Real-Time PCR to Quantify<i>Leishmania donovani</i>in Hamsters

Srivastava, Anuradha; Sweat, J. Mark; Azizan, Azliyati; Vesely, Brian; Kyle, Dennis E.

doi:10.1645/ge-3221.1

Cited by 13 publications

(14 citation statements)

References 35 publications

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“… Note: Bretagne et al (2001) , Nicolas et al (2002) , Bossolasco et al (2003) , Schonian et al (2003) , Schulz et al (2003) , Mary et al (2004) , Wortmann et al (2005) , Prina et al (2007) , Castilho et al (2008) , Deborggraeve et al (2008) , Van Der Meide et al (2008) , De Paiva Cavalcanti et al (2009) , Bezerra-Vasconcelos et al (2011) , Jara et al (2013) , Srivastava et al (2013) , Toz et al (2013) , Tellevik et al (2014) , Ghotloo et al (2015) , Zampieri et al (2016) . …”

Section: Resultsmentioning

confidence: 99%

A real-time PCR assay for quantification of parasite burden in murine models of leishmaniasis

Antonia

Wang

2018

PeerJ

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Eukaryotic parasites in the genus Leishmania place approximately 350 million people per year at risk of disease. In addition to their global health significance, Leishmania spp. have served as an important model for delineating basic concepts in immunology such as T-helper cell polarization. There have been many qPCR-based assays reported for measuring parasite burden in humans and animals. However, these are largely optimized for use in clinical diagnosis and not specifically for animal models. This has led several of these assays to have suboptimal characteristics for use in animal models. For example, multi-copy number genes have been frequently used to increase sensitivity but are subject to greater plasticity within the genome and thus may confound effects of experimental manipulations in animal models. In this study, we developed a sybr-green based quantitative touchdown PCR assay for a highly conserved and single-copy putative RNA-binding protein, DRBD3. With primers that share greater than 90% sequence identity across all sequenced Leishmania spp., we demonstrate that this assay has a lower limit of detection of 100 fg of parasite DNA for Leishmania major, L. donovani, L. venezuelensis, and L. panamensis. Using C57BL6/J mice, we used this assay to monitor parasite burden over 1 month of infection with two strains of L. major (Seidman and Friedlin), and L. venezeuelensis. These characteristics rival the sensitivity of previously reported qPCR based methods of parasite quantitation while amplifying a stable, single copy gene. Use of this protocol in the future will lead to improved accuracy in animal based models and help to tease apart differences in biology of host-parasite interactions.

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Section: Resultsmentioning

confidence: 99%

A real-time PCR assay for quantification of parasite burden in murine models of leishmaniasis

Antonia

Wang

2018

PeerJ

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“…Classically, this was done in leishmaniasis by limiting dilution (Lima et al 1997), a cumbersome technique. More recently, real-time PCR is being employed as an alternative to limiting dilution (Nicolas et al 2002; Srivastava et al 2013). Both limiting dilution and qPCR require animal sacrifice since tissue is obtained and processed for parasite quantification.…”

Section: Animal Modelsmentioning

confidence: 99%

Chemotherapy of leishmaniasis: present challenges

2017

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Cutaneous and visceral leishmaniasis are amongst the most devastating infectious diseases of our time, affecting millions of people worldwide. The treatment of these serious diseases rely on a few chemotherapeutic agents, most of which are of parenteral use and induce severe side-effects. Furthermore, rates of treatment failure are high and have been linked to drug resistance in some areas. Here, we reviewed data on current chemotherapy practice in leishmaniasis. Drug resistance and mechanisms of resistance are described as well as the prospects for applying drug combinations for leishmaniasis chemotherapy. It is clear that efforts for discovering new drugs applicable to leishmaniasis chemotherapy are essential. The main aspects on the various steps of drug discovery in the field are discussed.

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“…When compared to conventional PCR, qPCR represents a more efficient, sensitive and robust technique for the detection of amastigotes RNA in various tissues (spleen, skin, lymph, blood and bone marrow) and in different clinical groups [7][8][9]. In addition, qPCR is currently considered the most reliable method to evaluate the in vivo activity of compounds against visceral leishmaniasis, allowing effective disease monitoring, with increased speed and accuracy compared with standard methods [10,11].…”

Section: Introductionmentioning

confidence: 99%

In Vivo Evaluation of Leishmanicidal Activity of Benzophenone Derivatives by qPCR

Colombo¹,

Reis²,

Nunes³

et al. 2017

Med chem

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In many tropical areas, infections by Leishmania species are endemic and include visceral leishmaniasis (VL), which is often fatal if untreated. Outside India, VL treatment and control are based on long-term administration of highly toxic pentavalent antimonials. Previously, we described the synthesis and in vitro leishmanicidal activity of a series of nine benzophenone derivatives with low toxicity towards murine macrophages. Here in, we report the in vivo evaluation of the most promising active compounds of that series in an experimental model of established VL by L. (L). infantum chagasi in hamsters. Importantly, parasite DNA (amastigote form) quantification in infected tissues was performed by real time PCR, for improved detection accuracy and speed. Compounds 2-Hydroxy-4-O-(3,3-dimethyl)-allylbenzophenone (LFQM-117 (1)), 4-O-(3,3-Dimethyl)-allylbenzophenone (LFQM-120 (2)) and 4,4′-Di-methoxybenzophenone (LFQM-121 (3)) were administered as oral suspensions (50 mg/kg/day)

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Real-Time PCR to QuantifyLeishmania donovaniin Hamsters

Cited by 13 publications

References 35 publications

A real-time PCR assay for quantification of parasite burden in murine models of leishmaniasis

A real-time PCR assay for quantification of parasite burden in murine models of leishmaniasis

Chemotherapy of leishmaniasis: present challenges

In Vivo Evaluation of Leishmanicidal Activity of Benzophenone Derivatives by qPCR

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