Development of a real-time polymerase chain reaction assay for the quantification of Leishmania species and the monitoring of systemic distribution of the pathogen

Tupperwar, Nitin; Varanasi, Vineeth; Rath, Satyajit; Vaidya, Tushar

doi:10.1016/j.diagmicrobio.2007.12.013

Cited by 46 publications

(33 citation statements)

References 23 publications

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“…Our qPCR reaction achieved a detection limit of 0.01 pg of parasite DNA which corresponds to 0.12 parasites. In agreement, other authors have reported detection limits from 0.001 to 1 parasite and a dynamic range from 10 -2 to 10 7 , with an optimal range among 10 -2 to 10 4 in a ten-fold dilution (Hernandez et al, 1993;Mary et al, 2004;Nicolas et al, 2002;Francino et al, 2006;Ranasinghe et al, 2008;Tupperwar et al, 2008;de Paiva Cavalcanti et al, 2009;Boggiatto et al, 2010;Dantas-Torres et al, 2011). Accordingly, our qPCR quantification is within the expected detection range for Leishmania DNA.…”

Section: Discussionsupporting

confidence: 53%

Detection and quantification of Leishmania infantum in naturally and experimentally infected animal samples

Losada-Barragán

Cavalcanti

Umaña‐Pérez

et al. 2016

Veterinary Parasitology

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Section: Discussionsupporting

confidence: 53%

Detection and quantification of Leishmania infantum in naturally and experimentally infected animal samples

Losada-Barragán

Cavalcanti

Umaña‐Pérez

et al. 2016

Veterinary Parasitology

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“…As for ATL, a few qPCR assays that amplify multicopy DNA targets have been described (20,41,42). In terms of diagnostic sensitivity, our kDNA qPCR assay performed similarly to the qualitative PCR test based on the same primers.…”

Section: Discussionmentioning

confidence: 99%

Real-Time PCR Assay for Detection and Quantification of Leishmania (Viannia) Organisms in Skin and Mucosal Lesions: Exploratory Study of Parasite Load and Clinical Parameters

et al. 2013

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h Earlier histopathology studies suggest that parasite loads may differ between cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML) lesions and between acute and chronic CL. Formal demonstration requires highly sensitive detection and accurate quantification of Leishmania in human lesional tissue. In this study, we developed a quantitative real-time PCR (qPCR) assay targeting minicircle kinetoplast DNA (kDNA) to detect and quantify Leishmania (Viannia) parasites. We evaluated a total of 156 lesion biopsy specimens from CL or ML suspected cases and compared the quantitative performance of our kDNA qPCR assay with that of a previously validated qPCR assay based on the glucose-6-phosphate dehydrogenase (G6PD) gene. We also examined the relationship between parasite load and clinical parameters. The kDNA qPCR sensitivity for Leishmania detection was 97.9%, and its specificity was 87.5%. The parasite loads quantified by kDNA qPCR and G6PD qPCR assays were highly correlated (r ‫؍‬ 0.87; P < 0.0001), but the former showed higher sensitivity (P ‫؍‬ 0.000). CL lesions had 10-fold-higher parasite loads than ML lesions (P ‫؍‬ 0.009). Among CL patients, the parasite load was inversely correlated with disease duration (P ‫؍‬ 0.004), but there was no difference in parasite load according to the parasite species, the patient's age, and number or area of lesions. Our findings confirm that CL and recent onset of disease (<3 months) are associated with a high parasite load. Our kDNA qPCR assay proved highly sensitive and accurate for the detection and quantification of Leishmania (Viannia) spp. in lesion biopsy specimens. It has potential application as a diagnostic and follow-up tool in American tegumentary leishmaniasis.

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“…Mice infected with the parasites were treated with NTZ. All mice were sacrificed 1 month after the last treatment, and splenic and hepatic parasite burden were determined using real-time PCR based on a highly conserved gene GP63 in Leishmania, which is highly accurate and sensitive to detect and quantify Leishmania parasites (Tupperwar et al 2008). As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Quantitative real-time PCR was used to evaluate the splenic and hepatic parasite load from the treated mice infected with Leishmania as described elsewhere (Tupperwar et al 2008). Briefly, spleens and livers were weighted, and the extraction of DNA was carried out as a routine method.…”

Section: Quantitative Real-time Pcrmentioning

confidence: 99%

In vitro and in vivo antileishmanial efficacy of nitazoxanide against Leishmania donovani

et al. 2010

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Control of Leishmania infection relies primarily on chemotherapy, and the current drug available for treating leishmaniasis is limited. Nitazoxanide (NTZ) is a broad spectrum antiparasitic agent with activity against protozoa, nematodes, cestodes, and trematodes. In the present study, the in vitro antileishmanial efficacy of NTZ was evaluated by incubation of Leishmania donovani promastigotes with NTZ, indicating that NTZ can affect the ultrastructure of parasite promastigote and efficiently inhibit the parasite growth. Moreover, 200 microg/ml NTZ inhibited >90% of promastigotes growth, showing similar activity of the reference drug amphotericin B (P > 0.05). Therapeutic efficacy of NTZ against L. donovani-infected BALB/c mice demonstrated that oral NTZ produced a significant reduction of parasite burden in spleen and liver from L. donovani-infected mice, compared with the untreated mice (P < 0.05). These results indicated NTZ may be a novel therapeutic drug for leishmaniasis.

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Development of a real-time polymerase chain reaction assay for the quantification of Leishmania species and the monitoring of systemic distribution of the pathogen

Cited by 46 publications

References 23 publications

Detection and quantification of Leishmania infantum in naturally and experimentally infected animal samples

Detection and quantification of Leishmania infantum in naturally and experimentally infected animal samples

Real-Time PCR Assay for Detection and Quantification of Leishmania (Viannia) Organisms in Skin and Mucosal Lesions: Exploratory Study of Parasite Load and Clinical Parameters

In vitro and in vivo antileishmanial efficacy of nitazoxanide against Leishmania donovani

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