US-20260126447-A1 - METHODS OF DETECTING MICROFILARAE

Abstract

Provided herein are methods for detecting the presence of microfilariae in a blood sample. Generally, the methods include pouring a blood sample comprising erythrocytes onto a porous filter, wherein the porous filter captures microfilaria on a surface of the porous filter; drawing the erythrocytes through the porous filter, leaving a remaining sample on the surface of the porous filter; fluorescently labeling the remaining sample to form a labeled sample; washing the labeled sample; imaging the surface of the porous filter using an imaging system; and detecting the presence of microfilariae based on fluorescence in the images.

Inventors

• Pawel Slusarewicz
• Britt Ripley
• Alex Chen
• Eric HAUCK
• Kevin Calcotte

Assignees

• Parasight System Inc.

Dates

Publication Date

20260507

Application Date

20251107

Claims (20)

1. 1 . A method for detecting the presence of microfilariae in a blood sample comprising: pouring a blood sample comprising erythrocytes onto a porous filter, wherein the porous filter captures microfilaria on a surface of the porous filter; drawing the erythrocytes through the porous filter, leaving a remaining sample on the surface of the porous filter; fluorescently labeling the remaining sample to form a labeled sample; washing the labeled sample; imaging the surface of the porous filter using an imaging system; and detecting the presence of microfilaria based on fluorescence in the images.
2. 2 . The method of claim 1 , wherein fluorescently labeling the sample comprises adding a fluorophore.
3. 3 . The method of claim 1 , wherein the fluorophore is selected from fluorescein, DAPI, Texas red, rhodamine and Alexa fluor dyes.
4. 4 . The method of claim 1 , wherein fluorescently labeling the sample comprises adding fluorescent microbeads.
5. 5 . The method of claim 1 , wherein washing the labeled sample comprises washing with an alkaline, non-amine buffer system.
6. 6 . The method of claim 5 , wherein the buffer system is a sodium bicarbonate buffer system.
7. 7 . The method of claim 5 , wherein the buffer system comprises a thiol-containing component.
8. 8 . The method of claim 7 , wherein the thiol-containing component is cysteine.
9. 9 . The method of claim 1 , wherein the blood sample is lysed prior to pouring onto the filter.
10. 10 . The method of claim 1 , wherein the sample on the filter is lysed prior to imaging.
11. 11 . The method of claim 1 , wherein the sample is imaged using mono-or poly-chromatic light with no optical filtration.
12. 12 . The method of claim 1 , wherein magnification of the imaging system is between 0.25× and 10×.
13. 13 . A method for detecting the presence of microfilariae in a blood sample comprising: pouring the blood sample onto a porous filter that traps the microfilariae while allowing erythrocytes to flow through; drawing the sample through the filter to remove the sample; adding fluorescent microbeads; taking video or a sequence of photographs of the sample on the filter at low magnification using a optical system tuned to detecting bead fluorescence; and using computer vision algorithms to detect bead movement caused by the motion of any microfilariae present.
14. 14 . The method of claim 13 , wherein the sample on the filter is washed before imaging.
15. 15 . The method of claim 13 , wherein the diameter of the microbeads is between 10 and 60 μm.
16. 16 . The method of claim 13 , wherein the diameter of the microbeads is between 20 and 40 μm.
17. 17 . The method of claim 13 , wherein the fluorescent microbeads are added to the blood sample prior to pouring the blood sample onto the filter.
18. 18 . The method of claim 13 , wherein the fluorescent microbeads are added directly to the porous filter.
19. 19 . The method of claim 7 , wherein the blood sample is poured onto the porous filter subsequent to the fluorescent microbeads.
20. 20 . The method of claim 13 , wherein the fluorescent microbeads are added to the porous filter subsequent to the blood sample being poured.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS The present application claims priority to U.S. Provisional Ser. No. 63/717,563, filed Nov. 7, 2024, and U.S. Provisional Ser. No. 63/876,449 filed Sep. 5, 2025, the entirety of each of which is incorporated herein by reference. TECHNICAL FIELD The present disclosure generally relates to methods of detecting parasitic infections in biological samples. BACKGROUND Current diagnostic methods for detecting filarid infections rely on techniques such as microscopic examination, immunological, and molecular approaches, each presenting unique benefits and limitations. Microscopic examination, the simplest method, involves scanning small blood smears for the presence of microfilariae; however, its sensitivity is restricted due to the limited sample volume (about 20 μL). Techniques that increase blood sample size, like the examination of centrifuged hematocrit tubes and specialized counting chambers, can boost detection by concentrating larvae in specific blood layers. To further enhance sensitivity, two established tests, Knott's test and membrane filtration, process larger blood volumes (up to 1 mL) by hemolyzing blood and concentrating larvae either through centrifugation or fine filtering, respectively. These methods significantly raise the likelihood of detecting low-level infections but require laborious preparation and examination. Fluorescent staining methods, including histological dyes like methylene blue and advanced options like fluoresceinated lectins, enhance visibility, while real-time PCR using fluorescence enables non-microscopic detection of filarid DNA. Despite their effectiveness, these methods are time-consuming, labor-intensive, and require substantial laboratory expertise. Skilled personnel must carefully scan slides manually under high magnification, making the process challenging in busy clinical settings that demand prompt results. Although the natural wriggling motility of microfilariae can aid visual detection, most methods disrupt this movement due to osmotic shock, dehydration, or the use of fixatives, limiting its diagnostic utility. Consequently, these tests are often impractical for high-throughput clinical environments. Accordingly, a need exists to develop more streamlined methods for the detection of microfilariae in biological samples without requiring extensive manual processing, thereby offering a practical solution for rapid, reliable detection. SUMMARY Embodiments of the present disclosure generally relate to the detection of microfilariae in a biological sample. In one embodiment, the present disclosure concerns a method for detecting the presence of microfilariae in a blood sample comprising: pouring a blood sample comprising erythrocytes onto a porous filter, wherein the porous filter captures microfilaria on a surface of the porous filter; drawing the erythrocytes through the porous filter, leaving a remaining sample on the surface of the porous filter; fluorescently labeling the remaining sample to form a labeled sample; washing the labeled sample; imaging the surface of the porous filter using an imaging system; and detecting the presence of microfilaria based on fluorescence in the images. In another embodiment, the present disclosure concerns a method for detecting the presence of microfilariae in a blood sample comprising: pouring the blood sample onto a porous filter that traps the microfilariae while allowing erythrocytes to flow through; drawing the sample through the filter to remove the sample; adding fluorescent microbeads; taking video or a sequence of photographs of the sample on the filter at low magnification using a optical system tuned to detecting bead fluorescence; and using computer vision algorithms to detect bead movement caused by the motion of any microfilariae present. It is understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present disclosure as claimed. BRIEF DESCRIPTION OF THE DRAWINGS The present disclosure will be better understood from a detailed description of some example embodiments below in conjunction with the following drawings. FIG. 1 depicts an exemplary filter assembly for filtering of blood and capture of microfilariae. FIG. 2 depicts the movement of fluorescent beads elicited by microfilariae, according to one or more embodiments described herein. The upper panel shows successive frames from the raw video while the lower panel shows the differences between the pixels of adjacent frames following thresholding to increase the contrast. FIG. 3 depicts direct video imaging of moving microfilariae using fluorescence optics, according to one or more embodiments described herein. Consecutive frames from a raw video are shown in the upper panel with subtle mesh distortion caused by microfilarial movement indicated by the arrows. Microfilariae become apparent following inter-frame differencing