Background: Investigations of Plasmodium vivax are restricted to samples collected from infected persons or primates, because this parasite cannot be maintained in in vitro cultures. Contamination of P. vivax isolates with host leukocytes and platelets is detrimental to a range of ex vivo and molecular investigations. Easy-to-produce CF11 cellulose filters have recently provided us with an inexpensive method for the removal of leukocytes and platelets. This contrasted with previous reports of unacceptably high levels of infected red blood cell (IRBC) retention by CF11. The aims of this study were to compare the ability of CF11 cellulose filters and the commercial filter Plasmodipur at removing leukocyte and platelet, and to investigate the retention of P. vivax IRBCs by CF11 cellulose filtration.

The novel organometallic chloroquine analog ferroquine (SSR 97193) is effective against chloroquineresistant Plasmodium falciparum. The ex vivo efficacy of ferroquine against Plasmodium vivax isolates was tested. Ferroquine has a potent ex vivo effect on P. vivax schizont maturation (median 50% inhibitory concentration, 15 nM; n ‫؍‬ 42). No significant cross-sensitivity between ferroquine and other antimalarials was detected. This drug may be a suitable replacement for chloroquine in the treatment of drug-resistant P. vivax malaria.Plasmodium vivax malaria is an underrecognized cause of morbidity in tropical countries outside of Africa. The emergence of resistance to antimalarials and severe forms of the disease P. vivax malaria poses a significant global health threat (1, 3). Despite the spread of resistance, chloroquine (CQ) remains the most common first-line treatment for P. vivax malaria in pregnant and nonpregnant individuals, being inexpensive and relatively safe. Additionally, the extended half-life of CQ (ϳ30 days) suppresses the first relapse. Although CQresistant (CQR) P. vivax is generally sensitive to artemisinin therapy, the rapid elimination of artemisinin compounds will prevent relapses, a common feature of P. vivax malaria.Ferroquine (FQ) (SR97193; ferrocene CQ: 7-chloro-4{[2-(N,N-dimethylaminomethyl)]-N-methylferrocenylamino}quin-oline) a novel 4-aminoquinoline, is thought to have a metabolic profile similar to that of CQ (7) and possess antimalarial activity in the low nanomolar range when tested against CQR P. falciparum clones (2, 6, 9) and multidrug-resistant isolates of P. falciparum from the Thailand-Myanmar border (4). However, it is not known whether P. vivax is sensitive to FQ. This study aimed to examine the effect of FQ (SAR97193) and its demethylated metabolite (FQM; SR97213) on the schizont maturation of clinical isolates of P. vivax.One hundred ten P. vivax isolates were collected from October 2006 to April 2009 in the Mae Sot region of Tak Province in northwestern Thailand. All samples were collected from patients with acute P. vivax malaria (with a monospecies parasitemia of Ͼ100/500 white blood cells) attending the clinics of the Shoklo Malaria Research Unit (SMRU). After written consent was obtained, blood samples were collected by venipuncture in 5-ml lithium-heparinized tubes and arrived at the culture lab at SMRU within 5 h of collection at room temperature. Samples with more than 80% early trophozoites present were chosen for drug sensitivity testing. After platelets and leukocytes were removed from the isolates (17), P. vivax sensitivity was tested as previously described (14, 15). Stage-specific drug activity assays were carried out as previously described (16). The stage specificity experiment was conducted for CQ and FQ in duplicate wells on 10 isolates of P. vivax and P. falciparum. Plates were quality controlled by testing with a strain of P. falciparum (K1) with a known sensitivity profile.Dose-response curves and IC 50 (50% inhibitory concentration) values were calc...

Plasmodium species ex vivo sensitivity assay protocols differ in the requirement for leukocyte removal before culturing. This study shows that the presence of leukocytes significantly increases the 50% inhibitory concentration (IC 50 ) of P. vivax and P. falciparum to artesunate and chloroquine relative to results with the paired leukocyte-free treatment. Although leukocyte removal is not an essential requirement for the conduct of ex vivo assays, its use has important implications for the interpretation of temporal and spatial antimalarial sensitivity data.Plasmodium falciparum and Plasmodium vivax ex vivo sensitivity assays are an important adjunct to in vivo antimalarial resistance detection. Ex vivo antimalarial sensitivity testing (a specialized form of in vitro sensitivity testing) involves the phenotypic comparison of freshly isolated Plasmodium spp. cultured to a specified endpoint in the presence and absence of antimalarials. Ex vivo assays provide information on the intrinsic sensitivity of malaria parasites, free from confounding variations in patient immune status. Furthermore, in the case of P. vivax, ex vivo assays are also free from the problematic differentiation between relapse, reinfection, or recrudescence which is needed for in vivo testing.Since the development of ex vivo sensitivity antimalarial assays in the 1960s (20), there have been various modifications to the basic method, including changes in the blood medium mixture (BMM) composition (variations in hematocrit, culture medium used, and percent serum added), BMM volume (i.e., 50 l versus 200 l), and even incubation gas environment (candle jar versus commercial gas). Another important modification to ex vivo sensitivity antimalarial assays is the depletion of leukocytes from the patient blood sample prior to addition to the culture medium (26). It was supposed that the depletion of patient leukocytes and platelets removes the possibility that variations in host immune status (i.e., variations in leukocyte count) will confound the result of the sensitivity assay. Additionally, the removal of leukocytes aids in the microscopic examination of thick films which are used to assess the antimalarial effect on parasite development (26). Furthermore, thick and thin films made from filtered isolates produce "noisefree" images better suited for digital analysis. Despite the growing trend toward complete leukocyte depletion prior to culture (1, 8, 11, 13, 14, 17, 21-23, 25, 27), some groups still utilize patient samples with no or only partial depletion of leukocytes (removal of the buffy coat only) (5,6,15,16,28). Despite this major dichotomy in ex vivo assay methodology, it is not known if the removal of leukocytes influences the antimalarial sensitivity of the parasite or the ex vivo growth of P. vivax or P. falciparum. Consequently, the main objective of this study was to investigate whether the presence of leukocytes affects the IC 50 (50% inhibitory concentration) of chloroquine and artesunate against P. vivax and P. falciparum. It was also ...

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