Abstract
With the emergence of leishmaniasis in new regions around the world, molecular epidemiological methods with adequate discriminatory power, reproducibility, high throughput and inter-laboratory comparability are needed for outbreak investigation of this complex parasitic disease. As multilocus sequence analysis (MLSA) has been projected as the future gold standard technique for Leishmania species characterization, we propose a MLSA panel of six housekeeping gene loci (6pgd, mpi, icd, hsp70, mdhmt, mdhnc) for investigating intraspecific genetic variation of L. (Viannia) braziliensis strains and compare the resulting genetic clusters with several epidemiological factors relevant to outbreak investigation. The recent outbreak of cutaneous leishmaniasis caused by L. (V.) braziliensis in the southern Brazilian state of Santa Catarina is used to demonstrate the applicability of this technique. Sequenced fragments from six genetic markers from 86 L. (V.) braziliensis strains from twelve Brazilian states, including 33 strains from Santa Catarina, were used to determine clonal complexes, genetic structure, and phylogenic networks. Associations between genetic clusters and networks with epidemiological characteristics of patients were investigated. MLSA revealed epidemiological patterns among L. (V.) braziliensis strains, even identifying strains from imported cases among the Santa Catarina strains that presented extensive homogeneity. Evidence presented here has demonstrated MLSA possesses adequate discriminatory power for outbreak investigation, as well as other potential uses in the molecular epidemiology of leishmaniasis.
Highlights
Leishmaniasis, a vector-borne disease caused by protozoan parasites of genus Leishmania [1], represents one of the highest disease burdens among the neglected tropical diseases in developing nations [2]
In Brazil, beginning in 2005, an outbreak of human cutaneous leishmaniasis occurred in the southern Brazilian state of Santa Catarina, where the disease had not been observed previously as endemic
Many questions still remain regarding the outbreak, such as: is one main strain or various strains responsible for the outbreak; is the emergence of L. (V.) braziliensis in the region a recent event; and how are Santa Catarina strains related to other strains in Brazil? A wide range of molecular tools are available for the investigation of molecular epidemiology of leishmaniasis, but choosing which method and/or markers to use continues to be a challenge [12]
Summary
Leishmaniasis, a vector-borne disease caused by protozoan parasites of genus Leishmania [1], represents one of the highest disease burdens among the neglected tropical diseases in developing nations [2]. (Leishmania) donovani species complex [13,14], commonly employs multilocus microsatellite typing (MLMT) This technique has been proven to discriminate at the intra-species level [15] with high discriminatory power and is useful for determining outbreak strain origin when a database of MLMT strains is available for the Leishmania species of interest [13]. Dividing the isolates into many different genotypes reduces the statistical power of analyses involving epidemiological variables, such as clinical and demographic characteristics of the patient. Such reductions in statistical power greatly reduce the ability of researcher to conclude the relationship of factors like clinical form and disease virulence with a particular genotype
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