The 2022 Medical Laboratory Observer (MLO) State of the Industry (SOI) survey on molecular diagnostics revealed some intriguing insights on the use of molecular testing by U.S. labs for COVID-19 diagnosis and variant tracking, how labs are repurposing excess platform capacity for other in-house assays, and their use of technologies in conjunction with molecular testing (e.g., PCR/RT-PCR, NGS) for clinical diagnosis.
Survey respondents also voiced the impact of ongoing supply chain shortages, and shared challenges and solutions for quality control (QC) and quality assurance (QA), including effective handling of questionable results from molecular tests and interventions to reduce potential false positive test results.
This year, 254 lab professionals participated in the survey, with the majority (74%) of respondents in Lab Manager, Administrator, Supervisor, or Lab Director positions, and the majority working in hospital/health system labs (90%).
To bring the survey data to life, we have included commentary from U.S. lab professionals and molecular testing manufacturers on testing trends and best practices for success.
COVID-19 testing
This year’s respondents reported a relatively large drop in SARS-CoV-2 testing demand over the past 12 months, with 17% saying demand has decreased. Only 1% reported a COVID-19 testing volume decline in 2021.
Three-quarters of those surveyed (75%) said their labs perform an average of 0–100 COVID-19 molecular-based tests per day, 15% perform between 101–250, 5% perform between 251–500, 2% between 501–750, and 4% perform between 751–1,000+.
When asked what types of molecular diagnostic tests they use in their laboratories to test for COVID-19, the top three types reported were reverse transcriptase quantitative polymerase chain reaction (rRT-qPCR) at 64% (73% in 2021); rapid antigen tests at 56% (41% in 2021); and rapid molecular tests at 52% (46% in 2021). Far fewer respondents said they use reverse transcription loop-mediated isothermal amplification (RT-LAMP) at 7% (12% in 2021) or recombinase polymerase amplification (RPA) at 4% (7% in 2021).
“We have an organizational decision tree to guide providers for when to order each type of test in an effort to maintain supply availability as well as provide the best testing for each patient who comes in,” said Woodward.
COVID-19 variants
“I believe we are about three-quarters of the way through the game with COVID-19,” said Dr. Manoj Gandhi, Sr. Medical Director at Thermo Fisher Scientific. “We just came through the BA.4, BA.5 variants. It certainly wasn’t as bad as Omicron BA.1. As each of these waves go by, hopefully the severity of disease comes down. And that is what we’ve been seeing. The virus will try to do what it needs to do to continue. But at the same time, our immune system is no slouch either. Eventually, our immune system should win this game.”
When asked whether her lab is tracking COVID-19 variants, Woodward’s answer was “yes and no.” She states: “We continue to track all the COVID-19 in our hospital, and we are submitters to the CDC-LRN variant testing program as well. We do not keep official track of the variants though. Our City Health Department and CDC-LRN laboratories keep track of the variants, so we let them handle that piece.”
Radke’s lab serving Bellin Memorial Hospital and Bellin Health Oconto Hospital in Oconto, Wis. participates in variant tracking by submitting specimens to the Wisconsin State Laboratory of Hygiene, and they monitor trends on their sequencing dashboard.
“The scientific community has been working together the past two years to learn how to adapt to the new normal,” said Dr. Gandhi. A lot of bad things happened but a few good things came out too. One silver lining was the impact of molecular medicine that includes molecular diagnostics and the development of modern-day vaccines. Knowing the variants means we also must adapt some of the vaccines just as we do with influenza. That is why the surveillance part becomes so important because it will determine our future for vaccinations going forward.”
Variant surveillance
While COVID-19 rapid antigen home tests have made testing for SARS-CoV-2 more convenient for patients, it has had a negative impact on genomic surveillance from a sequencing perspective, according to Jeff Field, Chief Commercial Officer, Clear Labs:
Dr. Gandhi views surveillance as “more like a snapshot.” He states: “Some patients are still presenting to the doctor and getting PCR tests, so we have a good representation of what is circulating from a 30,000-foot level. We have gone from BA.1 to BA.2 to BA.4 to BA.5 in a matter of months and we have been able to track that all because of the molecular advancements we have today. Maybe in a small community a variant might come in and we might not know that small level of granularity, but I think overall we have a very good view of what is circulating.”
Wastewater sampling
When tracking COVID-19 variants on a large scale, Francisco Bizouarn, Market Development Manager, Digital Biology Group, Bio-Rad Laboratories, points to the benefits of wastewater-based epidemiology (WBE), which he says scientists use to detect and quantify small concentrations of pathogens coming from a building, a community, or a larger region. The collected data is sent to local authorities to provide early warning of indicators, such as disease outbreaks, days or even weeks before clinical symptoms appear.
According to Bizouarn, scientists have traditionally used quantitative PCR (qPCR) for WBE, but the downside is that wastewater samples contain inhibitors that can disrupt a PCR reaction and reduce the precision of qPCR. An alternative for testing is Droplet DigitalTM PCR (ddPCR) technology, which Bizouarn says has superior accuracy and sensitivity at low concentrations.
“The ddPCR technology separates a sample into tens of thousands of nanoliter-sized droplets that contain no more than a few nucleic acid strands in each droplet,” Bizouarn explains. “If a droplet contains the target nucleic acid sequence, that sequence will amplify, and a fluorescent probe will cause the droplet to light up. If the droplet does not contain the target sequence, the sequence will not amplify, and the droplet will stay dark.”
“The number of positive and negative droplets are counted, analyzed by Poisson distribution, and used to calculate the viral concentration in the original sample,” Bizouarn continued. “Since ddPCR technology takes a binary measurement of whether amplification occurred (independent of amplification efficiency) at all in each droplet, inhibitors have less impact on accuracy. This means ddPCR samples do not need to be purified as extensively (as qPCR), making this precise tool more sensitive to small quantities of pathogenic material.”
Excess analyzer capacity
“As demand for high-throughput SARS-CoV-2 testing decreases, labs can look at other ways to leverage the benefits of new analyzers,” said Allison McMullen, Roche Diagnostics molecular scientific partner. “In many cases, there are a wide variety of molecular assays that can be performed for other important infectious disease areas including other respiratory testing, such as influenza A/B and SARS-CoV-2, virology, sexual health, and transplant health.”
More than half of lab professionals surveyed (54%) said they have excess capacity in analyzers originally purchased to handle COVID-19 testing, compared with 47% last year. For those with excess capacity, 46% said they planned to add new tests to in-house offerings from among those that are currently sent out to reference labs and 10% said they would retire some analyzers.
Another 9% said they would use excess capacity in other ways, such as distributing the analyzers to other facilities; moving laboratory developed tests (LDT) assays to the FDA- approved versions of these analyzers; and keeping the equipment as back up in case COVID-19 test volumes surge again.
“It’s been a crazy few years,” said Blecker Shelly. “First the CoV-2 pandemic, then the Monkeypox outbreak, and continuing significant supply chain issues. It often seems as if no end is in sight.”
Blecker Shelly oversees the microbiology laboratory at Capital Health Medical Center Hopewell in Pennington, NJ and a molecular lab at Capital Health Regional Medical Center in Trenton, NJ. The molecular laboratory was formed in 2021 from a New Jersey Department of Health grant aimed at increasing COVID-19 testing in underserved areas and includes plans for a pilot program that provides testing services in the Trenton community via a mobile testing trailer.
“The grant is ending, and we have developed a business plan for bringing additional assays in house using existing equipment,” said Blecker Shelly. “First will be all the assays the Occupational Health department currently sends to a reference lab when onboarding new employees (e.g., MMR, etc.). One of our instruments in the molecular lab can perform that test menu with decreased turnaround time and cost, expediting onboarding of much needed hospital personnel.”
When looking at new applications for molecular testing platforms intended for COVID-19, Dr. Gandhi says the fundamental technology stays the same. And since it stays the same, it is very easy for a lab to implement it for other indications.
“Let’s say you have everything set up for COVID. You take the same infrastructure and add to the menu and say, ‘now I can do this not just for COVID but also for Monkeypox,’ for instance,” Dr. Gandhi explains. “The sample type might change – a nasal swab versus a lesion swab – but on the back end in the lab with just a few minor modifications you are essentially able to adapt the technology from one application to another relatively easily.”
“We purchased additional point-of-care (POC) analyzers to handle the increased COVID-19 testing requests. In the emergency and urgent care areas, these analyzers have been repurposed to run strep, RSV and flu testing,” said Woodward. “However, in the areas that were performing screening tests for surgeries and direct admits, there are no plans for those analyzers at this time.”
While some labs have the benefit of repurposing their analyzers, one of the molecular platforms Radke’s lab has in place for COVID-19 testing is limited in its testing scope as he explains:
“Unfortunately, an instrument we purchased to address COVID-19 testing volumes did not have a very expansive menu offering that fit in well with our other platforms so at this point we have actually placed this instrument into storage, hopefully never having to reinstate for COVID purposes.”
Molecular testing in general
With regards to non-COVID-19 molecular-based tests, three-quarters of those surveyed (75%) said their labs perform an average of 0–100 per day, 13% between 101–200, 5% between 201–300, 2% between 301–400, and 5% 400+ tests (down from 12% in 2021).
When asked if they use specific technologies with their molecular testing, the majority of respondents (79%) said they use PCR/RT-PCR. There was a large drop in those using next generation sequencing (NGS) compared with last year, at 7% down from 22% in 2021. Reported use of DNA/genetic testing declined sharply as well, at 6% this year, compared with 30% last year.
“We’re using RT-PCR and PCR,” Curtis commented. “The PCR is better for all, including asymptomatic patients, whereas the RT-PCR is ok for symptomatic. Both analyzers are fairly simple to use.”
“Today we really only employ PCR/RT-PCR and TMA molecular testing technologies on site,” said Radke. “We leave the flow, NGS, and other molecular testing for the big industry players.”
The survey results also showed decreases in the use of flow cytometry at 5% (12% in 2021), and liquid biopsy screening for cancer genes with bodily fluids at 3% (6% in 2021). A further 2% of respondents said they use other technologies in conjunction with their molecular testing, such as nucleic acid amplification testing (NAAT), transcription-mediated amplification (TMA), helicase dependent amplification (HDA), and isothermal amplification.
Next up in next generation sequencing
“We feel this is a curtain raiser for a lot of applications coming especially in the infectious disease space,” said Thwar. “For example, there are areas where super bugs are emerging, and this has been a threat even before the pandemic came. Not many technologies can provide that kind of granular detail on whether it is a super bug or not, if the bug is antibiotic resistant, how it is transmitted across different patient situations and related downstream events. That’s where sequencing can be a very effective tool.”
Field said most labs that perform NGS today are large, centralized labs that have the skilled labor to perform the manual library preparation, complex workflows, and “tedious pipetting” required of most NGS platforms. Once sequencing is performed, the lab must perform data analysis on the sequencing files, which Field says requires skilled scientists from a bioinformatics standpoint.
“Our vision is to liberate genomics to all,” said Field. “Clear Lab has a fully automated end-to-end solution where the lab technician loads the samples and reagents, pushes a button, walks away and 24 hours later, the lab gets the results. We make sequencing so easy anyone can do it. By overcoming all those adoption barriers, we can now bring genomics to the masses.”
Supply chain challenges
Supply challenges around molecular testing is a significant problem based on the survey results, with 85% of lab professionals saying a lack of sample-related products has impeded their testing capacity at times, up from 58% last year. Only 15% of respondents said they have adequate testing supplies to meet testing demands, down from 42% in 2021.
“The lab has become sort of a ‘supply chain enforcer,’ if you will, and for a while would only release blood tubes for specific requests and only when the orders were pre-placed in the computer. The staffing demand for these processes was huge. The number of phone calls for blood tests multiplied, the staff would have to check the computer for orders, beg for orders to be placed, and then individually release blood tubes when all parameters were met.”
About half of survey respondents said they are challenged with sourcing swabs/consumables (50%), winged blood collection sets (48%) and controls/reagents (46%), with reported shortages in this final category more than doubling since last year (up from 21% in 2021).
“(Our supplier) had us on allocation for quite a while, so we had to place standing orders for reagents, which incurs a large up-front cost,” said Curtis. “We never ran out of reagent, but sometimes inventory got pretty low before resupply.”
Other supply challenges include testing kits for SARS-CoV-2 (43% of respondents), transport media (38%), PPE (29%), other molecular testing kits (23%), pipettes (20%), contrast media for radiology (13%), and cannula syringes (5%). Among those surveyed, 10% said they are experiencing shortages of other supplies, with pipette tips and lab plastics among the most reported.
“In all areas of the lab, we continue to see reagents on eternal back order due to plastic and glass shortages,” Woodward commented. “Many tests have been completely substituted, or we have validated multiple types of tests, so that we can interchange them depending on reagent availability. This is creating a proficiency testing nightmare as we don’t have all types of testing available for proficiency testing at the time those samples arrive. So, there are lots of explanations occurring and extra comparison tests being completed to meet the laboratory standard requirements as well.”
In his lab, Radke has witnessed a “constant fluctuation of multiple different products.” He states:
“Overall, it seems supply chain issues have largely eased by comparison to the prior 18–24 months but between Pathology and Clinical Laboratory there are always items being impacted we wouldn’t have anticipated. Now we are hearing of national CO2 shortages that have us contemplating operational changes in Microbiology if we run out of CO2.”
When asked what advice she has for labs in maintaining a supply of testing products when challenged by supply chain issues, McMullen states:
“Laboratories should be specific in asking questions about how their manufacturers are planning ahead for minimal disruption to the supply chain. In addition to sharing how they plan to secure enough materials to meet the needs of laboratories, manufacturers should also be able to demonstrate how they use their own SARS-COV2 and influenza predictive modeling — or that of the Centers for Disease Control and Prevention — to anticipate demand.”
Molecular testing quality assurance
Again this year, MLO asked lab professionals questions related to molecular diagnostic testing quality controls and assurance. When faced with questionable results from molecular tests, 65% of those surveyed said they repeat the test with a different employee/equipment/test, 20% said they send the results to another lab for verification and to perform a second test; and 7% said they verify that all preanalytical steps were performed correctly.
“We handle questionable results first by looking at patient clinical information,” said Radke. “Depending on findings, we may repeat the same sample on a secondary molecular platform, review Ct values, melt curves, or RLU values. We assess for potential cross-contamination with other nearby positive samples and if necessary, we may suggest recollection.”
To reduce the number of potential false positive test results, 23% of respondents said they verify that all preanalytical steps were performed correctly; 22% refer to quality assurance program guidance; 21% repeat the test with another method and compare the results; and 19% repeat the test with the same sample and new extractions.
“We continue to recommend and utilize antigen testing as our first line testing in the emergency and urgent care areas because it is less likely to give a false positive due to prior infection,” said Woodward. “If the test comes out negative and the provider really suspects COVID-19 diagnosis, then we recommend either the 4-plex or a full respiratory panel for children.”
“False positives and questionable results and the actions taken when they happen are really platform or test dependent,” Blecker Shelly commented. “Most molecular testing platforms have some option to say the test didn’t work. When this happens, some platforms and tests will tell you to repeat testing, and others to request a different sample. Those are the typical actions taken.”
Radke says his lab adheres to general molecular best practices of frequent cleaning, sterile technique, handling in a biological safety cabinet (BSC), as appropriate, keeping samples capped and closed, multiple glove changes, etc. “Where it becomes more complicated to reduce false positives are at the sites collecting patient samples as we aren’t out there with direct oversight to make sure they are changing gloves frequently, packing and storing appropriately, etc.,” he added.
“Molecular tests are highly sensitive and specific,” said Rajasri Chandra, MS, MBA. “That said, occasional false positive results may occur.” She presents the following errors that may occur during the different stages of conducting molecular tests and steps that can be taken to minimize the chances of false positive results:
At preanalytical stage
Possible causes of false positive results:
- Mislabeling of specimens, contamination at the time of specimen collection or transportation
Steps to mitigate:
- Specimens should be collected in sterile vials and labeled immediately with two identifiers, properly packaged, and transported to the laboratory.
At the analytical stage
Possible causes of false positive results
- Reagent contamination, cross-contamination
Steps to mitigate:
- Perform a quality assurance check of every reagent lot received from suppliers before use.
- Mix reagents in a separate room that has positive air pressure to keep contaminants out while wearing a clean lab coat and gloves.
- Briefly centrifuge reagent tubes before opening to avoid the generation of aerosols.
- Aliquot reagents to avoid multiple freeze-thaws and reduce chance of contamination of master stocks.
- Prepare samples in a separate room, adding the sample to the reagent mix in a laminar flow hood.
- Ensure proper pipetting technique — aspirate and dispense accurate volumes and avoid splashing during liquid dispensing. Open and close all sample tubes and reaction plates carefully to avoid splashing of samples. Briefly spin the tubes/plates before opening to prevent aerosols when opening them.
- Include positive control to ensure the extraction and amplification process has been performed correctly and negative control or a non-template control (NTC) to verify the absence of contamination in reagents, consumables, and the environment.
- Follow good laboratory practice: wear fresh gloves when performing PCR work, perform aseptic cleaning of all work surfaces — bench tops, pipettors and all touch points — before and after PCR work.
Post-analytical stage
Possible cause of false positive results
- Transcription error at the time of reporting
Steps to mitigate
- Ensure all results and interpretations are reviewed prior to reporting.
With regards to preventing sample contamination, McMullen offers the following advice:
“The first step is to put measures in place that can lessen the possibility of contamination. It’s important to have appropriate molecular lab design, which separates pre- and post-PCR activities. Employing good lab techniques is essential. These include dedicated personnel, equipment, and consumables; good pipetting techniques; and appropriate controls that can identify potential contamination events. Quality monitoring programs are also key to help ensure accuracy. When questionable results arise, determining if the results make sense for that specific patient and looking for unexpected patterns of results may help guide next steps for troubleshooting.”
Molecular quality control challenges
Dr. Newton notes three major challenges that labs are facing when it comes to molecular testing quality control (QC):
1. Limited molecular testing standards: For many quantitative molecular assays (viral loads), there is a lack of clinical data to standardize how molecular tests are interpreted. Therefore, lab professionals have trouble differentiating between a magnitude of change that is clinically significant versus a magnitude of change that is simply variability intrinsic in the assay. Laboratorians also struggle to know how much intrinsic variability exists with their assays, as Dr. Newton explains:
“The criteria for quality control acceptance that laboratories use is often solely what is provided by the assay manufacturer. Therefore, the acceptable ranges for daily run QC are often broad, making it difficult to fail, because you don’t know for sure what magnitude of change is appropriate for clinical purposes, and how much intrinsic variability is acceptable for a particular assay.”
2. Lack of assay commutability: Unlike assays where there are in vitro diagnostic (IVD) solutions and international standards, most quantitative molecular infectious disease tests do not have commutability between labs for patient management and clinical study purposes.
“So, it is very difficult to efficiently compare results from one lab to another — and laboratory-developed tests can’t be compared at all,” said Dr. Newton. “This makes it difficult to do the studies to generate the data needed (as described above) to help determine what magnitude of change is clinically significant.”
3. Manual data management: From an operational standpoint, most molecular labs are using very manual, labor-intensive methods to manage their QC data. Without the utilization of more sophisticated tools, labs are restricted to performing only simple data collection and superficial analysis.
“As a result, most molecular laboratories generate basic Levey-Jennings charts to monitor QC trends over time, but really need to do more than that to determine if there are systematic issues with their test systems,” comments Dr. Newton. “The informatics and data management tools needed to conduct deeper analysis from a molecular standpoint are not yet widely available.”
When asked what steps labs can take to improve the quality of testing and results, Dr. Newton offers the following recommendations:
- Make assays commutable: Generate data or perform studies to assess and establish clinical performance, then share this data with other labs using data management tools, such as Unity.
- Prioritize QC: Perform thorough, daily QC information evaluation.
- Leverage standards: Use international standards as a reference where available. Use secondary standards that are traceable to an international standard, which can be helpful in optimizing assay performance.
- Assess assay robustness: Regardless of the QC data management tools being used (commercial, homegrown, etc.), labs should assess the robustness of their assays and know how much variation exists within each test so that the labs and clinicians understand how they perform and what changes are significant.
- Apply QC rules: Once a lab understands its test variability, it can start to apply appropriate QC rules depending on how much variability the assay has. This will optimize the error detection rate and minimize the false rejection rate.
Looking ahead
Dr. Gandhi recommends labs look to the near future when determining their molecular testing needs, starting with the 2022–2023 flu season. He states:
“It is going to be an interesting season because we have gone back to some sort of normalcy. Children are back in school, people are back in the workforce, masking has become optional, people are traveling internationally, countries have opened back up, etc. This might be the first season where we see what truly happens. Is flu going to come, and if so, with COVID still circulating, what is this flu season going to look like?”
He notes how COVID-19 and the flu present with very similar symptoms, which will make it challenging for healthcare providers to distinguish one from the other without testing. He stated:
“When you individually test for COVID or flu, you’re missing the situation where they can occur together. And that’s where I think some of the bigger issues will arise. If a patient has both COVID and flu together, we don’t know how that patient will do. Will they really fare worse than a patient with one or the other respiratory illness? That’s why we have multiplex tests to detect both COVID and flu in the same test.”
Long term, Dr. Gandhi says the lab community should look at how molecular diagnostics has evolved, how people have become more comfortable with the technology because of the experience gained during pandemic, and how it can be used beyond COVID-19 testing.
“With molecular testing, clinicians and patients don’t have to wait seven days to get a result from culture. So why wouldn’t I do it for other conditions and get my patients on the right treatment sooner? It opens a wide range of opportunities, such as testing of patients with GI infections, UTIs, STIs, etc. If a patient can be tested for a STI in a doctor’s office and get the results in 15 minutes, it could prevent them from spreading it to others. That is the promise of molecular diagnostics.”
