JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 2010, p. 3735–3737 Vol. 48, No. 10 0095-1137/10/$12.00 doi:10.1128/JCM.00898-10 Copyright ? 2010, American Society for Microbiology. All Rights Reserved.

    Accurate and Sensitive Detection of Plasmodium Species in Humans

    by Use of the Dihydrofolate Reductase-Thymidylate Synthase

    Linker Region †

    1222,3Naowarat Tanomsing,Mallika Imwong,* Sasikrit Theppabutr,Sasithon Pukrittayakamee, 1,41,4Nicholas P. J. Day,Nicholas J. White,and Georges Snounou5,6,7

    1Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Department of 2Clinical Tropical Medicine, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; The Royal Institute, Grand Palace, 34Bangkok, Thailand; Centre for Vaccinology and Tropical Medicine, Churchill Hospital, Oxford, United Kingdom; 5INSERM UMR S 945, De?partement de Parasitologie, Hoˆpital Pitie?-Salpeˆtrie`re, Paris, France; Universite? Pierre et 6Marie Curie, Paris, France; and Laboratory of Molecular and Cellular Parasitology, Department of 7Microbiology, Faculty of Medicine, National University of Singapore, Singapore

    Received 4 May 2010/Returned for modi，cation 6 May 2010/Accepted 5 August 2010

    A nested-PCR protocol based on the linker region of the Plasmodium dihydrofolate reductase-thymidylate

    synthase gene (dhfr-ts) was developed. This provides highly sensitive speci；c detection and identi；cation of the

    ；ve parasite species that infect humans.

    Thymidylate synthase (TS) and dihydrofolate reductase DQ517900, DQ514918-DQ514921, and XM_001615032), 8

    from P. malariae isolates (AY846633 and AY846634, (DHFR) are enzymes of the folate cycle that produce the

    2 -deoxythymidine-5 -monophosphate (dTMP) required for EF188271 to EF188273, and EF198109 to EF198111), 1

    from a P. brasilianum isolate (EF188274), 20 from P. ovale DNA synthesis. As these proteins have an essential role in the

    isolates (EU266601 to EU266618 and GQ250090 and cell cycle, they are relatively conserved between distantly re-

    GQ250091), and 4 from P. knowlesi isolates (GQ250087 to lated species. In several protozoans, the two enzymes are en-

    coded in a single gene, with a linker region between the two GQ250089 and XM_002258192). Sequence variations in the Plas-

    domains. Although this linker region is thought to control the modium dhfr domain have been investigated extensively because orientation of the TS and DHFR domains relative to each nonsynonymous mutations at de，ned residues are associated with other (8), it displays signi，cantly higher sequence diversity decreased susceptibility to antifolate antimalarial drugs (4–6, 9).

    than the relatively conserved enzymatic domains (12). Se- The detection and identi，cation to species level of malaria para- quence analysis of the published Plasmodium dhfr-ts sequences sites in ，eld samples are often carried out in separate PCR am- revealed that the different species are characterized by unique pli，cation reactions, generally based on the small-subunit rRNA linker sequences (Fig. 1), which are highly conserved between (ssrRNA) genes. Ampli，cation of the dhfr-ts linker has also been different isolates of the same species. The sequences ana- shown to be effective at detecting low numbers of P. falciparum

    lyzed (GenBank accession numbers are in brackets) in- parasites (13). We wished to ascertain whether PCR primers that cluded 9 from P. falciparum isolates (EU046228 to target the Plasmodium dhfr-ts genes could equally serve as a EU046231, EU046233, J03772, J04643, M22159, and means to detect and identify the ，ve parasite species that infect XM_001351443), 12 from P. vivax isolates (DQ517894 to humans with high sensitivity and accuracy.

     FIG. 1. Alignment of the DHFR-TS amino acids for the linker region of Plasmodium species that infect humans. The degenerate primers for the primary ampli，cation are located at the start of the dhfr and ts segments. The positions of the species-speci，c paired primers located within the linker region of each Plasmodium species are indicated by the black boxes, and ampli，cation protocols are in Table S1 in the supplemental material. PF, P. falciparum; PV, P. malariae; PM, P. vivax; PO, P. ovale; PK, P. knowlesi.

* Corresponding author. Mailing address: Department of Clinical

    Tropical Medicine, Faculty of Tropical Medicine, Mahidol University, 420/6 Rajvithi Rd., Bangkok 10400, Thailand. Phone: (66) 2 354 9172. Fax: (66) 2 354 9169. E-mail: noi@tropmedres.ac. † Supplemental material for this article may be found at http://jcm .asm.org/. Published ahead of print on 11 August 2010.

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3736 NOTES J. CLIN. MICROBIOL.

TABLE 1. Detection of minority parasite species in arti，cial TABLE 2. Detection of parasite species in ，eld samples amixed infections No. of isolates detected with: aSpecies Minority species detected with: Species (no. of parasite ssrRNA gene PCR dhfr-ts linker PCR ssrRNA gene PCR dhfr-ts linker PCR genomes/ l) in mixture 14 14 PF PM/PO/PK (1–10 of each) PM PO PK PM PO PK PV 16 16 2PM/PO/PK (10of each) 13 PM PO PK PM PO PK PM 13 5PF (10) PM/PO/PK 5 5 PF PM PO PK PF PM PO PK PO b(1–10 of each) 4 4 PK5PF PM PO PK PF PM PO PK PF (10) PM/PO/PK PF PV 6 6 2(10of each) PF PM 5 5 4PF PM PO PK PF PM PO PK PV (10) PM/PO/PK PV PO 1 1 (1–10 of each) 4aP. falciparum; PM, P. malariae; PV, P. vivax; PO, P. ovale; PK, P. PV (10) PM/PO/PK PF PM PO PK PF PM PO PK PF,2knowlesi. (10of each) b The genomic DNA from these four isolates was obtained from MR4.

aP. falciparum; PM, P. malariae; PV, P. vivax; PO, P. ovale; PK, P. PF,knowlesi. linker protocol were identical to those obtained with the ssrRNA gene protocol (data not shown) in that both methods speci，cally detected the different parasite species in aliquots We designed degenerate primers speci，c to Plasmodium that contained down to 1 to 10 parasite genomes. In order to parasites that would yield a fragment (ca. 1 kb) spanning the con，rm that the assays were speci，c to Plasmodium species, dhfr and ts domains from any Plasmodium species in a primary the nested reactions (genus and species speci，c) were applied ampli，cation reaction and in a seminested secondary ampli，- to DNA puri，ed from blood samples collected on admission cation (see Table S1 in the supplemental material) and a set of from a total of 45 febrile patients: 15 with active melioidosis, 15 ，ve primers pairs located in the linker region and speci，c to with active leptospirosis, and 15 with active scrub typhus. No each of the ，ve parasite species that infect humans. The P. ampli，cation was observed for any of these 45 samples or for ovale primers hybridize to sequences conserved between the puri，ed human genomic DNA (data not shown). two types P. ovale curtisi and P. ovale wallikeri (11). We then One of the more important advantages of ampli，cation- optimized the reaction conditions for ampli，cation (see Table based methods is the ability to detect mixed-species infections, S1 in the supplemental material). In order to assess the sensi- where one species often predominates numerically over the tivity and speci，city of these primer pairs, serial dilutions of other, so we produced arti，cial mixtures where the DNA tem- genomic DNA from the ，ve malaria parasite species that occur plate concentration from one parasite species was present in a in humans were prepared to serve as templates for PCR am- 100- to 10,000-fold excess over the DNA templates from a set pli，cation. For each species, two isolates were selected: P. of the other species. The minor species were detected in all falciparum 3D7 and K1, Thai isolates of P. vivax, P. malariae, cases (Table 1). Finally, the results from the two protocols and P. ovale, and two isolates of P. knowlesi (MRA456 and were fully concordant when applied to templates puri，ed from MRA457) obtained from the Malaria Research and Reference 60 samples collected from Thai patients infected by one or Reagent Resource Centre (MR4 [http://www.mr4.org/]). In other of the four parasite species or carrying mixed-species parallel, the same templates were ampli，ed using an estab- infections and four P. knowlesi isolates obtained from MR4 lished protocol based on the ssrRNA genes (10) that is now (Table 2). considered the “gold standard” for sensitivity and speci，city (1, Accurate detection and identi，cation of malaria parasites 2). In all cases, the primary ampli，cation reactions were initi- provides important epidemiological data that inform malaria ated with 1 l of the template genomic DNA, and 1 l of the control programs. The reliability and sensitivity of molecular resulting product was used to initiate the secondary ampli，ca- techniques make them ideally suited to gather this informa- tion. All ampli，cations were carried out in a total volume of 20 tion. We show that an ampli，cation protocol based on prim- l, in the presence of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, ers that target the dhfr-ts linker region equals the sensitivity 250 nM each oligonucleotide primer, 125 M deoxynucleoside and speci，city of Plasmodium detection protocols based on triphosphates, and 0.4 units of Taq polymerase (Invitrogen, ssrRNA genes. There are two advantages that this protocol has United States). The cycling parameters consisted of an initial over the classical ssrRNA-based protocols. First, the ampli，- denaturation step at 95?C for 5 min, annealing for 1 min at cation ef，ciency and speci，city are less prone to genetic vari- a temperature de，ned for each primer pair (58?C for the ations within a given species or to cross-hybridization between ssrRNA gene primers; see Table S1 in the supplemental ma- species (3, 7). Second, for the investigation of antifolate resis- terial for the dhfr-ts primers), and extension at 72?C for 1 min, tance, the product of the primary dhfr-ts ampli，cation can be followed by denaturation at 94?C for 1 min. After a given used to determine the presence of point mutations associated number of cycles (30 for the primary and 35 for the secondary with drug resistance in the samples that prove to be positive for ampli，cations), a ，nal cycle with a 5-min extension step was Plasmodium using the protocol presented above. carried out before product visualization on ethidium bromide- stained 2% agarose gels (BRL, United States). Negative con- We thank MR4 for the provision of parasite material. We are grate- trols (no-DNA template or DNA from heterologous species) ful to the Microbiology Department of the Mahidol-Oxford Tropical were used in each set of ampli，cations. Medicine Research Unit for DNA samples from patients with acute The sensitivity and speci，city of the nested-PCR dhfr-ts febrile illnesses used in the control group.

VOL. 48, 2010 NOTES 3737

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