Investigation of glucose-6-phosphate dehydrogenase (G6PD) deficiency prevalence in a Plasmodium vivax-endemic area in the Republic of Korea (ROK)

Abstract

Background: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most prevalent inborn disorder. This X-chromosome-linked recessive disease affects more than 400 million people globally, and is associated with haemolytic anaemia after medication with the anti-latent malaria drug, primaquine. To prevent malaria, the Republic of Korea (ROK) Army administers malaria chemoprophylaxis. Due to the previously low G6PD deficiency prevalence in the ROK, prior to primaquine administration, testing for G6PD deficiency was not mandatory. In this study, to evaluate the risk from malaria chemoprophylaxis in the ROK, G6PD deficiency prevalence was investigated.

Methods: Blood specimens from 1632 soldiers entering training camp for the 3^rd Infantry of the ROK Army were collected. CareStart™ Biosensor for G6PD and haemoglobin (Hb) was used to detect G6PD levels. G6PD variants using the DiaPlexC G6PD Genotyping kit (Asian type) and full-length sequencing were examined.

Results: Of 1632 blood specimens tested, none was observed to be G6PD deficient. The median value of all tested samples was 7.582 U/g Hb. An investigation of 170 G6PD DNA variants was analysed and categorized as partially low normal [n = 131, 30-80% (2.27-6.05 U/g Hb) of the median value], high [n = 3, > 150% (> 11.373 U/g Hb) of the median value], or normal [n = 36, 80-150% (6.05-11.373 U/g Hb) of the median value], and none was amplified by the DiaPlexC kit. Five silent mutations (C→T) in 131 partially low normal specimens were found at the 1311th nucleotide position by sequence analysis. Another 8 silent mutations (T93C) were also detected in 131 partially low normal specimens. Thus, it is inferred that these silent mutations could be related to G6PD activity.

Conclusions: This G6PD deficiency prevalence study, conducted among participants from the 3rd Infantry of the ROK Army, provided crucial evidence for the safety of malaria chemoprophylaxis. This study showed that the prevalence of G6PD deficiency among 1632 young soldiers was wholly absent. Although G6PD phenotypic mutations were not detected, many silent mutations (C1311T and T93C) were observed. Thus, it is inferred that malaria chemoprophylaxis is relatively safe against G6PD deficiency-mediated haemolytic anaemia. However, given the number of individuals whose G6PD were at the partially low normal range and the frequent detection of G6PD deficiency-related mutations, consistent monitoring of G6PD deficiency is needed.

Keywords: Glucose-6-phosphate dehydrogenase deficiency; Prevalence; Primaquine; Single nucleotide polymorphism.

Fig. 1

Schematic diagram of sample selection for enzymatic and genetic evaluation of G6PD deficiency. In accordance with IRB-approved (AFMC-17-IRB-023) protocol, blood samples were collected from soldiers who agreed to participate in the study. All 1632 blood samples were collected in two cities, Yangju (n = 853) and Paju (n = 779) and were screened using the CareStart G6PD and Hb POC test. Based on screened results, 134 samples that were below (30–80%; n = 131) and above (> 150%; n = 3) the G6PD median value underwent genetic analysis using a G6PD genotyping kit and full-length sequencing. Thirty-six normal-range G6PD samples were also tested. Paju recruitment training camp: n = 779/1007, consent rate = 77%; Yangju recruitment training camp: n = 853/1011, consent rate = 84%

Fig. 2

Comparison of two analytical methods for representing G6PD activity: Point Scientific G-6-PDH kit and CareStart G6PD Biosensor. Using two analytical methods (Pointe Scientific G-6-PDH kit and CareStart G6PD Biosensor), G6PD activity from 3 representative samples are presented. Significance was calculated using an unpaired, two-tailed t-test (mean ± SEM; n = 3 for both the Point Scientific and the POCT Analyzer tests)

Fig. 3

Distribution of G6PD activity. G6PD activity values for all 1632 participants fell within the normal range G6PD values (2.27–11.373 U/gHb), and the median value was 7.582 U/gHb

Fig. 4

Screening results from seven representative G6PD variants using the DiaPlexC G6PD genotyping Kit (Asian type). To detect 7 different G6PD variants, including Vanua Lava (383 T>C), Mahidol (487 G>A), Coimbra (592 C>T), Viangchan (871 G>A), Union (1360 C>T), Canton (1376 G>T), and Kalping (1388 G>A), 170 blood samples representing different ranges of G6PD activity level [n = 131 samples with partially low normal, 30–80% (2.27–6.05 U/g Hb) of the median; n = 3 samples with high, > 150% (> 11.373 U/g Hb) of the median; and n = 36 samples with normal, 80–150% (6.05–11.373 U/g Hb) of the median] (Among 170 samples, 82 samples were collected from Paju and 88 samples from Yangju) were screened with the one-step PCR method of the DiaPlexC kit. IC: internal control, WC: wild-type control, MC: mutant-type control

Fig. 5

Primer schematic for G6PD gene sequencing. For further SNP evaluation of G6PD (from exon 3 to exon 13), nested PCR and sequencing primers were designed

Fig. 6

Profiles of eight participants with mutations. Basic information (region, age, gender, and G6PD activity) of participants with a C1311T exon mutation or a T93C intron mutation