Relapse of imported Plasmodium vivax malaria is related to primaquine dose: a retrospective study

ABSTRACT: BACKGROUND: Relapsing Plasmodium vivax infection results in significant morbidity for the individual and is a key factor in transmission. Primaquine remains the only licensed drug for prevention of relapse. To minimize relapse rates, treatment guidelines have recently been revised to recommend an increased primaquine dose, aiming to achieve a cumulative dose of [greater than or equal to]6 mg/kg, i.e. [greater than or equal to]420 mg in a 70 kg patient. The aims of this study were to characterize the epidemiology of P. vivax infection imported into Queensland Australia, to determine the rates of relapse, to investigate the use of primaquine therapy, and its efficacy in the prevention of relapse. METHODS: A retrospective study was undertaken of laboratory confirmed P. vivax infection presenting to the two major tertiary hospitals in Queensland, Australia between January 1999 and January 2011. Primaquine dosing was classified as no dose, low dose (<420 mg), high dose ([greater than or equal to]420 mg), or unknown. The dose of primaquine prescribed to patients who subsequently relapsed was compared to patients who did not relapse. RESULTS: Twenty relapses occurred following 151 primary episodes of P. vivax infection (13.2%). Relapses were confirmed among 3/21 (14.2%), 9/50 (18.0%), 1/54 (1.9%)and 7/18 (38.9%) of patients administered no dose, low dose, high dose and unknown primaquine dose respectively. High dose primaquine therapy was associated with significantly lower rates of relapse compared to patients who were prescribed low dose therapy (OR 11.6, 95% CI 1.5- 519, p = 0.005). CONCLUSIONS: Relapse of P. vivax infection is more likely in patients who received low dose primaquine therapy. This study supports the recommendations that high dose primaquine therapy is necessary to minimize relapse of P. vivax.     

Therapeutic efficacy of 3 treatment protocols for non-complicated Plasmodium falciparum malaria, Antioquia, Colombia, 2002

High resistance of Plasmodium falciparum malaria to chloroquine poses malaria as a major public health problem in Colombia. In this context, the therapeutic response of uncomplicated P. falciparum malaria patients to chloroquine (CQ), sulfadoxine/pirymethamine (SDXP) and combined therapy (SDXP/CQ) was evaluated according to the WHO/PAHO protocols of 1998. The comparisons were based on a sample of 160 patients with uncomplicated P. falciparum malaria in Turbo and Zaragoza (Antioquia, Colombia). Patients were randomly assigned each of the treatment categories. The results were statistically similar in each municipality. In Turbo percentage of treatment failure was 87.5%, 22.2% and 22.6% for CQ, SDXP and SDXP/CQ, respectively, whereas in Zaragoza, the corresponding treatment failure was 77.7%, 26.5% and 12.1%. During follow up, 50% of subjects with late treatment failure were asymptomatic in Turbo, while 33.3% were asymptomatic in Zaragoza. A high level of treatment failure occurred with CQ monotherapy, while SDXP and SDXP/CQ had acceptable levels of failure, i.e., below 25%. The high percentage of late treatment failure in asymptomatic patients may contribute to increased risk of persistent transmission.     

Pyronaridine-artesunate versus chloroquine in patients with acute Plasmodium vivax malaria: a randomized, double-blind, non-inferiority trial

Background

New antimalarials are needed for P. vivax and P. falciparum malaria. This study compared the efficacy and safety of pyronaridine-artesunate with that of chloroquine for the treatment of uncomplicated P. vivax malaria.

Methods and Findings

This phase III randomized, double-blind, non-inferiority trial included five centers across Cambodia, Thailand, India, and Indonesia. In a double-dummy design, patients (aged >3–≤60 years) with microscopically confirmed P. vivax mono-infection were randomized (1∶1) to receive pyronaridine-artesunate (target dose 7.2∶2.4 mg/kg to 13.8∶4.6 mg/kg) or chloroquine (standard dose) once daily for three days. Each treatment group included 228 randomized patients. Outcomes for the primary endpoint, Day-14 cure rate in the per-protocol population, were 99.5%, (217/218; 95%CI 97.5, 100) with pyronaridine-artesunate and 100% (209/209; 95%CI 98.3, 100) with chloroquine. Pyronaridine was non-inferior to chloroquine: treatment difference −0.5% (95%CI −2.6, 1.4), i.e., the lower limit of the 2-sided 95%CI for the treatment difference was greater than −10%. Pyronaridine-artesunate cure rates were non-inferior to chloroquine for Days 21, 28, 35 and 42. Parasite clearance time was shorter with pyronaridine-artesunate (median 23.0 h) versus chloroquine (32.0 h; p<0.0001), as was fever clearance time (median 15.9 h and 23.8 h, respectively; p = 0.0017). Kaplan-Meier estimates of post-baseline P. falciparum infection incidence until Day 42 were 2.5% with pyronaridine-artesunate, 6.1% with chloroquine (p = 0.048, log-rank test). Post-baseline P. vivax or P. falciparum infection incidence until Day 42 was 6.8% and 12.4%, respectively (p = 0.022, log rank test). There were no deaths. Adverse events occurred in 92/228 (40.4%) patients with pyronaridine-artesunate and 72/228 (31.6%) with chloroquine. Mild and transient increases in hepatic enzymes were observed for pyronaridine-artesunate.

Conclusion

Pyronaridine-artesunate efficacy in acute uncomplicated P. vivax malaria was at least that of chloroquine. As pyronaridine-artesunate is also efficacious against P. falciparum malaria, this combination has potential utility as a global antimalarial drug.

Trial registration

Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00440999","term_id":"NCT00440999"}}NCT00440999     

Radical curative efficacy of five-day regimen of primaquine for treatment of Plasmodium vivax malaria in India

For over 4 decades the antimalarial program in India has been prescribing a 5-day primaquine regimen as an antirelapse therapy to treat Plasmodium vivax malaria. In view of conflicting reports on the effectiveness of this regimen in the Indian subcontinent, and the varying prevalence of P. vivax in various ecosystems in India, the antirelapse efficacy of this regimen was evaluated in Orissa, a malaria endemic state in eastern India where P. falciparum predominates. In 723 cases of P. vivax infection treated with chloroquine alone and followed up weekly for 1 yr, the prevalence of recurrence of parasitaemia with fever was 8.6%. Among another 759 P. vivax cases treated with chloroquine and a 5-day regimen of primaquine at 15 mg/day (adult dose), the recurrence of infection was 6.5%. The difference in recurrence was not significant (P = 0.53). It is important to note that in a great majority of cases of P. vivax in this area, infection did not recur even without treatment with primaquine. This finding, that the use of the 5-day primaquine regimen with chloroquine had no significant advantage over the use of chloroquine alone, undermines the rationale of using primaquine as an antirelapse drug in forested areas with a high prevalence of P. falciparum.     

Phenotypic diagnosis of primary immunodeficiencies in Antioquia,Colombia, 1994-2002

Recurrent infections are a frequent cause of medical visits. They can be due to a heterogeneous group of dysfunctions that increase the susceptibility to pathogenic and opportunistic microorganisms, such as immunological deficiencies. To define an opportune rational treatment and to guide the molecular diagnosis of primary immunodeficiency diseases, we establish a program for the phenotypic diagnosis of these illnesses in Antioquia, Colombia, including clinical and laboratory evaluations of patients who present recurrent infections with abnormal evolution. Between August 1, 1994 and July 31, 2002, phenotypic diagnosis of primary immunodeficiency was made in 98 patients. Similar to data reported in the literature, antibody deficiencies were the most frequent (40.8%), followed by combined deficiencies (21.4%). This phenotypic characterization has allowed for appropriate treatments for each patient and, in some cases, functional and molecular studies that can lead to a definite molecular diagnosis.     

Assessment of therapeutic response of Plasmodium vivax and Plasmodium falciparum to chloroquine in a Malaria transmission free area in Colombia

In order to determine the frequency of therapeutic failures to chloroquine (CQ) in patients with malaria due to either Plasmodium falciparum or P. vivax, and to explore the usefulness of a malaria-free city as a sentinel site to monitor the emergence of drug resistance, 53 patients (44 infected with P. vivax and 9 with P. falciparum) were evaluated at the Laboratory of Parasitology, Universidad del Valle in Cali, Colombia. Patients received 25 mg/kg of CQ divided in three doses over 48 h; they were followed during 28 days according to WHO/PAHO protocols. While therapeutic failures to CQ in the P. vivax group were not detected, the proportion of therapeutic failures in the P. falciparum group was high (78%) and consistent with the reports from endemic areas in Colombia. The diverse origin of cases presenting therapeutic failure confirmed that P. falciparum resistant to CQ is widespread in Colombia, and further supports the change in the national antimalarial drug scheme. Monitoring of drug resistance in malaria free areas would be useful to identify sites requiring efficacy evaluation, and in some situations could be the most appropriate alternative to collect information from endemic areas where therapeutic efficacy studies are not feasible.     

Open-label comparative clinical study of chlorproguanil-dapsone fixed dose combination (Lapdap) alone or with three different doses of artesunate for uncomplicated Plasmodium falciparum malaria

The objective of this study was to determine the appropriate dose of artesunate for use in a fixed dose combination therapy with chlorproguanil−dapsone (CPG−DDS) for the treatment of uncomplicated falciparum malaria.

Methods

Open-label clinical trial comparing CPG−DDS alone or with artesunate 4, 2, or 1 mg/kg at medical centers in Blantyre, Malawi and Farafenni, The Gambia. The trial was conducted between June 2002 and February 2005, including 116 adults (median age 27 years) and 107 children (median age 38 months) with acute uncomplicated Plasmodium falciparum malaria. Subjects were randomized into 4 groups to receive CPG–DDS alone or plus 4, 2 or 1 mg/kg of artesunate once daily for 3 days. Assessments took place on Days 0−3 in hospital and follow-up on Days 7 and 14 as out-patients. Efficacy was evaluated in the Day 3 per-protocol (PP) population using mean time to reduce baseline parasitemia by 90% (PC90). A number of secondary outcomes were also included. Appropriate artesunate dose was determined using a pre-defined decision matrix based on primary and secondary outcomes. Treatment emergent adverse events were recorded from clinical assessments and blood parameters. Safety was evaluated in the intent to treat (ITT) population.

Results

In the Day 3 PP population for the adult group (N = 85), mean time to PC90 was 19.1 h in the CPG−DDS group, significantly longer than for the +artesunate 1 mg/kg (12.5 h; treatment difference −6.6 h [95%CI −11.8, −1.5]), 2 mg/kg (10.7 h; −8.4 h [95%CI −13.6, −3.2]) and 4 mg/kg (10.3 h; −8.7 h [95%CI −14.1, −3.2]) groups. For children in the Day 3 PP population (N = 92), mean time to PC90 was 21.1 h in the CPG−DDS group, similar to the +artesunate 1 mg/kg group (17.7 h; −3.3 h [95%CI −8.6, 2.0]), though the +artesunate 2 mg/kg and 4 mg/kg groups had significantly shorter mean times to PC90 versus CPG−DDS; 14.4 h (treatment difference −6.4 h [95%CI −11.7, −1.0]) and 12.8 h (−7.4 h [95%CI −12.9, −1.8]), respectively. An analysis of mean time to PC90 for the Day 14 PP and ITT populations was consistent with the primary analysis. Treatment emergent, drug-related adverse events were experienced in 35.3% (41/116) of adults and 70.1% (75/107) of children; mostly hematological and gastroenterological. The nature and incidence of adverse events was similar between the groups. No dose-related changes in laboratory parameters were observed. Nine serious adverse events due to any cause occurred in five subjects including two cases of hemolysis believed to be associated with drug treatment (one adult, one child). One adult died of anaphylactic shock, not associated with investigational therapy.

Conclusions

CPG–DDS plus artesunate demonstrated advantages over CPG–DDS alone for the primary efficacy endpoint (mean time to PC90) except in children for the 1 mg/kg artesunate dose. Based on a pre-defined decision matrix, the primary endpoint in the child group supported an artesunate dose of 4 mg/kg. Secondary endpoints also supported a 4 mg/kg artesunate dose to take forward into the remainder of the development program.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00519467","term_id":"NCT00519467"}}NCT00519467     

Plasmodium falciparum malaria: rosettes are disrupted by quinine, artemisinin, mefloquine, primaquine, pyrimethamine, chloroquine and proguanil.

An assay was developed measuring the disruption of rosettes between Plasmodium falciparuminfected (trophozoites) and uninfected erythrocytes by the antimalarial drugs quinine, artemisinin mefloquine, primaquine, pyrimethamine, chloroquine and proguanil. At 4 hr incubation rosettes were disrupted by all the drugs in a dose dependent manner. Artemisinin and quinine were the most effective anti-malarials at disrupting rosettes at their therapeutic concentrations with South African RSA 14, 15, 17 and The Gambian FCR-3 P. falciparum strains. The least effective drugs were proguanil and chloroquine. A combination of artemisinin and mefloquine was more effective than each drug alone. The combinations of pyrimethamine or primaquine, with quinine disrupted more rosettes than quinine alone. Quinine may be an effective drug in the treatment of severe malaria because the drug efficiently reduces the number of rosettes.     

Effects of chloroquine and sulfadoxine/pyrimethamine on gametocytes in patients with uncomplicated Plasmodium falciparum malaria in Colombia

The effect of antimalarials on gametocytes can influence transmission and the spread of drug resistance. In order to further understand this relationship, we determined the proportion of gametocyte carriers over time post-treatment in patients with uncomplicated Plasmodium falciparum malaria who were treated with either chloroquine (CQ) or sulfadoxine/pyrimethamine (SP). The overall proportion of gametocyte carriers was high (85%) and not statistically significantly different between the CQ and SP treatment groups. However, an increased risk of carrying gametocytes on day 14 of follow up (1.26 95% CI 1.10-1.45) was found among patients having therapeutic failure to CQ compared with patients having an adequate therapeutic response. This finding confirms and extends reports of increased risk of gametocytaemia among CQ resistant P. falciparum.     

Performance of OptiMAL(R) in the diagnosis of Plasmodium vivax and Plasmodium falciparum infections in a malaria referral center in Colombia

Alternative, non-microscopic methods for the diagnosis of malaria have recently become available. Among these, rapid dipstick methods stand out. One such test, OptiMAL(R), is based on the immunochromatographic detection of Plasmodium lactate dehydrogenase (pLDH) and has the capacity to detect and distinguish infections caused by P. falciparum and Plasmodium sp. This capacity is particularly important in countries where different species of Plasmodium co-exist. In this study we evaluated the performance of OptiMAL(R) in an urban referral center for malaria diagnosis. Two sets of patients were included: one (n = 112) having predetermined infections with P. falciparum or P. vivax and individuals with negative blood smears; and another consisting of all eligible consecutive patients (n = 80) consulting for diagnosis at the referral center during one month. The overall diagnostic efficiency of OptiMAL(R) for both sets of patients was 96.9%. Efficiency was higher for P. vivax (98.1%) than for P. falciparum (94.9%). These results corroborate the diagnostic utility of OptiMAL(R) in settings where P. vivax and P. falciparum co-exist and support its implementation where microscopic diagnosis is unavailable and in circumstances that exceed the capacity of the local microscopic diagnosis facility.     

Efficacy of chloroquine plus primaquine treatment and pfcrt mutation in uncomplicated falciparum malaria patients in Rangamati, Bangladesh

A combination of chloroquine (CQ) and primaquine (PQ) had been used as the first-line treatment of uncomplicated Plasmodium falciparum malaria in Rangamati, Bangladesh until the end of 2004. Doctors or medical staffs had felt that CQ plus PQ had become less effective against uncomplicated falciparum malaria patients, but that it was more effective against the minority-indigenous patients than the Bengali patients. The efficacy of CQ plus PQ and the mutation status of the CQ resistance transporter (pfcrt) gene of infecting P. falciparum were, thus, investigated for 45 uncomplicated falciparum malaria patients in Rangamati in 2004. The total failure rate was 57.8%. One or two pfcrt sequences (CIETH and SMNTH at positions 72, 74-76, and 97, mutation underlined) with K76T mutation known to be related to CQ-resistant phenotype were detected in 38 patients' blood samples. Of the 38 patients, in total 15 patients (14/25 minority-indigenous and 1/13 Bengali patients) resulted in adequate clinical and parasitological response (ACPR). There was a statistically significant difference in ACPR rate between the minority-indigenous patients and the Bengali patients. P. falciparum with mutant or resistant pfcrt (pfcrt-resistant) was detected by PCR in blood samples on day 28 for 10 ACPR minority-indigenous patients but not for the only one Bengali ACPR patient, who all were infected with pfcrt-resistant P. falciparum on day 0. The minority-indigenous patients, but not Bengalis, are suggested to be often cured by CQ plus PQ, leaving a very few parasites detectable only by PCR, even when they are infected with pfcrt-resistant P. falciparum.     

Defining Plasmodium falciparum treatment in South West Asia: a randomized trial comparing artesunate or primaquine combined with chloroquine or SP

Introduction

Antimalarial resistance has led to a global policy of artemisinin-based combination therapy. Despite growing resistance chloroquine (CQ) remained until recently the official first-line treatment for falciparum malaria in Pakistan, with sulfadoxine-pyrimethamine (SP) second-line. Co-treatment with the gametocytocidal primaquine (PQ) is recommended for transmission control in South Asia. The relative effect of artesunate (AS) or primaquine, as partner drugs, on clinical outcomes and gametocyte carriage in this setting were unknown.

Methods

A single-blinded, randomized trial among Afghan refugees in Pakistan compared six treatment arms: CQ; CQ+(single-dose)PQ; CQ+(3 d)AS; SP; SP+(single-dose)PQ, and SP+(3 d)AS. The objectives were to compare treatment failure rates and effect on gametocyte carriage, of CQ or SP monotherapy against the respective combinations (PQ or AS). Outcomes included trophozoite and gametocyte clearance (read by light microscopy), and clinical and parasitological failure.

Findings

A total of 308 (87%) patients completed the trial. Failure rates by day 28 were: CQ 55/68 (81%); CQ+AS 19/67 (28%), SP 4/41 (9.8%), SP+AS 1/41 (2.4%). The addition of PQ to CQ or SP did not affect failure rates (CQ+PQ 49/67 (73%) failed; SP+PQ 5/33 (16%) failed). AS was superior to PQ at clearing gametocytes; gametocytes were seen on d7 in 85% of CQ, 40% of CQ+PQ, 21% of CQ+AS, 91% of SP, 76% of SP+PQ and 23% of SP+AS treated patients. PQ was more effective at clearing older gametocyte infections whereas AS was more effective at preventing emergence of mature gametocytes, except in cases that recrudesced.

Conclusions

CQ is no longer appropriate by itself or in combination. These findings influenced the replacement of CQ with SP+AS for first-line treatment of uncomplicated falciparum malaria in the WHO Eastern Mediterranean Region. The threat of SP resistance remains as SP monotherapy is still common. Three day AS was superior to single-dose PQ for reducing gametocyte carriage.

Trial Registration

ClinicalTrials.gov bold>     

Multidrug-resistant Plasmodium vivax associated with severe and fatal malaria: a prospective study in Papua, Indonesia

Background

Multidrug-resistant Plasmodium vivax (Pv) is widespread in eastern Indonesia, and emerging elsewhere in Asia-Pacific and South America, but is generally regarded as a benign disease. The aim of the study was to review the spectrum of disease associated with malaria due to Pv and P. falciparum (Pf) in patients presenting to a hospital in Timika, southern Papua, Indonesia.

Methods and Findings

Data were prospectively collected from all patients attending the outpatient and inpatient departments of the only hospital in the region using systematic data forms and hospital computerised records. Between January 2004 and December 2007, clinical malaria was present in 16% (60,226/373,450) of hospital outpatients and 32% (12,171/37,800) of inpatients. Among patients admitted with slide-confirmed malaria, 64% of patients had Pf, 24% Pv, and 10.5% mixed infections. The proportion of malarial admissions attributable to Pv rose to 47% (415/887) in children under 1 y of age. Severe disease was present in 2,634 (22%) inpatients with malaria, with the risk greater among Pv (23% [675/2,937]) infections compared to Pf (20% [1,570/7,817]; odds ratio [OR] = 1.19 [95% confidence interval (CI) 1.08–1.32], p = 0.001), and greatest in patients with mixed infections (31% [389/1,273]); overall p < 0.0001. Severe anaemia (haemoglobin < 5 g/dl) was the major complication associated with Pv, accounting for 87% (589/675) of severe disease compared to 73% (1,144/1,570) of severe manifestations with Pf (p < 0.001). Pure Pv infection was also present in 78 patients with respiratory distress and 42 patients with coma. In total 242 (2.0%) patients with malaria died during admission: 2.2% (167/7,722) with Pf, 1.6% (46/2,916) with Pv, and 2.3% (29/1260) with mixed infections (p = 0.126).

Conclusions

In this region with established high-grade chloroquine resistance to both Pv and Pf, Pv is associated with severe and fatal malaria particularly in young children. The epidemiology of P. vivax needs to be re-examined elsewhere where chloroquine resistance is increasing.     

Oxidant stress in malaria as probed by stable nitroxide radicals in erythrocytes infected with Plasmodium berghei. The effects of primaquine and chloroquine

Erythrocytes from normal mice and mice infected with the malarial parasite Plasmodium berghei reduce the water-soluble spin probes 2,2,6,6-tetramethylpiperidine-4-hydroxy-N-oxyl (TEMPOL), 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) and 2,2,6,6-tetramethylpiperidine-4-keto-N-oxyl (TEMPONE) at similar rates under both air and N2 atmospheres. The ESR signal of the lipid-soluble spin probe 5-doxyl-stearate is stable on incorporation into erythrocytes from normal mice. In contrast, parasitized red cells reduce this nitroxide probe, at a rate which increases with the level of parasitemia. Inhibitors of electron transport such as KCN and NaN3, increase the rate of reduction. We propose that nitroxide reduction occurs via the electron transport chain in the parasite. The antimalarial drug primaquine causes reduction of both water-soluble and lipid-soluble spin probes. This action of primaquine is independent of its ability to release H2O2 from oxyhemoglobin, and is ascribed to the ability of primaquine to accelerate flux through the hexose monophosphate shunt. The increased production of NADPH results in increased rates of reduction of the nitroxide radicals. Methylene blue, which also increases flux through the shunt, is even more effective than primaquine at reducing the nitroxides. Chloroquine has no such effect. Parasitized mice treated with chloroquine six hours prior to ESR measurements show less nitroxide reducing capacity than do untreated mice. Chloroquine is known to decrease flux through the hexose monophosphate shunt. The metabolic influences of the two antimalarial drugs are, thus, quite different.     

Comparative susceptibility of three important malaria vectors Anopheles stephensi, Anopheles fluviatilis, and Anopheles sundaicus to Plasmodium vivax

The 3 laboratory-colonized malaria vectors, i.e., Anopheles stephensi, An. sundaicus, and An. fluviatilis, were studied for their comparative susceptibility to Plasmodium vivax sporogony. There was no significant difference in oocyst and sporozoite recruitment by these 3 species, whereas the geometric mean (GM) of the oocyst number per midgut was significantly lower in An. fluviatilis as compared with that in the other 2 species. There was no difference in the GM of oocyst between An. stephensi and An. sundaicus. Adaptability to laboratory conditions and susceptibility to plasmodial infection suggest that An. fluviatilis and An. sundaicus can also be used as a vector model for vector-parasite interaction studies.     

Plasmodium vivax: Merozoites, invasion of reticulocytes and considerations for malaria vaccine development

Several Plasmodium vivax merozoite proteins have been characterized over the past few years, including two that bind specifically to reticulocytes. Here, Mare Galinski and John Barnwell examine P. vivax merozoites and constituent molecules that are involved in host cell selection and invasion, and that also are viewed as malaria vaccine candidates. They also discuss how knowledge of the reticulocyte-binding proteins furthers the development of a conceptual framework for malaria merozoite invasion at the molecular level, not only for P. vivax, but for all species of the parasite.     

Plasmodium vivax: isotopic, PicoGreen, and microscopic assays for measuring chloroquine sensitivity in fresh and cryopreserved isolates

In vitro susceptibility tests provide information on the intrinsic response of Plasmodium vivax to antimalarials, free from confounding factors such as host immunity or relapse. This study examined the utility of radioisotope and PicoGreen assays as alternatives to the traditional microscopic examination for assessing response of P. vivax to antimalarial drugs. There was no significant difference in the mean chloroquine IC(50) of P. vivax (n=40) as determined by the microscopic (33.4 ng/ml), isotopic (33.6 ng/ml), and PicoGreen (39.1 ng/ml) assays, respectively (F=0.239, df=2, 51, and p=0.788). However measurement of IC(50)s by the microscopic method was slightly more successful in producing valid assays (57%), compared to the isotopic (32.5%) and PicoGreen (45.5%) methods. In a paired comparison of 20 fresh and cryopreserved isolates as examined by the microscopic method, there were no significant differences between the mean IC(50) responses (T=1.58, df=15, and p=0.34). Detailed methodologies for the short time culture of field and cryopreserved P. vivax are described. Although the microscopic in vitro assay provides a useful method for characterizing the drug susceptibility phenotype of P. vivax isolates, its utility is limited by a laborious methodology and need for highly skilled microscopists. Future efforts should focus on further development of high throughput assays such as the PicoGreen assay as described in this study.     

The role of nitric oxide and lipid peroxidation in patients with Plasmodium vivax malaria

In this study, we investigated the role of nitric oxide metabolism and lipid peroxidation in patients with P. vivax malaria. The levels of nitrite and nitrate were analyzed using a procedure based on the Griess reaction and malondialdehyde levels which index of lipid peroxidation was determined by thiobarbituric acid reaction. The levels of nitrite/nitrate and malondialdehyde in patients were higher than controls and found to be statistically significant (p < 0.001). We performed this study to determine whether nitric oxide and lipid peroxidation is produced during blood-stage P. vivax malaria. This present study shows that lipid peroxidation occurs in P. vivax malaria. The levels of nitric oxide are associated with lipid peroxidation in this disease.