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

Glucose 6-phosphate dehydrogenase (G6PD) deficiency is the most common inherited enzymopathy. Identification of the G6PD deficiency through screening is crucial to preventing adverse effects associated with hemolytic anemia following antimalarial drug exposure. Therefore, a rapid and precise field-based G6PD deficiency diagnosis is required, particularly in rural regions where malaria is prevalent. The phenotypic diagnosis of the G6PD intermediate has also been a challenging issue due to the overlapping of G6PD activity levels between deficient and normal individuals, leading to a misinterpretation. The availability of an accurate point-of-care testing (POCT) for G6PD genotype diagnosis will therefore increase the opportunity for screening heterozygous cases in a low-resource setting. In this study, an allele-specific recombinase polymerase amplification (AS RPA) with clustered regularly interspaced short palindromic repeats-Cas12a (CRISPR-Cas12a) was developed as a POCT for accurate diagnosis of common G6PD mutations in Thailand. The AS primers for the wild type and mutant alleles of G6PD MahidolG487A and G6PD ViangchanG871A were designed and used in RPA reactions. Following application of CRISPR-Cas12a systems containing specific protospacer adjacent motif, the targeted RPA amplicons were visualized with the naked eye. Results demonstrated that the G6PD MahidolG487A and G6PD ViangchanG871A assays reached 93.62 and 98.15% sensitivity, respectively. The specificity was 88.71% in MahidolG487A and 99.02% in G6PD ViangchanG871A. The diagnosis accuracy of the G6PD MahidolG487A and G6PD ViangchanG871A assays was 91.67 and 98.72%, respectively. From DNA extraction to detection, the assay required approximately 52 min. In conclusion, this study demonstrated the high performance of an AS RPA with the CRISPR-Cas12a platform for G6PD MahidolG487A and G6PD ViangchanG871A detection assays and the potential use of G6PD genotyping as POCT.

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

BACKGROUND: Screening for G6PD deficiency in newborns can help prevent severe hemolysis, hyperbilirubinemia, and bilirubin encephalopathy, as recommended by the World Health Organization (WHO). It has been speculated that the presence of a high number of reticulocytes in newborns interferes with the diagnosis of G6PD deficiency since reticulocytes contain higher amounts of G6PD enzyme than mature erythrocytes. Therefore, the purposes of this study were to assess the effect of reticulocytosis in the determination of blood G6PD activity in Thai newborns by using a novel automated UV-based enzymatic assay and to validate the performance of this assay for the detection of G6PD deficiency in newborn samples. METHODS: The levels of reticulocytes and G6PD activity were measured in blood samples collected from 1,015 newborns. G6PD mutations were identified using TaqMan® SNP genotyping assay, PCR-restriction fragment length polymorphism (PCR-RFLP), and direct sequencing. The correlation between the levels of reticulocytes and G6PD activity was examined. The performance of the automated method was compared with that of the fluorescent spot test (FST) and the standard quantitative assay. RESULTS: The automated assay detected G6PD deficiency in 6.5% of the total newborn subjects compared to 5.3% and 6.1% by the FST and the standard method, respectively. The minor allele frequencies (MAFs) of G6PD ViangchanG871A, G6PD MahidolG487A, and G6PD UnionC1360T were 0.066, 0.005, and 0.005, respectively. The reticulocyte counts in newborns with G6PD deficiency were significantly higher than those in normal male newborns (p < 0.001). Compared with normal newborns after controlling for thalassemias and hemoglobinopathies, G6PD-deficient patients with the G6PD ViangchanG871A mutation exhibited elevated reticulocyte counts (5.82 ± 1.73%, p < 0.001). In a group of G6PD normal newborns, the percentage of reticulocytes was positively correlated with G6PD activity (r = 0.327, p < 0.001). However, there was no correlation between G6PD activity and the levels of reticulocytes in subjects with G6PD deficiency (r = -0.019, p = 0.881). The level of agreement in the detection of G6PD deficiency was 0.999, while the area under the receiver operating characteristic (AUC) curve demonstrated that the automated method had 98.4% sensitivity, 99.5% specificity, 92.4% positive predictive value (PPV), 99.9% negative predictive value (NPV), and 99.4% accuracy. CONCLUSIONS: We report that reticulocytosis does not have a statistically significant effect on the detection of G6PD deficiency in newborns by both qualitative and quantitative methods.

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

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 G6PD MahidolG487A was higher than wildtype patients (OR = 3.48, CI% 1.24-9.75, p = 0.018). Univariate and multivariate analyses confirmed that G6PD MahidolG487A 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.

An Observational Study of the Effect of Hemoglobinopathy, Alpha Thalassemia and Hemoglobin E on P. Vivax Parasitemia.

BACKGROUND: The protective effect of α-thalassemia, a common hematological disorder in Southeast Asia, against Plasmodium falciparum malaria has been well established. However, there is much less understanding of the effect of α-thalassemia against P. vivax. Here, we aimed to investigate the proportion of α-thalassemia including the impact of α-thalassemia and HbE on the parasitemia of P. vivax in Southeast Asian malaria patients in Thailand. METHODS: A total of 210 malaria patients, admitted to the Hospital for Tropical Diseases, Thailand during 2011-2012, consisting of 159 Myanmeses, 13 Karens, 26 Thais, 3 Mons, 3 Laotians, and 6 Cambodians were recruited. Plasmodium spp. and parasite densities were determined. Group of deletion mutation (--SEA, -α3.7, -α4.2deletion) and substitution mutation (HbCS and HbE) were genotyped using multiplex gap-PCR and PCR-RFLP, respectively. RESULTS: In our malaria patients, 17/210 homozygous and 74/210 heterozygous -α3.7 deletion were found. Only 3/210 heterozygous -α4.2 and 2/210 heterozygous--SEA deletion were detected. HbE is frequently found with 6/210 homozygotes and 35/210 heterozygotes. The most common thalassemia allele frequencies in Myanmar population were -α3.7 deletion (0.282), followed by HbE (0.101), HbCS (0.013), -α4.2 deletion (0.009), and --SEA deletion (0.003). Only density of P. vivax in α-thalassemia trait patients (-α3.7/-α3.7, --SEA/αα, -α3.7/-α4.2) but not in silent α-thalassemia (-α3.7/αα, -α4.2/αα, ααCS/αα) were significantly higher compared with non-α-thalassemia patients (p=0.027). HbE did not affect P. vivax parasitemia. The density of P. falciparum significantly increased in heterozygous HbE patients (p=0.046). CONCLUSIONS: Alpha-thalassemia trait is associated with high levels of P. vivax parasitemia in malaria patients in Southeast Asia.

Modulation of Malaria Phenotypes by Pyruvate Kinase (PKLR) Variants in a Thai Population.

Pyruvate kinase (PKLR) is a critical erythrocyte enzyme that is required for glycolysis and production of ATP. We have shown that Pklr deficiency in mice reduces the severity (reduced parasitemia, increased survival) of blood stage malaria induced by infection with Plasmodium chabaudi AS. Likewise, studies in human erythrocytes infected ex vivo with P. falciparum show that presence of host PK-deficiency alleles reduces infection phenotypes. We have characterized the genetic diversity of the PKLR gene, including haplotype structure and presence of rare coding variants in two populations from malaria endemic areas of Thailand and Senegal. We investigated the effect of PKLR genotypes on rich longitudinal datasets including haematological and malaria-associated phenotypes. A coding and possibly damaging variant (R41Q) was identified in the Thai population with a minor allele frequency of ~4.7%. Arginine 41 (R41) is highly conserved in the pyruvate kinase family and its substitution to Glutamine (R41Q) affects protein stability. Heterozygosity for R41Q is shown to be associated with a significant reduction in the number of attacks with Plasmodium falciparum, while correlating with an increased number of Plasmodium vivax infections. These results strongly suggest that PKLR protein variants may affect the frequency, and the intensity of malaria episodes induced by different Plasmodium parasites in humans living in areas of endemic malaria.