Development of a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) for clinical detection of Plasmodium falciparum gametocytes.
Plasmodium falciparum gametocytes are usually present in peripheral blood at a very low level, thus requiring a sensitive assay detection method. In this study, reverse transcription-loop-mediated isothermal amplification (RT-LAMP) was developed for clinical detection of P. falciparum gametocytes. Transcripts of Pfs16 of sexually committed ring and Pfs25 of mature gametocytes were detected by RT-LAMP in 82 clinical blood samples using nested RT-PCR as a gold standard. RT-LAMP demonstrated a detection limit of 1 parasitized red blood cell (RBC)/500microl of blood for both Pfs16 and Pfs25 transcripts. For Pfs16 transcript, RT-LAMP detected all 30 samples positive by nested RT-PCR (100% sensitivity) and 1 in 52 samples negative by nested RT-PCR (98.1% specificity). For Pfs25 transcript, RT-LAMP detected all 15 samples positive by nested RT-PCR (100% sensitivity) and none of 67 samples negative by nested RT-PCR (100% specificity). Negative predictive value (NPV) and positive predictive value (PPV) of RT-LAMP for detection of Pfs16 transcript were 100% and 96.8%, respectively, and 100% for both when employing Pfs25 transcript. Detection rate of Pfs16 and Pfs25 transcripts by RT-LAMP in microscopically gametocyte-negative samples was 91.7% and 29.2%, respectively. Compared with nested RT-PCR, RT-LAMP had a higher sensitivity but similar specificity, with the advantage of a shorter assay time. As RT-LAMP requires very basic instruments and the results can be obtained by visual inspection, this technique provides a simple and reliable tool for epidemiological studies of malaria transmission and in gametocyte-targeted control programmes.
Buates
Sureemas
S
Department of Microbiology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok, Thailand.
Bantuchai
Sirasate
S
Sattabongkot
Jetsumon
J
Han
Eun-Taek
ET
Tsuboi
Takafumi
T
Udomsangpetch
Rachanee
R
Sirichaisinthop
Jeeraphat
J
Tan-ariya
Peerapan
P
D000818:Animals; D000953:Antigens, Protozoan / Q000235:genetics; D016054:DNA, Protozoan / Q000032:analysis; D006801:Humans; D016778:Malaria, Falciparum / Q000175:diagnosis* / Q000469:parasitology; D008565:Membrane Proteins / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010963:Plasmodium falciparum / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification*; D011237:Predictive Value of Tests; D015800:Protozoan Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D000953:Antigens, Protozoan; D016054:DNA, Protozoan; D008565:Membrane Proteins; C071284:Pfs25 protein, Plasmodium falciparum; D015800:Protozoan Proteins; C068959:Pfs16 protein, Plasmodium falciparum