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PARTNERSHIPS FOR ENHANCED ENGAGEMENT IN RESEARCH (PEER)
Cycle 6 (2017 Deadline)


Mapping of pesticide residue and oocysts on vegetable and fruits using low-cost field based assays

PI: Basant Giri, chembasant@gmail.com, Kathmandu Institute of Applied Sciences
U.S. Partner: Toni Barstis, Saint Mary's College, Notre Dame, Indiana
Project Dates: December 2017 - January 2024


Project Overview:

Contamination of vegetables and fruits by pesticides and endoparasites is a major public health concern in developing countries, including Nepal. Pesticides are widely used worldwide to protect crops from pests, but their excessive and unmanaged use is harmful to humans and the ecosystem. Pesticides are well-known toxins that cause acute and delayed health effects, including disruption of the central and peripheral nervous systems and cancer. Similarly, according to the World Health Organization (WHO), about 8% of total deaths reported in Southeast Asia are caused by diarrhea. In Nepal, several instances of diarrheal outbreaks are reported every year. Human exposure to pesticides and parasites is due to contaminated food and water. Concerns over food pollution are rising in Nepal, but science-based understanding of the level of pollution is limited, due in part to the unavailability of reliable, economical, easy-to-use, and rapid field test methods to be used as important early warning tools for consumers. Conventional methods for determining pesticide residues on food involve sophisticated, time-consuming, expensive chromatographic methods that require advanced lab facilities and skilled operators. Similarly, endoparasite oocysts in food products are identified using expensive, high resolution optical microscopes, immunofluorescence-based microscopy, and polymerase chain reaction techniques that are not suitable for field screening of samples in developing countries. This project aims to develop (1) a paper-based pesticide residue assay and (2) smartphone-based oocyst assay methods. Both of these high throughput, low-cost, easy methods will be first developed and validated in the laboratory and later tested with vegetable samples at various locations across Nepal. It will be the first large-scale field testing of these methods with real samples. Other activities on the project will include providing training to students and government technicians, organizing workshops for concerned stakeholders, creating online map with field results, and disseminating information. Bringing reliable assay methods for pesticide residue and oocysts for public use with the possibility of commercialization will be the major output of the project. The USG-supported partner will provide technical advice and training for both the lab development and field testing components.

In addition, the team will involve undergraduate students from the Tri-Chandra Multiple Campus, Tribhuvan University. They will learn to use the newly developed assay methods and go into the field to test food pollution levels. In addition, two Master’s students from the Central Department of Environmental Science, Tribhuvan University, will be given the opportunity to carry out their thesis work in the framework of this project. The PI and his colleagues will share their field screening findings and involve the Plant Protection Directorate (PPD) of the Government of Nepal. PPD technicians will be trained in the new methods and be given some of the assay tools. Two workshops and one symposium will also be organized to share the results of the project.

See more by visiting PEER project website.

Summary of activities and impacts:

In this project, the major aim was to develop two types of devices/methods. The first one was a paper-based analytical device (PADs) to determine pesticide residue in vegetables and fruits. The second one was a smartphone-based microscope to detect (oo)cysts of Giardia and Cryptosporidium in vegetable and fruits samples. In addition, the project team had also proposed to train students and disseminate our findings. They organized several events, made efforts to establish new collaborations and engaged government and non-governmental agencies. By the end of the project the team did achieve their goals.

Research activities focused on the development of paper-based analytical device (PAD) for measuring pesticide residue on vegetable and fruits samples and a smartphone-based microscopy system for detecting the (oo)cysts of Giardia and Cryptosporidium. The team named the PAD as PesticidePAD which is about half of the business card in size. It utilizes enzyme inhibition assay to measure the presence of pesticides in samples. The PesticidePAD gives results in three forms: qualitative results, semi-quantitative results are obtained with the help a custom built smartphone android app. This app is available to download for free  and tells the user whether the sample vegetable is safe to consume or not safe to consume. It also tells whether the sample can be consumed after few days of quarantine.

The smartphone-based microscopy system, which the team custom designed and optimized, consists of a smartphone assembled with a ball lens and a microscope stage. The team optimized the ball lens, light source, and the stain. This microscopy system is able to detect and distinguish the cysts of two protozoan parasites - Giardia and Cryptosporidium and gives comparable results with a commercial brightfield and a fluorescence microscopes.

Sample collection and testing: the team collected more than 1124 samples of 18 types of vegetables and fruits from 9 different vegetable markets across Nepal. All these samples have been tested for pesticide residue levels and contamination of (oo)cysts. The sampling sites are: Kalimati Vegetable Market and Balkhu Vegetable Market – both from Kathmandu; Rudrayani Tarakari Collection Center, Dharke, Dhadhing; Agriculture product collection center, Dhusa-1, Charaundi, Dhading; Agriculture Product Market Center, Bharatpur, Chitwan; Butwal Agriculture Product Wholesale Bazar, Butwal; Rupandehi; Agriculture Product Market Center, Kapurkot, Salyan; Bulbule Chhetriya Krishi Upaj Bazar, Surkhet; Agriculture product wholesale market center, Kohalpur, Banke; Attariya agriculture product wholesale market, Attariya, Kailali. Samples from Kathmandu were collected covering three different seasons. From rest of the locations, samples were collected only one time each.

Publications and presentations: the team produced 15 technical documents which include 9 peer-reviewed research papers (3 already published, remaining under review, two lab modules, two patents (one submitted, another one under preparation) and curriculum of two short courses. Our researchers have made 16 technical presentations in domestic and international platforms showcasing the work under the project.

Capacity building: the PEER project played a crucial role for capacity development of the laboratory and researchers. As part of the support from the project, the project team obtained a solid Wax Printer, Centrifuges, Refrigerators, Micropipettes, Laminar Flow Hood, Fluorescence Microscope, Water Purification System and Weighing Balance. Project students and researchers will be using this equipment even after the completion of the project. During the course of the project period, the project team provided training opportunities for twelve students and researchers to work with the team, including thesis students, research interns, research associates. Some of these students are now pursuing higher studies in the U.S. and Europe and a few others secured jobs in Nepal. The project team have also provided training to more than 40 students from various colleges in Nepal as a part of short courses the team offered.

USG Partner & Supplemental Grant: the team hosted Prof. Toni Barstis from Saint Mary’s College (SMC), IN, USA supported as part of the supplemental PEER grant. The team conducted two workshops with Dr. Barstis. Participants of the workshop were KIAS researchers, and paper-based analytical device group (PADs group) from SMC who were on a month-long research experience trip supported by IRES, NSF. The workshop was useful for discussing various aspects of PesticidePADs and sharing experiences from both sides. Dr. Barstis provided training to PEER researchers in Kathmandu Institute of Applied Sciences and gave lectures in two highschools. Dr. Barstis has been awarded with Fulbright Global Scholar and the project team  we will be hosting her in Nepal.  

Collaboration: the project team are still actively collaborating with Dr. Toni Barstis from Saint Mary’s College, who is USG partner for this project. Seven students and faculty from her college visited our institute for one month in the summer of 2018 and were involved on research work. The team have now established active collaboration with Dr. Bishesh Khanal from NAAMI, Kathmandu and are expanding the work carried out during PEER project. Similarly, the team have established working collaboration with Mr. Prajwal Rajbhandari from Research Institute for Biotechnology and Bioscience, Kathmandu, Nepal. Good  contacts were established with a number of government agencies, university faculty, non-governmental research centers and private companies.

Events: the project team organized 10 events during the project period. About five hundred people participated in these events out of which 40% were female. The participants were government officials, university faculty and students, researchers etc.

Supplemental activity
 
The project received a supplementary grant aimed to validate the PesticidePAD method the team had developed for the detection of two groups of pesticides in vegetable samples and for training students and technicians on the new technology. During August 2022-July 2023 period the team optimized the paper analytical device for the detection of pesticide for re-confirmation of its performance. They visited government's Rapid Bioassay of Pesticide Residue (RBPR)'s laboratory to understand their protocol of testing pesticide in vegetable samples in local market and collaborated with NAAMII research institute to re-configure PesticidePAD analyzer android application in a new platform that could utilize machine learning algorithms for better results. The team  developed few dissemination materials to make people aware about the possible health impact from inappropriate use of pesticides and consuming contaminated vegetables. The team are testing  PesticidePAD and PesticidePAD analyzer with more samples and are planning to organize a workshop with government technicians as well as organize a training workshop for university/college students and faculty. 

 

Publications
:

1. Shrestha, R., Duwal, R., Wagle, S., Pokhrel, S., Giri, B., & Neupane, B. B. (2020). A smartphone microscopic method for simultaneous detection of (oo) cysts of Cryptosporidium and Giardia. PLOS Neglected Tropical Diseases, 14(9), e0008560.
2. Fuyal, M., & Giri, B. (2020). A Combined System of Paper Device and Portable Spectrometer for the Detection of Pesticide Residues. FOOD ANALYTICAL METHODS, 13, 1492–1502.
3. Giri, B., Pandey, S., Shrestha, R., Pokharel, K., Ligler, F. S., & Neupane, B. B. (2020). Review of analytical performance of COVID-19 detection methods. Analytical and bioanalytical chemistry,1-14.