DOI: 10.1542/peds.2010-1725 2011;127;e31-e38; originally published online Dec 6, 2010; Pediatrics

Graviss and Kim C. Smith Andrea T. Cruz, Abby M. Geltemeyer, Jeffrey R. Starke, Jaime A. Flores, Edward A.

Blood Test in ChildrenTBComparing the Tuberculin Skin Test and T-SPOT.

http://www.pediatrics.org/cgi/content/full/127/1/e31located on the World Wide Web at:

The online version of this article, along with updated information and services, is

rights reserved. Print ISSN: 0031-4005. Online ISSN: 1098-4275. Grove Village, Illinois, 60007. Copyright © 2011 by the American Academy of Pediatrics. All and trademarked by the American Academy of Pediatrics, 141 Northwest Point Boulevard, Elkpublication, it has been published continuously since 1948. PEDIATRICS is owned, published, PEDIATRICS is the official journal of the American Academy of Pediatrics. A monthly

. Provided by McGill University Library on January 14, 2011 www.pediatrics.orgDownloaded from

Comparing the Tuberculin Skin Test and T-SPOT.TBBlood Test in Children

WHAT’S KNOWN ON THIS SUBJECT: Interferon-�–release assaysare blood tests that offer more specificity for tuberculosis thanthe tuberculin skin test. The performance of this test in childrenstill is being studied.

WHAT THIS STUDY ADDS: This study adds more information onthe utility of interferon-�–release assays for children withtuberculosis disease and children in varying risk categories forlatent tuberculosis infection.

abstractBACKGROUND: Interferon-�–release assays (IGRAs) have been devel-oped for the diagnosis of tuberculosis infection, but few data are avail-able for children. There currently is no reference standard for thediagnosis of tuberculosis infection.

OBJECTIVE: To compare the performance of 1 IGRA, the T-SPOT.TB as-say with the tuberculin skin test (TST) in children with different epide-miologic risk factors for tuberculosis.

METHODS: We conducted a prospective study of 210 patients referredto 3 pediatric tuberculosis clinics, including those with no risk factorsfor tuberculosis (low risk, n � 27), risk factors but no identifiablesource case (intermediate risk, n� 78), contact with a known sourcecase (high risk, n � 74), and active disease (n � 31). Children weretested with TST and T-SPOT.TB. Concordance analyses were performed,and assay outcomes were modeled by multivariate logistic regression.

RESULTS: For 13 children with culture-confirmed tuberculosis dis-ease, sensitivity of TST and T-SPOT.TB was 77% and 92%, respectively,and concordance was 69%. For high-risk children, concordance was94% for BCG-unimmunized children and 88% for BCG-immunized chil-dren. For intermediate-risk children, concordance was 74% for BCG-unimmunized children and 33% for BCG-immunized children. For low-risk children, concordance was 74% for BCG-unimmunized childrenand 20% for BCG-immunized children. Multivariate analysis revealedthat contact with a source case was associated with T-SPOT.TB result,but age and BCG immunization were not.

CONCLUSIONS: T-SPOT.TB is comparable to the TST in the diagnosis oftuberculosis disease and identification of high-risk children with tu-berculosis infection and is more specific than the TST in children whohave received the BCG vaccine. Pediatrics 2011;127:e31–e38

AUTHORS: Andrea T. Cruz, MD, MPH,a Abby M. Geltemeyer,MD,b Jeffrey R. Starke, MD,a Jaime A. Flores, MD,b

Edward A. Graviss, PhD, MPH,c and Kim C. Smith, MD,MPHb

aDepartment of Pediatrics, Baylor College of Medicine, Houston,Texas; bDepartment of Pediatrics, University of Texas HealthSciences Center at Houston, Houston, Texas; and cMethodistHospital Research Institute, Center for Molecular andTranslational Human Infectious Diseases Research, Houston,Texas

KEY WORDStuberculosis, children, interferon-�–release assay, T-SPOT.TB

ABBREVIATIONSLTBI—latent tuberculosis infectionTST—tuberculin skin testNTM—nontuberculous mycobacteriaBCG—bacille Calmette-GuérinIGRA—interferon-�–release assayOR—odds ratioCI—confidence interval

This work was presented in part at the International UnionAgainst Tuberculosis and Lung Disease, North American Region;February, 22–24, 2007; Vancouver, British Columbia, Canada.

www.pediatrics.org/cgi/doi/10.1542/peds.2010-1725

doi:10.1542/peds.2010-1725

Accepted for publication Sep 20, 2010

Address correspondence to Andrea T. Cruz, MD, MPH, 6621Fannin St, Suite A210, MC 1-1481, Houston, TX 77030.E-mail: acruz@bcm.edu

PEDIATRICS (ISSN Numbers: Print, 0031-4005; Online, 1098-4275).

Copyright © 2011 by the American Academy of Pediatrics

FINANCIAL DISCLOSURE: Dr Graviss has received travel fundsfrom Cellestis, Ltd, Inc, and research funds and honoraria fromOxford Immunotec, Inc; Drs Flores and Smith were supported inpart by research funding from Oxford Immunotec, Inc; and DrsCruz, Geltemeyer, and Starke have indicated they have nofinancial relationships relevant to this article to disclose.

ARTICLES

PEDIATRICS Volume 127, Number 1, January 2011 e31. Provided by McGill University Library on January 14, 2011 www.pediatrics.orgDownloaded from

At least 9 million people develop tuber-culosis disease each year.1 The mosteffectiveway to prevent future cases ofdisease in nonendemic areas is toidentify and provide chemoprophylaxisto individuals with latent tuberculosisinfection (LTBI).2 An immunocompetentolder child or adult with untreatedLTBI has an �5% to 10% lifetime riskof developing tuberculosis disease,whereas an infant’s risk is as high as40% to 50%; most disease cases occurwithin 12 months of infection.3 It is es-timated that one-third of the globalpopulation4 and 4% of the US popula-tion5 have LTBI.

For over a century, the standard forthe diagnosis of LTBI and for assistingin making the diagnosis of tuberculo-sis disease has been the tuberculinskin test (TST). However, the TST lacksthe desired sensitivity and specificity.The negative predictive value can bediminished by false-negatives seen inyoung children and in immunocompro-mised individuals, with corticosteroidadministration, or in people with over-whelming tuberculosis disease.6 Con-versely, the positive predictive value ofthe TST can be decreased by false-positives caused by exposure to nontu-berculous mycobacteria (NTM)7 andcross-reactivity with the bacilleCalmette-Guérin (BCG) vaccine.8 TheTST is subject to interobserver vari-ability in the measurement of indura-tion,9 potential confusion about differ-ential cutoffs for TST positivity basedon risk factors, and boosting with se-rial TST placements.10

Interferon-�–release assays (IGRAs)detect interferon-� generated by Tcells in response to RD1 antigensfound in Mycobacterium tuberculosis.These blood tests offer several poten-tial advantages over TSTs, includinglack of boosting, 1 patient encounterfor testing (and a telephone call or an-other visit to review the results), uni-form standards for positivity, and lack

of cross-reactivity with theMycobacte-rium bovis–BCG and most NTM.11 Therole of IGRAs for children with LTBI orsuspected tuberculosis disease hasbeen poorly defined. A high proportionof failed test results, in part because ofage and immune status,12 has been de-scribed from some pediatric studies,13

and few studies have evaluated therole of IGRAs in the preschool-agedchild14–16 or in children with suspectedtuberculosis disease.16–19 The goal ofthis studywas to compare the diagnos-tic performance of an IGRA, theT-SPOT.TB, to the TST in children seenat US tuberculosis clinics.

METHODS

Study Population

Children (aged 1 month to 18 years)with LTBI or tuberculosis disease andchildren uninfected with tuberculosisseen in 3 pediatric tuberculosis clinics(Children’s Memorial Hermann, Lyn-don B. Johnson, and Ben Taub Generalhospitals) in Houston, Texas, from2005 to 2006, were recruited prospec-tively. Children on any tuberculosismedication for 2 or more months werenot eligible for enrollment. These clin-ics are the referral sites for local hos-pitals, health departments, and pedia-trician offices in the greater Houstonarea. The caregivers of all childrenwho met inclusion criteria were ap-proached. All patients were cared forby 1 or more clinicians (Drs Smith,Geltemeyer, Starke, and Cruz), whowere blinded to the results of theT-SPOT.TB. Informed consent was ob-tained from the child’s guardian, andassent was obtained for children aged7 years or older. Demographic, clini-cal, radiologic, and bacteriologic data,when applicable, were collected. Insti-tutional review board approval wasobtained from the University of TexasMedical School at Houston and BaylorCollege of Medicine and affiliated hos-pitals before study initiation.

Definitions

Children were classified as being unin-fected or having LTBI or tuberculosisdisease. Uninfected children had nor-mal physical examination, normalchest radiograph, and negative TST re-sults. Infected children were asymp-tomatic and had positive TST,20 normalexamination, and normal radiographresults. TSTs were considered positivefor all children who had results of 15mm or more, 10 mm or more for chil-dren with chronic medical problemsor exposure to people at high risk, and5 mm or more for children with sus-pected disease or who were immuno-compromised or children with identifi-able source cases. A source case was aperson with contagious tuberculosiswho was in contact with the child. BCG-vaccination status was confirmed by im-munization recordorpresenceof a scar.IGRAs were not used to define LTBI.

Children with tuberculosis diseasewas subcategorized as those with con-firmed or clinically diagnosed tubercu-losis. Children with confirmed tuber-culosis had a positive culture orpolymerase chain reaction result forMycobacterium tuberculosis. Clini-cally diagnosed case subjects were de-fined as children without positive my-cobacterial culture results who hadradiographic or clinical findings con-sistent with tuberculosis and at least 1or more of the following: (1) exposureto a known tuberculosis case; (2) apositive TST result (�5mm); or (3) his-topathologic findings compatible withtuberculosis (eg, caseating granulo-mas) and the exclusion of reasonablealternative diagnoses. Risk factors fortuberculosis were stratified as high(having known recent contact with asource case with tuberculosis), inter-mediate (eg, birth in or travel to a high-prevalence country of contact withadults with risk factors for tuberculo-sis),20 or low (no identifiable risk fac-tors). Some children without risk fac-

e32 CRUZ et al. Provided by McGill University Library on January 14, 2011 www.pediatrics.orgDownloaded from

tors were referred because of positiveTST results; for others, referral wasmade for exposure to suspectedsource cases who ultimately had alter-native diagnoses.

Procedures

All children had complete historiestaken and physical examinations per-formed. Chest radiographs were per-formed for children with positive TSTresults, for children with symptomsconsistent with tuberculosis disease,and/or for children with known con-tact with an adult source case withcontagious tuberculosis. Trained clinicor health department personnelplaced and interpreted Mantoux tests.Transverse induration was mea-sured at 48 to 72 hours and inter-preted according to the AmericanThoracic Society criteria.20 In 18 in-stances, the T-SPOT.TB and TST wereperformed simultaneously; for theremaining children, the TST wasplaced months previously. All chil-dren with tuberculosis disease werescreened for HIV.

The commercially available T-SPOT.TBassay (Oxford Immunotec, Oxford,United Kingdom) was performedwithin 5 hours of specimen collectionin the laboratory of 1 of the investiga-tors (Dr Smith) per manufacturer in-structions.21 Briefly, this assay used2 M tuberculosis–specific antigens,early secreted antigenic target 6-kDaprotein (ESAT-6) and culture filtrateprotein 10 (CFP10), to stimulateinterferon-� production inwashed andenumerated peripheral blood mono-nuclear cells; 8 mL of blood was drawnfrom children 10 years old or older and4 mL from children younger than 10years. Peripheral blood mononuclearcells were counted to ensure that astandardized cell number was addedin the assay to control for low T-cellvolumes. General T-cell reactivity wasconfirmed by a positive mitogen con-

trol (phytohemagglutinin). A negativecontrol was used to identify nonspe-cific cell activation. Spots werecounted manually by using a micro-scope and confirmed by using an auto-mated plate counter by the manufac-turer. Assays with 8 or more spotswere considered positive, and assayswith less than 5 spots were consid-ered negative. Borderline results(5–7 spots) were excluded from con-cordance analyses but were ana-lyzed separately. A subgroup analy-sis was performed for specimenswith 6 to 7 spots, because thesespecimens are sometimes consid-ered positive internationally.

Statistical Analysis

Sensitivity was calculated for childrenwith confirmed tuberculosis, concor-dance was assessed by percentageagreement, and outcomes were mod-eled by logistic regression. Invalid(failed) and borderline assays wereexcluded. The level of significance wasset to P � .05. Data were analyzed byusing Stata 10 (Stata, Inc, College Sta-tion, TX).

RESULTS

We were given consent for 215 chil-dren for this study; in 4 cases, veni-puncture was unsuccessful, 10 hadfailed and 7 had borderline T-SPOT.TBresults, and 1 child did not have a TST(Fig 1). Therefore, 193 children re-ceived paired results available for theTST and T-SPOT.TB, and 179 children re-ceived paired results that included TSTmeasurements. Children who receivedborderline or failed T-SPOT.TB resultswere included in the demographic de-scriptions (Table 1) but excluded fromsubsequent concordance analyses.Median age was 8.6 years (range: 1month to 18 years). Thirty-one (15%)children were diagnosed with tubercu-losis disease, 110 (51%) children hadLTBI as defined by their TST result (36of 110 [33%] of whom had recent con-tact with source case), and 74 (34%)children were uninfected (38 of 74[51%] of whom had recent contactwith a source case). Two childrenwerereceiving chemotherapy and 2 chil-dren were receiving long-term cortico-steroids for eosinophilic cystitis andinflammatory bowel disease (without

215 patients enrolled

1 TST not performed4 unsuccessful venipuncture

71 uninfected 31 diseased

13 culture-confirmed TB

108 infectedb

210 evaluable patientsa

18 clinically diagnosed TB

31 not exposed

40 exposedc

FIGURE 1Algorithm for patient inclusion. TB indicates tuberculosis. a Children with failed (10) or borderline (7)T-SPOT.TB results were excluded from subsequent concordance analyses, and 14 children had TSTresults documented simply as “positive” or “negative”; specific induration was unknown. b LTBI wasdefined by a positive TST results. c Exposed to source case with tuberculosis disease.

ARTICLES

PEDIATRICS Volume 127, Number 1, January 2011 e33. Provided by McGill University Library on January 14, 2011 www.pediatrics.orgDownloaded from

the use of tumor necrosis factor-� an-tagonists); no child was known to beinfected with HIV.

Table 2 lists the concordance of the TSTand the T-SPOT.TB assay. Overall con-cordance between assays was 69%. Al-though children who received a posi-tive T-SPOT.TB result tended to havelarger TST measurements, there wassubstantial overlap between TST mea-surements seen in children withT-SPOT.TB–positive and –negative as-says. Concordance between the 2 testswashigher for increasing TST indurationand for childrenwhowere deemed to be

uninfected; concordance was poor forchildren with LTBI who were vaccinatedwith BCG (Table 2) (Fig 2).

Five children younger than 2 years ofage were enrolled after being exposedto a source case. Four of 5 childrenreceived negative TST results, and 1child received an 11-mm result fromthe TST; none received BCG or had pos-itive T-SPOT.TB assay results. Fifty-fivechildren aged 24 to 59 months were en-rolled, and concordance between thetests was 79% (Table 2). These differ-ences were not statistically differentfrom concordance in older age groups.

Failed T-SPOT.TB results were ob-served in 10 children: inadequatemito-gen response in the positive controlsubjects (n � 7) and a high back-ground in the negative control well(n� 3). Seven children received posi-tive TST results; 1 child with clinicallydiagnosed tuberculosis disease hadfailed the T-SPOT.TB assay and had anegative TST result. Patients withfailed assays were younger than theremainder of the study population(4.7 vs 8.7 years; P � .02). Seven chil-dren received borderline (5–7 spots)T-SPOT.TB results; all received positiveTST results, and 5 of 7 children werevaccinated with BCG. There were nodifferences in age, immunocompe-tence, or disease status between chil-dren who received borderline resultsand the rest of the study population.The manufacturer recommendation isto repeat the T-SPOT.TB for childrenwho received borderline results, butinternationally, 6 to 7 spots often areconsidered a positive result. When thelatter cutoff (�6 spots) was used, con-cordance between the TST and theT-SPOT.TB was 69.7%.

Correlation Between T-SPOT.TB andthe TST for Children WithTuberculosis Disease

Thirteen (42%) children were diag-nosed with confirmed tuberculosisdisease; 18 (58%) children were clini-cally diagnosed with tuberculosis dis-ease (Table 3). Twenty children werediagnosed with intrathoracic disease,and 11 (35%) children were diagnosedwith extrathoracic disease (7 childrenwere diagnosed with lymphadenopa-thy, 2 with meningitis, and 2 with skel-etal disease). Using T-SPOT.TB and TSTtogether would have given a sensitivityof 100% for children diagnosed withconfirmed tuberculosis and 94% forchildren with clinically diagnosed dis-ease. Concordance between the TSTand T-SPOT.TB in children who werenot immunized with BCG and children

TABLE 1 Demographic Characteristics of the Study Population (N� 215)

Characteristic Uninfected(N� 74), n

Infected(N� 110), na

Disease(N� 31), n

Age0–23 mo 4 1 024–59 mo 31 26 860 mo to�10 y 16 35 6�10 y 23 48 17

GenderMale 36 54 14Female 38 56 17Race/ethnicityHispanic 48 67 20Non-Hispanic black 14 22 2Non-Hispanic white 9 10 4Asian 1 5 3Middle Eastern/Indian 2 2 0Other 0 4 2BirthplaceLow-prevalence regions 62 59 21United States 62 58 21England 0 1 0High-prevalence regions 12 51 10Latin America 11 41 8Asia 1 3 2Africa 0 4 0Middle East/India 0 3 0BCG statusVaccinated 12 56 10Not vaccinated 62 54 21Tuberculosis risk factorsKnown source case 39 36 11Household contact 21 19 7Nonresident family contact 10 9 2Nonfamily contact 8 8 2Immunosuppression 2 2 0TST resultsPositive 0 110 26Negative 74 0 5T-SPOT.TB resultPositive 0 47 23Negative 72 56 7Failed 2 7 1

Numbers here differ from those in subsequent tables, because these figures are for the entire study population, whereassubsequent tables may exclude some children with uninterpretable results.a Definitions of tuberculosis infected and tuberculosis uninfected were based on TST results.

e34 CRUZ et al. Provided by McGill University Library on January 14, 2011 www.pediatrics.orgDownloaded from

who were immunized with BCG was63% and 80%, respectively, for chil-dren with confirmed disease and 50%

and 80%, respectively, for childrenwith clinically diagnosed tuberculosis(Fig 2). Nine of 11 children with ex-

trapulmonary disease had positive TSTresults, and 8 children had positiveT-SPOT.TB assays. Both children withmeningitis had positive T-SPOT.TB as-says; 1 of 2 had positive TST results.

One patient with confirmed tuberculo-sis, an immunocompetent 18-year-oldwith septic arthritis and a normalchest radiograph, had a negativeT-SPOT.TB assay; his TST result was 10mm. An 11-year-old girl with pulmo-nary infiltrates and clinically diag-nosed tuberculosis had a failedT-SPOT.TB assay and a 0-mm TST result.Six children (mean age: 3.7 years)treated for clinically diagnosed tuber-culosis received negative T-SPOT.TBassays; all had TST results of 10 mmor more. Three of 6 children wouldnot have met the definition for clini-cally diagnosed tuberculosis had theT-SPOT.TB been used instead of the TST.

Correlation Between T-SPOT.TB andthe TST Based on the Level of Riskfor Infected and UninfectedChildren

Of 163 uninfected or infected childrenwith evaluable results, 71 were at highrisk for LTBI because of contact with aknown source case; of these children,34 had positive TST results and 30 hadpositive T-SPOT.TB assays. Concor-

TABLE 2 T-SPOT.TB Assay Results Stratified According to TST Size and Demographic Factors for 193Patients for Whom T-SPOT.TB Assays Were Interpretable

Characteristic Total, n TST�, n TSPOT.TB�, n Concordance, %

TST result0–4 mm 72 0 4 945–9 mm 2 1 1 10010–14 mm 45 45 13 29� 15 mm 60 60 42 68TST documented as positive 14 14 9 62Age0–23 mo 5 1 0 8024–59 mo 55 26 17 805 years to� 10 y 50 34 21 68� 10 y 83 59 31 63GenderMale 94 55 30 68Female 99 65 39 71Race/ethnicityHispanic 119 73 44 74Non-Hispanic black 35 22 11 69Non-Hispanic white 22 11 5 45Asian 8 7 6 88Middle Eastern/Indian 3 1 0 67Other 6 6 3 50Country of originHigh prevalence 58 48 21 53Low prevalence 135 72 48 76BCG vaccinationYes 66 55 20 47No 127 65 49 81Disease classUninfected 69 0 0 100Infected 94 94 46 46Disease 30 26 23 63Overall 193 120 69 69

Concordance analyses did not include patients with unavailable (n � 5), failed (n � 10), or borderline (n � 7) TST orT-SPOT.TB assay results.

0

10

20

30

40

50

60

70

80

90

100

Confirmeddisease

Clinicallydiagnosed

High risk Intermediaterisk

Low risk

% o

f p

atie

nts

Not BCG vaccinated - TST–positive

Not BCG vaccinated - T-SPOT.TB–positive

BCG vaccinated - TST–positive

BCG vaccinated - T-SPOT.TB–positive

FIGURE 2Performance of the T-SPOT.TB and TST stratified according to risk group and BCG-vaccination status.

ARTICLES

PEDIATRICS Volume 127, Number 1, January 2011 e35. Provided by McGill University Library on January 14, 2011 www.pediatrics.orgDownloaded from

dance for children who were not im-munized with BCG and children whowere immunized with BCG was 95%and 88%, respectively. Seventy chil-dren were at intermediate risk forLTBI. Fourteen children had positiveT-SPOT.TB assay results and 51 chil-dren had positive TST results. Concor-dance was 69% and 34% for childrenwho were not immunized with BCG andchildren who were immunized withBCG, respectively. Twenty-two childrenhad no documented risk factors for tu-berculosis. Of these children, 2 hadpositive T-SPOT.TB assay results and 9had positive TST results. Concordancewas 76% and 20% in children whowere not immunized with BCG and chil-dren who were immunized with BCG,respectively. This poor concordance inthe population of children who wereimmunized with BCG was across chil-dren who had low and intermediaterisk factors (Fig 2).

Predictors of T-SPOT.TB and TSTResults

BCG vaccination (OR [odds ratio]: 4.77[95% confidence interval (CI): 2.29–9.95), birth in high-prevalence nations(OR: 4.20 [95% CI: 1.96–8.99]), and pa-tient age (OR: 1.08 [95% CI: 1.02–1.14])were associated with a positive TST re-sult, whereas contact with an identifi-

able source case (OR: 0.48 [95% CI:0.26–0.91]) was not associated with apositive TST result. Only BCG vaccina-tion was independently associatedwith a TST result on multivariate anal-ysis (adjusted OR: 4.32 [95% CI: 1.02–18.35]). In univariate analyses wefound an association between an iden-tifiable source case with a T-SPOT.TBresult (OR: 3.52 [95% CI: 1.72–7.20]),whereas BCG vaccination (OR: 0.69 [95%CI: 0.37–1.31]), birth in high-prevalencenations (OR: 1.03 [95% CI: 0.54–1.95]),and patient age (OR: 1.02 [95% CI: 0.97–1.08]) were not associated withT-SPOT.TB results. Only contact with asource case was associated withT-SPOT.TB result on multivariate analy-ses (adjusted OR: 4.41 [95% CI:1.78–10.94]).

DISCUSSION

In this study we assessed the perfor-mance of a commercially availableIGRA in comparison to the TST in a pe-diatric population stratified accordingto epidemiologic risk factors and BCGreceipt. The T-SPOT.TB was compara-ble to the TST for children with tuber-culosis disease and children at highrisk for LTBI. For children at low riskfor LTBI, the T-SPOT.TB was more spe-cific than the TST. However, neithertest can rule out tuberculosis disease

or discriminate between tuberculosisdisease and infection. The 2 tests cor-related well for children at the ex-tremes of TST induration (�5 and�15mm) but showed poor concordancefor the group of children who had 10-to 14-mm TST results.

Previous studies have shown IGRAsensitivities in microbiologically con-firmed disease to be more than 80%for children,15,17,22 whereas IGRA sensi-tivities for adults with tuberculosis dis-ease have ranged from 55% to 100%.23

For children with clinically diagnosedtuberculosis disease, the TST tended tobe more sensitive than the T-SPOT.TB,which might, in part, be because somechildren only met the definition of clin-ically diagnosed tuberculosis diseasebecause the TST was part of the diag-nostic criteria. For children with sus-pected tuberculosis disease, the use ofboth tests would have an excellentcombined sensitivity. Clinically, if boththe TST and T-SPOT.TB results werenegative, tuberculosis would not beruled out, but it would be unlikely.

For children with identifiable contactwith an adult with contagious tubercu-losis, the T-SPOT.TB and the TST per-formed comparably in a population ofchildren who were well nourishedand not infected with HIV. In this high-risk population, especially childrenyounger than 5 years with a high riskof progression to tuberculosis dis-ease, sensitivity should be optimized atthe expense of specificity. However,indeterminate IGRA results also arehighest in this population. Some datahave shown that a positive IGRA resultwas more predictive than the TST inidentifying children at risk for pro-gressing to tuberculosis disease,24

which perhaps enables more specifictargeting of chemoprophylaxis. In ourstudy, there was an association be-tween contact with a source case anda positive T-SPOT.TB result. Lighter etal25 demonstrated that 1 IGRA, the

TABLE 3 Concordance and Sensitivity of TST and T-SPOT.TB Assays for Children WithMicrobiologically Confirmed (n� 13) and Clinically Diagnosed (n� 18) TuberculosisDisease

Disease Type/Result T-SPOT.TB Result Concordance, % Sensitivity

� � Total TST, % T-SPOT.TB, % P

Confirmeda 69 77 92 .59TST� 9 1 10TST� 3 0 3Total 12 1 13Clinically diagnosedb 63 94 65 .09TST� 10 6 16TST� 1 0 1Total 11 6 17Overall 63 87 77 .51TST� 19 7 26TST� 4 0 4Total 23 7 30

One child with clinically diagnosed tuberculosis had a negative TST and a failed T-SPOT.TB (failure of positive control) result.a Disease sites: 7 pulmonary, 2 meningeal, 2 skeletal, and 2 peripheral lymphadenopathy.b Disease sites: 12 pulmonary, 5 peripheral lymphadenopathy, and 1 pleural.

e36 CRUZ et al. Provided by McGill University Library on January 14, 2011 www.pediatrics.orgDownloaded from

QuantiFERON-TB Gold In-Tube assay,was specific for exposure in childrenfrom low-prevalence regions,25 andsome data from adults have shownthat IGRAs correlate more closely withdegree of exposure than the TST.26 Bothstudies reflect that, even for childrenat high risk, some positive TST resultsare likely because of either exposureto NTM species or BCG vaccination.However, it would be reasonable totreat for LTBI if test results for eitherare positive for children at high risk.27

For patients at intermediate risk(those who have tuberculosis risk fac-tors but no identifiable source case),T-SPOT.TB enables examination of therole of the BCG vaccine in children withpositive TST results.28 Most childrenwith intermediate risk factors in ourcohort were born abroad and receivedthe BCG vaccine. However, large stud-ies have shown that the impact of BCGimmunization during the neonatal pe-riod has minimal impact on TST sensi-tivity, especially 10 or more years afterimmunization.29,30 However, many chil-dren born abroad have TSTs beforeschool entry when only a few yearshave elapsed since BCG receipt. On thebasis of TST results alone, the majorityof children who were immunized withBCG and who were at intermediate riskwould have been treated for LTBI; two-thirds had a negative T-SPOT.TB result.For the school-aged BCG recipient at lowrisk for progression to disease, the ben-efit from chemoprophylaxis probably islow. In this population, the IGRA shouldbe given preference over the skin test,and consideration could be given to of-fering chemoprophylaxis only to chil-dren whose IGRA results are positive.27

For children at low risk (who should betested by neither TST nor IGRA), the en-hanced specificity of IGRAs can be usedto prevent unnecessary treatment forLTBI. In this population of children whohave not received the BCG vaccine, pos-itive TST results likely represent false-

positives after exposure to NTM spe-cies. Historically, the cutoff for TSTpositivity (�15 mm) in people withouttuberculosis risk factors20 was used todecrease the number of false-positivesfrom NTM species. However, study re-sults have indicated that exposure toNTMspecies can result in large TST indu-rations.7 The most recent study31 of NTMsensitization revealed that 17% of the USpopulation was sensitized toMycobacte-rium avium intracellulare; in contrast,only 4% of the US population had evi-dence of LTBI.5,32 In low-risk populations,optimizing specificity is crucial.

Failed IGRA results have been previ-ously reported in previous pediatricstudies to be as high as 17%13,14 andhave been inversely correlated withpatient age.12,33 Failed T-SPOT.TB re-sults were less frequent (5%) in thisstudy. In part, this may have been be-cause children younger than 2 yearswere infrequently enrolled. Previousstudies have shown that failed resultswere more common with enzyme-linked immunosorbent assays thanwith enzyme-linked immunosorbentspot–based tests.14 Borderline resultsalso were uncommon (3%) in ourstudy, and test performance did notchange when patients with borderlineresults were excluded or whose testresults were considered positive.

This study has limitations. One of thegreatest barriers to evaluation of IGRAperformance is the lack of a referencestandard for the diagnosis of LTBI;therefore, performance is best evalu-ated for children with culture-confirmed tuberculosis, a minority ofall children treated for tuberculosisdisease. Selection bias is possible, be-cause many of the children seen at ourclinics were identified via contact in-vestigations and were at high risk forLTBI, which could lead to overestima-tion of concordance between TST andT-SPOT.TB assays thatmight not be rep-licated in lower-risk settings. However,

this overestimation reflects the realityof active surveillance for tuberculosisseen in pediatric tuberculosis clinicsin industrialized nations. The exactamount of contact with source caseswas hard to quantify, which could po-tentially lead to classification bias.However, we did have the results of ahealth department contact investiga-tion and microbiologic results for thesource case. The study population didnot include large numbers of childrenin demographic categories that havebeen least studied (immunocompro-mised hosts, the youngest children[and most children at high risk], andchildren with microbiologically proventuberculosis disease). A characteristicscar was sometimes used as an indi-cator of BCG receipt when immuniza-tion historieswere unavailable. Becauseas many as 5% to 15% of children maynot develop a scar after BCG immuniza-tion,34 this may have led to classificationbias. For most children, IGRA followedthe TST, and boosting was possible; thisboosting has been described as early as3 days after TST,35 but the duration ofboosting is unclear.

CONCLUSIONS

The T-SPOT.TB assay was comparableto the TST for the identification of chil-dren with microbiologically confirmedtuberculosis disease and children athigh risk with LTBI. T-SPOT.TB wasmore specific than the TST for childrenwho were immunized with BCG andwho were at intermediate risk for LTBI,a population for which optimizingspecificity can decrease the need forunnecessary interventions.

ACKNOWLEDGMENTSFunding was obtained from Oxford Im-munotec, Inc, to defray the personnelcost of performing the assays and toreimburse families for time and travel.The investigators retained control ofstudy methodology, data collectionand analysis, and publication.

ARTICLES

PEDIATRICS Volume 127, Number 1, January 2011 e37. Provided by McGill University Library on January 14, 2011 www.pediatrics.orgDownloaded from

REFERENCES

1. World Health Organization. Global tubercu-losis control: 2009 report. Available at:http://whqlibdoc.who.int/publications/2009/9789241598866_eng.pdf. AccessedMarch 16, 2010

2. Broekmans JF, Migliori GB, Rieder HL, et al.European framework for tuberculosis con-trol and elimination in countries with a lowincidence: recommendations of the WorldHealth Organization (WHO), InternationalUnion Against Tuberculosis and Lung Dis-ease (IUATLD), and Royal Netherlands Tu-berculosis Association (KNCV) WorkingGroup. Eur Respir J. 2002;19(4):765–775

3. Marais BJ, Gie RP, Schaaf HS, Beyers N,Donald PR, Starke JR. Childhood pulmonarytuberculosis: old wisdom and new chal-lenges. Am J Respir Crit Care Med. 2006;173(10):1078–1090

4. Dye C, Scheele S, Dolin P, Pathania V, Ravigli-one MC. Consensus statement: global bur-den of tuberculosis: estimated incidence,prevalence, and mortality by country: WHOGlobal Surveillance and Monitoring Project.JAMA. 1999;282(7):677–686

5. Bennett DE, Courval JM, Onorato I, et al.Prevalence of tuberculosis infection in theUnited States population: the nationalhealth and nutrition examination survey,1999–2000. Am J Respir Crit Care Med.2008;177(3):348–355

6. Huebner RE, Schein MF, Bass JB Jr. The tu-berculin skin test. Clin Infect Dis. 1993;17(6):968–975

7. Haimi-Cohen Y, Zeharia A, Mimouni M,Soukhman M, Amir J. Skin indurations inresponse to tuberculin testing in patientswith nontuberculous mycobacterial lymph-adenitis. Clin Infect Dis. 2001;33(10):1786–1788

8. Wang L, Turner MO, Elwood RK, Schulzer M,FitzGerald JM. A meta-analysis of the effectof bacille Calmette Guerin vaccination ontuberculin skin testmeasurements. Thorax.2002;57(9):804–809

9. Pouchot J, Grasland A, Collet C, Coste J, Es-daile JM, Vinceneux P. Reliability of tubercu-lin skin test measurement. Ann Intern Med.1997;126(3):210–214

10. Menzies D. Interpretation of repeated tuber-culin tests: boosting, conversion, and rever-sion. Am J Respir Crit Care Med. 1999;159(1):15–21

11. Lewinsohn DA. Embracing interferon-gamma release assays for diagnosis of la-tent tuberculosis infection. Pediatr InfectDis J. 2009;28(8):674–675

12. Haustein T, Ridout DA, Hartley JC, et al. Thelikelihood of an indeterminate test result

from awhole-blood interferon-� release as-say for the diagnosis of Mycobacterium tu-berculosis infection in children correlateswith age and immune status. Pediatr InfectDis J. 2009;28(8):669–673

13. Connell TG, Curtis N, Ranganathan SC, But-tery JP. Performance of awhole-blood inter-feron gamma assay for detecting latent in-fection withMycobacterium tuberculosis inchildren. Thorax. 2006;61(7):616–620

14. Bergamini BM, Losi M, Valenti F, et al. Per-formance of commercial blood tests for thediagnosis of latent tuberculosis infection inchildren and adolescents. Pediatrics. 2009;123(3). Available at: http://www.pediatrics.org/cgi/content/full/123/3/e419

15. Bianchi L, Galli L, Moriondo M, et al.Interferon-gamma release assay improvesthe diagnosis of tuberculosis in children.Pediatr Infect Dis J. 2009;28(6):510–514

16. Connell TG, Ritz N, Paxon GA, et al. A three-way comparison of tuberculin skin testing,QuantiFERON-TB Gold, and T-SPOT.TB in chil-dren. PLoS One. 2008;3(7):e2624

17. Detjen AK, Keil T, Roll S, et al. Interferon-�release assays improve the diagnosis of tu-berculosis and nontuberculous mycobacte-rial disease in children in a country with alow incidence of tuberculosis. Clin InfectDis. 2007;45(3):322–328

18. Connell T, Bar-Zeev N, Curtis N. Early detec-tion of perinatal tuberculosis using awhole-blood interferon-� release assay. Clin InfectDis. 2006;42(11):e82–e85

19. Dewan PK, Grinsdale J, Kawamura LM. Lowsensitivity of a whole-blood interferon-� re-lease assay for detection of active tubercu-losis. Clin Infect Dis. 2007;44(1):69–73

20. American Thoracic Society. Targeted tuber-culin testing and treatment of latent tuber-culosis infection. MMWR Recomm Rep.2000;49(RR-6):1–51

21. Oxford Immunotec. T-SPOT.TB [package insert].Available at: http://www.oxfordimmunotec.com/eu/downloads/PI-TB96-IVD-UK-V8,%20English.pdf. AccessedMay 18, 2010

22. Liebeschuetz S, Bamber S, Ewer K, Deeks J,Pathan AA, Lalvani A. Diagnosis of tubercu-losis in South African children with a T-cell-based assay: a prospective cohort study.Lancet. 2004;364(9452):2196–2203

23. Pai M, Zwerling A, Menzies D. Systematicreview: T-cell-based assays for the diagno-sis of latent-tuberculosis infection: an up-date. Ann Intern Med. 2008;149(3):177–184

24. Bakir M, Millington KA, Soysal A, et al. Prog-nostic value of a T-cell-based, interferon-

gammabiomarker in childrenwith tubercu-losis. Ann InternMed. 2008;149(11):777–787

25. Lighter J, Rigaud M, Eduardo R, Peng C-H,Pollack H. Latent tuberculosis diagnosis inchildren by using the QuantiFERON-TB GoldIn-Tube Test. Pediatrics. 2009;123(1):30–37

26. Arend SM, Thijsen SF, Leyten EM, et al. Com-parison of two interferon-gamma releaseassays and tuberculin skin test for tracingTB contacts. Am J Respir Crit Care Med.2007;175(6):618–627

27. Mazurek M, Jereb J, Vernon A, LoBue P,Goldberg S, Castro K; IGRA Expert Commit-tee, Centers for Disease Control and Preven-tion. Updated guidelines for using inter-feron gamma release assays to detectMycobacterium tuberculosis infection:United States, 2010. MMWR Recomm Rep.2010;59(RR-5):1–25

28. Lewinsohn DA, Lobato MN, Jereb JA.Interferon-� release assays: new diagnostictests forMycobacterium tuberculosis infec-tion, and their use in children. Curr OpinPediatr. 2010;22(1):71–76

29. Farhat M, Greenaway C, Pai M, Menzies D.False-positive tuberculin skin tests: what isthe absolute effect of BCG and non-tuberculous mycobacteria? Int J TubercLung Dis. 2006;10(11):1192–1204

30. Sleiman R, Al-Tannir M, Dakdouki G, Ziade F,Assi NA, Rajab M. Interpretation of the tu-berculin skin test in bacille Calmette-Guérinvaccinated and nonvaccinated schoolchil-dren. Pediatr Infect Dis J. 2007;26(2):134–138

31. Khan K, Wang J, Marras TK. Nontuberculousmycobacterial sensitization in the UnitedStates: national trends over three decades.Am J Respir Crit Care Med. 2007;176(3):306–313

32. Khan K, Wang J, Hu W, Bierman A, Li Y, Gar-dam M. Tuberculosis infection in the UnitedStates: national trends over three decades.Am J Respir Crit Care Med. 2008;177(4):455–460

33. Connell TG, Tebruegge M, Ritz N, et al. Inde-terminate interferon-gamma release assayresults in children. Pediatr Infect Dis J.2010;29(3):285–286

34. Young TK, Mirdad S. Determinants of tuber-culin sensitivity in a child population cov-ered by mass BCG vaccination. Tuber LungDis. 1992;73(2):94–100

35. van Zyl-Smit RN, Zwerling A, Dheda K, Pai M.Within-subject variability of interferon-gamma assay results for tuberculosis andboosting effect of tuberculin skin testing: asystematic review. PLoS One. 2009;4(12):e8517

e38 CRUZ et al. Provided by McGill University Library on January 14, 2011 www.pediatrics.orgDownloaded from

DOI: 10.1542/peds.2010-1725 2011;127;e31-e38; originally published online Dec 6, 2010; Pediatrics

Graviss and Kim C. Smith Andrea T. Cruz, Abby M. Geltemeyer, Jeffrey R. Starke, Jaime A. Flores, Edward A.

Blood Test in ChildrenTBComparing the Tuberculin Skin Test and T-SPOT.

& ServicesUpdated Information

http://www.pediatrics.org/cgi/content/full/127/1/e31including high-resolution figures, can be found at:

References

http://www.pediatrics.org/cgi/content/full/127/1/e31#BIBLat: This article cites 32 articles, 18 of which you can access for free

Subspecialty Collections

http://www.pediatrics.org/cgi/collection/infectious_disease Infectious Disease & Immunity

following collection(s): This article, along with others on similar topics, appears in the

Permissions & Licensing

http://www.pediatrics.org/misc/Permissions.shtmltables) or in its entirety can be found online at: Information about reproducing this article in parts (figures,

Reprints http://www.pediatrics.org/misc/reprints.shtml

Information about ordering reprints can be found online:

. Provided by McGill University Library on January 14, 2011 www.pediatrics.orgDownloaded from