Title: HAEMOPHILUS
1HAEMOPHILUS BORDETELLA
2Haemophilus 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
3Clinical 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
4Clinical 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
5Clinical 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
6Clinical characteristics, cont.
H. ducreyi Chanchroid, cont. - can be
distinguished from syphilitic lesions that
are painless - presence of genital ulcers
increases risk of HIV infection
7Clinical 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
8Laboratory 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
9Laboratory 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
10http//www2.mf.uni-lj.si/mil/bakt2/bakt2.htm
11Laboratory 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
12Laboratory Diagnosis, cont.
- Culture
- Haemophilus require hemin (X factor) and NAD
- (V factor)
- Chocolate agar contains both
- 5 Sheep blood agar only contains hemin
13http//gold.aecom.yu.edu/id/micro/xvfactor.htm
14Laboratory Diagnosis, cont.
- Culture
- S. aureus produces NAD as a metabolic product
- - Haemophilus will satellite around colonies
- of S. aureus when growing on BAP
15http//www.petermp.dk/oerepodning.htm
16Laboratory 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
17http//www.uni-ulm.de/klinik/imi/mikrobio_2002/kra
nkenversorgung/ Diagnostik/Erreger/h_keim.htm
18Laboratory Diagnosis, cont.
- Identification
- Growth characterics on solid media
- Gram stain morphology
- X and/or V factor requirement
- Satelliting
- Porphyrin test
19Treatment
- 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
20Prevention
- 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
21CDC PHIL
22Bordetella 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
23Clinical 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)
24Clinical 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
25Clinical 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
26Clinical 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
27Laboratory 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
28Laboratory 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
29DFA for Bordetella
ASM Color Atlas of Bacteriology
30Laboratory 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
31Laboratory 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
32B. pertussis on Regan-Lowe agar
ASM Color Atlas of Bacteriology
33Gram stain of B. pertussis
http//www2.mf.uni-lj.si/mil/bakt2/bakt2.htm
34Regan-Lowe w/ antibiotics
Bordet-Gengou w/o antibiotics
ASM Color Atlas of Bacteriology
35Treatment
- Antimicrobial Susceptibility Testing and Therapy
- Not routinely performed because Erythromycin and
- Azithromycin are active and remain drugs of
choice
36Prevention
- 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
37Neisseria 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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40Gram stain of Neisseria
41Gram stain of Moraxella
http//www.labquality.fi/finnish/alustavat_tulokse
t/gramvarjays_pesake.htm
42- 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)
43- 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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45- 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
46Culture of Neisseria
http//www.bmb.leeds.ac.uk/mbiology/ug/ugteach/den
tal/tutorials/std/gccult.html
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48Culture of Moraxella
http//www.infek.lu.se/bakt/english/picture5.htm
49- 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
50- 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
51- 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