SENSITIVE AND SPECIFIC DETECTION OF TRYPANOSOMA-VIVAX USING THE POLYMERASE CHAIN-REACTION

Sensitive and specific detection of Trypanosoma vivax using the polyme rase chain reaction. Experimental Parasitology 85, 193-205. The nuclei c acid probes that are currently in use detect and distinguish Trypano soma vivax parasites according to their geographic origin. To eliminat e the need for using multiple DNA probes, a study was conducted to eva luate the suitability of a tandemly reiterated sequence which encodes a T. vivax diagnostic antigen as a single probe for detection of this parasite. The antigen is recognized by monoclonal antibody Tv27 curren tly employed in antigen detection ELISA (Ag-ELISA). A genomic clone wh ich contained a tetramer of the 832-bp cDNA sequence was isolated and shown to be more sensitive than the monomer. Oligonucleotide primers w ere designed based on the nucleotide sequence of the 832-bp cDNA inser t and used in amplifying DNA sequences from the blood of cattle infect ed with T. vivax isolates from West Africa, Kenya, and South America. The polymerase chain reaction (PCR) product of approximately 400 bp wa s obtained by amplification of DNA from all the isolates studied. The oligonucleotide primers also amplified DNA sequences in T. vivax-infec ted tsetse flies. Subsequently, PCR was evaluated for its capacity to detect T. vivax DNA in the blood of three animals experimentally infec ted with the parasite. T. vivax DNA was detectable in the blood of inf ected animals as early as 5 days post-infection. Blood and serum sampl es from the three cattle and from six other infected animals were also examined for the presence of trypanosomes and T. vivax-specific diagn ostic antigen. Trypanosomes appeared in the blood 7-12 days post-chall enge, while the antigenemia was evident on Days 5-20 of infection. Ana lysis of the data obtained in the three animals during the course of i nfection revealed that the buffy coat technique, Ag-ELISA, and PCR rev ealed infection in 42, 55, and 75% of the blood samples, respectively. PCR amplification of genomic DNA of T. vivax is thus superior to the Ag-ELISA in the detection of T. vivax. More importantly, both the T. v ivax diagnostic antigen and the gene encoding it are detectable in all the T. vivax isolates examined from diverse areas of Africa and South America. (C) 1997 Academic Press.