1. بسم الله الرحمن الرحیم

2. By Shervin Ghaffari Hoseini MD. PhD
Diagnosis of Malaria
By Shervin Ghaffari Hoseini
MD. PhD

3. Malaria is the world's most important parasitic disease, and kills more people than any other communicable disease except tuberculosis.
Each year million cases of malaria occur worldwide, and over one million people die

5. Laboratory diagnosis of malaria
Microscopic Diagnosis
Blood smear
Fluorescent Microscopy
Quantitative Buffy Coat (QBC ®)
Antigen Detection
Immunochromatographic Dipstick: RDT
Serology
IFA
ELISA
Molecular Diagnosis
PCR
Real time PCR

6. Malaria Blood Smear Remains the gold standard for diagnosis
Blood sample from vein or Puncture from finger pulp
New and clean slide
Both thin and thick films for all patients

7. Malaria Blood Smear Thin films: Dry Fix Stain Dry
Thick films:
Dry
Do not fix but dehemoglobinate
Stain
Staining methods:
Giemsa stain
Leishman's stain
Field’s stain

8. Malaria Blood Smear Thick films:
first step: Examination of a thick blood film
20 fold more concentrated
species identification difficult
minimum of 200 oil immersion fields
about 15 minutes for an experienced observer

9. Malaria Blood Smear Thin films: determine the species
entire thin film should be examined
about minutes for an experienced observer

10. Interpreting Thick and Thin Films
fixed RBCs, single layer
smaller volume
0.005 μl blood/100 fields
good species differentiation
requires more time to read
low density infections can be missed
THICK FILM
lysed RBCs
larger volume
0.25 μl blood/100 fields
more difficult to diagnose species
good screening test

11. Calculating Parasite Density - 1
Count the number of parasitized and nonparasitized RBCs in the same fields on thin smear
Count RBCs
# parasitized RBCs
total # of RBCs
% parasitemia =
X 100

12. Calculating Parasite Density -2
Count ≥ 200 WBCs on thick film
Assume WBC is 8000/l (or count it)
parasites counted
WBC counted
X WBC count/l
parasites/l =

13. Estimating Parasite Density Alternate Method
Count the number of asexual parasites per high-power field (HPF) on a thick blood film
Parasites observed
Percentage of red cells parasitized
10-20 per field 1
1-2 per field
0.1
1-2 per 10 fields
0.01
1-2 per 100 fields
0.001
1-2 per 1000 fields
0.0001
1-10 parasites per 100 HPF +
parasites per 100 HPF ++
1-10 parasites per each HPF
+++
> 10 parasites per each HPF
++++

14. Mixed infections are not uncommon.
Malaria Blood Smear
Prepare smears as soon as possible
Don not fix thick smear
time-honoured peripheral smear study
Identify P. falciparum in a dual infection with P. vivax
Mixed infections are not uncommon.

15. malarial pigment in circulating neutrophils and monocytes is useful
Malaria Blood Smear
negative test DOES NOT rule out malaria
Repeat tests
partial antimalarial treatment
sequestration of parasitised cells in deep vascular beds
malarial pigment in circulating neutrophils and monocytes is useful

16. Malaria Blood Smear Advantages
Distinguishes between species and life cycle stages
Parasitemia is quantifiable
Threshold of detection
thin film: 100 parasites/ 1 μ lit
thick film: parasites/ 1 μ lit
Simple and inexpensive

17. Malaria Blood Smear Disadvantages labor-intensive
equipment, training and supervision needed

18. Malaria Parasite Erythrocytic Stages
Ring form
Trophozoite
Schizont
Gametocytes

20. Fluorescent Microscopy
Fluorescent dyes detect RNA and DNA that is contained in parasites
Nucleic material not normally in mature RBCs
Stain thin film with acridine orange (AO)
Requires special equipment – fluorescent microscope
Staining itself is cheap
Sensitivities around 90%

21. Quantitative Buffy Coat (QBC ®)

22. Quantitative Buffy Coat (QBC ®)
Advantages
Useful for screening large numbers of samples
Quick, saves time
Due to larger volume of blood observed, method is more sensitive
diagnosis of other diseases such as Babesiosis, Trypanosomiasis and Filariasis is possible

23. Quantitative Buffy Coat (QBC ®)
Disadvantages
Species identification and quantification difficult: thick/thin films on QBC-positive samples is required
High cost of capillaries and equipment
Can’t store capillaries for later reference

24. An adapted light microscope for the viewing of QBC tubes.

25. Trophozoites of P. falciparum.

26. Malaria Antigen Detection
Immunologic assays to detect specific antigens
Commercial kits now available as immunochromatographic rapid diagnostic tests (RDTs), used with blood
P. falciparum histidine-rich protein 2 (PfHRP-2)
parasite LDH (pLDH)

27. Mode of action of common malaria RDT format

29. Malaria Antigen Detection - RDTs
Feature
PfHRP-2 tests
pLDH tests
Sensitivity/
Specificity*
Sensitivity %
Specificity %
Sensitivity P.f %
P.v %
Specificity P.f %
P.v %
Commercialcost/test**
Approximately US$ 0.60 –1.00
Approximately US$ 2.50
Commercial products
PATH falciparum Malaria IC Strip test – Program for Appropriate Technology in Health
MAKROmed™
Orchid ®
OptiMAL® - Flow, Inc.
Binax NOW ®ICT Malaria - Binax, Inc.
* Compared to microscopy, results from multiple studies
** Varies by size of order and vendor

30. Detection of Plasmodium antigens
A: HRP-2 (histidine-rich protein 2) (ICT)
B: pLDH (parasite lactate dehydrogenase)(Flow)
C: HRP-2 (histidine-rich protein 2) (PATH)

31. Antigen Detection Malaria Immunochromatographic Dipstick
OptiMAL Assay
P. falciparum specific
monoclonal antibody
Control
Plasmodium pan specific
monoclonal antibody

34. Malaria Antigen Detection - RDTs
Disadvantages
The use of the RDT does not eliminate the need for malaria microscopy
Cannot detect mixed infections
may not be able to detect infections with lower parasitemia
Cannot detect P. ovale and P. malariae
microscopy is needed to quantify parasitemia

35. Positive control: A panel of wells containing different antigens
Using quality control parasite dilutions to test the sensitivity of malaria RDTs: Producing and storing quality control dilutions of parasites obtained from field samples

36. Application of RDTs Potential uses Epidemics and emergencies
Inadequate or absent lab services, unskilled staff
Mobile clinics
self-diagnosis by travelers entering endemic areas
outbreak investigation and surveys of parasite prevalence

37. Para Sight F test

38. Malaria Serology – antibody detection
Methods
IFA
ELISA
Not practical for routine diagnosis of acute malaria because:
Delaied development of antibody
persistence of antibodies
Serology does not detect current infection but rather measures past experience

39. Malaria Serology Valuable epidemiologic tool in some settings
Useful for
Identifying infective donor in transfusion-transmitted malaria
Investigating congenital malaria, esp. if mom’s smear is negative
Retrospective confirmation of empirically-treated non-immunes

40. Indirect fluorescent antibody (IFA) test
Indirect fluorescent antibody (IFA) test. The fluorescence indicates that the patient serum being tested contains antibodies that are reacting with the antigen preparation (here, Plasmodium falciparum parasites).

41. Polymerase Chain Reaction (PCR)
Molecular technique to identify parasite genetic material
Uses whole blood collected in anticoagulated tube (200 µl) or directly onto filter paper (5 µl)

42. Polymerase Chain Reaction (PCR)
Advantages
PCR is a reference method. It is at least 10-fold more sensitive than microscopy.
Threshold of detection
0.1 parasite/µl if whole blood in tube
2 parasites/µl if using filter paper
more reliable for determining species in a mixed infection.
Can identify mutations – try to correlate to drug resistance
May have use in epidemiologic studies

43. Polymerase Chain Reaction (PCR)
Disadvantages
Parasitemia not quantifiable
Requires specialized equipment, reagents, and training

44. analysis of a PCR diagnostic test for species-specific detection of Plasmodium DNA.  PCR was performed using nested primers

45. Real-Time PCR potential to quantify parasitemia,
may detect multiple wavelengths in same tube identifying multiple species in one run
Needs further research and validation for malaria

46. Real-Time PCR

47. Quantitative Real-Time PCR

48. BCSH Guidelines for Quality Control
All malaria films should be examined by two observers
All new batches of Giemsa stain should be tested with a known P. vivax infection
ensure that Schüffner’s dots are stained
parasitised cells are decolourised.
Blood films for this purpose can be sealed in plastic slide boxes and frozen

49. BCSH Guidelines for Quality Control continue
All laboratories must ensure that new staff are adequately trained and maintain their skills:
Sets of mixed positive and negative thick and thin films should be available for examination
reference laboratories can often provide spare films for training purposes.
High quality photographs of malaria parasites should be available for reference

50. BCSH Guidelines for Quality Control continue
Websites can be used for on-going training.
(Centres for Disease Control and Prevention, USA)
(Royal Perth Hospital, West Australia, click on malaria information for learn and test yourself site)

52. Plasmodium falciparum
Infected erythrocytes: normal size M I
Gametocytes: mature (M)and
immature (I) forms (I is rarely
seen in peripheral blood)
Rings: double chromatin dots; appliqué forms;
multiple infections in same red cell
Schizonts: 8-24 merozoites
(rarely seen in peripheral blood)
Trophozoites: compact
(rarely seen in
peripheral blood)

53. Plasmodium vivax
Infected erythrocytes: enlarged up to 2X; deformed; (Schüffner’s dots)
Rings
Trophozoites: ameboid; deforms the erythrocyte
Schizonts: merozoites
Gametocytes: round-oval

54. “malariae - like parasite in vivax - like erythrocyte”
Plasmodium ovale
Infected erythrocytes: moderately enlarged (11/4 X); fimbriated; oval; (Schüffner’s dots)
“malariae - like parasite in vivax - like erythrocyte”
Trophozoites: compact
Rings
Gametocytes: round-oval
Schizonts: 6-14 merozoites;
dark pigment; (“rosettes”)

55. Plasmodium malariae
Infected erythrocytes: size normal to decreased (3/4X)
Trophozoite:
typical
band form
Trophozoite:
compact
Schizont:
6-12 merozoites;
coarse, dark pigment
Gametocyte:
round; coarse,
dark pigment