BIOMARKERS FOR TUBERCULOSIS AND HIV/AIDS

CROSS-REFERENCE TO PRIORITY APPLICATION

This application claims priority to U.S. Provisional Application No. 61/421 ,482, filed December 9, 2010, which is incorporated herein by reference in its entirety.

BACKGROUND

Diagnosing tuberculosis (TB) is a cumbersome task in countries where TB is endemic. The primary diagnostic test is obtaining sputum samples from patients and examining for acid- fast bacilli by microscopy. Multiple specimens and visits of the patient are required and this significantly increases the drop-out rate of patients who might be infected and thus leading to untreated TB. Unfortunately, the TB epidemic is not under control, making Mycobacterium tuberculosis (Mtb) the causative agent of TB a major health problem in where one-third of the world's population is latently infected. One out of every 10 infected TB patients will actually develop this disease, but this percentage increases significantly in those who are coinfected with both Mtb and HIV. Fortunately, TB, if caught early, can be treated, leading to fewer deaths. The drug regime is long and tedious consisting of 3 to 4 drugs over a period of 6 to 9 months, which leads to poor compliance and is the main cause of the emergence of single drug- resistant, multidrug resistant (MDR) and extensively drug-resistant (XDR) strains of Mtb.

SUMMARY

Provided herein are methods for determining the HIV status, the TB status and/or the purified protein derivative (PPD) status of a subject by measuring cytokine levels and utilizing predictive equations. Further provided are methods of treating HIV infection and/or TB infection.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1 A-C show HIV frequency plots of cytokines (PDGF ββ, SCF and eotaxin). Figures 2A-D show TB frequency plots of cytokines (GRO-a, MCP-3, TNF-β and MCSF).

Figures 3A-D show PPD+ vs. all others frequency plots of cytokines (MCP-3, LIF, CTAC and ICAM). Figure 4 shows PPD+ vs. TB- Frequency plots of cytokines (ICAM- 1 ).

Figure 5 shows PPD+ vs. healthy frequency plots of cytokines (ICAM- 1 ).

DETAILED DESCRIPTION

The present method provides for determining the HIV status, the TB status and/or the purified protein derivative (PPD) status of a subject by measuring cytokine levels and utilizing predictive equations. More specifically, the method includes diagnosing a subject as HIV+ or HIV- comprising (a) measuring the levels of eotaxin, stem cell factor (SCF), and platelet derived growth factor bb (PDGF ββ) in a biological sample from the subject and (b) computing a predictive value utilizing the following equation: p = 1 where z = 5.654 + 0.003*Eotaxin - 0.032*SCF - 0.001 *PDGF ββ,

1 + e ^~z wherein a value > 0.5 predicts HIV+, and a value < 0.5 predicts HIV-, thus diagnosing the subject as HIV+ or HIV-. Optionally, the method further comprises taking steps to initiate or alter treatment of the subject based on the determination.

As used herein, a biological sample is a sample derived from a subject and includes, but is not limited to, any cell, tissue or biological fluid. The sample can be, but is not limited to, peripheral blood, plasma, urine, saliva, gastric secretion or bone marrow specimens.

As used throughout, by subject is meant an individual. Preferably, the subject is a mammal such as a primate, and, more preferably, a human. Non-human primates include marmosets, monkeys, chimpanzees, gorillas, orangutans, and gibbons, to name a few. The term subject includes domesticated animals, such as cats, dogs, etc., livestock (for example, cattle, horses, pigs, sheep, goats, etc.) and laboratory animals (for example, ferret, chinchilla, mouse, rabbit, rat, gerbil, guinea pig, etc.). Veterinary uses and formulations for same are also contemplated herein.

Also provided is a method of diagnosing a subject as TB+ or TB- comprising (a) measuring the levels of macrophage colony stimulating factor (MCSF), tumor necrosis factor beta (TNFBeta), monocyte chemoattractant protein 3 (MCP3), and melanoma growth stimulating activity, alpha (GROalpha) in a sample from a subject and (b) computing a predictive value utilizing the following equation: p = 1 where z = 2.146 + 0.066*MCSF + 0.593*TNFp - 0.058*MCP3+0.012GROa,

1 + e - ^z

wherein a value > 0.5 predicts TB+, and a value < 0.5 predicts TB-, thus diagnosing the subject as TB+ or TB-. Optionally, the method further comprising taking steps to initiate or alter treatment of the subject based on the determination.

Also provided is a method of diagnosing a subject as PPD+ or not PPD+ comprising a) measuring the levels of leukemia inhibitory factor (LIF), MCP3, chemokine (C-C motif) ligand 27 (CTACK) and intercellular adhesion molecule 1 (ICAM- 1 ) in a sample from a subject and b) computing a predictive value utilizing the following equation:

P =_i_

l + e ^~z

where z = -0.61 1 - 0.055 *LIF + 0.009*MCP3 + 0.001 *CTACK -0.141 *ICAM 1/1000 wherein a value >0.5 predicts PPD+, and a value < 0.5 predicts not PPD+, thus diagnosing the subject as PPD+ or not PPD+. Optionally, the method further comprising taking steps to initiate or alter treatment of the subject based on the determination.

Also provided is a method of diagnosing a subject that is TB- as PPD+ or not PPD+ comprising a) measuring the levels of ICAM in a sample from a TB- subject and b) computing a predictive value utilizing the following equation:

p = 1 where z = 14.508 - 0.549*ICAM1 /1000

1 + e - ^z

wherein a value >0.5 predicts PPD+, and a value of <0.5 predicts not PPD+.

Table 1 sets forth identifying information for the proteins utilized in the predictive equations provided herein. Column 1 of Table 1 provides the name of the protein. Column 2 of Table 1 provides one or more aliases for each of the proteins. Therefore, it is clear that when referring to a protein, this also includes known alias(es) and any aliases attributed to the proteins listed in Table 1 in the future. Also provided in Table 1 are the GenBank Accession Nos. for the coding sequences (human mRNA sequences) (column 6) and the GenBank Accession Nos. for the human protein sequences (column 7). The nucleic acid sequences and protein sequences provided under the GenBank Accession numbers mentioned herein are hereby incorporated in their entireties by this reference. One of skill in the art would know that the nucleotide sequences provided under the GenBank Accession numbers set forth herein can be readily obtained from the National Center for Biotechnology Information at the National Library of Medicine (http://www.ncbi.nlm.nih. gov/entrez/query.fcgi?db=nucleotide).

Similarly, the protein sequences set forth herein can be readily obtained from the National Center for Biotechnology Information at the National Library of Medicine

( ^" http://www.ncbi. nlm.nih.gov/entrez/query.fcgi?db=protein). The nucleic acid sequences and protein sequences provided under the GenBank Accession numbers mentioned herein are hereby incorporated in their entireties by this reference. Further provided are the Entrez Gene numbers for the human genes (column 8). The information provided under the Entrez Gene numbers listed in Table 1 is also hereby incorporated entirely by this reference. One of skill in the art can readily obtain this information from the National Center for Biotechnology Information at the National Library of Medicine

(http://www.ncbi. nlm.nih.gov/entrez/querv.fcgi?db=gene).

These examples are not meant to be limiting as one of skill in the art would know how to obtain additional sequences for proteins and nucleic acids encoding the proteins listed in Table 1 from other species by accessing GenBank (Benson et al. Nucleic Acids Res. 2004 January 1 ; 32(Database issue); D23-D26), the EMBL Database (Stoesser et al., (2000) Nucleic Acids Res., 28, 19-23) or other sequence databases. One of skill in the art would also know how to align the sequences disclosed herein with sequences from other species in order to determine similarities and differences between the sequences set forth in Table 1 and related sequences, for example, by utilizing BLAST.

Table 1

Human

GenBank Human

Entrez Accession No. GenBank

Protein Alias Definition Gene for coding Accession No.

No. sequence/ for protein

mRNA

eotaxin CCL1 1 , Eosinophil NM_002986.2 NP 002977.1 6356

SCYA1 1 chemotactic protein

SCF SF, MGF, Stem cell factor; kit N 000899.4 NP_000890.1 4254

FPH2, KL- Iigand NM 003994.5 NP 003985.2 1, itl;

SHEP7;

kit-ligand Human

GenBank Human

Entrez Accession No. GenBank

Protein Alias Definition Gene for coding Accession No.

No. sequence/ for protein mRjNA

PDGF ββ PDGF2, PDGF-BB NM_002608.2 NPJ302599.1 5155 Homodimer SIS, SSV, (homodimer of NM 033016.2 NP 148937.1 of PDGF c-sis PDGF-B)

subunit b

MCSF CSF- 1 Colony stimulating NM 000757.5 NP 000748.3 1435 factor 1 NM ^" 72210.2 NP ^{" "} 757349.1

(macrophage) N ^{" "} 1 72212.2 NP] ^" 757351.1

TNFP TNFB, Tumor necrosis NM ^{" "} 000595.2 P ^" 000586.2 4049

TNFSF1 factor beta M ^{" "} 001 1597 NP] .001 153212.1

40. l ^"

MCP3 FIC, Monocyte NM 006273.2 NP_ 006264.2 6354

MARC; chemotactic protein-

NC28; 3

MCP-3;

SCYA6;

SCYA7

G Oa FSP, Melanoma growth NM 00151 1.2 NP 001502.1 2919

GROl, stimulating activity,

GROa, alpha

MGSA;

NAP-3,

SCYB 1 ,

MGSA-a

LIF CDF, DIA, Leukemia inhibitory NM_002309.3 NP_002300.1 3976

HILDA factor

CTAC ALP, ILC, Chemokine (C-C NM_006664.2 NP_006655.1 10850

CTAK, motif) ligand 27

PESKY,

ESK.INE,

SCYA27

ICAM BB2, Intracellular NM_000201.2 NP_000192.2 3383

CD54, adhesion molecule 1

P3.58

In the present methods, the levels of cytokines can be measured in picograms per milliliter (pg/ml) or micrograms per deciliter (μ§ ά\), for example. Protein levels or concentration can be determined by methods standard in the art for quantitating proteins, such as Western blotting, ELISA, ELISPOT, immunoprecipitation, immunofluorescence (e.g., FACS), immunohistochemistry, immunocytochemistry, etc., as well as any other method now known or later developed for quantitating protein in or produced by a cell. As utilized herein PPD means Purified protein derivative (PPD) tuberculin, TB means tuberculosis and HIV means human immunodeficiency virus. In the methods described herein, measuring the levels of the cytokines in the subject can be but is not necessarily performed by the individual that obtains the sample or the individual that computes the predictive values from the equations set forth herein. Also provided herein are methods of obtaining levels of cytokines in a sample from a subject in the form of numerical data, for example, via any means of data transmission, such as from a database, a laboratory report, a CD-ROM, electronic mail, etc. and entering the values into the predictive equations to obtain the HIV, TB and/or PPD status of the subject.

The methods set forth herein can be utilized to diagnose a subject as HIV+, TB+, HIV+/TB+, HIV-/TB+, HIV-TB-, HIV+/TB- PPD+, HIV+/TB-/PPD-, HIV-/TB-/PPD+, or HIV-/TB- PPD- . For example, and not to be limiting, levels of cytokines in the predictive HIV equation (eotaxin, SCF, PDFG ββ) and/or levels of cytokines in the predictive TB equation (MCSF, TNFBeta, MCP3, GROalpha) can be measured in a sample from a subject to determine the HIV and/or TB status of the subject. In addition, the levels of the cytokines in the predictive PDD equations (LIF, MCP3, CTACK and ICAM- 1 ) can be measured in a sample from a subject to determine the PPD status of the subject.

Once a diagnosis is made, for example, HIV+/TB+, the appropriate composition, for example, drug(s) or other therapy(ies) can be selected and administered for treatment of the co- infected subject. The composition can comprise, for example, a chemical, a compound, a small molecule, an aptamer, a drug, a protein, a cDNA, an antibody, a morpholino, a triple helix molecule, an siRNA, an shRNAs, an antisense nucleic acid or a ribozyme.

Compounds that decrease HIV infection and/or compounds that decrease tuberculosis infection can be utilized. Antiviral compounds useful in the treatment of HI V include, but are not limited to Combivir® (lamivudine-zidovudine), Crixivan® (indinavir), Emtriva®

(emtricitabine), Epivir® (lamivudine), Fortovase® (saquinavir-sg), Hivid® (zalcitabine),

Invirase® (saquinavir-hg), Kaletra® (lopinavir-ritonavir), Lexiva™ (fosamprenavir), Norvir®

(ritonavir), Retrovir® (zidovudine), Sustiva® (efavirenz), Videx EC® (didanosine), Videx®

(didanosine), Viracept® (nelfinavir) Viramune® (nevirapine), Zerit® (stavudine), Ziagen®

(abacavir), Fuzeon® (enfuvirtide) Rescriptor® (delavirdine), Reyataz® (atazanavir), Trizivir®

(abacavir-lamivudine-zidovudine) Viread® (tenofovir disoproxil fumarate), Agenerase®

(amprenavir) and combinations thereof. Compounds that can be used to treat tuberculosis include, but are not limited to, ethambutol, isoniazid, pyrazinamide, rifampicin, amikacin, kanamycin, capreomycin, viomycin, enviomycin, fluoroquinones (for example, ciprofloxacin, levofloxoacin and moxifloxacin), ethionamide, prothionamide, rifabutin, clarithromycin, linezoid, thioacetazone, thioridazine, arginine, vitamin D , R207910 and combinations thereof. Any combination of a compound(s) utilized to treat HIV and a compound(s) utilized to treat tuberculosis can be utilized to treat a subject coinfected with tuberculosis and HIV. Similarly, if the patient is HIV+/TB-, the appropriate drug(s) or other therapy(ies) to treat only HIV can be administered. Further, if the patient is HIV-/TB+, the appropriate drug(s) or other therapy(ies) to treat only tuberculosis can be administered.

Depending on the intended mode of administration, the composition can be in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, or suspensions, preferably in unit dosage form suitable for single administration of a precise dosage. The compositions will include a therapeutically effective amount of the compound described herein or derivatives thereof in combination with a pharmaceutically acceptable carrier and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, or diluents. By pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, which can be administered to an individual along with the selected compound without causing unacceptable biological effects or interacting in a deleterious manner with the other components of the pharmaceutical composition in which it is contained.

As used herein, the term carrier encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations. The choice of a carrier for use in a composition will depend upon the intended route of administration for the composition. The preparation of pharmaceutically acceptable carriers and formulations containing these materials is described in, e.g. ,

Remington's Pharmaceutical Sciences, 21 st Edition, ed. University of the Sciences in

Philadelphia, Lippincott, Williams & Wilkins, Philadelphia Pa., 2005. Examples of physiologically acceptable carriers include buffers such as phosphate buffers, citrate buffer, and buffers with other organic acids; antioxidants including ascorbic acid; low molecular weight

(less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN ^® (ICI, Inc.; Bridgewater, New Jersey), polyethylene glycol (PEG), and PLURONICS™ (BASF; Florham Park, NJ).

Compositions containing the compounds described herein or derivatives thereof suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.

These compositions may also contain adjuvants such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the action of microorganisms can be promoted by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Isotonic agents, for example, sugars, sodium chloride, and the like may also be included. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

Solid dosage forms for oral administration of the compounds described herein or derivatives thereof include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the compounds described herein or derivatives thereof is admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or (a) fillers or extenders, as for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders, as for example, carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, (c) humectants, as for example, glycerol, (d) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate, (e) solution retarders, as for example, paraffin, (f) absorption accelerators, as for example, quaternary ammonium compounds, (g) wetting agents, as for example, cetyl alcohol, and glycerol monostearate, (h) adsorbents, as for example, kaolin and bentonite, and (i) lubricants, as for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethyleneglycols, and the like.

Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and others known in the art. They may contain opacifying agents and can also be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner. Examples of embedding compositions that can be used are polymeric substances and waxes. The active compounds can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration of the compounds described herein or derivatives thereof include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents, and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1 ,3-butyleneglycol, dunethylformamide, oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil, sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols, and fatty acid esters of sorbitan, or mixtures of these substances, and the like.

Besides such inert diluents, the composition can also include additional agents, such as wetting, emulsifying, suspending, sweetening, flavoring, or perfuming agents.

Suspensions, in addition to the active compounds, may contain additional agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.

Compositions of the compounds described herein or derivatives thereof for rectal administrations are preferably suppositories, which can be prepared by mixing the compounds with suitable non-irritating excipients or carriers such as cocoa butter, polyethyleneglycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and therefore, melt in the rectum or vaginal cavity and release the active component. Dosage forms for topical administration of the compounds described herein or derivatives thereof include ointments, powders, sprays, gels and the like. The compounds described herein or derivatives thereof are admixed under sterile conditions with a

physiologically acceptable carrier and any preservatives, buffers, or propellants as may be required.

Throughout this application, by treat, treating, or treatment is meant a method of reducing the effects of an existing infection. Treatment can also refer to a method of reducing the disease or condition itself rather than just the symptoms. The treatment can be any reduction from native levels and can be, but is not limited to, the complete ablation of the disease or the symptoms of the disease. Treatment can range from a positive change in a symptom or symptoms of infection to complete amelioration of the an infection as detected by art-known techniques. For example, a disclosed method is considered to be a treatment if there is about a 10% reduction in one or more symptoms of the disease in a subject with the disease when compared to native levels in the same subject or control subjects. Thus, the reduction can be about a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

As utilized herein, by prevent, preventing, or prevention is meant a method of precluding, delaying, averting, obviating, forestalling, stopping, or hindering the onset, incidence, severity, or recurrence of infection. For example, if a subject is found to be HIV+/TB- PPD+, antimicrobial therapy can be administered prophylactically to prevent tuberculosis infection.

Administration can be carried out using therapeutically effective amounts of the agents described herein for periods of time effective to treat or prevent infection. The effective amount may be determined by one of ordinary skill in the art and includes exemplary dosage amounts for a mammal of from about 0.5 to about 200mg/kg of body weight of active compound per day, which may be administered in a single dose or in the form of individual divided doses, such as from 1 to 4 times per day. Alternatively, the dosage amount can be from about 0.5 to about 150mg/kg of body weight of active compound per day, about 0.5 to l OOmg/kg of body weight of active compound per day, about 0.5 to about 75mg/kg of body weight of active compound per day, about 0.5 to about 50mg/kg of body weight of active compound per day, about 0.5 to about 25mg/kg of body weight of active compound per day, about 1 to about

20mg/kg of body weight of active compound per day, about 1 to about lOmg/kg of body weight of active compound per day, about 20mg/kg of body weight of active compound per day, about l Omg/kg of body weight of active compound per day, or about 5mg/kg of body weight of active compound per day.

The terms effective amount and effective dosage are used interchangeably. The term effective amount is defined as any amount necessary to produce a desired physiologic response. Effective amounts and schedules for administering the agent may be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for administration are those large enough to produce the desired effect in which one or more symptoms of the disease or disorder are affected (e.g., reduced or delayed). The dosage should not be so large as to cause substantial adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the activity of the specific compound employed, the metabolic stability and length of action of that compound, the species, age, body weight, general health, sex and diet of the subject, the mode and time of administration, rate of excretion, drug combination, and severity of the particular condition and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosages can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.

Any appropriate route of administration may be employed, for example, parenteral, intravenous, subcutaneous, intramuscular, intraventricular, intracorporeal, intraperitoneal, rectal, or oral administration. Administration can be systemic or local. Pharmaceutical compositions can be delivered locally to the area in need of treatment, for example by topical application or local injection. Multiple administrations and/or dosages can also be used.

Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutations of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a method is disclosed and discussed and a number of modifications that can be made to a number of molecules including in the method are discussed, each and every combination and permutation of the method, and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific method steps or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed.

Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference in their entireties.

A number of embodiments have been described. Nevertheless, it will be understood that various modifications may be made. Accordingly, other embodiments are within the scope of the following claims. The following examples are exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.

EXAMPLES

Described herein is a diagnostic test that identifies circulating biomarkers for the differentiation and classification of the pathogenesis of TB and/or HIV in a population.

Currently, there are few, if any studies predicting biomarkers for both TB and HIV populations. Instead, the focus has been on predicting biomarkers for only TB or HIV populations. This is the first large scale study of circulating cytokines, chemokines and growth factors in a clinically relevant population. Out of the 50 cytokines, chemokines and growth factors tested, several were found to be candidates for new diagnostic tests to validate novel drug and vaccine candidates, and to identify patients with TB and/or HIV in which a diagnosis can be pronounced within days, and the appropriate drug regimen prescribed.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB) is a major health problem and it is estimated that one-third of the world's population is latently infected.

Typically, only 1 in 10 will develop the disease, but this percentage increases dramatically in those who are coinfected with Mtb and HIV. Most deaths from TB are avoidable by early diagnosis and treatment. However, more than 10% of HIViTB-coinfected persons may have a negative tuberculin skin test as a result of anergy. Here, we examined the profiles of 50 cytokines, chemokines and growth factors in the sera of 207 PPD-/HIV- (healthy controls), PPD+ HIV- (latent TB), HIV-/TB+ (active disease), HIV+/TB+ coinfected, and H1V+ PPD- patients from Peru. After estimating the univariate statistics for the cytokine intensity in each group, Analysis of Variance (ANOVA) was used to test for differences across groups. Once statistically significant differences between the groups were identified, Principal Component Analysis (PCA) was used to examine the ability of the cytokines to cluster the disease groups. A quadratic discriminant analysis procedure was used to test the capacity of the cytokines to discriminate between the five groups. Leave-One-Out-Cross- Validation (LOOCV) was used to examine the quality of the discrimination. The data showed that several cytokines, chemokines and growth factors tested were able to classify disease, or disease state. The biomarkers identified in this study are candidates that could be used to develop new TB and/or TB/HIV diagnostic tests.

Sera was collected in a blinded fashion from 207 patients from Peru and stored at -80°C until tested in a blinded fashion by using Bio-Rad's multiplex bead array approach based from the Luminex technology. After analysis the samples were un-blinded and categorized into their appropriate groups. Of these 207 patients, 34 were PPD- HIV (healthy controls) containing 15 males and 19 females aged 22 to 49, 44 were PPD+/HIV (latent TB) containing 21 males and 23 females aged 20 to 61 , 55 were HIV-1TB+ (active disease) containing 27 males and 28 females aged 19 to 61 , 58 were HIV+/TB+ (coinfected) containing 28 males and 30 females aged 22 to 55, and 16 were HIV+/PPD- containing 1 1 males and 5 females aged 18 to 49. Cytokine analysis

The Bio-Rad Bio-Plex Human Cytokine 27-Plex Panel (Catalog # 171 -A1 1 127) and Human Cytokine 23-Plex Panel (Catalog # 1 71 - A l 1 123) (Bio-Rad, CA) were performed on the Peru samples in triplicate according to the manufacturer's instructions. The 50 cytokines, chemokines and growth factors analyzed were IFN-a2, IL-l a, IL- 1 (3, IL- l ra, IL-2, IL-2ra, IL-3, IL-4, IL-5, IL-6, IL-7, 1L-8, IL-9, IL- 10, IL- 12 (p40), IL- 12 (p70), IL- 13, IL- 15, IL- 16, IL- 17, IL- 18, CTACK, Eotaxin, FGFbasic, G-CSF, GM-CSF, GRO-a, HGF, ICA -1 , IFN-y, IP- 10, L1F, MCP- 1 (MCAF), MCP-3, M-CSF, MEF, MIG, ΜΓΡ-l a, MIP-1 I3, B-NGF, PDGF bb, RANTES, SCF, SCGF-I3, SDF-l a, TNF-a, TNF-I3, TRAIL, VCAM- 1 and VEGF. Table 2 provides the results of the analysis. Healthy Latent TB HIV/TB HIV

(HIV TB ^" ) (HIV PPD ⁺ TB-) (HIV TB ^* ) (HIV ^* TB ^* ) (HIV ^* TB ) K-W Test

Median Range Median Range Median Range Median Range Median Range

IL-1 β 4.8 3.3-44.0 5.4 3.9-9.4 5.4 3.1-70.0 5.8 3.1-663.7 3.5 1.5-101.8 p<0.001

IL-1ra 222.7 115.0-1956 230.5 95.5-843.6 262.9 139.8-10944 210.7 62.9-40807 156.6 18.4-6303 p=0.002

IL-2 5.5 0.1-135.1 6.2 Ο.Ϊ-57Τ3 6.7 0.1-1019 1.9 0.1-10370 0.1 0.1-423.9 p<0.001

IL-4 26.5 18.1-49.9 24.2 13.7-37.3 23.2 13.7-77.4 27.5 9.7-878.3 15.7 4.8-154.0 p=0.001

IL-5 1.5 ^~" 0.2-91.8 1 1.1 0.1-9.3 3.3 0.8-68.1 1.8 0.2-44.0 p<0.001

IL-6 1Ϊ.3 ^' 5.4-143.4 ^~ 17.9 7.1-162.8 35.6 7.1-638.9 14.7 ^~ 2.9-2084 13.4 1)7-429.5 p<0.001

IL-7 ^~ 8A 3.9-457.1 12 2.9-34.1 17 2.8-40.6 7.2 ^" 1.2-205.8 ^' 7.9 4.4-22.3 p<0.001

IL-8 12.3 4.4-558.7 11.1 3.9-2340 25.5 3.8-31593 3.3 0.1-1230 5.3 0.2-31.0 p<0.001

IL-9 45 ^~ 6 15.2-33 .9 53.7 5.7-322 ^~ .5 59 21.5-13845 76.2 13.0-1050 11 p=0.002

IL-10 6.6 ^~ 1.0-1699 8.6 1.4-31.1 10.2 2.0-46.1 2.5 0.2-2348 4.1 0.2-168.8 p<0.001

IL-12 (p70) 6.6 ^" 2.2-602.0 ^~~ 7.3 2.1-23.5 8.2 2.7-66.6 5.7 o oT¾59 4.65 1.0-77.6 p=0.004

IL-13 20.6 5.9-1872 27.ϊ 8.0-69.8 29.1 11.5-88.9 24.7 1.0-1702 37.1 3.4-343.7 p=0.127

IL-15 8.9 0.1-64.5 7.7 0.1-25.2 9.8 0.1-490.5 1.5 ^~ 0.1-81.8 4.8 0.1-213.3 p<0.001

IL-17 6.9 0.1-49.3 6.5 0.1-34.8 9.9 0.1-77.4 ^{~ ' ~} 0.1 0.1-42.9 0.1 0.1-20.0 p<0.001

Eotaxin 95.3 2.2-853.6 111.1 13.4-791.3 78.9 13.4-2780 38.8 2.2-591.8 2.2 2.2-2291 P<6.OOI

FGF basic 33.6 1.0-96.4 32.1 1.0-189.4 40.3 1.0-189.4 35.7 1.0-162.9 39 1.0-94.9 p=0.809

G-CSF 33.3 20.7-88.6 30.1 19.7-51.5 36.1 21.7-145.50 38.9 20.9-2637 28.4 18.4-233.1 p<0.001

GM-CSF 21.9 0-138.1 16.8 0-67.7 9 0-466.8 25.6 0-28166 6.1 0-2204 p=0.008

IFN-Y 203.3 ^" 137.6-394.7 187.8 ^~ Ϊ08.5-4457ΐ 171 ^~ 4 111.2-912.1 203.2 90.2-20555 143 57.9-2385 p<0.001

IP-10 1189 ^{" "} 235.2-7129 1158 153.8-5708 1948 203.2-50939 1636 389.5-50939 2202 638.2-22808 p=0.027

MCP-1 3.2 0.1-237.9 0.9 0.1-76.0 0.1 0.1-76.0 0.1 0.1-556.4 0.1 0.1-139.8 p<0.001 IP-1a 20.3 13.8-295.2 18.6 13.5-191.2 21.5 13.5-1408 27.6 14.5-43.0 21 13.7-33.0 p<0.001

PDGF bb 7635 2359-16768 6817 1735-21149 5752 1735-19323 616.2 107.7-8489 245.9 57.1-11263 p<0.001

TNF-a 23.1 1.3-94.1 24.1 1.3-188.5 19.6 1.3-354.2 29.5 1.3-6126 6.8 1.3-2266 p=0.005

VEGF 125.4 1.7-656.5 200.7 7.0-1387 282.3 3.7-1387 0.1 0.1-343.0 33.6 0.1-312.0 p<0.001

IFN-a2 206.4 ^" 140.2-366.7 207.1 128.8-450.0 227.1 135.3-404.9 196.5 135.8-543.2 230.4 187.5-284.2 p=0.076

IL1-a 0.005 0.005-2.51 0.005 0.005-3.63 0.89 0.005-7.38 0.2 0.005-7.35 0.005 0.005-0.33 pO.001

IL-2ra 282.5 128.2-628.7 243.1 98.4-620.2 347.8 129.8-986.8 268.1 100.1-1334 418.8 232.1-1398 p<0.001

IL-3 143.3 2.1-462.5 96.1 2.1 -485.5 149.3 2.1-384.7 97.3 2.1-1191 137.7 23.5-1866 p=0.019

IL-12 (p40) 1043 223.2-2968 773.2 202.0-2534 11 17 175.0-2540 703.5 75.4-2560 1217 614.8-5284 p=0.003

IL-16 193.5 95.4-456.7 197.8 89.4-576.7 263.4 112.6-526.5 387 112.5-2433 439.8 155.7-1322 p<0.001

IL-18 157.7 68.1-363.5 119.7 40.9-407.3 252.6 83.9-1438 141.5 25.3-1077 189.9 73.6-428.6 p<0.001

CTACK ^" 1079 ^" 496.7-1983 1410 704.7-2038 1385 483.1-2302 1143 575.5-2422 1200 814.9-1860 p=0.005

GRO-a 153.4 4.3-370.3 171.2 4.3-440.5 348.3 116.4-1288 193.9 47.8-428.5 170.3 4.3-278.7 p<0.001

HGF 821.7 267.4-1439 938.5 405.8-1646 1416 513.4-4883 516 270.7-1221 584 371.4-2124 p<0.001

ICAM-1 30083 21596-30083 23009 19587-30083 30083 22208-30083 30083 8021-30083 30083 30083-30083 p<0.001

LIF 0.005 0.005-79.9 0.005 0.005-64.2 13.4 0.005-72.4 3.46 0.005-58.0 0.4 0.005-28.6 p<0.001

MCP-3 149.7 37.0-329.9 197.4 87.3-479.3 108.1 33.7-409.2 94 39.4-349.8 131.2 62.1-857.9 p<0.001

M-CSF 30.1 2.3-106.8 39.4 2.3-183.7 83.5 17.6-195.2 48.7 13.1-190.5 26.2 4.9-59.9 p<0.001

MIF 301.8 122.5-31600 334.9 138.2-3153 1191 199.1 -31600 1358 258.5-16105 1245 231.0-5110 p<0.001

MIG 1976 355.8-20401 1782 316.0-35285 0979 894.6-35285 1904 447.9-18522 4206 716.8-20205 p<0.001 β-NGF 5.5 2.4-9.2 5.5 3.0-13.6 5.8 2.3-10.4 6 2.6-13.6 6.4 3.2-9.2 p=0.634

SCF 114.7 ^" 54.4-211.3 ^" 102.1 34.7-179.8 109 46.8-195.8 83.6 35.5-153.6 86.8 50.3-265.4 p<0.001

SCGF-β 99.291 6277-251226 122,270 31672-251226 103,675 2184-230161 36,088 3852-251226 90,093 18417-251226 p<0.001

SDF-1a 1388 741.1-2749 1388 681.9-3744 1395 630.0-3168 1051 457.4-1985 1398 984.3-4004 p<0.001

TNF-β 0.005 0.005-9.7 0.005 0.005-36.5 6.9 0.005-51.3 2.48 0.005-34.6 0.005 0.005-2.6 p<0.001

TRAIL 334.3 128.3-714.8 283.5 83.9-973.5 335.5 56.5-778.4 290.4 65.0-694.4 425.2 219.2-1375 p=0.101

VCAM-1 26905 19221-26905 21245 9683-26903 26905 20284-26905 26905 7629-26905 26905 26905-26905 p<0.001

TABLE 2-Cytokine values were compared between the five groups using a Kruskal-Wallis test (non-parametric ANOVA). Data are given as median (minimum, maximum) since the values are not normally distributed and means and standard deviations would not be appropriate.

HIV LOGISTIC REGRESSION

Cytokine Odds Ratio 95% C. I. p value OR for δ=

GRO-ct 0.996 0.994, 0.999 0.003 0.670

IL2 1.000 0.999, 1.001 0.422

IL17 0.81 1 0.748, 0.880 <0.001 0.124*

SCF 0.977 0.967, 0.987 <0.001 0.091

IL12 p70 1.001 0.999, 1.003 0.373

PDGF ββ 0.999 0.999, 0.999 <0.001 0.905

Eotaxin 0.997 0.995, 1.000 0.045 0.741

IL4 1.008 0.993, 1.022 0.296

SDF- la 0.999 0.998, 0.999 <0.001 0.905

Of the 9 cytokines that looked promising in the First step, six of them are significantly predictive of the presence of HIV using logistic regression. Since HJV is coded as 0=no and l=yes, Odds Ratios of <1 imply that lower values are associated with presence of HIV. An Odds Ratio of >1 would imply that higher values are associated with the presence of HIV. The six significant cytokines were entered into a forward stepwise regression using the Likelihood ratio test to determine which variables entered the equation.

Multivariable Logistic Regression

Cytokine Odds Ratio 95% C. I. p value OR for 6=100

PDGF 0.999 0.999, 0.999 <0.001 0.905

SCF 0.969 0.947, 0.991 0.005 0.041

Eotaxin 1.003 1.000, 1.005 0.048 1.350

HIV LOGISTIC REGRESSION

The effect of eotaxin has changed direction in the multivariable equation. This probably represents a correction for over prediction with the first two variables. This equation predicts correctly predicts presence or absence of HIV in 95.6% of the patients. It predicts HIV- in 127 out of 132 individuals and HIV+ in 70 out of 74 individuals. The predictive equation is p = 1 where z = 5.654 + 0.003*Eotaxin - 0.032*SCF - 0.001 *PDGF ββ

1 + e ^"z

Values >0.5 predict HIV+

Examples:

Patient 3 z = 5.654 + 0.003*63.88 - 0.032* 144.91 - 0.001 *6648.05 = -5.530

p = ] /( 1 +e ^{+5 53} °) = 0.004 predicts HIV- (actual HI V-TB-)

Patient 187 z = 5.654 + 0.003*2.25 - 0.032*41.37 - 0.001 * 1270.15 = 3.067 p = l /(l+e ^{"3 067} ) = 0.956 predicts HIV+ (actual HIV+TB+)

Figures 1 A-C show H IV frequency plots of cytokines (PDGF ββ, SCF and eotaxin) found in the multivariable logistic regression table.

TB LOGISTIC REGRESSION

Cytokine Odds Ratio 95% C. Ϊ. p value OR for δ=]

11 j -a 10.205 2.644, 39.392 O. O1

M-CSF 1 .037 1.022, 1.052 <o.eoi 1 .433*

ΤΝΨ-β 1.896 1.319/2.725 0.001

M ^' CP-3 0.992 0.987, 0.997 0.003 0.923*

JL-Ι β i .003 0.991 , 1.016 0.617

G-CSF 1 .01 1 0.988, 1.035 0.?35

1L- 1S 1.003 1.001 , 1.006 0.013 1.030*

GRO-a 1 .01 1 1.006. 1.015 <0.001 1 .1 16*

P 1.043 1.009, 1.079 0.«H

Of the 9 cytokines that looked promising.in the first s¾p, seven of litem are significantly predictive of Die presence of HIV using logistic regression. Since TB :s coded as 0=no and l=yes, Odds Ratios of < 1 imply that lower values are tissociatcd with presence of TB. An Odds Ratio of > 1 would imply Ihnt higher vnlues are associated with the presence of TB. The seven significanl cyiolcincs were entered into a forward stepwise regression using the Likelihood ratio test to determine which variables entered the equation.

Multivariable .Logistic Regression

Cytokine. Odds Ratio 95% C. i. p value OR for 8=100

GRO-a 1.012 1 .002, 1.022 0.C21 1.128

CP-3 0.944 0.924, 0.964 <0.()0 l 0.560

TNF-p 1.809 1.276, 2.563 0.C01

MCS) ⁷ 1.06S 1 .024. 1 .1 15 0.(02 1.935

TB LOGISTIC REGRESSION

This equation correctly predicts presence or absence of TB in 90.7% of the patients. It predicts TB- in 42 out of 50 individuals (84%) and TB+ in 105 out of 112 individuals (94%). The predictive equation is: p = 1 ^" where z = 2.146 + 0.066*MCSF + 0.593*TNFp - 0.058*MCP3+0.012GROa 1 +e- ^z

Values > 0.5 predict TB+

Examples:

ID3 z=2.146 + 0.066*154.16 + 0.593*32.99-0.058*186.36 + 0.012*329.99 = 25.035

p=l(l+e ^"25035 )=0.999 predicts TB+ (actually HIV+TB+)

ID 137 z=2.146 + 0.066*40.45 + 0.593*0.01-0.058*152.2 + 0.012*192.49= -1.696

p=l/(l+e ^' ')=0.155 predicts TB- (actually H1V+TB-)

ID 193 z=2.146 +0.066*169.53 + 0.593*22.03-0.058*313.99 + 0.012*302.64= 11.819

P=l/(l+e 0.999 predicts TB+ (actually HIV-PPD+TB-)

Figures 2A-D show TB frequency plots of cytokines (GRO-a, MCP-3, TNF-β and MCSF)) found in the TB multivariable logistic regression table.

PPD+ vs. all others LOGISTIC REGRESSION

Cytokine Odds Ratio 95% C. I. p value

IP- 10/ 100 0.980 0.960, 1.00 0.052

MlP- l a 0.997 0.986, 1.008 0.583

IL12 (p40) 1 .000 0.999, 1.000 0.513

CTACK/100 1.096 1.012, 1 .188 0.025

LIF 0.937 0.894, 0.982 0.006

IL-3 0.998 0.995, 1.002 0.324

MCP-3 1.010 1.005, 1.014 <0.001

TNF-β 0.963 0.918, 1.009 0.1 12

ICAM- 1 /1000 0.838 0.777, 0.904 <0.001

VCAM-1/1000 0.831 0.764, 0.903 <0.001

Of the 10 cytokines that looked promising in the first step, six of them are significantly predictive of the presence of PPD+ using logistic regression. Since PPD+is coded as 0=no and l =yes, Odds Ratios of <1 imply that lower values are associated with presence of PPD+. An Odds Ratio of >1 would imply that higher values are associated with the presence of PPD+. The six significant cytokines were entered into a forward stepwise regression using the Likelihood ratio test to determine which variables entered the equation.

Multivariable Logistic Regression

Cytokine Odds Ratio 95% C. I. p value

LIF 0.946 0.908, 0.987 0.010

MCP-3 1 .009 1 .004, 1 .014 <0.001

CTACK/100 1.150 1.034, 1 .278 0.010

ICAM- 1/1000 0.868 0.808, 0.932 <0.001

PPD+ vs. all others LOGISTIC REGRESSION

This equation correctly predicts presence or absence of PPD+ in 83.0% of all patients. It predicts PPD+ in 20 out of 44 individuals (45%) and not PPD+ in 151 out of 162 individuals 93%). The predictive equation is p = 1 where z = -0.61 1 - 0.055*L1F + 0.009* CP3 + 0.001 *CTAC - 0.141 *ICAM/1000 1 + e ^"2

Values >0.5 predict PPD+ Examples:

ID 221 z = -0.61 1 - 0.055*0.005 + 0.009*479.29 + 0.001 * 1593.55 - 0.141 * 19.14 = 2.52

p = l/(l+e ^{"2 52} ) = 0.93 predicts PPD+ (actual H1V-PPD+TB-)

ID 154 z = -0.61 1 - 0.055*0.005 + 0.009* 1 13.37 + 0.001 * 1 132.81 - 0.141 *30.08 = -2.70

p = 1 /( 1 +e ² - ⁷⁰ ) = 0.06 predicts not PPD+ (actual HI V-TB-)

Figures 3A-D show PPD+ vs. all others frequency plots of cytokines (MCP-3, LIF,

CTAC and ICAM) found in multivariate logistic regression table.

PPD+ vs. TB- LOGISTIC REGRESSION

Cytokine Odds Ratio 95% C. I. p value

IP- 10/ 100 0.989 0.967, 1.01 1 0.313

lP- l a 0.996 0.984, 1.007 0.464

IL 12 (p40) 1.000 0.999, 1.000 0.201

CTACK 100 1 .205 1 .062, 1 .369 0.004

LIF 0.983 0.948, 1.019 0.343

1L-3 0.998 0.994, 1.002 0.265

MCP-3 1.005 1.000, 1.01 1 0.035

TNF-β 1.309 1.023, 1.675 0.032

ICAM- 1/1000 0.578 0.478, 0.698 <0,001

VCAM- 1/1000 0.551 0.449, 0.676 <0.001

Of the 10 cytokines that looked promising in the first step, five of them are significantly predictive of the presence of PPD+ using logistic regression. Since PPD+ is coded as 0=no and l =yes, Odds Ratios of < 1 imply that lower values are associated with presence of PPD+. An Odds Ratio of >1 would imply that higher values are associated with the presence of PPD+. The five significant cytokines were entered into a forward stepwise regression using the Likelihood ratio test to determine which variables entered the equation.

Multivariable Logistic Regression

Cytokine Odds Ratio 95% C. I. p value

ICAM- 1/1000 0.578 0.478, 0.698 <0.001

Only one variable enters the equation. The other variables do not provide additional information. This equation correctly predicts presence or absence of PPD+ in 83.0% of the patients who do not have TB. It predicts PPD+ in 36 out of 44 individuals (82%) and not PPD+ in 42 out of 50 individuals (84%). PPD+ vs. TB- LOGISTIC REGRESSION

The predictive equation is

p = 1 where z = 14.508 - 0.549*ICAM 1/1000

1 + e ^~z

Values >0.5 predict PPD+

Examples:

ID 221 z = 14.508 - 0.549* 19.14 = -5.181

p = l/(l+e ^{5 181} ) = 0.006 predicts not PPD+ (actual HIV-PPD+TB-)

ID 154 z =14.508 - 0.549* 19.14 = -2.006

p = l/(l+e ⁺²⁰⁰⁶ ) = 0.119 predicts not PPD+ (actual HIV-TB-)

Figure 4 shows PPD+ vs. TB- frequency plots of cytokines (ICAM-I)found in multivariable logistic regression table.

PPD+ vs. Healthy LOGISTIC REGRESSION

Cytokine Odds Ratio 95% C. I. p value

IL 6 1.008 0.992, 1.024 0.316

IL 7 0.996 0.985, 1.007 0.489

GM-CSF 0.989 0.970, 1.009 0.291

MCP-1 0.977 0.956, 0.999 0.037

VEGF 1.001 1.000, 1.003 0.129

IL-2ra 1.000 0.996, 1.004 0.920

CTACK 1.242 1.075, 1.434 0.003

ICAM-1/1000 0.620 0:513, 0.750 <0.001

MCP-3 1.009 1.002, 1.015 0.01 1

M-CSF 1.012 0.999, 1.025 0.079

TNF-β 1.278 0.982, 1.662 0.068

VCAM-1/1000 0.601 0.488, 0.741 <0.001

SCGF-β/ΙΟΟΟ 1.007 1.001 , 1.013 0.034

Of the 13 cytokines that looked promising in the first step, six of them are significantly predictive of the presence of PPD+ using logistic regression. Since PPD+ is coded as 0=no and l=yes, Odds Ratios of <1 imply that lower values are associated with presence of PPD+. An Odds Ratio of > 1 would imply that higher values are associated with the presence of PPD+. The six significant cytokines were entered into a forward stepwise regression using the Likelihood ratio test to determine which variables entered the equation.

Multivariable Logistic Regression

Cytokine Odds Ratio 95% C. I. p value

ICAM- 1/1000 0.620 0.513, 0.750 <0.001 This equation correctly predicts presence or absence of PPD+ in 80.8% of the patients. It predicts PPD+ in 37 out of 44 individuals (84%) and normal in 26 out of 34 individuals (76%). Figure 5 shows PPD+ vs. healthy frequency plots of cytokines found in multivariable logistic regression table.