This study was based on a cross-sectional model. Blood samples and data from medical records were included from March 2013 to April 2016 period. Inclusion criteria were patients (> 18 years old) of any sexes and skin color with severe (hospitalized) or uncomplicated (outpatient) malaria without any associated disease. Patients were randomly admitted at the Tropical Medicine Foundation Dr. Heitor Vieira Dourado (FMT-HDV), a reference center for infectious disease in Manaus, capital of the Amazonas state, Brazil. All patients were treated at the Clinical Research Ward (PES CLIN) of the hospital. All the patients included in the study are unrelated individuals. Patients with comorbidities, hemoglobinopathies, mixed Plasmodium infections, and viral infections were excluded. Patients were classified as uncomplicated or severe malaria as described previously (28,29), according to the World Health Organization (WHO) recommendations.
This research was initiated after approval by the Research Ethics Committee (CEP) of the FMT-HVD. To ensure patient welfare, all the patients included in the study signed the Free and Clarified Commitment Term (FICF), in compliance with CNS Resolutions 196/96.
The samples were from a larger study entitled “Clinical characterization of malaria complicated by Plasmodium vivax”, approved by the National Commission for Ethics in Research (CONEP), in June 2009, opinion nº. 343/2009, protocol nº 25.000.011.792 / 2009-15. This new project was approved by the Research Ethics Committee of (FMT-HVD) (study number 343/2009) entitled “Study of DUFFY Polymorphisms in patients infected with Plasmodium vivax” with opinion no. CAAE-0004.0.112.000-11 on 12/29/2011.
Approximately 0.5 mL of peripheral blood were collected in tube with an anticoagulant such as EDTA (ethylenediaminetetraacetic acid disodium salt) at a concentration of 1.5 mg / mL for blood counts. An aliquot of blood in 1.5 mL tubes were kept for extraction of nuclear DNA. An addition 0.6 mL was collected in a tube without anticoagulants for biochemical analyses. Immediately after blood collection, hematological determinations were performed on the automated counter - ABX Pentra 80 (Horiba Diagnostics, Montpellier, FR) and biochemistry was performed on a Beckman Coulter (Inc, CA, US).
The determination of parasitaemia was based on the count of asexual parasites per 200 white blood cells. The total number of leukocytes of each patient was used for the determination of the parasite density using the following formula (30):
Parasite density/μL = Number of parasites × Total white blood cells (WBC) counts
Number of white blood cells (WBC) counted
DNA was extracted from 200 µL of whole blood according to the QIAamp DNA Mini Kit (Qiagen, Hilden, DE) manufacturer's protocol (Cat No./ID 51304). After extraction, the DNA was quantified with a NanoDrop™ 2000 Spectrophotometer (Thermo Fisher Scientific, Massachusetts, EUA), and stored at -20ºC.
The DNA amplification step was divided into two: a conventional PCR for amplification of the sequence of interest, followed by restriction enzyme digestion by PCR-RFLP, one for Duffy blood group (Duffy PCR) and one for the GATA Box variant (GATA PCR).
Genotyping for Duffy blood system groups was performed with synthetic oligonucleotides FYAB1 (5 'TCC CCC TCA ACT GAG AAC TC 3') and FAB2 (5 'AAG GCT GAG CCA TAC CAG AC 3'). Amplified products were visualized on a 1.5% agarose gel stained with ethidium bromide. After PCR amplification was confirmed, the products were processed with BanI restriction enzyme digestion and incubated for at least 4 hours at 37ºC. The enzyme digestion product was electrophoresed on a 1.5% agarose gel stained with ethidium bromide for alleles discrimination.
Duffy genotypes as well as gene phenotypes were followed according to the FY (ISBT 008) Blood Group Alleles (31).
For verification of c.-67T>C SNP in the GATA Box promoter region, FY*01/FY*02 and FY*02/FY*02 genotype samples were used. Synthetic oligonucleotides FYN1 (5 'CAA GGC TGA CCC TA 3') and FYN2 (5 'CAT GGC ACC GTT TGG TTC AG 3') were used for the GATA PCR.
The GATA PCR product was treated with a StyI restriction enzyme and incubated for at least 4 hours at 37°C. The enzyme digestion product was observed on a 2.5% agarose gel. GATA normal genotypes appeared as 108 and 81bp bands, while GATA mutated showed an additional 61 bp band.
Samples with genotypes FY*01/FY*01 and FY*01/FY*02 were used to verify the SNPs c.265C >T and c.298G>A in the FY*01W.02 coding region and for Fyx.
The PCR product was treated with the restriction enzyme MspAI to verify the SNP c.265C>T and incubated for 4 hours at 60°C. For the SNP c.298G>A, the Mwol restriction enzyme was used and incubated for 4 hours at 37°C. Both were discriminated on an 8% polyacrylamide gel. The mutated genotypes for the SNP c.265C>T had an additional band of 161 bp and c.298G>A had an additional band of 343 bp.
For characterization of the variants, Real Time PCR (qPCR) was performed with the QuantStudio™ 3 Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific®) using TaqMan® probes specific for each polymorphism. The amplification reaction was performed for a final volume of 12uL/reaction, which contained 5uL of 2x TaqMan Universal Master Mix, 0.3uL of 20x SNP Genotyping Assay, 4.8uL of sterile water and 2.0uL of DNA (~ 100ng) of the sample. The G6PD variants analyzed in this project were chosen based on globally observed frequencies and their clinical importance according to WHO classifications. For qPCR Technique, we used the (A-) VAL68MET (202G>A) (rs1050828) and (A+) ASN126ASP (376A>G) (rs1050829) probes. To confirm the mutations found, amplification of the relevant DNA segments by PCR followed by DNA sequencing (ABI 3100, Applied Biosystems, Foster City, CA) (32) were performed.
Data were entered into a database using Graphpad-Prism 5.0 software (Graphpad Software, San Diego, CA-US) and IBM SPSS Statistics, version 19 (IBM Corp., Armonk, NY, US), organized by variable type. The analysis of qualitative or categorical variables of three or more groups was performed by the non-parametric Chi-square test (x2), corrected by the Mantel-Haenszel and Yates tests. The analysis of groups with only two categorical variables were performed with the Fisher's exact test. Confidence intervals of 95% and prevalence ratios were calculated for these variables. The Hardy-Weinberg equilibrium (HWE) was tested by comparing observed genotypes to expected genotypes frequencies by chi-square test.