Advantages of NAATs for medicine and public health include their high sensitivity and specificity, making them highly reliable for diagnosing active infections. They also allow for accurate detection of emerging variants, which is crucial for tracking the spread of new strains and informing public health interventions. However, the disadvantages of NAATs include their reliance on specialized equipment and skilled personnel, which can be resource-intensive and limit their accessibility in remote or resource-limited settings. Additionally, the turnaround time for some NAATs, particularly PCR, can be very long compared to point-of-care or self-tests, reducing or entirely removing their use for identifying currently infectious individuals in time to inform isolation procedures.

Advantages of antigen tests for medicine and public health include their rapid turnaround time, ease of use, and suitability for point-of-care or self-testing applications. These characteristics make them valuable for mass testing and frequent screening of asymptomatic individuals, which can help identify and isolate cases more quickly, reducing transmission rates. However, antigen tests generally have lower sensitivity and specificity than molecular tests, particularly in asymptomatic or early-stage infections, which can result in a higher rate of false negatives or positives. Additionally, antigen tests are more difficult to continuously monitor for performance against emerging variants, as compared to NAATs, which can limit their utility for monitoring the spread of new strains because antigen tests may need to be empirically confirmed to perform well whenever a new variant has a mutation in the protein where the antigen test detects.

Advances in technology have led to an explosion of new approaches to measure many antibodies at once, a process called multiplexing. Multiplexing technologies allow for the measurement of the serologic antibody repertoire within samples. Multiplexed testing that can evaluate many analytes simultaneously or technologies that can process many samples at a time are referred to as high-throughput assays and are increasingly useful for large-scale public health programs, such as serologic surveillance, which can be critical to monitor the spread of a virus across whole populations.

Bead-based antibody tests use microscopic beads coated with viral antigens to capture specific antibodies from a sample. A serum sample is applied to the assay and if there are antibodies in the sample that recognize the viral proteins coated on the beads, those particular beads will be detectable by an instrument. The most common of these is a technology called Luminex beads. These assays offer multiplexing capabilities, allowing for the simultaneous detection of antibodies against multiple viral proteins or variants. Bead-based assays can provide valuable information on the breadth and specificity of the immune response but require highly specialized equipment and trained personnel.²⁷

Phage immunoprecipitation sequencing or PhIP-Seq antibody testing is a high-throughput serology method that combines a technology called phage display with NGS to profile the antibody repertoire of an individual. This technique can provide a detailed understanding of the immune response to SARS-CoV-2 and inform vaccine development and evaluation efforts. PhIP-Seq is a new technology and exists primarily within academic research laboratories, but is increasingly being made available for public health testing.²⁸

Array-based antibody testing involves immobilizing viral antigens on a solid support, such as a glass slide or silicon chip, and probing the array with a sample to detect the presence of specific antibodies. This technology enables high-throughput, multiplexed analysis of antibody responses and can be used to monitor immune responses to different viral strains or vaccine formulations.^28,29

Antibody testing for medicine and public health offers the ability to assess past infections and immune responses, which can inform epidemiologic studies, vaccine development, and immunity-based surveillance efforts. However, antibody testing is limited in its utility for diagnosing active infections, and the potential for cross-reactivity with antibodies against other coronaviruses can complicate estimates of infection prevalence.

Sequencing for medicine and public health offers the ability to track and monitor viral variants, inform diagnostic and therapeutic development, and offer insights into viral transmission and evolution. By leveraging these strengths, sequencing technologies can contribute to a comprehensive understanding of the virus and help guide public health interventions.

However, sequencing is not suited for certain applications in COVID-19 testing. Although it is highly accurate and provides deep insights into the virus being detected, it is slower and much more costly than PCR or rapid antigen testing. Although it can be useful for large-scale surveillance, exemplified by its use in sequencing of wastewater for identification of SARS-CoV-2 genetic material, it is less useful for diagnostic use or public health screening and real-time surveillance for limiting outbreaks.

Nasopharyngeal swabs involve collecting samples from the back of the nasal cavity called the nasopharynx. This method is often considered the gold standard for testing for respiratory viruses, including COVID-19. Many respiratory viruses, including SARS-CoV-2, tend to grow well in the nasopharynx and thus a swab that collects a specimen from the nasopharynx has a high sensitivity to pick up the virus. However, the procedure can be uncomfortable for patients, often causing gagging or sneezing, and requires trained personnel to perform the swabbing correctly, ensuring a proper sample is collected.

Nasopharyngeal swabs are well-suited for both NAAT and antigen tests. They are ideal for hospital and clinical settings, where the highest sensitivity specimen collection is required for diagnosis and patient management and where healthcare professionals are present to perform the swabbing. During the early stages of the pandemic, nasopharyngeal swabs were the predominant specimen type used for COVID-19 testing. However, as the pandemic progressed, the extra burden on both the test recipient and the resources required to perform the nasopharyngeal swab caused these types of swabs to largely fall out of favor except in hospitals and doctors’ offices.

Nasal swabs involve collecting samples from the lower part of the nasal cavity or the nares. Nasal swabs have been considered less sensitive than a nasopharyngeal swab because the virus niche is more closely aligned with the nasopharynx than the nares, and thus a nasal swab may be slightly less likely to pick up virus in the nares. However, the much greater ease with which a nasal swab can be performed, including a self-swab, and the relative comfort of a nasal swab have caused nasal swabs to become a primary mode of sample collection. This is especially true for self-sampling for home testing where nasopharyngeal swabs are not practical.

Nasal swabs, like nasopharyngeal swabs, are suitable for both NAAT and antigen testing. They are commonly used in mass testing or screening programs, where ease of collection and patient comfort are prioritized and healthcare professional resources are limited.

Oropharyngeal swabs involve collecting samples from the back of the throat. Similar to nasopharyngeal swabs, oropharyngeal swabs may cause discomfort for patients and often require trained healthcare professionals to collect the samples correctly. During COVID-19 however, many parts of the world, particularly throughout Europe, encouraged oropharyngeal swabs for self-tests, either alone or in combination with self-collected nasal swabs, demonstrating that these types of swabs and specimens can be reliably performed at home by non-healthcare professionals, including people who are self-swabbing.³²

For COVID-19 testing, oropharyngeal swabs have slightly lower sensitivity over the duration of the infection. However, low-grade evidence suggests that the virus may be more readily detectable in the oropharynx very early in the infection.³³ Therefore, oropharyngeal swabs are usually used in combination with nasal or nasopharyngeal swabs. By sampling more than a single site, the performance of the combined swabs tends to be better than any single site alone. Despite data demonstrating that the addition of an oropharyngeal swab to a nasal swab improves overall sensitivity to detect the virus in an infected individual,³² and oropharyngeal swabs being deployed safely across Europe, in the United States, the FDA has discouraged the use of oropharyngeal swabs, warning that these swab types may pose too much of a safety hazard for home testing. This reflects a pattern of a much more conservative stance by the FDA in terms of bringing needed tools to individuals during a public health emergency, compared to many other nations.³⁴

Like nasopharyngeal and nasal swabs, oropharyngeal swabs are suitable for both NAAT and antigen-based testing.

Saliva samples are noninvasive and can be easily collected by the patients themselves. They offer a high level of patient comfort and convenience and are thus powerful specimen collection types for large-scale public health screening and surveillance. Saliva samples are commonly considered to have lower sensitivity overall, across the full course of an infection, compared to nasopharyngeal and nasal swabs, in part because the viral load in saliva can vary significantly across individuals. On the other hand, for COVID-19 testing, limited evidence suggests that saliva specimens may be superior to nasopharyngeal and nasal specimens during the initial 2 days of infection. Scientists hypothesize that the virus growth patterns and early kinetics may cause the virus to be abundant initially in the saliva before replicating to high enough levels in the nasopharynx or nares to be detectable.³³

Saliva samples are generally most suitable for NAAT, including PCR and LAMP assays, as well as some laboratory-based antigen tests. Although nasopharyngeal, nasal, and oropharyngeal swabs are often used for specimen collection for rapid antigen testing, saliva-based samples are generally not used for rapid antigen tests. As of this writing, there is one rapid NAAT authorized by the FDA for home use that is meant to be used with a saliva specimen.³⁵

The ease of saliva sample collection makes saliva specimens appropriate for use in large-scale public health screening programs where ease of collection, low-resource requirements, and patient comfort are prioritized. For example, the FDA authorized the first saliva-based COVID-19 laboratory PCR test developed by Rutgers University in April 2020, which was later used for widespread testing in various settings. Saliva-based collections also form the basis of the widely used SalivaDirect laboratory PCR test that benefited many school, university, and workplace testing programs in large part because of its simple saliva-based specimen collection. Of note, the SalivaDirect test also was an advancement because it replaced a major bottleneck in laboratory testing procedures, RNA extraction, with simple heating of the saliva specimen, making the laboratory test more resource friendly, particularly in the beginning of the pandemic when RNA extraction kits became the limiting resources for lab-based testing.³⁶

Exhaled breath is a promising albeit still unproven specimen and sample collection approach for COVID-19 testing. Exhaled breath collection involves, as the name suggests, collecting the exhaled breath from individuals. Methods and technologies for exhaled breath–based collection are currently under investigation.³⁷

In general, exhaled breath collection involves using a specialized device to collect and concentrate the exhaled breath. These devices can be portable or stationary, and they work by directing the exhaled breath through a filter or membrane that traps the aerosolized particles, including the virus. Once collected, the sample is then eluted off of the filter or membrane, usually by a test buffer or saline solution, and the eluted specimen in the buffer is analyzed for the presence of SARS-CoV-2
RNA or antigen using standard PCR or antigen tests, as well as other less conventional assays such as electrochemical or mass spectrometry–based tests.^36,38

Compared to other specimen collection methods such as swabs or saliva, breath-based collection has several potential advantages. It is noninvasive and ideally would not require the use of invasive techniques or specialized training for sample collection. Compared to a swab, breath-based collection has the potential to be more comfortable for patients than swabs, requiring only breathing out a number of times onto a collection device. Improved comfort of a noninvasive test might reduce anxiety associated with sample collection, which can be particularly useful for public health screening tests. Additionally, because of their novelty, breath-based collections use distinct supply chains compared to nasal swabs and could provide public health testing programs an alternative to traditional collection methods when swabs are in short supply, such as happened during the first months of the COVID-19 pandemic.

However, there are several challenges associated with breath-based collection for COVID-19. One key challenge is the variability in the concentration and amount of virus particles in exhaled breath, which can affect the sensitivity and accuracy of test results. Specifically, the kinetics of when virus particles exist in the exhaled breath as compared to when they can be detected on a nasal swab may be distinct and this could cause confusion when comparing the two methods. For example, early evidence suggests that virus load may be detectable in the breath earlier in the course of an infection compared to detection via nasal swab. However, the viral load may then drop out of the exhaled breath faster as the patient begins to clear the infection, compared to nasal swab. In such a situation, the sensitivity of exhaled breath as a collection modality may be higher at the beginning of an infection and lower toward the end of an infection, as compared to a nasal swab.³⁹ Significantly greater amounts of data are needed to make exhaled breath viable as a specimen type to be trusted and interpreted for clinical and public health purposes. Additionally, the collection efficiency may vary depending on factors such as the patient’s age, sex, and underlying health conditions, which can further impact the accuracy of the test results. As a relatively new approach, the potential for exhaled breath–based collection exists, but more research is needed to determine the utility and effectiveness of breath-based collection for COVID-19 testing in practice.

Self-collected samples, such as nasal swabs, oropharyngeal swabs, and saliva samples, offer several advantages for testing during a public health emergency, including increased patient comfort, reduced burden on healthcare professionals, and the potential for substantially increasing access to testing, particularly through at-home testing. However, self-collection can be difficult at first and may result in lower-quality samples or incorrect collection techniques for those not yet acquainted with self-testing, which could affect the tests’ performance.⁴⁰

Importantly, most data demonstrating reduced performance of self-collected samples as compared to healthcare professional–collected samples looked only at the initial attempt for an individual collecting their own sample. As individuals performed self-collections second, third, and fourth times however, the difference between self-collection and professional collection reduces toward no difference. Additionally, by now, many or most humans have performed a self-swab and thus the early data suggesting reduced accuracy with self-swabs may no longer be relevant. This reflects the challenges with how quickly data can become obsolete and, more generally, how well data are able to keep up with the changing nature of a pandemic such as COVID-19.^41,42

Self-collected samples are appropriate for large-scale testing programs. The lack of professional resource requirements makes them especially useful when rapid and frequent testing is required to monitor the spread of the virus in a community.^40,42

For public health programs, the ideal sample type should balance sensitivity, ease of collection, and patient comfort. Nasal swabs and saliva samples are particularly well-suited for these purposes because of their noninvasive nature, ease of collection, and suitability for self-collection. They are appropriate for large-scale testing, screening, and surveillance efforts.