Detection of mutant P2 adenosine transporter (TbAT1) gene in Trypanosoma brucei gambiense isolates from northwest Uganda using allele-specific polymerase chain reaction.
OBJECTIVE
To assess the application of allele-specific PCR (AS-PCR) as a fast, cheap and reliable method for detecting mutant TbAT1 associated with melarsoprol relapse in Trypanosoma brucei gambiense isolates from northwest Uganda.


METHODS
A total of 105 trypanosome isolates were analysed using SfaN1 restriction fragment length polymorphism (RFLP) and AS-PCR, the former used as the gold standard. Sensitivity, specificity, positive and negative predictive values of AS-PCR as well as agreement between the tests were determined.


RESULTS
Eleven trypanosome isolates had mutant TbAT1 while 94 exhibited the wild-type TbAT1 genes. There was a highly significant agreement between SfaN1 RFLP and AS-PCR with kappa and intra-class correlation values of 1.0. The sensitivity and specificity of AS-PCR were both 100%, while the positive and negative predictive values were found to be equal to 1.0. Cost and time analyses were performed and AS-PCR was 4.3 times cheaper than SfaN1 RFLP, in addition to the less time required for its execution.


CONCLUSIONS
AS-PCR should be the test of choice for screening for mutant TbAT1 in the ever-increasing numbers of field trypanosome isolates.
National Livestock Health Research Institute, Tororo, Uganda.
Barbara
B
Nerima
Enock
E
Matovu
George W
GW
Lubega
John C K
JC
Enyaru
D000483:Alleles; D000818:Animals; D002417:Cattle; D004351:Drug Resistance / Q000235:genetics*; D008549:Melarsoprol / Q000494:pharmacology*; D033703:Nucleoside Transport Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D012680:Sensitivity and Specificity; D014344:Trypanocidal Agents / Q000494:pharmacology*; D014347:Trypanosoma brucei gambiense / Q000235:genetics*; D014353:Trypanosomiasis, African / Q000235:genetics; D014454:Uganda
D033703:Nucleoside Transport Proteins; D014344:Trypanocidal Agents; C444719:adenosine transporter; D008549:Melarsoprol