38 results on '"Adaui V"'
Search Results
2. Putative markers of infective life stages in Leishmania (Viannia) braziliensis
- Author
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GAMBOA, D., VAN EYS, G., VICTOIR, K., TORRES, K., ADAUI, V., AREVALO, J., and DUJARDIN, J. -C.
- Published
- 2007
3. Proviral load and immune markers associated with human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in Peru
- Author
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Best, I., Adaui, V., Verdonck, K., González, E., Tipismana, M., Clark, D., Gotuzzo, E., and Vanham, G.
- Published
- 2006
4. Comparison of gene expression patterns among Leishmania braziliensis clinical isolates showing a different in vitro susceptibility to pentavalent antimony
- Author
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ADAUI, V., SCHNORBUSCH, K., ZIMIC, M., GUTIÉRREZ, A., DECUYPERE, S., VANAERSCHOT, M., DE DONCKER, S., MAES, I., LLANOS-CUENTAS, A., CHAPPUIS, F., ARÉVALO, J., DUJARDIN, J.-C, ADAUI, V., SCHNORBUSCH, K., ZIMIC, M., GUTIÉRREZ, A., DECUYPERE, S., VANAERSCHOT, M., DE DONCKER, S., MAES, I., LLANOS-CUENTAS, A., CHAPPUIS, F., ARÉVALO, J., and DUJARDIN, J.-C
- Abstract
Introduction. Evaluation of Leishmania drug susceptibility depends on in vitro SbV susceptibility assays, which are labour-intensive and may give a biased view of the true parasite resistance. Molecular markers are urgently needed to improve and simplify the monitoring of SbV-resistance. We analysed here the gene expression profile of 21 L. braziliensis clinical isolates in vitro defined as SbV-resistant and -sensitive, in order to identify potential resistance markers. Methods. The differential expression of 13 genes involved in SbV metabolism, oxidative stress or housekeeping functions was analysed during in vitro promastigote growth. Results. Expression profiles were up-regulated for 5 genes only, each time affecting a different set of isolates (mosaic picture of gene expression). Two genes, ODC (ornithine decarboxylase) and TRYR (trypanothione reductase), showed a significantly higher expression rate in the group of SbV-resistant compared to the group of SbV-sensitive parasites (P<0·01). However, analysis of individual isolates showed both markers to explain only partially the drug resistance. Discussion. Our results might be explained by (i) the occurrence of a pleiotropic molecular mechanism leading to the in vitro SbV resistance and/or (ii) the existence of different epi-phenotypes not revealed by the in vitro SbV susceptibility assays, but interfering with the gene expression patterns
- Published
- 2017
5. Controversial role of leishmania RNA virus as a determinant of pathogenicity in human leishmaniasis
- Author
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Valencia, B., primary, Jara, M., additional, Adaui, V., additional, Chantry, M., additional, Alba, M., additional, Ramos, A., additional, Arevalo, J., additional, Llanos-Cuentas, A., additional, and Boggild, A.K., additional
- Published
- 2014
- Full Text
- View/download PDF
6. Comparison of gene expression patterns amongLeishmania braziliensisclinical isolates showing a differentin vitrosusceptibility to pentavalent antimony
- Author
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ADAUI, V., primary, SCHNORBUSCH, K., additional, ZIMIC, M., additional, GUTIÉRREZ, A., additional, DECUYPERE, S., additional, VANAERSCHOT, M., additional, DE DONCKER, S., additional, MAES, I., additional, LLANOS-CUENTAS, A., additional, CHAPPUIS, F., additional, ARÉVALO, J., additional, and DUJARDIN, J.-C., additional
- Published
- 2010
- Full Text
- View/download PDF
7. American tegumentary leishmaniasis: is antimonial treatment outcome related to parasite drug susceptibility?
- Author
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Yardley V, Ortuño N, Llanos-Cuentas A, Chappuis F, De Doncker S, Ramirez L, Croft S, Arevalo J, Adaui V, Bermudez H, Decuypere S, and Dujardin J
- Abstract
Background. Antimonials are the first drug of choice for the treatment of American tegumentary leishmaniasis (ATL); however, their efficacy is not predictable, and this may be linked to parasite drug resistance. We aimed to characterize the in vitro antimony susceptibility of clinical isolates of Peruvian patients with ATL who were treated with sodium stibogluconate and to correlate this in vitro phenotype with different treatment outcomes. Methods. Thirty-seven clinical isolates were obtained from patients with known disease and treatment histories. These isolates were typed, and the susceptibility of intracellular amastigotes to pentavalent (SbV) and trivalent (SbIII) antimonials was determined. Results. We observed 29 SbV-resistant isolates among 4 species of subgenus Viannia, most of which exhibited primary resistance; isolates resistant only to SbIII; and 3 combinations of in vitro phenotypes: (1) parasites sensitive to both drugs, (2) parasites resistant to both drugs, and (3) parasites resistant to SbV only (the majority of isolates fell into this category). There was no correlation between in vitro susceptibility to both antimonials and the clinical outcome of therapy. Conclusion. Antimony insensitivity might occur in a stepwise fashion (first to SbV and then to SbIII). Our data question the definition of true parasite resistance to antimonials. Further studies of treatment efficacy should apply standardized protocols and definitions and should also consider host factors. Copyright © 2006 Infectious Diseases Society of America [ABSTRACT FROM AUTHOR]
- Published
- 2006
8. Evaluation of Candidate Gene and Viral Markers for HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis in Peruvian HTLV-1-Infected Individuals
- Author
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Talledo, M., Lopez, G., Huyghe, J., Verdonck, K., Gonzalez, E., Adaui, V., Ivan Best, Tipismana, M., Clark, D., Vanham, G., Gotuzzo, E., Camp, G., and Laer, L.
9. Comparison of gene expression patterns among Leishmania braziliensis clinical isolates showing a different in vitro susceptibility to pentavalent antimony
- Author
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ADAUI, V., SCHNORBUSCH, K., ZIMIC, M., GUTIÉRREZ, A., DECUYPERE, S., VANAERSCHOT, M., DE DONCKER, S., MAES, I., LLANOS-CUENTAS, A., CHAPPUIS, F., ARÉVALO, J., DUJARDIN, J.-C, ADAUI, V., SCHNORBUSCH, K., ZIMIC, M., GUTIÉRREZ, A., DECUYPERE, S., VANAERSCHOT, M., DE DONCKER, S., MAES, I., LLANOS-CUENTAS, A., CHAPPUIS, F., ARÉVALO, J., and DUJARDIN, J.-C
- Abstract
Introduction. Evaluation of Leishmania drug susceptibility depends on in vitro SbV susceptibility assays, which are labour-intensive and may give a biased view of the true parasite resistance. Molecular markers are urgently needed to improve and simplify the monitoring of SbV-resistance. We analysed here the gene expression profile of 21 L. braziliensis clinical isolates in vitro defined as SbV-resistant and -sensitive, in order to identify potential resistance markers. Methods. The differential expression of 13 genes involved in SbV metabolism, oxidative stress or housekeeping functions was analysed during in vitro promastigote growth. Results. Expression profiles were up-regulated for 5 genes only, each time affecting a different set of isolates (mosaic picture of gene expression). Two genes, ODC (ornithine decarboxylase) and TRYR (trypanothione reductase), showed a significantly higher expression rate in the group of SbV-resistant compared to the group of SbV-sensitive parasites (P<0·01). However, analysis of individual isolates showed both markers to explain only partially the drug resistance. Discussion. Our results might be explained by (i) the occurrence of a pleiotropic molecular mechanism leading to the in vitro SbV resistance and/or (ii) the existence of different epi-phenotypes not revealed by the in vitro SbV susceptibility assays, but interfering with the gene expression patterns
10. Comparison of gene expression patterns among Leishmania braziliensis clinical isolates showing a different in vitro susceptibility to pentavalent antimony
- Author
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ADAUI, V., SCHNORBUSCH, K., ZIMIC, M., GUTIÉRREZ, A., DECUYPERE, S., VANAERSCHOT, M., DE DONCKER, S., MAES, I., LLANOS-CUENTAS, A., CHAPPUIS, F., ARÉVALO, J., DUJARDIN, J.-C, ADAUI, V., SCHNORBUSCH, K., ZIMIC, M., GUTIÉRREZ, A., DECUYPERE, S., VANAERSCHOT, M., DE DONCKER, S., MAES, I., LLANOS-CUENTAS, A., CHAPPUIS, F., ARÉVALO, J., and DUJARDIN, J.-C
- Abstract
Introduction. Evaluation of Leishmania drug susceptibility depends on in vitro SbV susceptibility assays, which are labour-intensive and may give a biased view of the true parasite resistance. Molecular markers are urgently needed to improve and simplify the monitoring of SbV-resistance. We analysed here the gene expression profile of 21 L. braziliensis clinical isolates in vitro defined as SbV-resistant and -sensitive, in order to identify potential resistance markers. Methods. The differential expression of 13 genes involved in SbV metabolism, oxidative stress or housekeeping functions was analysed during in vitro promastigote growth. Results. Expression profiles were up-regulated for 5 genes only, each time affecting a different set of isolates (mosaic picture of gene expression). Two genes, ODC (ornithine decarboxylase) and TRYR (trypanothione reductase), showed a significantly higher expression rate in the group of SbV-resistant compared to the group of SbV-sensitive parasites (P<0·01). However, analysis of individual isolates showed both markers to explain only partially the drug resistance. Discussion. Our results might be explained by (i) the occurrence of a pleiotropic molecular mechanism leading to the in vitro SbV resistance and/or (ii) the existence of different epi-phenotypes not revealed by the in vitro SbV susceptibility assays, but interfering with the gene expression patterns
11. Diversity and dissemination of viruses in pathogenic protozoa.
- Author
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Heeren S, Maes I, Sanders M, Lye LF, Adaui V, Arevalo J, Llanos-Cuentas A, Garcia L, Lemey P, Beverley SM, Cotton JA, Dujardin JC, and Van den Broeck F
- Subjects
- Humans, Ecosystem, Peru epidemiology, Leishmaniasis, Cutaneous parasitology, Leishmania braziliensis genetics, Leishmania genetics
- Abstract
Viruses are the most abundant biological entities on Earth and play a significant role in the evolution of many organisms and ecosystems. In pathogenic protozoa, the presence of viruses has been linked to an increased risk of treatment failure and severe clinical outcome. Here, we studied the molecular epidemiology of the zoonotic disease cutaneous leishmaniasis in Peru and Bolivia through a joint evolutionary analysis of Leishmania braziliensis and their dsRNA Leishmania virus 1. We show that parasite populations circulate in tropical rainforests and are associated with single viral lineages that appear in low prevalence. In contrast, groups of hybrid parasites are geographically and ecologically more dispersed and associated with an increased prevalence, diversity and spread of viruses. Our results suggest that parasite gene flow and hybridization increased the frequency of parasite-virus symbioses, a process that may change the epidemiology of leishmaniasis in the region., (© 2023. The Author(s).)
- Published
- 2023
- Full Text
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12. Novel CRISPR-based detection of Leishmania species.
- Author
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Dueñas E, Nakamoto JA, Cabrera-Sosa L, Huaihua P, Cruz M, Arévalo J, Milón P, and Adaui V
- Abstract
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 × 100 (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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Dueñas, Nakamoto, Cabrera-Sosa, Huaihua, Cruz, Arévalo, Milón and Adaui.)- Published
- 2022
- Full Text
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13. UnCovid: A versatile, low-cost, and open-source protocol for SARS-CoV-2 RNA detection.
- Author
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Alcántara R, Peñaranda K, Mendoza-Rojas G, Nakamoto JA, Dueñas E, Alvarez D, Adaui V, and Milón P
- Subjects
- COVID-19 genetics, COVID-19 virology, Humans, SARS-CoV-2 isolation & purification, COVID-19 diagnosis, CRISPR-Cas Systems, Nucleic Acid Amplification Techniques methods, RNA, Viral analysis, RNA, Viral genetics, SARS-CoV-2 genetics
- Abstract
Here, we describe a detailed step-by-step protocol to detect SARS-CoV-2 RNA using RT-PCR-mediated amplification and CRISPR/Cas-based visualization. The optimized assay uses basic molecular biology equipment such as conventional thermocyclers and transilluminators for qualitative detection. Alternatively, a fluorescence plate reader can be used for quantitative measurements. The protocol detects two regions of the SARS-CoV-2 genome in addition to the human RNaseP sample control. Aiming to reach remote regions, this work was developed to use the portable molecular workstation from BentoLab. For complete details on the use and execution of this protocol, please refer to Alcántara et al., 2021., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)
- Published
- 2021
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14. A low-cost and open-source protocol to produce key enzymes for molecular detection assays.
- Author
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Mendoza-Rojas G, Sarabia-Vega V, Sanchez-Castro A, Tello L, Cabrera-Sosa L, Nakamoto JA, Peñaranda K, Adaui V, Alcántara R, and Milón P
- Subjects
- Chromatography, Affinity, Enzyme Assays, Molecular Typing, Transformation, Bacterial, Enzymes genetics, Enzymes isolation & purification, Enzymes metabolism, Escherichia coli genetics, Escherichia coli metabolism, Recombinant Proteins genetics, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism
- Abstract
Here, we describe a detailed step-by-step protocol for the expression, purification, quantification, and activity determination of key enzymes for molecular detection of pathogens. Based on previous reports, we optimized the protocol for LbCas12a, Taq DNA polymerase, M-MLV reverse transcriptase, and TEV protease to make it compatible with minimal laboratory equipment, broadly available in low- and middle-income countries. The enzymes produced with this protocol have been successfully used for molecular detection applications. For complete details on the use and execution of this protocol, please refer to Alcántara et al. (2021a, 2021b)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)
- Published
- 2021
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15. Unlocking SARS-CoV-2 detection in low- and middle-income countries.
- Author
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Alcántara R, Peñaranda K, Mendoza-Rojas G, Nakamoto JA, Martins-Luna J, Del Valle-Mendoza J, Adaui V, and Milón P
- Subjects
- Humans, Developing Countries, RNA, Viral genetics, Sensitivity and Specificity, Nucleic Acid Amplification Techniques, SARS-CoV-2 genetics, COVID-19 diagnosis
- Abstract
Low- and middle-income countries (LMICs) are significantly affected by SARS-CoV-2, partially due to their limited capacity for local production and implementation of molecular testing. Here, we provide detailed methods and validation of a molecular toolkit that can be readily produced and deployed using laboratory equipment available in LMICs. Our results show that lab-scale production of enzymes and nucleic acids can supply over 50,000 tests per production batch. The optimized one-step RT-PCR coupled to CRISPR-Cas12a-mediated detection showed a limit of detection of 10
2 ge/μL in a turnaround time of 2 h. The clinical validation indicated an overall sensitivity of 80%-88%, while for middle and high viral load samples (Cq ≤ 31) the sensitivity was 92%-100%. The specificity was 96%-100% regardless of viral load. Furthermore, we show that the toolkit can be used with the mobile laboratory Bento Lab, potentially enabling LMICs to implement detection services in unattended remote regions., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)- Published
- 2021
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16. Toxicity assessment of synthetic chalcones with antileishmanial potential in BALB/c mice.
- Author
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Cancino K, Castro I, Yauri C, Jullian V, Arévalo J, Sauvain M, Adaui V, and Castillo D
- Subjects
- Animals, Mice, Mice, Inbred BALB C, Antiprotozoal Agents therapeutic use, Chalcone, Chalcones toxicity, Leishmaniasis
- Abstract
Objective: To evaluate the toxicity of three synthetic chalcones administered intraperitoneally to BALB/c mice., Materials and Methods: The median lethal dose (LD50) was estimated by Dixon's Up-and-Down method. Subchronic toxicity of chalcones was evaluated at 20 and 40 mg/kg for 21 days. Behavioral, physiological, biochemical, and histological toxic effects were evaluated., Results: Chalcone 43 produced mucus in feces, visceral damage (liver) and alterations in organ coefficient (kidney, p = 0.037 and brain, p = 0.008) when compared to the control group. In addition, histological analysis showed that this chalcone produced edema, inflammation and necrosis in the evaluated organs, although there was no significant difference with the control. None of the biochemical parameters differed significantly between the treatment groups at 40 mg/kg dose and the control., Conclusions: The LD50 for all three chalcones was greater than 550 mg/kg of body weight. Chalcones 40 and 42 were found to be relatively non-toxic. Both can be considered safe for intraperitoneal application in BALB/c mice and, consequently, are potential candidates for use in the treatment of leishmaniasis.
- Published
- 2021
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17. Ecological divergence and hybridization of Neotropical Leishmania parasites.
- Author
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Van den Broeck F, Savill NJ, Imamura H, Sanders M, Maes I, Cooper S, Mateus D, Jara M, Adaui V, Arevalo J, Llanos-Cuentas A, Garcia L, Cupolillo E, Miles M, Berriman M, Schnaufer A, Cotton JA, and Dujardin JC
- Subjects
- Ecosystem, Forests, Genetic Speciation, Humans, Leishmania braziliensis pathogenicity, Leishmaniasis, Cutaneous epidemiology, Leishmaniasis, Cutaneous parasitology, Peru epidemiology, Phylogeography, Genome, Mitochondrial genetics, Host-Parasite Interactions genetics, Leishmania braziliensis genetics, Leishmaniasis, Cutaneous genetics
- Abstract
The tropical Andes are an important natural laboratory to understand speciation in many taxa. Here we examined the evolutionary history of parasites of the Leishmania braziliensis species complex based on whole-genome sequencing of 67 isolates from 47 localities in Peru. We first show the origin of Andean Leishmania as a clade of near-clonal lineages that diverged from admixed Amazonian ancestors, accompanied by a significant reduction in genome diversity and large structural variations implicated in host-parasite interactions. Within the Andean species, patterns of population structure were strongly associated with biogeographical origin. Molecular clock and ecological niche modeling suggested that the history of diversification of the Andean lineages is limited to the Late Pleistocene and intimately associated with habitat contractions driven by climate change. These results suggest that changes in forestation over the past 150,000 y have influenced speciation and diversity of these Neotropical parasites. Second, genome-scale analyses provided evidence of meiotic-like recombination between Andean and Amazonian Leishmania species, resulting in full-genome hybrids. The mitochondrial genome of these hybrids consisted of homogeneous uniparental maxicircles, but minicircles originated from both parental species. We further show that mitochondrial minicircles-but not maxicircles-show a similar evolutionary pattern to the nuclear genome, suggesting that compatibility between nuclear-encoded mitochondrial genes and minicircle-encoded guide RNA genes is essential to maintain efficient respiration. By comparing full nuclear and mitochondrial genome ancestries, our data expand our appreciation on the genetic consequences of diversification and hybridization in parasitic protozoa., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)
- Published
- 2020
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18. Application of CRISPR/Cas9-Based Reverse Genetics in Leishmania braziliensis : Conserved Roles for HSP100 and HSP23.
- Author
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Adaui V, Kröber-Boncardo C, Brinker C, Zirpel H, Sellau J, Arévalo J, Dujardin JC, and Clos J
- Subjects
- Endopeptidase Clp metabolism, Gene Editing, Gene Targeting, Genes, Protozoan, Heat-Shock Proteins metabolism, Leishmania braziliensis physiology, Leishmania major genetics, Leishmania major physiology, Mutation, Polymerase Chain Reaction, Protozoan Proteins metabolism, Thermotolerance, CRISPR-Cas Systems, Endopeptidase Clp genetics, Heat-Shock Proteins genetics, Leishmania braziliensis genetics, Protozoan Proteins genetics, Reverse Genetics
- Abstract
The protozoan parasite Leishmania ( Viannia ) braziliensis (L. braziliensis ) is the main cause of human tegumentary leishmaniasis in the New World, a disease affecting the skin and/or mucosal tissues. Despite its importance, the study of the unique biology of L. braziliensis through reverse genetics analyses has so far lagged behind in comparison with Old World Leishmania spp. In this study, we successfully applied a cloning-free, PCR-based CRISPR-Cas9 technology in L. braziliensis that was previously developed for Old World Leishmania major and New World L. mexicana species. As proof of principle, we demonstrate the targeted replacement of a transgene ( eGFP ) and two L. braziliensis single-copy genes ( HSP23 and HSP100 ). We obtained homozygous Cas9-free HSP23 - and HSP100 -null mutants in L. braziliensis that matched the phenotypes reported previously for the respective L. donovani null mutants. The function of HSP23 is indeed conserved throughout the Trypanosomatida as L. major HSP23 null mutants could be complemented phenotypically with transgenes from a range of trypanosomatids. In summary, the feasibility of genetic manipulation of L. braziliensis by CRISPR-Cas9-mediated gene editing sets the stage for testing the role of specific genes in that parasite's biology, including functional studies of virulence factors in relevant animal models to reveal novel therapeutic targets to combat American tegumentary leishmaniasis.
- Published
- 2020
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19. A Case-Control Study on the Association Between Intestinal Helminth Infections and Treatment Failure in Patients With Cutaneous Leishmaniasis.
- Author
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Martínez DY, Llanos-Cuentas A, Dujardin JC, Polman K, Adaui V, Boelaert M, and Verdonck K
- Abstract
Background: Endemic regions of cutaneous leishmaniasis (CL) and intestinal helminthiasis overlap. CL treatment with systemic pentavalent antimonial drugs (Sb
5+ ) fails in 10%-30% of patients. The study objective was to assess the etiological role of intestinal helminthiasis in CL treatment failure., Methods: An unmatched case-control study was done in 4 CL treatment sites in Peru in 2012-2015. Cases were CL patients with Sb5+ treatment failure; controls were CL patients with Sb5+ treatment success. Patients with a parasitologically confirmed CL diagnosis who had received supervised Sb5+ treatment and could be classified as cases or controls were eligible. The main exposure variables were intestinal helminthiasis and strongyloidiasis, diagnosed through direct examination, rapid sedimentation, Baermann, Kato-Katz, or agar culture of stool samples. Additional exposure variables were other infections (HIV, human T-lymphotropic virus 1, tuberculosis, hepatitis B, intestinal protozoa) and noninfectious conditions (diabetes, renal insufficiency, and immunosuppressive medication). Age, gender, CL history, probable exposure place, and Leishmania species were treated as potential confounders in multiple logistic regression., Results: There were 94 case and 122 control subjects. Overall, infectious and noninfectious comorbidities were frequent both among cases (64%) and controls (71%). The adjusted odds ratio (OR) for the association between any intestinal helminth infection and CL treatment failure was 0.65 (95% confidence interval [CI], 0.30-1.38), and the adjusted OR for the association between strongyloidiasis and CL treatment failure was 0.34 (95% CI, 0.11-0.92)., Conclusions: In the Peruvian setting, high Sb5+ treatment failure rates are not explained by intestinal helminthiasis. On the contrary, strongyloidiasis had a protective effect against treatment failure., (© The Author(s) 2020. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)- Published
- 2020
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20. Tegumentary leishmaniasis and coinfections other than HIV.
- Author
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Martínez DY, Verdonck K, Kaye PM, Adaui V, Polman K, Llanos-Cuentas A, Dujardin JC, and Boelaert M
- Subjects
- Animals, Argentina epidemiology, Brazil epidemiology, Disease Models, Animal, Humans, Skin pathology, Coinfection epidemiology, Coinfection parasitology, Leishmaniasis, Cutaneous epidemiology
- Abstract
Background: Tegumentary leishmaniasis (TL) is a disease of skin and/or mucosal tissues caused by Leishmania parasites. TL patients may concurrently carry other pathogens, which may influence the clinical outcome of TL., Methodology and Principal Findings: This review focuses on the frequency of TL coinfections in human populations, interactions between Leishmania and other pathogens in animal models and human subjects, and implications of TL coinfections for clinical practice. For the purpose of this review, TL is defined as all forms of cutaneous (localised, disseminated, or diffuse) and mucocutaneous leishmaniasis. Human immunodeficiency virus (HIV) coinfection, superinfection with skin bacteria, and skin manifestations of visceral leishmaniasis are not included. We searched MEDLINE and other databases and included 73 records: 21 experimental studies in animals and 52 studies about human subjects (mainly cross-sectional and case studies). Several reports describe the frequency of Trypanosoma cruzi coinfection in TL patients in Argentina (about 41%) and the frequency of helminthiasis in TL patients in Brazil (15% to 88%). Different hypotheses have been explored about mechanisms of interaction between different microorganisms, but no clear answers emerge. Such interactions may involve innate immunity coupled with regulatory networks that affect quality and quantity of acquired immune responses. Diagnostic problems may occur when concurrent infections cause similar lesions (e.g., TL and leprosy), when different pathogens are present in the same lesions (e.g., Leishmania and Sporothrix schenckii), or when similarities between phylogenetically close pathogens affect accuracy of diagnostic tests (e.g., serology for leishmaniasis and Chagas disease). Some coinfections (e.g., helminthiasis) appear to reduce the effectiveness of antileishmanial treatment, and drug combinations may cause cumulative adverse effects., Conclusions and Significance: In patients with TL, coinfection is frequent, it can lead to diagnostic errors and delays, and it can influence the effectiveness and safety of treatment. More research is needed to unravel how coinfections interfere with the pathogenesis of TL.
- Published
- 2018
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21. Association of the Endobiont Double-Stranded RNA Virus LRV1 With Treatment Failure for Human Leishmaniasis Caused by Leishmania braziliensis in Peru and Bolivia.
- Author
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Adaui V, Lye LF, Akopyants NS, Zimic M, Llanos-Cuentas A, Garcia L, Maes I, De Doncker S, Dobson DE, Arevalo J, Dujardin JC, and Beverley SM
- Subjects
- Antimony therapeutic use, Antiprotozoal Agents therapeutic use, Bolivia epidemiology, Cohort Studies, Drug Resistance, Humans, Leishmaniasis, Mucocutaneous epidemiology, Leishmaniasis, Mucocutaneous parasitology, Peru epidemiology, Treatment Failure, Leishmania braziliensis virology, Leishmaniasis, Mucocutaneous drug therapy, Leishmaniasis, Mucocutaneous virology, Leishmaniavirus classification, Leishmaniavirus genetics
- Abstract
Cutaneous and mucosal leishmaniasis, caused in South America by Leishmania braziliensis, is difficult to cure by chemotherapy (primarily pentavalent antimonials [Sb(V)]). Treatment failure does not correlate well with resistance in vitro, and the factors responsible for treatment failure in patients are not well understood. Many isolates of L. braziliensis (>25%) contain a double-stranded RNA virus named Leishmaniavirus 1 (LRV1), which has also been reported in Leishmania guyanensis, for which an association with increased pathology, metastasis, and parasite replication was found in murine models. Here we probed the relationship of LRV1 to drug treatment success and disease in 97 L. braziliensis-infected patients from Peru and Bolivia. In vitro cultures were established, parasites were typed as L. braziliensis, and the presence of LRV1 was determined by reverse transcription-polymerase chain reaction, followed by sequence analysis. LRV1 was associated significantly with an increased risk of treatment failure (odds ratio, 3.99; P = .04). There was no significant association with intrinsic Sb(V) resistance among parasites, suggesting that treatment failure arises from LRV1-mediated effects on host metabolism and/or parasite survival. The association of LRV1 with clinical drug treatment failure could serve to guide more-effective treatment of tegumentary disease caused by L. braziliensis., (© The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)
- Published
- 2016
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22. Quantitative Kinetoplast DNA Assessment During Treatment of Mucosal Leishmaniasis as a Potential Biomarker of Outcome: A Pilot Study.
- Author
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Jara M, Valencia BM, Adaui V, Alba M, Lau R, Arevalo J, Llanos-Cuentas A, and Boggild AK
- Subjects
- Adolescent, Adult, Aged, Aged, 80 and over, Amphotericin B administration & dosage, Antimony Sodium Gluconate administration & dosage, Biomarkers, Child, DNA, Protozoan genetics, DNA, Protozoan isolation & purification, Female, Humans, Male, Middle Aged, Pilot Projects, Real-Time Polymerase Chain Reaction, Treatment Outcome, Young Adult, Amphotericin B therapeutic use, Antimony Sodium Gluconate therapeutic use, DNA, Kinetoplast genetics, Leishmaniasis, Mucocutaneous parasitology
- Abstract
Mucosal leishmaniasis (ML) is a disfiguring manifestation of Leishmania (Viannia) infection. We evaluated parasite load (PL) over time as a potential biomarker of treatment outcome in ML. PL was assessed with kinetoplast DNA quantitative real-time polymerase chain reaction (kDNA-qPCR) at enrollment, days 14 and 21-28 of therapy and 3, 6, 12-18, and 18-24 months after treatment of ML and correlated to demographic, clinical, and parasitologic factors. Forty-four patients were enrolled: 30 men and 14 women. Enrollment PL differed significantly by causative species (P < 0.001), and was higher in patients with severe ML (nasal and laryngeal involvement) compared with those with only isolated nasal involvement (median = 1,285 versus 51.5 parasites/μg tissue DNA; P = 0.005). Two patterns of PL emerged: pattern 1 (N = 23) was characterized by a sequential decline in PL during and after therapy until kDNA was undetectable. Pattern 2 (N = 18) was characterized by clearance of detectable kDNA during treatment, followed by an increased PL thereafter. All patients who failed treatment (N = 4) demonstrated pattern 1. Leishmania (Viannia) braziliensis was overrepresented among those with pattern 2 (P = 0.019). PL can be quantified by cytology brush qPCR during and after treatment in ML. We demonstrate that treatment failure was associated with undetectable PL, and L. (V.) braziliensis infection was overrepresented in those with rebounding PL., (© The American Society of Tropical Medicine and Hygiene.)
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- 2016
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23. Quantification of Leishmania (Viannia) Kinetoplast DNA in Ulcers of Cutaneous Leishmaniasis Reveals Inter-site and Inter-sampling Variability in Parasite Load.
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Suárez M, Valencia BM, Jara M, Alba M, Boggild AK, Dujardin JC, Llanos-Cuentas A, Arevalo J, and Adaui V
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- Adolescent, Adult, Aged, Female, Humans, Leishmania classification, Leishmaniasis, Cutaneous pathology, Male, Middle Aged, Skin Ulcer pathology, Species Specificity, Young Adult, DNA, Kinetoplast genetics, Leishmania genetics, Leishmaniasis, Cutaneous parasitology, Parasite Load, Skin Ulcer parasitology
- Abstract
Background: Cutaneous leishmaniasis (CL) is a skin disease caused by the protozoan parasite Leishmania. Few studies have assessed the influence of the sample collection site within the ulcer and the sampling method on the sensitivity of parasitological and molecular diagnostic techniques for CL. Sensitivity of the technique can be dependent upon the load and distribution of Leishmania amastigotes in the lesion., Methodology/principal Findings: We applied a quantitative real-time PCR (qPCR) assay for Leishmania (Viannia) minicircle kinetoplast DNA (kDNA) detection and parasite load quantification in biopsy and scraping samples obtained from 3 sites within each ulcer (border, base, and center) as well as in cytology brush specimens taken from the ulcer base and center. A total of 248 lesion samples from 31 patients with laboratory confirmed CL of recent onset (≤3 months) were evaluated. The kDNA-qPCR detected Leishmania DNA in 97.6% (242/248) of the examined samples. Median parasite loads were significantly higher in the ulcer base and center than in the border in biopsies (P<0.0001) and scrapings (P = 0.0002). There was no significant difference in parasite load between the ulcer base and center (P = 0.80, 0.43, and 0.07 for biopsy, scraping, and cytology brush specimens, respectively). The parasite load varied significantly by sampling method: in the ulcer base and center, the descending order for the parasite load levels in samples was: cytology brushes, scrapings, and biopsies (P<0.0001); in the ulcer border, scrapings had higher parasite load than biopsies (P<0.0001). There was no difference in parasite load according to L. braziliensis and L. peruviana infections (P = 0.4)., Conclusion/significance: Our results suggest an uneven distribution of Leishmania amastigotes in acute CL ulcers, with higher parasite loads in the ulcer base and center, which has implications for bedside collection of diagnostic specimens. The use of scrapings and cytology brushes is recommended instead of the more invasive biopsy.
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- 2015
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24. A novel marker, ARM58, confers antimony resistance to Leishmania spp.
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Nühs A, Schäfer C, Zander D, Trübe L, Tejera Nevado P, Schmidt S, Arevalo J, Adaui V, Maes L, Dujardin JC, and Clos J
- Abstract
Protozoa of the Leishmania genus cause a variety of disease forms that rank at the top of the list of neglected tropical diseases. Anti-leishmanial drugs based on pentavalent antimony have been the mainstay of therapy for over 60 years and resistance against them is increasingly encountered in the field. The biochemical basis for this is poorly understood and likely diverse. No stringent correlation between genetic markers and antimony resistance has so far been shown, prompting us to use a functional cloning approach to identify markers of resistance. Using gene libraries derived from drug-resistant and drug-sensitive Leishmania braziliensis clinical isolates in a functional cloning strategy, we repeatedly selected one gene locus located on chromosome 20 whose amplification confers increased antimony (III) resistance in vitro to an otherwise sensitive L. braziliensis clone. The gene responsible for the effect encodes a previously hypothetical protein that we dubbed LbrARM58. It comprises four repeats of a domain of unknown function, DUF1935, one of them harbouring a potential trans-membrane domain. The gene is so far unique to the Leishmania genus, while a structurally related gene without antimony resistance functionality is also found in Trypanosoma spp. Overexpression of LbrARM58 also confers antimony resistance to promastigotes and intracellular amastigotes of the related species Leishmania infantum, indicating a conserved function in Old World and New World Leishmania species. Our results also show that in spite of their RNAi system, L. braziliensis promastigotes can serve as acceptor cells for episomally propagated cosmid libraries, at least for the initial stages of functional cloning efforts.
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- 2013
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25. An approach for interlaboratory comparison of conventional and real-time PCR assays for diagnosis of human leishmaniasis.
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Cruz I, Millet A, Carrillo E, Chenik M, Salotra P, Verma S, Veland N, Jara M, Adaui V, Castrillón C, Arévalo J, Moreno J, and Cañavate C
- Subjects
- Case-Control Studies, DNA blood, DNA chemistry, DNA, Kinetoplast analysis, DNA, Protozoan chemistry, Humans, Leishmania classification, Leishmania genetics, Polymorphism, Restriction Fragment Length, Quality Control, Sequence Analysis, DNA methods, Sequence Analysis, DNA standards, Species Specificity, DNA, Protozoan analysis, Leishmania isolation & purification, Leishmaniasis diagnosis, Polymerase Chain Reaction standards, Real-Time Polymerase Chain Reaction standards
- Abstract
Protozoa of the Leishmania genus are transmitted to humans by the bite of infected sandflies, and are the causative agents of leishmaniasis which ranges from cutaneous to visceral clinical forms. The definitive diagnosis of leishmaniasis has relied traditionally on parasite demonstration, either by microscopy or culture; in the last years, diagnosis based on PCR methods has overcome some drawbacks of traditional methods, increasing sensitivity and allowing using less invasive sampling for diagnosis. However, there are not defined protocols and almost each laboratory applies its own in-house method. Although there are several studies comparing the performance of different methods within the same laboratory, those addressing interlaboratory comparison are scarce, in spite of the growing number of collaborative projects between partners from different leishmaniasis endemic and non-endemic countries. In this work we propose a protocol for interlaboratory comparison of conventional and real-time PCR methods involving four participant laboratories from four different endemic regions in four continents; the protocol includes a quality control step and reduces the variability among the samples tested by each participant. A panel of 77 samples from human origin and 9 from different parasite strains was blindly tested by the participants, aiming to assess the sensitivity of the different methods as well as their usefulness for species identification. Real-time PCR methods targeting the kDNA minicircles returned the highest sensitivity, while both PCR targeting ITS-1 and further HaeIII digestion and a combined algorithm including hsp70 PCR and restriction fragment length polymorphism analysis were the most appropriate approaches for species identification., (Copyright © 2013 Elsevier Inc. All rights reserved.)
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- 2013
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26. Real-time PCR assay for detection and quantification of Leishmania (Viannia) organisms in skin and mucosal lesions: exploratory study of parasite load and clinical parameters.
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Jara M, Adaui V, Valencia BM, Martinez D, Alba M, Castrillon C, Cruz M, Cruz I, Van der Auwera G, Llanos-Cuentas A, Dujardin JC, and Arevalo J
- Subjects
- Adult, DNA, Kinetoplast analysis, DNA, Kinetoplast genetics, DNA, Protozoan analysis, DNA, Protozoan genetics, Female, Humans, Leishmaniasis pathology, Male, Middle Aged, Sensitivity and Specificity, Leishmania isolation & purification, Leishmaniasis parasitology, Mucous Membrane parasitology, Parasite Load methods, Real-Time Polymerase Chain Reaction methods, Skin parasitology
- Abstract
Earlier histopathology studies suggest that parasite loads may differ between cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML) lesions and between acute and chronic CL. Formal demonstration requires highly sensitive detection and accurate quantification of Leishmania in human lesional tissue. In this study, we developed a quantitative real-time PCR (qPCR) assay targeting minicircle kinetoplast DNA (kDNA) to detect and quantify Leishmania (Viannia) parasites. We evaluated a total of 156 lesion biopsy specimens from CL or ML suspected cases and compared the quantitative performance of our kDNA qPCR assay with that of a previously validated qPCR assay based on the glucose-6-phosphate dehydrogenase (G6PD) gene. We also examined the relationship between parasite load and clinical parameters. The kDNA qPCR sensitivity for Leishmania detection was 97.9%, and its specificity was 87.5%. The parasite loads quantified by kDNA qPCR and G6PD qPCR assays were highly correlated (r = 0.87; P < 0.0001), but the former showed higher sensitivity (P = 0.000). CL lesions had 10-fold-higher parasite loads than ML lesions (P = 0.009). Among CL patients, the parasite load was inversely correlated with disease duration (P = 0.004), but there was no difference in parasite load according to the parasite species, the patient's age, and number or area of lesions. Our findings confirm that CL and recent onset of disease (<3 months) are associated with a high parasite load. Our kDNA qPCR assay proved highly sensitive and accurate for the detection and quantification of Leishmania (Viannia) spp. in lesion biopsy specimens. It has potential application as a diagnostic and follow-up tool in American tegumentary leishmaniasis.
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- 2013
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27. Simultaneous infection with Leishmania (Viannia) braziliensis and L. (V.) lainsoni in a Peruvian patient with cutaneous leishmaniasis.
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Veland N, Valencia BM, Alba M, Adaui V, Llanos-Cuentas A, Arevalo J, and Boggild AK
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- Adult, Antimony Sodium Gluconate therapeutic use, Coinfection diagnosis, Coinfection drug therapy, DNA, Protozoan analysis, Female, Humans, Leishmania braziliensis isolation & purification, Leishmaniasis, Cutaneous drug therapy, Polymerase Chain Reaction, Polymorphism, Restriction Fragment Length, Protozoan Proteins analysis, Skin Ulcer drug therapy, Skin Ulcer parasitology, Skin Ulcer pathology, Coinfection parasitology, Leishmania braziliensis pathogenicity, Leishmaniasis, Cutaneous diagnosis
- Abstract
Conventional understanding suggests that simultaneous infection with more than one species of Leishmania is unlikely. In Peru, co-infections are clinically relevant because causative species dictates prognosis, treatment response, and follow-up. We describe a case of Leishmania (Viannia) braziliensis and L. (V.) lainsoni co-infection in a Peruvian patient with cutaneous leishmaniasis.
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- 2013
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28. Non-invasive cytology brush PCR for the diagnosis and causative species identification of American cutaneous leishmaniasis in Peru.
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Valencia BM, Veland N, Alba M, Adaui V, Arevalo J, Low DE, Llanos-Cuentas A, and Boggild AK
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- DNA metabolism, DNA, Kinetoplast metabolism, Humans, Paper, Peru, Polymorphism, Restriction Fragment Length, Sensitivity and Specificity, Skin chemistry, Skin microbiology, Skin Tests methods, Species Specificity, Specimen Handling methods, Leishmaniasis, Cutaneous diagnosis, Leishmaniasis, Cutaneous genetics, Polymerase Chain Reaction methods
- Abstract
Background: Traditional methods of detecting Leishmania from cutaneous lesions involve invasive diagnostic procedures, such as scrapings, which cause discomfort, require technical expertise, and carry risks of invasive procedures. We compared the performance of 2 novel, molecular-based non-invasive methods for the diagnosis of cutaneous leishmaniasis (CL)., Methods: Consecutive patients presenting to the Leishmania Clinic at the Hospital Nacional Cayetano Heredia were enrolled. PCR was performed on filter paper lesion impressions (FPLIs), cytology brushes, and lancets for detection of Leishmania DNA. Smears from lesion scrapings and leishmanin skin test were also performed. Outcome measures were sensitivity and specificity. Composite reference standard was any 2 of 5 tests positive. Species identification was performed by PCR assays of positive specimens., Results: Ninety patients with 129 lesions were enrolled, 117 of which fulfilled reference criteria for a diagnosis of CL. Of these 117 lesions, 113 were positive by PCR of lancets used for lesion scrapings versus 116 by PCR of FPLIs (p=0.930) or 116 by PCR of cytology brushes (p=0.930). Sensitivity and specificity of PCR on lancets were 96.6% [95% CI 93.3-99.9%] and 100%, respectively. Sensitivity and specificity of FPLI PCR were 99.1% [95% CI 97.4-100%] and 100%, respectively. Sensitivity and specificity of cytology brush PCR were 99.1% [95% CI 97.4-100%] and 100%, respectively. Giemsa-stained lesion smear and leishmanin skin test had inferior sensitivities at 47.9% [95% CI 38.9-57.0%] and 82.3% [95% CI 73.9-90.7%], respectively, compared to PCR of invasive or non-invasive specimens (p<0.001)., Conclusions: Cytology brush PCR constitutes a sensitive and specific alternative to traditional diagnostic assays performed on invasive specimens such as lesion scrapings. It performs comparatively to non-invasive FPLI PCR. This novel, rapid, and well-tolerated method has the potential for widespread use in the field and in pediatric populations where traditional specimen collection is difficult.
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- 2012
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29. Multilocus genotyping reveals a polyphyletic pattern among naturally antimony-resistant Leishmania braziliensis isolates from Peru.
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Adaui V, Maes I, Huyse T, Van den Broeck F, Talledo M, Kuhls K, De Doncker S, Maes L, Llanos-Cuentas A, Schönian G, Arevalo J, and Dujardin JC
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- Animals, Antimony therapeutic use, Antiprotozoal Agents therapeutic use, Genetic Variation, Genotype, Humans, Leishmania braziliensis pathogenicity, Leishmaniasis, Cutaneous drug therapy, Microsatellite Repeats, Parasitic Sensitivity Tests, Peru, Treatment Outcome, Antimony pharmacology, Antiprotozoal Agents pharmacology, Drug Resistance genetics, Leishmania braziliensis classification, Leishmania braziliensis drug effects, Leishmania braziliensis genetics
- Abstract
In order to understand the epidemiological dynamics of antimonial (Sb(V)) resistance in zoonotic tegumentary leishmaniasis and its link with treatment outcome, we analyzed the population structure of 24 Peruvian Leishmania braziliensis clinical isolates with known in vitro antimony susceptibility and clinical phenotype by multilocus microsatellite typing (14 microsatellite loci). The genetic variability in the Peruvian isolates was high and the multilocus genotypes were strongly differentiated from each other. No correlation was found between the genotypes and in vitro drug susceptibility or clinical treatment outcome. The finding of a polyphyletic pattern among the Sb(V)-resistant L. braziliensis might be explained by (i) independent events of drug resistance emergence, (ii) sexual recombination and/or (iii) other phenomena mimicking recombination signals. Interestingly, the polyphyletic pattern observed here is very similar to the one we observed in the anthroponotic Leishmania donovani (Laurent et al., 2007), hereby questioning the role of transmission and/or chemotherapeutic drug pressure in the observed population structure., (Copyright © 2011 Elsevier B.V. All rights reserved.)
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- 2011
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30. Comparative gene expression analysis throughout the life cycle of Leishmania braziliensis: diversity of expression profiles among clinical isolates.
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Adaui V, Castillo D, Zimic M, Gutierrez A, Decuypere S, Vanaerschot M, De Doncker S, Schnorbusch K, Maes I, Van der Auwera G, Maes L, Llanos-Cuentas A, Arevalo J, and Dujardin JC
- Subjects
- DNA, Protozoan chemistry, DNA, Protozoan genetics, Molecular Sequence Data, Protozoan Proteins biosynthesis, Protozoan Proteins genetics, Reverse Transcriptase Polymerase Chain Reaction, Sequence Analysis, DNA, Gene Expression Profiling, Gene Expression Regulation, Leishmania braziliensis genetics, Leishmania braziliensis growth & development
- Abstract
Background: Most of the Leishmania genome is reported to be constitutively expressed during the life cycle of the parasite, with a few regulated genes. Inter-species comparative transcriptomics evidenced a low number of species-specific differences related to differentially distributed genes or the differential regulation of conserved genes. It is of uppermost importance to ensure that the observed differences are indeed species-specific and not simply specific of the strains selected for representing the species. The relevance of this concern is illustrated by current study., Methodology/principal Findings: We selected 5 clinical isolates of L. braziliensis characterized by their diversity of clinical and in vitro phenotypes. Real-time quantitative PCR was performed on promastigote and amastigote life stages to assess gene expression profiles at seven time points covering the whole life cycle. We tested 12 genes encoding proteins with roles in transport, thiol-based redox metabolism, cellular reduction, RNA poly(A)-tail metabolism, cytoskeleton function and ribosomal function. The general trend of expression profiles showed that regulation of gene expression essentially occurs around the stationary phase of promastigotes. However, the genes involved in this phenomenon appeared to vary significantly among the isolates considered., Conclusion/significance: Our results clearly illustrate the unique character of each isolate in terms of gene expression dynamics. Results obtained on an individual strain are not necessarily representative of a given species. Therefore, extreme care should be taken when comparing the profiles of different species and extrapolating functional differences between them.
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- 2011
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31. Linking in vitro and in vivo survival of clinical Leishmania donovani strains.
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Vanaerschot M, Maes I, Ouakad M, Adaui V, Maes L, De Doncker S, Rijal S, Chappuis F, Dujardin JC, and Decuypere S
- Subjects
- Animals, Antimony Sodium Gluconate pharmacology, Drug Resistance, Female, Humans, Leishmania donovani growth & development, Leishmania donovani pathogenicity, Leishmaniasis, Visceral, Metals pharmacology, Mice, Nitroso Compounds pharmacology, Oxidative Stress drug effects, Stress, Physiological drug effects, Temperature, Leishmania donovani physiology
- Abstract
Background: Leishmania donovani is an intracellular protozoan parasite that causes a lethal systemic disease, visceral leishmaniasis (VL), and is transmitted between mammalian hosts by phlebotomine sandflies. Leishmania expertly survives in these 'hostile' environments with a unique redox system protecting against oxidative damage, and host manipulation skills suppressing oxidative outbursts of the mammalian host. Treating patients imposes an additional stress on the parasite and sodium stibogluconate (SSG) was used for over 70 years in the Indian subcontinent., Methodology/principal Findings: We evaluated whether the survival capacity of clinical L. donovani isolates varies significantly at different stages of their life cycle by comparing proliferation, oxidative stress tolerance and infection capacity of 3 Nepalese L. donovani strains in several in vitro and in vivo models. In general, the two strains that were resistant to SSG, a stress encountered in patients, attained stationary phase at a higher parasite density, contained a higher amount of metacyclic parasites and had a greater capacity to cause in vivo infection in mice compared to the SSG-sensitive strain., Conclusions/significance: The 2 SSG-resistant strains had superior survival skills as promastigotes and as amastigotes compared to the SSG-sensitive strain. These results could indicate that Leishmania parasites adapting successfully to antimonial drug pressure acquire an overall increased fitness, which stands in contrast to what is found for other organisms, where drug resistance is usually linked to a fitness cost. Further validation experiments are under way to verify this hypothesis.
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- 2010
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32. Targeted gene expression profiling in Leishmania braziliensis and Leishmania guyanensis parasites isolated from Brazilian patients with different antimonial treatment outcomes.
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Torres DC, Adaui V, Ribeiro-Alves M, Romero GA, Arévalo J, Cupolillo E, and Dujardin JC
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- Animals, Antiprotozoal Agents therapeutic use, Brazil, Drug Resistance genetics, Humans, Leishmania braziliensis isolation & purification, Leishmania guyanensis isolation & purification, Leishmaniasis, Cutaneous parasitology, Meglumine therapeutic use, Meglumine Antimoniate, Organometallic Compounds therapeutic use, Parasites genetics, Parasites isolation & purification, Treatment Outcome, Antimony therapeutic use, Gene Expression Profiling, Leishmania braziliensis genetics, Leishmania guyanensis genetics, Leishmaniasis, Cutaneous drug therapy
- Abstract
In Brazil, cutaneous leishmaniasis represents a serious public health problem, and chemotherapy is an important element of the clinical management of this disease. However, treatment efficacy is variable, a phenomenon that might be due to host and parasite (e.g., drug resistance) factors. To better understand the possible contribution of parasite factors to this phenomenon, we characterised 12 Leishmania braziliensis (LB) and 25 Leishmania guyanensis (LG) isolates collected from patients experiencing different antimonial treatment outcomes. For each isolate, promastigote cultures were grown in duplicate and were harvested at the late-log and stationary phases of growth. The RNA expression profiles of six genes encoding proteins with roles in antimony metabolism (AQP1, MRPA, GSH1, GSH2, TRYR and TDR1) were assessed by means of real-time quantitative PCR. Molecular data were compared to the clinical phenotypes. Within LB, we did not find statistically significant differences in the expression levels of the examined genes among isolates from patients with different treatment outcomes. In LG, GSH1 (encoding gamma-glutamylcysteine synthetase, gamma-GCS) was overexpressed in therapeutic failure isolates regardless of the growth curve phase. This finding reveals the predictive potential of promastigote expression curves for the prognosis of cutaneous leishmaniasis caused by LG in Brazil., (Copyright 2010 Elsevier B.V. All rights reserved.)
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- 2010
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33. Evaluation of host genetic and viral factors as surrogate markers for HTLV-1-associated myelopathy/tropical spastic paraparesis in Peruvian HTLV-1-infected patients.
- Author
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Talledo M, López G, Huyghe JR, Verdonck K, Adaui V, González E, Best I, Clark D, Vanham G, Gotuzzo E, Van Camp G, and Van Laer L
- Subjects
- Adolescent, Adult, Age Factors, Aged, Biomarkers, Female, HTLV-I Infections genetics, HTLV-I Infections virology, Humans, Japan, Male, Middle Aged, Peru, Predictive Value of Tests, Prognosis, Proviruses isolation & purification, Risk Factors, Viral Load, Young Adult, HLA Antigens genetics, HTLV-I Infections complications, Human T-lymphotropic virus 1 isolation & purification, Paraparesis, Tropical Spastic diagnosis, Polymorphism, Genetic, Tumor Necrosis Factor-alpha genetics
- Abstract
Human T-lymphotropic virus 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a complication that affects up to 5% of HTLV-1-infected individuals. Several host genetic and viral factors have been associated with the risk of HAM/TSP. The aim of this study was to evaluate the performance of a prognostic model for HAM/TSP developed in Japan in a Peruvian population of 71 HAM/TSP patients and 94 asymptomatic carriers (ACs). This model included age, proviral load (PVL), the presence of HLA-A*02 and HLA-Cw*08 alleles, SDF-1 +801, and TNF-alpha -863 polymorphisms, and viral subgroup. We describe frequencies for the four host genetic markers and demonstrate the presence of the HTLV-1 tax B subgroup in Peru. Using cross-validation, we show that the predictive ability of the prognostic model, as characterized by the area under the receiver-operating characteristic curve (AUC), does not differ from a model containing PVL only (both AUC = 0.74). We found some suggestive evidence of a protective effect of the HLA-A*02 allele but failed to replicate the associations with the other three genetic markers and with viral subgroup. A logistic model containing PVL, age, gender, and HLA-A*02 provided the best predictive ability in the Peruvian cohort (AUC = 0.79). J. Med. Virol. 82:460-466, 2010. (c) 2010 Wiley-Liss, Inc.
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- 2010
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34. Development and validation of a multiplex real-time PCR assay for simultaneous genotyping and human T-lymphotropic virus type 1, 2, and 3 proviral load determination.
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Moens B, López G, Adaui V, González E, Kerremans L, Clark D, Verdonck K, Gotuzzo E, Vanham G, Cassar O, Gessain A, Vandamme AM, and Van Dooren S
- Subjects
- Cell Line, DNA Primers chemistry, DNA Primers genetics, Deltaretrovirus Infections diagnosis, Deltaretrovirus Infections pathology, Genes, pX genetics, Genotype, Human T-lymphotropic virus 1 genetics, Human T-lymphotropic virus 1 isolation & purification, Human T-lymphotropic virus 2 genetics, Human T-lymphotropic virus 2 isolation & purification, Human T-lymphotropic virus 3 genetics, Human T-lymphotropic virus 3 isolation & purification, Humans, Proviruses genetics, Proviruses isolation & purification, Reproducibility of Results, Staining and Labeling methods, Deltaretrovirus Infections virology, Human T-lymphotropic virus 1 classification, Human T-lymphotropic virus 2 classification, Human T-lymphotropic virus 3 classification, Polymerase Chain Reaction methods, Proviruses classification, Viral Load
- Abstract
The human T-lymphotropic virus (HTLV) proviral load remains the best surrogate marker for disease progression. Real-time PCR techniques have been developed for detection and quantification of cosmopolitan HTLV type 1a (HTLV-1a) and HTLV-2. Since a growing level of diversity in subtypes and genotypes is observed, we developed a multiplex quantitative PCR for simultaneous detection, genotyping, and quantification of proviral loads of HTLV-1, 2, and 3. Our assay uses tax type-specific primers and dually labeled probes and has a dynamic range of 10(5) to 10 HTLV copies. One hundred sixty-three samples were analyzed, among which all of the different subtypes within each HTLV genotype could be detected. The performance of proviral load determination of our multiplex assay was compared with that of a previously published HTLV-1 singleplex quantitative PCR based on SYBR green detection, developed at a different institute. Linear regression analysis showed a statistically significant (P < 0.0001) and strong (r(2) = 0.87) correlation between proviral load values measured with the two distinct real-time PCR assays. In conclusion, our novel assay offers an accurate molecular diagnosis and genotyping, together with the determination of the proviral load of HTLV-infected individuals, in a single amplification reaction. Moreover, our molecular assay could offer an alternative when current available serological assays are insufficient.
- Published
- 2009
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35. Evaluation of a microculture method for isolation of Leishmania parasites from cutaneous lesions of patients in Peru.
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Boggild AK, Miranda-Verastegui C, Espinosa D, Arevalo J, Adaui V, Tulliano G, Llanos-Cuentas A, and Low DE
- Subjects
- Adolescent, Adult, Aged, Aged, 80 and over, Animals, Child, Child, Preschool, DNA, Protozoan analysis, Female, Humans, Infant, Male, Middle Aged, Polymerase Chain Reaction, Sensitivity and Specificity, Leishmania isolation & purification, Leishmaniasis, Cutaneous diagnosis, Skin parasitology
- Abstract
Traditional culture of Leishmania spp. is labor intensive and has poor sensitivity. We evaluated a microculture method for the diagnosis of cutaneous leishmaniasis in consecutive patients presenting to the Leishmaniasis Clinic at the Instituto de Medicina Tropical Alexander von Humboldt, Peru, for evaluation of skin lesions. Lesion aspirates were cultured in duplicate and parallel in traditional culture tubes containing modified Novy-MacNeal-Nicolle (NNN) medium or Roswell Park Memorial Institute medium 1640 with 10% fetal bovine serum (10% RPMI) and in 70-microl capillary tubes containing a mixture of lesion aspirate and 10% RPMI. For sensitivity analysis, the consensus standard was considered to be a positive result in any two of the following four tests: Giemsa-stained lesion smear, culture, kinetoplast DNA PCR, or leishmanin skin test. The outcome measures were sensitivity and time to culture positivity. Forty-five patients with 62 skin lesions were enrolled in the study, of which 53 lesions fulfilled the consensus criteria for a final diagnosis of cutaneous leishmaniasis. Of these 53 lesions, 39 were culture positive: 38 in capillary tubes, 29 in traditional culture tubes with modified NNN medium, and 19 in traditional culture tubes with 10% RPMI medium. The sensitivity of microculture was 71.7%, versus 54.7% for traditional culture with NNN (P, 0.038) and 35.8% with 10% RPMI (P, <0.001). The mean times to culture positivity were 4.2 days by microculture, 5.2 days in NNN, and 6 days in 10% RPMI (P, 0.009). We have demonstrated that microculture is a more sensitive and time-efficient means of isolating Leishmania parasites from cutaneous lesions than traditional culture.
- Published
- 2007
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36. Influence of Leishmania (Viannia) species on the response to antimonial treatment in patients with American tegumentary leishmaniasis.
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Arevalo J, Ramirez L, Adaui V, Zimic M, Tulliano G, Miranda-Verástegui C, Lazo M, Loayza-Muro R, De Doncker S, Maurer A, Chappuis F, Dujardin JC, and Llanos-Cuentas A
- Subjects
- Animals, Geography, Humans, Leishmania classification, Leishmania pathogenicity, Leishmaniasis, Cutaneous epidemiology, Meglumine Antimoniate, Peru epidemiology, Polymerase Chain Reaction, Polymorphism, Restriction Fragment Length, Species Specificity, Treatment Failure, Antimony therapeutic use, Antiprotozoal Agents therapeutic use, Leishmania isolation & purification, Leishmaniasis, Cutaneous drug therapy, Leishmaniasis, Cutaneous parasitology, Meglumine therapeutic use, Organometallic Compounds therapeutic use
- Abstract
Background: Pentavalent antimonials (SbV) are the first-line chemotherapy for American tegumentary leishmaniasis (ATL). There are, however, reports of the occurrence of treatment failure with these drugs. Few studies in Latin America have compared the response to SbV treatment in ATL caused by different Leishmania species., Methods: Clinical parameters and response to SbV chemotherapy were studied in 103 patients with cutaneous leishmaniasis (CL) in Peru. Leishmania isolates were collected before treatment and typed by multilocus polymerase-chain-reaction restriction fragment-length polymorphism analysis., Results: The 103 isolates were identified as L. (Viannia) peruviana (47.6%), L. (V.) guyanensis (23.3%), L. (V.) braziliensis (22.3%), L. (V.) lainsoni (4.9%), L. (Leishmania) mexicana (1%), and a putative hybrid, L. (V.) braziliensis/L. (V.) peruviana (1%). L. (V.) guyanensis was most abundant in central Peru. Of patients infected with the 3 former species, 21 (21.9%) did not respond to SbV chemotherapy. The proportions of treatment failure (after 12 months of follow-up) were 30.4%, 24.5%, and 8.3% in patients infected with L. (V.) braziliensis, L. (V.) peruviana, and L. (V.) guyanensis, respectively. Infection with L. (V.) guyanensis was associated with significantly less treatment failure than L. (V.) braziliensis, as determined by multiple logistic regression analysis (odds ratio, 0.07 [95% confidence interval, 0.007-0.8]; P=.03)., Conclusions: Leishmania species can influence SbV treatment outcome in patients with CL. Therefore, parasite identification is of utmost clinical importance, because it should lead to a species-oriented treatment.
- Published
- 2007
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37. Multilocus polymerase chain reaction restriction fragment--length polymorphism genotyping of Trypanosoma cruzi (Chagas disease): taxonomic and clinical applications.
- Author
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Rozas M, De Doncker S, Adaui V, Coronado X, Barnabé C, Tibyarenc M, Solari A, and Dujardin JC
- Subjects
- Animals, Chagas Disease epidemiology, Chagas Disease prevention & control, Chagas Disease transmission, DNA Primers, DNA, Protozoan analysis, Disease Reservoirs parasitology, Electrophoresis methods, Genotype, Host-Parasite Interactions, Humans, Insect Vectors parasitology, Mammals parasitology, Polymorphism, Restriction Fragment Length, South America epidemiology, Trypanosoma cruzi classification, Trypanosoma cruzi enzymology, Chagas Disease parasitology, Trypanosoma cruzi genetics, Trypanosoma cruzi pathogenicity
- Abstract
Background: Trypanosoma cruzi, the agent of Chagas disease, is subdivided into 6 discrete typing units (DTUs); their identification is important to understand clinical pleomorphism and track sylvatic DTUs that might (re-)invade domestic foci of the disease and jeopardize the running control programs., Methods: The genetic polymorphism of 12 loci was analyzed by multilocus polymerase chain reaction restriction fragment--length polymorphism (PCR-RFLP) analysis (MLP analysis) in a sample representative of the diversity within T. cruzi. We paid particular attention to genes involved in host-parasite relationships, because these may be prone to polymorphism as an adaptive answer to the immune selective pressure., Results: The results of MLP analysis were shown to agree with the current multilocus enzyme electrophoresis- and random amplified polymorphic DNA-based classification of T. cruzi in 6 DTUs, thereby providing a taxonomic validation of our method. Our data supported hypotheses of genetic recombination within T. cruzi. We demonstrated direct applicability of PCR-RFLP analysis to blood of mammal hosts and intestine content of vector insects. Domestic DTUs were encountered in wild animals, and, reciprocally, sylvatic DTUs were encountered in humans, raising questions about changes of transmission patterns., Conclusions: MLP analysis represents a new alternative to existing molecular methods for T. cruzi typing. It might offer an invaluable support to clinical and epidemiological studies and to control programs.
- Published
- 2007
- Full Text
- View/download PDF
38. SYBR Green-based quantitation of human T-lymphotropic virus type 1 proviral load in Peruvian patients with neurological disease and asymptomatic carriers: influence of clinical status, sex, and familial relatedness.
- Author
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Adaui V, Verdonck K, Best I, González E, Tipismana M, Arévalo J, Vanham G, Campos M, Zimic M, and Gotuzzo E
- Subjects
- Benzothiazoles, Cross-Sectional Studies, Diamines, Disease Progression, Humans, Leukocytes, Mononuclear virology, Organic Chemicals, Peru, Quinolines, Sensitivity and Specificity, Sex Factors, DNA, Viral isolation & purification, Human T-lymphotropic virus 1 isolation & purification, Paraparesis, Tropical Spastic virology, Proviruses isolation & purification, Reverse Transcriptase Polymerase Chain Reaction methods, Viral Load methods
- Abstract
To evaluate the human T-lymphotropic virus type 1 (HTLV-1) proviral DNA load in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and asymptomatic HTLV-1 carriers, a SYBR Green-based real-time quantitative polymerase chain reaction (qPCR) assay was developed. HTLV-1 proviral DNA in peripheral blood mononuclear cells (PBMCs) was quantified using primers targeting the pX region and the HTLV-1 copy number normalized to the amount of ERV-3 (Endogenous Retrovirus 3) cellular DNA. Thirty-three asymptomatic HTLV-1 carriers (ACs) and 39 patients with HAM/TSP were enrolled. Some participants were relatives of HAM/TSP cases (16 ACs and 7 patients with HAM/TSP). On multiple linear regression analysis, the authors found a significant association between clinical status and HTLV-1 proviral load (P < .01), but only among women. ACs showed a median proviral load of 561 copies per 104 PBMCs (interquartile range: 251-1623). In HAM/TSP patients, the median proviral load was 1783 (1385-2914). ACs related to HAM/TSP patients presented a relatively high proviral load (median 1152); however, the association between relatedness to a HAM/TSP patient and proviral load was not significant (P = .1). In HAM/TSP patients, no association was found between proviral load and disease duration, progression or severity. The fact that the effect of HAM/TSP upon the HTLV-1 proviral load differed between sexes and the finding of a high proviral load among asymptomatic relatives of HAM/TSP patients suggest that not yet identified genetic or environmental factors influence the pathogenesis of HTLV-1 infection.
- Published
- 2006
- Full Text
- View/download PDF
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