Malaria Detection and Identification
Introduction
Malaria is a global disease found in tropical and sub-tropical regions of the world. Despite international initiatives aimed at eradicating the disease, Malaria is still responsible for more than 600 000 deaths every year – most of them children.
Malaria is transmitted to humans via the Anopheles Mosquito. The mosquito bite introduces the parasite into the human’s blood via sporozoites in the saliva. The parasites then migrate to the liver, where they mature and reproduce. This is known as the exoerythrocytic phase. The organisms multiply in the liver in infected hepatocytes. These differentiate into thousands of merozoites, which rupture the host cell, infiltrating the blood and infecting red blood cells. This next stage is known as the erythrocytic stage of the life cycle. The parasite is able to leave the liver undetected as it envelopes itself in the liver cell membrane of the infected host.
Symptoms
The signs and symptoms of malaria are non-specific. Malaria is suspected clinically primarily on the basis of current or a recent history of fever. This pyrexia may be cyclical in nature corresponding to the parasite lifecycle however, there is no combination of signs or symptoms that reliably distinguishes malaria from other causes of fever.
Types of Malaria
There are five known types of malaria parasite: Plasmodium falciparum, Plasmodium Vivax, Plasmodium Ovale, Plasmodium Malariae and Plasmodium Knowlesi. The most severe is P. falciparum and the least common is P. Knowlesi.
Plasmodium Falciparum: found mainly in Africa, it's the most common type of malaria parasite and is responsible for most malaria deaths worldwide, though treatment does cure the infection.
Plasmodium Vivax: mainly found in Asia and South America, this parasite causes milder symptoms, but it can stay in the liver for years which can result in symptoms reoccurring if it isn’t treated properly.
Plasmodium Ovale: uncommon and usually found in West Africa.
Plasmodium Malariae: this is quite rare and usually only found in Africa.
Plasmodium Knowlesi: this is very rare and found in parts of southeast Asia.
These each have differences in appearance when viewed on a stained blood film which help identification, see table below:
Observation | P. Falciparum | P. Vivax | P. Malariae | P. Ovale |
|---|---|---|---|---|
| Size (RBC) | Not enlarged | Enlarged | Not enlarged | Enlarged |
| Shape (RBC) | Round, sometimes crenated | Round or oval | Round | Round or oval, often fimbriated |
| Colour (RBC) | Normal, but may become darker or may have a purple rim | Normal to pale | Normal | Normal |
| Stippling (RBC) | Maurer’s spots, appear as large red spots, loops and clefts | Schuffner’s dots, appear as small red dots, numerous | Ziemann’s dots, few tiny dots, rarely detected | Schuffner’s dots (James’s dots). Numerous small red dots |
| Pigment (RBC) | Black or dark brown | Seen as a haze of fine golden brown granules scattered through the cytoplasm | Black or brown coarse granules, scattered | Intermediate between P. Vivax and P. Malariae |
| Trophozoite (parasite) | Small, delicate, sometimes two chromatin dots, multiple rings commonly found | Relatively large, one chromatin dot, sometimes two, often two rings in one cell | Compact, one chromatin dot, single | Compact, one chromatin dot, single |
| Schizont (parasite) | Medium size | Large | Small | Medium |
| Gametocyte (parasite) | Crescent shaped | Spherical | Similar to P. Vivax, but smaller and less frequent | Like P. Vivax but smaller |
P. Falciparum - trophozoites and crescent shaped gametocyte
P. Vivax - Gametocyte (L) and trophozoite (R)
Testing for Malaria
The diagnosis of malaria is confirmed by blood tests which are either microscopic or non-microscopic tests. Microscopic tests involve staining and direct visualisation of the parasite under the microscope. The direct microscopic visualisation of the parasite on the thick and/or thin blood smears has been the accepted method for the diagnosis of malaria in most settings, from the clinical laboratory to the field surveys, for over a hundred years.
Careful examination of a well-prepared and well-stained blood film currently remains the “gold standard” for malaria diagnosis.
Microscopic Tests
High quality thick and thin films should be prepared and examined in every suspected case of malaria, regardless of the results of immunochromatographic rapid diagnostic tests (RDTs) or other means of screening for malarial antigen detection.
A thick film should be used to detect presence of malaria parasites and the thin film for identification of species. It is useful to prepare four thick films and four thin films so that two of each can be stained, leaving spare films to send to a reference centre and for further study if there is diagnostic difficulty.
Films should be made as soon as possible to avoid morphological alteration of parasites which occurs with storage of EDTA-anticoagulated blood.
After collection of the blood sample, the film is stained with Romanowski stains to examine the intracellular appearance of the red cells.
Thick Film
The thick smear of the correct density is one through which newsprint is barely visible. It is dried for 30 minutes and not fixed with methanol. This allows the red blood cells to be haemolysed and leukocytes and any malaria parasites present will be the only detectable elements. However, due to the haemolysis and slow drying, the plasmodia morphology can get distorted, making differentiation of species difficult. Thick smears are therefore used to detect infection, and to estimate parasite concentration.
Thick films are usually stained by the rapid Field’s technique or Giemsa’s stain for screening of parasites. The sensitivity of a thick blood film is 5-10 parasites/µl.
Thin Film
Thin smears allow one to identify malaria species (including the diagnosis of mixed infections), quantify parasitaemia, and assess for the presence of schizonts, gametocytes, and malarial pigment in neutrophils and monocytes. Thin blood films stained by Giemsa’s or Leishman’s stain are useful for specification of parasites and for the stippling of infected red cells. The optimal pH of the stain is 7.2.
Rapid Testing Kits (RTD’s)
Malaria rapid diagnostic tests (RDTs) supplement the diagnosis of malaria by providing evidence of the presence of malaria parasites in blood. RDTs are a used in addition to microscopy, particularly where good quality microscopy experience is not available.
The test works by detecting specific antigens (proteins) produced by malaria parasites in the blood of infected individuals. It is a lateral flow immuno-chromatographic antigen-detection tests, which works by capturing dye-labelled antibodies to produce a visible band on a strip of nitro-cellulose in a cassette casing.
With malaria RDTs, the dye-labelled antibody first binds to a parasite antigen, and the resultant complex is captured on the strip by a band of bound antibody, forming a visible line (T - test line) in the results window. A control line (C- control line) gives information on the integrity of the antibody-dye conjugate, but does not confirm the ability to detect parasite antigen.
Some kits can only detect one species (Plasmodium falciparum or P. vivax) while others detect multiple species (P. falciparum, P. vivax, P. malariae and P. ovale).
Detection via full blood counting
Because malarial parasites are found in the peripheral blood, they are known to interfere with the counting on full blood analysers. Manufacturers can therefore make use of these abnormalities to generate flags that can alert the operator to a potential malarial infection from a full blood count.
HORIBA Medical developed a Malaria flag on several instruments, the Microsemi CRP LC-667G and ABX Pentra XL80, Pentra XLR from signals found in the WBC channels.
This technology was further developed in a later series of instruments, the Yumizen H500 and Yumizen H550 instruments using machine learning techniques to create an S-score flag for P. Falciparum, P. Vivax and Dengue (the latter is a mosquito-borne virus that produces symptoms that are similar to malaria).
Other Tests
Some laboratories use quantitative buffy coat (QBC) on positive samples, however this is an expensive method.
PCR is another method, which is very sensitive and reliable when determining the species of malaria in a mixed infection. This is mainly used in reference labs.
Conclusion
The World Health Organisation (WHO) launched a Global Technical Strategy for malaria 2016 – 2030 in 2015 with the aim to reduce global malaria incidence and mortality rates by at least 90% by 2030. Technical solutions include the control of the mosquito vector, improved treatment and prophylaxis regimes, but also effective screening and diagnosis.
Access to laboratory testing and the availability of easy and rapid testing has an important role to play in achieving this goal.
Acknowledgements
This article is reproduced from the content of QSP Newsletters #24, #25 which are available here.
Authors: Kelly Duffy and Andrew Fisher of HORIBA UK Ltd.
References
https://www.ncbi.nlm.nih.gov/books/NBK555962/ (Bain, 2006; Bain et al, 2011)
https://www.who.int/teams/global-malaria-programme
Essential Haematology A.V .Hoffbrand & J.E.Pettit Moody AH, Chiodini PL.
Methods for the detection of blood parasites. Clin Lab Haematol 2000;22:189-201