Monday, August 4, 2008

This would be my second posting about my SIP. Today I will be talking about how do you detect presence of Dengue antibodies in patient’s serum.

Firstly I will discuss about the introduction of Dengue virus. Dengue is a flavivirus, it is found in large areas of the tropics and subtropics. Transmission is by mosquito, principally Aedes aesgypti and Aedes albopictus. Dengue virus infection causes a spectrum of clinical manifestations ranging from symptomatic to fatal hemorrhagic disease. Classic dengue is characterized by sudden onset of fever, intense headache, myalgia, arthralgia and rash. A dysphasic febrile course is common, as it is insomnia and anorexia with bitter or loss of taste. Dengue hemorrhagic fever and dengue shock syndrome are severe complications often associated with secondary dengue infection.

In endemic regions, patients diagnosed with dengue fever generally have secondary infection. Consequently, detection of antibodies to dengue is valuable procedure, particularly in second and subsequent infections where the occurrence of complications is high. Traditionally, haemagglutination-inhibition (HAI) titers have been used to classify infections as primary or secondary. The current definition depends on an assay of paired serum specimens separated in time by at least 7 days, although any acute specimen with an HAI titer ≥ 1:1280 is defined as coming from a patient with seconday flavirus infections.

My company uses this instrument called Panbio Dengue Duo Cassette. It is for the qualitative presumptive detection of IgM and IgG antibodies to dengue virus in human serum. The assay can be used for the presumptive differentiation between primary and secondary infection. This test should only be used for patients with signs and symptoms that are consistent with dengue virus infection. Positive results are presumptive and must be confirmed by virus isolation, paired serum analysis, antigen detection by immunohistochemistry or viral nucleic acid detection for conifrmation of dengue virus infection.

The sensitivity of this assay has been set so that in patients with primary dengue, IgM is positive while IgG is negative. In contrast, patients with secondary infections will have a postitive IgG result with or without postive IgM result.

The principle of this assay, is when present in the patient sample, dengue-specific IgM or IgG antibodies bind to anti-human IgM or IgG antibodies immobilizied in 2 lines across the cassette membrane. Colloidal gold complexes containing recombinant dengue 1-4 antigens are captured by the bound patients IgM or IgG to give visible pink line(s). a procedural control is included to indicate that the assay has been performed correcty.

By Ivan Ng
TG010605070B
12.15pm
4 August 2008 (Mon)

23 comments:

BMT said...

Can you explain more regarding paired-serum analysis?

Andika Putra
TGO1

tg01 group 2 said...

Hi Ivan,

1)What is the difference in the transmission of dengue virus between Aedes aesgypti and Aedes albopictus?

2)"...acute specimen with an HAI titer ≥ 1:1280 is defined as coming from a patient with seconday flavirus infections."

-Why is it from secondary flavirus infections but not primary?

Thankz!

Han Yang
TG01

THE CODEC 5 said...

Hi Ivan,
you mentioned your company uses "Panbio Dengue Duo Cassette". What then does your company use it for? (For example: aim to improve on its mode of action/accuracy etc)

Thanks mate.

Alexander Soo TG02
0608122H

tg01 group 2 said...

To: Andika Putra

paired-serum analysis is a method used to confirm the diagnosis od dengue virus. it is not done in my lab. All samples tested positive for antibodies corresponding to dengue virus will be sent out to hospitals such as SGH for confirmation. paired serum analysis is one of the example. its principle is similar as west blotting.

Reply from Ivan(TG01)

tg01 group 2 said...

To Alexander Soo

Panbio Dengue Duo Cassette is used by my company due to the fact that it can provide high true positive results. In confidence level of 95.5%, the relative serological sensitivity is 99.0% and the relative serological specificity is 98.6%. Furthermore it's reporducibility of reesults is 100%.

Reply from Ivan(TG01)

tg01 group 2 said...

To Han Yang

There is no difference between the transmission of the virus from the 2 insect species.Aedes albopictus is a vector for dengue fever in parts of Asia,chikungunya in Italy, Kerala. While Aedes aesgypti is mainly a vector in Africa and in the ropics worldwide.

because all specimens will be measured for their antibody levels.
Antibodies levels that are quantitated to be more than 1:1280 is considered high. Secondary infections will cause an increase or elvated production of antibodies, because of memory B cells present. Thus it is considered as secondary flavirus infection.

Reply from Ivan(TG01)

tg01 group 2 said...

Hi Ivan

You mention that some symptoms include myalgia and arthralgia.

May I know what are they?

Thank you
Ernest
TG01

tg01 group 2 said...

To Ernest

Myalgia means "muscle pain" and is a symptom of many diseases and disorders.Myalgia without a traumatic history is often due to viral infections.

arthralgia means joint pain.It is a symptom of injury, infection or illnesses.

Reply from Ivan(TG01)

BMT said...

hey ivan

how do you use the panbio dengue duo cassette? is it automated or do you have to programme it before you test the sample?

elyana
tg01
0606676e

THE CODEC 5 said...

Hi!

What is a dysphasic febrile course?
Can you explain? Thanks.

Lyn
TG02

SIP said...

Hi Ivan

When using the panbio dengue duo cassette, do you need to add any reagent(s) before the result can be seen?

Thanks!!

Justina
TG01
0605950E

tg01 group 2 said...

To Justina

It is a requirement to put 2 drops of buffer solution on to a designated place on the panbio dengue duo cassette. This will allow the results to be obtained in 5 mins. If not it may take about 30 mins.

Reply from Ivan(TG01)

tg01 group 2 said...

To Elyana

It is a fully manual equipment. I have to take 2 drops of serum from the patient's blood and put it on a designated position on the panbio dengue duo cassette. Then followed by 2 drops of buffer solution on the panbio dengue duo cassette. Finally wait for 5 mins and read the results.

Reply from Ivan(TG01)

tg01 group 2 said...

To Lyn

Dysplasia is a term used in pathology to refer to an abnormality in maturation of cells within a tissue. Febrile refers to fever which is a frequent medical sign that describes an increase in internal body temperature to levels above normal.

Reply from Ivan(TG01)

MadTechs said...

Hey Ivan,
you mentioned that the sensitivity of the assay has been set so that in patients with primary dengue, IgM is positive while IgG is negative right, how do you even set the sensitivity of the assay?

and how do you perform a procedural control?

much thanks.

Liyanah Zaffre
0607718D

tg01 group 2 said...

Hi Ivan

I would like to ask you what is a flavivirus, meaning what is the characteristics of the virus and why is dengue classified under such a category?

Thanx =)
Johan
TG01
0606637G

De Incredibles said...

Hey, how did the ratio of 1:1280 for secondary flavirus infection come about and what is the ration for the primary infection? Thanks ah. =)

Debbie
TG02

tg01 group 2 said...

To Debbie

The ratio of 1:1280 for secondary flavirus infection is like a reference value determined and developed by the manufacturers. Anything below the ratio of 1:1280 is considered as primary infection

Reply from Ivan(TG01)

tg01 group 2 said...

To Johan

Flavivirus is a genus of the family Flaviviridae. This genus includes the West Nile virus, dengue virus, Tick-borne Encephalitis Virus, Yellow Fever Virus, and several other viruses which may cause encephalitis.Flaviviruses share a common size (40-65 nm), symmetry (enveloped, icosahedral nucleocapsid), nucleic acid (positive-sense, single stranded RNA approximately 10,000-11,000 bases), and appearance in the electron microscope.

These viruses are transmitted by the bite from an infected arthropod (mosquito or tick) Human infections with these viruses are typically incidental, as humans are unable to replicate the virus to high enough titres to reinfect arthropods and thus continue the virus life cycle. The exceptions to this are Yellow fever virus and Dengue viruses which are well adapted to human and are not dependent upon any other hosts.

Reply from Ivan(TG01)

MadTechs said...

Hi Ivan!

i would like to clarify something:

there are gold colloidal complexes fixed in the cassette,

and these complexes contain antigens,

these antigens bind to the antibodies in the patient's sample,

and a buffer solution is added to facilitate optimal binding.

within 5 minutes, a visible pink line will be seen if the result is positive.

so.. is the above correct?

hmm.. then, about the titre, the cassette actually measures the antibody titre too?

thanks =)


Nor Liyana
0607927A

tg01 group 2 said...

To Nor Liyana

Yeah, your above procedure is right. the cassette does not measure the antibody titre. it once it detects there is above 1:1280 titre antibody, it will show a visible pink or red line at IgM which means secondary infection.

Reply from Ivan(TG01)

Ms_chew said...

Hi Ivan

Very good job. Clear and detailed explanation to your peers' question. Keep up the good job.

I have one question. Can the same system test out for the chikungunya infection?

tg01 group 2 said...

To Ms Chew

Panbio dengue duo cassette is only used as a qualitative presumptive detection of IgM and IgG antibodies to dengue virus in human serum.The assay can be used for the presumptive differentiation between primary and secondary infection. This test should only be used for patients with signs and symptoms that are consistent with dengue virus infection. Positive results are presumptive and must be confirmed by other methods listed in the post. Since chikungunya virus causes an illness with symptoms similar to dengue fever, it will give positive results in either primary or secondary infection. However Panbio dengue duo cassette cant differentiate between chikungunya virus and dengue virus. Thus another confirmatory test specific to the detection of the virus is required.

Reply from Ivan(TG01)