M A J O R A R T I C L E
Multidrug-Resistant Tuberculosis Not Due to
Noncompliance but to Between-Patient
Shashikant Srivastava,1Jotam G. Pasipanodya,1Claudia Meek,2Richard Leff,2and Tawanda Gumbo1
1Department of Medicine, University of Texas Southwestern Medical Center at Dallas; and2School of Pharmacy, Texas Tech University Health Science
(See the editorial commentary by Dartois, on pages 1827–9.)
(MDR-tuberculosis) emergence. The level of nonadherence associated with emergence of MDR-tuberculosis is
unknown. Performance of a randomized controlled trial in which some patients are randomized to nonadherence
would be unethical; therefore, other study designs should be utilized.
Methods.We performed hollow fiber studies for both bactericidal and sterilizing effect, with inoculum spiked
with 0.5% rifampin- and isoniazid-resistant isogenic strains in some experiments. Standard therapy was
administered daily for 28–56 days, with extents of nonadherence varying between 0% and 100%. Sizes of drug-
resistant populations were compared using analysis of variance. We also explored the effect of pharmacokinetic
variability on MDR-tuberculosis emergence using computer-aided clinical trial simulations of 10000 Cape Town,
South Africa, tuberculosis patients.
Results.Therapy failure was only encountered at extents of nonadherence $60%. Surprisingly, isoniazid- and
rifampin-resistant populations did not achieve $1% proportion in any experiment and did not achieve a higher
patients with perfect adherence would still develop MDR-tuberculosis due to pharmacokinetic variability alone.
Conclusions.These data, based on a preclinical model, demonstrate that nonadherence alone is not a sufficient
condition for MDR-tuberculosis emergence.
It is believed that nonadherence is the proximate cause of multidrug-resistant tuberculosis
Drug-susceptible tuberculosis is currently treated with
isoniazid, rifampin, and pyrazinamide as part of a directly
observed therapy–short-course strategy (DOTS). How-
ever, therapy success is threatened by the emergence of
multidrug-resistant tuberculosis (MDR-tuberculosis), de-
fined as simultaneous resistance to isoniazid and ri-
fampin. It is believed that most MDR-tuberculosis
arises due to poor adherence, hence the need for
a DOTS. The World Health Organization (WHO) has
built DOTS into the global approach for tuberculosis
control. However, the level of nonadherence associated
with the emergence of MDR-tuberculosis is unknown;
thus the program requires that all tuberculosis patients
be supervised by healthcare workers while swallowing
antibiotics. The WHO Director-General has called
DOTS the most important health breakthrough of past
Most studies that established the utility of directly
supervised therapy were retrospective and relied on
examining rates of MDR-tuberculosis before and after
implementation of DOTS programs [2, 3]. However,
DOTS has 5 components, including provision of an
adequate and standardized drug supply, which could lead
to reduction of MDR-tuberculosis rates independent of
adherence. Recently, a meta-analysis questioned the effi-
cacy of direct supervision by a healthcare worker .
Received 13 April 2011; accepted 22 July 2011.
Presented in part: Oral presentation, 3rd International Workshop on Clinical
Pharmacology of Tuberculosis Drugs, Boston, Massachusetts, 11 September 2010.
Correspondence: Tawanda Gumbo, MD, Office of Global Health, University of
Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, Texas 75390-
The Journal of Infectious Diseases
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MDR-tuberculosis Not Due to Nonadherence
d JID 2011:204 (15 December)
Moreover, despite .98% adherence, extensive emergence of
MDR-tuberculosis still occurs . We were interested in de-
termining whether poor adherence leads to therapy failure and
to MDR-tuberculosis emergence. Because DOTS is now ac-
cepted worldwide, it would be neither ethical nor desirable to
perform a randomized clinical study to answer these questions.
Therefore, we examined the questions in our hollow fiber system
(HFS) model of tuberculosis.
Mycobacterium tuberculosis H37Rv (ATCC 27294) with mini-
mum inhibitory concentrations (MICs) to isoniazid, rifampin,
and pyrazinamide of 0.06, 0.125, and 12.5 mg/L, respectively,
was used in the experiments. A rifampin-resistant isogenic
strain (ATCC 35838) with an MIC of .32 mg/L due to
a mutation at codon 531 Ser/Leu in the genes for the
b subunit of DNA-dependent RNA polymerase (rpoB) was
also used. We generated an isoniazid-resistant M. tuberculosis
H37Rv strain (MIC 5 2 mg/L), which has an AGC/GGC
mutation at codon 315 of the catalase-peroxidase gene (katG).
Storage and propagation of cultures were as described pre-
viously [6–9]. Cultures were grown in Middlebrook 7H9
broth supplemented with 10% Oleic acid, albumin, dextrose,
catalase (OADC) (herein termed ‘‘broth’’) at 37?C under 5%
CO2and shaking conditions. Day 4 cultures in log-phase
growth were then used for experiments.
The standarddefinitionofbactericidal activityis microbialkillof
rapidly multiplying bacilli by antibiotics, and the standard def-
inition of sterilizing activity is microbial kill of either semi-
dormant or slowly replicating bacilli in an acidic environment
[10, 11]. In the HFS, therapy failure was defined by presence of
positive culture at the end of the experiment. The extent of
nonadherence was defined as percentage of doses missed based
on the Centers for Disease Control and Prevention (CDC) reg-
imen 1, in which drugs are administered daily for 56 days .
However,in manyparts ofthe world, therapyis administered for
5 of 7 days each week (total 40 doses) during the initial phase of
therapy (5/7 regimen). WHO’s recommendation 2.1B is for
3 weekly doses that can be administered in a low-HIV setting
(thrice-weekly regimen) .
Hollow Fiber System
Technical details of our HFS model have been described pre-
viously [6–9]. Each HFS and its contents were incubated at 37?C
under 5% CO2. M. tuberculosis was inoculated into the peripheral
compartment of each HFS. Drug treatments were administered
daily via a computer-controlled syringe pump. We mimicked the
pharmacokinetics of isoniazid 300 mg daily, rifampin 600 mg
daily, and pyrazinamide 2 grams daily in patients. The HFS ach-
ieves decline of antibiotic concentration via dilution of the central
compartment with fresh media. We set dilution rates for the drug
with the smallest half-life, and achieved the longer half-lives by
syringe pumps. Drug concentrations achieved in each HFS were
measured at 5 time points during the first 24 hours. These con-
centrations were then modeled using the ADAPT 5 program ,
assuming a 1-compartment model with first-order elimination
[6–9]. Bacteria were sampled from the peripheral compartments,
then washed twice with normal saline to remove any carryover
drug, and cultured on Middlebrook 7H10 agar supplemented
resistant subpopulations, the samples were cultured on agar
supplemented with 0.2 mg/L isoniazid, 2.0 mg/L rifampin, or
300 mg/L pyrazinamide. In addition, we also examined for re-
sistance to 0.125 mg/L isoniazid or 0.0625 mg/L rifampin, based
on new susceptibility breakpoints that we have proposed . For
pyrazinamide, the agar was acidified to pH 5.8 and supplemented
with 10% fetal bovine serum . Cultures were incubated at 37?C
under 5% CO2, and colonies were counted starting 3 weeks after
We tested the hypothesis that the extent of nonadherence is di-
rectly proportional to the size of the drug-resistant M. tuberculosis
populations. For bactericidal-effect experiments, M. tuberculosis
on the fourth day of log-phase growth was inoculated into the
peripheral compartment of each HFS in which broth circulated.
This was done so that the M. tuberculosis would continue in log-
phase growth. For sterilizing effect on day 4, log-phase growth
cultures were cultured in broth acidified to pH 5.8 for 4 days of
peripheral compartment of each HFS with circulating acidified
First, each of 18 HFSs was inoculated with 20 mL of 6.5 log10
colony forming units (CFU) per milliliter of drug-susceptible
M. tuberculosis in log-phase growth. This mimics the volume of
a spherical tuberculosis abscess of approximately 2.2-cm radius
for each system. Therapy was administered daily until 0%, 20%,
40%, 60%, 80%, and 100% of total doses in a 28-day treatment
period was reached in each of 3 HFSs (Supplementary Table 1).
Cultures for total and drug-resistant M. tuberculosis were col-
semidormant bacilli were inoculated and nonadherence of 0%,
30%, 60%, 70%, 80%, and 100% was examined (Supplementary
Table 1), based on bactericidal-effect study results.
Next, we examined the effect of the nonadherence pattern on
resistance emergence. Emergence of drug-resistant mutants is
a stochastic process, which means that a drug-resistant sub-
population may simply fail to arise by chance. To exclude this
possibility, inoculum was premixed with 0.5% of isogenic
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MDR-tuberculosis Not Due to Nonadherence
d JID 2011:204 (15 December)