Publications by authors named "L Van Bockstal"

18 Publications

  • Page 1 of 1

Monosaccharides Dehydration Assisted by Formation of Borate Esters of α-Hydroxyacids in Choline Chloride-Based Low Melting Mixtures.

Front Chem 2020 7;8:569. Epub 2020 Jul 7.

Laboratory of Biomass and Green Technologies, University of Liege-Gembloux Agro-Bio Tech, Gembloux, Belgium.

The synthesis of 5-hydroxymethylfurfural (5-HMF) and 2-furfural (2-F) by hexoses and pentoses dehydration is considered as a promising path to produce materials from renewable resources. Low-transition-temperature mixtures (LTTMs) enable selective (>80%) dehydration of ketoses to furanic derivatives at moderate temperature (<100°C). However, aldoses dehydration generally requires higher temperatures and an isomerization catalyst. Chromium trichloride has been reported as one of the most efficient catalyst but its kinetic inertness could limit its performances below 100°C. Consequently, we investigate herein boric acid catalysis of aldoses dehydration in LTTMs based on choline halides and organic acids at 90°C. The limited activity of boric acid regarding furanic compounds synthesis (e.g., 5% 5-HMF yield and 23% glucose conversion after 1 h at 90°C with maleic acid) can be enhanced through tetrahydroxyborate esters (THBE) formation with α-hydroxyacids (e.g., 19% 5-HMF yield and 61% glucose conversion after 1 h at 90°C). THBE formation is however associated with HO generation favoring the appearance of side products (humins). We demonstrate that boric acid catalysis is not straightforward and that the use of THBE under moderate acidity should be further investigated to limit humins formation and promote furanic derivatives synthesis.
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http://dx.doi.org/10.3389/fchem.2020.00569DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7358950PMC
July 2020

Sand Fly Studies Predict Transmission Potential of Drug-resistant Leishmania.

Trends Parasitol 2020 09 23;36(9):785-795. Epub 2020 Jul 23.

Laboratory of Microbiology, Parasitology and Hygiene (LMPH), University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk, Belgium. Electronic address:

Leishmania parasites have the capacity to rapidly adapt to changing environments in their digenetic life cycle which alternates between a vertebrate and an invertebrate host. Emergence of resistance following drug exposure can evoke phenotypic alterations that affect several aspects of parasite fitness in both hosts. Current studies of the impact of resistance are mostly limited to interactions with the mammalian host and characterization of in vitro parasite growth and differentiation. Development in the vector and transmission capacity have been largely ignored. This review reflects on the impact of drug resistance on its spreading potential with specific focus on the use of the sand fly infection model to evaluate parasite development in the vector and the ensuing transmission potential of drug-resistant phenotypes.
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http://dx.doi.org/10.1016/j.pt.2020.06.006DOI Listing
September 2020

Interferon Alpha Favors Macrophage Infection by Visceral Species Through Upregulation of Sialoadhesin Expression.

Front Immunol 2020 9;11:1113. Epub 2020 Jun 9.

Laboratory of Microbiology, Parasitology and Hygiene (LMPH), Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Wilrijk, Belgium.

Type I interferons (IFNs) induced by an endogenous RNA virus or exogenous viral infections have been shown to exacerbate infections with New World Cutaneous parasites, however, the impact of type I IFNs in visceral infections and implicated mechanisms remain to be unraveled. This study assessed the impact of type I IFN on macrophage infection with and and the implication of sialoadhesin (Siglec-1/CD169, Sn) as an IFN-inducible surface receptor. Stimulation of bone marrow-derived macrophages with type I IFN (IFN-α) significantly enhanced susceptibility to infection of reference laboratory strains and a set of recent clinical isolates. IFN-α particularly enhanced promastigote uptake. Enhanced macrophage susceptibility was linked to upregulated Sn surface expression as a major contributing factor to the infection exacerbating effect of IFN-α. Stimulation experiments in Sn-deficient macrophages, macrophage pretreatment with a monoclonal anti-Sn antibody or a novel bivalent anti-Sn nanobody and blocking of parasites with soluble Sn restored normal susceptibility levels. Infection of Sn-deficient mice with bioluminescent promastigotes revealed a moderate, strain-dependent role for Sn during visceral infection under the used experimental conditions. These data indicate that IFN-responsive Sn expression can enhance the susceptibility of macrophages to infection with visceral promastigotes and that targeting of Sn may have some protective effects in early infection.
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http://dx.doi.org/10.3389/fimmu.2020.01113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7296180PMC
April 2021

Evaluation of a pan-Leishmania SL RNA qPCR assay for parasite detection in laboratory-reared and field-collected sand flies and reservoir hosts.

Parasit Vectors 2020 Jun 1;13(1):276. Epub 2020 Jun 1.

Laboratory of Microbiology, Parasitology and Hygiene, University of Antwerp, Wilrijk, Belgium.

Background: In eco-epidemiological studies, Leishmania detection in vectors and reservoirs is frequently accomplished by high-throughput and sensitive molecular methods that target minicircle kinetoplast DNA (kDNA). A pan-Leishmania SYBR green quantitative PCR (qPCR) assay which detects the conserved spliced-leader RNA (SL RNA) sequence was developed recently. This study assessed the SL RNA assay performance combined with a crude extraction method for the detection of Leishmania in field-collected and laboratory-reared sand flies and in tissue samples from hyraxes as reservoir hosts.

Methods: Field-collected and laboratory-infected sand fly and hyrax extracts were subjected to three different qPCR approaches to assess the suitability of the SL RNA target for Leishmania detection. Nucleic acids of experimentally infected sand flies were isolated with a crude extraction buffer with ethanol precipitation and a commercial kit and tested for downstream DNA and RNA detection. Promastigotes were isolated from culture and sand fly midguts to assess whether there was difference in SL RNA and kDNA copy numbers. Naive sand flies were spiked with a serial dilution of promastigotes to make a standard curve.

Results: The qPCR targeting SL RNA performed well on infected sand fly samples, despite preservation and extraction under presumed unfavorable conditions for downstream RNA detection. Nucleic acid extraction by a crude extraction buffer combined with a precipitation step was highly compatible with downstream SL RNA and kDNA detection. Copy numbers of kDNA were found to be identical in culture-derived parasites and promastigotes isolated from sand fly midguts. SL RNA levels were slightly lower in sand fly promastigotes (ΔCq 1.7). The theoretical limit of detection and quantification of the SL RNA qPCR respectively reached down to 10 and 10 parasite equivalents. SL RNA detection in stored hyrax samples was less efficient with some false-negative assay results, most likely due to the long-term tissue storage in absence of RNA stabilizing reagents.

Conclusions: This study shows that a crude extraction method in combination with the SL RNA qPCR assay is suitable for the detection and quantification of Leishmania in sand flies. The assay is inexpensive, sensitive and pan-Leishmania specific, and accordingly an excellent assay for high-throughput screening in entomological research.
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http://dx.doi.org/10.1186/s13071-020-04141-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7268266PMC
June 2020

Impact of clinically acquired miltefosine resistance by Leishmania infantum on mouse and sand fly infection.

Int J Parasitol Drugs Drug Resist 2020 08 1;13:16-21. Epub 2020 May 1.

Laboratory of Microbiology, Parasitology and Hygiene (LMPH), University of Antwerp, Universiteitsplein 1, B-2610, Wilrijk, Belgium. Electronic address:

Objectives: This study evaluated the implications of clinically acquired miltefosine resistance (MIL-R) by assessing virulence in mice and sand flies to reveal the potential of MIL-R strains to circulate.

Methods: Experimental infections with the MIL-R clinical Leishmania infantum isolate MHOM/FR/2005/LEM5159, having a defect in the LiROS3 subunit of the MIL-transporter, and its syngeneic experimentally reconstituted MIL-S counterpart (LEM5159) were performed in BALB/c mice and Lutzomyia longipalpis and Phlebotomus perniciosus sand flies. In mice, the amastigote burdens in liver and spleen were compared microscopically using Giemsa smears and by bioluminescent imaging. During the sand fly infections, the percentage of infected flies, parasite load, colonization of the stomodeal valve and metacyclogenesis were evaluated. The stability of the MIL-R phenotype after sand fly and mouse passage was determined as well.

Results: The fitness of the MIL-R strain differed between the mouse and sand fly infection model. In mice, a clear fitness loss was observed compared to the LiROS3-reconstituted susceptible strain. This defect could be rescued by episomal reconstitution with a wildtype LiROS3 copy. However, this fitness loss was not apparent in the sand fly vector, resulting in metacyclogenesis and efficient colonization of the stomodeal valve. Resistance was stable after passage in both sand fly and mouse.

Conclusion: The natural MIL-R strain is significantly hampered in its ability to multiply and cause a typical visceral infection pattern in BALB/c mice. However, this LiROS3-deficient strain efficiently produced mature infections and metacyclic promastigotes in the sand fly vector highlighting the transmission potential of this particular MIL-R clinical Leishmania strain.
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http://dx.doi.org/10.1016/j.ijpddr.2020.04.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7215113PMC
August 2020

Phenotypic adaptations of Leishmania donovani to recurrent miltefosine exposure and impact on sand fly infection.

Parasit Vectors 2020 Feb 22;13(1):96. Epub 2020 Feb 22.

Laboratory of Microbiology, Parasitology and Hygiene (LMPH), University of Antwerp, Antwerp, Belgium.

Background: Since the introduction of miltefosine (MIL) as first-line therapy in the kala-azar elimination programme in the Indian subcontinent, treatment failure rates have been increasing. Since parasite infectivity and virulence may become altered upon treatment relapse, this laboratory study assessed the phenotypic effects of repeated in vitro and in vivo MIL exposure.

Methods: Syngeneic Leishmania donovani lines either or not exposed to MIL were compared for drug susceptibility, rate of promastigote multiplication and metacyclogenesis, macrophage infectivity and behaviour in the sand fly vector, Lutzomyia longipalpis.

Results: Promastigotes of both in vitro and in vivo MIL-selected strains displayed a slightly reduced drug susceptibility that was associated with a reduced MIL-accumulation linked to a lower copy number (disomic state) of chromosome 13 harboring the miltefosine transporter (LdMT) gene. In vitro selected promastigotes showed a lower rate of metacyclogenesis whereas the in vivo derived promastigotes displayed a moderately increased growth rate. Repeated MIL exposure did neither influence the parasite load nor metacyclogenesis in the sand fly vector.

Conclusions: Recurrent in vitro and in vivo MIL exposure evokes a number of very subtle phenotypic and genotypic changes which could make promastigotes less susceptible to MIL without attaining full resistance. These changes did not significantly impact on infection in the sand fly vector.
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http://dx.doi.org/10.1186/s13071-020-3972-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7036194PMC
February 2020

Production of 5-Hydroxymethylfurfural from D-Fructose in Low-Transition-Temperature Mixtures Enhanced by Chloride Anions and Low Amounts of Organic Acids.

Chempluschem 2018 Dec 13;83(12):1135-1143. Epub 2018 Nov 13.

Laboratory of Biomass and Green Technologies, University of Liege, 2, Passage des Déportés, 5030, Gembloux, Belgium.

The use of safe and sustainable solvents able to solvate reagents and to catalyze their reactions at temperatures below 100 °C is an innovative strategy to develop future lignocellulosic biorefineries. Many low-transition-temperature mixtures (LTTMs) have been investigated for this purpose. Among them, natural deep eutectic solvents (NADESs) have been proposed as cheap and renewable alternatives to ionic liquids for the synthesis of bio-based chemical building blocks. We compare herein the ability of several organic acids/choline chloride/water LTTMs to perform D-fructose dehydration to 5-hydroxymethylfurfural (5-HMF). The addition of chloride salts as well as an increased proportion of choline chloride promotes 5-HMF formation, which seems to indicate a beneficial effect of chloride anions on D-fructose dehydration. Besides improving selectivity by at least 10 %, increasing the choline chloride/acid ratio could enhance the biodegradability of the LTTMs. Unlike other acidic components, maleic and citric acids are especially selective at early D-fructose conversion. Maleic acid was the most selective acidic component among the tested chemicals, achieving an 80 % 5-HMF molar yield in 1 h at 90 °C.
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http://dx.doi.org/10.1002/cplu.201800416DOI Listing
December 2018

Transmission potential of paromomycin-resistant Leishmania infantum and Leishmania donovani.

J Antimicrob Chemother 2020 04;75(4):951-957

Laboratory of Microbiology, Parasitology and Hygiene (LMPH), University of Antwerp, Antwerp, Belgium.

Objectives: Former studies demonstrated quick selection of paromomycin resistance for Leishmania infantum and Leishmania donovani accompanied by increased fitness. The present study aimed to interpret these findings in an epidemiological context by comparing infection of WT and experimentally derived paromomycin-resistant strains in the sand fly vector.

Methods: Depending on the Leishmania species, Lutzomyia longipalpis and Phlebotomus perniciosus or Phlebotomus argentipes sand flies were artificially infected with procyclic promastigotes of WT and paromomycin-resistant L. infantum (MHOM/FR/96/LEM3323-cl4) or L. donovani (MHOM/NP/03/BPK275/0-cl18). The infection rate and gut/stomodeal valve colonization were determined to monitor parasite phenotypic behaviour within the vector. The impact of the previously described gain of fitness in the vertebrate host on infectivity for the vector was assessed by feeding L. longipalpis on Syrian golden hamsters heavily infected with either WT or paromomycin-resistant parasites.

Results: WT and paromomycin-resistant Leishmania of both species behaved similarly in terms of infection and parasite location within the studied sand fly species. Blood feeding on infected hamsters did not reveal differences in acquisition of WT and paromomycin-resistant parasites, despite the higher organ burdens observed for the paromomycin-resistant strain. Strains remained resistant after passage in the vector.

Conclusions: Although paromomycin-resistant parasites show an increased parasite fitness in vitro and in laboratory rodents, the intrinsic infection potential of paromomycin-resistant parasites remains unaltered in the sand fly. Of importance is the fact that paromomycin-resistant Leishmania are able to complete development in the natural vectors and produce stomodeal infection with metacyclic forms, which clearly suggests their potential to spread and circulate in nature.
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http://dx.doi.org/10.1093/jac/dkz517DOI Listing
April 2020

In-depth comparison of cell-based methodological approaches to determine drug susceptibility of visceral Leishmania isolates.

PLoS Negl Trop Dis 2019 12 2;13(12):e0007885. Epub 2019 Dec 2.

Laboratory of Microbiology, Parasitology and Hygiene (LMPH), University of Antwerp, Antwerp, Belgium.

Monitoring the drug susceptibility of Leishmania isolates still largely relies on standard in vitro cell-based susceptibility assays using (patient-isolated) promastigotes for infection. Although this assay is widely used, no fully standardized/harmonized protocol is yet available hence resulting in the application of a wide variety of host cells (primary cells and cell lines), different drug exposure times, detection methods and endpoint criteria. Advocacy for standardization to decrease inter-laboratory variation and improve interpretation of results has already repeatedly been made, unfortunately still with unsatisfactory progress. As a logical next step, it would be useful to reach at least some agreement on the type of host cell and basic experimental design for routine amastigote susceptibility determination. The present laboratory study using different L. infantum strains as a model for visceral leishmaniasis species compared primary cells (mouse peritoneal exudate (PEC), mouse bone marrow derived macrophages and human peripheral blood monocyte derived macrophages) and commercially available cell lines (THP-1, J774, RAW) for either their susceptibility to infection, their role in supporting intracellular amastigote multiplication and overall feasibility/accessibility of experimental assay protocol. The major findings were that primary cells are better than cell lines in supporting infection and intracellular parasite multiplication, with PECs to be preferred for technical reasons. Cell lines require drug exposure of >96h with THP-1 to be preferred but subject to a variable response to PMA stimulation. The fast dividing J774 and RAW cells out-compete parasite-infected cells precluding proper assay read-out. Some findings could possibly also be applicable to cutaneous Leishmania strains, but this still needs cross-checking. Besides inherent limitations in a clinical setting, susceptibility testing of clinical isolates may remain problematic because of the reliance on patient-derived promastigotes which may exhibit variable degrees of metacyclogenesis and infectivity.
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http://dx.doi.org/10.1371/journal.pntd.0007885DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6907865PMC
December 2019

Impaired development of a miltefosine-resistant Leishmania infantum strain in the sand fly vectors Phlebotomus perniciosus and Lutzomyia longipalpis.

Int J Parasitol Drugs Drug Resist 2019 12 10;11:1-7. Epub 2019 Sep 10.

Laboratory of Microbiology, Parasitology and Hygiene (LMPH), University of Antwerp, Universiteitsplein 1, B-2610, Wilrijk, Belgium. Electronic address:

Objectives: To gain insight into the propagation of miltefosine (MIL) resistance in visceral leishmaniasis, this laboratory study explored development of resistant parasites with a defective miltefosine transporter (MT) in sand flies.

Methods: Infectivity, colonization of stomodeal valve and metacyclogenesis of a MIL-resistant (MIL-R) Leishmania infantum LEM3323 line with a defective MT were assessed in the natural sand fly vectors Phlebotomus perniciosus and Lutzomyia longipalpis. Given our recent description of partial drug dependency of the MT-deficient line, the impact of MIL pre-exposure on sand fly infectivity was explored as well.

Results: A significant reduction in sand fly infection, stomodeal valve colonization and differentiation into metacyclics (determined by a lower flagellum/cell body length ratio) was observed in both vectors for MIL-R as compared to the isogenic parent MIL-susceptible line. Re-introduction of the wildtype MT gene into MIL-R tended to partially rescue the capacity to infect sand flies. Pre-exposure to MIL did not alter infectivity of the MIL-R line.

Conclusion: The MIL resistant L. infantum LEM3323 line is significantly hampered in its development and transmissibility potential in two sand fly vector species. Additional studies are warranted to evaluate whether this applies to other visceral Leishmania parasites with acquired MIL-resistance.
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http://dx.doi.org/10.1016/j.ijpddr.2019.09.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6804374PMC
December 2019

Cysticercosis and taeniasis cases diagnosed at two referral medical institutions, Belgium, 1990 to 2015.

Euro Surveill 2019 Aug;24(35)

Department of Virology, Parasitology and Immunology, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.

BackgroundFew case reports on human infections with the beef tapeworm and the pork tapeworm, , diagnosed in Belgium have been published, yet the grey literature suggests a higher number of cases.AimTo identify and describe cases of taeniasis and cysticercosis diagnosed at two Belgian referral medical institutions from 1990 to 2015.MethodsIn this observational study we retrospectively gathered data on taeniasis and cysticercosis cases by screening laboratory, medical record databases as well a uniform hospital discharge dataset.ResultsA total of 221 confirmed taeniasis cases were identified. All cases for whom the causative species could be determined (170/221, 76.9%) were found to be infections. Of those with available information, 40.0% were asymptomatic (26/65), 15.4% reported diarrhoea (10/65), 9.2% reported anal discomfort (6/65) and 15.7% acquired the infection in Belgium (11/70). Five definitive and six probable cases of neurocysticercosis (NCC), and two cases of non-central nervous system cysticercosis (non-CNS CC) were identified. Common symptoms and signs in five of the definitive and probable NCC cases were epilepsy, headaches and/or other neurological disorders. Travel information was available for 10 of the 13 NCC and non-CNS CC cases; two were Belgians travelling to and eight were immigrants or visitors travelling from endemic areas.ConclusionsThe current study indicates that a non-negligible number of taeniasis cases visit Belgian medical facilities, and that cysticercosis is occasionally diagnosed in international travellers.
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http://dx.doi.org/10.2807/1560-7917.ES.2019.24.35.1800589DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6724463PMC
August 2019

Miltefosine enhances the fitness of a non-virulent drug-resistant Leishmania infantum strain.

J Antimicrob Chemother 2019 02;74(2):395-406

Laboratory of Microbiology, Parasitology and Hygiene (LMPH), University of Antwerp, Wilrijk, Belgium.

Objectives: Miltefosine is currently the only oral drug for visceral leishmaniasis, and although deficiency in an aminophospholipid/miltefosine transporter (MT) is sufficient to elicit drug resistance, very few naturally miltefosine-resistant (MIL-R) strains have yet been isolated. This study aimed to make a detailed analysis of the impact of acquired miltefosine resistance and miltefosine treatment on in vivo infection.

Methods: Bioluminescent versions of a MIL-R strain and its syngeneic parental line were generated by integration of the red-shifted firefly luciferase PpyRE9. The fitness of both lines was compared in vitro (growth rate, metacyclogenesis and macrophage infectivity) and in BALB/c mice through non-invasive bioluminescence imaging under conditions with and without drug pressure.

Results: This study demonstrated a severe fitness loss of MT-deficient parasites, resulting in a complete inability to multiply and cause a typical visceral leishmaniasis infection pattern in BALB/c mice. The observed fitness loss could not be rescued by host immune suppression with cyclophosphamide, whereas episomal reconstitution with a wild-type MT restored parasite virulence, hence linking parasite fitness to MT mutation. Remarkably, in vivo miltefosine treatment or in vitro miltefosine pre-exposure significantly rescued MIL-R parasite virulence. The in vitro pre-exposed MIL-R promastigotes showed a longer and more slender morphology, suggesting an altered membrane composition.

Conclusions: The profound fitness loss of MT-deficient parasites most likely explains the low frequency of MIL-R clinical isolates. The observation that miltefosine can reverse this phenotype indicates a drug dependency of the MT-deficient parasites and emphasizes the importance of resistance profiling prior to miltefosine administration.
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http://dx.doi.org/10.1093/jac/dky450DOI Listing
February 2019

Impact of primary mouse macrophage cell types on Leishmania infection and in vitro drug susceptibility.

Parasitol Res 2018 Nov 23;117(11):3601-3612. Epub 2018 Aug 23.

Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, Laboratory of Microbiology, Parasitology and Hygiene, University of Antwerp, Wilrijk, Belgium.

Primary mouse macrophages are frequently used to provide an in vitro intracellular model to evaluate antileishmanial drug efficacy. The present study compared the phenotypic characteristics of Swiss, BALB/c, and C57BL/6 mouse bone marrow-derived macrophages and peritoneal exudate cells using different stimulation and adherence protocols upon infection with a Leishmania infantum laboratory strain and two clinical isolates. Evaluation parameters were susceptibility to infection, permissiveness to amastigote multiplication, and impact on drug efficacy. Observed variations in infection of peritoneal exudate cells can mostly be linked to changes in the inflammatory cytokine profiles (IL-6, TNF-α, KC/GRO) rather than to differences in initial production of nitric oxide and reactive oxygen species. Optimization of the cell stimulation and adherence conditions resulted in comparable infection indices among peritoneal exudate cells and the various types of bone marrow-derived macrophages. BALB/c-derived bone marrow-derived macrophages were slightly more permissive to intracellular amastigote replication. Evaluation of antileishmanial drug potency in the various cell systems revealed minimal variation for antimonials and paromomycin, and no differences for miltefosine and amphotericin B. The study results allow to conclude that drug evaluation can be performed in all tested primary macrophages as only marginal differences are observed in terms of susceptibility to infection and impact of drug exposure. Combined with some practical considerations, the use of 24-h starch-stimulated, 48-h adhered, Swiss-derived peritoneal exudate cells can be advocated as an efficient, reliable, relatively quick, and cost-effective tool for routine drug susceptibility testing in vitro whenever the use of primary cells is feasible.
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http://dx.doi.org/10.1007/s00436-018-6059-4DOI Listing
November 2018

Evaluation of a Pan-Leishmania Spliced-Leader RNA Detection Method in Human Blood and Experimentally Infected Syrian Golden Hamsters.

J Mol Diagn 2018 03 31;20(2):253-263. Epub 2018 Jan 31.

Laboratory of Microbiology, Parasitology and Hygiene, University of Antwerp, Wilrijk, Belgium. Electronic address:

Several methods have been developed for the detection of Leishmania, mostly targeting the minicircle kinetoplast DNA (kDNA). A new RNA real-time quantitative PCR (qPCR) assay was developed targeting the conserved and highly expressed spliced-leader (SL) mini-exon sequence. This study compared the limits of detection of various real-time PCR assays in hamsters infected with Leishmania infantum, in spiked human blood, and in clinical blood samples from visceral leishmaniasis patients. The SL-RNA assay showed an excellent analytical sensitivity in tissues (0.005 and 0.002 parasites per mg liver and spleen, respectively) and was not prone to false-positive reactions. Evaluation of the SL-RNA assay on clinical samples demonstrated lower threshold cycle values than the kDNA qPCR, an excellent interrun stability of 97%, a 93% agreement with the kDNA assay, and an estimated sensitivity, specificity, and accuracy of 93.2%, 94.3%, and 93.8%, respectively. The SL-RNA qPCR assay was equally efficient for detecting Leishmania major, Leishmania tropica, Leishmania mexicana, Leishmania guayensis, Leishmania panamensis, Leishmania braziliensis, L. infantum, and Leishmania donovani and revealed similar SL-RNA levels in the different species and the occurrence of polycistronic SL-containing transcripts in Viannia species. Collectively, this single SL-RNA qPCR assay enables universal Leishmania detection and represents a particularly useful addition to the widely used kDNA assay in clinical studies in which the detection of viable parasites is pivotal to assess parasitological cure.
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http://dx.doi.org/10.1016/j.jmoldx.2017.12.003DOI Listing
March 2018

Miltefosine-resistant Leishmania infantum strains with an impaired MT/ROS3 transporter complex retain amphotericin B susceptibility.

J Antimicrob Chemother 2018 02;73(2):392-394

Laboratory of Microbiology, Parasitology and Hygiene (LMPH), University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk, Belgium.

Objectives: Increasing numbers of miltefosine treatment failures in visceral leishmaniasis therapy and reports of miltefosine resistance in the Indian subcontinent resulted in the recommendation to use liposomal amphotericin B as first-line therapy. Cross-resistance between miltefosine and amphotericin B has recently been documented, suggesting a role of mutations in the miltefosine transporter, a complex encoded by the MT and ROS3 genes. This study aimed to further explore the putative role of MT/ROS3 defects in the molecular basis of amphotericin B cross-resistance.

Methods: The susceptibility profiles of different miltefosine-resistant Leishmania infantum strains with well-characterized mutations in the transporter complex and the corresponding episomally restored susceptible parasite lines were determined using both the routine extracellular promastigote assay and the intracellular amastigote assay.

Results: In vitro amastigote and promastigote susceptibility testing of the two miltefosine-resistant and the episomally reconstituted L. infantum lines revealed full susceptibility to amphotericin B, despite the variable miltefosine susceptibility profile.

Conclusions: Mutations present in either the MT and/or ROS3 gene are not sufficient to elicit higher tolerance to amphotericin B. Additional synergistic adaptations may be responsible for the miltefosine/amphotericin B cross-resistance described earlier.
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http://dx.doi.org/10.1093/jac/dkx407DOI Listing
February 2018