Rapid in Vivo Detection of Isoniazid-Sensitive Mycobacterium Tuberculosis by Breath Test
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ARTICLE Received 19 Mar 2014 | Accepted 14 Aug 2014 | Published 23 Sep 2014 DOI: 10.1038/ncomms5989 Rapid in vivo detection of isoniazid-sensitive Mycobacterium tuberculosis by breath test Seong W. Choi1,*, Mamoudou Maiga2,*, Mariama C. Maiga2, Viorel Atudorei3, Zachary D. Sharp3, William R. Bishai2 & Graham S. Timmins1 There is urgent need for rapid, point-of-care diagnostic tools for tuberculosis (TB) and drug sensitivity. Current methods based on in vitro growth take weeks, while DNA amplification can neither differentiate live from dead organisms nor determine phenotypic drug resistance. Here we show the development and evaluation of a rapid breath test for isoniazid (INH)-sensitive TB based on detection of labelled N2 gas formed specifically from labelled INH by mycobacterial KatG enzyme. In vitro data show that the assay is specific, dependent on mycobacterial abundance and discriminates between INH-sensitive and INH-resistant 15 (S315T mutant KatG) TB. In vivo, the assay is rapid with maximal detection of N2 in exhaled 15 breath of infected rabbits within 5–10 min. No increase in N2 is detected in uninfected 15 animals, and the increases in N2 are dependent on infection dose. This test may allow rapid detection of INH-sensitive TB. 1 Department of Pharmaceutical Sciences, College of Pharmacy, University of New Mexico, Albuquerque, New Mexico 87131, USA. 2 Department of Medicine, Center for Tuberculosis Research, Johns Hopkins University, Baltimore, Maryland 21231, USA. 3 Department of Earth and Planetary Sciences, College of Pharmacy, University of New Mexico, Albuquerque, New Mexico 87131, USA. * These author contributed equally to this work. Correspondence and requests for materials should be addressed to G.S.T. (email: [email protected]). NATURE COMMUNICATIONS | 5:4989 | DOI: 10.1038/ncomms5989 | www.nature.com/naturecommunications 1 & 2014 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5989 acterially activated prodrugs are unusually well represented a O 15NH 15NH O O 15 among the first- and second-line TB drugs. These include 15NH NH C Bnot only established drugs such as isoniazid (INH)1, 2 2 3 KatG Beta ethionamide or pyrazinamide but also newly approved and H15N 4 + 15 developing agents such as the nitroimidazoles delamanid and scission NH PA824 (ref. 5). The selectivity of these agents arises from their N N N 15 · · specific activation by mycobacterial enzymes, usually to reactive N2-INH (1)IN-Hydrazyl INAcyl Diazene (2) intermediates, and is underlined by the major mode of resistance to these agents, with mutations in genes of their activating b –OOC H –OOC H 6 7 enzymes such as katG for INH , ethA for ethionamide , pncA + N + N2H2 2 8 9 – for pyrazinamide and ddn for nitroimidazoles . Since gene H COO– H COO inactivation may occur through a multiplicity of single-nucleotide polymorphisms (SNPs) or insertion/deletion (indel) events, c 2 N2H2 N2H4 + N2 nucleic acid amplification and SNP-indel detection approaches provide only partially predictive drug susceptibility data. Beyond Figure 1 | Production of N2 from KatG activation of isoniazid. 10–14 15 single-gene mutational resistance, multiple other alleles and (a) Production of labelled diazene from N2-hydrazyl-labelled INH; 15,16 other drugs may influence enzymatic activity of prodrug (b) oxidation of diazene to N2 by reaction with unsaturated carbon conversion, factors that may also limit nucleic acid-based bonds such as fumarate shown, rate constant 8 Â 102 M À 1 s À 1 (ref. 20); techniques for drug susceptibility testing. Despite the (c) disproportionation of diazene to N2 and hydrazine rate constant importance of prodrug activation, studies have been limited to 2.2 Â 104 M À 1 s À 1 (ref. 21). in vitro samples or bacterial culture, and at present there are no POC techniques to directly measure prodrug conversion and 15 of d N2 of 250 would indicate a 25% increase in the absolute enzymatic activity. 15 amount of N2 in a sample. This same principle is exploited by The mycobacterial enzyme KatG, which is responsible for INH other isotope ratio breath diagnostics including the urease breath activation, produces a range of INH-derived radicals that react test for Helicobacter pylori infection. with cellular components, especially the isonicotinoyl acyl radical 15 þ þ In this report, we describe the detection of N2 products of that adds covalently to NAD and NADP . The adducts INH activation that are specific for mycobacterial KatG, and test formed by these radicals are potent inhibitors of the key their specificity against other important lung bacterial pathogens mycobacterial targets. The first target of such inhibition to be that possess related peroxidase enzymes. By measuring the elucidated was 2-trans-enoyl-acyl carrier protein reductase 15 15 þ increase over baseline d N2 upon addition of the N2-hydrazyl (InhA) that binds INacyl-NAD adducts, tightly inhibiting - 17 INH (a method termed INH N here), we hypothesized that mycolic acid synthesis . Although other targets or reactive 15 IRMS detection of this N2 may allow sensitive measurement of species may play roles, the importance of these alternative INH activation by KatG. mechanisms compared with the widely accepted inhibition of InhA remains unclear18. The detection of degradation products of the INacyl-NAD þ Results adduct, such as 4-isonicotinoylnicotinamide in urine or other In vitro cultures of M. tuberculosis H37Rv or M. bovis Bacillus 15 fluids held great promise as a measure of INH prodrug Calmette–Gue´rin (BCG) were treated with N2-hydrazyl INH in conversion in TB, and hence determining KatG activity19. sealed tubes and portions of headspace gas collected, filtered and À 115 However, this appears to lack specificity for Mycobacterium analysed. Treatment with 1 mg ml N2-hydrazyl INH resulted 15 tuberculosis as 4-isonicotinoylnicotinamide was found in urine of in marked increases in d N2 that were dependent upon bacterial uninfected mice treated with INH, and in urine of TB patients density (CFU ml À 1; Fig. 2a). Next, we determined the correlation 19 15 15 even when they were culture-negative after treatment . between the accumulated d N2 and the dose of N2-hydrazyl Mycobacterial KatG activates INH by oxidation to a hydrazyl INH administered (Fig. 2b), and these experiments showed 15 radical that undergoes beta scission to form isonicotinoyl acyl sensitive IRMS detection of headspace d N2 following 15 À 1 radical. The other product of this beta-scission reaction, diazene, N2-hydrazyl INH doses of 0.1 mg ml , a concentration we has received little to no attention in the literature. To study diazene subsequently used throughout. The generation of headspace 15 production in KatG-expressing mycobacteria, we used doubly d N2 occurred rapidly (Fig. 2c), and plateau levels were reached 15 B N2-hydrazyl-labelled INH (1) to produce doubly labelled diazene in 1 h. Similar data were also observed with M. bovis BCG (Fig. 1a). Under physiologic conditions, this diazene rapidly (Fig. 3), another KatG-expressing mycobacterial species, although 20 15 undergoes either oxidation by unsaturated bonds (Fig. 1b) or generally lower levels of N2 production were observed 15 bimolecular disproportionation (Fig. 1c) to produce N2 (ref. 21). compared with M. tuberculosis H37Rv. These data confirmed Diazene is widely used synthetically in the stereospecific reduction our ability to measure mycobacterial KatG activity quantitatively 22 15 of a wide range of carbon–carbon double bonds . with IRMS monitoring of conversion of N2-hydrazyl INH to 15 15 This N2 produced from INH-derived diazene may be readily N2 using in vitro cultures of mycobacteria. 15 detected by isotope ratio mass spectrometry (IRMS), and its We then evaluated the specificity of our N2-hydrazyl INH 15 15 abundance is reported as d N2 where to N2 detection method for mycobacterial KatG activity. hÀÁ As may be seen in Fig. 4a, the common respiratory pathogens 15 15 15 14 14 d N2 ¼1;000Â N N= N N Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia samplei 15 15 ÀÁÀÁcoli did not produce N2 when treated with N2-hydrazyl INH. 15 15 =14 14 = 15 15 =14 14 15 15 À N N N N standard N N N N standard To determine whether our N2-hydrazyl INH to N2 detection method for INH prodrug conversion was specific for the 15 14 15 Atmospheric N is much lower in abundance than N mycobacterial KatG, we tested the production of N2 using an B 15 B ( 0.36%), hence N2 is very low in abundance ( 13 p.p.m.); M. tuberculosis strain harbouring a mutated KatG. This strain 15 therefore, even small amounts of N2 generation may be detected possessed the M. tuberculosis katG-S315T mutation that is known 15 through changes in d N2. For example, an increase in the value to profoundly decrease INH activation and result in drug 2 NATURE COMMUNICATIONS | 5:4989 | DOI: 10.1038/ncomms5989 | www.nature.com/naturecommunications & 2014 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5989 ARTICLE a * a 200 * * 3,000 * 150 * 2 2 N 2,000 N * 15 15 δ * δ 100 1,000 * gas gas 50 0 Increase in headspace Increase in headspace 0 0 105 106 107 108 0 105 106 107 108 CFU ml–1 CFU ml–1 b b 250 200 * * 200 2 2 N 150 N 100 15 15 δ δ 100 gas gas 0 50 * Increase in headspace Increase in headspace 0 0 1E–3 0.01 0.1 011E–3 0.01 0.1 15 –1 N2-hydrazyl-INH (mg ml ) 15 –1 N2-hydrazyl-INH (mg ml ) c * c 150 * 200 * 2 N 2 100 15 N δ 100 15 δ gas 50 gas 0 Increase in headspace Increase in headspace 0 01234 Incubation time (h) 0.00 0.25 0.50 0.75 1.00 15 Incubation time (h) Figure 2 | CFU, dose and time dependence of N2 production by 15 15 M.