Capacity for detection of avian influenza viruses representing a serious threat to human health (Bulgarian experience)

1. 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

2. Ministry of Health
Regional Health
Inspectorates
Regional Virology
Laboratories -9
(serological
diagnosis )
NCIPD
NRL “Influenza and
ARD”
Department of VirologyDepartment
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

3. 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.

4.  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.

5. 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.

6. 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.

7.  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

8.  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

9.  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

10. 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

11. In the name of science – for the human benefit!
Thank you for the
attention!