How to Make a Definite Diagnosis
Microbiological guidance for taking and handling specimens from individuals at risk of avian influenza
Investigation of cases can be discussed with Dr Maria Zambon (tel 020 8327 6269).
Link to PDF format (PDF, 82 KB)
This guidance is to assist in the decision making process of management and sampling of a possible case of avian influenza in the UK. Avian influenza has been in the news since 2004, with reports of fatal cases from East, South East Asia and Turkey. It is possible that enhanced awareness of avian influenza will lead to clinical enquiries regarding potential of H5N1 infection in travellers returning from affected areas. It should be remembered that the risk of avian to human transmission is very low.
There have been outbreaks of avian flu in the last 20 years with human infections. The outbreaks recorded since 1996 are given in table 1.
Table 1
| Year | Country | Influenza A | Cases | Deaths | Carriers | Human to human transmission | |
| 1996 | UK | H7N7 | 1 | 0 | Ducks | No | |
| 1997 | Hong Kong | H5N1 | 18 | 6 | Chicken, Ducks & Geese | Yes ( extremely limited dead-end transmission) | |
| 1999 | Hong Kong | H9N2 | 2 | 0 | Chicken | Uncertain | |
| 2003 | Hong Kong | H9N2 | 1 | 0 | Chicken | No | |
| 2003 | Hong Kong | H5N1 | 2 | 1 | Chicken | No | |
| 2003 | Netherlands | H7N7 | 89 | 1 | Chicken | Yes* | |
| 2003-2005 | Various | H5N1 | Current numbers are available from the Avian Influenza situation update page. | ||||
*Evidence of conjunctivitis in family members.
(modified from www.europa.eu.int/comm/health/ph_threats/com/Influenza/avian_influenza_en.htm)
This guidance is broadly divided into three sections;
clinical features of avian influenza,
risk assessment of individuals presenting with illness, and
testing, biosafety and transport of specimen.
The section on clinical features lists the signs and symptoms that are recognised to date in human cases from SE Asia from 1997-2004. It is essential to obtain a clear clinical history with appropriate epidemiological information including travel history and contact history. This information allows a risk assessment to be carried out. With clinical information and baseline laboratory tests, a probability of infection can be deduced. Based on the probability of infection, the risk category and appropriate biosafety level for handling specimens can be finalised.
Samples should be tested in the local/regional laboratories for appropriate causes of infection; in particular rapid tests for normal human influenza should be carried out as this is highly likely to be part of the differential diagnosis. If Influenza A is diagnosed in a high risk individual, then the sample may need to be transferred to the Virus Reference Division (VRD), HPA Colindale for further testing.
The probability of avian influenza infection should be considered in the context of appropriate clinical findings, relevant travel and contact history along with abnormal baseline investigations. The information that may be useful in assessing a case is given below.
Clinical Presentation
The exact clinical presentation in avian influenza is still unclear. In a report on 10 cases from Vietnam, the median incubation period is around 3 days (range 2 - 4 days) [1]. The median time to death after onset of symptoms is around 13 days [2]. The available information along with the presentation seen in the Hong Kong outbreak in 1997 is presented in the table 2. The main clinical features that have been reported are listed below.
a. Fever
b. Cough
c. Sore throat
d. Rhinorrhoea
e. Myalgia
f. Conjunctivitis
g. Watery diarrhoea
h. Severe unexplained respiratory illness
If there is no fever, it is highly unlikely that avian influenza infection has occurred.
Case definiton for suspected avian influenza (H5N1) infection.
Clinical Presentation
Fever ( > or = 38 °C) OR histroy of fever
AND respiratory symptoms ( cough or shortness of breath requiring hospitalization.)
AND
Travel and Contact History
- History of travel in the 7 days prior to onset of symptoms, to an area affected by avian influenza A (H5N1) ( see guidelines page) AND close contact (within 1 metre) with live or dead domestic fowl, wild birds or swine in any setting, including bird markets.
OR one of the following: - Close contact ( touching/speaking distance) with other case(s) of servere respiratory illness or unexplained death from the above areas)
- Part of a health care worker cluster of severe unexplained respiratory illness.
- A laboratory worker with potential exposure to influenza A (H5N1).
The list of countries where poultry are currently affected can be obtained from:
or via
The link given below gives a map of affected areas:
Baseline Investigations
The following baseline tests are recommended as they help in the risk assessment of the case.
a. Chest X-ray
b. Total and differential counts (for lymphopenia)
c. Liver function tests
Based on published data, these are highly likely to be abnormal in cases of avian influenza. If laboratory tests and CXR is normal, this is unlikely to be avian influenza
Table 2: Summary of clinical findings in cases of H5 avian influenza:
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Vietnam [1] | Thailand [2] | Hong Kong [3] | |||
| Year | 2004 | 2004 | 1997 | |||
| Number of patients described | 10 (M = 6, F = 4) |
5 (M =4, F = 1) |
12 (M=5, F =7) |
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| Age (Median) | 12.5 | 6.5 | 9 | |||
| Time from exposure to illness (Median) | 3 days | NA | NA | |||
| Time from exposure to illness (Range) | 2-4 days | NA | NA | |||
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% | % | % | ||
| Fever > 38 C | Yes | 100 | Yes | 100 | Yes | 100 |
| Cough | Yes | 100 | Yes | 100 | Yes | 67 |
| Shortness of breath | Yes | 100 | Yes | 100 | NA | |
| Hypoxia ( in those who died) | Yes | >90 | Yes | 100 | NA | |
| Crackles | Yes | 90 | NA | NA | ||
| Chest X-ray changes | Yes* | 100 | Yes | 100 | Yes | 58.3 |
| Sore throat | No | Yes | 80 | Yes | 33.3 | |
| Rhinorrhoea | No | Yes | 40 | Yes | 58.3 | |
| Myalgia | No | Yes | 40 | No | ||
| GIT symptoms (diarrhoea/loose stools, abdominal pain/vomiting) | Yes | 70 | No | Yes | 50 | |
| Conjunctivitis | No | No | No | |||
| Rash | No | No | No | |||
| Lymphopenia | Yes | 100 | Yes | 100 | Yes | 58.3 |
| LFT abnormality | Yes | 100 | Yes | 80 | Yes | 60 |
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(n=6) | (n=4) |
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| Death | Yes | 80 | Yes | 100 | Yes | 41.7 |
| Time to death after onset of illness (Median) | 9 days | 18 days | 7 days | |||
| Time to death after onset of illness (Range) | 6-17 days | 8-29 days | 5 -25 days | |||
* Chest X- ray abnormalities were non - specific and included diffuse, mulifocal or patchy infiltrates. some cases showed segmental or lobular consolidation with air bronchogrammes.
About the risk: should avian influenza be considered?
The next step is to assess the risk of being infected with avian influenza. In the presence of an appropriate travel and contact history, the probability of infection (a weighting given to clinical presentation and baseline investigations) is assessed. Then, the risk category for the individual is assessed. Respiratory sample processing should follow risk assessment.
Probability of Infection:
Clinical presentation and the results of baseline investigations are considered to measure the probability of infection. Table 3 gives the suggested probability of infection:
Table 3: Probability Of Infection
 LOW Increasing probability of infection HIGH |
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Suspect
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Possible
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Probable
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| Clinical Presentation |
Any 2 symptoms from (a - f)
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Suspect g
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Fever Severe respiratory illness or - Any other symptom
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| Baseline Investigations |
Normal
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Borederline
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Abnormal
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Risk Category:
In the presence of a:
? Positive travel history, AND
? Positive contact history,
Consult table 4 to find the risk category:
Table 4: Risk Category:
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Clinical presentation
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| Baseline investigation | Suspect | Possible | Probable |
| Normal | Low | Low | Low |
| Borderline | Low | Low | Medium |
| Abnormal | Low | Low | High |
Data is insufficient on the duration of virus shedding from the current outbreak. Figure 1 illustrates the viral shedding and antibody response to the infection following H5 influenza A infection.
Figure 1:
The most sensitive test is RT-PCR for the detection of viral RNA. Viral culture and antigen detection is useful in the first few days after onset of symptoms. Rapid EIA tests and immunofluorescence (IF) tests are aimed at the nucleoproteins of influenza A, and are capable of detecting avian influenza. Commercial IF reagents available in the UK are capable of detecting H5 influenza, and as DIF is the most commonly used rapid influenza diagnostic test used, it should be part of the investigation of a suspect case of avian influenza. The list of samples required and the tests that can be performed depending on the duration after onset of illness are given in table 5:
Table 5: Tests that can be performed on samples collected at different stages of illness
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Days from onset of illness | ||||||||||
| Detection of: | Tests recommended | Samples | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8-14 | >14 |
| Antigen | Immunofluorescence or Rapid EIA tests* |
NPA BAL ETA NTS |
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| Nucleic Acid | RT - PCR | NPA BAL ETA NTS |
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??? | |
| Virus | Culture | NPA BAL ETA NTS |
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| Sero-response | Neutralisation test | Acute sera | |
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| Neutralisation test | Conval- escent sera |
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NPA- nasopharyngeal aspirate; BAL-Broncheoalveolar lavage; ETA-Endotracheal aspirate; NTS-Nose and Throat swab
*- Directigen Flu A (Becton Dickinson), Quick Vue (Quidel)
If the travel history includes a geographical area where SARS could re-emerge, samples should also include EDTA blood, stool and urine samples in the acute stage. Please see guidance on SARS for further information. Local laboratories should be able to undertake tests for influenza in suspect avian influenza cases, using their influenza testing algorithms with consideration to biosafety ( see below). If influenza A is detected, and it is considered that avian influenza is a serious consideration, specimens can be referred to the Virus Reference Division (VRD) for specialist testing.
Handling specimens from a potentially infected individual:
Highly pathogenic avian influenza (H5N1 / H7N7) has been classified as a Containment Level 3 (CL3) organism by the advisory committee on dangerous pathogens (ACDP). The following guidance is for clinical laboratories - modified from ACDP guidance < http://www.hse.gov.uk/biosafety/diseases/avianflu.htm>
- A clinical risk assessment must be performed ( See above)
- Results of the risk assessment must be communicated to lab staff before work starts on the sample
- All staff handling specimens potentially containing these viruses should be given detailed nformation on hazard and instructions on measures to reduce risk of exposure to the agent
Containment Levels for Processing Clinical Samples:
- Low / Medium risk - CL2
- High risk - CL3
- Known positive - DEFRA Cat 4
(CL3 - either class I or class III biosafety cabinet can be used)
The containment levels and the tests that can be performed depending on the risk category are given in table 6:
Table 6: Containment levels by test and risk category
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Low/Medium risk | High risk |
| Immunofluorescence | CL2 | CL3 |
| Rapid tests | CL2 | CL3 |
| RT - PCR | CL2 | CL3* |
| Culture | CL2 | ** |
* Sample handling prior to inactivation for nucleic acid extraction.
** Wild type avian influenza H5N1 is an ACDP category 3 organism, but a DEFRA category 4 organism. However, DEFRA regulations take precedence on avian influenza work, hence work should only be performed in DEFRA 4 laboratories. If there is a strong suspicion that avian influenza strains (uncharacterised) are present, a high risk specimen should be handled at CL3 prior to confirmation that influenza A is present,
- In a known positive case, if a DEFRA level 4 facility is not available, samples have to be sent to a lab which has the facility. If the containment facility is not available in the regional laboratory, samples have to be sent to the Influenza laboratory at the Virus Reference Division, HPA, Colindale (contact number 020 8200 1295, or contact Dr Maria Zambon 020 8327 6269 for discussion about case).
- Risk of handling specimens for biochemical/hematological tests: virus shedding is primarily in the respiratory tract, and there is no evidence of viraemia or shedding in other body fluids. Hence there is no need for specialist handling of these samples.
Transport of specimens:
All samples should be transported as per UN 602, and instructions are available in http://www.iata.org/whatwedo/cargo/dangerous_goods/infectious_substances.htm
If specimens have to be transported to a regional or reference laboratory, specimens should be transported on wet ice. It is preferable to courier the specimen, after discussion with the receiving laboratory. Serum samples for serology can be sent to VRD through Hays Dx. All samples that are sent to VRD should be marked "For the attention of Dr. Maria Zambon", and should be sent only after telephone discussion with the lab personnel (contact number: 020 8327 6269).
References:
1. Hien TT, Liem NT, Dung NT, et al. Avian Influenza A (H5N1) in 10 Patients in Vietnam. N Engl J Med 2004,350:1179 - 1188.
2. Avian influenza A (H5N1). Wkly Epidemiol Rec 2004,79:65-76.
3. Yuen KY, Chan PK, Peiris M, et al. Clinical features and rapid viral diagnosis of human disease associated with avian influenza A H5N1 virus. Lancet 1998,351:467-471.
Maria Zambon
January 2006
Last reviewed: 28 August 2008