DENGUE

Laboratory diagnosis

Two Basic Methods

• Detection of Dengue Virus (Direct Methods)
• Isolation of Virus by Culture
• Detection of Viral Nucleic Acid
• Detection of Viral Antigens (NS1 Ag)
• Detection of Anti Dengue Antibodies (Indirect Methods)
• Serology Ig M
• Serology Ig G

ONSET OF ILLNESS

During first 4 to 5 days, Virus is detected in serum, plasma, Circulating blood cells and tissues Can be detected by Virus isolation, Nucleic acid detection and Antigen detection (NS1 Ag)

After the acute phase...?

• Antibody detection
• Antibody response to infection differs according to the immune status of the host.
• First antibody to appear - IgM
• Secondary antibody - IgG

Primary Dengue

Dengue infection in a person not previously infected with flavivirus nor immunized with a flavivirus vaccine. Primary antibody response (IgM - slow increase).

Detectable in

• 50% of patients - Day 3 to 5
• 80% of patients - Day 5
• 99% of patients - Day 10

IgM levels peak at 2 weeks after the onset of symptoms. Declines to undetectable levels over 2 to 3 months. Anti dengue serum IgG is detectable at low titres at the end of 1st week of illness, increasing slowly thereafter and is still detectable after several months and even for life.

• The physiological definition of a primary infection is a higher molar fraction of Ig M and a lower fraction of Ig G.

Secondary Dengue

Previous infection with a dengue virus/ vaccination with non-dengue flavivirus vaccine. Antibody titres rise rapidly ( IgG). IgG detectable at high levels in the acute stage appearing before or simultaneously with IgM, peaks at 2 weeks after the onset of symptoms and declines over 3-6 months. Early convalescent stage IgM levels are significantly lower in secondary infections than in primary.

• The physiological definition of a secondary case is characterized by a lower molar fraction of IgM and higher molar fraction of IgG.
• To distinguish primary and secondary - IgM/IgG antibody ratios are commonly used.

NS1 antigen ELISA - sensitivity 86% and specificity 100%

VIRUS ISOLATION

Limitations:

• During Viraemia only (early 5 days)
• Slow results (Days) vs. PCR (Hours)
• Expensive to maintain the technique
• Strict storage and transportation needs
• Heat-labile.
• Refrigeration(4-8 degree C) or wet ice for 24 hours Frozen at - 70 deg C, or more, for longer storage
• Never ever freeze in ordinary Freezer (-20C).