A versatile qPCR assay to quantify trypanosomatidic infections of host cells and tissues

Abstract

The majority of PCR-based detection systems for Leishmania spp. and Trypanosoma cruzi aim at high sensitivity and specificity, rather than an accurate parasite load quantification required for experimental infections in basic research and drug development. Here, we describe the use of a dual-labelled probe qPCR to detect and quantify intracellular Old World Leishmania spp. and T. cruzi amastigotes after in vitro and in vivo infection experiments. We show that quantification of parasite actin gene DNA relative to the host cell actin gene DNA accurately reflects the parasite load relative to the host cells and that qPCR quantification is highly sensible to drug-induced cell death. Furthermore, qPCR allows to determine parasite loads even after host cell detachment and/or rupture, important when comparing untreated versus drug-treated samples. The method is also suitable for the quantification of parasites from infected mouse tissue, making it suitable for drug testing and mutant phenotype analysis.

Synthetic analogs of an Entamoeba histolytica glycolipid designed to combat intracellular Leishmania infection

“…Since treatment with native Eh LPPG showed a parasite reducing effect, we next asked whether synthetic analogs Eh PIa C30:1 cis, Eh PIb C30:1 cis, Eh PIb C30:1 trans, and Eh PIb C28:0 had a similar impact on the intracellular L. major load. Therefore, we infected murine BMMs and human THP1 macrophages with L. major promastigotes (at stationary phase) and measured the intracellular Leishmania load using a Leishmania -specific TaqMan PCR 31 . We found that treatment of murine macrophages with αGalCer, Eh LPPG, Eh PIa C30:1 cis, or Eh PIb C30:1 cis led to significant inhibition of parasite infection compared with that in infected but untreated cells (αGalCer 1.0 µg/ml: 0.43 ± 0.1 [p = 0.003]; Eh LPPG 4.0 µg/ml: 0.64 ± 0.09 [p = 0.011]; Eh PIa C30:1 cis, 0.1 µg/ml: 0.75 ± 0.08 [p = 0.025]; Eh PIa C30:1 cis, 1.0 µg/ml: 0.39 ± 0.03 [p = 0.0001]; Eh PIa C30:1 cis, 10.0 µg/ml: 0.51 ± 0.09 [p = 0.002]; Eh PIb C30:1 cis, 0.1 µg/ml: 0.56 ± 0.081 [p = 0.002]; Eh PIb C30:1 cis, 1.0 µg/ml: 0.69 ± 0.09 [p = 0.018]; and Eh PIb C30:1 cis, 10.0 µg/ml: 0.61 ± 0.013 [p = 0.0001]) (Fig.…”

Antileishmanial Effects of Synthetic Eh PIb Analogs Derived from the Entamoeba histolytica Lipopeptidephosphoglycan

“…Here, we tested the antileishmanial activity of the newly synthesized compounds Eh-1, Eh-2, Eh-3, Eh-4, Eh-5, and Eh-6 against L. major-infected bone marrow-derived macrophages (BMDMs) and human THP1 cells. Relative parasite loads were determined after 48 h by real-time PCR using duplex TaqMan PCR (23). The synthetic analog Eh-2 caused a significant parasite load reduction at all tested concentrations (0.1 to 10 g/ml, P Ͻ 0.015) in murine and human cells.…”

“…Infected murine or human macrophages were treated with 0.1, 1.0, or 10 g/ml of compounds Eh-1 to Eh-6, as well as with 4.0 g/ml ␣-GalCer and 8.0 g/ml EhLPPG. After 48 h of treatment, murine or human macrophages were subjected to genomic DNA (gDNA) isolation (QIAmp gDNA kit; Qiagen) according to the manufacturer's instructions (23,44).…”

Phlebotomine sand flies in Southwest Germany: an update with records in new locations

“…For virus screening, extracted RNA samples were tested by pan-phlebovirus [38] and pan-flavivirus PCR [39]. In 2018, the 81 specimens were screened on Leishmania parasites with a quantitative polymerase chain reaction (qPCR) using the primers OWLeishkDNAfwd* (3′-GCT TTA GTG GGT TGG AGC CT-5′), OWLeishk-DNArev* (3′-TCA ACC CAA GAC CAC CAT CG-5′) and OWLeishkDNAProbe*: (6FAM)CGG GTG TCT TTG ATG ATG CTG GGG TGG GT(BHQ1) [40]…”