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Journal of Clinical Microbiology, July 2009, p. 2344-2346, Vol. 47, No. 7
0095-1137/09/$08.00+0     doi:10.1128/JCM.00924-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Confirmation of the First Hong Kong Case of Human Infection by Novel Swine Origin Influenza A (H1N1) Virus Diagnosed Using Ultrarapid, Real-Time Reverse Transcriptase PCR{triangledown}

Susanna K. P. Lau, Kwok-Hung Chan, and Cyril C. Y. Yip Tak-Keung Ng, Owen T. Y. Tsang, Patrick C. Y. Woo, and Kwok-Yung Yuen,*

Department of Microbiology
The University of Hong Kong
Queen Mary Hospital
Hong Kong

Department of Clinical Pathology
Princess Margaret Hospital
Hong Kong

Department of Medicine and Geriatrics
Princess Margaret Hospital
Hong Kong

Department of Microbiology
The University of Hong Kong
Queen Mary Hospital
Hong Kong

Received 12 May 2009/ Returned for modification 15 May 2009/ Accepted 20 May 2009


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INTRODUCTION
 
The emergence of novel swine origin influenza A (H1N1) virus (S-OIV), first recognized in Mexico, has affected 4,379 patients in 29 countries, with 49 deaths as of 10 May 2009 (2-5, 7, 8). Here we describe the first case of S-OIV infection in Hong Kong, as confirmed by LightCycler and ultrarapid reverse transcriptase PCR (RT-PCR) analysis.

A 25-year-old previously healthy and asymptomatic Mexican man arrived in Hong Kong from Mexico via Shanghai on 30 April 2009. He attended the hospital emergency room because of fever, productive cough, and myalgia on the same day. His temperature was 38°C. His total white cell count was 4.7 x 109/liter, with lymphopenia of 0.9 x 109/liter. Hemoglobin, platelet, liver, and renal function test results were normal. His chest radiograph was clear. The results of a test of his nasopharyngeal aspirate (NPA) for the presence of influenza A or B virus direct antigen were negative (9). The result from an RT-PCR test to detect the presence of the influenza A virus M gene was positive but that from an RT-PCR test to detect influenza A virus subtypes H1 (human) and H3 was negative. LightCycler and ultrarapid RT-PCR confirmed the presence of S-OIV. Oseltamivir therapy was started, and the symptoms subsided on the second day. A total of 12 specimens from close contacts, including passengers who had been in close proximity on the same flight and asymptomatic contacts at the same hotel, were negative upon testing.

RNA extraction was performed using a QIAamp virus RNA minikit (Qiagen) (6). All procedures involving clinical specimens and influenza virus were performed in a biosafety level 2 laboratory with biosafety level 3 practices (1). By means of conventional RT-PCR, an 83-bp fragment of the hemagglutinin (HA) gene of swine influenza A H1 virus was amplified using 0.5 µM primers (LPW9948 [5'-GGTAAATGTAACATTGCT-3'], corresponding to nucleotides 208 to 225, and LPW9949 [5'-ACAATGTAGGACCATGAGCTT-3'], corresponding to nucleotides 270 to 290) designed by multiple alignment of the HA gene sequences of swine H1 virus and S-OIV strain A/California/04/2009 (available in GenBank). These primers were designed to detect swine H1 virus but not human seasonal H1N1 or H3N2 viruses. Positive-control experiments with a swine H1 virus (A/SW/HK/294/09) and S-OIV (A/California/04/2009) and negative-control experiments with human H1N1 and H3N2 viruses were performed. RT was performed using a SuperScript III kit (Invitrogen). Each PCR mixture (25 µl) contained cDNA, PCR buffer (10 mM Tris-HCl [pH 8.3], 50 mM KCl, 2 mM MgCl2, 0.01% gelatin), 200 µM (each) deoxynucleoside triphosphates, and 1.0 U Taq polymerase (Boehringer). The mixtures were incubated at 95°C for 10 min and then amplified in 45 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s, followed by a final extension at 72°C for 5 min in an automated thermal cycler (Applied Biosystems).

Since early detection of this virus is crucial for timely implementation of infection control measures, we developed an ultrarapid RT-PCR assay which allows detection of S-OIV within 50 min by the use of the same PCR primers but with AmpliTaq Gold Fast PCR master mix, UP (2x) (Applied Biosystems). The mixtures were amplified using 45 cycles of 96°C for 3 s, 55°C for 3 s, and 68°C for 5 s and a final extension at 72°C for 10 s. Both conventional and ultrarapid RT-PCR analysis of RNA from the patient's NPA, S-OIV, and swine H1 virus positive controls, but not from seasonal H1N1 or H3N2 virus, showed bands of 83 bp. All of the positive-testing M and HA gene PCR products from the patient were confirmed by sequencing to be S-OIV.

Real-time RT-PCR assays were performed, using the same PCR primers, as described previously (6). cDNA was amplified in a 2.0 LightCycler (Roche) using 20-µl reaction mixtures containing FastStart DNA master SYBR green I mix reagent (Roche), 2 µl of cDNA, 3.5 mM MgCl2, and 0.5 mM primers at 95°C for 10 min followed by 50 cycles of 95°C for 10 s, 62°C for 5 s, and 72°C for 5 s. The melting temperatures of S-OIV and swine H1 virus were 79.9°C and 83.0°C, respectively (Fig. 1). The viral load in the nasopharyngeal secretion dropped from 2.3 x 106 copies per ml on day 1 of illness to an undetectable level on day 6 (Table 1). A total of 150 specimens that tested positive for seasonal influenza A H3N2 or H1N1 virus tested negative in both LightCycler and conventional RT-PCR assays. A cytopathic effect was observed in an MDCK cell line at 72 h after inoculation of the first NPA. The culture supernatant was strongly positive for H1 S-OIV by LightCycler RT-PCR, while the cells were strongly positive for influenza A virus nucleoprotein by immunofluorescence (Dako) (9). Complete gene sequencing of the H1 virus (GenBank accession no. GQ168606) showed 99.9% identity to S-OIV (A/California/04/2009).


Figure 1
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  		FIG. 1. LightCycler real-time RT-PCR assays for S-OIV and swine H1 virus. (A) Standard curve for quantitative analysis of S-OIV H1 gene. (B) An amplification plot of fluorescence intensity against the PCR cycle. The amplification curves of patient NPA containing S-OIV, swine H1 and negative controls are indicated. (C) Melting curve analysis of PCR products showing different melting temperatures for S-OIV (patient strain) and swine H1 virus A/SW/HK/294/09.


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  		TABLE 1. Viral load of patient specimens collected at different days after onset of illness by LightCycler RT-PCR

The ultrarapid and LightCycler assays described here can be used as rapid diagnostic tests to specifically detect S-OIV and other swine H1 viruses. Furthermore, the LightCycler assay may distinguish between novel S-OIV and other swine H1 viruses by determination of different melting temperatures. However, sequencing of longer or other gene segments should be required for more confident differentiation.


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ACKNOWLEDGMENTS
 
We thank Sik-To Lai, Sze Tsing Jonpaul Zee, Thomas So, and Kay Yan Tsang for patient management and provision of clinical data, Hoi-Wah Tsoi and Yi Huang for technical support, and York Chow, J. S. M. Peiris, and Vincent C. C. Cheng for support and advice.


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FOOTNOTES
 
* Phone: (852) 28554892, Fax: (852) 28551241, E-mail: kyyuen{at}hkucc.hku.hk Back

{triangledown} Published ahead of print on 27 May 2009. Back


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Journal of Clinical Microbiology, July 2009, p. 2344-2346, Vol. 47, No. 7
0095-1137/09/$08.00+0     doi:10.1128/JCM.00924-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



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