Primary HPV Testing in Cervical Cancer Screening (Part 5)

May 31, 2024

Welcome back to our ongoing series about the role of HPV testing in the early detection of cervical cancer. In this installment, we delve into the complex issues surrounding false negatives and false positives in primary HPV testing. 

The Challenge of False Negatives 

While HPV tests have significantly improved in sensitivity compared to traditional cytology-based screenings, they are not without their shortcomings. Notably, a considerable number of invasive cancers (9–10%) and high-grade squamous intraepithelial lesions (HSIL, 8.3–14%) may test negative for HPV with the currently available assays. There are several reasons behind these false negatives: 

  1. Non-HPV-related cancers: Adenocarcinoma, a less common form of cervical cancer, shows a variable rate of HPV negativity, which can range between 15% and 48% depending on its subtype. This makes detection challenging when solely relying on HPV testing. 
  1. Misclassification: Endometrial adenocarcinomas, often misdiagnosed as cervical cancer, could account for the range of 5% to 68% of HPV-negative primary cervical cancers. 
  1. Limited coverage: Current HPV tests may miss certain strains classified as ‘probably’ or ‘possibly’ carcinogenic by the WHO. Research including the ATHENA trial and other studies have highlighted this gap.1  
  1. ‘Hit and run’ hypothesis: In some cases, HPV infection initiates the cancer and then clears from the system, leading to an absence of detectable HPV DNA in cancer samples. 
  1. Sample and laboratory errors: Errors in sample collection or laboratory procedures can contribute to false-negative results. Challenges in collecting sufficient HPV DNA material may arise; this issue is equally relevant in traditional cytology-based screenings. The introduction of self-sampling techniques for HPV testing raises questions about the acceptability of the rate of self-collected samples that are deemed unsuitable for analysis. We plan to explore the intricacies of self-sampling in an upcoming blog post. Additional causes for false negatives may stem from other types of laboratory errors, such as insufficient procedural standards, inadequately trained staff or a lack of rigorous validation and quality control measures. 

Implications of False Negatives 

No screening test is perfect and attaining 100% sensitivity remains an unachievable goal. Patients must also be educated that while cervical cancer screening significantly lowers their risk, it does not eliminate it altogether. Furthermore, healthcare providers should mandate that laboratories employ FDA approved and rigourously validated methods as well as ensure stringent quality assurance and control protocols. 

The Dilemma of False Positives 

Within the realm of cervical cancer screening, a “false positive” doesn’t imply that HPV is absent. Instead, it suggests that the detected HPV infection has a low likelihood of progressing to cancer. As such, emphasizing sensitivity at the expense of specificity in HPV tests can lead to an elevated number of false positives. It is imperative to recognize that a single positive HPV test result does not automatically signify a high risk of developing cervical cancer. Factors contributing to these false positives include: 

  1. Testing for non-high-risk and/or marginally carcinogenic HPV types: The current inclusion of types like HPV66, which are only marginally carcinogenic, in the panel for high-risk HPVs has contributed to persistent false positives. Current evidence suggests that HPV 66 should not be definitively categorized as carcinogenic. Unfortunately, there is little economic incentive to rectify this issue, which continues to fuel the problem of false positives in HPV screening. 
  1. Cross-reactivity: Some assays may detect certain non-high-risk HPV types they are not designed to, leading to false positives that are highly unlikely to indicate a cancer risk. 
  1. Overtreatment in resource-limited settings: In the absence of appropriate triage tests to refine diagnoses, all patients testing positive for HPV may be referred to treatment, even when their HPV infections would have resolved on their own. This approach can subject numerous patients to unwarranted invasive procedures, along with their associated risks, such as the potential for premature birth. 


Cervical cancer screening is a nuanced and complex process. Although HPV-based screening has proven to be the most effective strategy so far, understanding its limitations is crucial for optimizing its efficacy. Thorough research and strict methodological standards are indispensable in meeting these challenges effectively. 

Stay tuned for our next blog post, where we will discuss the emerging subject of self-sampling methods in HPV testing. 

1 Wright TC Jr, Stoler MH, Behrens CM, Apple R, Derion T, Wright TL. The ATHENA human papillomavirus study: design, methods, and baseline results. Am J Obstet Gynecol. 2012;206(1):46.e1-46.e11. doi:10.1016/j.ajog.2011.07.024 

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