Discussion

Our studies largely corroborate those of Meyers et al and suggest that oxidizing agents, but not aldehydes, are able to inactivate HPV. However, 2 recently published companion studies ^{Reference Ozbun, Bondu and Patterson3,Reference Egawa, Shiraz and Crawford4} identified several limitations of the methodology followed by Meyers et al and our own that bring into question the validity of our results.

First, the HPV16 crude virus preps (CVPs) used in our assays do not meet common infection criteria of time-dependent viral gene expression and antibody neutralization. ^{Reference Christensen, Dillner and Eklund5} In the first hours after infection, E1^E4 mRNA should be undetectable, with a significant increase occurring over 48 hours. ^{Reference Ozbun6,Reference Culp and Christensen7} Like Meyers et al, ^{Reference Meyers, Ryndock, Conway, Meyers and Robison1} we only measured E1^E4 mRNA levels at 48 hours after infection. ^{Reference Meyers, Ryndock, Conway, Meyers and Robison1} Ozbun et al ^{Reference Ozbun, Bondu and Patterson3} showed that relatively high E1^E4 mRNA levels were detected immediately after inoculation with CVPs from raft cultures, with E1^E4 signal significantly decreasing over 48 hours. ^{Reference Ozbun, Bondu and Patterson3} In contrast, using refined (purified) HPV31 raft virus, E1^E4 levels were low at 0 hours after infection but significantly increased over 48 hours. These results raise the possibility that the E1^E4 signal from CVPs could be due to transfection of residual viral RNA rather than infection. In support of this, Ozbun et al ^{Reference Ozbun, Bondu and Patterson3} showed that despite treatment of CVP stocks with the nuclease benzonase, treatment with RNaseA blocked the E1^E4 signal. This finding suggests that benzonase treatment is inefficient in eliminating RNA contamination in CVPs, possibly due to the presence of residual cell debris. Additionally, they showed that CVPs, but not refined raft virus, were largely resistant to neutralization with well-characterized L1 genotype-specific monoclonal antibodies and that OPA treatment of refined raft virus, refined xenograft-derived HPV11 and recombinant quasivirus particles were inactivated by the HLD ortho-phalaldehyde as well as hypochlorite. ^{Reference Ozbun, Bondu and Patterson3}

Using in vitro studies, Egawa et al ^{Reference Egawa, Shiraz and Crawford4} showed that refined raft-derived HPV18 and HPV16 pseudovirus as well as mouse papillomavirus type 1 (MmuPV1) were inactivated by OPA, hydrogen peroxide, and hypochlorite. Additionally, in vivo studies demonstrated that treatment of MmuPV1 with OPA and hydrogen peroxide blocked lesion formation, supporting their in vitro results that aldehydes and oxidizing agents are effective at inactivating papillomaviruses. ^{Reference Egawa, Shiraz and Crawford4} Importantly, they found that OPA, hydrogen peroxide and hypochlorite were also effective against virus in the presence of exofoliated squames on fomites. ^{Reference Egawa, Shiraz and Crawford4}

One proposed disinfection mechanism for aldehydes is the cross linking of lysine residues in viral capsid proteins. Residual viral RNA would not be impacted by treatment with HLDs containing aldehydes or by neutralizing antibodies. We did not test whether the E1^E4 signal from our CVPs was sensitive to RNaseA nor did we confirm that viral infectivity could be neutralized with L1 monoclonal antibodies. Additionally, we did not utilize refined virus in our studies to test the effectiveness of CIDEX and CIDEX OPA as done by Ozbun et al and Egawa et al. Characterization of the CVPs was not included in the Meyers et al ^{Reference Meyers, Ryndock, Conway, Meyers and Robison1} study nor were results using refined virus.

Due to the limitations outlined above, coupled with the data presented in Ozbun et al and Egawa et al, ^{Reference Ozbun, Bondu and Patterson3,Reference Egawa, Shiraz and Crawford4} we cannot rule out the possibility that the E1^E4 signal detected after treatment with the aldehydes CIDEX and CIDEX-OPA was due to viral RNA contamination of the CVP stocks. However, hypochlorite solutions as well as oxidizing agents containing hydrogen peroxide (Oxivir, Sporox, Oxycide, Steris, Resert) are effective neutralizers of RNA contamination as well as many viruses. ^{Reference McDonnell and Russell8–Reference Fischer, Renevey, Thur, Hoffmann, Beer and Hoffmann10} This factor would explain why these oxidizing agents were effective at blocking the CVP E1^E4 signal. Based on the findings by Ozbun et al ^{Reference Ozbun, Bondu and Patterson3} and Egawa et al, ^{Reference Egawa, Shiraz and Crawford4} we believe that aldehydes (eg, OPA) as well as oxidizing agents are effective at inactivating HPV and can be used for reprocessing semicritical items.