# Tuberculin Skin Test Negativity Is Under Tight Genetic Control of Chromosomal Region 11p14-15 in Settings With Different Tuberculosis Endemicities

Aurélie Cobat, Christine Poirier, Eileen Hoal, Anne Boland-Auge, France de la Rocque, François Corrard, Ghislain Grange, Mélanie Migaud, Jacinta Bustamante, Stéphanie Boisson-Dupuis, et al.

► To cite this version:

Aurélie Cobat, Christine Poirier, Eileen Hoal, Anne Boland-Auge, France de la Rocque, et al. Tuberculin Skin Test Negativity Is Under Tight Genetic Control of Chromosomal Region 11p14-15 in Settings With Different Tuberculosis Endemicities. Journal of Infectious Diseases, 2014, 211 (2), pp.317 - 321. 10.1093/infdis/jiu446. cea-04580619

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# Tuberculin Skin Test Negativity Is Under Tight Genetic Control of Chromosomal Region 11p14-15 in Settings With Different Tuberculosis Endemicities

Aurélie Cobat, Christine Poirier, Eileen Hoal, Anne Boland-Auge, France de la Rocque, François Corrard, Ghislain Grange, Mélanie Migaud, Jacinta Bustamante, Stéphanie Boisson-Dupuis, et al.

► To cite this version:

Aurélie Cobat, Christine Poirier, Eileen Hoal, Anne Boland-Auge, France de la Rocque, et al. Tuberculin Skin Test Negativity Is Under Tight Genetic Control of Chromosomal Region 11p14-15 in Settings With Different Tuberculosis Endemicities. Journal of Infectious Diseases, 2014, 211 (2), pp.317 - 321. 10.1093/infdis/jiu446. cea-04580619

HAL Id: cea-04580619

https://cea.hal.science/cea-04580619v1

Submitted on 20 May 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Tuberculin Skin Test Negativity Is Under Tight Genetic Control of Chromosomal Region 11p14-15 in Settings With Different Tuberculosis Endemicities

Aurélie Cobat,<sup>1,2,3</sup> Christine Poirier,<sup>4</sup> Eileen Hoal,<sup>11</sup> Anne Boland-Auge,<sup>5</sup>

France de La Rocque,<sup>6</sup> François Corrard,<sup>6</sup> Ghislain Grange,<sup>7</sup>

Mélanie Migaud,<sup>7,9</sup> Jacinta Bustamante,<sup>7,9</sup> Stéphanie Boisson-Dupuis,<sup>7,9,12</sup>

Jean-Laurent Casanova,<sup>7,9,12,13</sup> Erwin Schurr,<sup>1,2,3</sup> Alexandre Alcaïs,<sup>7,9,10,12,a</sup>

Christophe Delacourt,<sup>8,a</sup> and Laurent Abel<sup>7,9,12,a</sup>

<sup>1</sup>McGill International TB Centre, <sup>2</sup>Department of Human Genetics, and <sup>3</sup>Department of Medicine, McGill University, Montreal, Canada; <sup>4</sup>Centre de Lutte Anti-Tuberculeuse, Centre Hospitalier Intercommunal de Créteil, Créteil, <sup>5</sup>National Center for Genotyping, Genomic Institute, CEA, Evry, <sup>6</sup>Association Clinique et Thérapeutique Infantile du Val de Marne, Saint Maur des Fossés, and <sup>7</sup>Laboratory of Human Genetics of Infectious Diseases, Necker Branch, INSERM U.1163, and <sup>8</sup>Pediatric Pneumology Unit, Necker-Enfants-Malades Hospital, AP-HP, <sup>9</sup>University Paris Descartes, Sorbonne Paris Cité, Imagine Institute, and <sup>10</sup>JRC, CIC, Necker, and Cochin Hospitals, Paris, France;

<sup>11</sup>Molecular Biology and Human Genetics, MRC Centre for Molecular and Cellular Biology, DST/NRF Centre of Excellence for Biomedical TB Research, Faculty of Health Sciences, Stellenbosch University, Tygerberg, South Africa; and <sup>12</sup>St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, and <sup>13</sup>Howard Hughes Medical Institute, Rockefeller University, New York, New York

A substantial proportion of subjects exposed to a contagious tuberculosis case display lack of tuberculin skin test (TST) reactivity. We previously mapped a major locus (TST1) controlling lack of TST reactivity in families from an area in South Africa where tuberculosis is hyperendemic. Here, we conducted a household tuberculosis contact study in a French area where the endemicity of tuberculosis is low. A genome-wide analysis of TST negativity identified a significant linkage signal (P < 3 × 10<sup>-5</sup>) in close vicinity of TST1. Combined analysis of the 2 samples increased evidence of linkage (P = 2.4 × 10<sup>-6</sup>), further implicating genetic factors located on 11p14-15. This region overlaps the TNFI locus controlling mycobacteria-driven tumor necrosis factor α production.

Received 2 March 2014; accepted 31 July 2014; electronically published 20 August 2014.

<sup>a</sup>A. A., C. D., and L. A. share senior authorship.

Correspondence: Laurent Abel, MD, PhD, Human Genetics of Infectious Diseases, Inserm U1163, Imagine Insitute, 24 Bd du Montparnasse, 75015 Paris, France (laurent.abel@inserm.fr).

**The Journal of Infectious Diseases®** 2015;211:317–21

© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact journals permissions@oup.com.

DOI: 10.1093/infdis/jiu446

**Keywords.** tuberculosis infection; tuberculin skin test; genome-wide linkage; human genetics.

*Mycobacterium tuberculosis* is the main cause of human tuber-
culosis, a disease that remains a major public health concern
worldwide. Despite the widespread use of BCG vaccine and spe-
cific control programs based on combinations of potent antibi-
otics, tuberculosis kills more individuals per year than any other
infectious agent. Progression to clinical tuberculosis is far from
inevitable, as only an estimated 10% of individuals infected by
*M. tuberculosis* develop clinical disease [1], whether during pri-
mary infection or later in life by reactivation of latent or second-
ary infection. There is accumulating evidence that human
genetics play an important role in explaining this large clinical
variability [1–3].

There is also interindividual variability at an earlier stage of
the infectious process, as approximately 30%-50% of subjects
with heavy exposure to *M. tuberculosis* in household studies
do not become infected [4]. This estimate is based on the diag-
nosis of *M. tuberculosis*-infected and uninfected persons by
means of the tuberculin skin test (TST), which measures indu-
ration on the skin after intradermal inoculation of tuberculin
(ie, *M. tuberculosis* purified protein derivative). Several family
studies have provided consistent evidence for an important
role of human genetics in the control of *M. tuberculosis* infec-
tion or TST reactivity [5, 6]. Indeed, by focusing on TST nega-
tivity (TST induration diameter, 0 mm vs >0 mm) in an area
hyperendemic for tuberculosis in South Africa, we recently
mapped *TST1*, located on chromosomal region 11p14, as a
major locus controlling this phenotype [7].

We report here results of a genome-wide linkage analysis (GWLA) of TST negativity in a household tuberculosis contact study conducted in a suburb of Paris, France. We identified a significant linkage signal (P value, approximately 2 × 10<sup>-5</sup>) in close vicinity of TST1. Combined analysis of the French and South African samples increased evidence of linkage at the TST1 locus (P value, approximately 2 × 10<sup>-6</sup>), consistent with the hypothesis that TST1 is a major locus controlling TST negativity.

## MATERIAL AND METHODS

A prospective study of household tuberculosis contacts was
conducted in the Val-de-Marne suburb of Paris, as detailed in
[8]. Briefly, Val-de-Marne is an area of low tuberculosis ende-
micity with an annual tuberculosis incidence of 22.1 cases per
# Tuberculin Skin Test Negativity Is Under Tight Genetic Control of Chromosomal Region 11p14-15 in Settings With Different Tuberculosis Endemicities

Aurélie Cobat,¹,²,³ Christine Poirier,⁴ Eileen Hoal,¹¹ Anne Boland-Auge,⁵

France de La Rocque,⁶ François Corrard,⁶ Ghislain Grange,⁷

Mélanie Migaud,⁷,⁹ Jacinta Bustamante,⁷,⁹ Stéphanie Boisson-Dupuis,⁷,⁹,¹²

Jean-Laurent Casanova,⁷,⁹,¹²,¹³ Erwin Schurr,¹,²,³ Alexandre Alcaïs,⁷,⁹,¹⁰,¹²,ᵃ

Christophe Delacourt,⁸,ᵃ and Laurent Abel⁷,⁹,¹²,ᵃ

¹McGill International TB Centre, ²Department of Human Genetics, and ³Department of Medicine, McGill University, Montreal, Canada; ⁴Centre de Lutte Anti-Tuberculeuse, Centre Hospitalier Intercommunal de Créteil, Créteil, ⁵National Center for Genotyping, Genomic Institute, CEA, Evry, ⁶Association Clinique et Thérapeutique Infantile du Val de Marne, Saint Maur des Fossés, and ⁷Laboratory of Human Genetics of Infectious Diseases, Necker Branch, INSERM U.1163, and ⁸Pediatric Pneumology Unit, Necker-Enfants-Malades Hospital, AP-HP, ⁹University Paris Descartes, Sorbonne Paris Cité, Imagine Institute, and ¹⁰JRC, CIC, Necker, and Cochin Hospitals, Paris, France;

¹¹Molecular Biology and Human Genetics, MRC Centre for Molecular and Cellular Biology, DST/NRF Centre of Excellence for Biomedical TB Research, Faculty of Health Sciences, Stellenbosch University, Tygerberg, South Africa; and ¹²St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, and ¹³Howard Hughes Medical Institute, Rockefeller University, New York, New York

A substantial proportion of subjects exposed to a contagious tuberculosis case display lack of tuberculin skin test (TST) reactivity. We previously mapped a major locus (TST1) controlling lack of TST reactivity in families from an area in South Africa where tuberculosis is hyperendemic. Here, we conducted a household tuberculosis contact study in a French area where the endemicity of tuberculosis is low. A genome-wide analysis of TST negativity identified a significant linkage signal (P < 3 × 10⁻⁵) in close vicinity of TST1. Combined analysis of the 2 samples increased evidence of linkage (P = 2.4 × 10⁻⁶), further implicating genetic factors located on 11p14-15. This region overlaps the TNFI locus controlling mycobacteria-driven tumor necrosis factor α production.

Received 2 March 2014; accepted 31 July 2014; electronically published 20 August 2014.

ªA. A., C. D., and L. A. share senior authorship.

Correspondence: Laurent Abel, MD, PhD, Human Genetics of Infectious Diseases, Inserm U1163, Imagine Insitute, 24 Bd du Montparnasse, 75015 Paris, France (laurent.abel@inserm.fr).

**The Journal of Infectious Diseases®** 2015;211:317–21

© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact journals permissions@oup.com.

DOI: 10.1093/infdis/jiu446

**Keywords.** tuberculosis infection; tuberculin skin test; genome-wide linkage; human genetics.

*Mycobacterium tuberculosis* is the main cause of human tuber-
culosis, a disease that remains a major public health concern
worldwide. Despite the widespread use of BCG vaccine and spe-
cific control programs based on combinations of potent antibi-
otics, tuberculosis kills more individuals per year than any other
infectious agent. Progression to clinical tuberculosis is far from
inevitable, as only an estimated 10% of individuals infected by
*M. tuberculosis* develop clinical disease [1], whether during pri-
mary infection or later in life by reactivation of latent or second-
ary infection. There is accumulating evidence that human
genetics play an important role in explaining this large clinical
variability [1–3].

There is also interindividual variability at an earlier stage of
the infectious process, as approximately 30%–50% of subjects
with heavy exposure to *M. tuberculosis* in household studies
do not become infected [4]. This estimate is based on the diag-
nosis of *M. tuberculosis*-infected and uninfected persons by
means of the tuberculin skin test (TST), which measures indu-
ration on the skin after intradermal inoculation of tuberculin
(ie, *M. tuberculosis* purified protein derivative). Several family
studies have provided consistent evidence for an important
role of human genetics in the control of *M. tuberculosis* infec-
tion or TST reactivity [5, 6]. Indeed, by focusing on TST nega-
tivity (TST induration diameter, 0 mm vs >0 mm) in an area
hyperendemic for tuberculosis in South Africa, we recently
mapped *TST1*, located on chromosomal region 11p14, as a
major locus controlling this phenotype [7].

We report here results of a genome-wide linkage analysis (GWLA) of TST negativity in a household tuberculosis contact study conducted in a suburb of Paris, France. We identified a significant linkage signal (P value, approximately 2 × 10⁻⁵) in close vicinity of *TST1*. Combined analysis of the French and South African samples increased evidence of linkage at the *TST1* locus (P value, approximately 2 × 10⁻⁶), consistent with the hypothesis that *TST1* is a major locus controlling TST negativity.

## MATERIAL AND METHODS

A prospective study of household tuberculosis contacts was
conducted in the Val-de-Marne suburb of Paris, as detailed in
[8]. Briefly, Val-de-Marne is an area of low tuberculosis ende-
micity with an annual tuberculosis incidence of 22.1 cases per
100 000 inhabitants. From April 2004 to January 2009, house-
hold contacts exposed to a patient with culture-confirmed pul-
monary tuberculosis were enrolled in the context of a general
screening procedure. A household contact was defined as any
person sharing the residence of a tuberculosis index case during
the 3 months preceding diagnosis of the case. The first screening
visit (V1) included clinical examination, performance of a TST
(ie, intradermal injection of 0.1 mL [5 tuberculin units] of tuber-
culin obtained from a human-pathogenic M. tuberculosis strain
[Tubertest; Sanofi Pasteur, Lyons, France]), chest radiography,
and a completion of standardized questionnaire to assess risk fac-
tors of M. tuberculosis infection, as detailed elsewhere [8]. These
investigations were repeated 8–12 weeks later (V2) in household
contacts for whom no infection was diagnosed at V1 [8].

We dichotomized the TST distribution, using a 0-mm indu-
ration as the threshold, to define a binary TST status (TST-bin).
Individuals with a discordant TST-bin status between V1 and
V2 were excluded. Prior to linkage analysis, we assessed the ef-
fect of relevant covariates on TST-bin by determining the infec-
tivity of the index case, based on the presence of cavitation on
chest radiography or the presence of bacilli in sputum smears,
age, sex, and estimated total exposure of the contact to the index
case (quantified as the number of days the contact spent with
the index case during the 3 months preceding the diagnosis
of tuberculosis). To account for the relatedness of individuals,
the impact of these covariates was assessed by estimating equa-
tion methods, as implemented in the R package GEEPACK
(http://cran.r-project.org/web/packages/geepack/index.html),
using a logit link function and an identity working correlation
matrix.

Genotyping of children and parents for the GWLA was per-
formed at the Centre National de Génotypage (Paris) with the
Illumina linkage V panel. Nonpolymorphic single-nucleotide
polymorphisms (SNPs) and SNPs with a call rate of <90%
were removed from the analysis. Linkage analysis was per-
formed on the quantitative Pearson residuals, obtained by ad-
justment of TST-bin on sex, age, level of exposure, and index
case infectivity. Model-free GWLA was performed by means
of the new maximum-likelihood binomial (MLB) method for
quantitative traits (nMLB-QTL v.3.0) [9, 10]. The MLB ap-
proach considers the sibship as a whole and does not make
any assumption about the distribution of the phenotype. The
test of linkage is a maximum-likelihood ratio test asymptotically
distributed as a 50:50 mixture of χ² distributions with 0 and 1
degree of freedom and can be expressed as a classic LOD score.
We used LOD scores of 3.6 and 2.2 as genome-wide significant
and suggestive thresholds, respectively, as described in [7].

RESULTS AND DISCUSSION

First, the analysis of covariates in the French cohort was per-
formed on 540 household tuberculosis contacts belonging to

155 pedigrees. Among them, 84 (15.6%) had a TST induration
size of 0 mm at both visits and were classified as having a neg-
ative TST-bin. The mean age (±SD) at the time of TST was 24.1
± 18.0 years, and the male to female ratio was 1.05. BCG vaccine
coverage was very high, with 13 individuals (2.6%) not vaccinat-
ed with BCG vaccine. The mean duration of exposure to the
index case (±SD) was 19.25 ± 21.6 days, and 449 household
contacts (83.1%) were exposed to an index case presenting
cavitation on chest radiography or bacilli in sputum smears.
Univariate and multivariate (ie, testing all covariates simultane-
ously) analysis showed that age had the strongest impact on
TST positivity (Table 1). Index case infectivity and total expo-
sure time to the index case were also significantly associated
with a positive TST result (Table 1).

Second, from the French sample, 97 nuclear families, includ-
ing 237 offspring, were informative for linkage analysis (ie,
composed of at least 2 genotyped sibs with a TST value). Char-
acteristics of the 237 offspring are shown in Supplementary
Table 1. Among them, only 4 had not received BCG vaccine,
eliminating BCG vaccination as a potential confounding factor
in our study. Results of the GWLA of TST-bin adjusted on the
covariates are presented in Figure 1A. Information content was
high across all autosomes, with mean genome-wide information
of approximately 90%. We identified 1 major locus, at position
14.3 Mb on chromosomal region 11p15, that reached the
threshold commonly used to claim genome-wide significance
(LOD score, 3.65; P = 2.08 × 10⁻⁵; Figure 1A). In addition, we
identified 18 weaker linkage peaks with a P value of <.01 (Sup-
plementary Table 2).

This major locus mapped at approximately 12 Mb from the *TST1* locus previously identified as controlling TST negativity in South Africa [7]. Considering the precision of location estimates in linkage studies of complex traits [11], this result is consistent with replication of the South African *TST1* locus in the French cohort. We therefore refined the linkage signal at chromosomal region 11p14-15 by performing a GWLA on the pooled samples (Figure 1B). Evidence of linkage for TST-bin increased in the analysis of the combined sample, compared with the analysis of each sample separately (Figure 1A; [7]), with a maximum LOD score of 4.54 at position 23.44 Mb (*P* = 2.40 × 10⁻⁶; Figure 1C). This linkage peak was preceded by a plateau from 11 to 20 Mb with LOD scores varying between 2.5 and 2.8. It is therefore difficult to precisely define the bounds of the confidence interval for the true location of *TST1*. Of note, we also observed suggestive evidence of linkage in the combined sample on chromosomal region 19q13 (LOD score, 2.85 at position 55.55 Mb), whereas only moderate evidence of linkage was observed in the analysis of each sample separately.

To further explore and refine the region of interest on chro-
mosome 11p14-15, we restricted our analysis to French and
South African families showing evidence of linkage (ie, those
with a LOD score of >0) in the region extending from 10 Mb
100 000 inhabitants. From April 2004 to January 2009, house-
hold contacts exposed to a patient with culture-confirmed pul-
monary tuberculosis were enrolled in the context of a general
screening procedure. A household contact was defined as any
person sharing the residence of a tuberculosis index case during
the 3 months preceding diagnosis of the case. The first screening
visit (V1) included clinical examination, performance of a TST
(ie, intradermal injection of 0.1 mL [5 tuberculin units] of tuber-
culin obtained from a human-pathogenic M. tuberculosis strain
[Tubertest; Sanofi Pasteur, Lyons, France]), chest radiography,
and a completion of standardized questionnaire to assess risk fac-
tors of M. tuberculosis infection, as detailed elsewhere [8]. These
investigations were repeated 8–12 weeks later (V2) in household
contacts for whom no infection was diagnosed at V1 [8].

We dichotomized the TST distribution, using a 0-mm indu-
ration as the threshold, to define a binary TST status (TST-bin).
Individuals with a discordant TST-bin status between V1 and
V2 were excluded. Prior to linkage analysis, we assessed the ef-
fect of relevant covariates on TST-bin by determining the infec-
tivity of the index case, based on the presence of cavitation on
chest radiography or the presence of bacilli in sputum smears,
age, sex, and estimated total exposure of the contact to the index
case (quantified as the number of days the contact spent with
the index case during the 3 months preceding the diagnosis
of tuberculosis). To account for the relatedness of individuals,
the impact of these covariates was assessed by estimating equa-
tion methods, as implemented in the R package GEEPACK
(http://cran.r-project.org/web/packages/geepack/index.html),
using a logit link function and an identity working correlation
matrix.

Genotyping of children and parents for the GWLA was per-
formed at the Centre National de Génotypage (Paris) with the
Illumina linkage V panel. Nonpolymorphic single-nucleotide
polymorphisms (SNPs) and SNPs with a call rate of <90%
were removed from the analysis. Linkage analysis was per-
formed on the quantitative Pearson residuals, obtained by ad-
justment of TST-bin on sex, age, level of exposure, and index
case infectivity. Model-free GWLA was performed by means
of the new maximum-likelihood binomial (MLB) method for
quantitative traits (nMLB-QTL v.3.0) [9, 10]. The MLB ap-
proach considers the sibship as a whole and does not make
any assumption about the distribution of the phenotype. The
test of linkage is a maximum-likelihood ratio test asymptotically
distributed as a 50:50 mixture of χ² distributions with 0 and 1
degree of freedom and can be expressed as a classic LOD score.
We used LOD scores of 3.6 and 2.2 as genome-wide significant
and suggestive thresholds, respectively, as described in [7].

RESULTS AND DISCUSSION

First, the analysis of covariates in the French cohort was per-
formed on 540 household tuberculosis contacts belonging to

155 pedigrees. Among them, 84 (15.6%) had a TST induration
size of 0 mm at both visits and were classified as having a neg-
ative TST-bin. The mean age (±SD) at the time of TST was 24.1
± 18.0 years, and the male to female ratio was 1.05. BCG vaccine
coverage was very high, with 13 individuals (2.6%) not vaccinat-
ed with BCG vaccine. The mean duration of exposure to the
index case (±SD) was 19.25 ± 21.6 days, and 449 household
contacts (83.1%) were exposed to an index case presenting
cavitation on chest radiography or bacilli in sputum smears.
Univariate and multivariate (ie, testing all covariates simultane-
ously) analysis showed that age had the strongest impact on
TST positivity (Table 1). Index case infectivity and total expo-
sure time to the index case were also significantly associated
with a positive TST result (Table 1).

Second, from the French sample, 97 nuclear families, includ-
ing 237 offspring, were informative for linkage analysis (ie,
composed of at least 2 genotyped sibs with a TST value). Char-
acteristics of the 237 offspring are shown in Supplementary
Table 1. Among them, only 4 had not received BCG vaccine,
eliminating BCG vaccination as a potential confounding factor
in our study. Results of the GWLA of TST-bin adjusted on the
covariates are presented in Figure 1A. Information content was
high across all autosomes, with mean genome-wide information
of approximately 90%. We identified 1 major locus, at position
14.3 Mb on chromosomal region 11p15, that reached the
threshold commonly used to claim genome-wide significance
(LOD score, 3.65; P = 2.08 × 10⁻⁵; Figure 1A). In addition, we
identified 18 weaker linkage peaks with a P value of <.01 (Sup-
plementary Table 2).

This major locus mapped at approximately 12 Mb from the
*TST1* locus previously identified as controlling TST negativity
in South Africa [7]. Considering the precision of location esti-
mates in linkage studies of complex traits [11], this result is con-
sistent with replication of the South African *TST1* locus in the
French cohort. We therefore refined the linkage signal at chro-
mosomal region 11p14-15 by performing a GWLA on the
pooled samples (Figure 1B). Evidence of linkage for TST-bin
increased in the analysis of the combined sample, compared
with the analysis of each sample separately (Figure 1A; [7]),
with a maximum LOD score of 4.54 at position 23.44 Mb
(*P* = 2.40 × 10⁻⁶; Figure 1C). This linkage peak was preceded
by a plateau from 11 to 20 Mb with LOD scores varying between
2.5 and 2.8. It is therefore difficult to precisely define the bounds
of the confidence interval for the true location of *TST1*. Of note,
we also observed suggestive evidence of linkage in the combined
sample on chromosomal region 19q13 (LOD score, 2.85 at po-
sition 55.55 Mb), whereas only moderate evidence of linkage
was observed in the analysis of each sample separately.

To further explore and refine the region of interest on chro-
mosome 11p14-15, we restricted our analysis to French and
South African families showing evidence of linkage (ie, those
with a LOD score of >0) in the region extending from 10 Mb
<table><caption>**Table 1.** Impact of Covariates on the Risk of Having a Tuberculin Skin Test (TST) Induration of >0 mm Among 540 Household Tuberculosis Contacts</caption><thead><tr><th rowspan="2">Covariates</th><th rowspan="2">TST Induration<br>>0 mm, No. (%)</th><th colspan="2">Univariate Analysis</th><th colspan="2">Multivariate Analysis</th></tr><tr><th>OR (95% CI)</th><th>P Value</th><th>OR (95% CI)</th><th>P Value</th></tr></thead><tbody><tr><td>Sex</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Male</td><td>235 (84.8)</td><td>...</td><td></td><td>...</td><td></td></tr><tr><td>Female</td><td>221 (84.0)</td><td>0.94 (.62-1.43)</td><td>.77</td><td>0.85 (.54-1.35)</td><td>.49</td></tr><tr><td>Age,<sup>a</sup> y, range (reference point)</td><td></td><td></td><td>2.5 x 10<sup>-10</sup></td><td></td><td>7.9 x 10<sup>-9</sup></td></tr><tr><td>0-10 (5)</td><td>113 (69.3)</td><td>...</td><td></td><td>...</td><td></td></tr><tr><td>10-20 (15)</td><td>91 (91.2)</td><td>2.80 (2.07-3.78)</td><td></td><td>2.94 (2.11-4.12)</td><td></td></tr><tr><td>20-30 (25)</td><td>80 (92.5)</td><td>4.31 (2.86-6.49)</td><td></td><td>4.60 (2.91-7.28)</td><td></td></tr><tr><td>>30 (40)</td><td>186 (90.3)</td><td>4.54 (2.84-7.27)</td><td></td><td>4.76 (2.81-8.07)</td><td></td></tr><tr><td>Exposure duration,<sup>a</sup> d, range (reference point)</td><td></td><td></td><td>.027</td><td></td><td>.04</td></tr><tr><td>0-3 (1)</td><td>133 (78.7)</td><td>...</td><td></td><td>...</td><td></td></tr><tr><td>3-15 (7)</td><td>131 (90.3)</td><td>1.45 (1.10-1.91)</td><td></td><td>1.52 (1.13-2.04)</td><td></td></tr><tr><td>>15 (30)</td><td>190 (85.2)</td><td>1.98 (1.10-3.58)</td><td></td><td>2.45 (1.29-4.65)</td><td></td></tr><tr><td>Index case infectivity<sup>b</sup></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>No</td><td>64 (71.9)</td><td>...</td><td></td><td>...</td><td></td></tr><tr><td>Yes</td><td>390 (86.9)</td><td>2.58 (1.47-4.53)</td><td>.001</td><td>3.20 (1.82-5.64)</td><td>5.8 x 10<sup>-5</sup></td></tr></tbody></table>

Abbreviations: CI, confidence interval; OR, odds ratio.

to 34 Mb. Our goal was to narrow the size of the region of interest by removing the noise randomly generated by unlinked families (eg, because of genetic heterogeneity). Results are shown in Figure 1D. While the height of the linkage peak has no more formal interpretation in that situation, its shape shows that the plateau observed between 11 and 20 Mb in Figure 1C is likely part of the region of interest. This region of interest, defined as displaying a LOD score of >2.2 in Figure 1C, extends from 11.6 Mb to 33.1 Mb and contains 194 genes (Supplementary Table 3). Of note, WT1 located at 32.4 Mb is the closest gene to rs2057178, a SNP found to be associated with pulmonary tuberculosis in several genome-wide association studies performed in different populations [12, 13]. Identification of genetic variant(s) underlying the linkage peak is ongoing following the same strategy (based on ultra-fine linkage disequilibrium mapping) as the one successfully used for pulmonary tuberculosis [3].

While the phenotype of interest was similar in the South African and French samples (ie, TST negativity, defined using a stringent cutoff of a 0-mm induration), the 2 studies were otherwise remarkably different. The South African study took place in an area hyperendemic for tuberculosis [7], where the transmission of *M. tuberculosis* occurs preferentially in the community, and with no requirement for the enrolled individuals to be household contacts of tuberculosis cases. By contrast, France is

a country with low tuberculosis endemicity, and the study design was a household tuberculosis contact study, which enabled us to take into account and quantify both the level of exposure and the index case infectivity. Beside the exposure context, the 2 studies also differed in terms of genetic background, as captured by the difference in ethnicities of subjects in each study. All the individuals studied in [7] belonged to the South African Coloured ethnic group, a unique highly admixed population resulting from the encounter of different founders from Africa, Europe, and Asia. By contrast, individuals in the present study belonged to several ethnic groups (white [48%], African [33%], Asian [6%], and mixed and other [13%]). Thus, replication of TST1 in such different settings suggests an important role of this locus in the control of TST negativity in humans.

The risk of developing tuberculosis for immunocompetent persons without TST reactivity despite sustained exposure to *M. tuberculosis* was previously shown to be extremely small [14]. Thus, persons with a TST induration of 0 mm are more likely not infected by *M. tuberculosis* than intrinsically deficient in mounting a delayed type hypersensitivity response. In the French cohort, the individual level of exposure to *M. tuberculosis* was variable. Some individuals were constantly exposed to a highly infectious case and are very likely naturally resistant to *M. tuberculosis* infection. Others were occasionally exposed to a moderately infectious case, and in this instance it is not

<footnote><sup>a</sup> Effect of continuous variables age and exposure was modeled using fractional polynomials (FP) as described elsewhere [7, 8]. For each variable, first degree (FP1) and second degree (FP2) FP models were fitted with power p for FP1 and p and q for FP2 chosen from -2, -1, -0.5, 0, 0.5, 1, 2, and 3, with 0 denoting log transformation. Among the 44 possible combinations, the model providing the smaller Akaike information criterion was selected. FP2 models with p = 1 and q = 1 and p = 0.5 and q = 2 were selected for age and exposure, respectively. For interpretability, category-based estimates are replaced with those from the regression model at relevant age and exposure length.</footnote>

<footnote><sup>b</sup> Defined as cavitation on the chest radiograph or bacillary in sputum smears.</footnote>
<table><caption>**Table 1.** Impact of Covariates on the Risk of Having a Tuberculin Skin Test (TST) Induration of >0 mm Among 540 Household Tuberculosis Contacts</caption><thead><tr><th rowspan="2">Covariates</th><th rowspan="2">TST Induration<br>>0 mm, No. (%)</th><th colspan="2">Univariate Analysis</th><th colspan="2">Multivariate Analysis</th></tr><tr><th>OR (95% CI)</th><th>P Value</th><th>OR (95% CI)</th><th>P Value</th></tr></thead><tbody><tr><td>Sex</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Male</td><td>235 (84.8)</td><td>...</td><td></td><td>...</td><td></td></tr><tr><td>Female</td><td>221 (84.0)</td><td>0.94 (.62-1.43)</td><td>.77</td><td>0.85 (.54-1.35)</td><td>.49</td></tr><tr><td>Age,<sup>a</sup> y, range (reference point)</td><td></td><td></td><td>2.5 x 10<sup>-10</sup></td><td></td><td>7.9 x 10<sup>-9</sup></td></tr><tr><td>0-10 (5)</td><td>113 (69.3)</td><td>...</td><td></td><td>...</td><td></td></tr><tr><td>10-20 (15)</td><td>91 (91.2)</td><td>2.80 (2.07-3.78)</td><td></td><td>2.94 (2.11-4.12)</td><td></td></tr><tr><td>20-30 (25)</td><td>80 (92.5)</td><td>4.31 (2.86-6.49)</td><td></td><td>4.60 (2.91-7.28)</td><td></td></tr><tr><td>>30 (40)</td><td>186 (90.3)</td><td>4.54 (2.84-7.27)</td><td></td><td>4.76 (2.81-8.07)</td><td></td></tr><tr><td>Exposure duration,<sup>a</sup> d, range (reference point)</td><td></td><td></td><td>.027</td><td></td><td>.04</td></tr><tr><td>0-3 (1)</td><td>133 (78.7)</td><td>...</td><td></td><td>...</td><td></td></tr><tr><td>3-15 (7)</td><td>131 (90.3)</td><td>1.45 (1.10-1.91)</td><td></td><td>1.52 (1.13-2.04)</td><td></td></tr><tr><td>>15 (30)</td><td>190 (85.2)</td><td>1.98 (1.10-3.58)</td><td></td><td>2.45 (1.29-4.65)</td><td></td></tr><tr><td>Index case infectivity<sup>b</sup></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>No</td><td>64 (71.9)</td><td>...</td><td></td><td>...</td><td></td></tr><tr><td>Yes</td><td>390 (86.9)</td><td>2.58 (1.47-4.53)</td><td>.001</td><td>3.20 (1.82-5.64)</td><td>5.8 x 10<sup>-5</sup></td></tr></tbody></table>

Abbreviations: CI, confidence interval; OR, odds ratio.

to 34 Mb. Our goal was to narrow the size of the region of interest by removing the noise randomly generated by unlinked families (eg, because of genetic heterogeneity). Results are shown in Figure 1D. While the height of the linkage peak has no more formal interpretation in that situation, its shape shows that the plateau observed between 11 and 20 Mb in Figure 1C is likely part of the region of interest. This region of interest, defined as displaying a LOD score of >2.2 in Figure 1C, extends from 11.6 Mb to 33.1 Mb and contains 194 genes (Supplementary Table 3). Of note, WT1 located at 32.4 Mb is the closest gene to rs2057178, a SNP found to be associated with pulmonary tuberculosis in several genome-wide association studies performed in different populations [12, 13]. Identification of genetic variant(s) underlying the linkage peak is ongoing following the same strategy (based on ultra-fine linkage disequilibrium mapping) as the one successfully used for pulmonary tuberculosis [3].

While the phenotype of interest was similar in the South African and French samples (ie, TST negativity, defined using a stringent cutoff of a 0-mm induration), the 2 studies were otherwise remarkably different. The South African study took place in an area hyperendemic for tuberculosis [7], where the transmission of *M. tuberculosis* occurs preferentially in the community, and with no requirement for the enrolled individuals to be household contacts of tuberculosis cases. By contrast, France is

a country with low tuberculosis endemicity, and the study design was a household tuberculosis contact study, which enabled us to take into account and quantify both the level of exposure and the index case infectivity. Beside the exposure context, the 2 studies also differed in terms of genetic background, as captured by the difference in ethnicities of subjects in each study. All the individuals studied in [7] belonged to the South African Coloured ethnic group, a unique highly admixed population resulting from the encounter of different founders from Africa, Europe, and Asia. By contrast, individuals in the present study belonged to several ethnic groups (white [48%], African [33%], Asian [6%], and mixed and other [13%]). Thus, replication of TST1 in such different settings suggests an important role of this locus in the control of TST negativity in humans.

The risk of developing tuberculosis for immunocompetent persons without TST reactivity despite sustained exposure to *M. tuberculosis* was previously shown to be extremely small [14]. Thus, persons with a TST induration of 0 mm are more likely not infected by *M. tuberculosis* than intrinsically deficient in mounting a delayed type hypersensitivity response. In the French cohort, the individual level of exposure to *M. tuberculosis* was variable. Some individuals were constantly exposed to a highly infectious case and are very likely naturally resistant to *M. tuberculosis* infection. Others were occasionally exposed to a moderately infectious case, and in this instance it is not

<footnote><sup>a</sup> Effect of continuous variables age and exposure was modeled using fractional polynomials (FP) as described elsewhere [7, 8]. For each variable, first degree (FP1) and second degree (FP2) FP models were fitted with power p for FP1 and p and q for FP2 chosen from -2, -1, -0.5, 0, 0.5, 1, 2, and 3, with 0 denoting log transformation. Among the 44 possible combinations, the model providing the smaller Akaike information criterion was selected. FP2 models with p = 1 and q = 1 and p = 0.5 and q = 2 were selected for age and exposure, respectively. For interpretability, category-based estimates are replaced with those from the regression model at relevant age and exposure length.</footnote>

<footnote><sup>b</sup> Defined as cavitation on the chest radiograph or bacillary in sputum smears.</footnote>
<figure><img src="image_2.png" /><figcaption>**Figure 1.** Genome-wide model-free linkage analysis of tuberculin skin test negativity (TST induration diameter, 0 mm vs >0 mm) in the French (A) and the combined French and South-African samples (B). Multipoint evidence of linkage expressed as a classical LOD score (black line; left y-axis) and the information content (red line; right y-axis) are plotted along the 22 autosomes. The 2 dotted horizontal lines indicate the formal cutoffs for genome-wide suggestive or significant linkage. C, Expanded view of the region with the highest LOD score in the combined analysis (y-axis) along chromosome 11 from 0 to 40 Mb (x-axis). The multipoint evidence of linkage (black line) and information content at marker positions (red line) are given. Left and right y-axes indicate the LOD score and information content, respectively. A blue diamond indicates the *TNF1* locus position. D, Multipoint evidence of linkage in the combined sample restricted to 114 families contributing to the linkage peak on 11p14-15. A family was considered as contributing positively to the linkage peak when the maximal positive LOD score contribution was greater than the absolute value of the maximal negative LOD score contribution within the 10–33-Mb interval on chromosome 11. A blue diamond indicates the *TNF1* locus position. Abbreviation: TNF, tumor necrosis factor.</figcaption></figure>

possible to distinguish between lack of exposure and resistance to infection. However, since lack of exposure is difficult to reconcile with a genetic cause, the most parsimonious

explanation is that the 11p14-15 locus reflects an innate mechanism of resistance to *M. tuberculosis* infection. Such a mechanism is an attractive working avenue for prevention as it could
<figure><img src="image_2.png" /><figcaption>**Figure 1.** Genome-wide model-free linkage analysis of tuberculin skin test negativity (TST induration diameter, 0 mm vs >0 mm) in the French (A) and the combined French and South-African samples (B). Multipoint evidence of linkage expressed as a classical LOD score (black line; left y-axis) and the information content (red line; right y-axis) are plotted along the 22 autosomes. The 2 dotted horizontal lines indicate the formal cutoffs for genome-wide suggestive or significant linkage. C, Expanded view of the region with the highest LOD score in the combined analysis (y-axis) along chromosome 11 from 0 to 40 Mb (x-axis). The multipoint evidence of linkage (black line) and information content at marker positions (red line) are given. Left and right y-axes indicate the LOD score and information content, respectively. A blue diamond indicates the *TNF1* locus position. D, Multipoint evidence of linkage in the combined sample restricted to 114 families contributing to the linkage peak on 11p14-15. A family was considered as contributing positively to the linkage peak when the maximal positive LOD score contribution was greater than the absolute value of the maximal negative LOD score contribution within the 10–33-Mb interval on chromosome 11. A blue diamond indicates the *TNF1* locus position. Abbreviation: TNF, tumor necrosis factor.</figcaption></figure>

possible to distinguish between lack of exposure and resistance to infection. However, since lack of exposure is difficult to reconcile with a genetic cause, the most parsimonious

explanation is that the 11p14-15 locus reflects an innate mechanism of resistance to *M. tuberculosis* infection. Such a mechanism is an attractive working avenue for prevention as it could
be an efficient target to protect persons from infection with highly drug resistant *M. tuberculosis* strains or those with a weakened acquired immunity arm, such as human immunodeficiency virus–infected subjects.

Interestingly, the major locus at chromosomal region 11p14-15 overlaps the *TNF1* locus identified for mycobacteria-driven tumor necrosis factor α (TNF-α) production by leukocytes in the South African family sample (Figure 1C and 1D) [15]. TNF is a key effector molecule of tuberculosis resistance that is critical for the sequestration of *M. tuberculosis* in infectious granuloma during latent *M. tuberculosis* infection [1]. Hence, the overlap of *TST1* with *TNF1* provides additional support for the interpretation of the *TST1* locus as conferring resistance to *M. tuberculosis* infection. In addition, the observation that the suggestive locus on chromosomal region 19q13 mapped in the vicinity of a locus also found to be linked to TNF production in the South African family sample (*P* < .01 from 58.1 to 59.1 Mb [15]) reinforces the hypothesis of a genetic connection between TST negativity and TNF production.

## Supplementary Data

Supplementary materials are available at *The Journal of Infectious Diseases* online (http://jid.oxfordjournals.org). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.

## Notes

**Acknowledgments.** We thank all members of the community who participated in this study and the Centre National de Génotypage for providing the genotypes.

**Financial support.** This work was supported by the Programme Hospitalier de Recherche Clinique (AOR 04 003), the Legs Poix, the French National Agency for Research, the European Research Council (ERC-2010-AdG-268777), the Rockefeller University, Institut National de la Santé et de la Recherche Médicale, Paris Descartes University, the St. Giles Foundation, the Canadian Institutes of Health Research, and the Government of Canada (Banting postdoctoral fellowship 112932 to A. C.).

**Potential conflicts of interest.** All authors: No reported conflicts.

All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

## References

1. O'Garra A, Redford PS, McNab FW, Bloom CI, Wilkinson RJ, Berry MP. The immune response in tuberculosis. Annu Rev Immunol **2013**; 31:475–527.

2. Casanova JL, Abel L. Genetic dissection of immunity to mycobacteria: the human model. Annu Rev Immunol **2002**; 20:581–620.

3. Grant AV, El Baghdadi J, Sabri A, et al. Age-dependent association between pulmonary tuberculosis and common TOX variants in the 8q12–13 linkage region. Am J Hum Genet **2013**; 92:407–14.

4. Marks SM, Taylor Z, Qualls NL, Shrestha-Kuwahara RJ, Wilce MA, Nguyen CH. Outcomes of contact investigations of infectious tuberculosis patients. Am J Respir Crit Care Med **2000**; 162:2033–8.

5. Cobat A, Barrera LF, Henao H, et al. Tuberculin skin test reactivity is dependent on host genetic background in Colombian tuberculosis household contacts. Clin Infect Dis **2012**; 54:968–71.

6. Cobat A, Gallant CJ, Simkin L, et al. High heritability of antimycobacterial immunity in an area of hyperendemicity for tuberculosis disease. J Infect Dis **2010**; 201:15–9.

7. Cobat A, Gallant CJ, Simkin L, et al. Two loci control tuberculin skin test reactivity in an area hyperendemic for tuberculosis. J Exp Med **2009**; 206:2583–91.

8. Aissa K, Madhi F, Ronsin N, et al. Evaluation of a model for efficient screening of tuberculosis contact subjects. Am J Respir Crit Care Med **2008**; 177:1041–7.

9. Alcais A, Abel L. Maximum-Likelihood-Binomial method for genetic model-free linkage analysis of quantitative traits in sibships. Genet Epidemiol **1999**; 17:102–17.

10. Cobat A, Abel L, Alcais A. The Maximum-Likelihood-Binomial method revisited: a robust approach for model-free linkage analysis of quantitative traits in large sibships. Genet Epidemiol **2011**; 35:46–56.

11. Roberts SB, MacLean CJ, Neale MC, Eaves LJ, Kendler KS. Replication of linkage studies of complex traits: an examination of variation in location estimates. Am J Hum Genet **1999**; 65:876–84.

12. Chimusa ER, Zaitlen N, Daya M, et al. Genome-wide association study of ancestry-specific TB risk in the South African Coloured population. Hum Mol Genet **2014**; 23:796–809.

13. Thye T, Owusu-Dabo E, Vannberg FO, et al. Common variants at 11p13 are associated with susceptibility to tuberculosis. Nat Genet **2012**; 44:257–9.

14. Rose DN, Schechter CB, Adler JJ. Interpretation of the tuberculin skin test. J Gen Intern Med **1995**; 10:635–42.

15. Cobat A, Hoal EG, Gallant CJ, et al. Identification of a major locus, TNF1, that controls BCG-triggered tumor necrosis factor production by leukocytes in an area hyperendemic for tuberculosis. Clin Infect Dis **2013**; 57:963–70.
be an efficient target to protect persons from infection with highly drug resistant *M. tuberculosis* strains or those with a weakened acquired immunity arm, such as human immunodeficiency virus–infected subjects.

Interestingly, the major locus at chromosomal region 11p14-15 overlaps the *TNF1* locus identified for mycobacteria-driven tumor necrosis factor α (TNF-α) production by leukocytes in the South African family sample (Figure 1C and 1D) [15]. TNF is a key effector molecule of tuberculosis resistance that is critical for the sequestration of *M. tuberculosis* in infectious granuloma during latent *M. tuberculosis* infection [1]. Hence, the overlap of *TST1* with *TNF1* provides additional support for the interpretation of the *TST1* locus as conferring resistance to *M. tuberculosis* infection. In addition, the observation that the suggestive locus on chromosomal region 19q13 mapped in the vicinity of a locus also found to be linked to TNF production in the South African family sample (*P* < .01 from 58.1 to 59.1 Mb [15]) reinforces the hypothesis of a genetic connection between TST negativity and TNF production.

## Supplementary Data

Supplementary materials are available at *The Journal of Infectious Diseases* online (http://jid.oxfordjournals.org). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.

## Notes

**Acknowledgments.** We thank all members of the community who participated in this study and the Centre National de Génotypage for providing the genotypes.

**Financial support.** This work was supported by the Programme Hospitalier de Recherche Clinique (AOR 04 003), the Legs Poix, the French National Agency for Research, the European Research Council (ERC-2010-AdG-268777), the Rockefeller University, Institut National de la Santé et de la Recherche Médicale, Paris Descartes University, the St. Giles Foundation, the Canadian Institutes of Health Research, and the Government of Canada (Banting postdoctoral fellowship 112932 to A. C.).

**Potential conflicts of interest.** All authors: No reported conflicts.

All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

## References

1. O'Garra A, Redford PS, McNab FW, Bloom CI, Wilkinson RJ, Berry MP. The immune response in tuberculosis. Annu Rev Immunol **2013**; 31:475–527.

2. Casanova JL, Abel L. Genetic dissection of immunity to mycobacteria: the human model. Annu Rev Immunol **2002**; 20:581–620.

3. Grant AV, El Baghdadi J, Sabri A, et al. Age-dependent association between pulmonary tuberculosis and common TOX variants in the 8q12–13 linkage region. Am J Hum Genet **2013**; 92:407–14.

4. Marks SM, Taylor Z, Qualls NL, Shrestha-Kuwahara RJ, Wilce MA, Nguyen CH. Outcomes of contact investigations of infectious tuberculosis patients. Am J Respir Crit Care Med **2000**; 162:2033–8.

5. Cobat A, Barrera LF, Henao H, et al. Tuberculin skin test reactivity is dependent on host genetic background in Colombian tuberculosis household contacts. Clin Infect Dis **2012**; 54:968–71.

6. Cobat A, Gallant CJ, Simkin L, et al. High heritability of antimycobacterial immunity in an area of hyperendemicity for tuberculosis disease. J Infect Dis **2010**; 201:15–9.

7. Cobat A, Gallant CJ, Simkin L, et al. Two loci control tuberculin skin test reactivity in an area hyperendemic for tuberculosis. J Exp Med **2009**; 206:2583–91.

8. Aissa K, Madhi F, Ronsin N, et al. Evaluation of a model for efficient screening of tuberculosis contact subjects. Am J Respir Crit Care Med **2008**; 177:1041–7.

9. Alcais A, Abel L. Maximum-Likelihood-Binomial method for genetic model-free linkage analysis of quantitative traits in sibships. Genet Epidemiol **1999**; 17:102–17.

10. Cobat A, Abel L, Alcais A. The Maximum-Likelihood-Binomial method revisited: a robust approach for model-free linkage analysis of quantitative traits in large sibships. Genet Epidemiol **2011**; 35:46–56.

11. Roberts SB, MacLean CJ, Neale MC, Eaves LJ, Kendler KS. Replication of linkage studies of complex traits: an examination of variation in location estimates. Am J Hum Genet **1999**; 65:876–84.

12. Chimusa ER, Zaitlen N, Daya M, et al. Genome-wide association study of ancestry-specific TB risk in the South African Coloured population. Hum Mol Genet **2014**; 23:796–809.

13. Thye T, Owusu-Dabo E, Vannberg FO, et al. Common variants at 11p13 are associated with susceptibility to tuberculosis. Nat Genet **2012**; 44:257–9.

14. Rose DN, Schechter CB, Adler JJ. Interpretation of the tuberculin skin test. J Gen Intern Med **1995**; 10:635–42.

15. Cobat A, Hoal EG, Gallant CJ, et al. Identification of a major locus, TNF1, that controls BCG-triggered tumor necrosis factor production by leukocytes in an area hyperendemic for tuberculosis. Clin Infect Dis **2013**; 57:963–70.
# EVIDENCE SUPPORTS THE HIGH BARRIER TO RESISTANCE OF DOVATO UP TO 5 YEARS¹⁻³

## CONFIDENCE IN DOVATO ACROSS TREATMENT SETTINGS⁴⁻⁹

Treatment-naïve resistance rates, with up to

3 years of evidence⁵⁻⁷

0% (n=0/1,885)*,⁴
REAL-WORLD EVIDENCE

0.1% (n=1/953)*,¹,²,⁵,⁷
RANDOMISED CONTROLLED TRIALS

Treatment-experienced resistance rates, with up to

5 years of evidence¹⁻³

0.03% REAL-WORLD EVIDENCE

0% (n=10/35,888)*,⁴
RANDOMISED CONTROLLED TRIALS

## >300,000 PEOPLE LIVING WITH HIV HAVE BEEN TREATED WITH DOVATO GLOBALLY¹⁰

DOVATO is supported by a wealth of evidence, with the outcomes of >40,000 people living with HIV captured within clinical trials and real-world evidence, including those with:⁴⁻⁹,¹¹,¹²

<figure><img src="image_3.png" /></figure>

NO PRIOR TREATMENT EXPERIENCE¹³

<figure><img src="image_4.png" /></figure>

NO BASELINE RESISTANCE TESTING¹³

<figure><img src="image_5.png" /></figure>

HIGH BASELINE VIRAL LOAD (>100,000 copies/mL and even >1M copies/mL)⁶,¹³

<figure><img src="image_6.png" /></figure>

LOW CD4 + COUNT (≤200 cells/mm³)¹³

■ Patients from phase III RCTs

■ Patients from unique real-world cohorts

<figure><img src="image_7.png" /></figure>

## IS IT TIME TO RECONSIDER THE VALUE OF THE 2ND NRTI?

LEARN MORE

<figure><img src="image_8.png" /></figure>

DOVATO is indicated for the treatment of Human Immunodeficiency Virus type 1 (HIV-1) infection in adults and adolescents above 12 years of age weighing at least 40 kg, with no known or suspected resistance to the integrase inhibitor class, or lamivudine.¹³

Adverse events should be reported. Reporting forms and information can be found at https://yellowcard.mhra.gov.uk/ or search for MHRA Yellowcard in the Google Play or Apple App store. Adverse events should also be reported to GSK on 0800 221441

### REFERENCES

1. Maggiolo F et al. BMC Infect Dis 2022; 22(1): 782.

2. Taramasso L et al. AIDS Patient Care STDs 2021; 35(9): 342–353.

3. Cicullo A et al. JAIDS 2021; 88(3): 234–237.

4. Viiv Healthcare. Data on File. REF-223795. 2024.

5. Cahn P et al. AIDS 2022; 36(1): 39–48.

6. Rolle C et al. Open Forum Infect Dis 2023; 10(3): ofad101.

7. Cordova E et al. Poster presented at 12th IAS Conference on HIV Science. 23–26 July 2023. Brisbane, Australia. TUPEB02.

8. De Wit S et al. Slides presented at HIV Glasgow. 23–26 October 2022. Virtual and Glasgow, UK. M041.

9. Llibre J et al. Clin Infect Dis 2023; 76(4): 720–729.

10. Viiv Healthcare. Data on File. REF-220949. 2024.

11. Rolle C et al. Poster presented IDWeek. 11–15 October 2023. Virtual and Boston, USA. 1603.

12. Slim J et al. Abstract presented IDWeek. 11–15 October 2023. Virtual and Boston, USA. 1593.

13. DOVATO. Summary of Product Characteristics. June 2023.

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©2024 Viiv Healthcare group of companies or its licensor. All rights reserved.

Intended for healthcare professionals only.

### ABBREVIATIONS

**3TC, lamivudine; CD4, cluster of differentiation 4; DTG, dolutegravir; FDA, United States Food and Drug Administration; FTC, emtricitabine; HIV, human immunodeficiency virus; ITT-E, intention-to-treat exposed; NRTI, nucleoside/nucleotide reverse transcriptase inhibitor; RCT, randomised controlled trial; RNA, ribonucleic acid; TAF, tenofovir alafenamide fumarate; TDF, tenofovir disoproxil fumarate; XTC, emtricitabine.

### FOOTNOTES

*Data extracted from a systematic literature review of DTG+3TC real-world evidence. Overlap between cohorts cannot be fully excluded.

**The reported rate reflects the sum-total of resistance cases calculated from GEMINI I and II (n=1/716, through 144 weeks), STAT (n=0/131, through 52 weeks), and D2ARLING (n=0/106, through 24 weeks).⁵⁻⁷

†GEMINI I and II are two identical 148-week, phase III, randomised, double-blind, multicentre, parallel-group, non-inferiority, controlled clinical trials testing the efficacy of DTG/3TC in treatment-naïve patients. Participants with screening HIV-1 RNA ≤500,000 copies/mL were randomised 1:1 to once-daily DTG/3TC (n=716, pooled) or DTG + TDF/FTC (n=717, pooled). The primary endpoint of each GEMINI study was the proportion of participants with plasma HIV-1 RNA <50 copies/mL at Week 48 (ITT-E population, snapshot algorithm).¹³

‡STAT is a phase IIIb, open-label, 48-week, single-arm pilot study evaluating the feasibility, efficacy, and safety of DTG/3TC in 131 newly diagnosed HIV-1 infected adults as a first line regimen. The primary endpoint was the proportion of participants with plasma HIV-1 RNA <50 copies/mL at Week 24.⁶

§2ARLING is a randomised, open-label, phase IV study designed to assess the efficacy and safety of DTG/3TC in treatment-naïve people with HIV with no available baseline HIV-1 resistance testing. Participants were randomised in a 1:1 ratio to receive DTG/3TC (n=106) or DTG + TDF/XTC (n=108). The primary endpoint was the proportion of participants with plasma HIV-1 RNA <50 copies/mL at Week 48.⁷ Results at week 24 of the study.

|| The reported rate reflects the sum-total of resistance cases calculated from TANGO (n=0/369, through 196 weeks) and SALSA (n=0/246, through 48 weeks).⁸,⁹

¶TANGO is a randomised, open-label, trial testing the efficacy of DOVATO in virologically suppressed patients. Participants were randomised in a 1:1 ratio to receive DOVATO (n=369) or continue with TAF-containing regimens (n=372) for up to 200 weeks. At Week 148, 298 of those on TAF-based regimens switched to DOVATO. The primary efficacy endpoint was the proportion of subjects with plasma HIV-1 RNA ≥50 copies/mL (virologic non-response) as per the FDA Snapshot category at Week 48 (adjusted for randomisation stratification factor).⁸,¹³

#SALSA is a phase III, randomised, open-label, non-inferiority clinical trial evaluating the efficacy and safety of switching to DTG/3TC compared with continuing current antiretroviral regimens in virologically suppressed adults with HIV. Eligible participants were randomised 1:1 to switch to once-daily DTG/3TC (n=246) or continue current antiretroviral regimens (n=247). The primary endpoint was the proportion of subjects with plasma HIV-1 RNA ≥50 copies/mL at Week 48 (ITT-E population, snapshot algorithm).⁹
# EVIDENCE SUPPORTS THE HIGH BARRIER TO RESISTANCE OF DOVATO UP TO 5 YEARS¹⁻³

## CONFIDENCE IN DOVATO ACROSS TREATMENT SETTINGS⁴⁻⁹

Treatment-naïve resistance rates, with up to

3 years of evidence⁵⁻⁷

0%

(n=0/1,885)*,⁴

0.1%

(n=1/953)*,¹,¹,¹,⁵⁻⁷

Treatment-experienced resistance rates, with up to

5 years of evidence¹⁻³

0.03%

(n=10/35,888)*,⁴

0%

(n=0/615)¹,⁸,⁹

REAL-WORLD EVIDENCE

RANDOMISED CONTROLLED TRIALS

## >300,000 PEOPLE LIVING WITH HIV HAVE BEEN TREATED WITH DOVATO GLOBALLY¹⁰

DOVATO is supported by a wealth of evidence, with the outcomes of >40,000 people living with HIV captured within clinical trials and real-world evidence, including those with:⁴⁻⁹,¹¹,¹²

<figure><img src="image_3.png" /></figure>

NO PRIOR TREATMENT EXPERIENCE¹³

<figure><img src="image_4.png" /></figure>

NO BASELINE RESISTANCE TESTING¹³

<figure><img src="image_5.png" /></figure>

HIGH BASELINE VIRAL LOAD (>100,000 copies/mL and even >1M copies/mL)⁶,¹³

<figure><img src="image_6.png" /></figure>

LOW CD4 + COUNT (≤200 cells/mm³)¹³

■ Patients from phase III RCTs

■ Patients from unique real-world cohorts

<figure><img src="image_7.png" /></figure>

## IS IT TIME TO RECONSIDER THE VALUE OF THE 2ND NRTI?

LEARN MORE

<figure><img src="image_8.png" /></figure>

DOVATO is indicated for the treatment of Human Immunodeficiency Virus type 1 (HIV-1) infection in adults and adolescents above 12 years of age weighing at least 40 kg, with no known or suspected resistance to the integrase inhibitor class, or lamivudine.¹³

Adverse events should be reported. Reporting forms and information can be found at https://yellowcard.mhra.gov.uk/ or search for MHRA Yellowcard in the Google Play or Apple App store. Adverse events should also be reported to GSK on 0800 221441

### REFERENCES

1. Maggiolo F et al. BMC Infect Dis 2022; 22(1): 782.

2. Taramasso L et al. AIDS Patient Care STDs 2021; 35(9): 342–353.

3. Cicullo A et al. JAIDS 2021; 88(3): 234–237.

4. Viiv Healthcare. Data on File. REF-223795. 2024.

5. Cahn P et al. AIDS 2022; 36(1): 39–48.

6. Rolle C et al. Open Forum Infect Dis 2023; 10(3): ofad101.

7. Cordova E et al. Poster presented at 12th IAS Conference on HIV Science. 23–26 July 2023. Brisbane, Australia. TUPEB02.

8. De Wit S et al. Slides presented at HIV Glasgow. 23–26 October 2022. Virtual and Glasgow, UK. M041.

9. Llibre J et al. Clin Infect Dis 2023; 76(4): 720–729.

10. Viiv Healthcare. Data on File. REF-220949. 2024.

11. Rolle C et al. Poster presented IDWeek. 11–15 October 2023. Virtual and Boston, USA. 1603.

12. Slim J et al. Abstract presented IDWeek. 11–15 October 2023. Virtual and Boston, USA. 1593.

13. DOVATO. Summary of Product Characteristics. June 2023.

### PRESCRIBING INFORMATION

Dovato Prescribing Information

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Viiv Healthcare, 980 Great West Road, Brentford, Middlesex, London, UK.

Viiv trademarks are owned by or licensed to the Viiv Healthcare group of companies.

Non-ViivV trademarks are owned by or licensed to their respective owners or licensors.

©2024 Viiv Healthcare group of companies or its licensor. All rights reserved.

Intended for healthcare professionals only.

### ABBREVIATIONS

**3TC**, lamivudine; **CD4**, cluster of differentiation 4; **DTG**, dolutegravir; **FDA**, United States Food and Drug Administration; **FTC**, emtricitabine; **HIV**, human immunodeficiency virus; **ITT-E**, intention-to-treat exposed; **NRTI**, nucleoside/nucleotide reverse transcriptase inhibitor; **RCT**, randomised controlled trial; **RNA**, ribonucleic acid; **TAF**, tenofovir alafenamide fumarate; **TDF**, tenofovir disoproxil fumarate; **XTC**, emtricitabine.

### FOOTNOTES

*Data extracted from a systematic literature review of DTG+3TC real-world evidence. Overlap between cohorts cannot be fully excluded.

**The reported rate reflects the sum-total of resistance cases calculated from GEMINI I and II (n=1/716, through 144 weeks), STAT (n=0/131, through 52 weeks), and D2ARLING (n=0/106, through 24 weeks).⁵⁻⁷

†GEMINI I and II are two identical 148-week, phase III, randomised, double-blind, multicentre, parallel-group, non-inferiority, controlled clinical trials testing the efficacy of DTG/3TC in treatment-naïve patients. Participants with screening HIV-1 RNA ≤500,000 copies/mL were randomised 1:1 to once-daily DTG/3TC (n=716, pooled) or DTG + TDF/FTC (n=717, pooled). The primary endpoint of each GEMINI study was the proportion of participants with plasma HIV-1 RNA <50 copies/mL at Week 48 (ITT-E population, snapshot algorithm).¹³

‡STAT is a phase IIIb, open-label, 48-week, single-arm pilot study evaluating the feasibility, efficacy, and safety of DTG/3TC in 131 newly diagnosed HIV-1 infected adults as a first line regimen. The primary endpoint was the proportion of participants with plasma HIV-1 RNA <50 copies/mL at Week 24.⁶

§2ARLING is a randomised, open-label, phase IV study designed to assess the efficacy and safety of DTG/3TC in treatment-naïve people with HIV with no available baseline HIV-1 resistance testing. Participants were randomised in a 1:1 ratio to receive DTG/3TC (n=106) or DTG + TDF/XTC (n=108). The primary endpoint was the proportion of participants with plasma HIV-1 RNA <50 copies/mL at Week 48.⁷ Results at week 24 of the study.

|| The reported rate reflects the sum-total of resistance cases calculated from TANGO (n=0/369, through 196 weeks) and SALSA (n=0/246, through 48 weeks).⁸,⁹

¶TANGO is a randomised, open-label, trial testing the efficacy of DOVATO in virologically suppressed patients. Participants were randomised in a 1:1 ratio to receive DOVATO (n=369) or continue with TAF-containing regimens (n=372) for up to 200 weeks. At Week 148, 298 of those on TAF-based regimens switched to DOVATO. The primary efficacy endpoint was the proportion of subjects with plasma HIV-1 RNA ≥50 copies/mL (virologic non-response) as per the FDA Snapshot category at Week 48 (adjusted for randomisation stratification factor).⁸,¹³

#SALSA is a phase III, randomised, open-label, non-inferiority clinical trial evaluating the efficacy and safety of switching to DTG/3TC compared with continuing current antiretroviral regimens in virologically suppressed adults with HIV. Eligible participants were randomised 1:1 to switch to once-daily DTG/3TC (n=246) or continue current antiretroviral regimens (n=247). The primary endpoint was the proportion of subjects with plasma HIV-1 RNA ≥50 copies/mL at Week 48 (ITT-E population, snapshot algorithm).⁹