Novel high resolution melting (HRM) and snapback assays for simultaneous detection and differentiation of Plasmodium ovale spp.

Accepted Manuscript Title: Novel High Resolution Melting (HRM) and Snapback Assays for Simultaneous Detection and Differentiation of Plasmodium ovale spp. Authors: Aline Lamien-Meda, Hans-Peter Fuehrer, Harald Noedl PII: DOI: Reference:

S0001-706X(18)30560-6 https://doi.org/10.1016/j.actatropica.2019.01.018 ACTROP 4905

To appear in:

Acta Tropica

Received date: Revised date: Accepted date:

4 May 2018 18 January 2019 18 January 2019

Please cite this article as: Lamien-Meda A, Fuehrer H-Peter, Noedl H, Novel High Resolution Melting (HRM) and Snapback Assays for Simultaneous Detection and Differentiation of Plasmodium ovale spp, Acta Tropica (2019), https://doi.org/10.1016/j.actatropica.2019.01.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Novel High Resolution Melting (HRM) and Snapback Assays for Simultaneous Detection and Differentiation of Plasmodium ovale spp.

of Specific Prophylaxis and Tropical Medicine, Medical University of Vienna,

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aInstitute

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Aline Lamien-Medaa*, Hans-Peter Fuehrerb, Harald Noedla,

Austria bInstitute

of Parasitology, University of Veterinary Medicine Vienna, Austria

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*Corresponding author at: Institute of Specific Prophylaxis and Tropical Medicine,

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Medical University of Vienna, Kinderspitalgasse 15, 1090 Vienna, Austria.

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Email address: [email protected] (A. Lamien Meda)

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Abstract

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Plasmodium ovale spp. are two of the six species of apicomplexan parasites belonging to the genus Plasmodium commonly causing disease in humans. A recent

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phylogeny study has identified both Plasmodium ovale species (P. ovale curtisi and P. ovale wallikeri) as two sympatric occurring species. The actual prevalence and

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clinical relevance of P. ovale spp. are likely underestimated due to low parasitemia and mixed infections, which pose a major challenge to microscopic diagnosis and are frequently undetectable using malaria Rapid Diagnostic Tests (RDTs). The aim of this work is to develop a HRM-based assay for simultaneous detection and differentiation of P. ovale wallikeri and P. ovale curtisi. Thirty three welldocumented P. ovale spp. samples from previous studies were used for this study. 1

The newly developed High Resolution Melting (HRM) assay targeting the apicoplast genome was highly specific to both P. ovale species. Adding a snapback tail at the 5’ end of the forward primer for a nested HRM PCR, increased the melting temperature (Tm) difference between the two species. To our knowledge this study reports the first direct HRM assay developed on the

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apicoplast genome, specific for both P. ovale species. This method provides added value to the WHO open request of developing new practical malaria diagnostic

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methods for the malaria elimination program and could contribute to a quick and

efficient diagnosis of low-level parasitemia, symptomatic or asymptomatic, as well as mixed or single P. ovale infections.

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melting; Snapback assay; Apicoplast; Malaria.

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Keywords: Plasmodium ovale curtisi; Plasmodium ovale wallikeri; High resolution

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1. Introduction

Human malaria is caused by Plasmodium parasites and transmitted through the bite

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of infective female Anopheles mosquitoes. Despite extended prevention and control efforts, 216 millon cases and 445 000 deaths were estimated in 2016 (WHO, 2017).

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Plasmodium falciparum, P. vivax, P. malariae, P. ovale curtisi, P. ovale wallikeri and P. knowlesi, are the six Plasmodium species causing human disease.

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Plasmodium ovale (P. ovale) was described in 1922 with tertian periodicity similar to P. vivax and with two developmental cycles (one in the human host and one in the mosquito vector). P. ovale is a relapsing parasite frequently causing asymptomatic infections with humans as primary natural host (Collins and Jeffery, 2005). However, severe P. ovale infections can also occur (Strydom et al., 2014).

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Until very recently P. ovale was considered to comprise a single species of apicomplexan parasites belonging to the genus Plasmodium and causing disease in humans. A recent phylogenetic study has placed both P. ovale species (P. ovale curtisi and P. ovale wallikeri) within the Plasmodium phylogeny as two sympatric

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occurring species (Sutherland et al., 2010). The presence and natural distribution of P. ovale in sub-Saharan Africa and islands of the Western Pacific is well documented (Collins and Jeffery, 2005; Lysenko and

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Beljaev, 1969). The parasite has also been reported from the Philippines, Papua

New Guinea, and some Indonesian islands (Bauffe et al., 2012; Gneme et al., 2013;

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Mueller et al., 2007) but with the upcoming of molecular diagnostic tools P. ovale

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parasites were also identified in regions where these pathogens had not been

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documented previously (e.g. Bangladesh) (Fuehrer et al., 2012, 2010; Starzengruber

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et al., 2010). The actual prevalence and clinical relevance of P. ovale spp. is likely underestimated due to the fact that they often occur in mixed infections with other

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malaria parasite species with low-level parasitemia, posing specific challenges to the microscopic detection in areas with low prevalence, and due to the reduced ability to

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detect P. ovale spp. using malaria Rapid Diagnostic Tests (RDTs) (Bigaillon et al.,

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2005; Collins and Jeffery, 2005; De Laval et al., 2010; Miller et al., 2015). Since the early 1990s numerous polymerase chain reaction (PCR) methods have been developed for malaria diagnosis. Several molecular methods, including

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standard (nested) PCR, loop-mediated isothermal amplification (LAMP), and quantitative real time PCR, have been developed to identify and discriminate human Plasmodium species (Bauffe et al., 2012; Chavatte et al., 2015; Chua et al., 2015; Fuehrer and Noedl, 2014; Mangold et al., 2005; Snounou et al., 1993). Among the three molecular methods listed above, nested-PCR has most widely been used in 3

diagnostic studies and malaria epidemiological surveillance, resulting in a major improvement of the original PCR protocol of Snounou et al. (Snounou et al., 1993) targeting the nuclear small subunit ribosomal rRNA (SSU rRNA) gene. However, nested and semi-nested PCR have the disadvantage of being labour-intensive and

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time consuming (Boonma et al., 2007; Haanshuus et al., 2013). The discrimination of P. ovale spp. by nested PCR has continuously been improved since the development of the original protocol and more recently, a number of

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quantitative real time PCR assays targeting the diagnosis of the two P. ovale species (P. ovale curtisi and P. ovale wallikeri) have been developed (Fuehrer and Noedl,

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2014; Joste et al., 2018; Snounou et al., 1993). Analysis of the genome of both

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parasites suggests that there are more genes that could be targeted to genetically

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discriminate them (Fuehrer and Noedl, 2014; Zaw and Lin, 2015).

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The caseinolytic protease C (clpc) is one of several highly conserved genes encoded

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by the plastid genome and possessing a conserved AAA domain belonging to the Walker super family of ATPases and GTPases (Arisue et al., 2012; Rathore et al.,

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2001). The clpc gene, also known as Hsp93, forms part of the Tic complex for protein import into plastids and helps supplement apicoplast translation for all apicoplast

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proteins that are not encoded on the apicoplast genome (Jackson-Constan et al., 2001; Waller and McFadden, 2005). The apicoplast is a semi-autonomous plastid organelle for which a wide range of genome copy numbers has been reported,

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ranging from one to fifteen (Matsuzaki et al., 2001; Ramya et al., 2007). It remains vital in making several essential molecules and has tremendous potential for therapeutic intervention (McFadden and Yeh, 2017). Recently, studies have reported potential clinical and latency duration differences between the two ovale species. In addition, there is an increasing number of reports 4

of misdiagnosed cases of P. ovale infections. This creates an urgent need for proper diagnostic tools for these sympatric parasites (Joste et al., 2018; Miller et al., 2015). Our study aimed to develop a High Resolution Melting (HRM) assay, targeting the clpc gene of the highly conserved apicoplast genome, to specifically detect and discriminate P.ovale curtisi and P. ovale wallikeri. As the ∆Tm between the two

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species was lower than 0.5°C, challenging the species differentiation, an additional snapback assay, nested of the HRM qPCR assay, was developed to even better

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discriminate both P. ovale species. The snapback primers are oligonucleotides

designed with additional bases at the 5’ terminus that are complementary to the extension product of the primer. The terminus of each single strand of the snapback

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2. Materials and methods

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hairpin loop (Wilton et al., 1998; Zhou et al., 2008).

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PCR products re-anneals or “snaps back” on its extension product thereby creating

2.1. Samples and DNA extraction

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A total number of 33 well-documented P. ovale positive samples originating from published studies conducted in Bangladesh and Ethiopia (Alemu et al., 2013;

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Fuehrer et al., 2012) and one sample each of the other Plasmodium species (P. falciparum, P. vivax, P. malariae, P. knowlesi) were used for this study. All samples were collected under approved protocols and after obtaining written informed

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consent. The parasite density of the selected samples and species diagnosis were originally established by microscopy and nested PCR performed on DNA extracted using a modified Chelex-based DNA extraction from filter paper (Alemu et al., 2013; Fuehrer et al., 2012).

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For our study, the filter paper (4 x 4 mm blood spots soaked overnight in 100 µl PBS at 4°C) DNA was extracted using illustra blood genomicPrep Mini Spin Kits (GE Healthcare, Buckinghamshire, UK) following the manufacturer’s protocol. The DNA was eluted with two times 50 µl of elution buffer and stored at -20°C.

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2.2. Target selection and primer design The apicoplast clpc gene sequences of the two P. ovale species (curtisi and wallikeri) were analysed in direct comparison to the same gene of other Plasmodium species

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affecting humans in order to identify regions that are specific to both P. ovale species only. The clpc gene sequences of P. ovale, P. falciparum and other Plasmodium

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species were obtained from GenBank (National Center for Biotechnology Information,

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Bethesda, MD). The selected fragment of the P. ovale clpc gene with P. ovale curtisi,

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P. ovale wallikeri and P. falciparum specific signatures are presented in Fig.1. The

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partial clpc gene sequences were aligned using the clustal W algorithm as implemented in the BioEdit software package version 7.2.6. After the identification

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and selection of a specific and conserved region, a pair of primers, specific to both P. ovale species was designed to amplify a 125 bp fragment, for the real time PCR-

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HRM assay (qPCR-HRM) using the Primer3 online tool (http://bioinfo.ut.ee/primer3-

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0.4.0/). For a further and better differentiation of both species, a snapback tail of 21 bp was designed manually and added to the 5’ end of the forward primer to be used in a nested PCR with the qPCR-HRM reverse primer.

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The specificity of the primers was checked using the Basic Local Alignment Search. The secondary structures of the primers and the anticipated PCR amplicons were studied using the DNA folding form of the Mfold Web Server (http://mfold.rna.albany.edu) (Fig. 2).

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All primers were synthesized by Eurofins MWG Synthesis GmbH (Ebersberg,

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Germany), and purified by reverse phase high-performance liquid chromatography.

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Fig. 1. Selected fragment of P. ovale clpc gene with P. ovale curtisi, P. ovale wallikeri

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and P. falciparum specific signatures: Portion of P. ovale clpc gene (647 bp);

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Fragment: (125 bp); POvaDif F (26 bp); POvaDif R (27 bp); Snapback tail (21 bp)

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with the corresponding bp on the fragment (blue selection). The selected region contains 5 single nucleotide differences (polymorphism) between P. ovale curtisi and

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P. ovale wallikeri (T:C, C:T, C:T, A:G, G:A, resulting to only 1 C:T nucleotide change). The snapback tail (21 bp) sequence matched 100% with P. ovale curtisi and

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showing T:C and C:T mismatches with P. ovale wallikeri. Two nucleotides (TT) not

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relevant to the extension product of the forward primer were added to the 5’ end of the snapback tail to prevent it from being extended once it anneals to its

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complementary sequence. 2.3. PCR and melting curve qPCR-HRM assay. The reaction was performed in 20 µl reaction volumes containing 500 nM of forward primer (POvaDif F), 500 nM of reverse primer (POvaDif R), 1x SsoFast EvaGreen Supermix (BioRad, Hercules, CA) and 4 µl DNA sample. The PCR was performed in a Roche LightCycler 480 qPCR system (Roche Diagnostics 7

GmbH, Mannheim, Germany) with an initial denaturation step at 95°C for 3 min, followed by 50 cycles of 95°C for 10 sec, 50°C for 20 sec and 60°C for 30 sec. The PCR products were then subjected to the following High Resolution Melting (HRM) program: denaturation at 95°C for 1 min, cooling to 50°C (held for 1 min) and continuous heating at 0.02°C/s with fluorescence acquisition from 50°C to 80°C.

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Snapback assay. The snapback reaction was optimized by varying the

concentration of the snapback forward primer (500-100 nM) and the reverse primer

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(200- 50 nM). 500 nM snapback forward primer and 50 nM of reverse primer were

identified as best concentrations for the PCR reaction (Fig. 2). The reaction mixture consisted of 1x SsoFast EvaGreen Supermix, and 2 µl of 1/10 diluted template from

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the qPCR-HRM assay in 20 µl reaction volume. The cycling conditions and the

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melting program were the same as in the qPCR-HRM assay.

Fig. 2. Mfold structure of the snapback fragment using Mfold Web Server (http://mfold.rna.albany.edu) (A), and detailed scheme (adapted from Zou et al., 8

2008) of the snapback primer principle with PCR products structures (B) with a saturated DNA dye. A snapback primer is a standard oligonucleotide carrying an unlabelled probe and an additional two base pairs mismatch. The unlabelled probe element is complementary to the extension product of the primer. Following an asymmetric PCR with an excess of the snapback primer, two structures, comprising

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the full-length amplicons and intramolecular hairpins of the excess snapback strand are formed. The fluorescence melting curve analysis produces two different melting

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temperatures with a low-temperature for the melting of snapback stem and high-

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temperature for the full-length amplicon.

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2.4. Positive control production

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After confirmation of the specificity of the design primers, plasmids containing the

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targeted clpc gene fragments (125 bp) of P. ovale curtisi and P. ovale wallikeri were

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synthetized by Eurofins Medigenomics GmbH. The cloning was done using pEX-A2 vector backbone via Type IIS restriction enzymes and using ampicillin as antibiotic.

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The identity of the insert was confirmed by sequencing.

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2.5. Assay sensitivity and specificity The P. ovale genesig® standard kit targeting a sequence within the plasmepsin gene was used to confirm the study sample diagnostics.

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The specificity of the method was evaluated in-situ with mix infection samples containing P. vivax, P. falciparum and P. malariae (Table 1 in Lamien-Meda et al., "submitted"), and also by using DNA from other Plasmodium sp. as well as from following organisms (using identical PCR conditions): Toxoplasma gondii, Leishmania infantum, Trypanosoma brucei, Trypanosoma cruzi, Babesia divergens, 9

Entamoeba histolytica, Cryptosporidium parvum, Giardia intestinalis, Enterocytozoon bieneusi, Enzephalitozoon cuniculi, Pneumocystis jirovecii, Echinococcus granulosus, Strongyloides stercoralis, Dirofilaria repens, Toxocara canis, and Ascaris suum. In addition, DNA extracts from 5 Plasmodium negative blood spots filter paper from human were tested. The assay was performed in duplicate with each DNA

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sample.

The limit of detection (LOD) of the HRM method was defined as the measurand

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concentration producing at least 95% positive replicates (Forootan et al., 2017).

The LOD was assessed by amplifying five different concentrations (10, 8, 6, 4 and 2

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copies/µl) of each plasmid of the two P. ovale, in two to three replicates on three

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separate occasions. The data from each PCR run were recorded and subjected to

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probit regression analysis using STATGRAPHICS Centurion XV Version 15.2.12

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software.

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Statistical analyses and boxplots of the melting temperatures (Tm) were performed using R version 3.4.2 (2017-09-28) via RStudio version Version 1.1.383. The Welch's

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unequal variances t-test was used to compare the difference between the Tm means

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of the amplicons of P. ovale curtisi and P. ovale wallikeri and the difference between

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the mean Tm of the snapback stem of P. ovale curtisi and P. ovale wallikeri.

3. Results 3.1. Samples Species identification of the selected 33 P. ovale spp. samples from two different geographical regions (sub-Saharan Africa and south Asia) was originally performed by microscopical analysis. Twenty one samples originated from Bangladesh and 14 10

from Ethiopia. Nested PCR was used to confirm species identification (Table 1 in Lamien-Meda et al., "submitted"). 3.2. Assay design and optimization A high resolution melting (HRM) qPCR was developed to specifically detect P. ovale

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species and simultaneously differentiate P. ovale curtisi from P. ovale wallikeri using a forward (POvaDif F) and a reverse (POvaDif R) primers (Fig. 1). The selected

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primer pair showed a high specificity for P. ovale species due to the presence of

mismatches at the extremities of both primers to other Plasmodium species, resulting in specific binding of the primers to P. ovale spp.

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The melting temperature (Tm) values were 71.26 ± 0.03°C for P. ovale curtisi and

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71.04 ± 0.02°Cfor P. ovale wallikeri (Fig. 1 in Lamien-Meda et al., "submitted"). The

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nucleotides differences resulted in a slight Tm difference between P. ovale curtisi and

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P. ovale wallikeri (∆Tm = 0.2°C) (Fig. 3, A, B,C). The melting of the nested PCR

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products with the snapback primer produced an additional melting temperature with a greater ∆Tm of 3.74°C. The following melting pairs were obtained with the snapback

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fragment and the amplicon: 58.6 ± 0.3°C, 72.0 ± 0.1°C for P. ovale wallikeri and 62.3

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± 0.1°C, 72.0 ± 0.1°C for P. ovale curtisi (Fig. 3, D).

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Fig. 3. Melting curves/ peaks of: (A) reference plasmid of P. ovale curtisi and P. ovale

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wallikeri with no amplification of Pf, Pv, Pm, Pk; (B) the normalized melting peaks of P.

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ovale curtisi (green) and P. ovale wallikeri (red); (C) samples: P. ovale wallikeri (red, Tm = 71.04 ± 0.02°C) and P. ovale curtisi (green, Tm = 71.26 ± 0.03°C); (D) the

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snapback melting curves differentiating P. ovale curtisi (green) to P. ovale wallikeri (red). Detailed melting curves of each P. ovale are presented in Lamien-Meda et al.,

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"submitted".

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3.3. Efficiency and limit of detection (LOD) of the assay The efficiency of the PCR with primers POvaDif F and POvaDif R was determined by amplifying a 10-fold serial dilutions (107 to 10 copies/µl) of plasmids containing the targeted fragments of P. ovale wallikeri and P. ovale curtisi. The efficiencies were 99.25 % for P. ovale curtisi (slope = -3.341, R2 = 0.996) and 99.66 % for P. ovale 12

wallikeri (slope = -3.330, R2 = 0.995). The calculated LODs at 95% confidence of the qPCR-HRM method using probit analysis were 8.90 (7.08 to 13.53) P. ovale curtisi copies/µl, and 9.61 (7.53 to 15.22) P. ovale wallikeri copies/µl. Considering one apicoplast genome copy in Plasmodium as described by (Ramya et al., 2007) the

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copy/µl are equivalent to the number of parasites/µl. 3.4. Samples genotyping and assay specificity

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The discrimination power of the assays was tested with the 33 P. ovale samples, one of each other human Plasmodium species (P. falciparum, P. vivax, P. malariae, P. knowlesi) and 16 other DNA samples. The melting temperatures of P. ovale spp.

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samples are illustrated in boxplots (Fig. 2 in Lamien-Meda et al., "submitted"). The

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Welch's unequal variances t-test showed that Tm of the amplicon P. ovale curtisi and

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P. ovale wallikeri were significantly different (p-value < 2.2e-16). Similarly, the

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snapback stem of P. ovale curtisi and P. ovale wallikeri were also significantly

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different.

Among the 33 P. ovale spp. samples, the nested PCR identified 15 as P. ovale

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wallikeri, 12 as P. ovale curtisi and 6 were just characterized as P. ovale. The nested

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PCR also showed that 39.4% (13/33 samples) of the samples were mix-infected with P. vivax (3%), P. falciparum (21%), P. falciparum / P. malariae (6%), and P. falciparum/ P. vivax/ P. malariae (9%). The HRM assay identified 15 samples as P.

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ovale wallikeri and 14 samples as P. ovale curtisi. Four samples that were not detected by the HRM method were identified a P. ovale wallikeri through the additional typing with the snapback primer. In total, the snapback method was able to characterize all samples, identifying 19 of them as P. ovale wallikeri and 14 as P. ovale curtisi. (Table 1 in Lamien-Meda et al., "submitted"). The assay was very 13

specific to P. ovale, and did not amplify human DNA and other human Plasmodium species (P. falciparum, P. vivax, P. malariae, P. knowlesi) or any of the sixteen other parasite DNAs. In general, the results of the HRM method were concurrent with those of the

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PrimerDesign P. ovale kit except for one sample that could not be detected with this kit (Table 1 in Lamien-Meda et al., "submitted").

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To confirm our results, the clpc fragments (640 bp) of a random selection of 12

samples were sequenced (GenBank Accession Nos.: MG911696 - MG911707). No point mutation was observed inside the PCR targeted 125-bp fragments for both P.

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ovale species and each sample was clustering exactly with the species defined by

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the developed methods (five P. ovale curtisi and seven P. ovale wallikeri) (Fig. 3 in

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Lamien-Meda et al., "submitted").

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4. Discussion

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A cost and time-effective and easy-to-perform real time High Resolution Melting (HRM) assay was developed to specifically detect P. ovale species and differentiate

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P. ovale wallikeri from P. ovale curtisi with a small ∆Tm (0.2°C). Nevertheless, depending on the intercalating dye used, the differences between the Tms of P.

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ovale wallikeri and P. ovale curtisi could decrease. In addition, when the initial parasite density is low, the melting peaks may be too flat, thereby creating a challenge for discrimination. To overcome this issue, an additional nested snapback method was developed to improve the genotyping. This snapback assay is used only when the HRM assay fails to provide the genotyping. The use of the snapback tail allows the production of a 14

second melting peak with lower melting temperature due to a smaller size of the double strain fragment formed by the snapback (Fig. 2). Using this approach together with a good quality of extracted DNA and a real time PCR instrument capable of running an accurate melting programme, it was possible to develop a user-friendly

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assay for the direct detection and typing of P. ovale species. The HRM assay was highly specific for P. ovale spp. and effective in assigning each of the tested samples to one of the two P. ovale species. In our approach, the

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genotyping of the 33 samples was achieved at 88% with the direct HRM assay. All the samples were further characterized by using the nested snapback assay with

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100% concordance with the nested PCR results. The nested snapback method

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produced a significant melting temperature difference between P. ovale wallikeri and

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P. ovale curtisi and could be used whenever P. ovale DNA content is low.

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Our findings were also confirmed by sequencing and analysing the clpc fragments of

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a set of 12 selected samples (Lamien-Meda et al., "submitted"). The sequence analysis and phylogenetic results were also in agreement with previous studies

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(Arisue et al., 2012; Chavatte et al., 2015; Hagner et al., 2007).

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The genome of the Plasmodium apicoplast was targeted in this study because it was reported to be highly conserved among each of the Plasmodium species with a wide range of copy number (1-15 for P. falciparum) (Matsuzaki et al., 2001; Waller and

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McFadden, 2005; Ramya et al., 2007). Though, the 15 copies could probably be from a schizont cell which contains several merozoites (6-14 for P. ovale) (CDC - DPDx, 2017). Our study confirms this high degree of conservation, especially for the Caseinolytic protease C (clpC) gene. The 125 bp fragment of the clpc gene targeted in the HRM assay development was found to be specific to P. ovale species.

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Our results were also fully in agreement with those obtained using conventional approaches based on nested PCRs (Fuehrer et al., 2012). While these conventional approaches use a lengthy process of three to four PCRs and PCR product separation by electrophoresis in agarose gel to define the P. ovale species, our HRM assay requires only one primer set and a single PCR run to diagnose a sample as P. ovale

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as well as to differentiate P. ovale wallikeri from P. ovale curtisi. A second PCR is

only used when the first fails to provide accurate genotyping. Our method is also cost

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effective as it uses no labelled probe unlike assays based on hydrolysis probes

(Calderaro et al., 2012; Miller et al., 2015; Oddoux et al., 2011; Phuong et al., 2014). Very recently Joste and collaborators developed a qPCR-HRM method targeting the

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18S RNA gene for differentiation of both P. ovale species (Joste et al., 2018). The

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authors concluded that their method was “fully efficient in Poc and Pow

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discrimination, after P. ovale spp. confirmed identification”. Our study uses a limited

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number of samples (33) from only two geographically different region (sub-Saharan

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Africa and south Asia) as compared to the 75 P. ovale samples in the study of Joste et al. (2018) all from sub-Saharan countries. Nevertheless, based on the online

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published clpc gene sequences from diverse regions, our methods is likely to detect ovale samples from other geographical locations.

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Our estimated LOD from Cq and melting curves (2 parasites/µl) was similar to that of Joste et al. (2018). But according the MIQE guidelines, only LODs ≥ 3 copies at 95%

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confidence should be considered (MIQE, 2009), meaning that our calculated LODs (9-10 copies/µl) are expressing a well sensitive HRM method. Our selected primer set was highly specific to P. ovale spp. while the primers used by Joste et al. (2018) was detecting all human Plasmodium species.

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In fact the snapback primer strategy is an unlabelled probes-based technology successfully used for pathogen genotyping (Gelaye et al., 2013; Sun et al., 2014; Zhou et al., 2008). This approach uses an asymmetric primer concentration with an excess of the snapback primer allowing the formation of the snapback tail which produces a lower melting temperature following the fluorescent melting curve

describe the use of snapback probes in malaria diagnosis.

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analysis within the Mfold structure (Fig. 2). To our knowledge, this study is the first to

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A limitation of this method is that a second PCR run with the snapback primer is

needed to better differentiate P. ovale wallikeri from P. ovale curtisi for some of the

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tested samples, when the initial parasite loads do not allow a clear separation of the

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two melting curves. However, the nested snapback assay also has the advantage of

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confirming the result of the first HRM assay without the need for sequencing. This is

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an important advantage in the diagnosis of very low levels of parasitemia in blood

5. Conclusions

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samples originating from asymptomatic individuals, which is often seen with P. ovale.

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The HRM assay is providing a fast, efficient, and easy-to-perform approach for

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epidemiological investigations into the two P. ovale species. Our snapback assay also provides a valid additional method for clear and unambiguous differentiation of P. ovale wallikeri from P. ovale curtisi and could contribute actively and efficiently to

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the accurate diagnosis of the two P. ovale species which often occur in mixed infections with other Plasmodium species. Although the full clinical and epidemiological implications of the differentiation between the two P. ovale species has not been defined, accurate species diagnosis remains a major factor in controlling and ultimately eliminating malaria.

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Declarations of interest: none

Acknowledgments

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We wish to thank all collaborators involved in collecting the samples used in the validation of the assay as well as all malaria patients participating in these studies. We

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also wish to thank our colleagues from the Epidemiology & Diagnostic of Zoonoses

Research Group, Institute of Specific Prophylaxis and Tropical Medicine, Medical

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University of Vienna, Austria for providing the Non-Plasmodium DNA.

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