Bryan L. Smith

Evidence of Artemisinin-Resistant Malaria in Western Cambodia

To the Editor: Although artemisinins are potent and rapidly acting antimalarial drugs, their widespread use for treating patients with Plasmodium falciparum malaria raises the question of emerging drug resistance.1,2 Artemisinin monotherapy should not be used in areas where malaria is endemic; it requires an extended administration period and may lead to treatment failure, most frequently because of problems with compliance. Recent reports of high failure rates associated with artemisinin-based combination therapy, as well as in vitro drug-susceptibility data, suggest the possibility of clinical artemisinin resistance along the Thai–Cambodian border.3,4 We studied the potential emergence of artemisinin resistance using .xa0.xa0.

Artemisinin Resistance in Cambodia: A Clinical Trial Designed to Address an Emerging Problem in Southeast Asia

BACKGROUNDnIncreasing rates of failure of artemisinin-based combination therapy have highlighted the possibility of emerging artemisinin resistance along the Thai-Cambodian border. We used an integrated in vivo-in vitro approach to assess the presence of artemisinin resistance in western Cambodia. This article provides additional data from a clinical trial that has been published in The New England Journal of Medicine.nnnMETHODSnNinety-four adult patients from Battambang Province, western Cambodia, who presented with uncomplicated falciparum malaria were randomized to receive high-dose artesunate therapy (4 mg/kg/day orally for 7 days) or quinine-tetracycline. Plasma concentrations of dihydroartemisinin, in vitro drug susceptibility, and molecular markers were analyzed. Cases meeting all the following criteria were classified as artemisinin resistant: failure to clear parasites within 7 days of treatment or reemergence of parasites within 28 days of follow-up; adequate plasma concentrations of dihydroartemisinin; prolonged parasite clearance; and increased in vitro drug susceptibility levels for dihydroartemisinin.nnnRESULTSnTwo (3.3%) of 60 artesunate-treated patients were classified as artemisinin resistant. Their parasite clearance times were prolonged (133 and 95 h, compared with a median of 52.2 h in patients who were cured). These patients had 50% inhibitory concentrations of dihydroartemisinin that were almost 10 times higher than the reference clone W2. Resistance did not appear to be mediated by the pfmdr1 copy number or selected PfATPase6 polymorphisms previously proposed to confer artemisinin resistance.nnnCONCLUSIONnArtemisinin resistance has emerged along the Thai-Cambodian border. The potentially devastating implications of spreading resistance to a drug that currently has no successor call for further studies of this emerging problem.nnnCLINICAL TRIAL REGISTRATIONnClinicalTrials.gov identifier NCT00479206.

Malaria and other vector-borne infection surveillance in the U.S. Department of Defense Armed Forces Health Surveillance Center-Global Emerging Infections Surveillance program: review of 2009 accomplishments

Vector-borne infections (VBI) are defined as infectious diseases transmitted by the bite or mechanical transfer of arthropod vectors. They constitute a significant proportion of the global infectious disease burden. United States (U.S.) Department of Defense (DoD) personnel are especially vulnerable to VBIs due to occupational contact with arthropod vectors, immunological naiveté to previously unencountered pathogens, and limited diagnostic and treatment options available in the austere and unstable environments sometimes associated with military operations. In addition to the risk uniquely encountered by military populations, other factors have driven the worldwide emergence of VBIs. Unprecedented levels of global travel, tourism and trade, and blurred lines of demarcation between zoonotic VBI reservoirs and human populations increase vector exposure. Urban growth in previously undeveloped regions and perturbations in global weather patterns also contribute to the rise of VBIs. The Armed Forces Health Surveillance Center-Global Emerging Infections Surveillance and Response System (AFHSC-GEIS) and its partners at DoD overseas laboratories form a network to better characterize the nature, emergence and growth of VBIs globally. In 2009 the network tested 19,730 specimens from 25 sites for Plasmodium species and malaria drug resistance phenotypes and nearly another 10,000 samples to determine the etiologies of non-Plasmodium species VBIs from regions spanning from Oceania to Africa, South America, and northeast, south and Southeast Asia. This review describes recent VBI-related epidemiological studies conducted by AFHSC-GEIS partner laboratories within the OCONUS DoD laboratory network emphasizing their impact on human populations.

Tafenoquine and NPC-1161B require CYP 2D metabolism for anti-malarial activity: implications for the 8-aminoquinoline class of anti-malarial compounds

BackgroundTafenoquine (TQ) is an 8-aminoquinoline (8AQ) that has been tested in several Phase II and Phase III clinical studies and is currently in late stage development as an anti-malarial prophylactic agent. NPC-1161B is a promising 8AQ in late preclinical development. It has recently been reported that the 8AQ drug primaquine requires metabolic activation by CYP 2D6 for efficacy in humans and in mice, highlighting the importance of pharmacogenomics in the target population when administering primaquine. A logical follow-up study was to determine whether CYP 2D activation is required for other compounds in the 8AQ structural class.MethodsIn the present study, the anti-malarial activities of NPC-1161B and TQ were assessed against luciferase expressing Plasmodium berghei in CYP 2D knock-out mice in comparison with normal C57BL/6 mice (WT) and with humanized/CYP 2D6 knock-in mice by monitoring luminescence with an in vivo imaging system. These experiments were designed to determine the direct effects of CYP 2D metabolic activation on the anti-malarial efficacy of NPC-1161B and TQ.ResultsNPC-1161B and TQ exhibited no anti-malarial activity in CYP 2D knock-out mice when dosed at their ED100 values (1xa0mg/kg and 3xa0mg/kg, respectively) established in WT mice. TQ anti-malarial activity was partially restored in humanized/CYP 2D6 knock-in mice when tested at two times its ED100.ConclusionsThe results reported here strongly suggest that metabolism of NPC-1161B and TQ by the CYP 2D enzyme class is essential for their anti-malarial activity. Furthermore, these results may provide a possible explanation for therapeutic failures for patients who do not respond to 8AQ treatment for relapsing malaria. Because CYP 2D6 is highly polymorphic, variable expression of this enzyme in humans represents a significant pharmacogenomic liability for 8AQs which require CYP 2D metabolic activation for efficacy, particularly for large-scale prophylaxis and eradication campaigns.

Prolonged Protection Provided by a Single Dose of Atovaquone-Proguanil for the Chemoprophylaxis of Plasmodium falciparum Malaria in a Human Challenge Model

BACKGROUNDnWe conducted a randomized, placebo-controlled, double-blind trial to establish the efficacy of atovaquone-proguanil to prevent malaria with the goal of simulating weekly dosing in a human Plasmodium falciparum challenge model.nnnMETHODSnThirty volunteers randomly received 1 of the following dose regimens: (1) 250 milligrams of atovaquone and 100 milligrams of proguanil (250/100 milligrams) 1 day prior to infectious mosquito challenge (day -1), (2) 250/100 milligrams on day 4 after challenge, (3) 250/100 milligrams on day -7, (4) 500 milligrams of atovaquone and 200 milligrams of proguanil (500/200 milligrams) on day -7 or, (5) 1000 milligrams of atovaquone and 400 milligrams of proguanil (1000/400 milligrams) on day -7. All regimens included matching placebo such that all volunteers received identical pill numbers. Six volunteers served as open-label infectivity controls. Volunteers underwent mosquito sporozoite challenge with P. falciparum 3D7 strain. Follow-up consisted of serial microscopy and close clinical monitoring for 90 days.nnnRESULTSnSix of 6 infectivity controls developed parasitemia as expected. Two of 5 evaluable volunteers receiving 250/100 milligrams 7 days prior to challenge and 1 of 6 volunteers receiving 1000/400 milligrams 7 days prior to challenge were microscopically diagnosed with malaria. All other volunteers were protected. Atovaquone exposure (area under the curve) during liver stage development was low in 2 of 3 volunteers with prophylactic failure (423 and 199 ng/mL × days compared with a mean for protected volunteers of 1903 ng/mL × days), as was peak concentration (165 and 81 ng/mL compared with a mean of 594 ng/mL in volunteers with prophylactic success). Elimination half-life was short in volunteers with prophylactic failure (2.4, 2.0, and 3.3 days compared with a mean of 4.1 days in volunteers with prophylactic success).nnnCONCLUSIONSnSingle-dose atovaquone-proguanil provides effective malaria chemoprophylaxis against P. falciparum challenge at dosing intervals supportive of weekly dosing. Postexposure prophylaxis 4 days after challenge was 100% effective.

Pharmacokinetics and Pharmacodynamics of Oral Artesunate Monotherapy in Patients with Uncomplicated Plasmodium falciparum Malaria in Western Cambodia

ABSTRACT Artemisinin-resistant malaria along the Thailand-Cambodian border is an important public health concern, yet mechanisms of drug action and their contributions to the development of resistance are poorly understood. The pharmacokinetics and pharmacodynamics of oral artesunate monotherapy were explored in a dose-ranging trial in an area of emerging artesunate resistance in western Cambodia. We enrolled 143 evaluable subjects with uncomplicated Plasmodium falciparum malaria in an open label study of directly observed artesunate monotherapy at 3 dose levels (2, 4, and 6 mg/kg of body weight/day) for 7 days at Tasanh Health Center, Tasanh, Cambodia. Clinical outcomes were similar among the 3 groups. Wide variability in artesunate and dihydroartemisinin concentrations in plasma was observed. No significant dose-effect or concentration-effect relationships between pharmacokinetic (PK) and parasite clearance parameters were observed, though baseline parasitemia was modestly correlated with increased parasite clearance times. The overall parasite clearance times were prolonged compared with the clearance times in a previous study at this site in 2006 to 2007, but this did not persist when the evaluation was limited to subjects with a comparable artesunate dose (4 mg/kg/day) and baseline parasitemia from the two studies. Reduced plasma drug levels with higher presentation parasitemias, previously hypothesized to result from partitioning into infected red blood cells, was not observed in this population with uncomplicated malaria. Neither in vitro parasite susceptibility nor plasma drug concentrations appeared to have a direct relationship with the pharmacodynamic (PD) effects of oral artesunate on malaria parasites. While direct concentration-effect relationships were not found, it remains possible that a population PK modeling approach that allows modeling of greater dose separation might discern more-subtle relationships.

A retrospective analysis of the protective efficacy of tafenoquine and mefloquine as prophylactic anti-malarials in non-immune individuals during deployment to a malaria-endemic area

BackgroundIn 2000/2001, the Australian Defense Forces (ADF), in collaboration with SmithKline Beecham and the United States Army, conducted a field trial to evaluate the safety, tolerability and efficacy of tafenoquine and mefloquine/primaquine for the prophylaxis of malaria amongst non-immune Australian soldiers deployed to East Timor (now called Timor Leste) for peacekeeping operations. The lack of a concurrent placebo control arm prevented an internal estimate of the malaria attack rate and so the protective efficacy of the study regimens was not determined at the time.MethodsIn a retrospective analysis of the trial results, the all species malaria attack rate was estimated for the prophylactic phase of the study which was defined as the period between administration of the first prophylactic dose and the first dose of post-deployment medication. First, the Plasmodium vivax attack rate was estimated during the prophylactic phase of the deployment by adjusting the observed P. vivax relapse rate during post-deployment to account for the known anti-relapse efficacies (or effectiveness) of the study medications (determined from prior studies). The all species malaria attack rate (P. vivax and Plasmodium falciparum) was then determined by adjusting the P. vivax attack rate based on the ratio of P. falciparum to P. vivax observed during prior ADF deployments to Timor Leste. This estimated all species malaria attack rate was then used as the ‘constant estimated attack rate’ in the calculation of the protective efficacy of tafenoquine and mefloquine during the prophylactic phase of the deployment.ResultsThe estimated attack rate during the prophylactic phase of the study was determined to be 7.88%. The protective efficacies of tafenoquine and mefloquine, with corresponding 95% confidence intervals (95% CI), were determined to be 100% (93%-100%) and 100% (79%-100%) respectively.ConclusionsThe protective efficacy of tafenoquine (200xa0mg per day for three days, followed by weekly 200xa0mg maintenance doses) is similar to that of the weekly standard of care (mefloquine, 250xa0mg).

Plasmodium falciparum Gametocyte Carriage Is Associated with Subsequent Plasmodium vivax Relapse after Treatment

Mixed P. falciparum/P. vivax infections are common in southeast Asia. When patients with P. falciparum malaria are treated and followed for several weeks, a significant proportion will develop P. vivax malaria. In a combined analysis of 243 patients recruited to two malaria treatment trials in western Cambodia, 20/43 (47%) of those with P. falciparum gametocytes on admission developed P. vivax malaria by Day 28 of follow-up. The presence of Pf gametocytes on an initial blood smear was associated with a 3.5-fold greater rate of vivax parasitemia post-treatment (IRRu200a=u200a3.5, 95% CI 2.0–6.0, p<0.001). The increased rate of post-treatment P. vivax infection persisted when correlates of exposure and immunity such as a history of malaria, male gender, and age were controlled for (IRRu200a=u200a3.0, 95% CI 1.9–4.7, p<0.001). Polymerase chain reaction (PCR) confirmed that only a low proportion of subjects (5/55 or 9.1%) who developed vivax during follow-up had detectable Pv parasites in the peripheral blood at baseline. Molecular detection of falciparum gametocytes by reverse transcriptase PCR in a subset of patients strengthened the observed association, while PCR detection of Pv parasitemia at follow-up was similar to microscopy results. These findings suggest that the majority of vivax infections arising after treatment of falciparum malaria originate from relapsing liver-stage parasites. In settings such as western Cambodia, the presence of both sexual and asexual forms of P. falciparum on blood smear at presentation with acute falciparum malaria serves as a marker for possible occult P. vivax coinfection and subsequent relapse. These patients may benefit from empiric treatment with an 8-aminoquinolone such as primaquine.

Intravenous Artesunate for the Treatment of Severe and Complicated Malaria in the United States: Clinical Use Under an Investigational New Drug Protocol

Context In the United States, the only U.S. Food and Drug Administrationapproved drug for treatment of severe malaria is quinidine gluconate, which has substantial adverse effects and limited availability. Contribution Among patients with severe malaria who could not receive quinidine, artesunate had an acceptable efficacy and safety profile. No deaths occurred, and most adverse events were considered to be due to malaria rather than artesunate. Caution The study was not a randomized trial of artesunate versus other therapies but rather an investigational new drug protocol. Implication In patients with severe malaria, artesunate seems to be an attractive alternative to quinidine. Approximately 1500 cases of malaria are reported annually in the United States (1). These cases are not endemic to the United States but occur in persons with a history of recent travel to or residence in malaria-endemic areas. Roughly 10% of U.S. malaria cases are considered severe by World Health Organization (WHO) criteria (2, 3), and most are caused by Plasmodium falciparum (47). Patients with severe malaria are at risk for life-threatening complications, including renal failure, the acute respiratory distress syndrome (ARDS), severe hemolytic anemia, and cerebral malaria. Among these, cerebral malaria is arguably the most dire because it can present rapidly, often with minimal warning signs, and has a mortality rate of 15% to 30% with treatment and 100% without treatment (2, 4, 810). The only U.S. Food and Drug Administration (FDA)approved treatment for severe malaria in the United States is quinidine, which is a stereoisomer of quinine that was once routinely used in hospitals as a cardiac antiarrhythmic medication (11, 12). With the development of newer antiarrhythmic agents, many hospitals have removed quinidine from their pharmacies (13, 14). Even when quinidine can be obtained, its intravenous regimen is complex and requires dosage adjustments and careful monitoring in an intensive care unit (ICU) (1517). Artemisinin derivatives have been studied for their antimalarial properties since the early 1970s (18), with a mechanism of action hypothesized to involve the production of oxygen radicals that interfere with parasite function (19). Among the artemisinins, artesunate is the most widely used because it can be delivered intravenously with a reliable pharmacokinetic profile (20, 21). Artesunate is a lifesaving drug that kills most malaria parasites very rapidly; further, any residual parasites are eliminated by postartesunate treatment with a follow-on oral antimalarial drug (2). Artesunate has been proven superior to quinine against malaria in 2 large studies in endemic regions (9, 10), and the drug is currently recommended as the first-line treatment for severe malaria by the WHO (2). In the developing world, artesunate is now being used in the initial treatment of severe and complicated malaria caused by P. falciparum (22). The product in use, however, is not manufactured according to current good manufacturing practice (CGMP) guidelines and is not FDA-approved for use in the United States (22). Since 2004, the Walter Reed Army Institute of Research has been developing a novel CGMP formulation of intravenous artesunate that has pharmacokinetics similar to preparations that do not follow CGMP (21, 23). This drug has been successfully tested in phase 1 (21, 24) and phase 2 (23) trials. In 2007, domestically manufactured CGMP artesunate from the Walter Reed Army Institute of Research was first made available to treating physicians and hospitals in the United States by the Centers for Disease Control and Prevention (CDC) for compassionate use under an investigational new drug (IND) protocol (25). To assess the safety, tolerability, and clinical benefit of intravenous artesunate among U.S. patients with severe and complicated malaria, we evaluated medical records for patients enrolled in this IND protocol from 1 January 2007 to 31 December 2010. Methods Design and Conduct We completed a retrospective analysis of clinical data from U.S. patients with severe or complicated malaria who received intravenous artesunate according to CDC protocol 76725, designated as protocol CDC-060, which was approved by the Institutional Review Board of the CDC. All patients or their legal representatives provided written informed consent before treatment. The protocol for the retrospective abstraction of data collected during CDC-060 was sponsored by the Office of the Surgeon General, Department of the U.S. Army, and was in accordance with CDC-060 protocol. Copies of all medical records for patients enrolled in CDC-060 (1 January 2007 to 31 December 2010) were sent from the treating hospitals to the CDC for data abstraction. Using these medicals records, trained clinical professionals from the U.S. Army and the CDC abstracted specific demographic and clinical information for the period beginning 24 hours before the patients first dose of artesunate through the time of ICU discharge or a maximum of 7 days. These data were deidentified and recorded onto data abstraction templates. Data in these templates were quality-assured by 2 separate medical professionals not involved in the initial abstraction and then finally presented to a clinical benefits end point committee (CBEC) consisting of at least 5 health care providers. All decisions about clinical benefit and safety outcomes for each patient were made by a majority vote of the CBEC. Results of the CBEC meetings were recorded in the templates and then entered into the database for analysis according to a predetermined statistical analysis plan. Treatments The Walter Reed Army Institute of Research supplied artesunate to the CDC as a sterile, dry-filled powder that was prepackaged with a phosphate buffer diluent for reconstitution. When a treating physician contacted the CDC malaria hotline (telephone: 770-488-7788 or 770-488-7100 after hours), artesunate was released if all CDC-060 enrollment criteria were met. Artesunate was shipped to the treating hospital rapidly from 1 of 9 CDC quarantine stations located across the United States. On receipt at the treating hospital, artesunate was to be reconstituted and administered in 4 weight-based doses (2.4 mg/kg), with the first dose infused immediately and the remaining 3 doses given at 12, 24, and 48 hours after the first. After their last dose, patients waited a minimum of 4 hours before commencing follow-on oral antimalarial therapy (not supplied by the CDC). The treating physician chose the follow-on antimalarial. Patients were to be treated in a hospital ICU, where they received appropriate concomitant medications and supportive treatments. However, other antimalarials were to be stopped during the period of artesunate administration. Patients To be eligible for artesunate treatment under CDC-060, a patient must have met at least 1 criterion in each of 3 groups of enrollment criteria, which were designated as A, B, and C. Group A criteria required microscopic confirmation of malaria or a strong clinical suspicion of severe malaria when microscopic diagnosis was unavailable. Group B criteria confirmed the patients need for intravenous treatment because of 1 or more of the following: inability to tolerate oral medications, high-density parasitemia (5%), or evidence of severe malaria (impaired consciousness, seizures, circulatory collapse or shock, pulmonary edema or ARDS, acidosis, acute renal failure, abnormal bleeding or disseminated intravascular coagulation, jaundice, or severe anemia with a hemoglobin level <70 g/L). Group C criteria verified the patients need for artesunate because of at least 1 of the following factors: problems with quinidine availability, inefficacy (parasitemia >10% of baseline value at 48 hours after starting intravenous quinidine), intolerance, or contraindications. Patients were excluded if they had a known allergy to artesunate. End Points Although efficacy was not formally assessed, changes in specific clinical benefit variables were treated as secondary outcomes. Clinical benefit end points included time to negative parasitemia, time to follow-on oral therapy, time to discharge from the ICU, and change in group B criteria versus baseline. For each group B criterion present at baseline, daily determinations were made about whether the criterion had resolved, had improved, had deteriorated, or was unchanged. Safety was assessed from data obtained from clinical assessments, laboratory measurements, adverse events (AEs), and deaths. Physical examination findings and AEs were coded according to the Medical Dictionary for Regulatory Affairs, version 14.0, and AE severity was graded based on the Common Terminology Criteria for Adverse Events, version 3.0. An AEs relationship to artesunate was determined by a majority consensus of the CBEC. Statistical Analysis Frequency tables were used to summarize categorical data, and continuous variables were summarized using descriptive statistics. The 95% CIs for outcomes and AE rates were reported. Changes in group B criteria were presented as the frequency and percentages of patients who had changes since baseline. Subgroup analyses of the data were done by segregating patients according to the following variables: baseline level of hepatic impairment (as measured by the Model for End-Stage Liver Disease formula) (26), baseline level of renal impairment (as measured by the estimated glomerular filtration rate) (27), and baseline presence of markers for cerebral malaria (such as seizures, impaired consciousness, or need for ventilator support). Additional subgroup analyses were done for patients with concurrent antimalarial use during artesunate administration and those with quinidine exposure at any time before or during artesunate administration. Results of the subgroup analyses were displayed using KaplanMeier survival curves and compared using the log-rank, Wilcoxon rank-sum,