Rapid diagnosis test versus blood

smear in Malaria
 Levels of parasitemia in different
group of malaria patients
Group Parasitemia (%) No parasites/µL blood
Subjects with positive blood smear 0.0001 – 0.0004 5 – 20
Patients with classical symptoms 0.0002 100
Patients with severe symptoms 0.2 10.000

Murray et al Clin.Micro.rev
 Microscopic examination

• Gold standard:
accordance to the level of parasite number
as causal of clinical symptoms (fever):
50 – 100/µL or 50.000 – 100.000/ml
• Detect parasites
• Can differentiate Plasmodium species and
stages
• Parasite count per µL
 Microscopic examination
• Accuracy depends on the person who
prepares and reads the malaria slides
• Requires long term experience
 Points of
blood smear preparation
• Finger prick blood
• Sufficient blood volume
• Reagents fulfill the standard criteria
• Clean glass object
Sequestration
BASOPHILIC STIPLING
PAPANHEIMER BODIES
 Parasite count
• Based on 1 microliter blood:
- 8.000 white blood cell (thick smear)
- 5.000.000 red blood cell (thin smear)
• Thick blood smear:
count parasite in 200 wbc, multiply by 40
• Thin blood smear:
count parasite in 1.000 erythrocyte and
multiply by 5.000.
• Percentage of infected erythrocyte:
number of parasite in 1 microliter/5.000.000 x 100%
 Weaknesses of microscopic
examination
• Low density of parasitemia
• Mixed infection
• Optimal morphology depends on preparation
of malarial blood smears
Digitalisasi pemeriksaan mikroskop
• Morfologi parasite ditransformasi ke bahasa
computer
• Dibuat alat digital yang dapat membaca secara
otomatis
• New test is required which has simple
procedure with accuracy of microscopic
level
 Rapid malaria test

• Is a test which essentially binds the

malaria antigens in the blood by using
monoclonal antibodies or Polyclonal
 Rapid malaria test
• Five kinds of antigen malaria:
1. PfHRP2 (P. falciparum histidine rich protein-2)
2. Plasmodium aldolase
3. Plasmodium lactate dehydrogenase (Pan-LDH,
Pf-LDH, Pv-LDH)
PfHRP-2 (Histidine Rich Protein-2)

• A water soluble protein that produced by

asexual stages and young gametocyte of P.
falciparum
• Detectable in the blood until 28 days of drug
administration (false positive)
 Plasmodium aldolase
• Is an enzyme of plasmodium from “glycolitic
pathway which is expressed by blood stages of
P. falciparum and non Pf
 Plasmodium aldolase
• Antibody monoclonal to Plasmodium aldolase
is pan-specific and utilize with the
combination of PfHRP2 as rapid diagnosis test
for Pf/Pv
Plasmodium lactate dehydrogenase (pLDH)

• The enzyme is found in all human plasmodia

(Pf, Pv, Pm dan Po)
• Can be made for various isomers pLDH which
is specific to those 4 species
• It is produced by asexual and sexual stages of
living plasmodium
 Rapid malaria test
• Developed in a variety of formats such as
dipstick, strip, card, tape or wells
• The procedure can vary between different test
kit
 Rapid malaria test
• Blood sample (2 to 50 µl) can be a finger tip
blood or blood with anti coagulant/plasma.
• The blood will be mixed with liquids which
contain components bufer hemolysis and
malaria specific antibody labelled with
colloidal gold so it can be seen if there is a
reaction
 Rapid malaria test
• When the target
antigen is present in the
blood, then it will form
a complex antigen
antibodies
• migrate in the strip to a
place where it's been
embedded in specific
antibodies against
antigens and antibodies
(control)
 Rapid malaria test
• Wash buffer is then added to remove
hemoglobin and cause the line formed by
antigen antibody complexes will appear.
 PfHRP2 PfHRP2/PMA pLDH

Control   

PfHRP2  

PMA

pLDH  

pfLDH 
 Rapid malaria test
• pLDH test capable of detecting the parasite
> 100 to 200 parasites/µl
• PfHRP2 tests able to detect > 40 parasites/µl
• Will be developed rapid test with sensitivity
as PCR: 1 – 10 of parasites/µl
Alonso Soto Tarazona et al
Evaluation of the Rapid Diagnostic Test OptiMAL for Diagnosis of Malaria
due to Plasmodium vivax
The Brazilian Journal of Infectious Diseases 2004;8(2):151-155

The sensitivityof the OptiMALÒ test progressively decreased when parasitemia

was lower than 1,000 parasites/microliter.

Table 2. Sensitivity of OptiMAL related to parasitemia levels

Parasitemia (parasites/ul) Number of patients Sensitivity

< 500 3 33%
501-1,000 6 83%
1,001-5,000 18 100%
5,001-10,000 3 100%
>10,000 9 100%
 Problems with rapid test
• A cross reaction with autoantibodies such as
rheumatoid factor so that can cause false
positive
 False negative
• Reported on severe infection where the
number of malaria parasites > 40,000/µL.
• Confirmation:
- microscopy
- repeat with diluted sample (NaCL 0.9%)
False negative
 False positive
• Persistent antigen due to parasite
sequestration

Peripheral Peripheral Placental Placental

Sens Spec Sens Spec

Peripheral ---- ---- 82% 86%

micros

Peripheral 96% 67% 95% 61%

RDT

Placental ---- ---- 95% 72%

RDT
 Multiple influence
• Stages of parasites
• Level of parasitemia
• The target antigen and the capture antibody
• Presistent antigenemia
• Sequstration of the parasites
• Present of auto antibodies
• Variation of batches
Malaria blood smear vs. Rapid tes
 Future Rapid diagnosis test
• Ultra/highly sensitive RDT:
- 10x lebih sensitive dari RDT (mendekati
PCR),
- P. falciparum asimtomatik malaria (densitas
parasite rendah)
- P. vivax masih dalam pengembangan
- Diperlukan dalam era eliminasi malaria
dimana jumlah parasite dalam darah
menurun
 Future Rapid diagnosis test
• Rapid test untuk P. knowlesi:
- bio marker spesifik P. knowlesi  PMT
(Phosfoethanolamine_N Methyltransferase)
- biosynthesis lipid
- tidak ditemukan pada host
- Ada pada Pf, Pv dan Pk
- larut dalam darah
PMT adalah bio marker berpotensi
dikembangkan untuk diagnostic Pk
• Rapid Diagnosis Test will become a more
important tools for malaria diagnosis in the
future due to practicability, improved
sensitivity and specificity in order to
differentiate plasmodium species