Malaria is a life-threatening disease caused by parasites that are transmitted to people through the bites of infected female Anopheles mosquitoes. It is one of the world’s leading causes of death, particularly among children in developing countries. Introduction Malaria Chronology in Malaria Epidemology Life cycle Pathogenesis and clinical feature Lab Test Treatment of Malaria Prevention of Malaria

1. Malaria
Technical class
24/2/23

2. Content
• Introduction Malaria
• Chronology in Malaria
• Epidemology
• Life cycle
• Pathogenesis and clinical feature
• Lab Test
• Treatment of Malaria
• Prevention of Malaria

3. Introduction Malaria
Kingdom: Protozoa
Phylum: Apicomplexa (sporozoa)
Class: Coccidea
Order: Haemospororida
Family: Plasmodiidae
Genus: Plasmodium
Species: P. malariae
• Malaria is a life-threatening disease caused by parasites that are transmitted to
people through the bites of infected female Anopheles mosquitoes. It is one of the
world’s leading causes of death, particularly among children in developing countries.
• Blood parasites of the genus Plasmodium.
• Four species are considered true parasites of humans
• Humans acts as a natural intermediate host: P. falciparum, P. vivax, P. ovale and P.
malariae.
• P. knowlesi is newer spp .

4. Chronology in Malaria
Millennia ago 18th century 1880 1885 1891 1898 1902 1984 1955 1976 1992
Charaka and Susruta
malaria a/w
mosquitoes
Louis Alphonse
Laveran identify
malarial parasite
Romanowsky
developed staining
method ME
Ronald Ross
demonstrate
Anopheles as vector
Italians named mal-
aria
Camillo Golgi
describe blood stage
Amigo and Grassi
described the life
cycle
Shortt and Garnham
exo-erythrocytic
stage in human
Malaria eradication
program
Trager and Jensen
cultured invitro
Failure of program
RTS,S developed by
GSK

5. Epidemiological classification of malaria depending on endemicity
Epidemiological classification of malaria depending on transmission
Epidemiology

6. Epidemiology
• P. falciparum is the most common species in India
• The major endemic areas in India are in the northeastern states, Andhra
Pradesh, Chhattisgarh, Gujarat, Jharkhand, Madhya Pradesh, Maharashtra,
Rajasthan and Odisha
• 22% of people reside in high transmission area (≥ 1 case per 1,000
population), 67% in low transmission area (0–1 case per 1,000 population)
and 11% in malaria free area
• Most affected>> Odisha (24%)>>92% of cases were due to P. falciparum
infection
• P. malariae (< 1%) >> restricted to Tumkur and Hassan districts of Karnataka
• P. ovale>>few cases from Odisha, Delhi, Assam, Gujarat and Kolkata

7. Life cycle
Causative agent:
• Anopheline culicifacies in rural areas,
• A. stephensi in urban areas
• A. fluviatilis in hilly areas.
• Others are A. minimus, A. philippinensis, A. sundaicus and A. maculatus
Definitive host:
• Mosquitoes
Intermediate host:
• Human

10. Pathogenesis and clinical feature
• Benign malaria
Febrile
paroxysm
Spleno-
megaly
Anaemia
Cold
stage
Hot
stage
Sweating
stage
normocytic normochromic anaemia.
factors attribute to the development of anaemia
• Parasite induced RBC destruction
• Lysis of RBC due to release of merozoites
• Splenic removal of both infected RBC and uninfected RBC
coated with immune complexes
• Bone marrow suppression
• Increased fragility of RBCs
• Autoimmune lysis of coated RBC
Splenomegaly
• massive proliferation of macrophages that engulf
parasitized and non parasitized coated RBCs.

11. Pathogenesis of falciparum malaria
 Sequestration of the parasites
• Cytoadherence
• Rosetting
• Deformability
 High level of parasitaemia:30–40% of total RBC are infected
 Other virulence factors like:
• Knob associated histidine rich protein II (HRP-II)
• Glycosyl phosphatidyl inositol (GPI):stimulates the host immune system to release cytokines
like IL-1, TNF and IFN-γ
PfEMP-1
• Microvascular
inflammation
• Obstruction
• Perivascular
leak

12. Complications of Falciparum malaria
• Cerebral malaria
• Pernicious malaria: blackwater fever, algid malaria and septicemic malaria
oBlack water fever: occurs following quinine treatment>>sudden intravascular hemolysis
followed by fever, hemoglobinuria and dark urine
oAlgid malaria: Characterized by cold clammy skin, hypotension, peripheral circulatory failure
and profound shock
oSepticemic malaria: Characterized by high degree of prostration, high grade fever with
dissemination of the parasite to various organs leading to multi organ failure
• Pulmonary edema and adult respiratory distress syndrome
• Hypoglycemia
• Renal failure: sequestration >acute tubular necrosis.
• Bleeding/disseminated intravascular coagulation
• Severe jaundice: results from hemolysis, hepatocyte injury and cholestasis
• Severe normochromic, normocytic anemia: hematocrit <15% or hemoglobin < 5 g/dL with
parasitemia level of more than 100,000/µL (> 2%)
• Acidosis: Results from accumulation of organic acids like lactic acid.

13. Laboratory Diagnosis
Microscopic tests:
Peripheral blood smear—Gold
standard
Fluorescence microscopy
(Kawamoto’s technique)
Quantitative buffy coat
examination
Non microscopic tests:
Antigen detection tests (RDTs)
or ICTs— detects parasitic
LDH, HRP-II, aldolase
Antibody detection—ELISA
Culture—RPMI 640 medium
Molecular diagnosis—PCR
using PBRK1 primer

14. Peripheral Blood Smear
Specimen:
• Peripheral blood >>ear lobe or by finger prick in older children & adults and
from the great toe in infants.
• Blood films should be prepared directly from the capillary blood.
• ethylene diamine tetra acetic acid (EDTA) anticoagulated blood, smears should
be made within an hour of collection of blood.
• pregnant women, cord blood and placental impression smears
• postmortem cases, smears from cerebral grey matter
Time for taking blood:
• few hours after the height of the paroxysm of fever and before taking
antimalarial drugs.
Frequency:
• at least twice daily until parasites are detected.

15. Types of peripheral blood smear
• For thick smear, a big drop of blood>> half inch square area >>dried>>kept in
distilled water in a koplin jar for 5–10 minutes>>dehemoglobinization
• For thin smear, a small drop of blood>>corner of a slide>>spread by another
slide at an angle of 45 ◦ and then is lowered to an angle of 30 ◦ and is pushed
gently to the left, till the blood is exhausted
• Thick smear—more sensitive
• Thin smear—speciation can be done
surface of a good thin film is:
• Even and uniform
• Consist of a single layer of RBCs
• “feathery tail end” is formed near the
center of the slide
• Margins of the film should not extend
to the sides of the slide

16. Thin smear
Advantages
• Intra RBC morphology of
parasite can be seen
• Species identification
• RBC morphology
• Mixed infection
• % of parasitized RBC
• Severity
• Know response to the treatment
Disadvantages
• Fixation of smear
• Low parasitaemia
• Less sensitive

17. Thick smear
Advantages
• More sensitive
• Rapid detection of parasites
• No fixation of smear
• Low parasitaemia can be
detected
Disadvantages
• Intra RBC morphology of
parasite can not be seen
• Cannot confirms Plasmodium
spp.

18. Romanowsky stain
• Methanol: fix the cells to slide
• Methylene blue : stain RNA,DNA - blue grey colour
• Eosin: stain haemoglobin - orange red
• Parasite cytoplasm : blue
• Nuclear material: purple red
• Pigment: depending on type and species

19. Romanowsky stain
Leishman’s Giemsa Field’s JSB
base Methanol based Water based Water based Water based
pretreatment dehaemoglobinisation
in thick smear
Methanol fixation in thin
film
Methanol fixation in
thin film
Methanol fixation in thin
film
content Methylene blue
Eosin
Methanol
Giemsa
Methanol
glycerol
Field A
Methylene blue Azur
Field B
Eosin
Buffered water
Solution I
Methyleneblue,
potassium dichromate,
sulfuric acid
Solution II
Eosin
pH Neutral 6.8-7.2 - -

20. Forms seen in peripheral blood smear examination

21. • Kawamoto technique

22. Schizonts
Character P. Falciparum P. vivax
Size of RBC
Number of merozoites
Arrangement of merozoites
Presence in peripheral
circularion
does not change
16-24 (upto 36)
asymmetrical
absent
Increases to become
twice the original size
14-20 (upto 24)
symmetrical in form of
rosette
present

23. Gametocytes
Character P. Falciparum P. vivax
Size F 10 to 12 µm ◊ 2 to 3 µm F 10 to 12 µm
M 8 to 10 µm ◊ 2 to 3 µm M 9 to 10 µm
Shape F cresentric
M sausage-shaped
F & M spherical
Nucleus F central, compact, surrounded by
malarial pigment
F peripheral, small,
compact, surrounded by
malarial pigment
M central, diffuse with malarial pigment
dispersed all over the cytoplasm
M central, diffuse with
malarial pigment dispersed
all over the cytoplasm
Infected RBC deformed, with its membrane stretched
over the gametocyte
enlarged

24. Ring forms
Character P. Falciparum P. vivax
Size 1.25 to 1.5 µm 2.5 µm
Cytoplasm Uniform thickness Thick opposite to
nucleus
Nucleus >1/ring 1 /ring
No of rings >1 ring/RBC 1
Location in RBC Inside as well accole forms Inside RBC

25. Quantification of parasites
• Thick smear is preferred to thin smear for quantification of parasitemia.
Quantification is helpful for:
• Assessing the severity of infection
• Monitoring the response to the treatment
• Detecting drug resistance of P. falciparum
By thick smear Number of parasites counted per 100 WBCs x Total WBC count (8000)/100
By thin smear Number of parasites counted per 100 RBCs x Total RBC count /100

26. Fluorescence microscopy
• Kawamoto technique >>fluorescent staining method>>demonstrating
malaria parasites.
• Blood smears are prepared on a slide and are stained with acridine -
orange and examined under a fluorescence microscope.
• Nuclear DNA – Green , Cytoplasmic RNA –red

27. Quantitative Buffy Coat Examination
• advanced microscopic technique for malaria diagnosis.
• consists of three basic steps
(1) concentration of blood by
centrifugation,
(2) staining with acridine orange stain
(3) examination under
ultraviolet (UV) light source

28. Quantitative Buffy Coat Examination
1.Crescent-shaped gametocytes (1) will appear near the
interface of the lymphocyte/monocyte and platelet layers.
2. A small number of (2) schizonts and (3) mature
trophozoites may appear in the granulocyte layer.
3.Ring-shaped (4) immature trophozoites will appear
throughout the red blood cell layer, with a concentration
near the interface with the granulocyte layer.

29. Quantitative Buffy Coat Examination
Advantages
• Useful for screening large sample
• faster (the entire tube can be
screened within minutes),
• more sensitive (at least as good as
a thick film),
• uses more blood (60 µL) than thick
smear and
• quantification is possible
Disadvantages
• expensive,
• less specific and
• speciation is difficult
• Cannot store
Interpretation
Acridine orange has a property of staining the nuclear DNA fluorescent brilliant
green. Normal RBCs don’t take up the stain (as they are anucleated).
However, parasitized RBCs appear as brilliant green dots. WBCs also take up the
stain

30. Antigen Detection by Rapid Diagnostic Tests
Parasite lactate dehydrogenase (pLDH):
• produced by trophozoites and gametocytes of all Plasmodium species
• differentiate pan malarial pLDH common to all species and pLDH specific
to P. falciparum
Parasite aldolase:
• Produced by all Plasmodium species
Plasmodium falciparum specific histidine rich protein-2 (Pf-HRP-II):
• produced by trophozoites and young gametocytes of P. falciparum
• Most of the kits>>combination of two antigens,P. falciparum specific
antigen (i.e. HRP2 or pLDH specific for P. falciparum) and other is a pan
malarial antigen (like aldolase or pan malarial pLDH)

31. Antigen Detection by Rapid Diagnostic Tests

32. Advantages
• Rapid
• simple to perform,
• no extra equipment or trained microscopist
• Sensitivity: > 90% sensitive at >100 parasites/µL, markedly reduced at <100
parasites/µL
• pLDH is produced by the viable parasites>>to monitor the response for treatment
Disadvantages
• expensive
• Cannot differentiate between the non falciparum malaria species
• HRP2 remains positive even after treatment
• False positive bands appear in rheumatoid arthritis factor positive cases
• The lower limit to detect HRP2 is 40 parasites/µL and pLDH is 100 parasites/µL.
Antigen Detection by Rapid Diagnostic Tests

34. Culture
• mainly used for preparation of malaria antigens
• RPMI 1640 medium(Roswell Park Memorial Institute and 1640 denotes
the number of passages)
• continuous flow system mixed with a thin layer of RBC and an overlay
medium consists of human serum maintained with 7% CO2 and 1–5% O2
• other media used are Delbecco’s modified Eagle medium (MEM), RPMI
1630, and Medium 199.

36. PCR
• detect a single P. falciparum in 20 µL of blood using PBRK1 primer
• 100 times more sensitive than that of thick blood smear
• Speciation can be done
• Drug resistance genes can be detected
• Useful tool for epidemiological study.

37. Website: nvbdcp.gov.in

38. Drug resistance in Malaria
P. falciparum malaria
• Chloroquine resistant
• Sulfadoxine-pyrimethamine
resistance
• Mefloquine resistance
P vivax malaria
• Sporadic cases of resistance to
chloroquine and/or primaquine
• Longer half life of drug
• Mutation of the parasite for
resistance
• Inadequate and irregular
usage of drug
• Poor compliance
• Host immunity
Chloroquine resistant
• mutations in the genes
encoding the transporter
proteins such as PfCRT (P.
falciparum chloroquine
transporter)
• PfMDR1 (P. falciparum
multidrug resistance gene
1).
sulfadoxine,
pyrimethamine and
proguanil resistant
• point mutation in DHFR

39. Nested PCR was done for detection
• pfcrt-o and pfmdr-1 for P. falciparum
• pvcrt-o, pvmdr-1 genes for P. vivax.
Gel picture showing band of pfcrt-o (Plasmodium
falciparum chloroquine resistant transporter-o) 280 bp
and pvcrt-o (Plasmodium vivax chloroquine resistant
transporter-o) 120 bp.

40. • Sample : 2ml edta blood
• DNA extraction: DNA Mini Kit (Invitrogen) spin column method
• Primer Design:
• :
•
Polymerase Chain Reaction
Nested PCR amplifications using a
PeqSTAR 96Xx Universal Gradient
PCR thermal cycler (Peqlab,
Germany).

41. In vitro drug sensitivity testing
• Antimalarial drug sensitivity testing was performed by invitro micro
test (Mark III)
• performed immediately after the collection of blood
• The test was considered valid and interpretable if 10% of the
parasites in the control well (drug free well) had developed into the
schizonts after 24–36 hours incubation.
• Isolates were considered resistant if they showed schizont
maturation at chloroquine concentrations 8 pmol/well (1.6 mmol/L
blood).
• To evaluate the drug-parasite response, the EC50 value (50%
inhibition) was calculated by HN Non Lin (V. 1.01 Beta) Software

42. Prophylaxis Against Malaria
• Chemoprophylaxis
• vector control strategies
• vaccine prophylaxis.

43. Chemoprophylaxis
• travelers going to endemic areas and as a short-term measure for
soldiers or police serving in highly endemic areas.
• Weekly regimen:
Chloroquine 300 mg or
proguanil 400 mg, or
mefloquine 250 mg
Chloroquine and mefloquine weekly regimens should be started 1 week
and 2 weeks before the travel respectively
• Daily regimen:
Doxycycline 100 mg
doxycycline daily regimen should be started 1 day before the travel

44. Vector Control Strategies
Anti-adult measures
• Residual spraying: Spraying the houses with residual insecticides such as
dichlorodiphenyl trichloroethane (DDT), malathion and fenitrothion is highly
effective against adult mosquito
• Space application of pesticide in the form of fog or mist by ultralow volume
method of pesticide dispersion
• Individual protection: Done by reduction of human mosquito contact by using
insecticide treated bed nets, repellents and protective clothing.
Antilarval measures
• Larvicide: Use of mineral oil or Paris green has been extensively used to kill
mosquito larvae and pupae
• Source reduction (to reduce the mosquito breeding site): Includes environmental
sanitation, water management and improvement of the drainage system.
• Biological larvicide: Gambusia affinis (fish) and Bacillus thuringiensis (bacteria)
can be used to kill the mosquito larva

45. Vaccination for malaria
• Pre-erythrocytic (sporozoites) vaccine
• prevent the entry of the parasite to liver
• Useful for people of hypoendemic area
• Trials are going on using vaccine candidates such as: −
PfCSP (circumsporozoite protein repeats) of Plasmodium falciparum
LSA: Liver specific antigen 1, 2, 3
SALSA: Sporozoite and liver stage antigen
SSP2: Sporozoite surface protein-2
STARP: Sporozoite threonine and asparagine protein
Duffy: Binding protein of P. vivax
RTS,S/AS01

46. RTS,S/AS01:
• repeat and T-cell epitope in the PfCSP (circumsporozoite protein of
Plasmodium falciparum),hepatitis B surface antigen (HBsAg)and a chemical
adjuvant (AS01) to boost the immune system response
• Intramuscular injection; the preferred injection site in children aged ≥ 5
months is the deltoid muscle
• 4 dose :strategy :three primary doses with a minimum interval of 4 weeks
between doses, followed by a booster dose approximately 12–18 months
after the third dose.
• 5-dose strategy: may be considered in areas with high seasonal malaria
transmission or areas with perennial malaria transmission with seasonal
peaks>> three primary doses administered at monthly intervals and two
annual booster doses administered prior to peak malaria transmission
season

47. Blood stage vaccine/erythrocytic vaccine
• help in preventing the disease thus, are useful for people of hyperendemic
areas of malaria
• trials are there using vaccine candidates such as
Pf EMP (P. falciparum erythrocytic membrane protein antigen) vaccine
Hsp70: Heat shock protein
SERA: Serine rich antigen
RESA: Ring infected erythrocyte surface antigen
ABRA: Acidic basic repeat antigen
HRP2: Histidine rich protein-2
Aldolase − Merozoite surface protein (MSP 1,2,3)
SPF 66

48. Transmission blocking/anti gametocyte vaccine
• inducing antigametocyte antibodies in >>passed to the mosquito while
they feed>>react with the gametocytic & other sexual stage antigens and
interfere with fertilization>>block the transmission of infection>> no
direct effect to the individuals.
• Vaccine candidates used are
Pfs230
Pfs45/48

49. THANK YOU