Transcript Salmonella

Salmonella
Infections
an update
Dr.T.V.Rao MD
Dr.T.V.Rao MD
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Salmonella
• Causes Infections in Humans and
vertebrates,
• Enteric Fever ( Typhoid fever )
• Gastroenteritis
• Septicemias,
• Carrier state a concern
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Salmonella
• A Very complex group
• Contains more > 2,000 spp
• Typed on the basis of Serotyping, and species
typing
• Divided into two groups
1 Enteric fever group
2 Food poisoning group – Septicemias.
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Key points
• There are more than 2000 different antigenic
types of Salmonella; those pathogenic to man
are serotypes of S. enterica.
• Most serotypes of S. enterica cause foodborne gastroenteritis and have animal
reservoirs.
• S. enterica serotypes Typhi and Paratyphi
cause typhoid fever.
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Enteric Fever
Typhoid Fever
• Caused by Salmonella typhi, and other
Groups called as Paratyphoid A, B, C
• Salmonella typhi - Causes Typhoid
• Salmonella Paratyphi A,B,C Causes
Paratyphoid fevers.
• Food Poison group
• Spread from Animals – Humans
• Causes Gastroenteritis – Septicemias,
Localized Infection
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Typhoid fevers are prevalent in many
regions in the World
Typhoid Mary Most Dangerous
Woman in America
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Typhoid Mary
• A famous example is
“Typhoid” Mary Mallon,
who was a food handler
responsible for infecting
at least 78 people,
killing 5. These highly
infectious carriers pose
a great risk to public
health.
Typhoid Mary
• "Typhoid Mary," real name Mary Mallon,
worked as a cook in New York City in the early
1900s. Public health pioneer Sara Josephine
Baker, MD, PhD tracked her down after
discovering that she was the common link
among many people who had become ill from
typhoid fever She was traced to typhoid
outbreaks a second time so she was put in
prison again where she lived until she died.
Morphology of Salmonella
• Gram negative
bacilli
• 1-3 / 0.5
microns,
• Motile by
peritrichous
flagella
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S.typhi with Flagella
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Bacteriology –Typhoid fever
• The Genus
Salmonella belong to
Enterobacteriaceae
• Facultative anaerobe
• Gram negative bacilli
• Distinguished from
other bacteria by
Biochemical and
antigen structure
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Different types of Salmonella
I - enterica
II - salamae
IIIa -arizonae
IIIb -diarizonae
IV - houtenae
V - bongori
VI - indica
Cultural Characters
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Aerobic / Facultatively anaerobic
Grows on simple media – Nutrient agar,
Temp 15 – 41ºc / 37º c
Colonies appear as large 2 -3 mm, circular, low
convex,
• On MacConkey medium appear
Colorless ( NLF )
Selective Medium - Wilson Blair Bismuth sulphide
medium. Produce Jet black colonies
H2 S produced by Salmonella typhi
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Enrichment Medium
Liquid Medium
• Selenite F medium
• Tetrathionate broth
• Above medium are used for
isolation of Salmonella from
contaminated specimens
• Particularly stool specimens..
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Identifying Enteric Organisms
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Isolates which are Non lactose fermenting
Motile, Indole positive
Urease negative
Ferment Glucose,Mannitol,Maltose
Do not ferment Lactose, Sucrose
Typhoid bacilli are anaerogenic
Some of the Paratyphoid form acid and gas
Further identification done by slide
agglutination tests
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Biochemical Characters
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Glucose ,Mannitol ,Maltose produce A/G
Salmonella typhi do not produce gas
Lactose/Salicin/sucrose not fermented.
Indole –
Methyl Red +
VP Citrate +
Urea –
H2S – produced by Salmonella typhi
Paratyphi A do not produce H2S
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Resistance of Salmonella
• 55º c – 1 hour
• 60º c – 15 MT
• Boiling ,Chlorination,
Pasteurization Destroy the
Bacilli.
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Antigenic structure of Salmonella
Two sets of antigens
• Detection by serotyping
• 1 Somatic or 0 Antigens contain long chain
polysaccharides ( LPS ) comprises of heat
stable polysaccharide commonly.
•
• 2 Flagellar or H Antigens are strongly
immunogenic and induces antibody formation
rapidly and in high titers following infection or
immunization. The flagellar antigen is of a dual
nature, occurring in one of the two phases.
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Salmonella
Antigenic Structure
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H – Flagellar antigens
O – Somatic antigen,
Vi – Surface antigen in some species only
H antigens also called flagellar antigens,
heat
labile protein,
• Boiling destroys antigenicity
• When mixed with Antiserum produces agglutination
and fluffy clumps are produced
• H antigens are strongly immunogenic Induces
antibodies rapidly,
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Antigens – Salmonella ( cont )
• O Antigens
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Forms integral part of Cell wall,
Like Endotoxin
0 Antigens unaffected by boiling.
When mixed with antiserum produce chalky clumps
are formed, take more time reaction, at high temp
50º – 55º c
• O antigens are less immunogenic. than H antigens
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Antigen (Vi) – Salmonella ( contd )
• Vi antigens
• Many strains in S.typhi covers the O antigensprevents agglutination.
• Resembles like K antigens
• Destroyed after boiling at 60º c / 1 hour.
• Vi a polysaccharide
• Acts as virulence factor, protects the bacilli against
Phagocytosis and activity of Complement
• Poorly immunogenic
• Low titer of antibodies are produced, Not diagnostic
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Classification of Salmonella
• Classified on the basis of
Kauffmann-White Scheme
• Structure of 0 and H antigens are
taken into consideration,
• More than 2000 species
characterized.
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Kauffmann – White scheme
• Serotype
0 antigens
1.Typhi
9,12,(Vi)
2 Paratyphi A 1,2.12
3 Paratyphi B 1,4,5,12
4 Typhimuruim 1,4,5,12
5 Enteritidis 1,9,12
H antigens
Phase 1
2
d
1,2
a
b
1,2
I
1,7
g m 1,2
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Antigenic Variation in
Salmonella
• May be phenotypic / Genotypic
• H to O = loss of Flagella
May be phase variation from I to
II
V to W variation
S to R variation
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Pathogenicity
• Salmonella are definite parasites to
humans.
• Eg S.typhi.
•
S.paratyphi A, B ,C
•
Other groups Salmonella
• The important clinical syndromes
1. Enteric fever, Septicemias,
gastroenteritis.
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Enteric Fever: S. typhi
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Ileocecal penetration
intraluminal multiplication
mononuclear response (macrophages)
Salmonella remains alive
2nd week - lymphoid hyperplasia (mesenteric
lymph nodes)
• back to bowel
Enteric Fever
Typhoid
• Typhoid – caused by S.typhi
• Paratyphoid Caused by
Paratyphi A,B,C
• Typhoid --- Like Typhus
• Infective dose ID50 / 107,
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Fever
• All the events coincides with Fever and other
signs of clinical illness
• From Gall bladder further invasion occurs in
intestines
• Involvement of peyr’s patches, gut lymphoid
tissue
• Lead to inflammatory reaction, and infiltration
with monocular cells
• Leads to Necrosis, Sloughing and formation of
chacterstic typhoid ulcers
Rashes in Typhoid
• May present with rash,
rose spots 2 -4 mm in
diameter raised discrete
irregular blanching pink
maculae's found in
front of chest
• Appear in crops of upto
a dozen at a time
• Fade after 3 – 4 days
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Events in a Typical typhoid Fever
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Pathology and Pathogenesis
• Bacilli enter through ingestion,
• Bacilli attach to Microvilli,ileal mucosa,
penetrate to Lamina propria and sub mucosa
• Phagocytosis by Polymorphs and
Macrophages
• Enters the mesenteric lymph nodes
• Enter the thoracic duct – Blood stream
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Infective Dose
• For human infections, the number of bacteria
that must be swallowed in order to cause
infection is uncertain and varies with the
serotype. In most of these the median
infective dose for most serotypes, including
Typhi, has varied from 106 to 109 viable
organisms. However, investigation of
outbreaks suggests that in natural infection
the infective dose might be fewer than 1000
viable organisms.
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Pathology and Pathogenesis
• Bacteremia Spread to Liver, Gall
bladder, Spleen, Bone marrow,
Lymph nodes, Lungs, Multiply in
kidneys
Once again spill into Blood stream
Causes clinical illness.
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Pathology and Pathogenesis
• Multiply abundantly in Gall bladder,
• Bile rich source of Bacteria
• Spill into Intestine, infects payers patches,
Lymph follicles
• Inflammation – Undergo necrosis, Slough off
• Typhoid ulcers
• Typhoid ulcers can cause perforation and
hemorrhage
• Duration of Illness 3 – 4 weeks
• Incubation 7 -14, ( 3-56 days )
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What is Enteric Fever
Typhoid Fever
• Enteric fever is caused by strains of S. Typhi or S.
Paratyphi A, B or C; although S. Paratyphi B,
which gene sequence analysis suggests is a
variant of S. Java, is more likely to cause nontyphoidal diarrhoea. The clinical features tend to
be more severe with S. Typhi (typhoid fever).
After penetration of the ileal mucosa the
organisms pass via the lymphatic's to the
mesenteric lymph nodes, whence after a period
of multiplication they invade the bloodstream via
the thoracic duct.
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Progress in Enteric Fever
• The liver, gall bladder, spleen, kidney and bone
marrow become infected during this primary
bacteraemic phase in the first 7-10 days of the
incubation period. After multiplication in
these organs, bacilli pass into the blood,
causing a second and heavier bacteraemia,
the onset of which approximately coincides
with that of fever and other signs of clinical
illness.
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Progress in Enteric Fever
• From the gall bladder, a further invasion
of the intestine results. Peyer's patches
and other gut lymphoid tissues become
involved in an inflammatory reaction,
and infiltration with mononuclear cells,
followed by necrosis, sloughing and the
formation of characteristic typhoid ulcers
occurs.
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Immunity in Typhoid
• Typhoid bacilli
are
Intracellular
pathogens
• Cell mediated
immunity is
crucial
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Diarrhea
Nausea
Vomiting
Stomach pain
Headache
Fever
Onset 12-72 hours after
infection
Clinical manifestation
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Head ache, malise,anorexia ,coated tongue
Abdominal discomfort,
Constipation / Diarrhea
Step ladder type fever,
Relative bradycardia,
A soft palpable spleen
Hepatomegaly
Rose spots appear
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Events in a Typical typhoid Fever
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Complications of Enteric fever
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Intestinal perforation,
Hemorrhage,
Circulatory collapse.
Bronchitis Bronchopneumonia,
Meningitis,
Cholecystitis,
Arthritis,Periostitis / Nephritis,
Osteomyletis,
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Relapses in Typhoid Fever
• Apparent recovery can be followed by
relapse in 5-10% of untreated cases.
Relapse is usually shorter and of milder
character than the initial illness, but can
be severe and may be fatal. Severe
intestinal haemorrhage and intestinal
perforation are serious complications
that can occur at any stage of the illness.
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Other complications
• Causes relapses in
particular to
patients treated
with
chloramphenicol.
• S.paratyphi
produce
septicemias.
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Typhoid carriers
• Salmonella enterica causes
approximately 16 million cases of typhoid
fever worldwide, killing around 500,000
per year. One in thirty of the survivors,
however, become carriers. In carriers the
bacteria remain hidden inside cells and
the gall bladder, causing new infections
as they are shed from an apparently
healthy host.
Carrier Stage in Typhoid Fever
• Most people infected with salmonella
continue to excrete the organism in their
stools for days or weeks after complete clinical
recovery, but eventual clearance of the
bacteria from the body is usual. A few patients
continue to excrete the salmonellae for
prolonged periods. The term chronic carrier is
reserved for those who excrete salmonellae
for a year or more.
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Carrier Stage in Typhoid Fever
• Chronic carriage can follow symptomatic
illness or may be the only manifestation of
infection. It can occur with any serotype, but
is a particularly important feature of enteric
fever: up to 5% of convalescents from typhoid
and a smaller number of those who have
recovered from paratyphoid fever become
chronic carriers, many for a lifetime.
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Carrier Stage in Typhoid Fever
• The bacilli are most commonly present in the
gall bladder, less often in the urinary tract,
and are shed in faeces and sometimes in
urine. The long duration of the carrier state
enables the enteric fever bacilli to survive in
the community in non-epidemic times and to
persist in small and relatively isolated
communities.
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Epidemiology
• Developed countries - Controlled.
• Water supply/ Sanitation /Economically
poor.
• S.typhi and S.paratyphi are prevalent in
India
• Previously Typhi are more common
Paratyphoid A on raise.
• Age 5 – 20 years, Sanitation
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Epidemiology
• Sanitation has great role
• Source an active patient or a Carrier shed the
Bacilli.
• Who are carriers.
Convalescent carrier 3 weeks to 3 months
Temporary carrier 3 months to 1 year
Chronic carrier
> 1 year,
Women attain more carrier stage
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Epidemiology (Contd)
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Bacilli persist in the Gall bladder and kidney
Food handlers spread the infection
Cooks great role
S.typhi and S.paratyphi in humans
S.para B in Animals,
Typhoid spread through
Water, Milk, Food
HIV patients potentially susceptible for Typhoid
disease.
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Bacteriological Diagnosis of Typhoid Fever
• Selective media, such as Deoxycholatecitrate agar or xylose-lysine Deoxycholate
agar, are used for the isolation of
salmonella bacteria from faeces. Fluid
enrichment media, such as Tetrathionate
or selenite broth, are also useful to
detect small numbers of salmonellae in
faeces, foods or environmental samples.
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Bacteriological Diagnosis of
Typhoid Fever
• Suspicious colonies from the culture
plates are tested directly for the
presence of Salmonella somatic (O)
antigens by slide agglutination and
subcultured to peptone water for the
determination of flagellar (H) antigen
structure and further biochemical
analysis.
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Bacteriological Diagnosis of
Typhoid Fever
• A presumptive diagnosis of salmonellosis
can often be made within 24 h of the
receipt of a specimen, although
confirmation may take another day, and
formal identification of the serotype
takes several more days. A negative
report must await the result of
enrichment cultures - at least 48 h.
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How we Diagnose Typhoid Fever
• Diagnosis is made by any blood, bone marrow
or stool cultures and with the Widal test
(demonstration of salmonella antibodies
against antigens O-somatic and H-flagellar ). In
epidemics and less wealthy countries, after
excluding malaria, dysentery or pneumonia, a
therapeutic trial time with chloramphenicol is
generally undertaken while awaiting the
results of Widal test and cultures of the blood
and stool.
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Laboratory Diagnosis of
Typhoid Fever
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1 Isolation of Bacilli. A Gold standard
2 Diagnosis for presence of Antibodies,
Positive Blood culture – A gold standard
Isolation from Feces and Urine ?
Detection of Antibodies Inconclusive.
Newer methods
Detection of antigen in Blood and Urine
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Blood Culture
1 st week Positive in 90 %
2 nd week Positive in 75 %
3 rd week Positive in 60 %
> 3 weeks positive in 25 %
Draw 5 – 10 cc of Blood by venipuncture.
ADD to 50 -100 ml of Bile broth.
Incubate at 37 c /Subculture in MacConkey
At regular intervals
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Blood Cultures in Typhoid Fevers
• Bacteremia occurs early
in the disease
• Blood Cultures are
positive in
1st week in 90%
2nd week in 75%
3rd week in 60%
4th week and later in 25%
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Castaneda’s method of
Blood Culture
• Double medium used Solid/Liquid medium in
the same Bottle.
• Bottle contains Bile broth/agar slant,
• For subculture the bottle is merely tilted.
• A subculture into MacConkey at regular
intervals,
• Reduces the chances of contamination
• Increases the chances of isolation.
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Salmonella on Mac Conkey's agar
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Salmonella on XLD agar
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Clot culture
• Clot cultures are more
productive in yielding
better results in
isolation.
• A blood after clotting,
the clot is lysed with
Streptokinase ,but
expensive to perform
in developing
countries.
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Bactec and Radiometric based methods
are in recent use
• Bactek methods in
isolation of Salmonella
is a rapid and sensitive
method in early
diagnosis of Enteric
fever.
• Many Microbiology
Diagnostic Laboratories
are upgrading to Bactek
methods
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Biochemical Characters
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Non Lactose fermenter,
Motile
Indole – MR + VP - Citrate +
Ferment Glu/Mal/Man
Do not ferment Lactose/Sucrose
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Slide agglutination tests
• In slide agglutination
tests a known serum
and unknown culture
isolate is mixed,
clumping occurs within
few minutes
• Commercial sera are
available for detection
of A, B,C1,C2,D, and E.
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Culturing other Specimens
• Feces Enrichment in Tetrathionate broth
and Selenite broth
• Culturing in MacConkey/DCA/Wilson
Blair medium – Large black colonies.
• Urine Culture – positive in 25 %
• Other samples
Bone Marrow,Bile,CSF/Sputum
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Serology
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WIDAL Test – Tube agglutination test.
Detects O and H antibodies
Diagnosis of Typhoid and Paratyphoid
Testing for H agglutinins in Dryers tubes, a
narrow tube floccules at the bottom
• Testing for O agglutinins in Felix tubes, Chalky
• Incubated at 37º c overnight
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Widal Test
• In 1896 Widal A professor of
pathology and internal
medicine at the University of
Paris (1911–29), he developed a
procedure for diagnosing
typhoid fever based on the fact
that antibodies in the blood
of an infected individual cause
the bacteria to bind together
into clumps (the Widal
reaction).
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Widal test
• S.typhi O and H tubes
• Paratyphi A/B H agglutinins only
• Common antigens O in all Factor sharing
12
• Significance
• I st week negative.
• Titers raise in 2nd week Raise of titers
diagnostic
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Diagnosis of Enteric Fever
Widal test
• Serum agglutinins raise abruptly during the 2nd or 3rd
week
• The Widal test detects antibodies against O and H
antigens
• Two serum specimens obtained at intervals of 7 – 10
days to read the raise of antibodies.
• Serial dilutions on unknown sera are tested against
the antigens for respective Salmonella
• False positives and False negative limits the utility of
the test
• The interpretative criteria when single serum
specimens are tested vary
• Cross reactions limits the specificity
Widal Test
• Single test not diagnostic.
• Paired samples tests
• Diagnostic.
O > 1 in 80
H > 1in 160
H agglutinins appear first
False positives in Unapparent infection,
Immunization
Previously infected
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Widal test
• Anamnestic response previous
infection and responding to
unrelated infection
• Other Diagnostic tests
CIE and ELISA
Detection of Circulating antigens
Co agglutination test.
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Limitation of Widal Test
• The Widal test is
time consuming
and often times
when diagnosis is
reached it is too
late to start an
antibiotic regimen.
• In spite of several
limitation many
Physicians depend
on Widal Test
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False Positive and Negative Reactions
with WIDAL Test
• The Widal test should be interpreted in
the light of baseline titers in a healthy
local population. This is especially
important when there is a high local
prevalence of non-typhoid salmonellosis.
The Widal test may be falsely positive in
patients who have had previous
vaccination or infection with S typhi.
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False Positive and Negative Reactions
with WIDAL Test
• Widal titers have also been reported in
association with the dysgammaglobulinaemia
of chronic active hepatitis and other
autoimmune diseases.64 '8 '9 False negative
results may be associated with early
treatment, with "hidden organisms" in bone
and joints, and with relapses of typhoid fever.
Occasionally the infecting strains are poorly
immunogenic.
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Diagnosis of Carriers and
Environments
• Fecal carriers by
isolation from
specimens. or Bile
aspirated.
• Sewer swabs
• Bacteriophage
typing
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Prophylaxis
• TAB vaccine
S.typhi 1,000 millions
S Paratyphi A,B 750 millions.
Injected subcutaneously 0.5 ml
at 4 – 6 weeks.
Live Oral Vaccine Typhoral
Mutant S.typhi strain Ty 2 1a Lacking enzyme UDP
galctose 4 epimerase 10 to9
Viable bacilli
Given orally 1 – 3 – 5 days
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Key points
• Antibiotics have no place in the
management of salmonella
gastroenteritis unless invasive
complications are suspected.
• Clean water, sanitation and hygienic
handling of foodstuffs are the keys to
prevention.
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Prevention
• Vi Polysaccharide vaccine
– Administered subcutaneously or intramuscular
– Confers protection seven days after injection
– Approximately 50% efficacy after three years
• Ty 21 vaccine
– Live attenuated strain of S. typhi
– Administered orally in capsule form
– Also available in liquid form which can be taken by
children as young as two years of age
Vaccines
• An Inject able vaccine Typhium Vi
• Contains purified Vi polysaccharide
antigen from S.typhi strain Ty2
• A single dose, subcutaneous route
• Given to children > 5 years
• Immunity lasts for 2- 3 years.
• Follow a booster
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Treatment
• Chloramphenicol 1948 /1970 resistance.
• Other Important drugs
Ampicillin
Amoxicillin,
Furazolidine
Cotromoxazole
Chloramphenical resistance /Mexico
Kerala
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Antimicrobial Therapy in Typhoid
• With prompt antibiotic therapy, more than
99% of the people with typhoid fever are
cured, although convalescence may last
several months. The antibiotic
chloramphenicol Some Trade Names
CHLOROMYCETIN
is used worldwide, but increasing resistance to
it has prompted the use of other antibiotics
BACTRIM
SEPTRAN
or ciprofloxacin
Other Drugs
• Fluroquinolones
Ciprofloxacillin,
Pefloxacillin
Ofloxacillin
Ceftazidime
Ceftriaxone /
Cefotoxaime
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Coalition against Typhoid
• Since May 2011, the
Coalition against Typhoid
(CaT) has featured monthly
articles in the WHO’s Global
Immunization Newsletters
(GIN). The articles, written
by CaT members from
around the world, highlight
important work being done
to accelerate adoption of
typhoid vaccines.
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Food Poisoning
• The laboratory diagnosis of bacterial food
poisoning depends on isolation of the causal
organism from samples of faeces or suspected
foodstuffs. The more common food-poisoning
serotypes, such as Enteritidis or Typhimuruim,
may be characterized more fully by phage typing
and antibiotic resistance typing (see above).
Strains can be differentiated further by plasmid
and pulsed-field gel electrophoresis typing so
that the isolates from patients may be matched
with those from the infected food and from a
suspected animal source.
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Salmonella Gastroenteritis
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Zoonotic disease
S.enteritidis
S.typhimurium
S.halder
S. agana
S.indiana
Contaminated poultry, Meat Milk, Milk products.
Enters the shells of the Intact eggs – Chicken feed,
and Fecal droppings.
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Nontyphoidal Salmonella
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General Incubation: 6 hrs-10 days; Duration: 2-7 days
Infective Dose = usually millions to billions of cells
Transmission occurs via contaminated food and water
Reservoir:
a) multiple animal reservoirs
b) mainly from poultry and eggs (80% cases from eggs)
c) fresh produce and exotic pets are also a source of contamination (>
90% of reptile stool contain salmonella bacterium); small turtles ban.
General Symptoms: diarrhea with fever, abdominal cramps, nausea and
sometimes vomiting
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Mechanism of Pathogenicity
Gastroenteritis
• ingestion
• absorbed to brush border of epithelial cells of
small intestine and colon
• migrate to lamina propria, Ileocecal
• multiply in lymphoid follicles
• Reticuloendothelial hyperplasia and
hypertrophy
Nontyphoidal Salmonella:
Gastroenteritis
• Incubation: 8-48 hrs ; Duration: 3-7 days for diarrhea &
72 hrs. for fever
• Inoculum: large
• Limited to GI tract
• Symptoms include: diarrhea, nausea, abdominal cramps
and fevers of 100.5-102.2ºF. Also accompanied by
loose, bloody stool; Pseudo appendicitis (rare)
• Stool culture will remain positive for 4-5 weeks
• < 1% will become carriers
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Nontyphoidal Salmonella:
Bacteremia and Endovascular Infections
• 5%
develop septicemia; 5-10% of septicemia patients
develop localized infections
• Endocarditis: Salmonella often infect vascular sites;
preexisting heart valve disease risk factor
• Arteritis: Elderly patients with a history of back/chest +
prolonged fever or abdominal pain proceeding
gastroenteritis are particularly at risk.
- Both are rare, but can cause complications that may lead
to death
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Salmonella Gastroenteritis
•
•
•
•
Can occur as cross infection
24 hours
Manifest with Diarrhea, omitting
Abdominal pain mucous and blood in
stools
• Last for 2 – 4 days
• Some times may lead to septicemias
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Diagnosis and Treatment
• Isolation by
culturing
• Rarely need
antibiotics.
• More frequent in
Developed
nations.
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Salmonella septicemias
• S.cholera suis
• Deep abscess,
Endocarditis
• Isolation from
Blood and Pus.
• Chloramphenicol
highly effective
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 Don’t eat raw or undercooked
food
 Cross-contamination of foods
should be avoided
 Do not prepare food or pour
water if you are infected with
the bacteria

Wash hands, kitchen surfaces, and utensils
with soap and water after they have come
in contact with raw meat or poultry

Wash hands after contact with animal
feces

Avoid direct/indirect contact between
reptiles and infants
Simple hand hygiene and washing
can reduce several cases of Typhoid
• The Programme Created by Dr.T.V.Rao MD for
Medical and Paramedical Students in the
Developing World
• Email
• [email protected]
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Reference:
www.Slideshare.com
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