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 xe2x80x9cDetection of various fungal pathogens in blood samplesxe2x80x9d 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 disintegrated by the preceding disintegration process, can be separated from the free cellular DNA in a quick and easy process, e.g. through centrifugation. During the subsequent lysis of the fungal cells isolated in this manner it can be safely assumed that there is no or only an insignificant amount of cellular DNA in the solution. The disintegration of the blood cells in step al) must thus be carried out in such a way that the fungal cells are not yet lysed. Since the fungal cell wall is much more complex than the cell wall of blood cells this can be ensured through adequate careful disintegration processes.
The procedural step a1) has a further advantage, namely that it releases any phagocyted fungi so that very small quantities of fungi can still be extracted from the whole blood, particularly for diagnostic purposes. With the new process it is no longer necessary for at least a few fungal cells to be freely dissolved in whole blood, it is more than sufficient if a few fungal cells are present after phagocytosis, e.g. in granulocytes or macrophages.
It is preferred if the following steps are carried out in step a):
a1.1) lysis of the erythrocytes by osmotic hemolysis;
a1.2) enzymatic disintegration of the leukocytes; and
a2.1) centrifugation of the whole blood treated in this manner and use of the pellet in the next procedural steps.
The advantage here is that although the blood cells are reliably dissolved in steps a1.1) and a1.2), the fungal cells themselves are not damaged yet. The subsequent centrifugation then ensures a very safe separation between the released cellular DNA and cell fragments of the blood cells on the one hand and the still predominantly intact fungal cells on the other. This also ensures that no fungal DNA is lost since fungal DNA is still present in the fungal cells in the pellet. Summing up, the advantages of the aforementioned steps are thus that on the one hand even minimal concentrations of fungal cells can be detected and on the other the isolated fungal DNA is at most only slightly contaminated with cellular DNA, permitting a high sensitivity and specificity in the subsequent diagnostic steps since the ratio of fungal to cellular DNA has been significantly improved in favor of fungal DNA.
In a further development it is then preferred if the following steps are carried out in step b):
b1.1) alkaline lysis and enzymatic treatment of the fungal cells.
It could be shown that this simple procedural step permits a safe disintegration of the fungal cells which are recovered from the pellet in procedural step a2.1).
It is furthermore preferable if the lysis of the erythrocytes in step a1.1) is carried out using a hypotonic solution, preferably with a final concentration of approx. 10 mM Tris pH 7.6, 5 mM MgCl2 and 10 mM NaCl, and if in step a1.2) the enzymatic digestion of the leukocytes is carried out in a solution having a final concentration of 200 xcexcg/ml proteinase K, 10 mM Tris pH 7.6, 10 mM EDTA pH 8.0, 50 mM NaCl and 0.2% SDS.
In a further development the solution from step a1.2) is incubated for 100-140 min, preferably 120 min, at 60-70xc2x0 C., preferably at 65xc2x0 C.
It has been found that in this way the blood cells can be reliably and completely disintegrated without damaging the fungal cells so that after these procedural steps both the fungal cells freely in solution and the phagocyted fungal cells in the initial blood remain relatively undamaged and can be separated by centrifugation without losing their DNA.
It is furthermore preferable if step b1.1) comprises the following steps:
incubation of the fungal cells present in the pellet from step a2.1) at 90-98xc2x0 C., preferably 95, for 5-15 min, preferably 10 min., in a solution with 50 mM NaOH,
neutralization with 1 M Tris-HCl pH 7.0,
enzymatic treatment with zymolyase for 50-70 min, preferably 60 min at 30-40xc2x0 C., preferably 37xc2x0 C.,
protein denaturation by incubation with Tris/EDTA at 60-70xc2x0 C., preferably 65xc2x0 C., for 10-30 min, preferably 20 min.
It has been found that these procedural steps permit a safe disintegration of the fungal cells with a subsequent complete release of fungal DNA so that the fungal DNA is then in solution and can be isolated from the cell fragments of the fungal cells.
It is preferred if in this connection step b2) comprises the following steps:
protein precipitation with 5 M potassium acetate, and
DNA precipitation of the supernatant in ice-cold isopropanol.
These procedural steps are very easy to be carried out, the protein is firstly precipitated by adding potassium acetate, the supernatant then removed and mixed with ice-cold isopropanol so that the DNA is precipitated. The precipitated DNA can then be used in the further procedural steps, i.e. to detect and identify a fungal infection.
Summing up the procedure as described up to now, the first advantage is that the fungal species can be generally detected on the basis of their DNA and can then be specifically identified. The fungal DNA is not extracted directly from the whole blood, rather there is firstly a separation of the fungal cells from cellular DNA to improve the sensitivity and specificity of the detection. In other words, if only very few fungal cells are present in the whole blood the very small quantity of fungal DNA extracted from this could be detected against the back-ground of cellular DNA present in a very high concentration, so that the separation mentioned above leads to great advantages in terms of sensitivity.
A further advantage of the new process is that phagocyted fungal cells are also available for detection purposes since the blood cells of the whole blood are initially disintegrated without damaging the fungal cells, irrespective of whether these are in solution or phagocyted. The fungal cells are only disintegrated following separation of the fungal cells from the cellular DNA and remaining cell debris of the blood cells. The method employed here ensures a complete disintegration with no damage to the fungal DNA despite the lightly complex fungal cell walls.
The object of the present invention is also a kit to carry out the process described above. Such a kit can contain a set of all basic solutions which are required for the aforementioned procedural steps. However, it is also possible to include in this kit only those basic solutions normally not found in a laboratory, e.g. the Tris buffer is dispensible, though at least the proteinase K and zymolyase are present in the kit.
The object of the present application is also the second step of the process, either isolated or in combination with the first step of the process described above, i.e. the detection of extracted fungal DNA. The fungal DNA to be detected is hereby preferably extracted and isolated as described above. However, it is also possible to obtain the fungal DNA by suitable density gradient centrifugation, specific precipitation steps with DNA probes, etc.
In all of these cases it is desirable to detect whether fungal DNA is actually present in that initial solution for further use.
Although the fungal DNA can be advantageously employed for the diagnosis of fungal infections, it can also be used differently. The fungal DNA can be obtained from agar plates or from the blood of animals which have been specifically infected with the fungal species in question. DNA probes can be cleaved from the fungal DNA obtained in this manner which can then be used for detection reactions or cloned in plasmids. Applications throughout the whole field of basic research, diagnostics, therapy, industrial gene technology, etc. are conceivable.
The new detection step should permit the recording of a number of various fungal species, even in low concentrations, in the initial solution in one single process so that it is possible to say whether there is a fungal infection at all very quickly and at an early stage in the diagnosis. Moreover, the new process should be able to be performed quickly and easily so that it can be employed in everyday work in hospitals by trained personnel.
The object is achieved in accordance with the invention in that the step to detect the extracted fungal DNA comprises the following steps:
c) amplification of at least a segment of the isolated fungal DNA; and
d) detection of the amplification product.
There are a number of standard methods with which even small amounts of an isolated DNA can initially be amplified and then detected. These methods are highly specific and very sensitive so that they provide the desired features for diagnosis, therapy, industrial gene technology, etc. Furthermore, these methods can be performed by trained personnel.
The amplification can be carried out either by PCR, cloning, DNA-dependent DNA-polymerases, etc., and the detection can be carried out by gel electrophoresis, optical density, staining with markers which bind specifically to nucleic acid, etc.
In a further development the amplification is carried out by means of a polymerase chain reaction (PCR) using two primers which bind to the DNA of a number of different fungal species, preferably to the fungal gene for the 18ssu-rRNA.
The advantage here is that a simple and in the meantime established method can be used to produce adequate amounts of DNA which is then easily detected, though the DNA can also further be used to identify the corresponding fungal species.
It is preferred if the nucleotide sequences SEQ ID-No: 1 and SEQ ID-No: 2 from the enclosed sequence listing are used as primers in step c).
The inventor of the present application surprisingly found that these two nucleotide sequences can be used as primers for various clinically relevant fungal species. The use of these two primers in the PCR reaction produces amplification products having a length of approx. 500 base-pairs, and are thus easy to be further processed since they can be easily detected by gel electrophoresis. In this way there is an identical detection process for all pathogenic fungal species since it was found that the two primer sequences bind to the various fungal DNAs.
It is preferred if the polymerase chain reaction is carried out with the following cycle:
c1) denaturation for 0.3-1 min, preferably for 0.5 min, at 90-96xc2x0 C., preferably at 94xc2x0 C.;
c2) hybridization for 0.5-1.5 min, preferably for 1 min, at 58-64xc2x0 C., preferably at 62xc2x0 C.;
c3) extension for 1.5-2.5 min, preferably for 2 min, at 68-75xc2x0 C., preferably at 72xc2x0 C.
It is preferred if a denaturation step of 5-9 min, preferably 3 min, at 90-96xc2x0 C., preferably 94xc2x0 C., is carried out before the start of the cycle steps.
It was found that the PCR is very reproducible and above all highly specific under the aforementioned conditions so that it can be ensured that the amplification products are in fact segments of the original fungal DNA.
It is furthermore preferred if in step d) the amplification products are detected by gel electrophoresis, preferably on an agarose gel, and are preferably stained with ethidium bromide.
The advantage of this is that a known and well established detection method is used to show that amplification products which indicate a fungal infection are in fact produced by the PCR reaction.
It is further preferred if a DNA sequence from the fungal gene for the 18ssu-rRNA is amplified.
The inventor of the present application found that this fungal gene displays a specific sequence segment in the various fungal strains and species which on the one hand is flanked by two binding regions for primers which are identical for all fungal strains and species, though on the other hand the sequence of this segment differs to such an extent for the various fungal strains and species that it can be used to detect individual fungal species and strains.
The invention further relates to the nucleotide sequences SEQ ID-No: 1 and SEQ ID-No: 2 from the enclosed sequence listing. It is preferable if these two nucleotide sequences are used to amplify a segment of fungal DNA.
The invention also relates to a kit to detect fungal DNA in a test solution, and the kit contains primers for a polymerase chain reaction binding to the DNA of a number of fungal species. The kit preferably contains the nucleotide sequences SEQ ID-No: 1 and SEQ ID-No: 2 from the sequence listing as primers.
The invention also relates to a kit to carry out the process described above.
The advantage of such a kit is that all necessary solutions, and in particular the necessary primers, can be put together so that they can be used for routine procedures. Thus these new kits can be used in everyday laboratory work to detect fungal species in test solutions. Moreover, this kit can be used to amplify certain segments of the proven fungal species which can then be used for further identification processes, for example.
The object of the present application is also the third step of the process, either isolated or in combination with the first and the second steps of the process described above, i.e. the identification of the fungal species using the extracted and detected fungal DNA.
The fungal identification of the species should be able to be carried out quickly and easily for diagnostic purposes so that it can be employed in everyday work in hospitals by trained personnel.
This object is achieved in accordance with the invention inasmuch as the following step is carried out:
e) identification of the nucleotide sequence sections of the DNA contained in the test solution characterizing the fungal species.
Since the detection process is carried out on the DNA level it is sufficient if certain segments of the fungal DNA are identified provided these segments are specific for the respective fungal species. Standard methods can be used by sequencing at least a part of the amplification product, investigating the melting behavior of the double strands, etc.
These processes can generally be performed quickly and easily so that they can also be carried out by trained personnel.
It is preferred if the fungal DNA or segments of the fungal DNA are hybridized with DNA probes which are specific for defined fungal strains and/or species in step e), the test of successful hybridization leading to an identification of the fungal species.
The advantage of this is that a very fast and simple hybridization process is used for identification. Specific probes are used which are specific to the respective fungal species so that they bind exclusively to the amplified segments of these fungal species. Such processes can be carried out on gels, for example, the hybrids being shown by staining. A further process consists of exploiting the optically different behaviors of single strands and double-stranded regions, e.g. optical density, dichroism or similar features.
It is furthermore preferred if the probes are labeled with digoxigenin for testing of successful hybridization and if the hybrids are detected using the Southern-Blot method, for instance.
This step further simplifies the process, the probes themselves are already provided with the corresponding marker which shows the formation of hybrids following hybridization, preferably by visually detectable dye reactions, using a well-known method.
It is preferred if one or more of the nucleotide sequences SEQ ID-No: 3 to SEQ ID-No: 8 from the enclosed sequence listing are used as DNA probes, the DNA probes being preferably used sequentially in the sequence SEQ ID-No: 3, SEQ ID-No: 8, SEQ ID-No: 6, SEQ ID-No: 7, SEQ ID-No: 4 and SEQ ID-No: 5 and if the respective hybridization is tested.
The individual fungal species can be tested in the order of their frequency by the sequential hybridization, thus significantly accelerating the detection process.
The invention further relates to the nucleotide sequences SEQ ID-No: 3-SEQ ID-No: 8 from the enclosed sequence listing.
The invention further relates to the use of one or more of these nucleotide sequences as DNA probes to identify fungal species.
The advantage here is that the 6 specified nucleotide sequences can specifically discriminate between all clinically relevant fungal species.
The nucleotide sequence SEQ ID-No: 3 detects the fungal species Candida albicans, SEQ ID-No: 4 detects Candida glabrata, SEQ ID-No: 5 detects Candida krusei, SEQ ID-No: 6 detects Candida tropicalis, SEQ ID-No: 7 detects Candida parapsilosis and SEQ ID-No: 8 detects fungal species belonging to the strain Aspergillus, particularly A. fumigatus, A. flavus, A. versiculor, A. niger, A. nidulans and A. terreus. 
The invention further relates to a kit to identify fungal species which contains DNA probes which hybridize to specific nucleotide sequence sections of the DNA of the respective fungal species. The kit preferably contains one or more of the nucleotide sequences SEQ ID-No: 3-SEQ ID-No: 8 from the enclosed sequence listing as DNA probes.
The invention further relates to a kit to carry out the process described above.
The advantage of this is that such a kit can be provided with either only the DNA probes or additionally with the adequate additional solutions so that all primary solutions, etc. necessary for everyday laboratory work can be taken directly from this kit. This considerably simplifies the detection or the identification of the respective fungal species since the individual primary solutions no longer have to be produced separately in the laboratory. However, it is also possible to include only the probes and possibly the primers and enzyme/primary solutions for the polymerase chain reaction in this kit so that apart from this, standard primary solutions can be used.
A process for detection of fungal cells in whole blood comprising the steps detailed above in an advantageous manner comprises the following steps:
isolation of predominantly intact fungal cells from whole blood,
extraction of DNA from the isolated fungal cells,
amplification of at least one segment of the isolated fungal DNA, preferably of at least one segment of the fungal gene for the 18ssu-rRNA,
detection of the amplification products according to a yes/no decision, and
assigning the amplification products to individual fungal species by hybridizing them with DNA probes being specific for defined fungal strains and/or species.
One of the main advantages of this summarized process is that only one amplification step is required amplifying a segment of the fungal DNA, preferably a segment of the fungal gene for the 18ssu-rRNA in such a way that the amplification products can be used for the yes/nor decision as well for the determination of the fungal species. The inventor of the present application has found that the fungal gene for the 18ssu-rRNA can at least partly be flanked by two primers being identical for all fungal species of interest on the one hand, the amplicon being different for the various fungal species on the other hand, so that it can be hybridized with different probes characteristic for the fungal species.
A single amplification step therefore is sufficient to answer the question whether there is a fungal infection at all and to answer the question which fungal species is concerned. The process is therefore extremely simple and can be easily carried out, specialized knowledge is not required, so that it can be carried out even by trained personnel.
Further advantages can be taken from the following description.
It is understood that the aforementioned features and those to be explained in the following can be used not only in the specified combinations but also in other combinations or alone without going beyond the scope of the present invention.
Examples of the performance of the individual procedural steps and the use of the process within the scope of a program of a clinical test program are quoted in the following description.
A particular advantage of the new process is the fact that it is performed using whole blood, i.e. no biopsy is required and no serum which first has to be produced is used. The fungal cells are firstly separated from the cellular DNA in the whole blood. This is necessary for the fungal DNA, which may only be present in very small quantities, not having to be detected against the background of the much higher concentration of cellular DNA. This also increases the specificity of the detection process. For this purpose the erythrocytes are initially lysed by osmotic hemolysis and the leukocytes are then enzymatically digested. These two procedural steps are selected in such a way that the fungal cells themselves are not yet damaged and that any phagocyted fungal cells are released undamaged so that they are also available for subsequent detection.