Laboratory Diagnosis

There are four species of the genus plasmodium responsible for the malarial parasite infections that commonly infect man, P.falciparum, P.vivax, P.malariae and P.ovale.  The most important of these is P.falciparum because it can be rapidly fatal and is responsible for the majority of malaria related deaths.

Malaria occurs in most tropical regions of the world with P.falciparum predominating in Africa, New Guinea and Haiti.  P.vivax is more common on the Indian sub-continent and Central America with the prevalence of these two infections roughly equal in Asia, Oceania and South America.  P. malariae is found in most endemic areas especially sub-Saharan Africa but much less frequently.  P. ovale is relatively unusual outside Africa although some cases are now being identified in other regions (eg. Southern States of India).  It is also important to recognise that with the relative ease and speed of modern travel and migration, "imported" cases of malaria may present in any country. Additionally so called "airport malaria" (see History section) has now been identified in a number of countries including the USA, UK, Belgium, and Switzerland.  Airport malaria is particularly dangerous since Clinicians may have little reason to suspect it, if the patient has had no recent travel to areas where malaria is endemic.  This may result in a delay before the correct diagnosis is made and which may lead to death before appropriate treatment can be initiated.  Small outbreaks of malaria may occur in countries considered free of the disease, such outbreaks are most likely the result of an infected person entering the country asymptomatic and where suitable mosquito vectors are present.

In recent years a number of new techniques based on the "dipstick" format, have become available for the diagnosis of malaria. These include the ICT-Malaria Pf, OptiMALr and the Kat-Quick kits.  The methods are based on the principle of the detection of plasmodial histidine rich protein-2 (HRP-2) or parasite-specific lactate dehydrogenase (pLDH) which is present in P.falciparum infections.  A number of reports claim sensitivities and specificities approaching 100% while other reports have claimed up to 6% cross reactivity with sera positive for rheumatoid factor.  Some of these "dipstick" methods have been extended to include screening for other forms of malaria but to date results have not been quite so impressive.

Dipstick tests have the potential of enhancing the speed and also the accuracy of diagnosing P. falciparum, particularly in non specialised laboratories where inexperienced or junior staff may be involved, since very little training is required for these techniques.  In this laboratory we have found the dipstick kits to be very useful screening or confirmatory tests, especially when there is difficulty in identifying scanty ring forms in blood films.  They have proved to be particularly useful out of hours when junior, less experienced staff have been on duty.  However dipstick methods are unable to indicate parasite load and in some countries the cost may be prohibitive.  A potential problem with these methods is that the circulating antigen may be detected for many days (up to 2 weeks in our laboratory) after the elimination of viable parasites from the circulation.  It must therefore be remembered that a positive test may not always be due to an active infection.  We would like to emphasise, that we regard these dipstick methods as useful additional tests to the long established method of examining thick and thin blood films (outlined below), which is still regarded as the "gold standard", NOT as replacement methods.  The highest density of malaria occurs in countries least able to afford sophisticated and expensive diagnostic tools.

Antibodies to malaria can be detected using enzymatic immunoassays or immunofluorescence techniques.  The antibodies to the asexual blood stages appear days to weeks after the infection and may persist for months.  Although useful in survey work or for screening blood donors and reducing wastage, they are of little value in the "acute" malaria situation.  (Vox Sanguinis. 73(3):143-8, 1997. Clin & Exp Immunol. 54(1):127-34, 1983.)

Other methods include the QBC II System, Becton-Dickinson's Quantitative Buffy Coat (QBC) method.  This involves centrifuging the patient's blood in special capillary tubes precoated with Acridine Orange (AO) in which parasite DNA is stained with AO.  A small precision moulded plastic float presses the parasitised red cells (which occupy the upper most part of the red cell column) against the wall of the tube, where they can be viewed by ultra violet light microscopy.  The sensitivity of this method is claimed to be very high with experienced users, although some reports suggest that young trophozoites of P. falciparum and P. vivax, could not be distinguished with any degree of certainty and that confirmatory blood films should always examined.  Additionally special equipment is required, which may preclude the method from being used in smaller centres.  (J Trop Med & Hygene. 96(4):245-8, 1993-Aug)

Another relatively new method is the polymerase chain reaction (PCR) which uses a non-isotopically labelled probe following PCR amplification.  It is possible to detect <10 parasites per 10uL of blood and PCR may yet prove to be a valuable addition to the examination of blood films for the diagnosis and speciation of malaria (Am J Trop Med & Hyg. 65(4):355-363. 2001-Oct).  Once again the special equipment required precludes all but the larger centres.  Some researchers have claimed that PCR (and Elisa) techniques are as sensitive as blood films, however they are infinitely more expensive, require specialised equipment and take a longer time to complete.

Examination of a thick blood film should be the first step since this has the advantage of concentrating the parasites by 20 fold in comparison to a thin film, although the parasites may appear distorted making species identification difficult.  If parasites are seen then the species should be confirmed by the examination of a thin film.  Ideally blood should be collected when the patient's temperature is rising.

Preparation of thick and thin blood films:

Thick films:- place a drop of blood in the middle of a clean microscope slide and with the corner of a second slide spread the drop until it is about 10-15mm in diameter.  The thickness should be such that it is just possible to see news print through it.  Thin films are made in the standard manner.  Allow the films to dry, do not leave on the bench in a laboratory which is not fly proofed otherwise the film will be eaten.

When the films are dry, fix and stain the thin films in the conventional manner but be careful about the pH of the stain, a slightly alkaline stain is recommended (pH 7.2) as an acid stain may fail to show the parasites.  When only a few thick films are to be stained it is best to use dilute Giemsa stain (1/20), using a staining jar so that the film is in an upright position, this will allow any debris to fall to the bottom of the jar.  Do not fix the sample prior to staining.  Stain for about 30 minutes, wash gently with clean water and allow to dry.  If available use a positive control.  When a large number of thick films require staininq, Field's stain is preferred because it is very quick.  Field's stain comprises two solutions; a polychrome methylene blue (A) and eosin (B).  The solutions are kept in covered staining jars.

1. Dip the dry but unfixed film into solution A for 1 or 2 seconds.
   
   
2. Remove from solution A and immediately rinse in clean water ( a 250ml beaker with water gently flowing into it is suitable).
   
   
3. Dip the film into solution B for 1 or 2 seconds.
   
   
4. Rinse in clean water for a few seconds.
   
   
5. Place in a vertical position to dry.

If films are old or too thick the red cells may not lyse completely in the brief staining time.  If this is likely dip the film in clean water for a few seconds or until the haemoglobin has dispersed before staining.  Instructions for preparing Field's stain can be found in many laboratory text books.

Under the microscope examine the thick film first, using an oil immersion or high dry lens to determine if parasites are present.  Be aware of the patient's platelet and leucocyte counts.  Malaria is usually associated with a normal or reduced leucocyte numbers.  A leucocytosis is only found in terminal cases.  Platelet numbers are moderately or markedly reduced in some 80% of patients with malaria.  Parasites may appear distorted if the patient has been treated or has had inadequate or ineffective prophylaxis.

Mixed infections are not uncommon.

The illustrations below show the characteristics of the four species.

Diagnostic Points

1. Red Cells are not enlarged.
2. Rings appear fine and delicate and there may be several in one cell.
3. Some rings may have two chromatin dots.
4. Presence of marginal or applique forms.
5. It is unusual to see developing forms in peripheral blood films.
6. Gametocytes have a characteristic crescent shape appearance.  However, they do not usually appear in the blood for the first four weeks of infection.
7. Maurer's dots may be present.

Diagnostic Points

1. Red cells containing parasites are usually enlarged.
2. Schuffner's dots are frequently present in the red cells as shown above.
3. The mature ring forms tend to be large and coarse.
4. Developing forms are frequently present.

Diagnostic Points

1. Ring forms may have a squarish appearance.
2. Band forms are a characteristic of this species.
3. Mature schizonts may have a typical daisy head appearance with up to ten merozoites.
4. Red cells are not enlarged.
5. Chromatin dot may be on the inner surface of the ring.

Diagnostic Points

1. Red cells enlarged.
2. Comet forms common (top right).
3. Rings large and coarse.
4. Schuffner's dots, when present, may be prominent.
5. Mature schizonts similar to those of P. malariae but larger and more coarse.