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Infdis/jiv312 PDF
Infdis/jiv312 PDF
molecules
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Shih-Jung Pan1,5, Asa Tapley1,2,3, John Adamson1, Tessa Little4, Michael
Urbanowski5, Keira Cohen1,6, Alexander Pym1, Deepak Almeida5, Afton Dorasamy1,
Emilie Layre7, David C. Young7, Ravesh Singh8, Vinod B. Patel9, Kristina
Wallengren10, Thumbi Ndung'u1,8,11,12, Douglas Wilson13, D. Branch Moody7,
William Bishai5,*
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KwaZulu-Natal Research Institute for Tuberculosis and HIV (K-RITH), Nelson R. Mandela School of
Medicine, University of KwaZulu-Natal, Durban, South Africa
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Center for Tuberculosis Research, Johns Hopkins University School of Medicine, Baltimore, Maryland,
USA
Division of Pulmonary and Critical Care Medicine, Department of Medicine, Brigham and Women's
Hospital, Boston, Massachusetts, USA
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Division of Rheumatology, Immunology and Allergy, Brigham and Womens Hospital, Harvard Medical
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School, Boston, Massachusetts, USA
8
HIV Pathogenesis Programme, Doris Duke Medical Research Institute, Nelson R. Mandela School of
Medicine, University of KwaZulu-Natal, Durban, South Africa
9
Department of Neurology, University of KwaZulu Natal, Mayville, Durban, South Africa
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Tuberculosis & HIV Investigative Network in KwaZulu-Natal (THINK), Durban, South Africa
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Max Planck Institute for Infection Biology, Chariteplatz, D-10117 Berlin, Germany
12
Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard
University, Cambridge, MA 02139, United States of America
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Department of Medicine, Edendale Hospital, Pietermaritzburg, University of KwaZulu-Natal, South
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Africa
*
Corresponding author: Johns Hopkins School of Medicine, CRB2 Room 108, 1550 Orleans Street,
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The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of
America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.
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ABSTRACT
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The lack of rapid, reliable diagnostic tests that correlate with bacterial load remains a
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tuberculosis. To develop tests based on products secreted by tubercle bacilli that are
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MS/MS), we demonstrated the presence of one or both mycobactins and/or
tuberculosinyladenosine in serum and whole lung tissues from infected mice and sputum,
cerebrospinal fluid, or lymph nodes from infected patients but not uninfected controls.
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Detection of the target molecules distinguished host infection status in 100% of mice
with both serum and lung as the target sample. In human subjects, we evaluated
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detection of the bacterial small molecules in multiple body compartments in three patient
three molecules in 90%, 71%, and 40% of TB patients sputum, cerebrospinal fluid, and
are difficult to diagnose even with culture, detection of one or more bacterial small
molecule was rapid and compared favorably to PCR-based detection. Secreted bacterial
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small molecules, which are detectable in serum, warrant further investigation as a means
MAIN TEXT
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resulting in 1.3 million deaths annually and infecting approximately one third of the
underscores the inadequacies of current public health control efforts. Major factors in the
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poor state of global tuberculosis control are the lack of rapid and specific point-of-care
experts regard the lack of validated markers as a major obstacle to conducting the clinical
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Consequently, there is an urgent need for effective new TB biomarkers both for
based on host immune responses with interferon- release assays, the majority of current
diagnostics for active TB are sputum-based. This limits their utility both in children, who
do not reliably produce sputum [2], and as treatment response markers because coughing
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and sputum production resolve many months before cure in most active TB patients.
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Moreover, since HIV co-infection and other forms of immunocompromise are common
risk factors for TB, diagnostics targeting host antibody or T cell responses may have
limitations in this significant sector of at-risk individuals [3]. For these reasons, we
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focused our studies on secreted, bacterial-derived small molecules which may enter
include mechanisms for harvesting host iron stores and for preventing phagosome
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maturation. M. tuberculosis acquires iron through the secretion and subsequent re-
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[4]. Species-specific differences in mycobactins T, S, and J, from M. tuberculosis, M.
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virulence [6]. Additionally, M. tuberculosis produces and sheds the terpene nucleoside 1-
are only found within the M. tuberculosis complex. However, the locus contains loss-of-
function mutations in M. bovis and M. bovis BCG, and it appears that M. tuberculosis
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may be the only complex member that produces 1-TbAd [8, 9]. These molecules--MBT,
cMBT, and TbAd--have shared properties that are attractive as diagnostic targets,
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including their abundant biosynthesis, export to the surface for shedding, and their
vivo. Also, their similar polarity and mass profiles facilitated the development of a single
detection.
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MBT and cMBT were purified from sterilized lipid extracts of M. tuberculosis H37Rv
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detection of the molecules using a collision-induced mass spectrometry (CID-MS)
protocol. For both MBT and cMBT, the method revealed three major molecular variants
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(Fig. 1a,b). 1-TbAd and an isomer recently identified as N6-TbAd were separately
purified (see Supplemental Methods), and their CID-MS profiles characterized (Fig. 1c)
fragment ion at m/z 148 (Fig. 1c). The structure of 1-TbAd used in this study was
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confirmed by NMR (Fig. 1d). Beyond these studies of purified, naturally derived
molecule we required detection of at least one parent ion and associated transition pair
with a signal-to-noise (S/N) ratio 3:1. For the purposes of analysis, we combined the
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two TbAd isomers into one category and did not differentiate between individual
infected BALB/c mice with a high-dose of the virulent M. tuberculosis H37Rv. Mice
implantation and at day 21 for evaluation of the final bacterial burden and presence of the
three targeted bacterial small molecules (BSM). The implantation and final bacterial
burdens were found to be typical for a high-dose aerosol infection (Supplemental Fig.
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1a), and we observed discrete pulmonary lesions in the lungs of mice 21 days post-
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LC-MS/MS evaluation revealed MBT in lung tissue and serum of 100% (7/7) M.
tuberculosis-infected mice (Table 1a and Fig. 1a-c). TbAd was detected in lung tissue
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of 100% (7/7) of infected mice, but in sera of only 43% (3/7). By contrast, we detected
cMBT in the serum of only one infected mouse and in no lung samples. The sensitivity
serum samples from 100% (6/6) of the uninfected mice were negative for all three
To test the utility of these BSM as markers of TB disease in humans, we analyzed sputum
patients were positive for TB by acid-fast bacilli (AFB) sputum smear, culture, and
GeneXpert testing. As shown in Table 1b, we detected MBT in 90% (9/10) of sputum
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samples from infected individuals and TbAd in 60% (6/10), and in none of ten uninfected
control sputa (90% sensitivity, 100% specificity for any BSM being positive). The one
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sputum sample negative for MBT, had a low bacterial burden as indicated by GeneXpert
and AFB smear. The S/N ratio of cMBT was below the detection threshold in this and
subsequent analyses.
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To determine whether MBT and TbAd levels may correlate with bacterial loads, we
compared signal intensities of these small molecules with other quantitative markers of
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M. tuberculosis abundance in the above sputum study. We correlated the ion counts per
second of the molecular ions for MBT and TbAd in each positive sputum sample with the
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AFB smear microscopy scores, mycobacterial growth indicator tube (MGIT) time-to-
positivity, and GeneXpert grade for each respective sputum sample. As may be seen in
approached significance in its correlation with GeneXpert grade. TbAd levels showed
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significant correlations with AFB smear microscopy and GeneXpert grades and
tuberculous meningitis, comparing them to control samples from patients with culture-
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confirmed cryptococcal meningitis. We were able to detect MBT in CSF from 71%
(10/14) of tuberculous meningitis cases and TbAd in 7% (1/14) of cases. There was no
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neoformans culture-positive CSF patient samples tested (Table 1d) (71% sensitivity,
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100% specificity for any BSM being positive). In 11 subjects with culture-confirmed
tuberculous meningitis who also had AFB smear performed on their CSF, 9/11 were
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positive for any BSM and 7/11 were AFB smear-positive (sensitivity of any BSM being
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Lastly we tested the BSMs in another paucibacillary form of TB, tuberculous
lymphadenitis. We obtained cervical lymph nodes from ten South African patients with
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lymphadenitis who underwent diagnostic excisions. In addition to histopathology and
mycobacterial culture, all samples were evaluated by AFB smear, GeneXpert, and LC-
while the other five had histology consistent with malignancy or reactive lymphadenitis
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with no evidence of caseation. We detected MBT in 40% (2/5) of the M. tuberculosis-
positive lymph nodes and TbAd in 20% (1/5) as shown in Table 1e. All M. tuberculosis-
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negative samples also tested negative for the target molecules (sensitivity 40%,
specificity 100% for any BSM being positive). AFB smear was positive in 20% (1/5) of
the tuberculous lymph nodes, while GeneXpert was positive in 100% (5/5).
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Interestingly, one of the patients with tuberculous lymphadenitis also provided a blood
sample which was GeneXpert positive and was BSM-positive (MBT positive/TbAd
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negative); however, the blood sample was culture-negative for M. tuberculosis. None of
the other nine patients had evidence of mycobacteremia by any of the three tests.
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Our studies demonstrate that two types of bacterial small molecules released by M.
tuberculosis, MBT and TbAd, may be found in blood, sputum, CSF, lung, and lymph
node samples from mice and human infected with M. tuberculosis. Evidence suggests
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that these two molecules play a role in TB virulence MBT in nutrient acquisition and
survival inside host cells [6] and TbAd in the resistance against macrophage killing [8].
So it is not surprising that they are actively produced during infection. Not only were
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these molecules found in infected mice and in sputum samples from infected human
patients, we were also able to detect them in human CSF and lymph node tissue in which
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the bacterial burden is low. The potential use of these bacterial small molecules in
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target BSMs in multiple forms of TB, we studied relatively small numbers of patients.
Hence the current data cannot predict the population sensitivity and specificity of this test
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in the clinical setting. Moreover, all sputum samples from TB patients in South Africa
were smear-positive whereas the control samples were obtained from the U.S. The fact
that no sputa from smear-negative TB patients were used and that the positive and control
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samples came from different countries might affect test accuracy. Additionally, our
the cost of TB clinical trials, simpler and less expensive methods of detection would be
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required for use in routine clinical settings. Although specific antibodies are not
immunoassays.
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shed less readily into other body compartments. These include proteins [10],
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lipoarabinomannan [11], dimycocerosates [12], and mycolic acids [13].
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blood, has been tested in several clinical trials as a TB biomarker. While in general
LAM antigen detection assays have been inferior to traditional TB diagnostics, some
the test may be valuable in certain sub-populations [14]. The introduction of nucleic
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acid amplification tests for TB has been an important advance during the past two
decades. In particular, the GeneXpert platform, which may deliver results in under two
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hours, has proven useful in TB control programs [15]. This and other nucleic-acid
amplification tests are limited in their ability to detect TB using blood and urine and also
to quantify disease burden over time since the DNA of killed bacteria remains detectable
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for many days despite effective therapy. Hence there remains an ongoing need for TB
biomarkers, similar to HIV viral load testing, which quantify disease progression or
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regression using readily available patient samples such as blood or urine. In this study
novel biomarkers for TB. Such markers would be valuable supplements to the currently
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NOTES
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Kingdom for providing advice and sample mycobactin extracts that aided our initial
method development. We thank Shaaretha Pelly for critical review of the manuscript.
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Health award AI097138 (W.B.), the Howard Hughes Medical Institute (W.B. and T.N.),
N.).
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Meetings or abstracts where this work has been presented. None
Corresponding author contact information. William Bishai, MD, PhD. Johns Hopkins
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School of Medicine, CRB2 Room 108, 1550 Orleans Street, Baltimore, MD 21287.
wbishai@jhmi.edu
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REFERENCES
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2. Swaminathan S, Rekha B. Pediatric tuberculosis: global overview and challenges.
Clin Infect Dis 2010;50 Suppl 3:S184-194.
3. Chen J, et al. Interferon-gamma release assays for the diagnosis of active
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tuberculosis in HIV-infected patients: a systematic review and meta-analysis. PLoS
ONE 2011;6:e26827.
9. Young, DC, et al. Terpene nucleosides are transformed in vivo and serve as specific
chemical markers for M. tuberculosis infection. Chemistry & Biology. In press.
10. Young BL, et al. The identification of tuberculosis biomarkers in human urine
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1991;266:9652-9660.
12. O'Sullivan DM, et al. Detection of Mycobacterium tuberculosis in sputum by gas
chromatography-mass spectrometry of methyl mycocerosates released by
thermochemolysis. PLoS ONE 2012;7:e32836.
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13. Shui G, et al. Mycolic acids as diagnostic markers for tuberculosis case detection in
humans and drug efficacy in mice. EMBO Mol Med 2012;4:27-37.
14. Gounder CR, et al. Diagnostic accuracy of a urine lipoarabinomannan enzyme-linked
immunosorbent assay for screening ambulatory HIV-infected persons for
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tuberculosis. J Acquir Immune Defic Syndr 2011;58:219-223.
15. Boehme CC, et al. Rapid molecular detection of tuberculosis and rifampin resistance.
New Engl J Med 2010;363:1005-1015.
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Table 1a.
Detection among mice, by
TB disease status (%)
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Sample Molecule targeted Infected Uninfected P-value
Serum Mycobactin T 7/7 (100) 0/6 (0) < 0.001
Carboxymycobactin 1/7 (0) 0/6 (0) 1.00
TbAd 3/7 (43) 0/6 (0) 0.192
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Lung Mycobactin T 7/7 (100) 0/6 (0) < 0.001
Carboxymycobactin 0/7 (14) 0/6 (0) 1.00
TbAd 7/7 (100) 0/6 (0) < 0.001
coefficient
Mycobactin T
AFB smear microscopy score 0.8739 0.0024
MGIT TTP time -0.2970 0.4069
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Table 1d.
Detection in patient cerebrospinal fluid samples,
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Table 1e.
Detection of M. tuberculosis in patient lymph node
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samples. Fraction Positive (%)
Sample Detection Method Infected Uninfected P-value
Lymph Node Any BSM 2/5 (40) 0/5 (0) 0.44
AFB smear 1/5 (20) 0/5 (0) 1.00
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GeneXpert 5/5 (100) 0/5 (0) 0.008
Culture 5/5 (100) 0/5 (0) 0.008
Blood Any BSM 1/5 (20) 0/5 (0) 1.00
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FIGURE LEGENDS
Figure 1.
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naturally-derived, TB-specific bacterial small molecules (BSM) illustrating the
characteristic ion intensity peaks with collision-induced LCMS in positive ion mode at
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concentrations of 10 nM (middle), and characteristic ion chromatograms of the product
ions detected by LCMS analysis from infected mouse lungs (bottom) for mycobactin T
isomeric N6-TbAd (c). For mycobactin T, the 15 methylene aliphatic side chain as
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shown gives a parent ion m/z of 881, but 17- (m/z 909) and 18-methylene (m/z 923)
forms are also produced. For carboxymycobactin, the aliphatic link as shown gives a
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parent ion m/z of 743 but the two-methylene aliphatic chain shown may extend to four
(m/z 771) or five methylenes (m/z 785) in length. For mouse infections, LCMS analyses
are shown from one processed lung tissue sample from a BALB/c mouse sacrificed 21
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days after aerosol infection with M. tuberculosis H37Rv. Infected mouse lungs showed
characteristic peaks for mycobactin T (a, bottom panel) and 1-TbAd (c, bottom panel),
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but carboxymycobactin was less abundant with only one ion transition observed above
the detection limit (b, bottom panel). Trace levels of N6-TbAd were observed eluting
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later than 1-TbAd in the infected mouse lung (c, bottom panel). The 1-TbAd used as
standard material in this study was fully characterized by mass spectrometry and nuclear
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