A simple double-site sandwich enzyme-linked immunosorbent assay (ELISA) for Plasmodium falciparum in vitro drug sensitivity tests based on measuring histidine-rich protein 2 (HRP2) is presented. The ELISA uses two commercial monoclonal antibodies and provides a drastically cheaper alternative to the test kits previously used in the HRP2 drug sensitivity test. The assay is simple to establish and perform. The sensitivity is comparable and the drug sensitivity results very closely match those obtained with the commercial ELISA kits (R 2 ‫؍‬ 0.979; P < 0.001; mean log difference at the 50% inhibitory concentration ‫؍‬ 0.07).Malaria in vitro drug sensitivity assays are largely based on measuring parasite growth in cultures exposed to different antimalarial drug concentrations. Enzyme-linked immunosorbent assays (ELISAs) can provide very sensitive measures of parasite growth by quantifying biomolecules produced during parasite development (1), such as histidine-rich protein 2 (HRP2) or parasite lactate dehydrogenase (3, 4). Although very convenient and user friendly, commercial test kits are relatively expensive. We therefore sought to develop a generic antigen capture HRP2 ELISA in order to drastically reduce the cost of the HRP2 malaria drug sensitivity assay.Study samples. The Plasmodium falciparum culture samples used for validating the new ELISA originated from 18 symptomatic adult outpatients at the malaria clinics in Mae Sot, So Oh, and Chedi Koh in Tak province, western Thailand. The study protocols were approved by the appropriate ethical review boards, and written informed consent was obtained from all study participants. The parasite densities ranged from 0.01 to 0.95% of infected red blood cells (geometric mean, 0.27%).Culture. The culturing was performed as previously described (5). In brief, the fresh P. falciparum parasite isolates were cultured in the presence of serial dilutions of antimalarial drugs (dihydroartemisinin [DHA], mefloquine [MEF], quinine [QNN], and chloroquine [CQ]) at 1.5% hematocrit in RPMI 1640 with 0.5% Albumax I (Gibco, Bangkok, Thailand) without freezing, washing, diluting, adding serum, or preculturing. After 72 h of culturing, the plates were frozen and stored at Ϫ20°C.Antibodies. Two commercial monoclonal antibodies (Immunology Consultants Laboratory Inc., Newberg, OR) directed against P. falciparum-specific HRP2 were employed: MPFM-45A (MPFM-55A), an immunoglobulin M (IgM) antibody used as the capture antibody, and MPFG-45P (MPFG-55P), a horseradish peroxidase-conjugated IgG antibody used as the indicator antibody.HRP2 double-site antigen capture ELISA. High-binding 96-well ELISA plates (Costar 3590; Corning Inc., NY) were coated with 100 l/well of a 1.0-g/ml solution of anti-HRP2 IgM antibody solution (MPFM-45A) in phosphate-buffered saline (PBS). Then, the plates were sealed and incubated overnight at 4°C. The supernatant was discarded, and plates were saturated for 2 h with 200 l/well of a 2% bovine serum albumin (Sigma-Aldrich, A9647) solution in PBS. The well supernatant...

With the spread of antimalarial drug resistance, simple and reliable tools for the assessment of antimalarial drug resistance, particularly in endemic regions and under field conditions, have become more important than ever before. We therefore developed a histidine-rich protein 2 (HRP2)-based drug sensitivity assay for testing of fresh isolates of Plasmodium falciparum in the field. In contrast to the HRP2 laboratory assay, the field assay uses a procedure that further simplifies the handling and culturing of malaria parasites by omitting centrifugation, washing, the use of serum, and dilution with uninfected red blood cells. A total of 40 fresh Plasmodium falciparum isolates were successfully tested for their susceptibility to dihydroartemisinin, mefloquine, quinine, and chloroquine (50% inhibitory concentration [IC50] = 3.43, 61.89, 326.75, and 185.31 nM, respectively). Results very closely matched those obtained with a modified World Health Organization schizont maturation assay (R2 = 0.96, P < 0.001; mean log difference at IC50 = 0.054).

In terms of drug resistance Bangladesh acts as an important gateway to the Indian Subcontinent. However, little is known about the current status of drug resistance in this country. The aim of this study was therefore to determine the therapeutic efficacy as well as in vitro drug sensitivity of quinine for 3 days plus a single dose of sulfadoxine/pyrimethamine (Q3F), an affordable alternative to the previously used chloroquine, for the treatment of uncomplicated falciparum malaria. Sixty-three patients were enrolled in the study; the overall cure rate in a 42-day follow-up after PCR adjustment was 87.3% (95% CI: 77.6-94.1). One patient was classified as early treatment failure (1.7%, 95% CI: 0.0-8.9%); 6 patients (10%; 95% CI: 3.8-20.5%) had late treatment failures within a median time of 27 days. HRP2 in vitro drug sensitivity tests were performed on all samples. Significantly higher (P = 0.008) in vitro IC(50)s for pyrimethamine in treatment failures reflect the somewhat compromised drug sensitivity to this drug. These data suggest that the combination of 3 days of quinine with a single dose of sulfadoxine/pyrimethamine is an interesting and affordable alternative as long as or whenever ACT is not available.

Drug resistance in Plasmodium falciparum has turned into a major problem throughout the malaria endemic regions of the world. Surveillance of antimalarial drug resistance is therefore essential for early changes in drug policies. In the course of this first major application of the new field-deployable histidine-rich protein 2 (HRP2) in vitro drug sensitivity assay, we investigated the current status of drug sensitivity to the standard antimalarial drugs dihydroartemisinin, mefloquine, quinine, and chloroquine in an area with an exceptionally high level of multidrug resistance in Thailand. The geometric mean of 50 and 90% inhibitory concentrations determined for 44 successfully tested samples by the HRP2 assay were comparable to earlier published results from the same area suggesting relatively little change in the local drug resistance pattern during the past years. The HRP2 field test was found to be highly sensitive and simple enough to be entirely performed in the field.

Particularly in Southeast Asia drug resistance has become a major constraint in the treatment of falciparum malaria. So far relatively little is known about the current status of drug resistance in Bangladesh. The aim of this study was therefore to determine the in vitro drug susceptibility of Plasmodium falciparum in south-eastern Bangladesh. In the HRP2 in vitro drug sensitivity assay the tested isolates demonstrated a relatively high sensitivity to dihydroartemisinine (IC50 = 1.33 nM; 95% CI: 1.08-1.63; IC90 = 2.65 nM; 95% CI: 2.13-3.29), mefloquine (IC50 = 11.26 nM, 95% CI: 9.75-13.0; IC90 = 19.55 nM, 95% CI: 15.73-24.29) and quinine (IC50 = 73.24 nM, 95% CI: 65.26-82.21; IC90 = 157.75 nM, (95% CI: 134.16-185.5) thus being significantly more sensitive to mefloquine and quinine than isolates from Thailand. Chloroquine (IC50 = 93.06 nM, 95% CI: 80.38-107.76; IC90 = 214.76 nM, 95% CI: 175.64-262.62) sensitivity was highly compromised with inhibitory concentrations reaching levels comparable to Thailand. Therefore this drug should not be used in the treatment of falciparum malaria in this region. Despite compromised in vitro drug sensitivity to sulfadoxine/pyrimethamine, in clinical studies the combination of sulfadoxine (IC50 = 40.46 microM, 95% CI: 31.15-51.97; IC90 = 173.48 microM, 95% CI: 120.78-249.17) and pyrimethamine (IC50 = 1.7 microM, 95% CI: 1.25-2.3; IC90 = 4.83 microM, 95% CI: 3.17-7.37) with quinine proved to be an interesting option for treating uncomplicated falciparum malaria in Bangladesh.