Group 3:

JIMENEZ, Jenny B.PINEDA, John PatrickLAXA, Kate Charossel

Tourniquet Test (Rumpel Leads Test)

Inflate the blood pressure cuff on the upper arm to a point midway between on the systolic and diastolic pressure for 5 minutes.

Release cuff and make an imaginary 2.5 cm2

or 1 inch just below the cuff, at the antecubital fossa.

Count the number of petechiae inside the box

A test is positive (+) when 20 or more petechiae per 2.5 cm2 or 1 inch per square are observed.

Confirmed diagnosis

A. Culture of the virus

Culture is positive within first 5 days (sensitivity <50%); can also culture from liver at autopsy.

B. Detection of the virus in tissue, serum or CSF by: 

Haemeagglutination Inhibition test > the gold standard for serological

diagnosis. However, because it is labour intensive and requires paired samples for interpretation, this test is now being used mainly for research purposes to differentiate between primary and secondary dengue infections.

Immunofluorescence assay 

Procedures: Direct:

>Add fluorescein-labeled antibody to patient tissue

>Wash and examine under fluorescent microscope.

Indirect:

>Patient serum is added to tissue containing known antigen.

>Wash and examine under fluorescent microscope.

IgM capture Enzyme-Linked Immunosorbent assay (ELISA)

most widely used serological test. The antibody titre is significantly higher in primary infections compared to secondary infections. Once the IgM is detectable, it rises quickly and peaks at about 2 weeks after the onset of symptoms and it wanes to undetectable levels by 60 days.

However in some patients, it may persist for more than 90 days. A positive result thus has to be interpreted and correlated cautiously with the clinical picture. If the dengue IgM test is the only available diagnostic test in the hospital, then establishing a negative early in the illness, and demonstrating a positive serology later will be essential to exclude false negative results.

Procedures: “Sandwich Technique”

Monoclonal or polyclonal antibody adsorbed on solid surface (bead or microtiter plate)

Patient serum added; if antigen is present in the serum, it binds to antibody coated bead or plate.

Excess labeled antibody (antibody conjugate) added; forms antigen-antibody labeled antibody “sandwich” (conjugate directed to another epitope of antigen being tested) 

Continuation of sandwich technique….

Substrate added, incubate and read absorbance.

Washing required between each step.

Direct relationship between absorbance and antigen concentration.

Indirect IgG ELISA test In primary and secondary dengue

infection, dengue IgG was detected in 100% of patients after day 7 of onset of fever.

Therefore, dengue IgG is recommended if dengue IgM is still negative after 7 days in secondary dengue.

Recommendation In order to establish serological

confirmation of dengue illness a seroconversion of dengue IgM needs to be demonstrated. Therefore, a dengue IgM should be taken as soon as the disease is suspected.

Dengue IgM is usually positive after 5-7 days of illness. Therefore, a negative IgM taken before 5-7 of illness does not exclude current dengue infection.

If the dengue IgM is negative before day 7, a repeat sample must be taken in the recovery phase to confirm the diagnosis.

IgM Dot Enzyme ImmunoassayA dengue rapid test that is a

commercially available kit for detection of dengue antibody.

* The yield of rapid tests was shown to be higher when samples were collected later in the convalescent phase of infection, with good specificity and could be used when ELISA facilities were not available. But the result had to be interpreted in the clinical context because of false positive and negative results. It is recommended that the dengue IgM Capture ELISA test could be done after a rapid test, to confirm the status. Serological tests for dengue have been shown to cross react with other flavivirus, non-flavivirus, previous vaccinations and connective tissue diseases.

C. Detection of genomic sequence of virus by RT-PCR (sensitivity >90% early;<10% in 7 days) still a research tool.

Reverse Transcriptase- Polymerase Chain Reaction (RT-PCR)

It is useful for the diagnosis of dengue infection in the early phase (<5 days of illness). It was shown to have a sensitivity of 100% in the first 5 days of disease, but reduced to about 70% by day 6, following the disappearance of the viraemia.

An additional advantage of RT-PCR is the ability to determine dengue serotypes.

Limitations of RT-PCR: It is only available in few centers with

facilities and trained personnel Expensive It requires special storage temperatures

and short transportation, time between collection and extraction.*In view of these limitations, the use of RT-PCR should only be considered for in-patients who present with diagnostic challenges in the early phase of illnesss.

D. WB and neutralization tests can be used to confirm acute infection if necessary.

Neutralization test the most sensitive and specific

method versus constant plaque reduction test.

Similar in principle and result of interpretation of Hemeagglutination inhibition.

Procedures: “Neutralization Reactions”

Antibody-binding > neutralization (antibody neutralizes toxin)

After binding, antibody is not available to react in indicator system.

Results:

>No agglutination or No hemolysis = positive reaction

>Agglutination or hemolysis = negative reaction

(antibody not bound in original reaction and is available to react

with indicator cells)

*Generally, positive control samples used in inhibition or neutralization tests show no reaction and negative control samples show a reaction (opposite of results in direct agglutination testing).

WBC decreased (2000-5000/uL) with toxic granulation of leukocytes and neutropenia; may have marked atypical lymphocytes.

E. Virus isolation the most definitive test for dengue

infection. It can only be performed in the lab equipped with tissue culture and other virus isolation facilities. It is useful only at the early phase of the illness. Generally, blood should be collected before day 5 of illness; e.g. before the formation of neutralizing antibodies.

Virus isolation… During the febrile illness, dengue virus

can be isolated from serum, plasma and leucocytes. It can also be isolated from post mortem specimens.

The monoclonal antibody immunofluorescence test is the method of choice for identification of dengue virus. It may take up to two weeks to complete the test and it is expensive.

F. Non-Structural Protein-1 (NS1 Antigen) a highly conserved glycoprotein that seems

to be essential for virus viability. Secretion of the NS1 protein is a hallmark of

flavivirus infecting mammalian cells and can be found in dengue infection.

This antigen is present in high concentrations in the sera of dengue infected patients during the early phase of the disease.

The detection rate is much better in acute sera of primary infection (75%-97.3%) when compared to the acute sera of secondary infection (60-70%). The sensitivity of NS1 antigen detection drops from day 4-5 of illness onwards and usually becomes undetectable in the convalescence phase.

Brazilian Journal of Infectious DiseasesDengue hemorrhagic fever and acute hepatitis: a case

report

Dengue fever is the world's most important viral hemorrhagic fever disease, the most geographically wide-spread of the arthropod-born viruses, and it causes a wide clinical spectrum of disease. We report a case of dengue hemorrhagic fever complicated by acute hepatitis. The initial picture of classical dengue fever was followed by painful liver enlargement, vomiting, hematemesis, epistaxis and diarrhea. Severe liver injury was detected by laboratory investigation, according to a syndromic surveillance protocol, expressed in a self-limiting pattern and the patient had a complete recovery. The serological tests for hepatitis and yellow fever viruses were negative. MAC-ELISA for dengue was positive.