Arbovirus is a term used to describe any virus transmitted by arthropod vectors such as mosquitos and ticks, with examples including West Nile virus and Eastern equine encephalitis, both of which can affect humans bitten by infected mosquitos. Surveillance programs, such as the one run by the Arbovirus Testing Lab at the New Hampshire Public Health Laboratories, provide valuable data for use in guiding the response to these mosquito-borne diseases.

Like many other laboratories around the world, the COVID-19 pandemic has forced the Arbovirus Testing Lab to make drastic changes to its mosquito surveillance program, adopting an automated, pooled sample testing workflow using an ASSIST PLUS pipetting robot. Laboratory supervisor Denise Bolton explained: “We realized that the surging demand for testing supplies, limited availability of PCR instrumentation and incredible strain on laboratory staff would continue for some time. We had been thinking about trying out a pooled testing model for arbovirus surveillance prior to the pandemic, and knew that it was time to implement this approach.”

Moving to pooled testing

After researching pooling methods and devising a protocol for the laboratory, a ‘proof of concept’ study was performed by testing combinations of WNV/EEE positive samples in varying concentrations. This demonstrated that even very low positive samples would still be detected in a pool of four specimens. “The validation showed really high accuracy and sensitivity, and we implemented the pooled testing procedure in summer 2020, initially constructing the sample pools manually. Mosquito processing is really labor intensive. There are many steps requiring uncapping and capping of tubes and pipetting of samples, and all must be performed while following strict safety protocols. The additional sample pooling step further complicated the workflow, and occupied over an hour of analyst time, requiring high levels of concentration and focus. Adding a manual pooling step at the end of the process was also quite risky from a quality standpoint, and I worried about the opportunity for analyst error.”

Reducing the opportunity for error

To minimize the potential for manual handling errors, the laboratory decided to automate the sample pooling process on an ASSIST PLUS pipetting robot. “I was already familiar with the ASSIST PLUS, as we use it for handling our COVID-19 samples,” said Denise. “The VIALAB software supplied with the instrument made it easy to write a pooling program, and the flexibility of the INTEGRA pipettes meant that there was no need to change our mosquito processing method. We use the ASSIST PLUS with an 8 channel VOYAGER adjustable tip spacing pipette to transfer aliquots from 2 ml tubes into a 96 well plate to create 200 µl pools comprising four samples each. Once this process is complete, the ASSIST PLUS adds lysis buffer directly to the sample pools, and the entire pool is used for nucleic acid extraction. This streamlines the process, saving time and reducing the use of plastic labware, since we aren't constructing high volume pools then transferring an aliquot for extraction. Using the ASSIST PLUS in this way has allowed us to automate two pipetting steps that were previously performed manually, actually reducing the need for user input.”

Saving time and money

The adoption of pooled testing for arbovirus surveillance has resulted in a 75 % saving in extraction and PCR reagents, and reduced the use of PCR instrumentation for the surveillance program. “Sample pooling with the ASSIST PLUS pipetting robot saves precious analyst time and, most importantly, gives us the confidence that the most complex step in the process is being performed without errors," Denise concluded.

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