Polymerase chain reaction detection of LeishmaniaDNA in skin biopsy samples in Sri Lanka where the causative agent of cutaneous leishmaniasis is Leishmania donovani

Ranasinghe, Shalindra; Wickremasinghe, Renu; Hulangamuwa, Sanjeeva; Sirimanna, Ganga; Opathella, Nandimithra; Maingón, R.; Chandrasekharan, Vishvanath

doi:10.1590/0074-02760150286

Cited by 32 publications

(49 citation statements)

References 38 publications

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“…We obtained PCR with sensitivity of 0.1 pg for specific subgenus reactions, and 1 pg for the reactions that amplify L. amazonensis and L. infantum kDNA sequences. These sensitivities were similar to those obtained in other studies using ITS1 primers [35, 37].…”

Section: Discussionsupporting

confidence: 90%

“…However, this technique is laborious, timeconsuming, difficult to process a large number of samples and does not allow the identification of genus and species [32, 33]. Alternatively, molecular techniques, such as PCR, are highly specific and more sensitive, allowing the detection of a single parasite depending on the used primer [34, 35]. The use of PCR for detection of Leishmania DNA in sand flies is a useful technique for the identification of putative vectors in different geographical areas [33, 36-39].…”

Section: Discussionmentioning

confidence: 99%

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Detection of multiple circulating Leishmania species in Lutzomyia longipalpis in the city of Governador Valadares, southeastern Brazil

Cardoso

Bento

Almeida

et al. 2018

Preprint

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28Background 29 Leishmaniasis encompasses a group of diverse clinical diseases caused by protozoan 30 parasites of the Leishmania genus. This disease is a major public health problem in 31 the New World affecting people exposed in endemic regions. The city of Governador 32 Valadares (Minas Gerais/Brazil) is a re-emerging area for visceral leishmaniasis, with 33 191 human cases reported from 2008 to 2017 and a lethality rate of 14.7%. The 34 transmission of the parasite occurs intensely in this region with up to 22% of domestic 35 dogs with positive serology for the visceral form. Lu. longipalpis is one of the most 36 abundant sand fly species in this area. Despite this scenario, so far there is no 37 information regarding the circulating Leishmania species in the insect vector Lutzomyia 38 longipalpis in this focus. 39 Methodology/Principal Findings 40 We collected 616 female Lutzomyia longipalpis sand flies between January and 41 September 2015 in the Vila Parque Ibituruna neighborhood (Governador 42 Valadares/MG), which is located on a transitional area between the sylvatic and urban 43 environments with residences built near a preserved area. After DNA extraction of 44 individual sand flies, the natural Leishmania infections in Lu. longipalpis were detected 45 by end-point PCR, using primers derived from kDNA sequences, specific for L. 46 (Leishmania) or L. (Viannia) subgenus. The sensitivity of these PCR reactions was 0.1 47 pg of DNA for each Leishmania subgenus and the total infection rate of 16.2% (100 48 positive specimens). Species-specific PCR detected the presence of multiple 49 Leishmania species in infected Lu. longipalpis specimens in Governador Valadares, 50 including L. amazonensis (n=3), L. infantum (n=28), L. (Viannia) spp. (n=20), 3 51 coinfections with L. infantum and L. (Viannia) spp. (n=5), and L. (Leishmania) spp 52 (n=44). 53 Conclusions 54 Our results demonstrate that multiple Leishmania species circulate in Lu. longipalpis 55 in Governador Valadares and reveal a potential increasing risk of transmission of the 56 different circulating parasite species. This information is a key factor for planning 57 surveillance and effective control strategies against leishmaniasis in this endemic 58 focus. 59 60 Author summary 61 Leishmaniasis is a neglected tropical disease transmitted to mammals by the 62 bite of sand flies infected with parasites of the Leishmania genus. This disease affects 63 millions of people in various regions of the world, including Brazil. The municipality of 64 Governador Valadares (Minas Gerais/Brazil) is a re-emergent focus of intense 65 transmission of leishmaniasis, with a high number of human cases and a high 66 prevalence of infected domestic dogs. To develop better leishmaniasis control 67 strategies for the region, we performed a surveillance study of Lu. longipalpis, the main 68 vector of visceral leishmaniasis in Brazil, and identified circulating species of 69Leishmania in this insect vector. We estimate that the natural infection rate of Lu. 70 longipalpis...

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Section: Discussionsupporting

confidence: 90%

Section: Discussionmentioning

confidence: 99%

Detection of multiple circulating Leishmania species in Lutzomyia longipalpis in the city of Governador Valadares, southeastern Brazil

Cardoso

Bento

Almeida

et al. 2018

Preprint

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“…A volume of 2μl of this extracted DNA was amplified with 100 pmols of previously described 50 set of primers LITSR/L5.8S that amplifies a 320 bp fragment of ITS1 region of Leishmania genus-specific DNA in the presence of 1.5 mM MgCl2, 25 mM Tris-HCL (pH 9.0), 25 mM NaCl, 200 μM each deoxynucleotide triphosphate, 50 units/ml Taq DNA polymerase (Promega) in a final volume of 10 μL. The PCR amplification cycles consisted of an initial denaturation at 95°C for 2 min, followed by 34 cycles of denaturation at 95°C for 20 s, annealing at 53°C for 30 s, and extension at 72°C for 1 min, with a final extension of 72°C for 6 min 50,51 . Extracted DNA from a pure culture was the positive control and no DNA sample was the negative control.…”

Section: Methodsmentioning

confidence: 99%

Early reduction in PD-L1 expression predicts faster treatment response in human cutaneous leishmaniasis

Dey

Senarathna

Somaratne³

et al. 2020

Preprint

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21 22 23 24 2 Cutaneous leishmaniasis (CL) is a disfiguring disease caused by infection with 25 Leishmania parasites and is characterised by parasitism of the dermis and chronic 26 inflammation. Whilst T cell responses to Leishmania are essential for both parasite 27 clearance and disease resolution they also drive inflammation, and clinical presentation 28 reflects the balance of these opposing activities 1 . Pentavalent antimonials (e.g. sodium 29 stibogluconate; SSG) remain the first line drugs for CL, even though treatment may be 30 protracted and painful. Although evidence from animal models indicates that an 31 effective clinical response to antimonials requires immune-drug synergy 2 , little is known 32 about how this operates in human disease. Here, we studied formalin fixed paraffin 33 embedded (FFPE) skin biopsies from patients in Sri Lanka with CL, at presentation 34 and during intra-lesional SSG treatment. Immune-targeted transcriptomics in a test 35 patient cohort indicated heightened immune checkpoint pathway expression at 36 presentation. We confirmed reduced PD-L1 and IDO1 protein expression on treatment 37 in a second validation cohort, using digital spatial profiling and quantitative 38 immunohistochemistry. PD-L1 and IDO1 expression on CD68 + monocytes / 39 macrophages was positively correlated with the degree of intracellular parasitism, as 40 determined by parasite-specific RNA FISH. Our data support a model whereby the 41 initial anti-leishmanial activity of antimonial drugs alleviates checkpoint inhibition of T 42 cell immunity, thus facilitating immune-drug synergism and clinical cure. We suggest a 43 need to evaluate shorter course SSG treatment and/or the use of checkpoint inhibition 44 as an adjunct host directed therapy (HDT) in CL. 45 46 One billion people are thought to be at risk of leishmaniasis, a group of diseases caused by 47 infection with protozoan parasites of the genus Leishmania and transmitted by phlebotomine 48 sand flies 3-5 . Approximately 600,000 -1 million new cases of CL occur, with a broad global 49 3 distribution, often leading to stigma and reduced life chances and placing a burden on health 50 services 6,7 . Treatment options for CL have changed little in over 70 years, since pentavalent 51antimonial drugs were first introduced, and there are scant new treatments on the horizon 8,9 . 52 Sri Lanka is endemic for CL 10 with the first autochthonous case being reported in 1992 11 . 53Sri Lankan CL is caused by Leishmania donovani zymodeme MON-37 12-14 , usually 54 associated with visceral leishmaniasis. Current treatment for CL in Sri Lanka involves 55 weekly intra-lesional or daily intra-muscular administration of SSG, with or without 56 cryotherapy, based on the site and size of the lesion and response to treatment. Cure often 57 takes many months, and some patients may fail to respond completely or withdraw from 58 treatment 15 . It is widely proposed that immune-drug synergy is required for fully effective 59 treatment in leishmaniasis and that host directed...

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“…Distribution of CL cases shown in Figure 1. with HaeIII enzyme (Thermoscienti c) over night at 37°C, according to the manufacturer's instructions and restriction fragments were analysed by gel electrophoresis in 3% metaphor gel [12].…”

Section: Materials Methodologymentioning

confidence: 99%

Cutaneous Leishmaniasis and/or Dermal Leishmanoid in Himachal Pradesh (India)

Lata¹,

Kumari²,

Das³

et al. 2020

Preprint

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Background: Visceral Leishmaniasis (VL) is in elimination phase in India while cases of Cutaneous Leishmaniasis (CL) are spreading to new foci in different parts of the country. In Himachal Pradesh, a foci of CL have been reported along Satluj River, but the causative agent poses a dilemma. To ascertain the Leishmania species from CL cases from Shimla, Kullu and Kinnaur districts of Himachal Pradesh, the present study was undertaken. Methods: A total of 28 CL patients registered in Department of Dermatology, Indira Gandhi Medical College (IGMC) and Hospital Shimla in 2018, were tested by rk39. Barring 16 cases undergoing treatment, 12 fresh cases were subjected to microscopic detection of Leishmania parasite, PCR and sequencing. Skin biopsies of 3-4 mm diameter were taken in culture medium and in formalin under anesthetic and sterile conditions from the border of the lesions. Imprints were prepared for the detection of Leishmania amastigotes. Biopsy samples were inoculated into different culture media (M199, RPMI 1640, and NNN) and were incubated at 22-24°C. Cultures were examined microscopically for the growth of promastigotes up to four weeks. Polymerase chain reaction (PCR) was performed to characterize leishmania parasite species.Results: Of 28 patients, one patient was found positive for by rK39 dipstick test. One imprint was found positive for leishmania amastigotes. Twelve biopsy DNA samples were subjected to PCR for Leishmania kDNA, of which all the 12 were found positive ITS1 Leishmania specific set of primers while eight were found positive with JW11/12 lesihmania species specific set of primers. Identification of Leishmania species was confirmed by PCR-RFLP and sequencing method. Of 12 Leishmania positive samples, six were identified as L. donovani, three L. tropica, two L. major and one remained unidentified.Conclusion: The detection of L. donovani from cutaneous leishmniasis patients is a significant finding leading towards existence of atypical leishmaniasis in Himachal Pradesh.

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Polymerase chain reaction detection of LeishmaniaDNA in skin biopsy samples in Sri Lanka where the causative agent of cutaneous leishmaniasis is Leishmania donovani

Cited by 32 publications

References 38 publications

Detection of multiple circulating Leishmania species in Lutzomyia longipalpis in the city of Governador Valadares, southeastern Brazil

Detection of multiple circulating Leishmania species in Lutzomyia longipalpis in the city of Governador Valadares, southeastern Brazil

Early reduction in PD-L1 expression predicts faster treatment response in human cutaneous leishmaniasis

Cutaneous Leishmaniasis and/or Dermal Leishmanoid in Himachal Pradesh (India)

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