Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-04-07
2004-06-22
Fredman, Jeffrey (Department: 1634)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S022100, C536S023100, C536S024310, C536S024320, C536S024330, C435S006120, C435S091200
Reexamination Certificate
active
06753421
ABSTRACT:
The present invention relates to novel probes and to mixtures of such probes, in addition to the design, construction and use of such novel probes or mixtures thereof for detecting a target sequence of one or more mycobacteria, which probes are capable of detecting such organism(s) optionally present in a test sample, e.g. sputum, laryngeal swabs, gastric lavage, bronchial washings, biopsies, aspirates, expectorates, body fluids (spinal, pleural, pericardial, synovial, blood, pus, bone marrow), urine, tissue sections as well as food samples, soil, air and water samples and cultures thereof. The invention relates in particular to novel probes and mixtures thereof for detecting the presence of one or more mycobacteria of the
Mycobacterium tuberculosis
Complex (MTC) and for detecting the presence of one or more mycobacteria other than mycobacteria of the
Mycobacterium tuberculosis
Complex (MOTT). The invention further relates to diagnostic kits comprising one or more of such probes. The probes of the present invention are surprisingly able to penetrate the cell wall of the mycobacteria, thus making possible the development of fast an easy-performed in situ protocols.
BACKGROUND OF THE INVENTION
Tuberculosis is a very life-threatening and highly epidemic disease which is caused by infection with
Mycobacterium tuberculosis.
Tuberculosis is presently the predominant infectious cause of morbidity and mortality world-wide, and is estimated to kill about three million people annually. WHO estimates that the annual number of new cases of tuberculosis will increase from 7.5 million in 1990 to 10.2 million in 2000, an escalation that will result in approximately 90 million new cases during this decade. It is furthermore estimated that 30 million people will die from tuberculosis during the 1990s, which equals one quarter of preventable deaths among adults.
The prevalence of tuberculosis has been very high in the poorer parts of the world such as Asia, Africa and South-America, but in recent years an increase has also been observed in industrialised countries. This appears to be due to an interaction of various factors including i.a. patterns of migration, poorly organised tuberculosis programmes and nutrition problems. Furthermore, a serious threat will arise from the emergence of new strains that are drug resistant or worse, multi-drug resistant.
Mycobacteria are often divided into
tuberculous
mycobacteria, i.e. mycobacteria of the
Mycobacterium tuberculosis
Complex (MTC), and non-tuberculous mycobacteria, i.e. mycobacteria other than those of the
Mycobacterium tuberculosis
Complex (MOTT). The MTC group comprises apart from
M. tuberculosis, M. bovis, M. africanum
and
M. microti.
Mycobacteria of the MOTT group are not normally pathogenic to healthy individuals but may cause disease in immunocompromised individuals, e.g. individuals infected with HIV. Clinical relevant mycobacteria of the MOTT group are in particular
M. avium, M. intracellulare, M. kansasii
and
M. gordonae,
but also
M. scrofulaceum, M. xenopi
and
M. fortuitum.
M. avium
and
M. intracellulare
together with
M. paratuberculosis
and
M. lepraemurium
constitute the
Mycobacterium avium
Complex (MAC). Extended with
M. scrofulaceum,
the group is named
Mycobacterium avium
-
intracellulare
-
scrofulaceum
Complex (MAIS).
It is well-known that treatment of mycobacterial infections with antibiotics may lead to the emergence of drug resistant strains. Many antibiotic drugs excert their effects by interfering with protein synthesis or with transcription. Studies of the molecular mechanisms underlying certain antibiotic resistance phenotypes in clinical mycobacterium isolates have revealed mutations in rRNA genes. The development of resistance because of mutation(s) located in the rRNA gene is likely to occur since slow-growing mycobacteria have only a single rRNA operon. All mycobacteria populations comprise a minority of drug resistant mutants that have arisen by spontaneous mutation. These mutated mycobacteria do normally not survive particularly well, but, when single-drug therapy is offered as treatment, the drug susceptible bacteria are killed, and only the resistant mutants will survive and multiply, and, thus at some point, constitute the majority of the mycobacteria population. The selection of drug resistant bacteria due to inadequate drug therapy leads to a state of so-called “acquired drug-resistance”. In contrast, “primary drug-resistance” is used to characterise a situation where drug-resistant mycobacteria can be isolated from a patient who has never been treated for mycobacterial infection, and has infected with drug-resistant mycobacteria from an individual suffering from infection with an acquired drug resistant bacterium.
Today, drug-resistance is determined primarily phenotypically by culturing clinical samples, in which presence of mycobacteria have been demonstrated, in the presence of the individual drugs. This is unfortunately a very slow and time-consuming procedure as the result of the drug-resistance studies depends on the growth rate of the mycobacteria, which are well-known to be slow. Thus, the result is not available until after several weeks.
Although the incidence of drug-resistance is, at least not yet, very common, it is nevertheless very important that resistant strains are identified and eradicated. Therefore, it is of major importance to find a reliable and rapidly performed method of diagnosing drug-resistance.
Presently, the detection of mycobacteria by microscopy is the most prevalent method for diagnosis. The sample (e.g. an expectorate) is stained for the presence of acid-fast bacilli using e.g. Ziehl-Neelsen staining. However, staining for acid-last bacilli does not provide the necessary information about the type of infection, only whether acid fast bacilli are present in the sample, and this is in itself not sufficient information for establishing a diagnosis. Samples positive for acid fast bacilli may subsequently be cultured in order to be able to perform species identification.
Since Ziehl-Neelsen staining cannot be used to determine whether the infection is caused by mycobacteria of the MTC group or mycobacteria other than mycobacteria of the MTC group, a positive staining frequently leads to very costly isolation of all the patients with suspected
M. tuberculosis
infection as well as treatment with medicaments to which the patient may not even respond.
Since the sensitivity of acid fast staining is only approximately 10
4
-10
5
per ml smear negative samples should also be cultured as culture-based tests are sensitive, and as it may be possible to detect 10-100 organisms per sample, but the result is not available before up to 8 weeks of culturing. Likewise, information about drug susceptibility is not available until after 1-3 weeks of further testing.
Different solid or liquid media (Loewenstein Jensen slants and Dubos broth) have traditionally been used for culturing of mycobacteria-containing samples. Newer media include ESP Myco Culture System (Difco), MB/BacT (Organon Teknika), BacTec (Becton Dickinson) and MGIT (Becton Dickinson). These test media are based on colourmetric or fluorometric detection of carbon dioxide or oxygen produced by mycobacterial metabolism, and adapted to automated systems for large scale testing.
Species identification is presently carried out following culturing using traditional biochemical methods or probe hybridisation assays (e.g. AccuProbe by Gen-Probe Inc, USA). There is, therefore, an increasing need for means allowing a more rapid distinction between mycobacteria of the MTC group and mycobacteria other than those of the MTC group, and for further species identification of those especially mycobacteria other than those of the MTC group.
A number of new attempts to replace the culture-based methods relies on molecular amplification technology. Several methods have emerged, among them the polymerase chain reaction (PCR), the ligase chain reaction and transcription mediated amplification. The basic principle of ampl
Lund Kaare
Mollerup Tina Anderson
Stender Henrik
Dako A/S
Fredman Jeffrey
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