Haematological profile of malaria patients with G6PD and PKLR variants (erythrocytic enzymopathies): a cross-sectional study in Thailand

Abstract

Background Glucose 6-phosphate dehydrogenase (G6PD) and pyruvate kinase (PKLR) deficiencies are common causes of erythrocyte haemolysis in the presence of antimalarial drugs such as primaquine and tafenoquine. The present study aimed to elucidate such an association by thoroughly investigating the haematological indices in malaria patients with G6PD and PKLRR41Q variants. Methods Blood samples from 255 malaria patients from Thailand, Myanmar, Laos, and Cambodia were collected to determine haematological profile, G6PD enzyme activity and G6PD deficiency variants. The multivariate analysis was performed to investigate the association between anaemia and G6PD MahidolG487A, the most common mutation in this study. Results The prevalence of G6PD deficiency was 11.1% (27/244) in males and 9.1% (1/11) in female. The MAFs of the G6PD MahidolG487A and PKLRR41Q variants were 7.1% and 2.6%, respectively. Compared with patients with wildtype G6PD after controlling for haemoglobinopathies, G6PD-deficient patients with hemizygous and homozygous G6PD MahidolG487A exhibited anaemia with low levels of haemoglobin (11.16 ± 2.65 g/dl, p = 0.041). These patients also exhibited high levels of reticulocytes (3.60%). The median value of G6PD activity before treatment (Day 0) was significantly lower than that of after treatment (Day 28) (5.51 ± 2.54 U/g Hb vs. 6.68 ± 2.45 U/g Hb; p < 0.001). Reticulocyte levels on Day 28 were significantly increased compared to that of on Day 0 (2.14 ± 0.92% vs 1.57 ± 1.06%; p < 0.001). PKLRR41Q had no correlation with anaemia in malaria patients. The risk of anaemia inpatients with G6PDMahidolG487A was higher than wildtype patients (OR = 3.48, CI% 1.24–9.75, p = 0.018). Univariate and multivariate analyses confirmed that G6PDMahidolG487A independently associated with anaemia (< 11 g/dl) after adjusted by age, gender, Plasmodium species, parasite density, PKLRR41Q, and haemoglobinopathies (p < 0.001). Conclusions This study revealed that malaria patients with G6PD MahidolG487A, but not with PKLRR41Q, had anaemia during infection. As a compensatory response to haemolytic anaemia after malaria infection, these patients generated more reticulocytes. The findings emphasize the effect of host genetic background on haemolytic anaemia and the importance of screening patients for erythrocyte enzymopathies and related mutations prior to anti-malarial therapy.

Effect of neonatal reticulocytosis on glucose 6-phosphate dehydrogenase (G6PD) activity and G6PD deficiency detection: a cross-sectional study

“…G6PD activity values of male and female newborns are expressed as the median ± interquartile range (IQR). The cut-off values of G6PD activity were defined as follows: deficiency < 30% and intermediate 30%—80% of the normal median [ 14 ]. Subjects with G6PD activity > 80% of the normal median were considered normal [ 12 ].…”

“…A quantitative G6PD enzymatic activity assay based on a spectrophotometric technique is the reference assay to detect G6PD deficiency and G6PD intermediates [ 11 ]. It was suggested by previous studies that the cut-off values for G6PD deficiency and intermediate G6PD should be activity values less than 30% and 30% to 60% of the adjusted male median (AMM), respectively [ 11 – 14 ]. The WHO has recommended that activity values less than 30% and 30%-80% of the normal median should be used instead [ 15 ].…”

Single-Drop Blood Detection of Common G6PD Mutations in Thailand Based on Allele-Specific Recombinase Polymerase Amplification with CRISPR-Cas12a

“…The G6PD variants are classified by the World Health Organization (WHO) into five classes based on G6PD activity and hemolytic levels . In Southeast Asia, the prevalence of G6PD deficiency was 11.0% in Thailand, 8.1% in Laos, 8.9% in Vietnam, 10.0% in Myanmar, and 15.8% in Cambodia . Currently, more than 186 mutations have been identified in the G6PD gene, of which nearly half are associated with reduced activity or stability of the G6PD enzyme .…”

“…Existing G6PD molecular genotyping methods are laborious and require specialized equipment and maintenance services, making them unsuitable for G6PD mutation screening in a low-resource environment. G6PD genotyping can be achieved in several ways, including polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), ,, Taq Man SNP assay, AS-PCR, reverse dot blot hybridization (RDB), amplification refractory mutation system, , gold nanoparticle-based test, high-resolution melting curve analysis (HRM), − and DNA sequencing. , In addition, a high-throughput multiplex AS-PCR-based assay is available for the diagnosis of G6PD mutations. DiaPlexC G6PD genotyping kits based on a single PCR step with gel electrophoresis are capable of identifying the G6PD mutations, which are specific to Asians , and Africans. , Recently, isothermal amplification methods using locked nucleic acids (LNAs) and the Yaku-Bonczyk principle primers for identifying substitution mutations for G6PD mutations at the point of care have been explored, as well.…”

“…Existing G6PD molecular genotyping methods are laborious and require specialized equipment and maintenance services, making them unsuitable for G6PD mutation screening in a low-resource environment. G6PD genotyping can be achieved in several ways, including polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), 4 , 6 , 9 Taq Man SNP assay, 9 AS-PCR, 21 reverse dot blot hybridization (RDB), 22 amplification refractory mutation system, 23 , 24 gold nanoparticle-based test, 25 high-resolution melting curve analysis (HRM), 26 − 28 and DNA sequencing. 4 , 29 In addition, a high-throughput multiplex AS-PCR-based assay is available for the diagnosis of G6PD mutations.…”