Improved Assessment of Plasmodium vivax Response to Antimalarial Drugs by a Colorimetric Double-Site Plasmodium Lactate Dehydrogenase Antigen Capture Enzyme-Linked Immunosorbent Assay

Abstract

The occurrence of Plasmodium vivax resistance to chloroquine has been reported in several countries of Asia and South America. However, the resistance of P. vivax is insufficiently documented for three reasons: it has received far less attention than P. falciparum; in vivo investigations are handicapped by the existence of hypnozoites, which make it difficult to distinguish between recrudescences due to drug failure and relapses due to dormant forms in the liver; and in vitro studies are greatly limited by the poor growth of P. vivax. We report on the adaptation to P. vivax of a colorimetric double-site Plasmodium lactate dehydrogenase antigen capture enzyme-linked immunosorbent assay previously developed for P. falciparum. The assay proved remarkably sensitive, as under optimal conditions it could detect P. vivax parasitemia levels as low as 10 ؊8 . The technique, which relies on the detection of protein synthesis by the parasite, yielded steep drug-response curves, leading to the precise determination of the 50% inhibitory concentrations for a high proportion of isolates. Chloroquine-resistant parasites were identified in an area where this phenomenon had been documented by in vivo methods. Thus, the results indicate that the in vitro susceptibility of P. vivax can now be monitored easily and efficiently. The data suggest that the threshold of resistance is similar to that of P. falciparum, i.e., in the range of 100 nM for chloroquine and 15 nM for pyronaridine. However, further studies are required to precisely define the cutoff for resistance and the sensitivity to each drug.The resistance of human malaria parasites to antimalarial compounds has become of considerable concern, particularly in view of the fast speed of emergence of resistant parasites, the fast spread of resistant parasites, and the shortage of novel classes of antimalarial drugs. The vast majority of studies have so far dealt with Plasmodium falciparum, the species responsible for most of the fatalities from malaria.However, over the past decade, several reports have documented the emergence of resistance of P. vivax, at least to chloroquine. Although P. vivax seldom kills, it is nevertheless as prevalent as P. falciparum and carries a morbidity load as large as that of P. falciparum (16,21).The first case of P. vivax resistance to chloroquine was reported from Papua New Guinea in 1989 (22). Further cases were observed as well in Indonesia (1,8,25,26,29,30), Myanmar (15, 17), India (12, 28), the Philippines (2), and Thailand (14), as well as in South America (11,18,23), thereby indicating that it may be a worldwide, yet underestimated, problem.The prevalence of P. vivax resistance is indeed most likely widely underestimated for two reasons: in contrast to P. falciparum, there is no method for the cultivation of P. vivax as effective as that for the cultivation of P. falciparum that can be used for in vitro studies; conversely, under in vivo conditions, the reemergence of parasites following treatment is usually attributed in primary health c...

Plasmodium vivax Susceptibility to Ferroquine

“…Until recently, it was assumed that P. vivax populations on the Thailand-Myanmar border were universally sensitive to non-antifolate antimalarials. However, a number of ex vivo studies on P. vivax in this region have reported an increased number of isolates with significantly reduced antimalarial sensitivity to CQ and mefloquine (8,10). Although our data show that most isolates are relatively sensitive to CQ, 1 of the 65 isolates tested for CQ had an IC 50 of 170 nM.…”

Determinants of In Vitro Drug Susceptibility Testing of Plasmodium vivax

“…Applying a criterion of selecting isolates with a majority of rings irrespective of the duration of the assay (i.e., quadrants B and C in Table 5) yielded a significantly higher population mean (142 nM). These data have relevance for the development of nonmicroscopic methods for determining the in vitro susceptibility of P. vivax, such as enzyme-linked immunosorbent sensitivity or DNA detection (5). The absence of chloroquine sensitivity at the trophozoite stage combined with minimal reinvasion and parasitemia, generally less than 0.5%, will both contribute to a poor signal-to-background ratio, which are likely to undermine the sensitivity and reproducibility of such assays (11).…”

Diagnosis and Treatment of Plasmodium vivax Malaria

“…Despite its limitations, the current schizont maturation assay has demonstrated utility in discriminating parasite populations with different degrees of CQR, [83][84][85] characterizing drug susceptibility profiles of P. vivax to commonly used antimalarial drugs, 86,87 and screening susceptibility to novel therapeutic agents. [88][89][90][91][92][93] Development of methods capable of sustaining P. vivax in continuous in vitro culture will transform the current ex vivo assay, accommodating cryopreservation of field isolates to reduce the reliance on the analysis of fresh isolates.…”