Diagnosis of cutaneous leishmaniasis and species discrimination of parasites by PCR and hybridization (original) (raw)
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Memórias do Instituto Oswaldo Cruz, 2009
The positivities of two methods for the diagnosis of localised cutaneous leishmaniasis (CL) were estimated in 280 patients enrolled in a clinical trial. The trial was conducted in an endemic area of Leishmania (Viannia) braziliensis and trial participants were patients with skin ulcers and positive leishmanin skin tests. Patients underwent aspirative skin punctures of the ulcerated lesions and lymph nodes for in vitro cultures, which were processed under field conditions at the local health centre. Skin lesion biopsies were tested at a reference laboratory using kinetoplastid DNA (kDNA)-PCR to detect DNA. The median time required to obtain a positive culture from the skin samples was seven days and the contamination rate of the samples was 1.8%. The positivities of the cultures from skin lesions, kDNA-PCR and the combination of the two methods were 78.2% (95% CI: 73-82.6%), 89.3% . We conclude that parasite culture is a feasible method for the detection of Leishmania in field conditions and that the combination of culture and PCR has a potential role for the diagnosis of CL in candidates for clinical trials.
Comparison of PCR Assays for Diagnosis of Cutaneous Leishmaniasis
Journal of Clinical Microbiology, 2006
Three PCR assays for diagnosing leishmaniasis were compared and validated against parasite cultures and microscopic evaluation of stained tissue smears using 92 specimens from suspected cases of cutaneous leishmaniasis (CL) in Israel and the West Bank. Samples from imported and locally acquired disease were examined. The kinetoplast DNA (kDNA) PCR showed the highest sensitivity (98.7%) of any assay, correctly diagnosing 77/78 of the confirmed positive samples, followed by the rRNA gene internal transcribed spacer 1 (ITS1) PCR (71/78 positive, 91.0% sensitivity) and then the spliced leader mini-exon PCR (42/78 positive, 53.8% sensitivity). Either parasite culture or microscopy alone detected 62.8% (49/78) or 74.4% (58/78) of the positive specimens, respectively, while culture and microscopy together improved overall sensitivity to 83.3% (65/78). Except for the kDNA PCR that had six false positives, all other assays were 100% specific. Further, restriction enzyme analysis of the ITS1 PCR product enabled identification of 74.6% of the positive samples, which included strains of Leishmania major (50.9%), Leishmania tropica (47.2%), and the Leishmania braziliensis complex (1.9%). This suggests that a PCR using kDNA should be used for the diagnosis of CL and that an ITS1 PCR can be reliably used for the diagnosis of CL when rapid species identification is needed.
Use of DNA-based diagnostic methods for human leishmaniasis in Minas Gerais, Brazil
Acta Tropica, 2001
DNA hybridisation was used to type 26 samples from lesions of human patients from the Rio Doce Valley (Minas Gerais, Brazil) clinically diagnosed as having cutaneous leishmaniasis, using kinetoplast DNA (kDNA) cloned mini-circle probes specific for the Leishmania mexicana and Leishmania braziliensis complexes. All samples were found to belong to the L. braziliensis complex. When biopsies were pressed directly onto touch blot membranes 38.5% of the samples were positive. The positivity and specificity obtained were both 100% when cultured blotted parasites were used. The results were confirmed by polymerase chain reaction (PCR) analysis using primers specific for the L. mexicana and L. braziliensis complexes.
Diagnosis of human visceral leishmaniasis by PCR using blood samples spotted on filter paper
Genetics and molecular research : GMR, 2004
The polymerase chain reaction (PCR) is a simple, rapid procedure that has been adapted for the diagnosis of leishmaniasis. In the present study, 85 blood samples and seven bone marrow aspirates from 85 patients with clinical symptoms suggestive of visceral leishmaniasis from the metropolitan region of Belo Horizonte in the Brazilian State of Minas Gerais were screened using molecular and serological techniques. Samples that were negative (N = 12) and positive (N = 19) in parasitological and serological tests were used as controls. Of the 85 samples analyzed by PCR, 61 (71.7%) showed the expected amplification products in agarose gels. However, when the technique was combined with molecular hybridization, 72 samples (83.5%) gave a positive signal on film. Nineteen patients with Leishmania parasites in bone marrow cultures (positive controls) showed PCR hybridization in whole-blood samples, as did the seven bone marrow aspirates positive for Leishmania. None of the negative controls r...
We evaluated the use of polymerase chain reaction (PCR) for diagnosis of American cutaneous leishmaniasis (ACL) in an area in Bahia, Brazil, where Leishmania braziliensis is endemic. Leishmania DNA was detected in 50 cases, yielding a positivity rate of 100%, which was higher than the rates for all of the other diagnostic methods studied—namely, the Montenegro skin test, anti-Leishmania serological testing, and microscopic examination of lesion biopsy specimens. These findings have led us to propose guidelines for the diagnosis of ACL that use PCR as the principal means of parasitological confirmation of cases. Leishmania, a protozoan parasite, is the cause of leish-maniasis, a human disease with diverse clinical mani-festations. An estimated 12 million people are currently infected with Leishmania species, whereas another 350 million people live at risk of infection [1]. American cutaneous leishmaniasis (ACL) is characterized by a cutaneous ulcer with elevated borders and a sharp c...
PCR detection of Leishmania in skin biopsies
The Journal of Infection in Developing Countries, 2009
Introduction: Cutaneous leishmaniasis (CL) is an endemic disease and one of the major health problems in Morocco. In 2006, the recorded total number of cases of CL was 3361, occurring predominantly in the rural population. A new and more sensitive diagnostic technique than current methods used is needed in this setting. The aim of this study was to assess the efficacy of polymerase chain reaction (PCR) to detect leishmanial parasites in skin biopsies of patients from different areas of endemicity in Morocco. Methodology: Biopsies from 26 patients with cutaneous ulcers suggestive of leishmaniasis were analysed by PCR using primers from the small subunit ribosomal gene. The ability of PCR to detect Leishmania was compared with smear-stained and in vitro culture. Results: PCR exhibited superior sensitivity (84,6%) compared with direct microscopy smear (69,2%) and in vitro culture (69,2%). Our PCR assay also showed good specificity (100%). Conclusions: PCR should be considered a valuable, sensitive, and faster diagnostic tool in the diagnosis of cutaneous leishmaniasis, especially for those patients with negative parasitologic examination.
The American Journal of Tropical Medicine and Hygiene, 2016
Microscopic examination is the standard method for diagnosis of cutaneous and mucocutaneous leishmaniasis despite its low sensitivity. This study compared the diagnosis efficacy of microscopic examination versus polymerase chain reaction (PCR)-based methods and DNA sequencing using whole blood and skin lesion samples from patients with suspected leishmaniasis. The presence of Leishmania was determined by microscopy and amplification of 18S ribosomal RNA gene from blood and skin samples of 22 patients. Twenty individuals were positive for leishmaniasis. Microscopic analysis identified 85%, whereas PCR identified 100% of positive cases from skin and 90% from blood. Cytochrome b gene (cyt-b) amplification and sequencing identified Leishmania guyanensis, Leishmania shawi, and Leishmania naiffi from skin and blood samples. This study demonstrated the usefulness of whole blood and molecular techniques for the diagnosis and species identification of leishmaniasis.
1998
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. Diluting crude extracts of skin biopsies was sufficient to eliminate sample inhibition yet maintain required sensitivity. Leishmania braziliensis was also detected by PCR in PBMCs of individuals with active cutaneous leishmaniasis. The simplifications described here significantly improve the feasibility of using the PCR in endemic countries as the primary method for detection and preliminary characterization of Leishmania in clinical specimens of cutaneous leishmaniasis.
Journal of Clinical Microbiology, 2009
Molecular methods such as PCR have become attractive tools for diagnosis of cutaneous leishmaniasis (CL), both for their high sensitivity and for their specificity. However, their practical use in routine diagnosis is limited due to the infrastructural requirements and the lack of any standardization. Recently, a simplified and standardized PCR format for molecular detection of Leishmania was developed. The Leishmania OligoC-TesT is based on simple and rapid detection using a dipstick with PCR-amplified Leishmania DNA. In this study, we estimated the diagnostic accuracy of the Leishmania OligoC-TesT for 61 specimens from 44 CL-suspected patients presenting at the leishmaniasis clinic of the Instituto de Medicina Tropical Alexander von Humboldt, Peru. On the basis of parasitological detection and the leishmanin skin test (LST), patients were classified as (i) confirmed CL cases, (ii) LST-positive cases, and (iii) LST-negative cases. The sensitivities of the Leishmania OligoC-TesT was...