Identification of H. influenzae Serotype
Haemophilus influenzae
can be encapsulated with one of six types of antigenically distinct
capsules which can be serotyped using antisera to each capsule (serotypes
a-f). H. influenzae may also be non-encapsulated and such strains that cannot be serotyped are
called H. influenzae nontypeable (NT).
Individual serotype-specific antisera for these major
serotypes are available commercially. A polyvalent antiserum that
recognizes all 6 serotypes is also available commercially.
It is not always practical to test for all serotypes for which antisera are
available in a laboratory. Testing algorithms may be set up in laboratories
with previous knowledge of whether or not a H. influenzae
serotype b (Hib) vaccination program has been implemented within that
particular geographic region. Modifications may be made to the testing
algorithm for any laboratory based on information regarding Hib vaccination status of the region.
It is essential that reference laboratories have the capacity to isolate,
identify, and characterize the serotype of isolates of H. influenzae. This valuable data
provides laboratories and public health authorities with the tools to
identify outbreaks controllable by vaccination campaigns and recognize serotypes causing sporadic disease.
SAST test algorithm for Areas without an Established Hib Vaccination
Program
If a Hib vaccination program has not been implemented in the country or
region from which the isolate originated, it is likely that the H. influenzae isolate is
serotype b and the isolate should first be tested for reactivity to serotype b antisera and a negative saline
control. If the isolate reacts positively with the serotype b antiserum with no agglutination in
saline, the isolate is identified as Hib.
However, if the isolate is non-reactive with the serotype b antiserum, and
if polyvalent antiserum is available, it should be tested with the
polyvalent antiserum. If positive, the isolate should then be tested with
the remaining monovalent antisera (a, c, d, e, and f) to determine the
serotype. If negative for all monovalent antisera and positive for hemin
and NAD growth requirements, then the isolate is considered NT.
SAST test algorithm for areas with an established Hib vaccination
program
If the isolate is from a country or region with an established Hib
vaccination program that has high Hib vaccine coverage, the isolate is
likely to be NT or a serotype other than b. In this case, the isolate should first be tested with the polyvalent antiserum, if
available, and a negative saline control. If positive for the polyvalent antiserum with no agglutination in
the saline, the isolate should then be tested with the remaining monovalent antisera (a, b, c, d,
e, and f) to determine the serotype. If the isolate is negative for the polyvalent antiserum
and/or the monovalent antisera, and requires hemin and NAD for growth, then
the isolate is considered NT.
A. Slide agglutination serotyping (SAST) test for serotyping H. influenzae isolates
Formalin-killed suspensions of H. influenzae should be used for
SAST testing rather than saline suspensions of living organisms to maintain
a safe working environment. A solution of 5% formalinized physiological
saline is sufficient to kill the bacteria. However, formalin is a carcinogen and must be stored and handled with great care. Alternatively,
work should be performed under a biosafety hood if formalin is not used.
Antisera should be stored in the refrigerator at 4°C and warmed to room temperature (25°C) before use. It
must be put back in the refrigerator as soon as testing is finished to prevent the loss of
binding activity of the antibody.
B. Performing the SAST test
1. Grow the isolate(s) to be tested for 18-24 hours on a CAP at 35-37°C
with ~5% CO2 (or in a
candle-jar).
2. Clean a glass slide with alcohol (optional if slides are pre-cleaned).
3. Divide the slide into equal sections (e.g., twelve 11 X 22 mm sections
on a standard 50 X 75
mm slide) with a liquid impermeable pen or a wax pencil.
· Each isolate will require as many sections on the slide as antisera that
will be tested (polyvalent and/or individual serotype-specific) as well as
a saline negative control.
4. In the lower portion of each of the sections of the glass slide
described in step (2), add 10 μl of the 5% formalinized saline with a
micropipettor.
· The instructions specify using a micropipettor with sterilized filtered
tips to measure the 10 ml of the 5% formalinized saline to suspend the
bacteria. The micropipettor will transfer precise and equal measurements
for a proper SAST reaction.
· If a micropipettor and tips are not available, sterile, disposable 10 ml
inoculation loops can be used to transfer 10 ml of the 5% formalinized
saline, but often do not deliver accurate amounts (between 5-10 ml).
5. Use a sterile, disposable 10 ml inoculating loop to collect a few
colonies from the surface of the overnight culture incubated on the CAP.
6. Suspend the bacteria in the 5% formalinized saline solution in the lower
portion of each of the sections of the slide. The suspension should be
moderately opaque Do not allow the cell suspension to dry before adding the
antisera.
· If the bacteria are difficult to suspend directly on the slide, make a
moderately milky suspension (comparable to McFarland 6.0 standard) of the
test culture in a small vial with 250 ml of 5% formalinized saline and briefly vortex the suspension to mix
and break up any pellets. Add 10 ml of this suspension to the lower portion of the slide.
7. In the upper portion of each of the sections of the glass slide
described in step (2), add 10 μl of the polyvalent and/or serotype-specific
antisera to be tested as well as unformalinized saline or phosphate
buffered saline (PBS) for a negative control with a micropipettor.
· DO NOT use the dropper provided with the antisera because it usually
delivers larger amounts than is necessary and can easily be contaminated.
· If a micropipettor and tips are not available, sterile, disposable 10 ml
inoculation loops can be used to transfer 10 ml of the antisera, but often
do not deliver accurate amounts (between 5-10 ml).
· Dispose of the tip or loop used to transfer the antisera to the slide in
a waste container after each use to avoid contamination of the antisera. If
the source of antisera is contaminated, a new vial must be used.
8. Gently tilt the slide to mix the cell suspensions with the antisera in
each section. Continue to gently rock the slide for 1 to 2 minutes to allow
the lower and upper portions to completely blend. Do not use a circular
motion while rocking, as it can cause the sections with different serogroup-specific antisera to run together and contaminate each other.
9. After 2 minutes, examine the SAST reactions under a bright light and
over a black background.
Use the rating system in Figure 7 to determine the
intensity of the agglutination reaction in each section of the slide. Disregard any agglutination that
occurs after the 2 minute time period.
10. Record the SAST results in the laboratory log book.
C. Reading the SAST results
Rating the intensity of the agglutination reaction
Agglutination occurs when the antisera bind to the bacterial cells causing
the cells to agglutinate or clump together, thus making the cell suspension appear clearer. The
intensity of the agglutination reaction may vary according to the density
of the cell suspension or the antisera used. A description on the intensity ratings shown in Figure 7 are listed
below.
4+ All of the cells agglutinate and the cell suspension appears clear
3+ 75% of the cells agglutinate and the cell suspension remains slightly
cloudy
2+ 50% of the cells agglutinate and the cell suspension remains slightly
cloudy
1+ 25% of the cells agglutinate and the cell suspension remains slightly
cloudy
+/- Less than 25% of the cells agglutinate and a fine granular matter
occurs
0 No visible agglutination; the suspension remains cloudy and smooth
Determining the serotype
· A positive result is designated by a 3+ or 4+ (strong agglutination) within 1-2 minutes.
· A negative result is designated by a 0 (saline), +/-, 1+ or 2+ (weak agglutination).
· The serotype is determined when a positive result occurs with the polyvalent antiserum and/or only one of the serotype-specific antisera and not with the saline.
· If a serotype is not determined, the isolate is considered NT. The following result combinations are all reported as NT:
o Agglutination in the saline, regardless of strong reactions with the polyvalent or other serotype-specific antisera, characterizes the culture as autoagglutinating.
o Agglutination with the polyvalent and/or more than one serotype-specific antisera in the absence of agglutination in saline characterizes the culture as polyagglutinating or cross-reactive.
o No agglutination with the polyvalent or any of the serotype-specific antisera or the saline characterizes the strain as non-reactive.
o Although rare, an isolate positive for the polyvalent antiserum, but negative for the serotype-specific antisera is considered NT.
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