Lab Diagnosis of Malaria Parasites

1
Lab Diagnosis of Malaria Parasites

• Dr Rajesh
• 21/11/07

2
Diagnosis of malaria

• Prompt and accurate diagnosis is the key
• Two diagnostic approaches
• Clinical diagnosis,
• Microscopic diagnosis, DIFFICULT
• Need for accurate and rapid diagnosis method
• RDTs comparable to those generally achieved by
  microscopy

3
Diagnostic Toolsfor Human Infections with Malaria

• Blood film examination
• Serology - IFA
• PCR

4
Microscopy advantages

• Sensitive. As low as 510 parasites per µl of
  blood typical microscopist might be more
  realistically placed at 100 parasites per µl of
  blood
• Informative. species and of the circulating
  stage, quantifications
• Inexpensive.
• Permanent record (the smears)

5
Microscopy disadvantages

• Labour-intensive and time -consuming,
• Unreliable tool
• Long delays

6
Types of Serological Assays Malaria
Antigen Detection Immunochromatographic Antibody
Detection Indirect Fluorescent Antibody Enzyme
immunoassays
7
Antigen Detection
8
Antigen Detection
9
(No Transcript)
10
(No Transcript)
11
Antigen DetectionMalaria Immunochromatographic
Dipstick
OptiMAL Assay
P. falciparum specific monoclonal antibody
Control
Plasmodium pan specific monoclonal antibody
12
Antigen DetectionMalaria Immunochromatographic
DipstickProblems

• Low sensitivity with parasites density lt100/ml

13
RDT disdvantages

• lack of sensitivity at low levels of
  parasitaemia
• inability to quantify parasite density
• inability to differentiate between P. vivax, P.
  ovale and P. malariae,
• Inability to differentiate between the sexual and
  asexual stages of the parasite
• persistently positive tests (for some antigens)
  in spite of parasite clearance following
  chemotherapy
• Relatively high cost per test.

14
Antigens

• Histidine-rich protein II ( HRP-II) is a
  water-soluble protein produced by trophozoites
  and young (but not mature) gametocytes of P.
  falciparum.
• Parasite lactate dehydrogenase (pLDH) is produced
  by asexual and sexual stages (gametocytes) of
  malaria parasites. cannot distinguish between P.
  vivax, P. ovale and P. malariae.
• Other antigen that are present in all four
  species are also targeted in kits that combine
  detection of the HRP-II antigen of P. falciparum
  together with that of an, as yet unspecified,
  pan-malarial antigen of the other species.

15
(No Transcript)
16
Antibody Detection
17
Antibody Detection
18
Other tests

• Microscopy using fluorochromes such as acridine
  orange, either on blood smears (34) or on
  centrifuged blood specimens (QBC technique) is
  expensive and requires special equipment and
  supplies (centrifuge and centrifuge tubes,
  special light sources and filters).
• PCR s more sensitive and specific than all other
  techniques. It is, however, a lengthy procedure
  that requires specialized and costly equipment
  and reagents, as well as laboratory conditions
  that are often not available in the field.

19
Indirect Fluorescent Antibody (IFA)
Microscope slide
20
Malaria IFA Test

• Sensitivity 98
• Specificity 99.5
• Sulzer et al, Am J Trop Med Hyg 196918199-205
• Sulzer et al, Bull Wld Hlth Org 197145375-379

21
P. malariae
22
P. malariae
23
P. malariae
24
P. falciparum
25
P. falciparum
26
P. falciparum
27
Malaria IFA TestInitial detection of antibodies

• Parasitemia precedes antibody
• P. vivax 2-6 days
• P. falciparum and P. malariae 4-6 days
• If parasitemia is suppressed by treatment, may
  develop detectable antibody

28
Malaria IFA TestDetermination of Infecting
Species

• Non-Immune
• Samples drawn 0-14 days post onset Highest titer
  was to the infecting species in 81
• Samples drawn 15-60 days post onset Highest
  titer was to the infecting species in 96

29
Malaria IFA TestDetermination of Infecting
Species

• Is possible in non-immune individuals with
  primary infection.
• Is NOT possible in immune individuals because
  their antibody response reflects multiple
  infections with multiple species.

30
Malaria IFA TestAntibody Persistence after
Treatment

• Non-Immunes (Vietnam Vets with Pv)
• 53 IFA negative at 6 mo. post-Rx
• 59 IFA negative at 12 mo. post-Rx
• Wilson et al, Am J Trop Med Hyg 197019401-404

31
Malaria IFA TestAntibody Persistence
32
Sensitivity of Tools forDiagnosis of Malarial
Infection

• Most sensitive
• Antibody detection
• 2. PCR
• Blood film examination
• RDT antigen dtection

33
Diagnosis of Untreated Acute Malaria

• Blood film examination
• PCR
• RDT antigen based

34
Diagnosis of Treated Recent Malaria

• Serology
• Blood film examination
• PCR

35
Diagnosis of Chronic Malaria

• Screen with serology
• If IFA positive
• May do blood film examination
• May do PCR

36
HRP2 antigen sensitivity
37
RDT advantages
38
Need for future

• Develop methods that permit quantification of
  parasite density with RDTs.
• Develop improved tests that reflect viable
  asexual parasitaemia only.
• Identify potential markers that predict the
  development of complications, treatment outcomes
  and/or drug resistance.
• Identify the gold standard against which
  malaria diagnostic tests should be assessed.