Jpn. J. Infect. Dis., 59, 2006

Laboratory and Epidemiology Communications

Rapid Subtyping of Influenza A Virus by Loop-Mediated Isothermal
Amplification: Two Cases of Influenza Patients Who Returned from Thailand
Naoko Nakagawa* and Masahiro Ito
Kobe Institute of Health, Kobe 650-0046, Japan
Communicated by Ichiro Kurane(Accepted March 23, 2006)

Influenza is an important infectious disease that is responsible for considerable morbidity and mortality in elderly andcompromised individuals (1,2). Once a patient has been
diagnosed, an anti-influenza virus drug such as oseltamivir
can be administered. Therefore, immunochromatography rapid
diagnosis tests are popularly used as diagnostic tools at
bedside. However, hemagglutinin (HA) subtypes of influenza
A virus are not detected by the immunochromatography tests,
and new subtypes of influenza A virus may emerge at anytime. When clinic patients are diagnosed as influenza Apositive by the immunochromatography tests and are foundto have just returned from countries where human avian
influenza cases have been reported, rapid virus subtyping isessential. We experienced 2 such cases and determined virus
subtypes rapidly by applying a reverse transcriptase (RT)loop-mediated isothermal amplification (LAMP) assay.
LAMP is a novel nucleic acid amplification method (3).
The specificity is extremely high, because 4 primers are usedrecognizing 6 distinct regions on the target DNA. This method
is faster than polymerase chain reaction, because amplification is carried out under isothermal conditions. LAMP has
been used for the rapid diagnosis of infectious agents including bacteria such as Mycobacterium tuberculosis (4) andDNA viruses such as herpes viruses (5,6). RT-LAMP has beenused for the detection of RNA viruses such as SARS
coronavirus (7,8). We established RT-LAMP for the detection of influenza A virus subtype 1 (AH1), subtype 3 (AH3)
and influenza B virus. The total procedure was completed
within 3 h, and the sensitivity was higher than that of theimmunochromatography test (9).
Case 1: A 30-year-old Japanese man, a resident of Bangkok,
Thailand, came back to Japan for summer vacation on Friday,
August 5, 2005. He was engaged in trading and had no history of contact with living or dead birds. On Saturday, August6, fever (temperature 38.5°C), sore throat and myalgia
developed, and on Sunday, August 7 he visited the hospital. He was diagnosed with influenza A by the immunochromatography test. Oseltamivir was administered, and thepatient returned home. The case was reported to Kobe HealthCenter on Monday, August 8. The nasopharyngeal sample
obtained for the immunochromatography test was kept in
the refrigerator, and transported to Kobe Institute of Healthfor further investigation (2:00 p.m.). RNA was extracted fromthe sample by QIAmp Viral RNA Mini Kit (Qiagen, Tokyo,Japan) in the BSL-3 laboratory. The specimen was examinedby RT-LAMP as previously described (9). The sample waspositive with subtype H3 specific primers. AH1 positive andAH3 positive controls were positive with subtype H1 and H3
specific primers, respectively. The negative control did not
respond to any primers (Figure 1). We immediately reported
to the hospital that the HA subtype of influenza A virus in thepatient’s sample was H3 (4:30 p.m.). Thereafter, the sample
was inoculated to Madin-Darby canine kidney (MDCK) cells
in 24-well plates and incubated at 35°C in the BSL-2 laboratory. On the 3rd day, a cytopathic effect was detected and theFig. 1. Influenza virus detection by RT-LAMP. Turbidity caused by theexistence of magnesium pyrophosphate, the amplification by-products
was detected by the real-time turbidimeter LA320-C (Y-axis). X-axis
shows time (min). (A) Absence of AH1 in the sample from Case 1. A
sample from Case 1 as well as positive controls (AH1, AH3) and anegative control were subjected to RT-LAMP using primers specificfor AH1. (B) Detection of AH3 in the sample from Case 1. A sample
from Case 1 as well as positive controls (AH1, AH3) and a negative
control were subjected to RT-LAMP using primers specific for AH3.

*Corresponding author: Mailing address: Kobe Institute of Health,
4-6, Minatojima-nakamachi, Chuo-ku, Kobe 650-0046, Japan.
Tel: +81-78-302-6260, Fax: +81-78-302-0894, E-mail: naoko_nakagawa@office.city.kobe.jp
200

virus was identified as influenza A virus H3 subtype byhemagglutination inhibition (HI) test. The viral antigenicity
was serologically similar to that of A/Wyoming/3/2003.
Case 2: A 31-year-old Japanese man who had traveled inThailand for 5 days came back to Japan on Monday, September 26, 2005. The next morning, he developed fever. In thelate afternoon on September 29, he visited the hospital withhigh fever, chilliness, shivering and sore throat. Leukocyte
count (6100/mm3) and C-reactive protein level (7.3 mg/dl)
were at normal levels. He was diagnosed with influenza A by
the immunochromatography test. The nasopharyngeal specimen was transported to Kobe Institute of Health, and RTLAMP was undertaken as described above. The patient’s
sample was positive with H3 specific primers. The virus wasisolated from sample-inoculated MDCK cells on the 4th day
and was identified as influenza A virus H3 type. The antigenicity was serologically similar to that of A/Wyoming/3/2003.
Thus, RT-LAMP was demonstrated to be a useful rapid
diagnostic and subtyping method for influenza viruses. By
designing primer pairs, RT-LAMP is applicable to the detection of other subtypes. Detection kits for H5 subtype are nowcommercially available. Human influenza by other subtypes
such as AH9 may also emerge at any time. RT-LAMP willserve as a useful diagnostic tool.

of influenza activity. Am. J. Epidemiol., 113, 227-235.
3. Notomi, T., Okayama, H., Masubuchi, H., Yonekawa,T., Watanabe, K., Amino, N. and Hase, T. (2000): Loopmediated isothermal amplification of DNA. Nucleic
Acids Res., 28, e63.
4. Iwamoto, T., Sonobe, T. and Hayashi, K. (2003): Loopmediated isothermal amplification for direct detectionof Mycobacterium tuberculosis Complex, M. avium, andM. intracellulare in sputum samples. J. Clin. Microbiol.,41, 2616-2622.
5. Ihira, M., Yoshikawa, T., Enomoto, Y., Akimoto, S.,
Ohashi, M., Suga, S., Nishimura, N., Ozaki, T.,
Nishiyama, Y., Notomi, T., Ohta, Y. and Asano, Y. (2004):
Rapid diagnosis of human herpesvirus 6 infection by
a novel DNA amplification method, loop-mediated isothermal amplification. J. Clin. Microbiol., 42, 140-145.
6. Okamoto, S., Yoshikawa, T., Ihira, M., Suzuki, K.,
Shimokata, K., Nishiyama, Y. and Asano, Y. (2004):Rapid detection of varicella-zoster virus infection by aloop-mediated isothermal amplification method. J. Med.
Virol., 74, 677-682.
7. Fujino, M., Yoshida, N., Yamaguchi, S., Hosaka, N., Ota,Y., Notomi, T. and Nakayama, T. (2005): A simple method
for the detection of measles virus genome by loopmediated isothermal amplification (LAMP). J. Med.
Virol., 76, 406-413.
8. Thai, H. T. C., Le, M. Q., Vuong, C. D., Parida, M.,
Minekawa, H., Notomi, T., Hasebe, F. and Morita,K. (2004): Development and evaluation of a novel
loop-mediated isothermal amplification of method for
rapid detection of severe acute respiratory syndrome
coronavirus. J. Clin. Microbiol., 42, 1956-1961.
9. Ito, M., Watanabe, M., Nakagawa, N., Ihara, T. andOkuno, Y.: Rapid detection and typing of influenza
A and B by loop-mediated isothermal amplification:comparison with immunochromatography and virus
isolation. J. Virol. Methods (in press).

This article appeared in the Infectious Agents SurveillanceReport (IASR), vol. 26, p. 303, 2005 in Japanese.
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