Development of a rapid detection method for the macrolide resistance gene in Mycobacterium avium using the amplification refractory mutation system-loop-mediated isothermal amplification method.

Macrolide antibiotics such as clarithromycin (CLR) and azithromycin are the key drugs used in multidrug therapy for Mycobacterium avium complex (MAC) diseases. For these antibacterial drugs, drug susceptibility has been correlated with clinical response in MAC diseases. We have previously demonstrated the correlation between drug susceptibility and mutations in the 23S rRNA gene, which confers resistance to macrolides. Herein, we developed a rapid detection method using the amplification refractory mutation system (ARMS)-loop-mediated isothermal amplification (LAMP) technique to identify mutations in the 23S rRNA gene of M. avium. We examined the applicability of the ARMS-LAMP method to genomic DNA extracted from six genotypes of M. avium clinical isolates. The M. avium isolates were classified into 21 CLR-resistant and 9 CLR-susceptible strains based on the results of drug susceptibility tests; the 23S rRNA genes of these strains were sequenced and analyzed using the ARMS-LAMP method. Sequence analysis revealed that the 9 CLR-sensitive strains were wild-type strains, whereas the 21 CLR-resistant strains comprised 20 mutant-type strains and one wild-type strain. Using ARMS-LAMP, no amplification from genomic DNAs of the 10 wild-type strains was observed using the mutant-type mismatch primer sets (MTPSs); however, amplification from the 20 mutant-type strain DNAs was observed using the MTPSs. The rapid detection method developed by us integrates ARMS-LAMP with a real-time turbidimeter, which can help determine drug resistance in a few hours. In conclusion, ARMS-LAMP might be a new clinically beneficial technology for rapid detection of mutations.IMPORTANCEMultidrug therapy for pulmonary Mycobacterium avium complex disease is centered on the macrolide antibiotics clarithromycin and azithromycin, and resistance to macrolides is an important prognosticator for clinical aggravation. Therefore, it is important to develop a quick and easy method for detecting resistance to macrolides. Drug resistance is known to be correlated with mutations in macrolide resistance genes. We developed a rapid detection method using amplification refractory mutation system (ARMS)-loop-mediated isothermal amplification (LAMP) to identify a mutation in the 23S rRNA gene, which is a macrolide resistance gene. Furthermore, we examined the applicability of this method using M. avium clinical isolates. The rapid method developed by us for detection of the macrolide resistance gene by integrating ARMS-LAMP and a real-time turbidimeter can help in detection of drug resistance within a few hours. Since this method does not require expensive equipment or special techniques and shows high analytical speed, it would be very useful in clinical practice.

A Rapid Screening Assay for Clarithromycin-Resistant Mycobacterium avium Complex Using Melting Curve Analysis with Nonfluorescent Labeled Probes.

Mycobacterium avium complex (MAC) thrives in various environments and mainly causes lung disease in humans. Because macrolide antibiotics such as clarithromycin or azithromycin are key drugs for MAC lung disease, the emergence of macrolide-resistant strains prevents the treatment of MAC. More than 95% of macrolide-resistant MAC strains are reported to have a point mutation in 23S rRNA domain V. This study successfully developed a melting curve assay using nonfluorescent labeled probes to detect the MAC mutation at positions 2058 to 2059 of the 23S rRNA gene (AA genotype, clarithromycin susceptible; TA, GA, AG, CA, AC, and AT genotypes, clarithromycin resistant). In the AA-specific probe assay, the melting peak of the DNA fragment of the AA genotype was higher than that of DNA fragments of other genotypes. Melting temperature (Tm) values of the AA genotype and the other genotypes were about 80°C and 77°C, respectively. DNA fragments of each genotype were identified correctly in six other genotype-specific probes (TA, GA, AG, CA, AC, and AT) assays. Using genomic DNA from six genotype strains of M. avium and four genotype strains of M. intracellulare, we confirmed that all genomic DNAs could be correctly identified as individual genotypes according to the highest Tm values among the same probe assays. These results indicate that this melting curve-based assay is able to determine MAC genotypes at positions 2058 to 2059 of the 23S rRNA gene. This simple method could contribute to the rapid detection of clarithromycin-resistant MAC strains and help to provide accurate drug therapy for MAC lung disease. IMPORTANCE Since macrolide antibiotics such as clarithromycin or azithromycin are key drugs in multidrug therapy for Mycobacterium avium complex (MAC) lung diseases, the rapid detection of macrolide-resistant MAC strains has important implications for the treatment of MAC. Previous studies have reported a correlation between drug susceptibility testing and the mutation of macrolide resistance genes. In this study, we developed a novel melting curve-based assay using nonfluorescent labeled probes to identify both clarithromycin-resistant M. avium and M. intracellulare with mutations in the 23S rRNA gene, which is the clarithromycin or azithromycin resistance gene. This assay contributed to not only the detection of MAC mutations but also the determination of all genotypes at positions 2058 to 2059 of the 23S rRNA gene. Furthermore, because nonfluorescent labeled probes are used, this assay is more easily and more immediately available than other methods.

Clinical and therapeutic features of pulmonary nontuberculous mycobacterial disease, Brazil, 1993-2011.

To identify clinical and therapeutic features of pulmonary nontuberculous mycobacterial (PNTM) disease, we conducted a retrospective analysis of patients referred to the Brazilian reference center, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil, who received a diagnosis of PNTM during 1993­2011 with at least 1 respiratory culture positive for NTM. Associated conditions included bronchiectasis (21.8%), chronic obstructive pulmonary disease (20.7%), cardiovascular disease (15.5%), AIDS (9.8%), diabetes (9.8%), and hepatitis C (4.6%).Two patients had Hansen disease; 1 had Marfan syndrome. Four mycobacterial species comprised 85.6% of NTM infections: Mycobacterium kansasii, 59 cases (33.9%); M. avium complex, 53 (30.4%); M. abscessus, 23 (13.2%); and M. fortuitum, 14 (8.0%). A total of 42 (24.1%) cases were associated with rapidly growing mycobacteria. In countries with a high prevalence of tuberculosis, PNTM is likely misdiagnosed as tuberculosis, thus showing the need for improved capacity to diagnose mycobacterial disease as well as greater awareness of PNTM disease prevalence.

Treatment of Mycobacterium avium-intracellulare complex lung disease with a macrolide, ethambutol, and clofazimine.

BACKGROUND: Mycobacterium avium-intracellulare (MAC) causes progressive lung disease. Recommended treatment regimens include a macrolide and a rifamycin, but drug intolerance and relapse after treatment is completed often limit successful therapy. METHODS: Consecutive individuals referred for treatment of MAC lung disease were treated with a regimen that included either clarithromycin, 500 mg bid, or azithromycin, 250 mg/d, on weekdays; ethambutol, 15 mg/kg/d; and clofazimine, 100 mg/d. The intention was to treat patients for a minimum of 12 months. The diagnosis of MAC lung disease was confirmed by multiple positive sputum culture findings in patients with typical symptoms and radiologic findings. RESULTS: Thirty patients (27 women and 3 men; mean age, 70 +/- 9.4 years [SD]) were treated. A total of 22 of the patients reported adverse effects from clarithromycin or azithromycin. Intolerance of clarithromycin resulted in the withdrawal of four patients before sputum conversion. The remaining patients continued treatment for an average of 10 months, and sputum findings converted to negative in all 26 patients (87%). One patient died of unrelated causes while still receiving therapy, and five patients (19%) relapsed an average of 17 months after treatment was completed. CONCLUSIONS: Treatment with a macrolide, ethambutol, and clofazimine was successful in 20 of 30 patients (67%) with MAC lung disease and is a reasonable alternative to rifamycin-containing regimens.