| Abstract|| |
Context: Establishment and kinetics of pandemic H1N1 2009 virus in Gujarat. Aims: To assess epidemiological and clinical profile of pandemic H1N1 cases and to correlate findings with signature features of pandemic agent. Settings and Design: Study was performed at a tertiary care hospital in Ahmedabad, Gujarat. Patients attending various hospitals across Gujarat for influenza-like illness were tested. Materials and Methods: Nasopharyngeal or nasal swabs collected in viral transport medium were subjected to real-time reverse transcriptase PCR using CDC-validated and WHO-approved protocols. Results: A total of 5750 samples were tested: 2620 tested positive for influenza A. 1710 were positive for pandemic H1N1 2009 virus: 910 males and 800 females with maximum positivity in the age group 18 to < 36 yrs (13.6% of total tested and 45.7% of pandemic H1N1 positive) with cough being the major clinical feature. Three distinct peaks of pandemic H1N1 2009 were observed, outnumbering seasonal flu A virus by 1.9:1. In most districts, a positivity of approximately 10-30% was observed. However, some districts showed very high positivity of >30% while some showed little incidence. This new virus is at low activity since October 2010. Conclusions: Pandemic H1N1 2009 virus is presently circulating in almost all parts of Gujarat but has been unable to replace seasonal influenza A completely. The present study corroborates the signature features for pandemic H1N1 as there is greater affection of younger age groups, appearance of successive peaks, higher transmissibility than seasonal influenza A, and differing impacts in different geographic regions.
Keywords: Eepidemiology, novel H1N1, pandemic H1N1 2009, rRTPCR, seasonal influenza
|How to cite this article:|
Sood NK, Dharsandia MV, Patankar MC, Vegad MM. Kinetics of pandemic influenza virus 2009 virus in Gujarat, Western India: An investigation of signature features. Ann Trop Med Public Health 2012;5:321-6
|How to cite this URL:|
Sood NK, Dharsandia MV, Patankar MC, Vegad MM. Kinetics of pandemic influenza virus 2009 virus in Gujarat, Western India: An investigation of signature features. Ann Trop Med Public Health [serial online] 2012 [cited 2018 Aug 15];5:321-6. Available from: http://www.atmph.org/text.asp?2012/5/4/321/102039
| Introduction|| |
The world witnessed the emergence of a novel influenza virus (H1N1) in early 2009 from Mexico,  which was later declared as pandemic (phase 6) by World Health Organization (WHO), on June 11, 2009.  It was different from the already circulating triple re-assortent H1N1 virus ,,,, with the following genetic segments: HA, NA, M, NS, and NP segments from the North American swine genetic lineage, PB2 and PA from North American Avian influenza lineage, and PB1 from classical human influenza A viruses. ,,,
This novel pandemic H1N1 2009 virus had the same lineages as triple re-assortent virus, except the NA and M genes that show a close homology to Eurasian swine influenza virus. This new pandemic virus, thus, has 30.6% gene population from the North American swine flu virus, 17.5% from the Eurasian swine flu virus, 17.5% from classical human flu virus, and 34.4% from the North American avian flu virus. ,
This new strain had the characteristics to fit into the signature features of pandemic potential-(i) Shift in the virus subtype, (ii) Successive pandemic waves, (iii) Shift of the highest death rate to younger population, (iv) Higher transmissibility than that of seasonal influenza, and (v) Differences in impact in different geographic regions. ,
On August 10, 2010, WHO announced pandemic H1N1 2009 to be in post-pandemic period. However, based on the knowledge about past pandemics, the H1N1 (2009) virus is expected to continue to circulate as a seasonal virus for some years to come. 
The present study was performed to assess the burden of this newly established virus in the population attending various hospitals across Gujarat, a major state in Western India and to correlate the epidemiological and clinical data of positive cases with the signature features of the pandemic agent. It also intends to provide a concise update on the current situation of pandemic H1N1 2009 virus infection in Gujarat.
| Materials and Methods|| |
The study was performed at a tertiary care hospital in Ahmedabad during the period of August 2009 and December 2010. A total of 5750 patients of all age groups attending various hospitals in Gujarat in 2009 and Central Gujarat, North Gujarat, and Kutch region (western Gujarat) in 2010 were tested for pandemic H1N1 2009 strain.
Demographic details and clinical history were obtained and recorded in laboratory request forms. Nasal swabs, nasopharyngeal swabs, or throat swabs were collected from the patients and transferred in viral transport media to the laboratory. All samples were checked for cold chain maintenance and quality of samples.  Samples were tested by real time reverse transcriptase PCR (rRTPCR) using CDC validated and WHO approved primers and probes sequences and rRTPCR protocols. 
RNA extraction was performed using QIAgen Mini RNA extraction kit. Extracted RNA was subjected to one step RTPCR reaction using 4 target sequences i.e. Influenza A (InfA), swine A (SwA), swine H1 (SwH1), and ribonucleoprotein (RNP). Master Mix was prepared by using Ag-Path one step RTPCR kit by Applied Biosystems.
Interpretation of the test results was done as follows:
Amplification in RNP target sequence was used as an internal quality control. A test was considered valid only if amplification in RNP was obtained.
Samples are routinely stored at-70 0 C after testing for a period of one year.
| Results|| |
A total of 5750 samples were tested as part of this study. Of the total patients tested, 29.7% (n = 1710) were found to be positive for pandemic H1N1 2009 virus, while 16% (n = 910) were positive for seasonal influenza A virus. A total of 2631 patients (45.7%) were positive for influenza A virus, out of which, 64.9% were positive for pandemic H1N1 2009 and 34.5% were positive for seasonal influenza A virus.
Of all the patients tested, 2410 were females and 3340 were males. From the total positives for pandemic H1N1 2009, 53% (n = 910) were males and 47% (n = 800) females. On the other hand, 59% (n = 545) males and 41% (n = 376) females were positive out of all the seasonal influenza A positive cases [Figure 1]. Upon analysis of the effect of flu on gender distribution, it was observed that pandemic H1N1 2009 is distributed almost equally amongst both genders, while seasonal flu affected males and females in a ratio of 1.5:1.
|Figure 1: Distribution of males and females showing the positivity for seasonal influenza A and pandemic influenza virus 2009|
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In our study, a majority of patients tested for flu cases were of 18 to <36 yrs age group (40.7%), followed by the age group 36 to 55 yrs (25.6%). It was observed that maximum positivity for pandemic H1N1 2009 virus belonged to the age group of 18 to <36 yrs (13.6%), followed by 36-55 yrs (8.7%). Out of the total positive for pandemic H1N1 2009, positivity was highest (45.7%) in the younger age group of 18 to <36 yrs, followed by the adult age group of 36-55 yrs (29.2%) [Figure 2].
|Figure 2: Age-wise distribution of laboratory-confirmed cases of pandemic influenza virus 2009 and seasonal influenza A virus between August 2009 to December 2010|
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A ratio of 2:1 for pandemic H1N1 2009 virus and seasonal influenza A virus was observed for the almost young and adult groups. While in the younger age group of <5 years and older age group of >55 years, a ratio of 1.3:1 and 1:1 was observed, respectively. The median age for seasonal flu observed was found to be 36 years while for pandemic H1N1 2009 virus, it was 34 years [Figure 2].
A month-wise analysis of our test record shows three distinct peaks of pandemic H1N1 2009 cases during August to October 2009, followed by December 2009 to January 2010 and August to September 2010 [Figure 3]. However, virus circulation is at a very low activity since October 2010 despite a good winter season during November and December in Gujarat.
|Figure 3: Month-wise distribution of laboratory-confirmed cases of pandemic influenza virus 2009 and seasonal influenza A virus between August 2009 to December 2010|
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This study as depicted in [Figure 4] also indicates the establishment of the pandemic H1N1 2009 virus along with seasonal influenza A viruses. It also indicates that the pandemic H1N1 2009 virus has outnumbered the seasonal influenza A virus by a ratio of 1.9:1 with ratios reaching as high as 4:1 in the month of December 2009. Conversely, in some months (March, April, and July) of year 2010, the seasonal flu activity is more than pandemic H1N1 2009. This study also proves the co-circulation of both pandemic H1N1 2009 virus and seasonal influenza A viruses.
|Figure 4: Correlation of pandemic influenza virus 2009 and seasonal influenza A cases over time|
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Out of 1710 laboratory-confirmed cases of pandemic H1N1 2009, clinical history was not available in only 4 cases. The major clinical feature in pandemic H1N1 2009 influenza cases was cough followed by fever, breathlessness, and sore throat. A very small number of cases presented with nasal catarrh [Table 1].
|Table 1: Clinical presentation of laboratory-confirmed pandemic influenza virus 2009 cases|
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Initially with the emergence of pandemic H1N1 2009 in Gujarat, we had tested samples from all the districts of Gujarat. We had received samples from Rajkot, Amreli, Jamnagar, Junagadh, and Porbandar till January 2010 and from Surat, Navsari, and Valsad till October 2009. We had tested samples from all other districts during August 2009 through December 2010.
Median positivity of 25% was observed in the study in Gujarat. In a majority of districts, a positivity of approximately 10-30% was observed for pandemic H1N1 2009. However, a few districts showed a very high positivity of >30%. It is evident from the [Table 2] that pandemic H1N1 2009 has established itself in a majority of the districts of Gujarat and has outnumbered seasonal influenza A (1.5:1 to 4:1) in all the districts, except a few. No cases were diagnosed with pandemic H1N1 2009 for instance, from districts like Bharuch, Dahod, Dangs, Porbandar, and Narmada.
|Table 2: Distribution of laboratory-confirmed seasonal influenza A and pandemic influenza virus 2009 cases in various districts of Gujarat|
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| Discussion|| |
Sitting astride the tropic of cancer, Gujarat is a major state situated in western region of India with 26 districts (provinces). It is a warm state with average to normal rainfall and a very short mild winter. There are large expanses of dry topography and of salt marshes.
In our study, 29.7% cases were found to be positive for pandemic H1N1 2009, which correlates well with the reported prevalence of pandemic H1N1 in other parts of India as indicated in an update by the Ministry of Health and Family Welfare  and 36% by a study from Israel.  Studies from Australia have reported a very high percentage of positivity (91%),  while a study from Eastern India and Pune, India showed a low positivity of 9.56% and 17.8%, respectively. , This study also indicates 16% positivity for seasonal influenza A virus, which is along the lines of the studies by Akhilesh Mishra from Pune, India that has also indicated a positivity of 16.3%.  Out of all positives for influenza A, pandemic H1N1 2009 has emerged as the major influenza A at 64% incidence. This coincides well with the study from Eastern India, in which, the positivity for pandemic H1N1 2009 cases is 74.35%. 
It is evident from the present study that pandemic H1N1 2009 has higher transmissibility than seasonal influenza A. It is also established that pandemic influenza 2009 has outnumbered seasonal influenza A in a ratio of 1:7 to 4:1 in majority of the districts of Gujarat. In few districts like Rajkot, Jamnagar, Banaskantha, and Amreli, pandemic H1N1 2009 has outnumbered seasonal influenza A more than double. In most of the other districts, it is 1.5 times higher than seasonal influenza A. This conforms to one of the parameters of signature features of pandemic agent that is higher transmissibility. 
Although this study shows that pandemic flu has outnumbered seasonal influenza A, co-circulation of pandemic influenza H1N1 2009 and seasonal influenza A is also well-established. This observation coincides with the study from Eastern India.  Another very significant observation during the study was that despite the establishment of pandemic H1N1, some months (March, April, and July) in the year 2010 showed a higher activity of seasonal influenza A over pandemic H1N1. This observation is also along the lines of the study from Eastern India.  This, however, is in sharp contrast to the studies reported in the Western countries where pandemic H1N1 has almost completely replaced seasonal influenza A. 
In this study, young and adult age groups (18-55 yrs) appear to be much more affected (66.3%) by pandemic H1N1 2009 than the elderly. The same has been reported by other studies from various parts of India  and from places abroad such as California, New York, Canada, Singapore, Japan etc.  However, the study from Eastern India indicates the age group of >55 years to be most affected.  One of the signature features for a pandemic agent is that it affects younger age groups  and our study corroborates this fact.
We shall now consider the gender correlation. In our study, both males and females are equally affected by pandemic H1N1 2009. This is close on the lines of other studies by Kelly  and Mukherjee. 
A month-wise analysis revealed the establishment of pandemic H1N1 2009 since August 2009 with three successive peaks, two during the rainy season (August and September 2009 and 2010) and one in the winter (December and January 2009). This also fits into the signature features of successive peaks for a pandemic agent.  The study from Eastern India and other parts of India also indicate increased activity during the rainy season and in the winter. , In the latter months of 2010, there is a drop seen in the sample size and drop in the detection of pandemic H1N1 2009 virus. This could be due to pacification of population towards the initial panic-like fears of pandemic H1N1 and the practice of traditional medicine for flu-like illness.
Out of the 26 districts in Gujarat, a total of 21 districts showed the circulation of pandemic influenza virus. In 5 districts namely Dahod, Dangs, Bharuch, Porbandar, and Narmada, no laboratory-confirmed pandemic H1N1 2009 cases could be established. There could be two reasons for Dahod and Dangs to remain pandemic flu-free. The first is that both these districts are tribal areas and are isolated from the mainstream population and depend on traditional wisdom for flu-like illness. The second reason could be the low sample size from these areas. Porbandar, Narmada, and Bharuch also are free of pandemic flu and this could be due to receipt of samples only in the year 2009 and that too a low sample size. A majority of the districts showed a positivity rate between 25-40%. A few districts like Navsari, Panchmahal, Amreli, Junagadh, and Jamnagar showed a very high rate of 40-50%. It could be due to relatively low sample size from these districts as compared to other districts. Our findings differ markedly from the studies of the West where the positivity is very high (>90%). , The above reasoning shows that there is a difference in impact in effect across the different geographic areas, which also strongly fits into the other signature feature of a pandemic agent. 
Regarding symptoms presentation, a majority of the patients had cough, fever, sore throat, breathlessness, and nasal catarrh. This is on the lines of studies from other parts of India, from the US, Canada, Australia, and New Zealand. ,,,, However, vomiting or diarrhea was not the presenting feature as shown in other studies. ,,
Finally, data regarding mortality of patients tested positive for pandemic H1N1 2009 was not available from the various hospitals. Consequently, the signature feature pertaining to the rate of mortality could not be studied.
We conclude that our study corroborates the signature features for pandemic H1N1 2009 as the study establishes affection of younger age groups more, the appearance of successive peaks, higher transmissibility than seasonal influenza A, and different impacts in different geographic regions. As mentioned earlier, a feature related to higher mortality could not be investigated due to lack of mortality data. Pandemic H1N1 virus is presently circulating in almost all parts of Gujarat. However, despite the elapse of 21 months since the circulation of pandemic H1N1 flu started, it has not been able to replace seasonal influenza A completely as observed in Western countries. There is a shift of influenza A from seasonal influenza A to pandemic H1N1 2009, but there is also co-circulation of seasonal influenza A virus in Gujarat. Also, both genders are equally affected by the pandemic H1N1 influenza.
| Acknowledgement|| |
The authors deeply acknowledge the technical support and training to carry out pandemic H1N1 flu testing using CDC validated and WHO approved protocols provided by National Centre for Disease Control (NCDC), New Delhi.
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Nidhi K Sood
10, Sterling Appartments-3, Sterling city, Bopal, Ahmedabad-380058, Gujarat
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]