Evaluation of a novel real-time PCR assay for the detection, identification and quantification of Plasmodium species causing malaria in humans.

BACKGROUND: The entry of PCR-based techniques into malaria diagnostics has improved the sensitivity and specificity of the detection of Plasmodium infections. It has been shown that humans are regularly infected by at least six different Plasmodium species. The MC004 real-time PCR assay for malaria diagnosis is a novel single-tube assay that has been developed for the purpose of simultaneously detecting all Plasmodium species known to infect humans, and discrimination between Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale wallikeri, Plasmodium ovale curtisi, Plasmodium knowlesi (including differentiation of three strains) and Plasmodium cynomolgi (including differentiation of three strains). Detection and identification of Plasmodium species relies on molecular beacon probe-based melting curve analysis. In addition, this assay might be used to quantify the parasitaemia of at least P. falciparum by calculating the level of parasitaemia directly from the Cq-value.
METHODS: The samples used in this study comprised reference samples, patient samples, and synthetic controls. The following analytical performance characteristics of the MC004 assay were determined: analytical specificity, limit of detection, the ability to detect mixed infections, and the potential to determine the level of parasitaemia of P. falciparum, including assessment of within-run and between-run precisions.
RESULTS: No false positive or false negative results were observed. The limit of detection of P. falciparum was 1 × 10-3 IU/mL (WHO standard). Mixed infections with P. falciparum and non-falciparum species were correctly identified. A calibration curve could be established to quantify the parasitaemia of at least P. falciparum. The within-run and between-run precisions were less than 20% CV at the tested parasitaemia levels of 0.09%, 0.16%, 2.15% and 27.27%.
CONCLUSION: Based upon the analytical performance characteristics that were determined, the MC004 assay showed performance suitable for use in clinical settings, as well as epidemiological studies.

Background

The genus Plasmodium consists of over 200 widely distributed species, of which at least six are now known to regularly infect humans: Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale wallikeri, Plasmodium ovale curtisi, Plasmodium knowlesi [1]. Previously, malaria was thought to be primarily caused by just the four species, P. falciparum, P. vivax, P. malariae and P. ovale, and only very rarely by other Plasmodium species. However, the entry of PCR-based techniques into malaria diagnostics has changed this view. It has led to the discovery of the existence of P. ovale wallikeri and P. ovale curtisi [2], and the demonstration that P. knowlesi is the predominant cause of malaria in regions of South East Asia. Interestingly, recent research indicates that also P. cynomolgi may be more common in South East Asia than previously suspected [3, 4]. In addition, human susceptibility to Plasmodium simium and Plasmodium brasilianum has been shown. Plasmodium simium is found throughout South America, while Plasmodium brasilianum seems restricted to a specific part of Brasil [5-7]. Globally, P. falciparum is responsible for the vast majority of malaria deaths and cases of severe malaria. Malaria caused by other Plasmodium species is not typically as life threatening, but can also be lethal. In particular, severe and fatal cases of malaria caused by P. vivax and P. knowlesi are well recognized [1].

The gold standard of malaria diagnosis is the examination of stained thick and thin blood films by light microscopy. The quantification of malaria parasites can be used to make clinical management decisions as well as monitoring response to treatment [8]. Microscopic diagnosis of malaria is prone to human error owing to its subjective nature. An inherent weakness of microscopy is the dependence on morphological features to distinguish Plasmodium species. Even under ideal conditions, reliable distinction of the infecting Plasmodium species can be very difficult, if not impossible. Particularly, P. vivax and P. ovale cannot always be easily differentiated based on morphology, distinguishing P. knowlesi from P. malariae can be very challenging, P. ovale wallikeri and P. ovale curtisi are morphologically identical, P. cynomolgi is morphologically indistinguishable from P. vivax, and P. simium and P. brasilianum cannot be distinguished by microscopy from P. vivax and P. malariae, respectively [9-11]. Also, the detection limit of microscopy is not always sufficient to detect low-density malaria infections. Submicroscopic infections are typically asymptomatic, but not benign. Such cases should be considered as chronic malaria, and should be treated accordingly. Untreated chronic malaria has significant health consequences for the individual, and, as it could potentially be transmitted, affects public health [12-15].

A plethora of polymerase chain reaction (PCR)-based nucleic acid amplification tests (NAATs) to detect, quantify and identify Plasmodium parasites in blood have been described in the literature. Rapid NAATs with a turnaround time of less than one hour are now available, and some NAATs can be used for high throughput of samples. In addition, NAATs are used to detect anti-malarial drug resistance markers. NAATs offer several significant advantages over standard microscopy. Importantly, the sensitivity of NAATs for the detection of Plasmodium parasites in blood is superior to any other diagnostic test. The general ability to detect far less than ten Plasmodium parasites per microlitre of blood provides the sensitivity to rule out malaria with high negative predictive value in clinical settings. In endemic settings, where the goal is to eradicate malaria, the required sensitivity to reliably detect submicroscopic infections can be met. Also, NAATs tend to be highly specific. Identification of Plasmodium species goes beyond what is possible with conventional microscopy. For example, for differentiating Plasmodium species with similar morphologies, detecting mixed Plasmodium infections, utilization of poor-quality or ambiguous samples, and when differentiation of Plasmodium species or strains is impossible based on morphology alone.

However, an important pitfall to be aware of is that NAATs not specifically designed to identify a certain Plasmodium species may fail to detect it or cause misidentification. Individual patient treatment may not always be affected, but such errors could ultimately prompt public health concerns by compromising malaria control and elimination.

Other concerns regarding the implementation of NAATs are the special equipment and reagents required, and the need for skilled laboratory technicians able to perform testing and to interpret the results of these relatively complex tests.

Furthermore, test characteristics vary widely, and although the World Health Organization (WHO) International Standard for P. falciparum [National Institute for Biological Standards and Control (NIBSC)-code 04/176] allows for comparison among the available assays, there is a lack of standardization and validation. As science and technology proceed, NAATs are bound to play an increasingly important role in malaria diagnosis, research, and epidemiology. At present, it is clear that NAATs cannot completely replace traditional microscopy [16-18].

The objective of the present study was to determine the analytical performance of the novel MC004 real-time PCR assay for malaria diagnosis (MRC Holland, Amsterdam, the Netherlands). This single-tube assay has been developed for the purpose of simultaneously detecting all Plasmodium species known to infect humans, and discrimination between P. falciparum, P. vivax, P. malariae, P. ovale wallikeri, P. ovale curtisi, P. knowlesi (including differentiation of three strains) and P. cynomolgi (including differentiation of three strains). In addition, this study shows how this assay might be used to estimate the parasitaemia of at least P. falciparum.

Methods

Analytical specificity study design

The analytical specificity of the assay was determined by testing samples received from reference institutes or referral laboratories [the Dutch Foundation for Quality Assessment in Medical Laboratories (SKML), UK NEQAS, Streeklab Haarlem—Laboratory for Medical Microbiology, the Biomedical Primate Research Centre, the London School of Hygiene & Tropical Medicine, and Labor Dr. Wisplinghoff]. In total, 40 reference samples were tested that included all seven Plasmodium species (P. falciparum, P. vivax, P. knowlesi, P. malariae, P. cynomolgi, P. ovale wallikeri, and P. ovale curtisi) at various levels of parasitaemia, and four different non-Plasmodium parasites: Babesia microti, Loa loa, Trypanosoma brucei rhodesiensis, and Leishmania donovani infantum. Specimen types included ethylenediaminetetraacetic acid (EDTA) whole blood, isolated DNA, and Giemsa-stained blood or bone marrow slides (Table 1). The results of the MC004 assay were compared with the results of the reference laboratory.

Table 1

Analytical specificity panel

Reference institute or referring laboratory	Description, if applicable the survey number is shown in parentheses	Specimen type	Level of parasitaemia
Dutch Foundation for Quality Assessment in Medical Laboratories (SKML) The Blood- and intestinal parasites scheme (n = 7)	P. falciparum (2019.1B)	Blood smear	1.9%
	P. falciparum (2019.3E)	EDTA whole blood	Unknown
	P. falciparum (2019.3F)	EDTA whole blood	Unknown
	P. vivax (2018.1G)	EDTA whole blood	Unknown
	P. ovale (2017.4B)	Blood smear	Unknown
	P. knowlesi (2016.3B)	Blood smear	4.2%
	Trypanosoma brucei rhodesiensis (2019.1A)	Blood smear	Not applicable
UK NEQAS (n = 3) The Blood parasitology scheme	P. malariae (5796)	Blood smear	Unknown
	Loa Loa (5211)	Blood smear	Not applicable
	P. falciparum + P. vivax (5844)	Blood smear	0.2%
UK NEQAS The Malaria molecular scheme (n = 8)	No parasites (5220)	EDTA whole blood	Not applicable
	No parasites (5222)	EDTA whole blood	Not applicable
	No parasites (5363)	EDTA whole blood	Not applicable
	No parasites (5364)	EDTA whole blood	Not applicable
	P. vivax (5365)	EDTA whole blood	Unknown
	P. vivax (5366)	EDTA whole blood	Unknown
	P. falciparum (5221)	EDTA whole blood	0.0002%
	P. knowlesi (5219)	EDTA whole blood	0.02%
Streeklab Haarlem—Laboratory for Medical Microbiology (n = 17)	P. falciparum	Blood smear	4.5%
	P. falciparum	Blood smear	1.3%
	P. falciparum	Blood smear	0.5%
	P. falciparum	Blood smear	9.9%
	P. falciparum	Blood smear	< 0.1%
	P. falciparum	Blood smear	Gametocytes
	P. vivax	Blood smear	Unknown
	P. vivax	Blood smear	Unknown
	P. vivax	Blood smear	Unknown
	P. ovale	Blood smear	Unknown
	P. ovale	Blood smear	Unknown
	Leishmania donovani infantum	Bone marrow smear	Not applicable
	Leishmania donovani infantum	Bone marrow smear	Not applicable
	No parasites	Blood smear	Not applicable
	No parasites	Blood smear	Not applicable
	P. knowlesi	Blood smear	Unknown
	Babesia microti	Blood smear	Not applicable
Biomedical Primate Research Centre (n = 2)	P. knowlesi (H strain)	Isolated DNA	Not applicable
	P. cynomolgi (M strain)	Isolated DNA	Not applicable
The London School of Hygiene & Tropical Medicine (n = 2)	P. ovale wallikeri	Isolated DNA	Not applicable
	P. ovale curtisi	Isolated DNA	Not applicable
Labor Dr. Wisplinghoff (n = 1)	P. knowlesi	EDTA whole blood	0.3%

MC004 assay technology

The MC004 assay (MRC-Holland, Amsterdam, the Netherlands) is a multiplex nucleic acid amplification test that detects mitochondrial Plasmodium DNA encompassing the cyclo-oxygenase 3 (COX-3), cyclo-oxygenase 1 (COX-1) and cytochrome b (CYTB) genes from Plasmodium species that cause malaria in humans. The assay provides a qualitative result for the presence/absence of Plasmodium DNA, and discriminates between 11 Plasmodium species/strains (P. falciparum, P. vivax, P. malariae, P. ovale wallikeri, P. ovale curtisi, P. knowlesi LT48, P. knowlesi ATCC 30153, P. knowlesi ATCC 30158 P. cynomolgi ATCC 30149, P. cynomolgi KJ569866.1 and P. cynomolgi KJ569868.1). The MC004 assay involves two main steps, which take place in a single tube: (1) asymmetric target amplification by two different primer sets (primer pair 1: 5ʹ-TCGCTTCTAACGGTGAACT-3ʹ/5ʹ-AAGCAAACACTAGCGGTGGAA-3ʹ and primer pair 2: 5ʹ-CAGTATAATATTGTAATTTGATCAGTATGAG-3ʹ/5ʹ-GGATATTGTATAAATGATGCTATATCAGGTA-3ʹ). Primer pair 1 was designed to amplify a 116 base pair fragment of all 11 Plasmodium species/strains. Primer pair 2 was designed to amplify a 212 base pair fragment of P. vivax, P. knowlesi, and P. cynomolgi only. See Table 2 for the precise annealing positions of the primers. Amplification can be monitored real-time in the FAM detection channel due to the presence of a nonspecific green fluorescent nucleic acid dye that becomes fluorescent upon binding to double stranded DNA. (2) detection and differentiation of the amplicons by molecular beacon probe-based melting curve analysis. The assay uses three different probes, which are either Texas red-labelled (identical to the sequence of P. knowlesi ATCC 30153), Cy5-labelled (identical to the sequence of P. falciparum), or Cy5.5 labelled (identical to the sequence of P. knowlesi LT48).

Table 2

Annealing positions of the forward and reverse primers of primer pairs 1 and 2

Plasmodium species	GenBank accession number	Annealing positions of the forward and reverse primers of primer pair 1a	Annealing positions of the forward and reverse primers of primer pair 2a
P. falciparum	LR605957.1	Forward: 339–357 Reverse: 434–454	Not applicable
P. vivax	KY923424.1	Forward: 1493–1511 Reverse: 1588–1608	Forward: 1941–1971 Reverse: 2122–2152
P. malariae	AB489194.1	Forward: 1560–1578 Reverse: 1655–1675	Not applicable
P. ovale wallikeri	HQ712053.1	Forward: 1038–1056 Reverse: 1133–1153	Not applicable
P. ovale curtisi	HQ712052.1	Forward: 1038–1056 Reverse: 1133–1153	Not applicable
P. knowlesi	LR701176.1	Forward: 340–358 Reverse: 435–455	Forward: 781–811 Reverse: 962–992
P. cynomolgi	AB444125.1	Forward: 1605–1623 Reverse: 1700–1720	Forward: 2051–2081 Reverse: 2232–2262

See main text for the primer sequences. The expected amplicon sizes of primer pairs 1 and 2 are 116 and 212 base pairs, respectively

aAccording to the deposited sequences in GenBank under the shown accession numbers

Specimen processing

The preferred specimen type is DNA extracted from 200 µL of (EDTA) human whole blood. DNA was isolated using the QIAamp DNA Blood Mini QIAcube Kit (Qiagen, Hilden, Germany) on a QIAcube instrument (Qiagen, Hilden, Germany) following the manufacturer’s specifications. DNA was eluted with 100 µL of elution buffer. Giemsa-stained blood smears were processed by pipetting 10 µL of 10 mM sodium hydroxide (NaOH pellets, 99%, Merck, Darmstadt, Germany) onto the surface of the blood smear. Subsequently, blood smear material was scraped off from the glass slide by making circular movements with a sterile lancet (Solofix, B Braun, Oss, Netherlands). The collected specimen was transferred to a sterile 2.0 mL tube (Eppendorf, Hamburg, Germany) that contained 100 µL of 10 mM sodium hydroxide. Processed specimen samples were stored at − 30 °C.

Amplification and detection

Each reaction was carried out in a final volume of 25 µL reaction mixture containing 23 µL of MC004 master mix (MRC-Holland, Amsterdam, the Netherlands) and 2 µL of template. The components of the master mix were: both forward primers (1.25 µM), both reverse primers (0.25 µM), all three probes (0.5 µM), fluorescent compound, dNTPs, reaction buffer, and Taq DNA polymerase. The following settings were used for the amplification step: 95 °C for 3 min, followed by 50 cycles of 95 °C for 15 s, 60 °C for 30 s and 68 °C for 40 s. The following settings were used for the melting curve step: a gradual temperature increase from 25 to 69.4 °C (0.4 °C per 5 s).

Controls

Positive and negative control samples were used to validate run validity. Each run included two negative controls: (1) with elution buffer added as template and (2) a fresh uninfected whole blood specimen processed as a separate sample. Positive control samples for each Plasmodium species come with the MC004 assay as provided by MRC Holland. The 1st WHO International Standard for P. falciparum DNA (NIBSC code: 04/176) diluted in EDTA whole blood (before DNA isolation) to a concentration of 1 × 10–3 IU/mL was used as a positive control for limit of detection. Each sample should produce an amplification curve, if no amplification curve is observed, this may be a sign of inhibition and renders the result invalid.

Instrumentation

The assay was performed on the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Runs were analysed using the accompanying Bio-Rad CFX manager 3.1 software (Bio-Rad, Hercules, CA, USA). Cq-values were determined by the software’s Cq Determination Mode (settings: Single Threshold set at 2000; Baseline Setting: Baseline Subtracted Curve Fit).

Melting temperatures were determined using the software’s default settings. The throughput is approximately 80 specimens for 1 operator in about 3.5 h. Up to 12 specimens can be processed, including DNA isolation using the QIAcube, within four hours of arrival of the specimens at the laboratory.

Limit of detection study design

The limit of detection of the MC004 assay was determined by preparing a tenfold serial dilution of the WHO International Standard for P. falciparum DNA (NIBSC, Hertfordshire, England) in uninfected EDTA whole blood to achieve a concentration range of 1 × 109 IU/mL to 1 × 10–9 IU/mL. To clarify, this WHO standard was assigned an arbitrary unitage in International Units (IU), unrelated to the level of parasitaemia or DNA copy number [19]. The lyophilized WHO standard, reconstituted in 500 µL of sterile water, was diluted in EDTA whole blood. Total volume of each dilution was 400 µL. DNA was isolated as described above, and samples were tested in three independent runs. The limit of detection was determined as the lowest concentration of P. falciparum detected in all three runs. In addition, similar tenfold serial dilutions of patient samples with known parasitaemia were tested: P. falciparum 1.3%, P. falciparum 0.2%, P. vivax < 0.1%, P. malariae 0.3%, P. ovale curtisi < 0.1%, and P. knowlesi ATCC 30158 0.3%. Samples were tested in two independent runs. The limit of detection was determined as the lowest concentration of the Plasmodium species detected in both runs.

Detection of mixed infections study design

Mixed Plasmodium infections were created by adding together two DNA samples, each containing a different Plasmodium species (P. falciparum and a non-falciparum species) 1:1 (equal volume). When applicable, the parasitaemia was determined by experienced microscopists (P. falciparum 0.2%, P. vivax 0.1%, P. ovale wallikeri 0.2%, P. malariae 0.4%, P. knowlesi ATCC 30158 0.3%, P. ovale curtisi and P. cynomolgi KJ569868.1 isolated DNA diluted 100 times with sterile water). In addition, a two-fold serial dilution (1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128) of a P. falciparum DNA sample (initial parasitaemia was 1.3%) in sterile water was prepared to create mixed infections of P. falciparum and a non-falciparum species (non-falciparum samples as described above).

Quantification of parasitaemia study design

To quantify parasite density, a calibration curve was established by plotting the microscopically determined parasite densities of ten samples from patients with P. falciparum infections as a function of the Cq-value. The parasite densities were: 0.1%, 0.1%, 0.4%, 0.8%, 1.3%, 1.5%, 3.7%, 23.7%, 31.8%, and 35.8%. Cq-values were determined in three independent runs. The exponential equation (y = a*e(b*x)) of the line of best fit through the 30 data points was determined using Microsoft Excel for Mac 2016, where ‘y’ is the parasitaemia (percentage of infected erythrocytes), ‘x’ is the Cq-value (threshold cycle), ‘a’ and ‘b’ are the dimensionless coefficients that define the equation, and ‘e’ is the base of the natural logarithm.

Precision of the quantification of the parasitaemia study design

Within-run and between-run precisions [standard deviations (SD) and coefficients of variation (CV)] for the quantification of the parasitaemia were determined using four clinical samples infected with P. falciparum. The parasitaemia levels of these four samples were estimated by real-time PCR to be around 0.1%, 0.2%, 2% and 27%, respectively. Precision was assessed by testing five replicates per run over five runs. Each run the samples were tested in different wells of the PCR plate. Within-run and between-run precision were calculated as described by Chesher, 2008 [20].

Results

Melting temperatures and melting curves

Probe melting temperatures for each Plasmodium species/strain are shown in Table 3. The observed maximum deviation of melting points was 1.2 °C. Figure 1 shows representative melting curves of P. falciparum and P. vivax (see Additional file 1: Figure S1 for the melting curves of all other Plasmodium species/strains).

Table 3

Melting temperatures of the Texas Red, Cy5 and Cy5.5 Labelled probes for 11 Plasmodium species/strains

Plasmodium species	Melting temperature (°C)Texas Red labelled probe	Melting temperature (°C)Cy5 labelled probe	Melting temperature (°C)Cy5.5 labelled probe
P. falciparum	42.4	54.4	None
P. vivax	49.2	40.4	51.6
P. malariae	39.6	33.6	None
P. ovale wallikeri	49.6	48.4	None
P. ovale curtisi	53.2	48.4	None
P. knowlesi LT48	49.2	40.8	62.8
P. knowlesi ATCC 30158	56.4	40.8	62.8
P. knowlesi ATCC 30153	56.4	40.8	52.4
P. cynomolgi ATCC 30149	56.0	40.4	57.6
P. cynomolgi KJ569866.1	56.4	40.8	54.8
P. cynomolgi KJ569868.1	50.8	40.8	58.8

Differences in probe melting temperatures enable distinction between the 11 Plasmodium species/strains

Fig. 1

Representative melting curves of P. falciparum and P. vivax. The x-axis shows the temperature (°C). The y-axis shows the negative derivative of fluorescence (RFU) with respect to temperature (T). The Plasmodium species is indicated above each melting curve diagram. Red curves correspond to the Texas Red labelled probe, purple curves to the Cy5 labelled probe, and brown curves to the Cy5.5 labelled probe. For the sake of clarity, the melting temperature thresholds were manually set at zero

Analytical specificity

Testing of samples that did not contain any parasites and samples that contained Leishmania donovani infantum, Loa loa, Trypanosoma brucei rhodesiensis, or Babesia microti, yielded no false positive results. Among the samples that did contain Plasmodium parasites, one discrepancy was observed between the result of the reference institute [see Table 1: SKML, P. vivax (2018.1G)] and the MC004 assay (P. knowlesi). This sample was further analysed by Sanger sequencing, and this confirmed the presence of P. knowlesi. No data regarding the origin of this specimen were available at the reference institute, therefore leaving the question of what might explain this anomaly open-ended. The analytical specificity panel contained one P. cynomolgi sample, which showed melting temperatures consistent with P. cynomolgi KJ569868.1. Five of the six P. knowlesi samples showed melting temperatures consistent with P. knowlesi LT48, the other one with P. knowlesi ATCC 30158. The one included mixed infection (UK NEQAS 5844, P. falciparum + P. vivax) was readily identified. Thus, among the 40 samples that were included in the specificity panel, no false positive or false negative results were observed with the MC004 assay.

Limit of detection

Limit of detection results are presented in Table 4. The 1st WHO International Standard for P. falciparum DNA (NIBSC code: 04/176) was detectable until a dilution of 10–12, which corresponds to a concentration of 1 × 10–3 IU/mL. The undiluted concentration of this standard has been described to correspond to 9.79 parasites per 100 red blood cells [21]. However, arguably, expressing the limit of detection as number of parasites per volume of blood would be misrepresentative, as the limit of detection determined by dilution series appears to be dependent on the starting concentration. Illustrative of this point are the dilutions of P. falciparum with a relatively high parasitaemia of 1.3%, which was still detectable at dilution 10–14, and of P. falciparum with a relatively low parasitaemia of 0.2%, which was detectable until dilution 10–5. Limits of detection of P. vivax, P. knowlesi ATCC 30158, and P. ovale curtisi were determined at dilution 10–4, that of P. malariae at dilution 10–7. Other Plasmodium species/strains were not tested due to lack of sample.

Table 4

Limit of detection

Plasmodium species	Undiluted parasite concentration	Highest dilution that consistently detected the Plasmodium species
P. falciparum (NIBSC)	1 × 109 IU/mL	10–12 (1 × 10–3 IU/mL)
P. falciparum	1.3%	10–14 (the highest dilution that was tested)
P. falciparum	0.2%	10–5
P. vivax	< 0.1%	10–4
P. malariae	0.3%	10–7
P. ovale curtisi	< 0.1%	10–4
P. knowlesi ATCC 30158	0.3%	10–4

Detection of mixed infections

The melting curves of the created mixed Plasmodium infections at parasitaemia levels ranging from 0.1 to 0.4% (P. falciparum with either P. vivax, P. ovale wallikeri, P. ovale curtisi, P. knowlesi ATCC 30158, or P. cynomolgi KJ569868.1) are shown in Fig. 2. All tested mixed Plasmodium infections could be visually identified by the presence of more than one melting peak in the Texas Red and/or Cy5 channels, shifted melting temperatures, a shoulder added to the original melting peak, or the presence of a Cy5.5 melting peak.

Fig. 2

Melting curves of mixed Plasmodium infections (P. falciparum with non-P. falciparum). The x-axis shows the temperature (°C). The y-axis shows the negative derivative of fluorescence (RFU) with respect to temperature (T). The type of mixed infection is indicated above each melting curve diagram. Red curves correspond to the Texas Red labelled probe, purple curves to the Cy5 labelled probe, and brown curves to the Cy5.5 labelled probe. The arrows indicate the differences between a mixed infection and a mono-infection with P. falciparum. For the sake of clarity, the melting temperature thresholds were manually set at zero

The melting curves of the created mixed Plasmodium infections using dilution 1:128 of P. falciparum (initial parasitaemia level of 1.3%) with either P. vivax, P. ovale wallikeri, P. ovale curtisi, P. knowlesi ATCC 30158, or P. cynomolgi KJ569868.1 are shown in Additional file 2: Figure S2. Mixed infections with P. vivax, P. ovale wallikeri, and P. ovale curtisi could be visually identified. Mixed infections with P. malariae, P. knowlesi ATCC 30158, and P. cynomolgi KJ569868.1 could be readily identified up to dilution 1:64. In these mixed infections, P. falciparum could just be detected at dilution 1:128 by a small melting peak of the Cy5-labelled probe.

Quantification of parasitaemia (P. falciparum)

The 30 Cq-values used to construct the calibration curve are shown in Additional file 3: Table S1. The calibration curve itself is shown in Fig. 3. The exponential line of best fit has the equation: parasitaemia equals 57,012 * e(− 0.615*Cq-value). Figure 3 illustrates that variation in Cq-values in the lower range has a greater impact than variation in Cq-values in the higher range with regard to the calculated parasitaemia. For example, the difference between the calculated parasitaemia from a Cq-value of 15 (5.6%) and 16 (3.0%) is 2.6%, while the difference between the calculated parasitaemia from a Cq-value of 20 (0.26%) and 21 (0.14%) is 0.12%. The most commonly used cut-off in clinical practice is a parasitaemia level of 2%, which corresponds to a Cq-value of 16.68.

Fig. 3

The calibration curve for calculating parasitaemia from Cq-value. By microscopy estimated P. falciparum parasitaemia (y-axis) is plotted as a function of the Cq-value (x-axis). The black curve through the white data points (see also Additional file 3: Table S1) is the exponential line of best fit with the equation: parasitaemia equals 57,012 * e(− 0.615*Cq-value). The red part of the figure indicates parasitaemia > 2% (Cq-values less than 16.68); the green part of the figure indicates parasitaemia of < 2% (Cq-values greater than 16.68)

Precision of the quantification of the parasitaemia (P. falciparum)

The results of the within-run and between-run precisions are summarized in Table 5. The within-run and between-run precisions at level 1 (average parasitaemia of 27.27%) were 4.6% and 7.8% CV, respectively. The between-run precisions equaled the within-run precision at level 3, 4 and 5 (average parasitaemia of 2.15%, 0.16% and 0.09%, respectively). This indicates stability under changing conditions.

Table 5

Within-run and between-run precisions of P. falciparum parasitaemia determined using the MC004 assay

Level	Average P. falciparum parasitaemia per level (%)	Within-run precisionSD/CV	Between-run precisionSD/CV
1	27.27	1.257/4.6	2.133/7.8
2	2.15	0.363/16.9	0.363/16.9
3	0.16	0.022/13.6	0.022/13.6
4	0.09	0.012/14.4	0.012/14.4

Five replicates per run were tested over five runs at four levels of parasitaemia. Parasitaemia was calculated from the Cq-value using the equation of the calibration curve (Fig. 3)

SD standard deviation, CV coefficient of variation

Discussion

No cross-reactivity was observed in the MC004 assay between the evaluated Plasmodium and non-Plasmodium species. The MC004 assay has been designed to specifically amplify Plasmodium DNA, and includes post-PCR melting curve analysis based on three different molecular beacon probes to determine specificity of the amplicons. Therefore, the occurrence of false-positive results in case of non-Plasmodium infections is deemed to be a highly unlikely possibility. This presumption is supported by the fact that cross-reactivity issues between Plasmodium and non-Plasmodium species have not been raised as a concern in previously published studies. However, cross-reactivity between different Plasmodium species is a well-known issue. In particular, the nucleotide sequences of P. vivax and P. knowlesi share high similarity, preventing specific PCR amplification in some assays. The same holds true for P. ovale curtisi and P. ovale wallikeri. This study shows these issues of cross-reactivity have been resolved by the MC004 assay. Arguably, cross-reactivity is the most likely explanation for the one discrepant result found with the MC004 assay (See Table 1, P. vivax (SKML sample 2018.1G). The result of MC004 was P. knowlesi, which was confirmed by Sanger sequencing), but it may also have been a clerical error or an oversight. The differentiation of P. knowlesi ATCC 30158 and P. knowlesi LT48 was verified, but no sample was available that contained P. knowlesi ATCC 30153. The only available P. cynomolgi sample showed melting temperatures consistent with strain KJ569868.1. Each Plasmodium species could be straightforwardly identified using the melting temperatures shown in Table 3. The MC004 assay cannot differentiate between P. vivax and P. simium, as their mitochondrial genomes differ at two nucleotides positioned outside the regions covered by the probes. Also, the genomes of P. malariae and P. brasilianum are very similar, and will have identical melting temperatures [22]. In clinical practice, this would not affect patient management. To reduce the probability of human error and to increase the speed of reporting results, species identification could be performed and reported automatically by programming the laboratory information management system, or semi-automatically by programming software like Microsoft Excel to return the species after entering the melting points.

The MC004 assay was capable of detecting 1 × 10–3 IU/mL of the 1st WHO International Standard for P. falciparum DNA (NIBSC code: 04/176), which is equivalent to a dilution of 10–12. The dilution series of two P. falciparum-infected whole blood samples (parasitaemia of 0.2% and 1.3%) shows that the limit of detection determined this way depends on the starting concentration (Table 4). Therefore, the detection limit was not expressed as number of parasites per volume of blood, but rather as the highest dilution of an arbitrary, relatively low, undiluted parasitaemia at which the Plasmodium species was consistently detected. Since there is no WHO standard available for other Plasmodium species than P. falciparum, a dilution series of clinical samples was used to assess the limit of detection for other Plasmodium species. Both the undiluted parasitaemia, ranging from < 0.1% to 0.3%, and the highest dilutions at which Plasmodium DNA was still detected, ranging from 10–4 to 10–7, were roughly comparable between P. falciparum, P. vivax, P. malariae, P. ovale curtisi and P. knowlesi ATCC 30158. No clinical sample was available to assess the limit of detection of P. cynomolgi. These limits of detection are regarded as sufficient to warrant further testing in both clinical and research settings.

Infections with more than one Plasmodium species are relatively rare in clinical practice, but relevant. Due to lack of sample, there was no opportunity to test actual samples from patients, besides the UK NEQAS sample (P. falciparum + P. vivax, See Table 1). Thus, mixed infections were mimicked by mixing DNA-isolates of P. falciparum and either P. vivax, P. ovale wallikeri, P. ovale curtisi, P. knowlesi ATCC 30158, or P. cynomolgi KJ569868.1. To further demonstrate the proof of principle that the MC004 assay can detect mixed infections, the amount of DNA of each Plasmodium species was kept roughly comparable, which was in the range of 0.1 to 0.4% parasitaemia (Fig. 2). An additional experiment, in which P. falciparum (with initial parasitaemia of 1.3%) was diluted up to 1:128, indicated that mixed infections may be missed if differences in DNA concentration between Plasmodium species are large (Additional file 2: Figure S2). P. falciparum is the most commonly identified species in our geographic area, and poses the greatest threat globally. Therefore, the focus was on mixed infections with P. falciparum, although any combination of two or even three Plasmodium species is theoretically possible. To what extent mixed infections are still detectable in cases where the difference between the Plasmodium species in parasitaemia are larger, or in case of infections with three Plasmodium species, remains to be verified by further studies, preferably empirically in the field. Features of the MC004 assay that specifically help to distinguish mixed infections with P. falciparum are the relatively large differences in melting temperatures of the Texas red and Cy5 labelled probes between P. falciparum and the other Plasmodium species, as well as the absence of a Cy5.5 melting temperature, which is only present in case of P. vivax, P. knowlesi or P. cynomolgi infections (Table 3).

One of the medical decision points to identify patients with severe or complicated malaria, particularly P. falciparum malaria, is more than two percent of the red blood cells parasitized [23, 24]. This arguably conservative threshold has traditionally been estimated by microscopy and is used rather than the WHO criterion of a parasitaemia estimate of greater than 4% [25]. A means to use the MC004 assay to estimate the level of parasitaemia based on the correlation between Cq-value and amount of Plasmodium DNA was sought out. The resulting calibration curve is shown in Fig. 3, which is traceable to by microscopy estimated levels of parasitaemia. To spare clinical sample and for sake of standardization, the WHO International Standard for P. falciparum DNA could be used to reestablish or verify the calibration curve when appropriate by assigning parasitaemia values to Cq-values. The performance of the MC004 assay when put into practice to estimate the parasitaemia of both P. falciparum and non-falciparum species at various levels and at different developmental stages, is to be shown empirically.

The within-run and between-run precisions close to the most relevant medical decision point of 2% parasitaemia (2.15%) and in the lower range (0.16% and 0.09%) and high range (27.27%) of less than 20% CV indicates robustness of the MC004 assay. While consistent results were obtained between runs and instruments, only one technician performed the testing, and one reagent lot MC004 was used. Nevertheless, considering the generally large variation in parasitaemia estimates by microscopy, precision is expected to improve with the MC004 assay.

Summary of limitations

This PCR protocol has limitations. First, differentiation between P. vivax and P. simium and between P. malariae and P. brasilianum is not possible, as the respective melting curves would be identical. Second, the detection of minority parasite populations in mixed infections may be compromised, if the differences in DNA concentration between Plasmodium species are too large. A methodological limitation of this study was the lack of field-collected parasites of each Plasmodium species or strain the PCR was designed to detect and identify, and of field-collected mixed infections.

Conclusions

The MC004 assay is a novel single-tube real-time PCR assay developed for the purpose of simultaneously detecting all Plasmodium species known to infect humans, and discrimination between P. falciparum, P. vivax, P. malariae, P. ovale wallikeri, P. ovale curtisi, P. knowlesi (including differentiation of three strains) and P. cynomolgi (including differentiation of three strains). This study shows there is sufficient evidence of validity and reliability to warrant further testing of the MC004 assay to detect, identify, and quantify Plasmodium species causing malaria in humans in both clinical and research settings. A separate paper reports on the application of the MC004 assay in clinical practice (Bergen et al. pers. commun.).

Additional file 1: Figure S1. Representative melting curves of P. ovale curtisi, P. ovale wallikeri, P. malariae, P. knowlesi strains and P. cynomolgi strains. The x-axis shows the temperature (°C). The y-axis shows the negative derivative of fluorescence (RFU) with respect to temperature (T). Red curves correspond to the Texas Red labelled probe, purple curves to the Cy5 labelled probe, and brown curves to the Cy5.5 labelled probe. For the sake of clarity, the melting temperature thresholds were manually set around -1000 RFU. Note that only P. vivax, P. knowlesi and P. cynomolgi show melting peaks of Cy5.5 labelled probes.

Additional file 2: Figure S2. Melting curves of mixed Plasmodium infections: P. falciparum (diluted 1:128) with non-P. falciparum. The x-axis shows the temperature (°C). The y-axis shows the negative derivative of fluorescence (RFU) with respect to temperature (T). The type of mixed infection is indicated above each melting curve diagram. Red curves correspond to the Texas Red labelled probe, purple curves to the Cy5 labelled probe, and brown curves to the Cy5.5 labelled probe. The arrows indicate the melting peaks of P. falciparum. Melting peaks not indicated are of the non-P. falciparum species. For the sake of clarity, the melting temperature thresholds were manually set at zero.

Additional file 3: Table S3. The 30 Cq-values used to construct the calibration curve and the corresponding parasitaemia of P. falciparum as estimated by microscopy.