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Published Online November 18, 2009

A 100 year update on diagnosis of tuberculosis

Ajit Lalvani* and Manish Pareek

Tuberculosis Research Unit, Department of Respiratory Medicine, National Heart and Lung
Institute, Imperial College London, London, UK, and Department of Infectious Disease
Epidemiology, Imperial College London, London, UK

Areas of agreement: IGRAs are more specific than TST for diagnosis of LTBI as
they are not confounded by previous bacille Calmette-Guerin (BCG) vaccination.
Assessing IGRA sensitivity in the absence of a gold standard for LTBI is
challenging. Studies have therefore used surrogate markers such as active TB
and correlation with degree of TB exposure in contact investigations. These
studies suggest that sensitivity of ELISpot is higher than TST while whole-blood
ELISA has similar sensitivity to TST. Recent longitudinal studies demonstrating
the prognostic power of these tests for development of active TB provide
definitive evidence that positive IGRA results reflect infection with dormant yet
viable bacilli.
Areas of controversy: Is the prognostic power of IGRAs greater than the TST?
What are the false-negative rates in immunocompromised individuals with LTBI
at high risk of progressing to active TB?

Accepted: October 7,
*Correspondence to: Ajit
Lalvani, Tuberculosis
Research Unit,
Department of
Respiratory Medicine,
National Heart and Lung
Institute, Imperial College
London, London, UK.
E-mail: a.lalvani@imperial.

Growing points: IGRAs have been incorporated into national guidelines,

although their optimal deployment in diagnostic algorithms is evolving. The
health economic benefits of utilizing IGRAs are increasingly recognized, partly
because their high specificity avoids unnecessary chemoprophylaxis in BCGvaccinated persons with false-positive TST results.
Areas timely for developing research: Current IGRAs are being improved and
next-generation tests, with improved sensitivity, could enable the reliable
exclusion of LTBI in immunocompromised individuals.

Keywords: latent tuberculosis/interferon-gamma release assays/tuberculosis


British Medical Bulletin 2010; 93: 6984


& The Author 2009. Published by Oxford University Press. All rights reserved.
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Background: Diagnosis and treatment of latent tuberculosis infection (LTBI) is a

cornerstone of tuberculosis (TB) control in the developed world. In the last
century, the tuberculin skin test (TST) was the only means of diagnosing LTBI.
ELISpot and whole-blood ELISA, collectively known as interferon-gamma release
assays (IGRAs), are promising new tools.

A. Lalvani and M. Pareek


The global epidemiology of latent TB infection

With an estimated one-third of the worlds population latently infected
with TB (LTBI),3 and therefore potentially at future risk of developing
active disease, increasing importance is being accorded to the effective
identification and treatment of individuals with LTBI, particularly in
low TB burden countries where latently infected migrants and recently
infected contacts of smear-positive cases account for the bulk of the TB
Data on TB natural history suggest that in the first 2 years following
initial infection with tubercle bacilli 5% of infected individuals will
progress to active disease;5 if the bacilli are brought under immune
control they may lie dormant for several decades with only a further
5 10% of infected immunocompetent people eventually going on to
develop active disease over their lifetime.5 As a result, the diagnosis
and treatment of LTBI will be most effective if it is specifically targeted
at those individuals with the highest risk of progression from LTBI to
active disease including: recently infected individuals and all those with
underlying immunosuppression.6

Methods of diagnosing LTBI

Making the diagnosis of LTBI is constrained by a low bacteria load
that makes it impossible to directly detect Mycobacterium tuberculosis
and a weak humoral response that makes serological testing unreliable.
As a consequence, for much of the previous century, diagnosis of LTBI
has been defined as a positive tuberculin skin test (TST) in an asymptomatic person exposed to tuberculosis with no other evidence of active
disease. The utility of the TST is that it detects a cutaneous delayedtyped hypersensitivity response to purified protein derivative (PPD),

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Globally, tuberculosis (TB) continues to be one of the leading infectious causes of morbidity and mortality accounting for an estimated
9 million cases and 2 million deaths per year.1 This failure to adequately control the global epidemic may partly reflect the slow progress
that has been made, over the last century, in developing innovative
diagnostic tools for TB.2 However, the potential to significantly
advance TB control now exists with the development of novel diagnostic modalities for latent TB infection (LTBI).

A 100 year update on diagnosis of tuberculosis infection

a mixture of over 200 M. tuberculosis proteins. However, the TST is

neither specific, as the antigens present in PPD cross-react with bacille
Calmette-Guerin (BCG) and environmental mycobacteria, nor sensitive, due to anergy in those individuals with a compromised immune
system (such as HIV, iatrogenic immunosuppression and children).
T-cell interferon gamma release assays

Performance of IGRAs in the diagnosis of LTBI

Objectively comparing the diagnostic performance of the IGRAs and the
TST is hindered by the lack of a gold standard reference test for LTBI
that makes it impossible to directly measure sensitivity and specificity. As
a result, investigators have utilized proxy markers of LTBI including:1
degrees of exposure to infectious cases,2 active TB as a surrogate for
LTBI and, to assess specificity,3 the presence of IGRA negativity in
healthy BCG-vaccinated individuals at low risk of TB infection due to the
absence of epidemiologic risk factors for tuberculosis exposure.

Clinical performance of IGRAs in immunocompetent

Correlation with TB exposure of IGRA results

An important facet of TB transmission is that the risk of acquiring

M. tuberculosis is primarily determined by the frequency, duration and
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T-cell interferon gamma release assays (IGRAs) have been developed as

an alternative immunodiagnostic approach to the TST for detecting
M. tuberculosis infection.7 Currently 2 ex vivo assays are available: the
ELISpot that directly counts the number of IFN-g secreting T-cells and
the whole-blood ELISA that measures the concentration of IFN-g
secretion. Commercially, the ELISpot assay is available as the
T-SPOTTM.TB (Oxford Immunotec, Abingdon, UK) and the ELISA as
the QuantiFERONTM -TB Gold In-Tube (Cellestis, Carnegie, Australia).
Both IGRAs incorporate two antigensearly secretory antigen target-6
(ESAT-6) and culture filtrate protein-10 (CFP-10)which are strong
targets of Th1 T-cells in M. tuberculosis infection but also deleted
from all strains of BCG and the majority of environmental mycobacteria (except M. Kansasi, M. szulgai, M. marinum, M. flavescens and
M. gastrii).8,9 Therefore, in comparison with the TST, T-cell responses
to these antigens are not confounded by prior BCG vaccination and
hence a more specific marker of TB infection.2

A. Lalvani and M. Pareek


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proximity of contact with an infectious source case.10 Hence, if a new

test is more sensitive and specific than the TST it should correlate more
closely with the level of exposure to M. tuberculosis and be independent of BCG status.11
Outbreak and contact-tracing investigations have made use of this
theory to compare the diagnostic accuracy of IGRAs and the TST11,12
and concluded that both IGRAs correlate either as well as, or better
than, the TST with levels of exposure to tuberculosis whilst also being
independent of BCG status. In one of the largest, and earliest, studies
of this type the ELISpot was employed in a large school outbreak investigation comprising 535 students.12 ELISpot results, but not TST
results, were independent of the BCG vaccination status, indicating
higher specificity. Overall, ELISpot also correlated better with tuberculosis exposure (based on proximity and duration of exposure to the
index case) than TST suggesting a higher diagnostic sensitivity for
LTBI.12 Other work from varying TB burden settings has also confirmed that the ELISpot significantly correlates with TB exposure.13 17
In a recent study of South African children, positive ELISpot and TST
results were found to be significantly associated with the degree of
exposure to smear-positive index cases.17
Studies have also confirmed that the ELISA significantly correlates
with exposure to TB.18 21 In a recent case control study from Nigeria,
a dose response relationship was found between bacillary load in the
sputum of index cases and ELISA positivity in child contacts;20 children who had been in contact with the most heavily smear-positive
index cases, rather than smear-negative adults, were more likely to be
TST and ELISA positive.20 However, in contrast, a study from a South
African township of children at high risk of LTBI does not find any significant relationship between ELISA positivity and levels of exposure.22
An important outcome of these studies is an assessment of the level
of concordance between the IGRAs and TST. In BCG-vaccinated populations, there are lower levels of agreement between IGRAs and the
TST. This is to be expected as the TST, but not IGRAs, are confounded
by BCG vaccination. Where the ELISpot assay has been studied, generally moderate to high levels of agreement with the TST have been
On the other hand, the concordance between the ELISA and the TST
appears to be more variable; across a variety of TB burden settings investigators have found agreement between the ELISA and TST ranges from
high to poor.13,18,19,21 Studies in non-BCG vaccinated children from
Australia and Spain, which are low TB burden countries, have found
that the ELISA remained negative in a significant proportion of children
with a positive TST.13,21 These data suggest that the ELISA may have a
lower sensitivity than the TST in diagnosing LTBI in children.

A 100 year update on diagnosis of tuberculosis infection

However, the significance of discordant TST/IGRA results cannot be

reliably elucidated by these cross-sectional studies and will require data
from longitudinal studies.

Active TB as a surrogate marker for LTBI

Clinical performance of IGRAs in immunocompromised

The strategy for diagnosis and treatment of LTBI is based on targeted
tuberculin testing, which targets persons at highest risk of progression
from LTBI to active tuberculosis. This group particularly includes
HIV-positive individuals and people with immune-mediated inflammatory diseases (such as inflammatory bowel disease and inflammatory
arthritides) who are iatrogenically immunosuppressed or receiving
anti-TNF therapy.25 In these populations with impaired cellular immunity, there is a need to assess the diagnostic performance of IGRAs as an
alternative to the TST, which is well-recognized to have poor sensitivity.

HIV-seropositive individuals

Following the early reports on the diagnostic performance of IGRAs in

HIV-positive individuals26,27 a number of studies have added to the
evidence base to show that the ELISpot has a higher sensitivity than
the TST.26 30 A recent German study of HIV-positive individuals
found that the ELISpot was more sensitive than the TST and also correlated with previous active TB unlike the ELISA or TST.30 Other
studies from high-burden settings in Africa have also found that the
ELISpot has a high sensitivity in both children and adults. Liebeschuetz
et al.27 conducted a prospective study in South African children with a
high prevalence of HIV coinfection, which showed that the sensitivity
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As infection with M. tuberculosis is a prerequisite for active TB

disease, this principle has been exploited as an alternative surrogate
marker for TB infection by several studies evaluating the diagnostic
sensitivity of IGRAs. Pai et al. 24 have recently systematically reviewed
these studies and concluded that the ELISpot had a pooled sensitivity
of 90% (range 832100%) which was significantly higher than the
pooled sensitivities of the second generation ELISA (78%, range
55288%) and the latest generation ELISA (QuantiFERON-Gold
in-tube, pooled 70%, range 64293%).

A. Lalvani and M. Pareek

Individuals with immune mediated inflammatory diseases

Published experience of the performance of the IGRAs in diagnosing

LTBI in immune mediated inflammatory diseases (IMID) is still fairly
limited and is mainly drawn from small-scale cross-sectional studies that
have concentrated on evaluating the concordance between the TST and
IGRAs rather than correlating IGRA results with risk factors for TB
infection. In general, these studies have found that agreement between

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of the ELISpot was higher than that for the TST and unaffected by
HIV infection, malnutrition and age under 3 years. Overall sensitivity
of ELISpot in children with culture confirmed, or highly probable, TB
was 83%, rising to 91% when combined with the TST.27 Conversely,
the TST alone had a sensitivity of only 36% in those with HIV infection. In a study looking at HIV-positive Zambian adults coinfected
with sputum smear-positive TB, ELISpot sensitivity was maintained at
92%.26 Subsequent studies with ELISpot have shown that the results
remain robust in HIV infection and independent of CD4 cell
count.27,29 31 Independent work by Clark et al.32 and Dheda et al.31
has found that CD4 cell counts do not affect the ELISpot performance
in HIV-positive individualsboth in terms of positivity and the frequency of indeterminate results.
However, the experience gained from using the ELISA in HIV-positive
individuals seems to be more inconsistent. Raby et al. 33 recently evaluated the ELISA in a cross-sectional study of Zambian adults with smearpositive TB, and found that the sensitivity of the ELISA in HIV-positive
individuals was 63%, which was significantly lower than the 84% sensitivity seen in HIV-negative individuals. In a South African study of
HIV-positive individuals comparing the IGRAs and TST in the diagnosis
of TB infection, the sensitivity of the ELISA (28%) was lower than that
for the ELISpot (61%) and the TST (41%).28 On the other hand,
Balcells et al.34 found that in HIV-positive adults the ELISA had a
higher positivity rate than the TST and correlated with TB exposure
whilst the TST did not. In addition, CD4 cell counts did not have an
adverse effect on ELISA results. However, other authors have found that
in advanced HIV where there are low CD4 cell counts, the performance
of the ELISA is compromised. Brock et al. 35 conducted a large crosssectional study of HIV-positive individuals in Denmark and found a significant association between low CD4 cell count and indeterminate
ELISA results. Subsequent studies have also confirmed that with declining CD4 count, false negative and indeterminate results become increasingly common, which may affect the diagnostic utility of the ELISA in
HIV-positive individuals.28,33,35,36

A 100 year update on diagnosis of tuberculosis infection

Specificity of IGRAs

To estimate the diagnostic specificity quantitatively, BCG-vaccinated

individuals at an ultra-low risk of LTBI due to the absence of epidemiologic risk factors for tuberculosis exposure have been studied. The
ELISA has been assessed in larger numbers of individuals in such
studies than the ELISpot. In a recent systematic review the specificity of
the ELISA ranged from 89 to 100% with a pooled specificity of 99%
for the second-generation ELISA and 96% for the latest generation,
in-tube ELISA.24 The pooled specificity of the ELISpot was also high:
93% (range 85 100%).24 On the whole, both IGRAs have consistently
been shown to have a higher specificity than the TST particularly in
BCG-vaccinated populations.

Reliability of the IGRAs in routine clinical use: indeterminate

Over the last few years the IGRAs have increasingly been incorporated into routine clinical use, which makes it imperative to evaluate
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the TST and IGRAs is, at best, fair in individuals with IMID and that
discordant TST-positive, IGRA-negative results are associated with prior
BCG vaccination.37 39 Conversely, recent work from Italy by Bartalesi
et al. 40 where individuals with IMID were screened for LTBI using the
ELISA and TST found a relatively higher concordance (k 0.55) and
less TST-positive, IGRA-negative discordancy, which possibly reflects
the low proportion of BCG-vaccinated individuals in their population.
Fewer studies have correlated IGRA and TST results in individuals
with IMID with risk factors for LTBI. In a study of 142 patients with
IMID, the ELISA was significantly more closely associated with the
presence of risk factors for LTBI than TST. In addition, a positive TST,
but not IGRA, was significantly associated with previous BCG vaccination.37 Martin et al.41 evaluated both IGRAs in individuals with
inflammatory arthritides and found that both the ELISpot and the
ELISA correlated with risk factors for LTBI. Data from a large Italian
study revealed that individuals who had been in close contact with
smear-positive TB patients were significantly more likely to have a
positive ELISA and TST result.40
On the basis of the current evidence, one can conclude that although
false-negative IGRA results may occur, the diagnostic sensitivity of
IGRAs is more robustly maintained than that of the TST in individuals
with IMID on immunosuppressive treatment.

A. Lalvani and M. Pareek

their reliability. Indeterminate results occur for both IGRAs2 and can
arise for many reasons but the most significant cause is a failed positive
control that usually reflects underlying cellular immune suppression.
Studies suggest that indeterminate results are relatively common with
the ELISA occurring in 5240% of cases with the second-generation
ELISA but less frequently with the newer in tube ELISA.42 44
Indeterminate ELISA results seem to be associated with extremes of
age (under 5 and over 80) and immunosuppression, especially HIV
infection.35 In contrast, indeterminate results are rarer with the
ELISpot occurring in 0 5.4% of tests undertaken.45

Clinical benefits from chemoprophylaxis for LTBI can only occur if

IGRA-positive contacts are truly at increased risk of subsequently progressing to active TB compared with IGRA-negative contacts.
Longitudinal data, which can answer this important question, have
recently begun to appear from studies that aim to assess the predictive
value of IGRAs (see Table 1).46 50 Aichelberg et al.50 explored the
prognostic power of a positive ELISA result in TB uninfected
HIV-positive individuals. Over a median follow-up period of 19
months, 3/37 individuals with a positive ELISA at baseline went on to
develop active TB whereas 0/738 with a negative ELISA subsequently
developed TB. A German study in 601 contacts found that a significantly higher proportion of untreated household contacts with a positive ELISA progressed to TB disease as compared with contacts with a
5 mm positive TST; all six incident cases were ELISA positive at
Bakir et al.47 conducted a large study of 908 child contacts in
Turkey. During the follow-up period of 1.3 years, 15 incident cases
occurred and the investigators found that contacts with a positive
ELISpot had a significantly increased risk of developing active TB as
compared with those contacts with a negative ELISpot. In this study,
TB incidence in the ELISpot positive contacts was similar to that in
TST positive contacts though the ELISpot predicted these from fewer
contacts tested.47 However, a high proportion of the children received
chemoprophylaxis which is likely to have resulted in the incidence rate
ratios being underestimated.
Hill et al. 48 followed up 2348 household contacts in the Gambia for
2 years. 11/649 ELISpot-positive individuals developed active disease
as compared with 14/843 TST-positive individuals; 10/1087 ELISpot

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Longitudinal data quantifying the prognostic value of IGRAs

for progression to active TB

British Medical Bulletin 2010;93

Table 1: Currently published studies that have evaluated the prognostic value of the IGRAs.
Study population


HIV adults








follow up


to ATB of

to ATB of

to ATB of





(5 mm)

(10 mm)



3/36 (8)


Not done


Period of


IGRA used

QFT-G in-tube

IGRA positive


TST (5 mm)

31/42 (74)

TST (10 mm)

Not done

et al.50

Household contacts:


QFT-G in-tube

66/601 (11)

243/601 (40)

110/601 (18)

6/41 (15)

5/219 (2)

5/90 (6)



Household contacts:




381/908 (42)

550/908 (61)

462/908 (51)


11/381 (3)

12/550 (2)

6/462 (1)


et al.47


Hill et al.48

Household contacts:
children & adults



649/1736 (37)

Not done

843/2230 (38)



Not done

14/843 (1.7)



Household contacts:



9/24 (38)

Not done

Not done

6/9 (67)

Not done

Not done


et al.49



Values in parenthesis represent percentage values.


A 100 year update on diagnosis of tuberculosis infection

Diel et al.46

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A. Lalvani and M. Pareek

Summary of clinical data

As the evidence base for the performance of IGRAs expands, one can
conclude that both IGRAs are not confounded by BCG vaccination
and are therefore more specific than the TST in the diagnosis of LTBI.
In LTBI, both tests are more sensitive than the TST, with the ELISpot
having a higher sensitivity than the ELISA.24 In children, the ELISpot
seems to be superior to the TST whilst the sensitivity of the ELISA
appears to be comparable to the TST although the data are inconsistent. Whilst both tests are relatively robust in HIV infection, the
ELISpot seems to perform better than the ELISA with a higher sensitivity and a lower proportion of indeterminate results, particularly
when CD4 counts are low. In individuals with IMID undergoing
immunosuppressive therapy, the performance of the ELISpot and
ELISA seem to be equivalent.

Impact of IGRAs on public health policy

As the experience with IGRAs has increased, they have become an integral part of national guidelines that recommend their use in the diagnosis of LTBI. European guidelines advise that the IGRA should be used
in individuals with a positive TST to confirm the diagnosis of LTBI
and as a direct replacement for the TST in persons in whom TST is
unreliable, e.g. in those with suppressed cellular immunity (see
Fig. 1).52 Conversely, in the US and Japan, it is recommended that the
IGRAs should completely replace the TST as the standard investigation
for LTBI in all individuals.53
Economics have been an important determinant in the diverse final
recommendations framed by different countries. Although the IGRAs

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negative and 11/1387 TST negative contacts developed TB.48 The

investigators concluded that neither the TST nor the ELISpot was prognostic of subsequent progression to tuberculosis. The reason for the
lack of prognostic power of both TST and ELISpot in the African study
is unclear but may relate to the highly endemic setting, in which de
novo community transmission outside the household may account for a
significant proportion of cases of tuberculosis.51
Longitudinal, predictive, data from these studies provide the initial
evidence to support the use of IGRAs in targeting chemoprophylaxis
for recent IGRA-positive contacts. This is promising as it may potentially prevent a similar number of cases as when the TST is used but
requiring the treatment of fewer contacts.

A 100 year update on diagnosis of tuberculosis infection

are more expensive than the TST, health economic analyses have found
that they are cost-effective as they reduce the number of individuals
needing unnecessary chemoprophylaxis and monitoring whilst on drug
therapy.54,55 The United Kingdom National Institute of Health and
Clinical Excellence (NICE) economic analysis concluded that the
two-step TST and confirmatory IGRA approach would be the most
cost-effective model and this was the basis of the recommendations
made for the UK and most other European countries.52
One consequence of the European/Canadian recommendations is that
immunocompetent individuals who are TST negative but who may
have been IGRA positive may not be identified as having LTBI and
hence left untreated. In contrast, the US/Japanese guidelines may result
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Fig. 1 Algorithm of all possible outcomes resulting from parallel IGRA and TST testing.

A. Lalvani and M. Pareek

in overtreatment of individuals who are IGRA positive/TST negative as

the risk of progressing to active TB in this group is not known. This
highlights the importance of prospective data that will quantify the predictive value of a positive IGRA result and the prognosis of contacts
where the IGRA and TST results are discordant.

Future directions for T-cell based TB testing

IGRAs represent a new paradigm with the potential to supersede the
century old TST and, in the process, revolutionize the diagnosis, and
thereby targeted chemoprophylaxis, of LTBI. The evidence base supporting their use has expanded dramatically over the last few years
which has resulted in them becoming an important diagnostic tool in
low prevalence settings. However, guidelines vary between countries,
which is likely to reflect some underlying uncertainty about the prognostic value of a positive IGRA result. As a consequence there is an
urgent need for further large, longitudinal studies to explicitly define
the prognostic value of positive IGRA results for the development of
active TB disease, particularly in high-risk groups and subgroups with
discordant IGRA and TST results. Data of this type would provide

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IGRAs have revolutionized the diagnosis of LTBI but they remain a

dynamic work in progress with attendant advantages and limitations.
As a diagnostic tool, IGRAs are unable to differentiate between active
and latent TB. In addition, longitudinal studies during treatment of
active and TB and LTBI have shown that serial IGRA testing cannot be
used for treatment monitoring or as test of cure.2 However, simultaneous measurement of IL-2 and IFN-g secretion by M. tuberculosis
specific T-cells correlate with therapeutic response and may allow treatment monitoring and potentially test of cure.56
Studies are also exploring whether measuring alternative, downstream chemokines secreted by IFN-g-activated macrophages such as
inducible protein 10 (IP-10), in combination with IFN-g, may serve as
a more amplified readout than IFN-g alone thereby resulting in higher
Although the current IGRAs have superior diagnostic sensitivity than
the TST, research indicates that next-generation assays will have an
even higher sensitivity without compromising specificity. This will be
achieved by including additional, novel, antigens such as RV3879c (in
ELISpotPLUS)58 alongside ESAT-6 and CFP-10.

A 100 year update on diagnosis of tuberculosis infection

a clear mandate for basing treatment decisions for LTBI on IGRA


Conflict of interest: Professor Lalvani is inventor for several patents

underpinning T-cell-based diagnosis. The Lalvani ELISpot was commercialized by an Oxford University spin-out company (T-SPOT.TB w,
Oxford Immunotec Ltd, Abingdon, UK) in which Oxford University
and Professor Lalvani have a minority share of equity.

AL is a Wellcome Senior Research Fellow in Clinical Science. MP is

undertaking a Medical Research Council Capacity Building Studentship.





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