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Five-Minute Fentanyl Assay in Development Takes Aim at Opioid Crisis


NEW YORK – University of Pennsylvania researchers have validated a gold nanoparticle-based lateral flow assay for fentanyl screening at the point of care.

Designed to provide results in five minutes wherever a patient is located, the test would fill an unmet clinical need by enabling quick identification of patients who have overdosed using the lethal drug, said test developer Ping Wang, an associate professor of pathology and laboratory medicine at the University of Pennsylvania.

Wang along with her colleagues completed a study published last week in Clinical Chemistry that describes the development and clinical validation of the test. Their lateral flow assay detected fentanyl and its metabolite, norfentanyl, with high specificity and sensitivity in samples that were also tested for comparison using liquid chromatography tandem mass spectrometry (LC-MS/MS).

The researchers believe that their lateral flow assay may be among the first that can detect fentanyl at 1 ng/mL and norfentanyl at 10 ng/mL, which are clinically relevant cut offs and suitable for use at the point of care. Other commercial lateral flow assays have higher cutoffs that yield more false-negative results during screening, Wang said.

The capability to detect norfentanyl is important, she noted, because fentanyl metabolizes rapidly, and assays that measure only fentanyl have a shorter window for accurate detection.

The UPenn group began developing the test after observing a startling surge in deaths from the fentanyl overdoses.

Fentanyl is a synthetic opioid that has been approved for treating severe pain. However, most recent cases of fentanyl-related overdose and death in the US are connected with the drug being sold illegally for its heroin-like effects.

For example, Philadelphia has witnessed "a significant rise" in unintentional drug overdose deaths, the majority of which stem from opioids, according to the city's Department of Public Health. In 2017, fentanyl surpassed heroin as the leading drug involved in overdose deaths there. The clinical need for a point-of-care diagnostic test became apparent to clinicians, emergency department physicians, and laboratorians at the UPenn's medical center and its laboratory last summer when there was a spike in the number of patients presenting with overdose symptoms.

According to Ceres Nanosciences Chief Business Officer Robert Barbero a point-of-care test "can give you a granular view" of whether a specific drug such as fentanyl has been used and when it has been used.

The National Institutes of Health has awarded Ceres Nanosciences a $224,750 grant to develop technology to enhance the detection of fentanyl in urine and oral fluids. The firm works with diagnostics companies to enhance the sensitivity of laboratory and lateral flow point-of-care tests, including those that detect fentanyl and its analogs, he said.

One of the challenges associated with abuse of fentanyl and other opioids is that people can more easily misuse drugs and go undetected in the absence of highly sensitive tests, Barbero said.

Such a lack of detection and confirmation is "one of the contributors to a crisis that we're experiencing right now," he added.

In her laboratory, Wang and her colleagues had recently begun using a high-throughput assay from Freemont, California-based Ark Diagnostics, cleared by the US Food and Drug Administration in June 2018, which detects both fentanyl and norfentanyl.

"The Fentanyl Assay available from Ark Diagnostics … is typically run in the central laboratory and can provide physicians quick results — approximately a 10-minute turnaround time," said Erin Celano, global marketing manager for Syva & Drug Testing Diagnostics at Siemens Healthineers, which distributes Ark's test. "Identifying more true positives — no matter the method — supports patients seeking addiction treatment," she said.

Many healthcare facilities do not currently have fentanyl testing on their assay menus, and that is hampering patients’ ability to seek addiction support, she said, adding that insurance providers often require a positive drug test to cover treatment costs, or patients can be denied admission completely.

In the context of a patient emergency from a fentanyl overdose, a high-throughput assay may not be as effective as a highly sensitive point-of-care assay, according to Wang.

Wang said use of a laboratory in such circumstances may slow down treatment because of a longer time-to-result and because testing is not done until after a patient has reached the emergency room. The time to receive a result from a central lab depends partly on how quickly a sample is transported from the emergency room to the lab, but it can take an additional hour to receive test results after the ambulance has reached the emergency department, she said.

Naloxone (Emergent BioSolutions' Narcan) has become a widely used treatment for opioid overdoses, and the US Centers for Disease Control and Prevention has suggested expanding its use as a safe and effective treatment for patients suspected of having overdosed on fentanyl.

"When you see somebody by the road, for example, and you are suspicious of a fentanyl overdose, you want to administer naloxone as quickly as possible but there are currently no FDA-cleared [point-of-care] tests you can use to confirm your suspicions," Wang said.

For an experienced emergency medical technician who is familiar with opioid overdose symptoms, the point-of-care test strip may be used for confirmation that the patient has taken fentanyl, Wang said. With that confirmation, the emergency medical technician can immediately alert the emergency department or hospital receiving the patient, she said.

That provides a medical benefit because "fentanyl-overdosed patients typically require a higher dose or multiple repeated doses of naloxone after arriving in the ER, compared with other opioids," Wang said.

She noted that for someone who is not familiar with opioid overdose symptoms, such as a family member or bystander who finds the patient and suspects they may have overdosed, "the strip would be very useful in pinpointing the offending agent and leading to subsequent naloxone administration."

The UPenn group designed its assay from off-the-shelf components, purchasing reagents and cellulose membranes and using them as a basis for the point-of-care platform. "We needed a platform that is inexpensive, easy to use at the point of care, and rapid, so we knew that a competitive assay that uses lateral flow technology would satisfy those criteria," Wang said.

In the UPenn competitive assay, fentanyl-bovine serum albumin competes with target fentanyl molecules in an attempt to bind to antibody-gold nanoparticle (Ab-AuNP) conjugates that are themselves bound to one red test line and one red control line. As fentanyl and norfentanyl concentration in a sample increases, the color intensity of the test line decreases until it disappears and indicates a positive test result.

In their study, the investigators collected urine samples from two cohorts of emergency department patients that they tested using the lateral flow assay and liquid chromatography tandem mass spectrometry (LC-MS/MS). One cohort consisted of 218 consecutive emergency department patients with urine drug-of-abuse screen orders, and the second cohort consisted of seven emergency department patients with a clinically suspected fentanyl overdose.

There was no cross-reactivity with amphetamine, cocaine, morphine, tetrahydrocannabinol, methadone, buprenorphine, naloxone, or acetaminophen, but the researchers cautioned that urine samples from patients taking the antipsychotic drug risperidone (Johnson & Johnson's Risperdal) may screen as false positive for fentanyl using the lateral flow assay. Confirmation of testing results using mass spectrometry is needed when a patient's prescription or ingestion history includes risperidone, they said.

In the 218 emergency department patients, the prevalence of cases with at least 1 ng/mL of fentanyl or 10 ng/mL of norfentanyl was 5.5 percent. The sensitivity of the lateral flow assay was 100 percent, and the specificity was 95 percent. Its positive and negative predictive values were 92.3 percent and 100 percent, respectively. Further, the concordance of the lateral flow assay with LC-MS/MS was 100 percent in the seven suspected fentanyl overdose cases.

Wang said that her group expects long term to seek FDA clearance that would enable broad clinical use of the assay. However, in the near term, the group is interested in conducting future studies in populations with higher fentanyl prevalence, larger sample sizes, and wider fentanyl and norfentanyl concentration distributions. Further, they are interested in investigating the clinical utility of the assay in settings other than the emergency department, such as in the field or in emergency response vehicles.

The researchers are also looking to optimize the assay to further reduce its time to result, Wang said. They are investigating use of saliva as a sample because it can be difficult to obtain urine samples from patients during emergencies.

Wang said that the group is applying for National Institutes of Health funding and seeking partnerships with commercial entities that could be interested in advancing the assay toward commercialization.