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• W2024337911 abstract "CME Accreditation Statement: This activity (“JMD 2014 CME Program in Molecular Diagnostics”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity (“JMD 2014 CME Program in Molecular Diagnostics”) for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. CME Accreditation Statement: This activity (“JMD 2014 CME Program in Molecular Diagnostics”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity (“JMD 2014 CME Program in Molecular Diagnostics”) for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Protozoan parasites of the family Trypanosomatidae are the causative agent of devastating diseases in humans and livestock that range from self-curing dermal lesions to fatality if not treated.1Srividya G. Kulshrestha A. Singh R. Salotra P. Diagnosis of visceral leishmaniasis: developments over the last decade.Parasitol Res. 2012; 110: 1065-1078Crossref PubMed Scopus (105) Google Scholar, 2Radwanska M. Emerging trends in the diagnosis of human African Trypanosomiasis.Parasitology. 2010; 137: 1977-1986Crossref PubMed Scopus (11) Google Scholar, 3Nunes M.C. Dones W. Morillo C.A. Encina J.J. Ribeiro A.L. Council on Chagas Disease of the Interamerican Society of CardiologyChagas disease: an overview of clinical and epidemiological aspects.J Am Coll Cardiol. 2013; 62: 767-776Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, 4Kennedy P.G. Clinical features, diagnosis, and treatment of human African trypanosomiasis (sleeping sickness).Lancet Neurol. 2013; 12: 186-194Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar Among the two genera, Leishmania and Trypanosoma, there are a multitude of species, each one resulting in a different disease manifestation. Many of the disease symptoms are common to other infections such as fever and malaise, making clinical diagnosis difficult. Correct diagnosis is essential because each agent responds to a different treatment. Thus, effective diagnosis is critical to treatment and control of these diseases. Diagnosis of infectious diseases has advanced significantly with the development of molecular diagnostics. Amplification of nucleic acid targets unique to the pathogen has made it possible to achieve high sensitivity and specificity.5Steensels D. Vankeerberghen A. De Beenhouwer H. Towards multitarget testing in molecular microbiology.Int J Microbiol. 2013; 2013: 121057Crossref PubMed Scopus (10) Google Scholar However, application of molecular detection of protozoan parasite infections has been challenging. Since the completion and publication of the genome sequences of three major pathogenic species of Typanosomatidae,6Ivens A.C. Peacock C.S. Worthey E.A. Murphy L. Aggarwal G. Berriman M. et al.The genome of the kinetoplastid parasite, Leishmania major.Science. 2005; 309: 436-442Crossref PubMed Scopus (1140) Google Scholar, 7Berriman M. Ghedin E. Hertz-Fowler C. Blandin G. Renauld H. Bartholomeu D.C. et al.The genome of the African trypanosome Trypanosoma brucei.Science. 2005; 309: 416-422Crossref PubMed Scopus (1327) Google Scholar, 8El-Sayed N.M. Myler P.J. Bartholomeu D.C. Nilsson D. Aggarwal G. Tran A.N. et al.The genome sequence of Trypanosoma cruzi, etiologic agent of Chagas disease.Science. 2005; 309: 409-415Crossref PubMed Scopus (1148) Google Scholar there has been great hope that the sequence information could be turned into tools for diagnosis. In fact, a great deal of work has been published to report molecular detection of parasites in blood or tissue biopsies1Srividya G. Kulshrestha A. Singh R. Salotra P. Diagnosis of visceral leishmaniasis: developments over the last decade.Parasitol Res. 2012; 110: 1065-1078Crossref PubMed Scopus (105) Google Scholar, 2Radwanska M. Emerging trends in the diagnosis of human African Trypanosomiasis.Parasitology. 2010; 137: 1977-1986Crossref PubMed Scopus (11) Google Scholar; yet few assays have achieved wide clinical use. One of the barriers is the low number of parasites that circulate in the blood, especially in the chronic phases when diagnosis by other means is also difficult. This low parasitemia places extreme demands on developing molecular methods that are highly sensitive. In an article in this issue of The Journal of Molecular Diagnostics, González-Andrade et al9González-Andrade P. Camara M. Ilboudo H. Bucheton B. Jamonneau V. Deborggraeve S. Diagnosis of trypanosomatid infections: targeting the spliced leader RNA.J Mol Diagn. 2014; 14: 400-404Abstract Full Text Full Text PDF Scopus (28) Google Scholar present a promising approach by targeting a nucleic acid element unique to trypanosomatids that is expressed with a high copy number in each cell. This group of protozoans developed mechanisms of gene expression that set them apart from all other eukaryotes. From a compact genome with open reading frames almost entirely uninterrupted by exons, the cells transcribe long polycistronic RNA molecules. In a subsequent splicing event, separate open reading frames are cut apart, and a 39-base spliced leader sequence transcribed from another location on the genome is ligated onto the 5′ end. Thus, every mature mRNA molecule carries an identical sequence behind the cap structure. González-Andrade et al9González-Andrade P. Camara M. Ilboudo H. Bucheton B. Jamonneau V. Deborggraeve S. Diagnosis of trypanosomatid infections: targeting the spliced leader RNA.J Mol Diagn. 2014; 14: 400-404Abstract Full Text Full Text PDF Scopus (28) Google Scholar estimated the number of copies in a cell to be at least 8600, suggesting the potential for high sensitivity of detection of this multicopy target. The unique sequence of the spliced leader makes its reverse-transcribed PCR amplification specific to the pathogen only limited by the probability of a 39-base sequence occurring at random in the human genome. González-Andrade et al9González-Andrade P. Camara M. Ilboudo H. Bucheton B. Jamonneau V. Deborggraeve S. Diagnosis of trypanosomatid infections: targeting the spliced leader RNA.J Mol Diagn. 2014; 14: 400-404Abstract Full Text Full Text PDF Scopus (28) Google Scholar reported analytical sensitivity down to 100 cells/mL and good sensitivity and specificity with clinical samples. Further, the conservation of the spliced leader sequence within genera suggests that assays can be designed that could target multiple species. As an approach to high-sensitivity molecular detection of trypanosomatids, the spliced leader target should be seen in the context of other targets that have been evaluated. Trypanosomatids possess another unusual nucleic acid target in their mitochondrion. As part of the RNA editing function in the mitochondrion, there is a 750-bp circular DNA that encodes guide RNAs. The thousands of copies of this minicircle are compacted with the larger mitochondrial genome in a structure called the kinetoplast. Within the minicircles there are two conserved sequences that have been exploited for the design of primers that amplify a product of approximately 120 bp in Leishmania10Smyth A.J. Ghosh A. Hassan M.Q. Basu D. De Bruijn M.H. Adhya S. Mallik K.K. Barker D.C. Rapid and sensitive detection of Leishmania kinetoplast DNA from spleen and blood samples of kala-azar patients.Parasitology. 1992; 105: 183-192Crossref PubMed Scopus (170) Google Scholar and another primer pair has been optimized for L. donovani-specific detection.11Singh N. Curran M.D. Rastogil A.K. Middleton D. Sundar S. Diagnostic PCR with Leishmania donovani specificity using sequences from the variable region of kinetoplast minicircle DNA.Trop Med Int Health. 1999; 4: 448-453Crossref PubMed Scopus (21) Google Scholar The assay was advanced to real-time PCR with the addition of SYBR Green dye12Nicolas L. Prina E. Lang T. Milon G. Real-time PCR for detection and quantitation of leishmania in mouse tissues.J Clin Microbiol. 2002; 40: 1666-1669Crossref PubMed Scopus (172) Google Scholar and achieved more-enhanced sensitivity for L. infantum parasites with the addition of a TaqMan probe.13Mary C. Faraut F. Lascombe L. Dumon H. Quantification of Leishmania infantum DNA by a real-time PCR assay with high sensitivity.J Clin Microbiol. 2004; 42: 5249-5255Crossref PubMed Scopus (314) Google Scholar The conserved region primers and a probe designed with degenerate bases have been used to achieve highly sensitive and multispecies detection.14Selvapandiyan A. Dey R. Nylen S. Duncan R. Sacks D. Nakhasi H.L. Intracellular replication-deficient Leishmania donovani induces long lasting protective immunity against visceral leishmaniasis.J Immunol. 2009; 183: 1813-1820Crossref PubMed Scopus (122) Google Scholar, 15Selvapandiyan A. Duncan R. Mendez J. Kumar R. Salotra P. Cardo L.J. Nakhasi H.L. A Leishmania minicircle DNA footprint assay for sensitive detection and rapid speciation of clinical isolates.Transfusion. 2008; 48: 1787-1798Crossref PubMed Scopus (26) Google Scholar With the use of this multispecies minicircle PCR reaction, human blood samples spiked with Leishmania were detected at the limit of detection González-Andrade et al9González-Andrade P. Camara M. Ilboudo H. Bucheton B. Jamonneau V. Deborggraeve S. Diagnosis of trypanosomatid infections: targeting the spliced leader RNA.J Mol Diagn. 2014; 14: 400-404Abstract Full Text Full Text PDF Scopus (28) Google Scholar reported for T. brucei of 100 cells/mL.16Grigorenko E. Fisher C. Patel S. Chancey C. Rios M. Nakhasi H.L. Duncan R.C. Multiplex screening for blood-borne viral, bacterial, and protozoan parasites using an OpenArray platform.J Mol Diagn. 2014; 16: 136-144Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar One advantage of the minicircle target is the durability of the 750-bp closed circular DNA and the high copy number per cell that contributes to recovery during DNA extraction. A disadvantage of the minicircle as a PCR target is the extensive variability of the sequences among these DNAs in a parasite, reducing the effective copy number. Outside the highly conserved regions chosen for the primers, there is little conserved sequence, which limits enhancements that could be made to the detection. An advantage of the spliced leader target is the absolute conservation of the sequence within a species so that all 8600 targets can be effectively amplified. Another genomic target that takes advantage of high copy number for sensitivity has been exploited for detection of another member of this parasite family, T. cruzi. This pathogen that causes American trypanosomiasis called Chagas disease, has a repetitive satellite DNA sequence present in the genome in so many copies that it represents 9% of the nuclear DNA. Early diagnostic application achieved highly sensitive detection with conventional PCR.17Moser D.R. Kirchhoff L.V. Donelson J.E. Detection of Trypanosoma cruzi by DNA amplification using the polymerase chain reaction.J Clin Microbiol. 1989; 27: 1477-1482Crossref PubMed Google Scholar Enhanced by the addition of a TaqMan-style probe, this target has been used for detection in real-time PCR reactions from spiked human blood16Grigorenko E. Fisher C. Patel S. Chancey C. Rios M. Nakhasi H.L. Duncan R.C. Multiplex screening for blood-borne viral, bacterial, and protozoan parasites using an OpenArray platform.J Mol Diagn. 2014; 16: 136-144Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar and patients with Chagas disease.18Piron M. Fisa R. Casamitjana N. Lopez-Chejade P. Puig L. Verges M. Gascon J. Gomez i Prat J. Portus M. Sauleda S. Development of a real-time PCR assay for Trypanosoma cruzi detection in blood samples.Acta Trop. 2007; 103: 195-200Crossref PubMed Scopus (281) Google Scholar Given the large number of copies of this target and the reliability that has been found for the PCR assay, it would be surprising if the spliced leader could surpass it as a molecular target. The biggest challenge of all of the PCR-based assays, including that described by González-Andrade et al,9González-Andrade P. Camara M. Ilboudo H. Bucheton B. Jamonneau V. Deborggraeve S. Diagnosis of trypanosomatid infections: targeting the spliced leader RNA.J Mol Diagn. 2014; 14: 400-404Abstract Full Text Full Text PDF Scopus (28) Google Scholar is optimizing a sample preparation method. These protozoan parasites can maintain an infection with fewer than one cell per milliliter of blood; suggesting that a means of concentrating the pathogens from a larger volume will be required. Separating the infected leukocytes from a larger volume of blood is one reported concentrating step.13Mary C. Faraut F. Lascombe L. Dumon H. Quantification of Leishmania infantum DNA by a real-time PCR assay with high sensitivity.J Clin Microbiol. 2004; 42: 5249-5255Crossref PubMed Scopus (314) Google Scholar The highly multicopy targets, such as the spliced leader sequence, once released into a lyzed blood sample could be detected in a small fraction of the lysate. This approach was found to detect fewer than one T. cruzi parasite in 20 mL of blood,19Sabino E.C. Lee T.H. Montalvo L. Nguyen M.L. Leiby D.A. Carrick D.M. Otani M.M. Vinelli E. Wright D. Stramer S.L. Busch M. NHLBI Retrovirus Epidemiology Donor Study-II (REDS-II) International ProgramAntibody levels correlate with detection of Trypanosoma cruzi DNA by sensitive polymerase chain reaction assays in seropositive blood donors and possible resolution of infection over time.Transfusion. 2013; 53: 1257-1265Crossref PubMed Scopus (36) Google Scholar representing possibly the highest sensitivity achieved for these parasites. Rapid and effective diagnosis of the many diseases caused by parasites of the Trypanosomatidae family would lead to better management of these diseases and relief of suffering. The active and innovative research exemplified by the work of González-Andrade et al,9González-Andrade P. Camara M. Ilboudo H. Bucheton B. Jamonneau V. Deborggraeve S. Diagnosis of trypanosomatid infections: targeting the spliced leader RNA.J Mol Diagn. 2014; 14: 400-404Abstract Full Text Full Text PDF Scopus (28) Google Scholar as well as others cited in this commentary, is the first step toward development of the diagnostic tools that are needed. Diagnosis of Trypanosomatid Infections: Targeting the Spliced Leader RNAThe Journal of Molecular DiagnosticsVol. 16Issue 4PreviewTrypanosomatids 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 5′ 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. 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