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• Introduction Malaria Epidemiology
• Malaria at KSA / Gulf area
• Blood Transfusion Malaria
• Study Objectives.
• Study Material and methods.
• Principle of Different Techniques.
• Results and discussion.

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• Introduction Malaria Epidemiology

Scientific studies on malaria made their first
significant advance in 1880, when a French army
doctor working in the military hospital
of Constantine in Algeria named Charles Louis
Alphonse Laveran observed parasites for the first
time, inside the red blood cells of people
suffering from malaria
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• Introduction Malaria Epidemiology

• Malaria is a preventable and treatable disease.
• It is a public health problem today in more than
  100 countries inhabited by some 2,400 million
  people -- 40 percent of the world's population.
• Malaria is estimated to cause 300- 500 million
  clinical cases and over one million deaths each
  year.
• Every 30 seconds, a child somewhere dies of
  malaria. In any given year, nearly ten percent of
  the global population will suffer a case of
  malaria.
• Most survive after an illness of 10-20 days.

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• Introduction Malaria Epidemiology

Global Distribution of Malaria (World Map)
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• Malaria at KSA / Gulf area

Malaria Endemic Area in KSA
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• Malaria at KSA / Gulf area

Source, MOH, Malaria Elimination Program - 2009
REPORTED MALARIA CASES IN KSA 1997/2008
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• Malaria at KSA / Gulf area

• In the Kingdom of Bahrain (1992 2001), 1572
  malaria cases (all of them were imported 85.6
  85.6 P. vivax and 14 P. falciparum) were
  reported Ismaeel et al. (2004).
• In Kuwait the following ranges of malaria were
  reported
• (1985 1990) 465 to 1,229 cases.
• (1992 1997) 684 to 1,379 cases.
• (1998 2000) 248 to 3939 cases.
• P . Vivax infection was detected in 71 and
  P. falciparum in 27
• of them.

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• Malaria at KSA / Gulf area

• In Saudi Arabia Al-Khalife (2003) reviewed
  records of the Malaria Referral Laboratory in
  Riyadh for the 6 years 1996 2001. He found
  that
• The annual mean number of positive slides was
  212 78. Positive thick films were reported at a
  rate of 18/100,000 among 137,402 potential blood
  donors screened during that period.
• Most slides were from Saudis (36.6), Sudanese
  (30.9), Indians (13.9),Pakistanis (8) and
  Yemenis (5). The type of malaria infection
  varied across the different nationalities,
  reflecting the pattern of endemicity, so that
  P.Falciparum represented 87.1 of infections in
  Saudis, 52.9 in Sudanese and 76.4 in Yeminis,
  while P. vivax represented 92.2 in Pakistanis
  and 88.1 in Indians.

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• Blood Transfusion Malaria

• The increased demand for blood transfusion has
  let to a worldwide increase of cases of
  transfusion malaria.
• An important factor of the risk is that the
  malaria parasites can survive in the
  refrigerator for a week and still be potent
  enough to cause an infection ( Olivier et
• al., 2002).

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• Blood Transfusion Malaria

• Guidelines of the US Food and Drug Administration
  ( FDA) and the American Association of Blood
  Banks (AABB) for deferral of blood donors at
  increased risk of malaria are to defer blood
  donation from travelers who are residents of
  non-malarious area and who have been in a
  malarious area for one year after their return
  (irrespective of the use of chemoprophylaxis) if
  they have been of malaria symptoms.
• Blood donation is also deferred from immigrants
  or visitors from malarious areas for three years
  after their departure from the malarious area if
  they have been asymptomatic, while persons who
  have had a diagnosis of malaria should be
  deferred for 3 years after becoming asymptomatic
  (AABB, 2002).

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• Study Objectives.

To identify and evaluate the best method(s) for
Malaria Diagnosis (The W.H.O recommends a
prompt and accurate diagnosis of Malaria).
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• Study Material and methods.

• 200 Malaria Patients from endemic area.
• 200 Healthy Blood Donors.

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• Study Material and methods.

Techniques under evaluation

• Thick and thin blood films
• Automated depolarization analysis.
• Malaria antibody test.
• Malaria antigen test (Two methods).
• Polymerase Chain Reaction (PCR).

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• Principle of Different Techniques.

The Blood Films

• The thick blood film provides sensitivity.
• The thin film is used for differentiation of
  species.

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• Principle of Different Techniques.

The Blood Films
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• Principle of Different Techniques.

The Automated Depolarization Analyzer

• It uses scattered Laser-light at different
  angles.
• There is optical differentiation color codes for
  the
• different white cell populations.
• The internalized malaria pigment (Haemozoin)
• causes unusual pattern of depolarizing
  leukocyte.

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• Principle of Different Techniques.

Malaria Antigen Test.

• Test is a direct enzyme-linked immunosorbent
  assay (ELISA) test using a specific monoclonal
  antibody.

• It recognizes the histidine-rich protein-2
  (HRP-2)1, a metabolic by product of the
  P.falciparum parasite, for specific detection of
  P. falciparum.

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• Principle of Different Techniques.

Malaria Antigen Test - 2

• It is an antigen immuno-capture ELISA test based
  on the detection of the specific intracellular
  antigen, lactate dehydrogenase (pLDH), a parasite
  enzyme present in infected erythrocytes.

• The test can detect all viable human malaria
  species in whole blood and can also be used with
  P.falciparum culture samples to measure metabolic
  inhibition during in vitro drug sensitivity assay.

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• Principle of Different Techniques.

Pan-Malaria Antibody Test.

• It is an antigen-based test, which detects
  antibodies to plasmodium species (P.falciparum ,
  P.vivax, P.ovale and P.malariae) in human serum.

• It is an indirect or sandwich ELISA test that
  utilizes a specific monoclonal antibody against
  Histidine - rich protein 2 (HRP-2).

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• Principle of Different Techniques.

Malaria Antibody Test.

• Specific antibodies and immunoglobulins spp.
  which produced when the plasmodium parasite
  infection occur.

• These antibodies are generally detectable
  shortly after the onset of infection.

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• Principle of Different Techniques.

Polymerase Chain Reaction(PCR)

• The test is based on the genetic composition of
  the parasite.
• It is both specific and sensitive.

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• Results and discussion.

Demographic data for the patient and control
groups.
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• Results and discussion.

Laboratory data pertaining to the patient group.
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• Results and discussion.

Laboratory data pertaining to selected control
group.
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• Results and discussion.

Sensitivity, specificity, positive predictive
value (PPV), negative predictive value (NPV) and
accuracy of different techniques for malaria
diagnosis.
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• Results and discussion.

Comparison of qualitative data for patient and
control groups.
HS highly significant statistically Plt0.001
(Chi-squar test).
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• Results and discussion.

A normal thick film (no malaria parasite seen).
Arrows indicate white blood cells.
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• Results and discussion.

A
B
C
Thick blood film showing ring form of P.
falciparum in a patient with a parasitic density
of 100,000/µL in peripheral blood A. White Blood
Cell (neutrophil) B. White Blood Cell
(Lymphocyte) C. P. falciparum rings forms
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• Results and discussion.

Normal depolarisation
Screen capture of sample analysis the leukocyte
differential graphic (upper left) shows an
extended neutrophil cluster (yellow events) that
is suggestive of sample ageing. Review of the
90?-depolarised (granularity) versus
90?-polarised (lobularity) plot (upper right)
reveals a normal discrete depolarising eosinophil
population (green events) with no other abnormal
depolarising events.  Blue Lymphocytes
Yellow Neutrophils Green Eosinophils Purple
Monocytes Brown Nucleated red blood cells.
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• Results and discussion.

Abnormal depolarisation
Screen capture of sample analysis the
differential graphic (upper left) shows normal
leukocyte subpopulation cluster locations
together with an abnormal population of events
(brown) below the instrument threshold. This
latter population may correspond to nucleated red
blood cells or platelet aggregates. Review of the
90-depolarised (granularity) versus
90-polarised (lobularity) plot (upper right)
reveals the presence of different abnormal
depolarizing events. The predominant abnormal
depolarising population is colour-coded purple
and almost certainly corresponds to monocytes
with internalised malaria pigment. In addition,
there are significant numbers of depolarising
green events that are not located in the position
associated with normal eosinophils. These
probably correspond to neutrophils with
internalised malaria pigment. A small number of
abnormal depolarising blue events is also noted,
but their cellular origin is unclear.  Blue
Lymphocytes Yellow Neutrophils Green
Eosinophils Purple Monocytes Brown Nucleated
red blood cells.
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• Results and discussion.

Samples showing occasional atypical depolarising
purple events  
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• Results and discussion.

Samples showing many atypical depolarising purple
events
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• Results and discussion.

Comparing sensitivity of different malaria
diagnostic tests Figure demonstrates that the
sensitivity of the blood film test (98.5) is
slightly lower than those of the automated
depolarization analyzer ELISA malaria antigen
test - 1 and ELISA pan malaria antigen test - 2,
all of which scored 100 on this parameter.
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• Results and discussion.

Automated Depolarized Method
Ag Method 1
Ag Method 2
Comparing accuracy of different malaria
diagnostic tests
Figure shows that the sensitivity of the blood
film test (99.25) is trivially lower than for
the automated depolarization analyzer ELISA
malaria antigen test - 1 and ELISA pan malaria
Antigen test which again registered 100 each.
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• Results and discussion.

Automated Depolarized Method
Ag Method 1
Ag Method 2
Comparing specificity and positive predictive
values of different malaria diagnostic tests
based on antigen detection.
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• Results and discussion.

Real-time PCR amplification plot, showing three
amplified samples for malaria parasites as well
as a positive and negative control.
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• Results and discussion.

• Beside low cost and highly sensitive thick
  blood film technique other methods might also
  be employed to diagnose malaria.
• The Automated depolarization analyzer was found
  to be a sensitive malaria diagnostic tool.

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• Results and discussion.

• The Automated depolarization analyzer was found
  to be a sensitive malaria diagnostic tool.
• During epidemiological surveys, misdiognosed
  cases of malaria infection could be successfully
  verified by using the Automated depolarization
  analyzer.
• The Automated depolarization analyzer was found
  to be cost effective, highly sensitive and more
  convenient technique.

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• Results and discussion.

• The ELISA antigen testing techniques were found
  to be very sensitive tools, and their sensitivity
  was comparable to the Automated depolarization
  analyzer in malaria diagnostic. The antibody
  ELISA test were establish to have poor
  sensitivity for malaria diagnosis.

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• Results and discussion.

The polymerase chain reaction (PCR)
technique was expensive but highly sensitive
technique which might be used to resolve any
discrepancy in malaria diagnosis.
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• Results and discussion.

CONCLUSION
According to the results of the current study,
different strategies for malaria patient and
blood donors in endemic and non-endemic areas of
Saudi Arabia may be adopted. For
patient-diagnosis of malaria in both endemic and
non-endemic areas, The Automated depolarization
analyzer is one of the other available and
reliable techniques. The thick blood film and the
antigen tests, with confirmation of the positive
results by PCR technique may be used.
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Thank you