WormAssay: a novel computer application for whole-plate motion-based screening of macroscopic parasites

Abstract

Lymphatic filariasis is caused by filarial nematode parasites, including Brugia malayi. Adult worms live in the lymphatic system and cause a strong immune reaction that leads to the obstruction of lymph vessels and swelling of the extremities. Chronic disease leads to the painful and disfiguring condition known as elephantiasis. Current drug therapy is effective against the microfilariae (larval stage) of the parasite, but no drugs are effective against the adult worms. One of the major stumbling blocks toward developing effective macrofilaricides to kill the adult worms is the lack of a high throughput screening method for candidate drugs. Current methods utilize systems that measure one well at a time and are time consuming and often expensive. We have developed a low-cost and simple visual imaging system to automate and quantify screening entire plates based on parasite movement. This system can be applied to the study of many macroparasites as well as other macroscopic organisms.

Figure 1. Dark field macroscopic imaging apparatus and computer application.

The video camera is positioned below the microtiter plate and the plate is recorded using the WormAssay software.

Figure 2. Microtiter plate images and application real-time preview.

(A) Video frames of 24-well plates of Brugia malayi were recorded using the apparatus. (B) Screen capture of the software's real-time preview user interface. Green circles indicate that the wells have been detected and that the program is ready to begin recording data. The blue outline in each well is the worm (or other well artifacts) and the red color indicates the worm's movements. Mean movement units are measured in real-time and are shown for each well using the Lucas-Kanade Optical Flow algorithm. Canonical well labels determined by the well finding algorithm are drawn in teal in the preview image by the application. (There is no movement in the 2 left columns because the worms are dead due to high drug concentrations.)

Figure 3. Dose-response curves of anthelmintic compounds used for validation.

of (A) albendazole (), (B) ivermectin () and (C) fenbendazole () determined using the proposed method with compounds at concentrations of , , , , and .

Figure 4. Simplified state machine of the plate detection and tracking logic.

The nodes indicate distinct states (or the start/end pseudostates) and the edges indicate transition conditions.

Figure 5. Schistosoma mansoni adult worms can also be assayed in 24- or 96-well plates using the WormAssay visual imaging system.

(A) Unprocessed video frame in a 96-well plate. (B) Screen capture of the software's user interface for those plates using the Consensus Voting Luminance Difference algorithm (see caption for Figure 2b).