During the last decade, growing efforts have focused on human papillomavirus (HPV) detection using liquid hybridization, conventional PCR, and real-time PCR-based methods to increase the overall proportion of patients participating in cervical cancer screening procedures. We proposed a new general HPV DNA real-time PCR on the Mx4000 (Stratagene) and LightCycler (Roche Diagnostics) systems usable for both cervical scrape specimens and urine samples. A linear range was obtained from 5 DNA copies to 8 log ₁₀ DNA copies/ml, and intra- and interassay variations were between 1.8 and 4%. Cervical carcinoma and HPV DNA screening was performed in 333 individual women referred for gynecological examination at the university hospitals of Angers and Brest and enrolled in the PapU study. Among cervical specimens ( n = 333), 45% were positive for HPV DNA, with a mean viral load at 5.00 log/ml (± 1.73). Among urine samples ( n = 177), 37% were positive with a significant 50-fold-lower mean viral load (3.77 ± 1.32 log/ml; P < 0.0001). Kappa agreement for HPV DNA between cervical and urine specimens was excellent (93%). Thus, we developed a highly sensitive and quantitative general HPV DNA real-time PCR method that allows mass screening of patients with HPV infection. The ongoing longitudinal and prospective multicenter PapU study should give us the opportunity to validate this method adapted to HPV DNA screening in urine samples in a larger population.

Background: The lung of patients with cystic fibrosis (CF) is particularly sensitive to Pseudomonas aeruginosa. This bacterium plays an important role in the poor outcome of CF patients. During the disease progress, first acquisition of P. aeruginosa is the key-step in the management of CF patients. Quantitative PCR (qPCR) offers an opportunity to detect earlier the first acquisition of P. aeruginosa by CF patients. Given the lack of a validated protocol, our goal was to find an optimal molecular protocol for detection of P. aeruginosa in CF patients. Methods: We compared two formerly described qPCR formats in early detection of P. aeruginosa in CF sputum samples: a qPCR targeting oprL gene, and a multiplex PCR targeting gyrB and ecfX genes. Results: Tested in vitro on a large panel of P. aeruginosa isolates and others gram-negative bacilli, oprL qPCR exhibited a better sensitivity (threshold of 10 CFU/mL versus 730 CFU/mL), whereas the gyrB/ecfX qPCR exhibited a better specificity (90% versus 73%). These results were validated ex vivo on 46 CF sputum samples positive for P. aeruginosa in culture. Ex vivo assays revealed that qPCR detected 100 times more bacterial cells than culture-based method did. Conclusion: Based on these results, we proposed a reference molecular protocol combining the two qPCRs, which offers a sensitivity of 100% with a threshold of 10 CFU/mL and a specificity of 100%. This combined qPCR-based protocol can be adapted and used for other future prospective studies.

Point-of-care syndromic PCR (POC SPCR) assays are useful tools for the rapid detection of the most common causative agents of community-acquired infections responsible for meningitis and encephalitis infections. We evaluated the performance characteristics of the new QIAstat-Dx^® Meningitis/Encephalitis panel (QS) compared to the laboratory reference methods and the POC SPCR Biofire^® FilmArray^® Meningitis Encephalitis Panel (FA). Viral (Enterovirus, Parechovirus, HSV-1, HSV-2, HHV-6, VZV) and bacterial (E. coli K1, H. influenzae, L. monocytogenes, encapsulated N. meningitidis, M. pneumoniae, S. agalactiae, S. pneumoniae, S. pyogenes) pathogens were suspended at low concentrations and tested with the POC SPCR systems. The reproducibility, analytical specificity, carryover contamination, interferences and clinical samples were evaluated. All samples tested positive with both QS and FA except for those containing the lowest concentrations of Enterovirus-D68-B3, Echovirus-30 and S. agalactiae which were only detected by FA. In terms of analytical specificity, we observed 3 false positive results out of 48 QS tests versus 1 out of 37 FA tests. For the other studied criteria, both QS and FA performed as expected. Our results suggest that the performance characteristics of QS are close to those of FA. A prospective multicenter study would be useful to complete the performances evaluation of QS.

The Human papillomaviruses (HPV) have existed in the human population since the archaic hominids. Over the course of human migration and evolution, HPVs have co-evolved with humans on all continents to become today the leading cause of cervical cancer. HPVs are classified by genera, species, genotype, lineage, sub-lineage and variants. Among more than 200 HPV genotypes, HPV16 is the most common and the most oncogenic at high-risk (HPV-HR). If viral oncogenesis is governed by numerous factors and mechanisms involving the virus and its host, the E6/E7 oncogenic proteins of HR-HPV play a central role and are always expressed in cervical cancers. Those of HPV16 have the greatest affinity for cellular proteins involved in cellular control, p53/E6 and pRb/E7. Some E6/E7 HPV16 mutants are associated with persistent infection, correlating with viral lineages and their ethnic and geographical origin. If the splicing of viral mRNAs encoding E6/E7 allows the overexpression of the E7 protein, which is an essential condition for the establishment of HR-HPV oncogenesis, other mechanisms contribute to strengthening it. On the one hand, the accidental integration of the viral genome, in particular the E1/E2 coding region, participates in the transformation of the infected cell. On the other hand, hypermethylation of the viral L1/L2 genomic regions is observed in the advanced stages of infection. Different methods for analyzing mutations, splice sites, integrations and methylations of the viral genome have been described. These virological markers could complete the detection of HR-HPV in the context of cervical cancer screening, currently recommended in France.