Semi-Quantitative, Duplexed qPCR Assay for the Detection of Leishmania spp. Using Bisulphite Conversion Technology

Abstract

Leishmaniasis is caused by the flagellated protozoan Leishmania, and is a neglected tropical disease (NTD), as defined by the World Health Organisation (WHO). Bisulphite conversion technology converts all genomic material to a simplified form during the lysis step of the nucleic acid extraction process, and increases the efficiency of multiplex quantitative polymerase chain reaction (qPCR) reactions. Through utilization of qPCR real-time probes, in conjunction with bisulphite conversion, a new duplex assay targeting the 18S rDNA gene region was designed to detect all Leishmania species. The assay was validated against previously extracted DNA, from seven quantitated DNA and cell standards for pan-Leishmania analytical sensitivity data, and 67 cutaneous clinical samples for cutaneous clinical sensitivity data. Specificity was evaluated by testing 76 negative clinical samples and 43 bacterial, viral, protozoan and fungal species. The assay was also trialed in a side-by-side experiment against a conventional PCR (cPCR), based on the Internal transcribed spacer region 1 (ITS1 region). Ninety-seven percent of specimens from patients that previously tested positive for Leishmania were positive for Leishmania spp. with the bisulphite conversion assay, and a limit of detection (LOD) of 10 copies per PCR was achieved, while the LOD of the ITS1 methodology was 10 cells/1000 genomic copies per PCR. This method of rapid, accurate and simple detection of Leishmania can lead to improved diagnosis, treatment and public health outcomes.

Laboratory diagnostics for human Leishmania infections: a polymerase chain reaction-focussed review of detection and identification methods

“…Moreover, detection of multiple species distinctly and concurrently requires multiplex PCR assays, and designing a single PCR cycling protocol to suit each primer pair can present assay constraints. The use of the novel bisulphite-conversion technique can mitigate such limitations [ 124 ].…”

“…The technique can give data on parasite load, which in turn provides information for prognosis and treatment, and is useful for epidemiological studies [ 186 , 187 ]. Bisulphite modification is a method for reducing the complexity of the genome prior to application of PCR that has been adapted to Leishmania detection in real-time PCR [ 124 , 188 ]. This assay achieved an analytical sensitivity of 10 genomic copies per real-time PCR reaction, and clinical sensitivity of 97.0% and specificity of 100.0%.…”

Comparison and clinical validation of qPCR assays targeting Leishmania 18S rDNA and HSP70 genes in patients with American Tegumentary Leishmaniasis

“…The targets commonly used for molecular diagnosis of Leishmania are directed to several regions of the ribosomal gene, such as the 18S rDNA, intergenic spacer sequences-ITS1 [16][17][18], conserved regions of the kDNA minicircles [19][20][21][22], and nuclear genes, such as the variable region of the hsp70 gene [23,24].…”

Evaluation of a kDNA-Based qPCR Assay for the Detection and Quantification of Old World Leishmania Species

“…Moreover, a pan- Leishmania assay can also be exploited in eco-epidemiological studies, including identification of vectors and reservoirs [ 24 ]. In recent years, only a few attempts to develop pan- Leishmania assays based on nucleic acids amplification (i.e., SL RNA, rDNA or kDNA minicircles) have been made [ 24 , 25 , 26 , 27 ]. We previously showed that qPCR-ML was able to detect Old World L. ( L .)…”