Latent tuberculosis infection (LTBI) is defined as an infection with Mycobacterium tuberculosis without any manifestation of active tuberculosis (TB). This is not stationary, but active state in which immune system of host and M. tuberculosis are balanced. Therefore, when host immune function is compromised, LTBI may progress to active TB disease. Early diagnosis and treatment of LTBI are considered to be the most effective strategy for reducing the incidence of TB in the population^1,2). Children are particularly vulnerable to development of severe disease and death following TB infection, and those with LTBI may serve as a reservoir for future transmission of TB disease^3,4).

There is no way to directly detect the presence of latent M. tuberculosis infection. Instead, diagnosis of LTBI relies on measurement of host immune responses to M. tuberculosis antigens. Until the early 2000's, the only test available for LTBI was the tuberculin skin test (TST)²⁾. Recently, two new diagnostic tests for LTBI have been approved, QuantiFERON–TB Gold (QFT) (Cellestis, a subsidiary of QIAGEN. Chadstone, Australia) and the T-SPOT.TB test (Oxford Immunotec Inc., Marlborough, MA, USA). Both tests are known as interferon-γ release assays (IGRAs) because they measure the release of interferon-γ from cells in vitro. Although it is unclear whether TST or IGRA is superior for detection of LTBI, the clinical importance of IGRA has been increasing⁵⁾. Moreover, confirmation of LTBI in young children lacking an evident contact history with active TB patients, but with positive results for TST performed as routine practice, is problematic in the clinical setting.

In this study, we compared the diagnostic performance of IGRA and TST in LTBI. The influence of age on IGRA results was investigated, and the diagnostic usefulness of IGRA in non-TB contact children with positive TST result was explored.

This study was performed to evaluate the performance of IGRA in diagnosing LTBI in children, particularly younger than 5 years of age, and non-TB contact children with positive TST results. We observed poor agreement between TST and IGRA results in young children. Interferon-γ response to various antigens was not decreased in children younger than 5 years of age and was not correlated with age. The incidence of indeterminate IGRA results was very low in this study.

The IGRA uses purified antigens from M. tuberculosis to stimulate peripheral blood lymphocytes to produce interferon-γ. A single specimen of peripheral blood is drawn and incubated overnight with specific antigens for M. tuberculosis. Interferon-γ production is then determined. A benefit of IGRAs is the avoidance of the booster phenomenon. TST results can be confounded by nontuberculous mycobacteria, history of BCG vaccination, competency of personnel administering the test, and reading of results. Despite these advantages of IGRA compared to the TST in the diagnosis of TB infection, the IGRA could not be actively used in children due to lack of accumulated clinical data. Previous studies conducted in Korean children mainly evaluated the value of TST and IGRA for TB contact screening in the limited situation with lacking a diagnostic gold standard^6,7,8,9). In this study, we focused on the performance of IGRA in children younger than 5 years old and analyzed the individual response of three IGRA antigens separately.

Previous studies comparing IGRA and TST have demonstrated that IGRA is more specific than, and at least as sensitive as, TST for detecting LTBI in children as well as adults^10,11). Other studies observed a strong correlation between positivity of IGRAs and a high TB exposure gradient, such as smear positivity, cavitary disease, and exposure time^12,13). However, IGRA has been shown to have variable levels of agreement with TST in pediatric populations. The kappa value of the 2 tests was 0.063–0.693 in the previous Korean studies^6,7,8,9), and was 0.196 in this study. Higher rates of discordance were reported in BCG-vaccinated than in non-BCG-vaccinated children in other studies^14,15). Therefore, IGRAs are mainly recommended for increasing test specificity in children who have received a BCG vaccine, and for increasing sensitivity for diagnosing LTBI in patients at high risk of developing progression from LTBI to active TB disease, using with the TST^16,17,18). Although the likelihood that prior BCG vaccination will interfere with the results of the TST has been shown to be minimal in a meta-analysis, only three of the 24 studies used in the analysis included children younger than 5 years of age, who were less than 1% of subjects in this study¹⁹⁾. As well as the results of this meta-analysis cannot be applied to young children, false positivity of TST due to BCG vaccination might be higher in young children because the time interval between vaccination and testing was shorter in young children. Although guidance from the Centers for Disease Control and Prevention (CDC) indicates a preference for TST over IGRA in children younger than 5 years old, recent studies' findings support the preferential use of IGRAs over TSTs for diagnosing LTBI in BCG-vaccinated children two years of age and older^20,21).

A main reason that IGRA is not recommended in children less than 5 years old is due to reports from previous studies suggesting an insufficient interferon-γ secretion response to the PHA being used as a positive control as well as to M. tuberculosis antigen in infants and children of this age group^22,23,24). Therefore, there are concerns about the applicability of these assays to newborns and infants since decreased Interferon-γ production in response to various stimuli has been described in newborns²⁵⁾. This would potentially decrease the sensitivity of the IGRA, and increase the chance of false-negative assay results, or require re-evaluation of lower threshold values for positive responses. However, false negativity in young children has also been observed in TST as well²⁶⁾. TSTs are frequently misleading, because the results are usually negative during the first few weeks of M. tuberculosis infection. On the other hand, the usefulness of IGRAs in the evaluation of infants with suspected perinatal TB was reported²⁷⁾. The IGRA performed well in children less than 5 years of age, and results did not appear to be adversely affected in very young children¹⁷⁾. Furthermore, there is no evidence that age adversely affects IGRA performance^17,28). Also in the current study, the values of interferon-γ released in response to nil, TB antigen, and PHA showed no significant differences between the age groups, and were not correlated with age.

Additionally, clinicians have been concerned that using IGRA in young children might yield a high proportion of indeterminate results. The frequencies of indeterminate IGRA results in children vary greatly among studies (range, 0%–17%)^{15,16,17,21,23,28)}. In a previous study, age younger than 5 years was significantly associated with indeterminate results in IGRA²³⁾. Although indeterminate results were more common among the youngest children, recent studies conducted in younger children showed very low rates (0.5%–0.7%)^17,21). In this study, the rate of indeterminate results was 2.0% (3 of 149). Indeterminate results in the early studies could be due to errors prior to analysis or clinical factors (e.g., low T-cell count, immunosuppression, or high background response due to recent viral infection). Moreover, most children with an indeterminate IGRA result were TST-negative and determined to be uninfected. Therefore, the risk of developing active TB among children with indeterminate results is very low^15,17,21,28).

An additional problem in diagnosing latent TB infection is that screening of potential contact is currently limited to those within a family or a group of close contacts. The CDC discourages the use of diagnostic tests for LTBI among populations at a low risk for infection with M. tuberculosis because of the high rates of false positive results²⁰⁾. In Korea, the incidence of tuberculosis remains high, and population influx from areas of high TB prevalence such as China and Southeast Asia continues. In this situation, it is difficult to interpret positive TST test results in patients with no history of TB contact. The IGRA may be used as an adjunct to TST in such instances, but current data are insufficient. A key determinant of TB infection is the amount of time spent sharing room air with the index case. Infection of parents or caregivers is an exclusively important source of infection in young children. In this case, children with positive TST results without history of contact with an active TB patient in their family may indicate that there is a likelihood of false positives in this population.

In this study, IGRA results were rechecked in 6 subjects including 5 patients diagnosed with LTBI. Although we could not provide any conclusion due to very low number of subjects, interferon-γ level on TB antigen and IGRA value were both elevated only in the first IGRA positive subject. On the other hand, interferon-γ level of TB antigen and IGRA value were both not elevated in first IGRA-negative and TST-positive subjects (defined as patients with LTBI in this study) as well as in both test-negative subject (control; not LTBI). IGRA follow-up testing might be helpful in decreasing the number of false positive of TST results in BCG-vaccinated children, but further study with a large number of subjects will be needed.

A limiting factor in this study was that we could investigate a relatively small number of children who had not been in contact with TB-infected adults, but had positive TST results, particularly children younger than 5 years (n=8). The number of children who underwent repeated IGRA testing was also very small (n=6). In addition, the proportions of subjects with data of TST result were different between TB-contact group and noncontact group. Therefore, the selection bias might be introduced into the analyses. However, the analyses were performed separately in each groups, rather than the comparison between the 2 groups. Therefore, we expect that this bias would not significantly influence on the main outcomes in this study. Furthermore, this study showed results consistent with those of recently performed, well-designed clinical studies, and provided multidirectional comparative analyses. The results of this study would be confirmed by further research with larger study populations. In conclusion, this study found that interferon-γ releasing response was not influenced significantly by age, and that serial IGRA testing may improve the accuracy of LTBI diagnosis in children.