Comparison of ELISA protocols measuring HPV16 IgG antibodies and evaluation of the association between HPV16 seropositivity and HPV DNA detection

Although cervical HPV infections are very common, seroconversion does not always occur. We compared two protocols based on two serum dilutions to measure human papillomavirus (HPV) type 16 seroreactivity and investigated if HPV DNA positivity was associated with HPV16 seropositivity. We also assessed if the association was influenced by co-infection with other HPV types and viral load. The data used are from Brazilian women participating in the Ludwig-McGill cohort study on the natural history of cervical HPV infection. The serology protocols were based on virus-like particles (VLPs) composed by the L1 or L1 and L2 proteins which are, respectively, the major and minor viral capsid proteins. Two serum dilutions were used: 1:10 and 1:50. HPV16 seroreactivity was expressed as normalized absorbance ratio (NAR). HPV DNA genotyping and viral load were evaluated by PCR-based methods. Correlation and agreement between serum dilutions of each protocol (L1 and L1+L2 VLP) were assessed by Pearson’s correlation (r) and Bland-Altman method, respectively. The performance of the different protocols was compared using the receiver operating characteristic (ROC) curve using the presence of HPV16 DNA as the gold standard. Linear regression was used to analyze the association between HPV16 seropositivity and the detection of HPV DNA infection with both protocols. The presence of HPV DNA was analyzed based on (1) specific HPV types more or less related to HPV16 and (2) HPV16 infection detected alone or in co-infection with other HPV types. The linear regression models presented above were also used on the entire cohort tested with VLP L1+L2 and serum dilution 1:10. The impact of age as a potential confounding factor or effect modifier was analyzed in this model. Finally, the association between HPV16 viral load and seroreactivity was analyzed using Pearson correlation. The magnitude of log10-NARs mean differences between serum dilutions and their standard deviations for each protocol (L1 and L1+L2 VLP) were -0,081 (0.123) and -0.026 (0.150) log units, respectively. The NARs obtained by the serum dilutions used (1:10 and 1:50) for each protocol were strongly correlated (r = 0.87 vs. 0.94, respectively). However, the use of L1+L2 VLPs increased the performance of the test to detect HPV16 IgG antibodies, especially with the 1:10 serum dilution [the highest ROC area (95% CI) = 0.7330 (0.6465 – 0.8495)]. The regression models showed that HPV16 seroreactivity was uniquely associated with the presence of HPV16 DNA and not with other HPV types. For example, the analyses with the protocol L1+L2 VLP and serum dilution 1:10 showed that HPV16 seroreactivity was associated with the presence of HPV16 DNA, β (95% CI) = 0.24 (0.14 - 0.34), and not to HPV31 or 35, β (95% CI) = 0.03 (-0.19 - 0.25), or HPV52, 67, 33 or 58, β (95% CI) = 0.15 (-0.04 - 0.34), compared to women infected with any other HPV type or negative. The analysis of the entire cohort shows that the association between HPV16 seroreactivity and HPV16 DNA infection was similar when the infection was present alone or in co-infection, β (95% CI) = 0.14 (0.07 - 0.21) and β (95% CI) = 0.11 (0.01 - 0.21), respectively, compared to those infected with any other HPV type or negative. Age was not a significant confounder nor an effect modifier in the analysis of the entire cohort. The HPV16 viral load was not correlated with HPV16 seroreactivity, r (95% CI) = -0.04 (-0.34 – 0.27); β (95% CI) = -0.01 (-0.08 – 0.06). In conclusion, the protocol with the higher correlation with HPV 16 positivity was that with the L1+L2 VLP and serum dilution 1:10. Only the presence of HPV16 DNA was associated with HPV16 seropositivity (no other HPV type), and it was not influenced by co-infection or viral load.

Keyphrases: Test immuno-enzymatique, HPV16 seroreactivity, HPV DNA infection, Virus-like particles, Enzyme-linked immunosorbent assay, IgG antibodies