Molecular Diagnosis of hMPV

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Molecular Diagnosis and Prevalence of Human

Metapneumovirus Infection among Egyptian Infants

Clinically Diagnosed with Acute Viral Bronchiolitis

WALID NABIL FOUAD

Department of Microbiology

Medical Research Institute

University of Alexandria

2011

BY

A PRESENTATION SEMINARFORA MASTER DEGREE THESIS PROTOCOL

Thesis Title:


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Supervisors:

1. Dr. Gamal El-Din Ahmed El-SawafProfessor, Department of Microbiology,

Medical Research Institute, University of Alexandria

2. Dr. Maged Mohammed Eissa

Professor, Department of Pediatrics,Faculty of Medicine, University of Alexandria

3. Dr. Abeer Abd El-Rahim Ghazal

Assistant Professor, Department of Microbiology,


4. Dr. Dalia El Sayed Metwally

Lecturer, Department of Microbiology,



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I. Background


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Bronchiolitis:

Acute, inflammatory lower respiratory

tract infection characterized by cough,

coryza (runny nose), fever, expiratory

wheezing, grunting, tachypnea (fast

breathing), retractions and air trapping.

Most common in children in the first two

years of life and a major cause of

hospitalization in that age group.

An estimated 25 in every 1,000 children will require hospitalization withbronchiolitis; for 1% to 2% of these children, infection will be severe

enough to require ventilatory support.


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Bronchiolitis is usually caused by respiratoryviruses including adenovirus, influenza andparainfluenza viruses.

Most infections, however, are due to humanrespiratory syncytial virus (hRSV).

Although numerous viral and bacterial agentswere found to cause bronchiolitis andpneumonia in young children, about 15%-34%of these illnesses were found to have noetiologic agent.

This observation has suggested that new undiscovered respiratoryinfectious agents are likely to exist and remain to be identified.


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Human Metapneumovirus:

Recently discovered respiratory viral

pathogen affecting human respiratory

epithelia.

Detected for the first time in 2001 by van

den Hoogen in the Netherlands.

CausesARIs in all age groups, especially infants

and young children.

Frequently found associated with other common

respiratory viruses (co-infection).


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Classification:

Single negative-stranded RNA Paramyxovirusbelonging to family

Paramyxovidiridae.


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Clinical Features:

Usually mild and similar to those ofhRSV infections with common

signs and symptoms including:

Fever

Cough

Rhinorrhea

Tachypnea

Severe infections usually cause acute bronchiolitis, asthma

exacerbation, and pneumonia.

Acute wheezing may be also associated with infection in some

children.


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Epidemiology:

Second most common cause of ARIs (after human respiratory

syncytial virus) in infants and young children.

Causative agent of infant bronchiolitis in 5-15% of cases in the US.

Detected in various parts of the world, with reports from North

America, Europe, Asia and Australia.

All children are virtually exposed to the virus by the age offive.

Reinfections are frequent and could lead to complications in

elderly and immunosuppressed hosts.


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Seasonal Distribution:

In temperate climates, hMPV circulates predominately in the late

winter and spring, and the peak of activity at any given location often

coincides with or follows the peak ofRSV activity.


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II. Aim of Work


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Two aims of the present work:

(1) Diagnosing hMPV infection

through molecular detection methods

(Real-time PCR Assay).

(2) Evaluating hMPV prevalence

among Egyptian infants clinically

diagnosed with acute bronchiolitis.


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III. Subjects and Methods


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1. Subjects:

After the approval of the Committee of Ethics of the Medical Research

Institute of Alexandria University, and under its guidelines, the present study

will be conducted on 100 infants attendingAlexandria University Childrens

Hospital of El-Chatby (Alexandria, Egypt), and clinically diagnosed with acute

viral bronchiolitis during the upcoming winter / spring season of2012-2013.

A- Inclusion Criteria:

Enrolled infants in the study will be diagnosed with acute bronchiolitis

through a pediatrician according to the following criteria: -

1. An age less than 2 years.2. A history of first attack of wheeze within the first year of life.

3. An absence of response to bronchodilators.


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B- Exclusion Criteria:

All the following subjects should be excluded from the study: -

1. Prematurely born or immunocompromised subjects.

2. Subjects with chronic pulmonary or congenital heart diseases.

3. Subjects whose parents/guardians refuse to enroll their children

in the study.

C- Data Collection:

After obtaining an informed consent from the parent of each child, the

data collected for each subject will include: -

1. Personal data.2. Full past medical history.

3. Treatment given prior to hospital admission.

4. Current clinical signs and symptoms.


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2. Methods:

The virus to be studied will be

diagnosed by detecting it in the

patients respiratory specimens using

a real-time, reverse-transcription

PCR assay.

For detecting the impact of co-infection, the

patients specimens will be also screened forcommon viral respiratory pathogens using a

qualitative indirect immunofluorescence

assay.


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Sample Collection:

From each child, two nasopharyngeal

sampleswill be collected:

1. One will be collected using a mucus

extractorand screened immediately through

an immunofluorescence assay for detectingcommon respiratory viruses, other than hMPV,

that might be involved in infection.

2. The second one will be collected using the

flexible aluminum-shafted Virocult swabs(Medical Wire and Equipment, Wiltshire, UK),

separated into aliquots and kept frozen at -70

C for further molecular analysis.


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Laboratory Investigations:

For each child, the following tests will be performed:

A. Immunofluorescence Screen Assay:

An IMAGEN respiratory screen kit (Oxoid, Hampshire, UK) will be used for

detecting any of the most common seven respiratory viruses in children,

including:

RSV

Influenza A and B

Parainfluenza viruses (1, 2, and 3)

Adenovirus


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Laboratory Investigations (Cont.):

B. Molecular PCR Assay:

A real-time, reverse transcription PCR assay will be performed for detecting

hMPV using the PrimerDesign PCR detection kit(PrimerDesign,

Southampton, UK), according to the manufacturers instructions.


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Molecular Diagnosis of hMPV