Haemophilus

TAXONOMY AND DESCRIPTION OF THE GENUS Back to top

Haemophilus spp. are members of the family Pasteurellaceae (51). While the number

of Haemophilus species described greatly exceeds the number of human pathogens, eight

species affecting humans currently included in this genus are H. influenzae, H. aegyptius, H.

ducreyi, H. pittmaniae, H. parainfluenzae, H. haemolyticus, H. parahaemolyticus, and H.

paraphrohaemolyticus. Aggregatibacter aphrophilus, Aggregatibacter

paraphrophilus, andAggregatibacter segnis were formerly included in the

genus Haemophilus but have recently been reclassified into the

genus Aggregatibacter (formerly Actinobacillus) based on molecular taxonomy (68).

Additionally, A. aphrophilus and A. paraphrophilus have been combined into the single

species A. aphrophilus (68). A description of the characteristics and epidemiology of the

newly reclassified A. aphrophilus species can be found in chapter 33 in this Manual. A

comprehensive review of the taxonomy of Haemophilus species has been provided by Kilian

(50) or can be located in reference 45.

In the absence of the recently reclassified species, significant genetic diversity still exists in

the Haemophilusgenus. The genomes of these species range in size from 1.8 Mb for H.

influenzae to 2.8 Mb for H. ducreyi (45). DNA-DNA hybridization studies demonstrate

significant heterogeneity between species; studies conducted by Burbach et al. as discussed

in reference 45 demonstrate binding ratios between H. influenzae and other species to range

from 10% (H. paracuniculus) to 70% (H. aegyptius). H. influenzae is most closely related

to H. aegyptius, with 90% homology, but is most distant from H. ducreyi, with only 18%

homology. Intraspecies heterogeneity is also significant, ranging from 50 to 100% in H.

influenzae and H. parainfluenzae strains (45).

Members of the Haemophilus genus are small, nonmotile, non-spore-forming, non-acid-fast,

pleomorphic gram-negative bacilli with fastidious growth requirements. Cells in this genus

are coccobacilli or short rods. The cell wall resembles those of other gram-negative bacilli but

contains fewer fatty acids than occur in other members of the Pasteurellaceae (35, 45, 51);

the lipopolysaccharide of Haemophilus is structurally different from those of members of

the Enterobacteriaceae (35, 45, 51). The fatty acid composition of the cell wall includesntetradecanoate

(14:0), 3-hydroxy-tetradecanoate (3-OH-14:0), n-hexadecanoate (16:0),

and hexadecanoate (16:1) (48). Fimbriae have been observed on the cell walls of certain

species of H. influenzaeand H. aegyptius (45). The genome of Haemophilus spp. is

characterized by a G+C content of 37 to 45% (45,51, 83).

Haemophilus spp. are facultatively anaerobic, with requirements for X and/or V factors for

growth. X factor is protoporphyrin IX, a metabolic intermediate in the hemin biosynthetic

pathway (45). V factor is composed of nicotinamide complexed as NAD or NADP. Both factors

are present in erythrocytes (“haemophilus” means “blood loving” in Greek). Requirements

for these compounds vary based on the species, with H. influenzae, H. aegyptius, and H.

haemolyticus requiring both X and V factors for growth, whereas others require only a single

factor (Table 1). Optimal growth occurs at 35 to 37°C in the presence of 5 to 7% CO2. All

species are CAMP reaction negative and produce alkaline phosphatase (46).

Organisms within the Haemophilus genus typically grow on chocolate agar, producing

colonies that are usually smooth, with a flat or convex shape. They are nonpigmented (i.e.,

buff or light tan) or slightly yellow and are 0.5 to 2.0 mm in diameter.

Certain Haemophilus spp. produce beta-hemolysis when grown on sheep blood agar plates

(Table 1). Growth in broth can vary between homogeneous and granular.

Species of Haemophilus, other than H. ducreyi, typically ferment a wide range of different

biochemical substrates. In particular, fermentation of glucose, sucrose, lactose, mannose,

and xylose are useful characteristics in the species identification of organisms in this genus.

Production of indole, ornithine decarboxylase, urease, catalase, and β-galactosidase plus the

ability to produce beta-hemolysis when grown on blood-containing media are other variable

properties of Haemophilus spp. that aid in the species identification of organisms in this

genus (Table 1).

Strains of H. influenzae may produce one of six distinct capsular polysaccharides or may be

nonencapsulated. Nonencapsulated H. influenzae strains are referred to as nontypeable

(NTHi) (33) (Fig. 1). The presence of polysaccharide capsular antigen provides the basis for

serotype designations, a to f. Capsular serotyping is based on the polysaccharide

composition of the capsular structure. Depending on the strain type, the capsule is composed

of ribose and fructose in the furanose ring and Glc, Gal, GlcNAc, or ManANAc in the pyranose

ring. The structures of the capsules belonging to each serotype can be found in reference 45.

Indole, ornithine decarboxylase, and urease production are the basis for a biotyping scheme

with both H. influenzae and H. parainfluenzae (30)

EPIDEMIOLOGY, PATHOGENESIS, AND

TRANSMISSION Back to top

Haemophilus influenzae may be found as part of the commensal bacterial flora of the

mucosal surfaces of the upper respiratory tracts (URT) of many healthy individuals (65).

Asymptomatic colonization of the URT with encapsulated strains of H. influenzae type b (Hib)

is rare, i.e., 2 to 5% of healthy children in the prevaccine era and significantly fewer

(~0.06%) following introduction of the pediatric Hib conjugate antigen vaccine, HIB, in the

early 1980s (61). In contrast, NTHi, together with strains of H. parainfluenzae, represents a

major portion of the cultured bacterial microbiota of the pharynxes and nasopharynxes of

>90% of healthy individuals (52,88). Clones of NTHi present in the URT differ when

asymptomatic carriers are compared to those with infection (76, 88). In asymptomatically

colonized individuals, the clones vary continuously, with a mean duration of carriage of 1 to

2 months (76). However, during infection, a single clonal group predominates.

The incubation period for H. influenzae is poorly understood. The presence of a concomitant

or preceding viral infection can potentiate infection. The colonizing bacteria invade the

mucosa and enter the bloodstream. The antiphagocytic nature of the Hib capsule and the

absence of the anticapsular antibody lead to increasing bacterial proliferation (65). When the

bacterial concentration exceeds a critical level, it can disseminate to various sites, including

the meninges, subcutaneous tissue, joints, pleura, pericardia, and lungs. The presence of

antibody, complement, and phagocytic cells determines the clearance of the bacteremia and

can influence dissemination (65).

Host defenses include activation of the alternate and classic complement pathways and

antibodies to the polyribosylribitol phosphate (PRP) capsule. Antibody to the Hib capsule

plays a primary role in conferring immunity. Newborns have a low risk of infection, likely

because of maternal antibodies acquired through colostrum. When these transplacental

antibodies to the PRP antigen wane, infants are at high risk of developing invasive H

influenzae disease, and their immune responses are low even after the disease (65).

Therefore, they are at high risk of repeat infections since prior episodes of H influenzae do

not confer immunity. By the age of 5 years, most children have naturally acquired

antibodies. The Hib conjugate vaccine induces protection by inducing antibodies against the

PRP capsule.

Colonization of the oral cavity by H. parainfluenzae and H. pittmaniae superior to the palatal

arches is normal.H. parahaemolyticus and H. haemolyticus colonization in healthy individuals

remains rare. Colonization of the cervix with H. ducreyi has been documented following

sexual intercourse.

CLINICAL SIGNIFICANCE Back to top

H. influenzae

Systemic infections caused by H. influenzae, such as meningitis, epiglottitis, orbital cellulitis,

and bacteremia, are usually caused by capsular type b strains and generally fall within

biotypes I and II of this species (69). Life-threatening Haemophilus infections, however,

have fortunately become exceedingly uncommon in developed countries since the

development and introduction of the pediatric HIB vaccine (14, 79, 91). When infections

caused by Hib occur today, it is usually in the setting of an unvaccinated child, although they

may also arise in both children and adults as a result of head trauma or cerebrospinal fluid

(CSF) leak or following a neurosurgical procedure. Biotype IV strains, at least in the pre-HIB

vaccine era, were often found to cause systemic infections in neonates as well as aggressive

infections of the genital tract in postpartum women (75,96).

The vast majority of H. influenzae infections today are caused by NTHi (33, 88). This

organism is an important cause of acute conjunctivitis, acute otitis media, acute maxillary

sinusitis, acute bacterial exacerbation of chronic bronchitis, and pneumonia (65). The

organism gains access to the site of infection by direct contiguous spread from its reservoir

in the URT. Spread via respiratory secretions, usually on the hands of patients, can lead to

conjunctival infection. Antecedent viral infections with resultant inflammation of the

Eustachian tubes and sinus ostea predispose to infection of the middle ear cavity and

maxillary sinuses, respectively, by compromising egress from and ingress to these closed

spaces (66). Establishment of infection in the lungs is facilitated by any condition that

diminishes mucociliary clearance of organisms from the respiratory tree (65,67, 86).

Examples include smoking, chronic obstructive pulmonary disease, viral infection, recurrent

bacterial infection, and physiological alterations, such as those that occur in individuals with

cystic fibrosis (65, 67, 86). Persons at risk for systemic NTHi infection include those with

functional or anatomic asplenism, sickle cell disease, complement deficiencies, Hodgkin’s

disease, congenital or acquired hypogammaglobulinemia, and T-cell immunodeficiency states

(e.g., human immunodeficiency virus infection). Rarely is NTHi documented to be a cause of

bacteremia. This may be due to the relative avirulence of the organism or the inadequacy of

conventional blood culture techniques in propagating this fastidious bacterium.

H. ducreyi

Chancroid is a sexually transmitted disease caused by H. ducreyi which is usually

characterized by the development of a single painful genital ulcer, with associated inguinal

lymphadenopathy occurring 2 to 7 days following exposure (23, 26, 60). Keratinocytes are

likely the first cell type encountered by H. ducreyi upon infection of human skin; thus, the

interaction between H. ducreyi and keratinocytes is likely important in establishing infection

(101). Chancroid occurs most often in developing countries, including much of Asia, Africa,

and Latin America. Epidemics of disease are associated with low socioeconomic status, poor

hygiene, prostitution, and drug abuse, and commercial sex workers are believed to serve as

reservoirs for H. ducreyi.Since 1987, reported cases of chancroid declined steadily until

2001. Since then, the number of cases reported has fluctuated from 17 to 55 cases. In 2003,

only 54 cases were reported to the CDC, with 24 of these cases from South Carolina. More

recently, in 2007, 23 cases of chancroid were reported in the United States, with only eight

states (California, Florida, Louisiana, Massachusetts, New York, North Carolina, Texas, and

Wisconsin) reporting 1 or more cases. Because of difficulties in establishing an etiologic

diagnosis of H. ducreyiinfection and limited resources in many countries of endemicity, the

true incidence of chancroid is unknown.

Other Haemophilus spp.

Haemophilus parainfluenzae remains the predominant species colonizing the URT, accounting

for fully 75% of the Haemophilus biota in the oral cavity and in the pharynx. Interestingly, H.

parainfluenzae does not routinely colonize the nasal cavity. H. parainfluenzae is thought to

account for at least some cases of acute otitis media, acute sinusitis, and acute bacterial

exacerbation of chronic bronchitis, although its role in these diseases is often inconclusive.

Infrequently, it has also been identified as a cause of subacute bacterial endocarditis. As is

the case with systemic infections due to NTHi, blood cultures are often negative in patients

with H. parainfluenzae endocarditis due to the fastidious nature of the pathogen and frequent

lysis of the organism, with high concentrations of sodium polyanethol sulfonate (5, 21, 37).

Haemophilus aegyptius, a distinct species of Haemophilus that closely resembles biotype III

strains of H. influenzae and which has been referred to as the Koch-Weeks bacillus, is an

important cause of acute purulent conjunctivitis (73). This disease, often called pinkeye,

occurs most often in younger children, especially those having extensive contact with other

children in closed settings, such as day care centers and grammar school classrooms. It is

characterized by the rapid onset of conjunctival inflammation, visual disturbance, and ocular

pain and pruritus. It often involves both eyes and is highly transmissible.

Brazilian purpuric fever, a condition that occurs most often in South America, is

characterized by rapid onset of high fevers, hypotension, diffuse cutaneous hemorrhaging,

and abrupt vascular compromise (4). The causative agent is often mistaken to be H.

aegyptius but is instead an organism that is classified in biogroup III of H. influenzae (4, 8).

These strains are characterized by the inability to ferment D-xylose activity, by a particular

pattern of their housekeeping genes, by a particular rRNA restriction pattern, and by

resistance to serum bactericidal activity, making them unique among other H.

influenzae biogroups (45).

Other Haemophilus species have only rarely been implicated as causes of infection in

humans, although lower respiratory tract infection, sinusitis, conjunctivitis, bacteremia,

meningitis, wound infections, peritonitis, arthritis, osteomyelitis, and brain abscess have

been documented in individual case reports or small case series.

SPECIMEN COLLECTION AND TRANSPORT AND

ORGANISM STORAGE Back to top

The collection of specimens for the diagnosis of Haemophilus infections is predicated on the

nature of the infection being evaluated. Details of specimen collection and transport can be

found in chapter 16. In patients suspected of having meningitis, blood and CSF cultures

should be performed. Middle ear fluid obtained by tympanocentesis is the specimen of choice

for patients with otitis media; however, in patients with perforated tympanic membranes and

otorrhea, an aseptically collected aspirate of middle ear fluid from the external auditory canal

is also satisfactory. In cases of maxillary sinusitis, direct sinus aspirates or middle meatal

swab specimens collected under endoscopic guidance should be obtained. Conjunctival swab

specimens are required in the evaluation of patients thought to

have Haemophilus conjunctivitis. In patients suspected of having bronchopulmonary

infections due to Haemophilus spp., specimens representative of lower respiratory tract

secretions should be obtained in such a way as to avoid contamination with oropharyngeal

commensal biota. This means that collection of optimal specimens, such as by

bronchoalveolar lavage or bronchial washing (less preferable) should be performed to

provide optimum specificity when evaluating patients suspected of

having Haemophilus bronchopulmonary infections. While collection of sputum and tracheal

aspirates is less invasive, distinguishing between pathogens and oral biota can be nearly

impossible by this means. When bacterial pneumonia is suspected, blood cultures should also

be obtained. Importantly, with one exception, nasal, nasopharyngeal, and nasal swab

specimens are of no value whatsoever in evaluating patients suspected of

having Haemophilus infections at any of these respiratory tract sites. The one possible

exception is in cystic fibrosis patients experiencing an exacerbation. In this setting, an

induced deep-cough specimen collected on a swab inserted into the posterior pharynx may

be rewarding (80). Inpatients suspected of having Haemophilus infections in normally sterile

sites, such as the pleural space, synovium, pericardium, or peritoneum, fluid aspirated

aseptically from the site of involvement represents the specimen of choice. Concomitant

blood cultures should also be performed.

Finally, specimens for culture of H. ducreyi should be collected from the margins of genital

lesions with a saline- or broth-moistened swab. The swab should be immediately transported

to the laboratory and plated without delay to avoid loss of organism viability. It is imperative

that health care providers inform the laboratory of the clinical suspicion of chancroid so that

appropriate media for culture of H. ducreyi can be employed. If extended transport is

required, swab specimens should be plated directly at the time of collection in the patient

care area or the specimen swab should be placed in transport medium containing hemin

(25). When refrigerated (4°C), the use of Amies transport medium has been demonstrated

to maintain the viability of H. ducreyi for up to 3 days. Alternatively, specially formulated

thioglycolate-hemin-based media containing albumin and glutamine can also be used to

preserve organism viability for transport taking longer than 3 days (25, 100). While optimal

cultivation of H. ducreyi is based on collection of ulcer materials, lymph node aspirates, pus,

and aspirates from buboes can also be submitted for culture, albeit with less sensitivity than

ulcer material. When cultivating H. ducreyi from these specimens, laboratories should

consider allowing clinicians to directly plate specimens to maintain optimal recovery.

Specimens for H. ducreyi nucleic acid amplification techniques should be collected using

standard collection techniques for nucleic acid amplification from genital specimens.

Long-term storage of Haemophilus spp. is usually accomplished by lyophilization or freezing

of isolates at −60°C to −80°C in tryptic soy broth with >10% glycerol or on porous beads

(Pro-Lab Diagnostics, Round Rock, TX).

Antigen Detection

Commercial immunochemical techniques are available for the detection of S. pneumoniae,

Streptococcus dysgalactiae, H. influenzae, and N. meningitidis directly from CSF and other

body fluids. While these techniques provide a rapid identification of the pathogen, they lack

sensitivity and specificity compared to Gram staining (59). Thus, the use of H.

influenzae antigen detection is of limited clinical value, and its use is generally discouraged.

However, in certain clinical contexts, such as in resource-constrained regions when the

prevalence of disease is high and routine culture is unreliable, antigen-based detection

methods may prove useful.

Molecular Techniques

Nucleic acid amplification assays, most notably assays predicated on PCR, have been

developed to detect H. influenzae directly in various clinical specimens, including CSF,

plasma, serum, and whole blood (74, 81). These techniques can be multiplexed to detect

other common bacterial causes of specific infectious disease entities, such as meningitis.

While publications cite variable detection sensitivities of these techniques, specificity is

generally excellent (16, 22, 92).

Studies have demonstrated that the accuracy of clinical diagnosis for chancroid ranges from

33% to 80% (24) and that culture is approximately 75% sensitive (54, 62), making it an

ideal candidate for molecular techniques. Molecular strategies have been developed to

directly detect H. ducreyi from clinical specimens. Primers for these assays have been

designed to amplify sequences from either the H. ducreyi 16S rRNA gene, the rrs (16S)-

rrl (23S) ribosomal intergenic spacer region, an anonymous fragment of cloned H.

ducreyi DNA, or the groEL gene, which encodes the H. ducreyi heat shock protein (54). One

strategy includes a chloroform extraction followed by a one-tube nested PCR directed to the

16S rRNA gene, with longer outer primers for annealing at a higher temperature and shorter

inner primers labeled with biotin and digoxigenin for binding with streptavidin and

colorimetric detection (98). The sensitivity of PCR directly from clinical specimens varies

among assays between 83 and 95% compared to culture or clinical diagnosis (54, 98). The

adaptations of molecular methods offer superior sensitivity for the diagnosis of chancroid;

they are clearly advantageous in areas where the organism is endemic, particularly where

testing by culture is difficult or impossible.

The use of molecular methods for the identification of other Haemophilus spp. directly from

clinical samples has proven difficult. The lack of both sensitivity and specificity has been

problematic. In clinical specimens, small numbers of organisms may be present, leading to

limitations in detection sensitivity. That is especially the case in patients

with Haemophilus bacteremia (9). To achieve adequate sensitivity, large volumes of blood or

CSF must be processed to achieve adequate sensitivity, creating laborious nucleic acid

extraction and concentration processes with little clinical relevance. Further complicating the

detection of Haemophilus from blood and CSF is the detection of agents following initiation of

therapy. While culture frequently becomes negative following administration of the first

appropriate dose of antimicrobials, patients can retain bacterial DNA in their blood or CSF for

at least 2 weeks following clearance of the organism, leading to false-positive reactions,

often making the interpretation of results challenging.

In certain other specimens, particularly specimens from the respiratory tract, commensal

strains ofHaemophilus spp. are often present, rendering positive results inconclusive.

Further, the presence of other commensals or use of antimicrobials prior to screening may

result in false-positive reactions, thus contributing to a lack of assay specificity. For these

reasons, the use of molecular detection techniques is not currently advocated for the

detection of Haemophilus spp. directly in clinical specimens until clinically significant

thresholds for molecular quantification of organisms from respiratory specimens are

achieved.

ISOLATION Back to top

Media

Optimum recovery of Haemophilus spp. in culture requires the use of enriched media that

support the growth of these fastidious bacteria. Media must contain at least 10 μg/ml of free

X and V factors. High concentrations of both the X and the V factor are found in whole blood,

most of it sequestered within erythrocytes. X factor is protoporphyrin IX and can be derived

from whole blood or can be added to bacteriological media using crystalline hemin; X factor

is readily available in traditional blood agar. V factor is composed of nicotinamide complexed

as NAD or NADP and is also readily available in blood. However, nicotinamide is not readily

bioavailable because of its intracellular location and the presence of NAD-glycohydrolase

enzymes in the blood. For the growth of Haemophilus spp. on solid media, crystalline hemin

and NAD must be added to a final concentration of 10 μg/ml, or the blood used in the

medium must be heated such that the red cells lyse and release free X factor and V factor

into the medium. The latter can be accomplished by adding blood to the basal medium as it

cools to 80°C after being autoclaved. This is referred to as “chocolatizing” blood. For optimal

growth of Haemophilus spp., a concentration of 5% chocolatized sheep blood should be

employed (51).

The optimum growth of Haemophilus spp., especially of more-fastidious species, such as H.

ducreyi and H. aegyptius, requires, in addition to the X and V factors, supplementation of

media with various other growth factors. Two commercially available supplements that

supply these growth factor requirements are IsoVitaleX (BD) and Vitox (Remel). These

compounds contain glucose, cystine, glutamine, adenine, thiamine, vitamin B12, guanine,

iron, and aminobenzoic acid and provide adequate supplementation for the growth of H.

ducreyi andH. aegyptius.

Enriched chocolate agar containing 5% lysed sheep red blood cells and supplemented with

1% IsoVitaleX or 1% Vitox represents one general-purpose medium that is commonly used

in clinical laboratories to effectively propagate Haemophilus spp. (Fig. 3). Another medium

that reliably supports the growth of Haemophilus spp. is Levinthal’s medium (58). Because it

is transparent, Levinthal’s medium offers the added benefit of permitting the detection of

colony iridescence, a property that is frequently associated with encapsulation (Fig. 4)

(12,72). One recent investigation found that a medium consisting of GC (BD or Remel) agar

base, 5% heated sheep red blood cells, and 1% yeast autolysate provided the best growth of

all Haemophilus spp. other thanH. ducreyi (77).

The use of selective media is also helpful in recovering H. ducreyi from genital tract

specimens (100). Selective media may include any of the following: GC agar base with 1%

IsoVitaleX, 5% fetal bovine serum, 1% hemoglobin, and 3 μg of vancomycin; GC agar base

with 5% Fildes enrichment, 5% horse blood, and 3 μg of vancomycin; 5% fresh rabbit blood

agar with 3 μg of vancomycin; or Mueller-Hinton agar with 5% chocolatized horse blood, 1%

IsoVitaleX, and 3 μg of vancomycin (100). Preferably, two different selective media are

employed (100).

Growth of Haemophilus spp. may also be achieved on 5% sheep blood agar by use of the

microsatellite phenomenon. With the microsatellite test, a single streak line of hemolysinproducing

Staphylococcus spp. is placed on an agar surface previously inoculated with a

specimen suspected of containing Haemophilus spp. The hemolysin produced by

the Staphylococcus species lyses the erythrocytes immediately adjacent to the streak line in

the medium, releasing sufficient concentrations of X factor (hemin) and V factor (NAD) into

the medium to supply the growth factor requirements

of Haemophilus spp. Staphylococcus also secretes NAD into the medium in proximity to the

streak line. Colonies of Haemophilus thus appear in a narrow zone adjacent to the

staphylococcal streak. This is referred to as “satelliting” growth (Fig. 6). Organisms other

than staphylococci can also produce the satellite phenomenon with Haemophilus, e.g.,

enterococci and yeast.

Although not a common cause of bacteremia, special techniques are not necessary for the

recovery ofHaemophilus spp. from blood specimens with modern, continuously monitoring

blood culture systems (36, 48). The broth medium used in such systems supports the growth

of Haemophilus spp. because the blood specimen itself supplies adequate concentrations of

both the X and V factors when the erythrocytes present in the specimen lyse as they come

into contact with the blood culture broth. However, the common practice of using such

systems for the culture of normally sterile body fluids, e.g., synovial, peritoneal, pericardial,

and pleural fluid, may be problematic insofar as these specimens may not contain sufficient

amounts of blood to supply the necessary levels of the X and V factors to support the growth

of Haemophilus spp. In situations where Haemophilus spp. is strongly suspected in such

specimens, blood culture bottles should be supplemented with at least 10 μg of both sterile

hemin and NAD/ml prior to inoculation with clinical specimens.

Following inoculation, solid media should be incubated at 35 to 37°C in a moist atmosphere

and in the presence of 5 to 7% CO2. Under these conditions, most Haemophilus spp. grow

within 24 to 48 h. When specimens for H. ducreyi and H. aegyptius are cultured, incubation

may be necessary for up to 5 days to allow sufficient time for the growth of these fastidious

organisms. Further, when technologists attempt to propagateH. ducreyi, plates should be

incubated at slightly lower temperatures, i.e., 30 to 33°C in 5% CO2 in a high-moisture

environment. Use of lower incubation temperatures will improve the recovery of H. ducreyi in

comparison to incubation temperatures of 35 to 37°C.

Colony Appearance

Colonies of Haemophilus spp. on suitable solid media, in general, are nonpigmented or

slightly yellow and flat to convex, and they have a diameter of 0.5 to 2 mm after 48 h of

incubation. Certain species of Haemophilusproduce beta-hemolysis (Table 1).

Colonies of H. influenzae on chocolate agar are smooth, low, convex, grayish, and

translucent. Encapsulated strains often have a mucoid appearance, while nonencapsulated

strains produce smaller, buff colonies (Fig. 3). Most strains of H. influenzae produce indole,

emitting a strong amine-like odor. Non-indole-producing strains emit a “mousy” odor.

Colonies are 1 to 2 mm in diameter and often grow within 24 h. Colonies grown on clear

media, such as Levinthal’s agar, demonstrate iridescence under obliquely transmitted light

(12, 72). Iridescence is most conspicuous with young colonies and disappears with age.

Iridescent colors may include yellow, red, green, or blue. Iridescence is more apparent with

capsular type b strains; nonencapsulated strains typically demonstrate a blue-green color

(Fig. 4).

Colonies of H. aegyptius reach a colony size of only ca. 0.5 mm after 48 h of growth.

Colonies are low, convex, and translucent, with a smooth, entire surface. On semisolid

media, “comet-like” colonies are produced.

Colonies of H. parainfluenzae are typically off-white to yellow and, like H. influenzae, 1 to 2

mm in diameter after 24 h of growth. The colony appearance is extremely varied, i.e., flat

and smooth, granular with serrated edges, or heaped up and wrinkled. Colonies exhibiting

the last morphology may be slid intact across the surface of the agar. The colony

morphology of H. parainfluenzae may change as the colonies age.

Colonies of H. haemolyticus are translucent, smooth, and convex and do not form satellites

aroundStaphylococcus. Colonies usually achieve a diameter of 0.5 to 1.5 mm after 24 h, with

a clear zone of beta-hemolysis surrounding each colony when the organism is grown on

blood agar; H. haemolyticus can lose its ability to cause hemolysis following serial subculture

on bacteriological media. The growth properties and colony morphology of H.

parahaemolyticus and H. paraphrohaemolyticus are similar to those of H. haemolyticus.

H. ducreyi grows poorly, regardless of the medium used, and frequently 3 to 5 days will pass

before growth appears. Colonies growing on chocolate agar are small, flat, gray, and

smooth. Larger colonies may be interspersed among small colonies but have the same

morphology. Growth on blood agar is poor, with a slight beta-hemolysis surrounding the

colonies. As with H. parainfluenzae, older colonies of H. ducreyi are cohesive and can be slid

across the agar.

IDENTIFICATION Back to top

The identification and differentiation of Haemophilus spp. are achieved through

determination of X and V factor requirements for growth, performance of the porphyrin test,

assessment of hemolysis, determination of carbohydrate fermentation patterns, and

production of indole, ornithine decarboxylase, urease, catalase, and β-galactosidase (Table

1). The pattern of X and V growth factor requirements and the porphyrin test provide

sufficient information for the presumptive species identification of selected Haemophilus spp.

Definitive species identification, however, requires assessment of the other phenotypic

characteristics listed above (Table 1). Alternatively, sequencing also provides excellent

identification of these organisms.

X and V Factor Growth Requirements

X and V factor requirements for the growth of Haemophilus spp. may be determined by swab

inoculation of a suspension of test organism equivalent in turbidity to a 0.5 McFarland

standard across the entire surface of a 100-mm petri dish containing tryptic soy agar. Filter

paper disks or strips impregnated with X factor, V factor, and the X and V factors (Remel or

BD) are then placed on the agar surface, and the plate is incubated for 20 to 24 h at 35°C in

an atmosphere of 5 to 7% CO2. The pattern of satellite growth around individual disks or

strips, in the absence of growth elsewhere on the plate, is used to define the growth factor

requirements of the test strain (Fig. 7). Tryptic soy agar is the preferred medium for use

when the X and V growth factor requirements for Haemophilus spp. are determined, as other

media may yield erroneous results (31, 34). Alternatively, tri-plates (Haemophilus ID II;

Remel) and quad-plates (Haemophilus ID quad; Remel) can be used to assess X and V

growth factor requirements. When performing X and V factor studies, care should be taken

to avoid carrying X factor along with the inoculum. This can result in erroneous identification

of H. influenzae as H. parainfluenzae.

that the test organism is X factor independent; when negative, the porphyrin test indicates

that the organism requires X factor. Since the vast majority of X-factorrequiring

Haemophilus spp. recovered in the clinical laboratory are H. influenzae, when the

porphyrin test is performed with a clinical isolate and found to be negative, it can be inferred

with a high likelihood that the organism is H. influenzae.

The porphyrin test is performed using commercially available ALA disks (Remel) or through

preparation of liquid porphyrin medium (49). To prepare liquid porphyrin medium, 2 mM ALA

and 0.8 mM MgSO4 in 0.1 M phosphate buffer (pH 6.9) are aliquoted in glass tubes with 0.5

ml of porphyrin medium in each tube. Tubes can be stored at 4°C for several months or for

years at -20°C. Tubes are inoculated with a loopful of freshly grown bacteria and incubated

for 4 h at 35°C in ambient air (51). Following incubation, the tubes are examined for brickred

fluorescence with a device, such as a Wood’s lamp, that emits a 360-nm-long-wave UV

light (Fig. 8). Tubes with questionable results may be reincubated for up to 24 h.

Alternatively, Kovacs’ reagent (0.5 ml) can be added to the liquid porphyrin medium, and the

tube can be shaken and observed for a red color in the lower water phase (51). A negativecontrol

tube lacking ALA should also be inoculated when the porphyrin test is performed to

rule out false-positive reactions due to the presence of indole. The porphyrin test has been

shown in several studies to outperform growth-factor-based methods for differentiation of H.

influenzae from non-H. influenzae species (63).

Commercial Biochemical Identification Systems

Several commercial identification systems have been developed to identify Haemophilus spp.

These systems employ a battery of conventional biochemical tests, frequently in a

miniaturized form, with results available in shorter time periods than with conventional

biochemical tests. The performance characteristics and identification accuracy of these

commercial systems are extremely variable (31, 63, 78, 94). The RapID NH system (Remel)

contains 11 biochemical reactions in a microwell tray. The reactions used for identification

ofHaemophilus include the production of urease, indole, ornithine decarboxylase, proline, and

gamma-glutamyl aminopeptidase, resazurin reduction, glucose and sucrose utilization,

nitrate reduction, and phosphate hydrolysis. The kit uses phosphate hydrolysis and nitrate

reduction reactions to identify an isolate as belonging to the genus Haemophilus and the

remaining reactions to identify the isolate to the species level and to determine the biotype

of H. influenzae. Although various results have been reported with the RapID NH system,

when used properly, >95% of clinical isolates of H. influenzae should be correctly identified

(31).

The BBL Crystal Neisseria/Haemophilus ID system (BD) and API NH kit (bioMerieux) provide

miniaturized biochemical identification schemes in a microwell tray, with results available in

≤5 h. The Crystal system employs 29 different growth substrates and is predicated on

measuring the substrate conversion chromogenically and fluorogenically after 5 h of

incubation. The API NH kit consists of 12 dehydrated substrates and a well to detect

penicillinase, and it permits the identification of Haemophilus spp., Neisseriaspp.,

and Moraxella spp. The test is performed by inoculating each well with an organism

suspension equivalent to a 4 McFarland turbidity standard prepared from 24-h colony growth

and incubating the plate for 2 to 2.25 h at 35°C. In addition to testing for penicillinase

production, it tests for the following biochemicals: glucose, fructose, maltose, saccharose,

ornithine decarboxylase, urease, lipase, alkaline phosphatase, β-galactosidase, proline

arylamidase, gamma-glutamyl aminotransferase, and indole (3, 64). Independent research

studies evaluating the performances of the Crystal Neisseria/Haemophilus and API NH kits

compared to accepted gold standards have not been published.

One instrument-based identification system has been developed for the species identification

of Haemophilusspp., the NH cards for use with the Vitek Legacy and Vitek 2 instruments

(bioMerieux). This system is based on colorimetric detection of preformed enzyme complexes

using chromogenic substrates. The database supporting these cards encompasses 27 taxa,

including Neisseria, Haemophilus, Actinobacillus, Campylobacter, Capnocytophaga,

Cardiobacterium, Eikenella, Gardnerella, Kingella, Moraxella, Oligella, and Suttonella species.

Studies with the Vitek 2 system using both collections of well-characterized stock strains and

clinical isolates have demonstrated identification accuracies of 90 to 95%, with results

varying by species (78, 94). In one recent study, >95% of isolates of H. influenzae, H.

segnis, H. parahaemolyticus, H. parainfluenzae, and H. actinomycetemcomitans were

correctly identified, while none of the test strains of H. haemolyticus were correctly identified

(78).

Molecular Identification

Several molecular methods, including 16S rRNA gene sequencing, PCR, and fluorescence insite

hybridization have been described in the literature as being effective tools for the species

identification of Haemophiluswhen performed on organisms recovered in culture

(17, 71, 74, 99).

Molecular targets for the detection and identification of Haemophilus spp. are numerous.

Previous studies have described the detection of H. influenzae using the cap locus (which

includes the capsule bexA) (1, 22, 57,70), the 16S rRNA gene (1, 74, 89, 97), the insertionlike

sequence (IS1016) (70), the fumarate reductase iron-sulfur gene B (frdB) (43), the

manganese-dependent superoxide dismutase (sodA) (13), and the outer membrane protein

P6 gene (ompP6) (1, 89, 95). Many of these targets are then combined with PCR (real time

or traditional), microarrays, or sequencing to identify the organism. Widely utilized for

sequencing, 16S rRNA frequently resolves the identities of strains to the species level;

however, identification of H. influenzae, H. aegyptius, and H. influenzae biogroup aegyptius

can be problematic due to the high degree of homology in their sequences. In these

instances, sequencing of other targets, such as ropD, can be considered, or the use of

combined sequencing and biochemical studies can be used. Other novel detection methods

are also being investigated. One report from Kalogianni et al. (44) describes a simple dipstick

test that may identify six pathogens and does not require special instrumentation. Universal

primers are used for PCR amplification of the 23S rRNA gene. The amplified product is then

labeled with biotin and hybridized to probes specific for the pathogens of interest and applied

to the strip. The buffer migrates along the strip by capillary action and binds nanoparticles,

producing a characteristic red line. The benefits of these types of technologies are that they

can readily be utilized outside large reference laboratories and can be performed in less time

than traditional sequencing. However, while microarray, sequencing, and immunoblotting

technologies are interesting, a product to detect Haemophilus nucleic acid from culture or

clinical specimens has yet to be approved by the U.S. Food and Drug Administration (FDA).

Problems in Identification

A significant challenge in the species identification of H. influenzae, H. aegyptius, and H.

influenzae biogroup aegyptius is a lack of biochemical diversity and sequence divergence

(45, 48, 51). Biochemical profiling and standard 16S rRNA gene sequencing fail to

adequately distinguish these organisms (47, 48), necessitating the use of alternate

sequencing targets, such as those mentioned in the previous section, or a combination of

sequencing and biochemical testing. This is problematic, since H. aegyptius lacks the

potential to cause Brazilian purpuric fever, while strains of H. influenzae biogroup aegyptius

cause the disease. Xylose fermentation by most H. influenzae isolates combined with the

lower growth rate of H. influenzae biotype aegyptius and the ability of H. aegyptius to

agglutinate human erythrocytes may be of some value in distinguishing these organisms

(47, 48, 51).

TYPING SYSTEMS Back to top

Capsular Serotyping and Biotyping

The capsular antigen of H. influenzae is a principal virulence determinant of this organism.

Six different capsular antigens have been recognized, each of which is characterized by a

distinct carbohydrate chemical composition and given a letter designation from a to f. Prior

to the introduction of the pediatric HIB vaccine, capsular type b strains were recovered from

human clinical material most often. However, today, at least among populations in which

there is widespread use of the HIB vaccine, non-b encapsulated strains occur with nearly

equal frequency. For this reason, it may be instructive to know the capsular serotypes of

strains ofH. influenzae recovered from clinical specimens, especially those representative of

invasive disease. This may be accomplished using both phenotypic and genotypic methods.

Capsular serotyping of H. influenzae is best accomplished by the use of a slide agglutination

assay which employs polyclonal antisera, specifically reactive with each of the six capsular

antigens (45, 53, 85). It is advisable to perform serotyping as soon as possible after isolation

of H. influenzae, as the amount of capsular antigen produced may diminish over time,

especially with repeated subculture. A thick, homogenous suspension of test organism is

prepared in saline, 1 to 2 drops are placed on a glass slide, and then a drop of type-specific

antisera is added. The antisera is mixed with the organism suspension, and then the glass

slide is rocked gently for ca. 1 min before being examined for the presence of clumping, an

indication of a positive reaction. The reagents for performing slide agglutination serotyping

of H. influenzae are commercially available in kit form from Remel and BD.

Alternatively, primary type-specific antibodies can be directly or indirectly detected with

fluorescent molecules, and binding of the antibody to the homologous capsular antigen can

be determined by fluorescence microscopy (84, 87). While these technologies remain viable,

reagents are frequently available on a research-use-only basis.

Whether a slide agglutination test or fluorescent antibodies are used to determine the

capsular serotype of an isolate of H. influenzae, positive- and negative-control strains should

always be processed simultaneously with clinical isolates as a means of validating test

results.

As noted above, based on three phenotypic properties, the production of indole, ornithine

decarboxylase, and urease, strains of H. influenzae and H. parainfluenzae can be

distinguished into multiple different biotypes (Table 2). Also, as outlined previously, at least

with H. influenzae, certain biotypes have been found to have specific disease associations.

Assessment of indole, ornithine decarboxylase, and urease production with clinical isolates

of H. influenzae and H. parainfluenzae can be accomplished using the conventional methods

described above (Fig. 9) or by use of commercially available miniaturized biochemical kit

systems (30). In one recent study which compared the API NH strip kit (bioMerieux) with the

IDS RapID NH system (Remel) and the NHI card (bioMerieux) as a means for determining

the biotypes of a large collection of recent clinical isolates of both H. influenzae and H.

parainfluenzae, the API NH kit yielded the most reliable results, correctly classifying the

biotypes of >97% of the strains tested (64).

Typing by Molecular Methods

Molecular methods have the advantage of enhanced specificity due to the use of

standardized techniques and a lack of false-positive reactions observed with nonencapsulated

strains in slide agglutination tests.

Capsular typing of H. influenzae can also be accomplished by use of various molecular

methods. Most such assays rely on the amplification of genes in the cap locus, the outer

membrane protein D gene (glpQ), the capsule-producing gene (bexA), the 16S rRNA gene,

and the insertion-like sequence (55). One algorithm was used for detection of the cap genes

to determine capsular serotypes a through f, while the capsule-producing gene, bcxA, was

used to separate strains that produce capsule from those that do not (55). Detection of

theompP2 (outer membrane lipoprotein P2) gene was used as a control. Using this system,

both a conventional PCR and a real-time PCR assay were found to be more sensitive than a

slide agglutination test for serotypingH. influenzae (55).

As with the Enterobacteriaceae, pulsed-field gel electrophoresis (PFGE) is considered the gold

standard for strain typing of Haemophilus. The method demonstrates excellent separation of

clones but is laborious and time-consuming (2, 10, 82). Other molecular methods for typing

have also been applied to Haemophilusspecies. Studies evaluating repetitive-element

sequence-based PCR using intergenic dyad sequence (IDS)-specific primers (IDS-PCR) for

nonencapsulated Haemophilus strains have been developed and demonstrate excellent

separation of NTHi strains (10). In one study evaluating the performance of IDS-PCR with 69

NTHi isolates, the assay demonstrated 65 different banding patterns that were

epidemiologically classified as fingerprints similar to those obtained by PFGE (10). Other

typing technologies applied to Haemophilus with a high degree of separation include

ribotyping, restriction fragment length polymorphism analysis, multilocus enzyme

electrophoresis, randomly amplified polymorphic DNA profile analysis, and multilocus

sequence typing (10). While all of these techniques have demonstrated excellent separation,

many are laborious and time-consuming (multilocus enzyme electrophoresis, PFGE, and

ribotyping), others produce overly complex banding patterns (restriction fragment length

polymorphism analyses), and others lack reproducibility (randomly amplified polymorphic

DNA profile analysis) (10). Multilocus sequence typing offers the advantage of superior

discriminatory power because it combines sequence typing of seven housekeeping genes

with results that can readily be compared between laboratories (48).

SEROLOGIC TESTS Back to top

Antibody tests have been developed for the detection of Haemophilus antibodies; however,

they are of little clinical value and are not readily available. Studies evaluating the

performances of the enzyme-linked immunosorbent assay (11) and immunofluorescent

assays (90) have been conducted; however, because immunity to Haemophilus is derived

from an eclectic combination of antibodies against the H. influenzae capsule and membrane

proteins, assays to detect a single class of antibody are of little value. Further complicating

the use of Haemophilus serologies is the individual variation in antibody level (many adults

have undetectable antibody levels), avidity, and persistence (65).

ANTIMICROBIAL SUSCEPTIBILITY TESTING Back to top

Resistance Rates

Haemophilus influenzae may produce one of two β-lactamases, TEM-1 and ROB-1. Both

enzymes are plasmid associated, extracellular, and produced constitutively in large amounts

(39).β-Lactamase-producing strains should be considered resistant to ampicillin and

amoxicillin, as these drugs typically have MICs of ≥128 μg/ml against these strains (29).

Currently, in the United States, ca. 25% of clinical isolates of NTHi produce β-lactamase

(38). Interestingly, this represents a slight decrease in rates, which peaked in the late 1990s

(38). One possible explanation for this decrease is the more common use, beginning in the

early 1990s, of non-β-lactam antimicrobials, such as the macrolides and fluoroquinolones, in

the empirical management of infections such as otitis media, sinusitis, and

bronchopulmonary infections, i.e., infections with which NTHi are most often associated. As a

consequence of this paradigm shift, β-lactams such as ampicillin and amoxicillin are used

less often, with resulting diminished selective pressure on the emergence and persistence of

β-lactamase-producing NTHi. β-Lactamase-producing strains of H. influenzae remain

susceptible to oral and parenteral cephalosporins and carbapenems (29, 32, 38, 40). They

are also susceptible to combination agents in which a β-lactamase inhibitor, such as

clavulanate, sulbactam, or tazobactam, is combined with a β-lactam agent (29,32, 38, 40).

Examples include amoxicillin-clavulanate, ampicillin-sulbactam, and piperacillin-tazobactam.

Strains of H. influenzae that fail to produce β-lactamase but for which the MICs of ampicillin

and amoxicillin are elevated have been described (56). These strains, which are often

referred to as β-lactamase negative and ampicillin resistant (BLNAR), have altered penicillin

binding proteins, which abrogates the binding of drugs such as ampicillin and amoxicillin to

their cell wall targets, in turn resulting in elevated MICs (42, 93). The activities of

cephalosporins are also diminished with such strains. If one uses an ampicillin or amoxicillin

MIC of ≥4 μg/ml to define resistance within Haemophilus spp., as is recommended by the

Clinical and Laboratory Standards Institute (CLSI) (20), the prevalence of BLNAR strains

of H. influenzae remains at levels of <1% in the United States (38).

Among other antimicrobials which are relevant to the management

of Haemophilus infections, with the exception of trimethoprim-sulfamethoxazole (TMP-SMX),

resistance rates remain at levels of <1% (29, 38). These antimicrobials include both oral and

parenteral cephalosporins, macrolides, fluoroquinolones, and tetracycline. TMP-SMX

resistance rates approach 20% (29, 38).

Susceptibility Test Methods

β-Lactamase production by Haemophilus spp. can be determined rapidly with either a

chromogenic cephalosporin spot test or an acidimetric penicillinase assay, as described

in chapter 70 in this Manual. Because the β-lactamases of H. influenzae are extracellular,

constitutive, and produced in large amounts, assuming the test is performed carefully and

with adequate positive and negative controls, both methods yield reliable results.

Disk diffusion susceptibility tests can be performed using Haemophilus test medium (HTM)

agar (41), with incubation of plates for 16 to 18 h at 35°C in 5 to 7% CO2, as described by

the CLSI (19). Zone diameter interpretive criteria have been developed for 39 different

antimicrobial agents (20). The details of disk diffusion susceptibility tests are presented

in chapter 68.

MICs can be determined with Haemophilus spp. by use of broth microdilution (BMD). The

CLSI advocates the use of HTM broth (41) when determining MICs by the BMD method (18).

Following inoculation, trays are incubated for 20 to 24 h in ambient air at 35°C prior to

determination of MICs; MIC interpretive criteria have been developed for 43 different

antimicrobial agents (20). The details of BMD MIC tests are presented inchapter 68. In

circumstances where HTM is not available or when equivocal results have been obtained with

this medium, BMD MICs can be determined with Haemophilus spp. using Mueller-Hinton

broth supplemented with 3 to 5% sterile lysed horse blood and 10 μg/ml NAD (27). The MIC

interpretive criteria for Haemophiluspromulgated by the CLSI for BMD tests in HTM can also

be applied to MICs determined by BMD in medium containing lysed horse blood.

Unfortunately, there exist almost no published data validating the use of the Etest method

with Haemophilus spp., and therefore, use of this method is not recommended for

susceptibility tests with this organism group.

Susceptibility tests with clinical isolates of H. ducreyi should not be attempted in routine

clinical microbiology laboratories, as standardized susceptibility test methods of proven

reliability for this organism have not yet been developed. Similarly, instrument-based

susceptibility tests for other Haemophilus spp., including H. influenzae, have not been

proven to be effective and are not recommended for testing this organism group.

Irrespective of the method used for performing susceptibility tests with Haemophilus spp., it

is essential that adequate quality control is applied using two H. influenzae quality control

strains, ATCC 49247 and ATCC 49766.

Susceptibility Testing Algorithm

Susceptibility testing with clinical isolates of Haemophilus spp. should be applied only to

those strains known to be of clinical significance. Further, in the vast majority of instances,

only a β-lactamase assay as a means for assessing the activities of ampicillin and amoxicillin

need be performed. The prevalence of BLNAR strains of H. influenzae and their resistance to

other agents that are commonly used to treat the types of infections with

which Haemophilus spp. are associated are simply too low to justify routine testing. One

exception might be resistance to TMP-SMX. This agent, however, is used almost exclusively

for oral therapy of community-acquired respiratory tract infections that are invariably

managed empirically without performance of laboratory studies aimed at elucidating the

specific cause of an individual patient’s infection. In other words, in settings where

knowledge of the activity profile of TMP-SMX versus H. influenzae could be of value, rarely, if

ever, is a patient isolate available for testing. In those rare circumstances when the

assessment of the activities of agents other than ampicillin or amoxicillin are found to be

warranted, either a disk diffusion susceptibility test or a BMD MIC test should be performed.

EVALUATION, INTERPRETATION, AND REPORTING OF

RESULTS Back to top

The genus Haemophilus is a diverse group of organisms which may exist as part of the

normal bacterial flora of healthy humans or may be associated with significant disease. As a

result, simple recovery of Haemophilusfrom a human clinical sample may not always indicate

that the organism is clinically significant. In the following three circumstances, recovery

of Haemophilus spp. in the laboratory is patho-gnomonic: (i) isolates from normally sterile

sites, including blood cultures, are compatible clinically with illness; (ii) H. ducreyi is

recovered from genital tract specimens obtained from patients with genital ulcers; and (iii)

isolates of H. aegyptius from conjunctival specimens are obtained from patients with

exudative inflammation of the conjunctiva.

Recovery of Haemophilus spp. from specimens that may be contaminated with commensal

microbial flora represents a situation in which the clinical significance of the isolate must be

questioned. This is often the case, for example, with isolates from respiratory tract sites. In

such instances, the quantity of organism recovered, both the absolute quantity and the

quantity of the isolate in comparison to quantities of other organisms recovered from the

specimen, is of limited value in assessing clinical significance. It may be helpful to try to

assess the quality of the specimen, as is possible, for example, with expectorated sputa and

endotracheal aspirates. It may also be instructive to compare the results of a given culture

with results obtained from previous and subsequent cultures from the same site. Generally

speaking, repetitive recovery of the same organism from multiple specimens representative

of a specific infectious disease process in an individual patient can be taken as an indication

of clinical significance. And finally, it must be recognized that in some cases, it simply is not

possible to know with certainty whether a given isolate of Haemophilus spp. is clinically

significant. In such instances, active dialogue with health care providers is encouraged.