Haemophilus


            Small, non-motile, non-sporing, Gram-negative bacilli that are parasitic on human beings and animals.  They are characterized by their requirement of one or both of two accessory growth factors present in blood, ie., X and V.  It was isolated in 1892 by Pfeiffer, hence known as Pfeiffer's bacillus. The genus name is derived from the Greekwords meaning 'blood loving'. H. influenzae is an exclusively human pathogen whose complete genome has sequenced. The important  species are   Haemophilus influenzae, H. aegyptius, H. ducreyi


Heamophilus influenzae
Morphology & Cultural characteristics - It exhibits pleomorphism i.e, it occurs as coccobacillary forms(1-2µm X 0.3-0.5µm) in sputum, in CSF from meningitis as long filamentous forms.  Strains from acute infections are often capsulated, it is stained with Loeffler's Methylene Blue or dilute Carbol Fuchsin for 5-15 min.
            It grows better in aerobic than in anaerobic conditions. Some require 10% CO2. It grows at an optimum temperature of 37°C and not below 20°C. It  has fastidious growth requirements. The accessory growth factors X and V present in blood are essential for growth. The heat stable factor X is hematin or other porphyrins required for the synthesis of cytochrome and other heme enzymes such as catalase and peroxidase involved in aerobic respiration. The factor V is heat labile, destroyed at 120°C in few minutes.  It is present in RBC, it is a coenzyme i.e. NAD/NADP which acts as hydrogen acceptor in the metabolism of the cell.
               It grows on blood agar scantily as V factor not available (it is inside RBC).  When Staph. aureus is streaked across a plate of blood agar, on which H. influenzae too inoculated.  After overnight incubation, the colonies of Haemophilus will be large and developed alongside with Staph.  This phenomenon is called satellitism. The lysed erythrocytes in the agar surrounding the S. aureus streak provide factor X, and staphylococcal cells themselves secrete factor V during logarithmic growth.  When blood agar is heated to 80-90°C, or boiled for a few minutes, the V factor is released from the RBC and this media is superior to blood agar for the growth of H. influenzae.  Clear transparent medium may be prepared by boiling and filtering a mixture of blood and nutrient broth ie., Levinthal's Medium or by adding a peptic digest of blood to nutrient agar - Fildes Agar, which is best for primary isolation  of  H. influenzae and gives a vast growth. Capsulated strains produce translucent colonies with a distinctive irridescence on Levinthal's agar.
Glucose and xylose are fermented with acid production. Catalase and oxidase reactions are positive. Nitrates are reduced to nitrites. On the basis of production of indole and urease, and ornithine decarboxylase activity H. influenzae is divided into 8 biotypes. H. influenzae is a delicate bacterium destroyed by heating  550C for 30minutes, refrigeration at 0-40C, drying and disinfectants. In culture the cells dies within 2-3 days due to autolysis. Cultures can be prepared in chocolate agar slopes for about a month. The culture can be lyophylised for a long term preservation.
Antigenic structure & Virulence factors – Some strains produce a capsule which is polysaccharide in nature. Most infections are caused by H. influenzae strains belonging to capsular serotype b(Hib). This capsule is a polymer of polyribosyl-ribitol phosphate(PRP). Antibodies to capsular material are protective. They promote complement dependent phagocytosis and killing of these organisms. These strains are associated with rapidly progressive and even life-threatening infections.
            The virulence is determined by the following factors :
  • Capsular polysaccharide – this helps the bacteria from phagocytosis.
  • Adherence – the lack of adherent capability in type b organisms, hence systemic infections. The presence of adherent capability by non capsulate strains explain the tendency of these strains to cause more localized infections.
  • Outer membrane proteins – contribute in adhesion and invasion of host tissues.
  • IgA1 protease - H. influenzae produces this enzyme and splits the antibody and inactivates it. Production of IgA1 protease allows pathogens to inactivate the primary antibody found on mucosal surfaces and thereby eliminate protection of the host by the antibody.
Pathogenecity - H. influenzae is an exclusively human pathogen. 25-80% healthy adults harbour non capsulate organisms in the nasopharynx and oropharynx. Diseases due to H. influenzae are of 2 groups : invasive and non invasive infections.
In  invasive, the bacillus acts as the primary pathogen, causing acute infections. The bacilli spread through blood, being protected  from phagocytes by their capsule. eg: meningitis, pneumonia, arthritis and epiglottitis. These infections are usually seen in children and are caused by the capsulated strains, type b accounts most cases.
1.      Meningitis : A serious diseases in which if untreated fatality rates upto 90%. The bacilli reach the meninges from the nasopharynx throuh the blood stream. The diseases is more common in children between 2 months and 3 years of age. This age incidence is because of the absence of bactericidal antibodies. Older children develop immunity. The  causative strain is Hib.
2.      Laryngoepiglottis : This disease is an acute inflammation of the epiglottis, seen in children above 2 years. If untreated death occours within hours. Tracheostomy is often neccesary to relieve respiratory obstruction caused by the grossly enlarged ulva. This is assoiated with bacteremia and blood cultures are usually positive.
3.      Pneumonia : Typically occurs in infants and is accoumpanied by empyema, meningitis. In older childrens and adults lobar pneumonia occours. This occur as primary infections due to capsulated strains, bronchopneumonia may occur as a secondary infections with the non-capsulated  strains.
In non invasive, the bacteria spread by local invasion along mucosal surface and cause secondary infections, usually of the respiratrory tracts. Eg : Sinusits, ear infections (otitis media). Non invasive infections are caused by non capsulate strains and are usually seen in  adults.
1.   Suppurative lesions : such as arthritis, endocarditis, pericarditis may result from  hematogenous dissemination. Otitis media(ear infection) occur by direct spread from the nasopharynx. Cellulitis in the buccal area is seen in young children.
2.   Bronchitis : Pimarily bronchi is involved, later infections spread to the alveoli of the lung and cause pneumonia. Patients cough sputum containing  mucus, pathogen and phagocytic cells. It is common in older people and is linked to smoking, air pollution, inhalation of coal dust etc.

Laboratary Diagnosis – The bacteria can be detected in CSF, blood, throat swab, sputum, pus, aspirates from joints, middle ears or sinuses etc. Presence of capsular polysaccharide antigens in CSF by demonstration of latex particle agglutination. For isolation, CSF should be plated on a blood agar or chocolate agar and incubated in an environment of 5-10% CO2 and high humidity. A strain of Staphylococcus should be streaked across the plate. After overnight incubatio at 370C, small opaque colonies appear and irridescence may be demonsterated on Levinthal's medium.

Blood cultures are often positive in laryngeoepiglottitis and pneumonia. Cultures can be done in nutrient broth as the patient's blood affords sufficient enrichment.
Sputum has to be homogenised by treatment with pancraetin or by shaking with sterile waer and glass beads for 15-30 minutes and then cultured.
Prophylaxis and treatment - Cefotaxime is the drug of choice for the Haemophilus meningitis. Amphicillin, cotrimoxazole were used for respiratory infection but as plasmid borne  resistance to the drug are common, amoxyllin -clavulanate is more effective.
Infection is transmitted by the respiratory route. Immunity is type specific. As large majority of serious infection are caused by type B strains, active immunization with Hib PRP vaccine is indicated. It is immunogenic in the older children and adults, but poorly to the children below 2 years. So  it is coupled with diphtheria and tetanus toxoids, and used in young children.

Haemophilus aegyptius  : causes a highly contagious form of conjuctivitis, ie., pink eye. It occurs in tropics. It responds to local sulfonamides or gentamicin. It is similar to non capsulated H. influenzae. This has haemagglutinating activity, no indole formation and no xylose fermentation. It grows slowly.
It also causes Brazilian Purpuric Fever(BPF), in which conjuctivitis leads to septicemia in infants with high fatality.
H. ducreyi: A short, ovoid bacillus with a tendency to occur end to end pairs or short chains.  The bacilli may be arranged in small groups or in parallel chain giving a “rail road track” appearance. It may be grown on fresh clotted rabbit blood, chorioallantoic membrane of chick embryo. It causes chancroid called soft chancre, which is a veneral disease.  It begins with the appearance of soft, painful lesion called chancres, which bleed easily, on the genitals 3-5 days after sexual exposure. The symptom is a burning sensation after urination. Chancre also occurs on the tongue and lips. It is highly infective. Medical personnel acquire lesions on the hands merely from contact with chancres. It spreads and forms enlarged masses of lymphatic tissues called buboes. These appear 1 week after infection, swell to great size, break through the skin, discharging pus to the surface.
Chancroid is diagnosed by identifying the organism in scrapings from lesions or in fluids from a bubo. Patients with chancroid also often have other STDs, thus a patient with a positive diagnosis for one STD should be tested for other STDs. No immunity. Treatment with tetracycline, erythromycin, sulfanilamide, or a combination of trimethoprim and sulfamethoxazole. With treatment lesions heal rapidly, but they often have deep scars with much tissue destruction.



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