Differential expression of var subgroups and PfSir2a genes in afebrile Plasmodium falciparum malaria: a matched case–control study

This study took place in the district of Manhiça (southern Mozambique) where community-based age-stratified cross-sectional surveys were conducted in May of 2012 to 2015. After giving written informed consent, participants were finger-pricked and their blood was used to detect P. falciparum infection by rapid diagnostic test (RDT), microscopy, quantitate real-time PCR (qPCR) and storage in RNAprotect. Plasmodium falciparum-infected individuals were defined as afebrile if their axillary temperature at the time of visit was < 37.5 °C without reporting having had fever in the preceding 24 h, and febrile if the axillary temperature was ≥ 37.5 °C at the time of visit or reported fever during the preceding 24 h. Febrile and afebrile cases were individually matched based on year of sample collection, age (0 to ≤ 5; > 5 to ≤ 15; > 15 to ≤ 25 and > 25 years) and parasite density (≤ 200, > 200 to ≤ 1000, and > 1000 parasites/µL). The Hospital Clínic (Barcelona, Spain) ethics review committee and National Mozambican Ethical Review Committee (Mozambique) approved the study.

Approximately 10 μL of finger-pricked blood were used to perform PfHRP2-based rapid diagnostic tests (SD BIOLINE Malaria Antigen P.f—05FK50) by trained technicians following the manufacturer’s instructions. Thin and thick blood smears were stained with Giemsa as described previously [13]. Two experienced microscopists independently read all slides, and a third reading was performed if there were discrepancies between the results of the first two. Plasmodium falciparum infections were also identified through qPCR from 25 μL of dried blood spots collected on filter papers. DNA was extracted using the QIAamp DNA Mini kit (Qiagen, Hilden, Germany), as per the manufacturer’s instructions. Parasite DNA amplification targeting the 18S rRNA gene was performed using the ABI PRISM 7500 HT Real-Time System (Applied Biosystems, Foster City, USA), following a method described elsewhere [14, 15]. Then, a standard curve was prepared on the foundation of an in vitro culture of the 3D7 strain, which contained known numbers of ring-infected erythrocytes. The standard curve was performed for each test in triplicate with five serial dilutions. Using the 18S rRNA gene as an amplification target, parasitaemia in the clinical samples were quantified and then subjected to extrapolation against the standard curve [16].

Extraction of RNA from P. falciparum infected blood (50 µL) preserved in 250 µL RNAprotect (Qiagen, Hilden, Germany) was performed using 700 µL of Trizol (Ambion, Life Technology, California, USA) following manual extraction with phenol/chloroform phase separation to elute total RNA. Extracted total RNA was subjected to DNase treatment using DNase Max kit (Qiagen, Hilden, Germany) at 37 °C for 30 min followed by reverse transcription of 400–800 ng of total RNA using PrimeScript™ RT Master Mix reagents (Takara, Shiga, Japan). To confirm the removal of gDNA or the synthesis of cDNA, qPCR was performed using primers targeting P. falciparum ubiquitin-conjugating enzyme (PF08_0085) on reverse transcription positive and negative controls (RNA samples without reverse transcriptase enzyme).

Transcript levels of var genes such as varA-exon2, varA-DBLα1 (varA-notDC3), varB group (varB-UTR region) and varC group (varC-UTR region) were assessed using degenerate primers and quantitative reverse transcriptase PCRs (RT-qPCRs) [7, 10, 17]. Domain cassette transcript levels were also determined using a set of primers targeting semi-conserved domains belonging to DC8 (CIDRα1.1), DC11 (CIDRβ2 + DBLγ7) and DC13 (CIDRα1.4) [7, 8]. The assessment of transcript level of genes involved in parasite multiplication (PfSir2a) and gametocyte mature stage (Pfs25) was also performed using previously described RT-qPCR primers [4, 18, 19]. PF08_0085 gene was used as a housekeeping gene [20]. In brief, 96-well plates containing individual 20 μL reactions were amplified in triplicates using a 7500 HT Real-Time System (Applied Biosystem, Foster City, USA). Each reaction mixture had 10 μL of 2X Power SYBR^® Green PCR Master Mix (Thermo Fisher Scientific, Warrington, WA, UK), 1 or 0.33 μM of each forward and reverse primers and 5 μL of template cDNA. The reaction volume was prepared with PCR-grade water. Amplifications were performed with a holding for 3 min at 50 °C, initial denaturation for 10 min at 95 °C, followed by 40 cycles of 95 °C for 15 s and 60 °C or 55 °C for 1 min. Samples with a Ct > 40 for any gene target were considered as not expressed. The specificity of primer pairs against human gDNA was also determined. The 7500 System SDS software v1.4 was used to analyse the collected data. A standard curve was then used to calculate the PCR efficiency of each primer from 7 log dilutions of the P. falciparum 3D7 gDNA, by using the formula (E = 10^−1/m), where “m” was the slope. The formula C/E^ΔCt was used to convert Ct values to copy numbers [9]. In the formula, “C” was the number of copies of the gene in the P. falciparum 3D7 genome, “E” was the efficiency of the PCR [7, 9] and ΔCt was the difference in Ct values between a sample and P. falciparum 3D7 reference gDNA loaded in each plate [21]. The relative transcript unit (RTU) of target genes was calculated by dividing the target gene transcript levels by PF08_0085 (housekeeping gene) transcript levels. Non-template controls were tested in every plate [8].

Wilcoxon matched-pairs signed rank test and McNemar’s Chi squared test were used to compare continuous and categorical variables, respectively. The association between being febrile or afebrile and log-transformed RTUs was tested by conditional logistic regression models adjusted by area of residence in order to account for differences in transmission intensities between areas. All statistical analyses were performed using R 3.3.2, and GraphPad Prism 5.0 (GraphPad Software) was used to generate graphs.