Comparison of three human papillomavirus DNA detection methods: Next generation sequencing, multiplex-PCR and nested-PCR followed by Sanger based sequencing.
To compare the diagnostic performance for HPV infection using three laboratorial techniques. Ninty-five cervicovaginal samples were randomly selected; each was tested for HPV DNA and genotypes using 3 methods in parallel: Multiplex-PCR, the Nested PCR followed by Sanger sequencing, and the Next_Gen Sequencing (NGS) with two assays (NGS-A1, NGS-A2). The study was approved by the Brazilian National IRB (CONEP protocol 16,800). The prevalence of HPV by the NGS assays was higher than that using the Multiplex-PCR (64.2% vs. 45.2%, respectively; P = 0.001) and the Nested-PCR (64.2% vs. 49.5%, respectively; P = 0.003). NGS also showed better performance in detecting high-risk HPV (HR-HPV) and HPV16. There was a weak interobservers agreement between the results of Multiplex-PCR and Nested-PCR in relation to NGS for the diagnosis of HPV infection, and a moderate correlation for HR-HPV detection. Both NGS assays showed a strong correlation for detection of HPVs (k = 0.86), HR-HPVs (k = 0.91), HPV16 (k = 0.92) and HPV18 (k = 0.91). NGS is more sensitive than the traditional Sanger sequencing and the Multiplex PCR to genotype HPVs, with promising ability to detect multiple infections, and may have the potential to establish an alternative method for the diagnosis and genotyping of HPV.
Tropical Medicine Foundation Dr. Heitor Vieira Dourado, University of State of Amazonas, Manaus, Brazil.|Center of Infectious Diseases Research, Federal University of Espírito Santo, Vitória, Brazil.
Allex Jardim
AJ
da Fonseca
Tropical Medicine Foundation Dr. Heitor Vieira Dourado, University of State of Amazonas, Manaus, Brazil.
Renata Silva
RS
Galvão
Center of Infectious Diseases Research, Federal University of Espírito Santo, Vitória, Brazil.
Angelica Espinosa
AE
Miranda
Tropical Medicine Foundation Dr. Heitor Vieira Dourado, University of State of Amazonas, Manaus, Brazil.
Luiz Carlos de Lima
LC
Ferreira
Department of Microbiology & Immunology, Epidemiology & Population Health, and Obstetrics, Gynecology & Women's Health, Albert Einstein College of Medicine, New York.|Department of Microbiology, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China.
Zigui
Z
Chen
D001938:Brazil; D002584:Cervix Uteri / Q000821:virology; D004279:DNA, Viral / Q000235:genetics*; D005260:Female; D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D027383:Papillomaviridae / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D030361:Papillomavirus Infections / Q000175:diagnosis*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014621:Vagina / Q000821:virology
D004279:DNA, Viral
diagnosis; diagnostic techniques; laboratory tests; obstetrical and gynecological; papillomavirus infections