Various molecular approaches for diagnosing Leishmania
infection have been described. For example, PCR methods that target minicircle kDNA can be highly sensitive for detecting Leishmania
parasites because of the abundance of minicircles in each kinetoplast (2
). However, high-level sequence polymorphism among minicircles is an impediment for species identification with protocols based solely on kDNA PCR. Targeting the rRNA-coding cluster poses other issues (7
). Highly sensitive approaches for identifying particular Leishmania
species (or a species complex/subgenus) have been described (8
). However, if multiple Leishmania
spp. need to be differentiated in a diagnostic laboratory, molecular approaches that require different PCR primers for each species have the potential for carryover contamination. Although strategies to minimize this potential have been developed (5
), contamination has been observed even when strict protocols were followed (20
Our goal was to develop a molecular approach for species-level discrimination that requires only one pair of PCR primers. We focused on the ITS region: the rRNA internal spacers are subject to less evolutionary pressure and show more sequence divergence than the coding regions and have been proposed as targets for molecular typing (1
). We identified a region of the ITS2 adequate for our diagnostic goal and designed generic PCR primers (LGITSF2/LGITSR2) to amplify this fragment from Leishmania
spp. associated with human infection. We found substantial differences in the ITS2 region spanned by these primers that generally allowed species identification.
During our analysis of the reference strains, we noted intraspecies variability in the AT, TC, TG, or GA microsatellite repeats, as have other investigators (12
), which initially hindered use of the ITS2 fragment for species identification. We also noted some distinct sequences because of a few transitions and transversions. In most instances, distinct sequences were found in clones obtained from the same isolate, which likely indicated the presence of multiple different ITS copies in the same genome. To exclude the possibility of erroneous incorporation of nucleotides (11
), we reamplified the DNA from some specimens by using Pfu
High-Fidelity DNA polymerase (Stratagene); the results were the same as those with the routinely used Taq
DNA polymerase (data not shown). During the development phase of the assay, we cloned reference strain amplicons to obtain the sequence of the entire diagnostic fragment for each species, and we demonstrated that partial sequences sufficed for species identification. Therefore, in the validation phase, when we tested clinical specimens, we used direct sequencing analysis, without cloning, and in some instances relied on partial sequence data.
The distribution of species among the clinical specimens [e.g., the disproportionate number of patients infected with L. (V.) panamensis] reflects travel and immigration patterns among U.S. civilians evaluated for leishmaniasis; almost all of the clinical specimens we analyzed were from skin lesions. Although the PCR results obtained with different combinations of the reverse primers LGITSR1 and LGITSR2 were comparable for the isolates tested (A, B, and C), we had insufficient numbers of specimens positive for L. (L.) amazonensis and L. (L.) aethiopica—the species that prompted us to design the alternative reverse primer (LGITS2R1)—to compare the sensitivities of the primer pairs. We correctly identified the species as L. (L.) amazonensis for seven cultured isolates (including two WHO reference strains). However, this species was not identified by the parasitologic or molecular approach for any clinical specimens (). For L. (L.) aethiopica, the analyses included three cultured isolates (including one from WHO) and two clinical specimens ( and ).
The overall agreement between the molecular and parasitologic approaches was >98% when the comparison, of necessity, was limited to specimens for which a species determination was accomplished by both approaches. The two discordant results (among 159 patients) remained unresolved: for one patient, L. (V.) guyanensis was identified by the molecular approach versus L. (V.) braziliensis by the parasitologic approach (culture followed by isoenzyme analysis), and for the other patient, L. (L.) tropica was identified versus L. (L.) major, respectively. No other methods were used to evaluate the specimens; both results were epidemiologically plausible (), and the possibility of coinfection or of variant strains could not be excluded.
In general, our molecular approach differentiated among Viannia
spp., namely, L. (V.) braziliensis
, L. (V.) guyanensis
, and L. (V.) panamensis
, on the basis of four SNPs that appear to be in linkage disequilibrium with the genes that code for the isoenzymes we used to differentiate them (i.e., 6PGDH and GPI). These species, each of which likely is heterogeneous, have been associated with phenotypic (clinical) differences, which underscores the importance of species-level diagnostic techniques (4
Our molecular approach did not differentiate L. (L.) infantum
from L. (L.) chagasi
, which many experts consider synonymous (18
). However, some distinguishing features in the ITS2 fragment were noted between the reference isolates of L. (L.) donovani
versus L. (L.) infantum/chagasi
, i.e., the length of a G stretch and, for the WHO L. (L.) donovani
strain from Asia, a G435T transversion. Similar variation has been reported previously for some L. (L.) donovani
). However, for several clinical specimens [four of the six classified as L. (L.) donovani
], the approach we used appears to have resulted in misclassification of L. (L.) infantum/chagasi
as L. (L.) donovani
. Additional experience with more specimens from more areas of endemicity is needed to ascertain the ability of this ITS2 molecular target to reliably distinguish L. (L.) donovani
from L. (L.) infantum/chagasi
Approximately half of the patients with negative cultures but positive PCR results had slides available that were positive by light microscopic examination (), thereby confirming the diagnosis of leishmaniasis and underscoring the plausibility of PCR positivity. In some instances, negative culture (versus molecular) results might have been attributable to fastidious or nonviable parasites in the specimens we received or to contamination of the cultures, e.g., because of suboptimal handling or delayed shipment to the CDC. Lack of PCR positivity for some of the culture-positive specimens might have been attributable to heterogeneous distributions of parasites in specimens, such that the parasite/DNA concentration in the portion analyzed by PCR was below the detection limit of the assay. Preliminary data from a 40-cycle SYBR green-based real-time PCR assay under development in our laboratory suggest that positive specimens with threshold cycle (CT) values around 35 could be negative by ITS2 PCR and/or culture (M. E. de Almeida, unpublished data).
A drawback of our approach is the need for DNA sequencing analysis, which is not yet available in most laboratories in areas of endemicity. However, the ITS2 fragment has cleavage sites for HinP1I or HhaI enzymes (GCGC) and MnlI enzyme (GAGG) that may have utility for identifying the species we evaluated except for those in the Viannia subgenus. A cleavage site for HphI (GGTGAN7) could be used to identify L. (V.) guyanensis but not L. (V.) braziliensis or L. (V.) panamensis. These cleavage sites could be explored as targets for restriction fragment length polymorphism analysis to distinguish among a few species by laboratories in which DNA sequencing analysis is not yet available.
In summary, our findings confirm our hypothesis that a region of the ITS2 gene can complement the characterization of Leishmania parasites at the species level. The approach we developed can be used as a diagnostic tool in reference laboratories that have adequate infrastructure to perform molecular characterization of pathogens.