What Is Haemophilus Influenzae?
Haemophilus influenzae was so named because of a supposed relationship to influenza and was designated haemophilic in virtue of its inability to grow-on culture medium without the addition of whole blood, or certain growth-promoting substances, termed X and V, present in blood. These growth factors, however, are not restricted to blood, but are present also in certain vegetable tissues.
If the term haemophilic were used in a broad sense to designate organisms which require blood for their growth it would embrace a number of heterogeneous species, and the generic term Haemophilus is therefore restricted to those organisms which are dependent on one or both of the growth factors required by Haemophilus influenzae.
Haemophilus influenzae was first described by Pfeiffer in 1892 as the cause of influenza in the 1889 to 1892 pandemic because of its constant presence in the 'characteristic purulent sputum in uncomplicated cases in absolutely pure culture and in almost incredible numbers'. In 1893, he was able to grow the bacillus on a blood-containing medium. Doubts about its causal relationship to influenza developed during and after the 1918-19 pandemic, mainly because other bacterial pathogens were being incriminated in the secondary influenzal pneumonias.
Only in 1933, however, did doubt become certainty with the discovery of the influenza virus A by Smith, Andrewes and Laidlaw. The view that there may be a special relationship between the virus and the bacillus found support in Shope's studies of swine influenza but the modern concept is that Haemophilus influenzae is a secondary pathogen on respiratory mucosa that has become susceptible to bacterial attack after the primary influenza virus infection. It seems to play a similar role in chronic bronchitis.
Later, Pittman demonstrated that Haemophilus influenzae is divisible into capsulate and non-capsulate strains and that the capsulate strains could be differentiated serologically into 6 types, some of which cross react with pileumococsal capsular antigens. Of these, type b, in particular, was implicated as a primary pathogen in acute purulent meningitis and laryngo-epiglottitis (croup), and helped to define the importance of this organism.
The great majority of strains of Haemophilus influenzae found in the healthy or diseased respiratory tract are non-capsulate and generally are pathogenic only in a secondary role. The minority of capsulate strains which may colonize the throats of a few healthy carriers may act as primary pathogens in the respiratory tract and meninges.
Haemophilus influenzae is a small Gram-negative bacillus. It shows considerable pleomorphism from cocco-bacillary forms which predominate in young cultures, and often as small clumps in sputum, to filamentous forms in older cultures and in the spinal fluid of cases of purulent meningitis.
Haemophilus influenzae needs good quality nutrient media for its growth but specifically it requires two nutritional factors, called X and V, which are present in blood. X factor, highly heat-resistant, is present in haemin and probably provides precursors for the haem-containing catalase, cytochromes, etc., which are required for the aerobic growth of the bacillus; V factor, which has been variously named co-enzyme I and II, di- and triphosphopyridine nucleotide (DPN and TPN) and nicotinamide adenine dinucleotide and its phosphate (NAD and NADP) is essential as a hydrogen acceptor in the cell's metabolism and is destroyed by heating at 120°C for a few minutes.
In ordinary blood agar, the V factor tends to be destroyed by enzymes from red blood cells; in chocolate agar (containing blood heated to 80 to 90°C). The V factor is released so that this latter medium, or a transparent medium containing blood extracts (Levinthal agar, Fildes' peptic digest agar), gives the best growth of Haemophilus influenzae. V factor is also produced by certain bacteria and yeasts : the staphylococcus can be used to demonstrate the dependence of Haemophilus influenzae on this growth factor by noting enlarged colonies of the bacillus around colonies of staphylococci in primary mixed cultures (satellitism) or by streaking a culture of staphylococcus across a plate inoculated with suspected Haemophilus influenzae.
Capsulate strains are best demon-strated by their colonial features on a transparent medium like Levinthal agar on which the colonies are mucoid, rather opaque and when viewed obliquely with transmitted light, characteristically iridescent. Typing of capsulate strains can be done by slide agglutination or by the capsule swelling reaction with monospecific antisera to types a to f.
Other haemophili that conform generically in their nutritional requirements for X or V factors or both are:
(a) The Koch-Weeks bacillus (Haemophilus aegyptius), requiring both X and V factors and closely resembling Haemophilus influenza.
(b) Haemophilus parainfluenzae, requiring only V factor and occasionally associated with subacute bacterial endocarditis, besides being a commensal in the upper respiratory tract.
(c) Haemophilus haemolyticus, also dependent on X and V factors and a commensal that may cause confusion in cultures of throat swabs because of its colonial and haemolytic resemblance to β-haemolytic streptococci.
(d) Haemophilus aphrophilus, requiring X factor and CO2.
(e) Haemophilus ducreyi, the causative organism of the venereal disease, chancroid or soft sore, with special nutritional requirements besides dependence on X factor. Clotted fresh rabbit blood is a good medium for its isolation.
In cases of Haemophilus influenzae meningitis, an early and rapid laboratory diagnosis may be life-saving because infants with fulminating infections may die within 24 hours of onset. Conversely, cases may have been treated with antimicrobial drugs for some days before admission to hospital and the infection drags into a subacute phase.
Smears of the centrifuged deposit of the cerebro-spinal fluid from any case of purulent meningitis must be stained by Gram's method with dilute carbol-fuchsin as the counterstain for haemophili which stain poorly and also with methylene blue. Careful search of stained smears may be needed to demonstrate the pathogen, which is most likely to be meningococcus, Haemophilus influenzae or pneumococcus. The urgency of a precise diagnosis lies in the need for the early administration of an appropriate antimicrobial drug, e.g. penicillin, or erythromycin and chloramphenicol in penicillin allergy, for meningococcus, chloramphenicol (usually with streptomycin or sulphadiazine) or ampicillin for Haemophilus influenzae or coliform bacilli, and penicillin for pneumococcus or other Gram-positive coccal pathogens.
When haemophilus meningitis is suspected, immediate verification may be obtained if type-b antiserum is available. Either a loopful of the antiserum may be mixed with the fresh deposit and the mixture examined microscopically for capsular swelling which demonstrates Haemophilus influenzae more easily than a Gram-stained film ; or the cerebrospinal fluid may be tested for antigen with the anti-serum in a capillary tube precipitin test or by counter electrophoresis. The deposit should be plated on ordinary blood agar and on chocolate agar incubated in a candle jar; it is emphasized that good quality nutrient agar base in the blood containing medium is essential for the growth of Haemophilus influenzae. When facilities are available, a few drops of the cerebrospinal fluid may be layered on the culture medium directly from the lumbar puncture needle.
Capsulation and satellitism are best demonstrated by sub-culture to a transparent medium and serological typing may be done locally or at a reference laboratory. Blood culture is advisable in meningitis cases and is essential for the diagnosis of acute laryngo-epiglottitis since the organisms are not easily cultured from the local lesion. For the isolation of haemophili from the sputum of cases of chronic bronchitis or bronchiectasis, the specimen must first be homogenized, smears stained by Gram's method, and cultures made on blood agar and chocolate agar.
Suspected haemophilus colonies may be confirmed by the application of disks containing X factor, V factor or both factors to different areas on a nutrient agar plate after it has been seeded from the colony. The laboratory procedures are similar for the culture of material from infections such as conjunctivitis, sinusitis and otitis media. Haemophilus strains are sensitive to refrigeration and neither specimens or cultures should be kept at 0-4°C.