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New approaches to diagnosis of parasitic diseases


Use of monoclonal antibodies and DNA probes for diagnosis of parasitic diseases
Development of monoclonal antibodies and specific antigens for diagnosis of African trypanosomiasis
Development of DNA probes for identification of African trypanosomes
Development of nonradioactive DNA probes for identification of African trypanosomes
Genotype analysis of trypanosomes
Use of monoclonal antibodies and DNA probes for theilerial strain/species characterization

Chairperson: J. David

Use of monoclonal antibodies and DNA probes for diagnosis of parasitic diseases

J. David

Rapporteur's summary

The lifecycle and geographic distribution of the species and subspecies of parasites causing visceral, cutaneous and mucocutaneous forms of leishmaniasis were described. It is important to distinguish these species and subspecies with regard to differences in pathogenicity.

Monoclonal antibodies were raised against purified membrane preparations of promastigotes and amastigotes. The number of species-specific monoclonal antibodies obtained was low (about 2% for promastigotes and 0.5% for amastigotes).

Using the monoclonal antibodies it was possible to:

(1) differentiate all the New World species and some of the Old World species

(2) identify amastigotes in tissues

(3)- detect promastigotes in the sandfly vector

(4) develop species-specific serological assays.

When some parasite antigens were isolated using the species-specific monoclonal antibodies, there was a loss of specificity in the purified antigens. This problem was alleviated by the use of a blocking assay in which crude antigen was adsorbed onto the plate and its reactivity with patients' sera blocked with the species-specific monoclonal antibody.

On the premise that there are repetitive sequences in kDNA, whole kDNA was purified from Leishmania. Whole kDNA preparations were used as the hybridization probe. It was possible in this way to detect as few as 100 organisms in tissue lesions by touch blot analysis and to distinguish L mexicana from L brasiliensis. Such kDNA probes could not be used to distinguish subspecies and there was a lot of cross-reactivity between Old World species. By shotgun cloning of kDNA minicircles and differential screening bacteria containing species-specific kDNA sequences were identified. Additional specificity was obtained by generating sequential Bal-31 deletions of the clones. The clones with inserts of 700 base pairs were not specific, whereas those of 400 base pairs were so specific that they could distinguish subspecies of L mexicana as well as specific isolates of the subspecies. This approach may be applicable to trypanosomes to achieve probes of selected specificities.

A similar approach was used to generate genomic probes specific to Plasmodium falciparum. In the blot test using parasitized whole blood, there were problems of high background. This was overcame by first mixing 100 microlitres of blood in 500 microlitres of lysis buffer containing Triton X100 and digesting the protein before application of the material to nitrocellulose filter paper. By so doing, the presence of P falciparum was detectable with DNA probes in blood samples where the parasitaemia was as low as 0.001%. Probes for the Brugia species of filaria have also been derived by shotgun cloning and used to detect parasites in the vector.

Points arising from the discussion

The value of antibody detection as a means of diagnosis was compared to demonstration of the presence of the causative organisms or their antigens. It was stressed that antibody detection could be useful in some instances, such as when it is difficult to demonstrate parasites or their antigens in certain epidemiological situations or in some eases of clinical diagnosis, e.g. splenomegaly due to L donovani. It was suggested that the sensitivity of the probes can be increased 100- to 1000-fold by the use of the SP6 plasmid.

The practicality of using nonradioactive probes was raised, as well as the problem of the high background and associated sensitivity. In some instances drug resistance can be related to amplification of certain genes and these genes could be used as hybridization probes.

Development of monoclonal antibodies and specific antigens for diagnosis of African trypanosomiasis

V. M. Nantulya

Rapporteur's summary

A major constraint to the serological diagnosis of African trypanosomiasis is the lack of defined reagents. Monoclonal antibodies were derived against membrane surface antigens of Trypanosoma brucei brucei, T vivax and T congolense by immunizing mice with in vitro propagated uncoated trypanosomes. These antibodies have been tested against several T b brucei, T b rhodesiense, T b gambiense, T evansi, T congolense, T simiae and T vivax isolates from East and West Africa by indirect immunofluorescence and ELISA. The antibodies against T b brucei reacted with all T b brucei, T b rhodesiense and T b gambiense isolates tested but not with the other species. Those against T vivax reacted with T vivax, while the antibodies against T congolense reacted with T congolense and T simiae. By sandwich ELISA, antibodies to T b brucei were used to detect circulating antigens in serum samples from cattle infected with T b brucei. Such antigens were detected 2 weeks after tsetse challenge. Thereafter, they were demonstrable in sera taken every third day even when parasites could not be detected in blood by microscopic examination of the blood buffy coat. The same monoclonal antibodies were also used to detect circulating antigens in sera from known eases of Rhodesian and Gambian sleeping sickness. These antigens were also detested in cerebrospinal fluid of some Rhodesian and Gambian sleeping sickness patients and in infected tsetse flies. The T vivax and T congolense monoclonal antibodies detected the corresponding antigens in sera of T vivax or T congolense-infected cattle. This test would appear to be more sensitive than microscopic examination of the blood buffy coat. Work is in progress to clone and express the genes encoding these antigens in bacteria for mass production of the antigens. An alternative approach involving peptide synthesis is also envisaged. The ultimate objective is to develop a simple field assay system for antigens and antibodies.

Points arising from the discussion

To distinguish the infecting trypanosomes species in field eases where there are multiple infections, sera can be tested against each of the antigens. False negative results in the antigen assay could arise from situations of antibody excess. So far, there has been no problem of false positives arising from hypergammaglobulinaemia in trypanosomiasis. The situation regarding other causes of hypergammaglobulinaemia is being investigated.

Sera from blood donors are now being analysed to establish the minimum serum dilution to be used in a screening assay. The reagents ire being tested against sera from patients with various infectious diseases to determine the level of specificity. Urine samples should also be tested for the presence of antigens.

Development of DNA probes for identification of African trypanosomes

J. Young

Rapporteur's summary

Cloned DNA hybridization probes can be used to detect trypanosomes and distinguish within and between species by two approaches: demonstration of the presence or absence of a particular repetitive sequence or detection of restriction enzyme site polymorphism around a sequence. The first approach is more easily applied because blood or-tissue samples can be used and propagation of trypanosomes is not necessary for DNA preparation.

Repetitive DNA sequences can be easily identified and cloned and can often be used to detect a very small number of parasites, either in the vertebrate host or in the insect vector. For example, repetitive minichromosome sequences cloned from Trypanosoma congolense can detect as few as 30 parasites. These probes appear to be specific for subgroups of T congolense. DNA probes that would define important characteristics of the trypanosomes such as infectivity to man, clinical characteristics or serodeme-specificity would be more valuable, but are more difficult to identify.

Development of nonradioactive DNA probes for identification of African trypanosomes

N. Massamba

Rapporteur's summary

The disadvantages of using radiolabeled probes, i.e. short halflife, expense and disposal problems, can be obviated by biotinylation of the probe. The DNA probes can be biotinylated by nick translation using either biotin-11 dUTP or biotin-11 dCTP, both of which are currently available commercially.

The hybridization reaction can then be visualized enzymatically using either avidin biotinylated peroxidase/alkaline phosphatase or streptavidin biotinylated peroxidase/alkaline phosphatase. The method can be used in all instances where radiolabeling is currently being employed, such as Southern blots, dot blots and colony hybridization. Adaptation for field use is possible since the stability of DNA probes would allow standardization of the assay. Efforts are being made to refine the technique by eliminating the high background and improving sensitivity.

Points arising from the discussion

The following problems associated with the use of nonradioactive detection systems were discussed:

(1) the lack of linear relationship between quantity applied and signal obtained when whole trypanosomes are used; this did not apply when purified DNA was used

(2) photosensitivity of the reagents and, therefore, need to standardize the reagents in each experiment.

A point was also raised regarding the comparative sensitivity of radioactive and nonradioactive probes. It was reported that the sensitivity of the two techniques is similar.

Genotype analysis of trypanosomes

P.A.O. Majiwa

Rapporteur's summary

One of the major impediments to the elucidation of the epidemiology of trypanosomiasis is the lack of appropriate tools with which to identify and characterize parasite populations at the molecular level. The feasibility of using the orthogonal-field-alternation gel electrophoresis (OFAGE) for karyotypic analysis of Trypanosoma congolense cloned populations has been explored. Karyotypic differences were demonstrated at the species and serodeme levels. Qualitative differences in gene expression were also demonstrated using variable surface glycoprotein-specific probes. The potential application of this technique in epidemiological studies was discussed.

Points arising from the discussion

An attempt should be made to resolve the large chromosomes which in the present system do not migrate appreciably. When certain repetitive hybridization probes are used on the chromosome blots, smears instead of discrete bands are observed. This problem might be overcame by decreasing the quantity of trypanosomes applied.

Use of monoclonal antibodies and DNA probes for theilerial strain/species characterization

P. Conrad

Rapporteur's summary

Classification of Theileria parva isolates continues to be a major epidemiological concern. Since the use of anti-schizont monoclonal antibodies alone does not appear to be adequate, the possible use of DNA probes and restriction endonuclease analysis of genomic DNAs is currently being investigated. A ganomic expression library prepared from T parva (Mariakani) piroplasm DNA was screened with anti-schizont monoclonal antibodies and two clones were identified which contain genes coding for schizont antigens. Preliminary experiments show that one of these probes hybridizes to DNA in preparations of lymphocytes infected with the Muguga and Mariakani isolates of T parva, but not to uninfected lymphoblasts. DNA has been prepared from lymphoblastoid cells infected with T p parva (Mariakani, Marikebuni and Muguga isolates), T annulata, T taurotragi and T p lawrencei-type isolates from buffalo. The objective of this approach is to identify gene probes that could be used:

(1) to better characterize isolates and identify closely related parasite populations that may correlate with the strain differences detected using in vitro cytotoxicity tests and in vivo cross-immunity trials

(2) to determine if the antigenic change undergone by T p lawrencei in carrier buffalo and during passage through cattle is the result of antigenic variation and/or genetic selection from a mixed parasite population.

Points arising from the discussion

A question was raised regarding the prospect, of using cell-mediated immunity as a means of typing theilerial strains. It was pointed out that such studies are already in progress.


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