Measurements of 5,6-Orthoquinone, a Surrogate for the Presumed Active Primaquine Metabolite 5-Hydroxyprimaquine, in the Urine of Cambodian Adults.

The active metabolites of primaquine, in particular 5-hydroxyprimaquine, likely responsible for the clearance of dormant hypnozoites, are produced through the hepatic CYP450 2D6 (CYP2D6) enzymatic pathway. With the inherent instability of 5-hydroxyprimaquine, a stable surrogate, 5,6-orthoquinone, can now be detected and measured in the urine as part of primaquine pharmacokinetic studies. This study performed CYP450 2D6 genotyping and primaquine pharmacokinetic testing, to include urine 5,6-orthoquinone, in 27 healthy adult Cambodians, as a preliminary step to prepare for future clinical studies assessing primaquine efficacy for Plasmodium vivax infections. The CYP2D6 *10 reduced activity allele was found in 57% of volunteers, and the CYP2D6 genotypes were dominated by *1/*10 (33%) and *10/*10 (30%). Predicted phenotypes were evenly split between Normal Metabolizer (NM) and Intermediate Metabolizer (IM) except for one volunteer with a gene duplication and unclear phenotype, classifying as either IM or NM. Median plasma primaquine (PQ) area under the curve (AUC) was lower in the NM group (460 h*ng/mL) compared to the IM group (561 h*ng/mL), although not statistically significant. Similar to what has been found in the US study, no 5,6-orthoquinone was detected in the plasma. The urine creatinine-corrected 5,6-orthoquinone AUC in the NM group was almost three times higher than in the IM group, with peak measurements (Tmax) at 4 h. Although there is variation among individuals, future studies examining the relationship between the levels of urine 5,6-orthoquinone and primaquine radical cure efficacy could result in a metabolism biomarker predictive of radical cure.

Update on Shigella and Nontyphoidal Salmonella Antimicrobial Drug Resistance: Implications on Empirical Treatment of Acute Infectious Diarrhea in Cambodia.

Information on causative diarrheal pathogens and their associated antimicrobial susceptibility remains limited for Cambodia. This study describes antimicrobial resistance patterns for Shigella and nontyphoidal Salmonella isolates collected in Cambodia over a 5-year period. Multidrug resistance was shown in 98% of Shigella isolates, with 70%, 11%, and 29% of isolates being resistant to fluoroquinolones, azithromycin, and cephalosporin, respectively. As many as 11% of Shigella isolates were resistant to nearly all oral and parenteral drugs typically used for shigellosis, demonstrating extreme drug resistance phenotypes. Although a vast majority of nontyphoidal Salmonella isolates remained susceptible to cephalosporins (99%) and macrolides (98%), decreased susceptibility to ciprofloxacin was found in 67% of isolates, which is notably higher than previous reports. In conclusion, increasing antimicrobial resistance of Shigella and nontyphoidal Salmonella is a major concern for selecting empirical treatment of acute infectious diarrhea in Cambodia. Treatment practices should be updated and follow local antimicrobial resistance data for the identified pathogens.

Prevalence of CYP2D6 Genotypes and Predicted Phenotypes in a Cohort of Cambodians at High Risk for Infections with Plasmodium vivax.

Clinical failure of primaquine (PQ) has been demonstrated in people with CYP450 2D6 genetic polymorphisms that result in reduced or no enzyme activity. The distribution of CYP2D6 genotypes and predicted phenotypes in the Cambodian population is not well described. Surveys in other Asian countries have shown an approximate 50% prevalence of the reduced activity CYP2D6 allele *10, which could translate into increased risk of PQ radical cure failure and repeated relapses, making interruption of transmission and malaria elimination difficult to achieve. We determined CYP2D6 genotypes from 96 volunteers from Oddor Meanchey Province, Cambodia, an area endemic for Plasmodium vivax. We found a 54.2% frequency of the *10 allele, but in approximately half of our subjects, it was paired with a normal activity allele, either *1 or *2. The prevalence of *5, a null allele, was 9.4%. Overall predicted phenotype percentages were normal metabolizers, 46%; intermediate metabolizers, 52%; and poor metabolizers, 1%.

Evaluation of the CareStart™ glucose-6-phosphate dehydrogenase (G6PD) rapid diagnostic test in the field settings and assessment of perceived risk from primaquine at the community level in Cambodia.

BACKGROUND: Primaquine is an approved radical cure treatment for Plasmodium vivax malaria but treatment can result in life-threatening hemolysis if given to a glucose-6-phosphate dehydrogenase deficient (G6PDd) patient. There is a need for reliable point-of-care G6PD diagnostic tests. OBJECTIVES: To evaluate the performance of the CareStart™ rapid diagnostic test (RDT) in the hands of healthcare workers (HCWs) and village malaria workers (VMWs) in field settings, and to better understand user perceptions about the risks and benefits of PQ treatment guided by RDT results. METHODS: This study enrolled 105 HCWs and VMWs, herein referred to as trainees, who tested 1,543 healthy adult male volunteers from 84 villages in Cambodia. The trainees were instructed on G6PD screening, primaquine case management, and completed pre and post-training questionnaires. Each trainee tested up to 16 volunteers in the field under observation by the study staff. RESULTS: Out of 1,542 evaluable G6PD volunteers, 251 (16.28%) had quantitative enzymatic activity less than 30% of an adjusted male median (8.30 U/g Hb). There was no significant difference in test sensitivity in detecting G6PDd between trainees (97.21%), expert study staff in the field (98.01%), and in a laboratory setting (95.62%) (p = 0.229); however, test specificity was different for trainees (96.62%), expert study staff in the field (98.14%), and experts in the laboratory (98.99%) (p < 0.001). Negative predictive values were not statistically different for trainees, expert staff, and laboratory testing: 99.44%, 99.61%, and 99.15%, respectively. Knowledge scores increased significantly post-training, with 98.7% willing to prescribe primaquine for P.vivax malaria, an improvement from 40.6% pre-training (p < 0.001). CONCLUSION: This study demonstrated ability of medical staff with different background to accurately use CareStart™ RDT to identify G6PDd in male patients, which may enable safer prescribing of primaquine; however, pharmacovigilance is required to address possible G6PDd misclassifications.

Dihydroartemisinin-piperaquine failure associated with a triple mutant including kelch13 C580Y in Cambodia: an observational cohort study.

BACKGROUND: Dihydroartemisinin-piperaquine has been adopted as first-line artemisinin combination therapy (ACT) for multidrug-resistant Plasmodium falciparum malaria in Cambodia because of few remaining alternatives. We aimed to assess the efficacy of standard 3 day dihydroartemisinin-piperaquine treatment of uncomplicated P falciparum malaria, with and without the addition of primaquine, focusing on the factors involved in drug resistance. METHODS: In this observational cohort study, we assessed 107 adults aged 18-65 years presenting to Anlong Veng District Hospital, Oddar Meanchey Province, Cambodia, with uncomplicated P falciparum or mixed P falciparum/Plasmodium vivax infection of between 1000 and 200,000 parasites per µL of blood, and participating in a randomised clinical trial in which all had received dihydroartemisinin-piperaquine for 3 days, after which they had been randomly allocated to receive either primaquine or no primaquine. The trial was halted early due to poor dihydroartemisinin-piperaquine efficacy, and we assessed day 42 PCR-corrected therapeutic efficacy (proportion of patients with recurrence at 42 days) and evidence of drug resistance from the initial cohort. We did analyses on both the intention to treat (ITT), modified ITT (withdrawals, losses to follow-up, and those with secondary outcomes [eg, new non-recrudescent malaria infection] were censored on the last day of follow-up), and per-protocol populations of the original trial. The original trial was registered with ClinicalTrials.gov, number NCT01280162. FINDINGS: Between Dec 10, 2012, and Feb 18, 2014, we had enrolled 107 patients in the original trial. Enrolment was voluntarily halted on Feb 16, 2014, before reaching planned enrolment (n=150) because of poor efficacy. We had randomly allocated 50 patients to primaquine and 51 patients to no primaquine groups. PCR-adjusted Kaplan-Meier risk of P falciparum 42 day recrudescence was 54% (95% CI 45-63) in the modified ITT analysis population. We found two kelch13 propeller gene mutations associated with artemisinin resistance--a non-synonymous Cys580Tyr substitution in 70 (65%) of 107 participants, an Arg539Thr substitution in 33 (31%), and a wild-type parasite in four (4%). Unlike Arg539Thr, Cys580Tyr was accompanied by two other mutations associated with extended parasite clearance (MAL10:688956 and MAL13:1718319). This combination triple mutation was associated with a 5·4 times greater risk of treatment failure (hazard ratio 5·4 [95% CI 2·4-12]; p<0·0001) and higher piperaquine 50% inhibitory concentration (triple mutant 34 nM [28-41]; non-triple mutant 24 nM [1-27]; p=0·003) than other infections had. The drug was well tolerated, with gastrointestinal symptoms being the most common complaints. INTERPRETATION: The dramatic decline in efficacy of dihydroartemisinin-piperaquine compared with what was observed in a study at the same location in 2010 was strongly associated with a new triple mutation including the kelch13 Cys580Tyr substitution. 3 days of artemisinin as part of an artemisinin combination therapy regimen might be insufficient. Strict regulation and monitoring of antimalarial use, along with non-pharmacological approaches to malaria resistance containment, must be integral parts of the public health response to rapidly accelerating drug resistance in the region. FUNDING: Armed Forces Health Surveillance Center/Global Emerging Infections Surveillance and Response System, Military Infectious Disease Research Program, National Institute of Allergy and Infectious Diseases, and American Society of Tropical Medicine and Hygiene/Burroughs Wellcome Fund.

Efficacy of two versus three-day regimens of dihydroartemisinin-piperaquine for uncomplicated malaria in military personnel in northern Cambodia: an open-label randomized trial.

INTRODUCTION: Emerging antimalarial drug resistance in mobile populations remains a significant public health concern. We compared two regimens of dihydroartemisinin-piperaquine in military and civilians on the Thai-Cambodian border to evaluate national treatment policy. METHODS: Efficacy and safety of two and three-day regimens of dihydroartemisinin-piperaquine were compared as a nested open-label evaluation within a malaria cohort study in 222 otherwise healthy volunteers (18% malaria-infected at baseline). The first 80 volunteers with slide-confirmed Plasmodium falciparum or vivax malaria were randomized 1:1 to receive either regimen (total dose 360 mg dihydroartemisinin and 2880 mg piperaquine) and followed weekly for up to 6 months. The primary endpoint was malaria recurrence by day 42. Volunteers with vivax infection received primaquine at study discharge with six months follow-up. RESULTS: Eighty patients (60 vivax, 15 falciparum, and 5 mixed) were randomized to dihydroartemisinin-piperaquine. Intention-to-treat all-species efficacy at Day 42 was 85% for the two-day regimen (95% CI 69-94) and 90% for the three-day regimen (95% CI 75-97). PCR-adjusted falciparum efficacy was 75% in both groups with nearly half (45%) still parasitemic at Day 3. Plasma piperaquine levels were comparable to prior published reports, but on the day of recrudescence were below measurable in vitro piperaquine IC50 levels in all falciparum treatment failures. CONCLUSIONS: In the brief period since introduction of dihydroartemisinin-piperaquine, there is early evidence suggesting declining efficacy relative to previous reports. Parasite IC50 levels in excess of plasma piperaquine levels seen only in treatment failures raises concern for clinically significant piperaquine resistance in Cambodia. These findings warrant improved monitoring of clinical outcomes and follow-up, given few available alternative drugs. TRIAL REGISTRATION: ClinicalTrials.gov NCT01280162.

Severe malaria in Battambang Referral Hospital, an area of multidrug resistance in Western-Cambodia: a retrospective analysis of cases from 2006-2009.

BACKGROUND: Despite recent malaria containment and control efforts leading to reduced incidence, Cambodia remains endemic for both Plasmodium vivax and multidrug-resistant Plasmodium falciparum malaria. Little has been reported in the peer-reviewed literature regarding the burden of severe malaria (SM) in Cambodia. METHODS: Medical records for all patients admitted to the Battambang Referral Hospital (BRH) with an admitting or discharge diagnosis of SM from 2006 to 2009 (suspected SM cases) were reviewed. Those meeting the case definition of SM according to retrospective chart review and investigator assessment of probable cases, based on published national guidelines available at the time, were analysed for trends in demographics, mortality and referral patterns. RESULTS: Of the 537 suspected SM cases at BRH during the study period, 393 (73%) met published WHO criteria for SM infection. Despite limited diagnostic and treatment facilities, overall mortality was 14%, with 7% mortality in children 14 and under, but 19% in adults (60% of cases). Cerebral malaria with coma was relatively rare (17%), but mortality was disproportionately high at 35%. Mean time to hospital presentation was five days (range one to 30 days) after onset of symptoms. While patients with delays in presentation had worse outcomes, there was no excess mortality based on treatment referral times, distance travelled or residence in artemisinin-resistance containment (ARC) Zone 1 compared to Zone 2. CONCLUSIONS: Despite limitations in diagnosis and treatment, and multiple confounding co-morbidities, mortality rates at BRH were similar to reports from other countries in the region. Interventions to improve access to early diagnosis and effective treatment, combined with modest improvements in intensive care, are likely to reduce mortality further. Patients referred from Zone 1 did not have excess mortality compared to Zone 2 ARC areas. A steep decrease in SM cases and deaths observed in the first half of 2009 has since continued, indicating some success from containment efforts despite the emergence of artemisinin resistance in this area.