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.

Vacuum sealing drainage: A novel treatment method for primary cutaneous Mycobacterium intracellulare infection.

The incidence of primary cutaneous Mycobacterium intracellulare infection is very low. We report a case of primary cutaneous M. intracellulare infection which presented as painful erythematous swelling of the right upper limb without systemic involvement. A novel technique of vacuum sealing drainage was successfully implemented after antimycobacterial treatment proved ineffective at the end of 3 months. Our technique also revealed some additional practical advantages.

Nontuberculous mycobacterial empyema in an immunocompetent child.

Nontuberculous mycobacterium (NTM) species are mycobacterial species other than those belonging to the Mycobacterium Tuberculosis complex and Mycobacterium leprae. There are very few reports of NTM in immunocompetent children causing empyema. In this article, we report a 9-year-old immunocompetent girl who presented with Mycobacterium avium-intracellulare empyema.

[Nontuberculous mycobacterial infections of the lung]. / Atypische Mykobakteriose der Lunge.

Nontuberculous mycobacterium (NTM) species are mycobacterial species other than those belonging to the Mycobacterium tuberculosis complex and M. leprae. NTM are generally free-living organisms that are ubiquitous in the environment. Pulmonary disease, especially in older persons with and without underlying lung disease, is caused primarily by M. avium complex (MAC) and M. kansasii. The symptoms and signs of MAC lung disease are variable and not specific, but include cough, malaise, weakness, dyspnoea, chest discomfort and occasionally hemoptoe. Two major clinical presentations include disease in those with underlying lung disease, primarily white, middle-aged or elderly men - often alcoholics and/or smokers with underlying chronic obstructive lung disease, patients in whom MAC develops in areas of prior bronchiectasis, and patients with cystic fibrosis; and those without known underlying lung disease, including non-smoking women over age 50 who have interstitial patterns on chest radiography. M. kansasii infections are endemic in cities with infected tap water. Symptoms of the M. kansasii lung disease resemble to tuberculosis. M. abszessus is the most pathogenic rapid growing Mycobacterium which causes pulmonary infection. The American Thoracic Society and Infectious Disease Society of America's diagnostic criteria for nontuberculous mycobacterial pulmonary infections include both imaging studies consistent with pulmonary disease and recurrent isolation of mycobacteria from sputum or isolated from at least one bronchial wash in a symptomatic patient. For treatment of MAC lung disease we recommend depending on severity and susceptibility testing a three to four drug treatment with a macrolide, rifampicin and ethambutol and for M. kansasii a treatment with Isoniazid, rifampicin and ethambutol. Surgical management only plays a role in rare and special cases. Treatment should be continued until sputum cultures are consecutively negative for at least one year.

Misidentification of Mycobacterium leprae as Mycobacterium intracellulare by the COBAS AMPLICOR M. intracellulare test.

Commercially available nucleic acid probe- and amplification-based systems for detection and differentiation of mycobacteria are widely used in clinical microbiology laboratories. Here we report two cases of human leprosy in which the COBAS AMPLICOR Mycobacterium intracellulare test led to false- positive results. Correct identification of Mycobacterium leprae was possible only by amplification and comparative sequence analysis of the 16S rRNA gene.

Cutaneous Mycobacterium avium intracellulare infection in an HIV+ patient mimicking histoid leprosy.

Cutaneous infections with Mycobacterium avium intracellulare (MAI) are uncommon in healthy patients but may arise in those with underlying immunocompromise, including patients with HIV. Their clinical manifestations are protean. We report an AIDS patient with a cutaneous MAI infection that clinically and histopathologically mimicked histoid leprosy, a presentation not previously described in this population.

[Diagnosis, therapy and prognosis of atypical mycobacterial infections--results of a retrospective study]. / Diagnostik, Therapie und Prognose atypischer Mykobakteriosen--Ergebnisse einer retrospektiven Studie.

The most important mycobacteria causing disease in humans are Mycobacterium tuberculosis and Mycobacterium leprae. These germs contrast the so-called atypical mycobacteria. The importance of the atypical mycobacteria was recognized in the fifties. Even if the quantity of atypical mycobacterial disease has increased during the last decades in Germany, it is still a rare disease today, but it is seen in patients with acquired immunodeficiency syndrome more often nowadays. In the period from 1st January 1986 til 31st December 1992 31 HIV-negative patients with a diagnosis of atypical mycobacterial disease have been seen in the department for lung diseases in the Thoraxklinik Heidelberg-Rohrbach. During the same period an atypical mycobacterial disease was diagnosed in 12 out of 413 HIV-positive patients (2.9%) of the AIDS-ambulance of the skin hospital of the University of Heidelberg. Most of the HIV-negative patients showed additional diseases which reduce the immunological resistance. In HIV-positive patients an atypical mycobacteriosis heralds a severe immunodeficiency. Because it is a rare disease and an exact diagnosis is difficult to establish there is a lack of controlled clinical trials and therefore detailed therapeutical guidelines do not exist. A therapeutical approach is also complicated by a lack of effective drugs. With disseminated disease in AIDS-patients, which is mostly caused by Mycobacterium avium-intracellulare, the therapy should be stopped, if there are any severe side-effects. The present results of therapy are still disappointing. In the future the management of atypical mycobacterial disease will be more important, because there is an increasing number of patients with acquired immunodeficiency.

Spindle cell pseudotumor due to Mycobacterium avium-intracellulare in patients with acquired immunodeficiency syndrome (AIDS). Positive staining of mycobacteria for cytoskeleton filaments.

A rare spindle-cell pseudotumor caused by Mycobacterium avium-intracellulare (MAI) that mimics a mesenchymal tumor, was recently reported (7,14). We report on three such pseudotumors in patients with the acquired immunodeficiency syndrome (AIDS), two involving lymph nodes and one involving the bone marrow. In the course of investigating the first-encountered example of this tumor for evidence of smooth-muscle origin of the spindle cells, it was noted that these cells stained positively for desmin by immunoperoxidase techniques (IPX), as did a variety of other cytoskeleton filaments of all sizes. Electron microscopic examination of one of these lesions revealed spindle cells containing lysosomes and large numbers of microorganisms compatible with MAI but no filaments or organelles suggestive of smooth-muscle cells. Further studies revealed that the typical lesions produced by MAI in patients with AIDS, namely aggregates of histiocytes or individual histiocytes laden with organisms, rather than the expansile spindle-cell pseudotumor, also strain strongly for cytoskeleton filaments, as do M. tuberculosis and Mycobacterium leprae. Awareness of the existence of this unusual manifestation of MAI infection in AIDS patients and its desmin positivity can avoid misdiagnosis of a primary or metastatic smooth-muscle neoplasm. The cell of origin appears to be the histiocyte.

Rapid and precise diagnosis of atypical mycobacterial infection by chemotaxonomical and immunological methods.

The species of 205 strains of acid fast bacteria isolated from swine and human mycobacteriosis were identified chemotaxonomically and numericaltaxonomically. The species of the isolates which were identified numericaltaxonomically as Mycobacterium avium intracellulare (MAI) complex were further classified by using both thin-layer chromatography of the antigenic glycopeptidolipids (GPL) from the bacteria and seroagglutination test devised by Schaefer. These MAI complex from swine fell into serotype 8 (45 strains), serotype 4 (32 strains), serotype 9 (9 strains) and untypable (9 strains), respectively. In contrast to swine, human isolates covered more wide ranges of serotypes such as serovar 7, 12, 16 besides serovar 4, 8 and 9. Furthermore, enzyme-linked immunosorbent assay (ELISA) which is based on the type specific glycolipid antigen and infected swine/human sera was applied to distinguish serological variants of the MAI complex. Of the fourteen cases in swine and five in human that had been typed by both the seroagglutination reaction and the thin layer chromatography (TLC) the thirteen in swine and two in human cases showed clear coincidence with the results of ELISA. The results demonstrated that enzyme-linked immunosorbent assay using infected sera was especially useful, and it was recommended from the sensitivity and rapidity as an adjunct to seroagglutination test and thin layer chromatography for the identification of serotypes of MAI complex.