title: Genetic Variability in Probe Binding Regions Explains False Negative Results of a Molecular Assay for the Detection of Dengue Virus.
abstract: Dengue fever is currently the most prevalent disease caused by mosquito-borne flaviviruses. Despite being potentially fatal, there are no specific antiviral therapies for Dengue virus (DENV) infections. Therefore, early, accurate, and rapid diagnosis plays an important role in proper patient management. In this study, we evaluated the performance of a probe-based real-time RT-PCR (rRT-PCR) assay against that of a conventional RT-PCR assay in three sample cohorts from Pakistan (n = 94) and Singapore (first cohort; n = 559, second cohort; n = 123). The Pakistan cohort also included a comparison with virus isolation. The rRT-PCR assay showed relatively lower overall sensitivity (20.2%) in the Pakistan cohort than that in first (90.8%) and second (80.5%) Singapore cohorts. Surprisingly, the overall sensitivity of rRT-PCR assay was lower compared with the virus isolation (26.6%) among Pakistan samples, indicating a high percentage (79.8%) of false negatives due to rRT-PCR assay. The analysis of sequences of failed and successful DENV isolates indicated mismatches in probe binding regions as the likely cause of rRT-PCR assay failure. Our observations testify the importance of utilizing a combination of methods for dengue diagnostics and surveillance. We emphasize that a thorough understanding of the genetic composition of local DENV populations as well as regular monitoring of the performance and reviewing of probe/primer sequences are essential to maintain a consistently high diagnostic accuracy of PCR-based assays.
authors.affiliation: 1 Environmental Health Institute , National Environment Agency, Singapore, Singapore .
authors.forename: Carmen
authors.initials: C
authors.lastname: Koo
authors.affiliation: 1 Environmental Health Institute , National Environment Agency, Singapore, Singapore .
authors.forename: Simrandeep
authors.initials: S
authors.lastname: Kaur
authors.affiliation: 1 Environmental Health Institute , National Environment Agency, Singapore, Singapore .
authors.forename: Zhi-Yong
authors.initials: ZY
authors.lastname: Teh
authors.affiliation: 1 Environmental Health Institute , National Environment Agency, Singapore, Singapore .
authors.forename: Helen
authors.initials: H
authors.lastname: Xu
authors.affiliation: 2 Department of Pathology Microbiology, Aga Khan University , Karachi, Pakistan .
authors.forename: Amna
authors.initials: A
authors.lastname: Nasir
authors.affiliation: 1 Environmental Health Institute , National Environment Agency, Singapore, Singapore .
authors.forename: Yee-Ling
authors.initials: YL
authors.lastname: Lai
authors.affiliation: 2 Department of Pathology Microbiology, Aga Khan University , Karachi, Pakistan .
authors.forename: Erum
authors.initials: E
authors.lastname: Khan
authors.affiliation: 1 Environmental Health Institute , National Environment Agency, Singapore, Singapore .
authors.forename: Lee-Ching
authors.initials: LC
authors.lastname: Ng
authors.affiliation: 1 Environmental Health Institute , National Environment Agency, Singapore, Singapore .
authors.forename: Hapuarachchige C
authors.initials: HC
authors.lastname: Hapuarachchi
mesh_terms: D001483:Base Sequence; D003716:Dengue Virus / Q000235:genetics* / Q000302:isolation & purification*; D005188:False Negative Reactions; D014644:Genetic Variation; D012367:RNA, Viral / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D065288:Serogroup
chemical_list: D012367:RNA, Viral
keywords: Dengue virus; False negatives; Genetic variability; Molecular diagnostics; Polymerase chain reaction