Analysis of Kinetoplast DNA from Mexican Isolates of Leishmania (L.) mexicana.

This study analyzed DNA minicircles of Mexican isolates of L. (Leishmania) mexicana to look for genetic differences between strains isolated from patients with diffuse cutaneous (DCL) and localized (LCL) leishmaniasis. The kDNA was analyzed using polymerase chain reaction (PCR), restriction fragment polymorphism analysis of the PCR products (PCR-RFLP) and the PCR products were sequenced. In the PCR with primers specific for the subgenus Leishmania, the Mexican isolates gave higher amplification products than the other L. mexicana complex strains and with specific primers for the L. mexicana complex they were poorly amplified. In the PCR-RFLP analysis with the Eco RV, Hae III, and Mbo I endonucleases, the Mexican isolates displayed similar restriction patterns, but different from the patterns of the other members of the L. mexicana complex. In the phylogenetic tree constructed, the kDNA sequences of the Mexican clones formed two groups including sequences of LCD or LCL clones, apart from the other L. mexicana complex members. These results suggest that the kDNA minicircles of the Mexican isolates are more polymorphic than the kDNA of other members of the L. mexicana complex and have different recognition sites for the restriction enzymes used in this study.

Molecular epidemiology of cutaneous leishmaniasis in Venezuela.

This paper discusses the utility of a set of primers (3J1, 3J2) designed from a repetitive nuclear deoxyribonucleic acid sequence for the diagnosis of Leishmania braziliensis infection in samples obtained from humans, insect vectors and mammalian reservoir hosts from different endemic areas in Venezuela. A high incidence of Leishmania (Viannia) braziliensis infection was found in the endemic areas studied. The sensitivity and specificity of the primers used were adequate for the identification of the natural vectors and reservoir hosts of L. (V.) braziliensis. The polymerase chain reaction was more sensitive than culture and stained smear examination in the diagnosis of cutaneous leishmaniasis, detecting 80% of cases compared to 42% and 72%, respectively.

Validation of the polymerase chain reaction for the diagnosis of human cutaneous leishmaniasis in north-west Colombia.

Leishmania species of the subgenus Viannia account for 88% of all cases of leishmaniasis recorded in Colombia. Correct diagnosis is essential as infection with members of this subgenus can produce disfiguring destruction of the mucosa. Several methods are available to diagnose leishmaniasis in clinical samples. More recently, the polymerase chain reaction (PCR) has been used, with varying sensitivities and specificities depending on the primers used. In this paper we report on the sensitivity and specificity of PCR primers B1/B2 used on clinical samples and compare their use to the conventional parasitological methods. PCR alone is more sensitive than any single conventional method used, but a combination of conventional methods produced comparable sensitivity. PCR is well suited for use in selected cases and as a test for mucosal leishmaniasis.

The method used to sample ulcers influences the diagnosis of cutaneous leishmaniasis.

Before beginning treatment for cutaneous leishmaniasis, parasitological confirmation of the disease is required. The most commonly used diagnostic procedures are microscopy and culture of samples taken from the active edge of the lesion. In this study, we compared the sensitivity of previous diagnostic procedures with the polymerase chain reaction (PCR), using smears taken from the edge of the lesion and its centre. The sensitivity was greater with smears taken from the centre of the lesion, both for microscopical examination (85%) and for PCR (81%), compared to those obtained from the edge of the lesion (69% and 58% respectively). When PCR was carried out on biopsy material from the edge of the lesion the sensitivity was 63%.

Isolation and characterization of Leishmania infantum cDNA encoding a protein homologous to eukaryotic elongation factor 1 gamma.

This paper reports the isolation and characterization of a complementary deoxyribonucleic acid clone showing sequence homology with genes coding for the eukaryotic elongation factor 1 gamma (EF-1 gamma). The clone encodes an open reading frame of 404 amino acids corresponding to a deduced molecular mass of 46.2 kDa. Database searches revealed 30-64% sequence identity between the Leishmania infantum EF-1 gamma and several eukaryotic homologues. Southern blot analysis indicated that 2 genes tandemly organized were present in the L. infantum genome. The 3' untranslated regions of these 2 genes differed in size. Southern hybridization and pulsed field gel electrophoresis showed that EF-1 gamma genes are highly conserved among members of the Leishmania genus and must be clustered in a single chromosomal locus.

Homologous minicircles in Leishmania donovani.

Leishmania minicircular deoxyribonucleic acid (DNA) is arranged into different classes according to sequence. These classes differ substantially in sequence, despite species- and genus-specific regions, and are present in widely different copy numbers within and between Leishmania strains. Homologous minicircles have been identified in different species of Leishmania by comparing sequences of known minicircles. However, it is possible to select for minicircles of the same class by amplifying Leishmania DNA with polymerase chain reaction primers from the conserved and variable regions. This approach was used with 2 different minicircle classes in the L. donovani complex. In all isolates tested it was possible to amplify minicircles of the selected class.

Leishmania (Viannia) spp. dissemination and tissue tropism in naturally infected dogs (Canis familiaris).

First evidence is presented for Leishmania (Viannia) spp. dissemination and tissue tropism in the domestic dog. Using PCR and histology, parasites were detected in the conjunctiva, lung, lymph nodes and ovaries of 2 naturally infected Peruvian dogs. The detection of parasites in the blood indicates that parasite dissemination to those organs may have been haematogenous.

PCR-based diagnosis of acute and chronic cutaneous leishmaniasis caused by Leishmania (Viannia).

We evaluated PCR methods for diagnosis of acute and chronic cutaneous leishmaniasis (CL) in an area of Colombia where Leishmania (Viannia) is endemic. The PCR method specifically amplified whole linearized minicircle kinetoplast DNA (kDNA) of the Leishmania subgenus Viannia from biopsy lysates. PCR products were detected in agarose gels. For 255 acute cases, this PCR method had greater sensitivity (75.7%) than each conventional method, i.e., microscopic examination of Giemsa-stained lesion scraping (46.7%), biopsy culture (55.3%), aspirate culture (46.3%), and the conventional methods combined (70.2%). Among 44 cases of chronic CL, amplification of biopsy DNA was more sensitive (45.5%) than the individual (4.5 to 27.7%) and combined (27.3%) conventional methods. The detection of kDNA in biopsies from chronic lesions was enhanced by a chemiluminescent dot blot hybridization, which produced a sensitivity of 65.8% when alone and 90.9% when in combination with DNA extraction of biopsy lysates (P < 0.001). Three biopsies from 84 skin lesions of other etiologies were falsely positive by PCR (specificity, 96.4%). PCR detected kDNA more frequently in biopsies (detection level, 83.9%) than in aspirates (74.7%) from 103 cases of acute CL. Among aspirates from 53 chronic cases of CL, the alternative methods, DNA extraction and hybridization, increased sensitivity from 41.5 to 56.6% (P > 0.05). This enhanced PCR method in chronic biopsies was so much more sensitive than conventional methods that it should be considered the preferred diagnostic method for chronic CL. These findings support the appropriate incorporation of PCR into diagnostic strategies for cutaneous leishmaniasis.

Cutaneous leishmaniasis caused by members of Leishmania braziliensis complex in Nayarit, State of Mexico

An epidemiological study was carried out in the northern Mexican state, Nayarit. Fourteen patients with possible cutaneous leishmaniasis skin lesions gave positive Montenegro skin tests. Biopsies were taken from the skin ulcer and analyzed by polymerase chain reaction (PCR) with specific primers for the Leishmania mexicana complex; however all biopsies were not amplified. PCR carried out with specific primers for the L. braziliensis complex resulted in the amplification of all patient DNA. DNA from 12 out of 14 biopsies gave positive amplification with primers species specific for L. (Viannia) braziliensis and hybridized with a species specific L. (V.) braziliensis probe. These results demonstrate the presence in Nayarit of at least two members of the L. braziliensis complex. Most of the cutaneous lesions were caused by L. (V.) braziliensis and two by another species belonging to the L. braziliensis complex. As far as we are aware, this is the first report of L. (V.) braziliensis in Nayarit. The main risk factor associated with the contraction of this disease in Nayarit is attributed to working on coffee plantations