Loss of the high-affinity pentamidine transporter is responsible for high levels of cross-resistance between arsenical and diamidine drugs in African trypanosomes

Treatment of many infectious diseases is under threat from drug resistance. Understanding the mechanisms of resistance is as high a priority as the development of new drugs. We have investigated the basis for cross-resistance between the diamidine and melaminophenyl arsenical classes of drugs in African trypanosomes. We induced high levels of pentamidine resistance in a line without the tbat1 gene that encodes the P2 transporter previously implicated in drug uptake. We isolated independent clones that displayed very considerable cross-resistance with melarsen oxide but not phenylarsine oxide and reduced uptake of [(3)H]pentamidine. In particular, the high-affinity pentamidine transport (HAPT1) activity was absent in the pentamidine-adapted lines, whereas the low affinity pentamidine transport (LAPT1) activity was unchanged. The parental tbat1(-/-) line was sensitive to lysis by melarsen oxide, and this process was inhibited by low concentrations of pentamidine, indicating the involvement of HAPT1. This pentamidine-inhibitable lysis was absent in the adapted line KO-B48. Likewise, uptake of the fluorescent diamidine 4',6-diamidino-2-phenylindole dihydrochloride was much delayed in live KO-B48 cells and insensitive to competition with up to 10 muM pentamidine. No overexpression of the Trypanosoma brucei brucei ATP-binding cassette transporter TbMRPA could be detected in KO-B48. We also show that a laboratory line of Trypanosoma brucei gambiense, adapted to high levels of resistance for the melaminophenyl arsenical drug melarsamine hydrochloride (Cymelarsan), had similarly lost TbAT1 and HAPT1 activity while retaining LAPT1 activity. It seems therefore that selection for resistance to either pentamidine or arsenical drugs can result in a similar phenotype of reduced drug accumulation, explaining the occurrence of cross-resistance.

Effects of HIV type-1 immune selection on susceptability to integrase inhibitor resistance

BACKGROUND: All site-specific interactions between HIV type-1 (HIV-1) subtype, human leukocyte antigen (HLA)-associated immune selection and integrase inhibitor resistance are not completely understood. We examined naturally occurring polymorphisms in HIV-1 integrase sequences from 342 antiretroviral-naive individuals from the Western Australian HIV Cohort Study and the Swiss HIV Cohort Study. METHODS: Standard bulk sequencing and sequence-based typing were used to generate integrase sequences and high-resolution HLA genotypes, respectively. Viral residues were examined with respect to drug resistance mutations and CD8(+) T-cell escape mutations. RESULTS: In both predominantly subtype B cohorts, 12 of 38 sites that mediate integrase inhibitor resistance mutations were absolutely conserved, and these included the primary resistance mutations. There were 18 codons with non-primary drug resistance-associated substitutions at rates of up to 58.8% and eight sites with alternative polymorphisms. Five viral residues were potentially subject to dual-drug and HLA-associated immune selection in which both selective pressures either drove the same amino acid substitution (codons 72, 157 and 163) or HLA alleles were associated with an alternative polymorphism that would alter the genetic barrier to resistance (codons 125 and 193). The common polymorphism T125A, which was characteristic of non-subtype B and was also associated with carriage of HLA-B*57/*5801, increased the mutational barrier to the resistance mutation T125K. CONCLUSIONS: Primary integrase inhibitor resistance mutations were not detected in the absence of drug exposure in keeping with sites of high constraint. Viral polymorphisms caused by immune selection and/or associated with non-subtype B might alter the genetic barrier to some non-primary resistance-associated mutations.

Prevalence and risk factors for carriage of multi-drug resistant Staphylococci in healthy cats and dogs

We investigated the distribution of commensal staphylococcal species and determined the prevalence of multi-drug resistance in healthy cats and dogs. Risk factors associated with the carriage of multi-drug resistant strains were explored. Isolates from 256 dogs and 277 cats were identified at the species level using matrix-assisted laser desorption ionisation-time of flight mass spectrometry. The diversity of coagulase-negative Staphylococci (CNS) was high, with 22 species in dogs and 24 in cats. Multi-drug resistance was frequent (17%) and not always associated with the presence of the mecA gene. A stay in a veterinary clinic in the last year was associated with an increased risk of colonisation by multi-drug resistant Staphylococci (OR = 2.4, 95% CI: 1.1˜5.2, p value LRT = 0.04). When identifying efficient control strategies against antibiotic resistance, the presence of mechanisms other than methicillin resistance and the possible role of CNS in the spread of resistance determinants should be considered.

Adherence as a Predictor of the Development of Class-Specific Resistance Mutations: The Swiss HIV Cohort Study

Background Non-adherence is one of the strongest predictors of therapeutic failure in HIV-positive patients. Virologic failure with subsequent emergence of resistance reduces future treatment options and long-term clinical success. Methods Prospective observational cohort study including patients starting new class of antiretroviral therapy (ART) between 2003 and 2010. Participants were naïve to ART class and completed ≥1 adherence questionnaire prior to resistance testing. Outcomes were development of any IAS-USA, class-specific, or M184V mutations. Associations between adherence and resistance were estimated using logistic regression models stratified by ART class. Results Of 314 included individuals, 162 started NNRTI and 152 a PI/r regimen. Adherence was similar between groups with 85% reporting adherence ≥95%. Number of new mutations increased with increasing non-adherence. In NNRTI group, multivariable models indicated a significant linear association in odds of developing IAS-USA (odds ratio (OR) 1.66, 95% confidence interval (CI): 1.04-2.67) or class-specific (OR 1.65, 95% CI: 1.00-2.70) mutations. Levels of drug resistance were considerably lower in PI/r group and adherence was only significantly associated with M184V mutations (OR 8.38, 95% CI: 1.26-55.70). Adherence was significantly associated with HIV RNA in PI/r but not NNRTI regimens. Conclusion Therapies containing PI/r appear more forgiving to incomplete adherence compared with NNRTI regimens, which allow higher levels of resistance, even with adherence above 95%. However, in failing PI/r regimens good adherence may prevent accumulation of further resistance mutations and therefore help to preserve future drug options. In contrast, adherence levels have little impact on NNRTI treatments once the first mutations have emerged.

A BAYESIAN APPROACH TO DOSE-RESPONSE ASSESSMENT AND DRUG-DRUG INTERACTION ANALYSIS: APPLICATION TO IN VITRO STUDIES

The considerable search for synergistic agents in cancer research is motivated by the therapeutic benefits achieved by combining anti-cancer agents. Synergistic agents make it possible to reduce dosage while maintaining or enhancing a desired effect. Other favorable outcomes of synergistic agents include reduction in toxicity and minimizing or delaying drug resistance. Dose-response assessment and drug-drug interaction analysis play an important part in the drug discovery process, however analysis are often poorly done. This dissertation is an effort to notably improve dose-response assessment and drug-drug interaction analysis. The most commonly used method in published analysis is the Median-Effect Principle/Combination Index method (Chou and Talalay, 1984). The Median-Effect Principle/Combination Index method leads to inefficiency by ignoring important sources of variation inherent in dose-response data and discarding data points that do not fit the Median-Effect Principle. Previous work has shown that the conventional method yields a high rate of false positives (Boik, Boik, Newman, 2008; Hennessey, Rosner, Bast, Chen, 2010) and, in some cases, low power to detect synergy. There is a great need for improving the current methodology. We developed a Bayesian framework for dose-response modeling and drug-drug interaction analysis. First, we developed a hierarchical meta-regression dose-response model that accounts for various sources of variation and uncertainty and allows one to incorporate knowledge from prior studies into the current analysis, thus offering a more efficient and reliable inference. Second, in the case that parametric dose-response models do not fit the data, we developed a practical and flexible nonparametric regression method for meta-analysis of independently repeated dose-response experiments. Third, and lastly, we developed a method, based on Loewe additivity that allows one to quantitatively assess interaction between two agents combined at a fixed dose ratio. The proposed method makes a comprehensive and honest account of uncertainty within drug interaction assessment. Extensive simulation studies show that the novel methodology improves the screening process of effective/synergistic agents and reduces the incidence of type I error. We consider an ovarian cancer cell line study that investigates the combined effect of DNA methylation inhibitors and histone deacetylation inhibitors in human ovarian cancer cell lines. The hypothesis is that the combination of DNA methylation inhibitors and histone deacetylation inhibitors will enhance antiproliferative activity in human ovarian cancer cell lines compared to treatment with each inhibitor alone. By applying the proposed Bayesian methodology, in vitro synergy was declared for DNA methylation inhibitor, 5-AZA-2'-deoxycytidine combined with one histone deacetylation inhibitor, suberoylanilide hydroxamic acid or trichostatin A in the cell lines HEY and SKOV3. This suggests potential new epigenetic therapies in cell growth inhibition of ovarian cancer cells.

TB research at UT-Houston--a review of cord factor: new approaches to drugs, vaccines and the pathogenesis of tuberculosis.

Tuberculosis remains a major threat as drug resistance continues to increase. Pulmonary tuberculosis in adults is responsible for 80% of clinical cases and nearly 100% of transmission of infection. Unfortunately, since we have no animal models of adult type pulmonary tuberculosis, the most important type of disease remains largely out of reach of modern science and many fundamental questions remain unanswered. This paper reviews research dating back to the 1950's providing compelling evidence that cord factor (trehalose 6,6 dimycolate [TDM]) is essential for understanding tuberculosis. However, the original papers by Bloch and Noll were too far ahead of their time to have immediate impact. We can now recognize that the physical and biologic properties of cord factor are unprecedented in science, especially its ability to switch between two sets of biologic activities with changes in conformation. While TDM remains on organisms, it protects them from killing within macrophages, reduces antibiotic effectiveness and inhibits the stimulation of protective immune responses. If it comes off organisms and associates with lipid, TDM becomes a driver of tissue damage and necrosis. Studies emanating from cord factor research have produced (1) a rationale for improving vaccines, (2) an approach to new drugs that overcome natural resistance to antibiotics, (3) models of caseating granulomas that reproduce multiple manifestations of human tuberculosis. (4) evidence that TDM is a key T cell antigen in destructive lesions of tuberculosis, and (5) a new understanding of the pathology and pathogenesis of postprimary tuberculosis that can guide more informative studies of long standing mysteries of tuberculosis.

Human mutations that confer paclitaxel resistance.

The involvement of tubulin mutations as a cause of clinical drug resistance has been intensely debated in recent years. In the studies described here, we used transfection to test whether beta1-tubulin mutations and polymorphisms found in cancer patients are able to confer resistance to drugs that target microtubules. Three of four mutations (A185T, A248V, R306C, but not G437S) that we tested caused paclitaxel resistance, as indicated by the following observations: (a) essentially 100% of cells selected in paclitaxel contained transfected mutant tubulin; (b) paclitaxel resistance could be turned off using tetracycline to turn off transgene expression; (c) paclitaxel resistance increased as mutant tubulin production increased. All the paclitaxel resistance mutations disrupted microtubule assembly, conferred increased sensitivity to microtubule-disruptive drugs, and produced defects in mitosis. The results are consistent with a mechanism in which tubulin mutations alter microtubule stability in a way that counteracts drug action. These studies show that human tumor cells can acquire spontaneous mutations in beta1-tubulin that cause resistance to paclitaxel, and suggest that patients with some polymorphisms in beta1-tubulin may require higher drug concentrations for effective therapy.

Clinical and microbiological aspects of linezolid resistance mediated by the cfr gene encoding a 23S rRNA methyltransferase.

The cfr (chloramphenicol-florfenicol resistance) gene encodes a 23S rRNA methyltransferase that confers resistance to linezolid. Detection of linezolid resistance was evaluated in the first cfr-carrying human hospital isolate of linezolid and methicillin-resistant Staphylococcus aureus (designated MRSA CM-05) by dilution and diffusion methods (including Etest). The presence of cfr was investigated in isolates of staphylococci colonizing the patient's household contacts and clinical isolates recovered from patients in the same unit where MRSA CM-05 was isolated. Additionally, 68 chloramphenicol-resistant Colombian MRSA isolates recovered from hospitals between 2001 and 2004 were screened for the presence of the cfr gene. In addition to erm(B), the erm(A) gene was also detected in CM-05. The isolate belonged to sequence type 5 and carried staphylococcal chromosomal cassette mec type I. We were unable to detect the cfr gene in any of the human staphylococci screened (either clinical or colonizing isolates). Agar and broth dilution methods detected linezolid resistance in CM-05. However, the Etest and disk diffusion methods failed to detect resistance after 24 h of incubation. Oxazolidinone resistance mediated by the cfr gene is rare, and acquisition by a human isolate appears to be a recent event in Colombia. The detection of cfr-mediated linezolid resistance might be compromised by the use of the disk diffusion or Etest method.

Molecular basis for class V beta-tubulin effects on microtubule assembly and paclitaxel resistance.

Vertebrates produce at least seven distinct beta-tubulin isotypes that coassemble into all cellular microtubules. The functional differences among these tubulin isoforms are largely unknown, but recent studies indicate that tubulin composition can affect microtubule properties and cellular microtubule-dependent behavior. One of the isotypes whose incorporation causes the largest change in microtubule assembly is beta5-tubulin. Overexpression of this isotype can almost completely destroy the microtubule network, yet it appears to be required in smaller amounts for normal mitotic progression. Moderate levels of overexpression can also confer paclitaxel resistance. Experiments using chimeric constructs and site-directed mutagenesis now indicate that the hypervariable C-terminal region of beta5 plays no role in these phenotypes. Instead, we demonstrate that two residues found in beta5 (Ser-239 and Ser-365) are each sufficient to inhibit microtubule assembly and confer paclitaxel resistance when introduced into beta1-tubulin; yet the single mutation of residue Ser-239 in beta5 eliminates its ability to confer these phenotypes. Despite the high degree of conservation among beta-tubulin isotypes, mutations affecting residue 365 demonstrate that amino acid substitutions can be context sensitive; i.e. an amino acid change in one isotype will not necessarily produce the same phenotype when introduced into a different isotype. Modeling studies indicate that residue Cys-239 of beta1-tubulin is close to a highly conserved Cys-354 residue suggesting the possibility that disulfide formation could play a significant role in the stability of microtubules formed with beta1- but not with beta5-tubulin.

Acquisition of a natural resistance gene renders a clinical strain of methicillin-resistant Staphylococcus aureus resistant to the synthetic antibiotic linezolid.

Linezolid, which targets the ribosome, is a new synthetic antibiotic that is used for treatment of infections caused by Gram-positive pathogens. Clinical resistance to linezolid, so far, has been developing only slowly and has involved exclusively target site mutations. We have discovered that linezolid resistance in a methicillin-resistant Staphylococcus aureus hospital strain from Colombia is determined by the presence of the cfr gene whose product, Cfr methyltransferase, modifies adenosine at position 2503 in 23S rRNA in the large ribosomal subunit. The molecular model of the linezolid-ribosome complex reveals localization of A2503 within the drug binding site. The natural function of cfr likely involves protection against natural antibiotics whose site of action overlaps that of linezolid. In the chromosome of the clinical strain, cfr is linked to ermB, a gene responsible for dimethylation of A2058 in 23S rRNA. Coexpression of these two genes confers resistance to all the clinically relevant antibiotics that target the large ribosomal subunit. The association of the ermB/cfr operon with transposon and plasmid genetic elements indicates its possible mobile nature. This is the first example of clinical resistance to the synthetic drug linezolid which involves a natural resistance gene with the capability of disseminating among Gram-positive pathogenic strains.

Significance of Increased Tissue Transglutaminase in Hormone Refractory Prostate Cancer

The progression of hormone responsive to hormone refractory prostate cancer poses a major clinical challenge in the successful treatment of prostate cancer. The hormone refractory prostate cancer cells exhibit resistance not only to castrate levels of testosterone, but also to other therapeutic modalities and hence become lethal. Currently, there is no effective treatment available for managing this cancer. These observations underscore the urgency to investigate mechanism(s) that contribute to the progression of hormone-responsive to hormone-refractory prostate cancer and to target them for improved clinical outcomes. Tissue transglutaminase (TG2) is a multifunctional pro-inflammatory protein involved in diverse physiological processes such as inflammation, tissue repair, and wound healing. Its expression is also implicated in pathological conditions such as cancer and fibrosis. Interestingly, we found that the androgen-independent prostate cancer cell lines, which lacked androgen receptor (AR) expression, contained high basal levels of tissue transglutaminase. Inversely, the cell lines that expressed androgen receptor lacked transglutaminase expression. This attracted our attention to investigate the possible role this protein may play in the progression of prostate cancer, especially in view of recent observations that its expression is linked with increased invasion, metastasis, and drug resistance in multiple cancer cell types. The results we obtained were rather surprising and revealed that stable expression of tissue transglutaminase in androgen-sensitive LNCaP prostate cancer cells rendered these cells independent of androgen for growth and survival by silencing the AR expression. The AR silencing in TG2 expressing cells (TG2-infected LNCaP and PC-3 cells) was due to TG2-induced activation of the inflammatory nuclear transcription factor-kB (NF-kB). Thus, TG2 induced NF-kB was found to directly bind to the AR promoter. Importantly, TG2 protein was specifically recruited to the AR promoter in complex with the p65 subunit of NF-kB. Moreover, TG2 expressing LNCaP and PC-3 cells exhibited epithelial-to-mesenchymal transition, as evidenced by gain of mesenchymal (such as fibronectin, vimentin, etc.) and loss of epithelial markers (such as E-cadherin, b-catenin). Taken together, these results suggested a new function for TG2 and revealed a novel mechanism that is responsible for the progression of prostate cancer to the aggressive hormone-refractory phenotype.

Surveillance of antimicrobial resistance of Streptococcus pneumoniae and Haemophilus influenzae in children with pneumonia

In order to identify optimal therapy for children with bacterial pneumonia, Pakistan's ARI Program, in collaboration with the National Institute of Health (NIH), Islamabad, undertook a national surveillance of antimicrobial resistance in S. pneumoniae and H. influenzae. The project was carried out at selected urban and peripheral sites in 6 different regions of Pakistan, in 1991–92. Nasopharyngeal (NP) specimens and blood cultures were obtained from children with pneumonia diagnosed in the outpatient clinic of participating facilities. Organisms were isolated by local hospital laboratories and sent to NIH for confirmation, serotyping and antimicrobial susceptibility testing. Following were the aims of the study (i) to determine the antimicrobial resistance patterns of S. pneumoniae and H. influenzae in children aged 2–59 months; (ii) to determine the ability of selected laboratories to identify and effectively transport isolates of S. pneumoniae and H. influenzae cultured from nasopharyngeal and blood specimens; (iii) to validate the comparability of resistance patterns for nasopharyngeal and blood isolates of S. pneumoniae and H. influenzae from children with pneumonia; and (iv) to examine the effect of drug resistance and laboratory error on the cost of effectively treating children with ARI. ^ A total of 1293 children with ARI were included in the study: 969 (75%) from urban areas and 324 (25%) from rural parts of the country. Of 1293, there were 786 (61%) male and 507 (39%) female children. The resistance rate of S. pneumoniae to various antibiotics among the urban children with ARI was: TMP/SMX (62%); chloramphenicol (23%); penicillin (5%); tetracycline (16%); and ampicillin/amoxicillin (0%). The rates of resistance of H. influenzae were higher than S. pneumoniae: TMP/SMX (85%); chloramphenicol (62%); penicillin (59%); ampicillin/amoxicillin (46%); and tetracycline (100%). There were similar rates of resistance to each antimicrobial agent among isolates from the rural children. ^ Of a total 614 specimens that were tested for antimicrobial susceptibility, 432 (70.4%) were resistant to TMP/SMX and 93 (15.2%) were resistant to antimicrobial agents other than TMP/SMX viz. ampicillin/amoxicillin, chloramphenicol, penicillin, and tetracycline. ^ The sensitivity and positive predictive value of peripheral laboratories for H. influenzae were 99% and 65%, respectively. Similarly, the sensitivity and positive predictive value of peripheral laboratory tests compared to gold standard i.e. NIH laboratory, for S. pneumoniae were 99% and 54%, respectively. ^ The sensitivity and positive predictive value of nasopharyngeal specimens compared to blood cultures (gold standard), isolated by the peripheral laboratories, for H. influenzae were 88% and 11%, and for S. pneumoniae 92% and 39%, respectively. (Abstract shortened by UMI.)^

Effects of butyrate on the colon cancer cell phenotype-chemosensitization and upregulation of proteins implicated in cancer progression

Colon cancer is the second leading cause of cancer mortality in the U.S. Surgery is the only truly effective human colon cancer (HCC) therapy due to marked intrinsic drug resistance. The inefficacy of therapies developed for metastatic HCC suggests that advances in colon cancer chemoprevention and chemotherapy will be needed to reduce HCC mortality. The dietary fiber metabolite butyrate (NaB) is a candidate cancer chemopreventive agent that inhibits growth, promotes differentiation and stimulates apoptosis of HCC cells. Epidemiological and experimental studies suggest that dietary fiber protects against the development of HCC, however, recent large prospective trials have not found significant protection. ^ The first central hypothesis of this dissertation project is that the diversity of phenotypic changes induced by NaB in HCC cells includes molecular alterations that oppose its chemopreventive action and thereby limit its efficacy. We investigated the effect of NaB on the expression/activity of epidermal growth factor receptor (EGFR) and cyclooxygenase-2 (COX-2) in HCC HT29 cells. NaB treatment induced a 13-fold increase in EGFR expression in concert with its chemopreventive action in vitro, i.e., induction of growth suppression and G1 arrest, apoptosis and a differentiated phenotype. NaB-induced EGFR was active based on multiple lines of evidence. The EGFR was: (1) heavily phosphorylated at Tyrosine (P-Tyr); (2) associated with the cytoskeleton; (3) localized at the cell surface, and activated in response to EGF; and (4) NaB treatment of the cells induced activation of the EGFR effector Erk1/2. NaB treatment also induced a 7-fold increase in COX-2 expression. The NaB-induced COX-2 was active based on significantly increased PGE2 production. ^ The second central hypothesis is that NaB treatment would render HCC cells more chemosensitive to chemotherapy agents based on the increased apoptotic index induced by NaB. NaB treatment chemosensitized HT29 cells to 5-FU and doxorubicin, despite increases in the expression of P-glycoprotein and a related drug resistance protein (MRP). ^ These results raise the intriguing possibility that the chemopreventive effects of fiber may require concomitant treatment with EGFR and/or COX-2 inhibitors. Similarly, NaB may be a rational drug to combine with existing chemotherapeutic agents for the management of advanced HCC patients. ^

Limited clinical benefit of minority K103N and Y181C-variant detection in addition to routine genotypic resistance testing in antiretroviral therapy-naive patients

OBJECTIVE: The presence of minority nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant HIV-1 variants prior to antiretroviral therapy (ART) has been linked to virologic failure in treatment-naive patients. DESIGN: We performed a large retrospective study to determine the number of treatment failures that could have been prevented by implementing minority drug-resistant HIV-1 variant analyses in ART-naïve patients in whom no NNRTI resistance mutations were detected by routine resistance testing. METHODS: Of 1608 patients in the Swiss HIV Cohort Study, who have initiated first-line ART with two nucleoside reverse transcriptase inhibitors (NRTIs) and one NNRTI before July 2008, 519 patients were eligible by means of HIV-1 subtype, viral load and sample availability. Key NNRTI drug resistance mutations K103N and Y181C were measured by allele-specific PCR in 208 of 519 randomly chosen patients. RESULTS: Minority K103N and Y181C drug resistance mutations were detected in five out of 190 (2.6%) and 10 out of 201 (5%) patients, respectively. Focusing on 183 patients for whom virologic success or failure could be examined, virologic failure occurred in seven out of 183 (3.8%) patients; minority K103N and/or Y181C variants were present prior to ART initiation in only two of those patients. The NNRTI-containing, first-line ART was effective in 10 patients with preexisting minority NNRTI-resistant HIV-1 variant. CONCLUSION: As revealed in settings of case-control studies, minority NNRTI-resistant HIV-1 variants can have an impact on ART. However, the sole implementation of minority NNRTI-resistant HIV-1 variant analysis in addition to genotypic resistance testing (GRT) cannot be recommended in routine clinical settings. Additional associated risk factors need to be discovered.

Detection and management of drug-resistant tuberculosis in HIV-infected patients in lower-income countries.

SETTING Drug resistance threatens tuberculosis (TB) control, particularly among human immunodeficiency virus (HIV) infected persons. OBJECTIVE To describe practices in the prevention and management of drug-resistant TB under antiretroviral therapy (ART) programs in lower-income countries. DESIGN We used online questionnaires to collect program-level data on 47 ART programs in Southern Africa (n = 14), East Africa (n = 8), West Africa (n = 7), Central Africa (n = 5), Latin America (n = 7) and the Asia-Pacific (n = 6 programs) in 2012. Patient-level data were collected on 1002 adult TB patients seen at 40 of the participating ART programs. RESULTS Phenotypic drug susceptibility testing (DST) was available in 36 (77%) ART programs, but was only used for 22% of all TB patients. Molecular DST was available in 33 (70%) programs and was used in 23% of all TB patients. Twenty ART programs (43%) provided directly observed therapy (DOT) during the entire course of treatment, 16 (34%) during the intensive phase only, and 11 (23%) did not follow DOT. Fourteen (30%) ART programs reported no access to second-line anti-tuberculosis regimens; 18 (38%) reported TB drug shortages. CONCLUSIONS Capacity to diagnose and treat drug-resistant TB was limited across ART programs in lower-income countries. DOT was not always implemented and drug supplies were regularly interrupted, which may contribute to the global emergence of drug resistance.