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Nucleic acid techniques and the detection of parasitic diseases
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22 Tracey, K.J.and Cerami, A. (1990) Ann. NY Acad. Sci. 587, 325-331 23 Kluger,M.J.(I 988) in Biome,-_hanismsRea~ulatlng Growth and Development(Steffens, G.A. and Rumsey, T.S., eds), pp 409-420, Kluwar Academic Publishers 24 Elsasser,T.H., Caperna, T.J. and Fayer, R. Proc. Sac. Exp. Biol. Med. (in press) 25 Scarborough, D.E. (1990) Metabolism 39,
108-11 I 26 Kenison,D.C., Elsasser,T.H. and Fayer,R.Am. J. Vet. Res.(in press) 27 Blalock,J.E.(1989) Physiol.Rev. 68, 1-32 28 Scarborough, D.E. (1990)Ann. NY Acad. Sci. 594, 169-187 29 Barry, T.N. et al. (1985) Aust. J. Biol. Sci. 38, 393-403 30 Porat,O. (1989) LymphokineRes. 8, 459-469
31 Miller, S.C. eta/. (1988) Mol. Cell. Biol. 8, 2295-230 I Ran Foyer is at the Zoonotic Diseases Laboratory and Ted Elsasser is at the Ruminant Nutrition Laboratory at the Livestock and Poultry Sciences Institute of the United States Department of Agriculture, 10300 Baltimore Boulevard, Beltsville, MD 20705-2350, USA.
Nucleic Acid Techniques and the Detection of Parasitic Diseases S.M, Wilson The detection of infectious diseasecausing organisms is important for the initiation of effective treatment, in monitoring response to therapy and in epidemiological studies of disease of human or animal hosts. In this article Stuart Wilson primarily considers parasitic diseases, but much can be applied to infectious diseases in general.
The simplest method of diagnosis is often by microscopic observation of parasites in blood films, '.~ools or after culture, in vitro. This method is sensitive but time consuming anti requires an experienced eye. As an alternative, many immunological methods of diagnosis have been developed, based either on the detection of circulating host antibodies to parasite antigens or the detection of parasite antigens themselves. These methods, while sensitive, have disadvantages I. Anti-parasite antibodies, when induced, may not be present until some time after the initiation of infection and conversely may persist long after the resolution of infection. The persistence of antibodies may be a problem in endemic ;areas; certain individuals may have a high level of reactive antibodies in the absence of the organism. Similarly, immunized individuals may have antibodies that confuse the diagnosis. Ideally, anti-parasite ~Lntibodies are detected by recognition of well-defined, purified antigen. Such antigens are difficult to obtain and the use of crude antigens can decrease the specificity of the assay because of epitopes shared by different parasite species. Similarly, detection of parasite antigens with polyclonal antibodies may suffer from nonspecific crossreactivity. It is hoped that the cloning and expression of genes that encode parasite-specific immuno~) 199 I, Elsevier Science Publishers Ltd, (UK) 0169 4707/91/$02.00
dominant peptides and the use of monoclonal antibodies will increase the specificity of these assays. Such methods have been used to detect a variety of parasitic diseases2 but, in those instances when an adequate immunodiagnostic test is not possible, direct detection of the parasite genetic material using nucleic acid probes or amplification techniques may provide an alternative. Detection of Parasites Using Nucleic Acid Probes Radiolabelled nucleic acid probes. Nucleic acid detection traditionally involves the autoradiographic localization of denatured, radiolabelled nucleic acid probes hybridized to membranebound, denatured target nucleic acids (Fig. l a). One advantage of nucleic acid probe-based assays is that a parasitic infection is always accompanied by the corresponding parasite nucleic acids. Furthermore, by careful selection of unique, invariant regions of the parasite genome, such probes can be specifically directed against reiterated, repetitive or multicopy sequences, thereby enhancing the sensitivity of detection. Kinetoplast DNA 3'4, tandem repeats 4's, parasite ribosomal RNA sequences 6 and total parasite genomes 7'8 have all been used as hybridization targets. As individual repetitive sequences may comprise I-10% of the total genomic DNA and there may be 10-50-fold more ribosomal RNA than genomic DNA per parasite, the advantages of using either as a target for a diagnostic probe are apparent. Cloned repeat sequences, synthetic oligonucleotides or labelled total parasite DNA have been used to detect
species of Leishmania, Trypanosoma, Echinococcus, Taenia, Fasciola, Onchocerca, Brugia and Plasmodium (for reviews see Refs 9, 10) but the sensitivities reported for such probes vary greatly. The reported sensitivity may depend on the type of probe, the method of sample preparation or the origin of the sample. For example, the sensitivities reported for cultured parasites or purified parasite DNA can be an order of magnitude higher than when clinical samples are investigated 11-13 Obviously the latter is an important consideration and some potentially promising diagnostic nucleic acid probes have proved to be less promising when tested in the field. Even so, some radiolabelled probes can be comparable to microscopy for malarial diagnosis4' 12.t3 and the resulting hybridization signals can be proportional to the number of parasites present 4'7'1~ which is important in quantitative diagnosis. A great advantage of nucleic acid probes is the ability to screen hundreds ~3 or thousands ~ of samples simultaneously, whereas microscopy is a skilled task requiring many minutes per slide. Nonradiolabelled nucleic acid probes. Radioactive labelling of nucleic acids is a versatile, simple and highly sensitive technique but it is of restricted use for routine diagnosis. Radiolabelled probes have to be used in restricted areas and their necessarily short half-lives make it difficult to incorporate them into simple detection kit format for use in routine diagnosis. This may explain why, despite exhaustive research on generating sensitive nucleic acid probes for many diseases, these assays are confined to research laboratories and have failed to be implemented in the general diagnostic laboratory.
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However, there are several nonisotopic alternatives to radiolabelling; the systems shown in Table I are marketed commercially. The simplest nonradioactive systems substitute chemical haptens for the isotopic label. The haptens most commonly used are the vitamin biotin Is and the plant steroid digoxigenin ~6. Both molecules can be covalently linked to nucleic acid bases and the hapten-modifled bases can subsequently be incorporated into a strand of DNA or RNA by commonly used DNA or RNA polymerases to form nucleic acid probes 17. Alternatively, these haptens can be chemically linked to the bases of the nucleic acid probe 17. Other chemical modifications, eg. sulphomodification ~8, can be used to generate different chemical haptens. After hybridization of the probes to target nucleic acids and the removal of unbound probe, the haptens bound to any hybridized probe can be detected by a number of systems. Biotin haptens can be detected using the protein avidin (from egg white) or streptavidin (from bacteria), both of which have a high affinity for biotin. Alternatively, biotin can be detected using an anti-biotin antibody. Similarly, other haptens (eg. digoxigenin and sulphomodified bases) are detected using anti-hapten antibody systems. If these hapten-binding proteins are in turn covalently linked to enzyme molecules (reporter enzymes), the whole target-probe-haptenhapten-binding protein-enzyme complex can be visualized by the action of the enzyme on substrate (Fig. I b). Alkaline phosphatase, horseradish peroxidase, 13-galactosidase and urease can be used as reporter enzymes although alkaline phosphatase and horseradish peroxidase are the most commonly used. Depending on the choice of substrate, the coloured product can be soluble for spectrophotometry or insoluble for visual detection. Such enzymes can even be coupled directly to the nucleic acid probe jg. As there is no need for a hapten or
hapten-binding protein, this direct enzyme labelling of the probe enables some incubation and washing steps to be omitted (Fig. I c). One disadvantage of this method is that the hybridization must be performed at lower temperatures to preserve the integrity of the enzyme. The specificity of the probe at these lower temperatures is maintained by using denaturants such as urea or formamide which lower the melting temperature of the nucleic acid probe while reversibly denaturing the enzyme component of the probe. Such alkaline phosphatase-labelled oligonucleotide probes have been used to detect Plasmodium in infected blood and have been used to monitor the clearance of parasites after therapy during which both the parasitemia and the hybridization signal declined 2°. Unfortunately these nonisotopic systems have proved to be of limited use in the development of routine, diagnostic nucleic acid probes because the sensitivities reported for these probes are usually lower than those for the corresponding 32p-labelled probes I 1.12. More recently, the substitution of colorimetric with chemiluminescent substrates has increased the potential sensitivities of many of these nonradiolabelled systems so that they approach or even surpass the sensitivity attainable by radioisotopes (see Box I).
In our laboratory we are investigating the potential of chemiluminescence and have used a digoxigenin-labelled repeat sequence and chemiluminescence to detect Leishmania parasites in tissuetouch blots from infected mice, with a sensitivity similar to that with the same probe labelled radioactively (Fig. 2). With the recent introduction of these sensitive and safe alternatives to radiolabelled probes, it is likely that we will see a rapid development of nucleic acid probes from research tools used solely in research laboratories to routine diagnostic assays applicable in the general diagnostic laboratory. F o r m a t for H y b r i d i z a t i o n
The time taken to perform an assay is often an important factor in its development. The traditional method of detection of membrane-bound nucleic acids using radiolabelled probes is a lengthy process. Hybridization is usually performed for 16h and the autoradiographic detection of the bound probe takes another 16-1 8 h. This would be a distinct disadvantage in the development of any diagnostic assay. However, there are several ways in which the time taken for these assays can be reduced. Nonisotopic probes reportedly exhibit less nonspecific binding to membranes and are often used at much higher concentrations than are isotopic probes ~s. This higher concentration of probe can serve to drive the hybridization reaction and increase the rate at which probe binds to the membranebound target. Thus the hybridization may be performed in 2-4h. Similarly, the visualization of these probes using chromogenic or chemiluminescent substrates is much more rapid and can be achieved in a similar time. Alternatively, to increase sensitivity and decrease hybridization times, single-
Table I. Nonradioactive systems for the detection of nucleic acid probes Hapten
Hapten-binding protein
Biotin
Avidin or streptavidin or anti-hapten antibody
DigoxJgenin Chemically modified bases, eg. sulphomodification Direct coupling of enzyme to probe
Anti-hapten antibody
Reporter enzyme
Substrate
Alkaline phospha~se or horseradish
Soluble or insoluble colour or chemiluminescence
peroxidase
Parasitology Today, vol. 7, no. a, 199 /
stranded DNA or RNA probes can be used w. These probes can be used at high concentrations in the hybridization reaction because, being single stranded, the target-probe hybridization on the membrane does not have to compete with probe-probe rehybridization in solution. Membrane-based hybridizations proceed more slowly than do solution hybridizations. The immobilization of the target nucleic acid onto a solid support alters the kinetics of the hybridization reaction and may reduce the amount of target accessible for detection at the membrane', surface. By adopting a solution hybridization methodology, the hybridization time can be greatly reduced and the reaction may proceed more efficiently; some method of post-hybridization capture of the target-probe hybrid onto a solid support may be necessary for quantitation of the reaction ~7. Such a solution hybridization and hybrid-capture protocol has been used to detect as little as 0. I pg of P. falciparum genomic DNA in minutes rather than hours 21. Typically, by using on,-= or more of these systems the assay times can be greatly reduced and the,. whole assay from hybridization to detection may be carried out in less than one day.
257 Box I. Chemiluminescent Substrates for Use With Nucleic Acid Probes
Enzyme-catalysed luminescence is not a new phenomenon. Since the 1960s the luminescent oxidation of luciferin by luciferase and of luminol by horseradish peroxidase have been studied. More recently similar substrates based on substituted dioxetanes have been developed for alkaline phosphatase. As both horseradish peroxidase and alkaline phosphatase are the most common enzymes used in nonradioactive, membrane-based nucleic acid detection systems, there is scope for replacing the chromogenic substrates for these enzymes with the chemiluminescent substrates (see below). One disadvantage of the latter is that the visualization of the signal is indirect and must be detected by exposure to photographic film. Such chemiluminescent substrates have been shown to be more sensitive than chromogenic substrates; this is due in part to the inhibition of enzyme activity by the insoluble chromogenic products and the use of enhancers in the chemiluminescent assay. Such enhancers can increase the intensity of the light emission by several orders of magnitude. The kinetics of light output from enhanced luminot and dioxetane systems do differ. The signal from the enhanced luminol system reaches a maximum after I-5 min and thereafter declines sharply. This means that the detection must be rapid but the maximum exposure is achieved very quickly. However, the signal from the enhanced dioxetane system begins to increase slowly, reaching a maximum after 60-90 min and, once at this maximum, declines only slowly over the next few hours. This increases the time needed for detection but means that the sensitivity can be increased by prolonged exposure and, if required, there is also time for multiple exposures. There are advantages and disadvantages to both of these chemiluminescent systems, depending on whether a rapid, repeatable or more sensitive system is desirable, but both have distinct advantages when compared to chromogenic or isotopic detection systems in that they combine sensitivity with safety. 0-0
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The polymerase chain reaction (PCR) (Ref. 22) is another alternative to the use of radiolabelled probes that relies on a completely different system of detection. From a nonspecific mass of DNA or RNA, defined fragments of usually 100-400 base pairs (bp) can be specifically amplified using oligonucleotide primers of defined sequence that bind to the target region to be amplified. This technique is very sensitive as the amplification is exponential until the concentration of nucleoddes, primers or enzyme becomes limiting. One cycle of PCR consists of thermal denaturation of the DNA, annealing of the oligonucleotide primers to the target DNA (one primer binding to each strand of DNA) at a lower, empirically determined temperature and then synthesis of DNA from each of these primers using a DNA polymerase. The DNA polymerase most widely used is Taq DNA polymerase, which was isolated from a thermophilic bacteria. This polymerase is stable at high temperatures, enabling many such cycles (3040) to be performed without addition of enzyme after each thermal denaturation step. This has enable.d automation
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of the cycling by microprocessor-controlled thermal cyclers of various design. Such cyclers can complete 25-30 cycles in under 3 h, though times as short as 0.25 h have been reported 23. The potential importance of PCR for the detection of parasitic diseases has been discussed by de Bruijn 24 and in some instances has already been realized. PCR has been used to detect Trypanosoma infections with a sensitivity greater than that with either microscopy or hybridization with radiolabelled DNA probes 2s. The PCR product is usually analysed by agarose gel electrophoresis followed by ethidium bromide staining and visualization with ultraviolet (uv) light. Re-
cently, colorimetric tests have been devised for PCR products 26-3°. These systems usually use modified primers, and either involve direct binding of the PCR product to a solid support and hybridization with specific oligonucleotide probes or the indirect binding of the PCR product and subsequent detection by virtue of modified primers. For example, a short nucleotide sequence that is recognized by a DNAbinding protein can be added to the end of the target-specific sequence of one of the primers. If the other primer used for the PCR has an attached biotin molecule then there are two systems that could be used for detection of this PCR product. The PCR product can be
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immobilized using either DNA-binding protein or avidin coated onto microtiter plates. Once immobilized, these products can be visualized using streptavidin 28'29 or DNA-binding protein enzyme conjugates27, respectively. These colorimetric systems can be adapted for a quantitative detection assay31 and lend themselves more easily to automation, both of which are important for screening many samples and for making a quantitative diagnosis. Although PCR is an exquisitely sensitive technique, this sensitivity can cause problems. Product from previous reactions can contaminate the laboratory environment and may act as a target for subsequent PCR. Thus contamination of a negative sample with a few molecules of product prior to PCR can lead to the generation of false positive results after PCR. The risk of sample contamination can be lessened by using positive displacement pipettes or aerosol-guarded disposable tips, by physically separating the areas used for sample preparation from those used for analysis of product and by uv sterilization of plastics or the reaction components themselves prior to PCR. Post- and pre-PCR sterilization methods have been published. The former modifies the PCR product chemically after PCR so that it cannot act as a target for subsequent PCR reactions 32 whereas the latter produces modified PCR product in the PCR itself which can be specifically degraded prior to subsequent PCR reactions 33. If the ultimate aim for PCR is a fully automated assay from sample preparation to analysis of product, thus enabling the simultaneous screening of many samples and the reduction of the chances of contamination, then these innovations will help towards that aim. More recently, alternative amplification systems involving the replication of a small RNA molecule by QJ3 replicase~4 and a transcription-based amplification system 35 have been reported. In the future these systems may become viable alternatives to PCR.
Sample Preparation Sample preparation is one factor that is often overlooked when describing an otherwise simple nucleic acid detection system, and the method of choice can sometimes affect the sensitivity of the assay 11'13. Often very crude samples can be used in immunodiagnosis whereas nucleic acid detection systems often require highly purified samples. Although simple methods do exist for the preparation of nucleic acids from many
b
Fig. 2. Detection of parasites in tissue-touch blots. The spleen (S) and liver (L) from a mouse infected with Leishmania donovani were touched repeatedly onto a nylon membrane. After denaturation, neutralization and uv-fixation of the DNA to the membrane, the membrane was probed with the radiolabelled complementary DNA (cDNA) clone Lmet 2 (10 6 cpmml-I), a L. donovani complexspecific probe that recognizes a series of repeat sequences in the parasite genome. Hybridization was for 16 h and the membrane was autoradiographed for 24 h (a). The membranes were then stripped of probe and reprobed with digoxigenin-labelled Lmet 2. Hybridization was for 16 h and the hybridized probe was detected with an anti-digoxigenin antibody conjugated to alkaline phosphatase after which the blot was overlayed with a dioxetane-based chemiluminescent substrate and exposed to photographic film for 4 h (b). There was no signal from the liver and spleen of an uninfected mouse with either probe (results not shown).
sources fT, some degree of expertise is required and the procedures can be lengthy and may involve the use of expensive, specialized equipment or the use of noxious, expensive chemicals. Clearly this is one aspect of nucleic acid detection methodology that must be addressed and progress is being made in this area. Solution hybridization methodologies can be simplified by hybridization directly in the chaotropic agent used for tissue solubilization36. Furthermore, such hybridizations may be performed at reduced temperatures (even room temperature), thus reducing the requirement for additional equipment. Where samples need to be applied to membrane supports, alkali denaturation and solubilization of crude tissue samples and the direct fixation of these samples to modified nylon membranes is extremely rapid and simple 37. Other methods involve the direct application of samples to the membrane. The dotting of blood samples7'~2'2°, tissuetouch blots 38 or the squashing of whole sandflies onto membranes prior to the detection of parasite infection by hybridization 4'39 have all been shown to be effective.
Often purified DNA is used as a template for PCR but this can be inconvenient and the preparation time consuming. However, small quantities of crude material can sometimes be used directly in the reaction, as in the PCR of material directly from small quantities of whole blood 4°. Other systems involve the capture of the infectious agent from crude samples by the use of specific antibodies coated onto solid supports 4~. After washing and lysis of the captured organisms by heating, the released nucleic acids can be used directly in PCR. Similarly, DNA can be captured from crude samples by absorption onto glass beads42 or filter discs43 before PCR.
Summary New developments in nucleic acid technology, such as PCR and the replacement of isotopic with safe, sensitive nonisotopic systems, witl enable nucleic acid detection techniques to progress from the research to the diagnostic laboratory. By using the most advanced and innovative technologies it is hoped that these systems will be developed into rapid, simple and yet sensitive assays which, in some instances, will become the methods of choice for the routine detection and diagnosis of disease-causing organisms. Acknowledgements I wish to thank Ruth McNerney for her technical assistance and William Ogunkolade and lain Frame for allowing us to use their radioactivity data for inclusion in Fig. 2. I also wish to thank the Overseas Development Administration for supporting this work and my colleagues for their constructive criticism.
References I Voller, A. and de Sauvigny, D. (1981)J. Irnrnunol. Methods 46, 1-29 2 Houba, V. (1980) Immunological Investigation of Tropical Parasitic D~seases,Churchill Livingstone 3 Labrada, L.A. and Smith, D.S. (1990) Parasitology Today 6, 30 4 Wirth, D.F. et al. (1986) Science 23~,, 975-979 5 Pettersson, U. and Hyppia, T. (I 985) Irnmunol. Today 6, 268-272 6 Waters, A.P. and McCutchan, T.F. (1990) Parasitology Today 6, 56-59 7 Pollack, Y. et al. (1985)Arn.j. Trap. Meal. Hyg. 34, 663-667 8 Ashall, F. et a/. (1988)J. Clin. Microbial. 26, 576-578 9 Barker, D.C. (1987) Parasitology Today 3, 177-184 I 0 Barker, D.C. (1989) Parasitology 99, 125-146 I I McLaughlin,G.L. et al. (1987)Am.J. Trap. Med. Hyg. 37, 27-36 12 Holmberg, M. et al. (1986) Bull. WHO 64, 579-585 13 Barker R.H., Jr et al. (1989) Am. J. Trap. Meal Hyg. 41,266-272
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14 Mucenski,C.M. et al. (1986) Am. J. Trap./vied. Hyg. 35, 912-920 15 Leary,J.J., Brigati, D.J. and Ward, D.C. (1983) Proc. Natl Acad. Sci. USA 80, 4045-4049 16 Heiles, B.J. et al. (1988) Biotechniques 6, 978-98 I 17 Keller, G.H. and Manak, M.M. (1989) DNA Probes, Macmillan Publishers 18 Poverenny, A.M. et al. (1979)/viol. Irnmunol. 16, 313-316 19 Renz,M. and Kurz, C. (1984) Nucleic Acids Res. 12, 3435-3444 20 Sethabutr, O. et al. (I 988) Am. J. Trap. MMed. Hyg. 39, 227-231 21 Chen, G-X. et al. ( 1991) Mol. Biochem. Parasitol. 44, 165-174 22 Saiki, R.K. et al. (1988) Science 239, 487-494 23 Wittwer, C,T., Fillmore, G.C. and Garling, D.J. (1991 ) Anal. Biochem. 186, 328-33 I 24 de Bruijn, M.H.L. (I 988) Parasitology Today 4, 293-295
25 Maser, D.R. et ol. (1989)J. Clin. MMicrobiol. 27, 1477-1482 26 Saiki,R.K.et al. (1989) Pr0c.Natl Acad. Sci. USA 86, 6230-6234 27 Lundeberg, J. et al. (1990) DNA Cell Biol. 9, 289-292 28 Kemp, D.J. et al. (1989) Proc. Natl Acad. Sci. USA 86, 2423-2427 29 Kemp, D.J. et al. (1990) Gene 94, 223-228 30 Inouye, S, and Hondo, R. ( 1990)J. Clin./Microbiol. 28, 1469-1472 31 Lundeberg, J., Wahlberg, J. and Uhlen, M. ( 1991) Biotechniques I 0, 68-75 32 Cimino, G.D. et al. (1991) Nucleic Acids Res. 19, 99-108 33 Longo, M.C., Berninger, M.S. and Hartley, J.L. (1990) Gene 93, 125-128 34 Lomeli, H. et al. (1989) Clin. Chem. 35, 1826-183 I 35 Kwoh, D.Y. et al. (I 989) Proc. Natl Acad. Sci. USA 86, I 173-1177
36 Thompson, J. and Gillespie, D. 0987) Anal. Biochern. 163, 281-29 I 37 Reed, K.C. and Matthaei, K.I. (1990) Nucleic Acids Res. 18, 3093 38 Wirth, D.F. and McMahon-Pratt, D. (1982) Proc. Natl Acad. Sci. USA 79, 6999-7003 39 Ready, P.D., Smith, D.F. and Killick-Kendrick, R. (1988) MMed. Vet. Entomol. 2, 109- I 16 40 Panaccio,M. and Lew, A. ( 1991) Nucleic Acids Res. 19, 1151 41 Jansen,R.W., Siegl,G. and Lemon, S.M.(I 990) Proc. Natl Acad. Sci. USA 87, 2867-287 I 42 Yamada, O. et al. (1990)J. Virot. /Methods 27, 203-210 43 Warhurst, D.C., Awad El Kariem, F.M. and Miles, M.A. (199 I) Lancet 337, 303-304 Stuart Wilson is at the London School of Hygiene and Tropical Medicine, Keppel Street, London W C I E 7HT, UK.
Boo£$ Books in Brief New Perspectives on Evolution by Leonard Warren and HiJrary Koprowski, Wiley-Liss, 1991. $49.95 (xii + 258 pages) ISBN 0 471 56068 5 With increasing interest in the evolutionary aspects of parasitology, parasitologists who are not experts in this field require a considerable amount of background information in order to understand the various arguments that are put forward. This Wistar Symposium, which represents the proceedings of a meeting held at the University of Pennsylvania in April 1990, will fill some of the gaps. There are fourteen chapters divided between four sections: Evolutionary Perspectives in Soviet Russia, Interplay of Org~,nisms and Environment, and two sections on the Impact of Recent Advances in Molecular Biology. The following review-type chapters are likely to be of interest to parasitologists: 'An Overview of Current Evolutionary Biology' (Ernst Mayr), 'New Perspectives on the Molecular Evolution of Genes and Genomes' (Daniel L. Hart1), 'Evolution of Transposable Elements in Drosophila' (Margaret G. Kidwell and Kenneth R. Peterson), 'Gene Structure and Evolutionary Theory' (Walter Gilbert), 'New Perspectives on Evolution Provided by Protein Sequences' (Russell F. Doolittle) and 'The Phylogenetic Significance of Sequence Diversity and Length Variations in Eukaryotic Small Subunit Ribosomal RNA Coding Regions' (Mitchell L. Sogin). In this context, the chapter on transposable elements in Drosophila would serve as a useful background for the article on site-specific retrotransposons
in trypanosomatid protozoa by Serap Aksoy, scheduled for publication in Parasitology Today in October.
Clinical Immunology edited by Jonathan Brostoff et al., Gower Medical Publishing, 1991. $34.95 (ix + 400 pages) ISBN 0 397 44563 6
Elsewhere in the book, there are chapters likely to be of interest to parasitologists: 'AIDS and HIV' by A.J. Pinching, 'Nutrition and Immunity' by R.K. Chandra, 'Present Vaccines' by J.R. Pattison and 'Future Vaccines' by D.J. Rowlands. As for the other books in this series, a slide atlas is also available.
Clinical Immunology is the third member
of a family of books produced by Gower Medical Publishing, of which the previous two, Immunology and Advanced Immunology, have become widely used textbooks. This book comes in the same attractive format with large pages and many clear, coloured illustrations. There are 30 chapters divided into nine sections with contributions from 39 authors. The stated intention is to describe the basic immunological features of each disease and to relate these to clinical features, and the book is largely aimed at medical students and clinicians. Parasites are covered rather briefly, mainly in the chapter 'Principles of Immunity to Infection' by John Playfair. This eleven-page chapter, which tends to concentrate on principles, covers all infectious agents, and parasites receive little more than passing attention; important diseases, such as malaria and toxoplasmosis, receive scant attention while schistosomiasis is not even mentioned. Elsewhere in the book, Chagas disease is described under Cardiovascular Diseases and leishmaniasis under Skin Disorders. Neither the index (which does not include malaria) nor the short, somewhat dated list of suggested reading is particularly informative for those who really want to know more about immunity and immunopathology of parasitic diseases.
The Pesticide Manual (gth edn) edited by C.R. Worthing and R.J. Hance, BCPC Publications, 199/. £ 7 5 . 0 0 (xlvii +
1200 pages) ISBN 0 948404 42 6 Previous editions of The Pesticide M a n u a l have been published every few years since 1968. Essentially this is a list of all available chemicals and microbial agents used for the control of crop pests and diseases, public health pests and animal ectoparasites, together with other substances such as plant growth regulators. Each compound is listed under its International Organization for Standardization (150) common name, with English spelling, and the data given include the molecular formula, derivatives, chemical structure and activity, plus a mass of other information including toxicity. Thus, deltamethrin is listed as a pyretroid insecticide effective against a wide range of pests, with nearly t w o pages devoted to its properties, formulation and toxicity and a useful note stating that its unofficial name, decamethrin, has been rejected. There are four indexes: Chemical Abstracts Service Registry Numbers, Molecular Formulae, Code Numbers (used to identify pesticides) and an Alphabetical List. Altogether, this book contains a wealth of practical information that should be invaluable to all those working with insecticides or other pesticides.
Parasitology Today, vol. 7, no. 9, 1991
22 Tracey, K.J.and Cerami, A. (1990) Ann. NY Acad. Sci. 587, 325-331 23 Kluger,M.J.(I 988) in Biome,-_hanismsRea~ulatlng Growth and Development(Steffens, G.A. and Rumsey, T.S., eds), pp 409-420, Kluwar Academic Publishers 24 Elsasser,T.H., Caperna, T.J. and Fayer, R. Proc. Sac. Exp. Biol. Med. (in press) 25 Scarborough, D.E. (1990) Metabolism 39,
108-11 I 26 Kenison,D.C., Elsasser,T.H. and Fayer,R.Am. J. Vet. Res.(in press) 27 Blalock,J.E.(1989) Physiol.Rev. 68, 1-32 28 Scarborough, D.E. (1990)Ann. NY Acad. Sci. 594, 169-187 29 Barry, T.N. et al. (1985) Aust. J. Biol. Sci. 38, 393-403 30 Porat,O. (1989) LymphokineRes. 8, 459-469
31 Miller, S.C. eta/. (1988) Mol. Cell. Biol. 8, 2295-230 I Ran Foyer is at the Zoonotic Diseases Laboratory and Ted Elsasser is at the Ruminant Nutrition Laboratory at the Livestock and Poultry Sciences Institute of the United States Department of Agriculture, 10300 Baltimore Boulevard, Beltsville, MD 20705-2350, USA.
Nucleic Acid Techniques and the Detection of Parasitic Diseases S.M, Wilson The detection of infectious diseasecausing organisms is important for the initiation of effective treatment, in monitoring response to therapy and in epidemiological studies of disease of human or animal hosts. In this article Stuart Wilson primarily considers parasitic diseases, but much can be applied to infectious diseases in general.
The simplest method of diagnosis is often by microscopic observation of parasites in blood films, '.~ools or after culture, in vitro. This method is sensitive but time consuming anti requires an experienced eye. As an alternative, many immunological methods of diagnosis have been developed, based either on the detection of circulating host antibodies to parasite antigens or the detection of parasite antigens themselves. These methods, while sensitive, have disadvantages I. Anti-parasite antibodies, when induced, may not be present until some time after the initiation of infection and conversely may persist long after the resolution of infection. The persistence of antibodies may be a problem in endemic ;areas; certain individuals may have a high level of reactive antibodies in the absence of the organism. Similarly, immunized individuals may have antibodies that confuse the diagnosis. Ideally, anti-parasite ~Lntibodies are detected by recognition of well-defined, purified antigen. Such antigens are difficult to obtain and the use of crude antigens can decrease the specificity of the assay because of epitopes shared by different parasite species. Similarly, detection of parasite antigens with polyclonal antibodies may suffer from nonspecific crossreactivity. It is hoped that the cloning and expression of genes that encode parasite-specific immuno~) 199 I, Elsevier Science Publishers Ltd, (UK) 0169 4707/91/$02.00
dominant peptides and the use of monoclonal antibodies will increase the specificity of these assays. Such methods have been used to detect a variety of parasitic diseases2 but, in those instances when an adequate immunodiagnostic test is not possible, direct detection of the parasite genetic material using nucleic acid probes or amplification techniques may provide an alternative. Detection of Parasites Using Nucleic Acid Probes Radiolabelled nucleic acid probes. Nucleic acid detection traditionally involves the autoradiographic localization of denatured, radiolabelled nucleic acid probes hybridized to membranebound, denatured target nucleic acids (Fig. l a). One advantage of nucleic acid probe-based assays is that a parasitic infection is always accompanied by the corresponding parasite nucleic acids. Furthermore, by careful selection of unique, invariant regions of the parasite genome, such probes can be specifically directed against reiterated, repetitive or multicopy sequences, thereby enhancing the sensitivity of detection. Kinetoplast DNA 3'4, tandem repeats 4's, parasite ribosomal RNA sequences 6 and total parasite genomes 7'8 have all been used as hybridization targets. As individual repetitive sequences may comprise I-10% of the total genomic DNA and there may be 10-50-fold more ribosomal RNA than genomic DNA per parasite, the advantages of using either as a target for a diagnostic probe are apparent. Cloned repeat sequences, synthetic oligonucleotides or labelled total parasite DNA have been used to detect
species of Leishmania, Trypanosoma, Echinococcus, Taenia, Fasciola, Onchocerca, Brugia and Plasmodium (for reviews see Refs 9, 10) but the sensitivities reported for such probes vary greatly. The reported sensitivity may depend on the type of probe, the method of sample preparation or the origin of the sample. For example, the sensitivities reported for cultured parasites or purified parasite DNA can be an order of magnitude higher than when clinical samples are investigated 11-13 Obviously the latter is an important consideration and some potentially promising diagnostic nucleic acid probes have proved to be less promising when tested in the field. Even so, some radiolabelled probes can be comparable to microscopy for malarial diagnosis4' 12.t3 and the resulting hybridization signals can be proportional to the number of parasites present 4'7'1~ which is important in quantitative diagnosis. A great advantage of nucleic acid probes is the ability to screen hundreds ~3 or thousands ~ of samples simultaneously, whereas microscopy is a skilled task requiring many minutes per slide. Nonradiolabelled nucleic acid probes. Radioactive labelling of nucleic acids is a versatile, simple and highly sensitive technique but it is of restricted use for routine diagnosis. Radiolabelled probes have to be used in restricted areas and their necessarily short half-lives make it difficult to incorporate them into simple detection kit format for use in routine diagnosis. This may explain why, despite exhaustive research on generating sensitive nucleic acid probes for many diseases, these assays are confined to research laboratories and have failed to be implemented in the general diagnostic laboratory.
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Parasitology Today, vol. 7, no. 9, 1991 b
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However, there are several nonisotopic alternatives to radiolabelling; the systems shown in Table I are marketed commercially. The simplest nonradioactive systems substitute chemical haptens for the isotopic label. The haptens most commonly used are the vitamin biotin Is and the plant steroid digoxigenin ~6. Both molecules can be covalently linked to nucleic acid bases and the hapten-modifled bases can subsequently be incorporated into a strand of DNA or RNA by commonly used DNA or RNA polymerases to form nucleic acid probes 17. Alternatively, these haptens can be chemically linked to the bases of the nucleic acid probe 17. Other chemical modifications, eg. sulphomodification ~8, can be used to generate different chemical haptens. After hybridization of the probes to target nucleic acids and the removal of unbound probe, the haptens bound to any hybridized probe can be detected by a number of systems. Biotin haptens can be detected using the protein avidin (from egg white) or streptavidin (from bacteria), both of which have a high affinity for biotin. Alternatively, biotin can be detected using an anti-biotin antibody. Similarly, other haptens (eg. digoxigenin and sulphomodified bases) are detected using anti-hapten antibody systems. If these hapten-binding proteins are in turn covalently linked to enzyme molecules (reporter enzymes), the whole target-probe-haptenhapten-binding protein-enzyme complex can be visualized by the action of the enzyme on substrate (Fig. I b). Alkaline phosphatase, horseradish peroxidase, 13-galactosidase and urease can be used as reporter enzymes although alkaline phosphatase and horseradish peroxidase are the most commonly used. Depending on the choice of substrate, the coloured product can be soluble for spectrophotometry or insoluble for visual detection. Such enzymes can even be coupled directly to the nucleic acid probe jg. As there is no need for a hapten or
hapten-binding protein, this direct enzyme labelling of the probe enables some incubation and washing steps to be omitted (Fig. I c). One disadvantage of this method is that the hybridization must be performed at lower temperatures to preserve the integrity of the enzyme. The specificity of the probe at these lower temperatures is maintained by using denaturants such as urea or formamide which lower the melting temperature of the nucleic acid probe while reversibly denaturing the enzyme component of the probe. Such alkaline phosphatase-labelled oligonucleotide probes have been used to detect Plasmodium in infected blood and have been used to monitor the clearance of parasites after therapy during which both the parasitemia and the hybridization signal declined 2°. Unfortunately these nonisotopic systems have proved to be of limited use in the development of routine, diagnostic nucleic acid probes because the sensitivities reported for these probes are usually lower than those for the corresponding 32p-labelled probes I 1.12. More recently, the substitution of colorimetric with chemiluminescent substrates has increased the potential sensitivities of many of these nonradiolabelled systems so that they approach or even surpass the sensitivity attainable by radioisotopes (see Box I).
In our laboratory we are investigating the potential of chemiluminescence and have used a digoxigenin-labelled repeat sequence and chemiluminescence to detect Leishmania parasites in tissuetouch blots from infected mice, with a sensitivity similar to that with the same probe labelled radioactively (Fig. 2). With the recent introduction of these sensitive and safe alternatives to radiolabelled probes, it is likely that we will see a rapid development of nucleic acid probes from research tools used solely in research laboratories to routine diagnostic assays applicable in the general diagnostic laboratory. F o r m a t for H y b r i d i z a t i o n
The time taken to perform an assay is often an important factor in its development. The traditional method of detection of membrane-bound nucleic acids using radiolabelled probes is a lengthy process. Hybridization is usually performed for 16h and the autoradiographic detection of the bound probe takes another 16-1 8 h. This would be a distinct disadvantage in the development of any diagnostic assay. However, there are several ways in which the time taken for these assays can be reduced. Nonisotopic probes reportedly exhibit less nonspecific binding to membranes and are often used at much higher concentrations than are isotopic probes ~s. This higher concentration of probe can serve to drive the hybridization reaction and increase the rate at which probe binds to the membranebound target. Thus the hybridization may be performed in 2-4h. Similarly, the visualization of these probes using chromogenic or chemiluminescent substrates is much more rapid and can be achieved in a similar time. Alternatively, to increase sensitivity and decrease hybridization times, single-
Table I. Nonradioactive systems for the detection of nucleic acid probes Hapten
Hapten-binding protein
Biotin
Avidin or streptavidin or anti-hapten antibody
DigoxJgenin Chemically modified bases, eg. sulphomodification Direct coupling of enzyme to probe
Anti-hapten antibody
Reporter enzyme
Substrate
Alkaline phospha~se or horseradish
Soluble or insoluble colour or chemiluminescence
peroxidase
Parasitology Today, vol. 7, no. a, 199 /
stranded DNA or RNA probes can be used w. These probes can be used at high concentrations in the hybridization reaction because, being single stranded, the target-probe hybridization on the membrane does not have to compete with probe-probe rehybridization in solution. Membrane-based hybridizations proceed more slowly than do solution hybridizations. The immobilization of the target nucleic acid onto a solid support alters the kinetics of the hybridization reaction and may reduce the amount of target accessible for detection at the membrane', surface. By adopting a solution hybridization methodology, the hybridization time can be greatly reduced and the reaction may proceed more efficiently; some method of post-hybridization capture of the target-probe hybrid onto a solid support may be necessary for quantitation of the reaction ~7. Such a solution hybridization and hybrid-capture protocol has been used to detect as little as 0. I pg of P. falciparum genomic DNA in minutes rather than hours 21. Typically, by using on,-= or more of these systems the assay times can be greatly reduced and the,. whole assay from hybridization to detection may be carried out in less than one day.
257 Box I. Chemiluminescent Substrates for Use With Nucleic Acid Probes
Enzyme-catalysed luminescence is not a new phenomenon. Since the 1960s the luminescent oxidation of luciferin by luciferase and of luminol by horseradish peroxidase have been studied. More recently similar substrates based on substituted dioxetanes have been developed for alkaline phosphatase. As both horseradish peroxidase and alkaline phosphatase are the most common enzymes used in nonradioactive, membrane-based nucleic acid detection systems, there is scope for replacing the chromogenic substrates for these enzymes with the chemiluminescent substrates (see below). One disadvantage of the latter is that the visualization of the signal is indirect and must be detected by exposure to photographic film. Such chemiluminescent substrates have been shown to be more sensitive than chromogenic substrates; this is due in part to the inhibition of enzyme activity by the insoluble chromogenic products and the use of enhancers in the chemiluminescent assay. Such enhancers can increase the intensity of the light emission by several orders of magnitude. The kinetics of light output from enhanced luminot and dioxetane systems do differ. The signal from the enhanced luminol system reaches a maximum after I-5 min and thereafter declines sharply. This means that the detection must be rapid but the maximum exposure is achieved very quickly. However, the signal from the enhanced dioxetane system begins to increase slowly, reaching a maximum after 60-90 min and, once at this maximum, declines only slowly over the next few hours. This increases the time needed for detection but means that the sensitivity can be increased by prolonged exposure and, if required, there is also time for multiple exposures. There are advantages and disadvantages to both of these chemiluminescent systems, depending on whether a rapid, repeatable or more sensitive system is desirable, but both have distinct advantages when compared to chromogenic or isotopic detection systems in that they combine sensitivity with safety. 0-0
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The polymerase chain reaction (PCR) (Ref. 22) is another alternative to the use of radiolabelled probes that relies on a completely different system of detection. From a nonspecific mass of DNA or RNA, defined fragments of usually 100-400 base pairs (bp) can be specifically amplified using oligonucleotide primers of defined sequence that bind to the target region to be amplified. This technique is very sensitive as the amplification is exponential until the concentration of nucleoddes, primers or enzyme becomes limiting. One cycle of PCR consists of thermal denaturation of the DNA, annealing of the oligonucleotide primers to the target DNA (one primer binding to each strand of DNA) at a lower, empirically determined temperature and then synthesis of DNA from each of these primers using a DNA polymerase. The DNA polymerase most widely used is Taq DNA polymerase, which was isolated from a thermophilic bacteria. This polymerase is stable at high temperatures, enabling many such cycles (3040) to be performed without addition of enzyme after each thermal denaturation step. This has enable.d automation
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of the cycling by microprocessor-controlled thermal cyclers of various design. Such cyclers can complete 25-30 cycles in under 3 h, though times as short as 0.25 h have been reported 23. The potential importance of PCR for the detection of parasitic diseases has been discussed by de Bruijn 24 and in some instances has already been realized. PCR has been used to detect Trypanosoma infections with a sensitivity greater than that with either microscopy or hybridization with radiolabelled DNA probes 2s. The PCR product is usually analysed by agarose gel electrophoresis followed by ethidium bromide staining and visualization with ultraviolet (uv) light. Re-
cently, colorimetric tests have been devised for PCR products 26-3°. These systems usually use modified primers, and either involve direct binding of the PCR product to a solid support and hybridization with specific oligonucleotide probes or the indirect binding of the PCR product and subsequent detection by virtue of modified primers. For example, a short nucleotide sequence that is recognized by a DNAbinding protein can be added to the end of the target-specific sequence of one of the primers. If the other primer used for the PCR has an attached biotin molecule then there are two systems that could be used for detection of this PCR product. The PCR product can be
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immobilized using either DNA-binding protein or avidin coated onto microtiter plates. Once immobilized, these products can be visualized using streptavidin 28'29 or DNA-binding protein enzyme conjugates27, respectively. These colorimetric systems can be adapted for a quantitative detection assay31 and lend themselves more easily to automation, both of which are important for screening many samples and for making a quantitative diagnosis. Although PCR is an exquisitely sensitive technique, this sensitivity can cause problems. Product from previous reactions can contaminate the laboratory environment and may act as a target for subsequent PCR. Thus contamination of a negative sample with a few molecules of product prior to PCR can lead to the generation of false positive results after PCR. The risk of sample contamination can be lessened by using positive displacement pipettes or aerosol-guarded disposable tips, by physically separating the areas used for sample preparation from those used for analysis of product and by uv sterilization of plastics or the reaction components themselves prior to PCR. Post- and pre-PCR sterilization methods have been published. The former modifies the PCR product chemically after PCR so that it cannot act as a target for subsequent PCR reactions 32 whereas the latter produces modified PCR product in the PCR itself which can be specifically degraded prior to subsequent PCR reactions 33. If the ultimate aim for PCR is a fully automated assay from sample preparation to analysis of product, thus enabling the simultaneous screening of many samples and the reduction of the chances of contamination, then these innovations will help towards that aim. More recently, alternative amplification systems involving the replication of a small RNA molecule by QJ3 replicase~4 and a transcription-based amplification system 35 have been reported. In the future these systems may become viable alternatives to PCR.
Sample Preparation Sample preparation is one factor that is often overlooked when describing an otherwise simple nucleic acid detection system, and the method of choice can sometimes affect the sensitivity of the assay 11'13. Often very crude samples can be used in immunodiagnosis whereas nucleic acid detection systems often require highly purified samples. Although simple methods do exist for the preparation of nucleic acids from many
b
Fig. 2. Detection of parasites in tissue-touch blots. The spleen (S) and liver (L) from a mouse infected with Leishmania donovani were touched repeatedly onto a nylon membrane. After denaturation, neutralization and uv-fixation of the DNA to the membrane, the membrane was probed with the radiolabelled complementary DNA (cDNA) clone Lmet 2 (10 6 cpmml-I), a L. donovani complexspecific probe that recognizes a series of repeat sequences in the parasite genome. Hybridization was for 16 h and the membrane was autoradiographed for 24 h (a). The membranes were then stripped of probe and reprobed with digoxigenin-labelled Lmet 2. Hybridization was for 16 h and the hybridized probe was detected with an anti-digoxigenin antibody conjugated to alkaline phosphatase after which the blot was overlayed with a dioxetane-based chemiluminescent substrate and exposed to photographic film for 4 h (b). There was no signal from the liver and spleen of an uninfected mouse with either probe (results not shown).
sources fT, some degree of expertise is required and the procedures can be lengthy and may involve the use of expensive, specialized equipment or the use of noxious, expensive chemicals. Clearly this is one aspect of nucleic acid detection methodology that must be addressed and progress is being made in this area. Solution hybridization methodologies can be simplified by hybridization directly in the chaotropic agent used for tissue solubilization36. Furthermore, such hybridizations may be performed at reduced temperatures (even room temperature), thus reducing the requirement for additional equipment. Where samples need to be applied to membrane supports, alkali denaturation and solubilization of crude tissue samples and the direct fixation of these samples to modified nylon membranes is extremely rapid and simple 37. Other methods involve the direct application of samples to the membrane. The dotting of blood samples7'~2'2°, tissuetouch blots 38 or the squashing of whole sandflies onto membranes prior to the detection of parasite infection by hybridization 4'39 have all been shown to be effective.
Often purified DNA is used as a template for PCR but this can be inconvenient and the preparation time consuming. However, small quantities of crude material can sometimes be used directly in the reaction, as in the PCR of material directly from small quantities of whole blood 4°. Other systems involve the capture of the infectious agent from crude samples by the use of specific antibodies coated onto solid supports 4~. After washing and lysis of the captured organisms by heating, the released nucleic acids can be used directly in PCR. Similarly, DNA can be captured from crude samples by absorption onto glass beads42 or filter discs43 before PCR.
Summary New developments in nucleic acid technology, such as PCR and the replacement of isotopic with safe, sensitive nonisotopic systems, witl enable nucleic acid detection techniques to progress from the research to the diagnostic laboratory. By using the most advanced and innovative technologies it is hoped that these systems will be developed into rapid, simple and yet sensitive assays which, in some instances, will become the methods of choice for the routine detection and diagnosis of disease-causing organisms. Acknowledgements I wish to thank Ruth McNerney for her technical assistance and William Ogunkolade and lain Frame for allowing us to use their radioactivity data for inclusion in Fig. 2. I also wish to thank the Overseas Development Administration for supporting this work and my colleagues for their constructive criticism.
References I Voller, A. and de Sauvigny, D. (1981)J. Irnrnunol. Methods 46, 1-29 2 Houba, V. (1980) Immunological Investigation of Tropical Parasitic D~seases,Churchill Livingstone 3 Labrada, L.A. and Smith, D.S. (1990) Parasitology Today 6, 30 4 Wirth, D.F. et al. (1986) Science 23~,, 975-979 5 Pettersson, U. and Hyppia, T. (I 985) Irnmunol. Today 6, 268-272 6 Waters, A.P. and McCutchan, T.F. (1990) Parasitology Today 6, 56-59 7 Pollack, Y. et al. (1985)Arn.j. Trap. Meal. Hyg. 34, 663-667 8 Ashall, F. et a/. (1988)J. Clin. Microbial. 26, 576-578 9 Barker, D.C. (1987) Parasitology Today 3, 177-184 I 0 Barker, D.C. (1989) Parasitology 99, 125-146 I I McLaughlin,G.L. et al. (1987)Am.J. Trap. Med. Hyg. 37, 27-36 12 Holmberg, M. et al. (1986) Bull. WHO 64, 579-585 13 Barker R.H., Jr et al. (1989) Am. J. Trap. Meal Hyg. 41,266-272
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14 Mucenski,C.M. et al. (1986) Am. J. Trap./vied. Hyg. 35, 912-920 15 Leary,J.J., Brigati, D.J. and Ward, D.C. (1983) Proc. Natl Acad. Sci. USA 80, 4045-4049 16 Heiles, B.J. et al. (1988) Biotechniques 6, 978-98 I 17 Keller, G.H. and Manak, M.M. (1989) DNA Probes, Macmillan Publishers 18 Poverenny, A.M. et al. (1979)/viol. Irnmunol. 16, 313-316 19 Renz,M. and Kurz, C. (1984) Nucleic Acids Res. 12, 3435-3444 20 Sethabutr, O. et al. (I 988) Am. J. Trap. MMed. Hyg. 39, 227-231 21 Chen, G-X. et al. ( 1991) Mol. Biochem. Parasitol. 44, 165-174 22 Saiki, R.K. et al. (1988) Science 239, 487-494 23 Wittwer, C,T., Fillmore, G.C. and Garling, D.J. (1991 ) Anal. Biochem. 186, 328-33 I 24 de Bruijn, M.H.L. (I 988) Parasitology Today 4, 293-295
25 Maser, D.R. et ol. (1989)J. Clin. MMicrobiol. 27, 1477-1482 26 Saiki,R.K.et al. (1989) Pr0c.Natl Acad. Sci. USA 86, 6230-6234 27 Lundeberg, J. et al. (1990) DNA Cell Biol. 9, 289-292 28 Kemp, D.J. et al. (1989) Proc. Natl Acad. Sci. USA 86, 2423-2427 29 Kemp, D.J. et al. (1990) Gene 94, 223-228 30 Inouye, S, and Hondo, R. ( 1990)J. Clin./Microbiol. 28, 1469-1472 31 Lundeberg, J., Wahlberg, J. and Uhlen, M. ( 1991) Biotechniques I 0, 68-75 32 Cimino, G.D. et al. (1991) Nucleic Acids Res. 19, 99-108 33 Longo, M.C., Berninger, M.S. and Hartley, J.L. (1990) Gene 93, 125-128 34 Lomeli, H. et al. (1989) Clin. Chem. 35, 1826-183 I 35 Kwoh, D.Y. et al. (I 989) Proc. Natl Acad. Sci. USA 86, I 173-1177
36 Thompson, J. and Gillespie, D. 0987) Anal. Biochern. 163, 281-29 I 37 Reed, K.C. and Matthaei, K.I. (1990) Nucleic Acids Res. 18, 3093 38 Wirth, D.F. and McMahon-Pratt, D. (1982) Proc. Natl Acad. Sci. USA 79, 6999-7003 39 Ready, P.D., Smith, D.F. and Killick-Kendrick, R. (1988) MMed. Vet. Entomol. 2, 109- I 16 40 Panaccio,M. and Lew, A. ( 1991) Nucleic Acids Res. 19, 1151 41 Jansen,R.W., Siegl,G. and Lemon, S.M.(I 990) Proc. Natl Acad. Sci. USA 87, 2867-287 I 42 Yamada, O. et al. (1990)J. Virot. /Methods 27, 203-210 43 Warhurst, D.C., Awad El Kariem, F.M. and Miles, M.A. (199 I) Lancet 337, 303-304 Stuart Wilson is at the London School of Hygiene and Tropical Medicine, Keppel Street, London W C I E 7HT, UK.
Boo£$ Books in Brief New Perspectives on Evolution by Leonard Warren and HiJrary Koprowski, Wiley-Liss, 1991. $49.95 (xii + 258 pages) ISBN 0 471 56068 5 With increasing interest in the evolutionary aspects of parasitology, parasitologists who are not experts in this field require a considerable amount of background information in order to understand the various arguments that are put forward. This Wistar Symposium, which represents the proceedings of a meeting held at the University of Pennsylvania in April 1990, will fill some of the gaps. There are fourteen chapters divided between four sections: Evolutionary Perspectives in Soviet Russia, Interplay of Org~,nisms and Environment, and two sections on the Impact of Recent Advances in Molecular Biology. The following review-type chapters are likely to be of interest to parasitologists: 'An Overview of Current Evolutionary Biology' (Ernst Mayr), 'New Perspectives on the Molecular Evolution of Genes and Genomes' (Daniel L. Hart1), 'Evolution of Transposable Elements in Drosophila' (Margaret G. Kidwell and Kenneth R. Peterson), 'Gene Structure and Evolutionary Theory' (Walter Gilbert), 'New Perspectives on Evolution Provided by Protein Sequences' (Russell F. Doolittle) and 'The Phylogenetic Significance of Sequence Diversity and Length Variations in Eukaryotic Small Subunit Ribosomal RNA Coding Regions' (Mitchell L. Sogin). In this context, the chapter on transposable elements in Drosophila would serve as a useful background for the article on site-specific retrotransposons
in trypanosomatid protozoa by Serap Aksoy, scheduled for publication in Parasitology Today in October.
Clinical Immunology edited by Jonathan Brostoff et al., Gower Medical Publishing, 1991. $34.95 (ix + 400 pages) ISBN 0 397 44563 6
Elsewhere in the book, there are chapters likely to be of interest to parasitologists: 'AIDS and HIV' by A.J. Pinching, 'Nutrition and Immunity' by R.K. Chandra, 'Present Vaccines' by J.R. Pattison and 'Future Vaccines' by D.J. Rowlands. As for the other books in this series, a slide atlas is also available.
Clinical Immunology is the third member
of a family of books produced by Gower Medical Publishing, of which the previous two, Immunology and Advanced Immunology, have become widely used textbooks. This book comes in the same attractive format with large pages and many clear, coloured illustrations. There are 30 chapters divided into nine sections with contributions from 39 authors. The stated intention is to describe the basic immunological features of each disease and to relate these to clinical features, and the book is largely aimed at medical students and clinicians. Parasites are covered rather briefly, mainly in the chapter 'Principles of Immunity to Infection' by John Playfair. This eleven-page chapter, which tends to concentrate on principles, covers all infectious agents, and parasites receive little more than passing attention; important diseases, such as malaria and toxoplasmosis, receive scant attention while schistosomiasis is not even mentioned. Elsewhere in the book, Chagas disease is described under Cardiovascular Diseases and leishmaniasis under Skin Disorders. Neither the index (which does not include malaria) nor the short, somewhat dated list of suggested reading is particularly informative for those who really want to know more about immunity and immunopathology of parasitic diseases.
The Pesticide Manual (gth edn) edited by C.R. Worthing and R.J. Hance, BCPC Publications, 199/. £ 7 5 . 0 0 (xlvii +
1200 pages) ISBN 0 948404 42 6 Previous editions of The Pesticide M a n u a l have been published every few years since 1968. Essentially this is a list of all available chemicals and microbial agents used for the control of crop pests and diseases, public health pests and animal ectoparasites, together with other substances such as plant growth regulators. Each compound is listed under its International Organization for Standardization (150) common name, with English spelling, and the data given include the molecular formula, derivatives, chemical structure and activity, plus a mass of other information including toxicity. Thus, deltamethrin is listed as a pyretroid insecticide effective against a wide range of pests, with nearly t w o pages devoted to its properties, formulation and toxicity and a useful note stating that its unofficial name, decamethrin, has been rejected. There are four indexes: Chemical Abstracts Service Registry Numbers, Molecular Formulae, Code Numbers (used to identify pesticides) and an Alphabetical List. Altogether, this book contains a wealth of practical information that should be invaluable to all those working with insecticides or other pesticides.