Browsing by Author "Garcia, Emilia"
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Publication Assembly of a Large Collection of Maxicircle Sequences and Their Usefulness for Leishmania Taxonomy and Strain Typing(Multidisciplinary Digital Publishing Institute (MDPI), 2022-06-15) Solana, Jose Carlos; Chicharro, Carmen; Garcia, Emilia; Aguado, Begoña; Moreno, Javier; Requena, Jose M; Ministerio de Ciencia e Innovación (España); Agencia Estatal de Investigación (España); Instituto de Salud Carlos III; Unión Europea. Fondo Europeo de Desarrollo Regional (FEDER/ERDF)Parasites of medical importance, such as Leishmania and Trypanosoma, are characterized by the presence of thousands of circular DNA molecules forming a structure known as kinetoplast, within the mitochondria. The maxicircles, which are equivalent to the mitochondrial genome in other eukaryotes, have been proposed as a promising phylogenetic marker. Using whole-DNA sequencing data, it is also possible to assemble maxicircle sequences as shown here and in previous works. In this study, based on data available in public databases and using a bioinformatics workflow previously reported by our group, we assembled the complete coding region of the maxicircles for 26 prototypical strains of trypanosomatid species. Phylogenetic analysis based on this dataset resulted in a robust tree showing an accurate taxonomy of kinetoplastids, which was also able to discern between closely related Leishmania species that are usually difficult to discriminate by classical methodologies. In addition, we provide a dataset of the maxicircle sequences of 60 Leishmania infantum field isolates from America, Western Europe, North Africa, and Eastern Europe. In agreement with previous studies, our data indicate that L. infantum parasites from Brazil are highly homogeneous and closely related to European strains, which were transferred there during the discovery of America. However, this study showed the existence of different L. infantum populations/clades within the Mediterranean region. A maxicircle signature for each clade has been established. Interestingly, two L. infantum clades were found coexisting in the same region of Spain, one similar to the American strains, represented by the Spanish JPCM5 reference strain, and the other, named "non-JPC like", may be related to an important leishmaniasis outbreak that occurred in Madrid a few years ago. In conclusion, the maxicircle sequence emerges as a robust molecular marker for phylogenetic analysis and species typing within the kinetoplastids, which also has the potential to discriminate intraspecific variability.Publication Evaluation of the Chagas VirClia® and Chagas TESA VirClia® for the Diagnosis of Trypanosoma cruzi Infection(Multidisciplinary Digital Publishing Institute (MDPI), 2023) García-Bermejo, Isabel; Molina Arana, David; Zaragoza Vargas, Gloria; Carrasco Fernández, Blanca; Garcia, Emilia; Nieto Martinez, Francisco Javier; Flores-Chavez, Maria; Instituto de Salud Carlos III; Fundación Mundo SanoChagas disease (CD), caused by the protozoan Trypanosoma cruzi, is an important problem of public health even in regions where it is not endemic. Spain ranks second worldwide in terms of imported cases of T. cruzi infection in the chronic phase. The diagnosis in this stage is made via the detection of antibodies against T. cruzi. Therefore, we aimed to evaluate the sensitivity and specificity of two fully automated chemiluminescence immunoassays, Chagas VirClia® (CHR), which uses a mixture of recombinant antigens, and Chagas TESA VirClia® (TESA), the first chemiluminescence assay based on excretion-secretion antigens of trypomastigotes, both designed in monotest format. A retrospective case-control study was performed using 105 well-characterized samples: 49 from patients with CD, 22 from uninfected individuals, and 32 from patients with other pathologies. Sensitivity was 98% for CHR and 92% for TESA. In contrast, the specificity in both was 100%. Cross-reactivity was observed in leishmaniasis (2/10). CHR meets the criteria to become a tool for serological screening, while TESA has the potential for confirmation and cross-reaction discrimination. The monotest format allows its application in laboratories with a small number of samples. The high specificity of both assays is useful in areas where leishmaniasis is endemic.Publication Evaluation of the Performance of the Loopamp Trypanosoma cruzi Detection Kit for the Diagnosis of Chagas Disease in an Area Where It Is Not Endemic, Spain(American Society for Microbiology (ASM), 2021-04-20) Flores-Chavez, Maria; Abras, Alba; Ballart, Cristina; Ibáñez-Pérez, Ismael; Perez-Gordillo, Pilar; Gállego, Montserrat; Muñoz, Carmen; Moure, Zaira; Sulleiro Igual, Elena; Nieto Martinez, Francisco Javier; Garcia, Emilia; Cruz, Israel; Picado, Albert; Foundation for Innovative New Diagnostics (Suiza); Instituto de Salud Carlos III; Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR); Institució Catalana de Recerca i Estudis Avançats; Barcelona Institute for Global HealthIn Spain, PCR is the tool of choice for the diagnosis of congenital Chagas disease (CD) and serology for diagnosing chronic CD. A loop-mediated isothermal amplification test for Trypanosoma cruzi DNA detection showed good analytical performance and ease of use. We aimed to evaluate the performance of the Loopamp Trypanosoma cruzi detection kit (Eiken Chemical Co. Ltd., Japan) (Tcruzi-LAMP) for congenital and chronic CD diagnosis using well-characterized samples. We included samples from 39 congenital and 174 chronic CD cases and from 48 uninfected children born to infected mothers and 34 nonchagasic individuals. The sensitivity, specificity, and accuracy of Tcruzi-LAMP were estimated using standard case definitions for congenital CD (positive result by parasitological or PCR tests or serology after 9 months of age) and chronic CD (positive serology by at least two tests). The Tcruzi-LAMP results were read by visual examination and a real-time fluorimeter. For congenital CD, Tcruzi-LAMP sensitivity was 97% for both types of reading; specificity was 92% by visual examination and 94% by fluorimeter. For chronic CD, sensitivity was 47% and specificity 100%. The accuracy in congenital CD was >94% versus 56% in chronic CD. The agreement of Tcruzi-LAMP with PCR tests was better in congenital CD (kappa, 0.86 to 0.91) than in chronic CD (kappa, 0.67 to 0.83). The Loopamp Trypanosoma cruzi detection kit showed good performance for the diagnosis of congenital CD. Tcruzi-LAMP, like PCR, can be useful for the screening and early diagnosis of congenital infection.Publication Loop-Mediated Isothermal Amplification Allows Rapid, Simple and Accurate Molecular Diagnosis of Human Cutaneous and Visceral Leishmaniasis Caused by Leishmania infantum When Compared to PCR.(Multidisciplinary Digital Publishing Institute (MDPI), 2021-03-16) Ibarra-Meneses, Ana Victoria; Chicharro, Carmen; Sanchez Herrero, Carmen; Garcia, Emilia; Ortega, Sheila; Ndung'u, Joseph Mathu; Moreno, Javier; Cruz, Israel; Carrillo, Eugenia; Federal Ministry of Education & Research (Alemania); Ministry of Foreign Affairs (Holanda); RETICS-Investigación colaborativa en Enfermedades Tropicales (RICET-ISCIII) (España); Unión Europea. Fondo Europeo de Desarrollo Regional (FEDER/ERDF)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™ Leishmania 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 L. infantum. A total of 230 DNA samples from cutaneous (CL) and visceral (VL) leishmaniasis cases and controls from Spain, characterized by Leishmania nested PCR (LnPCR) were tested by: (i) the Loopamp™ Leishmania 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 (Tp) obtained by real-time fluorimetry showed excellent concordance (C = 97.91%) and strong correlation (r = 0.799) with qPCR's cycle threshold (Ct). The performance of Loopamp is comparable to that of LnPCR and qPCR in the diagnosis of cutaneous and visceral leishmaniasis due to L. infantum. The excellent correlation between the Tp and Ct should be further investigated to determine the accuracy of Loopamp to quantify parasite load in tissues.Publication Molecular Diagnosis of Leishmaniasis in Spain: Development and Validation of Ready-To-Use Gel-Form Nested and Real-Time PCRs To Detect Leishmania spp(American Society for Microbiology (ASM), 2023-06-15) Chicharro, Carmen; Nieto Martinez, Francisco Javier; Miguelañez, Silvia; Garcia, Emilia; Ortega, Sheila; Peña Gallego, Ana; Rubio Muñoz, Jose Miguel; Flores-Chavez, Maria; Ministerio de Economía y Competitividad (España); Fundación Mundo SanoLeishmaniasis is an endemic parasitic disease in at least 98 countries. In Spain, it is considered a zoonosis caused by Leishmania infantum, with an annual incidence of 0.62 cases/100,000 inhabitants. The predominant clinical manifestations are the cutaneous (CL) and visceral forms (VL), and the diagnosis is performed by parasitological, serological, and molecular tests. At the WHO Collaborating Center for Leishmaniasis (WHOCCLeish), routine diagnostic tests are based on a nested PCR (Ln-PCR), culture, and serological tests. To simplify our PCR protocol, we aimed to develop and validate a ready-to-use nested gel-form PCR (LeishGelPCR) and a duplex real-time PCR (qPCR) that allowed simultaneous detection of Leishmania and mammalian DNA as an internal control (Leish-qPCR). Clinical validation was performed in 200 samples from the WHOCCLeish collection; 92 and 85 out of 94 and 87 samples were positive by LeishGelPCR and Leish-qPCR, respectively, showing a sensitivity of 98% in both approaches. The specificity was 100% for LeishGelPCR and 98% for Leish-qPCR. The limits of detection of both protocols were similar (0.5 and 0.2 parasites/reaction). Parasite loads in VL and CL forms were similar, although high loads were observed when invasive samples were tested. In conclusion, LeishGelPCR and Leish-qPCR showed excellent performance in the diagnosis of leishmaniasis. These new forms of 18S rRNA gene PCR are equivalent to Ln-PCR and can be introduced in the algorithm for CL and VL diagnosis. IMPORTANCE Although the gold standard for diagnosis of leishmaniasis is the microscopic observation of amastigotes, molecular techniques are becoming a cost-efficient alternative. Currently, PCR is a routine resource that is used in many reference microbiology laboratories. In this article, we have described two ways to improve the reproducibility and usability of the molecular detection of Leishmania spp. These new approaches could be introduced even in middle- and low-resource laboratories; one is a ready-to-use gel-form system of a nested PCR and the other is a real-time PCR. We show why molecular diagnosis is the best methodology to confirm a clinical suspicion of leishmaniasis with higher sensitivity than traditional methods, thus facilitating early diagnosis and timely treatment of human leishmaniasis.Publication Molecular typing of Leishmania infantum isolates from a leishmaniasis outbreak in Madrid, Spain, 2009 to 2012(European Centre for Disease Prevention and Control (ECDC), 2013-07-25) Chicharro, Carmen; Llanes-Acevedo, Ivonne Pamela; Garcia, Emilia; Nieto Martinez, Francisco Javier; Moreno, Javier; Cruz, Israel; Instituto de Salud Carlos IIILeishmaniasis is endemic in south-west Europe. Recent data point to the spread and (re-)emergence of this disease in previously endemic and non-endemic European countries. A recent example is the urban community outbreak of cutaneous and visceral leishmaniasis in the south-west of Madrid autonomous community, Spain, which began on 1 July 2009. A total of 446 cases associated to this outbreak were reported up to 31 December 2012. We show molecular typing data for 73 Leishmania infantum isolates obtained from January 2008 to July 2012 from different areas of Madrid, including those affected by the outbreak. Seven different genotypes were identified by combining data from two targets: the ribosomal internal transcribed spacers (ITS)-1 and -2 and the haspb (k26) gene. The results contribute to a better understanding of the parasite population circulating in the region, and indicate that most of the outbreak-associated isolates (22/31) were infected by parasites with the same combined genotype. Additional data from 82 L. infantum isolates typed as either MON-1 or MON-24 by isoenzyme analysis indicate that far from concluding that the outbreak was caused by a 'new' emerging genotype, further molecular typing-based surveillance studies are required to better understand the epidemiology of leishmaniasis in the region.Publication Parasitemia Levels in Trypanosoma cruzi Infection in Spain, an Area Where the Disease Is Not Endemic: Trends by Different Molecular Approaches(American Society for Microbiology (ASM), 2022-10-26) Flores-Chavez, Maria; Abras, Alba; Ballart, Cristina; Perez-Gordillo, Pilar; Gállego, Montserrat; Muñoz, Carmen; Moure, Zaira; Sulleiro, Elena; Nieto Martinez, Francisco Javier; Garcia, Emilia; Simon Mendez, Lorena; Cruz, Israel; Picado, Albert; Ibañez-Perez, Ismael; Foundation for Innovative New Diagnostics (Suiza); Instituto de Salud Carlos III; Fundación Mundo Sano; Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR)Trypanosoma cruzi infection has expanded globally through human migration. In Spain, the mother-to-child route is the mode of transmission contributing to autochthonous Chagas disease (CD); however, most people acquired the infection in their country of origin and were diagnosed in the chronic phase (imported chronic CD). In this context, we assessed the quantitative potential of the Loopamp Trypanosoma cruzi detection kit (Sat-TcLAMP) based on satellite DNA (Sat-DNA) to determine parasitemia levels compared to those detected by real-time quantitative PCRs (qPCRs) targeting Sat-DNA (Sat-qPCR) and kinetoplast DNA minicircles (kDNA-qPCR). This study included 173 specimens from 39 autochthonous congenital and 116 imported chronic CD cases diagnosed in Spain. kDNA-qPCR showed higher sensitivity than Sat-qPCR and Sat-TcLAMP. According to all quantitative approaches, parasitemia levels were significantly higher in congenital infection than in chronic CD (1 × 10-1 to 5 × 105 versus >1 × 10-1 to 6 × 103 parasite equivalents/mL, respectively [P < 0.001]). Sat-TcLAMP, Sat-qPCR, and kDNA-qPCR results were equivalent at high levels of parasitemia (P = 0.381). Discrepancies were significant for low levels of parasitemia and older individuals. Differences between Sat-TcLAMP and Sat-qPCR were not qualitatively significant, but estimations of parasitemia using Sat-TcLAMP were closer to those by kDNA-qPCR. Parasitemia changes were assessed in 6 individual cases in follow-up, in which trends showed similar patterns by all quantitative approaches. At high levels of parasitemia, Sat-TcLAMP, Sat-qPCR, and kDNA-qPCR worked similarly, but significant differences were found for the low levels characteristic of late chronic CD. A suitable harmonization strategy needs to be developed for low-level parasitemia detection using Sat-DNA- and kDNA-based tests. IMPORTANCE: Currently, molecular equipment has been introduced into many health care centers, even in low-income countries. PCR, qPCR, and loop-mediated isothermal amplification (LAMP) are becoming more accessible for the diagnosis of neglected infectious diseases. Chagas disease (CD) is spreading worldwide, and in countries where the disease is not endemic, such as Spain, the parasite Trypanosoma cruzi is transmitted from mother to child (congenital CD). Here, we explore why LAMP, aimed at detecting T. cruzi parasite DNA, is a reliable option for the diagnosis of congenital CD and the early detection of reactivation in chronic infection. When the parasite load is high, LAMP is equivalent to any qPCR. In addition, the estimations of T. cruzi parasitemia in patients living in Spain, a country where the disease is not endemic, resemble natural evolution in areas of endemicity. If molecular tests are introduced into the diagnostic algorithm for congenital infection, early diagnosis and timely treatment would be accomplished, so the interruption of vertical transmission can be an achievable goal.Publication Predicción de la Incidencia Mínima de Sida en España para el Período 1992–1995(Elsevier, 1993) Castilla, Jesús; Zunzunegui, María-Victoria; Garcia, Emilia[ES] Para estimar la incidencia mínima de SIDA en España entre los años 1992 al 1995 se ha obtenido del Registro Nacional, actualizado a junio de 1992, la incidencia anual de SIDA hasta el año 1991. Se ha corregido el retraso en la notificación de los casos diagnosticados desde julio de 1989, para aplicar después el método de retroproyección al total de casos y a cada categoría de transmisión por separado. Se asumió que no se han producido nuevas infecciones por el VIH después de 1991. Entre 1981 y 1995 se habrán diagnosticado más de 38000 casos de SIDA. La predicción mínima de la incidencia de SIDA presenta un aumento entre 1992 y 1995 para el total de casos y para todas las categorías de transmisión, salvo para los receptores de sangre y hemoderivados, y los hijos de madres de riesgo. La incidencia real será probablemente mayor que la estimada, por el efecto de las nuevas infecciones que se puedan producir y de la subnotificación de casos. [EN] In order to estimate minimum AIDS incidence in Spain between 1992 and 1995, annual AIDS incidence up to 1991 has been obtained from the June 1992 update of the National Register. Correction was made for reporting delays in cases diagnosed since July 1989, in order to run a subsequent back calculation on all cases and for each separate mode of transmission. It was assumed that no new HIV infections would appear after 1991. Since 1981 to 1995, more than 38,000 AIDS cases will have been diagnosed. Minimum AIDS incidence as forecast exhibits a rise between 1992 and 1995 for the total number of cases and for all categories of transmission, except for recipients of blood and blood products, and children of mothers at risk. Real incidence will probably prove higher than estimated owing to the effect of new infections which may arise, and to the underreporting of cases.Publication Validation of rK39 immunochromatographic test and direct agglutination test for the diagnosis of Mediterranean visceral leishmaniasis in Spain(Public Library of Science (PLOS), 2018-03-01) Bangert, Mathieu; Flores-Chavez, Maria; Llanes-Acevedo, Ivonne Pamela; Arcones, Carolina; Chicharro, Carmen; Garcia, Emilia; Ortega, Sheila; Nieto Martinez, Francisco Javier; Cruz, Israel; Instituto de Salud Carlos IIIBACKGROUND: 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.