Module 6

Malaria Rapid Diagnostic Testing

(RDT)

1
Learning Objectives

 Explain the significance of RDT in malaria control

 Describe the type of antigens detected

 Describe different RDT formats

 Explain the principle and general procedure of different RDT

 Describe principles of quality assurance in malaria RDT

2
Content Outline

 RDT and its Significance

 Antigens detected by malaria RDT
 RDT formats
 Principles and general procedure of malaria RDT
 Strength and Challenges of malaria RDT
 Choosing malaria RDT
 Quality Assurance of malaria RDT

3
 WHO Rec. & malaria Dx guidelines

Prompt parasitological confirmation of all malaria

suspected patients (all febrile patients)

Treatment solely on the basis of clinical suspicion should

only be considered when a parasitological diagnosis is

not accessible

10/07/20 4
 5

Malaria RDT

 Detects specific antigen derived from blood stage parasite

 Good alternative in the absence of microscopy

 Sensitivity & specificity of RDT is comparable to filed

microscopy

 Improves diagnosis and quality of patient care

10/07/20 Sindew M / EHNRI 5

Formats of RDT

1. Cassette –
 Expensive but safe and
easy to use
 Widely used

2. Card

3. Strip /Dipstick/

6
 7
Rapid Diagnostic Tests: Target Antigens
1. HRP II (Histidine-Rich Protein II)
- produced by P. falciparum only
- Target for Pf only RDTs
- persists after parasite death
2. pLDH (parasite Lactate Dehydrogenase enzyme)
- produced by all Plasmodium species
- D/t isomers of pLDH for each species
- Target for combination RDTs
- Closely reflects parasite viability

10/07/20 08:07 AM Sindew M / EHNRI 7

Cont…

3. pan-specific ( Aldolase) ~ found in all spp.

- Monoclonal antibodies are pan-specific

- Closely reflects parasite viability

10/07/20 8
 9
RDT mechanism of action …

• Dye-labeled antibody is pre-deposited on nitrocellulose strip.

• Bound Ab specific for target antigen (Ag) is bound on the test line

• Ab specific for dye labeled Ab is bound at the control line.

Test band
(bound Ab)
Control band
Dye labeled Ab (bound Ab)
unbound bound Ab
labeled Ab

10/07/20 08:07 AM Sindew M / EHNRI 9

 10

Mechanism …

• Blood is lysed with buffer to release more Ag

• Parasite antigen binds to the labeled antibody.

• Buffer flushes labeled Ag-Ab complex along the test strip.

buffer
Parasite Ag captured Parasitized
by labeled Ab blood

Blood and labeled Ab flushed along strip

10/07/20 08:07 AM Sindew M / EHNRI 10

 11

Mechanism …

• Some labeled Ag-Ab complex is trapped on the test line

• Excess labeled Ab is trapped on the control line

Labeled Ag-Ab Labeled Ab

complex captured by captured by
bound Ab at test line bound Ab of
control band

Captured labeled Captured

Ag-Ab complex labeled Ab

10/07/20 08:07 AM Sindew M / EHNRI 11

Strengths and Challenges of RDT

 Strengths
 Easy to use with minimal training
 Give rapid results & permitting immediate treatment in the site

 Do not rely electricity and special equipment

 Do not require refrigeration
 Uses whole blood

 Reinforce patient confidence in the diagnosis & health service

10/07/20 12
Cont…

Challenges

 Costs per test exceed microscopy

 Short shelf-life,

 Requires effective transportation, storage and distribution

systems

 Can’t estimate parasite density (qualitative test)

10/07/20 13
Cont…

 Intensity of test varies with parasite density

 Undetermined quality- Can be affected at manufacturing,

Testing, transport and storage

 Inability to distinguish new infection (HRP2 targeted RDTs)

10/07/20 14
Considerations in Choosing of Malaria RDT

 The type of plasmodium species found

 Accuracy (sensitivity and specificity).

 Shelf-life and temperature stability during transportation,

storage and use.

 Ease of use

 Cost

10/07/20 15
 The Type of Plasmodium spp found

WHO categorizes in to d/t zones

Zone 1. Pf only, sub-Saharan Africa and lowland Papua New

Guinea

Zone 2. Pf and non-Pf/Pv/ infections occurring commonly as single-

species infections - endemic areas in Asia, Americas and
Ethiopian

Zone 3. Areas with mainly Pv malaria- East Asia and central Asia

10/07/20 16
2. Accuracy (sensitivity and specificity):

 Sensitivity: True positive result as compared to reference

test( microscopy)

 Specificity: Ability of the test to exclude non-malaria patients

 > 95% is recommended

10/07/20 17
 3. Shelf life and stability

 Should be stored and transported based on the instructions

 Shelf life is usually 18 months (e.g. at least 15 months after

purchase)

 Longer shelf-life reduces pressure on supply chain and

wastage of expired tests

10/07/20 18
 4. Ease of use

 To be used in remote areas /e.g. at health posts/ an easy-to-use format

will be of greater importance. E.g. Cassettes.

5. Cost

 RDT are often more costly than microscopy

 should be considered when deciding purchase quantities

10/07/20 19
Types of Malaria RDT

Mono species RDT e.g. parachek

Multi species RDT e.g. CareStart

10/07/20 20
 Carestart test kit… procedures

Capillary blood Collection

1. Clean area to be pricked with an alcohol swab.

2. Squeeze the end of the fingertip and pierce with a sterile lancet.

3. Wipe away the first drop of blood

4. Gently squeeze the pipette provided, immerse the end in the

blood drop and gently release the pressure to draw blood up to

the black line/ring

10/07/20 21
Test procedure

1. Add 5μl of whole blood into

the sample well (small well).
2. Add two drops (60μl) of assay
buffer into the buffer well.
3. Read the test result in 20 min.

10/07/20 22
Test Procedure

10/07/20 23
Result reading and interpretation: Multi species

10/07/20 24
Result Reading…

In PfHRP2/PMA and the pLDH tests

 control line and pan specific lines positive

 Positive for non- Pf ( Pv, Pm and/or Po )

10/07/20 25
Result reading…

 Color change on all 3 lines

 Positive for Pf or mixed infection with non- Pf

10/07/20 26
Result reading…

 Control line and PfHRP2 or mono specific pLDH line

 Interpreted as Pf only

10/07/20 27
Points to remember when performing RDT

 Instructions should be strictly followed

 Blood-safety precautions

 Should be discarded if the envelope is punctured or damaged

 The test envelope should be opened only when it has reached

ambient temperature

 The RDT should be used immediately after opening

 RDT cannot be re-used

28
 Interpretation of RDT results

A negative test result does not A positive result does not

exclude malaria, because: always signify malaria illness

Insufficient parasites to give because:

a positive result. False positive b/c of post

RDT may have been treatment persistence

damaged

29
 Cont…

False-positive result:
 Illness may be caused by non
In individuals with high immunity

targeted spp to the RDTs there may be other causes of fever

Antigen persistency

30
 Quality assurance (QA) of malaria RDT

 This include
 Monitoring of the technical standard
 Minimize environmental abuse
 Training and onsite evaluation
 Interpretation by end-users.

31
QA of malaria RDT …

QA is intended to ensure high accuracy of tests by users

1. RDT introduction (planning)

2. Procurement

3. Lot testing: Pre and post purchase

4. Training and instructions for users

5. Use of results and community education

6. Storage and transport

32
Quality Assurance…

 Quality Control (QC)of malaria RDT

 Monitoring each steps of a test procedure to ensure that tests are

performed and interpreted correctly

33
QC can be monitored at three different stages.

1. Pre- Analytical Stage

 Store and transport RDT within temperature ranges

recommended by the manufacturer

 Check expiry date of RDT prior to use

 Check integrity of packages prior to use

34
2. Analytical Stage

 Ensure packages are opened only prior to testing

 Ensure tests are performed as per the manufacturer instruction

 Read results as per the manufacturer instruction only

 Avoid too much or too little blood and/or buffer

 Avoid using buffer from different box

35
3.Post Analytical Stage
 Reporting and recording
 E.g. for Carestart Pf/pan test result
 Pf
 Pf and pan
 Pan
 Avoid incorrect or misinterpretation of results
 Ensure that RDTs are not re-used.
 Ensure that all used RDTs and accessories are discarded
in a safe place for disposal

36
 Thank you!!!

10/07/20 37