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\nH. influenzae are small, pleomorphic, gram-negative bacilli or coccobacilli with random arrangements. H. influenzae is a fastidious organism which grows best at 35-37\u00b0C with ~5% CO2 (or in a candle-jar) and requires hemin (X factor) and nicotinamide-adenine-dinucleotide (NAD, also known as V factor) for growth. The standard medium used for growth of H. influenzae is a chocolate agar plate (CAP), which can be prepared with heat-lysed horse blood, a good source of both hemin and NAD, although sheep blood can also be used. Growth occurs on a CAP because NAD is released from the blood during the heating process of chocolate agar preparation (the heating process also inactivates growth inhibitors) and hemin is available from non-hemolyzed as well as hemolyzed blood cells. Alternatively, NAD can be included as a component of liquid H. influenzae growth media supplements, (available commercially or prepared in the laboratory), which are incorporated into the chocolate agar. H. influenzae appear as large, round, smooth, convex, colorless-to-grey, opaque colonies on a CAP (Figure 1). Encapsulated strains appear more mucoidal than non-encapsulated strains, which appear as smaller, compact grey colonies. No hemolysis or discoloration of the CAP is apparent. While H. influenzae produce a pungent indol smell, plates should not be opened in order to smell the cultures. H. influenzae cannot grow on an unsupplemented BAP. Prior to identification and characterization testing procedures, isolates should always be inspected for purity of growth and a single colony should be re-streaked, when necessary, to obtain a pure culture. For the following identification and characterization procedures, testing should be performed on 18-24 hour growth from a CAP at 35-37\u00b0C with ~5% CO2 (or in a candle-jar) (Figure 2).
\nThe following tests are recommended to confirm the identity of cultures that morphologically appear to be H. influenzae (Figure 3). H. influenzae can be identified using Kovac\u2019s oxidase test and determining the necessity of hemin and NAD as growth requirements. If the oxidase test is positive, hemin and NAD growth factor requirement testing should be performed. If the growth factor requirement test indicates that the isolate may be H. influenzae, serological tests to identify the serotype should be performed. This sequence of testing is an efficient way to save costly antisera and time. Additional methods for identification and characterization of H. influenzae using molecular tools are described in Chapter 10: PCR Methods and Chapter 12: Molecular Methods.
\nBiosafety Level 2 (BSL-2) practices are required for work involving isolates of H. influenzae, as this organism presents a potential hazard to laboratory personnel and the surrounding working environment. Please refer to Chapter 4: Biosafety in order to follow the guidelines that have been established for laboratorians working in BSL-2 facilities as many of the tests described in this chapter require opening plates with live cultures and are often performed outside of a biosafety cabinet (BSC).
\nFigure 1. H. influenzae colonies on a CAP
\nFigure 2. H. influenzae colonies on a CAP
\nFigure 3. Flow chart for identification and characterization of a H. influenzae isolate...view largerimage icon
\nFilter paper method
\nFigure 4. Kovac\u2019s oxidase test: a negative and positive reaction on filter paper.
\nPlate method
\nOrganism | \nRequirement for hemin (X factor) | \nRequirement for NAD (V factor) | \n?-hemolysis on horse blood agar | \n
---|---|---|---|
H. influenzae | \n+ | \n+ | \n\u2013 | \n
H. parainfluenzae1 | \n\u2013 | \n+ | \n\u2013 | \n
H. haemolyticus | \n+ | \n+ | \n+ | \n
H. parahaemolyticus | \n\u2013 | \n+ | \n+ | \n
H. aphrophilus2 | \n+ | \n\u2013 | \n\u2013 | \n
H. paraphrophilus1,2 | \n\u2013 | \n+ | \n\u2013 | \n
1H. parainfluenzae is ornithine decarboxylase positive, whereas H. paraphrophilus is negative.
2Although their requirements for hemin and NAD factor differ from each other, H. aphrophilus and H. paraphrophilus have recently been reclassified as a single species with a new genus: Aggregatibacter aphrophilus (3).
\nGrowth factor requirement procedure using paper disks and/or strips
\nFigure 5. Identification of hemin (X factor) and NAD (V factor) as growth requirements using paper disks. The top strain is only growing around the disk containing both hemin and NAD (black arrow), and is presumptively identified as H. influenzae.
\nReading the hemin and NAD paper disk and/or strip results
\nReading the Haemophilus ID Quad plate results
\nFigure 6. Growth pattern for H. influenzae on a Haemophilus ID Quad plate
\nQuality control of Quad plates
QC should be performed on each new lot of Quad plates before they are used for unknown isolates to ensure that they will support the proper growth of Haemophilus spp. Three plates from each new lot received should be tested using a well-characterized reference strain of H. influenzae, H. haemolyticus, and H. parahaemolyticus. One uninoculated plate from each new lot should also be tested in order to check for contamination of mold or other organisms in the laboratory and/or incubator. QC should be repeated on plates from a lot if they have been exposed to temperatures above 4\u00b0C or if there is reason to suspect that the plates have been contaminated since the initial QC was performed.
Procedure for quality control of Quad plates
\nReading the quality control test results
\nSAST 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.
\nRating 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
Figure 7. Rating the intensity of the agglutination reaction
\nDetermining the serotype
\nFollow the SAST testing procedure to QC each lot of antisera using all reference strains available in the laboratory. Record the results provided on the example QC sheet in Figure 8.
Reading the quality control test results
Passing test:
\nFailing test:
\n\n | A (strain number) | \n B (strain number) | \n C (strain number) | \n D (strain number) | \n E (strain number) | \n F (strain number) | \n NT (strain number) | \n
---|---|---|---|---|---|---|---|
A Lot# | \n ++++ | \n\u2013 | \n\u2013 | \n\u2013 | \n\u2013 | \n\u2013 | \n\u2013 | \n
B Lot# | \n \u2013 | \n++++ | \n\u2013 | \n\u2013 | \n\u2013 | \n\u2013 | \n\u2013 | \n
C Lot# | \n \u2013 | \n\u2013 | \n++++ | \n\u2013 | \n\u2013 | \n\u2013 | \n\u2013 | \n
D Lot# | \n\u2013 | \n\u2013 | \n\u2013 | \n++++ | \n\u2013 | \n\u2013 | \n\u2013 | \n
E Lot# | \n \u2013 | \n\u2013 | \n\u2013 | \n\u2013 | \n++++ | \n\u2013 | \n\u2013 | \n
F Lot# | \n \u2013 | \n\u2013 | \n\u2013 | \n\u2013 | \n\u2013 | \n++++ | \n\u2013 | \n
Poly Lot# | \n ++++ | \n++++ | \n++++ | \n++++ | \n++++ | \n++++ | \n\u2013 | \n
Saline | \n\u2013 | \n\u2013 | \n\u2013 | \n\u2013 | \n\u2013 | \n\u2013 | \n\u2013 | \n
Figure 8. Example QC sheet for testing antisera against all H. influenzae serotypes
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