title: Parasitological Confirmation and Analysis of Leishmania Diversity in Asymptomatic and Subclinical Infection following Resolution of Cutaneous Leishmaniasis.
abstract: BACKGROUND
  The contribution of individuals with subclinical infection to the transmission and endemicity of cutaneous leishmaniasis (CL) is unknown. Immunological evidence of exposure to Leishmania in residents of endemic areas has been the basis for defining the human population with asymptomatic infection. However, parasitological confirmation of subclinical infection is lacking.
  
  
  METHODS
  We investigated the presence and viability of Leishmania in blood and non-invasive mucosal tissue samples from individuals with immunological evidence of subclinical infection in endemic areas for CL caused by Leishmania (Viannia) in Colombia. Detection of Leishmania kDNA was conducted by PCR-Southern Blot, and parasite viability was confirmed by amplification of parasite 7SLRNA gene transcripts. A molecular tool for genetic diversity analysis of parasite populations causing persistent subclinical infection based on PCR amplification and sequence analysis of an 82bp region between kDNA conserved blocks 1 and 2 was developed.
  
  
  RESULTS
  Persistent Leishmania infection was demonstrated in 40% (46 of 114) of leishmanin skin test (LST) positive individuals without active disease; parasite viability was established in 59% of these (27 of 46; 24% of total). Parasite burden quantified from circulating blood monocytes, nasal, conjunctival or tonsil mucosal swab samples was comparable, and ranged between 0.2 to 22 parasites per reaction. kDNA sequences were obtained from samples from 2 individuals with asymptomatic infection and from 26 with history of CL, allowing genetic distance analysis that revealed diversity among sequences and clustering within the L. (Viannia) subgenus.
  
  
  CONCLUSIONS
  Our results provide parasitological confirmation of persistent infection among residents of endemic areas of L. (Viannia) transmission who have experienced asymptomatic infection or recovered from CL, revealing a reservoir of infection that potentially contributes to the endemicity and transmission of disease. kDNA genotyping establishes proof-of-principle of the feasibility of genetic diversity analysis in previously inaccessible and unexplored parasite populations in subclinically infected individuals.
authors.affiliation: Centro Internacional de Entrenamiento e Investigaciones Médicas-CIDEIM, Cali, Colombia.
authors.forename: Mariana
authors.initials: M
authors.lastname: Rosales-Chilama
authors.affiliation: Centro Internacional de Entrenamiento e Investigaciones Médicas-CIDEIM, Cali, Colombia.
authors.forename: Rafael E
authors.initials: RE
authors.lastname: Gongora
authors.affiliation: Centro Internacional de Entrenamiento e Investigaciones Médicas-CIDEIM, Cali, Colombia.
authors.forename: Liliana
authors.initials: L
authors.lastname: Valderrama
authors.affiliation: Centro Internacional de Entrenamiento e Investigaciones Médicas-CIDEIM, Cali, Colombia.
authors.forename: Jimena
authors.initials: J
authors.lastname: Jojoa
authors.affiliation: Centro Internacional de Entrenamiento e Investigaciones Médicas-CIDEIM, Cali, Colombia.
authors.forename: Neal
authors.initials: N
authors.lastname: Alexander
authors.affiliation: Centro Internacional de Entrenamiento e Investigaciones Médicas-CIDEIM, Cali, Colombia.
authors.forename: Luisa C
authors.initials: LC
authors.lastname: Rubiano
authors.affiliation: Centro Internacional de Entrenamiento e Investigaciones Médicas-CIDEIM, Cali, Colombia.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Cossio
authors.affiliation: Liverpool School of Tropical Medicine, Centre for Applied Health Research, Liverpool, United Kingdom.
authors.forename: Emily R
authors.initials: ER
authors.lastname: Adams
authors.affiliation: Centro Internacional de Entrenamiento e Investigaciones Médicas-CIDEIM, Cali, Colombia.
authors.forename: Nancy G
authors.initials: NG
authors.lastname: Saravia
authors.affiliation: Centro Internacional de Entrenamiento e Investigaciones Médicas-CIDEIM, Cali, Colombia.
authors.forename: María Adelaida
authors.initials: MA
authors.lastname: Gomez
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D058345:Asymptomatic Infections; D015139:Blotting, Southern; D002648:Child; D016000:Cluster Analysis; D003105:Colombia; D018509:DNA, Helminth / Q000235:genetics; D018105:DNA, Kinetoplast / Q000737:chemistry / Q000235:genetics; D005260:Female; D014644:Genetic Variation; D005838:Genotype; D006801:Humans; D007891:Leishmania / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D016773:Leishmaniasis, Cutaneous / Q000469:parasitology*; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D020733:RNA, Small Cytoplasmic / Q000032:analysis / Q000235:genetics; D017422:Sequence Analysis, DNA; D018271:Signal Recognition Particle / Q000032:analysis / Q000235:genetics; D055815:Young Adult
chemical_list: C115990:7SL RNA; D018509:DNA, Helminth; D018105:DNA, Kinetoplast; D020733:RNA, Small Cytoplasmic; D018271:Signal Recognition Particle