The laboratory-adapted gametocyte producing P. falciparum strains 3D7, Dd2 and W2mef were cultured in RPMI 1640 HEPES (Sigma Aldrich) supplemented with 5 g/L albumax II (Invitrogen), 92.6 mg/L L-glutamine (Sigma Aldrich), 500 μg/L gentamicin (Sigma Aldrich), 50 mg/L hypoxanthine (Sigma Aldrich). Culture medium was changed once daily. Cultures were maintained at 4–5% haematocrit and diluted with red blood cells when parasitaemias exceeded 5%. Cultures were incubated in an airtight cabinet (Nalgene, Model 53170120) at 37°C in an atmosphere with approximately 5% oxygen concentration. The low oxygen atmosphere was generated by gassing the cabinet with a gas mixture (1% O2; 95% N2; 4% CO2) at 10–15 kPa for 60 seconds each time it had been opened. To induce gametocyte development, the parasite culture was allowed to grow to maximum parasitaemia with gametocytes appearing approximately 10 days later.
For the present experiments, 10-fold dilution series of the parasite culture were prepared in freshly drawn whole blood. The starting gametocytaemia was determined by counting the number of red blood cells per 100 gametocytes on a thin film prepared from the initial parasite culture. Six experiments were conducted comparing the sensitivities of MF, and RT-PCR (4 experiments using 3D7, 1 experiment using W2mef and 1 experiment using Dd2 strain). The total blood volume prepared for each dilution was 1610 μL of which 100 μL were used for MF, 10 μL to prepare TBF, and 1500 μL for RNA extraction and RT-PCR.
Magnetic fractionations were conducted using a MidiMACS magnet, a MACS-multistand and LS columns (Miltenyi Biotech). The columns were placed into the magnet unit and primed with 0.7 mL sterile filtered magnetic fractionation buffer (MFB, PBS pH 7.4, containing 0.5 g/L bovine serum albumin and 0.0037 g/L EDTA) equilibrated to room temperature. 100 μL of blood from each dilution were suspended in 5 mL MFB in 15-mL centrifuge tubes (BD Biosciences) and incubated at room temperature for at least 10 min before the start of the fractionation. The flow rate through the column was regulated to 0.25 mL/min by attachment of a sterile syringe needle (BD Biosciences) with an inner diameter of 0.42 mm (26G) to the end of the column. After the cell suspension had passed through, the columns were washed with 2 × 1 mL MFB and removed from the magnet. The sterile needle was disconnected and the cells captured in the column were eluted into 15-mL centrifuge tubes (BD Biosciences) by washing with 1 × 5 mL MFB. These fractions were centrifuged at 400 g for 10 min (Sigma Laboratory Centrifuge, Model 4K15). The resulting cell pellet was resuspended in approximately 3 μL of PBS (pH 7.4) and spread on a microscope slide to form a circle of 0.5–1 cm in diameter. The slides were dried in an incubator (Sanyo, Model: MCO -15A) at 37°C for at least 30 min, fixed with methanol and stained with 5% Giemsa solution (Sigma-Aldrich) in PBS (pH 7.4) for 10 min. Leukocyte density was determined by counting the number of leukocytes in 100 high power fields across the MF preparation. Gametocyte density was determined by counting the number of gametocytes per leukocyte in 100 high power fields and assuming 8000 leukocytes per microliter of blood. If no gametocytes were detected in 100 fields, additional fields were examined until a gametocyte was seen or a maximum observation time of 60 min was reached.
Preparation of thick blood films
Thick blood films were prepared from 10 μL of blood from each dilution. The films were stained with 5% Giemsa stain without prior methanol fixation for 10 min and carefully rinsed with water. The films were evaluated using the same protocol as that used for the MF preparations.
Reverse transcriptase polymerase chain reaction
RT-PCR was conducted based on the methods of Mlambo et al , Babiker et al , and Menegon et al  for the gametocyte specific genes Pfs25 and Pfg377 using the same primer sequences as described in these publications. Total RNA was extracted from 1.5 mL blood from each dilution using the Qiagen Blood Kit following manufacturers instructions. RNA was eluted in 60 μL of RNAse free water. RNA was subjected to DNAse treatment (DNA-free Kit, Ambion Applied Biosystems). Total RNA was eluted with 60 μL of RNAse free water and DNAse treated using the Ambion DNA-free Kit (Ambion Applied Biosciences). The total RNA was quantified on a spectrophotometer (Nanodrop 1000, Thermo Fisher Scientific Inc.). RNA was subjected to reverse transcription directly without intermediate storage. cDNA was obtained from 5.5 μL of the total RNA eluate using reagents from a Superscript III first strand kit (Invitrogen) following manufacturers instructions with sequence specific primers Pfs25 -R (5'-AATTCTTACATTATAAAAAAGCATACTC-3') for the Pfs25 gene and Pfg377 -R3D1 (5'-GATGAAAGGGATATATCACCTCACAATGTG-3') and Pfg377 -R3R2 (5'-GTCATGATTTTCTTCTCCTTCGGATATGG-3') for the Pfg377 gene respectively.
PCR was conducted immediately following reverse transcription using reagents from the Platinum Taq Polymerase Kit (Invitrogen). The first PCR was conducted using 2 μL of cDNA template in 18 μL of master mix containing primers Pfs25 -R and Pfs25 -F (5'-ATCGATATGAATAAACTTTACAGTTTGTTTCT-3') for Pfs25 and Pfg377 -R3D1 and Pfg377 -R3R2 for Pfg377 respectively. The nested PCR was conducted using 2 μL of the product from the first PCR. For Pfs25 primers Pfs25 -1 (5'-TAATGCGAAAGTTACCGTGG-3') and Pfs25-2 (5'-TCCATCAACAGCTTTACAGG-3') were used. For Pfg377 primers Pfg377- R3D2 (5'-CCATAGGAATATTACACCATATCATGTG-3') and Pfg377 -R3R1 (5'-TATGGTGATAAATGAGGAGTGTCCCCTTAC-3') were used. The first PCR was conducted on two separate PTC 100 Thermocyclers (MJ Research Inc.) using the cycling conditions for each gene as described previously by Babicker and Menegon with 35 cycles each [13, 14]. No gels were run for the product from the first PCR and products were directly subjected to nested PCR. The nested PCR was conducted on a Rotorgene RG3000 thermocycler (Corbett Research) using the following cycling conditions for Pfs25 : Hold: 95°C, 5 min; Cycling: 95°C, 15s; 50°C, 20s; 72°C, 40s; for 40 cycles; Hold: 72°C, 30s; Melt: 72°C to 99°C; Hold: 40°C, 30s and for Pfg377 : Hold: 94°C, 5 min; Cycling: 94°C 35s, 55°C, 30s; 72°C, 60s for 40 cycles; Hold 72°C, 30s Melt: 72°C to 99°C; Hold: 40°C, 30s. PCR products were verified by melt curve analysis and gel electrophoresis in 1.5% agarose gel, containing ethidium bromide at 100 V for 60 min. The gels were visualized in a UV transluminator (Versadoc, Model 3000, Biorad).
Only dilutions with a calculated gametocyte density of < 100 μL-1 were used for comparison of the three techniques so that each of the six dilution series had one dilution in each of the following ranges: 101–102, 100–101, 10-1–100, and 10-2-10-1 μL-1. The limit of detection for each method in each dilution series was defined as the lowest gametocyte density where detection was positive. Detection limits were compared by using a nonparametric significance test (Wilcoxon's matched pairs test). The sensitivity of each technique for a given specified range of gametocyte density was defined as the fraction of gametocyte tests carried out in that range that were positive. In the case of RT-PCR a positive test was defined as the positive detection of at least one of the two genes being assayed.