Evaluation of a Pan-Leishmania Spliced-Leader RNA Detection Method in Human Blood and Experimentally Infected Syrian Golden Hamsters

Eberhardt, E.; Kerkhof, Magali Van den; Bulté, Dimitri; Mabille, Dorien; Bockstal, Lieselotte Van; Monnerat, Séverine; Alves, Fabiana; Mbui, Jane; Delputte, Peter; Cos, Paul; Hendrickx, Sarah; Maes, Louis; Caljon, Guy

doi:10.1016/j.jmoldx.2017.12.003

Cited by 22 publications

(36 citation statements)

References 31 publications

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“…Additionally, the MP kDNA qPCR provided higher Ct values on the field-collected sand flies, which most likely relates to mismatches of the reverse primer with the L. aethiopica kDNA fragment (S1 Fig) [17]. Earlier observations of a lower sensitivity for L. tropica (genetically very similar to L. aethiopica ) and L. mexicana [23] corroborates the limitations of this MP kDNA assay that was originally developed by Mary et al for detection of L. donovani [17]. The SL-RNA qPCR provided equal Ct values for various Leishmania species, demonstrating its suitability as a pan- Leishmania assay [23].…”

Section: Discussionmentioning

confidence: 98%

“…Hence, it would be highly informative to be able to specifically detect viable parasites. In our study, we evaluated whether the recently developed SL-RNA qPCR assay by Eberhardt et al [23] enables Leishmania detection in sand flies and skin tissue from CL infected animals. The targeted 39 bp SL-RNA sequence is conserved amongst Leishmania species and fulfils an essential function in RNA trans-splicing and polyadenylation processes [27].…”

Section: Discussionmentioning

confidence: 99%

“…We comparatively assessed the performance of four molecular diagnostic assays on field and laboratory infected sand flies and hyrax tissue samples. The JW kDNA [14,24] and SL-RNA qPCRs [23] showed the best performance using the laboratory L. major -infected L. longipalpis and field-collected L. aethiopica -infected P. pedifer sand flies. Both assays identified the same positive and negative samples, indicating that they have a similar analytical sensitivity and specificity.…”

Section: Discussionmentioning

confidence: 99%

“…Nucleic acid extracts of the sand fly and hyrax samples were subjected to three different real-time PCR approaches targeting: (i) kDNA/18S DNA in a multiplex Taqman probe assay (further referred to as ‘MP kDNA qPCR’ and ‘MP 18S qPCR’ respectively), (ii) kDNA in a SYBR Green assay with an alternate set of primers (‘JW kDNA qPCR’), and (iii) SL-RNA in a SYBR Green assay (‘SL-RNA qPCR’). The primers for the JW kDNA qPCR were adopted from Nicolas et al and the assay was carried out as explained in our previous study [14,24], while the other assays were performed as described by Eberhardt et al [23]. All extracts were 1:10 diluted to prevent qPCR inhibition and were run on a Step One Plus real-time qPCR system (Applied Biosystems, Life Technologies, Belgium).…”

Section: Methodsmentioning

confidence: 99%

“…The assay was able to detect eight Old- and New-World Leishmania species with equal threshold cycle (Ct) values and was validated on tissue samples of L. infantum infected hamsters, promastigote spiked human blood and blood nucleic acid extracts from visceral leishmaniasis patients. It appeared that the limit of detection (LoD) of the SL-RNA qPCR was one log lower than the LoD of a Taqman multiplex assay targeting kDNA [23].…”

Section: Introductionmentioning

confidence: 99%

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Evaluation of a pan-LeishmaniaSL-RNA qPCR assay for parasite detection in laboratory-reared and field-collected sand flies and reservoir hosts

Pareyn

Hendrickx

Girma

et al. 2020

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13Background 14 In eco-epidemiological studies, Leishmania detection in vectors and reservoirs is frequently 15 accomplished by high-throughput and sensitive molecular methods that target minicircle kinetoplast 16 DNA (kDNA). A pan-Leishmania SYBR Green quantitative PCR (qPCR) assay which specifically detects the 17 conserved spliced-leader RNA (SL-RNA) sequence has recently been developed. This study comparatively 18 assessed the SL-RNA assay performance for the detection of Leishmania in field and laboratory infected 19 sand flies and in tissue samples from hyraxes as reservoir hosts. 20 Principal findings 21 The qPCRs targeting SL-RNA and kDNA performed equally well on infected sand fly samples, despite 22 preservation and extraction under presumed unfavorable conditions for downstream RNA detection. 23 Nucleic acid extraction by a crude extraction buffer combined with a precipitation step was highly 24 compatible with downstream SL-RNA and kDNA detection. Copy numbers of kDNA were found to be 25 identical in culture-derived parasites and promastigotes isolated from sand fly midguts. SL-RNA levels 26 were approximately 3-fold lower in sand fly promastigotes (ΔCt 1.7). The theoretical limit of detection 27 and quantification of the SL-RNA qPCR respectively reached down to 10 -3 and 10 parasite equivalents. SL-28 RNA detection in stored hyrax samples was less efficient with some false negative assay results, most 29 likely due to the long-term tissue storage in absence of RNA stabilizing reagents. 30 Conclusion 31 This study shows that a crude extraction method in combination with the SL-RNA qPCR assay is suitable 32 for the detection and quantification of Leishmania in sand flies. The assay provides complementary 33 information to the standard kDNA assays, since it is pan-Leishmania specific and detects viable parasites, 34 a prerequisite for identification of vectors and reservoirs. 3 35 Author summary 36 In order to identify vectors and reservoirs of Leishmania, a large number of sand fly and animal tissue 37 samples needs to be screened, because the infection prevalence is generally low. Hence, sensitive low-38 cost methods are required for nucleic acid isolation and Leishmania detection. Most approaches amplify 39 DNA targets, in particular minicircle kinetoplast DNA (kDNA). Recently, a qPCR was developed that 40 detects the spliced-leader RNA (SL-RNA) sequence, which is conserved among various Leishmania 41 species and allows detection of viable parasites. We show that the SL-RNA qPCR is highly compatible 42 with a low-cost, crude extraction approach and performs equally well on laboratory and field infected 43 sand fly samples as kDNA qPCR assays. The assay can detect 10 -3 parasite equivalent in sand flies and 44 enables Leishmania quantification down to 10 parasites. We found that the copy number of SL-RNA is 3-45 fold lower in sand fly derived promastigotes compared to cultured promastigotes. SL-RNA detection in 46 hyrax tissue samples appeared less efficient, which is presumably due to long-term sto...

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