Gert Van der Auwera

Phylogeny of Leishmania species based on the heat-shock protein 70 gene

The 70kDa heat-shock protein (HSP70) is conserved across prokaryotes and eukaryotes, and the protein as well as its encoding gene have been applied in phylogenetic studies of different parasites. In spite of the frequent use of New World Leishmania species identification on the basis of restriction fragment length polymorphisms (RFLP) in the hsp70 gene, it was never sequenced extensively for studying evolutionary relationships. To fill this void we determined the nucleotide sequence of an 1380bp fragment of the coding region commonly used in RFLP analysis, from 43 isolates and strains of different geographic origins. Combination with previously determined sequences amounted to a phylogenetic analysis including 52 hsp70 sequences representing 17 species commonly causing leishmaniasis both in the New and Old World. The genus Leishmania formed a monophyletic group with three distinct subgenera L. (Leishmania), L. (Viannia), and L. (Sauroleishmania). The obtained phylogeny supports the following eight species: L. (L.) donovani, L. (L.) major, L. (L.) tropica, L. (L.) mexicana, L. (V.) lainsoni, L. (V.) naiffi, L. (V.) guyanensis and L. (V.) braziliensis, in some of which subspecies can be recognized: L. (L.) donovani infantum, L. (V.) guyanensis panamensis, and L. (V.) braziliensis peruviana. The currently recognized L. (L.) aethiopica, L. (L.) garnhami, and L. (L.) amazonensis did not form monophyletic clusters. These findings are discussed in relation to results from other genes and proteins, which have to be integrated in order to build a genetically supported taxonomy for the entire genus.

A Simplified and Standardized Polymerase Chain Reaction Format for the Diagnosis of Leishmaniasis

Abstract Background. Definite diagnosis of Leishmania infections is based on demonstration of the parasite by microscopic analysis of tissue biopsy specimens or aspirate samples. However, microscopy generally shows low sensitivity and requires invasive sampling. Methods. We describe here the development of a simple and rapid test for the detection of polymerase chain reaction-amplified Leishmania DNA. A phase 1 evaluation of the text was conducted in clinical samples from 60 nonendemic and 45 endemic control subjects and from 44 patients with confirmed cutaneous leishmaniasis (CL), 12 with mucocutaneous leishmaniasis (MCL), and 43 with visceral leishmaniasis (VL) from Peru, Kenya, and Sudan. Results. The lower detection limits of the assay are 10 fg of Leishmania DNA and 1 parasite in 180 µL of blood. The specificity was 98.3% (95% confidence interval [CI], 91.1%–99.7%) and 95.6% (95% CI, 85.2%–98.8%) for nonendemic and endemic control samples, respectively, and the sensitivity was 93.2% (95% CI, 81.8%–97.7%), 91.7% (95% CI, 64.6%–98.5%), and 86% (95% CI, 72.7%–93.4%) for lesions from patients with CL or MCL and blood from patients with VL, respectively. Conclusions. The Leishmania OligoC-TesT showed high specificity and sensitivity in clinical samples and was able to detect the parasite in samples obtained by less invasive means, such as blood, lymph, and lesion scrapings. The assay is a promising new tool for simplified and standardized molecular detection of Leishmania parasites.

Domestic animals and epidemiology of visceral leishmaniasis, Nepal.

Proximity of Leishmania donovani–positive goats is a risk factor for human infection.

Multilocus genotyping reveals high heterogeneity and strong local population structure of the Plasmodium vivax population in the Peruvian Amazon

BackgroundPeru is one of the Latin American countries with the highest malaria burden, mainly due to Plasmodium vivax infections. However, little is known about P. vivax transmission dynamics in the Peruvian Amazon, where most malaria cases occur. The genetic diversity and population structure of P. vivax isolates collected in different communities around Iquitos city, the capital of the Peruvian Amazon, was determined.MethodsPlasmodium vivax population structure was determined by multilocus genotyping with 16 microsatellites on 159 P. vivax infected blood samples (mono-infections) collected in four sites around Iquitos city. The population characteristics were assessed only in samples with monoclonal infections (n = 94), and the genetic diversity was determined by calculating the expected heterozygosity and allelic richness. Both linkage disequilibrium and the genetic differentiation (θ) were estimated.ResultsThe proportion of polyclonal infections varied substantially by site (11% - 70%), with the expected heterozygosity ranging between 0.44 and 0.69; no haplotypes were shared between the different populations. Linkage disequilibrium was present in all populations (IAS 0.14 - 0.61) but was higher in those with fewer polyclonal infections, suggesting inbreeding and a clonal population structure. Strong population differentiation (θ = 0.45) was found and the Bayesian inference cluster analysis identified six clusters based on distinctive allele frequencies.ConclusionThe P. vivax populations circulating in the Peruvian Amazon basin are genetically diverse, strongly differentiated and they have a low effective recombination rate. These results are in line with the low and clustered pattern of malaria transmission observed in the region around Iquitos city.

High Complexity of Plasmodium vivax Infections in Symptomatic Patients from a Rural Community in Central Vietnam Detected by Microsatellite Genotyping

Fourteen published and three newly identified polymorphic microsatellites were used to genotype 69 Plasmodium vivax samples obtained from 39 patients detected over a period of two years who lived in a rural community of central Vietnam. All samples were polyclonal with an average expected heterozygosity of 0.86. Among the 39 patients, 16 experienced 1–5 recurrent episodes of P. vivax malaria, most of them (83%) with a different genotype profile compared with previous infections. The minimal set of microsatellites required for differentiating the genotype profiles of the recurrent infections compared with the full set of 17 microsatellites was explored. A combination of five markers was sufficient to identify all recurrent infections with an unrelated or different genotype profile compared with all previous episodes.

Species Typing in Dermal Leishmaniasis

SUMMARY Leishmania is an infectious protozoan parasite related to African and American trypanosomes. All Leishmania species that are pathogenic to humans can cause dermal disease. When one is confronted with cutaneous leishmaniasis, identification of the causative species is relevant in both clinical and epidemiological studies, case management, and control. This review gives an overview of the currently existing and most used assays for species discrimination, with a critical appraisal of the limitations of each technique. The consensus taxonomy for the genus is outlined, including debatable species designations. Finally, a numerical literature analysis is presented that describes which methods are most used in various countries and regions in the world, and for which purposes.

Detection of Leptomonas sp. parasites in clinical isolates of Kala-azar patients from India

We report here nine unusual cases of Kala-azar, of which parasites were isolated and found by 18S rRNA gene sequencing to be most similar to Leptomonas species. One of these isolates was used to inoculate Balb/c mice; organs were collected and directly submitted to a genus-specific rDNA-ITS1 PCR analysis: this revealed the presence of both Leptomonas sp. and Leishmania donovani. Therefore, we conclude that there was a mixed infection of Leptomonas sp. and L. donovani in this isolate. We consider that mixed infection may be present in the patients themselves, Leptomonas persisting in them because of the immuno-suppression associated with Kala-azar.

PCR and direct agglutination as Leishmania infection markers among healthy Nepalese subjects living in areas endemic for Kala-Azar.

Objective  To compare a PCR assay and direct agglutination test (DAT) for the detection of potential markers of Leishmania infection in 231 healthy subjects living in a kala‐azar endemic focus of Nepal.

Real-Time PCR Assay for Detection and Quantification of Leishmania (Viannia) Organisms in Skin and Mucosal Lesions: Exploratory Study of Parasite Load and Clinical Parameters

ABSTRACT Earlier histopathology studies suggest that parasite loads may differ between cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML) lesions and between acute and chronic CL. Formal demonstration requires highly sensitive detection and accurate quantification of Leishmania in human lesional tissue. In this study, we developed a quantitative real-time PCR (qPCR) assay targeting minicircle kinetoplast DNA (kDNA) to detect and quantify Leishmania (Viannia) parasites. We evaluated a total of 156 lesion biopsy specimens from CL or ML suspected cases and compared the quantitative performance of our kDNA qPCR assay with that of a previously validated qPCR assay based on the glucose-6-phosphate dehydrogenase (G6PD) gene. We also examined the relationship between parasite load and clinical parameters. The kDNA qPCR sensitivity for Leishmania detection was 97.9%, and its specificity was 87.5%. The parasite loads quantified by kDNA qPCR and G6PD qPCR assays were highly correlated (r = 0.87; P < 0.0001), but the former showed higher sensitivity (P = 0.000). CL lesions had 10-fold-higher parasite loads than ML lesions (P = 0.009). Among CL patients, the parasite load was inversely correlated with disease duration (P = 0.004), but there was no difference in parasite load according to the parasite species, the patients age, and number or area of lesions. Our findings confirm that CL and recent onset of disease (<3 months) are associated with a high parasite load. Our kDNA qPCR assay proved highly sensitive and accurate for the detection and quantification of Leishmania (Viannia) spp. in lesion biopsy specimens. It has potential application as a diagnostic and follow-up tool in American tegumentary leishmaniasis.

Accurate and rapid species typing from cutaneous and mucocutaneous leishmaniasis lesions of the New World.

The heat-shock protein 70 gene (hsp70) has been exploited for Leishmania species identification in the Old and New World, using polymerase chain reaction (PCR) followed by restriction fragment length polymorphism analysis. Three new Leishmania-specific hsp70 PCRs were recently described, and we applied 2 of these on 89 clinical samples from a total of 73 Peruvian patients with either cutaneous or mucocutaneous leishmaniasis. The new PCRs on average showed a 2- to 3-fold improved sensitivity in the tested sample types (lesion biopsies, aspirates, and scrapings), for both genus detection and species typing, and were most successful in biopsies. Leishmania braziliensis, L. peruviana, and L. guyanensis were encountered. About one third of the L. braziliensis parasites contained 2 hsp70 alleles. This study is a paradigm for the implementation of a globally applicable upgraded tool for the identification of Leishmania directly on human specimens from cutaneous and mucocutaneous lesions in the New World.