Publications by authors named "Sheila Ortega"

6 Publications

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Loop-Mediated Isothermal Amplification Allows Rapid, Simple and Accurate Molecular Diagnosis of Human Cutaneous and Visceral Leishmaniasis Caused by When Compared to PCR.

Microorganisms 2021 Mar 16;9(3). Epub 2021 Mar 16.

WHO Collaborating Centre for Leishmaniasis, National Center for Microbiology, Instituto de Salud Carlos III, 28220 Majadahonda, Spain.

Loop-mediated isothermal amplification allows the rapid, sensitive and specific amplification of DNA without complex and expensive equipment. We compared the diagnostic performance of Loopamp™ Detection Kit (Eiken Chemical Co., Ltd., Tokyo, Japan) with conventional and real-time polymerase chain reaction (PCR) for human cutaneous and visceral leishmaniasis caused by . A total of 230 DNA samples from cutaneous (CL) and visceral (VL) leishmaniasis cases and controls from Spain, characterized by nested PCR (LnPCR) were tested by: (i) the Loopamp™ Detection Kit (Loopamp), run on Genie III real-time fluorimeter (OptiGene, UK); and (ii) real-time quantitative PCR (qPCR). The Loopamp test returned 98.8% (95% confidence interval-CI: 96.0-100.00) sensitivity and specificity of 97.7% (95% CI: 92.2-100) on VL samples, and 100% (95% CI: 99.1-100) sensitivity and 100.0% (95% CI: 98.8-100.0) specificity on CL samples. The Loopamp time-to-positivity () obtained by real-time fluorimetry showed excellent concordance (C = 97.91%) and strong correlation (r = 0.799) with qPCR's cycle threshold (). The performance of Loopamp is comparable to that of LnPCR and qPCR in the diagnosis of cutaneous and visceral leishmaniasis due to . The excellent correlation between the and should be further investigated to determine the accuracy of Loopamp to quantify parasite load in tissues.
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http://dx.doi.org/10.3390/microorganisms9030610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7999953PMC
March 2021

Prevalence of asymptomatic infection and associated risk factors, after an outbreak in the south-western Madrid region, Spain, 2015.

Euro Surveill 2019 May;24(22)

WHO Collaborating Centre for Leishmaniasis, National Center for Microbiology, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain.

BackgroundA large outbreak of leishmaniasis with 758 cutaneous and visceral leishmaniasis cases occurred in 2009 in Fuenlabrada, in the south-west of the Madrid region of Spain.AimWe aimed to determine the prevalence of asymptomatic infection after this outbreak, and its associated risk factors.MethodsA cross-sectional study of 804 healthy individuals living in Fuenlabrada who had no history of leishmaniasis, was conducted between January and July 2015. Asymptomatic infections were sought by either a combination of PCR, immunofluorescent antibody titre, and direct agglutination tests, or by whole blood stimulation assay (WBA) with interleukin-2 (IL-2) quantification.ResultsUsing the first approach, prevalence of asymptomatic individuals was 1.1% (9/804), while the second returned a value of 20.7% (143/804). Older age, being male, proximity to the park where the focus of infection was identified, and living in a detached house, were all strongly associated with the prevalence of asymptomatic infection.ConclusionsThe true number of infected individuals may be underestimated if only serological methods are used. The combination of WBA with IL-2 quantification may allow to better determine the prevalence of asymptomatic infection, which would be useful in establishing control measures and in quantifying their impact. In our study, the use of WBA with IL-2 quantification also helped establish the risk factors that influence exposure to and infection by .
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http://dx.doi.org/10.2807/1560-7917.ES.2019.24.22.1800379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6549460PMC
May 2019

Cellular Markers of Active Disease and Cure in Different Forms of -Induced Disease.

Front Cell Infect Microbiol 2018 13;8:381. Epub 2018 Nov 13.

WHO Collaborating Centre for Leishmaniasis, National Centre for Microbiology, Instituto de Salud Carlos III, Madrid, Spain.

Increased numbers of peripheral blood mononucleocytes (PBMC) and increased IFN-γ secretion following challenge of blood samples with soluble antigen (SLA), have been proposed as biomarkers of specific cell-mediated immunity, indicating that treatment of visceral leishmaniasis (VL) has been successful. However, infection may manifest as cutaneous leishmaniasis (CL), and less commonly as localized leishmanial lymphadenopathy (LLL) or mucosal leishmaniasis (ML). The present work examines the value of these biomarkers as indicators of cured leishmaniasis presenting in these different forms. Blood samples were collected before and after treatment from patients living in Fuenlabrada (Madrid, Spain), an endemic area recently the center of a leishmaniasis outbreak. All samples were subjected to -specific PCR, serological tests (IFAT and rK39-ICT), and the SLA-cell proliferation assay (SLA-CPA), recording PBMC proliferation and the associated changes in IFN-γ production. Differences in the results recorded for the active and cured conditions were only significant for VL. PCR returned positive results in 67% of patients with active VL and in 3% of those with cured leishmaniasis. Similarly, rK39-ICT returned a positive result in 77% of active VL samples . 52% in cured VL samples, and IFAT in 90% . 56%; in the SLA-CPA, PBMC proliferation was seen in 16% . 90%, and an associated increase in IFN-γ production of 14 and 84%, respectively. The present findings reinforce the idea that PBMC proliferation and increased IFN-γ production in SLA-stimulated PBMC provide biomarkers of clinical cure in VL. Other tests are urgently needed to distinguish between the cured and active forms of the other types of clinical leishmaniasis caused by .
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http://dx.doi.org/10.3389/fcimb.2018.00381DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6243388PMC
September 2019

Validation of rK39 immunochromatographic test and direct agglutination test for the diagnosis of Mediterranean visceral leishmaniasis in Spain.

PLoS Negl Trop Dis 2018 03 1;12(3):e0006277. Epub 2018 Mar 1.

WHO Collaborating Centre for Leishmaniasis, National Centre for Microbiology, Instituto de Salud Carlos III, Majadahonda, Spain.

Background: Visceral leishmaniasis (VL), the most severe form of leishmaniasis, is endemic in Europe with Mediterranean countries reporting endemic status alongside a worrying northward spread. Serological diagnosis, including immunochromatographic test based on the recombinant antigen rK39 (rK39-ICT) and a direct agglutination test (DAT) based on the whole parasite antigen, have been validated in regions with high VL burden, such as eastern Africa and the Indian subcontinent. To date, no studies using a large set of patients have performed an assessment of both methods within Europe.

Methodology/principal Findings: We selected a range of clinical serum samples from patients with confirmed VL (including HIV co-infection), Chagas disease, malaria, other parasitic infections and negative samples (n = 743; years 2009-2015) to test the performance of rK39-ICT rapid test (Kalazar Detect Rapid Test; InBios International, Inc., USA) and DAT (ITM-DAT/VLG; Institute of Tropical Medicine Antwerp, Belgium). An in-house immunofluorescence antibody test (IFAT), was included for comparison. Estimated sensitivities for rK39-ICT and DAT in HIV-negative VL patients were 83.1% [75.1-91.2] and 84.2% [76.3-92.1], respectively. Sensitivity was reduced to 67.3% [52.7-82.0] for rK39 and increased to 91.3% [82.1-100.0] for DAT in HIV/VL co-infected patients. The in-house IFAT was more sensitive in HIV-negative VL patients, 84.2% [76.3-92.1] than in HIV/VL patients, 79.4% [73.3-96.2]. DAT gave 32 false positives in sera from HIV-negative VL suspects, compared to 0 and 2 for rK39 and IFAT, respectively, but correctly detected more HIV/VL patients (42/46) than rK39 (31/46) and IFAT (39/46).

Conclusions/significance: Though rK39-ICT and DAT exhibited acceptable sensitivity and specificity a combination with other tests is required for highly sensitive diagnosis of VL cases in Spain. Important variation in the performance of the tests were seen in patients co-infected with HIV or with other parasitic infections. This study can help inform the choice of serological test to be used when screening or diagnosing VL in a European Mediterranean setting.
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http://dx.doi.org/10.1371/journal.pntd.0006277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5849364PMC
March 2018

Molecular detection of Leishmania infantum and Leishmania tropica in rodent species from endemic cutaneous leishmaniasis areas in Morocco.

Parasit Vectors 2017 Oct 2;10(1):454. Epub 2017 Oct 2.

Ecology and the Environment Laboratory L2E, (URAC 32, CNRST ERACNERS 06), Faculty of Sciences Semlalia, Cadi Ayyad University, Marrakesh, Morocco.

Background: Leishmaniasis remains a major public health problem in African nations, including Morocco, where little is known about the vertebrate reservoirs involved in the causal parasites' transmission cycles. The present study investigates the role of rodent species as potential reservoirs of Leishmania spp. in central Morocco, where both L. tropica and L. infantum have been reported.

Methods: Rodents were caught from 22 sites in central Morocco, by using Sherman metal traps, and identified morphologically. For each specimen, genomic DNA was extracted from different tissues using the Speed Tools DNA extraction Kit. Then, samples were PCR-analyzed, targeting the SSU rRNA gene to detect Leishmania spp. DNA, followed by amplification of the internal transcribed spacer 1 (ITS1) and its sequencing to identify the species.

Results: A total of 197 rodents belonging to ten species were captured and identified: Rattus rattus (40.61%), Mus musculus (25.38%), Apodemus sylvaticus (8.63%), Mus spretus (7.11%), Meriones shawi (5.58%), Rattus norvegicus (4.57%), Meriones libycus (3.05%), Mastomys erythroleucus (2.03%), Gerbillus campestris (2.03%) and Lemniscomys barbarus (1.01%). Molecular analysis revealed the presence of Leishmania species in 18 specimens: six R. rattus (out of 80 captured; 7.5%), 11 M. musculus (out of 50 captured; 22%), and one R. norvegicus (out of 9 captured; 11.11%).

Conclusions: To the best of our knowledge, L. infantum and L. tropica were identified in rodent species for the first time in Morocco. These findings suggest that rodent species may be involved in L. infantum and L. tropica transmission cycles in this country but that further studies are needed to confirm their role as reservoirs of Leishmania species in Morocco.
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http://dx.doi.org/10.1186/s13071-017-2398-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5625640PMC
October 2017

Application of real-time PCR for the detection of Strongyloides spp. in clinical samples in a reference center in Spain.

Acta Trop 2015 Feb 4;142:20-5. Epub 2014 Nov 4.

Department of Parasitology, National Centre for Microbiology, Instituto de Salud Carlos III, Majadahonda, Madrid 28220, Spain. Electronic address:

Strongyloidiasis is one of the major intestinal helminthic infections in humans with a worldwide distribution, affecting especially tropical and subtropical regions. This disease can occur without any symptoms or as a potentially fatal hyperinfection or disseminated infection. Definitive diagnosis of Strongyloides stercoralis infection relies mainly on demonstration of larvae in stool, but at present there is no gold standard for this diagnosis. Our main objective was to evaluate a real-time PCR targeting the 18S rRNA gene of Strongyloides spp. and to compare it with routine parasitological methods. DNA from Strongyloides venezuelensis was used to optimize PCR protocols obtaining an analytical sensitivity of 0.1 pg of parasite DNA per sample. Sensitivity and specificity of real-time PCR on fecal samples from 231 patients screened for suspected strongyloidiasis attending two hospitals in Madrid were 93.8% and 86.5%, respectively. No significant differences were found when comparing Ct-values of positive PCR between parasitological positive and negative samples. This study showed that real-time PCR is an effective tool for diagnosing strongyloidiasis and could be applied in association with parasitological methods in epidemiological studies in endemic areas. It would be also important to assess its performance in immunocompromised populations who are at risk of fatal disease.
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http://dx.doi.org/10.1016/j.actatropica.2014.10.020DOI Listing
February 2015
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