Hemophilus influenzae
last authored: Jan 2011, Julia Oliver
last reviewed: Feb 2011, Julie Louwen
Introduction
H. influenzae photomicrograph, courtesy of CDC PHIL
Haemophilus-like organisms were first described in the late 1800's by Koch, but the discovery of Haemophilus influenzae is usually attributed to Pfeiffer in 1892, during the influenza pandemic. In an effort to discover the cause of the worldwide outbreak, sputum isolates and lung tissue of those killed during the pandemic were analyzed. H. influenzae was isolated from these samples, and at the time, was believed to be the primary pathogen causing the illness. The species of the isolate was thus named "influenzae". In 1933 the human influenza virus was discovered to be the causative agent of influenza. It is generally believed that the Haemophilus influenzae found in the pandemic samples was a secondary opportunistic invader.
The Case of Ms Samuel
Ms Samuel brings her 2 month-old son into the clinic. She is concerned about vaccines, and asks you to describe the various pathogens that are vaccinated against.
- What do you know about Haemophilus vaccinations?
- What conditions does vaccination prevent?
Epidemiology
Haemophilus species are typically considered to be normal flora in the upper respiratory tract of humans. They are found in the nasopharynx of approximately 75% of healthy individuals. Normal flora H .influenzae are non-encapsulated. Less than 5% of healthy individuals harbour an encapsulated strain of Haemophilus influenzae, which serotypes as type b.
Classification and Characteristics
Bacteria of the genus Haemophilus are small, non-motile gram negative bacilli and coccobacilli, and are fastidious by nature. They characteristically require certain growth factors, namely heme (X factor) and nicotine adenine dinucleotide (NAD or V factor). These growth requirements are used in the clinical setting to identify suspect organisms to the species level. Haemophilus spp. grow best at a temperature between 35o and 37o Celsius, at a slightly alkaline pH of 7.6. Laboratory growth of H. influenzae is typically achieved in 5% CO2 atmospheric conditions on chocolate blood agar, which provides the previously mentioned X and V factors.
Haemophilus influenzae are typically classified into two groups: typeable and nontypeable. Bacteria which do not contain a capsule are considered nontypeable, while those that do are called typeable. Particular characteristics (proteins) found on the bacterial capsule are identified and bacteria are then grouped according to common characteristics. This mode of classification is called serotyping, and provides a much more specific method of identifying differences between bacteria, even within the same species. To date, there are six groups of Haemophilus influenzae: a, b, c, d, e and f. Type b (Hib) has historically been considered to be the most common cause of bacterial meningitis in young children ages 3 months to 6 years.
Virulence Factors
The encapsulated strains of Haemophilus spp. possess a polysaccharide capsule which prevents phagocytosis by the body’s immune cells, as well as other factors which aid in the attachment to host cells. Unlike other bacteria, the capsule of H. influenzae does not trigger the alternative complement pathway, and the bacteria are thus able to evade the immune system of the host on two levels. The presence of a capsule is directly linked to the virulence of the bacteria. Non-encapsulated strains may possess a variety of cell surface proteins, fimbriae and pili which may aid in host cell attachment.
Transmission and Infection
Transmission of H. influenzae is primarily through person to person contact via respiratory droplets contaminated with the bacteria. As this microorganism is normal flora of the upper respiratory tract, transmission can occur easily and infection can occur in a variety of ways. A person's own endogenous strains could colonize another area of the body, and become pathogenic in that area. Haemophilus influenzae could also become an opportunistic pathogen beginning in the upper respiratory tract in an immunocompromised host.
The use of the Haemophilus influenzae type b vaccine in children has virtually eradicated meningitis caused by the bacteria in many countries, but infections may still be seen in infants too young to be vaccinated or in other unvaccinated children.
Clinical Manifesations
A wide variety of infections and clinical manifestations may be seen with H. influenzae, and their severity varies depending upon whether the bacteria is of the encapsulated or non-encapsulated variety. Clinical manifestation is also dependent on factors such as location of infection, and the immune status of the host.
Non-encapsulated strains may cause the following:
Encapsulated strains may cause the following:
Diagnosis
A patient sample must be acquired. It is important to note that Haemophilus spp. are fastidious organisms and particular attention should therefore be paid to the collection, storage and transport of the specimen. This organism can be very susceptible to changes in environmental conditions such as temperature, humidity and atmosphere. If Haemophilus infection is suspected, care should be taken that the proper conditions are provided during recovery and transport, and that the specimen is cultured for growth as soon as possible.
Specimens should be cultured to chocolate agar plates and incubated in 5-10% CO2 at 35-37oC for 24- 72 hours where they will produce small or medium grey, mucoid (if encapsulated) colonies. A characteristic "mousy" odor is often detected.
Investigations may begin with the appearance of pale Gram negative coccobacilli or bacilli on a direct Gram stain. It should be noted that Haemophilus parainfluenzae may be pleomorphic with long filaments but it may also resemble H. influenzae. Laboratories may use a variety of identification schemes to determine the presence of Haemophilus in a specimen, and many use the fastidious growth requirements of the bacteria as a method of identification. Haemophilus influenzae requires supplementation with both hemin (X) and NAD (V) for growth, while Haemophilus parainfluenzae requires only NAD (V) supplementation.
One identification scheme involves plating the specimen to a blood agar plate and adding a streak line of Staphylococcus aureus, Streptococcus pneumoniae, or Neisseria species. The plate is then incubated at 5% CO2, 35oC, for 16-18 hours. S. aureus (and the other aforementioned microorganisms) creates NAD (V factor) as a metabolic byproduct. Since blood agar plates contain hemin (X factor), a presumptive identification can be made when bacterial colonies are seen satelliting around the S. aureus. A similar effect may be seen using manufactured discs of X, V and XV factors. If identification using discs is to proceed, the specimen should be planted to a Mueller Hinton, or TSA (trypticase soy agar) plate with the entire surface of the plate being inoculated. X, V, and XV factor discs should be applied, leaving a reasonable amount of space between the discs to prevent diffusion of the factors. Plates are incubated in CO2, and examined after 16-18 hours. Colonies which only surround the XV disc can be identified as Haemophilus influenzae, while those surrounding the XV and the V discs are Haemophilus parainfluenzae. A common test for confirmatory identification of Haemophilus influenzae is the porphyrin test. It can detect the presence of porphobilinogen synthase, an enzyme which converts d-aminolevulinic acid (ALA) to heme (X factor). Porphobilinogen and porphyrins are both products in the heme production reaction which can be detected as end products in the test.
Treatment
In the past, ampicillin was an effective treatment for H. influenzae infection, but many strains are now resistant to beta lactam antibiotics. Identification of Haemophilus influenzae therefore warrants testing for the production of beta lactamases. For type b encapsulated strains, cefuroxime is recommended for treatment as it has minimal side effects and has the ability to cross the blood brain barrier. For non-encapsulated strains causing upper respiratory infections, amoxicillin coupled with a beta lactamase inhibitor (e.g. clavulinic acid) is often employed.
Resources and References
Clinical Microbiology II, Class Lecture. NSCC, 2010
Clinical Microbiology II, Medical Bacteriology Laboratory Manual; Prepared by Robert Bethune. NSCC, 2010.
Forbes, B, Sahm, D. and Weissfeld, A. Bailey & Scott's Diagnostic Microbiology, 2007.
Koneman, E., Color Atlas and Textbook of Diagnostic Microbiology, 1992.
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