REVIEW doi: 10.1111/sji.

12567
..................................................................................................................................................................

Diagnosis of Tuberculosis in HIV Co-infected

Individuals: Current Status, Challenges and
Opportunities for the Future
P. M
endez-Samperio

Departamento de Inmunolog
ıa, Escuela Nacional
de Ciencias Biol
ogicas, IPN, M
exico, M
exico
Received 13 February 2017; Accepted in revised
form 7 May 2017
Correspondence to: Prof. P. M
endez-Samperio,
Departamento de Inmunolog
ıa, Escuela Nacional
de Ciencias Biol
ogicas, IPN, Prol. Carpio y Plan
de Ayala, CD M
exico 11340, M
exico. E-mail:
pmendezsamperio@gmail.com
Abstract
Tuberculosis (TB) remains one of the most important causes of death among
people co-infected with human immunodeficiency virus (HIV). The diagnosis of
TB remains challenging in HIV co-infected individuals, due to a high frequency
of smear-negative disease and high rates of extrapulmonary TB. Accurate, ease of
use and rapid diagnosis of active TB are critical to the World Health
Organization (WHO) End TB Strategy by 2050. Traditional laboratory
techniques do not provide rapid and accurate results to effectively manage
HIV co-infected patients. Over the last decade, molecular methods have provided
significant steps in the fight against TB. However, many HIV co-infected
patients do not have access to these molecular diagnostic tests. Given the costs
closely related with confirming a TB diagnosis in HIV patients, an overtreatment
for TB is used in this patient population. Nowadays, an estimated US $8 billion
a year is required to provide TB treatment, which is very high compared with
making an important strategy to improve the current diagnostic tests. This
review focuses on current advances in diagnosing active TB with an emphasis on
the diagnosis of HIV-associated TB. Also discussed are the main challenges that
need to be overcome for improving an adequate initial diagnosis of active TB in
HIV-positive patients.

TB in HIV-infected patients [11, 12]. Indeed, given timely

Introduction
Tuberculosis (TB) remains a major global public health
problem with an estimated 10.4 million newly emerging
active TB cases worldwide in 2016 [1]. Importantly, TB is
the leading infectious cause of death in HIV co-infected
patients [2, 3]. This is supported from a systematic review
of autopsy studies of HIV-infected adults and children [4–
6], highlighting that diagnosis of TB in HIV co-infected
individuals remains challenging for microbiologists. Meth-
ods available for the diagnosis of HIV-associated TB and
recommended by the World Health Organization (WHO)
include smear microscopy and mycobacterial culture [7, 8].
Although direct microscopy has the advantages of being a
rapid and inexpensive test with high specificity for TB
diagnosis, this method has poor sensitivity [9]. To date,
molecular diagnostic tests have the technical capacity to
overcome limitations of these conventional laboratory
techniques [10]. In this regard, nucleic acid amplification
tests (NAATs) developed to detect Mycobacterium tubercu-
losis (MTB)-complex nucleic acids, and urine lipoarabino-
mannan (LAM) lateral flow assay are used in a diagnosis of
diagnosis is the first step in providing care to this patient
population, new diagnostic tests are critical for timely
initiation of anti-TB treatment. This review describes
current progress in diagnosing active TB among HIV co-
infected patients and identified some of the highest
priorities for integration of new TB diagnostic tests into
routine use.
Current diagnostic tests in HIV-associated TB
In many countries, the diagnosis of HIV-associated TB
includes smear microscopy, mycobacterial culture, NAATs
and urine LAM assay (Table 1).
Smear microscopy
Acid-fast mycobacteria can be visualized on microscopic
examination of smears prepared from sputum. The most
conventional used method is examination of Ziehl-
Neelsen-stained slides under light microscopy. At present,
direct microscopy has the advantages of being an

76 Ó 2017 The Foundation for the Scandinavian Journal of Immunology

P. M
endez-Samperio Diagnosis of HIV-associated TB 77
..................................................................................................................................................................

Table 1 Summary of diagnostic tests for the diagnosis of HIV-associated TB and the detection of drug resistance.

Turnaround
Test Year endorsed by WHO time Advantages Limitations

Smear microscopy
Conventional Included in the WHO Same day Is inexpensive and rapid Lacks sensitivity
guidelines for many years technique
LED 2010 Same day More sensitivity than
conventional microscopy method
Mycobacterial culture
Culture with DST 2007 10-21 days Liquid culture is more Require biosafety level 3 laboratories and
sensitivity than solid culture results take time
NAATs
Xpert MTB/RIF 2010 Same day Simultaneous detection of RIF, Is expensive, and cannot distinguish
high sensitivity for smear-positive between live and dead bacilli
TB and a short time to diagnosis
LAMP 2016 Same day Is inexpensive Infrastructure requirements are necessary
First-line LPA 2008 1-2 days Detection of RIF and INH resistance Cost-effectiveness and resource
implications
Second-line LPA 2016 1-2 days Detection of fluoroquinolones and Cost-effectiveness and resource
second-line injectable drugs implications
Antigen detection test
LAM 2016 Same day A short time to diagnosis Lacks sensitivity

inexpensive and rapid technique with high specificity for
TB diagnosis in endemic areas. However, this technique
has the limitation of low and variable sensitivity (20–60%)
[13]. In this regard, fluorescent microscopy is more
sensitive and allows a more rapid screening of large
numbers of smears [14]. An important limitation of
fluorescent microscopy is that this test is relatively
expensive. However, the replacement of conventional
fluorescent light sources with fluorescent light-emitting
diode (LED) can significantly decrease cost [15]. Therefore,
an important recommendation of the WHO for smear
microscopy is the replacement of conventional fluorescent
light sources with LED microscopy.
Mycobacterial culture
MTB culture on solid agar or in liquid culture remains as
an important traditional test for diagnosis of TB. Solid
culture is less expensive than liquid culture. However,
liquid culture is faster and more sensitive [16]. Important
advantages of this diagnostic test are the availability of
strains for drug susceptibility testing (DST) and for
genotyping to identify transmission events or outbreaks.
Important limitations of mycobacterial culture are the need
for infrastructure to support the appropriate biosafety level
3 laboratories and rapid transport of samples to the
laboratory.
NAATs
NAATs have the potential to provide highly sensitive
detection of paucibacillary forms of TB. At present, these
molecular methods such as loop-mediated isothermal
amplification test (LAMP) and Xpert MTB/RIF are playing
an important role in the diagnosis of TB. These methods
rely upon isothermal amplification or real-time polymerase
chain reaction (PCR) techniques to rapidly identify the
presence of MTB and mutations associated with rifampicin
(RIF) resistance simultaneously.
Loop-mediated amplification test
The TB-loop-mediated isothermal amplification test assay
(Eiken Chemical Co., Tokyo Japan) is the current NAAT
temperature-independent method for amplifying DNA. An
important advantage of this test is in terms of cost due to
its result can be seen through a visual display that is easy to
read [17]. In contrast, an important limitation of this test is
that significant infrastructure requirements are necessary
[18]. Interestingly, data from Yuan et al. [19] showed in a
meta-analysis of 10 studies a sensitivity of 80% (78–83%)
and specificity of 96% (95–97%) to diagnose pulmonary
TB using LAMP in clinical samples. However, a study by
Nliwasa et al. [20] reported that among HIV-associated
patients a lower sensitivity of 65% (48–79%) was
observed.
Xpert MTB/RIF
At present, Xpert MTB/RIF is the most widely adopted
NAAT worldwide. The WHO endorsed Xpert MTB/RIF
(Cepheid Inc, CA, USA), in December 2010 [21]. Xpert
MTB/RIF assay is a cartridge-based real-time PCR auto-
mated system for rapid TB diagnosis and simultaneous
identification of RIF resistance [22–24]. The WHO has
recommended the use of this assay since 2013 as the initial

Ó 2017 The Foundation for the Scandinavian Journal of Immunology

78 Diagnosis of HIV-associated TB
..................................................................................................................................................................
diagnostic test in adults or children suspected of having
HIV-associated TB or multidrug-resistant TB [25]. Impor-
tant advantages of this test include simultaneous detection
of RIF resistance, the limited training/expertise and
relatively high sensitivity for smear-positive TB [26]. In
contrast, limitations of this diagnostic test are that Xpert
MTB/RIF does not distinguish between live and dead
bacilli, it requires high-quality samples, is very highly
priced in the private sector worldwide, and the clinical
sensitivity with smear-negative specimens is only 67%
reducing test impact [27–29]. In this regard, Scott et al.
[30] showed that sensitivities were lower among smear-
negative patients for diagnosing pulmonary TB on a single
sputum specimen in South Africa. These data were
supported by a publication from Theron et al. [31] which
demonstrated that the sensitivity of Xpert MTB/RIF was
good in smear-positive cases (95%), but was lower in
smear-negative cases (55%) using liquid culture as the
reference. In a latter publication, these authors demon-
strated similar results using bronchoalveolar lavage fluid
[32]. A more recent study reported that in smear-negative,
culture-positive TB patients, Xpert MBT/RIF had a pooled
sensitivity and specificity of 67% (range: 60%–75%) and
99% (range: 98%-99%), respectively [33]. The suboptimal
sensitivity of sputum-based diagnostics using the Xpert
MTB/RIF assay is due to the fact that this assay cannot be
used to rule out TB in HIV-positive patients as a
consequence to high rate of empiric TB treatment [34–
36]. In addition, DNA co-extraction of PCR inhibitors
from sputum may be at least in part, a cause of decreased
test sensitivity. Importantly, strategies to improve sensi-
tivity in TB diagnostic tests could be the use of DNA baits
to extract M. tuberculosis DNA, the detection of multicopy
targets, and the use of all of the extracted nucleic acid in
the PCR detection of MTB [37]. Furthermore, using
extrapulmonary samples sensitivity is highest for lymph
node biopsies but lower for pleural fluid (Table 2) [38, 39].
Although the Xpert MTB/RIF assay is a method with
known limitations, Albert et al. [40] have reviewed the
impact of Xpert MTB/RIF, and reported that this assay it
has become an important technique for the diagnosis of TB
with RIF resistance. At present, Xpert HIV-1 Qualitative
assay has been recommended by the WHO as an
Table 2 Xpert MTB/RIF sensitivity and specificity for TB detection in
different types of extrapulmonary samples.
Sensitivity, % Specificity, %
Sample type Pooled estimate Pooled estimate
Pleural fluids 46.4 99.1
34.0 98.0
Lymph nodes biopsies 83.1 93.6
96.0 93.0
Cerebrospinal fluid 80.5 98.0
Gastric aspirate 78.0 99.0
P. M
endez-Samperio
opportunity for integration of TB and HIV diagnosis
[41]. This test would certainly be very useful to achieve the
ambitious 90-90-90 targets: 90% of persons living with
HIV must be diagnosed, 90% of those diagnosed should be
on sustained therapy and 90% of those on therapy should
have an undetectable viral load [42]. Patients can access the
Xpert MTB/RIF test via the public sector and is available
in several resource-poor and TB-endemic countries at
concessional prices.
At present, the Xpert MTB/RIF Assay is performed by
purifying intact MTB from sputum, or by directly lysing
MTB bacteria in sputum followed by extraction of DNA.
As obtaining sputum samples may be very challenging,
and the sample volume of sputum is generally limited [43],
there is an important interest in using non-pulmonary
samples for the diagnosis of TB in HIV-infected patients.
In this context, Lawn et al. [44] showed that although
sputum Xpert MTB/RIF testing is more sensitive, urine
Xpert MTB/RIF testing is able to diagnostic more cases of
TB in HIV co-infected patients, indicating value of urine
samples for diagnosis of TB in these individuals.
Urine lipoarabinomannan lateral flow assay
Other promising molecular TB diagnostic test is detection
of structural MTB-specific molecules such as LAM, a
component of the cell wall of MTB that may be found in
urine of patients with active TB [45]. Urine LAM (Alere
Inc., Waltham, MA, USA) assay is a urine-based antigen
detection which can permit rapid initiation of therapy
which contribute to reduction in mortality in HIV co-
infected patients [46]. An important limitation of this
diagnostic test is that the application of this method may
be limited due to its lacks sensitivity for diagnosis of TB in
HIV-uninfected patients. However, data from Peter et al.
[47] indicated that the use of this method in high-burden
countries can reduce early mortality at 8 weeks in
hospitalized patients with severe immunodeficiency. There-
fore, an important recommendation of the WHO for this
method is its use for diagnosis of HIV-associated TB in
patients whose CD4 cell counts is <100 cells/ll [48], and
advanced immunodeficiency [49]. Interestingly, a study
from Shah et al. [50] reported that combining urine LAM
assay with Xpert MTB/RIF testing of urine could be an
important opportunity to improve the diagnosis of active
TB in HIV co-infected patients, indicating an important
use of urine as a specimen type for the diagnosis of HIV-
associated TB.
References
38 Challenges and future directions for improving
39
38
the diagnosis of active TB in HIV co-infected
39 patients
38
39
Development of feasible, faster, and more sensitive
molecular diagnostic tests to identify subclinical HIV
Scandinavian Journal of Immunology, 2017, 86, 76–82
P. M
endez-Samperio
..................................................................................................................................................................
co-infection is a good strategy to progress towards to end
TB epidemic [51–53]. Despite the increasing use of new
diagnostic tests for detection of TB has occurred in the past
decade, current molecular diagnostic tests are not suffi-
ciently rapid and accurate to manage HIV co-infected
patients. In order to overcome this, the molecular
technique Xpert MTB/RIF Ultra, a more sensitivity test
due to additional multicopy DNA targets, and using a
higher amount of extracted DNA, is currently under
clinical evaluation [54]. An important advantage of this
technique is that retains its ability to detect RIF resistance.
Given the Xpert MTB/RIF assay can detect 5% to 15% of
RIF-susceptible cases worldwide, a RIF-resistant Xpert
MTB/RIF result can be confirmed with a different line
probe assay (LPA). Nowadays, important research progress
in the field of detection of multidrug resistance in MTB has
been reported. In this regard, new multiplexed drug
susceptibility assays have been used due to drug resistance
in MTB occurs in several regions of the genome [55, 56].
At present, LPAs have been recommended for use by the
WHO for the detection of RIF and isoniazid (INH)
resistance in sputum smear-positive specimens and
mycobacterial cultures [57]. These assays represent an
important opportunity of reporting RIF and INH suscep-
tibility simultaneously [58, 59]. A second-LPA used for the
detection of resistance to anti-TB drugs includes probes to
detect resistance to the class of fluoroquinolones drugs [60],
and the WHO recommended it use to detect resistance in
patients diagnosed with resistance to RIF or for the
diagnosis of extensively drug-resistant [61]. Another
important molecular technology is the Abbott RealTime
MTB RIF/INH resistance, an assay for the detection of RIF
and INH resistant MTB in pulmonary specimens which is
under evaluation [62–64]. The main limitations of these
assays are that laboratory-trained staff in PCR, and
appropriate laboratory infrastructure is required. It is
important to consider that future evaluation studies will
need a broader range of designs to determine the impact of
these TB diagnostic tests on HIV co-infected patients [65].
Other promising molecular techniques which in the future
will impact a better TB diagnosis are based using the
whole genome sequencing for MTB and next-generation
sequencing [66–68]. The benefits of these diagnostic tests
over current techniques for the diagnosis of active TB
include a shorter time to diagnosis [69, 70], to combine TB
diagnosis and drug resistance profiling into one test [71], to
establish relapse or re-infection with MTB [72], and a
opportunity to identify whether there are the presence of
mixed strain infection within the same patient [73, 74].
However, these new diagnostic tests of TB have some
important challenges such as genomic DNA contains
repeat elements which can be hundreds to thousands of
base pairs in length, resulting in complete genomes [75,
76]. Also, another associated challenge is to determine
whether differences between sequences occur because of
Ó 2017 The Foundation for the Scandinavian Journal of Immunology
Diagnosis of HIV-associated TB 79
sequence error or variance. To overcome this, multiple
reads of the same sequence are required which can be
compared to a reference dataset.
On the other hand, it may be appropriate in future
development in diagnostic tests to have new biomarkers.
At present, investment in biomarker research has increased.
However, translation from biomarker discovery to clinical
tools has been poor. In this regard, improved detection of
the lipoglycan biomarker LAM could lead to a break-
through in urine-based antigen detection [77]. Another
approach to TB biomarkers is the analysis of mycobacteria-
specific lipid profiles. These lipids are known as mycobac-
terial lipidomics that can serve as diagnostic biomarkers. In
this regard, Branch Moody et al. [78] reported progress in
the lipidomic dissection of MTB. Interestingly, high
concentrations of the lipidomic 1-tuberculosinyladenosine
have been found in MTB, which have the potential to serve
as a diagnostic target [79, 80]. More recently, ongoing
research is focused on host transcriptional biomarkers, such
as mRNA signatures for TB disease risk [81–83]. It is
important to consider that new diagnostic tests for
detection of TB must be optimized for the use of different
specimen types including cerebrospinal fluid, lymph node,
sputum, and urine. In fact, biomarkers measurable in the
urine are of particular interest in the HIV-infected
population, because they represent an easy, quick, and
inexpensive method in diagnostic laboratory TB tests,
and overcome the critical challenge to collect enough
sputum samples for HIV patients with advanced
immunodeficiency.
It may be necessary to emphasize that a major obstacle
to the development of more sensitive diagnostic test for
detection of MTB is the cost-effectiveness and resource
implications of these methods, which must be optimized to
each country [84–86]. In this context, the price will have to
come down in the performance of molecular diagnostics for
human TB in order that many HIV co-infected patients
have access to these diagnostic tests. Nowadays, the TB
diagnostics research has more than 50 diagnostic compa-
nies and product developers which are involved in
developing new TB diagnostic technologies [87, 88].
However, they will require funding support to overcome
important challenges, such as the cost of machines/
maintenance [89]. This funding support would be cheaper
than US$1 billion which were spent worldwide on
traditional TB diagnostics annually [90], and would
certainly be less than the annual global financial burden
of TB treatment with an estimated of US$8 billion [91].
Conclusion
During the last decade, the use of new diagnostic tests for
TB has increased significantly worldwide. In particular,
new generation of molecular diagnostic tests has been used
towards the integration of TB and HIV diagnosis.
80 Diagnosis of HIV-associated TB
..................................................................................................................................................................
However, most TB patients and/or vulnerable populations
at increased risk of TB transmission still do not have access
to the current diagnostic strategies. As an international
health emergency, the TB epidemic requires interdisci-
plinary collaborative relationships among researchers across
different countries to have a TB rational sequencing data
platform. In addition, the next goal of the WHO Stop TB
Strategy for TB elimination will require rapid diagnostic
testing for active TB and drug susceptibility assays on
MTB strains [92, 93]. In particular, highly sensitive assays
capable of being performed on a range of specimen types
are required to reduce reliance on empiric treatment and
improve detection in highly vulnerable populations such as
people living with HIV. In the meantime, increased
investments are necessary to support the finding of new
biomarkers and translation into clinical applications. In
this regard, governments and/or philanthropic funders
must work together to commit sustained funds towards the
development of better and feasible TB diagnostic assays.
Acknowledgment
PMS is a researcher of COFAA, EDI and SNI.
Conflict of interest
The author declares no conflict of interest.
Author contributions
PMS contributed to the drafting and writing of the
manuscript.
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