Annex 1 - Laboratory and clinical diagnosis of dengue fever

An essential aspect of the laboratory diagnosis of dengue is proper collection, storage and shipment of specimens. Health workers should be informed of the appropriate procedures for collecting specimens. Blood samples are drawn from suspected DHF cases promptly after hospital admission or attendance at a clinic, shortly before discharge from the hospital and, if possible, 14-21 days after onset of disease. It is normally important to have an interval of 10 to 14 days between the first two samples to serologically diagnose primary dengue infections. This is not necessary for monoclonal antibody techniques or DOT enzyme immunoassay. An abbreviated case history should accompany each specimen to include the minimum information of name, address, age, sex, date of onset of illness, date of hospitalization and brief clinical findings.

Proof that the outbreak is caused by a dengue virus must be obtained as soon as possible after the first suspected cases have been recognized. Blood specimens can be collected in tubes, vials or on filter-paper discs or strips and sent with clinical data to a specialized laboratory. In addition, blood films should be made and sent to the nearest health centre laboratory to be stained for differential white-blood-cell and platelet estimation. A distinctive clinical laboratory finding is thrombocytopenia with haemoconcentration. Severe cases have a leakage of plasma manifested by a rising haematocrit value. Institutions providing care for DHF patients should have microhaematocrit equipment and microscopes for platelet estimation.

It is essential for health workers making a diagnosis by means of viral isolation to make arrangements with the appropriate virology laboratory prior to the collection of specimens. A factor favouring successful isolation is to take blood samples from patients early in the course of the disease. The virus can also be isolated from vector mosquitos and from autopsy material. Methods to confirm the presence of dengue virus include the inoculation of serum or plasma into mosquitos or mosquito cell cultures. The four different dengue virus serotypes can be identified by using immunofluorescence and type specific monoclonal antibody methods.

The objective of a laboratory-based surveillance system is to provide early and precise information to public health officials on four aspects of increased dengue activity: time, location, virus serotype and disease severity. The system allows early detection of dengue cases and thus improves the capacity of health officials to prevent and control the spread of dengue transmission.

Thrombocytopenia and haemoconcentration

Important laboratory findings are as follows:

Thrombocytopenia (blood platelets equal to or less than 100 000/mm³).

Haemoconcentration - Haematocrit increased by 20% or more of recovery/normal value. This provides evidence of plasma leakage.

The presence of fever, positive tourniquet test, thrombocytopenia, haemoconcentration and a rising haematocrit are sufficient to establish a clinical diagnosis of DHF. Anaemia or severe haemorrhage, pleural effusion (chest X-ray) and/or hypoalbuminemia serve as supporting evidence of plasma leakage.

The haematocrit/haemoglobin ratio has been proposed by some researchers as a useful tool to measure haemoconcentration. Normal figures fluctuate from 2.9 to 3.1; values of 3.2 are suspicious and those of 3.5 or more are considered conclusive for DHF.

Shock with a high haematocrit (except in patients with severe bleeding) and marked thrombocytopaenia support a diagnosis of DHF/DSS.

Thrombocytopenia is established as follows:

In the laboratory a direct count phase contrast microscope may be used per platelet count (normal: 200 000 - 500 000/mm³).

In practice, for outpatients, an approximate count from a peripheral blood smear is acceptable.

Normal persons: 4-10 platelets per oil-immersion field (an average reading from 10 fields is recommended) indicates an adequate platelet count.

Low: 2-3 platelets per oil - immersion field or less (i.e., < 100 000/mm³).

HI test

For the diagnosis of dengue infection, the haemagglutination inhibition (HI) test requires the collection of paired sera 10 to 14 days apart and endpoint titres against the four dengue serotypes to establish a fourfold seroconversion. While still very much the reference test for confirmation of dengue virus infections, the HI test is time consuming and requires careful optimization with each batch of haemagglutinins and erythrocytes used. This is related to the exquisite pH sensitivity of the dengue haemagglutinin binding reaction to the goose erythrocytes which are now commonly used in this assay. The requirement for fresh reagents which may not be stored for too long, makes this a difficult test to use in a decentralized system.

Guidelines for interpretation of results obtained by the HI test have been described by a WHO Technical Advisory Committee on Dengue Haemorrhagic Fever and are reproduced here:

To interpret high-fixed titres, laboratories should establish baseline titres for the local population taken at a time of little or no dengue transmission. Titres more than twice the standard deviation of the geometric mean may be presumed to indicate recent dengue infections.

CF test

The complement-fixation (CF) test may also be used in serological diagnosis wherever facilities for the test exist. This test is less sensitive than the HI test. Blood taken on filter-type is unsuitable for the CF test because it is haemolysed.

The CF test is useful since only anti-dengue IgG fixes complement with dengue antigens. A fourfold rise in CF antibody where the interval between the two sera samples is less than two weeks signifies a secondary seroresponse pattern.

Primary antibody response

The primary antibody response to dengue infection is characterized by slow evolution of HI antibody which is often relatively monotypic, with the absence of CF antibody until at least two weeks after onset of illness. Definitive characterization of primary response is established by demonstrating rising titres of anti-dengue IgM.

In practice, dengue HI antibody titre is generally less than 1:20 in serum obtained before the fourth day after onset of illness. There is a fourfold or greater increase in titre in convalescent specimens (1-4 weeks after onset), with antibody titre not greater than 1:1280.

Secondary antibody response

Secondary antibody response is characterized by a rapid evolution of HI and CF antibody. All antibodies are broadly reactive. Definite characterization of a secondary response is established by demonstrating rising titres of anti-dengue IgG.

Evidence of recent infection

In practice, HI antibody to dengue antigen(s) is less than 1:20 in serum obtained before the fifth day of illness with response equal to or higher than 1:2560 in convalescent serum, or HI antibody at least 1:20 in serum obtained before the fifth day after onset of illness, with rise to ³ 1:2560 in convalescent serum.

Regarding presumptive recent infection, the HI antibody is 1:1280 or greater in acute specimen without fourfold or greater antibody rise in convalescent specimen.

Other tests

Although the HI test has been successfully used for over 30 years for diagnosis of dengue, access to high titre haemagglutinin prepared from suckling mouse brain has been limited mainly to central, reference and research laboratories. This has created a dependence of peripheral health and vector control units upon diagnostic services offered by usually overloaded central laboratory services.

An alternative to the HI test is the DOT enzyme immunoassay. A cell culture is derived from dengue virus antigen dotted onto nitrocellulose membranes. This method is useful in field situations and peripheral areas. The IgM capture ELISA method also has been successfully applied in confirming a diagnosis of dengue infection.

It is important to remember that there are four dengue serotypes which may give rise to a primary dengue infection if a patient is infected for the first time by any one of the four serotypes, or to a secondary dengue infection if the patient has been infected by a different serotype at an earlier time. This complicates both diagnosis and interpretation of results of serological tests not specifically identifying IgM antibodies.

Dengue virus may be propagated in mosquitos. Intrathoracic inoculation of adult mosquitos or intracranial inoculation of Toxorhynchites mosquito larvae have been successfully used to detect the presence of dengue virus in patient sera. The growth of virus in these mosquitos is usually confirmed by detection of viral antigen in head squashes, using immunofluorescent antibody techniques. Mosquito cell lines derived from Aedes albopictus, Aedes pseudoscutellaris and Toxorhynchites may also be used to isolate virus from specimens of patients. The normal method of confirming the presence of virus is by using monoclonal antibodies in the immunofluorescence technique.

CASE INVESTIGATION REPORT ON DENGUE FEVER (DF) AND DENGUE HAEMORRHAGIC FEVER (DHF)

5. CLINICAL AND LABORATORY DIAGNOSIS

Findings:

Tests: