New Molecular Approach for the Detection of Kinetoplastida Parasites of Medical and Veterinary Interest.

Hacène Medkour,Bernard Davoust,Oleg Mediannikov,Marie Varloud

doi:10.3390/microorganisms8030356

Abstract

Kinetoplastids are protozoa containing a range of ubiquitous free_living species–pathogens of invertebrates, vertebrates and even some plants. Some of them are causative agents of canine vector-borne diseases. Their diagnosis is often missing in a gold standard. Here, we proposed a molecular approach for the diagnosis and study of Kinetoplastida. The TaqMan qPCR assays target the following genes: 24Sa LSU of Kinetoplastida, 28S LSU of Leishmania/ Trypanosoma spp., 5.8S rRNA of Trypanosoma spp., 18S SSU of Leishmania spp., kinetoplast minicircle DNA (kDNA) of L. donovani complex and kDNA of L. infantum, were designed, validated for their sensitivity (Se) and specificity (Sp) in silico and in vitro using a panel of known DNAs. They were then used to screen 369 blood samples (358 dogs, 2 equids, 9 monkeys). In addition, new 28S LSU primer sets are presented to use for Kinetoplastida’s identification by PCR/sequencing. All qPCRs showed consistently high analytical sensitivities and reproducibility. They detect approximately 0.01 parasite/ mL blood for the kDNA based- qPCRs and at least a single cell-equivalent of rDNA for the other systems. Based on the sequencing results, after screening, Se and Sp were: 0. 919 and 0.971, 0.853 and 0.979, 1.00 and 0.987, 0.826 and 0.995 for all of Kinetoplastida, Leishmania/ Trypanosoma, Trypanosoma, Leishmania spp. specific qPCRs, respectively. kDNA based qPCRs were more sensitive and specific (Se: 1.00; Sp: 0.997). PCR/sequencing allowed the detection of Kinetoplastids in animal blood samples such as L. infantum, L. guyanensis, T. congolense, T. evansi and Bodo spp. The molecular approach proposed here is useful for epidemiological studies, fundamental research such as screening for new Kinetoplastida species, diagnosis and therapeutic follow-up. In addition, researchers are free to choose the molecular tools adapted to their aims.

Highlights

Kinetoplastida are a group of flagellated protists belonging to the phylum Euglenozoa, characterized by the presence of a large massed DNA called kinetoplast, an organelle that stains like the nucleus, but is a specific part of the mitochondrion in which large amounts of extranuclear DNA are accumulated [1,2]
Trypanosoma and Leishmania are obligatorily dixenous, possess zoonotic or anthroponotic life-cycles and are transmitted by hematophagous insects [6,7]. These parasites cause devastating human diseases including human African trypanosomiasis due to Trypanosoma brucei, Chagas disease caused by Trypanosoma cruzi and the leishmaniases, which are attributable to about 20 species of Leishmania [8,9,10,11]
With the exception of the strictly anthroponotic gambiense form of human trypanosomiasis and infections caused by Leishmania tropica and L. donovani, for which humans are considered the main reservoir, diseases associated with trypanosomiasis are mainly zoonotic, with animal reservoirs playing a key role in maintaining endemicity [12,13,14]

Summary

Introduction

Kinetoplastida (or Kinetoplastea, as a class) are a group of flagellated protists belonging to the phylum Euglenozoa, characterized by the presence of a large massed DNA called kinetoplast ( the name), an organelle that stains like the nucleus, but is a specific part of the mitochondrion in which large amounts of extranuclear DNA are accumulated [1,2]. With the exception of the strictly anthroponotic gambiense form of human trypanosomiasis and infections caused by Leishmania tropica and L. donovani, for which humans are considered the main reservoir, diseases associated with trypanosomiasis are mainly zoonotic, with animal reservoirs playing a key role in maintaining endemicity [12,13,14] This includes L. infantum, the causative agent of the most common vector-borne protozoan disease worldwide [15], T. brucei and T. congolense, the causative agents of Nagana or a similar disease in Africa and Asia, as well as T. evansi, the etiological agent of surra and the so-called “mal de cadeiras” outside Africa. Animals are important reservoirs of T. cruzi in the Americas [16]

Methods

Results

Discussion

Conclusion