CASE

• 21yrear old male came with fever 10 days with

myalgia and yellow discoloration of skin. He was
apparently well 10 days back when he developed fever
continuous ,low grade without chills/ rigors subsided
on medication and generalized weakness
Past history: no significant history
Personal history: had constipation for 3 days along with
fever after which bowel habits were normal.
On examination : Patient conscious, oriented to time
place and person No pallor , No ictereus , No cyanosis ,
no lymphadenopathy or odema
Blood pressure : 130/80mmof Hg
Pulse : 98/ min
Temperature :100◦ F
Respiratory rate :20 / min
Systemic examination :
CVS, RS & CNS : within normal limits
PA examination : non tender , mild hepato-
spleenomegaly
COURSE IN THE HOSPITAL
• Investigations done :
Hb TC Platele Neutrop Lymp ESR
t hil hocyte
s
14.6 4000 75,000 73 25 40
11,500 1,50,00 59 34
0
Malaria/filaria : negative
Dengue IgM :negative
Leptospira IgM: positive , 21pan Biounits
LFT deranged : 4 fold rise in AST, ALT &
GGT & 20 fold rise in bilirubin
• Patient was started on crystalline penicillin
After 3 days fever did not subside ,
Blood culture was sent : S.Typhi grew after 16 hrs
of incubation
Susceptible to : azthromycin, ceftriaxone ,
cefuroxime, Chloramphenicol, cotrimoxazole
Resistant to : Ciprofloxacin, Ampicillin ,
Patient was treated with inj Ceftriaxone 2gm iv bd
for 14 days
 ENTERIC FEVER

Dr Pooja Rao
Associate Professor
KMC
LEARNING OBJECTIVES
1. Define Enteric fever and name the causative agent of Enteric fever
2. Discuss the mode of transmission of Salmonella spp.
3. Identify the virulence factor of Salmonella spp.
4. Discuss in detail pathogenesis of enteric fever
5. Discuss in detail the various specimen collection for diagnosis of
enteric fever.
6. Plan laboratory investigations for a patient presenting with clinical
manifestations of Enteric fever.
7. Discuss the serological tests performed for diagnosing Enteric fever
and its interpretation.
8.Understand the drug resistance in Salmonella Typhi
9. Describe the prevention of Enteric fever including the vaccines for
prevention of Enteric fever.
INTRODUCTION
 Enteric fever is a systemic infectious enteric disease.
 The term “Enteric fever” includes both typhoid &
paratyphoid fever.
 The organism classically responsible - S. enterica serotype Typhi
, Salmonella serotype Paratyphi A, Salmonella serotype
Paratyphi B, Salmonella Paratyphi C
 Other Non typhoidal Salmonellae: colonise animals and cause
food borne illness, gastroenteritis & septecemia.
 It continues to be a global health problem with an estimated 21
million cases worldwide & 600,000 deaths each year.
ANNUAL INCIDENCE OF TYPHOID FEVER IN 100,000
POPULATION
ANTIGENIC CHARACTERISATION BASED ON
KAUFFMANN- WHITE SCHEME.

Phase 1 "H" Phase 2 "H"

O"-group Serovar "O" antigens
antigens antigens

no phase 2
A S.Paratyphi A 1,2,12 a
antigen

B S. Paratyphi B 1,4,5,12 b 1,2

no phase 2
D S. Typhi 9,12,Vi d
antigen
Enterobacteriaciae family

MORPHOLOGY:

❑ Gram negative rods, non-spore

forming
❑ About 1-3 m x 0.5 m in size
❑ Motile – peritrichate flagella.
❑ Aerobic and facultative anaerobic
Antigenic structure
ANTIGENIC STRUCTURE:
H antigen
• Present on flagella
• Heat labile protein destroyed by boiling/treatment with alcohol.
• Strongly immunogenic.
• High titres following infection or immunisation.
• Appears in two phases
O antigen
• Phospholipids protein in a polysaccharide complex.
• Heat stable and identical to endotoxin.
• Adherence to the gut epithelium and resists phagocytosis.
 Vi – Antigen (Surface antigen)

• Polysaccharide antigen enveloping O antigen.

• Analogous to K antigen’s of coliforms.

• Heat labile and associated with virulence.

• Inhibits phagocytosis and bacterial lysis by alternate pathway

• Carrier state.
PATHOGENESIS
➢ Exclusively human disease
➢ Source of infection:- Faeces & urine of patients and carriers
➢ Mode of transmission:- feco-oral route

➢ Infective Dose
➢ 103 to 106 organisms.

➢ Incubation Period:7 to 14 days, but ranges from 5 to 21 days

VIRULENCE FACTORS
Vi Antigen:
❑ Possession of Vi antigen is associated with increased
infectivity and is antiphagocytic
Endotoxin
❑ The lipopolysaccharide in cell wall acts as endotoxin
Salmonella – Pathogenicity Island – 1:
❑ Groups of genes that are associated with pathogenicity located on
bacterial chromosome.

❑ Salmonella encode type III secretion system with in SPI-1 that is

required for bacterial mediated endocytosis and intestinal
epithelial invasion.

Salmonella Pathogenicity Island -2

❑ These second – type III secretion system that is necessary for
survival in the macrophage’s and establishment of infection is
encoded in Salmonella – pathogenicity island -2 (SP2).
HOST FACTORS:
Host defence’s against Salmonellae

Host factors Examples

Gastric factors Gastric acidity

Intestinal factors Intestinal motility

Normal intestinal flora
Mucous
Secretary antibodies
Genetic resistance to invasion
FACTORS INCREASING SUSCEPTIBILITY TO
SALMONELLOSIS:

Location of factor Specific condition

Stomach Achlorhydria
Gastric surgery

Intestine Antibiotic administration

Gastro intestinal surgery

Hemolytic Anemia Sickle cell anemia and other

Hemoglobinopathies

Impaired systemic Immunity In AIDS, Diabetes Mellitus, Patients on

Immunosuppressive drugs.
SEQUENCE OF EVENTS:-
Once in the small intestine

They adhere to the intestinal epithelial surface via M cells

Invade the intestinal epithelial cells by morphologically distinct process termed

bacteria-mediated endocytosis

Enters the submucosa(macrophage phagocytose) & travels to mesentric

nodes


 After a period of multiplication

Enter the blood stream via the thoracic duct
(transient primary bacteremia)

Liver , spleen, kidney, gallbladder & bone marrow

After a period of multiplication at these sites huge number of organisms
enter the blood stream
(secondary bacteremia)

Onset of clinical symptoms
CLINICAL FEATURES:
Acute non-complicated disease –
▪ Fever - Step-ladder pattern
▪ Disturbances of bowel function
▪ Headache, malaise , anorexia, cough, sore throat.
▪ 25% patient show rose spots on chest, abdomen and back during
second week of infection.
Rose spots
COMPLICATED DISEASE:

❖ 10% of typhoid patients

❖ Gastro intestinal bleeding(10-20%) & Intestinal perforation

seen in 3% of hospitalized cases.
.
❖Neurological manifestations: (2 -40%)Encephalopathy, Typhoid
meningitis, encephalomyelitis, Guillain-Barré syndrome, cranial or
peripheral neuritis & neuropsychiatric symptoms

❖Acute cholecystitis
❖Osteomyelitis & typhoid arthritis – sickle cell disease patients
❖Others: Hepatitis, myocarditis, pneumonia

S.paratyphi A & B- diarrhea, vomiting & entire intestinal tract

inflammation
❖ Patient continues to excrete S.typhi in faeces for several

weeks after recovery

❖ 1-3% become chronic carriers, which is defined as Salmonella

excretion in faeces or urine for 1 year after infection.

❖ 10-20% of patients treated with antibiotic suffer a relapse after

initial recovery, it usually occurs after a week or so after

stoppage of therapy. The blood culture is again positive, even

in the presence of high serum levels of H, O and Vi antibodies.

CHRONIC CARRIERS:
1)Chronic biliary carriage leading to chronic faecal carriage(faecal
carrier)
2) Chronic biliary carriage: infrequent but not invariably associated
with gall stones. S.Typhi invade stone & provide a site capable of
constant seeding. (biliary carrier)
3) Urinary tract abnormality leads to urinary carriage (urinary
carrier) but perhaps more dangerous than faecal carrier
Predisposing factors for carriers:
 Damaged organs: urinary schistosomiasis, renal calculi, biliary
tract abnormalities
 More common in women than men

 In middle & older age groups

 Genetic factors: In non secretors of ABH ( ABO blood group)

LABORATORY DIAGNOSIS:
Specimen Collection:

➢ Blood
➢ Bone marrow aspirate
➢ Stool
➢ Urine
➢ Bile
➢ Punch biopsy from skin rashes( Rose spot`s)
➢ Duodenal aspirate.
STAGE EXAMINATION RESULT
1st week Blood culture 90%
WIDAL test Low antibody titre
Stool & urine culture 5%
Blood culture >75%
2nd week
Widal test Low Titre Antibody
Stool & Urine culture 50%
Widal Test 100%
3rd week Blood culture 50%
Stool & Urine Culture 80%
Widal Test 100%
4th week Stool & Urine Culture 50- 90%
Blood Culture 5-10%
 BLOOD CULTURE
Blood culture is the main stay for the diagnosis of enteric fever,
bacteremia occurs in early disease and so blood culture is positive in 1st
week of fever.

Conventional blood culture –

Volume of blood
• 10 ml - school children and adults.
• 2-4ml- toddlers and pre school children.
BRAIN HEART INFUSION BROTH
Advantages:
 Cost effective

Disadvantages:
1. The contamination of blood culture reduces the isolation
rates for Salmonella Typhi.
2. Adequate amount of blood should be collected, because
reducing the blood volume, reduces the sensitivity of
culture.
3. Presence of antibiotics can give negative results.
BACT/ ALERT SYSTEM/ BACTEC SYSTEMS:

Positive blood culture bottles.

Grams stain + Processed as conventional blood culture.

Subcultured onto Blood agar, Mac Conkey`s agar & Chocolate agar
Advantages:
1) Rapid & more sensitive
2) risk of contamination is less

Disadvantages:
1)mostly not available in all microbiobiological setups
2)expensive
STOOL CULTURE
Stool sample (in acute patients) / rectal swab inoculated
into Cary Blair transport medium

Stool sample is cultured onto-

Less selective medium: Mac Conkey`s agar .
Highly selective medium: XLD, DCA, Wilson & Blair medium +
inoculated into enrichment media (Selenite F / Tetrathionate broth)
incubated for 6hrs

subcultured onto Mac Conkey`s agar & Wilson & Blair medium.
Advantage:
 Valuable in patients on antibiotics as the drug

does not eliminate the bacilli from the gut as

rapidly as it does from the blood.
Disadvantage:
 A positive faecal culture may occur in carrier Selenite F broth
as well as patients
URINE CULTURE:
Uncentrifuged urine samples- wet mount, Grams stain

Cultured onto Mac Conkey`s & CLED agar.

 The media are incubated at 370C for 18 hrs

CULTURE

TYPE OF MEDIA COLONY CHARACTERISTICS

Blood agar Non haemolytic smooth white
colonies
MacConkey agar NLF Smooth colonies, they are pale
yellow or colourless.

Deoxycholate Agar NLF with black centres

Xylose-Lysine-Deoxycholate Transparent red colonies with
Agar black centres
Wilson and Blair media Jet Black coloured colonies with
metallic sheen
XLD agar
WILSON & BLAIR`S MEDIUM
Microscopy:
Gram stain: Gram negative bacilli

Oxidase: negative
Catalase: positive
 Tests S.typhi S.paratyphi A S.paratyphi B

OF test Fermentative fermentative fermentative

Nitrate reduced reduced reduced

TSI & H2S K/A +H2S K/A K/A +H2S

Motility motile motile motile

Indole neg neg neg

Methyl red positive positive positive

VP neg neg neg

Citrate Not utilised Not utilised Utilised

PPA & Urease neg neg neg

 Tests S.typhi S.paratyphi A S.paratyphi B

Slide O9, Hd O2, Ha O4, Hb

agglutination
positive with

+ve
ANTIMICROBIAL SUSCEPTIBILITY TESTS:

Antimicrobial sensitivity tests are set up simultaneously by

Kirby Bauer disk diffusion method.
The antibiotics routinely used when the organism is an enteric
fever causing pathogen are:
 Ampillicin(10μg),
 Chloramphenicol(30μg),
 Cefuroxime(30μg),
 Ceftriaxone(30μg),
 Co-trimoxazole(25μg),
 Ciprofloxacin (5μg),
 Ofloxacin(5μg), according to CLSI guidelines.
Widal Test :
Definition : It is a tube agglutination test which is used for
serological diagnosis of enteric fever.
 The test detects antibodies against enteric fever group of
organisms in the patient`s serum.
Principle: Patient`s serum contains specific antibodies which
will react with Salmonella O & H antigens in the presence of
electrolytes.
Requirements:
1) Widal rack with Widal tubes-
2) Antigenic suspensions-O, H, AH, BH
3)Patient`s serum in serial dilutions.
4)Water bath

Procedure: Patient`s serum is taken in serial dilutions from1/80

to 1/640 & mixed with equal volumes of Salmonella H & O
antigens in agglutination tubes, incubated in water bath at
370C for 18 hrs. Control tubes containing antigen & normal
saline are set to check for auto agglutination.
OBSERVATION-

• H antigen – appear fluffy and floccular.

• O antigen – appear finely granular

Highest dilution producing agglutination is taken as titre.

It is possible to demonstrate rise in titres of agglutinins using

paired sera.

Absence of agglutination: Button formation

A titre of more than 1/80 and 1/160 for O and H antibodies is considered
significant

Interpretation
FACTORS AFFECTING WIDAL TEST:
 Vaccination
 Previous enteric fever may induce rise in titre of ‘H’
concentration when suffering from other unrelated illness, this
phenomenon is called anamnestic response.

 The interpretive criteria when a single specimen tested vary,

high O agglutinin titre of 1/160 or more is diagnostic of acute
infection.

 Endemic areas: Single elevated O antibody titre –

sensitivity: 50-90%
Specificity:70-99%
Single elevated H antibody titre – Sensitivity: 50-90%
Specificity: low
 A four fold rise in O & H antibody titre is found in fewer than
40% of culture positive patients. A single titre is significant only
if the baseline titre of the region is known.
 False positive test in case of non-typhoidal salmonella infection.
Advantages
➢ Inexpensive
➢Useful in 2nd & 3rd week of the infection

Disadvantages:
➢ Indirect method influenced by many factors and interpretation is
difficult.
➢ Not useful during the first stage.
➢ The results are interpreted carefully, with appropriate cut off values
for the determination of positive result in the particular locality or
area.
➢ In case of immunodeficient persons ,it is not very useful.
➢ The test has moderate sensitivity and specificity. It can lead to false
positive and negative results.
 False positive:
1. In case of previous vaccination. Widal test shows positive result.
2. Past infection.
3. Anamnestic response.
4. O antigen of salmonella cross react certain bacteria especially
members of family Enterobacterales.
.
False negative-
1. In early stages of infection.
2. Prior antibiotic therapy .
RAPID TESTS FOR DIAGNOSIS OF TYPHOID FEVER:
Typhidot M test: (Enzyme immunosorbent test), IDL TUBEX
TEST, dipstick assay:

It is a sensitive, specific, economical test.

Principle:

The presence of IgM and IgG antibodies produced by the patient

against a specific antigen on the outer membrane of Salmonella Typhi
are detected by incubating nitrocellulose strips dotted with the specific
antigen protein in the patients sera and control sera.

Advantages:

1. It can be useful in high areas of endemicity.

2. .
MOLECULAR METHODS:-

POLYMERASE CHAIN REACTION-

PCR for detection of S. Typhi in blood ,more rapid and

sensitive than standard culture methods

CARRIER DETECTION
 Culturing the organism from the specimen like faeces, urine,
sewer swabs, duodenal aspirate, blood of the suspected carrier.
 Faecal carriers can be identified by isolation of the bacillus from
faeces or from bile.
 Gelatin capsule string test is preferable for detection of
chronic faecal carrier.
 The tracing of carriers in cities may be accomplished by ‘sewer-
swab’ technique.(Moore swab)
 Membrane millipore filteration technique
 Vi Antibody determination has been used as screening technique
to identify carriers among food handlers and outbreak
investigation.
➢ Tube agglutination test ( by Felix)
DRUG RESISTANCE & TREATMENT

The emergence of MDR strains has reduced the choice of

antibiotics in many areas.
There are two categories of drug resistance
➢Resistance to first line antibiotics such as
chloramphenicol
Ampicillin Plasmid mediated
Trimethoprim-sulfamethoxazole

o Resistance to fluoroquinolone drugs - mutations in genes coding for

DNA gyrase enzyme.
EPIDEMIOLOGY & LOAD OF ENTERIC FEVER
PROBLEM IN INDIA

 Increasing incidence of paratyphoid fever compared to typhoid

fever has been observed in India .
 In India,drug resistance to S.typhi has been reported since
1960, followed by the first outbreak of multidrug resistant
S.typhi (MDRST) in Calicut . Since then, MDRST has appeared
throughout the world, especially in South America, the Indian
subcontinent, Africa and South East Asia.
 Resistance to commonly used antibiotics such as
chloramphenicol, ampicillin , cotrimoxazole, ciprofloxacin has
been reported from different parts of India in the last two
decades
 Resistance to 3rd generation of cephalosporins like ceftriaxone
which is used as DOC has also emerged.
 Azithromycin
PREVENTION OF TYPHOID FEVER

Enteric fever are inherently preventable & vulnerable to

eradication.
WASH
1)It is based on ensuring access to safe water & food handling
practices.
2)Health education should be given to raise public awareness.
3)Sanitation
PROPHYLAXIS

Typhoid vaccines:
It is recommended to
i)those living in endemic areas
ii)household contacts
iii)groups at risk of infection (school children, hospital staff)
iv) travellers proceeding to endemic areas
 Ty21a

 Vi CPS

 TAB

 Vi-rEPA
VACCINES
Vaccine name Type Route Dosage
Ty21a live Oral(enteric given on days
attenuated coated 1, 3, 5,and 7
capsules) with a booster
every 5 years
Parenteral Vi Killed 0.5-mL I.M Single booster
capsular every 2 years
polysaccharid
e vaccine (Vi
CPS)
Vi-rEPA Recombinant & I M Single booster
conjugate every 2 years

Parentral TAB Killed No longer used