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

Tariq A. Madani, El Tayb M E Abuelzein, Esam I. Azhar, Hussein M S Al-Bar, Huda Abu-Araki, Thomas Ksiazek

Research output: Contribution to journalArticle

3 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)1176-1180
Number of pages5
JournalJournal of Medical Virology
Volume86
Issue number7
DOIs
StatePublished - 2014

Fingerprint

Fever
Cell Culture Techniques
Viruses
Polymerase Chain Reaction
Cell Line
Flavivirus
Saudi Arabia
RNA Viruses
Viral RNA
Real-Time Polymerase Chain Reaction
Suspensions

Keywords

  • Alkhumra hemorrhagic fever virus
  • Cell culture, real time RT-PCR
  • Virus isolation

ASJC Scopus subject areas

  • Virology
  • Infectious Diseases
  • Medicine(all)

Cite this

Comparison of RT-PCR assay and virus isolation in cell culture for the detection of alkhumra hemorrhagic fever virus. / Madani, Tariq A.; Abuelzein, El Tayb M E; Azhar, Esam I.; Al-Bar, Hussein M S; Abu-Araki, Huda; Ksiazek, Thomas.

In: Journal of Medical Virology, Vol. 86, No. 7, 2014, p. 1176-1180.

Research output: Contribution to journalArticle

Madani, Tariq A. ; Abuelzein, El Tayb M E ; Azhar, Esam I. ; Al-Bar, Hussein M S ; Abu-Araki, Huda ; Ksiazek, Thomas. / Comparison of RT-PCR assay and virus isolation in cell culture for the detection of alkhumra hemorrhagic fever virus. In: Journal of Medical Virology. 2014 ; Vol. 86, No. 7. pp. 1176-1180.
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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.",
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AU - Madani, Tariq A.

AU - Abuelzein, El Tayb M E

AU - Azhar, Esam I.

AU - Al-Bar, Hussein M S

AU - Abu-Araki, Huda

AU - Ksiazek, Thomas

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N2 - 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.

AB - 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.

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