General Characteristics

The genus Haemophilus contains significant genetic diversity. Members of the genus are small, nontitle, pleomorphic gram-negative bacilli. The cells are typically coccobacilli or short rods. Species of the genus Haemophilus require protoporphyrin IX (a metabolic intermediate of the hemin biosynthetic pathway) referred to as X factor and the V factor, nicotine adenine dinucleotide (NAD) or NADP for in vitro growth. Haemophilus are facultative anaerobes enhanced in a 5% to 7% CO₂-enriched atmosphere. The morphologic and physiologic features of individual species are presented in the discussion of laboratory diagnosis. Aggregatibacter aphrophilus and Aggregatibacter paraphrophilus have been reclassified as a single species based on their multilocus sequence analysis (A. aphrophilus).

Epidemiology

As presented in Table 32-1, except for Haemophilus ducreyi, Haemophilus spp. normally inhabit the upper respiratory tract of humans. Asymptomatic colonization with H. influenzae type b is rare. Although H. ducreyi is only found in humans, the organism is not part of our normal flora, and its presence in clinical specimens indicates infection.

Among H. influenzae strains, there are two broad categories: typeable and nontypeable (NTHi). Strains are typed based on capsular characteristics. The capsule is composed of a sugar-alcohol phosphate (i.e., polyribitol phosphate) complex. Differences in this complex are the basis for separating encapsulated strains into one of six groups: type a, b, c, d, e, or f. H. influenzae type b (Hib) is most commonly encountered in serious infections in humans. Nontypeable strains do not produce a capsule and are most commonly encountered as normal inhabitants of the upper respiratory tract.

Although person-to-person transmission plays a key role in infections caused by Haemophilus influenzae and H. ducreyi, infections caused by other Haemophilus strains and species likely arise endogenously as a person’s own flora gains access to a normally sterile site. The colonizing organism invades the mucosa and enters the patient’s bloodstream. Encapsulated strains are protected from clearance from host phagocytes. Once in the circulation, the organism is able to spread to additional sites and tissues including the lungs, pericardium, pleura, and meninges.

Pathogenesis and Spectrum of Disease

Production of a capsule and factors that mediate bacterial attachment to human epithelial cells are the primary virulence factors associated with Haemophilus spp. In general, infections caused by Haemophilus influenzae are often systemic and life threatening, whereas infections caused by nontypeable (do not have a capsule) strains are usually localized (Table 32-2). The majority of serious infections caused by H. influenzae type b are typically biotypes I and II. The development and use of the conjugate vaccine in children since 1993 has reduced the infection rate by 95% in children younger than 5 years old in the United States.

The majority of H. influenzae infections are now caused by nontypeable strains (NTHi). Transmission is often via respiratory secretions. The organism is able to gain access to sterile sites from colonization in the upper respiratory tract. Clinical infections include otitis media (ear infection), sinusitis, bronchitis, pneumonia, and conjunctivitis. Immunodeficiencies and chronic respiratory problems such as chronic obstructive pulmonary disease may predispose an individual to infection with NTHi.

Chancroid is the sexually transmitted disease caused by H. ducreyi (see Table 32-2). The initial symptom is the development of a painful genital ulcer and inguinal lymphadenopathy. Although small outbreaks of this disease have occurred in the United States, this disease is more common among socioeconomically disadvantaged populations inhabiting tropical environments. Epidemics of disease are associated with poor hygiene, prostitution, drug abuse, and poor socioeconomic conditions.

Haemophilus spp. can be isolated from most clinical specimens. The collection and transport of these specimens are outlined in Table 5-1, with emphasis on the following points. First, Haemophilus spp. are susceptible to drying and temperature extremes. Therefore, specimens suspected of containing these organisms should be inoculated to the appropriate media immediately. Specimens susceptible to contamination with normal flora such as a lower respiratory specimen should be collected by bronchioalveolar lavage. In cases of pneumonia or cerebrospinal fluid (CSF) infection or suspected infection of any other normally sterile body fluid, blood cultures should also be collected.

Second, the recovery of H. ducreyi from genital ulcers requires special processing. The ulcer should be cleaned with sterile gauze moistened with sterile saline. A cotton swab moistened with phosphate-buffered saline is then used to collect material from the base of the ulcer. To maximize the chance for recovering the organism, the swab must be plated to special selective media within 10 minutes of collection.

Direct Detection Methods

Direct Observation

Gram stain is generally used for the direct detection of Haemophilus in clinical material (Figure 32-1). However, in some instances the acridine orange stain (AO; see Chapter 6 for more information on this technique) is used to detect smaller numbers of organisms that may be undetectable by gram staining.

To increase the sensitivity of direct Gram stain examination of body fluid specimens, especially CSF, specimens may be centrifuged (2000 rpm for 10 minutes) and the smear is prepared from the pellet deposited in the bottom of the tube. Most laboratories are now equipped with a cytocentrifuge (10,000 × g for 10 minutes) used for concentration of specimens. This is highly recommended over traditional centrifugation for non-turbid specimens. This concentration step can increase the sensitivity of direct microscopic examination from five to tenfold. Moreover, cytocentrifugation of the specimen, in which clinical material is concentrated by centrifugation directly onto microscope slides, reportedly increases sensitivity of the Gram stain by as much as 100-fold (see Chapter 71 for information on infections of the central nervous system).

Gram stains of the smears from clinical specimens must be examined carefully. Haemophilus spp. stain a pale pink and may be difficult to detect in the pink background of proteinaceous material often found in clinical specimens. Underdecolorization may result in misidentification of H. influenzae as either Streptococcus spp. or Listeria monocytogenes.

H. influenzae appears as pleomorphic coccobacilli or small rods, whereas the cells usually appear as long, slender rods. H. haemolyticus are small coccobacilli or short rods with occasional cells appearing as tangled filaments.

H. parainfluenzae produce either small pleomorphic rods or long filamentous forms, whereas H. parahaemolyticus usually are short to medium-length bacilli. Aggregatibacter aphrophilus is a very short bacillum but occasionally are seen as filamentous forms. H. ducreyi may be either slender or coccobacillary. Traditionally, H. ducreyi cells are described as appearing as “schools of fish.” However, this morphology is rarely seen in clinical specimens.

Table 32-3 presents Haemophilus influenzae and H. parainfluenzae biotypes.

TABLE 32-3

Differentiation of Haemophilus influenzae and H. parainfluenzae Biotypes

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Organism and Biotype	Indole	Ornithine Decarboxylase	Urease
H. influenzae
I	pos	pos	pos
II	pos	pos	neg
III	neg	pos	neg
IV	neg	pos	pos
V	pos	neg	pos
VI	neg	pos	neg
VII	pos	neg	neg
VIII