19006197v1 Full

medRxiv preprint doi: https://doi.org/10.1101/19006197.this version posted September 9, 2019.
The copyright holder for this preprint (which

was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-NC-ND 4.0 International license .
RISK6, a universal 6-gene transcriptomic signature of TB disease risk, diagnosis and

treatment response
Adam Penn-Nicholson1*, Stanley Kimbung Mbandi1*, Ethan Thompson2*, Simon C.

Mendelsohn1*, Sara Suliman1,3, Novel N. Chegou4, Stephanus T. Malherbe4, Fatoumatta
Darboe1, Mzwandile Erasmus1, Willem A. Hanekom1, Nicole Bilek1, Michelle Fisher1, Stefan H.
E. Kaufmann5,6, Jill Winter7, Melissa Murphy1, Robin Wood8, Carl Morrow8, Ildiko Van Rhijn3,
Branch Moody3, Megan Murray9, Bruno B. Andrade10, Timothy R. Sterling11, Jayne
Sutherland12, Kogieleum Naidoo13,14, Nesri Padayatchi13,14, Gerhard Walzl4, Mark Hatherill1,
Daniel Zak2, Thomas J. Scriba1, and the Adolescent Cohort Study team, GC6-74 Consortium,
The ScreenTB and AE-TBC teams, CAPRISA IMPRESS team, RePORT Brazil Consortium and
Peruvian Household Contacts Cohort study group.
*These authors contributed equally
Affiliations:
1
South African Tuberculosis Vaccine Initiative, Institute of Infectious Disease and Molecular
Medicine and Division of Immunology, Department of Pathology, University of Cape Town,
Cape Town, South Africa.

2
Center for Infectious Disease Research, Seattle, WA, USA.
3
Brigham and Women's Hospital, Division of Rheumatology, Immunity and Inflammation,
Harvard Medical School, Boston, USA.

4
DST-NRF Centre of Excellence for Biomedical Tuberculosis Research; South African Medical
Research Council Centre for Tuberculosis Research; Division of Molecular Biology and Human
Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South
Africa.
5
Max Planck Institute for Infection Biology, Berlin, Germany.
1
medRxiv preprint doi: https://doi.org/10.1101/19006197.this version posted September 9, 2019. The copyright holder for this preprint (which
6
Hagler Institute for Advanced Study at Texas A&M University, College Station, TX, USA.
7
Catalysis Foundation for Health, San Ramon, CA, USA.
8
Desmond Tutu HIV Centre, and Institute of Infectious Disease and Molecular Medicine (IDM),
University of Cape Town, Cape Town, South Africa.

9
Department of Global Health and Social Medicine, and Division of Global Health Equity,
Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA.
10
Instituto Gonçalo Moniz, Fundação Oswaldo Cruz, Salvador, Brazil.
11
Division of Infectious Diseases, Department of Medicine, Vanderbilt University School of
Medicine, Nashville, USA.

12
Vaccines and Immunity, Medical Research Council Unit, Fajara, the Gambia.
13
Centre for the AIDS Programme of Research in Africa, Durban, South Africa.
14
South African Medical Research Council-CAPRISA HIV-TB Pathogenesis and Treatment
Research Unit, Durban, South Africa.
Corresponding Author: Thomas J. Scriba. Email: thomas.scriba@uct.ac.za
Short title: RISK6 signature for TB
2
ABSTRACT
Improved tuberculosis diagnostics and tools for monitoring treatment response are urgently
needed. We developed a robust and simple, PCR-based host-blood transcriptomic signature,
RISK6, for multiple applications: identifying individuals at risk of incident disease, as a
screening test for subclinical or clinical tuberculosis, and for monitoring tuberculosis treatment.
RISK6 utility was validated by blind prediction using quantitative real-time (qRT) PCR in seven
independent cohorts.
Prognostic performance significantly exceeded that of previous signatures discovered in the
same cohort. Performance for diagnosing subclinical and clinical disease in HIV-uninfected and
HIV-infected persons, assessed by area under the receiver-operating characteristic curve,
exceeded 85%. As a screening test for tuberculosis, the sensitivity at 90% specificity met or
approached the benchmarks set out in World Health Organization target product profiles for non-
sputum-based tests. RISK6 scores correlated with lung immunopathology activity, measured by
positron emission tomography, and tracked treatment response, demonstrating utility as
treatment response biomarker, while predicting treatment failure prior to treatment initiation.
Performance of the test in capillary blood samples collected by finger-prick was noninferior to
venous blood collected in PAXgene tubes. These results support incorporation of RISK6 into
rapid, capillary blood-based point-of-care PCR devices for prospective assessment in field
studies.
3
INTRODUCTION
The “End Tuberculosis Strategy” of the World Health Organization (WHO) aims to reduce the
annual incidence of tuberculosis (TB) to less than 10 cases per 100,000 people by the year 20351.
To achieve this goal the primary proposed strategy is to increase and improve efforts to find and
treat individuals with active TB disease, to conduct universal screening of those at high risk, and
to provide preventive therapy to those at risk of progressing to active TB disease1. There is thus a
need for improved prognostic and diagnostic tests to identify those at risk of incident TB and
those with subclinical or active TB, for appropriate treatment. The provision and management of
TB treatment, as well as monitoring a patient’s response to treatment, also require much
improvement. The standard 6-month regimen of treatment appears to be unnecessarily long for
many patients with drug-susceptible TB, while insufficient to cure some patients, even in clinical
trials when treatment adherence is maximized2. Experimental regimens tested in recent clinical
trials have also been inadequate to cure treatment-refractory patients3. Collectively, these data
support the now accepted principle that TB exists in a pathophysiological spectrum that spans
several stages of infection, subclinical and active disease, including distinct stages of treatment
outcome. Achieving the “End Tuberculosis Strategy” clearly depends on approaches that can
place an individual into the stage of this spectrum such that clinical management is appropriate.
A universal, non-sputum biomarker capable of predicting progression to active TB, diagnosing
disease and monitoring the response to TB treatment would be a major advance in the efforts to
achieve the “End Tuberculosis Strategy”. We hypothesized that a single, parsimonious host-
blood transcriptomic signature can be developed for all three purposes with performance criteria
4
that meet the target product profiles for tests to predict TB progression4 and for a TB screening
test5 proposed by the WHO.
We sought to discover and validate a parsimonious and robust blood transcriptomic signature
with universal applicability for predicting incident TB, as a triage test for identifying those who
should be further investigated for TB disease, and for monitoring of TB treatment response. We
explicitly set out to develop this signature for ultimate translation to a hand-held point-of-care
platform and therefore performed all analyses, including signature training and performance
assessments in all validation cohorts, by quantitative RT-PCR, using a highly standardized
protocol and locked-down analysis algorithm. We assessed performance of RISK6 by blind
prediction as a prognostic test for incident TB, as a TB diagnostic in HIV-uninfected and HIV-
infected individuals, including individuals presenting with symptoms requiring investigation for
TB at primary health care centres, and as a treatment response biomarker. We also tested the
robustness of RISK6 and report performance of RISK6 measured in capillary blood samples
collected by finger-prick, facilitating the way for incorporation into point-of-care diagnostic
devices.
5
RESULTS
Prognostic performance of RISK6 in the Adolescent Cohort Study discovery cohort
The RISK6 signature was discovered on samples from adolescent progressors and controls
(Supplementary Figure 1a) by selecting the smallest set of transcripts with the best prognostic
performance based on qRT-PCR data, and comprises an ensemble of 9 transcript pairs formed
between three transcripts upregulated in progressors (GBP2, FCGR1B, and SERPING1), and
three transcripts downregulated in progressors (TUBGCP6, TRMT2A and SDR39U1), relative to
non-progressors (Supplementary Table 1 and Figure 1a-b). We first sought to determine if the
prognostic performance of RISK6 for incident TB in the discovery cohort was comparable to that
of the previously published ACS 16-gene signature6, consisting of 57 transcripts (PCR
primer/probe assays) or the ACS 11-gene version (48 PCR primer/probe assays), which was
developed for greater throughput in multiplex assays7. The PCR-based RISK6 and both ACS
signatures readily discriminated between Adolescent Cohort Study progressor and non-
progressor samples collected within 12 months of TB diagnosis (Figure 1c and Supplementary
Table 2). Interestingly, prognostic performance of RISK6, estimated by model fit (AUC 87.6%,
95%CI 82.8 - 92.4), was significantly better than ACS 16-gene (AUC 81.8%, 95%CI 75.1 - 88.6,
pROC analysis p = 0.024) and ACS 11-gene (AUC 82.2%, 95%CI 75.6 - 88.8, pROC analysis p
= 0.03). As observed previously with the 16-gene signature6, RISK6 also discriminated between
progressors and non-progressors using samples collected between 12 and 24 months before TB
disease diagnosis (AUC 74.0%, 95%CI 66.0 - 82.0), although discrimination was weaker than
observed on samples within a year of diagnosis (Figure 1d).
Validation of RISK6 prognostic performance in the GC6-74 cohort
6
We validated prognostic performance of the RISK6 signature for incident TB by blind prediction
on the independent GC6-74 cohorts of household TB contacts from South Africa, The Gambia
and Ethiopia, who either progressed to TB or remained asymptomatic8. RISK6 significantly
discriminated between GC6-74 progressors and non-progressors on samples collected within 12
months of incident TB diagnosis (AUC 70.6%, 95%CI 61.6 - 79.5) and those collected 12-24
months before TB diagnosis (AUC 67.6%, 95%CI 58.2 - 76.9, Figure 1e and Table 1). At a
sensitivity threshold of 75%, RISK6 achieved a specificity of 50.3% within 1 year of diagnosis
in the GC6-74 cohort, which does not meet the WHO target product profile for a test predicting
progression from tuberculosis infection to active disease4 (Table 2 and Supplementary Table
2).
Since RISK6 was discovered on a South African cohort it was important to determine if
performance varies by geography, since differences in population genetic structure, local
epidemiology and environment may influence blood biomarker performance9. We therefore also
assessed prognostic performance by country. Interestingly, when assessing samples collected
within 12 months of TB diagnosis, the AUC was highest for the Gambian cohort (AUC 76.3%,
95%CI 64.4% - 88.1%, Figure 1f and Table 1), while the AUC for the South African cohort was
similar to the entire, combined GC6-74 cohort (AUC 69.9%, 95%CI 55.5 - 84.2, Figure 1f).
Although the AUC for the smaller Ethiopian cohort (comprising 12 progressors) was also similar
(AUC 69.3%, 95%CI 39.4 - 99.2), the confidence intervals were very large and discrimination
between progressors and non-progressors was not significant (Figure 1f). RISK6 also validated
on samples from the entire 24 month period prior to TB diagnosis from these 3 GC6-74 cohorts
(Table 1).
7
Performance of RISK6 as a screening test in HIV-uninfected and HIV-infected individuals
Expression levels of the six transcripts in RISK6 differed most between adolescent progressors
and non-progressors at the time of TB diagnosis (Figure 1a). In light of this, we hypothesized
that RISK6 would also yield good performance as a screening or triage test for TB. Since HIV
infection is a major risk factor for TB and a large proportion of TB patients in settings endemic
for TB are HIV-infected10, we aimed to determine diagnostic performance in both HIV-infected
and uninfected individuals. We therefore compared diagnostic performance of RISK6,
benchmarked against the ACS 11-gene signature, in 112 HIV-uninfected (61 asymptomatic
controls and 51 TB cases) and 82 HIV-infected (40 asymptomatic controls and 42 TB cases)
adults from the Western Cape, South Africa. Excellent diagnostic performance of RISK6 was
seen in both HIV-uninfected (AUC 93.7%, 95%CI 87.9-99.4%) and HIV-infected persons (AUC
92.6%, 95%CI 86.8-98.5); performance was not different between the two groups (pROC
analysis p = 0.76, Figure 2a). By contrast, diagnostic performance of the ACS 11-gene signature
was better in HIV-uninfected (AUC 97.3%, 95%CI 93.7-100) than in HIV-infected persons
(AUC 87.9%, 95% CI 80.6-95.2); the 9% lower AUC in HIV-infected persons was significant
(pROC analysis p = 0.027, Figure 2b)RISK6 signature scores were higher in HIV-infected
controls compared to HIV-uninfected controls, suggesting an effect of underlying HIV infection
on RISK6 (Figure 2c). To understand the effects of underlying HIV infection on the RISK6
signature, we determined the difference in expression of each transcript between HIV-infected
and uninfected individuals. Expression of FCGR1B and GBP2, but none of the other transcripts,
was significantly higher in HIV-infected than uninfected controls, while no significant
differences were observed in TB cases (Figure 2d). At a sensitivity threshold of 90%, RISK6
8
achieved a specificity of 93.4% and 72.5% in HIV-uninfected and HIV-infected persons,
respectively (Table 3 and Supplementary Table 2).
Diagnostic performance of RISK6 as a screening test in patients with respiratory symptoms
We also determined RISK6 performance as a screening test in symptomatic adults enrolled into
the ScreenTB 11 and AE-TBC studies12,13. These adult participants presented at primary health
care clinics in Cape Town, South Africa with symptoms requiring investigation for TB including
coughing for >2 weeks and at least another symptom consistent with TB, and were enrolled prior
to the establishment of a TB or other disease diagnosis. RISK6 signature scores were measured
by blind prediction on PAXgene blood collected at presentation for care before treatment
initiation in (1) 76 patients with microbiologically-confirmed, definite TB, (2) 7 patients with
probable TB, and (3) 210 patients with other respiratory diseases (ORD) (Figure 2e, see
Supplementary Table 4 for diagnostic criteria). RISK6 discriminated between definite TB and
ORD patients with an AUC of 84.8% (95%CI 79.6-90.0, Figure 2f). At a sensitivity threshold of
90%, RISK6 achieved a specificity of 57.1% (95%CI 32.9-76.7) in these symptomatic patients
(Table 3 and Supplementary Table 2), which falls short of the WHO target product profile for
a community-based triage or referral test to identify people suspected of having TB5. RISK6
performance did not differ between HIV positive and negative participants (HIV-neg, n = 250:
AUC 85.4%, 95%CI 79.7-91.0; HIV-pos, n = 36: AUC 79.5%, 95%CI 65.1-94.0, data not
shown).
We unpacked the performance of RISK6 further in posthoc analyses performed after unblinding
of patient diagnostic status. We noted that a considerable number (n = 121) of ScreenTB and
9
AE-TBC participants had a record of least one previous episode of TB; this is typical of patients
presenting for TB investigation in such high-incidence settings 10. RISK6 discriminated between
definite TB and ORD among participants with no history of prior TB with an AUC of 87.2%
(95%CI 80.2-94.1%), whereas in those with a history of prior TB the AUC was 82.7% (95%CI
74.6-90.7%, Table 3, Figure 2g). Although these AUCs were not statistically different, the
specificities at a set sensitivity of >90% were markedly different at 75.0% and 37.8%,
respectively (Table 3).
We also applied RISK6 to published microarray datasets, using a risk score algorithm,
RISK6geo, adapted for application to microarray or RNA-seq data. RISK6geo discriminated
between TB cases and asymptomatic M.tb-infected controls with AUCs exceeding 90% in all
cohorts (Supplementary Table 3). Comparative performance characteristics of RISK6 and
RISK6geo on qRT-PCR data from the different cohorts in this study are also shown in
Supplementary Table 2.
Performance of RISK6 as a TB treatment monitoring biomarker
Diagnostic performance of RISK6 was also assessed in the Catalysis cohort of TB patients who
were studied during and after TB treatment14-16. RISK6 achieved an AUC of 93.5 (95%CI 85.5-
100) for discriminating between newly diagnosed TB cases and asymptomatic controls (Figure
3a). Next, we determined if RISK6 has utility as a biomarker for monitoring TB treatment. We
hypothesized that RISK6 scores, which are very high in patients with active disease, would
decrease rapidly during TB treatment such that samples collected after bacteriological cure can
be discriminated from the respective pre-treatment sample with high accuracy. We also
10
hypothesized that RISK6 would allow discrimination of cured patients from those with treatment
failure after 24 weeks of treatment. When measured by qRT-PCR in patients with bacteriological
cure in the Catalysis cohort, RISK6 scores decreased significantly during TB treatment, although
scores observed at the end of treatment were still significantly higher than those observed in
healthy controls (Figure 3b), as reported for the 16-gene ACS signature previously16. Despite
this, RISK6 significantly discriminated between samples collected pre-treatment and one week
after treatment initiation (AUC 79.5%, 95% CI 72.2-86.7), four weeks after treatment initiation
(AUC 77.4%, 95% CI 69.9-84.9) and end of treatment samples (AUC 88.1%, 95% CI 82.5-93.6;
(Figure 3c). Importantly, RISK6 was a strong predictor of treatment outcome and significantly
differentiated between the 78 patients with bacteriological cure and the 7 patients with treatment
failure at the time of TB diagnosis (AUC 77.1, 95% CI 52.9-100, Figure 3d), and at the end of
treatment (AUC 95.2, 95% CI 87.5-100, Figure 3d). These data are consistent with RISK6
detecting differences in inflammatory profiles before the initiation of treatment which predict the
outcome of treatment, while also detecting ongoing inflammation in those who fail treatment and
do not achieve bacteriological cure by 24 weeks. To address this further, we determined if blood
RNA signature scores were associated with in vivo pulmonary inflammation measured by 18F-
labeled fluorodeoxyglucose (18F FDG) PET-CT. Surprisingly, RISK6 scores directly correlated
metabolic activity in lung lesions as measured by total glycolytic activity index (TGAI)
(Spearman ρ = 0.66, p < 0.0001, Figure 3e), while signature scores correlated inversely with
Xpert Ct values (Spearman ρ = -0.60, p < 0.0001) and Mycobacterial Growth Indicator Tube
(MGIT) culture days-to-positivity values (Spearman ρ = -0.67, p < 0.0001) (data not shown).
11
Performance of RISK6 as triage test and TB treatment monitoring biomarker in South
American Cohorts
An important issue is how a biosignature that was trained and validated in African cohorts will
perform in geographically distinct populations. To address this, we assessed diagnostic
performance of RISK6 measured by qRT-PCR in cohorts from Peru and Brazil. In the Peruvian
cohort, RISK6 discriminated between culture-positive TB patients and QFT-negative with an
AUC of 91.5% (95%CI 86.2-96.9, Figure 4a) and between TB patients and QFT-positive
asymptomatic controls with an AUC of 89.6% (95%CI 83.5-95.7, Figure 4b). RISK6 also
achieved an AUC of 90.9% (95%CI 85.2-96.6) for discriminating between Brazilian culture-
positive TB patients and asymptomatic controls (Figure 4c). The minimum criteria for a
screening or triage test for TB, set out by the WHO, set the sensitivity at ≥90% at a specificity of
70%5. With sensitivity set at ≥90%, the specificities for RISK6 applied to the South African
HIV-negative (Cross-sectional and Catalysis cohorts) and HIV-positive cohorts far exceeded the
criteria, with specificities above 90% (Table 3 and Supplementary Table 2). Interestingly,
performance of RISK6 in the South American cohorts also met these criteria when
discriminating between TB cases and QFT-negative controls, but discrimination between TB
cases and QFT-positive asymptomatic controls fell short of the 70% specificity mark (Table 3
and Supplementary Table 2). It was notable that the RISK6 score threshold at which the
sensitivity was ~90% was quite variable, suggesting that the positivity cut-off for such a
transcriptomic signature may be different for different cohorts.
We also determined performance of RISK6 as a biosignature for monitoring TB treatment in the
Brazilian patients, all of whom achieved microbiological cure after 6 months of treatment.
12
RISK6 scores decreased significantly after 8 weeks of TB treatment and, unlike the Catalysis
cohort, scores observed in the Brazilian patients at the end of treatment had reached levels
observed in healthy controls (Figure 4d and e). RISK6 also significantly discriminated between
samples collected pre-treatment and 8 weeks after treatment initiation (AUC 67.5%, 95% CI
56.4-78.6), and end of treatment samples (AUC 87.4%, 95% CI 79.8-94.9, Figure 4f).
RISK6 as a treatment biomarker in HIV-infected patients with recurrent TB
The promising results from these treatment response studies prompted us to also evaluate if
RISK6 can monitor success of recurrent TB treatment in HIV-infected individuals on ART, who
participated in the randomized controlled IMPRESS trial. IMPRESS determined if a retreatment
regimen that contained moxifloxacin, instead of ethambutol, would improve TB retreatment
outcomes relative to the standard regimen17. No differences in RISK6 scores were observed
between the two treatment arms of the trial (data not shown). Consequently, all analyses were
performed with the treatment arms combined. RISK6 scores decreased upon treatment (Figure
5a) and could discriminate significantly between samples collected at the pre-treatment time
point (baseline) and those collected after the intensive phase of treatment, at 2 months (AUC
75.1%, 95%CI 66.5-83.8, Figure 5b). Discrimination between baseline samples and those
collected at the end of treatment, when all patients had achieved clinical cure, was better than
after 2 months of treatment (AUC 91.2%, 95%CI 86.0-96.3, Figure 5b), although inflammation
appeared to resolve further after the end of treatment, since RISK6 discriminated best between
baseline and samples collected 6-8 months after treatment completion (AUC 98.5%, 95%CI
96.5-100, Figure 5b). When measured at baseline (not shown) or the end of the intensive
treatment phase at 2 months (AUC 63.4%, 95%CI 48.2-78.5), RISK6 did not discriminate
13
significantly between patients who had sputum culture conversion and those who converted after
2 months (Figure 5c). It was not possible to determine if RISK6 could predict treatment failure
in this trial since all patients achieved bacteriological cure.
Although HIV infection causes immunodeficiency, it also drives chronic immune activation and
inflammation18-20 and induces expression of type I IFN response, including interferon stimulated
genes (ISGs)21,22. Successful antiretroviral therapy (ART) suppresses viral replication and
reduces plasma viral load (pVL), decreasing inflammation and immune activation, although not
to levels typical of HIV-uninfected persons23. Since RISK6 includes three IFN-inducible ISG
transcripts, we aimed to evaluate the effect of pVL on signature scores in the IMPRESS trial.
Eighty-five participants had pVL measurements, 36 with detectable viral loads (above 400
copies per mL) and 49 with undetectable viral loads (below 400 copies per mL); sixty of the
measurements were baseline samples and 25 were end-of-treatment samples. RISK6 signature
scores were significantly higher in samples with detectable pVL than those with undetectable
pVL (p = 0.0027, Figure 5d), showing that pVL is a confounder in ISG-containing
transcriptomic signatures, one that could affect many patients.
Robustness of the PCR-based RISK6 signature
An advantage of the pair-wise ensemble structure of RISK6 is that a signature score can be
calculated even if one or more transcript is not detected, for example due to a failure during PCR
amplification. To determine how robust the signature is to such missing data, we compared
diagnostic performance for discriminating between HIV-uninfected TB cases and asymptomatic
controls (Figure 2) by the full 6-gene RISK6 signature, which comprises nine pairs formed
14
between six transcripts, or after removing one, two, three or four, of these transcripts such that
every combination of the pairs was tested. Diagnostic performance was not affected by removal
of a single transcript, irrespective of transcript identity (AUC for full RISK6: 93.6, 95%CI 87.4-
99.7; average AUC for 5-transcript signature: 93.2, lower 95%CI bound: 85.5%, Figure 6a).
However, removal of two or more transcripts, especially when two or more of SERPING1,
SDR39U1 or TUBGCP6 were omitted, resulted in somewhat decreased performance of RISK6
(Average AUC for 4-transcript signature: 92.4, lower 95%CI bound: 80.1; average AUC for 3-
transcript signature: 91.4, lower 95%CI bound: 72.1). A very similar result was observed when
the same analysis was performed on the Brazilian cohort (Supplementary Figure 3). These
results show that RISK6 can tolerate one or even two missing transcripts without the diagnostic
performance being markedly eroded.
Effective deployment of transcriptomic signature tests such as RISK6 in community or primary
health care settings is dependent on successful translation of gene expression to methods that are
simple, cheap and rapid. An expensive and cumbersome component of any blood transcriptomic
assay is the procedure and cost of blood collection. Therefore, we sought to determine if RISK6
could be reliably measured in very small volumes of capillary blood collected by finger stick.
We compared discrimination between healthy controls and TB cases by RISK6, measured by
qRT-PCR in 20μL, 50μL or 100μL capillary blood, benchmarked against the typical 2.5mL
venous blood collected in PAXgene tubes. Among samples collected from the 49 participants,
the number of samples with one or more failed PCR reaction, where the amplification curve for
one transcript did not pass the QC threshold defined by Fluidigm, for the 20μL, 50μL and 100μL
capillary blood volumes was 4 (8%), 3 (6%) and 3 (6%), respectively. None of the 2.5mL venous
blood samples yielded failed PCR reactions. When failure of 1 of the 6 transcripts was tolerated,
15
RISK6 scores could be calculated for 98% (1 failed sample), 98% and 100% of the 20μL, 50μL
and 100μL capillary blood samples, respectively.
RISK6 signature scores measured on 2.5mL venous blood correlated strongly with those
measured on 20μL, 50μL or 100μL capillary blood samples (Spearman rho > 0.83; Figure 6b-
d). Diagnostic performance of RISK6 was statistically non-inferior when measured in 20μL,
50μL or 100μL capillary blood samples compared with venous blood; ROC analysis yielded
equivalent AUC curves (Figure 6b-d). These results show that RISK6 can be measured on very
small volumes of capillary blood collected by finger stick, which may be amenable to translation
to a point of care testing platform.
16
DISCUSSION:
We discovered and validated RISK6, a parsimonious and robust blood transcriptomic signature
with universal applicability for predicting incident TB, as a triage test for identifying individuals
with or without respiratory symptoms who should be further investigated for TB disease, and for
monitoring the response to TB treatment.
RISK6 identified individuals at risk of progression to incident TB and met or approached the
respective benchmarks set out in WHO target product profiles for incipient TB tests4. When
applied to samples collected within 1 year of TB diagnosis in the ACS discovery cohort, RISK6
met the minimum criteria for a test for progression to TB set out by the WHO and FIND4. At a
sensitivity of ≥75% a specificity of 82.8% was observed (Table 2 and Table 3). However, when
applied to samples collected within 1 year of TB diagnosis in the GC6-74 validation cohort, the
specificity at a sensitivity of ≥75% was 50.3%, which did not meet these criteria (Table 2).
Prognostic performance for incident TB of RISK6 was significantly better than that reported for
the previously described 16-gene ACS signature6, which was discovered by RNA-seq in the ACS
progressor and non-progressor cohort. RISK6 also validated in the independent GC6-74 cohort
of TB household contacts by blind prediction. Importantly, performance of RISK6 in distinct
cohorts from 3 different African countries was similar, although RISK6 did not significantly
discriminate between progressors and non-progressors from Ethiopia, likely due to the small
number of progressors, namely 12. The limitations of such small sample sizes for biomarker
validation is also evident from other biomarker studies on the GC6-74 cohort. It was notable that
the performance of RISK6 in the three GC6-74 cohorts was very similar to the previously
published RISK4 signature (Table 2), which was specifically developed as a “pan-African
signature”8. Recent studies showed that the 16-gene ACS signature, as well as two other small
17
diagnostic signatures24,25, did not validate in either one or both of the GC6-74 validation sub-
cohorts of Gambian or Ethiopian progressor and non-progressor TB household contacts8.
However, when the 16-gene ACS signature was measured in the full GC6-74 cohort from The
Gambia, comprising 30 progressors and 129 non-progressors6, the signature significantly
validated by blind prediction. These results highlight the value of longitudinal cohort studies
with sufficient incident TB cases to allow reliable assessment of prognostic performance of risk
signatures. It is critical that more such cohort studies be performed to increase our collective
capacity to develop, refine and validate such biomarkers.
A reliable and simple triage test to identify those who should be investigated more intensively
for subclinical or active TB disease is urgently needed to improve case finding strategies and
allow earlier diagnosis and treatment. RISK6 also performed well as a triage test in patients with
respiratory symptoms who presented for care. However, with 56% specificity at >90%
sensitivity, it did not meet the minimum criteria set out in the WHO target product profile (TPP)
for a referral test to identify people who may have TB5. However, in our post-hoc analyses the
specificity of RISK6 in differentiating between definite TB cases and ORD among patients with
no prior history of TB was 75% at a set sensitivity of >90% (Table 3), which met the WHO
target product profile (TPP). Data regarding the interval since the previous TB episode was often
unavailable, precluding analysis of this factor. This finding, along with the apparent effect of
chest radiographs on RISK6 scores, highlights the importance of including clinical and
epidemiological factors in studies of diagnostic biosignatures. Community-based case finding
studies and prevalence surveys have shown that a substantial proportion of microbiologically-
confirmed TB cases are asymptomatic26-28, highlighting the need for TB case finding in
asymptomatic communities. RISK6 showed excellent diagnostic performance in differentiating
18
between symptomatic TB cases and asymptomatic controls in four different case-control cohorts
from South Africa, Peru and Brazil. Application of RISK6 to the South African cohorts met or
exceeded the sensitivity and specificity criteria set out in the TPP for a screening or triage test5.
In the South American cohorts, however, these criteria were only met when TB cases were
compared to uninfected controls as determined by negative QuantiFERON tests. Whether this
reflects a real geographic, genetic, environmental or epidemiological difference between South
African and South American communities is not clear. For the Peruvian cohort RISK6
measurements were performed on RNA isolated from PBMC, which may have affected
diagnostic performance, although we showed that near-identical ROC AUC results were
observed when diagnostic performance of the ACS 11-gene signature was measured in whole
blood and PBMC7. It is noteworthy that diagnostic performance of RISK6 was higher in
Brazilian culture+smear+ TB cases (AUC 99.8%, 95%CI 99.4-100) than in culture+smear- TB
cases (AUC 90.5%, 95%CI 76.8-100) and that RISK6 scores correlated significantly with lung
lesion activity measured by PET in the South African Catalysis cohort. RISK6 scores also
decreased during disease resolution upon TB treatment and showed promise as a treatment
response biomarker. This reflects the opposite of the increasing inflammatory signals detected by
RISK6 during disease progression, as previously reported for transcriptomic signatures16. Our
findings strongly suggest that disease severity in TB cases play a role in performance of
transcriptomic signatures, and likely other biomarkers. Given the lines of evidence that the
signature tracks severity of disease and lung lesions, it should be noted that biomarker
performance in populations from different settings may be influenced by differences in study
design that may preferentially enrol patients with more or less severe disease, rather than
reflecting purely geography-associated differences. Larger and well-designed longitudinal
19
biomarker studies are necessary to investigate the performance characteristics of blood
biomarkers, such as RISK6, for classifying individuals with ambiguous respiratory phenotypes
that are difficult to diagnose, and for revealing which stage of the TB spectrum such individuals
may fall into.
Underlying HIV-infection did not significantly affect diagnostic or treatment response
performance of RISK6, which is crucial given the high prevalence of undiagnosed TB in people
living with HIV10. We acknowledge that the effect of HIV was not assessed in all of the
validation cohorts and more such analyses are necessary to definitively establish the effects of
underlying HIV infection on RISK6 performance. Regardless, other published blood-based
transcriptomic TB signatures showed reduced diagnostic performance in HIV-infected compared
to uninfected persons6,26-29. Since most transcriptomic TB signatures detect the elevation of ISG
expression during TB, this effect of HIV is not surprising given that strong Type I IFN responses
constitute the typical anti-viral response29. Persistent HIV viremia also drives chronic immune
activation30 which is characterised by high ISG expression. Our data show that HIV-infection
was associated with elevated RISK6 signature scores, but that expression levels of individual
transcripts in the signature were not dramatically modulated by HIV-infection. Although
discrimination between HIV-infected TB cases and controls was not diminished relative to HIV-
uninfected people, our results show that a different diagnostic test threshold would be required
for HIV-uninfected and HIV-infected populations. A limitation of our analyses of HIV effects is
that the clinical studies were not sufficiently powered to investigate the performance of RISK6 in
samples with detectable or high pVLs. The issue of limited power will be addressed in a
prospective, multicohort study currently underway in South Africa by performing a head-to-head
comparison of performance of RISK6 with other signatures, in both HIV-uninfected and HIV-
20
infected persons (clinicaltrials.gov NCT02735590). Our work suggests that underlying HIV
infection has a marked effect on performance of IFN response signatures, which requires further
examination. Of note, Esmail, Wilkinson and colleagues demonstrated that a transcriptomic TB
signature based on complement pathway genes may have greater utility in ART naïve HIV-
infected persons31. In their study, pVL did not affect circulating immune complexes, which were
associated with transcripts involved in the complement pathway.
We found that RISK6 scores correlated significantly with lung lesion activity measured by PET-
CT in TB patients of the Catalysis study who underwent TB treatment. RISK6 showed good
performance as a treatment response biomarker, decreasing in score during successful treatment
and showing very good discrimination between pre-treatment and post-treatment samples in
patients with clinical cure, even in patients with underlying HIV-infection. Similar utility as a
treatment response biomarker was observed in the Brazilian cohort to that observed in the South
African Catalysis cohort. Importantly, in the Catalysis cohort RISK6 significantly predicted
treatment failure prior to treatment initiation and differentiated between treatment failures and
cured patients with very high accuracy at the end of treatment. These findings suggest that
RISK6 detects inflammatory signals associated with the TB disease process in the lungs or other
affected sites and that resolution of these processes can be tracked by monitoring gene
expression in the blood. Our data provide proof of concept that RISK6 allows treatment
monitoring, as has been shown for a number of other transcriptomic signatures16,32-34.
We explicitly developed RISK6 with the ultimate objective of translation to a hand-held point-
of-care platform and therefore conducted all performance analyses, including the training and all
validation cohorts, by qRT-PCR using a standardized protocol and locked-down analysis
algorithm. The RISK6 score is computed based on an ensemble of nine transcript pairs using the
21
pair-ratio approach, which uses ratios of transcripts regulated in opposite directions during TB
progression, as previously described8,11,36,37. This pair-ratio feature of RISK6 eliminates the need
for standardisation of gene expression using reference (or housekeeper) transcripts, restricting
measurement of the signature to six primer-probes and simplifying data processing steps.
Importantly, RISK6 was measured by a highly standardized, locked-down protocol in our
studies. Consequently, RISK6 performance was not subject to the gene expression normalization
methods that are typically necessary to overcome reproducibility problems due to sample and
batch effects associated with microarray and RNA-sequencing data35,36. Regardless, to allow
measurement of RISK6 scores in public microarray or RNA-sequencing datasets, we also
provide the score computation algorithm, “RISK6geo”, which computes virtually equivalent
scores to the RISK6 algorithm from qRT-PCR data. Finally, we showed that RISK6 could be
measured on very small volumes of capillary blood collected by fingerstick, with no discernible
effect on signature performance.
Our results support work towards incorporation of RISK6 into rapid, capillary-blood-based
point-of-care devices for field evaluation in community and primary care settings and
implementation studies.
22
METHODS
We developed the transcriptomic signature of risk, RISK6, using samples collected from
participants of the Adolescent Cohort Study (Supplementary Figure 1a). RISK6 was then
applied to seven external validation cohorts to determine prognostic and diagnostic performance
and utility as a treatment response biomarker (Supplementary Figure 1b). Most of these cohorts
have been described previously 6,8,16,37,38.
Adolescent Cohort Study (ACS) (RISK6 discovery): The Adolescent Cohort Study, including
selection of progressors and non-progressors, was previously described6,8,37. Briefly, among
6,363 healthy adolescents from the Worcester region of the Western Cape, South Africa, who
were enrolled, 46 “progressors” were either TST or QuantiFERON TB-Gold In-Tube assay
(Qiagen) (QFT)-positive and developed microbiologically-confirmed intrathoracic disease
during 2 years of follow-up. Individuals who were TST or QFT-positive at enrolment and
remained healthy (no TB disease) during follow-up, and matched the progressors for age, gender,
ethnicity, school of attendance and prior history of TB disease, were included as “non-
progressors”. Participants were excluded if they developed tuberculosis disease within 6 months
of enrolment (or the first TST or IGRA-positive sample) to exclude early asymptomatic disease
that could have been present at the time of assessment, or if they were HIV-infected.
Longitudinally collected PAXgene samples were available from most participants at six-monthly
intervals. The Human Research Ethics Committee of the University of Cape Town approved the
study (045/2005) and all participants provided written full informed, while parents or legal
guardians provided written, informed consent. All research was performed in accordance with
relevant guidelines/regulations.
23
GC6-74 Cohort (Prognostic validation): The Grand Challenges 6–74 project was previously
described6,8,39. Briefly, HIV-uninfected household contacts of TB cases were longitudinally
followed for up to 2 years, with assessments at baseline, at 6 months and at 18 months. TB
progressors who developed microbiologically confirmed TB during follow-up were
retrospectively identified and matched 1:4 to healthy non-progressors. Individuals in whom TB
disease developed within 3 months of baseline were excluded. PAXgene samples collected from
26 Gambian progressors and 116 non-progressors, 41 South African progressors and 164 non-
progressors, and 12 Ethiopian progressors and 48 non-progressors were included. Participants
provided written, informed consent. Protocols were approved by the Joint Medical Research
Council and Gambian Government Ethics Review Committee, Banjul, The Gambia
(SCC.1141vs2), the Stellenbosch University Institutional Review Board (N05/11/187) and the
Armauer Hansen Research Institute (AHRI) / All Africa Leprosy, TB and Rehabilitation
Training Center (ALERT) Ethics Review Committee (P015/10). All research was performed in
accordance with relevant guidelines/regulations.
Cross-sectional TB cohort (CTBC, Diagnostic validation): Adults with newly diagnosed
active TB (sputum Xpert MTB/RIF-positive or liquid culture-positive) were recruited at primary
healthcare clinics in Worcester and Masiphumelele, South Africa. Asymptomatic community
controls were recruited from the Worcester or Masiphumelele areas. HIV-infection was
diagnosed with the Determine HIV1/2 test (Alere). Protocols were reviewed and approved by the
Human Research Ethics committee of the Faculty of Health Sciences at the University of Cape
24
Town (HREC 126/2006 and HREC 288/2008). All study participants provided written informed
consent and all research was performed in accordance with relevant guidelines/regulations.
In total, 114 HIV-uninfected adults (53 TB cases and 61 asymptomatic controls) and 86 HIV-
infected (45 TB cases and 41 asymptomatic controls) were enrolled. Blood was collected in
PAXgene tubes at diagnosis in TB cases and at enrolment in asymptomatic controls.
ScreenTB and AE-TBC cohorts (Diagnostic validation)
Adults aged >18 years who presented at primary health care clinics in Cape Town emergency or
medical wards of Tygerberg Hospital in Cape Town with respiratory symptoms compatible with
TB, including cough for at least 2 weeks and another symptom including fever, weight loss,
hemoptysis or night sweats, were screened for inclusion for the ScreenTB11 or the African-
European Tuberculosis Consortium (AE-TBC) studies12,13. Those who had TB treatment within
90 days, received immunosuppressive medication (ScreenTB) or quinolones or aminoglycosides
in the past 60 days (AE-TBC), had a record of alcohol or drug abuse or a haemoglobin level
<9g/dL (ScreenTB) or <10g/dL (AE-TBC), or who were pregnant or breastfeeding, where not
eligible. HIV-infection was not an exclusion criterion. Using a pre-defined TB classification
algorithm12 (Supplementary Table 4), patients with microbiologically confirmed pulmonary TB
were classified as having definite TB. Those with either a single positive sputum smear or with
chest radiographs that were compatible with pulmonary TB and who responded to TB treatment
were classified as probable TB. Patients whose sputum tested negative, and who were not started
on TB treatment were classified as having “other respiratory diseases” (ORD). These ORD
patients also did not have a TB diagnosis during 2 months further follow-up. We also performed
a post-hoc analysis after unblinding, where a subset of ORD patients with chest radiographs that
25
were compatible with pulmonary TB were analysed as a separate group. All study participants
provided written, informed consent. The documents for the ScreenTB and AE-TBC studies were
approved by the Health Research Ethics Committee at Stellenbosch University and all research
was performed in accordance with relevant guidelines/regulations. Blood was collected in
PAXgene tubes at enrolment, before treatment initiation.
Peruvian Household Contacts Cohort (Diagnostic validation)
Bacillus Calmette-Guérin (BCG)-vaccinated, HIV-uninfected Peruvian participants were
recruited through Socios En Salud (SES), an affiliate of Partners in Health from urban and peri-
urban settlements around Lima, Peru, as a case-control study. Participants included adults with
recently diagnosed microbiologically confirmed, culture-positive, drug-sensitive pulmonary TB
disease (active TB, n=48), and clinically asymptomatic household contacts of TB patients
assessed within two-weeks of diagnosing the index case. Household contacts were evaluated for
signs of TB disease at the time of enrolment, and were excluded if clinical symptoms of TB were
present. Healthy household contacts were assessed for M.tb infection using QuantiFERON TB-
Gold In-Tube (QFT) assay. Participants with QFT IFNγ responses >= 0.35 international units
(IU)/mL were considered latently Mtb infected (QFT-positive, n=49) and uninfected if QFT
IFNγ < 0.35 IU/mL (QFT-negative, n=47). Household contacts were evaluated for signs of TB
disease at the time of enrolment, and were excluded when clinical symptoms of TB were present.
The Institutional Review Board of the Harvard Faculty of Medicine and Partners Healthcare
(protocol number IRB16-1173), and the Institutional Committee of Ethics in Research of the
Peruvian Institutes of Health approved the study protocol. All adult study participants and
parents and/or legal guardians of minors provided informed consent, while minors provided
26
assent. All research was performed in accordance with relevant guidelines/regulations. Peripheral
blood mononuclear cells (PBMC) were isolated from 50mL of venous blood using ficoll and
cryopreserved at 5X106 cells/cryovial, then shipped to the Brigham and Women’s Hospital for
storage. RNA was extracted from 106 cells PBMCs using the RNeasy extraction kit (Qiagen).
RePORT-Brazil Cohort (Diagnostic validation and treatment response):
Regional Prospective Observational Research for Tuberculosis (RePORT)-Brazil is an ongoing
prospective cohort study at five participating centers in Brazil: three in Rio de Janeiro (Instituto
Nacional de Infectologia (INI), Clinica de Saude Rinaldo Delmare (Rochina), Secretaria de
Saude de Duque de Caxias (Caxias), one in Salvador (Instituto Brasileiro para Investigação da
Tuberculose), and one in Manaus (Fundação Medicina Tropical Dr. Heitor Vieira Dourado).
RePORT-Brazil enrols participants ≥18 years-old who initiate treatment for culture-confirmed
pulmonary TB, and their close contacts. Details of the protocol have been published
previously40-42. All participants provided written, informed consent and the protocol was
approved by the Ethics Committee of the Maternidade Climério de Oliveira, Salvador, Brazil.
All research was performed in accordance with relevant guidelines/regulations. Blood was
collected in PAXgene tubes at diagnosis in TB cases and at enrolment in contacts.
Capillary blood cohort: Twenty adults (18 years or older) with recently diagnosed,
microbiologically confirmed pulmonary TB, who were positive for either sputum MGIT or solid
culture, Xpert MTB/RIF, Xpert MTB/RIF Ultra, or smear microscopy within the preceding two
weeks and had received no more than two weeks of tuberculosis treatment were consecutively
recruited from ongoing TB diagnostic and treatment studies at the South African Tuberculosis
27
Vaccine Initiative (SATVI) field site. Twenty-nine healthy adults living in communities from the
Cape Winelands region were also enrolled. Individuals with anaemia (haemoglobin less than 8.0
g/dl) or any other acute or chronic disease were excluded from both groups but no screening for
HIV was performed. For each participant, 2.5mL of venous blood was collected into PAXgene
RNA tubes (Qiagen) while 20μl, 50μl or 100μL capillary blood was collected by fingerprick
sequentially using 20μl or 50μl Minivettes (Sarstedt) without anti-coagulant and immediately
transferred into 0.5mL microtubes (Sarstedt) containing PAXgene fluid at an equivalent ratio to
the manufacturer’s recommendations, i.e. 1μl blood: 2.76μl PAXgene fluid. Samples were mixed
by inversion (venous PAXgene tubes) or by flicking (capillary blood microtubes), incubated at
room temperature for two hours, and stored at -40°C. Participants provided written informed
consent and the protocol was approved by the Human Research Ethics committee of the Faculty
of Health Sciences at the University of Cape Town (HREC 812/2017). All research was
performed in accordance with relevant guidelines/regulations.
Catalysis treatment response cohort, “Catalysis” (TB treatment response in HIV-
uninfected patients): In total, 131 HIV-uninfected adults with newly diagnosed pulmonary TB,
as confirmed by sputum culture, were recruited at primary healthcare clinics in Cape Town; 101
completed the study. Disease pathology was quantified by positron emission tomography and
computerized tomography (PET-CT) imaging using 18F FDG at baseline, week 4 and week 24.
Total glycolytic activity index (TGAI) is a product of lesion volume and FDG uptake intensity
and represents the total inflammatory burden, as previously provided15,16. PAXgene tubes were
collected prior to the start of treatment and at one, four, and 24 weeks after treatment initiation.
Of the 101 sample sets sequenced for a transcriptomics analysis16, 84 patients met or exceeded
28
the WHO definition for cure after the standard six-month treatment (“cures”, had proven and
then maintained sputum culture negativity by month 6). Amongst these, 70 had RNA available
for qRT-PCR analysis. Eight patients did not achieve bacteriological cure (classified as
“treatment failures”, if the month 6 culture was still positive) and 7 had available RNA. None of
the treatment failures achieved culture negativity at any time point during treatment and 7 had
RNA for qRT-PCR analysis). The remaining 10 patients were probable cures (only final culture
was negative) or unevaluable (treatment response ambiguous) and were not included in any
analyses. Twenty-nine healthy controls were also enrolled from the same communities and 21
had RNA available for qRT-PCR analysis. All participants provided written, informed consent
and the protocol was approved by the Stellenbosch University Human Research Ethics
Committee (N10/01/013). All research was performed in accordance with relevant
guidelines/regulations.
IMPRESS trial cohort (Recurrent TB treatment response in HIV-infected patients): This
study was an open-label, randomized controlled trial, “Improving Retreatment Success”
(IMPRESS, clinicaltrials.gov, NTC02114684; SANCTR DOH-27-0414-4576), performed in
Durban, KwaZulu-Natal17. IMPRESS was designed to determine if a moxifloxacin-containing
24-week regimen, in which moxifloxacin was substituted for ethambutol, would improve TB
retreatment outcomes relative to the standard TB treatment regimen. The trial enrolled adults
with a previous history of TB disease who received a new diagnosis of drug-sensitive TB by
positive Xpert MTB/RIF (Cepheid) or sputum smear microscopy, or both. Sputum samples were
collected for culture testing every 2 weeks during the intensive phase of treatment and monthly
thereafter until successful treatment completion. Whole blood was collected in PAXgene tubes at
29
baseline, 7 days and 2, 6, 8 and 14 months after start of TB treatment. Sixty-three HIV-infected
patients had RNA available and were included in the analyses (44 early converters, with sputum
culture conversion before month 2; and 19 late converters, who converted after month 2). The
IMPRESS protocol was reviewed and approved by the University of KwaZulu-Natal Biomedical
Research Ethics Committee (BREC No. BFC029/13). The IMPRESS trial was also approved by
the Medicines Control Council of South Africa (MCC Ref:20130510). All research was
performed in accordance with relevant guidelines/regulations.
RNA extraction:
RNA was manually extracted from collected PAXgene Blood RNA tubes (Qiagen) with the
PAXgene blood RNA kit (Qiagen) according to the manufacturer’s instructions or on an
automated Tecan Freedom EVO 150 robotic platform with the Promega Maxwell SimplyRNA
kit, using a modified protocol. Manually extracted RNA was stored at -80°C, and later used for
transcriptomic analysis. For RNA extracted by robotic platform an aliquot was immediately used
for cDNA synthesis. For the Peruvian cohort, RNA samples were extracted from 106 PBMCs
using the RNeasy kit (Qiagen) according to manufacturers’ instructions, and blinded, frozen
aliquots of RNA were shipped to the University of Cape Town.
For the venous versus capillary blood comparison, RNA was isolated with the PAXgene blood
RNA kit (Qiagen) according to the manufacturer’s instructions with the following modifications:
capillary blood samples were washed in 400μl water (instead of 4ml) and homogenised by
pipetting to avoid loss of the small pellet; venous and capillary samples were eluted in 80μl and
40μl of PAXgene blood RNA kit elution buffer, respectively.
30
Gene expression:
cDNA was synthesized from extracted RNA using SuperScript II reverse transcriptase and pre-
amplified using a pool of specific TaqMan primer-probe sets for microfluidic qRT-PCR. Gene
expression of individual transcripts was then quantified by microfluidic qRT-PCR using either
96.96 or 192.24 Gene Expression chips on the BioMark HD (Fluidigm). An internal positive
control sample was run on every chip to monitor inter-chip gene expression consistency.
Discovery of a parsimonious prognostic signature of TB disease risk
We sought to develop a PCR-based signature comprising a small ensemble of transcript pairs
that each represent the ratio between one upregulated and one downregulated transcript in
progressors, relative to controls, as described previously8. This pair-ratio ensemble format
presents two advantages. Firstly, the up-down pairing provides a “self-standardisation” function
that eliminates the need for housekeeper transcript-based standardisation of RT-PCR cycle
threshold values. Secondly, the ensemble of pairs provides robustness to the signature since a
signature score can be calculated even if expression data for one transcript (and its pairs) is not
available, due to a failed PCR reaction, for example.
Discovery of RISK6 signature of TB disease risk (Supplementary Figure 1a) was performed
using all ACS cohort progressor/non-progressor samples collected within 360 days of TB disease
diagnosis6. We first identified exon junctions that were differentially expressed in RNA-
sequencing data from all progressors and matched non-progressors (published in 6 and available
on GEO: accession number GSE79362). We applied the random subsets approach, which
randomly selects a partition of half the samples with a quarter of the features, to train support
vector machines of all possible pairs of junctions using the Pair-Ratio approach. The Pair-Ratio
31
approach pairs transcripts that are regulated in opposite directions in progressors and non-
progressors. We identified transcript pairs that differentiated progressors and non-progressors
with the highest sensitivity and specificity on the remaining partition of samples not used for
fitting. This was repeated until pairs that comprised 84 unique exon junctions were identified,
such that these could be conveniently assayed, along with 12 housekeeper (reference) transcripts,
by microfluidic PCR in a 96-reaction format (Supplementary Figure 2).
Training RISK6, a prognostic PCR signature of TB disease risk
Taqman FAM-TAM primer-probe assays for each of the 84 exon junctions were used to measure
expression of all transcripts by microfluidic qRT-PCR using samples from the entire ACS
cohort. Delta Ct values were computed for each exon junction relative to the geometric mean of
the 12 reference transcripts. To train the best parsimonious signature, we evaluated fit of pair-
ratio ensembles consisting of either 10, 8, 6, 4 or 2 transcript pairs and evaluated their ability to
differentiate between progressors and non-progressors. An appreciable drop in area under the
receiver operating characteristic curve (ROC-AUC) was observed for the 2-transcript and 4-
transcript ensembles, compared to the 6-, 8- and 10-transcript pair ratio ensembles
(Supplementary Figure 2). As there was no significant difference in performance between the
6-, 8- and 10-transcript models, we selected the 6-primer model, which we termed RISK6, based
on performance and smallest functional ensemble size.
Statistical analysis:
32
The RISK6 signature score is calculated as follows (R-script available on Bitbucket:
https://bitbucket.org/satvi/risk6):
1. Measure the cycle thresholds (Cts) for the 6 primer-probe assays listed in Supplementary
Table 1, by qRT-PCR.
2. For each of the 9 transcript pairs, compute the difference in raw Ct, which produces the log-
transformed ratio of expression.
3. Compare the measured ratio to ratios in a look-up table for the given pair of transcripts.
4. Assign a corresponding score in the look-up table to the ratio. If the measured ratio is larger
than all ratios in the relevant column of the look-up table, then assign a score of 1 to the ratio.
5. Compute the average over the scores generated from the set of pairs. If any assays failed on
the sample, compute the average score over all ratios not including the failed assays. The
resulting average is the final score for that sample.
There is considerable interest in the biosignature field to apply such signatures to publicly
available microarray or RNA-sequencing data43(Gupta et al., BioRxiv,
https://doi.org/10.1101/668137). RISK6 scores can be computed from log10-transformed
microarray or RNA-sequencing data using the formula:
RISK6geo score = geometric mean (GBP2, FCGR1B, SERPING1) - geometric mean
(TUBGCP6, TRMT2A, SDR39U1)
where normalized log2-transformed mean fluorescence intensity or normalized read count values
of GBP2, FCGR1B, SERPING1, TUBGCP6, TRMT2A and SDR39U1 are used.
RISK6 scores can also be computed using this method from qRT-PCR data using the formula:
33
RISK6geo score = geometric mean (TUBGCP6, TRMT2A, SDR39U1) - geometric mean
(GBP2, FCGR1B, SERPING1)
where raw Ct values of GBP2, FCGR1B, SERPING1, TUBGCP6, TRMT2A and SDR39U1 are
used. Comparative performance characteristics of the RISK6 and RISK6geo signatures for the
different cohorts in this study are shown in Supplementary Table 2.
qRT-PCR gene expression data was quality controlled using scripts generated in R and signature
scores were calculated. All RISK6 scores, with the exception of those in the discovery cohort,
were generated by blinded laboratory personnel. Only once RISK6 score results were locked
down and, where appropriate, shared among collaborators, were group allocations unblinded for
performance analyses. ROC AUCs were generated and compared using the pROC44 and
verification45 packages in R. Statistical analyses were done using Mann Whitney U for
differences between two groups, Wilcoxon ranked sum and Kruskal-Wallis tests for differences
between three groups in GraphPad Prism v8. To generate spline plots that show temporal
changes in transcript expression between adolescent progressors and non-progressors, we
computed log2 fold change values between progressor and non-progressors transcript abundance
(measured by RNA-sequencing) as previously described37, and modeled these as a nonlinear
function of TimeToDiagnosis for the entire adolescent progressor/non-progressor cohort using
the smooth.spline function in R with three degrees of freedom. Ninety-nine percent confidence
intervals for the temporal trends were computed by performing 2000 iterations of spline fitting
after bootstrap resampling from the full dataset. The median difference and 95% CIs in
expression of RISK6 signature genes was computed from 1000 bootstrapped median Ct values
34
between HIV+ and HIV-individuals. Genes with 95% CI bounds above zero were considered
significant.
Data availability statement:
The RISK6 scores and associated clinical data for all cohorts are in Supplementary Tables 5-13.
35
References:
1. World Health Organization. The end TB strategy. (2014).
2. Churchyard, G. J. A stratified approach to tuberculosis treatment. Nature medicine
24, 1639–1641 (2018).
3. Imperial, M. Z. et al. A patient-level pooled analysis of treatment-shortening
regimens for drug-susceptible pulmonary tuberculosis. Nature medicine 24, 1708–
1715 (2018).
4. World Health Organization.FIND. Development of a Target Product Profile (TPP)
and a framework for evaluation for a test for predicting progression from
tuberculosis infection to active disease. (2017).
5. World Health Organization. High-priority target product profiles for new
tuberculosis diagnostics: report of a consensus meeting. (2014).
6. Zak, D. E. et al. A blood RNA signature for tuberculosis disease risk: a prospective
cohort study. The Lancet 387, 2312–2322 (2016).
7. Darboe, F. et al. Diagnostic performance of an optimized transcriptomic signature of
risk of tuberculosis in cryopreserved peripheral blood mononuclear cells.
Tuberculosis (Edinburgh, Scotland) 108, 124–126 (2018).
8. Suliman, S. et al. Four-gene Pan-African Blood Signature Predicts Progression to
Tuberculosis. American journal of respiratory and critical care medicine 197,
1198–1208 (2018).
9. MacLean, E. et al. A systematic review of biomarkers to detect active tuberculosis.
Nat Microbiol 4, 748–758 (2019).
10. World Health Organization. Global Tuberculosis Report 2018. (2018).
36
11. Manngo, P. M. et al. Prospective evaluation of host biomarkers other than interferon
gamma in QuantiFERON Plus supernatants as candidates for the diagnosis of
tuberculosis in symptomatic individuals. The Journal of infection 79, 228–235
(2019).
12. Chegou, N. N. et al. Diagnostic performance of a seven-marker serum protein
biosignature for the diagnosis of active TB disease in African primary healthcare
clinic attendees with signs and symptoms suggestive of TB. Thorax 71, 785–794
(2016).
13. Chegou, N. N. et al. Africa-wide evaluation of host biomarkers in QuantiFERON
supernatants for the diagnosis of pulmonary tuberculosis. Scientific reports 8, 2675–
12 (2018).
14. Malherbe, S. T. et al. A semi-automatic technique to quantify complex tuberculous
lung lesions on 18F-fluorodeoxyglucose positron emission
tomography/computerised tomography images. EJNMMI Res 8, 55–14 (2018).
15. Malherbe, S. T. et al. Persisting positron emission tomography lesion activity and
Mycobacterium tuberculosis mRNA after tuberculosis cure. Nature medicine 22,
1094–1100 (2016).
16. Thompson, E. G. et al. Host blood RNA signatures predict the outcome of
tuberculosis treatment. Tuberculosis (Edinburgh, Scotland) 107, 48–58 (2017).
17. Perumal, R. et al. A moxifloxacin-based regimen for the treatment of recurrent drug-
sensitive pulmonary tuberculosis: An open-label randomised controlled trial.
Clinical infectious diseases : an official publication of the Infectious Diseases
Society of America 362, 5:13 (2019).
37
18. Deeks, S. G., Tracy, R. & Douek, D. C. Systemic effects of inflammation on health
during chronic HIV infection. Immunity 39, 633–645 (2013).
19. Fraser, C. et al. Virulence and pathogenesis of HIV-1 infection: an evolutionary
perspective. Science 343, 1243727–1243727 (2014).
20. Joshi, A. et al. HIV-1 Env Glycoprotein Phenotype along with Immune Activation
Determines CD4 T Cell Loss in HIV Patients. J. Immunol. 196, 1768–1779 (2016).
21. McNab, F., Mayer-Barber, K., Sher, A., Wack, A. & O'Garra, A. Type I interferons
in infectious disease. Nat. Rev. Immunol. 15, 87–103 (2015).
22. Lehmann, C. et al. Increased interferon alpha expression in circulating plasmacytoid
dendritic cells of HIV-1-infected patients. J. Acquir. Immune Defic. Syndr. 48, 522–
530 (2008).
23. Klatt, N. R., Chomont, N., Douek, D. C. & Deeks, S. G. Immune activation and HIV
persistence: implications for curative approaches to HIV infection. Immunol. Rev.
254, 326–342 (2013).
24. Sweeney, T. E. & Khatri, P. Blood transcriptional signatures for tuberculosis
diagnosis: a glass half-empty perspective - Authors' reply. Lancet Respir Med 4, e29
(2016).
25. Maertzdorf, J. et al. Concise gene signature for point-of-care classification of
tuberculosis. EMBO molecular medicine 8, 86–95 (2016).
26. Onozaki, I. et al. National tuberculosis prevalence surveys in Asia, 1990-2012: an
overview of results and lessons learned. Trop. Med. Int. Health 20, 1128–1145
(2015).
38
27. Calligaro, G. L. et al. Effect of new tuberculosis diagnostic technologies on
community-based intensified case finding: a multicentre randomised controlled trial.
Lancet Infect Dis 17, 441–450 (2017).
28. Dowdy, D. W. et al. Designing and Evaluating Interventions to Halt the
Transmission of Tuberculosis. J. Infect. Dis. 216, S654–S661 (2017).
29. Singhania, A. et al. A modular transcriptional signature identifies phenotypic
heterogeneity of human tuberculosis infection. Nat Commun 9, 2308 (2018).
30. Herbeuval, J.-P. & Shearer, G. M. HIV-1 immunopathogenesis: how good interferon
turns bad. Clinical immunology (Orlando, Fla 123, 121–128 (2007).
31. Esmail, H. et al. Complement pathway gene activation and rising circulating
immune complexes characterize early disease in HIV-associated tuberculosis.
Proceedings of the National Academy of Sciences of the United States of America
115, E964–E973 (2018).
32. Cliff, J. M. et al. Distinct phases of blood gene expression pattern through
tuberculosis treatment reflect modulation of the humoral immune response. J. Infect.
Dis. 207, 18–29 (2013).
33. Bloom, C. I. et al. Detectable changes in the blood transcriptome are present after
two weeks of antituberculosis therapy. PLoS ONE 7, e46191 (2012).
34. Warsinske, H. C. et al. Assessment of Validity of a Blood-Based 3-Gene Signature
Score for Progression and Diagnosis of Tuberculosis, Disease Severity, and
Treatment Response. JAMA Netw Open 1, e183779 (2018).
39
35. SEQC/MAQC-III Consortium. A comprehensive assessment of RNA-seq accuracy,
reproducibility and information content by the Sequencing Quality Control
Consortium. Nat. Biotechnol. 32, 903–914 (2014).
36. Leek, J. T. et al. Tackling the widespread and critical impact of batch effects in
high-throughput data. Nat. Rev. Genet. 11, 733–739 (2010).
37. Scriba, T. J. et al. Sequential inflammatory processes define human progression
from M. tuberculosis infection to tuberculosis disease. PLoS pathogens 13,
e1006687 (2017).
38. Naidoo, A. et al. Effect of rifampicin and efavirenz on moxifloxacin concentrations
when co-administered in patients with drug-susceptible TB. J. Antimicrob.
Chemother. 72, 1441–1449 (2017).
39. Duffy, F. J. et al. A Serum Circulating miRNA Signature for Short-Term Risk of
Progression to Active Tuberculosis Among Household Contacts. Front Immunol 9,
(2018).
40. Hamilton, C. D. et al. RePORT International: Advancing Tuberculosis Biomarker
Research Through Global Collaboration. Clinical infectious diseases : an official
publication of the Infectious Diseases Society of America 61Suppl 3, S155–9
(2015).
41. Geadas, C. et al. Advances in basic and translational tuberculosis research:
Proceedings of the first meeting of RePORT international. in 102, 55–67 (2017).
42. van der Heijden, Y. F. et al. Building capacity for advances in tuberculosis research;
proceedings of the third RePORT international meeting. Tuberculosis (Edinburgh,
Scotland) 113, 153–162 (2018).
40
43. Warsinske, H., Vashisht, R. & Khatri, P. Host-response-based gene signatures for
tuberculosis diagnosis: A systematic comparison of 16 signatures. PLoS Med. 16,
e1002786 (2019).
44. Robin, X. et al. Display and Analyze ROC Curves [R package pROC version
1.13.0]. (2013).
45. Pocernich, M. Package ‘verification.’. In CRAN.R-project.org Internet (2015).
Funding:
Support for this study was provided by the Bill and Melinda Gates Foundation (grants
OPP1023483, OPP1065330, and Grand Challenges in Global Health (GC6-74 grant 37772)) and
the Strategic Health Innovation Partnerships (SHIP) Unit of the South African Medical Research
Council with funds received from the South African Department of Science and Technology.
The ACS study was also supported by BMGF GC12 (grant 37885) for QuantiFERON testing.
The IMPRESS trial was supported by the European and Developing Countries Clinical Trials
Partnership (EDCTP: TA.2011.40200.044), and by Bayer Healthcare (moxifloxacin donation).
The AE-TBC and ScreenTB projects were funded by the EDCTP with grant numbers
IP_2009_32040 and DRIA2014-311 respectively. RePORT Brazil is supported by the
Departamento de Ciência e Tecnologia (DECIT) - Secretaria de Ciência e Tecnologia (SCTIE) –
Ministério da Saúde (MS), Brazil [25029.000507/2013-07] the National Institutes of Allergy and
Infectious Diseases [U01-AI069923], and CRDF Global [DAA3-18-64151, DAA3-18-64152 and
DAA3-18-64153]. Work in Peru was supported by the Tuberculosis Research Unit Network
(U19 AI111224). SCM received training in research that was supported by the Fogarty
International Center of the National Institutes of Health under Award Number D43 TW010559.
The content is solely the responsibility of the authors and does not necessarily represent the
official views of the National Institutes of Health. F.D. was supported by the Margaret
McNamara educational grant for women in developing countries. The funders had no role in
study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Author contributions:
41
APN, ET, DZ and TJS conceived the study.

APN, SKM, WAH, SHEK, JW, RW, IVR, BM, MMurray, BBA, TRS, JS, GW, KN, NP, MH,
DZ and TJS implemented clinical studies, raised funds and/or provided the resources.
APN, ET, SKM, SCM, SS, NNC, STM, FD, ME, NB, MMurphy, IVR and BBA processed
samples, performed the experiments, and analyzed the data.
APN, SS, NNC, NB, MF, MM, CM provided operational and/or project management.
APN, ET, SKM, SCM, SS, NNC, STM, FD, JW, IVR, BM, MMurray, BBA, TRS, GW, MH,
DZ and TJS interpreted the results.
APN, ET, SKM, SCM, SS, DZ and TJS wrote the manuscript.
All authors have read and approved the manuscript.
Additional Information
Competing interests
APN, ET, WAH, DZ and TJS are co-inventors of a patent on RISK6. All other authors declare
no competing interests.
Consortium and team members
The Adolescent Cohort Study Team:

1
Fazlin Kafaar, 1Leslie Workman, 1Humphrey Mulenga, 1E. Jane Hughes, 1Onke Xasa, 1Ashley
Veldsman, 1Yolundi Cloete, 1Deborah Abrahams, 1Sizulu Moyo, 1Sebastian Gelderbloem,
1
Michele Tameris, 1Hennie Geldenhuys, 15Rodney Ehrlich, 16Suzanne Verver, 17Larry Geiter
The GC6-74 Consortium

4
Gillian F. Black, 4Gian van der Spuy, 4Kim Stanley, 4Magdalena Kriel, 4Nelita Du Plessis,
4
Nonhlanhla Nene, 4Teri Roberts, 4Leanie Kleynhans, 4Andrea Gutschmidt, 4Bronwyn Smith,
4
Andre G. Loxton, 4Gerhardus Tromp, 4David Tabb, 18Tom H.M. Ottenhoff, 18Michel R. Klein,
18 18 18 18
Marielle C. Haks, Kees L.M.C. Franken, Annemieke Geluk, Krista E van Meijgaarden,
18 19 19 20 20
Simone A Joosten, W. Henry Boom, Bonnie Thiel, Harriet Mayanja-Kizza, Moses
42
20 20 20 20
Joloba, Sarah Zalwango, Mary Nsereko, Brenda Okwera, Hussein Kisingo, 5Shreemanta
K. Parida, 5Robert Golinski, 5Jeroen Maertzdorf, 5January Weiner 3rd, 5Marc Jacobson, 21Hazel
21 21 21 21 21
Dockrell, Steven Smith, Patricia Gorak-Stolinska, Yun-Gyoung Hur, Maeve Lalor, Ji-
22 22 22 22 22
Sook Lee, Amelia C Crampin, Neil French, Bagrey Ngwira, Anne Ben-Smith, Kate
Watkins, 22Lyn Ambrose, 22Felanji Simukonda, 22Hazzie Mvula, 22Femia Chilongo, 22Jacky Saul,
22
Keith Branson, 1Hassan Mahomed, 1E. Jane Hughes, 1Onke Xasa, 1Ashley Veldsman, 1Katrina
Downing, 1Humphrey Mulenga, 1Brian Abel, 1Mark Bowmaker, 1Benjamin Kagina, 1William
Kwong Chung, 17Jerry Sadoff, 17Donata Sizemore, 17S Ramachandran, 17Lew Barker, 17Michael
17 17 17 23 23
Brennan, Frank Weichold, Stefanie Muller, Larry Geiter, Desta Kassa, Almaz Abebe,
23 23 24 24 25
Tsehayenesh Mesele, Belete Tegbaru, Debbie van Baarle, Frank Miedema, Rawleigh
25
Howe, Adane Mihret, 25Abraham Aseffa, 25Yonas Bekele, 25Rachel Iwnetu, 25Mesfin Tafesse,
25
Lawrence Yamuah, 12Martin Ota, 12Philip Hill, 12Richard Adegbola, 12Tumani Corrah, 12Martin
12 12 12 26 26
Antonio, Toyin Togun, Ifedayo Adetifa, Simon Donkor, Peter Andersen, Ida
Rosenkrands, 26Mark Doherty, 26Karin Weldingh, 27Gary Schoolnik, 27Gregory Dolganov, 27Tran
Van
The ScreenTB Consortium:

4
Petri Ahlers, 4Gian van der Spuy, 4Ilana van Rensburg, 4Hygon Mutavhatsindi, 4Portia Manngo,
4 4 4 12 12
Kim Stanley, Andriette Hiemstra, Shirley McAnda, Joseph Mendy, Awa Gindeh,
12
Georgetta Mbayo, 12Ebrima Trawally, 12Olumuyiwa Owolabi, 20Harriet Mayanja-Kizza, 20Mary
Nsereko, 20Anna-Rita Namuganga, 20Saudah Nambiru Kizito, 25Adane Mihret, 25Sosina Ayalew,
25 25 25 28 28
Rawleigh Howe, Azab Tarekegne, Bamlak Tessema, Emmanuel Nepolo, Joseph
28 28 28 21
Sheehama, Gunar Gunther, Azaria Diergaardt, Uapa Pazvakavambwa, Hazel Dockrell,
18 18 18 18 18
Tom Ottenhoff, Elisa Tjon Kon Fat, Shannon Herdigein, Paul Corstjens, Annemieke
Geluk
The AE-TBC Consortium:

4
Magdalena Kriel, 4Gian van der Spuy, 4Andre G. Loxton, 4Kim Stanley, 4Belinda Kriel, 4Leigh
A Kotzé, 4Dolapo O. Awoniyi, 4Elizna Maasdorp, 12
Olumuyiwa Owolabi, 12
Abdou Sillah,
12 12 12 12 12 20
Joseph Mendy, Awa Gindeh, Simon Donkor, Toyin Togun, Martin Ota, Harriet
20 20 20 20
Mayanja-Kizza, Ann Ritah Namuganga, Grace Muzanye, Mary Nsereko, Pierre Peters,
43
28
Marieta van der Vyver, 28Faustina N Amutenya, 28Josefina N Nelongo, 28Lidia Monye, 28Jacob
28 22 22 22
A Sheehama, Scholastica Iipinge, Amelia C Crampin, Felanji Simukonda, Alemayehu
Amberbir, 22Femia Chilongo, 22Rein Houben, 23Desta Kassa, 23Atsbeha Gebrezgeabher, 23Getnet
23 23 23 25
Mesfin, Yohannes Belay, Gebremedhin Gebremichael, Yodit Alemayehu, Rawleigh
25 25 25 25 18
Howe, Adane Mihret, Yonas Bekele, Bamlak Tessema, Lawrence Yamuah, Tom H.M.
18 18 18 18
Ottenhoff, Annemieke Geluk, Kees L.M.C. Franken, Paul L.A.M. Corstjens, Elisa M.
18 18 26
Tjon Kon Fat, Claudia J. de Dood, Jolien J. van der Ploeg-van Schip, Ida Rosenkrands,
26
Claus Aagaard,
5
Maria M. Esterhuyse, 21Jacqueline M. Cliff, 21Hazel M. Dockrell
The RePORT Brazil Team:

10 10 10 10
Bruno B. Andrade, Juan M. Cubillos-Angulo, Kiyoshi F. Fukutani, Laise Paixão,
10
Ricardo Khouri, 10Sayonara Melo, 10Alice Andrade, 10Jéssica Rebouças-Silva, 10Hayna Malta,
10 29 29 30 31
Artur T. L. Queiroz, Valeria C. Rolla, Solange Cavalcante, Betina Durovni, Marcelo
Cordeiro-Santos, 32Afranio Kritski, 32José R. Lapa e Silva, 11Marina C. Figueiredo
Peruvian Household Contacts Cohort Team:

3
Kattya Lopez Tamara, 33Kattya Lopez Tamara, 33Segundo R León, 33Leonid Lecca Garcia
The CAPRISA IMPRESS team:

13,14 13,14 13,14 13,14
Dhineshree Govender, Razia Hassan-Moosa, Anushka Naidoo, Rochelle Adams,
13,14
Natasha Samsunder, 13,14Lara Lewis
Affiliations
15
School of Public Health and Family Medicine, University of Cape Town, Cape Town, South
Africa
16
KNCV Tuberculosis Foundation, The Hague, and Amsterdam Institute of Global Health and
Development, Academic Medical Centre, Amsterdam, The Netherlands
17
Aeras, Rockville, MD, USA
18
Department of Infectious Diseases, Leiden University Medical Centre, Leiden, The
Netherlands
44
19
Tuberculosis Research Unit, Department of Medicine, Case Western Reserve University
School of Medicine and University Hospitals Case Medical Center, Cleveland, Ohio, USA
20
Department of Medicine and Department of Microbiology, College of Health Sciences, Faculty
of Medicine, Makerere University, Kampala, Uganda
21
Department of Immunology and Infection, Faculty of Infectious and Tropical Diseases, London
School of Hygiene & Tropical Medicine, London, United Kingdom
22
Karonga Prevention Study, Chilumba, Malawi
23
Ethiopian Health & Nutrition Research Institute, Addis Ababa, Ethiopia
24
University Medical Centre, Utrecht, The Netherlands
25
Armauer Hansen Research Institute, Addis Ababa, Ethiopia
26
Department of Infectious Disease Immunology, Statens Serum Institute, Copenhagen, Denmark
27
Department of Microbiology and Immunology, Stanford University, Stanford, California, USA
28
University of Namibia, Windhoek, Namibia
29
Instituto Nacional de Infectologia Evandro Chagas, Fundação Oswaldo Cruz , Rio de Janeiro,
Brazil
30
Secretaria Municipal de Saúde do Rio de Janeiro, Coordenação de Doenças Transmissíveis,
Rio de Janeiro, Brazil
31
Fundação de Medicina Tropical Doutor Heitor Vieira Dourado, Manaus, Brazil
32
Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Rio de
Janeiro, Brazil
33
Socios En Salud, Lima, Peru
45
Table 1: Performance of RISK6 signature in the GC6 cohort, compared to the ACS 16-
gene and RISK4 signatures, by blinded validation. Samples from 0-24 months before TB
diagnosis were included.
Gambia South Africa Ethiopia

AUC AUC AUC
p p p
(95% CI) (95% CI) (95% CI)
0.70 0.70 0.66
RISK6 <0.0001 0.0015 0.028
(0.59-0.81) (0.61-0.79) (0.46-86)
ACS 16- 0.67 0.72 0.60
0.0016 <0.0001 0.11
gene (0.56-0.78) (0.63-0.81) (0.41–0.79)
0.72 0.72 0.67
RISK4* 0.0054 0.0063 0.02
(0.55–0.88) (0.53-0.92) (0.50-0.83)
*In the smaller GC6-74 test set only, because RISK4 was discovered in the GC6-74 training set
(comprising Gambian and South African samples).
Table 2: Accuracy of the RISK6 signature benchmarked against the WHO target product
profile for prediction of incident TB
RISK6 Sensitivity, Case Control AUC
Cohort Specificity# AUC
Threshold# set to ≥75% samples samples 95%CI
ACS 82.8-
0.53 75.0% 82.8% 46 284 87.6%
(discovery)* 92.4%
61.6-
GC6-74* 0.35 75.0% 50.0% 50 372 70.6%
79.5%
*Samples collected within 1 year of TB diagnosis from participants from all three countries
combined.
#
Specificities and the RISK6 threshold are reported at a sensitivity of 75%, which is the
minimum criterion specified in the TPP for an incipient TB test4. At a sensitivity of 75%, the
minimum specificity as set out in this TPP should be ≥75%.
46
Table 3: Accuracy of the RISK6 signature benchmarked against the WHO target product
profile for a screening/triage test
Sensitivity,
RISK6 Cases, Controls, AUC
Cohort (comparison) set to Specificity# AUC
Threshold# n n 95%CI
≥90%
Cross- 86.8-
HIV+ 0.78 90.5% 72.5% 42 40 92.6%
sectional TB 98.5%
(Definite TB
vs 87.9-
HIV- 0.78 90.2% 93.4% 51 61 93.7%
asymptomatic 99.4%
controls)
A-priori 79.6-
0.55 90.8% 55.7% 76 210 84.8%
analysis 90.0%
ScreenTB $
Post-hoc
and AE-TBC:
analysis in 80.2-
Symptomatic 0.61 91.0 75% 37 128 87.2%
those without 94.1%
adults
previous TB
$
Post-hoc
(Definite TB
analysis in 74.6-
vs ORD) 0.46 92.3 37.8 39 82 82.7%
those with 90.7%
previous TB
Definite TB vs
RePORT- combined 85.2-
0.61 90.2% 73.7% 51 99 90.9%
Brazil QFT+ and 96.6%
QFT-
(Definite TB Definite TB vs 81.2-
0.61 90.2% 59.1% 51 22 88.6%
vs household QFT+ 96%
contacts) Definite TB vs 85.9-
0.61 90.2% 77.9% 51 77 91.5%
QFT- 97.1%
Definite TB vs
combined 85.5-
Peru 0.21 91.7% 65.6% 48 96 90.6%
QFT+ and 95.6%
QFT-
(Definite TB
Definite TB vs 83.5-
vs household 0.21 91.7% 57.1% 48 49 89.6%
QFT+ 95.7%
contacts)
Definite TB vs 86.2-
0.19 91.7% 74.5% 48 47 91.5%
QFT- 96.9%
Catalysis Cohort
85.5-
(Definite TB vs asymptomatic 0.39 90.0% 95.2% 87 21 93.9%
100%
controls)
#
Specificities and the RISK6 threshold are reported at a sensitivity of ~90%, which is the
minimum criterion specified in the TPP for an incipient TB test4. At a sensitivity of ~90%, the
minimum specificity as set out in this TPP should be ≥70%.
$
Posthoc performance assessed after sample unblinding.
47
48
FIGURE LEGENDS:
Figure 1: Discovery of the RISK6 signature. (a) Expression kinetics of the six transcripts in
RISK6 signature over time, measured by RNA-sequencing and expressed as log2 fold change
between matched adolescent progressors and controls and modelled as non-linear splines (dotted
lines). Light blue shading 95% CI for the temporal trends, computed by performing 2000 spline
fitting iterations after bootstrap resampling from the full dataset. Transcripts that are upregulated
during TB progression are on the left and those that are downregulated and on the right. (b)
RISK6 comprises nine pairs that each link a transcript that is upregulated during TB progression
with one that is downregulated, relative to healthy controls. Lines indicate pairing (refer to Table
1 for TaqMan primer-probe sets that match the transcripts). Transcripts that are upregulated in
progressors are in red nodes and those that are downregulated in progressors are in green. (c and
d) Receiver operating characteristic (ROC) curves depicting the performance (model fit) of
RISK6, relative to the ACS 11-gene7 and ACS 16-gene6 signatures, measured by qRT-PCR on
RNA from whole blood samples collected from participants of the ACS cohort within one year
of TB disease diagnosis (c), or 1-2 years before TB diagnosis (d). Shaded areas depict the 95%
CI. (e) Receiver operating characteristic (ROC) curves depicting prognostic performance, by
blind prediction of RISK6, measured by qRT-PCR on RNA from whole blood RNA collected
from participants of the GC6-74 cohort of household contacts. Shaded areas depict the 95% CI.
(f) Prognostic performance of RISK6 for incident TB in household contacts from South Africa,
The Gambia or Ethiopia, measured by qRT-PCR on RNA from whole blood RNA collected
within 1 year of TB diagnosis from participants of the GC6-74 cohort. Performance on 0-2 years
before TB diagnosis is shown in Table 1.
49
Figure 2: Diagnostic performance of RISK6 as a triage test. (a and b) ROC curves depicting
diagnostic performance of RISK6 (a) and the ACS 11-gene signature (b), for discrimination
between active TB cases and Mtb-infected controls in HIV-negative or HIV-positive individuals.
RISK6 was measured by qRT-PCR on RNA from whole blood RNA. (c) Comparison of RISK6
signature scores in Mtb-infected controls and active TB cases in HIV-negative or HIV-positive
individuals. P-values were calculated using the Mann-Whitney U test. Horizontal lines represent
medians; boxes represent the IQR and whiskers the range. Dots represent individual sample
scores. (d) Relative differences in RISK6 transcript expression levels between HIV-negative and
HIV-positive Mtb-infected controls (green) or active TB cases (orange). Dots depict medians and
error bars the 95% CI, calculated from 1000 bootstrapped Ct values. The dashed line represents
zero. (e) Comparison of RISK6 signature scores, by blind prediction, in patients with definite
TB (with rigorous microbiological conformation), patients with probable TB and in patients with
other respiratory diseases (ORD). Horizontal lines depict medians, boxes the IQR and the
whiskers the range. Violin plots depict the density of data points. P-values were computed by
Mann-Whitney U test. (f) Receiver operating characteristic (ROC) curve depicting
discrimination between prognostic performance, by blind prediction of RISK6, measured by
qRT-PCR in definite TB patients and patients with other respiratory diseases (ORD). Shaded
areas depict the 95% CI. (g) ROC curves depicting RISK6 discrimination between definite TB
and ORD participants of the ScreenTB and AE-TBC cohorts stratified into participants with no
history of prior TB (black), or in those with a history of prior TB (red). Shaded areas depict the
95% CI.
50
Figure 3. Treatment monitoring using the RISK6 signature in the Catalysis TB Treatment
Cohort. (a) ROC curve depicting diagnostic performance of RISK6 for discriminating between
active TB cases (irrespective of treatment outcome), sampled prior to treatment initiation
(baseline, n=87), and controls (n=21) from the Catalysis Cohort. Shaded areas depict the 95%
CI. (b) Comparison of RISK6 signature scores in cases (irrespective of treatment outcome) from
the Catalysis Cohort at baseline and week 1 or week 4 after treatment initiation and after
treatment completion (Post Rx). Also shown are the RISK6 signature scores in the healthy
controls. Horizontal lines depict medians, the boxes the IQR and the whiskers the range. Violin
plots depict the density of data points. The p-value, computed by Mann-Whitney U test,
compares RISK6 signature scores after treatment completion with those in controls. (c) ROC
curves depicting performance of RISK6 for discriminating between baseline (pre-treatment)
samples and samples collected after week 1, week 4 or completion of TB treatment. (d)
Prediction of treatment failure using RISK6. ROC curves depict discrimination between cases
with cure (n = 70) and those with treatment failure (n = 7) in samples collected at treatment
initiation (Baseline, red), at week 1 (blue), week 4 (green) and after treatment completion (Post
Tx, black). (e) RISK6 scores plotted versus total glycolytic activity index measured by PET-CT
for all available samples.
Figure 4. Diagnostic performance and treatment monitoring in South American cohorts.
(a-b) ROC curve depicting diagnostic performance of RISK6 for discriminating between (a)
culture-positive active TB cases (n = 48) and QuantiFERON-negative controls (n=47), or
between (b) culture-positive active TB cases (n = 48) and QuantiFERON-positive controls
(n=49) from the Peru Cohort. Shaded areas depict the 95% CI. (c) ROC curve depicting
51
diagnostic performance of RISK6 for discriminating between culture-positive active TB cases,
sampled prior to treatment initiation (baseline, n=51), and controls (n=99) from the RePORT-
Brazil Cohort. (d) Comparison of RISK6 signature scores in TB cases at baseline, week 8 after
treatment initiation and after treatment completion (Post Rx). Also shown are the RISK6
signature scores in healthy controls from Brazil. Horizontal lines depict medians, the boxes the
IQR and the whiskers the range. Violin plots depict the density of data points. The p-value,
computed by Mann-Whitney U test, compares RISK6 signature scores after treatment
completion with those in controls. (e) ROC curves depicting performance of RISK6 for
discriminating between healthy control samples and samples collected from TB cases before
treatment initiation (baseline), at week 8 after treatment initiation, or completion of TB treatment
(Post Rx). (f) ROC curves depicting performance of RISK6 for discriminating between baseline
samples from TB cases and samples collected 8 weeks after treatment initiation, or upon
completion of TB treatment (Post Rx).
Figure 5: Treatment monitoring using the RISK6 signature in the IMPRESS cohort. (a)
Comparison of RISK6 signature scores in HIV-infected TB cases (irrespective of treatment
outcome) from the IMPRESS cohort at baseline, month 2 after treatment initiation, at treatment
completion (EoRx) and 6-8 months after treatment completion (Post Rx). Horizontal lines depict
medians, the boxes the IQR and the whiskers the range. Violin plots depict the density of data
points. (b) ROC curves depicting performance of RISK6 for discriminating between baseline
(pre-treatment) samples and samples collected at month 2 after treatment initiation, at treatment
completion (EoRx) and 6-8 months after treatment completion (Post Rx). Shaded areas depict the
95% CI. (c) ROC curve depicting performance of RISK6, measured at 2 months on TB
52
treatment, for discriminating between TB cases who converted their sputum to negative by 2
months (early converters) and those who converted after 2 months. (d) Comparison of RISK6
signature scores in IMPRESS participants stratified by detectable (>400 RNA copies/mL
plasma) vs. undetectable plasma HIV load (<400 RNA copies/mL plasma). Horizontal lines
depict medians, the boxes the IQR and the whiskers the range. Violin plots depict the density of
data points. The effect size is the relative difference in median plasma viral load and the p-value
was calculated using Mann-Whitney U test.
Figure 6. Robustness of RISK6. (a) ROC AUC values for discrimination between HIV-
uninfected TB cases and asymptomatic controls in the CTBC cohort by the full 6-gene RISK6
signature (9 pairs formed between 6 transcripts, far left), or after removing 1, 2, 3 or 4 of the
transcripts such that every combination of the pairs (represented by individual blue dots) was
tested. The grey bar graph represents the mean and the error bar the 95% CI. (b-d) ROC curves
depicting performance of RISK6 for discriminating between TB cases and controls in the
capillary blood cohort. Green curves represent RISK6 measured on 2.5mL venous blood
collected in PAXgene tubes. Blue curves represent RISK6 measured on 20μL (b), 50μL (c) or
100μL (d) capillary blood collected by finger prick. Shaded areas depict the 95% CI. The
Spearman correlation coefficients and associated p-values for comparison of RISK6 scores
measured by 2.5mL venous blood versus each capillary blood volume are shown. Only
individuals with paired venous and capillary blood results were included in each comparison (i.e.
venous blood results in those with a missing capillary blood value were excluded from each
graph). ROC curves were compared using the pROC function in R and resulting p-values are
shown.
53
54

19006197v1 Full

Uploaded by

Copyright:

Available Formats

19006197v1 Full

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

19006197v1 Full

Uploaded by

Copyright:

Available Formats

medRxiv preprint doi: https://doi.org/10.1101/19006197.this version posted September 9, 2019.

The copyright holder for this preprint (which

RISK6, a universal 6-gene transcriptomic signature of TB disease risk, diagnosis and

Adam Penn-Nicholson1*, Stanley Kimbung Mbandi1*, Ethan Thompson2*, Simon C.

*These authors contributed equally

Medicine and Division of Immunology, Department of Pathology, University of Cape Town,

Cape Town, South Africa.

Harvard Medical School, Boston, USA.

University of Cape Town, Cape Town, South Africa.

Medicine, Nashville, USA.

Research Unit, Durban, South Africa.

Corresponding Author: Thomas J. Scriba. Email: thomas.scriba@uct.ac.za

Short title: RISK6 signature for TB

needed. We developed a robust and simple, PCR-based host-blood transcriptomic signature,

RISK6, for multiple applications: identifying individuals at risk of incident disease, as a

Prognostic performance significantly exceeded that of previous signatures discovered in the

HIV-infected persons, assessed by area under the receiver-operating characteristic curve,

positron emission tomography, and tracked treatment response, demonstrating utility as

TB treatment, as well as monitoring a patient’s response to treatment, also require much

A universal, non-sputum biomarker capable of predicting progression to active TB, diagnosing

test5 proposed by the WHO.

assessments in all validation cohorts, by quantitative RT-PCR, using a highly standardized

protocol and locked-down analysis algorithm. We assessed performance of RISK6 by blind

Prognostic performance of RISK6 in the Adolescent Cohort Study discovery cohort

three transcripts downregulated in progressors (TUBGCP6, TRMT2A and SDR39U1), relative to

of the previously published ACS 16-gene signature6, consisting of 57 transcripts (PCR

progressor samples collected within 12 months of TB diagnosis (Figure 1c and Supplementary

observed on samples within a year of diagnosis (Figure 1d).

Validation of RISK6 prognostic performance in the GC6-74 cohort

and Ethiopia, who either progressed to TB or remained asymptomatic8. RISK6 significantly

discriminated between GC6-74 progressors and non-progressors on samples collected within 12

performance varies by geography, since differences in population genetic structure, local

assessed prognostic performance by country. Interestingly, when assessing samples collected

Performance of RISK6 as a screening test in HIV-uninfected and HIV-infected individuals

for TB are HIV-infected10, we aimed to determine diagnostic performance in both HIV-infected

and uninfected individuals. We therefore compared diagnostic performance of RISK6,

controls compared to HIV-uninfected controls, suggesting an effect of underlying HIV infection

signature, we determined the difference in expression of each transcript between HIV-infected

was significantly higher in HIV-infected than uninfected controls, while no significant

achieved a specificity of 93.4% and 72.5% in HIV-uninfected and HIV-infected persons,

respectively (Table 3 and Supplementary Table 2).

Diagnostic performance of RISK6 as a screening test in patients with respiratory symptoms

respectively (Table 3).

RISK6geo, adapted for application to microarray or RNA-seq data. RISK6geo discriminated

cohorts (Supplementary Table 3). Comparative performance characteristics of RISK6 and

Performance of RISK6 as a TB treatment monitoring biomarker

Performance of RISK6 as triage test and TB treatment monitoring biomarker in South

perform in geographically distinct populations. To address this, we assessed diagnostic

cohort, RISK6 discriminated between culture-positive TB patients and QFT-negative with an

discriminating between TB cases and QFT-negative controls, but discrimination between TB

transcriptomic signature may be different for different cohorts.

We also determined performance of RISK6 as a biosignature for monitoring TB treatment in the

RISK6 as a treatment biomarker in HIV-infected patients with recurrent TB

participated in the randomized controlled IMPRESS trial. IMPRESS determined if a retreatment

regimen that contained moxifloxacin, instead of ethambutol, would improve TB retreatment

in this trial since all patients achieved bacteriological cure.

pVL (p = 0.0027, Figure 5d), showing that pVL is a confounder in ISG-containing

transcriptomic signatures, one that could affect many patients.

Robustness of the PCR-based RISK6 signature

Adam Penn-Nicholson1, Stanley Kimbung Mbandi1, Ethan Thompson2*, Simon C.