Phylum: Apicomplexa Class: Sporozoea Sub-class: Coccidia Order: Eucoccidia Sub-order: Haemosporina Family: Plasmodidae Genus: Plasmodium Sub-genus: Plasmodium Laverania Vinckeia ….... Species: vivax falciparum berghei malariae ovale

Causal Agents: There are approximately 156 named species of Plasmodium which infect various species of vertebrates.  Four are known to infect humans:.  P. falciparum, P. vivax , P. ovale  and P. malariae.

Distribution of Malarial Parasites P. vivax most widespread P. falciparum primarily tropics and subtropics P. malariae similar range as P. falciparum P. ovale

Malaria types Malignant Tertian Malaria Benign Tertian Malaria Benign QuartanMalaria

Anopheles mosqouite

Vectores in Iran An. Stephenciمهمترین و پایدارترین ناقل در ایران An. Superpictus فراگیرترین آنوفل در ایران An. Dethaliکوچکترین آنوفل در جنوب شرق ایران An. Culicifacies An. Fluviatilisخطرناکترین ناقل فالسیپاروم An. Maculipennisناقل اصلی درشمال و شمال غرب کشور An. Sacharovi ناقل اصلی درشمال و شمال غرب کشور

Life Cycle:                                                                                   Life Cycle

Geographic Distribution

شیوع مالاریا در ایران در سال 1381: 15378 مورد مثبت در کشور 24/0 در هزار نفر جمعیت:API 68/14درصد آن مربوط به پلاسمودیوم فالسیپاروم در سال 1380: 11614 مورد بیماری در منطقه جنوب شرقی کشور گزارش شده است.

Malaria Transmission natural (sporozoites/Anopheles) blood transfusions shorter incubation period fatality risk (P. falciparum) no relapses possible (vivax/ovale) syringe sharing congenital relatively rare although placenta is heavily infected

Clinical features 2-Fever ( Hot stage) 3- Sweating stage History of exposure: (mostly: past travel or residence in disease-endemic areas).  Since untreated malaria can progress to severe forms that may be rapidly (<24 hours) fatal, malaria should always be considered in patients who have a Paroxsym: 1-Chills ( Cold stage) 2-Fever ( Hot stage) 3- Sweating stage Other clinical features: splenomegaly, anemia, thrombocytopenia, hypoglycemia, pulmonary or renal dysfunction, and neurologic changes.

cold stage feeling of intense cold vigorous shivering, rigor lasts 15-60 min

hot stage intense heat dry burning skin throbbing headache lasts 2-6 hours

sweating stage profuse sweating declining temperature exhausted, weak  sleep lasts 2-4 hours

Malaria Paroxysm paroxysms associated with synchrony of merozoite release temperature is normal and patient feels well between paroxysms falciparum may not exhi-bit classic paroxysms continuous fever 24 hr periodicity tertian malaria quartan malaria

توضیح واژه ها Relapse Hypnozoite (Resting stage) Recrudescence Induced malaria Hemozoin

Stippling dots 1) Schuffner’s dots 2) Maurer’s dots 3) Ziemann’s dots 4) Jame’s dots Sticky phenomen( knobs)

Malignant Tertian Malaria Infections caused by P. falciparum can progress to severe; 1-Cerebral malaria 2-Black water fever ( severe anemia) 3-Acute renal failure 4- Algid malaria 5- Respiratory distress syndrome (pneumonic malaria) 6- Gastro-intestinal mlaria Complications of P. vivax malaria include splenomegaly (with, rarely, splenic rupture), and those of P. malariae include nephrotic syndrome.

Karunaweera et al (1992) PNAS 89:3200 sweating rigor TNF = tumor necrosis factor-a () proinflammatory cytokine (produced in response to malarial antigens?)

Immunity Anti-Parasite Immunity slow to develop short lived ‘premunition’ non-sterilizing lower parasitemia less symptoms Anti-Parasite Immunity immune response prevents merozoite invasion, eliminates infected erythrocytes, etc. Anti-Disease Immunity eg., neutralization of exo-antigens or toxic effects

Immunity in Malaria A) Natural immunity 1-Innate immunity 2- Genetic immunity B) Acquired immunity 1-Exo-Erythrocytic stage Premonition: prevents of super-infection no re-infection (stage specific). 2- Erythrocytic stage concomitant immunity (stage specific & strain specific ): Ab mediated immunity Ab dependent cell cytotoxicity (ADCC) Ab dependent phagocytosis Cellular immunity

Malaria Epidemiology Stable or Endemic Malaria ~constant incidence over several years includes seasonal transmission immunity and disease tolerance correlates with level of endemicity (especially adults) Unstable or Epidemic Malaria periodic sharp increase in malaria little immunity high morbidity and mortality Endemicity Levels: holo- hyper- meso- hypo-

Malaria laboratory diagnosis 1) Microscopic identification: -preparing thick and thin biood smear -comparison of plasmodium species 2- Quality Buffy Coat ( QBC) 3) Immunochromatographic methods 4)Antibody Detection 5) Molecular diagnosis techniques

Thin Blood Smear

Thick Blood Smear

A: Immature schizont in a thin blood smear. B: Mature schizont                                                              A B

Multiply infected red blood cells with appliqué forms in thin blood smears                                                              A B C

Ruptured schizonts in a thin blood smear.                                                    C D

Malaria antibody detection   The IFA procedure Blood stage Plasmodium species schizonts (meronts) are used as antigen Enzyme immunoassays have also been employed as a tool to screen blood donors, but are not recommended for clinical diagnosis due to limited sensitivity serologic testing is not practical for routine diagnosis of acute malaria. 

Antibody detection may be useful for: 1- screening blood donors 2- Fever of Unknown Origin 3- testing a patient who has been recently treated for malaria but in whom the diagnosis is questioned Species-specific testing is available for the four human species: P. falciparum, P. vivax, P. malariae, and P. ovale.  Cross reactions often occur between Plasmodium species and Babesia species. 

Usefulness of Antibody Detection in the Diagnosis of Malaria Parasites

Types of Serological Assays Malaria Antibody Detection: Indirect Fluorescent Antibody Enzyme immunoassays Antigen Detection: Immunochromatographic

Antibody Detection + = Antigen Patient’s serum Antigen-antibody contains specific and non-specific antibodies + = Antigen-antibody complex Antigen

Antigen-antibody- *antibody complex Antibody Detection *-labeled antibody to human antibody + Antigen-antibody- *antibody complex = Antigen-antibody complex

Indirect Fluorescent Antibody (IFA) Microscope slide

Enzyme Immunoassay (EIA/ELISA) substrate enzyme + _

ELISA Eight of the 8-kDa antigens were chemically synthesized and tested with a panel of defined sera in a FAST-ELISA. All sera were tested in triplicate against each synthetic protein. This plate shows the substrate development for one protein. For evaluation, the mean absorbance value for each serum was divided by the mean absorbance value for the positive reference serum to give a relative absorbance unit.

Antigen Detection + = Antigen-antibody complex Monoclonal antibody Antigen in patient’s serum

Antibody-antigen-antibody Antigen Detection Antigen-antibody complex = Antibody-antigen-antibody complex + Immobilized monoclonal antibody

Antigen Detection Malaria Immunochromatographic Dipstick Optimal Assay P. falciparum specific monoclonal antibody Control Plasmodium pan specific monoclonal antibody

Antigen Detection Malaria Immunochromatographic Dipstick Problems: Low sensitivity with parasites density <100/ml Currently only useful for detection of P. falciparum infections

Diagnostic Tools for Human Infections with Malaria Blood film examination Serology - IFA PCR

Malaria IFA Test Sensitivity = 98% Specificity = 99.5% Sulzer et al, Am J Trop Med Hyg 1969;18:199-205 Sulzer et al, Bull Wld Hlth Org 1971;45:375-379

P. malariae

P. malariae

P. malariae

P. falciparum

P. falciparum

P. falciparum

A: Positive IFA result with P. malariae schizont antigen.                                A A: Positive IFA result with P. malariae schizont antigen.

Malaria antibody detection for clinical diagnosis is performed using the indirect fluorescent antibody (IFA) test.  The IFA procedure can be used as a diagnostic tool to determine if a patient has been infected with Plasmodium.  Blood stage Plasmodium species schizonts (meronts) are used as antigen Because of the time required for development of antibody and also the persistence of antibodies, serologic testing is not practical for routine diagnosis of acute malaria.  Enzyme immunoassays have also been employed as a tool to screen blood donors, but are not recommended for clinical diagnosis due to limited sensitivity

Antibody detection may be useful for: 1- screening blood donors involved in cases of transfusion-induced malaria when the donor's parasitemia may be below the detectable level of blood film examination 2- testing a patient with a febrile illness who is suspected of having malaria and from whom repeated blood smears are negative ( Fever of Unknown Origin) 3- testing a patient who has been recently treated for malaria but in whom the diagnosis is questioned Species-specific testing is available for the four human species: P. falciparum, P. vivax, P. malariae, and P. ovale.  Cross reactions often occur between Plasmodium species and Babesia species. .

A: Positive IFA result with P. malariae schizont antigen.                                A A: Positive IFA result with P. malariae schizont antigen.

Rapid Diagnostic Tests(dipstickor test strip) (basedon the detection of antigens malaria parasites; Histiding- rich protein II)

Comparison of Plasmodium Species

Treatment Chloroquine Sulfadoxine-pyrimethamine (Fansidar) Mefloquine (Lariam) Quinine Doxycycline Artemisin derivatives

Malaria Control Reduce Human-Mosquito Contact Reduce Vector impregnated bed nets repellants, protective clothing screens, house spraying Reduce Vector environmental modificaton larvacides/insecticides biological control Reduce Parasite Reservoir diagnosis and treatment chemoprophylaxis