G6PD deficiency in Plasmodium falciparum and Plasmodium vivax malaria-infected Cambodian patients

Background Glucose-6-phosphate-dehydrogenase deficiency (G6PDd) rates are unknown in malaria-infected Cambodian patients. These data are key to a rational drug policy for malaria elimination of Plasmodium falciparum and Plasmodium vivax. Methods From September 2010–2012, a two-year survey of G6PDd and haemoglobinopathies assessed by quantitative enzyme activity assay and haemoglobin electrophoresis, respectively, was conducted in malaria-infected patients presenting to 19 health centres throughout Cambodia. Results A total of 2,408 confirmed malaria patients of mean age 26.7 (range 2–81) years were recruited from mostly western Cambodia (n = 1,732, 71.9%); males outnumbered females by 3.9:1. Plasmodium falciparum was present in 1,443 (59.9%) and P. vivax in 965 (40.1%) patients. Mean G6PD activity was 11.6 (CI 95%: 11.4-11.8) U/g Hb, G6PDd was present in 13.9% of all patients (335/2,408) and severe G6PDd (including WHO Class I and II variants) was more common in western (158/1,732, 9.1%) versus eastern (21/414, 5.1%) Cambodia (P = 0.01). Of 997/2,408 (41.4%) had a haemoglobinopathy. Mean haemoglobin concentrations were inversely related to age: 8.1 g/dL < five years, 8.7 g/dL five to 14 years, and 10.4 g/dL >15 years (P <0.001). Conclusions G6PDd prevalence, anaemia and haemoglobinopathies were common in malaria-infected patients. The deployment of primaquine in Cambodia should be preceded by primaquine safety studies paralleled with evaluations of easy to use tests to detect G6PDd.

Conclusions: G6PDd prevalence, anaemia and haemoglobinopathies were common in malaria-infected patients. The deployment of primaquine in Cambodia should be preceded by primaquine safety studies paralleled with evaluations of easy to use tests to detect G6PDd.

Background
Controlling malaria remains a significant global health challenge [1], especially in areas of low transmission which are seen as prime areas for malaria elimination [2]. The World Health Organization (WHO) has been urging countries for many years to use primaquine for both transmission blocking of Plasmodium falciparum, because it kills mature gametocytes, and as anti-relapse treatment against Plasmodium vivax by killing liver hypnozoites [3,4]. Primaquine is not used widely because of anxiety over its well-known propensity to cause acute haemolytic anaemia (AHA) in individuals with glucose-6-phosphate-dehydrogenase deficiency (G6PDd) [5], a scenario reported by senior Cambodian clinicians (Mey Bout Denis, Cambodian National Malaria Control Programme, CNM, personal communication), coupled with the current logistical and financial impossibility of offering G6PD screening to all malaria patients [6]. Nevertheless, the emergence of artemisinin resistance in Cambodia and other Southeast Asian countries has added considerable urgency to containing artemisinin resistant parasites by transmission blocking with primaquine [7]. Indeed, the WHO has recommended low dose primaquine (0.25 mg/kg stat) for P. falciparum without prior G6PD testing believing that AHA would be clinically mild [8].
Cambodia has low transmission of essentially falciparum and vivax malaria in an overall ratio of about 1:1 but there is seasonal and geographical variation. Approximately half of the population,~3,000,000, is estimated to be at risk of malaria [9] and public sector data from 2010 show an overall incidence of 4.07 cases/1,000 population (Cambodian National Malaria Control Programme, CNM Annual Reports 2000-2009), an historically low rate but one that is higher than neighbouring countries. Controlling malaria is a high priority for Cambodia and the Royal Cambodian government has committed itself to eliminating malaria by 2025.
G6PDd is a common, X-linked hereditary enzyme deficiency affecting approximately 400 million people worldwide [10], mainly in malaria-endemic regions [11]. G6PD is a key enzyme for protecting red cells against oxidant stress by allowing the production of NADPH from the hexose monophosphate pathway. G6PDd variants number about 400 and have differing amounts of G6PD enzyme activity that are classified broadly as very severe, severe or mild [12]. Hemizygote males and homozygote females are most and least commonly affected, respectively. Heterozygote females have mixed G6PD normal and deficient red cells and their total G6PD enzyme activity and susceptibility to haemolysis depends on the balance between the expression of the normal and abnormal X chromosomes [13]. Primaquine-induced AHA is dose dependent and inversely related to G6PD enzyme activity [14]. Thus, primaquine given to individuals with mild G6PDd (e g, African A-) tends to produce mild, self-limiting AHA [15][16][17][18] but greater AHA and longer times to haemoglobin recovery in severe G6PDd (e g, Asian or B-variants) [19][20][21][22].
To date, there has not been an estimate of the frequencies of G6PDd in malaria-infected patients seeking anti-malarial treatment in public health facilities. Such patients would be eligible to receive primaquine for either falciparum transmission blocking or weekly antirelapse primaquine but data on weekly primaquine in Cambodian G6PDd variants are currently lacking. Obtaining G6PDd data and its association with malaria, haemoglobinopathies and anaemia would be important for the Cambodian National Malaria Control Programme (CNM) to prioritize its anti-malarial drug policy and to conduct future research on the safety of primaquine. Results of a G6PDd survey in malaria patients are reported herein.

Study population and site
The study took place from 2010 to 2012 at 19 public health facilities from across Cambodia (Figure 1), which are involved in the National Network for Monitoring Anti-malarial Drug Resistance in Cambodia, collaboration between CNM and Institut Pasteur du Cambodge (IPC). Malaria diagnosis was achieved in febrile patients seeking treatment, either by microscopy of Giemsa-stained malaria blood films or by a malaria rapid diagnostic test (RDT) that detects P. falciparum and non-P. falciparum parasites (CareStart™, AccessBio, USA). Malaria-positive patients or their legal guardians were asked if they would be interested to join the study. If signed informed consent was obtained, patients were allocated a study number and had blood taken. The study protocol was reviewed and approved by the Ethics Committee of the Cambodian Ministry of Health (No 160 NECHR, 28 October, 2010).

Sample collection
Five ml of venous blood were collected into ACD-coated tubes, stored in a fridge before transport to IPC within 24 to 48 hours at +4°C in cool boxes. At IPC, repeat malaria blood films were made, stained with 3% Giemsa solution for 30 to 45 minutes, and checked for Plasmodium species by light microscopy. Blood samples were divided into two aliquots for (i) complete blood count (CBC), quantitative determination of G6PD activity and haemoglobin electrophoresis, and, (ii) in vitro antimalarial drug sensitivity testing and detection of molecular markers related to anti-malarial drug resistance.

Haematological parameters
The CBC was determined using a CellDyn 3200 ana-lyzer™ (Abbott, France) after daily standardization with three different controls of all the standard parameters.

Quantitative determination of G6PD activity
Determination of the G6PD enzyme activity was performed on the fresh blood within a maximum of 48 hours after sample collection, using the Trinity Biotech quantitative G6PD assay™ (Ref 345-UV, Trinity Biotech, USA) adapted on the Integra 400 analyzer™ (Roche Diagnostic, France), according to the manufacturer's instructions and as described previously [24]. The reliability of the results were monitored by calibration using three different enzyme activity controls provided by Trinity Biotech ( G6PD deficiency was classified according to the WHO classification expressing the G6PD enzyme activity as a percentage of the population defined mean, 11.8 U/g Hb for Cambodia [24]: (i) Class I: very severely deficient, ≤1% residual activity, ≤0.12 U/g Hb, (ii) Class II: severely deficient, >1-10% residual activity, >0.12-1.2 U/g Hb, (iii) Class III: mildly deficient, >10-60% residual activity, >1.2-7.1 U/g Hb, (iv) Class IV: normal activity, >60% to 150% residual activity, >7.1-17.7 U/g Hb, and (v) Class V: increased activity, >150% residual activity, >17.7 U/g Hb.

Data analysis
Data were entered and verified using Microsoft Excel 2010 software and analysed using MedCalc software (version 9.1.0.1, Mariakerke, Belgium) and XLSTAT for Windows XP (Addinsoft, Paris, France). Continuous variables were compared using an independent-sample analysis of variance (ANOVA) or Mann-Whitney test (skewed data). For categorical variables, Chi-squared or Fisher's exact tests were used to assess significant differences in proportions. All reported P-values are two-sided and were considered statistically significant if <0.05.
Variables with P-values <0.25 from the bivariate analyses were initially introduced into the model and removed following a backwards-stepwise selection procedure to leave only those variables those with a P-values <0.05 in the final model. Odds ratios (OR) and their 95% confidence intervals (CI) are reported for these significant explanatory variables. 12.0) with a range of two to 81 years, distributed as follows: (i) 0.5% < five years, (ii) 9.9% five to 14 years, and (iii) 89.6% >15 years. The male: female ratio was 3.9:1.

Discussion
For countries moving towards elimination of P. vivax and artemisinin-resistant P. falciparum, primaquine is an essential drug [3,29,30]. Primaquine is needed urgently in Cambodia but its safe introduction in the health system remains an important challenge due to the scarcity of G6PDd data, the lack of an inexpensive, reliable point-of-care test to detect G6PDd individuals [24] and the lack of data of any primaquine dose in malariainfected patients. The recent WHO recommendation that G6PD testing may not be needed for low dose primaquine [8] is not yet supported by substantial evidence from Cambodia and other countries that have severe forms of G6PDd, nor are there yet any data from