Development and Implementation of a Cervical Patch Sampling Approach for Cervical Screening and the Study of HPV Transmission
HPV is the causative agent behind 99.7% of Cervical cancers and is implicated in the pathogenesis of many other tumours such as Anal and Oropharyngeal tumours. Recently some studies have called into question the suitability of the techniques used in order to sterilise frequently used, reusable medical devices such as endoscopes or trans-vaginal ultrasound scan probes. These findings have real public health implications as in the case of trans-vaginal probes, the failure to disinfect these probes means that HPV could be transmitted to the cervix and the most vulnerable site for a transforming HPV infection i.e. the cervical transformation zone. To assess if these findings were correct, we utilised a novel Patch sampling approach to lift and sample the cervical surface epithelial cells that would be in contact with any vaginal medical device. Utilising this approach, we were able to quantify the infective viral titre that any device may come into contact with. By quantifying this infective titre, we were then able to utilise physiologically relevant viral particles generated using a RAFT culture system and an in vivo mouse model (MmuPV1) to first assess infectivity in vitro and then in vivo. Upon successful validation of these systems, we found that even at the highest clinical titres observed, the majority of high-level disinfectants had excellent activity against HPV. Interestingly we also found that in the case of the in vitro system the neutralised control utilised by other groups led to an experimental artefact being witnessed which led to this erroneous conclusion that HPV is resistant to certain disinfectants. The in vivo model also allowed to assess the role of fomites in the transmission of PV. Here we found that fomite transmission is indeed a real possibility and PV demonstrated a significant resistance to desiccation in our model systems. Past work has suggested a possible role for the viral protein E4 in successful transmission and survival of PV. To gain further insight on this aspect we generated a mutant E4KO virus using the RAFT culture system. Investigation of this mutant virus alongside the WT demonstrated that E4 has a role to play not only in survival of the virus but also in the early infection process surrounding a PV infection and perhaps also a role for improving the evolutionary fitness of PV via a possible interaction with APOBEC3. Alongside these observations around the E4 protein, we also assessed if E4 could be used to prognosticate on Cervical pre-cancerous lesions. Prior to investigating this we utilised our novel Patch sampling approach to assess if we could first use this technique to identify women at risk of having high-grade disease. Here we utilised the biomarker MCM and demonstrated that this approach certainly has clinical utility to identify high-grade lesions in their entirety in a non-invasive manner. Indeed, this approach in our pilot trial was found to be non-inferior to the current HPV triage test of liquid-based cytology and indeed other tests such as methylation that are being investigated for HPV triage. Moreover, by utilising machine learning we have now developed a trained algorithm that can analyse the image set created for each patient’ cervix and flag up areas of concern for review. Such automated analysis will lead to more high-throughput triage of these HPV positive women which frees up precious medical resources to be utilised in other areas. We are now assessing this dual marker approach of MCM/E4 in a large cohort of patients across 2 sites (Cambridge and Japan) to obtain high powered data that may lead to a change in the triage test being offered to HPV positive women.
hpv, screening, disinfection, triage, cervical cancer, machine learning
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