Grabias et al. Malar J (2019) 18:116 https://doi.org/10.1186/s12936-019-2743-9 Malaria Journal RESEARCH Open Access Sensitive real-time PCR detection of Plasmodium falciparum parasites in whole blood by erythrocyte membrane protein 1 gene amplification Bryan Grabias1, Edward Essuman1, Isabella A. Quakyi2 and Sanjai Kumar1,3* Abstract Background: Malaria remains a global public health problem responsible for 445,000 deaths in 2016. While micros- copy remains the mainstay of malaria diagnosis, highly sensitive molecular methods for detection of low-grade sub-microscopic infections are needed for surveillance studies and identifying asymptomatic reservoirs of malaria transmission. Methods: The Plasmodium falciparum genome sequence was analysed to identify high copy number genes that improve P. falciparum parasite detection in blood by RT-PCR. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1)-specific primers were evaluated for P. falciparum detection in hospital-based microscopically positive dried blood spots and field-acquired whole blood from asymptomatic individuals from Ghana. Results: PfEMP1 outperformed the Pf18S sequence for amplification-based P. falciparum detection. PfEMP1 primers exhibited sevenfold higher sensitivity compared to Pf18S primers for parasite genomic DNA. Probit analysis estab- lished a 95% detection threshold of 9.3 parasites/mL for PfEMP1 compared to 98.2 parasites/mL for Pf18S primers. The PfEMP1 primers also demonstrated superior clinical sensitivity, identifying 100% (20/20) of dried blood spot samples and 70% (69/98) of asymptomatic individuals as positive versus 55% (11/20) and 54% (53/98), respectively, for Pf18S amplification. Conclusions: These results establish PfEMP1 as a novel amplification target for highly sensitive detection of both acute infections from filter paper samples and submicroscopic asymptomatic low-grade infections. Keywords: Plasmodium falciparum erythrocyte membrane protein 1, Parasitemia, Real-time PCR, Genomic DNA Background falciparum, Plasmodium vivax, Plasmodium malar- In 2016, malaria caused approximately 445,000 deaths, iae, Plasmodium ovale, and Plasmodium knowlesi), P. primarily among young children in sub-Saharan Africa falciparum is the most common and the most lethal. [1]. The apicomplexan Plasmodium parasites responsi- Malaria also presents a public health challenge in the ble for malaria-related illness are transmitted through USA. While there have been no cases of autochtho- the bite of infected mosquitoes. Of the five recog- nous transmission of malaria in the USA since 1957 nized species that infect human beings (Plasmodium [2], increasing globalization and travel to areas where malaria is endemic still results in imported malaria cases in the Plasmodium. In 2015 alone, there were *Correspondence: Sanjai.Kumar@fda.hhs.gov 1 Laboratory of Emerging Pathogens, Division of Emerging 1186 of such travel-related malaria cases reported to and Transfusion Transmitted Diseases, Center for Biologics Evaluation the Center for Disease Control (CDC) [3] and the num- and Research, Food and Drug Administration, Silver Spring, MD 20993, ber of malaria hospitalizations from 2000 to 2014 far USA Full list of author information is available at the end of the article exceeded that of other common travel-related illnesses © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Grabias et al. Malar J (2019) 18:116 Page 2 of 9 [4]. Additionally, malaria is also a major risk to the children and serve as reservoirs of transmission in both blood supply in both endemic and non-endemic coun- low and high transmission endemic areas. tries [5–7]. The gold standard of malaria parasite detection is Methods direct visual confirmation of intraerythrocytic parasites Primers in a blood film. However, this method is time and labour- Primer oligonucleotides were purchased from Eurofin intensive and can only reach typical detection limits of Genomics (Louisville, Kentucky). The primer sequences 5000–150,000 parasites/mL of blood [8]. Several reports employed in this study are: 18S F: CTTA AC CTG CTA utilizing PCR-based methods to detect P. falciparum in ATTA GCG , 18S R: ATT CCT CGT TCA AGA TTA ATA blood have achieved far greater sensitivities than stand- ATT; EMP1 F: AAG AAAA CG AAT TAT TTG GGACA, ard blood smears [9, 10]. Given the lack of knowledge on EMP1 R: AGA AAC ATCA GTA TT CAAC GTT. SYBR the minimum number of parasites that could be present green RT-PCR mastermix was purchased from Bio-Rad in asymptomatic individuals and limitations in volume (Hercules, CA, USA). of blood used in detection assays, molecular methods of improved sensitivity are still needed to identify all Plas- Human blood samples modium infections in endemic areas. Plasmodium falciparum-infected blood smear posi- Typically, most nucleic acid-based assays rely upon tive samples from patients in Ghana used for this study the detection of pathogen specific 18S ribosomal RNA were collected on Whatman® FTA Elute cards (GE Lifes- (rRNA) sequences due to their universal presence, strong ciences, MA, USA) at Kpone Katamanso District Hospital inter- and intra-species sequence conservation, and the in the Greater Accra Region of Ghana, a malaria endemic relatively high abundance. While the given quantity of country which recorded about 10 million malaria cases 18S rRNA transcripts may be higher than the number of in the year 2015. The study site was developed for mul- gene copies present in an organism’s genome, RNA is typ- tidisciplinary malaria studies by the University of Ghana ically less stable and thus requires more stringent sample School of Public Health [13]. Kpone Katamanso district handling than genomic DNA (gDNA). Depending on the is a peri-urban district located in the coastal-savannah strain, P. falciparum parasites may possess only 5–8 gene zone that occupies an area of 215.4 km2 and has an esti- copies of the 18S ribosomal subunit [11]; therefore, the mated population of 50,225. While malaria transmission performance of assays that amplify and detect genomic intensity of the region varies seasonally, infections are sequences can be greatly enhanced with the identification acquired year-round. Study participants were 1 month to of novel biomarkers that are more abundant than Pf18S 16 years old children and asymptomatic adults who were sequences. In this report, PCR-based nucleic acid assays screened in the Kpone Katamanso District Hospital. utilizing primers specific to the P. falciparum 18S riboso- mal gene sequence and highly conserved regions of the Preparation of Plasmodium falciparum gDNA abundant (approximately 60 variants [12]) P. falciparum Plasmodium falciparum 3D7 parasites were cultured var. gene EMP1 are developed and characterized. Data in  vitro according to previously published protocols from analytical studies demonstrate that P. falciparum [14] utilizing O + human red blood cells purchased from detection using the PfEMP1 primer set consistently out- Interstate Blood Bank (Memphis, TN). Parasitaemia performs the traditional 18S primer set, detecting as lit- was determined by routine Giemsa staining of blood tle as 0.01 pg of Plasmodium gDNA and 9.3 parasites/mL smears. Final parasitaemia was measured and the num- compared to 0.07 pg and 98.2 parasites/mL for the Pf18S ber of parasitized red blood cells (pRBCs) per µL was primer set. The clinical performance of the PfEMP1 and obtained. Isolated ring stage P. falciparum parasites from Pf18S gene targets was determined in hospital-based culture were spiked into whole blood and serially diluted. blood film positive clinical malaria samples collected For spiked sample analysis, gDNA was extracted from on filter paper and in field acquired blood samples of 1  mL of whole blood at each parasite dilution. Alter- asymptomatic individuals from Ghana. PfEMP1 ampli- nately, approximately 100–200  µL of blood was utilized fication consistently outperforms Pf18S-based RT-PCR from capillary collection tubes collected from asympto- detection, identifying 100% (20/20) of dried blood spot matic blood smear negative adults from Ghana. For both samples and 70% (69/98) of asymptomatic individuals as spiked and Ghanaian blood samples, lysis of RBCs was positive versus 55% (11/20) and 54% (53/98), respectively, induced by adding saponin (Sigma, MO, USA) to a final for 18S amplification. In conclusion, a PfEMP1-based RT- concentration of 0.15%. Remaining cellular material was PCR test has applications in detecting acute filter paper sedimented by centrifugation at 16,000×g for 10 min and collected and low-grade asymptomatic infections in field washed three times with 1× PBS. gDNA from the pel- who have a potential to convert into severe malaria in let was obtained using the QIAmp DNA Blood Mini Kit Grabias et al. Malar J (2019) 18:116 Page 3 of 9 Fig. 1 Abundance of PfEMP1-specific amplicon in Plasmodium ▸ falciparum genome. Primers used in the RT-PCR assay amplify a region of 260 nucleotides located in the cytosolic acidic terminal segment (ATS) region of PfEMP1. Matching conserved sequences in the P. falciparum (3D7) genome were identified using an NCBI BLAST search. Chromosomes with homologous sequences are displayed in the figure. The conserved amplification region was found in 18 different loci reflecting significantly higher abundance than the 5 copies of the 18S gene in the P. falciparum genome. Black bars represent forward and reverse primer binding positions according to the manufacturer’s instructions and eluted in 60 µL ultrapure water. gDNA extracts from dried blood samples spotted onto W hatman® FTA Elute cards (were prepared according to the manufacturer’s instructions. Real‑time polymerase chain reaction Real-time amplification reactions were performed on a QuantStudio 3 RT-PCR instrument (Applied Biosystems, Foster City, CA, USA) using a SYBR Green mastermix from Biorad (CA, USA). The thermocycler protocol was: 95 °C for 5 min; a 40 repeat cycle of 95 °C for 15 s, 57 °C for 30 s, and data collection and elongation at 72  °C for 1 m. Cut-off threshold cycle values were determined for each RT-PCR plate run based upon the mean threshold of included negative (uninfected) control well results minus 1 standard deviation. All results are displayed as signal-to-cut-off ratios using a simple ΔCt method (Fold = 2(Mean Ct,cutoff − Mean Ct,sample). Probit analysis Approximately 40  mL of whole blood was spiked with differing concentrations of P. falciparum ring stage par- asites (ranging from 1 to 100  parasites/mL). Genomic DNA was isolated from 30 unique 1 mL aliquots drawn from these larger volumes. Extracted genomic DNA from each aliquot was tested by real-time PCR amplification utilizing both the Pf18S and PfEMP1 primer sets. The MedCalc statistical software package (MedCalc Software, Belgium) was used to perform probit regression on the RT-PCR data and determine the 95% reactivity threshold and corresponding confidence intervals. Results Sequence analysis of PfEMP1 gene amplicon in P. falciparum genome To identify a novel and abundant target sequence for RT- PCR-based detection of P. falciparum genomic DNA that would potentially outperform standard 18S amplifica- tion methods, the sequences of members of the PfEMP1 multi-gene family, which consists of approximately 60 Grabias et al. Malar J (2019) 18:116 Page 4 of 9 unique variants present per genome, were analysed. 64.5–131.9 parasites/mL) for the Pf18S primers (Fig. 3a) Specifically, primers were designed to amplify strongly and 9.3  parasites/mL (95% CI 6.36–17.8  parasites/mL) conserved gene regions that maximize the number for the Pf EMP1 primer set (Fig. 3b). of amplification targets. Primer sequences exhibited 91.3–100% identity to a maximum of 18 possible sites of Detection of P. falciparum in dried blood spots and serum amplification (Fig.  1) within the cytosolic acidic termi- samples from Ghana nal segment of PfEMP1. As this sequence resides on the To evaluate the clinical sensitivity of each primer set, intracellular side of the erythrocyte, there is substantially Ghanaian blood samples that were microscopically pos- less selective pressure for antigenic variation. A maxi- itive were assessed for P. falciparum reactivity by RT- mum of two base-pair mismatches far from the 3′ end of PCR. Dried blood spots (DBS) employ a small drop of each primer was allowed. Thus, when compared to the blood on filter paper and are highly utilized in endemic 5–8 available genomic copies of Pf18S ribosomal subunit areas due to their relative ease of use and stability at present, this PfEMP1-based RT-PCR assay would poten- room temperature [15]. Briefly, patient specimens were tially improve detection capability by offering a more collected on Whatman FTA-elute filter paper cards in abundant amplification target. volumes of approximately 10–20  μL and gDNA was isolated according to the manufacturer’s instructions. Limit of detection of P. falciparum gDNA Importantly, the sensitivity of any pathogen detection To compare the sensitivity of Pf18S and PfEMP1 as assay utilizing dried blood spots is generally lower than detection targets, both primers were evaluated in RT- whole blood due to the relatively small volume being PCR against purified parasite gDNA that was seri- sampled [16, 17]. To empirically estimate the difference ally diluted in ultrapure water. The Pf18S primer set in analytical sensitivity of the RT-PCR assay between detected as few as 0.07 pg of Pf gDNA (Fig. 2a) whereas gDNA extracted from dried blood spots versus whole the PfEMP1 primer set detected as little as 0.01 pg of Pf blood, gDNA was extracted from both 20  μL of para- gDNA (Fig. 2b) showing a sevenfold improved sensitivity site-spiked blood and 20  µL of unspiked, uninfected in detection of P. falciparum blood stage parasite gDNA. blood utilized as a negative control that were spotted onto FTA elute cards (Fig.  4a). The Pf18S primer set Determination of primer limit of detection (LOD) by probit only marginally detected the highest parasite concen- analysis tration tested (41,360 parasites/mL) while the PfEMP1 Approximately 40  mL of whole blood was spiked with primer set successfully reacted to as few as 2640 para- either 100, 10, 5, or 1 parasite/mL of P. falciparum blood- sites/mL. The superior sensitivity of PfEMP1 amplifica- stage parasite. gDNA was extracted from 30 unique tion was also confirmed by analysis of dried blood spots 1  mL aliquots taken from each stock 40  mL prepara- collected from individuals in a malaria endemic area tion and evaluated in RT-PCR using both Pf18S and of Ghana. The Pf18S primer set identified only 11/20 PfEMP1 primer sets. The MedCalc statistical software (55%) samples as positive, with 9/20 samples showing package was used to perform probit regression analysis no amplification at all (Fig. 4b). In contrast, the PfEMP1 to calculate the parasite concentration at the 95% detec- primer set successfully identified 20/20 (100%) blood tion threshold. The probit analysis demonstrates that smear positive samples as positive (Fig. 4b). the 95% detection limit is 98.2  parasites/mL (95% CI Fig. 2 Analytical sensitivity of Pf18S and PfEMP1 primers for serially diluted parasite genomic DNA (gDNA). A linear regression identifying the limit of detection for each primer set at the cutoff threshold of the assay. a The 18S primers detected as little as 0.07 pg. b PfEMP1 primers could detect 0.01 pg of parasite gDNA Grabias et al. Malar J (2019) 18:116 Page 5 of 9 Fig. 3 Determination of analytical sensitivity for Plasmodium falciparum-infected red blood cells by probit analysis. gDNA extracted from 30 independent 1 mL aliquots taken from the stock parasite dilutions and analysed by RT-PCR using both primer sets. Probit regression was performed using the MedCalc statistical software to identify the 95% reactive threshold. a The 18S primer set demonstrated a 95% reactivity of 98.2 parasites/ mL (95% CI 64.5–131.9). b PfEMP1 primer set demonstrated a 95% threshold of 9.3 parasites/mL (95% CI 6.36–17.8) Performance of both Pf18S and PfEMP1 primer sets was evaluated in individuals living in an endemic area for P. falciparum detection in serum was also evalu- of Ghana. Specifically, these blood samples reflect ated. Patient serum, as opposed to whole blood sam- asymptomatic adults who were diagnosed as negative ples which are typically assessed in parasite nucleic for P. falciparum infection by blood film microscopy. acid tests, are more commonly stored long-term [18]. Pf18S primers identified 53/98 (54.1%) of samples as Thus, adequate detection of P. falciparum from stored positive for P. falciparum gDNA whereas PfEMP1 serum samples could allow for more extensive retro- primers successfully detected 69/98 (70.4%) (Fig.  5, spective analyses on existing patient-acquired material. Table 1). These differences in sensitivity are statistically These results demonstrated that, while both primer significant as measured via McNemar’s test (P < 0.01). sets showed no amplification in the healthy adult sam- ples, only the EMP1 primer set successfully identified Discussion 5/7 (71.4%) sera from infected individuals as positive Blood-film microscopy is considered the gold-standard (Fig. 4c, d). for malaria diagnosis. While generally adequate for diag- nosis of acute malaria, microscopy lacks the sensitivity to detect low-grade asymptomatic infections as revealed by Detection of P. falciparum in asymptomatic blood smear the application of molecular tests that detect the parasite negative whole blood samples nucleic acid. Since submicroscopic malaria infections are The clinical performance of Pf18S and PfEMP1 primers important sources of further transmission, identification in detecting submicroscopic asymptomatic infections of this population group is critical for accurate estimation Grabias et al. Malar J (2019) 18:116 Page 6 of 9 Fig. 4 Detection of Plasmodium falciparum in dried blood spots from blood smear positive individuals in Ghana. a The 18S primer set detected only the highest parasite dilution (41,360 parasites/mL) while the PfEMP1 primer set detected the lowest parasite concentration tested corresponding to 2640 parasites/mL. b The 18S primer set identified 55% (11/20) smear positive samples as positive whereas the EMP1 primer set identified 100% (20/20) of smear positive samples as positive. gDNA was extracted from sera demonstrating high reactivity ( OD450 ≥ 1) against P. falciparum circumsporozoite protein measured in ELISA and amplified using both primer sets, c Pf18S primers failed to amplify from any of the sera samples. d PfEMP1 primer set identified 5/7 (71.4%) sera samples as positive for P. falciparum nucleic acid of malaria incidence rate in an endemic area. In semi- detection for the standard Pf18S amplification. Accord- immune individuals who live with chronic exposure to ingly, amplification of PfEMP1 identified 16% more malaria parasites, asymptomatic low-grade parasitaemia whole blood samples from asymptomatic adults who can persist for a year or more [19]. Failure to comprehen- were blood film negative as positive than standard Pf18S sively identify these individuals infected with low burden amplification establishing the superior efficacy of this parasitaemias that may not be detected by currently used molecule in detecting low grade parasite burdens missed microscopy, PCR assays, or Rapid Diagnostic Tests, dra- by other methods. matically reduces the effectiveness of Mass Screen and Results from analytical and clinical characterization Treat (MSAT) strategies targeting the infectious reservoir studies suggest that the PfEMP1-based RT-PCR assay of malaria [20, 21]. could provide a more comprehensive estimation of the In this work, the performance of an RT-PCR-based infectious reservoir of malaria and simultaneously allow screening assay for P. falciparum employing primers that better targeting of treatment and transmission-blocking amplify members of the PfEMP1 var gene family was interventions. In particular, this assay would reduce false- characterized. The data establish that PfEMP1 primers negative results associated with malaria screening assays exhibit a superior analytical sensitivity of 9.3  parasites/ of inadequate sensitivity in areas of less intense trans- mL in whole blood versus the 98.2 parasites/mL limit of mission where chronic parasite burdens are generally Grabias et al. Malar J (2019) 18:116 Page 7 of 9 collected as DBS is less sensitive than screening larger volumes of whole blood [16, 17]. Additionally, a recent study has shown that overall PCR reactivity of even high parasite burden DBS declines over time when not stored at − 20  °C [23]. These fundamental sample constraints necessitate a more sensitive diagnostic assay to ensure no loss of parasite detection in valuable field samples acquired in epidemiological or vaccine-related studies. The results demonstrate that PfEMP1-based RT-PCR amplification improved P. falciparum parasite detection of microscopically positive Ghanaian blood samples as DBS when compared to standard Pf18S amplification methods. Specifically, PfEMP1-mediated gene amplifi- cation demonstrated a sensitivity of 2460  p/mL versus Fig. 5 Detection of Plasmodium falciparum in asymptomatic blood smear negative individuals from Ghana. gDNA was extracted from a limit of detection of at least 41,360  parasites/mL for whole blood samples collected from children and adults who 18S-based detection. Additionally, PfEMP1 primers suc- exhibited no symptoms of malaria and had negative blood smears. cessfully amplified 100% of blood smear positive indi- Pf18S primers identified 53/98 (54.1%) of samples as positive for viduals from dried blood spot samples whereas Pf18S P. falciparum gDNA whereas PfEMP1 primers identified 69/98 amplification only detected 11/20 (55%) DBS samples as (70.4%) blood samples as positive. These differences are statistically significant when evaluated by McNemar’s test and demonstrate positive. Importantly, the failure of Pf18S amplification PfEMP1 primers possess superior sensitivity for the detection of low to consistently match results from gold standard blood burden parasitaemia and more comprehensive identification of smear microscopy of clinical samples collected as DBS infectious reservoir indicates a limitation in this blood collection method and inherent challenges in conducting large field studies in endemic areas. Cumulatively, these data demonstrate Table 1 Detection of  Plasmodium falciparum gDNA that RT-PCR amplification of PfEMP1 provides a supe- in asymptomatic individuals from Ghana rior method of parasite detection among DBS spots col- EMP1 Total lected in field sites and ensures that any potential losses of sensitivity are minimized when this widely used sam- + − ple format is employed. 18S The analytical and clinical performance studies estab- + 44 9 53 lish PfEMP1 as a superior biomarker for RT-PCR-based − 25 20 45 detection of both submicroscopic infections in samples Total 69 29 98 collected as whole blood and patent parasitaemias from samples collected as DBS. These results support the iden- tification of potentially novel abundant diagnostic mark- low [22]. Additionally, modeling studies have shown that ers as a means of improving the sensitivity of pathogen dramatic increases in the sensitivity of malaria diagnos- gDNA detection. While PfEMP1 is a well- known and tic assays, like that observed with PfEMP1-based ampli- characterized molecule associated with malaria patho- fication, are required to exert any substantial effect on genesis [24–26], no reports have thus far attempted to malaria transmission in regions of high intensity. Accord- harness this high copy multi-gene family as an ampli- ing to one report, application of higher sensitivity assays fication target in nucleic acid-based screening assays accelerates the interruption of malaria transmission in for malaria. All PfEMP1 variants are high-molecular an area. Accordingly, deployment of diagnostic assays of weight parasite proteins (200–450  kDa) inserted into superior sensitivity would render overall public health the membranes of P. falciparum-infected red blood cells. benefits and reduce the organizational and overhead PfEMP1 is the major constituent protein of knobs that costs of running programmes, such as MSAT campaigns are responsible for sequestration of infected RBCs in thus remaining a critical component of global malaria the peripheral vasculature that allows immune evasion eradication efforts [21]. of later-stage parasites [27, 28]. The conserved cytosolic Collection of blood samples as DBS on filter paper portion of PfEMP1 is anchored to the RBC cytoskeleton is highly amenable for field studies due to ease of sam- by lysine-rich membrane associated PHISTb (LyMP) ple collection and storage. However, detection of P. fal- [29, 30]. Although 60 variants of PfEMP1 exist, only a ciparum from the smaller volumes of blood typically single PfEMP1 is expressed during any given 48-h cycle. Grabias et al. Malar J (2019) 18:116 Page 8 of 9 All other unexpressed versions of parasite PfEMP1 are Authors’ contributions silenced by multiple different mechanisms such as his- BG, analysed data and wrote manuscript. EE collected the samples and performed the experiments. IAQ contributed with clinical samples and study tone modifications [31], promoter-intron ‘pairing’ [32], site information. SK conceptualized, designed the study and wrote the manu- and tethering of the var genes at the nuclear periphery script. All authors read and approved the final manuscript. [33]. Thus, while PfEMP1 may not serve as a superior Author details transcript for RNA-based amplification assays, the high 1 Laboratory of Emerging Pathogens, Division of Emerging and Transfusion number of gene copies of PfEMP1 in the malaria genome Transmitted Diseases, Center for Biologics Evaluation and Research, Food makes it an ideal candidate diagnostic marker for the and Drug Administration, Silver Spring, MD 20993, USA. 2 Department of Bio- logical Environmental and Occupational Health Sciences, School of Public detection of parasite DNA in blood. In this work, prim- Health, College of Health Sciences, University of Ghana, Legon, Ghana. ers were designed to amplify a strongly conserved gene 3 Laboratory of Emerging Pathogens, Division of Emerging and Transfusion sequence encoding the cytosolic acidic terminal seg- Transmitted Diseases, Center for Biologics Evaluation and Research, Food and Drug Administration, 10903 New Hampshire Ave. Building 52-72 Rm 5304, ment (ATS) region of PfEMP1. The finalized forward and Silver Spring, MD 20993, USA. reverse primers possess at least 91% identity to 18 dif- ferent loci within the P. falciparum genome which offer Acknowledgements We are grateful to all the Ghanaian individuals whose blood samples were more abundant targets of amplification than the available used in this study. We also thank the technicians for their assistance in pro- 5–8 genomic copies of the 18S ribosomal subunit gene. cessing the blood samples during sera collection. While the extracellular portion of PfEMP1 is subject to Competing interests immense selective pressure to evade the immune system, The authors declare that they have no competing interests. the cytosolic domain amplified in the assay and located in the second exon of the gene is significantly less diverse Availability of data and materials The datasets used and/or analysed during the current study are available from [34, 35]. the corresponding author per reasonable request. Consent for publication Conclusion There are no details on any individuals contained in this publication. Apart from developing countries, malaria also presents Ethics approval and consent to participate a challenge in industrialized countries where autochtho- This study protocol was approved by the Ghana Health Service ethical review nous transmission does not occur. In these countries, committee (GHS-ERC 06/05/16) and the FDA Research Involving Human Sub- malaria presents a significant public health challenge jects Committee (protocol #09-122B). All clinical samples used in this study were anonymized. through imported clinical cases and as transfusion transmitted malaria [4, 5]. Global malaria surveillance Funding is a priority of the World Health Organization, however, This work was funded by the intramural FDA grant and the Path malaria vac-cine initiative. reported cases are often incomplete, lacking confirma- tory testing, and many cases are unreported because of Publisher’s Note misdiagnosis. Most incidences of imported or trans- Springer Nature remains neutral with regard to jurisdictional claims in pub- fusion-related cases in nonendemic countries can be lished maps and institutional affiliations. traced to prior residents of malaria endemic areas [36]. 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