# Distinction of Plasmodium ovale wallikeri and Plasmodium ovale curtisi using quantitative Polymerase Chain Reaction with High Resolution Melting revelation

V. Joste, C. Kamaliddin, E. Kendjo, V. Hubert, N. Argy, S. Houze

► To cite this version:

V. Joste, C. Kamaliddin, E. Kendjo, V. Hubert, N. Argy, et al. Distinction of Plasmodium ovale wallikeri and Plasmodium ovale curtisi using quantitative Polymerase Chain Reaction with High Resolution Melting revelation. Scientific Reports, 2018, 8, pp.300. 10.1038/s41598-017-18026-1. hal-01696251

HAL Id: hal-01696251

https://hal.sorbonne-universite.fr/hal-01696251

Submitted on 30 Jan 2018

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
SCIENTIFIC REPORTS

<figure><img src="image_2.png" /></figure>

OPEN

# Distinction of *Plasmodium ovale wallikeri* and *Plasmodium ovale curtisi* using quantitative Polymerase Chain Reaction with High Resolution Melting revelation

V. Joste<fnref n="1" />,², C. Kamaliddin<fnref n="1" />,³, E. Kendjo<fnref n="1" />,⁴, V. Hubert<fnref n="1" />,³, N. Argy<fnref n="1" />,²,³ & S. Houzé<fnref n="1" />,²,³

*Plasmodium ovale curtisi* (*Poc*) and *Plasmodium ovale wallikeri* (*Pow*) have been described as two distinct species, only distinguishable by molecular methods such as PCR. Because of no well-defined endemic area and a variable clinical presentation as higher thrombocytopenia and nausea associated with *Pow* infection and asymptomatic forms of the pathology with *Poc* infection, rapid and specific identification of *Plasmodium ovale curtisi* and *Plasmodium ovale wallikeri* are needed. The aim of the study was to evaluate a new quantitative real-time PCR coupled with high resolution melting revelation (qPCR-HRM) for identification of both species. Results were compared with a nested-PCR, considered as a gold standard for *Pow* and *Poc* distinction. 356 samples including all human *Plasmodium* species at various parasitaemia were tested. The qPCR-HRM assay allowed *Poc* and *Pow* discrimination in 66 samples tested with a limit of detection evaluated at 1 parasite/µL. All these results were concordant with nested-PCR. Cross-reaction was absent with others blood parasites. The qPCR-HRM is a rapid and convenient technique to *Poc* and *Pow* distinction.

Received: 4 August 2017

Accepted: 28 November 2017

Published online: 10 January 2018

Malaria is triggered by an apicomplexan parasite, *Plasmodium* spp. 215 millions of cases and 429 000 deaths occurred in the world in 2015¹. Six *Plasmodium* species infect humans: *Plasmodium falciparum* (*Pf*), *Plasmodium vivax* (*Pv*), *Plasmodium malariae* (*Pm*), *Plasmodium knowlesi* (*Pk*) and *Plasmodium ovale* spp (*P. ovale* spp) including *Plasmodium ovale curtisi* (*Poc*) and *Plasmodium ovale wallikeri* (*Pow*). Although malaria transmission was absent, France was the most exposed non endemic country for malaria². Imported malaria cases diagnosed in France are reported to the National French Malaria Reference Center (FNMRC). In 2015, whereas *Pf* infection represented most of malaria cases (89%), *P. ovale* spp were involved in 6.8% of cases (4690 cases total)³, all contracted in Africa. Moreover, seven cases of *Plasmodium ovale* spp relapse (defined by malaria disease more than one year after the return from an endemic area) occurred in these patients (46% of the 15 observed relapses)³.

Malaria symptoms are nonspecific and the observation of thick and thin stained blood smears remained the reference method of diagnosis⁴,⁵. This technique is rapid and unexpensive, but required an experienced microscopist to ensure the quality of the result. However, species discrimination could be challenging, as *Poc* and *Pow* which cannot be reliably differentiated using this methodology.

Alternative ways to identify Plasmodium infection, such as Rapid Diagnosis Test (RDT) and PCR methods, have recently become available. Molecular biology diagnosis based methods are in constant expansion, in particular real-time methods. In the last decades, the malaria researcher community has developed several primers sets for species identification, to increase sensitivity in *Plasmodium* diagnosis.

<footnote n="1">¹National French Malaria Reference Center, Bichat-Claude Bernard Hospital, 75018, Paris, France. ²Parasitology and Mycology Laboratory, Bichat-Claude Bernard Hospital, APHP, 75018, Paris, France. ³UMR216-MERIT, COMUE Sorbonne Paris Cité, Faculté de Pharmacie de Paris, Paris Descartes University, Paris, 75006, France. ⁴National French Malaria Reference Center, Pitié Salpetrière hospital, 75013, Paris, France. Correspondence and requests for materials should be addressed to V.J. (email: valentin.joste@aphp.fr)</footnote>
<table><caption>**Table 1.** Median melting temperature and interquartile range for each species. We considered qPCR-Taqman (for *Pf*, *Pv*, *Pm* and *P. ovale* spp) and nested-PCR (for *Poc* and *Pow*) as the reference for species determination.</caption><thead><tr><th></th><th>Pf</th><th>Pv</th><th>Pm</th><th>Pow</th><th>Poc</th><th>Pk</th></tr></thead><tbody><tr><td>Median Tm1+/- interquartile range</td><td>72.892+/-0.100 (n=208)</td><td>74.798+/-0.100 (n=17)</td><td>73.926+/-0.179 (n=18)</td><td>71.165+/-0.2285 (n=31)</td><td>70.529+/-0.142 (n=34)</td><td>75.289 (n=2)</td></tr><tr><td>Median Tm2+/- interquartile range</td><td>75.396+/-0.112 (n=210)</td><td></td><td></td><td>73.387+/-0.14725 (n=33)</td><td>73.750+/-0.099 (n=36)</td><td></td></tr></tbody></table>

First, nested-PCR methods with agarose gel revelation were developed. Probe based real-time PCR assays such as Taqman have been developed later on, and increased sensitivity and specificity. However, the sample processing and the technique itself are time consuming and cannot be applied in routine diagnosis procedures.

Up to 2010, scientists and biologist referred to *Poc* and *Pow* as *P. ovale* spp⁶. It is now admitted that *Poc* and *Pow* are two distinct species and not subspecies⁷. *Poc* and *Pow* are minor forms of malaria, however severe disease forms and deaths have been reported⁸. Differences in clinical implication, specific geographic repartition and response to antimalarial therapy might also exist between *Poc* and *Pow*, but the respective implication of each has not been completely studied yet. In addition, *Poc* and *Pow* physiopathology is unknown, especially mechanisms leading to the dormant form hypnozoite formation and relapses⁹,¹⁰. In fact, some differences in the latency period of the hypnozoites were highlighted¹¹, higher thrombocytopenia and nausea with *Pow* was reported by another study¹². Yet, microscopic examination of blood smear does not allow differentiation of the two species, even if a recent study described a lack of discernible Schüffner's stippling in *Pow* samples¹³. Moreover, RDT efficiency was low and no differences in RDT positivity were found between *Poc* and *Pow*¹³. Therefore, differentiation of *Poc* and *Pow* remains challenging, and the development of convenient tools to differentiate *Poc* and *Pow* is crucial to investigate the burden on malaria infections of each species, as well as their clinical implication.

*Poc* and *Pow* can be differentiated based on differences in their DNA sequences. Nested-PCR technique has been developed¹⁴ as well as qPCR protocols¹⁵⁻¹⁸. However, nested-PCR techniques are time-consuming requiring a two-step amplification followed by a PCR product migration on agarose gel. The entire process takes almost a day, and needs a dedicated environment for post-PCR products' manipulation. Taqman probe based techniques are highly accurate and specific, but are expensive.

Here, we developed a new qPCR technique with High Resolution melting (HRM) (qPCR-HRM) revelation with the aim to reliably differentiate *Pow* and *Poc*, using samples from patients with imported malaria received at the FNMRC.

## Results

Of the 356 samples were included in the study period, 25 were negative for *Plasmodium* infection and 331 were positive according to qPCR-Taqman results (Supplementary Table S1): 219 were positive for *Pf*, 69 *Po* (subsequently identified as 33 *Pow*, and 36 *Poc* with nested PCR), 20 for *Pm*, 17 for *Pv*. 6 samples present mixed infection: 4P. ovale spp + Pf (subsequently identified for two *P. ovale* spp as *Poc*), 1P. ovale spp (subsequently identified as *Poc*) + Pm and 1P. ovale spp + Pm. In microscopy, among 343 observed by microscopy, we detected 46 negative, 6 *Plasmodium* spp, 182 *Pf*, 20 *Pm*, 70 *P. ovale* spp, 17 *Pv*, 1 *Pf* + Pm, 1 *Pf* + *P. ovale* spp.

For specificity analysis, we also included 6 other parasitological positive samples from blood parasites (*Toxoplasma gondii*, *Leishmania infantum*, *Loa loa*, *Onchocerca volvulus*, *Wuchereria bancrofti* and *Babesia divergens*).

**qPCR-HRM development.** *In silico* prevision. *In silico* alignment was performed from predictive PCR product using the primers chosen for qPCR-HRM analysis. Plasmo-1F and Plasmo-2R primers amplify a 160-based pair (bp) and 157 bp sequence for *Poc* and *Pow* respectively. As a sequence variation in predictive PCR product for *Poc* and *Pow* was observed, HRM reaction could highlight a difference between both species. Using the specific uMELT online tool for PCR product melting temperature (Tm) analysis, predicting melting temperature from the HRM phase based on an algorithm analyzing DNA bounds strength, Tm could be predicted for the amplified sequences (77.4 °C for *Pow* and 76.8 °C for *Poc*). Predicted Tm as well as theoretical derived melting curves for other *Plasmodium* species are displayed in Supplementary Figure S2.

*Distinction of P. ovale wallikeri and Plasmodium ovale curtisi.* Among the 69P. ovale spp samples confirmed by qPCR-Taqman, Tm values for each species were reproducible (see Table 1 for average Tm and interquartile interval for *Poc* and *Pow*). 32 *Pow* and 34 *Poc* were identified by qPCR-HRM and these results were compared with nested-PCR followed by an electrophoresis in agarose gel and species were determined with 100% of concordance (Supplementary Table S1). However, by qPCR-HRM, 3 samples were misidentified as *Plasmodium malariae*: two were *Poc* and one was *Pow* by nested-PCR.

Specific geographic repartition of *Poc* and *Pow* in the included samples is presented in Fig. 1 and in Supplementary Table S1. *Pow* seems to be overrepresented in Cameroon whereas *Poc* seems to be overrepresented in Central African Republic. *Poc* and *Pow* are co-endemic in Ivory Coast, Cameroon, Central African Republic, Mali, Congo.

**Mixed artificial samples with both Poc and Pow.** Both *Poc* and *Pow* were distinguished in artificial samples, except when *Pow* was present in a strong majority (ratio 80% *Pow*/20% *Poc* and 90% *Pow*/10% *Poc*). Derivative melt curves of those samples are presented in Supplementary Figure S3. Same conclusions were made for *Pf*, *Pv*, *Pm-Po* mixed infections which could not be detected in mixed artificial samples.
<figure><img src="image_3.png" /></figure>

● Plasmodium ovale wallikeri

● Plasmodium ovale curtisi

**Figure 1.** Map representing the origins of *P. ovale* spp samples included in the study. Orange represent *Poc* and purple *Pow*. Sign size is proportional to the number of samples from a given area. The smaller sign represents one sample, and the bigger one five samples. This map is modified from this URL: http://www.mapsopensource.com/africa-countries-outline-map-black-and-white.html which provided open source map with no permission required (https://creativecommons.org/licenses/by/3.0/).

**qPCR-HRM characteristics.** Repeatability and reproducibility. Repeatability was evaluated for *Poc* and *Pow* using three different parasite's densities. Repeatability was between 0.38 standard deviation (SD) and 0.49SD of Ct for *Poc* and, between 0.55 to 1.26 SD of Ct for *Pow*. Reproducibility was not evaluated as the assay was not quantitative. However, comparing the Tm for 10 samples on 10 distinct reactions, we observed a constant Tm in the different assay. Regarding *Poc*, coefficient variation was between 0.18 to 0.25% (70.90+/-0.132 to 70.90+/-0.162) for Tm1 and between 0.17 to 0.54% (73.98+/-0.13°C to 74.31+/-0.405°C) for Tm2. Regarding *Pow*, coefficient variation for Tm1 was between 0.20 to 0.47% (71.446+/-0.144°C to 41.434+/-0.314°C) and between 0.19 to 0.24% (73.708+/-0.144°C to 73.834+/-0.174°C) for Tm2.

**Sensitivity and specificity.** LOD was the lowest parasite concentration that had repeated positive signal detection with qPCR-HRM¹⁹ and was evaluated at 1 parasite/µL for both *Poc* and *Pow*.

No cross-reaction with *Toxoplasma gondii*, *Leishmania infantum*, *Loa loa*, *Onchocerca volvulus*, *Wuchereria bancrofti*, and *Babesia divergens* was detected with the qPCR-HRM technique. We observed significant amplification for *Toxoplasma gondii* and *Wuchereria bancrofti* but none of the Tm were specific to any *Plasmodium* (Supplementary Table S4).

As in silico provision for the ability of the qPCR-HRM to distinguish *P. ovale* spp from other *Plasmodium* species, we applied the qPCR-HRM on 356 *Plasmodium* positive samples to confirm experimentally in silico data. Concordance between the qPCR-HRM and the qPCR-Taqman FTD Malaria Differentiation was observed for 336 samples (93.6%). Among the 23 (6.4%) discordant samples, there were with qPCR-HRM 11 false negative (sensitivity = 97%), 5 undetected species mixed (only one of both species detected), and 7 false positive results with crossing reactions between *Pf* and *Pv* (2 cases), *P. ovale* spp and *Pm* (3 cases), *Pm* and *Pv* (2 cases). No false positive were reported (specificity of 100%). We could distinguish *Plasmodium* species because *Pf* and *P. ovale* spp displayed a profile pattern with two specific Tm (Table 1) whereas melting curves obtained with samples positive for *Pm*, *Pv* and *Pk* displayed a profile with one specific Tm. *Poc* and *Pow* melting curves derivative plot showed two distinct profiles with Tms specific of each (70.529°C and 73.750°C for *Poc*, and 71.165°C and 73.387°C for *Pow*) (Fig. 2). In our batch of sample, we described three *P. ovale* spp with one remaining Tm (2 *Poc* and 1 *Pow*) with no relation with the Ct (mean Ct equal to 25.755 +/- 2.188).

All isolated species, except *Pm*, are in good concordance between qPCR-HRM and qPCR-Taqman (Table 2).

## Discussion

The existence of the two species *Poc* and *Pow* has been known since 2010, but has currently no impact on clinical care of the patient. It is not possible to differentiate them by optical microscopy. However, some clinical and biological differences have been noticed, such as higher proportion of thrombocytopenia and nausea with *Pow*¹². It also appears that *Poc* is more frequently asymptomatic²⁰. For these reasons, a cost-effective qPCR-HRM method was developed for the differentiation of *Poc* and *Pow* species.

qPCR-HRM allowed us to discriminate *Poc* and *Pow* in confirmed *P. ovale* spp positive samples. To our knowledge, this is the first qPCR-HRM that allows *Poc* and *Pow* discrimination. LOD is similar to other qPCR-HRM already published²¹. Repeatability and reproducibility are really satisfying. From selected primer already published²², 18S RNA amplicons were aligned with the chosen primer to ensure the sequences divergence, and therefore the Tm variation, before setting the qPCR-HRM assay. The sequence presents noticeable variation between the *Pow* and *Poc* (98% consistency), and each parasites contains four to eight copies guaranteeing a good
<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /><figcaption>**Figure 2.** *Plasmodium ovale wallikeri* and *Plasmodium ovale curtisi* in derivative (a) and aligned melt curves (b) obtained after HRM phase in Viia7. X axis represents the temperature (°C). Y axis represent the fluorescence (derivative melt curve, a) or the aligned flurorescence (aligned melt curves, b). *Plasmodium ovale curtisi* profile is displayed in blue, and *Plasmodium ovale wallikeri* is displayed in orange.</figcaption></figure>

<table><caption>**Table 2.** Comparison between qPCR-Taqman, nested-PCR and qPCR-HRM results, by species, alone or in association.</caption><thead><tr><th rowspan="2">qPCR-Taqman/Nested-PCR</th><th colspan="9">qPCR-HRM</th></tr><tr><th>Pf</th><th>Pv</th><th>Pm</th><th>Poc</th><th>Pow</th><th>Pf+Po</th><th>Pm+Po</th><th>Negative</th><th>Total</th></tr></thead><tbody><tr><td>Pf</td><td>209</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>9</td><td>219</td></tr><tr><td>Pv</td><td>1</td><td>16</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>17</td></tr><tr><td>Pm</td><td>0</td><td>2</td><td>16</td><td>0</td><td>0</td><td>0</td><td>0</td><td>2</td><td>20</td></tr><tr><td>Poc</td><td>0</td><td>0</td><td>2</td><td>34</td><td>0</td><td>0</td><td>0</td><td>0</td><td>36</td></tr><tr><td>Pow</td><td>0</td><td>0</td><td>1</td><td>0</td><td>32</td><td>0</td><td>0</td><td>0</td><td>33</td></tr><tr><td>Pf+Po</td><td>3</td><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td><td>0</td><td>0</td><td>4</td></tr><tr><td>Pm+Po</td><td>0</td><td>0</td><td>1</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>2</td></tr><tr><td>Negative</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>25</td><td>25</td></tr><tr><td>Total</td><td>213</td><td>19</td><td>20</td><td>35</td><td>32</td><td>1</td><td>0</td><td>36</td><td>356</td></tr></tbody></table>

sensitivity to the PCR²³ but this also complicates quantitative approaches. *In silico* prediction shows us different predictive Tm for *Poc* and *Pow* that convinced us to test Plasmol-F and Plasmo2-R. In that *in silico* prediction, distinction between *P. ovale* spp and other *Plasmodium* species was also possible.

From 69 samples of *P. ovale* spp: 66 were identified by qPCR-HRM, and among those, concordance with nested PCR for species determination was perfect (100% concordance). This qPCR-HRM is fully efficient in *Poc* and *Pow* discrimination, after *P. ovale* spp confirmed identification: identification of other species than *Poc* or *Pow* don't have to be considered because we demonstrated false identification. qPCR methods have already been published to distinguish species of *P. ovale* spp using specific probes in qPCR-Taqman¹⁸ but we present here an innovative way of PCR revelation. In our study we highlighted that *Poc* and *Pow* are co-endemic in some African
Specificity was evaluated in two different way. First, we tested *Toxoplasma gondii*, *Leishmania infantum*, *Loa loa*, *Onchocerca volvulus*, *Wuchereria bancrofti*, and *Babesia divergens* patient positive blood samples from Bichat's hospital to prove the existence of *Poc* and *Pow* specific Tm. Second, we evaluated the ability of the qPCR-HRM of distinguish *P. ovale* spp from other *Plasmodium* species in clinical samples.

**Ethic approval.** Participants' consent was not required since samples were already collected for other medical purposes, and according to French legislations on non-interventional research.

## References

1. WHO | World Malaria Report 2016. WHO Available at: http://www.who.int/malaria/publications/world-malaria-report-2016/report/en/. (Accessed: 12th January 2017).

2. European Centre for Disease Prevention and Control. Annual Epidemiological Report 2016 - Malaria. [Internet]. Available at: https://ecdc.europa.eu/en/publications-data/malaria-annual-epidemiological-report-2016-2014-data. Cited 16 11 (2017).

3. Rapport d'activité - CNR Paludisme [Internet]. Available at: http://cnrpaludisme-france.org/pages/rapports-annuels/. Cited 2017 16 11 (2016).

4. Société de Pathologie Infectieuse de Langue Française *et al.* [Management and prevention of imported Plasmodium falciparum malaria (Revision 2007 of the 1999 Consensus Conference). Long text in French]. *Médecine Mal. Infect.* **38**, 68–117 (2008).

5. Recommandations Paludisme SPILF. [Internet]. Available at: http://www.infectiologie.com/fr/actualites/paludisme-rcp-2017-.n.html. Cited 2017 16 11 (2017).

6. Sutherland, C. J. *et al*. Two nonrecombining sympatric forms of the human malaria parasite *Plasmodium ovale* occur globally. *J. Infect. Dis.* **201**, 1544–1550 (2010).

7. Zaw, M. T. & Lin, Z. Two sympatric types of *Plasmodium ovale* and discrimination by molecular methods. *J. Microbiol. Immunol. Infect. Wei Mian Yu Gan Ran Za Zhi* **50**, 559–564 (2017).

8. Lau, Y.-L. *et al*. Acute respiratory distress syndrome and acute renal failure from *Plasmodium ovale* infection with fatal outcome. *Malar. J.* **12**, 389 (2013).

9. Markus, M. B. Do hypnozoites cause relapse in malaria? *Trends Parasitol.* **31**, 239–245 (2015).

10. Richter, J., Franken, G., Mehlhorn, H., Labisch, A. & Häussinger, D. What is the evidence for the existence of *Plasmodium ovale* hypnozoites? *Parasitol. Res.* **107**, 1285–1290 (2010).

11. Nolder, D. *et al*. An observational study of malaria in British travellers: *Plasmodium ovale wallikeri* and *Plasmodium ovale curtisi* differ significantly in the duration of latency. *BMJ Open* **3**, e002711 (2013).

12. Rojo-Marcos, G. *et al*. Comparison of Imported *Plasmodium ovale curtisi* and *P. ovale wallikeri* Infections among Patients in Spain, 2005–2011. *Emerg. Infect. Dis.* **20**, 409–416 (2014).

13. Phuong, M. S., Lau, R., Ralevski, F. & Boggild, A. K. Parasitological correlates of *Plasmodium ovale curtisi* and *Plasmodium ovale wallikeri* infection. *Malar. J.* **15**, 550 (2016).

14. Calderaro, A. *et al*. Genetic polymorphisms influence *Plasmodium ovale* PCR detection accuracy. *J. Clin. Microbiol.* **45**, 1624–1627 (2007).

15. Oguike, M. C. *et al*. *Plasmodium ovale curtisi* and *Plasmodium ovale wallikeri* circulate simultaneously in African communities. *Int. J. Parasitol.* **41**, 677–683 (2011).

16. Calderaro, A. *et al*. Accurate identification of the six human *Plasmodium* spp. causing imported malaria, including *Plasmodium ovale wallikeri* and *Plasmodium knowlesi*. *Malar. J.* **12**, 321 (2013).

17. Calderaro, A. *et al*. A new real-time PCR for the detection of *Plasmodium ovale wallikeri*. *PloS One* **7**, e48033 (2012).

18. Miller, R. H. *et al*. Characterization of *Plasmodium ovale curtisi* and *P. ovale wallikeri* in Western Kenya utilizing a novel species-specific real-time PCR assay. *PLoS Negl. Trop. Dis.* **9**, e0003469 (2015).

19. Bustin, S. A. *et al*. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. *Clin. Chem.* **55**, 611–622 (2009).

20. Fuehrer, H.-P. *et al*. *Plasmodium ovale* in Bangladesh: genetic diversity and the first known evidence of the sympatric distribution of *Plasmodium ovale curtisi* and *Plasmodium ovale wallikeri* in southern Asia. *Int. J. Parasitol.* **42**, 693–699 (2012).

21. Chua, K. H. *et al*. Development of High Resolution Melting Analysis for the Diagnosis of Human Malaria. *Sci. Rep.* **5**, 15671 (2015).

22. Shokoples, S. E., Ndao, M., Kowalewska-Grochowska, K. & Yanow, S. K. Multiplexed Real-Time PCR Assay for Discrimination of *Plasmodium* Species with Improved Sensitivity for Mixed Infections. *J. Clin. Microbiol.* **47**, 975–980 (2009).

23. Mercereau-Puijalon, O., Barale, J.-C. & Bischoff, E. Three multigene families in *Plasmodium* parasites: facts and questions. *Int. J. Parasitol.* **32**, 1323–1344 (2002).

24. Kassaza, K. *et al*. Detection of *Plasmodium* species by high resolution melt analysis of DNA from blood smears acquired in Southwestern Uganda. *J. Clin. Microbiol.*, https://doi.org/10.1128/JCM.01060-17 (2017).

25. Mace, K. E. & Arguin, P. M. Malaria Surveillance - United States, 2014. *Morb. Mortal. Wkly. Rep. Surveill. Summ. Wash. DC* 2002 **66**, 1–24 (2017).

26. Daniels, R. *et al*. Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria. *J. Vis. Exp. JoVE* e52839, https://doi.org/10.3791/52839 (2015).

27. Milbury, C. A., Li, J. & Makrigiorgos, G. M. COLD-PCR-Enhanced High-Resolution Melting Enables Rapid and Selective Identification of Low-Level Unknown Mutations. *Clin. Chem.* **55**, 2130–2143 (2009).

28. Singh, B. *et al*. A large focus of naturally acquired *Plasmodium knowlesi* infections in human beings. *The Lancet* **363**, 1017–1024 (2004).

29. Fuehrer, H.-P., Stadler, M.-T., Buczolich, K., Bloeschl, I. & Noedl, H. Two Techniques for Simultaneous Identification of *Plasmodium ovale curtisi* and *Plasmodium ovale wallikeri* by Use of the Small-Subunit rRNA Gene. *J. Clin. Microbiol.* **50**, 4100–4102 (2012).

30. Snounou, G. & Singh, B. Nested PCR analysis of *Plasmodium* parasites. *Methods Mol. Med.* **72**, 189–203 (2002).

31. Snounou, G., Viriyakosol, S., Jarra, W., Thaithong, S. & Brown, K. N. Identification of the four human malaria parasite species in field samples by the polymerase chain reaction and detection of a high prevalence of mixed infections. *Mol. Biochem. Parasitol.* **58**, 283–292 (1993).

32. Rougemont, M. *et al*. Detection of Four *Plasmodium* Species in Blood from Humans by 18S rRNA Gene Subunit-Based and Species-Specific Real-Time PCR Assays. *J. Clin. Microbiol.* **42**, 5636–5643 (2004).

33. Corpet, F. Multiple sequence alignment with hierarchical clustering. *Nucleic Acids Res.* **16**, 10881–10890 (1988).

34. Dwight, Z., Palais, R. & Wittwer, C. T. uMELT: prediction of high-resolution melting curves and dynamic melting profiles of PCR products in a rich web application. *Bioinforma. Oxf. Engl.* **27**, 1019–1020 (2011).

## Acknowledgements

French Malaria National reference center funded the experiments.
**Author Contributions**

V.J., V.H. and S.H. designed the experiment. V.J. performed the experiment. V.J. and V.H. analysed the data. V.J. and C.K. wrote the manuscript. S.H. supervised the study. V.J., C.K., E.K., V.H., N.A. and S.H. reviewed the manuscript.

**Additional Information**

**Supplementary information** accompanies this paper at https://doi.org/10.1038/s41598-017-18026-1.

**Competing Interests:** The authors declare that they have no competing interests.

**Publisher's note:** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

<figure><img src="image_6.png" /></figure>

**Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as 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 images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

© The Author(s) 2017