Developing, Validating and Standardizing Methodologies for the Use of PCR and PCR-ELISA in the Diagnosis and Monitoring of Control and Eradication Programmes for Trypanosomosis

Objective:

The general objective is to improve livestock production through effective control/eradication of livestock diseases. More specifically, the CRP aims to introduce molecular biological techniques for more effective diagnosis and surveillance of trypanosomosis.

Activities:

Ten Research contracts and five Agreement holders have been awarded. The first Research Coordination Meeting was held in Antwerp, Belgium, 26-30 March, 2001.The second Meeting took place in Rio de Janeiro, Brazil from 7 to 11 April 2003 and the third Meeting was held in Hanoi Vietnam from 20 to 24 June 2005.

The developmental areas of the CRP include:

  • The establishment of a standardised PCR test to detect trypanosomal DNA in blood samples. The ideal “golden” standard test would consist of a unique reaction using one set of primers amplifying the DNA of all pathogenic trypanosomes of mammals. Since this test is not readily available, in the first phase the PCR might consist of a species-specific test, applied with primers already available that have been shown to properly amplify the DNA of: T. vivax, T. brucei sensu lato, T. congolense (savanna type).
  • Modification of the technique to an easy-to-use, more sensitive, standardised format, which can handle larger numbers of samples (PCR-ELISA).
  • Development of a “pan-tryp” test that will amplify the DNA of all pathogenic trypanosomes of mammals. The test can be applied in specified geographical regions to assess trypanosomosis prevalence and assist decision makers in focusing and implementing appropriate disease control programmes.
  • Evaluation of the PCR-ELISA for the identification of sleeping sickness using human blood samples.
  • Application of a more sensitive technique to evaluate the importance and identify the presence of animal reservoirs of human trypanosomosis.
  • Improved monitoring of control/eradication programs with a more sensitive diagnostic technique with the result that a larger number of animals can be identified as infected and subsequently treated, and that animals no longer infected with parasites will be correctly identified.

The general and more specific themes of the research and development are summarized below.

Standardization of methods and the role of PCR
In line with determination of 'fitness for purpose' by the OIE, emphasis on test development must be placed on an exact determination of the role of the PCR. Elements of relative cost, sample collection and storage, sensitivity, specificity, the need for training, establishment of good laboratory facilities and contamination, should all be considered in the context of what exactly is required in any study at the national level. It was generally agreed that the PCR should be a component of any testing and not aim to totally replace existing assays.

Methods and Protocols
It was agreed that each PCR protocol (based on methods and primers used), should be written and that a standard format should be used. Model protocols from Dr. Peter-Henning Clausen have been received and passed on to participants. It was agreed that some basic background information should also be included with regard to reagent formulation, and Dr. G. Viljoen has sent this material which has been distributed to participant.

DNA Bank
A reference bank of DNA was set up at the at the FAO/IAEA laboratory, Seibersdorf. The DNA is from trypanosomes, however it would be useful to ssend control DNA from various livestock and insect species and DNA from relevant organisms likely to complicate the PCR diagnostic potential with regard to trypanosomosis. The strains selected were well characterized and “reliable” in terms of their pedigree Approximately one milligram of DNA was sent by mosty participants for each sample. The DNA was characterized with respect to the PCR products produced using standardized protocols an that the products would be sequenced to allow quality control.

GLP
The setting up of a PCR laboratory was fundamental. A good laboratory practice (GLP) document for PCR was developed.

Primers
Extensive discussions on which primers could be used to obtain products for use in various aspects of the study of trypanosomosis were made. Many papers presenting data are available from the contract and agreement holders through RCM reports and publications to show this work.

Comparative testing and sensitivity/specificity
The diagnostic sensitivity (DSn) and diagnostic specificity (DSp) of all tests including PCR is paramount in evaluation studies. There was a need to define sensitivity in terms of both analytical and diagnostic potential of the PCR. There are drawbacks to estimating analytical sensitivity from “spiked” samples by dilution of material. There is also a difficulty where field samples are examined and the effect of the matrix on PCR analysis. The stages of sampling and nucleic acid extraction are dominating features in examining diagnostic features.

Antibody assays (ELISA)
The indirect ELISA developed at Seibersdorf was used in the first 2 years of the studies. outlined. This assay features the use of pre-coated plates with either T. vivax or T. congolense, the antigens being totally denatured. The use of other ELISA systems to detect T. evansi was also noted.

PCR-ELISA
It was concluded that there was no direct benefits from the approach in most laboratories and that the advantages of high through put were not really required. There were discussions on the sensitivity aspects of the methods.

More Specific Developments:

  • The role of ITS primers and their use in diagnosis of a range of Trypanosomes is well developed. Publications and reports are included for reference.
  • Trypstick
    The development of a gel-independent stick method for visualising PCR products though a technical contract was reported. The method can be used where electrical power is not available and was reported as having a higher analytical sensitivity of detection of products than gels, which might allow improvement in the diagnostic sensitivity of tests and provide a more consistent approach. A ring test for the Trypstick was devised that will be completed by the end of 2005. Discussions on the development of specific Trypstick applications resulted in agreement that a device to detect trypanosomes through universal primers sets such as the ITS primers was desirable.

Participants:

[Download pdf]

Reports:

  • Report on the First RCM, Antwerp, Belgium, 26 –30 March 2001. [Download pdf]
  • Progress Report of the CRP. [Download pdf]
  • Second RCM, Rio de Janeiro, Brazil, 7–11 April 2003. [Download pdf, 4.7 MB]
  • Third RCM, Hanoi, Vietnam, 20–24 June 2005. [Download pdf]

Project Officer:

J.Crowther