The Pediatric Infectious Disease Journal Publish Ahead of ... of Tuberculin Skin... · Affiliations: aMalalties Infeccioses i Resposta Inflamatòria Sistèmica en Pediatria. Unitat

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The Pediatric Infectious Disease Journal Publish Ahead of Print

DOI: 10.1097/INF.0000000000002015

Performance of Tuberculin Skin Tests and Interferon-γ Release Assays in

Children Younger Than 5 years

Eneritz Velasco-Arnaiza,b

, MD, Antoni Soriano-Arandesc, MD, PhD, Irene Latorre

d,

PhD, Neus Altetc, MD, PhD, José Domínguez

d, PhD, Clàudia Fortuny

a,b,e,f, MD, PhD,

Manuel Monsonísg, MD,

Marc Tebruegge

h,i,j,k*, MD, MRCPCH, DTM&H, MSc, PhD,

and Antoni Noguera-Juliana,b,e,

f*, MD, PhD

Address correspondence to (and address for reprints): Antoni NOGUERA-

JULIAN, MD, PhD, Passeig de Sant Joan de Déu 2, 08950 Esplugues de Llobregat,

Barcelona (Spain), e-mail address: [email protected], Phone number: +34

93 280 40 00; fax number: +34 93 203 39 59

Abbreviated title: TST and IGRA in Children Younger Than 5 Years

Running head: TST and IGRA in Young Children

Affiliations: aMalalties Infeccioses i Resposta Inflamatòria Sistèmica en Pediatria.

Unitat d´Infeccions, Servei de Pediatria. Institut de Recerca Pediàtrica Hospital Sant

Joan de Déu; Barcelona, Spain. bDepartament de Pediatria, Universitat de Barcelona;

Barcelona, Spain. cInstitut Català de la Salut. Unitat de Tuberculosi Hospital

Universitari Vall d’Hebrón-Drassanes. Barcelona, Spain. dInstitut d’Investigació

Germans Trias i Pujol. CIBER Enfermedades Respiratorias. Universitat Autònoma de

Barcelona. Badalona, Spain. eCIBER de Epidemiología y Salud Pública (Ciberesp,

Spain). fRed de Investigación Transalacional en Infectología Pediátrica (RITIP, Spain).

gServei de Microbiologia. Hospital Sant Joan de Déu. Barcelona, Spain.

hAcademic Unit

of Clinical and Experimental Sciences, Faculty of Medicine & Global Health Research

Institute, University of Southampton, Southampton, United Kingdom. iDepartment of

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Copyright © 2018 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.

mailto:[email protected]

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Paediatric Infectious Diseases & Immunology, Evelina London Children’s Hospital,

Guy’s and St. Thomas’ NHS Foundation Trust, London, UK, United Kingdom. jGreat

Ormond Street Hospital Institute of Child Health, University College London, London,

UK. kDepartment of Paediatrics, The University of Melbourne, Parkville, Australia.

*Both authors share credit for senior authorship.

Conflicts of Interest and Source of Funding:

Dr. Tebruegge has received QuantiFERON-TB Gold assays at reduced cost for related

research projects from the manufacturer (Cellestis/Qiagen). The manufacturer had no

influence on the study design, data interpretation, writing of the manuscript or decision

to submit the data for publication. The remaining authors have no potential conflicts of

interest to disclose.

Dr. Domínguez is funded by the Miguel Servet program of the Instituto de Salud Carlos

III (Spain). This research was partially supported by different grants from the Instituto

de Salud Carlos III (PI 13/01546, PI 13/01740 and ICI14/00228), integrated in the Plan

Nacional de I+D+I and co-funded by the ISCIII-Subdirección General de Evaluación

and the Fondo Europeo de Desarrollo Regional (FEDER). Dr. Tebruegge was supported

by a Clinical Lectureship provided by the UK National Institute for Health Research and

funding provided by the Technology Strategy Board/Innovate U.K.

Key words: Bacillus Calmette-Guérin vaccine; infant; latent tuberculosis infection;

non-tuberculous mycobacteria; tuberculosis

Acknowledgements

The authors thank the patients and their families for kindly agreeing to take part in this

study.

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ABSTRACT

Background :Available data to assess the optimal diagnostic approach in infants and

pre-school children at risk of tuberculosis (TB) are limited.

Methods: We conducted a prospective observational study in children younger than 5

years undergoing assessment with both tuberculin skin tests (TST) and QuantiFERON-

TB Gold In-Tube® (QFT-GIT) assays at two tertiary TB Units in Barcelona, Spain.

Results: 383 children were included. One of 304 participants considered uninfected

developed active TB during follow-up (median [IQR]: 47 [30;48] months), compared

with none of 40 participants with latent TB infection (follow-up since completion of

anti-TB treatment: 42 [32;45] months). Overall test agreement between TST and QFT-

GIT was moderate (κ=0.551), but very good in children screened after TB contact

(κ=0.801) and in BCG-unvaccinated children (κ=0.816). Discordant results (16.8%, all

TST+/QFT-GIT negative) were mainly observed in new-entrant screening and in BCG-

vaccinated children. Children with indeterminate QFT-GIT results were on average

younger than those with determinate results (median age: 12 versus 30 months;

p<0.001). The sensitivity of TSTs and QFT-GIT assays in children with confirmed

active TB was 100% (95%CI: 79.4-100%) and 93.7% (95%CI: 69.8-99.8%),

respectively. In patients with latent TB infection or active TB there was no correlation

between age and antigen-stimulated interferon-gamma responses (r=-0.044, p=0.714).

Conclusions: In young BCG-unvaccinated children with recent TB contact a dual

testing strategy using TST and QFT-GIT in parallel may not be necessary. However,

TST+/QFT-GIT negative discordance is common, and it remains uncertain if this

constellation indicates TB infection or not. In active TB, QFT-GIT assays do not

perform better than TSTs.

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Introduction

Children younger than 5 years infected with Mycobacterium tuberculosis (MTB) are at

greater risk of progression to active tuberculosis (TB) and developing severe and

disseminated forms of TB than are adults. In infants the rate of progression is up to 40-

50% in the first 2 years after primary infection.1-3

In the absence of a gold standard, the diagnosis of latent tuberculosis infection (LTBI)

in infants and toddlers remains challenging, because of the limitations of current

immune-based diagnostic tests, the in vivo tuberculin skin test (TST) and ex vivo

interferon-gamma (IFN-γ) release assays (IGRAs).4,5

Moreover, the non-specific

clinical presentation of active TB and the comparatively low diagnostic yield of

microbiological investigations in this age group commonly result in delays in

establishing the diagnosis of active TB.2,6

Commercially available IGRAs detect circulating T-cells that produce IFN-γ in

response to stimulation with MTB-specific antigens that are absent from all Bacillus

Calmette-Guérin (BCG) vaccine strains and from most non-tuberculous mycobacteria

(NTM).3,7

Both TSTs and IGRAs indicate host sensitization to mycobacterial antigens

by detecting cell-mediated immune responses, which are critical in preventing

progression to active TB.8 The physiological immaturity of the immune system of

infants and toddlers not only results in a reduced ability to contain MTB infection (i.e.,

prevent progression),9 but may also result in impaired diagnostic accuracy of TSTs and

IGRAs.10,11

In many industrialized countries IGRAs have largely replaced TSTs as the main TB

screening tool in adults.12-14

The existing evidence regarding the optimal diagnostic

approach for children younger than 5 years at risk of LTBI is limited. Recent U.S.,

Canadian and European guidelines recommend the preferential use of TSTs irrespective

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of BCG vaccination status in this age group, and consider IGRAs as a complementary

tool to improve sensitivity and specificity.3,10,12-14

The Spanish Society of Pediatric

Infectious Diseases also recommends using TSTs as the first line investigation in

children younger than 5 years after TB contact.15

In those with a negative TST result,

performing an IGRA test is recommended. In patients with a positive TST result, IGRA

testing is considered unnecessary unless there is a history of prior BCG vaccination.

Recent shortages of purified protein derivative (the test substance used for TSTs) have

led to changes in TB screening practices, increasing the need for more robust data on

IGRAs in young children.16

The aim of this study was to evaluate the performance of TSTs and QuantiFERON-TB

Gold In-Tube (QFT-GIT) assays in the diagnostic evaluation of LTBI and active TB in

previously healthy children younger than 5 years.

Materials and methods

Study population

We performed a prospective observational study of children younger than 5 years at risk

of TB undergoing assessment at one of two tertiary Pediatric TB Units, the Drassanes-

Vall d’Hebrón (DVH) Unit in Barcelona City or the Hospital Sant Joan de Déu (SJD) in

Regió Sanitària Barcelona Sud. Both units are located in Catalonia, Spain and are

jointly covering a population of more than 2,9 million inhabitants, of which 15.8% are

younger than 15 years.17

In Catalonia, the incidence of TB gradually decreased from

21.6/100,000 in 2004 to 16.6/100,000 in 2014. In 2014 the TB incidence in children

aged 0 to 4 years was 12.4/100,000.17

BCG vaccination is not part of the routine

immunization program in Catalonia.

Children were eligible for participation if they were assessed for LTBI (either in the

context of contact tracing or as part of new entrant screening) or were investigated for

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suspected active TB, and underwent both TST and QFT-GIT testing simultaneously.

Children with a previous diagnosis of LTBI or active TB, chronic medical conditions

(including HIV infection) and/or receiving immunosuppressive therapy (including

corticosteroids) were excluded from participation. The study commenced at DVH in

January 2005 and at SJD in February 2012, and concluded in July 2015. The study was

approved by the institutional ethics review boards of both units. Written informed

consent was obtained from the parents/guardians of each participant.

Demographic characteristics (age, gender, country of birth, family origin, BCG

vaccination history and presence of BCG scar) were recorded on a standardized data

collection sheet. Participants were classified as BCG-vaccinated if they had a visible

scar in the deltoid region and/or a positive vaccination history. In addition, the primary

reason for performing the assessment was categorized as follows: a) clinical and/or

radiological suspicion of active TB, b) contact tracing following contact with a TB

index case, or c) new-entrant screening in immigrants and international adoptees from

high TB prevalence countries.

TST and IGRA testing

TSTs were performed by intradermal injection of 0.1 ml (2TU) of purified protein

derivative RT23 (Tuberkulin PPD RT23 SSI, Statens Serum Institut, Copenhagen,

Denmark), with results read after 48-72 hours. The cut-offs for a positive TST result

were defined according to national guidelines as follows: ≥5 mm of induration in

children assessed for clinically and/or radiologically suspected active TB and in children

assessed following TB contact, and ≥10 mm in children undergoing new-entrant

screening.15

QFT-GIT (Cellestis/Qiagen, Carnegie, Australia) assays were performed in

a fully-accredited routine diagnostic laboratory at each centre and interpreted according

to manufacturer's instructions.18

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Case definitions and follow-up

The diagnosis of active TB was made on the basis of epidemiological, clinical,

radiological and microbiological findings according to consensus criteria described

elsewhere,19

independent of TST and QFT-GIT results. Active TB cases were classified

as a) definite, microbiologically-confirmed cases when MTB was detected by culture

(Lowenstein-Jensen or liquid Middlebrook 7H9+OADC media) or molecular analysis

(AMPLICOR MTB-Test, Roche Molecular Systems, NJ, U.S.), and b) probable, non-

microbiologically-confirmed cases.

In children assessed following contact with a TB index case, LTBI was defined as the

absence of clinical and radiological signs of active TB in combination with a positive

TST and/or QFT-GIT result, independent of the BCG vaccination status.15

In accordance with national guidelines, in BCG-vaccinated children undergoing new-

entrant screening a positive TST result in combination with a negative QFT-GIT result

was interpreted as being the result of BCG vaccination, and the child was considered

uninfected. In the absence of BCG vaccination, a positive TST and/or QFT-GIT result

was considered to be evidence for LTBI.15

In both units children who undergo TB screening are routinely followed up clinically

every 3 to 6 months for at least 2 years. Repeat TB testing or radiological investigations

are not done routinely, but are performed if patients develop symptoms or signs

suggestive of active TB.

Statistical methods

Statistical analyses were carried out using SPSS version 21.0 (IBM Corp., Armonk, NY,

U.S.). Categorical variables are reported as proportions with 95% confidence intervals,

and continuous variables as medians with interquartile ranges (IQRs). Chi-square tests

and Fisher’s exact tests were used to compare qualitative variables, and Mann-Whitney

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U and Kruskal-Wallis tests for quantitative variables. Confidence intervals around

proportions were calculated with the ‘exact’ method. Spearman's Rho test was used to

analyze correlations between test results. Statistical significance was defined as a p-

value <0.05.

Total percentage agreement and Cohen's kappa coefficient (κ) with standard error (SE)

were used to quantify concordance between TST and QFT-GIT results; indeterminate

QFT-GIT results were excluded from this particular analysis. Strength of agreement was

defined as poor (κ≤0.2), fair (0.2<κ≤0.4), moderate (0.4<κ≤0.6), good (0.6<κ≤0.8) and

very good (κ>0.8). Sensitivity estimates of TST and QFT-GIT were based exclusively

on active TB cases.

Results

A total of 383 children were recruited into the study, comprising 207 at DVH and 176 at

SJD. The participants’ demographics, the reason for their assessment, and TST and

QFT-GIT results are summarized in Table 1. At assessment, the age distribution of the

cohort was: 0-6 months, n=22; 6-12 months, n=32; 12-24 months, n=84; 24-36 months,

n=94; 36-48 months, n=88; and 48-60 months, n=63.

Children vaccinated with BCG were on average older than those without a history of

BCG vaccination (median [IQR]: 37 [24;49] vs. 28 [16;40] months, p<0.001), and

mainly underwent assessment because of new entrant screening (66/97, 68.0%). BCG-

vaccinated children were diagnosed with LTBI more frequently than those without a

history of BCG vaccination (23/100, 23.0% vs. 16/269, 5.9%, p<0.001; BCG status

unknown in 14 cases).

A total of 304 children were classified as uninfected according to the study definitions.

In this subgroup the median follow-up period was 47 months (IQR: 30;48). In 7/304

participants (2.3%) the follow-up period was 12-24 months at the end of the study

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period (none followed up for less than one year). One patient (0.3%) in this subgroup

developed active TB during follow-up - a Pakistani boy initially assessed at 16 months

of age as part of new entrant screening. At that time the QFT-GIT result was negative

and the TST induration was 8 mm, which was attributed to prior BCG vaccination. Two

years later he presented with symptoms and radiological findings consistent with

intrathoracic TB (TST induration: 15mm; QFT-GIT not performed), but cultures were

negative. The only additional risk factor for TB identified between the two assessments

was a 2-month visit to Pakistan. The child fully recovered with 6 months of anti-TB

treatment.

All children diagnosed with LTBI (n=40) received chemoprophylaxis (isoniazid and

rifampicin for 3 months, n=28; or 6-9 months of isoniazid monotherapy, n=12) as per

national guidelines.15

None of these patients had significant drug-related adverse events

or developed active TB during the follow-up period (median [IQR] follow-up since

completion of anti-TB treatment: 42 [32;45] months).

Thirty-nine patients were diagnosed with active TB (n=32 intrathoracic disease; n=16

microbiologically-confirmed; all confirmed cases had fully drug-susceptible strains of

MTB). All children responded to standard anti-TB treatment (2-month induction phase

with three drugs until March 2009, four-drug regimen since then, followed by isoniazid

and rifampicin for a minimum of 4 months).15

The distribution and concordance between TST and QFT-GIT results according to the

reason for assessment, the BCG vaccination status and the final diagnosis are shown in

Table 2; complete TST and QTF-GIT results are shown in (Table Supplemental Digital

Content 1, http://links.lww.com/INF/D72). Overall, agreement between tests was

moderate (83.2%, κ=0.551). However, in children tested as part of contact tracing and in

children without a history of BCG vaccination the concordance was higher and the test

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agreement very good (94.4%, κ=0.801 and 94.6%, κ=0.816, respectively).

Discordance between TST and QFT-GIT results was observed in 62/369 participants

(16.8%; excludes n=14 with indeterminate QFT-GIT test results); all had a TST

positive/QFT-GIT negative (TST+/QFT-GIT-) result constellation. Discordant results

were mainly observed among BCG-vaccinated children (47/62, 75.8%; BCG status

unknown in one patient), in whom agreement between tests was poor (κ=0.190). Most

of these patients (47/62, 75.8%) were classified as uninfected according to the study

definitions. Among the 14 BCG-unvaccinated patients with discordant results the final

diagnoses were: NTM lymphadenitis (n=9, culture-confirmed in 4 cases; all recovered

fully without anti-TB treatment), LTBI (n=4) and probable active TB (n=1). The

diagnosis in the latter, who was investigated after recent TB contact, was based on a

positive TST (7mm induration) and hilar lymphadenopathy in combination with

pulmonary nodules on chest computer tomography imaging.

Of the 39 cases with active TB, 30 had concordantly positive TST and QFT-GIT results,

while five had concordantly negative results. Three cases had a TST+/QFT-GIT-

discordant result constellation; one case had a positive TST result and an indeterminate

QFT-GIT result. All concordantly negative and all discordant results occurred in

patients with probable disease. The five children with concordantly negative results all

had symptoms and radiological findings consistent with active TB, and responded

clinically to anti-TB treatment. In microbiologically-confirmed cases (n=16) TST

results were universally positive, and 15 cases had a positive QFT-GIT result; one had

an indeterminate QFT-GIT result (Table 2 and Table 3, Supplemental Digital Content 1,

http://links.lww.com/INF/D72). Therefore, the sensitivities of TSTs and QFT-GIT

assays in children with confirmed active TB were 100% (95%CI: 79.4-100%) and

93.7% (95%CI: 69.8-99.8%; p=0.388), and in children with confirmed or probable

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active TB combined 87.2% (95%CI: 72.6-95.7%) and 77.0% (95%CI: 60.7-88.9%;

p=0.221), respectively.

Indeterminate QFT-GIT results occurred in 14/383 (3.6%) children and universally were

due to inadequate IFN-γ responses in the positive control sample. Children with

indeterminate QFT-GIT results were significantly younger than those with determinate

(i.e. positive or negative) results [median age (IQR): 12 (6;19) vs. 30 (18;42) months,

p<0.001]. QFT-GIT indeterminate results were more common in children aged <2 years

(8.7%) than in older ones (0.8%; p<0.001).

In participants with LTBI and active TB the diameter of TST induration was similar

(median[IQR]: 14[10;17] vs. 15[10;18] mm, respectively; p=0.581). Also, the

background-corrected antigen-stimulated IFN-γ responses in the QFT-GIT assay did not

differ significantly between those two groups (median[IQR]: 1.37[0.20;10.72] vs.

4.96[1.26;10.50] IU/mL, respectively; p=0.241).

In the whole cohort there was a positive correlation between the diameter of the TST

induration and the magnitude of background-corrected antigen-stimulated IFN-γ

responses in the QFT-GIT assay (r=0.601, p<0.001). A positive correlation was also

observed between age and the diameter of TST induration (r=0.187, p<0.001).

Furthermore, there was a positive correlation between age and background-corrected

mitogen control responses in the QFT-GIT assay (r=0.263, p<0.001; Figure 1a). In

contrast, in children diagnosed with LTBI or active TB, background-corrected antigen-

stimulated IFN-γ responses were age-independent (r=-0.044, p=0.714; Figure 1b).

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Discussion

Most international guidelines on the management of children with suspected TB

highlight that IGRAs may perform worse in young children compared to adults, but also

acknowledge that the data to support this view remain relatively limited.10,12-14

A

growing number of studies have assessed the performance of IGRAs in children,

including seven meta-analyses.20-26

However, the interpretation of the findings of these

meta-analyses is complex because of the large heterogeneity of the studies included

(background TB prevalence rates, variations in participants’ ages, and varying

proportions of immunocompromised participants) and also methodological differences

(varying TST cut-offs and the range of microbiological investigations employed).

Importantly, our study is one of the largest studies specifically evaluating the

performance of TSTs and QFT-GIT assays in children younger than 5 years in a low-

prevalence setting to date.

Overall, 79.4% of children in our cohort were classified as uninfected. The observation

that only one (0.3%; 118.4 cases per 100,000 patient-years) of these children developed

active TB during a median follow-up period of 47 months is reassuring as the period of

greatest risk of progression to disease is within the first 2 years after primary

infection.1,4,27

In our cohort, the test agreement between TSTs and IGRAs was good in non-BCG-

vaccinated children and in children undergoing contact screening, which is consistent

with the findings reported by other pediatric studies in low TB burden countries.28-30

These results suggest that a dual immunodiagnostic testing strategy (i.e., use of both

tests simultaneously), an approach used by many pediatric specialists,5 may not be

necessary in these particular patient populations. The proportion of indeterminate IGRA

results (3.6%) in our cohort was relatively small compared to other pediatric studies,

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which have typically observed indeterminate IGRA results in >5-10% of the study

population.30-35

In keeping with other pediatric studies, a significant proportion (16.8%) of our study

populations had discordance between TST and IGRA results.36-38

All of these

participants had a TST+/QFT-GIT- result constellation. In the absence of a gold

standard for LTBI, it remains uncertain whether individuals with discordant TST and

IGRA results are TB-infected or TB-uninfected. Since TST+/IGRA- discordance is

more commonly observed in BCG-vaccinated children, some authors have argued that

this constellation reflects false-positive TST results caused by immune sensitisation

induced by the BCG vaccine. However, data from a large meta-analysis that included

more than 240,000 children immunised with BCG in the neonatal period suggest that

only 8.5% of vaccinees become TST-positive as a result of BCG vaccination.39

In

addition, the obvious confounder is that most children who are BCG-vaccinated are

from high TB burden countries or born to high-risk populations, meaning their TB

exposure risk is generally greater than that of BCG-unvaccinated children. Notably,

almost a quarter of the children with TST+/QFT-GIT- discordance in our study had not

been vaccinated with BCG, and consequently their positive TST result cannot be

attributed to vaccine-induced immune responses. It has been postulated that in some

cases TST+/IGRA- discordance may reflect false-positive TST results caused by

exposure to NTM; some evidence for this has been provided by a study that used an ex-

vivo NTM-sensitin ELISPOT assay.40

Interestingly, in our study some non-BCG-

vaccinated children with TST+/IGRA- discordance had microbiologically-confirmed

NTM lymphadenitis. However, compelling data from a recently published study using

detailed immunological analyses suggest that children with TST+/IGRA- discordance

are a heterogeneous group comprising both TB-infected and TB-uninfected

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individuals.41

Considering these uncertainties, most experts recommend to treat children

with risk factors for TB and discordant TST/IGRA results with chemoprophylaxis,

particularly as adverse events related to chemoprophylaxis are rare in childhood.5

We found no significant differences in TST indurations or antigen-stimulated IFN-γ

responses in the QFT-GIT assay between LTBI and active TB cases, confirming

previous observations that neither TSTs nor QFT-GITs can be used to discriminate

between LTBI and active TB.11,34,37,42

Also, our data confirm that QFT-GIT assays do

not have higher sensitivity than TSTs in children with active TB, which aligns with

previously published observations.29,34,35,43

We found no correlation between age and the magnitude of antigen-stimulated IFN-γ

responses in QFT-GIT assays in children with LTBI or active TB. This finding is

surprising considering existing data suggesting that T-cell-mediated immune responses

are less robust in young children because of incomplete immune maturation.44,45

Previous studies have highlighted that phytohaemagglutinin-induced IFN-γ (positive

control) responses in the QFT-GIT assay are significantly lower in young children than

in adults, in part contributing to the high proportions of indeterminate IGRA results in

children.30,31

Some authors have therefore argued that age-specific cut-offs should be

used for positive control responses, or suggested the use of alternative control

stimulants.30,31,36

In our cohort, we observed a positive correlation between age and

positive control responses, and found that indeterminate results were more common in

younger patients, universally as a result of inadequate positive control responses.

Therefore, while age adjustment is likely needed for the evaluation of positive control

responses, our data suggest that such adjustment may not be required for antigen-

stimulated IFN-γ responses. However, we can not rule out with certainty that the

absence of a correlation between age and antigen-stimulated IFN-γ responses was due to

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the relatively narrow age spectrum of our cohort.

Our study has a number of limitations in common with all previous studies investigating

IGRAs in children, namely the lack of a gold standard for LTBI, and the absence of

microbiological confirmation in a substantial proportion of active TB cases. Also, our

study did not include a control group of healthy children without known risk factors for

TB, and non-immigrant children were not routinely tested for HIV. However, the

prevalence of HIV infection in children and adolescents in Spain is estimated to be

below 5 per 100,000.46

Also, except for age, other factors known to be associated with

indeterminate QTF-GIT results, such as malnutrition and co-existing helminth

infections were not specifically investigated.47,48

Finally, it remains uncertain whether

our results can be extrapolated to the T-SPOT®.TB (Oxford/Immunotec, UK) or the

QuantiFERON®-TB Gold Plus assays (Cellestis/Qiagen, Australia).

Our study adds significantly to the knowledge base regarding the performance of TSTs

and IGRAs in infants and pre-school children in a low TB prevalence setting. In

children without prior BCG vaccination and those screened for LTBI as part of contact

investigations, there was very good agreement between both tests; in these patient

groups, a dual immunodiagnostic testing strategy may therefore not be necessary.

Similar to previous pediatric studies, a considerable proportion of the study population

had TST+/QFT-GIT- discordance, and it currently remains uncertain whether these

children are TB-infected or TB-uninfected. Although indeterminate QFT-GIT results

were relatively uncommon compared to other pediatric studies, we found that those

results occurred more commonly in children at the lower end of the age spectrum. QFT-

GIT assays did not have higher sensitivity than TSTs in children with probable and

confirmed active TB, highlighting that IGRAs should not replace TSTs as a first-line

adjunctive test in children with suspected active TB.

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Figure legends

Figure 1. Correlation between age and IFN-γ concentrations.

Correlation between age and (1a) background-corrected IFN-γ concentration in the

mitogen-stimulated (positive control) sample of the QFT-GIT assay in 173 study

participants in whom quantitative data were available, and (1b) background-corrected

IFN-γ concentration in the antigen-stimulated sample in the 72 participants diagnosed

with LTBI or active TB, with fitted regression line; the dotted line represents the QFT-

GIT cut-off for positivity (≥0.35 IU/mL). The values shown are Spearman’s correlation

coefficients (r) and their corresponding p-value.

List of Supplemental Digital Content

- Table 3, which has been submitted as a .pdf file.

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Table 1. Demographic characteristics and reason for assessment according to final diagnosis. Data are shown as number and percentage or

median and interquartile range; the p-values refer to differences between the three diagnostic groups.

Final diagnosis

n= 383 Uninfected n=304 (79.4) LTBI n=40 (10.4) TB n=39 (10.2) p-value

Male gender

Age in months

<2 years

Born in Spain

Born to immigrant family#

BCG vaccination&

196 (51.2)

29 (17;41)

141 (46.8)

278 (72.6)

222 (57.9)

100 (27.1)

158 (51.9)

29 (16;40)

118 (38.8)

226 (74.3)

165 (54.3)

72 (24.7)

20 (50.0)

44 (26;53)

8 (20.0)

18 (45.0)

32 (80.0)

23 (59.0)

18 (46.2)

28 (15;38)

15 (38.5)

34 (87.2)

25 (64.1)

5 (13.2)

0.682

0.002

0.066

<0.001

0.006

<0.001

Reason for assessment:

Suspected active TB*

Contact tracing

New-entrant screening

37 (9.7)

258 (67.4)

88 (22.9)

25 (8.2)

203 (66.8)

76 (25.0)

0

30 (75.0)

10 (25.0)

12 (30.8)

25 (64.1)

2 (5.1)

<0.001

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#Families’ origins: Spain, n=150 (39.2%); other European countries, n=16 (4.2%); South America, n=61 (15.9%); Africa, n=59 (15.4%); and

Asia, n=97 (25.3%).

&BCG vaccination status was known in 369 patients overall (n=292, n=39 and n=38 patients classified as uninfected, LTBI and TB, respectively).

*Clinically or radiologically suspected active TB.

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Table 2. Agreement between TST and QFT-GIT test results according to the reason for assessment, the BCG vaccination status and the final

diagnosis. Patients with indeterminate QTF-GIT test results (n=14) were excluded from the statistical analyses.

n#

Indeterminate

QTF-GIT results

Agreement

proportion (%)

κ (SE)&

QTF-GIT(+) QTF-GIT(-)

TST+ TST- TST+ TST-

Reason for assessment

Suspected active TB*

34 3 70.6 0.452 (0.122) 10 0 10 14

Contact tracing 249 9 94.4 0.801 (0.051) 35 0 14 200

New-entrant screening 86 2 55.8 0.199 (0.058) 11 0 38 37

BCG vaccination

No 259 10 94.6 0.816 (0.047) 39 0 14 206

Yes 98 2 52.0 0.190 (0.050) 15 0 47 36

Unknown 12 2 91.7 0.750 (0.232) 2 0 1 9

Final diagnosis

Uninfected 293 11 83.9 NC 0 0 47 246

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LTBI 38 2 68.4 NC 26 0 12 0

Active TB 38 92.1 0.725 (0.147) 30 0 3 5

Confirmed TB** 15 1 100.0 NC 15 0 0 0

Probable TB 23 86.9 0.685 (0.161) 15 0 3 5

Total 369 14 83.2 0.551 (0.046) 26 0 62 251

NC, not calculable (Cohen's kappa coefficient is not calculable when all TST or all QFT-GIT results are positive or negative in a group).

#Figures exclude patients with indeterminate QFT-GIT assay results.

&κ (SE), Cohen's kappa coefficient (standard error).

*Clinically or radiologically suspected active TB.

**Microbiologically-confirmed TB.

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