Performing Latex Agglutination Testing and RDT for Meningococcal
Meningitis
Several commercial kits are available for latex agglutination testing.
General recommendations and instructions for the detection of soluble bacterial antigens (capsular
polysaccharide) are provided below, but the manufacturer’s instructions included in the kit
should be followed precisely when using these tests. For best results, the supernatant of the
centrifuged CSF specimen should be tested as soon as possible.
If immediate testing is not possible, the CSF specimen can be refrigerated
(between 2-8°C) for several hours, or frozen at -20°C for longer periods.
It is imperative that the kits be kept refrigerated before use, but never
frozen, especially in tropical climates as the kits deteriorate at high
temperatures which may make the test results unreliable before the
expiration date of the kit.
Latex agglutination procedure for CSF
Follow the manufacturer’s instructions on the package insert for the
specific latex kit being used.
General instructions are listed below:
1. Centrifuge the CSF for 10-15 minutes at 1000 x g and collect
the supernatant. The sediment should be used for Gram stain and primary
culture.
2. Heat the CSF supernatant to be used for the test at 100°C for 3 minutes.
3. Shake the latex reagents gently until homogenous.
4. Place one drop of each latex reagent on a disposable card provided in
the kit or a ringed glass slide.
5. Add 30-50 μl of the supernatant of the CSF to each latex reagent.
6. Rotate by hand for 2-10 minutes. If available, mechanical rotation at
100 rpm is recommended.
Avoid cross-contamination when mixing and
dispensing reagents.
7. Examine the agglutination reactions under a bright light without
magnification.
Reading the latex agglutination results
-Positive reaction: agglutination (or visible clumping) of the latex
particles and slight clearing of the suspension occurs within 2-10 minutes
(Figure 4).
-Negative reaction: the suspension remains homogenous and slightly milky in
appearance.
Rapid diagnostic tests (RDTs)
A. RDT for meningococcal meningitis
RDTs have been developed for direct testing of CSF specimens without prior heat or centrifugation.
The test is based on the principle of vertical flow immunochromatography in which gold particles and nitrocellulose membranes are coated with monoclonal antibodies to capture soluble serogroup-specific polysaccharide antigens in the CSF. The test consists of 2 duplex paper sticks (also called dipsticks), which together enable identification of four serogroups of N. meningitidis (A, C, W135, and Y). RDT1 tests for serogroups A and W135/Y and RDT2 tests for serogroups C and Y.
RDTs can be produced in large quantities, are relatively inexpensive, and remain stable for weeks in hot weather if protected from humidity; therefore, they are practical for immediate testing of specimens obtained during adverse conditions. Initial evaluation using RDTs on stored CSF from patients in Niger showed correct identification of the meningococcal serogroup 97% of the time.
However, more recent studies under field conditions have shown similar specificity, but much lower sensitivity of 70% and, in contrast, similar sensitivity but much lower specificity.
RDT procedure for CSF
Follow the manufacturer’s instructions on the package insert. General instructions are listed below:
1. RDTs should be stored at 4°C in a moisture-proof bag until use.
2. Place the two dipsticks (RDT1 and RDT2) into two separate tubes (3 ml disposable plastic tubes are recommended) of 150-200 μl of CSF or a reference strain suspension in PBS, pH 7.2.
3. Record the chromatographic result on each strip after 10-15 minutes at room temperature (25°C).
Reading the RDT results
-Appearance of red lines on the dipsticks will indicate whether one of the four meningococcal serogroups has been detected in the CSF.
-The upper line on the dipstick is the positive control and should always be present.
-If the CSF is positive for one of the serogroups, a lower red line will also be present. The position of that red line indicates the specific serogroup based on the RDT that was tested.
See Figure 5 for result combinations for each serogroup.
-A negative result consists of a single upper pink control line only.
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