Since 1952, the SPA continues to represent the “gold standard” in determining viral concentration for infectious viruses [9], despite its multiple limitations. As indicated in the Edelman and Barletta report [10]:

…significant limitations of this method includes (i) the requirement for extensive hands-on time (≥5 h) for completion of the assay, at least 1.5 days to produce results, and the need for a large quantity of reagents and supplies; (ii) a limited dynamic range of one log (30–300 plaques/plate); (iii) media that are susceptible to environmental conditions (e.g., drying and aging), resulting in suboptimal bacterial growth, resulting in decreased phage infectivity; (iv) the dependence of accurate titers upon the viability of the host bacteria, with viability less than 100% causing a reduction in the true titer; (v) the inherent subjectivity and potential error when visually counting plaques; (vi) a lack of reproducibility in titer due to procedural errors from multiple dilution and pipetting steps; (vii) the detection of only functional phage rather than total phage DNA content in the preparation; and (viii) an extremely tedious and cumbersome procedure with a low throughput when assessing multiple clones.

PPE should be stored under controlled conditions in accordance with the manufacturer’s guidance [12]. Protective clothing manufacturers are not required to report the shelf life of their products, thereby bringing into question the performance of some PPE when subjected to long-term storage. The ability to quickly evaluate the performance of stockpiled PPE, including viral penetration resistance, is essential to making informed decisions regarding the distribution of PPE.

The ASTM F1671 test method typically requires 5 to 6 days to be completed. About half of the time is required to propagate the virus needed for both the initial fabric exposure and the SPA post-exposure. In addition, only a limited number of samples can be processed at a given time, making the assay inefficient and unsuitable in situations that require the testing of PPE on a large scale (e.g., multiple stockpiled production lots) or when results are needed quickly.

Also, before the ASTM F1671 can be initiated, a compatibility test is required to determine if the components of the PPE such as materials, adhesives, and coatings are compatible with Phi-X174 bacteriophage or the host bacterium, E. coli. This is necessary because this method is designed to measure only infective viral particles rather than the total viral load that penetrates the fabric. Therefore, this standard test method cannot be used to assess viral penetration on fabrics containing inhibitory agents or those pretreated with antimicrobial substances—techniques that are becoming more common [13–17]. As a consequence, potentially protective garments remain excluded by the limited applicability of the ASTM F1671, an aspect that requires additional considerations during PPE shortages.

The ASTM F1671 is also relatively expensive. Laboratories typically charge a few hundred dollars per sample, and several samples are generally required to obtain FDA approval. Among the factors that contribute to the high cost are the limited number of accredited laboratories authorized to perform the test, the time required to process the samples, the large amount of consumables required, and the costs associated with the disposal of the biohazardous waste produced during the test.

Due to the numerous limitations associated with the application of the SPA for quantification of infective virus, additional techniques should be considered for inclusion in the ASTM F1671 test method. Several viral detections and quantification methodologies have been proposed to improve the ASTM F1671 [18–22]. Among the alternative methods available for viral quantification, Quantitative Real-Time Polymerase Chain Reaction (qPCR) appears to be a promising methodology to address the limitations of the SPA in ASTM F1671. qPCR is a laboratory technique of molecular biology that allows for the quantification of different microbial agents present in a sample by amplification and detection of their genomic material.

qPCR is widely used in clinical laboratory research for several applications including biomedical research, criminal forensics [23, 24], and in the food industry to determine the microbial load in food or vegetable matter [25–27]. If deemed a valid technique for this application, replacing the SPA with the qPCR in the ASTM F1671 is expected to provide numerous benefits, which are summarized in Table 1. These benefits are explained below.

There is one key limitation to quantifying virus penetration of PPE fabric when using traditional qPCR compared to the SPA. The SPA is designed to detect only infectious virus whereas standard qPCR is unable to distinguish between infectious and non-infectious viral particles. In recent years however, a modification of the qPCR technique involving the use of a chemical reagent called Propidium Monoazide (PMA) has eliminated this limitation [34–37]. The use of PMA in qPCR has been extensively documented in food and environmental applications [38–44], and there are already kits commercially available for the detection and quantification of infectious microorganisms like bacteria and viruses [45–47]. The mechanism that allows PMA to differentiate between infectious and non-infectious viruses is based on the property of the molecule to bind to the nucleic acids of viruses with damaged capside (the protein shell surrounding the nucleic acid) and inhibit the PCR reaction. The criticism about the efficiency of PMA for infectious-noninfectious viral distinction is that the PMA is unable to differentiate viruses that have lost their infectivity due to damaged nucleic acids but whose capsid remains intact [34, 44]. In that regard, the simultaneous detection of several portions of a genome made possible by advancement in technology (multiplex analyses) allows the qPCR to overcome the legitimate concerns [48, 49]. However, it should be noted that different viruses have different shapes, stability profiles, and infectious potencies [50]; hence, claiming protective performance based exclusively on the detection of infectious PhiX-174 virus might not be representative of the protection provided in real circumstances. However, measuring total (infectious and non-infectious) penetration of PhiX-174 with the PMA reagent does allow for the adequate characterization of the barrier performance of PPE, which is the objective of the test method.