Comparison of RT-PCR assay and virus isolation in cell culture for the detection of alkhumra hemorrhagic fever virus

• Published in: Journal of medical virology Vol. 86; no. 7; pp. 1176 - 1180
• Main Authors: Madani, Tariq A., Abuelzein, El-Tayb M.E., Azhar, Esam I., Al-Bar, Hussein M.S., Abu-Araki, Huda, Ksiazek, Thomas G.
• Format: Journal Article
• Language: English
• Published: United States Blackwell Publishing Ltd 01.07.2014; Wiley Subscription Services, Inc
• Subjects: Alkhumra hemorrhagic fever virus; Animals; Bioassays; Cell culture; cell culture, real time RT‐PCR; Cell Line; Clinical Laboratory Techniques - methods; Encephalitis Viruses, Tick-Borne - genetics; Encephalitis Viruses, Tick-Borne - growth & development; Encephalitis Viruses, Tick-Borne - isolation & purification; Encephalitis, Tick-Borne - diagnosis; Flavivirus; Humans; Polymerase chain reaction; Predictive Value of Tests; real time RT-PCR; Real-Time Polymerase Chain Reaction - methods; Reverse Transcriptase Polymerase Chain Reaction - methods; Saudi Arabia; Sensitivity and Specificity; Virology; Virus Cultivation - methods; virus isolation; Saudi Arabia; cell culture, real time RT-PCR; virus isolation; Alkhumra hemorrhagic fever virus
• ISSN: 0146-6615; 1096-9071
• DOI: 10.1002/jmv.23755

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. J. Med. Virol. 86:1176–1180, 2014. © 2013 Wiley Periodicals, Inc.