Infection is one of the main causes of morbidity and mortality throughout the life of lung transplant recipients. The frequency of infection in lung transplantation is much higher than in other types of solid organ transplant (SOT).1 However, there are no tests that enable us to evaluate the specific risk of infection in a given patient. The immune cell functional assay (ImmuKnow®) is a noninvasive technique used to evaluate the global immune response. It has been approved by the United States Food and Drug Administration for monitoring immunosuppression in solid organ recipients. The assay quantifies production of adenosine triphosphate (ATP) in CD4+T lymphocytes when these are stimulated in vitro with phytohemagglutinin (PHA) and is both reproducible and affordable.2,3 In published studies, its usefulness for predicting infections varies with the type of transplant,4,5 and it could prove useful in lung transplantation.6–10 The objectives of this study were to evaluate the prognostic value of this assay for predicting infection other than cytomegalovirus (CMV) in lung transplant recipients. The predictive value was determined in the short term (the 2 months following testing) and in the medium term (the 6 months following testing).

Between months 6 and 12 post-transplantation, 23 of the 92 patients (25.0%) developed 29 non-CMV infections. No significant differences were found between patients who developed infection and those who did not, in terms of the demographic and clinical variables recorded (Table 1).

Infections occurred in the form of bacterial or fungal tracheobronchitis in 11 cases (3 cases of Aspergillus spp, 2 cases of Pseudomonas spp and one case each of Staphylococcus aureus, Streptococcus pneumoniae, Morganella spp, Enterococcus spp, Rhizopus spp and Mycobacterium gordonae), 8 cases of possible bacterial tracheobronchitis, and 2 cases of pneumonia (1 Pseudomonas spp and 1 unidentified fungus).

Other diagnoses were 3 cutaneous infections (2 herpes zoster and 1 olecranon bursitis due to Staphylococcus aureus), 2 esophageal candidiasis, 1 gastroenteritis due to Salmonella spp, 1 thoracotomy scar infection with Mycobacterium tuberculosis and 1 endocarditis due to Pseudomonas spp. Three of the 92 patients (3.3%) died before completing the study (1 septic shock secondary to pneumonia, 1 kidney failure, and 1 sudden death).

Mean ImmuKnow® values significantly increased from month 6 (142.45±88.60ng/mL) to month 12 (204.44±121.67ng/mL) after surgery (p=0.002) (Fig. 1).

Fig. 1.

Mean (± SD) ATP values on ImmuKnow® assay during the study period.

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Mean ImmuKnow® values were lower in patients who developed infection than in those who did not (111±58.5ng/mL vs. 155.38±96.40ng/mL, p=0.02). Trough serum tacrolimus levels were similar in both groups (9.70±2.81ng/mL vs. 10.90±3.99ng/mL, p=0.22), as were doses of mycophenolate or mycophenolic acid (1447.36±497.06mg vs. 1366.06±585.53mg, p=0.509) and corticosteroids (12.62±5.12mg vs. 11.07±5.41mg, p=0.164).

We observed a weak correlation between the dose of mycophenolate (mycophenolic acid) and ImmuKnow® values (r=−0.149, p=0.027). No correlation was observed for trough blood levels of tacrolimus (r=−0.069, p=0.28) or dose of corticosteroids (r=−0.037, p=0.560).

Mean ImmuKnow® values between 6 and 12 months post-transplant were similar in patients who received induction immunosuppressive treatment to those who did not (168.63±60.17ng/mL vs. 155.58±58.19ng/mL, p=0.294).

At 6 months post-transplant, a moderate immune response was detected in 14 of the 92 patients studied (15.2%) and a low response in 78 patients (84.8%). No patients showed a strong immune response. Only 1 of the 14 patients (7.1%) with moderate immune response developed an infection between 6 and 12 months after surgery compared with 22 of the 78 patients (28.2%) with low response. In the ROC curve analysis, the assay had a sensitivity of 95.7% and a specificity of 18.8% (AUC, 0.641; p=0.043). With an ATP cut-off of 225ng/mL, the PPV and NPV were 28.2%, and 92.9% respectively. Specificity improved at an ATP cut-off of<40ng/mL (86.36%) but sensitivity decreased (9.23%) (Fig. 2).

Fig. 2.

Flow diagram of patients who developed non-CMV infection and those who did not in the 6 months following ImmuKnow® testing, and ROC curve showing the sensitivity and specificity of the assay for prediction of non-CMV infection.

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Of the 273 assays performed, a moderate immune response was detected in 55 samples (20.2%) and a low response in 216 (79.1%). Two samples showed a strong immune response (0.7%). The rate of infection during the 2 months following testing was 0% in patients with strong or moderate immune response and 12.5% in those with a low response. ROC curve analysis revealed a sensitivity of 100% and a specificity of 22.5%, (AUC, 0.632; p=0.024) with a PPV of 12.5%, and NPV of 100%% at an ATP cut-off of 225ng/mL. Specificity improved at an ATP cut-off of<40ng/mL (88.89%) and sensitivity decreased (9.02%) (Fig. 3).

Fig. 3.

Flow diagram of assayed samples from patients who developed non-CMV infection or not in the 2 months following ImmuKnow® testing, and ROC curve showing the sensitivity and specificity of the assay29

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Between 6 and 12 months after surgery, 14 of the 92 patients studied (15.2%) had mean ATP values≥225ng/mL and 78 (84.8%) had mean ATP<225ng/mL. Acute rejection was recorded in 1 of the 14 patients with mean ATP≥225ng/mL (7.1%) and in 7 of the 77 patients with mean ATP<225ng/mL (9.1%, p=0.81). Similarly, there were no differences in respiratory function progress between the groups.

Little is known about the diagnostic value of the ImmuKnow® assay in lung transplantation.6–10 To our knowledge, this is the first multicenter prospective study to analyze the predictive value of ImmuKnow® for infections other than CMV in this type of transplant. We observed that patients with ImmuKnow® ATP values ≥225ng/mL had a very low risk of developing non-CMV infection both short term and medium term, with no increase in the acute rejection rate or worsening in the evolution of respiratory function. In the 2 months following performance of the assay, the NPV was 100%, and in the following 6 months it was 93%. However, the specificity and PPV were low. Therefore ImmuKnow® does not seem useful to predict episodes of non-CMV infection in lung transplant recipients, but could identify patients with a very low risk (ATP>225ng/mL) and help us define a target for an optimal immunosuppression.

The ImmuKnow® assay has been evaluated in lung transplant in a few studies with different designs to that of the present study. The novel aspects of our study are that it was designed to evaluate the sensitivity and specificity of the assay to predict which patients will go on to develop infections, that it included non-respiratory infections, and that it was a multicenter study. Bhorade et al.6 studied 143 samples from 57 lung transplant recipients. Like us, the authors observed that patients who developed infection had lower ImmuKnow® values, but unlike our study the assays were performed at the time of infection, not before. Husain et al.7 found that patients with bacterial pneumonia, viral infection, CMV disease, and fungal infection had lower ImmuKnow® values (also at time of infection) than uninfected patients and that ImmuKnow® values<100ng/mL were an independent risk factor for infection, with an odds ratio of 2.81. In a retrospective study, Shino et al.8 correlated ImmuKnow® values with findings from bronchoscopy. Their findings differed, in that ImmuKnow® values were similar in patients who developed infection and in those who did not. It should be noted that in this study, ImmuKnow® values were very high compared with those reported in other published studies.6,7,9,10 Even so, Shino et al.8 found that 40% of patients with ATP levels<225ng/mL developed an infection (odds ratio, 1.9).

This assay provides information on global T cell immunocompetence and risk of all types of opportunistic infection.7 However, Husain et al.7 observed than patients with CMV disease showed the lowest ImmuKnow® values, followed by those with fungal and then bacterial infections: this is probably because T cells are more involved in the control of CMV infection than in bacterial or fungal infections where innate immunity is the first line of defense. We therefore decided to study the predictive value of ImmuKnow® for infections other than CMV.

ImmuKnow® assay has been extensively studied in other types of SOT. Its value for predicting infection is controversial and varies with the organ transplanted. Discrepancies are probably due to differences in the incidence of infection between organs, immunosuppression protocols and study design.15 Overall, the sensitivity for predicting the risk of infection is high for liver transplant but lower for kidney transplant. In their meta-analysis, Ling et al.4 found that in 3 studies on liver transplant,16–18 sensitivity ranged from 81% to 100%, whereas in 3 studies on kidney transplant,19–21 sensitivity was between 21% and 68%. Specificity for both organs was similar, between 60% and 79% in liver transplant and between 74% and 100% in kidney transplant. In another meta-analysis, Rodrigo et al.5,16,18,22,23 found sensitivity to be 83% and specificity 75% for liver transplant. Huskey et al.21 also in a retrospective study with 583 renal transplant recipients at a single center, observed a lack of sensitivity and specificity as a predictive test for infection. Kowalski et al.15 observed in a meta-analysis with 504 SOT recipients that ATP levels around 280ng/mL may protect against allograft rejection and infection.

In our study, tacrolimus levels were no higher in patients who developed infection than in those who did not. Similarly, they did not correlate with ImmuKnow® levels. This finding is well documented6,24,25 and points to inappropriate monitoring of levels of immunosuppression based only on pharmacokinetic parameters. In addition to ImmuKnow®, other assays have been proposed for monitoring the immune response (eg, cytokine genetic polymorphisms, mixed lymphocyte reaction, enzyme-linked immunosorbent spot assay, Quantiferon assay or monitoring of nuclear factor of activated T cells-regulated gene expression), although they have not been implemented in clinical practice, since they involve complicated laboratory procedures or are subject to problems of reproducibility and cost. However, the main impediment to their use in clinical practice is the lack of prospective studies and randomized clinical trials.2,3,26–28 Ravaioli et al.25 performed a randomized controlled trial in liver transplant recipients in which they modified the dose of tacrolimus according to the ImmuKnow® result, with a 25% dose reduction if ATP levels were<130ng/mL and a 25% dose increase if they were>450ng/mL. With this strategy, patients developed fewer infections than in the control group (42% vs 54.9%) and had better 1-year survival (95% vs 82%). It would be interesting to perform a study with a similar design in lung transplant recipients, where infection plays an even more important role. In fact, reducing the number of infections is essential if we are to improve the outcomes of lung transplantation. Randomized clinical trials in which immunosuppressive therapy is adjusted according to ImmuKnow® or other immunological monitoring assays are needed.

Studies on acute rejection performed in liver transplantation4,16–18 have shown high specificity (94–100%) and low sensitivity (9–50%) for ImmuKnow® with a cut-off point of ATP>525ng/mL. The results are poorer in kidney transplantation, with a specificity of 65–80% and a sensitivity of 33–23%. In lung transplantation, Shino et al.8 reported a sensitivity of 45% and a specificity of 79% with this cut-off point. In our study, we cannot draw conclusions because only two samples out of 271 were above this limit; we did however observe the same acute rejection rate in patients with mean ImmuKnow® values of ATP≥225ng/mL and <225ng/mL during the study period.

With respect to pulmonary function, there were no differences between patients with ATP levels≥225ng/mL and patients with lower levels. However, follow-up was too short to draw conclusions on changes in pulmonary function.

The main limitations of the present study are those of multicenter observational studies. Although we attempted to collect all variables prospectively in order to minimize potential bias, some variables are difficult to record and analyze. For example, one physician may address a particular clinical situation differently from another, or there may be differences between centers in terms of prophylaxis, immunosuppression, and patient follow-up. Our study is also limited by the short follow-up period, although this was during a period when the patient is still very susceptible to infections, i.e., between 6 and 12 months after surgery. We excluded the first 6 months because some postoperative variables can also play a role in the development of infection (for example problems in bronchial anastomosis, leukopenia due to valganciclovir prophylaxis, and different fungal or bacterial prophylaxis between centers). Therefore, the predictive value of ImmuKnow® in these first 6 months is unclear. Finally, our study was limited by potential variability in the immunoassays used; we attempted to minimize this variability by using a central laboratory.

In conclusion, ImmuKnow® does not seem useful to predict episodes of non-CMV infection in lung transplant recipients, but could identify patients with a very low risk. ImmuKnow® could help us to adjust the immunosuppressive treatment, reducing the dose of immunosuppressants when the ImmuKnow® values are very low. However, this should be evaluated with clinical trials designed for this purpose.

VM, SGO, RC, VLP, JMC, JR, AS contributed to the design; VM, SGO, HS, RC, PU coordinated the study; VM, CB, MLM, PJM, JPO, VLP, RA, JMC, JMV, JR, RL, PU, JE, AS contributed to the data collection; SR, RC, ILA contributed to sample analysis; VM, SGO, HS, ILA, AM contributed to data analysis; VM wrote the manuscript; VM, VLP, JMC, DI, JR, PU, AS contributed to the revision of the manuscript.

This work was supported by Roche Farma S.A.

The authors have no conflict of interests related to this manuscript to disclose.