Plasmodium falciparum:Evaluation of Lactate Dehydrogenase in Monitoring Therapeutic Responses to Standard Antimalarial Drugs in Nigeria

Plasmodium falciparum, a unicellular parasite that causes human malaria, infects erythrocytes where it develops within a vacuole. The vacuolar membrane separates the parasite from the erythrocyte cytosol. Some secreted parasite proteins remain inside the vacuole, and others are transported across the vacuolar membrane. To identify the protein sequences responsible for this distribution we investigated the suitability of the green fluorescent protein and luciferase as reporters in transiently transfected parasites. Because of the higher sensitivity of the enzymatic assay, luciferase was quantified 3 days after transfection, whereas reliable detection of green fluorescent protein required prolonged drug selection. Luciferase was confined to the parasite cytosol in subcellular fractions of infected erythrocytes. When parasites were transfected with a hybrid gene coding for the cleavable N-terminal signal peptide of a secreted parasite protein fused to luciferase, the reporter protein was secreted. It was recovered with the vacuolar content and the erythrocyte cytosol. The results suggest that no specific protein sequences are required for translocation across the vacuolar membrane. The high local concentration of luciferase within the vacuole argues against free diffusion, and thus transport into the erythrocyte cytosol must involve a ratelimiting step.Plasmodium falciparum, the parasite that causes the most severe form of malaria, spends part of its life cycle in human erythrocytes. Here it resides within the so-called parasitophorous vacuole, which is bound by the parasitophorous vacuolar membrane (PVM).1 The vacuole constitutes a separate compartment in the infected red blood cell (iRBC) that is distinct from the cytosol of the parasite and from the cytosol of the erythrocyte, respectively (1). Most proteins secreted from P. falciparum are not released into an extracellular space but are transported to various destinations within the iRBC: the parasitophorous vacuole or the erythrocyte cytosol. Membranebound proteins are found in the PVM and erythrocyte plasma membrane (2, 3). It is completely unknown which information within a polypeptide chain determines whether a protein is transported across the PVM. Recently we described experimental evidence for a transport pathway that involves the release of secreted parasite proteins into the vacuolar space and translocation across the PVM in a subsequent step (4). This model infers that a sorting mechanism must operate within the vacuole that discriminates between vacuolar resident proteins and proteins destined for a location beyond the PVM (5). Sorting could involve two different principles: the retention of vacuolar proteins or, alternatively, the recognition of protein signals that mediate translocation across the PVM.A widely used experimental approach for the identification of protein targeting and sorting signals in eukaryotic cells is the fusion of putative signal sequences to reporter proteins and their subsequent localization in the transfected cell. Although...

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“…Cytosolic plasmodial lactate dehydrogenase was monitored as described in Ref. 20 using the MalStat™ assay (Flow, Inc.).…”

Section: Methodsmentioning

confidence: 99%

Luciferase, When Fused to an N-terminal Signal Peptide, Is Secreted from Transfected Plasmodium falciparum and Transported to the Cytosol of Infected Erythrocytes

Burghaus

Lingelbach

2001

Journal of Biological Chemistry

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“…pLDHbased RDTs appear to be slightly less sensitive than those detecting HRP2, but the antigen is rapidly cleared from the bloodstream, becoming undetectable at about the same time blood smears become negative after antimalarial therapy. [13][14][15] Thus, if sensitivity is adequate, the increased specificity of pLDH-based assays for acute malaria suggests that they may be better-suited for high-transmission areas, such as much of sub-Saharan Africa. With increasing advocacy for the implementation of RDTs, it is critical that optimal diagnostic strategies are identified.…”

Section: Introductionmentioning

confidence: 99%

COMPARISON OF HRP2- AND pLDH-BASED RAPID DIAGNOSTIC TESTS FOR MALARIA WITH LONGITUDINAL FOLLOW-UP IN KAMPALA, UGANDA

Hopkins¹,

Kambale²,

Kamya³

et al. 2007

The American Journal of Tropical Medicine and Hygiene

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Abstract. Presumptive treatment of malaria results in significant overuse of antimalarials. Malaria rapid diagnostic tests (RDTs) may offer a reliable alternative for case management, but the optimal RDT strategy is uncertain. We compared the diagnostic accuracy of histidine-rich protein 2 (HRP2)-and plasmodium lactate dehydrogenase (pLDH)-based RDTs, using expert microscopy as the gold standard, in a longitudinal study of 918 fever episodes over an 8-month period in a cohort of children in Kampala, Uganda. Sensitivity was 92% for HRP2 and 85% for pLDH, with differences primarily due to better detection with HRP2 at low parasite densities. Specificity was 93% for HRP2 and 100% for pLDH, with differences primarily due to rapid clearance of pLDH antigenemia after treatment of a previous malaria episode. RDTs may provide an effective strategy for improving rational delivery of antimalarial therapy; in Kampala, either test could dramatically decrease inappropriate presumptive treatments.

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“…[27][28][29] However, as with the panmalarial antibody of the ICT Pf/Pv test, the pLDH monoclonal antibodies of the OptiMAL test detect not only asexual parasites, but also gametocytes. 26,30,31 This suggests that in contrast to referral center findings reported to date, and as suggested by a preliminary African study, 30 it is possible that the high rates of posttreatment gametocytemia found after chloroquine or sulfadoxine-pyrimethamine in primary health centers in malaria-endemic areas may lead to gametocyte-associated persistence of pLDH despite clearance of asexual parasites, and as with the ICT Pf/Pv test, it may result in an unreliable ability to predict treatment outcome. 32 Studies of the ability of pLDH clearance to predict treatment outcome in primary health centers in endemic areas are required before pLDH rapid antigen tests can be advocated as prognostic tools in these settings.…”

Section: Discussionmentioning

confidence: 99%

Detection of histidine rich protein 2 and panmalarial ICT Malaria Pf/Pv test antigens after chloroquine treatment of uncomplicated falciparum malaria does not reliably predict treatment outcome in eastern Indonesia.

Tjitra

Suprianto

Dyer³

et al. 2001

The American Journal of Tropical Medicine and Hygiene

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Abstract. In regions with drug-resistant malaria, the ability to rapidly detect or predict treatment failure (TF) soon after a course of standard therapy for Plasmodium falciparum malaria would facilitate the prompt institution of secondline therapy. We thus evaluated longitudinally the ability of the ICT Malaria Pf/Pv immunochromatographic test to predict treatment outcome. Sixty-six Sumbanese Indonesians with uncomplicated falciparum malaria were treated with chloroquine and followed for 28 days by use of 1997 World Health Organization criteria for assessment of therapeutic efficacy of antimalarial drugs. The ICT Pf/Pv testing could be compared with microscopy in approximately half of the patients on each day of follow-up. Although strongly positive histidine rich protein 2 (HRP2) line intensities (equal to or greater than the control band) in convalescence were highly predictive of TF, any degree of positivity for the HRP2 and panmalarial antigens in convalescence was only moderately predictive of TF. Positive predictive values of the HRP2 and panmalarial antigens for TF were 76.9% and 87.0%, respectively, on Day 3, 82.4% and 87.5% on Day 7, and 78.9% and 78.9% on Day 14. Negative HRP2 and panmalarial antigen results in convalescence were even less predictive of an adequate clinical response, and false-negative HRP2 and panmalarial antigen test results were found in one-sixth (6 of 37) of recrudescent infections diagnosed by microscopy among patients with late treatment failure. To reliably predict treatment outcome with rapid antigen tests, further development appears necessary to improve sensitivity for viable asexual parasites while avoiding detection of both gametocytes and persistent antigen in convalescence.

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Plasmodium falciparum:Evaluation of Lactate Dehydrogenase in Monitoring Therapeutic Responses to Standard Antimalarial Drugs in Nigeria

Cited by 61 publications

References 5 publications

Luciferase, When Fused to an N-terminal Signal Peptide, Is Secreted from Transfected Plasmodium falciparum and Transported to the Cytosol of Infected Erythrocytes

Luciferase, When Fused to an N-terminal Signal Peptide, Is Secreted from Transfected Plasmodium falciparum and Transported to the Cytosol of Infected Erythrocytes

COMPARISON OF HRP2- AND pLDH-BASED RAPID DIAGNOSTIC TESTS FOR MALARIA WITH LONGITUDINAL FOLLOW-UP IN KAMPALA, UGANDA

Detection of histidine rich protein 2 and panmalarial ICT Malaria Pf/Pv test antigens after chloroquine treatment of uncomplicated falciparum malaria does not reliably predict treatment outcome in eastern Indonesia.

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