Evaluation of a rapid molecular algorithm for detection of pandemic influenza A (H1N1) 2009 virus and screening for a key oseltamivir resistance (H275Y) substitution in neuraminidase.
BACKGROUND
Rapid and specific molecular tests for identification of the recently identified pandemic influenza A/H1N1 2009 virus as well as rapid molecular tests to identify antiviral resistant strains are urgently needed.


OBJECTIVE
We have evaluated the performance of two novel reverse transcriptase polymerase chain reactions (RT-PCRs) targeting specifically hemagglutinin and neuraminidase of pandemic influenza A/H1N1 virus in combination with a conserved matrix PCR. In addition, we investigated the performance of a novel discrimination RT-PCR for detection of the H275Y resistance mutation in the neuraminidase gene.


METHODS
Clinical performance of both subtype specific RT-PCR assays was evaluated through analysis of 684 throat swaps collected from individuals meeting the WHO case definition for the novel pandemic influenza virus. Analytical performance was analyzed through testing of 10-fold serial dilutions of RNA derived from the first Dutch sequenced and cultured confirmed case of novel pandemic influenza infection. Specificity and discriminative capacities of the H275Y discrimination assay were performed by testing wild type and recombinant H275Y pandemic influenza.


RESULTS
121 throat swaps collected from April 2009 to July 2009 were positive by at least two out of three RT-PCRs, and negative for the seasonal H3/H1 subtype specific RT-PCR assays. 117 of these were tested positive for all three (Ct-values from 15.1 to 36.8). No oseltamivir resistance was detected.


CONCLUSIONS
We present a sensitive and specific approach for detection of pandemic influenza A/H1N1 2009 and a rapid RT-PCR assay detecting a primary oseltamivir resistance mutation which can be incorporated easily into clinical virology algorithms.
Erasmus MC, Department of Virology, 's Gravendijkwal 230, 3015 CE Rotterdam, The Netherlands.
E
E
van der Vries
M
M
Jonges
S
S
Herfst
J
J
Maaskant
A
A
Van der Linden
J
J
Guldemeester
G I
GI
Aron
T M
TM
Bestebroer
M
M
Koopmans
A
A
Meijer
R A M
RA
Fouchier
A D M E
AD
Osterhaus
C A
CA
Boucher
M
M
Schutten
D000465:Algorithms; D000998:Antiviral Agents / Q000494:pharmacology / Q000627:therapeutic use; D004196:Disease Outbreaks; D024882:Drug Resistance, Viral; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D007251:Influenza, Human / Q000188:drug therapy / Q000453:epidemiology* / Q000821:virology*; D016014:Linear Models; D009439:Neuraminidase / Q000235:genetics*; D053139:Oseltamivir / Q000494:pharmacology* / Q000627:therapeutic use; D017354:Point Mutation; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014764:Viral Proteins / Q000235:genetics
D000998:Antiviral Agents; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D014764:Viral Proteins; D053139:Oseltamivir; D009439:Neuraminidase