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Interferon- Assays for Tuberculosis

Is Anergy the Achilles' Heel?

^a San Francisco General Hospital, University of California, San Francisco, San Francisco, California
^b Portland Veterans Affairs Medical Center, Oregon Health and Sciences University, Portland, Oregon

Anergy in individuals with immunosuppressive conditions (e.g., HIV infection and in those undergoing immunosuppressive therapies) can result in false-negative tuberculin skin test (TST) results, reducing sensitivity for this test in those who are at risk for progression from latent to active tuberculosis (TB) (1–3). Because it is impossible to distinguish a true negative TST result from a lack of response due to generalized anergy, and because a negative TST result cannot definitively rule out TB infection, skin tests (e.g., with mumps and Candida antigens) were used in the past to assess the competence of the cellular immune response (2, 3). However, for various reasons, including inadequate standardization, poor reproducibility, and selective anergy to tuberculin in the setting of TB disease (1, 4, 5), anergy skin testing is currently not recommended (1, 4).

With the emergence of T-cell–based, in vitro IFN- assays for the detection of Mycobacterium tuberculosis infection, there is now considerable interest in evaluating their potential to replace the TST. Several studies have been conducted on the accuracy and utility of ELISA- and enzyme-linked immunospot (ELISPOT)–based IFN- assays, and these have been reviewed elsewhere (6, 7). In a study reported in this issue of the Journal (pp. 631–635), Ferrara and colleagues (8) performed the QuantiFERON-TB-Gold (QFT-Gold) test (Cellestis Ltd., Carnegie, Australia) in a series of 318 unselected, hospital-based patients in Italy. The QFT-Gold assay is an ELISA-based, U.S. Food and Drug Administration–approved assay that uses the early-secreted antigenic target 6 (ESAT-6), and culture filtrate protein 10 (CFP10) antigens. Of the 318 patients enrolled in the study, 65 (20%) were receiving immunosuppressive therapies, and a fairly high proportion (68 of 318 [21%]) of all patients had an indeterminate QFT-Gold result. Patients undergoing concomitant immunosuppressive therapies had significantly higher odds (odds ratio, 3.35; 95% confidence interval, 1.84, 6.08) of having indeterminate test results, as compared with patients who were not receiving immunosuppressive therapies. Furthermore, the IFN- assay produced a significantly higher proportion of indeterminate results in patients with a negative TST (29 vs. 7% in TST-positive patients) (8).

What is an indeterminate IFN- response? In general, an IFN- assay can be indeterminate if the positive mitogen (phytohemagglutinin, a potent stimulant of T-cell response) control in the assay elicits little or no IFN- release from the T cells, or if a very high background IFN- response is present when no antigen is present (nil control). The specific criteria for indeterminate results vary between assays. A negative IFN- response, in comparison to an indeterminate result, indicates little or no IFN- release from the T cells stimulated with TB antigens, in the presence of an adequate response to mitogen.

Why do indeterminate results occur? As suggested by Ferrara and colleagues, a low mitogen response may possibly reflect underlying anergy or inability of the cellular immune system to effectively mount a T-cell response. However, indeterminate results can occur in some perfectly healthy, immunocompetent individuals. Clearly, we need more data on the association between lack of response to mitogen and immunosuppression, and on whether this is a reproducible phenomenon that can be meaningfully interpreted. Indeterminate results can also occur due to improper assay techniques (e.g., time delays that lead to loss of viable T cells in the specimens and improper handling of specimens and antigens). Therefore, laboratories with insufficient experience with IFN- assays need to ensure adequate training and quality. Therefore, before concluding that a diminished response to mitogen reflects underlying immunosuppression, it may be prudent to repeat an indeterminate test to exclude technical errors.

The key message from the Italian study is that, like the TST, the performance of in vitro IFN- assays may potentially be affected by immunosuppression. Although indeterminate IFN- assay results have been reported earlier, none of the previous studies have reported such a large proportion of indeterminate results. The proportion of indeterminate results reported in previous published studies on the QFT-Gold assay ranged from 0.1 to 11% (9–12). This may be due to the fact that these studies recruited relatively fewer immunosuppressed patients (9–12). Although it is possible that technical errors contributed to the high indeterminate rate in the study by Ferrara and colleagues, this possibility is not consistent with the significant association that was seen between indeterminate results and immunosuppression.

A recent study from Denmark showed that 9 of 82 (11%) patients evaluated had indeterminate QFT-Gold results (12). Of these nine patients, three patients were HIV-positive, one patient was receiving immunosuppressive therapy, and two others had advanced TB. This study (12), although small, adds some support to the findings of the Italian study (8). Both studies highlight the importance and need for evaluating any new diagnostic tool in a variety of populations, to fully understand its limitations. There is a need for more such studies, particularly in patients with HIV infection or other immunosuppressive conditions (e.g., dialysis and transplantation), in those receiving immunosuppressive therapies, and in elderly or malnourished individuals (7). Because of differences between the immune systems of adults and children, studies should also evaluate IFN- assays in children (13). In theory, by controlling for the number of T cells in each assay, ELISPOT-based IFN- assays might be less affected by immunosuppression. Although limited, there is some evidence that the ELISPOT-based assay has utility in HIV-infected individuals (14). Further research is needed to verify this in clinical studies.

Although it is disappointing that the IFN- assay may be indeterminate in immunosuppressed patients, there may be some information that can be gleaned from indeterminate results. In contrast to the TST, which is simply negative, it is possible to distinguish an indeterminate response from a negative test result in IFN- assays. If lack of response to mitogen is shown to be consistently associated with immunosuppression, then indeterminate results can alert the clinician about anergy, and help to avoid the confusion between anergy and a true negative test result. Inclusion of mitogen controls, therefore, is helpful in two regards. First, it is the only indicator of adequate test performance, and second, it may reflect underlying immunosuppression. Because an indeterminate result can arise from either a poor mitogen response, or high background IFN- response, there may be some value in reporting specific information regarding the cause of the indeterminate result. Although an indeterminate IFN- result will not help a clinician to rule out TB, it might enable the clinician to take into account the strong likelihood of anergy, and prompt continued investigation to detect TB infection in vulnerable patients.

FOOTNOTES

Conflict of Interest Statement: None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

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