Leishmaniasis - 4 (8/9)

Question 1

Leishmaniasis is also known as

Answer 1

• orient boils
• Baghdad boils
• kala azar
• black fever
• sandfly disease
• DumDum fever
• espundia

Question 2

Leishmaniasis distribution

Answer 2

• 88 countries
  
  • tropical and sub-tropical countries
• 2 million new cases each year
• 12 million people infected / 52,000 deaths in 2010
• 350 million people at risk (will increase with global warming)
  
  • flies that spread the disease moving up the Italian peninsula
  • moving north as the earth warms up
• spectrum of diseases ranging from mild to severe
• due to militariy in Iraq etc. have gotten the disease in non-endemic sites
  
  • military gone into the area, go back home, bringing parasite with them
  • insect vector found in parts of the US
  • secondary foci - esp Southern US (eg Texas)

Question 3

Leishmaniasis

transmission

Answer 3

• parasite transmitted by phlebotomine sand flies
  
  • Phlebotomus in Africa, Asia, and Europe
  • Lutzomyia in Americas
• adult sandflies ~ 2mm in length
• adult sandflies characterized by hairy body and wings

Question 4

Leishmaniasis

transmission

Phlebotomus spp

Answer 4

• desert/semi-arid ecosystems
• some breed in peridomestic situations and enter human habitations
• flies transmitting in the old world
• some breed/propagate in areas surrounding houses, occassionally can transmit vector within human habitation
• moving into towns more and more, becoming more urban

Question 5

Leishmaniasis

transmission

Lutzomyia spp

Answer 5

• forest dwelling
  
  • new world, associated with forests
• disease transmission associated with humans living or working near forests

Question 6

Leishmaniasis

transmission

sandflies fly via

Answer 6

“hopping”

• short bursts of flight/few seconds of rest
• short flight range

Question 7

Leishmaniasis

transmission

sandflies: …. spread the disease

Answer 7

only females spread the diseae - males play no role in disease transmisson

• male flies don’t feed on any mammals - feed on phloem of plants
• only females feed on blood = only vectors for disease transmission

Question 8

Leishmaniasis

transmission

sandflies feed

Answer 8

most actively at twilight/night

Question 9

Leishmaniasis

transmission

sandflies are

Answer 9

pool feeders

• saw-like
• short mouthparts
• drink blood/lymph fluid from wound

Question 10

Leishmaniasis

transmission

parasite not in

Answer 10

insect salivary gland

• parasite found anterior portion of gut and pharynx
• transmitted to humans not from fly saliva in formation of wound BUT spread by vomit of the insect
  
  • parasite regurgitated by insect vector

Question 11

Leishmaniasis

transmission

zoonotic or anthroponotic

Answer 11

• zoonotic = animal to human
• anthroponotic = human to human
• different transmission cycles in different parts of the world (higher human density in some regions)
• generally in areas with sparse human populations = zoonotic transmission
• many humans = anthroponotic
• different flies have different preferences for what they feed on

Question 12

Leishmaniasis

transmission

zoonotic

Answer 12

• in Mediterranean and Latin America
• small rodents and canines (and humans)
  
  • rodents and canines are reservoirs in these regions

Question 13

Leishmaniasis

transmission

anthroponotic

Answer 13

• Indian subcontinent
• more people packed in so tend to have human-human transmission
• can still go through zoonotic cycle but in this region more associated with urban environment
• other methods of human to human transmissions:
  
  • blood transfusion
  • organ/bone marrow transplants
  • congenital
  • drug usage (needle sharing)

Question 14

Leishmaniasis

causative agent

Answer 14

• 21 different Leishmania species can infect humans
  
  • L. donovani
  • L. infantum
  • L. braziliensis
  • L. major
• some species are synonymous
  
  • L. infantum = L. chagasi (Old World vs New world)
    
    • old world anmes don’t tend to name location
• morphologically indistinguishable
  
  • hard to definitively diagnose because identical
  • hard to treat
• molecularly distinct
  
  • PCR
  • isoenzyme analysis
  • monoclonal antibodies

Question 15

Leishmaniasis

major forms of the parasite

(list)

Answer 15

• promastigote
  
  • non-infectious promastigotes
  • infectious metacycic promastigotes
• amastigote

Question 16

Leishmaniasis

major forms of the parasite

promastigote

Answer 16

• 15-20 μm in length
• flagellated
• motile
• common morphology in insect host
  
  • 2 types of promastigote form, both arise in insect vector
  • flagellum pocket - anterior of nucleus
  • flagellum - not attached to cell body like in epimastigote or trypomastigote
    
    • sticks straight out from body
  • kinetoplast - located in front of nucleus, near anterior end of cell body

promastigote not in T. brucei or T. cruzi

Question 17

Leishmaniasis

causative agent

2 types of promastigote

Answer 17

non-infectious promastigotes

• insect gut
• able to divide by binary fission

infectious metacyclic promastigotes

• attach/invade neutrophils/macrophages
• non-dividing (mechanism from going insect to mammalian host)
• inside the gut the non-infectious promastigote becomes infectious metacyclic promastigote
• metacyclic - form of the parasite that goes insect to human
  
  • generally true for most

Question 18

Leishmaniasis

major form of the parasite

amastigote

Answer 18

• 2-4 μm in diameter
• stumpy flagellated
• non-motile
• intracellular form
  
  • in phagolysosome of immune cells
• divides by binary fission
  
  • replicates within neutrophils and macrophages
    
    • in macrophage usually within vesicular compartment - membrane around them derived from host
    • can be transmitted back into the insect vector or in new neutrophils/macrophages
• many (500+) parasites in 1 host cell
• infectious - can invade macrophages
  
  • transmissable from humans to humans
• flagellum pocket - anterior of nucleus
• flagellum - very short (doesn’t normally extend beyond the cell body), projecting only slightly beyond flagellum pocket
• kinetoplast - located in front of the nucleus, near anterior endo f cell body

Question 19

Leishmaniasis
life cycle

(picture)

Answer 19

Question 20

Leishmaniasis

life cycle 1

picture

Answer 20

Question 21

Leishmaniasis

life cycle 1

Answer 21

• sand fly takes a blood meal
• metacyclic promastigotes regurgitated into victim’s bloodstream (pool of fluid it’s feeding from from bite)
  
  • regurgitation caused by the parasite…
• infected sandflies have dysfunctional stomodeal valve at foregut/midgut
  
  • valve that acts as boundary between foregut and midgut of insect vector’s digestive tract
• junction → damage caused by parasite
  
  • parasite in midgut, damages the valve between the 2
• valve can’t close → blood moves back into mouthpiece and wound
• when the insect vector sucks up blood/lymph that goes down the digestive tract, when it stops at the valve it goes back up, washing the parasite into the wound
• reflux reaction → expulsion of parasite from anterior of gut into bite wound
• exacerbated as parasite secretes promastigote secretory gel → physical barrier to fly feeding
  
  • gel acts as bung - promotes insect vector to suck and doesn’t feel as though it’s feeding
  • not much blood/lymph getting past this gel
    
    • getting more food than it thinkgs, so continues feeding
    • greater chance parasite going back into pool that insect vector is feeding on
• secreted gel (and insect saliva) may potentiate fly infectivity
  
  • gel and saliva contain elements that our immune systems will respond to
    
    • immune system responds to physical damage from cut, also gel and insect saliva → magnifies attraction at that site
• caused b promastigote’s secreted gel plus physical wound etc. = causes neutrophils to go to an area
  
  • the parasite invasdes the nuetrophils
    
    • always through neutrophil first, aters expression of neutrophil genes
    • does this to promote itself

Question 22

Leishmaniasis

life cycle 1.5

Answer 22

• metacyclic promastigote phagocytized by host neutrophils
• infectious form taken up by neutrophil
• use neutrophil to get into macrophages - 2 hypotheses​
  
  • both hypotheses lead to amastigote forms in macrophages
    
    • amastigotes multiply in macrophages
    • macrophages rupture, releasing amastigotes into bloodstream to continue cycle (infect new macrophages)
    • makes a long-lasting infection

Question 23

Leishmaniasis

life cycle 1.5

trojan horse hypothesis

Answer 23

trojan horse hypothesis (in L. major)

• infected neutrophils undergo apoptosis
• neutrophil apoptotic markers onto surface of neutrophils
• apoptotic cells phagocytosed by macrophages
• metacyclic promastigote cargo delivered into macrophages
  
  • in addition to taking up dying neutrophil also takes up metacyclic promastigotes
• once within the macrophage differentiates into amastigotes

Question 24

Leishmaniasis

life cycle 1.5

trojan horse hypothesis

(picture)

Answer 24

Question 25

Leishmaniasis

life cycle 1.5

hypothesis 2

Answer 25

• neutrophil becomes in fected
• metacyclic promastigote changes to amastigote which multiplies
• causes neutrophil to burst
• amastigotes into blood/lymph taken up by neutrophils
• from one cell type (doesn’t pack but only amplifies a few times) and neutrophil lyses to release amastigotes which are then taken up

Question 26

Leishmaniasis

life cycle 1.5

hypothesis 2

(picture)

Answer 26

Question 27

Leishmaniasis

life cycle 2

picture

Answer 27

Question 28

Leishmaniasis

life cycle 2

Answer 28

• back into the insect host
• sandfly takes a blood meal from an infected mammal, ingests macrophages with infected amastigotes
• macrophages rupture in insect vector gut
  
  • into midgut macrophage is digested and ruptures
  • releasing parasite amastigotes into the intracellular environment/lumen

Question 29

Leishmaniasis

life cycle 3

picture

Answer 29

Question 30

Leishmaniasis

life cycle 3

Answer 30

• in the midgut the amastigotes transform into non-infectious nondividing promastigotes
  
  • from decrease in temperature, increase in pH
• initally parasite is surrounded by mucus layer from the insect itself
  
  • parasite is trapped in this meshwork (peritrophic matrix)
    
    • peritrophic matrix = chitin and protein mesh secreted by the midgut epithelium that encloses the blood being digested
• various enzymes produced by insect and parasite will degrade away the matrix and release the noninfectious promastigote into the lumen
  
  • host and parasite chitinase
  • after a few days the parasite escapes from peritrophic matrix
• released parasite move toward anterior of midgut
  
  • attach to microvilli of insect epithelial cells via lipophosphoglycans (LPG) to prevent excretion in feces
  • the noninfectious promastigote then fires LPG that sticks to the epithelial layer of the gut wall
    
    • prevents the parasite being excreted

Question 31

Leishmaniasis

life cycle 4

picture

Answer 31

Question 32

Leishmaniasis

life cycle 4

Answer 32

• the promastigotes divide in the anterior midgut
• produce promastigote secretory gel
• some promastigotes form metacyclic promastigotes (infective)
• detach from midgut wall (LPG changes)

Question 33

Leishmaniasis

cell invasion

(general)

Answer 33

• Leishmania species are obligate intracellular parasites in the mammalian host
• infects cells with microbicidal capability - macrophages
• how do the parasites:
  
  • avoid instant killing?
  • replicate in a hostile environment?
  • establish chronic infections?
• have to overcome immune insults that our body will mount against infection
• may use immune systems to gain entry to mammalian cells

Question 34

Innate immune mechanisms

Answer 34

• complement-mediated lysis
• lysosomal pH
• lysosomal enzymes
• oxidative stress

Question 35

Innate immune mechanisms

complement-mediated lysis

picture

Answer 35

Question 36

Innate immune mechanisms

complement-mediated lysis

Answer 36

• /alternative activation pathway
• binding of early complement components to organism is followed by assembly of membrane attack complex (C5-C9)
• use this to get into the cell - so we can exploit this
• Leishmania parasites exploit this cascade to get into the parasite
• can overcome this and utilize it at the same time

Question 37

Innate immune mechanisms

lysosomal pH

picture

Answer 37

Question 38

Innate immune mechanisms

lysosomal pH

Answer 38

• phagosomal compartment is acified upon fusion with lysosomes to pH5
• microbes in a phagosome (a vacuole formed around a particle)
• once within the macrophage or neutrophil, must overcome the cellular mechanisms of those particular cell lines to destroy infectious organisms
• in neutrophil/macrophage vacular compartment becomes acidified
  
  • this is usually sufficient to damage membrane of infectious agent
  • downshift in pH
  • parasites use this to recognize the switch required in the life cycle
• these pockets are targeted by degradative enzymes
  
  • parasite resistant to all of these

Question 39

Innate immune mechanisms

lysosomal enzymes

Answer 39

• lysosomes contain an array of digestive enzymes including:
  
  • proteases
  • nucleases
  • lipases
• pockets (phagosomes) targeted by degradative enzymes
  
  • parasite resistant to all of these

Question 40

Innate immune mechanisms

oxidative stress

Answer 40

mechanisms by which our cells generate oxidative stress

• NADPH oxidase (Phox) complex on phagosomal membrane generates superoxide
  
  • leads to production of hydrogen peroxide and hypochlorite
  • breaks down to stuff iwth antimicrobial activities
• reactions with transition metals (eg iron) lead to the formation of hydroxyl radicals
• nitric oxide synthase generates nitric oxide which combines with superoxide to form peroxynitrite
  
  • this vacuole is targeted for something that makes nitric oxide with antimicrobial expression
    
    • these are amplified when come with superoxide to make something very damaging

parasite must overcome all of these systems

Question 41

The players of Leishmania infection

infective

Answer 41

metacyclic promastigotes

Question 42

The players of Leishmania infection

host target cells

Answer 42

neutrophils, then macrophages

Question 43

The players of Leishmania infection

mode of entry

Answer 43

• passive, opsonized phagocytosis
• doesn’t require energy
• opsonized - something that macrophages/neutrophils find attractive

Question 44

The players of Leishmania infection

host receptors

Answer 44

• CR1
• CR3

(complement receptors)

Question 45

The players of Leishmania infection

parasite molecules

Answer 45

• gp63 protease
  
  • zinc protease
• lipophosphoglycan (LPG)

Question 46

The players of Leishmania infection

site of replication

Answer 46

phagolysosome vacuole

Question 47

Infection involves

(2 ~steps)

Answer 47

• complement evasion
• host cell invasion

Question 48

Complement evasion

parasite surface

Answer 48

parasite surface changes during differentiation

replicative promastigote (non-infective)

• short LPG
• low level of GP64

metacyclic promastigote (infective)

• long LPG
• high level of GP63

Question 49

Complement evasion

LPG

Answer 49

• lipophosphoglycan
• sugar - fat phospho sugar mix
• coat all over the cell surface
• 3-5 million copies per cell
• contains
  
  • oligosaccharide cap
    
    • toward external environment
  • phosphoglycan region = trunk, can have branches
    
    • galactose-mannose-phosphate
    • side chains
    • galactosyl in replicative proastigotes
  • arabinosyl then caps then galactosyl in infective promastigotes
• galactosyl side chain binds to galectin
  
  • binds parasite to wall of insect gut
• capping prevents binding to galectin, facilitating detachment from wall of midgut
• replicative promstigote with short LPG side chains always have galacto side chain that can bind to galectin on epithelial layer of insect vector (galactose interacts with galectin, lets parasites stick to wall)
• into infectious metacyclic form - galactose is capped (sugar on the end) by arabinosyl that’s sufficient to inhibit the interaction of LPG with the galectin, releasing metacyclic off the epithelial layer

Question 50

LPG picture

Answer 50

Question 51

Answer 51

LPG

• pink = inside cell
• green dots = phosphoglycan
  
  • can have disc branches

Question 52

LPG picture - detailed

Answer 52

Question 53

Complement evasion

LPG - continued

difference between noninfectious promatigotes and

non-replicative infectious forms

Answer 53

• all linked by GPI anchor
  
  • way of packing molecules very close together on the surface
• apart from arabinosyl and galactose side chains, main difference is size
  
  • noninfectious promastigotes have short galactose mannose phosphate repeat sequences
  • significantly longer in non-replicative infectious forms
  • LPGs longer in infectious forms - more repeats

Question 54

Answer 54

• blue is more sugars

Question 55

Cutaneous leishmaniasis

pathology

Answer 55

• most common manifestation - 10 million cases
• doesn’t usually progress beyond this
• known as:
  
  • orient/bay sore
  • uta
  • Baghdad/Dehli/Kandahar/Lahore boil
  • city boil
• L. tropica in cities
• L. major in rural
• starts as raised, generally painless red lesion at site of bite
• can ulcerate/break the skin
• small spot to progressively larger lesion, eventually immune system will kick in and wipe it out

Question 56

Cutaneous Leishmaniasis

ulcer classified as wet or dry (crust formation)

Answer 56

• wet ulcer = L. major
  
  • ​remains open
• dry ulcer = L. tropica
• no pus formation unless get bacterial infection
• ulcer self heals
  
  • causes scarring
  • eg L. major
  • in about 6 months
  • usually treatment not provided because will heal on oyur own
    
    • drugs saved for more severe form of the disease
  • if face bitten will be given the drug to speed up healing
• once healed, individuals immune for life
  
  • once moutned appropriate immune response will be immune to that particular species
• in some individuals diffuse lesions develop
  
  • wet ulcer may disappear, reappear on different parts of the body later in time

Question 57

Diffusive Leishmaniasis

pathology

Answer 57

2 types

• diffuse cutaneous leishmaniasis (DCL)
• mucocutaneous leishmaniasis (MCL)

Question 58

Diffusive Leishmaniasis

diffuse cutaneous leishmaniasis (DCL)

Answer 58

• metastasizes over the whole body
• non-ulcerated, scaly lesions (nodular)
• generally associated with anergy (lack of immune response)
• amastigotes abundant in lesions
  
  • packed with macrophages and amastigotes
• eg L. aethiopica, L. mexicana
• can appear anywhere on the body

Question 59

Diffusive Leishmaniasis

mucocutaneous leishmaniasis (MCL)

Answer 59

• primarily L. braziliensis (3-5% of cases move from cutaneous to this kind of form)
  
  • most prevalent in Latin America
• metastasizes mucocutaneous junctoins
  
  • mouth/nose/soft palate or anus/genitalia
• can occur weeks/years after first degree infection
• tissue destroyed → severe disfigurement
• seconary bacterial infections common
• may affect eg breathing, prone to secondary infections

Question 60

Visceral Leishmaniasis

pathology

Answer 60

• starts as cutaneous lesion to visceral form
• known as
  
  • Kala-azar
  • DumDum fever
• most serious form
• 200-500,000 cases/50,000 deaths per year
• typically caused by
  
  • L. donovani (India/Pakistan)
  • L. infantum (Mediterranean) = L. chagasi (Latin America)
• high fatality rate
• darkening of the skin
• others highly disfiguring or may lead to death through secondary infection, but are not lethal itself

Question 61

Visceral Leishmaniasis

symptoms

Answer 61

• generalized infection of the reticuloendothelial system
• involves liver, spleen, bone marrow, lymph nodes
• characterized by
  
  • fever (sudden or gradually rising)
  • splenomegaly (enlargement of spleen)
  • hepatomegaly (enlargement of the liver)
  • enlarged lymph nodes/tonsils (→ anemia, leucopenia, thrombocytopenia)
• disease progression leads to malaise, tiredness, lassitude, weaness
• patient exhibits wasting syndrome despite good appetite
  
  • muscle wastage - musculature doesn’t develop to the same degree, remains extremely thin
• secondary infections are common
• kills by organ failure
  
  • ​affects organs associated with eg clearing blood and helping immune system = prone to secondar infections
    
    • pneumonia
    • tuberculosis
    • dysentery
• 50,000 deaths directly attributed to Leishmania
  
  • but if have visceral Leishmaniasis will die of secondary infection and won’t be attributed to Leishmania (which weakens the immune system)

Question 62

Leishmaniasis

disease severity depends on

Answer 62

immune status of the individual

Question 63

Th1-type response

Answer 63

• cellular immune response triggered - no/low antibody formation
• recover from infection, are immune (cutaneous)

Question 64

Th2-type response

Answer 64

• humoral immune response triggered, antibody formation occurs
• succumb to infection (visceral)
• Th2 by mistake = develops to more serious forms
• antibodies → succumb, more severe forms
• some of the more dangerous and highly infectious strains appear to be able to switch host’s immune response from something that will clear the parasitemia to something that will enhance the parasitemia
  
  • some strong L. donovani strains force host to switch from Th1 to Th2 response (mechanisms unknown)

Question 65

Leishmaniasis

diagnosis

Answer 65

1. epidemiological history
2. symptoms
3. definitive diagnosis - detection of parasites, seriology

Question 66

Leishmaniasis

diagnosis

epidemiological history

Answer 66

dermal and visceral both

• suspected
• geographical presence of parasite
• history of sandfly bite

Question 67

Leishmaniasis

diagnosis

symptoms - dermal

Answer 67

• chronic, painless lesion (CL)
• mucocutaneous lesion (MCL)
• scaly lesions (DCL)

Question 68

Leishmaniasis

diagnosis

symptoms - visceral

Answer 68

• prolonged fever
• splenomegaly
• hepatomegaly
• anemia
• leucopenia
• thrombocytopenia

Question 69

Leishmaniasis

diagnosis

definitive diagnosis - detection of parasites

dermal

Answer 69

amastigotes in scraping

• CL - incision of lesion
• dermal scraping examined by Giemsa staining/microscopy

amastigotes in aspirates

• CL - injectio saline in lesion
• examine aspirate following giemsa staining/microscopy

in vitro culturing of promastigotes from aspirates

• CL - injection saline in lesion
• transfer to tissue culture medium
• monitor for promastigote growth (~1 week)
• amastigote parasites break out in tissue culture of macrophage and spontaneously generate the promastigote form (grow out the form that grows in insect vector, but takes time)

inoculation of aspirates in model organism

• CL - injection saline in lesion
• transfer to model organism (hamster)
• monitor for clinical pathology (2-3 months)

Question 70

Leishmaniasis

diagnosis

definitive diagnosis - detection of parasites

visceral

Answer 70

amastigotes in bone marrow, aspirates, or biopsy

• CL- injection saline in lesion
• examine aspirate following Giemsa staining/microscopy

in vitro culturing of promastigotes from aspirates

• CL - injection saline in lesion
• transfer to tissue culture medium
• monitor for promastigote growth (~1 week)

Question 71

Leishmaniasis

diagnosis

definitive diagnosis - serology

dermal

Answer 71

Leishmanin skin test

• looks at person’s response to fixed parasites (dead) or parasitse in extracts
• intradermal injection of promastigotes
  
  • extracts or whole, killed
• monitor for local dealyed hypersensitivity (local redness)
• development of erythema if leish +ve
  
  • if some time in the past the person was infected - parsite may be wiped out but still be responding as if the parasite was there

Question 72

Leishmaniasis

diagnosis

definitive diagnosis - serology

visceral

Answer 72

serological test (direct agglutination, K39 dipstick)

direct agglutination

• fixed coloured parasites + patient serum
  
  • = anti-Leishmania antibodies → agglutinate fixed coloured parasites = blue/purple
  • (react with colored parasites to agglutinate them)

K39 dipstick

• patient serum applied to immunochromatographic dipstick
• dipstick contains antigen to K39, abundant amastigote surface antigen
  
  • K39 is a 39 amino acid repeat sequence
• K39 antigen on strip, let patient’s serum migrate
• if immune response will bind to K39 antigen and change color
• patient serum reacts to K39 if contains anti-Leishmania antibodies
• in figure lower band = control, 2 bands appear = infection

Question 73

Front line drugs against Leishmaniasis

pentavalent antimonial

Answer 73

drugs based on antimony - can draw comparisons with the arsenic-based drug for HAT

sodium stibogluconate (Pentostam) → GSK

meglumine antimoniate (Glucantime) → Sanofi S.A

• pro-drug
• administered in inactive state (pentavalent)
• activated (to trivalent state) only by amastigote
• introduced in 1945
• daily intravenous infusion (Pentostam) or intramuscular injections (Glucantime)
• 20mg/kg/day fro 10 days (CL)
• 20mg/kg/day for 30 days (VL/DCL/MCL)

Question 74

Front line drugs against Leishmaniasis

pentavalent antimonial

variation in species sensitivity

Answer 74

not all Leishmania species are the same

• those that cause the more severe form are more susceptible to these pentavalent antimonials
• L. donovani >*
• L. braziliensis* >
• L. mexicana* >
• L. major*

VL species > DCL/MCL species > CL species

Question 75

Front line drugs against Leishmaniasis

pentavalent antimonial

problems

Answer 75

high level of toxicity

• fatigue, nausea, muscle/joint pain, changes in ECG, cardiotoxicity, hepatic/renal dysfunction, shock, and (rarely) sudden death
• haevy metal = heavy metal poisining
• similar problems to melarsoprol

don’t always work

• may fail to clear parasitemia
• resistance in field
  
  • esp in India

Question 76

Front line drugs against Leishmaniasis

pentavalent antimonial

activation and uptake

Answer 76

• activation can occur inside/outside cell
• both Sb^V and Sb^III can be taken up by cell
  
  • prodrug = Sb^V
  • drug = Sb^III
• conversion from prodrug (pentavalent) to drug (trivalent) can only occur if amastigotes present
• don’t know where conversion occurs
  
  • surface of amastigote before drug goes into the cell
  • within the cell itself
  • could be both
• if activated outside know pentavalent can be taken in, as can trivalent

Question 77

Pentavalent antimonial

uptake

Answer 77

• Sb^III taken up by AQP (aquaglyceroporin)
  
  • same with melarsoprol
  • resistance toward antimonials have been associated with reduced uptake of trivalent form by AQP
• Sb^V - don’t know what’s used for uptake

Question 78

Pentavalent antimonials

Conversion/activation

Answer 78

• conversion outside pentavalent → trivalent
  
  • don’t know what mediates it
• Sb^v → Sb^III inside by reductases
  
  • ACR2 - arsenite reductase
  • TDR1 - thiol-dependent reductase

Question 79

Pentavalent antimonials

binding

Answer 79

• trivalent form binds conjugate agents - especially thiols
• trivalent form binds trypanothione, inhibits trypanothione-dependent enzymes
  
  • eg trypanothione reductase
  • which has major implications how these parasites:
    
    • respond to oxidative stress
    • synthesize DNA
  • heavy metal works the same as melarsoprol - sticks to thiols, messes up downstream pathways

Question 80

Resistance to antimonials

Answer 80

• 1980s antimonials resistance observed
• today 50-65% of VL cases in Bihar
• antimonials obsolete in India
• why?
  
  • unrestricted availability
  • incorrect doses
  • inconsistent quality
    
    • cut with other substances
    • may not be up to required therapeutic doses
• mechanism of resistance in the field is unknown
• lab approaches used to dissect pathways
  
  • resistance by parasites can be readily generated in the lab by growin cells on sub-lethal levels of the drug

Question 81

Resistance to antimonials

Amplification

Answer 81

• Sb^R Leishmania amplified specific genetic loci from genome → episome
  
  • amplify part of genome, throw it out to generate a plasmid
  • amplify up to give high copy number
  • very plastic genome
• sequence/mapping/biochemical studies shown that episome contains ABC transporter
  
  • localized to unknown organelle that’s very close to the flagella pocket
• in mammals ABC transporters involved in
  
  • multidrug resistance
  • heavy metal resistance
• Leishmania ABC transporter designated as MPRA (multipdrug resistance protein A)
• previously shown to function as metal efflux pump that recognizes metals conjugated to thiols
  
  • MRPA pumps metal-thiol conjugates into this organelle
  • trypanothione conjugates the antimonial, pumped via this mechanism into the uknown organelle
• overexpression in parasites causes resistance
  
  • higher level of MRPA leads to resistance to antimonials
  • can knock gene out - not essential to parasite genome, makes parasite more sensitive to the drug

Question 82

Resistance to antimonials

MRPA localization

Answer 82

MRPA localized to unknown organelle near flagella pocket

biochemical/functional analysis shows that MRPA

1. transports metals conjugated to thiols
2. over-expression causes resistance to antimonials
3. null mutants are sensitive to antimonials

Question 83

Resistance to antimonials

via sequestration

Answer 83

resistance to antimonials is via sequestratoin of trypanothione-drug conjugate

Question 84

Resistance to antimonials via sequestration of trypanothione-drug conjugate

antimonial-sensitive parasites

Answer 84

• pentavalent to trivalent form
• trivalent form interacts with trypanothil (TS) to form metal-thiol conjugate to hit the target enzymes (trypanothione-dependent enzymes)

Question 85

Resistance to antimonials via sequestration of trypanothione-drug conjugate

antimonial resistant parasites

Answer 85

in resistant cell lines

• get diversion away from the target
• because you amplify up MRPA gene - increases the amount of transporter such that the metal-thil conjugate isn’t getting to the target but is pumped out into the unknown organelle
• rate-limiting step is amount of TS2 (trypanothione)
• so is the level of trypanothione affected in resistant cell lines?

Question 86

Sb^R parasites

levels of trypanothione

Answer 86

• resistant parasites have elevated levels of trypanothione
  
  • Halmeur et al., 2002
• amplification of trypanothione synthesis genes
  
  • Grondin et al., 1997
  • Haimeur et al., 1999
• inhibitors of trypanothione synthesis (buthionine sulfoxice/eflornithine) can reverse resistance
  
  • Grondin et al., 1997
  • Haimeur et al., 1999
• high levels of trypanothione, enzymes in trypanothine synthesis amplified
• make more TS2 to bind out the drug
• use inhibitors that block synthesis of trypanothione to make cells sensitive to antimonial
• as well as upregulating transporter, upregulating trypanothione biosynthesis pathway to bind out lots of antimonial that can be cleared out of the cell into this organelle

Question 87

Resistance to antimonials via

Answer 87

sequestration of trypanothione-drug conjugate

AND

increased trypanothione synthesis

Question 88

Pentavalent antimonials

uptake and conversion

(picture)

Answer 88

Question 89

Antimonial sensitive parasites

(picture)

Answer 89

Question 90

Antimonial resistant parasites

(picture)

Answer 90

Question 91

Alternative drugs against Leishmaniasis

Answer 91

• amphotericin B
• miltefosine
• paromomycin

Question 92

Alternative drugs against Leishmaniasis

Amphotericin B

Answer 92

• polyene antibiotic
• originally developed as antifungal
• effective against antimonial resistant VL
• infusion, severe toxic side effects
• lipid formulation (AmBisome) have reduced toxicity
  
  • mycels = lipid globules into which you can insert drug = reduced toxicity
• amphotericin B binds to 24-substituted sterols (eg ergosterol) disrupting osmotic integrity of the membrane
  
  • binds sterols in parasite cell membrane = punches holes at point of sterol to make parasite cell leaky

Question 93

Alternative drugs against Leishmaniasis

miltefosine

Answer 93

• phosphocholine analogue
• developed in 1980-90s as anticancer drug, abandoned due to toxicity problems
• activity against Leishmania in vitro and in vivo (1987-1996)
• daily, oral administration for 28 days
• clinical trials for VL in Bihar State, India, >95% cure, including Sb(V) resistant cases
  
  • Registered in India & launched in 2003
• registered for VL/CL in Colombia, 90% cure: clinical trials in Brazil
• BUT it is teratogenic (causes malformations in embryos), cannot be used by women of child-bearing age
• mode of action – uncertain, possibly by inhibiting phospholipid formation, signal transduction or calcium homeostasis

Question 94

Alternative drugs against Leishmaniasis

paromomycin

Answer 94

• aminoglycosides antibiotic (related to kanamycin, neomycin)
• developed as antibacterial, used to treat Entamoeba and Cryptosporidium infections
• anti-leishmania activity reported in 1960s
• daily, oral administration for 20 days
• phase 2 trials for VL in India/Kenya, ~90% cure
• phase 3 trials ongoing/pharmaceutical manufacturer secured
• mode of action – uncertain. Possible effects in mitochondrion?

Question 95

Leishmaniasis

prevention and control

Answer 95

vector control

• insecticides and repellants
• screens and bednets
• difficult to carry out due to:
  
  • cost
  • re-emergence after cessation of spraying programs
  • diversity of epidemiology (rural vs urban)

control of reservoir species

• canine vaccines
• culling
• Mediterranean basin and Latin America
• try to control flies
• requires money and political will = problem