APPLIED MEDICAL BIOTECHNOLOGY: Course part 1
Chapter 1: malaria as an infectious human disease (world-wide)
1. Introduction: the disease Malaria
• Malaria worldwide
o = caused by protozoan parasites of the Plasmodium genus (~120 species)
o = most important infectious disease worldwide (number of malaria cases & deaths are huge (S2))
o 5 species infect humans
▪ * P. falciparum: most prevalent, causes most severe disease
▪ * P. vivax: prevalent outside Africa
▪ * P. malariae: less frequent
▪ * P. ovale: less frequent
▪ * P. knowlesi: parasite infecting monkeys and then jumps over to humans = zoonotic disease
o Who? → Mainly children, also pregnant women (and travelers) are at high risk
o Where? → Sub-Saharan Africa (= a tropical region):
▪ ~92% of all malaria cases
▪ ~20-35% of the “under-five” mortality
• → every 2 minutes, a child dies from malaria
▪ ~1,3% of gross domestic product of whole Africa
• Malaria symptoms
o 1) Severe fever peaks (systemic) every
▪ 36-48 hours (P. falciparum)
▪ 48 hours (P. vivax, P. ovale)
▪ 72 hours (P. malariae)
▪ = symptoms of uncomplicated malaria (no death)
o 2) Flu-like symptoms
▪ Central: headache
▪ Muscular: fatigue & pain
▪ Back: pain
▪ Skin: chills & sweating
▪ Respiratory: dry cough
▪ Spleen: enlargement
▪ Stomach: nausea, vomiting
▪ = symptoms of uncomplicated malaria (no death)
o 3) immunopathologies and complications: see further
▪ = symptoms of complicated malaria (risk of death)
• Malaria symptoms: cause of fever
o Parasite infects RBC → Multiplies in RBC → RBC bursts → parasitic molecules flow into the
bloodstream that can cause fever (fever peak = burst)
o → All parasites of P. vivax, P. malariae burst simultaneously → regular pattern of fever
o → Parasites of P. Falciparum burst not simultaneously → irregular pattern of fever
• Diagnosis:
o 1) By microscope of a thin blood smear or thick blood smear + staining
▪ Thin smear: visualize individual RBC infected with malaria
▪ Thick smear: can’t visualize individual RBC
▪ → morphology of the parasite can be used to discriminate between species (P. falciparum,
P. vivax, P. ovale, P. malariae)
o 2) PCR (expensive)
o 3) Rapid diagnostic test or antibodies
1
, ▪
= based on the detection of malaria antigen with antibodies
▪
1) Buffer (flushing agent) + parasitized blood + labelled Ab that catches parasite antigens →
run along the nitrocellulose strip
▪ 2) Formation of labelled Ab-Ag complex captured by bound AB of test band (if parasite is
present)
▪ 3) Formation of labelled Ab captured by bound AB of control band
• Diagnosis: Notes
o RDTs
▪ Lower sensitivity than thick smear microscopy
▪ But faster & more easy
o qPCR methods
▪ More sensitive
▪ But less practical
o Histidine-rich protein-2 (HRP-2) often used as malaria antigen for RDT
▪ but: increasing prevalence of HRP-2-deficient parasites
▪ → escape from diagnosis (and thus treatment)!
▪ → solution: use of other antigens
• Plasmodium parasites
o = protozoan parasites: unicellular, eukaryotic, no cell wall yeast: unicellular, eukaryotic, cellwall
o = part of Apicomplexa group: protozoan parasites with an apical complex
▪ = a complex of organelles for cell invasion (of hepatocytes, RBC,..)
o P. falciparum and P. vivax
▪ Genome has more than 5300 – 5400 genes
▪ P. falciparum: A+T content is very high (80% - 90 %)
2. Malaria lifecycle
• Malaria lifecycle: Involves 2 hosts: human & mosquito (zie verder)
• Preerythrocytic stage (hepatic / liver stage)
o Lifecycle: Human liver Stages: Exo-erythrocytic (hepatic) cycle (initial multiplication in liver)
▪ During blood meal, a malaria infected mosquito injects sporozoites into a human →
sporozoites migrate through blood to liver → infection of hepatocytes → multiplication into
schizonts containing merozoites → rupture of schizont & release of merozoites into blood
o Mosquito bite and invasion of the blood vessels
▪ 1) Mosquito bites through the skin until it bleeds (bloodvessel found) & sucks up blood
▪ 2) Mosquito injects saliva containing anti-coagulants to avoid blood clothing (& sporozoites)
o Mosquito: Anopheles species, including A. gambiae and A. funestus, can only transfer malaria
o The Sporozoite: Elongated cells with
▪ 1) Apical complex for cell invasion of hepatocytes
▪ 2) Circumsporozoite protein (CSP)
• Location: in micronemes and on surface coat
• Function: essential for motility vb migration to blood after injection in skin
• Skin -> blood
o 1) CSP produced at the tip & migrate along the sporozoite → movement of S
o 2) Then cleavage by cysteine protease posteriorly → which leaves a trail
• Blood -> liver
o CSP binds to glycosaminoglycans (“GAGs”) and LRP-1 (lipoprotein receptor-
related protein-1) in the liver (°)
• Main target for vaccination
o The Sporozoite: Invasion of hepatocytes (°)
2
, ▪ 1) Sporozoite arrives via blood in the liver & recognizes a Kupffer cell → enters Kupffer cell &
leaves at the basolateral side → to contact hepatocytes
▪ 2) The sporozoite migrates through nr of different hepatocytes until it finds a suitable
hepatocyte for multiplication into a schizont
▪ Opm: Kupffer cell = macrophage of the liver, in between endothelial cells
• Preerythrocytic stage (hepatic / liver stage)
o P. vivax and P. ovale form hypnozoites in the liver:
▪ 1) may remain dormant for many years
▪ 2) cause relapse infections by invading the bloodstreams, weeks or even years later
o Relapse ≠ recrudescence
▪ 1) Relapse (P. vivax & P. ovale): originates from hypnozoites (in the liver)
▪ 2) Recrudescence (P. falsiparum): originates from erythrocytic stage (in the RBC)
• Vb: antimalaria drugs or immune system eliminate the parasite → but a few
parasites remain in RBC circulating → continue to proliferate → 2 weeks later you
can have recrudescence & you are ill again
• Erythrocytic stage
o Lifecycle: Human blood Stages: Erythrocytic cycle (asexual multiplication in erythrocytes/RBC) (*)
▪ Merozoites infect RBC
• 1) Immature trophozoites (ring stage) → mature to mature trophozoites → mature
then into schizonts → rupture of schizont & release of merozoites → new
merozoites reinfect other RBC etc (*)
o → The blood stage parasites cause the symptoms of malaria
• 2) Some immature trophozoites (ring stage) → differentiate into gametocytes →
male and female gametocytes are ingested by a mosquito during blood meal
▪ Opm: no reinfection of other hepatocysts
• Gevolg: the exo-erythocytic cycle /hepatic cycle stays silent
o The Merozoite: resemble Sporozoite
▪ 1) Apical complex for cell invasion of RBC
▪ 2) But different shape (shorter, lemon shaped)
▪ 3) But not motile
o The Merozoite: Invasion of red blood cells
▪ 1) recognition through surface proteins
• MSP-1 (merozoite surface protein-1) → bind EB-3 (erythrocyte band 3 protein)
▪ 2) re-orientation of merzoite such that apical complex contacts RBC
• EBA-175 (erythrocyte binding antigen-175) → glycophorin A (determines ABO
bloodgroup)
▪ 3) Tight junction formation (initially 1 point, then a ring) + “motor”-driven invasion
• shedding of surface coat by a protease named SUB2
• parasites EBA-175 is connected to actin myosin system via linker proteins → like this
parasite moves inside the RBC, providing itself EN for invasion
▪ 4) Formation of a parasitophorous vacuole (= vacuole formed by RBC membrane)
▪ 5) proteolytic removal of adhesins by rhomboid
▪ 6) resealing of RBC membrane
▪ Opm: RBC cannot do endocytosis, thus the parasite does endocytosis itself in an active way
o The Merozoite: after invasion of red blood cells
▪ 1) The merozoite feeds itself on the content of the RBC vb cytosol (full of hemoglobine)
▪ 2) Hemoglobin ingestion into the food vacuole and degradation by proteases:
• Hemoglobin → large peptides + heme → small peptides → amino acids for protein
synthesis or efflux out of the parasite
3
, • Heme (containing Fe2+) is toxic for the parasite because it binds O2 → oxidative
reactions → kills parasite
• Heme (containing Fe2+) accumulation in neutral lipid nanospheres in the food
vacuole → crystallization into hemozoin crystals
o Reden: heme = toxic for the parasite because it binds O2 → oxidative
reactions → kills parasite
▪ 3) Transport of other nutrients from outside the RBC
• Vb: glucose, Ile, purine, panthotenate, phosphate
• → through native and new permeability pathways
• Erythrocytic stage
o 1) Invasion of red blood cells
o 2) Merozoite with a food vacuole with hemoglobine = Ring stage
o 3) When hemozoin is made = Trophozoite
o 4) When nucleus divide into multiple nuclei = Schizont
o 5) When nuclei segregate = Merozoite in the Schizont
o 6) When vacuole membrane degrades, schizont swells & bursts & releases merozoites and hemozoin
crystal in the circulation → pathogenesis
• Erythrocytic stage
o Outcome
▪ Parasitic clearance after infection → not ill
▪ Asymptomatic presence of parasite in blood → not ill
▪ Uncomplicated malaria → ill, but can be cured
▪ Severe complicated malaria (cerebral malaria, severe malarial anemia, placental malaria,
acidosis, ALI/ARDS, blackwater fever) → ill, often death
o The outcome is influenced by a lot of different factors:
▪ Immune evasion, immune stimulation, anti-parasite immunity, inflammation
▪ Metabolism, erythropoiesis, RBC destruction, blood flow/tissue oxygenation, vascular
integrity, (anti- coagulation,..
• Gametocytogenesis and mosquito stages
o Lifecycle: Mosquito stages: Sporogonous cycle (multiplication in the mosquito)
▪ In the mosquito’s stomach the male & female gametocytes leave RBC & mate generating a
zygote → zygote becomes motile & elongated (ookinete) → ookinete invades the midgut
wall of the mosquito where they develop into oocysts → oocyst proliferate/myosis into
haploid sporozoites → oocyst rupture & release sporozoites → sporozoites move to the
mosquito’s salivary glands → stap 1)
o Mosquito immune reaction
▪ Complement like proteins, e.g. TEP-1
▪ Function: they induce a melanisation (PO/polymerization reaction) to block parasite when its
migrating through epithelium
• Semi immunity
o People have no full immunity: always low parasite numbers remain present due to evasion of IS
o Acquired through continuous exposure (up to 5years) to the parasite
▪ Gevolg: mostly in adults of endemic region
• children (below 5y) (and tourists) are the most susceptible: first 6months
antibodies of mother after birth → then they disappear and don’t have semi-
immunity yet → children below 5y become ill → once 5y, semi-immunity rises
o Rapidly lost (e.g. in African students travelling to Europe)
• High transmission areas: central Africa → semi-immunity
• Lower transmission area: south Africa → no semi-immunity → adults also get severe malaria
4
Chapter 1: malaria as an infectious human disease (world-wide)
1. Introduction: the disease Malaria
• Malaria worldwide
o = caused by protozoan parasites of the Plasmodium genus (~120 species)
o = most important infectious disease worldwide (number of malaria cases & deaths are huge (S2))
o 5 species infect humans
▪ * P. falciparum: most prevalent, causes most severe disease
▪ * P. vivax: prevalent outside Africa
▪ * P. malariae: less frequent
▪ * P. ovale: less frequent
▪ * P. knowlesi: parasite infecting monkeys and then jumps over to humans = zoonotic disease
o Who? → Mainly children, also pregnant women (and travelers) are at high risk
o Where? → Sub-Saharan Africa (= a tropical region):
▪ ~92% of all malaria cases
▪ ~20-35% of the “under-five” mortality
• → every 2 minutes, a child dies from malaria
▪ ~1,3% of gross domestic product of whole Africa
• Malaria symptoms
o 1) Severe fever peaks (systemic) every
▪ 36-48 hours (P. falciparum)
▪ 48 hours (P. vivax, P. ovale)
▪ 72 hours (P. malariae)
▪ = symptoms of uncomplicated malaria (no death)
o 2) Flu-like symptoms
▪ Central: headache
▪ Muscular: fatigue & pain
▪ Back: pain
▪ Skin: chills & sweating
▪ Respiratory: dry cough
▪ Spleen: enlargement
▪ Stomach: nausea, vomiting
▪ = symptoms of uncomplicated malaria (no death)
o 3) immunopathologies and complications: see further
▪ = symptoms of complicated malaria (risk of death)
• Malaria symptoms: cause of fever
o Parasite infects RBC → Multiplies in RBC → RBC bursts → parasitic molecules flow into the
bloodstream that can cause fever (fever peak = burst)
o → All parasites of P. vivax, P. malariae burst simultaneously → regular pattern of fever
o → Parasites of P. Falciparum burst not simultaneously → irregular pattern of fever
• Diagnosis:
o 1) By microscope of a thin blood smear or thick blood smear + staining
▪ Thin smear: visualize individual RBC infected with malaria
▪ Thick smear: can’t visualize individual RBC
▪ → morphology of the parasite can be used to discriminate between species (P. falciparum,
P. vivax, P. ovale, P. malariae)
o 2) PCR (expensive)
o 3) Rapid diagnostic test or antibodies
1
, ▪
= based on the detection of malaria antigen with antibodies
▪
1) Buffer (flushing agent) + parasitized blood + labelled Ab that catches parasite antigens →
run along the nitrocellulose strip
▪ 2) Formation of labelled Ab-Ag complex captured by bound AB of test band (if parasite is
present)
▪ 3) Formation of labelled Ab captured by bound AB of control band
• Diagnosis: Notes
o RDTs
▪ Lower sensitivity than thick smear microscopy
▪ But faster & more easy
o qPCR methods
▪ More sensitive
▪ But less practical
o Histidine-rich protein-2 (HRP-2) often used as malaria antigen for RDT
▪ but: increasing prevalence of HRP-2-deficient parasites
▪ → escape from diagnosis (and thus treatment)!
▪ → solution: use of other antigens
• Plasmodium parasites
o = protozoan parasites: unicellular, eukaryotic, no cell wall yeast: unicellular, eukaryotic, cellwall
o = part of Apicomplexa group: protozoan parasites with an apical complex
▪ = a complex of organelles for cell invasion (of hepatocytes, RBC,..)
o P. falciparum and P. vivax
▪ Genome has more than 5300 – 5400 genes
▪ P. falciparum: A+T content is very high (80% - 90 %)
2. Malaria lifecycle
• Malaria lifecycle: Involves 2 hosts: human & mosquito (zie verder)
• Preerythrocytic stage (hepatic / liver stage)
o Lifecycle: Human liver Stages: Exo-erythrocytic (hepatic) cycle (initial multiplication in liver)
▪ During blood meal, a malaria infected mosquito injects sporozoites into a human →
sporozoites migrate through blood to liver → infection of hepatocytes → multiplication into
schizonts containing merozoites → rupture of schizont & release of merozoites into blood
o Mosquito bite and invasion of the blood vessels
▪ 1) Mosquito bites through the skin until it bleeds (bloodvessel found) & sucks up blood
▪ 2) Mosquito injects saliva containing anti-coagulants to avoid blood clothing (& sporozoites)
o Mosquito: Anopheles species, including A. gambiae and A. funestus, can only transfer malaria
o The Sporozoite: Elongated cells with
▪ 1) Apical complex for cell invasion of hepatocytes
▪ 2) Circumsporozoite protein (CSP)
• Location: in micronemes and on surface coat
• Function: essential for motility vb migration to blood after injection in skin
• Skin -> blood
o 1) CSP produced at the tip & migrate along the sporozoite → movement of S
o 2) Then cleavage by cysteine protease posteriorly → which leaves a trail
• Blood -> liver
o CSP binds to glycosaminoglycans (“GAGs”) and LRP-1 (lipoprotein receptor-
related protein-1) in the liver (°)
• Main target for vaccination
o The Sporozoite: Invasion of hepatocytes (°)
2
, ▪ 1) Sporozoite arrives via blood in the liver & recognizes a Kupffer cell → enters Kupffer cell &
leaves at the basolateral side → to contact hepatocytes
▪ 2) The sporozoite migrates through nr of different hepatocytes until it finds a suitable
hepatocyte for multiplication into a schizont
▪ Opm: Kupffer cell = macrophage of the liver, in between endothelial cells
• Preerythrocytic stage (hepatic / liver stage)
o P. vivax and P. ovale form hypnozoites in the liver:
▪ 1) may remain dormant for many years
▪ 2) cause relapse infections by invading the bloodstreams, weeks or even years later
o Relapse ≠ recrudescence
▪ 1) Relapse (P. vivax & P. ovale): originates from hypnozoites (in the liver)
▪ 2) Recrudescence (P. falsiparum): originates from erythrocytic stage (in the RBC)
• Vb: antimalaria drugs or immune system eliminate the parasite → but a few
parasites remain in RBC circulating → continue to proliferate → 2 weeks later you
can have recrudescence & you are ill again
• Erythrocytic stage
o Lifecycle: Human blood Stages: Erythrocytic cycle (asexual multiplication in erythrocytes/RBC) (*)
▪ Merozoites infect RBC
• 1) Immature trophozoites (ring stage) → mature to mature trophozoites → mature
then into schizonts → rupture of schizont & release of merozoites → new
merozoites reinfect other RBC etc (*)
o → The blood stage parasites cause the symptoms of malaria
• 2) Some immature trophozoites (ring stage) → differentiate into gametocytes →
male and female gametocytes are ingested by a mosquito during blood meal
▪ Opm: no reinfection of other hepatocysts
• Gevolg: the exo-erythocytic cycle /hepatic cycle stays silent
o The Merozoite: resemble Sporozoite
▪ 1) Apical complex for cell invasion of RBC
▪ 2) But different shape (shorter, lemon shaped)
▪ 3) But not motile
o The Merozoite: Invasion of red blood cells
▪ 1) recognition through surface proteins
• MSP-1 (merozoite surface protein-1) → bind EB-3 (erythrocyte band 3 protein)
▪ 2) re-orientation of merzoite such that apical complex contacts RBC
• EBA-175 (erythrocyte binding antigen-175) → glycophorin A (determines ABO
bloodgroup)
▪ 3) Tight junction formation (initially 1 point, then a ring) + “motor”-driven invasion
• shedding of surface coat by a protease named SUB2
• parasites EBA-175 is connected to actin myosin system via linker proteins → like this
parasite moves inside the RBC, providing itself EN for invasion
▪ 4) Formation of a parasitophorous vacuole (= vacuole formed by RBC membrane)
▪ 5) proteolytic removal of adhesins by rhomboid
▪ 6) resealing of RBC membrane
▪ Opm: RBC cannot do endocytosis, thus the parasite does endocytosis itself in an active way
o The Merozoite: after invasion of red blood cells
▪ 1) The merozoite feeds itself on the content of the RBC vb cytosol (full of hemoglobine)
▪ 2) Hemoglobin ingestion into the food vacuole and degradation by proteases:
• Hemoglobin → large peptides + heme → small peptides → amino acids for protein
synthesis or efflux out of the parasite
3
, • Heme (containing Fe2+) is toxic for the parasite because it binds O2 → oxidative
reactions → kills parasite
• Heme (containing Fe2+) accumulation in neutral lipid nanospheres in the food
vacuole → crystallization into hemozoin crystals
o Reden: heme = toxic for the parasite because it binds O2 → oxidative
reactions → kills parasite
▪ 3) Transport of other nutrients from outside the RBC
• Vb: glucose, Ile, purine, panthotenate, phosphate
• → through native and new permeability pathways
• Erythrocytic stage
o 1) Invasion of red blood cells
o 2) Merozoite with a food vacuole with hemoglobine = Ring stage
o 3) When hemozoin is made = Trophozoite
o 4) When nucleus divide into multiple nuclei = Schizont
o 5) When nuclei segregate = Merozoite in the Schizont
o 6) When vacuole membrane degrades, schizont swells & bursts & releases merozoites and hemozoin
crystal in the circulation → pathogenesis
• Erythrocytic stage
o Outcome
▪ Parasitic clearance after infection → not ill
▪ Asymptomatic presence of parasite in blood → not ill
▪ Uncomplicated malaria → ill, but can be cured
▪ Severe complicated malaria (cerebral malaria, severe malarial anemia, placental malaria,
acidosis, ALI/ARDS, blackwater fever) → ill, often death
o The outcome is influenced by a lot of different factors:
▪ Immune evasion, immune stimulation, anti-parasite immunity, inflammation
▪ Metabolism, erythropoiesis, RBC destruction, blood flow/tissue oxygenation, vascular
integrity, (anti- coagulation,..
• Gametocytogenesis and mosquito stages
o Lifecycle: Mosquito stages: Sporogonous cycle (multiplication in the mosquito)
▪ In the mosquito’s stomach the male & female gametocytes leave RBC & mate generating a
zygote → zygote becomes motile & elongated (ookinete) → ookinete invades the midgut
wall of the mosquito where they develop into oocysts → oocyst proliferate/myosis into
haploid sporozoites → oocyst rupture & release sporozoites → sporozoites move to the
mosquito’s salivary glands → stap 1)
o Mosquito immune reaction
▪ Complement like proteins, e.g. TEP-1
▪ Function: they induce a melanisation (PO/polymerization reaction) to block parasite when its
migrating through epithelium
• Semi immunity
o People have no full immunity: always low parasite numbers remain present due to evasion of IS
o Acquired through continuous exposure (up to 5years) to the parasite
▪ Gevolg: mostly in adults of endemic region
• children (below 5y) (and tourists) are the most susceptible: first 6months
antibodies of mother after birth → then they disappear and don’t have semi-
immunity yet → children below 5y become ill → once 5y, semi-immunity rises
o Rapidly lost (e.g. in African students travelling to Europe)
• High transmission areas: central Africa → semi-immunity
• Lower transmission area: south Africa → no semi-immunity → adults also get severe malaria
4
