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Sulfamethoxazole Susceptibility of Mycobacterium tuberculosis Isolates

from HIV-Infected Ugandan Adults with Tuberculosis Taking


Trimethoprim-Sulfamethoxazole Prophylaxis
Sam Ogwang,a,b Caryn E. Good,c Brenda Okware,a Mary Nsereko,a Michael R. Jacobs,c W. Henry Boom,d Charles M. Barkd,e
Uganda-Case Western Reserve University Research Collaboration, Kampala, Ugandaa; Joint Clinical Research Centre, Kampala, Ugandab; Department of Pathology, Case
Western Reserve University School of Medicine, Cleveland, Ohio, USAc; Tuberculosis Research Unit, Case Western Reserve University, Cleveland, Ohio, USAd; MetroHealth
Medical Center, Cleveland, Ohio, USAe

Additional drugs are needed for the treatment of multidrug-resistant tuberculosis (TB). Sulfamethoxazole has been shown to
have in vitro activity against Mycobacterium tuberculosis; however, there is concern about resistance given the widespread use of
trimethoprim-sulfamethoxazole prophylaxis among HIV-infected patients in sub-Saharan Africa. Thirty-eight of 40 Mycobacte-
rium tuberculosis isolates (95%) from pretreatment sputum samples from Ugandan adults with pulmonary TB, including HIV-
infected patients taking trimethoprim-sulfamethoxazole prophylaxis, were susceptible with MICs of <38.4 ␮g/ml.

A lternative drugs are urgently needed to treat multidrug-resis-


tant (MDR) tuberculosis (TB). Given the difficulties of new
drug development, repurposing currently licensed antibiotics is
they were resistant to isoniazid, rifampin, pyrazinamide, or
ethambutol.
Susceptibility testing was adapted from the Bactec MGIT 960
practical and efficient. Trimethoprim-sulfamethoxazole (SXT) is SIRE kit instructions (Becton Dickinson, Sparks, MD) and the
a fixed-dose drug combination used worldwide as treatment and CLSI approved standard M24-A2 (16). Briefly, pure M. tuberculo-
prophylaxis for multiple infections. Sulfamethoxazole (SMX) is in sis colonies grown on solid media (Löwenstein-Jensen slants or
the sulfonamide class of antibiotics, which were explored as an Middlebrook 7H10 plates; Becton-Dickinson) were inoculated
anti-TB treatment in the mid-20th century with early studies into 7-ml MGIT tubes supplemented with oleic acid-albumin-
showing potential value for the treatment of pulmonary and mil- dextrose-catalase (OADC) and polymyxin, amphotericin B, nali-
iary TB (1–5). More recently, Forgacs et al. reported defervescence dixic acid, trimethoprim, and azlocillin (PANTA) antibiotics, in-
of a patient with pulmonary TB who was initially treated with SXT cubated in the MGIT 960 until positive, diluted based on time
alone and also demonstrated in vitro susceptibility to SXT in 43 of since positive per SIRE instructions, and then 500 ␮l was used to
44 Mycobacterium tuberculosis isolates (6). These drug susceptibil- inoculate each of 8 MGIT tubes. Drugs were prepared from pow-
der (Sigma-Aldrich, St. Louis, MO) dissolved in dimethyl sulfox-
ity results were independently confirmed in laboratory strains (7,
ide (DMSO). Further dilution in DMSO was carried out to obtain
8) and in patient isolates demonstrating SMX to be the active
the drug concentrations in a standardized 100-␮l volume. The
agent with MICs within achievable serum levels (9, 10). In addi-
no-drug control was 100 ␮l DMSO. Eight MGIT 7-ml tubes were
tion, Alsaad and colleagues reported the use of SXT as part of a
inoculated for each isolate: undiluted no-drug control; rifampin, 1
combination regimen used to treat 10 patients with MDR-TB in ␮g/ml control; and SMX concentrations of 2.4, 4.8, 9.6, 19.2, 38.4,
the Netherlands (11). They also reported M. tuberculosis suscepti- and 76.8 ␮g/ml. An additional growth control tube was prepared
bility to SXT in 17 of 18 patients with TB-HIV coinfection; how- using 500 ␮l of a 1:100 dilution from the inoculum used in the
ever, only 1 was taking SXT prior to TB diagnosis (12). Given the other tubes. Growth was measured in the MGIT instrument in
development of drug resistance when active TB is treated with a drug-containing tubes in relation to the growth control tube to
single drug, there is concern for resistance to SMX among TB- determine drug susceptibility or nonsusceptibility. Quality con-
HIV-coinfected patients taking SXT prophylaxis. To address this trol strains for SMX and rifampin were Staphylococcus aureus
concern, we performed drug susceptibility testing (DST) on M. ATCC 29213 and Escherichia coli ATCC 25922, which were done
tuberculosis isolates obtained from pretreatment sputum speci- on each testing day, according to CLSI methods (17).
mens of HIV-infected patients taking SXT prophylaxis at the time
of diagnosis of active TB.
Sputum isolates used for this study were collected from pa- Received 12 May 2015 Returned for modification 15 June 2015
tients enrolled in the Kawempe Community Health Study Accepted 3 July 2015
(KCHS), a prospective cohort of adults with newly diagnosed pul- Accepted manuscript posted online 13 July 2015
monary TB and their household contacts (13, 14). Kawempe is Citation Ogwang S, Good CE, Okware B, Nsereko M, Jacobs MR, Boom WH, Bark
CM. 2015. Sulfamethoxazole susceptibility of Mycobacterium tuberculosis isolates
located in Kampala, Uganda, which is a high-TB-burden country from HIV-infected Ugandan adults with tuberculosis taking trimethoprim-
with an annual incidence of approximately 166 TB cases per sulfamethoxazole prophylaxis. Antimicrob Agents Chemother 59:5844 –5846.
100,000 population (15). The isolates included in this study were a doi:10.1128/AAC.01101-15.
convenience sample collected between November 2007 and July Address correspondence to Charles M. Bark, cmb148@case.edu.
2012, prior to the initiation of anti-TB treatment. Patients under- Copyright © 2015, American Society for Microbiology. All Rights Reserved.
went HIV testing and self-reported concomitant medications doi:10.1128/AAC.01101-15
were recorded at baseline. In addition, isolates were excluded if

5844 aac.asm.org Antimicrobial Agents and Chemotherapy September 2015 Volume 59 Number 9
Sulfamethoxazole Susceptibility of M. tuberculosis

available, inexpensive, and well-tolerated. Current in vitro and


clinical data support additional evaluation of SMX for the treat-
ment of TB. A phase 2 early bactericidal activity trial of SMX is a
reasonable next step (18).

ACKNOWLEDGMENTS
This study was funded in part by the National Institutes of Health Na-
tional Center for Research Resources through a CTSA KL2TR000440
grant, which also supports C.M.B. The Kawempe Community Health
Study was funded by the Tuberculosis Research Unit (grants N01-
AI95383 and HHSN266200700022C/N01-AI70022 from the National In-
stitutes of Health National Institute of Allergy and Infectious Diseases,
FIG 1 Distribution of sulfamethoxazole MICs among pretreatment M. tuber- awarded to W.H.B.).
culosis isolates obtained from patients taking or not taking trimethoprim- The contents are solely the responsibility of the authors and do not
sulfamethoxazole (SXT) at the time of TB diagnosis. necessarily represent the official views of the NIH.
We thank John L. Johnson, Case Western Reserve University, for crit-
ical review of the manuscript.
Pretreatment sputum M. tuberculosis isolates from 40 adults We declare no conflicts of interest.
with pulmonary TB were included in this study. Median age was
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