• HPV (Human Papillomavirus) 
  A highly prevalent virus responsible for almost all cervical cancers. The most critical high-risk genotypes are 16 and 18.
• HPV DNA screening 
  First-line test recommended by the WHO and European guidelines. It has a sensitivity of >90% and detects precancerous lesions at an early stage.
• Multiplex PCR 
  A technique that allows multiple HPV genotypes to be amplified and identified simultaneously in a single reaction. It is particularly useful in the presence of co-infections and for improving risk stratification.
• High-risk genotypes (HR-HPV) 
  These are the strains most frequently associated with precancerous lesions and tumours: HPV 16 and 18 in particular account for approximately 70% of invasive cervical carcinomas.
• Self-sampling
  Sample collection method performed by the patient at home. The latest meta-analyses confirm diagnostic efficiency similar to clinical sampling and significantly higher adherence rates.
• Clinical goal 
  Reduce incidence and mortality through early diagnosis, appropriate triage, and follow-up pathways based on genotypes and combined risk factors.

Persistent infection with Human Papillomavirus (HPV) is the necessary cause of cervical cancer【3】. There are over 200 genotypes of HPV, of which at least 14 are classified as high oncogenic risk because they are associated with the onset of precancerous lesions and cervical tumours. In particular, two genotypes – HPV16 and HPV18 – are responsible for approximately 70% of cervical cancer cases worldwide【4】.

Other high-risk types (e.g. HPV31, 33, 45, 52, 58 and others) contribute to the remaining 30% of cases. This aetiological knowledge has revolutionised prevention: today, prophylactic vaccines against the main oncogenic types (16, 18 and others) are widely used in girls and boys to prevent HPV infections, while cervical screening has evolved from traditional cytology (Pap test) to the detection of high-risk HPV DNA. The transition from Pap tests to HPV testing as the primary screening method is motivated by the latter's greater sensitivity in detecting precancerous cervical lesions. Clinical studies have shown that HPV DNA testing has significantly higher sensitivity (approximately >90%) in detecting CIN2+ cervical lesions than cytology, which has a sensitivity of around 50%【4】.

This translates into greater negative predictive value: a negative HPV test result offers a high guarantee that the woman will not develop significant lesions in the short to medium term, allowing screening intervals to be safely extended. On the other hand, the HPV test has lower clinical specificity than the Pap test, as many HPV infections are transient and will not progress to cancer. To maximise the benefits and reduce unnecessary interventions, screening programmes therefore adopt triage strategies for women who test positive for HPV (e.g. by means of reflex Pap tests or by identifying the most dangerous viral genotypes) – aspects that will be discussed in more detail below.

Polymerase Chain Reaction (PCR) has transformed molecular diagnostics by enabling the amplification and detection of viral DNA fragments, including HPV, with high sensitivity. In first-generation tests, however, this technology had some limitations in its application to HPV screening: for example, one of the most widely used tests, Hybrid Capture 2 (HC2), detects the presence of high-risk HPV DNA in a sample but does not distinguish between specific genotypes【3】.

Even many real-time PCR-based assays, despite being highly sensitive, required separate parallel reactions to detect different HPV types individually (using different specific probes)【3】. The lack of complete genotypic information was a limitation, as knowing the type of HPV present influences clinical management (consider the difference in risk between HPV16 and a less aggressive high-risk HPV) . New multiplex technologies overcome these limitations by allowing multiple target sequences to be amplified and detected simultaneously in a single reaction tube. In the field of HPV, this translates into the ability to test for the presence of all major high-risk genotypes with a single test, providing a specific genotype for each HPV detected. For example, nested multiplex PCR assays have been developed that can detect up to 40 different HPV genotypes in a single test【3】, using pairs of generic primers followed by specific (nested) primers for each type. Experimental studies have shown that an advanced multiplex approach can detect multiple HPV infections (co-infections) in a significant proportion of women: in highly endemic regions, more than 50% of HPV-positive patients had more than one high-risk genotype at the same time【3】. Detecting such co-infections is clinically useful, as the presence of multiple oncogenic types can increase the risk of lesions and is a phenomenon that needs to be monitored in the vaccine era. It has been hypothesised, for example, that with the elimination of types 16 and 18 through vaccination, other previously less common oncogenic types could increase their relative prevalence in populations (type replacement phenomenon)【3】.

A testing system capable of identifying all strains present therefore provides a complete picture of the virological risk for each individual patient. From a practical and logistical point of view, multiplex PCR diagnostics for HPV also offer advantages: they allow complete genotyping results to be obtained without having to perform additional tests on the sample, reducing laboratory time and costs. Furthermore, the automation of these assays on closed platforms improves standardisation and reduces the risk of cross-contamination between samples. A concrete example of multiplex innovation is the HPV Selfy test developed by Ulisse BioMed: a CE-IVD-marked multiplex real-time PCR assay capable of detecting and genotyping individually the 14 high-risk HPV genotypes relevant for screening【1】. This test has undergone extensive clinical validation on samples collected by healthcare professionals and on self-collected samples, proving to comply with the rigorous international criteria required for use in primary screening【1】.

Thanks to solutions of this type, clinicians receive a complete report indicating exactly which HR-HPV strains are present (e.g. “HPV16 positive, HPV18 negative, HPV52 positive”, etc.), which is valuable information for deciding the next step in the diagnostic-therapeutic pathway. It should be noted that the innovation also involves the portability and accessibility aspects of PCR. Thanks to compact platforms such as the Hyris System™ (which integrates the bCUBE™ portable device, the bAPP™ management app and specific reagents), it is now possible to perform multiplex PCR molecular tests for HPV even outside centralised laboratories. This means that diagnostics can be brought closer to the patient – for example, in peripheral clinics, health centres, developing countries or contexts without equipped laboratories – without sacrificing the sensitivity and specificity of advanced tests. The decentralisation of testing using systems such as the Hyris System™ allows for rapid responses, facilitating the timely treatment of precancerous lesions and helping to bridge the gap in access to diagnosis in different geographical areas.

To evaluate an HPV test intended for population screening, certain key performance indicators (KPIs) are essential. First and foremost is clinical sensitivity in detecting high-grade cervical lesions (CIN2/CIN3): a good HPV test must detect the vast majority of these precancerous lesions, exceeding a minimum threshold established by international consortia. According to the validation criteria of Meijer et al. adopted in Europe, a new HPV test must demonstrate sensitivity and specificity that are not inferior to reference tests (such as HC2 or GP5+/6+) in detecting CIN2+【5】.

In practice, the best tests currently available show sensitivity greater than 90% in detecting CIN2+ lesions (compared to approximately 50-60% for cytology)【4】. This high sensitivity is accompanied by an excellent negative predictive value (NPV): a negative HPV screening test provides protection for approximately 5 years, during which the probability of developing invasive cancer is extremely low (less than 1 in 1,000). This has made it possible to extend the interval between screenings (e.g. to 5 years) in HPV-negative women, while maintaining a good margin of safety. The clinical specificity of an HPV test, on the other hand, represents the ability to correctly identify the absence of disease (CIN2+ lesions). Since many HPV infections regress spontaneously, it is physiological for the HPV test to have lower specificity than cytology (which already detects cytological lesions, a sign of persistent infection). Here too, there are standards: the test must have a specificity that is not lower (at a defined interval) than the reference tests【5】. In practice, the specificity of HPV tests validated for screening typically ranges between 85% and 95%, depending on the age of the population and the context (some differences emerge, for example, between vaccinated and unvaccinated populations). A balance between high sensitivity and good specificity is important to ensure that screening brings net benefits: too many false positives (low specificity) would cause anxiety and unnecessary diagnostic procedures for many women. Another crucial KPI is the reproducibility of the test, both internally and between different laboratories. A test must provide consistent results when repeated on the same sample and must give similar results when performed in different centres. Validation protocols assess inter- and intra-laboratory agreement: the kappa coefficient of concordance for HPV positivity should ideally be very high (>0.5-0.6)【5】. This ensures that the test is robust and reliable, an essential requirement especially when planning large-scale programmes. Finally, the quality of test performance must be ensured by appropriate control systems. Every laboratory performing HPV screening should participate in External Quality Assessment (EQA) programmes specific to HPV testing【4】. In these schemes, panels of control samples are periodically sent to laboratories for analysis to verify that all centres are obtaining the expected results. For example, the WHO coordinates proficiency panels for HPV testing at an international level, and various networks (such as the European HPV DNA LabNet) provide EQA programmes that laboratories can join. EQA results allow any discrepancies in analytical performance between sites to be identified and corrected promptly【4】. In addition to external EQA, rigorous internal controls must be implemented: each analytical run must include positive and negative controls, and preferably each sample analysed should contain an internal control (e.g. a cellular gene) to confirm the presence of adequate biological material and the absence of PCR inhibitors. Compliance with international guidelines (such as IPVS standards and European/WHO guidelines) in terms of clinical validation of the test, quality control and reporting of results is an integral part of a reliable screening programme【5】. In recent years, the WHO has introduced a prequalification process for HPV tests intended for resource-limited countries, precisely to ensure that only tests that have passed independent assessments of quality, accuracy and robustness are used on a large scale【5】.

In summary, the adoption of innovations such as multiplex PCR must be accompanied by a robust quality assurance system in order to translate technological progress into real benefits for public health.

In the context of primary screening based on HPV testing, all women with positive tests require further investigation (triage) to determine the next diagnostic step. A fundamental triage approach concerns genotypes 16 and 18, recognised as the most oncogenic. Since HPV16 and 18 present the highest risk of progression to advanced cervical lesions, many guidelines recommend managing positivity for these genotypes differently from others. In practice, if a woman tests positive for HPV16 or 18, triage procedures often involve immediate referral for colposcopy, regardless of the outcome of any concomitant Pap test. This strategy is based on epidemiological evidence that HPV16 and 18 cause the majority of carcinomas and CIN3 lesions, making it less useful to wait for other findings【4】. For example, in HPV-based screening protocols adopted in Italy and other European countries, direct colposcopy is performed if the HPV test is positive for genotypes 16 or 18, while for other high-risk genotypes, a triage (reflex) Pap test is performed and only in the event of cytological abnormality is the woman referred for colposcopy.

Similarly, new international guidelines consider the possibility of incorporated molecular triage: in its 2021 recommendations, the WHO suggests that when an HPV test explicitly reports information on 16/18 (partial integrated genotyping), women who are 16/18 positive can go directly to treatment/colposcopy, while for other HPV-positive women, an additional triage step (such as VIA or cytology) can be used【6】. It is clear that the availability of tests capable of genotyping is crucial for implementing this differentiated triage. The main HPV tests approved for screening (e.g. Cobas® 4800, Abbott RealTime HR-HPV, BD Onclarity™ and others) include separate detection of HPV16 and HPV18 precisely to support immediate clinical decisions. A comprehensive multiplex test, such as HPV Selfy mentioned above, even provides information on all high-risk genotypes present. This means that in addition to identifying 16 and 18, it also allows you to know if the patient is positive for other types (e.g. HPV31, 52, 45, etc.), which is useful information for defining individual risk. For example, some studies have shown that there are differences in risk among other oncogenic genotypes (HPV33 and 31 carry a relatively higher risk than HPV56 or 51, for example): having the complete profile could allow for personalised surveillance schemes in the future. In any case, triage that isolates women with HPV16/18 is already an established and effective way of optimising resources, focusing second-level interventions (colposcopies, biopsies) on patients at higher risk.

A key factor in achieving the elimination of cervical cancer is increasing women's participation in screening. An innovative tool for improving coverage is the introduction of self-sampling of vaginal samples by women themselves. This method offers multiple advantages: it breaks down some of the logistical and psychological barriers associated with traditional gynaecological examinations, makes it possible to reach women who live in remote areas or who avoid medical examinations for cultural or personal reasons, and has proven to be highly appreciated by users.

The WHO recognises the potential of self-sampling and explicitly states in its guidelines that the use of self-sampling for HPV testing can make a decisive contribution to achieving the target of 70% global coverage by 2030【1】【6】. Numerous studies have investigated the effectiveness of self-sampling strategies, with very encouraging results: inviting women to perform HPV testing on self-collected samples at home can significantly increase uptake compared to standard invitations to attend a clinic. An international meta-analysis found that women are almost twice as likely to participate in screening if they are offered the option of self-sampling compared to the conventional approach【6】.

In practical terms, in various pilot programmes, sending a self-sampling kit to the homes of women who did not respond to the initial invitation led to a substantial recovery of these non-adherents (in some studies, adherence rose from 15-20% to over 50% thanks to the kit)【6】.

A crucial aspect is that the self-collected vaginal sample is suitable for molecular HPV analysis. There is now solid evidence on this point: HPV tests on self-collected samples show diagnostic accuracy comparable to that of samples collected by clinicians【1】. Comparative studies indicate that the sensitivity in detecting high-risk HPV infections is equivalent between self-samples and traditional cervical swabs, especially when using appropriate devices (e.g. flocked swabs, i.e. with a fibrillar surface, which collect more cells than cotton swabs)【1】. Based on these data, many jurisdictions are integrating self-sampling into organised screening programmes. A case in point is the Netherlands, which for several years now has been offering a nationwide self-sampling kit sent to the home as the primary screening alternative with an “opt-out” system, whereby the kit is automatically sent to those who have not had a Pap test【1】. Other countries, including Australia and the United Kingdom, have launched projects to incorporate self-sampling as a routine option. From an acceptability point of view, feedback from women is very positive. Surveys have found that most women who do not adhere to traditional Pap tests find self-sampling easy to use, less invasive and embarrassing than a gynaecological examination, and able to give a greater sense of control and privacy【6】.

This is particularly true for women who, for various reasons (fear, shame, past trauma, cultural barriers), avoided visiting the doctor: for many of them, self-testing is the solution that will finally enable them to participate in screening. Of course, it is essential to accompany the introduction of self-sampling with clear information campaigns and support for users, so that the sample is collected and sent correctly. Furthermore, on an organisational level, effective strategies must be chosen: studies have compared the “opt-in” approach (where women must request the kit) with the “opt-out” approach (direct delivery of the kit to the home without the need to request it). The latter generally achieves higher participation rates, despite being more expensive, while the opt-in model may be cheaper but with lower uptake【6】.

Each healthcare system will need to assess the cost-benefit balance in its own context. However it is implemented, self-sampling is a powerful tool for improving health equity, ensuring that even hard-to-reach populations can benefit from cervical cancer screening and prevention.

The historic goal of eliminating cervical cancer as a public health problem now seems more realistic than ever, thanks to the synergy between HPV vaccination and continuous improvements in screening programmes. In particular, advances in molecular diagnostics – with the development of increasingly sensitive, multiplex HPV tests that can also be used for self-sampling – are expanding the scope and impact of screening globally.

Multiplex PCR for HPV embodies these new frontiers: by enabling the accurate identification of all oncogenic viruses involved and supporting targeted triage (such as that for HPV16/18), it optimises the clinical management of positive patients and prevents more cases of cancer through timely intervention. At the same time, the integration of self-sampling and the possibility of performing tests even in decentralised settings thanks to portable devices are making screening more accessible and widespread, reducing disparities. However, it is essential to accompany these innovations with a robust quality assurance system and full adherence to guidelines: only validated and correctly applied tests will produce the expected benefits in terms of reducing incidence and mortality. Fortunately, the international community – from agencies such as the WHO to scientific societies – has defined clear standards and provides support so that every country can implement safe and effective HPV screening【5】.

On World Cervical Cancer Day, it is worth reiterating that innovation and quality must go hand in hand in this field. Ulisse BioMed's narrative strategy fits perfectly into this framework: through cutting-edge diagnostic solutions (such as the Hyris system and the HPV Selfy test) combined with a system vision focused on scientific validation and sustainability, we are making a concrete contribution to the fight against cervical cancer. Thanks to these advances, a future in which no woman has to die from uterine cancer – a future in which this cancer is truly eliminated – is becoming an increasingly achievable goal.