Laboratorians tin can lead a new era in rapid testing with expertise in quality control and result interpretation

For those exterior of the infectious diseases field, clinical microbiology might conjure an paradigm of microbiologists manipulating petri plates full of leaner. Although microbiologists withal work on the bench with organisms, culture-merely approaches have given way to molecular assays. In fact, most microbiology laboratories accept 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 accept 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 as well improved diagnosis of enteric pathogens, including C. difficile, and organisms that are not-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 straight 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 minor amounts of microbial genetic material.

These molecular methods are normally deemed moderate- or high-complication under CLIA and require extensive training, sterile technique, and post-analytical analysis that is not feasible in many laboratories. Virtually microbiology PCR assays take hours, but newer sample-to-reply assays are streamlined and involve minimal processing and easily-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 procedure.

Point-of-Care Testing: Not Just at the Patient Bedside

Innovations in molecular assays—especially on the signal-of-intendance 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 (i).

Admission to sensitive and rapid communicable diseases 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 about-patient diagnostic assays, POCT can exist performed virtually anywhere that possesses a valid CLIA document of waiver.

A waived test is defined as a uncomplicated assay that has low take chances 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 condition from waived to not-waived and prohibits a test from being performed as a waived test. Waived testing tin can exist performed in a moderate- or high-complication 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 starting time year of performing the assay and 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 grouping A Streptococcus (GAS) (2). These assays offer rapid, easy-to-use sample-to-answer options. Although their fast TAT enables patients to exist treated promptly, these assays take lower sensitivity and specificity than their laboratory molecular counterparts. In the case of influenza, a molecular assay should exist performed post-obit a negative influenza antigen-based examination 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, circuitous multistep testing, or gear up performing schedules. Molecular POC tests are now emerging that circumvent these obstacles. Molecular CLIA-waived POC tests are able to detect flu, respiratory syncytial virus (RSV), GAS, and a group of respiratory pathogens.

One instance 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 civilisation and take the added disadvantage of existence subjective and difficult to interpret. More sensitive methods, including culture and molecular-based tests, are recommended when an antigen exam yields a negative result because of the potential for this issue to exist a simulated 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 take demonstrated improved sensitivity compared to rapid antigen detection tests, eliminating the demand for secondary confirmation of negative results and resulting in significantly more than appropriate antibody use, including avoidance of antibiotic employ for viral infections (3–five).

The Role of Molecular POCT for Influenza and Respiratory Illnesses

Influenza, a seasonal respiratory virus, was responsible for an estimated 14 million to 21 meg medical visits in the United states alone since October 1, 2019, according to the Centers for Illness Command and Prevention (CDC). Unlike the vast bulk of respiratory pathogens, flu has an approved antiviral treatment. Unfortunately, for maximal effectiveness, this antiviral must be administered within 48 hours of symptom onset, requiring a doc visit and diagnosis inside that time frame. Oftentimes patients do non nowadays to their physicians until symptoms have worsened, narrowing the available time to treatment. Moreover, due to symptom overlap with other seasonal respiratory viruses, flu is difficult to diagnose based on clinical presentation solitary. This makes flu an ideal target for POCT.

Every bit 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 marketplace. These include Alere i Flu A&B, Accula Flu A/Flu B, BioFire FilmArray RP EZ, Xpert Xpress Flu, and cobas Liat Influenza A/B. These assays accept 15 minutes to 1 hr to run, and aside from Xpert Xpress Flu, just 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 1 of the leading causes of infant hospitalizations and likewise 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 role, and other patients are besides 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 proficient paw hygiene, and vesture gowns if they look to come in contact with blood or actual fluids.

For influenza, laboratories must follow droplet precautions. Patients must clothing masks when non in their assigned rooms, and healthcare workers should don confront masks when in the room of a patient with suspected or confirmed influenza. Based on current CDC recommendations, flu positive patients should be placed in individual rooms, and droplet precautions should exist implemented for 7 days after disease onset or after a total 24 hours following resolution of symptoms.

Because respiratory viruses cannot be distinguished on the ground of symptoms, ruling out or confirming influenza as presently 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 private 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 every bit quickly equally possible reduces the number of people exposed to this virus.

During influenza flavor a surge of patients visits urgent care, doc offices, and emergency departments. This leads to a shortage of space, healthcare workers, and PPE, for instance. Thus, rapid diagnosis of any respiratory illness can allow for shorter expect times and visit times, reducing the burdens on hospitals.

All-time Practices and Quality Control

Unlike viral culture, waived molecular testing poses minimal risks to the personnel performing the analysis. In guild for a test to be considered waived, a patient sample cannot be manipulated (diluted, centrifuged, etc.) in a way that is not specified past the manufacturer. This reduces the hazard of aerosols, spills, or exposures. Furthermore, many of these assays are airtight systems, meaning that the amplification and detection steps occur in a independent space. This prevents contagion of the environment with genetic material and organisms, farther mitigating the take chances. The greatest risk occurs during direct contact with a patient during specimen collection.

Test performing areas should be kept clean and organized to foreclose cross contagion. Surfaces should exist disinfected daily and likewise immediately disinfected following spills or visible contagion. As with whatsoever human specimen that would be candy in chemical science, hematology, or any clinical laboratory, all specimens should be handled using universal precautions and according to the notion that any sample might incorporate 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 co-ordinate 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 consequence can occur because of an instrument, specimen, or reagent. Specimens producing an invalid result should exist 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 establishment must ascertain a policy that follows good laboratory practices. Best practices include running daily external positive and negative QC, fifty-fifty in a CLIA-waived setting. Documentation of controls and results is recommended. When QC fails, patient results should non exist 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 command is incorporated into each sample while an external control—which should include a positive and negative sample—is run every bit private samples. The internal control tin can serve equally a processing control or control for that test. The internal command in molecular-based tests is often a Dna extraction control, which indicates whether or not a patient sample was properly extracted, a necessary footstep in club to receive a correct result. External controls evaluate whether an instrument provides right results (for instance, a positive external control is detected as positive) and should mimic patient specimens.

Limitations of Molecular POCT

Since both POCT and not-POCT molecular tests aren't able to distinguish betwixt live or dead organisms, they can't be used every bit 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 past a virus or group of viruses. These primers are designed to friction 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 Deoxyribonucleic acid 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 instance, the primer cannot bind, and that sequence volition not exist amplified, resulting in a simulated negative due to lack of detection of that sequence. Every bit 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 analysis failure and environmental cross contamination. For case, in clinics that administer influenza vaccine, contaminated instrumentation can produce fake positives. However, multiple studies take demonstrated that failure rate and environmental contagion is low. One report institute 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 organization was intentionally contaminated with influenza A/B-positive control material, this contagion was not plant to affect any of the negative command tubes in runs immediately after assessing organisation contamination, thus showing that the contamination did non bear upon the integrity of results (eight). Given the simplicity of the current molecular POCT with the sample-to-respond format, user variability, opportunities for contagion, and human being errors are minimized if protocols are followed.

Ane potential source of man error involves results reporting. Although POCT instrumentation provides a articulate positive, negative, or invalid event, the platforms are not usually interfaced to laboratory information systems, meaning that results must exist manually entered. Care must be taken to avert transcription or other information entry errors.

Space and cost limitations are as well 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 tin merely run ane sample at a fourth dimension. In a large emergency section or urgent care clinic, several instruments would be required to meet the demand for flu testing.

What's Adjacent for POCT

Sexually transmitted infections (STI) take garnered attention in the molecular POCT field as rapid diagnostics permit for prompt treatment and consultations with patients, who might otherwise exist lost to follow-upwardly. Given the public health concerns associated with STI, these tests actually need to exist accurate. Development of and investigations into such assays are already underway worldwide including a molecular POC test for Trichomonas, Chlamydia trachomatis, and Neisseria gonorrhoeae (ix).

Although there are few approved analytes for molecular POCT in the U.Southward., the ability to rapidly test and reply with constructive handling, when applicable, makes POCT an attractive methodology for a diversity of infectious diseases, including parasites, fungal infections, STI, and more than.

Molecular POCT is increasingly advantageous in resource-limited settings, which typically have lengthy TAT and not enough trained technologists to perform high-complication 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, hereafter studies are warranted to determine toll savings, antimicrobial usage, TAT, patient touch, and how to best implement in non-microbiology clinical laboratories and clinics.

Paige M.K. Larkin, PhD, 1000(ASCP) , is a clinical microbiology fellow at the Academy of California, Los Angeles (UCLA) Health System.

Omai B. Garner, PhD, D(ABMM), is an associate clinical professor, section chief of clinical microbiology, and manager of betoken-of-care testing at the UCLA Health System. +Email: [email protected]

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