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- Title
Molecular identification of two newly identified human pathogens causing leishmaniasis using PCR-based methods on the 3′ untranslated region of the heat shock protein 70 (type I) gene.
- Authors
Jariyapan, Narissara; Bates, Michelle D.; Bates, Paul A.
- Abstract
PCR-based methods to amplify the 3′ untranslated region (3′-UTR) of the heat shock protein 70 (type I) gene (HSP70-I) have previously been used for typing of Leishmania but not with Leishmania (Mundinia) martiniquensis and L. (Mundinia) orientalis, newly identified human pathogens. Here, the 3′-UTRs of HSP70-I of L. martiniquensis, L. orientalis, and 10 other species were sequenced and analyzed. PCR-Restriction Fragment Length Polymorphism (RFLP) analysis targeting the 3′-UTR of HSP70-I was developed. Also, the detection limit of HSP70-I-3′-UTR PCR methods was compared with two other commonly used targets: the 18S small subunit ribosomal RNA (SSU-rRNA) gene and the internal transcribed spacer 1 region of the rRNA (ITS1-rRNA) gene. Results showed that HSP70-I-3′-UTR PCR methods could be used to identify and differentiate between L. martiniquensis (480–2 bp) and L. orientalis (674 bp) and distinguished them from parasites of the subgenus Viannia and of the subgenus Leishmania. PCR-RFLP patterns of the 3′-UTR of HSP70-I fragments digested with BsuRI restriction enzyme successfully differentiated L. martiniquensis, L. orientalis, L. braziliensis, L. guyanensis = L. panamensis, L. mexicana = L. aethiopica = L. tropica, L. amazonensis, L. major, and L. donovani = L. infantum. For the detection limit, the HSP70-I-3′-UTR PCR method could detect the DNA of L. martiniquensis and L. orientalis at the same concentration, 1 pg/μL, at a similar level to the SSU-rRNA PCR. The PCR that amplified ITS1-rRNA was more sensitive (0.01 pg/μL) than that of the HSP70-I-3′-UTR PCR. However, the sizes of both SSU-rRNA and ITS1-rRNA PCR amplicons could not differentiate between L. martiniquensis and L. orientalis. This is the first report of using HSP70-I-3′-UTR PCR based methods to identify the parasites causing leishmaniasis in Thailand. Also, the BsuRI-PCR-RFLP method can be used for differentiating some species within other subgenera. Author summary: L. martiniquensis and L. orientalis, newly identified human pathogens, cause visceral leishmaniasis and cutaneous leishmaniasis in HIV-negative patients, respectively. However, both parasite species cause disseminated cutaneous leishmaniasis accompanying visceral leishmaniasis in HIV-positive patients. Species typing in leishmaniasis is important in diagnostics, epidemiology, and clinical studies. We show here that the 3′-UTR of HSP70-I region is a suitable target for PCR-based identification and discrimination between L. martiniquensis and L. orientalis. The technique is simple to perform and can be implemented in all settings where PCR is available. In species with similar PCR product size, the BsuRI-PCR-RFLP patterns of the 3′-UTR of HSP70-I fragments can be used for differentiating some species within other subgenera. However, where identification of species is essential or there is a travel history outside Thailand, sequencing of the HSP70-I-3′-UTR product or a similar discriminating target sequence is recommended. The PCR-based methods used in this study can also be applicable to the identification of Leishmania species obtained from vectors and reservoirs.
- Subjects
THAILAND; HEAT shock proteins; THEILERIA; LEISHMANIA mexicana; LEISHMANIASIS; CUTANEOUS leishmaniasis; VISCERAL leishmaniasis; RIBOSOMAL RNA
- Publication
PLoS Neglected Tropical Diseases, 2021, Vol 15, Issue 11, p1
- ISSN
1935-2727
- Publication type
Article
- DOI
10.1371/journal.pntd.0009982