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Updated: Jun 18, 2026
Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction
Published on: February 24, 2014
Determination of capsulation status in Haemophilus influenzae by multiplex polymerase chain reaction
Kevin Lee Nelson1, Arnold Lee Smith
1Center for Childhood Infections Seattle Children's Hospital Research Institute, Seattle, WA 98101, USA.
Abstract:
Since the introduction of Haemophilus influenzae type b conjugate vaccines, there have been concerns regarding the emergence of invasive non-type b strains. Serotyping of H. influenzae with commercially available reagents is subjective. Definitive characterization of the capsulation status can be performed by polymerase chain reaction (PCR) amplification of capsular genes. However, PCR amplification of the conserved export locus in the 2 known phylogenic lines of type b strains and detection of serotype conferring genes in each of the 6 serotypes require multiple assays. To rapidly screen multiple isolates, we devised a multiplex method using 15 primers, which produced a serotype-specific, distinct pattern of amplicons with reference-encapsulated H. influenzae. We applied this technique to a panel of 35 clinical isolates that had been serotyped as type a, c, d, e, or f by slide agglutination; 15 strains lacked capsular genes. Conversely, of 69 invasive isolates that were not serotypeable, all but 11 contained capsule genes. We conclude that this technique will be useful in screening recently isolated H. influenzae for capsulation status.
Insights
A new multiplex polymerase chain reaction (PCR) method rapidly screens Haemophilus influenzae (H. influenzae) capsulation status. This technique aids in identifying invasive non-type b strains, improving public health surveillance.
Area of Science:
- Microbiology
- Molecular Biology
- Public Health
Background:
- Haemophilus influenzae type b (Hib) conjugate vaccines have raised concerns about emerging invasive non-type b strains.
- Current serotyping methods for H. influenzae are subjective and lack definitive characterization.
- Polymerase chain reaction (PCR) offers definitive capsulation status but requires multiple assays for different serotypes.
Purpose of the Study:
- To develop a rapid multiplex PCR assay for simultaneous screening of H. influenzae capsulation status.
- To improve the efficiency of identifying invasive non-type b strains.
- To provide a reliable tool for epidemiological surveillance of H. influenzae.
Main Methods:
- A multiplex PCR assay was designed using 15 primers.
- The assay generated serotype-specific amplicon patterns with reference strains.
- The technique was validated on 35 clinical isolates previously serotyped and 69 non-serotypeable invasive isolates.
Main Results:
- The multiplex PCR assay successfully produced distinct, serotype-specific amplicon patterns.
- Of 35 isolates serotyped by slide agglutination, 15 lacked detectable capsular genes.
- Of 69 non-serotypeable invasive isolates, 58 contained capsule genes, indicating the assay's utility.
Conclusions:
- The developed multiplex PCR technique is effective for rapidly screening H. influenzae capsulation status.
- This method enhances the ability to identify and track invasive non-type b strains.
- The assay is a valuable tool for H. influenzae epidemiological surveillance and public health monitoring.
