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Plasmodium falciparum accounts for greater than 90% of malaria cases in sub-Saharan Africa. Successful development and presence of sexual forms (male and female gametocytes), which often persist at sub-microscopic levels, ensure continued malaria transmission. Mature forms of erythrocytic asexual and younger sexual stages of P. falciparum are sequestered in deep tissue by a process of cytoadherence, and at any time point of sample collection only the mature forms are detected.1–3
To fully understand the epidemiology of malaria, it is important to estimate gametocyte carriers in the human population who contribute to malaria transmission. In many instances, gametocyte load in peripheral blood can be sub-patent and sensitive techniques such as reverse transcription–polymerase chain reaction (RT-PCR) are required to detect gametocytes.4,5 An RT-PCR detection procedure has been developed based on a specific transcript, pfs25, that is produced by parasites when they undergo sexual development to produce gametocytes. The detection limit of such a method has been shown to be 1–2 gametocytes/µL in blood samples collected from humans.5 However, this method requires either immediate processing of samples or collection of blood in tubes containing an RNA stabilizer. Sub-patent and microscopic gametocytes have also been detected by Pfs25 mRNA real time quantitative nucleic acid–based sequence amplification with a sensitivity between 20 and 100 gametocytes/mL.6–8 However this method also requires collection of liquid blood and appropriate storage of samples to preserve intact RNA. Transporting liquid blood samples either at room temperature or frozen (preferably) adds yet another level of complexity for scaling up gametocyte detection assay for field studies. Genotype analyses of blood samples dried on filter papers is quite routine, and in a recent study dried blood on filter papers from P. yoelii-infected mice was used for cytokine analysis.9
We addressed the question of whether a filter paper–based approach would be feasible for RT-PCR analysis on P. falciparum-infected blood samples. We also validated this assay with samples collected from subjects in Macha, Zambia under field conditions. This report describes detection of gametocyte transcripts of P. falciparum from filter papers under both laboratory and field conditions. This assay thus simplifies the blood collection process and could be critical in studies on transmission dynamics and malaria epidemiology, obviating the need for freezing blood samples, which is often difficult under field conditions.
Plasmodium falciparum (NF54) parasites cultured according to the procedure of Trager and Jensen10 were diluted using 30% erythrocyte suspensions to yield approximately 104, 103, 102, 10, and 2 gametocytes/µL and aliquoted in tubes or spotted onto FT2 classic card filter paper (Whatman Maidstone, United Kingdom) or 903 filter paper (Schleicher and Schuell, Keene, NH) as summarized in Table 1
. Samples were stored under the stated conditions for three months before further analysis.
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Each blood sample suspension in a final volume of 500 µL of RNAlater was added to a QIAshredder column (Qiagen, Valencia, CA) and centrifuged at 14,000 rpm for 2 minutes at room temperature. Total RNA was extracted according to the instructions of the Qiagen handbook using the RNAeasy Minikit (Qiagen). Each sample was treated with RNAse-free DNAse (Qiagen) prior to elution of RNA from the column. Samples were suspended in 30 µL of RNAse-free water. Filter papers containing blood were resuspended in 500 µL of RNAlater, soaked for one hour at room temperature, and total RNA was extracted as above.
For the RT step, 3 µL of sample was added into a 17 µL of master mixture containing 500 µM of each dNTP, 10 units of RNase inhibitor, 1x RT buffer, 0.5 µM of antisense primer, (Pfs25R, 5'-gaattcTTACATTATAAAAAAGCATACTC-3'), and 4 units of Omniscript Reverse Transcriptase (Qiagen).5 The mixture was incubated at 37°C for 45 minutes to synthesize cDNA. Two microliters from the RT step was added to a 23-µL master mixture containing 100 µM of each dNTP, buffer (50 mM KCl, 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl2), 0.4 µM of Pfs25F (5'-atcgatATGAATAAACTTTACAGTTTGTTTCT-3') and Pfs25R, and 1.25 U of enzyme Taq polymerase. The PCR cycling conditions were an initial denaturation at 94°C for 2 minutes, 25 cycles of denaturation at 94°C for 30 seconds, annealing at 50°C for 35 seconds, and extension at 68°C for 2.5 minutes. Two microliters of the product of the first PCR was used as template for a nested PCR using a set of internal primers sense (25-1, 5'-TAATGCGAAAGTTACCGTGG-3') and anti-sense 25-2 (5'-TCCATCAACAGCTTTACA GG-3').5 The PCR cycling conditions were as described above with 30 cycles used instead of 25. The PCR products were resolved by electrophoresis on a 1% agarose gel, stained with ethidium bromide, and visualized with ultraviolet illumination.
Different conditions (Table 1
) were tested to determine optimum collection and storage of samples either as liquid in tubes or on different filter papers for RNA analysis. As summarized in Table 1, RNA transcripts were detected in blood samples that were either frozen immediately at –80°C (A) or kept overnight at 37°C then stored at –80°C (B) in the presence of RNAlater. We detected pfs25 transcripts at 103 gametocytes/µL in 50 µL of blood sample after the first round of PCR. With the nested PCR, transcripts were detected in 50-µL samples containing approximately 1–2 gametocytes/ µL, which is consistent with results of a previous study.5 Our analysis of filter papers showed that transcripts could be detected from dried blood samples on filter papers with a detection limit similar to that of blood frozen immediately at – 80°C. The 903 paper had a higher sensitivity compared with the FT2 paper, which was 2 logs lower than that of 903 paper (Table 1). More interestingly, RNA was obtained with or without RNAlater pretreatment on the 903 paper even when stored at 37°C for up to three months (D and H samples in Table 1).
After establishing that pfs25 transcripts could be obtained from filter papers with or without RNAlater, we validated this assay by analyzing 10 matched blood samples that were collected from the field under an ongoing malaria immune response study in Macha, Zambia. Table 2 provides a summary of asexual and gametocyte parasitemia by microscopy and corresponding RT-PCR results for Pfs25 transcripts. Six filter paper samples were positive for pfs25 transcripts. Curiously, only two of these were positive from the matching blood samples collected in tubes (Figure 1). This discordance could be explained by the fact that although samples on filter paper were dried immediately, the blood samples in tubes remained at 10–15°C for 6–8 hours (field study and transport back to the laboratory) prior to adding RNAlater and storage at –70°C, and this unavoidable delay could have compromised the stability of RNA. However, our study demonstrates that gametocyte transcript can be analyzed from blood collected on filter papers and this would be valuable especially in field studies where freezing blood for RNA analysis would be cumbersome and costly in resource poor settings. Filter papers offer a convenient source for storing specimens as there is no need for RNAlater pretreatment and no need to maintain a cold chain during shipment of samples. Although filter papers had been described for DNA extraction of P. falciparum,11 results presented in this study demonstrate that a similar procedure can be valuable in the detection of a specific gene transcript.
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Received July 9, 2007. Accepted for publication September 30, 2007.
Acknowledgments: We thank the Johns Hopkins Malaria Research Institute core facility for P. falciparum gametocyte cultures and Phil Thuma for facilitating Zambian field sample collection.
Financial support: Samples from Zambia were collected for a pilot project funded by the Johns Hopkins Malaria Research Institute under an approved protocol. Research in the laboratory of Nirbhay Kumar is supported by grants from the National Institutes of Health (NIH). The supply of human erythrocytes RBC is supported by NIH grant RR00052.
* Address correspondence to Nirbhay Kumar, Malaria Research Institute, Department of Molecular Microbiology and Immunology, Johns Hopkins University, Bloomberg School of Public Health, 615 North Wolfe Street, Baltimore, MD 21205. E-mail: nkumar{at}jhsph.edu 
Authors’ address: Godfree Mlambo, Yessika Vasquez, Ralph Le-Blanc, David Sullivan and Nirbhay Kumar, Malaria Research Institute, Department of Molecular Microbiology and Immunology, Johns Hopkins University, Bloomberg School of Public Health, 615 North Wolfe Street, Baltimore, MD 21205.
Reprint requests: Nirbhay Kumar, Malaria Research Institute, Department of Molecular Microbiology and Immunology, Johns Hopkins University, Bloomberg School of Public Health, 615 North Wolfe Street, Baltimore, MD 21205, Telephone: 410-955-7177, Fax: 410-955-0105, E-mail: nkumar{at}jhsph.edu.
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