Use of NASBA RNA amplification for detection of Mycobacterium leprae in skin biopsies from untreated and treated leprosy patients.

This study was performed to assess the value of NASBA RNA amplification of a 16S rRNA target for the detection of presumably viable Mycobacterium leprae in sections of skin biopsies from leprosy patients. The NASBA positivity rate was 90.4% (84/93) for untreated multibacillary (MB) patients [bacterial index (BI) > or = 2] and 16.7% (8/48) for the untreated paucibacillary (PB) patients (BI < 2). NASBA positivity showed a good concordance with the presence of solidly stained M. leprae [morphological index (MI)] in skin biopsies from leprosy patients, but no relationship could be demonstrated between the strength of the NASBA signals and the BI. Furthermore, the usefulness of the detection of 16S rRNA by NASBA to monitor the efficacy of leprosy treatment was investigated using an additional 154 biopsy specimens analyzed from 80 MB patients during the course of treatment. The NASBA positivity rate declined during treatment. A significant decrease was observed after only 1-3 months. These results favor the view that detection of RNA by NASBA may reflect the viability of M. leprae.

Colorimetric microwell plate hybridization assay for detection of amplified Mycobacterium tuberculosis DNA from sputum samples.

We developed a colorimetric microwell plate hybridization assay (CoMPHA) for the specific detection of 5'-biotinylated amplified Mycobacterium tuberculosis DNA. The optical densities of the CoMPHA corresponded to the initial amounts of purified template DNA. Here, we show that the CoMPHA is useful in distinguishing the PCR-positive and PCR-negative samples.

Assessment of mycobacterial viability by RNA amplification.

We investigated whether the presence of intact RNA is a valuable indicator of viability of mycobacteria with Mycobacterium smegmatis. M. smegmatis was exposed to various concentrations of rifampin and ofloxacin suspended in broth for different periods of time. The NASBA nucleic acid amplification system was used because of its rapid, sensitive, and specific detection of 16S rRNA. During drug exposure, the viability of the mycobacteria, expressed by the number of CFU, was compared with the presence of 16S rRNA as determined by NASBA and with the presence of DNA coding for 16S rRNA as determined by PCR. Both NASBA and PCR were shown to have a detection limit of approximately 5 x 10(2) CFU/ml. The intensity of the NASBA signal corresponded well with the number of CFU, and the lack of NASBA signal coincided with a loss of viability, which was reached after 3 days of exposure to bactericidal concentrations of both drugs. The presence of mycobacterial DNA, as determined by the intensity of the PCR signal, and the viability of M. smegmatis were not related, but an increase in the number of cells and intensity of PCR signal correlated well. Bacterial viability may thus be assessed by a rapid, sensitive, and specific, and semiquantitative technique by using NASBA. This system of viability testing provides the potential for rapid evaluation of drug susceptibility testing.

Nucleic acid sequence-based amplification (NASBA) for the identification of mycobacteria.

Nucleic acid sequence-based amplification (NASBA), an isothermal amplification technique for nucleic acids (NA), was investigated for the species-specific identification of mycobacteria. A set of primers was selected from a highly conserved region of the 16S rRNA sequence of mycobacteria sandwiching a variable sequence to perform amplification of mycobacterial RNA. Species-specific probes for the M. tuberculosis complex, M. avium-paratuberculosis, M. intracellulare and M. leprae were hybridized in-solution with the amplified nucleic acids of 10 pathogenic mycobacteria and 11 closely related bacteria, as well as with human-derived NA in an enzyme-linked gel assay (ELGA). Each probe was shown to hybridize specifically to the amplified single-stranded RNA of the corresponding species. Thirty-two clinical isolates of M. tuberculosis strains from different parts of the world were correctly identified by NASBA using the M. tuberculosis-complex-specific probe. In combination with the ELGA, NASBA could identify mycobacteria rapidly, i.e. in less than 6 h. The relative simplicity and rapidity of this technique makes it an attractive tool for species-specific identification of mycobacteria.

Simple colorimetric microtiter plate hybridization assay for detection of amplified Mycobacterium leprae DNA.

The detection of amplified products resulting from polymerase chain reactions (PCRs) remains a complicated process. To simplify the detection procedures, we developed a colorimetric microtiter plate hybridization assay for the specific detection of 5'-biotinylated PCR fragments of Mycobacterium leprae DNA. For this assay, an M. leprae DNA capture probe was made and immobilized on the wells of a microtiter plate. Hybridization of the biotin-labeled PCR fragments was detected through enzymatic color development. The resulting optical densities showed a logarithm-linear relationship with the amount of template DNA and corresponded to the intensity of the bands obtained through gel analysis and Southern blotting of the PCR products. The sensitivity of the assay was found to be 125 fg of genomic M. leprae DNA, or 20 lysed bacilli, revealing a detection limit similar to that of agarose gel analysis. The efficient coamplification of human DNA was used as a positive control for the presence of inhibitory substances in clinical material. For detection of human PCR products, a human DNA capture probe was also constructed for the colorimetric assay. This dual setup for hybridization, which thus detected both M. leprae and human DNA PCR products, was useful for ascertaining the presence of inhibiting substances in clinical specimens. All biopsy specimens (n = 10) from untreated patients with leprosy were positive. Apparently, this assay is more sensitive than microscopy, because biopsy specimens from half of the patients were negative upon histopathological examination. Biopsy specimens from three treated patients were negative, as were those from the three patients who did not have leprosy. We conclude that this colorimetric assay can replace agarose gel analysis and Southern hybridization, because it is as sensitive as those methods. Its advantages over conventional gel analysis and Southern hybridization are that it is less cumbersome and more rapid.

A simple colorimetric assay for detection of amplified Mycobacterium leprae DNA.

A colorimetric assay for the detection of PCR-products is described. The assay is based on amplification of DNA in the presence of digoxigenin-dUTP. After immobilization of the PCR products to a microtitre plate, amplified DNA could be detected colorimetrically. The sensitivity of this colorimetric assay was equal to gel-analysis allowing the detection of 100 fg of template DNA. Here, we show that it can be used to detect Mycobacterium leprae DNA in biopsy specimens from leprosy patients. The simplicity and the low degree of variation make this assay an alternative to gel-analysis.

The influence of excision repair on the distribution of N-propyl-N'-nitro-N-nitrosoguanidine-induced mutation in Escherichia coli.

To determine the influence of excision repair on the distribution of N-propyl-N'-nitro-N-nitrosoguanidine (PNNG)-induced mutation, we have analysed the DNA sequence changes of mutation induced at the lacI gene of Escherichia coli in a UvrB- strain. The mutagenic specificity was similar to that found in the wild-type strain. Base substitutions predominate; G:C----A:T transitions accounted for almost 75% of the mutational events, while G:C----T:A transversions constituted approximately 20% of the mutations. In both strains the distribution of G:C----A:T transitions was nonrandom, but the site specificity of the G:C----A:T transitions appear dissimilar, with the hot and cold sites occurring at different positions. A single - 1 frameshift was recovered in each strain but deletion events were absent in the excision-repair-deficient strain. The complexity of the sequence-specificity of PNNG might reflect the differential repair of n-propyl and iso-propyl adducts.

Mutational specificity of alkylating agents and the influence of DNA repair.

Alkylating treatments predominantly induce G: C = greater than A:T transitions, consistent with the predicted significance of the miscoding potential of the O6-alG lesion. However, the frequency and distribution of these events induced by any one compound may be diagnostic. SN1 agents that act via an alkyldiazonium cation, such as the N-nitroso compounds, preferentially generate G: C = greater than A:T transitions at 5'-RG-3' sites, while the more SN2 alkylsulfates and alkylalkane-sulfonates do not. The precise nature of this site bias and the possibility of strand bias are target dependent. The extent of this site bias and the contribution of other base substitutions are substituent size dependent. A similar 5'-RT-3' effect is seen for A:T = greater than G:C transitions, presumably directed by O4-alT lesions. The 5'-RG-3' effect, at least, likely reflects a deposition specificity arising from some aspect of helix geometry, although it may be further exaggerated by alkylation-specific repair. Excision repair appears to preferentially reduce the occurrence of ethylation-induced G:C = greater than A:T and A:T = greater than G:C transitions at sites flanked by A:T base pairs. This may be due to an enhancement of the helical distortion imposed by damage at such positions. A similar effect is not seen for methylation-induced mutations and in the case of propyl adducts, the influence of excision repair on the ultimate distribution of mutation cannot be as easily defined with respect to neighbouring sequence.

The complexity of nitrosoguanidine mutagenesis increases with size: observations of the mutational specificity of N-propyl-N'-nitro-N-nitrosoguanidine.

The mutational specificity of the monofunctional alkylating agent N-propyl-N'-nitro-N-nitrosoguanidine (PNNG) has been determined through the DNA sequence characterization of 109 LacI- mutations of Escherichia coli. The predominant mutation induced was the G:C----A:T transition (73%), presumably the result of O6-propylguanine damage. Transversions constituted 18% of the mutants, almost entirely due to G:C----T:A (9%) and A:T----C:G (8%) events. Two identical deletions, one single base pair frameshift and a tandem double base change were also recovered. In contrast, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was previously found to induce only transitions, again predominantly G:C----A:T events (98%). Moreover, the site specificity observed for PNNG-induced G:C----A:T transitions is quite distinct from that induced by MNNG. G:C----A:T transitions recovered following PNNG treatment do not appear to be influenced by neighbouring base sequence to the extent seen for MNNG.