Identification of Clinical Infections of Leishmania Imported into Australia: Revising Speciation with Polymerase Chain Reaction-RFLP of the Kinetoplast Maxicircle

Abstract

Leishmaniasis is a vector-borne disease caused by protozoan parasites of the Leishmania genus. In Australia, leishmaniasis is an imported disease that is presenting itself at increased rates because of international travel, the influx of immigrants, and deployment of military operations to endemic regions. Although Leishmania species are morphologically indistinguishable, there is a strong correlation between some causative species of leishmaniasis and the subsequent response to the treatments available and patient outcome. Consequently, identification of the infective species is imperative as misidentification can result in the administering of an ineffective drug. The aim of this study was to develop a simple diagnostic tool with high sensitivity and specificity, which is capable of detecting the presence of the parasite and accurately differentiating the causative species in question. Using the advantageous properties of the maxi-circle kinetoplast DNA, a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) targeting the ND7 gene was developed for the analysis of imported cases of human leishmaniasis in Australia. Designed as a dual analysis, concurrent PCR of Leishmania maxi-circle DNA and digestion with two separate enzymes (NlaIII and HpyCH4IV), this study provides an appraisal on 24 imported cases of leishmaniasis between 2008 and 2017. Five Leishmania species were reported, with members of the Viannia subgenus being the most common. The implementation of novel diagnostic procedures for leishmaniasis such as the one reported here is needed to establish a gold standard practice for the diagnosis and treatment of leishmaniasis.

Laboratory diagnostics for human Leishmania infections: a polymerase chain reaction-focussed review of detection and identification methods

“…The banding pattern produced can be used to identify a particular species, depending on the presence or absence of a restriction enzyme site in the PCR product derived from the target [ 226 ]. It is a relatively inexpensive technique compared to real-time PCR, and a multitude of gene targets can be utilised, including heat-shock protein 70, glycoprotein 63, kDNA, cysteine protease B, mini-exon or NADH dehydrogenase subunit 7 [ 121 , 227 – 231 ].…”

“…An ITS1-RFLP reported by Schönian et al [ 231 ] was able to differentiate most Leishmania species but was less useful for those species within the L. braziliensis complex [ 232 , 233 ]. Such findings can be problematic, as the clinical presentation of the MCL tropism is caused by more than one of these species, but the response to treatment differs between them [ 129 ].…”

“…Improvements in species discrimination using RFLP were achieved recently using the NADH dehydrogenase subunit 7 maxicircle gene target, where it was possible to discriminate L. braziliensis from other species within the L. braziliensis complex (Fig. 3 ) [ 231 ]. RFLP presents multiple difficulties, as it needs a relatively high parasite load, and is thus often paired with cell culture, and multiple restriction enzymes may have to be employed depending on the DNA target.…”

“…Digestion of in silico, culture and direct clinical samples enabled differentiation of Leishmania species. Reprinted with permission of Kaufer et al [ 231 ] …”

Identification of a conserved maxicircle and unique minicircles as part of the mitochondrial genome of Leishmania martiniquensis strain PCM3 in Thailand

“…selected genes, all coding regions) could be used as genetic markers to identify Leishmania species and strains and to depict phylogenetic relatedness [ 59 – 62 ]. For example, Kaufer et al used a variation on the Leishmania maxicircle sequences ( ND7 gene) to design PCR–RFLP assays to discriminate nine Leishmania species [ 63 ]. A recent study identified the origin of the intra-species L. donovani hybrid from Himachal Pradesh, India, that was derived from two independent L. donovani parents of the ISC1-Yeti clade from the Nepalese highlands using maxicircle phylogenetics, suggesting the usefulness for examination of intra-species diversity [ 64 ].…”