Haematology - Malaria Parasite

HELLOOOOOOO ALL~~ //

Am i supposed to blog this week? Anyway, i'm gonna talk about Malaria Parasite in my entry!~ It's a hot topic in my lab right now and everyone is taught to identify and differentiate between the vivax and falciparum species during roll call and CME. I shall only focus on explaining these two species which are more common in Singapore.


Name of test - Malaria Parasite Microscopic Examination (MPME)


Introduction

• Malaria is an infectious disease caused by the Plasmodia parasite.




• There are 4 identified species of this parasite: Plasmodium vivax, P. falciparum, P. ovale and P. malariae.




• It is transmitted by the female anopheles mosquito.

(Image taken from: http://www.lawestvector.org/)



• In fact, it can be treated in just 48 hours but it can be fatal if the diagnosis and treatment are delayed.




• It occurs in tropical countries especially in Africa and India as the tropics provide ideal breeding and living conditions for the anopheles mosquito.




• One child dies of malaria somewhere in Africa every 20 sec., and there is one malarial death every 12 sec somewhere in the world (quoted from the malaria site).




• Currently there's no vaccine available; Still developing.




• In Asia, the more common species encountered are P. vivax and P. falciparum.




• MPME provides information to confirm the diagnosis of a MP species. The test consists of both thick and thin smears.
  

Procedures & Principle of MPME

• To perform MPME, a EDTA blood tube is needed. (Alternative: finger-pricked blood)




• Giemsa stain is a differential stain used in the diagnosis of MP.




• It is a mixture of methylene blue and eosin and it differentially stains the RBC & platelets pink, WBC blue and MP purple.




• #1 - Thick smear: To screen for parasite




• A stick is used to spread the blood on the glass slide to a 50-cent coin area, yet thin enough to be seen through.




• It is air-dried for 30 mins. -> This allows the RBC to be hemolyzed.




• Leukocytes and any malaria parasites present are therefore, the only detectable elements.




• The thick smear is then de-hemoglobinised in water and stained with Giemsa.




• The WBC and MP pick up the stain and it is noticable under the microscope.




• However, its morphology is often distorted due to the hemolysed RBC, hence it is only used to detect infection and estimate the parasite density.




• Thick smear is more sensitive than the thin smear (as a larger volume of blood is screened), therefore it is easier to pick up low levels of infection.




• #2 - Thin smear: To identify the parasite species




• It is performed when thick smear shows positive MP infection.




• The thin smear in air-dried for 10 mins and fixed in methanol.




• The smear is covered with 10% Giemsa stain for 30 minutes, washed with distilled water, drained and dried.




• The stain is picked up even by the RBC, hence any parsite within the cell can be identified.




• It is used for species identification because the parasite appearance remains well preserved under the microscope.

Test Results

Thick smear - tiny purplish ring-like structure/comma observed in the hemolysed RBC -> +ve for Malaria

Thin smear - check several fields for infected RBC. Parasites in various forms can be observed e.g. early ring, late ring, early intermediate stage, late intermediate stage, presegmented, segmented (schizont), macrogametocyte & microgametocyte. More than 2 parasites can infect a cell and a person can suffer from multiple infection too. Below are some techniques to differentiate between the falciparum and vivax.

• RBC enlarged -> vivax


• RBC not enalrged -> falciparum


• Many RBC infected -> falciparum


• Schizont or growing trophozoite observed -> vivax


• Ring form observed -> small 'comma-like', sometimes two chromatin dots (like a headphone), often multiple rings in a cell, occuring at the tip of the cell -> falciparum


• Ring form observed -> relatively large, usually one chromatin dot with a thick blue ring (like a diamond ring) -> vivax


• Crescent shaped, central chromatin ('banana/rod-like') -> falciparum gametocyte


• Large central chromatin dot in RBC (without ring) -> falciparum (cerebral malaria)

Clinical Interpretation

MPME is used to screen for malaria parasite and identify the species by the use of a differential stain. Taking into consideration the limitations of the thick and thin smears, both of the smears are therefore necessary to make a definitive diagnosis. The various forms of MP require different drug therapy, hence the correct species diagnosis is vital in the recovery of a infected individual. In any case that MPME could not provide evidence to confirm the species, a Malaria Parasite PCR is done (this is not done in our lab; send-out to NUS). Meanwhile, if the patient condition is critical, the doctor in-charged may decide to dispense drug against falciparum as it is strong enough to even kill the vivax species.


Pictures (produced with permission from haematology section head)

-From Mr. J-

Mixed infection of vivax and falciparum.



The largest RBC is a vivax macrogametocyte. Schizont suggests vivax infection as it is absent in falciparum PBF.


Mostly falciparum parasites infection here.


Actual Diagnosis of Mr. J: Falciparum & Vivax Infection

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-From Miss. M-

Enlargement of RBC due to vivax infection is difficult to spot in anaemic patients. Hence no concluding statement could be made. Note that the RBC are anisopoikilocytes.


The RBC in the middle appears as enlarged, but it may be due to infection of a normal RBC in an anaemic patient. Therefore, it is better to check other fields for other signs or send-out the sample for a PCR testing.


Actual Diagnosis of Miss M: Vivax Infection

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Questions? ^_^
Reminder: Close all your windows between 5-10pm and early morning to prevent from being a MP victim!


-Pei Shan, TG02-