PROTOZOA Dr. SUHAEMI, SpPD, FINASIM

PENDAHULUAN

Protozoans

Protozoans are more diverse than all other eukaryotes. No longer classified in a single kingdom. Recently shown that there are at least

seven or more clades. May be more than 60 monophyletic

eukaryotic clades.

“Protozoa” is now used informally without implying phyletic relationship.

Protozoans

Protozoa Lack a cell wall Have at least one motile stage in life cycle Most ingest their food

Carry on all life activities within a single cell. Can survive only within narrow

environmental ranges. Very important ecologically. At least 10,000 species of protozoa are

symbiotic in or on other plants or animals. Relationships may be mutualistic, commensalistic,

or parasitic.

Cladogram of the Major Divisions of Organisms

Classification

Phylum: Class Genera:

Sarcomastigophora

Zoomastigophora

Trypanosoma, Leishmania, Giardia, Trichomonas

　Lobosea

Entamoeba, Naegleria, Acanthamoeba

Apicomplexa SporozoeaPlasmodium,Toxoplasma, Cryptosporidium, Isospora

Ciliophora Kinetofragminophorea Balantidium

Life Cycle Stages

The stages of parasitic protozoa that actively feed and multiply are frequently called trophozoites; in some protozoa, other terms are used for these stages. Cysts are stages with a protective membrane or thickened wall. Protozoan cysts that must survive outside the host usually have more resistant walls than cysts that form in tissues.

Ecological Niches in the Human Body:

1. Skin: Leishmania 2. Eye: Acanthamoeba 3. Mouth: Amoebae and flagellates (usually non-pathogenic) 4.Gut: Giardia, Entamoeba (and

invasion to liver), Cryptosporidium, Isospora, Balantidium 5. G.U. tract: Trichomonas

Protozoans

Protozoans are an extremely diverse assortment of unicellular eukaryotes.

leishmania

giardia

trichomonas

amoeba

MALARIA ORGANISMS

Malaria Pathogenesis and Clinical Presentation

Malaria Burden

Malaria kills 1.5 to 2.7 m people world wide every year

95% are due to P.falciparum In India P.falciparum up to 34% Case fatality rate is up to 9% Chloroquine resistance is major

concern Multi drug resistance emerged in

India

Plasmodium

Causative agent of malaria “bad air” Been around since ~3550 BC General life cycle

2 hosts Invertebrates – mosquitoes; technically the

definitive host because of sexual reproduction Vertebrates – reptiles, mammals or birds; asexual

reproduction here, intermediate host Gametocytes form in the blood of

vertebrates but fertilization occurs in the gut of the mosquito in the blood of the vertebrate, so vertebrates are the definitive host

Relapsing malaria

P. vivax and P. ovale hypnozoites remain dormant for months

They develop and undergoe pre-erythrocytic sporogeny

The schizonts rupture, releasing merozoites and produce clinical relapse

Life Cycle

Malaria Transmission Cycle

Parasite undergoes sexual reproduction in the mosquito

Some merozoites differentiate into male or female gametocyctes

Erythrocytic Cycle: Merozoites infect red blood cells to form schizonts

Dormant liver stages (hypnozoites) of P. vivax and P. ovale

Exo-erythrocytic (hepatic) Cycle: Sporozoites infect liver cells and develop into schizonts, which release merozoites into the blood

MOSQUITO HUMAN

Sporozoires injected into human host during blood meal

Parasites mature in mosquito midgut and migrate to salivary glands

Components of the Malaria Life Cycle

Mosquito Vector

Human Host

Sporogonic cycle

Infective Period

Mosquito bitesgametocytemic person

Mosquito bitesuninfected person

Prepatent Period

Incubation Period

Clinical Illness

Parasites visible

Recovery

Symptom onset

plasmodium

Life Cycle

Life Cycle (mosquito stages in orange):sporozoite in mosquito salivary glands injected during feeding sporozoite in blood invades hepatocyte trophozoite in hepatocyte mitotic division schizont in hepatocyte hepatocyte bursts merozoites in blood invade RBC trophozoite in RBC mitotic division schizont in RBC RBC bursts merozoites in blood reinvade RBC schizont or gametocyte in RBC gametocytes ingested by mosquito gametes in midgut fertilization zygote elongation ookinete penetrates midgut epithelium, meiotic and mitotic division oocyst containing sporozoites sporozoite migration in hemolymph sporozoites in salivary glands

200 mm

Anopheles Head

Female

Male

500 mm

Oocyst

Oocysts of Plasmodium sp. on the surface of an Anopheles sp.

Plasmodium Species

4 human plasmodium P falciparum P vivax P malariae P ovale

Plasmodium vivax

43% of the malaria world wide; not usually life threatening

Causes benign tertian malaraia, vivax malaria or tertian ague – fever every 48 hours

Found in temperate zones; mostly in Asia – 40% of American soldiers in Vietnam had this type

Blacks in tropical Africa are resistant May remain as hypnozoites in the liver,

relapse of up to 8 years later

P vivax (cont.)

Merozoites only infect young RBC called reticulocytes because they contain the right receptors on the surface

After the ring stage, they become active amoeba

Schüffner’s dots are stippling in the RBCs Hemozoin accumulated in RBC when

trophozoite is present 12-24 nuclie in mature schizont Some merozoites develop into gametocytes

Ratio of 2 macrogametocytes to 1 microgametocyte Macrogametocyte is almost is large as RBC Microgametocyte is not nearly as large Mature in about 4 days

10 mm

Sporozoites of Plasmodium vivax

Squash prep of an oocyst from an infected mosquito

Sporozoites develop in oocysts, migrate in the hemolymph to the salivary glands

Several thousand may be injected into the host by one mosquito during feeding

In host liver, each will penetrate into an hepatocyte and develop into an exoerythrocytic schizont by way of a trophozoite

10 mm

Exoerythrocytic Schizonts

Liver cells Exoerythrocytic schizont,

which is a single multinucleate cell Cytokinesis occurs, and

thousands of merozoites burst from the hepatocyte within 1-2 weeks post infection

Merozoites then infect erythrocytes

P. vivax and P. ovale, some schizonts develop into dormant hypnozoites May become active and cause

a relapse of the disease years after a supposed cure

10 mm

Trophozoites of P. vivax

Identified as P. vivax by the following features: Enlarged,

decolorized infected erythrocytes

Prominent Schüffner’s dots

Amoeboid shape of the troph

10 mm

Erythrocytic Schizonts of P. vivax

Merozoites invade host erythrocytes, most undergo schizogony

New merozoites burst out of the cell and immediately infect new cells

Large numbers of infected erythrocytes burst more or less simultaneously, causing a rapid rise in body temperature at 48-hour intervals

Plasmodium falciparum

50% of malaria world wide; most virulent strain

Causes malignant tertian, subtertian or estivoautumnal malaria

Concentrated in the tropics and subtropics

Exoerythrocyte stage in liver, more irregularly shaped

No relapse but can have recrudescence – develop symptoms years later due to resurgence of previously low, nondetectable levels of parasitemia (not to be confused with relapse)

P. falciparum (cont.)

Merozoites can infect any RBC, not age dependent Usually see early ring stage/gametocytes in the

blood smear Smallest ring stage of the 4

Can also bind uninfected RBC – forming a rosettes, can be hard to find in blood smears

RBC develop irregular blotches called Mauer’s clefts that are larger than Schüffner’s granules

Mature schizont is less symmetrical than others

Gametocytes take 10 days to develop and then can be seen in large numbers Cresent shaped and very distinct

10 mm

Troph of P. falciparum

Young signet ring stage

Diagnostic features are: High parasitemia Presence of only

signet ring trophozoites;

Double chromatin dots

Multiple infections in some cells

Absence of Schüffner’s dots

Schizogony results in new infected erythrocytes

10 mm

Erythrocytic schizonts of P. falciparum

This stage usually is not observed in peripheral blood, except in very heavy infections

Each schizont produces from 6 to 32 merozoites, with an average of 20 to 24, every 48 hours

Hemozoin pigment is clumped in the center of the infected RBC

Note that the merozoites are very small, and that the schizont usually does not fill up the RBC

10 mm

Gametocytes of P. falciparum

Macrogametocytes are elongate; nucleus less than one-half the length of the cell Microgametocytes may be shorter and more blunt-ended; lighter blue cytoplasm;

nucleus that is greater than one-half the length of the cell Gametes not produced until in the midgut of a mosquito

Differentiation of falciparum

P.falciparum gametocyte

P.vivax gametocyte

Differentiation of falciparum

P.falciparum shizont P.vivax shizont

Differentiation of falciparum

P.falciparum trophozite

P.vivax trophozite

Differentiation of falciparum

P.falciparum gametocyte

P.vivax gametocyte

Falciparum gametocytes

Male Female

Electron Micrographs

P.falciparum EM P.vivax EM

Falciparum invading RBC

Drug Rx. of falciparum

Chloroquine is not the drug of choice Should not be treated with single

drug Combination therapy is a must Weaker drugs like Proguanil are of no

avail Artemisinin based CT – ACT is the Rx.

of choice

The Anti-malarial Drugs

Artesunate, Artether, Artemether Mefloquine, Amodiaquine Quinine, Chloroquine Lumefantrine, Halofantrine, Proguanilchlor (chlorguanide) Sulfadoxin+Pyrimethmine, Dapsone Tetracyclines, Doxycyclin, Clindamycin

Today’s Watch Word

Combination Therapy (CT)

Artemisinin based Combination Therapy

(ACT)

What is CT ?

Anti-malarial combination therapy (CT) is the simultaneous use of two or more blood schizonticidal drugs with different biochemical targets in the parasites and independent modes of action.

What is ACT ?

Artemisinin-based combination

therapy (ACT) is an antimalarial

combination therapy with an

artemisinin derivative as one

component of the combination given

for at least 3 days.

What are Artemisinins ?

Artemisinin derivatives

Methyl Ether

Hemisuccinate

Ethyl Ether

Arteether Artemether

Artesunate

Dihydroartemisin

Qinghaosu ("ching-how-soo")

Why Artemisinins ?

Short half-life; hence good for combination

Rapid substantial reduction of the parasite biomass

Rapid resolution of clinical symptoms Effective action against multi-drug

resistant P. falciparum Reduction of gametocyte carriage No documented parasite resistance

yet Few reported adverse effects.

No Monotherapy

No Chloroquine for P.falcipatum

No Monotherapy with Artemisinin

ACT - WHO Guidelines

Technical Consultation on Anti-malarial Combination Therapy: Geneva, April 2001

Guidelines for the treatment of Malaria

WHO document – 266 page book – February 2006

Recommended Combinations

1. Artemether + Lumefantrine (Lumether)

2. Artesunate (3 days) + Amodiaquine

3. Artesunate (3 days) + Mefloquine

4. Artesunate (3 days) + SP

5. Amodiaquine + SP (as interim option)

β Artemether

Methyl ether of Artemisinin Effective Schizonticidal and

gametocidal drug Short half life 2 - 6 hours Interferes with the conversion of

Haem to non toxic hemozoin in the parasite

Not indicated in 1st trimester of preg.

β Artemether side effects

Very few and less troublesome Cough Body aches Abd pain, Nausea, Vomiting,

Anorexia Palpitations Dizziness, weakness Skin rash, itching

AL Dosage Schedule

PUBLIC SECTOR PRIVATE SECTOR

COARTEM® PREFERENTIAL PRICING FOR PUBLIC SECTOR: PRICE CHANGES BY 2005

Second line Combinations

1. Artesunate (7 days) + Tetracycline (7)

2. Artesunate (7 days) + Doxycycline (7)

3. Artesunate (7 days) + Clindamycin (7)

or

4. Quinine in place of AS + any of the above antibiotics for 7 days

What to give in pregnancy ? In 1st trimester

Quinine + Clindamycin 7 days

In 2nd and 3rd trimesters Any ACT combination as per rec. or Artesunate + Clindamycin 7 days or Quinine + Clindamycin 7 days

Lactating women same ACT

Complications of falciparum malaria

Coma - cerebral malaria, convulsions Renal failure – black water fever Hyperpyrexia, acute pulmonary

edema Hemolytic Jaundice, severe bleeding Hypovolemic shock, Hypoglycemia Metabolic acidosis, Coagulopathy, Severe anaemia, hyperparasitemia

Artemisinins parenteral

αβ Arteether – 150 mg (2ml) i.m od x 3 days or 3 mg/kg od i.m. x 3 days

Artesunate 2.4 mg/kg i.v. or i.m. given on admission (time = 0), then at 12 h and 24 h, then once a day

Artemether 3.2 mg/kg i.m. given on admission then 1.6 mg/kg per day is an acceptable alternative to quinine i.v infusions

Rectal artemisinins are not as effective

Quinine parenteral

A loading dose of quinine of 20 mg salt/kg bw. 10 mg/kg 8th hrly i.v infusion

Rate-controlled i.v. infusion is the preferred route of quinine admin.

If this cannot be given safely, then i.m. injection is a satisfactory alternative.

Rectal admin. is not effective Quinidine can substitute quinine

Quinine parenteral

A loading dose of quinine of 20 mg salt/kg bw. 10 mg/kg 8th hrly i.v infusion

Rate-controlled i.v. infusion is the preferred route of quinine admin.

If this cannot be given safely, then i.m. injection is a satisfactory alternative.

Rectal admin. is not effective Quinidine can substitute quinine

αβ ARTEETHER

150 mg (2 ml amp.) O.D.

intramuscular x 3 days =

Total 3 ampoules in a box

To be given I.M