The samples used in this study are part of a broad cross-sectional study that sought to describe the epidemiological pattern of malaria through spatial analysis of factors linked to the disease. The ethical and methodological aspects of this study were approved by the Ethics Committee of Research Involving Human Subjects of the Institute René Rachou/Fiocruz (CAAE: 70755617.8.0000.5091). Prior to their participation in the study, subjects were informed regarding the objectives and procedures of the study, and their voluntary participation was requested and confirmed through written formal consent. The study was performed in accordance with all relative guidelines and regulations.
Study design and data collection. This was an observational, cross-sectional study performed between March 2016 and September 2018. Blood samples were collected from individuals who sought public healthcare services in the cities of Boa Vista, Rorainópolis, and Pacaraima, in the state of Roraima, Brazil. The subjects were symptomatic and had sought treatment from the local public health services. The presence of Plasmodium spp. infection was confirmed by microscopy, based on Giemsa-stained thick blood smears, which were evaluated by well-trained microscopists according to the guidelines for malaria diagnosis of the Brazilian Ministry of Health. Following the confirmation of infection, patients positive for P. falciparum were invited to participate in the research (Supplementary data 1). The parasite density was determined as the number of asexual parasites observed per 200 leukocytes on a thick smear, with an estimated leukocyte count of 6,000 per µL. Subsequently, frozen blood samples were subjected to the SD-Bioline™ Malaria Ag Pf/Pf/Pv (Abbott, Inc.; Korea) test, which employs the proteins HRP2 and LDH as targets for the detection of P. falciparum. All samples were tested for malaria infection using a PET-PCR assay, as previously described by Lucchi et al. (2013)21. Firstly, infection by the Plasmodium genus was identified by the 18S ribosomal target, and then infection by P. falciparum was identified by amplification of the Pfr364 target from the primers described in Demas et al. (2011)22.
Nested PCR (nPCR) assays for amplification of the pfhrp2/3 and their flanking genes. DNA was extracted from 200 µL whole blood samples using the QIAamp DNA Mini Kit (QIAGEN, Minneapolis, MN, USA) according to the manufacturer's instructions. The DNA was eluted in 50 µL of elution buffer to perform molecular assays. To evaluate the presence of pfhrp2 (PF3D7_0831800) and pfhrp3 (PF3D7_1372200), nPCR was performed using the primers and conditions described in Abdallah et al. (2015)13. The flanking genes upstream and downstream to pfhrp2 (MAL7P1.230 and MAL7P1.228) and pfhrp3 (MAL13P1.475 and MAL13P1.485) were amplified for all negative samples, also using the conditions previously described13.
pfhrp2 multiplex quantitative PCR (qPCR). A second protocol for pfhrp2 amplification was performed using multiplex qPCR with the primers and probes described by Abdallah et al. (2015)13 and Schindler et al. (2019)23, respectively. The single-copy gene pfrnr2e2 (PF3D7_1015800) was employed as an internal control for the reaction. The reactions were prepared with 1X GoTaq Probe master mix (Promega), 300 nM of each primer and 150 nM of each probe (VIC-labeled probe for pfhrp2 and FAM-labeled for pfrnr2e2), 2 µL sample DNA (~ 10 ng), and RNAse-free water to a final volume of 10 µL. The thermal cycling process was conducted under the following conditions: 50°C for 2 min, 95°C for 10 min, followed by 45 cycles of 95°C for 15 s, 56°C for 30 s, and 60°C for 30 s.
pfhrp2 sequencing and analysis. A conventional PCR was performed using the conditions previously described for the sequencing of the histidine and alanine repetitive regions of pfhrp224. The PCR products were purified using the QIAquick PCR purification kit (QIAGEN, Chatsworth, CA, USA) and subsequently analyzed in the ABI 3730xL DNA Analyzer system (Thermo Fisher Scientific, Waltham, MA, USA). The nucleotide sequences were aligned and translated into amino acids using the Mega X software25 and the amino acid repeat types were identified using the numerical code described by Baker et al. (2005; 2010)24,26.
msp2 genotyping and genetic variation. The central region of msp2 (IC and FC27) was genotyped by nPCR using primers and reaction conditions previously described in Snounou et al. (1999)27 and Liljander et al. (2009)28 to assess infection complexity (presence of multiple genetically distinct parasites). The reactions were subsequently analyzed by capillary electrophoresis using the ABI 3730xL DNA Analyzer system, with the products analyzed using GeneMapper™ software version 4.1 (Thermo Fisher Scientific, Waltham, MA, USA). For the analysis, the minimum peak height was set at 200 arbitrary fluorescence units (rFU), and a threshold of one-third the height of the predominant peak (the peak with the greatest fluorescence) was established to exclude any potential artifacts.
HRP2 immunological assays. The presence of HRP2 antigen in the blood was assessed by enzyme-linked immunosorbent assay (ELISA) using a commercially available kit (Quantimal Celisa PfHRP2 Assay kit, Cellabs, Cat number: KM8). The positivity cut-off value was determined by subtracting the average optical density (OD) at (450 nM) analyzed on Varioskan LUX Multimode Microplate Reader (Thermo Fisher Scientific, Waltham, MA, USA) of the negative controls from the blank (containing only the reagents and RPMI) and adding one unit. The reactivity index (RI) value was calculated from the ratio between the mean ODs of the samples in duplicates and the cut-off value. To correct variations between experiments, a normalization factor was calculated as the ratio between the OD of the positive control in each experiment and the mean of the readings. Finally, the RI was divided by the corrected value for the day of the experiment, obtaining the normalized index.