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Title:	Detection and molecular characterisation of influenza virus and respiratory syncytial virus in Universiti Kebangsaan Malaysia Medical Centre
Authors:	Wong Kon Ken (P47229)
Supervisor:	Md. Mostafizur Rahman, Prof. Dr.
Keywords:	Influenza virus Respiratory syncytial virus Public health
Issue Date:	30-Sep-2014
Description:	Jangkitan pernafasan merupakan masalah kesihatan awam utama yang berleluasa di seluruh dunia. Jangkitan ini mudah tersebar di kalangan komuniti serta menyebabkan kadar morbiliti dan mortaliti yang tinggi. Agen penyebab utama jangkitan ini adalah virus pernafasan dan ia boleh menjangkiti pesakit pada semua peringkat umur. Agen virus pernafasan yang paling ketara adalah virus influenza dan "Respiratory Syncytial Virus" (RSV). Kajian ini dijalankan untuk mengesan dan mencirikan secara molekular virus influenza dan RSV yang diambil daripada pesakit di Pusat Perubatan Universiti Kebangsaan Malaysia (PPUKM) dari Januari 2009 hingga Disember 2011. Sejumlah 2781 spesimen swab tekak dan aspirat cecair nasofarink telah dikumpul daripada pesakit yang disyaki jangkitan virus pernafasan dan dengan diagnosis influenza-like illness (ILI). Spesimen diuji dengan ujian kultur sel dan asai imunofluoresens (IFA) untuk mengesan jangkitan influenza and RSV. Sejumlah 505 spesimen digunakan untuk menguji keberkesanan "real-time reverse transcriptase PCR" (rRT-PCR) rekaan secara dalaman. Dengan teknik konvensional ujian kultur sel dan IFA, 33 (1.19%) spesimen adalah positif terhadap virus influenza A dan 42 (1.51%) adalah positif terhadap RSV. Tiada virus influenza B yang dikesan dalam kajian ini. Ujian rRT-PCR dapat mengesan 189 daripada 505 (37.43%) spesimen yang positif termasuk 65 positif terhadap virus influenza A dan 124 positif terhadap RSV. Walaubagaimanapun, hanya 12 daripada 505 (2.38%) kes positif yang dapat dikesan dengan teknik konvensional. Tambahan pula, rRT-PCR mampu mengesan 19 spesimen positif terhadap kedua-dua jangkitan virus influenza A dan RSV yang tidak dapat dikesan dengan kaedah konvensional. Sensitiviti untuk ujian rRT-PCR rekaan dalaman adalah 100% untuk kedua-dua virus influenza A dan RSV dan spesifisiti adalah 88% dan 77% untuk virus influenza A dan RSV, masing-masing. Identifikasi virus influenza A dengan penjujukan dan analisa filogenetik telah dijalankan dan kesemua 65 virus influenza A telah dikelaskan dalam tiga genotaip yang dinamakan sebagai H1N1 (n=40), H3N2 (n=22) dan H5N1 (n=3). Selain daripada itu, 124 RSV juga telah dikenalpasti dengan penjujukan dan analisa filogenetik dan dikelaskan ke dalam 3 genotaip iaitu GA5 (n=51), GA2 (n=43) dan GA7 (n=30). Kes positif untuk virus influenza A dan RSV telah dianalisa dengan faktor umur, jantina, kaum dan simptom klinikal pesakit. Tiada perbezaan yang signifikan dalam hubungan di antara jantina, kaum dan simptom klinikal pesakit untuk kedua-dua jangkitan virus influenza A dan RSV. Walaubagaimanapun, dapatan kajian menunjukkan bahawa kanak-kanak di bawah umur 3 tahun adalah lebih mudah dijangkiti RSV, manakala jangkitan virus influenza A berlaku pada semua peringkat umur pesakit. Ujian rRT-PCR rekaan dalaman ini dapat mengesan jangkitan virus pernafasan dalam masa yang lebih singkat dengan sensitiviti yang lebih tinggi. Ini dapat membantu dalam pengurusan pesakit melalui penjimatan kos dan pengurangan masa pengurusan. Dapatan kajian ini membuka peluang baharu untuk pengesanan virus pernafasan dan meningkatkan repositori kajian epidemiologi molekul jangkitan virus. Analisa filogenetik dalam kajian ini membekalkan maklumat yang baharu mengenai genom virus influenza A dan RSV di Malaysia. Ini dapat membantu dalam menentukan sumber jangkitan dan pemilihan strain untuk pembangunan vaksin yang sesuai.,Respiratory infections are major public health problem because of morbidity and mortality, worldwide occurrence, and ease of spread in the community. Major causal agents of the infections are respiratory viruses which play predominant role in spreading the infections to all age groups. The most common respiratory viral agents are influenza virus and respiratory syncytial virus (RSV). The present study was undertaken for the detection and molecular characterisation of influenza virus and RSV in Universiti Kebangsaan Malaysia Medical Centre (UKMMC) from January 2009 to December 2011. A total of 2781 throat swabs and nasopharyngeal aspirates specimens were obtained from patients suspected of respiratory viruses infections or presenting as influenza-like illness (ILI). The specimens were processed by cell culture and immunofluorescent assay (IFA) for detection of influenza virus and RSV. As result, 33 (1.19%) specimens were positive for influenza virus A and 42 (1.51%) were positive for RSV. Influenza B was not detected in this study. A total of 505 specimens were evaluated by in-house developed real-time reverse transcriptase-PCR (rRT-PCR). The rRT-PCR was able to identify 189 of 505 (37.43%) positive specimens including 65 and 124 specimens for influenza A virus and RSV respectively. However, only 12 of 505 (2.38%) positive cases were identified by conventional method. In addition, rRT-PCR could identify 19 specimens with coinfections of influenza A virus and RSV but none was detected by conventional method. Sensitivity of the in-house developed rRT-PCR was 100% for both influenza A virus and RSV and specificity was 88% and 77% for influenza A virus and RSV, respectively. Identified influenza A virus was sequenced and phylogenetic analysis was performed. It was observed that all the 65 positive influenza virus A strains were classified into three genotypes namely H1N1 (n=40), H3N2 (n=22) and H5N1 (n=3). Similarly identified 124 RSV isolates were sequenced and phylogenetic analysis carried out and placed in genotype GA5 (n=51), GA2 (n=43) and GA7 (n=30). Positive cases for influenza virus A and RSV were analysed on the basis of patients' age, gender, ethnicity and clinical symptoms. There was no significant difference among gender, ethnicity and clinical symptoms with influenza A virus and RSV infections. However, it was observed that children below 3 years old were more prone to RSV infection and influenza A virus infection affected all age groups. In-house developed rRT-PCR diagnosed respiratory viruses in shorter time with a high level of sensitivity. These advantages help in managing patients by saving cost and reduce hospitalisation stay. The results provide new opportunity for diagnosis of respiratory viruses and their molecular epidemiological study. Phylogenetic analysis of the present study provides new information on the genomic nature of influenza A virus and RSV in Malaysia. This would help in determining the origins of the viruses and selection of an appropriate vaccine strain.,PhD
Pages:	158
Call Number:	tesis WA20.50W837d 2014 9HUKM
Publisher:	UKM, Kuala Lumpur
Appears in Collections:	Faculty of Medicine / Fakulti Perubatan

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