WO2019122427A2 - Diagnosis of infection by detecting rna in a sample - Google Patents

Abstract

The present invention relates to a method for identifying an infection agent and/or a method of a diagnosing the presence of an infectious agent, the method comprising the detecting of RNA originating from the infectious agent in the serum of an individual, detecting RNA contained in extracellular vesicles (EVs) released from cells of the individual, in particular from blood cells.

Description

Diagnosis of Infection by Detecting RNA in a Sample

Technical Field

The present invention relates to methods and kits for diagnosis, for assessing virulence of an infection and/or for assessing the parasitaemia of an infection.

Background Art and Problems Solved by the Invention

Malaria remains one of the most serious public health problems facing tropical areas. More than 400 million people were infected worldwide in 2015 by the parasites called Plasmodium, killing approximately 1 million thereof, mainly children. However, recent studies have found that the mortality may be underestimated due to poor diagnostics. Due to massive international effort to eradicate malaria the number of malaria cases decreased sharply. Most victims still die because the disease is not diagnosed in time by health workers. To further complicate the diagnosis, co-infections with viruses and bacteria are common in endemic regions. Although co-infections are known to be potentially fatal, too often a proper diagnosis is only possible at autopsy. Most of the severe cases of malaria are caused by Plasmodium falciparum ( Pf ) in subsaharian part of Africa. Besides P. f several plasmodium species can infect human including P. vivax (P.v.), P. ovale, P. malariae and P. knowlesi. Vivax malaria is the predominant Plasmodium spp. outside African countries and accounts for 14-80 million cases of clinical malaria each year with more than 70% of the infections in Asia and the Americas (WHO 2015). Diagnosis of malaria is first based on clinical symptoms. However, all symptoms and signs of uncomplicated malaria are non-specific and are observed in several other febrile conditions. In non-endemic region, malaria continues to pose challenges in diagnosis and management and remains an infrequently encountered infection for many physicians. Noteworthy, a significant amount of cases is found in developed countries mainly amongst travelers. It is estimated that 25-30 million individuals travel annually from Europe to areas with malaria. The WHO reported an increase in the number of imported malaria cases culminating at 15,500 cases in the year 2000 in Europe.

The gold standard diagnostic test for malaria is still light microscopy of thin and thick blood smears. The sensitivity is estimated at 100 parasites per mΐ of blood. Unfortunately, microscopy relies strongly on the skills of the operator. More convenient are the Rapid Diagnostic Tests (RDTs) which detect P.f. antigens with a sensitivity of 200 parasites per mΐ. Upon repeated infections, adults develop a partial immunity keeping the parasitemia below the detection level of microscopy. Since those asymptomatic carriers are still able to transmit the parasites, they constitute a reservoir for transmission and create a significant public health concern of reintroducing malaria in areas that have competent vectors and climatic conditions such as in southern USA and Europe. Furthermore, a typical parasitemia in P. v. infection is relatively low, often requiring the use of thick smears to concentrate the blood for reliable diagnosis.

Therefore, more sensitive technologies have to be developed to improve the detection and/or the clinical management of malaria and/or other infections. Furthermore, it is an objective to provide methods and kits for diagnosis, for assessing parasitemia, virulence of infection and possibly occurrence of resistance against treatment. It is an objective to provide other methods of diagnosis, which may be accomplished with different test protocols and/or simplified test procedures, and which are preferably suitable to overcome limitations of existing tests.

The present invention addresses the problems depicted above.

Summary of the Invention

Remarkably, the inventor found that RNA of infectious agents is present in samples taken from an individual and can be detected in a sample for diagnosing infection. The RNA is generally found in extracellular vesicles (EV) found in body fluids, such as blood and/or serum.

In an aspect, the present invention provides a method of diagnosis, comprising detecting in a sample taken from an individual RNA originating from and/or associated with an infectious agent and/or with a disease.

In an aspect, the present invention provides a method for diagnosing whether an individual is infected from an infectious agent (and/or suffers from a disease), the method comprising: analyzing whether a sample taken from said individual comprises an RNA originating from and/or associated with said infectious agent (and/or disease), and, finding infection when said RNA is detected. In an aspect, the present invention provides a method for diagnosing a disease and/or an infection, the method comprising detecting in a sample taken from an individual one or more RNA molecules originating from and/or associated with the infectious agent causing said infection.

In an aspect, the present invention provides a method for diagnosing an infection, the method comprising detecting in the blood and/or plasma of an individual one or more RNA molecules originating from and/or associated with the infectious agent causing said infection.

In an aspect, the present invention provides a method for assessing the virulence of an infection, the method comprising detecting RNA related to one or more virulence factors in a sample, and determining from the presence of RNA related to said one or more virulence factors, the virulence of the infection.

In an aspect, the present invention provides a method for detecting whether an infectious agent is virulent, the method comprising detecting, in the blood and/or plasma of an individual, one or more RNA molecules originating from and/or associated with the infectious agent causing said infection, wherein a sequence of said RNA molecules is associated with the virulence of the pathogens.

In an aspect, the present invention provides a kit for diagnosis, the kit comprising means for detecting the occurrence of RNA form an infectious agent in a sample of an individual.

In an aspect, the present invention provides a kit for assessing the virulence of an infection.

In an aspect, the present invention provides the RNA contained in a sample of an individual, preferably a blood and/or serum sample, for diagnosing the presence of an infection and/or of an infectious agent.

In an aspect, the present invention provides the use of reverse transcriptase for producing DNA from an RNA isolated and/or obtained from a serum sample.

In an aspect, the invention provides a method for assessing the parasitaemia of an infection of an individual by an infectious agent, the method comprising: quantifying an RNA contained in extracellular vesicles (EVs) present in a sample taken from said individual, wherein said RNA originates from and/or is associated with said infectious agent and/or disease, and, wherein a comparatively high amount of said RNA is an indication of a comparatively higher parasitaemia.

In an aspect, the invention provides a method for assessing and/or detecting the presence of resistance towards treatment of an infection in an individual, the method comprising the step of: detecting in a sample taken from said individual an RNA contained in extracellular vesicles (EVs) present in said sample, wherein said RNA encodes a product conferring resistance to one or more particular treatments, and, finding resistance when said RNA is detected.

Further aspects and preferred embodiments are provided in the appended claims and the detailed description of the preferred embodiments provided herein after. Further features and advantages of the invention will become apparent to the skilled person from the description of the preferred embodiments given below. These embodiments are provided by way of examples and the present invention is not intended to be limited to the particular embodiments.

Brief Description of the Drawings

Figure 1 shows the distribution of iRBC EV (extracellular vesicles) small RNAs. The RNAs, including human and P. falciparum RNA were obtained from the iRCB EV as described in the example. Segments of the bar indicate the percent of reads attributed to each RNA biotype among all RNA reads that mapped perfectly to known sequences, averaged across 3 biological replicates is shown.

Figure 2 shows the types of Types of Plasmodium genes in the EVs by RNA-Seq.

Figure 3 shows that Plasmodial RNA are contained inside the EVs. The RNAs are protected from proteinase K digestion, EVs were treated with proteinase K (5 mg/ml, close symbols) or untreated (open symbol) at 55°C. At times indicated aliquots were removed and the RNA was isolated and the expression of PFAOl lOw (similar results obtained with other plasmodial RNAs are not shown). Figure 4 shows a fraction from elution profile of conditioned media collected from iRBC culture as measured by absorbance at 280 nm. In Fig. 4, the fractions from conditioned media were assayed for PFA01 lOw RNA.

Figures 5A and 5B the size analysis of plasma RBC-derived EVs by Nano Flow Cytometry (nanoFCM) of healthy (5A) and malaria infected (5B) individuals. Infected individuals are seen to have more and larger EVs.

Figure 6 shows the size of EVs obtained from different groups of individuals (patients from Senegal as compared to healthy controls and patients from the Hospital in Fribourg). The analysis of the NanoFCM (Figs 5A and 5B) demonstrate a change in EVs size associated with fever in a cohort of 50.

Figures 7 shows standard curve for plasmodial 28S rRNA was determined by serial dilution of plasmid DNA, for the purpose of detecting the 28S rRNA (similar results obtained with other plasmodial RNAs are not shown).

Figures 8 is a correlation between 28S rRNA isolated from plasma of malaria patients in accordance with an embodiment of the invention vs parasitemia determined by microscopy. Quantity of 28 S rRNA was determined using the standard curve shown in Fig. 7. (similar results obtained with other plasmodial RNAs are not shown).

Figure 9 shows the characteristics of qRT-PCR amplification curves, which plot fluorescence signal versus cycle number. The amplification curves for four samples of malaria infected plasma patients as well as a one non-infected individual are shown. The amount of target (28S rRNA) is quantified by measuring the Ct value and using the standard curve as shown in Fig. 7 to determine starting copy number.

Figure 10 shows the relative parasite load quantification by qPCR in plasma samples of 50 individuals admitted at the Fe Dantec Hospital in Dakar, Senegal. Two groups were generated according to the detection or not of P. falciparum by light microscopy of thin blood smear. Each patient is represented by a circle (similar results obtained with other plasmodial RNAs are not shown). Detailed Description of the Preferred Embodiments

In some embodiments, the invention provides a method of diagnosis and to a kit for conducting the diagnosis.

In a preferred embodiment, the invention relates to detecting RNA in a sample of an individual for the purpose of diagnosis. The individual is preferably a mammal, more preferably a human or a domestic animal, most preferably a human.

Preferably, said RNA is contained and/or comprised in extracellular vesicles (EVs) present in said sample. Preferably, said EVs have a size of 50-350 nm, preferably 90-300 nm, most preferably 100-250 nm.

Interestingly, said RNA is protected within said EVs from degradation, in particular RNase degradation, such that the samples may be handled without particular precaution until RNA is separated from said EVs.

In an embodiment, said RNA is selected from mRNA, riRNA, tRNA, miRNA, piRNA, Y- RNA, Metazoan Signal Recognition Particle (SRP) RNA, U-RNA, Vault-RNA, snoRNA, and pre-mature RNA.

In an embodiment, the method comprises detecting in the sample, such as a blood and/or plasma of an individual one or more RNA molecules originating from and/or associated with the infectious agent causing said infection.

In an embodiment, said RNA contained in said EV has less than 300 nucleotides and/or is a fragment of a larger RNA molecule, said fragment having less than 1000 nucleotides, preferably than 300 nucleotides.

In a preferred embodiment, said sample is a bio fluid, for example selected from blood, serum, plasma, urine, and saliva. Preferably, the sample is selected from blood, plasma and serum, preferably serum.

In a preferred embodiment, the sample, preferably said blood and/or serum sample, has been obtained previously from said individual.

In an embodiment, the method is an in vitro and/or ex vivo method.

In another embodiment, the method comprises taking a sample, such as a biofluid sample, preferably a blood and/or serum sample from said individual.

For the purpose of the present specification, the expression "comprise" and its various grammatical forms are intended to mean "include, amongst other". It is not intended to mean "consists only of'.

In an embodiment, said infectious agent is a protozoan parasite and said RNA is protozoan RNA.

In an embodiment, said infectious agent is a parasite infecting red blood cells (RBCs) of said individual and/or of a mammalian host.

In an embodiment, said infectious agent is selected from the group consisting of: Bartonella henselae, Borrelia burgdorferi, Brucella melitensis, Chlamydia psittaci, Chlamydia trachomatis, Chlamydophila pneumoniae, Francisella tularensis, Legionella pneumophila, Listeria monocytogenes, Mycobacterium leprae, M. tuberculosis, Neisseria meningitidis, Nocardia asteroides, N. brasiliensis, N. caviae, Rhodococcus equi, Salmonella typhi, Yersinia pestis; fungi including: Cryptococcus neoformans, Histoplasma capsulatum, Pneumocystis jirovecii; Parasites including: Babesia diver gens, B. bigemina, B. equi, B. microfti, B. duncani, Blastocystis spp, Entamoeba histolytica, Giardia lamblia, Plasmodium falciparum, P. vivax, P. malariae, P. ovale, P. knowlesi, Toxoplamsa gondii, Trichomonas vaginalis, Trypanosoma brucei, T cruzi.

In an embodiment, said infectious agent is selected from protozoan parasites, preferably from Babesia and Plasmodium. In an embodiment, the infectious agent is Plasmodium, preferably selected from P. falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi.

In an embodiment, said infectious agent is Plasmodium, and the method comprises detecting one or more RNA selected from the RNAs nos 1-126 in Table 2. In an embodiment, the method and/or kit of the invention are for diagnosing any one selected from the group consisting of: bartonellosis, Lyme disease, borreliosis, brucellosis, psittacosis, sexually transmitted infections, Pneumonia, tularemia, legionnaires’ disease, listeriosis, leprae, meningitides, nocardiosis, cellulitis, typhoid fever, plague, malaria, toxoplamosis, Chagas disease, sleeping sickness, cryptococcosis, histoplasmosis.

The RNA in said sample may be detected as appropriate and depending on the type of sample and the RNA to be identified. Hereinbelow, exemplary embodiments for detecting the RNA in the sample are described without limiting the scope of the invention.

In an embodiment, the method of the invention comprises the steps of separating the EVs comprising said RNA from the sample and subsequently, isolating the RNA from the EVs.

In particular, the method preferably comprises steps for removing material other than said EVs, such as solids (cells, cellular debris), but possibly at least part of dissolved molecules from said sample, and obtaining purified EVs.

Preferably, the method comprises purifying said EVs in said sample, and subsequently, purifying the RNA from said EVs.

In an embodiment, the method of the invention comprises isolating RNA contained in the sample, preferably the plasma sample, preferably after removal of cells and/or cellular debris from a blood sample.

In some embodiments, the sample is selected from a blood, blood plasma and blood serum sample. In case the sample is a blood sample, and in other types of samples, the method of the invention comprises removing cells and cellular debris from the sample, for example by centrifugation. In case of a blood sample, this yields a plasma sample devoid of blood cells, such as red blood cells (RBCs) and cellular debris.

In an embodiment, the method of the invention comprises producing plasma and/or serum from a blood sample taken from said individual. In an embodiment, said sample is a blood sample, and wherein said method comprises preparing a plasma sample from said blood sample. Plasma samples may be obtained as is conventional, for example by centrifugation and/or filtration.

In an embodiment, the method of the invention comprises separating said EVs comprising said RNA from said plasma sample.

In an embodiment, the method of the invention comprises filtering a blood and/or serum sample, preferably before detecting RNA in said sample. Filtering of a serum sample preferably further purifies and/or concentrates the EVs containing the RNA. Accordingly, if the sample is a serum sample, the method may comprise the step of filtering said serum sample. If the sample is a blood sample, the method preferably comprises preparing a serum sample (and/or separating cellular debris and cells from the blood sample) and filtering the resulting supernatant.

In an embodiment, the method of the invention comprises separating said RNA from said EVs, and detecting said RNA once said RNA has been separated from said EVs. For example, the RNA may be isolated from EVs by commercially available RNA isolation kits, such as miRNeasy kit (Qiagen, Hilden, Germany).

In order to detect a particular RNA molecule suitable to achieve diagnosis, the method preferably comprises detecting whether said particular RNA molecule is present in said sample. Said particular RNA molecule is preferably characteristic for the infectious agent and/or the disease to be diagnosed.

There are many ways of specifically detecting any particular RNA molecule or RNA fragment present in a sample. In an embodiment, reverse transcriptase and PCR (polymerase chain reaction) may be conducted.

In an embodiment, the method of the invention comprises exposing RNA in the sample to reverse transcriptase, preferably so as to produce complementary DNA (cDNA).

In an embodiment of the method of the invention, said one or more RNA molecules are detected by reverse transcription of RNA contained in said sample, so as to obtain DNA derived from said RNA, and by detecting the presence of said DNA derived from said RNA.

In an embodiment, the method of the invention comprises exposing DNA obtained from the RNA contained in said sample to primers, preferably primers capable of biding to a strand of said DNA. Preferably, said primers are specific to and/or complementary to RNA of said infectious agent, or to a stretch thereof, or the primers are specific and/or complementary to DNA obtained from said RNA by reverse transcriptase, for example

In an embodiment, the method of the invention comprises amplifying a nucleotide sequence associated with and/or derived from the infectious agent, for example by polymerase chain reaction (PCR), preferably amplifying a DNA obtained from RNA contained in said sample. The RNA can then be detected by detecting whether DNA has been an amplified, and possibly by identifying the DNA (sequencing, restriction enzyme assay, etc)

In an embodiment, the method of the invention comprises sequencing a nucleic acid molecule contained in said sample and/or obtained from said sample, for example obtained by reverse transcription and/or amplification and/or cloning.

In an embodiment, the method of the invention comprises comparing the obtained sequences with sequences of said infectious agent and/or detecting an infection if said obtained sequences can be attributed to a sequence of said infectious agent by sequence comparison and/or alignment tools.

In an embodiment, the method of the invention comprises sequencing a nucleic acid molecule contained in said sample and/or obtained from said sample, for example obtained by reverse transcription and/or amplification and/or cloning.

In an embodiment, the method of the invention comprises exposing RNA of said sample to an agent capable of binding said RNA and detecting said binding.

In some embodiments, that the invention encompasses detecting and/or identifying RNA in a sample without a reverse transcriptase and/or without a PCR or other amplification or cloning step, for example based on methods disclosed in the art. For example, such assays are disclosed in US2015/0010903, for example in paragraph [0090] and/or Examples 7 and 8 of this reference, which are expressly incorporated herein by reference.

In an embodiment, the sample comprising the RNA and/or the RNA isolated and/or purified from said sample may be exposed to an oligonucleotide linked to a marker, or said RNA may be reacted with a marker element, such as biotin or a fluorescent moiety. In a subsequent step, the oligonucleotide linked to the marker may be detected and/or isolated. If the RNA was reacted with a marker or label, a subsequent step comprises exposing the labelled RNA to an oligonucleotide specifically binding to the labelled RNA, wherein the oligonucleotide may comprise a moiety allowing separation and/or isolation, and detecting the labelled RNA, thereby detecting RNA originating from an infectious agent. The use of labelling may, of course, also be used in combinations with detection methods based on reverse transcription and/or amplification and/or cloning as is known from the art. Furthermore, the specificity is achieved by providing oligonucleotides binding specifically to a particular RNA molecule due to complementary base pairing, such that the oligonucleotides and/or primers bind specifically to an RNA or a stretch thereof, said RNA originating from the infectious agent and being preferably characteristic to a particular infectious agent. For example, the oligonucleotide binds specifically to any one of the sequences disclosed in this application in a method for diagnosing Plasmodium infection.

In methods and/or kits for diagnosing malaria and/or an infection by Plasmodium, the reactive agents are preferably selected so as to suitable to detect RNA that is specific to Plasmodium. On the other hand, when another infectious agent is to be diagnosed, the reactive agents for detecting the parasite are preferably selected and designed so as to be specific for the parasite in question.

For the purpose of the present specification, diagnosing a disease, for example "malaria", is equivalent to detecting in said sample (RNA of) an infectious agent causing said disease, in particular a Plasmodium species, in the case of malaria. Diagnosing cat scratch disease, for example, is equivalent to detecting the RNA originating from and/or associated with an infectious agent causing cat scratch disease, such as Bartonella henselae.

In some embodiments, the present invention provides a method and/or the kit for assessing the virulence of an infection. The kit preferably comprises reactants suitable to conducing the method of assessing the virulence of an infection, such as oligo- and/or polynucleotides capable of hybridizing with RNA to be detected and/or to be quantified.

In accordance with some embodiments, certain RNA associated with virulence is detected (e.g. using the primers associated with particular virulence factors). If detected, the invention encompasses making conclusion with respect to the virulence of infection. The determination of virulence allows determining the urgency and kind of treatment, based on the virulence as assessed in accordance with the invention, for example. Furthermore, the invention encompasses associating the amount of RNA of the infectious agent detected with virulence, where more RNA may be related to higher virulence, for example. In an embodiment, the method and/or kit is suitable to detect the presence of RNA related to the VAR genes, for example, mRNA encoding the products of the var genes, or fragments of such detecting RNA, e.g. mRNA, encoding PfEMPl (P. falciparum erythrocyte membrane protein 1) and/or a variant thereof, and/or a fragment of such RNA, or RNA otherwise associated with the var genes.

In some embodiments, the present invention provides a method and/or the kit for assessing and/or detecting the presence of resistance. In particular, it is detected whether or not the infectious agent, such as a Plasmodium strain, for example, possesses and/or expresses a gene that confers resistance to one or more treatment, in particular to treatment by administration of one or more small molecules, such as artemisinin and derivates, chloroquine, quinine and derivatives, sulfonamides, amodiaquine, proguanil, atovaquone, doxycycline and the like. In an embodiment, the method of the invention comprises detecting mRNA encoding a gene product that confers such resistance, or a fragment of such an mRNA, or other types of RNA associated with resistance. In the experimental section, the inventor detected the presence of parasite-derived mRNAs coding for proteins involved in drug resistance such as PFEl l50w (multidrug resistance), PFLl4l0c (ABC transporter, multidrug resistance-associated protein 2). As well as PFKELCH13, which is involved in artemisinin resistance. Preferably, said RNA is present in said EVs, and the description for detecting the RNA from the sample as given above applies directly to the detection of RNA associated with resistance and/or with virulence.

In an embodiment, the of the invention kit preferably comprising means, tools, facilities, items and/or equipment for conducting the diagnosis. Such items preferably comprise test tubes, solvents, buffer solutions, and reactive agents, such as primers and the like, suitable to detect the RNA in the sample. In an embodiment, the kit comprises oligo- and/or polynucleotide molecules capable of hybridizing with said RNA to be detected or with DNA obtained from said RNA, preferably by reverse transcription. For example, said oligo-or polynucleotide molecules are primers capable of hybridizing with said RNA or with DNA obtained from said RNA.

In some embodiments, the kit and methods may be used for diagnosis for example upon occurrence of one or more of the following symptoms including but not limited to: fever, sweats and chills, change in cough or new cough, sore throat or new mouth sore, shortness of breath, nasal congestion, stiff neck, burning or pain with urination, unusual vaginal discharge or irritation, increased urination, redness, soreness, or swelling in any area, including surgical wounds and ports, diarrhea, vomiting, pain in the abdomen or rectum, new onset of pain, changes in skin, urination, and mental status.

The methods and kits of the invention have manifold advantages compared to existing diagnostic tests. It is noted that EVs are present in the plasma and are thus detectable from plasma in accordance with the invention even if the infectious agent is present in cells (i.e. RBCs) and thus not present in the plasma. Taking the diagnosis of malaria as an example, compared to PCR testing of blood samples for the presence of plasmodial DNA, the invention has the advantage that RBCs do not need to be lysed, as the parasites do not need to be separated from the RBCs. Accordingly, in an embodiment of the method of the invention, a lysis step is absent and/or the kit of the invention lacks an agent for lysing blood cells. Certain stages of Plasmodium species (or other parasites) may be confined to particular tissues. For example, it is known that parasitized red blood cells (pRBCs) are sequestered in cerebral micro-circulation (cerebral malaria), such that they may not be visible in peripheral blood smears. In these situations, RNA that is present in EVs in serum is still detectable, since it is not linked to the location of the parasite.

Further features and advantages of the invention will become apparent to the skilled person from the description of the Examples below. These examples are provided by way of examples and the present invention is not intended to be limited to the particular examples nor any embodiment as shown in the figures. Theories and explanations presented in order to explain the surprising results are not intended to limit the scope of the appended claims. Examples

Here I collected EVs derived from in vitro cultures of P. falciparum and performed RNA-Seq to characterize for the first time the small RNA content of iRBCs derived EVs. I found several species of host as well as plasmodial RNAs.

Characterization and properties of extracellular vesicles isolated by differential centrifugation

To investigate the potential regulatory RNAs present in EVs, I collected EVs from P. falciparum iRBCs cultures and purified the vesicles by ultracentrifugation. To verify the vesicular structure of the isolated EVs, I used transmission electron microscopy (TEM). Analysis by TEM revealed that EVs have a size of 100-250 nm and demonstrated an intact lipid bilayer. The morphology is consistent with EVs as previously reported measuring about 150 nm in size as observed by TEM and the nanoparticle tracking system. Larger EVs observed by the nanoparticle tracking system might be the result of aggregate formation. The purity of the EV preparations was determined by western blotting with a panel of antibodies. EVs were enriched in the lipid raft protein stomatin as well as the parasite protein RESA, while Bip a plasmodium protein specifically expressed in endoplasmic reticulum was absent.

To further define the EV cargoes, I isolated total RNA from EVs and intact iRBC cells. The quantity and quality of isolated RNA were determined using an Agilent Bioanalyzer. In contrast to intact cells, total RNA bioanalyzer profiles indicated that iRBC EVs contain minor amounts of 18S and 28S rRNA species. Furthermore, the RNA composition is different in EVs in comparison with intact cells, with enrichment in small RNAs below 300 nt in length in the vesicles.

Extracellular vesicles derived from iRBCs contain small RNAs

Next to decipher the RNA content of EVs, I generated small RNA libraries of the samples for small RNA-Seq from three biological replicates of EVs derived from iRBCs. The same 3D7 parasite strain was cultured in RBCs of three different donors.

Small RNA sequencing of the EV libraries yielded a total of 83’319’ 134 raw reads that were pre-processed and trimmed to remove adapter sequences using sRNAbench to identify high quality reads that were considered for further analysis. After processing for adapter and unmapped sequences, the number of reads was reduced to 26’647’563, 8’l26’655 and 11’925’918 respectively. The total number of reads mapping to the human genome was 43’689’670, whereas the total number of reads mapping to the P. falciparum genome was 3’0l0’466. Therefore, the proportion of P. falciparum reads was 6.4 % versus 93.6 % of human (Table 1).

Table 1 Sequencing statistics

Sample 1 Sample 2 Sample 3 Average

Sequencing reads 68003555 33846106 35423492 45757717

QC passed reads 51798945 14610329 16909860 27773044

Mapped to human genome 24968040 7274347 11447283 14563223

Mapped to Plasmodium falciparum 1679523 852308 478635 1003488 genome

Identified miRNAs 219 87 276

Since small RNAs have regulatory functions, I focused our analysis on the identification of small RNAs. As expected miRNAs and tRNAs were widely and abundantly expressed. Then I performed a detailed quantification of annotated RNAs to functionally categorize the human small RNAs. The average across all three samples was calculated to obtain a representative profile of biological diversity between donors. Of total reads, the miRNAs were the most abundant small RNA species with 42.3 %, followed by 34.79 % of tRNAs, 8 % piRNA, 7 % Y RNA, 3.60 % Metazoa Signal Recognition Particle (SRP) RNA, 2.17 % U-RNA, 1.17 % Vault RNA, 0.48 % snoRNA and 0.14 % pre-mature RNA (Fig. 1).

P. falciparum iRBCs release plasmodial RNAs.

To investigate whether plasmodial RNA is released in the supernatant, I labelled newly transcribed RNAs in tightly synchronized iRBCs. Ring stage parasites were incubated with 5- ethynyl uridine (EU), which is incorporated into RNA during transcription. First, I monitored the cells for active transcription and, only iRBCs were positive for EU as measured by flow cytometry. Then, I collected the supernatants and freshly transcribed RNA was detected by flow cytometry in the supernatant with more than 80% of events being positive for freshly synthesized RNAs. Since RBCs are transcriptionally inactive, I assume that the labelled RNA is derived from the parasites. This experiment gave us strong confidence that EVs contain plasmodial RNA. In order to identify the plasmodial sequences, I aligned our RNA-Seq results generated previously to the P. falciparum genome (Plasmodium 6.1). I found that several plasmodial RNAs including tRNAs, rRNAs and mRNAs were present in EVs. In total, I identified 126 RNAs mapping to the Plasmodium genome of which 28 were identified in all the 3 samples (Table 2). Most of the reads mapped to chromosome 1 (10.7 %), chromosome 5 (33.1 %), chromosome 7 (33.6 %) and chromosome 13 (9.42 %), only a minor amount mapped to mitochondrial and apicoplast DNAs (0.75 and 0.30 %, respectively). Then, I normalized by the length of the DNA, excluding mitochondrial and apicoplast DNAs since they are present in several copies per cell. Chromosome 1, 5 and 7 keep the highest number of mapped RNAs. Most of the RNA transcripts are derived from structural RNAs. There are a few putative novel ncRNAs, however their abundance is low. I have found mostly rRNA, tRNA and snoRNA in the three replicates from Plasmodium (Figure 2). In addition, a large amount of transcripts coding for proteins exported to the RBC cytosol such as RESA, ETRAMP and mRNAs from the PHIST family including Mal7Pl.l72 and PF0 0137 were present. Furthermore, EVs contain a large amount of apicoplast tRNAs and mitochondrial rRNAs (Fig. 5e), including tRNA-GLu-l, tRNA-Serl, tRNA-Metl, tRNA-Prol, tRNA-Argl, tRNA-Trpl, tRNA-Arg3, tRNA-Cysl, pre-tRNA-PrOl, tRNA-SelCysl. Interestingly several RNAs were involved in drug resistance, such as PFEl l50w (multidrug resistance protein 1), PFLl4l0c (ABC transporter, multidrug resistance-associated protein 2), PF13 0238 (Kelch- 13).

I then compared the RNA EV expression profile with stage specific RNA profile of the blood stage and found that EV RNAs are mostly derived from genes with peak expression in merozoites and early rings. Very few RNAs are derived from other stages. Importantly, there was no direct correlation of the gene expression levels between iRBC and EV transcriptome.

Table 2: P. falciparum RNA Sequences identified in samples

PF3D7_1371300 28S ribosomal RNA Pf3D7_13_v3:2802944-2807159

40S ribosomal protein S26

PF3D7_0217800 (RPS26) Pf3D7_02_v3:734271-734595

40S ribosomal protein S4,

PF3D7_1105400 putative Pf3D7_ll_v3:235596-237097 PF3D7_1447000 40S ribosomal protein S5 Pf3D7_14_v3:1929930-1931379 PF3D7_0531800 5.8S ribosomal RNA Pf3D7_05_v3:1292051-1292210 PF3D7_0725800 5.8S ribosomal RNA Pf3D7_07_v3:1086212-1086371 PF3D7_1418500 5S ribosomal RNA Pf3D7_14_v3:779376-779495 PF3D7_1418600 5S ribosomal RNA Pf3D7_14_v3:780005-780124 PF3D7_1418700 5S ribosomal RNA Pf3D7_14_v3:780915-781034

60S ribosomal protein L26,

PF3D7_0312800 putative Pf3D7_03_v3:534238-535238 PF3D7_1244300 ACEA small nucleolar RNA U3 Pf3D7_12_v3:1857848-1858090 PF3D7_1246200 actin I (ACT1) Pf3D7_12_v3:1920783-1921914 PF3D7_1200700 acyl-CoA synthetase (ACS7) Pf3D7_12_v3:61555-64336 acyl-CoA synthetase,

PF3D7_1477900 pseudogene (ACSlb) Pf3D7_14_v3:3209128-3211574 choline/ethanolaminephosphot

PF3D7_0628300 ransferase, putative (CEPT) Pf3D7_06_v3:1170812-1173696 chromodomain-helicase-DNA- binding protein 1 homolog,

PF3D7_1023900 putative (CHD1) Pf3D7_10_v3:997474-1007461 conserved Plasmodium

membrane protein, unknown

PF3D7_0404600 function Pf3D7_04_v3:247730-260147 conserved Plasmodium

membrane protein, unknown

PF3D7_1221900 function Pf3D7_12_v3:872360-874940 conserved Plasmodium protein,

PF3D7_0606000 unknown function Pf3D7_06_v3:250611-253803 conserved Plasmodium protein,

PF3D7_0609700 unknown function Pf3D7_06_v3:413651-419781 conserved Plasmodium protein,

PF3D7_0716200 unknown function Pf3D7_07_v3:712468-714967 conserved Plasmodium protein,

PF3D7_1024800 unknown function Pf3D7_10_v3:1034684-1039079 conserved Plasmodium protein,

PF3D7_1237900 unknown function Pf3D7_12_v3:1577180-1580747 conserved Plasmodium protein,

PF3D7_1329500 unknown function Pf3D7_13_v3:1252058-1257973 conserved Plasmodium protein,

PF3D7_1404800 unknown function Pf3D7_14_v3: 163989-166854 conserved Plasmodium protein,

PF3D7_1451200 unknown function Pf3D7_14_v3:2094339-2099736 conserved protein, unknown

PF3D7 0818000 function Pf3D7 08 v3:821333-822452 mal_mito_3 cytochrome b (CYTB) M76611:3491-4622

DNA polymerase epsilon

PF3D7_1234300 subunit b, putative Pf3D7_12_v3:1433819-1435995

DNA/RNA-binding protein Alba

PF3D7_0814200 1 (ALBA1) Pf3D7_08_v3:687339-688086 early transcribed membrane

PF3D7_1001500 protein 10.1 (ETRAMP10) Pf3D7_10_v3:81415-81739 early transcribed membrane

PF3D7_1102800 protein 11.2 (ETRAMP11.2) Pf3D7_ll_v3: 131702-131987 early transcribed membrane

PF3D7_1401400 protein 14.1 (ETRAMP14) Pf3D7_14_v3:53410-53734 PFC10_API0028 elongation factor (tufA) PFC10_API_IRAB:19443-20676 PF3D7_0730900 EMPl-trafficking protein (PTP4) Pf3D7_07_v3:1325374-1331813 PF3D7_1302000 EMPl-trafficking protein (PTP6) Pf3D7_13_v3: 112791-113815 PF3D7_1471100 exported protein 2 (EXP2) Pf3D7_14_v3:2905162-2906565 glyceraldehyde-3-phosphate

PF3D7_1462800 dehydrogenase (GAPDH) Pf3D7_14_v3:2559097-2560347

GTPase-activating protein,

PF3D7_1114200 putative Pf3D7_ll_v3:542329-544765 PF3D7_0708400 heat shock protein 90 (HSP90) Pf3D7_07_v3:381591-384614 histone H2A variant, putative

PF3D7_0320900 (H2A.Z) Pf3D7_03_v3:875212-876295 interspersed repeat antigen

PF3D7_0501400 (FIRA) Pf3D7_05_v3:74508-79842 PF3D7_0220000 liver stage antigen 3 (LSA3) Pf3D7_02_v3:796751-801586 PFC10_API0060 molecular chaperone (Clp (C?)) PFC10_API_IRAB:16196-18497 mRNA-decapping enzyme

PF3D7_1032100 subunit 1, putative (DCP1) Pf3D7_10_v3:1293498-1297071 origin recognition complex

PF3D7_1203000 subunit 1 (ORC1) Pf3D7_12_v3: 167705-171275 PF3D7 1464600 phosphatase, putative Pf3D7 14 v3:2619678-2624187

Plasmodium exported protein

(hypl6), unknown function

PF3D7_1001900 (PfJ23) Pf3D7_10_v3:99378-100361

Plasmodium exported protein

PF3D7_0831500 (PHIST), unknown function Pf3D7_08_v3:1353455-1354669

Plasmodium exported protein

PF3D7_0402100 (PHISTb), unknown function Pf3D7_04_v3:123587-125464

Plasmodium exported protein

PF3D7_0424600 (PHISTb), unknown function Pf3D7_04_v3:1113344-1114486 polyadenylate-binding protein,

PF3D7_1224300 putative (PABP) Pf3D7_12_v3:988627-991255 PF3D7_1011800 PRE-binding protein (PREBP) Pf3D7_10_v3:455397-458817 PF3D7_0517300 pre-mRNA-splicing factor (SRI) Pf3D7_05_v3:724680-725751 PF3D7_1121700 protein GCN20 (GCN20) Pf3D7_ll_v3:820072-822520 PF3D7 1442900 protein transport protein SEC7, Pf3D7 14 v3:1753509-1763664 putative (SEC7)

rab specific GDP dissociation

PF3D7_1242800 inhibitor (rabGDI) Pf3D7_12_v3:1805379-1807356 PF3D7_0419600 ran binding protein 1, putative Pf3D7_04_v3:870736-871873 ring-infected erythrocyte

PF3D7_1149200 surface antigen Pf3D7_ll_v3:1976406-1979804 ring-infected erythrocyte

PF3D7_0830200 surface antigen (RESA) Pf3D7_01_v3:98818-102282 PF3D7_0823200 RNA-binding protein, putative Pf3D7_08_v3:1022628-1023846 PF3D7_0104900 RNase MRP Pf3D7_01_v3:218516-218824 PF3D7_1418800 signal recognition particle RNA Pf3D7_14_v3:782048-782361 skeleton-binding protein 1

PF3D7_0501300 (SBP1) Pf3D7_05_v3:68929-70113 small nucleolar RNA Mel8S- PF3D7_0309400 Uml356 Pf3D7_03_v3:401457-401576 PF3D7_1109000 small nucleolar RNA snoR07 Pf3D7_ll_v3:381153-381259 PF3D7_1304800 small nucleolar RNA snoR16 Pf3D7_13_v3:243654-243772 PF3D7_1424300 small nucleolar RNA snoR26 Pf3D7_14_v3:975240-975336 PF3D7_1424700 small nucleolar RNA snoR29 Pf3D7_14_v3:981587-981671 transcription factor with AP2

PF3D7_0730300 domain(s) (AP2-L) Pf3D7_07_v3:1297458-1301454 translocon component PTEX150

PF3D7_1436300 (PTEX150) Pf3D7_14_v3:1479776-1482758 PF3D7_0714700 tRNA Asparagine Pf3D7_07_v3:670794-670866 PF3D7_0411600 tRNA Glutamic acid Pf3D7_04_v3:518153-518225 PF3D7_0527700 tRNA Glutamic acid Pf3D7_05_v3:1150233-1150306 PF3D7_0203500 tRNA Glutamine Pf3D7_02_v3: 164268-164340 PF3D7_1370200 tRNA Glycine Pf3D7_13_v3:2783765-2783836 PF3D7_1339200 tRNA Proline Pf3D7_13_v3:1576925-1576997 tryptophan/threonine-rich

PF3D7_0830500 antigen (TryThrA) Pf3D7_08_v3:1296133-1298328 PF3D7_1213100 U1 spliceosomal RNA Pf3D7_12_v3:576416-576580 PF3D7 1137000 U2 spliceosomal RNA Pf3D7 11 v3: 1461300-1461498

U3 small nucleolar

ribonucleoprotein protein

PF3D7_1461200 IMP3, putative (IMP3) Pf3D7_14_v3:2489697-2490249 PF3D7_0830200 unspecified product Pf3D7_08_v3:1284577-1287755 PFC10_API0002:rRNA unspecified product PFC10_API_IRAB:235-1662 PFC10_API0003:tRNA unspecified product PFC10_API_IRAB:1708-1779 PFC10_API0004:tRNA unspecified product PFC10_API_IRAB:1808-1880 PFC10 API0007:tRNA unspecified product PFC10 API IRAB:2049-2121 PFC10_API0008:tRNA unspecified product PFC10_API_IRAB:2141-2213

PFC10_API0009:tRNA unspecified product PFC10_API_IRAB:2218-2292

PFC10_API0010:rRNA unspecified product PFC10_API_IRAB:2334-5029

PFC10_API0023:tRNA unspecified product PFC10_API_IRAB:18895-18966

PFC10_API0024:tRNA unspecified product PFC10 _ API_I RAB:18979-19049

PFC10_API0045:tRNA unspecified product PFC10_API_IRAB:27534-27604

PFC10_API0046:tRNA unspecified product PFC10_API_IRAB:27611-27684

PFC10_API0048:tRNA unspecified product PFC10_API_IRAB:27776-27868

PFC10_API0049:tRNA unspecified product PFC10_API_IRAB:27881-27965

PFC10_API0050:tRNA unspecified product PFC10_API_IRAB:27970-28058

PFC10_API0053:tRNA unspecified product PFC10_API_IRAB:28288-28359

PFC10_API0057:rRNA unspecified product PFC10_API_IRAB:29203-29430 mal_rna_14:rRNA unspecified product (LSUA) M76611:5025-5201 mal_rna_18:rRNA unspecified product (LSUD) M76611:5771-5854 mal_rna_17:rRNA unspecified product (LSUE) M76611:5576-5771 malmito_rna_LSUG:r

RNA unspecified product (LSUG) M76611:282-389

malmito_rna_RNAll:

rRNA unspecified product (RNA11) M76611:5283-5340 malmito_rna_RNA12:

rRNA unspecified product (RNA12) M76611:4886-4945 mal_rna_9:rRNA unspecified product (RNA2) M76611:1696-1763 mal_rna_10:rRNA unspecified product (RNA3) M76611:1829-1910 malmito_rna_RNA4:r

RNA unspecified product (RNA4) M76611:4624-4696 malmito_rna_RNA5:r

RNA unspecified product (RNA5) M76611:4715-4802 malmito_rna_RNA6:r

RNA unspecified product (RNA6) M76611:4807-4865 malmito_rna_RNA7:r

RNA unspecified product (RNA7) M76611:5201-5283 mal rna 19:rRNA unspecified product (RNA8) M76611:5854-5955 121 malmito_rna_9:rRNA unspecified product (RNA9) M76611:71-125

122 mal rna ll:rRNA unspecified product (SSUA) M76611:1915-2023

123 malmito_SSUB:rRNA unspecified product (SSUB) M76611:389-505

124 mal_rna_15:rRNA unspecified product (SSUD) M76611:5378-5446

malmito rna SSUF:rR

125 NA unspecified product (SSUF) M76611:5446-5507

126 PF3D7 0906600 zinc finger protein, putative Pf3D7 09 v3:320810-323342

RNA nos: no. 2: MAL5 18S rRNA; no. 3: MAL7_l8S:rRNA; no. 12: PF11 0065; no. 20:

PF3D 7_ 12_U 3 snoRN A; no. 22 : MAL12P1.7, PFL0035c; no. 69: PFAOl lOw; no. 72: PF14 0183; no. 73: PFE0065w; no. 74: PFC0392c; no. 75: PF11 0102; no. 76:

PFD3D7 13_snoRl 6; no. 77: PF3D7_l4_snoR26; no. 78: PF3D7_l4_snoR29; no. 79: PF07 0126; no. 80: PF14 0344; no. 81 : MAL7_tRNA_Asp 1 ; no. 82: MAL4_tRNA_Glul ; no. 83: MAL5_tRNA_Glul ; no. 84: pre-tRNA-Gln-l; no. 85: MAL 13_tRNA_Gly 1 ; no. 86: MAL 13_tRNA_Pro 1 ; no. 87: PF08 0003.

Plasmodial extracellular RNAs are associated with EVs

The extracellular RNAs found in the plasma can be associated with proteo-lipid complexes and the complexes containing RNAs might be co-purified with EVs during ultracentrifugation. To exclude the possibility that plasmodial RNA molecules originate from protein-lipid complexes present in our EV preparation or in the conditioned medium derived from in vitro P. falciparum iRBCs, I treated EVs with proteinase K²⁰. In fact, proteolytic digestion of the protein complex stabilizing the plasmodial RNAs would release the RNAs and render them sensitive to degradation. However, RNAs protected inside a vesicular structure would not be expected to show sensitivity to proteolysis. I treated EVs with or without proteinase K at 55 °C. The samples were incubated for 0, 15, 30 and 45 min followed by RNA extraction and quantification by qPCR. In fact, MAL5 18S, 28S rRNA and PFAOl lOw were protected from degradation inside EVs, as illustrated in Figure 3 for PFAOl lOw.

To further confirm that the plasmodial RNAs are associated with EVs, I performed size exclusion chromatography, which allows efficient separation of vesicles from protein lipid complexes that are smaller and elute later than vesicles. For this purpose, I cultured iRBCs and collected the conditioned supernatant after 48 hours. I isolated 14 fractions by chromatography and most of the proteins were eluted in the latest fractions (9-12), only a minor portion was eluted in the fraction 5 that corresponds to the EV fractions. I can assume based on our standards that most of the proteins in the supernatant are albumin, gamma- globuline. I then isolated total RNA including small RNAs from each isolated fraction and performed qPCR. As miR45la, all the tested plasmodial RNAs were detected in fraction 5, which corresponds to the EV fraction. Figure 4 illustrates this finding with the results obtained for PFAOl lOw. Altogether these experiments demonstrated that Plasmodium RNAs are located in EVs.

Development and testing of a qRT-PCR assay

The next step is to establish a RT-PCR assay capable to quantify the level of extracellular plasmodial RNAs. I tested the primer efficiency and sensitivity of the primer pairs for PFAOl lOw in dilution curves using pGEM-T Easy plasmids containing the cloned target fragments as template. The primers used are shown in Table 3 below. These experiments demonstrated that the pair was between 90-110% efficient (figure not shown). Next, I determined the minimal amount of plasma that has to be used for detection of extracellular RNA. I was able to detect reliably, miR45la in 10 mΐ of plasma, the RNA was extracted from different amount of plasma and miR45la was quantified by RT-PCR. There is a good correlation between serum amount and detection (figure not shown).

Table 3: Primers for qRT-PCR assays

Flow cytometry quantitation of plasma RBC-derived EVs

Although flow cytometry is often used to quantify and characterize EVs in the plasma, traditional flow cytometers are only capable of measuring particles down to 500 nm, which is significantly larger than the average and median sizes of plasma EVs. Our collaborators have developed a Nano flow cytometer (Nano FCM) that can detect particles of a size below 100 nm as determined by analysis of liposomes and beads (Danielson, K. M. et al. Diurnal Variations of Circulating Extracellular Vesicles Measured by Nano Flow Cytometry. PLoS One 11, e0l44678, doi:lOT37l/joumal.pone.0144678 (2016)). NanoFCM can be used to detect EVs in healthy individual plasmas (figure not shown). Our collaborators have screened our plasma samples coming from healthy donors and malaria patients in Senegal. During P.f. infection the shape and size of EVs changes dramatically as revealed by NanoFCM (Figures 5A and 5B). The increase in size was particularly well correlated with fever (Figure 6).

Detection of 28S rRNA in the plasma of malaria patients

I isolated RNA from plasma of malaria patients and quantified the exRNAs for plasmodial EV genes by qPCR. First, a standard curve is done by using a serial dilution of a plasmid containing the amplicon of 28S rRNA (Figure 7). I, then quantified the RNA in patient samples. Remarkably, the plasmodial R As were detected in patients with Pf infection and show a correlation with the parasitemia as determined by microscopy of thick smears (Figure 8). However, it is interesting to note that a major limitation is that only immature stage of the parasites are visible in bloodstream by microscopy, while mature parasites are sequestered in the capillaries. Therefore, the exact parasite biomass might be underestimated by microscopy. Our data are very promising and show that our assay is more sensitive than microscopy.

Figure 9 shows a typical amplification plot by qPCR that demonstrates the presence of the 28S rRNA only in patients infected with P . falciparum as illustrated by the square, diamond, cross and triangle, whereas the samples derived from non-infected individuals remain negative (circles). Next, I collected about 50 plasma samples from Senegal. The RNA was extracted from EVs, followed by reverse transcription, the cDNA was quantified by qPCR. I divided the samples into 2 groups based on positive or negative detection by light microscopy of thick smears. Our approach detected plasmodial RNAs in all the microscopy positive samples. In addition, I detected parasites in three negative samples, suggesting that our approach is more sensitive than microscopy.

Material and Methods

Cell culture of parasites

The P. falciparum strain 3D7 was used for this study. Parasites were kept in fresh type 0+ human red blood cells, suspended at 4% hematocrit in HEPES -buffered RPMI 1640 containing 10 % (w/v) heat inactivated human serum, 0.5 ml Gentamycin, 2.01 g sodium bicarbonate and 0.05 g Hypoxanthine at pH 6.74. Prior to culture, the complete medium was depleted from extracellular vesicles and debris by ultracentrifugation at 100’ 000 g for 1 hour. The parasite cultures were maintained in a controlled environment at 37°C in a gassed chamber at 5% C0₂ and 1% 0₂.

Synchronization of parasites

In order to obtain highly synchronized parasite cultures I performed a combination of Percoll and sorbitol. Parasites at 42 - 45 hours post-invasion were purified using a 70% Percoll. Fresh blood was added to the isolated schizonts. After 8 hours, 5 % sorbitol was used to eliminate the remaining schizonts to yield highly synchronized rings.

Purification of EVs

EVs from iRBCs were isolated from cell culture supernatants as described³. In brief, cell culture supernatants of Plasmodium falciparu -lnfcctcd RBCs were collected. Cells and cellular debris were removed from the supernatant by centrifugation at 600 g, 1600 g, 3600 g and finally 10000 g for 15 min. To further concentrate the EVs, the supernatant was filtered through a Vivacell 100 filter (100 kDa molecular weight cut off; Sartorius). Then, the concentrated supernatant was pelleted at 100000 g, the pellet resuspended in PBS and layered on top of a 60 % sucrose cushion and spun at lOOOOOg for 16 hours. The interphase was collected and washed with PBS twice at 100000 g for 1 hour to yield EVs.

Isolation of RNA and gene expression studies in BMEC-1

Total RNA was isolated from EVs and BMEC-l using miRNeasy kit (Qiagen, Hilden, Germany). The concentration and integrity of total RNA was measured using a NanoDrop- 1000 spectrophotometer (Thermo Scientific, Wilmington, DE). Size distribution was determined by Agilent 2100 Bioanalyzer with the Agilent Small RNA Chip (Agilent Technologies, Santa Clara, CA).

For Plasmodial RNAs, reverse transcription reactions were performed with 1 pg total RNA using M-MLV Reverse Transcriptase kit (Promega) after DNase I treatment (Invitrogen). Mature miR-45la and selected miRNAs were detected by quantitative RT-PCR (qRT-PCR) using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). Small nuclear RNA U6 (RNU6) (for miR-45la) and 18S rRNA and EF1 (for mRNAs) were used as reference genes for relative quantitation using the 2^L- Ct method.

Metabolic labeling of RNA and imaging

Ring stage parasites at a parasitemia of about 5 % (3D7), (15 hours post-invasion) were incubated at a final concentration of 0.5 mM 5-ethynyl uridine (EU) for 30 h. EVs were isolated from the conditioned medium as described above with exception of the sucrose gradient. The cells and EVs were subsequently fixed with 4% para-formaldehyde and permeabilized with 0.1% Triton-X-lOO. EU incorporated EV RNA was detected using Click chemistry according to the manufacturer’s protocol (Invitrogen, Cl 0329) and nuclei were counterstained using Hoechst 33342 and analyzed with a MACSQuant VYB flow cytometer. Flow cytometry data acquisition was performed using FlowJo X (Tree Star, Ashland, OR).

Size exclusion chromatography

Experiments to determine possible presence of serum-derived Ago2-miRNA complexes were performed essentially as follows: Sephacryl S-500 resin (GE Healthcare) was packed in a chromatography column (0.9 A~ 30 cm, 19.1 mL bed volume). Before injection, the column was equilibrated with 25 mL of PBS solution at 0.5 mL/min at room temperature. The column was injected with 0.5 mL of undiluted fresh serum or purified EVs and eluted at 4°C for approximately 1 h with PBS solution (pH 7.4) at a flow rate of 0.5 mL/min. A total of 25 fractions of 1 mL each were collected. Fractions were stored at 4 °C before use. Protein molecular weight standards included BSA (67 kDa; GE Healthcare) and tyrosine (0.181 kDa; Sigma- Aldrich).

Gene ontology analysis

ClueGO was used to find over-represented GO terms in the categories“biological process”, “cellular component” and molecular function”. Benjamini-Hochberg correction was performed for multiple testing-controlled P values. The GO analysis was conducted using Cytoscape 3.2.1 software⁶³ with the ClueGO⁶⁴ plugin. The GO analysis was conducted using a two-sided hypergeometric test with Bonferroni correction. The GO term levels were from five to ten. The minimum number of genes to form a cluster was set at three, while the minimum percentage of genes covered by our data set against the database was set at 7%. The rest of the settings were left as defaults.

RNA-Seq

Each sequencing library was constructed from 2 ng of isolated and treated plasma RNA. All libraries were uniquely bar-coded with index primer for multiplexing into sequencing lanes. The small RNA libraries were prepared and amplified using the NEBNext small RNA Library Prep Set (New England BioLabs, Ipswitch, MA, USA) following manufacturer instruction. The amplified libraries were resolved on a 10% Novex TBE gel (Life technologies) for size selection and the 140 to 160 nucleotide bands that correspond to adapter- ligated constructs derived from the 21 to 40 nucleotide RNA fragments were excise and recovered in DNA elution buffer. The average size distribution of each library was determined using Agilent Bioanalyzer with High Sensitivity Chip Kit (Agilent, Santa Clara, CA, USA) and quantified on ABI 7900HT Fast RT-PCR instrument using the KAPA Library Quantification kit according to the manufacture’s protocol (Kapa Biosystems, Woburn, MA, USA). Each library was adjusted to final concentration of 2 nM, pooled, and sequenced on an Illumina HiSeq 2000 or MiSeq sequencer for single read 50 cycles at the Center for Cancer Computational Biology at Dana-Farber Cancer Institute.

Sequence Analysis

The BCL files were de-multiplexed using CASAVA vl.82, and the adaptor sequences within the read sequences were trimmed by FastX-Toolkit (http ://hannonlab. cshk edu/ fastx toolkit) . The processed sequences were filtered for small RNAs greater than 16 nucleotides in length. The sequences were then aligned, quantified and annotated using sRNABench 1.0 pipeline⁶⁵. Briefly, the pipeline implemented hierarchical sequence mapping strategy that first mapped and remove spike-in library, contaminants, and rRNA before sequentially mapped to known mature miRNA, tRNA, snoRNA and piRNA onto the human genome sequence (hgl9) using Bowtie2 ⁶⁶ with parameters that allow for 1 mismatch in seed alignment (-N 1), try two set of seeds (-R 2), and set the length of seed substrings to be 16 (-L 16). Mapped small RNA species was quantified to read counts and normalized to RPM as described in sRNABench. Detected species were mapped to mature miRNA only and not precursor miRNA. Reads derived from microRNA with multiple copies in the genome were summed together, and read counts from sample duplicates were aggregated by mean for where it is applicable. All statistical analysis was performed using R version 3.2.

Claims

Claims

1. A method for diagnosing whether an individual is infected from an infectious agent and/or suffers from a disease, the method comprising:

detecting in a sample taken from said individual an RNA contained in extracellular vesicles (EVs) present in said sample, wherein said RNA originates from and/or is associated with said infectious agent and/or disease, and,

finding infection and/or disease when said RNA is detected.

2. The method of claim 1, wherein said sample is a bio fluid, for example selected from blood, serum, plasma, urine, and saliva, preferably serum.

3. The method of any one of the preceding claims, wherein said EVs have a size of 50- 350 nm, preferably 90-300 nm, most preferably 100-250 nm.

4. The method of any one of the preceding claims, wherein said RNA is selected from mRNA, riRNA, tRNA, miRNA, piRNA, Y-RNA, Metazoan Signal Recognition Particle (SRP) RNA, U-RNA, Vault-RNA, snoRNA, and pre-mature RNA.

5. The method of any one of the preceding claims, wherein said RNA contained in said EV has less than 300 nucleotides and/or is a fragment of a larger RNA molecule, said fragment having less than 300 nucleotides.

6. The method of any one of the preceding claims, wherein said infectious agent is a protozoan parasite and said RNA is protozoan RNA.

7. The method of any one of the preceding claims, wherein said infectious agent is a parasite infecting red blood cells (RBCs) of said individual and/or of a mammalian host.

8. The method of any one of the preceding claims, wherein said infectious agent is selected from the group consisting of:

Bartonella henselae, Borrelia burgdorferi, Brucella melitensis, Chlamydia psittaci, Chlamydia trachomatis, Chlamydophila pneumoniae, Francisella tularensis, Legionella pneumophila, Listeria monocytogenes, Mycobacterium leprae, M. tuberculosis, Neisseria meningitidis, Nocardia asteroides, N. brasiliensis, N. caviae, Rhodococcus equi, Salmonella typhi, Yersinia pestis;

fungi including: Cryptococcus neoformans, Histoplasma capsulatum, Pneumocystis jirovecii;

Parasites including: Babesia divergens, B. bigemina, B. equi, B. microfti, B. duncani, Blastocystis spp, Entamoeba histolytica, Giardia lamblia, Plasmodium falciparum, P. vivax,

P. malariae, P. ovale, P. knowlesi, Toxoplamsa gondii, Trichomonas vaginalis, Trypanosoma brucei, T cruzi.

9. The method of any one of the preceding claims, which is for diagnosing any one selected from the group consisting of: bartonellosis, Lyme disease, borreliosis, brucellosis, psittacosis, sexually transmitted infections, Pneumonia, tularemia, legionnaires’ disease, listeriosis, leprae, meningitides, nocardiosis, cellulitis, typhoid fever, plague, malaria, toxoplamosis, Chagas disease, sleeping sickness, cryptococcosis, histoplasmosis.

10. The method of any one of the preceding claims, comprising separating and/or purifying EVs from said sample.

11. The method of any one of the preceding claims, wherein said sample is a blood sample, and wherein said method comprises preparing a plasma sample from said blood sample.

12. The method of claim 11, comprising separating said EVs comprising said RNA from said plasma sample.

13. The method of any one of the preceding claims, comprising separating said RNA from said EVs, and detecting said RNA once said RNA has been separated from said EVs.

14. The method of any one of the preceding claims, wherein said one or more RNA molecules are detected by reverse transcription of RNA contained in said sample, so as to obtain DNA derived from said RNA, and by detecting the presence of said DNA derived from said RNA.

15. A method for assessing the parasitaemia of an infection of an individual by an infectious agent, the method comprising:

quantifying an RNA contained in extracellular vesicles (EVs) present in a sample taken from said individual, wherein said RNA originates from and/or is associated with said infectious agent and/or disease, and,

- wherein a comparatively high amount of said RNA is an indication of a comparatively higher parasitaemia.

16. A method for assessing the presence of resistance towards treatment of an infection in an individual, the method comprising the step of:

- detecting in a sample taken from said individual an RNA contained in extracellular vesicles (EVs) present in said sample, wherein said RNA encodes a product conferring resistance to one or more particular treatments, and,

finding resistance when said RNA is detected.

17. A kit for diagnosis, the kit comprising means for detecting the occurrence of RNA form an infectious agent in a sample of an individual.

18. The kit of claim 16, for conducting the method of any one of claims 1-14.