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`Patent Office
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`EP 2 351 857 A1
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`(12)
`
`EUROPEAN PATENT APPLICATION
`
`(43) Date of publication:
`03.08.2011 Bulletin 2011/31
`
`(51) Int CL:
`C 120 1/68 (2005“)
`
`(21) Application number: 10190717.8
`
`(22) Date of filing: 14.02.2006
`
`
`(84) Designated Contracting States:
`AT BE BG CH CY CZ DE DK EE ES FI FR GB GR
`HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI
`SK TR
`
`(30) Priority: 17.02.2005 IT RM20050067
`25.05.2005 US 137934
`16.06.2005 US 691186 P
`10.02.2006 US 351799
`
`(62) Document number(s) of the earlier application(s) in
`accordance with Art. 76 EPC:
`06735068.6 / 1 885 877
`
`(72) Inventors:
`0 Milkonyan, Hovsep
`Princeton, NJ 08540 (US)
`0 Cannas, Angela
`l-09031, Arbus (IT)
`0 Tomei, Louis, David
`l-00030, Home (IT)
`0 Umansky, Samuil, R.
`Princeton, NJ 08540 (US)
`
`(74) Representative: Clarke, Victoria et al
`Mintz Levin Cohn Ferris Glovsky and
`Popeo Intellectual Property LLP
`Alder Castle
`10 Noble Street
`
`(71) Applicant: TrovaGene, Inc.
`London EC2V 7JX (GB)
`San Diego, CA 92121 (US)
`
`
`(54)
`
`Compositions and methods for detecting pathogen specific nucleic acids in urine
`
`The invention is based upon the discovery that
`(57)
`small nucleic acids from non-viral pathogens are able to
`cross the kidney and are present in urine of a subject
`when the subject, is infected with the non-viral pathogen.
`These transrenal DNAs are especially prevalent at small-
`
`ersizes under aboutSOO bp). Thusthe invention provides
`compositions and methods for the diagnosis of infection
`of a subject with non- viral pathogens through the detec-
`tion of transrenal nucleic acids from those pathogens in
`the urine of the subject.
`
`8 Healthy Individuals
`
`
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`20 TB —infected + 1 Positive Control
`
`
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`Printed by Jouve, 75001 PARlS (FR)
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`EP2351857A1
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`EP 2 351 857 A1
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`Description
`
`BACKGROUND OF THE INVENTION
`
`[0001] There are curre ntlythree types of in Vitro diagnostic systems widely used forthe detection of non—viral pathogens.
`These are direct culture of the pathogenic agentfrom the biological sample; immunological assays based on the detection
`of products or antigens of the infectious agent; and indirect immunological assays that can detect antibodies produced
`against the infectious agent during infection.
`[0002]
`In the first system, the principal disadvantage is that the biological sample must be considered to be at risk for
`the transmission of the pathogenic agent. In the second and third systems, the disadvantages include sample retrieval
`that is often invasive and potentially infective sample when collected. In the third system, one major disadvantage is
`that there is often little possibility of discriminating between past and current infections.
`[0003] More recently, molecular diagnostic methods have been developed based on the detection ofthe nucleic acids
`of the pathogenic agent in the blood or plasma samples, or in the cell cultures, taken from the patient. These assays
`are generally much more sensitive than the immunological assays. However, they may require the presence of special
`equipment and qualified personnel. Furthermore, the biological samples - in the case of plasma, blood, or cell cultures
`- are difficult to store unaltered, except under controlled temperature conditions, and are considered to be biohazardous
`to personnel who handle them.
`[0004] Recently, molecular diagnostic methods based on transrenal DNA (Tr—DNA) have been described for monitoring
`the progress of allogeneic transplants, to diagnose the sex of a fetus, and to detect the presence of tumor markers.
`(Botezatu et al. Clinical Chemistry 46(8):1078-84 (2000); Su et al. Ann. NY Acad. Sci. 1022:81-89 (2004)) For example,
`U.S. Patent No. 6,251,638 describes an analytical method for detecting male fetal DNA in the urine of pregnant women.
`U.S. Patent No. 6,287,820 describes a system aimed at the diagnosis of tumors, particularly of adenocarcinomas of the
`colon and pancreas. U.S. Patent No. 6,492,1 44 teaches that the Tr-DNA nucleic-acid analysis method may be used to
`monitor the progress of allogeneic transplants. The presence oftransrenal DNA in urine,
`in the form of nucleic-acid
`fragments of fewerthan 1000 base pairs was also described in Al-Yatama et al. (2001), Prenat Diagn, 21 :399-402; and
`Utting, M., et al. (2002), Clin Cancer Res, 8:35—40. Keiko Koide, et al., Prenat Diagn, 2005; 25: 604—607; Mengjun Wang,
`et al.., Clinical Chemistry, 2004, 50: 211-213; Y.-H. Su, et al., J. Mol. Diagn., 2004,6:101-107.
`[0005] The presence of transrenal DNA has been explained through the apoptosis phenomenon. During cell death
`most of the nuclear DNA is converted into nucleosomes and oligomers (Umansky, S.R., et al. 1982, Biochim. Biophys.
`Acta 655:9—17), which are finally digested by macrophages or neighboring cells. However, a portion of this degraded
`DNA escapes phagocytic metabolism, and can be found in the bloodstream (Lichtenstein, A.V., et al. 2001, Ann NY
`Acad Sci, 945:239-249), and, as confirmed in the above-indicated patents, also in urine.
`[0006] The application of this system to pathogenic microorganism infections has never been studied. Previously, it
`was only known that prokaryotic DNA could be isolated from urine sediment that contained bacteria (Frasier, et al. 1992,
`Acta Virol, 36:83-89). During a pathogenic infection, prokaryotes and parasites are generally ingested by the cells of the
`immune system, such as macrophages and dendritic cells. The prokaryotes are then dissolved by the phagolysosome
`vesicles. The prokaryotic DNA is then released by the cell and a portion of this DNA enters the bloodstream in either of
`two ways. Eitherthe ingesting cell becomes apoptotic and breaks apart (Navarre, W.V. 2000; Cell Microbiol 2:265-273);
`orthe phagolysosome vesicles release the fragments of the prokaryote (including the fragmented DNA) into the blood-
`stream (Friedlander, AM. 1978, Infect Immune 22:1 48—1 54). However, these fragmented nucleic acids have never been
`detected in the urine of the infected subject.
`[0007] The instant invention describes a method of detecting the presence of non-viral pathogens in a subject through
`the detection of DNA sequences from those pathogens in the urine of the subject.
`
`SUMMARY OF THE INVENTION
`
`[0008] The invention provides compositions and methods for the diagnosis of an infection of a subject through the
`detection of the presence of pathogens by the presence of pathogen nucleic acids in a urine sample of the subject. The
`methods of the invention are also used to validate the diagnosis of an infection of a subject through the detection of the
`presence of pathogen nucleic acids in a urine sample of the subject, wherein a previous diagnosis has already been
`performed by a method of this invention or another diagnosis method. The compositions and methods of the invention
`are also used to determine the efficacy of a treatment of non-viral pathogen infection in a subject by detecting the
`presence or absence of a nucleic acid of the non—viral pathogen in the urine of the subject. More specifically, the
`compositions and methods ofthe invention are used to identify a drug resistant non-viral pathogenic infection in a subject
`by detecting the presence or absence of a nucleic acid of the non—viral pathogen in the urine of the subject after the
`subject has been treated for the infection with a drug. The compositions and methods of the invention are also used to
`determinethe likelihood of pathologyfromthe infection ofthe subject by a non-viral pathogen in asubject at risktherefrom.
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`More specifically, the compositions and methods of the invention may be used to genotype a non-viral pathogen which
`has infected a subject by detecting and genotyping nucleic acids from the pathogen in the urine of the subject.
`[0009] The invention provides a method of isolating a nucleic acid from urine, the method comprising providing urine
`from a subject; separating cells and cell debris from the urine by filtration or centrifugation; adding EDTA and Tris-HCI
`to the urine; adding a chaotropic salt to the urine; adding a resin, wherein the resin bindsthe nucleic acid in the presence
`of the chaotropic salt; removing the resin from the urine; and eluting the nucleic acid from the resin; thereby isolating
`the nucleic acid from urine. In one embodiment of the method of isolating a nucleic acid from urine, the concentration
`of EDTA and Tris-HCI after it is added to the urine is about 10 mM, and pH between about 8.0 and about 8.5.
`[0010]
`In another embodiment of the method of isolating a nucleic acid from urine, the filtration is performed with a
`filter with a pore size between about 0.1 pm and about 5.0 pm.
`[0011]
`In another embodiment of the method of isolating a nucleic acid from urine, the chaotropic salt is guanidine
`isothiocyanate. In one aspect of this embodiment, the guanidine isothiocyanate has a concentration after being added
`to the urine of at least about 3 M, and at most about 6 M.
`[0012]
`In another embodiment of the method of isolating a nucleic acid from urine, the resin is constructed of silica.
`[0013]
`In another embodiment of the method of isolating a nucleic acid from urine, the subject is a mammal. In one
`aspect of this embodiment, the mammal is a human.
`[0014] The invention also provides a method of detecting a non-viral pathogen in a subject comprising detecting the
`presence of a nucleic acid from the non-viral pathogen in urine of the subject, wherein the non-viral pathogen is selected
`from Helicobacterpylori, Mycobacterium tuberculosis, Bacillus anthracis, Plasmodium species and Lelshmania species.
`In one embodiment of the method of detecting a non—viral pathogen in a subject, the method furthercomprises the step
`of quantitating the nucleic acid.
`[0015]
`In another embodiment ofthe method of detecting a non-viral pathogen in a subject, the detecting is performed
`by a method selected from polymerase chain reaction (PCR), nested PCR, semi—nested PCR, hybridization, Single—
`Strand Conformation Polymorphism analysis (SSCP), ligase chain reaction (LCR), strand displacement amplification
`(SDA), and pairing with molecular probes that are specific forthe non-viral pathogen.
`[0016]
`In another embodiment of the method of detecting a non-viral pathogen in a subject, the subject is a mammal.
`In one aspect of this embodiment, the mammal is a human.
`[0017] The invention also provides a method for diagnosing a Helicobacter pylori infection in a subject, comprising
`detecting the presence of a Helicobacter pylori nucleic acid in a urine sample from the subject, thereby diagnosing the
`Helicobacrerpy/oriinfection. In one embodiment of the method for diagnosing a Helicobacterpyloriinfection in a subject,
`the method further comprises the step of quantitating the nucleic acid.
`[0018]
`In another embodiment of the method for diagnosing a Helicobacterpylori infection in a subject, the detecting
`is performed by a method selected from PCR, nested PCR, semi-nested PCR, hybridization, SSCP, LCR, SDA, and
`pairing with molecular probes that are specific for the non—viral pathogen. In one aspect of this embodiment, a primer
`set is used in the method, and the primer set comprises a forward primer and a reverse primer. Optionally, the forward
`primer is selected from SEQ ID NOs: 5, 8,10,11,12 and 15 and the reverse primer is selected from SEQ ID NOs: 6, 7,
`9,13,14,16 and 17.
`[001 9]
`In anotherembodiment ofthe method fordiagnosing a Helicobacterpyloriinfection in asubject, the Helicobacter
`pylorinucleic acid comprises transrenal DNA.
`[0020]
`In another embodiment of the method for diagnosing a Helicobacterpylori infection in a subject, the subject is
`a mammal. In one aspect of this embodiment, the mammal is a human.
`[0021]
`In another embodiment of the method for diagnosing a Helicobacterpyloriinfection in a subject, the method
`further comprises the step of genotyping the Helicobacter pylori which infected the subject. In one aspect of this em-
`bodiment, the Helicobacter pylori is genotype for the presence of a cag pathogenicity island. In another aspect of this
`embodiment, the Helicobacfer pylori is genotyped for the presence of a vacA genotype selected from s1/m1, s1m2,
`s2m1 and $2m2.
`
`[0022] The invention also provides a method for diagnosing a Mycobacterium tuberculosis infection in a subject,
`comprising detecting the presence of a Mycobacterium tuberculosis nucleic acid in a urine sample from the subject,
`wherein the Mycobacterium tuberculosis nucleic acid has crossed the kidney barrier and is from cells outside a urinary
`tract of the subject, thereby diagnosing the Mycobacterium tuberculosis infection. In one embodiment of the method for
`diagnosing a Mycobacterium tuberculosis infection in a subject, the method further comprises the step of quantitating
`the nucleic acid.
`
`In another embodiment of the method for diagnosing a Mycobacterium tuberculosis infection in a subject, the
`[0023]
`detecting is performed by a method selected from PCR, nested PCR, semi—nested PCR, hybridization, SSCP, LCR,
`SDA, and pairing with molecular probes that are specific for the non-viral pathogen. In one aspect of this embodiment,
`a primer set is used forthe method, wherein the primer set comprises aforward primer and a reverse primer. Optionally,
`the forward primer is selected from SEQ ID NOs: 34, 37 and 39 and the reverse primer is selected from SEQ ID NOs:
`35, 36, 38 and 40.
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`In another embodiment of the method for diagnosing a Mycobacterium tuberculosis infection in a subject, the
`[0024]
`Mycobacten'um tuberculosis nucleic acid comprises transrenal DNA.
`[0025]
`In another embodiment of the method for diagnosing a Mycobacterium tuberculosis infection in a subject, the
`subject is a mammal. In one aspect of this embodiment, the mammal is a human.
`[0026] The invention also provides a method for diagnosing a Bacillus anthracis infection in a subject, comprising
`detecting the presence of a Bacillus anthracis nucleic acid in a urine sample from the subject, thereby diagnosing the
`Bacillus anthracisinfection. In one embodiment of the method for diagnosing a Bacillus anthracis infection in a subject,
`the method further comprises the step of quantitating the nucleic acid.
`[0027]
`In another embodiment of the method for diagnosing a Bacillus anthracis infection in a subject, the detecting
`is performed by a method selected from PCR, nested PCR, semi-nested PCR, hybridization, SSCP, LCR, SDA, and
`pairing with molecular probes that are specific for the non-viral pathogen. In one aspect of this embodiment, a primer
`set is used for the method, wherein the primer set comprises a forward primer and a reverse primer. Optionally, the
`forward primer is selected from SEQ ID NOs: 41,43, 45,47,48 and 50 and the reverse primer is selected from SEQ ID
`NOs: 42,44,46 and 49.
`[0028]
`In another embodiment of the method for diagnosing a Bacillus anthracis infection in a subject, the Bacillus
`anthracis nucleic acid comprises transrenal DNA.
`[0029]
`In another embodiment of the method for diagnosing a Bacillus anthracis infection in a subject, the subject is
`a mammal. In one aspect of this embodiment, the mammal is a human.
`[0030] The invention also provides a method for diagnosing a Plasmodium species infection in a subject, comprising
`detecting the presence of a Plasmodium species nucleic acid in a urine sample from the subject, thereby diagnosing
`the Plasmodium species infection. In one embodiment of the method for diagnosing a Plasmodium species infection in
`a subject, the method further comprises the step of quantitating the nucleic acid.
`[0031]
`In another embodiment of the method for diagnosing a Plasmodium species infection in a subject, the detecting
`is performed by a method selected from PCR, nested PCR, semi-nested PCR, hybridization, SSCP, LCR, SDA, and
`pairing with molecular probes that are specific for the non-viral pathogen. In one aspect of this embodiment, a primer
`set is used for the method, wherein the primer set comprises a forward primer and a reverse primer. Optionally, the
`forward primer is selected from SEQ ID N03: 18, 21 and 23 and the reverse primer is selected from SEQ ID N03: 19,
`20 and 22.
`
`In another embodiment of the method for diagnosing a Plasmodium species infection in a subject, the Plas-
`[0032]
`modium species nucleic acid comprises transrenal DNA.
`[0033]
`In another embodiment of the method for diagnosing a Plasmodium species infection in a subject, the subject
`is a mammal. In one aspect of this embodiment, the mammal is a human.
`[0034] The invention also includes a method for diagnosing a Leishmania species infection in a subject, comprising
`detecting the presence of a Leishmania species nucleic acid in a urine sample from the subject, thereby diagnosing the
`Leishmania species infection. In one embodiment of the method for diagnosing a Leishmania species infection in a
`subject, the method further comprises the step of quantitating the nucleic acid.
`[0035]
`In another embodiment of the method for diagnosing a Leishmania species infection in a subject, the detecting
`is performed by a method selected from PCR, nested PCR, semi-nested PCR, hybridization, SSCP, LCR, SDA, and
`pairing with molecular probes that are specific for the non-viral pathogen. In one aspect of this embodiment, a primer
`set is used for the method, wherein the primer set comprises a forward primer and a reverse primer. Optionally, the
`forward primer is selected from SEQ ID N03: 26, 28, 30, 32 and 51 and the reverse primer is selected from SEQ ID
`NOs: 24, 25, 27, 29,31 and 33.
`[0036]
`In another embodiment ofthe method for diagnosing a Leishmania species infection in asubject, the Leishmania
`species nucleic acid comprises transrenal DNA.
`[0037]
`In another embodiment of the method for diagnosing a Leishmania species infection in a subject, the subject
`is a mammal. In one aspect of this embodiment, the mammal is a human.
`[0038] The invention also provides acomposition for use in the detection of Helicobacterpylori, wherein the composition
`comprises a nucleic acid sequence selected from SEQ ID NOs: 5-17. In one embodiment of the composition for use in
`the detection of Helicobacterpylori, the composition consists of a nucleic acid sequence selected from SEQ ID NOs: 5—17.
`[0039] The invention also provides a composition for use in the detection of Plasmodium species, wherein the com-
`position comprises a nucleic acid sequence selected from SEQ ID NOs: 18—23. In one embodiment of the composition
`for use in the detection of Plasmodium species, the composition consists of a nucleic acid selected from SEQ ID NOs:
`18-23.
`
`[0040] The invention also provides a composition for use in the detection of Leishmania species, wherein the com—
`position comprises a nucleic acid sequence selected from SEQ ID NOs: 24-33 and 51.
`In one embodiment of the
`composition for use in the detection of Leishmania species, the composition consists of a nucleic acid selected from
`SEQ ID NOs: 24-33 and 51.
`
`[0041] The invention also provides a composition for use in the detection of Mycobacterium tuberculosis, wherein the
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`composition comprises a nucleic acid sequence selected from SEQ ID NOs: 34-40. In one embodiment of the composition
`for use in the detection of Mycobacterium tuberculosis, the composition consists of a nucleic acid selected from SEQ
`ID NOs: 34-40.
`
`[0042] The invention also provides a composition for use in the detection of Bacillus anthracis, wherein the composition
`comprises a nucleic acid sequence selected from SEQ ID NOs: 41 —50. In one embodiment of the composition for use
`in the detection of Bacillus anthracis, the composition consists of a nucleic acid selected from SEQ ID NOs: 41 -50.
`[0043] The invention also provides a kit for detecting Helicobacter pylori in a urine sample from a subject in need
`thereof, comprising at least one forward primer selected from SEQ ID NOs: 5, 8, 10, 11, 12 and 15 and at least one
`reverse primer selected from SEQ ID NOs: 6,7, 9, 13, 14, 16 and 17, either in the same or separate packaging, and
`instructions for its use.
`
`[0044] The invention also provides a kit for detecting Mycobacterium tuberculosis in a urine sample from a subject in
`need thereof, comprising at least one forward primer selected from SEQ ID N03: 34, 37 and 39 and at least one reverse
`primerselected from SEQ ID NOs: 35, 36, 38 and 40, eitherin the same orseparate packaging, and instructionsforits use.
`[0045] The invention also provides a kit for detecting Bacillus anthracis in a urine sample from a subject in need
`thereof, comprising at least one forward primer selected from SEQ ID NOs: 41,43,45,47,48 and 50 and at least one
`reverse primer selected from SEQ ID NOs: 42,44,46 and 49, either in the same or separate packaging, and instructions
`for its use.
`
`[0046] The invention also provides a kit for detecting Plasmodium species in a urine sample from a subject in need
`thereof, comprising at least one fonNard primer selected from SEQ ID N03: 18, 21 and 23 and at least one reverse
`primer selected from SEQ ID NOs: 19,20 and 22, either in the same or separate packaging, and instructions for its use.
`[0047] The invention also provides a kit for detecting Leishmania species in a urine sample from a subject in need
`thereof, comprising at least one forward primer selected from SEQ ID NOs: 26, 28, 3 0, 32 and 51 and at least one
`reverse primer selected from SEQ ID N03: 24, 25, 27, 29, 31 and 33, either in the same or separate packaging, and
`instructions for its use.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`[0048]
`
`Figure 1 is a photograph of PCR reactions performed on human urine with primers specifcfor human b-actin resolved
`by electrophoresis.
`Figure 2 is a photograph of PCR reactions performed on rabbit urine with primers specifc for rabbit b-globin resolved
`by electrophoresis.
`Figure 3A is a photograph of semi—nested PCR reactions performed on human urine with primers specifc for H.
`pylori ureA sequences resolved by electrophoresis.
`Figure BB is a photograph of semi-nested PCR reactions performed on human urine with primers specifc for H.
`pylori cagA sequences resolved by electrophoresis.
`Figure 4 is a photograph of semi-nested PCR reactions performed on human urine with primers specie for Plasmo-
`dium falciparum sequences resolved by electrophoresis.
`Figure 5 is a photograph ofsemi-nested PCR reactions performed on human urine with primers specifcforLeishmania
`infantum sequences resolved by electrophoresis.
`Figure 6 is a photograph of semi—nested PCR reactions performed on human urine from TB infected and healthy
`patients with primers specifc for Mycobacterium tuberculosis sequences resolved by electrophoresis.
`Figure 7 is a photograph of semi-nested PCR reactions performed on the supernatants and pellets of centrifuged
`hum an urinefrom TB infected and healthy patients before and aftertreatment with primers specifc forMycobacterium
`tuberculosis sequences resolved by electrophoresis.
`Figure 8 shows photographs of semi-nested PCR reactions performed on human urine from TB infected patients
`with primers specific for Mycobacterium tuberculosis sequences of 129/67 bp (left panel) and 330/69 bp (right panel)
`resolved by electrophoresis.
`Figure 9 is a photograph of semi-nested PCR reactions performed on human urine from TB actively infected patients
`and patients two months after treatment for TB with primers specifc for Mycobacterium tuberculosis sequences
`resolved by electrophoresis.
`Figure 10 is a photograph of semi-nested PCR reactions performed on rabbit urine from B. anthracis infected and
`uninfected subjects with primers specifc for Bacillus anthracis sequences resolved by electrophoresis.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`[0049] The invention is based, in part, upon the discovery described herein that pathogenic nucleic acids are detectable
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`in the urine of a subject infected with the pathogen. In some instances, nucleic acids from pathogenic organisms cross
`the kidney barrier and pass into the urine of mammals. The nucleic acids which cross the renal barrier, termed herein
`as transrenal nucleic acids, and more specifically transrenal DNA tend to be shorter than 1,000 bp in length, but are
`preferably less than 500 bp in length, and more preferably shorter than 250-300 bp in length or shorterthan 250 bp in
`length. While it had been shown in the past that DNA from mammalian cells, cancerous and non—cancerous which were
`hypothesized to die by apoptosis in the body of a subject may be able to cross the renal barrier, this is the first time it
`has been shown that the DNA of pathogens also crosses this barrier. Further, nucleic acids from pathogens found in
`the kidney would shed pathogen specific nucleic acids into the urine without crossing the kidney barrier. There may be
`other mechanisms by which pathogen specific nucleic acids pass into the urine of a subject infected with the pathogen.
`[0050] Based on this discovery, the invention provides compositions and methods forthe detection of non-viral path-
`ogens in the urine of a subject. The compositions and methods of the invention are used to detect transrenal-nucleic
`acids derived from pathogenic microorganisms which traverse the renal barrier and are present in the urine of a subject.
`The invention includes methods for the isolation, amplification and detection of these transrenal nucleic acids. The
`invention also includes compositions which may be used to isolate, amplify these transrenal nucleic acids.
`[0051] One method of the invention forthe isolation of urinary DNA from a subject includes the following steps. Urine
`is first centrifuged or filtered to separate cells and cell debris from the urine. EDTA/Tris HCI is then added to the urine
`to bind bivalent ions and to adjust pH to approximately pH 8. To this solution is then added a chaotropic salt, for example
`guanidine isothiocyanate to a final concentration of at least 3 M. Then a silica resin is added to the urine to which DNA
`binds. This resin is then washed, and the DNA eluted from it, thereby isolating the trans renal DNA.
`[0052] DNA isolated from the urine of a subject may then be amplified in orderto be detected. Amplification methods
`include polymerase chain reaction (PCR), nested PCR, semi-nested PCR, Single-Strand Conformation Polymorphism
`analysis (SSCP), ligase chain reaction (LCR) and strand displacement amplification (SDA). Detection of transrenal DNAs
`is also performed through hybridization of at least one labeled probe.
`[0053] The amplification and detection of urinary nucleic acids from non-viral pathogens may be used to detect the
`presence of these pathogens, and thus diagnose the infection of a subject with these pathogens and to diagnose
`pathologies associated with the infection of a subject by these pathogens. Further, the detection of these pathogens
`may be used to monitortreatment of the infections and pathologies associated with these pathogens. Also, detection of
`these pathogens may be used to suggest treatments for a subject in which the pathogens are detected. These treatments
`may be used to relieve symptoms which are associated with the detection of pathogens or diagnosis with infection of
`the pathogens.
`[0054]
`Non—viral pathogens which are able to be detected by the presence of their nucleic acids in mammalian urine
`include bacteria and parasites. Bacterial species which are detectable using the compositions and methods of the
`invention include Helicobacter pylori (H. pylori), Mycobacterium tuberculosis (M. tuberculosis, MTB or TB), and Bacillus
`anthracis (B. anthracis). Parasite species which are detectable using the compositions and methods of the invention
`include Plasmodium species including Plasmodium falciparum and Leishmania species.
`
`Detection of the Presence of Pathogenic Microorganisms
`
`Bacteria
`
`Helicobacter pylori
`
`[0055] Most cases of peptic ulcer disease, gastric mucosa associated lymphoid tissue (MALT) lymphoma and cancer
`of the distal stomach are complications of Helicobacter pylori infection. (Axon AT. Gut. 1999 Jul; 45 Suppl 1:|1-4).
`However, most H. pylori-positive individuals remain symptom free throughout their life. Symptoms associated with H.
`pyloriinfection include abdominal discomfort, weight loss, poor appetite, bloating, burping, nausea and vomiting. Awidely
`accepted explanation of this phenomenon is that the outcome of H. pylori colonization is determined by combination of
`several factors including the genotypes of the bacteria and the host and the environmental cofactors such as diet and
`smoking. For the successful medical management of the infection it is very important the development of diagnostics
`tools to direct the decision concerning the treatment of the carriers. The test should give information about the genotype
`of the bacteria in the carrier.
`
`[0056] Colonizing human stomach H. pylori strains are genetically very diverse. Observed diversity is attributed to
`both horizontal transfer of genetic material between coexisting strains as well as to the instability of the bacterial genome.
`Polymorphism occurs due to point mutations, substitutions, insertions, deletions that may involve one or more genes
`(Akopyanz, N.S., et al. 1992. Nucleic Acids Res. 20:5137-5142; Achtman, M., et al. 1999. Mol. Microbiol. 32:459-470;
`Tomb, J.—F., et al. 1997. Nature. 388:539—547; Alm, R.A., et al. 1999. Nature. 397:176—180; Kleanthous, H., et al. 1991.
`Mol. Microbiol. 5:2377-2389; Kersulyte, D., et al. 1998. Gene. 223:175-186.) Therefore, important initial step for the
`development of genetic test is the selection of specific genetic markers of H. pylori associated with clinical symptoms.
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`There are numerous publications concerning the genetic markers associated with elevated virulence of the bacteria and
`the disease.
`
`A major genetic determinant of H. pylorivirulence is the cag pathogenicity island (cag PAI) (Blaser, M. J., et
`[0057]
`al. 1995. Cancer Res. 55:2111-2115; Cover, T. L., et al. 2001. Helicobacter pylori pathogenesis. Academic Press, San
`Diego, Calif.; Mobley, H. L. 1996. Am. J. Med. 100:2S—11S.), a 40—kb region of chromosomal DNA that is present in
`some H. pyloristrains but absent from others. The cag PAI encodes a type IV secretion system and an immunodominant
`antigen, CagA, which is translocated into gastric epithelial cells.
`In comparison to infection with cag PAI-negative H.
`pylori strains, infection with cag PAI-positive strains is associated with an increased severity of gastric mucosal inflam-
`mation, an increased risk for development of peptic ulceration, and an increased risk of gastric cancer (Ho, S. A., et al.
`1991. J. Clin. Microbiol. 29:2543-2549). cagA is present in only 60-70% of prloriisolates in Western countries. Over
`80% of patients with ulcers harbor cagA positive strains (Atherton JC. Gut. 1997 Jun;40(6):701-3; Tummuru MKR, et
`al. Infect Immun 1993;61:1799—809; Atherton JC, et al. Curr Microbiol. 1999 Oct;39(4):211—8.).
`[0058]
`In contrast to cagA, the gene encoding the vacuolating cytotoxin (vacA) is present in all H. pyloristrains analyzed
`so far. But only 50% of them produce an active vacuolating cytotoxin. Production and cytotoxicity of the vacA protein is
`determined by its structure. There are two polymorphic regions in thevacA gene: the region encodingthe signal sequence
`with two distinct types of s1 and s2, and midregion marked as m1 and m2. Existence of combination of all s and m types
`was registered except 52/m1. Most active genotype of vacA gene is shown to be s1/m1 followed by s1/m2. Very little
`activity or no activity was seen forthe type s2/m2. Studies in US demonstrated that over 90% of patients with duodenal
`ulcer disease had vacA 31 strains and those with 32 strains were at the level of uninfected patients. There is very close
`genetic linkage between cagA and vacA s1/m1 genotypes.
`In this pair of genetic markers vacA is considered to be
`surrogate forthe cagA (Cover L, T et al. J Biol Chem 1994;269:10566-73; Graham DY, and Yamaoka Y. Disease-specific
`Helicobacter pylori virulence factors: the unfulfilled promise.Helicobacter. 2000;5 Suppl 1:83-9; discussion 827-31).
`[0059] There are many other H. pylori genetic markers studied such as: 168 rRNA gene, the random chromosome
`sequence, the 26-kDa species-specific antigen (SSA) gene, the urease A (ureA) gene, and the urease C (ureC) gene
`(Valentine, J. L., et al. 1991. J. Clin. Microbiol. 29:689-695; Hammer, M., T. et al. 1992. J. Clin. Microbiol. 30:54-58;
`Clayton, C. L., et al. 1992. J. Clin. Mi