SYBR green-based detection of Leishmania infantum DNA using peripheral blood samples

Ghasemian, Mehrdad; Gharavi, Mohammad Javad; Akhlaghi, Lame; Mohebali, Mehdi; Meamar, Ahmad Reza; Aryan, Ehsan; Oormazdi, Hormozd; Ghayour, Zahra

doi:10.1007/s12639-014-0452-4

Cited by 9 publications

(8 citation statements)

References 27 publications

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“…A different SYBR green based reaction demonstrated only 79% sensitivity targeting another kDNA minicircle sequence [ 21 ], rising to 94.7% sensitivity when targeting a genomic single copy polymerase gene by Taqman chemistry [ 33 ]. 95.7% sensitivity and 100% sensitivity are also reported with parasitologically confirmed patients in two recent SYBR green reaction based studies targeting kinetoplast DNA [ 34 , 35 ].…”

Section: Discussionmentioning

confidence: 73%

Real-time PCR in detection and quantitation of Leishmania donovani for the diagnosis of Visceral Leishmaniasis patients and the monitoring of their response to treatment

et al. 2017

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Sustained elimination of Visceral Leishmaniasis (VL) requires the reduction and control of parasite reservoirs to minimize the transmission of Leishmania donovani infection. A simple, reproducible and definitive diagnostic procedure is therefore indispensable for the early and accurate detection of parasites in VL, Relapsed VL (RVL) and Post Kala-azar Dermal Leishmaniasis (PKDL) patients, all of whom are potential reservoirs of Leishmania parasites. To overcome the limitations of current diagnostic approaches, a novel quantitative real-time polymerase chain reaction (qPCR) method based on Taqman chemistry was devised for the detection and quantification of L. donovani in blood and skin. The diagnostic efficacy was evaluated using archived peripheral blood buffy coat DNA from 40 VL, 40 PKDL, 10 RVL, 20 cured VL, and 40 cured PKDL along with 10 tuberculosis (TB) cases and 80 healthy endemic controls. Results were compared to those obtained using a Leishmania-specific nested PCR (Ln-PCR). The real time PCR assay was 100% (95% CI, 91.19–100%) sensitive in detecting parasite genomes in VL and RVL samples and 85.0% (95% CI, 70.16–94.29%) sensitive for PKDL samples. In contrast, the sensitivity of Ln-PCR was 77.5% (95% CI, 61.55–89.16%) for VL samples, 100% (95%CI, 69.15–100%) for RVL samples, and 52.5% (95% CI, 36.13–68.49%) for PKDL samples. There was significant discordance between the two methods with the overall sensitivity of the qPCR assay being considerably higher than Ln-PCR. None of the assay detected L. donovani DNA in buffy coats from cured VL cases, and reduced infectious burdens were demonstrated in cured PKDL cases who remained positive in 7.5% (3/40) and 2.5% (1/40) cases by real-time PCR and Ln-PCR, respectively. Both assays were 100% (95% CI, 95.98–100) specific with no positive signals in either endemic healthy control or TB samples. The real time PCR assay we developed offers a molecular tool for accurate detection of circulating L. donovani parasites in VL, PKDL and RVL patients, as well as being capable of assessing response to treatment. As such, this real time PCR assay represents an important contribution in efforts to eliminate VL.

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Section: Discussionmentioning

confidence: 73%

Real-time PCR in detection and quantitation of Leishmania donovani for the diagnosis of Visceral Leishmaniasis patients and the monitoring of their response to treatment

et al. 2017

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“…6e9 This assay also performs well for L. donovani and was applied for parasite detection in mice, 5,10 hamsters, 11 and human samples. 2,12 However, the heterogeneity of kDNA minicircles so far precluded the development of a pan-Leishmania real-time quantitative PCR (qPCR) assay. 2 Also, the number of kDNA minicircles differs between parasites, hindering uniform quantification.…”

mentioning

confidence: 99%

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

Eberhardt

Kerkhof

Bulté

et al. 2018

The Journal of Molecular Diagnostics

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Several methods have been developed for the detection of Leishmania, mostly targeting the minicircle kinetoplast DNA (kDNA). A new RNA real-time quantitative PCR (qPCR) assay was developed targeting the conserved and highly expressed spliced-leader (SL) mini-exon sequence. This study compared the limits of detection of various real-time PCR assays in hamsters infected with Leishmania infantum, in spiked human blood, and in clinical blood samples from visceral leishmaniasis patients. The SL-RNA assay showed an excellent analytical sensitivity in tissues (0.005 and 0.002 parasites per mg liver and spleen, respectively) and was not prone to false-positive reactions. Evaluation of the SL-RNA assay on clinical samples demonstrated lower threshold cycle values than the kDNA qPCR, an excellent interrun stability of 97%, a 93% agreement with the kDNA assay, and an estimated sensitivity, specificity, and accuracy of 93.2%, 94.3%, and 93.8%, respectively. The SL-RNA qPCR assay was equally efficient for detecting Leishmania major, Leishmania tropica, Leishmania mexicana, Leishmania guayensis, Leishmania panamensis, Leishmania braziliensis, L. infantum, and Leishmania donovani and revealed similar SL-RNA levels in the different species and the occurrence of polycistronic SL-containing transcripts in Viannia species. Collectively, this single SL-RNA qPCR assay enables universal Leishmania detection and represents a particularly useful addition to the widely used kDNA assay in clinical studies in which the detection of viable parasites is pivotal to assess parasitological cure.

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Section: Discussionmentioning

confidence: 80%

“…In this study, Real Time PCR assay was able to detect 0.1 parasite per reaction with a detectable melt curve. The limit of detection of this analysis is comparable and, in some cases, better than other currently available dye and probe-based Real Time PCR, while most of them were studied for VL diagnosis from blood or other more invasive biological samples[27,28,31,32]. It is noticeable that the diagnosis using urine showed 100% sensitivity which is same as it showed using blood and bone marrow specimens.The road map of World Health Organization (WHO) for neglected tropical diseases (NTD) aims to eradicate VL including PKDL from 75 endemic countries by 2030 by emphasizing (a) research on the relationship among the human, vector, parasite and reservoir, (b) development of more specific and user-friendly diagnostic test, and (c) introduction of faster, more effective, safer, cost…”

mentioning

confidence: 79%

Real-Time PCR-based diagnosis of human visceral leishmaniasis using urine samples

Rahim

Karim²,

Rahman

et al. 2022

Preprint

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Background: Diagnosis of visceral leishmaniasis (VL) through the detection of its causative agents namely Leishmania donovani and L. infantum is traditionally based on Giemsa-stained smears of bone marrow, spleen aspirates, liver or lymph node. Collection of these samples involve invasive procedures and carries the risk of fatal hemorrhage especially during splenic aspiration. Earlier, we reported a Polymerase Chain Reaction (PCR)-based diagnosis of L. donovani in peripheral blood using a novel set of PCR primers with absolute specificity (Khatun et al. 2017). Using the same set of primers and PCR conditions, here we describe diagnosis of L. donovani from urine, for a non-invasive, rapid and safe diagnosis. Methods: Diagnosis of L. donovani was carried out using urine samples collected from clinically diagnosed VL patients (n=23) of Bangladesh in Real Time PCR. Test results were validated by comparing blood samples from the same set of patients. Sensitivity and specificity of this diagnosis was analyzed using retrospective bone marrow samples, collected earlier from confirmed VL patients (n=19) (Khatun et al. 2017). Results: The method showed 100% sensitivity in detecting VL in urine and corresponding blood samples and bone marrow samples, as well as 100% specificity in control groups. Conclusion: Urine-based diagnosis could be a patient-friendly, non-invasive approach for VL detection with precision and perfection.

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SYBR green-based detection of Leishmania infantum DNA using peripheral blood samples

Cited by 9 publications

References 27 publications

Real-time PCR in detection and quantitation of Leishmania donovani for the diagnosis of Visceral Leishmaniasis patients and the monitoring of their response to treatment

Real-time PCR in detection and quantitation of Leishmania donovani for the diagnosis of Visceral Leishmaniasis patients and the monitoring of their response to treatment

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

Real-Time PCR-based diagnosis of human visceral leishmaniasis using urine samples

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