Evaluation of a point-of-care molecular detection device for Leishmania spp. and intercurrent fungal and mycobacterial organisms in Peruvian patients with cutaneous ulcers

Kariyawasam, Ruwandi; Valencia, Braulio M.; Lau, Rachel; Shao, Eric; Thompson, Courtney; Stevens, Michael H.; Kincaid, Leah; Castillo, Ana Luz Quispe Del; Cruz-Arzapalo, Lloysi O; Llanos‐Cuentas, Alejandro; Boggild, Andrea K.

doi:10.1007/s15010-021-01673-y

Cited by 7 publications

(8 citation statements)

References 35 publications

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“…It is known that the sensitivity of a multiplex PCR assay is reduced with increased numbers of target genes in the reaction [45]. In a recent study describing a Palm PCR assay coupled to agarose gel electrophoresis for Leishmania spp., detection in cutaneous ulcers achieved a specificity and a sensitivity of 90% and 91.7%, respectively, when tested in lab conditions [32]. In the field, the same assay showed a sensitivity of 100% and a specificity of 25% [32].…”

Section: Discussionmentioning

confidence: 94%

“…To our knowledge, this is the first study that associates convective PCR technology with lateral flow detection for Leishmania parasites detection and identification. The unique study describing an ultra-fast PCR for CL diagnosis used an E-gel reader electrophoresis system for the detection of Leishmania viannia and some mimickers such as fungal or mycobacterial infections [32]. Moreover, it is the fastest PCR-based technology method so far that could be used for the concomitant detection and identification of Leishmania parasites.…”

Section: Discussionmentioning

confidence: 99%

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Handheld Ultra-Fast Duplex Polymerase Chain Reaction Assays and Lateral Flow Detection and Identification of Leishmania Parasites for Cutaneous Leishmaniases Diagnosis

Bel Hadj Ali,

Saadi-Ben Aoun,

Hammami

et al. 2023

Pathogens

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Early and accurate detection of infectious diseases is a key step for surveillance, epidemiology and control, notably timely disease diagnosis, patient management and follow-up. In this study, we aimed to develop handheld ultra-fast duplex PCR assays coupled to amplicon detection by lateral flow (LF) immunoassay to deliver a rapid and simple molecular diagnostic test for concomitant detection and identification of the main Leishmania parasites encountered in Tunisia. We selected two DNA targets to amplify L. major/L. tropica and L. infantum/L. tropica groups of species DNAs, respectively. We optimized the experimental conditions of a duplex ultra-fast PCR. The amplification is performed using a portable Palm convection PCR machine within 18 min, and the products are detected using an LF cassette within 10 min. The test allows the identification of the infecting species according to the position and number of test lines revealed. Tested on a selection of DNAs of representative Leishmania strains of the three studied species (N = 37), the ultra-fast duplex PCR–LF showed consistent, stable and reproducible results. The analytical limit of detection of the test was 0.4 pg for L. major, 4 pg for L. infantum and 40 pg for L. tropica.

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Section: Discussionmentioning

confidence: 94%

Section: Discussionmentioning

confidence: 99%

Handheld Ultra-Fast Duplex Polymerase Chain Reaction Assays and Lateral Flow Detection and Identification of Leishmania Parasites for Cutaneous Leishmaniases Diagnosis

Bel Hadj Ali,

Saadi-Ben Aoun,

Hammami

et al. 2023

Pathogens

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“…The presence of bacteria in the ulcer border and 'pain' and 'pruritus' had no influence on wound closure, the presence of 'secretion' and 'burning sensation' delayed epithelialization time but not total healing time. Kariyawasam et al [135] observed that in 61% of CL patients, there was the co-colonization fungal genera such as Malassezia, Aspergillus, Candida and Cladosporium. In about 50% of the patients, the fungal infections were responsible for inflammation, and fungal-bacterial infections complicated the diagnosis of CL.…”

Section: Targeting Secondary Bacterial Infectionsmentioning

confidence: 99%

“…Kariyawasam et al . [135] observed that in 61% of CL patients, there was the co-colonization fungal genera such as Malassezia, Aspergillus , Candida and Cladosporium . In about 50% of the patients, the fungal infections were responsible for inflammation, and fungal–bacterial infections complicated the diagnosis of CL.…”

Section: Multiple Nano-drug Delivery Systems For Simultaneous Leishma...mentioning

confidence: 99%

Nanomedicine-based strategies to improve treatment of cutaneous leishmaniasis

et al. 2022

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Nanomedicine strategies were first adapted and successfully translated to clinical application for diseases, such as cancer and diabetes. These strategies would no doubt benefit unmet diseases needs as in the case of leishmaniasis. The latter causes skin sores in the cutaneous form and affects internal organs in the visceral form. Treatment of cutaneous leishmaniasis (CL) aims at accelerating wound healing, reducing scarring and cosmetic morbidity, preventing parasite transmission and relapse. Unfortunately, available treatments show only suboptimal effectiveness and none of them were designed specifically for this disease condition. Tissue regeneration using nano-based devices coupled with drug delivery are currently being used in clinic to address diabetic wounds. Thus, in this review, we analyse the current treatment options and attempt to critically analyse the use of nanomedicine-based strategies to address CL wounds in view of achieving scarless wound healing, targeting secondary bacterial infection and lowering drug toxicity.

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“…Another test development concerns the simplified and standardized detection of PCR-amplified DNA of Leishmania using an oligochromatographic dipstick format (i.e., the commercially available Leishmania OligoC-TesT kit), which showed high diagnostic sensitivity and specificity in clinical samples ( Deborggraeve et al, 2008 ; Espinosa et al, 2009 ). The advent of user-friendly portable devices such as Palm PCR ™ ( Kariyawasam et al, 2021 ), real-time fluorimeters [e.g., for real-time monitoring of the amplification profile of loop-mediated isothermal amplification (LAMP) reactions ( Ibarra-Meneses et al, 2018 ; Dixit et al, 2021 )], and the Bento Lab ® , a mobile DNA laboratory setup ( Kambouris et al, 2020 ), have opened the possibility to ease implementation of field-applicable molecular methods for use at POC to facilitate early diagnosis of leishmaniasis.…”

Section: Introductionmentioning

confidence: 99%

Novel CRISPR-based detection of Leishmania species

Dueñas

Nakamoto²,

Cabrera-Sosa³

et al. 2022

Front. Microbiol.

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Tegumentary leishmaniasis, a disease caused by protozoan parasites of the genus Leishmania, is a major public health problem in many regions of Latin America. Its diagnosis is difficult given other conditions resembling leishmaniasis lesions and co-occurring in the same endemic areas. A combination of parasitological and molecular methods leads to accurate diagnosis, with the latter being traditionally performed in centralized reference and research laboratories as they require specialized infrastructure and operators. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) systems have recently driven innovative tools for nucleic acid detection that combine high specificity, sensitivity and speed and are readily adaptable for point-of-care testing. Here, we harnessed the CRISPR-Cas12a system for molecular detection of Leishmania spp., emphasizing medically relevant parasite species circulating in Peru and other endemic areas in Latin America, with Leishmania (Viannia) braziliensis being the main etiologic agent of cutaneous and mucosal leishmaniasis. We developed two assays targeting multi-copy targets commonly used in the molecular diagnosis of leishmaniasis: the 18S ribosomal RNA gene (18S rDNA), highly conserved across Leishmania species, and a region of kinetoplast DNA (kDNA) minicircles conserved in the L. (Viannia) subgenus. Our CRISPR-based assays were capable of detecting down to 5 × 10 −2 (kDNA) or 5 × 10 0 (18S rDNA) parasite genome equivalents/ reaction with PCR preamplification. The 18S PCR/CRISPR assay achieved pan-Leishmania detection, whereas the kDNA PCR/CRISPR assay was specific for L. (Viannia) detection. No cross-reaction was observed with Trypanosoma cruzi strain Y or human DNA. We evaluated the performance of the assays using 49 clinical samples compared to a kDNA real-time PCR assay as the reference test. The kDNA PCR/CRISPR assay performed equally well as the reference test, with positive and negative percent agreement of 100%. The 18S PCR/CRISPR assay had high positive and negative percent agreement of 82.1% and 100%, respectively. The findings support the potential applicability of the newly developed CRISPR-based molecular tools for first-line diagnosis of Leishmania infections at the genus and L. (Viannia) subgenus levels.

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Evaluation of a point-of-care molecular detection device for Leishmania spp. and intercurrent fungal and mycobacterial organisms in Peruvian patients with cutaneous ulcers

Cited by 7 publications

References 35 publications

Handheld Ultra-Fast Duplex Polymerase Chain Reaction Assays and Lateral Flow Detection and Identification of Leishmania Parasites for Cutaneous Leishmaniases Diagnosis

Handheld Ultra-Fast Duplex Polymerase Chain Reaction Assays and Lateral Flow Detection and Identification of Leishmania Parasites for Cutaneous Leishmaniases Diagnosis

Nanomedicine-based strategies to improve treatment of cutaneous leishmaniasis

Novel CRISPR-based detection of Leishmania species

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