PREVALENCE

OF

RESPIRATORY

NASOPHARYNGEAL

AND

VIRAL

OROPHARYNGEAL

PATHOGENS SPECIMENS

IN AND

CLINICAL OUTCOMES IN YOUNG CHILDREN PRESENTING WITH SEVERE

ACUTE

RESPIRATORY

INFECTIONS

AT

KENYATTA

NATIONAL HOSPITAL

A RESEARCH PROPOSAL FOR DISSERTATION SUBMITTED IN PART FULFILLMENT OF THE DEGREE OF MASTERS OF MEDICINE (MMED) IN PAEDIATRICS AND CHILD HEALTH, UNIVERSITY OF NAIROBI

PRINCIPAL INVESTIGATOR: ROSEMARIE GACHIE- LOPOKOIYIT H58/68446/2011 CO-INVESTIGATORS: 1. PROF. AGGREY WASUNNA, 2. PROF. ELIZABETH MALECHE OBIMBO 3. DR. GRACE IRIMU 4. DR. BASHIR ADMANI 5. DR. JOSHUA MOTT

DECLARATION I certify that this is my original work and has not been presented for any academic program in any other academic institution. Signed……………………………… Date: ………………………………. Dr. Rosemarie Gachie-Lopokoiyit MBChB SUPERVISORS DECLARATION I hereby certify that this dissertation has been submitted to the Department of Paediatrics with my approval. 1. Prof Aggrey Wasunna MMed (Paeds) F. Neon Professor of Pediatrics and Neonatology Signed……………………………… Date: ………………………………. 2. Prof Elizabeth Maleche Obimbo MMed (Paeds), MPH (Epid), CPulm (Paeds) Associate Professor of Pediatrics and Respiratory Medicine Signed……………………………… Date: ………………………………. 3. Dr. Grace Irimu MMed (Paeds), PhD Senior Lecturer, Department of Pediatrics and Child Health UON Signed……………………………… Date: ………………………………. 4. Dr. Bashir Admani MMed (Paeds) Fellow Pediatric Nephrology Lecturer, Department of Pediatrics and Child Health UON Signed……………………………… Date: ………………………………. 5. Dr. Joshua Adam Mott (PhD) Influenza Program Director, Center for Disease Control and Prevention Signed……………………………… Date: ………………………………. All the Department of Pediatrics and Child Health, School of Medicine, University of Nairobi

ii

DEDICATION This book is dedicated to my husband and children without whose support this would not have been possible.

iii

ACKNOWLEDGEMENTS I would firstly like to thank all of my supervisors who have guided me throughout this study. I would like to acknowledge KEMRI/CDC through Dr. Joshua Mott who provided the all the funding and technical support. Of special mention is Dr. Henry Njuguna who has been a mentor proving critical supervision and guidance throughout this process. I would also like to thank the data team at KEMRI/CDC including Gideon Emukule and especially Geofrey Arunga for all the assistance with data analysis. Finally, I would like to acknowledge my research assistants Conrad Maseno and Benjamin Matei who were instrumental in data collection.

iv

TABLE OF CONTENTS DECLARATION ................................................................................................. ii DEDICATION.................................................................................................... iii ACKNOWLEDGEMENTS .................................................................................iv TABLE OF CONTENTS..................................................................................... v LIST OF TABLES..............................................................................................vii LIST OF FIGURES...........................................................................................viii LIST OF ABBREVIATIONS ..............................................................................ix DEFINITIONS .....................................................................................................x SEVERE ACUTE RESPIRATORY ILLNESS (SARI) CASE DEFINITION ............ x DEFINITION OF OUTCOMES ............................................................................. x

ABSTRACT.........................................................................................................xi Background .......................................................................................................... xi Objectives ............................................................................................................. xi Design, Setting and Participants ............................................................................ xi Methods................................................................................................................ xi Results................................................................................................................. xii Conclusion ........................................................................................................... xii

BACKGROUND AND LITERATURE REVIEW............................................... 2 1. INTRODUCTION .............................................................................................. 2 2. EPIDEMIOLOGY.............................................................................................. 3 2.1 MAGNITUDE OF ACUTE RESPIRATORY INFECTIONS ................................... 3 2.2 SEASONALITY OF VIRAL RESPIRATORY INFECTIONS ................................. 4 3. ETIOLOGY OF ACUTE RESPIRATORY INFECTIONS................................... 4 3.1 VIRUSES AND ACUTE RESPIRATORY INFECTIONS ....................................... 5 3.2 ETIOLOGY OF ACUTE RESPIRATORY INFECTIONS IN HIGH RISK GROUPS ........................................................................................................................ 10 4. DETECTION OF VIRUSES IN THE UPPER AIRWAY AMONG PATIENTS PRESENTING WITH SEVERE ACUTE RESPIRATORY INFECTIONS ............ 11 5. SPECIMEN TYPE AND RESPIRATORY VIRUS DETECTION ...................... 14 6. IDENTIFICATION OF RESPIRATORY VIRAL PATHOGENS....................... 16

JUSTIFICATION.............................................................................................. 17 RESEARCH QUESTION.................................................................................. 18 Primary objective................................................................................................. 18 Secondary objectives ............................................................................................ 18

METHODS........................................................................................................ 19 STUDY DESIGN ................................................................................................. 19 STUDY SITE ....................................................................................................... 19 STUDY POPULATION ....................................................................................... 20 Inclusion criteria ............................................................................................................. 20 Exclusion criteria ............................................................................................................ 20

v

DEFINITION OF OUTCOMES ........................................................................... 20 SAMPLE SIZE CALCULATION ......................................................................... 20 STUDY PROCEDURES....................................................................................... 22 1. Epidemiological and Clinical Data Collection ........................................................... 22 2. Specimen collection and laboratory testing ................................................................ 23 3. Laboratory procedure nucleic acid extraction and rtRT-PCR .................................... 25 DATA MANAGEMENT ...................................................................................... 26 Data collection ................................................................................................................ 26 Data analysis................................................................................................................... 26 Data quality assurance .................................................................................................... 26

STUDY LIMITATIONS.................................................................................... 27 ETHICAL CONSIDERATIONS ....................................................................... 27 Autonomy ............................................................................................................ 27 Informed Consent ................................................................................................ 27 Confidentiality ..................................................................................................... 28 Safety .................................................................................................................. 28

DATA SHARING PLAN ................................................................................... 28 RESULTS.......................................................................................................... 29 Clinical presentation ............................................................................................ 29 Patient Outcomes ................................................................................................. 29 Prevalence of Viruses in Children with SARI ........................................................ 31 Spectrum of Respiratory Viruses in SARI ............................................................. 32 Monthly Variation of Respiratory Viruses Isolated in SARI................................... 34 Patient Outcomes and Viral status ........................................................................ 34

DISCUSSION .................................................................................................... 36 CONCLUSION.................................................................................................. 40 RECOMMENDATIONS ................................................................................... 40 REFERENCES.................................................................................................. 41 APPENDIX 1..................................................................................................... 48 INFORMED CONSENT FORM (ENGLISH) ....................................................... 48 INFORMED CONSENT FORM (SWAHILI)........................................................ 51

APPENDIX 2..................................................................................................... 54 SARI CASE ENROLMENT QUESTIONNAIRE: ................................................. 54 SARI case follow-up form.................................................................................... 59

APPENDIX 3..................................................................................................... 61 Measurement of axillary temperature ................................................................... 61 Measurement of weight ........................................................................................ 61 Measurement of arterial oxygen saturation ........................................................... 62 Measurement of Height ........................................................................................ 62

APPENDIX 4..................................................................................................... 64 Measurement of left mid upper arm circumference ............................................... 64

APPENDIX 5 BUDGET AND TIMELINE ....................................................... 65 APPENDIX 6: KNH/ UoN Ethics and Research Committee Approval Letter and Study registration certificate. ............................................................................ 67

vi

LIST OF TABLES Table 1 Summary of studies of viral isolates in respiratory infections; frequency and most common virus isolated............................................................................... 9 Table 2 Relationship between viral isolates from upper airways and SARI; Evidence from case control studies ................................................................................. 13 Table 3 Sensitivity and specificity of different specimen types and tests for identification of respiratory viruses .................................................................. 15 Table 4: Descriptive Characteristics of the Study Population ................................. 30 Table 5: Presence of Respiratory Viral Co-infections N=281................................... 31 Table 6 Spectrum of Viral Isolates n=177................................................................ 33 Table 7 Patient Outcomes N=281 ............................................................................ 35 Table 8 Association between outcome and viral infections N=281........................... 35 Table 9 Budget ....................................................................................................... 65 Table 10 Timeline ................................................................................................... 66

vii

LIST OF FIGURES Figure 1 Prevalence of Respiratory Viruses in SARI ............................................... 31 Figure 2 Spectrum of Respiratory Viruses in SARI.................................................. 32 Figure 3 Monthly Variation in Respiratory Viruses Isolated in SARI....................... 34 Figure 4 Digital Thermometer ................................................................................. 61 Figure 5 Measurement of Mid upper arm circumference.......................................... 64 Figure 6 KNH/UoN Ethics and Research Committee Approval Letter Page 1 of 2... 67 Figure 7 KNH/UoN Ethics and Research Committee Approval Letter Page 2 of 2... 68 Figure 8 Study Registration Certificate.................................................................... 69

viii

LIST OF ABBREVIATIONS AdV

Adenovirus

CDC

Centers for Disease control and prevention

FluA

Influenza A virus

FluB

Influenza B virus

HIV

Human Immunodeficiency Virus

HMPV

Human metapneumovirus

KEMRI

Kenya Medical Research Institute

KNH

Kenyatta National Hospital

MUAC

Mid upper arm circumference

NPS

Nasopharyngeal swab

OPS

Oro-pharyngeal swab

PIV

Parainfluenza Virus

PCR

Polymerase chain reaction

RSV

Respiratory syncytial virus

rtRT-PCR

Real time Reverse transcription PCR

SARI

Severe acute respiratory illness

VTM

Viral transport Media

ix

DEFINITIONS SEVERE ACUTE RESPIRATORY ILLNESS (SARI) CASE DEFINITION This study will adapt the SARI case definition as defined by the World Health Organization Interim Global Surveillance Standards for Influenza 2012 which defines SARI as an acute respiratory infection with presence of ALL the following 4 factors:  History of fever OR measured fever of ≥ 38 C° AND  Presence of cough AND  Onset of illness within the last 14 days AND  Requires hospitalization DEFINITION OF OUTCOMES Outcomes of hospitalization include: Good outcome  Discharge after 5 days of treatment Poor outcomes  Prolonged hospital stay (greater than 5 days)  Mechanical ventilation / Intensive Care Unit (ICU) Admission  Death

x

ABSTRACT Background Acute respiratory infections

are one of the leading causes of under-5 mortality

worldwide with the majority of these deaths occurring in the developing countries. Viruses have been shown to contribute significantly to the burden of respiratory infections (1). Improved diagnostic tests have improved viral yield but remain costly and as such, there is limited data on respiratory virus burden in resource poor settings such as Kenya. Objectives To determine the prevalence and spectrum of respiratory viral pathogens from nasopharyngeal and oropharyngeal specimens among children aged 2-59 months hospitalized with severe acute respiratory illness (SARI) at Kenyatta National Hospital To describe and compare the clinical outcomes of children with SARI with and without respiratory viral pathogens isolated. Design, Setting and Participants This hospital based short longitudinal survey was conducted at Kenyatta National Hospital (KNH), the main referral hospital in Kenya. Participants were children aged 2 months to 59 months hospitalized in the general pediatric wards with severe acute respiratory illness (SARI) as defined by an adapted WHO SARI case definition. Methods Patients were enrolled consecutively and nasopharyngeal and oropharyngeal swabs tested using polymerase chain reaction for adenovirus(AdV), respiratory syncytial virus (RSV), human metapneumovirus (HMPV), influenza A and B (FluA and FluB) xi

and parainfluenza viruses (PIV) 1 to 3. Good outcomes was discharge ≤5days while poor outcomes included prolonged hospital stay> 5 days, admission in the intensive care unit or death. Results We enrolled 281 patients with a median age of 10 months (IQR, 6-13), 54.8% were male. Respiratory viruses were detected in 177 of the 281 participants giving a prevalence of 63.0% [95% CI, 57.1-68.6]. Adenovirus (AdV) was the most commonly detected (37.7%) followed by respiratory syncytial virus (RSV) (19.2%) and parainfluenza 3 (PIV 3) (13.5%). Co-infections with more than one virus were present in 50 (17.8%) cases. Poor outcomes were similar in children with viral isolates and without viral infection (Odds Ratio (OR): 1.139, 95%CI: 0.694-1.871) p=0.606. Conclusion The majority of SARI cases had a virus detected, mainly adenovirus . Viral coinfections were not unusual most of which were co- infections with adenovirus. More than half of the patients with SARI had a prolonged hospital stay but ICU admission and death were rare outcomes of hospitalization.

xii

BACKGROUND AND LITERATURE REVIEW 1. INTRODUCTION Acute respiratory infections are a major killer of children under 5 years in developing countries. Improved molecular diagnostic techniques have shown that viruses are associated with respiratory disease. Respiratory viruses including: influenza viruses, parainfluenza viruses, respiratory syncytial virus and adenoviruses are commonly identified in cases presenting with respiratory infections (1). Previous influenza pandemics underpin the role of viruses in causing respiratory disease mortality. In the 1918 influenza pandemic, a third of the world’s population was infected with influenza and of those infected approximately 20-40 million died. The 1957 Asian and 1968 Hong Kong influenza pandemics resulted in 1-4 million deaths each (2). Due to rapid mutation of influenza viruses, avian influenza (A/H5N1) first detected in 1997 in Hong Kong, continues to have pandemic potential. Avian influenza virus A(H5N1) is highly virulent and lethal with more than half of infected cases resulting in death. Other respiratory viruses including coronaviruses have been associated with increased respiratory morbidity and mortality. Severe Acute Respiratory Syndrome (SARS), caused by a coronavirus was first detected in China in 2002 and later spread to 37 different countries within a few weeks. Following these outbreaks, viral associated acute respiratory illnesses have now been appreciated to be a major public health concern. Pandemic influenza occurs when new highly infective influenza viruses emerge following genetic re-assortment (antigenic shift). Genetic re-assortment occurs when two different influenza viruses that affect different species infect a host cell and during replication their genetic material is exchanged resulting in a new

2

haemaglutinin glycoprotein (an antigenic site) to which the general population has no immunity. Seasonal outbreaks of influenza occur when viruses also undergo mutations during replication resulting in minor changes in the haemagluttinin antigenic sites (antigenic drift). When this happens, the binding of neutralizing antibodies acquired from previous influenza infections is reduced (3). 2. EPIDEMIOLOGY 2.1 MAGNITUDE OF ACUTE RESPIRATORY INFECTIONS

Acute respiratory infections, including pneumonia are the most common illnesses affecting all age groups with children particularly at increased risk of morbidity and mortality. In 2000 approximately 156 million new episodes of childhood clinical pneumonia occurred globally, more than 95% of these were in developing countries. About 9% of these pneumonia cases were severe enough to require hospital admission (4). In 2010, it was estimated that 7·6 million deaths occurred in children younger than 5 years of age globally. Of these deaths, 4.9 million (64·0%) were attributable to infectious causes of which pneumonia was the leading cause of death estimated at 1.1 million deaths (14·1%) (5). The majority of these deaths (47%) occurred in subSaharan Africa. In Kenya, pneumonia is estimated to cause 15% of childhood deaths (6). Many of the etiologies of pneumonia are vaccine preventable or can be treated with inexpensive antibiotics (7). With the increased use of pneumococcal and haemophilus influenza type B vaccines and the advancement in diagnostic capacity, viruses are emerging as an important cause of respiratory illnesses. Some of these respiratory viruses have effective vaccines available and the use of these vaccines could substantially reduce the burden of respiratory disease. Influenza vaccine use has been associated with marked

3

reduction in respiratory illnesses associated hospitalization and episodes of febrile acute otitis media (8). Influenza vaccine has also been shown to decrease respiratory disease related morbidity and mortality in the first 6 months of life among infants whose mothers’ received influenza vaccine during pregnancy (9). Most of these studies have been conducted in the developed world and limited data is available in sub-Saharan Africa. The modes of spread of respiratory viruses include: contact spread, endogenous spread, droplet contact spread, airborne spread, common vehicle spread and vector spread. Direct and indirect contact spread plays a major role in spread of RSV and other viruses including influenza. Influenza appears to be spread mainly by airborne droplet nuclei. This mode of transmission is suggested by the explosive spread of the virus after annual introduction of the virus during influenza season. 2.2 SEASONALITY OF VIRAL RESPIRATORY INFECTIONS

Respiratory viral infections have been shown to have various seasonal peaks in temperate countries. RSV peaks between late autumn and early spring. A study done in Kilifi, Kenya noted a seasonal variation for RSV with peaks in the first quarter of the year not associated with the rainy season (10). Influenza in temperate climates usually peaks during the colder months and vaccination is advocated for all people above 6 months of age just before the onset of the cold season. In Kenya influenza has been shown to circulate all year round (11). 3. ETIOLOGY OF ACUTE RESPIRATORY INFECTIONS Etiologic agents of acute respiratory illness are not only widespread but also associated with varying degrees of severity of illness. In Kenya, the distribution of etiologic pathogens associated with acute respiratory illness is likely to change

4

following the introduction and widespread use of Haemophilus influenzae vaccine in 2001 and Streptococcus pneumoniae vaccine in 2011. Similar observations in changing distribution of etiologic agents of respiratory disease have been seen elsewhere (12). Introduction of pneumococcal vaccine (13 valent) in Massachusetts, USA was associated with disappearance of the vaccine serotypes and replacement with penicillin nonsusceptible non-vaccine serotypes (13) 3.1 VIRUSES AND ACUTE RESPIRATORY INFECTIONS

Respiratory viral infections may elicit both upper and lower respiratory tract symptoms. The mechanisms for these symptoms are still unclear. With improved sensitivity and specificity of diagnostic tests and in part, widespread use of vaccines targeting bacterial etiologies of respiratory disease, respiratory viruses have been shown to play an important role in the etiology of pediatric respiratory infections including pneumonia (14). In western countries, respiratory viral infections are a leading cause of hospitalization with peaks seen during winter months. RSV and influenza have been associated with more severe illness. Co-infections are common and studies have shown that up to a third of children have evidence of viral-bacterial co-infection (15). Viral infections result in physical damage to respiratory cells and may lead to opportunistic adherence of bacteria and this has been shown in respiratory cells infected with influenza A virus, respiratory syncytial virus and adenovirus in both in vitro and in vivo models. Preceding viral infections facilitate Streptococcus pneumoniae and HiB infections especially of the upper respiratory tract (16). RESPIRATORY SYNCYTIAL VIRUS

RSV is an important cause of respiratory infection worldwide with infants and young children being at highest risk of severe illness. It is the leading cause of respiratory viral disease in children under 1 year of age and has been identified in 15-20% of 5

cases of lower respiratory tract infections including pneumonia and bronchiolitis in children admitted to hospital in developing countries (17). Virtually all children have at least one episode of RSV infection by their fifth year. RSV induces feeble and fleeting immunity and thus infections tend to recur. However with increase in age, severity of RSV infections decreases (18). It is estimated that RSV is directly or indirectly responsible for over 600,000 deaths worldwide annually (19). Pretorius et al in South Africa conducted a large study of SARI and found RSV to be the most common viral SARI associated pathogen in children under 5 years (20). The findings by Berkley et al in Kenya were similar with RSV prevalence at 34% in patients with severe or very severe pneumonia (21). Studies done at Kenyatta National Hospital, Kenya’s leading referral hospital, showed that the prevalence of respiratory viral infections was over 50% in pediatric patients with lower respiratory tract infections. The spectrum of etiologic viruses has varied over time. In studies conducted in KNH, prevalence of RSV increased from 20.8% in 1985 to 28.8% in 2009 as determined by viral cultures and direct immunofluorescence (22, 23). RSV infects through the upper respiratory tract, especially the nasopharynx and the eyes. Incubation is about 3-5 days. Infection of the bronchiolar epithelium results in mucosal inflammation and edema. Subsequent necrosis leads to sloughing off of the epithelium with formation of intraluminal plugs made up of cellular debris and mucus. These plugs lead to a ball valve effect with hyperinflation of the distal airways and alveoli (bronchiolitis).

Immunity against RSV infection requires both

neutralizing antibodies and cytotoxic T cell responses. Exuberant cytotoxic T cell responses may augment the clinical disease (24). Studies have indicated that the inflammatory response is responsible for severity of infection with RSV (25). RSV causes upper respiratory infections such as acute otitis media and croup but more

6

commonly are bronchiolitis and pneumonia. Signs of upper respiratory infections precede those of lower respiratory infections by a few days. Subsequent bacterial co infection is common (26). INFLUENZA

In 2008, influenza infection was estimated to cause 1-2 million cases of severe acute lower respiratory tract infections and up to 111,500 deaths. Almost all of these deaths occurred in developing countries (27). Studies have shown that influenza vaccination not only offers protection against seasonal influenza but also influenza-related bacterial otitis media in children (16). Influenza viruses are transmitted via droplets, which contaminate the mucosa of the respiratory tract mainly targeting columnar epithelial cells. Once a cell is infected the virus RNA gets into the host cell’s nucleus where transcription and translation occurs. New virions are released by budding. The duration from infection to shedding of virus is as short as 6 hours however incubation period is usually 1-2 days. Shedding may continue up to 2 weeks after infection especially in children, the elderly and immunocompromised persons. The most frequent complication of influenza is pneumonia. Secondary bacterial pneumonia is most common but primary influenza pneumonia is more severe. Influenza may exacerbate underlying heart or lung disease such as asthma and other chronic conditions (3) HUMAN METAPNEUMOVIRUS (HMPV)

Discovered in 2001,in the Netherlands, HMPV has since been shown to be not only widespread but also to cause a similar spectrum of disease to RSV (28). In a study done in Nashville, USA, 20% of all previously virus-negative lower respiratory tract illnesses were attributable to HMPV (29). HMPV has been shown to be of increasing

7

importance in lower respiratory tract infections. In Madagascar, Hoffman et al found HMPV to be associated with 23.9% of community acquired pneumonia cases in children 2- 59 months (30). The pathophysiology appears to be similar to RSV with the virus showing a tropism for

respiratory

epithelium.

The

cytopathic

effects

are

morphologically

indistinguishable from those induced by RSV. It may cause a range of illnesses from mild upper respiratory infections to croup, bronchiolitis and asthma. Duration of symptoms may be approximately 7 days with virus being shed for 1 to 2 weeks (31) PARAINFLUENZA AND ADENOVIRUS

Other important viral etiologies of respiratory illness include parainfluenza and adenovirus viruses (4). Parainfluenza viruses have a similar spectrum of disease as RSV though infection is associated with fewer hospitalizations (32). Parainfluenza replicates in the nasopharyngeal epithelium spreading to the lower respiratory tract in 1 to 3 days (26). Adenoviruses cause not only respiratory infections, but also gastrointestinal, central nervous system and ocular infections. It has been shown to establish latent infections in tonsillar and adenoid tissue and as such identification of the virus from upper respiratory tract specimens may be due to carriage (33). Table 1 summarizes 6 studies of respiratory viruses and the most common viruses isolated.

8

Table 1 Summary of studies of viral isolates in respiratory infections; frequency and most common virus isolated. AUTHOR

SPECIMEN TYPE

CLINICAL CONDITION

Wafula et al < 5 years 1985 KNH n=835

NP aspirate

Pneumonia, Bronchiolitis, 451 (54%) URTI, Croup, Bronchospasm, Bronchitis, RSV (20.8%) Mixed Diagnosis

< 5 years

NP aspirate

Pneumonia, LTB

Sang et al 2009 KNH n= 285

AGE

FREQUENCY OF VIRAL ISOLATE (%) & MOST COMMON ISOLATE (%)

bronchiolitis, 154 (54%) RSV 49(28.8%)

Berkley et al 1 day - 12 Nasal wash yrs. 2010 Kilifi n=759

Severe pneumonia and very 425 (56%) severe pneumonia RSV 260 (34%)

Hoffman et al 2-59 mths 2012 Madagascar n=295

CAP, other ALRI’s, URTI 212 (71.8%)* with cough, URTI without HMPV cough, CAP(23.9%)

NP swab

Pretorius et al 3 mths - 5 NP aspirate, SARI NP swab, OP 2012 South years swab Africa

-

4,666 (57.1%) Rhinovirus 2034 (24.9%)**

n= 8173 RSV (14.3%) Iwane et al 5

Discharge ≤ 5

Virus

days

days n(%)

Virus

111 (62.7)

66 (37.3)

177

No Virus

62 (59.6)

42 (40.4)

104

Total

173

108

281

Total

35

DISCUSSION We found that at Kenyatta National Hospital there is a high prevalence of respiratory viruses in nasopharyngeal and oropharyngeal specimens collected from children hospitalized with SARI. Adenovirus was the most common virus detected in almost 40 % of cases. Viral co-infections were not unusual and most of which were coinfections with adenovirus. More than half of the patients with SARI had a prolonged hospital stay but ICU admission and death were rare outcomes. Tests such as PCR have improved yield from upper airway specimens for patients with respiratory illnesses (50). Nasopharyngeal aspirates have the highest yield followed by nasopharyngeal wash, but combining nasopharyngeal and oropharyngeal swabs gives comparable results with less discomfort (42-47). Interpretation of these data, however, may be a challenge due to the high sensitivity of these tests. In addition when evaluating the clinical significance of our findings we must consider what is known about carriage of virus in healthy children. Case control studies have shown that nasopharyngeal carriage of pathogenic viruses is rare ranging from 0-5% (Table 2) varying with each virus. Some viruses such as HMPV had no positive controls(21,40.41). Therefore these viruses when detected, were likely pathogenic including RSV, HMPV and influenza. Adenoviruses however, are known to be carried in latent form in the tonsils and adenoids (39). Therefore presence of adenovirus in nasopharyngeal and oropharyngeal specimens may be difficult to interpret. This study demonstrated a prevalence of respiratory viruses in nasopharyngeal and oropharyngeal specimens of children with SARI of 63%, which is slightly higher than that seen in other studies done in Kenya with prevalence ranging from 54-56%

36

(21,22,23). This difference could be partly attributed to different sensitivity of diagnostic tests used. In the study done by Wafula et al in 1985 and Sang et al in 2008 also conducted at KNH, viral culture and immunofluorescence were used to detect presence of respiratory viruses. These diagnostic tests have been shown to have lower sensitivity than rtRT-PCR which was used in our study (table 3). Berkley et al in Kilifi used rtRT-PCR and found a prevalence of 56%. These authors used nasal washes, which may explain the decreased yield compared to the combination of NPS and OP swab used in our study (43,46). Their study also found that children with viruses detected were younger (7.5 months) than those with no viruses (11.3 months). This was unlike our findings where the median age of those with viruses was slightly older (11 months) than those without viruses ( 10 months), which could be explained by the low prevalence of RSV in our study, which has been shown to be highly seasonal. In addition, when adenovirus was excluded from analysis, the median age of those with viruses decreased to 9 months consistent with the Kilifi study. Overall, both of these highly sensitive respiratory studies showed that respiratory viruses were more common in infants compared to older children. The most common viral isolate was adenovirus at 37.7%. This is much higher than other local and regional studies. Berkley et al found AdV in 3.8% of isolates while Pretorius et al in South Africa isolated AdV in 13.3%. Some factors that may have contributed to our increased detection include the higher yield of our specimens, the broader case definition and local and regional variation. Berkley et al used nasal washes and the WHO case definitions for severe and very severe pneumonia (46,47). While AdV is associated with carriage, we cannot exclude that it was responsible for SARI in our children.

37

We isolated RSV in almost 20% of our children, which was consistent with other local and regional studies where ranges were 14-34% (20,21). This has shown that RSV is an important pathogen in our population. The prevalence of PIV 3 (13.5%) was almost double that found in the studies by Berkley et al and Pretorious et al where the prevalence was approximately 4 % in both. It is possible that our specimens were collected during the PIV 3 season, which would be better defined if this study was carried out over a one year period. Concurrent infections with multiple viruses were present in 17.8% of the patients in our study. Pretorius et al found similar rates of co-infections with 17.4% of cases having more than 1 virus isolated. Berkley et al found viral co-infections in 9.8% of cases which again could be attributed to the specimen type these authors used. We found that almost all co-infections were with AdV, which again due to common occurrence of carrier states may be difficult to interpret. It was noted that during the study period of 6 months there was marked variation for the prevalence of RSV which showed an upsurge in the first quarter of the year. This is similar to what was found by Nokes et al in Kilifi (10). This was less marked for influenza which also showed a slight upsurge in the first quarter of 2014. While influenza is known to be seasonal in temperate countries local studies have shown that it circulates all year round (11). This may be more clearly demonstrated in a study spanning a whole year. Our study evaluated the association of poor outcome with viral status. There was no evidence that presence of respiratory viruses was associated with poor outcome OR 1.139 (95%CI, 0.694-1.871). This is possibly due to the high prevalence of AdV, which has been shown to be carried in the respiratory tract and also due to the low

38

prevalence of viruses such as RSV and FluA which have been shown in other studies to be associated with severe disease (5,9,21). However in our analysis excluding AdV did not alter the impact of viral status on outcome. This study was powered to determine prevalence of respiratory viruses, not to determine outcomes therefore the power of this study may not have been sufficient to analyze outcomes. The strengths of this study included high quality laboratory analysis with strong quality control systems. We also included a comprehensive set of viruses in our analysis. Our study had several limitations. Many of the deaths, which occurred soon after admission, may have been missed. As a prevalence study this research was not designed to assess the respiratory viruses for causality therefore we were not able to determine the role of the different viruses including adenovirus in causation of SARI.

39

CONCLUSION 1. Children aged 2-59 months with SARI at KNH had a high prevalence (63%) of respiratory viral pathogens with 17.8% having multiple respiratory viral pathogens. 2. A wide spectrum of respiratory viral pathogens were identified , the most prevalent being adenovirus (37.7%), respiratory syncytial virus (19.2%) and parainfluenza (16.0%) followed by influenza (7.1%) and HMPV(4.3%). 3. Prolonged hospital stay was common among the study population (55.9%). 4. Short term in-hospital mortality for SARI was low and children rarely required intensive care.

RECOMMENDATIONS A cohort study may be useful to determine the role of adenovirus in SARI. This study should be carried out for a period of 1 year to capture viruses that are highly seasonal.

40

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47

APPENDIX 1 INFORMED CONSENT FORM (ENGLISH)

Title: Prevalence and of respiratory viral pathogens and outcome in young children presenting with severe acute respiratory infections in Kenyatta National Hospital Principle Investigator:

Dr. Rosemarie Gachie-Lopokoiyit University of Nairobi, Dept. Of Pediatrics and Child Health TEL: 0721 216 289

Co- Investigators : 1. Prof. A. Wasunna, 2. Prof. E. Obimbo, 3. Dr. G. Irimu, 4. Dr. A. Bashir 5. Dr. J. Mott Investigator Note:

Thank you for agreeing to read this form. It offers information

about this study, which will help you decide if you will take part in it, or not. Appropriate translation will be carried out in the language you are most comfortable with. Introduction:

Viral infections contribute significantly to the burden of acute

respiratory infections including pneumonia. Some of these viruses such as influenza virus have vaccines and drugs available. Better understanding of the specific viruses, 48

which cause these illnesses, will help clinicians to better manage these illnesses in the future. Procedure:

If you agree to be part of this study, I will ask you detailed questions

about your child’s illness. I will then take specimens from their nose and mouth using a cotton swab, which can be uncomfortable to the child but will not harm them. These will be taken to KEMRI/CDC for testing. After 5 days I will take details from the file of laboratory tests done and the final diagnosis. Benefits:

Findings of this study will be interpreted to you, the hospital

management team and the University of Nairobi. Any results, which may help in management of your child, will be availed immediately they are obtained. This will also help doctors in the future when planning treatment for patients with a similar illness. Confidentiality:

If you agree to be part of this study, the information you give

will be held in strict confidence and only used for the purpose of the study Reassurance: The management of your child will in no way affected by your decision to decline or to participate in this study. The cost will in no way change through this study. At any time you are free to drop out of the study without penalty. Ethical consideration:

I have been granted approval from the Research and

Ethics Committees to conduct this study. Inquiries on ethical considerations can be gotten from: Prof. A.N. Guantai, Secretary, KNH/UON-ERC,

49

Kenyatta National Hospital, Hospital Rd, along Ngong Rd, P.O.Box 20723, Nairobi Tel: (020) 726300-9 Fax: 725272 To indicate that you understand the conditions of this study and that you consent to participate in it, please sign or put your thumbprint in the space provided below. I,

confirm that the study has

been fully explained to me and I give full consent to participate in it. Signature/thumbprint Investigator’s signature Date

50

INFORMED CONSENT FORM (SWAHILI)

Kichwa: Kuenea kwa magonjwa ya kifua /mapafu yanayo sababishwa na virusi kwa watoto wachanga katika hospitali kuu ya Kenyatta Daktari Mkuu:

Dr. Rosemarie Gachie-Lopokoiyit University of Nairobi, Dept. Of Pediatrics and Child Health TEL: 0721 216 289

Madaktari Wahusika : 1. Prof. A. Wasunna, 2. Prof. E. Obimbo, 3. Dr. G. Irimu, 4. Dr. A. Bashir 5. Dr. J. Mott Tunawashukuru kwa kukubali kusoma hii nakali. Inaelezea kielelezo cha chunguzi huu ambao utasaidia kwa uamuzi utakao uchukua kushiriki katika zoezi hili au ukatae. Kielelezo:

Virusi vinaweza sababisha uambukizaji wa aina ya magonjwa kama

homa ya mapafu ambao ni mzigo kwa jamii. Aina ya virusi kama influenza zinatiba. Kuelewa kila aina ya virusi vinavyosababisha magonjwa tofauti tofauti itasaidia madaktari kumudu kila aina ya virusi siku za usoni.

51

Utaratibu:

Ukikubali mtoto kuwa mmoja wapo wa zoezi hili itabidi nikuulize

maswali kwa kina juu ya mtoto wako kuhusu hali ya udhaifu wa afya yake. Baadaye nitachukuwa chembechembe kutoka kwa mapua na midoma yao nikitumia pamba ambapo huiende ikawafanya wahisi usumbufu kidogo lakini haita wadhuru. Hizi chembechembe zitapelekwa KEMRI/CDC kwa uchunguzi. Baada ya kufanyiwa uchunguzi na kimaabra nitachukuwa kwa ustadi yaliyomo yote na kufanya matibabu ya mwisho. Faida: Matokeo ya zoezi hili yatatafsiriwa kwa usimamizi wa timu na chua kikuu cha Nairobi. Matokeo yatakayo kuwa ya manufaa kwa malezi ya mtoto wako yatatloewa kwako ili yasaidie familia. Zaidi ya yote pia, yatasaidia madaktari kwa mpangilio wa kutibu wagonjwa walio na magonjwa kama hayo. Uadilifu/Uaminifu: Ukikubali kushiriki katika zoezi hili habari yoyote utakoyatoa kwa minajili ya zoezi hili, iatchukuwa kwa uadilifu na kuwekwa kwa siri ili itumike kwa lengo la zoezi hili peekee. Hakikisho:

Uamuzi wako wa kushiriki au kutoshiriki katika zoezi hili hautaadhiri

maelezi ya mtoto wako. Kilia mtu ako huru kubakia katika zoezi hili au kujiondoa wakati wowote. Maadili yanayozingatiwa:

Nimepewa kibali na The Research and Ethics

Committee , kamati ya uchunguzi na maadili kuendesha zoezi hili. Kwa maswali ama maoni zaidi unaweza pata kupitia kwa:Prof. A.N. Guantai, Secretary, KNH/UON-ERC, Kenyatta National Hospital, 52

Hospital Rd, along Ngong Rd, P.O.Box 20723, Nairobi Tel: (020) 726300-9 Fax: 725272 Mimi,

nathibitisha kwamba zoezi hili

limefanywa kwa kina na kupewa maelezo yote. Ninakubali kushiriki Sahihi Sahihi ya Daktari Mkuu Tarehe

53

APPENDIX 2 SARI CASE ENROLMENT QUESTIONNAIRE: STUDY TITLE: PREVALENCE OF RESPIRATORY VIRAL PATHOGENS AND CLINICAL OUTCOMES IN YOUNG CHILDREN PRESENTING WITH SEVERE ACUTE RESPIRATORY INFECTIONS IN KENYATTA NATIONAL HOSPITAL Patient enrolment questionnaire

Interviewer’s initials: _______ Patient’s unique identification number _ _ _ / _ _ _ _ Interview date |_D_|_D_|_M_|_M_|_Y_|_Y_|_Y_|_Y_| PART ONE: Case classification 1. Does the patient meeting the SARI surveillance case definition? Yes 

No  Don’t Know 

If yes, confirm that all the following in the case definition are met  Has an acute respiratory infection Yes 

No  Don’t Know 

 Has history of fever or measured fever ≥38 0CYes 

No  Don’t

Know   Has cough

Yes 

No  Don’t Know 

 Onset within the last 14 days

Yes 

No  Don’t Know 

 Requires hospitalization Yes 

No  Don’t Know 

PART TWO: Demographic Information 2. Date of Birth (DOB) |_D_|_D_|_M_|_M_|_Y_|_Y_|_Y_|_Y_| If DOB is unknown, please enter age: years___________ or months if < 1 year _______________ 3. Age-group: >2 days < 2 months 

≥ 2 months