Multilocus microsatellite typing of Leishmania infantum isolates in monitored Leishmania/HIV coinfected patients

Particle and Fibre Toxicology

Roser Fisa³  Montserrat Portús³  Esteve Ribera¹  Anne-Laure Bañuls²  Liliana Montoya³  Cristina Riera³  Mallorie Hide²  Míriam Tomás-Pérez³ 

[1] Infectious Diseases Department and Microbiology Department, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain;MIVEGEC, UMR IRD 224-CNRS 5290-Université de Montpellier, Montpellier, France;Laboratory of Parasitology, Faculty of Pharmacy, Universitat de Barcelona, Avda Joan XXIII s/n, Barcelona, 08028, Spain

关键词: Reinfection;    Relapse;    Phenetic analyses;    Genetic diversity;    Microsatellites;    HIV+;    Leishmania infantum;   

Others  :  1222198
DOI  :  10.1186/s13071-015-0989-9

 received in 2015-02-10, accepted in 2015-07-06,  发布年份 2015

【 摘 要 】

Background

Leishmania infantum is the main etiological agent of both visceral and cutaneous clinical forms of leishmaniasis in the Mediterranean area. Leishmania/HIV coinfection in this area is characterized by a chronic course and frequent recurrences of clinical episodes. The present study using Multilocus Microsatellite Typing (MLMT) analysis, a highly discriminative tool, aimed to genetically characterize L. infantum isolates taken from monitored Leishmania/HIV coinfected patients presenting successive clinical episodes.

Methods

In this study, by the analysis of 20 microsatellite loci, we studied the MLMT profiles of 25 L. infantum isolates from 8 Leishmania/HIV coinfected patients who had experienced several clinical episodes. Two to seven isolates per patient were taken before and after treatment, during clinical and non-clinical episodes, with time intervals of 6 days to 29 months. Genetic diversity, clustering and phenetic analyses were performed.

Results

MLMT enabled us to study the genetic characteristics of the 25 L. infantum isolates, differentiating 18 genotypes, corresponding to a genotypic diversity of 0.72. Fifteen genotypes were unique in the total sample set and only 3 were repeated, 2 of which were detected in different patients. Both clustering and phylogenetic analyses provided insights into the genetic links between the isolates; in five patients isolates showed clear genetic links: either the genotype was exactly the same or only slightly different. In contrast, the isolates of the other three patients were dispersed in different clusters and some could be the result of mixing between populations.

Conclusions

Our data indicated a great MLMT variability between isolates from coinfected patients and no predominant genotype was observed. Despite this, almost all clinical episodes could be interpreted as a relapse rather than a reinfection. The results showed that diverse factors like an intrapatient evolution over time or culture bias could influence the parasite population detected in the patient, making it difficult to differentiate between relapse and reinfection.

【 授权许可】

   
2015 Tomás-Pérez et al.

【 图 表 】

Fig. 1.

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【 参考文献 】

• [1]Alvar J, Aparicio P, Aseffa A, Den Boer M, Cañavate C, Dedet JP, Gradoni L, Ter Horst R, López-Vélez R, Moreno J. The relationship between leishmaniasis and AIDS: the second 10 years. Clin Microbiol Rev. 2008; 21:334-59.
• [2]Control of the Leishmaniases. In: WHO Technical Report Series. World Health Organization, Geneva; 2010: p.949.
• [3]Morales MA, Cruz I, Rubio JM, Chicharro C, Cañavate C, Laguna F, Alvar J. Relapses versus reinfections in patients coinfected with Leishmania infantum and human immunodeficiency virus type 1. J Infect Dis. 2002; 185:1533-7.
• [4]Kuhls K, Chicharro C, Cañavate C, Cortés S, Campino L, Haralambous C, Soteriadou K, Pratlong F, Dedet JP, Mauricio I, Miles M, Schaar M, Ochsenreither S, Radtke O, Schönian G. Differentiation and gene flow among European populations of Leishmania infantum MON-1. PLoS Negl Trop Dis. 2008; 2: Article ID e261
• [5]Gelanew T, Kuhls K, Hurissa Z, Weldegebreal T, Hailu W, Kassahun A, Abebe T, Hailu A, Schönian G. Inference of population structure of Leishmania donovani strains isolated from different Ethiopian visceral leishmaniasis endemic areas. PloS Negl Trop Dis. 2010; 4: Article ID e889
• [6]Pratlong F, Dereure J, Deniau M, Marty P, Faraut-Gambarelli F, Dedet JP. Enzymatic polymorphism during Leishmania/HIV co-infection: a study of 381 Leishmania strains received between 1986 and 2000 at the international cryobank in Montpellier France. Ann Trop Med Parasitol. 2003; 97:47-56.
• [7]Gállego M, Pratlong F, Riera C, Muñoz C, Ribera E, Fisa R, Rioux JA, Dedet JP, Portús M. Isoenzymatic identification of Leishmania isolates from repeated clinical human leishmaniasis episodes in Catalonia (Spain). Trans R Soc Trop Med Hyg. 2002; 96:45-7.
• [8]Chicharro C, Jiménez MI, Alvar J. Iso-enzymatic variability of Leishmania infantum in Spain. Ann Trop Med Parasitol. 2003; 97:57-64.
• [9]Martín-Sánchez J, Gramiccia M, Di Muccio T, Ludovisi A, Morillas-Márquez F. Isoenzymatic polymorphism of Leishmania infantum in southern Spain. Trans R Soc Trop Med Hyg. 2004; 98:228-32.
• [10]Toth G, Gaspari Z, Jurka J. Microsatellites in different eukaryotic genomes: survey and analysis. Genome Res. 2000; 10:967-81.
• [11]Ochsenreither S, Kuhls K, Schaar M, Presber W, Schönian G. Multilocus microsatellite typing as a new tool for discrimination of Leishmania infantum MON-1 strains. J Clin Microbiol. 2006; 44:495-503.
• [12]Schönian G, Kuhls K, Mauricio IL. Molecular approaches for a better understanding of the epidemiology and population genetics of Leishmania. Parasitology. 2011; 138:405-25.
• [13]Piarroux R, Azaiez R, Lossi AM, Reynier P, Muscatelli F, Gambarelli F, Fontes M, Dumon H, Quilici M. Isolation and characterization of a repetitive DNA sequence from Leishmania infantum: development of a visceral leishmaniasis polymerase chain reaction. Am J Trop Med Hyg. 1993; 49:364-9.
• [14]Fisa R, Riera C, Ribera E, Gállego M, Portús M. A nested polymerase chain reaction for diagnosis and follow-up of human visceral leishmaniasis patients using blood samples. Trans R Soc Trop Med Hyg. 2002; 96:191-4.
• [15]Goudet J, Perrin N, Waser P. Tests for sex-biased dispersal using bi-parentally inherited genetic markers. Mol Ecol. 2002; 11:1103-14.
• [16]Goudet J. FSTAT (Version 1.2): a computer program to calculate F-statistics. J Hered. 1995; 86:485-6.
• [17]Nei M, Chesser RK. Estimation of fixation indices and gene diversities. Ann Hum Genet. 1983; 47:253-9.
• [18]Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987;4:406-25.
• [19]Chevenet F, Brun C, Bañuls AL, Jacq B, Christen R. TreeDyn: towards dynamic graphics and annotations for analyses of trees. BMC Bioinformatics. 2006;7:439.
• [20]Pritchard JK, Stephens M, Donnelly P. Inference of population structure using multilocus genotype data. Genetics. 2000; 155:945-59.
• [21]Evanno G, Regnaut S, Goudet J. Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Mol Ecol. 2005; 14:2611-20.
• [22]Garnier S, Alibert P, Audiot P, Prieur B, Rasplus JY. Isolation by distance and sharp discontinuities in gene frequencies: implications for the phylogeography of an alpine insect species, Carabussolieri. Mol Ecol. 2004; 13:1883-97.
• [23]Chicharro C, Alvar J. Lower trypanosomatids in HIV/AIDS patients. Ann Trop Med Parasitol. 2003; 97:75-8.
• [24]Hide M, Marion E, Pomares C, Fisa R, Marty P, Bañuls AL. Parasitic genotypes appear to differ in leishmaniasis patients compared with asymptomatic related carriers. Int J Parasitol. 2013; 43:389-97.
• [25]Cortés S, Maurício IL, Kuhls K, Nunes M, Lopes C, Marcos M, Cardoso L, Schönian G, Campino L. Genetic diversity evaluation on Portuguese Leishmania infantum strains by multilocus microsatellite typing. Infect Genet Evol. 2014; 26:20-31.
• [26]Montoya L, Gállego M, Gavignet B, Piarroux R, Rioux JA, Portús M, Fisa R. Application of microsatellite genotyping to the study of a restricted Leishmania infantum focus: different genotype compositions in isolates from dogs and sand flies. Am J Trop Med Hyg. 2007; 76:888-95.
• [27]Campino L, Santos-Gomes G, Pratlong F, Dedet JP, Abranches P. The isolation of Leishmania donovani MON-18, from an AIDS patient in Portugal: possible needle transmission. Parasite. 1994; 1:391-2.
• [28]Cruz I, Morales MA, Noguer I, Rodríguez A, Alvar J. Leishmania in discarded syringes from intravenous drug users. Lancet. 2002; 359:1124-5.
• [29]Gouzelou E, Haralambous C, Antoniou M, Christodoulou V, Martinković F, Živičnjak T, Smirlis D, Pratlong F, Dedet JP, Özbel Y, Toz SÖ, Presber W, Schönian G, Soteriadou K. Genetic diversity and structure in Leishmania infantum populations from southeastern Europe revealed by microsatellite analysis. Parasit Vectors. 2013; 5:342. BioMed Central Full Text
• [30]Molina I, Fisa R, Riera C, Falcó V, Elizalde A, Salvador F, Crespo M, Curran A, López-Chejade P, Tebar S, Pérez-Hoyos S, Ribera E, Pahissa A. Ultrasensitive real-time PCR for the clinical management of visceral leishmaniasis in HIV-Infected patients. Am J Trop Med Hyg. 2013; 89:105-10.
• [31]Motoie G, Ferreira GE, Cupolillo E, Canavez F, Pereira-Chioccola VL. Spatial distribution and population genetics of Leishmania infantum genotypes in São Paulo State, Brazil, employing multilocus microsatellite typing directly in dog infected tissues. Infect Genet Evol. 2013; 18:48-59.
• [32]Bulle B, Millon L, Bart JM, Gállego M, Gambarelli F, Portús M, Schnur L, Jaffe CL, Fernandez-Barredo S, Alunda JM, Piarroux R. Practical approach for typing strains of Leishmania infantum by microsatellite analysis. J Clin Microbiol. 2002; 40:3391-7.
• [33]Jamjoom MB, Ashford RW, Bates PA, Kemp SJ, Noyes HA. Towards a standard battery of microsatellite markers for the analysis of the Leishmania donovani complex. Ann Trop Med Parasitol. 2002; 96:265-70.
• [34]Rossi V, Wincker P, Ravel C, Blaineau C, Pages M, Bastien P. Structural organization of microsatellite families in the Leishmania genome and polymorphisms at two (CA)n loci. Mol Biochem Parasitol. 1994; 65:271-82.