MALARIA
D r. A r v i n d N a t a r a j a n
Additional Professor
Dept of Microbiology
SDUMC
Introduction
Causative agent
Life cycle : Definitive host
Intermediate host
Infective form
Clinical features
Complications
Laboratory Diagnosis
Specimen collection
Microscopy
QBC
Serology
Molecular methods
Culture
Treatment
Prevention & Control
Summary
INTRODUCTION
Malaria
Mal - Bad aria - air
Oldest disease of mankind
Caused by Plasmodium species
Kingdom : Protozoa
Phylum : Sporozoa
❖ Plasmodium falciparum
❖ Plasmodium vivax
❖ Plasmodium malariae
❖ Plasmodium ovale
❖ Plasmodium knowlesi
Parasite present in the Blood - RBC’s
Scientists Contribution
Alphonse Laveran discovered the causative
agent - Plasmodium inside the RBC
Sir Ronald Ross described the sexual cycle
of the parasite in the female Anopheles
mosquito
Life Cycle
Plasmodium complete its life cycle in 2 hosts
Definitive host : Female Anopheles mosquito - Sexual cycle
( Sporogony )
Intermediate host : Human - Asexual cycle - Schizogony
Infective form : Sporozoites
Mode of Transmission : Bite of infected female Anopheles Mosquito
Rarely :
Blood transmission / Through placenta - Merozoites
Asexual cycle :
• Pre erythocytic schizogony
• erythocytic schizogony
• Gametogony
Human - Intermediate host
1st Stage : Pre erythrocytic schizogony - Liver ( Exo erythrocytic / Intrahepatic / Tissue
stage )
Sporozoites - Bite of female Anopheles mosquito
enters Blood & reach
Liver - hepatic cell
Trophozoites
transforms
Pre erythrocytic Schizont - contains several merozoites
cell Rupture
Merozoites
Invade RBCs - Erythrocytic Schizogony 2nd stage - Ring –Trophozoite - Schizont
Relapse :
Sporozoites - Hynozoites - Liver cell
P,vivax / P. ovale
Mosquito : Definitive host - Sexual cycle
During Blood meal - Mosquito takes gametocytes
Fusion of Male microgamete with Female microgamete
Zygote
transforms
Ookinete in the midgut
penetrate stomach wall
Oocyst
sporogony - sporozoites
Rupture of Oocyst
release
Sporozoites – Migrate to Salivary glands
bites human
Life cycle continues
CLINICAL FEATURES
Benign Malaria : - 4 species
❑ Febrile Paroxysm : Cold stage , Hot stage & Sweating stage
❑ Anemia
❑ Splenomegaly
Malignant Tertian fever : Plasmodium falciparum
Virulence factors -
PfEMP -1 ( [Link] erythrocyte membrane protein 1 ) helps in
o Cytoadherence
o Rosetting
o Deformability of RBCs - Sequestration of the parasites
Complications of P. falciparum
❑ Cerebral Malaria : Occlusion of brain capillaries - cerebral Anoxia
❑ Pernicious malaria :
➢ Black water fever
➢ Algid malaria &
➢ Septicemic malaria
Complications of P. falciparum
Pernicious malaria : Black water fever, Algid malaria & Septicemic
malaria
Black water fever : Intra vascular hemolysis
Occurs following quinine treatment
Mechanism : Auto immune mechanism - Immune complex formation
followed by complement mediated massive destruction
C/F : Fever, hemoglobinuria & dark colored urine
Algid Malaria : Cold clammy skin , hypotension & shock
Septicemic malaria : Dissemination of parasites to various organs ..
high fever ,
Complications of P. falciparum ..Continued
• Pulmonary Oedema & ARDS
• Hypoglycemia
• Renal failure
• DIC
• Severe Jaundice
• Acidosis
• Tropical Spenomegaly Syndrome - Endemic areas –abnormal immunologic response to repeated
malarial infections
Immunity against Malaria : Innate & Acquired immunity
Resistance to [Link]
• Sickle cell disease
• Hb C & E
• fetal hemoglobin
• Thalassemia
• Hereditary Ovalocytosis
• RBC with G6PD deficiency
Resistance to P. vivax
• Duffy negative RBC
RELAPSE RECRUDESCENCE
Seen in [Link] & [Link] Seen in P falciparum & P malariae
Sporozoites – remain P falciparum : Persistence of drug
Dormant Hynozoites resistant parasites even after completion of
treatment
P malariae : long term survival of RBC
stages at low undetectable levels in blood
National Programmes to Control Malaria :
❑ National Malaria Control Programme – 1953
❑ National Malaria Eradication Programme -1958
❑ Roll Back Malaria 2000
World Malaria Day - 25th April
LABORATORY DIAGNOSIS
Specimen Collection : Blood collected few hours after the fever spike
Microscopy :
• Peripheral smear : Thin smear & Thick smear
• Fluorescent Microscopy
• Quantitative Buffy coat analysis
Serology :
• Antigen Detection tests - PLDH, HRP ---ICT
• Antibody detection -ELISA
Molecular methods : DNA Probes - Target gene PBRK1
Culture : RPMI 1640 medium
LABORATORY DIAGNOSIS
Microscopy : Peripheral smear to detect malarial parasites in
blood
Simple & Gold standard test
2 types : Thin smear & Thick smear
Stains used : Leishmans / Giemsa / Fields / Wrights or JSB
JSB – Jaswanth Singh Bhattacharya
Peripheral smear
Advantages :
• Simple, Rapid & cheap
• Thick smear more sensitive : 5-10 parasites/ µl of blood
• Quantification of parasitemia - prognosis
• Demonstration of malarial pigments *
• Speciation of malarial parasite - Thin smear
Disadvantages :
▪ Requires Experienced microscopist
▪ Thin smear less sensitive : 200 parasites/ µl of blood
* Malarial pigments ( Hemozoin pigment) : Product of hemoglobulin
metabolism –hematin, excess protein & iron porphyrin
Differences between Thin smear & Thick smear
FEATURES THIN SMEAR THICK SMEAR
Volume of blood small drop Big drop
RBCs - Dehemoglobinised
Sensitivity 5-10 parasites / µl 200 parasites / µl
Speciation Possible Not possible
Detection of Malarial parasites
Plasmodium falciparum
Multiple ring Gametocyte -
Crescent
Plasmodium vivax
Schizont Gametocyte
Plasmodium malariae
Band form
FEATURES P Falciparum [Link] [Link] [Link]
Ring form Multiple rings
Accole
Double dot
Gametocyte Banana shape
Larger than RBC
Late Band forms
trophozoite
Stipplings Maurers cleft Schuffners dots Ziemanns dots James dot
RBC s RBCs of all ages Young RBCs Old RBCs Young RBCs
infected
Malarial Dark brown Yellowish Dark brown Dark Yellowish
pigments brown brown
Fever Malignant Tertian Benign Tertian Benign Quartan Ovale tertian
periodicity fever fever fever malaria
Fluorescent Microscopy : Kawamoto technique :
Blood smear are stained with acridine orange & examined under
Fluorescent Microscopy.
Quantitative Buffy coat examination : QBC
3 steps :
▪ Concentration of blood by centrifugation
▪ Staining with acridine orange
▪ Examination under ultraviolet light source
Interpretation : Acridine orange stains nuclear DNA fluorescent brilliant green
Normal RBCs don’t takeup stain - ???
Parasitised RBC appear Brilliant green
Advantages :
• Very rapid
• More sensitive - 5-10 parasites/ µl of blood
• Quantification possible
Disadvantages :
Expensive , less specific & speciation very difficult
SEROLOGY
Detection of Antigen by Rapid Diagnostic tests :
❖ Parasite Lactate dehydrogenase ( pLDH )
❖ Plasmodium falciparum specific Histidine Rich protein-2 ( Pf-HRP-2)
❖ Parasite Aldolase
Principle : Immunochromatography
Advantages :
• Rapid diagnosis 15 –20 mins
• Experienced microscopist not required
• pLDH is produced by viable parasite – Prognosis
Disadvantages :
• kits costly
• Cannot differentiate between Non falciparum malarial
species
• Gametocytes cannot be detected
• Lower limit : HRP 2 detection limit is 40 parasites/ µl
pLDH detection limit is 100 parasites/ µl
• HRP remains positive even after treatment
Detection of Antibodies :
• ELISA
• IFA
• IHA
Used for epidemiological survey
Screening for infected blood donors
Molecular Methods :
• DNA probes - highly sensitive can detect parasites 10/ µl
• Target gene – PBRK1
• Speciation possible
• Can detect drug resistance gene
Culture :
Not used for routine diagnosis…
Preparation of antigens / vaccines
RPMI 1640 Medium : Roswell Park Memorial Institute
TREATMENT
• Chloroquine
• Chloroquine resistant areas - Artemisinin
• Treatment failure cases - Quinine with Doxycycline / Tetracycline
Chemoprophylaxis :
• Chloroquine – weekly regimen
• Doxycycline - Daily regimen
Prevention & Control : Vector control Strategies
Important weapon to control malaria
Anti adult measures :
Residual spraying : DDT, Malathion
Space application of pesticide in the form of fog/ mist
Individual protection : Mosquito nets/ coils/ repellents
Antilarva measures :
Larvicide : Mineral oil / Paris green
Source reduction : environmental sanitation/ drainage
system
Biological larvicide : Gambusia fish
Bacillus thuringiensis
MALARIAL VACCINES
Pre Erythrocytic vaccine :
Sporozoite vaccine
Prevents entry of parasite to the liver
Blood stage vaccine / Erythrocytic vaccine
Merozoite surface protein vaccine
Helps in preventing the disease
Antigametocyte vaccine / Transmission blocking vaccine
Thank you
