Douglas C. Barker

Diagnosis of New World leishmaniasis : specific detection of species of the Leishmania braziliensis complex by amplification of kinetoplast DNA

We have sequenced single kinetoplast DNA minicircles from three species and part of a minicircle from the fourth major species within the Leishmania braziliensis complex. Alignment of these sequences with each other and with those of other kinetoplastids allowed the selection of a pair of oligonucleotides suitable as primers in a polymerase chain reaction which is highly specific for the Leishmania braziliensis complex. The reaction is capable of detecting less than one femtogramme of kinetoplast DNA. It has been tested with crude specimens from South American leishmaniasis patients, potential wild animal reservoirs and sandfly vectors. The tests indicate that these primers are suitable for diagnosis of leishmaniasis and potentially useful in epidemiological surveys.

Rapid and sensitive detection of Leishmania kinetoplast DNA from spleen and blood samples of kala-azar patients

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.

PCR-Based Diagnosis of Acute and Chronic Cutaneous Leishmaniasis Caused by Leishmania (Viannia)

ABSTRACT 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 immune response and PBMC subsets in canine visceral leishmaniasis before, and after, chemotherapy

Peripheral blood mononuclear cell subsets, in vitro lymphoproliferative response to leishmanial antigen, and Leishmania-specific serum antibody levels were examined in 11 dogs, naturally infected with L. infantum, and 9 healthy control dogs. A decrease in the percentage of CD4+ T-cells and an increase in the proportion of gammadelta T-cells and sIgG+ B-cells were observed during canine visceral leishmaniasis (CVL). These changes may be responsible for the marked humoral response and the absence of in vitro lymphoproliferation to mitogen and specific parasite antigens. This possibility was supported by the analysis of these subsets after treatment with amphotericin B. One month after therapy, a significant increase in the percentage of CD4+ T-cells and a decrease of gammadelta T-cells and sIgG+ B-cells were observed. At the same time, the lymphocyte blastogenesis assay with leishmanial antigen was positive and the levels of specific antibodies to Leishmania were significantly lower than before the treatment. Five months after therapy, lymphocyte proliferative response to LSA disappeared, antibody and lymphocyte subsets levels returned to those observed during CVL. Therapeutic failure in CVL is associated with the inability of antileishmanial drugs to completely revert the profound immunodepression induced by the infection and prevent relapse.

The use of DNA probes in the identification of leishmanias: discrimination between isolates of the Leishmania mexicana an L. Braziliensis complexes☆

Kinetoplast DNA (kDNA), initially characterized by buoyant density, from ten reference isolates of the Leishmania braziliensis and L. mexicana complexes has been radio-actively labelled and used as hybridization probes. Filters containing endonuclease digested, electrophoresed, Southern transferred fragments of kDNA from reference and other isolates sent to us for DNA typing have been tested for kDNA sequence homology. We record a complete lack of sequence homology between kDNA of any isolate of the L. braziliensis complex and kDNA of any isolate of the L. mexicana complex. L. b. braziliensis, L. b. guyanensis and L. b. panamensis have kDNA sequences in common with each other and with a number of test isolates from Brazil, Panama, Venezuela and Peru. L. b. panamensis (1.695 g/ml) can be separated from L. b. braziliensis or L. b. guyanensis (1.691-1.693 g/ml) by differences in buoyant density of kDNA. L. m. mexicana and L. m. pifanoi have kDNA sequences in common with each other but kDNA of L. m. amazonensis has insignificant homology with kDNA of other reference isolates of the L. mexicana complex. We conclude that the kDNAs of species of the L. mexicana complex are sufficiently different from kDNA of species of the L. braziliensis complex to make kDNA sequence homology identification a feasible proposition.

Detection of Leishmania braziliensis in naturally infected individual sandflies by the polymerase chain reaction

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.

Detection of Leishmania (Viannia) braziliensis complex in wild mammals from Colombian coffee plantations by PCR and DNA hybridization

The small mammal fauna of coffee plantations in SW Colombia was surveyed to determine which of the species present were infected with parasites of the Leishmania (Viannia) braziliensis complex and might therefore act as reservoirs of human cutaneous leishmaniasis. Fifty animals of seven different species were captured. Tissue samples were taken from the ears of specimens from each of the seven species. Thirty three samples were analysed by polymerase chain reaction (PCR) using oligonucleotide primers directed against conserved regions of L. (V) braziliensis complex kinetoplast DNA. Three of the samples (two from mouse opossums Micoureus demerarae, and one from a pygmy rice at Microryzomys minutus) gave positive results based on PCR analysis. When the samples were subjected to DNA hybridization (dot blot) analysis using the B18 (L. (V.) braziliensis complex-specific) probe, a total of ten specimens belonging to six species (the opossums M. demerarae and Didelphis marsupilalis, the rodents Melanomys caliginosus, Mi. minutus and Rattus rattus, and a rabbit Sylvilagus brasiliensis) gave positive results, indicating that all these animals had flies of species occurring in the same habitat by allowing them to feed on infected animals.

The origin and evolution of the Leishmania donovani complex as inferred from a mitochondrial cytochrome oxidase II gene sequence.

Members of the Leishmania donovani complex are parasites of the reticulo-endothelial system that are often associated with serious epidemics of a life threatening disease known as visceral leishmaniasis or kala-azar. Twenty-two Leishmania isolates representative of the geographical range of the parasite were analysed for sequence variations in their cytochrome oxidase II gene. In performing phylogenetic analysis, the maximum parsimonious, neighbour joining and maximum likelihood trees were congruent and produced a tree that differentiated between two clades conforming to the current classification of the species complex into two species: Leishmania donovani and Leishmania infantum. Furthermore, the molecular haplotypes were concordant, in general, with the isoenzyme data of the complex. The donovani isolates from the Sudan that possessed the most ancestral sequence were of a single haplotype that significantly resembled the sequence of Leishmania major. Our sequence data tallied with a general neutral model of sequence evolution with manifestations of weak selection. The data allowed an approximate dating of the origin of the complex to a period contemporary to or predating the spread of modern humans out of Africa.

Plasticity of gp63 gene organization in Leishmania (Viannia) braziliensis and Leishmania (Viannia) peruviana.

The genomic organization of gp63 genes in 4 and 7 isolates of Leishmania braziliensis and L. peruviana, respectively was studied by RFLP analysis with 3 restriction enzymes (Bgl I, Sal I and Apa I). Our results showed a marked polymorphism among isolates. Some characters were specific to L. braziliensis or to L. peruviana, and others specific to the respective biogeographical populations of L. peruviana. The average minimum copy number of gp63 genes was found to be higher in L. braziliensis (71) than in L. peruviana (46), suggesting that deletion of gp63 genes might be partially involved in the size decrease of the chromosome bearing gp63 genes, observed between those 2 species (from 700 to 610 kb). Our results may suggest the existence of at least 2 arrays of heterologous gp63 repeats, varying in relative copy number between L. braziliensis and L. peruviana, and among isolates of the latter species. Rearrangement of the gp63 genes was observed during long-term in vitro maintenance of a reference strain of L. braziliensis. These observations document the existence of a dynamic gp63 gene organization in Leishmania of the braziliensis complex.

A focus of mucocutaneous leishmaniasis in Três Braços, Bahia, Brazil: characterization and identification of Leishmania stocks isolated from man and dogs

The characterization and identification to species and subspecies of 20 stocks of Leishmania isolated from the region of Três Braços, Bahia, Brazil, are described: 17 stocks were from patients and three from dogs. The following techniques were used (i) biological (growth in culture, hamster tissues and phlebotomine gut), (ii) biochemical (isoenzyme and kinetoplast DNA analysis) and (iii) immunological (using monoclonal antibodies). All except two stocks belong to the L. braziliensis complex. One of these two corresponded to L. mexicana amazonensis but the other, while clearly in the mexicana complex, showed slight differences from the L. mexicana amazonensis reference strain on isoenzyme analysis. Two stocks from different lesions in the same patient and with different growth characteristics in hamster tissues were both identified as L. braziliensis braziliensis. All the fully characterized stocks of the L. braziliensis complex were identified as L. braziliensis braziliensis. L. braziliensis guyanensis was not identified. Dog and human stocks of L. braziliensis braziliensis were indistinguishable. From these findings and other evidence, L. braziliensis braziliensis seems to be the predominant species transmitted in Três Braços.