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Sand Fly Studies Predict Transmission Potential of Drug-resistant Leishmania

2020; Elsevier BV; Volume: 36; Issue: 9 Linguagem: Inglês

10.1016/j.pt.2020.06.006

ISSN

1471-5007

Autores

Lieselotte Van Bockstal, Sarah Hendrickx, Louis Maes, Guy Caljon,

Tópico(s)

Trypanosoma species research and implications

Resumo

Drug resistance represents a major threat for the control of visceral leishmaniasis (VL).Monitoring the spread of resistance is essential to adjust control interventions and safeguard current and future drugs.Sand fly infection studies represent a useful laboratory tool to assess the transmission potential of resistant phenotypes.The resistance-conferring genetic alterations determine transmissibility by the vector.The possibility of recombination between different Leishmania strains and species in the vector may result in the formation of transmissible hybrids. 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. 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. Leishmaniasis currently affects 12 million people and poses an infection risk to one billion people. Visceral leishmaniasis (VL) is the most severe form, causing over 20 000 deaths each year (https://www.who.int/leishmaniasis/en/). It is one of the world's most important neglected diseases affecting poor populations in developing countries [1.Burza S. et al.Leishmaniasis.Lancet. 2018; 392: 951-970Abstract Full Text Full Text PDF PubMed Scopus (563) Google Scholar]. VL can be treated with only a small number of drugs, all of which show moderate to severe side-effects, and only a few promising novel drug leads have recently entered clinical development [2.Chakravarty J. Sundar S. Current and emerging medications for the treatment of leishmaniasis.Expert. Opin. Pharmacother. 2019; 20: 1251-1265Crossref PubMed Scopus (48) Google Scholar,3.Hendrickx S. et al.Need for sustainable approaches in antileishmanial drug discovery.Parasitol. Res. 2019; 118: 2743-2752Crossref PubMed Scopus (15) Google Scholar]. The further expansion of drug resistance (see Glossary) remains a major threat and is already reported for all commonly used drugs [4.Ponte-Sucre A. et al.Drug resistance and treatment failure in leishmaniasis: a 21st century challenge.PLoS Negl. Trop. Dis. 2017; 11e0006052Crossref PubMed Scopus (321) Google Scholar]. The application of particular drugs for canine leishmaniasis may further accelerate the development of resistance through zoonotic transmission [5.Reguera R.M. et al.Current status on prevention and treatment of canine leishmaniasis.Vet. Parasitol. 2016; 227: 98-114Crossref PubMed Scopus (57) Google Scholar]. Because antileishmanial therapy is much less effective in HIV-positive patients and in other severely immunocompromised individuals, no sterile immunity is achieved, rendering these patients reservoirs of drug-resistant parasites [6.Akuffo H. et al.New insights into leishmaniasis in the immunosuppressed.PLoS Negl. Trop. Dis. 2018; 12e0006375Crossref PubMed Scopus (39) Google Scholar]. For this reason, it is important to monitor the development and spread of resistance against current and future antileishmanial chemotherapy [7.Hendrickx S. et al.Evaluating drug resistance in visceral leishmaniasis: the challenges.Parasitology. 2016; 145: 453-463Crossref PubMed Scopus (29) Google Scholar]. Beyond these considerations, and receiving much less attention, the evaluation of the impact of resistance on parasite fitness in the sand fly vector may be a useful laboratory method to assess the spreading potential and epidemiological impact of such a phenotype. The current fitness studies are mostly limited to the mammalian host and in vitro promastigote growth and differentiation [8.Garcia-Hernandez R. et al.Fitness of Leishmania donovani parasites resistant to drug combinations.PLoS Negl. Trop. Dis. 2015; 9e0003704Crossref PubMed Scopus (23) Google Scholar], while fitness in the sand fly and transmission capacity to the mammalian host are indeed largely overlooked. Previous studies have shown that drug resistance to antimonials (Sb) [9.Vanaerschot M. et al.Antimonial resistance in Leishmania donovani is associated with increased in vivo parasite burden.PLoS One. 2011; 6e23120Crossref PubMed Scopus (46) Google Scholar], paromomycin (PMM) [10.Hendrickx S. et al.Evidence of a drug-specific impact of experimentally selected paromomycin and miltefosine resistance on parasite fitness in Leishmania infantum.J. Antimicrob. Chemother. 2016; 71: 1914-1921Crossref PubMed Scopus (24) Google Scholar, 11.Hendrickx S. et al.Experimental induction of paromomycin resistance in antimony-resistant strains of L. donovani: outcome dependent on in vitro selection protocol.PLoS Negl. Trop. Dis. 2012; 6e1664Crossref PubMed Scopus (38) Google Scholar, 12.Shaw C. et al.Genomic and metabolomic polymorphism among experimentally selected paromomycin-resistant Leishmania donovani strains.Antimicrob. Agents Chemother. 2019; 64e00904-19Crossref PubMed Scopus (6) Google Scholar, 13.Bhandari V. et al.Elucidation of cellular mechanisms involved in experimental paromomycin resistance in Leishmania donovani.Antimicrob. Agents Chemother. 2014; 58: 2580-2585Crossref PubMed Scopus (51) Google Scholar], and miltefosine (MIL) [10.Hendrickx S. et al.Evidence of a drug-specific impact of experimentally selected paromomycin and miltefosine resistance on parasite fitness in Leishmania infantum.J. Antimicrob. Chemother. 2016; 71: 1914-1921Crossref PubMed Scopus (24) Google Scholar,14.Rai K. et al.Relapse after treatment with miltefosine for visceral leishmaniasis is associated with increased infectivity of the infecting Leishmania donovani strain.mBio. 2013; 4e00611-13Crossref PubMed Scopus (50) Google Scholar] in Leishmania can result in alterations to some aspects affecting parasite fitness [15.Vanaerschot M. et al.The concept of fitness in Leishmania.in: Ponte-Sucre A. Padrón-Nieves M. Drug Resistance in Leishmania Parasites. Springer, 2018: 341-366Crossref Scopus (1) Google Scholar] while studies in the vector are only recently emerging. Parasite fitness is a complex phenomenon involving several factors that assure parasite survival, reproduction, and transmission (Figure 1) [15.Vanaerschot M. et al.The concept of fitness in Leishmania.in: Ponte-Sucre A. Padrón-Nieves M. Drug Resistance in Leishmania Parasites. Springer, 2018: 341-366Crossref Scopus (1) Google Scholar]. The Leishmania life cycle in the vector includes a series of interactions that, in many cases, are species-specific. This species-specificity is driven by several molecular factors that allow the parasite to infect, survive, and multiply within the sand fly midgut and be transmitted to a suitable vertebrate host (Figure 2, Key Figure) [16.Dvorak V. et al.Parasite biology: the vectors.in: Bruschi F. Gradoni L. The Leishmaniases: Old Neglected Tropical Diseases. Springer, 2018: 31-77Crossref Scopus (24) Google Scholar, 17.Sacks D. Kamhawi S. Molecular aspects of parasite–vector and vector–host interactions in leishmaniasis.Annu. Rev. Microbiol. 2001; 55: 453-483Crossref PubMed Scopus (267) Google Scholar, 18.Ramalho-Ortigao M. et al.Sand fly-Leishmania interactions: long relationships are not necessarily easy.Open Parasitol. J. 2010; 4: 195Crossref PubMed Scopus (24) Google Scholar]. According to their ability to support late-stage development of different Leishmania species under experimental conditions, sand flies have been subdivided into two categories [19.Kamhawi S. Phlebotomine sand flies and Leishmania parasites: friends or foes?.Trends Parasitol. 2006; 22: 439-445Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar,20.Myskova J. et al.A lipophosphoglycan-independent development of Leishmania in permissive sand flies.Microbes Infect. 2007; 9: 317-324Crossref PubMed Scopus (79) Google Scholar]. Some sand flies are vectors for specific species of Leishmania, and are called 'specific' vectors, while others support the full development of a broad range of Leishmania species and are thus called 'permissive' vectors [16.Dvorak V. et al.Parasite biology: the vectors.in: Bruschi F. Gradoni L. The Leishmaniases: Old Neglected Tropical Diseases. Springer, 2018: 31-77Crossref Scopus (24) Google Scholar]. Parasites need to survive and multiply in the vector to complete their development, for which several barriers must be overcome. Besides the physiological barriers, sand flies mount an immune response upon Leishmania infection through the immune deficiency (IMD) pathway [21.Louradour I. et al.CRISPR/Cas9 mutagenesis in Phlebotomus papatasi: the immune deficiency pathway impacts vector competence for Leishmania major.mBio. 2019; 10e01941-19Crossref PubMed Scopus (9) Google Scholar] with the induction of antimicrobial peptides [22.Boulanger N. et al.Characterization of a defensin from the sand fly Phlebotomus duboscqi induced by challenge with bacteria or the protozoan parasite Leishmania major.Infect. Immun. 2004; 72: 7140-7146Crossref PubMed Scopus (98) Google Scholar,23.Telleria E.L. et al.Bacterial feeding, Leishmania infection and distinct infection routes induce differential defensin expression in Lutzomyia longipalpis.Parasit. Vectors. 2013; 6: 12Crossref PubMed Scopus (36) Google Scholar]. Somewhat surprisingly, no major production of reactive oxygen species seems to be induced by the parasite [24.Diaz-Albiter H. et al.Reactive oxygen species-mediated immunity against Leishmania mexicana and Serratia marcescens in the phlebotomine sand fly Lutzomyia longipalpis.J. Biol. Chem. 2012; 287: 23995-24003Crossref PubMed Scopus (59) Google Scholar]. For its survival, Leishmania strives to manipulate different aspects of the sand fly's physiology and immunity [18.Ramalho-Ortigao M. et al.Sand fly-Leishmania interactions: long relationships are not necessarily easy.Open Parasitol. J. 2010; 4: 195Crossref PubMed Scopus (24) Google Scholar].Figure 2Key Figure. Overview of Leishmania–Sand Fly Interactions and Factors Determining Parasite Fitness.Show full captionGraphical representation of the different parasite stages in the sand fly vector (numbers 1–6) and the essential factors determining parasite fitness (capital letters A–I). The various physiological and immunological players known to be involved in the parasite–sand fly interaction are indicated in the legend. Adapted from [18.Ramalho-Ortigao M. et al.Sand fly-Leishmania interactions: long relationships are not necessarily easy.Open Parasitol. J. 2010; 4: 195Crossref PubMed Scopus (24) Google Scholar].View Large Image Figure ViewerDownload Hi-res image Download (PPT) Graphical representation of the different parasite stages in the sand fly vector (numbers 1–6) and the essential factors determining parasite fitness (capital letters A–I). The various physiological and immunological players known to be involved in the parasite–sand fly interaction are indicated in the legend. Adapted from [18.Ramalho-Ortigao M. et al.Sand fly-Leishmania interactions: long relationships are not necessarily easy.Open Parasitol. J. 2010; 4: 195Crossref PubMed Scopus (24) Google Scholar]. Parasites first adapt to the changed temperature and increase in pH upon moving from the mammalian host to the sand fly midgut which triggers transformation into procyclic promastigotes [25.Dostalova A. Volf P. Leishmania development in sand flies: parasite–vector interactions overview.Parasit. Vectors. 2012; 5: 276Crossref PubMed Scopus (164) Google Scholar]. After a blood meal, parasites are surrounded by the peritrophic matrix (PM) [26.Pruzinova K. et al.Comparison of blood meal digestion and the peritrophic matrix in four sand fly species differing in susceptibility to Leishmania donovani.PLoS One. 2015; 10e0128203Crossref PubMed Scopus (28) Google Scholar] and need to overcome the digestive enzymes that may inhibit early growth [17.Sacks D. Kamhawi S. Molecular aspects of parasite–vector and vector–host interactions in leishmaniasis.Annu. Rev. Microbiol. 2001; 55: 453-483Crossref PubMed Scopus (267) Google Scholar]. Surface and secreted phosphoglycans protect against proteolytic damage [27.Secundino N. et al.Proteophosphoglycan confers resistance of Leishmania major to midgut digestive enzymes induced by blood feeding in vector sand flies.Cell. Microbiol. 2010; 12: 906-918Crossref PubMed Scopus (29) Google Scholar], enabling promastigotes to thrive in the digestive midgut environment [27.Secundino N. et al.Proteophosphoglycan confers resistance of Leishmania major to midgut digestive enzymes induced by blood feeding in vector sand flies.Cell. Microbiol. 2010; 12: 906-918Crossref PubMed Scopus (29) Google Scholar]. Several studies additionally described that Leishmania can modulate the levels of insect digestive enzymes [28.Telleria E.L. et al.Trypsin-like serine proteases in Lutzomyia longipalpis – expression, activity and possible modulation by Leishmania infantum chagasi.PLoS One. 2010; 5e10697Crossref PubMed Scopus (46) Google Scholar,29.Dostálová A. et al.The midgut transcriptome of Phlebotomus (Larroussius) perniciosus, a vector of Leishmania infantum: comparison of sugar fed and blood fed sand flies.BMC Genomics. 2011; 12: 223Crossref PubMed Scopus (24) Google Scholar]. The peritrophic matrix creates a physical barrier that may also prevent colonization. It was shown that the PM is broken down by sand fly-derived chitinases [30.Sádlová J. Volf P. Peritrophic matrix of Phlebotomus duboscqi and its kinetics during Leishmania major development.Cell Iissue Res. 2009; 337: 313Crossref PubMed Scopus (32) Google Scholar], enabling escape from the PM and the subsequent critical period of defecation since the excretion of the digested blood meal may result in loss of infection [17.Sacks D. Kamhawi S. Molecular aspects of parasite–vector and vector–host interactions in leishmaniasis.Annu. Rev. Microbiol. 2001; 55: 453-483Crossref PubMed Scopus (267) Google Scholar]. Leishmania parasites have circumvented this obstacle in two ways: (i) by secreting a myoinhibitory peptide that arrests hindgut peristalsis and avoids excretion [31.Vaidyanathan R. Isolation of a myoinhibitory peptide from Leishmania major (Kinetoplastida: Trypanosomatidae) and its function in the vector sand fly Phlebotomus papatasi (Diptera: Psychodidae).J. Med. Entomol. 2005; 42: 142-152Crossref PubMed Google Scholar] and (ii) by inserting their flagella between microvilli of the midgut epithelium [20.Myskova J. et al.A lipophosphoglycan-independent development of Leishmania in permissive sand flies.Microbes Infect. 2007; 9: 317-324Crossref PubMed Scopus (79) Google Scholar,32.Bates P.A. Leishmania sand fly interaction: progress and challenges.Curr. Opin. Microbiol. 2008; 11: 340-344Crossref PubMed Scopus (83) Google Scholar,33.Volf P. Myskova J. Sand flies and Leishmania: specific versus permissive vectors.Trends Parasitol. 2007; 23: 91-92Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar]. This attachment in the midgut is stage-dependent, being limited to the nectomonad and leptomonad forms and playing a major determining role in vector competence for Leishmania species [16.Dvorak V. et al.Parasite biology: the vectors.in: Bruschi F. Gradoni L. The Leishmaniases: Old Neglected Tropical Diseases. Springer, 2018: 31-77Crossref Scopus (24) Google Scholar]. In specific vectors, such as Phlebotomus papatasi, the attachment is controlled by galectin which serves as a receptor for lipophosphoglycan (LPG) on the surface of L. major [34.Kamhawi S. et al.A role for insect galectins in parasite survival.Cell. 2004; 119: 329-341Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar]. In contrast, attachment in permissive sand flies does not require LPG since even parasites deficient in LPG develop well [16.Dvorak V. et al.Parasite biology: the vectors.in: Bruschi F. Gradoni L. The Leishmaniases: Old Neglected Tropical Diseases. Springer, 2018: 31-77Crossref Scopus (24) Google Scholar,20.Myskova J. et al.A lipophosphoglycan-independent development of Leishmania in permissive sand flies.Microbes Infect. 2007; 9: 317-324Crossref PubMed Scopus (79) Google Scholar]. Whilst overcoming these barriers, parasites need to complete up to five different promastigote stages while moving anteriorly, before transforming into the infective metacyclic promastigote stage [19.Kamhawi S. Phlebotomine sand flies and Leishmania parasites: friends or foes?.Trends Parasitol. 2006; 22: 439-445Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar,35.Rogers M.E. The role of Leishmania proteophosphoglycans in sand fly transmission and infection of the mammalian host.Front. Microbiol. 2012; 3: 223Crossref PubMed Scopus (65) Google Scholar]. It has long been assumed that Leishmania parasites only undergo asexual clonal amplification; however, studies have indicated that genetic exchange occurs in the vector, resulting in the generation of hybrids [36.Inbar E. et al.The mating competence of geographically diverse Leishmania major strains in their natural and unnatural sand fly vectors.PLoS Genet. 2013; 9e1003672Crossref PubMed Scopus (58) Google Scholar, 37.Romano A. et al.Cross-species genetic exchange between visceral and cutaneous strains of Leishmania in the sand fly vector.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 16808-16813Crossref PubMed Scopus (42) Google Scholar, 38.Sadlova J. et al.Visualisation of Leishmania donovani fluorescent hybrids during early stage development in the sand fly vector.PLoS One. 2011; 6e19851Crossref PubMed Scopus (47) Google Scholar, 39.Volf P. et al.Increased transmission potential of Leishmania major/Leishmania infantum hybrids.Int. J. Parasitol. 2007; 37: 589-593Crossref PubMed Scopus (90) Google Scholar]. This interspecific hybridization may increase the fitness of parasite progeny, enabling acquisition of combined (resistance) traits and development in new sand fly vectors [39.Volf P. et al.Increased transmission potential of Leishmania major/Leishmania infantum hybrids.Int. J. Parasitol. 2007; 37: 589-593Crossref PubMed Scopus (90) Google Scholar]. Making the specific vector–Leishmania interaction even more complex, the gut microbiome is essential for parasite survival. Indeed, antibiotic-mediated perturbation of the midgut microbiome renders sand flies unable to support Leishmania growth and metacyclogenesis, identifying the midgut microbiome as a critical factor for disease transmission to the mammalian host [40.Louradour I. et al.The midgut microbiota plays an essential role in sand fly vector competence for Leishmania major.Cell. Microbiol. 2017; 19e12755Crossref Scopus (36) Google Scholar,41.Kelly P.H. et al.The gut microbiome of the vector Lutzomyia longipalpis is essential for survival of Leishmania infantum.mBio. 2017; 8e01121-16Crossref PubMed Scopus (72) Google Scholar], which occurs during the blood-feeding process [42.Bates P.A. Transmission of Leishmania metacyclic promastigotes by phlebotomine sand flies.Int. J. Parasitol. 2007; 37: 1097-1106Crossref PubMed Scopus (321) Google Scholar]. By production of promastigote secretory gel (PSG) in the anterior midgut, a physical blockage is created resulting in what is called a 'blocked fly' [43.Rogers M.E. et al.Transmission of cutaneous leishmaniasis by sand flies is enhanced by regurgitation of fPPG.Nature. 2004; 430: 463-467Crossref PubMed Scopus (197) Google Scholar]. This blockage hampers efficient blood feeding affecting the vectorial capacity (Box 1) and leads to regurgitation of PSG together with insect microbiota [44.Dey R. et al.Gut microbes egested during bites of infected sand flies augment severity of leishmaniasis via inflammasome-derived IL-1beta.Cell Host Microbe. 2018; 23: 134-143 e6Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar] and exosomes [45.Atayde V.D. et al.Exosome secretion by the parasitic protozoan Leishmania within the sand fly midgut.Cell Rep. 2015; 13: 957-967Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar], enhancing the disposition of metacyclic promastigotes and infection of mammalian host skin thereby creating a substantially different interaction as compared to experimental needle-based infections. This reflux is further facilitated by a malfunctioning stomodeal valve due to damage by parasite chitinases [25.Dostalova A. Volf P. Leishmania development in sand flies: parasite–vector interactions overview.Parasit. Vectors. 2012; 5: 276Crossref PubMed Scopus (164) Google Scholar,42.Bates P.A. Transmission of Leishmania metacyclic promastigotes by phlebotomine sand flies.Int. J. Parasitol. 2007; 37: 1097-1106Crossref PubMed Scopus (321) Google Scholar,46.Rogers M.E. et al.Leishmania chitinase facilitates colonization of sand fly vectors and enhances transmission to mice.Cell. Microbiol. 2008; 10: 1363-1372Crossref PubMed Scopus (52) Google Scholar].Box 1Aspects of Vectorial Capacity Impacted by Leishmania InfectionThe parasite and its vector undergo a coevolution that will drive natural selection for the adaptation and counter-adaptation of both organisms [94.Caljon G. et al.Alice in microbes' land: adaptations and counter-adaptations of vector-borne parasitic protozoa and their hosts.FEMS Microbiol. Rev. 2016; 40: 664-685Crossref PubMed Scopus (16) Google Scholar] thereby impacting on the vectorial capacity of the sand fly. For example, infection will affect the feeding behavior resulting in a more efficient transmission of parasites [35.Rogers M.E. The role of Leishmania proteophosphoglycans in sand fly transmission and infection of the mammalian host.Front. Microbiol. 2012; 3: 223Crossref PubMed Scopus (65) Google Scholar]. In nature, driven by an evolutionary need to lay as many batches of eggs as possible, sand flies take multiple blood meals hence promoting Leishmania replication and augmenting vector infectivity [95.Serafim T.D. et al.Sequential blood meals promote Leishmania replication and reverse metacyclogenesis augmenting vector infectivity.Nat. Microbiol. 2018; 3: 548-555Crossref PubMed Scopus (59) Google Scholar]. From the observation that only engorged females are able to mature and lay eggs, Oliveira and colleagues suggested that the blood meal itself reduced the probability of survival since egg-laying is causing stress and exhaustion that can culminate in death [96.de Oliveira E.F. et al.Experimental infection and transmission of Leishmania by Lutzomyia cruzi (Diptera: Psychodidae): aspects of the ecology of parasite-vector interactions.PLoS Negl. Trop. Dis. 2017; 11e0005401PubMed Google Scholar]. The effect of infection by Leishmania on the longevity and fecundity of sand flies has not yet been fully clarified [19.Kamhawi S. Phlebotomine sand flies and Leishmania parasites: friends or foes?.Trends Parasitol. 2006; 22: 439-445Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar] although some researchers found that infection of Lutzomyia longipalpis by L. infantum reduced survival and life expectancy, fertility and fecundity [97.Agrela I.F. Feliciangeli M.D. Effect of Leishmania spp infection on the survival, life expectancy, fecundity and fertility of Lutzomyia longipalpis sl and Lutzomyia pseudolongipalpis.Mem. Inst. Oswaldo Cruz. 2015; 110: 611-617Crossref PubMed Scopus (6) Google Scholar]. The parasite and its vector undergo a coevolution that will drive natural selection for the adaptation and counter-adaptation of both organisms [94.Caljon G. et al.Alice in microbes' land: adaptations and counter-adaptations of vector-borne parasitic protozoa and their hosts.FEMS Microbiol. Rev. 2016; 40: 664-685Crossref PubMed Scopus (16) Google Scholar] thereby impacting on the vectorial capacity of the sand fly. For example, infection will affect the feeding behavior resulting in a more efficient transmission of parasites [35.Rogers M.E. The role of Leishmania proteophosphoglycans in sand fly transmission and infection of the mammalian host.Front. Microbiol. 2012; 3: 223Crossref PubMed Scopus (65) Google Scholar]. In nature, driven by an evolutionary need to lay as many batches of eggs as possible, sand flies take multiple blood meals hence promoting Leishmania replication and augmenting vector infectivity [95.Serafim T.D. et al.Sequential blood meals promote Leishmania replication and reverse metacyclogenesis augmenting vector infectivity.Nat. Microbiol. 2018; 3: 548-555Crossref PubMed Scopus (59) Google Scholar]. From the observation that only engorged females are able to mature and lay eggs, Oliveira and colleagues suggested that the blood meal itself reduced the probability of survival since egg-laying is causing stress and exhaustion that can culminate in death [96.de Oliveira E.F. et al.Experimental infection and transmission of Leishmania by Lutzomyia cruzi (Diptera: Psychodidae): aspects of the ecology of parasite-vector interactions.PLoS Negl. Trop. Dis. 2017; 11e0005401PubMed Google Scholar]. The effect of infection by Leishmania on the longevity and fecundity of sand flies has not yet been fully clarified [19.Kamhawi S. Phlebotomine sand flies and Leishmania parasites: friends or foes?.Trends Parasitol. 2006; 22: 439-445Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar] although some researchers found that infection of Lutzomyia longipalpis by L. infantum reduced survival and life expectancy, fertility and fecundity [97.Agrela I.F. Feliciangeli M.D. Effect of Leishmania spp infection on the survival, life expectancy, fecundity and fertility of Lutzomyia longipalpis sl and Lutzomyia pseudolongipalpis.Mem. Inst. Oswaldo Cruz. 2015; 110: 611-617Crossref PubMed Scopus (6) Google Scholar]. Overall, assessing parasite fitness is challenging and requires a combination of in vitro and in vivo tools. Drug resistance in Leishmania can result in alterations to various aspects of parasite fitness [15.Vanaerschot M. et al.The concept of fitness in Leishmania.in: Ponte-Sucre A. Padrón-Nieves M. Drug Resistance in Leishmania Parasites. Springer, 2018: 341-366Crossref Scopus (1) Google Scholar], which may be drug-, species-, and stage-dependent [47.Hendrickx S. et al.Transmission potential of paromomycin-resistant Leishmania infantum and Leishmania donovani.J. Antimicrob. Chemother. 2019; 75: 951-957Crossref Scopus (4) Google Scholar]. Although the generation of resistant strains in the laboratory can provide a lot of information, its relevance should be considered with caution. Firstly, laboratory strains that have been cultured for several years should be avoided, and resistance selection should preferably be on strains that are representative of the field, that is, recent clinical isolates [48.Moreira D. et al.Impact of continuous axenic cultivation in Leishmania infantum virulence.PLoS Negl. Trop. Dis. 2012; 6e1469Crossref PubMed Scopus (50) Google Scholar]. Secondly, it was shown that selection using promastigotes can lead to different phenotypes than when exposing intracellular amastigotes either in vitro or in vivo [11.Hendrickx S. et al.Experimental induction of paromomycin resistance in antimony-resistant strains of L. donovani: outcome dependent on in vitro selection protocol.PLoS Negl. Trop. Dis. 2012; 6e1664Crossref PubMed Scopus (38) Google Scholar]. The 'field' relevance of experimentally induced resistant strains may therefore be debatable since clinical resistance is selected in a complex host immunological setting (considering immunocompromised states, coinfections and malnutrition) and involves vector transmission through sand flies, two aspects that are insufficiently represented in laboratory selection systems [15.Vanaerschot M. et al.The concept of fitness in Leishmania.in: Ponte-Sucre A. Padrón-Nieves M. Drug Resistance in Leishmania Parasites. Springer, 2018: 341-366Crossref Scopus (1) Google Scholar]. A major drawback of studying resistant clinical isolates is the lack of the parent sensitive strains as references [7.Hendrickx S. et al.Evaluating drug resistance in visceral leishmaniasis: the challenges.Parasitology. 2016; 145: 453-463Crossref PubMed Scopus (29) Google Scholar]. In the past, subsets of resistant and susceptible strains with a different genetic background were compared to try to circumvent this issue [49.Laurent T. et al.Epidemiological dynamics of antimonial resistance in Leishmania donovani: genotyping reveals a polyclonal population structure among naturally-resistant clinical isolates from Nepal.Infect. Genet. Evol. 2007; 7: 206-212C

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