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HAEMOPHILUS BORDETELLA

HAEMOPHILUS BORDETELLA. Haemophilus sp. Organism Reservoir Transmission H. influenzae Normal flora of human Person-to-person, droplets; upper resp. tract sometimes endogenous H. ducreyi Not normal flora; only Person-to-person; sexual present during infection contact

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HAEMOPHILUS BORDETELLA

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  1. HAEMOPHILUS BORDETELLA

  2. Haemophilus sp. Organism Reservoir Transmission H. influenzae Normal flora of human Person-to-person, droplets; upper resp. tract sometimes endogenous H. ducreyi Not normal flora; only Person-to-person; sexual present during infection contact Other Haemophilus spp. Normal flora of human Spread of endogenous strain upper resp. tract to non-resp. tract sites; less common than H. influenzae

  3. Clinical characteristics H. influenzae Major virulence factor is polyribitol phosphate capsule - enhance resistance to phagocytosis - serologic typing based on antigenic characteristics - six capsule types: a, b, c, d, e, or f - type b is the most commonly associated with serious human infection - infections are often systemic and life-threatening: meningitis, epiglottitis, cellulitis with bacteremia, septic arthritis, and pneumonia Also produce factors that promote attachment to human epithelial cells

  4. Clinical characteristics, cont. H. influenzae Non-typeable strains do not produce a capsule - virulence mediated through attachment (pili, etc.) - infections are typically less serious and more localized: otitis media, sinusitis, conjuctivitis, and bronchitis - pneumonia and bacteremia in adults with underlying medical conditions - isolated from patients with cystic fibrosis

  5. Clinical characteristics, cont. H. ducreyi Virulence factors also uncertain but probably include capsule, pili and toxins involved in attachment and penetration human epithelial cells Etiologic agent of chanchroid - genital lesions beginning as tender papules that progress to painful ulcers with several satellite lesions - regional lymphadenitis - primarily occurs in lower socioeconomic groups in tropical areas

  6. Clinical characteristics, cont. H. ducreyi Chanchroid, cont. - can be distinguished from syphilitic lesions that are painless - presence of genital ulcers increases risk of HIV infection

  7. Clinical characteristics, cont. Other Haemophilus spp. Mainly low virulence, opportunistic pathogens Cause infections similar to H. influenzae but much less common H. aphrophilus is an uncommon cause of brain abscesses and endocarditis - H of HACEK; subacute bacterial endocarditis

  8. Laboratory Diagnosis • Specimen collection • Can be isolated from most clinical specimens • - relatively high bacterial load in blood • of children with bacteremia • Susceptible to drying and temperature extremes • For H. ducreyi, specimen should be plated within • 10 min. of collection

  9. Laboratory Diagnosis, cont. • Direct detection • Gram stain: most are small, faintly staining, • gram-negative coccobacilli • H. ducreyi often described as “school of fish” • - mostly seen in lymph node specimens

  10. http://www2.mf.uni-lj.si/~mil/bakt2/bakt2.htm

  11. Laboratory Diagnosis, cont. • Direct detection • Latex agglutination can be performed on CSF or urine • - can be falsely-positive for recent vaccinees • - sensitivity is equivalent to Gram stain

  12. Laboratory Diagnosis, cont. • Culture • Haemophilus require hemin (X factor) and NAD • (V factor) • Chocolate agar contains both • 5% Sheep blood agar only contains hemin

  13. http://gold.aecom.yu.edu/id/micro/xvfactor.htm

  14. Laboratory Diagnosis, cont. • Culture • S. aureus produces NAD as a metabolic product • - Haemophilus will satellite around colonies • of S. aureus when growing on BAP

  15. http://www.petermp.dk/oerepodning.htm

  16. Laboratory Diagnosis, cont. • Culture, cont. • Growth conditions: • Haemophilus spp.: 35 – 37°C, 5-10% CO2, 3 days • H. ducreyi: 33 – 35°C, 5-10% CO2, 7 days • - also require supplemented media • Colony morphology: • Small and translucent • Exude a “mouse nest” odor

  17. http://www.uni-ulm.de/klinik/imi/mikrobio_2002/krankenversorgung/http://www.uni-ulm.de/klinik/imi/mikrobio_2002/krankenversorgung/ Diagnostik/Erreger/h_keim.htm

  18. Laboratory Diagnosis, cont. • Identification • Growth characterics on solid media • Gram stain morphology • X and/or V factor requirement • Satelliting • Porphyrin test

  19. Treatment • Antimicrobial Susceptibility Testing and Therapy • Routine testing can be performed using disk diffusion • or broth dilution • Special supplemented media required • Beta-lactamase testing routinely performed • Test panel limited because of lack of resistance to • later generation cephalosporins • Cefotaxime or Ceftriaxone are drugs of choice

  20. Prevention • Vaccine • Routine vaccination with protein-polysaccharide • conjugated vaccine (Hib) • Significant reduction of serious, life-threatening • infections in children • Recommended starting at 2 months of age

  21. CDC PHIL

  22. Bordetella sp. Organism Reservoir Transmission B. pertussis Not normal flora; only Person-to-person; airborne present during infection transmission via cough B. parapertussis Not normal flora; only Person-to-person; airborne present during infection transmission via cough B. bronchiseptica Normal flora of animal Exposure to contaminated upper resp. tract droplets following close (dogs, cats, rabbits) contact with animals

  23. Clinical characteristics • B. pertussis and B. parapertussis • - cause URT infections in humans with almost identical • symptoms, epidemiology and therapeutic management • - Pertussis (whooping cough) • - optimal recovery requires special culture media • B. bronchiseptica • - opportunistic infection in compromised patients with • history of close animal contact (pneumonia, bacteremia, • UTI, meningitis, endocarditis)

  24. Clinical characteristics, cont. • Epidemiology • Pertussis primarily caused by B. pertussis, rarely by • B. parapertussis; former cause more severe disease • - higher infection rates and increased duration • of symptoms • Prior to vaccine, epidemic disease occurred in 2 – 5 • cycles; still occurs in unvaccinated populations • Adults and adolescents can serve as reservoirs and • transmit to unvaccinated or vaccinated with waning • immunity

  25. Clinical characteristics, cont. Pathogenesis Multiple virulence factors with various functions Adhesion Fimbriae Filamentous hemagglutinin Toxicity Pertussis toxin Adenylate cyclase toxin Tracheal cytotoxin Outer membrane inhibits host lysozyme Siderophore production to circumvent host iron sequestering

  26. Clinical characteristics, cont. Spectrum of disease Catarrhal Mild cold Several weeks Paroxysmal Severe coughing 1 to 4 weeks “Whooping” Convalescent  Symptoms Months Symptoms in adults tend to be milder and are misdiagnosed as bronchitis; also tend to be mixed with other pathogens

  27. Laboratory Diagnosis • Specimen collection • Nasopharyngeal wash or swab (Calcium alginate or • dacron on a flexible wire shaft) • Swabs should be immediately inoculated onto media • and direct smears made at the bedside • Swabs not directly inoculated should be placed in • transport if time to lab is extended

  28. Laboratory Diagnosis, cont. • Direct detection • DFA of smear using polyclonal Abs against B. pertussis • and B. parapertussis • Sensitivity is limited (50 – 70% at best), so should always • be used in conjunction with culture • PCR methods (home-brew and commercial assays) are • increasing in use and are replacing culture as gold standard • - specificity has been an issue

  29. DFA for Bordetella ASM Color Atlas of Bacteriology

  30. Laboratory Diagnosis, cont. • Culture • Historical gold standard • Selective media required • Bordet-Gengou • - Potato infusion agar with glycerol and sheep blood • Methicillin or cephalexin • Regan-Lowe • - Charcoal agar with 10% horse blood • Cephalexin

  31. Laboratory Diagnosis, cont. • Culture, cont. • 35 – 37°C, 5 – 10% CO2, hold for 10 – 12 days • - most isolates are detected in 3 – 5 days • Colonies are small, shiny; resemble mercury drops • Gram stain shows small, faintly staining gram negative • coccobacilli • - confirm identity with DFA reagents • - can distinguish between B. pertussis and • B. parapertussis

  32. B. pertussis on Regan-Lowe agar ASM Color Atlas of Bacteriology

  33. Gram stain of B. pertussis http://www2.mf.uni-lj.si/~mil/bakt2/bakt2.htm

  34. Regan-Lowe w/ antibiotics Bordet-Gengou w/o antibiotics ASM Color Atlas of Bacteriology

  35. Treatment • Antimicrobial Susceptibility Testing and Therapy • Not routinely performed because Erythromycin and • Azithromycin are active and remain drugs of choice

  36. Prevention • Vaccine • Whole-cell vaccines have been used historically • - adverse reactions and waning immunity • Acellular vaccines have been developed and include • booster doses for older children and adults

  37. Neisseria and Moraxella • General characteristics • Gram-negative diplococci, oxidase-positive • Epidemiology • Table 45-1 • Pathogenesis • Table 45-2 • Other Neisseria are saprophytes

  38. Gram stain of Neisseria

  39. Gram stain of Moraxella http://www.labquality.fi/finnish/alustavat_tulokset/gramvarjays_pesake.htm

  40. Laboratory Diagnosis • Specimen collection and transport • No special considerations for Moraxella • Pathogenic Neisseria are sensitive to drying and temp extremes • Swabs are acceptable for GC culture if plated in 6 hrs. • best method for GC culture is direct inoculation • Describe JEMBEC plates • Blood cultures as per routine, although Neisseria inhibited by high conc of SPS • Specimen processing • JEMBEC should be incubated as soon as received in lab • Body fluids should be kept at RT or 37C before culture (not cold) • Vol >1 ml should be concentrated and plate the sediment (e.g. joint fluid or CSF)

  41. Laboratory detection • Direct detection • Gram stain • shows GN diplococci for both genera; Moraxella tend to be bigger and fatter • GNDCs in PMNs from the urethral discharge of symptomatic male is diagnostic for GC • Normal vaginal and rectal flora has GNDCs so diagnosis requires confirmation • Antigen detection • not recommended; poor sensitivity • Molecular detection • Amplified methods are more sensitive than non-amplified methods • Increased detection of GC overall • Can test for CT at the same time • Cannot be used as evidence in medico-legal cases • We use B-D Viper automated instrument

  42. Laboratory detection • Culture • Media of choice • N. meningitidis, Moraxella and saprophytic Neisseria grow well on BAP, CAP • GC requires enriched CAP on primary culture • Selective media have been developed to inhibit normal flora and allow N. meningitidis and GC to grow • Modified Thayer-Martin • IsoVitaleX, colistin, nystatin, vanco, trimethoprim • Martin Lewis is similar • Incubation conditions and duration • 35-37C, 3 - 7% CO2, humid, 72 hrs • this CO2 conc can be achieved in incubator or candle jar • Colony appearance

  43. Culture of Neisseria http://www.bmb.leeds.ac.uk/mbiology/ug/ugteach/dental/tutorials/std/gccult.html

  44. Culture of Moraxella http://www.infek.lu.se/bakt/english/picture5.htm

  45. Laboratory detection • Approach to identification • Biochemicals • Moraxella: glucose -, maltose -, lactose -, butyrate disk +, ox + • GC: g +, m -, l –, ox + • NM: g +, m +, l –, ox + • Saprophytes: + + + or any other combo • Culture confirm and ID must be unequivocal in abuse cases • Saprophytes are not routinely identified (i.e. from respiratory cultures • Serotyping • Mening: A, B, C, Y, W135

  46. Susceptibility testing and therapy • Moraxella • testing not routinely performed because many options available • beta-lactams; b-l/b-lactamase inhib; cephs; macrolides; quinolones; bactrim • GC • routinely not performed because most labs use molecular so no isolate • resistance is a Public Health issue so surveillance mechanisms exist • penicillin resistance is widespread • ceftriaxone resistance not documented • quinolone resistance is emerging problem • N. meningitidis • not routinely performed; resistance rare • pen, cephs

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