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.

Open-Label Trial of Amikacin Liposome Inhalation Suspension in Mycobacterium abscessus Lung Disease.

BACKGROUND: Mycobacterium abscessus is the second most common nontuberculous mycobacterium respiratory pathogen and shows in vitro resistance to nearly all oral antimicrobials. M abscessus treatment success is low in the presence of macrolide resistance. RESEARCH QUESTION: Does treatment with amikacin liposome inhalation suspension (ALIS) improve culture conversion in patients with M abscessus pulmonary disease who are treatment naive or who have treatment-refractory disease? STUDY DESIGN AND METHODS: In an open-label protocol, patients were given ALIS (590 mg) added to background multidrug therapy for 12 months. The primary outcome was sputum culture conversion defined as three consecutive monthly sputum cultures showing negative results. The secondary end point included development of amikacin resistance. RESULTS: Of 33 patients (36 isolates) who started ALIS with a mean age of 64 years (range, 14-81 years), 24 patients (73%) were female, 10 patients (30%) had cystic fibrosis, and nine patients (27%) had cavitary disease. Three patients (9%) could not be evaluated for the microbiologic end point because of early withdrawal. All pretreatment isolates were amikacin susceptible and only six isolates (17%) were macrolide susceptible. Eleven patients (33%) were given parenteral antibiotics. Twelve patients (40%) received clofazimine with or without azithromycin as companion therapy. Fifteen patients (50%) with evaluable longitudinal microbiologic data demonstrated culture conversion, and 10 patients (67%) sustained conversion through month 12. Six of the 33 patients (18%) demonstrated mutational amikacin resistance. All were patients using clofazimine or clofazimine plus azithromycin as companion medication(s). Few serious adverse events occurred for ALIS users; however, reduction of dosing to three times weekly was common (52%). INTERPRETATION: In a cohort of patients primarily with macrolide-resistant M abscessus, one-half of the patients using ALIS showed sputum culture conversion to negative findings. The emergence of mutational amikacin resistance was not uncommon and occurred with the use of clofazimine monotherapy. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT03038178; URL: www. CLINICALTRIALS: gov.

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.

Laboratory detection of bacterial pathogens and clinical and laboratory response of syndromic management in patients with cervical discharge: A retrospective study.

Background Cervical discharge as part of cervicitis and pelvic inflammatory disease is a cause of significant morbidity in sexually active women worldwide. Non-gonococcal and non- chlamydial bacterial pathogens are becoming more prevalent. Aims This study aims to determine bacterial pathogens causing cervical discharge using culture and/or polymerase chain reaction and assess the clinical and laboratory response to the conventional syndromic kit regimen established by the World Health Organisation. Methods A retrospective review of records of women with cervical discharge over one year period. Culture and/or polymerase chain reaction results of endocervical swabs of various bacterial pathogens at baseline and after four weeks of treatment with syndromic kit regimen were recorded. Results A total of 70 case records were reviewed for clinical details, out of which results of bacterial culture and polymerase chain reaction were available for 67 cases. Infectious aetiology was found in 30 (44.7%) patients with Ureaplasma species being the most common organism isolated on culture (18, 26.8%) and polymerase chain reaction (25, 37.3%), respectively. Polymerase chain reaction for Chlamydia trachomatis and Mycoplasma hominis was positive in ten (14.9%) and four (6%) cases, respectively. None of the patients showed positive culture for Neisseria gonorrhoeae. Coinfection was seen in eight (11.9%) patients with the majority showing Chlamydia trachomatis and Ureaplasma spp. coinfection (five patients). Forty one cases (58.5%) received tab. cefixime 400 mg and tab. azithromycin one gram stat (kit 1), while 29 cases (43.3%) received tab. cefixime 400 mg stat, tab. metronidazole 400 mg and cap. doxycycline 100 mg, both twice daily for 14 days (kit 6). Minimal to no clinical improvement with treatment was seen in 14 out of 32 cases (44%) at the end of four weeks with the conventional kit regimen. Post-treatment culture and/or polymerase chain reaction were positive in nine out of 28 cases (32.1%) with Ureaplasma spp. being the most common. Limitations Retrospective study design, small sample size and fewer cases with follow-up data were the main limitations. Conclusion Ureaplasma spp. was the most common infectious cause of cervical discharge in our patients. Treatment given as part of syndromic management led to a clinical and microbiological response in around half and two-third cases, respectively.

The efficacy of azithromycin in pityriasis rosea: a randomized, double-blind, placebo-controlled trial.

BACKGROUND: Macrolides are prescribed in the treatment of pityriasis rosea despite conflicting results of the limited number of studies evaluating their role in its treatment. AIM: A randomized double-blind placebo-controlled trial was conducted to evaluate the effect of azithromycin on the clinical course of pityriasis rosea. METHODS: Seventy patients of pityriasis rosea were given either azithromycin (n=35) or placebo (n=35) and were followed-up at 2, 4 and 6 weeks. Pruritus was assessed in both groups using the visual analogue scale (VAS) . Change in the pityriasis rosea severity score (PRSS) and in the VAS were recorded as outcome measures and were compared statistically. RESULTS: The decrease in PRSS from baseline through 2, 4 and 6 weeks within both treatment (P<0.001) and placebo (P<0.001) arms was found to be statistically significant; however, this change was not significantly different in the two groups (P=0.179). Similarly, the decrease in VAS was found to be statistically significant within both groups (P<0.001); however, the change was comparable between the two groups (P<0.937). Analysis by Fisher's exact test did not find a significant difference between the two groups for PRSS and VAS. CONCLUSION: Azithromycin is not effective in pityriasis rosea and the use of macrolides for this disease should not be encouraged in clinical practice.

The antibiotic resistance arrow of time: efflux pump induction is a general first step in the evolution of mycobacterial drug resistance.

We hypothesize that low-level efflux pump expression is the first step in the development of high-level drug resistance in mycobacteria. We performed 28-day azithromycin dose-effect and dose-scheduling studies in our hollow-fiber model of disseminated Mycobacterium avium-M. intracellulare complex. Both microbial kill and resistance emergence were most closely linked to the within-macrophage area under the concentration-time curve (AUC)/MIC ratio. Quantitative PCR revealed that subtherapeutic azithromycin exposures over 3 days led to a 56-fold increase in expression of MAV_3306, which encodes a putative ABC transporter, and MAV_1406, which encodes a putative major facilitator superfamily pump, in M. avium. By day 7, a subpopulation of M. avium with low-level resistance was encountered and exhibited the classic inverted U curve versus AUC/MIC ratios. The resistance was abolished by an efflux pump inhibitor. While the maximal microbial kill started to decrease after day 7, a population with high-level azithromycin resistance appeared at day 28. This resistance could not be reversed by efflux pump inhibitors. Orthologs of pumps encoded by MAV_3306 and MAV_1406 were identified in Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium marinum, Mycobacterium abscessus, and Mycobacterium ulcerans. All had highly conserved protein secondary structures. We propose that induction of several efflux pumps is the first step in a general pathway to drug resistance that eventually leads to high-level chromosomal-mutation-related resistance in mycobacteria as ordered events in an "antibiotic resistance arrow of time."

ICAAC update on opportunistic infections.

AIDS: Recent research on the effects of opportunistic infections (OIs) on HIV replication suggests that the OIs can actually increase HIV replication through the production of cytokines. Treatment options for thrush include antifungal treatments for early episodes and reserving fluconazole for patients with difficult to treat infections. Other studies show that prevention and treatment of Mycobacterium avium complex (MAC) increases survival rates. Drugs and dosages are described.^ieng

Azithromycin and clarithromycin.

Azithromycin and clarithromycin are alternatives to conventional macrolides in the routine treatment of many dermatologic, upper respiratory, and lower respiratory tract infections. In this role as alternative therapy, they are better tolerated, less toxic, and more convenient to take, although at a greater cost to the patient. This dosing convenience is an important consideration for the clinician; as shown by Nelson, patient compliance ranges from 95% with once-daily dosing to 58% with four-times-a-day dosing. Thus, less frequent dosing with both drugs as well as the shorter course of therapy possible with azithromycin may be therapeutically advantageous. In addition to their role as alternatives to conventional macrolide therapy, azithromycin and clarithromycin extend the spectrum of macrolides and offer new therapeutic options for H. influenzae, MAC in AIDS, MOTT, and leprosy. Finally, experimental therapy may extend their use for additional opportunistic infections, such as toxoplasmosis and cryptosporidiosis.

Azithromycin and clarithromycin: overview and comparison with erythromycin.

Azithromycin and clarithromycin are erythromycin analogues that have recently been approved by the FDA. These drugs inhibit protein synthesis in susceptible organisms by binding to the 50S ribosomal subunit. Alteration in this binding site confers simultaneous resistance to all macrolide antibiotics. Clarithromycin is several-fold more active in vitro than erythromycin against gram-positive organisms, while azithromycin is 2- to 4-fold less potent. Azithromycin has excellent in vitro activity against H influenzae (MIC90 0.5 microgram/ml), whereas clarithromycin, although less active against H influenzae (MIC90 4.0 micrograms/ml) by standard in vitro testing, is metabolized into an active compound with twice the in vitro activity of the parent drug. Both azithromycin and clarithromycin are equivalent to standard oral therapies against respiratory tract and soft tissue infections caused by susceptible organisms, including S aureus, S pneumoniae, S pyogenes, H influenzae, and M catarrhalis. Clarithromycin is more active in vitro against the atypical respiratory pathogens (e.g., Legionella), although insufficient in vivo data are available to demonstrate a clinical difference between azithromycin and clarithromycin. Superior pharmacodynamic properties separate the new macrolides from the prototype, erythromycin. Azithromycin has a large volume of distribution, and, although serum concentrations remain low, it concentrates readily within tissues, demonstrating a tissue half-life of approximately three days. These properties allow novel dosing schemes for azithromycin, because a five-day course will provide therapeutic tissue concentrations for at least ten days. Clarithromycin has a longer serum half-life and better tissue penetration than erythromycin, allowing twice-a-day dosing for most common infections. Azithromycin pharmacokinetics permit a five-day, single daily dose regimen for respiratory tract and soft tissue infections, and a single 1 g dose of azithromycin effectively treats C trachomatis genital infections; these more convenient dosing schedules improve patient compliance. Azithromycin and clarithromycin also are active against some unexpected pathogens (e.g., B burgdorferi, T gondii, M avium complex, and M leprae). Clarithromycin, thus far, appears the most active against atypical mycobacteria, giving new hope to what has become a difficult group of infections to treat. Gastrointestinal distress, a well known and major obstacle to patient compliance with erythromycin, is relatively uncommon with the new macrolides. Further clinical data and experiences may better define and expand the role of these new macrolides in the treatment of infectious diseases.

Activities of sparfloxacin, azithromycin, temafloxacin, and rifapentine compared with that of clarithromycin against multiplication of Mycobacterium avium complex within human macrophages.

The activities of sparfloxacin, azithromycin, temafloxacin, and rifapentine against two virulent strains of the Mycobacterium avium complex isolated from patients with AIDS were evaluated in a model of intracellular infection and were compared with that of clarithromycin. Human monocyte-derived macrophages were infected with the M. avium complex at day 6 of culture. The intracellular CFU was counted 60 min after inoculation. The intracellular and supernatant CFU was counted on days 4 and 7 after inoculation. The concentrations used, which were equal to peak levels in serum, were 10 micrograms of rifapentine per ml (MICs for the two strains, 4 and 16 micrograms/ml), 4 micrograms of clarithromycin per ml (MICs, 8 and 4 micrograms/ml), 1 microgram of azithromycin per ml (MICs, 32 and 16 micrograms/ml), 4 micrograms of temafloxacin per ml (MICs, 2 and 16 micrograms/ml), and 1 microgram of sparfloxacin per ml (MICs, 0.5 and 2 micrograms/ml). Compared with controls on day 7 after inoculation, clarithromycin (P less than 0.001), sparfloxacin (P less than 0.001), and azithromycin (P less than 0.001 for the first strain, P less than 0.02 for the second) slowed intracellular replication. Rifapentine (P less than 0.001) and temafloxacin (P less than 0.001) slowed intracellular replication of the first strain but not of the second strain. Azithromycin plus sparfloxacin was as effective as sparfloxacin alone. In this macrophage model, sparfloxacin or clarithromycin (difference not significant) exhibited a better efficacy than rifapentine, azithromycin, or temafloxacin against intracellular M. avium complex infection.

Activities of various macrolide antibiotics against Mycobacterium leprae infection in mice.

We evaluated the activities of several macrolide antibiotics against M. leprae infections in mouse footpads. Erythromycin and azithromycin were inactive, while both roxithromycin and clarithromycin were found to be consistently active and, in fact, bactericidal. By both methods, clarithromycin was found to be superior to roxithromycin, a finding which, at least in part, may be a consequence of the higher levels of clarithromycin at the site of infection.