Assessing the cost-benefit effect of a plasmodium falciparum drug resistance mutation on parasite growth In vitro

Plasmodium falciparum mutations associated with antimalarial resistance may be beneficial for parasites under drug pressure, although they may also cause a fitness cost. We herein present an in vitro model showing how this combined effect on parasite growth varies with the drug concentration and suggest a calculated drug-specific cost-benefit index, indicating the possible advantage for mutated parasites. We specifically studied the D-to-Y change at position 1246 encoded by the pfmdr1 gene (pfmdr1 D1246Y) in relation to amodiaquine resistance. Susceptibilities to amodiaquine, desethylamodiaquine, and chloroquine, as well as relative fitness, were determined for two modified isogenic P. falciparum clones differing only in the pfmdr1 1246 position. Data were used to create a new comparative graph of relative growth in relation to the drug concentration and to calculate the ratio between the benefit of resistance and the fitness cost. Results were related to an in vivo allele selection analysis after amodiaquine or artesunate-amodiaquine treatment. pfmdr1 1246Y was associated with decreased susceptibility to amodiaquine and desethylamodiaquine but at a growth fitness cost of 11%. Mutated parasites grew less in low drug concentrations due to a predominating fitness cost, but beyond a breakpoint concentration they grew more due to a predominating benefit of increased resistance. The cost-benefit indexes indicated that pfmdr1 1246Y was most advantageous for amodiaquine-exposed parasites. In vivo, a first drug selection of mutant parasites followed by a fitness selection of wild-type parasites supported the in vitro data. This cost-benefit model may predict the risk for selection of drug resistance mutations in different malaria transmission settings.Swedish International Development Agency, Department for Research Cooperation (SIDA/SAREC) [SWE-2005-027/2006-2007, SWE-2005-027/2008-2009]info:eu-repo/semantics/publishedVersio

Point-of-care mobile digital microscopy and deep learning for the detection of soil-transmitted helminths and Schistosoma haematobium

Background: Microscopy remains the gold standard in the diagnosis of neglected tropical diseases. As resource limited, rural areas often lack laboratory equipment and trained personnel, new diagnostic techniques are needed. Low-cost, point-of-care imaging devices show potential in the diagnosis of these diseases. Novel, digital image analysis algorithms can be utilized to automate sample analysis. Objective: Evaluation of the imaging performance of a miniature digital microscopy scanner for the diagnosis of soil-transmitted helminths and Schistosoma haematobium, and training of a deep learning-based image analysis algorithm for automated detection of soil-transmitted helminths in the captured images. Methods: A total of 13 iodine-stained stool samples containing Ascaris lumbricoides, Trichuris trichiura and hookworm eggs and 4 urine samples containing Schistosoma haematobium were digitized using a reference whole slide-scanner and the mobile microscopy scanner. Parasites in the images were identified by visual examination and by analysis with a deep learning-based image analysis algorithm in the stool samples. Results were compared between the digital and visual analysis of the images showing helminth eggs. Results: Parasite identification by visual analysis of digital slides captured with the mobile microscope was feasible for all analyzed parasites. Although the spatial resolution of the reference slide-scanner is higher, the resolution of the mobile microscope is sufficient for reliable identification and classification of all parasites studied. Digital image analysis of stool sample images captured with the mobile microscope showed high sensitivity for detection of all helminths studied (range of sensitivity = 83.3-100%) in the test set (n = 217) of manually labeled helminth eggs. Conclusions: In this proof-of-concept study, the imaging performance of a mobile, digital microscope was sufficient for visual detection of soil-transmitted helminths and Schistosoma haematobium. Furthermore, we show that deep learning-based image analysis can be utilized for the automated detection and classification of helminths in the captured images.Peer reviewe

Influence of consecutive-day blood sampling on polymerase chain reaction-adjusted parasitological cure rates in an antimalarial-drug trial conducted in Tanzania

We assessed the influence that consecutive-day blood sampling, compared with single-day blood sampling, had on polymerase chain reaction (PCR)-adjusted parasitological cure after stepwise genotyping of merozoite surface proteins 2 (msp2) and 1 (msp1) in 106 children in Tanzania who had uncomplicated falciparum malaria treated with either sulfadoxine-pyrimethamine or artemether- lumefantrine; 78 of these children developed recurrent parasitemia during the 42-day follow-up period. Initial msp2 genotyping identified 27 and 33 recrudescences by use of single- and consecutive-day sampling, respectively; in subsequent msp1 genotyping, 17 and 21 of these episodes, respectively, were still classified as recrudescences; these results indicate a similar sensitivity of the standard single-day PCR protocol - that is, 82% (27/33) and 81% (17/21), in both genotyping steps. Interpretation of PCR-adjusted results will significantly depend on methodology. © 2007 by the Infectious Diseases Society of America. All rights reserved

The CoESCA station at BESSY: Auger electron–photoelectron coincidences from surfaces demonstrated for Ag MNN

In this work, we present the CoESCA station for electron–electron coincidence spectroscopy from surfaces, built in a close collaboration between Uppsala University and Helmholtz-Zentrum Berlin at the BESSY II synchrotron facility in Berlin, Germany. We start with a detailed overview of previous work in the field of electron–electron coincidences, before we describe the CoESCA setup and its design parameters. The system is capable of recording shot-to-shot resolved 6D coincidence datasets, i.e. the kinetic energy and the two take off angles for both coincident electrons. The mathematics behind extracting and analysing these multi-dimensional coincidence datasets is introduced, with a focus on coincidence statistics, resulting in fundamental limits of the signal-to-noise ratio and its implications for acquisition times and the size of the raw data stream. The functionality of the CoESCA station is demonstrated for the example of Auger electron–photoelectron coincidences from silver surfaces for photoelectrons from the Ag 3d core levels and their corresponding MNN Auger electrons. The Auger spectra originating from the different core levels, 3d and 3d could be separated and further, the two-hole state energy distributions were determined for these Auger decay channels

Plasmodium falciparum multidrug resistance protein 1 and artemisinin-based combination therapy in Africa

Plasmodium falciparum response mechanisms to the major artemisinin-based combination therapies (ACTs) are largely unknown. Multidrug-resistance protein (MRP)-like adenosine triphosphate (ATP)-binding cassette transporters are known to be related to multidrug resistance in many organisms. Therefore, we hypothesized that sequence variation in pfmrp1 can contribute to decreased parasite sensitivity to ACT. Through sequencing of the pfmrp1 open reading frame for 103 geographically diverse P. falciparum infections, we identified 27 single-nucleotide polymorphisms (SNPs), of which 21 were nonsynonymous and 6 synonymous. Analyses of clinical efficacy trials with artesunate-amodiaquine and artemether-lumefantrine detected a specific selection of the globally prevalent I876V SNP in recurrent infections after artemether-lumefantrine treatment. Additional in silico studies suggested an influence of variation in amino acid 876 on the ATP hydrolysis cycle of pfMRP1 with potential impact on protein functionality. Our data suggest for the first time, to our knowledge, the involvement of pfMRP1 in P. falciparum in vivo response to ACT.info:eu-repo/semantics/publishedVersio

A Malaria Diagnostic Tool Based on Computer Vision Screening and Visualization of Plasmodium falciparum Candidate Areas in Digitized Blood Smears

Peer reviewe

A deep sequencing tool for partitioning clearance rates following antimalarial treatment in polyclonal infections

BACKGROUND AND OBJECTIVES: Current tools struggle to detect drug-resistant malaria parasites when infections contain multiple parasite clones, which is the norm in high transmission settings in Africa. Our aim was to develop and apply an approach for detecting resistance that overcomes the challenges of polyclonal infections without requiring a genetic marker for resistance. METHODOLOGY: Clinical samples from patients treated with artemisinin combination therapy were collected from Tanzania and Cambodia. By deeply sequencing a hypervariable locus, we quantified the relative abundance of parasite subpopulations (defined by haplotypes of that locus) within infections and revealed evolutionary dynamics during treatment. Slow clearance is a phenotypic, clinical marker of artemisinin resistance; we analyzed variation in clearance rates within infections by fitting parasite clearance curves to subpopulation data. RESULTS: In Tanzania, we found substantial variation in clearance rates within individual patients. Some parasite subpopulations cleared as slowly as resistant parasites observed in Cambodia. We evaluated possible explanations for these data, including resistance to drugs. Assuming slow clearance was a stable phenotype of subpopulations, simulations predicted that modest increases in their frequency could substantially increase time to cure. CONCLUSIONS AND IMPLICATIONS: By characterizing parasite subpopulations within patients, our method can detect rare, slow clearing parasites in vivo whose phenotypic effects would otherwise be masked. Since our approach can be applied to polyclonal infections even when the genetics underlying resistance are unknown, it could aid in monitoring the emergence of artemisinin resistance. Our application to Tanzanian samples uncovers rare subpopulations with worrying phenotypes for closer examination

The usefulness of rapid diagnostic tests in the new context of low malaria transmission in zanzibar.

BACKGROUND\ud \ud We assessed if histidine-rich-protein-2 (HRP2) based rapid diagnostic test (RDT) remains an efficient tool for Plasmodium falciparum case detection among fever patients in Zanzibar and if primary health care workers continue to adhere to RDT results in the new epidemiological context of low malaria transmission. Further, we evaluated the performance of RDT within the newly adopted integrated management of childhood illness (IMCI) algorithm in Zanzibar.\ud \ud METHODS AND FINDINGS\ud \ud We enrolled 3890 patients aged ≥2 months with uncomplicated febrile illness in this health facility based observational study conducted in 12 primary health care facilities in Zanzibar, between May-July 2010. One patient had an inconclusive RDT result. Overall 121/3889 (3.1%) patients were RDT positive. The highest RDT positivity rate, 32/528 (6.1%), was found in children aged 5-14 years. RDT sensitivity and specificity against PCR was 76.5% (95% CI 69.0-83.9%) and 99.9% (95% CI 99.7-100%), and against blood smear microscopy 78.6% (95% CI 70.8-85.1%) and 99.7% (95% CI 99.6-99.9%), respectively. All RDT positive, but only 3/3768 RDT negative patients received anti-malarial treatment. Adherence to RDT results was thus 3887/3889 (99.9%). RDT performed well in the IMCI algorithm with equally high adherence among children <5 years as compared with other age groups.\ud \ud CONCLUSIONS\ud \ud The sensitivity of HRP-2 based RDT in the hands of health care workers compared with both PCR and microscopy for P. falciparum case detection was relatively low, whereas adherence to test results with anti-malarial treatment was excellent. Moreover, the results provide evidence that RDT can be reliably integrated in IMCI as a tool for improved childhood fever management. However, the relatively low RDT sensitivity highlights the need for improved quality control of RDT use in primary health care facilities, but also for more sensitive point-of-care malaria diagnostic tools in the new epidemiological context of low malaria transmission in Zanzibar.\ud \ud TRIAL REGISTRATION\ud \ud ClinicalTrials.gov NCT01002066

Plasmodium falciparum drug resistance phenotype as assessed by patient antimalarial drug levels and Its association With pfmdr1 polymorphisms

Background. Multidrug-resistant Plasmodium falciparum is a major threat to global malaria control. Parasites develop resistance by gradually acquiring genetic polymorphisms that decrease drug susceptibility. The aim of this study was to investigate the extent to which parasites with different genetic characteristics are able to withstand individual drug blood concentrations. Methods. We analyzed 2 clinical trials that assessed the efficacy and effectiveness of artemether-lumefantrine. As a proof of concept, we used measured day 7 lumefantrine concentrations to estimate the concentrations at which reinfections multiplied. P. falciparum multidrug resistance gene 1 (pfmdr1) genotypes of these parasites were then correlated to drug susceptibility. Results. Reinfecting parasites with the pfmdr1 N86/184F/D1246 haplotype were able to withstand lumefantrine blood concentrations 15-fold higher than those with the 86Y/Y184/1246Y haplotype. Conclusions. By estimating drug concentrations, we were able to quantify the contribution of pfmdr1 single-nucleotide polymorphisms to reduced lumefantrine susceptibility. The method can be applied to all long-half-life antimalarial drugs, enables early detection of P. falciparum with reduced drug susceptibility in vivo, and represents a novel way for unveiling molecular markers of antimalarial drug resistance.Swedish Development Cooperation Agency-Department for Research Cooperation (SIDA-SAREC) [SWE 2004-3850, Bil-Tz 16/9875007059, SWE-2009-165]; World Health Organization MIM-TDR [[A60100] MAL IRM 06 03]; Goljes Foundation; Swedish medical research council [K2010-56X-21457-01-3]; Wellcome Trust of Great Britai