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Fulltext KASP: a genotyping method to rapid identification of resistance in Plasmodium falciparum

Alvarez-Fernandez A, Bernal MJ, Fradejas I, Martin Ramírez A, Md Yusuf NA, Lanza M, et al.

Malar J, 2021 Jan 06;20(1):16.
PMID: 33407529 DOI: 10.1186/s12936-020-03544-7

BACKGROUND: The emergence and spread of anti-malarial resistance continues to hinder malaria control. Plasmodium falciparum, the species that causes most human malaria cases and most deaths, has shown resistance to almost all known anti-malarials. This anti-malarial resistance arises from the development and subsequent expansion of Single Nucleotide Polymorphisms (SNPs) in specific parasite genes. A quick and cheap tool for the detection of drug resistance can be crucial and very useful for use in hospitals and in malaria control programmes. It has been demonstrated in different contexts that genotyping by Kompetitive Allele Specific PCR (KASP), is a simple, fast and economical method that allows a high-precision biallelic characterization of SNPs, hence its possible utility in the study of resistance in P. falciparum.
METHODS: Three SNPs involved in most cases of resistance to the most widespread anti-malarial treatments have been analysed by PCR plus sequencing and by KASP (C580Y of the Kelch13 gene, Y86N of the Pfmdr1 gene and M133I of the Pfcytb gene). A total of 113 P. falciparum positive samples and 24 negative samples, previously analysed by PCR and sequencing, were selected for this assay. Likewise, the samples were genotyped for the MSP-1 and MSP-2 genes, and the Multiplicity of Infection (MOI) and parasitaemia were measured to observe their possible influence on the KASP method.
RESULTS: The KASP results showed the same expected mutations and wild type genotypes as the reference method, with few exceptions that correlated with very low parasitaemia samples. In addition, two cases of heterozygotes that had not been detected by sequencing were found. No correlation was found between the MOI or parasitaemia and the KASP values of the sample. The reproducibility of the technique shows no oscillations between repetitions in any of the three SNPs analysed.
CONCLUSIONS: The KASP assays developed in this study were efficient and versatile for the determination of the Plasmodium genotypes related to resistance. The method is simple, fast, reproducible with low cost in personnel, material and equipment and scalable, being able to core KASP arrays, including numerous SNPs, to complete the main pattern of mutations associated to P. falciparum resistance.
Malaria diagnosis using a combined system of a simple and fast extraction method with a lyophilised Dual-LAMP assay in a non-endemic setting

Martín Ramírez A, Barón Argos L, Lanza Suárez M, Carmona Rubio C, Pérez-Ayala A, Hisam SR, et al.

Pathog Glob Health, 2024 Feb;118(1):80-90.
PMID: 37415348 DOI: 10.1080/20477724.2023.2232595

Malaria is a parasitic disease distributed in tropical areas but with a high number of imported cases in non-endemic countries. The most specific and sensitive malaria diagnostic methods are PCR and LAMP. However, both require specific equipment, extraction procedures and a cold chain. This study aims to solve some limitations of LAMP method with the optimization and validation of six LAMP assays, genus and species-specific, using an easy and fast extraction method, the incorporation of a reaction control assay, two ways (Dual) of result reading and reagent lyophilization. The Dual-LAMP assays were validated against the Nested-Multiplex Malaria PCR. A conventional column and saline extraction methods, and the use of lyophilized reaction tubes were also assessed. A new reaction control Dual-LAMP-RC assay was designed. Dual-LAMP-Pspp assay showed no cross-reactivity with other parasites, repeatability and reproducibility of 100%, a significant correlation between parasite concentration and time to amplification and a LoD of 1.22 parasites/µl and 5.82 parasites/µl using column and saline extraction methods, respectively. Sensitivity and specificity of the six Dual-LAMP assays reach values of 100% or close to this, being lower for the Dual-LAMP-Pm. The Dual-LAMP-RC assay worked as expected. Lyophilized Dual-LAMP results were concordant with the reference method. Dual-LAMP malaria assays with the addition of a new reaction control LAMP assay and the use of a fast and easy saline extraction method, provided low limit of detection, no cross-reactivity, and good sensitivity and specificity. Furthermore, the reagent lyophilization and the dual result reading allow their use in most settings.
Fulltext Biotic and abiotic variables influencing plant litter breakdown in streams: a global study

Boyero L, Pearson RG, Hui C, Gessner MO, Pérez J, Alexandrou MA, et al.

Proc Biol Sci, 2016 Apr 27;283(1829).
PMID: 27122551 DOI: 10.1098/rspb.2015.2664

Plant litter breakdown is a key ecological process in terrestrial and freshwater ecosystems. Streams and rivers, in particular, contribute substantially to global carbon fluxes. However, there is little information available on the relative roles of different drivers of plant litter breakdown in fresh waters, particularly at large scales. We present a global-scale study of litter breakdown in streams to compare the roles of biotic, climatic and other environmental factors on breakdown rates. We conducted an experiment in 24 streams encompassing latitudes from 47.8° N to 42.8° S, using litter mixtures of local species differing in quality and phylogenetic diversity (PD), and alder (Alnus glutinosa) to control for variation in litter traits. Our models revealed that breakdown of alder was driven by climate, with some influence of pH, whereas variation in breakdown of litter mixtures was explained mainly by litter quality and PD. Effects of litter quality and PD and stream pH were more positive at higher temperatures, indicating that different mechanisms may operate at different latitudes. These results reflect global variability caused by multiple factors, but unexplained variance points to the need for expanded global-scale comparisons.
Fulltext Riparian plant litter quality increases with latitude

Boyero L, Graça MAS, Tonin AM, Pérez J, J Swafford A, Ferreira V, et al.

Sci Rep, 2017 09 05;7(1):10562.
PMID: 28874830 DOI: 10.1038/s41598-017-10640-3

Plant litter represents a major basal resource in streams, where its decomposition is partly regulated by litter traits. Litter-trait variation may determine the latitudinal gradient in decomposition in streams, which is mainly microbial in the tropics and detritivore-mediated at high latitudes. However, this hypothesis remains untested, as we lack information on large-scale trait variation for riparian litter. Variation cannot easily be inferred from existing leaf-trait databases, since nutrient resorption can cause traits of litter and green leaves to diverge. Here we present the first global-scale assessment of riparian litter quality by determining latitudinal variation (spanning 107°) in litter traits (nutrient concentrations; physical and chemical defences) of 151 species from 24 regions and their relationships with environmental factors and phylogeny. We hypothesized that litter quality would increase with latitude (despite variation within regions) and traits would be correlated to produce 'syndromes' resulting from phylogeny and environmental variation. We found lower litter quality and higher nitrogen:phosphorus ratios in the tropics. Traits were linked but showed no phylogenetic signal, suggesting that syndromes were environmentally determined. Poorer litter quality and greater phosphorus limitation towards the equator may restrict detritivore-mediated decomposition, contributing to the predominance of microbial decomposers in tropical streams.