Molecular and Biochemical Parasitology 99 (1999) 143–148

Short communication

Detection of low level Plasmodium falciparum gametocytes usingreverse transcriptase polymerase chain reaction

Hamza A. Babiker a,b,*, Amel Abdel-Wahab c,d, Salah Ahmed c, Suad Suleiman d,Lisa Ranford-Cartwright a, Richard Carter a, David Walliker a

a Institute of Cell, Animal and Population Biology, Uni6ersity of Edinburgh, Edinburgh, UKb Malaria Administration, Ministry of Health, Khartoum, Sudan

c Department of Biochemistry, Faculty of Medicine, Uni6ersity of Khartoum, Khartoum, Sudand Tropical Medicine Research Institute, National Institute for Research, Khartoum, Sudan

Received 8 October 1998; accepted 30 November 1998

The polymerase chain reaction (PCR) is widelyused to detect and identify low numbers of bloodforms of different species of human malaria para-sites [1]. The method has demonstrated a highprevalence of sub-microscopic asymptomaticPlasmodium falciparum infections among inhabi-tants of malaria-endemic countries [2–5]. Absenceof patent parasites from the peripheral blood isoften associated with sub-patent parasitaemiasthat can last for several months, even when thepossibility of reinfection can be ruled out [5,6].However, it is not known whether such sub-patentlong lasting infections can produce sexual formsand thus be capable of infecting mosquitoes.Some studies have indicated that the minimumdensity of gametocytes capable of infectingmosquitoes can be remarkably low (reviewed byCarter and Gwadz [7]), and it has been demon-strated that sexual forms are infectious to

mosquitoes even when they occur at levels notdetectable by microscopy [8]. Here we describe areverse transcriptase polymerase chain reaction(RT-PCR) for detection of gametocytes of P.falciparum.

Unlike asexual parasites, the mature gameto-cytes of P. falciparum are metabolically relativelyinactive [9]. However, almost immediately maturegametocytes are activated to undergo gametogen-esis, either upon ingestion by a mosquito or uponstimulation in vitro, they enter a period of intenseprotein synthesis [10]. Among the proteins whichare produced rapidly and in large amounts follow-ing such activation, is the zygote and ookinetesurface protein Pfs25 [10]. This protein and, as weshow here, its mRNA molecules are specific to theactivated gametocyte and to the zygote andookinete stages into which it transforms. BothPfs25 and its mRNA are totally absent from theasexual stage parasites of P. falciparum. We haveexploited this property of the mRNA of Pfs25 inthe activated gametocytes of P. falciparum in or-

* Corresponding author. Tel.: +44-131-6508658; fax: +44-131-6673210; e-mail: H.Babiker@ed.ac.uk.

0166-6851/99/$ - see front matter Published by Elsevier Science B.V.

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H.A. Babiker et al. / Molecular and Biochemical Parasitology 99 (1999) 143–148144

der to detect the presence of the mature gameto-cytes by RT-PCR. The method, which combinesRT-PCR and nested PCR, can readily detect 1–2gametocytes ml−1 blood with total specificity withrespect to asexual stages.

The P. falciparum parasites used in this workwere: (i) clones 3D7 and HB3, both cultured toproduce mature gametocytes [11,12] (ii) clone 3D7cultured to produce predominantly asexual forms[13,14]; and (iii) clones 3D7-F12 and T9-94 whichhad been cultured for prolonged periods and didnot produce visible gametocytes (Read D. andCarter R. unpublished data [15]).

For 3D7 and HB3 gametocytes, parasites weregrown in culture for 14 days. The gametocytedensity ranged between 10 000 and 50 000 maturegametocytes ml−1 blood (40% hematocrit erythro-cytes in mixture of RPMI/non-immune heat inac-tivated serum). A sample of 200 ml of culture wasadjusted to 40% hematocrit, and maintained atroom temperature for 30 min to allow maturegametocytes to undergo exflagellation and gameteformation. The induction of gametogenesis stimu-lates production of mRNA of the Pfs25 gene,expressed during gamete and zygote formation[16,17].

For preparation of RNA, infected erythrocyteswere first lysed using hypotonic lysis buffer (RedBlood Cell Lysis Buffer, Boehringer). Four hun-dred microliters of the lysis buffer were added to50 ml of the parasitised erythrocytes (40% haemat-ocrit) and mixed by inversion. The mixture wasrocked for 10 min at room temperature, thencentrifuged at 1000×g for 5 min. The superna-tant was discarded and the pellet containing para-site material was suspended in 200 ml PBS (136.8mM NaCl, 2.68 mM KCl, 8.1 mM Na2HPO4,1.47 mM KH2PO4, pH 7.4). Parasite RNA waspurified using an RNA isolation kit (High PureRNA Isolation Kit, Boehringer). The method wascarried out as described by the manufacturer withsome modifications. The preparation was firstthoroughly mixed in 400 ml of High Pure lysisbinding buffer (4.5 M guanidine hydrochloride, 50mM Tris–HCl, 30% Triton X-100 pH 6.6) whichinactivates RNases. It was placed on a glass filtercolumn and centrifuged at 8000×g for 15 s. Inthe presence of chaotropic salt, nucleic acids bind

specifically to the surface of glass fibres, the bind-ing conditions being optimised for RNA. In orderto remove carry-over contaminating DNA, thepreparation was incubated, with 180 U RNasefree DNase (Boehringer) in DNase incubationbuffer (0.9 M NaCl, 18 mM Tris–HCl, 9 mMMnCl2, pH 7.0), at 37°C for 4 h.

Bound RNA was purified from cellular impuri-ties by two step washing. Five hundred microlitersof wash buffer 1 (5 M guanidine hydrochloride,20 mM Tris–HCl pH 6.6 and 37.5% ethanol)were added to each preparation, which were thencentrifuged at 8000×g for 15 s. A similar washwas carried out with wash buffer 2 (20 mM NaCl,2 mM Tris–HCl pH 7.5 and 80% ethanol). Athird wash with buffer 2 (200 ml) was carried outfollowed by centrifugation at 13 000×g for 2 minto remove residual washing buffer. Finally theRNA was eluted in 60 ml nuclease-free steriledouble distilled water. The RNA preparationswere incubated at 75°C for 9 min to inactivateexcess DNase.

RT-PCR was carried out to detect mRNA ofthe Pfs25 gene [15]. Two microliters of each RNApreparation were used in 20 ml premix reactionswith primers Pfs25-1 and Pfs25-2 recognising sitesat the 5% and 3% ends of the gene respectively (seelegend to Fig. 1), and rTth recombinant bifunc-tional enzyme (Perkin-Elmer). The reaction mix-ture consisted of 0.45 mM of each primer, 75 mMeach of dGTP, dATP, dCTP and dTTP, 2.5 mMmanganese acetate and 2 U of rTth enzyme inbuffer (50 mM Bicine, 115 mM K acetate, 8% w/vglycerol). Reverse transcription of RNA and sub-sequent amplification of the synthesized cDNAtook place in a single tube under the followingconditions: 50°C, for 45 min; 94°C for 2 min forone cycle; 94°C for 30 s; 50°C for 35 s; and 68°Cfor 2 min and 30 s for 25 cycles; and 68°C for 10min and 4°C, holding temperature, for one cycle.Simultaneously another 2 ml of RNA per 20 mlpremix were subjected to conventional PCR withthe same primers but omitting the reverse tran-scription step, to detect any carry-over genomicDNA that might have co-purified with RNA. ThePCR temperature conditions were 94°C for 30 s,50°C for 35 s and 68°C for 2 min and 30 s for 25cycles; and 68°C for 10 min and 4°C, holding

H.A. Babiker et al. / Molecular and Biochemical Parasitology 99 (1999) 143–148 145

temperature, for one cycle. A volume of 10 ml ofeach of the RT and the corresponding PCRproduct of each RNA preparation were analysedby an agarose electrophoresis.

Results for the gametocyte-producing and asex-ual 3D7 cultures and for T9-94 are shown in Fig.1. The 3D7 gametocyte culture gave an RT-PCRproduct (lane 3). The 3D7 culture grown forasexual multiplication with occasional gameto-cytes gave a faint RT-PCR product (lane 5) indi-cating the presence of a few copies of Pfs25 RNAmolecules; thus, while the parasites in this culturewere predominantly asexual, it appeared thatsome gametocytes were being produced. CloneT9-94 (non-gametocyte-producing) gave noproduct with RT-PCR (lane 7), showing that inthese parasites there was no mRNA derived fromthe Pfs25 gene. No conventional PCR productswere obtained from any of these RNA samples(lanes 4, 6 and 8), showing that they did notcontain contaminating genomic DNA. Resultssimilar to those of T9-94 were obtained with thesecond non-gametocyte-producing parasite 3D7-F12 (not shown).

In order to establish the sensitivity of the RT-PCR, samples of 3D7 and HB3 gametocyte cul-tures were subjected to 10-fold dilutions usinguninfected erythrocytes at 40% haematocrit innon-immune heat-inactivated serum before RNAwas prepared. The diluted parasites were firstexamined using primers Pfs25-1 and Pfs25-2, and

samples that gave no RT-PCR product were thensubjected to another round of PCR using nestedprimers Pfs25-3 and Pfs25-4, inner to Pfs25-1 andPfs25-2 (see legend to Fig. 2). Two microliters ofthe RT-PCR product and the corresponding con-ventional PCR (test for carry-over genomic DNA)were added to 40 ml premix containing 100 nMeach primer, 75 mM each dNTPs and 0.5 U ofTaq DNA polymerase. The PCR conditions were94°C for 35 s, 42°C for 1 min and 65°C for 2 minfor 30 cycles, and 65°C for 10 min and 4°C,holding temperature, for one cycle.

Fig. 2 show the results of eight samples ofdifferent densities of HB3 gametocytes suspendedin human RBCs. An RT-PCR product was seenin the highest gametocyte densities when the outerprimers alone were used, and in densities up to0.005 gametocytes ml−1 blood when nested PCRwas used. The nested products for dilutions up to0.05 gametocytes ml−1 blood had similar intensi-ties, even though they were from samples contain-ing decreasing numbers of gametocytes. Thisindicates that they had originated from consider-able quantities of cDNA in each sample, beyondthe exponential phase of the PCR amplification.No products were seen with conventional PCRcontrols in which the reverse transcription condi-tions were absent, with the exception of dilution 3in which a band was detected following nestedPCR (Fig. 2B, lane 4). This indicated that DNasetreatment had not completely removed genomic

Fig. 1. Detection of gametocytes of P. falciparum using RT-PCR. Total RNA purified from parasite cultures was subjected toRT-PCR and conventional PCR using Pfs25 primers Pfs25-1 (5%-TCCATCAACAGCTTTACAGG-3%) and Pfs25-2 (5%-TAATGC-GAAAGTTACCGTGG-3%). Lane 1: molecular size markers. Lane 2: negative control (no DNA). Lane 3: 3D7 gametocyte-produc-ing culture, RT-PCR. Lane 4: 3D7 gametocyte-producing culture, conventional PCR. Lane 5: 3D7 asexual culture, RT-PCR. Lane6: 3D7 asexual culture, conventional PCR. Lane 7: T9-94, RT-PCR. Lane 8: T9-94, conventional PCR.

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Fig. 2.

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DNA, although it appeared that few copies wereleft, dilutions 4–8 did not give a PCR productunder these conditions.

The very low limit of detection was repro-ducible with three independent sets of serial dilu-tion of 3D7 and HB3 gametocyte cultures. Wecalculate from these experiments that a minimumof 520 gametocytes ml−1 blood could be detectedusing the outer primers. With the nested PCR thelevel of detection ranged between 0.005 and 1.5gametocytes ml−1 blood, and we therefore take aconservative figure of 1–2 gametocytes ml−1

blood as the limit of detection of this technique.This would be equivalent to 1–2 gametocytes per8000 white blood cells (WBCs), a typical WBCcount in an malaria endemic country [18]. Thiswould require an experienced microscopist to ex-amine around 1000 fields of a well-stained thickblood smear (0.1 ml), which could take at least 50min.

We have carried out preliminary work on sam-ples of P. falciparum from patients with patent aswell as undetectable gametocytes. One sample wascollected from a patient (G1) in Khartoum, Su-dan, with a patent gametocytaemia of 3500gametocytes ml−1 blood (0.07%). A total 2 ml ofvenous blood were collected in an anticoagulant-free tube and incubated at room temperature for30 min to allow gametocytes to undergo exflagel-lation, then immediately stored at −40°C. TotalRNA was prepared using the RNA isolation kitas described above, using 200 ml of lysate ob-tained by thawing the sample and homogenisingthe clotted erythrocytes. At the same time, totalRNA was prepared from a gametocyte culture of3D7 (:97 mature gametocytes ml−1 blood). RT-PCR and conventional PCR carried out on theabove RNA preparations did not give a visible

amplified product using outer primers alone.However, following nested PCR with the innerprimers, the RT-PCR samples of both 3D7 andG1 gave a visible product, while no conventionalPCR product was detected in either preparation.The failure of the first round of RT-PCR toproduce an amplified product from G1, despite itsrelatively high numbers of gametocytes, couldhave been due to the clotting of the erythrocytesin this preparation which probably trapped thegametocytes; following homogenisation, it is pos-sible that only few gametocytes were available forRNA extraction. In other field material, 2 out of5 samples collected from patients withchloroquine-resistant recrudescent parasitaemias[19], all containing high density asexual forms andno visible gametocytes, gave an RT-PCR productfor Pfs25 following nested PCR, indicating thatsmall numbers of gametocytes were present.

In summary, we have demonstrated that RT-PCR can be used for detection of sub-microscopiclevels of gametocytes of P. falciparum. The RT-PCR product was derived from cDNA and notgenomic DNA that co-purified with the RNApreparation. The combination of RT-PCR andnested PCR allows detection of very low numbersof gametocytes (1–2 ml−1) in infected blood. Thelimit of detection is below that reported for detec-tion of asexual blood forms using nested PCR [1],and this is probably due to an abundance of Pfs25mRNA in gametocytes. In nature, P. falciparumgametocyte carriers generally harbour less than100 gametocytes ml−1 blood, and there is evidencethat 1–10 gametocytes ml−1 blood are sufficientto establish infections in mosquitoes [7]. Thus, themethod reported here should be suitable for de-tection of carriers of low numbers of gametocytes,who undoubtedly play a major role in malariatransmission.

Fig. 2. RT-PCR and conventional PCR of diluted samples of P. falciparum clone HB3. A. Results using outer primers Pfs25-1 andPfs25-2. Lanes 1 and 9: molecular size markers. Lane 2: negative control. Lanes 3 and 4: original culture, 52 000 gametocytes ml−1

blood. Lanes 5 and 6: 5200 gametocytes ml−1 blood. Lanes 7 and 8: 520 gametocytes ml−1 blood. Lanes 10 and 11: 52 gametocytesml−1 blood. Lanes 12 and 13: 5.2 gametocytes ml−1 blood. Lanes 14 and 15: 0.52 gametocytes ml−1 blood. Lanes 3, 5, 7, 10, 12,14: RT-PCR. Lanes 4, 6, 8, 11, 13, 15: conventional PCR. B. Results using the nested primers Pfs25-3 (5%-CAGATGAGTGGT-CATTTGG-3%) and Pfs25-4 (5%-AATGAGCATTTGGTTTCTCC-3%). Lanes 1 and 9: molecular size markers. Lane 2: negativecontrol. Lanes 3 and 4: 52 gametocytes ml−1 blood. Lanes 5 and 6: 5.2 gametocytes ml−1 blood. Lanes 7 and 8: 0.52 gametocytesml−1 blood. Lanes 10 and 11: 0.052 gametocytes ml−1 blood. Lanes 12 and 13: 0.0052 gametocytes ml−1 blood. Lanes 14 and 15:0.00052 gametocytes ml−1 blood. Lanes 3, 5, 7, 10, 12, 14: RT-PCR. Lanes 4, 6, 8, 11, 13, 15: conventional PCR.

H.A. Babiker et al. / Molecular and Biochemical Parasitology 99 (1999) 143–148148

Acknowledgements

This work was supported by the WellcomeTrust (grant No. 054597/Z/98/Z), the UK Medi-cal Research Council and a WHO-TDR TrainingAward to Amel Abdel-Wahab. We thank Abdel-Muhsin A. Abdel-Muhsin for helping with thefield work and Emilia Rappoccciolo for sometechnical advice.

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