Evaluation of the Idaho Technology LightCycler PCR for the direct detection of Mycobacterium tuberculosis in respiratory specimens.

SETTING: The rapid detection of Mycobacterium tuberculosis (TB) in clinical samples is an important goal. The LightCycler heralds an advance in thermal cycle technology combining rapid cycle DNA amplification with fluorimetry, eliminating the need to perform amplification and product analysis separately. OBJECTIVES: To evaluate the LightCycler for direct detection of M. tuberculosis complex in respiratory specimens. To evaluate a DNA extraction method based on Chelex 100 resin, heating and ultrasonication for the prevention of endogenous inhibitions in respiratory samples. DESIGN: DNA was extracted from sputum samples using the Chelex method and polymerase chain reaction (PCR) for TB performed with the LightCycler. RESULTS: For 88 sputum samples positive by microscopy and culture for M. tuberculosis, 95% were PCR-positive. None of the five sputum samples that were smear-negative but culture-positive for M. tuberculosis, the 79 culture-negative sputum samples and the 29 sputum samples that were culture-positive for mycobacteria other than TB yielded positive PCR results. PCR inhibitors were not detected in any of the samples. CONCLUSION: The LightCycler proved a simple, reproducible and rapid system, reducing the time to result from weeks (culture) or days (conventional PCR) to hours. The Chelex 100 resin method produced good results for the smear-positive specimens. However, a larger study is required to determine the efficacy of the method with smear-negative specimens and for specimens known to contain endogenous inhibitors.

The relationship between the in vitro drug susceptibility of opportunist mycobacteria and their in vivo response to treatment.

SETTING: It is generally accepted that qualitative drug susceptibility tests established and validated for Mycobacterium tuberculosis are not applicable to opportunist (non-tuberculous) mycobacteria. Previous studies have shown that in vitro antimicrobial susceptibilities for opportunist mycobacteria, performed by the method of modal resistance (MR), correlate poorly with clinical response. Minimum inhibitory concentration (MIC) determination may provide better correlation with predicted clinical response than the conventional MR results. OBJECTIVE: To determine the relationship between quantitative in vitro sensitivity results for opportunist mycobacteria and their in vivo response to treatment. DESIGN: MICs were performed radiometrically with the Bactec TB-460 system; 35 M. avium complex isolates, 29 isolates of M. malmoense and 16 isolates of M. xenopi were tested. RESULTS: Susceptibility results were analysed in comparison with therapeutic outcome by Fisher's exact probability test. Only one significant association was found; in vitro resistance to ethambutol correlated with treatment failure for M. malmoense infections (P = 0.027). There were no other significant correlations between in vitro results and treatment outcome. CONCLUSION: Prediction of treatment outcome from in vitro susceptibility tests continues to be a problem in infections with opportunist mycobacteria.

Susceptibilities of Mycobacterium malmoense determined at the growth optimum pH (pH 6.0).

SETTING: Pulmonary disease caused by Mycobacterium malmoense is increasing. Conventional in vitro antimicrobial susceptibilities correlate poorly with response to treatment for this organism. Radiometrically determined minimum inhibitory concentrations (MICs) allow quantitative susceptibility testing for non-tuberculous mycobacteria. The M. avium complex (MAC) has been investigated extensively with this approach, and clear interpretative criteria have been established at pH 6.8. However, there has been little work with the acidophilic M. malmoense, which grows poorly at pH 6.8. OBJECTIVE: To determine whether MICs at pH 6.0 provide results compatible with the interpretative criteria established for the MAC. DESIGN: MICs were performed in Middlebrook PZA medium (pH 6.0) and 7H12 medium (pH 6.8) for ten strains of M. malmoense. RESULTS: MICs can be determined at pH 6.0 for M. malmoense using the criteria adopted for the M. avium complex. CONCLUSION: The low optimal pH of M. malmoense suits this organism for growth in acid conditions. As with MAC, M. malmoense multiplies within macrophages in vivo, and MICs determined at pH 6.0 may reflect in vivo activity. The combination of radiometric MIC testing at optimal growth pH and interpretation based on pharmacokinetic parameters may be helpful in designing therapeutic regimens.

Epidemiological typing of Klebsiella pneumoniae by pyrolysis mass spectrometry.

Thirteen isolates of ceftazidime-resistant Klebsiella pneumoniae from a suspected cross-infection outbreak involving patients on an intensive care unit and a haematology ward were examined in pyrolysis-mass spectrometry (Py-MS), along with eight concurrent non-outbreak-associated clinical isolates of klebsiellae as controls. Py-MS showed tight clustering of the suspected outbreak isolates, suggesting cross-infection with a single strain. Non-outbreak isolates were clearly distinct from one another and from the outbreak strain. The results confirm that Py-MS is a powerful tool for rapid strain comparison in investigations of cross-infection incidents.

Pyrolysis mass spectrometry: a predictor of clinical response to treatment in pulmonary opportunist mycobacterial infection: preliminary work with M. malmoense.

Pyrolysis mass spectrometry (Py-MS) yields data reflecting overall cell composition. The changes in composition induced by treatment with rifampicin and ethambutol, alone and in combination, were investigated for a collection of seven strains of Mycobacterium malmoense from pulmonary infections. Two strains, both from patients that had responded to therapy with this combination, showed large changes in composition from control, untreated cultures. The difference was particularly marked for the ethambutol treated cultures. Four strains, all from patients who had failed to respond to therapy with this combination, showed minimal changes in composition for all treatments. The remaining strain also showed minimal treatment-induced change, but, for this patient, therapy with the combination had proved successful. Minimum inhibitory concentrations (MICs) were determined radiometrically. All strains showed MICs < 0.5 microgram/mL for rifampicin (sensitive) and of 8 micrograms/mL for ethambutol (resistant). MIC results did not correlate with clinical response, whereas the Py-MS results correlated with clinical response for six of the seven isolates. Py-MS may have a role in predicting effective therapy for this problem group.

A Mycobacterium malmoense-specific DNA probe from the 16S/23S rRNA intergenic spacer region.

Mycobacterium malmoense was first described in 1977. It is now recognized as an opportunistic human pathogen which can be difficult to identify using standard methods. M. malmoense may be underestimated as the causative agent of clinical disease because of the recognized difficulties in its primary cultivation and identification. In this study, the nucleotide sequence of the 16S/23S rRNA intergenic spacer region from five clinical isolates of M. malmoense has been determined, in order to develop a PCR-based DNA probe assay to facilitate the early identification of this organism. The DNA sequence generated was utilized to design an oligunucleotide probe that specifically hybridizes with M. malmoense. The ability of this DNA probe to detect geographically distinct M. malmoense isolates was investigated. The value of this DNA probe was realized by its ability to differentiate three isolates of the Mycobacterium avium complex, which had been misidentified as M. malmoense using conventional biochemical methods.

Comparison of solid media for cultivation of anaerobes.

Two commercial agar media for the cultivation of anaerobes were compared with four other media for their ability to support the growth of a wide range of anaerobes from clinical specimens of subgingival plaque. Fastidious anaerobe agar (FAA, Lab M) and anaerobe agar (GAA, Gibco) allowed better growth of the pure cultures than the other media. FAA recovered the highest numbers of bacteria from subgingival plaque specimens which were composed predominantly of anaerobes. GAA performed poorly with these samples. It is concluded that FAA seemed to be superior to the other media tested for the cultivation and recovery of anaerobes.