Parasitological Confirmation and Analysis of Leishmania Diversity in Asymptomatic and Subclinical Infection following Resolution of Cutaneous Leishmaniasis.

Mariana Rosales-Chilama,Nancy G Saravia,Luisa C Rubiano,Liliana Valderrama,María Adelaida Gomez,Jimena Jojoa,Emily R Adams,Neal Alexander,Alexandra Cossio,Rafael E Gongora,Paul Andrew Bates

doi:10.1371/journal.pntd.0004273

Abstract

BackgroundThe 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.MethodsWe 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.Principal FindingsPersistent 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.ConclusionsOur 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.

Highlights

Asymptomatic dermal or visceral leishmaniasis (VL) constitute a variable and sometimes high proportion of the naturally exposed population in endemic foci of Leishmania transmission, ranging from 17 to 91% of incident infections [1,2,3]
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 cutaneous leishmaniasis (CL) caused by Leishmania (Viannia) in Colombia
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

Summary

Introduction

Asymptomatic dermal or visceral leishmaniasis (VL) constitute a variable and sometimes high proportion of the naturally exposed population in endemic foci of Leishmania transmission, ranging from 17 to 91% of incident infections [1,2,3]. Parasite persistence and viability have been demonstrated after treatment and clinical resolution of disease [8,9], supporting the possibility that subclinically infected individuals can accumulate to constitute an important, unrecognized proportion of the population in endemic foci. Re-activation of prior infection, whether clinically apparent or subclinical, is a potential source of incident disease. The contribution of individuals with subclinical infection to the transmission and endemicity of cutaneous leishmaniasis (CL) is unknown.

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Conclusion