No abstract

Studies were conducted from 1986 through 1993 to further define the geographic distribution and relative importance of different species of Leishmania as a cause of leishmaniasis in Peru. Patients with a clinical diagnosis of cutaneous and/or mucosal or diffuse cutaneous leishmaniasis were enrolled at the Naval Medical Research Institute Detachment (NAMRID) Laboratory in Lima, the Tropical Disease Clinic at San Marcos University Daniel A. Carrión, the Central Military Hospital, and a Ministry of Health hospital in Cusco, Peru. Clinical features, lesion aspirates, and biopsy tissue were obtained from each patient. All specimens were collected and assayed separately, including multiple specimens from some of the same patients for Leishmania parasites by inoculating aliquots of either aspirates or biopsy tissue suspensions onto Senekji's blood agar medium. Stocks of Leishmania isolates were used to prepare promastigotes to produce extracts for identifying the Leishmania species by the cellulose acetate electrophoresis enzyme technique. A total of 351 isolates of Leishmania were obtained from 350 patients who were infected primarily in the low and high jungle of at least 15 different Departments of Peru. Of the 351 isolates, 79% were identified as L. (V.) braziliensis, 7% as L. (V.) guyanensis, 10% as L. (V.) peruviana, 2% as L. (V.) lainsoni, and 1.7% as L. (L.) amazonensis. The clinical form of disease varied depending on the species of Leishmania, with L. (V.) braziliensis being associated most frequently with cutaneous, mucosal ulcers and mixed cutaneous and mucosal disease, and L. (V) peruviana, L. (V.) guyanensis, L. (V.) lainsoni with cutaneous lesions.Leishmania (L.) amazonensis was isolated from six patients, three with cutaneous lesions, one with mucosal lesions, and two with diffuse cutaneous lesions. Among all of the leishmaniasis cases, males were affected more frequently, and cases occurred among patients less than 10 to more than 51 years of age. These data further defined the geographic distribution and the relative frequency of Leishmania species associated with different clinical forms of leishmaniasis in Peru.

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.The leishmaniases are a group of illnesses of the skin, oral and respiratory mucosae and the reticuloendothelium caused by protozoa of the genus Leishmania. Of these, the cutaneous form is the most widespread, afflicting primarily rural and periurban populations exposed to the infected sand fly vector. Cutaneous leishmaniasis (CL) is most frequently diagnosed by clinical evaluation, either alone or in combination with the leishmanin skin test. Clinical evaluation usually suffers from lack of standardization (13,38) and is hampered by the fact that even fairly typical acute lesions can be confused with other dermatological problems, such as sporotrichosis (9). The leishmanin skin test is highly sensitive but lacks specificity when employed in areas of endemicity because it cannot distinguish acute lesions from past infection. A definitive, laboratory diagnosis of mucosal or cutaneous leishmaniasis traditionally requires either the visualization of amastigotes or the isolation of replicative Leishmania from lesions (24). The most widely employed laboratory methods for CL are microscopic examination of lesion scrapings, biopsy impression smears, and histopathology. The most sensitive conventional diagnostic methods, culture of lesion biopsies and aspirates, are available only in reference laboratories. Even these less available, more sensitive methods ...

Following sequence analysis of a Leishmania donovani kinetoplast DNA (kDNA) minicircle, we have developed synthetic oligonucleotides for use in the polymerase chain reaction (PCR). With these primers, we have amplified L. donovani kDNA from splenic aspirates and blood samples taken from kala-azar patients. Treatment of the samples for PCR requires only limited DNA purification by lysis in SDS, digestion with proteinase K, phenol extraction and ethanol precipitation of the resulting nucleic acid. We have obtained amplified product routinely with DNA prepared from the equivalent of 2.5-25 microliters of splenic aspirate or of 50-500 microliters of blood from infected patients. In dilution experiments a visible product has been obtained on amplification of DNA from the equivalent of 2.5 x 10(-7) microliters of splenic material. We therefore propose the amplification of L. donovani kDNA by PCR as a rapid and highly sensitive method for the diagnosis of kala-azar.

Parasitologic confirmation of cutaneous leishmaniasis is obligatory before chemotherapy can be considered. Direct microscopic examination of scrapings taken from indurated borders of ulcers has been routinely used as primary method of diagnosis. In this report we compared the sensitivity of examination of dermal scrapings taken from the bottoms of ulcers (BDS) with that of dermal scrapings taken from indurated active margins of lesions (MDS) in a total of 115 patients. The sensitivities of the microscopic examination were 90.4 and 78.3% for BDS and MDS samples, respectively. When the PCR method was used with a group of 40 patients, we also observed a higher sensitivity when BDS samples were examined (80.8% in BDS samples versus 57.7% in MDS samples). The improvement of the diagnostic sensitivity in the BDS samples appears to be related to the higher parasite load and more easily detectable morphology of amastigotes in the centers of the ulcers. Other parasitologic diagnostic methods, such as culture and histopathologic examination of biopsies, are less sensitive (67.5 and 64.3%, respectively). Aspirate culture, however, was shown to be the most sensitive method for the diagnosis of patients with chronic ulcers. When microscopic examinations of both MDS and BDS samples are combined, the sensitivity of diagnosis may rise up to 94%. We therefore recommend this method as a primary routine procedure for diagnosis of cutaneous leishmaniasis.

No abstract

Characterisation of the telomeres of Leishmania is important for understanding many aspects of the parasitic life of this primitive protozoan and for the completion of the physical map and sequencing of the genome. After sequencing more than 300 telomere-derived clones from Leishmania braziliensis and Leishmania major, a conserved 100 bp sequence was identified immediately adjacent to the telomere at the chromosome end and was named LCTAS (Leishmania conserved telomere-associated sequence). The LCTAS contains two conserved sequence boxes, and is present in all Leishmania species studied. The organisation of the LCTAS in the telomeric region differs between L. braziliensis and L. major: in L. major the LCTASs are tandemly repeated, while in L. braziliensis the LCTAS is present as a single copy per end. Two additional TASs with 1.6 kb and 274 bp repeat structures, which are apparently different to LCTAS, were isolated and mapped onto a L. braziliensis 250 kb multicopy minichromosome and the L. major chromosome 1, respectively. An unusual feature in L. braziliensis is that the telomeric repeats are often comprised of a novel tandem repeat CCCTAACCCGTGGA. A 'slippage' mechanism for LCTAS formation is proposed in this study as an alternative way for the synthesis and maintenance of telomeres and subtelomere regions.

The natural infection of sandflies by Leishmania in wild-caught specimens was studied, using the polymerase chain reaction (PCR)-hybridization technique. The PCR was carried out using 2 oligonucleotides (primers 3J1 and 3J2) derived from a repetitive nuclear DNA sequence. The primers support the enzymatic amplification of a fragment of approximately 500 bp, present in the nuclear DNA of Leishmania braziliensis. The expected band was observed in 5 of 65 sandflies containing flagellates. After hybridization with a species-specific probe, we confirmed natural infection by L. braziliensis. The technique allowed the identification of Lutzomyia gomezi and Lu. panamensis as vectors of L. braziliensis in an endemic area of cutaneous leishmaniasis in Urama, Puerto Cabello district in Venezuela. As far as we are aware, this work constitutes the first report of natural infection of Lu. panamensis with L. braziliensis in the study area. We also demonstrate that PCR-hybridization is a suitable approach to establish the Leishmania-sandfly relationship and will be useful in epidemiological studies of leishmaniasis in endemic areas.