The sensitivity of FDA approved IGRAs is less than ideal, and typically no better than the sensitivity of the TST 
. This pilot study was designed to investigate the use of other cytokines as markers of Mtb
infection and to evaluate the potential impact of their use on detection sensitivity. Cytokine responses to individual Mtb
antigens and antigen combinations were compared.
Each of three multiplexed assays demonstrated that in addition to IFN-γ, multiple cytokines are released in response to Mtb antigens with significantly greater responses among patients with CCTB than among control subjects. The in-house MMIA and microarray demonstrated that IFN-γ, IL-2, IL-6, and IL-8 responses to the combination antigen ESAT-6+CFP-10+TB7.7 were greater among patients than among controls. In addition, the microarray data demonstrated significantly greater MCP-1, MIP-1α, and MIP -1β responses among patients. The custom commercial MMIA confirmed that multiple cytokines were released in response to Mtb antigens, and that IFN-γ, IL-2, IL-6, IL-8, IP-10, MCP-1, MIP-1β, and TNF-α responses to ESAT-6+CFP-10+TB7.7 were significantly greater for patients than for controls. Four of these cytokines appear to be excellent candidates as diagnostic markers of Mtb infection; sensitivity and specificity were 100% with IL-2, IL-6, IP-10, and MIP-1β. In contrast, IFN-γ sensitivity with the custom commercial MMIA was 91.6% and specificity was 100%.
One CCTB patient failed to demonstrate an IFN-γ response with any of the Mtb antigens using multiple test formats including QFT-G. The subject had a robust IFN-γ response to mitogen, no other evidence of immune dysfunction, nor any unusual IFN-γ measurements to suggest laboratory errors. In stark contrast to the lack of IFN-γ response, this subject's IL-2, IL-6, IL-8, IP-10, and MIP-1β responses to ESAT-6+CFP-10+TB7.7 were greater than corresponding mean responses plus three standard deviations for controls. This observation demonstrates that the use of other cytokines in addition to IFN-γ or as alternatives to IFN-γ can improve sensitivity for detecting Mtb infection.
Plausible reasons for false-negative IGRA results include: 1) measurement errors 
, 2) host-mediated inhibition of IFN-γ release 
, 3) drug or disease induced immune suppression 
, 4) genetic defects in IFN-γ production 
, and 5) Major Histocompatibility Complex (MHC) determined restriction in the ability to recognize presented epitopes 
. The potential for improving diagnostic sensitivity by assessing responses of multiple cytokines has been suggested previously 
. By assessing both IP-10 response and IFN-γ response, Ruhwald et al. noted an increase in detection rate to 90% (i.e., detection by either cytokine) from 83% and 81%, respectively, for IP-10 or IFN-γ alone. Individual cytokines have been found to be down-regulated in some patients with tuberculosis, suggesting that assessment of multiple cytokines may reduce the possibility of a negative response with any single analyte 
The amount of cytokine in unstimulated plasma from patients with CCTB was generally not significantly different from the amount in unstimulated plasma from control subjects. However, background levels of IP-10, MCP-1, and MIP-1β measured with the custom MMIA were statistically higher for patients than for controls. This observation has also been made for IP-10 and MCP-3 previously 
. The specificity and sensitivity of this observation for Mtb
infection should be further investigated. If these cytokines are consistently elevated without antigen stimulation in Mtb
infected persons, as opposed to those with other infections, they could serve as the basis for a more rapid test for Mtb
infection. If antigen stimulation is not required, detection of elevated cytokine levels in the urine may be of diagnostic value 
IP-10 has been targeted as a potential diagnostic marker for tuberculosis in other studies 
. In conjunction with our results, these studies provide clear evidence that IP-10 is produced in nanogram amounts when fresh blood samples from most sensitized persons are stimulated with mycobacterial antigens. Azzurri et al. noted an association between unstimulated IP-10 plasma levels and disease activity, as well as response to treatment 
. In a pilot study, Syed Ahamed Kabeer et al. found that tuberculosis treatment was associated with a significant decrease in IP-10 response to certain peptides representing ESAT-6 and CFP-10 
. Other promising indicators of Mtb
infection include antigen-induced MCP-2, MCP-3, and IL-1RA responses 
The antigen or combination of antigens used to stimulate blood can have a dramatic impact on the magnitude of the cytokine response observed. Among sensitized persons, the number of epitopes in an antigen and Major Histocompatibility Complex (MHC) restrictions determines the number of T-lymphocyte clones that will be stimulated by the antigen, and in turn, affects the amount of IFN-γ released upon stimulation 
. For example, among CCTB patients, IFN-γ responses measured by ELISA to ESAT-6+CFP-10 or ESAT-6+CFP-10+TB7.7 were greater than the response to any of the individual antigens alone. However, the IFN-γ response to ESAT-6+CFP-10+TB7.7 was not significantly greater than the response to ESAT-6+CFP-10. This trend toward higher response to the combination antigens continued for other cytokines measured with the in-house MMIA. For this reason, and because ESAT-6+CFP-10+TB7.7 is used in the QuantiFERON®-TB Gold In-Tube test, only responses to ESAT-6+CFP-10+TB7.7 were measured with the custom commercial MMIA.
Cytokine levels vary considerably from individual to individual, so the range of values observed with the multiplexed assays was rather large. Despite these large variations and the relatively small sample sizes, significant differences between CCTB patients and controls were observed, and measurements of IL-2, IL-6, IP-10, and MIP-1β responses allowed complete separation of the two groups. The dot plots and histograms ( and ) illustrate the multiple sizeable cytokine responses and the pattern seen among patients. For IP-10 and MIP-1β, the magnitude of the responses to Mtb antigens was so large compared to background cytokine levels that subtraction of the background had little effect on the calculated responses. This has two implications. First, the complexity and cost of testing may be reduced by screening only Mtb-antigen stimulated plasma for characteristically high cytokine concentrations. If increased concentrations of IP-10 or MIP-1β are found in the Mtb-antigen stimulated plasma, a more sensitive and complex assay could be performed that would take into account the background cytokine concentrations. Second, significant cytokine responses may be detectable with shorter incubation times, thus allowing for more rapid assessments.
Differences are often seen in the magnitude of cytokine concentrations measured with different ELISAs or different multiplexed immunoassay systems 
. These differences are primarily due to varying antibody affinities for the assay targets, as well as differences in the platforms employed. We used three multiplexed immunoassay systems that assessed some of the same cytokines with presumably different capture and detection antibodies. In all three systems, differences in IL-2, IL-6, IL-8, and IFN-γ responses between CCTB patients and controls were significant. In two systems, differences in MCP-1 and MIP-1β responses were significant. TNF-α responses were significantly greater among patients versus controls using the in-house MMIA and commercial MMIA, but not when using the microarray. However, TNF-α concentrations were generally low and highly variable, which may explain why the differences in TNF-α response were not significant for all three assays.
Limitations of this study stem from the examination of small subsets of subjects with each of the different multiplexed immunoassays. However, even with small numbers of subjects significant differences in cytokine responses were demonstrated between CCTB patients and controls. While differences in demographic and clinical characteristics for the patient and control populations were observed (listed in ), it is unlikely that these explain the differences in cytokine responses seen. Subtracting the background cytokine concentration (in the Nil plasma) from the concentration in the antigen stimulated plasma adjusts for demographic and clinical factors that may contribute to nonspecific release of cytokines. The observed differences in unstimulated concentrations for some cytokines (including IFN-γ) is likely due to elevated background levels of cytokine associated with acute or chronic illness, including tuberculosis. Another limitation is the lack of adequate diagnostic standards to confirm all forms of Mtb
infection, including the most common, which is latent infection. We addressed this limitation by comparing responses among patients with confirmed Mtb
infection (i.e., among patients with tuberculosis confirmed by recovery of Mtb
by culture) and control subjects at low-risk of Mtb
exposure who were presumed to be uninfected. Clearly, immunologic differences exist between latent Mtb
infection and infection manifesting as active disease, but specific immunologic differences have been difficult to demonstrate 
. Larger epidemiologically based studies will be required to identify and confirm these differences. Multiplexed assays, which can be readily automated for high-throughput, can facilitate these larger studies.
MMIAs, which are also referred to as suspension arrays, are gaining wide acceptance as tools for research and diagnostic assays 
. These assays are performed in 2 to 4 hours, similar to the time required to perform ELISAs. However, in addition to measuring more than one analyte per test, MMIAs require smaller sample volumes (4 µL in this study), less amounts of reagent and operator time, and cost less than multiple ELISAs. These three dimensional assays have a detection range 1–2 logs broader than ELISAs, so fewer sample dilutions are necessary. Already a simpler, less expensive instrument is available that is not dependent on the fluidics system of a flow cytometer (MAGPIX®, Luminex Corporation). This and similar systems are more compact and portable and show potential for use in developing regions of the world where both reduction in assay cost and time delay between diagnosis and treatment are important. As multiplexed assays that show potential for detection of Mtb
infection are optimized, they can be incorporated onto other platforms that are fully automated or designed for point-of-care diagnostics.
In conclusion, multiple cytokines are released when whole blood samples from patients with CCTB are stimulated with specific Mtb antigens. Compared with measurement of IFN-γ alone, multiplexed assays can improve diagnostic sensitivity for Mtb infection by assessing multiple cytokine responses simultaneously, using smaller sample volumes and reducing the cost of testing compared to ELISAs and other methods. The use of combinations of specific Mtb antigens may also increase diagnostic sensitivity by increasing the magnitude of cytokine responses as compared to individual antigens. Larger studies are needed to confirm the clinical utility of multiplexed assays that examine the cytokines identified in this study as potential diagnostic markers of Mtb infection and to examine the possibility of differentiating infection with disease from latent infection.