The document discusses malaria, which is caused by Plasmodium parasites transmitted via mosquito bites. It outlines the life cycle and species of Plasmodium that cause malaria in humans. The presentation summarizes techniques used to study malaria parasites in vitro, including culturing isolates, examining blood smears, and assessing drug sensitivity. Genetic analysis of field isolates by PCR targeting msp1 and msp2 genes showed changes in alleles present over time in culture. In vitro culture allows studying parasite growth and drug resistance profiles.

1. Presented by: Km.Manorama
M.tech(biotechnology)
Department of Bioscience and Biotechnology.
Banasthali University

2.  Malaria -Malaria is a life-threatening mosquito-borne blood
disease caused by a Plasmodium parasite. It is transmitted to
humans through the bite of female Anopheles mosquito.
 The term malaria is a combination of two Italian words “mala
aria” meaning “foul air,”.
 plasmodium species that are responsible for causing malaria:
1. P. falcipaum
2. P. vivax
3. P. malariae
4. P. knowlesi
5. P. ovale

3.  In India, approximately 1.5 million cases of malaria are
reported each year, of which 50% are due to P.falciparum
 P.falciparum causes both “mild” and “severe” form of clinical
malaria.
 Clinical signs and symptoms of malaria:
 High fever, headache, severe chills, profuse sweating and
general body pains.
 Some patients may have vomiting, cough or diarrhoea.
 Severe manifestations of malaria also vary and include
anaemia, hypoglycaemia, hypotension, spontaneous bleeding,
acute kidney failure, coma and multiple organ failure.

4.  The life cycle is almost the same
for all the five species that infect
human
 Stages of malaria parasite life
cycle:
1. Infection of a human with
sporozoites
2. asexual reproduction
3. sexual reproduction
The two first stages take place
exclusively into the human body,
while the third one starts in the
human body and is completed
into the mosquito organism.

6.  In vitro techniques:
 Preparation of culture medium for the cultivation of
parasite. (RPMI medium, complete and incomplete medium,
serum, RBCs for culture)
 Blood smear preparation and examination. (thick and thin
blood films)
 Estimation of the percentage of erythrocytes infected with
P.falciparum.
 Calculation A= number of infected erythrocytes/ number of
erythrocytes ×100


7.  Synchronization of parasite: Malaria parasites consists
merozoites, ring-stages, trophozoites and schizonts stages.
 synchronization should be done several times until the ring-
stage predominates in the cultures.
 Schizont maturation inhibition assay (SMI): To study in
vitro sensitivity of P.falciparum of field isolates, 96 wells
micro well plate were used. These plates were coat with drug
according to the protocol given by WHO.
 Genomic DNA extraction from dried blood spots.

8.  Polymerase chain reaction
1. 18s PCR: it is genus specific PCR.
2. PCR assay for P.falciparum genotyping.
 P.falciparum positive samples were analyzed by PCR
amplification of central polymorphic region of MSP2 (FC27
and 3D7/IC allelic families), and block 2 of MSP1 (K1,
MAD20 and RO33 allelic families) using nested- PCR in
accordance to the genotyping protocol.

9. Gene
Polymorphic
genetic markers
(family specific)
Primer name &sequence
MSP1 K1 M1-F:AAATGAAGAAGAAATTACTACAAAAGGTGC
M1-R:GCTTGCATCAGCTGGAGGGCTTGCACCAGA
MAD20 M1-F:AAATGAAGGAACAAGTGGAACAGCTGTTAC
M1-R:ATCTGAAGGATTTGTACGTCTTGAATTACC
RO33 M1-F:TAAAGGATGGAGCAAATACTCAAGTTGTTG
M1-R:CATCTGAAGGATTTGCAGCACCTGGAGATC
MSP2 FC27 M2-F:AATACTAAGAGTGTAGGTGCAATGCTCCA
M2-R:TTTTATTTGGTGCATTGCCAGAACTTGAAC
3D7/IC M2-F:AGAAGTATGGCAGAAAGTAACCTCTACT
M2-R:GATTGTAATTCGGGGGGATTCAGTTTGTTCG
Table 1: Sequences of the oligonucleotide primers used to genotype msp1 and msp2 gene in
P.falciparum parasite.

10. Species primers Sequence (5`-3`)
Size(bp) of
PCR product
Plasmodium spp. rPLU5
rPLU6
CCTGTTGTTGCCTTAAACTTC
TTAAAATTGTTGCAGTTAAAACG
1100
P.falciparum rFAL1
rFAL2
TTAAACTGGTTTGGGAAAACCAAATATATT
ACACAATGAACTCAATCATGACTACCCGTC
205
P.vivax rVIV1
rVIV2
CGCTTCTAGCTTAATCCACATAACTGATAC
ACTTCCAAGCCGAAGCAAAGAAAGTCCTTA
120
P.malariae rMAL1
rMAL2
ATAACATAGTTGTACGTTAAGAATAACCGC
AAAATTCCCATGCATAAAAAATTATACAAA
144
Table 5: Primer sequences of 18S PCR.

11.  In Vitro culture:
 In in vitro culture monitoring we monitored two cryopreserved samples (RKL-9
and 3D7). In the first step we revived the cryopreserved isolates and monitored
their growth in culture.
Microscopic identification
The thin blood smears prepared were stained with JSB I and JSB II and
thereafter observed under microscope at 100X under oil immersion.

12. The following asexual stages of parasite are observed under
microscope:
Rings
Trophozoite
Schizonts
RING
SCHIZONT
TROPHOZOIETS

13. % parasitemia in the reference strain 3D7.
Fig 1: Percent parasitemia of 3D7, at zero day parasitemia was 0.8% and reached
upto 5-6% on the 4th day. The culture was sub- cultured on 4th day.
0
1
2
3
4
5
6
1 3 5 7 9 11 13 15
%parasitemia
No. of days in culture
% parasitemia

14. % parasitemia in the field isolates RKL-9
Fig 2: Percent parasitemia of RKL-9, at zero day parasitemia was 0.5% and reached up to 5-
6% on the 5th day. The culture was sub- cultured on 5th day.
0
2
4
6
1 3 5 7 9 11 13 15
%parasitemia
No. of days in culture
%parasitemia

15. BLANK
CQ
MWT10
CQ MWT11 CQ RNC59 CQ RNC60 CQ 3D7
CQ RKL-
9
BLANK
A 100 63 80 80 50 70
B 61 60 78 4 28 70
C 65 52 70 1 5 66
D 56 48 64 00 00 34
E 37 30 60 00 00 26
F 5 00 3 00 00 15
G 00 00 00 00 00 00
H 00 00 00 00 00 0
IC50 88.179 192.259 229.972 25 27.120 137.181
Table 2 : Micro test plate is coated by CQ and dosing of samples RKL-9, 3D7, RNC 59, RNC 60, MWT 10 and MWT 11.

17. IC50
Fig 3: Inhibition concentration (IC50) of chloroquine resistance and sensitive strains with
different field isolates. Six field isolates were examined (MWT10, MWT11, RNC59,
RNC60, 3D7, RKL-9) three of them were found chloroquine sensitive (MWT10,
RNC59 and 3D7) and rest were chloroquine resistance (MWT11, RNC60 and RKL-9).
0
50
100
150
200
250
IC50(nM)
samples
Ic50

18. DAYS
Template
DNA
Locus /strain
Msp1 Msp2
MAD 20 K1 RO33 FC27 3D7/IC
DAY 0
MWT10 400
MWT 11 200,300 400,320,250 500
MWT MIX 200,300 400,320,250
DAY 30
MWT 10 100 400
MWT 11 200,270 400,300,200 500
MWT MIX 200,270 400,300,200
DAY 60
MWT 10 210 250 100
MWT 11 100
MWT MIX 100
1.Allelic diversity of P.falciparum MSP1 and MSP2
Table 3: Base pair ranges observed per allelic family in msp1 and msp2.

19. DAYS
Template
DNA
Locus /strain
Msp1 Msp2
MAD 20 K1 RO33 FC27 3D7/IC
DAY 0
RNC 59 200
RNC 60 500
RNC MIX 200 500
DAY 30
RNC 59
RNC 60 400 400
RNC MIX 400 400
DAY 60
RNC 59
RNC60
RNC MIX 400 500
Table 4: Base pair ranges observed per allelic family in msp1 and msp2.

20. MSP1 and MSP2
Fig 4: Gel picture showing the amplification of P. falciparum msp1, msp2 for 30th day.
Lane 1and 15 shows 100bp DNA ladder, lane 2-5 shows msp1 allelic family MAD20,
lanes 6-9 show allelic family K1, lanes 10 and 13 show allelic family RO33, lanes 16 and
19 show msp2 allelic family IC/3D7, lanes 20-24 show family FC27.

21. 18S PCR
Fig 5: Gel picture showing the 18s PCR assay for detection of species identification for
Delhi samples.

22.  In this study, we observed that the field isolates of
P.falciparum positive by RDT are also positive by 18S PCR
and microscopy.
 The drug sensitivity of these cultures with chloroquine drug
was assessed their IC50 values. From this experiment we
concluded that the P.falciparum can be cultivated in vitro if
desirable conditions are provided.
 The parasitemia increased after each day and is subcultured
when the parasitemia is reaches 5-6%. It could be also seen by
drug sensitivity assay that P.falciparum is resistant to drugs
such as chloroquine. Two strains 3D7 and RKL-9 are drug
sensitive.

23.  We also analysed the genetic variations in respect to MSP1
(block 2), MSP2 (central repeat region, block 3) in field
isolates collected from different endemic state of India. At
the beginning of the PCR assay for msp1 and msp2 we
found some alleles for both the genes. But after in vitro
cultivation of mixed cultures for a period of 60 days we sow
a different set of alleles also emerging for msp1 and msp2.
This could be because of the dormant parasite clone in the
mixed culture which gets activated after few days of
passaging in culture. Hence the change in the appearance of
alleles after the PCR assay.