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When can one assert that someone has the disease "malaria"? There are several problems and the question has still not been fully resolved. The demonstration of malaria parasites in the blood is essential, but insufficient in itself. Many people will develop an acquired immunity after several years of exposure, and may harbour parasites without exhibiting symptoms. The degree of parasitaemia may help, but there is no absolute criterion (the higher the parasitaemia, the more chance that malaria is in fact the diagnosis). There are patients with malaria for whom the thick smear is negative (luckily this is rare in a good laboratory). There are no pathognomonic clinical signs. An accurate diagnosis is becoming more and more important, in view of the increasing resistance of P. falciparum and the high price of alternatives to chloroquine.
No single clinical sign permits the diagnosis of malaria. Yet malaria must always be considered in cases of fever in the tropics. Since the symptoms can be quite diverse, a clinical diagnosis is unreliable in itself. Microscopic confirmation of the diagnosis is often not possible in many regions and situations. It is of the greatest importance that other important diagnoses are ruled out before instituting a blind anti-malaria therapy. All too often fever is considered as malaria without considering alternative diagnoses.
The presence of parasites does not rule out an additional diagnosis: e.g. someone with fever may well have some malaria parasites in a thick smear, but this does not rule out meningitis or pyelonephritis. Chronic carriers are people who, in spite of the fact that they have malaria parasites in their blood, have no symptoms of this. When such people develop another infection their symptoms are often attributed to the malaria parasites in their blood, although these are not responsible. The absence of parasites in a single preparation does not rule out malaria, but does make the diagnosis of P. falciparum highly improbable. Where there is strong clinical suspicion it is best to repeat the test 12h later.
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A thick smear concentrates the parasites 10 to 25 times. It is rather more difficult to interpret than a thin smear preparation and often does not permit species identification.
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A thick smear contains no intact red blood cells (haemolysis due to the distilled water used in the staining). If a thick smear is positive, a thin smear should be examined. Sometimes parasitaemia is estimated in a thick smear and expressed as +, ++, +++. This form of record is of course quite subjective and confusing and is best avoided. If the thick smear is positive, it is then best to count the percentage of parasited cells in a thin smear preparation. The parasitic density can also be roughly determined in a thick smear, by counting the number of parasites per 200 leukocytes and multiplying this by 30. It is assumed that on average there are 6000 leukocytes per µl blood and that there is one leukocyte per 500 red blood cells. For example: 5 parasites per leukocyte (1000 parasites for every 200 leukocytes) corresponds to a density of 30,000 parasites per µl. Roughly 30,000 parasites per µl corresponds to a parasitaemia of 1% (a moderately anaemic person). If the thick smear is found to be negative in a reliable laboratory, and if there is nevertheless strong suspicion of malaria, the test is repeated every 12 hours for 48 hours. One great disadvantage of the thick smear method is that reliable technical expertise is needed which should be monitored (e.g. quality control). The argument that a lab technician has carried out the test for years and thus has plenty of experience, is absolutely no guarantee of quality or reliability. The test also requires plenty of time if the parasitaemia is low, or before a negative result can be concluded.
This shows the presence of undistorted parasites. The thin blood smear permits identification and also calculation of the parasitaemia (% of parasitised red blood cells). This is necessary to start appropriate therapy (P. vivax is treated differently from P. falciparum). If the parasite cannot be identified it is regarded as a P. falciparum as a safety precaution. Mixed infections do occur. For staining, Giemsa is used with a slightly alkaline pH. It is a good habit to prepare the buffer solutions each day in the morning (interaction with CO2 from the atmosphere changes the pH of older solutions). Phosphate buffers are mostly used.
An alternative to Giemsa is acridine orange, but the day-to-day use of this technique is quite unpleasant and it is necessary to have a special microscope (ocular filter, halogen light, interference filter above the condenser). [This latter filter restricts transmission to some narrow spectral bands].
P. falciparum infection is characterised by:
P. vivax preferably penetrates young (therefore large) erythrocytes.
P. ovale is often found in a thin smear preparation in rather oval-shaped, sometimes distorted red blood cells.
P. malariae trophozoites sometimes have a typical band shape. The mature schizonts have a daisy head appearance.
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In well-stained preparations the nuclei of the parasites are always stained red and the cytoplasm blue. The presence of malaria pigment is very characteristic of the older stages of Plasmodium sp. P. falciparum often contains a single black dot. P. vivax often contains countless fine golden yellow/brown specks of malaria pigment. In P. ovale and P. malariae the pigment inclusions are many and brownish black. Countless fine red spots in the red blood cell (Schüffner’s dots) can be seen in P. vivax and P. ovale (the more mature the parasite, the more dots). In P. ovale the dots are sometimes called James’s dots. Sometimes a few flecks can be observed in P. falciparum (Maurer’s dots or clefts). P. malariae almost never exhibits dots (Ziemann’s dots). The visibility of these dots depends to a great extent on the acidity (pH) with which the thin slide preparation is stained (slightly alkaline: pH = 8 is best). The acidity is important because blood smears are usually stained for haematological tests with a slightly acid pH. With such a stain, the dots will not be seen clearly if at all.
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Note: Ratio gametocytes/trophozoites
In an infected person, countless parasites can often be seen in the peripheral blood. Since gametocytes are the only forms which are responsible for transmission in nature, it is remarkable how few gametocytes are generally found. Often the number of gametocytes is only a small percentage of the number of trophozoites. Yet higher gametocyte densities must lead to higher transmission. Why gametocytes occur in such low proportions compared to the number of trophozoites is still not clear. Generally there are more female than male gametocytes, certainly early on in an infection. This is explained by the fact that one male gametocyte can form 8 viable male gametes, unlike the female gametocyte. This is in fact the case when infections are monoclonal, yet in mixed infections the ratio would have to be 1/1 according to population genetics. Later in the infection agglutinating antibodies are produced which immobilise male gametes and thus inhibit their function. Agglutination of female gametes has no effect on their function. To compensate for this the parasite produces more male gametocytes later in infection, possibly under the influence of increasing concentrations of erythropoietin. The latter hormone increases as anaemia increases. A single haploid clone of P. falciparum can produce both male and female gametocytes. Precisely how this works is not clear. Plasmodium falciparum gametocytes need 7-10 days for maturation. The sex ratio is determined by the erythropoietin content 7-10 days before they mature. It is interesting to note that P. falciparum exhibits increased infectibility in humans with sickle cell anaemia, regardless of the gametocyte density. This is explained by the chronically increased erythropoietin in this disorder, which leads to a higher percentage of male gametocytes. Nevertheless much study is still needed before the details will be fully understood.
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Note: Inclusions in red blood cells
A special glass capillary tube is filled with 20 µl blood (a droplet via a finger prick). The inner side of this tube is coated with anticoagulants and the dye acridine orange. Acridine orange will bind to the DNA in the nuclei of the malaria parasites, and also to ribosomal RNA. Afterwards the tube is centrifuged (10,000 g x 5 minutes). The blood cells are thus separated according to density. The buffy coat is the part of the centrifuged blood which contains platelets and white blood cells (buff = pale yellow). In the tube is a longitudinal plastic float with the same density as the buffy coat. The float serves to spread the buffy coat and adjacent cells and press them in a thin layer against the wall. Since parasitised red blood cells are lighter than non-parasitised ones and heavier than white blood cells, infected red blood cells will be found on top of the red cell column, just below the white blood cells, right against the buffy coat. The parasites in this layer can be observed using a fluorescence microscope. However, be aware that Howell-Jolly bodies (nuclear residues) may look similar to parasites. This technique is much quicker than reading thick or thin smears but requires training and appropriate apparatus. Nevertheless, its use can considerably reduce the workload of the laboratory staff especially in larger hospitals where many samples are processed every day. No species identification can be obtained using QBC. With QBC there is quite wide inter-observer variability. The specially prepared disposable tubes need to be available as well as a microhaematocrit centrifuge and a microscope with a UV-lens. The thin tubes sometimes break.
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Several antigen detection tests have been developed (Parasight®, Malaquick®, ICT Malaria Pf® [Immunochromatographic Test]). The material for the test kit consists of a simple strip (similar to urine dipsticks) and some dropper bottles with reagents. These tests detect the histidine-rich protein, the PfHRP-II antigen. The abbreviation stands for Plasmodium falciparum Histidine Rich Protein. This protein is a constituent of the nodules on the membranes of red blood cells which are infected by P. falciparum. The test makes use of two antibodies which are specific for the PfHRP-II antigen. Both are attached to a paper strip. One of the antibodies is coupled to colloidal gold and applied to the place where the blood sample is to be applied. The second antibody is fixed elsewhere on the strip, in a band where the test result is read. Approximately 10 µl blood is applied (a droplet from a finger prick. Some blood may also be applied via a capillary tube containing EDTA). The red blood cells are lysed. If there is PfHRP-II antigen in the blood, this binds to the antibodies labelled with gold. After administration of a buffer the gold-labelled antibodies migrate with the capillary flow along the test strip and then cross the band containing the second antibody. If the blood sample is positive the antigen antibody complex labelled with PfHRP-II binds to the second antibody and a clear purple band is produced. This does not occur with a negative sample. A control band must always be visible.
The test is quite quick and simple to carry out and needs no technical apparatus. The sensitivity is 90-95% for parasitaemia of more than 100 parasites/µl. Low parasitaemia is thus often missed. The test remains positive so long as there is still antigen in the blood (that is even if the parasites have already disappeared due to adequate therapy). The ICT Malaria Pf® test can only detect P. falciparum. The presence of rheumatoid factor may lead to a false positive result (a problem with Parasight®). P. vivax does not cause cross reactivity. The chief problem, however, is the high price. A curiosity: using Parasight® malaria antigen was found in Egyptian mummies, which is an argument for the presence of this infection in antiquity.
An antigen detection test has also been developed for P. vivax. Using the latter test both P. falciparum and P. vivax can be detected, as well as mixed infections, on the same paper strip (ICT Malaria P.f/P.v®). The result is the presence of one or more horizontal stripes on the strip (as with a urine dipstick). In mixed infections the presence of P. vivax may not be discovered. In view of the simplicity of the test, this technique should in future be of benefit to frequent travellers in isolated tropical regions. It should be noted that these tests do not produce any quantitative information (including no parasitaemia). This is important in regions where chronic carriers are common.
The test "OptiMAL" is based on the detection of parasitic lactate dehydrogenase (pLDH). This quick test (10 minutes) consists of a dipstick coated with monoclonal antibodies to pLDH. Differentiation of the parasite species is based on antigenic differences between various pLDH isoforms. The pLDH is only produced by live parasites (also by gametocytes). The specificity and sensitivity of the test varies from study to study. The test may be positive due to the presence of circulating gametocytes when these people have been clinically cured and no longer have trophozoites or schizonts.
Serology can only be carried out in reference hospitals and is of no importance for the individual diagnosis in acute fever. The antibodies are positive from the tenth day, so at the beginning of the attack they will be negative. The presence of antibodies only shows that there has been contact with the parasite. This does not mean that there is sufficient immunity. There will be high titres of antibodies in the tropical hyperreactive splenomegaly syndrome. Malaria type IgG antibodies penetrate the placenta and will give the neonate temporary and partial protection against malaria during the first months of life. Antibodies after infection remain positive for a longer time.
Signs of haemolysis include yellow serum, dark urine while faeces have a normal colour, high LDH and low haptaglobin. Often there is also thrombocytopenia. Sometimes there is malaria pigment in white blood cells. The percentage of neutrophils containing malaria pigment is a measure of the severity of the situation (e.g. more than 15% is a high percentage). Monocytes which contain malaria pigment have little clinical prognostic value.
In endemic regions fever, muscle pain, or even generally feeling unwell are often attributed to "malaria". An anti-malaria treatment is then instituted, without obtaining confirmation of the diagnosis or often even without considering alternative diseases. The argument given is that such a treatment can do no harm, that the diagnosis of malaria is always probable because the disease is common, and that this is a good strategy for first-line care. Each of these arguments can be defended to a certain extent, but in this way often useless and sometimes expensive treatments with potential side effects are administered. Not recognising and treating other diseases (borreliosis, rickettsiosis, kidney infections, amoebic liver abscess, pneumonia, septicaemia and so on) is a daily reality in many tropical regions. The over-diagnosis of malaria often leads to under-diagnosis of other treatable disorders. It is sometimes stated that fever which does not disappear after three days adequate therapy, is not malaria. The problem with this attitude is of course "adequate": the problem of drug-resistant malaria and malaria accompanied by complications (e.g. septicaemia).
