Diagnostic procedures vary not only according to the tools available, but often even more to what one wishes to know.
If owners report disease problems in their cattle herd or flock of small ruminants, in a region where tsetse flies are known to occur, one is not really interested in knowing which species of trypanosome is or are causing the disease. It is enough to confirm that the disease is indeed trypanosomosis, as the control measures are generally similar for all species. Direct parasitological diagnosis in wet blood films or buffy coat preparations may be all that is needed for this purpose. Even though such methods are not the most sensitive and a proportion of the infections will be missed, as long as a sufficient number of animals is examined, the diagnosis of trypanosomosis will be reached. Once the disease has thus been confirmed, clinical diagnosis of individual animals is indicated, unless treatment of the whole herd appears to be preferable. Control measures should be instituted as fast as possible, and no time should be lost by sending specimens to a distant laboratory. So-called “pen-side testing” is needed in this case.
For mechanically transmitted trypanosomosis in herds in tsetse-free regions, it is usually enough to confirm that the disease is caused by trypanosomes. A more precise diagnosis is usually even less important here; for instance there is only one species of trypanosome causing surra in camel herds. For animals such as horses, which are kept individually, or at least not usually in large numbers, a more sensitive method is preferred, or, if none is available, direct parasitological methods should be carried out repeatedly over a length of time, to increase the chances of finding the trypanosomes (or the reliability of a negative result).
On the other hand, in research, such as epidemiological surveys, requirements may be far more precise. Assessment of the seasonal occurrence of different trypanosome species demands the most sensitive and specific method applicable in the circumstances in which the researcher has to work. The accuracy of the work will be increased if two or more methods are used that can complement each other. Thus, if the investigator can fall back on a fairly sophisticated laboratory, it may be possible to use the potentially very sensitive polymerase chain reaction (however, at present, available reagents are so specific that they will not detect all different types of a trypanosome species and reagents are not [yet] available for all the known types, see further on). Sanitary surveillance associated with eradication schemes (for example in the case of dourine) or the prevention of the introduction of trypanosomosis (dourine, surra) across national borders, also presents particular requirements (the sensitivity of the test used is essential in this case).
In regions where the disease is known to occur, clinical symptoms and post-mortem lesions are important indications, especially in combination with the history of the disease and the region in which it occurs. However, symptoms and lesions of trypanosomosis are never pathognomonic (which means specific for the disease), and suspicion has to be confirmed by other means. Clinical signs and PM lesions are reviewed in Chapter 2.
Microscopical diagnosis. Direct methods: wet blood films (=fresh blood preparation). A simple technique is to examine fresh blood between a coverslip and a slide with the microscope, using medium magnification (usually a dry objective of 40x or even less, and eyepieces of 5–10x). Trypanosomes are seen either directly, moving between the blood cells, or indirectly, as they cause the blood cells to move. The lower the magnification, the larger the field (and the quantity of blood) observed, and the faster the examination; the preferred approach depends on the experience of the operator.
Fresh preparation of lymph. Trypanosomes, particularly T. vivax, are sometimes found in lymph collected from a lymph node, when they are not found in blood. The opposite may also occur however, and the procedure is not commonly carried out.
The lymph is usually collected from a swollen prescapular lymph node, and examined between slide and coverslip, like blood. The presence of trypanosomes is usually only seen indirectly, by the movements of the lymphocytes, because the great density of the lymphocytes will obscure the trypanosomes.
Thick blood film. A drop of blood is applied on a clean slide and spread out with the corner of another slide, or with a match or a needle, to produce a circular area to a thickness such that, when dry, the hands of a watch or small print can be seen through the film. One may also let the drop run over the inclined slide until the appropriate thickness is attained. Next, the film is thoroughly dried, and in the laboratory stained without fixation, usually with Giemsa stain. Giemsa stain is aqueous and removes the haemoglobin by lysing the red blood cells. Fixation with methanol is not carried out, as it would prevent lysis, which is necessary in order to see through the several layers of blood cells, which otherwise would obscure the parasites. If lysing and staining are done before the blood has thoroughly dried, the film may be washed off the slide. On the other hand, if many days elapse between the preparation of the film and lysis and staining, lysis may be incomplete, especially if the film has been exposed to heat or sun. It may help to lyse such films in distilled (or at least clean) water before staining, but it may difficult to obtain satisfactory results. Examination is carried out under the microscope, preferably using an oil immersion objective of 40–50x. Specific diagnosis of the trypanosomes is sometimes possible using the 100x oil immersion, but is usually difficult or impossible because the process of lysis distorts the parasites.
Thin blood smears. These are made as in the case of blood smears to detect other blood parasites. They are fixed by methanol and stained with Giemsa stain, or with one of the more recent fast stains such as Diff-Quik, RAL 555, Field's stain, which have the advantage of acting much faster than Giemsa. They are read using an oil immersion objective (40–50x for scanning, 100x for identification of trypanosomes). The morphology of the different species has been indicated in Chapter 1 (p. 22).
Thin smears of lymph. Lymph aspired from a prescapular lymph node, instead of being examined as a fresh preparation (or after a fresh preparation has been found positive), can also be made into a thin smear, fixed and stained, which will make specific identification possible. The smears should be very thin, as the many lymphocytes, which are also stained, complicate the visualization of the parasites. For this reason thick lymph smears are not suitable for diagnosis, lymphocytes cannot be lysed as can red cells.
Concentration methods: huffy coat examination (also called the Woo method). It may be useful to discuss here first the constituents of blood. Blood consists of a liquid, the plasma, in which blood cells are suspended. There are three main kinds of blood cells, the red cells (erythrocytes), white cells (leukocytes, of which there are several types) and thrombocytes (or platelets, responsible for starting the process of blood clotting). If a blood sample is taken into an ordinary glass tube, the blood will coagulate, clot, as one of the blood proteins, fibrinogen, changes under the influence of the platelets into insoluble fibrin, which forms a mesh of strands in which the blood cells are trapped. This mesh contracts and the serum (= plasma without fibrinogen) separates.
Clotting of blood can be prevented by adding an anticoagulant, such as sodium citrate, heparin or EDTA. If such blood is left alone in a tube, the cells which are slightly heavier than the plasma, sediment into a column beneath the plasma. This process can be much accelerated and the separation improved by spinning the tube in a centrifuge at high speed. It will then be seen that most of the white cells settle at a slower rate than the red cells, as their specific gravity is a little less than that of the red cells, and form the so-called buffy coat between the much thicker red cell layer and the column of plasma (Figures 9 and 10). The specific gravity of trypanosomes, which are free in the plasma, is also slightly less than that of the red cells and so they tend to concentrate at the limit of plasma and the buffy coat, as well as in the buffy coat. (This also depends on the species, the specific gravity of T. congolense is almost the same as that of the red cells, and they are thus less concentrated.)
This concentration technique has been standardized by the use of microhaematocrit tubes and a specially designed centrifuge (Figure 11). The tubes are capillary tubes, which can be obtained treated inside with an anticoagulant or, if untreated, an anticoagulant must be added to the blood before it is taken up in the tubes. The rotor plate, which can take many tubes of different animals at the same time, is spun at high speed (12 000 rpm) for five minutes.
The components of blood
Blood sample mixed with anticoagulant and allowed to stand when cells and plasma separate
Blood components after centrifugation
The capillary tubes are open at both ends. Blood is taken up at one end, until about three-quarters of the length of the tube is filled. Filling occurs by capillary attraction and practice will show how to apply and incline the tube to achieve this. It is important not to have the blood column interrupted by air. The other end of the tube is then sealed over a burner (taking care not to char the blood) or by special wax or plasticine (Figure 12). The tubes are then placed in the grooves (individually identified by a number corresponding to the number of the blood sample) of the rotor plate, with the sealed end outwards (to prevent the blood from being thrown out during centrifugation); the cover is closed and screwed down, and the timing is set for five minutes. After centrifugation, the tubes are removed, care being taken that it remains known to which animal each of the tubes corresponds.
Sealing haematocrit tubes
The packed cell volume (PCV) value (= the length of the column of concentrated cells, expressed as a percentage of the total length of the blood column) can be read directly in a special reader, which can be individually adjusted for the length of the blood column in each tube; the PCV gives a valuable indication on the presence and degree of anaemia.
The tube is then examined for the presence of trypanosomes, either by direct examination of the buffy coat/plasma junction, or after making a smear of this area.
Direct examination. The tube is placed on a microscope slide in a slot formed by sticking two pieces of glass to the slide 1.5 mm apart (Figure 13). Immersion oil is placed on the capillary tube, to fill the space between the glass bars and the tube, over the region of the tube where the parasites, if present, are concentrated and will be visible (buffy coat, and buffy coat/plasma junction). The best results are obtained with a special oil immersion objective lens, magnifying 40–60 times, which has an extended working length so that one can focus deeply into the tube as it is rotated during examination, and with a special substage condenser providing phase contrast illumination. This is the classical Woo method.
Section through a slide adapted to hold haematocrit tubes
Darkground/phase contrast buffy coat technique. The haematocrit tube is snapped 1 mm below the buffy coat/red cell junction, to include the upper layer of red cells. Clean and precise snapping can be obtained by marking the spot with a small glass saw or diamond pen. The contents, including the first centimetre above the buffy coat, is then gently expressed on to a clean slide, so that some plasma is included with the buffy coat. (The tube may also be snapped in this second site, and the contents of the intermediate section thus obtained is expressed on the slide.) The expressed contents are carefully mixed and then examined under a coverslip, using a special condenser providing dark ground phase contrast background illumination, which renders the trypanosomes much more visible. The material can also be treated as a smear, to be fixed and stained for specific identification of the parasites.
Advantages and disadvantages (compared to the classical Woo method)
A comparison of these various parasitological techniques has shown that their sensitivity differs to some extent according to the trypanosome species concerned, but that in general the descending order of sensitivity is dark ground/buffy coat - classical Woo method - thick film - wet film - thin film.
Lysis and centrifugation. By hypotonic shock or even better the use of appropriate concentrations of certain detergents it is possible to obtain lysis of the red cells, without affecting the mobility of the trypanosomes, which can then be concentrated by centrifugation. Sodium dodecyl sulphate (SDS) has been reported as particularly suitable, in a concentration of 0.12 percent. Larger volumes of blood can be examined by this method than by the haematocrit method. Equal volumes of blood (e.g. 5 ml), to which an anticoagulant has been added, and PSG buffer containing 0.24 percent of SDS are mixed and left at ambient temperature for at least ten minutes. The mixture is then centrifuged for ten minutes at 2.250 g. The sediment is examined between slide and coverslip, if necessary after adding a minute quantity of PSG.
Use of experimental animals. Low or subpatent (= not patent, not observable by microscopical examination of blood) parasitaemias of certain species and strains of the pathogenic trypanosomes can be revealed by injecting laboratory animals with the blood to be examined. The sensitivity of this method varies according to the species or even strain present and the susceptibility of the experimental animals used. The last column of Table 2 (Chapter 1) indicates experimental animals that are susceptible to each trypanosome species. It should however be realized that within each trypanosome species there are important strain differences, in this respect too. Also, there may be important differences in the susceptibility to a given trypanosome species or strain between inbred laboratory strains of an experimental animal species.
The two species of experimental animals most commonly used are laboratory rats and mice. They can be bred and maintained by diagnostic laboratories, provided that strict attention is paid to housing, hygiene, correct feeding, and breeding..
A blood sample, with anticoagulant, from the suspected domestic animal is injected in one or several rats or mice. This is mostly done by the intraperitoneal route (in the abdominal cavity), using 1 or 2 ml for a mouse and up to 5 ml in rats. The technique of inoculation is easily mastered after a demonstration and a little practice. The blood of the rodent is then examined frequently by snipping off a minute piece of the tip of the tail and transferring the resulting drop of blood to a slide for microscopical examination under a coverslip. Regular examination (preferably daily, at least twice a week) should continue for at least two months after inoculation.
Some more details are given below for the different trypanosome species.
T. congolense. Rats and mice are commonly used, although rabbits and guinea pigs may also be considered.
Some strains of T. congolense are readily established in rodents, whereas others do not easily infect them. In some cases the less virulent strains have long incubation periods, up to two weeks or more and some may not establish in rodents at all or the parasitaemia may be fleeting and short lived.
Generally speaking, rats are more easily infected than mice and should be used for isolating strains of known low pathogenicity.
T. simiae. Only rabbits may be susceptible to the species and not all strains establish themselves in this host. Some strains are virulent for monkeys and it was in this animal that the species was first discovered, hence its name.
After passage through rabbits or monkeys, T. simiae often becomes less virulent for domestic pigs and loses its polymorphic characteristics, existing as the long stout form only.
T. godfreyi. This trypanosome apparently does not infect laboratory animals.
T. vivax. With rare exceptions, this species does not infect any of the laboratory animals. There have been (very few) reports of adaptation of T. vivax to rodents by serial passages and other methods, and rare strains will grow well in laboratory rodents straight away after isolation. In general the use of laboratory animals is not suitable for the diagnosis of T. vivax.
T. brucei. Rats and mice are very susceptible to most strains of T. brucei brucei (and T. brucei rhodesiense, much less T. brucei gambiense) and the blood of apparently healthy cattle in tsetse country may prove to be infected at a subpatent level with this trypanosome when blood is injected into rodents. But even with this species there is a range of sensitivity; some strains appear in the blood after 48 hours or less, rapidly reaching enormous numbers and killing the rodent in a few days, others take longer to appear and may cause a disease lasting many weeks.
Rapid serial passage through rodents of the same species, i.e. transferring infected blood to the next animal as soon as the trypanosomes appear, increases the virulence for the rodent species to a maximum which kills rapidly. At the same time the parasite loses its polymorphic characteristics so that it shows virtually only the long slender type. As the polymorphism is lost, so is the ability to infect tsetse flies. (It is in this way that T. evansi is believed to have originated from T. b. brucei, following rapid serial mechanical passages by biting flies in camels.)
Laboratory rodents are used to reveal the almost true prevalence of T. brucei in domestic or wild animals; one single trypanosome is often enough to establish infection in a rat or mouse. Only T. brucei gambiense will often not readily establish in rats or mice at the first isolation, although it may adapt to rodents after passage through other hosts, such as monkeys.
T. evansi. Strains of this species are infective for laboratory rodents in a similar manner as T. b. brucei and T. brucei rhodesiense, so that rat and mouse inoculation is used to reveal subpatent infections with this parasite.
T. equiperdum. Even in the normal equine host, the parasites are exceedingly scanty in the circulating blood, and isolation in laboratory rodents is rarely successful and not normally used for diagnosis. However, once adapted to rodents following rapid serial passages, it becomes indistinguishable from rodent-adapted strains of T. brucei and T. evansi.
General. As mentioned above, strains of trypanosomes that give fleeting low parasitaemias in rodents can usually be adapted to them by making serial passages in rodents as soon as the parasites are seen. Gradually the parasitaemias become higher and the incubation period shorter.
A word should be said here about the possibility of parasitological diagnosis of trypanosomes in the biological vectors, Glossina spp. It is used only in epidemiological studies, not in routine diagnosis. An operator skilled in dissecting tsetse flies can tentatively determine the infection rates of tsetse flies with different trypanosome species by microscopical examination of the various parts of the digestive tract (see Table 1).
Uncertainties remain, however:
22 Using appropriate molecular techniques on the parasites in the various parts of the digestive tract (PCR, nuclear probes), the investigator can acquire more certainty but, as stated before, specific molecular tools are not (yet) available for all types of every trypanosome species.
20 The parasitological diagnosis of dourine is discussed in Chapter 5, p. 138, as this venereal disease is a special case.
21 The sensitivity depends to some extent on the species concerned, T. brucei and T. vivax, being larger and the latter also more mobile, are more easily noticed than T. congolense.
Serological tests. The aim of serological tests is to detect specific antibodies (which are blood proteins belonging to the immunoglobulins), developed by the host against the infection or, inversely, to demonstrate the occurrence of circulating parasitic antigens in the blood by the use of characterized specific antibodies. The detection of antibodies indicates that there has been infection, but as antibodies persist for some time (weeks, sometimes months) after all trypanosomes have disappeared from the organism (either by drug treatment or self-cure) a positive result is no proof of active infection. On the other hand, circulating trypanosomal antigens are eliminated quickly after the disappearance of the trypanosomes, and their presence therefore shows almost always that live trypanosomes are present in the animal.
Validation and standardization of procedures is important if one hopes to compare results obtained in various countries and by various teams. Validation and standardization of some tests are coordinated by the Joint FAO/IAEA Animal Health and Production Division in Austria. The World Organization for Animal Health (OIE) in Paris issues the Manual of standards for diagnostic tests and vaccines, which should be consulted for further details. Serological tests are mainly used as tools for research, for monitoring trypanosomosis control programmes and for surveys, not so much for the routine diagnosis of the disease in the field, and we will indicate only the principles here.
Antibody-detecting tests. Many different types of serological tests have been in use for many decades, and there is little point in enumerating them all. We will mention and describe the general principles of some of the most commonly used, the indirect immunofluorescent test, the indirect ELISA and, particularly for the detection of antibodies to T. evansi, a card agglutination test. Variations of these and other tests are used for specific scientific purposes. It is quite possible that other tests will become routine in the near future, such as agglutination assays using latex beads coated with either antigen or antibody; time will tell.
The indirect fluorescent antibody (IFA) test
Figure 14 shows the principle of the test.
There are quite a number of possible pitfalls associated with this test, and it is carried out only in the laboratory by qualified laboratory personnel.
Immunofluorescent antibody test
The indirect enzyme-linked immunosorbent assay (ELISA). The principle of this test is in fact very similar to that of the IFA test. The binding of anti-trypanosomal antibodies to the antigen is shown by a conjugate of antibovine (if the test serum is bovine) immunoglobulins labelled with an enzyme, which can be visualized by adding an appropriate chromogenic substrate (i.e. the interaction between enzyme and substrate will create a colour). The use of substrate can be compared to the use of UV rays to visualize the fluorescent conjugate in the IFA test.
Usually solubilized antigens obtained from disrupted trypanosomes are used (instead of smears containing whole trypanosomes), and the soluble antigens are coated in the wells of microtrays (“stuck” on the surface of the well, as it were). Each microtray contains many (usually 96) wells. This makes it possible to process many sera at the same time, using multichannel pipettes. Only small quantities of sera and conjugate are used. The test results can be read visually, but this introduces an element of subjectivity, and a special ELISA reading instrument will quickly give the optical density (OD) of each well (showing quantitatively the intensity of the interaction between the enzyme and the substrate), thus helping to speed up the processing of large numbers of sera. The ELISA lends itself to standardization and automation.
Nevertheless, the use of antigens derived from whole trypanosomes means that the test is not necessarily more species-specific than the IFA test. More specific results can be obtained by the use of characterized species-specific antigens, which are produced by molecular engineering. But, as stated before, such a level of specificity may not be required in the field. Even using such specific antigens, it may not be possible to distinguish between very closely related trypanosomes, such as T. evansi and T. equiperdum, and even T. brucei, in the horse family, or T. brucei and T. evansi in other animals.
Card agglutination test. This has been developed from a commercial test for the diagnosis of human sleeping sickness (the Testryp® CATT), into a commercial kit for T. evansi, CATT test T. evansi®. For the detection of antibodies to surra (T. evansi infection) serum samples are mixed on a plastic card with fixed and stained trypanosomes as antigen and the test is positive when the antigen agglutinates. A titre can be determined by serial dilutions of the serum.
The great advantage of this test is that in principle it is easy to carry out even in the field. Its specificity and sensitivity appear to need further evaluation, and in the experience of the author reading the test results is not always easy.
Antigen-detecting tests (Ag-ELISA). These tests have been developed for the detection of circulating trypanosomal antigens. The surface antigens of trypanosomes are variable; only one or two of the many different variants are present in the blood at any one time and, unless one would possess mixtures of antibodies to all the possible variants, their detection is not reliable. Therefore, the tests that have been developed are based on so-called monoclonal antibodies against invariable (internal) antigens. This needs some explanation.
Among the constituents of the immune system are various types of lymphocytes. Each of the lymphocytes that manufacture antibodies in response to an infection only makes one type of antibody, directed against only one antigen of the infectious organism. In fact, antibodies are so specific that they only fit on a particular site of an antigen (a so-called epitope), like a key in a lock. By genetic engineering it is possible to fuse single lymphocytes with cancer cells of a mouse; single-fused (hybridized) cells will grow (because of their cancerous component) in cell culture or in mice, and produce a single type of antibody, a so-called monoclonal antibody. Such monoclonal antibodies in principle react very specifically with one antigen only, or rather fit in one particular shape of epitope only.
The principle of detecting circulating antigens using monoclonal antibodies is again quite simple (but there are many problems).
The wells of a microplate are coated with a monoclonal antibody, which is specific for an invariable (non-surface) antigen of a trypanosome species. Serum of the animal to be tested is allowed to react in one of the wells, and if there is the corresponding circulating antigen, its presence in the well after washing off the test serum can be shown by finishing off the test with a conjugate of the same monoclonal antibody marked with a suitable enzyme, and the presence of the enzyme is shown by use of the suitable chromogenic substrate, as in the normal ELISA.
Tests based on this principle, using monoclonal antibodies supposedly specific for the various subgenera, species or types of pathogenic trypanosomes, have been widely tested and distributed to National Agricultural Research Systems (NARS) in Africa for AAT diagnosis and elsewhere for the diagnosis of surra and T. evansi infections. It has however become apparent recently that the sensitivity of this type of test is not as high as was claimed, and even that positive results are not reliable, at least with the monoclonal antibodies that were in common use. On the one hand false positive results may occur, on the other hand there are cross-reactions between species.
The fact that the sensitivity of this test is often low and irregular can easily be understood, as the antigens detected are only released in the blood stream when the trypanosomes die and disintegrate. Thus, in the early stage of the disease, before the first peak of antibodies causes massive lysis of the parasites, the trypanosomes are healthy, and the test is negative. Thereafter it is likely that the result is negative during each peak of healthy parasites covered by a new type of surface antigen, and only becomes positive when the immune system has caught up and produced the appropriate antibody, causing the death of this particular trypanosome variant, and thus the release of internal antigens. The sensitivity for T. vivax is particularly low, considerably lower than the buffy coat technique for the detection of parasites.
The fact that even positive results are not reliable and not specific is more difficult to explain. On the one hand it should not be forgotten that the reaction between an antibody and an epitope is like that of a key in the three-dimensionally appropriate lock. The same three-dimensional configuration sometimes exists on a very different protein of a very different organism. But on the other hand it appears also likely that the monoclonal antibodies that were selected for the test, although they were promising in initial laboratory tests, were released at too early a stage for evaluation and distribution in the field. Fortunately field evaluation has successfully detected their shortcomings. False positive results and cross-reactions appear to be particularly frequent with the Ag-ELISA for T. congolense and that for the T. brucei group.
Until monoclonal antibodies are developed which give more reliable results, it appears best not to use these tests but, as they had already been widely advertised and distributed, some mention of these problems was required here.
Molecular tests. The principle of molecular tests is the demonstration of the occurrence of sequences of nucleotides, which are specific for a trypanosome subgenus, species or even type or strain. Nucleotides are the constituents of DNA (deoxyribonucleic acid), the molecules which constitute the genes on the chromosomes in the cell nucleus. A positive result indicates active infection with the trypanosome for which the sequences are specific, as parasite DNA will not persist for long in the host after all live parasites have been eliminated. These tests are not only suitable for detecting parasites in the mammalian host, but also in the insect vector. The general principle of these tests will be shortly explained here, but as they can only be carried out reliably in well-equipped laboratories by specifically trained staff, and are still mainly research tools, no technical details will be given.
It seems appropriate to recall first the general structure of DNA, which contains the basic genetic information for all living organisms (except for some viruses which possess only RNA, another form of nucleic acid). DNA occurs as a double helix (= screw-shaped coil, or a spiral staircase), made up of two strands of nucleotides or bases, which are linked together by hydrogen bonds. There are only four different nucleotides, adenine (A), thymine (T), cytosine (C) and guanine (G). All genetic information (the genetic code) depends on the linear sequence of these four bases. The bonds between the two strands either connect A to T or C to G, i.e. the only possible base pairs are A-T and C-G.
The hydrogen bonds between base pairs can be broken by heating DNA, resulting in separation of the strands. The bonds will be restored when the sample is cooled down.
This basic knowledge will make it easy to understand the first of the two main molecular methods for the diagnosis of disease.
DNA-probes (nucleic acid probes).25 The sample to be examined is heated to separate the two strands of DNA (this is also called denaturing of DNA), and these are fixed to a membrane, so that they cannot recombine again on cooling. A probe is then added. A probe consists of a linear sequence of nucleotides of a certain length, which has been prepared to correspond with a similar sequence of nucleotides in one of the strands of the parasite which the test is meant to detect. The probe will link (hybridize) with that part of the parasite DNA strand which is the mirror image of the base sequence of the probe. Depending on the sequence of DNA that has been selected for the probe, the test can be more or less specific, certain sequences are common to all species of a subgenus (and thus will for example not allow to distinguish between T. brucei brucei, T. brucei gambiense, T. brucei rhodesiense, T. evansi and T. equiperdum, but indicate the presence of trypanosomes of the subgenus Trypanozoori), while other sequences are so specific that they only occur in each species, or subspecies, or even type. Whether hybridization has occurred or not is demonstrated by showing that the probe remains fixed to the sample after washing. For this it is of course necessary to “label” the probe, and this can be done by incorporating radioactive isotopes in the probe molecule, and showing that the radioactivity persists. The method is suitable for simultaneously processing large numbers of samples.
Unfortunately, radio-isotopes are not suitable for use in field laboratories. The procedure is long and involves quite a number of steps, but the main reasons are that special training is essential for working with radioactive materials and a special infrastructure for the safe disposal of radioactive waste is necessary; another reason is that the radio-isotope normally used (32P) decays quickly (has a short half-life) and frequent delays in transport and storage (such as customs) mean that the probe is often out of date by the time it arrives at the site where it is to be used. Because of this, radioactive DNA probes are not used for routine diagnosis, and remain essentially research tools.
For these reasons DNA-probes have been developed without a radioactive label, but labelled for instance with an enzyme, which can be demonstrated by an ELISA. However, such probes mostly have a considerably lower sensitivity than the radioactive ones, and are as or even more complex, involving many steps.
It should be realized that DNA-probes for trypanosomosis are not commercially available, and a laboratory that intends to use this technique either has to prepare its own probes or obtain them from other laboratories such as ILRI.
The polymerase chain reaction (PCR). This is another molecular method of detecting parasite DNA. It is based on the use of an enzyme, DNA polymerase, which amplifies (multiplies, copies) sequences of DNA bases, until sufficient material is produced to be detected. It does so by polymerization (“sticking together”) of nucleic acids. Parasite DNA is denatured (separated by heat into the two single strands). Two “primers” are used, which are short sequences of nucleotides (one for each DNA strand), each constructed so as to be complementary to a specific site on one of the two single parasite DNA strands. The primers attach to the sites for which they are complementary and DNA polymerase then starts to reproduce the rest of each complementary sequence which follows from that primer. This occurs in opposite directions until the entire sequence of double-stranded DNA between the primers has been doubled (as a complementary strand is produced from each primer). The polymerase can of course only do its work when nucleic acids are added to the test material. The cycle is then repeated, the two double-stranded DNA sequences are again denatured, the primers attach again, the polymerase amplifies, etc. In the end, the PCR product is submitted to electrophoresis and the bands are detected by special staining.
This procedure is extremely sensitive, as even minute quantities of parasite DNA can be amplified into a detectable quantity if the number of cycles is sufficiently high. It can also be highly specific, or less so, depending on the primers available for the reaction. Some primers will amplify a piece of DNA that is specific for a subspecies, type or even strain.
Important advantages of PCR over DNA probes are the greater sensitivity and the fact that no radioactive isotopes are needed. But there are also many pitfalls and disadvantages. Just to mention a few: false negative results may for instance be obtained if the specificity of the primers is too high, e.g. many infections by T. vivax are not recognized by existing primers, which do not recognize all types of the species. (The situation is better for T. [Nannomonas] congolense, as there are at present specific primers for the savannah, riverine-forest, Kilifi and Tsavo types of T. congolense, and also for T. [N]) simiae.) Whole blood contains factors which inhibit the PCR, another reason for false negative results.26 Strict spatial separation of the various steps of the PCR procedure is required, and a number of controls have to be used, as otherwise there is a considerable danger of obtaining false positive results by contamination of samples with other, non-relevant DNA. These and several other possible causes of false positive and false negative results prevent the use of PCR as a routine in the field, or even in laboratories which have not been adequately set up and equipped for this purpose. The technical staff has to be adequately trained and has to be aware of the possible pitfalls. Also, the test is relatively expensive, mainly because the special polymerase used for the test, which is thermostable (= can withstand the repeated high temperatures needed for the cycles of denaturing DNA), has been patented and is expensive. On the other hand, as for DNA probes, there are no PCR kits or primers commercially available for trypanosomes. Each laboratory has to make its own primers, adapted to its intentions, or rely on those that have been made by other laboratories (and by no means cover all possible requirements).
In order to detect and avoid false positive results, it is possible to combine PCR with the use of DNA probe technology: a suitable DNA probe can tell whether the amplified PCR product is indeed what was expected.
23 It is in principle possible in well-equipped and well-staffed laboratories to make conjugates which are not commercially available, directed against immunoglobulins of other animal species, but it is not an easy procedure.
24 It should, however, be noted that it may not be impossible to develop eventually so-called “pen-side” (or “crush-side”) ELISAs, which should give a positive or negative visible result on a specially prepared and impregnated stick or paper (“dip-stick tests”). Such tests, perhaps not always very satisfactory, exist for certain other diseases; for some, especially diseases of poultry, the market is so extensive and rewarding that they have been commercialized.
25 Some nucleic acid probes use RNA instead of DNA, but we will discuss here only DNA probes.
26 It has been reported that the PCR for the detection of active T. vivax infections in blood only detects parasitaemias of over 1 000 trypanosomes per ml. This is rather similar to the sensitivity of parasitological techniques. PCR carried out on the pellet resulting from plasma centrifugation is very sensitive (parasitaemias of some ten trypanosomes/ml are detected). DNA purification is even more sensitive, but requires a commercial kit and is more expensive and time-consuming.