Ethics statement
Dogs and equids from Algeria in this study were examined with the assistance and acceptance of their owners. Blood samples were collected by veterinarians according to the good practices of veterinary medicine. Risk assessment was submitted to and approved by the ethics committee and decision board of the veterinary practitioners from the wilayas of the North of Algeria. These institutions are affiliated with the Algerian Ministry of Agriculture and Rural Development (Directions des Services Vétérinaires). Protocol of the study was also approved by the scientific college (Procès-Verbal du CSI N°6, 2018) at the Veterinary Science Institute, University Constantine 1, Algeria. To facilitate field work, collaborations were established with veterinary doctors and their assistants working in these establishments.
Dogs from Cote d’Ivoire were sampled after obtaining the verbal consent of their owners. The blood samples were taken from radial vein by veterinarians (French and Ivorian) with the ethical responsibility to ensure the animal care in accordance with Articles 433-434; Chapter 2 of the Ivoirian Penal Code.
Dog’s blood samples from French Guiana were collected by veterinarians according to the good practices of veterinary medicine. Article R.214-88 of the French Rural Code and Sea Fishing (Decree No. 2013–118 of 1 February 2013 on the protection of animals used for scientific purposes) excludes these acts from the scope of applications for authorization granted by the Minister responsible for research. Red howler monkeys were hunted by two Amerindian hunters and intended for consumption by the family. The hunters applied the provisions of the prefectural decree regulating the quotas of species that can be taken by a person in the department of Guiana (No. 583/DEAL of April 12, 2011). Blood samples has been taken at the moment of hunting.
Primers and probe’s designs
Custom protocol and in silico validation
First, for each PCR essay, the target gene was chosen and a fasta file was constructed from the sequences available in GenBank database. Sequences were aligned using BioEdit v 7.0.5.3 software [21] to reveal conserved areas suitable as target regions for specific primers and probes. This region was submitted in Primer3 software v. 0.4.0 (http://primer3.ut.ee/), in order to determine a valuable candidate primers and probes and the selection was based on the criteria for primer and probe design.
Settings for the PCR primers and probes were in accordance with the guidelines as described by Apte and Daniel [22] and as recommended by Invitrogen™ and Applied Biosystems™. Melting temperatures (T), secondary structures and the possibility for primer-dimers were tested using the free online software Oligo Analyzer 3.1 [23]. All primer and probe sequences were also checked for their specificity in NCBI BLAST nucleotide sequence similarity search [24]. They were also checked within the DNA databases of metazoans (taxid:33208), vertebrates (taxid:7742), bacteria (taxid:2), Canidae (taxid:9608), Felidae(taxid:9682) and humans (taxid:9605). This has been done for all possible combinations of forward-reward and probe-reward of each PCR system. Primers were synthetized by Eurogentec (Liège, Belgium) and the hydrolysis probe by Applied Biosystems™.
Specificity-based principles of oligonucleotide design
Quantitative TaqMan real-time PCR (qPCR) for Kinetoplastida parasites detection
The conserved region from the 28S rRNA gene, more exactly the 24S alpha subunit of most Kinetoplastida class, especially Bodonida and Trypanosomatida orders, has been targeted for the primers and probe design. The primers called F LSU 24a, F LSU 24a and a probe (P LSU 24a) were proposed (Table 1). The choice of this gene (28S) was based on its availability in GenBank for almost all Kinetoplastida families and its high level of conservation [28].
TaqMan qPCR targeting Leishmania–Trypanosoma spp.
Among the Kinetoplastida parasites, several members from Leishmania and Trypanosoma genera are involved in human and animal pathology [17]. Another conserved region from the 28S rRNA gene has been chosen to design primers and probe (Table1) to amplify most species belonging to Leishmania and Trypanosoma genus. Oligonucleotides were designed in order to not amplify other members of Kinetoplastida.
TaqMan qPCR assays for Leishmania spp. and for Trypanosoma spp.
As described in a previous study (Medkour et al. submitted), two qPCRs assays had been developed: i) qPCR for Leishmania spp. targeting the 18S rRNA gene of most Leishmania species affecting humans and animals: L. donovani, L. infantum/chagasi, L. major, L. tropica, L. guyanensis, L. amazonensis, L. braziliensis, L. mexicana, L. siamensis, L. aethiopica, L. tarentolae, L. alderi, L. gymnodactyli, L. hoogstraali and possible new species; ii) qPCR for Trypanosoma spp. targeting about 85 pb-long fragments from the 5.8S rRNA gene of the most species threating humans, pets, livestock and wildlife: T. brucei brucei, T. brucei gambiense, T. brucei rhodesiense, T. congolense,T. evansi, T. vivax, T. cruzi, T.theileri, T. equiperdum, T. rangeli, T. simiae, T. godfreyi, T. minasense and a large number of potential new species.
TaqMan qPCR for canine and human visceral leishmaniasis diagnosis (L. donovani, L. infantum):
The conserved region of the Leishmania kDNA minicircle (kDNA), recognized for its high sensitivity (several 1000’s-fold repeated sequence) [29], was elected to design the specific primers targeting a 175 pb fragment from the kDNA of L. infantum and L. donovani. Two labeled probes were also designed from the same region: i) the VIC-labeled probe for the detection of both L. infantum and L. donovani kDNA; ii) the FAM-labeled probe confined only for L. infantum kDNA (Table 1).
Run protocols
The qPCR reactions were carried out in a final volume of 20 µl, containing 5 µl of DNA template, 10 µl of Master Mix Roche (Eurogentec). The concentration of each primer per reaction was 0.5 µl, 0.5 µl of both UDG and each probe. Finally, the volume was completed to 20 µl using ultra-purified water DNAse-RNAse free. The TaqMan cycling conditions included two hold steps at 50°C for 2 min followed by 95°C for 15 min, and 40 cycles of two steps each (f 95° C for 30 s and 60° C for 30 s). The qPCR amplification was performed in a CFX96 Real-Time system (Bio-rad Laboratories, Foster City, CA, USA) after activating the readers of the dyes (FAM and/or VIC) used in each qPCR system.
Conventional PCR primers sets design, amplification protocol and sequencing
The 28S rRNA was used to design a primer set in order to complete the molecular identification of Kinetoplastids. Primer pair combinations amplify from 550 to 1300 pb-fragments long of the 28S rRNA gene with a variable specificity among the species belonged into Kinetoplastida class (Table 1).
To identify Leishmania species other than L. donovani/infantum, a primer pair targeting 550 pb-fragments of the 18S rRNA of Leishmania spp. has been used (Medkour et al. submitted), as well as universal primer pairs targeting Leishmania spp. intergenic transcribed spacers 1 and 2 (ITS1 and ITS2) [30][31]. In addition, primers ITS1CF and ITS1 BR, previously designed to amplify the ITS1 partial gene of African trypanosomes [32] have been used in this approach.
PCR reactions were performed in a total volume of 50 µl, consisting of 25 µl of AmpliTaq Gold master mix, 18 µl of ultra-purified water DNAse-RNAse free, 1 µl of each primer and 5µl of DNA template. The thermal cycling conditions were: incubation step at 95°C for 15 minutes, 40 cycles of one minute at 95°C, 30s for the annealing at a different melting temperature for each PCR assays (Table 1), 1 min for elongation time at 72°C followed by a final extension for five minutes at 72°C (Table 1). PCR amplification was performed in a Peltier PTC-200 model thermal cycler (MJ Research Inc., Watertown, MA, USA). The results of amplification were visualized by electrophoresis on 2% agarose gel. The purification of PCR products was performed using NucleoFast 96 PCR plates (Macherey Nagel EURL, Hoerdt, France) according to the manufacturer’s instructions. The amplicons were sequenced using the Big Dye Terminator Cycle Sequencing Kit (Perkin Elmer Applied Biosystems, Foster City, CA, USA) with an ABI automated sequencer (Applied Biosystems).
PCR systems validation
The specificity and sensitivity of all PCR assays were tested in silico using primer-BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and validated in vitro using, i) DNAs of cultivated Leishmania species: L. infantum MCAN/ES/98/LLM-877 (WHO international reference strain), L. donovani (MHOM/IN/00/DEVI), L. major (MHOM/IL/81/Friedlin) and L. guyanensis previously detected on red howler monkey from French Guiana (MK782154) [33]; ii) for Trypanosoma 5.8S based qPCR system: DNA panel from cultivated Trypanosoma species: T. congolense IL 3000, T. evansi, T. vivax, T. brucei brucei, T. brucei gambiense and T. brucei gambiense biyiamina groupe II, T. cruzi CL Brenner, and DNA of uncultivated T. congolense detected on French dog died few weeks after return from 3 months mission in Cote d’Ivoire; iii) for Leishmania-Trypanosoma 28S based qPCR: All the DNAs cited above; iv) for Kinetoplastida 28S based PCRs: All the DNAs cited above, DNA of Leptomonas sp. isolated on flea Ctenocephalides felis from Senegal and DNA of Bodo sp. from blood of an Algerian dog. All PCR systems were tested for their specificity using several arthropods of laboratory-maintained colonies as well as human, monkey, donkey, horse, cattle, mouse and dog DNAs for all the PCR systems. DNA collections used to test the sensitivity and specificity of PCR systems are summarized in the Table S1.
Sensitivity and efficiency of TaqMan assays (quantification)
To assess the analytical sensitivity of the generic real-time PCR assays (28S Leishmania-Trypanosoma spp., 18S Leishmania spp. and Leishmania donovani/ infantum kDNA based qPCR (VIC-labeled probe)), a standard curve was established using L. donovani DNA; 5 ml of serial dilutions, ranging from 10000 to 0.001 parasites was introduced into reaction tubes for each qPCR system. The standard curve concentration was expressed as parasite/ml (par/ml).
To assess the sensitivity of the 28S based qPCR for Kinetoplastida and the 5.8S based qPCR for Trypanosoma spp., we used blood of French dog died after T. congolense infection, for which the number of parasites was determined by microscopical counting on blood smear (Fig.S1). The number of T. congolense visualized was 1.06 E6 parasites/ mL of blood. Twelve-fold dilutions of this DNA (initial concentration 1.06 E6 parasites/ mL) were made for standard curve analysis. Also, the standard curve concentration was expressed as parasite/mL (par/mL).
PCR tools validation by sample screening and identification of Kinetoplastida on biological samples
An already-existing collections of animal-blood samples was screened by qPCR assays assessed in this study. This includes:
- 42 dogs from Cote d’Ivoire (April 2018) from the study of (Medkour et al. submitted);
- 218 dogs, one horse and one donkey from Kabylia, northern Algeria (May 2018) from the previous study of Medkour et al,[34]. The samples were collected after obtaining a verbal consent from dog owners by a veterinarian specialist. Risk assessment was submitted to and approved by the ethics committee and decision board of the veterinary practitioners from the wilayas of the North of Algeria. These institutions are affiliated with the Algerian Ministry of Agriculture and Rural Development (Directions des Services Vétérinaires).
- 98 dogs sampled in 2016 and 9 red howler monkeys sampled in 2014 from French Guiana from the previous studies [35] and [33].
The genomic DNA was extracted from 200 mL-blood of each sample after enzymatic digestion with proteinase K, using the QIAGEN DNA tissues extraction kit (QIAGEN, Hilden, Germany), following the manufacturer’s recommendations. The extracted DNA was eluted in a total volume of 200 µl and stored at – 20°C until use.
Samples were screened by qPCRs: 28S Kinetoplastida, 28S Leishmania-Trypanosoma spp., 5.8S Trypanosoma spp., 18S Leishmania spp. and the qPCR targeting the kDNA of L. donovani complex. Positive samples for of L. donovani complex were tested to L. infantum using the L. infantum kDNA based qPCR;
All samples found positive by qPCR were subjected to conventional PCR/sequencing of ~550 pb-fragments of 28S gene of Kinetoplastida. Leishmania-positive samples were tested in PCR/sequencing of partial ITS2 and 18S rRNA genes. Trypanosoma-positive samples have also been tested by the ITS1 based-PCR and amplicons were sequenced (Table 1).
Determination of assay performance characteristics
Here, the performances of qPCR assays were evaluated in the absence of gold standard test. The true positive samples were determined: the sample was considered true positive if at least one of the sequences was obtained by PCR/sequencing of a part of 28S rRNA for Kinetoplastida, 18S rRNA and/or ITS2 for Leishmania spp. and/or ITS 1 for Trypanosoma spp. (Table 1). By using this criterion to determine the true positive samples, we reduced the possibility of false positives and qPCR assays will be evaluated as much as possible for their specificity. One of the limitations is the difficulties to take decisions in cases found positive by qPCR and negative in standard PCR that may be linked to a sequencing defect, for X or Y reasons, e.i. low quantity of DNA in the sample, not related to failure in specificity of the qPCR.
Statistical analysis
After set of databases Microsoft Excel® program (Microsoft Corp., Redmont, USA), a descriptive study of the Kinetoplastida of infections was performed. The statistical analysis was conducted using XLSTAT Addinsoft version 2018.7 (Data Analysis and Statistical Solution for Microsoft Excel, Paris, France).
To determine assay performance characteristics for each test, Prevalence (Pr), Correct classification (Cc) and Misclassification (Mc), Se, Sp, False positive rate (FP), False negative rate (PN), Positive and negative Predictive Value (PPV & NPV), calculated for each test.
To measure the agreement of qPCR assays according to sequencing (defined as gold standard in this study), one could simply compute the percentage of cases for which both qPCR and sequencing results agree. This statistic has nevertheless an important weakness. It does not account for agreement randomly occurring. On the other hand, Cohen’s Kappa measures agreement, while removing random effects, thus ensuring good reproducibility. Cohen’s Kappa (k) measures agreement was used to evaluate the relevance of each qPCR assay according to the sequencing results as established by Landis and Koch (1977).