- Email: [email protected]
Asymptomatic Leishmania infantum infection in blood donors living in an endemic area, northeastern Italy
Journal Pre-proof
Asymptomatic Leishmania infantum infection in blood donors living in an endemic area, northeastern Italy Margherita Ortalli , Alessandra Mistral De Pascali , Serena Longo , Nadia Pascarelli , Andrea Porcellini , Deborah Ruggeri , Vanda Randi , Anna Procopio , Maria Carla Re , Stefania Varani PII: DOI: Reference:
S0163-4453(19)30291-9 https://doi.org/10.1016/j.jinf.2019.09.019 YJINF 4345
To appear in:
Journal of Infection
Accepted date:
13 September 2019
Please cite this article as: Margherita Ortalli , Alessandra Mistral De Pascali , Serena Longo , Nadia Pascarelli , Andrea Porcellini , Deborah Ruggeri , Vanda Randi , Anna Procopio , Maria Carla Re , Stefania Varani , Asymptomatic Leishmania infantum infection in blood donors living in an endemic area, northeastern Italy, Journal of Infection (2019), doi: https://doi.org/10.1016/j.jinf.2019.09.019
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd on behalf of The British Infection Association.
Highlights:
Most leishmanial infections remain asymptomatic in immunocompetent individuals
Leishmaniasis is endemic in the province of Bologna, northeastern Italy
We tested 240 blood donors in the Bologna province to detect asymptomatic Leishmania infection
Highly sensitive serological test, ie Western Blot, was employed in combination with a real time PCR assay
The prevalence of asymptomatic Leishmania infection in the Bologna province was 12.5 %
Asymptomatic Leishmania infantum infection in blood donors living in an endemic area, northeastern Italy
Margherita Ortalli1, Alessandra Mistral De Pascali1, Serena Longo1, Nadia Pascarelli2, Andrea Porcellini2, Deborah Ruggeri2, Vanda Randi2, Anna Procopio3, Maria Carla Re1,4* and Stefania Varani1,4 *
1
Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, 40100,
Italy 2
Centro Regionale Sangue Emilia-Romagna, Maggiore Hospital, Bologna, 40100, Italy
3
Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia-Romagna, Brescia, 25100,
Italy 4
Unit of Clinical Microbiology, Regional Reference Centre for Microbiological Emergencies
(CRREM), St. Orsola-Malpighi University Hospital, Bologna, 40100, Italy * These authors contributed equally to this work Correspondence to: Stefania Varani, St. Orsola-Malpighi University Hospital, Giuseppe Massarenti n. 9 40138, Bologna, tel. +39 051 2143013, email: [email protected] ORCID: 0000-0003-0862-4937 Declaration of interest: none Keywords: Asymptomatic Leishmania infection, blood donors, Western Blot, PCR
Abstract Objectives. Human leishmaniasis can be severe and fatal, yet in the Mediterranean region only a small percentage of infections progress to clinical disease. We evaluated the percentage of asymptomatic Leishmania infection in the Bologna province, northeastern Italy. Methods. We examined the presence of specific antibodies by Western Blot (WB) and parasitic DNA by real time PCR in peripheral blood of 240 blood donors residing in the Bologna province. Results. Anti-Leishmania IgG were detected by WB in 27 subjects (11.2%, 95% CI 7%-15%), while Leishmania kinetoplast DNA was detected in peripheral blood specimens of 4 out of 240 donors (1.7%, 95% CI 0.2%-3.2%). Overall, the prevalence of Leishmania infection in the blood donor cohort was 12.5%, thus indicating an elevated cumulative exposure to the Leishmania parasite in the examined municipality. Conclusions. Our results suggest that a surveillance system for monitoring Leishmania infection in blood donors and/or strategies of protozoan inactivation in whole blood should be taken into consideration in areas with circulation of the Leishmania parasite.
Introduction The Leishmania spp. protozoa are sandfly-borne intracellular parasites that are responsible for the leishmaniases, a complex of diseases with important clinical implication [1]. In humans, the major clinical forms are cutaneous, mucocutaneous and visceral leishmaniasis, the latter being fatal if untreated. Most leishmanial infections remain asymptomatic in immunocompetent individuals; for example, the ratio of subclinical to clinical cases of Leishmania infantum infection has been reported to be around 50:1 in Spain [2]. Human leishmaniasis is endemic in Italy, with an increase of cases in the last two decades due to disease spreading within traditionally endemic regions and to the appearance of autochthonous cases in previously non-endemic areas such as northern continental Italy [3-4]; as an example, an outbreak of visceral leishmaniasis has been recorded in the province of Bologna, northeastern Italy [5]. Since little is known on the circulation of the parasite in the abovementioned area, which was previously regarded as low risk area for leishmaniasis, we aimed to evaluate the prevalence of asymptomatic Leishmania infection in blood donors living in the Bologna province. Serum samples were evaluated for the presence of specific anti-Leishmania antibodies by Western Blot (WB) assay, while buffy coats from peripheral blood specimens were analyzed for the presence of Leishmania DNA by PCR.
Materials and methods Setting and study cohort We conducted a retrospective serological study in healthy Italian individuals volunteering for blood donation. The study cohort included blood donors residing in Valsamoggia, an Italian municipality counting 30,071 citizens in the Bologna province, Emilia-Romagna region (northeastern Italy, Fig.1). Between April 2014 and March 2015, peripheral blood samples were collected from 240 blood donors; 174 males and 66 females with a median age of 47 years (range 22-70). None of the donors had previously shown clinical manifestation of visceral or cutaneous leishmaniasis. All subjects enrolled in the study were asymptomatic at the time of blood donation, as requested by the Italian National Transfusion Network rules [6]. Blood samples were obtained from the Regional Blood Centre of Emilia-Romagna, Maggiore Hospital of Bologna, stored at 4°C and sent to the Unit of Microbiology (St. Orsola-Malpighi University Hospital, Bologna, Italy) within 5 days from the collection. Blood samples were centrifuged and both sera and buffy coats were stored at -80°C. The study was conducted in accordance with the Declaration of Helsinki. Generic written informed consent for storage and future use for research purpose was obtained from each blood donor before sampling. Blood samples were coded and retrospectively analyzed by serological and molecular assays. As samples were anonymized, no further consent to participate to this study could be requested to blood donors, according to the Italian Data Protection Authority (Authorization n. 9/2016). Serological detection of Leishmania infection Serological testing was performed by the Leishmania WESTERN BLOT IgG (LDBio Diagnostics®, Lyon, France). The WB technique provides detailed antibody responses to two antigens. In details, leishmanial 14-kDa and 16-kDa proteins of L. infantum are electrotransferred onto a nitrocellulose membrane and probed with patients’ serum. The positivity of the p14 and/or p16 bands is considered a sensitive and specific evidence of anti-Leishmania IgG in the tested serum as antibodies against p14 and/or p16 antigens were detected in 100% of patients with visceral
leishmaniasis and exhibited minimal cross-reactivity with antibodies directed against other infectious agents [7]. Molecular detection of Leishmania infection DNA was extracted from buffy coats using the QIAsymphony DSP Virus/Pathogen kit and the QIAsymphony SP/AS instrument, Qiagen® (Hilden, Germany). The nucleic acid extraction kit was employed according to the manufacturer’s instructions, while an in-house molecular method was used to detect leishmanial DNA in buffy coats from peripheral blood specimens. In details, nucleic acids were extracted from 300 µL buffy coat and DNA was eluted in 200µL of elution buffer and amplified employing a PCR targeting leishmanial kinetoplast (k)DNA [8]. Primers (15 pmol of RV1
5’-CTTTTCTGGTCCTCCGGGTAGG-3’,15
pmol
of
RV2
5’-
CCACCCGGCCCTATTTTACACCAA-3’) were synthesized by PrimmBiotech (Milan, Italy) and 50 pmol of TaqMan probe (FAM-TTTTCGCAGAACGCCCCTACCCGC-TAMRA) were synthesized by IDTDNA (Leuven, Belgium). β2-microglobulin real-time PCR assay was run simultaneously as a control of amplification of the extracted DNA. The real-time PCR assays were performed by employing the Real Time PCR Cycler Rotor Gene 3000 (Qiagen, Hilden Germany). Real time PCR was considered positive for Leishmania DNA when an amplification curve at threshold cycle (tC) < 40 was present. In case of positive PCR test, real time PCR procedure was repeated using new DNA extracts from the original specimen to rule out potential contamination; only samples with confirmed positive PCR results were considered true positive.
Results In total, 30 of 240 individuals tested positive at the serological and/or molecular screening for Leishmania infection. None of the individuals included in the study had a medical history of visceral or cutaneous leishmaniasis. Results by WB analysis showed the presence of specific antibody to 14 and/or 16 kDa proteins, indicative of anti-Leishmania IgG in 27 blood samples (11.2 %, 95% CI 7% -15%, Table 1). In two donors, sera were capable of detecting both bands, while antibodies against p14 or p16 were found in 9 and 16 serum samples, respectively. Circulating Leishmania kDNA was detected in buffy coats from 4 out of 240 blood donors (1.7%, 95% CI 0.2%-3.2%). Median tC value for the PCR positive samples was 37.5 (range, 37.0-39.8). Only one donor showed simultaneously a positivity by serological (WB reactivity to p14) and molecular methods employed in the study. Overall, the prevalence of asymptomatic Leishmania infection in the studied population was 12.5 % (95% CI 8.3%-16.7%). No significant difference was observed by age or sex in the prevalence rate of asymptomatic Leishmania infection (Table 1 and data not shown).
Discussion In this study, we screened blood samples from 240 donors living in an area where L. infantum is endemic and we detected an asymptomatic Leishmania infection in 12.5% of tested individuals, which indicates an important cumulative exposure to the parasite in the examined municipality. Leishmania-specific antibodies were revealed by WB in serum samples obtained from 27 donors, which showed antibody to 14 and/or 16 kDa proteins. We considered a WB test as positive if one of these bands was is present; in fact, anti-p14 and anti-p16 showed less than 2% cross-reactivity with antibodies directed against other infectious agents [7]. On the other hand, the presence of the parasite was only detected in blood samples of 4 donors (1.7%) by kDNA amplification, suggesting low levels of circulating parasites. Epidemiological studies from Mediterranean countries show a highly variable prevalence (from <1 0% to 53%) of asymptomatic Leishmania infection [4 and Table 2], probably due to a fluctuating circulation of the parasite in different areas and to a diversity of methods employed for testing; commonly used serological methods, such as immunoenzymatic tests, direct agglutination test and immunofluorescence tests can be inaccurate and underestimate the infection rates in asymptomatic carriers. On the other hand, the leishmanin skin test can specifically detect T-cell mediated responses with high sensitivity, but antigen preparation has not been standardized for universal use [3]. As using a combination of tests has been reported to increase the capacity to detect asymptomatic Leishmania infection [1], we employed a highly sensitive serological test, ie WB [7] in combination with a real time PCR assay. In this study, no significant difference was observed by sex in the prevalence rate of asymptomatic Leishmania infection, while clinical disease is overall predominant in men [1] including the study area [5]; our findings contradict the assumption that higher frequency of visceral leishmaniasis in male than female reflects an increased exposure of men to sandflies. Thus, factors other than sandfly exposure should be considered in triggering progression from asymptomatic infection to overt disease.
Our results are in line with previous evidence reporting that many cases of Leishmania infection are asymptomatic in the Mediterranean Europe [2, 4, 10-12, 14, 16, 17, 19, 21, 22]. Some patients with subclinical infection can harbor viable parasites throughout life and may develop reactivation to full-blown visceral leishmaniasis if immunosuppression occurs thereafter [23]. Visceral and cutaneous leishmaniasis are compulsory notifiable diseases in Italy and confirmed cases are obligatorily reported by local public health departments to the regional health authorities. As no leishmaniasis cases were reported to the Regional Health Authority among blood donors from the Emilia-Romagna region (around 4,500,000 inhabitants) up to August 2019, we assume that none of the asymptomatic leishmanial infection that we detected in blood donors in 2014-2015 progressed to clinical disease. Cases of possible L.infantum transmission by blood donation have been reported. However, pre-storage leukodepletion, which is mandatory in Italy since January 2016, is greatly effective in reducing the risk of Leishmania transmission by parasitemic blood donors [24]. Strategies that reduce live parasites in blood supplies are under study and should be taken into consideration in endemic areas. Several methods for inactivation of Leishmania parasites such as riboflavin and ultraviolet light [25], amatosalen and UVA illumination [26], and thiazole orange [27] have been shown to be effective when applied to different blood components in in vitro studies. In addition, the employment of amatosalen and ultraviolet light as well as riboflavin and ultraviolet light effectively eliminated leishmanial DNA in apheresis platelet units from single asymptomatic infected blood donors [28, 29]. However, these findings need to be confirmed by further studies before the above-mentioned strategies could be considered for routine use. This study has some limitations. Firstly, the examined cohort exclusively comprised healthy adults, thus the results cannot be generalized to the overall population of the Emilia-Romagna region (Italy), secondly, the study was conducted on a limited number of blood donors. As a third point, no gold standard method is currently available to detect asymptomatic leishmanial infection [3], thus rendering difficult to compare results from different studies on this matter.
Even if our study was conducted in a limited number of blood donors samples and no symptomatic Leishmania infection has been so far recorded in subjects who received blood products in Italy, the geographic spread of Leishmania suggests that a surveillance system for monitoring this parasitic infection in blood donors and/or strategies that inactivate the parasite in whole blood should be taken into consideration in endemic areas.
Acknowledgments This work was supported by Lab P3 funds from the Emilia-Romagna Region (Italy) and by RFO 2010-2016 funds from the University of Bologna. We thank Giorgio Galletti and Annalisa Santi for support in preparation of the study area map.
Conflict of interest The authors declare no conflict of interest.
References 1. WHO. Control of the leishmaniases: report of a meeting of the WHO Expert Committee on the
Control
of
Leishmaniases,
Geneva,
22-26
March
2010.
Available
from:
http://apps.who.int/iris/bitstream/10665/44412/1/WHO_TRS_949_eng.pdf 2. Moral L, Rubio E M, Moya M. A leishmanin skin test survey in the human population of l’Alicanti region (Spain): implications for the epidemiology of Leishmania infantum infection in southern Europe. Trans R Soc Trop Med Hyg 2002; 96: 129-32. 3. Gradoni L, Rogelio L V, Mourad M. Regional Office for Europe. Manual on case management and surveillance of the leishmaniases in the WHO European Region. WHO 2017. Available from: http://www.euro.who.int/en/publications/abstracts/manual-on-casemanagement-and-surveillance-of-the-leishmaniases-in-the-who-european-region-2017 4. Moriconi M, Rugna G, Calzolari M, Bellini R, Albieri A, Angelini P, et al. Phlebotomine sand-fly borne pathogens in the Mediterranean Basin: Human leishmaniasis and phlebovirus infections. PLoS Negl Trop Dis 2017; 11: e0005660. 5. Varani S, Cagarelli R, Melchionda F, Attard L, Salvadori C, Finarelli A C, et al. Ongoing outbreak of visceral leishmaniasis in Bologna Province, Italy, November 2012 to May 2013. Euro Surveill 2013;18: 20530. 6. Ministerial Decree DM, November 2, 2015, “Disposizioni relative ai requisiti di qualità e sicurezza del sangue e degli emocomponenti” . Available from: https://www.avis.it/userfiles/file/GU%20SG%20n_300%20del%2028-122015_SO_069%20(1).pdf 7. Mary C, Lamouroux D, Dunan S, Quilici M. Western blot analysis of antibodies to Leishmania infantum antigens: potential of the 14-kD and 16-kD antigens for diagnosis and epidemio- logic purposes. Am J Trop Med Hyg 1992;47: 764-71.
8. Mary C, Faraut F, Lascombe L, Dumon H. Quantification of Leishmania infantum DNA by a Real-Time PCR Assay with High Sensitivity. J Clin Microbiol 2004; 42: 5249–5255. 9. Garrote J I, Gutiérrez M P, Izquierdo R L, Dueñas M A, Zarzosa P, Cañavate C, et al. Seroepidemiologic study of Leishmania infantum infection in Castilla-Leon, Spain. Am J Trop Med Hyg 2004;71: 403-6. 10. Riera C, Fisa R, Udina M, Gállego 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: 102-10. 11. Riera C, Fisa R, López-Chejade P, Serra T, Girona E, Jiménez M, et al. Asymptomatic infection by Leishmania infantum in blood donors from the Balearic Islands (Spain). Transfusion 2008;48: 1383-9. 12. Pérez-Cutillas P, Goyena E, Chitimia L, De la Rúa P, Bernal L J, Fisa R, et al. Spatial distribution of human asymptomatic Leishmania infantum infection in southeast Spain: a study of environmental, demographic and social risk factors. Acta Trop 2015;146:127-34. 13. Ibarra-Meneses A V, Carrillo E, Nieto J, Sánchez C, Ortega S, Estirado A, et al. Prevalence of asymptomatic Leishmania infection and associated risk factors, after an outbreak in the south-western Madrid region, Spain, 2015. Euro Surveill 2019;24. 14. Le Fichoux Y, Quaranta J F, Aufeuvre J P, Lelievre A, Marty P, Suffia I, 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: 1953-7. 15. Scarlata F, Vitale F, Saporito L, Reale S, Vecchi V L, Giordano S, et al. Asymptomatic Leishmania infantum/chagasi infection in blood donors of western Sicily. Trans R Soc Trop Med Hyg 2008;102: 394-6.
16. Biglino A, Bolla C, Concialdi E, Trisciuoglio A, Romano A, Ferroglio E. Asymptomatic Leishmania infantum infection in an area of northwestern Italy (Piedmont region) where such infections are traditionally nonendemic. J Clin Microbiol 2010;48: 131-6. 17. Maritati M, Trentini A, Michel G, Bellini T, Almugadam S, Hanau S, et al. Subclinical Leishmania infection in patients with rheumatic diseases under biological drugs. Infection 2018;46: 801-809. 18. Colomba C, Saporito L, Polara V F, Barone T, Corrao A, Titone L. Serological screening for Leishmania infantum in asymptomatic blood donors living in an endemic area (Sicily, Italy). Transfus Apher Sci 2005;33: 311-4. 19. Kyriakou D S, Alexandrakis M G, Passam F H, Kourelis T V, Foundouli P, Matalliotakis E, et al. Quick detection of Leishmania in peripheral blood by flow cytometry. Is prestorage leucodepletion necessary for leishmaniasis prevention in endemic areas? Transfus Med 2003;13: 59-62. 20. Diza E, Kansouzidou A, Gerou S, Vezyri E, Metallidis S, Antoniadis A. Leishmaniases in Northern Greece: seroprevalence of the infection and incidence of the disease during the period 2001-2006. Eur J Clin Microbiol Infect Dis 2008;27: 997-1003. 21. Šiško-Kraljević K, Jerončić A, Mohar B, Punda-Polić V. Asymptomatic Leishmania infantum infections in humans living in endemic and non-endemic areas of Croatia, 2007 to 2009. Euro Surveill 2013;18: 20533. 22. Ferroglio E, Battisti E, Zanet S, Bolla C, Concialdi E, Trisciuoglio A, et al. Epidemiological evaluation of Leishmania infantum zoonotic transmission risk in the recently established endemic area of Northwestern Italy. Zoonoses Public Health 2018; 65: 675-682. 23. van Griensven J, Carrillo E, López-Vélez R, Lynen L, Moreno J Leishmaniasis in immunosuppressed individuals. Clin Microbiol Infect 2014;20: 286-99.
24. Jimenez-Marco T, Fisa R, Girona-Llobera E, Cancino-Faure B, Tomàs-Pèrez M, Berenguer D, et al. Transfusion transmitted Leishmaniasis: a practical review. Transfusion 2016; 26: 45-51. 25. Cardo L J, Rentas F J, Ketchum L, Salata J, Harman R, Melvin W, et al. Pathogen inactivation of Leishmania donovani infantum in plasma and platelet concentrates using riboflavin and ultraviolet light. Vox Sang 2006;90: 85-91. 26. Eastman R T, Barrett L K, Dupuis K, Buckner F S, Van Voorhis W C. Leishmania inactivation in human pheresis platelets by a psoralen (amotosalen HCI) and longwavalength ultraviolet irradiation. Transfusion 2005;45: 1459-63. 27. Wagner S J, Skrichenko A, Salata J, O’Sullivan A M, Cardo L J. Inactivation of Leishmania donovani infantum and Trypanosoma cruzi in red cell suspensions with thiazole orange. Transfusion 2008; 48: 1363-7. 28. Jimenez-Marco T, Fisa R, Riera C, Girona-Llobera E, Sedeno M, Saura A, et al. Pathogen inactivation technology applied to a blood component collected from an asymptomatic carrier of Leishmania infantum: a case report. Vox Sang 2012; 103: 356-8. 29. Jimenez-Marco T, Riera C, Fisa R, Girona-Llobera E, Sedeno M, Goodrich R P, et al. The utility of pathogen inactivation technology: a real-life example of Leishmania infantum inactivation in platelets from a donor with an asymptomatic infection. Blood Transfus 2012; 10: 536-41.
1. Table 1. Rates of asymptomatic Leishmania infection among blood donors (n=240) residing in the Bologna Province (northeastern Italy) by Western Blot and real time PCR.
Variables
Results WB Pos
WB Neg
PCR Pos
PCR Neg
No.
%
No.
%
No.
%
No.
%
27
11.2
213
88.7
4
1.7
236
98.3
Male (174)
21
12.1
153
87.9
4
2.3
170
97.7
Female (66)
6
9.1
60
90.9
0
0
66
100
No. of subjects (240) Sex
WB; Western Blot. PCR; polymerase chain reaction. Pos; positive. Neg; negative. No.; number.
Table 2. Prevalence of asymptomatic Leishmania infection in Southern Europe. Prevalence/year a
Country Serology
PCR
Spain
T-cell memory response
Reference
53%, LST (2002)
Moral et al. [2]
General population (L’Alacantì region)
Garrote et al. [9]
General population (Castilla-Leon region) Blood donors (Balearic Islands)
5%, EIA (2004) 2% EIA, 8% WB (2004)
22% (2004)
22%, LST (2004)
Riera et al. [10]
6% (2008)
11%, LST (2008)
Riera et al.
3%, WB (2008) 8% (2015) 2%, EIA (2015) 0.1% IFA, 1% DAT (2019) France
0% (2019)
13%, WB (1999)
Italy 1%, IFA (2008)
7%, WB (2010)
36%, only IFApositiveb (2008) 4% (2010)
8% (2018) 0%, EIA (2005) Greece
15%, IFA+WB (2003) 3%, IFA (2008)
Study cohort
2% (2003)
21%, WBA (2019)
[11]
Blood donors (Balearic Islands)
Pérez-Cutillas. [12]
Blood donors (Murcia region)
IbarraMeneses et al. [13]
Healthy individuals (Madrid province)
Le Fichoux et al. [14]
Blood donors (Marseille province)
Scarlata et al. [15]
Blood donors (Sicily)
Biglino et al. [16]
General population (Piedmont)
Maritati et al. [17] Colomba et al. [18] Kyriakou et al. [19] Diza et al. [20]
Healthy individuals (Emilia-Romagna region) Blood donors (Sicily) Blood donors (Crete) General population (northern Greece) General population (Croatia)
SiskoKraljevic et al. [21] a Year of the article’s publication (only data published after 1999 are reported). b Only IFA positive samples were tested by PCR and leishmanial DNA was detected in 36% of this subgroup. DAT: direct agglutination test; EIA: enzyme immunoassay; IFA: immunofluorescent assay; LST: Leishmania skin test; Ref.: References; WB: Western Blotting; WBA: whole blood stimulation with soluble Leishmania antigen. Balkans
11%, EIA (2013)
Figure legend
Fig.1 Geographical location of the Valsamoggia municipality in the Bologna Province, northeastern Italy. The Valsamoggia municipality is depicted in red.
Asymptomatic Leishmania infantum infection in blood donors living in an endemic area, northeastern Italy Margherita Ortalli , Alessandra Mistral De Pascali , Serena Longo , Nadia Pascarelli , Andrea Porcellini , Deborah Ruggeri , Vanda Randi , Anna Procopio , Maria Carla Re , Stefania Varani PII: DOI: Reference:
S0163-4453(19)30291-9 https://doi.org/10.1016/j.jinf.2019.09.019 YJINF 4345
To appear in:
Journal of Infection
Accepted date:
13 September 2019
Please cite this article as: Margherita Ortalli , Alessandra Mistral De Pascali , Serena Longo , Nadia Pascarelli , Andrea Porcellini , Deborah Ruggeri , Vanda Randi , Anna Procopio , Maria Carla Re , Stefania Varani , Asymptomatic Leishmania infantum infection in blood donors living in an endemic area, northeastern Italy, Journal of Infection (2019), doi: https://doi.org/10.1016/j.jinf.2019.09.019
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd on behalf of The British Infection Association.
Highlights:
Most leishmanial infections remain asymptomatic in immunocompetent individuals
Leishmaniasis is endemic in the province of Bologna, northeastern Italy
We tested 240 blood donors in the Bologna province to detect asymptomatic Leishmania infection
Highly sensitive serological test, ie Western Blot, was employed in combination with a real time PCR assay
The prevalence of asymptomatic Leishmania infection in the Bologna province was 12.5 %
Asymptomatic Leishmania infantum infection in blood donors living in an endemic area, northeastern Italy
Margherita Ortalli1, Alessandra Mistral De Pascali1, Serena Longo1, Nadia Pascarelli2, Andrea Porcellini2, Deborah Ruggeri2, Vanda Randi2, Anna Procopio3, Maria Carla Re1,4* and Stefania Varani1,4 *
1
Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, 40100,
Italy 2
Centro Regionale Sangue Emilia-Romagna, Maggiore Hospital, Bologna, 40100, Italy
3
Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia-Romagna, Brescia, 25100,
Italy 4
Unit of Clinical Microbiology, Regional Reference Centre for Microbiological Emergencies
(CRREM), St. Orsola-Malpighi University Hospital, Bologna, 40100, Italy * These authors contributed equally to this work Correspondence to: Stefania Varani, St. Orsola-Malpighi University Hospital, Giuseppe Massarenti n. 9 40138, Bologna, tel. +39 051 2143013, email: [email protected] ORCID: 0000-0003-0862-4937 Declaration of interest: none Keywords: Asymptomatic Leishmania infection, blood donors, Western Blot, PCR
Abstract Objectives. Human leishmaniasis can be severe and fatal, yet in the Mediterranean region only a small percentage of infections progress to clinical disease. We evaluated the percentage of asymptomatic Leishmania infection in the Bologna province, northeastern Italy. Methods. We examined the presence of specific antibodies by Western Blot (WB) and parasitic DNA by real time PCR in peripheral blood of 240 blood donors residing in the Bologna province. Results. Anti-Leishmania IgG were detected by WB in 27 subjects (11.2%, 95% CI 7%-15%), while Leishmania kinetoplast DNA was detected in peripheral blood specimens of 4 out of 240 donors (1.7%, 95% CI 0.2%-3.2%). Overall, the prevalence of Leishmania infection in the blood donor cohort was 12.5%, thus indicating an elevated cumulative exposure to the Leishmania parasite in the examined municipality. Conclusions. Our results suggest that a surveillance system for monitoring Leishmania infection in blood donors and/or strategies of protozoan inactivation in whole blood should be taken into consideration in areas with circulation of the Leishmania parasite.
Introduction The Leishmania spp. protozoa are sandfly-borne intracellular parasites that are responsible for the leishmaniases, a complex of diseases with important clinical implication [1]. In humans, the major clinical forms are cutaneous, mucocutaneous and visceral leishmaniasis, the latter being fatal if untreated. Most leishmanial infections remain asymptomatic in immunocompetent individuals; for example, the ratio of subclinical to clinical cases of Leishmania infantum infection has been reported to be around 50:1 in Spain [2]. Human leishmaniasis is endemic in Italy, with an increase of cases in the last two decades due to disease spreading within traditionally endemic regions and to the appearance of autochthonous cases in previously non-endemic areas such as northern continental Italy [3-4]; as an example, an outbreak of visceral leishmaniasis has been recorded in the province of Bologna, northeastern Italy [5]. Since little is known on the circulation of the parasite in the abovementioned area, which was previously regarded as low risk area for leishmaniasis, we aimed to evaluate the prevalence of asymptomatic Leishmania infection in blood donors living in the Bologna province. Serum samples were evaluated for the presence of specific anti-Leishmania antibodies by Western Blot (WB) assay, while buffy coats from peripheral blood specimens were analyzed for the presence of Leishmania DNA by PCR.
Materials and methods Setting and study cohort We conducted a retrospective serological study in healthy Italian individuals volunteering for blood donation. The study cohort included blood donors residing in Valsamoggia, an Italian municipality counting 30,071 citizens in the Bologna province, Emilia-Romagna region (northeastern Italy, Fig.1). Between April 2014 and March 2015, peripheral blood samples were collected from 240 blood donors; 174 males and 66 females with a median age of 47 years (range 22-70). None of the donors had previously shown clinical manifestation of visceral or cutaneous leishmaniasis. All subjects enrolled in the study were asymptomatic at the time of blood donation, as requested by the Italian National Transfusion Network rules [6]. Blood samples were obtained from the Regional Blood Centre of Emilia-Romagna, Maggiore Hospital of Bologna, stored at 4°C and sent to the Unit of Microbiology (St. Orsola-Malpighi University Hospital, Bologna, Italy) within 5 days from the collection. Blood samples were centrifuged and both sera and buffy coats were stored at -80°C. The study was conducted in accordance with the Declaration of Helsinki. Generic written informed consent for storage and future use for research purpose was obtained from each blood donor before sampling. Blood samples were coded and retrospectively analyzed by serological and molecular assays. As samples were anonymized, no further consent to participate to this study could be requested to blood donors, according to the Italian Data Protection Authority (Authorization n. 9/2016). Serological detection of Leishmania infection Serological testing was performed by the Leishmania WESTERN BLOT IgG (LDBio Diagnostics®, Lyon, France). The WB technique provides detailed antibody responses to two antigens. In details, leishmanial 14-kDa and 16-kDa proteins of L. infantum are electrotransferred onto a nitrocellulose membrane and probed with patients’ serum. The positivity of the p14 and/or p16 bands is considered a sensitive and specific evidence of anti-Leishmania IgG in the tested serum as antibodies against p14 and/or p16 antigens were detected in 100% of patients with visceral
leishmaniasis and exhibited minimal cross-reactivity with antibodies directed against other infectious agents [7]. Molecular detection of Leishmania infection DNA was extracted from buffy coats using the QIAsymphony DSP Virus/Pathogen kit and the QIAsymphony SP/AS instrument, Qiagen® (Hilden, Germany). The nucleic acid extraction kit was employed according to the manufacturer’s instructions, while an in-house molecular method was used to detect leishmanial DNA in buffy coats from peripheral blood specimens. In details, nucleic acids were extracted from 300 µL buffy coat and DNA was eluted in 200µL of elution buffer and amplified employing a PCR targeting leishmanial kinetoplast (k)DNA [8]. Primers (15 pmol of RV1
5’-CTTTTCTGGTCCTCCGGGTAGG-3’,15
pmol
of
RV2
5’-
CCACCCGGCCCTATTTTACACCAA-3’) were synthesized by PrimmBiotech (Milan, Italy) and 50 pmol of TaqMan probe (FAM-TTTTCGCAGAACGCCCCTACCCGC-TAMRA) were synthesized by IDTDNA (Leuven, Belgium). β2-microglobulin real-time PCR assay was run simultaneously as a control of amplification of the extracted DNA. The real-time PCR assays were performed by employing the Real Time PCR Cycler Rotor Gene 3000 (Qiagen, Hilden Germany). Real time PCR was considered positive for Leishmania DNA when an amplification curve at threshold cycle (tC) < 40 was present. In case of positive PCR test, real time PCR procedure was repeated using new DNA extracts from the original specimen to rule out potential contamination; only samples with confirmed positive PCR results were considered true positive.
Results In total, 30 of 240 individuals tested positive at the serological and/or molecular screening for Leishmania infection. None of the individuals included in the study had a medical history of visceral or cutaneous leishmaniasis. Results by WB analysis showed the presence of specific antibody to 14 and/or 16 kDa proteins, indicative of anti-Leishmania IgG in 27 blood samples (11.2 %, 95% CI 7% -15%, Table 1). In two donors, sera were capable of detecting both bands, while antibodies against p14 or p16 were found in 9 and 16 serum samples, respectively. Circulating Leishmania kDNA was detected in buffy coats from 4 out of 240 blood donors (1.7%, 95% CI 0.2%-3.2%). Median tC value for the PCR positive samples was 37.5 (range, 37.0-39.8). Only one donor showed simultaneously a positivity by serological (WB reactivity to p14) and molecular methods employed in the study. Overall, the prevalence of asymptomatic Leishmania infection in the studied population was 12.5 % (95% CI 8.3%-16.7%). No significant difference was observed by age or sex in the prevalence rate of asymptomatic Leishmania infection (Table 1 and data not shown).
Discussion In this study, we screened blood samples from 240 donors living in an area where L. infantum is endemic and we detected an asymptomatic Leishmania infection in 12.5% of tested individuals, which indicates an important cumulative exposure to the parasite in the examined municipality. Leishmania-specific antibodies were revealed by WB in serum samples obtained from 27 donors, which showed antibody to 14 and/or 16 kDa proteins. We considered a WB test as positive if one of these bands was is present; in fact, anti-p14 and anti-p16 showed less than 2% cross-reactivity with antibodies directed against other infectious agents [7]. On the other hand, the presence of the parasite was only detected in blood samples of 4 donors (1.7%) by kDNA amplification, suggesting low levels of circulating parasites. Epidemiological studies from Mediterranean countries show a highly variable prevalence (from <1 0% to 53%) of asymptomatic Leishmania infection [4 and Table 2], probably due to a fluctuating circulation of the parasite in different areas and to a diversity of methods employed for testing; commonly used serological methods, such as immunoenzymatic tests, direct agglutination test and immunofluorescence tests can be inaccurate and underestimate the infection rates in asymptomatic carriers. On the other hand, the leishmanin skin test can specifically detect T-cell mediated responses with high sensitivity, but antigen preparation has not been standardized for universal use [3]. As using a combination of tests has been reported to increase the capacity to detect asymptomatic Leishmania infection [1], we employed a highly sensitive serological test, ie WB [7] in combination with a real time PCR assay. In this study, no significant difference was observed by sex in the prevalence rate of asymptomatic Leishmania infection, while clinical disease is overall predominant in men [1] including the study area [5]; our findings contradict the assumption that higher frequency of visceral leishmaniasis in male than female reflects an increased exposure of men to sandflies. Thus, factors other than sandfly exposure should be considered in triggering progression from asymptomatic infection to overt disease.
Our results are in line with previous evidence reporting that many cases of Leishmania infection are asymptomatic in the Mediterranean Europe [2, 4, 10-12, 14, 16, 17, 19, 21, 22]. Some patients with subclinical infection can harbor viable parasites throughout life and may develop reactivation to full-blown visceral leishmaniasis if immunosuppression occurs thereafter [23]. Visceral and cutaneous leishmaniasis are compulsory notifiable diseases in Italy and confirmed cases are obligatorily reported by local public health departments to the regional health authorities. As no leishmaniasis cases were reported to the Regional Health Authority among blood donors from the Emilia-Romagna region (around 4,500,000 inhabitants) up to August 2019, we assume that none of the asymptomatic leishmanial infection that we detected in blood donors in 2014-2015 progressed to clinical disease. Cases of possible L.infantum transmission by blood donation have been reported. However, pre-storage leukodepletion, which is mandatory in Italy since January 2016, is greatly effective in reducing the risk of Leishmania transmission by parasitemic blood donors [24]. Strategies that reduce live parasites in blood supplies are under study and should be taken into consideration in endemic areas. Several methods for inactivation of Leishmania parasites such as riboflavin and ultraviolet light [25], amatosalen and UVA illumination [26], and thiazole orange [27] have been shown to be effective when applied to different blood components in in vitro studies. In addition, the employment of amatosalen and ultraviolet light as well as riboflavin and ultraviolet light effectively eliminated leishmanial DNA in apheresis platelet units from single asymptomatic infected blood donors [28, 29]. However, these findings need to be confirmed by further studies before the above-mentioned strategies could be considered for routine use. This study has some limitations. Firstly, the examined cohort exclusively comprised healthy adults, thus the results cannot be generalized to the overall population of the Emilia-Romagna region (Italy), secondly, the study was conducted on a limited number of blood donors. As a third point, no gold standard method is currently available to detect asymptomatic leishmanial infection [3], thus rendering difficult to compare results from different studies on this matter.
Even if our study was conducted in a limited number of blood donors samples and no symptomatic Leishmania infection has been so far recorded in subjects who received blood products in Italy, the geographic spread of Leishmania suggests that a surveillance system for monitoring this parasitic infection in blood donors and/or strategies that inactivate the parasite in whole blood should be taken into consideration in endemic areas.
Acknowledgments This work was supported by Lab P3 funds from the Emilia-Romagna Region (Italy) and by RFO 2010-2016 funds from the University of Bologna. We thank Giorgio Galletti and Annalisa Santi for support in preparation of the study area map.
Conflict of interest The authors declare no conflict of interest.
References 1. WHO. Control of the leishmaniases: report of a meeting of the WHO Expert Committee on the
Control
of
Leishmaniases,
Geneva,
22-26
March
2010.
Available
from:
http://apps.who.int/iris/bitstream/10665/44412/1/WHO_TRS_949_eng.pdf 2. Moral L, Rubio E M, Moya M. A leishmanin skin test survey in the human population of l’Alicanti region (Spain): implications for the epidemiology of Leishmania infantum infection in southern Europe. Trans R Soc Trop Med Hyg 2002; 96: 129-32. 3. Gradoni L, Rogelio L V, Mourad M. Regional Office for Europe. Manual on case management and surveillance of the leishmaniases in the WHO European Region. WHO 2017. Available from: http://www.euro.who.int/en/publications/abstracts/manual-on-casemanagement-and-surveillance-of-the-leishmaniases-in-the-who-european-region-2017 4. Moriconi M, Rugna G, Calzolari M, Bellini R, Albieri A, Angelini P, et al. Phlebotomine sand-fly borne pathogens in the Mediterranean Basin: Human leishmaniasis and phlebovirus infections. PLoS Negl Trop Dis 2017; 11: e0005660. 5. Varani S, Cagarelli R, Melchionda F, Attard L, Salvadori C, Finarelli A C, et al. Ongoing outbreak of visceral leishmaniasis in Bologna Province, Italy, November 2012 to May 2013. Euro Surveill 2013;18: 20530. 6. Ministerial Decree DM, November 2, 2015, “Disposizioni relative ai requisiti di qualità e sicurezza del sangue e degli emocomponenti” . Available from: https://www.avis.it/userfiles/file/GU%20SG%20n_300%20del%2028-122015_SO_069%20(1).pdf 7. Mary C, Lamouroux D, Dunan S, Quilici M. Western blot analysis of antibodies to Leishmania infantum antigens: potential of the 14-kD and 16-kD antigens for diagnosis and epidemio- logic purposes. Am J Trop Med Hyg 1992;47: 764-71.
8. Mary C, Faraut F, Lascombe L, Dumon H. Quantification of Leishmania infantum DNA by a Real-Time PCR Assay with High Sensitivity. J Clin Microbiol 2004; 42: 5249–5255. 9. Garrote J I, Gutiérrez M P, Izquierdo R L, Dueñas M A, Zarzosa P, Cañavate C, et al. Seroepidemiologic study of Leishmania infantum infection in Castilla-Leon, Spain. Am J Trop Med Hyg 2004;71: 403-6. 10. Riera C, Fisa R, Udina M, Gállego 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: 102-10. 11. Riera C, Fisa R, López-Chejade P, Serra T, Girona E, Jiménez M, et al. Asymptomatic infection by Leishmania infantum in blood donors from the Balearic Islands (Spain). Transfusion 2008;48: 1383-9. 12. Pérez-Cutillas P, Goyena E, Chitimia L, De la Rúa P, Bernal L J, Fisa R, et al. Spatial distribution of human asymptomatic Leishmania infantum infection in southeast Spain: a study of environmental, demographic and social risk factors. Acta Trop 2015;146:127-34. 13. Ibarra-Meneses A V, Carrillo E, Nieto J, Sánchez C, Ortega S, Estirado A, et al. Prevalence of asymptomatic Leishmania infection and associated risk factors, after an outbreak in the south-western Madrid region, Spain, 2015. Euro Surveill 2019;24. 14. Le Fichoux Y, Quaranta J F, Aufeuvre J P, Lelievre A, Marty P, Suffia I, 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: 1953-7. 15. Scarlata F, Vitale F, Saporito L, Reale S, Vecchi V L, Giordano S, et al. Asymptomatic Leishmania infantum/chagasi infection in blood donors of western Sicily. Trans R Soc Trop Med Hyg 2008;102: 394-6.
16. Biglino A, Bolla C, Concialdi E, Trisciuoglio A, Romano A, Ferroglio E. Asymptomatic Leishmania infantum infection in an area of northwestern Italy (Piedmont region) where such infections are traditionally nonendemic. J Clin Microbiol 2010;48: 131-6. 17. Maritati M, Trentini A, Michel G, Bellini T, Almugadam S, Hanau S, et al. Subclinical Leishmania infection in patients with rheumatic diseases under biological drugs. Infection 2018;46: 801-809. 18. Colomba C, Saporito L, Polara V F, Barone T, Corrao A, Titone L. Serological screening for Leishmania infantum in asymptomatic blood donors living in an endemic area (Sicily, Italy). Transfus Apher Sci 2005;33: 311-4. 19. Kyriakou D S, Alexandrakis M G, Passam F H, Kourelis T V, Foundouli P, Matalliotakis E, et al. Quick detection of Leishmania in peripheral blood by flow cytometry. Is prestorage leucodepletion necessary for leishmaniasis prevention in endemic areas? Transfus Med 2003;13: 59-62. 20. Diza E, Kansouzidou A, Gerou S, Vezyri E, Metallidis S, Antoniadis A. Leishmaniases in Northern Greece: seroprevalence of the infection and incidence of the disease during the period 2001-2006. Eur J Clin Microbiol Infect Dis 2008;27: 997-1003. 21. Šiško-Kraljević K, Jerončić A, Mohar B, Punda-Polić V. Asymptomatic Leishmania infantum infections in humans living in endemic and non-endemic areas of Croatia, 2007 to 2009. Euro Surveill 2013;18: 20533. 22. Ferroglio E, Battisti E, Zanet S, Bolla C, Concialdi E, Trisciuoglio A, et al. Epidemiological evaluation of Leishmania infantum zoonotic transmission risk in the recently established endemic area of Northwestern Italy. Zoonoses Public Health 2018; 65: 675-682. 23. van Griensven J, Carrillo E, López-Vélez R, Lynen L, Moreno J Leishmaniasis in immunosuppressed individuals. Clin Microbiol Infect 2014;20: 286-99.
24. Jimenez-Marco T, Fisa R, Girona-Llobera E, Cancino-Faure B, Tomàs-Pèrez M, Berenguer D, et al. Transfusion transmitted Leishmaniasis: a practical review. Transfusion 2016; 26: 45-51. 25. Cardo L J, Rentas F J, Ketchum L, Salata J, Harman R, Melvin W, et al. Pathogen inactivation of Leishmania donovani infantum in plasma and platelet concentrates using riboflavin and ultraviolet light. Vox Sang 2006;90: 85-91. 26. Eastman R T, Barrett L K, Dupuis K, Buckner F S, Van Voorhis W C. Leishmania inactivation in human pheresis platelets by a psoralen (amotosalen HCI) and longwavalength ultraviolet irradiation. Transfusion 2005;45: 1459-63. 27. Wagner S J, Skrichenko A, Salata J, O’Sullivan A M, Cardo L J. Inactivation of Leishmania donovani infantum and Trypanosoma cruzi in red cell suspensions with thiazole orange. Transfusion 2008; 48: 1363-7. 28. Jimenez-Marco T, Fisa R, Riera C, Girona-Llobera E, Sedeno M, Saura A, et al. Pathogen inactivation technology applied to a blood component collected from an asymptomatic carrier of Leishmania infantum: a case report. Vox Sang 2012; 103: 356-8. 29. Jimenez-Marco T, Riera C, Fisa R, Girona-Llobera E, Sedeno M, Goodrich R P, et al. The utility of pathogen inactivation technology: a real-life example of Leishmania infantum inactivation in platelets from a donor with an asymptomatic infection. Blood Transfus 2012; 10: 536-41.
1. Table 1. Rates of asymptomatic Leishmania infection among blood donors (n=240) residing in the Bologna Province (northeastern Italy) by Western Blot and real time PCR.
Variables
Results WB Pos
WB Neg
PCR Pos
PCR Neg
No.
%
No.
%
No.
%
No.
%
27
11.2
213
88.7
4
1.7
236
98.3
Male (174)
21
12.1
153
87.9
4
2.3
170
97.7
Female (66)
6
9.1
60
90.9
0
0
66
100
No. of subjects (240) Sex
WB; Western Blot. PCR; polymerase chain reaction. Pos; positive. Neg; negative. No.; number.
Table 2. Prevalence of asymptomatic Leishmania infection in Southern Europe. Prevalence/year a
Country Serology
PCR
Spain
T-cell memory response
Reference
53%, LST (2002)
Moral et al. [2]
General population (L’Alacantì region)
Garrote et al. [9]
General population (Castilla-Leon region) Blood donors (Balearic Islands)
5%, EIA (2004) 2% EIA, 8% WB (2004)
22% (2004)
22%, LST (2004)
Riera et al. [10]
6% (2008)
11%, LST (2008)
Riera et al.
3%, WB (2008) 8% (2015) 2%, EIA (2015) 0.1% IFA, 1% DAT (2019) France
0% (2019)
13%, WB (1999)
Italy 1%, IFA (2008)
7%, WB (2010)
36%, only IFApositiveb (2008) 4% (2010)
8% (2018) 0%, EIA (2005) Greece
15%, IFA+WB (2003) 3%, IFA (2008)
Study cohort
2% (2003)
21%, WBA (2019)
[11]
Blood donors (Balearic Islands)
Pérez-Cutillas. [12]
Blood donors (Murcia region)
IbarraMeneses et al. [13]
Healthy individuals (Madrid province)
Le Fichoux et al. [14]
Blood donors (Marseille province)
Scarlata et al. [15]
Blood donors (Sicily)
Biglino et al. [16]
General population (Piedmont)
Maritati et al. [17] Colomba et al. [18] Kyriakou et al. [19] Diza et al. [20]
Healthy individuals (Emilia-Romagna region) Blood donors (Sicily) Blood donors (Crete) General population (northern Greece) General population (Croatia)
SiskoKraljevic et al. [21] a Year of the article’s publication (only data published after 1999 are reported). b Only IFA positive samples were tested by PCR and leishmanial DNA was detected in 36% of this subgroup. DAT: direct agglutination test; EIA: enzyme immunoassay; IFA: immunofluorescent assay; LST: Leishmania skin test; Ref.: References; WB: Western Blotting; WBA: whole blood stimulation with soluble Leishmania antigen. Balkans
11%, EIA (2013)
Figure legend
Fig.1 Geographical location of the Valsamoggia municipality in the Bologna Province, northeastern Italy. The Valsamoggia municipality is depicted in red.