Polyhexamethylene Biguanide Reduces High-Risk Human Papilloma Virus Viral Load in Cervical Cell Samples Derived from ThinPrep Pap Test

Abstract

Human papilloma virus (HPV) infection and its progression still represent a great medical challenge worldwide. Clinical evidence has demonstrated the beneficial effects of polyhexamethylene biguanide (PHMB) on HPV clinical manifestations; however, evidence of the effect of this molecule on HPV viral load is still lacking. In this in vitro study, 13 ThinPrep Papanicolaou (Pap) tests were treated with a PHMB solution (0.10 g/100 mL) for 2 h. We observed no cytological changes but a significant reduction in the viral load of high-risk (HR) HPV after PHMB treatment, also revealing a dose-dependent antiviral effect. In addition, by stratifying the obtained results according to HR-HPV genotype, we observed a significant reduction in the viral load of HPV 16, P2 (56, 59, 66), 31, and P3 (35, 39, 68) and a strong decrease in the viral load of HPV 45, 52, and P1 (33, 58). Overall, 85% of the analyzed cervical cell samples exhibited an improvement in HPV viral load after PHMB exposure, while only 15% remain unchanged. For the first time, the data from this pilot study support the activity of PHMB on a specific phase of the HPV viral lifecycle, the one regarding the newly generated virions, reducing viral load and thus blocking the infection of other cervical cells.

Keywords: HPV; PHMB; ThinPrep Pap test; cervical cell samples; viral load.

Figure 1

Flow chart of the experimental design of the in vitro study. Each sample of scraped cervical cells was put into a ThinPrep Pap test vial and divided into two aliquots to perform the cytological analysis and the analysis of the HPV viral load both before and after treatment with PHMB solution.

Figure 2

Effects of PHMB treatment (1:50 dilution) on HPV viral load in cervical cell samples. The 24-h treatment with 1:50 diluted PHMB solution induced a slight decrease in HPV viral load, evaluated both as ct (A) and as fold change (B), however, without reaching statistical significance.

Figure 3

Effects of PHMB (stock solution) treatment on HPV viral load of scraped cervical cell samples. The 2-h treatment with PHMB (0.10 g/100 mL) significantly reduced HPV viral load, evaluated both as ct (A) and fold change (B). In addition, by dividing samples according to HR-HPV genotype (C), PHMB significantly reduced viral load of HPV-16, P2, P3, and 31 and strongly reduced viral load of HPV 45, 52, and P1 compared to pretreatment condition. Overall, by evaluating the percentage of improved and unchanged cervical cell samples (D) in terms of viral load, the HPV viral load was reduced in 85% of the analyzed Thin Prep Pap test samples after exposure to PHMB solution (nonparametric Mann–Whitney U test, * p-value < 0.05; ** p-value < 0.01; **** p-value < 0.0001).

Figure 4

Proposed mechanisms of action of PHMB treatment on HPV viral particles. Our data support the mechanism of action of PHMB against a specific phase of the HPV life cycle, as shown in this figure. Considering the experimental conditions, this molecule seems to be able to act on the newly generated virions outside of the cervical cells, thus reducing the detected viral load and blocking the infection of other cells. These data allow us to consider the use of PHMB alone or in combination with natural molecules that may act on other phases of the viral lifecycle, such as virus entry, replication and penetration, as also reported in the review by Laganà and colleagues [30].