Expression profiling of the Leishmania life cycle: cDNA arrays identify developmentally regulated genes present but not annotated in the genome.

As genomic sequencing of Leishmania nears completion, functional analyses that provide a global genetic perspective on biological processes are important. Despite polycistronic transcription, RNA transcript abundance can be measured using microarrays. To provide a resource to evaluate cDNA arrays, we undertook 5' expressed sequence tag analysis of 2183 full-length randomly selected cDNAs from Leishmania major promastigote (days 3, 7, 10 of culture in vitro), and lesion-derived amastigote libraries. PCR-amplified inserts from 1830 of these cDNA representing 1001 unique genes were spotted onto microarrays, and compared internally with PCR-amplified open reading frames (ORFs) from 904 genes representing 842 unique genes annotated in the L. major genome. Microarrays were screened with RNA from procyclic, metacyclic and amastigote populations of L. major. Redundant clones on the array gave highly reproducible results, providing confidence in identification of stage-specific gene expression. Four hundred and thirty unique (i.e. non-redundant) stage-specific genes were identified. A higher percentage of stage-specific gene expression was observed in amastigotes ( approximately 35%) compared to metacyclics ( approximately 12%) for both cDNAs and ORFs, but cDNAs provided a richer source of regulated genes than currently annotated ORFs from the Leishmania genome. In mapping cDNAs onto the Leishmania genome, we noted that approximately 42% aligned to regions not recognised as genes using current predictive annotation tools. These genes are highly represented in our stage-specific genes, and therefore represent important drug targets and vaccine candidates. Careful annotation of cDNAs onto the Leishmania genome will be important before producing the next generation of oligonucleotide arrays based on annotated genes of the genomic sequencing project.

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.