Dataset Information

Glutamate dehydrogenase: a novel candidate to diagnose Plasmodium falciparum through rapid diagnostic test in blood specimen from fever patients.

ABSTRACT: In recent years, Plasmodium falciparum histidine-rich protein 2 gene deletion has been reported in India. Such isolates are prone to selective transmission and thus form a challenge to case management. As most of the rapid malaria diagnostic tests are based on the detection of HRP2 protein in the blood, we attempted to use Glutamate Dehydrogenase (GDH) as a biomarker for the diagnosis of P. falciparum. Recombinant PfGDH was successfully cloned, expressed and purified using the Ni-NTA approach. Polyclonal antibodies were raised against full-length rPfGDH and its peptides. Antibodies for rPfGDH showed a strong immune response against the recombinant protein. However, antibody showed no affinity towards the peptides, which suggests they failed as antigen. Antibodies for rPfGDH significantly detected the GDH in human blood specimens. This is the first report where P. falciparum GDH was detected in malaria cases from various parts of India. The raised polyclonal antibodies had shown an affinity for PfGDH in quantitative ELISA and are capable to be exploited for RDTs. This research needs further statistical validation on a large number and different sample types from candidates infected with P. falciparum and other species.

SUBMITTER: Kori LD

PROVIDER: S-EPMC7156408 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Glutamate dehydrogenase: a novel candidate to diagnose Plasmodium falciparum through rapid diagnostic test in blood specimen from fever patients.

Kori Lokesh D LD Valecha Neena N Anvikar Anupkumar R AR

Scientific reports 20200414 1

In recent years, Plasmodium falciparum histidine-rich protein 2 gene deletion has been reported in India. Such isolates are prone to selective transmission and thus form a challenge to case management. As most of the rapid malaria diagnostic tests are based on the detection of HRP2 protein in the blood, we attempted to use Glutamate Dehydrogenase (GDH) as a biomarker for the diagnosis of P. falciparum. Recombinant PfGDH was successfully cloned, expressed and purified using the Ni-NTA approach. P ...[more]

PMID: 32286365

Similar Datasets

Project description:BackgroundPlasmodium falciparum strains with mutations/deletions of the genes encoding the histidine-rich proteins 2/3 (pfhrp2/3) have emerged during the last 10 years leading to false-negative results in HRP2-based rapid diagnostic tests (RDTs). This can lead to unrecognized infections in individuals and to setbacks in malaria control in endemic countries where RDTs are the backbone of malaria diagnostics and control.Case descriptionHere the detection of a pfhrp2/3-negative P. falciparum infection acquired in Ethiopia by a 63-year old female traveller is presented. After onset of symptoms during travel, she was first tested negative for malaria, most probably by RDT, at a local hospital in Harar, Ethiopia. Falciparum malaria was finally diagnosed microscopically upon her return to Germany, over 4 weeks after infection. At a parasite density of approximately 5387 parasites/µl, two different high-quality RDTs: Palutop + 4 OPTIMA, NADALRMalaria PF/pan Ag 4 Species, did not respond at their respective P. falciparum test lines. pfhrp2/3 deletion was confirmed by multiplex-PCR. The patient recovered after a complete course of atovaquone and proguanil. According to the travel route, malaria was acquired most likely in the Awash region, Central Ethiopia. This is the first case of imported P. falciparum with confirmed pfhrp2/3 deletion from Ethiopia.ConclusionHRP2-negative P. falciparum strains may not be recognized by the presently available HRP2-based RDTs. When malaria is suspected, confirmation by microscopy and/or qPCR is necessary in order to detect falciparum malaria, which requires immediate treatment. This case of imported P. falciparum, non-reactive to HRP2-based RDT, possibly underlines the necessity for standardized, nationwide investigations in Ethiopia and should alert clinicians from non-endemic countries to the possibility of false-negative RDT results which may increase in returning travellers with potentially life-threatening infections.

Project description:BackgroundRapid diagnostic tests (RDTs) have become the most common diagnostic tool for detection of Plasmodium falciparum malaria, in particular in remote areas. RDT blood spots provide a source of parasite DNA for molecular analysis. In this study, the utility of RDTs for molecular analysis and the performance of different methods for whole genome amplification were investigated.MethodsPositive P. falciparum RDTs were collected from Kayin, Myanmar from August 2014 to January 2016. The RDT samples were stored for 6 months, 9 months, 20 months, 21 months, and 32 months before DNA extraction and subsequent molecular analysis of P. falciparum kelch 13 (pfkelch13) mutations, P. falciparum multidrug resistance 1 (pfmdr1), and P. falciparum plasmepsin 2 (pfplasmepsin2) gene amplification. In addition, performance of four whole genome amplification (WGA) kits were compared, including REPLI-g®, MALBACTM, PicoPLEX®, and GenomePlex®, for which DNA quantity and quality were compared between original DNA and post-WGA products.ResultsThe proportion of successful amplification of the different molecular markers was similar between blood spots analysed from RDTs stored for 6, 9, 20, 21, or 32 months. Successful amplification was dependent on the molecular markers fragment length (p value < 0.05): 18% for a 1245 bp fragment of pfkelch13, 71% for 364 bp of pfkelch13, 81% for 87 bp of pfmdr1, 81% for 108 bp of pfplasmepsin2. Comparison of the four WGA assay kits showed that REPLI-g®, MALBACTM, and PicoPLEX® increased the quantity of DNA 60 to 750-fold, whereas the ratio of parasite DNA amplification over human DNA was most favourable for MALBAC®. Sequencing results of pfkelch13, P. falciparum chloroquine resistance transporter (pfcrt), P. falciparum dihydrofolate reductase (pfdhfr) and six microsatellite markers assessed from the post-WGA product was the same as from the original DNA.ConclusionsBlood spots from RDTs are a good source for molecular analysis of P. falciparum, even after storage up to 32 months. WGA of RDT-derived parasite DNA reliably increase DNA quantity with sufficient quality for molecular analysis of resistance markers.

Project description:In the Greater Mekong Subregion in Southeast Asia, malaria elimination strategies need to target all Plasmodium falciparum parasites, including those carried asymptomatically. More than 70% of asymptomatic carriers are not detected by current rapid diagnostic tests (RDTs) or microscopy. An HRP2-based ultrasensitive RDT (uRDT) developed to improve the detection of low-density infections was evaluated during prevalence surveys within a malaria elimination program in a low-transmission area of eastern Myanmar. Surveys were conducted to identify high-prevalence villages. Two-milliliter venous blood samples were collected from asymptomatic adult volunteers and transported to the laboratory. Plasmodium parasites were detected by RDT, uRDT, microscopy, ultrasensitive qPCR (uPCR), and multiplex enzyme-linked immunosorbent assay (ELISA). The sensitivity, specificity, and predictive positive and negative values of RDT and uRDT were calculated compared to uPCR and ELISA. Parasite and antigen concentrations detected by each test were defined using uPCR and ELISA, respectively. A total of 1,509 samples, including 208 P. falciparum-positive samples were analyzed with all tests. The sensitivity of the uRDT was twofold higher than that of RDT, 51.4% versus 25.2%, with minor specificity loss, 99.5% versus 99.9%, against the combined reference (uPCR plus ELISA). The geometric mean parasitemia detected by uRDT in P. falciparum monospecific infections was 3,019 parasites per ml (95% confidence interval [95% CI], 1,790 to 5,094; n = 79) compared to 11,352 parasites per ml (95% CI, 5,643 to 22,837; n = 38) by RDT. The sensitivities of uRDT and RDT dropped to 34.6% and 15.1%, respectively, for the matched tests performed in the field. The uRDT performed consistently better than RDT and microscopy at low parasitemias. It shows promising characteristics for the identification of high-prevalence communities and warrants further evaluation in mass screening and treatment interventions.

Project description:BackgroundAntigen-detecting rapid diagnostic tests (RDTs) have been recommended by the World Health Organization for use in remote areas to improve malaria case management. Lactate dehydrogenase (LDH) of Plasmodium falciparum is one of the main parasite antigens employed by various commercial RDTs. It has been hypothesized that the poor detection of LDH-based RDTs is attributed in part to the sequence diversity of the gene. To test this, the present study aimed to investigate the genetic diversity of the P. falciparum ldh gene in Thailand and to construct the map of LDH sequence diversity in P. falciparum populations worldwide.MethodsThe ldh gene was sequenced for 50 P. falciparum isolates in Thailand and compared with hundreds of sequences from P. falciparum populations worldwide. Several indices of molecular variation were calculated, including the proportion of polymorphic sites, the average nucleotide diversity index (π), and the haplotype diversity index (H). Tests of positive selection and neutrality tests were performed to determine signatures of natural selection on the gene. Mean genetic distance within and between species of Plasmodium ldh was analysed to infer evolutionary relationships.ResultsNucleotide sequences of P. falciparum ldh could be classified into 9 alleles, encoding 5 isoforms of LDH. L1a was the most common allelic type and was distributed in P. falciparum populations worldwide. Plasmodium falciparum ldh sequences were highly conserved, with haplotype and nucleotide diversity values of 0.203 and 0.0004, respectively. The extremely low genetic diversity was maintained by purifying selection, likely due to functional constraints. Phylogenetic analysis inferred the close genetic relationship of P. falciparum to malaria parasites of great apes, rather than to other human malaria parasites.ConclusionsThis study revealed the global genetic variation of the ldh gene in P. falciparum, providing knowledge for improving detection of LDH-based RDTs and supporting the candidacy of LDH as a therapeutic drug target.

Dataset Information

Glutamate dehydrogenase: a novel candidate to diagnose Plasmodium falciparum through rapid diagnostic test in blood specimen from fever patients.

Publications

Glutamate dehydrogenase: a novel candidate to diagnose Plasmodium falciparum through rapid diagnostic test in blood specimen from fever patients.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets