Updated diagnosis and graft involvement for visceral leishmaniasis in kidney transplant recipients: a case report and literature review

Purpose Visceral leishmaniasis (VL) has become a rising concern to transplantation teams, being associated with graft dysfunction and reduced survival of renal transplant recipients. Here, we describe a case of VL occurring in a kidney transplant (KT) recipient in Italy, a country in which Leishmania infantum is endemic and we reviewed the literature on the clinical course and diagnosis of VL in KT recipients residing or travelling to southern Europe. Results The VL case was diagnosed 18 months after transplant and 28 days after the onset of symptoms by quantitative PCR (qPCR) on peripheral blood. A graft biopsy showed renal involvement, and PCR performed on graft tissue displayed the presence of Leishmania DNA. The retrospective confirmation of Leishmania-positive serology in a serum sample collected before transplantation, as well as the absence of anti-Leishmania IgG in the graft donor strongly suggest that reactivation of a latent parasitic infection caused VL in the current case. Conclusion VL is often underdiagnosed in transplant recipients, despite the presence of latent Leishmania infection being reported in endemic countries. This case report, as well as the literature review on leishmaniasis in KT recipients, underline the importance of rapid VL diagnosis to promptly undergo treatment. Serology is scarcely sensitive in immunocompromised patients, thus molecular tests in peripheral blood should be implemented and standardized for both VL identification and follow-up.


Introduction
Visceral leishmaniasis (VL) is an infectious disease caused by protozoa of the Leishmania donovani complex, and associated with considerable morbidity and mortality [1]. VL is also a known complication of solid organ transplantation (SOT) [2]; the prevalence of VL among SOT recipients in endemic areas is up to 0.9% [3,4]. In these patients, VL can occur either ex novo or through reactivation of a pre-existing infection induced by the immunosuppressive drugs. In kidney transplant (KT) recipients, leishmaniasis often includes acute interstitial nephritis with moderate inflammation and infiltration of lymphocytes, plasma cells and macrophages, and can lead to graft dysfunction [5]. VL occurring in SOT is a severe event, being associated with frequent relapses and a mortality rate that exceeds 20% [6].
Here, we report a VL case in a KT recipient residing in central Italy, where L. infantum circulates. We diagnosed and monitored VL by quantitative PCR (qPCR) and Marco Busutti and Alessandro Deni contributed equally to this work. retrospectively screened pre-transplant samples of the index patient and the graft donor by serology. We also briefly reviewed the literature on the clinical course and diagnosis of VL in KT recipients residing or travelling to southern Europe. A 36-year-old Caucasian male underwent pre-emptive kidney transplantation at the Nephrology, Dialysis and Renal Transplant Unit, University Hospital of Bologna, Italy, for end-stage renal disease (ESRD) secondary to autosomal dominant polycystic kidney disease (ADPKD). The graft was available from a living donor (a first-degree relative). Induction therapy with high dose steroids and anti-IL2r monoclonal antibodies (basiliximab) was first administered after transplant, followed by maintenance therapy with prednisone, tacrolimus and mycophenolic acid. No major complications were reported, and graft function was stabilized with serum creatinine (sCreat) levels at 2 mg/dl (eGFR CKD-EPI 42 ml/min).

Case presentation
Approximately 18 months after transplant, the patient was admitted following a routine follow-up appointment during which he complained of night sweat and persistent mild fever in the previous 3 weeks. Symptoms had been empirically treated with oral antibiotics by the patient's general practitioner, with no benefit. Laboratory examination showed pancytopenia (WBC 2370/mmc, Hb 10.5 g/dl, PLT 76.000/ mmc), renal impairment (sCreat 3.7 mg/dl, eGFR CKD-EPI 19 ml/min), elevated C-reactive protein (CRP 12 mg/ dl), and elevated ferritin (902 ng/ml). 18F-FDG PET/C was performed, showing splenomegaly and splenic hypercaptation. Since a discrepancy between the donor's and the recipient's serostatuses for human cytomegalovirus (CMV) was known (D + /R −), a primary CMV infection was initially suspected, and mycophenolic acid was suspended at admission. As CMV DNAemia tested negative, the suspicion of CMV disease was dismissed.
Two real-time (rt)PCR assays targeting the small-subunit ribosomal (r)RNA gene and the leishmanial kinetoplast (k)DNA, respectively, were simultaneously performed on peripheral whole blood as described by Varani et al. [7]. Quantification of parasitic kinetoplast (k)DNA was also performed. At diagnosis, the parasite load was 9360 parasite equivalents/ml (Fig. 1a-c). VL serology was carried out by rK39-based immunochromatographic test (ICT; Rapydtest, Diagnostic International Distribution S.p.A, Milan, Italy), by enzyme-linked immunosorbent assay (ELISA, Vircell, Granada, Spain) and by immunofluorescence assay (IFA, BioMérieux, Marcy-l'Étoile, France). The sample tested positive on ELISA and negative on the ICT, while the IFA returned a title of 1:80, corresponding to an indeterminate result.
As the renal function worsened, a graft biopsy was performed. Histological examination of the biopsy showed diffuse interstitial fibrosis/tubular atrophy with moderate chronic interstitial inflammation and glomerulosclerosis in almost 50% of glomeruli associated to chronic vascular damage (Fig. 1d). CD1a staining was negative and parasite amastigotes were not detected at histology nor at electron microscopy, while parasitic kDNA was identified by rtPCR in renal tissue (data not shown). Bone marrow biopsy was also carried out, showing myelodysplasia, which was consistent with VL. Unfortunately, this sample was not sent to the Microbiology Unit and kDNA rtPCR was not performed.
Anti-leishmanial therapy with liposomal amphotericin B (L-Amb) was carried out (eight infusions; 5 mg/kg/dose) with good response. During treatment, both thrombocytopenia and leukopenia rapidly improved, and a decrease of sCreat levels was also observed after a brief initial increase, likely due to L-AmB-related nephrotoxicity. rtPCR for Leishmania tested negative on peripheral blood within 3 months after the first L-AmB infusion ( Fig. 1a-c). Graft function partially recovered, with sCreat stabilizing at 2.1-2.5 mg/dl at 15 months after VL diagnosis and further decreasing to 1.8-2.0 mg/dl at 42 months; at this time the patient was also free from VL relapses, with no Leishmania DNA detected in peripheral blood samples. Administration of mycophenolic acid was not reinstated after treatment.
The serostatus for Leishmania in the index patient and the graft donor was also retrospectively analysed by testing sera that were collected immediately before the transplant with the Leishmania Western Blot IgG (LDBio Diagnostics ® , Lyon, France), a sensitive method to detect specific IgG in individuals with VL or asymptomatic Leishmania infection [9,10]. The test revealed the presence of anti-leishmanial IgG in the patients' serum immediately before the transplant, while the donor's serum tested negative.

Methods
We conducted a review of case reports and case series published in the MEDLINE (PubMed) database between 1990 and 2021 using the following search terms: "Leishmania", "transplant" and their derivatives, and excluding results relative to non-European countries, cutaneous leishmaniasis and haematological transplants. Furthermore, the bibliographies of extant case series were searched for relevant articles. The results included articles in English, French and Spanish.

Epidemiology and transmission
VL is an underrecognized and misdiagnosed disease among transplant recipients [6]. According to our review of the extant literature (Table 1), 61 VL cases (including the current case) have been reported in KT recipients in southern Europe, or in patients who travelled there, since 1990. Most cases (n = 23) were reported in Spain, which is among the foremost countries in performing organ transplantation and in which L. infantum is endemic [11], followed by France (n = 15), Italy (n = 12, including the case of this study) and Greece (n = 5); Turkey reported two cases, and Portugal and Malta reported one VL case each. Finally, two cases were described in patients residing in non-endemic countries, but reporting travelling to Spain. Thus, VL is a threat for SOT recipients in areas where leishmaniasis is endemic.
In the examined case, reactivation of a latent infection was strongly suggested by the retrospective serological analysis of the patient's and the donor's sera, which were collected before transplantation; this may be of importance since we recently observed the presence of a latent Leishmania infection in 16% of ESRD patients in haemodialysis treatment [44]. These findings call for further evaluation of the clinical utility of pre-transplant screening strategies, which are currently not recommended for this parasitic infection [4].

VL onset and diagnosis
Development of VL is considered a late event post-transplantation, with an average VL onset at 18 months after transplants [6]. Accordingly, in southern Europe, 45 (75%) KT recipients who developed VL did so at least 6 months after the transplant (Table 1): this includes the examined case, who progressed into VL at around 18 months after receiving the graft. Timely diagnosis of VL is critical in SOT recipients, but leishmaniasis is often overlooked in differential diagnosis, leading to treatment delay. Antinori et al. [6] reported a mean time to diagnosis of 30 days from the onset of symptoms, with peaks of up to 5 months. Time to diagnosis is seldom reported in studies of VL in KT, with only 13 (21%) case reports from southern Europe stating it (data not shown). In line with previous studies [6,45], the current VL case was diagnosed 8 days after hospital admission, corresponding to 28 days after the symptoms' onset.
In immunocompromised patients, diagnostic tools for VL exhibit variable performances, with serological tests showing lower sensitivity than in immunocompetent individuals and non-invasive direct diagnosis, such as molecular testing on peripheral blood, being of great value [46][47][48][49]. Nevertheless, microscopy on bone marrow samples has been the most used diagnostic method (53 cases, 87%), with PCR in peripheral or bone marrow blood carried out in 12 (20%) VL cases in KT recipients described in southern Europe, including the current case (Table 1).

Renal involvement in VL
Renal involvement is frequent in human leishmaniasis and it is associated with increased mortality. Clinical features are diverse, mainly represented by urinary abnormalities (proteinuria, haematuria, and pyuria) and acute kidney injury (AKI), which is reported in 4-46% of VL cases. Tubular and glomerular dysfunction have also been reported, although less frequently [5,[50][51][52][53].
Renal injury can be the consequence of direct kidney involvement, but can also be caused or exacerbated by concomitant events.
Direct Leishmania-induced renal damage mainly results from immunological phenomena, such as the deposition of immune complexes, activation of T cells, up-regulation of adhesion molecules, inflammatory processes, but can also be caused by parasite proliferation in the kidney tissue [54,55] Beside VL, the other causes that can contribute to the development of renal damage in VL patients are drug toxicity, presence of associated infections and haemodynamic abnormalities [51,53,56]. Histological examination of graft biopsies in KT recipients with VL is seldom described ( Table 1); findings range from the absence of parasites [39] to diffuse interstitial inflammation and abundant parasites [35] to complete occlusion of renal vasculature [34]. The examined case shows interstitial fibrosis, tubular atrophy and glomerulosclerosis (seven out of 13 glomeruli) at histology; these chronic lesions may be related to the prolonged damage caused by the parasitic infection, considering the long-time interval between VL onset and histological evaluation. The presence of leishmanial DNA in the kidney tissue sustains this hypothesis, even though no amastigotes were detected in the lesions.

VL treatment and monitoring
Treatment of VL in SOT recipients is mostly based on case reports or small case series. A high dose L-Amb (total dose of 40 mg/kg) is considered the therapy of choice in immunocompromised patients with VL; doses of immunosuppressive drugs should be decreased during VL treatment whenever possible [1]. In the examined case, immunosuppressants were decreased and the response to specific anti-leishmanial therapy was rapid.
qPCR on peripheral blood is considered the most useful technique for monitoring the efficacy of VL treatment and for identifying relapses in immunocompromised patients [1,46]. In the current case, the employment of qPCR for the patient's follow-up allowed the observation of an effective response to anti-parasitic therapy with a rapid drop of parasitaemia, with no Leishmania DNA detected in peripheral blood samples for the entire monitoring period (48 months) (Fig. 1a-c and data not shown). Importantly, leishmanial DNA can be detected in immunocompromised patients after treatment without clinical significance, thus caution should be taken to identify VL relapse merely on a positive PCR test [46]. qPCR should be used, with raised levels of parasitic DNA predicting relapses. It is also important to underline that qPCR results should be combined with clinical and laboratory signs to evaluate whether the patient undergo a clinically evident relapse and requires VL treatment.

Conclusions
VL must be suspected among the opportunistic infections that can develop in SOT patients who reside in (or travelled to) Leishmania-endemic countries, including southern Europe. As VL exhibits high mortality in KT patients, prompt VL diagnosis and early anti-parasitic treatment are essential. PCR in peripheral blood appears to be effective for VL identification and follow-up; nevertheless, standardization and validation of a consensus protocol for molecular diagnosis and parasite load estimation is still lacking as well a consensus for screening strategies for SOT candidates in endemic regions.