- Email: [email protected]
Diagnosis of Trypanosomatid Infections
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The Journal of Molecular Diagnostics, Vol. -, No. -, - 2014
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Diagnosis of Trypanosomatid Infections: Targeting the Spliced Leader RNA Pablo González-Andrade,* Mamady Camara,y Hamidou Ilboudo,z Bruno Bucheton,x Vincent Jamonneau,zx and Stijn Deborggraeve* From the Biomedical Sciences Department,* Institute of Tropical Medicine, Antwerp, Belgium; the National Sleeping Sickness Control Program,y Ministry of Health and Public Hygiene, Conakry, Guinea; the Centre International de Recherche Développement sur l’Elevage en zones Subhumides (CIRDES),z BoboDioulasso, Burkina Faso; and the Institut de Recherche pour le Développement,x Unité Mixte de Recherche IRD-CIRAD 177, Montpellier, France Accepted for publication February 26, 2014.
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Address correspondence to Stijn Deborggraeve, Institute of Tropical Medicine, Nationalestraat 155, 2000 Antwerpen, Belgium. E-mail: [email protected].
Trypanosomatids transcribe their genes in large polycistronic clusters that are further processed into mature mRNA molecules by trans-splicing. During this maturation process, a conserved spliced leader RNA (SL-RNA) sequence of 39 bp is physically linked to the 50 end of the pre-mRNA molecules. Trypanosomatid infections cause a series of devastating diseases in man (sleeping sickness, leishmaniasis, Chagas disease) and animals (nagana, surra, dourine). Here, we investigated the SL-RNA molecule for its diagnostic potential using reverse transcription followed by real-time PCR. As a model, we used Trypanosoma brucei gambiense, which causes sleeping sickness in west and central Africa. We showed that the copy number of the SL-RNA molecule in one single parasitic cell is at least 8600. We observed a lower detection limit of the SL-RNA assay in spiked blood samples of 100 trypanosomes per milliliter of blood. We also proved that we can detect the trypanosome’s SL-RNA in the blood of sleeping sickness patients with a sensitivity of 92% (95% CI, 78%e97%) and a specificity of 96% (95% CI, 86%e99%). The SL-RNA is thus an attractive new molecular target for next-generation diagnostics in diseases caused by trypanosomatids. (J Mol Diagn 2014, -: 1e5; http://dx.doi.org/10.1016/j.jmoldx.2014.02.006)
Trypanosomatids are early eukaryotic protozoans that possess unique molecular features. Genes typically do not harbor introns but are arranged in large polygenic clusters that are transcribed polycistronically.1 Polycistronic premRNA is further processed into functional monocistronic mRNA molecules by trans-splicing and polyadenylation. During this RNA maturation process, a conserved spliced leader RNA (SL-RNA) molecule is donated to the 50 end of each individual mRNA.2 The SL-RNA sequence of the trypanosomatids can be 39 to 41 nucleotides long and carries methyl groups on the first four ribose moieties and on the first and fourth bases following the conserved 7-methylguanosine (m7G).2,3 Among the trypanosomatids are three major human pathogens: Trypanosoma brucei, T. cruzi, and Leishmania spp. Their trans-splicing mechanisms have been extensively investigated for understanding eukaryotic cell biology, but the SL-RNA has never been explored as a diagnostic target, although this molecule has several attractive diagnostic
features. It is a short, noncoding RNA sequence that is Q5 conserved, but unique, for each species and is present in each mRNA molecule in the cell. Importantly, mRNA is considered to be the best surrogate marker for viable organisms,4,5 because it is rapidly degraded after death of the organism with a typical half-life of 3 minutes.6 Here, we describe the first study that explores the trypanosomatid SL-RNA molecule for its diagnostic potential. As a model, we used sleeping sickness, a devastating disease that is endemic in sub-Saharan Africa and caused by the T. brucei subspecies gambiense and rhodesiense. The trypanosomes are transmitted to humans by tsetse flies and invade the brain, which in most cases is fatal if not treated in time. T. brucei gambiense is associated with the chronic form of sleeping sickness in west and central Africa, whereas Supported by the Research Foundation Flanders (FWO) (grant number 1501413N). Disclosures: None declared. Q2
Copyright ª 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jmoldx.2014.02.006
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González-Andrade et al T. brucei rhodesiense causes acute sleeping sickness in east Africa. Here, we show that the T. brucei SL-RNA is an excellent target molecule for the detection of the trypanosomes in clinical specimens.
Materials and Methods Oligonucleotides Oligonucleotides were synthesized and purified using highperformance liquid chromatography by Integrated DNA Technologies (IDT, Leuven, Belgium). Nucleic acid sequences were as follows: SL-RNA molecule, 50 -AACUAACGCUAUUAUUAGAACAGUUUCUGUACUAUAUUG30 ; SL-RNAespecific primer for reverse transcription and real-time PCR (cSL), 50 -CAATATAGTACAGAAACTG-30 ; forward primer for real-time PCR (SL-F), 50 -AACTAACGCTATTATTAGAA-30 .
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T. brucei gambiense (LiTat 1.3), T. brucei rhodesiense (AnTat 25.1), T. brucei brucei (Lister 427), T. evansi (Kazakstan), T. equiperdum (OVI), and T. vivax (ILRAD700) were propagated in OF1 mice or HsdCpb:WU rats (Harlan Laboratories, Horst, The Netherlands). Trypanosomes were separated from the blood using diethylaminoethyl cellulose chromatography7 followed by repeated centrifugation and sediment washes with PBS glucose [38 mmol/L Na2HPO4$H2O, 2 mmol/L NaHPO4, 80 mmol/L glucose, 29 mmol/ L NaCl (pH 8.0)]. T. congolense (Savannah type, KTT/ MSORO/M7), T. rangeli (ScTb), and Leishmania donovani (BPK282/0cl4) were propagated in vitro as described previously.8e10 In vitroecultured parasites were harvested by repeated centrifugation and washing in PBS glucose. Parasite concentrations in PBS glucose were measured using Uriglass counting chambers (Menarini, Vienna, Austria).
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result in the card agglutination test for trypanosomiasis (CATT) for antibody detection in blood,12 and trypanosomes were detected in their blood by the mini anion exchange centrifugation technique.13 Endemic controls did not show antibodies against T.b. gambiense in the CATT and in the immune trypanolysis assay with LiTat 1.3, LiTat 1.5, and LiTat 1.6 T.b. gambiense parasites.14 For RNA isolation, 2 mL of blood was taken in PAXgene Blood RNA tubes (PreAnalytiX, Hombrechtikon, Switzerland) from patients and controls, and transported at 20 C to Centre International de Recherche-Développement sur l’Elevage en Zone Subhumide (Bobo-Dioulasso, Burkina-Faso). On arrival, samples were immediately stored at 80 C until the RNA was isolated.
RNA Isolation and cDNA Preparation Total RNA from laboratory-cultured parasites and spiked human blood samples were extracted using TRIzol (Ambion, Austin, TX). Briefly, 10 volumes of TRIzol reagent and two volumes of chloroform were added to the sample, followed by centrifugation at 12,000 g for 15 minutes. The RNA in the aqueous phase was precipitated in isopropanol, washed in 75% ethanol, and then dissolved in 50 mL of diethylpyrocarbonate-treated water. Total RNA from the blood of pa- Q8 tients and controls was extracted with the PAXgene Blood RNA kit (PreAnalytiX), and RNA was eluted in 80 mL of diethylpyrocarbonate-treated water. Copy DNA (cDNA) of the SL-RNA was prepared using the SL-RNAespecific cSL primer and SuperScript II Reverse Transcriptase (Invitrogen/ Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions.
Real-Time PCR SL-RNA cDNA levels were measured by real-time PCR in the LightCycler 480 PCR system (Roche Applied Science, Penzberg, Germany). Two and a half microliters of cDNA were mixed with 0.25 mmol/L cSL and SL-F primers in a 25-mL reaction containing 1 SensiMix SYBR green NoRox (Bioline, London, UK). Thermal cycling conditions were 10 minutes incubation at 95 C, followed by 40 cycles of 94 C for 15 seconds, 50 C for 15 seconds, and 60 C for 15 seconds with data acquisition. Post-amplification melting curves were recorded from 45 C to 95 C, with increments of 0.1 C per second. Genomic DNA contamination in the RNA samples was verified by subjecting the RNA to realtime PCR without cDNA preparation.
Thirty-six blood samples from sleeping sickness patients and 49 blood samples from endemic controls were analyzed with the SL-RNA assay. Patients and controls were recruited in the main human African trypanosomiasis foci along the Guinean coast (Forecariah, Dubreka, and Boffa) between 2007 and 2009 as reported by Ilboudo et al11 in 2012. This study is part of a larger project aiming to improve human African trypanosomiasis diagnosis for which approval was obtained from the World Health Organization (Research Ethics Review Committee) and from the ethical committee of the Institut de Recherche pour le Développement (Comité Consultatif de Déontologie et d’Ethique). All participants were informed of the objectives of the project in their own language and signed a written informed consent form. For participants <18 years of age, informed consent was obtained from their parents. Sleeping sickness patients had a positive
Mean threshold cycle (Ct) values of duplicate samples in realtime PCR were calculated with one-sigma SD (s). Sensitivities and specificities of the SL-RNA assay in sleeping sickness patients and endemic controls were calculated at a 95% CI using Wilson’s score. Sensitivity is defined as the
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Figure 1 Melting peak of reverse-transcribed and PCR-amplified SL-RNA in total RNA from 2 million T.b. gambiense parasites. Non-reversetranscribed (NRT) total RNA was used as a negative control.
proportion of confirmed sleeping sickness patients who are positive by the index test, and specificity as the proportion of endemic controls who are negative by the index test.
Results Proof-of-Concept The SL-RNA sequences of T.b. gambiense and T.b. rhodesiense were aligned in silico, together with those from the closely related T.b. brucei, T. equiperdum, T. evansi, T. congolense, T. vivax, T. rangeli, T. cruzi, and L. donovani Q9 using Clustal Omega (Supplemental Figure S1). The alignment shows a Trypanozoon-specific central region from bp 11 to 20 and a hyperconserved region from bp 21 to 39. The Trypanozoon subgenus consists of the human infective T. brucei gambiense and T. brucei rhodesiense, as well as the animal pathogens T. brucei brucei, T. evansi, and T. equiperdum. The cSL primer used during reverse transcription of the SL-RNA is complementary to the 19-bp hyperconserved sequence. The additional primer that is used during PCR is situated adjacent and has its 30 region in the Trypanozoon-specific region. To deliver the proof of concept, we extracted total RNA from 2 million T.b. gambiense parasites and detected the SL-RNA molecules by reverse transcription and real-time PCR. The melting peak Q10 of the amplified cDNA showed a melting temperature of ½F1 67 C (Figure 1), which corresponds to the in silicoepredicted melting temperature of the 39-bp T.b. gambiense SLRNA sequence (66.5 C). Non-reverse-transcribed total RNA did not show any amplified DNA during the real-time PCR. Using standards of chemically synthesized SL-RNA molecules and the equation
g RNA=ðlength 340Þ 6:023 1023 Znumber
ð1Þ
of RNA molecules;
we have estimated the number of SL-RNA molecules per parasite. We found that there are at least 8600 (s Z 1080) SL-RNA molecules in one single trypanosome.
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311 312 313 314 315 316 317 318 319 320 321 322 323 Figure 2 Amplification curves and melting peaks (inset) of the SL-RNA 324 in 10-fold serial dilutions of T.b. gambiense parasites in human blood. 325 Q15 Expressed as parasites per milliliter of blood. 326 327 Analytical Sensitivity 328 329 We determined the analytical sensitivity by applying the SL330 RNA assay to RNA extracts from naive human blood sam331 332 ples spiked with known numbers of T.b. gambiense parasites, 333 ranging from 1,000,000 to 10 parasites per milliliter (p/mL) 334 of blood in a 10-fold dilution series. All samples were pre335 pared in duplicate, and the analytical sensitivity of the assay 336 was defined as the lower dilution that showed a positive test 337 result in both duplicates. The analytical sensitivity of the SL338 RNA assay is 100 p/mL of blood (Figure 2). Mean Ct values ½F2 339 of the duplicate samples were 29.0 (s Z 0.80) for 1,000,000 340 p/mL, 33.35 (s Z 0.98) for 100,000 p/mL, 36.35 (s Z 0.80) 341 for 10,000 p/mL, and >40 for 1000 and 100 p/mL. Non342 spiked blood remained negative in the assay. 343 344 345 Analytical Specificity 346 347 To determine the analytical specificity of the SL-RNA 348 assay, we tested 10 pg of total RNA from different Trypanosoma species and from Leishmania (Figure 3). Our assay ½F3 349 350 only detects the parasites of the Trypanozoon subgenus T. 351 vivax; Leishmania showed a minor amplification signal, and 352 the SL-RNA of T. rangeli and T. congolense were not 353 amplified at all. These observations are fully consistent with 354 the in silico alignments of the SL-RNA sequences from 355 these organisms (Supplemental Figure S1). 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 Figure 3 Melting peaks of reverse-transcribed and PCR-amplified total 371 Q16 RNA (10 pg) from different trypanosomatids. 372 print & web 4C=FPO
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Patients and Controls Out of the 36 human African trypanosomiasis patients, 33 were positive in the SL-RNA assay, whereas 47 of 49 endemic controls remained negative. This corresponds with a test sensitivity of 92% (95% CI, 78%e97%) and a test specificity of 96% (86%e99%). No DNA contamination was observed in the analyzed RNA samples.
Discussion
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Technological advancements are continuously expanding our portfolio of diagnostic tests for infectious diseases. In parallel with the search for more accurate technologies to detect infectious agents, there is a need to provide the best target molecule for such a diagnostic test. Here, we show that the spliced leader RNA is an excellent target for accurate detection of trypanosomatids in blood samples. The proof of concept was delivered for sleeping sickness, but the idea can be exploited for Chagas disease and leishmaniasis, as well as for important animal diseases such as nagana and surra. Furthermore, nematodes share a similar transsplicing mechanism to process their mRNA,15 and a similar approach can thus be used to detect the SL-RNA of parasitic nematodes in man and animals. We reverse transcribed the 39-bp SL-RNA RNA of T.b. gambiense and analyzed the cDNA by real-time PCR in experimentally prepared samples and in blood samples from sleeping sickness patients. Despite the very short length, the assay was highly efficient in detecting trypanosomes and did not show any amplification artifacts such as primer dimers. Crucial steps during the optimization of the assay were efficient RNA extractions without genomic DNA contamination, the annealing temperature of the oligonucleotides during reverse transcription, and the annealing and elongation temperatures during real-time PCR. In blood samples spiked with known numbers of T.b. gambiense, we were able to detect the parasite’s SL-RNA in as few as 100 parasites per milliliter of blood. This is in the same range as DNA detection methods such as PCR and LAMP, which target multicopy DNA sequences of T. brucei,16e18 and a nucleic acid sequenceebased amplification assay, which targets the ribosomal RNA.19 However, the major advantage of our SL-RNA assay is that we target the parasite’s mRNA, which is considered to be a better marker for viable organisms than DNA or rRNA.4,5 Analytical specificity experiments showed that the assay did not detect RNA from genetically closely related Trypanosomatidae. In silico analysis of our primers did not indicate any potential crossreaction with other microorganisms that are endemic in sleeping sickness foci and cause diseases with similar clinical symptoms, such as malaria parasites for fever and meningococcal bacteria for neurological symptoms (data not shown). We also verified whether we could detect the T.b. gambiense SL-RNA in the blood of patients with sleeping sickness. Our SL-RNA assay showed a sensitivity of 92%
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and specificity of 96% in 36 patients and 49 endemic controls from Guinea. The two endemic controls that showed positive SL-RNA test results may be true sleeping sickness cases that were missed by the reference tests, although this is unlikely because the serological reference tests make use of three different immunodominant antigens, the LiTat 1.3, LiTat 1.5, and LiTat 1.6 variant surface glycoproteins. Because the SLRNA assay detects the Trypanozoon subgenus, we cannot exclude a transient infection with T.b. brucei, which is not detected by the serological reference tests used in this study. T.b. brucei is generally not infectious to man but has been reported in atypical human infections.20 Although not observed during the optimization and the analytical sensitivity and specificity experiments, we cannot exclude amplification artifacts in the SL-RNA assay. By including standards of chemically synthesized SL-RNA in the assay, we showed that the number of SL-RNA molecules in one single T.b. gambiense cell is at least 8600. This is consistent with what we theoretically expect based on previous studies. The T. brucei genome project reported 9000 protein coding genes,1 and a subsequent transcriptome study showed that 75% of these genes generate 1 to 10 mRNAs per cell.21 Trypanosomatids are responsible for several diseases in man, animals, and even plants.22 Sleeping sickness, leishmaniasis, and Chagas disease have the worst reputation because they cause >100,000 deaths each year. Accurate diagnosis of these diseases is challenging because of the extremely low parasite load in the blood of patients. Nucleic acidebased tests are often considered to be the future reference methods because they generally show high sensitivity and specificity.16 In this study, we have shown for the first time the great potential of the SL-RNA as a marker for trypanosomatids in the blood of patients. This is an important new step in expanding our biomarker portfolio for diagnosing diseases caused by trypanosomatids, as well as by other pathogens sharing a similar trans-splicing mechanism such as nematodes.
Acknowledgments We thank Dr. Büscher for the support throughout the project and Q12 critical reading of the manuscript; Prof. Jan Van den Abbeele, Dr. Vincent Delespaux, and Eliane Tihon for providing the T. congolense strain; and Prof. Jean-Claude Dujardin and Dr. Manu Van Aerschot for providing the L. donovani strain.
Supplemental Data Supplemental material for this article can be found at http://dx.doi.org/10.1016/j.jmoldx.2014.02.006.
References 1. Berriman M, Ghedin E, Hertz-Fowler C, Blandin G, Renauld H, Bartholomeu DC, et al: The genome of the African trypanosome Trypanosoma brucei. Science 2005, 309:416e422
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RNA Diagnostics for Trypanosomatids 2. Perry KL, Watkins KP, Agabian N: Trypanosome mRNAs have unusual “cap 4” structures acquired by addition of a spliced leader. Proc Natl Acad Sci U S A 1987, 84:8190e8194 3. Bangs JD, Crain PF, Hashizume T, McCloskey JA, Boothroyd JC: Mass spectrometry of mRNA cap 4 from trypanosomatids reveals two novel nucleosides. J Biol Chem 1992, 267:9805e9815 4. Keer JT, Birch L: Molecular methods for the assessment of bacterial viability. J Microbiol Methods 2003, 53:175e183 5. Cangelosi GA, Weigel KM, Lefthand-Begay C, Meschke JS: Molecular detection of viable bacterial pathogens in water by ratiometric prerRNA analysis. Appl Environ Microbiol 2010, 76:960e962 6. von Gabain A, Belasco JG, Schottel JL, Chang AC, Cohen SN: Decay of mRNA in Escherichia coli: investigation of the fate of specific segments of transcripts. Proc Natl Acad Sci U S A 1983, 80:653e657 7. Lanham SM, Godfrey DG: Isolation of salivarian trypanosomes from man and other mammals using DEAE-cellulose. Exp Parasitol 1970, 28:521e534 8. Coustou V, Guegan F, Plazolles N, Baltz T: Complete in vitro life cycle of Trypanosoma congolense: development of genetic tools. PLoS Negl Trop Dis 2010, 4:e618 9. Deborggraeve S, Coronado X, Solari A, Zulantay I, Apt W, Mertens P, Laurent T, Leclipteux T, Stessens T, Dujardin JC, Herdewijn P, Büscher P: T. cruzi OligoC-TesT: a simplified and standardized polymerase chain reaction format for the diagnosis of Chagas disease. PLoS Negl Trop Dis 2009, 3:e450 10. Vanaerschot M, Maes I, Ouakad M, Adaui V, Maes L, De Doncker S, Rijal S, Chappuis F, Dujardin JC, Decuypere S: Linking in vitro and in vivo survival of clinical Leishmania donovani strains. PLoS One 2010, 5:e12211 11. Ilboudo H, Berthier D, Camara M, Camara O, Kabore J, Leno M, Keletigui S, Chantal I, Jamonneau V, Belem AM, Cuny G, Bucheton B: APOL1 expression is induced by Trypanosoma brucei gambiense infection but is not associated with differential susceptibility to sleeping sickness. Infect Genet Evol 2012, 12:1519e1523 12. Magnus E, Vervoort T, Van Meirvenne N: A card-agglutination test with stained trypanosomes (C.A.T.T.) for the serological diagnosis of
The Journal of Molecular Diagnostics
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13.
14.
15. 16. 17. 18.
19.
20.
21.
22.
T.b. gambiense trypanosomiasis. Ann Soc Belg Méd Trop 1978, 58: 169e176 Camara M, Camara O, Ilboudo H, Sakande H, Kaboré J, N’Dri L, Jamonneau V, Bucheton B: Sleeping sickness diagnosis: use of buffy coats improves the sensitivity of the mini anion exchange centrifugation test. Trop Med Int Health 2010, 15:796e799 Jamonneau V, Bucheton B, Kaboré J, Ilboudo H, Camara O, Courtin F, Solano P, Kaba D, Kambire R, Lingue K, Camara M, Baelmans R, Lejon V, Büscher P: Revisiting the immune trypanolysis test to optimise epidemiological surveillance and control of sleeping sickness in west Africa. PLoS Negl Trop Dis 2010, 4:e917 Nilsen TW: Trans-splicing of nematode premessenger RNA. Annu Rev Microbiol 1993, 47:413e440 Deborggraeve S, Büscher P: Molecular diagnostics for sleeping sickness: where’s the benefit for the patient? Lancet Infect Dis 2010, 10:433e439 Deborggraeve S, Büscher P: Recent progress in molecular diagnosis of sleeping sickness. Expert Rev Mol Diagn 2012, 12:719e730 Mitashi P, Hasker E, Ngoyi DM, Pyana PP, Lejon V, Van der Veken W, Lutumba P, Büscher P, Boelaert M, Deborggraeve S: Diagnostic accuracy of Loopamp Trypanosoma brucei detection kit for diagnosis of human African trypanosomiasis in clinical samples. PLoS Negl Trop Dis 2013, 7:e2504 Mugasa CM, Schoone GJ, Ekangu RA, Lubega GW, Kager PA, Schallig HD: Detection of Trypanosoma brucei parasites in blood samples using real-time nucleic acid sequence-based amplification. Diagn Microbiol Infect Dis 2008, 61:440e445 Deborggraeve S, Koffi M, Jamonneau V, Bonsu FA, Queyson R, Simarro PP, Herdewijn P, Büscher P: Molecular analysis of archived blood slides reveals an atypical human Trypanosoma infection. Diagn Microbiol Infect Dis 2008, 61:428e433 Kolev NG, Franklin JB, Carmi S, Shi H, Michaeli S, Tschudi C: The transcriptome of the human pathogen Trypanosoma brucei at singlenucleotide resolution. PLoS Pathog 2010, 6:e1001090 Lopes AH, Souto-Padrón T, Dias FA, Gomes MT, Rodrigues GC, Zimmermann LT, Alves e Silva TL, Vermelho AB: Trypanosomatids: odd organisms, devastating diseases. Open Parasitol J 2010, 4:30e59
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Supplemental Figure S1 Alignment of the SL-RNA sequences from T.b. gambiense (GenBank; http://www.ncbi.nlm.nih.gov/genbank; accession number U68270.1), T.b. rhodesiense (K02946.1), T.b. brucei (AF012853.1), T. evansi (EU180706.1), T. equiperdum (EU180668), T. congolense (U68268.1), T. vivax (U682699.1), T. cruzi (AF506368.1), T. rangeli (AJ012419), and L. donovani (AF531425.2), and annealing positions of the primers during reverse transcription (primer cSL) and PCR amplification (primers cSL and SL-F).
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Diagnosis of Trypanosomatid Infections: Targeting the Spliced Leader RNA Pablo González-Andrade,* Mamady Camara,y Hamidou Ilboudo,z Bruno Bucheton,x Vincent Jamonneau,zx and Stijn Deborggraeve* From the Biomedical Sciences Department,* Institute of Tropical Medicine, Antwerp, Belgium; the National Sleeping Sickness Control Program,y Ministry of Health and Public Hygiene, Conakry, Guinea; the Centre International de Recherche Développement sur l’Elevage en zones Subhumides (CIRDES),z BoboDioulasso, Burkina Faso; and the Institut de Recherche pour le Développement,x Unité Mixte de Recherche IRD-CIRAD 177, Montpellier, France Accepted for publication February 26, 2014.
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Address correspondence to Stijn Deborggraeve, Institute of Tropical Medicine, Nationalestraat 155, 2000 Antwerpen, Belgium. E-mail: [email protected].
Trypanosomatids transcribe their genes in large polycistronic clusters that are further processed into mature mRNA molecules by trans-splicing. During this maturation process, a conserved spliced leader RNA (SL-RNA) sequence of 39 bp is physically linked to the 50 end of the pre-mRNA molecules. Trypanosomatid infections cause a series of devastating diseases in man (sleeping sickness, leishmaniasis, Chagas disease) and animals (nagana, surra, dourine). Here, we investigated the SL-RNA molecule for its diagnostic potential using reverse transcription followed by real-time PCR. As a model, we used Trypanosoma brucei gambiense, which causes sleeping sickness in west and central Africa. We showed that the copy number of the SL-RNA molecule in one single parasitic cell is at least 8600. We observed a lower detection limit of the SL-RNA assay in spiked blood samples of 100 trypanosomes per milliliter of blood. We also proved that we can detect the trypanosome’s SL-RNA in the blood of sleeping sickness patients with a sensitivity of 92% (95% CI, 78%e97%) and a specificity of 96% (95% CI, 86%e99%). The SL-RNA is thus an attractive new molecular target for next-generation diagnostics in diseases caused by trypanosomatids. (J Mol Diagn 2014, -: 1e5; http://dx.doi.org/10.1016/j.jmoldx.2014.02.006)
Trypanosomatids are early eukaryotic protozoans that possess unique molecular features. Genes typically do not harbor introns but are arranged in large polygenic clusters that are transcribed polycistronically.1 Polycistronic premRNA is further processed into functional monocistronic mRNA molecules by trans-splicing and polyadenylation. During this RNA maturation process, a conserved spliced leader RNA (SL-RNA) molecule is donated to the 50 end of each individual mRNA.2 The SL-RNA sequence of the trypanosomatids can be 39 to 41 nucleotides long and carries methyl groups on the first four ribose moieties and on the first and fourth bases following the conserved 7-methylguanosine (m7G).2,3 Among the trypanosomatids are three major human pathogens: Trypanosoma brucei, T. cruzi, and Leishmania spp. Their trans-splicing mechanisms have been extensively investigated for understanding eukaryotic cell biology, but the SL-RNA has never been explored as a diagnostic target, although this molecule has several attractive diagnostic
features. It is a short, noncoding RNA sequence that is Q5 conserved, but unique, for each species and is present in each mRNA molecule in the cell. Importantly, mRNA is considered to be the best surrogate marker for viable organisms,4,5 because it is rapidly degraded after death of the organism with a typical half-life of 3 minutes.6 Here, we describe the first study that explores the trypanosomatid SL-RNA molecule for its diagnostic potential. As a model, we used sleeping sickness, a devastating disease that is endemic in sub-Saharan Africa and caused by the T. brucei subspecies gambiense and rhodesiense. The trypanosomes are transmitted to humans by tsetse flies and invade the brain, which in most cases is fatal if not treated in time. T. brucei gambiense is associated with the chronic form of sleeping sickness in west and central Africa, whereas Supported by the Research Foundation Flanders (FWO) (grant number 1501413N). Disclosures: None declared. Q2
Copyright ª 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jmoldx.2014.02.006
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González-Andrade et al T. brucei rhodesiense causes acute sleeping sickness in east Africa. Here, we show that the T. brucei SL-RNA is an excellent target molecule for the detection of the trypanosomes in clinical specimens.
Materials and Methods Oligonucleotides Oligonucleotides were synthesized and purified using highperformance liquid chromatography by Integrated DNA Technologies (IDT, Leuven, Belgium). Nucleic acid sequences were as follows: SL-RNA molecule, 50 -AACUAACGCUAUUAUUAGAACAGUUUCUGUACUAUAUUG30 ; SL-RNAespecific primer for reverse transcription and real-time PCR (cSL), 50 -CAATATAGTACAGAAACTG-30 ; forward primer for real-time PCR (SL-F), 50 -AACTAACGCTATTATTAGAA-30 .
Parasites
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T. brucei gambiense (LiTat 1.3), T. brucei rhodesiense (AnTat 25.1), T. brucei brucei (Lister 427), T. evansi (Kazakstan), T. equiperdum (OVI), and T. vivax (ILRAD700) were propagated in OF1 mice or HsdCpb:WU rats (Harlan Laboratories, Horst, The Netherlands). Trypanosomes were separated from the blood using diethylaminoethyl cellulose chromatography7 followed by repeated centrifugation and sediment washes with PBS glucose [38 mmol/L Na2HPO4$H2O, 2 mmol/L NaHPO4, 80 mmol/L glucose, 29 mmol/ L NaCl (pH 8.0)]. T. congolense (Savannah type, KTT/ MSORO/M7), T. rangeli (ScTb), and Leishmania donovani (BPK282/0cl4) were propagated in vitro as described previously.8e10 In vitroecultured parasites were harvested by repeated centrifugation and washing in PBS glucose. Parasite concentrations in PBS glucose were measured using Uriglass counting chambers (Menarini, Vienna, Austria).
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result in the card agglutination test for trypanosomiasis (CATT) for antibody detection in blood,12 and trypanosomes were detected in their blood by the mini anion exchange centrifugation technique.13 Endemic controls did not show antibodies against T.b. gambiense in the CATT and in the immune trypanolysis assay with LiTat 1.3, LiTat 1.5, and LiTat 1.6 T.b. gambiense parasites.14 For RNA isolation, 2 mL of blood was taken in PAXgene Blood RNA tubes (PreAnalytiX, Hombrechtikon, Switzerland) from patients and controls, and transported at 20 C to Centre International de Recherche-Développement sur l’Elevage en Zone Subhumide (Bobo-Dioulasso, Burkina-Faso). On arrival, samples were immediately stored at 80 C until the RNA was isolated.
RNA Isolation and cDNA Preparation Total RNA from laboratory-cultured parasites and spiked human blood samples were extracted using TRIzol (Ambion, Austin, TX). Briefly, 10 volumes of TRIzol reagent and two volumes of chloroform were added to the sample, followed by centrifugation at 12,000 g for 15 minutes. The RNA in the aqueous phase was precipitated in isopropanol, washed in 75% ethanol, and then dissolved in 50 mL of diethylpyrocarbonate-treated water. Total RNA from the blood of pa- Q8 tients and controls was extracted with the PAXgene Blood RNA kit (PreAnalytiX), and RNA was eluted in 80 mL of diethylpyrocarbonate-treated water. Copy DNA (cDNA) of the SL-RNA was prepared using the SL-RNAespecific cSL primer and SuperScript II Reverse Transcriptase (Invitrogen/ Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions.
Real-Time PCR SL-RNA cDNA levels were measured by real-time PCR in the LightCycler 480 PCR system (Roche Applied Science, Penzberg, Germany). Two and a half microliters of cDNA were mixed with 0.25 mmol/L cSL and SL-F primers in a 25-mL reaction containing 1 SensiMix SYBR green NoRox (Bioline, London, UK). Thermal cycling conditions were 10 minutes incubation at 95 C, followed by 40 cycles of 94 C for 15 seconds, 50 C for 15 seconds, and 60 C for 15 seconds with data acquisition. Post-amplification melting curves were recorded from 45 C to 95 C, with increments of 0.1 C per second. Genomic DNA contamination in the RNA samples was verified by subjecting the RNA to realtime PCR without cDNA preparation.
Thirty-six blood samples from sleeping sickness patients and 49 blood samples from endemic controls were analyzed with the SL-RNA assay. Patients and controls were recruited in the main human African trypanosomiasis foci along the Guinean coast (Forecariah, Dubreka, and Boffa) between 2007 and 2009 as reported by Ilboudo et al11 in 2012. This study is part of a larger project aiming to improve human African trypanosomiasis diagnosis for which approval was obtained from the World Health Organization (Research Ethics Review Committee) and from the ethical committee of the Institut de Recherche pour le Développement (Comité Consultatif de Déontologie et d’Ethique). All participants were informed of the objectives of the project in their own language and signed a written informed consent form. For participants <18 years of age, informed consent was obtained from their parents. Sleeping sickness patients had a positive
Mean threshold cycle (Ct) values of duplicate samples in realtime PCR were calculated with one-sigma SD (s). Sensitivities and specificities of the SL-RNA assay in sleeping sickness patients and endemic controls were calculated at a 95% CI using Wilson’s score. Sensitivity is defined as the
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Figure 1 Melting peak of reverse-transcribed and PCR-amplified SL-RNA in total RNA from 2 million T.b. gambiense parasites. Non-reversetranscribed (NRT) total RNA was used as a negative control.
proportion of confirmed sleeping sickness patients who are positive by the index test, and specificity as the proportion of endemic controls who are negative by the index test.
Results Proof-of-Concept The SL-RNA sequences of T.b. gambiense and T.b. rhodesiense were aligned in silico, together with those from the closely related T.b. brucei, T. equiperdum, T. evansi, T. congolense, T. vivax, T. rangeli, T. cruzi, and L. donovani Q9 using Clustal Omega (Supplemental Figure S1). The alignment shows a Trypanozoon-specific central region from bp 11 to 20 and a hyperconserved region from bp 21 to 39. The Trypanozoon subgenus consists of the human infective T. brucei gambiense and T. brucei rhodesiense, as well as the animal pathogens T. brucei brucei, T. evansi, and T. equiperdum. The cSL primer used during reverse transcription of the SL-RNA is complementary to the 19-bp hyperconserved sequence. The additional primer that is used during PCR is situated adjacent and has its 30 region in the Trypanozoon-specific region. To deliver the proof of concept, we extracted total RNA from 2 million T.b. gambiense parasites and detected the SL-RNA molecules by reverse transcription and real-time PCR. The melting peak Q10 of the amplified cDNA showed a melting temperature of ½F1 67 C (Figure 1), which corresponds to the in silicoepredicted melting temperature of the 39-bp T.b. gambiense SLRNA sequence (66.5 C). Non-reverse-transcribed total RNA did not show any amplified DNA during the real-time PCR. Using standards of chemically synthesized SL-RNA molecules and the equation
g RNA=ðlength 340Þ 6:023 1023 Znumber
ð1Þ
of RNA molecules;
we have estimated the number of SL-RNA molecules per parasite. We found that there are at least 8600 (s Z 1080) SL-RNA molecules in one single trypanosome.
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311 312 313 314 315 316 317 318 319 320 321 322 323 Figure 2 Amplification curves and melting peaks (inset) of the SL-RNA 324 in 10-fold serial dilutions of T.b. gambiense parasites in human blood. 325 Q15 Expressed as parasites per milliliter of blood. 326 327 Analytical Sensitivity 328 329 We determined the analytical sensitivity by applying the SL330 RNA assay to RNA extracts from naive human blood sam331 332 ples spiked with known numbers of T.b. gambiense parasites, 333 ranging from 1,000,000 to 10 parasites per milliliter (p/mL) 334 of blood in a 10-fold dilution series. All samples were pre335 pared in duplicate, and the analytical sensitivity of the assay 336 was defined as the lower dilution that showed a positive test 337 result in both duplicates. The analytical sensitivity of the SL338 RNA assay is 100 p/mL of blood (Figure 2). Mean Ct values ½F2 339 of the duplicate samples were 29.0 (s Z 0.80) for 1,000,000 340 p/mL, 33.35 (s Z 0.98) for 100,000 p/mL, 36.35 (s Z 0.80) 341 for 10,000 p/mL, and >40 for 1000 and 100 p/mL. Non342 spiked blood remained negative in the assay. 343 344 345 Analytical Specificity 346 347 To determine the analytical specificity of the SL-RNA 348 assay, we tested 10 pg of total RNA from different Trypanosoma species and from Leishmania (Figure 3). Our assay ½F3 349 350 only detects the parasites of the Trypanozoon subgenus T. 351 vivax; Leishmania showed a minor amplification signal, and 352 the SL-RNA of T. rangeli and T. congolense were not 353 amplified at all. These observations are fully consistent with 354 the in silico alignments of the SL-RNA sequences from 355 these organisms (Supplemental Figure S1). 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 Figure 3 Melting peaks of reverse-transcribed and PCR-amplified total 371 Q16 RNA (10 pg) from different trypanosomatids. 372 print & web 4C=FPO
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Patients and Controls Out of the 36 human African trypanosomiasis patients, 33 were positive in the SL-RNA assay, whereas 47 of 49 endemic controls remained negative. This corresponds with a test sensitivity of 92% (95% CI, 78%e97%) and a test specificity of 96% (86%e99%). No DNA contamination was observed in the analyzed RNA samples.
Discussion
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Technological advancements are continuously expanding our portfolio of diagnostic tests for infectious diseases. In parallel with the search for more accurate technologies to detect infectious agents, there is a need to provide the best target molecule for such a diagnostic test. Here, we show that the spliced leader RNA is an excellent target for accurate detection of trypanosomatids in blood samples. The proof of concept was delivered for sleeping sickness, but the idea can be exploited for Chagas disease and leishmaniasis, as well as for important animal diseases such as nagana and surra. Furthermore, nematodes share a similar transsplicing mechanism to process their mRNA,15 and a similar approach can thus be used to detect the SL-RNA of parasitic nematodes in man and animals. We reverse transcribed the 39-bp SL-RNA RNA of T.b. gambiense and analyzed the cDNA by real-time PCR in experimentally prepared samples and in blood samples from sleeping sickness patients. Despite the very short length, the assay was highly efficient in detecting trypanosomes and did not show any amplification artifacts such as primer dimers. Crucial steps during the optimization of the assay were efficient RNA extractions without genomic DNA contamination, the annealing temperature of the oligonucleotides during reverse transcription, and the annealing and elongation temperatures during real-time PCR. In blood samples spiked with known numbers of T.b. gambiense, we were able to detect the parasite’s SL-RNA in as few as 100 parasites per milliliter of blood. This is in the same range as DNA detection methods such as PCR and LAMP, which target multicopy DNA sequences of T. brucei,16e18 and a nucleic acid sequenceebased amplification assay, which targets the ribosomal RNA.19 However, the major advantage of our SL-RNA assay is that we target the parasite’s mRNA, which is considered to be a better marker for viable organisms than DNA or rRNA.4,5 Analytical specificity experiments showed that the assay did not detect RNA from genetically closely related Trypanosomatidae. In silico analysis of our primers did not indicate any potential crossreaction with other microorganisms that are endemic in sleeping sickness foci and cause diseases with similar clinical symptoms, such as malaria parasites for fever and meningococcal bacteria for neurological symptoms (data not shown). We also verified whether we could detect the T.b. gambiense SL-RNA in the blood of patients with sleeping sickness. Our SL-RNA assay showed a sensitivity of 92%
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and specificity of 96% in 36 patients and 49 endemic controls from Guinea. The two endemic controls that showed positive SL-RNA test results may be true sleeping sickness cases that were missed by the reference tests, although this is unlikely because the serological reference tests make use of three different immunodominant antigens, the LiTat 1.3, LiTat 1.5, and LiTat 1.6 variant surface glycoproteins. Because the SLRNA assay detects the Trypanozoon subgenus, we cannot exclude a transient infection with T.b. brucei, which is not detected by the serological reference tests used in this study. T.b. brucei is generally not infectious to man but has been reported in atypical human infections.20 Although not observed during the optimization and the analytical sensitivity and specificity experiments, we cannot exclude amplification artifacts in the SL-RNA assay. By including standards of chemically synthesized SL-RNA in the assay, we showed that the number of SL-RNA molecules in one single T.b. gambiense cell is at least 8600. This is consistent with what we theoretically expect based on previous studies. The T. brucei genome project reported 9000 protein coding genes,1 and a subsequent transcriptome study showed that 75% of these genes generate 1 to 10 mRNAs per cell.21 Trypanosomatids are responsible for several diseases in man, animals, and even plants.22 Sleeping sickness, leishmaniasis, and Chagas disease have the worst reputation because they cause >100,000 deaths each year. Accurate diagnosis of these diseases is challenging because of the extremely low parasite load in the blood of patients. Nucleic acidebased tests are often considered to be the future reference methods because they generally show high sensitivity and specificity.16 In this study, we have shown for the first time the great potential of the SL-RNA as a marker for trypanosomatids in the blood of patients. This is an important new step in expanding our biomarker portfolio for diagnosing diseases caused by trypanosomatids, as well as by other pathogens sharing a similar trans-splicing mechanism such as nematodes.
Acknowledgments We thank Dr. Büscher for the support throughout the project and Q12 critical reading of the manuscript; Prof. Jan Van den Abbeele, Dr. Vincent Delespaux, and Eliane Tihon for providing the T. congolense strain; and Prof. Jean-Claude Dujardin and Dr. Manu Van Aerschot for providing the L. donovani strain.
Supplemental Data Supplemental material for this article can be found at http://dx.doi.org/10.1016/j.jmoldx.2014.02.006.
References 1. Berriman M, Ghedin E, Hertz-Fowler C, Blandin G, Renauld H, Bartholomeu DC, et al: The genome of the African trypanosome Trypanosoma brucei. Science 2005, 309:416e422
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RNA Diagnostics for Trypanosomatids 2. Perry KL, Watkins KP, Agabian N: Trypanosome mRNAs have unusual “cap 4” structures acquired by addition of a spliced leader. Proc Natl Acad Sci U S A 1987, 84:8190e8194 3. Bangs JD, Crain PF, Hashizume T, McCloskey JA, Boothroyd JC: Mass spectrometry of mRNA cap 4 from trypanosomatids reveals two novel nucleosides. J Biol Chem 1992, 267:9805e9815 4. Keer JT, Birch L: Molecular methods for the assessment of bacterial viability. J Microbiol Methods 2003, 53:175e183 5. Cangelosi GA, Weigel KM, Lefthand-Begay C, Meschke JS: Molecular detection of viable bacterial pathogens in water by ratiometric prerRNA analysis. Appl Environ Microbiol 2010, 76:960e962 6. von Gabain A, Belasco JG, Schottel JL, Chang AC, Cohen SN: Decay of mRNA in Escherichia coli: investigation of the fate of specific segments of transcripts. Proc Natl Acad Sci U S A 1983, 80:653e657 7. Lanham SM, Godfrey DG: Isolation of salivarian trypanosomes from man and other mammals using DEAE-cellulose. Exp Parasitol 1970, 28:521e534 8. Coustou V, Guegan F, Plazolles N, Baltz T: Complete in vitro life cycle of Trypanosoma congolense: development of genetic tools. PLoS Negl Trop Dis 2010, 4:e618 9. Deborggraeve S, Coronado X, Solari A, Zulantay I, Apt W, Mertens P, Laurent T, Leclipteux T, Stessens T, Dujardin JC, Herdewijn P, Büscher P: T. cruzi OligoC-TesT: a simplified and standardized polymerase chain reaction format for the diagnosis of Chagas disease. PLoS Negl Trop Dis 2009, 3:e450 10. Vanaerschot M, Maes I, Ouakad M, Adaui V, Maes L, De Doncker S, Rijal S, Chappuis F, Dujardin JC, Decuypere S: Linking in vitro and in vivo survival of clinical Leishmania donovani strains. PLoS One 2010, 5:e12211 11. Ilboudo H, Berthier D, Camara M, Camara O, Kabore J, Leno M, Keletigui S, Chantal I, Jamonneau V, Belem AM, Cuny G, Bucheton B: APOL1 expression is induced by Trypanosoma brucei gambiense infection but is not associated with differential susceptibility to sleeping sickness. Infect Genet Evol 2012, 12:1519e1523 12. Magnus E, Vervoort T, Van Meirvenne N: A card-agglutination test with stained trypanosomes (C.A.T.T.) for the serological diagnosis of
The Journal of Molecular Diagnostics
-
13.
14.
15. 16. 17. 18.
19.
20.
21.
22.
T.b. gambiense trypanosomiasis. Ann Soc Belg Méd Trop 1978, 58: 169e176 Camara M, Camara O, Ilboudo H, Sakande H, Kaboré J, N’Dri L, Jamonneau V, Bucheton B: Sleeping sickness diagnosis: use of buffy coats improves the sensitivity of the mini anion exchange centrifugation test. Trop Med Int Health 2010, 15:796e799 Jamonneau V, Bucheton B, Kaboré J, Ilboudo H, Camara O, Courtin F, Solano P, Kaba D, Kambire R, Lingue K, Camara M, Baelmans R, Lejon V, Büscher P: Revisiting the immune trypanolysis test to optimise epidemiological surveillance and control of sleeping sickness in west Africa. PLoS Negl Trop Dis 2010, 4:e917 Nilsen TW: Trans-splicing of nematode premessenger RNA. Annu Rev Microbiol 1993, 47:413e440 Deborggraeve S, Büscher P: Molecular diagnostics for sleeping sickness: where’s the benefit for the patient? Lancet Infect Dis 2010, 10:433e439 Deborggraeve S, Büscher P: Recent progress in molecular diagnosis of sleeping sickness. Expert Rev Mol Diagn 2012, 12:719e730 Mitashi P, Hasker E, Ngoyi DM, Pyana PP, Lejon V, Van der Veken W, Lutumba P, Büscher P, Boelaert M, Deborggraeve S: Diagnostic accuracy of Loopamp Trypanosoma brucei detection kit for diagnosis of human African trypanosomiasis in clinical samples. PLoS Negl Trop Dis 2013, 7:e2504 Mugasa CM, Schoone GJ, Ekangu RA, Lubega GW, Kager PA, Schallig HD: Detection of Trypanosoma brucei parasites in blood samples using real-time nucleic acid sequence-based amplification. Diagn Microbiol Infect Dis 2008, 61:440e445 Deborggraeve S, Koffi M, Jamonneau V, Bonsu FA, Queyson R, Simarro PP, Herdewijn P, Büscher P: Molecular analysis of archived blood slides reveals an atypical human Trypanosoma infection. Diagn Microbiol Infect Dis 2008, 61:428e433 Kolev NG, Franklin JB, Carmi S, Shi H, Michaeli S, Tschudi C: The transcriptome of the human pathogen Trypanosoma brucei at singlenucleotide resolution. PLoS Pathog 2010, 6:e1001090 Lopes AH, Souto-Padrón T, Dias FA, Gomes MT, Rodrigues GC, Zimmermann LT, Alves e Silva TL, Vermelho AB: Trypanosomatids: odd organisms, devastating diseases. Open Parasitol J 2010, 4:30e59
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Supplemental Figure S1 Alignment of the SL-RNA sequences from T.b. gambiense (GenBank; http://www.ncbi.nlm.nih.gov/genbank; accession number U68270.1), T.b. rhodesiense (K02946.1), T.b. brucei (AF012853.1), T. evansi (EU180706.1), T. equiperdum (EU180668), T. congolense (U68268.1), T. vivax (U682699.1), T. cruzi (AF506368.1), T. rangeli (AJ012419), and L. donovani (AF531425.2), and annealing positions of the primers during reverse transcription (primer cSL) and PCR amplification (primers cSL and SL-F).
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