NRL Influenza and Acute

Respiratory Diseases

Capacity for detection of avian influenza viruses

representing a serious threat to human health

(Bulgarian experience)

2015, Avian Influenza Preparedness Workshop, ECDC, Stockholm

Svetla Angelova, PhD

Ministry of Health

Regional Health Inspectorates

Regional Virology Laboratories -9

(serological diagnosis )

NCIPD

NRL “Influenza and ARD”

Department of Virology Department

Epidemiology and

CD Surveillance

NRL “Influenza and ARD”

• Routine seasonal influenza surveillance

• Enhanced surveillance for Influenza A (H5N1) in humans

GPs Hospitals

Part of National Centre of

Infectious and Parasitic Diseases

(NCIPD).

Recognized by WHO as National

Influenza Center (NIC).

The only laboratory responsible

for the application of modern

influenza diagnosis in humans,

A(H5N1) avian influenza virus

diagnosis included.

(sentinel provider

network data)

http://grippe.ncipd.org

CAPACITY FOR DETECTION AND IDENTIFICATION OF A(H5N1)

• In Bulgaria there is a National Influenza Pandemic Plan. According to it,

additional funds for technical equipment and influenza virus molecular

diagnostics consumables are gain.

• Since May 2009 CDC primers, probes, positive controls and kits for Real

Time RT-PCR assays for the new subtype A (H1, H3, H3v, H5, H7) have been

available in NRL.

• Staff with good qualification in molecular biology techniques.

• NRL participates in the External Quality Control for human influenza A

and B, avian A(H5N1) and A(H7N9) and pandemic A(H1N1)2009 viruses,

organized by different international institutions :

Institute Düsseldorf, Germany (INSTAND) – genome detection of influenza

viruses (2005-2015);

Centre for Health Protection, Hong Kong SAR, China (WHO control)-

genome detection of influenza viruses (2007-2015).

Centre of Disease Control and Prevention, Atlanta (2014)

QСMD, Glasgow (2013, 2015)

Based on successful performance (100% in the last WHO EQAP panel) in the WHO External

Quality Assessment Program, NRL in Bulgaria has capacity to detect avian A(H5N1) and

A(H7N9) viruses with RT-PCR analysis.

Direct detection and typing of influenza A/B viruses by Real Time

RT-PCR (BSL-2 conditions)

Subtyping of influenza viruses type A (H1, H3, H3v, H5, H7);

determination of the line of type B viruses (B/Victoria, B/Yamagata)

by Real Time RT-PCR (BSL-2 conditions)

Isolation of influenza viruses in cell cultures (only human

Influenza A/B viruses; lack of BSL-3 conditions for isolating viruses

with high pathogenic potential)

Rapid tests for influenza antigens detection

Monitoring the sensitivity of influenza viruses to antivirals by Real

Time RT-PCR (detecting H275Y mutation)

Sequencing the haemagglutinin gene, phenotypic susceptibility

characterization of influenza viruses to antiviral agents (MUNANA

test) - at WHO-CC, London.

DIAGNOSTIC METHODS APPLIED IN NRL

NRL sends the isolated strains to WHO-CCs

London for confirmation and detailed

characterization.

Clinical specimens:

Nasal and throat swabs

Bronchoalveolar lavage fluid

Endotracheal aspirate

Pleural fluid

Sputum

Post-mortem tissue

Type A

Sub-type H5 RT-PCR:5 hours

Preliminary

result

A/H5+/H7+

probable case

Type B

Sub-types

H3/H1

Sub-type H7

Lineage B

•Yamagata

•Victoria

NRL sends A/H5+/H7+ probable specimens to

WHO-CCs London for confirmation and

detailed characterization.

Samples from patients

with respiratory diseases

in close contact with ill or

dead birds, their family

members or travellers in

countries with registered

avian flu cases.

•RT - PCR used to screen - BSL-2 facility

Avian Influenza Viruses Laboratory Diagnostic

RNA extraction /personnel protection/

Testing for H5 alone can create

problems: test for flu A, B and

H5 (preferably also H1 and H3).

Coupling of clinical and epi to lab

data is essential for interpretation

of results.

Out

breaks

(year)

Total

number of

analyzed

samples

(from suspect

A(H5N1)

patients)

(n=28)

A H1

pdm H3 H5 B *RNP Results

Past

(2006)

26

3 positive

3

positive

-

-

-

+

3

A(H1N1)

pdm

Recent

(2015)

2

-

-

-

-

2 positive

+

2

B (Yam)

RT-PCR results from testing suspected A(H5N1) patients in

relation with past (2006) and recent (2015) avian influenza

outbreaks in Bulgaria

*sample quality controls (Human RNase P (RNP) gene);also- negative, positive controls

Hadzhiolova, T., Pavlova, S., Kotseva, R. (2008). Laboratory investigation of the

first suspected human cases of infection with avian influenza A (H5N1) virus in

Bulgaria. Euro surveillance: 13(30), 717-727.

Close contact with ill or dead birds (25 among our

A(H5N1) suspected patients met these criteria )

History of travel to a country with influenza H5N1

documented in poultry, wild birds, and/or humans,

AND had at least one potential exposure (i.e. sick

poultry) during travel (2 patients)

Close contact (approach within 3 feet) of an ill

patient who was confirmed or suspected to have

H5N1(1 patient)

Worked with live influenza H5N1 virus in a

laboratory

EXPOSURES

Observation for symptoms within 10 days after

possible exposition.

Consultation with a specialist in infectious diseases

at the occurrence of symptoms of the disease.

Laboratory testing of sick persons suspected of avian

influenza to confirm the diagnosis.

Triage of ill patients; Isolation

Evaluate for antiviral prophylaxis.

FOLLOW UP OF EXPOSURES

Ask patients about recent exposure and

contact with humans or animals that may

have had avian influenza A (H5N1) virus

infection

Sampling person(s) meeting these criteria

(Local lab/NRL)

Treating Suspected Patients

Follow up Suspected Patients

Health care activities in outbreaks regions

Health care service

Network - local RHI

- information letter to:

GPs, hospitals, local

laboratories

- detailed sampling

procedure

National Pandemic Influenza

Preparedness Plan

– recover and published in 2013.

Underlies better communication

and better information of the

population in case of a new

influenza pandemic and better

cooperation between different

organizations.

Crisis Communication/ crisis plans

Limit human infections:

Use PPE around sick

animals and humans

Antiviral prophylaxis in

at-risk individuals

Isolate and treat

human cases

Educate the public

Develop preparedness

and response plans

In connection with approved National

Influenza Pandemic Preparedness

Plan NRL received the necessary

equipment and tests for application of

diagnostic tests for influenza viruses.

FOLLOWING THE OUTBREAKS

Several workshops (local/national/international level) were organized focused

on specific topics related to the pandemic influenza threat and avian viruses

• TRIAVNA (22-23.06.2015)- funding by SECID and Ministry of Health

• transferring knowledge among the trained specialists (human and veterinarian)

Training courses/meetings

Lessons/plans

NCIPD established Zoonoses with Epidemic Potential and New Infections

Reference Center to monitor the current epidemiological situation.

involving epidemiologists, microbiologists and veterinary specialists

(exchanging protocols, scientific ideas and allowed the control of Influenza in

Bulgaria.

web information recovery

regularly on the website of the

NCIPD http://grippe.ncipd.org

epidemiological update of human

infection with avian influenza and

other serious cross-border threats to

health

Kits/technical equipment

In the name of science – for the human benefit!

Thank you for the

attention!