Viral genotyping gave a positive result for infections by a single HPV genotype for 363 patients (48 %), 394 patients (52 %) were, instead, positive for multiple infections, 127 women (13.7 %) were negative for the viral genotypes present in the kit that was used (tab.1). Thirty-six samples analyzed, from all the samples (3.9 %), were inadequate; these patients and negative women were excluded from the study group.
At the histologic examination, carried out after the biopsy, 475/758 patients (62.6 %) presented one low-grade precancerous lesion (CIN 1); 63 women (8.3 %) had one moderate-grade lesion (CIN 2), 100 women (13.2%) one sever-grade lesion (CIN 3); 5 women (0.6%) squamous carcinomas (SCC). Finally, 113 (14,9%) patients had a negative histological diagnosis (Table 1).
1. Single HPV infection
The single infections in our study group were 363(48%). The typology of lesion with the highest percentage of single infection was SCC with 100%, followed by CIN3 with 84%, CIN2 with 60,3% and finally negatives and CIN1 respectivelly with 49,5% and 37,8%.
The most frequent genotype of single infections was genotype 16, present in 153/363cases (42.1%), followed by genotype 31 with 29/363 cases (7.9%) then genotype 18 with 13 cases (3.6%), and finally genotypes 33 and 45, both with 7 cases (1.9%).
These genotypes, all at high risk, are responsible for 97.6% (82/84 cases) of CIN3, 89.4% (34/38 cases) of the cases of CIN2, 61.8% (73/118 cases) of the cases of CIN1 and we found 16/32 cases (50%) negative at histology (Table 2). Genotype 16 was responsible for 85.7 % of the cases of CIN3, genotype 31 for 6.56 %, genotypes 18 and 35 for 2.63%, and genotypes 45 and 81 for 1.3 % of the cases.
In the present study the patients with one single HPV 16 infection had a higher incidence (85, 3%) of CIN2+ with respect to those with other genotypes (17, 8%). In particular, the presence of the HPV16 genotype in our study was associated with a 10 times greater risk of developing a high grade lesion (CIN2 +) (OR=10.04; IC95%=5.66-17.83). Thus, also in our clinical study, genotype 16 was at the highest oncogenic risk.
2. Multiple HPV infections
There were 394 (52%) multiple infections in our study. The typology of the lesion with the highest percentage of multiple infections was CIN1 with a frequency of 62.1%, and negative cases were 50,5%. The percentage decreases decisively in CIN2 (39.7%) and progressively in CIN3 (16%), reaching zero in carcinoma exclusively characterized by single infections (Fig.1).
There were 17 high risk (HR) genotypes that were isolated 666 times (Table 3), and 19 at low risk (data not shown).
We calculated the incidence of each genotype for histologic lesion, the four most diffused HPV genotypes were HPV 16, 51, 59 and 31 (Table 4), also in multiple types infections the most common genotype was HPV 16 , which was found 150 times, and was significatively higher in patients with CIN2/CIN3, 71.4% (OR=4,85; IC 95%=2.39-9,78).
We analyzed the differences between single and multiple types infections with HPV16, the patients with single infections had a higher incidence of CIN2+ (83.3%) with respect to those with multiple infections (71.4%).
The ORs of the single infections of HPV 16 associated with CIN2+ were more than those of HPV16 multiple infections (Table 5).
We studied genotype 16 in particular and we divided the infections into “16mHPV infection”, the cases in which genotype 16 was the first of the strains present (e.g.: 16, 31, 52), and in “m16HPV infection”, the cases in which genotype 16 was among the strains that made up the infection ( e.g: 52, 16, 31).
There were 100 women (66,6%) with 16mHPV infections, in 64 cases we had an infection with 2 genotypes, in 19 cases with 3 genotypes, in 13 cases with 4 genotypes and in 4 cases with 5 genotypes (Table 6).
In the 50 cases (33.3%) of m16HPV infection we had: 11 cases of infection with 2 genotypes, 32 cases with 3 genotypes, 4 cases with 4 genotypes and 5 cases with 5 genotypes.
We then correlated genotype 16 with the number of strains and histological diagnosis (Table 7).
In the 16mHPV group we had 12 cases of CIN3, 13 cases of CIN2, 65 cases of CIN1 and 10 negative cases.
In the m16HPV group we had 3 cases of CIN3, 2 cases of CIN2, 35 cases of CIN1, and 10 negative cases. Only in this group, 1 CIN3 was caused by an infection with 2 genotypes, the other lesions were caused by 3 or more genotypes.
We calculated the OR of the 16mHPV infection with respect to m16HPV for CIN2/CIN3, 16mHPV infection was significative (p<0.05) with an OR=3 (IC% 1.07-8.39). (Table 8)
When the prevalence of the combinations between the genotypes was studied, we found that in 16mHPV infection HPV16, 18 and HPV 16, 31 were the most common combinations of multiple types infection (50%) and the most frequent in CIN2/CIN3. Genotype 16 was present in 94% of the cases of CIN3 (15/16) of multiple types infections and they were all lesions with two genotypes.
From the analysis of the OR, 16mHPV infection with 2 genotypes, with respect to the infections with 3 or more genotypes, was significative with an OR= 7.94 (IC% 2.55-24.73) (Table 8).