Source of samples
Cross-sectional surveys, including blood collection, were conducted in 2012 and 2013 in 200 randomly selected households at each of three districts located in different epidemiological settings: Kihihi, Kanungu District, a rural area with relatively low transmission intensity in southwestern Uganda; Walukuba, Jinja District, a peri-urban area with moderate transmission intensity in central Uganda; and Nagongera, Tororo District, a rural area with high transmission intensity in eastern Uganda. Of note, since the time of this study transmission has decreased considerably in Walukuba, likely due its peri-urban characteristics, and in Nagongera, associated with regular rounds of indoor residual spraying of insecticide since 2014 . Participants for this study were children ages 6 months to15 years who were full-time residents of the recruited households, selected as previously described . This study was approved by the Makerere University Research and Ethics Committee, the Uganda National Council of Science and Technology, and the University of California, San Francisco Committee on Human Research.
Sample collection and malaria diagnosis
Blood was obtained by finger prick for thick blood smears, malaria RDTs and drying on filter paper for molecular studies. Thick blood smears were stained with 2% Giemsa for 30 minutes and read by laboratory technologists at the field sites. Parasite densities were calculated by counting the number of asexual parasites per 200 leukocytes (or per 500 leukocytes, if the count was <10 asexual parasites/200 leukocytes), assuming a leukocyte count of 8,000/μL. For quality control, all slides were read by a second microscopist, and discrepancies resolved by a third microscopist at the field sites. In addition, all positive blood smears with parasite densities ≤20,000/µL based on the field readings were re-read by an expert microscopist in Kampala; confirmation of parasitaemia was required for inclusion in the final analyses. RDTs (SD BIOLINE Malaria Ag Pf, a PfHRP2-based test from Standard Diagnostics Inc; Suwon City, Republic of Korea) were performed immediately after blood collection following manufacturer’s instructions. Samples for study were all those that were positive for malaria parasites by microscopy but negative by RDT.
Plasmodium species identification
DNA was extracted using Chelex100, as previously described . Species identification was performed by nested species-specific PCR with primers specific for the 18S small subunit ribosomal DNA gene of all human plasmodial species, as previously described . PCR reactions were performed in 25 µl containing 1x standard Taq buffer (New England Biolabs), 200 µM deoxynucleoside triphosphates, 200 µM of each primer, 2 µl of template DNA (from Chelex extraction or the prior cycle of PCR), and 1 unit of Taq polymerase (New England Biolabs). All reactions included negative controls (water) and positive controls, obtained from the Biodefense and Emerging Infections Research Resources Repository (BEI U.S).
PCR products were resolved by electrophoresis on 2% agarose gels stained with ethidium bromide and visualized by UV illumination. Sizes of amplicons were identified based on comparison with standard fragments of known size.
Detection of deletions in pfhrp2 and pfhrp3 genes
To identify deletions, we PCR-amplified fragments spanning exon 1, the intron, and exon 2 of the pfhrp2 and pfhrp3 genes, as previously described . P. falciparum Dd2 strain DNA was a negative control for pfhrp2 and a positive control for pfhrp3. P. falciparum HB3 strain DNA was a negative control for pfhrp3 and a positive control for pfhrp2. All PCR reactions were performed in duplicate. PCR products were separated and visualized on 2% agarose gels. In the event of discordant replicates, reactions were repeated, and the result recorded was that seen in multiple assays. Deletions were identified by the absence of amplification of pfhrp2/3 in the setting of successful amplification of ribosomal DNA in the sample and amplification of pfhrp2/3 in positive control DNA.
Multiplicity of infection (MOI)
MOI, the number of different parasite genotypes co-existing within a host, is a metric of transmission dynamics [23, 24]. To determine MOI, the 3D7 and FC27 alleles of the merozoite surface protein-2 (msp2) gene, which each have extensive size polymorphism, were amplified as previously described . Amplicons were identified on 2% agarose gels, the size of products was compared to standards on densitometric digitized gel images analysed by GelCompar II software (Applied Maths NV Belgium), and the number of differently sized amplicons in each sample was determined.
Bead-based immunoassay for detection of PfHRP2 protein
As the absence of gene amplification does not definitively prove the presence of a gene deletion, we also assessed expression of PfHRP2 in study samples. Unfortunately, adequate material was available for this analysis for only 39 of the 56 RDT-negative P. falciparum samples. For these assays recombinant PfHRP2 (Microcoat Biotechnologie GmbH, Bernried am Starnberger See, Germany) was used as a positive control and blood from persons not infected with malaria as a negative control. PfHRP2 levels was quantified using a bead-based immunoassay with a MAGPIX instrument (Luminex Corp., Austin, TX), as previously described [26-28]. Briefly, the bead-based HRP2 immunoassay, which relies on antigen capture, is capable of detecting PfHRP2 at sub-picogram levels, allowing fast processing and screening of large numbers of samples. As in prior studies, the cut-off for a positive PfHRP2 antigen result was the mean plus three standard deviations based on a panel of 92 antigen negative blood samples .