JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 2010, p. 2972–2974 Vol. 48, No.

8
0095-1137/10/$12.00 doi:10.1128/JCM.00363-10
Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Quantitative Analysis of a Urine-Based Assay for Detection of

Lipoarabinomannan in Patients with Tuberculosis䌤
Maunank Shah,1 Neil A. Martinson,1,2 Richard E. Chaisson,1 Desmond J. Martin,3
Ebrahim Variava,4 and Susan E. Dorman1*
Johns Hopkins University School of Medicine, Baltimore, Maryland1; Perinatal HIV Research Unit, University of the Witwatersrand,
Soweto, South Africa2; Toga Laboratories, Johannesburg, South Africa, and Department of Medical Virology, University of
Pretoria, South Africa3; and Tshepong Hospital, Northwest Department of Health, Klerksdorp, South Africa4
Received 22 February 2010/Returned for modification 18 May 2010/Accepted 2 June 2010

Urinary lipoarabinomannan (LAM) detection is a promising approach for rapid diagnosis of active tuber-
culosis (TB). In microbiologically confirmed TB patients, quantitative LAM detection results increased pro-
gressively with bacillary burden and immunosuppression. Patients with disseminated TB and/or advanced HIV
are target populations for whom urine LAM detection may be particularly useful.

Effective control of tuberculosis (TB) is hindered by the lack
of rapid, accurate diagnostic modalities. Current tools have
serious limitations when applied to HIV-infected patients, and
clinical diagnosis can be challenging (2, 6, 8, 13). Sputum
smear microscopy is widely utilized to diagnose active TB but
has impaired sensitivity and detects less than half of HIV-TB
coinfection cases (2, 6, 8). Mycobacterial culture is the labo-
ratory standard for diagnosis but is not widely available in
resource-constrained settings and can take weeks to determine
a positive result.
The detection of lipoarabinomannan (LAM), a 17.5-kDa
glycolipid component of the mycobacterial cell wall, is an at-
tractive approach to diagnosing active TB (1, 7, 9, 15, 16).
LAM is released from metabolically active mycobacteria and is
detectable intact in urine (1, 7). The Clearview TB enzyme-
linked immunosorbent assay (ELISA) (“urine LAM test”; In-
verness Medical Innovations, Waltham, MA) is a direct anti-
gen sandwich immunoassay in a 96-well-plate format that
provides both quantitative and qualitative results. Our group
and others have recently reported on the qualitative diagnostic
accuracy of this urine LAM test (9, 10, 16). Urine LAM test
sensitivity, while imperfect, appears to be higher than that of
sputum smear microscopy, and the test performs with a high
positive predictive value in populations with high HIV and TB
prevalence (9, 12, 16). However, quantitative urine LAM test
results have not been studied fully. Quantitative analysis allows
a more complete understanding of test performance and may
offer insight into optimal test usage. Preclinical data and lim-
ited regression modeling from clinical studies have shown that
quantitative test results positively correlate with increasing ba-
cillary burden (1, 16). We therefore examined the relationship
between quantitative urine LAM test results and TB charac-
teristics among patients with culture-confirmed TB.
A full description of the study design has been published
previously (16). Briefly, we conducted a nested prospective
* Corresponding author. Mailing address: Johns Hopkins University
Center for Tuberculosis Research, CRB2, Room 1M-12, 1550 Orleans
Street, Baltimore, MD 21231. Phone: (410) 502-2717. Fax: (410) 955-
0740. E-mail: dsusan1@jhmi.edu.
䌤
Published ahead of print on 9 June 2010.
cohort study at three hospitals in South Africa to evaluate the
diagnostic accuracy of the urine LAM test. Hospitalized adult
TB suspects were enrolled after informed consent. Study-di-
rected testing included sputum smear microscopy for acid-fast
bacilli (AFB), sputum mycobacterial culture, mycobacterial
blood cultures, HIV and CD4 testing, urine LAM testing using
the Clearview TB ELISA kit, and additional mycobacterial
cultures (e.g., from enlarged lymph nodes) when clinically in-
dicated. Participants had a follow-up study visit at 2 months.
Participants with at least one positive AFB smear or mycobac-
terial culture positive for Mycobacterium tuberculosis were de-
termined to have “confirmed TB” and were included in the
current analysis. Individuals with M. tuberculosis isolated from
a blood culture (mycobacteremia) were considered to have
disseminated disease. Pulmonary TB (PTB) was defined as the
presence of one or more sputa positive for AFB by microscopy
and/or positive for M. tuberculosis by culture. Localized ex-
trapulmonary TB was defined as isolation of M. tuberculosis
from pleural culture or fine-needle aspiration of a lymph node
but not from other respiratory specimens or blood. Quantita-
tive urine LAM test results were expressed as optical density
(OD) readings. The final sample OD was determined by sub-
tracting the OD of the negative control from the sample read-
ing, with a minimum value of 0 (i.e., a value of 0 was reported
if the OD of the negative control was greater than the OD of
the sample). An OD above 0.1 was considered positive per
manufacturer specifications. Multiple linear regression models
were used to determine predictors of higher quantitative urine
LAM test results. Wilcoxon rank sum and Kruskal-Wallis tests
were used to compare nonnormal continuous outcomes; chi-
square (␹2) tests were used to compare categorical outcomes.
A P value of ⱕ0.05 was considered statistically significant, and
95% confidence intervals (95% CI) were used. Statistical cal-
culations were performed using Stata 10.1 (StataCorp). This
study was approved by the institutional review boards of Johns
Hopkins University School of Medicine and the University of
Witwatersrand.
Full characteristics of the study population are published
elsewhere (16). Overall, 499 participants were enrolled, and
193 (39%) had confirmed TB (185 with positive cultures for M.

2972
VOL. 48, 2010 NOTES 2973

TABLE 1. Optical density by type of disease (determined by site) TABLE 2. Optical density by sputum AFB smear microscopy status
among participants with confirmed TB among participants with sputum culture-positive pulmonary TB
No. of cases with No. of cases with
Sputum AFB
positive urine positive urine
Type of disease (no. of OD median OD mean smear microscopy OD median OD mean
LAM test result LAM test result
cases) (IQR) (SD) status (no. of (IQR) (SD)
(% sensitivity, (% sensitivity,
cases)c
95% CI) 95% CI)

Localized extrapulmonary 0.01 (0.01–0.06) 0.09 (0.16) 1 (20, 1–72) Negative (88) 0.13 (0.02–0.62)a,b 0.60 (0.93) 47 (53, 0.42–0.64)
TB (5)a
Pulmonary TB only (145)b 0.13 (0.02–0.70) 0.63 (0.97) 78 (54, 45–62) Positive
Mycobacteremia only (18)c 0.64 (0.14–1.43) 0.99 (1.02) 14 (78, 52–94) Any grade (73) 0.33 (0.052–1.71)b 0.94 (1.15) 47 (64, 0.52–0.75)
Pulmonary TB with 1.4 (0.43–2.8) 1.6 (1.1) 21 (84, 64–96) Grade 1⫹ (13) 0.18 (0.1–1.3)a 0.68 (0.89) 10 (77, 0.46–0.94)
mycobacteremia (25) Grade 2⫹ (15) 0.26 (0.03–2.8)a 1.13 (1.3) 10 (67, 0.38–0.88)
P value 0.0001d —e 0.003f Grade 3⫹ (45) 0.38 (0.048–1.8)a 0.95 (1.18) 27 (60, 0.44–0.74)
a
Four of five participants had M. tuberculosis isolated from pleural cultures, a
P ⫽ 0.18 for comparison of AFB-negative, AFB 1⫹, AFB 2⫹, and AFB 3⫹
and 1 of 5 had M. tuberculosis isolated from a lymph node. The urine LAM test results by the Kruskal-Wallis test.
was positive in the participant with M. tuberculosis isolated from the lymph node b
P ⫽ 0.03 for comparison of AFB-negative and AFB-positive results by the
but negative in the 4 participants with localized pleural TB. Wilcoxon rank sum test.
b c
M. tuberculosis in sputum but not blood. Grading per WHO guidelines (http://wwwn.cdc.gov/dls/ila/documents/lstc2
c
M. tuberculosis in blood but not sputum. .pdf).
d
Kruskal-Wallis test of equality between types of disease.
e
—, analysis of variance (ANOVA) was not performed, due to nonnormal
distribution.
f
Chi-square (␹2) test comparing proportions of positive urine LAM test results (P ⫽ 0.0001) (Table 3). The median OD increased progres-
(sensitivities) between types of disease.
sively from 0.10 (IQR, 0.02 to 0.5) to 0.20 (IQR, 0.03 to 0.8) to
0.42 (IQR, 0.04 to 1.0) to 1.3 (IQR, 0.2 to 2.8) in those with
CD4 counts of ⬎150, 101 to 150, 50 to 100, and ⬍50, respec-
tuberculosis and 8 with positive AFB smears only); 6/193 (3%)
tively (P ⫽ 0.0001).
confirmed TB cases were on recent TB treatment (less than 2
Multiple linear regression models were used to further ex-
months) at time of enrollment. Among the confirmed TB
plore factors associated with higher quantitative urine LAM
cases, the median age was 34 years, 65% were female, and 87%
test results. Covariates included in the models were age, sex,
(167/193) had documented HIV infection, of which 9% (15/
HIV status, mortality at 2 months, TB treatment, mycobacte-
167) were on antiretroviral therapy (ART) at the time of en-
remia, sputum smear positivity, fever, Karnofsky score, chest X
rollment. TB manifestations ranged from localized to dissem-
ray (CXR) cavitation, hypoxia, pulse, and CD4 strata. Adjust-
inated disease (Table 1). Localized extrapulmonary TB was
ing for all other factors, individuals with mycobacteremia had
diagnosed in 5 cases (3%), PTB without mycobacteremia (M.
an average OD that was 0.64 OD units higher than that for
tuberculosis in sputum but not blood) in 145 cases (75%),
individuals with negative blood cultures (P ⫽ 0.002); individ-
mycobacteremia without PTB (M. tuberculosis in blood but not
uals with smear-positive pulmonary TB had an average OD
sputum) in 18 cases (9%), and combined PTB with mycobac-
that was 0.33 OD units higher than that for individuals with
teremia (M. tuberculosis in blood and sputum) in 25 cases
smear-negative pulmonary TB (P ⫽ 0.05). Among HIV-in-
(13%). The overall LAM test sensitivity was 59% (95% CI, 52
fected patients, individuals with CD4 counts of ⬍50 had an
to 66) in participants with confirmed TB, and the specificity
average OD that was 1.05 OD units higher than that for indi-
was 96% (95% CI, 91 to 99) among individuals without TB.
viduals with CD4 counts of ⬎150 (P ⬍ 0.0001). No other
Among participants with confirmed TB, the range of quanti-
covariates were significantly associated with higher quantita-
tative LAM test results was 0 to 4.87 (median, 0.19; interquar-
tive urine LAM test results.
tile range [IQR], 0.03 to 1.3).
Taken together, our findings show that quantitative urine
Among participants with confirmed TB, there was a signif-
LAM test results correlate with degree of bacillary burden in
icant difference in median OD for those with more localized
patients with microbiologically confirmed TB. Further, the ab-
disease compared to those with disseminated disease (P ⫽
solute urine LAM test OD increased dramatically as immuno-
0.0001) (Table 1). The median OD increased progressively
from 0.01 (IQR, 0.01 to 0.06) to 0.13 (IQR, 0.02 to 0.70) to 0.64
(IQR, 0.14 to 1.43) to 1.4 (IQR, 0.43 to 2.8) in those with TABLE 3. Optical density by CD4 status among participants with
localized extrapulmonary TB, PTB alone, mycobacteremia HIV infection and confirmed TB
without PTB, and combined PTB with mycobacteremia, re-
No. of cases with
spectively. Among 161 participants with sputum culture-posi- CD4 count OD median OD mean positive urine LAM
tive PTB, those that were AFB smear positive had higher ODs (no. of cases) (IQR) (SD) test results (%
than those that were smear negative (median ODs of 0.33 sensitivity, 95% CI)
versus 0.13, respectively; P ⫽ 0.03) (Table 2). There was a ⬎150 (63) 0.10 (0.02–0.5) 0.42 (0.72) 32 (51, 0.38–0.64)
trend toward higher OD with increasing grades of smear pos- 101–150 (16) 0.2 (0.03–0.8) 0.65 (0.96) 9 (56, 0.30–0.80)
itivity (median ODs of 0.13 [IQR, 0.02 to 0.62], 0.18 [IQR, 0.1 50–100 (28) 0.42 (0.04–1.0) 0.72 (0.9) 20 (71, 0.51–0.87)
to 1.3], 0.26 [IQR, 0.03 to 2.8], and 0.38 [IQR, 0.048 to 1.8] for ⬍50 (60) 1.3 (0.2–2.8) 1.4 (1.12) 51 (85, 0.73–0.93)
P value 0.0001a —b 0.00c
smear-negative, smear-positive grade 1⫹, smear-positive grade
2⫹, and smear-positive grade 3⫹ cases, respectively; P ⫽ 0.18).
a
Kruskal-Wallis test of equality between CD4 strata.
b
—, ANOVA was not performed, due to nonnormal distribution.
Among confirmed TB patients with HIV infection, the urine c
Chi-square (␹2) test comparing proportions of positive urine LAM test re-
LAM test OD increased with decreasing strata of CD4 count sults (sensitivities) between CD4 strata.
2974 NOTES
suppression progressed among HIV-coinfected TB patients,
probably due to higher mycobacterial burden and greater like-
lihood of disseminated TB. Strikingly, the median quantitative
urine LAM test result was over 10-fold higher than the current
threshold for test positivity in those with CD4 counts less than
50 and 5-fold higher than this threshold in those with CD4
counts between 51 and 100. Usage of quantitative urine LAM
test results may thus offer additional clinical insight into the
degree of TB disease severity that cannot be gleaned from
qualitative results alone.
To date, studies of the qualitative accuracy of the urine
LAM test have yielded varied results. In heterogeneous pop-
ulations with low HIV prevalence, the sensitivity has ranged
from 13 to 51% (3, 4, 10, 12, 14). Alternatively, sensitivity rises
among HIV-positive patients and could reach 67 to 85% in
those with low CD4 counts (9, 16). This quantitative analysis
provides evidence that urine LAM test performance may be
best in patients with disseminated TB disease and high myco-
bacterial disease burden. HIV-infected TB suspects with ad-
vanced immunosuppression, a group in which sputum micros-
copy is of low yield (5, 8, 11, 17), may be a target population for
whom the urine LAM test would be particularly useful. Of
note, the combination of LAM testing and sputum smear mi-
croscopy may have an additive benefit in this target group (16).
While the urine LAM test is unlikely to stand alone for defin-
itive TB diagnostic testing, its usage is attractive as a rapid
diagnostic modality that complements smear microscopy for
HIV-prevalent, resource-constrained settings where mycobac-
terial culture is unavailable.
This work was funded in part by the Doris Duke Charitable
Foundation and the United Kingdom Government through a grant
from the Department for International Development (DFID). Ad-
ditional support was provided by NIH grants AI48526, AI01637,
and T32AI007291-18.
Clearview TB ELISA kits were generously provided by Inverness
Medical Innovations, which had no role in the study design, imple-
mentation, or analysis, the manuscript writing, or the decision to sub-
mit a manuscript for publication.
REFERENCES
1. Boehme, C., E. Molokova, F. Minja, S. Geis, T. Loscher, L. Maboko, V.
Koulchin, and M. Hoelscher. 2005. Detection of mycobacterial lipoarabino-
mannan with an antigen-capture ELISA in unprocessed urine of Tanzanian
J. CLIN. MICROBIOL.
patients with suspected tuberculosis. Trans. R. Soc. Trop. Med. Hyg. 99:893–
900.
2. Colebunders, R., and I. Bastian. 2000. A review of the diagnosis and treat-
ment of smear-negative pulmonary tuberculosis. Int. J. Tuberc. Lung Dis.
4:97.
3. Daley, P., J. S. Michael, P. Hmar, A. Latha, P. Chordia, D. Mathai, K. R.
John, and M. Pai. 2009. Blinded evaluation of commercial urinary lipoarabi-
nomannan for active tuberculosis: a pilot study. Int. J. Tuberc. Lung Dis.
13:989–995.
4. Dheda, K., V. Davids, L. Lenders, T. Roberts, R. Meldau, D. Ling, L. Brunet,
R. van Zyl Smit, J. Peter, C. Green, M. Badri, L. Sechi, S. Sharma, M.
Hoelscher, R. Dawson, A. Whitelaw, J. Blackburn, M. Pai, and A. Zumla.
2010. Clinical utility of a commercial LAM-ELISA assay for TB diagnosis in
HIV-infected patients using urine and sputum samples. PLoS One 5:e9848.
5. El-Sadr, W. M., and S. J. Tsiouris. 2008. HIV-associated tuberculosis: diag-
nostic and treatment challenges. Semin. Respir. Crit. Care Med. 29:525–531.
6. Getahun, H., M. Harrington, R. O’Brien, and P. Nunn. 2007. Diagnosis of
smear-negative pulmonary tuberculosis in people with HIV infection or
AIDS in resource-constrained settings: informing urgent policy changes.
Lancet 369:2042.
7. Hamasur, B., J. Bruchfeld, M. Haile, A. Pawlowski, B. Bjorvatn, G. Kalle-
nius, and S. B. Svenson. 2001. Rapid diagnosis of tuberculosis by detection
of mycobacterial lipoarabinomannan in urine. J. Microbiol. Methods 45:
41–52.
8. Havlir, D. V., H. Getahun, I. Sanne, and P. Nunn. 2008. Opportunities and
challenges for HIV care in overlapping HIV and TB epidemics. JAMA
300:423.
9. Lawn, S. D., D. J. Edwards, K. Kranzer, M. Vogt, L. G. Bekker, and R. Wood.
2009. Urine lipoarabinomannan assay for tuberculosis screening before an-
tiretroviral therapy diagnostic yield and association with immune reconsti-
tution disease. AIDS 23:1875–1880.
10. Mutetwa, R., C. Boehme, M. Dimairo, T. Bandason, S. S. Munyati, D.
Mangwanya, S. Mungofa, A. E. Butterworth, P. R. Mason, and E. L. Corbett.
2009. Diagnostic accuracy of commercial urinary lipoarabinomannan detec-
tion in African tuberculosis suspects and patients. Int. J. Tuberc. Lung Dis.
13:1253–1259.
11. Perkins, M. D., and J. Cunningham. 2007. Facing the crisis: improving the
diagnosis of tuberculosis in the HIV era. J. Infect. Dis. 196(Suppl. 1):S15.
12. Peter, J., C. Green, M. Hoelscher, P. Mwaba, A. Zumla, and K. Dheda. 2010.
Urine for the diagnosis of tuberculosis: current approaches, clinical applica-
bility, and new developments. Curr. Opin. Pulm. Med. 16:262–270.
13. Reid, M. J., and N. S. Shah. 2009. Approaches to tuberculosis screening and
diagnosis in people with HIV in resource-limited settings. Lancet Infect. Dis.
9:173.
14. Reither, K., E. Saathoff, J. Jung, L. T. Minja, I. Kroidl, E. Saad, J. F.
Huggett, E. N. Ntinginya, L. Maganga, L. Maboko, and M. Hoelscher. 2009.
Low sensitivity of a urine LAM-ELISA in the diagnosis of pulmonary tu-
berculosis. BMC Infect. Dis. 9:141.
15. Sada, E., D. Aguilar, M. Torres, and T. Herrera. 1992. Detection of li-
poarabinomannan as a diagnostic test for tuberculosis. J. Clin. Microbiol.
30:2415–2418.
16. Shah, M., E. Variava, C. B. Holmes, A. Coppin, J. E. Golub, J. McCallum,
M. Wong, B. Luke, D. J. Martin, R. E. Chaisson, S. E. Dorman, and N. A.
Martinson. 2009. Diagnostic accuracy of a urine lipoarabinomannan test for
tuberculosis in hospitalized patients in a high HIV prevalence setting. J.
Acquir. Immune Defic. Syndr. 52:145–151.
17. Swaminathan, S., C. Padmapriyadarsini, and G. Narendran. 2010. HIV-
associated tuberculosis: clinical update. Clin. Infect. Dis. 50:1377–1386.