METHOD OF DETECTING INVASIVE FUNGI ACCORDING TO MORPHOLOGY THEREOF BASED ON CONTRAST STAINING, AND KIT FOR SAME

Abstract

A method of detecting invasive fungi according to morphology thereof based on contrast staining, including: sterilizing and storing the necessary equipment aseptically; drawing 1 ml of venous blood from a tested subject's elbow vein; dripping one drop of the venous blood, prior to coagulation, into an ampoule containing 0.8 ml of a detection reagent under an aseptic environment; gently shaking the ampoule until the drop of venous blood is evenly distributed; leaving the ampoule to stand for 20 minutes to form a stained solution; sterilizing or disinfecting a microscope slide and a cover slip; dripping one drop of the stained solution on the microscope slide prepared under aseptic condition; observing the sample sequentially with 4×, 10× and 40× objective lenses and a 100× oil-immersion lens; magnifying with a 5 million pixel eyepiece; displaying an image of the sample on computer screen using a high-resolution imaging software for observation and record.

Claims

1. A method of detecting invasive fungi according to morphology thereof based on contrast staining, comprising: step 1: sterilizing equipment necessary for the detection of the invasive fungi; and storing the equipment aseptically; step 2: drawing 1 ml of venous blood from an elbow vein of a tested subject using a 1-2 ml standardized syringe; step 3: dripping one drop of the venous blood, prior to coagulation, into a 5 ml ampoule containing 0.8 ml of a detection reagent under an aseptic environment; gently shaking the 5 ml ampoule until the drop of the venous blood is evenly distributed within the 5 ml ampoule; and leaving the 5 ml ampoule to stand for 20 minutes to allow the detection reagent to stain the drop of the venous blood to form a stained solution; wherein the detection reagent is prepared by: dissolving 0.8-1.5 g of C.I. Direct Blue 1 powder in 100 ml of aseptic physiological saline to form a C.I. Direct Blue 1 stain solution; filtering and disinfecting the C.I. Direct Blue 1 stain solution by a disposable needle filter; placing the C.I. Direct Blue 1 stain solution in a sterile ampoule; and dispensing and packing the C.I. Direct Blue 1 stain solution into 5 ml ampoules in an amount of 0.8 ml per ampoule; step 4: sterilizing or disinfecting a microscope slide and a cover slip; wherein the step of sterilizing or disinfecting the microscope slide and the cover slip is performed according to either one of the following: v. immersing the microscope slide and the cover slip in a 75% alcohol solution for more than 3 hours; heating the microscope slide and the cover slip with an alcohol burner for disinfection at the time of slide preparation; and cooling the microscope slide and the cover slip in an aseptic sealed container with a diameter of greater than 100 mm until the staining in step 3 is completed; or vi. directly disinfecting the microscope slide and the cover slip on a clean bench with ultraviolet light; and storing the microscope slide and the cover slip aseptically until the staining in step 3 is completed; step 5: drawing the stained solution from the 5 ml ampoule by a 1 ml standardized disposable sterile syringe or sterile dropper; dripping one drop of the stained solution on the microscope slide prepared in step 4 under aseptic condition; and placing the cover slip over the microscope slide; and step 6: upon placing the cover slip over the microscope slide, observing the drop of the stained solution on the microscope slide sequentially with a 4× objective lens, a 10× objective lens, a 40× objective lens, and a 100× oil-immersion lens; magnifying with a 5 million pixel eyepiece; displaying an image of the drop of the stained solution on the microscope slide on a screen of a computer using a high-resolution imaging software; and observing and recording the drop of the stained solution on the microscope slide on the computer.

2. A kit detecting invasive fungi according to morphology thereof based on contrast staining, comprising a detection reagent formed by mixing a C.I. Direct Blue 1 powder with an aseptic physiological saline in a weight ratio of (0.8-1.5):100.

3. A kit detecting invasive fungi according to morphology thereof based on contrast staining, comprising a detection reagent formed by mixing a C.I. Direct Blue 1 powder with an aseptic physiological saline in a weight ratio of 1:100.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0033] FIG. 1 is a flow diagram according to the present application.

[0034] FIG. 2 is an image of the blood of a normal human subject (without fungi) after staining by the C.I. Direct Blue 1 stain.

[0035] FIG. 3 is an image of hyphae without staining.

[0036] FIG. 4 is an image of hyphae after staining by the C.I. Direct Blue 1 stain.

[0037] FIG. 5 is an image of a sample of body fluid culture under the microscope in a morphological detection of fungi. The sample was cultured for two weeks and directly smeared.

[0038] FIG. 6 is an image of a sample of body fluid culture which was obtained from the same individual and prepared in the same way as that shown in FIG. 5, except that this sample was subjected to the detection method according to the present application.

[0039] FIG. 7 is an image of a blood sample from an individual who had been confirmed to carry fungi in the blood. The sample was subjected to the detection method according to the present application.

[0040] FIG. 8 is an image of a body fluid sample in a galactomannan antigen detection (GM test) of fungi.

[0041] FIG. 9 is an image of a body fluid sample which was obtained from the same individual and prepared in the same way as that shown in FIG. 8, except that this sample was subjected to the detection method according to the present application.

[0042] FIG. 10 is an image obtained by the method of detecting whether fungi are present in the pleural effusion according to the present application.

[0043] FIG. 11 is an image obtained by the method of detecting whether fungi are present in the cerebrospinal fluid according to the present application.

[0044] FIG. 12 is an image of provoked leukocytes.

[0045] In the figures:

1—normal erythrocyte; 2—normal leukocyte; 3—normal platelet; 4—hypha; 5—abnormal erythrocyte; 6—spore and sporozoite; 7—provoked leukocyte; 8—fiber exudates; 9—crystal.

DETAILED DESCRIPTION

[0046] Embodiments of the present application are described below with reference to the accompanying drawings. However, these embodiments are not intended to constitute limitations on the present application, but are merely examples of the present application. Meanwhile, the following description would render the advantages of the present application clearer and easier to understand.

[0047] In the following examples, C.I. Direct Blue 1 (CAS No.: 2610-05-1; trade name: Chicago Sky Blue; Product No.: Sigma—C8679) was purchased from Sigma-Aldrich Trading Co., Ltd. (Shanghai, China). C.I. Direct Blue 1 has a chemical formula of:

##STR00001##

[0048] Referring to FIG. 1, the present application provides a method of detecting invasive fungi according to morphology thereof based on contrast staining, including:

Step 1: sterilizing equipment necessary for the detection of the invasive fungi; and storing the equipment aseptically;
Step 2: drawing about 1 ml of venous blood from an elbow vein of a tested subject using a 1-2 ml standardized syringe;
Step 3: dripping one drop of the venous blood, prior to coagulation, into a 5 ml ampoule containing 0.8 ml of a detection reagent (diluted at 15-fold) under an aseptic environment; gently shaking the 5 ml ampoule until the drop of the venous blood is evenly distributed within the 5 ml ampoule; and leaving the 5 ml ampoule to stand for 20 minutes to allow the detection reagent to stain the drop of the venous blood to form a stained solution; [0049] wherein the detection reagent is prepared by: [0050] dissolving 0.8-1.5 g of C.I. Direct Blue 1 powder in 100 ml of aseptic physiological saline to form a C.I. Direct Blue 1 stain solution; filtering and disinfecting the C.I. Direct Blue 1 stain solution by a disposable needle filter; placing the C.I. Direct Blue 1 stain solution in a sterile ampoule; and dispensing and packing the C.I. Direct Blue 1 stain solution into 5 ml ampoules in an amount of 0.8 ml per ampoule;
Step 4: sterilizing or disinfecting a microscope slide and a cover slip; [0051] wherein the step of sterilizing or disinfecting the microscope slide and the cover slip is performed according to either one of the following: [0052] iii. immersing the microscope slide and the cover slip in a 75% alcohol solution for more than 3 hours; heating the microscope slide and the cover slip with an alcohol burner for disinfection at the time of slide preparation; and cooling the microscope slide and the cover slip in an aseptic sealed container with a diameter of greater than 100 mm until the staining in step 3 is completed; or [0053] iv. directly disinfecting the microscope slide and the cover slip on a clean bench with ultraviolet light; and storing the microscope slide and the cover slip aseptically until the staining in step 3 is completed;
Step 5: drawing the stained solution from the 5 ml ampoule by a 1 ml standardized disposable sterile syringe or sterile dropper; dripping one drop of the stained solution on the microscope slide prepared in step 4 under aseptic condition; and placing the cover slip over the microscope slide; and
Step 6: upon placing the cover slip over the microscope slide, observing the drop of the stained solution on the microscope slide sequentially with a 4× objective lens, a 10× objective lens, a 40× objective lens, and a 100× oil-immersion lens; magnifying with a 5 million pixel eyepiece; displaying an image of the drop of the stained solution on the microscope slide on a screen of a computer using a high-resolution imaging software; and observing and recording the drop of the stained solution on the microscope slide on the computer.

[0054] Preferably, the detection reagent is formed by mixing a C.I. Direct Blue 1 powder with the aseptic physiological saline in a weight ratio of (0.8-1.5):100. More preferably, the detection reagent is formed by mixing a C.I. Direct Blue 1 powder with the aseptic physiological saline in a weight ratio of 1:100.

[0055] As shown in FIGS. 2, 6 and 7, when being stained by the C.I. Direct Blue 1 stain, significant difference was shown in the morphological features between the normal erythrocytes and the abnormal erythrocytes in the blood. Based on the cell morphology, one skilled in the art may conveniently and rapidly determine whether fungi or spores are present in the blood.

[0056] As shown in FIGS. 3 and 4, the difference between the stained hyphae and the non-stained hyphae under the microscope was very significant. Based on the results observed under the microscope, one skilled in the art not only may rapidly determine whether fungi are present in the blood of the tested subject, but also may preliminarily determine the approximate quantity of bacteria in the blood of the tested subject.

[0057] As shown in FIGS. 5, 6, 8 and 9, the method according to the present application was effective in the detection of fungi and might be used for detecting whether fungi are present in the blood. Meanwhile, since the method according to the present application only requires about 20 minutes of staining time to accurately determine whether fungi are present in the tested body fluid, as compared to the various existing detection methods, the method according to the present application may significantly shorten the detection time and cost, increase the detection accuracy, and possess good social benefits and market prospect.

[0058] As shown in FIG. 7, the method according to the present application could clearly detect the presence of fungi in the body of the fungal infected subject. Therefore, the method according to the present application is effective in detecting fungi and worthy for popularization.

[0059] As shown in FIGS. 9, 10, 11 and 12, the method according to the present application might be widely adopted in the detection of fungi. It may be used not only in the detection of whether fungi are present in the blood, but also in the detection of whether fungi are present in other fluids (such as pleural effusion, cerebrospinal fluid, and synovial fluid). Meanwhile, the method according to the present application may also be adopted in other experiments (such as the leukocyte provocation test shown in FIG. 12).

[0060] In order to more clearly explain the characteristics of the method and the kit according to the present application, and further illustrate the difference between the samples stained by the C.I. Direct Blue 1 stain and those not stained by the C.I. Direct Blue 1, the inventor of the present application conducted a comparison on the same sample with and without being stained, and the results were as follows:

TABLE-US-00001 TABLE 1 Comparison between blood sheet stained by C.I. Direct Blue 1 contrast stain and blood sheet not stained by C.I. Direct Blue 1 contrast stain. Stained by C.I. Direct Blue 1 Not Contrast Stained Contrast Stain Cell Uniform color, consistent Clear cell contours, clear Color with the color of difference in color depth erythrocyte Hypha Not visualized Hyphae and spores could be clearly visualized Spore Sporozoites distributed Sporozoites distributed at at the bottom of the the bottom of the microscope microscope slide could slide could be clearly visualized not be visualized Infected Infected leukocytes could Infected leukocytes could Cell not be visualized be visualized Erythro- Spikes on the surface of Spikes on the surface of cyte erythrocytes could not be erythrocytes could be clearly visualized visualized Cell Statically Dynamically - various cell Move- functional states of the ment infected cells could be observed dynamically Other Stainable structures at the Stainable structures at the bottom of the microscope bottom of the microscope slide could not be slide could be contrast visualized stained and visualized

[0061] Other portions which have not been specifically described in the present application belong to the prior art.