Previous studies have indicated that the conventional tests used for the identification of mycobacteria may (i) frequently result in erroneous identification and (ii) underestimate the diversity within the genus Mycobacterium. To address this issue in a more systematic fashion, a study comparing phenotypic and molecular methods for the identification of mycobacteria was initiated. Focus was given to isolates which were difficult to identify to species level and which yielded inconclusive results by conventional tests performed under day-today routine laboratory conditions. Traditional methods included growth rate, colonial morphology, pigmentation, biochemical profiles, and gas-liquid chromatography of short-chain fatty acids. Molecular identification was done by PCR-mediated partial sequence analysis of the gene encoding the 16S rRNA. A total of 34 isolates was included in this study; 13 of the isolates corresponded to established species, and 21 isolates corresponded to previously uncharacterized taxa. For five isolates, phenotypic and molecular analyses gave identical results. For five isolates, minor discrepancies were present; four isolates remained unidentified after biochemical testing. For 20 isolates, major discrepancies between traditional and molecular typing methods were observed. Retrospective analysis of the data revealed that the discrepant results were without exception due to erroneous biochemical test results or interpretations. In particular, phenotypic identification schemes were compromised with regard to the recognition of previously undescribed taxa. We conclude that molecular typing by 16S rRNA sequence determination is not only more rapid (12 to 36 h versus 4 to 8 weeks) but also more accurate than traditional typing.
Mycobacterium genavense" is a proposed new species recently reported to cause disseminated infections in 18 patients with AIDS in Europe. We have recovered "M. genavense" as slowly growing fastidious mycobacteria in blood cultures of seven patients with AIDS. In the original studies of "M. genavense," the fastidious organism grew only in BACTEC 13A vials. The Seattle, Washington, isolates of "M. genavense" also failed to grow when subcultured from 13A vials to routine solid media, but dysgonic colonies were produced on Middlebrook 7H11 agar supplemented with mycobactin J. The mycolic acid pattern of patients' isolates closely resembled that of the type strain of Mycobacterium simiae when analyzed by one-and two-dimensional thin-layer chromatography and by high-performance liquid chromatography. Whole-cell fatty acid analyses by gas-liquid chromatography distinguished the isolates from M. simiae but misidentified them as Mycobacterium fortuitum. Sequence determinations of the hypervariable regions of the 16S rRNA gene indicate that these organisms belong to the recently proposed new species "M. genavense." Growth from Middlebrook 7H11 agar supplemented with mycobactin J consistently yielded positive tests for catalase (semiquantitative and at 68°C), pyrazinamidase, and urease which enable mycobacteriology laboratories to presumptively identify "M. genavense" without nucleic acid analyses. The failure of "M. genavense" to grow on conventional mycobacterial solid media suggests that mycobacterial blood cultures should include a broth medium incubated for at least 8 weeks.
Three children with human immunodeficiency virus infection and invasive infection with Mycobacterium genavense are reported. Fever spikes, abdominal cramps and distension, diarrhea or ileus, and anemia were the predominant symptoms in the severely immunodeficient patients (CD4 lymphocytes < 0.04 x 10(9)/l). Numerous acid-fast bacilli were readily detectable by microscopy in stool samples and in lymph node biopsies, but cultures for mycobacteria remained negative. Mycobacterium genavense should be sought when invasive non-tuberculous mycobacteriosis is suspected and mycobacterial cultures from blood or other sites show limited growth. Multiple-drug regimens including amikacin, ethambutol, rifampin, and clarithromycin may be of benefit in controlling the infection, as observed in two patients.
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