Ethics statement and sample preparation
This study was approved by the ethics committee of the Armed Forces Medical Command (AFMC) of the ROK (Approval No. AFMC-17-IRB-023). Written informed consent was obtained from each participant to collect a 5-ml blood sample. Using educational materials, study objectives and activities were explained to all soldiers who were admitted to recruitment training camps in Paju and Yangju, which are malaria-endemic areas where the military administers malaria chemoprophylaxis. Afterward, a written study explanation and a research agreement form were distributed to obtain participant consent.
This study was conducted at the Department of Infectious Disease Research of the Armed Forces Medical Research Institute from April 2017 to January 2018. EDTA-preserved venous blood samples were obtained from 1,632 soldier participants who resided in the endemic area (Paju recruitment training camp: n=779/1007, consent rate=77%; Yangju recruitment training camp: n=853/1011, consent rate=84; Figure 1).
Determination of G6PD deficiency
Point-of-care (POC)-based G6PD and Hb testing by venipuncture was performed with CareStart™ Biosensor 1 (Cat No: BBA-E00182; AccessBio, New Jersey, USA) according to the manufacturer’s instructions. Two blood samples, both 10 ul volume, were collected: one was transferred to an Hb strip and the other was spotted onto parafilm and then transferred to a G6PD strip. After 5 minutes, the CareStart™ Biosensor 1 kit produced estimates of Hb (g/dL) and G6PD (U/dL) concentrations. All samples were tested in duplicate. To validate the reliability of this POC-based test, we also evaluated G6PD activity using the Glucose-6-Phosphate Dehydrogenase Reagent Set (Pointe Scientific, Canton, USA) as reference test. For the reference test, we added 10 ul of blood to 1 ml of R1 reagent to a labeled cuvette, and mixed thoroughly to suspend erythrocytes; we then incubated the mixture at room temperature for 5–10 min. After adding 2 ml of R2 reagent to the incubated sample, we placed the cuvette in a 37ºC-water bath for 5 min, and then measured absorbance (A1) at 340 nm. Next, we checked absorbance (A2) at 340 nm after another 5 min incubation. Using these absorbance values, we calculated ΔA per min as [(A2-A1)/5]. By assigning measurements to the following formula [G6PD (U/g Hb) = △A per min X [(100 X 3.01) / (0.01 X 6.22 X Hb (g/dl))] X TCF, we calculated G6PD in U/g Hb. [100 = factor to convert activity 100 ml, 3.01 = total reaction volume (ml), 0.01 = sample volume (ml), 6.22 = millimolar absorptivity of NADPH at 30 nm, Hb (g/dl) = hemoglobin concentration for each specimen, TCF = temperature correction factor (1 at 37°C)]
DNA extraction and nested PCR diagnosis
Genomic DNA was extracted from 200 ul of whole blood using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany), according to manufacturer instructions. Purified DNA samples were diagnosed using nested polymerase chain reaction (PCR) and the G6PD gene was amplified using specific primers . The first PCR round was performed under the following conditions: 94°C for 1 min, followed by 38 cycles at 94°C for 12 s, 65°C for 30 s, and 68°C for 6 min, and a final extension at 68°C for 10 min. The amplification reaction was carried out in 20 ul volume reactions, including primer volumes (1 ul of forward and reverse primer, 5 pmol/ul), 10 ul of PCR master mix (Takara Bio Inc., Shiga, Japan), and 3 ul of DNA template. The amplified products from the first round were subjected to a second PCR round with primers (Figure 5). The second-round amplification reaction was carried out in 20-ul volume reactions, including primers [1 ul of forward and reverse primer, 5 pmol/ul), 10 ul of PCR master mix (Takara Bio Inc., Shiga, Japan), and 3 ul of DNA template. The second PCR round was performed under the following conditions: 94°C for 1 min, followed by 35 cycles at 94°C for 12 sec, 62°C for 25 sec, and 72°C for 3 min, and a final extension at 72°C for 5 min. Each fragment was analyzed with 2 sequencing primers (Figure 5).
Analysis of G6PD variants
We performed genotyping on 170 blood samples representing different ranges of G6PD activity level (n=131 samples with 30%–80% (2.27–6.05 U/g Hb) of the median; n=3 samples with >150% (>11.373 U/g Hb) of the median; and n=36 samples within the normal range (4.6–13.5 U/g Hb). G6PD variants were detected using the DiaPlexC G6PD Genotyping Kit (Asian type; SolGent, Daejeon, ROK), which specifically screens for the seven representative Asian variants of the G6PD gene via one-step PCR. The variants each produce PCR products of different sizes: 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). PCR mixtures were prepared with 2 ul of genomic DNA (internal control, wild-type control, or mutation control) and 23 ul of master mix (12.5 µl polymerase premix, 2 µl primer premix, and 8.5 µl distilled water). The PCR cycling conditions were as follows: initial denaturation at 95°C for 15 min, 30 amplification cycles (denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec, elongation at 72°C for 40 sec), and a final extension at 72°C for 5 min. PCR products were resolved by electrophoresis on 1% agarose gel, stained with SYBR Safe DNA Gel Stain (Thermo Fisher Scientific, Waltham, MA, USA), and visualized using FluorChem FC3 (ProteinSimple, Santa Clara, CA, USA).
G6PD amplification and G6PD variant sequencing
Of the 170 blood samples genotyped for G6PD,we performed further sequencing on the 134 samples that represented the lower and upper bounds of G6PD activity (n=131 samples with 30%–80% median activity and n=3 samples with >150% median activity). Primer sets used for G6PD amplification and sequencing are listed in Figure 5. Each fragment was analyzed with 2 sequencing primers (Figure 5). Confirmed sequences were analyzed with a wild type sequence using BioEdit v7.0.5 software.
Data are presented as means and standard errors. Statistical analyses were performed using GraphPad Prism version 7 (GraphPad Software, La Jolla, CA, USA). The significance of pairwise comparisons was determined using the Student’s t-test, and p <0.05 was considered significant.