 2 days in both ACT and ACT-primaquine arms. However, multiple clone infections persisted until day 14 post treatment. Our data suggest the presence of genetically diverse persisting parasite populations after ACT treatment. Although we did not demonstrate clinical treatment failures after ACT and the viability and transmissibility of persisting ring stage parasites remain to be shown, these findings are of relevance for the interpretation of parasite clearance transmission dynamics and for monitoring drug effects in Plasmodium falciparum parasites

Predicting the environmental suitability for onchocerciasis in Africa as an aid to elimination planning

Recent evidence suggests that, in some foci, elimination of onchocerciasis from Africa may be feasible with mass drug administration (MDA) of ivermectin. To achieve continental elimination of transmission, mapping surveys will need to be conducted across all implementation units (IUs) for which endemicity status is currently unknown. Using boosted regression tree models with optimised hyperparameter selection, we estimated environmental suitability for onchocerciasis at the 5 × 5-km resolution across Africa. In order to classify IUs that include locations that are environmentally suitable, we used receiver operating characteristic (ROC) analysis to identify an optimal threshold for suitability concordant with locations where onchocerciasis has been previously detected. This threshold value was then used to classify IUs (more suitable or less suitable) based on the location within the IU with the largest mean prediction. Mean estimates of environmental suitability suggest large areas across West and Central Africa, as well as focal areas of East Africa, are suitable for onchocerciasis transmission, consistent with the presence of current control and elimination of transmission efforts. The ROC analysis identified a mean environmental suitability index of 0.71 as a threshold to classify based on the location with the largest mean prediction within the IU. Of the IUs considered for mapping surveys, 50.2% exceed this threshold for suitability in at least one 5×5-km location. The formidable scale of data collection required to map onchocerciasis endemicity across the African continent presents an opportunity to use spatial data to identify areas likely to be suitable for onchocerciasis transmission. National onchocerciasis elimination programmes may wish to consider prioritising these IUs for mapping surveys as human resources, laboratory capacity, and programmatic schedules may constrain survey implementation, and possibly delaying MDA initiation in areas that would ultimately qualify

Global, regional, and national disability-adjusted life-years (DALYs) for 359 diseases and injuries and healthy life expectancy (HALE) for 195 countries and territories, 1990–2017: A systematic analysis for the Global Burden of Disease Study 2017

Background How long one lives, how many years of life are spent in good and poor health, and how the population's state of health and leading causes of disability change over time all have implications for policy, planning, and provision of services. We comparatively assessed the patterns and trends of healthy life expectancy (HALE), which quantifies the number of years of life expected to be lived in good health, and the complementary measure of disability-adjusted life-years (DALYs), a composite measure of disease burden capturing both premature mortality and prevalence and severity of ill health, for 359 diseases and injuries for 195 countries and territories over the past 28 years. Methods We used data for age-specific mortality rates, years of life lost (YLLs) due to premature mortality, and years lived with disability (YLDs) from the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2017 to calculate HALE and DALYs from 1990 to 2017. We calculated HALE using age-specific mortality rates and YLDs per capita for each location, age, sex, and year. We calculated DALYs for 359 causes as the sum of YLLs and YLDs. We assessed how observed HALE and DALYs differed by country and sex from expected trends based on Socio-demographic Index (SDI). We also analysed HALE by decomposing years of life gained into years spent in good health and in poor health, between 1990 and 2017, and extra years lived by females compared with males. Findings Globally, from 1990 to 2017, life expectancy at birth increased by 7·4 years (95% uncertainty interval 7·1–7·8), from 65·6 years (65·3–65·8) in 1990 to 73·0 years (72·7–73·3) in 2017. The increase in years of life varied from 5·1 years (5·0–5·3) in high SDI countries to 12·0 years (11·3–12·8) in low SDI countries. Of the additional years of life expected at birth, 26·3% (20·1–33·1) were expected to be spent in poor health in high SDI countries compared with 11·7% (8·8–15·1) in low-middle SDI countries. HALE at birth increased by 6·3 years (5·9–6·7), from 57·0 years (54·6–59·1) in 1990 to 63·3 years (60·5–65·7) in 2017. The increase varied from 3·8 years (3·4–4·1) in high SDI countries to 10·5 years (9·8–11·2) in low SDI countries. Even larger variations in HALE than these were observed between countries, ranging from 1·0 year (0·4–1·7) in Saint Vincent and the Grenadines (62·4 years [59·9–64·7] in 1990 to 63·5 years [60·9–65·8] in 2017) to 23·7 years (21·9–25·6) in Eritrea (30·7 years [28·9–32·2] in 1990 to 54·4 years [51·5–57·1] in 2017). In most countries, the increase in HALE was smaller than the increase in overall life expectancy, indicating more years lived in poor health. In 180 of 195 countries and territories, females were expected to live longer than males in 2017, with extra years lived varying from 1·4 years (0·6–2·3) in Algeria to 11·9 years (10·9–12·9) in Ukraine. Of the extra years gained, the proportion spent in poor health varied largely across countries, with less than 20% of additional years spent in poor health in Bosnia and Herzegovina, Burundi, and Slovakia, whereas in Bahrain all the extra years were spent in poor health. In 2017, the highest estimate of HALE at birth was in Singapore for both females (75·8 years [72·4–78·7]) and males (72·6 years [69·8–75·0]) and the lowest estimates were in Central African Republic (47·0 years [43·7–50·2] for females and 42·8 years [40·1–45·6] for males). Globally, in 2017, the five leading causes of DALYs were neonatal disorders, ischaemic heart disease, stroke, lower respiratory infections, and chronic obstructive pulmonary disease. Between 1990 and 2017, age-standardised DALY rates decreased by 41·3% (38·8–43·5) for communicable diseases and by 49·8% (47·9–51·6) for neonatal disorders. For non-communicable diseases, global DALYs increased by 40·1% (36·8–43·0), although age-standardised DALY rates decreased by 18·1% (16·0–20·2). Interpretation With increasing life expectancy in most countries, the question of whether the additional years of life gained are spent in good health or poor health has been increasingly relevant because of the potential policy implications, such as health-care provisions and extending retirement ages. In some locations, a large proportion of those additional years are spent in poor health. Large inequalities in HALE and disease burden exist across countries in different SDI quintiles and between sexes. The burden of disabling conditions has serious implications for health system planning and health-related expenditures. Despite the progress made in reducing the burden of communicable diseases and neonatal disorders in low SDI countries, the speed of this progress could be increased by scaling up proven interventions. The global trends among non-communicable diseases indicate that more effort is needed to maximise HALE, such as risk prevention and attention to upstream determinants of health. Funding Bill & Melinda Gates Foundation