Molecular Point-of-Care Testing in Clinical Laboratories

LABORATORIANS CAN LEAD A NEW ERA IN RAPID TESTING WITH EXPERTISE IN QUALITY CONTROL AND RESULT INTERPRETATION
For those outside of the infectious diseases field, clinical microbiology might conjure an image of microbiologists manipulating petri plates full of bacteria. Although microbiologists still work on the bench with organisms, culture-only approaches have given way to molecular assays. In fact, most microbiology laboratories have decreased or discontinued using routine viral culture, a time-consuming and labor-intensive process.
Molecular microbiology has revolutionized the virology field in particular. These methods have cut down turnaround times (TAT) from weeks to mere hours, increased the sensitivity and specificity of viral detection, and allowed for quantification of viral load. Molecular assays have also improved diagnosis of enteric pathogens, including C. difficile, and organisms that are non-culturable using routine culture methods such as Toxoplasma gondii, Bartonella, and Leishmania.
Molecular microbiology approaches are based on detecting targeted portions of microbial genetic material, either DNA or RNA, that have been extracted directly from a patient sample. With molecular assays utilizing polymerase chain reactions (PCR), targeted genetic material is amplified, making a large number of copies so that an instrument can detect even very small amounts of microbial genetic material.
These molecular methods are normally deemed moderate- or high-complexity under CLIA and require extensive training, sterile technique, and post-analytical analysis that is not feasible in many laboratories. Most microbiology PCR assays take hours, but newer sample-to-answer assays are streamlined and involve minimal processing and hands-on time. These simplified molecular assays enable more technologists and laboratories to perform these tests outside of clinical microbiology laboratories, increasing their utilization and the number of personnel involved in the process.
POINT-OF-CARE TESTING: NOT JUST AT THE PATIENT BEDSIDE
Innovations in molecular assays—especially on the point-of-care testing (POCT) front—have spread this testing from molecular diagnostics laboratories into clinical microbiology laboratories—and now into general laboratories and even clinics and exam rooms (1).
Access to sensitive and rapid infectious disease diagnostic assays is essential for accurate diagnosis, effective treatment, and timely infection control, making POCT vital to reducing TAT. Although people think of POCT as near-patient diagnostic assays, POCT can be performed virtually anywhere that possesses a valid CLIA certificate of waiver.
A waived test is defined as a simple assay that has low risk for erroneous results. In using a POC test, the manufacturer’s protocol must be followed exactly. Any modification, whether a specimen source (such as nasal versus nasopharyngeal) or specimen handling (manually diluting a specimen before loading), changes the CLIA status from waived to non-waived and prohibits a test from being performed as a waived test. Waived testing can be performed in a moderate- or high-complexity lab environment. Laboratories must maintain training records for personnel performing the assay regardless of the complexity of the lab. Further, they must assess competency for all operators twice during the first year of performing the assay and then annually thereafter.
FROM ANTIGEN-BASED TO MOLECULAR PLATFORMS IN POCT
In the microbiology field, clinics have long used POCT that detects antigens or antibodies for infections such as influenza, mononucleosis, and group A Streptococcus (GAS) (2). These assays offer rapid, easy-to-use sample-to-answer options. Although their fast TAT enables patients to be treated promptly, these assays have lower sensitivity and specificity than their laboratory molecular counterparts. In the case of influenza, a molecular assay should be performed following a negative influenza antigen-based test due to false negatives occurring in high-prevalence populations.
Licensed technologists perform high-complexity molecular assays in molecular or microbiology laboratories. Although incredibly valuable, these assays suffer from increased TAT resulting from specimen transport delays, batch testing, complex multistep testing, or set performing schedules. Molecular POC tests are now emerging that circumvent these obstacles. Molecular CLIA-waived POC tests are able to detect influenza, respiratory syncytial virus (RSV), GAS, and a group of respiratory pathogens.
One example of the shift from antigen-based to molecular diagnostics in the POCT setting involves detecting GAS, which is responsible for an estimated 15%– 30% of sore throats in pediatric patients. While rapid antigen-based assays enable providers to make diagnoses in clinics, these assays lack sensitivity and specificity compared to conventional bacterial culture and have the added disadvantage of being subjective and difficult to interpret. More sensitive methods, including culture and molecular-based tests, are recommended when an antigen test yields a negative result because of the potential for this result to be a false negative.
Molecular GAS POCT enables a clinician to provide, or exclude, a diagnosis and administer treatment while a patient is in a clinic. Moreover, molecular assays have demonstrated improved sensitivity compared to rapid antigen detection tests, eliminating the need for secondary confirmation of negative results and resulting in significantly more appropriate antibiotic use, including avoidance of antibiotic use for viral infections (3–5).
THE ROLE OF MOLECULAR POCT FOR INFLUENZA AND RESPIRATORY ILLNESSES
Influenza, a seasonal respiratory virus, was responsible for an estimated 14 million to 21 million medical visits in the United States alone since October 1, 2019, according to the Centers for Disease Control and Prevention (CDC). Unlike the vast majority of respiratory pathogens, influenza has an approved antiviral treatment. Unfortunately, for maximal effectiveness, this antiviral must be administered within 48 hours of symptom onset, requiring a physician visit and diagnosis within that time frame. Often patients do not present to their physicians until symptoms have worsened, narrowing the available time to treatment. Moreover, due to symptom overlap with other seasonal respiratory viruses, influenza is difficult to diagnose based on clinical presentation alone. This makes influenza an ideal target for POCT.
As previously discussed, antigen-based influenza POCT is popular in outpatient and emergency department settings, but lacks sensitivity (50%–90%) compared to molecular methods. The first molecular influenza POC test was approved in 2015, and since then several waived molecular POC tests have entered the market. These include Alere i Influenza A&B, Accula Flu A/Flu B, BioFire FilmArray RP EZ, Xpert Xpress Flu, and cobas Liat Influenza A/B. These assays take 15 minutes to 1 hour to run, and aside from Xpert Xpress Flu, only process one sample at a time.
More influenza POC tests now are incorporating RSV into their panels. Although RSV does not have a treatment, this virus is one of the leading causes of infant hospitalizations and also is problematic in the elderly, so identifying it is essential. Since RSV symptoms and seasonality overlap with influenza and other seasonal respiratory viruses, a molecular assay is necessary for diagnosing and managing this illness.
Molecular influenza testing has been shown to prevent unnecessary hospitalizations and antibiotic prescriptions, allow antivirals to be administered before patients are discharged, and directly guide isolation precautions (6). While the benefit to patients is evident, the advantages to medical staff, testing personnel, overall hospital function, and other patients are also significant.
CDC establishes guidelines for patient precautions based on the suspected infectious agent. Standard precautions are observed for all patients. For most seasonal respiratory viruses, including rhinovirus, healthcare professionals follow contact precautions, meaning that staff wear gloves when in contact with a patient, practice good hand hygiene, and wear gowns if they expect to come in contact with blood or bodily fluids.
For influenza, laboratories must follow droplet precautions. Patients must wear masks when not in their assigned rooms, and healthcare workers should don face masks when in the room of a patient with suspected or confirmed influenza. Based on current CDC recommendations, influenza positive patients should be placed in private rooms, and droplet precautions should be implemented for 7 days after illness onset or after a full 24 hours following resolution of symptoms.
Because respiratory viruses cannot be distinguished on the basis of symptoms, ruling out or confirming influenza as soon as possible is crucial. If a patient is negative for influenza, droplet precautions might not be necessary. This would reduce the strain on availability of individual rooms and usage of personal protective equipment (PPE). On the other hand, if a patient is positive for influenza, the ability to provide that diagnosis, appropriately treat, and discharge that patient as quickly as possible reduces the number of people exposed to this virus.
During influenza season a surge of patients visits urgent care, physician offices, and emergency departments. This leads to a shortage of space, healthcare workers, and PPE, for example. Thus, rapid diagnosis of any respiratory illness can allow for shorter wait times and visit times, reducing the burdens on hospitals.
BEST PRACTICES AND QUALITY CONTROL
Unlike viral culture, waived molecular testing poses minimal risks to the personnel performing the assay. In order for a test to be considered waived, a patient sample cannot be manipulated (diluted, centrifuged, etc.) in a way that is not specified by the manufacturer. This reduces the risk of aerosols, spills, or exposures. Furthermore, many of these assays are closed systems, meaning that the amplification and detection steps occur in a contained space. This prevents contamination of the environment with genetic material and organisms, further mitigating the risk. The greatest risk occurs during direct contact with a patient during specimen collection.
Test performing areas should be kept clean and organized to prevent cross contamination. Surfaces should be disinfected daily and also immediately disinfected following spills or visible contamination. As with any human specimen that would be processed in chemistry, hematology, or any clinical laboratory, all specimens should be handled using universal precautions and according to the notion that any sample might contain infectious pathogens. Testing personnel are required to wear appropriate PPE, including disposable gloves that should be changed between runs. In addition, test reagents must be stored and handled according to the manufacturer’s instructions.
The current CLIA-waived molecular POC tests are qualitative assays, meaning that they only provide a positive or negative result. In some cases, an invalid result can occur because of an instrument, specimen, or reagent. Specimens producing an invalid result should be repeated.
Quality control (QC) confirms that an assay is functioning as expected by the manufacturer. According to CLIA regulations, QC must be performed according to the manufacturer’s instructions for waived testing. If the manufacturer does not define QC, the testing institution must define a policy that follows good laboratory practices. Best practices include running daily external positive and negative QC, even in a CLIA-waived setting. Documentation of controls and results is recommended. When QC fails, patient results should not be reported to avoid incorrect results. The problem should be identified and corrected before proceeding with patient samples.
QC metrics often include external and internal controls. An internal control is incorporated into each sample while an external control—which should include a positive and negative sample—is run as individual samples. The internal control can serve as a processing control or control for that test. The internal control in molecular-based tests is often a DNA extraction control, which indicates whether or not a patient sample was properly extracted, a necessary step in order to receive a correct result. External controls evaluate whether an instrument provides correct results (for example, a positive external control is detected as positive) and should mimic patient specimens.
LIMITATIONS OF MOLECULAR POCT
Since both POCT and non-POCT molecular tests aren’t able to distinguish between live or dead organisms, they can’t be used as a test of cure and might produce false positives due to residual nucleic content from past infections. On the other hand, false negatives can occur due to viral genomic shifts and drifts, which is a limitation of all molecular assays. This was observed in 2014-2015 for clades of influenza A H3N2. Molecular assays use primers, which target specific areas of genetic material that are encoded by a virus or group of viruses. These primers are designed to match a conserved region of DNA or RNA, depending on the type of virus.
When the targeted genetic material is present, the primers bind to the DNA or RNA segment and that region is amplified and detected by the assay. However, when genetic changes occur, such as insertions or deletions, the primer might no longer match the viral genetic material. In this case, the primer cannot bind, and that sequence will not be amplified, resulting in a false negative due to lack of detection of that sequence. As such, new molecular POC tests will need to be developed to address novel viruses.
The increased sensitivity coupled with use by non-molecular laboratory personnel poses a risk of assay failure and environmental cross contamination. For instance, in clinics that administer influenza vaccine, contaminated instrumentation can produce false positives. However, multiple studies have demonstrated that failure rate and environmental contamination is low. One study found that the average failure rate for the Liat GAS assay was 6.6%, while environmental contamination was not detected after performing the assay on swabs on the instrumentation weekly (7). In another study where the cobas Liat system was intentionally contaminated with flu A/B-positive control material, this contamination was not found to affect any of the negative control tubes in runs immediately after assessing system contamination, thus showing that the contamination did not impact the integrity of results (8). Given the simplicity of the current molecular POCT with the sample-to-answer format, user variability, opportunities for contamination, and human errors are minimized if protocols are followed.
One potential source of human error involves results reporting. Although POCT instrumentation provides a clear positive, negative, or invalid result, the platforms are not usually interfaced to laboratory information systems, meaning that results must be manually entered. Care must be taken to avoid transcription or other data entry errors.
Space and cost limitations are also a concern. Molecular POC tests are more expensive than antigen-based tests, but have an increased sensitivity and specificity. Although molecular instruments are typically compact, many platforms can only run one sample at a time. In a large emergency department or urgent care clinic, several instruments would be required to meet the demand for influenza testing.
WHAT’S NEXT FOR POCT
Sexually transmitted infections (STI) have garnered attention in the molecular POCT field as rapid diagnostics allow for prompt treatment and consultations with patients, who might otherwise be lost to follow-up. Given the public health concerns associated with STI, these tests really need to be accurate. Development of and investigations into such assays are already underway worldwide including a molecular POC test for Trichomonas, Chlamydia trachomatis, and Neisseria gonorrhoeae (9).
Although there are few approved analytes for molecular POCT in the U.S., the ability to rapidly test and respond with effective treatment, when applicable, makes POCT an attractive methodology for a variety of infectious diseases, including parasites, fungal infections, STI, and more.
Molecular POCT is increasingly advantageous in resource-limited settings, which typically have lengthy TAT and not enough trained technologists to perform high-complexity assays. Moreover, molecular testing closer to patient care, whether in  generalized hospital laboratories or in emergency departments, mitigates the challenges faced with molecular testing in centralized clinical microbiology laboratories as previously discussed. With novel POCT on the horizon, future studies are warranted to determine cost savings, antimicrobial usage, TAT, patient impact, and how to best implement in non-microbiology clinical laboratories and clinics.