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A Project By 2010 A/L Batch


1. Discuss the microbiological and/or immunological basis of the following,
1.1 Herpes simplex virus causes recurrent disease in some patients
1.2 Acute renal failure in leptospirosis

First encounter with HSV gives rise to primary infection(formation
of vesicles which will give rise to ulcers)
Usually primary infection is asymptomatic.
After primary HSV remains latent, Virus entering via skin or mucus
membrane reaches sensory ganglia via sensory nerves and remains
dormant without replicating.
HSV expresses latent associated transcript (LAT) RNA
which regulates the host cell genome and interferes with the
natural cell death mechanism
thereby maintains host cell and preserve the reservoir of virus.
HSV1 and HSV2 establish latency in the trigeminal and sacral ganglia
Virus can get reactivated by illnesses such as cold and influenza,
eczema, stress or exposure to bright sunlight.
Virus reaches skin via sensory nerves and causes recurrent infection.
In HSV1 and HSV2 recurrent infection occurs in the distribution of
maxillary or mandibular branches of trigeminal nerve and sacral
nerves respectively.

Leptospirosis is a zoonosis caused mainly by Leptospira interrogans
Its a biphasic disease; leptospiraemic phase and leptospiriuremic
(immune) phase.
Leptospira enter via abraded skin or intact mucus membrane.
Virlulent organisms access to blood stream via lymphatics, resulting
in leptospiremia and spread to all organs.
If the host survives the acute infection septicemia remains until the
development of antibodies followed by rapid immune clearance.
After clearance, leptospira tend to remain in immunologically
privileged sites sanctuary sites; liver kidney brain
Leptospira contains antigenic lipoprotein, lipopolysaccharides and
endotoxins that may account for kidney injury leading to acute renal
LipL32 is specifically important in renal failure.
They act on toll like receptors(TLR 2) in proximal tubular cells and
activate nuclear factor NF-k
This stimulates synthesis of inflammatory mediators
(interleukins,TNF,CCL2/MCP1) and
causes interstitial nephritis, tubular necrosis and impaired capillary
permeability results in renal failure.
Leptospirosis associated with renal and liver failure is known as
Weils disease

2. A male school teacher having fever for 1 weekend found to be anemic with
enlarged liver. List 3 parasitic infections in order of priority in the
differential diagnosis of the above patient who has never been abroad.
Malaria Leishmaniasis Ameobiasis

3. Briefly describe the pathological changes and pathogenic mechanisms
associated with cerebral malaria.
Cerebral malaria is caused by plasmodium falciparum infection
In its life cycle, during blood meal malaria infected female
anopheles inoculate sporozoites into human host.
Then sporozoites infect liver cells and in liver cells they mature into
Schizonts rupture and release merozoites
Merozoites invade any stage of RBC and develop,
after ring stage they leave peripheral circulation and enter vessels
of the deep oragans

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It is called sequestration
And due to infected RBC s knobby appearance they can adhere to
other RBC and endothelial cells and
this is called as rosette formation
These rosette can block small vessels in the brain and cause
microvascular obstruction
So due to decreased blood flow to brain cells, cells undergo
anaerobic respiration due to hypoxia and result in increased
production of lactic acid
At the same time there will be releasing of merozoites from
ruptured schizonts
Those released merozoites and parasitic products will stimulate
endothelial cells and mononuclear cells to produce reactive
nitrogen species which cause endothelial damage
And also it will stimulate monocytes and lymphocytes to produce
cytokines like TNF
Cytokines also cause endothelial damage resulting fluid leakage
Due to rosette formation and microvascular obstruction, capillaries
are dilated and cerebral tissue is markedly congested
Due to increased fluid leakage cerebral tissue becomes oedematous
Due to malaraial pigment deposition, cerebral cortex is dusky gray
or brown in colour
Due to capillary obstruction and endothelial damage there will be
petechial hemorrhage in perivascular tissue

4.1 Describe the physiological basis of fever in infections.
Toxins from bacteria such as endotoxins act on monocytes,
macrophages and kupffer cells to produce cytokines
Cytokines act as endogenous pyrogens
So cytokines such as IL-1, IL-6, TNF can act independently to
produce fever
These cytokines act on vascular receptors in the thermoregulatory
center of the hypothalamus
Stimulate production of prostaglandin E
Prostaglandin act on vasomotor centre and activate sympathetic
nervous system
Increased sympathetic stimulation causes cause vasoconstriction in
skin vessels
Reduced heat dissipation occurs.
Prostaglandin also raise temperature set point above normal
stimulating body heat generating mechanisms and increased heat
Increased heat generation and reduced dissipation causes fever

4.2 Name 1 bacterial and 1 parasitic infection that presents as acute fever in
Sri Lanka.
Bacterial- Typhoid fever
Parasitic- Malaria

4.3 For each of the infection listed above name the specimen/s, appropriate
microbiological and parasitological investigations for definitive diagnosis.
Blood culture- 1
urine culture- 2
stool culture- 3
Blood smear stained with giemsa stain both thick and thin smears

4.4 For one of the infections listed in 4.2
1. List the therapeutic objectives
To destroy bacteria
To relieve symptoms and complications
To reduce transmission
Prevent emergence of carrier stages
Reduce occurrence of endemics

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2. Explain how you would select an antimicrobial based on efficacy and
Antimicrobial must cover the spectrum of infectious agents
Side effects has to be low and benefits must be outweighed than
side effects
Drugs which are not toxic and high therapeutic index drugs are
Infectious agent must be sensitive to the antimicrobial
So in typhoid now 3
generation cephalosporin can be used
3. Describe the mechanism of action
Cephalosporins inhibit cell wall synthesis of bacteria
by competitively inhibiting the penicillin binding proteins
crosslinking of peptidoglycan
Therefore it is an bactericidal drug

5.1 What do you understand by the term sepsis syndrome?

Sepsis syndrome or SIRS (Systemic Inflammatory Response
Syndrome) is systemic level of acute inflammation,
that may or may not be due to infection,
and is generally manifested as a combination of vital sign
abnormalities, such as fever/hypothermia, tachycardia and
It is a systemic response to a wide variety of clinical insults.
In sepsis syndrome there is also evidence of altered organ perfusion
with at least one of the following features : hypoxaemia, elevated
lactate, oliguria, altered mental status.
Risk factors for SIRS would be extremes of age, indwelling
lines/catheters, immunocompromised states, malnutrition,
alcoholism, malignancy, diabetes, cirrhosis and genetic
Overall mortality from SIRS is high and the mortality rate is
proportionate to the no. of failed organs.
Sever sepsis is SIRS associated with organ dysfunction, hypoperfusion
or hypotension and the marker is acute organ dysfunction.

5.2 What are the characteristic features of this condition?

2 of the following 4 features should be present :
Temp>38.3 or <36.0 C
Tachypnea (RR > 20 or Minute Volume > 10L ) / PaCO
Tachycardia (HR > 90, in the absence of intrinsic heart
WBC > 10,000/mm3 or <4000/mm3 OR > 10% band forms
on differential count.

In severe Sepsis, 1 of the following should also be there :
Altered mental status
Systolic BP<90mmHg or fall of >40mmHg from baseline
Impaired gas exchange
Metabolic acidosis

5.3 List 2 important cytokines that are responsible for the
pathophysiology of this condition

IL 1

5.4 What is the primary reason for the above cytokines in the sepsis
The above cytokines are produced in response to the
microbial signals.
About 70% of cases are due to endotoxins produced by
gram (-) bacteria.
These endotoxins are bacterial wall lipopolysaccharides
consisting of a toxic fatty acid core (lipid A), and a complex
polysaccharide coat (O antigen) unique for each species.

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Peptidoglycan/lipoechoic acids in gram (+) bacteria can also
elicit this.
Toxins such as TSST 1 (Toxic Shock Syndrome Toxin 1)
produced by S. aureus (gram (+) cocci), Group B
Streptococci Toxin can also cause this.
The free lipopolysaccharides bind to CD14 receptor in
monocytes, macrophages, neutrophils.
It circulates in blood bound to a lipopolysaccharide binding
plasma protein.
Engagement of CD14 results in intracellular signaling
pathways being activated (TLR -4) resulting in activation of
mono nuclear cells.
The macrophages present the antigen to the T helper cells
which activates the T helper cells.
These in turn activates the macrophage again which triggers
them to secrete TNF and IL 1
Both of these cytokines which are the early mediators, act
on endothelial cells and other cells to produce additional
cytokines. (IL6, IL8)
These are the late mediators.
These induce adhesion molecules and other factors such as
prostaglandins, coagulation factors are also induced.
These result in systemic vasodilatation, intravascular
volume depletion and endothelial injury.

6.1 List 2 lab diagnostic methods that could be used for Plasmodium
vivax malaria
Light microscopic diagnosis
1. PCR technique

6.2 Write advantages and disadvantages of each of the above methods.

Light Microscopy
Advantages :
o Can be performed under field conditions.
o Requires inexpensive equipment (microscope, glass
slides and reagents)
o Results available in 15 20 minutes useful in OPD
o Detection of any species of Plasmodia are possible
in a single test
o Detects all parasitic stages present in peripheral
o Sensitivity is high (If analyst is trained)
o Can monitor the response of a patient to therapy.

o May give false negatives if the parasite density is
very low or if parasites are absent in peripheral
o Required trained personnel to identify parasites
o Sensitivity and specificity are high but varies :
At low incidence rates
At very low parasite densities
On the skill of the analyst
Lab conditions
o Relies on electricity
PCR technique
o Highly sensitive
o Highly specific
o Can be diagnosed even with a very low parasite density

o Requires expensive lab equipment and reagents
o Requires good laboratory conditions
o Might give false positives even after cure of the patient,
because parasite DNA might remain in circulation for
several days to a week after successful treatment and
termination of infection.


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7. A 55 year old male who underwent an emergency bowel surgery
developed fever on the 3
post-operative day. He has an indwelling
urinary catheter and an IV cannula in situ.

7.1 List 4 likely infections causing fever in this patient.(8)
Surgical site infection
Urinary tract infection
IV line associated infection and bacteremia
Hospital acquired pneumonia

7.2 Name 2 likely microorganisms responsible for each of the infections
listed above in 7.1 (32)
Surgical site infection Staphylococcus aureus, Escherichia coli
Urinary tract infection Escherichia coli, Pseudomonas
IV line associated infection & bacteremia Staphylococcus
aureus, Staphylococcus epidermidis
Hospital acquired pneumonia Staphylococcus aureus,
Pseudomonas aeruginosa

7.3 Name the relevant specimen required for microbiological diagnosis
of each of the infections listed in 7.1 (20)
Surgical site infection blood for culture and ABST
Urinary tract infection urine for culture and ABST
IV line associated infection & bacteremia blood for culture and
Hospital acquired pneumonia expectorated sputum for
culture, ABST, gram stain

7.4.1 List the therapeutic objectives (10)
Elimination of the causative organism with a suitable
antimicrobial drug or a combination
Symptomatic relief (antipyretics, bronchodilators)
Enhancing patients immunity (nutrition, deficient vitamins)
Prevent recurrences (surgical wound hygiene)

7.4.2 State the principles on which you would select the appropriate
antimicrobial/s (20)
Specificity activity of the drug should match the infecting
organisms.(narrow spectrum)
Empirical therapy before identification of causative organism
must be reasonably broad spectrum.
Pharmacokinetic factors chosen drug must reach the site of
infection in adequate amounts. (Eg. crossing the blood brain
Patient factors previous allergy to the drug, impaired routes of
elimination (renal disease)

7.5 2 days later, the patient develops tachycardia (130 beats/min) and
an increased respiratory rate (40/min). His white cell count was
/L with 90% neutrophils. His urine output was decreased. What is
the complication the patient has developed? (10)
Systemic inflammatory response syndrome secondary to
infection (or sepsis)

8. A 6 year old girl presents with anemia.

8.1 Name 2 parasitic infections that could be the cause of anemia in this
patient. (10)

Hook worm infestation

8.2 Explain the pathophysiological basis for the anemia in each of the
parasitic infections listed in 8.1 (30)

The released merozoites from infected hepatocytes infect the
The intraerythrocytic parasites either continue asexual
reproduction, producing more merozoites infecting more RBC
or give rise to gametocytes.
The new merozoites escape to the blood stream by lysing the

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This destroys large numbers of red cells causing hemolytic

Hook worm infestation
Adult worms of Necator americanus live in the duodenum and
upper jejunum, where they are found in large numbers in
severe disease.
They attach firmly to the mucosa using their buccal plate and
feed on host blood.
They secrete molecular inhibitors of coagulation factors causing
bleeding from the attachment site.
They also degrade host haemoglobin.
The resulting massive loss of blood from the body causes an
iron deficiency anemia.

9.1 List 5 infectious diseases where blood culture is useful in the diagnosis. (5)

in any infection with bacteraemia
Enteric fever
Bacterial endocarditis

9.2 Describe in detail how you would obtain blood for culture & transport it to
laboratory including universal precautions. (30)

first obtain the necessary equipment to perform the blood culture
- Blood culture bottles
- Hypodermic syringes and needles
- Sterile gloves
- Tourniquet
- Alcohol
- Sterile gauze pads
- 10% povidone iodine (if allergic 4% chlorhexidine)
Then check the identity of the patient and explain the procedure
to the patient
Next check the blood culture bottles for any contamination (see
whether they are clear)
Wash hands using soap and water thoroughly and dry on a sterile
Wear the sterile gloves taking care not to contaminate
Select an appropriate venepuncture site
Clean the site by applying 70% alcohol concentrically rubbing from
inwards to outwards. Allow this to dry
Next clean the site using 10% povidone iodine in a concentric
fashion as above from inwards to outwards
Allow at least 2 minutes to dry
While waiting for this site to dry, remove the protective covering
from the bottle top and wipe the exposed diaphragm using an
alcohol based disinfectant and allow it to dry
Then perform the venepuncture using a dry, sterile needle and
syringe and draw at least 20 ml of blood
While drawing blood take precautions to prevent sharps injuries
such as avoiding hand to hand passage and maintaining a safe
distance from others when drawing blood
Inoculate 5 ml of blood into each blood culture
Do not change needles for this purpose and always place the
culture bottle on a flat surface while inoculating blood
Once the bottle has been inoculated, gently shake the contents so
that it has been mixed
Carefully dispose of the used needles and syringes into the sharps
bin (Do not recap or break needles in the process)
Then remove the gloves and wash hands
Label the bottle with the relevant patient details
Fill the laboratory request forms
Keep the samples at room temperature before transporting them
as soon as possible to the lab

9.3 From the steps you have mentioned in 9.2, select & write the steps that
are taken to ensure asepsis.(aseptic precautions) (10)


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Wash hands using soap and water thoroughly and dry on a sterile
Wear the sterile gloves taking care not to contaminate
Disinfecting the skin over the venepuncture site by applying 70%
alcohol concentrically rubbing from inwards to outwards and allowing
this to dry
Cleaning the site using 10% povidone iodine in a concentric fashion as
above from inwards to outwards and allowing at least 2 minutes to
Performing venepuncture using a dry sterile needle

9.4 From the steps you have mentioned in 9.2, select & write the steps that are
taken to prevent occupational exposure to blood borne pathogens. (universal
precautions) (10)

Wash hands using soap and water
Wearing sterile gloves
Taking precautions to avoid needle stick injuries such as avoiding
hand to hand passage, maintaining safe distance from colleagues
while using sharps
Not changing needles to inoculate the blood
Placing the culture bottles on a flat surface before inoculating
Disposing used needles and sharps into sharps bins without recapping
or breaking needle
Washing hands after removing the gloves

9.5 Outline the principles of antimicrobial therapy for one of the infectious
diseases you have listed in 9.1. (25)

Before initiating any antimicrobial therapy, it is necessary to make a
microbial diagnosis of the condition as precisely as possible
For this purpose it is necessary to obtain a CSF sample by performing
a lumbar puncture
The sample is sent to the microbiological department for gram
staining and culture
Also blood samples must be taken for blood cultures (and to
determine plasma glucose)
Next it is necessary to consider whether antimicrobial therapy is
really necessary
For this purpose criteria such as diagnosis, severity of the disease,
onset of the disese and the natural history of the disease are
Since a meningitis is a serious life threatening disease of acute onset
with many neurological sequale if left untreated, a diagnosis of
meningitis usually necessitates treatement
Removal of any barriers to the cure must be taken in order to ensure
that antimicrobial drugs can reach the site of infection to exert their
Once all the above has been performed, it is necessary to select a
drug/drugs to use in this situation
In the case of meningitis, empirical/best guess therapy is given soon
after the relevant samples have been taken
The drugs used for empirical therapy are selected based on factors
such as the site of infection, the likely organisms to be causing the
infection as well the resistance patterns of these organisms to various
Once the microbial diagnosis arrives, it is necessary to narrow the
spectrum of the antibiotic to target the identified agent
The selection drugs in this case should be based on
- Pharmacokinetic factors
- Whether the selected drug can reach the site of
infection in adequate amounts to exert its action
- Patient factors
Renal and Hepatic Function
Pregnancy and Lactation
History of allergy
History of recent antibiotic use
- Microbial factors
- Resistance to various antimicrobials

Once a drug is selected based on the above factors, it should be
administered at the
- Optimal dosage (to prevent development of resistant

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- In the appropriate dosage forms
- At the optimal frequency (intermittent dosing is
- Via the appropriate route (parenteral considering the
severity of meninigitis)
- Correct duration (to prevent the development of
resistant organisms
Clinical and plasma monitoring must be done while the drug is being
Finally the resolving of the infection must be confirmed clinically or
microbiologically before the patient is discharged

9.6 Based on the principles you have mentioned in 1.5,
9.6.1Choose an antimicrobial/s that is effective in the infectious disease
discussed in 9.5, giving reasons for your choice. (15)
9.6.2Mention, of administration duration of therapy (5)

9.6.1) Cefotaxime
This is a 3
generation cephalosporin
Which acts as an inhibitor of bacterial cell wall synthesis and
therefore acts as a bactericidal drug
It has a very good gram negative cover and is effective against
common bacterial agents causing meningitis such as H.influenzae
type B (noncapsulated) and Neisseria meningitides as well as being
effective against gram positive organisms such as Streptococcus
It has a good distribution throughout the body tissues and passes
through the blood brain barrier, meaning that it can reach the
meninges in order to exert its antimicrobial action
It is available in the parenteral dosage form which is the dosage
form of drugs used in meningitis due to its severity
It is safe to use in children and its excretion is not affected by liver
failure (but it is affected by renal failure)
It can also be used during pregnancy since it does not cause any
harmful effects to the foetus even though it crosses the placenta
In addition to the above, cefotaxime does not have many significant
side effects except for beta lactam hypersensitivity which must be
tested for before administration of the drug Intravenous Route 14 days

10. A 34 year old patient presents to the OPD of the NHSL with the history
blood & mucus diarrhoea of 2 weeks duration. He has vague abdominal pain
& anorexia.

10.1 List 2 likely parasites that cause blood & mucous diarrhea. (5)

Entamoeba histolytica
Trichuris trichiura

10.2 Describe the pathogenic mechanism for the blood & mucus diarrhoea
for each parasite mentioned in 10.1

Entamoeba histolytica

The trophozoite form of the organism present in the intestinal
lumen penetrates the colonic mucosa in between the glandular
by the production of tissue lytic substances which degrade the
protein and polysaccharide components of the mucus layer
They then attach to the epithelial cells of the surface of the gut and
induce apoptosis in these cells.
(this results in the production of ulcers with pin head centre and
raised edges)
Sometimes the invasion is superficial and results in the formation of
flask shaped ulcers
The trophozoites then penetrate the submucosal layer of the
intestine and multiply rapidly causing lysis of the surrounding tissue
and blood vessels
Causing the leaking of blood into the lumen

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Trophozoites also alter the mucus composition of the mucous
secreted In goblet cells, thereby increasing the mucus secretion
This results in the production of a blood and mucus diarrhoea

Trichuris trichiura

Once the embryonated egg of the organism is ingested, the
activated larvae hatches out from the weakened egg shell in the
upper small intestine and penetrates through the intestinal villus
A spear like projection in the anterior extremity allows the worm to
penetrate and embed its whip like anterior portion into the
intestinal mucosa of the host
In this process it releases various chemicals to liquefy the adjacent
mucosal cells
As a result of penetration of blood vessels at the site of attachment
of these worms, blood leaks into the intestinal lumen giving rise to
blood and mucus diarrhoea
This occurs especially in heavy infections of the parasite

10.3 Taking one of the parasites mentioned above as an example,
10.3.1 Describe the collection of stool sample & its transport to the
laboratory. (20)
10.3.2. Outline the laboratory procedures you would carry out on the stool
sample collected in 10.3.1, and the expected findings to arrive at a definitive
diagnosis. (20)

10.3.1 Entamoeba histolytica

The patient should defecate into a clean and dry bed pan without
any disinfectant
If this is not possible the patient should defecate into a clean bowl
or even a banana leaf
A selected portion of the stool from the mucoid and bloody area is
transferred into a clean and wide mouthed disposable container
with a lid (Eg : Ice cream container)
This should be transported as soon as possible to the laboratory
If any delay occurs a suitable transport medium should be used

Light microscopy using a wet smear for demonstration of
trophozoites or cysts must be performed within 30 min
Iron haematoxylin stains are used for better visualization
If negative, three repeat samples are viewed under the
microscope under high concentrations
If a trophozoite is present it would consist of outer
ectoplasm which is wide and clear and inner endoplasm
which is granular.
It also contains a round nucleus with a cartwheel
appearance and also contains ingested RBCs
If a cyst is present it is round and surrounded by a wall and
consists of 2 -4 nuclei with undifferentiated cytoplasm
containing sausage shaped chromatoid bodies

10.4 For each of the parasites listed in 10.1, list one anti microbial drug
effective for the treatment & give reasons for your selection. (20)

Entamoeba histolytica - Metronidazole

Metranidazole can kill the trophozoites of Entamoeba
histolytica and can therefore act against the active
organisms present within the lumen of the gut
It is well distributed after oral and rectal administration and
reaches effective concentrations within the gut and liver
where trophzoites are usually found
It has only a few unwanted effects at therapeutic doses
including gastrointestinal disturbances, metallic taste,
dizziness, headache and disulfuram reaction

Trichuris trichiura - Mebendazole
Mebendazole acts on both the larvae and the adult worm as
well as on the ova of the organism
It causes inhibition of mitochondrial enzymes and inhibits
glucose uptake by the parasites and therefore causes
hypoglycaemia in them

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It is poorly absorbed in the gut meaning that it can remain
there in order to exert its action
It does not cause any significant systemic toxicity in active
clinical use even in the presence of anaemia and
malnutrition which are common in patients infected with
these parasites

10.5 What advice would you give to the patient in order to,
10.5.1 prevent re-infection in this patient
10.5.2 prevent spread of this infection from the patient to the community. (10)

10.5.1 Since these infections are mainly transmitted by the faeco
oral route, reinfection can be prevented by
1) Food safety
This involves proper cleaning of hands before preparing or
consuming food and after toilet use,
washing vegetables whose edible portions grow within the
soil and
covering food to prevent contamination by flies and
cockroaches which can be used as mechanical vectors.
Children should not be allowed to play in contaminated soil
as they may accidentally ingest the eggs of the parasite

2) Water safety
Only boiled water or water that has been sterilized properly
using iodine salts or other suitable agents should be taken
for consumption

10.5.2 Spread of infection to the community can be
prevented by
1) Preventing soil contamination
Proper sanitary facilities should be used and there should be
sanitary disposal of waste in places where eggs do not come
into contact with humans.
Drainage systems and water lines should be cleaned and
maintained in a proper manner so as to prevent
contamination with infected waste
2) Screening and treatment
It is important that individuals who have contact with foodstuffs
prior to consumption such as chefs and restaurant carriers are
screened for the condition since they may be an active source for
the spread of infection.
Once infection has been confirmed in these patients they should be
treated as soon as possible

3) Supportive measures
Health education regarding these diseases can be given to the
general public to create awareness which would reduce
In addition to this public health measures such as providing
decontaminated drinking water and sanitary facilities would
also prevent the spread of the disease

Insect control is also important and would prevent spread of
this infection since they act as mechanical vectors