SENSITIVE MONITOR OF ANTI-VIRAL THERAPY FIELD OF THE INVENTION [01] The invention relates to improved methods of detecting virus in and evaluating antiviral treatment of infected patients.

BACKGROUND OF THE INVENTION [02] Advancements in the treatment of viral infections, such as highly active anti-retroviral therapy (HAART) treatment of human immunodeficiency virus type-1 (HIV-1), have resulted in long-term suppression of virus replication in infected individuals.

Suppression of virus often lasts for years, but the virus is not eradicated. Evidence that long-term suppression does not lead to virus eradication is that once anti-viral therapy is withdrawn, the virus rebounds to levels similar to those in the patient prior to treatment. For instance, HIV-1 positive patients experience a return of virus to pre- treatment levels when withdrawn from HAART. (Carrasco and Tyring (2001) Dermatol. Cli71. 19 (4): 757-772). Further, rebound of virus levels in HIV-1 infected patients withdrawn from HAART is rapid. Patients with plasma HIV-1 viral loads below the limit of detection undergo an increase in plasma levels of HIV-1 RNA from 50 to 500 copies per milliliter over a period of 6 to 15 days following withdrawal of HAART. The HIV-1 RNA levels further increase to or above pre-therapy levels in all patients within 21 days of stopping therapy. (Harrigan et al. (1999) AIDS 13 (8): F59- 62).

[03] Virus continues to replicate in patients taking advanced anti-viral treatments although the virus is suppressed to undetectable levels in the plasma or serum. In a study of eleven HIV-1 infected patients undergoing a HAART treatment regimen and having plasma viral loads below 50 HIV-1 RNA copies per milliliter, four acquired drug resistance mutations. (Elbeik et al. (2001) J. Hum. Virol. 4 (6): 317-328). The accumulation of drug resistant virus in patients with undetectable virus levels is not surprising. Retroviruses, such as HIV-1, have a low fidelity polymerase that reverse transcribes the viral RNA genome into DNA. This low fidelity polymerase introduces mutations into the viral genome during replication. Mutations that confer drug resistance are selected in the microenvironment of the treated patient and cause failure of the anti-retroviral therapy.

[04] In fact, failure of HAART therapy of HIV-1 infected patients is often associated with accumulation of drug resistance mutations in the viral genome. Fifty eight percent of patients failing a HAART treatment including a protease inhibitor, nelfinavir, acquired mutations in the protease gene. (Roge et al., HIV Med. (2003) 4 (1) : 38-47).

Forty percent of antiretroviral drug-naive HIV-infected'patients starting HAART exhibited viral breakthrough at 44 months. Virological failure was partly associated with resistance related mutations. (Van Vaerenbergh et al. (2002) AIDS Res. Hunz.

Retroviruses 18 (6): 419-426}.

[05] Methods that more readily detect these mutations will help to maintain low viral loads in treated patients. HIV-1 infected patients whose drug treatment therapy is altered upon detection of drug resistance mutations are more likely to sustain suppression of viral replication. Thirty-one and a third percent of HIV-1 infected patients in a drug therapy program that included genotyping detectable virus and altering the drug therapy in response to the development of resistance mutations in the virus achieved undetectable virus levels six months after initiation of therapy. These undetectable levels of virus were maintained during follow-up. In contrast, 14% of patients whose virus was not genotyped and whose therapy regimen was static maintained HIV-1 RNA levels below the limit of detection over six months. (Clevenbergh et al. (2000) Antivir. Ther. 5 (1) : 65-70).

[06] Virus is commonly detected and monitored in a patient's plasma or serum. Detecting virus in plasma or serum is minimally invasive and can provide accurate results.

Virus is also detected in specific blood cell types that are latent reservoirs for viral replication. These cells are useful to test for virus because they are a potential source of ongoing viral replication. Latent reservoirs of HIV-1 include latently infected CD4+ T cells and monocyte/macrophage cells (Chun et al. (1997) Proc. Natl. Acad.

Sci. (1997) 94: 13193-13197; Garbuglia et al. (2001) J. Chemother. 13 (2): 188-194).

A latent reservoir of Hepatitis C virus (HCV) is in the peripheral blood mononuclear cells. (Zignego, et al. (1995) J. Med. Virol. 47 (1) : 58-64). A latent reservoir of Epstein Barr virus (EBV) is B cells. (Schwarzmann et al. (1998) Int. J. Mol. Med.

1 (1): 137-42). Detecting viral nucleic acids associated with plasma, serum, or latent reservoirs of viral replication are frequently not sufficiently sensitive to detect levels of viral replication in patients that are long-term suppressors of virus. Thus other cell types have been used as indicators of viral replication and to monitor viral replication when viral nucleic acid loads are no longer detectable. One such method of detecting HIV-1 replication in patients with below detectable virus levels is to count CD8+/CD38++ T cells. This cell count has been used as an indication of residual viral replication. (Tilling et al. (2002) AIDS 16 (4): 589-596). Hess et al. found that erythrocyte-associated HIV-1 RNA was found in 80 of 82 chronically infected HIV patients (Hess et al. (2002) Lancet 359: 2230) [07] There is a continuing need in the art for a highly sensitive method to detect and genotype virus of infected patients. Development of such a method will improve monitoring of anti-viral therapy in infected patients and improve tailoring of anti-viral treatments.

BRIEF SUMMARY OF THE INVENTION [08] In one embodiment of the invention there is provided a method of measuring the effectiveness of anti-viral therapy in a patient. Red blood cells of a patient undergoing anti-viral therapy are obtained. The viral nucleic acids associated with the red blood cells are amplified. The amplified viral nucleic acids are quantified to determine a quantified viral nucleic acid value. The anti-viral therapy is evaluated based on the quantified viral nucleic acid value.

[09] In a further embodiment of the invention there is provided a method for deciding treatment of a patient on anti-viral therapy. Red blood cells are obtained from a patient. The viral nucleic acids associated with the red blood cells are amplified. The amplified viral nucleic acids are quantified to determine a quantified nucleic acid value. The decision of whether to modify the anti-viral therapy of the patient or not can be based on the quantified nucleic acid value.

[10] In the above methods, the effectiveness of the anti-viral therapy or the decision to modify the therapy that the patient is receiving can be based on the quantified nucleic acid value obtained from a single patient sample or two or more sample may be obtained at different points in time (for example, a sample before treatment and a sample after or during treatment, or two or more samples obtained at different times in the course of treatment). Comparison of the quantified nucleic acid values of the two or more samples can be useful to determine if the viral load in the patient is increasing, decreasing or remaining constant.

[11] In a another embodiment of the invention there is provided a method for detecting the development of viral resistance to an anti-viral therapy. Red blood cells of a patient undergoing anti-viral therapy are obtained. The viral nucleic acids associated with the red blood cells are amplified. The detected viral nucleic acids are quantified to determine a quantified viral nucleic acid value. The viral resistance to the anti-viral therapy is determined based on the quantified viral nucleic acid value.

[12] In yet another embodiment of the invention there is provided a method for genotyping virus in an infected patient on anti-viral therapy. Red blood cells are isolated from a patient. Viral nucleic acids associated with the red blood cells are amplified.

Sequence information is obtained for the amplified viral nucleic acids. The virus is genotyped.

[13] In any of the above methods, the red blood cells may be in the form of isolated red blood cells or may be in a blood sample that comprises red blod cells, for example, a whole blood sample, a diluted whole blood sample, a non-serum fraction sample, a diluted non-serum fraction sample. The methods of the invention are applicable to many different viral infections and are particularly suitable for monitoring of HIV-1, HCV, HBV or SARS infections.

DETAILED DESCRIPTION OF THE INVENTION Viruses [14] The methods of the invention evaluate anti-viral therapies. Any virus can be treated with an appropriate anti-viral therapy. Viruses that are the subject of the anti-viral therapy can include, for example, human immunodeficiency virus type 1 (HIV-1), human immunodeficiency virus type 2 (HIV-2), hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), severe acute respiratory syndrome virus (SARS), West Nile virus (WNV), human T cell lymphotropic virus type 1 (HTLV-1), human T cell lymphotropic virus type II (HTLV-2), human papilloma virus (HPV), herpes viruses, Epstein-Barr virus (EBV), and varicella virus. Other viruses are known in the art. Preferably for applying the method of the invention, the virus is HIV-1, HCV, HBV, WNV, or SARS.

Obtaifzifng Whole Blood and Red Blood Cells [15] Viruses in whole blood or associated with red blood cells are detected to evaluate anti-viral therapies. If the viruses are associated with red blood cells, the red blood cells can be present, for example, in whole blood, the non-serum fraction of blood, or isolated red blood cell samples. Thus, each of these samples can be used to amplify and quantify the viral nucleic acids associated with red blood cells.

[16] If the red blood cells obtained from a patient are in whole blood or if whole blood is used to detect the viral nucleic acids, a whole blood sample is one that contains all the cells and fluids of circulating blood in the patient. Whole blood typically contains all fractions of blood including red blood cells, white blood cells, platelets, and plasma.

Whole blood can be obtained from a patient, for example, by venipuncture.

[17] If red blood cells from a patient are used in the methods, they may be in the non- serum fraction of blood. The non-serum fraction of blood contains all components of whole blood except the serum. The serum is the liquid portion of whole blood and comprises approximately 95% water. The non-serum fraction of blood thus contains blood cells, e. g., red and white blood cells, and platelets. The non-serum fraction of blood may also contain clotting factors, enzymes, salts, and other factors known to be contained in blood but that exclude serum. A method of obtaining the non-serum fraction of blood is to obtain whole blood and to sediment the whole blood. Whole blood may be sedimented by centrifugation at 1500 x g for 3 minutes. The serum is then removed from the sedimented whole blood. The remaining non-serum portion of the blood remains and can be used in the method. Other methods of obtaining a non- serum fraction of blood are well-known in the art.

[18] If red blood cells obtained from the patient are used in the methods, the red blood cells may be isolated. Red blood cells are isolated by any method known in the art.

These methods include, for example, inverted centrifugation, reconstitution of frozen or thawed blood, passage through a nylon column, sedimentation in dextran, hydroxyethyl starch (HES), or Ficoll-Hypaque, and washing with physiological saline solution. These and other suitable methods are further described in"Blood component therapy, "ed. American Association of Blood Banks, Twentieth Century<BR> Press, Inc. , Chicago, U. S. A. , 1969. Red blood cells can also be isolated by passage through de-fatted and bleached Egyptian cotton. Red blood cells pass through the Egyptian cotton and are collected while other blood cell types adhere to the cotton.

See, for example, U. S. Patent 4,130, 642.

[19] Red blood cells can also be isolated by centrifugation techniques. Whole blood samples are centrifuged at approximately 1700xg for 30 min., which sediments the red blood cells. The plasma supernatant is removed from the sedimented red blood cells and the red blood cells are washed. U. S. Patent 4,448, 888 provides further details of this red blood cell isolation technique. Another centrifugation technique used to isolate red blood cells is described in U. S. Patent 5,550, 060. Three , centrifugation steps are performed in this technique. First, centrifugation is performed at 20 to 200xg for about 5 min. Second, centrifugation is performed at 2,000 to 6, 000xg for about 20 min. Finally, centrifugation is performed at greater than 8, 000xg for about 5 min. The red blood cells sediment on the bottom of the centrifuge tube and other cell components are removed.

[20] Isolated red blood cells are an essentially pure population of red blood cells. An essentially pure population of red blood cells is one in which at least 80% of the cells in the population are red blood cells. Preferably, at least 85 or 90% of the cells in the population are red blood cells. More preferably, at least 95, 97,98, or 99% of the cells in the population are red blood cells.

[21] Viral nucleic acids in whole blood or associated with red blood cells can be amplified.

If the viral nucleic acids are associated with red blood cells, the viral nucleic acids can be amplified following separation from the red blood cells or while still associated with the red blood cells.

[22] Other methods of obtaining red blood cells are well known and within the level of skill of one in the art.

Viral Nucleic Acids Associated witli Red Blood Cells [23] Viral nucleic acids associated with red blood cells can be amplified in the methods.

Viral nucleic acids associated with red blood cells describes, for example, viral nucleic acids that are directly or indirectly attached to the surface of red blood cells.

Viral nucleic acids associated with red blood cells typically attach to the surface of red blood cells indirectly. A non-limiting example of indirect attachment of viral nucleic acids to the surface of the red blood cells is via immune complexes. Immune complexes are formed when virus particles, which contain viral nucleic acids, are bound by antibodies that recognize an epitope on the virus and the antibodies are bound by complement. The immune complex attaches to receptors for complement on the surface of red blood cells, associating the viral nucleic acids with the red blood cell. Any viral nucleic acid that becomes attached to a red blood cell through intermediary molecules or that becomes directly attached to the red blood cell is associated with red blood cells.

Diluting [24] The whole blood or red blood cells of the patient can be diluted prior to amplification of viral nucleic acids. The whole blood or red blood cells can be diluted at any ratio that allows detection of the viral nucleic acids. For example, the ratio can be at least 1: 4 up to 1: 1000 or more, for example the dilution ratio can be 1: 4,1 : 5,1 : 8,1 : 10, 1: 50, 1 : 100, 1: 250,1 : 500, 1 : 750, or 1: 1000 of whole blood or red blood cells to diluent. Preferably, the whole blood or red blood cells are diluted at a ratio of between 1 : 4 and 1: 100; more preferably, the dilution is at a ration of 1: 4 or 1: 5. The diluent can be any buffer known in the art, such as saline, phosphate-buffered saline, Tris-buffered saline, serum of an uninfected individual, a non-calcium containing electrolyte solution, or Ringers solution. Preferably, the diluent is saline.

Amplifying [25] Viral nucleic acids can be amplified by any means known in the art. Polymerase Chain Reaction (PCR) is a well-known amplification technique that can be used in the claimed methods. PCR techniques are taught, for example, in Innis et al., eds. PCR Protocols: A Guide to Methods and Amplification (Academic Press, Inc. , San Diego, CA, 1990) and are disclosed in U. S. Patents 4,683, 202 and 4,965, 188. PCR amplification requires the use of a polymerase. Polymerases that can be used in PCR amplification include Thermus aquaticus (Taq) polymerase (U. S. Patents 4,889, 818 and 5,352, 600), Themlococcus litoralis (Vent) polymerase (U. S. Patents 5, 210,036 and 5,322, 785), Pyrococcus furiosus (Pfu) polymerase (U. S. Patents 5,545, 552 and 5,948, 663), Therrnus tlaennophilus (Tth) polymerase (U. S. Patent 5,192, 674), Thermococcus gorgonarius (Tgo) polymerase, and variants of these enzymes.

Variants of these enzymes are typically mutants which have improved fidelity or an increased rate of polymerization. Variants also include mixtures of more than one of these enzymes which also have greater fidelity and rates of polymerization.

[26] The polymerases may also be modified to prevent polymerization of nucleic acid products that are a result of non-specific annealing of primer to template. These modifications inactivate the polymerase until it is exposed to a sufficiently high temperature. A non-limiting example of a modification of a polymerase is binding of an antibody to the polymerase. When the antibody-bound polymerase is exposed to a sufficiently high temperature the antibody is denatured and released from the polymerase. The sufficiently high temperature also exceeds the temperature that allows non-specific priming of primer to template. Polymerases modified by antibody binding are described in U. S. Patents 5,587, 287 and 5,338, 671.

[27] Viral nucleic acids can also be amplified by reverse transcription PCR (RT-PCR).

RT-PCR is described in U. S. Patents 5,322, 770,5, 310, 652, and 5,561, 058. RT-PCR is commonly used to amplify viruses having RNA genomes. First, a copy DNA (cDNA) is reverse transcribed from the viral RNA. The cDNA copy of the viral genome can then be amplified using PCR. Enzymes that can be used to reverse transcribe viral RNA genomes include Moloney murine leukemia virus (MoMLV) reverse transcriptase (disclosed in U. S. Patents 5,017, 492 and 5,668, 005), Avian Myeloblastosis Virus (AMV) reverse transcriptase, and variants thereof. The variants of these enzymes typically have been mutated for improved fidelity.

[28] Other amplification methods that produce DNA copies of the viral genome can be used in the methods of the invention. These methods include strand displacement amplification (SDA) and ligase chain reaction (LCR). SDA is disclosed in U. S.

Patent 5,422, 252. LCR is disclosed in European patents EP-A-320 308 and EP-A-439 182. Polymerases used in these methods include Klenow, T7, T4, and E. coli polymerase I.

[29] It is also possible to amplify viral nucleic acids using methods that produce multiple RNA copies of viral nucleic acids. These amplification reactions include transcription mediated amplification (TMA), disclosed in U. S. Patent 5, 399, 491. TMA is an amplification reaction in which an RNA viral genome is reverse transcribed to cDNA.

The cDNA copy of the viral RNA genome is used as a template to transcribe multiple RNA copies of the cDNA using an RNA polymerase. Suitable RNA polymerases for use in TMA include T7, T3, SP6, Thermus, and baculovirus RNA polymerase.

[30] Primers are also used to amplify the viral nucleic acids. The primers anneal to nucleotide sequences within the viral genome and are used to produce an initial copy of the viral genome. The primers can also anneal to the initial copy of the viral genome or subsequent copies of the viral genome during later amplification steps.

[31] A primer can anneal to a nucleotide sequence in the viral nucleic acid molecule along its entire length or a primer can anneal to a nucleotide sequence in the viral nucleic acid molecule along only a portion of its length. If only a portion of the primer anneals to a nucleotide sequence in the viral nucleic acid molecule then the portion that does not anneal to a nucleotide sequence in the viral nucleic acid molecule (i. e. , non-annealing portion) can contain a recognition site for an RNA polymerase. The non-annealing portion in this example is useful in TMA methods for production of multiple RNA copies of the viral nucleic acids from cDNA. The non-annealing portion of the primer may alternatively contain sequences that encode recognition sites for restriction endonucleases, hybridize to probes on a solid support, or hybridize to linkers. These, and other, non-annealing sequences can be used to isolate and manipulate the amplified viral nucleic acids. Preferably, the non-annealing portion of the primer is at the 5'region of the primer.

[32] The annealing portion of the primer can be perfectly or substantially complementary to a nucleotide sequence in the viral nucleic acid sequence. If the annealing portion of the primer is perfectly complementary to the viral nucleic acids then each nucleotide in the primer is the exact complement of each nucleotide in the viral nucleotide sequence. If the annealing portion of the primer is substantially complementary to the viral nucleotide sequence then at least one nucleotide in the primer is not the perfect complement of at least one nucleotide in the viral nucleic acid sequence. Preferably no more than 10% of the nucleotides in the annealing portion of the primer lack complementarity to nucleotides in the viral nucleic acid sequence. Preferably no more than 7%, 5%, 3%, 2%, or 1% of the nucleotides in the primer lack perfect complementarity to a nucleotide of the target nucleotide sequence. Nucleotides in the annealing portion of the primer may not be perfectly complementary to nucleotides in the viral nucleic acid sequence because a nucleotide in the primer is not complementary to a nucleotide in the viral nucleic acids, e. g., a T and a C, because the primer is missing nucleotides opposite nucleotides in the viral nucleic acid sequence, or because the primer contains nucleotides in addition to nucleotides in the viral nucleic acid sequence.

[33] If amplification requires the use of two primers, e. g., PCR, the primers must anneal to opposite strands of the viral nucleic acids and be separated by a number of base pairs that is sufficiently close to allow formation of an amplification product. The primers can anneal to opposite strands of the viral nucleic acids separated by a number of base pairs that allows formation of the desired product in the reaction. Preferably, the primers anneal to opposite strands of the viral nucleic acids separated by no more than 5,000, 1,000, 500,400, 300,200, 150, or 100 base pairs. More preferably, the primers anneal to opposite strands of the viral nucleic acids separated by no more than 500, 400,300, 200,150, or 100 base pairs.

[34] The viral nucleic acids of a single virus can be amplified in the methods. It is also possible to amplify the viral nucleic acids of more than one virus. For example, the viral nucleic acids of 2,3, or 5 viruses can be amplified in the methods. Preferably, if the viral nucleic acids of more than one virus are amplified, the viral nucleic acids are those of HIV-1 and HCV.

[35] When the viral nucleic acids of more than one virus are amplified they can be amplified simultaneously in a single reaction vessel or separately in different reaction vessels. If the viral nucleic acids of more than one virus are amplified separately in different reaction vessels, the viral nucleic acids of different viruses can be amplified at the same time or at different times.

[36] Other methods of amplifying viral nucleic acids are well known in the art. All of these methods can be readily practiced by one of skill in the art.

Detecting [37] The amplified nucleic acids can be detected by any means known in the art.

Amplified nucleic acids can be detected by electrophoresing the amplified products on a gel and visualizing the electrophoresed products (e. g. , by staining with ethidium<BR> bromide or non-fluorescent unsymmetrical cyanine dyes, e. g. , SYBRs green, and visualizing under ultraviolet light). Detection can also be performed by electrophoresing the amplified products on a gel, transferring the electrophoresed products to a membrane and interrogating the membrane with labeled probes, e. g., Northern blot, Southern blot, or dot blot analysis.

[38] Detection can also be performed in solution or on a solid support. If detection is performed in solution, a probe is added to amplification products in an amplification mixture. The probes are then detected in the amplification mixture solution. If detection is performed on a solid support, the amplified products are attached to the solid support, probes are added, and the probes are detected. Solid supports include, but are not limited to, beads, coverslips, arrays, and tubes. Other detection methods are known in the art.

[39] The probes used to detect the amplified products are typically perfectly complementary or substantially complementary to a sequence of the amplified products. Perfectly complementary probes are probes in which each nucleotide in the probe is the exact complement of each nucleotide in the amplified product.

Substantially complementary probes have a nucleotide sequence that contains one or more nucleotides that are not perfectly matched to the nucleotide sequence of the amplified product. Preferably, a substantially complementary probe contains no more than 10% nucleotides along its length that are not complementary to the amplified product. More preferably, a substantially complementary probe contains no more than 8%, 6%, 5%, 4%, 2%, or 1% mismatches relative to the amplified product.

[40] The probes can comprise a label that detects the amplified product. The label can be any molecule which emits a signal. The label can be, for example, fluorescent, enzymatic (e. g. alkaline phosphatase or horseradish peroxidase), radioactive (e. g., 33P, 32p, 3sS, or l25I) chemiluminescent (e. g., acridinium ester, hemicyanine, or rhodamine labels), a chromophore (e. g., rhodamine, flourescein, monobromobimane, pyrene trisulfonates or Lucifer yellow), or electrochemiluminescent (e. g., tris (2,2'- bipyridine) ruthenium (II)).

[41] The probes are detected by any means known in the art. Radioactive probes can be detected, for example, on autoradiographic film, phosphorimaging cassettes, or in scintillation counters. Fluorescent probes are detected, for example, by spectroscopy or fluorometry. Enzymatic probes can be detected by providing substrates converted by the enzyme that produce a color or luminescent change (e. g., 5-bromo, 4-chloro, 3- indolylphosphate (BCIP)/nitroblue tetrazolium (NBT) can be provided to probes labeled with alkaline phosphatase and 3,3, 5, 5'-tetramehtylbenzidine (TMB) can be provided to detect probes labeled with horseradish peroxidase). Chemiluminescent probes can be detected on autoradiographic film, phosphorimaging cassettes, or a luminometer. Chromophores are detected, for example, by spectroscopy.

Electrochemiluminescent probes are detected, for example, using an Origin tricorder (Igen) subsystem that reads electrochemiluminescent signal.

Quafztifyzfzg [42] Amplified viral nucleic acids are quantified in the methods. Viral nucleic acids can be quantified absolutely or relatively. If the viral nucleic acids are quantified absolutely the actual quantity of viral nucleic acid present per volume of blood is determined. The units of viral nucleic acids present can be, for example, a nanogram or gram quantity of viral nucleic acids in the volume of blood, e. g., mL blood. The units of viral nucleic acids can also be represented by the number of copies of the viral genomic nucleic acids present in a volume of blood, e. g., copy number of the viral genomic nucleic acids in the volume of blood. Other representations of the absolute quantity of viral nucleic acid per volume of blood are also known.

[43] A nonlimiting example of a method of absolute quantification of viral nucleic acids is performed by comparing the detected level of probe hybridized to amplified nucleic acid in the patient sample to the detected level of probe hybridized to amplified nucleic acid standards containing known quantities of viral nucleic acid. The known standards provide a basis through which the absolute quantity of viral nucleic acid is determined.

[44] The viral nucleic acids can also be determined relatively. If the viral nucleic acids are determined relatively, the viral nucleic acids are assigned a fold or relative expression level compared to, for example, an internal standard or a designated sample in the assay.

[45] The steps of amplifying and quantifying or amplifying, detecting, and quantifying can be performed in separate reaction vessels or in a single reaction vessel. If the steps of amplifying and quantifying are performed in the same reaction vessel then the steps may be performed as a real-time amplification assay. A reaction mixture for real time amplification typically includes both the reagents for amplification of a target nucleic acid and a probe that detects the amplification products. Each time an amplification product is produced a probe that emits a signal is detected. Several well-known commercially available kits are sold for use in real time amplification. These kits include the TaqMan' (Applied Biosystems), QuantiTect Probe (Qiagen), and MasterAmp (Epicentre) kits.

[46] The steps of amplifying and quantitating can also be performed in a single tube in a target capture followed by transcription mediated amplification (TMA) assay. The target capture followed by TMA assay separates viral nucleic acids from blood components, amplifies the viral nucleic acids, and detects the amplified viral nucleic acids in a single vessel. Greater detail of this assay is provided in the Methods.

[47] Other methods in which amplifying and quantifying can be performed in a single reaction vessel are known and can be practiced by one of ordinary skill in the art.

[48] Other methods of quantifying are well known and within the level of skill of one in the art.

Viral Nucleic Acids [49] The viral nucleic acids that are amplified in the methods include DNA, cDNA, RNA or cRNA polynucleotides. HIV-1, HIV-2, HAV, HCV, SARS, WNV, HTLV-1, and HTLV-2 have single-stranded positive sense RNA viral genomes. HBV, HPV, EBV, VZV, and herpesviruses have double-stranded DNA genomes. Other viral nucleic acids may be single-stranded negative sense RNA, single-stranded positive sense DNA, or double-stranded RNA. Any of these viral nucleic acids can be amplified and detected in the methods. Viral nucleic acids also include any amplification product of a virus genome produced in the methods and any cell-or virus-produced copy of the virus genome.

Viral Nucleic Acid Value [50] The viral nucleic acid value is the absolute or relative amount of virus that is quantified in the methods. If the viral nucleic acid value quantified in the methods is an absolute amount of virus, then the viral nucleic acid value will be a known quantity of virus or viral genomes per volume of blood, e. g., ng viral nucleic acids/mL blood or copies of viral genomes/mL blood. If the viral nucleic acid value quantified is a relative amount of virus then the viral nucleic acid value will be expressed as a greater or lesser fold amount of virus compared to, e. g., a control or a designated sample in which the viral nucleic acids are detected. The quantified nucleic acid value is related to the viral load. A decrease in the quantified viral nucleic acid value indicates a corresponding decrease in the viral load. An increase in the quantified viral nucleic acid value indicates a corresponding increase in the viral load. Thus, the quantified viral nucleic acid value can be used as a surrogate for the viral load.

Obtaining Sequence Information [51] The sequence of viral nucleic acids can further be obtained in the methods. The sequence of the viral nucleic acids can be used to genotype the viruses or to detect or confirm that the viruses have acquired a mutation that confers drug resistance.

Previously identified mutations can be detected in the viral nucleic acids as well as new, previously unidentified mutations.

[52] Sequence information for a known mutation can be obtained using probes that hybridize to the nucleic acid sequence containing the mutated nucleotide. Sequence information for a known mutation can also be detected using restriction fragment length polymorphism methods if the mutation alters a restriction enzyme recognition site. Sequence information for a known mutation can also be obtained in an extension reaction. A primer having a 3'nucleotide that is complementary to only the wild-type or mutant nucleotide at the site of the suspected mutation is subjected to an extension reaction with a polymerase. If the primer does not extend, the nucleotide opposite the 3'-most nucleotide of the primer is not complementary to the 3'-most nucleotide of the primer.

[53] A new mutation in the viral nucleic acids can also be determined. Sequence information for new mutations in viral nucleic acids can be determined by standard sequencing methods known in the art. These methods include the dideoxy sequencing method and sequencing by hybridization on nucleotide arrays. Dideoxy sequencing methods can be performed manually or can be automated, for example, using an ABI Prism 377 DNA sequencer. A portion of the viral nucleic acids suspected of containing a mutation can be sequenced or the entire viral genome can be sequenced to detect a mutation. These sequencing methods can also be used to detect a known mutation in the viral genome. For example, HIV-1 patients using protease inhibitors as anti-viral therapy frequently accumulate mutations in the protease gene.

Sequencing of the protease gene may thus be performed.

[54] Other methods of determining the sequence of known and unknown nucleic acids are well known in the art and can be optionally be performed in the methods.

Resistant Viral Strains [55] Any viral strain resistant to anti-viral therapy can be detected by methods of the invention. A resistant viral strain is a viral strain that has acquired a mutation or mutations that make it unresponsive or less responsive to an anti-viral therapy. The mutation can be a mutation previously identified with viral resistance or a mutation not previously associated with viral resistance. Known mutations in the reverse transcriptase gene of HIV-1 associated with resistance to nucleoside and nucleotide reverse transcriptase inhibitors result in amino acid substitutions in the reverse transcriptase polypeptide as follows: M41L, T69D, K70R, L74V, V75T, M184V, M184I, T215Y, T215F, K219E (Stuyver et al. (1997) Antimicrob. Agents G'lzefnother.

41 : 284-291), A62V, V75I, F77L, F116Y, Q151M (Iversen et al. (1996) J. Virol.

70: 1086-1090), E44D (Montes et (2002) J. Med. Virol. 66: 299-303), K65R (Selrni et al. (2001) J. Biol. Chem. 276: 48466-72), D67N (de Ronde et al. (2001} J. Virol.

75 : 595-602), Y115F (Cases-Gonzales et al. (2000) J. Biol. Cherrz. 275 : 19759-67), Vu 181 (Walter et al. (2002) Antimicrob. Agents Cliemotlier. (2002) 46 : 89-94) ), and L210W (Hooker et al. (1996) J. Virol. 70: 8010-8). Known mutations in the reverse transcriptase gene of HIV-1 associated with virus resistance to nonnucleoside reverse transcriptase inhibitors result in the following amino acid substitutions in the sequence of the reverse transcriptase polypeptide: L100I, Y181C, L187F, Y188H (Buckheit et al. (1999) Antimicrob. Agents Chemot11er. 43 : 1827-34), K103N, V108I, Y188L, Y188C, G190A, G190E, G190S, P225H, (Bachelor et al. (2001) J. Virol.

75 : 4999-5008), V106A (Larder (1992) Antimicrob. Agents Chemother. 36 : 2664- 2669), and P236L (Gerondelis et al. (1999) J. Virol. 73 : 5803-13). Known mutations in the protease gene of HIV-1 associated with virus resistance to protease inhibitors result in the following amino acid substitutions in the protease polypeptide : D30N, G48V, V82A, L90M (Winters et al. (1998) J. Virol. 72: 5303-6), M46I, V82T, (Schock et al. (1996) J. Biol. Chem. 271 : 31957-63), M46L, V82S (Mahalingham et al. (1999) Eur. J. Biochem. 263 : 238-45), 150V (Maquire et al. (2002) J. Virol.

76: 7398-406), V82F and I84V (Todd et al. (2000) J. Virol. 39 : 11876-83).

[56] Mutations in the HCV genome have also been identified that cause resistance to interferon alpha therapy. These mutations cause amino acid substitutions in the nonstructural-5A (NS5A) protein at residues 2350-2370 (Sarrazin et al-. (2002) J.

Virol. 76: 11079-90).

Viral Load [57] Viral load is a measure of how much viral genomic nucleic acid is present in the blood of an infected patient. Several numerical representations can be used to describe viral load. Viral load can be represented as the number of copies of viral genomes in a specified volume of infected patient blood, e. g., copies/mL blood. Viral load can also be represented by the number of international units of virus genomes per volume of infected patient blood, e. g., IU/ml blood. An international unit is a standard that normalizes the results obtained from different testing methods used to determine viral load values. In an HCV infected individual there are approximately 2,000, 000 copies HCV per 800,000 IU. Viral load can also be represented as a weight of viral nucleic acid per volume of infected patient blood, e. g. , ng virus/mL blood.

Any other known representation that describes viral load may be used.

[58] Viral load is determined by several methods. One nonlimiting method of determining viral load is by amplifying viral nucleic acids from blood or a portion of the blood such as the plasma or red blood cells. The amplified viral nucleic acids are labeled and quantitated. Viral load can also be determined in a branched DNA (bDNA) assay. The bDNA assay has steps of attaching viral nucleic acids to a solid support with capture probes., attaching target probes to the captured viral nucleic acids, and attaching detectable molecules to the target probes. The detectable molecules are detected and quantitated. Other methods of determining viral load are also known in the art.

Effectiveness of Therapy [59] The methods of the invention can be used to determine the effectiveness of an anti- viral therapy. Effectiveness of an anti-viral therapy can be evaluated depending on how long a patient has been in an anti-viral treatment regimen. If a patient has begun a first anti-viral therapy after diagnosis with the virus or has begun a new anti-viral therapy that is different from a prior anti-viral therapy, the therapy can be evaluated as effective if it causes the patient's viral load to decrease. An effective anti-viral therapy causes a patient's viral load to decrease by at least about 10% relative to viral load prior to the first or new antiviral therapy. Preferably, an effective therapy causes a patient's viral load to decrease by 20%, 25%, 30%, 40%, 50%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%. Alternatively, the effectiveness of anti-viral therapy can be determined based on the quantified nucleic acid value of a single patient sample. Standard nucleic acid values can be determined for each particular virus as indicating effectiveness of anti-viral treatment, e. g. , a nucleic acid value of 10 copies/ ml indicates effective therapy or in some cases an undetectable nucleic acid value indicates effective therapy.

[60] Effectiveness of antiviral therapy can also be evaluated throughout the course of a continuing antiviral therapy for a patient. The antiviral therapy is effective if the patient's viral load does not increase. If the patient's viral load does not increase it may remain relatively the same throughout the course of antiviral treatment or it may decrease during the course of antiviral treatment. If the viral load remains relatively the same it will increase by a factor of, for example, no more than 100-fold, preferably no more than 50-, 40-, 25-, 20-, 10-, 5-, 3-, or 2-fold relative to the most recent prior viral load measurement.

[61] The present invention therefore includes a method of measuring the effectiveness of anti-viral therapy in a patient comprising: obtaining a first sample comprising red blood cells of a patient receiving anti-viral therapy; amplifying viral nucleic acids associated with the red blood cells in said first sample; quantifying the amplified viral nucleic acids from said first sample to determine a first quantified viral nucleic acid value; and evaluating the anti-viral therapy based on the first quantified viral nucleic acid value. The method can further include the steps of obtaining a second sample comprising red blood cells of the patient, wherein said second sample is obtained after the patient has received further anti-viral therapy; amplifying viral nucleic acids associated with the red blood cells in said second sample; quantifying the amplified viral nucleic acids from said second sample to determine a second quantified viral nucleic acid value; comparing said first and second quantified viral nucleic acid values; and evaluating the anti-viral therapy based on the comparison.

[62] Further anti-viral therapy can be additional, continuing therapy of the same type as the patient received at the time that the first sample was obtained, or it can be a different therapy.

[63] The present invention also includes a method for deciding whether to modify the treatment of a patient on anti-viral therapy comprising: obtaining a first sample comprising red blood cells of a patient receiving anti-viral therapy; amplifying viral nucleic acids associated with the red blood cells in said first sample; quantifying the amplified viral nucleic acids in said first sample to determine a first quantified nucleic acid value; and deciding whether or not to modify the anti-viral therapy of the patient based on the first quantified viral nucleic acid value. The method may include the further steps of obtaining a second sample comprising red blood cells of said patient, wherein said second sample is obtained after the patient has received further anti-viral therapy; amplifying viral nucleic acids associated with the red blood cells in said second sample; quantifying the amplified viral nucleic acids from said second sample to determine a second quantified viral nucleic acid value; comparing said first and second quantified viral nucleic acid values; and deciding whether to modify the anti- viral therapy based on the comparison. The decisions for modifying the anti-viral therapy will be based on the effectiveness of the current anti-viral therapy which effectiveness can be determined as described above. Thus, if the comparison indicates that the quantified viral nucleic acids value has decreased by at least about 10 % from the first sample to the second sample, the anti-viral therapy may be determined to be effective and no modification of the therapy is warranted.

Alternatively, if the quantified viral nucleic acids value has increased by no more than 100-fold, the anti-viral therapy may be determined to be effective and no modification of the therapy is warranted. These criteria are most appropriate for determining the course of therapy for those patients having a low viral load (e. g., a viral load that is undetectable in a serum or plasma sample from the patient). Other criteria may be more suitable by patients in different situations and a competent medical practicioner can readily determine the appropriate criteria for any particular patient.

[64] Effectiveness of antiviral therapy can also be evaluated by the presence of a mutation or mutations in the viral genome that confers resistance to at least one molecule of the antiviral therapy. The mutation or mutations are detected by sequencing the viral genome. The mutations can have been previously associated with resistance to the drug or may be identified as a new mutation associated with resistance to the drug.

Anti-viral therapy [65] An anti-viral therapy is any physician recommended regimen designed to curtail viral replication in an infected patient or to improve the physical well-being of the infected patient. Anti-viral therapies include drug treatments designed to interfere with viral replication. These drug treatments can, for example, inhibit the activity of virally encoded enzymes, inhibit virus entry into cells, or inhibit the interaction of viral proteins with other virally encoded proteins or the interaction of viral proteins with cellular proteins.

[66] Anti-viral therapy can also include holistic medicines, or changes in diet, exercise, or sleep recommended by a physician.

Patients [67] The methods of the invention detect viral nucleic acids associated with red blood cells of a patient. A patient is any organism that is infected with a virus and that is treated with anti-viral therapy. The patient can be an organism infected with the virus through normal behaviors, e. g. , without experimental intervention. The patient can also be an organism infected with virus for experimental research purposes. If the patient is infected with virus for experimental purposes it may be a model to study virus replication and to test anti-viral therapies. The patient can be a mouse, rat, pig, cow, monkey, gorilla, chimpanzee, ape, gibbon, cat, dog, or human. Preferably the patient is a human.

[68] The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention.

EXAMPLES Example 1 [69] Detection of HCV in whole blood [70] To determine whether viral nucleic acids can be detected in whole blood, a known quantity of HCV-infected serum was added to a set of whole blood samples. Each sample contained HCV negative whole blood (Bloodsource, Sacramento) diluted at a 1: 2, 1 : 4, or 1: 8 ratio in serum. The serum was a mixture of HCV positive and HCV negative serum (SeraCare, Oceanside) sufficient to provide a final concentration of 50 HCV genome copies/mL in each diluted sample. Negative control whole blood samples were also prepared. The negative control samples contained whole blood diluted at a 1: 2,1 : 4, or 1: 8 ratio in HCV negative serum. The samples were subjected to a target capture followed by TMA assay to determine the presence or absence of HCV nucleic acids. The results of the assay are shown in Table 1.

[71] Table 1 Sam le composition Whole Negative HCV TCR Whole Internal Internal Analyte Analyte Analyte blood serum control (L) blood control control ALU S/CO result () L) (pL) 100 dilution ALU result copies/niL | L). Whole blood samples 250 0 0 400 1: 2 312 Invalid 27,177 0.70 Invalid 250 0 0 400 1: 2 0 Invalid 41,369 1.07 Invalid 125 125 0 400 1: 4 190,127 Valid 6,440 0.17 Nonreactive 125 125 0 400 1: 4 198,841 Valid 4,653 0.12 Nonreactive 62.5 187.5 0 400 1: 8 182,295 Valid 12,317 0.32 Nonreactive 62.5 187.5 0 400 1: 8 177,027 Valid 8, 880 0. 23 Nonreactive 250 0 250 400 1: 2 478 Invalid 12,551 0.32 Invalid 250 0 250 400 1: 2 609 Invalid 17,334 0.45 Invalid 125 125 250 400 1: 4 203,899 Valid 288,594 7.44 Reactive 125 125 250 400 1: 4 189, 557 Valid 427, 102 11.01 Reactive 62.5 187.5 250 400 1: 8 178,431 Valid 436,172 11.24 Reactive 62.5 187.5 250 400 1: 8 175,069 Valid 406,214 10.47 Reactive Serum controls 0 500 0 400 Serum 32,273 Invalid 8,068 0.21 Nonreactive control 0 500 0 400 Serum 166,572 Valid 5, 791 0. 15 Nonreactive control 0 250 250 400 Serum 161,465 Valid 226,140 5.83 Reactive control 0 250 250 400 Serum 172, 052 Valid 413,380 10. 65 Reactive control The internal control ALU was required to have a minimum value of 77,978 to be valid. The analyte ALU was required to have a minimum value of 38,801 to be valid. TCR is the amount of target capture reagent added to the blood sample composition. S/CO is a signal to cutoff ratio. A signal to cutoff ratio of greater than or equal to 1 was designated as reactive. A reactive result indicates that HCV was detected. A nonreactive result indicates that HCV was not detected.

[72] The data indicated that HCV could be detected in a whole blood sample diluted at a ratio of 1: 4 or 1: 8 at concentrations of HCV as low as 50 viral genome copies/mL.

Example 2 [73] Whole blood is more sensitive than plasma to test for viral nucleic acids.

[74] Example 1 demonstrated that whole blood could be used to detect low levels of viral nucleic acids. It was next determined whether whole blood was a more sensitive sample than plasma for viral nucleic acid detection. Whole blood samples of HIV-1 positive individuals were obtained (ProMedDx, Norton). Each sample was divided into two portions. The first portion was used to test for the presence of HIV-1 in whole blood. The second portion was sedimented to obtain the plasma fraction of the whole blood and the plasma was tested for the presence of HIV-1. Each of the whole blood and plasma portions were diluted at ratios of 1: 10,1 : 100, or 1: 1000 or were undiluted (neat) prior to testing. HIV-1 was detected in the samples using the target capture followed by TMA assay. The results for the assay are shown in Table 2.

[75] Table 2 Internal Analyte Internal Analyte control control Dilution RLU Result RLU S/CO Result RLU Result RLU S/CO Result ProMedDx sample 1=HIV negative Whole blood Plasma Neat 613 Invalid 8, 288 0. 20 Invalid 177, 788 Valid 7, 021 0. 17 Nonreactive Neat 0 Invalid 7, 753 0. 19 Invalid 196, 314 Valid 7, 877 0. 19 Nonreactive ProMedDx sam 1 2HIV ositiv (SO. viral'enozne co ies/mL lasma),,. Whole blood Plasma Neat 630 Invalid 7, 388 0. 18 Invalid 178, 272 Valid 8, 258 0. 20 Nonreactive Neat 0 Invalid 125, 690 3. 03 Invalid 183, 600 Valid 5, 924 0. 14 Nonreactive 1 : 10 156, 303 Valid 346, 653 8. 36 Reactive 163, 257 Valid 6, 668 0. 16 Nonreactive 1 : 10 164, 408 Valid 640, 359 15. 45 Reactive 173, 317 Valid 10, 411 0. 25 Nonreactive 1 : 100 180, 726 Valid 63, 821 1. 54 Reactive 184, 269 Valid 8, 582 0. 21 Nonreactive 1 : 100 187, 991 Valid 10, 683 0. 26 Nonreactive 183, 148 Valid 12, 599 0. 30 Nonreactive 1 : 1000 195, 792 Valid 9, 643 0. 23 Nonreactive 202, 217 Valid 10, 798 0. 26 Nonreactive 1 : 1000 192, 583 Valid 9, 037 0. 22 Nonreactive 194, 465 Valid 8, 454 0. 20 Nonreactive CP C + 3w'-15, 7539'ori, 0g'* + 4 < < = oM am. I . 9 v noin eJmL'lasm) Whole blood Plasma Whole bloodPlasma Neat 317 Invalid 7, 906 0. 19 Invalid 168, 480 Valid 512, 978 12. 37 Reactive Neat 821 Invalid 6, 996 0. 17 Invalid 162, 009 Valid 521, 633 12. 58 Reactive 1 : 10 145, 456 Valid 489, 717 11. 81 Reactive 167, 630 Valid 479, 361 11. 56 Reactive 1 : 10 161, 233 Valid 487, 701 11. 76 Reactive 190, 484 Valid 461, 864 11. 14 Reactive 1 : 100 171, 580 Valid 505, 762 12. 20 Reactive 198, 239 Valid 502, 517 12. 12 Reactive 1 : 100 166, 744 Valid 462, 542 11. 16 Reactive 192, 536 Valid 498, 503 12. 02 Reactive 1 : 1000 180, 129 Valid 434, 735 10. 49 Reactive 202, 927 Valid 472, 517 11. 40 Reactive 1 : 1000 206, 000 Valid 472, 362 11. 39 Reactive 197, 359 Valid 455, 419 10. 99 Nonreactive The internal control RLU was required to have a minimum value of 85,219 to be valid. The analyte RLU was required to have a minimum value of 41,458 to be valid. RLU is relative light units. S/CO is a signal to cutoff ratio. A signal to cutoff ratio of greater than or equal to 1 was designated as reactive. A reactive result indicates that HIV was detected. A nonreactive result indicates that HIV was not detected.

[76] HIV-1 could not be detected in the diluted or undiluted plasma fraction of ProMedDx sample 2, which contains low levels of HIV-1 RNA at 50 genome copies/mL. In contrast, HIV-1 was detected in whole blood at dilutions of 1: 10 and 1: 100. Thus testing whole blood for HIV-1 nucleic acids detects low levels of virus more accurately than does plasma and is a more sensitive method of testing for viral nucleic acids.

[77] When HIV-1 was present in patient samples at a much higher concentration, 15,539 copies/mL in ProMedDx sample 3, HIV-1 is detected in both plasma and whole blood.

[78] To confirm these results, additional patient samples were tested for the presence of HIV-1. Whole blood patient samples were obtained from 63 HIV positive patients.

Twenty-seven of the 63 patients had viral loads below the limit of detection (<75 copies/mL) in a branched DNA assay. The samples were divided into two portions.

The first portion was diluted at a ratio of 1: 5 and tested for the presence of HIV-1 (whole blood 1: 5). The second portion was sedimented to obtain the serum fraction.

The serum fraction was diluted 1: 5 and tested for the presence of HIV-1 (serum 1: 5).

A target capture followed by TMA assay was used to detect HIV-1 in each of the diluted samples. Table 3 shows the results of the assay.

[79] Table 3 Whole blood 1 : 5 Serum (neat or 1 : 5) Analyte IC Analyte Sample 1C ALU 1C result ALU S/CO Analyte result IC ALU result ALU S/CO Analyte result V, pationt simie 100 patil nta plesv 0 r i s v s v ; 100 104, "95S Valid 906, 404 27. "3t Reactive 15, 605 "Valid 34, "28z 1. "0 Reactive 103 161, 32 Valid 840, 134 25. 3 Reactive 61, 241 Valid 500, 123 15. 08Reactive 104. 109, 984 Valid 462, 891 13. 96 Reactive 43, 395 Valid 228, 53 6. 89 Reactive 105 127, 168Valid 943, 222 28. 4'Reactive 85, 220 Valid 801, 242 24. 17Reactive 106 147, 192Valid 587, 44 (17. 7= Reactive 100, 353 Valid 133, 76 4. 03Reactive 107 123, 417 Valid 403, 813 12. 18Reactive 142, 015 Valid 473, 34 14. 28 Reactive 108 Valid 259, 611 7. 83Reactive 0 Valid 878, 022 26. 48 Reactive 109 92, 57 Valid 923, 14 27. 84 Reactive 78, 185Valid 457, 151 13. 7 Reactive 110 Valid 137, 541 4. 15 Reactive 137, 282Valid 970, 139 29. 26 Reactive 112 C Valid 45, 83 1. 53Reactive Valid 944, 207 31. 49Reactive l 113 155, 14 Valid 1, 057, 649 32. 07Reactive 145, 666 Valid 891, 606 29. 7 Reactive 114 159, 864 Valid 4, 44 0. 15Nonreactive 138, 866 Valid 8, 246 0. 28NonReactive 115'165, 882Valid 1, 036, 437 34. 57Reactive 140, 371 Valid 830, 632 27. 7 Reactive "k . r ,.. r r 117 182, 959 alid 935, 89 24. 06Reactive 150, 525 Valid 607, 453 15. 61 Reactive 118 131, 403Valid 1, 145, 279 29. 44Reactive 138, 148Valid 953, 155 24. 50 Reactive . s., . v "w> W w M M g g g F. YLT' S ]'h'a3) 1t, a nR 121 144, 957Valid 1, 160, 932 29. 84 reactive"131, 744Valid 937, 709 28. 43 reactive 122 138, 633Valid 1, 023, 792' 31. 04Reactive 107, 973Valid 977, 466 29. 64Reactive 123 129, 744 Valid 1, 262, 657 32. 46 Reactive 9, 762 Valid 149, 240 4. 52 Reactive 124 144, 721Valid 1, 173, 599 30. 17 eactive 155, 486Valid 809, 40 24. 5 Reactive 125 12, 715Valid 338, 155 8. 69Reactive 132, 13 Valid 211, 38 6. 41Reactive 126 27, 836Valid 243, 105 7. 37Reactive 114, 323Valid 534, 409 16. 2 Reactive 127 164, 05 Valid 208, 991 6. 3 Reactive 126, 33 Valid 485, 329 12. 48Reactive 128 148, 787Vatid 501, 731 15. 21Reactive 136, 192Va ! id 240, 515 7. 29 Reactive 129 Valid 1, 162, 117 35. 23 eactive 134, 85 Valid, 943, 085 28. 59 eactive 130 178, 735Valid 13, 780 0. 42NonReactive 136, 112Valid 3, 700 0. 11 NonReactive 131 163, 323Valid 824, 324 21. 19Reactive 145, 086 Valid 527, 569 13. 56Reactive 132 144, 211Valid 695, 473 17. 88Reactive 125, 697Valid 730, 548 18. 78Reactive 133 117, 572Valid 1, 133, 872 29. 15Reactive 115, 486Valid 998, 034 25. 65Reactive 134 123, 75 Valid 1, 184, 643 30. 45Reactive 130, 805 Valid 974, 131 25. 04 Reactive 135 121, 01 Valid 813, 313 20. 91Reactive 144, 878Valid 50, 801 1. 31Reactive 136 106, 406Valid 1, 238, 006 31. 82Reactive 133, 928Valid 960, 602 24. 69Reactive 137 119, 685Valid 1, 052, 34 27. OSReactive 126, 532Valid 941, 37 24. 2 Reactive 138 125, 52, Valid 1, 107, 795 28. 48Reactive 141, 315Valid 1, 020, 298 26. 23 Reactive 139 140, 870Valid 1, 134, 043 29. 15Reactive 133, 515Valid 862, 352 22. 17Reactive 140 149, 207 Valid 19, 061 0. 49NonReactive 141, 651 Valid 1, 874 0. 05NonReactive pop ; !) a p t (r,, y 144 177, 055 Valid 658, 432 17. 55 Reactive 153, 886 alid 502, 58 13. 40 Reactive 145 145, 802 Valid 936, 874 24. 97 Reactive 143, 092 Valid 926, 628 24. 7 Reactive 146 180, 04 Valid 1, 032, 891 27. 53 Reactive 151, 577 Valid 922, 925 24. 60 Reactive 147 173, 15 Valid 525, 056 13. 99 Reactive 125, 13 Valid 960, 205 25. 59 Reactive 148 178, 29'Valid 1, 135, 367 30. 26 Reactive 171, 95'Valid 967, 663 25. 79 Reactive 149 192, 437Valid 1, 135, 829 30. 27 Reactive 157, 575 Valid 77 36, 2. 0 Reactive =mown 151 157, 10 Valid 1, 017, 992 27. 13 Reactive 152, 24 (Valid 866, 045 23. 0E Reactive 152 150, 133Valid 497, 433 13. 26 Reactive 146, 805Valid 710, 053 18. 93 Reactive 153 156, 887Valid 836, 792 22. 30 Reactive 143, 626Valid 527, 391 14. 06 Reactive 154 204, 463 Valid 1, 089, 011 29. 03 Reactive 158, 308Valid 935, 87 24. 9 Reactive "f t. , "rp c a 160 17263= Valid 116153a 33. 23 Reactive 16158'Valid 97843'28. 0 (Reactive 161 146970 Valid 1258987 36. 02 Reactive 182611 Valid 960958 27. 50 Reactive 165 15515 alid 114528 28. 71 Reactive 131576 alid 1054818 26. 45 Reactive 166 202228 Valid 1288291 32. 30 Reactive 171323 alid 1029030 25. 80 Reactive . .., : 167 (rerun) 158763Valid 27371 0. 73NonReactive 156827Valid 2403 0. 06NonReactive 168 177429Valid 88343 22. 15 Reactive 142512 Valid 713718 17. 89 Reactive 169 175045 Valid 491 0. 12 NonReactive 161854 alid 219329 5. 50 Reactive 169 (rerun) 156478 Valid 376026 9. 96 Reactive 166594 Valid 107698 2. 85 Reactive 172 17743 Valid 1014551 26. 88 Reactive 155221 Valid 964775 25. 5 Reactive 173 182809 Valid 1166096 30. 89 Reactive 165294 Valid 92139 24. 4 Reactive 174 176308Valid 1082941 28. 69 Reactive 16877 (Valid 494965 13. 11 Reactive 175 172119Valid 1276678 33. 82 Reactive 149172 alid 987151 26. 15 Reactive , e ls ne ative atieint ; sam les..., . . __. 141 179, 492 Valid 4, 797 0. 14 NonReactive 139, 782 Valid 3, 001 0. 08NonReactive 142 174, 393 Valid 4, 584 0. 12 onReactive 135, 290Valid. 3, 036 0. 08FonReactive 155 162, 312 Valid 3, 1551 0. 08 NonReactive 150, 63 ^ Valid 5, 001 0. 13NonReactive 156 172, 361 Valid 3, 11 0. 08NonReactive 151, 921 Valid 3, 318 0. 09NonReactive 157 186, 255 Valid 2, 53 0. 07NonReactive 160, 338 Valid 1, 926 0. 05NonReactive 162 204790 Valid 5667 0. 16 NonReactive 172383Valid 4828 0. 14NonReactive 163 201933Valid 6969 0. 2 NonReactive 174506 Valid 4193 0. 12 onReactive 164 182811 Valid 6691 0. 17NonReactive 172278Valid 3953 0. llNonReactive 170 181557 Valid 3222 0. 08 onReactive 148191 alid 225 0. 06NonReactive 171 181368 Valid 3523 0. 09NonReactive 133138VaIid 2319 0. 06NonReactive [80] Both the serum and whole blood for 55 of the 63 HIV-1 patients tested positive, i. e., reactive, for HIV-1 RNA. Eight samples (116,119, 120,143, 150,158, 159, and 167; shaded) did not test positive for HIV-1 RNA in both the serum and whole blood. In each of these eight samples, HIV-1 RNA was detected in whole blood only. These eight samples were also identified as having viral loads below the limit of detection (<75 copies/mL) in the branched DNA assay. Thus the whole blood samples were again more useful at detecting HIV-1 in patient samples with a lower level of HIV-1 RNA. These data demonstrate that whole blood testing is more sensitive at detecting the presence of viral nucleic acids.

[81] It will be noticed that sample 169 initially tested positive for HIV-1 RNA in serum but negative in whole blood. Sample 169 was retested (rerun) and was found to be positive in both the serum and whole blood portions.

Example 3 [82] Testing isolated red blood cells for the presence of viral nucleic acids.

[83] Whole blood is comprised of two major components, plasma and red blood cells. The plasma occupies approximately 55% of the total blood volume and the red blood cells occupy approximately 47% of the total blood volume. As demonstrated in Example 2, low levels of viral nucleic acids were detected in a whole blood sample of a patient but not in comparably diluted plasma samples from the same patient. If the viral nucleic acids cannot be detected in the plasma fraction of the whole blood, the viral nucleic acids must be associated with another blood component. Although not wishing to be bound by theory, the red blood cells occupy the majority of the remainder of the blood volume and may be the expected the source of the amplified viral nucleic acids. The red blood cells also outnumber other cell types in the blood to such an extent that the amount of virus detected in whole blood is too high to be associated with any other cell type.

[84] Isolated red blood cells are tested for association with viral nucleic acids. Red blood cells are isolated from whole blood using Ficoll-Hypaque sedimentation. The red blood cells sediment to the bottom of the Ficoll-Hypaque and are removed from the other cell fractions of the blood. The red blood cell fraction is washed in saline to a final volume of 1.5 mL. One hundred milliliters of the washed red blood cells is removed and diluted with 0.9 mL diluent. One half milliliter of the diluted red blood cells is used to detect viral nucleic acids.

Methods [85] Target capture followed by TMA assay [86] The target capture followed by TMA assay was performed using the Procleix (E) SV- 1/HCV Assay (Gen-Probe Incorporated; Chiron Corporation), which comprises three steps: target capture, amplification, and hybridization protection.

[87] Target capture is performed manually, or in an automated system. First, 400 p-L of vortexed target capture reagent is added to each of a set of reaction tubes. Five hundred microliters of test or control sample is added to each reaction tube containing the target capture reagent. The reaction tubes are transferred to a rack and covered with sealing cards. Each rack of reaction tubes is vortexed for a minimum of 20 seconds to mix the sample and the target capture reagent. The rack is then incubated for approximately 20 minutes at approximately 60°C and then at room temperature for 14 to 20 minutes. The viral nucleic acids attached to the target capture reagent are separated from the remainder of the sample by transferring the rack to a separation bay for 9 to 20 minutes and aspirating the supernatant from each tube. The target capture reagent is washed twice in 1 mL wash solution.

[88] The washed target capture reagent containing the viral nucleic acids is directly used in the amplification reaction. Seventy-five microliters of amplification reagent is added to the bottom of each of the reaction tubes and 200 pL of oil is added to overlay the amplification reaction mixture. The tubes are covered with sealing cards and are vortexed a minimum of 20 seconds to resuspend the target capture reagent. The reaction tubes are incubated at approximately 60°C for about ten minutes and then are transferred to approximately 41. 5°C for 9 to 20 minutes. While the tubes are at 41. 5°C, 25 fiL of enzyme reagent is added to each tube. The tubes are shaken to mix the reagents and incubated at approximately 41. 5°C for about an hour. The tubes are then removed from the water bath and are directly used for the hybridization protection assay.

[89] One hundred microliters of probe reagent is added to each tube. The probe reagent is mixed with the amplification reagents by vortexing a minimum of 20 seconds. The tubes are then incubated at approximately 60°C for about 15 minutes. The selection reagent is next added to each tube. Two hundred fifty microliters of selection reagent is added and is mixed into the reaction mixture by vortexing at least 20 seconds. The tubes are incubated again at 60°C for approximately 10 minutes. Incubating in a water bath at a temperature of 23°C4°C then cools the tubes. The tubes are transferred to a luminometer and the probes are detected.