Post-pandemic influenza A (H1N1) virus detection by real-time PCR and virus isolation

  • Zaki, Ali Mohamed (Medical Microbiology and Immunology Department, Faculty of Medicine, Ain Shams University) ;
  • Taha, Shereen El-Sayed (Medical Microbiology and Immunology Department, Faculty of Medicine, Ain Shams University) ;
  • Shady, Nancy Mohamed Abu (Pediatrics Department, Faculty of Medicine, Ain Shams University) ;
  • Abdel-Rehim, Asmaa Saber (Internal Medicine Department, Faculty of Medicine, Ain Shams University) ;
  • Mohammed, Hedya Said (Chest Diseases Department, Faculty of Medicine, Ain Shams University)
  • Received : 2018.12.17
  • Accepted : 2018.12.28
  • Published : 2019.03.31


Influenza A (H1N1) virus caused a worldwide pandemic in 2009-2010 and still remains in seasonal circulation. Continuous surveillance activities are encouraged in the post pandemic phase to watch over the trend of occurrence every year, this is better to be done by a rapid and sensitive method for its detection. This study was conducted to detect proportions of occurrence of influenza A virus (H1N1) in patients with influenza-like illness. Samples from 500 patients with influenza or influenza-like clinical presentation were tested by real-time reverse transcription polymerase chain reaction (RT-PCR) and virus tissue culture. Among the total 500 participants, 193 (38.6%) were females and 307 (61.4%) males. Seventy-one patients (14.2%) were positive for H1N1 virus infection with real-time RT-PCR while 52 (10.4%) were positive by tissue culture. Non-statistically significant relation was found between age and gender with the positivity of H1N1. Sensitivity and specificity of real-time RT-PCR was 98.08% and 95.54%, respectively, in comparison to virus isolation with accuracy 95.8%. This study showed that H1N1 virus was responsible for a good proportion of influenza during the post-pandemic period. Real-time RT-PCR provides rapidity and sensitivity for the detection of influenza A virus (H1N1) compared with virus isolation and thus it is recommended as a diagnostic tool.


influenza A virus;H1N1;post pandemic;real-time RT-PCR and tissue culture;seasonal influenza

MSMHBQ_2019_v55n1_25_f0001.png 이미지

Fig. 1. Amplification plot of H1N1 positive samples.

Table 1. Relation between of influenza A (H1N1) infection and gender

MSMHBQ_2019_v55n1_25_t0001.png 이미지

Table 2. Distribution of influenza A (H1N1) infection by age group

MSMHBQ_2019_v55n1_25_t0002.png 이미지

Table 3. Relation between of influenza A (H1N1) infection and age

MSMHBQ_2019_v55n1_25_t0003.png 이미지

Table 4. Clinical manifestations of influenza in the infected population

MSMHBQ_2019_v55n1_25_t0004.png 이미지

Table 7. The performance of real time RT-PCR in relation to conventional viral culture

MSMHBQ_2019_v55n1_25_t0005.png 이미지

Table 5. Comorbid conditions in the infected population

MSMHBQ_2019_v55n1_25_t0006.png 이미지

Table 6. Seasonal distribution of influenza A (H1N1) virus infection

MSMHBQ_2019_v55n1_25_t0007.png 이미지


  1. Amaravathi KS, Sakuntala P, Sudarsi B, Manohar S, Nagamani R, and Rao SR. 2015. Clinical profile and outcome of recent outbreak of influenza A H1N1 (swine flu) at a tertiary care center in Hyderabad, Telangana. Ann. Trop. Med. Public Health 8, 267-271.
  2. Angione SL, Inde Z, Beck CM, Artenstein AW, Opal SM, and Tripathi A. 2013. Microdroplet sandwich real-time RT-PCR for detection of pandemic and seasonal influenza subtypes. PLoS One 8, e73497.
  3. Behzadi MA, Ziyaeyan M, and Alborzi A. 2016. A diagnostic one-step real-time reverse transcription polymerase chain reaction method for accurate detection of influenza virus type A. Arch. Med. Sci. 12, 1286-1292.
  4. Centers for Disease Control and Prevention. 2002. Prevention and control of influenza. Morb. Mortal. Wkly. Rep. 51, 1-31.
  5. Chauhan N, Narang J, Pundir S, Singh S, and Pundir CS. 2013. Laboratory diagnosis of swine flu: a review. Artif. Cells Nanomed. Biotechnol. 41, 189-195.
  6. Cheung PH, Chan C, and Jin D. 2017. To announce or not to announce: What is known about the 2016-2017 influenza season in Hong Kong? Emerg. Microbes Infect. 6, e78.
  7. Chudasama RK, Patel UV, Verma PB, Amin CD, Savaria D, Ninama R, and Fichadiya N. 2011. Clinico-epidemiological features of the hospitalized patients with 2009 pandemic influenza A (H1N1) virus infection in Saurashtra region, India (September, 2009 to February, 2010). Lung India 28, 11-16.
  8. Chudasama RK, Patel UV, Verma PB, Banerjee A, Buch P, and Patel P. 2013. A two wave analysis of hospitalizations and mortality from seasonal and pandemic 2009 A (H1N1) Influenza in Saurashtra, India: 2009-2011. Ann. Med. Health Sci. Res. 3, 334-340.
  9. Dee S and Jayathissa S. 2010. Clinical and epidemiological characteristics of the hospitalized patients due to pandemic H1N1 2009 viral infection: Experience at Hutt hospital, New Zealand. N. Z. Med. J. 123, 45-53.
  10. Demicheli V, Jefferson T, Rivetti D, and Deeks J. 2000. Prevention and early treatment of influenza in healthy adults. Vaccine 18, 957-1030.
  11. Dhakad S, Mali PC, Kaushik S, Lal AA, and Broor S. 2015. Comparison of multiplex RT-PCR with virus isolation for detection, typing and sub-typing of influenza virus from influenza-like illness cases. Indian J. Med. Microbiol. 33, 73-77.
  12. Dominguez-Cherit G, Lapinsky SE, Macias AE, Pinto R, Espinosa-Perez L, de la Torre A, Poblano-Morales M, Baltazar-Torres JA, Bautista E, Martinez A, et al. 2009. Critically ill patients with 2009 influenza A (H1N1) in Mexico. JAMA 302, 1880-1887.
  13. Dudley JP. 2009. Age-specific infection and death rates for human A H5N1avian influenza in Egypt. Euro Surveill. 14, 19198.
  14. Espy MJ, Uhl JR, Sloan LM, Buckwalter SP, Jones MF, Vetter EA, Yao JDC, Wengenack NL, Rosenblatt JE, Cockerill FR, et al. 2006. Real-time PCR in clinical microbiology: applications for routine laboratory testing. Clin. Microbiol. Rev. 19, 165-256.
  15. Gohar MK. 2012. Rapid diagnosis of influenza virus infection in different age groups. Egypt. J. Med. Microbiol. 21, 115-126.
  16. Hien TT, Liem NT, Dung NT, San LT, Mai PP, van Vinh Chau N, Suu PT, Dong VC, Mai LTQ, Thi NT, et al. 2004. Avian influenza A (H5N1) in 10 patients in Vietnam. N. Engl. J. Med. 350, 1179-1188.
  17. Hiergeista A, Stenderb S, and Kastlb L. 2016. Sensitive and reliable detection of influenza A (H1N1), influenza A (H3N2) and influenza B by commercial real-time PCR assays: RIDA$^{(R)}$GENE flu and RIDA$^{(R)}$GENE flu LC2.0 real-time PCR. Available on (accessed on Mar. 3, 2016).
  18. Jagannatha Rao SR, Rao MJ, Swamy N, and Umapathy BL. 2011. Profile of H1N1 infection in a tertiary care hospital. Indian J. Pathol. Microbiol. 54, 323-325.
  19. Jain B, Singh AK, Dangi T, Verma AK, Dwivedi M, Mohan M, Singh KP, and Jain A. 2014. Feasibility of real time PCR over cell culture in diagnosis of influenza virus infection: An experience of grade I viral diagnostic laboratory of developing country. Int. J. Appl. Sci. Biotechnol. 2, 97-102.
  20. Jordi RI, Rodriguez A, and Ibanez P. 2009. Intensive care adult patients with severe respiratory failure caused by influenza A (H1N1) in Spain. Crit. Care 13, 148.
  21. Kar SS, Selvaraj K, Ramalingam A, and Roy G. 2016. Time trend and predictors of lab positivity among suspected cases in the post pandemic phase of H1N1: An observation from a tertiary care hospital, South India. AMJ 9, 92-102.
  22. Khattab AM, Wagih KM, and Tag Eldin AMA. 2014. A study of the outcome of confirmed avian flu and swine flu cases admitted to Abbassia Chest Hospital between 2006 and 2010. Egypt. J. Bronchol. 8, 70-78.
  23. Krafft AE, Russell KL, Hawksworth AW, McCall S, Irvine M, Daum LT, Connoly JL, Reid AH, Gaydos JC, and Taubenberger JK. 2005. Evaluation of PCR testing of ethanol-fixed nasal swab specimens as an augmented surveillance strategy for influenza virus and adenovirus identification. J. Clin. Microbiol. 43, 1768-1775.
  24. Kumar A, Zarychanski R, and Pinto R. 2009. Critically ill patients with 2009 influenza A (H1N1) infection in Canada. JAMA 10, 1496.
  25. Lopez RP, Catalan P, Giannella M, Garcia de Viedma D, Sandonis V, and Bouza E. 2011. Comparison of real-time RT-PCR, shell vial culture, and conventional cell culture for the detection of the pandemic influenza A (H1N1) in hospitalized patients. Diagn. Microbiol. Infect. Dis. 69, 428-431.
  26. Mehta AA, Kumar VA, Nair SG, Joseph FK, Kumar G, and Singh SK. 2013. Clinical profile of patients admitted with swine-origin influenza a (H1N1) virus infection: An experience from a tertiary care hospital. J. Clin. Diagn. Res. 7, 2227-2230.
  27. Mu YP, Zhang ZY, Chen XR, Xi XH, Lu YF, Tang YW, and Lu HZ. 2010. Clinical features, treatment and prognosis of the initial cases of pandemic influenza H1N1 2009 virus infection in Shanghai China. QJM 103, 311-317.
  28. Muscatello DJ, Barr M, Thackway SV, and MacIntyre CR. 2011. Epidemiology of influenza-like illness during pandemic (H1N1) 2009, New South Wales, Australia. Emerg. Infect. Dis. 17, 1240-1247.
  29. National Institute For Communicable Diseases, NICD, Influenza NICD. 2017. Recommendation for the diagnosis, prevention, management and public health response Influenza Guidelines. Available on (accessed on Dec. 12, 2018).
  30. Peaper DR and Landry ML. 2014. Rapid diagnosis of influenza: State of the art. Clin. Lab. Med. 34, 365-385.
  31. Puvanalingam A, Rajendiran C, Sivasubramanian K, Ragunanthanan S, Suresh S, and Gopalakrishnan S. 2011. Case series study of the clinical profile of H1N1 swine flu influenza. J. Assoc. Physicians India 59, 14-18.
  32. Puzelli S, Valdarchi C, Ciotti M, Dorrucci M, Farchi F, Babakir-Mina M, Perno CF, Donatelli I, Rezza G, and the Italian ARI Study Group. 2009. Viral causes of influenza-like illness: Insight from a study during the winters 2004-2007. J. Med. Virol. 81, 2066-2071.
  33. Ruest A, Michaud S, Deslandes S, and Frost EH. 2003. Comparison of the directigen flu A+B test, the quickvue influenza test, and clinical case definition to viral culture and reverse transcription-PCR for rapid diagnosis of influenza virus infection. J. Clin. Microbiol. 41, 3487-3493.
  34. Sadeq R, Mohtady H, Sabah SB, Gohar M, Amer F, Atef S, and Rabia R. 2014. Virological diagnosis of influenza viruses in Zagazig University Hospitals. J. Hum. Virol. Retrovirol. 1, 00012.
  35. Samra T, Pawar M, and Yadav A. 2011. One-year experience with H1N1 infection: Clinical observation from a tertiary care hospital in Northern India Indian. Indian J. Community Med. 36, 241-243.
  36. Schweiger B and Biere B. 2009. TaqMan real-time PCR zur detektion von porcinen. Influenza A/H1N1-viren. [TaqMan real-time PCR for the detection of porcine influenza A/H1N1 viruses. [In German]. Berlin: Robert Koch-Institute. Available on,templateId=raw,property=publicationFile.pdf/Schweinegrippe_PCR.pdf (accessed on 3 May 2009).
  37. Seifi SJ and Ghannad MS. 2014. Research article comparison of real-time polymerase chain reaction and conventional cell culture for detection of influenza A in Tabriz, Iran. Avicenna J. Clin. Microbiol. Infect. 1, e21034.
  38. Singh M and Sharma S. 2013. An epidemiological study of recent outbreak of influenza A H1N1 (swine flu) in Western Rajasthan region of India. J. Med. Allied Sci. 3, 48-52.
  39. Torres JP, O'Ryan M, Herve B, Espinoza R, Acuna G, Manalich J, and Chomali, M. 2010. Impact of the novel A (H1N1) influenza during the 2009 autumn-winter season in large hospital settings in Santiago, Chile. Clin. Infect. Dis. 50, 860-868.
  40. Tulloch F, Correa R, Guerrero G, Samaniego R, Garcia M, Pascale JM, Martinez A, Mendoza Y, Victoria G, de Lee MN, et al. 2009. Profile of the first cases hospitalized due to A (H1N1) influenza 2009 in Panama City, Panama. May-June 2009. J. Infect. Dev. Ctries 3, 811-816.
  41. Vemula SV, Zhao J, Liu J, Wang X, Biswas S, and Hewlett I. 2016. Current approaches for diagnosis of influenza virus infections in humans. Viruses 8, 96.
  42. Vontas A, Plakokefalos E, Krikelis V, and Manouras A. 2015. Comparative analysis of real time RT-PCR and virus isolation for detection and subtyping of A(H1N1)pdm09 influenza virus. Internet J. Microbiol. 13, DOI: 10.5580/IJMB.34912.
  43. Waleed A. 2010. Master thesis. Study of avian flu confirmed cases admitted in Al-Abbassia Chest Hospital between 2006-2008. Alzhar University, Cairo, Egypt.
  44. Webby RJ and Webster RG. 2003. Are we ready for pandemic influenza? Science 302, 1519-1522.
  45. Whiley DM, Bialasiewicz S, Bletchly C, Fauxa CE, Harrower B, Goulde AR, Lambert SB, Nimmoc GR, Nissen MD, and Sloots TP. 2009. Detection of novel influenza A(H1N1) virus by real-time RT-PCR. J. Clin. Virol. 45, 203-204.
  46. WHO. 2009. Weekly epidemiological record No. 41, (Oct. 9, 2009). Available on (accessed on Oct. 16, 2011).
  47. WHO. 2010. H1N1 in post-pandemic period: Director-general's opening statement at virtual press conference (Aug. 10, 2010). Available on (accessed on July 1, 2013).
  48. WHO. 2016. Influenza (Seasonal). Fact sheet Nov. 2016. Available on http:/ (accessed on Dec. 19, 2016).
  49. Zambon M, Hays J, Webster A, Newman R, and Keene O. 2001. Diagnosis of influenza in the community: relationship of clinical diagnosis to confirmed virological, serologic, or molecular detection of influenza. Arch. Intern. Med. 161, 2116-2122.
  50. Zitterkopf NL, Leekha S, Espy MJ, Wood CM, Sampathkumar P, and Smith TF. 2006. Relevance of influenza A virus detection by PCR, shell vial assay, and tube cell culture to rapid reporting procedures. J. Clin. Microbiol. 44, 3366-3367.
  51. Laguna-Torres VA, Gomez J, Ocana V, Aguilar P, Saldarriaga T, Chavez E, Perez J, Zamalloa H, Forshey B, Paz I, et al. 2009. Influenza-like illness sentinel surveillance in Peru. PLoS One 4, e6118.