1

Introduction
 2

Members of the genes haemophilus are small, pleomorphic,

gram-negative coccobacilli. These organisms form part of the indigenous
flora of the mucous membrane of the human upper respiratory tract and
mouth. Most members of the genus are non-pathogenic or opportunistic;
however, some species such as H. influenzae causes several diseases ranging
from chronic respiratory to serious, invasive infections. H. influenza type b
(Hib) was the single most important of meningitis in children in US before
the availability of vaccines.

The word haemophilus means “blood loving” and refers to the growth
requirement of these organism for one or both of two factors present in
blood – X and V factors. The X factor is protoporphyrin or hemin. The heat
stable X factor is necessary for synthesizing iron containing respiratory
enzymes, cytochrome, cytochrome oxidase, catalase, and peroxidase. The
heat labile V factor , nicotinamide adenine dinucleotide (NAD), is a
coenzyme involved in oxidation-reduction reaction.

Effective infectious disease surveillance systems provide basic

information on incidence and geographic distribution of known infectious
agents. This information, needed at local, national, and international levels,
is necessary for detecting new or re-emerging threats, documenting
antimicrobial resistance, and developing new treatments and vaccines.18
Surveillance data can also be used to change clinical management of disease,
update treatment guidelines and lists of essential drugs, educate prescribers,
and guide infection control policies. Surveillance of pathogen prevalence
and drug-resistance patterns requires laboratory facilities arranged within a
network designed to share, analyze, and interpret these data.
Haemophilus influenzae is an important cause of acute, recurrent and
persistent infection of the human respiratory tract. It is found in 8% of
pneumonias. More than 95% of the invasive infections are associated with
 3

type b encapsulated H. influenzae, which accounts for a third of all bacterial

pneumonias among 4-month to 4-year old children. The incidence of this
bacterium is reported to be 5 – 10 cases per 100,000 persons in USA of
which 95% are caused by type b. The unencapsulated strains also play an
important role in chronic bronchitis, and are the second cause of pneumonia
in adults. These strains are also an important cause of lower respiratory tract
infections in patients with cystic fibrosis.12
Nontypeable Haemophilus influenzae is the most common bacterial
cause of exacerbations of COPD. Chronic obstructive pulmonary disease
(COPD) causes 165,000 deaths, 10 million office visits, and 2 million
hospital admissions annually in the United States, and these are frequently
related to exacerbations owing to bacterial infection. Antibiotic therapy is
effective in accelerating recovery from exacerbations and preventing early
clinical deterioration. Therefore, antibiotic therapy is used widely for the
treatment of exacerbations of COPD. As a result, the antimicrobial agents
used to treat exacerbations should be active against strains of H. influenzae
that are present in the lower airways.13
Haemophilus influenzae colonizes the human nasopharynx.
Nontypeable H. influenzae (NTHi) can cause a variety of infections,
including otitis media, sinusitis, conjunctivitis, bronchitis, and pneumonia.
Children are frequent carriers of H. influenzae in the nasopharynx, and the
rate of carriage is high in infancy. The colonization may subsequently lead
to the development of infectious diseases caused by H. influenzae. It has also
recently been reported that β-lactamase negative ampicillin-resistant
(BLNAR) strains have increased in some countries, although the global
prevalence still remains low.14
In the diagnostic view, the reason for lack of data on burden of
haemophilus influenzae in developing countries was mainly failure of
detection of this fastidious organism in the laboratory. Specimens can be
 4

collected as sputum, throat swab, ear, conjunctival scrapings, CSF, blood

and other body fluids.

Most laboratories speciate the genus haemophilus on the basis of

hemolysis and X and V factor requirements. Biochemical tests are useful for
species identification and for differentiation of biotypes. 15 Additional useful
methods are commercial identification system, Immunoserologic and
serologic identification, identification by molecular methods, epidemiologic
typing systems etc.

Vaccines constitute the most significant preventive measure. Several

polysaccharide vaccines were approved in 1985 for the immunization of
older children, but these are not effective for infants, the group at highest
risk. Protien polysaccharide conjugate vaccines have been licensed for
children 2 months of age and older.15 These vaccines are reduced the
incidence of Hib disease dramatically.

The intent of the present study is to determine the prevelance of

Haemophilus influenzae in patients suffering from respiratory tract
infection.
 5

Aims And Objectives

 6

1. To study the prevalence of haemophilus influenzae in respiratory tract

infection.

2. To study the antibiotic sensitivity pattern of haemophilus influenzae.

3. To monitor drug resistance strains and helps to introduce appropriate

antibiotic therapy for respiratory tract infection.
 7

Review of Literature
 8

A study was conducted on Nasopharyngeal Carriage of Haemophilus

influenzae, and observed that the multidrug resistant nasopharyngeal H.
influenzae in young healthy children may act as reservoir.¹
The study on Haemophilus influenzae shows the historical landmarks
that have led to our present-day understanding of H. influenzae
pathogenicity, the concerns about antibiotic resistance, the features of the
host immune response to H. influenzae, and the introduction of Hib vaccine.²
The study summarise that Non-typeable H. influenzae is an important
cause of respiratory tract infections in children and adults. Most strains are
susceptible to amoxicillin/clavulanate, fluoroquinolones and the newer
macrolides. Research in the next decade promises substantial progress in the
challenge of developing vaccines for nontypeable H. influenzae.³
The studies states that, In developing countries Nontypeable strains of
H. influenzae are also responsible for many cases of pediatric pneumonia.
Currently approximately 30% of nontypeable H. influenzae strains are beta-
lactamase-positive and can inactivate susceptible penicillins, ampicillin and
amoxicillin. Most second generation oral cephalosporins are active against
beta-lactamase-producing H. influenzae, third generation oral
cephalosporins, however, have particularly good efficacy against H.
influenzae.4
The study on H. influenzae observed that, 60% of patients with
pneumonia caused by type b H. influenzae are more in 50 years old, 30% to
40% are alcoholics, and 30% to 40% have chronic pulmonary disease or
other concurrent illness. The risk of secondary infection in children who
come into contact with patients infected with type b H. influenzae amounts
to approximately 2.1%. 5
 9

A study on Identification of strains on recurrent H. influenzae

infection in patients with chronic respiratory tract infections, observed that
early recurrence and late recurrence of recurrent H. influenzae infections
occur in a different mechanism.6
A study on Haemophilus influenzae in the throats of healthy infants
with different feeding methods suggest that the colonization of H. influenzae
in the throat was inhibited by the presence of breast milk.7
A study conducted on lower respiratory tract infection by H.
influenzae observed that the isolation rate of H. influenzae could be
improved by Modified Columbia Chocolate Agar ( ICCA ).8

Introduction:

Haemophilus influenzae is an obligate human parasite that is

transmitted from person to person by way of respiratory route. It is reported
that 30% to 50% of all children carry the bacillus asymptomatically in the
nasopharynx.generally as avirulent, nonencapsulated organism.25
Haenzophilus infuenzae causes a variety of community acquired respiratory
tract infections, including acute otitis media, sinusitis, bronchitis, and
pneumonia.17
Chronic bronchitis is an important health problem in our country. It
constitutes 30 percent patient seen in chest clinics and accounts for 1-2.5%
of admission in hospital all over India.16

There is also increasing evidence that non capsulated H. influenzae is

an inportant patho-gen of pneumonia and other lower respiratory tract
infections in developing countries. Strep. pneumoniae and H. influenzae are
the most frequent bacterial agents of acute respiratory infections (ARI) in
children in developing countries.30
 10

Microbiological diagnosis of these haemophilus infection is often

hampered by an inability to recover the organism on primary plated media
due to the frequency with which it is over grown by the commensal
microbial flora of the upper respiratory tract.

History:

Haemophilus influenzae is widespread in its distribution among the

human population. It was first isolated by PFEIFFER during the influenza
pandemic of 1890. It was mistakenly thought to be the cause of the disease
influenza, and it was named accordingly. Probably, H. influenzae was an
important secondary invader to the influenza virus in the 1890 pandemic, as
it has been during many subsequent influenza epidemics. In pigs, a
synergistic association between swine influenza virus and Haemophilus
suis is necessary for swine influenza. Similar situations between human
influenza virus and H. influenzae have been observed in chick embryos and
infant rats.19

The casual relationship between this bacillus ansd human influenza

couldnot be substantiated and was finally disproved when SMITH,
ANDREWES and LAIDLAW (1993) isolated the influenza virus.24

Haemophilus influenzae has long been recognised as a major cause of

serious infection and mortality in children less than 5 years old. Prior to the
introduction of Haemophilus influenzae type b (Hib) immunisation, the
incidence of a child suffering an invasive Haemophilus infection was 20-
50/100,000 in industrialised countries and up to ten times higher in
developing regions. The introduction of a Hib vaccine programme results in
a rapid and dramatic decline in the incidence of Hib infection in the
susceptible childhood population. For example, within two years of the
introduction of routine Hib vaccination of infants in the UK, the risk of
 11

serious Hib infection had fallen from 1:600 to 1:30,000 by 5 years of age.
Many other European countries have introduced, or are in the process of
introducing, a routine Hib immunisation programme. Because the
epidemiology of Haemophilus influenzae infection is changing so
dramatically, it is opportune to review Haemophilus influenzae as it was
perceived in the pre-vaccine era (the past) and during vaccine
implementation (the present), and how its role may change in the post-
vaccination era (the future). This review will summarise the historical
landmarks that have led to our present-day understanding
of Haemophilus influenzae pathogenicity, the concerns about antibiotic
resistance, the features of the host immune response to Haemophilus
influenzae, and the introduction of the Hib vaccine. Furthermore, the
possible importance of this organism in the future will be discussed.20

During the 40 year period between 1946 and 1986 the incidence of
HiB increased 4 times over, and while some scientists speculate that the use
of antibiotics may have caused a resistant strain of this bacteria, but another
aspect to be looked at is the widespread use of vaccines. Like Polio, Hib
increased it's incidents dramatically with widespread use of vaccines, which
could be due in part to some ingredients in vaccines. While vaccine
manufacturers include ingredients that are adjuvants which help to trigger
immune response, many vaccines also include ingredients which actually
suppress the immune system which could give bacterium and viral particles
which are normally dormant in the human body, the opportunity to attack
cells without the bodies natural defense mechanisms fighting back in a
healthy fashion. Hib was first discovered in the 1890's during an influenza
outbreak. If HiB is not treated immediately, the death rate is typically around
5 to 10% with Neurological damage in as high as 30% of it's victims.21
 12

History shows that Haemophilus influenzae type B was the leading

cause of bacterial meningitis in children in the United States in the mid-
1980s and earlier. It caused between 12,000 and 20,000 cases a year, leading
to about 500 deaths each year. Doctors treated children infected with
Haemophilus influenzae with antibiotics and while survival rates were good
for a child who had been diagnosed early, 30 percent of children who
developed meningitis and then recovered experienced lingering neurological
effects.22

Haemophilus influenzae type b (Hib) disease is

estimated to cause 3 million cases of meningitis and severe
pneumonia and approximately 386,000 deaths worldwide
per year in children aged <5 years .23

Haemophilus influenzae is the first freeliving organism

whose complete genome has been sequenced.24

Colonial morphology:

Small (0.5-1.0 mm), ß hemolytic, smooth, translucent, grayish,

convex with entire edge and a “mousy” odor; encapsulated strains larger (1-
3 mm) and more mucoid, with tendency to coalesce.

Morphology:

• They are gram-negative coccobacilli

• Pleomorphic in shape
• Size is usually small(0.3-0.5×0.3-0.5µm)
• Non-sporing
• Non-motile
Antigenic structure:

There are three major surface antigen:

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• Capsular polysaccharide antigen

• Outer membrane protein (OMP)

• Lipooligosaccharide (LOS)

Classification:

The organism H. influenzae are devided in to six types, designated

a, b, c, d, e and f on the basis of antigenic differences in their capsular
material.25

According to certain biochemical properties; Indole, Urease, and

Ornithine Decarboxylase reactions H. influenzae is subdivided in to eight
biotypes. They mentioned with their characteristics as follows.15

Biotypes Indole Urease Activity Ornithine

Production Decarboxylase
Activity
Biotype I + + +
Biotype II + + -
Biotype III - + -
Biotype IV - + +
Biotype V + - +
Biotype VI - - +
Biotype VII + - -
Biotype VIII - - -

Pathogenesis:

Naturally-acquired disease caused by H. influenzae seems to occur in

humans only. In infants and young children (under 5 years of age), H.
influenzae type b causes bacteremia and acute bacterial meningitis.
 14

Occasionally, it causes epiglottitis (obstructive

laryngitis), cellulitis, osteomyelitis, and joint infections. Nontypable H.
influenzae causes ear infections (otitis media) andsinusitis in children, and is
associated with respiratory tract infections(pneumonia) in infants, children
and adults.19

H. influenzae is an exclusively human pathogen. It is not naturally

pathogenic for animals but intraperitoneal inoculation of large doses is fatal
in mice, guinea pigs and rabbits.24

Diseases due to H. influenzae may be considered under two groups,

invasive and non-invasive diseases. In the first group, bacillus acts as a
primary pathogen, causing acute invasive infection. In the second group, the
bacillus spreads by local invasion along mucosal surfaces and causes
secondary or superadded infections.

Virulence:

H. influenzae does not produce any demonstrable exotoxins The direct

role ofendotoxin in meningitis or bacteremia is unclear, although the Gram-
negative bacterium's outer membrane lipooligosaccharide (LOS) is thought
to play a role in inflammation associated with otitis media. All virulent
strains produceneuraminidase and an IgA protease, but the role of these
extracellular enzymes in invasion is unproven. Fimbriae increase the
adherence of bacteria to human mucosal cells in vitro, and they are required
for successful colonization of the nasopharynx. The Anton antigen, as
defined in red blood cells, appears to be the receptor.

Virulence, at least in the case of bacteremia and meningitis, is directly

related to capsule formation. Virtually all of these infections are caused by
the type b serotype, and its capsular polysaccharide, containing ribose,
ribitol and phosphate, is the proven determinant of virulence. The capsule
 15

material is antiphagocytic, and it is ineffective in inducing the alternative

complement pathway, so that the bacterium can invade the blood or
cerebrospinal fluid without attracting phagocytes or provoking an
inflammatory response and complement-mediated bacteriolysis. For this
reason, anticapsular antibody, which promotes both phagocytosis and lysis
of bacteria, is the main factor in immune defense against H.
influenzae infections. 19

Mechanism:

Factors contributing to the pathogenicty of H. influenzae including

colonization, the first event are poorly understood. Clearly, the type b
capsular polysaccharide , a polymer of ribosyl ribose phosphate , is the
major virulence determinant of Hib.

Haemophilus influenzae

Nasopharyngitis or URT infectio

Sinusitis and otitis media

Pneumonia

Bacteremia

Joint infection or meningitis

 polysaccharide capsule (major virulance)

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Encapsulated organism

Epithelium of the nasopharynx

Blood capillaries

Capsule resist phagocytosis

Complement mediated

Non-immune host

 Nonencapsulated (less invasive)

Inflammatory response

Clinical features:

Diseases of children:

Colonization of the nasopharynx usually preceds infection

with Hib. From this site the organism may extend to local tissues, or they
may invade the blood stream to cause meningitis, epiglotitis, cellulitis, septic
arthritis, pneumonia, or osteomyelitis. Prior to the introduction of vaccines,
Hib caused about 20,000 invasive infections annually. This number has
reduced substantially as a result of immunization programs.

Pneumonia: The true incidence of H. influenzae pneumonia

in infants and children is unknown because of the difficulties in making a
 17

precise bacteriologic diagnosis. Sputum is often unobtainable in children

less than 4 years of age. Furthermore, the presence of this organism in
sputum is not necessarily abnormal or relevant to the etiology of pneumonia

Diseases of adults:

The noncapsulated isolates of H. influenzae that commonly

colonize the nasopharyngeal region are associated with acute sinusitis and
bronchitis in adult who usually have compromised host defenses. H.
influenzae has also been associated with invasive disease in adults. These
include otitis media, sinusitis and exacerbations of COPD, chronic
bronchitis and bronchiectasis.

H. influenzae may be a nosocomial pulmonary pathogen

spread by equipment or person to person contact. Community acquired
pneumonia is probably infrequently caused by H. influenzae. Since H.
influenzae is normally found in the upper respiratory tract, it is difficult to
prove as the cause unless appropriate specimens are taken.

Lab diagnosis:

Specimen collection:

Depending on the associated disease, H.influenzae may be

recovered from the upper and lower respiratory tract, ear, conjunctival
scrapings, cerebrospinal fluid, blood, and other body fluids. Including
synovial and pleural.

In respiratory tract infections, usually collect sputum,

purulent nasal discharges, scrapings from RT …etc.

The collected specimens immediately sent to the

laboratory. If delaying the sample should be kept in transport media.
 18

Specimens should be left at room temperature but processed as soon as

possible because the haemophilus organisms are susceptible to drying and
chilling.

Direct examination (Microscopy):

Gram staining may be performed on the centrifuged

sediment of body fluids(sediment prepared by conventional centrifugation,
or on slides prepared by cytocentrifugation). The second method is most
sensitive.

Examine the slide for the typical pleomorphic gram-

negative coccobacilli of H. influenzae. Slides must be examined carefully
because the organism frequently stain lightly and may not be observed or
mistaken for debris.

Culture:

Direct gram stains of the lesion exudates may reveal

gram-negativec coccobacillary organisms in tangled chains or in parallel
arrays, problems with sensitivity and specificity have been reported.

Detection of the organism in a blood culture or any other body fluid is

the most confirmatory method of establishing the diagnosis.

Slide agglutination with type-specific antisera is used for

serotyping H influenzae. In one study, molecular typing with PCR was
found to be more accurate than slide agglutination serotyping.

Seventy to 90% of patients with epiglottitis have positive blood

culture results. However, to avoid laryngospasm, perform venipuncture and
cultures of the inflamed epiglottitis only after the airway has been secured.31

Cultural characteristics:
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Media used:

• Blood agar.
• Chocolate agar.
• Levinthal agar (selective media).
• Complex media.
An antiserum agar medium was evaluated to determine its usefulness
and accuracy in the study of prevalence of haemophilus influenzae type b
(HIB) in the pharynx of infants and young children.the effects of varying the
concentration of ingredients were demonstrated. The medium could be store
at 4 °c for periods up to 12 weeks with no loss of effectiveness.26

H. influenzae isolates (N=194) were simultaneously grown on

chocolate agar (CA) with and
without isovitalex (IVX). Average colony size of H. influenzae on CA with
IVX (CA-IVX) was larger only by 0.10 cm (range 0.05 to 0.16 cm)
compared to CA alone.27

A study was conducted on antibiotic resistance in respiratory tract

isolates of H. influenzae , the collected samples were sent to the laboratory
on Amies Charcol swabs.28

A method was devised to test the growth-promoting ability of a broth

medium. The "dilute to extinction" method determines the inoculum required
to develop heavy turbidity in a broth with overnight incubation. A statistical
method using Poisson distribution was used to show that a single
Haemophilus cell can develop heavy turbidity in an optimal broth. The
dilute to extinction method was used to evaluate the shelf life of stored
media, to titrate the growth factor requirements of Haemophilus, and to
evaluate the use of purified hemin and nicotinamide adenine dinucleotide in
a broth medium for the growth of Haemophilus.29
 20

Growth requirements:

• Media contains X and V factors.

• Optimum temperature 35-37 °C.
• Optimum ph is 7.4.
• Facultative anaerobes.

Haemophilus influenzae is differentiated from

other Haemophilus species primarily by its growth requirements for both
hemin (called factor X in the old literature) and nicotinamide adenine
dinucleotide (NAD or factor V). H. influenzae strains normally grow well in
rich media such as brain heart infusion (BHI), suitably supplemented with
hemin and NAD (sBHI), and on sBHI or chocolate agar plates. Many
isolates classified as H. influenzae differ in their other nutritional
requirements, for a purine and for specific amino acids. Thus, although a
number of defined media have been described, their use often leads to
frustration. The medium MMB, described by Klein and Luginbuhl, is
simplest to prepare; when supplemented with a small amount of casamino
acids it reproducibly gives good growth.32

Viability and storage:

The viability of Haemophilus spp. preserved for 5 to 12 months at

−70°C. The following media were used: Laboratoire de Santé Publique du
Québec (LSPQ) preservation medium, trypticase soy broth with 10°C
(vol/vol) glycerol and 40°C (vol/vol) horse serum (TSBG), and Levinthal's
broth (LB) medium. Three clinical isolates of both H. influenzae and H.
parainfluenzae were used. After 5 months no differences in viability were
observed between strains preserved in TSBG and strains preserved in LB,
but a significant loss of viability was observed in strains preserved in LSPQ
medium. No significant changes in antimicrobial susceptibility were
 21

observed after 5-month storage in any medium. After 12 months, TSBG

appeared to be the most suitable cryopreservation medium for the six strains
tested. We conclude that TSBG represents a good medium for the
maintenance of Haemophilus spp. at −70°C for up to 1 year.33

Biochemical reactions:

Fermentation of glucose, sucrose, and lactose is important for

species identification. These tests are performed in phenol red broth base,
containing 1% of the respective carbohydrates and supplemented with X and
V factors after autoclaving.34

Sugar fermentation test: Result

Glucose +
Sucrose -
Lactose -
Mannose -
Catalase +
Oxidase +
Nitrate reduction +
H2S production -
Phosphatase +
Indole production 21-79% +
Urease 80-89% +
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Treatment and prevension: