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Experimental Parasitology 118 (2008) 41–46

A multiplex PCR that discriminates between Trypanosoma bruceibrucei and zoonotic T. b. rhodesiense

Kim Picozzi a, Mark Carrington b, Susan C. Welburn a,*

a Centre for Infectious Diseases, College of Medicine and Veterinary Medicine, Royal (Dick) School of Veterinary Science,

The University of Edinburgh, Edinburgh EH25 9RG, UKb Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK

Received 3 November 2006; received in revised form 27 May 2007; accepted 31 May 2007Available online 12 June 2007

Abstract

Two subspecies of Trypanosoma brucei s.l. co-exist within the animal populations of Eastern Africa; T. b. brucei a parasite which onlyinfects livestock and wildlife and T. b. rhodesiense a zoonotic parasite which infects domestic livestock, wildlife, and which in humans,results in the disease known as Human African Trypanosomiasis (HAT) or sleeping sickness. In order to assess the risk posed to humansfrom HAT it is necessary to identify animals harbouring potentially human infective parasites. The multiplex PCR method describedhere permits differentiation of human and non-human infective parasites T. b. rhodesiense and T. b. brucei based on the presence orabsence of the SRA gene (specific for East African T. b. rhodesiense), inclusion of GPI-PLC as an internal control indicates whether suf-ficient genomic material is present for detection of a single copy T. brucei gene in the PCR reaction.� 2007 Elsevier Inc. All rights reserved.

Index Descriptors and Abbreviations: Human African sleeping sickness; T. b. rhodesiense; Trypanosomiasis; Epidemiology; Zoonoses; Diagnosis; Mult-iplex; PCR; Polymerase chain reaction; Whatman FTA; DNA; Serum Resistance Associated; SRA; Phospholipase C, GPI-PLC

1. Introduction

Control of human sleeping sickness, caused by eitherTrypanosoma brucei rhodesiense or T. b. gambiense is com-plicated by the fact that both parasites which cause diseasein man exist in animals. In the case of T. b. rhodesiense

across East Africa, the human infective parasite exists ina wide variety of domestic and wildlife species which actas long term reservoirs of disease (Welburn et al., 2006);for T. b. gambiense the animal reservoir appears more con-servative with infection in non-human hosts being limitedto pigs. These human infective parasites co-exist in all spe-cies, other than man, with the morphologically identicalparasite species T. b. brucei which only infects non-humanmammalian hosts. All subspecies of T. brucei are able to

0014-4894/$ - see front matter � 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.exppara.2007.05.014

* Corresponding author. Fax: +44 131 651 3903.E-mail address: sue.welburn@ed.ac.uk (S.C. Welburn).

co-exist with a range of other morphologically distinct try-panosomes which are pathogenic only to animals; T. vivax,T. congolense and T. simiae (in pigs). Recently a new PCRmethod, ITS-PCR has enabled differentiation to the specieslevel of all trypanosomes circulating in an animal host bymeans of a single PCR reaction, which is based on identi-fication of Internal Transcribed (ITS) regions of the para-site genome (Cox et al., 2005). Previously this wouldrequire single PCR reactions based on primers designedfor individual species. Aside from reducing costs, this offersa significant breakthrough in differentiating the variousspecies circulating in animals and in our understandingthe molecular epidemiology of Trypanosomiasis in live-stock and wildlife but does not differentiate T. b. brucei

from T. b. rhodesiense. Differentiation of the subspeciesof Trypanosoma brucei s.l. has recently become possiblewith the observation that one of the two human infectivesubspecies, T. b. rhodesiense, carries a gene that protectsthe parasite from lysis in the human host, called the Serum

42 K. Picozzi et al. / Experimental Parasitology 118 (2008) 41–46

Resistance Associated gene (SRA) (De Greef et al., 1989,1992; Xong et al., 1998; Vanhamme et al., 2003). This genehas not been found in any other species of trypanosomeand has been developed as a diagnostic marker for theidentification of T. b. rhodesiense infections (Welburnet al., 2001), indeed the gene has never been identified inany T. b. brucei genetic background as analysed by RFLP(Welburn and Odiit, 2002). The SRA gene has been shownto be a ubiquitous maker for T. b. rhodesiense parasitesthroughout East Africa, both within the human host andthe domestic animal reservoir (Welburn et al., 2001; Gib-son et al., 2002; Radwanska et al., 2002).

PCR has improved the sensitivity and accuracy of diag-nosing trypanosome infections significantly when com-pared to the direct observation of parasites withininfected blood (Solano et al., 1999). Currently the detectionlimit of PCR is thought to be 1 trypanosome in 10 ml ofcattle blood (Masake et al., 2002) or 25 trypanosomes/mlof human blood (Kanmogne et al., 1996). Inhibitory fac-tors present in human blood are believed to cause the dis-crepancy between bovine and human studies (Contaminet al., 1995).

For identification of T. brucei s.l. universal Trypanozo-on primers are directed against a 177 bp satellite repeat ofwhich there are several thousand targets within the parasitegenome (Sloof et al., 1983; Moser et al., 1989) and thisPCR methodology enables detection of parasite DNAwithin a fraction of a genome (Artama et al., 1992). Con-cerns over primer specificity of these primers (Solanoet al., 2002) have been now addressed by redesigning thegeneric primers to amplify a smaller target within the samerepeated sequence (Becker et al., 2004) but a number ofproblems arise when relating sensitivity at this level forT. brucei s.l. to that of detection at single copy level, suchas for (SRA) for T. b. rhodesiense. Diagnosis of T. b. rho-

desiense hinges on the presence or absence of the SRA genein previously identified T. brucei s.l. infections but infec-tions within the animal reservoir are often chronic withvery low levels of parasitaemias (Katunguka-Rwakishaya,1996) in which case the accuracy of a PCR test based onlyon the absence or detection of a single copy gene is notdefinitive. A negative PCR result may indicate the absenceof SRA or simply indicate that too little genomic materialis present.

In order to overcome this problem we have developed a‘‘multiplex’’ PCR. This is a PCR reaction that contains twodifferent sets of primers, so that there are two differentproducts amplified within the same reaction from the samestarting material, one targeting the genus of parasite inquestion, the second being sub species specific (Zarlengaand Higgins, 2001). Within each reaction, the PCR targetsboth SRA (the diagnostic gene for T. b. rhodesiense), and asingle copy gene found within T. brucei s.l., namely a phos-pholipase C (GPI-PLC) (Hereld et al., 1988; Carringtonet al., 1989; Mensa-Wilmot et al., 1990). A positive ampli-fication of GPI-PLC indicates sufficient T. brucei s.l. geno-mic material is present to detect a single copy gene and the

presence or absence of SRA determines whether T. b. rho-

desiense is present.

2. Materials and methods

2.1. Parasite material from animals

T. brucei s.l. stocks were either collected in Tororo,south east Uganda, between 1988 and 1991, or Soroti dis-trict, south central Uganda, in 2001 and have been fullydescribed elsewhere (Hide et al., 1994; Tilley et al., 2003;Welburn et al., 2001). Trypanosoma congolesense stocksisolated from cattle: Sikuda 124 was isolated from a cowfrom Sikuda, Tororo District, Uganda 1990 and T. congo-

lense 1/148 originally isolated from a cow in Nigeria(Young and Godfrey, 1983), their identity previously con-firmed by species specific PCR (Picozzi et al., 2003). The T.

evansi and T. vivax stocks have been described elsewhere(Boid et al., 1989; Leeflang et al., 1976).

2.2. Human blood samples

Blood was collected onto FTA cards from T. b. rhodes-iense patients in Serere Health Centre, Soroti and T. b.

gambiense patients in Omugo Health Centre, Arua, northwest Uganda.

2.3. DNA extraction

Extraction of DNA and preparation of FTA cards wascarried out as previously described (Welburn et al., 2001;Picozzi et al., 2003).

2.4. PCR primers (Table 1)

The primers for the GPI-PLC gene were designed toamplify a 324 bp fragment located centrally within thegene. There are VSG genes with some sequence identityto the SRA gene but the SRA gene contains an internaldeletion when compared with these VSG genes (Campilloand Carrington, 2003). Two pairs of primers were designedto amplify the SRA gene across this deleted region, ensur-ing a clear size distinction between the SRA amplicon andany VSG amplicon. The first pair of primers (02 and 03)were highly specific for the SRA gene while the second pair(651 and 652) were a modification of primers previouslydescribed (Welburn et al., 2001; Picozzi et al., 2005) whichamplified both SRA and a VSG (Campillo and Carrington,2003).

2.5. Single and multiplex PCR reaction conditions

A 25 ll PCR reaction routinely contained: 1.5 U Hot-Star Taq, 1.25 ll of Rediload dye (Invitrogen), with a finalconcentration of 3 mM MgCl2 and 0.2 lM of each primer.Amplification conditions were as recommended in the Qia-gen Multiplex PCR handbook; an initial denaturation step

K. Picozzi et al. / Experimental Parasitology 118 (2008) 41–46 43

of 95 �C for 15 min was followed by 30–40 cycles of 94 �Cfor 30 s, 63 �C for 90 s, 72 �C for 70 s, with a final extensionstep of 10 min.

2.6. Analysis of PCR products

PCR products were separated by agarose (1.5%) electro-phoresis containing ethidium bromide (0.2 lg/ml) and thenvisualised on a UV transilluminator. Sequencing of PCRproducts was carried out by Lark Technologies.

2.7. Real time PCR

Real-time PCR was performed on a DNA Engine Opti-con (GRI); the amplification mixture was a 2· QuantiTectSYBR Green PCR mastermix containing 3 mM MgCl2 andprimer concentration was 0.2 lM with 10 ng of genomicDNA. The amplification programme was as above with adata acquisition step after the extension stage of each cycle.The crossing threshold (CT) of the reactions was calculatedautomatically by the Opticon software, being the point atwhich signal surpasses background noise and begins toincrease.

3. Results

3.1. Amplification optimisation and target specificity

Reactions were optimised across a temperature gradientand 63 �C was identified as the optimum annealing temper-ature for all reactions. Products obtained from the amplifi-cation of a number of known T. b. brucei and T. b.

rhodesiense stocks with the GPI-PLC and SRA primerswere isolated and sequenced in order to confirm the speci-ficity of the reaction. The GPI-PLC and SRA ampliconshad the expected sequences (EMBL Accession Nos.X13292 (Carrington et al., 1989) and AF097331 (Milnerand Hajduk, 1999)).

3.2. Amplification bias

The three sets of primers were compared in terms ofamplification efficiency by real time PCR. The crossingthreshold for each of the ten T. brucei s.l. stocks amplified(five T. b. brucei, two T. b. gambiense and three T. b. rho-

desiense) was initiated by 10 ng of DNA, with each reactioncarried out in triplicate. A discrepancy between the effi-ciency of the two sets of SRA primers was observed, with02/03 entering the initial logarithmic stage of the reaction8 cycles later than 651/652. When amplifying T. b. rhodes-

iense the CT with either 651/652 (SRA) or 657/658 (GPI-

PLC) primer pairs varied by 0.3 cycles. Greater variationwas observed when amplifying T. b. brucei or T. b. gamb-

iense due to the presence of VSG genes with homology toSRA.

When primers 651/652 (SRA) and 657/658 (GPI-PLC)were combined in a multiplex reaction the CT of 657/658

differed on average by 0.93 cycles from the single PCR,irrespective of the origins of the T. brucei s.l. The same istrue for primers 651/652 (SRA) when the target materialwas T. b. rhodesiense. There was no adverse effect onPCR amplification whether the reaction contained a singleor double set of primers.

3.3. Amplification specificity

The specificity of the reaction was investigate by screen-ing 70 fully characterised stocks of T. brucei s.l. collectedfrom both cattle and humans in Tororo District, Uganda,between 1989 and 1991. There was a 100% correlationbetween the results generated by the multiplex PCR andthe human infectivity status of the samples (Fig. 1). In allcases the GPI-PLC gene was amplified, with T. b. rhodes-

iense parasites distinguished by the presence of an addi-tional amplicon for SRA gene. Stabilates of T.

congolense, T. brucei s.l., T. vivax and T. evansi were alsoscreened to further assess the specificity of the PCR andthe identification of human infective parasites (see Table2). It may be concluded that GPI-PLC is a Trypanozoonspecific marker with amplification observed only in T. bru-cei s.l. and T. evansi (Fig. 1). In total the multiplex reactionwas found to be completely accurate when tested againstover 158 characterised parasite stocks. The multiplexPCR described here was designed to differentiate T. b. rho-

desiense from other members of the trypanozoon family.While the genetic marker used as the internal control isfound in all members of this family the distinction for T.

b. rhodesiense is based on the presence or absence of theSRA gene. We acknowledge that in theory those sampleswithin which the internal control alone is amplified maybe either T. b. brucei, T. b. gambiense or T. evansi, howevergiven both the host range and geographical distribution ofthese parasites the assumption that the absence of the SRAamplicon indicates the presence of T. b. brucei is made.

3.4. DNA preparation and host genetic material

Reactions were carried out in triplicate using DNAextracted from both procyclic and bloodstream forms ofT. b. brucei and T. b. rhodesiense, as well as DNA fromFTA cards prepared from the infected blood with parasi-taemias of 4 · 106; no variation in the results was found.No amplification was observed in the negative controlsfor human, bovine and mouse DNA (Fig. 1). The multiplexPCR also clearly differentiated between T. b. gambiense

and T. b. rhodesiense in the infected blood of two sleepingsickness patients (Fig. 1).

3.5. Amplification sensitivity

Following the approach of MacLeod et al. (1997), themultiplex PCR was assessed for its ability to detect a singlecopy gene within a single parasite. Initial experiments withgenomic DNA diluted to the equivalent of a single genome

Table 1PCR primers

Name Sequence Tm (�C) Product size (bp)

SRA gene detection

651 GAA GAG CCC GTC AAG AAG GTT TG 60.2 669652 TTT TGA GCC TTC CAC AAG CTT GGG 60.402 GGA GCC AAA ACC AGT GGG CAC ATC 63.8 23903 AAG TAG CGC TGTC CTGT AGA CGC TTC 60.4

PLC gene detection

657 CGC TTT GTT GAG GAG CTG CAA GCA 62.1 324658 TGC CAC CGC AAA GTC GTT ATT TCG 60.4

Table 2Additional isolates screened to assess the specificity of the multiplex PCR

Stock Country ofOrigin

Source No. ofsamples

PLC SRA

T. congolense Uganda Bovine 4 � �T. b. rhodesiense Uganda Human 14 + +T. b. rhodesiense Uganda Bovine 5 + +T. b. brucei Uganda Bovine 10 + �T. evansi Various Various 14 + �T. vivax Nigeria Bovine 1 � �

Fig. 1. Multiplex PCR products generated from trypanosome infected blood. Lanes 1 and 26 contain a 100 bp standard ladder, the double strength bandbeing 1 Kb (Bioline), Lanes 13 and 20 are H2O controls, Lanes 2–4 T. b. brucei (stock Buteba 135) as procyclic, blood stream form (bsf) and bsf spottedonto FTA cards at 1 · 106 parasites per ml, respectively. Lanes 5–7 are T. b. rhodesiense (stock D. Obwang) as above. Lane 8, T. b. gambiense (stockTSW83); Lane 9 T. evansi (stock Kenya 1793); Lane 10 T. vivax (stock Y486); Lanes 11 and 12, T. congolense (stocks 1/148 and Sikuda 124, respectively).Lanes 14–16 are T. b. brucei (stocks Papol 371, Bumanda 25 and Mela 3); Lanes 17–19 are T. b. rhodesiense (stocks M. Apollo, Ug I, U89/3); Lane 21infected blood from a patient with T. b. gambiense; Lane 22 infected blood from a T. b. rhodesiense patient. Lanes 23–25 are negative controls of cow,mouse and human genomic DNA. All samples were amplified through 35 cycles and each reaction contained 1 ng of DNA, except Lanes 21 and 22 both ofwhich were amplified from FTA cards through 40 cycles. The PLC band is 324 bp, SRA is 669 bp and the pVSG >1 Kb.

Table 3The detection of single trypanosomes using the multiplex PCR

Samples screened No. of samples PLC SRA Accuracy (%)

T. b. rhodesiense 19 13 13 72T. b. brucei 8 6 0 75Blanks 12 1 0 8.3

44 K. Picozzi et al. / Experimental Parasitology 118 (2008) 41–46

(0.12 pg) per reaction (Borst et al., 1982), resulted in repro-ducible differentiation between the T. brucei subspecies.

To determine if this amplification would be successfulwith a single trypanosome, procyclic stocks of Buteba135 and D. Obwang (T. b. brucei and T. b. rhodesiense,respectively) were diluted in 10 ll of Cunningham’s med-ium across a 96-well plate in a checkerboard manner, bothhorizontally and vertically. Each well was examined usingan inverted microscope and cross checked by two indepen-

dent observers for the presence of single procyclic trypano-somes. Nineteen T. b. rhodesiense and eight T. b. bruceiwere successfully isolated in this way and subjected toPCR amplification. Negative controls were taken fromtwelve wells where no trypanosomes were visible. Theresult (Table 3) show that a single copy gene from singletrypanosomes can be detected in 73% of reactions. Thepositive PCR signal generated from one of the blank reac-tions is assumed to be due to the presence of a single over-looked trypanosome. However, given that only 8.3% ofblanks produced a positive signal it is assumed that theamplification products were generated by trypanosomesrather than extraneous contaminating DNA.

K. Picozzi et al. / Experimental Parasitology 118 (2008) 41–46 45

4. Discussion

Multiplex PCR has been used to identify several parasitespecies including Plasmodium species (Padley et al., 2003)and to differentiate between Cyclospora and Eimeria spe-cies (Orlandi et al., 2003). Such PCRs are based on thedetection of the 18S small-subunit RNA gene, combiningthe identification of genus and species specific portions ofthis gene to produce a multiplexed reaction. A multiplexPCR used to identify Schistosoma mansoni infections inhost snails combined detection of trematode mitochondrialDNA with species specific primers directed against 18SrRNA (Jannotti-Passos et al., 1997).

ITS regions have also been used to identify different try-panosome species (Njiru et al., 2004; Desquesnes et al.,2001; Cox et al., 2005) but this PCR is unable to differen-tiate between the subspecies of T. brucei s.l. The multiplexPCR used in the present work was specifically developedfor the differentiation of T. b. brucei from T. b. rhodesiense

infections by detection of the SRA gene together with acontrol to confirm that sufficient genetic material was pres-ent for accurate diagnosis. This test was specificallydesigned for material collected on Whatman FTA cards;each PCR was seeded from a single disc cut from the cardswhich irreversibly bind DNA.

The multiplex PCR was assessed to ensure that therewas no amplification bias within the reaction. The bindingability of primers directed against the GPI-PLC gene wasunaffected by the presence of other primer sets within thePCR reaction mix. There was no significant differencebetween the primers CT regardless of the conditions. TheSRA primers selected for the multiplex reactions (651/652) do however occasionally amplify VSG genes thatshare homology with the SRA gene. Whilst the size ofthe SRA and VSG products can be clearly differentiatedby electrophoresis, real time PCR using SYBR green isunable to differentiate between these products as the CT

is measured as an increase in overall fluorescence ratherthan being target specific. The observed variation in CT

for this primer set with T. brucei s.l., when in a singleand multiplexed reactions, other than for T. b. rhodesiense,does not indicate a bias for the amplification of GPI-PLC

but rather the background presence of a transient target forwhich the reaction has not been optimised.

There is a highly reproducible relationship between theamount of starting material and the point at which PCRamplification enters the logarithmic phase of the reaction(Ginzinger, 2002). The difference in CT of 0.3 cyclesbetween the SRA and GPI-PLC amplifications whenamplifying T. b. rhodesiense demonstrates the similaritybetween the copy numbers of these genes. This ensures thatthe amplification of GPI-PLC identifies T. b. rhodesiense

when sufficient parasitic material is present within the sam-ple to successfully amplify the SRA gene if present.

In summary, we have developed a simple single stepmultiplex PCR that can identify T. b. rhodesiense basedon the recognition of the SRA gene. However, rather than

simply depending on the presence or absence of this targetgene as the defining parameter of this PCR, we have incor-porated the detection of a single copy control gene to con-firm that sufficient parasite genomic material would havebeen present within the reaction to provide a positiveresponse. Since identification of reservoirs of infection ofT. b. rhodesiense hinges on the presence or absence of theSRA gene in an individual animal previously identified ashaving been infected with T. brucei s.l. infections andbecause infections within the animal reservoir often exhibitvery low levels of parasitaemia (<10 parasite per ml wholeblood, Welburn pers.com.) the accuracy of a PCR testbased only on the absence or detection of a single copygene cannot be regarded as definitive. A negative PCRresult may indicate the absence of SRA or simply indicatethat too little genomic material is present. The methodol-ogy described here solves this problem and enables accu-rate determination of reservoir status in field materials(Picozzi et al., 2005; Kaare et al., 2007).

Acknowledgments

We thank Florence Achom and Ian Anderson for thecollection of patient samples in Uganda. This work wassupported by funding from the Cunningham Trust, theUK Department for International Development (K.P.and S.C.W.), WHO (S.C.W.) and the Leverhulme Trust(S.C.W. and M.C.).

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