Sung Jae Shin

Clinical Significance of Differentiation of Mycobacterium massiliense from Mycobacterium abscessus

RATIONALE Mycobacterium massiliense has been recognized as a separate species from Mycobacterium abscessus; however, little is known regarding the clinical impact of this differentiation. OBJECTIVES To compare clinical features and treatment outcomes between patients with M. abscessus lung disease and those with M. massiliense lung disease. METHODS We performed molecular identification of stored clinical isolates of M. abscessus complex and compared clinical characteristics and treatment outcomes between 64 patients with M. abscessus lung disease and 81 patients with M. massiliense lung disease. MEASUREMENTS AND MAIN RESULTS The clinical and radiographic manifestations of disease caused by each species were similar. Standardized combination antibiotic therapy, including a clarithromycin-containing regimen in combination with an initial 4-week course of cefoxitin and amikacin, was given to 57 patients (24 with M. abscessus and 33 with M. massiliense) for more than 12 months. The proportion of patients with sputum conversion and maintenance of negative sputum cultures was higher in patients with M. massiliense infection (88%) than in those with M. abscessus infection (25%; P < 0.001). Inducible resistance to clarithromycin (minimal inhibitory concentrations ≥ 32 μg/ml) was found in all tested M. abscessus isolates (n = 19), but in none of the M. massiliense isolates (n = 28). CONCLUSIONS Treatment response rates to combination antibiotic therapy including clarithromycin were much higher in patients with M. massiliense lung disease than in those with M. abscessus lung disease. The inducible resistance to clarithromycin could explain the lack of efficacy of clarithromycin-containing antibiotic therapy against M. abscessus lung disease.

Macrolide Treatment for Mycobacterium abscessus and Mycobacterium massiliense Infection and Inducible Resistance

RATIONALE Macrolides, such as clarithromycin (CLR) and azithromycin (AZM), are frequently the only oral antibiotics that are active against Mycobacterium abscessus and M. massiliense infections. OBJECTIVES To compare the activity of CLR and AZM in experimental models. METHODS We compared the treatment efficacies of CLR and AZM and determined the correlation between efficacy and induced erythromycin ribosome methyltransferase gene (erm)(41) expression in experimental models of M. abscessus and M. massiliense infections. MEASUREMENTS AND MAIN RESULTS In all tested M. abscessus isolates, a high level of inducible CLR resistance developed (minimal inhibitory concentration [MIC] on Day 3 versus Day 14; P < 0.001). Whereas the AZM MIC increased on Day 14 (P < 0.01 versus Day 3), the level was significantly lower than the CLR MIC on Day 14 (P < 0.001). However, the MICs of CLR and AZM for the M. massiliense isolates did not change. Compared with CLR, AZM presented greater antibiotic activity against M. abscessus in vitro, ex vivo, and in vivo (P < 0.05), whereas both macrolides were comparably effective against M. massiliense. In M. abscessus infection, the level of erm(41) expression was higher after exposure to CLR than after exposure to AZM (P < 0.001). Experiments using an erm(41)-knockout M. abscessus mutant and an M. massiliense transformant expressing M. abscessus erm(41) confirmed that erm(41) was responsible for inducible CLR resistance. CONCLUSIONS CLR induces greater erm(41) expression and thus higher macrolide resistance than AZM in M. abscessus infection. AZM may be more effective against M. abscessus, whereas both macrolides appear to be equally effective against M. massiliense.

Clinical Significance of the Differentiation Between Mycobacterium avium and Mycobacterium intracellulare in M avium Complex Lung Disease

BACKGROUND Mycobacterium avium and Mycobacterium intracellulare are grouped together as the M avium complex; however, little is known about the clinical impact of this species differentiation. This study compared the clinical features and prognoses of patients with M avium and M intracellulare lung disease. METHODS From 2000 to 2009, 590 patients were given a new diagnosis of M avium complex lung disease; 323 (55%) had M avium lung disease, and 267 (45%) had M intracellulare lung disease. RESULTS Compared with the patients with M avium lung disease, the patients with M intracellulare lung disease were more likely to have the following characteristics: older age (64 vs 59 years, P = .002), a lower BMI (19.5 kg/m² vs 20.6 kg/m², P < .001), respiratory symptoms such as cough (84% vs 74%, P = .005), a history of previous treatment for TB (51% vs 31%, P < .001), the fibrocavitary form of the disease (26% vs 13%, P < .001), smear-positive sputum (56% vs 38%, P < .001), antibiotic therapy during the 24 months of follow-up (58% vs 42%, P < .001), and an unfavorable microbiologic response after combination antibiotic treatment (56% vs 74%, P = .001). CONCLUSIONS Patients with M intracellulare lung disease exhibited a more severe presentation and had a worse prognosis than patients with M avium lung disease in terms of disease progression and treatment response. Therefore, species differentiation between M avium and M intracellulare may have prognostic and therapeutic implications.

Intermittent Antibiotic Therapy for Nodular Bronchiectatic Mycobacterium avium Complex Lung Disease

RATIONALE Although intermittent, three-times-weekly therapy is recommended for the initial treatment of noncavitary nodular bronchiectatic Mycobacterium avium complex (MAC) lung disease, supporting data are limited. OBJECTIVES To evaluate the clinical efficacy of intermittent therapy compared with daily therapy for nodular bronchiectatic MAC lung disease. METHODS A retrospective cohort study of 217 patients with treatment-naive noncavitary nodular bronchiectatic MAC lung disease. All patients received either daily (n = 99) or intermittent therapy (n = 118) that included clarithromycin or azithromycin, rifampin, and ethambutol. MEASUREMENTS AND MAIN RESULTS Modification of the initial antibiotic therapy occurred more frequently in the daily therapy group than in the intermittent therapy group (46 vs. 21%; P < 0.001); in particular, ethambutol was more frequently discontinued in the daily therapy group than in the intermittent therapy group (24 vs. 1%; P ≤ 0.001). However, the rates of symptomatic improvement, radiographic improvement, and sputum culture conversion were not different between the two groups (daily therapy vs. intermittent therapy: 75 vs. 82%, P = 0.181; 68 vs. 73%, P = 0.402; 76 vs. 67%, P = 0.154, respectively). In addition, the adjusted proportion of sputum culture conversion was similar between the daily therapy (71.3%; 95% confidence interval, 59.1-81.1%) and the intermittent therapy groups (73.6%; 95% confidence interval, 62.9-82.2%; P = 0.785). CONCLUSIONS These results suggest that intermittent three-times-weekly therapy with a macrolide, rifampin, and ethambutol is a reasonable initial treatment regimen for patients with noncavitary nodular bronchiectatic MAC lung disease. Clinical trial registered with www.clinicaltrials.gov (NCT 00970801).

Thiopurine Drugs Azathioprine and 6-Mercaptopurine Inhibit Mycobacterium paratuberculosis Growth In Vitro

ABSTRACT The in vitro susceptibility of human- and bovine-origin Mycobacterium paratuberculosis to the thioupurine drugs 6-mercaptopurine (6-MP) and azathioprine (AZA) was established using conventional plate counting methods and the MGIT 960 ParaTB culture system. Both 6-MP and AZA had antibacterial activity against M. paratuberculosis; isolates from Crohns disease patients tended to be more susceptible than were bovine-origin isolates. Isolates of Mycobacterium avium, used as controls, were generally resistant to both AZA and 6-MP, even at high concentrations (≥64.0 μg/ml). Among rapidly growing mycobacteria, Mycobacterium phlei was susceptible to 6-MP and AZA whereas Mycobacterium smegmatis strains were not. AZA and 6-MP limited the growth of, but did not kill, M. paratuberculosis in a dose-dependent manner. Anti-inflammatory drugs in the sulfonamide family (sulfapyridine, sulfasalazine, and 5-aminosalycilic acid [mesalamine]) had little or no antibacterial activity against M. paratuberculosis. The conventional antibiotics azithromycin and ciprofloxacin, used as control drugs, were bactericidal for M. paratuberculosis, exerting their killing effects on the organism relatively quickly. Simultaneous exposure of M. paratuberculosis to 6-MP and ciprofloxacin resulted in significantly higher CFU than use of ciprofloxacin alone. These data may partially explain the paradoxical response of Crohns disease patients infected with M. paratuberculosis to treatment with immunosuppressive thiopurine drugs, i.e., they do not worsen with anti-inflammatory treatment as would be expected with a microbiological etiologic pathogen. These findings also should influence the design of therapeutic trials to evaluate antibiotic treatments of Crohns disease: AZA drugs may confound interpretation of data on therapeutic responses for both antibiotic-treated and control groups.

Rapid and Reliable Method for Quantification of Mycobacterium paratuberculosis by Use of the BACTEC MGIT 960 System

ABSTRACT A simple method for the enumeration of viable Mycobacterium paratuberculosis cells was developed and evaluated using the MGIT 960 culture system. For each of 12 M. paratuberculosis strains isolated from either cattle or humans, single-cell suspensions of M. paratuberculosis cells were adjusted to an optical density at 600 nm of 1.00 (107.6 to 108.2 cells/ml), and serial dilutions were prepared. Standard curves were established by relating the MGIT time-to-detection data to the log10 CFU for these suspensions using standard plate counting and BACTEC 460 results as reference methods. Universal and strain-specific standard quantification curves were generated. A one-phase exponential decay equation best fit the universal standard curve and strain-specific curves (R2 of 0.96 and >0.99, respectively). Two subgroups within the universal curves were distinguished: one for laboratory-adapted strains and the other for recently isolated low-passage bovine strains. The predictive errors for log10 estimations using the universal standard curve, each subgroups standard curve, and strain-specific curves were ±0.87, ±0.45, and ±0.31 log10 units, respectively. CFU estimations by all three standard curves were highly reproducible, regardless of the M. paratuberculosis strain or inoculum volume. In comparison with the previously described BACTEC 460 M. paratuberculosis counting method, quantification with MGIT 960 was less expensive, more rapid, more accurate, and more sensitive (<10 CFU). This MGIT counting method has broad applications for studies requiring the quantification of viable M. paratuberculosis cells, such as drug susceptibility testing or environmental survival studies.

Enhanced Efficacy of Therapeutic Cancer Vaccines Produced by Co-Treatment with Mycobacterium tuberculosis Heparin-Binding Hemagglutinin, a Novel TLR4 Agonist

Effective activation of dendritic cells (DCs) toward T helper (Th)-1 cell polarization would improve DC-based antitumor immunotherapy, helping promote the development of immunotherapeutic vaccines based on T-cell immunity. To achieve this goal, it is essential to develop effective immune adjuvants that can induce powerful Th1 cell immune responses. The pathogenic organism Mycobacterium tuberculosis includes certain constitutes, such as heparin-binding hemagglutinin (HBHA), that possess a strong immunostimulatory potential. In this study, we report the first clarification of the functions and precise mechanism of HBHA in immune stimulation settings relevant to cancer. HBHA induced DC maturation in a TLR4-dependent manner, elevating expression of the surface molecules CD40, CD80, and CD86, MHC classes I and II and the proinflammatory cytokines IL-6, IL-12, IL-1β, TNF-α, and CCR7, as well as stimulating the migratory capacity of DCs in vitro and in vivo. Mechanistic investigations established that MyD88 and TRIF signaling pathways downstream of TLR4 mediated secretion of HBHA-induced proinflammatory cytokines. HBHA-treated DCs activated naïve T cells, polarized CD4(+) and CD8(+) T cells to secrete IFN-γ, and induced T-cell-mediated cytotoxicity. Notably, systemic administration of DCs that were HBHA-treated and OVA(251-264)-pulsed ex vivo greatly strengthened immune priming in vivo, inducing a dramatic regression of tumor growth associated with long-term survival in a murine E.G7 thymoma model. Together, our findings highlight HBHA as an immune adjuvant that favors Th1 polarization and DC function for potential applications in DC-based antitumor immunotherapy.

Efficient Differentiation of Mycobacterium avium Complex Species and Subspecies by Use of Five-Target Multiplex PCR

ABSTRACT Infections caused by the Mycobacterium avium complex (MAC) are on the rise in both human and veterinary medicine. A means of effectively discriminating among closely related yet pathogenetically diverse members of the MAC would enable better diagnosis and treatment as well as further our understanding of the epidemiology of these pathogens. In this study, a five-target multiplex PCR designed to discriminate MAC organisms isolated from liquid culture media was developed. This MAC multiplex was designed to amplify a 16S rRNA gene target common to all Mycobacterium species, a chromosomal target called DT1 that is unique to M. avium subsp. avium serotypes 2 and 3, to M. avium subsp. silvaticum, and to M. intracellulare, and three insertion sequences, IS900, IS901, and IS1311. The pattern of amplification results allowed determination of whether isolates were mycobacteria, whether they were members of the MAC, and whether they belonged to one of three major MAC subspecies, M. avium subsp. paratuberculosis, M. avium subsp. avium, and M. avium subsp. hominissuis. Analytical sensitivity was 10 fg of M. avium subsp. paratuberculosis genomic DNA, 5 to 10 fg of M. avium subsp. avium genomic DNA, and 2 to 5 fg of DNA from other mycobacterial species. Identification accuracy of the MAC multiplex was evaluated by testing 53 bacterial reference strains consisting of 28 different mycobacterial species and 12 nonmycobacterial species. Identification accuracy in a clinical setting was evaluated for 223 clinical MAC isolates independently identified by other methods. Isolate identification agreement between the MAC multiplex and these comparison assays was 100%. The novel MAC multiplex is a rapid, reliable, and simple assay for discrimination of MAC species and subspecies in liquid culture media.

Therapeutic Drug Monitoring in the Treatment of Mycobacterium avium Complex Lung Disease

RATIONALE Little is known regarding the application of therapeutic drug monitoring for treatment of Mycobacterium avium complex (MAC) lung disease. OBJECTIVES To evaluate drug interactions of multidrug regimens and clinical usefulness of therapeutic drug monitoring in the management of MAC lung disease. METHODS A total of 130 patients with MAC lung disease and 60 patients with Mycobacterium abscessus complex lung disease were enrolled in this study. All of the MAC patients were treated with multidrug regimens that included clarithromycin (CLR), rifampin (RIF) or rifabutin (RFB), and ethambutol (EMB), and the plasma drug concentrations of CLR, RIF, and EMB were measured. MEASUREMENTS AND MAIN RESULTS Peak plasma CLR concentrations were lower in patients with MAC lung disease who received daily (median, 0.3 μg/ml) or intermittent (median, 0.2 μg/ml) therapy with CLR in conjunction with RIF in both groups, compared with those diagnosed with M. abscessus complex lung disease who received CLR without RIF (median, 3.8 μg/ml; P < 0.05). The proportion of patients with MAC lung disease who received daily therapy and whose plasma CLR levels were below the target range of 2 μg/ml was 97% (96 of 99), and this rate was 100% (21 of 21) among patients with MAC lung disease who received intermittent therapy. The peak plasma drug concentrations and the peak plasma drug concentration/minimal inhibitory concentration ratios of CLR, RIF, and EMB did not differ between patients with unfavorable treatment outcomes and those with favorable outcomes. CONCLUSIONS Low plasma CLR concentrations were common in patients treated for MAC lung disease. However, there was no association between low plasma CLR concentrations and treatment outcomes. Therefore, therapeutic drug monitoring may not be beneficial in managing the therapy of patients with MAC lung disease.

Improved sensitivity of diagnosis of tuberculosis in patients in Korea via a cocktail enzyme-linked immunosorbent assay containing the abundantly expressed antigens of the K strain of Mycobacterium tuberculosis.

ABSTRACT Tuberculosis (TB) is the leading cause of death from a single infectious agent in Korea. In this study, we compared the proteins present in culture filtrates from Mycobacterium tuberculosis strain K, which is the dominant clinical isolate in Korea, with those present in culture filtrates from M. tuberculosis H37Rv. Several differences in expression were detected between the two strains for those proteins with a molecular mass of <20 kDa. ESAT-6, HSP-X, and CFP-10 were found to be abundantly expressed in the strain K culture filtrates by liquid chromatography-electrospray ionization-time of flight mass spectrometry. The serodiagnostic potentials of recombinant antigens rESAT-6, rHSP-X, and rCFP-10 and two native antigens (Ag85 and PstS1) were evaluated by Western blot analysis and enzyme-linked immunosorbent assay (ELISA) using sera collected from 46 TB patients with active disease and 46 healthy controls. As for our ELISA results, HSP-X was superior to the other antigens in terms of sensitivity when a single antigen was employed. The results of a receiver operator characteristic analysis revealed that a cocktail ELISA using all five antigens was significantly more sensitive (77.8%) than the use of a single antigen and offered equivalent specificity; moreover, it produced the largest area under the curve (0.91 versus 0.55 to 0.87). Therefore, a cocktail ELISA containing abundantly expressed antigens enhances the sensitivity of a single antigen and can be a useful diagnostic tool for the detection of active TB.