Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0024530 (malaria)
 44,886 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Reliable analytical techniques to test growth-promoting and antimalarial efficacy on plasmodia are very important. Flow cytometry (FCM) offers the possibility to study developmental stages of intraerythrocytic growth of malaria parasites using nucleic acid staining. To analyze the growth of Plasmodium falciparum SYBR Green I was introduced as an intercalating dye with FCM for the 488nm line of an argon laser. Procedures employing FCM, including fixatives, dye concentrations, dilution buffer, and staining period, were optimized to simplify the method. FCM as described here allows parasitemia and parasites of different stages to be quantified according to the DNA content. The proportion of parasitized erythrocytes estimated by FCM and the Giemsa method agreed with determination by parasite lactate dehydrogenase. The protocol was extended to merozoite counting as a sensitive assay of growth inhibition of the parasite.
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PMID:Plasmodium falciparum: development and validation of a measure of intraerythrocytic growth using SYBR Green I in a flow cytometer. 1901 30

Phage-displayed chicken single-chain antibody fragment libraries can provide useful diagnostic and research reagents. Using avian immunoglobulin genes simplifies the construction of such repertoires since far fewer primer sets are required to access the avian antibody repertoire than is the case with mice or humans. Libraries constructed using mRNA from an immune source are enriched in affinity-matured sequences and consequently need not be as large as "universal" non-immune repertoires to have a reasonable probability of yielding high-affinity binders. Repertoires focused on a number of defined targets can be constructed using lymphocyte mRNA from chickens immunized with a mixture of several different antigens. This approach was evaluated with the aim of economically and rapidly deriving immunodiagnostic reagents for malaria, trypanosomiasis, and malignant catarrhal fever, all of which are important to health or food security in Africa. Two chickens were each immunized with a mixture comprised of recombinantly expressed histidine-rich protein, the aldolase and the lactate dehydrogenase of Plasmodium falciparum, the variant surface glycoprotein of Trypanosoma sp., and purified malignant catarrhal fever virus, a herpesvirus that causes an economically important disease of cattle and other ruminants. Immune responses to each of the individual antigens were determined by extracting egg-yolk IgY and testing for antigen-specific antibodies in ELISA. The chicken splenocytes were then recovered, RNA was extracted, and after reverse transcription, the immunoglobulin VH and VL regions were amplified by PCR and joined via a single glycyl residue for surface expression on a collection of filamentous bacteriophages. The resulting display library was then screened by panning to isolate binders. The immunized chickens did not, however, respond equally well to all the different antigens, nor was it possible to derive antibody fragments against all the targets. These limitations notwithstanding, several useful binders with the potential to be used in malaria diagnosis were obtained.
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PMID:Single-chain antibody fragments from a display library derived from chickens immunized with a mixture of parasite and viral antigens. 1910 14

L-Malate dehydrogenase (PfMDH) from Plasmodium falciparum, the causative agent for the most severe form of malaria, has shown remarkable similarities to L: -lactate dehydrogenase (PfLDH). PfMDH is more closely related to [LDH-like] MDHs characterized in archae and other prokaryotes. Initial sequence analysis and identification of critical amino acid residues involved in inter-subunit salt-bridge interactions predict tetrameric structure for PfMDH. The catalytically active recombinant PfMDH was characterized as a tetramer. The enzyme is localized primarily in the parasites cytosol. To gain molecular insights into PfMDH/PfLDH relationships and to understand the quaternary structure of PfMDH, dimers were generated by mutation to the potential salt-bridge interacting sites. The R183A and R214G mutations, which snapped the salt bridges between the dimers and resulted in lower dimeric state, did not affect catalytic properties of the enzyme. The mutant dimers of PfMDH were active equally as the wild-type PfMDH. The studies reveal structure of PfMDH as a dimer of dimers. The tetrameric state of PfMDH was not essential for catalytic functions of the enzyme but may be an evolutionary adaptation for cytosolic localization to support its role in NAD/NADH coupling, an important metabolic function for survival of the malaria parasite.
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PMID:Analysis of quaternary structure of a [LDH-like] malate dehydrogenase of Plasmodium falciparum with oligomeric mutants. 1918 66

Rapid diagnosis is prerequisite for effective treatment and reducing mortality and morbidity of malaria. This study was taken up to compare the efficacy of various methods available, i.e., thick and thin smear, quantitative buffy coat (QBC), plasmodium lactate dehydrogenase and aldolase in blood of patient. A total of 411 samples were collected from patients presenting with classic symptoms of malaria. For traditional microscopy; thick and thin smears were prepared and stained with Leishman's stain, taking thick smear as gold standard, thin smear had a sensitivity and specificity of 54.8% and 100%, respectively. QBC and antigen detection was done using commercially available kits; out of 411 samples, QBC and Malariagen were positive in 66 and 62 cases, with a sensitivity of 78% and 75%, respectively. Leishman's thick smear, although cost effective, is difficult to interpret for inexperienced microscopists; so if facilities are available, QBC should be used for routine diagnosis. In places where facilities are not available, rapid, simple and easy to interpret antigen detection test can be used despite low sensitivity.
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PMID:A comparative study of blood smear, QBC and antigen detection for diagnosis of malaria. 1933 12

There is a need for more objective and quantitative tools to replace microscopy in malaria diagnosis. Emphasis has recently been placed on alternative methods such as immunochromatography-based rapid tests. However, these tests provide only qualitative results. Two bio-molecules, parasite lactate dehydrogenase (pLDH) and histidine-rich proteins (HRPs), that are released by the intra-erythrocytic stages of the parasite offer certain specific characteristics that could potentially improve malaria diagnosis. In this paper, we describe a protocol for a unified sandwich ELISA that allows for the separate but concurrent measurement of pLDH and HRP biomolecules in aliquots taken from the same samples. Freshly drawn blood from a healthy unexposed adult male was used to serially dilute in vitro cultivated and synchronized ring stage Plasmodium falciparum parasites. Commercially available ELISA formats were modified to allow for the measurement of pLDH and HRP from aliquots of the same samples. The pLDH and HRP levels in the samples spiked with known numbers of infected red blood cells (iRBCs) were measured, and the values were used to generate standard graphs. The standard graphs were used to estimate the numbers of iRBCs in test samples. Serially diluted recombinant proteins were similarly used to generate a calibration curve, allowing for the expression of test results in nanograms of their respective recombinant protein. Levels of pLDH and HRPs were determined by using 1) P. falciparum culture material (cells and medium) 2) P. falciparum infected human blood (N = 6) samples, and 3) plasma from P. falciparum-infected patient (N = 22) samples. The parasite density of all culture and infected patient samples was also estimated by microscopy. Both pLDH and HRP levels correlated positively with the parasite density assessed by microscopy: Pearson correlation coefficient pLDH (r = 0.754, P < 0.0001, 95% CI: 0.47-0.89); HRP (r = 0.552, P < 0.007, 95% CI: 0.16-0.79). The HRPs seem to be released in larger quantities than pLDH (in a ratio of ~1 pLDH:~6 HRP), making the detection of HRP in culture material, blood, and plasma easier. The modified ELISA assay with quantitative measurement of pLDH and HRPs may provide a valuable tool for malaria research and patient management.
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PMID:Unified parasite lactate dehydrogenase and histidine-rich protein ELISA for quantification of Plasmodium falciparum. 1934 68

The objective of this study is to develop and evaluate a simple, cheap, and stable positive control for the quality control and quality assurance (QA) of rapid diagnostic tests (RDT) for the diagnosis of malaria. Plasmodium falciparum in vitro culture of known parasite concentrations was dried on a protein saver card, that is, dried blood spots (DBSs). The cards were stored at temperatures ranging from 27 to 60 degrees C from 1 day up to 6 months. Antigens were subsequently eluted from the card giving final concentrations ranging from 30 000 parasites to 300 parasites/microL and tested for stability against RDT based on the antigens parasite lactate dehydrogenase (pLDH), aldolase, and histidine-rich protein 2 (HRP-2). HRP-2 antigens were stable throughout the whole study and yielded positive results irrespective of parasite concentration, storage duration, or temperature, although band intensity differences could be observed when high parasites were compared with low parasite densities. Aldolase was able to generate positive signals for up to 4 weeks irrespective of the storage conditions. Thereafter, intensities decreased proportionally to increasing temperature and storage duration. Thirty thousand parasites per liter could give a signal up to 16 weeks when stored at a temperature of maximum 45 degrees C. However, densities of 300 parasites/microL were not able to generate a signal during the study. pLDH, the least stable of the 3 antigens, was not able to generate a signal after 1 week of storage. The DBS method yields a very stable positive control for quality control and QA of RDTs based on HRP-2. RDTs based on aldolase may also benefit from this method although to a lesser extent because that particular antigen is less stable in the DBS system.
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PMID:Development of a stable positive control to be used for quality assurance of rapid diagnostic tests for malaria. 1937 69

Although highly accurate rapid diagnostic tests (RDT) for Plasmodium falciparum [based on identification of histidine-rich protein-2 (PfHRP2)] have been developed, the accuracy of non-falciparum tests is relatively poor. Recently, a Plasmodium vivax-specific RDT [based on identification of species-specific lactate dehydrogenase (PvLDH)] became available, which along with PfHRP2 may improve malaria diagnosis by identifying the species correctly. A cross-sectional hospital-based study was designed to evaluate the diagnostic accuracy of FalciVax, a commercially available PfHRP2- and PvLDH-based RDT (index test), using malaria microscopy as a reference standard. All consecutive inpatients who presented with fever underwent both the index test and the reference standard. The study sample included 657 patients and the overall sensitivity and specificity of the RDT for diagnosis of any malarial species were 92.9% and 98.4%, respectively. The diagnostic accuracy estimates for correct species identification were lower (sensitivity 91.8%, specificity 96.8%). The accuracy of the PvLDH test to detect P. vivax was low (sensitivity 76.6%, specificity 98.1%).
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PMID:Accuracy of a multispecies rapid diagnostic test kit for detection of malarial parasite at the point of care in a low endemicity region. 1947 76

Plasmodium knowlesi has a relatively broad host range extending to humans, in whom it causes zoonotic malaria. Recent studies have shown that human infection with P. knowlesi is widely distributed in forested areas of Southeast Asia. In the present study, we evaluated commercial rapid diagnostic tests (RDTs) for human malaria to assess their reactivity and sensitivity in detecting P. knowlesi parasites using blood samples obtained from infected monkeys. The blood samples were assayed using two commercial RDTs based on immunochromatographic assays: (i) the OptiMAL-IT, designed to detect parasite lactate dehydrogenase (pLDH) of both P. falciparum and other plasmodia, and (ii) the Entebe Malaria Cassette (MC), designed to detect P. falciparum-specific histidine-rich protein 2 (PfHRP2) and P. vivax-specific pLDH. Interestingly, when the P. knowlesi-infected blood samples were examined with the RDTs, OptiMAL test results were interpreted as falciparum malaria-positive, while Entebe MC test results were interpreted as vivax malaria-positive. The sensitivities of both tests in detecting P. knowlesi parasite were similar to those for P. falciparum and higher than P. vivax. Thus, commercial RDTs based on detection of pLDH should be used with great caution, and should not replace conventional microscopy in the diagnosis of suspected cases of P. knowlesi malaria.
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PMID:Cross-reactivity in rapid diagnostic tests between human malaria and zoonotic simian malaria parasite Plasmodium knowlesi infections. 1952 97

The emergence and wide dissemination of drug-resistant malarial parasites underscore the need to prevent post-transfusion malaria. In Nigeria, as in most of sub-Saharan Africa, however, blood donors are not routinely screened for malarial infection. Recently, 391 consecutive potential blood donors in a malaria-endemic area of south-western Nigeria were each checked for malarial parasitaemia using three methods: microscopy (all samples), OptiMAL (315 samples) and/or the Clinotech Malaria Cassette (142 samples). OptiMAL detects parasite-specific lactate dehydrogenase whereas the Clinotech test detects the surface proteins of merozoites and sporozoites. Microscopy revealed parasitaemias in 79 (20.2%) of the potential donors, the levels of parasitaemia varying from 34 to 6289 asexual parasites/microl (mean=445/microl). The prevalence of malarial parasitaemia, as detected by microscopy, was significantly higher during the rainy season than in the dry season (27.3% v. 5.5%; P<0.0001). There was no significant association between patent parasitaemia and fever (i.e. an axillary temperature > or =37.5 degrees C), blood group, gender or anaemia. The corresponding prevalences of malarial parasitaemia detected using the rapid diagnostic tests were 3.8% (12/315) for OptiMAL and 57.8% (82/142) for the Clinotech. With the results of the microscopy used as the 'gold standard', OptiMAL gave a sensitivity of only 16.0% but a specificity of 98.5%. The corresponding values for the Clinotech tests were 69.2% and 50.0%, respectively. It would clearly be beneficial to include screening for malaria parasitaemia in the routine investigation of potential blood donors in Nigeria, especially during the rainy season, when the risk of transfusion-transmitted malaria appears relatively high.
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PMID:Blood banking in a malaria-endemic area: evaluating the problem posed by malarial parasitaemias. 1958 9

The malaria parasite thrives on anaerobic fermentation of glucose for energy. Earlier studies from our laboratory have demonstrated that a cytosolic malate dehydrogenase (PfMDH) with striking similarity to lactate dehydrogenase (PfLDH) might complement PfLDH function in Plasmodium falciparum. The N-terminal glycine motif, which forms a characteristic Rossman dinucleotide-binding fold in the co-substrate binding pocket, differentiates PfMDH (GlyXGlyXXGly) from other eukaryotic and prokaryotic malate dehydrogenases (GlyXXGlyXXGly). The amino acids lining the co-substrate binding pocket are completely conserved in MDHs from different species of human, primate and rodent malaria parasites. Based on this knowledge and conserved domains among prokaryotic and eukaryotic MDH, the role of critical amino acids lining the co-substrate binding pocket was analyzed in catalytic functions of PfMDH using site-directed mutagenesis. Insertion of Ala at the 9th or 10th position, which converts the N-terminal GlyXGlyXXGly motif (characteristic of malarial MDH and LDH) to GlyXXGlyXXGly (as in bacterial and eukaryotic MDH), uncoupled regulation of the enzyme through substrate inhibition. The dinucleotide fold GlyXGlyXXGly motif seems not to be responsible for the distinct affinity of PfMDH to 3-acetylpyridine-adenine dinucleotide (APAD, a synthetic analog of NAD), since Ala9 and Ala10 insertion mutants still utilized APADH. The Gln11Met mutation, which converts the signature glycine motif in PfMDH to that of PfLDH, did not change the enzyme function. However, the Gln11Gly mutant showed approximately a 5-fold increase in catalytic activity, and higher susceptibility to inhibition with gossypol. Asn119 and His174 participate in binding of both co-substrate and substrate. The Asn119Gly mutant exhibited approximately a 3-fold decrease in catalytic efficiency, while mutation of His174 to Asn or Ala resulted in an inactive enzyme. These studies provide critical insights into the co-substrate binding pocket of PfMDH, which may be important in design of selective PfMDH/PfLDH inhibitors as potential antimalarials.
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PMID:Structure and function of Plasmodium falciparum malate dehydrogenase: role of critical amino acids in co-substrate binding pocket. 1977 85


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