Seroprevalence of Leishmania infection among asymptomatic renal transplant recipients from southern Spain

Seroprevalence of Leishmania infection among asymptomatic renal transplant recipients from southern Spain E.K. Elmahallawy, E. Cuadros-Moronta, M.C. Li ebana-Martos, J.M. Rodr ıguez-Granger, A. Sampedro-Mart ınez, A. Agil, J.M. NavarroMari, J. Bravo-Soto, J. Gutierrez-Fern andez. Seroprevalence of Leishmania infection among asymptomatic renal transplant recipients from southern Spain. Transpl Infect Dis 2015: 17: 795–799. All rights reserved Abstract: Background. The aim of this article is to assess the seroprevalence of Leishmania infection among asymptomatic renal transplant recipients in a population in the south of Spain. Methods. Serum samples were screened for immunoglobulin-G antibodies against Leishmania with an indirect fluorescent antibody test. Results. Of 625 examined serum samples, 30 (4.8%) samples were positive for Leishmania antibodies. Thirteen samples showed titers of 1:80, 15 samples showed titers of 1:160, and 2 samples showed titers of 1:320. None of the patients with positive serology to Leishmania showed signs or symptoms compatible with leishmaniasis. Conclusion. The prevalence of Leishmania infection found among asymptomatic renal transplant patients reinforces the need for attention in evaluation of these patients in endemic areas. E.K. Elmahallawy 1 , E. CuadrosMoronta 2 , M.C. Li ebana-Martos 2 , J.M. Rodr ıguez-Granger 2 , A. Sampedro-Mart ınez 2 , A. Agil 3 , J.M. Navarro-Mari 2 , J. Bravo-Soto 4 , J. Gutierrez-Fern andez 5 1 Department of Zoonotic Diseases, Faculty of Veterinary Medicine, Sohag University, Sohag, Egypt, 2 Service of Microbiology, University Hospital Virgen de las Nieves, Granada, Spain, 3 Department of Pharmacology and Neurosciences Institute (CIBM), Faculty of Medicine, University of Granada, Granada, Spain, 4 Service of Nephrology, University Hospital Virgen de las Nieves, Granada, Spain, 5 Department of Microbiology, Faculty of Medicine, University of Granada, Granada, Spain Key words: seroprevalence; leishmaniasis; renal transplant recipient Correspondence to: Javier Mois es Rodr  ıguez-Granger, Microbiology Service Hospital, University Virgen de las Nieves, Avenida de Fuerzas Armadas s/n, 18014 Granada, Spain Tel: +34 667446814 Fax: +34 958 02 00 10 E-mail: [email protected] Received 18 March 2015, revised 20 July 2015, accepted for publication 4 August 2015 DOI: 10.1111/tid.12444 Transpl Infect Dis 2015: 17: 795–799 Leishmaniasis refers to a group of vector-borne diseases caused by an opportunistic intracellular protozoan parasite of the genus Leishmania, belonging to the family Trypanosomatidae, order Kinetoplastida (1). The disease is associated with 3 main manifestations: (i) cutaneous leishmaniasis, (ii) mucocutaneous leishmaniasis, and (iii) visceral leishmaniasis (VL) also known as kala-azar (2). VL is considered to be a wellestablished zoonotic disease in the area of the Mediterranean basin, especially in southwestern Europe, where approximately 700 cases per year appear and the clinical form is frequently associated to immunosuppression (3, 4). The actual prevalence of leishmaniasis in Spain is not completely known (5); however, in our geographical area of southern Spain, studies published in 1996 of the Alpujarra region (Granada province) and Axarquia region (Malaga province) of rural populations, using the leishmanin skin test (LST) (test cell immunity), reported average positivity rates >40% in both studies 795 ©2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Transplant Infectious Disease, ISSN 1398-2273 (6, 7). Nationwide, more recent studies in areas close to ours, such as the study performed on blood donors from Ibiza (Balearic Islands, Spain) in 2004, indicated a high rate of asymptomatic infection on the island, and another study in 2008 from Balearic Islands confirms this high prevalence (8, 9). Apart from these studies, the most studied populations have been human immunodeficiency virus (HIV)- infected individuals and intravenous drug users in our environment (southern Spain) (10–12). Not many publications have reported the known prevalence of Leishmania in special groups, such as transplant recipients, oncology patients, or other immunodeficient non-HIV groups, in Spain and the Mediterranean region. Leishmaniasis is not a common disease among transplant recipients, but with this growing pool of transplant survivors and increasing migration dynamics, the numbers of infected cases among transplant recipients are steadily increasing, especially among renal transplant recipients mainly in southern Europe (13), which is considered to be an additional challenge (14, 15). Transmission of VL to this specific population occurs through different routes: Leishmania transmission by sandflies after transplantation, when the specific group of population is living in an endemic area or makes a short stay in an endemic area represents a route of transmission (16, 17). Also, it may have originated from a donor with an undiagnosed infection, or a recrudescence or latent infection in a previously infected recipient during a period of immunosuppression. Infection can also be acquired through blood transfusion (18). Aim of study Considering these findings, the present study was undertaken to assess the seroprevalence of Leishmania infection among asymptomatic renal transplant recipients in Granada (Southern Spain) while they are tracked post transplant. Material and methods This study was approved by the Ethical Committee of Research of the University Hospital Virgen de las Nieves (Granada, Spain), before initiation of data collection. Serum samples from 625 individual renal transplant patients were collected for 1 year (April 2013–April 2014). Age, gender, cause of the transplant, survival time after transplantation, and type of immunosuppressive therapy were recorded. Any individuals with fever or infectious disease were excluded from the study. All patients were living in neighboring Granada provinces for at least the last 10 years, for which our hospital is a referral center for renal and liver transplantation. To investigate Leishmania infection, peripheral blood was collected into sterile Vacutainer TM tubes. Samples were centrifuged at 4°C and stored at 80°C until serological tests were performed. All patients had a low level of immunosuppression at the time of blood sampling. Serum samples were screened for leishmaniasis with indirect fluorescent antibody (IFA) test. The IFA test for determination of antibodies against Leishmania infantum (Leishmania IFI IgG Kit, Vircell, Santa Fe, Granada, Spain) was performed according to the manufacturer’s instructions. When samples were positive with a titer of 1:80, a 2fold serum dilution was done until samples reached 1:1280 dilution. As shown in the kit instructions, IFA test is considered positive with promastigotes fluorescence, with a cutoff dilution of 1:40 owing to the endemicity of the area of study. This result is considered as indeterminate in this assay (19). Results During the study period, 625 patients with a mean time of 10 years from transplantation to the last review were recruited at their transplant consultation. Median age distribution was 49 years (range 11–81 years). According to gender, 225 (36%) were female and 400 (64%) were male. Regarding the distribution by race, the Caucasian race was predominant at 621 (99.4%), within which 17 were of Romani ethnicity, and 4 (0.6%) were black. The etiology of renal failure that caused the transplantation was as follows: glomerular disease 174 (27.8%), unknown 116 (18.6%), cystic disease 98 (15.7%), interstitial disease 84 (13.4%), vascular disease 81 (13%), diabetes 27 (4.3%), and 45 other causes (6.1%). IFA testing was performed on 625 serum samples; 30 samples were positive, 13 showed titers of 1:80, 15 showed titers of 1:160, and 2 showed titers of 1:320. Fourteen additional samples showed titers of 1:40, considered as indeterminate. The characteristics of patients with Leishmania antibodies in relation to the variable studied are shown in Table 1. The prevalence was 4.8%. The immunosuppressive treatments after transplantation for patients with positive serology included the following regimens: anti-thymocyte globulin (administered only during the first 3 months after 796 Transplant Infectious Disease 2015: 17: 795–799 Elmahallawy et al: Leishmania in renal transplant 13993062, 2015, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tid.12444 by Universidad De Granada, Wiley Online Library on [19/03/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License transplantation) +mycophenolate mofetil (MMF) + prednisone in 10 patients (30.3%); tacrolimus +MMF + prednisone in 8 patients (2.6%); prednisone +MMF + basiliximab in 5 patients (16.6%); tacrolimus (once daily) +basiliximab +MMF +prednisone sodium in 4 patients (13.3%); and basiliximab +cyclosporine + MMF +prednisone in 3 patients (10%). All patients had a low level of immunosuppression at the time of blood sampling. No differences existed between the immunosuppressive treatment among patients with negative serology and positive serology. No patient developed leishmaniasis disease during the study period or to date. Discussion Although VL is a rare disease among transplant patients, the current situation has been changing, and the number of published cases has increased in recent years (20). Worldwide, >100 VL cases following transplantation have been reported, predominantly described in renal transplantation (15, 19, 20). The prevalence of asymptomatic infections of Leishmania species varied from 0.6% to 71% in endemic areas (21). To detect asymptomatic carriers, direct method (polymerase chain reaction [PCR], culture, microscopic examination) or indirect methods (IFA test, direct agglutination test, enzyme-linked immunosorbent assay [ELISA] with single or crude Leishmania antigens, Western blot [WB], or immunochromatographic techniques) can be used, but no gold standard test is available to identify asymptomatic infection with high sensitivity and specificity (21, 22). The prevalence of Leishmania infection in our study is in agreement with previous studies in different geographical areas of our country, Balearic Islands and Castilla Le on, with results of prevalence of 3.1% and 4.9% in blood donors and in the general population respectively (9, 23). More recent studies, like the one published in the province of Seville on a small population that included intravenous drug users and non-drug injectors, where the frequency of Leishmania seropositivity was high, showed prevalence of infection by Leishmania of 24% using PCR-ELISA. This study showed that a remarkable proportion of asymptomatic Leishmania-seropositive individuals at risk for parenterally transmited infections carry Leishmania kinetoplast DNA (kDNA) in blood (11). Some studies have been made in southern Spain in asymptomatic HIV population diagnosing by amplificaCharacteristics of patients (n=30) grouped by antibody titer against Leishmania infantum Antibody titer (n) 1:80 (n=13) 1:160 (n=15) 1:320 (n=2) Age in years (range) 44.15 (29–66) 49.33 (29–67) 45.50 (43–48) Gender Male 6 (46.2) 9 (60) 2 (100) Female 7 (53.8) 6 (40) 0 (0) Race Mediterranean white 13 (100) 14 (93.3) 2 (100) Nordic white –1 (6.7) – Renal failure type Unknown 4 (30.8) 3 (20) 1 (50) Glomerular 5 (38.5) 3 (20) – Vascular 2 (15.4) 1 (6.7) – Systemic 2 (15.4) –– Interstitial –2 (13.3) 1 (50) Cystic –3 (20) – Diabetes –2 (13.3) – Survival time in years (range) 2.64 (1.58–3.72) 2.20 (0.3–3.67) 1.52 (0.44–2.61) All patients had a low level of immunosuppression. Table 1 Transplant Infectious Disease 2015: 17: 795–799 797 Elmahallawy et al: Leishmania in renal transplant 13993062, 2015, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tid.12444 by Universidad De Granada, Wiley Online Library on [19/03/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License tion of kDNA from peripheral blood showing a prevalence of 30.8% (28/92) infection by Leishmania. No patients showed positive results by other techniques, such as ELISA, WB, or LST. Conversely, patients with a negative PCR result showed prevalence values, respectively, of 3.5%, 2.4%, and 4.3% when tested by ELISA, WB, and LST (12). In other zones endemic for L. infantum, such as Brazil, studies have recently been published that raise interest in the detection of asymptomatic infection in the transplant population living in endemic areas, as well as the need to use more sensitive techniques for screening this population group, such as the research of Clemente et al. (19) in 2014 on asymptomatic Leishmania infection among liver donors and recipients. The results of this study show a prevalence of Leishmania infection of 1.5% using serological techniques, which increased to 7.5%, 8.9%, and 5.9%, respectively, in blood samples, liver samples, and splenic samples, using molecular methods (19). The results obtained in our study (4.8%) using a unique serological technique may be underestimated, because the IFA test has less sensitivity than other indirect assays (ELISA) (21). The use of 2 serological methods could slightly increase the proportion of asymptomatic carriers detected (24, 25). On the other hand, antibody detection may not reflect a chronic infection, but more a recent contact with parasite followed by cure; PCR assay would have greater sensitivity (26) than the indirect method, and reflects actual parasitism rather than a previous infection by Leishmania. Different studies highlight the added value of using a combination of tests (molecular and serological) to increase the capacity to detect asymptomatic Leishmania infections (21, 22). At present, it is not possible to predict exactly when and who among the asymptomatically infected people will develop VL disease (27). Reasons causing an infection to remain asymptomatic or progress to VL are likely the result of a complex interaction between environment, parasite, and host-related factors (28). Studies of the risk of progression from infection to disease have yielded contradictory results based on serological status; however, the data revealed strong associations between the magnitude of positive serology and risk of progressing to symptomatic VL (29). International guidelines for the management of transplant recipients recommend specific serology in donors and recipients from endemic areas, regardless of laboratory test limitations and availability (30). Given that patients, such as transplant recipients, are susceptible to develop leishmaniasis, and the clinical manifestations in these patients can be serious, it seems advisable to perform a screening of serological status in this population group and a more intense monitoring of patients with positive serology with high titers to prevent possible development of disease. Acknowledgements: Support: E.K. Elmahallawy has a PhD scholarship (number 736) from Erasmus Mundus Scholarship Program (ELEMENT Action 1 First call). Dedication: We dedicate this article to Dr. Juan Bravo-Soto in the year of his retirement as head of kidney transplants in University Hospital Virgen de Las Nieves, in appreciation of his excellent work and professional dedication to patients. References 1. Clem A. A current perspective on leishmaniasis. J Glob Infect Dis 2010; 2: 124–126. 2. Herwaldt BL. Leishmaniasis. Lancet 1999; 354: 1191–1199. 3. Ready PD. Leishmaniasis emergence in Europe. Euro Surveill 2010; 15: 19505. 4. Fernandez-Guerrero ML, Aguado JM, Buzon L, Barros C, Montalban C. Visceral leishmaniasis in immunocompromised hosts. Am J Med 1987; 83: 1098–1102. 5. Botet J, Port us M. La leishmaniosis en la Espa~ na Peninsular. Revisi on hist orico-bibliogr afica (1912–1985). Rev San Hig Publica 1993; 67: 255–266. 6. Acedo S anchez C, Martin S anchez J, V elez Bernal ID, et al. Leishmaniasis eco-epidemiology in the Alpujarra region (Granada province, southern Spain). Int J Parasitol 1996; 25 (3): 303–310. 7. Morillas F, S anchez Tabasco F, Oca~ na J, et al. Leishmaniasis in the focus of the Axarquia region, Malaga province, southern Spain: a survey of the human, dog, and vector. Parasitol Res 1996; 82 (6): 569–570. 8. Riera C, Fisa R, Udina M, Gallego M, Portus M. Detection of Leishmania infantum cryptic infection in asymptomatic blood donors living in an endemic area (Eivissa, Balearic Islands, Spain) by different diagnostic methods. Trans R Soc Trop Med Hyg 2004; 98: 102–110. 9. Riera C, Fisa R, L opez-Chejade P, et al. Asymptomatic infection by Leishmania infantum in blood donors from the Balearic Islands (Spain). Transfusion 2008; 48: 1383–1389. 10. Pineda JA, Gallardo JA, Mac ıas J, et al. Prevalence of and factors associated with visceral leishmaniasis in human immunodeficiency virus type1-infected patients in southern Spain. J Clin Microbiol 1998; 36 (9): 2419–2422. 11. Mart ın-S anchez J, Pineda JA, Morillas-M arquez F, Garc ıa-Garc ıa JA, Acedo C, Mac ıas J. Detection of Leishmania infantum kinetoplast DNA in peripheral blood from asymptomatic individuals at risk for parenterally transmitted infections: relationship between polymerase chain reaction results and other Leishmania infection markers. Am J Trop Med Hyg 2004; 70 (5): 545–548. 12. Garc ıa-Garc ıa JA, Mart ın-S anchez J, G allego M, et al. Use of noninvasive markers to detect Leishmania infection in 798 Transplant Infectious Disease 2015: 17: 795–799 Elmahallawy et al: Leishmania in renal transplant 13993062, 2015, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tid.12444 by Universidad De Granada, Wiley Online Library on [19/03/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License asymptomatic human immunodeficiency virus-infected patients. J Clin Microbiol 2006; 44 (12): 4455–4458. 13. Ma DD, Concannon AJ, Hayes J. Fatal leishmaniasis in renaltransport patient. Lancet 1979; 2: 311–312. 14. Antinori S, Schifanella L, Corbellino M. Leishmaniasis: new insights from an old and neglected disease. Eur J Clin Microbiol Infect Dis 2012; 31: 109–118. 15. Simon I, Wissing KM, Del Marmol V, et al. Recurrent leishmaniasis in kidney transplant recipients: report of 2 cases and systematic review of the literature. Transpl Infect Dis 2011; 13: 397–406. 16. Vinhal FA, Afonso-Cardoso SR, Silva AG, et al. A typical presentation of cutaneous leishmaniasis after renal transplantation. Nephrol Dial Transplant 2007; 22: 3674. 17. Clemente WT, Couto CA, Ribeiro DD, de Medeiros Chaves Franca M, Sanches MD. An atypical course of visceral leishmaniasis (Kala-azar) in a liver transplant recipient. Transplantation 2007; 83: 368–369. 18. le Fichoux Y, Quaranta JF, Aufeuvre JP, et al. Occurrence of Leishmania infantum parasitemia in asymptomatic blood donors living in an area of endemicity in southern France. J Clin Microbiol 1999; 37: 1953–1957. 19. Clemente WT, Rabello A, Faria LC, et al. High prevalence of asymptomatic Leishmania spp among liver transplant recipients and donors from an endemic area of Brazil. Am J Transplant 2014; 14: 96–101. 20. Bouchekoua M, Trabelsi S, Ben Abdallah T, Khaled S. Visceral leishmaniasis after kidney transplantation: report of a new case and a review of the literature. Transplant Rev 2014; 28: 32–35. 21. Michel G, Pomares C, Ferrua B, Marty P. Importance of worldwide asymptomatic carriers of Leishmania infantum (L. chagasi) in human. Acta Trop 2011; 119 (2–3): 69–75. 22. Srivastava P, Gidwani K, Picado A, et al. Molecular and serological markers of Leishmania donovani infection in healthy individuals from endemic areas of Bihar, India. Trop Med Int Health 2013; 18: 548–554. 23. Garrote JI, Gutierrez P, Lopez Izquierdo R, et al. Seroepidemiologic study of Leishmania infantum infection in Castilla-Le on, Spain. Am J Trop Med Hyg 2004; 71: 403–406. 24. Alborzi A, Pourabbas B, Shahian F, Mardaneh J, Pouladfar GR, Ziyaeyan M. Detection of Leishmania infantum kinetoplast DNA in the whole blood of asymptomatic individuals by PCRELISA and comparison with other infection markers in endemic areas, southern Iran. Am J Trop Med Hyg 2008; 79 (6): 839–842. 25. de Gouv^ ea VL, de Assis TS, Orsini M, et al. Combined diagnostic methods identify a remarkable proportion of asymptomatic Leishmania (Leishmania) chagasi carriers who present modulated cytokine profiles. Trans R Soc Trop Med Hyg 2008; 102 (6): 548–555. 26. Mary C, Faraut F, Drogoul MP, et al. Reference values for Leishmania infantum parasitemia in different clinical presentations: quantitative polymerase chain reaction for therapeutic monitoring and patients follow-up. Am J Trop Med Hyg 2006; 75 (5): 858–863. 27. Gidwani K, Kumar R, Rai M, Sundar S. Longitudinal seroepidemiologic study of visceral leishmaniasis in hyperendemic regions of Bihar, India. Am J Trop Med Hyg 2009; 80: 345–346. 28. Singh OP, Hasker E, Sacks D, Boelaert M, Sundar S. Asymptomatic Leishmania infection: a new challenge for Leishmania control. Clin Infect Dis 2014; 58 (10): 1424–1429. 29. Hasker E, Malaviya P, Gidwani K, et al. Strong association between serological status and probability of progression to clinical visceral leishmaniasis in prospective cohort studies in India and Nepal. PLoS Negl Trop Dis 2014; 8 (1): e2657. 30. Schwartz BS, Mawhorter SD; AST infectious Diseases Community of Practice. Parasitic infections in solid organ transplantation. Am J Transplant 2013; 13 (Suppl 4): 280–303. Transplant Infectious Disease 2015: 17: 795–799 799 Elmahallawy et al: Leishmania in renal transplant 13993062, 2015, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tid.12444 by Universidad De Granada, Wiley Online Library on [19/03/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License