Thierry Laurent

Gene Expression Analysis of the Mechanism of Natural Sb(V) Resistance in Leishmania donovani Isolates from Nepal

ABSTRACT Control of visceral leishmaniasis (VL) is being challenged by the emergence of natural resistance against the first line of treatment, pentavalent antimonials [Sb(V)]. An insight into the mechanism of natural Sb(V) resistance is required for the development of efficient strategies to monitor the emergence and spreading of Sb(V) resistance in countries where VL is endemic. In this work, we have focused on the mechanism of natural Sb(V) resistance emerging in Nepal, a site where anthroponotic VL is endemic. Based on the current knowledge of Sb(V) metabolism and of the in vitro trivalent antimonial [Sb(III)] models of resistance to Leishmania spp., we selected nine genes for a comparative transcriptomic study on natural Sb(V)-resistant and -sensitive Leishmania donovani isolates. Differential gene expression patterns were observed for the genes coding for 2-thiol biosynthetic enzymes, gamma-glutamylcysteine synthetase (GCS) and ornithine decarboxylase (ODC), and for the Sb(III) transport protein aquaglyceroporin 1 (AQP1). The results indicate that the mechanism for natural Sb(V) resistance partially differs from the mechanism reported for in vitro Sb(III) resistance. More specifically, we hypothesize that natural Sb(V) resistance results from (i) a changed thiol metabolism, possibly resulting in inhibition of Sb(V) activation in amastigotes, and (ii) decreased uptake of the active drug Sb(III) by amastigotes.

Molecular Dipstick Test for Diagnosis of Sleeping Sickness

ABSTRACT Human African trypanosomiasis (HAT) or sleeping sickness is a neglected disease that affects poor rural populations across sub-Saharan Africa. Confirmation of diagnosis is based on detection of parasites in either blood or lymph by microscopy. Here we present the development and the first-phase evaluation of a simple and rapid test (HAT-PCR-OC [human African trypanosomiasis-PCR-oligochromatography]) for detection of amplified Trypanosoma brucei DNA. PCR products are visualized on a dipstick through hybridization with a gold-conjugated probe (oligochromatography). Visualization is straightforward and takes only 5 min. Controls both for the PCR and for DNA migration are incorporated into the assay. The lower detection limit of the test is 5 fg of pure T. brucei DNA. One parasite in 180 μl of blood is still detectable. Sensitivity and specificity for T. brucei were calculated at 100% when tested on blood samples from 26 confirmed sleeping sickness patients, 18 negative controls (nonendemic region), and 50 negative control blood samples from an endemic region. HAT-PCR-OC is a promising new tool for diagnosis of sleeping sickness in laboratory settings, and the diagnostic format described here may have wider application for other infectious diseases.

Nucleic Acid Sequence-Based Amplification with Oligochromatography for Detection of Trypanosoma brucei in Clinical Samples

Molecular tools, such as real-time nucleic acid sequence-based amplification (NASBA) and PCR, have been developed to detect Trypanosoma brucei parasites in blood for the diagnosis of human African trypanosomiasis (HAT). Despite good sensitivity, these techniques are not implemented in HAT control programs due to the high cost of the equipment, which is unaffordable for laboratories in developing countries where HAT is endemic. In this study, a simplified technique, oligochromatography (OC), was developed for the detection of amplification products of T. brucei 18S rRNA by NASBA. The T. brucei NASBA-OC test has analytical sensitivities of 1 to 10 parasites/ml on nucleic acids extracted from parasite culture and 10 parasites/ml on spiked blood. The test showed no reaction with nontarget pathogens or with blood from healthy controls. Compared to the composite standard applied in the present study, i.e., parasitological confirmation of a HAT case by direct microscopy or by microscopy after concentration of parasites using either a microhematocrit centrifugation technique or a mini-anion-exchange centrifugation technique, NASBA-OC on blood samples had a sensitivity of 73.0% (95% confidence interval, 60 to 83%), while standard expert microscopy had a sensitivity of 57.1% (95% confidence interval, 44 to 69%). On cerebrospinal fluid samples, NASBA-OC had a sensitivity of 88.2% (95% confidence interval, 75 to 95%) and standard microscopy had a sensitivity of 86.2% (95% confidence interval, 64 to 88%). The T. brucei NASBA-OC test developed in this study can be employed in field laboratories, because it does not require a thermocycler; a simple heat block or a water bath maintained at two different temperatures is sufficient for amplification.

Universal PCR assays for the differential detection of all Old World Leishmania species.

For the epidemiological monitoring and clinical case management of leishmaniasis, determination of the causative Leishmania species gains importance. Current assays for the Old World often suffer from drawbacks in terms of validation on a geographically representative sample set and the ability to recognize all species complexes. We want to contribute to standardized species typing for Old World leishmaniasis. We determined the ribosomal DNA internal transcribed spacer 1 sequence of 24 strains or isolates, and validated four species-specific polymerase chain reactions (PCRs) amplifying this target. They discriminate L. aethiopica, L. tropica, L. major, and the L. donovani complex, use the same cycling conditions, and include an internal amplification control. Our PCRs amplify 0.1xa0pg of Leishmania DNA, while being 100% specific for species identification on an extensive panel of geographically representative strains and isolates. Similar results were obtained in an endemic reference laboratory in Kenya. Species could also be identified in clinical specimens. The presented PCRs require only agarose gel detection, and have several other advantages over many existing assays. We outline potential problems, suggest concrete solutions for transferring the technique to other settings, and deliver the proof-of-principle for analyzing clinical samples.

Fluorogenic Assay for Molecular Typing of the Leishmania donovani Complex: Taxonomic and Clinical Applications

We describe a new fluorogenic assay for the identification of species and intraspecies groups within the Leishmania donovani complex. The assay combined (1) 2 polymerase chain reactions targeting the 2 cysteine proteinase b isogenes and (2) a fluorescence-resonance energy transfer/melting curve analysis of the polymorphisms within a 31-nt region. All strains within the L. donovani complex were distinguished from L. tropica, L. major, and L. aethiopica, and 5 distinct groups were identified within the L. donovani complex. Discrepancies were observed with the present taxonomy on the basis of isoenzyme analysis and concerned East African strains, which suggests the need for a systematic reevaluation of the taxonomy. The capacity to type parasites directly from clinical samples was demonstrated with blood and bone marrow samples. This rapid and high-throughput alternative for molecular diagnosis and epidemiological studies of visceral leishmaniasis could be adapted for use with other Leishmania species.

Identification of Old World Leishmania spp. by specific polymerase chain reaction amplification of cysteine proteinase B genes and rapid dipstick detection

We used the cysteine proteinase B (cpb) gene family of the trypanosomatid genus Leishmania as a target to develop rapid, specific, and easy-to-use polymerase chain reaction (PCR) tests to discriminate Leishmania infantum, Leishmania donovani, Leishmania tropica, Leishmania aethiopica, and Leishmania major. Identification of all 5 Old World species and validation of intraspecies variability are features lacking in other species-specific PCRs. Amplicon analysis was done on agarose gels and was further simplified by using an oligochromatography dipstick to detect L. infantum and L. donovani products. Because the analytical sensitivity is lower than that of certain other species- and genus-specific PCRs, our assays are especially valuable for use on cultured isolates or directly on cryostabilates. As such, they can be implemented by research and health centers having access to culturing, DNA isolation, and PCR.

Leishmania OligoC-TesT as a Simple, Rapid, and Standardized Tool for Molecular Diagnosis of Cutaneous Leishmaniasis in Peru

ABSTRACT Molecular methods such as PCR have become attractive tools for diagnosis of cutaneous leishmaniasis (CL), both for their high sensitivity and for their specificity. However, their practical use in routine diagnosis is limited due to the infrastructural requirements and the lack of any standardization. Recently, a simplified and standardized PCR format for molecular detection of Leishmania was developed. The Leishmania OligoC-TesT is based on simple and rapid detection using a dipstick with PCR-amplified Leishmania DNA. In this study, we estimated the diagnostic accuracy of the Leishmania OligoC-TesT for 61 specimens from 44 CL-suspected patients presenting at the leishmaniasis clinic of the Instituto de Medicina Tropical Alexander von Humboldt, Peru. On the basis of parasitological detection and the leishmanin skin test (LST), patients were classified as (i) confirmed CL cases, (ii) LST-positive cases, and (iii) LST-negative cases. The sensitivities of the Leishmania OligoC-TesT was 74% (95% confidence interval (CI), 60.5% to 84.1%) for lesion aspirates and 92% (95% CI, 81.2% to 96.9%) for scrapings. A significantly higher sensitivity was observed with a conventional PCR targeting the kinetoplast DNA on the aspirates (94%) (P = 0.001), while there was no significant difference in sensitivity for the lesion scrapings (88%) (P = 0.317). In addition, the Leishmania OligoC-TesT was evaluated for 13 CL-suspected patients in two different peripheral health centers in the central jungle of Peru. Our findings clearly indicate the high accuracy of the Leishmania OligoC-TesT for lesion scrapings for simple and rapid molecular diagnosis of CL in Peru.