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Vanessa Adaui - One of the best experts on this subject based on the ideXlab platform.
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quantification of leishmania viannia kinetoplast dna in ulcers of cutaneous leishmaniasis reveals inter site and inter sampling variability in Parasite Load
PLOS Neglected Tropical Diseases, 2015Co-Authors: Milagros Suarez, Braulio M Valencia, Marlene Jara, Milena Alba, Andrea K Boggild, Jeanclaude Dujardin, Alejandro Llanoscuentas, Jorge Arevalo, Vanessa AdauiAbstract: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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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
Journal of Clinical Microbiology, 2013Co-Authors: Marlene Jara, Braulio M Valencia, Milena Alba, Vanessa Adaui, Dalila Martinez, Carlos Castrillon, Maria Do Socorro Pires E Cruz, Israel CruzAbstract: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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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
Journal of clinical microbiology, 2013Co-Authors: Marlene Jara, Braulio M Valencia, Milena Alba, Vanessa Adaui, Dalila Martinez, Carlos Castrillon, Israel Cruz, Maria Cruz, Gert Van Der Auwera, Alejandro Llanos-cuentasAbstract: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 (
Marlene Jara - One of the best experts on this subject based on the ideXlab platform.
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quantification of leishmania viannia kinetoplast dna in ulcers of cutaneous leishmaniasis reveals inter site and inter sampling variability in Parasite Load
PLOS Neglected Tropical Diseases, 2015Co-Authors: Milagros Suarez, Braulio M Valencia, Marlene Jara, Milena Alba, Andrea K Boggild, Jeanclaude Dujardin, Alejandro Llanoscuentas, Jorge Arevalo, Vanessa AdauiAbstract: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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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
Journal of Clinical Microbiology, 2013Co-Authors: Marlene Jara, Braulio M Valencia, Milena Alba, Vanessa Adaui, Dalila Martinez, Carlos Castrillon, Maria Do Socorro Pires E Cruz, Israel CruzAbstract: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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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
Journal of clinical microbiology, 2013Co-Authors: Marlene Jara, Braulio M Valencia, Milena Alba, Vanessa Adaui, Dalila Martinez, Carlos Castrillon, Israel Cruz, Maria Cruz, Gert Van Der Auwera, Alejandro Llanos-cuentasAbstract: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 (
Milena Alba - One of the best experts on this subject based on the ideXlab platform.
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quantification of leishmania viannia kinetoplast dna in ulcers of cutaneous leishmaniasis reveals inter site and inter sampling variability in Parasite Load
PLOS Neglected Tropical Diseases, 2015Co-Authors: Milagros Suarez, Braulio M Valencia, Marlene Jara, Milena Alba, Andrea K Boggild, Jeanclaude Dujardin, Alejandro Llanoscuentas, Jorge Arevalo, Vanessa AdauiAbstract: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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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
Journal of Clinical Microbiology, 2013Co-Authors: Marlene Jara, Braulio M Valencia, Milena Alba, Vanessa Adaui, Dalila Martinez, Carlos Castrillon, Maria Do Socorro Pires E Cruz, Israel CruzAbstract: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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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
Journal of clinical microbiology, 2013Co-Authors: Marlene Jara, Braulio M Valencia, Milena Alba, Vanessa Adaui, Dalila Martinez, Carlos Castrillon, Israel Cruz, Maria Cruz, Gert Van Der Auwera, Alejandro Llanos-cuentasAbstract: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 (
Braulio M Valencia - One of the best experts on this subject based on the ideXlab platform.
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quantification of leishmania viannia kinetoplast dna in ulcers of cutaneous leishmaniasis reveals inter site and inter sampling variability in Parasite Load
PLOS Neglected Tropical Diseases, 2015Co-Authors: Milagros Suarez, Braulio M Valencia, Marlene Jara, Milena Alba, Andrea K Boggild, Jeanclaude Dujardin, Alejandro Llanoscuentas, Jorge Arevalo, Vanessa AdauiAbstract: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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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
Journal of Clinical Microbiology, 2013Co-Authors: Marlene Jara, Braulio M Valencia, Milena Alba, Vanessa Adaui, Dalila Martinez, Carlos Castrillon, Maria Do Socorro Pires E Cruz, Israel CruzAbstract: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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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
Journal of clinical microbiology, 2013Co-Authors: Marlene Jara, Braulio M Valencia, Milena Alba, Vanessa Adaui, Dalila Martinez, Carlos Castrillon, Israel Cruz, Maria Cruz, Gert Van Der Auwera, Alejandro Llanos-cuentasAbstract: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 (
Otacilio C Moreira - One of the best experts on this subject based on the ideXlab platform.
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memory impairment in chronic experimental chagas disease benznidazole therapy reversed cognitive deficit in association with reduction of Parasite Load and oxidative stress in the nervous tissue
PLOS ONE, 2021Co-Authors: Glaucia Vilarpereira, Constanca Britto, Otacilio C Moreira, Leda Castano Barrios, Andrea Alice Da Silva, Angelica Martins Batista, Isabela Resende Pereira, Hilton Antonio Mata Dos Santos, Joseli LannesvieiraAbstract:Memory impairment has been associated with chronic Chagas disease (CD), a neglected tropical disease caused by the protozoan Parasite Trypanosoma cruzi. In degenerative diseases, memory loss has been associated with increased oxidative stress, revealed as enhanced lipid peroxidation, in the cerebral cortex. Benznidazole (Bz), a trypanocidal drug efficient to reduce blood Parasite Load in the acute and chronic phases of infection, showed controversial effects on heart disease progression, the main clinical manifestation of CD. Here, we evaluated whether C57BL/6 mice infected with the Colombian type I T. cruzi strain present memory deficit assessed by (i) the novel object recognition task, (ii) the open field test and (iii) the aversive shock evoked test, at 120 days post infection (dpi). Next, we tested the effects of Bz therapy (25mg/Kg/day, for 30 consecutive days) on memory evocation, and tried to establish a relation between memory loss, Parasite Load and oxidative stress in the central nervous system (CNS). At 120 dpi, T. cruzi-infected mice showed memory impairment, compared with age-matched non-infected controls. Bz therapy (from 120 to 150 dpi) hampered the progression of habituation and aversive memory loss and, moreover, reversed memory impairment in object recognition. In vehicle-administered infected mice, neuroinflammation was absent albeit rare perivascular mononuclear cells were found in meninges and choroid plexus. Bz therapy abrogated the infiltration of the CNS by inflammatory cells, and reduced Parasite Load in hippocampus and cerebral cortex. At 120 and 150 dpi, lipid peroxidation was increased in the hippocampus and cortex tissue extracts. Notably, Bz therapy reduced levels of lipid peroxidation in the cerebral cortex. Therefore, in experimental chronic T. cruzi infection Bz therapy improved memory loss, in association with reduction of Parasite Load and oxidative stress in the CNS, providing a new perspective to improve the quality of life of Chagas disease patients.
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monitoring the Parasite Load in chronic chagas disease patients comparison between blood culture and quantitative real time pcr
PLOS ONE, 2018Co-Authors: Daniella Alchaar Davila, Lucia Maria Da Cunha Galvao, Giovane Rodrigo Sousa, Constanca Britto, Otacilio C Moreira, Egler ChiariAbstract:BACKGROUND: Despite the improvements in diagnostic tools for detection of Trypanosoma cruzi in human blood samples, the isolation of Parasite from bloodstream in the chronic phase of Chagas disease is challenging. Thus, there is an increasing interest in the development of strategies that allow an accurate monitoring of the Parasite Load in bloodstream of Chagas disease patients. Given that, the comparison of a classical diagnostic method such as blood culture and multiplex quantitative real-time PCR (qPCR) was few explored so far. Therefore, this study aimed to compare the detection and quantification of T. cruzi Load in the circulating blood of patients with chronic Chagas disease, using blood culture and qPCR techniques. METHODS⁄PRINCIPAL FINDINGS: The multiplex real-time quantitative PCR assay (qPCR) based on TaqMan technology was evaluated in 135 blood samples from 91 patients with chronic Chagas disease presenting indeterminate (asymptomatic, n = 23) and cardiac (chronic cardiomyopathy, n = 68) forms, in comparison with the classical blood culture (BC) technique. The total positivity of qPCR and BC was 58.5% and 49.6%, respectively. The median Parasite Load of all positive patients was 1.18 [0.39-4.23] par. eq.⁄mL, ranging from 0.01 to 116.10 par. eq.⁄mL. We did not find significant differences between T. cruzi Load with age and distinct clinical manifestations of patients. CONCLUSIONS/SIGNIFICANCE: Our data suggest that qPCR can be an auxiliary tool for studies that require T. cruzi isolation from the bloodstream of patients with chronic Chagas disease, after the establishment of a Parasite Load cut-off that guarantees a relative success rate of Parasite isolation using BC technique.
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Exploring the Parasite Load and molecular diversity of Trypanosoma cruzi in patients with chronic Chagas disease from different regions of Brazil.
PLoS neglected tropical diseases, 2018Co-Authors: Ícaro Rodrigues-dos-santos, Constanca Britto, Myllena F. A. D. Melo, Liane De Castro, Alejandro Marcel Hasslocher-moreno, Pedro Emmanuel Alvarenga Americano Do Brasil, Andréa Silvestre De Sousa, Otacilio C MoreiraAbstract:Chagas disease is still a major public health issue in many Latin American countries. One of the current major challenges is to find an association between Trypanosoma cruzi discrete typing units (DTUs) and clinical manifestations of the disease. In this study, we used a multilocus conventional PCR and quantitative real time PCR (qPCR) approaches to perform the molecular typing and Parasite Load quantification directly from blood specimens of 65 chronic Chagas disease patients. All patients were recruited at the same health center, but their place of birth were widely distributed in different geographic regions of Brazil. Of the 65 patients, 35 (53.8%) presented positive amplification by real time qPCR, being 20 (30.7%) with the clinical indeterminate form and 15 (23.1%) with the cardiac form of the disease. The Parasite Load median for all positive patients was 2.54 [1.43-11.14] Parasite equivalents/mL (par. Eq./mL), with the Load ranging from 0.12 to 153.66 par. Eq./mL. Noteworthy, the Parasite Load was significantly higher in patients over 70 years old (median 20.05 [18.29-86.86] par. Eq./mL). Using guanidine-EDTA blood samples spiked with reference T. cruzi strains, belonging to the six DTUs, it was possible to genotype the Parasite up to 0.5 par. Eq./mL, with high specificity. Of the patients with positive qPCR, it was possible to identify the T. cruzi DTU in 28 patients (80%). For the remaining patients (20%), at least a partial result was obtained. Analysis of specimens showed prevalences of TcVI, TcII and mixed infection TcVI+TcII equal to 40%, 17.1% and 14.3%, respectively. In addition, two patients were infected by TcV, and one patient was coinfected by TcIII+TcVI, These last three patients were in stage A of chronic chagasic cardiomyopathy (CCC), and they were born at the Bahia State (northeast region of Brazil). When T. cruzi genotypes were compared with the Parasite Load, more elevated Parasite Loads were observed in patients infected by TcII in general (Parasite Load median of 7.56 par. Eq./mL) in comparison to patients infected by TcVI (median of 2.35 par. Eq./mL). However, while the frequency of CCC was 50% in patients infected by TcVI and TcV, only 16.7% of patients infected by TcII evolved to CCC. Taking together, our results contribute to update the epidemiological knowledge of T. cruzi DTUs in Brazil, and highlight the age of patient and infection by TcII as important features that lead to the observation of higher Parasitemia levels.
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Usefulness of real time PCR to quantify Parasite Load in serum samples from chronic Chagas disease patients
Parasites & Vectors, 2015Co-Authors: Myllena F. A. D. Melo, Otacilio C Moreira, Priscila Tenório, Virginia Lorena, Izaura Lorena-rezende, Wilson Oliveira Júnior, Yara Gomes, Constanca BrittoAbstract:Background Inconclusive results of serological diagnosis in Chagas disease have an important impact on blood banks worldwide, reflecting in the high number of discarded bags or in an increased transmission by blood transfusion. Molecular techniques such as qPCR have been used for diagnosis and to monitor Trypanosoma cruzi Load in peripheral blood samples. A promising perspective refers to the possibility of Parasite DNA detection in serum, taking advantage in using the same samples collected for serological screening. Methods In order to evaluate the effectiveness of a qPCR strategy for detecting and quantifying T. cruzi DNA in serum, we selected 40 chronic Chagas disease patients presenting different clinical manifestations: Cardiac (23), Digestive (4), Mixed form [cardiodigestive] (7), and asymptomatic (6). Twenty seronegative individuals from non-endemic areas were included as controls. Samples were extracted using QIAamp DNA mini kit (QIAGEN) and qPCR was performed in a multiplex format with TaqMan probes for the nuclear satellite DNA of T. cruzi and for the human RNase P gene. In addition, DNA migration to serum during blood coagulation was assessed using a commercial exogenous control (Exo IPC, Applied Biosystems) in a separate qPCR reaction. Results The comparative duplex qPCR analysis revealed that, even with an increase in Ct values, it was possible to detect all DNA targets in serum. In addition, the same linearity range for T. cruzi quantification (from 10^5 to 0.5 par. eq./mL) between serum, blood or culture samples ( T. cruzi epimastigotes – Cl Brener strain) was found. When patient samples were evaluated, no significant differences in Parasite Load between the distinct clinical manifestations were found for both blood and serum samples. Moreover, median values of Parasite burden were 1.125 and 1.230 par. eq./mL for serum and blood, respectively. Using serology as gold standard, we found 95% sensitivity for T. cruzi detection in serum and 97.5% for blood, and 100% specificity for both samples. Conclusions Taken together, our data indicate the potential of using serum samples for molecular diagnosis and Parasite Load quantification by qPCR, suggesting its use in reference laboratories for the diagnosis of Chagas disease patients.
