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Abstract. Polymerase chain reaction (PCR)–based detection of New World Leishmania from different ... more Abstract. Polymerase chain reaction (PCR)–based detection of New World Leishmania from different types of clinical specimens has been further streamlined for field use by simplifying sample preparation and modifying published protocols. A multiplex PCR reaction was developed that allows simultaneous detection of the Leishmania genus and identification of the L. braziliensis complex. For typing isolates in culture, we found that simply boiling diluted cultured strains was sufficient preparation for the PCR. We have demonstrated that Leishmania parasites can be reliably detected from boiled dermal scrapings, instead of the more invasive skin biopsies often used as PCR specimens. The PCR of dermal scrapings yielded a sensitivity of 100 % and a specificity of 100%, as compared with microscopic examination. In a study population, PCR was more sensitive than classic diagnostic techniques. The PCR detection of Leishmania in biopsies and peripheral blood mononuclear cells (PBMCs) was invest...
The American Journal of Tropical Medicine and Hygiene, 1999
Leishmania chagasi, the causative agent of visceral leishmaniasis (VL) in the Americas, has recen... more Leishmania chagasi, the causative agent of visceral leishmaniasis (VL) in the Americas, has recently been associated with atypical cutaneous leishmaniasis (ACL) in Central America; however, little comprehensive information about this disease is available. Clinical, epidemiologic, and parasitologic characteristics of 252 ACL cases and 44 VL cases in Nicaragua were analyzed. Visceral leishmaniasis is primarily associated with malnourished children less than five years of age, whereas ACL is found predominantly in children greater than five years of age and young adults. Genetically similar parasites are associated with both disease manifestations. The sand fly Lutzomyia evansi, in addition to Lu. longipalpis, may be involved in transmission of L. chagasi to humans. Our results indicate that ACL is more prevalent than previously thought, affecting up to 10% of a local population. The fact that the same parasite appears to cause both ACL and the potentially fatal visceral disease suggests that the host immune response is critical in determining the outcome of L. chagasi infection. The public health implications of the widespread presence of L. chagasi are discussed.
We have developed a PCR assay for one-step differentiation of the three complexes of New World Le... more We have developed a PCR assay for one-step differentiation of the three complexes of New World Leishmania (Leishmania braziliensis, Leishmania mexicana, and Leishmania donovani). This multiplex assay is targeted to the spliced leader RNA (mini-exon) gene repeats of these organisms and can detect all three complexes simultaneously, generating differently sized products for each complex. The assay is specific to the Leishmania genus and does not recognize related kinetoplastid protozoa, such as Trypanosoma cruzi, Trypanosoma brucei, and Crithidia fasciculata. It correctly identified Leishmania species with a broad geographic distribution in Central and South America. The sensitivity of the PCR amplification ranged from 1 fg to 10 pg of DNA (0.01 to 100 parasites), depending on the complex detected. Crude extracts of cultured parasites, prepared simply by boiling diluted cultures, served as excellent templates for amplification. Crude preparations of clinical mate-rial were also tested...
Journal of clinical microbiology, 1998
We have developed a PCR assay for one-step differentiation of the three complexes of New World Le... more We have developed a PCR assay for one-step differentiation of the three complexes of New World Leishmania (Leishmania braziliensis, Leishmania mexicana, and Leishmania donovani). This multiplex assay is targeted to the spliced leader RNA (mini-exon) gene repeats of these organisms and can detect all three complexes simultaneously, generating differently sized products for each complex. The assay is specific to the Leishmania genus and does not recognize related kinetoplastid protozoa, such as Trypanosoma cruzi, Trypanosoma brucei, and Crithidia fasciculata. It correctly identified Leishmania species with a broad geographic distribution in Central and South America. The sensitivity of the PCR amplification ranged from 1 fg to 10 pg of DNA (0.01 to 100 parasites), depending on the complex detected. Crude extracts of cultured parasites, prepared simply by boiling diluted cultures, served as excellent templates for amplification. Crude preparations of clinical material were also tested....
The American journal of tropical medicine and hygiene, 1998
Polymerase chain reaction (PCR)-based detection of New World Leishmania from different types of c... more Polymerase chain reaction (PCR)-based detection of New World Leishmania from different types of clinical specimens has been further streamlined for field use by simplifying sample preparation and modifying published protocols. A multiplex PCR reaction was developed that allows simultaneous detection of the Leishmania genus and identification of the L. braziliensis complex. For typing isolates in culture, we found that simply boiling diluted cultured strains was sufficient preparation for the PCR. We have demonstrated that Leishmania parasites can be reliably detected from boiled dermal scrapings, instead of the more invasive skin biopsies often used as PCR specimens. The PCR of dermal scrapings yielded a sensitivity of 100% and a specificity of 100%, as compared with microscopic examination. In a study population, PCR was more sensitive than classic diagnostic techniques. The PCR detection of Leishmania in biopsies and peripheral blood mononuclear cells (PBMCs) was investigated. Dil...
Parasite Immunology, 2007
In humans, Leishmania chagasi parasites can produce subclinical infections, atypical cutaneous le... more In humans, Leishmania chagasi parasites can produce subclinical infections, atypical cutaneous leishmaniasis (ACL) and visceral leshmaniasis that is potentially fatal if not treated in a timely fashion. L. chagasi parasites that cause both ACL and visceral disease appear to be genetically similar, which suggests that host factors such as the immune response play an important role in controlling infection. We evaluated the immunologic response in ACL using peripheral blood mononuclear cells (PBMCs) of 37 subjects divided into three groups: (i) active ACL cases, (ii) asymptomatic cases and (iii) persons with no history of Leishmania infection. The supernatants of stimulated PBMCs were analysed for production of IL-10, IFN-gamma and IL-2. Robust production of IL-10 in response to Leishmania stimulation was observed in active ACL cases, compared to low levels in asymptomatic cases and negative controls. Serum IgE levels, measured by ELISA, were not significantly different among the three groups. In addition, ACL cases displayed depressed levels of all cytokines in response to mitogen. Thus, this first characterization of the immune response in ACL suggests a role for IL-10 as well as partial immunosuppression.
American Journal of Tropical Medicine and Hygiene, 2014
Abstract. Polymerase chain reaction (PCR)–based detection of New World Leishmania from different ... more Abstract. Polymerase chain reaction (PCR)–based detection of New World Leishmania from different types of clinical specimens has been further streamlined for field use by simplifying sample preparation and modifying published protocols. A multiplex PCR reaction was developed that allows simultaneous detection of the Leishmania genus and identification of the L. braziliensis complex. For typing isolates in culture, we found that simply boiling diluted cultured strains was sufficient preparation for the PCR. We have demonstrated that Leishmania parasites can be reliably detected from boiled dermal scrapings, instead of the more invasive skin biopsies often used as PCR specimens. The PCR of dermal scrapings yielded a sensitivity of 100 % and a specificity of 100%, as compared with microscopic examination. In a study population, PCR was more sensitive than classic diagnostic techniques. The PCR detection of Leishmania in biopsies and peripheral blood mononuclear cells (PBMCs) was invest...
The American Journal of Tropical Medicine and Hygiene, 1999
Leishmania chagasi, the causative agent of visceral leishmaniasis (VL) in the Americas, has recen... more Leishmania chagasi, the causative agent of visceral leishmaniasis (VL) in the Americas, has recently been associated with atypical cutaneous leishmaniasis (ACL) in Central America; however, little comprehensive information about this disease is available. Clinical, epidemiologic, and parasitologic characteristics of 252 ACL cases and 44 VL cases in Nicaragua were analyzed. Visceral leishmaniasis is primarily associated with malnourished children less than five years of age, whereas ACL is found predominantly in children greater than five years of age and young adults. Genetically similar parasites are associated with both disease manifestations. The sand fly Lutzomyia evansi, in addition to Lu. longipalpis, may be involved in transmission of L. chagasi to humans. Our results indicate that ACL is more prevalent than previously thought, affecting up to 10% of a local population. The fact that the same parasite appears to cause both ACL and the potentially fatal visceral disease suggests that the host immune response is critical in determining the outcome of L. chagasi infection. The public health implications of the widespread presence of L. chagasi are discussed.
We have developed a PCR assay for one-step differentiation of the three complexes of New World Le... more We have developed a PCR assay for one-step differentiation of the three complexes of New World Leishmania (Leishmania braziliensis, Leishmania mexicana, and Leishmania donovani). This multiplex assay is targeted to the spliced leader RNA (mini-exon) gene repeats of these organisms and can detect all three complexes simultaneously, generating differently sized products for each complex. The assay is specific to the Leishmania genus and does not recognize related kinetoplastid protozoa, such as Trypanosoma cruzi, Trypanosoma brucei, and Crithidia fasciculata. It correctly identified Leishmania species with a broad geographic distribution in Central and South America. The sensitivity of the PCR amplification ranged from 1 fg to 10 pg of DNA (0.01 to 100 parasites), depending on the complex detected. Crude extracts of cultured parasites, prepared simply by boiling diluted cultures, served as excellent templates for amplification. Crude preparations of clinical mate-rial were also tested...
Journal of clinical microbiology, 1998
We have developed a PCR assay for one-step differentiation of the three complexes of New World Le... more We have developed a PCR assay for one-step differentiation of the three complexes of New World Leishmania (Leishmania braziliensis, Leishmania mexicana, and Leishmania donovani). This multiplex assay is targeted to the spliced leader RNA (mini-exon) gene repeats of these organisms and can detect all three complexes simultaneously, generating differently sized products for each complex. The assay is specific to the Leishmania genus and does not recognize related kinetoplastid protozoa, such as Trypanosoma cruzi, Trypanosoma brucei, and Crithidia fasciculata. It correctly identified Leishmania species with a broad geographic distribution in Central and South America. The sensitivity of the PCR amplification ranged from 1 fg to 10 pg of DNA (0.01 to 100 parasites), depending on the complex detected. Crude extracts of cultured parasites, prepared simply by boiling diluted cultures, served as excellent templates for amplification. Crude preparations of clinical material were also tested....
The American journal of tropical medicine and hygiene, 1998
Polymerase chain reaction (PCR)-based detection of New World Leishmania from different types of c... more Polymerase chain reaction (PCR)-based detection of New World Leishmania from different types of clinical specimens has been further streamlined for field use by simplifying sample preparation and modifying published protocols. A multiplex PCR reaction was developed that allows simultaneous detection of the Leishmania genus and identification of the L. braziliensis complex. For typing isolates in culture, we found that simply boiling diluted cultured strains was sufficient preparation for the PCR. We have demonstrated that Leishmania parasites can be reliably detected from boiled dermal scrapings, instead of the more invasive skin biopsies often used as PCR specimens. The PCR of dermal scrapings yielded a sensitivity of 100% and a specificity of 100%, as compared with microscopic examination. In a study population, PCR was more sensitive than classic diagnostic techniques. The PCR detection of Leishmania in biopsies and peripheral blood mononuclear cells (PBMCs) was investigated. Dil...
Parasite Immunology, 2007
In humans, Leishmania chagasi parasites can produce subclinical infections, atypical cutaneous le... more In humans, Leishmania chagasi parasites can produce subclinical infections, atypical cutaneous leishmaniasis (ACL) and visceral leshmaniasis that is potentially fatal if not treated in a timely fashion. L. chagasi parasites that cause both ACL and visceral disease appear to be genetically similar, which suggests that host factors such as the immune response play an important role in controlling infection. We evaluated the immunologic response in ACL using peripheral blood mononuclear cells (PBMCs) of 37 subjects divided into three groups: (i) active ACL cases, (ii) asymptomatic cases and (iii) persons with no history of Leishmania infection. The supernatants of stimulated PBMCs were analysed for production of IL-10, IFN-gamma and IL-2. Robust production of IL-10 in response to Leishmania stimulation was observed in active ACL cases, compared to low levels in asymptomatic cases and negative controls. Serum IgE levels, measured by ELISA, were not significantly different among the three groups. In addition, ACL cases displayed depressed levels of all cytokines in response to mitogen. Thus, this first characterization of the immune response in ACL suggests a role for IL-10 as well as partial immunosuppression.
American Journal of Tropical Medicine and Hygiene, 2014