Journal of Infectious Diseases Advance Access published June 29, 2015

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1 Journal of Infectious Diseases Advance Access published June 29, Leishmania-RNA virus presence in L. guyanensis parasites increases the risk of firstline treatment failure and symptomatic relapse Eliane Bourreau a, Marine Ginouves b, Ghislaine Prévot b, Mary-Anne Hartley c, Jean- Pierre Gangneux d,h, Florence Robert-Gangneux d,h, Julie Dufour e, Dominique Sainte Marie e, Antoine Bertolotti e, Francine Pratlong f, Ricardo Martin c, Frédéric Schütz g, Pierre Couppié b,e, Nicolas Fasel c, Catherine Ronet c a Immunologie des leishmanioses, Institut Pasteur de Guyane, Cayenne, French Guiana b Equipe EPaT-EA3593 (Epidémiologie des Parasitoses Tropicales en Guyane), Antenne Guyane de l Université des Antilles et de la Guyane, Cayenne, French Guiana c Department of Biochemistry, University of Lausanne, Epalinges, Switzerland d Laboratoire de Parasitologie-Mycologie du CHU de Rennes, Rennes, France e Service de Dermatologie, Centre Hospitalier de Cayenne, Cayenne, French Guiana f University Montpellier 1, Laboratory of Parasitology-Mycology, Hospital University Centre (C.H.R.U.) of Montpellier & French National Reference Centre for Leishmaniasis; CNRS IRD University Montpellier 1&2 (UMR "MIVEGEC"), Montpellier, France g Swiss Institute of Bioinformatics, University of Lausanne, Switzerland h INSERM U1085 IRSET (Institut de Recherches en Santé Environnement Travail), Université Rennes 1, Rennes, France Corresponding author: Dr. Catherine Ronet, Department of Biochemistry, University of Lausanne, Chemin des Boveresses, 155, CH-1066, Epalinges, Switzerland, Phone: , catherine.ronet@unil.ch The Author Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please journals.permissions@oup.com.

2 2 Footnotes: 1. We declare no competing interests. 2. Financial support: This work was supported by Bill and Melinda Gates Foundation Grand Challenges Explorations [No. OPP to CR and MAH], Pierre Mercier Foundation to CR, Guiana Pasteur Institute located in Cayenne to EB, the French Ministry for Higher Education and Research to EB and by the grant FNRS [N. 3100A /1, N to NF]. 3. This worked was presented during the following conference: - Scientific Symposium of the Institute Pasteur International Network, September 2014 Paris, France. - Amazonian Conference on Emerging and Infectious Diseases, September 2014, Cayenne, French Guiana 4. Corresponding author: Dr. Catherine Ronet Department of Biochemistry, University of Lausanne, Chemin des Boveresses, 155, CH-1066, Epalinges, Switzerland Phone: catherine.ronet@unil.ch

3 3 ABSTRACT Treatment failure and symptomatic relapse are major concerns in American tegumentary leishmaniasis (ATL). Such complications are seen frequently in L.guyanensis infections where patients respond variously to first-line anti-leishmanials and are more prone to develop chronic cutaneous leishmaniasis. The factors underlying this pathology, however, are unknown. Recently, we reported that a dsrna virus (LRV1) nested within L.guyanensis parasites is able to exacerbate experimental murine leishmaniasis by inducing a hyperinflammatory response. This report investigates the prevalence of LRV1 in human L.guyanensis infection and its effect on treatment efficacy as well as its correlation to symptomatic relapses after the first-line treatment. In our cohort of 75 patients diagnosed with primary-localised ATL, the prevalence of LRV1+ infection was elevated to 58%. All patients infected with LRV1-negative L.guyanensis were cured after one (22/31 i.e.71%) or two (31/31 i.e.100%) doses of pentamidine. Oppositely, 12 out of the 44 LRV1-positive patients presented with persistent infection and symptomatic relapse required extended therapy and the use of second-line drugs. Finally, LRV1 presence was associated with a significant increase in intra-lesional inflammatory markers. In conclusion, LRV1 status in L.guyanensis infection is significantly predictive (p=0.0009) of first-line treatment failure and symptomatic relapse, and could stand to guide therapeutic choices in ATL.

4 4 INTRODUCTION Neotropical cutaneous leishmaniases (American tegumentary leishmaniases, ATL) are well known to cause particularly disfiguring inflammatory skin lesions that are variously responsive to recommended first-line therapies and prone to symptomatic relapse [1, 2]. ATL is most common in the Amazonian basin with the predominant causative species being L. braziliensis, L. guyanensis and L. panamensis. Between 5 and 10% of these infections progress to chronic disease, where skin lesions form at sites distant to the sand fly inoculum, sometimes infesting and destroying the soft tissues of nasopharyngeal mucosa. These aggressive symptomatic outcomes have a distribution, which strongly correlates to that of the parasite species, and thus implies the presence of parasite-intrinsic virulence factors. We recently demonstrated that the presence of a cytoplasmic dsrna virus (Leishmania RNA virus, LRV1) residing endosymbiotically within L. guyanensis (L.g) parasites acts as an aggravating factor in an experimental murine model. Here, the dsrna genome of LRV triggered a potently inflammatory anti-viral response, which worsened lesional swelling and prolonged parasite survival [3-5]. LRV is composed of a 5.3 kb dsrna genome, which encodes a non-enveloped capsid and an RNA-dependent RNA polymerase. It belongs to the Totiviridae family[6], a group of dsrna viruses known to infect several species of the animal kingdom including arthropods [7], fish [8], bats [9], yeast [10], as well as molds (Helminthosporium sp.[11], and Aspergillus [12]), and several human pathogens (Trichomonas vaginalis [13], Giardia lamblia [14]). Recently, an inflammatory anti-viral immune response to the Trichomonavirus within Trichomonas vaginialis has been linked to increased clinical complications during trichomoniasis including susceptibility to preterm birth and HIV infection [15]. The abundant prevalence of microbial viruses nested within pathogenic species and their arthropod vectors creates great evolutionary potential for similar hyper-pathogenic partnerships [16].

5 5 So far, LRV has been isolated from various Leishmania species which are commonly associated to the occurrence of chronic, disseminated, diffuse or muco-cutaneous lesions [17] such as L. guyanensis [5, 18], L. braziliensis [19] and L. aethiopica [20]. For instance, L. g parasites have a tendency to metastasize, forming satellite papules and adenopathy in draining lymph nodes [2, 21], sometimes evolving into chronically inflamed lesions, which are prone to treatment failure and symptomatic relapse [1, 22, 23]. While the role of LRV1 as an aggravating factor during L,g infection has been convincingly tested in a murine model [5], no studies have yet investigated its role in human disease or treatment efficacy. More importantly, the prevalence of LRV1 endosymbiosis in L.g is completely unknown. In this study, a cohort of 75 human patients diagnosed with uncomplicated acute cutaneous leishmaniasis (ACL) caused by L.g was investigated for the prevalence of LRV co-infection and its association to treatment failure or symptomatic relapse. We observed a 58 percent prevalence of LRV1 in our cohort of L.g infected patients, which could be significantly correlated to increased intra-lesional inflammation, first-line treatment failure and increased risk of symptomatic relapse. METHODS Patients Ethical approval was granted based on the human experimentation guidelines of the Comité consultatif sur le traitement de l'information en matière de recherche (CCTIRS: ), the Commission nationale de l informatique et des libertés (CNIL: DR ) as well as the regulations of Cayenne Hospital ( Adult patients diagnosed with cutaneous leishmaniasis contracted in French Guiana were enrolled into the study under informed consent at the Cayenne hospital in French Guiana (88) or at the Rennes hospital in France (16) patients from people returning after their stay stationed in French Guiana as previously described) [1].

6 6 Inclusion criteria for the first part of the study (LRV1 prevalence analysis) selected only newly diagnosed patients who had never previously received anti-leishmanials and reported a lesion history of less than 4 months. HIV testing was conducted on an opt-in basis, and was a requirement for pre-selection, where only HIV-negative patients were included in order to maintain immunocompetence within the cohort (Figure Supplementary 1). Only patients who attended all monthly follow-up visits (until diagnosed as healed ) were included in the final analysis (21 patients excluded). Relapses were recoded sporadically on the patient s decision to return to the clinic over the course of 1-year after their initial diagnosis. To avoid an underestimation of relapse, all the patients that did not return to hospital were contacted by phone to confirm the absence of disease recurrence after the initial treatment. Further only patients infected with L. guyanensis species were selected given that other Leishmania species cohorts were too small to be considered (8 patients excluded, of which 7 had L. braziliensis and 1 L. amazonensis). All 104 patients enrolled under these criteria (92 adult males and 12 adult females in the age range of 18 to 63 years old) were tested for the presence of LRV within lesional biopsies. LRV1-status was then used to split the cohort into LRV1+ and LRV1- groups for statistical analysis of treatment responsiveness and symptomatic relapse. Overall, 75 patients met the eligibility criteria for inclusion in the second part of the study. These patients underwent extensive clinical examination, including localization, measurement and enumeration of lesions. Further analysis of leishmanial diagnostics, typing, quantification of inflammatory mediators was performed on a 3-mm punch biopsy in the lesional border before the beginning of treatment. Leishmanial diagnostics Initial diagnostics adhered to the local standard guidelines. When possible, serous dermal fluid was extracted from the lesion border to produce a smear for microscopic analysis by May-Grünwald Giemsa staining. This was accompanied by a 3mm intra-lesional punch biopsy, which was split into 2 segments: one for starting an in vitro parasite culture (on NNN

7 7 medium or RPMI containing 10% fetal calf serum or Schneider medium) and the other for qrt-pcr analysis of LRV and inflammatory markers. Phylogenic RFLP-PCR analysis was performed on parasite cultures based on a conserved isoenzyme polymorphism as previously described [24]. Leishmania RNA virus and inflammatory markers detection in biopsies Total RNA was isolated as previously described using the RNeasy minikit (Qiagen) [27]. First strand cdna was synthetized from total RNA using first strand cdna synthesis kit (Superscript III first strand synthesis system, Invitrogen). qrt-pcr was performed on a 7300 Real Time System (Applied Biosystems). FAM-MGB-labeled primer/probe sets for LRV, KMP11 as endogenous control were designed with Primer Express software from Applied Biosystems. The L. guyanensis strain MHOM/BR/75/M4147 and the L. panamensis strain MHOM/PA/1971/LS94 were used as positive and negative controls respectively. Primer sequences are the following: KMP11-F 5 -GAGCACACGGAGAAGATCAAC A-3 ; KMP11-R 5 -CAAGCAGCTCAGCGAACTTG-3 ; KMP11-MGB probe 5 -FAM-CTCGGAGCACTTCAA-3, LRV-F 5 -GAGTGGGAGTCCCCCACAT-3, LRV-R 5 -TGGATACAACCAGACGATTGCT-3, LRV MGB probe 5 -FAM CATTTATGTAGTTCCT-3. Inflammatory markers were analysed using the succeeding primers: FAM-MGB labeled primer/ probe sets for CCL5 (Hs m1), CXCL-10 (Hs m1), IL-6 (Hs m1), TNF- (Hs m1) and -actin (Hs m1 used as endogenous control) were purchased from Applied Biosystems. The default ABI 7300 thermal cycling conditions were used: 10min at 95 C followed by 40 cycles (15 sec at 95 C and 1 min at 60 C). Leishmania RNA virus detection in parasite isolates L. guyanensis parasites isolated from biopsies of patients infected in French Guiana were collected in the Leishmania reference Centers of Cayenne (16 isolates), Montpellier (21

8 8 isolates, BioBank number BB ) and the Institut Pasteur de Guyane (17 isolates, Collection number n 3). Parasite cultures were expanded in vitro in complete Schneider s medium and were tested for LRV by dot-blot using an antibody recognizing dsrna irrespective of its underlying sequence (J2- English & Scientific Consulting), as previously described.[19] Similar results were obtained with dot blots and RT-PCR analysis, using pimers described above. Anti-leishmanial treatment strategy Treatment was administered according to standard local guidelines. In Cayenne s hospital, this comprised of the first-line therapy of pentamidine isethionate (Pentacarinat ) administered in a single injection of 7 mg/kg. In the event that lesions showed no sign of healing after 1 month, a second pentamidine treatment at the same dose was administered. If lesions persisted for a further month after the second administration, patients would be classified as unresponsive to first-line treatment and then hospitalized to receive a 20 day course of Meglumine antimonate (Glucantime ) at 20 mg/kg/day. Statistical analysis. Given the small sample sizes, a Fisher's exact test determined significant differences in clinical outcome between LRV1+ and LRV1- groups. Student s t-tests found significant correlations between cytokine production and LRV1-status. RESULTS Members of the L. guyanensis (L.g) species are endemic to the Northern Amazonian basin [24], which includes the patient catchment area of Cayenne Hospital in French Guiana, from where our cohort was recruited. Cutaneous leishmaniasis was diagnosed using routine local diagnostic techniques on lesional exudate. Before treatment, a 3mm intra-lesional punch

9 9 biopsy was performed on a total of 104 patients fitting the selection criteria (HIV-negative, 4 months or less with visible lesion and no previous history of leishmaniasis) (Supplementary Figure 1). Parasite phylogeny was determined by RFLP-PCR in order to restrict our study to L.g species. As expected, the vast majority (96/104 patients, i.e. >90%) of infections conformed to the L.g genotype. The remaining 8% were either L. braziliensis (7 patients) or L. amazonensis (1 patient) and formed a too limited cohort to be considered for further analysis. A further 21 patients failed to complete the required follow-up visits or adhere to treatment guidelines, obliging their exclusion. Thus, 75/104 patients qualified for inclusion in the clinical cohort (Supplementary Figure 1). Lesional tissue was investigated for the presence of LRV1 by qrt-pcr amplifying a previously described conserved region of viral dsrna, controlled to the parasitic housekeeping gene, kmp11 [19]. Of the 75 patients, 44 tested positively for LRV1 (58% prevalence) (Figure 1). To ensure that our results were not skewed by restricted intralesional parasite numbers, parasite cultures isolated from 54 patients infected in French Guiana and preserved in the Leishmania reference centers collection were also investigated for LRV1 presence by immunoblotting using an antibody, which recognizes dsrna irrespective of its sequence [19]. Interestingly, 47/54 cultures (i.e. 87%) tested positively for LRV1 by immunoblotting (Figure 1). Taken together, LRV1 presence across all samples and techniques amounted to a high prevalence in French Guyana ranging from 58 % for lesions to 87% for isolates. In the early stages of infection under which patients were enrolled (4 months or less with evidence of a cutaneous lesion), no differences of age, sex, anatomical lesion location or appearance could be related to either LRV1+ or LRV1- patient groups (Table 1). As mentionned in the material section, all enrolled patients were HIV- and were not suffering of co-morbid affections. After this initial investigation, and without regard to their LRV1 status, all patients were admitted for standard first-line anti-leishmanial therapy with pentamidine isethionate (intramuscular injections of 7 mg/kg). If active lesions persisted in the follow-up appointment 1 month later, a second round of therapy was administered. Patients who were

10 10 not cured by this second dosage of pentamidine, and whose lesions persisted for a third month, were offered meglumine antimoniate (20 mg/kg/day during 21 days). Significant differences in treatment patterns were observed between patients from LRV1+ and LRV1- groups (Figure 2). During the initial period treatment, while the majority of infections (71/75 patients, 95%) were clinically resolved after one or two doses of pentamidine, the remaining 4 patients (5%) who developed persistent lesions were all from the LRV1+ group (Figure 2A). Three of these 4 patients were cured by administration of the second-line drug, meglumine antimoniate. Thus amounting to an overall 99% cure rate over the 3-month treatment period. The last non-responding patient was eventually cured several months later by a repeated administration of pentamidine. All 74 patients cured within the 3-month treatment window were followed over the subsequent year to monitor for disease reactivation or other complications. During this follow-up, 12 of the 74 patients reported a reactivation of the disease after a 3-6 month period without symptoms (Figure 2B). Thus, including the patient unresponsive to meglumine antimoniate, 13 out of the 75 patients of our cohort presented with relapsed disease. Notably, each of these 13 patients was infected by LRV1+ L.g parasites (13 on 44 LRV+ patients, i.e. 30% of LRV+ patients). No symptomatic relapse was observed in any of the LRV1- L.g infections. The correlation between LRV1 presence and symptomatic relapse was highly significant (Fisher s exact test, p=0 0009). These 13 patients necessitated additional administrations of pentamidine isethionate (1 dose for 9/12 patients i.e. 75%, 2 doses for 1/12 patients i.e. 8%) or meglumine antimoniate (for 2/12 patients i.e. 17%) to achieve symptomatic resolution. All 75 patients remained symptom-free over the following 12- months. At a macroscopic level, LRV1+ L.g and LRV1- L.g infected patients had similar frequencies of lymphangitis and adenopathy (Supplementary Figure 2). Interestingly, the LRV1+ L.g infected patients had a tendency (albeit not statistically significant) to develop more clinically palpable signs of lymphangitis, or/and adenopathy (Supplementary Figure 2). In addition,

11 11 significant differences were revealed in the levels of intra-lesional inflammatory markers.which are in accordance with our previously described murine model of L.g infection. LRV+ L.g lesions, and this irrespectively of their ability to relapse or not, had higher levels of CXCL-10 and IL-6 transcripts compared to their LRV1- counterparts (Figure 3). Because the relapsing patients with increased inflammatory markers were all infected by LRV1+ L.g, this observation supports our experimental hypothesis that LRV1 presence is associated to clinical signs of inflammation and is able to act as a virulence factor in leishmaniasis. DISCUSSION The viral endosymbiont, LRV, was discovered in the cytoplasm of L.guyanensis parasites several decades ago [18, 25], but its potential as a clinical determinant and aggravating factor in cutaneous leishmaniasis was only recently hypothesized [5]. No epidemiological studies have yet described its geographical prevalence or correlation to human disease. Albeit on a small-scale, this is the first study to investigate the prevalence of LRV1 in L.guyanensis infection in an endemic region and substantiate its proposed pathogenic role in human infection. LRV1 was detected and verified by a variety of techniques on freshly isolated lesional biopsies and parasite cultures. Our study found a high LRV1 prevalence in randomly selected samples from the patient catchment area of the Cayenne Hospital in French Guiana as well as in patients returning from this area (recruited at the Rennes Hospital in France). It is interesting to note that the LRV1 prevalence is higher when tested on parasites isolates than on biopsies. The LRV1-typing results obtained in our French Guiana cohort were then used to investigate the association between LRV1 and the occurrence of inflammatory leishmanial pathology, first-line treatment failure and symptomatic relapse. For this, we selected only

12 12 immunocompetent patients infected by L. guyanensis. Our results showed that LRV1 presence in human L. guyanensis infections was significantly associated to an increased disseminate and intra-lesional inflammation. Strikingly, no LRV1- L.g infected patients in our cohort experienced first-line treatment failure or symptomatic relapse. The 30% of patients experiencing such complications all had LRV1+ L.g infections. These patients had a higher tendency to develop clinical and molecular signs of inflammation and required additional antileishmanial treatments to resolve their lesions. As during the follow up period, the patient enrollment was passive and based on the patient s decision to return to the hospital, all the other patients were contacted directly by phone to definitively confirm the absence of relapse. Cutaneous leishmaniasis caused by L. guyanensis has long been associated to macroscopic signs of inflammation and the occurrence of infectious metastasis in French Guiana: observations that were attributed to the particular resistance of L. guyanensis parasites to conventional anti-leishmanial treatment [26]. Our studies, however, found no significant difference in drug sensitivity between LRV1+ and LRV1- L.g parasites after in vitro exposure to various concentrations of pentamidine isethionate (data not shown). Similarly, no links between LRV1 presence and L. braziliensis drug resistance were established in the companion paper by Adaui et al. In addition, these authors also demonstrated that resistance of LRV1 bearing strains is not linked to a preferential co-segregation of parasite nuclear or kineotoplast genes. Quantification of parasites/biopsy and LRV1/parasite by qrt-pcr revealed a wide degree of variability among the LRV1+ L.g isolates, preventing an analysis of a correlation between the parasite number, the ratio of LRV1/parasite and a specific disease outcome (data not shown). Further, LRV1/parasite within LRV1+ isolates did not correlate to variations in total parasite number. Intralesional parasite burden is known to fluctuate widely during the course of human leishmaniasis according to numerous and unpredictable immunological parameters and thus, variability in total parasite number is anticipated due to variations in the disease timeframe from when the biopsies were taken (i.e. 15 days to 4 months of infection). Thus,

13 13 LRV1+ L.g strains were significantly correlated to treatment failure and relapse irrespective of the level of LRV1/parasite. This observation indicates that LRV1 has a low threshold of pathology in human infection, where even the parasites hosting smaller LRV1 burdens are able to negatively influence disease outcome. Intrestingly, the LRV1 presence in Leishmania parasites not only impacts L. guyanensis infection, since a companion report by Adaui et al. describes an association between the LRV1 presence in L. braziliensis and treatment failure. Whether and how the LRV1 burden is influenced in an in vivo infection is worth further investigation. Taken together, the data from this study shows that the LRV1-status in L.g parasites could be predictive of clinical complications such as first-line treatment failure, increased inflammation and symptomatic relapse. Thus, LRV diagnostics could guide treatment strategies, to better predict, avoid and manage the complications of metastatic leishmaniasis. Fundings This work was supported by Bill and Melinda Gates Foundation Grand Challenges Explorations No. OPP to CR and MAH,, Pierre Mercier Foundation to CR, Guiana Pasteur Institute located in Cayenne to EB, the French Ministry for Higher Education and Research to EB and by the grant FNRS[ N. 3100A /1, N ] to NF. Acknowledgements Stephen Beverley (Washington University, St Louis, USA) and Jean-Claude Dujardin (Institute of Tropical Medecine Antwerp, Belgium) are acknowledged for discussions and communications of unpublished data. We also thank the Pôle Intégré de Recherche Clinique from the Pasteur Institute, notably Nathalie Jolly, who facilitated the ethical approval and Pr Pascal Launois for helpful discussion. The authors acknowledge Stéphane Simon, Loic Taglignani and Patrick Lami for their contribution regarding the Leishmania parasite cultures.

14 14 FIGURE LEGENDS. Figure 1. Prevalence of LRV in parasites isolated from cutaneous leishmaniasis patients in French Guiana. The presence of LRV was determined by qrt-pcr in 75 biopsies from patients diagnosed with cutaneous leishmaniasis due to L. guyanensis (left panel) and 54 L. guyanensis parasite isolates preserved in Leishmania reference centers (middle panel). The LRV1 prevalence is presented as percentage in fresh lesions (left panel), in isolates (right panel) Figure 2. LRV is associated to clinical relapse, and increased intra-lesional inflammation A: Initial Period- Newly diagnosed immunocompetent adult patients infected with L. guyanensis and presenting with cutaneous lesion(s) were monitored for treatment responsiveness according to the treatment guidelines: one dose of 7mg/kg pentamidine (followed by a second dose if unresponsive after 1 month). Administration of the second-line treatment, 20mg/kg/day for 20 days of meglumine antimoniate (Glu) was indicated for lesions unresponsive to 2 doses of pentamidine. Results are expressed in percentage and patient numbers are indicated above bars. B: Follow-up Period - Disease relapse was monitored in previously cured patients presented in A. Results are expressed in percentage and patient numbers are indicated above bars. Statistical analysis p=0.009 via a Fisher's exact test. Figure 3. LRV is associated to an increased intra-lesional inflammation Transcripts of inflammatory cytokines CCL5 (A), CXCL10 (B), IL-6 (C) and TNF- (D) were measured by qrt-pcr in total RNA extracted from intra-lesional biopsies (8 LRV1+ and 8

15 15 LRV1-). Values of the different markers were normalized to endogenous levels of actin mrna and are represented as arbitrary units. Data are means +/- SEM. *p <0.05 as calculated by a two sample, two-sided Student s t test. Table1. Clinical data for patients with ACL due to LRV1+ or LRV1- L. guyanensis parasites.

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19 LRV status (%) lesions (n=75) isolates (n=54) LRV+ LRV- LRV+ LRV- Figure 1

20 A Initial period B Follow-up period LRV1- L. guyanensis LRV1+L. guyanensis LRV1- L. guyanensis LRV1+ L. guyanensis patients (%) Glu 1 2 Glu Pentamidine Treatment(s) Pentamidine Treatment(s) 4 % patient No relapse relapse No relapse relapse Figure 2 Figure 2

21 A CCL5 C IL * B Relative expression Relative expression CXCL10 * D LRV1+ TNFα 0.0 LRV1+ LRV1- LRV1+ LRV1- Relative expression Relative expression LRV1+ LRV1- LRV1- Figure 3

22 Characteristic LRV1+ LRV1- Patients Age (median) Sex, M/F (number) 41/3 25/6 Number of lesions, 1 lesion 1with satellite papule(s) 2 lesions 3 lesions and more Localisation of lesions, % of patient (Number of patient) 57 (25) 18 (8) 11 (5) 14 (6) 42 (13) 10 (3) 39 (12) 10 (3) Number of patient Face 5 2 Neck 2 2 Trunk 2 2 Arm, wrist Hand 2 1 Leg, foot unlocalised 5 5 Table 1