Differentiation ofOesophagostomum bifurcumfromNecator americanusby PCR using genetic markers in spacer ribosomal DNA

Molecular and Cellular Probes (1997) 11, 169–176

Differentiation of Oesophagostomum bifurcum from Necator americanus by PCR using genetic markers in spacer ribosomal DNA A. Romstad,1,3 R. B. Gasser,1∗ J. R. Monti,1 A. M. Polderman,2 P. Nansen,3 D. S. S. Pit2,4 and N. B. Chilton1 1

Department of Veterinary Science, The University of Melbourne, 250 Princes Highway, Werribee, Victoria 3030, Australia, 2Department of Parasitology, Faculty of Medicine, University of Leiden, PO Box 9605, 2300 RC Leiden, The Netherlands, 3 Danish Centre for Experimental Parasitology, Royal Veterinary and Agricultural University, Bu¨lowsvej 13, DK-1870 Frederiksberg C, Denmark, and 4Ministry of Health, Dapaong, Togo (Received 21 October 1996, Accepted 26 November 1996) Oesophagostomiasis in humans due to infection with Oesophagostomum bifurcum (nodular worm) is of major human health significance in northern Togo and Ghana where Necator americanus (human hookworm) also exists at high prevalence. However, very little is known about the transmission patterns of O. bifurcum, partly due to the difficulty in differentiating O. bifurcum from N. americanus at some life-cycle stages using morphological features. To overcome this limitation, a molecular approach utilizing genetic markers in the second internal transcribed spacer (ITS-2) of ribosomal (r) DNA was developed. The ITS-2 sequence of each species was determined, and specific oligonucleotide primers were designed to the regions of greatest sequence difference between the species. Utilizing these primers, rapid PCR assays were developed for the specific amplification of DNA of O. bifurcum or N. americanus, which have the potential to confirm the identity of eggs from faeces and larvae from the intestine or environment. The application of species-specific PCR has important implications for studying the epidemiology and population  1997 Academic Press Limited biology of O. bifurcum.

KEYWORDS: Oesophagostomum bifurcum, Necator americanus, parasitic nematodes, species identification, internal transcribed spacer, ribosomal DNA, specific PCR. INTRODUCTION Infection of humans with the nodular worm Oesophagostomum bifurcum (Nematoda: Strongylida) causes the formation of granulomata and caseous nodules in the large intestine (oesophagostomiasis; ‘Tumeur de Dapaong’ or ‘Koun Koul’) as a result of the presence of encysted larvae in the wall of the large intestine.1,2 O. bifurcum occurs at high prevalence (40–80%) in regions of northern Togo and Ghana

and co-exists with the human hookworm, Necator americanus.3,4 Despite the medical significance of O. bifurcum in these geographical regions of Africa, there is a lack of information available on the epidemiology and population biology of the parasite, which has mainly been a consequence of the inability to accurately identify some life-cycle stages of the parasite.4 Although adult worms of O. bifurcum (harboured in

∗Author to whom all correspondence should be addressed.

0890–8508/97/030169+08 $25.00/0/ll960094

 1997 Academic Press Limited

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the large intestine) have characteristic morphological features, the eggs shed in human faeces cannot be differentiated from those of hookworms. Infective (third stage) larvae which emerge from eggs in the faeces of infected humans can be identified to the genus level (Oesophagostomum), and free-living (first and second stage) larvae found in soil or on vegetation cannot be reliably differentiated from those of other Oesophagostomum spp. commonly infecting cattle and pigs.5 There have been attempts to improve methods for the specific identification of O. bifurcum eggs, such as the use of monoclonal antibodies, but this approach has so far been unsuccessful due to cross-reactivity of the antibodies with antigens on eggs from heterologous parasite species (Polderman et al., unpublished). DNA technology has been used successfully for the identification of parasites where morphology is unreliable.6,7 Ribosomal (r)DNA sequences have been shown to provide genetic markers for species or strains of parasitic helminths.8–11 Recently, it was demonstrated that internal transcribed spacer (ITS) rDNA provides markers to identify species of strongylid nematodes of veterinary importance.12–15 The use of such markers in PCR-based assays has allowed single eggs and larvae to be identified to the species level.13,15 These studies provide the basis for the present study, the aims of which were to define species markers in the ITS rDNA of O. bifurcum and N. americanus from Togo, and to develop PCR-based strategies for the specific amplification of DNA of either parasite species.

MATERIALS AND METHODS Parasites and isolation of genomic DNA The parasites and DNA samples used in this study are listed in Table 1. Adult worms of O. bifurcum and N. americanus were obtained in northern Togo (Naki-Est, Region des Savannes) from patients after treatment with pyrantel pamoate as described previously.3 Individual adult parasites were repeatedly washed in physiological saline, pH 7·3, identified morphologically5,16,17 and kept at −20°C until required for DNA isolation. Also obtained were eight other parasite species, namely Ancylostoma duodenale, Strongyloides stercorialis, Ascaris lumbricoides, Trichuris trichiura (nematodes), Hymenolepsis nana (a cestode), Schistosoma mansoni (a trematode), Giardia intestinalis and Entamoeba histolytica (protozoa). Genomic DNA was isolated by a method of sodium dodecyl sulfate and proteinase

K treatment, phenol/chloroform extraction and ethanol precipitation, purification over WizardTM DNA Clean-Up columns (Promega) and elution into 40 ll H2O.

Amplification by PCR Regions of rDNA were amplified by PCR.18 A diagram of the rDNA transcriptional unit and location of primers used in this study are shown in Fig. 1. PCR conditions were optimized by titration of MgCl2 and dNTP concentrations, as well as varying PCR annealing temperatures, cycle numbers and times. PCR reactions (50 ll) were performed with 10 m Tris– HCl, pH 8·4; 50 m KCl; 3·0 m MgCl2; 250 l each of dATP, dCTP, dGTP and dTTP; 100 pmol or 200 pmol of each primer (unlabelled or labelled with c33P-ATP using T4 kinase, Promega) using 2 U Taq polymerase (Perkin Elmer Cetus) under the following conditions: 94°C, 30 s (denaturation); 55°C, 30 s (annealing); 72°C, 30 s (extension) for 30 cycles (480 Thermocycler, Perkin Elmer Cetus). A 1 ll (0·25 pg to 4 ng) aliquot of genomic DNA of O. bifurcum or N. americanus DNA was added to each PCR reaction. Samples without genomic DNA (no-DNA controls) were included in each amplification run. Also, control-DNA samples derived from human blood or faeces (from a healthy person with no history of parasite infection) and from eight other species of parasites were subjected to the same amplification procedure as for O. bifurcum and N. americanus DNA. PCR products were run on 1·5–2·5% agaroseTBE (65 m Tris–HCl, 27 m boric acid, 1 m EDTA, pH 9) gels, stained with ethidium bromide and photographed with Polaroid 667 film (Kodak). For radiolabelled PCR products, agarose gels were dried onto Whatman 3MM filter paper and exposed to autoradiographic film (RP2, Agfa) for 12 h. This was carried out to confirm the specificity of products, the PCR and conditions.

DNA cycle sequencing The ITS+ region, comprising the ITS-1, 5.8S gene and the ITS-2, was amplified by PCR (from individual worm samples Ob10, Ob11, Ob12, Ob14, Na9, Na10 and Na13) using the NC5-NC2 primer set (see Fig. 1) and purified using WizardTM PCR-Prep columns (Promega). Then, the second internal transcribed spacer (ITS-2) was sequenced in both orientations from each ITS+ PCR product using primers NC1 and NC2 (see Fig. 1). A 1 ll sample (approx. 2·5–5·0 ng DNA) of the purified product was sequenced19 using a

PCR identification of O. bifurcum and N. americanus Table 1.

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DNA samples used in this study

Species

Stage

Oesophagostomum bifurcum

Single Single Single Single

Necator americanus

Control samples Ancylostoma duodenale Strongyloides stercorialis Ascaris lumbricoides Trichuris trichiura Hymenolepis nana Giardia intestinalis Entamoeba histolytica

DNA sample code

Origin

adult adult adult adult

Ob10 Ob11 Ob12 Ob14

Togo, Togo, Togo, Togo,

Single adult Single adult Single adult

Na9 Na10 Na13

Togo, patient 2 Togo, patient 2 Togo, patient 2

Single adult Multiple infective larvae Single adult Multiple adults Segment from adult Pooled trophozoites (culture) Pooled trophozoites (culture)

Ad-PP Ss-RP Al-SN Tt-JA Hn-AI Gi-RA Eh-ET

Borneo South Australia USA Guatemala Japan South Australia Germany

ITS-1

18S NC5>

5.8S

ITS-2


patient patient patient patient

1 1 1 1

28S

NC1> OB> NA>

Fig. 1. Schematic representation of part of the rDNA transcriptional unit and relative locations of primers (NC5, NC1, OB, NA and NC2) used in this study (not to scale). ITS-2 was amplified with NC1: 5′-ACGTCTGGTTCAGGGTTGTT-3′ (forward) and NC2: 5′-TTAGTTTCTTTTCCTCCGCT-3′ (reverse), and ITS+ was amplified with NC5: 5′-GTAGGTGAACCTGCGGAAGGATCATT-3′ (forward) and NC2. Primers OB and NA were designed to regions within the ITS-2.

commercial cycle sequencing kit ( fmolTM kit, Promega). The following cycling conditions were used: 95°C for 5 min (initial time delay); then 95°C, 30 s (denaturation), 55°C, 30 s (annealing), 70°C, 30 s (extension) for 30 cycles. The 5′ and 3′ ends of the ITS2 sequences were determined by comparison with those of a range of other species of strongylid nematodes.12–15,19,20 Sequence alignments were performed manually with reference to a secondary structure model for the ITS-2 pre-rRNA (Chilton et al., unpublished).

RESULTS Sequence analysis For both O. bifurcum and N. americanus, there was no variation in length or composition of the ITS2 sequences among samples. Some polymorphism, defined herein as multiple bases detected at a sequence position, was detected in the ITS-2 sequence of each species. For O. bifurcum, ITS-2 sequences were derived from each of the four samples (Ob10, Ob11, Ob12 and Ob14). Sequence poly-

morphism was detected at six different positions in the ITS-2 (Fig. 2, alignment positions 99, 171, 177, 187, 194 and 257). The ITS-2 of O. bifurcum was 216 bp in length, and the G+C content was 42%. The ITS-2 sequences for N. americanus were determined using each of the three samples (Na9, Na10 and Na13). Polymorphism in the sequences for N. americanus was observed at positions 52, 136 and 174 (Fig. 2). The ITS-2 region was 325 bp in length, and the G+C content was 43%. Subsequently, the ITS-2 sequences for O. bifurcum and N. americanus were aligned over a length of 329 bp (Fig. 2). The two species differed at 155 nucleotide positions (47%) in the alignment, 52 of which represented single-base substitutions. Of these, 73% were transitions between either purines (A↔G) (n=19) or pyrimidines (C↔T) (n=19) and 27% were transversions (i.e., substitutions between a purine and a pyrimidine).

Development of specific PCR for O. bifurcum or N. americanus Given the size difference in the ITS-2 between O. bifurcum and N. americanus, the size of the PCR

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O. bifurcum N. americanus

TATA--AATA CTACAGTGTG GCTTGTGA-- ---------- ---------- -----CACTG AACGATAATA CTACAGTGTA GCTTGTGGAC AGTACTCTCA CCGAGTATTG TYGAACACTG

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O. bifurcum N. americanus

TTTGTCGAAC GATGCTTAC- ---------- -ATT-TA-VT GTG------- ATCCTCGTTC TTTGTCGAAC GGTACTTGCT CTGTACTACG CATTGTATAC GTGTTCAGCA ATTCCCGTT-

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O. bifurcum N. americanus

TAGATAAGAA ATATAT-TGC AACAGGTAT- ---------- ---------- YTTGGTRCAA TAAGTGAAGA ACACAYGTGC AACATGTGCA CGCTGTTATT CACTACGTTA GTTRGC-TAG

O. bifurcum N. americanus

T---CCRA-G -ATR-CA-CG G--------- -ATGTCGTGA CC---TCGTT G-TCACTGTC TTTACTAACG TATGATAGCG GTGCATACTG TATGACATGA ACATATCGTT GTTCACTGTT

240

O. bifurcum N. americanus

AAA-GCG-TT TAGCGAY-TA AGAAT---GC TATGGCGGGG -CC-TGTAT- -GACAAC--T TAATTCGCTC TCGCGACTTA TGAGCGTGGT TGAA-CGGAG ACAATGTGAA GGACAACGAT

300

O. bifurcum N. americanus

GCGGTTTCAT --------GT CATTTGCAA GTT-CGCCAT GTGGATGTGT CATTTGCAA

OB–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––Π 180

NA–––––––––––––––––––––––––Π

Alignment position 52 99 136 171 174 177 187 194 257

329

Species

Bases detected in sequence (sample)

N. americanus O. bifurcum N. americanus O. bifurcum N. americanus O. bifurcum O. bifurcum O. bifurcum O. bifurcum

C/T(Na10); T(Na9, Na13) A/G(Ob10); A/C(Ob11, Ob14); A(Ob12) C/T(Na9); C(Na10, Na13) C/T(Ob10, Ob12); T(Ob11, Ob14) A/G(Na10, Na13); G(Na9) A/G(Ob10, Ob11, Ob12, Ob14) A/G(Ob10, Ob12); A(Ob11, Ob14) A/G(Ob10, Ob12); G(Ob11, Ob14) C/T(Ob14); C(Ob10, Ob11, Ob14)

Fig. 2. Alignment of the ITS-2 rDNA sequences of O. bifurcum and N. americanus. The locations of the forward primers OB (5′-TATATTGCAACAGGTATTTTGGTAC-3′) and NA (5′ATGTGCACGCTGTTATTCACT-3′) are indicated. Polymorphism within the ITS-2 sequences (designated with IUPAC codes) are shown in the accompanying table. Sequences have been deposited in the EMBL, GenBank and DDBJ databases (accession numbers Y11733 and Y11734).

product could be considered a genetic marker to distinguish the two species. However, the ITS-2 PCR product generated for O. bifurcum using the primer set N1-NC2 is similar in size to those from a variety of strongylid nematodes.13–15 Thus, size alone is not a reliable genetic marker for species identification. For this reason, primers unique for each species were designed to the ITS-2 and assessed in PCR. Primers OB and NA were designed to central regions of the ITS-2 sequences corresponding to the region of major sequence difference between the two species (Fig. 2, alignment positions 130 to 180). The primer sets OBNC2 and NA-NC2 were evaluated for their specificity in PCR using optimum conditions (as described above using 100 pmol of each primer). The primer set NC1NC2, known to amplify ITS-2 from different species of strongylid nematodes12–15,19–23 but not from vertebrate host DNA, ascarid, enoplid or protozoan parasites (unpublished data), was used as a control in PCR to amplify Oesophagostomum or Necator DNA from each sample (PCR products of 310 bp and 450 bp, respectively). As expected, the primer set OB-NC2 amplified a PCR product of approximately 220 bp

from O. bifurcum but not from N. americanus DNA (Fig. 3). Conversely, the primer set NA-NC2 amplified a product from N. americanus (approx. 260 bp) but not from O. bifurcum DNA (Fig. 3). The specificity of the PCR using OB-NC2 or NA-NC2 was also evaluated using the control DNA samples derived from various other parasites and from a ‘negative’ faecal sample. No amplification products were generated from any of these samples (results not shown). Sequence analysis (using NC2) of the PCR products amplified from O. bifurcum and N. americanus DNA using primer set OB-NC2 and NA-NC2, respectively, proved them to be partial ITS-2 sequences of the appropriate species. Finally, titration experiments were conducted to determine the ‘sensitivity’ of the PCR using the primer set OB-NC2 or NA-NC2. The lowest quantity of Oesophagostomum or Necator DNA reproducibly detectable by amplification using each primer set was found to be approximately 0·6 pg. This was also the case in the presence of excess DNA (2 ng) from the heterologous species (data not shown). Having demonstrated the specificity of primers OB and NA, a single tube ‘three-primer’ PCR procedure

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Fig. 3. PCR amplification from genomic DNA of O. bifurcum or N. americanus. Approximately 4 ng of DNA from individual adult worms of O. bifurcum (lanes 1–3) or N. americanus (lanes 4–6) and control samples without DNA (no-DNA) (lanes 7–9) were subjected to PCR using primer sets NC1-NC2 (lanes 1, 4 and 7) and OB-NC2 (lanes 2, 5 and 8) or NA-NC2 (lanes 3, 6 and 9). M=UX174/HaeIII molecular size marker (bp).

was also established. Instead of using only two primers (at 100 pmol each), three primers (2 forward, 1 reverse) were directly incorporated into a PCR reaction. Two PCR reaction mixes were prepared and evaluated, namely the Ob-mix (with primer combination NC1-OB-NC2) and the Na-mix (with primer combination NC1-NA-NC2). The concentrations of the primers in these reaction mixes were 100 pmol for NC1 and NC2, and 200 pmol for OB or NA. The rationale for this approach was that the primer set NC1-NC2 would amplify the entire ITS-2 (plus flanking sequence) from DNA of Oesophagostomum (310 bp product) or Necator (450 bp product) in each sample tube, and that the specific primer (OB or NA) would prime internally within the ITS2 together with primer NC2 to produce specific products (Fig. 4). The Fig. 4. Specific PCR amplification from genomic DNA (4 ng) of O. bifurcum and N. americanus. (a) Ethidium bromide-stained agarose gel showing products amplified from DNA of O. bifurcum (lanes 1 and 4) and N. americanus (lanes 2 and 5), no-DNA controls (lanes 3 and 6) using primer set NC1-OB-NC2 (Ob-mix; lanes 1–3) or NC1-NA-NC2 (Na-mix; lanes 4–6). Autoradiograph of agarose gel showing products amplified from three individual adults of O. bifurcum (lanes 1–3) and N. americanus (lanes 4–6), no-DNA control (lane 7) using (b) primer set NC1-OB-NC2 (Obmix) or (c) NC1-NA-NC2 (Na-mix). Primers endlabelled with c33P-ATP prior to PCR amplification. M=UX174/ HaeIII molecular size marker (bp).

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Fig. 5. PCR amplification using (a) primer set NC1-OB-NC2 (Ob-mix) or (b) primer set NC1-NA-NC2 (Na-mix). Control DNA samples from the parasites A. duodenale, S. stercorialis, A. lumbricoides, T. trichiura, H. nana, S. mansoni, G. intestinalis, E. histolytica (lanes 1–8, respectively), and human DNA (lane 9), faecal sample from a human with no history of gastro-intestinal parasitism (lane 10), O. bifurcum (lane 11), N. americanus (lane 12) and a sample without DNA (lane 13). M=UX174/HaeIII molecular size marker (bp).

specificity of the PCR (and conditions) was assessed using DNA from O. bifurcum, N. americanus and a range of control samples (Fig. 5). PCR amplification was carried out using approximately 4 ng genomic DNA (1/50–1/100th of the DNA isolated from an adult worm). Upon amplification with the Ob-mix (Fig. 5a), strong bands Ob-310 bp and Ob-220 bp (Fig. 4) were produced from O. bifurcum DNA, whereas only a single band of 450 bp was produced from N. americanus DNA. Amplification of N. americanus DNA with the Na-mix (Fig. 5b) produced strong bands Na-450 bp and Na-260 bp, while only a single band of 310 bp was produced from O. bifurcum DNA. Faint bands of 620 bp (in O. bifurcum) and 900 bp (in N. americanus) were present in PCR products amplified from O. bifurcum and N. americanus using the Ob-mix and Na-mix, respectively, which probably represented ITS-2 duplex molecules (Fig. 4). Another faint non-specific band of 170 bp was detected in N. americanus using the Na-mix (Fig. 4). For each reaction mix, the specificity of the PCR was then evaluated using the control DNA samples derived from various other parasites and from a ‘negative’ faecal sample. On ethidium bromide-stained agarose gels, a strong band of approximately 310 bp (an NC1-NC2 product) was amplified from Ancylostoma duodenale DNA using each PCR reaction mix (Fig. 5). This was expected as A. duodenale is a strongylid nematode, as are O. bifurcum and N. americanus. Autoradiography of the same agarose gels (using radiolabelled PCR products) revealed an additional faint band of approximately 310 bp in the Hymenolepis nana sample, which was most likely the result of non-specific amplification (results not shown). Over-exposed autoradiographs did not detect any other bands in any of the control samples (not

shown). Then, bands Ob-220 bp and Na-260 bp were individually excised from the agarose gels, purified over spin columns and subjected to direct cycle sequencing19 using NC2. The sequence analysis confirmed that bands Ob-220 bp and Na-260 bp were amplified from the appropriate regions of rDNA of O. bifurcum and N. americanus, respectively. Finally, the ‘sensitivity’ of the PCR using the Obmix or Na-mix was determined by titration of genomic DNA (data not shown). The lowest quantity of O. bifurcum or N. americanus DNA reproducibility amplified using each PCR mix was found to be approximately 1·3 pg. The lower ‘sensitivity’ of the ‘three-primer’ PCR compared with that using OB-NC2 or NA-NC2 is presumed to be the result of competition between the NC1 primer and the specific primer for amplification with NC2. The presence of excess DNA from the heterologous species (2 ng) in the sample decreased the lowest level of detectability of parasite DNA to approximately 60 pg in the three-primer PCR.

DISCUSSION This study demonstrated that O. bifurcum could be distinguished from N. americanus using genetic markers in ITS-2 rDNA. The size difference and the magnitude of sequence difference (47%) in ITS-2 sequences between the two species was substantially greater than the sequence variation within each species (<1%). These findings imply that the ITS-2 sequence is useful for the delineation of these two parasites. Some degree of sequence polymorphism (two or more different bases at a particular position in the sequence) was detected in the sequences derived

PCR identification of O. bifurcum and N. americanus

from single worms of both O. bifurcum and N. americanus. For example, sequence polymorphism was detected at the alignment position 177 in the ITS-2 of O. bifurcum (Fig. 2). At this position, samples Ob11 and Ob14 had a strong A (faint G), and Ob10 and Ob12 had a strong G (faint A). Given that position 177 created a recognition site for the endonuclease RsaI (GTAC; between alignment positions 175–178) when an A was present, ‘incomplete’ digestion with this endonuclease would reflect the existence of the different sequence types. Comparison of the sequence data with a previous restriction analysis indicated that the varying intensities of the A and G at position 177 were reflected in the level of digested ITS-2 PCR product on agarose gels.24 While the ITS-2 products of Ob11 and Ob14 digested almost to completion with RsaI, a higher proportion of undigested PCR product remained for samples Ob10 and Ob12. The previous PCR–RFLP analysis24 also supported the existence of polymorphism in the ITS-2 sequence of N. americanus. The polymorphism (A/G) at alignment position 174 of the ITS-2 of N. americanus (Fig. 2) was within an AluI recognition site (AGCT) when an A was present. While an A and a G were present in a similar intensity at this position in the ITS-2 of samples Na10 and Na13, only a G existed in the sequence of Na9. PCR–RFLP analysis revealed that a proportion of the ITS-2 PCR products of Na10 and Na13 was digested by AluI, while the product of Na9 remained undigested.24 Polymorphism in the ITS, reflecting the existence of two or more sequence variants in the population of rDNA copies within a species and within a single individual, appears to be the consequence of mutational changes occurring during DNA replication.26 It has been reported in individual organisms from a range of species,10,13,19,21,26,27 and has been confirmed by cloning and mutation detection procedures.13,28 The polymorphisms detected in the ITS-2 sequences in this study are unlikely to have been due to artefactual nucleotide misincorporation during PCR, because they were present at the same positions in the ITS-2 sequence, irrespective of the sequencing run and primer used, using PCR products generated on different days. Furthermore, the rate of artefactual nucleotide misincorporation during the PCR is random and has been shown to be very low in sequences of less than 700 bp in length.29,30 Utilizing the genetic markers defined in the ITS-2 rDNA, specific PCR assays were developed for the differentiation of O. bifurcum from N. americanus. Given the limitation of current coprological methods for diagnosis of human oesophagostomiasis,5 such assays have considerable promise for the identification of eggs of O. bifurcum derived from faecal

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samples, especially since there is no evidence for significant variation in the ITS-2 sequence among different life-cycle stages of species.13,15,19 Previous studies indicate clearly that the ‘sensitivity’ of a PCR assay should suffice for the amplification from single eggs.13,15,19 Another possible application may be in the diagnosis of human cases with arrested larval development4 by the detection of parasite-specific ITS rDNA in faecal or surgical biopsy samples derived from the large intestine. This warrants investigation. The accurate identification of different life-cycle stages of O. bifurcum also has important implications for studying the epidemiology of the parasite in Togo and Ghana.4 Little is known about the parasite’s mode of transmission. It is likely that humans become infected through ingestion of infective-third stage larvae from the environment, which can remain viable even after desiccation.4 Larvae of the ‘strongylid nematode type’ are frequently found in environmental samples (soil and sludge) and on vegetation (Polderman, personal observations), but it has not been possible to determine their species identity because of distortion/deformation as a consequence of desiccation. Another aspect is that it is not known whether monkeys harbour O. bifurcum and therefore act as reservoir hosts for human infection.4,16 It has also been postulated that other animals (perhaps pigs or dogs) may act as transport hosts for eggs or larvae of O. bifurcum, which resist gastrointestinal passage and are disseminated via faeces into the environment (Polderman, personal observations). Again, such stages may represent a source for human infection. Hence, the application of specific PCR for the accurate identification of O. bifurcum eggs, larvae or DNA from environmental samples or faecal samples from potential transport or reservoir hosts should allow such epidemiological questions to be addressed for the first time. The application of a PCR assay will depend on the ‘sensitivity’ (i.e. lowest level of genomic DNA required for successful amplification) needed. If it were the case that the sensitivities of the assays developed herein (0·6 pg or 60 pg of genomic DNA) were not adequate for some applications, a nested PCR strategy could be rapidly adapted.

ACKNOWLEDGEMENTS Thanks to Henrik Bøgh and P. Bloch for discussions. The authors are grateful to J. Blotkamp and S. Baeta who assisted in the collection of the O. bifurcum and N. americanus samples. Thanks to R. Andrews, P. Prociv, R. Putland (Australia), A. Ito (Japan), S. Nadler (USA), G. Greer (Guatemala), J. Allan, P. Craig (UK), E. Tannich (Germany) and B. Vennervald (Denmark) for providing control samples. Funding support was provided by the Australian Research

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Council, the University of Melbourne (Collaborative Program), the Department of Industry, Science and Tourism, and the Danish National Research Foundation.

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