title: Comparison of RT-PCR assay and virus isolation in cell culture for the detection of Alkhumra hemorrhagic fever virus.
abstract: Alkhumra hemorrhagic fever virus (AHFV) is an emerging flavivirus that was isolated originally from Saudi Arabia in 1994-1995. The main tests used for the detection of AHFV are the real time (rt) RT-PCR and virus isolation in cell culture. In the present study the detection of AHFV by rtRT-PCR was compared with virus isolation in BHK-21, HEp-2, and LLC-MK2 cell lines. AHFV suspensions grown in BHK-21, HEp-2, and LLC-MK2 cell lines were serially diluted 10-fold from 10(-1) to 10(-11) . Samples from each dilution were used to inoculate four cell culture tubes and were also examined by the rtRT-PCR for AHFV RNA. Fifteen non-inoculated cell culture samples (five from each cell line) were included blindly in both tests. Thus, a total of 132 AHFV-positive and 15 negative control samples were tested. The rtRT-PCR could detect the viral RNA in all diluted specimens up to and including the 10(-10) dilution (40 specimens for each cell line), whereas, cell cultures were positive in 70% of specimens for BHK-21, 65% for LLC-MK2, and 45% for HEp-2 at this dilution. None of the three cell cultures nor the rtRT-PCR was positive at 10(-11) dilution. The specificity and positive predictive values of virus isolation compared to rtRT-PCR were each 100%, whereas the negative predictive values were 29.4% for BHK-21, 26.3% for LLC-MK2, and 18.5% for HEp-2. In conclusion, the rtRT-PCR is more sensitive than virus isolation for detecting AHFV.
authors.affiliation: Faculty of Medicine, Department of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia.
authors.forename: Tariq A
authors.initials: TA
authors.lastname: Madani
authors.forename: El-Tayb M E
authors.initials: el-TM
authors.lastname: Abuelzein
authors.forename: Esam I
authors.initials: EI
authors.lastname: Azhar
authors.forename: Hussein M S
authors.initials: HM
authors.lastname: Al-Bar
authors.forename: Huda
authors.initials: H
authors.lastname: Abu-Araki
authors.forename: Thomas G
authors.initials: TG
authors.lastname: Ksiazek
mesh_terms: D000818:Animals; D002460:Cell Line; D019411:Clinical Laboratory Techniques / Q000379:methods*; D004669:Encephalitis Viruses, Tick-Borne / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D004675:Encephalitis, Tick-Borne / Q000175:diagnosis*; D006801:Humans; D011237:Predictive Value of Tests; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012529:Saudi Arabia; D012680:Sensitivity and Specificity; D014776:Virus Cultivation / Q000379:methods
keywords: Alkhumra hemorrhagic fever virus; cell culture, real time RT-PCR; virus isolation