MYCOPLASMA PNEUMONIAE

# Introduction:

• Mycoplasmas are the smallest microbes capable of free-living in the environment and self replicating on
artificial culture media.
• They resembles to viruses in certain properties, like size and they are also filterable by bacterial filters.
• They lack rigid cell wall, instead have a triple layered cell membrane containing sterol.

# History:

• Nocard and Roux (1898) were the first to isolate Mycoplasma as a filterable and highly pleomorphic
microorganism from bovine pleuropneumonia (PPLO)
• Later it was termed as Mycoplasma
• Eaton’s agent: it refers to the most pathogenic species, i.e. Mycoplasma pneumoniae, which was first
isolated by Monroe Eaton (1944).

# Morphology:

• They are very small pleomorphic cells and range in size from 0.2 to 0.8 mm in diameter which may
range from spherical through coccoid, coccobacillary, ring and dumbbell forms, to short and long
branching, beaded or segmented filaments.
• The filaments are slender, of varying lengths and show true branching.
• Mycoplasmas are gram-negative but are better stained by Giemsa stain.
• Mycoplasmas do not possess spores, flagella or fimbria.
• Some Mycoplasmas, including M. pneumoniae, exhibit a gliding motility on liquid covered surfaces.

# Cultural Characteristics:
• Most Mycoplasmas are facultative anaerobes.
• They grow within a temperature range of 22–41°C
• Media for cultivating Mycoplasma are enriched with 20% horse or human serum and yeast extract.
The high concentration of serum is necessary as a source of cholesterol and other lipids
• Most Mycoplasma colonies are hemolytic.
• Transportation medium: PPLO broth (cholesterol, serum enriched, albumin, phenol red/
tetrazolium)

# Morphology on agar:

• Mycoplasmas are spherical to filamentous cells with no cell walls.

• Fried-egg-shaped colonies are seen on agar.
• M. pneumoniae are small and betahemolytic.

# Biochemical Tests:

• Catalase and Oxidase positive.

• Mycoplasmas are mainly fermentative. Most Mycoplasmas use glucose (or other carbohydrates) or
arginine as a major source of energy.
• Urea is not hydrolyzed.
# classification based on the on oxygen and sterol requirement:

• Mycoplasmataceae - Mycoplasma pneumoniae

• Acholeplasmataceae - does not require sterol
• Spiroplasmataceae – usually attack animals, require little sterol, facultative anaerobe
• Anaeroplasmataceae – strict anaerobes, requires little amount of sterol

# Resistance:

• Mycoplasmas are killed by heating at 56°C for 30 minutes.

• M. pneumoniae can grow in the presence of 0.002% methylene blue in agar, while many other
species are inhibited.
• Antiseptic solutions, such as chlorhexidine and cetrimide inhibit the growth of Mycoplasmas.
• They are resistant to penicillin and cephalosporin as well as to lysozymes that act on the bacterial
cell walls but are sensitive to tetracycline and many other antibiotics that inhibit protein synthesis.

# Pathology:

1. Virulence factor:
• Rich glycoprotein and protein in plasma membrane also acts as one of the virulence factor.
• M. pneumoniae produces a unique virulence factor known as Community Acquired Respiratory
Distress Syndrome (CARDS) toxin.
• The CARDS toxin most likely aids in the colonization and pathogenic pathways of M. pneumoniae,
leading to inflammation and airway dysfunction.
• While M. pneumoniae primarily lives on the surface of the respiratory epithelial cells, it can invade
tissues and replicate intracellularly.
• The endocytosis of M. pneumoniae by the host cells could aid in the establishment of a latent or
chronic disease state.
• It may also facilitate the bacterium in evading an immune response and interfere with the efficacy of
certain drug therapies

2. Pathogenesis:

• Source of infection: respiratory droplets

• Mode of transmission: person to person from respiratory droplets
• Route of entry: nasopharyngeal
• Clinical manifestations:

o Bulluos myringitis
o Meningoencephalitis
o Skin rashes
o Myocarditis, pericarditis
o Reactive arthritis
o Hemolytic anemia and hypercoagulopathy.

# Laboratory diagnosis:

Specimens:
• Throat swabs
• Nasopharyngeal swabs
 • Sputum,
• Tracheal aspirate
• Lung tissue specimens.

Diagnostic feature: fried egg appearance

Transportation medium: PPLO broth (cholesterol, serum enriched, albumin, phenol red/ tetrazolium)

Tetrazolium reduction test: Colonies of M. pneumoniae appear red when these are flooded with solution of
tetrazolium compound which is colourless. M. pneumoniae reduces tetrazolium (colourless) to red coloured
compound (formazan).

Dienes test: A block of agar containing the colony is cut and placed on a slide. It is covered with a cover slip
on which has been dried an alcoholic solution of methylene blue and azure. And the Mycoplasmas stains azure
blue and exhibit the typical fried egg appearance.

Other tests:
‣ Polymerase chain reaction amplification
‣ Antigen detection techniques
‣ DNA probes
‣ Serological tests (specific and anti specific antigenic tests)

Treatment:
‣ Tetracyclines
‣ Erythromycin
‣ Doxycyclin
‣ Macrolite

Rickettsial diseases: Pathogenesis, Typhus fever group, Spotted fever group

Rickettsial diseases: Pathogenesis, Typhus fever group, Spotted fever group

Rickettsia

Classification:
▪ Order: Rickettsiales
▪ Tribe: Rickettsiae
▪ Family: Rickettsiaceae
▪ Genera: Rickettsia, Orientia, Ehrlichia
Introduction:
▪ Rickettsiae are obligate, intracellular, small Gram-negative bacilli.
▪ It multiplies within the cytoplasm of eukaryotic cells.
▪ Size: 0.3×1-2 µm.
▪ Genome: 1-1.5 million base pairs
▪ Rickettsiae are primary pathogens of arthropods like:
▪ Lice
▪ Fleas
▪ Ticks
▪ Mites
▪ Transmitted to humans by these arthropod vectors.
 ▪ Rickettsiae were originally thought to be a virus because:
▪ Have small size
▪ Stain poorly with Gram stain
▪ Grows only in the cytoplasm of eukaryotic cells
▪ Obligate intracellular parasites
▪ Rickettsiae are bacteria because:
▪ Have Gram-negative cell wall
▪ Contain both DNA and RNA
▪ Contain enzymes for Kreb cycle
▪ Contain ribosomes for protein synthesis
▪ Susceptible to antibiotics
Morphology of Rickettsiae:
▪ They are small Gram-negative coccobacilli.
▪ Size: 0.3-0.6 to 0.8-2 µm.
▪ Non-motile
▪ Non-capsulated
▪ Stains poorly with Gram stain
▪ Stains well with these stains:
▪ Deep red with Machiavello and Gimenez stain
▪ Bluish purple with Giemsa and Castaneda stain
Culture characteristics of Rickettsiae:
▪ Rickettsiae do not grow in cell-free media.
▪ Most Rickettsia grows in the cytoplasm inside the cell.
▪ Rickettsia causing spotted fever grows in the nucleus of the cell.
▪ Cell lines:
▪ HeLa, Hep2, Detriot-6, mouse fibroblasts, and other continuous cell lines.
▪ Chick embryo:
▪ Grows in the yolk sac of 5-6 days old chick embryo.
Human Infections Caused by Rickettsia:
Bacteria Diseases
Epidemic or louse-borne typhus; relapsing louse-
Rickettsia prowazekii borne typhus or Brill-Zinsser disease
Rickettsia typhi Endemic or flea-borne murine typhus
Rickettsia rickettsiae Rocky Mountain spotted fever
Rickettsia akari Rickettsial pox
Rickettsia conori Boutonneuse fever

Antigenic Structure of Rickettsia:

1. Group-specific antigen:
▪ It is the soluble antigen.
▪ It is present on the surface of the organisms.
▪ From the repeated washings and centrifugation, it can be extracted.
2. Species- or strain-specific antigen:
▪ It is present in the cell wall of the bacteria.
3. Alkali-stable polysaccharide antigen:
 ▪ It is a surface antigen.
▪ It is present in some species of Rickettsia and some strains of Proteus (Proteus OX19, OX2 and
OXK).
▪ This sharing of antigen form the basis of the Weil-Felix test.
Pathogenesis of Rickettsioses
▪ Human rickettsiosis occurs when an infected tick or mite bites on the surface of the skin or through the
fecal-oral route from the ingestion of infected louse or flea feces.
▪ Infection occurs after the Rickettsiae invade the vascular endothelium, multiply in the cytoplasm, and
spread via the bloodstream to different parts of the body.
▪ It then infects the vascular endothelium and smooth muscle cell with an increase in immune-effector
responses.
▪ The disseminated infection increases the permeability of vascular cells and fluids are assembled in the
interstitial surroundings.
▪ It leads to a decrease in blood volume which might cause hypovolemic shock and death.
▪ Other affected vital organs are the brain, liver, lungs, heart, and adrenal glands causing diseases like
meningoencephalitis and interstitial pneumonitis.
▪ Infection can also occur by inhalation of the organisms or their feces which mainly occurs in laboratory
workers due to poor hygienic practices.
▪ When the kidney is infected, acute renal failure may occur due to a decrease in the perfusion of fluid.
▪ The vascular leakage of plasma into the alveolar spaces leads to gas exchange in the lungs and causes
hypoxemia.
▪ Cellular immunity of the host cell is important for inhibition of intracellular rickettsiae by killing it in
endothelial cells with the inducible nitric oxide production.
▪ The inducible nitric oxide is activated by the cytokines gamma interferon and tumor necrosis factor.
▪ Natural killer cells also produce immune responses against rickettsial disease and the humoral immune
response produces antibodies against the outer membrane protein and prevents reinfection.
▪ Vasculitis may lead to:
▪ Increased vascular permeability with consequent edema
▪ Loss of blood volume
▪ Hypoalbuminemia
▪ Reduced osmotic pressure
▪ Hypotension
Typhus Fever Group:
▪ Epidemic or louse-borne typhus caused by Rickettsia prowazekii
▪ Relapsing louse-borne typhus or Brill-Zinsser disease caused by prowazekii.
▪ Endemic or flea-borne murine typhus caused by Rickettsia typhi.
1. Epidemic or Louse-borne Typhus:
▪ It is caused by Rickettsia prowazekii.
▪ It is transmitted by the human body louse Pediculus humanus corporis causing the acute febrile
illness.
▪ It is named after the scientist Von Prowazek. He died of typhus fever while studying this
disease.
▪ prowazekii is an invasive bacterium.
▪ It leads to vasculitis by multiplying in the endothelial cells of blood vessels.
▪ The average incubation period is 8 days whereas it may vary low as 2-3 days.
▪ Characteristics of epidemic typhus:
 ▪ High fever
▪ Severe headache
▪ Chills
▪ On the 4 or 5th day, the petechial or macular rash appears
th

▪ The rashes first start to appear on the trunk.

▪ Without affecting the face, palms, and sole it then spreads to the extremities.
▪ In nearly, 40 % of patients rashes are seen.
▪ The patient may become stuporous and delirious if they are left untreated.
▪ In the disease process, the cloudy state of consciousness appears. The name typhus is
derived from it. The meaning of tyhus is cloud or smoke.
▪ Complications:
▪ Myocarditis
▪ Central nervous system (CNS) dysfunction
▪ Mortality rate is as high as 60% in old or immunocompromised persons.
2. Relapsing or Recrudescent Typhus:
▪ Example of a recrudescent case of typhus fever is Brill-Zinsser disease.
▪ This condition was seen in the patients who were cured of the disease or the patients who
were treated with antibiotics.
▪ Even after the antibiotic treatment, the recurrence of typhus fever has re-emerged after
many months, years and decades.
▪ It is because of the persistence of prowazekii in the body tissues which re-emerges later.
▪ Primary reservoir of epidemic typhus: Human
▪ If a person is suffering from typhus fever from brill-Zinsser disease, and when lice feed on it,
it will be infected with prowazekii.
▪ Vector of epidemic typhus: Body louse ( humanus corporis )
▪ Pubic louse does not transmit it.
▪ Occasionally transmission may occur by head louse (humanus capitis )
▪ In the alimentary tract of louse prowazekii, lives and multiplies.
▪ Within the 3-5 days of infection, the bacteria gets excreted in feces.
▪ After the infection, lice die.
▪ During the blood meal when the rickettsia-harboring louse bites the human, infection is
transmitted.
▪ During the feeding, lice defecate.
▪ The contaminated louse feces gets inoculated into the minute lesion of the bite wound when
the host scratches on it.
▪ From there the bacteria reach the circulation, multiplies and cause rickettsemia.
▪ Infection may also be transmitted rarely through the conjunctiva or inhalation of aerosols of
dry louse feces.
3. Endemic or Flea-borne Murine Typhus:
▪ It is caused by typhi.
▪ It has a short duration and the disease is milder than epidemic typhus.
▪ Incubation period: 7 to 14 days.
▪ Symptoms:
▪ Fever
▪ Headache
▪ Malaise
▪ Myalgia
▪ In about 50% of infected patients, rash develops on the 3rd to 5th day of infection.
 ▪ Rash appears on the chest and abdomen.
▪ It may spread to palms and soles.
▪ May last up to 3 weeks in the untreated course.
▪ Reservoirs: Rat ( Rattus rattus ), mice, and cat
▪ Humans are the accidental hosts.
▪ Vectors for transmission of disease: Rat flea (Xenopsylla cheopis ) or cat flea (Ctenocephalides
felis).
▪ It is transmitted from rats to rat by the rat flea.
▪ It is transmitted to humans accidentally by the feces of infected fleas.
▪ When the fleas feed on the mice, cat, or natural host, it becomes infected.
▪ It then transmits the disease to humans by biting.
▪ At the site of the bite, inoculation occurs.
▪ Disease transmission also may occur by:
▪ Cat flea felis
▪ Inoculation or inhalation of aerosolized infectious specimens
▪ Ingestion
▪ Contaminated food with infected rat urine or flea feces
Spotted Fever Group:
1. Rocky Mountain spotted fever caused by Rickettsia rickettsiae.
2. Rickettsialpox caused by R. akari
▪ Boutonneuse fever caused by R. conori
▪ Kenyatick-bite fever,
▪ African tick typhus
▪ The Mediterranean spotted fever
▪ Indian tick typhus
▪ Marseilles fever
1. Rocky Mountain Spotted Fever:
▪ Incubation period: 7 days
▪ Symptoms:
▪ Fever
▪ Severe headache
▪ Chills
▪ Myalgia
▪ Development of rash three or more days
▪ At first, a rash develops on the wrist, ankles, palms, and soles.
▪ It then spreads to the trunk.
▪ In the early stage, the rash is maculopapular but in the later stage, it becomes
petechial and hemorrhagic.
▪ Complications:
▪ Respiratory failure
▪ Encephalitis
▪ Renal failure
▪ Patient may die within 5 days of onset of symptoms.
▪ Ticks are the host, reservoir, and vector of rickettsiae.
▪ Vectors:
▪ Woodtick (Dermacentor andersoni )
▪ American dog tick (Dermacentor variabilis)
▪ Lone star tick (Amblyomma Americana ).
 ▪ When the tick bites the human, it gets transmitted by saliva.
2. Rickettsial Pox:
▪ It is caused by akari.
▪ It is a milder form of infection.
▪ Natural reservoir: Common home mouse ( Mus musculus )
▪ By the bite of mouse mite (Liponyssoides sanguineus ), R. akari Iis transmitted from mouse to
mouse.
▪ Incubation period: 7 days
▪ Papule develops at the site of the bite which progresses to ulcer and leads to eschar
formation.
▪ In 3-10 days fever, headache, malaise, and myalgia develop.
▪ After the emergence of fever, a popular vesicular rash appears un 3-4 days.
▪ Recovery starts after the illness of 10-14 days.
▪ Without treatment complete healing of rash takes 2-3 weeks.
What are the conditions/ characteristics that differentiate Rickettsial pox from other rickettsial infections?
▪ Presence of eschar at the site of the bite
▪ Presence of vesicular pustular eruption
Diagnosis of Rickettsioses
▪ The initial diagnosis is based on the clinical observation by immunohistochemical detection by taking
skin biopsies of skin lesions for antigen detection.
▪ Serological tests are done to confirm the diagnosis using serum samples but the antibodies might not
be detected at the initial stage of infection.
▪ Rickettsiosis is difficult to diagnose in an artificial culture therefore, they are isolated in viable eukaryotic
host cells such as embryonated eggs, tissue cultures, antibiotic-free cell cultures, and many susceptible
laboratory animals.
▪ Rickettsia is grown on cell lines like HeLa, Hep2, Detriot-6, and mouse fibroblasts for their maintenance,
and for isolation, they are cultivated in developing 5 to 6 days old chick embryos.
▪ Laboratory animals like Guinea pigs and mice are used for the isolation of Rickettsia species from animal
specimens.
▪ For the diagnosis of Rocky Mountain spotted fever, a direct fluorescent antibody test is used as it is a
rapid and specific method for confirmation.
▪ IFA and ELISA are newly developed serological tests for the early diagnosis of Rickettsial disease.

Chlamydia trachomatis- An Overview

Habitat of Chlamydia trachomatis
1. It is an obligate intracellular human pathogens.
2. Humans are the only natural host.
3. It cannot survive outside of a eukaryotic host.
4. Chlamydia trachomatis is transmitted by oral, vaginal or anal sex, and can also be transmitted
from mother to newborn during a vaginal delivery.
5. They can cause discharge from the penis, pain and burning during urination, infection or
inflammation in the ducts of testicles, and tenderness or pain in the testicles.
115K
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Morphology of Chlamydia trachomatis
1. It is a weak Gram-negative bacteria.
 2. It also contains LPS, which helps cause damage to the host’s body.
3. It lacks a peptidoglycan cell wall.
4. It lacks muramic acid that is found in the cell walls of most other bacteria.
5. It is non-sporing.
6. They are non-motile.
7. It has a coccoid or rod shape.
8. They exist in two morphological forms: small infectious elementary bodies 300 nm – 400 nm
in diameter and larger replicating reticulate bodies 800 nm – 900 nm in size.
9. The elementary body is the dispersal form, which is analogous to a spore.
10. The dispersal form is about 0.3 um in diameter.
11. The reticulate body divides through binary fission at approximately 2-3 hours per generation.
12. The reticulate body has an incubation period of 7-21 days in the host.
13. The reticulate body contains no cell wall and is detected as an inclusion in the cell.
.
Genome of Chlamydia trachomatis
1. It have an extra-chromosomal plasmid.
2. It has a genome that consists of 1,042,519 nucleotide base pairs.
3. It has approximately 894 likely protein coding sequences.
4. All the isolates are about 7,500 nucleotides long.
5. It has eight open reading frames computer-predicted to code for proteins of more than 100
amino acids.
Cultural Characteristics of Chlamydia trachomatis
• Chlamydia trachomatis is an obligate intracellular bacterium that grows within a mammalian
host cell for survival.
• The bacteria cannot be cultured via conventional cultural methods on bacteriological medium.
This also makes C. trachomatis a difficult organism to grow and maintain in most
laboratories.
• Until 1965, inoculation in the yolk sac of the embryonated eggs was the only method for the
isolation and propagation of the organism.
• Nowadays, however, the tissue culture system has been developed that allows easier
laboratory culture and maintenance of most Chlamydia species.
• The tissue system can be used for the isolation, culture and purify large quantities of Chlamydia
species from clinical and laboratory stock cultures.
• Laboratory works related to C. trachomatis are to be performed by following appropriate
safeguards as aerosols might be produced during centrifugation and sonication posing a
great risk of laboratory infections.
• C. trachomatis can be isolated from various clinical samples like urethral, urogenital and rectal
swabs.
• McCoy monolayer cells are infected with C. trachomatis to prepare frozen stocks of amplified
clinical isolates.
• The incubation of the chlamydial cultures occurs within the optimal temperature of 35°C to
37°C.
• The cells of Chlamydia reproduce by binary fission in the cytoplasmic vacuole surrounded by
the cytoplasmic membrane.
 Pathogenesis of Chlamydia trachomatis
• Chlamydia are acquired by direct contact with mucous membranes or abraded skin, that is, by
sexual contact or by direct inoculation into the eye in the case of trachoma or neonatal
conjunctivitis.
• Two forms of the organism are needed for infection and disease to occur: the infectious,
extracellular form called an elementary body (EB) and the noninfectious but metabolically
active intracellular form called a reticulate body (RB).
• Receptors for EBs are primarily restricted to nonciliated columnar, cuboidal, and transitional
epithelial cells, which are found on the mucous membranes of the urethra, endocervix,
endometrium, fallopian tubes, anorectum, respiratory tract, and conjunctivae.
• Infection is initiated by attachment of EBs to the apical surfaces of epithelial cells of the
conjunctiva, respiratory, gastrointestinal, or urogenital tracts, followed by entry by receptor-
mediated endocytosis.
• The EBs quickly modify their early endosomal membrane to exit the endosomal pathway,
thereby avoiding fusion with lysosomes and traffic on microtubules to the peri-Golgi/ nuclear
hof region.
• The EB-containing endosomes of C. trachomatis then fuse homotypically with one another to
form their one nascent microcolony called an inclusion.
• The EBs then transform into RBs, the chromosome becomes relaxed and transcriptionally
active, and metabolic growth and binary fission ensue to generate progeny.
• RBs obtain their nutrients from the epithelial cytoplasm using at least two strategies: (1)
insertion of chlamydial proteins (Incs) into the inclusion membrane, some of which likely have
autotransporter functions and (2) insertion of a unique appendage through the inclusion
membrane.
• RBs are osmotically fragile and do not survive outside their inclusion nor can they bind to
epithelial cells. Thus, to perpetuate the infectious process, RBs must mature back into
infectious EBs before escaping from infected cells.
• The inclusion membrane may then fuse with the host plasma membrane to release chlamydiae,
or the host cell, depleted of nutrients and energy, may lyse.
• Luminal C. trachomatis progeny are released at the apical surfaces of polarized columnar
epithelial cells to spread canalicularly to the upper genital tract, whereas the invasive LGV
serovars are released at the basal domain into the submucosa enroute to the regional lymph
nodes.
• Infection of epithelial mucosal cells with C. trachomatis has been shown to generate several
cytokines, including interleukin-1α (IL-1α), IL-6, IL-8, GRO-α and granulocyte–macrophage
colony stimulating factor (GM–CSF), which generate and sustain an inflammatory response.
• Inflammatory mediators and chemokines produced in infected epithelial cells serve as initial
triggers for an influx of leukocytes including neutrophils, natural killer cells, dendritic cells,
monocytes, and lymphocytes.
• Infected epithelial cells and early infiltrating natural killer cells activate antigen presenting cells
into programming the cell-mediated immune response.
• As the host immune response develops, active sites of infection show an infiltration of
lymphocytes, plasma cells, and macrophages.
• IFNs, in particular IFN-α, play an important part in the immune response to chlamydial
infection by inhibiting intracellular replication at the RB stage through tryptophan depletion.
 • However, this may result in continuing secretions of chlamydial antigens, leading to further
sensitization and also induction of persistent non-replicating infection.
• Resistance to infection and clearance of primary infection are due in large part to T-cell
function, with both CD4 and CD8 cells having a protective role.
• MOMP-specific T-helper 1 (TH1) CD4 cells may have an important role in immunity, while
Hsp60-specific TH2 CD4 cells are associated with the pathological sequelae of persistent
infection.
• A chlamydial heat-shock protein (hsp 60), elicits antibody responses that are associated with
the damaging sequelae of C. trachomatis infections in both the eye and genital tract.
• A period of chronic inflammation ensues, with the development of sub-epithelial follicles, and
this leads eventually, in some cases, to fibrosis and scarring.
• Follicles contain typical germinal centres, consisting predominantly of B lymphocytes, with T
cells, mostly CD8+, in the parafollicular region.
• The inflammatory infiltrate between follicles comprises plasma cells, dendritic cells,
macrophages, and polymorphonuclear leucocytes, with T and B lymphocytes .
• The scarring process is responsible for much of the morbidity associated with C. trachomatis, in
both the genital tract and the eye.
• Studies of gene expression at the site of ocular infection have shown the importance of innate
immune pathways and NK (natural killer) cell activation, and suggest that matrix
metalloproteinases 7 and 9 play an important role in the scarring process.
• It is particularly likely to be seen after repeated infections.
• Polymorphisms in immune response genes encoding tumour necrosis factor-α, interferon-γ
and interleukin-10 are associated with the development of severe scarring following ocular C.
trachomatis infection.
• Fibrosis is seen at a late stage, typically in trachoma and pelvic inflammatory disease.
Clinical Manifestations of Chlamydia trachomatis
A. Trachoma
• It is a chronic keratoconjunctivitis that begins with acute inflammatory changes in the
conjunctiva and cornea and progresses to scarring and blindness.
• The C. trachomatis serovars A, B, Ba, and C are associated with clinical trachoma.
• Trachoma is transmitted through direct contact (fingers and fomites) with discharges from the
eyes of the infected patients or indirect contact through contaminated clothes or flies.
• The incubation period for chlamydial conjunctival infection is 3–10 days.
• The earliest symptoms of trachoma are lacrimation, mucopurulent discharge, conjunctival
hyperemia, and follicular hypertrophy.
• Acute infection presents as a follicular conjunctivitis, with congestion and oedema affecting
both the palpebral and bulbar conjunctivae.
• There is papillary hyperplasia, giving the palpebral conjunctiva a velvety appearance.
• In hyperendemic areas, infection tends to be more pronounced in the upper lid.
• Follicles rupture to leave shallow pits termed Herbert’s pits.
• Keratitis develops in the cornea.
• Recurrent infection leads to conjunctival scarring or cicatrization which may occur at sclera-
conjunctiva junction (limbal scarring) or on palpebral conjunctiva and new vessel formation
(pannus).
 • Palpebral conjunctival scarring (cicatrisation) leads to in-turning of the eyelashes (entropion),
which scrape the bulbar corneal surface (trichiasis).
• It is the cycle of recurrent infection, with conjunctival scarring and pannus extending over the
cornea, which results in impaired vision or blindness.
B. Genital infections
• C. trachomatis serovars D–K cause sexually transmitted diseases, and may also produce
infection of the eye (inclusion conjunctivitis).
Infection in men
• Genital infection in men presents most commonly as non-gonococcal urethritis (NGU).
• C. trachomatis is detectable in the urethra of up to 50% of men with symptomatic non-
gonococcal urethritis.
• The incubation period is 7–21 days, compared to 2–5 days for gonorrhoea.
• Patients present with a history of dysuria, usually accompanied by a mild to moderate
mucopurulent urethral discharge, acute epididymitis.
• Patients present with unilateral scrotal pain, swelling and tenderness, often accompanied by
fever.
• Proctitis and proctocolitis may occur in men and women, although these infections appear to
be most common in men who have sex with men.
Infection in women
• C. trachomatis typically infects the columnar epithelial cells of the endocervix.
• Infection is associated with a mucopurulent discharge from the cervix visible on speculum
examination, and with hypertrophic cervical ectopy that tends to bleed on contact.
• C. trachomatis has been implicated as a cause of the urethral syndrome, characterized by
dysuria, frequency and sterile pyuria.
• The clinical manifestations include bartholinitis, cervicitis, endometritis, perihepatitis, salpingitis,
and urethritis.
• Spread to the peritoneum may result in perihepatitis (the Curtis–Fitz-Hugh syndrome), which
may be confused with acute cholecystitis in young women.
• Many women, if untreated, will go on to develop serious long-term sequelae of infection such
as PID, infertility and ectopic pregnancy..
C. Neonatal inclusion conjunctivitis (ophthalmia neonatrum)
• The incubation period for chlamydial infection is significantly longer (6–21 days).
• Chlamydial ophthalmia presents with a watery ocular discharge, which may progress to a
purulent conjunctivitis with marked periorbital oedema.
• Because the conjunctiva at birth lacks a lymphoid layer, follicles do not develop initially but
may be seen after 3–6 weeks.
• Unlike trachoma, the lower conjunctival surface is more heavily infected than the upper.
• Swelling of the infant’s eyelid, hyperemia, and purulent discharge characterize the condition
• Conjunctival scarring and corneal vascularization occurs in untreated infections of long
duration
D. Infant pneumonia
• Infant pneumonia caused by C. trachomatis is seen in infants between 4 and 16 weeks of age.
• The incubation period is variable but usually takes 2–3 weeks after birth.
 • Pneumonitis develops when organisms present in the conjunctiva pass down the nasolacrimal
duct into the pharynx.
• The condition is characterized by respiratory symptoms, such as rhinitis with cough and
wheezing
• Infection via the Eustachian tube may cause otitis media.
E. Adult inclusion conjunctivitis
• Adult inclusion conjunctivitis results from the infection with C. trachomatis strains associated
with genital infection (A, B, Ba, and D–K).
• This infection is more frequently seen in sexually active adults.
• The condition can also occur in neonates.
• A uniocular and less commonly binocular red eye, ocular discharge, marked hyperemia,
papillary hypertrophy, and a predominant follicular conjunctivitis are the important
manifestations.
• The condition if untreated progresses to a chronic remittent course, keratitis, and possible iritis.
F. Reactive arthritis
• Arthritis occurring with or soon after non-gonococcal urethritis is termed ‘sexually acquired
reactive arthritis’.
• It is believed that Reiter syndrome (urethritis, conjunctivitis, polyarthritis, and mucocutaneous
lesions) is initiated by genital infection with C. trachomatis.
G. Lymphogranuloma venereum (LGV)
• C. trachomatis serovar Ll, L2, L2a, L2b and L3 are the agents of lymphogranuloma venereum
(LGV).
• The clinical course of LGV can be divided into three stages:
1. Primary stage
• Painless papule, ulcer or vesicle develops on the penis or vulva after an incubation period of 3
days to 6 weeks.
• The primary lesion is self-limiting and may pass unnoticed by the patient
2. Secondary stage
• This occurs some weeks after the primary lesion.
• It may involve the inguinal lymph nodes, or the anus and rectum.
• LGV proctitis occurs in those who practice receptive anal intercourse, probably due to direct
inoculation.
• The cardinal feature of the inguinal form of LGV is painful, usually unilateral, inguinal and/or
femoral lymphadenopathy (bubo).
• Enlarged lymph nodes are usually firm and often accompanied by fever, chills, arthralgia and
headache.
• Small discrete areas of necrosis surrounded by proliferating epithelioid and endothelial cells,
which may enlarge to form stellate abscesses that may coalesce and break down to form
discharging sinuses.
• In women, signs include a hypertrophic suppurative cervicitis, backache and adnexal
tenderness.
• Clinical features of anorectal disease include a purulent anal discharge, pain and bleeding due
to an acute haemorrhagic proctitis or proctocolitis, often with fever, chills and weight loss.
• Enlarged inguinal nodes may also be palpable.
 3. Third stage
• Occurs in untreated cases, especially in women and homosexual men.
• Chronic untreated LGV leads to fibrosis, which may cause lymphatic obstruction and
elephantiasis of the genitalia in either sex due to impaired lymphatic drainage or rectal
strictures, rectosigmoid obstruction and fistula formation
• Esthiomene-the vulva, scrotum or penis may undergo edematous granulomatous hypertrophy.
Laboratory Diagnosis of Chlamydia trachomatis
Specimen
• Urethral discharge, cervix swab, rectum, oropharynx, and conjunctiva swab are the frequently
collected specimens.
• In addition, other specimens such as, blood, urine, respiratory secretions, sputum, lung, and
other tissues are collected and examined.
• Pus from bubo is also useful for diagnosis of LGV.
Microscopy
• Demonstration of chlamydial inclusion bodies stained by Giemsa, Castaneda, Machiavello,
Gimenez stains or Lugol’s iodine.
• C. trachomatis infections of conjunctiva, urethra, and cervix are diagnosed by demonstration of
typical reniform inclusion bodies surrounding the nucleus.
• Direct Immunofluorescence test (DIF) is used as for direct detection of inclusion bodies in
clinical material, particularly from the genital tract and eye.
• In DIF, fluorescent tagged monoclonal antibodies directed against group-specific LPS antigen
or species-specific MOMP antigens are added.
Culture
• Isolation of C. trachomatis in cell cultures is the more specific method for diagnosis of C.
trachomatis infection.
• Centrifugation of specimens onto cycloheximide treated McCoy or HeLa cell monolayers,
followed by incubation and then staining with a fluorescent monoclonal antibody or with a
vital dye, to detect inclusions, has been widely used for the diagnosis
of C. trachomatis infection.
Antigen detection
• Two general approaches have been used to detect chlamydial antigens in clinical specimens:
direct immunofluorescence staining with fluorescein-conjugated monoclonal antibodies and
enzyme-linked immunosorbent assays.
• In both assays, antibodies are used that have been prepared against either the chlamydial
MOMP or the cell wall LPS.
• The DFA uses monoclonal antibodies directed against a species-specific antigen on the
chlamydial MOMP.
• The EIA detects the presence of genus-specific antigens extracted from EBs in the specimen.
Nucleic Acid-Based Tests
• Nucleic acid probe tests most commonly measure the presence of a species-specific sequence
of 16S ribosomal RNA.
• The advantage of these tests is that the nucleic acid does not have to be amplified, making the
tests rapid and relatively inexpensive; however, these tests are relatively insensitive for the
detection of small numbers of chlamydiae.
 • Various methods available are:
• Polymerase chain reaction (PCR)
• Ligase chain reaction (LCR)
• Transcription-mediated amplification (TMA)
• Strand displacement assay (SDA).
Antibody Detection
• Serologic testing is of limited value in the diagnosis of C. trachomatis urogenital infections in
adults because the test cannot differentiate between current and past infections.
• C. trachomatis IgM antibody is the ‘gold standard’ for the diagnosis of chlamydial pneumonia
in babies.
• Antibody tests for the diagnosis of LGV can be helpful.
• Infected patients produce a vigorous antibody response that can be detected by complement
fixation (CF), microimmunofluorescence (MIF), or enzyme immunoassay (EIA).
Treatment of Chlamydia trachomatis Infection
• Tetracyclines and macrolides are the mainstay of treatment.
• Tetracyclines (eg, doxycycline) are commonly used in nongonococcal urethritis and in
nonpregnant infected women.
• Azithromycin is effective and can be given to pregnant women.
• Ophthalmia neonatorum and neonatal pneumonia due to C. trachomatis should be treated
with erythromycin.
• Erythromycin may be administered orally and topically for treatment of ophthalmia
neonatorum.
• Systemic erythromycin is effective treatment in severe cases.
• Recommended treatment for LGV is doxycycline or erythromycin.
• Azithromycin has been used successfully in some cases.
• Ocular infection can be effectively treated with a single oral dose of azithromycin.
Prevention and Control of Chlamydia trachomatis Infection
• Health education and condom promotion, especially for the youngest sexually active age
groups may help to reduce the incidence of genital C. trachomatis infection.
• Periodic screening of high risk groups, such as young women having multiple sex partners.
• Use of barrier methods of contraception such as condoms.
• Chlamydia conjunctivitis and genital infections are prevented through the use of safe sex
practices and the prompt treatment of symptomatic patients and their sexual partners.
• The blindness associated with advanced stages of trachoma can be prevented only by prompt
treatment of early disease and the prevention of re-exposure.
• Screening of mother giving birth to a child for Chlamydial infections.

Bordetella pertussis: characteristics,

virulence factors, pathogenesis, symptoms,
treatment and vaccine
May 11, 2021 Gaurab Karki Bacteriology 0
Bordetella pertussis: characteristics, virulence factors, pathogenesis, symptoms, treatment and vaccine

Bordetella pertussis
▪ Bordetella pertussis (Bordet-Gengou Bacillus; formally known as Hemophilus pertussis)

Morphology of Bordetella pertussis :

▪ The Bordetella spp are small, gram-negative coccobacilli with slight pleomorphism measuring 0.2-
0.3 μm by 0.5 to 1.0 μm.
▪ They appear singly, in pairs, and in small clusters.
▪ On primary isolation, cells are uniform in size, but in subcultures they become quite pleomorphic
and filamentous, and thick bacillary forms are common.
▪ It is nonmotile and non-sporing.
▪ Bipolar metachromatic staining may be demonstrated with toluidine blue.
▪ Capsules may be demonstrable in young, freshly isolated cultures only by special stains.
▪ In culture films, the bacilli tend to be arranged in loose clumps, with clear spaces in between
giving a ‘thumb print’ appearance.
▪ Freshly isolated strains of B. pertussis have fimbriae.

Cultural Characteristics of Bordetella pertussis:

▪ It is an obligate aerobe.
 ▪ The optimum temperature for growth is 35-36°C.
▪ It does not require X and V factors for its growth.
▪ Complex media are necessary for primary isolation.
▪ The medium in common use is the Bordet-Gengou medium (potato-blood-glycerol agar).
▪ Primary isolation of B. pertussis requires the addition of charcoal, ion exchange resins, or 15-20
percent blood to neutralize growth-inhibiting effects.
▪ Potatoes impart a high starch content to the medium that neutralizes toxic materials.
▪ Glycerol acts as a stabilizing agent. Charcoal blood agar is a useful medium.
▪ The plates are incubated at 35-36°C in a moist environment (e.g., a sealed plastic bag).
▪ After incubation for 48-72 hours, colonies on Bordet-Gengou medium are small, dome shaped,
smooth, opaque, viscid, greyish white, refractile and glistening, resembling ‘bisected pearls’ or
‘mercury drops’.
▪ Colonies are surrounded by a hazy zone of hemolysis. Confluent growth presents an ‘aluminium
paint’ appearance.
▪ Subcultures of B. pertussis may be obtained on less exacting media, e.g. nutrient agar to which
charcoal or starch has been added.

Biochemical Reactions of Bordetella pertussis:

▪ It is biochemically inactive.
▪ It does not ferment sugars, form indole, reduce nitrates, utilize citrate or split urea.
▪ It produces oxidase and usually catalase also.

Antigenic Constituents and Virulence Factors of Bordetella pertussis:

▪ B. pertussis is a delicate organism.

▪ It can be killed by heating at 55°C for 30 minutes, drying and disinfectants.
▪ Outside the body it can survive for five days on glass, three days on cloth and a few hours on
paper.
▪ The organism is usually sensitive to ampicillin and erythromycin and these drugs have a
reasonable therapeutic record.
▪ B. pertussis produces a number of factors that are involved in the pathogenesis of disease.
▪ 1. Agglutinogens
▪ Bordetellae possess genus specific and species specific surface 14 agglutinogens
associated with the capsular K antigens or fimbriae.
▪ Factors 1 to 6 are found only in strains of B. pertussis, all of which carry Factor I and
one or more of the other factors.
▪ All three mammalian species of bordetellae has common Factor 7.
▪ Factor 12 is specific’ for B. bronchiseptica and Factor 14 for B. parapertussis.
▪ Agglutinogens promote virulence by help- ing bacteria to attach to respiratory
epithelial cells.
▪ They are useful in serotyping strains and in epidemiological studies.
▪ On the basis of the agglutinogens Bordetellae carry they are classified into various
types.
▪ 2. Pertussis toxin (PT)
▪ PT, also known as lymphocytosis-promoting factor, pertussigen, histamine-
sensitizing factor and islet-activating factor, has a wide spectrum of biologic activity.
 ▪ Pertussis toxin promotes lymphocytosis, sensitization to histamine, and enhanced
insulin secretion.
▪ PT is expressed on the surface of the bacillus and secreted into the surrounding
medium.
▪ PT has a molecular weight of 117,000 and is a classic A-B toxin(dissociated into A
and B subunits) made up of 6 polypeptide chains consisting of a A subunit, toxic
subunit (SI) and five binding subunits (S2 to S5; two S4 subunits are present in each
toxin molecule).
▪ B unit consists of the remaining 5 polypeptide chains binds the toxin to the target
cells and helps A unit to cross the membrane.
▪ It can be toxoided. Pertussis toxoid is the major component of acellular pertussis
vaccines.
▪ It is apparently responsible for many of the clinical signs and symptoms of
pertussis, as well as the relative and absolute lymphocytosis observed during the
clinical illness.
▪ Antibody against PT is also protective in animal models of pertussis. It can be
toxoided.
▪ The major component of acellular pertussis vaccines is PT toxoid.
▪ Antibody to PT can protect mice against intranasal, intra- peritoneal or
intracerebral challenge.
▪ Outside the B. pertussis cells, the filamentous hemagglutinin and pertussis toxin are
found which are secreted proteins .
▪ 3. Filamentous Hemagglutinin (FHA)
▪ The filamentous hemagglutinin and pertussis toxin are secreted proteins and are
found outside of the B. pertussis cells.
▪ It is a protein that acquired its name through its ability to agglutinate erythrocytes.
▪ It mediates adhesion to ciliated epithelial cells.
▪ FHA is used in acellular pertussis vaccines along with PT toxoid.
▪ Antibodies against filamentous hemagglutinin are protective.
▪ FHA and PT hemagglutinins also promote secondary infection by coating other
bacteria such as Haemophilus influenzae and or Streptococcus pneumoniae and assisting
their binding to respiratory epithelium besides facilitating adhesion of B. pertussis to
respiratory epithelium.
▪ This potential “piracy of adhesins” by other organisms may contribute to secondary
bacterial invasion in pertussis .
▪ 4. Adenylate Cyclase (AC)
▪ All mammalian Bordetellae but not B. avium produce adenylate cyclase .
▪ At least two types of AC are known, only one of which has the ability to enter target
cells and act as a toxin.
▪ This is known as AC toxin (ACT).
▪ It has the ability to enter target cells (leukocytes) and act as a toxin. It can be
activated by eukaryotic calcium-dependent regulatory protein, calmodulin.
▪ Inside the cell, after activation by calmodulin, this enzyme synthesizes cAMP(as
pertussis toxin does) which is responsible for the biological effects such as
interfering with leukocyte functions (inhibition of phagocytosis and chemotaxis).
▪ 5. Heat Labile Toxin (HLT) or Dermonecrotic Toxin
 ▪ The heat–labile toxin (HLT) produced by all species of Bordetella appears to be a
cytoplasmic protein it is a heat- labile toxin it is dermonecrotic and at high doses,
this toxin causes fatal reactions in mice.
▪ Role in disease is unknown.
▪ 6. Tracheal Cytotoxin (TCT)
▪ Tracheal cytotoxin is a low-molecular-weight cell wall peptidoglycan monomer that
has a specific affinity for ciliated epithelial cells.
▪ It induces ciliary damage in hamster tracheal ring cultures and inhibition of DNA
synthesis in the ciliated respiratory epithelial cells, resulting in accumulation in the
lungs of mucus, bacteria and inflammatory debris leading to severe cough.
▪ The disruption of ciliary function may also contribute to the secondary bacterial
infections.
▪ The toxin also stimulates the release of the cytokine interleukin-l, which leads to
fever.
▪ 7. Lipopolysaccharide (Heat-Stable Toxin)
▪ It is present in all bordetellae and exhibits features of gram-negative bacterial
endotoxins.
▪ Their role in the disease process is unknown.

Pathogenesis of Bordetella pertussis:

▪ Whooping cough is predominantly a pediatric disease.
▪ Whooping cough in 95 percent of cases is caused by B. pertussis.
▪ B. parapertussis causes about 5 percent of the cases and by B. bronchiseptica very infrequently
(0.1%).

Stages of Whopping cough Disease:

▪ In human beings, after an incubation period of about 1-2 weeks, the disease takes a protracted
course comprising three stages-the catarrhal, paroxysmal and convalescent -each lasting
approximately two weeks.
▪ 1. Prodromal or Catarrhal Stage
▪ The first stage, the catarrhal stage, resembles a com- mon cold, with serous
rhinorrhea, sneezing, malaise, anorexia, and low grade fever.
▪ Clinical diagnosis in the catarrhal stage is difficult.
▪ During this stage, large numbers of organisms are sprayed in droplets, and the
patient is highly infectious but not very ill.
▪ This is unfortunate as this is the stage at which the disease can be arrested by
antibiotic treatment.
▪ 2. Paroxysmal Stage
▪ After 1 to 2 weeks, the paroxysmal stage begins.
▪ As the catarrhal stage advances to the paroxysmal stage, the cough increases in
intensity and comes on in distinctive bouts.
▪ During the paroxysm, the patient is subjected to violent spasms of continuous
coughing, followed by a long inrush of air into the almost empty lungs, with a
characteristic whoop (hence the name).
 ▪ The paroxysms of coughing may be so severe that cyanosis, vomiting and
convulsions follow, completely exhausting the patient.
▪ 3. Convalescent Stage
▪ After 2 to 4 weeks, the paroxysmal stage is followed by convalescence stage, during
which the frequency and severity of coughing gradually decrease but secondary
complications can occur.
▪ The disease usually lasts 6-8 weeks though in some it may be very protracted.
▪ Complications:
▪ Subconjunctival hemorrhage, subcutaneous emphysema, inguinal hernia or rectal
prolapse due to pressure effects during the violent bouts of coughing.
▪ Respiratory (bronchopneumonia, lung collapse)- Respiratory complications are self-
limited, the atelectasis resolving spontaneously.
▪ Neurological (convulsions, coma.)-neurological complications may result in
permanent sequelae such as epilepsy, paralysis, retardation, blindness or deafness.
Epidemiology of Bordetella pertussis:
▪ Whooping cough is predominantly a pediatric disease, the incidence and mortality being highest
in the first year of life.
▪ Maternal antibodies do not seem to give protection against the disease.
▪ Immunization should, therefore, be started early.
▪ The disease is commoner in the female than in the male at all ages.
▪ It is worldwide in distribution.
▪ It occurs in epidemic form periodically but the disease is never absent from any community.
▪ The source of infection is the patient in the early stage of the disease.
▪ Infection is transmitted by droplets and fomites contaminated with oropharyngeal secretions.
▪ Whooping cough is one of the most infectious of bacterial diseases and nonimmune contacts
seldom escape the disease.
▪ The secondary attack rates are highest in close household contacts.
▪ The disease is often atypical.
▪ In adolescents and adults and may present as bronchitis.
▪ They may serve as a source of infection in infants and children.
▪ Natural infection confers protection though it may not be permanent, and second attacks have
been reported.
Laboratory Diagnosis of whooping cough:
▪ Blood changes in the disease are distinctive and helpful in diagnosis.
▪ Pertussis typically causes an elevated white cell count, sometimes in excess of 50,000 cells/ μl
(normal range=4500-11000 white blood cells/μl during the latter part of the catarrhal or early
paroxysmal phase.
▪ A marked leukocytosis occurs, with relative lymphocytosis (total leukocytic counts 20,000-30,000
per/ μl with 60-80 percent lymphocytes).
▪ The erythrocyte sedimentation rate is not increased, except when secondary infection is present.
Three lab diagnosis methods are available:
1. Isolation of B. pertussis by culture from a pernasal swab;
2. Identification of the organism in a smear from a pernasal swab by immunofluorescence
microscopy;
3. Serological demonstration of specific antibodies in the patient’s serum.
1. Microscopy:
▪ Microscopic diagnosis depends on demonstration of the bacilli in respiratory secretions by the
fluorescent antibody technique.
2. Specimen Collection and Transport:
▪ Though the disease is mainly in the lower respiratory tract, the organism can be recovered
readily from the nasopharynx.
▪ ‘Cough plates’ and postnasal swabs are unsatisfactory because of overgrowth by commensal
bacteria.
▪ The optimal diagnostic specimen is a naso-pharyngeal aspirate.
▪ i. The Cough Plate Method
▪ Here a culture plate is held about 10-15 cm in front of the patient’s mouth during
about of spontaneous or induced coughing so that droplets of respiratory exudates
impinge directly on the medium.
▪ This has the advantage that specimen is directly inoculated at the bedside.
▪ ii. The Postnasal (Peroral) Swab
▪ Secretions from the posterior pharyngeal wall are collected with a cotton swab on a
bent wire passed through the mouth.
▪ Salivary contamination should be avoided.
▪ A West’s postnasal swab may be conveniently employed.
▪ Cotton swabs should not be used because they contain fatty acids that are toxic
to B. pertussis so it is preferable to use dacron or calcium alginate swabs for
specimen collection.
▪ iii. The Pernasal Swab
▪ A sterile swab on a flexible wire is passed gently along the floor of the nose until it
meets resistance.
▪ The swab, which will collect mucopus, is withdrawn and either plated immediately
on charcoal blood agar, or placed in transport medium.
▪ The use of transport medium reduces the isolation rate.
▪ A single swab may yield a negative culture, but isolation rates of up to 80 percent
may be achieved by taking specimens on several successive days.
▪ The pernasal swab has generally replaced the cough plates or post- nasal swabs
that were used in the past.
3. Culture:
▪ The swab is inoculated immediately on charcoalhorse blood agar and Bordet-Gengou medium
both with and without methicillin or cephalexin and incubated for at least seven days before
being discarded as negative.
▪ The specimen may be transported in Regan-Lowe semi-solid medium if delay in transport is
unavoidable.
▪ Plates are incubated in high humidity at 35-36°C and colonies appear in 48-72 hours.
▪ Typical ‘bisected pearl’ colonies appearing after 3-5 days must be investigated further.
4. Identification:
▪ Identification is confirmed by microscopy and slide agglutination with specific antisera.
▪ Immunofluorescence is useful in identifying the bacillus in direct smears of clinical specimens
and of cultures.
5. Detection of Bacterial Antigens:
▪ Bordetella antigens may be detected in serum and urine in tests with specific antiserum.
▪ Alternatively, bacteria in nasopharyngeal secretions are labelled with fluorescein-conjugated
antiserum and examined by ultraviolet microscopy.
▪ This method has the theoretical advantage, compared with culture, of detecting dead
bordetellae.
6. Polymerase Chain Reaction (PCR):
 ▪ Polymerase chain reaction (PCR) is used for the detection of bordetella DNA in nasopharyngeal
specimens, by the use of various primers with a sensitivity of 80 percent to 100 percent.
7. Serology:
▪ Rise in antibody titer may be demonstrated in paired serum samples by ELISA, agglutination.
▪ Complement fixation, immunoblotting, indirect hemagglutination, and toxin neutralization.
▪ Demonstration of specific secretory IgA antibody in nasopharyngeal secretions by ELISA has
been proposed as a diagnostic method in culture negative cases.
Treatment of whooping cough:
▪ B. pertussis is susceptible to several antibiotics (except penicillin).
▪ The drug of choice is erythromycin (or one of the newer macrolides such as clarithromycin),
which may reduce the severity of the illness if given before the paroxysmal stage.
▪ Chloramphenicol and cotrimoxazole are also useful.
▪ Treatment with pertussis immunoglobulin has been tried, but with limited success.
▪ Prophylaxis:
▪ Preventing the spread of infection by isolation of cases is seldom practicable as
infectivity is highest in the earliest stage of the disease when clinical diagnosis is
not easy.
▪ Treatment and Quarantine:
▪ Antibiotics and immunoglobulins currently available are not very effective for the
treatment of patients or the protection of contacts.
▪ Control of the disease by quarantine is unrealistic.
Vaccination for whooping cough :
▪ Specific immunization with killed B. pertussis vaccine has been found very effective.
▪ It is of utmost importance to use a smooth phase I strain for vaccine production.
▪ The vaccines in general use are suspensions of whole bacterial cells, killed by heat or chemicals.
▪ Adsorption of the bacteria on to an adjuvant, such as aluminium hydroxide, enhances the
immune response (particularly important with factor3) and also causes fewer adverse reactions.
▪ B. pertussis acts as an adjuvant for the toxoids (diphtheria and tetanus toxoid) producing better
antibody response.
▪ Infants and young children should be kept away from cases.
▪ Those known to have been in contact with whooping cough may be given prophylactic antibiotic
(erythromycin or ampicillin) treatment for 10 days to prevent the infecting bacteria to become
established.
▪ The best protection that can be given to an infant is to administer a booster dose of DPT/DT to
his siblings before he is born.
▪ Adverse Reactions:
▪ Pertussis vaccination may induce reactions ranging from local soreness and fever
to shock and neurological complications like convulsions and encephalopathy.
▪ Provocation poliomyelitis is a rare complication.
▪ Contraindications:
▪ If severe complications such as encephalopathy, seizures. ‘shock or hyperpyrexia
develop following the vaccine, subsequent doses of the vaccine are
contraindicated.
▪ Routine pertussis vaccination is not advisable after the age of seven years as
adverse reactions are likely and the risk of severe disease is low.
▪ Acellular Pertussis Vaccine:
▪ Acellular vaccines containing the protective components of the pertussis bacillus
(PT. FHA. agglutinogens 1, 2. 3) first developed in Japan, cause far fewer reactions,
particularly in older children.
▪ Both whole cell and acellular vaccines have a protection rate of about 90 percent.