Pathogenesis

Benign malaria: 3 stages----afebrile intervals-----periodicity of paroxysm of fever (tertian or quartan) Typical periodicity is absent in non-immune individuals Infection with-----P.vivax----chronic course with periodic relapse P. ovale------generally mild P. malariae-----less severe but may cause nephrotic syndrome in children

Cont..
1- Fever: probably due to IL-1 & TNF- released by macrophages 2- Anemia: parasite destroy large no. of RBCs---hemolytic anemia------results extreme fatigue and weakness Causes:- (a)haemolysis of infected & uninfected RBCs, (b) Dyserythropoiesis, (c) depletion of folate stores, (d) splenomegaly---erythrocyte sequestration & haemodilution

Cont..
3- Brown malarial pigment----Hb derivative--released from ruptured shizonts---discolors liver, spleen and bone marrow 4- Phagocytic defense mechanism-----marked hyperplasia of phagocytic cells throughout the body----splenomegalyalso liver in long standing cases 5- Immunity---gradually develops---untreated patient survive---episodes become less severe

Cont..
Plasmodia differ greatly in their ability to multiply in RBCs Pv--- prefer to invade young RBCs 1-2 % Pm---- prefer to invade older RBCs Pf---- invade all ages of RBCs 5-8% or even more

Cont..
Malignant Malaria----P.falciparum---if untreated life threatening complications develop Pernicious malaria---complex life threatening complications---occurs in heavy infections, >5% RBCs are infected.

Pathogeneis of P.falciparum
1- Erythrocytic schizogony---occurs in deeper capillaries of internal organs (brain, heart, liver, spleen, lungs, intestine, placenta and bone marrow)----obstruction & blood flow to organs

Cont.
2- Alteration on RBCs membrane---surface changes of deformed prasitised RBCs---make them sticky to adhere one another on capillary endothelium-----sequestration of infected RBCs in capillaries of internal organs------congestion, hypoxia, blockage & inflammation 3- High level parasitaemia----30-40% RBCs may infected.

Complications of Falciparum malaria

1- Cerebral malaria- involvement of brain--commonest cause of death----manifested by coma or confusion.
surface changes in RBCs---late schizonts secrete proteins----make it sticky----promote aggregation of RBCs in capillary endothelial cells-----causes capillary plugging due to accumulated RBCs and malarial pigment------results in anoxia, ischaemia and haemorrhage

Complications of Falciparum malaria

2- Algid malaria- symptoms include circulatory collapse (peripheral)---hypotension, hypodermia, rapid thready pulse & cold skin------also intestinal involvement----syndrome is thought due to adrenal damage (post mortem-----adrenal are congested, necrotic and haemorrhagic)

Complications of Falciparum malaria

3- Septicaemic malariamultisystemic infection----acute lung injury occurs---often develop shock and bacterial coinfection Microscopically---(a) blood shows high degree of parasitaemia (b) Alveolar capillaries & coronary blood vessels---congested and filled with parasitised erythrocytes.

Complications of Falciparum malaria

4- Black water fever (haemoglobinuria)infrequent ----in those who have repeated infections and inadequate quinine therapy---characterized by vomiting & prostation with passage of dark red or blakish urine. Pathogenesis involves---intravascular haemolysis with destruction of RBCs by Igs----leading to haemoglobinaemia & hemoglobinuria. repeated attacks---hypersensitivity develop

Immunity against malaria

Some individuals resistant to falciparum malaria due to inheritance of selected genes ---1- Haemoglobin S gene; Sickle cell---pf does not multiply 2- Thalassaemia genes; 3- G6PD deficiency genes; as this enzyme is necessary for respiration of pf

Immunity
4- Ovalocytosis gene ; deletion of eryt. Band 3 gene---RBCs are rigid resistant to invasion 5- Malnutrition and Iron deficiency 6-Infants are immune coz fetal Hb (Hb-F)

Congenital malaria
Transplacental infection
Can be all 4 species Commonly P.v. and P.f. in endemic areas P.m. infections in nonendemic areas due to long persistence of species

Neonate can be diagnosed with parasitemia within 7 days of birth or longer if no other risk factors for malaria (mosquito exposure, blood transfusion) Fever, irritability, feeding problems, anemia, hepatosplenomegaly, and jaundice

Laboratory Diagnosis of Malaria

Malaria Diagnosis
Clinical Diagnosis Malaria Blood Smear Fluorescent microscopy Antigen Detection Serology Polymerase Chain Reaction

Clinical Diagnosis
Hyperendemic and holoendemic areas Laboratory resources not needed Fever or history of fever Sensitivity ranges from poor to high Often has poor specificity and predictive values Overlap with other syndromes

Malaria Blood Smear

Remains the gold standard for diagnosis
Giemsa stain distinguishes between species and life cycle stages parasitemia is quantifiable

Threshold of detection
thin film: 100 parasites/l thick film: 5 -20 parasites/l

Requirements: equipment, training, reagents, supervision Simple, inexpensive yet labor-intensive Accuracy depends on laboratorian skill

Interpreting Thick and Thin Films

THICK FILM

THIN FILM
fixed RBCs, single layer smaller volume 0.005 l blood/100 fields good species differentiation requires more time to read low density infections can be missed

lysed RBCs larger volume 0.25 l blood/100 fields blood elements more concentrated good screening test positive or negative parasite density more difficult to diagnose species

Recognizing Malaria Parasites

Inside a red blood cell

Blue cytoplasm

One or more red chromatin dots

Recognizing Erythrocytic Stages:

Schematic Morphology
Blue Cytoplasm RING Red Chromatin Brown Pigment TROPHOZOITE

SCHIZONT

GAMETOCYTE

Malaria Parasite Erythrocytic Stages

Ring form

Schizont

Trophozoite

Gametocytes

Plasmodium falciparum
Infected erythrocytes: normal size

Rings: double chromatin dots; appliqu forms; multiple infections in same red cell

Gametocytes: mature (M)and immature (I) forms (I is rarely seen in peripheral blood) Schizonts: 8-24 merozoites (rarely seen in peripheral blood)

Trophozoites: compact (rarely seen in peripheral blood)

Plasmodium vivax
Infected erythrocytes: enlarged up to 2X; deformed; (Schffners dots)

Rings Schizonts: 12-24 merozoites

Trophozoites: ameboid; deforms the erythrocyte Gametocytes: round-oval

Plasmodium ovale
Infected erythrocytes: moderately enlarged (11/4 X); fimbriated; oval; (Schffners dots) malariae - like parasite in vivax - like erythrocyte

Trophozoites: compact Rings Schizonts: 6-14 merozoites; dark pigment; (rosettes) Gametocytes: round-oval

Plasmodium malariae
Infected erythrocytes: size normal to decreased (3/4X)

Trophozoite: compact

Trophozoite: typical band form

Schizont: 6-12 merozoites; coarse, dark pigment

Gametocyte: round; coarse, dark pigment

Species Differentiation on Thin Films

Feature Enlarged infected RBC Infected RBC shape Stippling infected RBC Trophozoite shape Chromatin dot Mature schizont Gametocyte round Mauer clefts small ring, often double rare, 12-30 merozoites crescent shape P. falciparum P. vivax + round, distorted Schuffner spots P. ovale + oval, fimbriated Schuffner spots round none small ring, compact single 6-12 merzoites compact, round P. malariae

large ring, large ring, amoeboid compact single large

12-24 4-12 merozoites merozoites large, round large, round

Species Differentiation on Thin Films

P. falciparum Rings P. vivax P. ovale P. malariae

Trophozoites

Schizonts

Gametocytes

Species Differentiation on Thick Films

Feature Uniform trophozoites Fragmented trophozoites Compact trophozoites Pigmented trophozoites Irregular cytoplasm Stippling (RBC ghosts) Schizonts visible Gametocytes visible very rarely occasionally + + often usually + + often usually often usually P. falciparum + ++ + + + + P. vivax P. ovale P. malariae

Estimating Parasite Density

Alternate Method
Count the number of asexual parasites per high-power field (HPF) on a thick blood film
+ ++ +++ ++++ 1-10 parasites per 100 HPF 11-100 parasites per 100 HPF 1-10 parasites per each HPF > 10 parasites per each HPF

Fluorescent Microscopy
Modification of light microscopy Fluorescent dyes detect RNA and DNA that is contained in parasites Nucleic material not normally in mature RBCs Kawamoto technique
Stain thin film with acridine orange (AO) Requires special equipment fluorescent microscope Staining itself is cheap Sensitivities around 90%

Quantitative Buffy Coat (QBC )

Fluorescent microscopy after centrifugation AO-coated capillary is filled with 50-100 l blood Parasites concentrate below the granulocyte layer in tube May be slightly more sensitive than light microscopy but some reports of 55-84%

Quantitative Buffy Coat (QBC )

Useful for screening large numbers of samples Quick, saves time Requires centrifuge, special stains 3 main disadvantages
Species identification and quantification difficult High cost of capillaries and equipment Cant store capillaries for later reference

Malaria Serology antibody detection

Immunologic assays to detect host response Antibodies to asexual parasites appear some days after invasion of RBCs and may persist for months Positive test indicates past infection Not useful for treatment decisions

Malaria Serology antibody detection

Valuable epidemiologic tool in some settings Useful for
Identifying infective donor in transfusion-transmitted malaria Investigating congenital malaria, esp. if moms smear is negative Diagnosing, or ruling out, tropical splenomegaly syndrome Retrospective confirmation of empirically-treated non-immunes

Malaria Antigen Detection

Immunologic assays to detect specific antigens Commercial kits now available as immunochromatographic rapid diagnostic tests (RDTs), used with blood
P. falciparum histidine-rich protein 2 (PfHRP-2) parasite LDH (pLDH)

Monoclonal and polyclonal antibodies used in antigen (Ag) capture test Species- and pan-specific Ab Cannot detect mixed infections Cross reactivity with rheumatoid factor reportedly corrected

Detection of Plasmodium antigens: pLDH (parasite lactate dehydrogenase)

Detection of Plasmodium antigens

A: HRP-2 (histidine-rich protein 2) (ICT) B: pLDH (parasite lactate dehydrogenase)(Flow) C: HRP-2 (histidine-rich protein 2) (PATH)

Malaria Antigen Detection - RDTs

Feature Test principle PfHRP-2 tests Use of monoclonal (Ab) pLDH tests Use of monoclonal and polyclonal Ab Detects a parasite enzyme, lactate dehydrogenase

Detects a histidine rich protein of P.f.

pLDH is found in sexual and Water-soluble protein is released from parasitized RBCs asexual forms

Not present in mature gametocytes

Differentiation between malarial species is based on antigenic differences between pLDH isoforms

Malaria Antigen Detection - RDTs

Feature Advantages PfHRP-2 tests Threshold for parasite detection as low as 10 parasites/l (but sensitivity drops at < 100 parasites /l) pLDH tests Threshold for parasite detection 100 parasites/l Can detect all species which infect humans

Does not cross react with other Can differentiate between P.f. species P.v., P.o., P.m. and non-falciparum malaria Does not cross react with human LDH Positive only in viable parasites, potentially useful for monitoring success of treatment

Malaria Antigen Detection - RDTs

Feature Disadvantages PfHRP-2 tests Some tests only detect P.f. pLDH tests Cannot differentiate between non-falciparum species

Cannot detect mixed infections Cannot detect mixed infections Sensitivity and specificity decreases < 100 parasites/l Can remain positive up to 14 days post treatment, in spite of asexual and sexual parasite clearance, due to circulating antigens Sensitivity and specificity decreases < 100 parasites/l

Polymerase Chain Reaction (PCR)

Molecular technique to identify parasite genetic material Uses whole blood collected in anticoagulated tube (200 l) or directly onto filter paper (5 l)
100% DNA is extracted 10% blood volume used in PCR reaction

Polymerase Chain Reaction (PCR)

Threshold of detection at CDC
0.1 parasite/l if whole blood in tube 2 parasites/l if using filter paper

Definitive species-specific diagnosis now possible Can identify mutations try to correlate to drug resistance Parasitemia not quantifiable May have use in epidemiologic studies Requires specialized equipment, reagents, and training

Real-Time PCR
New technique based on fluorescence Promising because it has potential to quantify parasitemia, decreases contamination, may detect multiple wavelengths in same tube identifying multiple species in one run, saves hands-on time

Needs further research and validation for malaria

Treatment
Quinine Chloroquine Primaquin Pyrimethamine +salfadoxine Fanssidar Newer drugs; halofantrine, artemisinin

Prevention & Control

Mosquito repellants and bed nets Biological control of mosquito (Gambiens) Proper treatment of malaria patient Vaccines; 3 types of vaccines are under trial 1- Anti-sporozoite vaccine; aimed to prevent 1st step of human infection: blocking invasion of hepatocytes

Vaccines Cont
2- Vaccines against asexual form; surface Ag of trophozoites and schizonts: characterized and cloned-----under trail 3- Antigametocyte vaccine; aim to control malaria transmission: under trial

Vaccines in use
1- Spf 66; widely tested synthetic vaccine :contains epitope of Ag present on sporozoite & asexual stage---moderate reduction in malaria during field trials in Colombia, Tanzania & Gambia 2- vaccines using candidate molecules including antigametocyte vaccine; breaking malaria transmission