RESEARCH ARTICLE Leishmaniasis and tumor necrosis factor alpha antagonists in the Mediterranean basin. A switch in clinical expression Pau Bosch-Nicolau1, Maria Ubals2, Fernando Salvador1, Adrián Sánchez-Montalvá1, Gloria Aparicio2, Alba Erra3, Pablo Martinez de Salazar4, Elena Sulleiro4, Israel Molina ID1* 1 Department of Infectious Diseases, Hospital Universitari Vall d’Hebron, PROSICS Barcelona, Universitat Autònoma de Barcelona, Spain, 2 Department of Dermatology, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Spain, 3 Department of Rheumatology, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Spain, 4 Department of Clinical Microbiology, Hospital Universitari Vall d’Hebron, PROSICS Barcelona, Universitat Autònoma de Barcelona, Spain a1111111111 * imolina@vhebron.net a1111111111 a1111111111 a1111111111 a1111111111 Abstract Background OPEN ACCESS Tumor necrosis factor alpha (TNF-α) blockers are recognized as a risk factor for reactivation Citation: Bosch-Nicolau P, Ubals M, Salvador F, of granulomatous infections. Leishmaniasis has been associated with the use of these Sánchez-Montalvá A, Aparicio G, Erra A, et al. drugs, although few cases have been reported. (2019) Leishmaniasis and tumor necrosis factor alpha antagonists in the Mediterranean basin. A switch in clinical expression. PLoS Negl Trop Dis Methodology 13(8): e0007708. https://doi.org/10.1371/journal. pntd.0007708 We performed a retrospective observational study including patients with confirmed leish- Editor: Abhay R Satoskar, Ohio State University, maniasis acquired in the Mediterranean basin that were under TNF-α blockers therapy at UNITED STATES the moment of the diagnosis. Patients diagnosed in our hospital from 2008 to 2018 were Received: May 13, 2019 included. Moreover, a systematic review of the literature was performed and cases fulfilling the inclusion criteria were also included. Accepted: August 15, 2019 Published: August 30, 2019 Principal findings Copyright: © 2019 Bosch-Nicolau et al. This is an open access article distributed under the terms of Forty-nine patients were analyzed including nine cases from our series. Twenty-seven the Creative Commons Attribution License, which (55.1%) cases were male and median age was 55 years. Twenty-five (51%) patients were permits unrestricted use, distribution, and under infliximab treatment, 20 (40.8%) were receiving adalimumab, 2 (4.1%) etanercept, reproduction in any medium, provided the original author and source are credited. one (2%) golimumab and one (2%) a non-specified TNF-α blocker. Regarding clinical pre- sentation, 28 (57.1%) presented as cutaneous leishmaniasis (CL), 16 (32.6%) as visceral Data Availability Statement: All relevant data are within the manuscript and its Supporting leishmaniasis (VL) and 5 (10.2%) as mucocutaneous leishmaniasis (MCL). All VL and MCL Information files. patients were treated with systemic therapies. Among CL patients, 13 (46.4%) were treated Funding: The author received no specific funding with a systemic drug (11 received L-AmB, one intramuscular antimonials and one miltefo- for this work. sine) while 14 (50%) patients were given local treatment (13 received intralesional pentava- Competing interests: The authors have declared lent antimonials, and one excisional surgery). TNF-α blockers were interrupted in 32 that no competing interests exist. patients (65.3%). After treatment 5 patients (10.2%) relapsed. Four patients with a CL (3 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007708 August 30, 2019 1 / 16 Leishmaniasis in patients treated with tumor necrosis factor alpha antagonists initially treated with local therapy maintaining TNF-α blockers and one treated with miltefo- sine) and one patient with VL treated with L-AmB maintaining TNF-α blockers. Conclusions This data supports the assumption that the blockage of TNF-α modifies clinical expression of leishmaniasis in endemic population modulating the expression of the disease leading to atypical presentations. According to the cases reported, the best treatment strategy would be a systemic drug and the discontinuation of the TNF-α blockers therapy until clinical resolution. Author summary Tumor necrosis factor alpha (TNF-α) blockers are widely used in numerous inflammatory diseases such rheumatoid arthritis, psoriasis or inflammatory bowel diseases. They have been recognized as a risk factor for reactivation of granulomatous infections. Although few cases have been reported, Leishmaniasis has been associated with the use of these drugs. Leishmania infantum is the main causative agent of leishmaniasis in Southern Europe and is prone to produce the visceral form. However, TNF-α has been implicated in the initial events of the infection mediating the disease expression. In our series, we have observed a surprisingly high proportion of cutaneous form (32.6%) and muco-cuta- neous form (10.2%). Clinical outcome observed in this series is also unusual. Four cases (14.3%) with cutaneous leishmaniasis who received local therapy relapsed. Among patients with visceral leishmaniasis, one patient who maintained TNF-α blockers therapy relapsed despite etiological treatment. This data supports the assumption that the blockage of TNF-α modifies clinical expression of leishmaniasis leading to atypical presentations. According to the cases reported we proposed as best treatment strategy a systemic drug and the discontinuation of the TNF-α blockers therapy until clinical resolution. Introduction Tumor Necrosis Factor-α (TNF-α) is a crucial cytokine in the inflammatory cascade by acti- vating the type 1 T helper (Th1) immune response, enhancing the activity of the macrophages and essential for the formation and maintenance of granulomas [1]. Since TNF-α has been implicated in numerous immune-mediated disorders, the blockage of this cytokine has been studied as a therapeutic strategy against such diseases. Nowadays, the anti-TNF based therapy is widely used and approved for the treatment of chronic inflamma- tory conditions as rheumatoid arthritis, polyarticular juvenile idiopathic arthritis, plaque pso- riasis and psoriatic arthritis, ankylosing spondylitis and inflammatory bowel diseases [2]. The first approved TNF-α blocker was etanercept (Enbrel) in May 1998 followed by infliximab (Remicade) in November 1999, adalimumab (Humira) in December 2002, certolizumab (Cin- zia) in April 2008 and golimumab (Simponi) in April 2009. Since their first use, the TNF-α blockers were recognized as a risk factor for reactivation of granulomatous infections such as tuberculosis, intracellular infections such as salmonellosis or listeriosis and other opportunistic fungal or viral infections [3]. PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007708 August 30, 2019 2 / 16 Leishmaniasis in patients treated with tumor necrosis factor alpha antagonists Leishmaniasis is a parasitic granulomatous infection and it is endemic to South America, South Asia, Africa and South Europe. The protozoon is an obligate intracellular parasite of mononuclear phagocytic system cells. The clinical spectrum of leishmaniasis comprises sub- clinical (asymptomatic), localized (cutaneous) and disseminated infection (cutaneous, mucosal and visceral). Its clinical expression is determined on one hand by the species and zimodeme of the parasite and on the other hand by host factors and immune response [4]. Leishmaniasis has been associated with the use of TNF-α blockers, but only few cases have been reported in the literature, mainly in the Mediterranean basin [5,6]. We report nine more cases related to the use of TNF-α blockers and systematically review the published cases acquired in the Mediterranean basin. We also analyze their clinical presentation and discuss the relationship with immunomodulatory therapy. Finally, a therapeutic approach is discussed. Methods We carried out a retrospective observational study including patients with a diagnosis of leish- maniasis in its different forms. All patients were under TNF- α blockers and were diagnosed in our center between 2008 and 2018. We also performed MEDLINE search using the terms Leishmania, leishmaniasis, TNF-α inhibitors, TNF-α blockers, anti-TNF-α, adalimumab, inflixi- mab, etanercept, certolizumab and golimumab. No language or time restrictions were applied. A manual search of the references of the selected manuscript was also performed. In order to be included in the analysis, cases had to be diagnosed based on direct observa- tion of amastigotes and/or positive Leishmania polymerase chain reaction (qPCR) of blood, bone marrow or skin samples. DNA extraction from blood and bone marrow samples was carried out from 110 μl with sil- ica–membrane technology (NucliSens easyMAG. Biomerieux. France) and eluted in 110 μl according to the manufacturer´s instructions. The extraction protocol from skin biopsy was made in a Magcore Compact (RBC Bioscience. Taiwan) and eluted in 100 μl according to the manufacturer´s instructions. A duplex qPCR targeted to kinetoplast minicircle DNA of Leish- mania sp and human RNase P gene (Taq Man Human RNase P detection reagent; Applied Biosystems) were performed using the primers and probe described previously [7]. Cycling conditions were a first step of 15 minutes at 95˚C followed by 45 cycles at 95˚C for 15 seconds and 55˚C for 1 minute. Amplifications were carried out in a CFX Real-Time PCR detection system (Bio-Rad, Hercules, CA). Leishmania infantum identification was performed by restric- tion fragment length polymorphism of the internal transcribed spacer regions (ITS-RFLP) in a reference laboratory. Patients were classified based on the clinical form as follows: VL when compatible signs and symptoms and blood or bone marrow samples had a positive culture or Leishmania poly- merase chain reaction (qPCR). MCL when compatible signs and symptoms and mucose sam- ples had a positive culture or Leishmania qPCR. CL when compatible signs and symptoms and cutaneous samples had a positive culture Leishmania qPCR. We assessed the patients’ com- plete medical records: demographic data, underlying disease, TNF- α blockers therapy, specific treatment and outcomes were recorded. Cure was defined as an absence of clinical signs after a minimum follow-up of 1 year after leishmanicidal treatment discontinuation. Unrelated death was established when a death with no relation to the infection or its treatment occurred during follow-up. Relapses were defined as reappearance of clinical signs after treatment discontinua- tion during the first year of follow-up unless relapse occurring after this period can be microbi- ologically demonstrated. Those cases without relevant information or not well identified were excluded from analysis. PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007708 August 30, 2019 3 / 16 Leishmaniasis in patients treated with tumor necrosis factor alpha antagonists Statistical analysis Categorical variables are expressed as percentages, and numerical data as the mean±SD for variables with a normal distribution or the median (IQR) for those with a skewed distribution. Categorical variables were compared with the chi-square test or Fisher exact test, and continu- ous variables with the Student t or the Mann-Whitney U test, depending on distribution. All statistical tests were 2-tailed, and significance was set at P < .05. Statistical analyses were per- formed using SPSS version 20.0 (SPSS, Inc., Chicago, IL, USA). Ethics statement Due to its retrospective design, oral consent was obtained by phone contact from the included patients. The study was approved by the Ethics Committee of Vall d’Hebron Research Institute. Results A total of 33 publications were retrieved in our search including forty cases that fulfilled inclu- sion criteria [8–39]. Thus, a total of 49 cases were analyzed including our 9 cases (Table 1). Twenty-seven (55.1%) cases were male and the median age was 55 (range 7–80) years. Twenty-five (51%) patients were under infliximab treatment at the moment of leishmaniasis diagnosis, twenty (40.8%) were receiving adalimumab, two (4.1%) were receiving etanercept, one (2%) golimumab and another one (2%) received a non-specified TNF-α blocker. From greater to lesser frequency, the underlying disease was psoriatic arthritis in twelve (24.5%) cases, rheumatoid arthritis in twelve (24.5%) cases, ankylosing spondylitis in nine (18.4%) cases, Crohn’s diseases in five (10.2%) cases, plaque psoriasis in five (10.2%) cases and rheuma- toid arthritis with psoriasis, ulcerative colitis, juvenile idiopathic arthritis, giant cell arthritis, seronegative arthritis and folliculitis decalvans in one case each other. All patients were diag- nosed in European hospitals and probable place of infection was Spain in thirty-three (67.3%) cases followed by Greece in five (10.5%) cases, Italy in four (8.2%) cases, France in two (4.1%) cases, Malta in two (4.1%) cases, Algeria in two (4.1%) cases and Turkey in one (2%) case. Regarding the clinical presentation, CL was the most frequent form (28 patients, 57.1%), eighteen cases presenting as a solitary ulcerative lesion and ten cases including one attended in our hospital who presented multifocal lesions (Fig 1). Sixteen (32.6%) patients presented VL and five (10.2%) patients had MCL, three in the nasal cavity (Fig 2), one as hyperplasic lesions around perianal mucosa and one as an infiltrative tumor involving upper lip, hard palate and nasal septum. Bone marrow aspirate was performed in one patient with MCL and one patient with CL. Although no amastigotes forms were observed, the patient with MCL had a positive Leishmania RT-PCR on the bone marrow sample. Identification to the level of species could be performed in eighteen patients. L. infantum was identified in seventeen cases and one was reported as L. donovani complex. All MCL and VL patients were treated with a systemic therapy. Fifteen (71.4%) of them were treated with liposomal amphotericine B (L-AmB), including one patient treated in com- bination with intralesional pentavalent antimonials, three (14.3%) patients were treated with parenteral pentavalent antimonials and one (4.8%) patient with miltefosine. Among CL cases, thirteen (46.4%) patients received a systemic treatment; eleven were given L-AmB, one intra- muscular antimonials and one miltefosine. Local treatment was given to fourteen (50%) patients, thirteen received intralesional pentavalent antimonials, one combined with surgery and another with cryotherapy and one patient was treated with surgical excision of the lesion. Only one patient defaulted and did not receive any treatment. PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007708 August 30, 2019 4 / 16 Table 1. Clinical and microbiological characteristics of patients with leishmaniasis associated to TNF-α antagonist treatment in the Mediterranean Basin. Patient Author, year Country Anti TNF Other IS Sex-Age Disease Clinical Number Specie Sample Diagnostic Treatment Outcome Comment Number and (Region) form of lesions reference 1 Romanı́- Spain Infliximab - M-55 PA VL - Leishmania Bone DO+ Parenteral Sb Cure Costa (Catalonia) sp. Marrow 2004 (8) 2 Fabre France Infliximab Azatioprine F-53 RA VL - L. infantum Bone DO+ L-AmB Cure 2005 (9) (Languedoc- Corticosteroids MON-1 Marrow PCR+ 15.4mg/Kg Rousillon) Anti-TNF Stopped 3 Bagalas Greece Etanercept Cyclosporin F-60 RA VL - Leishmania Bone DO+ L-AmB 50mg/ Dead Death was due to 2006 (10) (Central Corticosteroids sp. Marrow Kg respiratory Macedonia) superinfection 4 Bassetti Italy Adalimumab MTX F-69 RA VL - Leishmania Bone DO+ L-AmB 18mg/ Cure 2006 (11) (Liguria) Corticosteroids sp. Marrow PCR+ Kg Anti-TNF Stopped 5 Tektonidou Greece Infliximab MTX 12.5mg/ M-45 PA VL - Leishmania Bone PCR+ L.Amb 18mg/ Cure 2008 (12) (Atica) w sp. Marrow Kg Pred 7.5g/d Anti-TNF Stopped 6 De Italy Infliximab MTX 7.5mg/w M-63 PA VL - Leishmania Bone DO+ L-Amb Cure Leonardis (Emilia AZA (different sp. Marrow Anti-TNF 2008 (13) Romagna) times) Stopped 7 Garcia- Spain Infliximab MTX M-55 AS VL - L. donovani Bone DO+ Parenteral Sb Cure Vidal (Catalonia) Corticosteroids complex Marrow Anti-TNF 2009 (14) Stopped 8 Jeziorski France Infliximab MTX 10mg/w F-7 JIA VL - L. infantum Bone DO+ L-Amb 24mg/ Relapse Relapsed 2009 (15) (Languedoc- Marrow Kg 26months after Roussillon) L-Amb cessation with a MCL form. 9 Xynos Greece Infliximab MTX 10mg/w F-71 GCA VL - Leishmania Bone DO+ L-Amb 21mg/ Cure 2009 (16) (Atica) Corticosteroids sp. Marrow PCR+ Kg PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007708 August 30, 2019 10 Moreno Spain Infliximab MTX 15mg/w F-72 RA VL - Leishmania Bone DO+ L-AmB 21mg/ Cure 2010 (17) (Valencian Pred 5–10 mg/ sp. marrow Kg Comunity) d Duodenal Anti-TNF biopsy Stopped 11 Moltó Spain Adalimumab MTX 20 mg/w M-60 RA VL - Leishmania Bone DO+ L-AmB 30mg/ Cure 2010 (18) (Catalonia) Pred 5–10 mg/ sp. Marrow Kg d Anti-TNF Stopped 12 Kritikos Greece Infliximab - F-77 RA VL - Leishmania Bone DO+ L-AmB Cure 2010 (19) (Atica) sp. Marrow Anti-TNF Stopped 13 Erre Italy Adalimumab MTX 10mg/w F-71 RA VL - Leishmania Bone DO+ L-Amb Cure 2010 (20) (Sardinia) sp. Marrow Anti-TNF Stopped 14 Khan Malta Adalimumab MTX F-74 PA VL/CL - L. infantum Bone DO+ L-Amb 30mg/ Cure Episode 2010 (21) Marrow PCR+ Kg considered as Skin Intralesional visceralization of biopsy Sb a CL form Anti-TNF Stopped 5 / 16 Leishmaniasis in patients treated with tumor necrosis factor alpha antagonists (Continued ) Table 1. (Continued) Patient Author, year Country Anti TNF Other IS Sex-Age Disease Clinical Number Specie Sample Diagnostic Treatment Outcome Comment Number and (Region) form of lesions reference 15 Besada Spain Etanercept MTX 20mg/w F-80 PA VL - L. infantum Bone DO+ L-AmB 21mg/ Cure 2013 (22) (Valencian Marrow PCR + Kg Comunity) Anti-TNF Stopped 16 Berry Spain Adalimumab MTX 20mg/w F-69 RA VL - L. infantum Bone DO+ L.AmB 40mg/ Cure 2013 (23) (Andalusia) Marrow PCR+ Kg Spleen Anti-TNF Biopsy Stopped 17 Garcı́a- Spain Infliximab MTX 15mg/w F-66 RA MCL 1 L. Infantum Mucosa DO+ L-AmB/Sb Cure Castro (Andalusia) Pred 2.5mg/d MON-183 biopsy Culture + Anti-TNF 2011 (24) Stopped 18 Garcı́a- Italy Adalimumab MTX 10mg/w M-49 AS MCL 1 L. infantum Mucosal DO+ L-AmB 15mg/ Cure González (Tuscany) M-pred 4mg/d biopsy Culture+ Kg 2012 (25) Anti-TNF Stopped 19 Neumayr Spain Adalimumab - M-53 PA MCL 1 L. infantum Skin PCR+ Miltefosine Cure 2013 (26) (Balearic biopsy Islands) 20 Juzlova Turkey/ Infliximab MTX 15mg/w M-44 CD MCL 1 L. infantum Perianal DO+ Intramuscular Dead Death was due to 2014 (27) Croatia Biopsy PCR+ MgA fatal arrhythmia as side effect of antimony 21 Bosch- Spain Infliximab - M-53 PA MCL/ 1 L. Infantum Mucosal DO L-AmB 40mg/ Cure Episode (Fig 2) Nicolau (Catalonia) VL biopsy +(biopsy) Kg considered as a 2019 Bone PCR + Anti-TNF reactivation of a marrow Stopped previous CL occurred 4 years before 22 Baltà-Cruz Spain Infliximab MTX 15mg/w F-56 RA CL 1 L. infantum Skin DO+ Intralesional Relapse MC form treated 2009 (28) (Catalonia) followed Pred 3.75 mg/d Biopsy PCR+ Sb with L-AmB/ IM Adalimumab Surgery Sb. Anti-TNF PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007708 August 30, 2019 Stopped Finally cured 23 Mueller Spain Infliximab - M-31 AS CL 1 L. Infantum Skin DO+ Miltefosine Relapse C form treated 2009 (29) (Balearic MON 1 Biopsy PCR+ Anti-TNF with intralesional Islands) Culture + Stopped Sb. Anti-TNF Stopped Finally cured 24 Schneider Algeria Adalimumab MTX F-51 AS CL 3 L. infantum Skin PCR+ L-AmB 21mg/ Cure 2009 (30) Biopsy Kg Anti-TNF Stopped 25 Xynos Greece Infliximab MTX 10mg/w M-55 AS CL Several Leishmania Skin DO+ L-AmB 21mg/ Cure Presented several 2009 (16) (Atica) sp. Biopsy Kg crusted lesions on Anti-TNF face Stopped 26 Hakimi Algeria / Infliximab - M-50 AS CL 3 L. infantum Skin DO+ Intralesional Cure 2010 (31) France MON-1 Biopsy PCR+ MgA (Languedoc- Culture + Anti-TNF Rousillon) Stopped 6 / 16 Leishmaniasis in patients treated with tumor necrosis factor alpha antagonists (Continued ) Table 1. (Continued) Patient Author, year Country Anti TNF Other IS Sex-Age Disease Clinical Number Specie Sample Diagnostic Treatment Outcome Comment Number and (Region) form of lesions reference 27 Romero- Spain Adalimumab - F-61 RA CL 6 L. infantum Skin PCR+ None - Lost to follow-up Maté (Madrid) biopsy 2013 (32) 28 Romero- Spain Adalimumab MTX 20mg/w F-59 PA CL 1 L. infantum Skin PCR+ Intralesional Cure Maté (Madrid) biopsy MgA 2013 (32) 29 Hernández- Spain Infliximab - M-50 P CL 1 Leishmania Skin DO+ L-AmB 40mg/ Cure Torres (Murcia) sp. biopsy PCR+ Kg 2013 (33) Anti-TNF Stopped 30 Català Spain Infliximab Leflunomide M-33 PA CL 1 Leishmania Skin DO+ Intralesional Cure 2014 (34) (Catalonia) 20mg/d sp. biopsy Culture + MgA Pred 5mg/d + Cryotherapy Anti-TNF Stopped 31 Micallef Malta Adalimumab MTX F-45 SNA CL 1 Leishmania Skin PCR+ Parenteral Sb Cure 2014 (35) sp. biopsy Anti-TNF Stopped 32 Català Spain Adalimumab - F-59 RA CL 1 Leishmania Skin DO+ L-AmB 40mg/ Cure 2015 (36) (Catalonia) sp. biopsy Kg Anti-TNF Stopped 33 Marcoval Spain Adalimumab - M-35 CD CL 1 Leishmania Skin PCR+ Intralesional Relapse L-Amb Finally 2017 (37) (Catalonia) sp. biopsy MgA cured 34 Marcoval Spain Infliximab - F-55 CD CL 1 Leishmania Skin PCR+ Intralesional Relapse L-Amb Finally 2017 (37) (Catalonia) sp. biopsy MgA cured 35 Alcover Spain Not specified - M-55 RA+P CL 12 L. infantum Skin DO+ L-Amb 40mg/ Cure Presented 2018 (38) (Balearic biopsy PCR+ Kg numerous skin Islands) Culture+ Anti-TNF ulcers Stopped PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007708 August 30, 2019 36 Martı́nez- Spain Adalimumab - F-34 P CL 1 Leishmania Skin DO+ Intralesional Cure Doménech (Valencian sp. biopsy MgA 2019 (39) Comunity) Anti-TNF Stopped 37 Martı́nez- Spain Adalimumab - M-46 AS CL 1 Leishmania Skin DO+ Intralesional Cure Doménech (Valencian sp. biopsy PCR+ MgA 2019 (39) Comunity) Anti-TNF Stopped 38 Martı́nez- Spain Adalimumab - F-40 P CL 1 Leishmania Skin DO+ Intralesional Cure Doménech (Valencian sp. biopsy PCR+ MgA 2019 (39) Comunity) 39 Martı́nez- Spain Adalimumab MTX M-71 PA CL 2 Leishmania Skin DO+ Intralesional Cure Doménech (Valencian sp. biopsy PCR+ MgA 2019 (39) Comunity) Anti-TNF Stopped 40 Martı́nez- Spain Adalimumab - M-53 P CL 1 Leishmania Skin PCR+ Intralesional Cure Doménech (Valencian sp. biopsy MgA 2019(39) Comunity) Anti-TNF Stopped (Continued ) 7 / 16 Leishmaniasis in patients treated with tumor necrosis factor alpha antagonists Table 1. (Continued) Patient Author, year Country Anti TNF Other IS Sex-Age Disease Clinical Number Specie Sample Diagnostic Treatment Outcome Comment Number and (Region) form of lesions reference 41 Martı́nez- Spain Adalimumab - M-22 FD CL 2 Leishmania Skin DO+ Intralesional Cure Doménech (Valencian sp. biopsy PCR+ MgA 2019(39) Comunity) 42 Bosch- Spain Infliximab - M-42 AS CL 4 L. Infantum Skin DO+ L-AmB 20mg/ Cure Presented (Fig 1) Nicolau (Catalonia) biopsy PCR + Kg numerous skin 2019 Anti-TNF ulcers Stopped 43 Bosch- Spain Infliximab Deflazacort M-67 AS CL 1 Leishmania Skin DO+ Surgery Cure Nicolau (Catalonia) 6mg/d sp. biopsy 2019 44 Bosch- Spain Infliximab Azatioprine F-54 CD CL 1 Leishmania Skin DO+ Intralesional Cure Nicolau (Catalonia) sp. biopsy PCR + MgA 2019 45 Bosch- Spain Golimumab - M-71 PA CL 1 Leishmania Skin DO+ L-AmB 20mg/ Cure Nicolau (Catalonia) sp. biopsy Kg 2019 46 Bosch- Spain Infliximab - M-67 UC CL 1 Leishmania Skin DO+ L-AmB 24mg/ Cure Nicolau (Catalonia) sp. biopsy Kg 2019 Anti-TNF Stopped 47 Bosch- Spain Infliximab - M-50 PA CL 2 Leishmania Skin PCR+ L-AmB 20mg/ Cure Nicolau (Catalonia) sp. biopsy Kg 2019 48 Bosch- Spain Infliximab Azatioprine M-42 CD CL 1 Leishmania Skin DO+ L-AmB 16mg/ Cure L-AmB Nicolau (Catalonia) sp. biopsy Kg discontinued 2019 Anti-TNF after 4 doses Stopped 49 Bosch- Spain Adalimumab M-74 P CL 2 Leishmania Skin PCR+ L-AmB 20mg/ Cure Nicolau (Catalonia) sp. biopsy Kg 2019 Anti-TNF PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007708 August 30, 2019 Stopped MTX: Metrotexate, Pred: prednisone, M-Pred: Metylprednisolone, M: male, F: female, PA: Psoriatic Arthritis, RA: Rheumatoid Arthritis, AS: Ankylosing Spondylitis, JIA: Juvenile Idiopathic Arthritis, SNA: Sero-Negative Arthritis, GCA: Giant cell arteritis, CD: Crohn’s disease, UC: Ulcerative Colitis, P: Psoriasis, FD: Foliculitis decalvans, VL: visceral leishmaniasis, CL: cutaneos leishmaniasis, MCL: mucucutaneous leishmaniasis, DO: Direct Observation, PCR: Polymerase Chain Reaction, Sb: Sodium tibogluconate, MgA: Meglumine antimoniate; L-AmB: Liposamal Amphotericine B, TNF: Tumor necrosis Factor. https://doi.org/10.1371/journal.pntd.0007708.t001 8 / 16 Leishmaniasis in patients treated with tumor necrosis factor alpha antagonists Leishmaniasis in patients treated with tumor necrosis factor alpha antagonists Fig 1. Patient with multiple cutaneous leishmania lesions. https://doi.org/10.1371/journal.pntd.0007708.g001 In thirty-two (65.3%) cases TNF-α blockers therapy was interrupted. After treatment, four (8.2%) patients with CL diagnosis relapsed. Three of these cases were initially treated with local medication and anti-TNF-α was not stopped. After relapse, the three patients received systemic treatment and anti-TNF-α therapy was discontinued in one patient, all achieving clinical cure. Another relapsing patient was initially treated with miltefosine and finally cured with local antimonials therapy. TNF-α blocker therapy was discontinued in both treatment courses. The last relapsing patient was a VL treated with L-AmB and TNF-α blockers were not stopped as her rheumatic disease was active presenting a MCL form 20 months after. When Fig 2. Patient with a mucocutaneous leishmaniasis. https://doi.org/10.1371/journal.pntd.0007708.g002 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007708 August 30, 2019 9 / 16 Leishmaniasis in patients treated with tumor necrosis factor alpha antagonists Fig 3. Leishmaniasis form, treatment and outcomes of all included patients. CL: cutaneous leishmaniasis, S: solitary lesion; Mx: multiple lesions; L-AmB: liposomal amphotericine-B; Sb S: systemic sodium tibogluconate; MTF: miltefosine; Mg IL: intralesional meglumine antimoniate; Surg: surgery; TNF: tumor necrosis factor; Ø no treatment, ¥ deceased. Arrows represent patients who relapsed and its path shows patient initial treatment and TNF-alpha blocker management. https://doi.org/10.1371/journal.pntd.0007708.g003 comparing clinical cure of CL patients, although statistical significance could not be reached cure ratios were 92.3% vs. 78.6% (p = 0.6) when patients received systemic treatment or not and 94.1% vs 70% (p = 0.13) when TNF-α blockers therapy could be stopped or not. Finally, the patient who did not receive any treatment was lost to follow up. Two (4.1%) patients died: one patient after a bacterial superinfection in relation to his immunosuppression and the other patient as a result of a fatal arrhythmia during his treatment with systemic antimonials. Case presentation, treatment and outcomes are represented in Fig 3. Discussion According to the published data, TNF-α blockers based therapy seems to be associated with a higher risk of infections, at times with a worse outcome [3]. Nevertheless their efficacy in the management of many chronic inflammatory diseases has reaffirmed its use, representing one of the major breakthroughs in the treatment of these diseases [40]. L. infantum is the major causative agent of leishmaniasis in Southern Europe and the most frequent zymodeme is MON 1 [41]. Even though it has been suggested that there are dermato- tropic and viscerotropic strains, L. infantum is prone to produce the visceral form of disease and less frequently the cutaneous form. Although different gens have been proposed to con- tribute to the viscerlalization process, its mechanisms still remains unclear due to the lack of good animal models [42]. It is also well known that in L. infantum endemic areas there is a frequent natural challenge to Leishmania parasites, producing circulating parasitemia in the host during an undefined non-permanent time-span. Some authors have referred to this subclinical form as “asymptom- atic carriage”. It means that these patients without symptoms have parasite load only detectable by highly sensitive methods such as RT-PCR [43,44]. PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007708 August 30, 2019 10 / 16 Leishmaniasis in patients treated with tumor necrosis factor alpha antagonists In southern Europe the prevalence of asymptomatic carriers is remarkably variable (from 0.5% to 48%) depending on the test used for detection [45]. Those data may suggest that there are natural and frequent challenges between host and parasite in endemic areas, and that an effective immune system is required to control clinical expression. While it seems that in mammalian hosts Leishmania species may be able to infect and develop within non-hematopoietic cells such as fibroblasts it is mostly inside monocyte/mac- rophage lineage cells where the parasite replicates and develops long-time survival [46]. Since these cells have a central role in both innate response and acquired immune response as para- site antigen presenting, Leishmania interferes in an extremely complex manner with the host immunological response [47]. The role of TNF-α in the pathogenesis of the disease has been widely studied in both humans and animal models. As innate TNF-α dependant mechanisms drive cell mediated immunity by activation of CD4+ and CD8+ T cells, TNF-α is known to be fundamental in the initial control of the infection together with other cytokines such as IL-12 and IFN-γ [48]. These cytokines mediate the development of an effective CD4+ Th1 response which is critical to control the infection [49,50]. Furthermore, TNF-α and IFN-γ are responsible for activating leishmanicidal activity of macrophage which is characterized by an increased production of free oxygen radicals and nitric oxide (NO) as well as inducing infected cells apoptosis [51,52]. In CL, a polarized Th1 response with low Th2 cytokines has been related to infection resis- tance and disease outcome. Besides, Th1 and Th2 pathways may also modify disease expres- sion [53]. However, in patients presenting disseminated diseases such as VL, immune response seems to be a net suppressive-type response. Rather than an inert Th1 type response, a Th2 / Th1 type response associated with the production of down-regulating cytokines such as IL-10 and TGF-β seems to be responsible of the persistent inflammation, the hallmark of clinical leishmaniasis [54,55]. Moreover, regulatory T- and Th17- cells appear to play an important role in susceptibility and disease resistance [56]. Hence, TNF-α has been implicated in the initial events of the infection, in direct leishmanici- dal activity and thus controlling the multiplication of the parasite, developing effective acquired immunity for long term control of the disease as well as mediating the disease expression [47]. The classical VL in Mediterranean countries has been found primarily in children and HIV/AIDS infected patients with poor immune status. Nowadays VL cases in adults are also diagnosed among non-VIH immunosuppressed patients [57]. Moreover, CL is less frequent than VL in some Mediterranean countries as Spain although it may be underreported [58]. Besides, MCL is considered exceptional in the Old World [43]. Anyhow, in our series the pro- portion of CL and MCL it is surprisingly high. Clinical outcome observed in this series is unusual. The natural course of CL caused by L. infantum is benign and trends to heal spontaneously. In fact, if lesions are less than 5 cm in diameter and are localized in areas where there is no risk of disfiguring or disabling, only a local wound care is recommended [59]. Four out of twenty-eight (14.3%) cases of CL reported in this series relapsed despite etiological treatment. Despite of the natural viscerotropism of L. infantum, the main acquisition of this infection is through the skin. Besides, in endemic areas there is a high exposure to Leishmania. Although some studies report up to 48% of prevalence [60], there are only limited cases of CL reported. Consequently, it may be assumed that within immune-competent individuals, the immunity of the host is capable to control the infection, even before that it causes any clinical symptoms. The reactivation of a latent parasitic form from a granulomatous lesion could be a feasible explanation for those cases. Clinical data from patient number 21 supports this assumption. This patient was diagnosed with a MCL form (Fig 2) 4 years after a CL in his left ankle demon- strated by PCR amplification for Leishmania kinetoplastid sequences which healed PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007708 August 30, 2019 11 / 16 Leishmaniasis in patients treated with tumor necrosis factor alpha antagonists spontaneously. Because of the latency between the first episode and the second one and the appearance of a distant lesion far from the initial one, the episode was thus considered as a reactivation in the context of the TNF-α blockers therapy. Previous series including patients with Old and New world leishmania species showed no con- sensus regarding treatment in patients under TNF-α blockers [26]. In this series only including leishmaniasis acquired at Mediterranean basin countries, besides all patients with VL and MCL were given a systemic therapy, treatments differ considerably. Seventeen (80.9%) patients received L-AmB at different doses ranging from 25.4mg/Kg to 50mg/Kg divided in 5 to 10 doses, three patients received pentavalent antimonials and one miltefosine. L-AmB is usually recom- mended as first-choice treatment of VL offering similar cure-rates than pentavalent antimonial [61]. Especially, considering that pentavalent antimonials present a worse toxicity profile and major toxicities such cardiac arrhythmias or prolonged QTc interval that can lead to death as in case number 20 of our series [62]. Although miltefosine has successfully been used treating VL, when it comes to L. infantum, a growing number of treatment failures have been published so further investigation is needed before its systematic recommendation [63]. Concerning CL treat- ment, approach disparities are even greater. Half patients received systemic treatment with differ- ent therapies and dose disparity and the other half received local treatment. As mentioned before, TNF-α plays a key role in the process of controlling infection that ranges from limiting the replication of the parasite up to eliciting an effective adaptative response. TNF-α blockers therapy could modulate the immunological response to a less effective control of the parasite, hence allowing the infection reactivation or more evident disease expression of newly acquired infections. To this end, patients under treatment with TNF-α blockers have less effective immune mediated mechanisms to control and eradicate a parasite challenge, ending in a higher chance to develop classical and non-classical L. infantum presenting forms, that otherwise it would have caused a transient parasitemia or an aborted local replication. Likewise, the course and outcome of the disease may be directly affected by the inhibition of TNF- α. Therefore, dis- continuation of the anti TNF-α therapy seems to play an important role in the treatment success. Although miltefosine is an excellent option for the New World CL, evidence is scarce for the treatment of L. infantum infection [64]. Thus, it seems appropriate to limit its use when other treatment options are not available or have failed. According to the cases reported, in our opinion the best treatment strategy would be a sys- temic treatment and the discontinuation of the TNF-α blockers therapy. L-AmB has proved to have the best safety profile and compelling evidence of its efficacy in immunosuppressed patients. One of the major concerns is the reintroduction of TNF-α blockers. Taking into account published information, it seems reasonable reintroducing them once clinical cure has been achieved ensuring close clinical follow-up and blood RT-PCR. Although there is scarce information for its recommendation, etanercep or certolizumab have been suggested as a ther- apeutical option instead of re-introducing other anti-TNF-α monoclonal antibodies due to its possible lower risk of reactivating leishmaniasis [65–66]. This study has the limitations of any retrospective review study. Although is one of the big- gest series published on this topic, the scarce number of patients analyzed limits the strength of the recommendations. We agree with existing reports on the fact that despite of not being a common disease in our area, leishmaniasis is likely underreported [58]. Finally, with the results extracted from this study it is not possible to assess the risk of developing clinical leish- maniasis during anti TNF-α therapy. However, there is enough supporting data for the biolog- ical plausibility of the influence of the use of such therapy in the appearance of new cases of clinical leishmaniasis with a switch in the disease expression and outcome. The blockage of TNF-α could determine the incapacity to control and eradicate the parasite within the granu- loma playing an important role in increasing the risk of progression to clinical disease. PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007708 August 30, 2019 12 / 16 Leishmaniasis in patients treated with tumor necrosis factor alpha antagonists Conclusions The increase in the use of TNF-α antagonist has been associated with the emergence of new cases of leishmaniasis. The blockage of TNF-α favors the reactivation of latent leishmaniasis modulating its expression and worsening its clinical outcome. Once the leishmaniasis is confirmed, systemic drug treatment and the discontinuation of the TNF-α blockers therapy until clinical recovery seems to be the best therapeutic approach when possible. Otherwise, those patients receiving such therapy and coming from endemic areas require a close monitoring in order to detect early forms and start adequate treatment. Prospective studies and more participation on declaring existing cases in the adverse events notification system is required in order to assess the risk of leishmaniasis and other opportu- nistic diseases related to the use of anti TNF-α treatment more accurately. Supporting information S1 Checklist. STROBE checklist. (DOCX) Author Contributions Conceptualization: Pau Bosch-Nicolau, Fernando Salvador, Adrián Sánchez-Montalvá, Israel Molina. Formal analysis: Pau Bosch-Nicolau, Israel Molina. Investigation: Pau Bosch-Nicolau, Maria Ubals, Fernando Salvador, Adrián Sánchez- Montalvá. Project administration: Israel Molina. 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