Nucleic acid detection

RNA is heat-labile and therefore specimens for nucleic acid detection must be
handled and stored according to the procedures described for virus isolation.

4.3.2.1. RT-PCR

Since the 1990s, several reverse transcriptase-polymerase chain reaction (RT-PCR)

assays have been developed. They offer better sensitivity compared to virus isolation
with a much more rapid turnaround time. In situ RT-PCR offers the ability to detect
dengue RNA in paraffin-embedded tissues.

All nucleic acid detection assays involve three basic steps: nucleic acid extraction and
purification, amplification of the nucleic acid, and detection and characterization of
the amplified product. Extraction and purification of viral RNA from the specimen
can be done by traditional liquid phase separation methods (e.g. phenol, chloroform)
but has been gradually replaced by silica-based commercial kits (beads or columns)
that are more reproducible and faster, especially since they can be automated using
robotics systems. Many laboratories utilize a nested RT-PCR assay, using universal
dengue primers targeting the C/prM region of the genome for an initial reverse
transcription and amplification step, followed by a nested PCR amplification that is
serotype-specific (14). A combination of the four serotype-specific oligonucleotide
primers in a single reaction tube (one-step multiplex RT-PCR) is an interesting
alternative to the nested RT-PCR (15). The products of these reactions are separated
by electrophoresis on an agarose gel, and the amplification products are visualized as
bands of different molecular weights in the agarose gel using ethidium bromide dye,
and compared with standard molecular weight markers. In this assay design, dengue
serotypes are identified by the size of their bands.

Compared to virus isolation, the sensitivity of the RT-PCR methods varies from 80%
to 100% and depends on the region of the genome targeted by the primers, the
approach used to amplify or detect the PCR products (e.g. one-step RT-PCR versus
two-step RT-PCR), and the method employed for subtyping (e.g. nested PCR, blot
hybridization with specific DNA probes, restriction site-specific PCR, sequence
analysis, etc.). To avoid false positive results due to non-specific amplification, it is
important to target regions of the genome that are specific to dengue and not
conserved among flavi- or other related viruses. False-positive results may also occur
as a result of contamination by amplicons from previous amplifications. This can be
prevented by physical separation of different steps of the procedure and by adhering
to stringent protocols for decontamination.

4.3.2.2. Real-time RT-PCR

The real-time RT-PCR assay is a one step assay system used to quantitate viral RNA
and using primer pairs and probes that are specific to each dengue serotype. The use
of a fluorescent probe enables the detection of the reaction products in real time, in a
specialized PCR machine, without the need for electrophoresis. Many real-time RT-
PCR assays have been developed employing TaqMan or SYBR Green technologies.
The TaqMan real-time PCR is highly specific due to the sequence-specific
hybridization of the probe. Nevertheless, primers and probes reported in publications
may not be able to detect all dengue virus strains: the sensitivity of the primers and
probes depends on their homology with the targeted gene sequence of the particular
virus analyzed. The SYBR green real-time RT-PCR has the advantage of simplicity in
primer design and uses universal RT-PCR protocols but is theoretically less specific.

Real-time RT-PCR assays are either singleplex (i.e. detecting only one serotype at a
time) or multiplex (i.e. able to identify all four serotypes from a single sample). The
multiplex assays have the advantage that a single reaction can determine all four
serotypes without the potential for introduction of contamination during manipulation
of the sample. However the multiplex real-time RT-PCR assays, although faster, are
currently less sensitive than nested RT-PCR assays. An advantage of this method is
the ability to determine viral titre in a clinical sample, which may be used to study the
pathogenesis of dengue disease (16).

4.3.2.3. Isothermal amplification methods

The NASBA (nucleic acid sequence based amplification) assay is an isothermal

RNA-specific amplification assay that does not require thermal cycling
instrumentation. The initial stage is a reverse transcription in which the single-
stranded RNA target is copied into a double-stranded DNA molecule that serves as a
template for RNA transcription. Detection of the amplified RNA is accomplished
either by electrochemiluminescence or in real-time with fluorescent-labelled
molecular beacon probes. NASBA has been adapted to dengue virus detection with
sensitivity near that of virus isolation in cell cultures and may be a useful method for
studying dengue infections in field studies (17).

Loop mediated amplification methods have also been described but their performance
compared to other nucleic acid amplification methods are not known (18).

4.3.3. Detection of antigens

Until recently, detection of dengue antigens in acute-phase serum was rare in patients
with secondary infections because such patients had pre-existing virus-IgG antibody
immunocomplexes. New developments in ELISA and dot blot assays directed to the
envelop/membrane (E/M) antigen and the non-structural protein 1 (NS1)
demonstrated that high concentrations of these antigens in the form of immune
complexes could be detected in patients with both primary and secondary dengue
infections up to nine days after the onset of illness.

The NS1 glycoprotein is produced by all flaviviruses and is secreted from mammalian
cells. NS1 produces a very strong humoral response. Many studies have been directed
at using the detection of NS1 to make an early diagnosis of dengue virus infection.
Commercial kits for the detection of NS1 antigen are now available, though they do
not differentiate between dengue serotypes. Their performance and utility are
currently being evaluated by laboratories worldwide, including the WHO/TDR/PDVI
laboratory network.

Fluorescent antibody, immunoperoxidase and avidin-biotin enzyme assays allow

detection of dengue virus antigen in acetone-fixed leucocytes and in snap-frozen or
formalin-fixed tissues collected at autopsy.

4.3.4. Serological tests

4.3.4.1. MAC-ELISA

For the IgM antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA)

total IgM in patients' sera is captured by anti- chain specific antibodies (specific to
human IgM) coated onto a microplate. Dengue-specific antigens, from one to four
serotypes (DEN-1, -2, -3, and -4), are bound to the captured anti-dengue IgM
antibodies and are detected by monoclonal or polyclonal dengue antibodies directly or
indirectly conjugated with an enzyme that will transform a non-coloured substrate
into coloured products. The optical density is measured by spectrophotometer.

Serum, blood on filter paper and saliva, but not urine, can be used for detection of
IgM if samples are taken within the appropriate time frame (five days or more after
the onset of fever). Serum specimens may be tested at a single dilution or at multiple
dilutions. Most of the antigens used for this assay are derived from the dengue virus
envelope protein (usually virus-infected cell culture supernatants or suckling mouse
brain preparations). MAC-ELISA has good sensitivity and specificity but only when
used five or more days after the onset of fever. Different commercial kits (ELISA or
rapid tests) are available but have variable sensitivity and specificity. A
WHO/TDR/PDVI laboratory network recently evaluated selected commercial
ELISAs and first-generation rapid diagnostic tests, finding that ELISAs generally
performed better than rapid tests.

Cross-reactivity with other circulating flaviviruses such as Japanese encephalitis, St

Louis encephalitis and yellow fever, does not seem to be a problem but some false
positives were obtained in sera from patients with malaria, leptospirosis and past
dengue infection (10). These limitations have to be taken into account when using the
tests in regions where these pathogens co-circulate. It is recommended that tests be
evaluated against a panel of sera from relevant diseases in a particular region before
being released to the market. It is not possible to use IgM assays to identify dengue
serotypes as these antibodies are broadly cross-reactive even following primary
infections. Recently, some authors have described MAC-ELISA (Figure 4.3) that
could allow serotype determination but further evaluations are required (19).

Figure 4.3

Principle of a MAC-ELISA test.

4.3.4.2. IgG ELISA

The IgG ELISA is used for the detection of recent or past dengue infections (if paired
sera are collected within the correct time frame). This assay uses the same antigens as
the MAC-ELISA. The use of E/M-specific capture IgG ELISA (GAC) allows
detection of IgG antibodies over a period of 10 months after the infection. IgG
antibodies are lifelong as measured by E/M antigen-coated indirect IgG ELISA, but a
fourfold or greater increase in IgG antibodies in acute and convalescent paired sera
can be used to document recent infections. Test results correlate well with the
haemagglutination-inhibition test. An ELISA inhibition method (EIM) to detect IgG
dengue antibodies (20) is also used for the serological diagnosis and surveillance of
dengue cases. This system is based in the competition for the antigen sites by IgG
dengue antibodies in the sample and the conjugated human IgG anti-dengue.

This method can be used to detect IgG antibodies in serum or plasma and filter-paper
stored blood samples and permits identification of a case as a primary or secondary
dengue infection (20,21,22). In general, IgG ELISA lacks specificity within the
flavivirus serocomplex groups. Following viral infections, newly produced antibodies
are less avid than antibodies produced months or years after infection.

Antibody avidity is used in a few laboratories to discriminate primary and secondary

dengue infections. Such tests are not in wide use and are not available commercially.

4.3.4.3. IgM/IgG ratio

A dengue virus E/M protein-specific IgM/IgG ratio can be used to distinguish primary
from secondary dengue virus infections. IgM capture and IgG capture ELISAs are the
most common assays for this purpose. In some laboratories, dengue infection is
defined as primary if the IgM/IgG OD ratio is greater than 1.2 (using patient's sera at
1/100 dilution) or 1.4 (using patient's sera at 1/20 dilutions). The infection is
secondary if the ratio is less than 1.2 or 1.4. This algorithm has also been adopted by
some commercial vendors. However, ratios may vary between laboratories, thus
indicating the need for better standardization of test performance (8).
4.3.4.4. IgA

Positive detection for serum anti-dengue IgA as measured by anti-dengue virus IgA
capture ELISA (AAC-ELISA) often occurs one day after that for IgM. The IgA titre
peaks around day 8 after onset of fever and decreases rapidly until it is undetectable
by day 40. No differences in IgA titres were found by authors between patients with
primary or secondary infections. Even though IgA values are generally lower than
IgM, both in serum and saliva, the two methods could be performed together to help
in interpreting dengue serology (22,23). This approach is not used very often and
requires additional evaluation.

4.3.4.5. Haemagglutination-inhibition test

The haemagglutination-inhibition (HI) test (see Figure 4.4) is based on the ability of
dengue antigens to agglutinate red blood cells (RBC) of ganders or trypsinized human
O RBC. Anti-dengue antibodies in sera can inhibit this agglutination and the potency
of this inhibition is measured in an HI test. Serum samples are treated with acetone or
kaolin to remove non-specific inhibitors of haemagglutination, and then adsorbed
with gander or trypsinized type O human RBC to remove non-specific agglutinins.
Each batch of antigens and RBC is optimized. PH optima of each dengue
haemagglutinin requires the use of multiple different pH buffers for each serotype.
Optimally the HI test requires paired sera obtained upon hospital admission (acute)
and discharge (convalescent) or paired sera with an interval of more than seven days.
The assay does not discriminate between infections by closely related flaviviruses
(e.g. between dengue virus and Japanese encephalitis virus or West Nile virus) nor
between immunoglobulin isotypes. The response to a primary infection is
characterized by the low level of antibodies in the acute-phase serum drawn before
day 5 and a slow elevation of HI antibody titres thereafter. During secondary dengue
infections HI antibody titres rise rapidly, usually exceeding 1:1280. Values below this
are generally observed in convalescent sera from patients with primary responses.

Figure 4.4

Haemagglutination-inhibition assay.

4.3.5. Haematological tests

Platelets and haematocrit values are commonly measured during the acute stages of
dengue infection. These should be performed carefully using standardized protocols,
reagents and equipment.
A drop of the platelet count below 100 000 per L may be observed in dengue fever
but it is a constant feature of dengue haemorrhagic fever. Thrombocytopaenia is
usually observed in the period between day 3 and day 8 following the onset of illness.

Haemoconcentration, as estimated by an increase in haematocrit of 20% or more

compared with convalescent values, is suggestive of hypovolaemia due to vascular
permeability and plasma leakage.