Cytokine Release Assays as Tests for Exposure to Leishmania, and for Confirming Cure from Leishmaniasis, in Solid Organ Transplant Recipients

Spain has one of the world’s largest pools of organ donors and is a global leader in terms of the number of transplants it performs. The current outbreak of leishmaniasis in Fuenlabrada (in the southwest of the region of Madrid, Spain) has involved 600 clinical cases since late 2009 (prevalence 0.2%). It may therefore be wise to monitor the town’s transplanted population for Leishmania infantum; its members are immunosuppressed and at greater risk of infection and relapse following treatment. The present work examines the use of cytokine release assays to determine the prevalence of Leishmania infection in this population, and to confirm recovery following treatment for visceral leishmaniasis (VL). The humoral and cellular immune responses to L. infantum were characterized in 63 solid organ transplant (SOT) recipients from Fuenlabrada, 57 of whom reported no previous episode of VL (NVL subjects), and six of whom had been cured of VL (CVL subjects). Seventeen subjects (12 NVL and 5 CVL) showed a patent lymphoproliferative response to soluble Leishmania antigen (SLA). Stimulation of peripheral blood mononuclear cell cultures and of whole blood with SLA led to the production of different combinations of cytokines that might serve to confirm Leishmania infection or recovery from VL and help prevent cured patients from relapsing into this serious condition.

tion for Leishmania infantum; its members are immunosuppressed and at greater risk of infection and relapse following treatment. The present work examines the use of cytokine release assays to determine the prevalence of Leishmania infection in this population, and to confirm recovery following treatment for visceral leishmaniasis (VL). The humoral and cellular immune responses to L. infantum were characterized in 63 solid organ transplant (SOT) recipients from Fuenlabrada, 57 of whom reported no previous episode of VL (NVL subjects), and six of whom had been cured of VL (CVL subjects). Seventeen subjects (12 NVL and 5 CVL) showed a patent lymphoproliferative response to soluble Leishmania antigen (SLA). Stimulation of peripheral blood mononuclear cell cultures and of whole blood with SLA led to the production of different combinations of cytokines that might serve to confirm Leishmania infection or recovery from VL and help prevent cured patients from relapsing into this serious condition.

Author Summary
We have used cytokine release assays to determine the prevalence of Leishmania infantum infection in solid organ transplant (SOT) recipients living in an area where the organism is

Introduction
In Spain, leishmaniasis is an endemic zoonosis caused by Leishmania infantum. The national mean annual incidence is 0.45 cases/100,000 inhabitants, similar to that traditionally reported for the capital, Madrid (0.5 cases/100,000 inhabitants) [1]. A few years ago, an unusual, large outbreak of leishmaniasis was noted in the southwest of the Region of Madrid, and disease incidence in the town of Fuenlabrada is currently 21.54/100,000. Since 2009, more than 100 cases of visceral leishmaniasis (VL) have been recorded every year in Fuenlabrada; similar figures are expected for the coming years [2].
Immunosuppression is a major risk factor in the appearance of overt clinical leishmaniasis; it can also alter the presentation of the disease and response to treatment [3]. Indeed, the worldwide number of cases of VL in recipients of solid organ transplants (SOT) has quadrupled since the 1990s [4] (77% of those affected received a kidney transplant, the most commonly performed transplant operation). Most of these cases occurred in the Mediterranean basin, particularly in Spain, which has one of the world's largest pool of organ donors and is among the foremost in performing SOTs.
Very little is known, however, about the relationship between leishmaniasis and transplants, and the prevalence of asymptomatic leishmaniasis is unknown [5]. During the recent outbreak in Fuenlabrada, eight out of the total 130 SOT recipients residing in the area developed VL (6.15%); the risk for these patients is therefore around 30 times that faced by immunocompetent individuals.
Recently, molecular and serological analyses of liver transplant recipients in an area of Brazil where leishmaniasis is endemic revealed a high prevalence of asymptomatic infection [6]. However, measurement of the cell-mediated immune response is critical when estimating exposure to pathogens causing intracellular infections. Immunity to leishmaniasis is associated with a strong Th1-type immune response, as demonstrated by a positive delayed type hypersensitivity (DTH) reaction in the Leishmanin skin test (LST), along with lymphocyte proliferation and the production of high levels of IFN-γ and TNF-α [7] following stimulation with soluble leishmania antigen (SLA).
T-cell-based interferon-γ (IFN-γ) release assays have been developed as alternatives to DTH tests [8]. For example, the whole-blood gamma interferon release assay (IGRA) has recently been reported capable of detecting subclinical infection in healthy individuals living in an area where VL is endemic [9]. Although some studies have been published on the use of IGRA in tuberculosis and cytomegalovirus infection [10,11], the literature contains nothing on its use to detect Leishmania infection in SOT recipients. The aim of the present work was to test cytokine release assays as a means of determining the prevalence of Leishmania infection in SOT recipients, and to confirm recovery following treatment for VL. Assessing the exposure to Leishmania and the immunological memory of SOT recipients living in an area highly endemic for leishmaniasis should throw light on the infection rate in this population, help prevent those treated for VL from relapsing, and reveal the epidemiological features of this disease in the immunosuppressed within the context of an outbreak.

Population
Sixty three SOT (kidney, liver and heart) recipients were enrolled in the present study. All were aged 18 years or older, had undergone transplant surgery between 2005 and 2013 at the 12 de Octubre University Hospital, and resided in the town of Fuenlabrada. Fifty seven subjects had experienced no previous episode of VL or compatible symptomology (NVL subjects), and six had been cured of visceral leishmaniasis (CVL subjects).

Ethics statement
Recruitment and sample collection were performed in accordance with Good Clinical Practice guidelines. The study was approved by the ethics Committee of the 12 de Octubre University Hospital. All subjects gave their written informed consent to be included in the study.

Immunosuppressive treatment of SOT subjects
Recipients of a graft from a non-heart beating donor (30% of all SOT recipients) underwent induction therapy with intravenous (IV) rabbit anti-thymocyte globulin (ATG-Fresenius) (1.25 mg/kg daily for 5-7 days) and a calcineurin inhibitor (CNI) from day 6. Patients at high immunological risk received induction therapy with ATG for 1-3 days plus CNI from day 0. Basiliximab (20 mg on days 0 and 4) was provided to patients at high risk of CNI-related nephrotoxicity owing to advanced age or pre-transplant comorbidities. Immunosuppression was maintained with tacrolimus (0.1 mg/kg daily), mycophenolate mofetil (500-1000 mg twice daily) or mycophenolic acid (360 mg twice daily), and prednisone (0.5 mg/kg daily with progressive tapering beyond day 20 or 30). Perioperative prophylaxis consisted of a single dose of 2 g of IV cefazolin. Trimethoprim-sulphamethoxazole (160/800 mg 3 times weekly) or monthly IV pentamidine was provided as prophylaxis for Pneumocystis jirovecii pneumonia for the first nine months. Patients at high risk of cytomegalovirus disease were administered IV gancyclovir (5 mg/kg daily) or oral valgancyclovir (900 mg daily) for the first three months.
Preparation of soluble L. infantum antigen for stimulation Leishmania infantum antigen extract was prepared from promastigote stationary phase parasite cultures (JPC strain, MCAN/ES/98/LLM-722). SLA was obtained from parasites by washing in 1x phosphate-buffered saline (PBS) and centrifuging at 1000 g for 20 min at 4°C. The supernatant was removed and the pellet resuspended in lysis buffer (50 mM Tris/5 mM EDTA/HCl, pH 7; 1 ml for every 10 9 parasites). The latter was then subjected to three rapid freeze/thaw cycles followed by three 20 s 40W pulses with a sonicator, and centrifuged at 27,000 g for 20 min at 4°C. The supernatants were then collected, aliquoted and stored at -80°C until use. Protein quantification was performed using the Bradford method employing the Bio-Rad Protein Assay kit (Bio-Rad, USA).

Culture and stimulation of peripheral blood mononuclear cells
Peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation through Ficoll-Hypaque (Rafer, Spain). The collected cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated foetal bovine serum, 100 IU/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, 50 μM 2-mercaptoethanol, and 1 mM sodium pyruvate. They were then plated in 96 well-plates and kept with RPMI 1640 medium alone (unstimulated) or in RPMI 1640 supplemented with 10 μg/ml phytohaemagglutinin type M (PHAM) (Sigma-Aldrich, USA) (positive control), or with SLA (10 μg/ml). All were kept in a humidified, 5% CO 2 atmosphere at 37°C for 5 days. Cell proliferation was measured by bromodeoxyuridine incorporation using the Cell Proliferation Biotrak ELISA kit (General Electric Healthcare Life Sciences, UK). The results are shown as a stimulation index (SI) for each mitogen or antigen. The cut-off for positive lymphoproliferation was calculated as the mean+3 SD (standard deviation) of the SI for 45 Leishmania-exposed but completely negative SOT subjects: SI = 2.76. The supernatants of the in vitro cell cultures were collected and stored at -20°C for cytokine quantification.

Whole blood stimulation
Blood samples (9-10 ml) were collected from patients in tubes containing heparin. Aliquots (500 μl) of whole blood were incubated in tubes with 10 μg/ml SLA or 5 μg/ml PHAM as a positive control. An additional tube for which no stimulation was provided was included as a negative control. After adding the antigens and mixing thoroughly, the tubes were incubated at 37°C for 24 h. After centrifugation at 2000 g for 10 min, the supernatant plasma were collected and stored at -20°C for cytokine analysis.

Antibody detection methods
An enzyme-linked immunosorbent assay (ELISA) was used to detect antibodies to SLA. Briefly, 96-well plates (NuncMaxisorp Immuno Plates, USA) were coated with 100 μl/well of 10 μg/ml SLA and left overnight at 4°C. The plates were then washed three times with PBS, 0.1% Tween 20 (PBS-T), pH7.4 and blocked with 200 μl/well of PBS containing 0.1% Tween 20 and 3% BSA for 1 h at 37°C. After washing with PBS-T, diluted blood plasma (1/200 in PBS-T) was added (100 μl/well) and incubated for 2 h at 37°C. The plates were then washed with PBS-T and 100 μl/well of 1/5000-diluted HRP-conjugated anti-human Ig (Invitrogen, USA) were added for 30 min at 37°C. All plates were then developed with 100 μl/well of Sigma Fast o-phenylene diamine dihydrochloride (OPD) tablets (Sigma, USA) for 20 min. The reaction was stopped with 50 μl/well of 2N HCl, and absorbance measured at 492 nm. All tests were performed in duplicate and the mean value recorded. The cut-off for seropositivity was calculated as described above at 0.132.
Finally, rK39-ICT dipsticks were purchased from Leti Laboratories and the corresponding test performed following the manufacturer's instructions.

DNA isolation and real time PCR
DNA isolation and real time PCR (qPCR) were performed as described by Cunha et al. [12]. Briefly, DNA was extracted from 100 μL of peripheral blood by conventional phenol-chloroform extraction and eluted in 100 μl sterile distilled water. For qPCR, 1000 nM of R223 and 500 nM of R333 primers (Sigma-Aldrich, USA) for the small subunit rRNA (SSUrRNA) sequence were used [13]. Total DNA was used as a template in touchdown qPCR reactions involving the LightCycler FastStart DNA Master SYBR Green I kit (Roche Applied Science, Switzerland) [14].

NNN culture of PBMCs
PBMCs (2x10 5 ) from all SOT recipients were cultured in Novy-MacNeal-Nicolle medium. The presence/absence of Leishmania was checked every week up to 4 weeks.

Statistical analysis
Immunological data were tested for normality using the Shapiro-Wilk test. Means were compared using the Mann-Whitney U test. The Spearman's rank test was used to seek correlations between immunological variables. Significance was set at p<0.05. All calculations were performed using GraphPad Prism 5.0 software (GraphPad Software, USA).

Immunological response and parasite load
The in vitro cell proliferation assay with PHAM stimulation revealed the functional capacity of the PBMCs to mount a response in all immunosuppressed individuals. Some 21.05% of the NVL subjects (12/57) mounted a positive lymphoproliferative response to SLA stimulation (Fig 1) (hereinafter referred to as NVLpl+ subjects). The IFAT results revealed just two of these same 57 subjects to have mounted a positive serological response; the ELISA and rK39-ICT tests were positive in one of these latter two subjects. This person (i.e., positive in all three serological tests) also showed a strong lymphoproliferative response to SLA (SI 14). The subject who was serologically positive by IFAT alone showed no lymphoproliferative response and developed VL within a few days of testing (treatment was provided within 2 weeks of the original blood sampling), and for that reason was excluded for further analysis (S1 Fig).
The SLA-stimulated PBMC cultures returned positive results for five out of six CVL subjects. IFAT detected anti-Leishmania antibodies in two of these subjects, while the ELISA and rK39-ICT tests detected the same in only one. (Note: The single CVL subject who showed no lymphoproliferative response showed no cytokines in the supernatants of SLA-stimulated PBMC cultures or plasma [see below], and produced no antibodies against Leishmania. This subject relapsed four months after sampling and was therefore considered not cured, and excluded from further analysis).
No Leishmania DNA was detected in any blood sample from any SOT recipient. Absence of residual parasite DNA could be due to the reduced sample size, endemicity of the area, PCR sensitivity or episodic presence of parasite in blood. Neither were any NNN cultures positive for parasites.

the correlation between IFN-γ and granzyme B.
Non-specific stimulation of the PBMCs with PHAM led to an efficient Th1 response that was similar in the NVL lp+ , NVL lp-and CVL subjects (Table 1). IL-10 production was however, significantly higher in the NVL lp+ subjects than in the NVL lp-subjects (P = 0.0097).
Quantification of cytokines in plasma from stimulated whole blood SLA-stimulated whole blood from NVL lp+ subjects produced significantly more IFN-γ (P = 0.0004), TNF-α (P = 0.0469), and IL-2 (P = 0.0028) than that of SLA-stimulated whole blood from NVL lp-subjects (Fig 4). In these NVL lp+ subjects, IFN-γ production correlated with IL-2 (r = 0.616, P = 0.018) (Fig 5). Similarly, CVL subjects with a positive lymphoproliferative response showed significantly higher concentrations of IFN-γ and TNF-α in plasma  (P = 0.0101 and P = 0.0467 respectively) than the NVL lp-subjects. No differences in IL-10 concentration were seen between the three different groups of subjects (Fig 4). Stimulating whole blood with SLA for 24 h did not induce production of IL-17A, IL-5 or IL-4 (S1 Table). No significant differences were found between NVL lp+ and CVL subjects.
Interestingly, in the NVL lp+ subjects, a strong correlation was found between the lymphoproliferative capacity of SLA-stimulated PBMCs and the production of IFN-γ in SLA-stimulated whole blood (r = 0.881, P = 0.0002) (Fig 6).
PHAM stimulation of whole blood led to similar levels of cytokine production in all subjects, with the exception of TNF-α, which was higher in the CVL subjects than in the NVL lp+ subjects (P = 0.0061) ( Table 2), and higher in the NVL lp+ subjects than in the NVL lp-subjects (P = 0.0225).

Discussion
The present results show that tests for the cell-mediated immune response are crucial for detecting exposure to L. infantum; serological tests alone are insufficient. This is especially true  Table 1. Cytokine and granzyme B production after PBMC stimulation with PHAM for 5 days. IFN-γ, granzyme B, TNF-α, IL-10, IL-5, IL-17A, IL-2 and IL-4 concentrations (pg/ml) were measured in culture supernatants of 12 NVL lp+ subjects, 7 NVL lp-subjects, and 5 CVL subjects.

SOT
IFN-γ (pg/ml) TNF-α (pg/ml) IL-10 (pg/ml) Granzyme B (pg/ml) IL-17A (pg/ml) IL-2(pg/ml) IL-4 (pg/ml) when trying to detect evidence of exposure in immunosuppressed individuals, who are at greater risk of developing clinical leishmaniasis after becoming infected, or relapsing after treatment for VL. Few studies have examined the exposure of a large population of SOT recipients living in an area with a high level of endemic leishmaniasis. To our knowledge, this is the first work to study the cell-mediated immunological response to Leishmania in such subjects. In early post-transplant patients, parasites have been detected in the bone marrow [15] and liver [6], the consequence of dormant infections. Although, it is unusual to find parasites in the  [6]. In the present work no subject was detected with leishmanial DNA in the blood. However, Leishmania-specific serology was detected in 2 of the 57 NVL subjects (3.50%). In areas with high transmission rates, the tests commonly used to detect exposure are in vitro antigen recall experiments and the Leishmanin skin test (LST). These tests show good agreement (98-100%) [16] [17]. However, LST testing cannot be performed in Europe since there is no registered commercial reagent for use with humans. Whole-blood IGRAs, offer alternatives. In fact, in intracellular tuberculosis infections, IGRAs generally have a better detection rate than the tuberculin skin test (TST) [18]. In the present work, supernatants from cultures of SLA-stimulated PBMCs from CVL subjects showed a patent lymphoproliferative response and strong production of IFN-γ and TNFα. The response of the NVL lp+ subjects was very similar. The same responses have been widely reported in immunocompetent cured patients [19,20] and asymptomatic individuals [21,22]. The NVL lp+ subjects can thus be considered asymptomatic. This shows that, in spite of their drug-induced immunosuppression, some SOT recipients in contact with Leishmania can mount a specific Th1 response against the parasite.
One published work has recently suggested the measurement of granzyme B as a new marker for asymptomatic individuals and those clinically cured of VL and cutaneous leishmaniasis [23]. In the present work, granzyme B production strongly increased after SLA stimulation in the NVL lp+ and CVL subjects. Further, a correlation was seen between IFN-γ, TNF-α and granzyme B production in supernatants from SLA-stimulated PBMC cultures from NVL lp+ subjects. This is the first study to show granzyme B can be used as a marker of an immunological response to Leishmania in immunosuppressed individuals.
SLA stimulation of PBMCs from NVL lp+ subjects led to significant increases in IL-5, IL-17A and IL-10 production. Correlations between IL-10/IL-5 and IFN-γ have previously been reported in asymptomatic subjects [21,24]. Further, IL-17A production has been reported to increase in exposed but healthy, resistant subjects who did not develop VL [25]. The relationship between IL-17A and protection against leishmaniasis in SOT recipients deserves further study.
Proof of the accuracy of IGRA tests for screening exposed individuals in areas with endemic leishmaniasis already exists [26][27][28]. However, clinical experience with whole blood stimulation assays to detect Leishmania infection is limited. In fact, this is the first study to assess the predictive capacity of this assay in a large cohort of SOT recipients. Interestingly, a strong, positive correlation was detected between the in vitro SLA lymphoproliferative SI and IFN-γ production in SLA-stimulated whole blood from NVL lp+ subjects, showing that cytokine assays can be used to test immunological status. In addition, SLA-stimulated whole blood from these NVL lp+ subjects showed increases in IFN-γ, TNF-α and IL-2. It has been reported that wholeblood cells from the majority of patients with active VL, and those cured of it, as well as 24% of healthy controls, produce significantly elevated levels of IFN-γ [26]. An increase in TNF-α and an absence of IL-10 after SLA stimulation in healthy controls has also been noted [28]. However, this is the first time that differences in IL-2 between NVL lp+ and NVL lp-subjects have been reported. Certain combinations of cytokines may therefore serve as markers of exposure and of cure. Currently, donor screening for leishmaniasis is not performed; the present results suggests that both organ donors and recipients should be screened for this infection. The present results confirm the usefulness of the whole blood stimulation assay and of IFNγ/ TNF-α analysis for following up SOT recipients treated for VL; they could help confirm cure and prevent relapses. Indeed, the tests performed detected one recipient who had recently become infected who developed active leishmaniasis in the following days.

Conclusions
The present results highlight the need to use tests that detect the cell-mediated immune response when screening for asymptomatic subjects in areas with endemic leishmaniasis. They also show that PBMC cultures and/or whole blood assays can be used to search for leukocyte cytokine production as a marker of infection. The production of IFN-γ, TNF-α, granzyme B, IL-5 and IL-10 by SLA-stimulated PBMCs, and of IFN-γ, TNF-α and IL-2 by SLA-stimulated whole blood, could be used to indicate exposure to leishmaniasis, especially for patients subjected to induced immunosuppression. The combination of serological and cell-based tests could help determine the true size of the epidemic of leishmaniasis affecting Fuenlabrada, and indeed of those affecting other areas.