8/6/2019 Comparative Detection of Plasmodium Vivax and Plasmodium Falciparum

1/20

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

8/6/2019 Comparative Detection of Plasmodium Vivax and Plasmodium Falciparum

2/20

(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

3/20

(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

4/20

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

8/6/2019 Comparative Detection of Plasmodium Vivax and Plasmodium Falciparum

5/20

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.

8/6/2019 Comparative Detection of Plasmodium Vivax and Plasmodium Falciparum

6/20

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

7/20

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

8/20

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.

8/6/2019 Comparative Detection of Plasmodium Vivax and Plasmodium Falciparum

9/20

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.

8/6/2019 Comparative Detection of Plasmodium Vivax and Plasmodium Falciparum

10/20

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

8/6/2019 Comparative Detection of Plasmodium Vivax and Plasmodium Falciparum

11/20

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.

8/6/2019 Comparative Detection of Plasmodium Vivax and Plasmodium Falciparum

12/20

8/6/2019 Comparative Detection of Plasmodium Vivax and Plasmodium Falciparum

13/20

8/6/2019 Comparative Detection of Plasmodium Vivax and Plasmodium Falciparum

14/20

8/6/2019 Comparative Detection of Plasmodium Vivax and Plasmodium Falciparum

15/20

8/6/2019 Comparative Detection of Plasmodium Vivax and Plasmodium Falciparum

16/20

D

Is

c

us

s

Io

n

DNA of P. falciparum in saliva

and urine samples of infected

individuals for potential

malaria diagnosis.

8/6/2019 Comparative Detection of Plasmodium Vivax and Plasmodium Falciparum

17/20

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.

8/6/2019 Comparative Detection of Plasmodium Vivax and Plasmodium Falciparum

18/20

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.

8/6/2019 Comparative Detection of Plasmodium Vivax and Plasmodium Falciparum

19/20

how malarial DNA istransported to saliva and

urine of malaria patients?

8/6/2019 Comparative Detection of Plasmodium Vivax and Plasmodium Falciparum

20/20

Matur Nuwun..