Articles

Novel lipoarabinomannan point-of-care tuberculosis test

for people with HIV: a diagnostic accuracy study
Tobias Broger*, Bianca Sossen*, Elloise du Toit, Andrew D Kerkhoff, Charlotte Schutz, Elena Ivanova Reipold, Amy Ward, David A Barr,
Aurélien Macé, Andre Trollip, Rosie Burton, Stefano Ongarello, Abraham Pinter, Todd L Lowary, Catharina Boehme, Mark P Nicol,
Graeme Meintjes†, Claudia M Denkinger†

Summary
Lancet Infect Dis 2019; Background Most tuberculosis-related deaths in people with HIV could be prevented with earlier diagnosis and
19: 852–61 treatment. The only commercially available tuberculosis point-of-care test (Alere Determine TB LAM Ag [AlereLAM])
Published Online has suboptimal sensitivity, which restricts its use in clinical practice. The novel Fujifilm SILVAMP TB LAM (FujiLAM)
May 30, 2019
http://dx.doi.org/10.1016/
assay has been developed to improve the sensitivity of AlereLAM. We assessed the diagnostic accuracy of the FujiLAM
S1473-3099(19)30001-5 assay for the detection of tuberculosis in hospital inpatients with HIV compared with the AlereLAM assay.
See Comment page 794
*Contributed equally
Methods For this diagnostic accuracy study, we assessed biobanked urine samples obtained from the FIND Specimen
†Contributed equally
Bank and the University of Cape Town Biobank, which had been collected from hospital inpatients (aged ≥18 years)
FIND, Geneva, Switzerland
with HIV during three independent prospective cohort studies done at two South African hospitals. Urine samples
(T Broger MSc, were tested using FujiLAM and AlereLAM assays. The conduct and reporting of each test was done blind to other test
E Ivanova Reipold PhD, results. The primary objective was to assess the diagnostic accuracy of FujiLAM compared with AlereLAM, against
A Macé PhD, S Ongarello PhD, microbiological and composite reference standards (including clinical diagnoses).
C Boehme MD,
C M Denkinger MD);
Department of Medicine, Findings Between April 18, 2018, and May 3, 2018, urine samples from 968 hospital inpatients with HIV were
Faculty of Health Sciences evaluated. The prevalence of microbiologically-confirmed tuberculosis was 62% and the median CD4 count was
(B Sossen MBChB, 86 cells per µL. Using the microbiological reference standard, the estimated sensitivity of FujiLAM was 70·4%
C Schutz MBChB, A Ward MBChB,
R Burton MBChB,
(95% CI 53·0 to 83·1) compared with 42·3% (31·7 to 51·8) for AlereLAM (difference 28·1%) and the estimated
Prof G Meintjes PhD), specificity of FujiLAM was 90·8% (86·0 to 94·4) and 95·0% (87·7–98·8) for AlereLAM (difference –4·2%). Against
Wellcome Center for Infectious the composite reference standard, the specificity of both assays was higher (95·7% [92·0 to 98·0] for FujiLAM vs
Diseases Research in Africa, 98·2% [95·7 to 99·6] for AlereLAM; difference –2·5%), but the sensitivity of both assays was lower (64·9% [50·1 to 76·7]
Institute of Infectious Disease
and Molecular Medicine
for FujiLAM vs 38·2% [28·1 to 47·3] for AlereLAM; difference 26·7%).
(B Sossen, C Schutz, A Ward,
Prof G Meintjes), and Division of Interpretation In comparison to AlereLAM, FujiLAM offers superior diagnostic sensitivity, while maintaining
Medical Microbiology specificity, and could transform rapid point-of-care tuberculosis diagnosis for hospital inpatients with HIV. The
(E du Toit PhD,
Prof M P Nicol MBChB),
applicability of FujiLAM for settings of intended use requires prospective assessment.
University of Cape Town,
Cape Town, South Africa; Funding Global Health Innovative Technology Fund, UK Department for International Development, Dutch Ministry
National Health Laboratory
of Foreign Affairs, Bill & Melinda Gates Foundation, German Federal Ministry of Education and Research, Australian
Service, Cape Town, South
Africa (E du Toit, Prof M P Nicol); Department of Foreign Affairs and Trade, Wellcome Trust, Department of Science and Technology and National
Division of HIV, Infectious Research Foundation of South Africa, and South African Medical Research Council.
Diseases and Global Medicine,
Zuckerberg San Francisco
Copyright © 2019 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY
General Hospital, University of
California, San Francisco, 4.0 license.
San Francisco, CA, USA
(A D Kerkhoff MD); Wellcome Introduction been identified as an urgent unmet clinical need by
Trust Liverpool Glasgow Centre
Tuberculosis is the leading infectious cause of death WHO.3
for Global Health Research,
University of Liverpool, globally and remains the most common cause of The Alere Determine TB LAM Ag assay (AlereLAM;
Liverpool, UK (D A Barr MBChB); mortality in people with HIV, causing an estimated Abbott, Chicago, IL, USA) detects the presence of the
FIND, Cape Town, South Africa 300 000 deaths in 2017.1 Most tuberculosis-related deaths mycobacterial cell wall component, lipoarabinomannan
(A Trollip PhD); Southern
in people with HIV could be prevented with earlier (LAM), in a urine sample. However, in a meta-analysis
African Medical Unit, Médecins
sans Frontières, Cape Town, diagnosis and treatment.1 Extrapulmonary tuberculosis by WHO,4,5 the sensitivity of this test was only 45% in
South Africa (R Burton); Public is more common in people with HIV who are severely people with HIV, with higher sensitivity (56%) in
Health Research Institute immunocompromised than in immunocompetent patients with CD4 counts equal to or less than 100 cells
Center, New Jersey Medical
School, Rutgers University,
people and therefore sputum might not represent the per µL. Despite suboptimal sensitivity, the test reduces
Newark, NJ, USA ideal diagnostic sample.2 Furthermore, independent of mortality when implemented for immunocompromised
(Prof A Pinter PhD); and location of disease, producing a sputum sample is often hospital inpatients with HIV.6 On this basis, WHO
Department of Chemistry and difficult for patients with advanced HIV who are recommends the use of AlereLAM for people with HIV
severely ill. As a result, non-sputum-based tests have and a CD4 count equal to or less than 100 cells per µL

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Alberta Glycomics Centre,

Research in context University of Alberta,
Edmonton, AB, Canada
Evidence before this study (lipoarabinomannan or LAM) AND (Fuji*), but no articles were (Prof T L Lowary PhD)
WHO recommended the use of the rapid, point-of-care identified. Correspondence to:
Alere Determine TB LAM Ag assay (AlereLAM) for the diagnosis Dr Claudia M Denkinger, FIND,
Added value of this study
of tuberculosis in people with HIV. This recommendation was 1202 Geneva, Switzerland
This is the first study to assess the accuracy and diagnostic yield [email protected]
informed by a Cochrane systematic review and meta-analysis of
of the FujiLAM assay for the diagnosis of active tuberculosis.
12 cross-sectional or cohort studies that showed a relatively
Diagnostic accuracy was compared with rigorously defined
low pooled sensitivity of 45% (95% CI 29–63) and specificity of
microbiological and composite reference standards in three
92% (80–97) against a microbiological reference standard.
cohorts of hospital inpatients with HIV. The findings from this
In a subgroup of patients with HIV and CD4 counts less than or
study show that FujiLAM is substantially more sensitive than
equal to 100 cells per µL, pooled sensitivity was 56% (41–70).
AlereLAM, while maintaining specificity, for the diagnosis of
We searched PubMed for articles published between Feb 5, 2015, active tuberculosis in hospital inpatients with HIV.
and Sept 17, 2018, evaluating the diagnostic accuracy or
diagnostic yield of AlereLAM using the search terms (tuberculosis Implications of all the available evidence
or TB) AND (lipoarabinomannan or LAM) AND (test OR assay OR Considering the substantially improved sensitivity of FujiLAM
antigen OR Ag OR lateral flow assay* OR urine antigen OR point compared with AlereLAM and the high diagnostic yield compared
of care) AND (accuracy OR sensitivity OR specificity OR yield OR with sputum-based diagnostics, the FujiLAM assay has the
diagnos* OR screening). Our search yielded an additional potential to substantially improve rapid diagnosis of tuberculosis
23 relevant studies, which confirmed the moderate clinical in patients with HIV who are admitted to hospital and potentially
sensitivity of AlereLAM. The search also identified two people with HIV in the general population. Since AlereLAM has
randomised trials that demonstrated reduced mortality with demonstrated survival benefit, FujiLAM might potentially further
AlereLAM point-of-care testing for tuberculosis among severely reduce tuberculosis-related mortality in people with HIV.
ill inpatients with HIV. These findings will inform a WHO policy review for
lipoarabinomannan-based diagnostic tests of active tuberculosis.
The novel urine-based Fujifilm SILVAMP TB LAM (FujiLAM) Further research, including prospective and operational studies
assay was developed to overcome the limited sensitivity of on the FujiLAM assay in settings of intended use and in additional
AlereLAM and increase the diagnostic yield of rapid urinary patient populations, including outpatients with HIV, populations
lipoarabinomannan testing. On Sept 17, 2018, we did a second without HIV, and paediatric populations, are needed.
PubMed search using the search term (tuberculosis or TB) AND

and in those defined as seriously ill according to WHO of hospital inpatients with HIV, in whom the AlereLAM
criteria (respiratory rate >30 breaths per min, body assay is recommended for use.
temperature >39°C, heart rate >120 beats per min, or
unable to walk unaided).4 A more sensitive, rapid urine- Methods
based test could widen the indication for testing and Study participants
improve the diagnosis of tuberculosis and associated In this diagnostic accuracy study, we assessed urine
outcomes in people with HIV.3 samples from the FIND Specimen Bank and the University For more on the
A novel urine-based assay, Fujifilm SILVAMP TB LAM of Cape Town Biobank obtained from inpatients FIND Specimen Bank see
https://www.finddx.org/
(FujiLAM; Fujifilm, Tokyo, Japan), has been developed (aged ≥18 years) with HIV, collected in three independent specimen-bank/
that also detects lipoarabinomannan on an instrument- prospective cohort studies (two unpublished and one
free platform, with results available in less than 1 h. published2,11) done at two district hospitals in South Africa
This assay combines a pair of high affinity monoclonal (appendix). These cohorts were selected for inclusion on
antibodies directed towards largely Mycobacterium the basis of the availability of frozen urine samples for a
tuberculosis-specific lipoarabinomannan epitopes7–9 and a full cohort of hospital inpatients with HIV in tuberculosis
silver-amplification step10 that increases the visibility of endemic settings, in whom a comprehensive work-up was
test and control lines. This enables the detection of done to identify tuberculosis or alternative diagnoses.
urinary lipoarabinomannan concentrations that are Standard national guidelines12 for tuberculosis and HIV
approximately 30 times lower than that detected by management were used across all three cohorts. Cohort 1
AlereLAM and improved analytical specificity compared included adults with tuberculosis symptoms who were
with AlereLAM, which in contrast, uses conventional able to produce sputum, and were enrolled regardless
lateral flow immunoassay technology and polyclonal of CD4 count on admission to Khayelitsha Hospital
antibodies.7 (Cape Town, South Africa) between Feb 22, 2017, and
In this study, we aimed to assess the diagnostic August 31, 2017. Patients with extrapulmonary disease
accuracy of FujiLAM for the detection of active without respiratory symptoms were excluded. Cohort 2
tuberculosis compared with AlereLAM in three cohorts included adults with HIV who were admitted to medical

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wards at GF Jooste Hospital (Cape Town, South Africa) scale card and any line identified on the test line was
between June 6, 2012, and Oct 4, 2013, regardless of CD4 deemed positive.
count, their ability to produce sputum, or whether or not Both AlereLAM and FujiLAM were independently read
they reported tuberculosis symptoms.2,11 Study staff by two readers masked to the test results of the index or
systematically attempted to collect urine, blood, and two comparator test, respectively, patient status, and all other
sputum samples for testing within 24 h of hospital test results. After interpretation of the initial independent
admission. Cohort 3 included adults with HIV with a CD4 test, readers compared results and, in case of discordance,
count equal to or less than 350 cells per µL in whom re-inspected the test strip to establish the final consensus
tuberculosis was considered the most likely diagnosis at result (through mutual agreement) that was used for
presentation, who were admitted to Khayelitsha Hospital analysis. In the case of FujiLAM or AlereLAM assay
failure, the test was repeated once.
between Jan 16, 2014, and Oct 19, 2016. All cohorts excluded
patients who were already receiving tuberculosis therapy For reference standard testing, the specimens were
(appendix). In cohorts 1 and 2, enrolment was done processed using standardised protocols at centralised
accredited laboratories of the South African National
consecutively. In cohort 3, patients were randomly enrolled
Health Laboratory Service (Cape Town, South Africa). The
using a dice on a daily basis after all potentially eligible
patients were identified. In all cohorts, patients were number of samples tested and testing flow for each cohort
enrolled on admission to hospital. Sputum, blood, and are shown in the appendix.
urine specimens for M tuberculosis reference standard Sputum collection in all cohorts was done by an
experienced nurse or trained clinical research worker
testing were collected at enrolment and additional clinical
samples were obtained during hospital admission and at and sputum induction was done, when required, as
follow-up. Follow-up was 8 weeks for cohort 1, and described previously.11 Reference standard testing was
See Online for appendix 12 weeks for cohorts 2 and 3 (appendix). done on all available sputum specimens and included
All studies were approved by the Human Research Xpert MTB/RIF (Xpert; Cepheid, Sunnyvale, USA;
Ethics Committee of the University of Cape Town testing pre-dated rollout of Xpert Ultra MTB/RIF), smear
(Cape Town, South Africa). Written informed consent was fluorescence microscopy after auramine O staining,
obtained from patients, as per study protocols. Study mycobacteria growth indicator tube liquid culture
participation did not affect standard of care. This study is
(Becton Dickinson, Franklin Lakes, NJ, USA) and solid
reported in accordance with the Standards for Reporting ofculture on Löwenstein-Jensen medium. The presence of
Diagnostic Accuracy Studies guidelines.13 Retrospective M tuberculosis complex in solid and liquid culture was
urine lipoarabinomannan testing was supervised by the confirmed with MPT64 antigen detection or MTBDRplus
study sponsor (FIND, Geneva, Switzerland) and was done line probe assays (Hain Lifesciences, Nehren, Germany).
at the University of Cape Town. Blood cultures from all participants were done in
BACTEC Myco/F Lytic culture vials (Becton Dickinson)
Procedures and WHO prequalified in-vitro diagnostic tests were
Frozen urine aliquots of unprocessed urine were thawed used for HIV testing (rapid diagnostic tests) and CD4 cell
to ambient temperature and mixed manually. Samples counting (flow cytometry). For urinary Xpert testing,
that were not immediately used for testing were stored at 20–40 mL urine was centrifuged and following removal
4°C for a maximum of 4 h. of supernatant the pellet was re-suspended in the
AlereLAM testing was done according to the manu-​ residual urine volume and a 0·75 mL sample was used
facturer’s instructions.Briefly, 60 µL urine was applied for testing.2 For cohorts 2 and 3, additional respiratory
to the sample pad. After 25 min, test strips were read and non-respiratory samples such as pleural fluid,
using the test’s reference scale card for grading. In cerebrospinal fluid, and tissue fine needle aspirates were
parallel, testing with the FujiLAM was done according obtained, when clinically indicated, and tested
to manufacturer’s instructions using urine from the using MGIT culture or Xpert. Clinical information and
same aliquots. The five-step test procedure (figure 1; FujiLAM and AlereLAM results were not available to the
See Online for video video) took 50–60 min from sample collection to result. assessors of the reference standard at the time of testing.
Briefly, urine was added to the reagent tube up to the Before data analysis, clinical investigators, who were
indicator line (approximately 200 µL), mixed, and masked to index test results, categorised patients as
incubated for 40 min at ambient temperature. After having definite tuberculosis, possible tuberculosis, not
mixing again, two drops of urine were added to the test tuberculosis, and unclassifiable using a combination of
strip. Following this, button two was immediately clinical and laboratory findings (appendix). Patients with
pressed to release a reducing agent for silver ampli-​ definite tuberculosis had microbiologically confirmed
fication. After the Go Next colour indicator mark turned M tuberculosis (any culture or any Xpert positive result for
orange (within 3–10 min), button 3 was pressed to M tuberculosis during admission). Patients defined as not-
release a silver-ion solution to activate the silver tuberculosis had negative microscopy, cultures, and Xpert
amplification reaction. The result was read within test results for M tuberculosis (and at least one non-
10 min. The FujiLAM assay does not use a reference contaminated negative culture result), had not started

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tuberculosis treatment, and were alive or had improvement Tuberculosis test device
in clinical tuberculosis symptoms at 3 months’ follow-up.
Sample port
Patients defined as possible tuberculosis did not satisfy 2 drops Push completely
Button to Go next Button to
the criteria for definite tuberculosis, but had clinical or release silver release
radiological features suggestive of tuberculosis and were ion reagent for 3 1 2 reducing
amplification reagent for
started on tuberculosis treatment. Patients that did not fall amplification
into any of these categories were defined as unclassifiable
and were removed from the main analyses (appendix).
Go-next colour indicator and control and test line reading window
Statistical analysis
Tuberculosis test procedure
For the primary analysis, we calculated the point estimates
60 min from sample collection to result
and 95% CIs for the sensitivity, specificity, positive
predictive value, negative predictive value, positive Tuberculosis
likelihood ratio, and negative likelihood ratios of FujiLAM negative
and AlereLAM assays by comparison with a microbiological
reference standard and a composite reference standard. Line Tuberculosis
Definite tuberculosis versus not-tuberculosis diagnostic positive
classifications were used to allocate patients into reference 1. Add urine to 2. Incubate for 3. Add two drops 4. On orange
standard positive versus reference standard negative the tube 40 min at position 1 press 3
and press 2 5. Interpret result
groups. The possible tuberculosis group was deemed
negative within a microbiological reference standard but Tuberculosis test principle
positive within a composite reference standard. Diagnostic
accuracy was determined separately for each cohort as per Gold particle Silver particle
protocol. In a sensitivity analysis, unclassifiable patients 0·05 µm 10 µm
were included to assess the effect of exclusions on Au Au Au
1st
diagnostic accuracy (appendix). Heterogeneity between anti- 2nd
cohorts was assessed using Cochran’s Q test (appendix).15 body anti-
MTX-LAM body
We did a post-hoc analysis to estimate pooled sensitivity Test-line
Test line Test line
antigen
and specificity across cohorts and CD4 strata, using a
Au-conjugated Formation of the Silver formation
Bayesian bivariate random-effects model to account
primary antibody sandwich immune- around the Au
for differences in study design.16 Simple pooling captures MTX-LAM complex through particle amplifies
estimates, as planned a priori, are presented in the in patient urine binding to the band intensity
immobilised
appendix with 95% CIs based on Wilson’s score method.17 secondary antibody
The 95% CIs of the sensitivity and specificity differences
of the three cohorts for FujiLAM compared with Figure 1: Fujifilm SILVAMP TB LAM test device, procedure, and principle
One antibody binds to tetra-arabinoside and hexa-arabinoside structures in the arabinan domain of
AlereLAM was computed using Tango’s score method.18
lipoarabinomannan and the other antibody targets MTX-Man capping motifs of lipoarabinomannan
The difference between two tests was considered to be (MTX-Man refers to mannose caps further modified with a 5-methylthio-D-xylofuranose residue).7–9
significant if the 95% CIs did not overlap. Cohen’s κ Au=gold. C=control line. MTX-LAM=5-methylthio-D-xylofuranose-lipoarabinomannan. T=test line.
statistic19 was used to calculate agreement of positive and
negative results between the two independent readers of Role of the funding source
the lipoarabinomannan tests. The funders of the study had no role in study design,
In an additional post-hoc analysis, we used the total data collection, data analysis, data interpretation, or
number of microbiologically confirmed tuberculosis writing of the manuscript. The corresponding author
patients, (defined as the detection of M tuberculosis by had full access to all the data in the study and had final
culture or Xpert in at least one clinical specimen of any responsibility for the decision to submit for publication.
type) to calculate the comparative diagnostic yield of
a single FujiLAM, AlereLAM, sputum Xpert (version G4), Results
urine Xpert, and sputum smear microscopy test from We evaluated urine samples between April 18, 2018, and
samples collected within the first 24 h of presentation May 3, 2018. Of the 1840 patients included in the three
(appendix). This analysis only included patient samples independent cohort studies, 1188 patients were eligible
from cohort 2, because the systematic collection of blood, for retrospective urinary lipoarabinomannan testing. Of
urine, and two sputum diagnostic samples was attempted the 1188 eligible patients, 220 patients were excluded
whenever possible in all patients in this cohort within the from the main analysis because of unavailability of a
first 24 h of admission. urine sample (n=93), failed FujiLAM tests (n=6), or
The study protocol and statistical analysis plan are unclassifiable diagnostic status (n=121; figure 2). The
available in the appendix. All data analysis was done primary reasons for which patients were deemed
using R (version 3.5.1) and Matlab (version 2017b). unclassifiable were death before diagnosis (n=62) and

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loss to follow-up where a vital status or an improvement

1840 potentially eligible patients from three in clinical status was required for diagnosis (n=17;
independent cohort studies screened
140 patients included in cohort 1 appendix). Of the 968 patients included in the main
1018 patients included in cohort 2 analysis (96 patients from cohort 1, 364 patients from
682 patients included in cohort 3
cohort 2, and 508 patients from cohort 3), 600 (62%) were
classified as definite tuberculosis, 91 (9%) as possible
652 patients not eligible tuberculosis, and 277 (29%) as not-tuberculosis (table;
31 patients from cohort 1 excluded appendix). The microbiological reference standard for
28 HIV negative
3 HIV status unknown tuberculosis diagnosis was based on a total of 6397 culture
598 patients from cohort 2 excluded and Xpert tests (mean 6·2 tests per patient) and included
404 HIV negative
5 HIV status unknown
3261 tests on sputum samples and 3136 tests on non-
165 pre-existing tuberculosis sputum samples (appendix). 236 (24%) of 968 patients
15 refused could not provide a sputum sample. Definite tuberculosis
5 transferred to another hospital
3 readmissions diagnosis was based on the results from non-sputum
1 died samples for 117 (20%) of 600 patients. Most patients were
23 patients from cohort 3 excluded
1 HIV negative
young immunocompromised adults (median age
18 CD4 count >350 cells per µL 35 years [IQR 30–42]), with a median CD4 count of
2 CD4 count unknown 113 cells per µL (IQR 40–262) in cohort 1, 153 cells per µL
2 withdrew consent
(53–313) in cohort 2, and 59 cells per µL (23–122) in
cohort 3. 439 (45%) of 968 patients had a history of
1118 patients with HIV eligible for retrospective previous tuberculosis treatment and all patients in
urinary lipoarabinomannan testing
109 patients from cohort 1 cohort 1 and 3 and 329 (90%) of 364 patients in cohort 2
420 patients from cohort 2 had a positive WHO symptom screen for tuberculosis.
659 patients from cohort 3 Overall, compared with the microbiological reference
standard, the sensitivity of FujiLAM was 70·4% (95% CI
220 patients excluded 53·0–83·1) and 42·3% (31·7–51·8) for AlereLAM
13 patients from cohort 1 (difference 28·1%; figure 3). In comparison to the
10 unclassifiable
3 no urine sample
microbiological reference standard, the highest FujiLAM
56 patients from cohort 2 sensitivity was observed in cohort 3 (81·0% [76·9–84·5]),
46 unclassifiable which enrolled patients with more advanced HIV-related
9 no urine sample
1 missing index test immunosuppression (ie, more patients with a CD4 count
151 patients from cohort 3 less than 100 cells per µL) than cohort 2 (65·9%
65 unclassifiable
81 no urine sample
[57·7–73·3]) and cohort 1 (59·6% [45·3–72·4]). The
5 missing index test sensitivity of both assays was higher in patients with lower
CD4 counts. In patients with a CD4 count less than
100 cells per µL, FujiLAM had a sensitivity of 84·2%
968 patients eligible and tested
96 patients from cohort 1 (71·4–91·4) compared with 57·3% (42·2–69·6) for
47 definite tuberculosis AlereLAM (difference 26·9%). For patients with a CD4
3 possible tuberculosis
46 not tuberculosis
count of more than 200 cells per µL sensitivity was 44·0%
364 patients from cohort 2 (29·7–58·5) for FujiLAM and 12·2% (4·6–23·7) for
138 definite tuberculosis AlereLAM (difference 31·8%).
37 possible tuberculosis
189 not tuberculosis Using the composite reference standard, the sensitivity
508 patients from cohort 3 of both assays was slightly lower than that compared with
415 definite tuberculosis
51 possible tuberculosis
the microbiological reference standard (64·9% [50·1–76·7]
42 not tuberculosis for FujiLAM vs 38·2% [28·1–47·3] for AlereLAM;
difference 26·7%). Since the 95% CIs of the differences
around sensitivity between FujiLAM and AlereLAM did
not overlap, FujiLAM was considered to have significantly
600 definite tuberculosis 91 possible tuberculosis 277 not tuberculosis
higher sensitivity than AlereLAM for all analyses, with the
exception of cohort 1, in which the 95% CIs overlapped
691 CRS positive 277 CRS negative due to the small sample size (figure 3).
Compared with the microbiological reference standard,
the specificity of FujiLAM was 90·8% (95% CI 86·0 to 94·4)
600 MRS positive 368 MRS negative
and 95·0% (87·7 to 98·8) for AlereLAM, with no significant
difference (–4·2%). Using the composite reference
Figure 2: Study flow diagram
Details of patients are provided in the appendix. CRS=composite reference standard. MRS=microbiological standard, overall estimates of specificity were increased to
reference standard. 95·7% (92·0 to 98·0) for FujiLAM and 98·2% (95·7 to 99·6)

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for AlereLAM, with no significant difference (–2·5%).

Cohort 1 Cohort 2 Cohort 3 All patients
Using the composite reference standard, specificity of the (n=96) (n=364) (n=508) (n=968)
FujiLAM assay was lower among patients with CD4 counts
Age, years 35 (31–43) 36 (29–42) 35 (30–43) 35 (30–42)
equal to or less than 100 cells per µL (91·2%, 95% CI 83·1
Sex
to 96·3) than those with CD4 counts of 101–200 cells
Women 51 (53%) 218 (60%) 254 (50%) 523 (54%)
per µL (97·8% [95% CI 90·6 to 99·9]) and higher than
Men 45 (47%) 146 (40%) 254 (50%) 445 (46%)
200 cells per µL (98·2% [93·8 to 99·9]; figure 3). Eight of
Positive WHO tuberculosis 96 (100%) 329 (90%) 508 (100%) 933 (96%)
the 11 FujiLAM false positive samples, using the composite symptom screen
reference standard, were from patients with CD4 counts
History of tuberculosis 52 (54%) 162 (45%) 225 (44%) 439 (45%)
equal to or less than 100 cells per µL. Additional
Antiretroviral therapy 64 (67%) 153 (42%) 177 (35%) 394 (41%)
information on the 11 FujiLAM false positive results is
CD4 count, cells per µL 113 (40–262) 153 (53–313) 59 (23–122) 86 (33–190)
available in the appendix.
Diagnosis
Using the composite reference standard, the positive
Definite tuberculosis 47 (49%) 138 (38%) 415 (82%) 600 (62%)
predictive value for the three different cohorts ranged
Possible tuberculosis 3 (3%) 37 (10%) 51 (10%) 91 (9%)
from 90·6–99·4% for FujiLAM and 93·8–100·0% for
Not tuberculosis 46 (48%) 189 (52%) 42 (8%) 277 (29%)
AlereLAM. The negative predictive value ranged from
CD4 count, cells per µL
24·8–71·8% for FujiLAM and 13·7–62·5% for AlereLAM.
0–100 44 (46%) 135 (37%) 337 (66%) 516 (53%)
Positive likelihood ratios ranged from 8·9–18·5 for
101–200 19 (20%) 82 (23%) 115 (23%) 216 (22%)
FujiLAM and 13·8–17·3 AlereLAM and negative
>200 30 (31%) 145 (40%) 56 (11%) 231 (24%)
likelihood ratios ranged from 0·3–0·4 for FujiLAM and
0·6–0·7 for AlereLAM (appendix). Unknown 3 (3%) 2 (1%) 0 5 (1%)

All patients in cohort 2 (n=420) were eligible for the Outcome at 3 months
analysis of diagnostic yield (figure 2). Among the Died within 3 months 1 (1%) 19 (5%) 85 (17%) 105 (11%)
420 eligible patients, only 153 (36%) could produce a Alive 58 (60%) 336 (92%) 416 (82%) 810 (84%)
sputum sample within the first 24 h of admission, whereas Lost to follow-up 0 9 (2%) 7 (1%) 16 (2%)
418 (100%) were able to provide a urine sample, as No follow-up 37 (39%) 0 0 37 (4%)
described previously11 (appendix). 141 (34%) of 420 patients Data are median (IQR), or n (%).
had microbiologically confirmed tuberculosis. 84 (60%) of Table: Demographic and clinical characteristics
141 tuberculosis cases could be diagnosed with rapid tests
using samples collected in the first 24 h of admission:
37 (26%) from sputum Xpert and 59 (42%) from urine rate for AlereLAM on the first attempt was 0·4% (four of
Xpert using 1 mL urine. 57 (40%) of 141 tuberculosis 1095 tests) and all four repeat tests provided a result on
diagnoses were not achieved in the first 24 h and were the second attempt.
established by mycobacterial culture on any specimen Inter-reader agreement was high for both FujiLAM and
collected at any point during patient admission, diagnosed AlereLAM tests (appendix): 97·0% (938 of 967 reads;
by Xpert using concentrated samples from 20–40 mL κ coefficient 0·94) for FujiLAM, and 96·7% (934 of
urine or diagnosed by Xpert testing of specimens collected 966 reads; κ coefficient 0·92) for AlereLAM.
after the first 24 h. The additional specimens collected for
culture and Xpert testing included ascitic fluid, blood, Discussion
urine, sputum, cerebrospinal fluid, gastric lavage, pus, or In this study of 968 hospital inpatients with HIV in a
pleural fluid (appendix). high-burden setting, the FujiLAM point-of-care assay
Figure 4 shows the diagnostic yield of FujiLAM and identified a significantly higher proportion of patients
AlereLAM compared with other rapid diagnostic tests with tuberculosis than did the AlereLAM assay, while
done within the first 24 h of hospital admission. 91 (65%) maintaining comparable specificity. In all sub-analyses,
of 141 tuberculosis cases could have been diagnosed the sensitivity of FujiLAM was significantly higher (range
within a few hours of presentation with FujiLAM, 22–35%) than AlereLAM. FujiLAM had the highest
compared with 61 (43%) of 141 cases with AlereLAM. sensitivity (84·2%) in patients with the highest risk of
A combination of sputum Xpert and FujiLAM within the mortality (CD4 ≤100 cells per µL), which was 26·9%
first 24 h of admission would have been able to diagnose higher than that with AlereLAM. Combined with sputum
102 (72%) of 141 microbiologically confirmed cases. Xpert, FujiLAM could diagnose nearly three-quarters of
A combination of sputum smear microscopy and microbiologically confirmed tuberculosis within 24 h of
FujiLAM would have yielded 98 (70%) of 141 diagnoses. hospital admission. The meta-analysis4,5 that formed the
Overall, 18 (2%) of 1095 FujiLAM tests failed on the basis of WHO recommendations for the use of AlereLAM
first attempt. Of the 15 tests that could be repeated, reported an overall sensitivity of 45% in patients
three failed on the second attempt resulting in a total with HIV, which is similar to the AlereLAM sensitivity
overall error rate of 1·9% (21 of 1110 tests). FujiLAM observed in this study (42%), suggesting that our study
failure rates are summarised in the appendix. The error populations were similar to the populations included

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A
Test n TP FP FN TN Sensitivity (95% CI) Specificity (95% CI)

MRS FujiLAM 968 455 33 145 335 70·4% (53·0 to 83·1) 90·8% (86·0 to 94·4)
AlereLAM 968 268 18 332 350 42·3% (31·7 to 51·8) 95·0% (87·7 to 98·8)
Difference 28·1% –4·2%
CRS
FujiLAM 968 477 11 214 266 64·9% (50·1 to 76·7) 95·7% (92·0 to 98·0)
AlereLAM 968 281 5 410 272 38·2% (28·1 to 47·3) 98·2% (95·7 to 99·6)
Difference 26·7% –2·5%

B
MRS
Cohort 1 FujiLAM 96 28 4 19 45 59·6% (45·3 to 72·4) 91·8% (80·8 to 96·8)
AlereLAM 96 15 1 32 48 31·9% (20·4 to 46·2) 98·0% (89·3 to 99·6)
Difference 27·7% (16·9 to 41·8) –6·2% (–17·6 to 3·9)
Cohort 2 FujiLAM 364 91 18 47 208 65·9% (57·7 to 73·3) 92·0% (87·8 to 94·9)
AlereLAM 364 61 7 77 219 44·2% (36·2 to 52·5) 96·9% (93·7 to 98·5)
Difference 21·7% (14·7 to 29·7) –4·9% (–9·3 to –1·0)
Cohort 3 FujiLAM 508 336 11 79 82 81·0% (76·9 to 84·5) 88·2% (80·1 to 93·3)
AlereLAM 508 192 10 223 83 46·3% (41·5 to 51·1) 89·2% (81·3 to 94·1)
Difference 34·7% (30·1 to 39·5) –1·0% (–9·4 to 7·2)
CRS
Cohort 1 FujiLAM 96 29 3 21 43 58·0% (44·2 to 70·6) 93·5% (82·5 to 97·8)
AlereLAM 96 15 1 35 45 30·0% (19·1 to 43·8) 97·8% (88·7 to 99·6)
Difference 28·0% (17·5 to 41·7) –4·3% (–15·8 to 5·9)
Cohort 2 FujiLAM 364 103 6 72 183 58·9% (51·5 to 65·9) 96·8% (93·2 to 98·5)
AlereLAM 364 64 4 111 185 36·6% (29·8 to 43·9) 97·9% (94·7 to 99·2)
Difference 22·3% (15·8 to 29·4) –1·1% (–4·9 to 2·6)
Cohort 3 FujiLAM 508 345 2 121 40 74·0% (69·9 to 77·8) 95·2% (84·2 to 98·7)
AlereLAM 508 202 0 264 42 43·3% (38·9 to 47·9) 100·0% (91·6 to 100)
Difference 30·7% (26·2 to 35·3) –4·8% (–15·8 to 4·0)

C
MRS
0–100 cells per μL FujiLAM 516 332 20 49 115 84·2% (71·4 to 91·4) 85·0% (75·8 to 91·7)
AlereLAM 516 221 8 160 127 57·3% (42·2 to 69·6) 94·1% (88·3 to 97·7)
Difference 26·9% –9·1%
101–200 cells per μL FujiLAM 216 83 9 49 75 60·6% (44·4 to 72·5) 89·6% (78·5 to 98·1)
AlereLAM 216 35 7 97 77 26·4% (15·2 to 38·9) 92·8% (69·2 to 99·9)
Difference 34·2% –3·2%
>200 cells per μL FujiLAM 231 37 4 46 144 44·0% (29·7 to 58·5) 97·0% (92·5 to 99·3)
AlereLAM 231 10 3 73 145 12·2% (4·6 to 23·7) 97·2% (88·4 to 100)
Difference 31·8% –0·2%
CRS
0–100 cells per μL FujiLAM 516 344 8 76 88 80·6% (72·0 to 86·7) 91·2% (83·1 to 96·3)
AlereLAM 516 226 3 194 93 53·1% (40·7 to 63·6) 96·6% (91·0 to 99·3)
Difference 27·5% –5·4%
101–200 cells per μL FujiLAM 216 91 1 66 58 55·7% (39·9 to 67·6) 97·8% (90·6 to 99·9)
AlereLAM 216 41 1 116 58 25·3% (15·3 to 35·6) 97·8% (90·6 to 99·9)
Difference 30·4% 0·0%
>200 cells per μL FujiLAM 231 39 2 71 119 35·5% (22·4 to 50·4) 98·2% (93·8 to 99·9)
AlereLAM 231 12 1 98 120 11·3% (2·3 to 28·7) 98·9% (95·0 to 100)
Difference 24·2% –0·7%

0 50 100 0 50 100
Sensitivity Specificity

Figure 3: Sensitivity and specificity of FujiLAM versus AlereLAM against MRS and CRS
Sensitivity and specificity of FujiLAM and AlereLAM assays for all cohorts combined (A), by cohort (B), and by CD4 count (C). Sensitivity and specificity estimates for (A) and (C) were based on analysis
using a bivariate random-effects model. AlereLAM=Alere Determine TB LAM Ag assay. CRS=composite reference standard. FP=false positive. FN=false negative. FujiLAM=Fujifilm SILVAMP TB LAM
assay. MRS=microbiological reference standard. TP=true positive. TN=true negative.

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in the WHO meta-analysis. Collectively, these results

A Patients with microbiologically confirmed B Patients with microbiologically confirmed
suggest that, if implemented in clinical practice and tuberculosis (n=141) tuberculosis and CD4 ≤100 cells per µL (n=74)
linked with appropriate treatment, the FujiLAM point-of-
care assay might be able to save lives by enabling earlier 20
7

diagnosis of HIV-associated tuberculosis in a large

7
proportion of hospital inpatients.6,20,21 11
4
The point estimates of FujiLAM specificity were lower
1 4
than those for AlereLAM. Although the differences in 3
2 1 2
25
specificity between FujiLAM and AlereLAM were not 30 1
significant, the reduced specificity of both AlereLAM and 10 6
4 5 5 5
7
FujiLAM could be partly explained by the use of an 1
2 2
imperfect reference standard that lacks complete 11 4
sensitivity. The existing reference standard is especially 2
limited in its ability to identify tuberculosis in immuno-​ 26 7
compromised patients with HIV,22 since these patients
Diagnostic yield Diagnostic yield
are more likely to have paucibacillary disease or extra-
Urine FujiLAM 64·5% (91/141) Urine FujiLAM 79·7% (59/74)
pulmonary tuberculosis than immunocompetent patients, Urine AlereLAM 43·3% (61/141) Urine AlereLAM 64·9% (48/74)
making diagnosis more difficult. An imperfect reference Urine Xpert 41·8% (59/141) Urine Xpert 58·1% (43/74)
standard could disproportionally affect a more sensitive Sputum Xpert 26·2% (37/141) Sputum Xpert 24·3% (18/74)
Sputum smear microscopy 19·1% (27/141) Sputum smear microscopy 18·9% (14/74)
test and result in increased false positives (ie, lower Tuberculosis cases missed 18·4% (26/141) Tuberculosis cases missed 9·5% (7/74)
specificity with the more sensitive FujiLAM assay). The
Figure 4: Diagnostic yields for cohort 2
decreasing specificity observed with decreasing CD4 cell
Number of microbiologically confirmed tuberculosis diagnoses in all patients (A), and patients with CD4 counts
count in this study and the improved specificity observed of equal to or less than 100 cells per µL (B), detected by each diagnostic test on samples obtained within 24 h of
with the composite reference standard in comparison to hospital admission. Numbers represent the number of tuberculosis cases diagnosed by a given assay or assays.
the microbiological reference standard further supports Tuberculosis cases missed includes diagnoses made by positive mycobacterial culture on any specimen collected at
any point during patient admission or diagnoses made on the basis of Xpert testing of any specimen collected after
this explanation. Cross-reactivity of the antibodies used in the first 24 h of hospital admission. AlereLAM=Alere Determine TB LAM Ag assay. FujiLAM=Fujifilm SILVAMP TB
FujiLAM to common urinary tract pathogens and LAM assay.
fast-growing non-tuberculous mycobacteria has been
excluded in previous studies.7
Our study has limitations that indicate further research of enrolment. Six of the nine FujiLAM positive patients
is warranted. FujiLAM testing was done in a research who died were not started on tuberculosis treatment;
laboratory setting using biobanked specimens collected assuming the assay had 100% specificity, these patients
from hospital inpatients. Although no technical reason might have been true positives and thus might not have
exist as to why the test would perform differently in fresh died if they were treated for tuberculosis.
versus frozen samples, this needs to be investigated. A The inclusion of patients from three cohorts from
previous study23 suggests that early morning urine similar inpatient settings with different pretest probability
collection could further improve the sensitivity of urinary of tuberculosis (appendix) provided an overview of the
lipoarabinomannan-based tuberculosis testing. This performance of FujiLAM in hospital inpatients with
aspect could have important implications for clinical advanced HIV. However, the heterogeneity of these
practice and should be addressed in future studies. cohorts is also a limitation. As a result, we presented
FujiLAM has the potential to be implemented as a true results per cohort and by CD4 strata. Grouped analysis
point-of-care assay, but the feasibility of this approach with a Bayesian bivariate random-effects model was used
and its effect on patient outcomes requires prospective to account for heterogeneity across the cohorts.
assessment in relevant clinical settings. Both AlereLAM Renal tuberculosis infection has been proposed as the
and FujiLAM cannot discern drug-resistant tuberculosis main cause of urinary lipoarabinomannan antigenuria
from drug-sensitive tuberculosis and therefore it is and positive AlereLAM results.25 However, in this study,
important that these rapid diagnostic tools are the more sensitive FujiLAM assay detected a number of
supplemented with sample collection for drug urine Xpert-negative patients in whom renal tuberculosis
susceptibility testing. is unlikely since Xpert would detect intact M tuberculosis
Difficulties with regard to the assignment of diagnostic bacteria in urine. This suggests that other mechanisms,
categories have been reported in previous literature24 and such as passage of lipoarabinomannan or lipoara-​
10% of all eligible patients could not be classified in this binomannan fragments through the glomerular basement
study. The higher sensitivity of FujiLAM compared with membrane, potentially exacerbated by HIV-associated
AlereLAM was maintained when the unclassifiable nephropathy, might be more important than originally
group was included in a sensitivity analysis (appendix). proposed. This is supported by findings from our
18 of the 121 unclassifiable patients had positive FujiLAM previous study,26 which showed that blood and urine
results and nine of these 18 patients died within 3 months lipoara-​binomannan concentrations correlate in patients

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with tuberculosis, and that of a 2018 study,7 which found the conceptualisation of this work, Anna Mantsoki for data
that low urine lipoarabinomannan concentrations are management, and the clinical and laboratory teams at the partner sites
for their efforts in the implementation, conduct, and timely completion
detectable in immunocompetent HIV-negative patients of the study. This work was funded by Global Health Innovative
with tuberculosis. The use of the more sensitive FujiLAM Technology Fund (grant number G2015-201), UK Department for
assay could help to increase the mechanistic understanding International Development (grant number 300341-102), the Dutch
of how lipoarabinomannan enters urine. Ministry of Foreign Affairs (grant number PDP15CH14), the Bill &
Melinda Gates Foundation (grant number OPP1105925), the Australian
The diagnostic yield analysis showed that only a minority Department of Foreign Affairs and Trade (grant number 70957),
of patients were able to produce sputum within the first and the German Federal Ministry of Education and Research through
24 h of admission. Although this has been demonstrated Kreditanstalt für Wiederaufbau. The cohort 2 study was funded by
in other studies of hospital inpatients,27,28 the percentage of the Wellcome Trust (088590 and 085251). GM was supported by the
Wellcome Trust (098316 and 203135/Z/16/Z), the South African
patients able to provide sputum was particularly low in Research Chairs Initiative of the Department of Science and Technology
cohort 2,11 despite substantial efforts to obtain the sample and National Research Foundation of South Africa (grant number
by a trained nurse, including access to sputum induction 64787), NRF incentive funding (UID: 85858), and the South African
facilities. The inability to provide a sputum sample is likely Medical Research Council through its Tuberculosis and HIV
Collaborating Centres Programme, with funds received from the
a reflection of the severity of illness in this cohort and will National Department of Health (RFA#SAMRC-RFA-CC:TB/HIV/
be less pronounced in outpatients with HIV and AIDS-01-2014). BS received salary support from the Wellcome Trust
tuberculosis symptoms, who also typically have higher (grant number 088316). CS received funding from the South African
CD4 cell counts.29 Studies of tuberculosis diagnostic Medical Research Council through the National Health Scholarship
Programme. ADK received funding from the National Institute of
assessments often exclude patients who cannot produce Allergy and Infectious Diseases (grant number T32 AI060530).
sputum, which can, particularly in the case of inpatients The opinions, findings and conclusions expressed in this manuscript
who are severely ill, lead to a biased study population and reflect those of the authors alone.
test assessment and exclude the population that would References
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Declaration of interests point-of-care, urine-based lipoarabinomannan testing to guide
TB, EI, AM, SO, AT, CB, and CMD are employed by the Foundation for tuberculosis treatment initiation in HIV-positive hospital inpatients:
Innovative New Diagnostics (FIND). FIND is a not-for-profit foundation a pragmatic, parallel-group, multicountry, open-label, randomised
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agreements with several private sector companies that design diagnostics diagnosis. J Clin Microbiol 2018; 56: e01338–18.
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