This document discusses malaria diagnostics, emphasizing the significance of accurate and timely detection for effective treatment and epidemiological purposes. It reviews various diagnostic methods, including microscopy, rapid diagnostic tests (RDTs), and molecular techniques, highlighting their advantages and limitations. The document also underlines the importance of adhering to guidelines for laboratory confirmation and monitoring of malaria cases.

1. Diagnostics for Malaria
Abdullatif Sami Al Rashed
Clinical Microbiology Resident.
King Fahd Hospital of the University.
Teaching Assistant, Department of Microbiology, Imam Abdulrahman
Bin Faisal University, Dammam, Saudi Arabia.

2. Objectives
To Present an up-to-date summary of
the best microbiology practice related
to malaria diagnostics

3. Introduction
• Prompt and accurate diagnosis of malaria is very
critical for implementation of appropriate
treatment to reduce associated morbidity and
mortality.
• Accurate detection of malaria is also important
for:
– Epidemiological screening and surveillance to inform
malaria control strategies,
– Research purposes in testing efficacy of antimalarial
drugs and vaccines, and
– Blood bank screening.

4. Introduction
• Malaria is caused by protozoan parasites of
the genus Plasmodium
https://www.moh.gov.sa/Ministry/MediaCenter/Public
ations/Documents/2018-07-29-002.pdf

5. Introduction
• Characteristics of a useful malaria diagnostic tool include:
The ability to definitively establish presence or absence of infection
Determine which species of malaria is/are present
Quantify parasitemia
Detect low-level parasitemia
Monitoring of response to antimalarial therapy (including
detection of recrudescence or relapse).
Thus far, there is no single
malaria diagnostic tool that
meets all of these criteria.

6. When To Suspect Malaria
• In general, malaria should be suspected in the
setting of:
Fever (temperature
≥37.5°C)
Relevant
epidemiologic
exposure (residence
in or travel to an
area where malaria
is endemic
+
World Health Organization. Guidelines for the treatment of malaria, 3rd ed, WHO, Geneva 2015

7. When To Suspect Malaria
• In malaria-endemic areas, malaria should also
be suspected in children <5 y with palmar
pallor or hemoglobin concentration <8 g/dL.
World Health Organization. Guidelines for the treatment of malaria, 3rd ed, WHO, Geneva 2015

8. When To Suspect Malaria
UK malaria treatment guidelines, 2016

9. Diagnostic Approaches
• Suspected malaria should be confirmed with a
parasitologic diagnosis whenever possible.
• If parasite-based diagnostic tools are NOT
readily available and there is clinical suspicion
for malaria, especially Plasmodium falciparum
infection, it is reasonable to make a
presumptive diagnosis of malaria and initiate
empiric therapy.
World Health Organization. Guidelines for the treatment of malaria, 3rd ed, WHO, Geneva 2015

10. Parasite-Based Diagnosis
• Clinical tools for parasite-based diagnosis
include:
1. Microscopy (visualization of parasites in stained
blood smears) and
2. Rapid diagnostic tests (RDTs; which detect
antigen or antibody).
Guideline: the laboratory diagnosis of malaria. General Haematology Task Force of the British Committee for Standards in Haematology.
• Smear examination via light microscopy is
the standard tool for diagnosis of malaria;
• RDTs should be used if microscopy is not
readily available.

11. Parasite-Based Diagnosis
• Molecular techniques for detection of genetic
material are limited to reference labs and
research settings
Guideline: the laboratory diagnosis of malaria. General Haematology Task Force of the British Committee for Standards in Haematology.

12. MICROSCOPY

13. Detection of parasites on Giemsa-stained
blood smears by light microscopy is the
gold standard tool for diagnosis of
malaria
World Health Organization. Guidelines for the treatment of malaria, 3rd ed, WHO, Geneva 2015

15. In Expert Hands:
• The sensitivity of microscopy can be
excellent, with detection of malaria
parasites at densities as low as 4 to 20
parasites/mcL of blood (approximately
0.0001 to 0.0005 percent parasitemia)
Dowling MA et al. A comparative study of thick and thin blood films in the diagnosis of scanty
malaria parasitaemia.

16. Drawbacks of Microscopy
1. Labor intensive and requires substantial training
and expertise.
2. The sensitivity and specificity of malaria
microscopy in resource-limited settings is often
below levels achievable in reference or research
laboratories.
3. Microscopy cannot reliably detect very low
parasitemia (<5 to 10 parasites/mcL) or cases
where the majority of the parasite biomass is
sequestered (eg, in the case of placental
sequestration during pregnancy)
Kilian AH et al; Malaria diagnosis: memorandum from a WHO meeting.

17. Notes in Blood Smear Preparation
1. Capillary blood from a fingerprick (or earlobe or
infant heelstick) or anticoagulated venous blood
may be used for malaria smear.
2. Smears should be prepared as soon as possible
after blood collection to avoid alteration in
parasite morphology and staining properties.
3. Malaria parasites are best seen under 100x
magnification using the oil immersion objective
lens; smear evaluation should include
examination of at least 200 to 500 fields or
examination for 20 to 30 minutes.
Kilian AH et al; Malaria diagnosis: memorandum from a WHO meeting.

18. If malaria is suspected and the initial smear is
NEGATIVE, additional smears should be
prepared and examined over the subsequent
48 to 72 hours (White NJ. N Engl J Med)
The CDC recommends repeating
a thick and thin smear every 12
to 24 hours for a total of three
sets before ruling out malaria
http://www.cdc.gov/malaria/diagnosis_treatment/clinicians1.html
After confirmed diagnosis, serial smears should
be examined to monitor the parasitological
response and ensure resolution of infection.
http://www.cdc.gov/malaria/diagnosis_treatment/clinicians1.html

20. RAPID DIAGNOSTIC TEST
IMMUNOCHROMATOGRAPHIC
TESTS
(RDT)

21. RDTs for detection of malaria parasite antigens are
increasingly important diagnostic tools in resource-limited
endemic settings due to their accuracy and ease of use.
They require no electricity or laboratory infrastructure, give
results within 15 to 20 minutes, and can be performed
successfully even by health workers with limited training.
RDTs provide a qualitative result but cannot provide
quantitative information regarding parasite density.

22.  The approach to RDT selection depends on the
epidemiology of infection and goals for control in
the region where the test is used.
 In regions where infection is primarily caused by P.
falciparum, use of an assay that detects P. falciparum
only may be sufficient and cost-effective.
 In regions where falciparum and non-falciparum
parasites coexist or where P. vivax predominates, use
of an RDT that can detect and distinguish between
these species is warranted

23. Histidine-
rich protein
2
(HRP2)
Plasmodiu
m lactate
dehydroge
nase
(pLDH)
Aldolase
RDT TYPES

24. HRP2 RDT
• Histidine-rich protein 2 is part of a family of P.
falciparum histidine-rich proteins that is only
produced by P. falciparum
• Use of HRP2-based RDTs is appropriate in
regions where P. falciparum is the
predominant species
– (eg, much of sub-Saharan Africa).
• Combination tests may be useful in areas
endemic for multiple Plasmodium species.
Rakotonirina et al, 2007
If the parasite density of >100
parasite/μl, sensitivity is 95% (95%
Confidence Interval 93.5 – 96.2%) and
specificity is 95.2%, (95% CI 93.4 –
99.4 %) (Abba K et al., 2011)

25. Drawbacks of HRP2 RDT
1. Detectable HRP2 antigen may persist in the
bloodstream for a few days to several weeks
after parasitemia is no longer present.
– In the absence of good quality confirmatory
microscopy, there is currently no way to distinguish
HRP2 positive test resulting from a new infection or
persistent infection (ie, resulting from treatment
failure) from antigenemia persisting from a recently
treated infection.
– Some endemic countries' national guidelines therefore
advise that a positive HRP2 result should be
considered to represent a new infection only if the
specimen was drawn 7 to 14 days or longer following a
previously treated infection
Rakotonirina et al, 2007

26. Drawbacks of HRP2 RDT
2. Deletions in the genes encoding for HRP2
(and the similar HRP3 protein), leading to
false-negative RDT results, have been
identified in P. falciparum parasites from the
Peruvian Amazon; therefore, HRP2-based
tests are not reliable for infections contracted
in this area.
– More recently, reports have emerged of pfhrp2
and pfhrp3 mutations or deletions in Africa and
India leading to false-negative RDT results in
some cases
Rakotonirina et al, 2007

27. Drawbacks of HRP2 RDT
3. Rarely, false-negative HRP2 results may occur
at very high levels of antigenemia or
parasitemia due to a prozone-like effect
Rakotonirina et al, 2007

28. pLDH RDT
pLDH is a
terminal
enzyme in the
malaria parasite
glycolytic
pathway that is
produced all
Plasmodium
species.
Two types of pLDH-based
RDTs are available:
Target a
conserved
pLDH element
in all human
malaria
species (non
specific)
Target species-
specific
regions that
distinguish P.
falciparum or
P. vivax.
Rakotonirina et al, 2007

29. Serum pLDH levels correlate with
parasite density and become
undetectable at the same time
blood smears become negative
following antimalarial therapy
(may be used for monitoring
following treatment and also to
diagnose treatment failure)
pLDH-based RDTs are less
sensitive than HRP2-based tests
for detection of P. falciparum
infection, especially at relatively
low parasite densities (<500
parasites/mcL)
Sensitivity 93.2% (95% CI 88 –
96.2 %)
Advantages: Disadvantages:
(Abba K et al., 2011)(Houzé S et al., 2009)
pLDH RDT

30. Aldolase is an enzyme in the malaria parasite glycolytic
pathway that is conserved across all human malaria species.
Serum aldolase levels correlate with parasite density and
become undetectable with clearance of parasitemia.
For detection of P. falciparum, the sensitivity of aldolase-
based assays is generally lower than that of HRP2-based
assays.
For detection of non-falciparum infections, the sensitivity of
aldolase and pLDH assays is comparable.
Aldolase RDT
TDR/World Health Organization & FIND. Malaria Rapid Diagnostic Test Performance: Summary
results of WHO product testing of malaria RDTs: Round 1-4 2008-2012. Geneva, Switzerland,
TDR/WHO, FIND, CDC. 2012

32. One RDT has been approved by the US
Food and Drug Administration (FDA):
BinaxNOW Malaria. The test is a
combination assay with antibodies for
detection of HRP2 and aldolase.

33. Screening Blood Donors?
• RDTs should not be used for screening
donated blood, since the thresholds of
detection are not sufficiently sensitive to
detect low parasite density. (Murray CK et al., 2008; Seed CR et al., 2005)
• Malaria antibody detection for by using the
indirect fluorescent antibody (IFA) test is used
for screening blood donors involved in cases
of transfusion-induced malaria to overcome
the undetectable level of parasitemia by blood
film examination & RDTs.
Rakotonirina et al, 2007

34. MOLECULAR TESTS

35. Use of molecular tests for malaria detection is
generally limited to reference laboratories and
is primarily for research and epidemiologic
purposes.
The CDC offers PCR confirmation of species and
identification of drug resistance mutations for
malaria cases diagnosed in the United States
http://www.cdc.gov/malaria/features/ars.html

36. Molecular Testing
• Nested PCR is the most sensitive and specific
nucleic acid amplification technology.
• Commonly used PCR assays target genus-
specific and species-specific sequences of the
18S small-subunit ribosomal RNA,
circumsporozoite surface protein (a nuclear
gene encoding a cysteine protease), and the
cytochrome b gene
Mixson-Hayden T et al., 2010

38. SUMMARY OF THE GUIDELINES

39. Microscopy
Saudi Guidelines CDC Yellow book WHO UK guidelines
Laboratory confirmation is obtained by demonstration
of malaria parasites in blood films (thick and thin film).
This remains the gold standard method for the
diagnosis, identification of Plasmodium
species and estimation of parasite density.
WHO

40. RDT
Saudi Guidelines CDC Yellow book WHO UK guidelines
• A Rapid Diagnostic Test (RDT) is an alternate test to confirm the
diagnosis of malaria infection by detecting specific malaria
antigens in the patient’s blood. (Saudi)
• Both positive and negative RDT results must always be confirmed
by microscopy. (CDC)
• Because of a lack of expertise in many UK labs, particularly out of
hours, rapid diagnostic tests (RDTs) based upon detection of
parasite antigens, are now commonly used in addition to blood
slides. Although slightly less sensitive than good quality blood films
examined by experienced microscopists, they are easier for the
non-expert to use to detect falciparum infections and are useful as
an initial screen if expertise in reading slides is not immediately
available. (UK)

41. Molecular
Saudi Guidelines CDC Yellow book WHO UK guidelines
• PCR techniques are used in reference laboratories to
determine the species of malaria but are not sufficiently
standardized or validated to use for routine clinical diagnosis.
(UK)
• Use of PCR testing is encouraged to confirm the species of
malaria parasite and detect mixed infections. (CDC)
• Techniques to detect parasites nucleic acid are highly
sensitive and very useful for detecting mixed infections, at
low parasite densities that are not detectable by conventional
microscopy or RDT, and in estimating drug resistance and
epidemiological studies. however these tests have no role in
clinical management of malaria or routine diagnostivc testing
(WHO)

42. References
• Goswami ND, LoBue PA. INFECTIOUS AGENT. CDC
Yellow Book 2020: Health Information for
International Travel. 2019 May 14;87(6):355.
• Lalloo DG, Shingadia D, Bell DJ, Beeching NJ, Whitty
CJ, Chiodini PL. UK malaria treatment guidelines
2016. Journal of Infection. 2016 Jun 1;72(6):635-49.
• World Health Organization (WHO): Guidelines for the
treatment of malaria, 3rd edition (2015)
• NATIONAL MALARIA DRUG POLICY SAUDI ARABIA