**4. Viral antigen detection from saliva**

The United States' Food and Drug Administration (FDA) has approved the use of antigen testing for the detection of SARS-CoV-2 in the summer of 2020 to increase the national testing capacity [6, 16]. Antigen tests are immunoassays that are commonly used in the diagnosis of respiratory pathogens such as influenza [17]. Antigen test are designed to detect the presence of a specific viral antigen, which is defined as a toxin or other foreign substance which induces an immune response [16, 17]. Antigen test are currently approved for nasopharyngeal swab specimens however studies suggest saliva samples may be just as effective in detecting viral antigens [6, 16, 18]. When compared to PCR test, antigen testing is relatively inexpensive, and most test results are available in 15 minutes or less. Antigen test in general are less sensitive than RT-PCR test as well as other nucleic acid amplification test (NAATs) [19]. Alternatively, RT-PCR can amplify and detect minute levels of nucleic acid that cannot be cultured which in this case suggest the presence of viral nucleic acids does not signify contagiousness [20]. Both antigen and nucleic acid tests are optimal when the patient is at their viral load peek [6, 16, 20]. More data is needed to guide the use of antigen tests on asymptomatic individuals and to determine if those who were at one time diagnosed positive for SARS-CoV-2 remain infectious.

The advantage of antigen testing is its convenience and accessibility in the use screening high-risk congregate settings such as primary or secondary educational environments, as well as correctional facilities [16, 21]. Repeat testing could quickly identify infection, therefore allowing implementation of patient quarantine and other preventive measure. However, health care professionals need to understand the limitations of antigen testing [19, 20]. Specifically, the testing factors and analytical performance characteristics, such as sensitivity, specificity, and accurate positive and negative predicted values. The "Holy Grail" for SARS-CoV-2 testing remains to be RT-PCR or some form of nucleic acid amplification testing [21]. Nucleic acid testing should be used to confirm an antigen test to avoid inconsistent and inaccurate results test performance may vary based on specimen choice, quality of specimen, the presence of transport medium, and the amount of time required for transport [20, 21]. Since antigen test are typically less sensitive than NAAT testing, negative results can occur while RT-PCR tests may return a positive result [19]. This may occur is specimen sample is collected early before symptom onset or late in the infection [19, 20]. The specificity of antigen tests is as high as NAAT testing, reducing the likelihood of false positives [16, 21]. False positive will still occur, particularly in communities where prevalence of infection is low [16, 20, 21]. The CDC recommends testing professionals establish infection prevalence for antigen testing based on a rolling average, using the positivity rate of their own SARS-CoV-2 testing over the previous 7–10 days, while considering the clinical and epidemiological context of the person or community being tested [6, 16].

Despite the debated advantages and disadvantages of antigen testing, the concept of saliva-based antigen testing for SARS-CoV-2 detection gained traction due to the ease of use for the patient and potential for rapid turn around time in laboratory processing to support public health efforts. However, as noted in the previous section on saliva-based PCR testing, working with such a viscous material posed difficulty in automation integration prior to the addition of homogenization into the workflow. Antigen testing faced similar difficulties when using saliva for large scale testing, the viscous patient samples required further processing prior to automation integration.

In an attempt to mitigate variations in saliva viscosities and allow for sample integration into high-throughput liquid handler reliant workflows, several protocols were developed to dissociate the saliva samples while maintaining intact antigen for detection [6, 22]. These protocols recommend various combinations of heating and enzyme digestion; heating greater than 60 degrees centigrade for as long as an hour or incubating with Proteinase K as an enzymatic digestion [23]. Reports have found these techniques to be somewhat effective in permitting antigen detection from saliva samples, however inconsistencies have also been scored [18, 20, 23]. Heating can denature the viral proteins and RNA, rendering them undetectable, and enzymes such as Proteinase K is very costly as well as cause degradation of targeted proteins through excessive digestion [20]. Not to mention the suggested incubation periods as great as an hour extends the amount of time required to have a patients' result.

Just as with saliva-based PCR testing, homogenization was proposed as a method for efficient sample disruption [24]. Viscosity in homogenized saliva samples has been shown to be greatly reduced to amounts that are similar to those found in water. Allowing for ease in pipetting and increase throughput using automation and liquid handlers [15]. The various forces found in homogenization are only required for small amounts of processing time, as short as 5 seconds per sample without generating any extra heat during the processing, maintaining the integrity of the antigens targeted. In contrast to other proposed methods for saliva processing in antigen detection, additional enzymes are not required, saving costs and without any needed incubation steps, also saving valuable time during testing.

### **5. Improving laboratory safety with homogenization**

During the COVID-19 global pandemic, safety of all individuals involved in the care of COVID-19 patients as well as laboratory and clinical staff involved in testing for SARS-CoV-2 became a top priority. Given the highly virulent nature of SARS-CoV-2 and the lack of knowledge and treatments we had available, it was essential to neutralize the virus during laboratory testing while preserving the diagnostic capacity of all assays [5]. Employing viral neutralization techniques in the diagnostic workflow was a critical step in increasing the number of facilities available to process COVID-19 patient samples, supporting increased public health testing efforts.

Techniques involving thermal inactivation, chemical neutralization or degradation, enzymatic digestion, and mechanical disruption of samples were all proposed as potential solutions to laboratory safety when handling potential COVID-19 positive patient samples [5, 6]. However, given the global strain on the plastics and chemical reagents needed to complete many of these neutralization steps, the authors felt it was prudent to examine the potential of mechanical sample dissociation in the form of homogenization and its effect on virus neutralization [2, 3, 8]. Ultimately, it was shown that following 30 seconds of homogenization, 98% of the

#### *The Utility of Mechanical Homogenization in COVID-19 Diagnostic Workflows DOI: http://dx.doi.org/10.5772/intechopen.97110*

virus in any given sample was inactivated, while still preserving the genetic material for adequate PCR detection [2, 3]. This finding supported expanding the implementation of homogenization in the COVID-19 diagnostic workflow because it could be done both in the laboratory setting, as well as the location of sample collection provided the homogenized sample would be properly refrigerated and transferred for PCR detection within the next 12 hours [2–4, 15].

The mechanical lysis of the SARS-CoV-2 particles in a potentially infectious sample permitted these samples to be processed in a BSL-2 facility, supporting the expansion of laboratory testing facilities equipped to process COVID-19 samples [2, 3, 8]. Without a proven neutralization step, such as mechanical homogenization, all COVID-19 samples would have to be processed in BSL-3 facilities due to the potential risk of exposure to infectious virus. While it is still recommended that the homogenization procedure occur in a biosafety cabinet within a BSL-2 facility, the procedure provides sufficient viral lysis to improve safety when handling potentially infected patient samples and allows additional laboratories to assist with testing in a cost-effective manner [2, 3, 8, 15].
