Padronização e validação de técnica de qPCR para detecção dos HPVs de alto risco 16 e 18 em pacientes com doença inflamatória intestinal

Abstract

The Human papillomaviruses (HPVs) are double-stranded DNA viruses with great genetic diversity cause skin and mucous membrane infections. Some of the virus’ types are linked to several forms of cancer, such as cervical and anal cancers, with the types 16 and 18 being the most common. However, data on the prevalence of anal HPV infection are scarce, and data on anal infection in patients with inflammatory bowel disease (IBD), such as Crohn's disease and ulcerative colitis, are even scarcer. This population may be at special risk due to constant inflammation, atypical immune response, and frequent use of immunosuppressive drugs, which are known to be associated with the development of neoplasia. This highlights the importance of detecting HPV in this population. HPV infection can occur even in the absence of detectable lesions, and molecular techniques such as PCR are remarkably useful in identifying the pathogen and differentiating its types. Therefore, the objective of this study is to standardize a quantitative PCR (qPCR) reaction for the detection of HPV 16 and 18, and to validate the protocol in clinical samples from patients with IBD. For standardization, variations in the concentration of primers and probes, thermal cycling conditions, as well as other qPCR parameters were tested, in addition to in silico tests of the primers and probes. Quantified samples known to be positive for HPV types 16 and 18 were used to construct a standard concentration curve in order to evaluate parameters related to the performance of the assay, such as the efficiency, sensitivity and linearity of the reaction. Different commercial DNA extraction kits were tested in order to select the most suitable one for the samples used. To this end, the genetic material extracted from two samples was quantified and assessed for its purity. After the reaction was standardized, samples collected from 183 patients, 133 with IBD and 50 healthy patients (control group) were used for validation. The best DNA extraction result was achieved using the Qiaamp DNA blood mini kit (Quiagen). Specificity tests showed that the primers were specific for both the types tested. The sensitivity of the reaction for detecting HPV 16 and 18 is up to 10 copies/µL. The efficiency and r² calculated for the HPV 16 detection reaction were 92.032 and 0.985, respectively. For the HPV 18 detection reaction, the efficiency was 93.055 and the r² was 0.986. Of the 183 samples used, 9 were positive for some type of HPV, 2 from the control group and 7 from the IBD group. However, the difference between the two groups was not significant (p>0.05). Finally, it should be emphasized that it is important to generate more data on HPV infection in patients with IBD, so that better prophylactic measures and health guidelines can be developed for this population.