The Effect of Age on Whole Blood Interferon-Gamma Release AssayResponse among Children Investigated for Latent Tuberculosis Infection

Elena Critselis, MPH, Virginia Amanatidou, MD, PhD, Garyfallia Syridou, MD, PhD, Nikos P. Spyridis, MD, PhD,

Mersini Mavrikou, MD, Nikos G. Papadopoulos, MD, PhD, and Maria N. Tsolia, MD, PhD

Objective To evaluate the effect of age upon QuantiFERON-TB Gold-In-Tube (QFT-IT) assay outcome amongchildren examined for latent tuberculosis infection (LTBI).Study design A cross-sectional study was conducted among 761 children (mean age � SD: 7.84 � 4.68 years)evaluated for LTBI. Participants were examined with both tuberculin skin test and QFT-IT (Cellestis, Australia) andcategorized into 4 age groups. Multivariate logistic and linear regressions were used to evaluate the associationbetween selected demographic and patient characteristics upon the qualitative and quantitative QFT-IT outcomes.Agreement between the tuberculin skin test and QFT-IT within groups was evaluated with the k statistic.Results QFT-IT indeterminate results occurred more frequently among young children (8.1%; P < .0001) and chil-dren (2.7%; P = .025) than adolescents (0.7%). Among QFT-IT positive patients, infants had higher mean (�SD)interferon-gamma (IFNg) concentration than adolescents. QFT-IT positive (vs negative) outcome was associatedwith origin from a high tuberculosis endemicity setting (AOR = 4.54; 95% CI, 3.22-6.25) and lack of previous BacilleCalmette Guerin immunization (AOR = 2.70; 95% CI, 1.89-3.85), but not patient age (AOR = 0.96; 95% CI, 0.92-0.99). However, among QFT-IT positive patients, the IFNg concentration was inversely associated with patientage (P = .009) and positively with mitogen response (P = .0002). Agreement between tests was not significantly dif-ferent between younger and older children in the different risk groups.ConclusionsQualitative QFT-IT assay results are not affected by patient age. However, indeterminate results oc-cur more frequently among younger children. Among patients with LTBI the quantitative QFT-IT result (ie, IFNg) isinversely associated with patient age. (J Pediatr 2012;161:632-8).

See editorial, p 581

uberculosis (TB) persists globally as an important public health problem both in adults and children.1 It has been es-

T timated that about one-third of the world’s population has been infected with Mycobacterium tuberculosis.2 The prev-alence of latent tuberculosis infection (LTBI) is notable not only in resource poor settings, but also in areas with a low

disease burden. In particular, it is estimated that approximately 1 million children and adolescents in the United States haveasymptomatic TB infection.3 Early diagnosis and treatment of LTBI is vitally important for disease control and eradication.4

For approximately 1 century, the tuberculin skin test (TST) has been used as the sole immunodiagnostic test for the detectionofM tuberculosis infection. The recently developed interferon-gamma release assays (IGRAs) have been a breakthrough in thediagnosis of tuberculosis infection in adults and children.5-7 Compared with the TST, IGRAs have enhanced specificity forM tuberculosis infection because the result is unaffected by previous Bacille Calmette Guerin (BCG) immunization or infectionwithmost nontuberculous mycobacteria.8-10 In addition, preliminary studies have indicated that IGRAsmay have a higher pos-itive predictive value for the development of TB compared with the TST among close contacts of patients with TB.11,12 How-ever, it has been suggested that young age may have a negative effect on the performance of IGRAs, mainly because of frequentindeterminate results due to lower interferon-gamma (IFNg) production in response to the mitogen control.13-17 Concernsabout lower sensitivity of IGRAs to detect M tuberculosis infection among young children also have been expressed.13,15

Our primary aim was to evaluate the effect of multiple factors, including age, on the qualitative (positive/negative) and quan-titative (IFNg concentration) whole blood IGRA response in children investigated for LTBI. In addition, the agreement

between IGRA and TST was compared between children and adolescents inthe study population and according to risk of TB infection.

From the Second University Department of Pediatrics,P. and A. Kyriakou Children’s Hospital, National andKapodistrian University of Athens School of Medicine,Athens, Greece

Funded by the Second University Department of Pedi-atrics, National and Kapodistrian University of AthensSchool of Medicine. V.A. has received a fellowship awardfrom the European Society for Pediatric Infectious Dis-eases. The authors declare no conflicts of interest.

0022-3476/$ - see front matter. Copyright ª 2012 Mosby Inc.

All rights reserved. 10.1016/j.jpeds.2012.04.007

BCG Bacille Calmette Guerin

IFNg Interferon-gamma

IGRA Interferon-gamma release assay

LTBI Latent tuberculosis infection

QFT-IT QuantiFERON-TB Gold-In-Tube

TB Tuberculosis

TST Tuberculin skin test

632

Vol. 161, No. 4 � October 2012

Methods

A cross-sectional study was conducted among children eval-uated for TB infection who were referred to the outpatientTuberculosis Clinic at the P. & A. Kyriakou Children’s Hos-pital in Athens, Greece, during the period from January 2007through July 2010. The study protocol was approved by theHuman Research and Ethics Committee of the P. & A.Kyriakou Children’s Hospital and informed consent forstudy participation was requested from the legal guardiansof all eligible participants. Demographic and socioeconomiccharacteristics, as well as complete personal and family histo-ries in relation to TB exposure, were recorded. All patientswere evaluated clinically and tested with both the TST andQuantiFERON-TB Gold-In-Tube (QFT-IT) (Cellestis Lim-ited, Carnegie, Victoria, Australia) assay.

TSTTSTwas performed with an intradermal injection of 0.1mL or2 tuberculin units PPD RT 23 (Statens SerumInstitut, Copen-hagen, Denmark) in the forearm. A single trained TB clinicstaff member, blinded to the study objectives, evaluated thetransverse diameter of the induration 48-72 hours laterwith the palpation method. TST was considered positive if$5 mm among nonimmunized and$10 mm among immu-nized childrenwith a positive contact history. TheTST cut-offsfor children without contact exposure, but who had high riskfor infection was$10 mm and for those at low risk was$15mm, regardless of BCG immunization history.18

All children with a positive contact history were tested withboth the TST and QFT-IT concomitantly in our clinic. Uni-versal (not targeted) TST screening of children is currentlyconducted in our settings by pediatricians in either the pri-vate or public sector. Screening of immigrants also is per-formed similarly. As a result, a considerable number ofchildren belonging to both high risk or low risk groups forTB infection, had the TST performed during screening andwere referred to our clinic for evaluation. In such cases, theQFT-IT test was performed within 7 days following theTST. Children who had a TST performed >7 days prior toenrollment were excluded from study participation to avoidpossible boosting of QFT-IT outcome by prior TST.19

QFT-IT AssayThe whole blood was processed for the QFT-IT assay within2 hours of sampling, according to manufacturer’s instruc-tions. Onemilliliter of whole blood was drawn into 3 separatetubes. The first tube contained heparin (negative control), thesecond phytohemagglutinin as a mitogen (positive control),and the third the M tuberculosis specific antigens ESAT-6,CFP-10, and TB7.7. Each tube was shaken vigorously andwas incubated at 37�C for 24 hours within 2 hours from col-lection. Following centrifugation, the supernatants werestored at �70�C. The enzyme-linked immunosorbent assaymethod was used to measure the amount of IFNg produced,and the amount released after stimulation with TB antigens

was calculated by subtracting the amount of IFNg releasedin the control tube. A positive QFT-IT result ($0.35IU/mL) was calculated according to the manufacturer’s soft-ware. Indeterminate results were defined as those with IFNglevels <0.35 IU/mL and either mitogen minus nil values<0.5 IU/mL or nil values >8.0 IU/mL. Because the QFT-IT as-say does not accurately assess IFNg values >10 IU/mL, highervalues were treated as 10 IU/mL in all analyses. Response tomitogen (phytohemagglutinin) was considered positive ifIFNg concentration (minus nil) was >0.5 IU/mL. In addition,negative values after mitogen stimulation were evaluated asnil values and values >15 IU/mL were treated as 15 IU/mLin all analyses.17

Case Group DefinitionsLTBI was identified in asymptomatic children with positiveTST and/or QFT-IT, as well as chest radiographic findings,which were either normal or indicative of healed fibroticchanges and/or calcifications.18 Participants were assignedinto 3 groups based on the presence of risk factors for infec-tion: (1) known exposure to an adult with active TB disease;(2) other children at high risk for TB infection20 (negativecontact history) because either they or their parents wereborn in a high prevalence country21 (>24 cases/100 000population) or if they had recently travelled to such a destina-tion. Children with TST positive first degree family memberswere also characterized as being at high risk for TB infection;and (3) children at low risk for TB infection were defined asthose with no known risk factors for TB infection.21

Statistical AnalysesThe Kolmogorov–Smirnov test was used to test for the nor-mality of the distributions of continuous variables. Continu-ous variables with non-normal distributions were comparedbetween age groups with the nonparametric Mann–WhitneyU test. The basis of comparison was adolescents aged $10years. Categorical variables were compared with the c2 testor Fisher exact test in cases where sample sizes did not exceed5 patients. Ordinal TST values were compared with theWilcoxon rank sum test. The percent concordance betweenthe TST and IGRA was computed and comparisons weremade between age groups. The kappa (k) statistic was com-puted to evaluate the agreement between the 2 tests exam-ined. The criterion of significance was P < .05. TheMantel–Haenszel method was used to calculate OR and95% CI for categorical variables between age groups. Univar-iate logistic regression was used to evaluate the associationbetween selected demographic and patient characteristicsupon a positive (vs negative) IGRA outcome. Independentvariables with a Wald P value of <.05 were entered stepwiseinto the multivariate regression model for the evaluation ofthe factors associated with IGRA outcome following adjust-ment for sex. Linear regression was used to evaluate the asso-ciation between the aforementioned variables and thequantitative IGRA response, mitogen response, and TSTsize among patients with positive IGRA outcome. Statistical

633

THE JOURNAL OF PEDIATRICS � www.jpeds.com Vol. 161, No. 4

analyses were undertaken with SAS v. 9.2 (SAS Institute,Cary, North Carolina).

Results

The study source population consisted of 769 children andadolescents #18 years of age; 761 children and adolescents(99%) were eligible and participated in the study. Amongthe study population, 163 (21.4%) patients had known expo-sure to an adult index case with TB, 257 (33.8%) had highrisk for TB infection but with unknown exposure to an indexcase, and 341 (44.8%) had low risk for TB infection. Themean age (�SD) of study participants was 7.84 � 4.68 years.Approximately one-quarter of the study population (n = 198,26.0%) was aged <5 years, of which approximately one-third(n = 74; 37.3%) were aged <2 years, and 35.6% (n = 271)were aged $10 years.

Overall, 29% (n = 221) of patients had a positive QFT-ITassay result; 3% (n = 23) of study participants had QFT-ITindeterminate results and could not be investigated further.In most of the latter patients (16/23, 70%), the indeterminateresult was due to a low mitogen response.

Characteristics of the Study PopulationThe characteristics of the study population are shown inTable I. Children of younger age did not significantly differfrom their older counterparts with regards to their birth ina TB endemic country. However, compared with adolescentcounterparts, younger children were more likely to be bornof parents originating from high TB endemic countries. Theobserved differences may reflect increasing immigrationrates from highly endemic countries to Greece, particularlyduring recent years. As expected, BCG immunization wasmore common among older children because BCGimmunization is administered at the age of 6 years inGreece. Exposure to an adult index case was identified in

Table I. Demographic and patient characteristics of children

Ages <2 yearsn = 74 P value*

Ages 2-4 yn = 124

Male sex, n (%) 35 (47.3%) .436† 62 (50.0%Child’s birth place, n (%)High TB prevalence country 9 (12.2%) .430† 17 (13.7%

Parents’ origin, n (%)High TB prevalence country 39 (52.7%) <.0001† 50 (40.3%

BCG immunization, n (%) 14 (18.9%) <.0001† 22 (17.7%Exposure to TB, n (%)Known exposure to TB 25 (33.8%) .018† 37 (29.8%At high risk for TB infection 25 (33.8%) .520† 40 (32.2%At low risk for TB infection 24 (32.4%) .009† 47 (37.9%

TST size, n (%) <.0001x

<5 mm 22 (29.7%) <.0001z 39 (31.4%5-9 mm 21 (28.4%) <.0001z 24 (19.4%10-15 mm 23 (31.1%) .004z 53 (42.7%>15 mm 8 (10.8%) <.0001z 8 (6.4%

*All comparisons were made with the group of adolescents $10 years of age.†c2 P value.zFisher exact test P value.xWilcoxon rank sum exact P value.

634

a greater proportion of younger children. A greaterproportion of older children and adolescents were at lowrisk for TB infection.

QFT-IT Qualitative Outcome and QuantitativeResponse According to AgeThe comparisons of the proportions of positive, negative,and indeterminate QFT-IT outcome, respectively, betweenage groups are shown in Table II. Overall, children 5-9years of age more frequently had a QFT-IT positive resultcompared with adolescents. QFT-IT indeterminate findingsoccurred more often among young children compared withadolescents (Table II).Among QFT-IT (+) patients, mean production of IFNg

upon stimulation with TB specific antigens was higher amongyoung (9.49 � 4.70 IU/mL; P = .132) and school-aged chil-dren (9.26 � 5.16 IU/mL; P = .062) compared with adoles-cents (7.61 � 5.42 IU/mL). In contrast, mean TST size wassignificantly smaller in infants (11.83 � 4.90 mm; P = .004)and young children (13.45 � 4.20 mm; P = .005) comparedwith adolescents (15.81 � 3.50 mm) (Table III).

Concordance of QFT-IT and TST According to Ageand Risk of TB InfectionAmong children with known exposure to a TB index case,agreementbetweenQFT-IT andTSTwas greater among youn-ger children (Table IV). In particular, in young children withknown exposure to a TB index case who were not BCGimmunized, the agreement between tests was excellent, andtheir concordance did not differ from that observed amongolder children (c2 P = .150). In addition, the agreementbetween QFT-IT and TST was diminished among childrenat high risk for TB infection who were not BCG immunized.However, both the agreement and concordance of these testresults did not differ significantly by age in this patientgroup (c2 P = .236).

investigated for LTBI (n = 761)

earsP value*

Ages 5-9 yearsn = 92 P value*

Ages ‡10 yearsn = 271

) .658† 141 (48.3%) .330† 142 (52.4%)

) .579† 43 (14.7%) .707† 43 (15.9%)

) .019† 120 (41.1%) .002† 77 (28.4%)) <.0001† 108 (37.0%) <.0001† 200 (73.8%)

) .046† 45 (15.4%) .104† 56 (20.7%)) .635† 111 (38.0%) .042† 81 (29.9%)) .033† 136 (46.6%) .496† 134 (49.4%)

<.0001x <.0001x

) <.0001z 28 (9.6%) .348z 20 (7.4%)) <.0001z 34 (11.6%) .017z 16 (5.9%)) .191z 150 (51.4%) .712z 135 (49.8%)) <.0001z 80 (27.4%) .016z 100 (36.9%)

Critselis et al

Table II. Comparison between age groups of qualitative QFT-IT outcomes among children investigated for LTBI(n = 761)

QFT-IT resultAges <2 years

n = 74 P value*Ages 2-4 years

n = 124 P value*Ages 5-9 years

n = 292 P value*Ages ‡10 years

n = 271

Overall (n = 761)Negative, n (%) 53 (71.6%) 83 (66.9%) 175 (59.9%) 206 (76.0%)Positive, n (%) 18 (24.3%) .734† 31 (25.0%) .433† 109 (37.3%) <.0001† 63 (23.2%)Indeterminate, n (%) 3 (4.0%) .058z 10 (8.1%) <.0001z 8 (2.7%) .028z 2 (0.7%)

Known exposure to TB (n = 163)Negative, n (%) 17 (68.0%) 25 (67.6%) 24 (53.3%) 32 (57.1%)Positive, n (%) 7 (28.0%) .287† 10 (27.0%) .204† 21 (46.7%) .522† 23 (41.1%)Indeterminate, n (%) 1 (4.0%) .466z 2 (5.4%) .338z 0 (0.0%) .593z 1 (1.8%)

At high risk for TB infection (n = 257)Negative, n (%) 16 (64.0%) 22 (55.0%) 43 (38.7%) 53 (63.8%)Positive, n (%) 8 (32.0%) .911† 14 (35.0%) .653† 62 (55.8%) .0007† 28 (34.6%)Indeterminate, n (%) 1 (4.0%) .243z 4 (10.0%) .010z 6 (5.4%) .010z 0 (0.0%)

At low risk for TB infection (n = 341)Negative, n (%) 20 (83.3%) 36 (76.6%) 108 (79.4%) 121 (90.3%)Positive, n (%) 3 (12.5%) .226† 7 (14.9%) .604† 26 (19.1%) .011† 12 (9.0%)Indeterminate, n (%) 1 (4.2%) .252z 4 (8.5%) .013z 2 (1.5%) .348z 1 (0.7%)

*All comparisons were made with the group of adolescents $10 years of age.†c2 P value.zFisher exact test P value.

October 2012 ORIGINAL ARTICLES

Mitogen Response According to AgeYounger children were more likely to have lower mean mito-gen response compared with adolescents (8.09� 5.52 IU/mLvs 10.54 � 4.65 IU/mL; P = .0002) (Table III). In themultivariate analysis, a positive mitogen responsecorrelated only with age (AOR = 1.16; 95% CI, 1.07-1.26)and with neither BCG immunization (AOR = 1.06; 95%CI, 0.56-1.99) nor with origin from a high TB endemicitysetting (AOR = 1.32; 95% CI, 0.70-2.49).

Factors Associatedwith the QFT-IT AssayOutcomeUnivariate logistic regression analyses indicated that a posi-tive (vs negative) QFT-IT assay outcome was associatedwith younger age, parents’ origin from a high TB endemicitysetting, and negative history of BCG immunization. Mitogen

Table III. Comparison between age groups of QFT-IT quantchildren with positive QFT-IT outcome (n = 221)

Age <2 yearsn = 18 P value*,†

Agen

Overall (n = 221)QFT-IT result, mean IU/mL � SD 10.42 � 5.21 .058 9.4TST size, mean mm � SD 11.83 � 4.90 .004 13.4Mitogen response, mean IU/mL � SDz 8.09 � 5.52 .0002 8.2

Known exposure to TB (n = 61)QFT-IT result, mean IU/mL � SD 10.70 � 4.97 .224 11.TST size, mean mm � SD 11.43 � 5.97 .137 13.3Mitogen response, mean IU/mL � SDz 7.42 � 5.56 .096 9.0

At high risk for TB infection (n = 112)QFT-IT result, mean IU/mL � SD 11.90 � 3.86 .140 9.6TST size, mean mm � SD 12.75 � 4.33 .097 13.6Mitogen response, mean IU/mL � SDz 8.80 � 5.75 .021 8.0

At low risk for infection (n = 48)QFT-IT result, mean IU/mL � SD 5.81 � 7.96 .832 7.1TST size, mean mm � SD 10.33 � 4.93 .103 13.2Mitogen response, mean IU/mL � SDz 8.03 � 5.36 .015 7.8

*All comparisons were made with the group of adolescents $10 years of age.†Mann Whitney U-test.zCalculated among the entire study population (n = 761).

The Effect of Age on Whole Blood Interferon-Gamma Release AsTuberculosis Infection

response was only marginally associated with the qualitativeQFT-IT assay outcome (OR = 1.04; 95% CI, 1.00-1.07). Themultivariate logistic regression analysis indicated that the or-igin of the parents from a high TB endemicity setting andnegative history of BCG immunization were independentlyassociated with a positive QFT-IT outcome (Table V).Therefore, the qualitative outcome of the QFT-IT assay wasnot independently associated with patient age among thestudy population examined.Among patients with a positive assay outcome, the quan-

titative QFT-IT response was inversely associated with pa-tient age (b: �0.24; SE: 0.09; P = .009). Moreover, amongthis patient group, the quantitative QFT-IT response was as-sociated with lack of prior BCG immunization (b: 1.93; SE:0.75; P = .011), as well as the patient’s mitogen response

itative response, TST size, and mitogen response among

2-4 years= 31 P value*,†

Age 5-9 yearsn = 109 P value*,†

Age ‡10 yearsn = 63

9 � 4.70 .132 9.26 � 5.16 .062 7.61 � 5.425 � 4.20 .005 15.42 � 3.65 .475 15.81 � 3.507 � 5.50 <.0001 10.15 � 5.14 .445 10.54 � 4.65

0 � 5.20 .134 11.26 � 3.96 .032 7.55 � 5.600 � 5.85 .313 16.19 � 4.02 .519 15.61 � 3.973 � 5.39 .896 9.25 � 5.18 .964 9.39 � 4.24

7 � 3.76 .681 8.83 � 5.27 .820 8.36 � 5.684 � 3.43 .022 15.56 � 3.43 .345 15.82 � 3.019 � 5.48 .0006 10.26 � 5.52 .230 11.24 � 4.66

6 � 5.52 .534 8.66 � 5.51 .101 6.03 � 4.479 � 3.35 .076 14.46 � 3.78 .151 16.17 � 3.903 � 5.66 .001 10.35 � 4.80 .601 10.61 � 4.74

say Response among Children Investigated for Latent 635

Table IV. Comparison of concordance and agreement between QFT-IT and TST among children investigated for LTBI

Overall*n = 738

Ages 0-4 years*n = 185

Ages 5-18 years*n = 553

Concordance, % k (P value) Concordance (%) k (P value) Concordance (%) k (P value)

Known exposure to TB (n = 163)BCG non-immunized 93.6% (88/94) 0.87 (.0001) 96.4% (53/55) 0.91 (<.0001) 89.7% (35/39) 0.79 (<.0001)BCG immunized 53.8% (35/65) 0.20 (.010) 75.0% (3/4) 0.50 (.248) 52.4% (32/61) 0.18 (.014)

At high risk for TB infection (n = 257)BCG non-immunized 77.0% (87/113) 0.40 (<.0001) 70.3% (26/37) 0.41 (.002) 80.3% (61/76) 0.36 (.0002)BCG immunized 39.1% (52/133) 0.08 (.039) 9/23 (39.1%) 0.03 (.795) 39.1% (43/110) 0.08 (.036)

At low risk for TB infection (n = 341)BCG non-immunized 87.4% (174/199) 0.57 (<.0001) 90.3% (56/62) 0.57 (<.0001) 86.1% (118/137) 0.57 (<.0001)BCG immunized 50.0% (67/134) 0.06 (.081) 4/4 (100.0%) NC 48.5% (63/130) 0.06 (.097)

*Population excludes participants with QFT-IT indeterminate results.

THE JOURNAL OF PEDIATRICS � www.jpeds.com Vol. 161, No. 4

(b: 0.27; SE: 0.07; P = .0002). It is noteworthy, though, thatamong this patient group, TST size was significantly associ-ated with children’s age (b: 0.25; SE: 0.07; P = .0002) andprior BCG immunization (b: 2.10; SE: 0.56; P = .0002).

The occurrence of a QFT-IT indeterminate result was in-versely associated with children’s age (b: �0.16; AOR: 0.85;95% CI, 0.77-0.95) and positively associated with originfrom a high TB endemicity setting (b: 0.88; AOR: 2.44;95% CI, 1.04-5.62).

Comparison of QFT-IT and TST Performanceamong Infants Under 1 Year of AgeThe proportion of young infants (n = 19) with QFT-IT posi-tive (n = 7; 36.8%; P = .440) and indeterminate (n = 1; 5.3%;P= .321) results did not differ from adolescents. Among thosewith LTBI, mean (�SD) QFT-IT response (13.81 � 2.35IU/mL) was greater than among adolescents (P = .010). How-ever, mean (�SD) mitogen response (11.97 � 5.76 IU/mL)did not differ from that of adolescents (P = .252). Further-more,mean (�SD)TSTfindingswere lower among this groupcompared with adolescents (11.00� 5.94mm vs 15.24� 3.76mm; P = .046). Lack of agreement between the QFT-IT andTST was found among BCG immunized infants. In contrast,among nonimmunized patients, the agreement between theQFT-IT and TST was good (90.0% [9/10]; k = 0.74).

Discussion

Because a large number of children and adolescents wereevaluated for LTBI at our tertiary care referral center ina low TB endemicity setting, it was possible to evaluate the

Table V. Logistic regression analyses for the assessment of demqualitative performance of QFT-IT in the total study populat

Positive (vs negative) QFT-IT outcome

Univa

b S

Age (y) �0.04 0.0Parents’ origin from a high TB endemicity setting (vs low) 1.40 0.1No BCG immunization (vs immunization) 0.80 0.1Mitogen response (IU/mL) 0.03 0.0

636

differential effect of age on the results of a whole bloodIGRA. The study findings indicate that young patient agedoes not adversely affect IGRA response among childreninvestigated for LTBI.Among the overall study population, the qualitative (pos-

itive vs negative) outcome of the QFT-IT assay was not asso-ciated with age among children investigated for LTBI.However, it is noteworthy that indeterminate QFT-IT assayfindings occurred more frequently among younger children.Furthermore, among children with a positive QFT-IT assayoutcome, young children were able to produce IFNg effec-tively when stimulated with TB specific antigens. In fact,younger age was associated with higher mean concentrationsof IFNg production. This age effect might reflect the fact thatinfection with M tuberculosis was acquired more recently inthe younger children. Finally, the concordance betweenTST and QFT-IT did not differ significantly between patientage groups, particularly among children with known expo-sure to an adult case. Agreement between tests was excellentamong both older and younger children nonimmunized withBCG. Thus, these findings provide evidence supporting theuse of QFT-IT for the diagnosis of LTBI among youngerchildren.Although neonates have weak Th1 lymphocyte responses

and lower IFNg production, it has been shown that a degreeof plasticity exists and certain antigens can elicit notable Th1responses.22 Neonatal immunization with the BCG vaccineinduces a strong Th1 response to BCG; consequent IFNgproduction upon T-lymphocyte stimulation is comparableto adults.23,24 BCG vaccination at birth induces a Th1 mem-ory response of similar magnitude with vaccination later in

ographic and patient characteristics as associated with theion (n = 761)

riate analysis Multivariate analysis

E OR (95% CI) b SE OR (95% CI)

2 0.96 (0.92-0.99) - - -7 4.00 (2.94-5.55) 1.74 0.20 4.54 (3.22-6.25)7 2.22 (1.59-3.12) 0.74 0.20 2.70 (1.89-3.85)2 1.04 (1.00-1.07) - - -

Critselis et al

October 2012 ORIGINAL ARTICLES

life.23 The BCG vaccine is a live bacterium and recent reportshave indicated that the ability to activate multiple patternrecognition receptors (PRRs) on antigen presenting cellsplays an important role.22 Similarly, it is possible that infec-tion with M tuberculosis also may provide a strong antigenicstimulus enabling infants and young children (at least thosewho are latently infected and do not have active disease) tomount a strong Th1 cell response and effectively produceIFNg when their T lymphocytes are stimulated ex vivowith TB specific antigens. In a recent study conducted inUganda among household contacts of adults with culturepositive TB, it was shown that young childrenmount a robustIFNg response that is comparable to adults.25

In contrast, among patients with a positive QFT–IT result,younger age was significantly associatedwith smaller TST size.In addition, among this patient group prior BCG immuniza-tion was associated with larger TST size. Exposure to nontu-berculous mycobacteria and BCG immunization may in partaccount for the larger TST findings observed in older childrenand adolescents. Furthermore, a positive QFT-IT outcomewas inversely associated with prior BCG immunization.Hence, BCG immunization may provide protection againstinfection with M tuberculosis. However, although several po-tential confounding variables were controlled for in the pres-ent investigation, the observed associationmay be attributablepartly to population bias. Thus, further studies aimed atassessing the association between BCG immunization andQFT-IT outcome in pediatric populations are necessary.

The study findings corroborate with previous reports indi-cating that younger age is associated with a low mitogenresponse.13,16,17 In addition, among children with a positiveQFT-IT outcome, TB-specific mean IFNg response was asso-ciated with the mitogen response. Moreover, indeterminateIGRA results are reported to range between 0% and 17% insimilar studies.26 The present findings indicate that althougha relatively low rate (3%)of indeterminate results occur amongchildrenwith LTBI, the rate is higher among younger children.Blood samples are transferred and processed within less than 2hours to our laboratory and may have contributed to the lowrate of indeterminate results observed.27 Although a lowmito-gen response in younger children is associatedwith indetermi-nate results, it is possible that other factors alsomay play a role.In particular, drawing adequate blood volumesmay be techni-cally difficult in younger children, and this may affect the per-formance of the test in the younger age groups.

A strength of the present study is comparing the QFT-ITqualitative outcome and quantitative response between chil-dren and adolescents. A population bias, however, may havebeen introduced because a number of children without riskfactors, some of whomhad a positive BCG immunization his-tory, were referred to our clinic following a positive or ques-tionable TST finding during screening. This could result in anoverestimation of true TST positivity rates expected amongthe general population. However, such an event would likelyonly lead to an underestimation of the true agreement be-tween the TST and QFT-IT assays. Although a considerablesample of children aged younger than 2 years were evaluated

The Effect of Age on Whole Blood Interferon-Gamma Release AsTuberculosis Infection

(n = 78), the current findings need to be confirmed amonglarger study populations. A further limitation is that QFT-IT response values exceeding 10 IU/mL could not be assessedas the particular assay is reported to be inaccurate above thisrange. Finally, the study findings cannot be extrapolated topatients with active TB disease because only children investi-gated for LTBI were included in the present study.The present findings indicate that the qualitative outcome

(positive vs negative) of the whole blood IGRA result, as wellas the amount of IFNg produced, is not adversely affected byyoung age among children investigated for LTBI. These re-sults provide support for the application of whole bloodIGRA assays in young children being evaluated for LTBI. n

The authors thank Flora Metsou and Kyriaki Benetatou (health visi-tors), who are part of the TB clinic staff, for their contribution to patientcare and data management. The authors also thank Maria Tsagarakifor her contribution in the laboratory work.

Submitted for publication Oct 11, 2011; last revision received Feb 23, 2012;

accepted Apr 13, 2012.

Reprint requests: Maria N. Tsolia, MD, PhD, Second University Department of

Pediatrics, P. and A. Kyriakou Children’s Hospital, National and Kapodistrian

University of Athens School of Medicine, Athens, 11527 Greece. E-mail:

matsolia@ath.forthnet.gr

References

1. Global tuberculosis control: a short update to the 2009 report. Geneva:

World Health Organization; 2009 (WHOHTM/TB/2009.426).

2. Dye C, Scheele S, Dolin P, Pathania V, Raviglione MC. Consensus state-

ment. Global burden of tuberculosis: estimated incidence, prevalence,

and mortality by country. JAMA 1999;282:677-86.

3. Bennett DE, Courval JM, Onorato I, Agerton T, Gibson JD, Lambert L,

et al. Prevalence of tuberculosis infection in the United States popula-

tion: the national health and nutrition examination survey, 1999-2000.

Am J Respir Crit Care Med 2008;177:348-55.

4. Horsburgh CR. Priorities for the treatment of latent tuberculosis infec-

tion in the United States. N Engl J Med 2004;350:2060-7.

5. Lalvani A, Millington KA. T cell-based diagnosis of childhood tubercu-

losis infection. Curr Opin Infect Dis 2007;20:264-71.

6. Centers for Disease Control and Prevention. Updated Guidelines

for Using Interferon Gamma Release Assays to Detect Mycobacte-

rium tuberculosis Infection, United States. MMWR 2010;59(No.

RR-5):2-3.

7. Lewisohn DA, Lobato MN, Jereb JA. Interferon-gamma release assays:

new diagnostic tests for Mycobacterium tuberculosis infection, and their

use in children. Curr Opin Pediatr 2010;22:71-6.

8. Tsolia MN, Mavrikou M, Critselis E, Papadopoulos NG, Makrinioti H,

Spyridis NP, et al. Whole blood interferon-g release assay is a useful

tool for the diagnosis of tuberculosis infection particularly among

BCG- immunized children. Pediatr Infect Dis J 2010;29:1137-40.

9. Cruz AT, Geltemeyer AM, Starke JR, Flores JA, Graviss EA, Smith KC.

Comparing the tuberculin skin test and T-SPOT.TB blood test in chil-

dren. Pediatrics 2011;127:e31-8 [Epub 2010 Dec 6].

10. Bianchi L, Galli L, MoriondoM, Veneruso G, Becciolini L, Azzari C, et al.

Interferon-gamma release assay improves the diagnosis of tuberculosis

in children. Pediatr Infect Dis J 2009;28:510-4.

11. Bakir M, Millington KA, Soysal A, Deeks JJ, Efee S, Aslan Y, et al. Prog-

nostic value of a T-cell-based, interferon-gamma biomarker in children

with tuberculosis contact. Ann Intern Med 2008;149:777-87.

12. Diel R, Loddenkemper R, Niemann S, Meywald-Walter K, Nienhaus A.

Negative and positive predictive value of a whole-blood IGRA for de-

veloping active TB–an update. Am J Respir Crit Care Med 2011;183:

88-95.

say Response among Children Investigated for Latent 637

THE JOURNAL OF PEDIATRICS � www.jpeds.com Vol. 161, No. 4

13. Lighter J, Rigaud M, Eduardo R, Peng CH, Pollack H. Latent tuberculo-

sis diagnosis in children by using the QuantiFERON-TB gold in-tube

test. Pediatrics 2009;123:30-7.

14. Haustein T, Ridout DA, Hartley JC, Thaker U, Shingadia D, Klein NJ,

et al. The likelihood of an indeterminate test result from a whole-blood

interferon-gamma release assay for the diagnosis of Mycobacterium tu-

berculosis infection in children correlates with age and immune status.

Pediatr Infect Dis J 2009;28:669-73.

15. NicolMP, DaviesMA,Wood K, Hatherill M,Workman L, Hawkridge A,

et al. Comparison of T-SPOT.TB assay and tuberculin skin test for the

evaluation of young children at high risk for tuberculosis in a community

setting. Pediatrics 2009;123:38-43.

16. Bergamini BM, Lois M, Valenti F, D’Amico R, Meccugni B, Meacci M,

et al. Performance of commercial blood tests for the diagnosis of latent

tuberculosis infection in children and adolescents. Pediatrics 2009;123:

e419-24.

17. Connell T, Tebruegge M, Ritz N, Bryant P. Indeterminate interferon g

release assay results in children. Pediatr Infect Dis J 2010;29:285-6.

18. American Academy of Pediatrics. Tuberculosis. In: Pickering LK, ed.

Red book: 2009 report of the committee on infectious diseases.

28th ed. Elk Grove Village, IL: American Academy of Pediatrics;

2009. p. 680-700.

19. Van Zyl-Smit RN, Pai M, Peprah K, Meldau R, Kieck J, Juritz J, et al.

Within-subject variability and boosting of T-cell interferon-gamma re-

sponses after tuberculin skin testing. Am J Respir Crit Care Med 2009;

180:49-58.

638

20. Pediatric Tuberculosis Collaborative Group. Targeted tuberculin skin

testing and treatment of latent tuberculosis infection in children and

adolescents. Pediatrics 2004;114(Suppl 4):1175-201.

21. World Health Organization. Global tuberculosis control: World Health

Organization report 2011. Available at: http://whqlibdoc.who.int/

publications/2011/9789241564380_eng.pdf. Accessed January 17, 2012.

22. Philbin VJ, Levy O. Developmental biology of the innate immune re-

sponse: implications for neonatal and infant vaccine development.

Pediatr Research 2009;65(5 Pt 2):98R-105R.

23. Marchant A, Goetghebuer T, Ota MO, Wolfe I, Ceesay SJ, De Groote D,

et al. Newborns develop a Th1-type immune response toMycobacterium

bovis bacillusCalmette-Guerin vaccination. J Immunol 1999;163:2249-55.

24. Vekemans J, Amedei A, Ota MO, D’Elios MM, Goetghebuer T, Ismaili J,

et al. Neonatal bacillus Calmette-Guerin vaccination induces adult-like

IFN-gamma production by CD4+ T lymphocytes. Eur J Immunol

2001;31:1531-5.

25. Lewinsohn DA, Zalwango S, Stein CM, Mayanza-Kizza H, Okwera A,

Boom WH, Mugerwa RD, Whalen CC. Whole blood interferon-

gamma responses to Mycobacterium tuberculosis antigens in young

household contacts of persons with tuberculosis in Uganda. PLoS One

2008;3:e3407.

26. Ling DI, Zwerling AA, Steingart KR, Pai M. Immune-based diagnostics

for TB in children: what is the evidence? Pediatr Respir Rev 2011;12:9-15.

27. Herrera V, Yeh E, Murphy K, Parsonnet J, Banaei N. Immediate incuba-

tion reduces indeterminate results for QuantiFERON-TB Gold-In-Tube

assay. J Clin Microbiol 2010;488:2672-6.

Critselis et al