Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-05-29
2003-08-12
Whisenant, Ethan (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023100, C536S024300
Reexamination Certificate
active
06605439
ABSTRACT:
The present invention generally relates to a process for detection of fungal cells in clinical material.
Such processes are known from practice, and are usually based on cultivation of fungal species from clinical material on adequate nutrient media.
The speed and sensitivity of identification are greatly affected by the cultivation, e.g. in Petri dishes, and detection on the basis of the colonies which have grown or not, particularly on account of the slow growth of the fungal species. An invasive fungal infection can thus often only be diagnosed by an extremely complicated biopsy of an organ or even only after the death of a patient.
The interest in a process for detection of fungal cells must be seen in relation to the fact that particularly over the past few years, fungal pathogens have become very important for immunosuppressed patients as significant nosocomial pathogens. Invasive fungal infections have increased considerably following bone marrow transplantation (BMT), though also after liver, kidney, pancreas and heart or heart-lung transplantation. In 1994, for example, there was an accumulation of infections, particularly Aspergillus infections, in French BMT centers which resulted in closure of these centers for a number of months.
Apart from patients with organ transplants, patients suffering from cancer, particularly after chemotherapy or surgery, patients with burns and patients in surgical and neonatal intensive care units are being increasingly affected by invasive fungal pathogens. As soon as an organ system or even a number of organ systems are affected in these groups of patients, the mortality rate for these infectious complications rises to between 80 and 100%.
The success of a therapy can only be improved in such cases through an early diagnosis. On account of the disadvantages associated with the standard detection methods mentioned above, there have been intensive attempts to permit an early, safe diagnosis of a systemic fungal infection.
Although new techniques based on molecular biology methods have already permitted a more sensitive diagnosis and thus partly an earlier detection and treatment of the infectious diseases for a number of other pathogens, this has as yet been impossible for fungal pathogens.
However, the publication “Detection of various fungal pathogens in blood samples” in: EBMT 1995, Vol. 15, Suppl. 2, March 1995, Abstract Book, Abstract 432, P. 103, already describes the amplification of a DNA segment of a fungal gene using the PCR method so as to identify a number of fungal pathogens in blood. Through an additional hybridization with species-specific oligonucleotides, a differentiation between various fungal strains was also possible.
The most important problems during the detection of fungal infections using molecular biology techniques are caused by the very complex composition of the fungal cell walls, which have made time-consuming and expensive extraction methods necessary until now.
A further problem is the fact that an increasing number of fungal species can trigger dangerous infections in immunosuppressed patients, leading to the necessity of the recording and detection of a whole range of different fungal species and strains in these patients. Since therapies differ for various fungal species it is necessary to not only record all fungal species but also to differentiate between and identify these species.
In view of the above, it is an object of the present invention to improve the process mentioned above to permit an early diagnosis of a fungal infection.
The process should be as quick and easy as possible, it should record as many fungal species as possible and it should also be able to identify these in a further stage of development.
The process to detect fungal pathogens in clinical material found in accordance with the invention comprises the following steps:
1.) extraction of fungal DNA from whole blood; and
2.) detection of extracted fungal DNA.
By abandoning the traditional cultivation of fungal species from clinical material and turning to the fungal DNA itself a process has been developed which permits a very early detection of fungal infections with a high sensitivity through detection of the fungal-specific DNA. Since the detection can be performed on the DNA level, at least partially highly sensitive, well established and fast processes can be used, leading to great advantages over known methods in terms of sensitivity and speed. It should be remembered that the release and purification of the fungal DNA must be performed not only quickly but also almost completely and relatively purely to guarantee a high sensitivity and permit a rapid diagnosis.
Moreover, the new process should be sensitive for a number of fungal species, and the fungal species should also be identified in the least possible procedural steps.
This further object is achieved in the aforementioned new process through the following procedural step which is carried out in addition to or in place of procedural step 2.):
3.) determination of the fungal species from the extracted fungal DNA.
This process has the further advantage that the diagnosis of the specific fungal infection on the DNA level can be carried out very quickly and displays a high specificity, since, for example, known sequencing processes can be used.
A number of problems had to be overcome in the individual steps of the new process, some of which arose from the composition of the fungal cell wall and from the fact that a large number of the fungal cells are not freely dissolved in whole blood but are located in a number of blood cell populations following phagocytosis, especially in granulocytes and macrophages.
The object of the present application is thus also the isolated first step in the process, namely the extraction of fungal DNA from whole blood. Although this process can be used advantageously in diagnostics to detect a fungal infection in a patient, it can also be employed wherever fungal DNA is required for other further processing.
Using the new process for example, fungal DNA can be extracted from the blood of animals which have been specifically infected by the fungal species in question. DNA probes can be cut out from the fungal DNA obtained in this manner which can then be used for detection reactions or be cloned in plasmids. Applications throughout the whole field of basic research, diagnostics, therapy, industrial gene technology, etc. are conceivable.
The process for the extraction of fungal DNA should be able to reliably extract fungal DNA even with very small quantities of fungi in whole blood. Furthermore, this step of the process should be able to be performed quickly and easily so that it can be employed in everyday work in hospitals by trained personnel.
This object is achieved in accordance with the invention in that the step of extracting fungal DNA from whole blood comprises the steps of:
a) isolation of predominantly intact fungal cells from whole blood; and
b) extraction of DNA from the isolated fungal cells.
The object of the invention is completely achieved in this manner. The new process now comprises two stages, comprising isolation of fungal cells from whole blood in the first step and then extracting of DNA from these isolated fungal cells in the second step so that the extracted DNA can be used to detect and if necessary identify fungal infections. This two-stage process above all improves the specificity since only small amounts of any interfering other DNA are present in the second step of the process.
It is preferred if the process comprises the following procedural steps:
a1) disintegration of the blood cells in whole blood;
a2) isolation of predominantly intact fungal cells from cellular DNA;
b1) disintegration of isolated fungal cells; and
b2) isolation of fungal DNA.
This leads to a very rapid and safe separation of fungal DNA from cellular DNA. The cellular DNA is released in this initial solution by lysis of blood cells in whole blood so that the fungal cells which up to then have not been, or at least have not been completely dis
Eberhard-Karls Universität Tübingen Universitätsklinikum
Knobbe Martens Olson & Bear LLP
Tung Joyce
Whisenant Ethan
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