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. 2024 Jan 19;19(1):e0296708. doi: 10.1371/journal.pone.0296708

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

Arkasha Sadhewa 1,‡,*, Alina Chaudhary 2,‡, Lydia V Panggalo 3, Angela Rumaseb 1, Nabaraj Adhikari 2, Sanjib Adhikari 2, Komal Raj Rijal 2, Megha Raj Banjara 2, Ric N Price 1,4,5, Kamala Thriemer 1, Prakash Ghimire 2, Benedikt Ley 1,‡, Ari Winasti Satyagraha 3,6,‡
Editor: Walter R J Taylor7
PMCID: PMC10798449  PMID: 38241389

Abstract

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

Introduction

Plasmodium vivax (P. vivax) causes 4.9 to 14.3 million clinical episodes annually [1, 2] and has become the dominant Plasmodium species outside of sub-Saharan Africa. Globally, more than 3.3 billion people are at risk of infection with P. vivax [1]. In contrast to most human Plasmodium species, P. vivax and P. ovale form dormant liver stages (hypnozoites), that can reactivate weeks to months after the initial infection causing recurrent episodes of malaria (relapses) [3, 4]. The risk and frequency of relapses vary depending on the strain [3], with those in equatorial regions relapsing more frequently, associated with a significant health and economic burden [5, 6].

Radical cure of P. vivax ensures the clearance of both the asexual blood-stage parasites that cause febrile illness (schizontocidal treatment) and clearance of dormant liver stages (hypnozoites) that can cause relapsing infections. Since schizontocidal antimalarial drugs have no activity against hypnozoites, radical cure requires a combination of schizontocidal and hypnozoitocidal agents. Depending on geographic location, chloroquine (CQ) or artemisinin combination therapies (ACT) are used as schizontocidal treatment [7]. The only currently licensed hypnozoitocides are the 8-aminoquinolines (8AQs) primaquine (PQ) and tafenoquine (TQ) [8, 9]. Although well tolerated in most recipients, 8AQs can induce severe haemolysis in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency, a common enzymopathy found in malaria endemic areas [10].

G6PD deficiency (G6PDd) is caused by mutations in the G6PD gene, located on the X-chromosome. It affects between 400 to 500 million people worldwide and impairs the red blood cells’ (RBC) ability to regulate cellular redox potential whilst ensuring their survival against oxidative stressors [11]. Males have one X-chromosome and are either hemizygous G6PD normal (the vast majority having >70% G6PD activity) or G6PD deficient (the majority having <30% G6PD activity) [12, 13]. Females have two copies of the gene and can be G6PD homozygous normal, G6PD heterozygous, or G6PD homozygous deficient [14]. In heterozygous females, G6PD deficient and G6PD normal RBC populations exist with varying proportions [15]. G6PD activities of heterozygous females range from close to 0% to almost normal activity with the majority of females having intermediate activities between 30% to 70% of normal activity [12].

In the presence of strong oxidants, such as 8AQs, G6PD deficient RBCs can haemolyse, resulting in a severe drop in haemoglobin (Hb) concentrations leading to haemodynamic instability and potentially life-threatening acute kidney injury [16]. The World Health Organization (WHO) therefore recommends that all patients are tested for G6PDd prior to administration of either PQ or TQ [7, 17]. Diagnosing individuals with intermediate G6PD activity is also recommended to exclude these patients from administration of TQ [18] and to guide the use of short-course high-daily dose PQ regimens [19, 20]. Diagnosing intermediate deficiency is currently only possible using quantitative testing, due to the low inherent diagnostic thresholds of available qualitative tests [17].

Over the last few years several handheld devices (biosensors) have been introduced to the market capable of measuring G6PD activity quantitatively or semi-quantitatively at point-of-care. The semi-quantitative STANDARD G6PD Test (SD Biosensor, RoK; “Biosensor”) has excellent performance under laboratory and field conditions [21–23] and has now been rolled out for routine testing in seven countries [24]. The Biosensor’s reproducibility and repeatability under lab conditions is excellent [25], however anecdotal evidence suggests lower repeatability when the test is applied under field conditions.

As the incidence of malaria declines, the proportion of patients with P. vivax infection in remote areas increases [26, 27]. Delivering quantitative G6PD measurements at these remote communities is challenging [24], hence patients diagnosed with P. vivax are referred to higher level health care facilities for a G6PD measurement, although the rate of referral is often low [28]. An alternative option to referring patients to the closest health facility, is collection and transport of the patients’ blood to the health facility instead. Collected blood can then be tested by Biosensor and the results communicated back to the treating clinician to inform suitable treatment. The stability of G6PD activity of samples stored at 4°C has been evaluated in the past with spectrophotometry [21] and was found to remain stable over several days, though this has not yet been assessed using a Biosensor.

The objective of this study was to assess whether the repeatability (variation of measurement results if the same sample is tested repeatedly under the same conditions) and reproducibility (variation of measurement results if the same sample is tested repeatedly under different conditions) of the STANDARD G6PD Test (Biosensor) can be improved by modifying test procedures and assess the impact of delayed testing on recorded G6PD activity and Hb concentration. In addition, the manufacturer-recommended cut-off was evaluated against the site-specific thresholds of two field sites.

Methods

Overview

In a Pilot Study several variations of the Biosensor’s standard measurement method were trialled at the Menzies School of Health Research, Darwin, Australia. The Standard Method recommended by the manufacturer and the method with the least variability in recorded activity were subsequently assessed at field sites in Malinau Regency, Indonesia, and Kailali District, Nepal. While ethical approval in Indonesia was provided to collect capillary and venous blood from the same participant, Nepalese ethics limited blood collection to either venous or capillary blood. Accordingly, twice as many participants were enrolled in Nepal compared to Indonesia, while the number of measurements performed was the same (Fig 1). Written informed consent was collected from all participants or their legal guardians prior to enrolment.

Fig 1. Schematic workflow of the pilot study in Australia, and the field studies in Indonesia and Nepal.

Fig 1

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Ethics

Ethical approval was obtained from the Human Research Ethics Committee (HREC) of the Northern Territory Health, Australia (Menzies HREC 22–4346), the Atma Jaya Catholic University Research Ethics Committee, Indonesia (No. 0008A/III/PPPE.PM.10.05/09/2022) and the Institute of Science and Technology, Tribhuvan University Institutional Review Committee, Nepal (No. IRC/IOST60/079/080).

Assay procedures

  • Standard Method (recommended by the manufacturer, Fig 2): A single use plastic pipette (Ezi tube, Fig 3) was used to transfer 10 μL of capillary blood from a finger prick to the lysis buffer (included in the test kit) immediately after collection. The blood buffer solution was mixed and 10 μL of the solution was then transferred to the test membrane of a single use test device inserted into the Biosensor. The Biosensor provides normalized G6PD activity (in U/g Hb) and quantifies Hb concentration (in g/dL) within 2 minutes of sample application.

Fig 2. Illustrations of components and step-by-step guide of the STANDARD G6PD Test (SD Biosensor, RoK) from Adhikari et al (2022) [29].

Fig 2

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Fig 3. The Ezi tube, a single use plastic pipette included in the Biosensor test kit.

Fig 3

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When the tip is inserted into a blood sample, 10 μL are collected through capillary action. The black line indicates 10μL.

Four variations to the standard method were developed, considering practical relevance in remote field setting, the use of stored blood (capillary and venous) and the use of additional equipment such as micropipettes:

  • Method 1: Capillary blood was collected in K2 EDTA microtainer tubes (BD, USA; 500 μL) and stored at 4°C; the sample was tested within 60 hours of collection. Prior to testing, the tube was brought to room temperature and 15 μL of blood were transferred from the EDTA tube using a standard micropipette to the surface of a sealing film (Parafilm M, Amcor, USA). Ten μL of blood were collected from the sealing film and testing procedure then followed the standard method.

  • Method 2: was the same as Method 1, but venous blood instead of capillary blood was collected in a K2 EDTA vacutainer tube (BD, USA; 3mL).

  • Method 3: was same as Method 2, but the reservoir of the Ezi tube was not squeezed when dispensing the blood buffer solution onto the test membrane of the test device. Instead, the tip of the Ezi tube was placed on the membrane, allowing the buffer blood solution to flow onto the membrane through capillary action.

  • Method 4: was the same as Method 2, only a calibrated micropipette was used instead of the Ezi tube.

Pilot study

Variation and repeatability of an assay is relative to the absolute values measured. The lower the absolute “true” value is, the smaller the absolute assay specific variation will be. Considering the inherent background noise of any assay, variation is better measured in samples with higher G6PD activities. The Standard Method and each of the four modified methods were performed in 10 duplicates on three adult participants known not to be G6PD deficient as defined by at least two distinct assays. Twenty finger prick capillary samples (two pricks per finger, 10 μL of capillary blood each, defined as paired samples) were collected from each participant. An additional 400 μL of capillary blood were collected from one of the finger pricks into a K2 EDTA microtainer tube (BD, USA), and 3 mL of venous blood were collected into a K2 EDTA vacutainer tube (BD, USA). Any two consecutive measurements of blood collected from microtainers or vacutainers were defined as paired measurements.

Field study

In the Indonesian field study two drops of capillary blood (each ≥20 μL, defined as paired samples) from two distinct finger pricks were collected from participants above the age of six years (Fig 1). In addition, 3 mL of venous blood were collected from the same participants into a K2 EDTA vacutainer (BD, USA), stored in 4°C, and tested twice by the biosensor (paired sample). In Nepal the same procedures were followed, however only paired capillary or venous blood samples were collected from each participant, therefore twice as many participants as in Indonesia were enrolled.

Spectrophotometry (Reference method)

G6PD activity of all Indonesian participants was measured in duplicate from venous blood stored in EDTA tubes by reference spectrophotometry using a commercial kit (Cat. No. G7583-180, Pointe Scientific, USA), executed at 37°C, read at 340 nm (Biowave II UV/Vis, Biochrom WPA, UK), following kit manufacturer instructions [30]. Ten μL of venous blood were mixed thoroughly with 1 mL of R1 reagent that had been reconstituted with lyse reagent (Cat. No. G7583-LYS). After incubation at room temperature for 5 minutes, 2 mL of R2 reagent were added. The absorbance of the mixture was read at 340 nm after incubation for 5 minutes at 37°C, and again after another 5-minute incubation at the same temperature. G6PD activity was calculated from the difference of measured absorbance and normalized by Hb reading using the formula provided by the manufacturer. Hb levels were measured with a Hb 301 device (Hemocue, USA) done at the same time as reference testing [31]. G6PD deficient, intermediate, and normal controls (Cat. Nos. HC-108DE, HC-108IN, and HC-108, respectively) were measured daily to assure the quality of reference spectrophotometric readings (ACS Analytics, USA).

Statistical analysis and sample size calculation

All data were recorded on case report forms and digitalized using EpiData version 3.1 (EpiData Association, Denmark). Data were organized and analysed using STATA versions 13, 15, and 17 (Stata Corp, USA) [32].

The adjusted male median (AMM) was calculated per country from all male participants recruited through the prospective health facility-based surveillance, using the results from the Biosensor Standard Method [33].

To compare the repeatability of the Standard Method and the modified methods, the absolute difference between paired measurements was calculated for each method. In the Pilot Study, the median absolute differences for all methods were compared using the Kruskal-Wallis test. In the Field Study, the median absolute differences of the field-assessed methods were compared using the Wilcoxon signed-rank test (matched samples from Indonesia) or the Mann-Whitney U test (unmatched samples from Nepal). The median absolute differences of each field-assessed methods were compared between the two sites using the Mann-Whitney U test (S1 Table).

In Indonesia the correlation between the Biosensor and spectrophotometry readings was quantified by matching the mean of paired Biosensor readings with the mean of duplicate spectrophotometry readings and calculating the Spearman’s rank correlation coefficient (rs). The difference in absolute readings was assessed using the Wilcoxon signed-rank test and Bland-Altman plot analysis. The same procedures were followed to assess the correlation and significance of differences between paired readings of the Biosensor Standard Method and modified method. The proportions of deficient individuals were compared between methods using the McNemar’s test for correlated proportions. The median absolute differences of Hb measurements were analysed with the same statistical tests as G6PD activity, and the agreements analysed by calculating Pearson’s correlation (r), paired Student T-test, and Bland-Altman plot analysis.

The primary objective of this study was to assess the repeatability of different methods. Repeatability was defined as the difference between paired measurements. To identify a minimal and clinically relevant difference of 0.5 U/g Hb with 80% power and 95% confidence, assuming a standard deviation of 1.5 U/g Hb and a minimal correlation coefficient of r≥0.65 required recruitment of 52 participants per site. Assuming procedural errors in more than 10% of all participants we enrolled 60 participants for each method at each site to allow for a site-specific analysis using a two-sided approach.

Results

Pilot study

Three healthy G6PD normal adult volunteers were enrolled into the Pilot Study between the 4th and 10th of August 2022. Repeatability did not differ significantly between the five Biosensor methods (p = 0.381). The median absolute difference between paired measurements observed when using the Standard Method was 0.5 U/g Hb (interquartile range [IQR]: 0.2 to 0.9, total range: 0.0 to 3.1). Method 3 generated the smallest median absolute difference (0.3 U/g Hb, IQR: 0.2 to 0.4, total range: 0.0 to 2.8) (Table 1 and Fig 4). The Standard Method and Method 3 were subsequently moved forward for evaluation under field conditions.

Table 1. Comparison of median absolute difference between paired measurements per Biosensor method from the pilot study.

Method Name Blood Collection Sample Application Median absolute difference between paired measurements (IQR, total range) in U/g Hb
Standard Method Capillary Ezi Tube 0.5 (0.2–0.9, 0.0–3.1)
Method 1 Capillary EDTA Ezi Tube 0.3 (0.2–0.8, 0.0–3.9)
Method 2 Venous EDTA Ezi Tube 0.5 (0.2–0.7, 0.0–3.7)
Method 3 Venous EDTA Ezi Tube (not squeezed) 0.3 (0.2–0.4, 0.0–2.8)
Method 4 Venous EDTA Micropipette 0.5 (0.1–0.9, 0.0–4.5)
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Fig 4. Absolute differences between paired measurements in U/g Hb for each Biosensor method used in the pilot study.

Fig 4

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Field study

Baseline participant data

In Indonesia 60 participants were enrolled between the 24th and 28th of November 2022, in Nepal 120 participants were enrolled between the 25th of January and 13th of March 2023 (S1 Data). Site-specific AMM by Biosensor Standard Method was calculated to be 6.1 U/g Hb (IQR: 5.3 to 7.5) in Indonesia and 6.1 U/g Hb (IQR: 5.3 to 7.1) in Nepal (Fig 5). None of the Indonesian participants and only 1 (0.8%) Nepalese participant was G6PD deficient (<30% AMM) according to the Biosensor Standard Method. One (1.7%) and 10 (8.3%) participants had intermediate G6PD activity (30–70% AMM) in Indonesia and Nepal, respectively (Table 2).

Fig 5. Histogram of G6PD activity as measured by the Biosensor of female and male participants from Indonesia (Standard Method, n = 60) and Nepal (Standard Method n = 60, and Method 3 n = 60).

Fig 5

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Vertical lines denote (from left to right) 30%, 70%, and 100% of AMM.

Table 2. Demography of the field study populations.
Indonesia Nepal
Male 15 51
Female 45 69
Total 60 120
Median age in years (range) 35 (10 to 81)† 32 (8 to 90)
Median Hb in g/dL (IQR)* 15.9 (14.6 to 17.1) 14.3 (13.2 to 16.2)
AMM (IQR)* 6.1 (5.3 to 7.5) 6.1 (5.3 to 7.1)
G6PD Deficient (Activity <30% AMM)*
    Male (%) 0 1 (2.0%)
    Female (%) 0 0
G6PD Intermediate (Activity 30–70% AMM)*
    Male (%) 0 4 (7.8%)
    Female (%) 1 (2.2%) 6 (8.7%)
G6PD Normal (Activity >70% AMM)*
    Male (%) 15 (100.0%) 46 (90.2%)
    Female (%) 44 (97.8%) 63 (91.3%)
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†Age data from 59/60 participants.

*Based on the Biosensor Standard Method

Biosensor repeatability of G6PD measurements

Repeatability of measurements by the Standard Method and Method 3 did not differ in Indonesia (p = 0.425) and Nepal (p = 0.330); Table 3 and Fig 6. The median absolute differences of the Standard Method (0.3 U/g Hb, IQR: 0.1 to 0.5) and Method 3 (0.3 U/g Hb, IQR: 0.1 to 0.6) were similar when combining results from both countries (p = 0.713); Table 3 and Fig 6.

Table 3. Comparison of median absolute difference between paired Biosensor G6PD activity measurements between methods and sites.
Median absolute difference between paired measurements (IQR) in U/g Hb
Standard Method Method 3 p-value (Standard Method vs Method 3)
Indonesia 0.2 (0.1 to 0.4) 0.3 (0.1 to 0.5) 0.425
Nepal 0.4 (0.2 to 0.6) 0.3 (0.1 to 0.6) 0.330
Indonesia + Nepal 0.3 (0.1 to 0.5) 0.3 (0.1 to 0.6) 0.713
p-value (Indonesia vs Nepal) 0.025 0.680
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Fig 6.

Fig 6

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Boxplot of absolute differences between paired Biosensor G6PD activity readings per method (in U/g Hb) from field studies in Indonesia (left), Nepal (middle), and both sites (right).

The repeatability of the Standard Method differed significantly between measurements done in Indonesia (0.2 U/g Hb, IQR: 0.1 to 0.4) and in Nepal (0.4 U/g Hb, IQR: 0.2 to 0.6); p = 0.025; Table 3. However, the repeatability for Method 3 did not differ between countries: 0.3 U/g Hb (IQR: 0.1 to 0.5) in Indonesia and 0.3U/g Hb (IQR: 0.1 to 0.6) in Nepal (p = 0.680); Table 3 and Fig 6.

Biosensor repeatability of Hb measurements

The repeatability of Hb readings did not differ in Indonesia (p = 0.262), however, in Nepal the Hb median absolute difference of the Standard Method (0.8 g/dL, IQR:0.3 to 1.3) was significantly higher than that of Method 3 (0.3 g/dL, IQR: 0.2 to 0.9); p = 0.006, S3 Table, S1 Fig.

Biosensor G6PD readings after time delay and comparison to spectrophotometry

In Indonesia G6PD activity was measured immediately by the Standard Method, while Method 3 was delayed by a median of 307 minutes (IQR: 275 to 333 minutes) and spectrophotometry by a median of 348 minutes (IQR 133 to 395 minutes); S2 Fig. The median activity by the Standard Method was 6.4 U/g Hb (IQR: 6.0 to 7.0) compared to 6.7 U/g Hb (IQR: 6.2 to 7.3) by Method 3, with a difference of 0.4 U/g Hb (IQR: 0.2 to 0.7); p = 0.005, S3 Fig.

The median activity by spectrophotometry was 10.8 U/g Hb (IQR: 9.3 to 12.2), significantly higher than that derived by the Standard Method (p<0.001) and Method 3 (p<0.001). The correlation between both Biosensor methods (rs = 0.795; p<0.001) was better than that between spectrophotometry and the Biosensor Standard Method (rs = 0.451; p<0.001).

Each observation was categorised as deficient (<30%), intermediate (30–70%), or normal (>70%). Categories did not differ between Standard Method or Method 3; Fig 7B. However, when the categories derived from the Biosensor were compared to those from spectrophotometry readings (S2 Table), one individual categorized as deficient by spectrophotometry was categorized as intermediate by the Biosensor, and two individuals that were classified as intermediate by spectrophotometry were categorized as normal by the Biosensor (red dots, Fig 7A). Overall, there was no significant difference between the proportions of deficient and intermediate individuals categorized by the Biosensor vs spectrophotometry (p = 0.317 and p = 0.157 respectively).

Fig 7.

Fig 7

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

Biosensor vs Hemocue Hb readings. Matched Hb readings from Biosensor (Standard Method and Method 3) and Hemocue were only assessed in Indonesia (S4 Fig). The mean Hb concentration by the Biosensor Standard Method was 15.9 g/dL (95%CI: 15.4 to 16.3) compared to 16.6 g/dL (95%CI: 16.2 to 17.0) by the Biosensor Method 3 (p<0.001). Both Biosensor measurements were significantly higher than the paired readings of the Hemocue (mean: 13.5 g/dL, 95%CI: 13.2 to 13.9); p<0.001.

Discussion

Our study demonstrated that the repeatability of the Biosensor was consistent irrespective of type of blood (capillary or venous) and the sample application method used. In the pilot study, Method 3 was the method with the least variability and therefore selected for field assessment. In the field study, the repeatability of G6PD activity measurements did not vary between methods at either site. When repeatability of G6PD activity measurements of either method were compared between sites, there was no statistically significant difference for Method 3; while the observed difference for the Standard Method was statistically significant, the observed difference is unlikely to be clinically relevant (Table 3). The repeatability of Hb readings from capillary samples showed significantly greater variation compared to venous samples in Nepal but not in Indonesia (S1 Table and S1 Fig), however, this variation did not impact on the repeatability of the G6PD activity.

In the Indonesian field study, G6PD activity by the Standard Method was significantly lower compared to the paired reading of Method 3 performed more than five hours later. However, the median difference was less than 0.5 U/g Hb and thus unlikely to be of clinical relevance.

The readings of both Biosensor methods were well correlated, while the correlation between spectrophotometry and the Biosensor was lower, irrespective of the applied method. Since repeatability and the definition of 100% activity in Indonesia and Nepal were very similar this suggests a degree of imprecision with the reference method and underlines the level of standardization the Biosensor offers irrespective of end user [25, 34].

The AMM was calculated and defined as site-specific 100% G6PD activity based on results from the Biosensor Standard Method [33]. In both countries 100% activity was calculated as 6.1 U/g Hb, an activity that corresponds to the current cut-off for 70% activity which the manufacturer recommends to define the upper limit of intermediate enzyme activity. A recent study from Cambodia reports a site-specific AMM of 6.4 U/g Hb, very close to the manufacturer’s 70% cut-off and almost matching our observations [35]. If this observation is confirmed in other settings, this could suggest that the current recommended thresholds may be too conservative.

In the Indonesian cohort, the concentration of Hb measured by the Biosensor was compared to that measured by the Hemocue. The mean differences were 2.3 g/dL and 3.1 g/dL respectively for the Standard Method and Method 3 compared to the Hemocue, with the Biosensor generating the higher readings. A Bland-Altman plot analysis showed that this difference was consistent and not due to extreme outliers (S5 Fig). Past evaluations of the Biosensor have shown differences of less than 0.4 g/dL when compared to the Hemocue Hb201+ [36] or CBC [21, 22]. If the now observed discrepancy is confirmed in other settings, a re-evaluation of the Biosensor Hb readings against a more robust reference may be needed.

Our study has a number of limitations. Only one laboratory and two field sites were involved, with one operator and one machine per site, a design that does not allow to determine the impact of end-users on performance and repeatability. Our study also used different sample application methods on different blood source types, thus the effects of method and blood source on repeatability could not be separated.

In conclusion, there was no significant advantage of modifying the current recommended test procedures, nor was there any difference in readings between capillary and venous blood sampling. The currently recommended cut-off activities to define normal G6PD activities might be conservative, and may lead to misclassification of a proportion of G6PD normal individuals as having intermediate activity. If confirmed in other settings, patients eligible for effective radical cure might be withheld the right treatment. Whilst the Biosensor has the potential to facilitate universal definitions of G6PD deficiency in remote areas [34], the observed difference in Hb readings between the Biosensor and Hemocue warrants further investigation at other sites.

Supporting information

S1 Fig

Boxplot of absolute difference per paired Biosensor Hb readings per method from field studies in Indonesia (left) and Nepal (right).

(TIF)

pone.0296708.s001.tif (704.1KB, tif)
S2 Fig. Boxplot of the time differences (in minutes) between blood collection and G6PD activity measurement for Biosensor Standard Method (capillary blood), Biosensor Method 3 (venous EDTA blood), and spectrophotometry (venous EDTA blood).

(TIF)

pone.0296708.s002.tif (278.6KB, tif)
S3 Fig. Bland-Altman plot comparing G6PD activity readings by the Biosensor Standard Method against the Biosensor Method 3; the green dashed line marks the mean difference and the area shaded in grey depicts the 95% limits of agreement.

(TIF)

pone.0296708.s003.tif (385KB, tif)
S4 Fig

Scatterplot of Hb readings by the Biosensor using the Standard Method vs Hemocue (left, r = 0.895, p<0.001); and by the Biosensor using the Standard Method vs Method 3 (right, r = 0.881, p<0.001).

(TIF)

pone.0296708.s004.tif (793.8KB, tif)
S5 Fig

Bland-Altman plots comparing Hb readings by the Biosensor Standard Method (left) and Method 3 (right), both against Hemocue; the green dashed line marks the mean difference and the areas shaded in grey depicts the 95% limits of agreement.

(TIF)

pone.0296708.s005.tif (928.8KB, tif)
S1 Table. Summary of groupings and comparisons to assess the repeatability of G6PD measurements by Biosensor.

Definition of a group: the absolute differences of all paired measurements taken using the same method at one site.

(DOCX)

pone.0296708.s006.docx (16.2KB, docx)
S2 Table. Summary of Hb, AMM, and G6PD status of Indonesian study population based on reference spectrophotometry.

(DOCX)

pone.0296708.s007.docx (14.1KB, docx)
S3 Table. Comparison of the median absolute difference between paired Biosensor Hb readings between methods and sites.

(DOCX)

pone.0296708.s008.docx (14.5KB, docx)
S1 Data. Corresponding database.

(XLSX)

pone.0296708.s009.xlsx (30.5KB, xlsx)

Acknowledgments

We would like to thank all participants who allowed us to collect blood samples. We are thankful to all staff involved in this study, including Mr. Umesh Bajgain, laboratory technologist at Tikapur Hospital, Tikapur, Kailali and Mr. Janak Raj Joshi, laboratory technologist at Malakheti Hospital, Malakheti, Kailali for their help during entire study period.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This work has received grant funding from the Australia-Indonesia Institute of the Department of Foreign Affairs and Trade of Australia (AII202100069) awarded to BL. AS is supported by Charles Darwin International PhD Scholarships (CDIPS). Publication costs were funded in part by the Division of Teaching of the Menzies School of Health Research

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Decision Letter 0

Walter RJ Taylor

24 Nov 2023

PONE-D-23-30837Field assessment of the operating procedures of a semi-quantitative G6PD Biosensor to improve repeatability of routine testingPLOS ONE

 Dear Dr. Sadhewa,

Thankyou for submitting your paper. I have a number of comments and suggestions to complement those of the two reviewers. Most are comparatively minor but I do thin you need to be more critical in the Discussion.

Abstract

Line 34 – suggest you add by Biosensor measured G6PD activity

Line 45 – worth saying that the most patients were G6PD normal

Introduction

Line 58 – this sentence implies that some blood stage drugs have antihypnozoite action. I am not aware of any but please include the name of the drug you think has such an action.

Line 77 – a reference for acute kidney injury is required

Lone 99 – not everyone will know the difference between repeatability and reproducibility. Perhaps, these can be defined in the Methods section.

Methods

The methods are a little difficult to follow and I would suggest that more detail is added to Figure 1 to show exactly what was done in the field trial – which method and timing and how each method was analysed i.e. intra group comparison vs. intergroup comparison.

Was there not also an intragroup comparison in Nepal – you took 2 finger pricks per patients?

Line 163 – there is no information on how patients were selected. Did any of them have malaria or other illnesses?

Line 171 – G6PD activity was measured from a venous sample with variable delay. Was pipetting involved in the spectro measurement? If so, we need to know the details to compare with Method 3.

Line 172 – can we assume this was also at 40C and for how long?

Line – 195 – often with Bland Altman analyses, clinicians predefine limits. The authors may wish to do this for the G6PD activity and haemoglobin concentrations. Also, it is conventional to add 95% confidence intervals around the mean difference on the Figures. Can this be done, please?

Results

Line 255 – you mean compared to the HemoCue 301?

Line 263 – with this large difference between the spectrophotometric result and the Biosensor, to me this justifies a Bland Altman analysis between the spectro and standard method with the Figure in the main paper.

Line 270 – I would suggest the G6PD results are integrated in Table 2.

Discussion

Line 306 – did you assess the AMM with the spectrophotometric method?

Line 310 – We need more details about how the manufacturer arrived at higher AMM to justify the statement that their cut off choice is conservative.

Line 315 – so which method is correct? In previous evaluations did the Biosensor also have high Hbs? A mean of 16 g/dL seems quite high which is why it is important to know who was recruited and from where.

Y axis of S5 needs units - g/dL.

Line 317 – does this sentence colour the conclusion in line 310 regarding the SD Biosensor cut off being conservative?

323 – precision. To me this is the same as repeatability. Suggest one or other term is used as the non-specialist may not appreciate that they are the same.

Line 329 – clinical implications?

Line 331 – HemoCue is not a reference method

Limitations

This was quite small study. A full blood count, the reference method for measuring the haemoglobin concentration, was not done. Whilst not essential, genotyping would have been very useful. Most patients were G6PD normal. Applicability to G6PDd and heterozygous females?

General points

The Discussion needs to suggest why we see large differences between the Biosensor and the specto reading and the Biosensor and HemoCue for the Hb. What are the clinical implications of these findings? Are we going to give tafenoquine to the wrong patient?

Ideas for future research?

What about the Biosensor – fit for purpose?

As per one of the reviewers, please critically compare and contrast your findings with the Cambodian study – Adhikari et al. 2022.

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Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #1: Field assessment of the operating procedures of a semi-quantitative G6PD Biosensor to improve repeatability of routine testing—is an important study and offers valuable insights on the scope of Biosensor comparing the findings from two countries with a reference method: Spectrophotometry. The article is well written. I have few comments below for authors’ consideration.

On methods: Could the study have recruited a more heterogenous group of participants based on their G6PD status so that it could have generated the measurement for various categories including repeatability?

Also add an explanation on why Indonesia had only 60 participants compared to 120 in Nepal?

Statistical analysis

Explain why data were analyzed using different versions of STATA.

Discussion

Line 295: More explanation needed on this. Does this imply Hb measurement by biosensors are more variable and is not suitable to rely on?

Line 298: Add if it was statistically significant?

Line 301 to 305: Does it imply the limitations of the reference method?

Line 306-310: The discrepancy of AMM based categories and manufacturer's recommendations have been previously shown in Cambodia. Suggest discussing the previous findings.

Line 328-331: Suggest adding with the more recent study from Cambodia that showed discrepancy in manufacturer's recommendation.

In conclusion, authors have well summarized the findings with implications for future studies. But there is also a need to improve the currently deployed SD biosensor owing to some of the user identified shortcomings of the machine highlighted in Cambodian field studies. This also means newer versions could improve these limitations (this may have been overlooked or the manufacturer may have been complacent about it) including the need for more competition from different manufacturers.

Minor:

Figure 3 legend: thorough? Should it be through?

Reviewer #2: This study is interesting and explore different techniques and result of biosensor, and it was also interesting to compare Hb result between biosensor and hemocue. However, there are some questions that need to address :

1. This study used 4 modification techniques but did not mentioned the background of using those different methods, kindly explain.

2. no G6PD deficient sample included in pilot study and only 1 G6PD def samples included in field study, will the result be different if G6PD deficient samples included? please justify why the author only use G6PD normal samples in pilot study.

3. Why did the author use hemocue for normalized Hb reading from spectrophotometry? is this the standard one? please justify and add information about this.

**********

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Reviewer #2: No

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PLoS One. 2024 Jan 19;19(1):e0296708. doi: 10.1371/journal.pone.0296708.r002

Author response to Decision Letter 0

10 Dec 2023

Author’s note: The lines cited in the responses below refer to the clean version of the revised manuscript (Manuscript.docx).

Response to comments from the Editor

Abstract

Point 1. Line 34 – suggest you add by Biosensor measured G6PD activity

Response 1. We have clarified that it was measured by the Biosensor in the abstract (line 33: “The adjusted male median (AMM) of the Biosensor Standard Method readings was defined as 100% activity.”

Point 2. Line 45 – worth saying that the most patients were G6PD normal

Response 2. We have added this information as follows (lines 37-38): “One Nepalese participant had <30% activity, one Indonesian and 10 Nepalese participants had intermediate activity (≥30% to <70% activity).”

Introduction

Point 3. Line 58 – this sentence implies that some blood stage drugs have antihypnozoite action. I am not aware of any but please include the name of the drug you think has such an action.

Response 3. We have changed the respective sentence as follows (line 58-59): “Since schizontocidal antimalarial drugs have no activity against hypnozoites, radical cure requires a combination of schizontocidal and hypnozoitocidal agents.”

Point 4. Line 77 – a reference for acute kidney injury is required

Response 4. We have added a respective reference (reference 16, line 77).

Point 5. Line 99 – not everyone will know the difference between repeatability and reproducibility. Perhaps, these can be defined in the Methods section.

Response 5. We have added definitions in the objectives (lines 99-101): “The objective of this study was to assess whether the repeatability (variation of measurement results if the same sample is tested repeatedly under the same conditions) and reproducibility (variation of measurement results if the same sample is tested repeatedly under different conditions)…”

Methods

Point 6. The methods are a little difficult to follow and I would suggest that more detail is added to Figure 1 to show exactly what was done in the field trial – which method and timing and how each method was analysed i.e. intra group comparison vs. intergroup comparison.

Response 6. We have added a supplementary table (S1_Table.docx) to clarify the groupings and comparisons of the absolute differences of the paired G6PD activity measurements, referred to at line 208 in the text.

Point 7. Was there not also an intragroup comparison in Nepal – you took 2 finger pricks per patients?

Response 7. No, the G6PD measurements done from the two fingerpricks were paired measurements. The absolute difference between these pairs was compared between methods.

Point 8. Line 163 – there is no information on how patients were selected. Did any of them have malaria or other illnesses?

Response 8. The aim of this study was to assess the Biosensor’s repeatability by comparing the difference between paired measurements. Any underlying condition of the patient was not relevant for this analysis. Accordingly, we did not collect information regarding malaria or other illnesses.

Point 9. Line 171 – G6PD activity was measured from a venous sample with variable delay. Was pipetting involved in the spectro measurement? If so, we need to know the details to compare with Method 3.

Response 9. Yes, there were multiple pipetting steps involved. We have added further details on the spectrophotometry procedures to the methods section (lines 185-191): “Ten µL of venous blood were mixed thoroughly with 1 mL of R1 reagent that had been reconstituted with lyse reagent (Cat. No. G7583-LYS). After incubation at room temperature for 5 minutes, 2 mL of R2 reagent were added. The absorbance of the mixture was read at 340 nm after incubation for 5 minutes at 37 °C, and again after another 5-minute incubation at the same temperature. G6PD activity was calculated from the difference of measured absorbance and normalized by Hb reading using the formula provided by the manufacturer. Hb levels were measured with a Hb 301 device (Hemocue, USA) done at the same time as reference testing.”

Point 10. Line 172 – can we assume this was also at 40C and for how long?

Response 10. Incubation temperature was at 37 °C for 5 minutes prior to the first measurement, then another 5 minutes before measuring for the second time. We have altered this section accordingly (kjndly see response 9 and lines 187-189): “The absorbance of the mixture was read at 340 nm after incubation for 5 minutes at 37 °C, and again after another 5-minute incubation at the same temperature.”

Point 11. Line 195 – often with Bland Altman analyses, clinicians predefine limits. The authors may wish to do this for the G6PD activity and haemoglobin concentrations. Also, it is conventional to add 95% confidence intervals around the mean difference on the Figures. Can this be done, please?

Response 11. We have added in the captions of Figures S3 and S5 that the grey shaded areas are 95% limits of agreement (lines 502-504 and 510-512, respectively). This was an exploratory study and accordingly, we did not predefine any limits of difference for the Bland-Altman analyses.

Results

Point 12. Line 255 – you mean compared to the HemoCue 301?

Response 12. (Now lines 278-280): This sentence refers to repeatability and does not involve a comparison with a reference method. To assess repeatability, we compared paired Hb readings within the Standard Method and Method 3. We explain how we calculated repeatability within the methods (lines 202-203): “To compare the repeatability of the Standard Method and the modified methods, the absolute difference between paired measurements was calculated for each method.” In the results section we again emphasize that we talk about repeatability (lines 278-280): “The repeatability of Hb readings did not differ in Indonesia (p=0.262), however, in Nepal the Hb median absolute difference of the Standard Method (0.8 g/dL, IQR:0.3 to 1.3) was significantly greater than that of Method 3 (0.3 g/dL, IQR: 0.2 to 0.9); p=0.006, S1 Table, S1 Fig.” .

Point 13. Line 263 – with this large difference between the spectrophotometric result and the Biosensor, to me this justifies a Bland Altman analysis between the spectro and standard method with the Figure in the main paper.

Response 13. (Now line 286) We included a scatterplot with 30, 70, and 100% AMM lines (Figure 7) to illustrate correlation and categorization agreement instead of a Bland-Altman analysis. A direct comparison of different assays is not informative as has been demonstrated by Pfeffer et al [1].

Point 14. Line 270 – I would suggest the G6PD results are integrated in Table 2.

Response 14. We have added the spectrophotometry results, which were only done in Indonesia, in supplementary Table S2, referred to in line 295 in the text.

Discussion

Point 15. Line 306 – did you assess the AMM with the spectrophotometric method?

Response 15. Yes, we did. Kindly see the recently added Table S2. Kindly see our reply to Point 13 above, given that absolute values for G6PD activity differ by assay, a direct comparison of AMM by spectrophotometry and Biosensor is not informative.

Point 16. Line 310 – We need more details about how the manufacturer arrived at higher AMM to justify the statement that their cut off choice is conservative.

Response 16. We do not know how the manufacturer arrived at the suggested definitions. We had enquired with the manufacturer but did not get a clear reply. We agree that this point requires clarification but think this is beyond the scope of this article. We do however report that the site-specific cut-offs we calculated for Indonesia and Nepal suggest fairly conservative definitions by the manufacturer.

Point 17. Line 315 – so which method is correct? In previous evaluations did the Biosensor also have high Hbs? A mean of 16 g/dL seems quite high which is why it is important to know who was recruited and from where.

Response 17. (Now line 342) The Hemocue is probably the most widely used point-of-care device to measure haemoglobin levels, but it is not the reference method (this is complete blood count (CBC)). Previous Biosensor evaluations were cited in line 343 and did not show a significant difference between Hb measurement by Biosensor and Hemocue or CBC. However, these evaluations did not include absolute numbers of Hb levels, only comparisons in the form of Bland-Altman analyses. We agree that a mean of 15.9 g/dL in this study is quite high and is significantly higher than the mean as measured by Hemocue. This will require additional follow up as we have clarified in the conclusion (lines 358-359): “…the observed difference in Hb readings between the Biosensor and Hemocue warrants further investigation…”

Point 18. Y axis of S5 needs units - g/dL.

Response 18. We have revised Figure S5 accordingly.

Point 19. Line 317 – does this sentence colour the conclusion in line 310 regarding the SD Biosensor cut off being conservative?

Response 19. (Now line 343) Following discussion among the authors we have realized that the study design did not allow us to distinguish between a machine inherent erroneous Hb reading and an end user error as stated in the limitations lines 347-349. We have therefore removed the respective sentence.

Point 20. Line 323 – precision. To me this is the same as repeatability. Suggest one or other term is used as the non-specialist may not appreciate that they are the same.

Response 20. (Now line 349) We have replaced the word “precision” with “repeatability”.

Point 21. Line 329 – clinical implications?

Response 21. We added a possible implication of the manufacturer-recommended cut-off being too conservative (lines 356-357): “If confirmed in other settings, patients eligible for effective radical cure might be withheld the right treatment.”

Point 22. Line 331 – HemoCue is not a reference method

Response 22. (Now lines 359). We replaced “the reference method” with “Hemocue”.

Limitations

Point 23. This was quite small study. A full blood count, the reference method for measuring the haemoglobin concentration, was not done. Whilst not essential, genotyping would have been very useful. Most patients were G6PD normal. Applicability to G6PDd and heterozygous females?

Response 23. We have added our sample size considerations to the methods (lines 219-225): “The primary objective of this study was to assess the repeatability of different methods. Repeatability was defined as the difference between paired measurements. To identify a minimal and clinically relevant difference of 0.5 U/g Hb with 80% power and 95% confidence, assuming a standard deviation of 1.5 U/g Hb and a minimal correlation coefficient of r≥0.65 required recruitment of 52 participants per site. Assuming procedural errors in more than 10% of all participants we enrolled 60 participants for each method at each site to allow for a site-specific analysis using a two-sided approach.” We have justified the large proportion of phenotypically G6PD normal participants (lines 162-165): “Variation and repeatability of an assay is relative to the absolute values measured. The lower the absolute “true” value is, the smaller the assay specific absolute variation will be. Considering the inherent background noise of any assay, variation is better measured in samples with higher G6PD activities.”

General points

Point 24. The Discussion needs to suggest why we see large differences between the Biosensor and the specto reading and the Biosensor and HemoCue for the Hb. What are the clinical implications of these findings? Are we going to give tafenoquine to the wrong patient?

Response 24. Whether the evaluated assay is suitable to guide tafenoquine based radical cure is best answered by assessing the device’s performance (sensitivity and specificity). This has been done repeatedly [2] and was not part of this study. In this study we assessed whether we could improve repeatability, highly relevant when introducing the device into routine care. We therefore do not comment on the assay suitability to guide radical cure.

Point 25. Ideas for future research?

Response 25. Plenty! Amongst others we conclude with the following statement (lines 357-359): “Whilst the Biosensor has the potential to facilitate universal definitions of G6PD deficiency in remote areas [33], the observed difference in Hb readings between the Biosensor and the Hemocue warrants further investigation at other sites,”

Point 26. What about the Biosensor – fit for purpose?

Response 26. Our study focused on several aspects of the Biosensor. As stated in our concluding paragraph (lines 352-359), we found that the Biosensor gave consistent results regardless of procedure modifications, blood type, or time delay. However, the concerns with the manufacturer-recommended cut-off and the observed high Hb readings warrant further investigations.

Point 27. As per one of the reviewers, please critically compare and contrast your findings with the Cambodian study – Adhikari et al. 2022.

Response 27. We have added a section where we compare our findings to those of Adhikari et al (lines 333-336): “A recent study from Cambodia report a site-specific AMM of 6.4 U/g Hb, very close to the manufacturer’s 70% cut-off [34]. If this observation is confirmed in other settings, it suggests that the current recommended threshold may be too conservative.”

Response to comments from Reviewer 1

Point 1. Could the study have recruited a more heterogenous group of participants based on their G6PD status so that it could have generated the measurement for various categories including repeatability?

Response 1. Variation and repeatability in G6PD measurement are relative to the measured absolute G6PD activity. In individuals with lower G6PD activity, the observed variation will be smaller and harder to distinguish from the inherent background noise of the assay. In line with our aim to assess repeatability, we only recruited G6PD normal individuals for the pilot study and did not use any G6PD activity based inclusion criteria in the field studies. We have added this explanation to the Methods section, lines 162-165: “Variation and repeatability of an assay is relative to the absolute values measured. The lower the absolute “true” value is, the smaller the absolute assay specific variation will be. Considering the inherent background noise of any assay, variation is better measured in samples with higher G6PD activity.”

Point 2. Also add an explanation on why Indonesia had only 60 participants compared to 120 in Nepal?

Response 2 We have added the following section at the beginning of the methods (lines 112-116): “While ethical approval in Indonesia was provided to collect capillary and venous blood from the same participant, Nepalese ethics limited blood collection to either venous or capillary blood. Accordingly, twice as many participants were enrolled in Nepal compared to Indonesia, while the number of measurements performed was the same (Fig. 1).”

Point 3. Explain why data were analyzed using different versions of STATA.

Response 3. Different versions of STATA were used between the author’s personal computer (version 13) and the work computer used (versions 15). There was a centralized update from version 15 to 17 in our institution’s computers while preparing this manuscript.

Point 4. Line 295: More explanation needed on this. Does this imply Hb measurement by biosensors are more variable and is not suitable to rely on?

Response 4. (Now line 321): The sentence “The repeatability of Hb readings from capillary samples showed significantly greater variation compared to venous samples in Nepal but not in Indonesia (S1 Table and S1 Fig.), however, this variation did not impact on the repeatability of the G6PD activity.” refers to comparison of variability between the two Biosensor methods. The implication was that one method was more variable than the other in measuring Hb, but only in the Nepali field study.

Point 5. Line 298: Add if it was statistically significant?

Response 5. The difference was statistically significant but unlikely of any practical significance. We have clarified this. The revised sentence now reads (lines 324-326): “In the Indonesian field study, G6PD activity by the Standard Method was significantly lower compared to the paired reading of Method 3 performed more than five hours later. However, the median difference was less than 0.5 U/g Hb and thus unlikely to be of clinical relevance.”

Point 6. Line 301 to 305: Does it imply the limitations of the reference method?

Response 6. (Now lines 327-331) Yes. As explored in Pfeffer et al’s meta-analysis of the reference method [1] it has been shown that G6PD measurements using the reference method vary due to the multiple brands of assay kits and differences in spectrophotometry instruments.

Point 7. Line 306-310: The discrepancy of AMM based categories and manufacturer's recommendations have been previously shown in Cambodia. Suggest discussing the previous findings.

Response 7. We have added a section where we compare our findings to those of Adhikari et al (lines 335-337): “A recent study from Cambodia report a site-specific AMM of 6.4 U/g Hb, very close to the manufacturer’s 70% cut-off and almost matching our observations. If this observation is confirmed in other settings, it suggests that the current recommended threshold may be too conservative.”

Point 8. Line 328-331: Suggest adding with the more recent study from Cambodia that showed discrepancy in manufacturer's recommendation.

Response 8. We have added comparison in lines 335-337, kindly see response to Point 7 above.

Point 9. Figure 3 legend: thorough? Should it be through?

Response 9. Thank you for pointing out the mistake. It has been revised accordingly (line 159): “When the tip is inserted into a blood sample, 10 µL are collected through capillary action.”

Response to comments from Reviewer 2

Point 1. This study used 4 modification techniques but did not mentioned the background of using those different methods, kindly explain.

Response 1. We have added the explanation to the Methods section, lines 136-138: “Four variations to the standard method were developed, considering practical relevance in remote field setting, including the use of stored blood (capillary and venous) and additional equipment such as micropipettes:”

Point 2. No G6PD deficient sample included in pilot study and only 1 G6PD def samples included in field study, will the result be different if G6PD deficient samples included? please justify why the author only use G6PD normal samples in pilot study.

Response 2. Variation and repeatability in G6PD measurement are relative to the measured absolute G6PD activity. In individuals with lower G6PD activity, the observed variation will be smaller and harder to distinguish from the inherent background noise of the assay. In line with our aim to assess repeatability, we only recruited G6PD normal individuals for the pilot study and did not use any G6PD activity based inclusion criteria in the field studies. We have added this explanation to the Methods section, lines 162-165: “Variation and repeatability of an assay is relative to the absolute values measured. The lower the absolute “true” value is, the smaller the absolute assay specific variation will be. Considering the inherent background noise of any assay, variation is better measured in samples with higher G6PD activities.”

Point 3. Why did the author use hemocue for normalized Hb reading from spectrophotometry? is this the standard one? please justify and add information about this.

Response 3. The gold standard for Hb measurement is complete blood count (CBC) but the field site in Indonesia was remote and did not have access to a CBC machine. It is common practice to replace CBC by Hemocue instead since the device shows consistent good performance. We have added the following reference to support this argument [3] (reference 31 in the manuscript).

References

1. Pfeffer DA, Ley B, Howes RE, Adu P, Alam MS, Bansil P, et al. Quantification of glucose-6-phosphate dehydrogenase activity by spectrophotometry: A systematic review and meta-analysis. PLOS Medicine. 2020;17(5):e1003084. doi: 10.1371/journal.pmed.1003084.

2. Zobrist S, Brito M, Garbin E, Monteiro WM, Clementino Freitas S, Macedo M, et al. Evaluation of a point-of-care diagnostic to identify glucose-6-phosphate dehydrogenase deficiency in Brazil. PLOS Neglected Tropical Diseases. 2021;15(8):e0009649. doi: 10.1371/journal.pntd.0009649.

3. Jain A, Chowdhury N, Jain S. Intra- and inter-model reliability of Hemocue Hb 201+ and HemoCue Hb 301 devices. Asian J Transfus Sci. 2018;12(2):123-6. doi: 10.4103/ajts.AJTS_119_17. PubMed PMID: 30692796; PubMed Central PMCID: PMCPMC6327767.

Decision Letter 1

Walter RJ Taylor

18 Dec 2023

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

PONE-D-23-30837R1

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Acceptance letter

Walter RJ Taylor

10 Jan 2024

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Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Fig

    Boxplot of absolute difference per paired Biosensor Hb readings per method from field studies in Indonesia (left) and Nepal (right).

    (TIF)

    pone.0296708.s001.tif (704.1KB, tif)
    S2 Fig. Boxplot of the time differences (in minutes) between blood collection and G6PD activity measurement for Biosensor Standard Method (capillary blood), Biosensor Method 3 (venous EDTA blood), and spectrophotometry (venous EDTA blood).

    (TIF)

    pone.0296708.s002.tif (278.6KB, tif)
    S3 Fig. Bland-Altman plot comparing G6PD activity readings by the Biosensor Standard Method against the Biosensor Method 3; the green dashed line marks the mean difference and the area shaded in grey depicts the 95% limits of agreement.

    (TIF)

    pone.0296708.s003.tif (385KB, tif)
    S4 Fig

    Scatterplot of Hb readings by the Biosensor using the Standard Method vs Hemocue (left, r = 0.895, p<0.001); and by the Biosensor using the Standard Method vs Method 3 (right, r = 0.881, p<0.001).

    (TIF)

    pone.0296708.s004.tif (793.8KB, tif)
    S5 Fig

    Bland-Altman plots comparing Hb readings by the Biosensor Standard Method (left) and Method 3 (right), both against Hemocue; the green dashed line marks the mean difference and the areas shaded in grey depicts the 95% limits of agreement.

    (TIF)

    pone.0296708.s005.tif (928.8KB, tif)
    S1 Table. Summary of groupings and comparisons to assess the repeatability of G6PD measurements by Biosensor.

    Definition of a group: the absolute differences of all paired measurements taken using the same method at one site.

    (DOCX)

    pone.0296708.s006.docx (16.2KB, docx)
    S2 Table. Summary of Hb, AMM, and G6PD status of Indonesian study population based on reference spectrophotometry.

    (DOCX)

    pone.0296708.s007.docx (14.1KB, docx)
    S3 Table. Comparison of the median absolute difference between paired Biosensor Hb readings between methods and sites.

    (DOCX)

    pone.0296708.s008.docx (14.5KB, docx)
    S1 Data. Corresponding database.

    (XLSX)

    pone.0296708.s009.xlsx (30.5KB, xlsx)

    Data Availability Statement

    All relevant data are within the manuscript and its Supporting Information files.

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