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HAEMOPHILUS BORDETELLA. Haemophilus sp. OrganismReservoirTransmission H. influenzae Normal flora of humanPerson-to-person, droplets; upper resp. tractsometimes endogenous H. ducreyi Not normal flora; onlyPerson-to-person; sexual present during infectioncontact

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HAEMOPHILUS BORDETELLA

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HAEMOPHILUS

BORDETELLA


Haemophilus sp.

OrganismReservoirTransmission

H. influenzaeNormal flora of humanPerson-to-person, droplets;

upper resp. tractsometimes endogenous

H. ducreyiNot normal flora; onlyPerson-to-person; sexual

present during infectioncontact

Other Haemophilus spp.Normal flora of humanSpread of endogenous strain

upper resp. tractto non-resp. tract sites; less

common than H. influenzae


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


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


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


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


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


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


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


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


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


Laboratory Diagnosis, cont.

  • Culture

  • Haemophilus require hemin (X factor) and NAD

  • (V factor)

  • Chocolate agar contains both

  • 5% Sheep blood agar only contains hemin


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


Laboratory Diagnosis, cont.

  • Culture

  • S. aureus produces NAD as a metabolic product

  • - Haemophilus will satellite around colonies

  • of S. aureus when growing on BAP


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


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


http://www.uni-ulm.de/klinik/imi/mikrobio_2002/krankenversorgung/

Diagnostik/Erreger/h_keim.htm


Laboratory Diagnosis, cont.

  • Identification

  • Growth characterics on solid media

  • Gram stain morphology

  • X and/or V factor requirement

  • Satelliting

  • Porphyrin test


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


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


CDC PHIL


Bordetella sp.

OrganismReservoirTransmission

B. pertussisNot normal flora; onlyPerson-to-person; airborne

present during infectiontransmission via cough

B. parapertussisNot normal flora; onlyPerson-to-person; airborne

present during infectiontransmission via cough

B. bronchisepticaNormal flora of animalExposure to contaminated

upper resp. tractdroplets following close

(dogs, cats, rabbits)contact with animals


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)


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


Clinical characteristics, cont.

Pathogenesis

Multiple virulence factors with various functions

AdhesionFimbriae

Filamentous hemagglutinin

ToxicityPertussis toxin

Adenylate cyclase toxin

Tracheal cytotoxin

Outer membrane inhibits host lysozyme

Siderophore production to circumvent host iron sequestering


Clinical characteristics, cont.

Spectrum of disease

CatarrhalMild coldSeveral weeks

ParoxysmalSevere coughing1 to 4 weeks

“Whooping”

Convalescent SymptomsMonths

Symptoms in adults tend to be milder and are misdiagnosed

as bronchitis; also tend to be mixed with other pathogens


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


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


DFA for Bordetella

ASM Color Atlas of Bacteriology


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


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


B. pertussis on Regan-Lowe agar

ASM Color Atlas of Bacteriology


Gram stain of B. pertussis

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


Regan-Lowe

w/ antibiotics

Bordet-Gengou

w/o antibiotics

ASM Color Atlas of Bacteriology


Treatment

  • Antimicrobial Susceptibility Testing and Therapy

  • Not routinely performed because Erythromycin and

  • Azithromycin are active and remain drugs of choice


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


Neisseria and Moraxella

  • General characteristics

    • Gram-negative diplococci, oxidase-positive

  • Epidemiology

    • Table 45-1

  • Pathogenesis

    • Table 45-2

      • Other Neisseria are saprophytes


Gram stain of Neisseria


Gram stain of Moraxella

http://www.labquality.fi/finnish/alustavat_tulokset/gramvarjays_pesake.htm


  • 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)


  • 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


  • 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


Culture of Neisseria

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


Culture of Moraxella

http://www.infek.lu.se/bakt/english/picture5.htm


  • 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


  • 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


  • Prevention

    • Vaccine available for A, C, Y, W135

      • military recruits, college students, asplenics > 2 y.o.

    • Chemoprophylaxis with rifampin, cipro, or ceftriaxone for close contacts of patients with meningococcal disease

      • no chemo prophylaxis for pneumococcal mening

    • Eye antibiotics for neonates to prevent gonococcal eye infections


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