Treatment response of cutaneous leishmaniasis due to Leishmania aethiopica to cryotherapy and generic sodium stibogluconate from patients in Silti, Ethiopia.

Cutaneous leishmaniasis in Ethiopia is caused mainly by Leishmania aethiopica. In this study, the response of L. aethiopica to sodium stibogluconate (SSG) and liquid nitrogen in Silti has been investigated. Patients were divided into two groups by the treating physician and were treated with either liquid nitrogen or SSG. Punch biopsy samples were collected from 54 patients with mean age of 20.61 (± 9.87 SD) years for histological characterization. The histological spectrum found to be type-1, type-2, type-3 and type-4 were 37.0%, 3.7%, 37.0% and 22.2% respectively. One hundred and three patients with a mean age of 18.4 (± 11.7 SD) years were treated with liquid nitrogen. The mean duration of the lesions before the onset of treatment was 8.5 months (± 6.7 SD). Of the 103 patients 80.6% (83/103) were cured, 13.6% (14/103) were dropouts and 5.8% (6/103) did not respond. Twenty patients with a mean age of 19.55 (+1.64 SD) years were treated with Pentostam on conventional dose. Of the 20 patients 85.0% (17/20) were cured, 10.0% (2/20) were unresponsive and 5.0% (1/20) were dropouts. The per protocol cure rate for cryotherapy and Pentostam was 93.3% and 89.5% respectively. Hence, liquid nitrogen can be used as one of the treatment options especially in resource poor settings.

Leishmania aethiopica: development of specific and sensitive PCR diagnostic test.

PCR has proved useful for rapid diagnosis and typing of Leishmania. Lack of specificity to discriminate between species and/or sensitivity to detect from clinical samples has always been an issue. Previously developed primers either require PCR-RFLP analysis for Leishmania aethiopica discrimination or lack sensitivity to detect L. aethiopica from clinical samples. Here we report the development and validation of L. aethiopica specific PCR primers (V5F/V10R) based on cysteine protease B (cpb), a multicopy and polymorphic gene of Leishmania. V5F/V10R primers differentiate L. aethiopica from Leishmania tropica, Leishmania major, Leishmania donovani and Leishmania infantum by direct PCR. In addition, they are sensitive enough to detect L. aethiopica from biopsy samples. The primers can be very useful for epidemiological studies, species typing and diagnosis of L. aethiopica directly from clinical samples. Implementation of these primers in routine L. aethiopica diagnosis can improve detection rate, save time, money and labor required for culturing Leishmania.

Outbreak of cutaneous leishmaniasis in Silti woreda, Ethiopia: risk factor assessment and causative agent identification.

An outbreak of skin lesions was reported in June 2005 in the district of Silti woreda, 150 km south of Addis Ababa, by the Christian Children's Fund (CCF) and confirmed to be cutaneous leishmaniasis (CL) by our group from the Armauer Hansen Research Institute in July 2005. A house-to-house survey of 1907 residents in three kebeles of Silti woreda conducted in April 2006 showed a prevalence of 4.8%. RFLP analysis of the internal transcribed spacer RNA (ITS1) showed that Leishmania aethiopica was the causative agent. In the survey, it was found that the age group 11-20 years was the most affected. Environmental factors such as proximity of the house to the gorge where hyraxes reside, presence of the plants Adhatoda schimperiana and Acacia spp. in the compound and sharing the same room with domestic animals were significantly associated with developing CL. The prevalence of active disease was higher in Kibet town (10.4%) compared to the rural kebeles. The identified risk factors of CL in the area need further study. The appearance of leishmaniasis in Silti, which was not known to be endemic for the disease, underlines the need to initiate a leishmaniasis control program in Ethiopia to limit its expansion.

Phylogenetic and primary sequence characterization of cathepsin B cysteine proteases from the oxymonad flagellate Monocercomonoides.

Cysteine proteases are crucial for general lysosomal function and for the pathogenic mechanisms of many protistan parasites. Cathepsin B cysteine proteases are currently defined by the presence of the "occluding loop" motif and have been best characterized from humans and their parasites. Though related to a variety of pathogenic excavate flagellates, oxymonads are themselves commensals. While studying this cell biologically aberrant protist lineage, we identified 11 different cathepsin B homologues. These were found to be expressed, at comparable levels to common house-keeping genes, such as elongation factor 1-alpha, alpha-tubulin, beta-tubulin, and glyceraldehyde phosphate dehydrogenase. Primary structure examination of the cathepsin B homologues identified putative signal peptide sequences, and the pre-, pro-, and mature domains of the protein. However, the occluding loop motif was either partially or entirely absent. Comparative genomics, sequence alignment, and phylogenetics of cathepsin sequences from across the diversity of eukaryotes demonstrated that absence of the occluding loop is not a feature exclusive to oxymonads, but is relatively common, suggesting that the "occluding loop" should no longer be used as the defining feature of the cathepsin B subfamily. Overall, this report identifies an abundant protein family in oxymonads, and provides insight both into the evolution and classification of cathepsin B cysteine proteases.

Leishmania aethiopica: identification and characterization of cathepsin L-like cysteine protease genes.

There is limited information on the biology and pathogenesis of Leishmania aethiopica, causative agent of cutaneous leishmaniasis (CL) in Ethiopia. In this study we have identified and characterized two cathepsin L-like cysteine protease genes, Laecpa and Laecpb, from L. aethiopica. The predicted amino acid sequence of Laecpa and Laecpb is more than 75% identical with homologous cathepsin L-like cysteine protease genes of other Leishmania species and less than 50% identical with human cathepsin L. Laecpa is expressed predominantly in the stationary, and to a lower level, during the amastigote stage while Laecpb is specifically expressed in the stationary stage of L. aethiopica development. Phylogenetic analysis showed that the two genes are grouped into separate clades which are the result of gene duplication. The isolation of these genes will be useful in developing Leishmania species specific diagnostics for molecular epidemiological studies and serves as a first step to study the role of cysteine proteases in L. aethiopica pathogenesis.

Identification, sequencing and expression of peroxidoxin genes from Leishmania aethiopica.

Cutaneous leishmaniasis (CL) is a painful, disfiguring and debilitating disease prevalent in Ethiopia and other countries around the world. In Ethiopia, CL is primarily caused by Leishmania aethiopica and less often by L. tropica and L. major. The intracellular survival mechanisms of Leishmania parasites are still not well understood. Recently a new family of antioxidant enzymes called peroxidoxins have been identified that play an important role in parasite survival. In this study, we have identified two distinct peroxidoxin genes (Pxn1 and Pxn2) that are part of a multi-gene family in L. aethiopica. Protein sequence analysis showed that Pxn1 and Pxn2 are highly homologous to peroxidoxins from other Leishmania species. We have found that L. aethiopica Pxn1 is predominantly expressed in amastigotes and stationary phase promastigotes, whereas Pxn2 is constitutively expressed in the different stages of the parasite. This pattern of RNA expression is consistent with patterns seen in some Leishmania species, but not all. Data from this study will be helpful in enhancing vaccine strategies and drug studies targeted towards peroxidoxins.

Leishmania aethiopica: strain identification and characterization of superoxide dismutase-B genes.

This study was performed to characterize the genes that code for superoxide dismutase (SOD) in Leishmania aethiopica. It involved three main steps: specimen collection and parasite isolation, species identification, and molecular characterization of the SOD genes. Out of 20 skin slit specimens cultured and processed from suspected cutaneous leishmaniasis patients enrolled in the study, five (25%) were found to be positive for motile promastigotes. Isoenzyme electrophoresis and PCR-RFLP results confirmed that the isolates were L. aethiopica. Superoxide dismutase-B (SODB) genes were identified from L. aethiopica for the first time. Iron superoxide dismutase-B genes amplified from promastigotes of L. aethiopica (LaeFeSODB) were similar in size to the SODB genes of other Leishmania species. Nucleotide sequences of LaeFeSODB1 showed 95.4, 93.5, and 97.3% identity with L. donovani SODB1 (LdFeSODB1) L. major SODB1 (LmFeSODB1) and L. tropica SODB1 (LtrFeSODB1), respectively. Similarly, LaeFeSODB2 showed 95.9 and 94.1 and 97.6% identity with LdFeSODB2 and LmFeSODB2 and LtrFeSODB2, respectively. On the other hand, predicted amino acid sequence comparison indicated that LaeFeSODB1 had 91.3, 89.8, and 93.9% identity with LdFeSODB1, LmFeSODB1, and LtrFeSODB1, respectively. The difference in nucleic acid sequence of LaeFeSODB from that of LmFeSODB and LtrFeSODB can be utilized to develop specific molecular methods that help differentiate these species in places where there is an overlap in the distribution of these species. In addition, the data provide information about the situation of L. aethiopica with respect to SODB genes.