Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of...
Reexamination Certificate
2001-12-07
2003-12-16
Swartz, Rodney P (Department: 1645)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
C424S009100, C424S009200, C424S184100, C424S185100, C424S190100, C424S234100, C424S248100, C435S041000, C435S252100, C435S253100, C435S863000, C530S300000, C530S350000
Reexamination Certificate
active
06664096
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention generally relates to improved diagnosis and treatment of mycobacterial infections. In particular, the invention provides methods for using spent culture supernatant, components of the spent culture supernatant, or cell extracts to enhance cultivation of mycobacteria, or to revive dormant mycobacteria bacilli.
2. Background of the Invention
Tuberculosis (TB) is a leading infectious killer worldwide with 8 million new cases and 2 million deaths a year (WHO Report on the Tuberculosis Epidemic, 2000). One third of the world population is latently infected with
Mycobacterium tuberculosis.
The success of
M. tuberculosis
as a human pathogen relates to its remarkable ability to persist for long periods of time in the face of immunity and chemotherapy.
The current TB treatment is suboptimal, requiring a minimum of 6 months using the WHO recommended treatment regimen (DOTS, Directly Observed Treatment, Shortcourse), which consists of 4 drugs isoniazid, rifampin, pyrazinamide and ethambutol used in combination (WHO Report on the Tuberculosis Epidemic, 2000). Such lengthy treatments are expensive, and are likely to be problematic in terms of patient compliance. In addition, one potentially catastrophic effect of the lengthy therapy is the development of drug-resistant TB.
This lengthy treatment is thought to be due to the presence of a population of dormant bacilli in vivo that are not effectively killed by current TB drugs (McKinney et al., 1998). Dormant bacilli can be demonstrated in the Cornell mouse model of dormancy (McCune et al, 1966), where mice infected with tubercle bacilli were treated with INH and PZA for 2 months, at which time no viable bacilli were demonstrable in the tissues as judged by colony forming units (CFU); yet disease relapsed with viable yet drug susceptible bacilli after cessation of treatment for 3 months in one third of mice or in almost all mice given immunosuppressing steroids. This suggests that the drugs are unable to eliminate dormant bacilli completely and that although the dormant bacilli do not form colonies on plates they are not dead and can revive and cause disease when the immune system is compromised.
The unresponsiveness of dormant or nongrowing bacilli to DOTS is phenotypic or physiologic but not genetic, so that when the dormant bacilli revive and start growing they become susceptible to TB drugs again. Therefore, agents that cause dormant bacilli to revive and resuscitate so that they respond to treatment are potential modulators of drug activity in the host and can be used for improved treatment of the disease by potentially shortening the treatment time.
The current diagnosis of tuberculosis still relies on culture of the
M. tuberculosis
organism as the definitive method of diagnosis. However,
M. tuberculosis
grows very slowly and it takes several weeks for the primary isolation of the bacilli from clinical specimens for confirmation of the disease. Current clinical diagnosis uses solid media such as Lowenstein-Jensen medium, 7H10 or 7H11 agar medium and liquid 7H12B medium as in BACTEC460 machine for primary isolation of the bacilli from clinical specimen. In general, the liquid 7H12B based medium is more sensitive in terms of primary isolation of positive cultures from clinical specimens. However, the current medium for isolation of
M. tuberculosis
from clinical specimens is not optimal. Even with liquid 7H12B medium in the presence of growth enhancing agent POES (polyoxyethelene stearate) (Becton Dickinson, Sparks, Md., U.S. Pat. No. 4,769,332), the isolation rate is about 80%, and some 20% samples which later prove to be containing the bacilli are not easily detected. Agents that can improve the primary isolation sensitivity and enhance the growth of
M. tuberculosis
should improve the ability to diagnose TB.
SUMMARY OF THE INVENTION
The present invention provides media and methods for enhancing the cultivation of mycobacteria, or reviving (resuscitating) dormant bacilli from mycobacterium species. The media and methods utilize mycobacterial products from the
M. tuberculosis
complex. By “
M. tuberculosis
complex” we mean
M. tuberculosis
complex organisms which include
M. tuberculosis, Mycobacterium bovis
, including the vaccine strain BCG, and
Microbacterium microti
. The products include cell extracts, early-stationary-phase culture supernatant (ESPSN), and stationary phase culture supernatant (SPSN), either crude or as substantially purified components from these sources. The products may be used to enhance the growth of mycobacterial species that are difficult to culture, and/or to effect the resuscitation of dormant mycobacteria bacilli.
It is an object of this invention to provide a supplemented medium for culturing mycobacterium species, for example
Mycobacterium tuberculosis, Mycobacterium paratuberculosis,
and
Mycobacterium leprae.
The medium comprises a cell extract from
M. tuberculosis
complex, or at least one product from
M. tuberculosis
complex (e.g. a component of early-stationary-phase culture supernatant (ESPSN), of stationary phase culture supernatant (SPSN), or of a cell extract) and a suitable culture medium. The cell extract or substantially purified product exhibits resuscitation activity for dormant bacilli of the mycobacterium species, or the ability to enhance the growth of the mycobacterium species. The substantially purified product may be a phospholipid or a component of a phospholipid, such as phosphotidyl-L-serine, dioleoyl phosphotidyl-L-serine, phosphotidylcholine, phosphotidylethanolamine, tuberculostearic acid, arachidonic acid, and fatty acids. Alternatively, the substantially purified product may be a protein or a fragment of a protein, e.g. protein Rv1147c (accession number F70875 in the National Institute of Health's Entrez Protein Database), a peptide corresponding to SEQ ID NO. 1, a peptide corresponding to SEQ ID NO. 2, a peptide corresponding to SEQ ID NO. 3, a peptide corresponding to SEQ ID NO. 4 and a peptide corresponding to SEQ ID NO. 5. Suitable culture media to be supplemented include 7H12B, 7H9, 7H10, 7H11, Sauton's medium, Dubos medium, and egg-based media (for example Lowenstein-Jensen medium). In addition, a mixture of substantially purified component of ESPSN or SPSN may be utilized, e.g. a mixture of phospholipids and/or proteins and peptides.
In addition, isolated and sterilized ESPSN or SPSN of
M. tuberculosis
complex can itself be utilized as a culture medium, or combined with a suitable fresh culture medium or other fresh nutrients to produce supplemented culture medium. Further, substantially purified components of ESPSN, SPSN, or mycobacterium cell extracts that exhibit resuscitation activity for dormant bacilli of the mycobacterium species may be added.
In another aspect, the present invention provides a method for reviving dormant mycobacterium bacilli of, for example
Mycobacterium tuberculosis, Mycobacterium paratuberculosis,
and
Mycobacterium leprae.
According to the method, dormant bacilli are exposed to a cell extract or at least one substantially purified product of
M. tuberculosis
complex (e.g. a component of early-stationary-phase culture supernatant (ESPSN), of stationary phase culture supernatant (SPSN), or of a cell extract). The cell extract and substantially purified product exhibit resuscitation activity for dormant bacilli of the mycobacterium species, and the cell extract or product is present in sufficient quantity to effect revival of the dormant bacilli. The substantially purified product may be a phospholipid or component thereof, e.g. phosphotidyl-L-serine, dioleoyl phosphotidyl-L-serine, phosphotidylcholine, or phosphotidylethanolamine, tuberculostearic acid, arachidonic acid, and fatty acids. Alternatively, the substantially purified product may be a protein or a fragment of a protein, e.g., protein Rv1147c, a peptide corresponding to SEQ ID NO. 1, a peptide corresponding to SEQ ID NO. 2, a peptide corresponding to SEQ ID NO. 3, a peptide corre
Swartz Rodney P
Whitham Curtis & Christofferson, P.C.
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