WO1997030176A1

WO1997030176A1 - Diagnostic assay for plasmodium falciparum

Info

Publication number: WO1997030176A1
Application number: PCT/AU1997/000076
Authority: WO
Inventors: Allan James Saul; Qin Cheng
Original assignee: Queensland Institute of Medical Research QIMR
Current assignee: QIMR Berghofer Medical Research Institute
Priority date: 1996-02-13
Filing date: 1997-02-12
Publication date: 1997-08-21
Anticipated expiration: 1998-08-13
Also published as: AUPN803796A0

Abstract

The present invention relates generally to a diagnostic assay procedure and more particularly to an assay procedure for Plasmodium falciparum malaria parasite. The present invention also provides a diagnostic kit for detecting P. falciparum. The present invention permits for the first time a highly sensitive and quantitative assay for the malaria parasite and greatly facilitates diagnostic and therapeutic regimums for this debilitating disease. The assay of the present invention is particularly useful for monitoring and screening the efficacy of a malarial vaccine.

Description

DIAGNOSTIC ASSAY FOR PLASMODIUM FALCIPARUM

Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description. Sequence Identity Numbers (SEQ ID NOs.) for the nucleotide sequences referred to in the specification are defined following the bibliography.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

The rapidly increasing sophistication of recombinant DNA technology is greatly facilitating research and development in medicine and in allied health fields. However, the diagnosis and treatment of a broad range of tropical diseases has not routinely benefitted from this developing technology. A particularly important and debilitating tropical disease is malaria caused by the malaria parasite, Plasmodium falciparum.

The detection and enumeration of malaria parasites in the blood is essential for managing the treatment of individuals infected with malaria and for epidemiological surveys required for malaria control programs. Microscopic examination is inexpensive and does not require exotic or labile reagents. However, its efficiency is highly dependent on the skill of the trained microscopist. Under ideal conditions, with a proficient operator, microscopic examination has a lower limit of detection of about 20 parasites per microlitre. These limits can only be achieved with a highly trained and proficient microscopist with an extended period of examination. For example, in local practice, two microscopists examine a thick film for 15 minutes each before scoring it as parasite negative. In field situations using 5 minute examination at 100 high power fields, a practical limit of detection is of the order of 40 parasites per microlitre. These levels of detection are comparable to the disease threshold in people with little or no prior malaria exposure. In people deliberately infected with malaria for experimental or therapeutic purposes, approximately 50% were ill on the day before the parasite could be detected (1;2).

Many people travel from areas not endemic for malaria to malarious areas. For example, many Australians travel for business or pleasure to countries such as Papua New Guinea in the South West Pacific, or to countries in South East Asia; people from Europe travel to Africa and people from North America travel to Africa and central America. Although relatively few of these will contract malaria (there are about 1000 cases annually in Australia from this source), many become sick with fever of unknown etiology within a short time of returning to their home. In this case, a major diagnostic imperative is to exclude malaria as a source of the disease, to enable other treatment procedures to be implemented.

Large numbers of tests are done every year for this purpose in developed countries. Although standard microscopy is the only test routinely available, it is a very poor test for this purpose since it has a high false negative rate in the target population. To be useful, a test needs a routine detection level at least 10 fold greater than microscopy (i.e. a detection threshold of <1 parasite per microlitre). Quite commonly, people returning from endemic areas, who get sick, will take anti-malarial drugs prior to clinical examination. In that case, to positively eliminate malaria as a cause of illness, a malaria test needs to be much more sensitive since it needs to be able to detect low frequencies of dead or dying parasites (i.e. a detection limit of <0.1 parasite per microlitre). In recent years, tests other than microcopy have been proposed (3-11). These include revised microscopic tests such as the QBC test from Becton Dickinson and a number of Dip-stick tests for rapid non-microscopic diagnosis. However, these have a detection limit similar to or only marginally greater than conventional microscopy. Tests based on DNA detection and amplification have also been proposed, but none have been commercially developed. Although several of these are able to achieve a high sensitivity, complex and time consuming detection procedures make them difficult and expensive to use in a routine pathology laboratory and take too long to produce a result able to influence the course of treatment.

In accordance with the present invention, a new quantitative polymerase chain reaction based test for malaria is provided. This test is readily able to detect a single parasite and the sensitivity is limited only by sampling considerations. It is 1000 fold more sensitive than microscopy, detection is rapid and the assay inexpensive. The inclusion of an internal control allows positive verification that the test is valid and a comparison of the relative intensity of the internal standard and the test bands gives a measure of the parasite density. The assay of the present invention is particularly useful in determining the efficacy of a malaria vaccine thus reducing the need for expensive field trials based on natural challenge.

Accordingly, one aspect of the present invention contemplates a method for detecting Plasmodium falciparum in a biological sample, wherein said method comprises isolating DNA from said biological sample wherein said DNA would include P. falciparum DNA if present in said biological sample and subjecting said isolated DNA to polymerase chain reaction (PCR) using at least one set of primers from a conserved sequence of a multicopy DNA family in the P. falciparum genome and then detecting the product of said PCR wherein the presence of said PCR product in indicative of the presence of P. falciparum.

Preferably, the conserved sequence is the subtelemeric variable open reading frame which is abbreviated herein to "STEVOR". Surprisingly, although this family of sequences is polymorphic, short conserved regions have been detected which permit PCR primers to be identified. The use of this multicopy family gives a substantial increase in sensitivity compared with amplification of single copy genes.

In a particularly preferred embodiment, the primers are oligonucleotides selected from the list comprising or consisting of SEQ LD NO:l to SEQ LD NO:6, inclusive. These primers are identified herein in Table 1. However, the present invention extends to other suitable sequences within STEVOR.

According to a preferred embodiment, there is provided a method for detecting P. falciparum in a biological sample, said method comprising isolating DNA from said biological sample wherein said DNA would include P. falciparum DNA if present in said biological sample and subjecting said isolated DNA to at least one round of PCR using sets of primers capable of amplifying STEVOR sequences on P. falciparum and then detecting the presence of the PCR product wherein the presence of said product is indicative of the presence of P. falciparum.

Preferably, the primers are selected from the list comprising or consisting of SEQ LD NO: 1 to SEQ LD NO:6, inclusive.

Generally, it is preferred to conduct two or more rounds of PCR. In this case, for example, a first round may be conducted using a first set of primers selected from SEQ LD NO: 1 to SEQ LD NO:6 and the second or subsequent round(s) conducted using a set of primers also selected from SEQ LD NO: 1 to SEQ LD NO:6. Both sets of primers may be the same or different and it is particularly preferred that the second set of primers be nested between the first set of primers.

According to this preferred embodiment, there is provided a method for detecting P. falciparum in a biological sample, said method comprising isolating DNA from said biological sample wherein said DNA would include P. falciparum DNA if present in said biological sample and subjecting said isolated DNA to a first round PCR using a set of primers capable of amplifying STEVOR sequences on P. falciparum and then subjecting the resulting PCR product to a second round PCR wherein at least one of the PCR primers used in the second round PCR is nested between the primers used in the first round PCR and then detecting the presence of the second round PCR product wherein the second round PCR product is indicative of P. falciparum.

Preferably, the primers are selected from SEQ LD NO: 1 to SEQ LD NO:6.

The biological sample used in accordance with the present invention comprises any source of blood cells potentially parasitized with P. falciparum. Generally, whole blood is used from a human subject.

For high sensitivity, there are two problems to be overcome. First, the need to sample an adequate volume of blood to ensure parasites are present. Second, the ability to amplify the trace parasite DNA in a huge excess of human DNA present in the blood sample. One solution to this latter problem is to deplete the human DNA by first removing or decreasing the number of white blood cells. Although this embodiment is encompassed by the present invention, it does tend to add to the complexity of the subject assay. Accordingly, in a preferred embodiment, the PCR technique is modified which reduces or avoids the need for extra sample preparation steps to be undertaken. In this particularly preferred embodiment, an assayable amount of blood is taken such as from about 10 μl to about 50 ml, preferably from about 20 μl to about 20 ml, more preferably from about 100 μl to about 10 ml and most preferably around about 100 μl. The red blood cells are lysed and the total DNA extracted in an appropriate volume of water. Conveniently, approximately 1 ml of water is used. Such a solution contains a very high concentration and may require the addition of higher taq polymerase and magnesium ion concentration. In any event under these conditions, a detection limit is achieved of about 10 parasites per ml or about 1000 fold greater sensitivity than conventional microscopy.

Although the preferred biological sample comprises whole blood it extends to any preparation with may contain P. falciparum. Although PCR is the preferred detection means, the present invention extends to any nucleic acid-based detection means such as nucleic acid hybridisation techniques. The invention further encompasses the use of different assay formats of nucleic acid-based detection means, including restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), single-strand chain polymorphism (SSCP), amplification and mismatch detection (AMD), interspersed repetitive sequence polymerase chain reaction (LRS-PCR), inverse polymerase reaction (iPCR) and reverse transcription polymerase chain reaction (RT-PCR), amongst others.

Where the detection means is a nucleic acid hybridisation technique, the Plasmodium target nucleic acid may be labelled with a reporter molecule capable of producing an identifiable signal (e.g. a radioisotope such as ³ P or ^3SS or a biotinylated molecule).

The present invention extends to all lines, sub-species and varieties of P. falciparum and may be modified to permit distinction between variants of the malaria parasite.

The assay protocol of the present invention may also include an internal control to facilitate measuring the relative amount of parasite DNA in the blood sample. For example, plasmid DNA carrying the STEVOR sequence may be amplified and its concentration determined using any convenient means, such as UV spectroscopy. Conveniently the plasmid DNA control is added to the blood before extraction of total DNA.

The present invention further contemplates a kit for detecting P. falciparum in a biological sample, said kit comprising in compartmentalised form a first container adapted to contain a set of primers based on a STEVOR sequence, optionally a second container adapted to contain a second set of primers based on a STEVOR sequence wherein at least one primer in the second set is nested between the primers of the first set, a third container adapted to contain reagents for PCR and optionally a fourth container to receive the biological sample. Other containers may be included in order to facilitate the PCR. The kit may also include electrophoretic apparatus, detecting reagents and instructions for use. Another aspect of the present invention is directed to the use of primers based on the STEVOR sequence in a PCR to detect P. falciparum DNA in a biological sample.

Still a further aspect of the present invention contemplates an agent comprising one or more sets of primers based on the STEVOR sequence for use in a PCR to detect P. falciparum DNA in a biological sample.

The present invention is further described by the following non-limiting Figures and/or Examples.

In the figures:

Figure 1 is a photographic representation showing amplification of the STEVOR sequence from 13 laboratory lines of P. falciparum.

Figure 2 is a photographic representation showing a comparison of the amplification of the STEVOR sequence with a single copy gene (MSA-1 pgl9). PCR reaction mixtures contained: 1) 200pg; 2) 20pg; 3) 2pg; 4) 0.2pg; 5) 0.02pg; 6) 0.002pg; 7) 0.0002pg; and 8) Opg of total P. falciparum DNA, respectively.

Figure 3 is a photographic representation showing standards for the detection of malaria parasites in blood. DNA in 3ml blood samples containing 300, 100, 30, 10 or 0 parasitised red cells per ml was extracted and amplified as detailed in Example 2. The resulting amplified DNA after separation by gel electrophoresis gives a 188bp product derived from the parasite DNA and a 133bp product from the internal control. The total amount of DNA amplified is approximately constant, hence, the ratio of parasite to control band is an indicator of the number of parasites present in the sample.

Figure 4 is a photographic representation of amplified DNA extracted from the blood of a volunteer infected with malaria. The volunteer was infected on day 0 and samples taken on day 1 and then twice daily (day 2 to day 9, morning (am) and afternoon/evening (pm) sample). Duplicate assays at each time point are shown on this figure. A trace of parasites were detected on day 1, day 2 am and pm samples, with a big increase in the amount of parasite DNA (upper band) in the pm sample from day 4. Parasites were detected by microscopy on day 9.

Figure 5 is a graphical representation showing parasite growth curves for blood induced infections in volunteers, measured every 24 hours. Numbers in the legend represent the volunteers.

EXAMPLE 1

Ethical considerations

Protocols for preparing the inoculum, and for recruiting and infecting volunteers, were reviewed and approved by the Queensland Institute for Medical Research Ethics Committee Brisbane, Queensland, Australia and the Healthy Volunteer Studies Research Ethics Sub- Committee, Lothian Health Board, Edinburgh.

EXAMPLE 2 Inoculum

Volunteers had no serological evidence of infection with syphilis, hepatitis A, B, C, HTLV1 or HIV and Ross River Virus. Hepatitis G and parvovirus were not detected by PCR. Full blood counts and liver function tests were normal.

Laboratory-reared Anopheles stephensi mosquitoes were infected with P. falciparum clone 3D7A (a chloroquine sensitive line) by membrane feeding on a blood meal containing gametocytes. The parasites were cultured in vitro under standard gametocyte culture conditions (13, 14), harvested after 14 and/or 7 days and mixed with fresh red blood cells and heat inactivated serum (supplied by the Edinburgh and South East Scotland Blood Transfusion Service, UK) to form the infectious blood meal. Five day old mosquitoes, previously starved for 48 hours, were fed on this material through a membrane feeder (13, 14) and an uninfected blood meal was given 4 days later to increase the number of sporozoites in the salivary glands (15). Ten and fourteen days after the infectious blood meal, mosquitoes were fed on the volunteers until all, or nearly all, mosquitoes had taken a full blood meal. The fed mosquitoes were then killed with chloroform and dissected to assess the extent and level of sporozoite infection in the salivary glands.

On day zero, one volunteer (volunteer 1) was exposed to the bite of 22 mosquitoes of which 7 were sporozoite positive. On day 2, the volunteer was exposed to the bite of 3 more mosquitoes, 2 of which were sporozoite positive. Another volunteer (volunteer 2) was exposed to the bite of 25 mosquitoes on day zero of which 3 were sporozoite positive and a further 8 on day 2, 6 of which were sporozoite positive. Parasitaemia in both volunteers was followed daily by microscopy and PCR from day 4 to day 21 after infection.

A unit of blood (500 ml) was taken from both volunteers 6 hours after they became ill and developed high fever (13 and 14 days after the initial mosquito bites respectively) when both were microscopically positive. The parasitaemia in the blood before processing was determined by counting the ratio of parasites to leucocytes on thick films, and using the measured leucocyte and red cell concentrations to calculate the proportion of red cells infected. The figure was used as the basis of calculating parasite inocula sizes.

Volunteers were treated with standard doses of oral chloroquine immediately after blood donation. Leucocytes were removed using a BPF4™ leucocyte filter (Pall Biomedical Products Corp., East Hills, NY). The red blood cells were mixed with Glycerolyte 57 Solution (Baxter, Deerfield, LL) and cryopreserved in 1 ml aliquots following a protocol from the American Association of Blood Banks Technical Manual. Over the following 3 months, both volunteers recorded their temperature daily. They were repeatedly checked for seroconversion to the diseases mentioned above over 12 months.

EXAMPLE 3 Primers and PCR conditions

Primers corresponding to the conserved regions in STEVOR were chosen to achieve high sensitivity. The primers are shown in Table 1. To avoid spuriously amplified human DNA, it is preferable to perform a nested PCR. Although the conditions for PCR may be varied, a particularly useful methodology employed a first round PCR using primers P5, P18, P19 and P20.

The DNA contained control plasmid DNA prior to total DNA extraction. The first round 100 μl reaction included 20 μl of the 1 ml DNA solution isolated from blood; 150 ng of P5, P18, P20 and 200 ng of P19; 0.02 mM dNTPs (Promega, Madison, WI), 7.5 mM Mg⁺⁺, 2.5U Taq polymerase (Perkin Elmer, Norwalk, CT) was performed for 22 cycles (93 °C, 30 sec, 50°C, 50 sec and 72°C, 30 sec) using a DNA Engine (MJ Research, Watertown, MS). Primers P17 were used in a 50 μl second round PCR. This was performed using 2 μl of the first round reaction with 75ng of each primer, 3.5 mM Mg⁺⁺, 0.2mM dNTPs and 1.25U Taq polymerase with cycling conditions 93 °C, 30 sec, 55 °C, 50 sec and 72 °C, 30 sec for 40 cycles.

Five to 10 μl of PCR product was loaded on a 3% w/v agarose (FMC BioProducts, Rockland, ME) gel and electrophoresed for 25 min at 120 volts. Following electrophoresis, a digital fluorescence image of the gel was captured with a NovaLine gel documentation system (Novex, Australia) and the relative fluorescence (F_R) in the parasite band compared with the total fluorescence (parasite+internal control) was calculated.

EXAMPLE 4

Comparison of sensitivity of PCR of single copy gene and of STEVOR sequences

The sensitivity of amplification of STEVOR was compared to that of the gp 19 fragment of the single copy gene MSA-1. The primers used are shown in Table 1.

A 50μl reaction containing 2μl of serially diluted DNA extracted from cultured parasites, 75ng of primers P17 and P24 or P. falciparum MSA-1 gpl9 primers, 4mM MgCl₂, 200μM dNTPs (Promega) and 1.25U Taq polymerase (Perkin Elmer/Cetus) was used. The cycling conditions were: 95 °C 30 sec, 55 °C 50 sec and 72°C 30 sec for 40 cycles. Five μl of the PCR product was loaded on a 3% w/v agarose gel containing 25 ng/ml ethidium bromide, electrophoresed and visualised on a UV transilluminator. Following electrophoresis, a fluorescence image of the gel was captured with a NovaLine gel documentation system (Novax). This uses a CCD camera with a HOYA HMC 46 mm O(G) filter to capture a digital image. The image was stored as a TLF file and the relative fluorescence of each band determined using ImageQuant software (Molecular Dynamics).

Using these conditions, the primer pair P17 and P24 amplified a single band of 188bp from 0.002 pg of parasite DNA following one round of 40 cycles of amplification, which is equivalent to one tenth of a parasite (Figure 2). This band was not observed when amplifying from uninfected human DNA or P. vivax infected samples. PCR of the MSA-1 gene detected 2pg of parasite DNA. This was 1000 fold less sensitive than the PCR of STEVOR (Figure 2).

Because the target sequence is present in many copies, PCR detection based on this sequence family is much more sensitive than PCR of single copy genes. Figure 1 demonstrates the difference in sensitivity obtained using a single round of PCR with primers hybridising to the STEVOR sequences and to a single copy gene, PfMSP 1. Furthermore, the assay of the present invention amplified DNA from all 13 P. falciparum lines tested (Figure 1) which indicates that the assay will be useful world wide.

EXAMPLE 5

Preparation of a standard curve for the high sensitivity detection of parasites

Red blood cells infected with ring stage parasites of the 3D7 line of P. falciparum were added to 3 ml aliquots of freshly drawn human blood. DNA was isolated from the 3 ml samples using a modified salting method originally described by La iri and Numberger (12). Samples were diluted to 10ml using lOmM Tris pH7.6, lOmM KC1, lOmM MgCl₂ and 2mM EDTA, and red blood cells lysed by adding lOOμl of 10% v/v saponin. Parasites were pelleted and washed with the same buffer twice (14,500xg for 15 min). A volume of 1.6 ml of the above buffer containing 0.4M NaCl was added to the parasite pellet plus lOOμl of 20% w/v sodium dodecyl sulphate and incubated at 55 °C for 5-1 Omin. An amount of 0.0045pg of the plasmid DNA was added to the solution. After adding 0.6ml of 6M NaCl the samples were spun at 2400xg for 5 min. Supematants were transferred to fresh tubes and DNA precipitated by adding 2 vol of ethanol. After washing with 75% v/v ethanol DNA was air dried and redissolved in 1ml of water.

Twenty microlitres of the 1 ml DNA solution isolated from blood, 200ng of P19 with 150ng each of primers 20, 18 and 5, and the above described PCR reagents were used in a lOOμl reaction volume of first round PCR (22 cycles of 95°C 30sec, 50°C 50sec and 72°C for 30 sec). Two microlitres of the first PCR product were transferred to a second round PCR tube where primers 17 and 24 were used and 40 cycles performed (95°C 30 sec, 55°C 50sec and 72°C for 30 sec). Five microlitres of the PCR product was electrophoresed and the intensity of the bands quantitated as described above. An amount of 30 parasites in 3ml of blood were detected reproducibly giving a 188bp product. The internal control DNA that was co-processed and co-amplified with the parasite DNA, gave a band of 133 bp. As shown in Figure 3, the relative brightness of the control and test bands varied depending on the number of parasites present in the blood sample. In uninfected blood where only human DNA was present, the control bands is very bright. As the parasitaemia increased, the control band became weaker and then disappeared. The ratios between these bands at different parasitaemia were measured in triplicates from each of 3 individuals (n=9), and are shown in Table 2.

EXAMPLE 6 High sensitivity detection of parasites in an infected volunteer

Blood from a volunteer infected by the injection of 3000 parasitized red blood cells was collected twice daily. The parasitaemia in each blood sample was measured by the assay described in this patent and by conventional microscopy.

As shown in Figure 4, the parasitaemia was just detectable on day 2, then increased over the next 6 days. As expected for synchronized parasites there was a substantial increase in parasitaemia on every other day. Parasites were first detected by microscopy on day 12.5 at approximately 20,000 parasites per ml when the volunteer was treated. Parasite DNA levels fell rapidly, but could still be detected for 6 days following treatment. EXAMPLE 7 Internal control

5 The assay can use internal control DNA for measuring the relative amount of parasite DNA in the blood sample. Specifically, one internal control molecule was engineered by cloning the 7H8/6 sequence with a deletion from the Bglll to AccI sites (from basses -13 to 99 and 154 to 300 numbered according to GeneBank sequence PFA7H86) into pUC18. DNA from the M13 phage containing the 7H8/6 insert was amplified in a PCR with the P5 and P17 primers and the

10 product further amplified with the P5 and P19 primers. This product was cut with restriction enzymes Bgl II and Accl into three fragments. The 3' and 5' fragments were purified by preparative electrophoresis, ends filled, and ligated. PCR amplification of the ligated product with primers P5 and P19 gave a 253 bp product which was cloned into the vector pUClδ and transfected into K coli. Plasmid DNA was extracted from E. coli cultures, purified, linearised

15 with BamHI, and the concentration determined by UV spectroscopy. In routine assays, 9 x 10^"16g of plasmid DNA (30 molecules) are added to a PCR mixture.

EXAMPLE 8 Blood stage challenge infections 0

After the donors of the infected blood had been followed for 12 months with no evidence of any illness as judged by non-specific indicators such as fever or by seroconversion to the specific pathogens tested during screening, five healthy males aged 30 to 64 years were individually infected over a period of 6 months. Volunteers 1 and 2 were the same volunteers who had

25 undergone mosquito infections 1 year earlier and were negative to PCR at the time of reinfection. Volunteers 3-5 did not have a history of malaria.

Parasites were thawed using solutions licensed for clinical use. An amount of 200 μl of 12% w/v saline was added dropwise to 1 ml of thawed infected blood, left for 5 min, and a further

30 10 ml of 1.6% w/v saline was added dropwise. The parasitized blood was centrifuged for 5 min at 400 x g, the supernatant removed and 10 ml 0.9% w/v saline added dropwise. The cell pellet was washed two more times with 0.9% saline and resuspended in normal saline for injection. No leucocytes were detected by microscopy in 3 μl of thawed, packed washed red cells. Removal of plasma components was monitored by adding ¹²⁵I labelled protein A to a vial of cells immediately after thawing. Following the final wash, remaining ¹²⁵I had been reduced by a factor of 10⁶.

Volunteer 1 received 3000 of the volunteer's stored infected red cells on day zero. Volunteers 2-5 received 3000, 3000, 6000 and 300 stored infected red cells, originally collected from volunteer 2. In this stored blood, there were 400 infected cells per μl of packed cells. All volunteers were followed at least daily by PCR and after day 6 of infection, by microscopy as well. Ten ml of venous blood was taken at 24 hour intervals for volunteer 2 and 12 hour intervals for the rest of the volunteers. Volunteers 1 to 3 were treated with chloroquine after they became positive by microscopy (day 8 to day 9) whereas volunteers 4 and 5 were treated when parasitaemia, measured by PCR, first reached 500 to 1000 parasites per ml.

The viability of thawed parasites was investigated by a limiting dilution assay followed by PCR. The thawed inoculum was plated in 96-well plates at a theoretical 30, 10, 3, 1, 0.3 and 0.1 parasites/well based on post-thawed haematocrit counts and the microscopically determined parasitaemia prior to freezing. 100 μl of culture medium containing 5 μl of uninfected red cells was added and the plates were incubated for 9 days. Half of each culture was transferred to a new plate and lysed by adding 100 μl of 0.1 saponin in TKMl solution. Parasites were pelleted by centrifuging the plate, then resuspended in 100 μl TKMl and re-pelleted. Each pellet was then resuspended in 20 μl of H₂O and transferred to a well of a PCR plate. The plate was heated at 95 °C for 10 min. Thirty μl of PCR stock solution was added to each well containing 75 ng of primers P17 and P24, 1.25U Taq and 0.005 pg control DNA. Forty cycles of PCR were performed using the conditions of the second round of the nested PCR. Reaction products were electrophoresed on a 3% w/v agarose gel and the number of positive and negative wells used to calculate the number of viable parasites. Using the assay, parasites (about 10 per ml) were first detected on day 5.5 (Volunteer 2) and 6.5 (volunteer 1) after the first bites of sporozoite-infected mosquitoes. Parasitaemias increased until day 12.5 (volunteer 1) or 13.5 (volunteer 2) when blood was collected and volunteers treated. Parasites were first detected by thick film microscopy concurrent with symptoms at day 12.5 in both subjects. Parasitaemias at the blood collection, determined by microscopy, were 19 per μl (volunteer 1) and 290 per μl (volunteer 2). Both volunteers became microscopically negative on days 15.5, 2-3 days after the treatment. Five volunteers were infected with various number of parasites. By the limiting dilution assay, the recovered parasites were fully viable. Volunteers 1 to 4, infection with 3000 to 6000 parasites, gave similar parasite growth curves (Fig. 5). The curve for volunteer 5 who received 1/10 the inoculum parallelled the other volunteers but was delayed by 2 days. As expected from an inoculum consisting entirely of circulating ring stage parasites, the parasitaemia in the volunteers showed partial synchrony. The largest increase in parasitaemia occurred on odd days with a similar increase, or even a decrease on even days (e.g. day 6) as mature parasites sequestered.

To take account of this growth pattern, the growth rates and the effective starting parasitaemia were determined using a maximum likelihood fit to a model assuming that the inoculum had two broods of parasites separated by 24 hours and that parasites older than 24 hours would be sequestered. Thus, this program allows estimates of growth rates where combination of synchronous parasites and sequestration results in decreased circulating parasitaemia on certain days. After taking synchrony and sequestration in to account, all volunteers had a constant growth rate over the period of the infections of 15.5±2.8, 15.6±2.8, 13.0±3.0 and 17.2±3.2 fold per cycle (48 hours) for volunteers 2, 3, 4 and 5 respectively, with 39% of parasites dividing in the first 24 hours and the remainder over the second 24 hours. The estimated starting parasitaemia derived from the curve fitting was 37% lower than the estimate based on both microscopic counts and on the number of viable parasites determined by limiting dilution in the inoculum. However, all three measures have uncertainties. In particular, errors in calculating the growth rates and starting parasitaemia are negatively correlated. Growth rates calculated assuming a starting parasitaemia based on the microscopic estimate were 12.1±0.1, 12.2±0.1, 9.3±0.6 and 13.9±0.4 fold per cycle, respectively.

Volunteers 1, 2 and 3 were treated with chloroquine in the evening of day 8 (volunteers 1 and 2) and the morning of day 9 (volunteer 3) when symptoms occurred. Parasites were confirmed by thick film examinations in the three volunteers. Parasitaemia decreased rapidly after treatment. Volunteers 4 and 5 were treated on day 6 and 8, respectively, when parasitaemia first reach 500 to 1000 per ml. No symptoms were noted by these volunteers prior to and during the treatment.

EXAMPLE 9

Test modifications

As the basic test has such high sensitivity, there is an opportunity to trade off sensitivity for ease of use. For example, where a sensitivity close to the disease threshold is required, more rapid DNA extraction methods may be employed from small blood volumes rather than those used in the Examples.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. TABLE 1

Primer Sequence SEQ LD NO:

P5 gggaattcTTTATTTGATGAAGATG* 1

P17 ACATTATCATAATGA(C/T)CCAGAACT 2

P18 TTTC(A C)TCACCAAACATTTCTT 3

P19 AATCCACATTATCATAATGA 4

P20 CCGATTTTAACATAATATGA 5

P24 GTTTCCAATAATTCTTTTTCTATC 6

* sequence homologous to the members of STEVOR family are in upper case

TABLE 2 Relative intensity of the parasite band and parasitaemia

Ratio % (P/P+C)* Parasitaemia

MEAN ± SD (per ml)

O ± O 0

50.92 ± 16.11 10

72.35 ± 7.73 30

86.63 ± 4.66 100

97.04 ± 2.96 300

* P. Intensity of the parasite band; C: Intensity of the control band

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12. Lahiri, D.K. and Numberger, Jr J. I. (1991) Nucleic Acids Research 19(19): 5444.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: THE COUNCIL OF THE QUEENSLAND INSTITUTE OF MEDICAL RESEARCH

(ii) TITLE OF INVENTION: DIAGNOSTIC ASSAY FOR PLASMODIUM

FALCIPARUM

(iii) NUMBER OF SEQUENCES: 6

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: DAVLES COLLISON CAVE

(B) STREET: 1 LITTLE COLLINS STREET

(C) CITY: MELBOURNE

(D) STATE: VICTORIA

(E) COUNTRY: AUSTRALIA

(F) ZLP: 3000

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: LBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: PCT International

(B) FILING DATE: 12-FEB-1997

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: PN8037 (AU)

(B) FILING DATE: 13-FEB-1996

(viii) ATTORNEY/AGENT INFORMATION: (A) NAME: HUGHES DR, E JOHN L

(C) REFERENCE/DOCKET NUMBER: EJH EK (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: +61 3 9254 2777

(B) TELEFAX: +61 3 9254 2770

(2) INFORMATION FOR SEQ LD NO: 1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ LD NO:l :

GGGAATTCTT TATTTGATGA AGATG 25

(2) INFORMATION FOR SEQ LD NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ LD NO:2:

ACATTATCAT AATGA(C/T)CCAG AACT 24 (2) INFORMATION FOR SEQ LD NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ LD NO:3:

TTTC(A/C)TC ACC AAAC ATTTCT T 21

(2) INFORMATION FOR SEQ LD NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ LD NO:4:

AATCCACATT ATCATAATGA 20 (2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ LD NO: 5:

CCGATTTTAA CATAATATGA 20

(2) INFORMATION FOR SEQ LD NO:6:

(i) SEQUENCE CHARACTERISTICS.

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ LD NO:6:

GTTTCCAATA ATTCTTTTTC TATC 24

Claims

1. A method for detecting Plasmodium falciparum in a biological sample said method comprising isolating DNA from said biological sample and subjecting said isolated DNA to polymerase chain reaction (PCR) using at least one set of primers from a conserved sequence of a multicopy DNA family in the P. falciparum genome and then detecting the product of said PCR wherein the presence of said PCR product in indicative of the presence of P. falciparum.

2. A method according to claim 1 wherein the multicopy DNA family is the subtelemic variable open, reading frame (STEVOR).

3. A method according to claim 2 wherein at least one set of primers comprises oligonucleotides selected from SEQ LD NO: l to SEQ LD NO:6.

4. A method according to claim 1 or 2 or 3 wherein at least two rounds of PCR are performed.

5. A method for detecting P. falciparum in a biological sample, said method comprising isolating DNA from said biological sample and subjecting said isolated DNA to a first round PCR using a set of primers capable of amplifying STEVOR sequences on P. falciparum and then subjecting the resulting PCR product to a second round PCR wherein at least one of the PCR primers used in the second round PCR is nested between the primers used in the first round PCR and then detecting the presence of the second round PCR product wherein the second round PCR produce is indicative of P. falciparum.

6. A method according to claim 5 wherein the primers for each round of PCR are selected from SEQ LD NO:l to SEQ LD NO:6.

7. A method according to claim 1 or 5 wherein the biological sample comprises any source of blood cells potentially parasitized with P. falciparum.

8. A method according to claim 7 wherein the biological sample is whole blood.

9. A method according to claim 1 or 5 comprising the additional step of removing or decreasing the number of while blood cells in the biological sample prior to isolating DNA.

10. A kit for detecting P. falciparum in a biological sample, said kit comprising in compartmentalised form a first container adapted to contain a set of primers based on a STEVOR sequence, optionally a second container adapted to contain a second set of primers based on a STEVOR sequence wherein at least one primer in the second set is nested between the primers of the first set, a third container adapted to contain reagents for PCR and optionally a fourth container to receive the biological sample.

11. A kit according to claim 10 wherein the primers are oligonucleotides selected from SEQ LD NO: l to SEQ LD NO:6, inclusive.

12. Use of primers based on STEVOR sequence in a PCR to detect P. falciparum in a biological sample.

13. Use according to claim 12 wherein the primers are oligonucleotides selected from SEQ LD NO: l to SEQ LD NO:6, inclusive.

14. An isolated oligonucleotide comprising a nucleotide sequence selected from SEQ LD NO:l to SEQ LD NO:6, inclusive.

15. An agent for use in detecting P. falciparum in a biological sample said agent comprising at least two oligonucleotides of claim 14.