During the 1889-1892 influenza pandemic, Pfeiffer observed and cultured from sputum seized a small bacillus,Bacillus influenzae and made the etiologic agent of “flu” or “influenza”; it showed the presence of blood necessary for the cultivation of this bacterium and invents blood agar.
A few years earlier, in 1883 in Egypt by Koch in 1886 in the USA by Weeks, was observed and grown in exudate purulent conjunctivitis bacterium Mycobacterium tuberculosis-Weeks, reported in a treaty of 1889 under the nameBacillus aegyptius.
The name of the genus Haemophilus was proposed in 1917. In 1939, A.Lwoff proposes stripping “Hemophilae”and creating the kind Moraxella, followed in 1952 by Moreno-Lopez proposing the genus Bordetella.
Until 1933, when the discovery of the causative agent of influenza, H. influenzae remained, sometimes with doubts, the bacteria suspected to be responsible for the influenza.In 1930 Miss Pittman highlights the existence of capsulated strains, serotypes features and shows the predominance of type b in meningitis and other acute suppurative infections.
I – GENERAL CHARACTERISTICS OF GENRE:
The general characteristics of bacteria of genus Haemophilus are: small Gram-negative bacilli or coccobacilli with a very accentuated polymorphism (elongated shapes); immobile, non-spore forming, sometimes encapsulated, aerobic and facultative anaerobic, demanding factors in the blood, factor X or protoporphyrin IX or protohème and / or V factor (NAD or NADP), whose growth is also favored by circles complex, the optimum temperature of 35-37 ° C;with a nitrate reductase, having a reaction of the catalase and oxidase variables; using carbohydrates by a fermentative process; are obligate parasites of the mucous membranes of humans and animals; the G + C% of the DNA is between 37 and 44 mol.
II – TAXONOMY AND NOMENCLATURE:
The Haemophilus is placed (Kilian, Bergey’s Manual 1984) in the family Pasteurellaceae with Pasteurella andActinobacillus genres.
Haemophilus influenzae is the type species of the genus that contains sixteen species of animal and human origin.
Among the sixteen species described, 3 require X and V factors (H. influenzae, H. aegyptius and H. Haemolyticus), 2 do not require that the factor X (H and H haemoglobinophilus. Ducreyi) and 10 require only Factor V (H. parainfluenzae, H. parahaemolyticus, H. paraphrohaemolyticus, H. pleuropneumoniae, H. paracuniculus, H. paraphrophilus, H. segnis, H. parasuis, H. paragallinarum, H. aviwn). H. aphrophilus has a requirement for variable X factor.
III – HABITAT AND EPIDEMIOLOGY:
Haemophilus are part of the normal flora of the mucous membranes of the upper respiratory tract and oral cavity of humans; they may also be isolated in the digestive tract and at the vaginal mucosa.
Two species, H. aegyptius and H. ducreyi are not met in healthy subjects. By against H. influenzae, H. haemolyticus, H. parainfluenzae, H. parahaemolyticus, H. paraphrohaemolyticus, H. aphrophilus, H. and H. paraphrophilus segnisoccupy a variable position in the different ecological niches.
Haemophilus represent 11% of the pharyngeal flora of a normal subject; the dominant species is H. parainfluenzae, H. influenzae is present in smaller quantities and is more common in children. Porting H. influenzae affects 75% of young children and 35% of adults and older children, but there are wide variations in the pharyngeal colonization by capsulated strains of type b. In a normal population the capsulated strains are encountered in less than 5% of children (half of the stem being of type b); adults, porting is usually less than 0.50%.
H. influenzae is rarely encountered in the oral mucosa, saliva and on the surface of the vaginal mucosa. H.haemolyticus is located in the pharynx.
H. parainfluenzae is more ubiquitous and colonizes the oral and pharyngeal mucosa; it is also present in the stool and the vaginal cavity. In saliva, H. parainfluenzae may be in a large amount (1107 bacteria per ml). Other species, mostly dependent V H. parahaemolyticus, H. paraphrohaemolyticus, H. segnis, H. paraphrophilus have the same ecological preferences (pharyngeal and oral mucosa), but are less common. In the mouth, the tooth surface (plaque, dental area, gingival pocket) is the preferred site for H. parainfluenzae, H. segnis, H. and H. aphrophilus paraphrophilus.
H. influenzae type b is one of the three main bacterial species responsible primitive meningitis. It occupies in our country second behind the meningococcus. In the United States of America, in first place and the risk for a child to have a meningitis H. influenzae during the first five years of his life is estimated at 1/500. This risk is 1/1 500 in Britain. The disease affects children aged 2 months to 3 years with a peak incidence between 6 months and 1 year.Strains are usually of biotype I.
Meningococcal H. influenzae is a sporadic infection and there is no real epidemics such as those observed with meningitis. However, recent studies have shown that Haemophilus meningitis should be considered infectious, with a risk equivalent to that of cerebrospinal meningitis. When invasive event in a community or a family, there is always a significant increase in the number of carriers of capsulated strains. Other invasive demonstrations distribution by age, identical to that of meningitis except less frequent epiglottitis affecting older children 2 to 7 years.
Dissemination of encapsulated strains is by droplets dispersed during breathing or direct contact, intimate, with secretions from a patient or a carrier; the bacteria do not survive desiccation in the external environment.
IV – PATHOGENICITY NATURAL:
H. influenzae is a pyogenic bacterium responsible for varied sometimes severe infections observed at all ages but most commonly in children. It is either acute systemic infections caused by invasive strains capsulated type b or acute or chronic bacterial infections without caused by unencapsulated strains in which H. influenzae sometimes plays a secondary role.
A – In children:
Infections H. influenzae are rare in the neonatal period; it is the same puerperal infections recognized, however, since the beginning of the century. Officials strains are usually unencapsulated. Meningeal localization is exceptional at this age.
After two months of age invasive manifestations are the most common and most serious.
Meningitis H. influenzae are common in children aged 3 months to 3 years.
The classic form is usually preceded by signs of infection of the upper respiratory tract (pharyngitis, sinusitis or otitis media) contemporary or not a viral infection. There rarely are devastating forms. Mortality is less than 10% but neurological sequelae are common, observed in 20-30% of cases. The risk of complications and sequelae is even higher than the bacterial concentration (or soluble antigens) in the
CSF is significant and lasting. The bacteria is H. influenzae type b.
Epiglottitis that occurs in older children (2 to 7 years) is less common than meningitis.
Other locations are observed with variable frequency during systemic infection: arthritis (and more rarely osteitis or osteomyelitis), otitis media, cellulitis, pericarditis, pneumonia with or without empyema epididymoorchitis. In children older infections H. influenzae deficiency can occur during the field, immune deficiency, leukemia, cancer.
Otitis media with H. influenzae is most often a localized infection, without bacteremia and the unencapsulated strains in 80% of cases. It is the same with other infections of the ENT and bronchopulmonary infections. Conjunctivitis H.influenzae occurs in two forms: sporadic cases caused by encapsulated or not, and stem outbreaks located in countries with hot climates (North Africa, southern USA) usually caused by Mycobacterium tuberculosis-Weeks, H.aegyptius, unencapsulated H. close influenzae biotype III.
B – Adults:
All the previously described manifestations in children can be observed in adults. The land will play a predominant role in the occurrence of infection with H. influenzae. The meningitis H. influenzae represent from 1 to 10% of purulent meningitis of the adult. They are more readily observed in the elderly or when predisposing causes: head injury, agammaglobulinemia, diabetes, alcoholism, splenectomy, other H. intercurrent illness. influenzae rarely causes recurrent meningitis (mainly pneumococcus). The strains are usually unencapsulated.
Pulmonary and bronchopulmonary locations are the most frequent. It is either pneumonia with bacteremia or bronchopulmonary infection in chronic bronchitis. In this case, the loss of defense capabilities of the bronchial mucosa allows bronchial colonization by bacteria of the upper respiratory tract. Acute exacerbation of chronic disease is accompanied by the proliferation alone or with other bacteria H. influenzae usually not encapsulated. H.influenzae plays with pneumococcal an important role in acute sinusitis.
As against infections with bacteremia are uncommon in adults (epiglottitis, pericarditis, endocarditis, arthritis, cellulitis). Biliary infections, appendicular, urinary tract, prostate, genital and gynecological have been described.
V – PATHOPHYSIOLOGY AND FACTOR VIRULENCE:
Haemophilus influenzae develops different products to various degrees involved in pathogenicity of this species. All strains of H. influenzae (as S. pneumoniae, N. meningitidis and Neisseria gonorrhoeae) produce an enzyme, IgA protease, extracellular, specific for human IgA subclass of IgA1 role of IgA1 proteases is not yet elucidated.
From recent discovery pili H. influenzae play a still imperfectly known role. They are responsible for adherence to epithelial cells but does not appear to play a key role in the colonization of the mucosa and invasion.
Among the bacterial components, lipopolysaccharide and outer membrane proteins are important antigenic components for epidemiological studies.
The capsule polysaccharide antigens are the essential support for invasive virulence events. The capsulated strains were classified by Miss Pittman 6 serotypes a, b, c, d, e, and f. Serotype b is the most common; it is due to a polyribosylribitol phosphate PRP, which antigen, purified, is used as a vaccine.
Antibodies against PRP antibodies that are protective gradually appear in children. Fothergill and Wright showed in 1932 the relationship between the absence of serum bactericidal activity and the incidence of meningitis H.influenzae type b. Later the same correlation was established between the PRP antibodies and the occurrence of meningitis, more frequent between 3 months and 3 years. The period between the end of the passive protection conferred by maternal antibodies and antibody acquisition to adequate security is favorable to the development of meningitis.
VI – BACTERIOLOGICAL DIAGNOSIS:
A – The specimens:
They are either monomicrobial products obtained by puncture (CSF, joint, pleural fluid) or polymicrobial products such as bronchial secretions and levies in ENT. These must be made by avoiding or limiting contamination by oropharyngeal flora. Cultivation of bronchial secretions is after homogenization of the sample and is accompanied by a quantitative approach of bacteria. Blood cultures are useful in all cases of febrile evolution. He will think aboutH. influenzae in certain unusual and rare circumstances (urine, pus deep, …)
B – Direct examination:
In a pathological or a culture, H. influenzae is as small Gram negative bacillus (0.3-0.4 x 1-1.5 u, m) short, usually coccobacillary. But it should be noted that the frequency of polymorphisms in the observation of microscopic preparation can make a casual observer puzzled. These are immobile bacilli unsporulated and in some cases having a capsule.
Direct examination is useful for monomicrobial products and meningitis during the coccobacillary appearance and Gram-negative bacteria polymorphism are good guidance. Reading is more difficult in products often polymicrobial as sputum, things to look for are plenty of coccobacilli or bacilli purposes (finer than enterobacteria) accompanying polynuclear and polymorphism.
C – Culture – Identification characters:
Belonging to the genus Haemophilus based on the requirement factor growth X and V present in red blood cells.
Factor V, NADP dehydrogenase is a coenzyme. NAD is thermolabile and inactivated by heating 30 minutes at 120 ° C. Factor V is intraglobulaire, is present in the tissues and is synthesized by most bacterial species.
Factor X is haemin which enters into the composition of iron-containing respiratory enzymes (cytochromes, cytochrome oxidase, catalase, peroxidase). This compound is essential to bacteria lacking the enzyme of the acid processing chain delta amino levulinic protoporphyrin. Anaerobically needs H. X factors influenzae are very small or even zero.
The X factor diffuses from intact red blood cells and is released after heating.
The culture media must contain the X and V factors Blood agar with a streak of S. aureus produces a provision of factor V allows the cultivation by
satellitism phenomenon. Agar cooked blood is obtained by moderate heating
causing the release of X and V from the red blood cells. Chocolate agar is a complex nutrient medium containing hemin which is added NAD.
The usual nutrient media can be supplemented with factor X and V (Fildes, NAD, hemin, yeast extract).
Search Haemophilus in polymicrobial products from the respiratory tract using selective media prepared by adding antibiotic bacitracin, for example (this was the first use of penicillin by Fleming in 1929).
The optimal cultivation temperature is 35-37 ° C. Incubation in a humid atmosphere promotes growth and some species require CO2-enriched atmosphere (H. aphrophilus, H. paraphrophilus}.
Different types of colonies have been described in H. influenzae. The encapsulated strains give mucous colonies, large, tend to spread or smooth colonies, round at regular edges, curved, easily separable, iridescent on transparent medium oblique light. Non-encapsulated strains are either smooth colonies, neighboring the previous, smaller, or more rarely without iridescence rough guy, hard to take, which is the usual appearance of H colonies.parainfluenzae.
In liquid medium S strains give a homogeneous disorder, R strains form a granular deposit without changing the clarity of the environment.
Certain strains of H. indole influenzae emit odor.
D – Identification:
The first stage of Haemophilus identification is the identification of the requirement of factor X and / or V. Different methods are used to explore this requirement: satellitism phenomenon on blood agar with a streak of Staphylococcus, supplemented medium with one and / or another factor, discs with one and / or another factor deposited on the surface of an agar medium. Any contribution of parasite growth factor (especially X from middle to blood, blood culture bottle, …) should be carefully avoided, as it is important to perform these tests using nutrient media containing no traces of these factors. The test exploring the synthesis of porphyrin from delta-aminolevulinic acid helps to provide certainty as to the dependence factor X (test porphyrin).
Identification is completed by the study of biochemical characteristics presented in Tables 1 and II. The fermentation of glucose, with or without gas production is constant; the use of xylose and ribose H. influenzae and the sucrose and fructose by H. parainfluenzae are good differential characters.
Only demanding cash only factor V and H. aphrophilus have a test to positive ONPG.
According to three biochemical characteristics, ornithine decarboxylase, urease and indole production, Kilian proposed biotypes currently 8 in number for H. 3 influenzae and H. parainfluenzae. These characters can be searched with the usual media supplemented or not, or using micro method (API 10 E gallery with a heavy inoculum).
The determination of encapsulated strains of serotype is performed by slide agglutination, swelling of the capsule, co-agglutination or electrophoresis.
The study of factors needs X and / or V is the first step in the differential diagnosis. The CO2 strict requirement is another element of the differential diagnosis.
Useful biochemical characteristics to distinguish the species of Haemophilus and neighboring or isolated in identical situations bacteria are shown in Tables I and II.
E – Classification: biotypes, serotypes
Various markers may be used to characterize the strains of H. . The distribution of biotypes influenzae is different depending on the origin of strains: capsulated invasive strains usually belong to biotype I, the strains present in the mucous membranes of the upper respiratory biotype II.
Among the six serotypes defined by Pittman, serotype b is almost always the one isolated from invasive manifestations. Other markers such as subtypes defined by the electrophoretic profile of the outer membrane or LPS proteins allow complete epidemiological studies.
F – Quick Diagnosis:
Rapid diagnosis is based on research of polysaccharide antigens (soluble antigens) in biological fluids, CSF, serum, urine. This search can be made by various techniques, electrophoresis, agglutination of latex particles coated with antibodies, co-agglutination, ELISA. It concerns only the type b H. influenzae and indications are systemic infections in children.
VIII – OTHER SPECIES HAEMOPHILUS:
Biochemical characteristics of other Haemophilus species are presented in Table 1 and II.
– H. aegyptius (Koch-Weeks bacillus) – This case must be regarded as a variety of hemagglutinating H. influenzaebiotype III, but all biotypes III are H. aegyptius. This bacterium is responsible for conjunctivitis in hot countries. In 1984 occurred in Brazil, in children, an epidemic of fulminant purpura can be fatal and consecutive to a purulent conjunctivitis. Conjunctivitis and severe illness known as “Brazilian purpuric fever” are due to H. aegyptius.
H. parainfluenzae. Rarely responsible for infections, mainly in adults, this species was isolated during endocarditis.Cases of meningitis and systemic infections are rare.
H. haemolyticus, H. parahaemolyticus, H. paraphrohaemolyticus. These species were isolated in very rare cases of endocarditis and liver abscesses.
H. aphrophilus and H. paraphrophilus. Both species are responsible for endocarditis (more frequently than H. parainfluenzae) and brain abscess.
IX – TREATMENT AND PREVENTION:
A – Antibiotic Sensitivity and Treatment:
Different species are usually resistant to lincosamides (character can be used as a diagnostic aid) and low sensitivityin vitro to macrolides.
H. influenzae is sensitive to the main families of antibiotics: penicillin (ampicillin), cephalosporins (Ie, the second and especially the third generation), aminoglycosides, chloramphenicol, tetracycline, trimethoprim, sulfa drugs, rifampicin and quinolones. In the 1970s was seen the emergence of resistant strains in particular ampicillin by production of a plasmid beta-lactamase TEM-type (identical to that observed in E. coli and many species of Enterobacteriaceae, inhibited by clavulanic acid). This resistance is not always detected by standard susceptibility testing (agar diffusion) and it is essential to seek, specifically by an appropriate technique, beta-lactamase production for all strains isolated in pathogenic situation (use of chromogenic cephalosporin, or acidometric or microbiological method). More rarely, the resistance is due to a ROB-1 enzyme.
It is sometimes due to a change of the permeability of the bacterial cell wall.
The ampicillin resistance concerns 12 to 20% of the strains isolated in our country. Chloramphenicol resistance is rare (3%) by production of a chloramphenicol acetyl transferase plasmid origin. Resistance also relates tetracycline, kanamycin and trimethoprim. There are invasive strains multiresistant to ampicillin and chloramphenicol.
H. parainfluenzae has the same sensitivity as H. influenzae but resistant strains, in particular to ampicillin are more frequent.
The treatment uses different active antibiotics. Ampicillin is the antibiotic of choice in the treatment of meningitis administered parenterally in high doses. The emergence of strains resistant to ampicillin did change regimens and give ampicillin for chloramphenicol or more frequently, a third-generation cephalosporin with good meningeal dissemination.
B – Prophylaxis:
It is conceivable against infection with H. influenzae type b with the aim of eliminating carriage among the isolated individual or in a community and increasing the body’s defenses by vaccination of susceptible individuals. These two objectives were the subject of numerous studies, but doubts remain as to their appropriateness and effectiveness.
Chemoprophylaxis, not used in our country, may appeal to rifampin orally. Vaccination is the use of the polysaccharide type b or polyribosylribitol (PRP) obtained purified. The response after vaccination is highly dependent on the age and the synthesis of antibodies is low antipolysaccharidiques 18 months. Improvements can develop a vaccine, usable and effective for 18 months, by coupling PRP with proteins such as diphtheria toxoid or tétamiques or membrane proteins of Neisseria meningitidis
I – TAXONOMIC POSITION:
In 1889 Ducrey described the bacterium causes chancroid, currently known as the Haemophilus ducreyi. This infection was individualized for syphilis in 1852, but the growing difficulties bacillus Ducrey have long bases the diagnosis the only clinical data.
H. ducreyi is a homogeneous species and belongs to the genus Haemophilus.
However, recent studies of DNA-DNA hybridization showed that this species was removed from other species ofHaemophilus.
II – HABITAT AND EPIDEMIOLOGY:
H. ducreyi is a bacterium strictly adapted to humans and has never been found in the environment. It causes chancroid, a sexually transmitted disease that is endemic in tropical and subtropical regions of Asia and Africa.Europe and France disease disappeared for several decades; she was occassionnellement observed in ports.
The infection is observed again since the 1970s Besides isolated cases, chancroid evolving in epidemics in populations of immigrant workers from Africa the most. In most cases, the infection occurs in contact with prostitutes.Transmission is by direct contact and requires breaking the skin. It has not been demonstrated in healthy carriers (male or female). The disease is much more common in men.
Infections H. ducreyi are in many developing countries the main cause of genital ulcer, more common than syphilis.In industrialized countries the most common cause is infection with Herpes Virus followed by infection withTreponema pallidum.
III – PATHOGENICITY:
A – pathogenic natural power:
After incubation for varying lengths, short, less than one week, or longer than 15 to 30 days or more, the damage begins with a pustule which evolves in the form of painful ulceration, not hardened at elevated and irregular edges, purulent. Chancroid is much more frequently observed in humans and sits mostly on the genital skin (coronal sulcus, sleeve) more rarely on the glans. There may be multiple ulcerations resulting from a self-inoculation. The chancre may be accompanied by a satellite lymphadenopathy (inguinal bubo) progressing to fistula after softening.
The infection is very rarely seen in women, and the lesions are localized to the skin of the genitals.
B – Experimental pathogenicity:
The self-inoculation into the skin of the patient from his own injury was completely abandoned as a diagnostic tool.Animal experimental pathogenic power has been sought intradermally in rabbits. Strain virulence is highly variable and decreases over conservation.
IV – BACTERIOLOGICAL DIAGNOSIS:
A – pathological Product:
The wood is collected at the canker after debridement at the base of the ulcer with a lancet or a swab or puncture in the inguinal bubo. Conservation and transportation are avoided and the product must be cultured immediately.
B – Direct examination:
Microscopic examination of the smear is done after staining with methylene blue or Giemsa. Gram staining does not correctly display bacillus Ducrey. Playback can be complicated by the presence of associated flora. H. ducreyi is as short bacillus (0.5 x 1.5-2 microns (im) with rounded ends, bipolar staining intra- and extra-cellular, evoking a safety pin. Bacteria can be isolated or grouped chain (appearance bicycle chain) or shoal. Direct examination is positive in 50-80% of cases.
C – Culture:
H. ducreyi is one of the difficult cultivation of bacteria or “fastidious bacteria”. In addition the frequent presence of other bacteria in the sample requires the use of m ilieux selective. Different solid media have been proposed, blood agar (human, rabbit, fetal bovine), chocolate agar Isovitalex.
Enrichment hemin and serum (10-20%) improves crop yields. The addition of vancomycin (3 mg / 1) and polymyxin (7.5 mg / 1) performs a selective medium (PNT). It is necessary to use two media (selective and non-selective) in isolation. The best basal media are the Columbia agar, agar polp and GC medium for gonorrhea.
Incubation is carried out at 33-35 ° C in an atmosphere of 5 to 10% CO2 and saturated humidity.
The colonies visible after 2-5 days of incubation are small, semi-opaque, gray-yellow, not mucous membranes, which during the attempted removal, slide intact on the agar.
Pathological product during harvesting can be inoculated in 1 ml of serum (human, rabbit or fetal bovine) and incubated at 35 ° C for 2-3 days. This achieves an enrichment bacillus Ducrey which after staining, takes the form of long chains of bipolar staining bacilli with the bicycle chain appearance.
D – Identification:
The species is poor identification criteria. H. ducreyi is a demanding species X factor and its requirements are high (200 mg / 1), higher than those of other Haemophilus. The need for X factor is difficult to explore the satellitism around a disk-deficient medium as for Other Haemophilus, but it may be by the test porphyrin (porphyrin synthesis research from the delta aminolevulinic acid).
The usual identification characters are predominantly negative; the main positive features are the reduction of nitrate to nitrite; the presence of a-alkaline phosphatase and the oxidase test carried out with the tetra-methyl-pphénylènediamine.
H. ducreyi has amino peptidase but not glycosidases.
The electrophoretic profile of the outer membrane proteins can define subtypes used for epidemiological studies.
The diagnosis is based on the particular aspect of the bacillus, crop requirements and adverse biochemical characters.
The differential diagnosis should be considered for slow-growing colonies as some corynebacteria (positive Grain).
V – TREATMENT AND PREVENTION:
Treatment involves local care and systemic antibiotics: successfully used antibiotics are trimethoprim – sulfamethoxazole, streptomycin, tetracycline, the étrythromycine.
There are strains resistant to ampicillin (for production of beta-lactamase), tetracycline, chloramphenicol, sulfonamides, aminoglycosides. This resistance is plasmid-borne.
Prophylaxis is that of sexually transmitted diseases. There is no superinfection immunity.