WO2016120396A1

WO2016120396A1 - Combined plasmodium-rna virus detection method

Info

Publication number: WO2016120396A1
Application number: PCT/EP2016/051824
Authority: WO
Inventors: Lieselotte CNOPS; Kevin ARIËN
Original assignee: INSTITUUT VOOR TROPISCHE GENEESKUNDE
Current assignee: INSTITUUT VOOR TROPISCHE GENEESKUNDE
Priority date: 2015-01-29
Filing date: 2016-01-28
Publication date: 2016-08-04
Anticipated expiration: 2017-07-29

Abstract

The present invention in general relates to a combined Plasmodium-RNA virus detection method. More in particular, the present invention relates to a combined detection method for Plasmodium in combination with Ebolaviruses and/or Arboviruses, such as for example Dengue Viruses, Chikungunya Viruses, or West Nile viruses. Furthermore, the present invention relates to kits comprising primers and/or probes for performing the combined Plasmodium-RNA virus detection method according to this invention.

Description

COMBINED PLASMODIUM-RNA VIRUS DETECTION METHOD

FIELD OF THE INVENTION The present invention in general relates to a combined Plasmodium-RNA virus detection method. More in particular, the present invention relates to a combined detection method for Plasmodium in combination with Ebolaviruses and/or Arboviruses, such as for example Dengue Viruses, Chikungunya Viruses, or West Nile viruses. Furthermore, the present invention relates to kits comprising primers and/or probes for performing the combined Plasmodium-RNA virus detection method according to this invention.

BACKGROUND TO THE INVENTION

Many infectious diseases are prevalent in the tropical and subtropical regions where ecological, geographical and socioeconomic factors facilitate their propagation. These tropical pathogens include a high diversity of species such as bacteria, fungi, parasites, viruses,.... However, many of these tropical pathogens provoke similar clinical symptoms, thereby making diagnosis by classical clinical observations very difficult. Nevertheless, early, rapid and accurate diagnosis is crucial in patient management and control of these tropical diseases, and in order to institute effective control measures: from timely therapeutic intervention to effective treatment. Correct diagnosis can mostly only be determined through reliable laboratory-confirmed detection and identification of tropical pathogens in clinical samples.

For example, malaria, caused by parasitic protozoans of the Plasmodium type causes symptoms that typically include fever, fatigue, vomiting and headaches, at least in the early stages of the disease. Similar symptoms are, however, found in patients infected with RNA viruses such as haemorrhagic fever viruses (e.g. Ebola virus) and Arboviruses (e.g. Dengue virus, Chikungunya virus, West Nile virus, ...), therefore these disorders can easily be mistaken from one another. However, each type of causing pathogen requires its own adapted containment measures and treatment regime and it is thus essential to distinguish Plasmodium infections from RNA virus infections in order to combat these pathogens.

Not only in terms of containment measures or treatment regime, but also in terms of structural characteristics, Plasmodium parasites and RNA viruses differ substantially, rendering it difficult to analyse both type of species using one and the same detection method. For example, Plasmodium parasites have a DNA genome, and can be detected using DNA-specific detection methods, while RNA viruses have an RNA genome, which can only be detected using RNA-specific detection methods. Therefore, combining the various assays (e.g. DNA vs RNA analysis) for parallel screening of tropical diseases is not a feasible approach given the high diversity of the protocols with many limitations associated with each pathogen. This is for example evident from several scientific publications relating to the combined detection of pathogens in the diagnosis of haemorrhagic fevers, being limited to the detection of a given type of pathogen (i.e. viruses), with an improvement in pathogen coverage, but not in species coverage (i.e. viruses, parasites, bacteria,... ) (Palacios et al., 2006, Drosten et al., 2002, Pang et al., 2014).

Furthermore, references aimed at improving species coverage, make use of separate detection reactions for each type of species (Tan et al., 2014, Venter et al., 2014, Waggoner et al., 2014, commercial FastTrack assay) and/or make use of microarray assays or Lab-On-Chip approaches (Palacios et al., 2007, Venter et al., 2014). For example, Palacios et al., 2007 provides a microarray assay for the simultaneous detection of viruses, bacteria, parasites, and fungi. Also Venter et al., 2014 uses a microarray chip assay with hybridization. Tan et al., 2014 provides a Lab-On-Chip approach for allowing the rapid identification of up to 26 tropical pathogens (including parasites, bacteria and viruses). Such microarrays and chips still require specialized detection equipment and expertise for data interpretation and have not found their place yet as diagnostic tool in clinical practice. On the other hand, improvement of species coverage has also been reported using separate detection reactions for each type of species (i.e. viruses, parasites, bacteria, ...). For example in Tan et al., 2014 pathogen detection was split in 2 chip versions to be subjected to 2 different multiplex reactions; i.e. a DNA chip and an RNA chip. Waggoner et al., 2014 uses 2 different extraction and detection methods, i.e. an RNA extraction/detection method for Dengue virus (RNA virus) and a DNA extraction/detection method for Plasmodium detection (DNA species). In the commercial FastTrack detection kit, simultaneous detection of RNA and DNA species is achieved using whole nucleic acid extracts. However, the detection limit for Plasmodium species is rather high in the FastTrack kit (i.e. +/- 100 copies/ml based on plasmid DNA) and the sensitivity even further decreased (2-log difference) in the multiplex detection using clinical samples (compared to the single-plex detection.

It was thus an object of the present invention to provide a diagnostic assay capable of distinguishing Plasmodium infections from RNA virus infections starting from a single sample, which can be performed using equipment available in diagnostic laboratories, such as for example PCR or real-time PCR devices, and which has a good sensitivity for Plasmodium species. As a solution to the problems mentioned above, the inventors have now developed a combined Plasmodium-RNA virus PCR based detection method including a single RNA extraction step, a reverse transcription step, and a multiplex PCR assay. In addition, the inventors have surprisingly found that the combined Plasmodium-RNA virus detection method according to this invention, has a significantly improved detection (4-10 Ct lower, which correlates to an improvement of 1 - to 3-logs) for Plasmodium species compared to a single- plex Plasmodium detection assay, and is thus much more sensitive in terms of Plasmodium detection, as further detailed in the examples. This finding was very surprising, since Plasmodium species have a DNA genome, and it was thus not expected that by applying an RNA extraction method, an improved detection could be established. In contrast, a person skilled in the art, would have expected that further to the teaching of the publications mentioned above, a DNA extract or a whole nucleic acid extract, rather than an RNA extract could improve the detection of the Plasmodium species (having a DNA genome). Furthermore, the combined detection method of the present invention also allows the simultaneous detection of Plasmodium and filoviruses (e.g. Ebolavirus), which has up till now not been achieved.

SUMMARY OF THE INVENTION In a first aspect, the present invention provides a combined Plasmodium-RNA virus detection method comprising the steps of:

(a) providing a sample, suspected of containing at least one Plasmodium species and/or at least one RNA virus;

(b) performing an RNA extraction method on said sample of step (a);

(c) performing a reverse transcription step on said extract of step (b);

(d) performing a multiplex PCR-based assay on the sample obtained in step (c), using one or more Plasmodium specific primers and one or more RNA virus specific primers; and

(e) analysing the presence of said Plasmodium species and/or RNA virus in said sample, based on the outcome of said multiplex PCR-based assay of step (d).

In a particular embodiment, said multiplex PCR-based assay of step (d) is a real-time PCR assay further comprising one or more Plasmodium specific probes and one or more RNA virus specific probes.

In yet a further embodiment, said Plasmodium species is selected from the list comprising: Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malahae, and Plasmodium knowlesi. In another particular embodiment, said at least one RNA virus is selected from the list comprising: Filoviruses (e.g. Ebolaviruses) and one or more Arboviruses; and wherein said one or more RNA virus specific primers are selected from the list comprising one or more Filovirus-specific primers (e.g. Ebolavirus specific primers) and one or more Arbovirus specific primers. ln yet a further embodiment, said one or more Arboviruses are selected from the list comprising: Flaviviruses (e.g. Dengue viruses, or West Nile viruses) or Alphaviruses (e.g. Chikungunya viruses); and wherein said one or more Arbovirus specific primers are selected from the list comprising one or more Flavivirus specific primers (e.g. Dengue virus specific primers, or West Nile virus specific primers) and one or more Alphavirus specific primers (e.g. Chikungunya virus specific primers).

The present invention thus in particular provides a combined Plasmodium-RN/\ virus detection method comprising the steps of:

(a) providing a sample, suspected of containing at least one Plasmodium species and/or at least one RNA virus, selected from the list comprising Dengue virus, Chikungunya virus and West Nile virus;

(b) performing an RNA extraction method on said sample of step (a);

(c) performing a reverse transcription step on said extract of step (b);

(d) performing a multiplex PCR-based assay on the sample obtained in step (c) using

- one or more Plasmodium specific primers,

- one or more Dengue virus specific primers,

- one or more Chikungunya virus specific primers, and

- one or more West Nile virus specific primers; and

(e) analysing the presence of said Plasmodium species and/or RNA viruses in said blood sample, based on the outcome of said multiplex PCR-based assay of step (d).

More in particular, the present invention provides a combined Plasmodium-RN/\ virus detection method comprising the steps of:

(a) providing a sample, suspected of containing at least one Plasmodium species and/or at least one RNA virus, selected from the list comprising Ebolavirus, Dengue virus,

Chikungunya virus and West Nile virus;

(b) performing an RNA extraction method on said sample of step (a);

(c) performing a reverse transcription step on said extract of step (b);

- one or more Plasmodium specific primers,

- one or more Ebolavirus specific primers,

- one or more Dengue virus specific primers,

- one or more Chikungunya virus specific primers, and

- one or more West Nile virus specific primers; and

(e) analysing the presence of said Plasmodium species and/or RNA virus in said blood sample, based on the outcome of said multiplex PCR-based assay of step (d). ln a further aspect the present invention provides a kit for performing a combined Plasmodium- RNA virus detection method according to claim 1 ; said kit comprising:

- one or more Plasmodium specific primers, and

- one or more RNA virus specific primers selected from the list comprising one or more Ebolavirus specific primers and one or more Arbovirus specific primers.

In a particular embodiment, said kit according to the present invention may further comprise:

- one or more Plasmodium specific probes, and

- one or more RNA virus specific probes selected from the list comprising Ebolavirus specific probes and/or one or more Arbovirus specific probes.

In a particular embodiment, said one or more Arbovirus specific primers are selected from the list comprising:

- one or more Dengue virus specific primers,

- one or more Chikungunya virus specific primers, and

- one or more West Nile virus specific primers.

In yet a further particular embodiment, said one or more Arbovirus specific probes are selected from the list comprising:

- one or more Dengue virus specific probes,

- one or more Chikungunya virus specific probes, and

- one or more West Nile virus specific probes.

In the methods and kits of the present invention, said one or more Plasmodium specific primers are preferably selected from the list comprising SEQ ID N°1 -4. Furthermore, said one or more RNA virus specific primers are preferably selected from the list comprising SEQ ID N° 7-8, SEQ ID N° 1 1 -12, SEQ ID N° 14-17, SEQ ID N° 19-20 and SEQ ID N° 22-23. Furthermore, said one or more Plasmodium specific probes are preferably selected from the list comprising SEQ ID N° 5-6. Finally, said one or more RNA virus specific probes are preferably selected from the list comprising SEQ ID N° 9-10, SEQ ID N° 13, SEQ ID N° 18, SEQ ID N° 21 and SEQ ID N° 24. DETAILED DESCRIPTION OF THE INVENTION

As already indicated herein before, the inventors have developed a combined Plasmodium- RNA virus PCR-based detection method including an RNA extraction step, a reverse transcription step, and a multiplex PCR-based assay. Said combined Plasmodium-RNA virus detection method was found to have a significantly lower detection limit for Plasmodium species compared to a single-plex Plasmodium detection assay, and is thus much more sensitive in terms of Plasmodium detection. Hence, in a first aspect, the present invention provides a combined Plasmodium-RNA virus detection method comprising the steps of:

(b) performing an RNA extraction method on said sample of step (a);

(c) performing a reverse transcription step on said extract of step (b);

In the context of the present invention, an RNA extraction method is to be considered as a method aimed at specifically enriching the RNA fraction of a sample (total RNA extraction), and hence, does not encompass methods aimed at enriching the DNA fraction specifically or whole nucleic acid extraction methods. It is generally known that any RNA extraction method will co-purify DNA to some extend (usually around 1 -10%). Such DNA 'contamination' is usually removed by DNAse treatment, however, since the method of the present invention, includes the detection of DNA species i.e. Plasmodium, the RNA extraction method of the present invention does not include a further DNAse treatment. A suitable method for RNA extraction in the context of the present invention is for example the use of RNA-selective binding silica membranes to bind RNA using fast-spin columns or vacuum assisted procedures, followed by water/buffer-assisted elution from the membranes, such as for example in the QIAamp Viral RNA kit. More specifically, the sample is first lysed under highly denaturing conditions to inactivate RNases and to ensure isolation of intact (viral) RNA. Buffering conditions are then adjusted to provide optimum binding of the RNA to the silica membrane, and the sample is loaded onto the silica membrane-containing spin column. The RNA binds to the membrane, and contaminants are efficiently washed away. High-quality RNA is eluted in a special RNase-free buffer or in RNase-free water. The purified RNA is free of protein, nucleases, and other contaminants and inhibitors, but includes low amounts of co- purified DNA. Other suitable RNA extraction technologies are organic extraction methods with a phenol-containing solution or magnetic particle methods using particles or beads. RNA extraction methods can be performed manually or can be partially or completely automated and even be integrated into the RT-PCR system.

While the method of the present invention is preferably performed on an RNA extract, to improve sensitivity of the Plasmodium species detection, the method could alternatively be performed on a whole nucleic acid extract as it encompasses a RNA extraction and DNA extraction.

The method according to the invention is a 'combined' detection method, i.e. capable of simultaneously detecting Plasmodium and RNA virus templates in a sample. This means that the method has the potential of detecting more than one target template in a sample at the same time. If the sample contains only one target template (e.g. only Plasmodium), then the method of course detects that one template only, the primers and probes specific for other templates are then not used. The phrase "simultaneously detecting" as used herein indicates that a plurality, i.e. more than 1 , different targets may be detected in one and the same analysis. For that purpose, the method according to the invention provides a different primer pair for each nucleic acid template to be amplified.

As used herein, the term "primer" refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of nucleic acid sequence synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, i.e. in the presence of different nucleotide triphosphates and a polymerase in an appropriate buffer ("buffer" includes pH, ionic strength, cofactors etc.) and at a suitable temperature. One or more of the nucleotides of the primer can be modified for instance by addition of a methyl group, a biotin or digoxigenin moiety, a fluorescent tag or by using radioactive nucleotides. In the context of the present invention, the term 'sample' is meant to include a variety of sample types obtained from an organism. The term encompasses bodily fluids such as blood, blood components, saliva, serum, plasma, urine and other liquid samples of biological origin, solid tissue biopsy, tissue cultures, or supernatant taken from cultured patient cells. In the context of the present disclosure, the biological sample is typically a bodily fluid such as blood, serum, plasma, CSF, urine, .... The biological sample can be processed prior to assay, e.g., to remove cells or cellular debris. The term encompasses samples that have been manipulated after their procurement, such as by treatment with reagents, solubilization, sedimentation, or enrichment for certain components. Although, identification of the species may be accomplished by amplification of a target nucleic acid sequence and detection of the resulting DNA by visualisation using gel electrophoresis and DNA-binding fluorescent dyes, preferably, the target is detected using real-time analysis methods. Therefore, in a particular embodiment, said multiplex PCR-based assay of step (d) is a real-time PCR assay further comprising one or more Plasmodium specific probes and one or more RNA virus specific probes.

The method and kits of the present invention are suitable for detecting any type of Plasmodium infection, in as far as specific primers and/or probes suitable for detecting and/or distinguishing said specific Plasmodium species are used. In a preferred embodiment, said Plasmodium species is selected from the list comprising: Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malahae, and Plasmodium knowlesi.

The method and kits of the present invention are suitable for detecting any type of RNA virus infection, in as far as specific primers and/or probes suitable for detecting and/or distinguishing said specific RNA viruses are used. In a preferred embodiment, said RNA virus is selected from the list comprising: Ebolaviruses and one or more Arboviruses; and said one or more RNA virus specific primers are thus preferably selected from the list comprising one or more Ebolavirus specific primers and one or more Arbovirus specific primers. Even more in particular, said one or more Arboviruses are selected from the list comprising: Dengue viruses, Chikungunya viruses and West Nile viruses; and said one or more Arbovirus specific primers are thus preferably selected from the list comprising one or more Dengue virus specific primers, one or more Chikungunya virus specific primers and one or more West Nile virus specific primers.

A particularly interesting combination assay relates to the simultaneous detection of Plasmodium species, Dengue viruses, Chikungunya viruses and West Nile viruses. Hence, in a particular embodiment, the present invention provides a combined Plasmodium-RNA virus detection method comprising the steps of:

(b) performing an RNA extraction method on said sample of step (a);

(c) performing a reverse transcription step on said extract of step (b);

- one or more Plasmodium specific primers,

- one or more Dengue virus specific primers,

- one or more Chikungunya virus specific primers, and

- one or more West Nile virus specific primers; and (e) analysing the presence of said Plasmodium species and/or RNA viruses in said blood sample, based on the outcome of said multiplex PCR-based assay of step (d).

Another particularly interesting combination assay relates to the simultaneous detection of Plasmodium species, Ebolavirus, Dengue viruses, Chikungunya viruses and West Nile viruses. Hence, in a particular embodiment, the present invention provides a combined Plasmodium-RNA virus detection method comprising the steps of:

(a) providing a sample, suspected of containing at least one Plasmodium species and/or at least one RNA virus, selected from the list comprising Ebolavirus, Dengue virus, Chikungunya virus and West Nile virus;

(b) performing an RNA extraction method on said sample of step (a);

(c) performing a reverse transcription step on said extract of step (b);

- one or more Plasmodium specific primers,

- one or more Ebolavirus specific primers,

- one or more Dengue virus specific primers,

- one or more Chikungunya virus specific primers, and

- one or more West Nile virus specific primers; and

(e) analysing the presence of said Plasmodium species and/or RNA virus in said blood sample, based on the outcome of said multiplex PCR-based assay of step (d).

In a further aspect the present invention provides a kit for performing a combined Plasmodium- RNA virus detection method according to claim 1 ; said kit comprising:

- one or more Plasmodium specific primers, and

- one or more RNA virus specific primers selected from the list comprising one or more

Ebolavirus specific primers and one or more Arbovirus specific primers.

Said kit may further comprise instructions for use of said kit.

- one or more Plasmodium specific probes, and

- one or more Dengue virus specific primers,

- one or more Chikungunya virus specific primers, and

- one or more West Nile virus specific primers. ln yet a further particular embodiment, said one or more Arbovirus specific probes are selected from the list comprising:

- one or more Dengue virus specific probes,

- one or more Chikungunya virus specific probes, and

- one or more West Nile virus specific probes.

Although any specific primers and probes are suitable for use in the methods and kits of the present invention, said one or more Plasmodium specific primers are preferably selected from the list comprising SEQ ID N°1 -4. Furthermore, said one or more RNA virus specific primers are preferably selected from the list comprising SEQ ID N° 7-8, SEQ ID N° 1 1 -12, SEQ ID N° 14-17, SEQ ID N° 19-20 and SEQ ID N° 22-23. Furthermore, said one or more Plasmodium specific probes are preferably selected from the list comprising SEQ ID N° 5-6. Finally, said one or more RNA virus specific probes are preferably selected from the list comprising SEQ ID N° 9-10, SEQ ID N° 13, SEQ ID N° 18, SEQ ID N° 21 and SEQ ID N° 24.

EXAMPLES

The Plasmodium-RNA virus combination RT-PCR test according to this invention aims to detect Plasmodium in combination with haemorrhagic fever viruses (e.g. Ebola virus) and/or arboviruses (dengue virus, Chikungunya virus, West Nile virus,... ) in one real-time reverse- transcriptase polymerase chain reaction (RT-PCR). To our knowledge, a diagnostic molecular test that combines the detection of Ebola virus and/or arboviruses with Plasmodium is not yet described. Such a new test will be of clinical relevance in the context of diagnosis of tropical fevers. The test is innovative because it combines the detection of genomic DNA from a blood parasite (Plasmodium) with the detection of viral RNA (Ebola virus, dengue virus, Chikungunya virus, ...) when using an RNA extraction method.

Materials & Methods

Sample

Clinical samples from febrile patients with an arboviral infection (serum) or a Plasmodium falciparum infection (EDTA-coagulated whole blood) were used. No clinical samples were available from Ebola virus infected patients, hence synthetic gene fragment constructs (gBlock, IDT) and Ebola virus Zaire (EBOV) inactivated reference material were used. RNA extraction

Total RNA from 140 μΙ of sample (serum, blood, inactivated reference material) was extracted by QIAamp viral RNA mini kit (Qiagen Benelux, Venlo, The Netherlands) and eluted into 80 μΙ buffer according to the manufacturer's instructions. DNA extraction

DNA was extracted from 200 μΙ blood with Qiagen DNA mini kit (Qiagen Benelux, Venlo, The Netherlands) according to manufacturer's guidelines. DNA was eluted with 100 μΙ elution buffer. Total nucleic acid extraction

Extraction of total nucleic acids from serum was done on the MagNa Pure LC (Roche) with the MagNA Pure LC Total Nucleic Acid (NA) Isolation-High Performance kit (Roche).

Single-plex real-time PCR

Plasmodium falciparum

The real-time PCR was performed as described by Cnops et al., 2010. In this study, the P. falciparumlP. vivax duplex reaction (Pf/Pv) was run in a 25 μΙ reaction using 5 μΙ of DNA with 1 x Perfecta qPCR Supermix (Quanta Biosciences). PCR was run for 2 min at 95 °C followed by 50 cycles of 15 sec at 95 °C and 60 sec at 60 °C on the SmartCycler II (Cepheid Benelux, Bouwel, Belgium). See table 1 , for the sequence of the primers and probes used in this assay.

Single-plex real-time RT-PCR

One-step RT-PCRs were performed on the SmartCycler II (Cepheid Benelux) in a 25 μΙ reaction volume using 5 μΙ RNA and iTaq universal Probes one step kit (Biorad Laboratories, Temse, Belgium). The RT-PCR program consisted of a RT-reaction of 10 min at 50 °C and a denaturation step of 5 min at 95 °C, followed by 50 cycles of 10 sec at 95 °C and 30 sec at 60 °C. See table 1 , for the sequence of the primers and probes used in this assay.

Ebola Virus (EBOV)

The sequences of the primers and EBOV and SUDV probes were used as described by Gibb, et al., 2001 . See table 1 , for the sequence of the primers and probes used in this assay.

Dengue virus 1 (DENV1)

The sequences of the DENV1 primers and probes were used as described by Johnson et al., 2005. See table 1 , for the sequence of the primers and probes used in this assay.

Chikungunya virus (CHIKV)

Two different sets of primers and one probe according to the protocol of Panning et al. 2008 were used to detect the general and Ocean islands epidemic CHIKV strain in a duplex RT-PCR. See table 1 , for the sequence of the primers and probes used in this assay.

West Nile virus (WNV)

Two different sets of primers and probes, one according to the protocol of Linke et al. 2007 (linWNV) and one with adapted sequences (lotWNV), were used to detect WNV in a duplex RT-PCR. See table 1 , for the sequence of the primers and probes used in this assay. Table 1 : Sequence of primers and probes used in the single-plex and combined Plasmodium- RNA virus detection method of the invention.

Name 5'-3' sequence SEQ ID N°

FW: falc CTAG G TG TTG G ATG AAAG TG TT AA 1

FW: viv GACTAGGCTTTG G ATG AAAG ATTTT A 2

FW: PAN-Plasmo GTTAAGGGAGTGAAGACGATCAGA 3

REV: PAN-Plasmo AACCCAAAGACTTTGATTTCTCATAA 4

PR: Falc (FAM) AGTCATCTTTCG AG GTG ACTTTTAG ATTG CT 5

PR: Viv-MGB (VIC) ATAAACTCCGAAGAGAAAATTC 6

FW: EBOGP1 D TGGGCTGAAAAYTGCTACAATC 7

REV: EBOGP1 D CTTTGTGMACATASCGGCAC 8

PR: EBOGP1 DZ CTACCAGCAGCGCCAGACGG

9

(FAM)

PR: EBOGP1 DS TTACCCCCACCGCCGGATG

10

(HEX)

FW: DEN-1 F CAAAAGGAAGTCGTGCAATA 1 1

REV: DEN-1 C CTGAGTGAATTCTCTCTACTGAACC 12

PR: DEN-1 P (FAM) CATGTGGTTGGGAGCACGC 13

FW: ChikSII CCGACTCAACCATCCTGGAT 14

REV: ChikAsll GCAGACGCAGTGGTACTTCCT 15

FW: ChikSI TGATCCCGACTCAACCATCCT 16

REV: ChikAsl GGCAAACGCAGTGGTACTTCCT 17

PR: ChikP (FAM) TCCGACATCATCCTCCTTGCTGGC 18

FW: lotWNV GCTGACAAACTTAGTAGTGTT 19

REV: lotWNV GCGTTTTAGCATATTGAC 20

PR: lotWNV GCC CTC CTG GTT TCT TAG ACA T 21

FW: linWNV CCTGTGTGAGCTGACAAACTTAGT 22

RV: linWNV GCGTTTTAGCATATTGACAGCC 23

PR: linWNV CCTGGTTTCTTAGACATCGAGATCTCGTG 24 EXAMPLE 1: RT-PCR for the simultaneous detection of Plasmodium and a RNA virus

An equal volume of the single PCR reaction mixture of Plasmodium with the single RT-PCR reaction mixture of respectively EBOV, DENV1 , or CHIKV was mixed and used for real-time RT-PCR analysis with 5 μΙ_ of Plasmodium DNA-extract, Plasmodium RNA-extract, and EBOV, DENV1 , and CHIKV RNA extracts. The PCR was run on the Smart Cycler II, with the same thermocycling conditions as in the single RT-PCRs.

Results

Ebola virus DNA (gBlock, IDT) and P. falciparum and P. vivax DNA extracted from whole blood were successfully detected in the Ebola-Plasmodium combination RT-PCR (Table 2). Furthermore, both P. falciparum and P. vivax detection was significantly improved in terms of sensitivity (lower Ct value) in the combined detection assay versus the single-plex detection assay of both species. Table 2: combined detection of EBOV RNA and Plasmodium DNA

Plasmodium DNA and RNA extracted from a P. falciparum positive whole blood sample and EBOV RNA spiked in whole blood before RNA extraction, were analyzed by the single PCR for Plasmodium and Ebola, respectively. Again both P. falciparum and P. vivax detection (in the mo no- infected samples) was significantly improved in terms of sensitivity (> 10 Ct lower, ~ 3- log difference, underlined in Table 3) in the combined detection assay versus the single-plex detection assay of both species. Furthermore, analysis of a sample comprising DNA of P. vivax and RNA of EBOV in the combination Plasmodium/Ebola virus RT-PCR assay, revealed that both species were successfully detected (grey highlighting in Table 3), indicating that the assay according to this invention is suitable for the combined detection of Plasmodium and RNA viruses.

Table 3: combined detection of EBOV RNA and Plasmodium DNA and RNA

Plasmodium DNA and RNA extracted from a P. falciparum positive whole blood sample and CHIKV RNA or DENV1 RNA extracted from serum, were successfully detected by the Plasmodium-C KV or Plasmodium-DENV\ combination RT-PCR (Table 4 and Table 5). Again, P. falciparum detection (in the mono-infected samples) was significantly improved in terms of sensitivity (> 10 Ct lower, ~ 3-log difference, underlined in Table 4 and Table 5)) in the combined P/asmod/^'i/m/Chikungunya virus and PlasmodiumlDengue Virus detection assays versus the single-plex detection assays. Table 4: combined detection of CHIKV RNA and Plasmodium DNA and RNA

Table 5: combined detection of DENV RNA and Plasmodium DNA and RNA

Also, combined detection of different RNA viruses (filoviruses and arboviruses) was successful. RNA extracted from serum samples positive for CHIKV or DENV1 and EBOV (synthetic DNA) were detected by the Ebola-arbovirus combination RT-PCR (Table 6, Table 7).

Table 6: combined detection of CHIKV RNA and Ebola synthetic DNA

Table 7: combined detection of DENV RNA and Ebola synthetic DNA

In all tested combinations, the negative water control was always negative indicating that there was no non-specific reaction between the primers and probes of the combined pathogens. Conclusion

Plasmodium positive clinical blood samples can be detected in combination with RNA viruses such as ebolaviruses and arboviruses in a combination RT-PCR test after the use of a single RNA extraction method. This test enables a rapid differential diagnosis between Plasmodium species and RNA viruses in a single combination test and will be useful for the clinical management of tropical fevers.

EXAMPLE 2: RT-PCR for the simultaneous detection of Plasmodium and a RNA virus

Results

Combined detection of Plasmodium with a filovirus species (EBOV) and a flavivirus species (DENV) (Table 8) or of Plasmodium with a filovirus species (EBOV), a flavivirus species (DENV), and an alphavirus species (CHIKV) (Table 9) was successful.

In addition, the multiplex RT-PCR was successfully performed on RNA, DNA or total nucleic acids (NA) extracts, and can thus be performed on a single extract. RNA extraction gave the best results (> 4 Ct lower, at least 1 -log difference, underlined in Table 9). Table 8: combined detection of Plasmodium total NA and DNA, EBOV RNA and synthetic DNA, and DENV total NA and RNA

Combination RT-PCR P/asmod/^'um-Ebola-DENV

Ct ct ct ct ct ct Ct Ct

EBOV SUDV Pf Pv DENV1 DENV2 DENV3 DENV4

Water control 0 0 0 0 0 0 0 0

EBOV (synthetic) DNA 28,31 0 0 0 0 0 0 0

EBOV (spiked serum) Total NA 18,84 0 0 0 0 0 0 0

Pf-positive serum Total NA 0 0 30,9 0 0 0 0 0

Pf/Pv PC DNA 0 0 31 ,6 35,7 0 0 0 0

DENV2-positive serum Total NA 0 0 0 0 0 31 ,63 0 0

DEN2+DEN3 PC RNA 0 0 0 0 0 22,46 26,3 0

Table 9: combined detection of Plasmodium total NA and DNA, EBOV RNA and synthetic DNA, and DENV and CHIKV total NA and RNA.

Table 9-Continued:

*below threshold

Finally, analytical sensitivity tests on serial dilutions of a P. falciparum-pos l\ve sample demonstrated that in the combined multiplex detection method (P/asmod/i/m-Ebola-Dengue) a better sensitivity was seen when RNA extracts were tested, than when DNA extracts of the same sample where used. The limit of detection in the combined multiplex method was 0.6 parasites/ L for RNA extracts and 6 parasites/ L for DNA extracts.

REFERENCES

Drosten et al., 2002. Rapid Detection and Quantification of RNA of Ebola and Marburg Viruses, Lassa Virus, Crimean-Congo Hemorrhagic Fever Virus, Rift Valley Fever Virus, Dengue Virus and Yellow Fever Virus by Real-Time Reverse Transcription-PCR. Journal of Clinical Microbiology. Vol. 40, N°7, pg. 2323-2330.

Palacios et al., 2006. MassTag Polymerase Chain Reaction for Differential Diagnosis of Viral Hemorrhagic Fevers. Emerging Infectious Diseases. Vol. 12, N°4, pg. 692 - 695.

Pang et al., 2014. Comprehensive Multiplex One-Step Real-Time TaqMan qRT-PCR Assays for Detection and Quantification of Hemorrhagic Fever Viruses. PLOS One, Vol. 9 Issue 4, e95635. Cnops L, Jacobs J, Van Esbroeck M. Validation of a four-primer real-time PCR as a diagnostic tool for single and mixed Plasmodium infections. Clin Microbiol Infect. 2011 Jul;17(7):1 101 -7.

Gibb TR, Norwood DA Jr, Woollen N, Henchal EA. Development and evaluation of a fluorogenic 5' nuclease assay to detect and differentiate between Ebola virus subtypes Zaire and Sudan. J Clin Microbiol. 2001 Nov;39(1 1 ):4125-30.

Johnson BW, Russell BJ, Lanciotti RS. Serotype-specific detection of dengue viruses in a fourplex real-time reverse transcriptase PCR assay. J Clin Microbiol. 2005 Oct;43(10):4977- 83.

Panning M, Grywna K, van Esbroeck M, Emmerich P, Drosten C. Chikungunya fever in travelers returning to Europe from the Indian Ocean region, 2006. Emerg Infect Dis. 2008 Mar;14(3):416-22. Linke S, Ellerbrok H, Niedrig M, Nitsche A, Pauli G. Detection of West Nile virus lineages 1 and 2 by real-time PCR. J Virol Methods. 2007 Dec;146(1 -2):355-8.

Claims

1 . A combined Plasmodium-RNA virus detection method comprising the steps of:

(b) performing an RNA extraction method on said sample of step (a);

(c) performing a reverse transcription step on said extract of step (b);

2. A method according to claim 1 , wherein said multiplex PCR-based assay of step (d) is a real-time PCR assay further comprising one or more Plasmodium specific probes and one or more RNA virus specific probes.

3. A method according to anyone of claims 1 -2, wherein said Plasmodium species is selected from the list comprising: Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malahae, and Plasmodium knowlesi.

4. A method according to anyone of claims 1 -3, wherein said at least one RNA virus is selected from the list comprising: Ebolaviruses and one or more Arboviruses; and wherein said one or more RNA virus specific primers are selected from the list comprising one or more Ebolavirus specific primers and one or more Arbovirus specific primers.

5. A method according to claim 4, wherein said one or more Arboviruses are selected from the list comprising: Dengue viruses, Chikungunya viruses and West Nile viruses; and wherein said one or more Arbovirus specific primers are selected from the list comprising one or more Dengue virus specific primers, one or more Chikungunya virus specific primers and one or more West Nile virus specific primers.

6. A combined Plasmodium-RNA virus detection method as defined in claim 1 comprising the steps of:

(b) performing an RNA extraction method on said sample of step (a);

(c) performing a reverse transcription step on said extract of step (b); (d) performing a multiplex PCR-based assay on the sample obtained in step (c) using

- one or more Plasmodium specific primers,

- one or more Dengue virus specific primers,

- one or more Chikungunya virus specific primers, and

- one or more West Nile virus specific primers; and

7. A combined Plasmodium-RNA virus detection method as defined in claim 1 comprising the steps of:

(b) performing an RNA extraction method on said sample of step (a);

(c) performing a reverse transcription step on said extract of step (b);

- one or more Plasmodium specific primers,

- one or more Ebolavirus specific primers,

- one or more Dengue virus specific primers,

- one or more Chikungunya virus specific primers, and

- one or more West Nile virus specific primers; and

8. A kit for performing a combined Plasmodium-RNA virus detection method according to claim 1 ; said kit comprising:

- one or more Plasmodium specific primers, and

9. The kit according to claim 8, further comprising:

- one or more Plasmodium specific probes, and

10. The kit according to claim 8; wherein said one or more Arbovirus specific primers are selected from the list comprising:

- one or more Dengue virus specific primers,

- one or more Chikungunya virus specific primers, and

- one or more West Nile virus specific primers.

1 1 . The kit according to claim 9; wherein said one or more Arbovirus specific probes are selected from the list comprising:

- one or more Dengue virus specific probes,

- one or more Chikungunya virus specific probes, and

- one or more West Nile virus specific probes.

12. The method according to anyone of claims 1 -7 or the kit according to anyone of claims 8- 1 1 , wherein said one or more Plasmodium specific primers are selected from the list comprising SEQ ID Ν -4.

13. The method according to anyone of claims 1 -7 or the kit according to anyone of claims 8- 1 1 , wherein said one or more RNA virus specific primers are selected from the list comprising SEQ ID N° 7-8, SEQ ID N° 1 1 -12, SEQ ID N° 14-17, SEQ ID N° 19-20 and SEQ ID N° 22-23.

14. The method according to claim 2 or the kit according to anyone of claim 9, wherein said one or more Plasmodium specific probes are selected from the list comprising SEQ ID N° 5-6.

15. The method according to claim 2 or the kit according to anyone of claim 9, wherein said one or more RNA virus specific probes are selected from the list comprising SEQ ID N° 9-10, SEQ ID N° 13, SEQ ID N° 18, SEQ ID N° 21 and SEQ ID N° 24.