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Development of loop-mediated isothermal amplification (LAMP) assay using SYBR safe and gold-nanoparticle probe for detection of Leishmania in HIV patients.

ABSTRACT: Asymptomatic leishmaniasis cases have continuously increased, especially among patients with HIV who are at risk to develop further symptoms of cutaneous and visceral leishmaniasis. Thus, early diagnosis using a simple, sensitive and reliable diagnostic assay is important because populations at risk mostly reside in rural communities where laboratory equipment is limited. In this study, the highly sensitive and selective determination of Leishmania infection in asymptomatic HIV patients was achieved using dual indicators (SYBR safe and gold-nanoparticle probe; AuNP-probe) in one-step LAMP method based on basic instruments. The assay can be simply evaluated under the naked eye due to clear interpretation of fluorescent emission of LAMP-SYBR safe dye-complex and colorimetric precipitate of specific AuNP-probes. The sensitivities and specificities of fluorescent SYBR safe dye and AuNP-probe indicators were equal, which were as high as 94.1 and 97.1%, respectively. Additionally, detection limits were 10² parasites/mL (0.0147 ng/µL), ten times more sensitivity than other related studies. To empower leishmaniasis surveillance, this inexpensive one-step SYBR safe and AuNP-LAMP assay is reliably fast and simple for field diagnostics to point-of-care settings, which can be set up in all levels of health care facilities including resource limited areas, especially in low to middle income countries.

SUBMITTER: Ruang-Areerate T

PROVIDER: S-EPMC8190085 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Leishmaniasis is a neglected tropical disease that is caused by an obligate intracellular protozoan of the genus Leishmania. Recently, an increasing number of autochthonous leishmaniasis cases caused by L. martiniquensis and the novel species L. siamensis have been described in Thailand, rendering an accurate diagnosis of this disease critical. However, only a few laboratories are capable of diagnosing leishmaniasis in Thailand. To expand leishmaniasis diagnostic capabilities, we developed a simple colorimetric loop-mediated isothermal amplification (LAMP) technique for the direct detection of Leishmania DNA.LAMP was performed for 75 min using four primers targeting the conserved region of the18S ribosomal RNA gene, and the DNA indicator used was malachite green (MG). To simulate crude samples, cultured promastigotes of L. siamensis were mixed with blood or saliva. Also, clinical samples (blood, saliva, and tissue biopsies) were obtained from patients with cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL). All samples were boiled for 10 min and introduced directly into the LAMP reaction mixture without DNA purification.The use of MG resulted in an unambiguous differentiation of positive and negative controls. For L. siamensis, the detection limit was 10(3) parasites/mL or 2.5 parasites/tube. Saliva, tissue biopsies, and whole blood were indicative of active Leishmania infection, and their direct usages did not adversely affect the detection limit. In addition, this LAMP assay could detect DNA from multiple Leishmania species other than L. siamensis and L. martiniquensis, including L. aethiopica, L. braziliensis, L. donovani and L. tropica.The simplicity and sensitivity of LAMP in detecting active Leishmania infection could enable the rapid diagnosis of leishmaniasis, thereby facilitating the survey and control of leishmaniasis in Thailand. However, our limited number of samples warranted a further validation with a larger cohort of patients before this assay could be deployed.

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Development of loop-mediated isothermal amplification (LAMP) assay using SYBR safe and gold-nanoparticle probe for detection of Leishmania in HIV patients.

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