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International Journal of Infectious Diseases 32 (2015) 8793

Contents lists available at ScienceDirect

International Journal of Infectious Diseases


journal homepage: www.elsevier.com/locate/ijid

Review

Imaging in tuberculosis
Evangelia Skoura a, Alimuddin Zumla b, Jamshed Bomanji a,*
a

Institute of Nuclear Medicine, University College Hospitals NHS Trust, London NW1 2BU, UK
Division of Infection and Immunity, Centre for Clinical Microbiology, University College London, and NIHR Biomedical Research Centre,
University College London Hospitals, London, UK

A R T I C L E I N F O

Article history:
Received 14 November 2014
Received in revised form 28 November 2014
Accepted 1 December 2014
Corresponding Editor: Eskild Petersen,
Aarhus, Denmark
Keywords:
Pulmonary tuberculosis
Extrapulmonary tuberculosis
Computed tomography
Positron emission tomography
Fluorodeoxyglucose
Magnetic resonance imaging

S U M M A R Y

Early diagnosis of tuberculosis (TB) is necessary for effective treatment. In primary pulmonary TB, chest
radiography remains the mainstay for the diagnosis of parenchymal disease, while computed
tomography (CT) is more sensitive in detecting lymphadenopathy. In post-primary pulmonary TB, CT
is the method of choice to reveal early bronchogenic spread. Concerning characterization of the infection
as active or not, CT is more sensitive than radiography, and 18F-uorodeoxyglucose positron emission
tomography/CT (18F-FDG PET/CT) has yielded promising results that need further conrmation. The
diagnosis of extrapulmonary TB sometimes remains difcult. Magnetic resonance imaging (MRI) is the
preferred modality in the diagnosis and assessment of tuberculous spondylitis, while 18F-FDG PET shows
superior image resolution compared with single-photon-emitting tracers. MRI is considered superior to
CT for the detection and assessment of central nervous system TB. Concerning abdominal TB, lymph
nodes are best evaluated on CT, and there is no evidence that MRI offers added advantages in diagnosing
hepatobiliary disease. As metabolic changes precede morphological ones, the application of 18F-FDG PET/
CT will likely play a major role in the assessment of the response to anti-TB treatment.
2015 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/).

1. Introduction
Tuberculosis (TB) remains a global emergency despite substantial investment in health services over the past two decades.
Patients with sputum-negative pulmonary TB (PTB) and extrapulmonary TB (EPTB) are difcult to diagnose and may be missed
at all points of care. Diagnostic imaging is challenging because
signs of TB may mimic those of other diseases such as neoplasms or
sarcoidosis. Clinical signs and symptoms in affected adults can be
non-specic and a high level of pre-test clinical suspicion based on
history is fundamental in the diagnostic work-up. The global
impact of TB is extremely important, considering that an estimated
9.0 million people developed TB in 2013 and 1.5 million died from
the disease, according to the recent World Health Organization
(WHO) global tuberculosis report 2014.
Early diagnosis promotes effective treatment and leads to a
reduced onward transmission of TB. This article gives a review of
imaging patterns of chest TB as may be detected on conventional
radiography and computed tomography (CT). The main aim is to
* Corresponding author.
E-mail address: jamshed.bomanji@uclh.nhs.uk (J. Bomanji).

improve the radiologists familiarity with the spectrum of imaging


features of this disease in order to facilitate timely diagnosis.
Furthermore, we consider the emerging role of alternative
methods of imaging, such as magnetic resonance imaging (MRI),
which can be helpful and highly accurate for a better denition of
some of the signs of TB.
Although new imaging methods are now being used, conventional radiography remains the initial modality for suspected PTB and
for mass screening purposes.1 CT and MRI are the modalities of choice
for the evaluation of specic body parts.1 Positron emission
tomography/computed tomography with the use of 18F-uorodeoxyglucose (18F-FDG PET/CT) is a non-invasive imaging method that
has been used widely for the differentiation of malignant from
benign lesions. However, 18F-FDG also accumulates in inammatory
cells such as neutrophils, activated macrophages, and lymphocytes at
the site of inammation or infection.2 Consequently, 18F-FDG uptake
is observed in PTB, in tuberculoma, and in other TB-related lesions.3,4
Using PET/CT, pulmonary and extrapulmonary TB involvement is
assessed simultaneously, with time- and cost-saving implications.
Although any organ of the body can be involved, the lung
remains the most commonly involved organ in TB. The imaging
appearances of TB are described below for both pulmonary and
extrapulmonary involvement.

http://dx.doi.org/10.1016/j.ijid.2014.12.007
1201-9712/ 2015 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

E. Skoura et al. / International Journal of Infectious Diseases 32 (2015) 8793

88

2. Pulmonary tuberculosis
Classically, PTB can be divided into a primary and a postprimary pattern, each presenting with characteristic radiological
features. In practice, however, it is very difcult to draw distinct
lines between these radiographic patterns, and there is considerable overlap in the radiological manifestations.5
2.1. Primary tuberculosis
Primary TB is due to rst-time exposure to Mycobacterium
tuberculosis. At radiology, primary PTB manifests as four main
entities parenchymal disease, lymphadenopathy, pleural effusion, and miliary disease or any combination thereof.1
Chest radiography continues to be the mainstay of diagnosis.
Typically, parenchymal disease manifests as consolidation in any
lobe, with predominance in the lower and middle lobes.6 In these
cases, the bacterial infections are much more likely to be the cause
of such radiological features and hence the ndings are nonspecic, although primary infection should be suspected in
individuals at risk of exposure to TB. Multilobar consolidation
can be seen in almost 25% of cases.1 In approximately two-thirds of
cases, the parenchymal lesion resolves without sequelae on
conventional radiography.6 In the remainder, a radiological scar
persists that can be calcied in up to 15%, while persistent masslike opacities called tuberculomas are seen in approximately 9% of
cases.6 Frequently, the only radiological evidence suggestive of
previous TB is the so-called Ranke complex: the combination of a
parenchymal scar, calcied or not (Ghon lesion), and calcied hilar
and/or paratracheal lymph nodes.5 Destruction and brosis of the
lung parenchyma result in the formation of traction bronchiectasis
within the brotic region.5
The most common abnormality in children is lymph node
enlargement, which is seen in 9095% of cases; by comparison, in
adults the percentage reaches up to 43%.7 Right paratracheal and
hilar lymph nodes are the most common sites of nodal involvement, although involvement is bilateral in about a third of cases. CT
is more sensitive than plain radiography in detecting tuberculous
lymphadenopathy. It reveals nodes often measuring more than
2 cm, with a very characteristic, but not pathognomonic, rim sign
that consists of a low-density centre, representing caseous
necrosis, surrounded by a peripheral enhancing rim due to
granulomatous inammatory tissue.8,9
In contrast to lymphadenopathy, the prevalence of radiographically detectable parenchymal involvement is signicantly lower in
children up to 3 years old (51%) than in older children, among whom
the prevalence is similar to the reported percentage in adults
(80%).7,8 Also, evolution to cavitary disease is rare in children.5

Figure 1. Arrows indicate a mildly


tuberculosis.

18

Concerning the role of 18F-FDG PET/CT, two distinct patterns of


PTB have been described: (1) the lung pattern, related to a
restricted and slight hypermetabolic infection, with 18F-FDG
uptake in areas of lung consolidation  cavitation surrounded by
micronodules and mild uptake within lymph nodes, and (2) the
lymphatic pattern, related to a systemic and intense infection, with
more enlarged and 18F-FDG-avid hilar and mediastinal lymph
nodes.10
A limitation to the use of 18F-FDG PET/CT for the assessment of a
single pulmonary nodule, especially in endemic areas, is the
inability to distinguish tubercular from malignant lesions
(Figure 1).11 Studies investigating the diagnostic value of dual
time-point 18F-FDG PET/CT imaging have shown limited promise,
but further investigations in larger series of patients are
warranted.12,13
2.2. Post-primary tuberculosis
Post-primary PTB is one of the many terms (including
reactivation, secondary, or adulthood) applied to the form of TB
that develops and progresses under the inuence of acquired
immunity.5 The most common radiographic manifestation of postprimary PTB is focal or patchy heterogeneous, poorly dened
consolidation involving the apical and posterior segments of the
upper lobes and the superior segments of the lower lobes
(Figure 2).14,15 In the majority of cases, more than one pulmonary
segment is involved.6 Cavitation, the radiological hallmark of PTB,
is radiographically evident in 2045% of patients (Figure 3), while
air-uid levels in the cavity occur in 10% of cases.14,15 Cavitation
may progress to endobronchial spread and results in a typical treein-bud distribution of nodules in addition to cavitation; this is
considered a reliable marker of active TB.16 High-resolution CT is
the method of choice to reveal early bronchogenic spread, with 2to 4-mm centrilobular nodules and sharply marginated linear
branching opacities around terminal and respiratory bronchioles
(tree-in-bud sign).16 The tree-in-bud sign is the constellation of
small centrilobular nodules and concomitant branching opacities,
which mimics the branching pattern of a budding tree.17 The
centrilobular nodules are peripheral, spare the subpleural lung,
and denote the inammatory lesions in the bronchioles and
peribronchial alveoli.16,17 Hilar or mediastinal lymphadenopathy
is uncommon in post-primary PTB, seen in only 510% of patients
(Figure 4).18,19
Although pulmonary tuberculomas are most often the result of
healed primary PTB, a pulmonary tuberculoma is the main or only
abnormality on chest radiographs in approximately 5% of patients
with reactivation.20 The CT scan shows a round or oval granuloma,
measuring from 0.4 to 5 cm in diameter, with a wall lined by

F-FDG avid right lower lobe nodule measuring 1.5 cm (SUVmax 2). The differential diagnosis for this nodule would include cancer or

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Figure 2. Trans-axial CT section showing a patchy, heterogeneous, poorly dened consolidation with cavitating lesion in the upper lobe of the right lung.

inammatory granulomatous tissue or encapsulated by connective


tissue.21 Tuberculomas can cavitate, while calcication is found in
2030% of them.21 In 80% of cases, satellite lesions are observed in
the immediate vicinity of the main lesion.5 Because of increased
glucose metabolism caused by active granulomatous inammation,
tuberculomas may accumulate 18F-FDG.22 Maximum standardized
uptake values (SUVmax) tend not to be signicantly different for
tuberculous and malignant lesions.23,24 One study has suggested
that unlike 18F-FDG PET, the 11C-choline PET scan can help to
differentiate between lung cancer and tuberculoma, because
tuberculoma shows low tracer uptake on 11C-choline PET scan.25

and/or nodal disease, pleural effusion has been reported to be the


only radiographic nding indicative of primary PTB in approximately 5% of adult cases.26 Pleural effusion is usually unilateral and
on the same side as the primary focus of PTB, while complications
such as effusion, empyema, and bronchopleural stula are rare.6 The
CT scan of patients with post-primary pleural effusion typically
shows smooth thickening of visceral and parietal pleura.28
Ultrasonography often demonstrates a complex septated effusion.6
Fibrothorax with diffuse pleural thickening, but without pleural
effusion on CT, suggests inactivity.29 The 18F-FDG PET/CT scan may
demonstrate diffusely intense 18F-FDG uptake in thickened pleura
that can be confused with pleural mesothelioma.30

2.3. Radiological patterns in primary and/or post-primary PTB


2.4. Differentiation between active and inactive TB
Miliary pulmonary disease affects between 1% and 7% of
patients with all forms of TB.6 It is usually seen in the elderly,
infants, and immunocompromised persons.6 Initially, standard
radiographs are normal in 2540% of cases.26 CT can demonstrate
miliary disease before it becomes radiographically apparent, and
its characteristic ndings consist of innumerable 1- to 3-mmdiameter nodules randomly distributed throughout both lungs,
often associated with intra- and interlobular septal thickening.14,27
The nodules usually resolve within 26 months with treatment,
without scarring or calcication; however, they may coalesce to
form focal or diffuse consolidation.6
A pleural effusion is seen in approximately one-fourth of
patients with primary PTB and in 18% of post-primary PTB.26
Although, usually observed in association with parenchymal

TB makes its presence felt on imaging long after the resolution


of disease. Sometimes a question that needs to be answered is
whether the infection is active or not. Active disease is in general
characterized by the presence of centrilobular nodules, tree-in-bud
pattern, thick-walled cavities, consolidation, miliary nodules,
pleural effusions, or necrotic lymphadenopathy.1 Resolution to
thin-walled smooth cavities, brosis, and parenchymal, nodal, or
pleural calcications often denotes inactive disease.1
Chest radiographs may be normal or show only mild or nonspecic ndings in patients with active disease.26 The diagnosis of
PTB with radiography is initially correct in only 49% of all cases:
34% for primary and 59% for post-primary PTB.26 On the other
hand, CT can correctly diagnose 91% of cases of PTB and correctly

Figure 3. Left panel: CT image showing a 1.6  1.2-cm cavitating lesion (arrow) in the upper lobe of the right lung. Right panel: this lesion shows mild 18F-FDG uptake on the
PET scan (small arrow) (SUVmax 2.2). Bottom left panel: Fused 18F-FDG PET/CT image showing the same cavitating avid lesion (arrow).

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E. Skoura et al. / International Journal of Infectious Diseases 32 (2015) 8793

Figure 4. Left panel: Multiple intensity projection image showing 18F-FDG uptake in the mediastinal and bilateral hilar lymph nodes (arrows). Right panel: Multiple fused
trans-axial section 18F-FDG PET/CT images showing hilar and mediastinal lymph nodes (arrows).

characterize 80% of patients with active disease and 89% with


inactive disease.31
CT is more sensitive than radiography in the detection and
characterization of both parenchymal disease and mediastinal
lymphadenopathy.9,32 In a study that compared the two methods,
high-resolution CT showed cavities in 58% of patients with active
PTB, whereas chest radiographs in only 22%.32 The diagnosis of active
PTB was based on positive acid-fast bacilli in sputum and changes on
serial radiographs obtained during treatment.32 CT may also show
pleural disease that is not evident on chest radiography and be
helpful in the evaluation of pleural complications.33
CT features predictive of highly infectious/active PTB include
the following:34 (1) consolidation involving the apex or the
posterior segment of the right upper lobe or the apico-posterior
segment of the left upper lobe, (2) consolidation involving the
superior segment of the right or left lower lobe, (3) a cavity lesion,
(4) clusters of nodules, and (5) absence of centrilobular nodules.
High-resolution CT is better than chest radiography in predicting
active PTB, with a sensitivity of 96% versus 48%.35
It has been reported that 18F-FDG PET is able to differentiate
active PTB from old or inactive disease, as active tuberculoma has
signicantly higher SUVmax values compared with inactive
tuberculoma.3 When a SUVmax of 1.05 (at 60 min) was used as
the cut-off, the sensitivity and specicity were 100% and 100%,
respectively.3 A recent study concluded that 18F-FDG PET/CT has
the potential to become a tool for monitoring the treatment
response in selected cases of EPTB or multidrug resistance.36 An
interesting study of patients with radiographic lesions suggestive
of old healed TB aimed to gather information on the metabolic
status of TB lesions using 18F-FDG PET/CT imaging.37 The authors
showed that patients with old healed TB lesions with a higher
SUVmax may be at higher risk of active TB.37 Further investigation is
needed to conrm these results.

3. Extrapulmonary tuberculosis
Despite recent advances in imaging, the diagnosis of extrapulmonary involvement sometimes remains difcult.38 The

imaging of some frequent extrapulmonary sites of TB is reviewed


below.
3.1. Musculoskeletal tuberculosis
Approximately 50% of cases of skeletal TB involve the spine.6
Spondylodiscitis, also known as Potts disease, is the most
common form.39 The infection begins in subchondral bone and
spreads slowly to the intervertebral disk space and the adjacent
vertebral bodies, commonly in the lower dorsal and upper lumbar
spine.40 Failure to identify and treat these areas of involvement at
an early stage may lead to serious complications such as vertebral
collapse, spinal compression, and spinal deformity.38 Plain
radiography is normal early in the disease.1 The rst sign may
be demineralization of the endplates with resorption and loss of
dense margins. As the disease progresses, radiography will show
progressive vertebral collapse with anterior wedging and gibbus
formation.1 MRI is the preferred imaging modality in the
diagnosis and assessment of tuberculous spondylitis.41,42 Because of the often multifocal nature of spinal TB, the MRI imaging
of the entire spinal column could be more effective in the early
diagnosis of the disease.43 In cases of spinal TB, the spinal cord is
susceptible to myelopathy secondary to compression from an
epidural abscess.6 The collapsed vertebra along with the
epidural collection/abscess is also best evaluated on MRI.6 After
antibiotic administration is initiated, repeat imaging is advised at
approximately 4-week intervals or at any time if neurological
deterioration occurs.44
As tubercular lesions demonstrate high 18F-FDG uptake, 18FFDG PET/CT is a promising technique for the diagnosis of spinal
infection (Figures 5 and 6).4548 An interesting nding was that
63.6% of patients with spinal TB had clinically occult noncontiguous multifocal skeletal involvement at the time of
whole-body 18F-FDG PET/CT scan.49
Besides the spine, any part of the musculoskeletal system can
become involved, but the large joints of the lower limbs are most
commonly affected. Imaging ndings in musculoskeletal TB are
often non-specic. MRI is the most sensitive modality for early
diagnosis and complete delineation of the disease.1

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Figure 5. CT (left panel) and 18F-FDG PET/CT fused images (right panel): trans-axial and sagittal sections. Moderate to intense 18F-FDG uptake is seen in a paravertebral soft
tissue mass lesion extending from the level of T7T10 vertebrae with associated lytic sclerotic changes in T7T9 vertebrae and collapse of the T8 vertebra (arrows). The lesion
inltrates into the spinal canal at the level of the T8 vertebra and involves the spinal cord (small arrow). The lesion is seen to extend along the left costal margin, with faint 18FFDG uptake and foci of calcication, likely representing a cold abscess (courtesy of Prof. B.R. Mittal).

3.2. Central nervous system (CNS) tuberculosis

3.3. Abdominal tuberculosis

TB of the CNS is a highly devastating form of the disease. Various


forms of involvement of the CNS are observed: parenchymal,
meningeal, calvarial, spinal, or any combination thereof.1 MRI is
generally considered superior to CT in detecting and assessing CNS
TB.50 Parenchymal involvement is most frequently seen in the form of
a tuberculoma, which may be single or multiple. In the paediatric age
group it is seen more frequently in the cerebellum, whereas in adults
it has a predilection for the cerebral hemispheres and basal ganglion.
The appearance of a tuberculoma varies on MRI depending on its
stage of maturation.50,51 A non-caseating granuloma is hyperintense
on T2 and hypointense on T1 and shows solid enhancement, while a
solid caseating granuloma is usually hypointense on both T1 and T2
images. On CT, tuberculomas appear as round or lobulated soft tissue
masses with varying attenuation and homogeneous or ring
enhancement.1 Miliary TB is often associated with TB meningitis
and presents as small (<2 mm) foci of hyperintensity on T2
acquisitions, while after gadolinium administration, T1 images show
numerous, round, small, homogeneous, enhancing lesions.50 Contrast-enhanced MRI is also superior to CT for the evaluation of
meningitis and its complications, including hydrocephalus.1

Abdominal lymphadenopathy is the most common manifestation of abdominal TB, seen in 5566% of patients, and may or may
not be associated with other abdominal organ involvement.52
Abdominal lymph nodes are best evaluated on CT, which reveals
enlarged nodes with hypoattenuating centres and hyperattenuating enhancing rims.52,53 On MRI, the appearance is typically
hypointense on T1 images, whereas on T2 images the signal is
generally hyperintense or with peripheral low-intensity signal.1
Hepatic TB can be classied into local, miliary TB, or
tuberculomas.54 Miliary TB is the most common form of liver TB
and is a part of generalized disease; innumerable small nodules are
found on the liver which may or may not be seen on CT, but on
ultrasound usually present as bright liver or spleen patterns in the
form of a diffuse increase in echogenicity.54
Calcication in the hepatic region on plain radiography may
occasionally be seen in local hepatic TB.54 On CT, liver tuberculoma
appears as a non-enhancing, central, low-density lesion with a
slightly enhancing peripheral rim, while liver calcications can
also be demonstrated. MRI offers no added advantage in
diagnosing hepatobiliary TB.55

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E. Skoura et al. / International Journal of Infectious Diseases 32 (2015) 8793

Figure 6. Multiple intensity projection image (left panel) and fused trans-axial section 18F-FDG PET/CT images (right panel) showing 18F-FDG uptake in multiple lymph nodes
(SUVmax 6.8) (porta hepatis, portacaval, para-aortic, retroperitoneal, bilateral internal iliac, left external iliac, and left inguinal (arrows)) and heterogeneous 18F-FDG uptake in
the grossly enlarged spleen (SUVmax 10.2) (arrow) and in bones (T8 vertebra coupled with paravertebral soft tissue uptake (SUVmax 17.6), L3 vertebral body anteriorly (SUVmax
11.6), and the right sacral alae (SUVmax 6.7) (arrows)) (courtesy of Dr A. Alshammari).

Few reports are available on 18F-FDG PET/CT imaging in


abdominal TB, showing that the appearance of abdominal TB is
non-specic and varied (Figure 6).5658
4. Assessment of treatment response
This is potentially the most important clinical application of 18FFDG PET/CT in TB. During anti-TB treatment, some bacillus-negative
tuberculomas do not decrease in size and may even increase, making
it difcult for the physician to decide whether or not to modify
treatment. In these cases, 18F-FDG PET/CT imaging may help, as the
changes in glycolytic activity within the inammatory lesion,
measured by 18F-FDG uptake, correlate well with the clinical
markers of response.49 Several studies have conrmed the value of
18
F-FDG PET/CT in the follow-up and evaluation of the treatment
response, especially in patients with extrapulmonary involvement
and when drug resistance is prevalent.57,5963 In pulmonary and
extrapulmonary TB, a decrease of approximately one-third in
SUVmax has been reported after 1 month of anti-TB treatment when
there is a good response.60 Initial data has shown that SUVmax (both
early and delayed) of involved lymph nodes and the number of
involved lymph node basins are signicantly higher in nonresponders than in responders.64 These ndings warrant further
conrmation in larger cohorts of patients.
After 4 months of anti-TB treatment 18F-FDG PET/CT can also
evaluate the treatment response in patients with high sensitivity and
specicity, using the value of 4.5 as the SUVmax cut-off.62
Other authors have aimed to monitor the metabolic changes in
spinal TB during the course of therapy.49 The mean changes in
SUVmax at various time points from baseline to 6, 12, and
18 months, from 6 to 12 months, from 6 to 18 months, and from 12 to
18 months were calculated and found to be highly signicant (pvalue <0.001).49 18F-FDG PET/CT also shows encouraging results for
the prognosis and detection of residual disease in patients with
spinal infection, particularly when MRI is unconvincing in distinguishing between degenerative changes and infection.65
5. Tuberculosis in HIV patients
The diagnosis of active PTB is a major challenge, especially
in individuals with severe immunosuppression, such as those

co-infected with HIV. Such patients characteristically demonstrate an atypical radiographic pattern, for example middle and
lower lung involvement, absence of cavity formation, presence
of lymphadenopathy and pleural effusions, or a miliary pattern.38
The radiographic appearance of HIV-associated PTB has been
found to be dependent on the level of immunosuppression at the
time of overt disease.66 Radiological manifestations in patients
with a CD4 T-lymphocyte count of <200/mm3 show a higher
incidence of mediastinal or hilar lymph node enlargement, a lower
prevalence of cavitation, and often extrapulmonary involvement
compared with HIV patients with a CD4 T-lymphocyte count of
200/mm3.66 A study performed to determine the CT spectrum of
PTB in HIV patients showed nodular opacities (in 78.5% of cases),
consolidation (46.4%), lymphadenopathy (35.7%), pleural effusion
(35.7%), ground glass opacity (21.4%), and cavitation (21.4%).67
Other authors have reported that features of post-primary PTB are
patchy consolidation with involvement at unusual sites.68 Cavitation is less common at lower CD4 counts. Patients with severe
immunosuppression have an increased incidence of miliary
pulmonary disease, with diffuse, randomly distributed nodules
on CT.69 Mediastinal and hilar lymphadenopathy occurs in 7577%
of cases and is more commonly seen in HIV-positive than in HIVnegative patients.68 Extrapulmonary localizations are frequent in
HIV-infected patients and may involve brain, pericardium,
gastrointestinal tract, peritoneum, and genitourinary tract.70
Currently there are no data to support the use of 18F-FDG PET/
CT in this patient group.
Funding: None.
Ethical approval: Ethical approval was not required.
Conict of interest: All authors have no competing interests to
declare.
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