DNA differential diagnosis of taeniasis and cysticercosis by multiplex PCR

Abstract

Multiplex PCR was established for differential diagnosis of taeniasis and cysticercosis, including their causative agents. For identification of the parasites, multiplex PCR with cytochrome c oxidase subunit 1 gene yielded evident differential products unique for Taenia saginata and Taenia asiatica and for American/African and Asian genotypes of Taenia solium with molecular sizes of 827, 269, 720, and 984 bp, respectively. In the PCR-based detection of tapeworm carriers using fecal samples, the diagnostic markers were detected from 7 of 14 and 4 of 9 T. solium carriers from Guatemala and Indonesia, respectively. Test sensitivity may have been reduced by the length of time (up to 12 years) that samples were stored and/or small sample volumes (ca. 30 to 50 mg). However, the diagnostic markers were detected by nested PCR in five worm carriers from Guatemalan cases that were found to be negative by multiplex PCR. It was noteworthy that a 720 bp-diagnostic marker was detected from a T. solium carrier who was egg-free, implying that it is possible to detect worm carriers and treat before mature gravid proglottids are discharged. In contrast to T. solium carriers, 827-bp markers were detected by multiplex PCR in all T. saginata carriers. The application of the multiplex PCR would be useful not only for surveillance of taeniasis and cysticercosis control but also for the molecular epidemiological survey of these cestode infections.

FIG. 1.

(A) Multiplex PCR with mtDNA prepared from causative agents of taeniasis or cysticercosis. Lanes 1 to 10, T. saginata; lane 11, a mixture of T. saginata and T. asiatica; lanes 12 to 16, T. asiatica; lanes 17 to 25, American/African genotype of T. solium; lanes 26 to 32, Asian genotype of T. solium. The lane numbers correspond to the lanes shown in Table 1. (B) Confirmation by multiplex PCR of cysticerci developed in immunodeficiency mice. The cysticerci originated from the eggs in lane 11 of panel A. Lanes 1 to 4, 6 to 8, 10, 12, 13, and 16, T. asiatica; lanes 5, 9, 11, 14, and 15, T. saginata. M, DNA size markers (100-bp ladder [Promega]).

FIG. 2.

Detection limit of target gene by multiplex PCR with DNA samples prepared from taeniid eggs. Target cox1 fragments were amplified by a dose-dependent fashion (indicated by an arrow). Lane 1, positive control with mtDNA from a T. saginata proglottid, lanes 2 to 8, 5, 10, 20, 50, 10², 10³, and 10⁴ eggs/g of feces, respectively.

FIG. 3.

Differential diagnosis of tapeworm carriers by multiplex and nested PCR with copro-DNA. Diagnostic markers with molecular sizes of 720, 984, and 827 bp were detected from tapeworm carriers with the T. solium American/African genotype from Guatemala (lanes 1 to 7), and nested PCR results with Tsol/Amer and Rev primers products are shown in lanes 8 to 14. The worm carriers with the Asian genotype of T. solium from Indonesia and T. saginata are shown in lanes 15 to 18 and lanes 19 to 23, respectively. Lanes 24 to 30 are from negative controls. M, DNA size markers.