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Detection of histidine rich protein & lactate dehydrogenase of Plasmodium falciparum in malaria patients by sandwich ELISA using in-house reagents.

ABSTRACT: BACKGROUND & OBJECTIVES:Despite major control efforts, malaria remains a major public health problem that still causes high mortality rate worldwide especially in Africa and Asia. Accurate and confirmatory diagnosis before treatment initiation is the only way to control the disease. The present study was undertaken to develop reagents using sandwich ELISA for simultaneous detection of PfHRP2 (Plasmodium falciparum histidine rich protein) and PfLDH (P. falciparum lactate dehydrogenase) antigens in the proven malaria cases. METHODS:The antibodies were raised against two epitopes of PfHRP2 protein and three unique and unexplored epitopes of PfLDH protein. These antibodies were able to detect PfHRP2 and PfLDH antigens in culture supernatant and parasitized RBC lysate of P. falciparum, respectively up to 50 parasites/?l. The in-house reagents were tested in 200 P. falciparum positive patients residing in Baghpat district of Uttar Pradesh in northern India. RESULTS:Microsphere (PLGA) with CpG ODN were used to generate high titre and high affinity antibodies against selected peptides of PfHRP-2 and pLDH antigen in mice and rabbit. The peptide specific peak titre varied from 12,800 - 102,400 with an affinity ranging 0.73 - 3.0 mM. The indigenously developed reagents are able to detect PfHRP2 and PfLDH antigens as low as 75 parasites/?l of blood with a very high sensitivity (96-100%) and specificity (100%). INTERPRETATION & CONCLUSIONS:The study highlight the identification of unique epitopes of PfHRP2 and PfLDH, and the generated antibodies against these antigens were used for quantitative estimation of these two antigens using sandwich ELISA. No corresreactivity with P. vivax infected patients was observed with the sera.

SUBMITTER: Verma P

PROVIDER: S-EPMC3978991 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Detection of histidine rich protein & lactate dehydrogenase of Plasmodium falciparum in malaria patients by sandwich ELISA using in-house reagents.

Verma Priyanka P Biswas Sukla S Mohan Teena T Ali Shakir S Rao D N DN

The Indian journal of medical research 20131201 6

<h4>Background & objectives</h4>Despite major control efforts, malaria remains a major public health problem that still causes high mortality rate worldwide especially in Africa and Asia. Accurate and confirmatory diagnosis before treatment initiation is the only way to control the disease. The present study was undertaken to develop reagents using sandwich ELISA for simultaneous detection of PfHRP2 (Plasmodium falciparum histidine rich protein) and PfLDH (P. falciparum lactate dehydrogenase) an ...[more]

PMID: 24521645

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Project description:BackgroundMalaria rapid diagnostic tests (RDTs) are precious tools to diagnose malaria. Most RDTs used currently are based on the detection of Plasmodium falciparum histidine-rich protein 2 (PfHRP2) in a patient's blood. However, concern has been raised in recent years that deletion of pfhrp2 in the parasite could affect the accuracy of PfHRP2-based RDTs. In addition, genetic variation in pfhrp2 might influence the accuracy and sensitivity of RDTs. In this study, the genetic variation in pfhrp2 and pfhrp3 in Myanmar P. falciparum isolates was analysed.MethodsBlood samples were collected from malaria patients who were infected with P. falciparum in Mandalay, Naung Cho, Tha Beik Kyin, and Pyin Oo Lwin, Upper Myanmar between 2013 and 2015. The pfhrp2 and pfhrp3 were amplified by nested polymerase chain reaction (PCR), cloned and sequenced. Genetic variation in Myanmar pfhrp2 and pfhrp3 was analysed using the DNASTAR program. Comparative analysis of Myanmar and global pfhrp2 and pfhrp3 isolates was also performed.ResultsOne-hundred and two pfhrp2 and 89 pfhrp3 were amplified from 105 blood samples, of which 84 pfhrp2 and 56 pfhrp3 sequences were obtained successfully. Myanmar pfhrp2 and pfhrp3 showed high levels of genetic variation with different arrangements of distinct repeat types, which further classified Myanmar pfhrp2 and pfhrp3 into 76 and 47 haplotypes, respectively. Novel amino acid changes were also found in Myanmar pfhrp2 and pfhrp3, but their frequencies were very low. Similar structural organization was shared by Myanmar and global pfhrp2 and pfhrp3, and differences in frequencies of repeat types and lengths were also observed between and among global isolates.ConclusionLength polymorphisms and amino acid substitutions generated extensive genetic variation in Myanmar pfhrp2 and pfhrp3. Comparative analysis revealed that global pfhrp2 and pfhrp3 share similar structural features, as well as extensive length polymorphisms and distinct organizations of repeat types. These results provide a better understanding of the genetic structure of pfhrp2 and pfhrp3 in global P. falciparum populations and suggest useful information to develop RDTs with improved quality.

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

Detection of histidine rich protein & lactate dehydrogenase of Plasmodium falciparum in malaria patients by sandwich ELISA using in-house reagents.

Publications

Detection of histidine rich protein & lactate dehydrogenase of Plasmodium falciparum in malaria patients by sandwich ELISA using in-house reagents.

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