Variation in Glucose-6-Phosphate Dehydrogenase activity following acute malaria.

Primaquine and tafenoquine are the only licensed drugs with activity against Plasmodium vivax hypnozoites but cause haemolysis in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Malaria also causes haemolysis, leading to the replacement of older erythrocytes with low G6PD activity by reticulocytes and young erythrocytes with higher activity. Aim of this study was to assess the impact of acute malaria on G6PD activity. Selected patients with uncomplicated malaria were recruited in Bangladesh (n = 87), Indonesia (n = 75), and Ethiopia (n = 173); G6PD activity was measured at the initial presentation with malaria and a median of 176 days later (range 140 to 998) in the absence of malaria. Among selected participants (deficient participants preferentially enrolled in Bangladesh but not at other sites) G6PD activity fell between malaria and follow up by 79.1% (95%CI: 40.4 to 117.8) in 6 participants classified as deficient (<30% activity), 43.7% (95%CI: 34.2 to 53.1) in 39 individuals with intermediate activity (30% to <70%), and by 4.5% (95%CI: 1.4 to 7.6) in 290 G6PD normal (≥70%) participants. In Bangladesh and Indonesia G6PD activity was significantly higher during acute malaria than when the same individuals were retested during follow up (40.9% (95%CI: 33.4-48.1) and 7.4% (95%CI: 0.2 to 14.6) respectively), whereas in Ethiopia G6PD activity was 3.6% (95%CI: -1.0 to -6.1) lower during acute malaria. The change in G6PD activity was apparent in patients presenting with either P. vivax or P. falciparum infection. Overall, 66.7% (4/6) severely deficient participants and 87.2% (34/39) with intermediate deficiency had normal activities when presenting with malaria. These findings suggest that G6PD activity rises significantly and at clinically relevant levels during acute malaria. Prospective case-control studies are warranted to confirm the degree to which the predicted population attributable risks of drug induced haemolysis is lower than would be predicted from cross sectional surveys.

Introduction

In Asia, Oceania, the Horn of Africa, and the Americas, Plasmodium vivax is the predominant cause of malaria, with an estimated 7–14 million cases of malaria each year [1,2]. P. vivax forms dormant liver stages (hypnozoites), that can reactivate weeks to months after the initial infection causing multiple relapses and febrile illness [3]. The only available compounds with activity against hypnozoites are primaquine (PQ) and tafenoquine (TQ) [4]. Both of these 8-aminoquinoline compounds cause oxidative stress that can result in severe haemolysis in patients with glucose-6-phosphate-dehydrogenase (G6PD) deficiency, an inherited enzymopathy, affecting approximately 400 million people globally [5,6].

G6PD is an essential enzyme in the pentose phosphate pathway, the only pathway for red blood cells (RBCs) to maintain their redox potential by reducing nicotinamide adenine dinucleotide phosphate (NADP+) to NADPH [7]. The G6PD gene is located on the X-chromosome, hence males are hemizygous and can be either G6PD deficient or normal, whereas females can be homozygous deficient, homozygous normal, or heterozygous for the gene [7]. The latter results in intermediate enzyme activity determined by the embryonic process of X-chromosome inactivation (lyonization) [5]. Mature RBCs do not have a nucleus and depend on the G6PD generated during erythropoiesis, hence reticulocytes and young RBCs have the greatest G6PD activity which decreases with the age of the RBC [7]. The risk of drug induced haemolysis is determined by the patients G6PD variant, the degree of enzyme activity, the degree of lyonization in females, the age of the red cell population, and the degree of exposure [8,9].

Malarial parasitaemia also causes haemolysis, arising from loss of both parasitized and unparasitized red blood cells [10,11]. In a recent study from Bangladesh G6PD activities differed significantly between patients with acute malaria and healthy individuals from the same area, whereas there was no significant difference in G6PD activity between individuals with and without a history of malaria in a case control study [12]. These findings suggest that Plasmodium spp. parasitaemia affects G6PD activity independent of any protective effect of G6PD deficiency from acute malaria [12-15]. In this longitudinal study we have quantified the change in G6PD activity before and after an acute episode of malaria in the same individuals.

Methods

Ethics statement

Ethical approval was provided by the Human Research Ethics Committee of the Northern Territory, Australia (HREC), the Ethical Review Committee of the ICDDR, B (Bangladesh), the Medical and Health Research Ethics Committee (Indonesia), and the National Research Ethics Review Committee (Ethiopia). Written informed consent was collected from all participants prior to enrolment, in case of minors written informed consent was collected from a legal guardian and written informed assent was also collected in case the minor was above 11 years of age (S1 Table).

Enrolment with malaria

Antimalarial clinical efficacy trials were conducted in Bandarban, eastern Bangladesh [16] (ClinicalTrials.gov Identifier: NCT02389374), Timika in southern Papua province, Indonesia (ClinicalTrials.gov Identifier: NCT02787070), and Arba Minch, in southern Ethiopia [17] (ClinicalTrials.gov Identifier: NCT01814683). At all three sites patients with uncomplicated malaria were enrolled following informed written consent from the patient or their legal guardian, pregnant and lactating women were excluded. In Indonesia and Bangladesh patients were infected with either P. falciparum or P. vivax, whereas in Ethiopia only patients with P. vivax were enrolled (S2 Table).

At enrolment a minimum of 3ml of venous blood were collected, hemoglobin (Hb) was measured, and malaria diagnosis was confirmed by blood film examination. All slides were read by two independent microscopists and the mean of both readings was recorded. Whenever discordant results were obtained, slides were re-read at a reference laboratory and the expert reading was considered as final. Remaining venous blood was stored in EDTA at 4–8°C and transported to a reference laboratory within 48 hours [18]. Hb was again measured immediately prior to spectrophotometry in the laboratory (S3 Table).

Patients were treated according to study protocols and monitored daily until aparasitemic and then followed weekly for 6 weeks in Bangladesh, 6 months in Indonesia, and 12 months in Ethiopia (S2 Table).

Repeat G6PD assessment during convalescence

A subset of patients enrolled into the original trials, who had been tested for G6PD activity, were contacted and those consenting to further investigation were enrolled into the longitudinal study. In Indonesia and Ethiopia all eligible individuals were approached. For logistical reasons, in Bangladesh a sample of 100 individuals (55%) with the lowest G6PD activity at baseline were selected, and patients who could not be contacted or declined to participate were replaced. Patients consenting to further investigation had a venous blood sample collected for repeat blood film examination or rapid diagnostic test for malaria and quantitative G6PD assay. Patients with peripheral parasitaemia by microscopy or RDT as well as patients with self-reported history of malaria within the last 90 days, or major blood loss were excluded (S3 Table and S1 Fig).

Assessment of G6PD activity

G6PD activity was measured on temperature-controlled spectrophotometers. At each site the same equipment and assay method was applied to assess the same individuals during an acute episode of malaria and again during follow up. In Bangladesh and Indonesia, measurements were done on a Shimadzu 1800 (Shimadzu, Japan) spectrophotometer and in Ethiopia a Humalyzer 3000 (Human, Germany) was used. Assay kits were supplied by Randox Laboratories (PD410, UK) in Bangladesh, and by Trinity Biotech (345-A, Ireland) in Indonesia and Ethiopia. Each measurement was done in duplicate, and whenever these differed by more than 15% a third measurement was undertaken. If all 3 measurements differed by more than 15% the sample was re-tested in duplicate. All G6PD results were normalized by Hb measured directly prior to spectrophotometry using a Hemocue 301, (Hemocue, Sweden). Other laboratory methods and follow up periods are presented in S3 Table.

G6PD normal and G6PD deficient controls were run throughout the clinical trial in Bangladesh (September 2014 until March 2015) and again during the period of convalescence review (March 2017 to May 2017). In Indonesia and Ethiopia G6PD normal, intermediate, and deficient controls were run in regular intervals throughout the duration of the study (October 2016 to May 2018 and January 2017 to October 2017, respectively) (S2 Fig and S4 Table).

Data management and statistical analysis

Data collected at enrolment were extracted from existing databases, including sex, age, G6PD activity, Hb concentration (at baseline and day 7), and species of infection.

The primary endpoint was G6PD activity measured during acute malaria and follow up. To pool enzyme activity across sites, individual G6PD activities were normalized and expressed as a fraction of 100% G6PD activity, defined as the adjusted male median in the local population (AMM) [19]. Since participants in Bangladesh were selected purposively 100% G6PD activity was defined as 7.03U/gHb based on results from a previous cross-sectional survey conducted in the same area using the same laboratory methods [20]. In Indonesia and Ethiopia 100% G6PD activity was defined as the AMM based on the follow up measurements. G6PD activity was categorized using the following cutoff values: <30% activity was defined as G6PD deficient, ≥30% to <70% as intermediate activity and ≥70% activity as G6PD normal.

The change in normalized G6PD activity (quantified as a percentage of the AMM) was analyzed using a linear mixed-effects model, with G6PD activity as the dependent variable, the categorical time point of measurement (malaria or follow up) and study site (Bangladesh, Ethiopia, and Indonesia) as fixed effects and the individual participant as random effect. The analysis was repeated stratifying by the species of initial infection. Since G6PD activity is normalized by Hb, changes in activity are vulnerable to associated changes in Hb concentration. Analysis was therefore repeated adjusting for the time-varying covariate, Hb concentrations (in g/dL), measured at the malaria episode and follow-up.

To assess whether the observed difference in activities was due to effects from regression to the mean (rtm), rtm effects were calculated considering 30% and 70% activity as cut-offs and the follow up measurement when participants were aparasitemic as baseline activity. The calculated rtm effect was then compared to the observed effect [21].

To assess within-individual agreement in classification of deficient, intermediate, and normal G6PD activity using measurements taken at time of malaria and follow up, the extended McNemmars test for correlated proportions was performed on the whole study population and stratified by species.

It was assumed that in the absence of haemolysis or major blood loss, G6PD activity does not vary significantly over time, and therefore G6PD activity during follow up reflects an individual’s G6PD activity at steady state. As a proxy for drug induced haemolysis the change in Hb between the day when Pq radical cure was first administered and seven days later was considered. Linear regression analysis was used to determine whether activity during steady state or the G6PD activity during the malaria episode was predictive of drug induced haemolysis. Hb concentration on day 7 was the dependent variable and G6PD activity during malaria and follow up were considered exposures after controlling for sex, parasite density, and Hb at treatment start [10,22]. Since day 7 Hb was not recorded for Indonesian patients this cohort was excluded from this analysis.

Results

Overall, 335 participants were included in the paired analysis, representing 27.4% of the 1,221 patients enrolled into the original clinical trials. In Bangladesh out of the planned 100 individuals 87 patients could be contacted and their identity verified (26.0%), while 75 (22.4%) patients from Indonesia and 173 (51.6%) patients from Ethiopia were included. In Bangladesh 33.3% (29/87) had a P. vivax infection, in Indonesia 49.3% (37/75) had a P. vivax infection and in Ethiopia only patients with P. vivax infection were enrolled (Table 1). The baseline characteristics were similar between the patients included in the analysis and the overall cohort, except for Hb readings in Indonesia and parasite density in Ethiopia.

Table 1

Characteristics of selected and non-selected participants per study site.

	Bangladesh		Indonesia		Ethiopia
	Excluded cohort	Selected Cohort	Excluded cohort	Selected Cohort	Excluded cohort	Selected Cohort
Number	94	87	591*	75	201	173
Females (%)	21 (22.3)	25 (28.7)	312 (52.8)	35 (46.7)	98 (49.5)	78 (45.1)
P.falciparum (%)	61 (64.9)	54 (62.1)	313 (52.3)	38 (50.7)	0 (0.0)	0 (0.0)
P.vivax (%)	26 (27.7)	29 (33.3)	261 (46.7)	37 (49.3)	198 (100.0)	173 (100.0)
Mixed infection (%)	7 (7.4)	4 (4.6)	24 (4.0)	0 (0.0)	0 (0.0)	0 (0.0)
Geometric mean (95% of range) parasite density for P.falciparum and mixed infections	3,924 (80–80,000)	8,903 (80–960,000)	4,532 (200–76,000)	5,314 (360–84,000)	-	-
Geometric mean parasite density (per μl) (95% of range) for P.vivax	2,920 (360–26,600)	2,507 (80–28,720)	3,796 (160–36,000)	2,236 (80–18,440)	15,433 (889–167,500)	9,143 (667–117,500)
Geometric mean parasite density (per μl) (95% of range) for mixed infections	6,124 (200–460,000)	24,878 (667–185,000)	894 (0–1,040)	0 (0–0)	-	-
Mean Hb in g/dL (95%CI) on day of enrolment	12.5 (12.1–12.9)	12.8 (12.3–13.3)	11.9 (11.8–12.1)	12.6 (11.9–13.2)	13.2 (13.0–13.4)	13.3 (13.0–13.4)
Median G6PD activity at enrolment in U/gHb (IQR)	8.8 (8.3–9.5)	7.5 (6.2–9.5)	-	10.3 (9.7 to 10.9)	10.8 (9.3–12.2)	11.5 (10.6 to 12.8)
100% G6PD activity (in U/gHb)**	7.03		9.24		12.13

*the clinical trial comprised of 420 participants and was complemented by an observational arm, G6PD activity was only measured in the selected cohort

** from literature in Bangladesh [23] and calculated from convalescent subset in Indonesia and Ethiopia

The median time between presentation with malaria and follow up varied significantly between sites: 887 days in Bangladesh (range: 790 to 998), 168 days in Indonesia (range: 140–168), and 176 days in Ethiopia (range: 169–183) (S2 Fig). Overall, the mean Hb concentration was lower at acute presentation compared to follow up (mean difference: -0.9 g/dL, 95% confidence interval [95%CI]: -1.1 to -0.6, p<0.001). This trend was apparent for patients enrolled in Bangladesh (-1.3 g/dL, 95%CI: -1.7 to -0.9, p<0.001) and Ethiopia (-1.1 g/dL, 95%CI:-1.3 to -0.9, p<0.001), but was not significant in Indonesia (0.2 g/dL, 95%CI: -0.5 to 0.9, p = 0.555).

Changes in acute and convalescent G6PD activity

Based on results from a previous cross sectional study in the same population 100% G6PD enzyme activity was defined as 7.03 U/gHb in Bangladesh [23]; based on the G6PD measurements during follow up 100% activity was defined as 9.24U/gHb in Indonesia, and 12.13 U/gHb in Ethiopia (Table 1). When comparing the same individual’s G6PD activity during malaria and again during follow up, enzyme activity was -10.4% (95% confidence interval [95%CI]: -13.9 to -6.9) lower during follow up. The decrease in activity from malaria to follow-up was greatest in those classified as G6PD deficient at follow-up (<30% activity) (-79.1%, 95%CI: -117.8 to -40.4), compared to -43.7% (95%CI: -53.1 to -34.2) in those with intermediate deficiency at follow-up (≥30% to <70% activity), and -4.5% (95%CI: -7.6 to -1.4) in those who were G6PD normal at follow-up (≥70% activity) (Fig 1).

Fig 1

Change in G6PD activity between the malaria episode and follow up, stratified by G6PD status at the time of follow up.

Only one male patient with P. vivax mixed infection from Bangladesh was deficient and his activity did not change between enrolment and follow up (0.7U/gHb at both time points), while activity decreased by -95.0% (95%CI: -126.5 to—63.5) in the 5 deficient patients with P. falciparum infection. Among the 17 intermediate patients with P. vivax mono or mixed infection, activity was -49.4% (95%CI: -61.1 to -37.8) lower at the time of follow up, while activity dropped by -39.2% (95%CI: -52.8 to -25.6) in the 22 intermediate patients with P. falciparum mono-infection (Table 2).

Table 2

Percentage Change (95% Confidence Intervals) in G6PD activity between acute malaria and follow up.

Sites	Species	Deficient*	Intermediate*	Normal*
All Countries	All species (95%CI, n):	-79.1 (-117.8 to -40.4, 6)	-43.7 (-53.1 to -34.2, 39)	-4.5 (-7.6 to -1.4, 290)
	P. vivax mono and mixed infections (95%CI, n)	-0.3 (n = 1)	-49.4 (-61.1 to -37.8, 17)	-1.4 (-4.6 to 1.7, 225)
	P. falciparum mono-infection (95%CI, n)	-95.0 (-126.5 to -63.5, 5)	-39.2 (-52.8 to -25.6, 22)	-15.2 (-23.0 to -7.3, 65)
Bangladesh	All species (95%CI, n)	-79.1 (-117.8 to -40.4, 6)	-48.5 (-58.9 to -38.0, 33)	-30.7 (-39.1 to -22.3, 48)
	P. vivax mono and mixed infections (95%CI, n)	0.3 (n = 1)	-56.3 (-70.2 to -42.3, 14)	-34.1 (-48.6 to -19.6, 18)
	P. falciparum mono-infection (95%CI, n)	-95.0 (-144.9 to -45.1, 5)	-42.7 (-60.7 to -24.7, 19)	-28.7 (-40.2 to -17.2, 30)
Ethiopia	All species (95%CI, n)	NA (n = 0)	1.2 (n = 1)	3.6 (1.0 to 6.1, 172)
	P. vivax mono and mixed infections (95%CI, n)	NA (n = 0)	1.2 (n = 1)	3.6 (1.0 to 6.1, 171)
	P. falciparum mono-infection (95%CI, n)	NA (n = 0)	NA (n = 0)	NA (n = 0)
Indonesia	All species (95%CI, n)	NA (n = 0)	-21.0 (-36.4 to -5.5, 5)	-6.4 (-14.0 to 1.2, 70)
	P. vivax mono and mixed infections (95%CI, n)	NA (n = 0)	-26.7 (-34.2 and -19.3, 2)	-9.2 (-21.4 to 3.0, 35)
	P. falciparum mono-infection (95%CI, n)	NA (n = 0)	-17.0 (-37.6 and -25.0 and 11.6, 3)	-3.6 (-13.6 to 6.5, 35)

*Defined during follow up

The calculated rtm effect for deficient individuals was -60.9% (95%CI: –71.4 to -50.5, p<0.001) at the 30% activity threshold and -31.4% (95%CI: -36.8 to -25.9, p<0.001) at the 70% threshold.

The greatest mean difference between initial presentation with malaria and follow up was observed in Bangladesh (-40.9%, 95%CI: -48.1 to –33.4), followed by Indonesia (-7.4%, 95%CI: -14.6 to -0.2), while in Ethiopia G6PD activity was slightly lower during acute malaria compared to follow up (3.6%, 95%CI: 1.0 to 6.1); Fig 2.

Fig 2

Box and Whiskers plot of median normalized G6PD activity during malaria and follow up per study site among selected participants.

The country specific drop in activity corresponded to the proportion of participants with decreased activities enrolled. In Bangladesh 33 (37.9%) individuals were categorized with intermediate G6PD deficiency and 6 (6.9%) with severe deficiency. The corresponding proportions were significantly lower in Indonesia (6.7% and 0.0%) and Ethiopia (0.6% and 0.0% respectively). The proportions of participants categorized as normal, intermediate, and deficient varied significantly between time points (p<0.001). Overall, 4 of the 6 participants (66.7%) classified as G6PD deficient during the follow up were categorized as G6PD normal and 1 (16.7%) as G6PD intermediate at time of their acute presentation. Among 39 individuals classified as having intermediate activity, 34 (87.2%) were categorized as G6PD normal during acute malaria, whereas none were categorized as G6PD deficient. Conversely of the 290 individuals categorized as G6PD normal during follow up, 3 (1.0%) participants were classified as G6PD intermediate during the malaria episode. All 3 participants were enrolled in Indonesia and had between 60% and 70% activity during enrolment. The shift in categories was significant in individuals presenting with P. vivax and P. falciparum infection and in Bangladesh (Table 3).

Table 3

Participants categorized by G6PD status during acute malaria and follow up stratified by species and site.

			G6PD activity during follow up
			Deficient (%)	Intermediate (%)	Normal (%)	Total (%)
G6PD activity during acute malaria	All species, n = 347	Deficient	1 (16.7%)	0 (0.0%)	0 (0.0%)	1 (0.3)
		Intermediate	1 (16.7%)	5 (12.8%)	3 (1.0%)	9 (2.7)
		Normal	4 (66.7%)	34 (87.2%)	287 (99.0%)	325 (97.0)
		Total (%)	6 (1.8)	39 (11.6)	290 (86.6)	335 (100.0, p<0.001)
	P. vivax mono and mixed infections, n = 247	Deficient	1 (100.0%)	0 (0.0%)	0 (0.0%)	1 (0.4)
		Intermediate	0 (0.0%)	1 (5.9%)	3 (1.3%)	4 (1.7)
		Normal	0 (0.0%)	16 (94.1%)	222 (98.7%)	238 (97.9)
		Total (%)	1 (0.4)	17 (7.0)	225 (92.6)	243 (100.0, p = 0.003)
	P. falciparum mono-infection, n = 100	Deficient	0 (0.0%)	0 (0.0%)	0 (0.0%)	0 (0.0)
		Intermediate	1 (20.0%)	4 (18.2%)	0 (0.0%)	5 (5.0)
		Normal	4 (80.0%)	18 (81.8%)	65 (100.0%)	95 (95.0)
		Total (%)	5 (5.4)	22 (23.9)	65 (70.7)	92 (100.0, p<0.001)
	Bangladesh, n = 87	Deficient	1 (100.0)	0 (0.0)	0 (0.0)	1 (1.2)
		Intermediate	1 (25.0)	2 (50.0)	1 (25.0)	4 (4.6)
		Normal	4 (4.9)	31 (37.8)	47 (57.3)	82 (94.3)
		Total (%)	6 (6.9)	33 (37.9)	48 (55.2)	87 (100.0, p<0.001)
	Ethiopia, n = 172	Deficient	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
		Intermediate	0 (0.0)	1 (100.0)	0 (0.0)	1 (0.6)
		Normal	0 (0.0)	0 (0.0)	172 (100.0)	172 (99.4)
		Total (%)	0 (0.0)	1 (0.6)	172 (99.4)	173 (100.0, p = 1.00)
	Indonesia, n = 75	Deficient	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
		Intermediate	0 (0.0)	2 (50.0)	2 (50.0)	4 (5.3)
		Normal	0 (0.0)	3 (4.2)	68 (95.8)	71 (94.7)
		Total (%)	0 (0.0)	5 (6.7)	70 (93.3)	75 (100.0, p = 0.655)

Deficient: <30% G6PD activity, Intermediate: ≥30% to <70% G6PD activity, Normal: ≥70% G6PD activity

In total 30 patients from Bangladesh and 172 patients from Ethiopia had a Hb concentration measured at enrolment and seven days after starting PQ based radical cure. Their Hb dropped by a mean of 0.1 g/dL (95%CI: -0.3 to 0.0) in the first seven days of treatment. A 10% increase in G6PD activity at enrolment was associated with a drop in day 7 mean Hb of -0.04 g/dL (95%CI: -0.13 to 0.06, p = 0.471) and a 10% increase in G6PD activity during steady state was associated with a decrease of 0.02 g/dL (95%CI: -0.11 to 0.07, p = 0.611).

Discussion

Our study highlights that G6PD activity in the same individual can differ significantly when measured during acute presentation with malaria and after recovery. Overall an individual’s G6PD activity was 10% greater when measured during an acute episode of malaria compared to when remeasured during follow up. When G6PD status was categorized according to an individual’s enzyme activity during follow up, two thirds of participants with severe deficiency and almost 90% of those with intermediate deficiency had normal enzyme activity (>70%) at the time of presentation with malaria. The overall change in activity was more pronounced for patients with P. vivax infection, however the differences in an individual’s G6PD status were apparent in patients presenting with P. vivax and those presenting with P. falciparum infection. Among selected participants the greatest change in G6PD activity was observed in Bangladesh where a large proportion of deficient and intermediate individuals were included, followed by Indonesia where two out four participants with intermediate activities had normal activities when revisited. In Ethiopia only one individual had reduced G6PD activity during follow up, a 25-year-old male with 61% enzyme activity during malaria and 62% during follow up. Whilst the overall mean G6PD activity in Ethiopian participants was 4% higher during follow up compared to during the malaria episode, this modest rise was not clinically relevant. This is also reflected when considering the change in in G6PD category between enrolment and follow up. There was a significant difference in participants G6PD status between enrolment and follow up, however this only reached statistical significance in Bangladesh where 45% of participants were G6PD deficient during follow up. In contrast only 1% of participants in Ethiopia and 7% in Indonesia had enzyme activities <70% during follow up.

G6PD is essential for the production of glutathione, which binds free radicals and protects cells from oxidative stress [7,9,24]. Since mature RBCs do not have a nucleus, they depend on the G6PD synthesised during erythropoiesis, hence enzyme activity is highest in reticulocytes and young RBCs, and subsequently falls as erythrocytes age [7]. During an acute P.vivax or P.falciparum infection, loss of RBCs occurs from haemolysis of both parasitized and unparasitized erythrocytes [10,25-27]. The ratio of infected to uninfected RBCs lost is estimated at 1:34 for P.vivax and 1:8 for P. falciparum [28]. While the underlying mechanisms for the loss in unparasitized RBCs is not fully understood [29], it has been attributed to oxidative membrane damage induced by the malaria parasite [30]. G6PD plays a crucial role in maintaining RBC redox potential and reducing oxidative stress, thus as G6PD activity decreases with RBC age, older cells are preferentially removed from the peripheral blood [8]. Furthermore parasite induced hemolysis results in a decrease in Hb concentration and oxygen delivery that triggers reactive erythropoiesis and production of young RBCs with higher G6PD activity [10], hence the mean age of the hosts RBC population decreases and the overall G6PD activity increases.

The observation that G6PD activity increases during acute malaria is not unexpected, however the degree to which this occurred in our study was surprising with potentially important public health implications. The prevalence of G6PD deficiency is highest in P. vivax endemic areas and can result in healthcare providers being reluctant to prescribe radical cure due to fears of drug induced haemolysis [31,32]. Our findings suggest that in patients presenting with P. vivax malaria the prevalence of severe and intermediate G6PD deficiency is considerably lower than that predicted from cross sectional surveys; if confirmed these differences will influence population-based risk and cost -benefit estimates for different test and treat strategies for primaquine radical cure [33]. Our findings also support previous models proposing a delay in starting hypnozoitocidal treatment start until the end of schizontocidal treatment (once the most deficient RBCs have been lysed), to reduce drug induced haemolysis [34].

We were unable to determine whether G6PD activity during malaria or follow up was predictive of haemolytic risk following PQ treatment, since only two (1.0%) out of the 202 participants with Hb measured on baseline and day 7 who received 14-day primaquine had intermediate activities during malaria and none were deficient. The degree to which transient increases in G6PD activity alter the risks of drug induced haemolysis will require further prospective clinical studies.

Current WHO guidelines recommend testing for G6PD deficiency prior to starting primaquine so that individuals at greatest risk of haemolysis can be given alternative treatment [35]. Our findings raise the possibility that aparasitemic individuals identified as severely or intermediate G6PD deficient may not necessarily be at high risk of drug induced haemolysis during malaria. A total of 34 out of 39 participants classified as intermediate deficient when aparasitemic had normal activities when enrolled with malaria, 4 out of 6 participants diagnosed as deficient when aparasitemic were G6PD normal when tested during acute malaria. Conversely 287 out of 290 individuals categorized as G6PD normal during follow up had no change in G6PD status when presenting with malaria. All three participants with normal G6PD activities during malaria and intermediate activities during follow up, had borderline G6PD activity (60% to 70%) at the first measurement and the mean increase in G6PD activity during follow up was less than 20%.

In a recent study from Cambodia, G6PD activity was measured over an 8-week period in patients with P. vivax malaria treated with weekly PQ [36]. G6PD activity increased between the first and second PQ dose, with the greatest change in activity observed in deficient patients. G6PD activity did not change markedly throughout the rest of the treatment period irrespective of G6PD status, likely due to the weekly PQ doses continuously hemolyzing RBCs with low G6PD levels [37]. G6PD activity was not measured again following completion of treatment and subsequent return to steady state, so the baseline normal state of these participants could not be determined.

In our study 12% of males had intermediate G6PD activity (30%-70%) recorded during follow up (S5 Table); this was most apparent in Bangladesh where patients with lowest G6PD activities were purposively selected. The G6PD gene is located on the X-chromosome and males are thus either hemizygous deficient or normal [8], however intermediate activities have been reported from previous cross-sectional surveys [23,38,39] and may reflect the AMM (100% activity) being calculated from heterogenous populations with G6PD variants associated with different severity.

Our study has several important limitations. First, the study adopted an opportunistic design, revisiting individuals who had been enrolled into previous antimalarial clinical trials. Since only patients initially presenting with malaria were enrolled and there were no aparasitemic controls, one cannot definitively infer that initial elevations in G6PD activity were attributable to the initial peripheral parasitaemia. Spectrophotometry procedures at the study sites could have changed, systematically altering either rounds of G6PD measurement which in Bangladesh occurred more than 2 years after the initial presentation [40]. Reassuringly quality control procedures suggested consistent testing throughout the study period at all three sites. There was a small but significant trend in deficient control isolates in Indonesia, but this would not have affected the outcome since none of the patients enrolled were severely deficient at either time point. Secondly, G6PD activity was analyzed after normalizing for Hb concentration, hence changes in the Hb between the first and second measurement could have impacted the observed changes in G6PD activity. However, after adjusting for Hb concentration in the multivariable analysis the estimated changes in G6PD activity levels between the acute malaria episode and follow-up were still apparent. Thirdly, the time to follow up differed significantly between sites. Haematological recovery following acute malaria usually occurs within 28–63 days [41,42], although after haemolytic events it can take significantly longer for the age distribution of RBC to return to steady state [43]. The primary objective of the current study was to resample patients following complete haematological recovery and during a period when the patients were aparasitaemic with no recent history of major blood loss or malaria within the previous 3 months, and this was achieved in all study sites with a minimum time to resampling of 140 days.

Fourth our analysis did not include genetic confirmation of the diagnosis of individuals with G6PD deficiency. More than 230 clinically relevant G6PD variants have been described [8,44] that confer different degrees of enzymatic deficiency. The observed changes in enzyme activity associated with acute haemolysis may differ between G6PD variants. For example, for individuals with the Mediterranean variant almost all RBCs, including young cells, are severely deficient and thus parasite or drug induced haemolysis will have a minimal impact on the overall activity, whereas for individuals with milder variants, G6PD activity would be more likely to normalize. [45,46]. Hence observed changes in enzyme activity may differ significantly by region.

Finally, the observed difference in activities could have arisen from regression to the mean, with extreme values measured during follow up becoming less extreme when measured again during acute malaria [21]. However, at both the 30% and 70% definitions for G6PD deficiency the calculated rtm effect was below the observed effect. Keeping the small sample size of deficient individuals in mind, this suggests a true trend.

Our study is confounded by heterogeneity between study sites, selection bias (specifically in Bangladesh), differences in prevalence of G6PD deficiency, potentially known local variants, and potential regression to the mean. Despite these limitations, the changes in G6PD activity were apparent in varying degrees across all three sites and highlight that a substantial proportion of individuals with severe and intermediate G6PD deficiency were diagnosed as G6PD normal when presenting with acute malaria. Subsequent prospective case-control studies, with molecular confirmation of genetic variants, are warranted to confirm and quantify these findings in other endemic settings.

In conclusion, our study highlights that an individual’s G6PD activity is significantly higher during acute malaria, particularly those with intermediate or severe G6PD deficiency. If these findings are confirmed, it raises the possibility that an individuals’ G6PD status is not static, and thus the risk of drug induced haemolysis when presenting with malaria cannot be assumed from measuring an individual’s enzyme activity when aparasitaemic. From a public health perspective, the population attributable risks of drug induced haemolysis may be significantly lower than that predicted from cross sectional surveys.

Ethical boards and protocol numbers.

(DOCX)

Click here for additional data file.

Study details on studies from which patients were recruited.

* This trial is complemented by an observational arm with a total sample size of 420 that is not registered with Clinicaltrials.gov. *** Not applicable for complementary arm **this site is part of a multi-center trial, however measurements relevant to this study were only performed at this site.

(DOCX)

Click here for additional data file.

Site specific procedures.

(DOCX)

Click here for additional data file.

Quality control for spectrophotometry.

(DOCX)

Click here for additional data file.

Sex distribution per G6PD status based on G6PD activity during follow up.

(DOCX)

Click here for additional data file.

Consort chart.

(TIF)

Click here for additional data file.

Results of quality control testing througout the study period at the study sites.

Legend: top horizontal line: line of best fit for G6PD normal controls, center horizontal line: line of best fit for G6PD intermediate controls (not done in Bangladesh), lowest horizontal line: line of best fit for G6PD deficient controls; vertical line (Bangladesh) follow up period where no testing was done

(TIF)

Click here for additional data file.

Days between enrolment and follow up.

(TIF)

Click here for additional data file.

Underlying database.

(XLS)

Click here for additional data file.

1 Dec 2021

Dear Dr Ley,

Thank you very much for submitting your manuscript "Variation in Glucose-6-Phosphate Dehydrogenase activity following acute malaria" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Hans-Peter Fuehrer

Deputy Editor

PLOS Neglected Tropical Diseases

Hans-Peter Fuehrer

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: “A subset of patients enrolled in the original trials were reviewed again 6 to 33 months after enrolment.” This statement is rather vague. Please indicate clearly how participants in the study were selected. Please provide a “consort chart” illustrating how the study participants were assembled? It may be necessary to provide separate charts for each site.

Please state clearly inclusion/exclusion criteria? If the criteria differ between sites, please provide a table with the criteria for each site?

“Since participants in Bangladesh were selected purposively …” I can’t follow this argument – please explain?

Reviewer #2: Methods; line 116: clarify better for the different studies how the subset were selected for the follow-up. In Ethiopia/Indonesia, was it just based on participant contactability? This matters given importance of the AMM estimates on the results. Also please clearly state the participants’ health status at the 2nd time point.

To avoid misinterpretation, it would be good to emphasise that the rates of deficiency seen at the two time points are based on selected sample groups: “among selected patients” (only G6PD normal indivs recruited in Ethiopia/Indonesia at first timepoint; G6PD deficient patients preferentially included in Bangladesh). E.g. line 57 in Abstract, Fig 1 legend, Line 243 “selectively enrolled”.

Reviewer #3: The design is not ideal to address the stated objectives as there are no control cases in the study.

The statistics presented are misleading and in some places inappropriately used to substantiate the authors conclusions

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: Table 1: please explain what you mean by “unselected cohort”? I assume you mean excluded? Please highlight the this excluded cohort in the consort chart?

“The time between presentation with malaria and follow up ranged between 790 and 998 days in Bangladesh, 140 and 168 days in Indonesia, and 169 and 183 days in Ethiopia.” Please provide a more detailed statistical description of the distribution of F/U dates. Pls. consider providing a Figure to illustrate the distributions.

“The greatest difference between acute presentation and follow up was observed in Bangladesh” not only was this difference greatest in Bangladesh. The time window between 1st and 2nd measurement was also by the widest. Perhaps the extended duration explains the differences in enzyme activity? Pls. explain why you think this is not the case?

“Figure 2: Change in G6PD activity between malaria and follow up, stratified by G6PD status during follow up” Pls. rephrase the title? … between the malaria episode and follow up, stratified by G6PD status at the time of follow up

Pls consider a similar analysis as presented in Fig 2 instead of stratified G6D status stratified by site? (at a minimum include table S5 in the main text.)

“The estimated mean change in Hb associated with an increase of 10 % units in G6PD activity was -0.05 g/dL (95% CI: -0.14 to 0.05, p=0.336) at enrolment and -0.03 g/dL (95% CI: -0.11 to 0.06, p=0.560) at follow up.” This observation could be interesting but seems to be difficult to understand the way it is presented. The change in enzyme activity is over the time period from malaria episode to F/U date? Why don’t you report the Hb change over the same period?

Reviewer #2: The implications of the results are most significant for P. vivax patients. Would there be statistical power to allow a subanalysis with only Pv patients?

Supplementary figure 1: suggest including control recommended ranges to help interpret the variation seen. Could also add control manufacturer names to plot titles.

Reviewer #3: The results are clearly presented but the analysis is misleading

A major limitation in the analysis is the fact that the analysis (change in G6PD activity) per Plasmodium species is not presented, and the fact that the one site with only P.vivax infections does not support their conclusions is not discussed.

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: Discussion

“Activity increased by 11% overall, ranging by almost 80% in deficient individuals, 45% in

intermediate individuals, and 5% in individuals categorized as G6PD normal during follow up.” This sentence can be interpreted in different ways. Either the enzyme activity increased during dollow up or the individuals were categorized as normal during follow up. Please consider rewriting to provide more clarity?

“during health” is poor English – pls. rephrase?

“Over 80% of study participants diagnosed with less than 70% activity during follow up would have

been denied primaquine radical cure if they had not been re-tested.” In this sentence the temporal relationship between the first test and the second test is the wrong way around. Fewer readers may be confused if you would consider rephrasing? “Over 80% of study participants diagnosed with less than 70% enzyme activity during follow up would have been denied the radical cure if health care providers would rely solely on the follow-up test instead of the test result at the time of the acute malaria episode”.

“Reassuringly quality control procedures suggested reliable testing throughout the study period at all three sites. ‘ perhaps the keyword here is “consistent” rather than “reliable”?

Please discuss the advantages/disadvantages of combining data from different sites when the data are so different. Isn’t that comparing “apples with oranges”?

Conclusions:

“Our findings have important public health implications for identifying populations at risk of haemolysis, as well as test and treat strategies for radical cure.” This sentence is meaningless – why waste the time of the reader? Please clearly state the implications!

Reviewer #2: (No Response)

Reviewer #3: The conclusions are not supported by the data presented.

The limitations are not clearly described

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: Abstract

“Patients with uncomplicated malaria were recruited” should be Patients with uncomplicated vivax malaria …

“At the time of follow up” … please indicate the median follow-up time?

“… this may have implications for risk of drug induced haemolysis.” Many readers focus on the conclusions provided in the abstract. The conclusions as the are currently written are not helpful. Please indicate which implications do you have in mind? Are they good, bad, or neutral? You write in the discussion “in patients presenting with malaria the prevalence of severe and intermediate G6PD deficiency is considerably lower than that predicted from cross sectional surveys” which frequently guide prescription practices. Your study suggests that these prescription practices may be overly cautious. Please dare to be controversial.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: (No Response)

Reviewer #2: This is a very worthwhile study that carries important implications for assessing population-level P. vivax treatment risks. The manuscript is clearly presented, and the interpretation of results a good representation of the presented data.

Patients’ genetic variants were not characterised. If available, this would have been valuable additional data to help interpret the observations: authors could perhaps mention this in their discussion. It matters because the global diversity of G6PD mutations means that the trend observed in this study from these 3 sites may not be replicated everywhere. E.g. would this same difference be visible in patients with the Mediterranean mutation? Or is the effect less pronounced in patients where even young RBCs have low G6PD activity?

Minor comments:

Line 50: “replacing”: implies direct causation, suggest “leading to replacement of…”

Lines 87-90: for clarity, suggest listing references mid-sentence

Line 112: not sure S2 table is right one to link to here?

Line 164: day 7 testing not previously mentioned; Methods implies all testing was on day 0.

Line 170: typo: “Norther Territory”

Line 269: “support previous model ?recommendations”?

Line 286 and 301: check wording

Reviewer #3: In this article the authors present very interesting data on the difference in G6PD activity in individuals when malaria positive versus when malaria negative. The data is interesting and the sheer number of cases for which they have this matched data could be useful. There are however some deficiencies in the data that need to be clarified and perhaps some issues in the manner in which the data is presented that should be resolved. The presentation of the statistics seems to be somewhat leading and possibly not necessarily supported by the data presented.

Major limitation of the study design:

The study recruited the patients as malaria positive and then conducted a follow-up visit with a wide ranging period of time between the two visits. The study does not have a control arm of healthy individuals at the time of recruitment and time of follow-up. This is significant because all of the significant changes in G6PD status arise from the one site, Bangladesh, which noticeably (i) had the widest time gap between malaria and follow-up visits and (ii) was the only site to use the Randox assay and (iii) it is the one site where “ In Bangladesh participants were chosen to include the fraction with lowest G6PD activities during enrolment” so these will have had most samples that were on or close to the G6PD deficient/intermediate/normal thresholds. A lot of the differences may arise from slight differences in the Randox assay lots, the individuals/details of how the tests were run or differences in how the specimens were handled which would not be identified by the test controls. The AMM from the follow-up visits were used to determine the percent activity across both malaria and follow-up visits, so any bias resulting for the time lag would accentuate those observed. Without a control arm this cannot be resolved. These limitations should be specifically called out as they represent a significant limitation to the analysis.

Noticeably the one site, Ethiopia where only P.vivax cases were collected showed the median G6PD activity increases in the follow-up. Which leads to another major limitation with regards to how the data is presented. The authors make their claims based on combined P.vivax and P.falciparum infections while their discussion is only relevant to P.vivax infections. It is surprising that the authors do not present the data disaggregated by species. Specially as the inherent (and not mentioned) assumption that both species have the same hematological effects is not addressed. The 5 samples that are G6PD deficient in the follow-up and not during the malaria infection, were all from Bangladesh and presumably (based on the discussion) in P.falciparum infections.

Specifically:

Major limitations:

1. The issue that there is no control arm for in this study should be clearly called out as it is a major limitation given the reasons mentioned above

2. The authors should present the change in G6PD activity disaggregated by species, P.falciparum and P.vivax and discuss the hemolytic risks according to the P.vivax results.

a. If there are differences observed here they should be discussed and the relevance to hemolytic risk also updated accordingly

3. In the abstract and discussion the authors should clarify that the significant drop in G6PD activity leading to different classifications almost entirely arise from the Bangladesh data (which has the above mentioned biases and limitations) and not from all three sites. The discussion should explain why the trend is only observed at one of the three sites, and specifically not observed in the site with only P.vivax infections (Ethiopia). The way the data is presented is misleading in that it suggests that this an observation across all sites.

4. Abstract lines 61 to 63: “67% (4/6) of deficient and 89% (36/41) of…” is an example of misleading statistics. Most of these cases (the 4 and 36) arise from the Bangladesh study in which all except one deficient and 4 intermediates were normal and so they could only go one way in the follow up visit. The Ethiopia and Indonesia study included all normal by FST so again this sample set was bias to only have switches towards intermediate or deficient cases.

5. Author summary lines 40-42: “ …85% (40 / 47) of individuals ineligible for standard treatment during follow up, were eligible for treatment during malaria.” This is again misleading (assuming all the other issues are OK) since actually the 36 with 30-70% G6PD activity are eligible in most countries to 14 day primaquine.

6. As per the above statement in the discussion lines 279-280, 70% is not the threshold for withholding primaquine, only those with G6PD activity below 30% would have primaquine withheld according to WHO guidelines.

7. Line 282: “All three participants with lower G6PD activities during malaria compared to follow up,…” this seems to be misleading since their supplemental data and their Ethiopia data (Figure 1) which is relevant since these are all P.vivax cases there are than 3 cases lower G6PD activities compared to follow up.

--------------------

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Figure Files:

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3 Feb 2022

Submitted filename: Rebuttal_v0.5.docx

Click here for additional data file.

18 Mar 2022

Dear Dr Ley,

Thank you very much for submitting your manuscript "Variation in Glucose-6-Phosphate Dehydrogenase activity following acute malaria" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Dear Authors,

Reviewer 3 lists several limitations of this study Please critically discuss.

BW

Hans-Peter Fuehrer

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Hans-Peter Fuehrer

Deputy Editor

PLOS Neglected Tropical Diseases

Hans-Peter Fuehrer

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Dear Authors,

Reviewer 3 lists several limitations of this study Please critically discuss.

BW

Hans-Peter Fuehrer

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: (No Response)

Reviewer #2: The methods are clearly presented.

Reviewer #3: (No Response)

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: (No Response)

Reviewer #2: Results and associated figures are clear

Reviewer #3: (No Response)

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: (No Response)

Reviewer #2: The conclusions correspond well to the results described and reflect their public health significance. The authors have added substantially to their discussion of the study limitations which was needed. These detailed limitations also now lay out for readers the challenge to carry out robust follow-on studies using prospective study designs in diverse epidemiological settings.

Reviewer #3: (No Response)

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: Modification made to Line 203-204: this is now less clear, suggest simplifying to “10.4% higher during follow-up compared to at the time of acute malaria infection”

Reviewer #3: (No Response)

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The authors have addressed all of my suggestions adequately.

Reviewer #2: The authors have significantly revised the manuscript to address the previous reviews, which has improved it; the paper will make a valuable contribution to the literature with potentially important public health implications.

Reviewer #3: The authors have tried to address many of the issues raised by the reviewers. Several concerns still remain.

1. Bangladesh data set: There are several concerns with this data set and how it is presented. The follow-up data set has a median (and mean) G6PD value approximately 2.5 U/gHb lower than the acute malaria set and almost 2.0 U/g Hb lower than that of 100% normal used to determine the percent change in activity. In contrast the malaria and healthy G6PD medians are within 0.5 U/g Hb of each other and equally close to that of the authors 100% normal. This has several implications: (i) the Bangladesh data set is an outlier compared to the other two, and extremely unusual, (ii) using a 100% normal G6PD activity level that is 2 U/gHb or 40% greater than the population median does not make, by definition, any sense and will only exacerbate any difference with the acute malaria cases where the median was above the 100% normal, (iii) the bias generated by this will significantly and disproportionately affect any pooled analysis by species as presented by the authors in Table 2.

2. Bangladesh data set: The authors only included G6PD deficient cases identified during the follow-up visit, and no deficient cases identified during the acute malaria phase, so inherently any analysis of the deficient cases will be biased and misleading. As the authors also point out, none of these were confirmed by sequencing, so it is just as likely that these are an artefact of the low G6PD values generated by the reference assay during this period. The controls seem to work well so possibly there was a specimen integrity issue. An indication that this sample set is strange is the data in supplementary table 5 with more “intermediate” males than females.

3. Indonesia and Ethiopia: Given the outlier characteristics of the Bangladesh data set it is worth (and even in absence of that) looking at the G6PD levels per species per site and Ethiopia and Indonesia pooled. When this is done, at least by median G6PD values there is no significant difference in G6PD values between acute and follow-up visits, maybe a slight decrease for Pf (in Indonesia) and slight increase for Pv (in BOTH Indonesia and Ethiopia). By presenting the data the way it is presented in Table 2, rather than by species by site, as it would typically be done in this kind of analysis, this is not shown.

4. Indonesia and Ethiopia: When only looking at these two data sets, the only changes in status is for 6 individuals, 3 of which are males so actually no change in status, and all males and females would have received primaquine. These changes in status maybe attributed to variation in reference testing

5. There is no biological or pharmacological sense in pooling the analysis across both species. Pv infects almost primarily reticulocytes and at lower parasite densities compared to Pf which infects a broader range of red blood cells and at higher parasite density. These differences are likely to have an impact on the relative effect of infection on G6PD activity levels, which their data (Indonesia and Ethiopia) actually seems to show. Notably (even in Bangladesh) all clinically relevant changes in G6PD activity are in Pf.

6. None of the studies included deficient cases in the acute phase of malaria which was when the study participants were recruited. So any analysis based on these classifications is inherently biased. Changes in G6PD activity per site per species across all study participants and the statistical significance should be shown. And possibly pooled for Ethiopia and Indonesia.

--------------------

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

6 Apr 2022

Submitted filename: Rebuttal2_v0.4.docx

Click here for additional data file.

8 Apr 2022

Dear Dr Ley,

We are pleased to inform you that your manuscript 'Variation in Glucose-6-Phosphate Dehydrogenase activity following acute malaria' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Hans-Peter Fuehrer

Deputy Editor

PLOS Neglected Tropical Diseases

Hans-Peter Fuehrer

Deputy Editor

PLOS Neglected Tropical Diseases

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26 Apr 2022

Dear Dr Ley,

We are delighted to inform you that your manuscript, "Variation in Glucose-6-Phosphate Dehydrogenase activity following acute malaria," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Editorial, Viewpoint, Symposium, Review, etc...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript will be published online unless you opted out of this process. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases