Week 4 - Leishmaniasis Flashcards
How many countries is Leish distributed over?
88
how many new cases each year?
2 million
how many people are infected each year?
12 million
how many deaths are there per year?
52,000
how many people are at risk each year? what may cause this to rise
350 million
global woarming
what is the parasite transmitted by?
Sand flies
how big are sand flies?
2mm in length
what are adult sand flies characterised by?
hairy body and wings
Generally Phlebotomus sp are found in which ecosystems?
desert / semi arid
Lutzomyia sp are found in which ecosystem?
forest
why is the term sand fly misleading?
they do not fly, rather hop as short bursts of flight only
which gender spread the disease?
only females
when do sandflies most actively feed?
nocturnal and dusk
what sort of feeders are sand flies?
saw feeders. their saw like mouth parts create a wound which allows blood to well up in to a pool
suck the blood and lymph from that pool
where is the parastie located inthe sand fly?
anterior portion of gut and pharynx
how does the sand fly transmit the parasite?
regurgitates it up
transmission of leish can be both ___ and ___
zoonotic
anthroponotic
Zoonotic transmission is common in which 2 places? and which animals are reservoirs?
mediterranean
latin america
canines and rodents
generally whether the transmission cycle is zoonotic or antrhoponotic depends on which factor?
the human density in that area
What are 4 other mechanisms of transmission of leish?
blood transfusion
organ / bone marrow transplant
congenital - mother to foetus
drug usage - needle sharing
how many different species of Leishmania are there and how many can infect humans?
30
21 can infect humans
What has molecular typing demonstrated about some o fthe Leishmania species?
that in the old and new world the species are synonymous
How can you distinguish Leishmania species?
morphologcically indistinguishable
can only distinguish by molecular level
What are the 2 forms of the parasite?
promastigote
amastigote
where do you find the promastigote?
insect vector
key features of the promastigote?
flagellum sticks straight out of the body
kinetoplast in front of nucleus
what are the 2 types of promastigote?
non infectious promastigote
infectious metacyclic promastigote
The non - infectious promastigote is found where? can it divide?
insect gut
binary fission
the infectious metacyclic promastigote is found where? can it divide?
human macrophages and neutrophils
non - dividing
also found in the insect gut - transformed from non-infectious promastigote
what does the term metacyclic generally mean?
go from the insect to human
Key features of the amastigote?
intracellular
infectious
invade macrophages and neutrophils - binary fission
non motile
when the amastigote is inside the macrophage what are they like?
inside a vesicular compartment - essentially have a membrane around them which is derived from the host
which form of the parasite is transmitted back from humans into the insect?
amastigote
Life cycle :
the sand fly takes a meal and starts to regurgitate ___ _____ form parasites into the host
infectious metacyclic promastigote
What causes the regurgitation mechansim in the sand fly?
the parasite
dysfunctional stomedeal valve at foregut/midgut junction
when the insect feeds the blood goes down into the gut and because the valve doesnt close properly it washes back up into the host bringing up metacyclic promastigote
The whole process of regurgitating the parasite is exacerbated by what?
parasite secretes promastigote secretory gel that acts like a bung and promotes the insect to feed for longer
increases the chance of the parasite being transferred into the pool of blood if the feeding time is longer
why do the insect saliva and promastigote secretory gel contain elements that our immune system will respond to?
Our immune system responds to the damage done by the insect feeding and also responds to the secretory gel and insect saliva so this magnifies the attraction of neutrophils to that site.
Want more neutrophils for the metacyclic promastigote to infect
How does the metacyclic promastigote form invade neutrophil cells?
phagocytosed
What does the parasite do to the neutrophil cells?
alters their gene expresssion to promote itself
why doesnt the parasite want to stay in the neutrophil cells?
because they are short lived
what are the 2 hypotheses as to how the parasite gets form the neutrophils into macrophages?
trojan horse
simple one
explain the trojan horse hypothesis
Infects neutrophil
causes the neutrophil to undergo apoptosis
apoptotic marjers are then exposed on the cell surface of the neutrophil
the macrophages recognise the apoptotic markers and consume the dying cell
phagocytose the neutrophil along with the metacyclic promastigote inside the neutrophil
what happens to the metacyclic promastigote once it is inside the macrophage?
undergoes differentiation into an amastigote and then starts dividing
in the trojan horse hypothesis what is the trojan horse?
neutrophil
what is the simple hypothesis as to how the metacyclic promastigotes get into macrophages?
Whilst the metacyclic is inside the neutrophil undergoes differentiation into amastigote
amastigotes start to divide and rupture the neutrophil
the amastigotes are released into the blood stream and then taken up by the macrophages
What is key about the 2 hypothesis as to how the metacyclic promastigotes get out of the neutrophils?
they both lead to amastigotes form in the macrophage where they can divide and rupture the macrophage and then reinfect other cells
how long can leishmania infection last?
6 months
why is it likely we have these 2 different hypotheses?
because we are dealing with 21 different leish species
how does the parasite get back into the insect vector?
sand fly takes a blood meal from an infected person and may take up a macrophage with amastigotes in it. once inside the sand fly the macrophage is digested and amastigotes are released
what happens to the amastigotes when they are inside the insect vector after the macrophage has ruptured?
once in the digestive tract of the insect vector the amastigotes decrease in temperature and increase in pH causes the amastigotes to transform into non-infectious promastigotes
what do the non infectious promastigotes do in the insect vector?
covered in a mucous layer that is derived from the insect itself, which contains a protein mesh
oarasite is trapped inside this peritrophic matrix
with time various enzymes produced byt the insect and parastie witll degrade away this matrix and the non infectious promastigote is released into the lumen
when the non infectious promastigote is in the insect vector and has been released from the matrix into the lumen what occurs?
using a LPG molecule will stick to the epithelial layer of the gut wall which prevents the parasite from being excreted
non infectious promastigotes divide by binary fission and colonises insect gut wall
release the promastigote sseceretory gel to damage the valve
transformation into metacyclic promastigotes
what happens to the metacyclic promastigotes in the insect?
they detach from the wall
and congregate around where the valve is found
life cycle complete
Leish species are obligate ____ parasites in the mammalian host
intracellular
which cells do leish species inffect?
microbicidal capability - macrophages and neutrophils
which mechanism does the parasite exploit to get into the macrophages and neutrophils?
complementary cascade
what is the complement mediated lysis (alternative activation pathway)?
binding of early complement components to organism is followed by assembly of membrane attack complex
when the parasite is inside the neutrophil cell it has to overcome the mechanisms this cell uses to destroy foregin cells, what is it facing? (3)
lysosomal pH
lysosomal enzymes
oxidative stress
Explain the lysosmal pH the parasite has to overcome?
phagosomal compartment is acidified upon fusion with lysosomes to pH 5
most infectious agents this is sufficient to damage the membrane
leishmania parasite utilises this change in pH to recognise it is inside a mammalian host cell and transforms into an amastigote
explain the lysosomal enzymes the parasite has to overcome?
lysosomes contain an array of digestive enzymes including proteases, nucleases, and lipases.
parasite appears to be resistant to all of these
explain the oxidative stress the parasite has to overcome?
NADPH oxidase (Phox) complex on phagosomal membrane generates superoxide leading to the production of hydrogen peroxide and hypochloride
reaction with transitionmetals lead to formation of hydroxyl radicals
nitric oxide synthase generates nitric oxide which combines with superoxide to form peroxynitrite
The player is Leishmania infection:
Infective - ??? Host target cells - ?? Mode of entry - ?? Host receptors - ?? Parasite molecules implicated in the uptake - ?? Site of replication - ??
metacyclic promastigotes neutrophils then macrophages passive, opsonised phagocytosis CR1, CR3 gp63 protease, lipophosphoglycan (LPG) phagolysosome vacuole
the parasite cells surface changes during differntiation from non-infectious promastigote (replicative) to the infectious metacyclic promastigote, explain (2)
Replicative promastigote has short LPG and low levels of GP63
Infective metacyclic promastigote has long LPG and high levels of GP63
what is the LPG similar to?
the variant surface glycoprotein of t.brucei but have to remember this is not a protein it is a sugar, a fat phosphor sugar mix
So the LPG plays a part in attaching the promastigote to the insect gut wall, explain the structure of LPG further
LPG consists of several domains.
It has a phosphoglycan region that contains repeated elements.
The galactosyl side chain binds to gelactin and this binds parasite to the wall of the insect gut
the side chains of the metacyclic promastigote are capped with a galactose residue which prevents it from binding to gelactin and this facilitates detachment form the wall of the mid gut
the whole LPG molecule is linked by a ____ anchor which is way of doing what?
GPI anchor
packing many molecules closely together on the cell surface
why should the parasite bother to make the change of its cell when it changes the promastigote form? (2)
it causes a detachment from the lumen so the parasite can be transmitted
the longer LPG coat prevents the membrane complex from getting access to the parasite cell membrane so it cant puncture holes through it
so what happens to C3 on the parasite?
during the complement cascade C3 is broken down into C3a and C3b
the parasite binds C3b component in the LPG layer and this C3b is partially degraded by zinc protease to be inactivated so it cannot be involved in the complement cascade any more
complement cascade is now modified
what happens to the iC3b on the parasite?
essentially coats itself with the inactivated C3b componenet which then attracts neutrophils and macrophages.
This iC3b is still attracted to complement receptors 1 and 3 (CR1, CR3) and the complement receptors on the macrophage bind to iC3b and promote the entry of the parasite into the macrophage via phagocytosis
Cell invasion the parasite uses complement and phagocytosis occurs, how do we know this process is phagocytotic?
pseudopodia form to start the engulfment process.
we know this is phagocytotic because you can block this process using an appropriate drug which will prevent this process from occurring.
also use cell biology marker and see the marker forming around the parasite
what makes the parasite more susceptible to phagocytosis?
iC3b
once the metacyclic promastigote is inside the phagosome what occurs?
lysosome vesicles fuse with the phagosome and some of these vesicles will result in a drop in the pH of the lumen to pH 5
the promastigote responds to the drop in pH and differentiates into the amastigote form which then undergoes binary fission and colonises that cell
How does the parasite modulate host cell signalling and essentially overcome the oxidative stress?
Leish sp inhibits protein kinase C activity and the NADPH oxidase (Phox) complex is no longer formed. This reduces respiratory burst (ie oxidative stress)
How else does the parasite modulate host cell signalling so the inflammatory responses are reduced?
Leish activates SHP1 a tyrosine phosphatase
SHP1 causes dephosphoylation of Jak (Janus kinase)
The transcription factors STAT is not phosphorylated and remains in cytosol
change in gene expression as STAT can no longer access nucelus
Inflammatory responses are supressed
not all macrophages in our body are the same how does Leish respond to this?
dermal macrophages lack the respiratory burst machinery and promastigotes have enhance ability to differentiate as amastigotes in these cells compared to other populations and so some of the parasites have a preference for invading dermal layer macrophages.
Which is the most common pathology of Leish?
cutaneous leishmaniasis
out of the 12 million reported leish cases how many are cutaneous?
10 million
cutaneous lesih starts out how?
raised, generally painless red lesion at site of the bit and can ulcerate.
what are the 2 most common species that are associated with cutaneous leish pathology?
l. tropica - cities
l. major - rural
Generally all the other pathologies of leish start off as which pathology?
cutaneous leish
In cutaneous leish ulcers that form can be classified as 2 types, what are they and which species are they commonly associated with?
L.major - wet ulcer
L.tropica - dry ulcer
Is there puss formation in cutaneous leish?
no only get pus if there is a secondary infection
Do you need medication for cutaneous leish?
they will eventuallly self heal and once heled you have immunity against that leish species for life
When in cutaneous leish would you be provided medication?
if the ulcer is on your face for cosmetic reasons and also to speed up healing / reduce scarring
In __% of cases the lesion can disappear and you get ___ leish
5
diffuse
what are the 2 types of diffuse leishmania?
diffuse cutaneous (DCL) mucocutaneous (MCL)
Describe the pathology of diffuse leishmaniasis (4)
- -metastasizes over whole body
- -non-ulcerated, scaly lesions
- -generally associated with anergy (lack of immune response)
- -amastigotes abundant lesions
Describe the pathology of mucocutaneous leishmaniasis (5)
- –metastasizes mucocutaneous junctions (open or scaly)
- -can occur weeks/years after primary infection
- -tissue destroyed and severe disfiguremnet
- -secondary bacterial infection common
- -most prevalent in latin america
- -3-5% cutaneous will progress to this type
what is the most serious pathology of leish?
visceral leishmaniasis — kala azar — dum dum fever
what is a characteristic symptom of visceral leish?
darkening of the skin
which is the pathology of leishmaniasis that kills people?
visceral. 50,000 per year
the other forms of the disease are not lethal, they are highly disfiguring but may cause death due to secondary infections
In visceral leish the parasite invades which organs?
reticuloendothelial system
liver spleen bone marrow lymph nodes
what symptoms are visceral leishmaniasis commonly characterised by? (4)
fever spenomegaly heptomegaly enlarged lymph nodes wasting symptoms
In leishmaniasis disease severity depends on the immune status of the individual, what are the 2 immune responses that can occur?
TH1
TH2
what is a TH1 type response?
cellular immune response triggered
no/low antibody formation
recover from infection and are immune CL
what is a TH2 type response?
humoral immune response triggered antibody formation occurs
succumb to infection (VL)
what is the problem we have with our immune system and strong l.donovani strains?
force host to switch from TH1 (that would clear parasitemia) to Th2 (does not clear parasitemia favours the parasite) response by an unknown mechanism
if you have a suspected case of leishmaniasis what 2 things do you look to diagnose?
for visceral and dermal - geographical presence of parasite
history of sand fly bite
what are the symptoms for the 3 types of dermal leish?
chronic painless leision (CL) mucocutaneous lesion (MCL) scaly lesions (DCL)
what are the symptoms for visceral leish?
prolonged fever
spenomegaly
aneamia
leucopenia
definitive diagnosis - detection of parasites
what are the four ways to detect the parasites in dermal leish?
amastigotes in scraping
amastigotes in aspirates
invitro culturing of promastigotes from aspirates
innoculation of aspirates in model organism - hamster
definitive diagnosis - detection of parasites
what are the 2 ways to detect the parasite in visceral leish?
amastigotes in bone marrow or biopsy
invitro culturing of promastigotes from aspirates
seriology diagnosis
immunodiagnostics - method for dermal leish?
leishmanian skin test
- —intradermal injection of killed promastigotes
- —monitor for local delayed hypersensitivity
- —development of erythema if leish +ve
seriology diagnostics
immunodiagnostics - visceral (2)
- —serological test direct agglutination
- —-K39 dipstick
what is the frontline drug treatment against leish?
pentavalent antimonial
Pentavalent antimonial is a ___ ___ that is administered in its inactive state. It can only be activated by ____ form.
prodrug
amastigote
Which species are more susceptible to pentavalent antimonial?
Luckily the species that cause the more severe form of the disease appear to be slightly more susceptible than the species which cause CL
How is pentavalent antimonial administered to the patient?
intramuscular or intravenous
what are the problems associated with pentavalent antimonial?
heavy metal drug
toxicity - fatigue, nausea, joint pain
may fail to clear parasiteimia - resistance in the field
how does pentavalent antimonial work?
activation can occur inside/outside the cell we dont know where as both forms of the drug can enter the cell
has to be converted to its trivalent form by amastigote
what does the trivalent antimonial do?
binds and conjugate with various targets, the trivalent form can inhibit trypanothione which has major implications for how these parasites can synthesise DNA and respond to oxidative stress
where do the most resistant cases to antimonial occur?
Bihar 50-65%
why are the parasites resistant (3)?
unrestricted availability - no public health system
incorrect dosages - dont have standards / monitoring
inconsistent quality
the mechansim of resistance in the field is unknown but how do leish parasites become resistant to antimonials in the lab?
growing cells on sub lethal levels of drug
ABC transporter
see slides for antimonial resistance specifics
see slides for antimonial resistance specifics
what are 3 alternative drugs for leishmaniasis?
amphotericin B
Miltefosine
paromomycin
Amphotericin B
Was initially developed as an _____ ____, however it was discovered to be toxic. Played around with the formulation and placed the drugs in ____, these are ___ globules which you insert the drug into. This drastically ___ toxicity.
How does this drug function?
antifungal agent
mycells
lipid
reduced
Binds to 24-substituted sterols in the parasite membrane and essentially punches holes in the membrane, disrupting osmotic integrity of the membrane
Miltefosine
Developed as an __ ___, but was abandoned due to toxicity problems. It is the only drug which can be administered _____. Significant drawback of this drug is that it is ____ and therefore cannot be used by women of childbearing age.
The mode of action is uncertain
anti cancer drug
orally
tetratigenic (causes malformations in embryos)
Paromomycin
developed as an _____ to treat infections.
still undergoing clinical trials and so not clinically used unless all other drugs have failed.
Administered ___
antibacterial
orally
Prevention and control of reservoir species? (2)
canine vaccinations
culling in brazil of canines
What are 3 types of vector control?
insecticides
repellents
bed screens / nets
Why is vector control brilliant in theory but practice more difficult?
because it requires money and political will
