Malaria-Mission Impossible

DEFINITION

AETIOLOGY & TAXONOMY

EPIDEMIOLOGY

LIFE CYCLE

MORPHOLOGY

CLINICAL SIGNS

PATHOGENESIS AND IMMUNOLOGY

DIAGNOSIS

CONTROL ( VACCINE DEVELOPMENT)

TREATMENT

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DEFINITION

A protozoan disease caused by Plasmodium

species of the phylum Apicomplexa.

Transmitted by the bite of infected female

anopheline mosquitoes.

It is characterized by periodic paroxysm with

shaking chills, high fever, heavy sweating.

Anemia and splenomegaly may also occur in

cases. 4

AETIOLOGY

Four species of Plasmodium cause malaria in human. P. vivax (benign tertian malaria) P. ovale (benign tertian malaria) P. malariae (quartan malaria) P. falciparum (malignant tertian malaria) Each species has its own morphologic, biologic, pathogenic, and clinical characteristics.

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TAXONOMY

Kingdom: Protista

Sub-Kingdom: Protozoa

Phylum: Apicomplexa

Class: Sporozoasida

Order: Eucoccidiorida

Family: Plasmodiidae

Genus: Plasmodium

Specie: P. falciparum 6

EPIDEMIOLOGY

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EPIDEMIOLOGY

Malaria is the third leading cause of death due to infectious disease.

It affects 300- 500 million people annually worldwide and accounts for over 100million deaths, mainly in African children under the age of 5yrs. A child in Africa dies every 30 seconds of malaria.

Endemic around the tropics and sub-tropics although it is world wide in distribution.

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FEMALE ANOPELES MOSQUITO 9

TRANSMISSION

DISTRIBUTION OF PLASMODIUM FALCIPARUM

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DISTRIBUTION OF PLASMODIUM VIVAX

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WATERSHEDS OF THE AFRICAN CONTINENT

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Mosquitoes are aquatic insects

Populationdensity

LIFE CYCLE

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LIFE CYCLE CONT’D

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PLASMODIUM MORPHOLOGY

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PLASMODIUM ANATOMY

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EX-FLAGELLATION OF THE MICROGAMETOCYTEOF A MALARIA PARASITE IN MOSQUITO

STOMACH

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PORTION OF AN INFECTED MOSQUITO STOMACH.

NOTE NUMEROUS OOCYSTS ON OUTER WALL.

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SPOROZOITES OF MALARIA IN INFECTEDMOSQUITO STOMACH PREPARATION

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Light Micrograph SEM

ERYTHROCYTIC STAGES OF MALARIA:ALL INFECTIONS BEGIN WITH THE RING STAGE

REGARDLESS OF THE SPECIES

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Ring stage

CLINICAL SIGNS & SYMPTOMSCLINICAL SIGNS & SYMPTOMS

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Cold stage

MALARIAL PAROXYSM Cold stagefeeling of intense cold vigorous shivering lasts 15-60 minutes Hot stage intense heat dry burning skin throbbing headache lasts 2-6 hours

Sweating stage profuse sweating declining temperature exhausted and weak →sleep lasts 2-4 hours

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UNCOMPLICATED MALARIA Uncomplicated malaria is defined as:

Symptomatic infection with malaria parasitaemia without signs of severity and/or evidence of vital organ dysfunction.

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SEVERE MALARIA

Severe malaria is defined as symptomatic malaria in a patient with P. falciparum asexual parasitaemia with one or more of the following complications: Cerebral malaria (unrousable coma not attributable to other causes). Generalised convulsions (> 2 episodes within 24 hours) Severe normocytic anaemia (Ht<15% or Hb < 5 g/dl) Hypoglycaemia (glood glucose < 2.2 mmol/l or 40 mg/dl ) Metabolic acidosis with respiratory distress (arterial pH < 7.35 or bicarbonate <

15 mmol/l)* Fluid and electrolyte disturbances Acute renal failure (urine <400 ml/24 h in adults; 12 ml/kg/24 h in children) Acute pulmonary oedema and adult respiratory distress syndrome* Abnormal bleeding Jaundice Haemoglobinuria Circulatory collapse, shock, septicaema (algid malaria) Hyperparasitaemia (>10% in non-immune; >20% in semi-immune)

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RELAPSE

A specific attack that it is up to months or even years after the primary attacks.Tachysporozoite grow in the hepatic cell and multiply to form exoerythrocytic schizonts and then invade RBCs to clinic malaria. Bradysporozoites in the liver spend a rest and sleeping times of months or even years , then they start develop in exoerythrocytic stage and erythrocytic stage. at this time, the patient occurs paroxysm , showing as periodic fever like the primary attacks, it is called Relapse.

NB: Relapse only occurs in P.vivax. 30

Paroxysms (acute febrile episodes) associated with synchrony coincide with the Merozoite release.Temperature is normal and patient feels well.P. falciparum may not exhibit classic paroxysms (continuous fever)

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Plasmodium falciparumPlasmodium falciparum

Plasmodium vivax Plasmodium ovalePlasmodium vivax Plasmodium ovale

Plasmodium malariaePlasmodium malariae

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Chronic Asymptomatic

Infection

PlacentalMalaria

Anemia

InfectionDuring

Pregnancy

Developmental Disorders;

Transfusions;Death

LowBirth weight

IncreasedInfant

Mortality

Non-severeAcute Febrile

disease

Cerebral

Malaria

Death

CLINICAL PICTURECLINICAL PICTURE

COMPLICATIONS OF MALARIA :CEREBRAL MALARIA

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COMPLICATIONS OF MALARIA :

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Child with severe malaria anaemia in conjunction with acidosis and

respiratory distress Pulmonary Edema

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Hepato-splenomegalyHepato-splenomegaly

survivors partially immunedoften with splenomegaly

MALARIAL HAEMOGLOBINURIA

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Clinical Picture :

Patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency may develop intravascular haemolysis and haemoglobinuria precipitated by Primaquine and other oxidant drugs, even in the absence of malaria.Haemoglobinuria associated with malaria (“blackwater fever”) is uncommon and malarial haemoglobinuria usually presents in adults as severe disease with anaemia and renal failure.

PATHOGENESIS &

IMMUNOLOGY

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SUSCEPTIBILITY TO MALARIA, ANTIBODY PRODUCTION, AND LETHALITY

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PATHOGENESIS

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The functions of most of the metabolic products of P. falciparum are not known. It is known that they are resistant to treatment with proteases.

P. falciparum uses hemoglobin as a source of energy and produces hemozin to digest hemoglobin, this toxin is stored in the pigment of P. falciparum .

This pigment has been linked to over production of tumor necrosis factor alpha, the gamma interferon and interleukin-1.

These are important and natural parts of human immune systems, but over production can lead to fever and the destruction of healthy host cells.

Information for the following slides adapted from: Chen, Q., M. Schlichtherle, and M. Wahlgren. 2000. Molecular aspects of severe malaria. Clin. Microbiol. Rev. 13:439-450

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TOXICITY AND PATHOGENICITY

Infection by P. falciparum also radically changes the cell membrane of red blood cells.

The membrane of infected cells becomes rigid and the parasite creates channels through the membrane in order to transport nutrients into the cell.

Protein components of the cell membrane are digested by the parasite and are replaced by the “knobs” (electron rich protrusions of ~100 micrometers).

The knobs are used to bind to uninfected RBCs and to the walls of veins and arteries. This is known as rosetting and can lead to some of the most severe complications of malaria, including cerebral malaria, where such rosettes occur in the brain.

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There are five receptors on RBC which are thought to be involved with the formation of rosettes. They include blood group antigens A and B, CD-36, compliment receptor 1 and HS-like GAGs (heparin sulfate glycosaminoglycans).

Rosettes formed in blood types A and B are larger, tighter and stronger than those formed in persons with O type blood. Blood type A is most often affected by severe malaria.

P. Falciparum also binds using knobs to both IgG and IgM.

The reason for having accumulations of IgM is not precisely known, but it is theorized that such accumulations hinder the access of antibodies specific to infected cells and thus help malaria to evade the immune system.


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ANTIGENIC VARIATION Malaria has many tools to evade the immune system. P. falciparum has a

very high degree of antigenic variation, making it difficult for the immune system to recognize malaria. P. falciparum has two different ways in which to vary which antigens it expresses.

The first way in which this might occur is during the sexually reproducing stage in the lifecycle when P. Falciparum recombines genetic material. This has unlimited potential to change the genome of P. Falciparum.

The second way in which antigenic variation can occur is through variable genes and point mutations during asexually reproducing stages of the lifecycle. P. Falciparum o has several families of variable antigenic genes.

These are var family, the rosettin/ rif family, and the p60 family. With such a large amount of variability available to malaria it is no

wonder that it can successfully evade the immune system and cause many recurring infections if not properly treated.


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VAR FAMILY

There are ~40-50 genes in the var family with a few exception they are extremely variable. The var genes are scattered throughout the chromosomes, but concentrated on the 4, 7, and 12 chromosomes.

Using the high variability in these regions at least 2% of individuals vary their antigenic expression each generation. These genes are thought to be involved with resistance to chloroquine and to help P. falciparum evade the host’s immune system.

Mutations at this sight are found in 100% of all resistant strains of P. falciparum. The efficacy of the resistance is greater when a mutation also occurs at a sight known as pfmdr1 (P. falciparum multidrug resistance gene).


Dorsey, G., M. R. Kamya, A. Singh, and P. J. Rosenthal. 2001. Polymorphisms in the Plasmodium falciparum pfcrt and pfmdr-1 genes and clinical response to chloroquine in Kampala, Uganda. J. Infect. Dis. 183:1417-1420.

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CROSS OVER AND VAR FAMILY

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DRUG-RESISTANT MALARIA

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Red - chloroquine resistantGreen - chloroquine sensitiveBlack - chloroquine and mefloquine resistant

ENTRY OF SPOROZOITES INTO PARENCHYMAL CELLS OF THE LIVER

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TRANSMISSION EM OF MEROZOITE ENTERING A RED CELL.

NOTE POINTS OF ATTACHMENT

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MECHANISMS OF RED CELL INVASIONBY PLASMODIUM

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TRANSMISSION EM: RBC INFECTED WITH P. FALCIPARUM

51N = Nucleus; F = food vacuole

“Knobs” of histidine-rich protein.Points of attachment to endothelial cell

DIAGNOSIS

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DIAGNOSIS

LIGHT MICROSCOPYRAPID DIAGNOSTIC TESTSEROLOGY: ELISA KITS- MOLECULAR TECHNIQUES: PCR (18S rRNA

Assay, Nested PCR Assay, real-time PCR), LAMP.

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Trophozoites

GametocyteSch

izont

Cytoplasm

Stippling

Vacuole

Nucleus/chromatin dot

Dr.

Osenium®

LABORATORY DIAGNOSIS OF MALARIA

Plasmodium falciparum

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Diagnostic Points: Small, regular, fine to

fleshy cytoplasm Infected RBCs not

enlarged Numerous, multiple

infection is common Ring, comma, marginal

or accole forms are seen; often have double chromatin dots

Maurer’s dots not clearly visible

CCMOVBD

Multiple infection

Marginal form

Double chromatin

LABORATORY DIAGNOSIS OF MALARIA

56Rapid diagnostic tests detect malaria antigens

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Plastic cassette format of RDT

RAPID DIAGNOSTIC TESTS DETECT MALARIA ANTIGENS

CONTROL AND

VACCINE DEVELOPMENT

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RTS,S VACCINE - PRE- ERYTHROCYTIC VACCINE

Hybrid containing the central repeats and most of the C-terminal of the CSP fused with hepatitis B surface antigen

Complex adjuvant mixture AS02 Completely protected six out of seven volunteers Field study in The Gambia showed good short-term

protection A clinical trial in Mozambique and Tanzania showed

delay of infection and reduction in incidence of severe malaria in young children

The vaccine advanced to Phase III trial.

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Liver Stage

SporozoitesPre-erythrocytic

Stage

PRE- ERYTHROCYTIC STAGE VACCINES

How they work:Generates Ab response against sporozoites

and prevents them from invading the liverPrevents intra-hepatic multiplication by

killing parasite-infected hepatocytes Intended Use:

Ideal for travelers - protects against malaria infection

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Merozoites

Asexual

Erythrocytic

Stage

ASEXUAL ERYTHROCYTIC STAGE VACCINES

How they work:Elicit antibodies that will inactivate

merozoites and/or target malarial Ag expressed on RBC surface

Inhibit development of parasite in RBCs Intended Use:

Morbidity reduction in endemic countries

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Malaria: Plasmodium Life Cycle

Sexual

Stage

SEXUAL STAGE VACCINES

How they work:Induces Ab against sexual stage AgPrevents development of infectious

sporozoites in salivary glands of mosquitoes

Prevent or decrease transmission of parasite to new hosts

Intended Use: Decreased malaria transmission

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VACCINE PORTFOLIO

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Ad5 CSP/LSA/TRAP

Pre-ClinicalEvaluation

Phase 1+/- Challenge

Phase 1bendemic

Phase 2bendemic Phase 3

MSP-2in ISCOM

PvR IIin AlOH

PvR IIin ASO2

MSP-2in ISA 720

RTS,Sin ASO1

RTS,S in ASO2

Pfs-16

MSP-4

LSA-1in ASO1

LSA-1in ASO 2

MSP-1Cin Alum-CPG

CP2.9in ISA 720

RTS,S in ASO2

DevelopmentManufactureAd5 CSP/LSA/TRAP

AMA-1Cin ISA 720

Pfs-16

MSP-5

Ad5 MSP-AMA 1

MSP-4

AMA-1in ASO1

AMA-1in ASO2

RECENT LANDMARKS IN MALARIA GENOMES - SEQUENCING

2002: Complete genome sequence of P. falciparum A partial sequence of rodent parasite, P. berghei

2005:sequences of several other rodent parasites P. vivax (a human malaria parasite) P. knowlesi (primarily a monkey parasite)

+ sequence of:Human genomeAnopheles mosquito

New Candidates for drug and vaccine pipeline68

OTHER VACCINE AVENUES Several antigens expressed during the blood

stream and liver stage of P. falciparum have been shown to elicit an immune response in humans.

The study showed that liver stage antigen 3 was highly immunogenic and a good candidate for use in a vaccine to prevent the invasion of RBC by P. falciparum. Immune memory of the antigens (especially LSA3) lasted up to 9 months when tested in chimpanzees.

Information for this slide from: Pouniotis DS, Proudfoot O, Minigo G, Hanley JC, Plebanski M. Long-Term Multiepitopic Cytotoxic-T-Lymphocyte Responses Induced in Chimpanzees by Combinations of Plasmodium falciparum Liver-Stage Peptides and Lipopeptides Infection and Immunity, August 2004, p. 4376-4384, Vol. 72, No. 8 69

VACCINATING MOSQUITOES

In mosquitoes, there are proteins on the surface of gametes and ookinets that may prove useful in formulating a vaccine that protects mosquitoes from infection.

Antibodies to these proteins prevent the parasite from taking up residence in the midgut of mosquitoes and forming oocysts. However, in order for such vaccines to reach mosquitoes they must be combined with efforts to vaccinate people living in endemic areas.

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PARATRANSGENESIS Paratransgenesis is the manipulation of

symbiotic bacteria such as E. coli to make the host immune to a pathogen.

Bacteria are engineered to produce proteins or peptides that either block binding of or kill parasites.

Several bacteria known to live in the anopheles midgut including Escherichia, Pseudomonas , and bacillus .

When fed with E. coli that produced antibodies to P. berghei, Anopheles mosquitoes showed a reduction in oocyst formation of 95%. 71

Transgenic mosquitoes expressing bee venom known as Phospholipidase A2 have also been shown to resist oocyst formation by up to 87%. Synthetic molecules have also been studied as ways of reducing susceptibility.

Anopheles mosquitoes with a synthetic gene expressing SM1 peptide were found to have 82% reduction in formation of oocysts.

Information on this slides from Michael A. Riehle, Prakash Srinivasan, Cristina K. Moreira and Marcelo Jacobs-Lorena. Towards genetic manipulation of wild mosquito populations to combat malaria: advances and challenges. The Journal of Experimental Biology 206, 3809-3816 (2003)

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CHALLENGES FOR MALARIA VACCINE

Four antigenetically distinct malaria speciesEach has ~6,000 genesFirst gene was only identified in 1983

Immunity in malaria is complex and immunological responses and correlates of protection are incompletely understood.

Identifying and assessing vaccine candidates takes time and is expensive

There is no clear ‘best approach’ for designing a malaria vaccine

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OTHER CONTROL METHODS

Biological ControlMosquito fishes (Gambusia affinis) have been

found to be predatory on the anopheles larvae.

Chemical Control Spray insecticides: DDVP and so on.Use mosquito nets, screen, or mosquito

repellents to protect the person from mosquito bites.

Physical Control: Eradicate the breeding places of mosquitoes. 74

TREATMENT

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ACTION OF ANTIMALARIAL DRUG IN THE ACTION OF ANTIMALARIAL DRUG IN THE DIFFERENT LIFE STAGES OF THE MALARIA DIFFERENT LIFE STAGES OF THE MALARIA

PARASITEPARASITE

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Tissue SchizontocidesTissue Schizontocides•PrimaquinePrimaquine•PyrimethaminePyrimethamine•TetracyclineTetracycline•ProguanilProguanil

Anti-relapse (P.vivax)•PrimaquineBlood Schizontocides

•Chloroquine•Sulfadoxine/Pyrimethamine•Quinine•Quinidine•Artemisinins

GametocycideGametocycidePrimaquinePrimaquine

SporontocidSporontocideses•PrimaquinePrimaquine•PyrimethaminePyrimethamine•ProguanilProguanil

Symptomatic and supportive treatment

Aetiologic treatment

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SYMPTOMATIC AND SUPPORTIVE TREATMENT

High fever, convulsion, cerebral edema, black water fever, etc.

Keep warm for shaking chill; Physical and chemical deffervescence

methods for high fever, such as ice bag, air condition.

Corticosteroid may be given , if necessary. Diazepam and wintermin for convulsion.

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AETIOLOGIC TREATMENT

Falciparum easily treated before complications as no relapses and no para-erythrocytic stage

Chloroquine is treatment of choice for sensitive strains of Plasmodia (merozoites)

Primaquine (Hypnozoites) Mefloquineorquinine and doxycycline (Chloroquine

resistant strains of falciparum) Atovaquone and proguanil (Malarone) for Chloroquine

resistance of P. falciparum. Artemether and lumefantrine (newer)

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CONCLUSION

Malaria is not inevitable, it can be eradicated, the

mission is possible if only we devote ourselves to quality research and we never give-

up.

Oseni Saheed Oluwasina (2013) 80

Malaria-Mission Impossible

Health & Medicine

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