Dynamics of G6PD activity in patients receiving weekly primaquine for therapy of Plasmodium vivax malaria.

BACKGROUND: Acute Plasmodium vivax malaria is associated with haemolysis, bone marrow suppression, reticulocytopenia, and post-treatment reticulocytosis leading to haemoglobin recovery. Little is known how malaria affects glucose-6-phosphate dehydrogenase (G6PD) activity and whether changes in activity when patients present may lead qualitative tests, like the fluorescent spot test (FST), to misdiagnose G6PD deficient (G6PDd) patients as G6PD normal (G6PDn). Giving primaquine or tafenoquine to such patients could result in severe haemolysis.
METHODS: We investigated the G6PD genotype, G6PD enzyme activity over time and the baseline FST phenotype in Cambodians with acute P. vivax malaria treated with 3-day dihydroartemisinin piperaquine and weekly primaquine, 0·75 mg/kg x8 doses.
RESULTS: Of 75 recruited patients (males 63), aged 5-63 years (median 24), 15 were G6PDd males (14 Viangchan, 1 Canton), 3 were G6PD Viangchan heterozygous females, and 57 were G6PDn; 6 patients had α/β-thalassaemia and 26 had HbE. Median (range) Day0 G6PD activities were 0·85 U/g Hb (0·10-1·36) and 11·4 U/g Hb (6·67-16·78) in G6PDd and G6PDn patients, respectively, rising significantly to 1·45 (0·36-5·54, p<0.01) and 12·0 (8·1-17·4, p = 0.04) U/g Hb on Day7, then falling to ~Day0 values by Day56. Day0 G6PD activity did not correlate (p = 0.28) with the Day0 reticulocyte counts but both correlated over time. The FST diagnosed correctly 17/18 G6PDd patients, misclassifying one heterozygous female as G6PDn.
CONCLUSIONS: In Cambodia, acute P. vivax malaria did not elevate G6PD activities in our small sample of G6PDd patients to levels that would result in a false normal qualitative test. Low G6PDd enzyme activity at disease presentation increases upon parasite clearance, parallel to reticulocytosis. More work is needed in G6PDd heterozygous females to ascertain the effect of P. vivax on their G6PD activities. TRIAL REGISTRATION: The trial was registered (ACTRN12613000003774) with the Australia New Zealand Clinical trials (https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=363399&isReview=true).

Introduction

Glucose-6-phosphate dehydrogenase deficiency (G6PDd), the most common inherited red blood cell (RBC) enzymopathy[1], limits the rate of glutathione reduction (GSSG to 2GSH) and, consequently, the ability of RBCs to counter oxidant stress. GSH is produced by coupled redox reactions, involving the G6PD catalysed conversion of glucose-6-phosphate to 6-phospholgluconate in the pentose phosphate shunt, NADP+ to NADPH which then regenerates GSH via glutathione reductase[2]. In G6PDd individuals, infections (e.g. pneumonia, typhoid fever), drugs [e.g. the 8-aminoquinolines, primaquine (PQ) and tafenoquine (TQ)], and fava beans [3,4,5,6,7,8] are well established causes of oxidant-related acute haemolysis (AH) that may necessitate a blood transfusion [7,9,10,11,12] and be complicated by acute kidney injury[10,13].

PQ- and TQ-induced AH is a major concern for malaria control programmes, especially in the more severe SE Asian G6PD variants like Viangchan (a WHO Class II variant)[14] because reducing the substantial burden of vivax malaria requires attacking its hypnozoite reservoir with PQ or TQ; indeed, hypnozoites are the dominant (>80%) source of recurrent vivax infections (relapses)[15]. Accordingly, the reliable diagnosis of G6PDd is fundamental to the safe administration of the haemolytic 8-aminoquinolines.

In this respect, a specific issue is that of women who are heterozygous for G6PD deficiency because their G6PD activity ranges from fully deficient (i.e., <30% of normal) to normal (i.e., > 80% of normal)[16]. Heterozygotes with intermediate enzyme activities (30%-70%) may screen as normal with qualitative tests and be given 8-aminoquinoline therapy with the attendant risk of AH; two G6PD Mahidol heterozygotes were transfused following exposure to 1 mg/kg of daily PQ[11]. This risk justifies the obligatory measurement of G6PD activity before administering single-dose TQ whose long mean terminal elimination half-life of 16 days may result in prolonged AH[17].

The fluorescent spot test (FST) has been the standard qualitative screening test for the past 50 years. Although simpler and less expensive than quantitative testing, it requires a UV lamp, laboratory skills, and a cold chain for the necessary reagents, explaining partly its limited use in resource-limited, endemic settings. The FST and recently introduced, point-of-care, qualitative rapid diagnostic tests (RDTs) can reliably detect G6PD activities of < 30% activity but they have not been fully validated for detecting heterozygous females having ≥ 30% of normal activity and fail to detect G6PDd with increasing G6PD activity[18,19,20]. A 30% of normal cut off excludes effectively hemizygous males, homozygous females, and fully deficient heterozygous females from exposure to 8-aminoquinolines[21].

Where possible and practised, G6PD status is usually determined when febrile patients first present to clinics and are diagnosed with acute P. vivax malaria. Physicians must interpret G6PD test findings in weighing the decision to prescribe an 8-aminoquinoline. Regardless of G6PD status, acute malaria causes intra- and extravascular haemolysis and, therefore, stimulates erythropoiesis and increases the reticulocyte count. On the other hand, the reticulocyte count may be decreased through the selective destruction by P. vivax and disturbed erythropoiesis. This is highly relevant to G6PD status because reticulocytes have much greater (~3–8 fold) G6PD activity than mature red cells[22,23,24] and, therefore, a G6PDd patient with reticulocytosis may be misclassified as G6PDn and receive, without appropriate supervision, daily primaquine (0.25/0.5/1.0 mg/kg/d) or single-dose TQ (300 mg in an adult). Getting the G6PD diagnosis right is crucial for avoiding potential harm.

As part of a clinical trial of weekly administered primaquine to vivax-infected, G6PDd and G6PDn patients, we assessed the ability of the FST to diagnose correctly G6PDd at presentation and measured the G6PD activity and reticulocyte count at baseline and over time to ascertain how they are affected by P. vivax and its treatment.

Materials and methods

Ethics statement

Ethical approvals were obtained from the National Ethical Committee for Health Research of the Cambodian Ministry of Health, Phnom Penh (ref: 225 NECHR), and the ethical review board of the Western Pacific Regional Office of WHO, Manila, Philippine (ref: 2011. 08. CAM. 01. MVP). All patients or their legal guardians gave written or verbal consent to join this study.

The trial was registered (ACTRN12613000003774) with the Australia New Zealand Clinical trials on 3/1/13 (https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=363399&isReview=true).

Trial design, study site and conduct

Study methods were detailed previously[9]. Briefly, from January 2013 to January 2014, 75 non-pregnant Cambodians aged > 1 year (y) with uncomplicated P. vivax were treated with dihydroartemisinin piperaquine (DHAPP, target dose of DHA 2 mg/kg/d) on Day (D) 0, 1 and 2 and eight doses (0.75 mg/kg) of weekly primaquine (D0–D49): (i) 10–17 kg, 7.5 mg, (ii) 10–25 kg, 15 mg, (iii) 26–35, 22.5 mg, (iv) 36–45 kg, 30 mg, (v) 46–55 kg, 37.5 mg, (vi) 56–75 kg, 45 mg, and (vii) ≥76 kg, 60 mg.

Key laboratory investigations were: (i) vivax parasitaemia (40 x number of vivax parasites/200 white cells on a Giemsa stained thick film), (ii) reticulocyte counts (thin blood film), (iii) Hb concentration (HemoCue AB, Ängelholm, Sweden), (iv) Hb electrophoresis [19], (v) G6PD genotype by polymerase chain reaction [19], and (vi) G6PD activity, full blood count and routine biochemistry (D0, 7, 28, 56). G6PD status was assessed by the fluorescent spot test (FST) at baseline.

We adjusted G6PD enzyme activity in thalassaemic patients (D0, 7, 28 & 56) by taking into account their low mean corpuscular volume (MCV), which results in increased numbers of RBCs/g Hb and an artificially high G6PD activity[25] (correction factor = mean MCVthalassaemia/mean MCVnormal Hb). The lower and upper limits of normal for manually measured reticulocyte counts were defined as 0·4–2·3% [26].

Data management and statistical methods

Clean, double entered data were analysed using Stata v14 (Stata Corporation, Texas, USA). For the enzyme analysis, four values from G6PDn patients were inappropriately low (probably related to delayed analysis) and excluded and 39 values were missing due to loss to follow up or were not done.

Proportional data between groups were compared using chi squared or Fisher’s exact test, as appropriate. Normally distributed data were analysed by paired (within groups) or unpaired ‘t’ (between groups) tests and skewed data by the corresponding non-parametric tests. Data distribution was assessed using the sktest command in Stata. The sensitivity and specificity of the FST to diagnose G6PDd patients were calculated against the G6PD genotype as the “gold standard.” Using backward stepwise multivariate regression, we examined the effects of age, sex, illness days, thalassaemia, and baseline values of temperature, parasitaemia, haemoglobin and reticulocyte count on the mean baseline G6PD activity. Linear mixed effects modelling was used to assess the independent effects of age, sex, illness days, baseline splenomegaly, thalassaemia, baseline reticulocyte count and parasitaemia, the fall in Hb (baseline—nadir Hb) and G6PD activity over time. The relationship between two continuous variables was determined by simple linear regression to determine the Pearson correlation coefficient.

Results

Patient baseline characteristics

75 patients with microscopically-confirmed P. vivax mono-infections were enrolled into the study and 67 completed follow up to D56. 80% of patients were young adult males (< 30 y) and 20% were patients aged 5 –< 18y (Table 1). The 18 G6PDd patients comprised 15 hemizygous males (14 G6PD Viangchan and 1 G6PD Canton) and 3 G6PD heterozygous Viangchan females. Mean baseline Hb, reticulocyte counts, corpuscular volume and red cell distribution width were similar between the G6PDd and G6PDn arms (Table 1). With treatment, there was an initial decrease in the mean Hb, that was significantly greater (p<0.001) in the G6PDd vs. G6PDn patients (S1 Fig), followed by a sustained rise in Hb. However, one G6PDd male had a sustained fall in Hb and was transfused.

Table 1

Patients’ baseline characteristics.

Parameter	G6PD deficient n = 18	G6PD normal n = 57	P value
Age years	25 (5–56)	24 (7–63)	0.95
Aged < 18 years*	5 (27.8)	10 (17.5)	0.34
Male sex	15 (83.3)	48 (84.2)	0.93
Weight kg	54 (20–56)	53 (14–88)	0.83
Days ill	2 (0–8)	3 (0–13)	0.14
Primaquine dose mg/kg/d	0.74 (0.65–0.78)	0.73 (0.53–0.98)	0.43
G6PD activity †
G6PD activity all patients	0.85 (0.1–1.36) ‡	11.38 (6.67–16.78)	<0.0001
G6PD activity % normal§	7.08 (0.83–11.33)	94.83 (55.58–139.83)
G6PD activity normal haemoglobin	0.77 (0.1–1.2)	11.23 (6.9–14.8)
G6PD activity thalassaemia	1.03 (0.72–1.36)	11.57 (6.67–16.78)
Haemoglobin parameters
Haemoglobin g/dL†	12.94 (9.6–16)	13.26 (9–16.3)	0.48
Reticulocyte count %†	1.86 (0.6–3.8)	1.5 (0.5–4.5)	0.10
Normal haemoglobin	11/17 (64.7)	31/57 (54.4)	0.60
Heterozygous HbE	5/17 (29.4)	20/57 (35.1)
Homozygous Hb E	0/17	1/57 (1.75)
Alpha thalassaemia	1/17 (5.9)	1/57 (1.75)
Beta thalassaemia	0/17	4/57 (7.1)
Mean corpuscular volume (MCV) fL	87.5 (71–97)	84 (64–98)	0.15
High MCV (> 95 fL)	2/16 (12.5)	2 (3.5)	0.20
Red cell distribution width (RDW) %	12.3 (11–16.4)	12.9 (11–15.7)	0.83
High RDW (> 14.5%)	2/16 (12.5)	2/54 (3.7)	0.22
Parasite data
Vivax parasitaemia/μL	6,420 (159–9,326)	8,300 (220–59,542)	0.13

* Proportional data are shown as N (%)

† mean (range), other continuous data are median (range)

‡ includes 2 heterozygous females with measured baseline activities of 0.9 and 1.18 U / g Hb

§ In Cambodia, the median G6PD activity of a normal population is 12 U / g Hb

Baseline G6PD activity and performance of the fluorescent spot test

The FST detected correctly all 15 hemizygous males and two of the three heterozygous females whilst two genotypically G6PDn patients (one female and one male) were classified as G6PD deficient for a sensitivity of 17/18, 94.4 (95% confidence interval: 72.7–99.8)% and a specificity of 55/57, 96.5 (87.9–99.6)%.

There was no correlation between the baseline G6PD activity and baseline reticulocyte counts for all patients combined, r = -0.81 (p = 0·28), and by G6PD status: (i) G6PDn r = 0.54 (p = 0.19), (ii) G6PDd r = -0.09 (p = 0.36). However, baseline G6PD activity was significantly correlated (r = 0.87, p = 0.007) with baseline temperature only in the G6PDn patients; r = 0.003 (p = 0.96) in the G6PDd patients (S2 Fig).

Time course of G6PD activity

In both G6PDd and G6PDn patients, the mean G6PD activity followed broadly changes in the reticulocyte counts and was relatively steady, peaking on D7 [Fig 1 (males) and Fig 2 (females)]. G6PD activity increased in 42 (13 G6PDd) patients, fell in 19 (2 G6PDn) and remained unchanged in one; nine patients (21.4%) with increases in G6PD activity had concomitant decreases in reticulocyte counts (S1 Table). By linear mixed effects modelling, G6PD activity was explained only by changes in reticulocyte counts (Table 2); there was no significant effect of thalassaemia/Hb E (S3 Fig).

Fig 1

Time course of G6PD activity and reticulocyte counts in male vivax-infected patients given weekly primaquine.

Panel A: G6PD activity. Panel B: reticulocyte counts.

Fig 2

Time course of G6PD activity and reticulocyte counts in female vivax-infected patients given weekly primaquine.

Panel A: G6PD activity. Panel B: reticulocyte counts.

Table 2

Significant factors independently associated with G6PD activity over time and reticulocyte counts over time by multivariable analysis.

Parameter	Coefficient	95% Confidence interval	P value
G6PD activity over time
Reticulocyte count over time	0.29	0.039–0.52	0.024
Reticulocyte counts over time
G6PD deficient activity over time	-0.028	-0.051–-0.005	0.015
Baseline reticulocyte count	0.48	0.36–0.61	<0.001
Female sex	0.34	0.05–0.63	0.021

Compared to D0, mean D7 activities were significantly higher in the G6PDd arm (1·65 vs. 0·90 U/g Hb, p = 0·02) with a trend in the G6PDn arm (12·09 vs 11·46 U/g Hb, p = 0·06). The mean absolute changes were similar but the mean relative increases were greater in G6PDd patients: 83·3% (0·75/0·9) vs. 5·5% (0·63/11·46). One heterozygous female (Fig 2) had an absolute increase of 4·37 U/g Hb to 5·54 U/g Hb, for a 4·7-fold increase over baseline. The D7-D0 percent change in G6PD activity positively and significantly correlated (Fig 3) with the D7-D0 percent change in reticulocyte counts in the G6PDn arm (p < 0.001) but not in the G6PDd arm (p = 0.81). By D56, the mean G6PD activities were not significantly different vs. D0 (p = 0·44).

Fig 3

Relationship between the percentage changes in G6PD activity and percentage changes in reticulocyte counts between Day 7 and baseline in the two G6PD groups.

Panel A: G6PD normal patients: the Pearson correlation coefficient, r, was 0.14, adjusted R2 0.34 (p<0.001). Panel B: G6PD deficient patients: r = 0.063, aR2 = -0.063 (p = 0.64).

Reticulocyte counts

The mean baseline reticulocyte count was 1·56% (range of 0·5–4·5%) and was not significantly different by G6PD status (Table 1). However, there was a significant correlation [r = 0.64 (p = 0.001)] with the baseline temperatures only in the G6PDd patients (S4 Fig). Over time, reticulocytes peaked on D14 in the G6PDd hemizygous males (Fig 1) and on D7 in the G6PDn group and in the G6PDd heterozygous females (Fig 2).

Discussion

This is the first study detailing changes in G6PD enzyme activity in pheno- and genotypically characterised patients with P. vivax infection treated with single weekly PQ dose of 0·75 mg/kg for eight weeks, per a decades-old WHO recommendation. We found that baseline G6PD activity was independent of the reticulocyte count, as reported previously [27], but, over 56 days, changes in G6PD activity correlated with changes in the reticulocyte counts.

On day 7, we observed a modest increase in absolute G6PD activity in the G6PDn and G6PDd patients that correlated with an increase in reticulocyte counts although about a fifth had a concomitant fall in reticulocyte count. We interpret the reticulocytosis as resulting from two factors: reticulocyte destruction by P. vivax ceases and the erythropoietic activity of the bone marrow is no longer suppressed by malaria infection.

After day 7, there are fluctuations in G6PD levels in individual patients but the mean G6PD level is stable. The reticulocyte response in the G6PDd group was longer, peaking at D14, and was ~2-fold greater compared to the G6PDn group due to the additional haemolysis of vulnerable RBCs after the first and second doses of PQ.

One aim of this study was to determine whether, in a real-life situation, a G6PDd patient may be misclassified as G6PDn, because of a high baseline G6PD activity, and thus be exposed to the danger of AH when receiving a full course of PQ. Despite the limited size of the studied population, our work shows that at no point in time did the level of G6PD activity in the G6PDd hemizygous males get close to the normal range and, in contrast to the G6PDn patients, their baseline G6PD activity was unaffected by fever. Accordingly, the FST performed well in the G6PDd males with mostly G6PD Viangchan. This is consistent with several studies in SE Asia of the FST and G6PD RDTs, demonstrating their high sensitivities in malaria patients [28] and healthy individuals with enzyme activities < 30% activity [18,29] with a spectrum of mostly WHO class II G6PD variants.

One of the three heterozygous females was diagnosed as normal by the FST. Her baseline G6PD activity was measured as 1·18 U/g Hb (~10% of normal) but on D7 her activity was 5·54 U/g Hb (46% of normal) and remained almost as high thereafter. Her D0 value may have been spuriously low for technical reasons and closer to her D7 value (~50% normal activity); if that were the case, the FST would be expected to classify her as normal. Other studies have reported the mis-diagnosis of G6PD heterozygous females by the FST and G6PD RDTs with declining test sensitivities, as low as ~70%, with G6PD activities between 30—< 60% in G6PDd heterozygous females [18, 28,29,30] and in an in vitro system of G6PD enzyme inhibition of red blood cells [20]. The skewed X-inactivation in G6PD heterozygous females results in a spectrum of phenotypes and limits the usefulness of qualitative tests and puts such women at risk of drug-induced haemolysis; only DNA analysis can identify all heterozygotes[1] but this is impractical in the field. The advent of point-of-care Biosensors that measure G6PD activity is a step forward because they can enumerate the ratio of G6PDn and G6PDd RBCs [31] in an individual heterozygote. Adequate health worker training and patient counselling on the clinical features of haemolysis are crucial when PQ, TQ and G6PD testing are rolled out.

The 8-week course of weekly PQ was associated with good Hb recovery in all but one of the G6PDd patients. We attribute this to their state of compensated haemolysis that is supported by the substantial increases (>80%) in G6PD activity, indicating a younger red cell population during this period of time, in keeping with a sustained increase in reticulocyte counts. A concomitant rise in G6PD activity and reticulocyte count has also been documented in G6PD A- African Americans challenged with 30 mg of daily primaquine and that continued primaquine administration resulted in an “equilibrium/resistance” phase[32,33]. Similarly, our data suggest that the young red cells of G6PD Viangchan (and the single patient with G6PD Canton) have sufficiently high G6PD activity to confer “resistance” to haemolysis in the face of continued PQ dosing. The G6PD A- and our data contrast with the findings of Pannacciulli et al who showed that continued haemolysis occurred in G6PDd Mediterranean males given two courses of 30 mg of daily primaquine (30mg x7d) separated by a week to allow for a reticulocyte response[34].

Our study was limited by the small number of G6PDd patients, the very low number of G6PDd heterozygous females, almost all of whom had one G6PD variant—G6PD Viangchan. More data are needed in more G6PDd variants to support or refute our findings and provide more data on heterozygous females to guide clinicians in the field. Studies are also needed in falciparum-infected patients, given the evolving notion of giving radical cure to falciparum patients in areas of mixed vivax-falciparum transmission[35], and so determine the risk and clinical consequences of missing a diagnosis of G6PDd in patients receiving daily primaquine. We did not have a no primaquine arm which would have given us useful information on the effects of malaria itself and its treatment on the changes in G6PD activity and reticulocyte counts over time.

In conclusion, our small study of mostly G6PD Viangchang hemizygous males with acute P. vivax malaria have little risk of being misdiagnosed as G6PD normal by qualitative tests in our region. Although this may also apply to heterozygous females and other variants with higher mean activity e.g. G6PD A-, confirmation should be sought from more research.

Haemoglobin changes over time in males and females.

Panel A: males. Panel B: females.

(TIF)

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The relationship between the baseline G6PD activity and the baseline temperature.

A significant association was seen only in the G6PD normal patients: the Pearson correlation coefficient was 0.87 and the adjusted R2 was 0.11 (p = 0.007). The Pearson correlation coefficient in the G6PD deficient patients was 0.003 (p = 0.96).

(TIF)

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Glucose-6-phopshate dehydrogenase activity in patients with and without thalassaemia.

Panel A: G6PD normal patients. Panel B: G6PD deficient patients.

(TIF)

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The relationship of the baseline reticulocyte count and baseline temperature in patients with acute Plasmodium vivax malaria by G6PD status.

The Pearson correlation coefficients were 0.64 (p = 0.001) and -0.12 (p = 0.24) in the G6PD deficient and normal patients, respectively.

(TIF)

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Listings of G6PD enzyme activity and their changes between Day 0 and Day 7.

(XLSX)

Click here for additional data file.

18 May 2021

Dear Dr. Taylor,

Thank you very much for submitting your manuscript "Dynamics of G6PD activity in patients receiving weekly primaquine for therapy of Plasmodium vivax 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.

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

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: The objectives of the study are not as clearly stated as desired. It is assumed (but unclear) that the primary objective is hypothesis-testing for the FST diagnosis question, but the authors do not explicitly state this. If the primary objective is hypothesis-testing, the study does not seem adequately powered and the sample population not large enough. That is, the authors claim that the FST qualitative test accurately diagnoses individuals with G6PD Viangchan but this conclusion is based on only 15 geno- and phenotypically confirmed G6PDd males and 3 heterozygous female patients. Further, the authors need to provide a justification for the sample size. The authors should consider whether or not it is even appropriate to test this hypothesis for inclusion in this manuscript. Regarding the reticulocyte portion of the study, as this is an exploratory analysis, the sample size is acceptable. Additionally, while the authors stated which statistical methods they employed, explicit mention of the assumptions tested (and how) is requested, as well as mentioning in the figure legends which statistical test was performed and for which variables the parametric assumptions were not met. Finally, critical detail on the regression model building is requested; specifically, which variables were included in the initial full model, what was the process of elimination, what was the P-value cutoff for elimination and re-entry, were there any confounding variables and if so how was it dealt with

Reviewer #2: The objectives were not clear:

1. In both the abstract and introduction, the authors state that one of the goals of the study is to “assess the impact of acute vivax malaria and of its treatment on G6PD activity levels” however, to assess the impact of vivax malaria on G6PD activity, don’t you need an uninfected comparison group? I didn’t see any data on the impact of malaria on G6PD in this study, so perhaps it should be added or this goal removed.

2. Since you are assessing the performance of a diagnostic test in the setting of an active malaria infection, would it be helpful to use ROC curves to assess the performance of the test? Sensitivity and specificity?

3. Would it be helpful to compare the performance of the test in uninfected individuals to see if it is changed by malaria infection?

4. To what extent was drug treatment versus the disease itself driving the changes in G6PD activity observed over time? Is there data available for patients who did not receive primaquine?

5. I’m surprised that G6PD activity has to be adjusted for MCV. I would expect its concentration in the cytoplasm to be the same, even if the RBC is smaller, and therefore its concentration relative to Hb would be unchanged, even if the activity per RBC is lower.

Reviewer #3: Methods are sound

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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: Though it is not immediately clear to this reviewer and required close scrutiny of the text and table/figure legends, the authors appear to have followed their analysis plan. However, the order of results is somewhat confusing, as the authors flip-flop the order from the Introduction. That is, in the Intro the authors first discuss the performance of the FST then the reticulocyte/G6PD activity studies but then in the Results they present the retic/G6PD activity first and FST performance second--this should be corrected. Furthermore, figures and tables require revision. First, none of the figures have titles on the y-axis; each graph should be able to stand alone. Second, this reviewer requests "A" and "B" designations for two-panel figures. Third, it is requested that all correlation coefficients regardless of statistical significance be presented in the manuscript. Fourth, it is not clear what is meant by the bolded outcome variables in Table 2--what exactly does the variable Reticulocyte Dynamics mean, is it continuous or categorical, etc. In general, it is preferred to see the marker of central tendency as median with 95% CI if the authors are concerned that their population is not normally distributed or large enough to assume normality (which it is not).

Reviewer #2: 6. Table 2 is unclear, and I could not find a definition for the factor “Reticulocyte dynamics” or the outcome variable “G6PD activity dynamics” or the outcome variable “Reticulocyte dynamics”.

Reviewer #3: Yes, except spaghetti plots are hard to look at.

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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: For the FST performance, this reviewer is concerned that the authors are over-generalizing their conclusion based on limited sample size. Furthermore, the limitations section of this study requires additional limitations and more extensive discussion. Here it may be prudent to discuss published sensitivity and specificity of the FST, whether there are known correlations between quantitative and qualitative testing (that is, at what quantitative threshold of enzyme activity does the FST correctly diagnose a patient). Discussion on this second point would be helpful for framing the FST performance results in the context of a small sample size.

Reviewer #2: 7. In the discussion, you refer to the safety and tolerability of the primaquine course and refer to one patient that needed transfusion. If safety is an endpoint in the study, please add that to the methods and results.

Reviewer #3: Yes

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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: 8. AHA can also refers to autoimmune hemolytic anemia, so perhaps better not to use it as an abbreviation for acute hemolytic anemia.

9. If the goal of the study was to assess the performance of the test at baseline, then are the additional time points necessary?

Reviewer #3: I'm not sure that very dense spaghetti plots are the best way to represent this data. I would favor mean or median over time for each group with error bars representing 95% CI or interquartile range. Box plots at each time point would also be much easier on the eyes.

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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 current study addressed two clinical questions: 1) whether diagnosis by FST is impacted by acute malarial infection in G6PDd males and G6PDhet females, and 2) how do reticulocyte dynamics change and influence G6PD activity during malaria infection/treatment in G6PDd and G6PDn individuals. While the limitations of the first point are discussed below, it is worth mentioning that the retic and enzyme activity dynamics data and subsequent discussion are strong and quite compelling. This portion of the paper adds an important piece of information to the field, and is helpful for understanding the underlying retic/enzyme dynamics during acute malaria infection and treatment.

To the first question, the authors claim that the evidence presented supports the notion that diagnosis by FST is not affected. Out of 75 total patients, 15 were G6PDd males and 3 G6PDhet females, and the FST correctly diagnosed all 15 males, and of the three females, two were classified as deficient while one was diagnosed as normal, and the authors cite “technical reasons” to explain a low reading on Day 0 and that it is more likely that she was ~5 U/ g Hb. It would have been useful to include calculated sensitivities and specificities (both ~99-100%) as the major point of the study was to examine test characteristics in this population, and would have been even more helpful to include published test characteristics of FST in different G6PD variant populations, as well as the lower limit of detection of the FST test (if that data exist). That is, at what activity level (in U/ g Hb) does the FST give a qualitative diagnosis of G6PD deficiency. Even if it is true that the single patient’s Day 0 activity is falsely low, it is an over-generalization to state that this study presents clear evidence that FST does not misdiagnose G6PD heterozygous females. Perhaps the authors should consider re-framing this conclusion. Furthermore, there seems to be a disconnect between the framing of the clinical problem in the Background section and the ensuing Discussion of results. Frankly, I was expecting the sample population to be more highly enriched for G6PD heterozygous females, and I am concerned that the target population was inappropriate to answer the proposed question. While this may be due to a possible heterozygous resistance to clinically-significant malaria infection or decreased access to treatment for Cambodian women, it is a significant limitation of the study that receives inadequate discussion. If it is not possible to enrich the sample with female heterozygotes due to a low volume seen at this clinic or for whatever reason, this would be helpful to mention while responding to these comments. Further, and with regard to the presentation of results, it would have been helpful to address the major clinical question earlier in the manuscript with the reticulocytes and G6PD connection occurring later.

Regarding the reticulocyte and G6PD activity data, this was highly interesting and helpful to understand the increase in G6PD activity in the peri-infection/initiation of therapy period, though it is clear that the relationship between reticulocyte dynamics and G6PD activity is nuanced.

Specific Comments:

1. It would be helpful to designate which of the comparisons were non-parametric, and which assumptions specifically were violated. Furthermore, if the authors are significantly concerned about sample size, it might be helpful to display central tendencies as medians + 95% CIs, especially if the sample population is not normally distributed or sufficiently large enough to approach normality. In general, I would advocate for median w/ 95% CIs.

2. It would be helpful to include “A” and “B” designations for two-part figures, as well as titles for the y-axis. As it stands, no y-axis is labeled. Overall, the graphs cannot stand alone and can be difficult to interpret.

3. Which WHO classification does the Viangchan mutation belong to? Would we expect clinical manifestations at baseline and a corresponding reticulocytosis, or were the baseline retic counts expected? It would be helpful to illuminate these expectations for the reader.

4. The authors claim that G6PDd retics might have higher G6PD activity levels compared to G6PDn retics as evidenced by a higher relative increase in activity at Day 7 in G6PDd compared to G6PDn with a smaller increase in retic counts. Has this been reported previously or is there other evidence to support this claim?

5. Is it known what is the activity level threshold to trigger hemolysis with the study anti-malarials?

6. Typically, the abbreviation “AHA” is reserved for “autoimmune hemolytic anemia,” not “acute hemolytic anemia.” I would re-consider this usage.

7. In Line 143, is this larger drop statistically significant? While it is stated that the antimalarial therapy was well-tolerated and only one patient required transfusion, it is clinically useful to recognize the hemolytic risks of these therapies.

8. In the regression models, did the authors adjust for confounding? If so, how was this dealt with statistically? Were any indicators not included in the full regression model?

9. Table 2 is difficult to understand. It is presumed that these are three reduced (final) models and the outcome variable is in bolded text. However, it is very unclear what “reticulocyte dynamics” means in the context of the model? What type of variable is it? How was it defined? What does it mean exactly? What predictor variables were included in the initial full models?

10. In Figure 3 title, please include the correlation coefficient for G6PDd.

11. It would be of major benefit to enrich the clinical sample with female heterozygotes and would substantiate the preliminary findings from just three female hets

Reviewer #2: Summary: The authors measured the performance of a qualitative test for G6PD activity and found that it was accurate even in the setting of an active P vivax infection.

Reviewer #3: The authors have performed a study evaluating G6PD levels in subjects with acute vivax malaria and also its impact on the receipt of treatment. I have no concerns regarding the methodology, and to my knowledge this is novel. It asks and addresses a question that matters. The introduction and discussion are reasonable.

I only have one minor suggestion of relevance. I'm not sure that very dense spaghetti plots are the best way to represent this data. I would favor mean or median over time for each group with error bars representing 95% CI or interquartile range. Box plots at each time point would also be much easier on the eyes.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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28 Jul 2021

Submitted filename: response to reviewers-G6PD_v0.5.docx

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28 Jul 2021

Dear Dr. Taylor,

We are pleased to inform you that your manuscript 'Dynamics of G6PD activity in patients receiving weekly primaquine for therapy of Plasmodium vivax malaria' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

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3 Sep 2021

Dear Dr. Taylor,

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