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8/6/2019 Comparative Detection of Plasmodium Vivax and Plasmodium Falciparum
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Comparative detection of Plasmodium
vivax and Plasmodium falciparum DNAin saliva and urine samples from
symtomatic malaria patients in a low
endemic area
Pattakorn Buppan, Chaturong
Putaporntip, Urassaya Pattanawong,
Sunee Seethamchai, Somchai
Jongwutiwes
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(1) Background
Plasmodium
falciparumPlasmodium vvax
human malaria accounting for
300-500 million cases and 130-145
million infections
per annum,
diagnosis of acute
malaria infection in routine laboratory
peripheral blood smears under light
microscope.
8/6/2019 Comparative Detection of Plasmodium Vivax and Plasmodium Falciparum
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(1) Background (cont)
Failure to detect cryptic P.
falciparum infections.
Recent studies at Zambia :
PCR MSP 2 & dihydrofolate
reductase
Why use saliva and urine?
detected of the small subunit
ribosomal RNA gene (SSU
rRNA).
8/6/2019 Comparative Detection of Plasmodium Vivax and Plasmodium Falciparum
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M
e
t
h
o
d
s
Study Area :
Malaria clinic at Ta Song Yang District in TakProvince ,Bangkok, Thailand.
Patients and sample collection :
Cross-sectional study Juni-July 2008
Inclusion criteria : febrile patients (oral
T emperature > 37.5C) of any age group.
Exclusion criteria : those having previous
anti-malarial treatment or presence of clinical
signs and symptoms of severe malaria
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100 malaria positive individuals giemsa stained thick blood film
20
febrile illness after entry and stay forest Area.
2 ml venous blood + EDTA
Laboratory at Chulalongkorn University stored at -40 0 C until use.
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After blood sample collection (prior toanti-malarial treatment)
1-2 ml (saliva), 20 ml (urine)
Half volumeice Laboratory (+ 2 volums ofabsolute ethanol 250C 35 0C) DNAextraction
8/6/2019 Comparative Detection of Plasmodium Vivax and Plasmodium Falciparum
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Microscopy :
Both thin & thick blood smears stained with10 % Giemsa solution.
Thin blood film examined for at least 200
microscopist fields.Thick blood film for at least 200
leukocytes, using a 100 objective
DNA Extraction
DNA isolate was extracted from 200 ml of
blood, saliva and urine samples using Qiagen
DNA Mini Kit.
8/6/2019 Comparative Detection of Plasmodium Vivax and Plasmodium Falciparum
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PCR- based Detection
Detection P. palciparum & P.vivax
Nested PCR.
1 2 ml of DNA was added to a total volume
of 20 ml amplification reaction+primer.
2
Thirty cycles (94C for 40 s, 60C for 30
s and 72C for1 min).
3 One ml of PCR product DNA template secondary primer.
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4 PCR product were sparated in 1 % and 2 %agarose gels for primary and nested PCR
5 Staining with ethidium bromide Visualized under a UV light.
6 Fragments of SSU rRNA gene ofP.falciparum 452 bp and P. vivax 416 bp.
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Data Analysis..
Data nested PCR gold standard.
indices : number true positive (TP), number of true
negative (TN), number of false positive (FP) and number
of false negative (FN).
Sensitivity was expressed as TP/(TP+FN) and specificity
as TN/(TP+FP).
Accuracy calculate (TP/TN)/number of all tests.
Kappa statistics
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R
E
S
U
L
T
s
1. Microscopy diagnosis and nested PCR detection of
Plasmodium falciparum and P. vivax in blood, saliva and
urine samples.
2. Diagnostic performance of microscopy and nested PCR
assays using DNA templates from saliva and urine samples.
3. Comparison of nested PCR assays using blood, saliva
and urine samples with microscopy results as reference.
4. Relationship between parasite density andpositive tests by nested PCR of saliva and urine samples
from malaria patients with single infections.
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8/6/2019 Comparative Detection of Plasmodium Vivax and Plasmodium Falciparum
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8/6/2019 Comparative Detection of Plasmodium Vivax and Plasmodium Falciparum
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D
Is
c
us
s
Io
n
DNA of P. falciparum in saliva
and urine samples of infected
individuals for potential
malaria diagnosis.
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PCR-based detection of P. falciparum in saliva
and urine samples compared with
microscopy has revealed a high
Thailand
Specificity (100 %)
sensitivity of 74.1%
for saliva and
44.4% for urine
Gambia
specificity (97%-98%)
sensitivity : moderate to
low (73% for saliva and
32% for urine samples)
Importantly, this study demonstrated for the first time that P. vivax can
also be detected in both saliva and urine samples of patients at a
comparable diagnostic performance for P.falciparum.
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Note
Proper preservation of clinical specimens
improvement of detection method is of
primary importance before saliva and urine
samples can be reliably applied for
alternative diagnosis of malaria parasites
or evaluation of malaria control measures
such as vaccine efficacy.
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how malarial DNA istransported to saliva and
urine of malaria patients?
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Matur Nuwun..