G6PD Deficiency at Sumba in Eastern Indonesia Is Prevalent, Diverse and Severe: Implications for Primaquine Therapy against Relapsing Vivax Malaria

Safe treatment of Plasmodium vivax requires diagnosis of both the infection and status of erythrocytic glucose-6-phosphate dehydrogenase (G6PD) activity because hypnozoitocidal therapy against relapse requires primaquine, which causes a mild to severe acute hemolytic anemia in G6PD deficient patients. Many national malaria control programs recommend primaquine therapy without G6PD screening but with monitoring due to a broad lack of G6PD deficiency screening capacity. The degree of risk in doing so hinges upon the level of residual G6PD activity among the variants present in any given area. We conducted studies on Sumba Island in eastern Indonesia in order to assess the potential threat posed by primaquine therapy without G6PD screening. We sampled 2,033 residents of three separate districts in western Sumba for quantitative G6PD activity and 104 (5.1%) were phenotypically deficient (\u3c4.6U/gHb; median normal 10U/gHb). The villages were in two distinct ecosystems, coastal and inland. A positive correlation occurred between the prevalence of malaria and G6PD deficiency: 5.9% coastal versus inland 0.2% for malaria (P\u3c0.001), and 6.7% and 3.1% for G6PD deficiency (P\u3c0.001) at coastal and inland sites, respectively. The dominant genotypes of G6PD deficiency were Vanua Lava, Viangchan, and Chatham, accounting for 98.5%of the 70 samples genotyped. Subjects expressing the dominant genotypes all had less than 10% of normal enzyme activities and were thus considered severe variants. Blind administration of anti-relapse primaquine therapy at Sumba would likely impose risk of serious harm

Assessment of Point-of-Care Diagnostics for G6PD Deficiency in Malaria Endemic Rural Eastern Indonesia.

BACKGROUND:Patients infected by Plasmodium vivax or Plasmodium ovale suffer repeated clinical attacks without primaquine therapy against latent stages in liver. Primaquine causes seriously threatening acute hemolytic anemia in patients having inherited glucose-6-phosphate dehydrogenase (G6PD) deficiency. Access to safe primaquine therapy hinges upon the ability to confirm G6PD normal status. CareStart G6PD, a qualitative G6PD rapid diagnostic test (G6PD RDT) intended for use at point-of-care in impoverished rural settings where most malaria patients live, was evaluated. METHODOLOGY/PRINCIPAL FINDINGS:This device and the standard qualitative fluorescent spot test (FST) were each compared against the quantitative spectrophotometric assay for G6PD activity as the diagnostic gold standard. The assessment occurred at meso-endemic Panenggo Ede in western Sumba Island in eastern Indonesia, where 610 residents provided venous blood. The G6PD RDT and FST qualitative assessments were performed in the field, whereas the quantitative assay was performed in a research laboratory at Jakarta. The median G6PD activity ≥ 5 U/gHb was 9.7 U/gHb and was considered 100% of normal activity. The prevalence of G6PD deficiency by quantitative assessment (<5 U/gHb) was 7.2%. Applying 30% of normal G6PD activity as the cut-off for qualitative testing, the sensitivity, specificity, positive predictive value, and negative predictive value for G6PD RDT versus FST among males were as follows: 100%, 98.7%, 89%, and 100% versus 91.7%, 92%, 55%, and 99%; P = 0.49, 0.001, 0.004, and 0.24, respectively. These values among females were: 83%, 92.7%, 17%, and 99.7% versus 100%, 92%, 18%, and 100%; P = 1.0, 0.89, 1.0 and 1.0, respectively. CONCLUSIONS/SIGNIFICANCE:The overall performance of G6PD RDT, especially 100% negative predictive value, demonstrates suitable safety for G6PD screening prior to administering hemolytic drugs like primaquine and many others. Relatively poor diagnostic performance among females due to mosaic G6PD phenotype is an inherent limitation of any current practical screening methodology

Field assessment of the operating procedures of a semi-quantitative G6PD Biosensor to improve repeatability of routine testing.

In remote communities, diagnosis of G6PD deficiency is challenging. We assessed the impact of modified test procedures and delayed testing for the point-of-care diagnostic STANDARD G6PD (SDBiosensor, RoK), and evaluated recommended cut-offs. We tested capillary blood from fingerpricks (Standard Method) and a microtainer (BD, USA; Method 1), venous blood from a vacutainer (BD, USA; Method 2), varied sample application methods (Methods 3), and used micropipettes rather than the test's single-use pipette (Method 4). Repeatability was assessed by comparing median differences between paired measurements. All methods were tested 20 times under laboratory conditions on three volunteers. The Standard Method and the method with best repeatability were tested in Indonesia and Nepal. In Indonesia 60 participants were tested in duplicate by both methods, in Nepal 120 participants were tested in duplicate by either method. The adjusted male median (AMM) of the Biosensor Standard Method readings was defined as 100% activity. In Indonesia, the difference between paired readings of the Standard and modified methods was compared to assess the impact of delayed testing. In the pilot study repeatability didn't differ significantly (p = 0.381); Method 3 showed lowest variability. One Nepalese participant had <30% activity, one Indonesian and 10 Nepalese participants had intermediate activity (≥30% to <70% activity). Repeatability didn't differ significantly in Indonesia (Standard: 0.2U/gHb [IQR: 0.1-0.4]; Method 3: 0.3U/gHb [IQR: 0.1-0.5]; p = 0.425) or Nepal (Standard: 0.4U/gHb [IQR: 0.2-0.6]; Method 3: 0.3U/gHb [IQR: 0.1-0.6]; p = 0.330). Median G6PD measurements by Method 3 were 0.4U/gHb (IQR: -0.2 to 0.7, p = 0.005) higher after a 5-hour delay compared to the Standard Method. The definition of 100% activity by the Standard Method matched the manufacturer-recommended cut-off for 70% activity. We couldn't improve repeatability. Delays of up to 5 hours didn't result in a clinically relevant difference in measured G6PD activity. The manufacturer's recommended cut-off for intermediate deficiency is conservative

Demographic, malaria and G6PDd prevalence data by gender and ecosystem in western Sumba.

<p>n = sample number.</p><p>¹ Population number was obtained from central agency statistic from each district.</p><p>² Microscopy diagnosed.</p><p>³ One subject experienced mixed infection of <i>P</i>. <i>falciparum</i> and <i>P</i>. <i>vivax</i>.</p><p>⁴ Hb < 10 g/dl.</p><p>⁵ G6PD activities ≤4.6 U/gHb.</p><p>⁶ Samples screened as G6PD deficient but declined, absent or failed to be DNA extracted.</p><p>Demographic, malaria and G6PDd prevalence data by gender and ecosystem in western Sumba.</p

A) Schematic work flow of the study in western Sumba in 2012 and B) map of G6PD deficiency and malaria prevalences in study sites in Sumba.

<p>Small inlet showed the map of Indonesia and where Sumba island is located.</p

Impact of Hb level on G6PD activities.

<p>Blue line represents those samples having extremely high G6PD activity and some degree of anemia (R² = 0.08) and red line represents those having normal Hb level (R² = 0.48) and t-value = 28.6 (p<0.001).</p

Scatter plot showing the relationship between age and G6PD activity in Sumba.

<p>Scatter plot showing the relationship between age and G6PD activity in Sumba.</p

Scatter plot of G6PD activities of G6PD deficient and normal subjects from Trinity quantitative versus kinetics assays measured in U/g Hb.

<p>The different colours represented different variant (V in either normal, heterozygous and hemizygous mutants. VL Het, VC Het and CT Het represented heterozygous females having Vanua Lava, Viangchan and Chatham variant respectively. VL Hem, VC Hem and CT Hem represented hemizyogous males having Vanua Lava, Viangchan and Chatham variant respectively.</p

Enzymatic parameters from purified G6PD from study subjects in western Sumba according to genotype.

<p>* median of the samples analyzed;</p><p>**cannot be calculated because the interval between the data are too small;</p><p>CI is confidence interval</p><p>Enzymatic parameters from purified G6PD from study subjects in western Sumba according to genotype.</p

Fig 7 -

Scatterplot of G6PD activity as measured by the Biosensor using the Standard Method vs spectrophotometry (left); and by the Biosensor using the Standard Method vs Method 3 (right). The diagonal line denotes the line of equality. The red horizontal and vertical lines, from the point of origin outwards, mark the 30%, 70%, and 100% of the AMM (normal G6PD activity). Individuals whose G6PD categorization changed when measured by the Biosensor and spectrophotometry were marked as red dots.</p