Colourimetric signal amplification of in situ hybridization assay for human papillomavirus DNA detection in cytological samples.

Signal amplification of In Situ Hybridization (ISH) can be obtained by Catalyzed Reporter Deposition (CARD), the catalyzed deposition of biotinylated tyramide at the location of the labelled probe. We analyzed 156 cervico/vaginal samples in order to evaluate whether ISH-CARD improves conventional ISH technique in detecting Human Papillomavirus (HPV) on cytological smears and can be used in clinical practice. 126 patients were clinically suspected to have a squamous intraepithelial lesions (SIL) and 30 were negative control-patients. We performed a HPV test screening on these 156 patients with both methods. The screening positive cases were tested for the presence of subtypes of HPV: 6/11, 31/33/51 and 16/18, using only the ISH-CARD. We found a significant increase of positivity with the amplified system, ISH: positive = 50 cases, ISH-CARD: positive = 120 cases, even in many cases cytologically negative. Many cases showed coinfections with different types of HPV. We observed overlapping results in some cell lines harbouring specific HPV subtypes (SiHa, CaSki and HeLa cells), whereas there was no reaction in the C33A cells, negative for HPV presence. This method enables the detection and the typing of HPV in cytological smears, represents a useful tool for clinical purposes and even identifies occult HPV infections.