Tut 2- Serodiagnosis Of An Acute Viral Illness

Question 1

Which Ig isotypes would you detect anti-dengue antibodies in the blood of an infected person?

Answer 1

IgM and IgG

Question 2

How early (days) can antibodies be detected against dengue after onset of symptoms?

Answer 2

Antibodies against dengue can be detected in most patients five days after onset of symptoms, and IgG can be detected for many years after an infection.

Question 3

What type of RNA virus is Dengue virus?

Answer 3

Flavivirus

Four serotypes of dengue viruses have been described - dengue 1, 2, 3 and 4. Each of the 4 serotypes is capable of causing the full spectrum of clinical manifestations following DENV infection.

Question 4

What is the reservoir for Dengue virus?

Answer 4

Humans; non-human primates such as monkeys maintain the virus in limited forest settings of Asia and Africa.

Larvae develop in artificial water-holding containers close to or inside people’s homes (such as buckets, tyres, pot-plant bases, roof gutters, rainwater tanks).

Question 5

What is the mode of transmission for Dengue virus?

Answer 5

Transmission is via the bite of an infective female mosquito, principally Ae. aegypti. Ae. aegypti is a highly domesticated urban mosquito found in the tropics and subtropics

Question 6

Can Dengue virus be found in Australia?

Answer 6

Yes. In Australia it’s geographical distribution is currently confined to parts of Queensland.

Also close by on Torres Strait islands,

Question 7

Humans are the preferred source of blood meals for which species of Dengue virus and what time of the day/night has increased biting activity?

Answer 7

Ae. aegypti

Day-biting species, with increased biting activity for 2 hours after sunrise and several hours before sunset.

Question 8

Which species of Dengue virus is confined to Torres Strait islands?

Answer 8

Ae. albopictus

Question 9

What are the breeding grounds out in nature where Ae. albopictus can be found?

Answer 9

Ae. albopictus breeds in artificial containers and some naturally occurring sites such as tree holes and coconut shells. Adults prefer to rest in heavily-shaded outdoor sites; and the female takes blood from a range of mammals.

Question 10

What is the incubation period for Dengue virus?

Answer 10

The illness typically starts from 4 to 7 days after a person is bitten by an infected mosquito, but ranges from 3-14 days.

The extrinsic incubation period (EIP) (the incubation period in the mosquito) is from 8-12 days, although shorter EIPs (as low as 5 days) have been reported, leading to explosive outbreaks.

Question 11

What is the infectious period for Dengue virus?

Answer 11

There is no direct person-to-person transmission of dengue (apart from through blood transfusion). A person with dengue can transmit the virus to mosquitoes from shortly before the onset of symptoms (and febrile period) to the cessation of symptoms: usually 3-5 days. However, to reliably trace possible infectiousness to local vectors, a longer duration of viraemia is assumed, from one day before until 12 days after the onset of symptoms in the case. An infected mosquito can transmit dengue until it dies.

Question 12

What clinical presentation and outcome is experienced from infection by Dengue virus?

Answer 12

Infection with DENV can produce a wide clinical spectrum of disease, ranging from a mild febrile illness through to a severe, even fatal condition such as dengue haemorrhagic fever (DHF) or severe dengue. The clinical syndrome experienced can be influenced by both age and immunological status

Typical symptoms of classical dengue include the sudden onset of fever (up to 40°C) accompanied by headache, retro-orbital pain, muscle pains in back and limbs, and rash (erythematous, maculopapular or petechial).

Other symptoms include lethargy, weakness, depression, anorexia, taste aberrations (e.g., an unpleasant metallic taste), sore throat, cough, vomiting, abdominal pain and possibly minor haemorrhagic manifestations such as epistaxis, menorrhagia, haematuria and gingival bleeding.

Hospitalisation may be required depending on signs of severity such as dehydration, bleeding or comorbidities. Hepatitis is a frequent complication. DHF and dengue shock syndrome (DSS) manifest generally as plasma leakage leading to shock and can be fatal, and occur more frequently among children and young adults.

Question 13

Is there treatment for Dengue virus?

Answer 13

There is no specific treatment for dengue, and care is largely supportive. Oral rehydration and analgesia are routinely used. Intravenous rehydration is the therapy of choice for severe cases, and can ensure that the case fatality rate remains below 1% for these severe cases.

Question 14

Does recovery from infection of one DENV serotype provide infection for lifelong immunity against all serotypes?

Answer 14

Recovery from infection with one DENV serotype provides lifelong immunity against that serotype but only short-term protection against other serotypes.

There is increased risk of DHF in secondary dengue virus infections with a different serotype to the primary infection which is thought to be due to differences in immune responses between primary and secondary dengue virus infections.

Question 15

What is the disease occurrence and public health significance for DENV?

Answer 15

There has been a global resurgence of dengue in the last three decades, with an estimated annual average of 96 million clinical cases occurring in recent years.

Approximately 2.5 billion people live in areas at risk for epidemic transmission of dengue, most of these in countries of South East Asia and central and South America.

The global burden of DHF has been estimated at hundreds of thousands of cases each year with case-fatality rates between 1-20% depending on access to effective management. Most fatal cases are children and young adults.

Question 16

What is some travel advice for travellers to endemic countries that should be advised to take precautions to prevent DENV?

Answer 16

• Ensuring hotel (or any other accommodation) rooms are free of mosquitoes by closing window screens, using insecticide sprays indoors, using bed nets if no window screens
• Wearing light coloured, long sleeved clothing in urban or residential areas to minimise skin exposure to day-biting mosquitoes
• Wearing permethrin impregnated fabrics
• Using an appropriate mosquito repellent containing DEET or picaridin on all exposed skin, and applying frequently and thoroughly according to the manufacturer’s recommendations
• Seeking medical advice, as soon as practicable, if they become unwell with a high fever during or soon after travel.

Question 17

What laboratory definitive evidence is performed?

Answer 17

• Isolation of dengue virus
• Detection of dengue virus by nucleic acid testing
• Detection of dengue non-structural protein 1 (NS1) antigen in blood by EIA
• IgG seroconversion or a significant increase in antibody level or a fourfold or greater rise in titre to dengue virus, proven by neutralisation or another specific test
• Detection of dengue virus-specific IgM in cerebrospinal fluid, in the absence of IgM to Murray Valley encephalitis, West Nile virus / Kunjin, or Japanese encephalitis viruses.

Question 18

Wha type of laboratory tests are recommended for symptoms up to 9 days from onset of symptoms?

Answer 18

PCR and/or NS1 (non-structural protein 1) and serology

PCR and NS1 are likely to be negative after 7 days, but detections are possible for longer. Serology is likely to be negative in the first 5 days, but it is often helpful to have the earlier blood to show seroconversion

Question 19

What type of laboratory tests are recommended for symptoms of DENV from day 10 onwards?

Answer 19

Serology. (May need to be repeated)

Question 20

The pattern of DENV virus, antigen and antibody production in secondary infections are different from primary infections. Explain.

Answer 20

In secondary infections the predominant immunoglobulin is IgG, and IgM levels are lower than in primary infections. Both antigen detection and PCR are less sensitive in secondary dengue. For people who have previously been infected with dengue there is a particular need to avoid subsequent infections. Public Health laboratories should note that diagnoses of secondary episodes of dengue can be problematic and laboratories should seek expert advice.

Question 21

What are some advantages for using non-structural protein 1 (NS1) to detect DENV?

Answer 21

Detection of non-structural protein 1 (NS1) can provide a sensitive and specific alternative to PCR and is also useful for dengue diagnosis during the first week of illness (as early as 1 day post-infection) particularly before the development of dengue-specific IgM or IgG.

Due to its specificity, the non-structural antigen (NS1) test can be used to differentiate dengue from other suspected flavivirus infections.

Question 22

What are some disadvantages for using non-structural protein 1 (NS1) to detect DENV?

Answer 22

• As an antigen test it cannot detect past (historical) infections (> 18 days).
• The NS1 antigen is usually less labile and thus less susceptible to suboptimal storage and transport conditions.
• It does not differentiate between dengue serotypes.

Question 23

What is NS1?

Answer 23

NS1 detection is by enzyme immunoassay either in plate assay or on lateral flow immunoassays (rapid antigen tests).

Question 24

What are some advantages for using PCR to diagnose DENV?

Answer 24

PCR can provide a rapid result (within a day of receipt) and allows the infecting serotype to be identified. Its sensitivity is high (80-100%) in detecting virus during the acute phase of the disease

Question 25

What are some disadvantages for using PCR to diagnose DENV?

Answer 25

viral RNA may be affected by transport and storage conditions. Therefore specimens of whole blood, serum, plasma and tissues for PCR need to be refrigerated at 4 to 8oC

Question 26

What are type of serology testing can be used for DENV detection?

Answer 26

Enzyme linked immunosorbent assay (ELISA)

Question 27

What are some advantages for using ELISa for DENV detection?

Answer 27

Enzyme linked immunosorbent assay (ELISA) kits for DENV antibodies can provide a rapid result but are not specific for dengue; a positive result could indicate a cross-reaction or a recent flavivirus infection other than dengue. IgG ELISA is used for detection of recent or past dengue infections in acute and convalescent paired sera but is not specific for dengue and should be interpreted carefully within the context of the clinical illness and exposure history.

Question 28

In Australia, much of the testing of human sera for evidence of DENV infection in state pathology and private diagnostic laboratories involves the use of commercially-available antibody kits. Are these tests sensitive enough to detect/measure IgM and IgG?

Answer 28

Detection of IgM by enzyme immunoassay (EIA) is usually positive if samples are taken 5 days or more after onset of fever, and it persists for several months. Dengue IgG appears shortly afterwards and persists indefinitely. Whilst these kits measure antibodies to DENV, flavivirus IgG is highly cross-reactive, so the tests will also be positive for other flavivirus infections. IgM is more specific, but still shows substantial cross-reactivity with IgM due to other flavivirus infections.

Question 29

What does A significant rise in IgG or seroconversion indicate?

Answer 29

recent infection, but is not specific for dengue unless confirmed by a specific IgG test such as neutralizing antibody titres. Neutralizing titres can also be used to identify the infecting serotype and to distinguish between primary and secondary dengue. In the latter situation the acute serum will contain antibody to the previously infecting serotype, while the convalescent serum will demonstrate a rise in antibody to the currently infecting serotype. Advice on diagnosis of suspected secondary dengue should be sought from diagnostics experts.

Question 30

Why would a GP test for both Dengue and Ziva virus?

Answer 30

- Zika is a virus that is closely related to DENV - Both are spread by mosquitoes with similar transmission cycles - Symptoms are similar including fever, rash, myalgia, and arthralgia.

Question 31

What tests are preferred for suspected DENV or Zika?

Answer 31

For patients with suspected dengue or Zika virus disease, nucleic acid amplification tests (NAATs) are the preferred method of diagnosis. Immunoglobulin M (IgM) antibody testing can identify additional infections and remains an important tool for the diagnosis of these diseases, but interpreting the results is complicated by cross-reactivity, and determining the specific timing of infection can be difficult.

Question 32

When should testing for DENV or Zika be performed?

Answer 32

For symptomatic nonpregnant persons, dengue and Zika virus NAATs should be performed on serum collected ≤7 days after symptom onset. Dengue and Zika virus IgM antibody testing should be performed on NAAT-negative serum specimens or serum collected >7 days after onset of symptoms. For symptomatic pregnant women, serum and urine specimens should be collected as soon as possible within 12 weeks of symptom onset for concurrent dengue and Zika virus NAATs and IgM antibody testing. Positive IgM antibody test results with negative NAAT results should be confirmed by neutralizing antibody tests when clinically or epidemiologically indicated, including for all pregnant women.

Question 33

What does Haemagglutination inhibition test for?

Answer 33

Will detect IgG and IgM

Question 34

What does ELISA test for?

Answer 34

Will detect IgG Detect DENV NS1 antigen (I.e will detect presence of virus)

Question 35

What does Immunofluorescence assay (IFA) detect?

Answer 35

Will detect IgM

Question 36

What is the rational for selecting multiple tests for testing against both DENV and Zika?

Answer 36

Antibody tests are designed to detect anti-DENV or anti-ZIKV antibodies and each test is meant to detect antibody to one or the other virus, not both. However, the relatively high degree of antigenic similarity between DENV and ZIKV – both viruses belong to the Flavivirus family – means that cross-recognition may occur i.e. antibodies induced by infection with DENV may be detected in the anti-ZIKV test, and vice-Versa.

Question 37

Describe how Haemagglutination inhibition (HI) is able to assess antigenic similarity between DENV and Zika.

Answer 37

The HI test assesses the degree of antigenic similarity between the two viruses using a scale based on greater dilutions of antibodies. The HI test is performed using a microtiter plate. The microtiter plate contains rows and columns of wells (i.e., cups) where RBCs, influenza virus and antibodies (developed against a comparison virus, such as a vaccine virus) are mixed. Dilutions are marked across the top of the microtiter plate. These dilutions function as a scale for assessing antigenic similarity and immune response. By testing the ability of greater dilutions of antibody to prevent hemagglutination, scientists measure how well those antibodies recognize and bind to (and therefore inactivate) an influenza virus. The higher the dilution, the fewer antibodies are needed to block hemagglutination and the more antigenically similar the two viruses being compared are to each other. The highest dilution of antibody that results in hemagglutinin inhibition is considered a virus’s HI titer. Higher HI titers are associated with greater antigenic similarity. Greater antigenic similarity suggests that vaccination would produce an immune response against the test virus.

Question 38

What did the results show between Zika antigen and DENV antigen using HI testing?

Answer 38

There is a high degree of antigen similarity between DENV and Zika (they both belong to flavivirus family) and the result showed that both ZIKV and DENV were detected. Results show high cross reactivity

Question 39

What was the result of the anti-ZIKV IgG ELISA test- positive or negative?

Answer 39

Negative

Question 40

What was the result of the anti-DENV IgG ELISA test- positive or negative?

Answer 40

Negative

Question 41

Which test is better to detect primary and secondary DENV infection?

Answer 41

HI testing to be used for (HI) test to distinguish between primary and secondary dengue infections. To determine secondary infection, the IgG ELISA criteria will perform better

Question 42

The Immunofluorescence assay (IFA) to detect IgM was used to detect ZIKV/DENV. What was the result?

Answer 42

Positive IgM result for DENV only

Question 43

What did the DENV NS1 antigen ELISA show?

Answer 43

A positive result.

Question 44

What is Haemagglutination and how is is able to be utilised to detect the presence of virus particles?

Answer 44

Many viruses (for example influenza virus) have an envelope protein on the virion surface called the haemagglutinin, or HA, which binds to the sialic acid, N-acetylneuraminic acid molecules on the surface of red blood cells. When these viruses are mixed with red blood cells, they are interconnected to form a lattice of agglutinated red cells held together by virus particles. This property can be utilised to detect the presence of virus particles.

Question 45

What does agglutination look like in a test well?

Answer 45

Because each virus particle (virion) can bind to multiple red blood cells, a clump of cells will begin to form. To quantify the amount of virus present, serial dilutions of the sample is added to a fixed amount of red blood cells. The higher the dilution at which haemagglutination still occurs, the more virus was present in the original sample. The last dilution that shows complete haemagglutination activity is the haemagglutination (HA) titre.

Question 46

If virus is not detected and there is no evidence of agglutination, how will the red blood cells look in the test well?

Answer 46

If virus is not present the red cells will not form a lattice but instead will sink to the bottom of the test well and form a ‘button’.

Question 47

What do antibodies do in response to infection wth virus against the viral haemagglutinin protein and how is this relevant to HI testing?

Answer 47

Antibodies will prevent haemagglutinin. – and this is the basis of the HI test. In the HI test, antibodies bind to the viral HA molecule and by doing so, prevent the HA from being able to bind to sialic acid on red blood cells.

Question 48

How do you determine the amount of anti-HA antibody in a patients serum sample?

Answer 48

To determine the amount of anti-HA antibody in a patient serum sample, the serum is serially diluted (beginning at 1:2) and then added to the wells of a plate, together with a standard amount virus. The virus/antibody mixture is allowed to incubate for 30-60 minutes, then red blood cells are added to the wells. The test is interpreted 45 minutes later. If anti-HA antibodies are present, the binding of HA to sialic acid on the RBC is blocked. No agglutination lattice is formed, and the indicator red cells fall to the bottom of the well, forming a ‘button’. A ‘button’ therefore indicates the presence of anti-viral antibody. The result is expressed as the greatest serum dilution at which haemagglutination is inhibited.