In addition to the irreproducibility of the MY09/11 primer synthesis, amplification efficiency has been shown to vary systematically among the HPV genotypes when known target quantities of the genotype are analyzed and when amplification with the MY09/11 primer system is compared to that with another consensus PCR system (17
). Analysis of the alignment of the MY09/11 primer binding regions for 19 of the 23 sequenced genital HPV genotypes (Table ) revealed more destabilizing mismatches for the genotypes shown in our laboratories and others to amplify with poor efficiency (e.g., HPV types 26, 52, and 55) relative to the number of mismatches for the HPV types that amplified well (e.g., HPV types 16, 18, and 33). The efficiency of amplification appeared to be related to the number, position, and stability of the mismatch (data not shown). As expected, primers with greater than four mismatches to the target sequence tended to be less efficient (e.g., HPV types 42 [MY09], 26 [MY09], and 59 [MY11]). Primers with less than four mismatches overall but with one or more mismatches at the 3′ end of the oligonucleotide also tended to segregate with the less efficiently amplified HPVs (e.g., types HPV 39 [MY09], 45 [MY09], and 55 [MY09]).
The MY09/11 consensus primers were redesigned in an attempt to correct both the irreproducibility of the degenerate primer synthesis and to increase the sensitivity of amplification to a 10-copy endpoint for each of the HPV genotypes commonly found in the genital tract. The primer sequences resulting from this analysis are shown in Table . The upstream primer pool, designated PGMY11, contains five oligonucleotide primers. The downstream primer pool, designated PGMY09, contains a total of 13 oligonucleotide primers. The amplification parameters were reoptimized in the presence of the new primer pools. The overall increase in stability of the new primers to their target sequences required a reduction in the total final MgCl2 concentration to 4 mM. This primer pool accommodates the efficient amplification of the following HPV genotypes to at least a sensitivity of 10 genomes per PCR: 6, 11, 16, 18, 26, 31, 33, 35, 40, 45, 51, 52, 56, and 59 (data not shown). Several other HPV genotypes, including HPV types 39, 42, 53, 54, 55, 58, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73, IS39, CP8304, CP6108, MM4, MM7, and MM8, were amplified as well as or better with PGM09/11 than with MY09/11, as determined from comparison of endpoint dilution amplifications (data not shown).
To verify the results of the analytic analyses, we conducted a parallel comparison of the MY09/11 and PGMY09/11 amplification systems with 262 cervical specimens. Of the 262 cervical specimens, a total of 15 were excluded from further analysis due to poor or no β-globin amplification, indicating either a lack of sufficient cellular material for PCR or the presence of polymerase inhibitors. Thirteen of these samples were negative for β-globin amplification by both the PGMY09/11 and the MY09/11 amplification systems, while two samples were excluded because of a lack of β-globin amplification by the PGMY09/11 system only. The general summary results for HPV prevalence for the remaining 247 samples are presented in Table . The overall percent agreement between the two methods was 91.5%, with a kappa value of 0.83 (P < 0.001). There was an increase in overall HPV prevalence with the PGMY09/11 system relative to that with the MY09/11 system (62.8 and 55.1%, respectively). Of the 21 samples with discordant HPV results, 20 were positive with the PGMY09/11 system only and 1 was positive with the MY09/11 system only (McNemar's χ2 = 17.19; P < 0.001). The additional positive specimens detected by the PGMY09/11 system comprised 17 samples with single infections with HPV types 16, 18 (2 samples), 35, 42, 51, 52, 54 (2 samples), 55, 59, 66 (four samples), MM7, and MM8 and 3 samples with multiple infections containing HPV type 51 and 42, HPV types 31, 54, and 66, and HPV types 33, 45, and 6. The one sample called positive only with the MY09/11 system contained HPV-31. The most notable differences between the two primer systems were seen when the abilities of the two systems to detect specific types as part of multiple infections were compared. The overall proportion of multiple infections detected with the MY09/11 primer system was 46 of 136 (33.8%), whereas that with the PGMY09/11 primer system was 62 of 155 (40%). A summary of the type-specific positive results is presented in Fig. . Figure demonstrates graphically the absolute increase in the rate of detection of specific HPV types. The following cancer-associated HPV types were detected at least 25% more often with the PGMY09/11 primer system: HPV types 26, 35, 45, 52, 55, 59, 68, 73, and MM7. The following non-cancer-associated types were also detected at least 25% more often with the PGMY09/11 primer system: HPV types 42, 54, and 66. Table shows a comparison of HPV detection results when categorized by cancer risk group as HPV negative, high-risk type positive (positive for at least one of the following HPV types: 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 55, 56, 58, 59, 68, 73, MM4, and MM7), or low-risk type positive (positive for at least one of the following HPV types without concomitant coinfection with high-risk HPV types: HPV types 6, 11, 40, 42, 53, 54, 57, 66, and MM8). The agreement between the PGMY09/11 and MY09/11 amplification systems by risk group is 88.7% (kappa = 0.81; P < 0.001). The PGMY09/11 primers were more likely to reclassify samples into a higher risk group category compared with the risk assignment based on HPV typing with the MY09/11 primers (Stuart-Maxwell χ2 = 20.45; P < 0.001).
TABLE 3 Overall agreement in results for HPV with MY09/11 and PGMY09/11 for 247 cervicovaginal lavagespecimensa
FIG. 1 HPV type-specific positive results with PGMY09/11 and MY09/11. The total number of positive results by HPV type are plotted: open bar, detected with MY09/11 only; shaded bar, detected with both primer sets; stippled bar, detected with PGMY09/11 only. (more ...)
TABLE 4 Agreement in HPV risk group assignment with MY09/11 and PGMY09/11 for 247 cervicovaginal lavagespecimensa