Leishmaniasis in the Sudan: Parasite Characterization and Phylogeny

The study concentrated mainly on characterization of Sudanese Leishmania isolates; and the phylogenetic relation between isolates from different clinical forms in addition to the relation between these isolates and reference leishmania strains. The study also included attempts to identify parasite factors associated with PKDL in Sudan. In order to achieve the mentioned aims, more than 120 Sudanese leishmania isolates were cultured from different clinical forms of the disease. Isolated parasites were primarily characterized by their isoenzyme profiles compared with reference strains.64 isolates were characterized using isoenzyme: 8 were identified as L.donovani, 9 as L.infantum, 44as L.archibaldi and 3 as L.major. Interestingly L.archibaldi was strongly associated with VL and was the main parasite isolated from PKDL. Total genomic DNA was extracted and kDNA PCR and genomic PCR were performed. The kDNA was performed using species-specific primers followed by restriction fragment length polymorphism (RFLP) with AluI restriction enzyme. The total genomic DNA was analysed using 2 sets of primers; RH1, RH2 and SG1, SG2. The first set was used to amplify parasite sequences by PCR while the second set was used to amplify the gp63 genes then followed by RFLP and hybridisation technique. Radiolabeled probes were used to hybridize the digested genomic DNA in 2 ways; after gp63 amplification and restriction enzyme digestion, and after restriction enzyme digestion without gene amplification. Gp63 PCR analysis of selected 29 isolates identified 26 as L.archibaldi and 3 as L.major. Differential display technique was used for RNA in order to determine the differentially expressed candidate genes between VL/PKDL paired isolates. Cloning and sequencing were used to achieve full descriptions of these differentially expressed genes and northern blot was done to ensure that genes were not false expressed genes. The results obtained in this study suggest that L.archibaldi is the major cause of VL, PKDL and ML and also associated with CL infection. Another important finding in this study is that the PKDL infection is caused by the same parasite that caused the VL form. The high incidence of PKDL in Sudan might be L.archibaldi infection

Synthesis, Characterization, and Antileishmanial Activity of Certain Quinoline-4-carboxylic Acids

Leishmaniasis is a fatal neglected parasitic disease caused by protozoa of the genus Leishmania and transmitted to humans by different species of phlebotomine sandflies. The disease incidence continues to increase due to lack of vaccines and prophylactic drugs. Drugs commonly used for the treatment are frequently toxic and highly expensive. The problem of these drugs is further complicated by the development of resistance. Thus, there is an urgent need to develop new antileishmanial drug candidates. The aim of this study was to synthesize certain quinoline-4-carboxylic acids, confirm their chemical structures, and evaluate their antileishmanial activity. Pfitzinger reaction was employed to synthesize fifteen quinoline-4-carboxylic acids (Q1-Q15) by reacting equimolar mixtures of isatin derivatives and appropriate α-methyl ketone. The products were purified, and their respective chemical structures were deduced using various spectral tools (IR, MS, 1H NMR, and 13C NMR). Then, they were investigated against L. donovani promastigote (clinical isolate) in different concentration levels (200 μg/mL to 1.56 μg/mL) against sodium stibogluconate and amphotericin B as positive controls. The IC50 for each compound was determined and manipulated statistically. Among these compounds, Q1 (2-methylquinoline-4-carboxylic acid) was found to be the most active in terms of IC50

Screening and Characterization of RAPD Markers in Viscerotropic <i>Leishmania</i> Parasites

<div><p>Visceral leishmaniasis (VL) is mainly due to the <i>Leishmania donovani</i> complex. VL is endemic in many countries worldwide including East Africa and the Mediterranean region where the epidemiology is complex. Taxonomy of these pathogens is under controversy but there is a correlation between their genetic diversity and geographical origin. With steady increase in genome knowledge, RAPD is still a useful approach to identify and characterize novel DNA markers. Our aim was to identify and characterize polymorphic DNA markers in VL <i>Leishmania</i> parasites in diverse geographic regions using RAPD in order to constitute a pool of PCR targets having the potential to differentiate among the VL parasites. 100 different oligonucleotide decamers having arbitrary DNA sequences were screened for reproducible amplification and a selection of 28 was used to amplify DNA from 12 <i>L. donovani</i>, <i>L. archibaldi</i> and <i>L. infantum</i> strains having diverse origins. A total of 155 bands were amplified of which 60.65% appeared polymorphic. 7 out of 28 primers provided monomorphic patterns. Phenetic analysis allowed clustering the parasites according to their geographical origin. Differentially amplified bands were selected, among them 22 RAPD products were successfully cloned and sequenced. Bioinformatic analysis allowed mapping of the markers and sequences and priming sites analysis. This study was complemented with Southern-blot to confirm assignment of markers to the kDNA. The bioinformatic analysis identified 16 nuclear and 3 minicircle markers. Analysis of these markers highlighted polymorphisms at RAPD priming sites with mainly 5′ end transversions, and presence of inter– and intra– taxonomic complex sequence and microsatellites variations; a bias in transitions over transversions and indels between the different sequences compared is observed, which is however less marked between <i>L. infantum</i> and <i>L. donovani</i>. The study delivers a pool of well-documented polymorphic DNA markers, to develop molecular diagnostics assays to characterize and differentiate VL causing agents.</p></div

RAPD profiles obtained with primers OP-O7 (A) and OP-O13 (B) to illustrate examples of monomorphic and polymorphic profiles.

<p>Laboratory codes of the strains are indicated for each lane. SD: Sudan; TN: Tunisia; M: 100 bp DNA size marker. Indicated sizes are in bp.</p

Panel of <i>Leishmania</i> strains used for screening of RAPD markers.

<p>WHO that summarizes Host, geographical origin, year of isolation and laboratory code is presented together with pathology and zymodeme code whenever available. MON– corresponds to zymodeme code attributed by the reference center in Montpellier. The table also gathers study codes assigned to some of the isolates in other studies: D21, D28, D29, D31 and D32: strains used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109773#pone.0109773-Mauricio1" target="_blank">[20]</a>; DON-39 and ARC-43: strains used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109773#pone.0109773-Kuhls1" target="_blank">[21]</a>; Devi, H9, LRC-L57, ADDIS 164: strains used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109773#pone.0109773-Jamjoom1" target="_blank">[18]</a>; Devi, GEBRE1 and KA-Jeddah: strains used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109773#pone.0109773-Thiel1" target="_blank">[53]</a>; DON-81and ARC-43 (LG11): strains used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109773#pone.0109773-Kuhls2" target="_blank">[28]</a>; DON-09, DON-31, DON-39 and ARC-11: strains used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109773#pone.0109773-Chocholov1" target="_blank">[25]</a>. Country abbreviations are shown as specified by WHO recommendations (SD: Sudan; TN: Tunisia; ET: Ethiopia; SA: Saudi Arabia; KE: Kenya; IN: India). ND: Not Determined; CL: cutaneous leishmaniasis; VL: visceral leishmaniasis; PKDL: Post Kala azar Dermal Leishmaniasis.</p><p>Panel of <i>Leishmania</i> strains used for screening of RAPD markers.</p

Selected features characterizing the cloned RAPD markers.

a<p>: Non coding Sequence;</p>b<p>: Overlap with a coding sequence;</p>c<p>: Matching with a coding sequence;</p>d<p>: Minicircle sequence;</p><p>* Imperfect Microsatellite: one mutation in one repeat;</p><p>**Imperfect Microsatellite: one mutation in two repeats;</p><p>*** Imperfect Microsatellite: one mutation in three repeats; (d): a 58 bp deletion associated to the microsatellite; NA: not applicable; −: no microsatellite observed or no mutations at priming site; +: presence of mismatch at priming site.</p><p>Selected features characterizing the cloned RAPD markers.</p

Comparative inter-species analysis of mutations within the RAPD priming sites.

<p>Comparative inter-species analysis of mutations within the RAPD priming sites.</p

Evolution of Plasmodium falciparum drug resistance genes following artemisinin combination therapy in Sudan

Background Malaria control efforts in Sudan rely heavily on case management. In 2004, health authorities adopted artemisinin-based combination therapies (ACTs) for the treatment of uncomplicated malaria. However, some recent surveys have reported ACT failure and a prevalent irrational malaria treatment practice. Here we examine whether the widespread use of ACT and failure to adhere to national guidelines have led to the evolution of drug resistance genes. Methods We genotyped known drug resistance markers (Pfcrt, Pfmdr-1, Pfdhfr, Pfdhps, Pfk13 propeller) and their flanking microsatellites among Plasmodium falciparum isolates obtained between 2009 and 2016 in different geographical regions in Sudan. Data were then compared with published findings pre-ACT (1992–2003). Results A high prevalence of Pfcrt76T, Pfmdr-1-86Y, Pfdhfr51I, Pfdhfr108N, Pfdhps37G was observed in all regions, while no Pfk13 mutations were detected. Compared with pre-ACT data, Pfcrt-76T and Pfmdr-1-86Y have decayed, while Pfdhfr-51I, Pfdhfr-108N and Pfdhps-437G strengthened. Haplotypes Pfcrt-CVIET, Pfmdr-1-NFSND/YFSND, Pfdhfr-ICNI and Pfdhps-SGKAA predominated in all sites. Microsatellites flanking drug resistance genes showed lower diversity than neutral ones, signifying high ACT pressure/selection. Conclusions Evaluation of P. falciparum drug resistance genes in Sudan matches the drug deployment pattern. Regular monitoring of these genes, coupled with clinical response, should be considered to combat the spread of ACT resistance