Article Figures & Data
Figures
- FIG. 1.
Sample processing and assay work flow schematic. Microscopy-positive samples were amplified directly in the pan-species assay, and positive samples were subsequently tested in the speciation assay. Microscopy-negative samples were first grouped into pools of four and then amplified in the pan-species assay; the individual constituents of positive pools were then retested in the pan-species assay, and positive samples were subsequently tested in the speciation assay.
- FIG. 2.
Real-time PCR output from masked validation of pan-species and speciation assays using artificial test samples. Panel A shows the output of real-time PCR testing with 5 artificial test pools (pools A to E) in the pan-species assay. Each pool contained 2 μl of DNA from 10 samples. Pool A contained a total of 1.111 ng/μl, divided among 4 samples, and pool B contained 1 ng/μl of DNA in a single sample. Pools C, D, and E contained DNA at total concentrations of 0.1, 0.01, and 0.001 ng/μl, respectively. Panel B shows the output observed for the speciation real-time PCR assay when tested on a sample with 0.01 ng/μl of P. falciparum plasmid (Pf), 0.000001 ng/μl of P. ovale plasmid (Po), and 2.5 ng/μl of human DNA (Hu). Delta Rn, fluorescent signal generated relative to background fluorescence.
Tables
- TABLE 1.
Primer and probe sequences
Reagent Sequencec Reference or source Primersa Pan-species forward GTT AAG GGA GTG AAG ACG ATC AGA TA 38 Pan-species reverse AAC CCA AAG ACT TTG ATT TCT CAT AAG 38 P. falciparum 18S rDNA forward ATT GCT TTT GAG AGG TTT TGT TAC TTT 46 P. falciparum 18S rDNA reverse GCT GTA GTA TTC AAA CAC AAT GAA CTC AA 46 P. ovale 18S rDNA forward CCG ACT AGG TTT TGG ATG AAA GAT TTT T 39 P. ovale 18S rDNA reverse CAA CCC AAA GAC TTT GAT TTC TCA TAA 46 P. malariae 18S rDNA forward AGT TAA GGG AGT GAA GAC GAT CAG A 46 P. malariae 18S rDNA reverse CAA CCC AAA GAC TTT GAT TTC TCA TAA 46 Human GAPDH forward CCT CCC GCT TCG CTC TCT This study Human GAPDH reverse GCT GGC GAC GCA AAA GA This study P. falciparum LDH forward ACG ATT TGG CTG GAG CAG AT 36 P. falciparum LDH reverse TCT CTA TTC CAT TCT TTG TCA CTC TTT C 36 MGB probesb Pan-species VIC-TCG TAA TCT TAA CCA TAA AC 38 P. falciparum FAM-CAT AAC AGA CGG GTA GTC AT 46 P. ovale VIC-CGA AAG GAA TTT TCT TAT T 38 P. malariae FAM-ATG AGT GTT TCT TTT AGA TAG C 46 Human GAPDH VIC-CCT CCT GTT CGA CAG TCA GCC GC This study TaqMan probeb (P. falciparum LDH) FAM-GTA ATA GTA ACA GCT GGA TTT ACC AAG GCC CCA-TAMRA 36 - TABLE 2.
Study subject demographics (n = 182)
Characteristic Value Mean (SD) age (yr) 27.5 (5.3) Mean (SD) gestational age (wk) at enrollment 18.9 (2.8) No. (%) of subjects with gravidity Primigravid 47 (25.8) Secundigravid 26 (14.3) Multigravid 109 (59.8) No. (%) of subjects HIV infected 5 (2.7) Mean (SD) total no. of study visits 6.3 (1.4) Mean (SD) no. of malaria treatments received 2.5 (0.8) No. (%) of visits to subjects who always slept under an insecticide-treated bed net during the previous 2 wka 531 (66.6) ↵ a Bed net use was assessed for 797 regular study visits.
- TABLE 3.
Comparison of results of microscopy and real-time PCR for malaria detection
Microscopy result No. of subjects with real-time PCR result Total no. of subjects Positive Negative Positive 74 102 176 Negative 35 1,057 1,092 Total 109 1,159 1,268
