Haemophilus

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Haemophilus 1 Haemophilus

Definition:

Definition Small to medium-sized coccobacilli or rods, markedly pleomorphic Gram -negative, non motile, non sporeforming and non acid-fast Aerobic and facultatively anaerobic Require one or both of two accessory growth factors – factor X and factor V Haemophilus 2

Definition :

Definition Chemo - organotrophic Sugars are attacked fermentatively Oxidase and catalase positive Nitrate is reduced to nitrite Obligate parasites of humans and animals The G+C content of DNA is 37–44 mol% The type species is H. influenzae Haemophilus 3

History :

History 1892 -- Pleiffer --- Haemophilus influenzae From the sputum of patients suffering from epidemic influenza 1883 -- Koch------ H. aegyptius Bacillus causing conjunctivitis in Egypt Weeks -----first isolated Koch –Weeks bacilli Now considered a biotype of Hemophilus infuenzae Haemophilus 4

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Haemophilus 5 Pleiffer & Koch

Species in the genus :

Species in the genus H. influenzae H. parainfluenzae H. haemolyticus H. aegyptius H. intermedius H.aphrophilus H. paraphrophilus H. ducreyi Haemophilus 6

Growth factor requirements :

Growth factor requirements 7 Haemophilus major criteria by which the genus Haemophilus is defined Species Requirement of factor X Requirement of factor V Hemolysis H.influenzae + + - H.parainfluenzae - + - H.ducreyi + - - H.hemolyticus + + + H.aphrophilus H. paraphrophilus + - - + - - H.parahemolyticus - + + H.segnis - + -

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H.aphrophilus & H. paraphrophilus are co 2 loving H.aphrophilus losses X factor requirement after initial isolation Now it come under separate genus Aggregatibacter Haemophilus 8

Habitat :

Habitat Obligate parasites of the mucous membranes of humans Inhabit the human upper respiratory tract and mouth May also be isolated from the intestinal tract and vagina Haemophilus 9

Role in normal human flora:

Role in normal human flora Humans are the hosts of various hemophili In the mouth, hemophili are found on the tongue, palate, cheeks, teeth, in dental plaque & in saliva H. influenzae Constitutes10 percent of the hemophili found in the pharynx Absent from the hemophilus flora of the oral cavity Haemophilus 10

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Hemophili Site H. parahaemolyticus Pharynx and oral cavity H. haemolyticus Nasopharyngeal flora of 1% of the population H. parainfluenzae 75 % of the hemophili isolated from the nasopharynx and mouth Found in the normal vaginal flora H. segnis ,H. paraphrophilus and H. aphrophilus Predilection for teeth Haemophilus 11

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The upper respiratory tract of the neonate is sterile, but rapidly becomes colonized by a wide variety of organisms, including NCHI Rate of nasopharyngeal colonization reaches 34 percent at 1 year 44 percent at 2 years By the age of 5–6 years, over 50 percent of children are colonized Haemophilus 12

Oropharyngeal carriage of Hib:

Oropharyngeal carriage of Hib Uncommon during the first 6 months of life 3–5 percent among children aged 3–5 years Factors influencing colonization rates: Age , ethnic origin , day-care attendance , close family contact with a case of invasive Hib disease , season of the year & recent antibiotic therapy Hib immunization of infants results in reduced pharyngeal carriage of Hib Giving Hib vaccine to a child does not rapidly terminate carriage Haemophilus 13

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Hemophili Carrier rate NCHI 50-80 % H. influenzae type b 2-4% H. influenzae type ( a,c,d,e,f ) 1 -2% Haemophilus 14

Growth factor requirements :

Growth factor requirements Specific growth factor requirements major criteria by which the genus Haemophilus is defined They will not grow in the absence of certain factors that are present in blood Haemophilus or ‘blood-loving H. influenzae requires two accessory growth factors heat-stable growth promoting substance present in red blood cells (X factor) heat-labile vitamin-like substance (V factor) Haemophilus 15

X factor:

X factor Hemin -dependent strains unable to convert δ- aminolaevulinic acid (ALA) to protoporphyrin Fildes (1924) named hemin as X factor and suggested it plays a role as an enzyme in respiration Factor X is required for the synthesis of catalase , peroxidase & cytochrome oxidase Haemophilus 16

X factor :

X factor Iron -containing compounds hemes or protoporphyrin IX, can satisfy a requirement for X factor Hemin sources hemoglobin– haptoglobin complexes, heme–hemopexin and heme –albumin The requirement for X-factor is substantially reduced during anaerobic growth and strains might be erroneously regarded as X-factor independent Haemophilus 17

V factor:

V factor Present in the tissues of plants and animals Synthesized by most bacterial species other than H. influenzae V-factor requirement can be satisfied by NAD + or by precursors of this coenzyme V-factor in blood is not available to hemophili it is largely intracellular blood of many animal species contains NADase activity Heating blood agar to about 75°C releases V-factor from red cells inactivates NADase activity Haemophilus 18

V factor:

V factor Staphylococcus aureus and some fungi, release V-factor during growth. This diffuses into the surrounding medium Any V-factor-requiring species of Haemophilus will show satellitism Bacteria showing satellitism Some strains of streptococci Neisseria Diphtheroids Haemophilus 19

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Haemophilus 20 This organism would be identified as H. influenzae because it is using both X and V factors. Nutrient Agar with discs

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21 This organism would be identified as H. parainfluenzae because it is using V factor only

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Haemophilus 22

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Haemophilus 23 Haemophilus influenzae

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Haemophilus 24 CSF morphology sputum

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Haemophilus 25 Haemophilus influenzae is a nonmotile , Gram-negative, rod-shaped bacterium. The rounded ends of short (0.5-1.5 micrometres ) bacilli make many appear round, hence the term coccobacilli

Physical requirements for growth :

Physical requirements for growth The optimum temperature 35–37°C The minimum temperature for growth 20–25°C Raised carbon dioxide tension improves the growth Carbon dioxide should be added to the atmosphere for the primary isolation Haemophilus 26

CULTURE MEDIA:

CULTURE MEDIA BA with Staph streak CA Fildes a g ar Levinthal a g ar Selective medium: CA with bacitracin 300m g /l Differential & selective media: Medium of Roberts Addition of bacitracin makes it selective Contains sucrose & phenol red to differentiate from parainfluenzae HI does not ferment sucrose so form colourless colonies Haemophilus 27

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Antiserum agar Columbia agar supplemented with yeast extract, hemin , NAD, bacitracin , and type b antiserum Colonies of H. influenzae type b can be readily discerned in mixed cultures from respiratory material since they have an obvious halo of antigen–antibody precipitate Haemophilus 28

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Haemophilus 29 Colonies of Haemophilus influenzae on chocolate agar (left) and no growth on blood agar (right).

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Haemophilus 30

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Haemophilus 31

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Haemophilus 32

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Haemophilus 33

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Haemophilus 34

Colony morphology :

Colony morphology On blood agar translucent, low convex or flat pinpoint colonies with satellitism & seminal smell Chocolate agar, Levinthal’s agar, or Fildes ’ agar Noncapsulate strains greyish , transparent, smooth and low convex or flat with a slightly splayed-out entire edge Attain a size of 0.5–0.8 mm after incubation for 24 h at 37°C Enlarges to 1–1.5 mm by 48 Capsulate strains form larger, more opaque, smooth, mucoid colonies 3–4 mm in diameter, which may appear to coalesce Haemophilus 35

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Hemolysis of horse blood agar differentiates H. influenzae from H. haemolyticus H. parainfluenzae from H. parahaemolyticus Haemophilus 36

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Haemophilus 37

Colonies of capsulate strains grown on clear media :

Colonies of capsulate strains grown on clear media Iridescent when examined by obliquely transmitted light Iridescence is due to dispersion of light by the arrangement of organisms in a grid-like pattern This phenomenon is best observed in 18-h cultures Haemophilus 38

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Produce indole , which gives the cultures a characteristic pungent smell H. paraphrophilus and H. aphrophilus Pit the blood agar Misidentified as Eikenella corrodens Hemolysis H. haemolyticus and H. parahaemolyticus Haemophilus 39

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H. influenzae strain Rd was the first bacterium for which the entire DNA sequence was determined H. influenzae is naturally transformable Haemophilus 40

Biochemical reactions :

Biochemical reactions Catalase + ve Oxidase + ve Nitrate is reduced to nitrite Glucose & xylose fermented 8 biotypes based on Indole production Ornithine decarboxylase activity Urease activity Biotype 1 isolated from most meningitis cases Haemophilus 41

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Haemophilus 42 Biotype 1 2 3 4 5 6 7 8 Indole production + + _ - + - + - Urease activity + + + + _ - - - Ornithine decarboxylase + _ _ + + + - -

Identification :

Identification A presumptive Haemophilus spp. isolate should first be tested for its X- and V-factor requirements Demonstrated by the absence of growth on media deficient in X and V factor except near paper disks or strips impregnated with X, V, or X plus V factors Consistent results will be obtained only if a small inoculum is used, to avoid carry over of the factors. Haemophilus 43

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V-factor requirement of strains can be inferred by demonstrating satellitism near a streak of S. aureus . As V factor is heat-labile, media in which all the constituents have been autoclaved will reliably be free of it. Most of the basic fluid and solid media contain traces of X factor, as do some brands and batches of peptone Haemophilus 44

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Porphyrin test Haemophilus 45 Provides an accurate and reliable method for determining X-factor requirement

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Haemophilus 46

Porphyrin test:

Porphyrin test Provides an accurate and reliable method for determining X-factor requirement A substrate of ALA and MgSO 4 in 0.1 M phosphate buffer pH 6.9 is used. A loopful of growth from a 24 h chocolate agar culture is added to 0.5 ml of substrate and the mixture is incubated for 4 h at 37°C. Porphyrin production is demonstrated by the development of a red fluorescence when the mixture is exposed to a Wood’s lamp (maximum emission 360 nm). Haemophilus 47

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Haemophilus 48 Alternatively, the presence of porphobilinogen can be demonstrated after incubation for 24 h by adding 0.5 ml of Kovac’s reagent A red color in the aqueous phase indicates the presence of porphobilinogen .

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Haemophilus 49

Biochemical tests :

Biochemical tests Carbohydrate utilization can be studied in phenol red broth base supplemented with 1 percent of the carbohydrate and 10 mg/l each of NAD + & hemin Read after incubation for 24 h Haemophilus 50

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Haemophilus 51 H.influenzae H.aegyptius H.haemolyticus H.parainfluenzae H.parahaemolyticus H.aphrophilus H.paraphrophilus H.segnis H.ducreyi Glucose, acid + + + + + + + - - Glucose , gas - - +/- +/- +/- + + - - Lactose, acid - - - - + + - - Sucrose acid - - - + + + + - - Mannose ,acid - - - + - + + - - Xylose ,acid + - +/- - - - - - -

Hemagglutination :

Hemagglutination Most strains of H.influenzae & many strains of H.aegyptius agglutinate red cells Tested in parallel with red cells of humans(group O), guinea pig, fowl, pig & sheep Test should be done at 4 0 c as they elute on warming to 20 0 c Reaction is mannose resistant Haemophilus 52

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Centrifuge red cells Wash thrice in saline(.85%) Resuspend to 3% in saline Harvest the growth ( buffered CA ) in saline to a high concentration (10 11 bacteria / ml) Mix a large drop of dense bacterial suspension with a large drop of 3% suspension of erythrocytes in a depression on a white tile chilled to 4 0 c Haemophilus 53

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Rock the tile to & fro for 13 mnts while keeping it at 4 0 c Then look for the presence of fine or coarse clumping of red cells Observe a control mixture of erythrocytes in saline to rule out auto agglutination Haemophilus 54

Antigenic structure:

Antigenic structure Capsule Pili Lipo -oligosaccharide Outer-membrane proteins Haemophilus 55

Capsule :

Capsule There are six antigenic types of capsule in H. influenzae , designated a–f ( Pittman 1931) Each one is composed of a linear polymer of disaccharide units Capsules of types a, b, c, and f contain phosphodiester linkages and are teichoic acids Type d and e contain glycosidic linkages and are true polysaccharides Haemophilus 56

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Types a and b differ from the others in containing the 5-carbon component ribitol Type b is unique as both sugars are pentoses ----ribose & ribitol The type b capsule consists of a phosphodiester -linked polymer of polyribosyl ribitol phosphate (PRP) PRP cross-reacts with ………..Streptococcus pneumoniae, Streptococcus pyogenes , Staphylococcus aureus , Staphylococcus epidermidis , Enterococcus faecium and Escherichia coli K100 Haemophilus 57

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Hib disease is associated with a lack of antibodies to polyribosyl ribitol phosphate. Infants less than 2 years old have little or no anti capsular antibodies once maternal antibodies decline Immunization of infants with PRP fails to elicit protective levels of antibody,  due to the T-cell-independent nature of the immune response to polysaccharide antigens. The conjugation of PRP to a protein carrier stimulates anti-PRP antibodies in young infants and is the basis of current Hib vaccines Haemophilus 58

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Hib from the nasopharynx usually express pili Do not express pili Isolates from blood and cerebrospinal fluid Following serial subculture Piliated H. influenzae bind to erythrocytes Haemophilus 59

Lipo-oligosaccharide :

Lipo -oligosaccharide The major constituent of the outer membrane is lipo -oligosaccharide (LOS) The lipid A backbone is similar to enterobacterial lipid A, but with a simpler fatty acid composition Antibodies to LOS have been demonstrated after infection but are not protective. Haemophilus 60

Outer-membrane proteins :

Outer-membrane proteins The outer membrane of H. influenzae contains 10–20 different protein The major OMPs are P1 ,P2 -- with porin activity P4 ,P5 ,P6 ---- involved in heme uptake Infant rat bacteremic model Antibodies to OMP P1, ,P5 &P6 were shown to be partially protective against Hib Haemophilus 61

Preservation and storage:

Preservation and storage Stored for a short time on chocolate agar slopes in screw-capped bottles or tubes Slopes should be heavily inoculated, incubated overnight at 37°C and stored at room temperature.  culture will remain viable for up to 4–6 weeks. Long-term storage maintained at –70°C in tryptic soy broth supplemented with 15–20 percent glycerol. Lyophilisation Haemophilus 62

Typing methods:

Typing methods Capsule typing Biotyping OMP subtyping DNA-based typing and characterization Haemophilus 63

Typing methods:

Typing methods Capsule typing Differentiated into capsulate and noncapsulate strains Six antigenically distinct types of capsule Detected by Latex agglutination , Staphylococcal co-agglutination, Counter current immune elctrophoresis , Immunoflurescence microscopy, DNA probes with cap genes Haemophilus 64

Biotyping :

Biotyping H. influenzae and H. parainfluenzae can each be subtyped on the basis of three biochemical tests (for indole , urease , and ornithine decarboxylase ) into eight biotypes Most clinical isolates ---- biotypes (I, II, III, and IV) Majority of NCHI are biotype II or III. Haemophilus 65

OMP subtyping :

OMP subtyping Sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) of OMPs has formed the basis for subtyping Hib . 13 subtypes Haemophilus 66

DNA-based typing and characterization :

DNA-based typing and characterization Restriction endonuclease analysis (REA) of chromosomal DNA PCR-based methods Ribotyping has been used to discriminate NCHI strains & Hib Haemophilus 67

Bacteriocins :

Bacteriocins Most H. influenzae type b produce a bacteriocin ‘ hemocin ’ Active against capsulate H. influenzae strains of other types, NCHI, other Haemophilus spp & certain enterobacteria (E. coli ) Not active against gram-positive bacteria Act by inhibiting DNA synthesis Haemophilus 68

Diseases caused by H. influenzae :

Diseases caused by H. influenzae Invasive Non invasive Meningitis Epiglottitis Pneumonia Cellulitis ( buccal & periorbital region) Osteomyelitis Septic arthritis Conjunctivitis Endocarditis , Pericarditis Otitis media Sinusitis A/C exacerbation of Chronic obstructive airway disease Haemophilus 69

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Haemophilus 70

Meningitis symptoms :

Meningitis symptoms Haemophilus 71

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Haemophilus 72

Clinical spectrum of H. influenzae infections:

Clinical spectrum of H. influenzae infections 73 Haemophilus Type of infection Age group strains Invasive 90% children <4 years 90% H. influenzae Type b Neonatal& maternal infections >90% by noncapsulate strains Non invasive respiratory Children & adults >90% by noncapsulate strains

Virulence factors in invasive disease:

Virulence factors in invasive disease The type b capsule major virulence factor aids colonization protects against phagocytosis and complement-mediated lysis Fimbriae – attachment to epithelial cells IgA proteases Lipopolysaccharide OMP P2 Haemophilus 74

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Haemophilus 75

Potentiation of invasion:

Potentiation of invasion Host genetic factors Intercurrent viral illness Immunosuppression Organism penetrates through the submucosa of nasopharynx & establishes systemic infection through blood stream Haemophilus 76

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Risk factors for Hib disease are mainly socioeconomic and include Overcrowding Attendance at day-care centers Chronic illness Lack of access to good health care facilities Haemophilus 77

Diseases caused by NCHI :

Diseases caused by NCHI Respiratory infections in patients with chronic bronchitis, bronchiectasis or cystic fibrosis Acute otitis media & acute and chronic sinusitis Community-acquired pneumonia & conjunctivitis Neonatal meningitis & septicemia Strains of NCHI biotype IV are a significant cause of urogenital and neonatal infections Haemophilus 78

Diseases caused by NCHI :

Diseases caused by NCHI In NCHI meningitis there are predisposing factors such as head injury, with or without a cerebrospinal fluid leak, otitis media or sinusitis, and the infection is presumed to result from direct extension Haemophilus 79

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H. influenzae biogroup aegyptius is associated with an acute form of seasonal conjunctivitis seen mainly in hot climates and in children. It has been implicated as the cause of BPF ( Brazilian purpuric fever) Haemophilus 80

Complication of meningitis:

Complication of meningitis Subdural effussions Brain abscess Pericarditis Septic arthritis Localised infection at other sites Haemophilus 81

Lab diagnosis:

Lab diagnosis Specimens CSF  never be refrigerated Blood Sputum Joint aspirate Ear swab Conjunctival swab Pus aspirates from cellulitis & osteomyelitis Haemophilus 82

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blood innoculated on BHIB Visual examintion cannot be relied upon to indicate positive growth in BHIB bottles Routine sub culture should be done unless an automated system is used Blood cultures are often positive in cases of laryngo epiglottitis , pneumonia & meningitis Haemophilus 83

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Large variations in rates of isolation occur when different parts of sputum are sampled So sputum sample should be well homogenised by treating with pancreatin or shaking with sterile water & glass beads for 15 – 30 mnts Haemophilus 84

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Isolation from lower respiratory tract may be the result of contamination with colonising flora from the upper respiratory tract so try direct bronchoscopy & lavage The presence of a predominance of H.influenzae on direct plating, or in large numbers( 10 6 /ml) from cultures of dilutions of homogenised purulent sputum will support a pathogenic role Haemophilus 85

SPECIMEN COLLECTION AND TRANSPORT :

SPECIMEN COLLECTION AND TRANSPORT Haemophilus spp. are very susceptible to drying and temperature extremes Specimens must be plated immediately Haemophilus 86

SPECIMEN PROCESSING :

SPECIMEN PROCESSING DIRECT DETECTION METHODS Direct Observation Gram stain Acridine orange stain Haemophilus 87 Body fluid specimens are centrifuged (2000 rpm for 10 minutes) and the smear is prepared from the resulting pellet  will increase sensitivity by five fold to tenfold

Antigen Detection :

Antigen Detection Detection of H. influenzae type b capsular polysaccharide directly in clinical specimens, such as CSF and urine, can be performed using commercially available particle agglutination assays PCR Haemophilus 88

CULTIVATION :

CULTIVATION Media of Choice BA with Staph streak, CA A selective medium  horse blood– bacitracin agar, used for isolation of H . influenzae from respiratory secretions. Designed to prevent overgrowth of H. influenzae by mucoid Pseudomonas Haemophilus spp. will not grow on Mac- Conkey agar. Haemophilus 89

Incubation Conditions and Duration :

Incubation Conditions and Duration Growth is stimulated by 5% to10 % carbon dioxide Grow within 24 hours Cultures are held for 72 hours 90

APPROACH TO IDENTIFICATION :

APPROACH TO IDENTIFICATION Traditional identification criteria include the requirement for X and V factors for growth Porphyrin test 91

Antibiotic susceptibility:

Antibiotic susceptibility H. influenzae is commonly susceptible in vitro to Ampicillin , Cefotaxime , ceftriaxone , cefixime , cefpodoxime , cefaclor , cefuroxime , Chloramphenicol , Tetracycline, Aminoglycosides , Rifampicin , Meropenem , Ciprofloxacin , Sulphonamides , Clarithromycin , Azithromycin Haemophilus 92

Ampicillin resistance:

Ampicillin resistance Production of a β- lactamase is the most common mechanism Haemophilus 93 β- lactamase Year TEM-1 β- lactamase 1974 ROB-1 1981 VAT-1 1994

BLNAR strains :

BLNAR strains β- lactamase negative Ampicillin resistant strains Mecahanism : decreased binding to PBP 3A & 3B Haemophilus 94

Chloramphenicol resistance:

Chloramphenicol resistance First noted in 1976 Two mechanisms Plasmid-mediated  chloramphenicol acetyl transferase Decreased uptake of chloramphenicol by the cell  loss of an outer-membrane protein Haemophilus 95

Resistance to co-trimoxazole:

Resistance to co- trimoxazole Varies from region to region, but in some countries now exceeds 50 percent Resistance to β- lactams , macrolides and co- trimoxazole is more frequently observed in respiratory isolates than in blood culture isolates. Resistance rates also tend to be higher in NCHI than in H. influenzae type b Haemophilus 96

Antimicrobial susceptibility testing:

Antimicrobial susceptibility testing Haemophilus test medium (HTM) Mueller–Hinton agar + 50mg NAD+50 mg hematin and yeast extract 5 g NCCLS specifies the use of HTM Haemophilus 97

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Haemophilus 98 The British Society for Antimicrobial Chemotherapy (BSAC) stipulates the use of Iso-Sensitest agar supplemented with 5 percent defibrinated horse blood and 20 mg/l NAD

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E-test ---an antimicrobial agent gradient-coated plastic test strip which allows MIC determinations on solid media--------can be used The detection of antimicrobial resistance genes by the use of DNA probes or PCR is now possible Haemophilus 99

Treatment :

Treatment Meningitis & epiglottitis DOC Cefotaxime \ ceftriaxone Ampicillin + chloramphenicol Duration : 1-2 weeks Noninvasive infections Oral antibiotics- Amoxiclav , Clarithromycin , Ciprofloxacin, Azithromycin 20-35% of NCHI resistant to Ampicillin Haemophilus 100

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For children Cefotaxime is given as 200mg/kg/day, divided six hourly Ceftriaxone is 75 to 100 mg/kg, divided 12 hourly Adult doses Ceftriaxone , 2g every 12 hours Cefotaxime 2g every 4 to 6 hours The usual duration of therapy is 7 to 10 days . Haemophilus 101

Prevention & Control:

Prevention & Control Currently four conjugate vaccines are available PRP-D is not recommended or licensed for children under 12 months of age. HbOC and PRP-T are also available in combination with DTP and DTaP vaccines Widespread use of H influenzae type b vaccine has reduced the incidence of H influenzae type b meningitis in children by over 95%. The vaccine reduces the carrier rates for H influenzae type b. Haemophilus 102

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Conjugate PRP- HbOC CRM197, a nontoxic diphtheria toxin PRP-OMP Outer membrane protein complex of Neisseria meningitidis PRP-T Tetanus toxoid PRP-D diphtheria toxoid Haemophilus 103

Conjugate Hib Vaccines:

Conjugate Hib Vaccines Commercial preparations PRP-D ProHIBIT HbOC Hibtiter PRP-T ActHIB , OmniHIB , TriHIBit PRP-OMP PedvaxHIB , COMVAX

Haemophilus influenzae type b Vaccine:

Haemophilus influenzae type b Vaccine Vaccination at <6 weeks of age may induce immunologic tolerance to Hib antigen Minimum age 6 weeks Minimum interval 4 weeks for primary series doses

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Vaccine 2 months 4 months 6 months 12-18 months HbOC + + + + PRP-T + + + + PRP-OMP + + + Haemophilus 106

Haemophilus influenzae type b Vaccine Delayed Vaccination Schedule:

Haemophilus influenzae type b Vaccine Delayed Vaccination Schedule Children starting late may not need entire 3 or 4 dose series Number of doses child requires depends on current age All children 15-59 months of age need at least 1 dose

Haemophilus influenzae type b Vaccine Detailed Schedule for Unimmunized Children:

Vaccine Age at 1st Dose (months) Primary series Booster HbOC /PRP-T PRP-OMP PRP-D 2-6 7-11 12-14 15-59 2-6 7-11 12-14 15-59 15-59 3 doses, 2 m apart 2 doses, 2 m apart 1 dose 1 dose 2 doses, 2 m apart 2 doses, 2 m apart 1 dose 1 dose 1 dose 12-15 months 12-18 months 2 months later -- 12-15 months 12-18 months 2 months later -- Haemophilus influenzae type b Vaccine Detailed Schedule for Unimmunized Children --

Haemophilus influenzae type b Vaccine Vaccination following invasive disease:

Haemophilus influenzae type b Vaccine Vaccination following invasive disease Children <24 months may not develop protective antibody after invasive disease Vaccinate during convalescence Complete series for age

Haemophilus influenzae type b Vaccine Use in older children and adults:

Haemophilus influenzae type b Vaccine Use in older children and adults Consider for high risk persons: asplenia , immunodeficiency, HIV infection One pediatric dose of any conjugate vaccine

Chemoprophylaxis :

Chemoprophylaxis Rifampicin 20 mg / kg ( max 600 mg) for 4 days Haemophilus 111

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Rifampin prophylaxis is recommended for all household members including adults when there has been contact with an index case of H. influenzae type b disease by a household member who is younger than 48 months and whose immunization status with the conjugate vaccine is incomplete, or is an immunocompromised child Haemophilus 112

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A contact is defined as a child who is a household member or who has spent 4 or more hours each day with the index case for at least 5 of the 7 days preceding the day that the index case was hospitalized. Chemoprophylaxis is not recommended when all household contacts younger than 48 months have completed their vaccination. Haemophilus 113

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Rifampin should be given within 7 days after the index patient is hospitalized. The index patient should also be given rifampin if treated with regimens other than cefotaxime or ceftriaxone . Chemoprophylaxis is usually provided just before discharge from the hospital Unvaccinated siblings of the index case who are less than 10 years should be given chemoprophylaxis Haemophilus 114

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Rifampin prophylaxis is indicated for all attendees and personnel at a daycare center or nursery when two or more cases of invasive H.influenzae type b disease have occurred within 60 days if incompletely immunized children attend the facility. Chemoprophylaxisis not indicated for a single case at a daycare center or nursery. Haemophilus 115

Haemophilus ducreyi :

Haemophilus ducreyi Ducrey (1889) demonstrated bacilli in smears prepared from the exudates of soft genital sores ( chancroid ) Bezancon et al. (1900) succeeded in isolating the organisms on nutrient agar plus 30 percent whole fresh rabbit blood Haemophilus 116

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DNA–RNA hybridization have been used to clarify the taxonomic position of H. ducreyi . The results suggest that H. ducreyi is a member of the Pasteurellaceae No formal renaming has been proposed Haemophilus 117

Morphology :

Morphology The organisms  intracellularly or extracellularly , and appear as pleomorphic coccobacilli or short rods. Haemophilus 118

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Gram variable Bipolar staining Arranged in parrallel chains  school of fish or rail road track appearance Haemophilus 119

Cultural characteristics and growth requirements :

Cultural characteristics and growth requirements Fastidious organism Grows slowly on chocolate agar Use of two media is optimal Gonococcal agar-based and a Mueller–Hinton-based medium can be used To this add 1 percent IsoVitalex 5 percent chocolatized horse blood or 1 percent hemoglobin 5 percent fetal calf serum Haemophilus 120

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Media made selective by the addition of vancomycin 3 mg/l Fresh plates with a moist surface should be used Incubated for 3–4 days at 33–36°C with added moisture and carbon dioxide Haemophilus 121

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Can also be grown on Fresh clotted rabbit blood Chorioallantoic membrane of chick embryo Thioglycollate – hemin -based transport medium can be used for transportation Haemophilus 122

Colony morphology :

Colony morphology Varies with the medium used. After incubation for 24 h, the colonies are small, yellow-gray, translucent or semi-opaque. Cultures often appear ‘mixed’ with a variety of colonial sizes and appearance. The colonies are very cohesive and can be pushed intact across the agar surface. It is extremely difficult to emulsify the colonies. Haemophilus 123

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Causes soft chancre Tender nonindurated irregular ulcer on the genitalia Disease is localised Spread only to the regional lymph nodes Haemophilus 124

Treatment :

Treatment Azithromycin 1g single dose Ceftriaxone 250 mg i.m single dose Ciprofloxacin 500mg b.d for 3 days Erythromycin 500mg tds for 7 days Haemophilus 125

Other Haemophilus spp :

Other Haemophilus spp Mainly of low virulence, opportunistic pathogens Cause infections similar to H. influenzae but much less common Constitute the Hacek group causing sub acute bacterial endocarditis H.parainfluenzae : urethritis , purperal infections,acute pharyngitis ,neonatal sepsis H.aphrophilus : dental abscess, brain abscess, sinusitis pneumonia Haemophilus 126

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Haemophilus 127 Thank you