Abstract:BACKGROUND Leishmaniasis caused by Leishmania martiniquensis infection has
been reported in human and domestic animals of Martinique Island, Germany,
Switzerland, USA, Myanmar and Thailand. The peculiar clinical features of
disseminated cutaneous and visceral forms co-existence render the urgent need of
specific diagnostic tool to identify the natural sand fly vectors for effective
prevention and control strategies. Loop-mediated isothermal amplification (LAMP)
of 18S rRNA gene as well as polymerase chain reac… Show more
“…martiniquensis from ‘ L . siamensis ’ (MHOM/TH/2010/TR) but the size of PCR products could not differentiate parasites in other subgenera [ 28 ]. For the ITS1-PCR method, it is not suitable for the discrimination of ‘ L .…”
Section: Discussionmentioning
confidence: 99%
“…orientalis ) and L . martiniquensis infection as it generates the same size of products [ 1 , 28 , 47 ].…”
Section: Discussionmentioning
confidence: 99%
“…L . orientalis ), but the size of the PCR products could not differentiate these parasites from other subgenera [ 28 ]. In 2012, Requena and colleagues analyzed the 3′untranslated region (3′UTR) of Leishmania HSP70-type I ( HSP70-I ) genes from 24 strains representing eleven Leishmania species following PCR amplification [ 29 ].…”
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.
“…martiniquensis from ‘ L . siamensis ’ (MHOM/TH/2010/TR) but the size of PCR products could not differentiate parasites in other subgenera [ 28 ]. For the ITS1-PCR method, it is not suitable for the discrimination of ‘ L .…”
Section: Discussionmentioning
confidence: 99%
“…orientalis ) and L . martiniquensis infection as it generates the same size of products [ 1 , 28 , 47 ].…”
Section: Discussionmentioning
confidence: 99%
“…L . orientalis ), but the size of the PCR products could not differentiate these parasites from other subgenera [ 28 ]. In 2012, Requena and colleagues analyzed the 3′untranslated region (3′UTR) of Leishmania HSP70-type I ( HSP70-I ) genes from 24 strains representing eleven Leishmania species following PCR amplification [ 29 ].…”
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.
“…23,40 Our results differed from those reported by Adams et al 10 with a LoD of 1 × 10 2 parasites/mL for L. donovani and Sriworarat et al 14 with a LoD of 1 × 10 3 parasites/mL for Leishmania siamensis in whole blood samples. In addition, a report by Tiwananthagorn et al 58 concluded that a PCR-kDNA assay was more sensitive than PCR-ITS and LAMP with the 18S rRNA gene. 42 Therefore, a comprehensive study to evaluate these parameters in a vast set of Leishmania species causing CL in the New World is needed.…”
Loop-mediated isothermal amplification (LAMP) is ideal for the detection of DNA as it is a quick and easy-to-perform test that does not require complex or sophisticated equipment or infrastructure. However, the application of this technique in the detection of DNA has not been comprehensively analyzed to date (analytical validation). Our objective was to evaluate the sensitivity and analytical specificity (anticipated reportable range [ARR], the limit of detection [LoD], and accuracy) of LAMP targeting the 18S rRNA gene in the diagnosis of six New World species. We then applied the validated LAMP assay across 50 samples of sandflies and 50 direct smears from a recent outbreak of cutaneous leishmaniasis in Colombia to determine its diagnostic performance. The LAMP assay exclusively amplified the DNA of spp., and an ARR of between 1 × 10 and 1 × 10 equivalent parasites/mL was determined. An LoD of 1 × 10 equivalent parasites/mL was established and there was no statistically significant variation in terms of accuracy. Finally, a sensitivity of 100% in direct smears and sandflies samples was calculated and a specificity of 90.9% for direct smears using microscopy as reference and 96.8% for sandflies using real-time polymerase chain reaction as reference were determined. To our knowledge, this is the first attempt to analytically validate a LAMP test to detect DNA, which showed good diagnostic potential from sandflies and direct smear samples.
“…A similar study also experimentally validated the potential field applicability of the MG-LAMP for detection of L . martiniquensis DNA in sand flies in Thailand; however, no Leishmania DNA was detected by LAMP and PCR when applied to 380 field-caught flies, probably due to low infection rate in the study area [79]. Additionally, the performance of the MG-LAMP was further validated using purified DNA from sand fly pools and was found to be of potential use for the entomological surveillance of CL in Colombia with 100% sensitivity and 96.8% specificity [80].…”
Section: Molecular Xenomonitoring/surveillance Potentials Of Lamp Formentioning
Leishmaniasis, caused by protozoan parasites of the Leishmania genus, represents an important health problem in many regions of the world. Lack of effective point-of-care (POC) diagnostic tests applicable in resources-limited endemic areas is a critical barrier to effective treatment and control of leishmaniasis. The development of the loop-mediated isothermal amplification (LAMP) assay has provided a new tool towards the development of a POC diagnostic test based on the amplification of pathogen DNA. LAMP does not require a thermocycler, is relatively inexpensive, and is simple to perform with high amplification sensitivity and specificity. In this review, we discuss the current technical developments, applications, diagnostic performance, challenges, and future of LAMP for molecular diagnosis and surveillance of Leishmania parasites. Studies employing the LAMP assay to diagnose human leishmaniasis have reported sensitivities of 80% to 100% and specificities of 94% to 100%. These observations suggest that LAMP offers a good molecular POC technique for the diagnosis of leishmaniasis and is also readily applicable to screening at-risk populations and vector sand flies for Leishmania infection in endemic areas.
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