International Journal of Current Medical And Applied Sciences, 2017, March, 14(1),01-05.

ORIGINAL RESEARCH ARTICLE

Utility of Urinary Lipoarabinomannan (LAM) Antigen

Detection Assay for Diagnosis of Pediatric Pulmonary
Tuberculosis.
Swapna Kanade1, Shreshtha Tiwari2, Gita Nataraj3 & Preeti Mehta4
1Associate Professor, 2Resident, 3Professor, 4Professor & Head, Department of Microbiology, Seth GSMC &
KEM Hospital, Parel, Mumbai [MS], India; 400012.
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Abstract:
Objective: To determine the utility of lipoarabinomannan detection assay in urine for diagnosing pediatric pulmonary
tuberculosis.
Method: A prospective cross-sectional study was carried out on 100 children less than 14 years of age, with strong
clinical suspicion and radiological evidence suggestive of pulmonary Tuberculosis. One fresh early morning urine
R
sample (approximately 5ml) was collected. The LAM assay was performed using the CLEARVIEW TB ELISA, Alere
Medicals, U.S.A, according to the manufacturer’s package insert. Sputum samples/ gastric lavage were collected. Direct
smears and culture on Lowenstein Jensen media was performed.
Results: Of the 100 children included in the study, culture was positive in 31, microscopy was positive in 13 and LAM
assay in 19 children. When compared with culture, sensitivity and specificity of LAM assay was 45.16% and 92.75%
respectively and the PPV and NPV was 73.68% and 79.1% respectively. Of the 71 children who received ATT, LAM assay
was positive in 17, culture was positive in 31 and microscopy in 13. When intension to treat was used as the standard,
LAM assay had sensitivity, specificity, PPV and NPV of 23.94%, 93.10%, 89.47% and 33.33%. All that were positive by
microscopy were also positive by culture.
Conclusion: The present study reinforces better case detection by combining urinary LAM assay with microscopy and
culture for diagnosis of pediatric pulmonary tuberculosis. Considering high specificity, it can be used as a very usefull
‘add-on’ test to rule out TB in endemic countries.
Keywords: Urine, LAM, Pediatric pulmonary tuberculosis.

Introduction:
Pediatric pulmonary tuberculosis (PPTB) remains a
major cause of morbidity and mortality worldwide,
particularly in developing countries [1]. Though the
actual burden of pediatric tuberculosis is not known due
to diagnostic difficulties, it has been assumed that in
India, it accounts for 10 -15% of total TB cases [2]. In
children, it is important to detect TB early and treat
effectively since young children are more likely to
develop severe disseminated disease as compared to
adults. Childhood TB is a sentinel event, indicating on
going transmission of TB within communities [3].
The diagnosis of pulmonary TB [PTB] in children
remains an unmet challenge in clinical practice
throughout the world today. The reason include but are
not limited to the non-specific signs / symptoms, low
bacillary load in PPTB, the method used to recover
samples, and the inherent limitations of the diagnostic
tests themselves [4]. Diagnosis is usually based on
clinical and radiological manifestations and results of
Tuberculin skin test, all of which lacks specificity and
sensitivity.

Address for correspondence:

Dr. Swapna Kanade,
Access this Article Online
Associate Professor,
Department of Microbiology, Website:
Seth GSMC & KEM Hospital,
Mumbai [MS], India 400012. www.ijcmaas.com
Email: swapnakanade71@gmail.com
How to cite this article:
Swapna Kanade, Shreshtha Tiwari, Gita Nataraj & Preeti Mehta : Utility Subject:
of Urinary Lipoarabinomannan (LAM) Antigen Detection Assay for Medical Sciences
Diagnosis of Pediatric Pulmonary Tuberculosis.: International Journal
of current Medical and Applied sciences; 2017, 14(1),01-05. Quick Response Code

IJCMAAS,E-ISSN:2321-9335,P-ISSN:2321-9327. Page | 01
 Swapna Kanade, Shreshtha Tiwari, Gita Nataraj & Preeti Mehta
As per International standards of TB care,
microbiological confirmation should be sought in all
children suspected of having pulmonary TB, through
examination of respiratory specimen for smear
microscopy, Xpert MTB / RIF assay and / or culture
[5]. Sputum microscopy with a sensitivity of 15% and
culture positivity of 30--40%, bacteriological diagnosis
of tuberculosis in children is difficult in settings with
limited resources [6]. Nucleic acid amplification test
(NAAT) offers enormous potential for accurate rapid
diagnosis, but only commercials kits have been
validated and are trustworthy for replicable results
but are expensive [7]. The specimens required for
diagnosis of PPTB includes expectorated / induced
sputum and gastric lavage which are difficult to obtain
from children.
Antigen detection tests provide direct evidence of
active disease, thus allowing immediate initiation of
treatment [8]. A number of mycobacterial antigens can
be detected in the urine of the patients with
pulmonary TB, but most promising of these is the cell
wall polysaccharide lipoarabinomannan (LAM) [9]. It
is a 17.5k D glycolipid found in the cell wall of
mycobacterium and it accounts for upto 15% of the
total bacterial weight. It serves as an immunogenic
virulence factor that is released from metabolically
active or degrading bacterial cells during TB infection.
Detection of LAM antigen was originally described
using serum but this test was limited due to immune
complex formation [10,11]. LAM is filtered unchanged
by the kidneys. Urinary excretion of LAM occurs
independent of the anatomical location of the
infection. It is excreted in variable amounts ranging
from 0.5 to several hundred ng/ml. Being heat stable,
it does not get readily degraded in clinical specimens,
increasing its suitability as a diagnostic target [12].
Detection of LAM in urine has several advantages.
Urine sample is simple to collect and the infection
control measures required for processing urine
specimen are far fewer than sputum specimen.
Hence, this study was carried out to determine the
utility of lipoarabinomannan detection assay in urine
for diagnosing pediatric pulmonary tuberculosis.
Material and Method:
Institutional ethics committee permission was
obtained. A prospective, experimental study was
carried out over a period of one year in a tertiary care
teaching hospital. 100 children less than 14 years of
age and with strong clinical suspicion of pulmonary
tuberculosis whose parents/ guardians provided
written informed consent were included in the study.
Symptoms that were likely to suggest a diagnosis of
pulmonary TB included, cough >= 2 weeks and / or
weight loss of at least 10% of healthy body weight or
no weight gain in 3 months and / or symptoms of
fever for >= 2 weeks or one measured temperature
above 38.50C and radiological evidence suggestive of
pulmonary TB. Children who were on anti-TB therapy
(ATT) for pulmonary/ extra-pulmonary TB or those
who had received ATT within the last 6 months were
excluded from the study. Based on the results of
Logic Publications @ 2017, IJCMAAS, E-ISSN: 2321-9335,P-ISSN:2321-9327.
laboratory tests, radiological findings and clinical
suspicion, the treatment was initiated by the clinician.
Each child was followed up at the end 15 days. Those
started on ATT were followed at the end of 2 months
and 6 months to note any change in treatment
strategy/ resolution of symptoms such as fever, cough,
loss of appetite, documented evidence of gain in
weight, result of a repeat CXR at end of completion of
ATT and repeat microscopy results.
One fresh early morning urine sample (approximately
5ml) was collected by clean catch technique from each
child. From children > 5 years of age and who could
expectorate, two sputum specimens were collected in
sterile wide mouthed screw capped containers as per
Revised National Tuberculosis Control programme
[RNTCP] protocol [13]. In younger children and those
who could not expectorate, gastric lavage (GL) was
collected by the paediatrician on 3 consecutive days in
sterile, wide mouthed, screw capped containers
having sodium bicarbonate 100 mg for 5–10 ml of GL
to neutralize the acidic pH [14].
Microbiology workup:
LAM ELISA assay on urine
Urine was refrigerated immediately after collection
and processed within 24 hours of collection. One ml of
urine was placed in a 1.5 ml micro-centrifuge tube and
heated at 95°C for 30 min. After cooling to room
temperature, it was centrifuged at 10,000 rpm for 15
min. Supernatant was collected into a new labelled
tube and stored at −20°C for subsequent ELISA testing.
The LAM assay was performed using the CLEARVIEWR
TB ELISA, Alere Medicals, U.S.A, according to the
manufacturer’s package insert [15]. Whole procedure
was done in microtitre strips. Optical density [OD] was
read immediately at 450 nm using Microelisa plate
reader (Bio-Rad Laboratories). As recommended by
the manufacturer, the sample was reported as positive
if OD was at least 0.1 above the signal of the negative
control.
Any single sample from the subject showing acid fast
bacilli on microscopy or growth on LJ medium
suggestive of MTB was considered as positive sample
for analysis. Treatment initiated by the clinician was
also noted.
Direct smears were prepared from all respiratory
samples, and stained by Ziehl Neelsen method. The
results were recorded as per RNTCP protocol [13]. All
specimens were decontaminated with NALC-NaOH
method and concentrated by centrifuging at 3000 g for
15 minutes. The supernatant was carefully discarded
into a disinfectant container. 1 ml of the supernatant
was retained with the sediment which was then
vortexed to mix properly. Two loopfuls of this mixture
was inoculated on LJ medium and incubated
aerobically at 37oC [16]. All cultures were read daily
for the first week for detecting contamination and
rapidly growing mycobacterium species and then
weekly thereafter, till growth was detected or 8 weeks
whichever was later. If mycobacterial growth was
detected, it was identified as MTB/ NTM using SD
BIOLINE TB Ag MPT 64 Rapid® .
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 Logic Publications @ 2017, IJCMAAS, E-ISSN: 2321-9335,P-ISSN:2321-9327.

Statistical analysis:
100 clinically suspected PPTB cases were included. Two trained technicians performed the processing of urine and
respiratory sample separately, unaware of the result of other. Data was entered in SPSS version 15.0. Performance
of LAM assay was reported as sensitivity and specificity at 95% confidence interval. Two reference standards were
used for comparison; culture positivity and intention to treat.
Results:
Of 100 consecutive, clinically suspected PPTB cases included in the study, 36 were less than 5 years of age. Gender
ratio was observed to be equal. Gastric lavage was obtained from 41 and expectorated sputum from 59 children.
All respiratory specimens were processed and examined using microscopy and culture and urine specimens were
tested by LAM ELISA assay. Of the 100 children included in the study, culture was positive in 31, microscopy was
positive in 13 and LAM assay in 19 children. All the culture isolates were identified as MTBC by “SD Bioline MPT 64
Rapid” test. In clinically suspected cases of PPTB, the sensitivity of LAM, culture and microscopy was 19%, 31%
and 13% respectively.
Table 1: Comparison of LAM assay and microscopy with culture and Intension to treat (ITT).
Culture ITT
[Intension to treat]
Positive Negative Yes (n=71) No (n=29)
(n=31) (n=69)
Urinary LAM assay
Positive (n=19) 14 (45.16%) 5 (7.25%) 17 (23.94%) 2 (6.90%)
Negative (n=81) 17 (54.84%) 64 (92.75%) 54 (76.06%) 27 (93.10%)
Sputum Microscopy
Positive (n=13) 13 (41.94%) 0 (0%) 13 (18.30%) 0 (0%)
Negative (n=87) 18 (58.06%) 69 (100%) 58 (81.70%) 29 (100%)

Concordant results by LAM assay and culture (Table 1) were observed in 78 children with 14(45.16%) testing
positive both by culture and LAM assay and 64 (92.75%) testing negative by both. Discordance was observed in 22
of which five were LAM positive and culture negative and 17 were LAM negative and culture positive.

Table 2: Sensitivity and specificity of urinary LAM assay and sputum microscopy.
Culture
Test Sensitivity
Urinary LAM assay 45.16%
Sputum Microscopy 41.94%
When compared with culture (Table 2), sensitivity and
specificity of LAM assay was 45.16% and 92.75%
respectively. The PPV was 73.68% and NPV was
79.1%.
Of the 100 children enrolled in the study, 29 received
only antibiotics (Amoxicillin or Cloxacillin and / or
Amikacin) and responded. Of these, none were acid
fast microscopy positive or culture positive. However,
two of these were LAM assay positive. 71 children
received ATT. LAM assay was positive in 17 (23.94%)
of these cases while culture was positive in 31
(43.66%) and microscopy in 13 (18.31%) cases.
When ITT was used as the standard, LAM assay had
sensitivity, specificity, PPV and NPV of 23.94%,
93.10%, 89.47% and 33.33%. All that were positive by
microscopy were also positive by culture.
Discussion:
Definitive diagnosis of pediatric pulmonary TB using
conventional modalities of microscopy and culture has
given unsatisfactory yield. In the present study done
on 100 children with clinical presentation strongly
suggestive of PTB, performance of LAM assay in urine
specimens has been evaluated against culture on LJ
medium and intention to treat (ITT) by
antituberculosis treatment. This is one of the few
International Journal of Current Medical And Applied Sciences [IJCMAAS], Volume: 14, Issue: 1.
ITT
Specificity Sensitivity Specificity
92.75% 23.94% 93.10%
100% 18.30% 100%
studies which have included ITT as standards for
defining performance of the different diagnostic tests
[17].
A number of mycobacterial antigens like LAM,
mycobacterial sonicates, extracted glycolipids, PPD,
Antigen 5 (38 KDU), cord factor (trehalose
dimycolate), etc. can be used in antigen detection
assays for diagnosis of TB. LAM is the cell wall
polysaccharide released by lysis of metabolically
active bacteria in large quantity. Approximately 15 mg
of LAM is secreted per gram of bacteria in culture [18].
Development of high tissue concentration of LAM at
anatomic sites of disease results in systemic
antigenaemia. LAM has a similar molecular size to
myoglobin and is filtered unchanged by the kidneys.
Successful detection of LAM in urine is not associated
with heavy proteinuria, indicating its excretion in
kidney having normal glomerular function. It is heat
stable and so does not readily degrade in clinical
specimens [19].
The sensitivity of LAM was found to be 45.16% and
specificity was 92.75%. Higher specificity ranging
from 87% to 99% has been reported in various studies
[20,21]. But variable sensitivity has also been reported
ranging from 13% -37% [21,22] and 40% -60%
[20,23,24]. 80.3% sensitivity has been reported by
 Swapna Kanade, Shreshtha Tiwari, Gita Nataraj & Preeti Mehta
Boehme et al [25]. The disparity in sensitivity and
specificity observed in various studies can be due to
poor quality of sample in pediatric age group and
variable techniques used for culture. In a meta-
analysis done, sensitivity of 14% (4-38%) in non HIV
infected individuals and 47% in HIV infected patients
was found with a specificity of more than 96% [26].
Five children had culture negative and LAM positive
results. One child showed improvement with ATT.
Two of these responded to antibiotics and two expired
during follow up period. False positive LAM results
could be due to contamination of urine with normal
commensal flora in the perineum e.g. Candida or due
to colonization with NTM [21]. Contamination of
reagents could be another possible explanation for
false positivity.
17 children were culture positive but LAM negative.
This could be due to low sensitivity of LAM assay itself.
Immune stimulation by LAM and subsequent
formation of large immune complexes which cannot
pass through the glomerular membrane might result
in LAM negative urine sample [27]. Some data suggest
that concentration of LAM spiked into urine samples is
stable for the first two hours but deteriorate after
this [28].
It is clear that LAM ELISA cannot be used as a
standalone test but has a potentially important role as
an add-on tests to microscopy in diagnosis of PPTB. In
our study, microscopy as alone detected 13 cases
while LAM alone detected 19 cases. But if LAM is
considered as add-on test to microscopy, the
combination of two tests detected 22 cases. Of these
21 were positive by culture. However, further studies
using a larger sample size are required to ascertain its
efficiency. Though Urinary LAM has low sensitivity, it
demonstrated high specificity. Detection of LAM from
urine samples promise less cumbersome diagnostic
technique in children from whom obtaining gastric
lavage samples is difficult. Thus this might support its
role as an adjunct test in diagnosis of pediatric TB.
Microscopy is a rapid screening test which detects
104-105 bacilli/ ml of sample [29]. Culture on solid/
liquid media is highly specific and is considered the
gold standard test for diagnosis. It can detect 10-100
viable bacilli/ml. PCR can detect ~10 bacilli/ ml in
respiratory samples which makes it a highly sensitive
diagnostic tool. Detection limit of LAM ELISA has not
been quantified by manufacturers.
The limitation of this study is that it was a prospective
study enrolling 100 clinically and radiologically
suspected cases of PTB in children. However no
control group was included to reflect upon the
predictive values of these tests. The collection of
specimen was dependent on the pediatrician. Thus,
there was no control over the quality of specimen and
transport to laboratory. Smear preparation after
concentration of the specimen and the use of
fluorescent / LED microscopy could have increased
smear positivity. Use of liquid culture systems might
have improved the culture yield. Drug susceptibility
Logic Publications @ 2017, IJCMAAS, E-ISSN: 2321-9335,P-ISSN:2321-9327.
testing was not performed for the case of treatment
failure.
Children usually acquire TB from adults. Thus TB in
children reflects the ongoing transmission in the
community and indirectly reflects on the performance
of national programs. This emphasizes the need for
diagnosing the disease in children more accurately
and rapidly to benefit both the affected child and
community. RNTCP emphasizes on acid fast
microscopy for diagnosis of PTB. In the present study,
the sensitivity of smear microscopy in children was
only 13 %. This limits its utility as a diagnostic tool in
children. Though culture remains the gold standard,
its long turnaround time doesn’t have much value in
immediate patient care. As per International
standards, microscopy and molecular tests are advised
for diagnosis of PPTB [5]. But considering very high
cost of molecular tests, it may not be sustainable in
long future in resource constrained settings.
Conclusion:
Urinary LAM assay alone may not be a useful stand-
alone test due to its low sensitivity, but it has a
potential to be used as a very useful ‘add-on’ test.
When combined with microscopy and culture, it can
prove to be a supportive test for diagnosis in
childhood TB. With proven high specificity of this test,
it can be used to rule out TB in endemic countries.
Acknowledgement:
The authors would like to acknowledge the
operational research grant of RNTCP and funds from
Diamond Jubilee Society Trust, Seth GSMC & KEM
hospital, Mumbai.
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Conflict of interest: None declared
Source of funding: RNTCP and funds
from Diamond Jubilee Society Trust, Seth
GSMC & KEM hospital, Mumbai.