Molecular diagnosis of leishmaniosis in the Paraná state of southern Brazil (original) (raw)
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Journal of the European Academy of Dermatology and Venereology, 2008
Background The diagnosis of cutaneous leishmaniasis (CL) is traditionally based on microscopic demonstration of amastigote forms in tissue biopsies or smears. However, this method usually presents low sensitivity, and in atypical forms, CL may be overlooked because of similarity to other dermal diseases. Thus, it is necessary to apply specific diagnostic methods as polymerase chain reaction (PCR). Objective To evaluate the possible advantage of PCR in the diagnosis and species identification of CL in patients with atypical clinical presentation. Methods Fifty-one patients clinically suspected of CL with positive and negative controls were tested. After microscopic examination, extraction of DNA was performed on their smears and analysed by two specific PCR assays for diagnosis and species identification. For these methods, conserved and variable regions of kinetoplastic DNA (KDNA) of Leishmania species have been amplified, respectively. Atypical forms of CL were evaluated among PCRpositive patients. Results PCR results were positive in 37 out of 51 cases (72.5%), among whom microscopic examination revealed Leishmania amastigotes in only 3 (5.9%). Among these patients, 10 (27%) had atypical presentation of CL; using speciesspecific primers, 6 patients had Leishmania major , 3 had Leishmania tropica and 1 patient had no species diagnosis. None of the samples of other dermal diseases revealed positive results (specificity, 100%). All patients were successfully treated by CL-specific drug regimens. Discussion The results showed that KDNA PCR methods have a higher sensitivity compared with microscopic method. Moreover, PCR could identify the parasite species for specific therapy. Microscopic method had low sensitivity and less value in chronic and atypical CL cases.
2002
PCR-based approaches targeting kinetoplast DNA were evaluated for the diagnosis of American cutaneous leishmaniasis (ACL) in regions of endemicity in northeastern Brazil. A total of 119 cutaneous biopsy specimens from patients with ACL and nonleishmaniasis cutaneous lesions were studied. Two PCR-based systems were used; one was specific for the subgenus Viannia, and the other was specific for the genus Leishmania. The PCR specific for the subgenus Viannia had a sensitivity of 95.4%, whereas the genus-specific PCR detected the target DNA in 88.2% of the samples tested. The specificities of the assays, determined with samples from a group with nonleishmaniasis cutaneous lesions, was 100%. The results of the conventional tests indicate that the sensitivities of the PCR-based methods were significantly higher than those of smear examination, histological staining, and isolation by culture (P < 0.05). Antibodies specific for Leishmania braziliensis were detected by indirect immunofluorescence in 82.9% of the patients tested. Parasites were isolated from 40 of 86 patients (46.5%). Sixty-seven percent of dermal scrapings and 66.2% of stained tissue sections were positive by microscopy. Amplified products from the subgenus-specific PCR hybridized with the Leishmania panamensis minicircle, confirming infection consistent with L. braziliensis. The evidence available at present incriminates L. braziliensis as the only causative agent of ACL in the state of Pernambuco in Brazil.
This study evaluated the applicability of kDNA-PCR as a prospective routine diagnosis method for American tegumentary leishmaniasis (ATL) in patients from the Instituto de Infectologia Emílio Ribas (IIER), a reference center for infectious diseases in São Paulo -SP, Brazil. The kDNA-PCR method detected Leishmania DNA in 87.5% (112/128) of the clinically suspected ATL patients, while the traditional methods demonstrated the following percentages of positivity: 62.8% (49/78) for the Montenegro skin test, 61.8% (47/76) for direct investigation, and 19.3% (22/114) for in vitro culture. The molecular method was able to confirm the disease in samples considered negative or inconclusive by traditional laboratory methods, contributing to the final clinical diagnosis and therapy of ATL in this hospital. Thus, we strongly recommend the inclusion of kDNA-PCR amplification as an alternative diagnostic method for ATL, suggesting a new algorithm routine to be followed to help the diagnosis and treatment of ATL in IIER. INTRODUCTION American tegumentary leishmaniasis (ATL) presents different clinical manifestations in Latin America, including mucosal leishmaniasis (ML), cutaneous localized leishmaniasis (CL), disseminated leishmaniasis (LCD), and diffuse leishmaniasis (DCL). These manifestations are caused by seven different species of Leishmania: Leishmania (Leishmania) amazonensis, L. (Viannia) braziliensis, L. (V.) guyanensis, L. (V.) lainsoni, L. (V.) naiffi, L. (V.) lindenberg, and L. (V.) shawi 7,24 .
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.
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...
Journal of clinical microbiology, 1994
The aim of this study was to assess the efficacy of PCR methodology in establishing the diagnosis of cutaneous leishmaniasis in patients from areas of endemicity in Venezuela. Biopsies from 233 patients with cutaneous ulcers suggestive of leishmaniasis were analyzed by PCR, employing oligonucleotides directed against conserved regions of kinetoplast DNA (kDNA), and the PCR products were then hybridized to nonradioactively labeled, species-specific, cloned kDNA fragments. The ability of PCR to detect Leishmania cells was compared with those of the conventional methodologies: skin testing with killed promastigotes (Montenegro test), examination of Giemsa-stained biopsy smears, and in vitro culture of biopsy tissue. The PCR-hybridization technique detected the presence of Leishmania cells in 98% of patients clinically diagnosed as having leishmaniasis and also positive by the Montenegro skin test. In comparison, leishmania positivity was found in only 42% of cultures and 64% of biopsy ...
2011
BACKGROUND: American cutaneous leishmaniasis is a disease with a wide variety of clinical manifestations that is expanding throughout Brazil, the state of Mato Grosso do Sul constituting a significant endemic area. OBJECTIVES: To evaluate the clinical, epidemiological and laboratory characteristics of patients with American cutaneous leishmaniasis. Patients were recruited among those attending the Maria Aparecida Pedrossian Teaching Hospital of the Federal University of Mato Grosso do Sul, Brazil. METHODS: This was a cross-sectional, observational study conducted using a descriptive and analytical approach. Data from patients suspected of having American cutaneous leishmaniasis who were receiving care at this institute between 1998 and 2008 and were referred to the institute's parasitology laboratory for confirmation of diagnosis were evaluated retrospectively. Clinical and laboratory criteria were taken into consideration for the inclusion of patients to the study. RESULTS: Forty-seven patients were included in the study, the majority of whom were male and between 45 and 59 years of age. Most had the cutaneous form of the disease with a single, ulcerated lesion on exposed areas of the body, which had generally been present for periods of less than six months. Mucosal involvement increased with age and was highest in patients who had sought medical care at a later stage. The Montenegro skin test showed the highest sensitivity. Finding the parasite was more difficult in older lesions. CONCLUSION: Suspicion of the disease at an early stage is of extreme importance for a precise diagnosis. A combination of parasitological and immunological tests renders laboratory diagnosis more reliable.