INT J TUBERC LUNG DIS 18(2):216219

2014 The Union

http://dx.doi.org/10.5588/ijtld.13.0464
[A version in French of this article is available from the Editorial Offce in Paris and from the Union website www.theunion.org]
Chest X-ray vs. Xpert

MTB/RIF assay for the diagnosis

of sputum smear-negative tuberculosis in Uganda
C. Wekesa,* B. J. Kirenga,

M. L. Joloba,

F. Bwanga,

A. Katamba,

M. R. Kamya

*

Medical Research Council/Uganda Virus Research Institute, Uganda Research Unit on AIDS, Entebbe,

Department
of Medicine, College of Health Sciences, Makerere University, Kampala,

Department of Medical Microbiology,
College of Health Sciences, Makerere University, Kampala, Uganda
Correspondence to: Clara Wekesa, MRC/UVRI Uganda Research Unit on AIDSMSU, P O Box 49, Entebbe, Kampala,
Uganda. Tel: (+256) 772 342 181. Fax: (+256) 041 432 1137. e-mail: clara.wekesa@gmail.com
Article submitted 28 June 2013. Final version accepted 7 October 2013.
SETTI NG: An out-patient clinic in a country with high
rates of tuberculosis-human immunodeficiency virus (TB-
HIV) co-infection.
DESI GN: Cross-sectional analytical study of 123 adults
with chronic cough and no previous anti-tuberculosis
treatment. Demographic, clinical, chest X-ray (CXR) and
GeneXpert

MTB/RIF data were collected. Proportions

of TB diagnoses using both tests were calculated and
compared using an unpaired t-test.
RESULTS: Sixty-six patients (53.7%) were female and
35 (28.5%) tested positive for HIV; 21 (17.1%) were
Xpert-positive, while 51 (42.5%) had CXR suggestive of
TB (P = 0.0018), of whom only 15 (29.4%) were Xpert-
positive. CXR was suggestive of pulmonary TB in 15
(71.4%) of the 21 patients with a positive Xpert test.
CONCLUSI ONS: The majority of the sputum smear-
negative patients did not have TB on single Xpert testing.
CXR gave an overestimate of sputum smear-negative
TB cases.
KEY WORDS: pulmonary tuberculosis; Xpert MTB/RIF;
sputum smear-negative PTB
TUBERCULOSIS (TB) remains of public health con-
cern, particularly in highly endemic regions.
1
It ac-
counts globally for 3 million deaths annually, 85% of
which are in sub-Saharan Africa.
2
Pulmonary TB
(PTB) is primarily diagnosed by identifcation of
Mycobacterium tuberculosis using sputum smear mi-
croscopy. Advanced human immunodefciency virus
(HIV) infection reduces the diagnostic accuracy of
this method as it reduces sputum positivity, creating a
diagnostic dilemma in resource-limited settings.
3
Cli-
nicians often depend on alternative diagnostic algo-
rithms based on patient symptoms and chest X-ray
(CXR) fndings, which have low sensitivity.
4,5
This
may lead to either over- or underdiagnosis of PTB,
with huge consequences on patient outcomes and use
of limited resources.
CXR cannot be used alone to diagnose smear-
negative PTB, as no single radiological lesion patho-
gnomonic of PTB exists.
6
Other non-tuberculous in-
fections can present in similar fashion, and in the era
of HIV/AIDS (acquired immune-defciency syndrome),
radiological PTB lesions can take on a variety of pat-
terns.
7
There is also considerable inter- and intra-
observer variation in the interpretation of CXR fnd-
ings between radiologists and physicians.
8
Sample culture, the gold standard diagnostic tech-
nique, has high accuracy and can be used to identify
drug-resistant disease.
4
Culture is a lengthy procedure,
hindering point-of-care decisions. It requires high lev-
els of expertise and specialised infrastructure to en-
sure biosafety.
4
These reasons limit its use in resource-
constrained settings.
The GeneXpert

MTB/RIF assay (Cepheid, Sunny-

vale, CA, USA) has been shown to have an accuracy
comparable to that of culture. The sensitivity of a sin-
gle Xpert result in smear-negative, culture-positive
cases is 72.5%; the sensitivity increases with repeated
testing.
9
Xpert has been found to be a cost-effective
method for smear-negative TB diagnosis in compari-
son to traditional methods in resource-limited set-
tings.
10
Xpert is an automated real-time polymerase
chain reaction assay. The sample processing time is
short; it is not labour-intensive and does not require
special expertise. Xpert detects both the presence of
M. tuberculosis in sputum and resistance to rifampicin
(RMP), the latter of which is a marker for multidrug-
resistant TB (MDR-TB). Xpert has been endorsed by
the World Health Organization (WHO), particularly
in HIV-infected persons and MDR-TB suspects. It
may be an important tool for the diagnosis of smear-
negative TB, particularly in HIV-endemic areas.
Uganda is a high TB burden country with a high
prevalence of HIV; 55% of TB patients have dual
TB-HIV coinfection.
11
The high HIV prevalence rate
S U MMA R Y
X-ray vs. Xpert for smear-negative TB diagnosis 217
creates a disproportionate number of sputum-negative
cases. High rates of undiagnosed PTB due to smear-
negative disease play a key role in TB transmission
at community level,
12
making it pertinent to isolate
potential sources of TB.
13
Early diagnosis improves
treatment outcomes and reduces the risk of primary
TB resistance.
Due to logistical challenges in resource-constrained
settings, widespread use of Xpert in primary health
care units is diffcult. Its rollout in such settings
should target specifc patient categories. The use of
Xpert, together with existing diagnostic tools such as
CXR, in diagnosing smear-negative TB has not been
evaluated in a resource-limited setting such as Uganda;
this may provide a more effcient diagnostic algorithm
for clinicians.
The aim of the present study was to compare the
proportions of presumptive PTB patients diagnosed
with smear-negative TB using CXR and Xpert. We
hope our fndings will help clarify the role of CXR
in the diagnosis of smear-negative TB in the era of
Xpert technology.
METHODS
Design and setting
This was a cross-sectional analytical study of 123 adults
conducted at an out-patient clinic in the Mulago Na-
tional Referral Hospital, Kampala, from December
2011 to March 2012. The clinic is the initial entry
point for ambulatory patients, and receives on aver-
age 20 000 patients per month. Patients in need of
specialised care are referred to specialist clinics in the
hospital. The unit is open every weekday from 8 am
to 12 pm. There is an accredited microbiology labo-
ratory within the facility, with staff trained in good
clinical and laboratory practice. Routine tests include
smear microscopy using Ziehl-Neelsen stain and HIV
testing. Over 500 sputum smear microscopy tests are
performed each month.
Study eligibility
Patients included in the study were aged 18 years
and had productive cough of 2 weeks. Patients were
also required to have two negative sputum smear mi-
croscopy results at the clinic laboratory for inclusion.
Patients with a history of anti-tuberculosis treatment
and documented chronic pulmonary disease were ex-
cluded. All enrolled patients provided written in-
formed consent. Patient recruitment was conducted
according to randomised time blocks each day.
Patient assessment
Participants were subjected to an interviewer-
a dministered case report form (CRF) to collect rele-
vant socio-demographic information and presenting
symptoms. Demographic information included age,
sex and employment status. Symptoms recorded
i ncluded productive cough as evidenced by the ability
to expectorate, fever as reported by the patient as an
unpleasant sensation of warmth/heat, evening sweats
as reported by the patient as drenching sweats most
evenings of the week, weight loss and loss of appetite
as reported by the patient.
A physical examination (with special emphasis on
the respiratory system) was performed for every par-
ticipant by a doctor and relevant fndings were en-
tered into the CRF. Consolidation was detected by
the presence of crackles and/or presence of bronchial
breathing on auscultation. Pleural effusion was de-
tected by presence of stony dull percussion note over
the chest wall accompanied by auscultation fndings
of bronchial breathing. Lymphadenopathy was mea-
sured as presence of lymph node(s) 0.5 cm diameter
in the cervical area, 1 cm diameter in the axillary
area and 2 cm diameter in the inguinal area.
Early morning sputum samples sent to the out-
p atient laboratory were used for Xpert testing.
14

The sputum sample was mixed with the Xpert sam-
ple treatment reagent at a ratio of 1:2 and trans-
ferred into the sample chamber of the cartridge. All
subsequent processing steps were fully automated, in
which the sample underwent concentration, amplif-
cation and detection of the rpoB DNA segment. Re-
sults were interpreted as negative if no M. tuberculo-
sis was detected and positive if M. tuberculosis or
RMP resistance was detected, as documented in the
evaluation form.
HIV-1 testing was performed in accordance with
the Ministry of Health algorithm using two rapid
tests.
15
Antero-posterior CXRs were performed in
the hospital radiology department and read by a TB
clinician with training in CXR interpretation. CXR
fndings of interest were the presence of fbrosis, doc-
umented as reticulo-linear opacities; cavities, docu-
mented as walled hollow structures within the lungs;
consolidation, documented as heterogeneous or ho-
mogeneous opacifcation of the lung feld without a
meniscus; pleural effusion, documented as homoge-
neous opacifcation of the lung feld with the pres-
ence of meniscus; hilar adenopathy, documented as
mediastinum occupying more than a third of the
transthoracic diameter or the presence of circular
homogeneous opacities in the hilar area; and miliary
disease, documented as the presence of micro-nodules
distributed in all lung felds. All CXRs with pathol-
ogy were grouped up as abnormal and all CXRs
with no pathology were grouped as normal. A CXR
diagnosis of likely TB was made based on the pres-
ence of cavitary disease, miliary disease, pleural effu-
sion and widespread infltrates.
Data analysis
We calculated the proportion of sputum smear-
n egative TB suspects who tested positive on Xpert
and represented it as a percentage. We calculated the
218 The International Journal of Tuberculosis and Lung Disease
proportion of CXRs suggestive of sputum smear-
negative TB and represented it as a percentage. We
compared these two proportions as categorical data
using the
2
test.
Ethics
The study was approved by the Research and Ethics
Committee, Makerere University School of Medicine.
Informed consent was obtained from study partici-
pants, including consent to retrieve HIV and CD4
cell count records. Participants were free to withdraw
from the study at any time without penalty. No speci-
men other than that stated in the protocol was taken
from the participants. Patients diagnosed with TB
were transferred to the hospitals anti-tuberculosis
treatment unit.
RESULTS
During the study period, the facility performed a to-
tal of 681 sputum smear microscopy tests, of which
83.1% were negative for PTB. Of 132 patients sam-
pled, 123 were enrolled into the study (Figure). Sixty-
six patients (53.7%) were female, 35 (28.5%) tested
positive for HIV. Ninety-eight patients (79.7%) had
previous antibiotic use without resolution of symp-
toms. Fever (62.6%), chest pain (61.8%) and night
sweats (43.9%) were the most common symptoms in
addition to cough (Table 1).
Figure Study profile. CXR = chest X-ray; Xpert = GeneXpert

MTB/RIF; + = positive;
= negative.
Table 1 Baseline patient characteristics (N = 123)
n (%)
Male 57 (46.3)
Female 66 (53.7)
Median age, years [range] 31 [1966.8]
HIV-positive 35 (28.5)
Previous antibiotic use 98 (79.7)
Symptoms
Haemoptysis 26 (21.1)
Fever 77 (62.6)
Chest pain 76 (61.8)
Loss of appetite 42 (34.1)
Night sweats 54 (43.9)
Significant weight loss 43 (35)
HIV = human immunodeficiency virus.
Table 2 Radiological features observed among patients with
abnormal CXR (n = 51)
CXR pathology n (%)
Consolidation 45 (88.2)
Cavitary disease 6 (11.8)
Micronodules 3 (5.9)
Hilar adenopathy 2 (3.9)
Pleural effusion 5 (9.8)
CXR = chest X-ray.
Table 3 Diagnostic yield of gene of CXR and GeneXpert

(N = 123)
CXR status
M. tuberculosis
Detected Not detected Total
Abnormal 15 36 51
Normal 6 66 72
Total 21 102 123
CXR = chest X-ray.
Tuberculosis diagnosis using Xpert and chest X-ray
Twenty-one patients (17.1%, 95% confdence inter-
val [CI] 11.424.7) were Xpert-positive, while 51
(42.5%, 95%CI 33.150.3) had a CXR consistent
with TB. The diagnostic proportions of CXR vs.
Xpert were statistically signifcantly different (P =
0.0018). Of the 51 patients with CXR suggestive of
PTB, 15 (29.4%) were Xpert-positive. CXR was sug-
gestive of PTB in 15 (71.4%) of the 21 patients with
positive Xpert results (Table 2). None of the patients
with confrmed PTB were resistant to RMP. The
most common CXR abnormality was consolidation
(88.2%), followed by cavitation (11.8%) and pleural
effusion (9.8%; Table 3).
DISCUSSION
The prevalence of smear-negative TB was found to be
17.1%. This fgure is comparable to that found in the
multi-centred study conducted to validate the Xpert
test.
9
A study performed at Mulago Hospital to as-
sess the aetiology of pulmonary infections among
smear-negative HIV adults showed a similar preva-
lence.
16
However, studies conducted in Malawi have
X-ray vs. Xpert for smear-negative TB diagnosis 219
shown a much higher prevalence of smear-negative
TB.
17
We attributed the failure to demonstrate higher
prevalence to the inability to increase the diagnostic
accuracy of the Xpert test by repeat testing due to f-
nancial constraints. We also acknowledge our inability
to perform sample culture to confrm TB diagnosis.
The use of fuoroquinolones, which could potentially
have affected prevalence, was not evaluated. Studies
have shown that the use of fuoroquinolones delays
the diagnosis of TB and affects CXR patterns.
18
We
also considered the possibility that the majority of
the patients presenting had unresolved pneumonia
and thus tested negative on Xpert.
The proportion of participants with CXR fndings
suggestive of PTB was three times that using Xpert.
This could imply that the diagnosis of smear-negative
TB using CXR could lead to overdiagnosis. The re-
sulting implications include unnecessary exposure to
drug toxicity and overutilisation of limited resources
such as drugs. The high proportion of smear-negative
TB diagnoses by CXR can be explained by the fact
that other conditions may present features similar to
those of TB on CXR.
In this study, all of the patients with PTB con-
frmed on Xpert tested susceptible to RMP, indicating
no primary MDR-TB among new smear-negative
cases. Drug resistance studies performed in south-
western Uganda have reported similarly low rates
(1.6%) using Xpert,
19
as has a WHO report released
in 2012 that showed MDR-TB rates of 1% in Uganda
among new PTB cases.
20
The study also noted that the majority of the pa-
tients with chronic cough did not have a diagnosis of
TB. We attributed this fnding to the probable diag-
nosis of unresolved pneumonia in most of these pa-
tients, the possibility of previous fuoroquinolone use
that partially treated the TB, and the relatively low
accuracy of Xpert in diagnosing smear-negative dis-
ease, coupled with the inability to perform more than
one Xpert assay per sample.
CONCLUSION
The majority of ambulatory patients with chronic
cough and negative sputum microscopy did not have
PTB on single Xpert testing. CXR was found to over-
estimate the number of smear-negative TB cases.
Acknowledgements
The authors thank the participants, study team and investigators
for their invaluable contribution in various ways. Special recogni-
tion goes to the Aereus Microbiology Laboratory, medical staff at
the Mulago Assessment Centre, and to all the staff members of the
Department of Medicine, Mulago Hospital, Kampala, Uganda.
This work was supported and funded by the Fogarty AIDS Inter-
national Training and Research Program, Case Western University
Reserve, Cleveland, OH, USA.
Confict of interest: none declared.
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X-ray vs. Xpert for smear-negative TB diagnosis i
CONTEXTE : Consultation externe dans un pays taux
lev de confection tuberculose-virus de limmuno-
dficience humaine (TB-VIH).
SCHMA : Analyse transversale de 123 adultes prsen-
tant une toux chronique sans traitement antituberculeux
pralable. Recueil de donnes dmographiques et cli-
niques, de radios pulmonaires (CXR) et de tests Gene-
Xpert

. Le taux de diagnostic de TB par les deux examens

a t calcul et compar grce un test t non appari.
RSULTATS : Des 123 patients, 66 (53,7%) taient des
femmes et 35 (28,5%) taient sropositives pour le VIH ;
21 (17,1%) taient positifs au test Xpert tandis que 51
(41,5%) avaient une CXR suggrant une TB (P =
0,0018). Sur les 51 patients ayant une CXR suspecte,
seulement 15 (29,4%) avaient un test Xpert positif.
Par contre, la CXR suggrait une TB pulmonaire chez
15 (71,4%) des 21 patients dont les tests Xpert taient
positifs.
CONCLUSI ON : La majorit des patients dont les frottis
de crachats taient ngatifs avaient galement un test
Xpert ngatif. Par contre, la CXR surestimait la prva-
lence de la TB chez les patients dont le frottis de cra-
chats tait ngatif.
MARCO DE REFERENCI A: Un consultorio de atencin
ambulatoria en un pas con alta incidencia de coinfec-
cin por tuberculosis e por el virus de la inmunodefi-
ciencia humana (TB-VIH).
MTODOS: Se llev a cabo un estudio transversal ana-
ltico de 123 adultos con tos crnica, sin antecedente
de tratamiento antituberculoso y dos baciloscopias de
esputo negativas. Se recogieron los datos demogrficos y
clnicos y los resultados de la radiografa de trax (CXR)
y del anlisis por el sistema GeneXpert

de diagnstico
de la TB. Se calcularon las proporciones de casos di-
agno s ticados por ambos mtodos y se compararon me-
diante una prueba de la t para datos independientes.
RESULTADOS: De los participantes, 66 pacientes fueron
mujeres (53,7%) y 35 obtuvieron un resultado positivo
frente al VIH (28,5%); 21 pacientes tuvieron un resul-
tado positivo con el mtodo Xpert (17,1%) y 51 presen-
taron CXR indicativas de TB (42,5%; P = 0,0018). De
los 51 pacientes con CXR en favor del diagnstico
de TB, solo 15 presentaron resultados positivos con la
prueba Xpert (29,4%). Adems, las CXR fueron indica-
tivas de TB pulmonar en 15 de los 21 pacientes con re-
sultados positivos en Xpert (71,4%).
CONCLUSI N: La mayora de los pacientes con baci-
loscopias negativas del esputo no obtuvo resultados en
favor de TB con una prueba nica del sistema Xpert. La
CXR ofreci una sobrestimacin de los casos de TB con
examen de esputo negativo.
R S U M
R E S U ME N