Epidemiology, Clinical Characteristics, Diagnostic Work Up, and Treatment Options of Leishmania Infection in Kidney Transplant Recipients: A Systematic Review

Current knowledge on Leishmania infection after kidney transplantation (KT) is limited. In order to offer a comprehensive guide for the management of post-transplant Leishmaniasis, we performed a systematic review following the latest PRISMA Checklist and using PubMed, Scopus, and Embase as databases. No time restrictions were applied, including all English-edited articles on Leishmaniasis in KT recipients. Selected items were assessed for methodological quality using a modified Newcastle–Ottawa Scale. Given the nature and quality of the studies (case reports and retrospective uncontrolled case series), data could not be meta-analyzed. A descriptive summary was therefore provided. Eventually, we selected 70 studies, describing a total of 159 cases of Leishmaniasis. Most of the patients were adult, male, and Caucasian. Furthermore, they were frequently living or travelling to endemic regions. The onset of the disease was variable, but more often in the late transplant course. The clinical features were basically similar to those reported in the general population. However, a generalized delay in diagnosis and treatment could be detected. Bone marrow aspiration was the preferred diagnostic modality. The main treatment options included pentavalent antimonial and liposomal amphotericin B, both showing mixed results. Overall, the outcomes appeared as concerning, with several patients dying or losing their transplant.


Introduction
Leishmaniasis represents one of the most neglected infectious diseases worldwide, largely affecting individuals with disadvantaged social backgrounds residing in less economically developed countries, often afflicted by malnutrition, poor residency conditions, and generalized lack of health care resources [1,2]. The term Leishmaniasis encompasses a group of parasite-associated diseases with cutaneous, mucocutaneous, or visceral manifestations, primarily caused by obligate intracellular protozoa of the genus Leishmania [3,4]. Although most cases remain outside formal registries, Leishmaniasis is currently endemic in more than 80 countries, particularly in the tropics, subtropics, and southern regions of Europe, with 350 million people at risk and 1.6 million new infections every year [2][3][4][5][6]. Given the ongoing global climate and environmental changes, it is likely that the geographic range of the vectors of Leishmania and the areas in the world where Leishmania can be found will further expand. Leishmaniasis is usually acquired through the bite of a (especially in non-endemic regions), the increased susceptibility to viral infections with overlapping manifestations, and the frequent occurrence of drug-related myelotoxicity, immunosuppressed subjects often experience a delay in diagnosis and treatment. Bone marrow aspiration (BMA), usually carried out to investigate possible causes of pancytopenia, represents the cornerstone of the diagnostic work up. A polymerized chain reaction (PCR) can also be used for diagnosis as much as for the definition of Leishmania species. Serological tests for antigens or antibodies detection are generally performed to screen individuals at risk or to confirm donor-derived infections [13]. For many years, pentavalent antimonial has been widely adopted as a first-line treatment of VL. Given the high incidence of adverse events (up to 34% in some series), it is being replaced by liposomal amphotericin B, which seems to be more effective and better tolerated [7].
To date, no meta-analyses or systematic reviews on Leishmania infection after KT have been published. Therefore, we aimed to comprehensively review available literature on epidemiology, clinical characteristics, diagnostic work up, and treatment options of Leishmaniasis in KT recipients.

Materials and Methods
We conducted a systematic review according to the latest Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) Checklist. PubMed, Embase, and Scopus were searched in March 2022 for any papers (including congress abstracts) reporting on patients with Leishmaniasis after KT. No time limits were applied. The following keyword combinations were used: "Leishmania AND kidney transplant", "Leishmania AND renal transplant", "Leishmaniasis AND kidney transplant", or "Leishmaniasis AND renal transplant". Only manuscripts edited in English were considered.
Two different groups of authors performed the primary (EF and GS) and secondary (FB and CA) searches. Disagreements between the two groups were resolved by discussion with a third author (AG) and the senior author (MF). Duplicates and non-English articles were removed. The remainder were screened out by reading the titles and abstracts. All items potentially describing cases of patients developing Leishmaniasis after KT were assessed in full text whilst items reporting on patients with pre-transplant diagnosis of Leishmania infection were excluded. Only original contributions reporting on Leishmaniasis in KT recipients were considered. An additional search of reference lists was performed by SD and RC. If available, the following data were collected and transferred to a dedicated database: recipient country of origin and travel activity to endemic regions, patient ethnicity, sex, and age, donor type, immunosuppression, time from transplant to Leishmaniasis onset, time from symptoms onset to final diagnosis, Leishmania species, symptoms, diagnostic work up, treatment, outcomes, Leishmaniasis-specific survival, graft survival, and irreversible graft disfunction. Extracted data were transferred to a dedicated anonymized database for analysis purposes.
Selected studies were assessed for methodological quality using a tool based on a modification of the Newcastle-Ottawa Scale as proposed by Murad et al. [14]. As suggested by the authors, questions 5 and 6 of the original questionnaire were not considered, since they were mostly relevant to cases of drug-related adverse events. Rather than using an aggregate score, we made an overall judgement considering the questions deemed most critical in the specific clinical scenario. Accordingly, the quality of the studies was classified as low, average, or high, depending on their scoring in the questionnaire: respectively, 0-2, 3-4, or 5-6 points out of a total of 6 points.
Our systematic review considered a large majority of single case reports and some small retrospective case series. No meta-analysis could be performed as the small case series are composed of heterogeneous patients, making any summary measures meaningless. To compactly describe the literature, we reported the number for the categorical variables and the range for the continuous ones. The tables must also be considered as a compact way of describing the results from the literature. No inferences can be drawn from this study. Furthermore, as a potential limitation of the present work, we recognize the possibility that some cases of Leishmaniasis in a KT setting may have been omitted since they could have been included in papers or congress abstracts referring to solid organ transplant recipients in general. The statistical methods were assessed by an expert in biomedical statists (CA). The review was not registered.

Included Studies
A flow diagram summarizing included articles and selection processes is depicted in Figure 1. The number of reports preliminarily retrieved using each of the keyword combinations previously mentioned was 954. In more detail: Leishmania AND renal transplant, 149 (58 from PubMed, 47 from Scopus, and 44 from Embase); Leishmania AND kidney transplant, 161 (53 from PubMed, 37 from Scopus, and 71 from Embase); Leishmaniasis AND renal transplantation, 318 (102 from PubMed, 102 from Scopus, and 114 from Embase); Leishmaniasis AND kidney transplantation, 326 (97 from PubMed, 151 from Scopus, and 78 from Embase). After duplicate (n = 755) and non-English articles (n = 8) were removed, a pool of 191 items remained for further evaluation. Following the inclusion criteria previously described and after reviewing papers by title and abstract, 83 articles were identified. Studies not reporting original cases of Leishmaniasis after KT were excluded (n = 13). No additional reports were found through searches of references. Eventually, 70 papers were selected. No randomized clinical trials, prospective controlled studies, or prospective uncontrolled studies were identified. At the end of the process, we included 66 retrospective case reports and 4 retrospective case series. According to the modified Newcastle-Ottawa Scale, 11 items were classified as low-quality [7,[15][16][17][18][19][20][21][22][23][24], 34 as average-quality , and 25 as high-quality studies [4,10,. In total, our analysis includes 159 cases of Leishmania infection after KT. The main characteristics and qualitative evaluations of the studies meeting the criteria for the systematic review are described in Table 1. Abbreviations: A, average-quality; CL, cutaneous Leishmaniasis; H, high-quality; L, low-quality; MCL, mucocutaneous Leishmaniasis; M-U-R-CS, multi-center uncontrolled retrospective case series; R-CR, retrospective case-report; S-C-R-CS, single-center controlled retrospective case series; S-U-R-CS, single-center uncontrolled retrospective case-series; VL, visceral Leishmaniasis.

Epidemiology
No articles reported on the total number of KT performed over the same period in which Leishmania infections were diagnosed and treated. Consequently, no estimate of cumulative incidence or prevalence could be calculated.
Sex and age at diagnosis were recorded for 157/159 (98.7%) and 156/159 (98.1%) subjects, respectively. Eventually, we were able to identify 34 female and 123 male KT recipients with Leishmania infection.
Overall, the time between transplant and Leishmaniasis onset ranged from a few days to 246 months.
Information on secondary prophylaxis was omitted in virtually all the studies included. On the contrary, data regarding relapse episodes after first-line and second-line treatments were reported for 159 and 50 KT recipients, respectively. First-line and secondline treatments with treatment-specific outcomes (including relapses) are summarized in Tables 5 and 6. Table 5. Summary of first-line treatment options and treatment-related outcomes of the case reports and case series of Leishmania infection after kidney transplantation (summaries based on individual cases should not be considered as an estimate of the "real world").
Abbreviations: NA, not available; SAE, serious adverse event. Table 6. Summary of second-line treatment options and treatment-related outcomes of the case reports and case series of Leishmania infection after kidney transplantation (summaries based on individual cases should not be considered as an estimate of the "real world").

Discussion
It is well-known that solid organ transplant recipients are more prone to opportunistic infections than the general population [13]. Over the last two decades, we have witnessed a considerable rise in the cases of Leishmaniasis among KT patients. Such a concerning phenomenon is mostly due to a wider diffusion of the parasite, as well as the exponential increase in the number of patients at risk of the disease. Undoubtedly, the massive migrations from rural to urban areas, the savage requalification processes of rural and suburban zones, and the opportunity to easily travel from and to endemic regions have greatly contributed to expanding the geographical distribution of the sandfly vectors of Leishmania. Nevertheless, the increasing prevalence of acquired immunodeficiency conditions observed in both less and more economically developed countries as a result of expanding HIV contagion and widespread transplant activity have played a significant role [2,3,5,6].
Unfortunately, the studies included in our systematic review failed to provide information regarding the incidence and prevalence of Leishmania infection among the populations enrolled, thus limiting the epidemiological value of the present analysis. However, considering the progressive increase in the number of transplants performed worldwide, current achievements in long-term recipient and allograft survival, the wider use of powerful immunosuppressive agents, and the regained awareness of the transplant community, it is reasonable to expect that the incidence of Leishmaniasis will rise considerably. In this regard, it is paramount to promptly develop national and international registries for the implementation of infection control strategies and formal outcomes assessment.
Details on donor ethnicity were completely omitted in all the studies included in the review. On the contrary, recipient heritage was described in about half of the cases. Apparently, most of the patients developing post-transplant Leishmaniasis were Caucasian [4,7,10,19,22,25,26,[28][29][30]33,35,[37][38][39]42,43,45,52,58,61,66,68,70,73,75,80]. However, there is scarce evidence of any actual association between ethnicity and Leishmania infection susceptibility among transplanted and non-transplanted subjects. Carrasco-Antón et al. [10] reported an association between sub-Saharan African ethnicity and VL in the general population, possibly explained by genetic predisposition, but the role of genetic factors in posttransplant VL remains to be determined. More likely, the perceived disproportion in the prevalence of Leishmania infection among different ethnic groups reflects the fact that most reports were produced by authors residing in the Mediterranean basin or possible disparities among different minorities in their access to the KT waiting list [82].
Available data seem to suggest that Leishmania infection is more frequent among middle aged male KT recipients [22]. However, the reduced incidence or prevalence of the disease observed among pediatric, elderly, or female recipients may be due to differences in the numbers of KT performed in these subgroups of patients rather than actual differences in infection susceptibility [83]. Accordingly, no sex-related differences in susceptibility have been confirmed in the general population.
We found that the time between transplantation and the onset of Leishmania infectionrelated symptoms was extremely variable. Nonetheless, most patients developed the disease as a late post-transplant complication [3,62]. This observation highlights the need for a high index of suspicion during all the phases of the post-transplant follow-up, particularly in the long term, as prolonged exposure to immunosuppression may progressively increase the risk of infection. In addition, it confirms that the donor-recipient route (via the allograft) has a marginal impact on the transmission of the parasite in solid organ transplant setting.
In the studies included in our research, VL was the predominant form of the disease reported after KT, with only a few cases of MCL or CL [21,[32][33][34]37,45,50,58,62,68]. This finding confirms that KT patients are at an increased risk of severe infectious complications compared to the general population and suggests that the combination of end-stage renal disease and drug-induced immunosuppression can significantly impair Leishmania-specific immune response .
In the general population, CL and MCL represent the prevalent forms of the disease. However, in the transplanted population, a striking predominance of VL has been reported, also in individuals infected by less aggressive Leishmania species. Left untreated, VL is associated with a lethality rate as high as 95%. As such, systemic treatment is recommended in immunocompromised hosts. Main therapeutic agents for Leishmaniasis after KT described in the literature were pentavalent antimonial and amphotericin B [82]. Pentavalent antimonial (namely, sodium stibogluconate and meglumine antimoniate) explicate their anti-Leishmania action inhibiting DNA topoisomerase function, glycolytic activity, and fatty acid beta-oxidation, thus inducing metabolic imbalance and structural modifications of the parasite's membrane. The main limitations of pentavalent antimonial use are the frequent occurrence of drug-related side effects (cardiotoxicity, bone marrow suppression, nephrotoxicity, acute pancreatitis, and abnormal liver function tests), intravenous route of administration, and increasing global resistance [89]. Amphotericin B is a polyene antifungal compound with a broad range of activity against yeasts, molds, and protozoa, including Leishmania. Amphotericin B binds to the ergosterol of the fungal cell membrane, leading to ion leakage and cell death [90]. Exhibiting a better safety profile, liposomal amphotericin B has progressively replaced amphotericin B deoxycholate, and it is currently used as a first-line treatment of VL in patients with impaired renal function [3]. As demonstrated in the general population, our analysis suggests that liposomal amphotericin B is more effective than pentavalent antimonial in KT recipients [3]. Another emerging option for the treatment of VL is the alkyl-lysophospholipid miltefosine [41,91]. There is still limited knowledge of the mechanism of action of miltefosine, but experimental models support the hypothesis that it may trigger and enhance programmed cell death (apoptosis) in both metazoans and protozoans. Some authors also believe that miltefosine may act by inhibiting phosphatidyl choline synthesis during the processes involved in the formation of the cell membrane [92]. The efficacy and safety of miltefosine for the treatment of VL have been primarily evaluated by Sundar and Ollario in non-transplanted patients. They showed that miltefosine administration was overall well tolerated, with the occurrence of mild gastrointestinal side effects and few episodes of vomiting, diarrhea, or acute nephrotoxicity [93]. Current experience in a KT setting remains anecdotal and not convincing [41]. Awaiting additional data, it seems reasonable to restrict the use of miltefosine to KT patients not suitable for pentavalent antimonial or amphotericin B. On the contrary, available evidence suggests wide administration of amphotericin B as a first-line treatment of post-transplant VL. Proper identification of Leishmania species could improve treatment-related outcomes, particularly for those patients with limited response to first-line therapy.
Despite recent advancements in diagnostics and therapeutics, the outcomes of KT recipients with Leishmaniasis remain concerning, particularly in the case of systemic disease. As a matter of fact, our analysis shows that about 25% of the patients developing Leishmania infection during the post-transplant course eventually died, regardless of the treatment received [29,43,44,58,81]. The occurrence of allograft loss [8,30,41,64,72,74,78] or irreversible allograft dysfunction [8,22,30,41,55,64,65,72,73,78,81] was also concerning, and further emphasizes the need for optimized diagnosis and treatment.

Conclusions
To the best of our knowledge, this is the first systematic review on Leishmania infection after KT. Due to the lack of properly designed studies and large populations databases, performing a meaningful meta-analysis was not possible. Nevertheless, we herein reported a comprehensive and updated reference that could serve as a basis for further research projects, hopefully guiding the clinicians involved in the care of this complex group of patients in the case of suspected or overt Leishmaniasis. The institution of formal national and international registries is vital for the optimization of both management and outcomes.