Transcript H.flu,Bordettela (2010).ppt

H.Flu
Ali Somily MD
HAEMOPHILUS SPECIES
• General Characteristics
• Haemophilus influenzae
 Historical perspective
 Virulence factors
 Clinical manifestations of
Haemophilus influenzae
infections
• Infections Associated with
Other Haemophilus Species
• Laboratory Diagnosis
• Specimen processing and
isolation
• Microscopic morphology
• Colony morphology
Laboratory identification
Treatment
Pasteurellaceae
• Pasteurellaceae.
– Haemophilus
– Pasteurella
• Gramnegative
– Pleomorphic, coccoid to rod-shaped cells
• Nonmotile and aerobic or facultatively
anaerobic
• Nitrites from nitrates
• Oxidase and catalase positive.
IDENTIFICATION OF
HAEMOPHILUS SPECIES
Haemophilus are associated with
humans
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Haemophilus influenzae
Haemophilus parainfluenzae
Haemophilus haemolyticus,
Haemophilus
parahaemolyticus,
Haemophilus aphrophilus,
Haemophilus
paraphrophilus,
Haemophilus
paraphrohaemolyticus,
Haemophilus aegyptius,
Haemophilus segnis, and
Haemophilus ducreyi.
• Six species are
associated with animals
• 3 species of uncertain
status.
Haemophilus
• Greek words meaning "blood-lover."
• Require preformed growth factors present in blood:
– X factor (hemin, hematin)
– V factor (nicotinamideadenine dinucleotide [NAD]).
• Small, gram-negative bacillus (coccobacillus)/ X and/or V
• Para- require V factor only
• Production of hemolysis on 5% horse's or rabbit's blood agar
• Although certain species are also hemolytic on a sheep's blood
agar plate,
• Not grow in pure culture on sheep's blood
Satellitism
• A phenomenon that helps in the recognition of Haemophilus
species that require V factor is satellitism.
• Satellitism occurs when an organism such as Staphylococcus
aureus, Streptococcus pneumoniae, or Neisseria species
produces V factor (NAD) as a byproduct of its metabolism.
• The Haemophilus organisms isolate obtains X factor from the
sheep's blood agar and V factor from one of these organisms.
• On sheep's blood agar plate, tiny colonies of Haemophilus
organisms may be seen growing or engaging in "satellitism"
around the V factorproducing organism
• Except for H. aphrophilus and H ducreyi, all clinically
significant Haemophilus species require V factor for growth
and display this unusual growth pattern
• Upper respiratory tract 10% of this normal bacterial
flora in adults consists of Haemophilus spp.
• H. parainfluenzae and nonencapsulated H. influenzae.
• Of the two, H. parainfluenzae is the predominant
species.
• Begin in infancy with encapsulated strains 2% to
6%
• Day care centers, colonization may reach as high as
60%.
• Nonencapsulated strains of H. influenzae in healthy
children average 2% of the normal bacterial flora.
Haemophilus influenzae
Historical perspective
• "flu," is a viral infectious disease characterized by acute inflammation of the upper airways.
• Symptoms
• inflammation of the mucous membranes lining the nose
(coryza), headache,
• bronchitis, and severe generalized muscle pain (myalgias).
• Haemophilus influenzae named/pandemic between 1889 and
1890.
• Influenza was caused by a virus
• The actual role of H. influenzae was that of a secondary
invader.
Virulence factors
• H. influenzae, /pathogenic potential. /virulence
factors/invasiveness :
• Capsule
• IgA proteases
• Outer membrane proteins and lipo
• oligosaccharide (LOS)
• Adherence
Capsule
• The serologic grouping of H. influenzae into six serotypes, a,
b, c, d, e, and f, is based on the capsular polysaccharide.
• Most invasive infections caused by serotype b (Hib)
– Primarily in young children.
• The serotype b capsule is a unique polymer composed of
ribose, ribitol, and phosphate.
• Role in the pathogenesis of invasive disease.
• Antiphagocytic property and anticomplementary activity
• Not all strains of H. influenzae are encapsulated.
• Two patterns of disease
1. Invasive disease / encapsulated (typable) strains
predominantly of serotype b in which bacteremia
(hematogenous spread) plays a significant role.
– ie meningitis, cellulitis, arthritis, and epiglottitis,
– pediatric population
2. Localized infection / contiguous spread of nonencapsulated
(nontypable) strains /close proximity to the respiratory tract.
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Ie pneumonia and sinusitis
in the adult population.
Meningitis in the adult population, I”C, debilitated person
Neonatal sepsis and invasive lower respiratory tract infections in
children.
Clinical manifestations of
Haemophilus influenzae infections
• INFECTIONS CAUSED BY ENCAPSULATED
(TYPABLE) STRAINS
• Meningitis
– Until recently serotype b has been a common cause of
pediatric meningitis in children between the ages of 3
months and 6 years.
– Blood stream invasion and bacteremic spread follow
colonization, invasion, and replication of this organism in
the respiratory mucous membranes.
– Headache, stiff neck, and other meningeal signs are usually
preceded by mild respiratory disease.
Epiglottitis
• Serotype b is the most common cause of this
syndrome.
• The manifestations include
– Rapid onset
– Acute inflammation
– Intense edema that may cause complete airway
obstruction, requiring an emergency tracheostomy.
• Peak of 2 and 4 years.
What is the diagnosis
Haemophilus influenzae
• Cultures problematic.
• Dangerous to collect in
these severely ill
patients
• Acute obstructive
epiglottitis
• Blood cultures are usually
positive
• Does not cause acute
pharyngitis
• Will not grow on sheep blood
agar because X factor (hemin)
and V factor (NAD)
Epiglotitis
HiB-vaccine
• Polysaccharide-protein conjugate immunogenic in young infants
• T-cell stimulation: enhanced response and
memory
• PRP-T used across Canada, only product
combined with aP,D,T, IPV
• At 2,4,6,18mos.
• Arthritis
• The bacteremic spread of
serotype b is the most
common cause of arthritis
seen in children younger
than 2 years of age.
• Cellulitis
• Serotype b is the usual
culprit in cases of cellulitis
in children younger than 2
years of age.
• Cheek
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Rapid onset
Pain
Edema
Reddishblue color on the
inflamed area.
• Upper extremities.
• Pharyngitis and pneumonia.
• Pneumonia in children is usually caused by Hib.
• The mean age at infection is approximately 14
months.
• A significant decrease in invasive disease among
children.
• It still remains a problem in the elderly and debilitated population who have not been vaccinated
INFECTIONS CAUSED BY NONENCAPSULATED
(NONTYPABLE) STRAINS
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Otitis media
Bronchitis
Sinusitis
Pneumonia in elderly patients
Genital tract infections.
Haemophilus ducreyi
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Chancroid
A highly communicable sexually transmitted disease.
IP 4 to 14 days,
A nonindurated, painful lesion with an irregular edge
develops, generally on the genitalia or perianal areas.
• Penis ,the labia or the vagina .
• Suppurative (forming or discharging pus), enlarged,
draining, inguinal lymph nodes (buboes) are common
in the majority of infected patients
Endocarditis.
• Haemophilus parainfluenzae,
• H. aphrophilus,
• H. paraphropilus
Laboratory Diagnosis
• Specimen processing and isolation
• Common sources include
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Blood,
CSF
Middle-ear exudate,
Joint fluids
Upper and lower respiratory tract
Swabs from conjunctivae,
Vaginal swabs
Abscess drainage.
Die rapidly in clinical specimens;
• Prompt transportation and processing
Clinical specimens from areas of the
human body
• Colonies on chocolage agar translucent, moist,
smooth, and convex,
• A distinct "mousy" or "bleach-like" odor.
• Haemophilus influenzae a more grayish-appearing
colony.
• The encapsulated larger and more mucoid .
• Gram-negative pleomorphic coccobacilli on
chocolate agar,
• No growth on sheep's blood agar in pure culture.
• Haemophilus species do not grow on MacConkey
agar .
Laboratory identification
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Testing for growth factors (X and V)
Ttraditional biochemicals
Hemolysis on rabbit's or horse's blood agar,
Oxidase, and catalase.
The porphyrin test is an alternative method for the determination of X factor
requirements.
Commercial systems
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Minitek System
RapID-NH
HNID
NHI
Latex agglutination and coagglutination tests
Of all the species that require V factor, H. segnis is the only organism that is
oxidase negative.
The porphyrin
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Treatment
• Life-threatening
– Cefotaxime, ceftriaxone, or
cefuroxime.
– Trimethoprimsulfamethoxazole,
– Imipenem, and ciprofloxacin.
– Increased resistance to
ampicillin
– Possibility of resistance to
chloramphenicol,
• Non-life-threatening
– Amoxicillin-clavulanate
– Oral second- or thirdgeneration cephalosporin,
– Trimethoprimsulfamethoxazole,
– Ampicillinsulbactam.
• H. ducreyi,
– Erythromycin,
– Ceftriaxone,Fluoroquinolone
• H. aphrophilus
– Penicillin and gentamicin,
– Cephalothin and gentamicin.
Beta-lactamase-positive
BORDETELLA PERTUSSIS
Taxonomy
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Pertussis "violent cough”
Small, gram negative coccobacilli or short rods.
The genus Bordetella consists of four species:
B. pertussis
B. parapertussis
B. bronchiseptica
B. avium.
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Obligate aerobes, grow in 35 C to 37 C
Pathogenesis
• B pertussis
• B. parapertussis
– Responsible for the majority of Bordetella infections in
humans.
• B.bronchiseptica
• B.avium
– Animal pathogens.
• B.bronchiseptica
– An opportunistic human pathogen, causing pneumonia and
wound infections.
Virulence factors(1)
1. Pertussis toxin (PT )* :
– A-B type exotoxin which inhibits the function of
phagocytes.
– known as histamine-sensitizing factor, lymphocytosispromoting factor, or islet-activating protein.
– This toxin has ADP-ribosylating activity causing
elevation of cAMP levels in the affected tissue and is
responsible for pronounced lymphocytosis
2. Filamentous hemagglutinin (FHA)* :mediates bacterial
adherence to ciliated cells and agglutinate erythrocytes .
3. Fimbriae *
4. Pertactin (PRN):* adherence factor.
Virulence factors(2)
5. Tracheal cytotoxin (TCT)*:
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inhibit DNA synthesis, production of interleukin-1 and
nitric oxide and ultimately kills tracheal epithelial cells
6. Adenylate cyclase toxin ) ACT (
– is taken up by the host cell, activated by Ca dependent
calumodulin and elevates intracellular c-AMP level of
the host cell.
Virulence factors(3)
7. Dermonecrotic toxin (DNT):
– causes vascular smooth muscle contraction.
8. Endotoxin
– Gram-negative bacterium, Bordetella pertussis possesses
LPS lipopolysaccharide (Endotoxin) in its outer
membrane ,induces fever in the host via IL-1 and TNFa .
Transmission
1. Aerosol:
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its highly communicable in the early catarrhal stage
before the paroxysmal cough stage.
2. Direct contact
CLINICAL MANIFESTATIONS
Clinical manifestation
• Incubation period 1 to 3 wks
Catarrhal Stage
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1-2 weeks
Coryza
Sneezing
Low grade fever
Occasional cough
Paroxysmal Stage
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1-6 weeks
Paroxysmal attacks-rapid coughs
Thick mucus
Whoops, possibly cyanosis, vomiting
Well in between
< 6 months maybe no Whoop
• Symptoms may be atypical in infants and
partially immunized children and previously
immunized adolescents and adults.
Convalescent Stage
• Gradual, cough leaves 3-6 weeks
• May have paroxysms with resp infections
months later
• Adolescents and Adults less severe- 7% of
cough illness
• Adults are carriers
Laboratory Findings
• Leukocytosis with lymphocyte predominance.
• Low blood glucose in pediatric pertussis patients.
• Pulmonary consolidation and pneumonia is present in
20% of hospitalized patients
Complication
• Secondary infections, such as otitis media and
pneumonia.
• Physical sequelae :aspiration during whooping and
vomiting.
• Subconjunctival and scleral hemorrhages, petechiae,
epistaxis, CNS hemorrhages ,pneumothorax, hernias,
and rectal prolapse.
• In adults it may precipitatee angina pectoris.
Complications
1. Pneumonia
(11.2%<6mons)
2. Seizures
3. Encephalopathy
4. Death
5. Hospitalization
(all cases reported to CDC 1997-2000)
5.2 %
0.8 %
0.1 %
0.2 %
20 %
Diagnisis
 Pertussis can be diagnosed by culture, DFA ,PCR or by
ELISA.
• Culture of B pertussis was of limited sensitivity in all studies
but it still the gold standard.
• The material should be collected using Dacron or calcium
alginate nasopharyngeal (NP) swabs (cotton inhibits growth of
the organism) or by NP aspiration or bronchial wash.
•  Throat swabs are not recommended because of the
overgrowth of oral flora
• Charcoal-horse blood agar transport medium is recommended
Diagnisis
• Most current data support the use of charcoal-horse
blood agar for culture supplemented with cephalexin
(40mg/L (to impair growth of normal flora .
• The charcoal is added to bind fatty acids that are toxic
to the organism
• Other medias e.g. Regan-Lowe, Bordet-Gengou .
• Isolation of the organism is most successful in the
early stages of the disease.
Diagnisis
• B.pertussis and parapertussis are small fastidious
gram negative coccobacilli.
• They are slow growing, colonies becoming visible
after 2 to 4 days incubation at 35oC in air or low CO2
with increased humidity.
• Specimen must be examined daily for 7 days to
identify the slow-growing, shiny, convex, haemolytic
colonies
• Parapertussis colonies are yellowish.
C:Bordet-Gengou agar
DFA
Regan-Lowe
Suggestive colonies of Bordetellae
Gram Stain
GN coccobacilli or short rods
Other bacteria
DFA or LATEX for
B. pertussis or parapertussis
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Subculture to BAP ,CA
and Bordetellae plates
)-( or equivocal
B. pertussis should not
grow on BAP and CA plates
Diagnosis
• Serology is mostly done by ELISA with purified
antigens of B pertussis, such as (PT), (FHA), or
pertactin to detect IgG or IgA but it's still not optimal.
• Only PT is specific for B pertussis, whereas FHA
and pertactin are also expressed by other members
Bordetella.
• DFA is widely used with the culture to detect B.
pertussis in the nasopharyngeal smears or from
culture.
• Serum antibodies are useful epidemiologically, but
less so during the acute illness.
Diagnosis
• Detection of Bordetella- DNA by PCR is both
sensitive and specific, fast and able to detect a
carrier/infection state in asymptomatic individuals .
• Although there are significant advantages to the use
of PCR, it is important to culture the organism
because strain variation, antibiotic resistance, and
phenotypic features will be missed by the PCR.
• Undiagnosed cases provide a reservoir for serious
infections in young infants, who are unvaccinated or
whose vaccinations are not yet fully protective.
Prevention
Pertussis Vaccines
• The development of the whooping cough vaccine in
the 1930s has made whooping cough an uncommon
disease in developed countries.
• In countries where the vaccine is not used whooping
cough is an important cause of mortality in children,
with an estimated 51,000,000 cases and 600,000
deaths annually.
 Whole Cell Vaccine
• A major limitation to whole cell vaccine use is the associated
reactogenicity.
• Produces local and systemic reactions such as pain, swelling,
fever, anorexia and vomiting
• 20% of the children that receive the whole cell vaccine
experience mild side effects.
• About 0.1% of infants experience convulsions
• 1 in 150,000 may develop severe or irreversible brain damage.
• Efficacy ranged from 0 to 100% depending on the vaccine
preparation used, the type of study, and the case definition.
• At present, whole cell pertussis vaccine is used in adults only
under special circumstances for control of hospital outbreaks
 Acellular Vaccine
• This vaccine has fewer side effects than the whole
cell vaccine
• Contains one to five of pertussis components such as
pertussis toxoid, FHA, pertactin, and types 2 and 3
fimbriae, but all preparations contain PT.
• Given in combination with vaccines against
diphtheria and tetanus (DTaP(
• Recently, developed a vaccine that combines the
DTaP with vaccine against Haemophilus influenzae
type b meningitis (Hib). This vaccine is called
DTaP/Hib .
CLINICAL MICROBIOLOGY REVIEWS,
Apr. 2005, p. 326–382 Vol. 18, No. 2
• Although vaccination is highly effective for young
children, immunity diminishes in many adolescents
and adults
 Pertussis vaccination has reduced the numbers of
notified cases by more than 95%.however, older
children, adolescents, and adults represent the
majority of pertussis cases and pose a significant risk
to infants too young to be vaccinated.
Approximate immunity after
expusure to infection or
Treatment
Supportive Care
Infants with pertussis are at greatest risk of
complications
Monitoring of vital signs; frequent nasotracheal
suctioning; and provision of oxygen and parenteral
hydration and nutrition.
Treatment
Specific Therapy
• There is preliminary evidence that pertussis
immune globulin may be useful in reducing
the severity of disease, especially in infants.
• Erythromycin, shortens the period of
communicability due to its ability to enter the
respiratory tract. But does not reduce
symptoms except when given during the early
paroxysmal period or before for 14 days.
• TMP-SMX is an alternative
Public health issues
• Report to the local health authority
• Isolation: respiratory isolation for known
cases. Suspected cases should be removed
from the presence of young children until the
patients have received at least 5 days of the 14
days treatment.
• Suspected cases who do not receive antibiotics
should be isolated for 3 wks.
Public health issues
• Quarantine: inadequately immunized household
contacts less than 7 years of age should be excluded
from schools, day cares and public gatherings for 21
days after the last exposure or until the cases and
contacts have received 5 days of the 14 days course
of treatment
• Protection of contacts: under 7 years who have not
received the vaccine should be given a dose as soon
after exposure as possible.
• 14 day course of erythromycin for close contacts,
regardless of immunization and age is recommended.
Epidemic measures
• A search for unrecognized cases is indicated to
protect preschool children from exposure and to
ensure adequate preventive measures for exposed
children less than 7 years of age.
• Accelerated immunization with the first dose at 4-6
wks of age, and the second and third doses at 4 wks
intervals, may be indicated, immunizations should be
completed for those whose schedule is incomplete.