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UNIVERSITI PUTRA MALAYSIA

DETECTION OF VIRUSES IN NASOPHARYNGEAL ASPIRATES OF CHILDREN ADMITTED WITH LOWER RESPIRATORY TRACT

INFECTIONS AT HOSPITAL SERDANG, SELANGOR, MALAYSIA

MOHAMMADREZA ETEMADI

FPSK(m) 2011 44

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DETECTION OF VIRUSES IN NASOPHARYNGEAL ASPIRATES OF

CHILDREN ADMITTED WITH LOWER RESPIRATORY TRACT INFECTIONS AT HOSPITAL SERDANG, SELANGOR, MALAYSIA

By

MOHAMMADREZA ETEMADI

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirements for the Degree of Master of Science

July 2011

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Abstract of thesis presented to the Senate of University Putra Malaysia in fulfillment of the requirement for the degree of Master of Science.

DETECTION OF VIRUSES IN NASOPHARYNGEAL ASPIRATES OF CHILDREN ADMITTED WITH LOWER RESPIRATORY TRACT

INFECTIONS AT HOSPITAL SERDANG, SELANGOR, MALAYSIA

By

MOHAMMADREZA ETEMADI

July 2011

Chair: Professor Norlijah Othman, MBBS, MRCP

Faculty: Medicine and Health Sciences

Acute lower respiratory tract infections (ALRTIs) continue to be the most important

cause of infant and young children mortality worldwide, most of them occuring in

developing countries including Southeast Asia and Africa. The role of viruses as major

causative agents of ALRTIs in children is increasingly becoming more evident by using

sensitive molecular detection methods. The aim of the study was to assess the

epidemiology of respiratory viral infections among children less than five years of ages

hospitalized with ALRTIs to the Hospital Serdang using conventional and molecular

detection methods and to correlate these findings with demographic and clinical

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features of the patients in order to determine further common viral atiologic agents. A

cross-sectional study was conducted from June until December 2009 among children

hospitalized with ALRTI. Nasopharyngeal aspirates were collected from 165 patients

based on pre-determined inclusion and exclusion criteria. Direct immunofluorescence

assay (DFA) was performed to screen the samples for the presence of respiratory

syncytial virus (RSV.), human metapneumovirus (HMPV), parainfluenzavirus 1-3 (PIV

1-3), influenza virus type A and B (IFV A & B), and human adenoviruses (HAdV).

Negative samples tested by DFA were followed by shell vial culture (SVC), as a

supplementary test to enhance the detection of these eight viruses. Viral genomes

(RNA/DNA) were extracted and subsequently reverse transcription was done on RNA

extracts in order to perform diagnosis using molecular methods. Hemi-nested multiplex

RT-PCR was applied for detection of RSV, HMPV, IFV-A and B, PIV 1,2,3, and 4,

human rhinoviruses (HRV), human enteroviruses (HEV) and human coronaviruses

(HCoV) 229E and OC43. In addition, the presence of human bocavirus (HBoV) and

human adenoviruses (HAdV) was investigated separately by nested PCR method. The

positive samples using either method were subjected to isolation by cell culture. Vero,

HEp-2, HeLa and MRC-5 cell lines were used for isolation of RSV, HAdV and HRV.

Selected samples of patients diagnosed with RSV, HRV/HEV, and HAdV were

subjected to the sequencing and molecular typing. Mycoplasma serology and bacterial

culture were performed on blood samples. At the end of the hospitalization, the

children’s hospital chart was reviewed to collect demographic, clinical, laboratory and

radiological investigation data using standardized protocol. The association of

demographic, clinical features, hematologic factors, radiographic findings, hospital

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course and severity of disease with infections due to different viruses was studied.

Aetiologic agents including virus and/or bacteria were detected in 158 (95.8%) of the

patients. Single virus was detected in 114 (67.9%) patients; 46 (27.9%) were co-

infected with different viruses including double-virus infections in 37 (22.4%) and

triple-virus infections in 9 (5.5%) cases. Approximately 70% of samples were found

positive using conventional methods as compared with 96% using molecular methods.

A wide range of respiratory viruses was detected in the study. RSV (50.3%), with

predominance of group B (GB3 genotype), played a major role among hospitalized

children. The results of this survey also showed significant burden of HRV infections

(32.7%). Phylogenetic study of the VP4/VP2 region confirmed the broad genetic

diversity of circulating HRV. HRV-A strains represented the majority of the detections,

22/36 (61%). Recently discovered HRV-C group was substantially implicated as

etiological agent among studied patients, 14/36 (39%). Other etiological agents

including HAdV (serotypes 1, 2, 3, and 6), HMPV, IFV-A, PIV 1-3, HBoV, HCoV-

OC43 and HEV (B, C, and D species) were detected in 14.5, 9.6, 9.1, 4.8, 3.6, 2.4 and

1.8 percent of the samples, respectively. Ninety percent of the cases occurred in

children less than 2 years. The majority of RSV infections occurred in children less than

six months as compared with other virus groups. However, HRV was mainly detected

in the second half of the infancy. The most common clinical presentations of ALRTI,

among hospitalized children infected with the studied viruses were cough (96%),

fever (85%), rhinorrhea (83%), difficulty in breathing (84%), tachypnea, chest wall

crepitations (93%) and recession (80%). Children were admitted after a mean duration

of three days. However, it was significantly earlier for HRV (1.9 days) than RSV (4.0

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days) infections. Fever was a prominent feature of RSV and IFV infections. In this

study, HRV-C infected children were more likely to have wheezing/rhonchi as

compared with HRV-A. The results of the investigation also showed that antibiotics

were administerd in majority of the patients 136/165, (82%). HRV-infected patients

were less likely to receive antibiotics compared with RSV patients. The results of the

study suggested that respiratory viral agents significantly contributed to the aetiology of

ALRTIs among hospitalized children. Our results demonstrated the potential usefulness

of molecular detection methods compared with conventional methods for the diagnosis

of ARTIs among hospitalized children. In this study, newly discovered viruses

including HMPV, HBoV and HRV-C were reported for the first time in Malaysia. Our

study also highlighted that the epidemiology and clinical features were specified to

certain viral agents studied.

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Abstrak tesis yang telah dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan ijazah Master Sains

PENGESANAN VIRUS-VIRUS DALAM ASPIRAT NASOFARIKS KANAK-KANAK DENGAN JANGKITAN PERNAFASAN BAHAGIAN BAWAH YANG

DIMASUKKAN KE DALAM HOSPITAL SERDANG, SELANGOR, MALAYSIA

Oleh

MOHAMMADREZA ETEMADI

Julai 2011

Pengurusi: Profesor Norlijah Othman, MBBS, MRCP

Fakulti: Perubatan dan Sains Kesihatan

Jangkitan pernafasan bahagian bawah akut (ARLTIs) merupakan penyebab utama

kematian di kalangan bayi dan kanak-kanak di seluruh dunia, kebanyakannya berlaku di

negara membangun termasuk Asia Tenggara dan Afrika. Peranan virus sebagai agen

penyebab ARLTIs di kalangan kanak-kanak semakin jelas dengan menggunakan

kaedah pengesanan molekular yang sensitif. Tujuan kajian adalah untuk menilai

epidemiologi jangkitan virus sistem pernafasan di kalangan kanak-kanak kurang

daripada lima tahun yang dimasukkan ke Hospital Serdang disebabkan ARLTIs

menggunakan kaedah pengesanan konvensional dan molekular serta

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menghubungkaitkan penemuan ini dengan ciri-ciri demografik dan klinikal pesakit

untuk menentukan kebiasaan etiologik agen virus dengan lebih mendalam. Kajian

keratan rentas dilakukan dari Jun sehingga Disember 2009 di kalangan kanak-kanak

hospital disebabkan ARLTIs. Aspirat nasofarinks dikumpulkan daripada 165 pesakit

berdasarkan kriteria pra penentuan inklusi dan eksklusi. Ujian imunofloresense secara

langsung (DFA) dilakukan pada sampel untuk mengesan kehadiran virus respirasi

sinsitium (RSV.), metapneumovirus manusia (HMPV), virus parainfluenzavirus 1-3

(PIV 1-3), virus influenza jenis A and B (IFV A & B), dan adenovirus manusia

(HAdV). Sampel negatif diuji oleh DFA diikuti kultur shell vial (SVC) sebagai ujian

tambahan untuk meningkatkan pengesanan kelapan-lapan virus ini. Genom virus

(RNA/DNA) diekstrak dan diikuti dengan transkripsi berbalik ke atas ekstrak RNA

untuk menjalankan diagnosis menggunakan cara molekular. Hemi-nested multiplex RT-

PCR telah digunakan untuk mengesan RSV, HMPV, IFV-A dan B, PIV 1,2,3, dan 4,

rhinovirus manusia (HRV), enterovirus manusia (HEV), dan coronavirus manusia

(HCoV) 229E dan OC43. Tambahan pula, kehadiran bocavirus manusia (HBoV) dan

adenovirus manusia (HAdV) telah disiasat berasingan menggunakan cara nested PCR.

Sampel positif menggunakan kaedah-kaedah ini dilanjutkan kepada isolasi oleh sel

kultur. Sel Vero, HEp-2, HeLa dan MRC-5 telah digunakan untuk isolasi RSV, HAdV

and HRV. Sampel pesakit terpilih yang mengandungi RSV, HRV/HEV, dan HAdV

telah dirujuk kepada jujukan dan molekular typing. Serologi mycoplasma dan kultur

bakteria telah dijalankan ke atas sampel darah. Di akhir penghospitalan, carta hospital

kanak-kanak diselidik untuk mengumpul data demografi, klinikal dan makmal

menggunakan kaedah yang dipiawaikan. Hubungkait antara demografi, ciri-ciri klinikal,

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faktor hematologi, penemuan radiografi, laporan hospital, dan keterukkan jangkitan

disebabkan oleh kajian pelbagai virus telah dinilai menggunakan kaedah statistik. Agen

aetiologi termasuk virus dan/atau bakteria dikesan pada 158 (95.8%) pesakit. Virus

tunggal telah dikesan pada 114 (67.9%) pesakit; 46 (27.9%) pesakit telah dijangkiti oleh

beberapa virus yang berlainan termasuk jangkitan dua-virus pada 37 (22.4%) pesakit

dan jangkitan tiga-virus di dalam 9 (5.5%) kes. Dianggarkan 70% sampel telah dikesan

positif menggunakan kaedah konvensional berbanding 96% menggunakan kaedah

molekular. Virus sistem pernafasan yang meluas telah dikesan di dalam kajian ini. RSV

(50.35%) dengan predominan kumpulan B (genotip GB3), memainkan peranan utama

di kalangan kanak-kanak yang dimasukkan ke dalam hospital. Keputusan tinjauan ini

juga menunjukkan bebanan yang signifikan oleh jangkitan HRV (32.7%). Kajian

filogenetik kawasan VP4/VP2 telah mengesahkan diversiti genetik edaran HRV yang

luas. Strain HRV-A mewakili majoriti pengesanan 22/36 (61%). Baru-baru ini

penemuan kumpulan HRV-C telah dibabitkan sebagai agen etiologi di kalangan pesakit-

pesakit yang di kaji 14/36 (39%). Agen etiologi lain termasuk HAdV (serotip 1,2,3,

dan 6), HMPV, IFV-A, PIV 1-3, HBoV, HCoV-OC43 dan HEV (spesis B, C, dan D)

telah dikesan masing-masing di dalam 14.5, 9.6, 9.1, 4.8, 3.6, 2.4 and 1.8 peratus

sampel. Sembilan puluh peratus kes berlaku pada kanak-kanak kurang daripada dua

tahun. Majoriti jangkitan RSV terdiri daripada kanak-kanak kurang dari enam bulan

berbanding kumpulan virus lain. Walaubagaimanapun, HRV terutamanya dikesan pada

separuh masa kedua kehamilan. Kebiasaannya, laporan klinikal ALRTIs di kalangan

kanak-kanak hospital yang dijangkiti dengan virus yang dikaji merupakan batuk (96%),

demam (85%), rhinorrhea (83%), kesukaran bernafas (84%), tachypnea, krepitasi

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dinding dada (93%), dan retraksi (80%). Kanak-kanak telah dimasukkan ke hospital

pada purata masa 3 hari. Walaubagaimanapun, ia signifikan lebih awal bagi jangkitan

HRV (1.9 hari) berbanding RSV (4.0 hari). Demam merupakan ciri-ciri utama oleh

jangkitan RSV dan IFV. Dalam kajian ini, kanak-kanak yang dijangkiti oleh HRV-C

lebih cenderung menghadapi wheezing/ronki berbanding HRV-A. Keputusan kajian

menunjukkan bahawa antibiotik yang dimasukkan ke dalam majoriti pesakit-pesakit

136/165, (82%). Pesakit-pesakit yang dijangkiti HRV kurang memerlukan antibiotic

berbanding dengan pesakit RSV. Ini merupakan kajian pertama ke atas epidemiologi

dan etiologi oleh panel virus sistem pernafasan di Malaysia yang lengkap. Keputusan

menunjukkan agen virus sistem pernafasan menyumbang secara signifikan atiologi

ALRTIs di kalangan kanak-kanak yang dimasukkan ke hospital. Keputusan kami

menunjukkan potensi penggunaan kaedah pengesanan molekular berbanding kaedah

konvensional untuk mendiagnos ALRTIs di kalangan kanak-kanak yang dimasukkan ke

hospital. Dalam kajian ini, penemuan baru virus-virus termasuk HMPV, HBoV dan

HRV-C telah dilaporkan pertama kali di Malaysia.

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ACKNOWLEDGEMENTS

I am heartily offered my regards and blessings to all my supervisory members, whose

encouragement, guidance and support from the initial to the final level enabled me to

develop an understanding of the subject.

I am grateful to the members of the Department of Medical Microbiology and

Parasitology, Department of Pediatrics, Department of Community Health and

Department of Imaging, Faculty of Medicine and Health Sciences, University Putra

Malaysia for their companionship. I would like to express special thanks to all my

labmates and members of the Clinical and Molecular Virology Laboratory and Medical

Bacteriology Laboratory for their kind support during the study.

The author would like to acknowledge the Departmnet of Pediatrics, Hospital Serdang,

the head of the Department, all the doctors and nurses for their close co-operation to

recruit the patients and provide the nasopharyngeal aspirates for the study.

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I certify that an Examination Committee has met on 4 July 2011 to conduct the final examination of Mohammadreza Etemadi on his thesis entitled ″″″″Detection of Viruses in Nasopharyngeal Aspirates of Children Admitted with Lower Respiratory Tract Infections to Hospital Serdang, Malaysia″ in accordance with Universities and University College Act 1971 and the Constitution of the Universiti Putra Malaysia [P.U.(A) 106] 15 March 1998. The committee recommends that the student be awarded the Master of Science.

Members of the Examination Committee were as follows:

Chong Pei Pei, Ph.D Associate Professor Faculty of Medicine and Health Sciences University Putra Malaysia (Chairman) Farida Fatema @ Faida Jamal, MBBS, MSc. FRCPath Professor Faculty of Medicine and Health Sciences University Putra Malaysia (Internal Examiner) Zaiton Ahmad, MD, MMed Lecturer Faculty of Medicine and Health Sciences University Putra Malaysia (Internal Examiner) Che Illina Che Ishak, MD, Ph.D Professor Faculty of Medicine University Kebangsaan Malaysia (External Examiner)

_____________________________

NORITAH OMAR, PhD Associate Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia

Date: 23 August 2011

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This thesis was submitted to the Senate of University Putra Malaysia and has been accepted as fulfillment of the requirement for the degree of Master of Science. The members of the Superivisory Committee were as follows: Norlijah Othman, MBBS, MRCP Professor Faculty of Medicine and Health Sciencesa University Putra Malaysia (Chairman) Zamberi Bin Sekawi, MD, MPath Associate Professor Faculty of Medicine and Health Sciences University Putra Malaysia (Member) Lye Munn Sann, MBBS, DrPH Professor Dato’ Faculty of Medicine and Health Sciences University Putra Malaysia (Member) Mohd Zaini Abd Hamid, MD, MMed Lecturer Faculty of Medicine and Health Sciences University Putra Malaysia (Member)

HASANAH MOHD GHAZALI, PhD Professor and Dean

School of Graduate Studies Universiti Putra Malaysia

Date: 15 September 2011

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DECLARATION

I declare that the thesis is my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously, and is not concurrently, submitted for any other degree at University Putra Malaysia or at any other institution.

MOHAMMADREZA ETEMADI

Date: 4 July 2011

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TABLE OF CONTENTS Page ABSTRACT iii ABSTRAK vii ACKNOWLEDGMENTS xi APPROVAL xii DECLARATION xiv LIST OF TABLES xix LIST OF FIGURES xxi LIST OF ABBREVIATIONS xxiii CHAPTER

1� INTRODUCTION ................................................................................................................ 1�

2� LITERATURE REVIEW .................................................................................................... 9�2.1� Respiratory Syncytial Virus (RSV.) ............................................................................. 10�

2.1.1� Virus Properties ....................................................................................................10�2.1.2� Epidemiology ........................................................................................................11�2.1.3� Age Distribution and Seasonality .........................................................................12�2.1.4� Clinical Features ...................................................................................................13�2.1.5� Complications .......................................................................................................14�2.1.6� Risk Factors ..........................................................................................................14�2.1.7� Laboratory Diagnosis ............................................................................................15�

2.2� Human Metapneumovirus (HMPV) ............................................................................. 17�2.2.1� Virus Organization ................................................................................................17�2.2.2� Epidemiology ........................................................................................................18�2.2.3� Age Distribution and Seasonality .........................................................................18�2.2.4� Clinical Features ...................................................................................................20�2.2.5� Complications .......................................................................................................20�2.2.6� Risk Factors ..........................................................................................................21�2.2.7� Laboratory Diagnosis ............................................................................................22�

2.3� Parainfluenza Viruses (PIVs) ....................................................................................... 23�2.3.1� Virus Organization ................................................................................................23�2.3.2� Epidemiology ........................................................................................................23�2.3.3� Clinical Features ...................................................................................................25�2.3.4� Laboratory Diagnosis ............................................................................................26�

2.4� Human Rhinoviruses (HRV) ........................................................................................ 27�2.4.1� Virus Organization ................................................................................................27�2.4.2� Antigenic and Genetic Diversity ...........................................................................27�2.4.3� Epidemiology ........................................................................................................28�2.4.4� Age Distribution and Seasonality .........................................................................29�

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2.4.5� Clinical Features ...................................................................................................29�2.4.6� Complications .......................................................................................................30�2.4.7� Risk Factors ..........................................................................................................31�2.4.8� Laboratory Diagnosis ............................................................................................31�

2.5� Influenza viruses (IFV) ................................................................................................ 32�2.5.1� Virus Organization ................................................................................................32�2.5.2� Antigenicity...........................................................................................................33�2.5.3� Epidemiology ........................................................................................................33�2.5.4� Age Distribution and Seasonality .........................................................................34�2.5.5� Clinical Features ...................................................................................................35�2.5.6� Complications .......................................................................................................36�2.5.7� Risk Factors ..........................................................................................................36�2.5.8� Laboratory Diagnosis ............................................................................................37�

2.6� Human Adenovirus (HAdV) ........................................................................................ 38�2.6.1� Virus Organization ................................................................................................38�2.6.2� Virus Characterization ..........................................................................................38�2.6.3� Epidemiology ........................................................................................................39�2.6.4� Clinical Features ...................................................................................................40�2.6.5� Laboratory Diagnosis ............................................................................................40�

2.7� Human Bocavirus (HBoV) ........................................................................................... 41�2.7.1� Virus structure .......................................................................................................41�2.7.2� Epidemiology ........................................................................................................42�2.7.3� Clinical Features ...................................................................................................42�2.7.4� Laboratory Diagnosis ............................................................................................44

3� MATERIALS AND METHODSError! Bookmark not defined........................................48�3.1� Study Design and Location .......................................................................................... 48�3.2� Participant Recruitment ................................................................................................ 48�3.3� Inclusion Criteria .......................................................................................................... 49�3.4� Exclusion Criteria ......................................................................................................... 50�3.5� Definition of Final Diagnosis ....................................................................................... 50�3.6� Data Collection ............................................................................................................. 51�

3.6.1� Demographic and Clinical Data ............................................................................51�3.6.2� Laboratory Data ....................................................................................................52�

3.7� Warnings and Precautions ............................................................................................ 53�3.8� Collection and Transportation of Nasopharyngeal Aspirates (NPA.) .......................... 54�3.9� Processing of NPA ....................................................................................................... 54�3.10� Immunological Assays ................................................................................................. 55�

3.10.1� Direct Immunofluorescence Assay (DFA) ...........................................................55�3.10.2� Shell Vial Culture (SVC) ......................................................................................58�

3.11� Conventional Cell Culture ............................................................................................ 60�3.11.1� Cell Lines ..............................................................................................................60�3.11.2� Virus Culture .........................................................................................................61�

3.12� Molecular Diagnostic Methods .................................................................................... 62�3.12.1� Validation of PCR Assay ......................................................................................63�3.12.1� Genome Extraction from NPA..............................................................................66�

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3.12.2� cDNA Synthesis ....................................................................................................67�3.12.3� Multiplex RT-PCR ................................................................................................68�3.12.4� Hemi-nested Multiplex PCR .................................................................................69�3.12.5� Interpretation of Multiplex RT-PCR .....................................................................70�3.12.6� Detection of Human Bocavirus .............................................................................70�3.12.7� Detection of Human Adenovirus ..........................................................................71�3.12.1� Agarose Gel Electrophoresis.................................................................................72�

3.13� Molecular Typing ......................................................................................................... 72�3.13.1� PCR Gel Purification ............................................................................................74�3.13.2� Sequencing ............................................................................................................75�3.13.3� Analysis of Sequences ..........................................................................................75�

3.14� Statistical Analysis ....................................................................................................... 76�3.15� Ethical Clearances ........................................................................................................ 78�

4� RESULTS………………………………………………………………………………...79 �4.1� Study Population .......................................................................................................... 79�4.2� Detection of Viruses using Conventional Methods...................................................... 83�

4.2.1� Direct Immunofluorescence Assay (DFA) ...........................................................83�4.2.1� Shell Vial Culture (SVC) ....................................................................................886�4.2.2� Conventional Cell culture for RSV .......................................................................88�4.2.3� Conventional Cell Culture for HAdV ...................................................................90�4.2.4� Conventional Cell Culture for HRV .....................................................................92�

4.3� Detection of Viruses using Molecular Methods ........................................................... 95�4.3.1� Optimization of Multiplex PCR Assay .................................................................95�4.3.2� Detection of Viruses by Multiplex PCR assay .....................................................98�4.3.3� Detection of Human Rhinoviruses ......................................................................100�4.3.4� RSV Sub grouping into A and B.........................................................................102�4.3.5� Detection of Human Bocavirus ...........................................................................104�4.3.6� Detection of Human Adenovirus ........................................................................106�

4.4� Conventional Methods versus Molecular Methods .................................................... 109�4.4.1� Phylogenetic Analysis of RSV Isolates ..............................................................112�4.4.1� Phylogenetic Analysis of HRV Isolates ..............................................................119�

4.5� Occurrence of Respiratory infections by Viral Aetiology ......................................... 124�4.5.1� Viral Infections ...................................................................................................124�4.5.2� Non-viral Infections ............................................................................................125�

4.6� Multiple Viral Infections ............................................................................................ 129�4.7� Monthly Distribution of Viral Infections ................................................................... 131�4.8� Demographic Features................................................................................................ 133�

4.8.1� Demographic Features of RSV, HRV, IFV and HMPV Infections ....................133�4.8.2� Demographic Characteristics of HAdV, HBoV and HBoV Infections ..............137�4.8.3� Demographic Characteristics of HRV-A and HRV-C Infections .......................138�

4.9� Clinical Features ......................................................................................................... 141�4.9.1� Clinical Features of RSV, HRV, IFV-A and HMPV ..........................................141�4.9.1� Clinical Features of HAdV, PIVs and HBoV .....................................................146�4.9.1� Clinical Features of Single vs. Multiple Viruses ................................................148�4.9.1� Clinical Features of HRV-A vs. HRV-C Viruses ...............................................150�

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4.10� Laboratory and Chest X-Ray Findings....................................................................... 152�4.10.1� Laboratory and CXR Findings of RSV, HRV, IFV-A and HMPV Infections ...152�4.10.2� Laboratory and CXR Findings of HAdV, PIVs, HBoV Infections ....................158�4.10.3� Laboratory and CXR Findings of Total Single and Multiple Viral Infections ...160�4.10.4� Laboratory and CXR Findings of HRV-A and HRV-C Infections.....................162�

4.11� Hospital Course .......................................................................................................... 165�4.11.1� Hospital Course of RSV, HRV, IFV-A, & HMPV Infections ............................165�4.11.2� Hospital Course of HAdV, PIVs & HBoV Infections ........................................170�4.11.3� Hospital Course of Single and Multiple Viral Infections ...................................172�4.11.4� Hospital Course of HRV-A and HRV-C Infections ...........................................174�

5� DISCUSSION ................................................................................................................... 177�5.1� Comparison of the methods........................................................................................ 177�5.2� Phylogenetic Study ..................................................................................................... 183�5.3� Prevalence of the respiratory agents........................................................................... 186�5.4� Multiple Viral Infections ............................................................................................ 193�5.5� Demographic Features................................................................................................ 195�5.6� Clinical Features ......................................................................................................... 198�5.7� Laboratory and X-Ray Findings ................................................................................. 202�5.8� Hospital Course .......................................................................................................... 205�

6� CONCLUSION, LIMITATIONS AND RECOMMENDATIONS ............................. 209�6.1� Conclusion .................................................................................................................. 209�6.2� Limitations and Shortcomings ................................................................................... 213�6.3� Recommendations for Future Studies ........................................................................ 214��

REFERENCES/BIBLIOGRAPHY 217

APPENDICES

Appendix A 242

Appendix B 244

Appendix C 249

BIODATA OF STUDENT 251