HAEMOPHILUS

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HAEMOPHILUS BORDETELLA
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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
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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
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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
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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
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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
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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
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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

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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

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

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Laboratory Diagnosis, cont.

• Culture
• Haemophilus require hemin (X factor) and NAD
• (V factor)
• Chocolate agar contains both
• 5 Sheep blood agar only contains hemin

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http//gold.aecom.yu.edu/id/micro/xvfactor.htm
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Laboratory Diagnosis, cont.

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

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http//www.petermp.dk/oerepodning.htm
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Laboratory Diagnosis, cont.

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

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http//www.uni-ulm.de/klinik/imi/mikrobio_2002/kra
nkenversorgung/ Diagnostik/Erreger/h_keim.htm
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Laboratory Diagnosis, cont.

• Identification
• Growth characterics on solid media
• Gram stain morphology
• X and/or V factor requirement
• Satelliting
• Porphyrin test

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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

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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

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CDC PHIL
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Bordetella sp.
Organism Reservoir Transmission B.
pertussis Not normal flora only Person-to-person
airborne present during infection transmissio
n 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
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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)

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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

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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
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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
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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

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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

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DFA for Bordetella
ASM Color Atlas of Bacteriology
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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

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Laboratory Diagnosis, cont.

• Culture, cont.
• 35 37C, 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

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B. pertussis on Regan-Lowe agar
ASM Color Atlas of Bacteriology
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Gram stain of B. pertussis
http//www2.mf.uni-lj.si/mil/bakt2/bakt2.htm
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Regan-Lowe w/ antibiotics
Bordet-Gengou w/o antibiotics
ASM Color Atlas of Bacteriology
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Treatment

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

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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

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Neisseria and Moraxella

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

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Gram stain of Neisseria
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Gram stain of Moraxella
http//www.labquality.fi/finnish/alustavat_tulokse
t/gramvarjays_pesake.htm
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• 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 gt1 ml should be concentrated and plate the
  sediment (e.g. joint fluid or CSF)

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• 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

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• 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

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Culture of Neisseria
http//www.bmb.leeds.ac.uk/mbiology/ug/ugteach/den
tal/tutorials/std/gccult.html
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Culture of Moraxella
http//www.infek.lu.se/bakt/english/picture5.htm
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• 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

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• 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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• Prevention
• Vaccine available for A, C, Y, W135
• military recruits, college students, asplenics gt
  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