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

• Gram negative– 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

• 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 (nicotinamide adenine 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 factor producing 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 respi ratory tract 10% of this normal bacterial flora in adults consists of Haemophilus spp.

• H. parainfluenzae and nonen capsulated 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 influenzaeHistorical perspective

• "flu," is a viral infectious disease characterized by acute in flammation 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 non encapsulated (nontypable) strains /close proximity to the respiratory tract.– 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– Rapid onset– Pain– Edema– Reddish blue color on the

inflamed area.

• Upper extremities.

• Pharyngitis and pneumonia. • Pneu monia 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 debili

tated population who have not been vaccinated

INFECTIONS CAUSED BY NONENCAPSULATED (NONTYPABLE) STRAINS

• Otitis media

• Bronchitis

• Sinusitis

• Pneumonia in elderly patients

• Genital tract infections.

Haemophilus ducreyi

• 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

– 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• 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 de termination of X factor

requirements. • Commercial systems

– Minitek Sys tem – 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• .

Treatment

• Life-threatening– Cefotaxime, ceftriaxone, or

cefuroxime. – Trimethoprim-

sulfamethoxazole, – Imipenem, and ciprofloxacin. – Increased resistance to

ampicillin– Possibility of resistance to

chloramphenicol,

• Non-life-threatening – Amoxicillin-clavulanate– Oral second- or third-

generation cephalosporin, – Trimethoprim-

sulfamethoxazole, – Ampicillin sulbactam.

• H. ducreyi, – Erythromycin, – Ceftriaxone,Fluoroquinolone

• H. aphrophilus – Penicillin and gentamicin, – Cephalothin and gentamicin.

Beta-lactamase-positive

BORDETELLA PERTUSSIS

Taxonomy• 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.• Obligate aerobes, grow in 35oC to 37oC

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, lymphocytosis-promoting 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)*: – 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: – 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

• 1-2 weeks

• Coryza

• Sneezing

• Low grade fever

• Occasional cough

Paroxysmal Stage

• 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 5.2 % (11.2%<6mons)

2. Seizures 0.8 %

3. Encephalopathy 0.1 %

4. Death 0.2 %

5. Hospitalization 20 % (all cases reported to CDC 1997-2000)

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 agarRegan-Lowe

DFA

Suggestive colonies of BordetellaeGram Stain

GN coccobacilli or short rods Other bacteria

DFA or LATEX forB. pertussis or parapertussis

(+) (-) or equivocal

Subculture to BAP, CA and Bordetellae plates 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.

PreventionPertussis 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

vaccination

TreatmentSupportive 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.

TreatmentSpecific 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.