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Title page 1 
Title: An exploratory analysis of  C-X-C motif chemokine ligand 10 as a new biomarker 2 
of asymptomatic Leishmania infantum infection in Solid-Organ Transplant Recipients.  3 
 4 
Running title: CXCL10 in Leishmania infection of SOT recipients 5 
 6 
Authors: 7 
Nerea Carrasco-Antón1a, Ana Victoria Ibarra-Meneses1b, Eugenia Carrillob*, 8 
Mario Fernández-Ruiza, Pilar Hernández-Jiméneza, José María Aguadoa, Javier 9 
Moreno2b, Francisco López-Medrano2b 10 
aUnit of Infectious Diseases, University Hospital 12 de Octubre, Instituto de Investigación 11 
Hospital 12 de Octubre (imas12), Department of Medicine, School of Medicine, 12 
Universidad Complutense, Madrid, Spain. CIBERINFEC 13 
bWHO Collaborating Centre for Leishmaniasis, Spanish National Center for 14 
Microbiology, Instituto de Salud Carlos III, Majadahonda, Spain. CIBERINFEC 15 
1These first authors contributed equally to this article. 16 
2These senior authors contributed equally to this article. 17 
 18 
Nerea Carrasco-Antón.  E-mail: nereacarrascoanton@gmail.com 19 
Ana V. Ibarra-Meneses. E-mail: ana.ibarrameneses@gmail.com   20 
Eugenia Carrillo. E-mail: ecarrillo@isciii.es   21 
 Mario Fernández-Ruiz. E-mail: mario_fdezruiz@yahoo.es 22 
Pilar Hernández-Jiménez. E-mail: pilihj@hotmail.com  23 
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José M. Aguado. E-mail:  jaguadog1@gmail.com 24 
Javier Moreno. E-mail: javier.moreno@isciii.es  25 
Francisco López-Medrano. E-mail: flmedrano@yahoo.es  26 
 27 
Corresponding author contact information: Eugenia Carrillo, WHO Collaborating 28 
Centre for Leishmaniasis, Spanish National Center for Microbiology, Instituto de Salud 29 
Carlos III, Majadahonda, Spain. E-mail: ecarrillo@isciii.es.  30 
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Footnote Page 38 
  39 
Current affiliations and addresses for authors whose affiliations have changed since 40 
completion of the study: 41 
1aNerea Carrasco-Anton. Internal Medicine-Sepsis Unit, IIS-Fundación Jiménez Díaz, 42 
UAM, Av. Reyes Católicos 2, 28040, Madrid, Spain. 43 
1bAna V. Ibarra-Meneses. Département de Pathologie et Microbiologie, Faculté de 44 
Médecine Vétérinaire, Université de Montréal, Canada; Research Group on Infectious 45 
Diseases in Animal Production (GREMIP), Faculty of Veterinary Medicine, University 46 
of Montreal, Canada. 47 
 48 
 49 
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Abstract—175 words 50 
Objective: Sensitive and less laborious assays are needed to detect asymptomatic 51 
Leishmania among solid organ transplant (SOT) recipients. Using SLA-stimulated 52 
plasma from SOT recipients living where an outbreak of Leishmania infantum occurred, 53 
we examined potential biomarkers to identify asymptomatic Leishmania infections. 54 
Methods: Concentrations of cytokines/chemokines in plasma from whole blood 55 
stimulated with specific Leishmania antigen (SLA) were compared against infection 56 
status as determined by a currently used cell proliferation assay. 57 
Results: Twenty-six percent (13/50) of the SOT recipients had a cell proliferation assay 58 
(CPA) indicating asymptomatic infection, and showed higher processed plasma C-X-C 59 
motif chemokine ligand 10 (CXCL10 or IP-10) concentrations than did non-infected 60 
subjects (median 2272.0 pg/ml [IQR-1570-2772] vs. 18.2 pg/ml [IQR 1-150.1]; 61 
p<0.0001). CXCL10 showed a sensitivity of 93% and a specificity of 95% compared to 62 
CPA. In addition, we demonstrated that the number of asymptomatic infections detected 63 
using CXCL10, decreased with distance from a park at the centre of the mentioned 64 
outbreak.  65 
Conclusion: CXCL10 in plasma from SLA-stimulated blood could be a robust biomarker 66 
of asymptomatic L. infantum infection in solid organ transplant recipients.  67 
 68 
Keywords: Solid-Organ Transplant recipients, Leishmania, leishmaniasis, biomarkers, 69 
asymptomatic infection, IP-10, CXCL10. 70 
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Text -2319 words- 71 
Introduction 72 
Visceral leishmaniasis (VL) is a potentially serious complication for recipients of solid 73 
organ transplants (SOT). The mortality attributable to such infection is thought to be 74 
around 3% and the relapse rate may exceed 25% (1, 2). After infection, progression to 75 
clinical VL depends on the balance between multiple factors that promote or prevent the 76 
multiplication and expansion of parasites in the body. The complexity of the response 77 
makes it difficult to predict the outcome of the infection, but it is known that most people 78 
infected with Leishmania remain asymptomatic (3). Up to date, there are only three 79 
relevant well-driven works studying the prevalence of asymptomatic leishmaniasis in 80 
SOT recipients (4), who can be at risk of developing clinical VL. Nowadays, there is not 81 
a formal recommendation for the treatment of SOT recipients with an asymptomatic 82 
Leishmania infection, although the beneficial impact of antiparasitic is widely discussed. 83 
There is no single universally accepted assay to identify asymptomatic infection. 84 
Conventional serological tests for leishmaniasis show limited sensitivity when used with 85 
immunocompromised patients (5). The detection of Leishmania-specific cell-mediated 86 
immunity, however, may offer a more accurate assessment, even indicating the 87 
prevalence of asymptomatic infection (asymptomatic subjects are those from an endemic 88 
area of VL with a detectable immune response, or parasitaemia, in the absence of signs 89 
or symptoms of active disease) (3, 5, 6). The Leishmanin Skin Test (LST) provides for 90 
such detection, and it has been used to study the prevalence of Leishmania infection in 91 
the field (7-9). However, its associated side effects, and the failure of its makers to follow 92 
the principles of good manufacturing practice, have caused its use in some countries to 93 
be abandoned. The cell proliferation assay (CPA), performed with peripheral blood 94 
mononuclear cell (PBMC) cultures stimulated with soluble L. infantum antigen (SLA), 95 
6 
 
can be used instead (10). However, while this can confirm asymptomatic Leishmania 96 
infection in SOT recipients, it is laborious and time-consuming (11). A further alternative 97 
is the whole blood stimulation assay (WBA), an easy, rapid test that can be used to 98 
monitor SOT recipients treated for VL, and for detecting asymptomatic Leishmania 99 
infection (12, 13). However, it relies on interferon- (IFN-) as a marker, and more 100 
sensitive and specific markers have recently been described (11). These include the 101 
Interferon-γ-induced protein 10 (IP-10 or CXCL10), the monokine induced by IFN-γ 102 
(MIG or CXCL9), and monocyte chemotactic protein 1 (MCP-1 or CCL2), all of which 103 
are produced at much higher concentrations in plasma from SLA-stimulated whole blood 104 
- at least that of immunocompetent patients (14-17). It is possible that they may also be 105 
useful for detecting asymptomatic infection in SOT recipients, who are of course 106 
immunosuppressed. The present work examines the chemokine profile of plasma from 107 
SLA-stimulated whole blood as a means of identifying asymptomatic Leishmania 108 
infection in SOT recipients.  109 
In earlier work, it was found that symptomatic cases of VL among members of the general 110 
population, and among SOT recipients, were more numerous with increasing proximity 111 
to the semi-urban park at the centre of the 2009 outbreak in Fuenlabrada (Madrid, Spain) 112 
(18). The present work examines whether this relationship also holds true for SOT 113 
recipients with asymptomatic Leishmania infection as identified using a proposed test 114 
based on CXCL10 as a marker. 115 
 116 
Methods 117 
Ethics statement 118 
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This study was approved by the institutional Ethics Committee of the Hospital 119 
Universitario 12 de Octubre.  All subjects gave their written, informed consent to be 120 
included.  121 
Study design and setting 122 
The study population included 50 adult patients (≥18 years of age) who had undergone 123 
SOT (kidney, liver or heart) at the Hospital Universitario 12 de Octubre (Madrid, Spain) 124 
(the reference centre for SOT in the Madrid Region's southwest) between 2005 and 2013, 125 
and whose usual place of residence was Fuenlabrada (where the above-mentioned 126 
outbreak occurred). Sample collection was performed between October 2012 and October 127 
2013. None of these subjects had any symptoms of leishmaniasis or previous history of 128 
the disease. 129 
Immunosuppression and prophylaxis regimens 130 
Those SOT recipients at high risk of graft rejection received induction therapy with rabbit 131 
anti-thymocyte globulin (rATG) for 1-3 days after transplantation. Basiliximab was also 132 
administered to those at high risk of calcineurin inhibitor-related nephrotoxicity. A 133 
maintenance immunosuppressive regimen was followed, based on tacrolimus (0.1 mg/kg 134 
daily), mycophenolate mofetil (500-1000 mg twice daily) or mycophenolic acid (360 mg 135 
twice daily), and prednisone (0.5 mg/kg daily with tapering off after the first month post-136 
transplantation). Supplementary Table 1 shows the treatment received by each patient.  137 
Preparation of specific Leishmania antigen 138 
Antigen extract from promastigote stationary phase parasite cultures (JPC strain, 139 
MCAN/ES/98/LLM-722) was used for the preparation of SLA for stimulation purposes 140 
as described by Aleka et al (19). 141 
Laboratory analysis 142 
Routine hematological parameters were determined. 143 
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Cell proliferation assay to detect asymptomatic subjects 144 
All blood samples were subjected to CPA within 24 h of collection to determine which 145 
of the SOT recipients had an asymptomatic infection. Peripheral blood mononuclear cells 146 
were isolated from whole blood and resuspended in RPMI-1640 supplemented with 10% 147 
fetal calf serum, and cultivated with SLA for 5 days (20). The lymphoproliferative 148 
response of each subject was then determined by bromodeoxyuridine incorporation using 149 
the Cell Proliferation Kit (GE Healthcare Life Sciences, UK), following the 150 
manufacturer’s instructions. Results were expressed in the form of a stimulation index 151 
(absorbance of stimulated cells/unstimulated cells).  152 
Detection of new biomarkers in asymptomatic subjects 153 
To search for new biomarkers of asymptomatic infection, whole blood samples (9-10 mL) 154 
were collected in heparinized tubes. Aliquots (500 μL) were incubated with 10 μg/mL 155 
SLA or phytohemagglutinin (PHA-M) as a positive control. A third unstimulated tube 156 
was used as a negative control. All tubes were incubated at 37°C for 24 h. After 157 
centrifugation at 2000 g for 10 min, the plasma was collected and the concentrations of 158 
the chemokines CXCL10, CXCL9 and CCL2 were determined using the BD Cytometric 159 
Bead Array Human Flex Set (Becton Dickinson Biosciences, San Diego, CA, USA) 160 
following the manufacturer’s instructions, subtracting the background levels measured 161 
inthe negative control samples (nonstimulated tube). IFN-γ, interleukin (IL)-2 and tumor 162 
necrosis factor-α (TNF-α), which have been reported as markers of leishmaniasis 163 
infection in other populations, were examined in the same way for comparative purposes. 164 
Serological and molecular tests 165 
Also, for comparative purposes, a routinely used enzyme-linked immunosorbent assay 166 
(ELISA) was used to detect antibodies to SLA (11). Plasma samples were also subjected 167 
to immunofluorescent antibody titer (IFAT) analyses using 2 × 105 L. infantum 168 
9 
 
promastigotes in PBS per well (MCAN/ES/98/LLM-722), as previously described (11). 169 
The threshold title for positivity was set at 1:80. The rK39 immunochromatographic test 170 
(rK39-ICT) was performed using the dipstick format Kalazar Detect Rapid test (InBIOS 171 
International, Seattle, WA). Antibody detection was performed with plasma samples 172 
according to the manufacturer's instructions. In addition, routinely used real-time PCR 173 
(qPCR) targeting the small subunit ribosomal RNA (SSUrRNA) genes of Leishmania 174 
was performed using DNA isolated from 200 µl of peripheral blood with a commercial 175 
extraction column, as described by Cruz et al (21).  176 
CXCL10 profile and proximity to the park at the centre of the outbreak 177 
To determine the relationship between asymptomatic infection as determined by CXCL10 178 
(the marker that returned the clearest results in the above assays) and the distance between 179 
the semi-urban park where the outbreak begun, each SOT recipient's home was located 180 
on a map and the shortest linear distance to the border of the park measured using an 181 
online mapping tool (Google Maps, Google Inc., Mountain View, CA, USA).    182 
Statistical analysis  183 
Quantitative data are shown as medians with interquartile ranges (IQR). The normality of 184 
the distribution of continuous variables was assessed using the Shapiro-Wilk test. 185 
Biomarker concentrations were compared using the non-parametric Mann-Whitney U 186 
test. All tests were two-tailed. Significance was set at p<0.05.  The area under the receiver 187 
operating characteristic (ROC) curve, diagnostic cut-off values, sensitivity and 188 
specificity, and the Youden J statistic (J = sensitivity + specificity - 1), were all calculated. 189 
All calculations were performed using SPSS v.20.0 (IBM Corp., Armonk, NY, USA) or 190 
Graph Pad Prism v.7.02 (GraphPad Software Inc., La Jolla, CA, USA). 191 
 192 
Results 193 
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Clinical characteristics of the study cohort 194 
Table 1 summarizes the demographic and clinical characteristics of the SOT recipients 195 
(40 [80%] had a kidney transplant, 8 [16%] a liver transplant, and 2 [4%] a heart 196 
transplant).  Most recipients were male (76%).  The mean age at transplantation was 50.2 197 
± 13.8 years. The mean hemoglobin was 13.4g/dL. The total number of leukocytes, 198 
neutrophils, and lymphocytes was 6.84, 4.52, and 1.46 x 103cell/ml, 199 
respectively. And the platelet count was 188 x 103cell/ml.  200 
Capacity of the tested biomarkers to indicate asymptomatic Leishmania infection in 201 
SOT recipients 202 
Fourteen of the 50 (28%) SOT recipients returned a positive CPA test (SI ≥3.44); these 203 
subjects were classified as having an asymptomatic infection (Fig. 1A). Those infected 204 
by L. infantum had significantly higher levels of IFN-γ, IL-2, TNF-α, CXCL9 and CCL2 205 
(p<0.0001 for all) in plasma from SLA-stimulated whole blood compared to those with 206 
no asymptomatic infection (i.e., with a negative CPA result). However, those with 207 
asymptomatic infection had much higher CXCL10 concentrations that did non-infected 208 
subjects (Fig. 1B: median 2272 pg/ml [IQR 1441-3433] vs. 18.22 pg/ml [IQR 1-150.10]). 209 
The area under the ROC curve for the detection of asymptomatic infection by CXCL10 210 
was 0.9644 (95%CI 0.91-1.00; p<0.0001); the cut-off was 762.5 pg/ml. Sensitivity was 211 
93% (95%CI 77.23-99.15) and specificity 95% (95%CI 87.23-98.57) (Fig. 1C) - higher 212 
than for all other cytokines tested - and the Youden J value was 0.92 (Table 2).  213 
 214 
Figure 1.  (A) The cell proliferation assay (CPA) identified asymptomatic subjects (AS) 215 
and non-infected subjects (NI). (B) Production of CXCL10 in plasma from SLA-216 
stimulated whole blood in both types of subject. Box-whisker plots show medians, 217 
interquartile ranges, and min/max values. ****p<0.0001. (C) Receiver operating 218 
11 
 
characteristic curve analysis showing the sensitivity and specificity of CXCL10 for 219 
detecting asymptomatic subjects. 220 
 221 
By way of comparison, no Leishmania DNA was detected in any blood sample from any 222 
SOT recipient. IFAT and rK39-ICT tests detected anti-Leishmania antibodies in just one 223 
asymptomatic subject, while ELISA did so in just four (Table 3). 224 
Spatial determinants of CXCL10 profiles  225 
In agreement with a previous finding that the incidence of VL is greater among SOT 226 
recipients living <1000 m from the park than among those living further away, the present 227 
subjects with a positive CPA result (indicative of asymptomatic L. infantum infection) 228 
lived closer to the park than did those with a negative result (861 m ± 621 m vs. 1390 m 229 
± 581 m; p=0.04) (Figure 2). The home-park distance was then compared between SOT 230 
recipients with and without a positive CXCL10 response in plasma from SLA-stimulated 231 
whole blood according to the established cut-off value (≥762.5 pg/mL) (Table 2). As 232 
expected, those with a positive CXCL10 response lived significantly closer than those 233 
with a negative response (1011 m ± 581 m vs. 1380 m ± 784 m; p=0.0408). A significant 234 
difference was also found with respect to IL-2 positivity (861 m ± 447 m vs. 1390 m ± 235 
782 m; p=0.0247). No significant relationship was seen between the home-park distance 236 
and a positive IFN- or CXCL9 response (1235 m ± 531 m vs. 1350 m ± 807 m; p=0.2531; 237 
1235 m ± 531 m vs. 1350 m ± 827 m; p=0.2768).   238 
 239 
Figure 2. Location of the SOT recipients' homes and relationship with CXCL10 240 
(p=0.0408).  241 
 242 
Discussion  243 
12 
 
In the present work, the detection of IFN-γ, IL-2, and CXCL9 in plasma from SLA-244 
stimulated whole blood (i.e., in samples that returned a positive CPA result) yielded good 245 
sensitivity and specificity values (Table 2). However, CXCL10 was the most efficient 246 
biomarker (sensitivity 93%, specificity 95%) for identifying the asymptomatic population 247 
among the SOT recipients. A previous study performed in the same geographical area 248 
reported IL-2 to show good diagnostic accuracy in the detection of subjects with 249 
asymptomatic infection among blood donors and healthy volunteers (12, 22). To our 250 
knowledge, this is the first work to investigate the use of CXCL10 as a marker of 251 
Leishmania infection in SOT recipients. In work by other authors, the expression of 252 
CXCL10 in stimulated plasma was shown to reveal latent tuberculosis infection, and, in 253 
non-stimulated plasma from SOT recipients, it was reported to have potential as a 254 
biomarker of cytomegalovirus-induced inflammation (23, 24).  255 
The present results reveal an inverse correlation between the home-park distance and the 256 
number of CXCL10-positive results. Those SOT recipients with an asymptomatic 257 
infection according to their CXCL10 result lived closer to the park than did those who 258 
were identified as not infected. In a previous paper, our group reported that living <1000 259 
m from the park was an independent risk factor for SOT recipients developing clinical 260 
leishmaniasis (18). We also previously described the usefulness of IL-2 for establishing 261 
the true prevalence of asymptomatic infection in an immunocompetent population living 262 
in the area bordering the park (22). In that study, proximity to the park was measured as 263 
the distance from the latter to the primary healthcare centers where patients were attended 264 
to - not the home address of the subjects as it was in the present work.  265 
The present study suffers from a number of limitations. The sample size is small, affecting 266 
the statistical power available for detecting differences between groups (although an 267 
intense effort was made to identify every single SOT recipient living in the affected area 268 
13 
 
during the study period; it is unlikely that any was missed). It is also possible that the 269 
subjects were asymptomatically infected before 2009, a period for when no such records 270 
are available. The nature of the 2009 outbreak (wild transmission cycle in contact with 271 
people) strongly suggests that natural immunity in the study area was little developed or 272 
absent (25). 273 
In conclusion, CXCL10 detected in plasma from SLA-stimulated whole blood may act as 274 
a useful tool for identifying asymptomatic L. infantum-infection in SOT recipients. This 275 
finding might hold true in other endemic areas in which immunocompromised hosts act 276 
as a reservoir of disease.  If so, determining CXCL10 concentrations in such plasma could 277 
be of much use in the control of leishmaniasis. 278 
Acknowledgments 279 
Thanks are owed to all the patients who took part in this study.  280 
Funding 281 
This study was co-funded by the World Health Organization (APW-2012/271093-O), and 282 
the Spanish Ministry of Science and Universities (via Proyecto Integrado de Excelencia 283 
[PIE] 13/00045).  Additional funding was provided by the Instituto de Salud Carlos III 284 
via project PI18CIII/00028 and DTS16CIII/00010, and via the Red de Enfermedades 285 
Tropicales, Subprograma RETICS del Plan Estatal de I+D+I 2013-2016, which is co-286 
supported by FEDER “Una Manera de Hacer Europa” funds via project 287 
RD16CIII/0003/0002. M.F.R. holds a “Miguel Servet” (CP18/00073) research contract 288 
from the Spanish Ministry of Science and Innovation, Instituto de Salud Carlos III. The 289 
funders had no role in the design of the study, data collection, or the interpretation of the 290 
results. 291 
Conflict of Interest 292 
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The authors have declared no competing interest.  293 
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Legends 392 
Figure 1.  (A) The cell proliferation assay (CPA) identified asymptomatic subjects (AS) 393 
and non-infected subjects (NI). (B) Production of CXCL10 in plasma from SLA-394 
stimulated whole blood in both types of subjects. Box-whisker plots show medians, 395 
interquartile ranges and min/max values. ****p<0.0001. (C) Receiver operating 396 
characteristic curve analysis showing the sensitivity and specificity of CXCL10 for 397 
detecting asymptomatic subjects. 398 
Figure 2. Location of the SOT recipients' homes and relationship with CXCL10 399 
(p=0.0408).  400 
 401 
 402 
 403