Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-11-28
2003-10-14
Siew, Jeffrey (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S091520, C536S022100, C536S024300, C536S024330
Reexamination Certificate
active
06632607
ABSTRACT:
The present invention relates to the field of drug-resistant mycobacteria.
The present invention relates to probes, primers, methods and kits comprising the same for the detection of mycobacterial nucleic acids in biological samples.
Identification of most clinically relevant Mycobacterium species, in particular of Mycobacterium tuberculosis is tedious and time consuming due to culture-procedures which can take up to 6 weeks. Rapid diagnosis of Mycobacterium infection is very important since the disease might be life-threatening and highly contagious. Only recently some methods—all making use of one or another amplification process—have been developed to detect and identify Mycobacterium species without the need for culture (Claridge et al., 1993). Most of these methods are still in evaluation and their benefit in routine applications remains questionable. Moreover, these methods do not solve the problem of Mycobacterium drug-resistance detection which still relies on culture.
Since the frequency of multidrug resistance in tuberculosis is steadily increasing (Culliton, 1992), it is clear now that early diagnosis of
M. tuberculosis
and the rapid recognition of resistance to the major tuberculosis are essential for therapy and an optimal control of the resurgent epidemic.
The antibiotics used for treatment of
M. tuberculosis
infections are mainly isoniazid and rifampicin either administered separately or as a combination of both. Occasionally, pyrazinamide, ethambutol and streptomycin are used: other classes of antibiotics like (fluoro)quinolones may become the preferred tuberculostatics in the future.
Since most multidrug resistant mycobacteria also lost susceptibility to rifampicin, rifampicin-resistance is considered to be a potential marker for multidrug resistant tuberculosis. For this reason, the detection of resistance to rifampicin might be of particular relevance.
For the majority of the
M. tuberculosis
strains examined so far, the mechanism responsible for resistance to rifampicin (and analogues like rifabutin) has been elucidated. Rifampicin (and analogues) block the RNA polymerase by interacting with the &bgr;-subunit of this enzyme. Telenti et al. (1993a) found that mutations in a limited region of the &bgr;-subunit of the RNA polymerase of
M. tuberculosis
give rise to insensitivity of the RNA polymerase for rifampicin action. This region is limited to stretch of 23 codons in the
rpo
B gene. The authors describe 17 amino acid changes provoking resistance to rifampicin (Telenti et al., 1993b). These amino acid changes are caused by point mutations or deletions at 15 nucleotides or 8 amino acid codons respectively scattered over a stretch of 67 nucleotides or 23 amino acid codons.
Telenti et al. (1993a and b) described a PCR-SSCP method to screen for the relevant mutations responsible for rifampicin resistance (SSCP refers to single-strand conformation polymorphism). SSCP analysis can be performed either by using radio-activity or by using fluorescent markers. In the latter case sophisticated and expensive equipment (an automated DNA-sequencing apparatus) is needed. The SSCP approach described has also other limitations with respect to specificity and sensitivity which might impede its routine use. Specimen can only be adequately analysed directly if a significant load of bacteria (microscopy score: >90 organisms/field) is observed microscopically and on crude DNA samples strand-separation artifacts may be observed which complicate the interpretation of the results.
Kapur et al. (1994) describe 23 distinct
rpo
B alleles associated with rifampicin resistance. In addition to the mutations described by Telenti et al. (1993a), some new mutant
rpo
B alleles are described. However, the most frequently occurring alleles remain the same as those described before.
In
M. leprae,
the molecular basis for rifampicin resistance was described by Honoré and Cole (1993). Here too, resistance stemmed from mutations in the
rpo
B gene, which encodes the beta subunit of RNA polymerase of
M. leprae.
Only a limited number of resistant
M. leprae
strains (9) were analysed, and in most of them (8/9) resistance was due to a mutation affecting the Ser-425 residue.
Clinically important mycobacteria other than
M. tuberculosis
and
M. leprae
often show an innate, be it variable, resistance to rifampicin. This is the cause for
M. avium
and
M. intracellulare,
human pathogens for which only limited treatment options are available. Guerrero et al. (1994) compared the
rpo
B-gene sequences of different
M. avium
and
M. intracellulare
isolates with that of
M. tuberculosis.
Differences are present at the nucleotide level but a full amino acid identify was found with rifampicin-sensitive
M. tuberculosis.
These findings suggest that another mechanism of resistance, possibly a permeability barrier, applies for
M. avium
and
M. intracellulare.
The specific detection of point mutations or small deletions can elegantly be approached using hybridization procedures such as the reverse hybridization assay. However, the complexity observed in the relevant part of the
rpo
B gene does not allow a straightforward probe development. As will be exemplified further, it was one of the objects of the present invention to design a specific approach allowing the detection of most if not all mutations found so far in a fast and convenient way without the need for sophisticated equipment.
The mechanism of resistance to isoniazid (INH) is considerably more complex than that for rifampicin. At least two gene products are involved in INH-resistance. First, there is catalase-peroxidase which is believed to convert INH to an activated molecule. Hence, strains which do not produce catalase-peroxidase by virtue of a defective or deleted
kat
G gene are not anymore susceptible to INH (Zhang et al., 1992; Stoeckle et al., 1993). In this context it should be mentioned that the association between INH-resistance and the loss of catalase activity was already noted in the fifties (Middlebrook, 1954 a and b; Youatt, 1969).
The second molecule involved is the
inh
A gene product, which is believed to play a role in the mycolic acid biosynthesis. It is postulated that the activated INH molecule interacts either directly or indirectly with this product and probably prevents proper mycolic acid biosynthesis. This hypothesis is based on the recent observation that overexpression of the wild type
inh
A gene or a particular amino acid change (S94A) in the
inh
A gene product confers resistance to INH (Banerjee et al., 1994).
In short, and somewhat simplified we can state that in certain
M. tuberculosis
strains resistance to INH might be mediated by:
the loss of catalase-peroxidase activity
the presence of certain amino acid changes in the
inh
A protein
the expression level of the wild type
inh
A protein
Also, other mechanisms might be involved in conferring resistance to INH and related drugs. The importance of these factors in the total spectrum of INH-resistance mechanisms has yet to be assessed. This issue can be addressed by means of DNA probe techniques if reliable DNA probes can be developed from the available DNA-sequences of the
kat
G gene (EMBL n° X68081) and
inh
A gene (EMBL n° U02492) of
M. tuberculosis.
These probe-tests could then also be applied for detection of drug resistance in biological samples.
For the detection of resistance to streptomycine and (fluoro)quinolones the same approach as for rifampicin can be followed. Resistance to these antibiotics is also induced by point mutations in a limited region of one or more genes. Point mutations in the gyrase gene confer resistance to (fluoro)quinolones (EMBL n° L27512). Streptomycin resistance is correlated with mutations in either the 16S rRNA gene or the gene of a ribosomal protein S12 (
rps
L) (Finken et al., 1993: Douglas and Steyn, 1993; Nair et al., 1993).
Resistance due to nucleotide changes in the
kat
G,
rpo
B and
rps
L genes have been described in international application WO 93;22454. For each of the different genes in
M
De Beenhouwer Hans
Jannes Geert
Machtelinckx Lieve
Portaels Françoise
Rossau Rudi
Innogenetics N.V.
Siew Jeffrey
Sterne Kessler Goldstein & Fox P.L.L.C.
Tung Joyce
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