Association of G6PD status and haemolytic anaemia in patients receiving anti-malarial agents: a systematic review and meta-analysis

Abstract

Background

Some anti-malarial drugs often cause haemolytic anaemia in glucose-6-phosphate-dehydrogenase deficiency (G6PDd) patients. This study aims to analyse the association of G6PDd and anaemia in malaria patients receiving anti-malarial drugs.

Methods

A literature search was performed in major database portals. All studies searched using keywords with Medical Subject Headings (MeSH) were included, without date or language restriction. Pooled mean difference of haemoglobin and risk ratio of anaemia were analysed using RevMan.

Results

Sixteen studies comprising 3474 malaria patients that included 398 (11.5%) with G6PDd were found. Mean difference of haemoglobin in G6PDd/G6PD normal (G6PDn) patients was − 0.16 g/dL (95% CI − 0.48, 0.15; I² 5%, p = 0.39), regardless of the type of malaria and dose of drugs. In particular with primaquine (PQ), mean difference of haemoglobin in G6PDd/G6PDn patients with dose < 0.5 mg/kg/day was − 0.04 (95% CI − 0.35, 0.27; I² 0%, p = 0.69). The risk ratio of developing anaemia in G6PDd patients was 1.02 (95% CI 0.75, 1.38; I² 0%, p = 0.79).

Conclusion

Single or daily standard doses of PQ (0.25 mg/kg/day) and weekly PQ (0.75 mg/kg/week) did not increase the risk of anaemia in G6PDd patients.

Background

In 2021, according to the World Health Organization (WHO), approximately 241 million malaria cases occurred worldwide. Although there was a significant decrease of 20 million malaria cases compared to 2010, data from the last three years showed a trend of increasing incidence and relative to the 2019 data, a steep increase of fourteen million malaria cases occurred in 2021[1].

Efforts to eradicate malaria worldwide still face serious challenges in establishing an accurate diagnosis, adequate access to diagnostic tests, and accessible anti-malarial drugs. Misdiagnosis and mistreatment of malaria, in particular the dormant stage (hypnozoite) in the latency of Plasmodium vivax malaria, can lead to prolonged transmission and onset of resistance to countermeasures against both the parasite and its mosquito vector [2, 3]. The only available anti-malarial agents against latent malaria, the 8-aminoquinolines (primaquine and tafenoquine) cause potentially serious haemolytic anaemia in glucose-6-phosphate-dehydrogenase deficient (G6PDd) patients, and hence, very often imposes a critical barrier to safe access [4, 5].

In particular, for primaquine (PQ) treatment, the WHO recommends that G6PD status be assessed prior to high-dose PQ treatment (≥ 0.5 mg/kg). The recommended dose of PQ in all vivax and ovale malaria with G6PD normal (G6PDn) status is 0.25–0.5 mg/kg/day for 14 days, whereas in G6PD deficiency (G6PDd) patients, 0.75 mg/kg/week for eight weeks is recommended [6, 7]. Unfortunately, not all countries have adequate resources to screen patients for G6PD. Many physicians rationally decline to offer PQ to G6PD-unknown due to reasonable fear of causing harm. However, the decision likely leads to patients suffering multiple acute attacks with morbidity, risk of mortality, and onward transmission [3].

Many studies have been reported on G6PDd, anti-malarial drugs, and haemolytic anaemia. Two previous systematic reviews showed an increased risk of anaemia in G6PDd patients treated with PQ. However, the overall population was not specifically malaria patients [8, 9]. Based on previous evidence, we conducted this systematic review to evaluate the association of G6PDd and anaemia in malaria patients receiving anti-malarial drugs. These findings help inform the decisions of physicians and policymakers on G6PD screening in malaria-endemic areas.

Methods

This systematic review and meta-analysis were conducted based on The Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) guidelines. The protocol of this review was registered at the Prospective Register of Systematic Reviews (PROSPERO) with code number CRD42021227813.

Searching strategy

The literature search was conducted in July 2021 using six databases: PubMed/ Medline®, Embase®, Cochrane Central Register of Controlled Trials (CENTRAL®), EBSCOhost/CINAHL®, ProQuest®, and Scopus®, along with snowballing method from study references and hand-searching from the electronic university library and local journals or databases. All observational and experimental studies were included in this review. The keywords that were used included “malaria,” “glucose-6-phosphate-dehydrogenase/ G6PD”, “antimalarial” (including the names of antimalarial drugs used by the patients: primaquine, tafenoquine, amodiaquine, pyrimethamine, proguanil, atovaquone, artemisinin, lumefantrine, doxycycline, clindamycin), “anemia”, “hemoglobin”, and “hemolysis”, with MeSH terms in English without time of study or language restriction. Grey literature, such as abstracts, proceedings, and academic thesis, were included in the literature search. The list of keywords from respective databases is shown in Table 1.

Table 1 Searching for articles in databases

Inclusion and exclusion criteria

Studies included in this systematic review and meta-analysis had to meet the following criteria: (1) adult male or female patients with confirmed malaria infection (Plasmodium falciparum, Plasmodium vivax, and/or mixed infection); (2) treatment with anti-malarial agents known to cause haemolysis occurred; and (3) patients underwent evaluation of G6PD status, anaemia, and/or haemolysis evaluation. Study subjects with only paediatrics and/or normal G6PD level were excluded from the systematic review. No animal, in-vitro, or healthy volunteer studies were included.

Malaria diagnosis and species of infection were defined by positive thin and thick blood film examination by microscopy on day 0. Definition of G6PD deficiency, anaemia and/or haemolysis, and haemoglobin level were determined based on standard examination in respective studies. The use of anti-malarial agents included drugs such as primaquine, tafenoquine, amodiaquine, pyrimethamine, proguanil, atovaquone, artemisinin, lumefantrine, doxycycline, and clindamycin.

Data extraction

Literature search was conducted by AP, whereas both title/abstract screening and full-text reading were reviewed by two independent investigators (SS, AP), with the help of Covidence® online software. Any disagreements during the process were discussed and resolved by a third independent investigator (EJN).

Data extraction comprised of author’s name, year of study, country, study drugs and dose, number of participants, gender, mean age, type of malaria infection, method of study, and duration of follow-up. The outcomes of the study were anaemia, level of haemoglobin, or haemolysis status, depicted in the mean difference of haemoglobin or risk ratio of anaemia. When unavailable, data on the outcomes were described in the text review.

Quality assessment

Selected studies were critically reviewed based on the type of studies. Randomized controlled trials (RCTs) were assessed using Cochrane risk-of-bias tool for randomized trials (RoB 2)[10], and non-randomized trials were assessed with Risk of Bias in Non-Randomized Studies—of Interventions (ROBINS-I)[11], whereas observational cohort studies were assessed with Newcastle–Ottawa Scale (NOS) checklist for cohort studies [12]. RoB 2 quality was classified into low, with some concerns, or high risk of bias; ROBINS-I was classified into low, moderate, serious, critical risk of bias, or no information; and NOS for cohort studies was classified into high-quality study, high risk of bias, and very high risk of bias studies. When the data to assess the risk of bias was unclear, attempts were made to contact the authors for clarification. The quality of evidence was assessed by using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach [13].

Statistical analysis

Statistical analysis was performed using RevMan® version 5.4.1. Data were presented in tables, and the results of the analysis were presented in the forest plot (when possible) or text review. In the absence of statistical heterogeneity, a fixed-effects model was used; otherwise, a random-effects model was performed in more heterogeneous trials. When available, the values were reported as mean differences in haemoglobin level and risk ratio of anaemia as aggregated data, with a 95% confidence interval (95% CI) and p-value. Heterogeneity was identified using the I² statistic, in which value I² 0%-40%: might not be important, 30%-60%: moderate heterogeneity, 50%-90% substantial heterogeneity, and 75%-100%: considerable heterogeneity.

Results

The initial literature search found 1533 articles from databases and manual searching. After excluding duplicates, 1097 articles were screened based on their title and abstract. A total of 1046 articles were excluded, leaving 51 articles for full-text reading and assessment of eligible articles. Thirty-five studies were excluded, as depicted in Fig. 1. Eleven articles did not include haemoglobin level and/or anaemia and/or haemolysis status as an outcome; one study was done only on the paediatric population; 5 studies were done on healthy subjects, using anti-malarials as prophylaxis; 9 studies excluded G6PDd patients; 2 studies were preliminary studies, and 2 studies were cross-sectional studies. In summary, 16 articles were included in this systematic review. The publication year for the articles ranged from 1981 to 2021, most were published in English, and one article was in Portuguese.

Fig. 1

Study selection flow diagram

Sixteen selected articles comprised 3,415 malaria patients treated with studied anti-malarial agents, and 400 (11.7%) had G6PDd. Seven articles reported only malaria caused by P. falciparum, five articles reported only malaria caused by P. vivax, and the rest studied malaria caused by both parasites and/or mixed infection [14,15,16,17,18]. Searching was done for anti-malarials not limited only to PQ, but most studies (14 articles) [14,15,16,17, 19,20,21,22,23,24,25,26,27,28,29] reported the study of PQ, and the two remaining studies [18, 20] reported the use of other anti-malarials: chlorproguanil–dapsone–artesunate (CDA), chlorproguanil–dapsone (CPG–DDS), sulfadoxine-pyrimethamine (SP), and chloroquine (CQ). However, the study population with CDA, CPG-DDS, SP, and CQ only was not included in the meta-analysis due to the limited study. There was only one study for each drug; therefore the meta-analysis for non-PQ drugs could not be performed. The dose of PQ used in each article was various. Some of the studies used more than one regimen dose for PQ; four studies used a single PQ dose of 0.25 mg/kg, one study used a single 15 mg PQ, one study used a single PQ dose of 0.25–0.5 mg/kg, one study used a single PQ dose of 0.75 mg/kg, three studies used daily 0.25 mg/kg/day PQ for fourteen days, three studies used daily 0.25–0.5 mg/kg PQ for seven to fourteen days, one study used daily 15 mg PQ for fourteen days, one study used PQ dose of ≥ 0.5 mg/kg/day for fourteen days, and two studies used PQ 0.75 mg/kg/weekly for 8 weeks. The duration of treatment follow-up also varied, ranged from 7 to 30 days, depending on the species involved.

All selected articles reported outcomes of anaemia or haemolysis, but only one half of them reported the outcomes as mean difference of haemoglobin level between G6PDn and G6PDd patients, risk ratio of anaemia, or both, and hence, not all articles could be further analysed in the meta-analysis [19,20,21,22, 24, 25, 27, 28]. The outcomes of the other one half portion of the selected articles were presented as mean or median haemoglobin level, haemolysis incident, rate of anaemia, or mean percentage of fractional haemoglobin reduction [14,15,16,17,18, 23, 26, 29]. G6PD deficiency was established through various methods, such as by biochemical phenotyping examination (qualitative or quantitative) followed by genotyping assessment, phenotyping or genotyping examination only, or even not mentioned in the article. The summary of articles is shown in Table 2.

Table 2 Summary of characteristics of study participants

Risk of bias assessment

The selected RCTs were evaluated for risk of bias with ROB 2 and showed some concerns, low, and high risk of bias (Table 3). Studies with high risk of bias indicated concerns in missing outcome data, deviation from intended intervention, or process of randomization. Among non-RCTs, only three studies presented as high quality, whereas others had moderate or high risk of bias, mainly in the area of identification of participants and assessment of the outcomes [25, 28, 29]. Complete risk of bias assessment figures were depicted in Tables 3, 4, and 5.

Table 3 Risk of bias of randomized controlled trial studies with ROB 2

Table 4 Risk of bias of non-randomized studies for intervention with ROBINS-I

Table 5 Risk of bias of cohort studies with NOS

Mean difference of haemoglobin level

Data of mean difference of haemoglobin level before and after anti-malarials administration in G6PDd and G6PDn patients were only available in seven studies comprising nine groups that could be further analysed in the forest plot. The mean difference of haemoglobin in G6PDd patients is higher than the G6PDn patients [− 0.70 (95% CI − 1.91, 0.50) vs. 0.27 (95% CI 0.09, 0.45)]. When both groups were further analysed, the gap of mean difference between G6PDd and G6PDn group was lower in the G6PDd group -0.16 (95% CI − 0.48, 0.15); however, it was not statistically nor clinically significant (Fig. 2). When analysed separately according to whether the infection was caused by P. falciparum only or P. vivax and/or mixed infection, the mean difference of haemoglobin level showed a slight gap between each group [0.1 (95% CI − 0.27, 0.48) and − 0.56 (95% CI − 1.03, − 0.08)].

Fig. 2

Mean difference of haemoglobin level in G6PD deficient and G6PD normal patients receiving primaquine based on the type of malaria. Data of mean difference was presented as g/dL, standard error (SE), and 95% confidence interval of mean difference. Differences in haemoglobin level were measured between day 0 (baseline) and day 7 or 28, depending on the type of infection and study design. Doses of primaquine vary. SE standard error, G6PD Glucose-6-phosphate-dehydrogenase; ^†analysis of study within 7 days of observation; ‡analysis of study within 28 days of observation; *analysis of study with all doses of primaquine included in the study

The analysis of seven articles (nine study groups) in Fig. 2 were performed with variance in drug doses and duration of follow-up. Analysis of the mean difference in haemoglobin level in the respective two aspects was done. Both groups, with duration of follow-up of seven days or 28 days, showed a slight gap in mean difference, which was not statistically nor clinically significant [0.21 (95% CI − 0.51, 0.09) and − 0.16 (95% CI-0.94, 0.62)] (Figs. 3 and 4).

Fig. 3

Mean difference of haemoglobin in G6PD deficient and G6PD normal patients receiving primaquine with a duration of follow-up of seven days (D0 and D7). Data of mean difference was presented as g/dL, standard error (SE), and 95% confidence interval of mean difference. Doses of primaquine vary. SE standard error, G6PD Glucose-6-phosphate-dehydrogenase; ^†analysis of study within 7 days of observation; *analysis of study with all doses of primaquine included in the study

Fig. 4

Mean difference of haemoglobin in G6PD deficient and G6PD normal patients receiving primaquine with a duration of follow-up of 28 days (D0 and D28). Data of mean difference was presented as g/dL, standard error (SE), and 95% confidence interval of mean difference. Doses of primaquine vary. SE standard error, G6PD Glucose-6-phosphate-dehydrogenase; ‡analysis of study within 28 days of observation

Figure 5 showed a forest plot of mean difference on studies with administration of PQ dose < 0.5 mg between G6PDd and G6PDn group with value − 0.04 (95% CI − 0.35, 0.27). This value was lower compared to the result by Poirot [25] who studied a group with PQ dose administration > 0.5 mg/kg/day in G6PDd and G6PDn patients [mean difference − 2.75 (95% CI − 4.22, − 1.28) and − 0.86 (95% CI − 1.1, − 0.62), respectively].

Fig. 5

Mean difference of haemoglobin in G6PD deficient and G6PD normal patients with administration of low primaquine dose < 0.5 mg/kg/day. Data of mean difference was presented as g/dL, standard error (SE), and 95% confidence interval of mean difference. Doses of primaquine vary. SE standard error, G6PD Glucose-6-phosphate-dehydrogenase, PQ primaquine; †analysis of study within 7 days of observation; ¹analysis of study with primaquine dose ≤0.25 mg/kg/day; ²analysis of study with primaquine dose 0.25–0.5 mg/kg/day

The risk of developing anaemia after anti-malarial therapy

Three studies [19,20,21,22, 27] evaluated the risk of developing anaemia after a single dose or a daily dose of PQ in the G6PDd and G6PDn group with an overall risk ratio of 1.02 (95% CI 0.75, 1.38) (Fig. 6).

Fig. 6

Risk ratio of developing anaemia in G6PD deficiency and G6PD normal before and after single dose or daily primaquine treatment. Data of mean difference was presented as g/dL, standard error (SE), and 95% confidence interval of mean difference. Doses of primaquine vary. SE standard error, G6PD Glucose-6-phosphate-dehydrogenase; ^†analysis of study within 7 days of observation

Discussion

In the study protocol, many anti-malarials were included to evaluate their effect on the G6PDd population, however, most of the studies were done using primaquine and only a small number of studies used other anti-malarials, such as tafenoquine, chlorproguanil–dapsone–artesunate (CDA), chlorproguanil–dapsone (CPG–DDS), sulfadoxine-pyrimethamine (SP), chloroquine; therefore, we only examined the pooled effect of primaquine. 16 studies on malaria patients treated with anti-malarial agents were analyzed. Among 3,474 patients, 11.5% of them are G6PD deficient. Most studies evaluated PQ as the investigational drug and excluded severe malaria infection. PQ is one of the drugs to be wary of due to the high risk of haemolytic anaemia [4, 30].

Regardless of the type of Plasmodium species and the administered anti-malarial drugs, forest plot of seven studies showed a small mean difference of haemoglobin level between G6PDd and G6PDn group (− 0.16 g/dL) (Fig. 2). Other studies not included in the meta-analysis had similar outcomes. One study with PQ in South Africa [23] measured the median haemoglobin level and found that G6PDd subjects reached a lower haemoglobin level compared to G6PDn subjects, however, its nadir point was 11.5 g/dL and it was clinically harmless. Other studies in single low dose PQ in P. falciparum infection also highlighted no difference in mean haemoglobin level or percentage of mean relative haemoglobin reduction between G6PDd and G6PDn subjects [22, 24].

A study by Llanos-Cuentas compared the haemolytic effect of primaquine and tafenoquine. The results showed that the decrease in haemoglobin levels occurred in the normal G6PD group and did not occur in the G6PDd group, although the number of subjects in the G6PDd group was very low. Decreased haemoglobin levels occurred in the tafenoquine and primaquine groups but were self-limited without requiring special treatment [15].

Based on these studies, it was presumed that anti-malarial treatment in non-severe malaria patients would be unlikely to cause a clinically significant side effect of anaemia. Analysis of three studies on PQ also did not show an increased risk ratio of developing anaemia in G6PDd patients, hence, confirming the safety of PQ (Fig. 6).

Almost half of the articles included in this review were performed in P. falciparum infection only [19,20,21,22,23,24, 29]. Based on the subgroup analysis of forest plot, the mean difference of haemoglobin level between P. falciparum and P. vivax and/or mixed infection was not statistically nor clinically significant. Other studies on P. vivax that were not included in the forest plot showed various outcomes [15,16,17,18, 26]. A multinational RCT with low dose PQ (15 mg) and single-dose tafenoquine observed no significant decrease of haemoglobin level in the G6PDd group [15]. In contrast, one cohort in Brazil with higher dose PQ (0.5 mg/kg/day, seven days) displayed a lower mean of haemoglobin level in G6PDd group [16]. However, this study had small number of study subjects and a relatively short duration of follow-up (3 days), therefore, further higher quality study is needed. Two other studies in P. vivax and/or mixed infection revealed an increased risk of haemolysis and severe anaemia in PQ administration, nevertheless, they did not perform a comparison to the G6PDn group [17, 26].

Instead of the type of the Plasmodium species, one presumably important condition that might add the risk of anaemia was the accumulation of administered dose of PQ. When PQ was given in a standard low dose (≤ 0.5 mg/kg/day), the result of the forest plot for the mean difference of haemoglobin level between G6PDd and G6PDn groups was not statistically nor clinically significant. Results from other studies with administration of PQ dose < 0.5 mg/kg/day) (not included in the forest plot) also disclosed a consistent outcome [15, 23]. When given a higher dose (0.5 mg/kg/day) of PQ, subjects showed a significant decrease in mean haemoglobin level and a high rate of severe anaemia in G6PDd patients up to 50% [16, 17]. Studies with PQ dose > 0.5 mg/kg/day in G6PDd patients also showed a significant fall in haemoglobin level and an increased risk of haemolysis up to 30% [25, 26]. There were two studies that used 0.75 mg/kg/week of PQ for 8 weeks. Both studies showed a decline in mean haemoglobin level during the first week, which eventually went up to a normal level in the third and fourth week of drug treatment. The author concluded that the administration of a low dose of PQ (< 0.5 mg/kg/day) and a weekly dose of PQ (0.75 mg/kg/week for eight weeks) in G6PDd patients did not increase the risk of anaemia.

The change of mean difference of haemoglobin level also showed no significant decrease during seven or 28-days follow-up period, indicating that anaemia as a side effect in the administration of anti-malarial was not hazardous in both short and long-term evaluation. Haemolytic anaemia caused by G6PDd is usually a self-limited condition, and anaemia is usually resolved when the precipitating drugs are discontinued.

Among sixteen studies in this review, half were classified as low risk of bias. Some pooled analyses showed high heterogeneity and, therefore, subgroup analysis was performed which exhibited significant results and lower heterogeneity. The funnel plot could not be arranged because of the lack of studies in meta-analysis.

This review is the first systematic review of the effect of G6PD status, which includes only malaria patients receiving anti-malarial agents. A particular strength of this study is that all of the included study was high-quality cohort and randomized controlled trials with before and after endpoints, thus, the authors are confident in this study’s ability to demonstrate a causal relationship between drug administration and haemoglobin level. In the table displaying the summary of findings (Table 6), the certainty of the evidence was downgraded one level due to the serious risk of inconsistency and imprecision. Based on GRADE evaluation, the outcome of this study showed a high and moderate certainty of evidence. Currently, no publication bias was suspected for the outcome included in this review.

Table 6 Summary of the findings

There were several limitations in this study, i.e., heterogenous species of malaria parasites, various type and dose of administered drugs, small sample size in G6PD groups, and diverse method of outcomes measurement. Moreover, the G6PD status was not assessed genotypically, and phenotypes can be variable across different mutations. Higher quality clinical trials regarding the use of anti-malarial agents in G6PDd patients and their side effect on haemoglobin levels are recommended.

Based on this systematic review and meta-analysis, it was concluded that in G6PDd patients, the side effect of anaemia in single, daily low-dose (0.25 mg/kg/day), and weekly PQ was not statistically and clinically significant, and therefore, a massive screening of G6PD status might not be relevant in all populations. Several studies on the safety of anti-malarial regimens concerning G6PDd patients are ongoing (clinicaltrials.gov NCT03529396; NCT05044637; NCT05096702).

Conclusion

Single low dose (0.25 mg/kg) or daily standard low dose of PQ (0.25 mg/kg/day) and weekly PQ (0.75 mg/kg/week for eight weeks) does not increase the risk of anaemia in G6PDd patients.