Enteric fever

UNDER GARADUATE STUDENT’S LECTUER ON
BY
GUNJAL PN
ASSIST. PROF.
DEPT OF MICROBIOLOGY
DVVPF’S MEDICAL COLLEGE & HOSPITAL
AHMENDAGAR
7/8/2021 DEPT OF MICROBIOLOGY 1

Competencies
• Following are the competencies for this theory
class :
• M 3.3
• M3.4
• M8.15

Learning Objectives
• At the end of the session, the students will be able to
understand:
• What is the term “Enteric Fever”
• Etiological agents responsible to cause “Enteric Fever”
• Pathogenesis and clinical manifestations of “Enteric Fever”.
• Laboratory Diagnosis for “Enteric Fever”
• Treatment and Prophylaxis for “Enteric Fever”
• Prevention and control

Introduction
• Although Salmonella spp can cause a wide spectrum of
clinical illness there are four major syndromes, each
with its own diagnostic and therapeutic problems,
which are considered separately.
• These are
• Enteric fever
• Gastro-enteritis
• Bacteremia with or without metastic infection, and
• Asymptomatic carrier state.

Enteric Fever
• Enteric fever is a potentially fatal multisystem
illness caused by Salmonella typhi (Typhoid
Fever) and Salmonella paratyphi A, B and C
(Paratyphoid fever).

Enteric Fever
• Enteric Fever syndrome is an acute systemic illness
characterized by fever, headache, and abdominal discomfort.
• Classically produced by S. typhi – refereed – Typhoid fever.
• S. paratyphi A, S. paratyphi B (S. schottmuelleri) , and S.
paratyphi C (S. hirschefeldii) – similar but less severe clinical
syndrome- refereed – Paratyphoid fever.
• Humans are only natural reservoir hosts.
• Incubation period – 1-10 days.

Enteric Fever –Pathogenesis
• Infective dose – 103 to 09 viable organisms.
• Route of Transmission – Food and water borne infections.
• Organisms ingested in water and other drinks may be carried
through the stomach relatively rapidly, and evade the effect of
gastric acid.
• Similarly the administration of antacids, or gastric resection,
reduces the infective dose.
• Bacteria within food particles also evade the action of
stomach acids.

Host factors
• Age specific isolation rates for salmonellae, as for some other gut
pathogens, are higher for children less than 1 year of age than for
any other age group.
• Higher proportion of infections are investigated in this age group.
• RISK FACTOR –
• These promote transmission include the conditions that decrease
• Stomach acidity (<1 year age, antacid ingestion or achlorhydria or
prior Helicobacter pylori infection)
• Intestinal integrity (inflammatory bowel disease, prior to GIT
surgery or suppression of intestinal flora by antibiotics).

Initiation of Infection – Salmonella
• Once Salmonella enter the lumen of intestine able to tolerate
action of digestive bile – need to compete with prevailing gut flora –
adhere to the gut mucosa and multiply.
• Certain serotypes such S. typhimurium expresses type-1 fimbriae,
which enable them to adhere to α-mannose-containing molecules
on the microvilli of the ileal mucosa.
• S. typhimurium and other have pathogenicity islands that can
encode binding sites/receptors for its adhesions inside the host
cells unlike fimbriae, which require host-derived binding sites
located in the intestinal wall.
• Attachment to host mucosa followed by degeneration of microvilli
to form breaches in the cell membrane through which salmonellae
enter the intestinal epithelial cells.

Initiation of Infection
• For certain strains, further multiplication in these cells and
macrophages of Peyer’s patches follows.
• Some penetrate into submucosa and pass to the local
mesenteric lymph nodes.
• All clinical manifestations of infection with salmonellae,
including diarrhoea, begin after ileal penetration.
• For strains of S. typhi infection involves the invasion of the
bloodstream and various organs.

Enteric Fever
• Entry through epithelial cells (M cells) – lining the intestinal
mucosa – Salmonella can trigger the formation of membrane
ruffles on the cell membarane of M cells.
• These ruffles reach out and engulf the attached organisms
inside a large vesical, this process of uptake is called
“Bacteria-Mediated-Endocytosis (BME)”.
• This is mediated by specialised type III secretion system.
• Following entry, the bacilli remain inside the vacuoles in the
cytoplasm.

Enteric Fever
• Entry into macrophages – Salmonellae containing vacuoles cross
the epithelial layer to reach submucosa, where they are
phagocytosed by the macrophages.
• Survival inside the macrophages – S. typhi induces certain
alterations on its surface (in LPS), so it is no longer susceptible to
the lysosomal enzymes of macrophages.
• Primary Bacteremia – Salmonellae contained inside the
macrophages spread via the lymphatics to enter the bloodstream
(transient primary bacteremia).
• Spread – Bacilli then disseminate throughout the
reticuloendothelial spread via the lymphatics to enter the
bloodstream (transient primary bacteremia).
• Secondary bacteremia – Occurs from the seeded organs, which
leads to the onset of clinical disease.

Enteric Fever
• ONSET – Interval between ingestion
of the organisms and the onset of
illness varies with the size of
infecting dose.
• Can range from 3 to 50 days, usually
about 2 weeks.
• Onset is usually insidious.
• Early symptoms are often vague:
• Dry cough,
• Epistaxis associated with anorexia,
• A dull continuous headache,
• Abdominal tenderness and
discomfort are uncommon and early,
• Many complain of constipation.

Enteric Fever
• PROGRESSION – In untreated cases –
temperature shows a step ladder rise over the
first week of illness, remains high for 7-10 days.
Then falls lysis during the third or fourth week.
• PHYSICAL SIGNS INCLUDE:
• Relative bradycardia at the height of fever,
• Hepatomegaly,
• Splenomegaly,
• Often rash of “Rose spots” 2-4 mm in diameter,
• Slightly raised discrete irregular balancing pink
macule most often found on front of chest.
• Appear in crops upto dozen at a time and fade
after 3 to 4 days, leaving no scar- characteristic
of but not specific fro enteric fever.

Enteric Fever
• RELAPSE – Apparent recovery can be followed by relapse in 5-10%
of untreated cases.
• Relapse – usually shorter- mild- than initial illness but can be severe
and may be fatal.
• Severe intestinal haemorrhage and intestinal perforation are
serious complications but can occur at any stage of the illness.
• COMPLICATIONS – Gastrointestinal bleeding and intestinal
perforation can occur mostly in 3rd or 4th week.
• NEUROLOGICAL MANIFESTATIONS: Rare- include- meningitis,
cerebellar ataxia and neuropsychiatric symptoms (described as “
muttering delirium” or “coma vigil”) such as paranoid psychosis,
hysteria, delirium and aggressive behaviour.

Epidemiology
• HOST: Humans are only natural hosts for typhoid salmonellae.
• TRANSMISSION: By ingestion of contaminated food and water
• PREVALANCE: As per WHO, estimated 11-21 million cases with 1.2-1.6
lakh deaths annually worldwide.
• Compared to 6 million cases and 54000 deaths of paratyphoid annually.
• India bears major burden of the disease with >6million cases annually.
• INCIDENCE: Varies between countries –
• Highest : (>100 cases per 1 lack population per year)in South central and
Southeast Asia.
• Moderate: (10-100 cases per 1 lack) in rest Asia, Africa, Latin America.
• Low: (<10 cases per 1 lack) in other parts of the world.

Epidemiology
• LOCALITY AND AGE: Enteric fever is –
• More common in urban than in rural area.
• More common among young children and in adolescents than
in adults.
• Factors: favouring transmission include:
• Poor sanitation and improper cleaning of drinking water.
• Contaminated water, food and drinks.
• Lack of hand hygiene and toilet access.
• Evidence of prior H.pylori infection.

Epidemiology
• TYPHI Vs PARATYPHI:
• S. typhi infection is more common than S. paratyphi A (ratio
4:1).
• However S. paratyphi A appears to be increase in India: due to
increased vaccination against S. typhi.

Epidemiology
• CARRIAGE: Untreated patients become carriers and excrete S. typhi in
feces or urine.
• TYPES OF CARRIERS:
• 1. FECAL CARRIER: Bacilli multiplies in gallbladder and excreted in feces.
Fecal carriers are more common.
• 2. URINARY CARRIERS: Multiplication takes place in kidneys and bacilli are
excreted in urine. Urinary carriers are rare.
• DURATION OF SHEDDING: Carriers continue to shed the bacilli in feces
and urine for :
• Temporary carriers: Shed bacilli S. typhi in feces upto 3 months ; upto 10%
of untreated patients excrete S. typhi.
• Chronic carriers: They shed S. typhi in either urine or stool for >1 yr; seen
upto 2-5% of patients.

Epidemiology
• Chronic carriers:
• It occurs in about 1-4 % of infected pts. Chronic carriage is
more common in:
• Women, infants and old age
• Biliary tract abnormalities which leads to increased feceal
excretion.
• Abnormalities of the urinary tract and associated S.
haematobium infection of the bladder- leads to increased
urinary excretion.

Epidemiology
• Food handlers and Cooks:
• Converted to chronic carriers are dangerous, can excrete the
bacilli for many years.
• Best known example: Marry Mallon (Typhoid Marry).
• A New York based cook gave rise to more than 1,300 cases
during her lifetime causing several outbreaks.

Typhoid Marry

Bacteriology
• Salmonella typhi is most imp. Member of the genus Salmonella.
Causes fatal disease – Typhoid.
• Eberth in 1880 first observed the typhoid bacillus in mesenteric
lymph nodes & spleen in fatal cases of typhoid fever.
• Gaffkey 1884 successfully isolated the organism.
• Hence called Eberth - Gaffkey bacillus or Eberthela typhi.
• Salmon & Smith 1885 isolated the American-hog-cholera bacillus
(S. cholerasuis).
• Therefore the name of first author the term “Salmonella”.

Salmonella
• The genus Salmonella
includes :
• Gram Negative , motile
bacilli.
• Parasitize the intestines of
many vertebrate animals.
• Lead to Enteric fever,
• Gastroenteritis,
• Septicemia with or without
focal suppuration &
• Carrier state.

Picture of Gram Negative Bacillus – Staining
Grams Staining Technique.

Cultural characteristics
• Enrichment media –
• Selenite F Broth .
• Tetrathionate Broth. For 12-
18 hrs.
• Selective Media –
• MacConkey’s Agar
• DCA
• Salmonella – Shigella Agar.
• Wilson & Blair Agar.
• Colonies –
• Large, 2 –3 mm, Circular, Smooth.
• Colorless / NLF on Mac Conkey’s
and DCA.
• On Wilson & Blair Agar –
• Jet black colonies due to
production of H2S by S. typhi, S.
paratyphi B.
• Except S. paratyphi A and other
which do not form H2S produce
green colonies.
Colonies on Mac Conkey’s
Medium NLF

Biochemical reactions
G L M S MR VP Ci U Indole H2S
+ -- + -- + -- + -- -- +
1.S. typhi -- anaerogenic.
2.S. typhi ,S.paratyphi A – may be citrate negative.
3.S.paratyphi A & S. cholerasuis – H2S negative.
H2S production seen in 3rd tube with Alkaline slant – reaction by
Salmonella spp given on TSI

Antigenic classification – Kauffmann-
White Scheme
Kauffmann-White antigenic classification of Salmonella
Serogroup Serotype name O Ag* Vi Ag H Ag*
New Old Phase 1 Phase 2
2 A S. paratyphi A 1,2,12 - a (1,5)
4 B S. paratyphi B 1,4,(5),12 - b 1,2
S. typhimurium 1,4, (5),
12
- i 1,2
7 C1 S. paratyphi C 6,7 + c 1,5
S. cholerasuis 6,7 - c 1,5
9 D1 S. typhi 9, 12 + d -
S. enteritidis 1,9,12 - g, m (1,7)

Molecular Classification
• Based on DNA hybridization studies, the genus Salmonella consist
of two species –
• 1. Salmonella enterica, & 2. S. bongori
•
• Within species S. enterica there are 6 subspecies namely enterica,
salamae, arizonae, diarizonae, houtenae and indica
• Each subspecies further differentiated into serogroups based on
presence of Somatic(O) Antigen and Flagellar (H) Antigen as
described in Kauffmann-White Scheme.
• Most of the pathogenic typhoidal and non-typhoidal salmonellae
are placed into species enterica and subspecies enterica.
• Nomenclature : Salmonella species enterica subspecies enterica
serotype S. typhi.

Antigenic Structure
• Based on important Ag placed on cell wall of
Salmonella are classified.
• 1. Somatic O Antigen
• 2. Flagellar H Antigen
• 3. Surface Envelop Antigen (Vi) – found in
some species.

Difference Between Somatic (O) Antigen and Flagellar (H) Antigen
Somatic (O) Antigen Flagellar (H) Antigen
Part of Cell wall Lipopolysaccharide (LPS) Made up of protein Flagellin, confers
motility.
In Widal test, O Ag of S. typhi is used In Widal test, H antigens of S. paratyphi A
and B are used
Less immunogenic More immunogenic
O Abs appear early, disappears early;
indicates recent infection
H Abs appears late, disappears late;
indicates convalescent stage
O ag and O Ab combination forms
compact, granular, chalky clumps.
Agglutination takes place slowly
Optimum temp for agglutination is 550C
H Ag reacts with H Ab forms large, fluffy,
clumps.
Agglutination takes place rapidly
Optimum temp is 370C for agglutination.
Sero grouping of salmonellae based on O
Ag
Sero grouping are differentiated into
serotypes based on H Ag.

Antigenic Structure
• Surface Envelop Antigen (Vi):
• Surface polysaccharide or capsular Ag covering the O
Ag.
• Named so as believed to be related with virulence of
organism.
• Expressed on few serotypes S. typhi, S. paratyphi C
• Poorly immunogenic, Ab titre is low, not used for
diagnosis.
• Hence not employed in Widal test.

Laboratory Diagnosis
• Type of specimen to be collected depends on the
duration of illness.
• The preferred specimen(s) to be collected are:
• First week of illness: Blood for culture,
• Bone marrow for culture or
• Duodenal aspirate for culture.
• Second week of illness: Serum for serology (Widal)
• Third / Fourth week of illness: Urine and stool culture.

Laboratory diagnosis of Enteric fever
– Isolation of bacillus – culture.
– Demo. of Ab – Widal test.
– Demo. of circulating Ag.
– Other laboratory tests.
STAGE EXAMINATION RESULT (%) POSITIVE
1st week. Blood Culture. 95.
Blood picture. Leucopenia with relative
lymphocytosis.
2nd week. Blood Culture . 40-50.
Widal Test. Low titre antibody.
3rd week. Widal Test. 100.
Blood Culture. 15 -20.
4th week. Widal Test. 100.
Stool & Urine Culture. 90.
Blood Culture. 5 -10.

A. Culture
• Specimen – Blood, feces, urine, BM, bile, rose spots.
• 1. Blood culture - 5 – 10 ml of blood
Inoculated in 50 –100 ml Bile broth.
Overnight incubation at 370C.
S/c on MAC & DCA.
Overnight incubation at 370C.
NLF Colonies.
Tested for motility & biochemical reactions.
Identification is confirmed by agglutination with antisera.
Ref. Center for Salmonella – National Salmonella reference center,
located at central research institute, Kasauli.

NOTE –
When Salmonellae are not isolated - In 1st S/C,
S/C should be made on every alternate day.
Culture should be declared negative only after incubation for 10 days.
Significance of Blood Culture –
1.Blood Cultures are positive in
90% cases in 1st week.
75% cases in 2nd week.
60% cases in 3rd week.
25% cases till pyrexia subsides.
2. After treatment with Chloramphenicol, Blood cultures
rapidly become negative.

Castaneda’s Method –
• Double medium containing Bile
broth & agar slope in same bottle.
• For S/C, bottle is tilted & bile broth
allowed to run over agar slope.
• Advantages –
• Eliminates possibility of
contamination during S/C.
• Safety.
• Economy.

2. Clot culture –
• 5 ml Blood.
• Collected in sterile Test Tube &
allowed to clot.
• Clot broken with sterile glass rod &
inoculated in bile broth.
• Advantages of Clot Culture –
• Higher rate of isolation as bactericidal
action of serum is eliminated.
• Serum becomes available for Widal
test.

3.Feces Culture –
• Feces collected in sterile container.
• Enrichment in Selenite F broth /
Tetrathionate broth for 12 – 18 hrs.
• Plating on MAC, DCA & W&B Media
- Identification.
• Advantage –
• Patients on antibiotic treatment,
blood culture may be negative but
feces culture may be positive.
• Limitation –
• Feces culture is positive in patients
as well as in carriers.

4. Urine Culture –
• Midstream urine sample is collected.
• Centrifuge deposit is inoculated in enrichment &
selective media.

B. Demo. Of Antibodies : Widal Test –
• Test for measurement of H & O Abs. for typhoid & paratyphoid
bacilli in patient’s sera.
• Tubes used for test
• Dreyer’s Tube – Narrow tube, conical bottom for ‘H’
agglutination.
• Felix’s Tube – Short tube, round bottom for ‘O’ agglutination.
• Serum in varying dilutions (From 1:20 to 1:320) is taken in
different tubes.
• Mixed with equal volume of Ag. (TO, TH, AH, BH) tubes are
incubated in water bath at 370C over night or some
recommends 50-550C for two hrs. & examined for agglutination.
• ‘O’ agglutination – Compact, granular, chalk powder.
• ‘H’ agglutination – Loose, fluffy cotton wool.

Interpretation of Widal test –
• Stage of disease – Abs. Appear
by the end of 1st week.
• Rise steadily till 3 – 4 wks. &
• Afterwards decline gradually.
• 2. Demo. of rise in titer by
testing paired sera is more
significant than reporting results
on single or first sample.
• 3. Significant levels -
• ‘O’ Abs. – 1:100 or More.
• ‘H’ Abs. – 1:200 or More.

Interpretation of Widal test –
• 4. Prior disease, inapparent infection Or Immunisation
can be a cause of production of Abs. (false +ve test).
• 5. Prior infection / immunization can lead to
anamnestic reaction(renewed rapid production of an
antibody on the second encounter with the same antigen).
• In this response is transitory. In enteric fever, it is
sustained.
• 6. Fimbrial Ag can cause false +ve reaction.
• 7. Treatment with chloramphenicol & other antibiotics
in early stage causes poor antibody response.

C. Demonstration of Circulating Antigen (Ag)
• Antigen specific for typhoid bacillus is present in blood & urine of
patient in early stages.
• It is demonstrated by ‘Coagglutination Test’.
• Staph. aureus (Cowan I strain) which contains protein A is
stabilized with formaldehyde and coated with S. typhi Abs.
• When 1% suspension of such sensitized staphylococcal cells
mixed on a slide with serum from patient in first wk. of typhoid
fever.
• The typhoid Ag present in serum combines with the Ab attached
to staphylococcal cells producing visible agglutination two mins.
• Advantages –
• Rapid, Sensitive & specific,
• Positive in 1st week of disease.

D. Other Laboratory Tests –
• Leucopenia with relative lymphocytosis.
• Diazo Test of urine.
• Test is positive between 5th – 14th Days.

Diagnosis of Carriers –
• Imp. for epidemiological & public health
problems.
• Diagnosis of fecal carriers.
• Culture of feces & bile.
• Diagnosis of urinary carriers.
• Repeated culture of urine.
• Demo. Of Vi Abs.
• Sewer swab technique.
• Culture of gauze pad left in sewers & drains.
• Filtration of sewage through Millipore membrane
& culture of membrane.

Prophylaxis
• Typhoid fever can be effectively controlled by general
measures like improvement in sanitation and provision
of protected water supply.
• Vaccination of travellers against typhoid
recommended, but does not remove need for good
hygiene.

Vaccines
• Parenteral TAB Vaccine – Heat killed whole cell S. typhi/ S.
paratyphi A & B : it is no longer in use due to significant side
effects.
• Parenteral Vi Polysaccharide Vaccine – Composed of Purified
Vi Capsular polysaccharide Ag derived from S. typhi strain Ty2.
• Dosage: Single dose 25ug of Vi Ag – IM or SC – Protection – 2
yr.
• Age : After 2 yrs of Age.
• Vi Ag elicits T independent IgG Ab response that is not
boosted by additional dose of vaccine.

• Two new typhoid vaccine are introduced –
• Typhoral – The oral one.
• Live attenuated vaccine, containing stable strain of S.
typhi strain Ty2 1a,lacking enzyme UDP- galactose -4-
epimerase (Gal E mutant).
• On ingestion, it initiates infection but “Self-destructs”
after 4-5 cell divisions and not inducing any illness.(Due
to lack of Gal E enzyme).
• The vaccine is enteric coated capsule containing 109
viable lyophilised mutant bacilli.

• The course consist one capsule orally, an hour before
meal with a glass of water or milk - On days 1, 3 and 5.
• No antibiotic should be taken during this period.
• Protective Immunity : starts after 7th day of last dose
and lasts for 4 yrs.
• Boosters : Recommended after every 3 yrs. For people
residing in endemic areas and every year for travelers
proceeding to endemic areas.

Drug Resistance in Salmonella –
• Chloramphenicol resistant
strains of S. typhi were
reported from Mexico & Kerala
(India) in 1972 .
• R factor confers multiple drug
resistance in Salmonellae .
• MDR Salmonellae are known to
cause septicaemia, meningitis
& pyogenic infections
particularly in neonates.

Multidrug Resistant (MDR) S. typhi
• Defined as – Resistant to chloramphenicol,
ampicillin and cotrimoxazole. MDR emerged since
1989 in China and Southeast Asia including India.
• NAR Strains : (Nalidixic Acid Resistant) – due to
increased use of fluoroquinolones to treat MDR in
1990s, strain reduced susceptibility to ciprofloxacin
have emerged in India, other regions.
• Resistance to Ceftriaxone : It has been reported
recently both ESBL and AmpC producing S. typhi
have been detected.
• Old is Gold : Many strains revert to susceptibility to
Amoxicillin, chloramphenicol, cotrimoxazole as were
not for long time.

Treatment of Enteric Fever
Drug of Choice Alternative drug
Empirical Treatment This treatment is given
before
ABST report is available.
Ceftriaxone 1-2 g/day for
7-14 days
Azithromycin-1g/day oral for 5
days
Fully susceptible Susceptible to all drugs Given for enteric fever
Ciprofloxacin – 500 mg
twice a day oral for 5-7
days
Amoxicillin
Chloramphenicol
Cotrimoxazole
MDR Ciprofloxacin - Ceftriaxone
Azithromycin
NAR Ceftriaxone Azithromycin, Ciprofloxacin –
750 mg twice a day oral for 10-
14 days.
Carriers Ampicillin or Amoxicillin +
Probenecid for 6 wks.
Cotrimoxazole or Ciprofloxacin

Expected Questions
• Write assay on:
• What is enteric fever, mention etiological
agents causing enteric fever and the
pathogenesis of enteric fever.
• Laboratory diagnosis of enteric fever.
• Treatment and vaccination for enteric fever.

Expected Questions
• Write Short note on:
• Typhoid carrier.
• Drug resistance in salmonellae.
• Widal test
• Castaneda’s Blood culture system

MCQ
• S. typhi is the causative agent of typhoid fever. The infective dose is
• A. one bacillus
• B. 103 to 109 bacilli
• C. 1 to 10 bacilli
• D. 1010 to 1012 bacilli
• In patient with typhoid, diagnosis after 15 days of onset of fever is best done by
• A. Blood culture
• B. Stool Culture
• C. Urine culture
• D. Widal test
• Antibodies against which of the following Ag appear early following infection with
S. typhi
• A. Vi
• B H
• C. O
• D. Capsular
• Answers – 1. B , 2-B, 3- C

Thank you!

Enteric fever

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