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ABSTRACTCritical to the search for new anti-leishmanial drugs is the availability of high-throughput screening (HTS) methods to test chemical compounds against the relevant stage for pathogenesis, the intracellular amastigotes. Recent progress in automated microscopy and genetic recombination has produced powerful tools for drug discovery. Nevertheless, a simple and efficient test for measuring drug activity against Leishmaniaclinical isolates is lacking. Here we describe a quantitative colorimetric assay in which the activity of a Leishmanianative enzyme is used to assess parasite viability. Enzymatic reduction of disulfide trypanothione, monitored by a microtiter plate reader, was used to quantify the growth of Leishmaniaparasites. An excellent correlation was found between the optical density at 412 nm and the number of parasites inoculated. Pharmacological validation of the assay was performed against the conventional alamarBlue method for promastigotes and standard microscopy for intracellular amastigotes. The activity of a selected-compound panel, including several anti-leishmanial reference drugs, demonstrated high consistency between the newly developed assay and the reference method and corroborated previously published data. Quality assessment with standard measures confirmed the robustness and reproducibility of the assay, which performed in compliance with HTS requirements. This simple and rapid assay provides a reliable, accurate method for screening anti-leishmanial agents, with high throughput. The basic equipment and manipulation required to perform the assay make it easy to implement, simplifying the method for scoring inhibitor assays.

Molecular tools, such as real-time nucleic acid sequence-based amplification (NASBA) and PCR, have been developed to detect Trypanosoma bruceiparasites 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. brucei18S rRNA by NASBA. The T. bruceiNASBA-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. bruceiNASBA-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.

ABSTRACTCurrently available methods for the diagnosis of cutaneous leishmaniasis (CL) have low sensitivities or are unable to quantify the number of viable parasites. This constitutes a major obstacle for the diagnosis of the disease and for the study of the effectiveness of treatment schedules and urges the development of improved detection methods. In this study, quantitative nucleic acid sequence-based amplification (QT-NASBA) technology was used to detect and quantify Leishmaniaparasites in skin biopsy samples from CL patients. The assay is based on the detection of a small subunit rRNA (18S rRNA), which may allow for the detection of viable parasites. The QT-NASBA assay was evaluated using in vitro-cultured promastigotes and amastigotes and 2-mm skin biopsy samples from Old and New World CL patients. The study demonstrated that the lower detection limit of the QT-NASBA was two parasites per biopsy sample. Parasites could be quantified in a range of 2 to 11,300,000 parasites per biopsy sample. The QT-NASBA could detect levels of parasites 100-fold lower than those detected by conventional PCR. Test evaluation revealed that the QT-NASBA had a sensitivity of 97.5% and a specificity of 100% in the present study. The QT-NASBA is a highly sensitive and specific method that allows quantification of both Old and New World Leishmaniaparasites in skin biopsy samples and may provide an important tool for diagnosis as well as for monitoring the therapy of CL patients.

ABSTRACTA quantitative nucleic acid sequence-based amplification (QT-NASBA) assay for the detection of Plasmodiumparasites has been developed. Primers and probes were selected on the basis of the sequence of the small-subunit rRNA gene. Quantification was achieved by coamplification of the RNA in the sample with one modified in vitro RNA as a competitor in a single-tube NASBA reaction. Parasite densities ranging from 10 to 108Plasmodium falciparumparasites per ml could be demonstrated and quantified in whole blood. This is approximately 1,000 times more sensitive than conventional microscopy analysis of thick blood smears. Comparison of the parasite densities obtained by microscopy and QT-NASBA with 120 blood samples from Kenyan patients with clinical malaria revealed that for 112 of 120 (93%) of the samples results were within a 1-log difference. QT-NASBA may be especially useful for the detection of low parasite levels in patients with early-stage malaria and for the monitoring of the efficacy of drug treatment.

Two nonculture methods, in situhybridization and immunoperoxidase staining with monoclonal antibodies, were compared for the detection of Hemophilus influenzaein 184 sputa. For in situhybridization, a biotin-labeled probe of total genomic DNA of H influenzaetype b was prepared that hybridizes specifically with H influenzae, H parainfluenzae, H hemolyticus, and H parahemolyticusDNA. Immunoperoxidase staining was done with monoclonal antibody 8BD9 directed against outer membrane protein P6 of H influenzae.Both techniques detected Hemophilusin sputum equally well and were superior to culture: all 30 sputum samples culture-positive for H influenzaewere positive on both nonculture tests, and 13 additional positive sputum samples were detected from which Hemophiluswas not cultured. The higher sensitivity of the nonculture tests was mainly attributed to culture failure because of overgrowth of H influenzaeby other bacteria, especially in patients with cystic fibrosis. The immunoperoxidase staining technique appeared slightly easier and quicker to perform than the in situhybridization test. For the in situDNA hybridization probe, DNA can be prepared from any strain of H influenzae. Theimmunoperoxidase test requires monoclonal antibody 8BD9 but has a higher specificity than the hybridization technique. Both techniques can be reliably applied, especially for the detection of Hemophilus in sputum of patients with cystic fibrosis.