Abstract

Haemophilus influenzae (formerly called Pfeiffer's bacillus or Bacillus influenzae) is a Gram-negative, coccobacillary, facultatively anaerobic pathogenic bacterium belonging to the "Pasteurellaceae" family. "H. influenzae" was first described in 1892 by Richard Pfeiffer during an influenza pandemic.

The bacterium was mistakenly considered to be the cause of influenza until 1933 when the viral cause of influenza became apparent, and is still colloquially known as 'bacterial influenza'. "H. influenzae" is responsible for a wide range of localized and invasive infections. This species was the first free-living organism to have its entire genome sequenced.

Serotypes

In 1930, two major categories of "H. influenzae" were defined: the unencapsulated strains and the encapsulated strains. Encapsulated strains were classified on the basis of their distinct capsular antigens. There are six generally recognized types of encapsulated "H. influenzae": a, b, c, d, e, and f.

Genetic diversity among unencapsulated strains is greater than within the encapsulated group. Unencapsulated strains are termed nontypable (NTHi) because they lack capsular serotypes; however, they can be classified by multilocus sequence typing. The pathogenesis of "H. influenzae" infections is not completely understood, although the presence of the capsule in encapsulated type b (Hib), a serotype causing conditions such as epiglottitis, is known to be a major factor in virulence. Their capsule allows them to resist phagocytosis and complement-mediated lysis in the nonimmune host. The unencapsulated strains are almost always less invasive; they can, however, produce an inflammatory response in humans, which can lead to many symptoms. Vaccination with Hib conjugate vaccine is effective in preventing Hib infection, but does not prevent infection with NTHi strains.

Diseases

Most strains of "H. influenzae" are opportunistic pathogens; that is, they usually live in their host without causing disease, but cause problems only when other factors (such as a viral infection, reduced immune function or chronically inflamed tissues, e.g. from allergies) create an opportunity. They infect the host by sticking to the host cell using trimeric autotransporter adhesins.

Naturally acquired disease caused by "H. influenzae" seems to occur in humans only. In infants and young children, "H. influenzae" type b (Hib) causes bacteremia, pneumonia, epiglottitis and acute bacterial meningitis. On occasion, it causes cellulitis, osteomyelitis, and infectious arthritis. It is one cause of neonatal infection.

Due to routine use of the Hib conjugate vaccine in the U.S. since 1990, the incidence of invasive Hib disease has decreased to 1.3/100,000 in children. However, Hib remains a major cause of lower respiratory tract infections in infants and children in developing countries where the vaccine is not widely used. Unencapsulated "H. influenzae" strains are unaffected by the Hib vaccine and cause ear infections (otitis media), eye infections (conjunctivitis), and sinusitis in children, and are associated with pneumonia.

Diagnosis

Clinical features may include initial symptoms of an upper respiratory tract infection mimicking a viral infection, usually associated with fevers, often low-grade. This may progress to the lower respiratory tract in a few days, with features often resembling those of a wheezy bronchitis. Sputum may be difficult to expectorate and is often grey or creamy in color. The cough may persist for weeks without appropriate treatment. Many cases are diagnosed after presenting chest infections do not respond to penicillins or first-generation cephalosporins.

Clinical diagnosis of "H. influenzae" is typically performed by bacterial culture or latex particle agglutinations. Diagnosis is considered confirmed when the organism is isolated from a sterile body site. In this respect, "H. influenzae" cultured from the nasopharyngeal cavity or sputum would not indicate "H. influenzae" disease, because these sites are colonized in disease-free individuals. However, "H. influenzae" isolated from cerebrospinal fluid or blood would indicate "H. influenzae" infection.

Diagnosis | Culture

Bacterial culture of "H. influenzae" is performed on agar plates, the preferable one being chocolate agar, with added X (hemin) and V (nicotinamide adenine dinucleotide) factors at 37 °C in a CO-enriched incubator. Blood agar growth is only achieved as a satellite phenomenon around other bacteria. Colonies of "H. influenzae" appear as convex, smooth, pale, grey or transparent colonies.

Gram-stained and microscopic observation of a specimen of "H. influenzae" will show Gram-negative coccobacillus. The cultured organism can be further characterized using catalase and oxidase tests, both of which should be positive. Further serological testing is necessary to distinguish the capsular polysaccharide and differentiate between "H. influenzae" b and nonencapsulated species.

Although highly specific, bacterial culture of "H. influenzae" lacks in sensitivity. Use of antibiotics prior to sample collection greatly reduces the isolation rate by killing the bacteria before identification is possible. Beyond this, "H. influenzae" is a finicky bacterium to culture, and any modification of culture procedures can greatly reduce isolation rates. Poor quality of laboratories in developing countries has resulted in poor isolation rates of "H. influenzae".

H. influenzae will grow in the hemolytic zone of "Staphylococcus aureus" on blood agar plates; the hemolysis of cells by "S. aureus" releases factor V which is needed for its growth. "H. influenzae" will not grow outside the hemolytic zone of "S. aureus" due to the lack of nutrients such as factor V in these areas. Fildes agar is best for isolation. In Levinthal medium, capsulated strains show distinctive iridescence.

Diagnosis | Latex particle agglutination

The latex particle agglutination test (LAT) is a more sensitive method to detect "H. influenzae" than is culture. Because the method relies on antigen rather than viable bacteria, the results are not disrupted by prior antibiotic use. It also has the added benefit of being much quicker than culture methods. However, antibiotic sensitivity testing is not possible with LAT alone, so a parallel culture is necessary.

Diagnosis | Molecular methods

Polymerase chain reaction (PCR) assays have been proven to be more sensitive than either LAT or culture tests, and highly specific. However, PCR assays have not yet become routine in clinical settings. Countercurrent immunoelectrophoresis has been shown to be an effective research diagnostic method, but has been largely supplanted by PCR.

Interaction with "Streptococcus pneumoniae"

Both "H. influenzae" and "S. pneumoniae" can be found in the upper respiratory system of humans. In an "in vitro" study of competition, "S. pneumoniae" always overpowered "H. influenzae" by attacking it with hydrogen peroxide and stripping off the surface molecules "H. influenzae" needs for survival.

When both bacteria are placed together into a nasal cavity, within 2 weeks, only "H. influenzae" survives. When either is placed separately into a nasal cavity, each one survives. Upon examining the upper respiratory tissue from mice exposed to both bacteria species, an extraordinarily large number of neutrophils (immune cells) was found. In mice exposed to only one of the species, the neutrophils were not present.

Lab tests showed neutrophils exposed to dead "H. influenzae" were more aggressive in attacking "S. pneumoniae" than unexposed neutrophils. Exposure to dead "H. influenzae" had no effect on live "H. influenzae".

Two scenarios may be responsible for this response:

1. When "H. influenzae" is attacked by "S. pneumoniae", it signals the immune system to attack the "S. pneumoniae"

2. The combination of the two species triggers an immune system response that is not set off by either species individually.

It is unclear why "H. influenzae" is not affected by the immune response.

Signs and symptoms

Pneumonia occurs when the lungs become infected, causing inflammation (swelling). Symptoms of pneumonia usually include:

- Fever (but older people may have lower than normal body temperature)

- Cough

- Shortness of breath

- Chills

- Sweating

- Chest pain that comes and goes with breathing

- Headache

- Muscle pain

- Excessive tiredness

- Nails may turn blue from lack of oxygen

Treatment

"Haemophilus influenzae" produces beta-lactamases, and it is also able to modify its penicillin-binding proteins, so it has gained resistance to the penicillin family of antibiotics.

In severe cases, cefotaxime and ceftriaxone delivered directly into the bloodstream are the elected antibiotics, and, for the less severe cases, an association of ampicillin and sulbactam, cephalosporins of the second and third generation, or fluoroquinolones are preferred. (Fluoroquinolone-resistant "Haemophilus influenzae" have been observed.)

Macrolide antibiotics (e.g., clarithromycin) may be used in patients with a history of allergy to beta-lactam antibiotics. Macrolide resistance has also been observed.

Serious complications

The serious complications of HiB are brain damage, hearing loss, and even death.

Prevention

Effective vaccines for "Haemophilus influenzae" Type B have been available since the early 1990s, and is recommended for children under age 5 and asplenic patients. The World Health Organization recommends a pentavalent vaccine, combining vaccines against diphtheria, tetanus, pertussis, hepatitis B and Hib. There is not yet sufficient evidence on how effective this pentavalent vaccine is in relation to the individual vaccines.

Hib vaccines cost about seven times the total cost of vaccines against measles, polio, tuberculosis, diphtheria, tetanus, and pertussis. Consequently, whereas 92% of the populations of developed countries was vaccinated against Hib as of 2003, vaccination coverage was 42% for developing countries, and only 8% for least-developed countries.

The Hib vaccines do not provide cross-protection to any other "Haemophilus influenzae" serotypes like Hia, Hic, Hid, Hie or Hif.

Sequencing

"H. influenzae" was the first free-living organism to have its entire genome sequenced. Completed by Craig Venter and his team at The Institute for Genomic Research - one of the institutes now part of the J. Craig Venter Institute. "Haemophilus" was chosen because one of the project leaders, Nobel laureate Hamilton Smith, had been working on it for decades and was able to provide high-quality DNA libraries. The genome consists of 1,830,140 base pairs of DNA in a single circular chromosome that contains 1740 protein-coding genes, 2 transfer RNA genes, and 18 other RNA genes. The sequencing method used was whole-genome shotgun, which was completed and published in "Science" in 1995.

Likely protective role of transformation

Unencapsulated "H. influenzae" is often observed in the airways of patients with chronic obstructive pulmonary disease (COPD). Neutrophils are also observed in large numbers in sputum from patients with COPD. The neutrophils phagocytize "H. influenzae", thereby activating an oxidative respiratory burst. However instead of killing the bacteria the neutrophils are themselves killed (though such an oxidative burst likely causes DNA damage in the "H. influenzae" cells). The lack of killing of the "H. influenzae" appears to explain the persistence of infection in COPD.

"H. influenzae" mutants defective in the "rec1" gene (a homolog of "recA") are very sensitive to killing by the oxidizing agent hydrogen peroxide. This finding suggests that "rec1" expression is important for "H. influenzae" survival under conditions of oxidative stress. Since it is a homolog of "recA", "rec1" likely plays a key role in recombinational repair of DNA damage. Thus "H. influenzae" may protect its genome against the reactive oxygen species produced by the host’s phagocytic cells through recombinational repair of oxidative DNA damages. Recombinational repair of a damaged site of a chromosome requires, in addition to "rec1", a second homologous undamaged DNA molecule. Individual "H. influenzae" cells are capable of taking up homologous DNA from other cells by the process of transformation. Transformation in "H. influenzae" involves at least 15 gene products, and is likely an adaptation for repairing DNA damages in the resident chromosome (as suggested in Transformation (genetics)#Transformation, as an adaptation for DNA repair).

Vaccines that target unencapsulated "H. influenzae" serotypes are in development.