Background
Malaria has exerted the greatest genetic pressure on the human genome in recent times, resulting in the evolutionary selection of genetic mutations that confer protection against the disease [
1‐
4]. Glucose-6-phosphate dehydrogenase (G6PD) is an X-linked recessive hereditary disorder that currently affects 200–400 million people worldwide, with over 160 mutations identified [
3‐
6] and there is pronounced geographical overlap between areas of G6PD deficiency prevalence and malaria endemicity [
2,
7‐
15]. The G6PD gene codes for an enzyme responsible for catalyzing nicotidamine adenine dinucleotide phosphate (NADP+) to its reduced form, NADPH, in the pentose phosphate pathway. Among G6PD variants with reduced enzyme activity, several phenotypic effects have been described, and are classified by the WHO as: enzyme deficiency with chronic non-spherocytic anaemia (class I, <10% activity); severe enzyme deficiency (class II, <10% activity); moderate/mild enzyme deficiency (class III, 10-60% activity); very mild or no enzyme deficiency (class IV, >60-100% activity); and increased enzyme activity (class V, >150% activity) [
16]. Erythrocytes with insufficient G6PD are thus unprotected against oxidative injury, and individuals with G6PD deficiency may develop haemolytic anaemia in response to a number of stresses, including infection and exposure to medications such as the 8 amino-quinoline, primaquine [
17].
Primaquine has received renewed interest in the context of malaria eradication. The drug is recommended as presumptive anti-relapse treatment of
Plasmodium vivax and
Plasmodium ovale infection due to its activity against hypnozoites. Furthermore, it remains the only readily-available drug that actively clears mature
P. falciparum gametocytes [
18‐
22]. Given the risk of haemolysis in G6PD deficient individuals, and the genetic and phenotypic variability of G6PD deficiency across geographic areas where primaquine treatment is considered, estimation of G6PD enzyme function prior to drug administration is recommended [
23]. At present, however, primaquine therapy without prior determination of G6PD enzyme function, perhaps due to a lack of reliable tests, is thought to be common [
24].
One possible reason for the current lack of a standard diagnostic test is that the majority of methods for assessing G6PD deficiency have shortcomings for field use in tropical countries [
25‐
27] (see Table
1). In 2003, a novel enzymatic method to detect G6PD deficiency was developed [
28], based on the WST8 tetrazolium salt and the 1-methoxy PMS hydrogen carrier. The assay has reduced light sensitivity, and is easily interpretable, both quantitatively and qualitatively. In 2010 Kuwahata
et al. reported a version of this method, optimized for use in a 96-well plate format using dried bloodspots in filter paper, which was successfully tested as an in-field mass-screening tool for G6PD deficiency in the Solomon Islands [
25]. The aims of this current study were to further validate the WST8/1-methoxy-PMS test by comparison with a commercially available enzymatic reference test and to assess the test’s robustness for field use.
Table 1
Available tests for determination of G6PD deficiency and their use in field settings
DNA sequence analysis of the G6PD gene. | Extremely reliable. Primers are used to check whether the G6PD gene contains a mutation. | Requires training, and equipment. Genotype does not correlate with enzyme function and the risk of haemolysis. Female heterozygous have unpredictable phenotype due to X chromosome lyonization. Only one mutation can be analysed with one primer (>160 mutations exist). |
Brilliant cresyl blue decolouration test | Involves the action of G6PD and NADPH diaphorase. A deficiency of either one of these enzymes on RBCs would result in the brilliant cresyl blue remaining unchanged in the test. | Laborious processes; requires technical skill, and has low sensitivity. |
Methaemoglobin reduction test | Based on the oxidation of Hb to MetHb by sodium nitrate and the subsequent enzymatic reconversion to Hb in the presence of methylene blue. | Laborious, qualitative and low sensitivity. Does not enable identification of heterozygous deficient females. |
Formazan ring method | Uses the principle of the MTT-Linked spot test. When G6PD is present at normal levels, MTT is reduced to a purple insoluble formazan derivative, and results in a specific diameter of discolouration. | Prone to misdiagnosis.Ring thickness may be affected by exogenous factors. |
Sephadex gel MTT-PMS method | Mostly used in Asia, and predecessor in concept, of the WST8/1-methoxy PMS test. | Reacts with haemoglobin; is light sensitive and water insoluble. It is of a qualitative nature. |
Fluorescent spot test (FST) | ICSH-recommended method. | Its cut-off value for G6PD deficiency determination is only 10-20% of the normal G6PD activity, which excludes patients with moderate enzyme deficiency and increases the risk of false-normal diagnosis. |
BinaxNOW® rapid test | Rapid test format: Overcomes issues of technical skill, sophisticated equipment and reliability. | It is highly dependent on temperature-sensitive kinetic enzymatic reactions. This limits its use to areas with temperatures between 18 and 25C. Potential cost. |
CareStart™ test | RDT format. Qualitative chromatographic test, based in the reduction of colourless nitro blue tetrazolium dye to dark colour formazan. Long-term temperature stability. | Potential cost. |
R&D® enzymatic test (reference) | Both depend on the conversion of NADP + to NADPH by G6PD. NADPH converts colourless tetrazolium salt into a coloured formazan, while NADP + does not. | Enzymatic gold standard. Requires various temperature-dependent incubations. |
WST8/1-methoxy PMS test (test under validation) | Evaluated in this work. Advantages: no reaction with haemoglobin, lower light sensitivity. |
Methods
Study site & sample selection
The study was conducted in seven sub-counties (Nagongera, Paya, Kirewa, Kisoko, Petta, Mulanda, and Rubongi) in Tororo district, an area with very high malaria transmission intensity in Uganda. In August-September 2010, the study area was mapped and a census survey carried out. Households within 2 km of a health facility were included in the sampling frame. Children under the age of five years were recruited from randomly selected households and were enrolled into a cohort study (Clinical Trials registration number NCT01024426) if they met the following inclusion criteria: 1) age < 5 years, 2) agreement of parents or guardians to provide informed consent, 3) no intention to move during the follow-up period. Clinical and laboratory evaluations were conducted at enrolment and repeated every six months over the period of follow-up. Blood samples collected from cohort study participants at follow-up visits conducted in July and August 2011 were used for the G6PD study.
Laboratory procedures
Blood samples were collected by finger-prick, onto 3MM filter paper, and were dried at ambient temperature. Samples were then stored at room temperature in zip-lock bags containing silica desiccant beads, and assayed within 24-72 h. The remainder of the sample was stored for various time periods and temperature/illumination conditions for further evaluation. Additionally, haemoglobin values were obtained using a HaemoCue B analyser. In parallel, two sets of internal controls were generated to calibrate the assay. A commercial standard reagent of known G6PD activity (Trinity Biotech Normal Control) was used to create a panel of normal, moderate, and severe deficiency (100%, 30% and 10% activity respectively), as well as a no-enzyme control (0%). The second set of internal controls was generated from human blood from two volunteers with normal G6PD activity, and followed the procedure described for the field-adapted test [
25]. Each set of controls were spotted onto 3MM filter paper (Whatman), and stored under the same conditions as the samples. Blood spots and controls were tested by both the optimized WST8/1-methoxy PMS assay, and by the commercially available standard R&D® test. Results were evaluated both visually and quantitatively.
WST8/1-methoxy PMS assay
The principle of the WST8/1-methoxy PMS method depends on reducing hydrogen from NADPH converting WST8 to WST8-formazan in the presence of the hydrogen carrier 1-methoxy-PMS. This reaction yields a strong easily detectable orange colour, with colour intensity directly proportional to G6PD activity. After a 2 hr incubation at room temperature, samples with normal G6PD activity show strong orange colour, deficient samples show faint colour (moderate deficiency likely to represent heterozygotes) or no colour (severe deficiency & negative controls).
Two stock solutions were prepared: a working mix, and a control mix. The working mix contained 50 mM G6P (Roche), 4 mM NADP (Merck Pty Ltd), 1M Tris–HCl pH 7.2-7.5, and 100 mM MgCl2 (Sigma-Aldrich). The control mix contained all reagents in the concentrations described above, but lacked NADP and G6P. Mixes for assay development consisted of 0.5 mL of WST8/1-methoxy PMS (Dojindo Laboratories), 0.5 mL of working stock solution, and 19mL of distilled water for every 96-well plate. Negative controls were generated on site as described in previous studies [
25]
.
A 1.5 mm diameter disc was punched out from each blood spot sample and placed inside a single well of the 96-well flat bottom microplate. Samples were assessed in duplicate. Plates were incubated for 2 h at ambient temperature, and were then inspected by eye by two different observers for qualitative analysis. For quantitative analysis, the optical density was quantified in a microplate reader (Multiskan EX, Thermo scientific) at wavelength OD450-594 nm. G6PD levels were determined in reference to the control panels.
Reference assay: standard quantitative G6PD assay (R&D® diagnostics)
The R&D® colourimetric test was used for validation [
29,
30]. In this test, the resulting NADPH reacts with a colour reagent in which a formazan salt (nitrotetrazolium blue) is produced, generating a visually detectable purple colour. The resulting OD (measured at 550 nm), is proportional to the level of G6PD present in the dried sample. The assay was performed using 96-well plates and dried blood-spots in filter paper as per manufacturers instructions. The same sets of controls were used for both assays, and their robustness tested under various temperature, storage and light conditions.
Experiments to assess assay robustness
Storage
Storage of 150 dried blood spots (FPBS) prior to development of the assay was done at 24°C and 4°C and tested at days 1, 2, 4, 5, 9, and 10 post-collection. Working mixes were stored at 24, 4, and −20°C, and tested at weeks 1,2 and 3.
Reaction stability
Control assays were developed for 2 hrs at 3 different temperatures (37°C, 24°C and 10°C) to determine whether the kinetics of the assay was affected by temperature. Given the identification of limitations related to storage at room temperature of blood-spots in filter paper previously reported [
25], a selection of samples were assayed 24 h after collection of the sample, and frozen at −20°C immediately after absorbance was quantified. They remained frozen for 1, 2, 3 and 4 weeks before G6PD assessment was carried out again both qualitatively and quantitatively.
Light
The 2 hr development of assays for G6PD determination was done under various light conditions: in the dark, scattered light (indoors), and direct exposure to sunlight (outdoors).
Filter paper use
Assays for filter-paper saturation with blood-spots were done for both the WST8/1-methoxy PMS assay and the standard test, to assess whether or not significant differences in saturation could affect G6PD level determination. Such assessment was done by perforating 5–6 blood spots with different levels of saturation from the filter paper, and comparing the final quantitative readout.
For all measurements, the same preliminary experiments to those carried out with the WST8/1-methoxy PMS assay were reproduced with the standard reference test.
Sample size
The sample size was computed based on G6PD deficiency prevalence previously calculated by two independent studies in Kampala (16%) [
31,
32]. To validate the WST8/1-methoxy PMS method by comparison to the reference test with 80% power, the minimum number of samples calculated was 108. The final number of samples compared was 122, and a further 113 samples were evaluated by the WST8/1-methoxy PMS method alone.
Data entry and statistical analysis
Data regarding clinical evaluations, and G6PD assay outcomes were double-entered and validated. Visual analysis was done independently by two observers. Agreement scores between observers, G6PD level visual determination, and quantitative data were produced, and analysed with STATA version 11 (STATA Corporation, College Station, TX). For analysis of the use of the two tests, a contingency table was produced and sensitivity, specificity, PPV and NPV were calculated. A Receiver Operating Characteristic (ROC) curve was calculated. Potential characteristics that could affect G6PD deficiency assessment including gender, age, haemoglobin levels, and prevalence of anaemia were tested by univariate and multivariate regression analysis. Agreement between observers regarding qualitative G6PD activity levels by the WST8 assay, and the R&D reference test was determined by calculating a weighted kappa (Kw) value. A p- value < 0.05 was considered as statistically significant.
Ethics
Ethical approval to perform the G6PD assay validation was obtained from the London School of Hygiene and Tropical Medicine Ethics Committee (application no. 010/361). The use of human participant samples from the ACT PRIME study was under ethical approval of the Makerere University School of Medicine Research and Ethical Committee (no. 2010–108), the Ugandan National Council for Science and Technology (no. HS 794), the LSHTM Ethics Committee (no. 5779), and the University of California San Francisco (no. 006160).
Discussion
Susceptibility of G6PD deficient individuals to haemolysis caused by anti-malarial drugs such as primaquine and other 8-aminoquinolines is a concern for worldwide efforts for malaria eradication, given the geographical overlap between malaria-endemic areas and those populations with high prevalence of G6PD deficiency [
12,
13,
27]. While primaquine administration without G6PD screening for confirmed malaria cases is thought to be relatively common, ethical issues regarding the use of the drug are regaining attention as wider community use is considered. At present, a main limitation for wide-scale implementation of G6PD screening is the lack of a robust, low-cost and rapid test that can accurately classify the majority of samples obtained from individuals in a steady state (ie. not suffering from haemolytic anaemia at the time of test), and that enables testing of a large number of samples simultaneously. In 2003, Tantular and Kawamoto published a simple screening method for detection of G6PD deficiency based on enzymatic activity, with improved performance and reagent stability compared to its predecessors [
28]. Additionally, the method offers the advantage of enabling both qualitative and quantitative assessment of G6PD levels based on the NADPH concentration in the test, which yields strong colouration. Since its description in 2003, the assay has been used in various settings, including Thailand [
12] and Suriname [
26]. In 2010, Kuwahata
et al. successfully optimized the WST8/1-methoxy PMS assay for field use by adapting it to a 96-well plate format, and dried blood-spots in filter paper. The optimized test was successfully used to determine G6PD deficiency prevalence in Isabel Province, Solomon Islands [
25]. Given the observations previously described regarding the performance of the WST8 test, the aims of this study were to validate the WST8 method in relation to a standard reference enzymatic test (commercially available R&D); and to identify operational shortcomings and advantages of this test for use in field and resource-limited settings.
The assay was found to be easy to use, with low use of consumables, and low requirements for sophisticated equipment, as well as being less time consuming than the reference test. On average, processing time for a 96-well plate worth of samples took 10 minutes of active processing and a 2 hr waiting period for development, while the R&D test took 1 hour of active processing. The WST8 test was not overly affected by temperature variation, and the temperature range within which accurate G6PD classification was possible, includes temperatures generally observed in tropical areas. Similarly, the test was less sensitive to scattered light in the laboratory than previously reported for other tests. Nevertheless, from our observations, we suggest avoiding unnecessary exposure of the test and the reagents to light for extended periods. In terms of storage of assay mixes and reagents, our conclusions are similar to those previously reached by Kuwahata
et al. and confirm that long-term storage is advantageous for assay transport and assay use in field settings where assays may need to be run in the field, and subsequently tested in a central laboratory. This is likely, as a major limitation for storage is that enzyme degradation occurs in blood spots in filter papers limiting the time they can be held prior to testing. Previously, Kuwahata
et al. determined that accurate G6PD classification could be done by the WST8 method on filter papers stored at ambient temperature for no more than five days, or alternatively at 4°C for up to 10 days. This is similar to the findings of this study for storage of samples at 4°C, yet storage of samples at room temperature for more than four days led to G6PD level misclassification. It is thus recommended that the samples be tested within 48-72h following sample collection, given that degradation time may vary slightly among different settings after this time frame. An alternative is the possibility to freeze assayed plates at −20°C for quantitation at a later time point. Although this requires freezing facilities, it would allow subsequent mass testing of samples for confirmation of visual readings. A key observation from this and previous studies [
30], is that a control panel, which comprises positive controls and various levels of relative G6PD concentrations, should be stored in similar conditions to those of samples to be tested, as this will reduce the risk of misclassification. Two other observations, common to spectrophotometric assessments with FPBS were that both blood spot saturation and bubbles in the microplate wells can adversely affect reactions leading to aberrant readings and underestimation of G6PD levels. Overall, it was found that the WST8 assay offered major advantages in relation to other currently-used G6PD screening tests in its suitability for field use.
Importantly the assay also performed well. In comparison with the standard reference test, the WST8 test had 72% sensitivity, 98% specificity, and an AUC value of 0.904. The sensitivity of the test was only with misclassifications corresponding to samples with values between normal enzyme activity and mild G6PD deficiency i.e. individuals not at risk of severe haemolytic anaemia after treatment with primaquine (ie. >30% enzyme activity as defined by the WHO). Current tests, including the ICSH recommended fluorescent spot test (FST) method, report a sensitivity value as low as 32% [
27,
34‐
36]. In this context, the WST8 test enabled accurate identification of a wide range of G6PD enzyme levels. The study also showed a good inter-observer reliability (qualitative assessment) with very good agreement in relation to the quantitative classification though numbers of observers and samples were relatively few.
A known confounder for G6PD tests is haemoglobin concentration. This may be potentially attributed to the fact that in patients with haemolytic anaemia, older erythrocytes are haemolysed, while the remaining reticulocytes have normal or near-normal enzyme activity. Previous G6PD screening studies have therefore suggested that G6PD testing must be done in parallel with haemoglobin measurements [
25,
33], or that inbuilt haemoglobin normalization must be considered for accurate determination of status [
30]. In this study, baseline haemoglobin measurements in G6PD normal and G6PD-deficient children were significantly different by univariate analysis. The lower haemoglobin levels in children with normal G6PD in this study may be attributable to G6PD deficiency being associated with a protective effect against infections that may result in anaemia, however, the study was not powered to test this effect. Importantly, no severe deficiency was detected in this study population using both the reference and WST8 tests, which is in agreement with the expected G6PD A- prevalent genotype in Africa [
5,
8].
In conclusion, the WST8 test offers some important advantages in comparison to other tests for G6PD deficiency assessment in large-scale screening studies and public health interventions where primaquine administration is being considered. As demonstrated by this study, G6PD screening using the WST8 assay can be easily nested into other public health interventions, which is advantageous for its inclusion in malaria elimination programmes contemplating the use of primaquine. Additionally, the high comparability of quantitative and qualitative G6PD estimates between the WST8- and the standard colorimetric tests used for diagnosis in hospital settings, suggest that the WST8 test would be a relatively safe basis for clinical decisions. This would obviously depend on existing clinical and laboratory capacity in any facility and require some adaptation to single sample testing [
37]. No individuals with severe deficiency were identified in this study. Although this is a limitation in terms of validation of the test, a previous study carried out in the Solomon Islands with the WST8 test [
25], enabled the identification of severely G6PD deficient individuals, as well as a range of G6PD activities similar to the one reported here. In order to fully assess the capacity of the test in various field settings, further studies in various geographical locations where diverse G6PD genotypes are prevalent, would be advantageous. A further key observation is the need for parallel haemoglobin determination, emphasized in previous G6PD deficiency assessments [
25,
26,
30,
33]. This is likely to be of general benefit both in assessing the interpretability of the test and also may indicate other causes of anaemia and any required treatment. Overall, the WST8 test has a considerable potential as a diagnostic tool prior to primaquine administration in malaria-endemic areas as a point of care test and/or as a screening tool for assessing G6PD prevalence in large-scale screening studies in areas contemplating primaquine deployment.
Acknowledgements
We would like to express our gratitude to the Infectious Disease Research Collaboration, and the ACT PRIME and Tororo lab teams, particularly Florence Nankya, Rita Kabuleta Luswata, Joseph Wadamba, and Andrew Walakira for their contributions to the logistics of this study. We thank Eric Becha and Simon P. Kigozi for advice on data analysis. We are very grateful to all participants and their families. We thank James McCarthy and Melissa Kuwahata, and George Reclos for their advice on the use of the WST8/1-methoxy PMS test and the R&D reference tests respectively. ACE and CD are supported by a grant from the Wellcome Trust (090558 & 091924 respectively). MDN was supported by an LSHTM grant to carry out this study. The ACT PRIME study was supported by the ACT Consortium, which is funded through a grant from the Bill and Melinda Gates Foundation to the London School of Hygiene & Tropical Medicine. This research was previously presented at the 2012 ‘Challenges in Malaria Research, Progress towards elimination’ meeting in Basel, Switzerland.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
MDN, ACE, SS, CD wrote and edited the manuscript. MDN carried out the G6PD assay experiments and testing. MDN, ACE, CD carried out data analysis. CMS, DD, SG, PT and SS facilitated and supported the survey. MDN, EM, DN, LO, ES carried out sample collection and/or clinical assessment of the children in the study cohort. CD, ACE, and SS conceived the study. CD, ACE, SS, and MDN contributed to experiment design. ACE, SS, CD were involved in supervised field coordination. All authors read and approved the final manuscript.