EPIDEMIOLOGY OF INFLUENZA VIRUS AND METAGENOMIC ANALYSIS OF VIRUSES IN HUMAN RESPIRATORY TRACT SPECIMENS

Abstract

Acute respiratory infections are the cause of immense clinical problem worldwide, affect both adults and children every seasonal cycle. These respiratory tract symptoms involved both upper and lower respiratory tract. Many studies found that viruses are generally associated with the infection, and Influenza viruses are the one that find to be one of the most common viruses that cause respiratory illness. Overall, this thesis aimed to detect antibodies against influenza A(H1N1)pdm09, between July 2009 and November 2010, after its emergence in 2009. Seroprevalence among general population and medical personnel was established in three cross-sectional studies at the end of each wave of the pandemic in Khon Kaen province, Thailand, by performing HI assay. Sample of 255 medical personnel and 307 members of the general population were collected after the first peak of outbreak during second week of December 2009. The results of the HI test demonstrated that HI titers among Healthcare personnel (48%) were higher than general population (36%) due to the higher occupational risk, while only two of the 100 stored sera from 2008 contained antibodies (HI titers ≥40) against pandemic influenza. Furthermore, serum specimens from medical personnel were collected after the end of each wave, for another two consecutive waves. After the second wave in June 2010, 397 serum samples were collected, and 366 serum samples were collected after the third wave of the pandemic in December 2010. The positive HI titers after the second and third waves were 22.4%, and 25.7%, respectively. Also, the medical personnel (n=146) who had received the influenza A(H1N1)pdm09 monovalent vaccine between February and March 2010 showed significant higher GMT when compare to unvaccinated groups. This showed that annual influenza vaccination can induce an immune response against the virus and is the most effective way to prevent against influenza infection. These findings show that seasonal influenza strain in Chumphae and the predominant influenza strain from each wave was influenza A(H1N1)pdm09. These results also represent the severity of the attack rate in each wave. To investigate seasonal pattern, distribution of influenza cases in patients (n=17,416) diagnosed with influenza-like-illness (ILI) during June 2009- July 2014. All samples were subjected to real-time RT-PCR, as laboratory confirmation, for detection of influenza viruses. Results during the period showed that influenza virus type A predominated over type B. The seasonal pattern of influenza activity in Thailand tended to peak during rainy season, although no significant correlation with environmental factor such as average temperature, average relative humidity, and rainfall. We found that influenza activity in Thailand usually presents as an annual cycle, except during the 2009 pandemic and its aftermath, when the circulation of influenza A(H1N1)pdm09 caused the normal pattern to break. Most likely this was the result of the Thai population’s lack of immunity toward the new pandemic strain. At the emergence of influenza A(H1N1)pdm09 in 2009, the mean age of infected patients was 19 years, which correlates with other studies that have reported that influenza A(H1N1)pdm09, in contrast to seasonal influenza, specifically targets younger age groups. This observation of influenza’s annual incidence pattern provides a better understanding of its occurrence, suggesting that vaccination campaigns should be started before the influenza season begins in order to reduce transmission. Recently, metagenomic sequencing is one of the method that being explore as a tool in clinical diagnosis because its potential for identifying known or new viruses without specific primers. These advantages include speed and the ability to generate large volumes of sequencing data, but sensitivity of deep sequencing is still in question when compare to specificity and sensitivity of PCR primer and probe. The last part involves comparing efficiency of next generation sequencing approach and real time diagnostic RT-PCR of respiratory viruses in a cohort of Children (n=81) with respiratory disease in Thailand. Results revealed that next generation sequencing approach was at least as sensitive as diagnostic real time RT-PCR for rhinovirus and human metapneumovirus and maybe less sensitive than real time RT-PCRs for entero- and bocavirus detection. Moreover, an advantage of using a next-generation sequencing approach to detect viruses in clinical specimens is that it can also be used to obtain information regarding the virus species and/or type of virus. Considering its declining cost and development for increasing sequencing depth, this approach, combined with bioinformatics analysis, can be an alternative method for virus identification in clinical and public health setting in future.