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An Overview of Diagnostic Methods in Malaria

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An Overview of Diagnostic Methods in Malaria Powered By Docstoc
					Rapid and accurate diagnosis
is key to effective treatment
and management of malaria.

Conventional approaches

1. Clinical assessment
2. Light Microscopy
1. CLINICAL ASSESSMENT

 Done by trained health workers in areas
 where microscopy is not available

 Based on signs and symptoms and
 history of travel to a malaria-endemic
 area

  Not very specific (50% accurate, WHO)
  Conventional methods of malaria diagnosis

2. LIGHT MICROSCOPY
 “gold standard”
 based on demonstration of parasites in blood films
 It is specific and enables the identification of parasite at the
 species level
 It is quantitative, allows monitoring of response to treatment
 requires high degree of expertise for reliable results
 needs special equipment (microscope) and good reagents
 labor-intensive and time consuming
Malaria Microscopy Training
     Competency based training:
      Should be able to identify, differentiate,& count the
 four species of malaria parasite

 have to achieve 80% grade to pass the course

     2- weeks for medical technologists
     5- weeks for barangay microscopists
Intensive microscopy training
          activities
1. Fluorescent microscopy
        Quantitative Buffy Coat (QBC), Kawamoto, BCP
2. Molecular Techniques (Nucleic Acid Probes)
        Polymerase Chain Reaction (PCR)
        DNA hybridization
3. Immunological Techniques
    A. Antibody detection
           Serology - ELISA, IFAT
    B. Antigen detection
           RDTs (Rapid Diagnostic Tests)
   Staining of malaria parasites
    with a fluorochrome dye
Conventional microscopy
“made easy” or facilitated

Improved sensitivity (>90%)
when     parasitemia is
more than     100
parasites/ul
                              RPH Image Collection
Needs special equipment      Estimation of
and reagents (Paralens       parasitemia is not
objective)                   possible
Staining is not permanent,   Difficulty in species
QC/QA not possible           identification
Safety hazards in            False(+) due to
centrifuging blood (QBC)     Howell-Jolly bodies
Acridine orange is           Relatively expensive
hazardous and needs
proper disposal
detect specific parasite nucleic acid sequences

1.   DNA hybridization


2.   Polymerase Chain Reaction
              (PCR)
Advantages:
Highly sensitive – can detect as low as 5 parasites/ul
   blood with 100% specificity
May be a tool for research, quality control checks on
   microscopic diagnosis or to determine distribution of
   important genes


Disadvantages:
Expensive, needs special equipment and facilities
Labor-intensive
Can’t distinguish between viable and non-viable
parasites; past and present infection
 Serology

Antibody detection
For retrospective study , particularly for epidemiological
purposes, and for tracing asymptomatic infections in
blood donors
Measures prior exposure, but not necessarily current
infection
Most commonly used techniques:
    ELISA – Enzyme-Linked Immunosorbent Assay
    IFAT – Indirect Fluorescent Antibody Test
MALARIA RAPID DIAGNOSTIC TESTS
            (RDTs)
Also called “dipsticks” or “malaria rapid diagnostic
devices”.
Lateral flow immuno-chromatographic antigen-
detection tests, in dipstick or cassette format
Detect antigens (or proteins) produced by the
malaria parasites in human blood.
Some RDTs detect P. falciparum only
(Ex. Paracheck Pf ).
Some detect P. falciparum + P.vivax or P.malariae
or P. ovale (OptiMAL, ICT).
Single test takes about 10-20 minutes.
They come in cassette, dipstick or card formats.
                  Mechanism of action

1. Dye-labeled specific antibody (Ab) is pre-deposited on
   nitrocellulose strip or in a plastic well. Ab specific for
   target antigen (Ag) is bound to the strip in test line, and Ab
   specific for labeled Ab, or Ag is bound at the control line.

                                            Test band
                                            (bound Ab)
                                                    Control band
                                   Labeled Ab       (bound Ab)
  unbound         bound Ab
  labeled Ab
                 Mechanism of action

2. Blood is lysed with buffer to release more proteins (Ag).
   Parasite antigen, if present, binds to the labeled
   antibody. Buffer flushes the labeled antigen-antibody
   complex along the test strip.

                              buffer
   Parasite Ag captured                Parasitised
   by labeled Ab                       blood




                                  Blood and labeled Ab flushed
                                  along strip.
                 Mechanism of action

3. 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 is captured                               captured by
 by bound Ab of test                               bound Ab of
 band                                              control band


           Captured labeled           Captured
           Ag-Ab complex              labeled Ab
               Target Antigens
1. HRP II (Histidine-Rich Protein II)
        - produced by P. falciparum only
        - persists after parasite death
2. pLDH (parasite Lactate Dehydrogenase enzyme)
       - produced by all 4 Plasmodium species
        - asexual and sexual stages
        - closely reflects parasite viability
        - ? potential for monitoring treatment efficacy
        - Pv, Po, Pm-specific Mabs developed
3. Aldolase
        - pan-specific
        - closely reflects parasite viability

				
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Description: An overview of the diagnostic methods in malaria.