Table 1.
Reference strains of Leishmania and other trypanosomatids used in this study.
Table 2.
Primers, probes and standard curve parameters for the targets used in the conventional and real time PCR assays.
Fig 1.
Sensitivity comparison of conventional PCR and qPCR for HSP70 target.
(A) Electrophoresis results of cPCR for L. (V.) braziliensis and L. (V.) guyanensis: 1: ladder 100 pb; 2: Negative control; 3 to 10: serial dilution from 104 to 10−3 par. eq./reaction of L. (V.) braziliensis, respectively; 11: Negative control; 10 to 19: serial dilution from 104 to 10−3 par. eq./reaction of L. (V.) guyanensis, respectively. (B) Electrophoresis results of cPCR for L. (L.) amazonensis and L. (V.) panamensis. 21: Negative control; 22 to 29: serial dilution from 104 to 10−3 par. eq./reaction of L. (L.) amazonensis respectively; 30: Negative control; 31 to 38: serial dilution from 104 to 10−3 par. eq./reaction of L. (V.) panamensis respectively. (C) Ct values of qPCR reaction with HSP70 target for L. (V.) braziliensis, L. (V.) guyanensis, L. (L.) amazonensis, L.(V.) panamensis. OBS: The same serial dilutions from 104 to 10−3 Par. Eq./reaction, used in cPCR, were used in qPCR reactions, to determinate the dynamic extension of detection.
Fig 2.
Dynamic extension of L. (V.) braziliensis detection.
(A) Standard curve for the targets 18S rDNA and HSP70. (B) Standard curve for the targets 18S rDNA and HSP70 in multiplex with RNAse P internal control. Serial dilutions of human DNA mixed with parasite DNA in a 1:9 proportion were assayed for all curves.
Table 3.
Inclusivity assay for Leishmania species and Exclusivity assay for other trypanosomatids.
Table 4.
Parasite loads in ATL clinical samples using qPCR assays targeting HSP70 and 18S rDNA.
Fig 3.
Normalized parasite load distribution on positive patient samples.
(A) Parasite load distribution on the 59 positive patient samples for the HSP70 target. (B) Parasite load distribution on the 77 positive patient samples for the 18S rDNA target. (C) Direct comparison between Leishmania parasite load quantification by qPCR reactions with 18S rDNA versus the HSP70 target. In this graph, parasite load of the 59 positive patient samples for both targets are plotted.
Fig 4.
Agreement of parasite load quantification using 18S rDNA and HSP70.
A. Parasite load plot (18S rDNA versus HSP70). B. Bland-Altman bias (difference) plot analysis for the agreement degree between parasite load quantification using 18S rDNA and HSP70 as targets.
Table 5.
Sensitivity and specificity of Leishmania detection by real time PCR (qPCR) in comparison to microscopy.