Malaria Flashcards
Malaria work up - starting paragraph
A parasitic disease of human caused by Plasmodium spp.
* P. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi.
* Generally asymptomatic for 12-35 days (incubation period) until the erythrocytic stage of parasite lifecycle.
Clinical history esp travel history, prophylaxis.
Delayed processing can also affect red cell + parasite morphology, making dx more difficult.
Aim for for EDTA collection at peak of fever
Film features that raise suspicion for malaria include anaemia, thrombocytopenia, reactive lymphs & toxic neuts.
Diagnostic tools for malaria
- Coagulopathy (common in setting of P. falciparum)
*haemolysis - Liver dysfunction – elevated bilirubin, transaminitis and/ or cholestatic
At my lab we perform two tests available to screen in addition to blood film examination:
- ICT testing
- Malaria DNA amplification (LAMP)
Thick and thin films are both then used for the identification of malarial parasites after positive ICT and/or positive malarial amplification assay.
THIN - confirmation of presence of malaria and identification
THICK - useful when there is a low parasite denisty,
(gold standard by an expert user)
Thin film is reported on ALL malaria test request.
PCR - VIDRL to confirm species
Thick films
Preparation involves mechanical lysis of RBCs so that parasites can be seen independent of cell structures (not fixed before staining)
Sample: Whole blood in EDTA (within 3-4 hrs from collection)
Principle: Concentrates parasites & Allows examination of larger blood volume over shorter period of time . sensitivity inc 10-20x compared to thin films
Method: Make 4 slides fresh
One drop of blood on slide + spread in circular motion over 2 cm size
Correct thickness = just possible to read the print (10 font size) of a page through smear
Air dry
Dilute Giemsa’s stain (2 copies)
Place slides in dilute Giemsa’s stain for 20 mins
Dip slides in tap water 2 times to remove excess stain
Allow to drain vertically + air dry
Keep 2 copies in reserve
Interpret: Examined for at least 5-10 mins
For identification only, not speciation
WHO recommends that at least 100 fields (each containing 20 WBC) be screened before saying negative . (5 mins)
QC: Internal: Compared to positive control
External: RCPA – virtual microscopy: 2 samples, 2x per year
Thin Film
Used to speciation
3-4 slides, Giemsa stain on fixed slide- buffered water to pH 7.2 (specific for malaria)
Examined for at least 10-15 mins
*To exclude malaria, blood films must be examined on 3 separate occasions to accounts for periodicity of release of parasites
Malaria Morphology
MALARIA IMMUNOCHROMATOGRAPHY TEST (ICT)
Sample: Blood (venous, capillary) in EDTA OR fingerprick
Rapid whole blood test which detects Plasmodium falciparum HRP II protein and a pan malarial antigen common to 4 species.
Principle: Detection of circulating P. falciparum histidine-rich protein 2 (HRP-2) & pan-malarial Ag common to all 4 species (Pf, Pv, Po, Pm)- Rapid Diagnostic Test
Method: Ag detection by immunochromatographic lateral flow (lateral diffusion system) 2 Ab immobilised in 2 separate lines along test strip
P. falciparum specific: HRP-2
Pan-malarial Ag: Plasmodium LDH/ aldolase
Whole blood (15ul) is added to sample pad impregnated with colloidal gold-labelled Ab directed towards these 2 malarial Ag
Malarial Ag in positive sample bind to gold-coupled Ab in the pad and the immune complexes formed migrate along pad where they are captured by immobilised Ab to HRP-II antigen (line T1) or antibodies to pan-malarial antigen (T2).
Positive sample: a pink line forms at either T1 and/or T2.
Negative sample: no pink line forms.
Immobilised control antibody captures control conjugate, forming the control line.
**Screening test only – should be interpreted with gold standard of morph; though detects P falciparum with >90% sensitivity still need film
Limitations of ICT
negative test does not exclude infection with malaria, as low levels of parasitaemia may be below LOD
Pf HRP-II antigen +/- pan-malarial antigen in the blood may still be detectable following treatment even when parasites are no longer visible on film.
Final diagnosis should be based on a correlation of test results with other clinical and laboratory
findings.
Malaria DNA Amplification Assay
performed on the illumipro-10™
Qualitative invitro diagnostic test for the direct detection of DNA of Plasmodium sp. in human EDTA whole blood.
Utilizes loop-mediated isothermal DNA amplification (LAMP) technology to detect Plasmodium sp. DNA by targeting 214bp sequence of a mitochondrial DNA noncoding region of the Plasmodium genome.
DOES NOT distinguish between plasmodium species.
Isothermal DNA amplification –> byproduct of magnesiums pyrophosphate –> forms white precipitate –> turbid reaction solution –> reaction solution absorbance are monitored/calculate.
No DNA = no change in sample absorbance.
sensitivity of 2
parasites/ul for P.Falciparum and 0.125 parasites/ul for
P.Vivax.
Malaria identification procedure steps
- DNA amplification assay preferred initial test to screen for presence of Plasmodium infection
> if +ve then proceed - ICT assist with identifying or excluding P.falciparum following a positive amplification assay result as well as to confirm a positive result.
(is the initial test in regional labs)
IF positive make 4 thick and thin films for species identifcation.
