Abstract:
The inventive subject matter relates to a method for the diagnosis of  Orientia tsutsugamushi  infection by measuring the increased or decreased expression of specific human genes following infection by microarray or polymerase chain reaction analysis. The method employs the creation of gene modulation profiles in patients suspected to be infected with  O. tsutsugamushi  and comparing the profiles with a pre-determined profile of genes known to modulate in response to  O. tsutsugamushi  exposure and infection. The method permits the early detection of  O. tsutsugamushi  infection and diagnosis of scrub typhus earlier than currently available methods. The method also permits mid-course monitoring of disease progression with greater detail than currently available methods.

Description:
BACKGROUND OF INVENTION  
       [0001]     1. Field of Invention  
         [0002]     The inventive subject matter relates to a method of diagnosing Rickettsial diseases by analysis of modulation of host gene expression. The method contemplates the use of microarray technology for the detection and analysis of gene up or down regulation in response to bacterial infection.  
         [0003]     2. Description of Related Art  
         [0004]     The disease scrub typhus, caused by the Gram negative bacteria  Orientia  (formerly  Rickettsia )  tsutsugamushi  is one of the most common rickettsial diseases and can cause up to 35% mortality if left untreated (1, 2). The bacterial pathogen accounts for up to 23% of all febrile episodes in endemic areas of the Asia-Pacific region. Geographic distribution of the disease occurs principally within an area of about 13 million square kilometers and includes Pakistan, India and Nepal in the west to Japan in the east and from southeastern Siberia, China and Korea in the north to Indonesia, Philippines, northern Australia and the intervening Pacific islands in the south. During World War II, more than 5,000 cases of scrub typhus were reported among U.S. troops and 30,000 cases for Japanese troops. Scrub typhus ranked only behind malaria as the most important arthropod borne infectious disease. More recently, scrub typhus was the second leading cause of fevers of unknown origin among U.S. personnel during the Vietnam conflict.  
         [0005]     Because of the relatively high mortality rate in untreated patients, the rising prevalence of drug resistant strains, and the lack of vaccines against the organism, early detection of exposure and infection is becoming increasingly important. For this reason, simple and accurate methods are important for early detection and effective treatment of the disease. However, despite the global public health importance of scrub typhus, currently available diagnostic methods are inadequate. Diagnosis of scrub typhus is generally based on the clinical presentation and history of the patient. Because of similarities in symptomatology, however, differentiation of scrub typhus from other febrile diseases, such as leptospirosis, murine typhus, malaria, dengue fever and viral hemorrhagic fevers, is often difficult especially early after infection.  
         [0006]     In order to overcome the short-comings in scrub typhus diagnosis, significant research effort has been devoted to developing accurate laboratory diagnostic methods for scrub typhus. The currently available assays are typically seriologically-based and include indirect-fluorescence assay (IFA), indirect immunoperoxidase assay (IIP), enzyme-linked immunosorbent assay (ELISA) and dot blot assays. These assays, however all suffer from the requirement of requiring the availability of antigen which typically entails growing rickettsiae grown in host cells or preparing extracts of purified bacteria as well as the availability of antibody in patient sera (3-10). Additionally, the assay methods are time consuming to perform and offer limited insight into serotypes not represented by the panel of available antigen.  
         [0007]     A problematic hurdle in the design of sensitive and accurate diagnostic assays is ensuring the assay&#39;s effectiveness early after infection. In currently available and employed antibody-based assays, sensitivity requires a suitable number of bacteria in tissue samples. Typically, adequate levels of bacterial load to meet the required threshold are not found, especially early after an infection. Likewise, detection of seroconversion is also not an effective diagnostic method early after exposure and infection since no detectable, specific antibody would be present.  
         [0008]     Other confounding issues in designing suitable assays include the fact that Orientia strains exhibit significant antigenic differences thereby complicating assay antigen selection for use in available scrub typhus serodiagnostic procedures. For example, the major outer membrane protein (vOmp) of  O. tsutsugamushi  is an important serodiagnostic antigen but varies from 53-63 kDa even among isolates from the same country (11). Furthermore, both unique and cross-reactive domains exist in different homologs that potentially necessitating the use of multiple strains in scrub typhus diagnostic test design. Additionally, the list of scrub typhus serotypes is incomplete.  
         [0009]     Polymerase chain reaction (PCR) amplification of  O. tsutsugamushi  genes has been demonstrated to be a reliable diagnostic method for scrub typhus (12, 13). PCR permits the rapid identification of distinct genetypes that are associated with  Orienta  serotypes (12, 14-18). However, despite the advantages of PCR, significant disadvantages include the requirement for sophisticated instrumentation and labile reagents to conduct the assays that are often not available in rural medical facilities. Additionally, PCR procedures are highly susceptible to false positive results due to inadvertent carry-over of nucleic acid material. This is particularly prevalent in field settings or in facilities that are not fully equipped to conduct PCR laboratory procedures.  
         [0010]     A solution to the paucity of early diagnostic methods is to monitor the expression of host response genes in response to infection. Early after exposure to an infectious organism, host responsiveness to infection is manifested by modulation of specific gene expression. Some genes are differentially expressed very early after infection thus permitting the construction of unique gene expression profiles that are exhibited early after infection of human cells, such as peripheral blood mononuclear cells (PBMC). The patterns or profiles of gene expression would thus enable the differentiation of exposure by pathogens and toxins, including  Bacillus anthracis, Yersinia pestis, Brucella melitensis , botulinum toxin, staphylococcal exotoxins A and B (SEB, SEA), lipopolysaccharide (LPS), cholera toxin, Venezuelan equine encephalitis virus (19). Furthermore, it has been previously shown that specific human genes modulate up or down in response to bacterial infection (20).  
         [0011]     Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) is capable of sensitively measuring changes in gene expression from collected host cell RNA. By designing primer sets specific to a limited number of genes, known to have altered expression following infection, molecular-based assays can be devised to diagnosis and monitor infection early after infection by direct assessment of gene modulation.  
         [0012]     Although measurement of changes in gene expression by RT-PCR is a valuable diagnostic strategy, the method suffers from the disadvantages associated with PCR in that it is often not suitable for high-throughput screening of large numbers of genes. A more convenient method of measuring gene expression changes is by hybridizing amplified RNA onto cDNA microarrays containing large numbers of double-stranded sequences of important host genes. A number of computer programs are available to accurately analyze and transform the ensuing gene expression data into useful and reproducible gene expression profiles.  
         [0013]     Microarrays are well suited for high-throughput detection of thousands of differentially expressed genes in a single experiment (21). The method allows for the characterization of the cascade of cellular signaling and concomitant interrelated host gene expression profiles following infection by specific pathogens or toxins (22, 23). Therefore, data from cDNA microarrays provides the ability to quickly and accurately assess and monitor the changes in gene expression profiles specific to infection by specific pathogenic organisms. Microarrays can also be used to evaluate genomic differences between virulent and nonvirulent strains of a species (24).  
         [0014]     Therefore, in order to improve early diagnosis of scrub typhus, an aspect of this invention is the diagnosis of  O. tsutsugamushi  early after exposure and infection by the measurement of specific host gene expression profile. The invention, therefore, will give diagnosticians the ability to diagnosis  O. tsutsugamushi  days or weeks earlier than previously possible with a concomitantly greater likelihood of accuracy in disease etiology. Additionally, the care provider will be able to accurately monitor the course of the disease, thereby facilitating the selection of effective drug regimens.  
       SUMMARY OF INVENTION  
       [0015]     Current methods for the detection and diagnosis of scrub typhus, caused by the rickettsial organisms  Orientia tsutsugamushi  early after infection are inadequate. An object of this invention is a method for diagnosis of  O. tsutsugamushi  early after exposure and infection to the organism and the monitoring of disease course by the modulation of expression of specific host cell genes.  
         [0016]     A further object of the invention is the diagnosis of  O. tsutsugamushi  by polymerase chain reaction with low background due to amplification of contaminating DNA. 
     
    
     BRIEF DESCRIPTION OF DRAWINGS  
       [0017]      FIG. 1 . Comparison of expression of interferon induced protein using mRNA from  O. tsutsugamushi  infected and uninfected leukocytes by real-time polymerase chain reaction.  
         [0018]      FIG. 2 . Comparison of expression of 2′-5′ oligoadenylate synthetase 3 using mRNA from  O. tsutsugamushi  infected and uninfected leukocytes by real-time polymerase chain reaction. 
     
    
     DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS  
       [0019]     Diagnosis of the disease scrub typhus caused by  O. tsutsugamushi  early after exposure of individuals to the bacteria is difficult due to a lack of available assay methods. Current methods for the diagnosis of scrub typhus rely on detection of serum conversion, which is not possible until significant time has elapsed after exposure or the direct detection of the organism which requires a considerable incubation period following exposure.  
         [0020]     Analysis of human gene expression profiles has become an increasingly important mode of predicting disease onset and for monitoring disease progression. Following exposure to external insults, such as infectious organisms or toxins, some cellular genes are modulated to increase or decrease expression. Specific cell perturbations can result in precise gene modulation profiles that are predictive for a specific external insult. The current invention capitalizes on this phenomenon by monitoring gene expression early after exposure of human cells to  O. tsutsugamushi  by measuring mRNA encoding the gene product or by measuring the genes protein product itself. Analysis of the gene modulation profile of cells is highly predictive of prior exposure and infection with  O. tsutsugamushi . Therefore, an aspect of the invention is the detection and measurement of changes in gene expression following exposure and infection by  Orientia tsutsugamushi.    
         [0021]     Analysis of human gene expression can therefore be a predictor of infection by specific microorganisms. The general approach, therefore, of evaluating changes in human gene expression can be utilized as an effective diagnostic tool very early after infection, when other currently available methods are not effective. The approach can be used alone or in tandem with other methods, therefore, to follow progression of the disease state through treatment.  
         [0022]     Diagnosis of infection is operationally carried out by initially measuring changes in gene expression in response to infection. Any semi-quantitatively or quantitative procedure can be used to measure changes in expression. A number of methods can be used to measure gene expression. Gene expression profiles can be measured by antibody-based methods, such as enzyme-linked immunosorbent assay (ELISA). In ELISA, a specific quantity of extracted cell protein is immobilized which is then exposed to antibody specific for genes suspected of modulation. The expression of the specific genes are normalized to the expression of a house-keeping gene. Antibody-based assays, however, suffer from the inherent requirement of antibody to selected antigens of interest and time-intensity required to conduct the assay. Therefore, alternative approaches include molecular assay methods.  
         [0023]     Measuring changes in gene expression by reverse transcriptase polymerase (RT-PCR) chain reaction is best conducted by constructing primer sets containing at least one of the primers to the mRNA splice site. This aspect of the invention significantly increases specificity and therefore reliability of diagnosis by reducing the amplification of contaminating DNA.  
         [0024]     Alternatively, or in addition to RT-PCR, labeled cDNA copies of mRNA from the infected human cells can be exposed to complimentary DNA copies of specific genes attached to glass microarray chips and the bound cDNA quantitated. Use of microarrays permits the convenient analysis of large numbers of genes in a single experiment. RT-PCR can also be used, in conjunction with microarray analysis, to either confirm results or to more accurately determine the relative degree of modulation of target genes. Evaluation of gene modulation profiles is conducted by computer program analysis.  
         [0025]     Any semi-quantitatively or quantitative procedure can be used that accurately measures changes in host cell gene expression following bacterial exposure and infection. Regardless of the specific method used, the general approach in all methods employs the following steps: 
        a. obtaining leukocytes from blood samples from patients potentially exposed to  O. tsutsugamushi;       b. extracting total RNA or protein from the leukocytes;     c. measuring gene products of a panel of important host genes by molecular, antibody-based or other methods;     d. normalizing the expression of the important host genes in the potentially infected cells to that in uninfected cells;     e. analyzing the pattern or profile of gene modulation by computer program.        
 
         [0031]     Based on the gene modulation profile, a diagnosis early after exposure and infection is made by comparing the profile detected with that associated with the profile associated with  O. tsutsugamushi  infection. Since this method permits diagnosis much earlier after infection than other available assay methods, early, and presumably more efficacious, antibiotic treatment can be instituted. Additionally, regular re-evaluation of expressed genes during disease progression permits real-time evaluation of the effectiveness of the drug treatment regimen and modification of treatment methods, if needed. To more clearly describe the invention, the following examples are given.  
       EXAMPLE 1  
     Detection of Gene Expression in the in PBLs by Hybridization of Gene Products to Microarray Chips  
       [0032]     Peripheral blood lymphocyte (PBL) were utilized as the source of RNA in order to examine the gene expression modulation in response to infection with  O. tsutsugamushi . Other cell types, however could be used including purified peripheral blood mononuclear cells or subpopulations such as T-cells, B-cells and macrophages.  
         [0033]     PBLs were obtained from whole blood from healthy individuals are drawn into cell preparation tubes containing anti-clotting agents, such as citrate. The tubes are inverted 8 to 10 times and centrifuged at 1,500×g for 30 minutes at room temperature. Plasma is then removed and the PBLs carefully removed. After washing the cells in phosphate buffered saline (PBS) the cells are suspended in RPMI 1640 media, supplemented with 2.5 mM L-glutamine, 25 mM HEPES and 7.5% human serum. The cells are then re-centrifuged and subsequently re-suspended in RPMI media until used.  
         [0034]     The PBLs are exposed to  O. tsutsugamushi  by exposing the cells to the bacteria for 30 minutes in 500 μl of RPMI. Same numbers of cells are also incubated without the addition of bacteria for use in the preparation of control RNA. After 30 minutes, the cells are washed with media and re-suspended in 48 ml of complete media (RPMI supplemented with supplemented with 2.5 mM L-glutamine, 25 mM HEPES and 7.5% human serum). Five ml of the re-suspended cells was then added to flasks containing 20 ml of the RPMI media supplemented with 2.5 mM L-glutamine, 25 mM HEPES and 7.5% human serum. At specific times after infection the cultured cells are scraped off and the RNA prepared utilizing Trizol® (Invitrogen, Calsbad, Calif.). After preparation of RNA the RNA was treated with DNase to remove remaining amounts of contaminating DNA. The resulting RNA was stored at −80° C. until required.  
         [0035]     RNA of control and  O. tsutsugamushi  infected cells was reverse transcribed with oligo dT to systhesize cDNAs. The cDNA was then labeled with either Cy3 or Cy5. The reference RNA (UNIVERSAL HUMAN REFERENCE RNA, Strategene, Calif.) was labeled with the dye not used in the labeling of sample cDNA. Labeled cDNA was permitted to hybridize at 42° C. overnight to glass chips containing approximately 7,680 cDNA gene sequences. The cDNA clones were spotted onto poly-L-lysine-coated slides using an OmniGrid arrayer® (GeneMachines, San Carlos, Calif.). After hybridization, the bound labeled cDNA was scanned and the image was analyzed using a GenePix® (Molecular Devices Corporation, Union City, Calif.) computer program. Normalization of induced expression, following  O. tsutsugamushi  infection, was conducted by comparing expression of RNA from sample PBLs to reference RNA from uninfected PBLs and subtracting from the spot intensity the background intensity to produce a channel-specific value. The data were filtered for signal intensities and background of 2.0-fold in both channels. The raw data were converted into log2 data.  
         [0036]     For each time period, data from 2 to 4 separate experiments are obtained and the data from these multiple experiments are subjected to a 2-way ANOVA analysis. The GeneSpring® (version 5.0)(Silicon Genetics, San Carlos, Calif.) and Partek Pro® (version 5.0) (Partek, Inc, St. Charles, Mo.) are used to visualize and analyze the data, which is shown in tabular form in Table 1. Table 1 shows the mean results of two experiments using samples from three donors. A determination of up or down modultion was predicated on relative expression, in arbitrary units, either above or below a baseline level. A measurement of 1.000 would indicate no change in expression. As seen in Table 1, some genes, such as Tyrosine 3-monooxygenase, actually exhibited a large increase in expression then a precipitous decline in expression with a presumed return to baseline expression. Based on this kinetic profile, the gene was scored as up-modulated. PCR confirmatory analysis supported this contention. Gene expression that varied significantly across samples were identified as well as consistent patterns of gene expression modulation in PBLs exposed to  O. tsutsugamushi . In some arrays, a scatter-plot smoother (e.g. Lowess algorithm) was employed (25).  
         [0037]     If the PBLs had been obtained from presenting patients, early treatment, prior to that capable using currently available methods, can be initiated. Diagnosis is made by comparing and contrasting the gene modulation profile of the obtained PBLs with the expected gene induction following infection with  O. tsutsugamushi.    
         [0038]     Follow-up, confirmatory diagnostic assays, such as RT-PCR or ELISA and other antibody-based assays for the detection of bacterial antigen, can be undertaken in order to give further assurance of infection and strain identification. Furthermore, additional assays, during the course of the disease, by microarray analysis or by other traditional diagnostic methods using fresh PBLs, can be undertaken to monitor the disease progression and effectiveness of treatment.  
                                                                                                                           TABLE 1                                       Relative Expression (arbitrary units)                Cells from uninfected   Cells from infected donor           donor (control)   (target cell)            Gene   Time (hr)            (up/down modulated)   1   4   8   18   1   4   8   18                    Mylein basic Protein   0.966   0.931   0.697   0.956   2.723   0.305   0.576   0.523       (down)       Lymphotoxin alpha (up)   —   —   —   —   1.33   1.148   1.230   5.807       FK506 binding protein (up)   —   —   —   —   1.140   4.654   2.145   1.656       Interferon induced protein   —   —   —   —   0.830   2.234   9.999   2.353       with tetracopeptide repeats       2 (IFIT2) (up)       Chemokine receptor 7 (up)   0.894   0.813   1.302   0.821   2.614   3.367   0.958   1.025       Never-in-Mitosis gene a   —   —   —   —   12.415   2.014   0.776   3.230       relaed kinase (NIMA) (up)       Chemokine ligand 3   —   —   —   —   3.100   1.723   10.812   1.863       (CCL3) (up)       Transcription factor 12   —   —   —   —   0.0   1.044   4.133   7.138       (up)       Minichromosome maint   —   —   —   —   1.534   0.785   5.204   4.484       deficient 3 associted       protein (up)       NADH dehydrogenase Fe—S   —   —   —   —   1.352   2.191   1.494   3.909       Protein 3       Zinc finger protein 147   —   —   —   —   2.091   2.028   1.440   7.780       (ZNF 147) (up)       Chemokine ligand 8   —   —   —   —   0.616   1.069   16.370   6.687       (CCL8) (up)       2′-5′ oligoadenylate synt 3   —   —   —   —   0983   3.945   8.410   3.401       (up       TP inducible gene   0.939   0.876   1.015   0.982   0.588   2.208   1.577   0.510       (TP53TG3) (down)       Junction plakoglobin (JUP)   —   —   —   —   22.99391   1.445   2.654   1.909       (up)       Viperin (cig5) (up)   —   —   —   —   2.868   0.666   8.112   4.033       Replication Protein A2 (up)   0.971   0.914   0.954   0.955   1.721   0.969   0.938   1.043       G protein signaling 1   0.800   0.896   0.404   0.935   0.218   1.006   5.104   1.104       (RGS1) (up)       Apoptosis-related cysteine   0.956   0.546   1.141   0.932   0.858   1.491   1.055   1.023       protease (CASP7) (up)       Tyrosine 3-   0.973   0.918   0.786   0.758   3.447   0.472   0.659   0.660       monooxygenase/tryptophan       5-monooxygenase       activation protein, beta       polypeptide (up)       Polymerase gamma 2,   0.746   0.936   0.840   0.721   1.631   0.738   0.657   1.069       accessory subunit (up)       Enhancer of zeste homolog   0.934   0.903   1.068   0.865   1.699   0.942   0.906   1.091       2 (EZH2) (up)                  
 
       EXAMPLE 2  
     Analysis of Gene Modulation by Polymerase Chain Reaction  
       [0039]     Gene modulation can be determined by quantitative reverse transcriptase polymerase chain reaction (RT-PCR). RT-PCR analysis can also be used alone or in tandem with other methods, such as microarray analysis, in order to confirm the results obtained by that method. In this embodiment, human monocytic cells or PBLs are obtained from potentially  O. tsutsugamushi  infected patients. Whole blood from healthy individuals are drawn into cell preparation tubes containing anti-clotting agents, such as citrate. The tubes are inverted 8 to 10 times and centrifuged at 1,500×g for 30 minutes at room temperature. Plasma is then removed and the PBLs carefully removed. After washing the cells in phosphate buffered saline (PBS) the cells are suspended in RPMI 1640 media, supplemented with 2.5 mM L-glutamine, 25 mM HEPES and 7.5% fetal calf serum. The cells are then re-centrifuged and subsequently re-suspended in RPMI media until used.  
         [0040]     A specific example of how the PCR is practiced in the diagnosis of  O. tsutsugamushi  is illustrated by the following example. Freshly isolated human leukocytes are either used uninfected or infected with  O. tsutsugamushi  at MOI of 1.0 for 45 minutes at 35° C. The leukocytes, both infected and uninfected, were culture at 35° C. in 5% CO 2 . After incubation, infected and uninfected leukocytes were collected at 1 hour, 4 hours, 8 hours or 18 hours after infection. Total RNA was then obtained according to the Trizo® method (Invitrogen, Carlsbad, Calif.) followed by treatment with DNase. The concentration of total RNA in each sample was initially quantitated spectrophotometrically at 260 nm. The first strand cDNA synthesis was performed in 100 μl reaction volumes containing 15 μg of total RNA from each sample and 2 μl of oligo dT or random primers is added to total RNA and the mixture heated at 70° C. for 5 minutes then cooled on ice for 3 minutes. Following the addition of the first strand buffer, dNTP, DTT and reverse transcriptase, the final mixture was incubated at 42° C. for 50 minutes and then shifted to 70° C. for 10 minutes. Specific primers that flank the mRNA splicing sites of 658 genes were designed using a primer designing algorithm, GeneLooper™, (GeneHarbor, Inc, Rockville, Md.). The primers were designed to provide a uniform annealing temperature of 62° C. and amplification products of 300-350 bp. Synthesized cDNA was semi-quantitated by PCR in “comparing groups” composed of cDNA derived uninfected and infected leukocytes. Semi-quantitation was normalized to the house-keeping gene glyceraldehyde 3-phosphate dehydrogenase. Synthesized cDNA was stored at −80° C. until needed.  
         [0041]     After production of a cDNA copy of the RNA by reverse transcriptase, primers to selected targets are used to amplify specific target genes sequences. Primer sets are designed such that at least one primer member of a primer set is complementary to the sequence encoding the splice site of the target mRNA. Targeting of primer sequences complementary to splice junctions ensures that amplification of sequences will not occur using genomic DNA as template. Thus background amplification due to amplification of remaining DNA, despite treatment of RNA with DNase will be minimized.  
         [0042]     PCR analysis was performed using the i-cycler™ (BioRad, Hercules, Calif.) using cDNA from infected and uninfected leukocytes. PCR was performed using the light cycler DNA master SYBR green I® kit (Rocke Diagnositcis, Indianapolis, Ind.) in 20 μl reaction volumes using cDNA from infected and uninfected comparing groups, after addition of dNTPs, PCR buffer, forward and reverse primers and Taq polymerase. The PCR mixture was incubated at 94° C. for 3 min followed by 32 cycles of 3 step amplification at 94° C. for 30 seconds, 62° C. for 30 seconds and 72° C. for 1 minute. A 72° C. hold step was performed for 5 minutes following PCR cycling. PCR amplified products of reverse transcribed RNA can be visualized by first separating the products by electrophoresis on 1% agarose gel. Semi-quantitation of the PCR gel image data was then performed using gel analyzing software (GelPicAnalyzer™, GeneHarbor, Inc., Rockville, Md.). Background was subtracted and the values normalized to the amplified house-keeping gene (i.e. glyceraldehyde 3-phosphate dehydrogenase). The results of these studies are illustrated in Table 2.  
         [0043]     The open reading frame (ORF) of two genes, interferon induced protein and 2′-5′ oligoadenylate synthetase 3, were used to develop a fluorogenic-based PCR assay. For the assay PCR probes were labeled with 6-carboxyfluorescein (FAM) and 6-carboxytetramethyl-rhodamine (TAMRA) at the 5′ end and 3′ ends, respectively. This permits monitoring of specific PCR product over time.  
         [0044]     PCR reactions were conducted in 25-50 μl reaction mixtures containing 1×TaqMan Universal PCR Master Mix®), 0.02 μM of each primer, 0.1 μM probe and 1 μl of diluted cDNA template. After incubation at 50° C. for 2 min and denaturation at 95° C. for 10 minutes the reaction is permitted to proceed for 45 to 60 cycles with denaturation at 94° C. for 15 seconds and extension at 60° C. for 1 minute. The 18 S rRNA gene was included as an endogenous reference and the comparative C t  (threshold cycles) method applied using arithmetic formulas. By this method, the amount of target was normalized to that of the 18S RNA. Real-time RT-PCR assays are performed in triplicate for each sample and a mean value and standard deviation calculated for relative RNA expression levels. The results of studies using interferon induced protein and 2′-5′ oligoadenylate synthetase 3 as target genes is shown in  FIG. 1  and  FIG. 2 , respectively. As can be seen in  FIG. 1  and  FIG. 2 , a marked shift in the number of cycles required for visualization of product is clearly evident in the mRNA from infected (i.e. approximately 14 to 15 cycles) verses uninfected cells (i.e. 30 to 31 cycles) in these two genes. In both situations, the 18 S RNA was used to normalize the raw PCR reaction data.  
         [0045]     Analysis of only interferon induced protein and 2′-5′ oligoadenylate synthetase 3 from patients suspected of infection, compared mRNA from uninfected leukocytes, is required in order to make a determination of prior infection. However, analysis of these genes in combination with other genes that were evaluated in Tables 1 and 2 would provide a more accurate analysis of and determination of prior infection.  
                                                                                                                                             TABLE 2                                       Sample   Up/down   Donor 1   Donor 2   Donor 3            Gene   number   regulated   Control   Infected   Control   Infected   Control   Infected               Mylein Protein zero   1   down   1,124     338   8,434   5,441    4,384    3,176           2   down   10,663     3,781   7,744   7,338    3,725    2,872       Lymphotoxin alpha   1   up   —   —   6,812   11,698   11,077   28,815           2   up      0    8,618   1,085   12,586   —   —       FK506 binding protein   1   up   —   —   3,065   10,803   15,877   21,439           2   up   3,065   11,137   11,796   14,793   —   —       Interferon induced protein   1   up   —   —   5,114   15,063   15,707   27,989       with tetratricopeptide       repeats 2 (IFIT2)           2   up   4,228   16,620   0   963   —   —       Chemokine receptor 7   1   up   3,358    9,751   20,227   37,167   11,462   14,687       (CCR7)           2   up   6,430   16,156   15,070   20,887    8,731   17,533       Never in mitosis gene a-   1   up   —   —   502   8,625   13,677   22,057       related kinase 3           2   up   —   —   13,626   20,958   —   —       Chemokine ligand 3   1   up   —   —   58   883   15,266   22,297       (CCL3)           2   up   —   —   9,548   11,990   —   —       Transcription factor 12   1   up   —   —   9,280   13,089     874   16,857           2   up   9,280   13,442   9,390   14,878   —   —       Minichromosome maint   1   up   —   —   3,622   4,104    5,898   18,194       deficient 3 associated       protein           2   up   2,814   11,120   7,971   9,619   —   —       NADH dehydrogenase Fe—S   1   up   —   —   10,323   17,684   16,501   24,291       Protein 3           2   up   10,323    18,230   9,482   14,189   —   —       Zinc finger protein 147   1   up   —   —   16,446   20,901   10,352   25,411       (ZNF 147)           2   up      0   16,180   2,872   13,498   —   —       Chemokine ligand 8   1   up   —   —   12,444   19,140    4,229   15,296       (CCL8)           2   up   4,092    5,282   433   15,670   —   —       2′-5′ oligoadenylate synt 3   1   up   —   —   2,596   14,787    3,084   18,794           2   up   4,092   18,919   0   9,299   —   —       TP inducible gene   1   down   1,838    1,138   8,458   3,481    2,761    2,490       (TP53TG3)           2   down   6,970    2,757   5,104   6,629   21,700   14,857                        Sample   Up/down   Donor 1   Donor 2   Donor 3            Gene   number   regulated   Control   Treatment   Control   Treatment   Control   Treatment               Junction plakoglobin (JUP)   1   up      1      1   6,355   4,833      1    3,179           2   up      1    7,559   0   7,731      1      1       Viperin (cig5)   1   up   —   —   16,014   19,723   20,773   27,480           2   up   8,512   15,357   16,670   24,589   —   —       Replication Protein A2   1   up   6,054   34,550   22,858   23,905   17,386   23,297       (RPA2)           2   up   3,015    4,644   8,226   6,428   —   —       G protein signaling 1   1   up   17,053    26,272   18,312   21,610   11,945   11,805       (RGS1)           2   up   18,486    26,449   —   —   —   —       Apoptosis-related cysteine   1   up   4,471   15,834   3,086   4,124      0.0   10,148       protease (CASP7)           2   up   13,281    14,530   —   —   —   —       Tyrosine 3-   1   up   29,277    35,740   23,164   25,013   18,072   26,616       monooxygenase/tryptophan       5-monooxygenase       activation protein, beta       polypeptide           2   up   5,228   14,320   1,524   2,567   —   —       Polymerase gamma 2,   1   up      0.0   11,309   16,683   19,821   15,972   21,288       accessory subunit           2   up   14,467    20,818   1,148   8,158   —   —       Enhancer of zeste homolog   1   up   13,362    14,065   17,005   22,224   18,024   22,360       2 (EZH2)           2   up   5,470   19,153   5,953   9,207   —   —                  
 
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         [0071]     Having described the invention, one of skill in the art will appreciate in the claims that many modifications and variations of the present invention are possible in light of the above teachings. It is therefore, to be understood that, within the scope of the claims, the invention may be practiced otherwise than as specifically described.