Blood Parasite Load as an Early Marker to Predict Treatment Response in Visceral Leishmaniasis in Eastern Africa.

BACKGROUND: To expedite the development of new oral treatment regimens for visceral leishmaniasis (VL), there is a need for early markers to evaluate treatment response and predict long-term outcomes. METHODS: Data from 3 clinical trials were combined in this study, in which Eastern African VL patients received various antileishmanial therapies. Leishmania kinetoplast DNA was quantified in whole blood with real-time quantitative polymerase chain reaction (qPCR) before, during, and up to 6 months after treatment. The predictive performance of pharmacodynamic parameters for clinical relapse was evaluated using receiver-operating characteristic curves. Clinical trial simulations were performed to determine the power associated with the use of blood parasite load as a surrogate endpoint to predict clinical outcome at 6 months. RESULTS: The absolute parasite density on day 56 after start of treatment was found to be a highly sensitive predictor of relapse within 6 months of follow-up at a cutoff of 20 parasites/mL (area under the curve 0.92, specificity 0.91, sensitivity 0.89). Blood parasite loads correlated well with tissue parasite loads (ρ = 0.80) and with microscopy gradings of bone marrow and spleen aspirate smears. Clinical trial simulations indicated a > 80% power to detect a difference in cure rate between treatment regimens if this difference was high (> 50%) and when minimally 30 patients were included per regimen. CONCLUSIONS: Blood Leishmania parasite load determined by qPCR is a promising early biomarker to predict relapse in VL patients. Once optimized, it might be useful in dose finding studies of new chemical entities.

Efficacy and Safety of AmBisome in Combination with Sodium Stibogluconate or Miltefosine and Miltefosine Monotherapy for African Visceral Leishmaniasis: Phase II Randomized Trial.

BACKGROUND: SSG&PM over 17 days is recommended as first line treatment for visceral leishmaniasis in eastern Africa, but is painful and requires hospitalization. Combination regimens including AmBisome and miltefosine are safe and effective in India, but there are no published data from trials of combination therapies including these drugs from Africa. METHODS: A phase II open-label, non-comparative randomized trial was conducted in Sudan and Kenya to evaluate the efficacy and safety of three treatment regimens: 10 mg/kg single dose AmBisome plus 10 days of SSG (20 mg/kg/day), 10 mg/kg single dose AmBisome plus 10 days of miltefosine (2.5mg/kg/day) and miltefosine alone (2.5 mg/kg/day for 28 days). The primary endpoint was initial parasitological cure at Day 28, and secondary endpoints included definitive cure at Day 210, and pharmacokinetic (miltefosine) and pharmacodynamic assessments. RESULTS: In sequential analyses with 49-51 patients per arm, initial cure was 85% (95% CI: 73-92) in all arms. At D210, definitive cure was 87% (95% CI: 77-97) for AmBisome + SSG, 77% (95% CI 64-90) for AmBisome + miltefosine and 72% (95% CI 60-85) for miltefosine alone, with lower efficacy in younger patients, who weigh less. Miltefosine pharmacokinetic data indicated under-exposure in children compared to adults. CONCLUSION: No major safety concerns were identified, but point estimates of definitive cure were less than 90% for each regimen so none will be evaluated in Phase III trials in their current form. Allometric dosing of miltefosine in children needs to be evaluated. TRIAL REGISTRATION: The study was registered with ClinicalTrials.gov, number NCT01067443.

Corrigendum to 'Duplex quantitative Reverse-Transcriptase PCR for simultaneous assessment of drug activity against Leishmania intracellular amastigotes and their host cells' [Int. J. Parasitol. Drugs Drug Resist. 4 (2014) 14-19].

[This corrects the article DOI: 10.1016/j.ijpddr.2013.11.001.].

Duplex quantitative Reverse-Transcriptase PCR for simultaneous assessment of drug activity against Leishmania intracellular amastigotes and their host cells.

Currently available drugs for treatment of Leishmania infections are highly toxic and drug resistance to first line therapies has been observed. New, safer and more effective drugs are urgently needed to improve clinical resolution of the disease and reduce the risks associated with it. High-throughput screening of new compounds against cultured promastigotes is easy to perform, but the results are poorly predictive of in vivo efficacy. Intra-macrophage amastigote models provide a better proxy of the clinically relevant stage of disease and should be routinely implemented in the search for new anti-leishmanial agents, despite being labor intensive. This study describes the use of a duplex quantitative Reverse-Transcriptase PCR (qRT-PCR) for assessment of drug activity against Leishmania intracellular amastigotes and their host cells. The assay simultaneously quantifies Leishmania 18S ribosomal RNA and the human ß2-microglobulin (ß-2M) mRNA, used for monitoring drug cytotoxicity and test performance. Accurate determination of parasite viability by the newly developed qRT-PCR was confirmed by parallel assessment of compound performance against standard microscopy. Highly reproducible anti-leishmanial activities were obtained with a set of structurally- and pharmacologically-diverse compounds, whose toxicity against host cells correlated with a low ß-2M amplification. Sensitive and versatile, this duplex qRT-PCR offers a valuable tool for assessment of drug activities against Leishmania amastigotes and their host cells.

Safety and efficacy of single dose versus multiple doses of AmBisome for treatment of visceral leishmaniasis in eastern Africa: a randomised trial.

BACKGROUND: Anti-leishmanial drug regimens that include a single dose AmBisome could be suitable for eastern African patients with symptomatic visceral leishmaniasis (VL) but the appropriate single dose is unknown. METHODOLOGY: A multi-centre, open-label, non-inferiority, randomized controlled trial with an adaptive design, was conducted to compare the efficacy and safety of a single dose and multiple doses of AmBisome for the treatment of VL in eastern Africa. The primary efficacy endpoint was definitive cure (DC) at 6 months. Symptomatic patients with parasitologically-confirmed, non-severe VL, received a single dose of AmBisome 7.5 mg/kg body weight or multiple doses, 7 times 3 mg/kg on days 1-5, 14, and 21. If interim analyses, evaluated 30 days after the start of treatment following 40 or 80 patients, showed the single dose gave significantly poorer parasite clearance than multiple doses at the 5% significance level, the single dose was increased by 2·5 mg/kg. In a sub-set of patients, parasite clearance was measured by quantitative reverse transcriptase (qRT) PCR. PRINCIPAL FINDINGS: The trial was terminated after the third interim analysis because of low efficacy of both regimens. Based on the intention-to-treat population, DC was 85% (95%CI 73-93%), 40% (95%CI 19-64%), and 58% (95%CI 41-73%) in patients treated with multiple doses (n = 63), and single doses of 7·5 (n = 21) or 10 mg/kg (n = 40), respectively. qRT-PCR suggested superior parasite clearance with multiple doses as early as day 3. Safety data accorded with the drug label. CONCLUSIONS: The tested AmBisome regimens would not be suitable for VL treatment across eastern Africa. An optimal single dose regimen was not identified. TRIALS REGISTRATION: www.clinicaltrials.govNCT00832208.

Simple colorimetric trypanothione reductase-based assay for high-throughput screening of drugs against Leishmania intracellular amastigotes.

Critical 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 Leishmania clinical isolates is lacking. Here we describe a quantitative colorimetric assay in which the activity of a Leishmania native 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 Leishmania parasites. 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.

Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of Leishmania infantum in canine leishmaniasis based on cysteine protease B genes.

We developed a Leishmania infantum specific LAMP assay that was carried out using a set of, six primers targeting the cysteine protease B multi copy gene of L. infantum. Our result shows that we, successfully detect the L. infantum DNA and that amplification is specific as no cross reaction was seen, with L. major, L. tropica, L. turanica, L. aethiopica, L. tarentolae, L. gerbilii, Trypanosoma cruzi or, human genomic DNA. When compared to conventional cpb based PCR, the sensitivity of LAMP assay, was higher with a detection limit of 50 fg/µl of genomic L. infantum parasite DNA. Accurate and rapid, diagnosis of canine leishmaniasis (CanL) is an important issue that allows early treatment and, prevents transmission. Our developed LAMP assay was used to evaluate occurrences of Leishmania infantum in seventy five (75) dogs from the field. Blood samples were used to perform LAMP assay, classical PCR, IFAT and microscopy that was used as gold standard. The IFAT in addition to, microscopy, are the basic techniques used for CanL diagnosis at the School of Veterinary Medicine, where we obtained our samples. Compared to molecular methods, the serology (IFAT) test shows the, best sensitivity (88.57%) with, however, a much lower specificity (52.5%) due to a relatively high, number of false-positive results (22 animals). The PCR assay shows a low sensitivity (37.14%) and, specificity around (82.5%). Our LAMP assay shows a suitable sensitivity (54%) and a good specificity, (80%), with however, positive (70%) and negative (66%) predictive values. Furthermore, the best, positive likelihood ratio (LR+) was obtained by LAMP assay (2.7). This technique presents the highest, kappa value (with a fair agreement of 0.34). Moreover, the relative stability of the reagents indicates, that LAMP may be a good alternative to a conventional PCR, especially under field conditions. Finally in, a brief cost evaluation, the LAMP assay compares favorably with other molecular diagnostic tests. This, is the first study that evaluates the L. infantum specific LAMP alongside other diagnostics tools for, CanL. Our results indicate a suitable sensitivity and specificity for the developed LAMP assay that could, has usefulness application on dogs and human L. infantum diagnosis.

Dynamics of parasite clearance in cutaneous leishmaniasis patients treated with miltefosine.

Parasite loads were quantified in repeated skin biopsies from lesions of 2 patients with Old-World cutaneous leishmaniasis (CL) caused by Leishmania major and L. infantum during and after treatment with miltefosine. Miltefosine induced a rapid therapeutic effect on both infections with an initial decline of parasites of ∼1 log/week for the L. major infection. These observations illustrate the usability of quantifying parasite loads in skin lesions as a pharmacodynamic measure and quantitative descriptor of drug effect for CL supporting clinical assessment.

Comparison of short-term and long-term protocols for stabilization and preservation of RNA and DNA of Leishmania, Trypanosoma, and Plasmodium.

Molecular tools continue to be important in the prevention and control of parasitic diseases. However, using these techniques directly in the field remains a major challenge. Therefore, the preservation of clinical samples collected from endemic field areas for later analysis remains an important preanalytical process. This study aimed at identifying a suitable protocol for stabilization and preservation of RNA and DNA in bioclinical specimens for Trypanosoma, Leishmania, and Plasmodium research. Both spiked and unspiked blood samples were preserved in 7 protocols (different media; storage temperatures). Samples were evaluated for possible degradation of DNA and RNA along the storage duration up to the 10th week. Nucleic acid targets were assessed as follows: (i) Trypanosoma and Plasmodium RNA analysis was done using real-time nucleic acid sequence-based amplification (RT-NASBA) for 18S rRNA and for stage-specific Pfs25 mRNA, respectively; (ii) Trypanosoma DNA assessment analysis was conducted by using a conventional PCR for 18S rDNA; (iii) Leishmania RNA analysis was performed with a quantitative NASBA for 18S rRNA and Leishmania DNA assessment with an RT-PCR for 18S rDNA. Findings suggested that a newly developed L3™ buffer proved to be reliable and suitable for both short- and long-term preservation of parasite nucleic acid material. This buffer is envisaged to be suitable for utilization in field situations where resources are limited.

Diagnostic accuracy of the Leishmania OligoC-TesT and NASBA-Oligochromatography for diagnosis of leishmaniasis in Sudan.

BACKGROUND: The Leishmania OligoC-TesT and NASBA-Oligochromatography (OC) were recently developed for simplified and standardised molecular detection of Leishmania parasites in clinical specimens. We here present the phase II evaluation of both tests for diagnosis of visceral leishmaniasis (VL), cutaneous leishmaniasis (CL) and post kala-azar dermal leishmaniasis (PKDL) in Sudan. METHODOLOGY: The diagnostic accuracy of the tests was evaluated on 90 confirmed and 90 suspected VL cases, 7 confirmed and 8 suspected CL cases, 2 confirmed PKDL cases and 50 healthy endemic controls from Gedarif state and Khartoum state in Sudan. PRINCIPAL FINDINGS: The OligoC-TesT as well as the NASBA-OC showed a sensitivity of 96.8% (95% CI: 83.8%-99.4%) on lymph node aspirates and of 96.2% (95% CI: 89.4%-98.7%) on blood from the confirmed VL cases. The sensitivity on bone marrow was 96.9% (95% CI: 89.3%-99.1%) and 95.3% (95% CI: 87.1%-98.4%) for the OligoC-TesT and NASBA-OC, respectively. All confirmed CL and PKDL cases were positive with both tests. On the suspected VL cases, we observed a positive OligoC-TesT and NASBA-OC result in 37.1% (95% CI: 23.2%-53.7%) and 34.3% (95% CI: 20.8%-50.9%) on lymph, in 72.7% (95% CI: 55.8%-84.9%) and 63.6% (95% CI: 46.6%-77.8%) on bone marrow and in 76.9% (95% CI: 49.7%-91.8%) and 69.2% (95% CI: 42.4%-87.3%) on blood. Seven out of 8 CL suspected cases were positive with both tests. The specificity on the healthy endemic controls was 90% (95% CI: 78.6%-95.7%) for the OligoC-TesT and 100% (95% CI: 92.9%-100.0%) for the NASBA-OC test. CONCLUSIONS: Both tests showed high sensitivity on lymph, blood and tissue scrapings for diagnosis of VL, CL and PKDL in Sudan, but the specificity for clinical VL was significantly higher with NASBA-OC.

Phase II evaluation of sensitivity and specificity of PCR and NASBA followed by oligochromatography for diagnosis of human African trypanosomiasis in clinical samples from D.R. Congo and Uganda.

BACKGROUND: The polymerase chain reaction (PCR) and nucleic acid sequence-based amplification (NASBA) have been recently modified by coupling to oligochromatography (OC) for easy and fast visualisation of products. In this study we evaluate the sensitivity and specificity of the PCR-OC and NASBA-OC for diagnosis of Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense human African trypanosomiasis (HAT). METHODOLOGY AND RESULTS: Both tests were evaluated in a case-control design on 143 HAT patients and 187 endemic controls from the Democratic Republic of Congo (DRC) and Uganda. The overall sensitivity of PCR-OC was 81.8% and the specificity was 96.8%. The PCR-OC showed a sensitivity and specificity of 82.4% and 99.2% on the specimens from DRC and 81.3% and 92.3% on those from Uganda. NASBA-OC yielded an overall sensitivity of 90.2%, and a specificity of 98.9%. The sensitivity and specificity of NASBA-OC on the specimens from DRC was 97.1% and 99.2%, respectively. On the specimens from Uganda we observed a sensitivity of 84.0% and a specificity of 98.5%. CONCLUSIONS/SIGNIFICANCE: The tests showed good sensitivity and specificity for the T. b. gambiense HAT in DRC but rather a low sensitivity for T. b. rhodesiense HAT in Uganda.

Accordance and concordance of PCR and NASBA followed by oligochromatography for the molecular diagnosis of Trypanosoma brucei and Leishmania.

OBJECTIVE: To evaluate the repeatability and reproducibility of four simplified molecular assays for the diagnosis of Trypanosoma brucei spp. or Leishmania ssp. in a multicentre ring trial with seven participating laboratories. METHODS: The tests are based on PCR or NASBA amplification of the parasites nucleic acids followed by rapid read-out by oligochromatographic dipstick (PCR-OC and NASBA-OC). RESULTS: On purified nucleic acid specimens, the repeatability and reproducibility of the tests were Tryp-PRC-OC, 91.7% and 95.5%; Tryp-NASBA-OC, 95.8% and 100%; Leish-PCR-OC, 95.9% and 98.1%; Leish-NASBA-OC, 92.3% and 98.2%. On blood specimens spiked with parasites, the repeatability and reproducibility of the tests were Tryp-PRC-OC, 78.4% and 86.6%; Tryp-NASBA-OC, 81.5% and 89.0%; Leish-PCR-OC, 87.1% and 91.7%; Leish-NASBA-OC, 74.8% and 86.2%. CONCLUSION: As repeatability and reproducibility of the tests were satisfactory, further phase II and III evaluations in clinical and population specimens from disease endemic countries are justified.

Sensitivity and specificity of the Leishmania OligoC-TesT and NASBA-oligochromatography for diagnosis of visceral leishmaniasis in Kenya.

OBJECTIVE: To estimate the sensitivity and specificity of the OligoC-TesT and nucleic acid sequence-based amplification coupled to oligochromatography (NASBA-OC) for molecular detection of Leishmania in blood from patients with confirmed visceral leishmaniasis (VL) and healthy endemic controls from Kenya. METHODS: Blood specimens of 84 patients with confirmed VL and 98 endemic healthy controls from Baringo district in Kenya were submitted to both assays. RESULTS: The Leishmania OligoC-TesT showed a sensitivity of 96.4% (95% confidence interval [CI]: 90-98.8%) and a specificity of 88.8% (95% CI: 81-93.6%), while the sensitivity and specificity of the NASBA-OC were 79.8% (95% CI: 67-87%) and 100% (95% CI: 96.3-100%), respectively. CONCLUSION: Our findings indicate high sensitivity of the Leishmania OligoC-TesT on blood while the NASBA-OC is a better marker for active disease.

Development of a reverse transcriptase loop-mediated isothermal amplification (LAMP) assay for the sensitive detection of Leishmania parasites in clinical samples.

Here we describe a generic, reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP) assay, for the identification of Leishmania species from clinical samples. LAMP is an isothermal reaction recently developed as a point-of-care diagnostic tool. Primers were designed in the conserved region of the 18S ribosomal RNA (rRNA) gene; amplification was visualized by the pre-amplification addition of fluorescent detection reagent (FDR) and a simple UV lamp. By using a reverse-transcriptase step, the system detected infections between 10 and 100 parasites per mL. The assay was tested on a range of nucleic acid extracts from Leishmania species, visceral leishmaniasis (VL) patients from Sudan, and cutaneous leishmaniasis (CL) patients from Suriname. The sensitivity of RT-LAMP from the blood of VL patients was 83% (N = 30) compared with microscopy of bone-marrow and lymph-node aspirates; for CL patients the observed sensitivity was 98% (N = 43). The potential to use LAMP as a diagnostic tool for leishmaniasis is discussed.

Simplified molecular detection of Leishmania parasites in various clinical samples from patients with leishmaniasis.

BACKGROUND: Molecular methods to detect Leishmania parasites are considered specific and sensitive, but often not applied in endemic areas of developing countries due to technical complexity. In the present study isothermal, nucleic acid sequence based amplification (NASBA) was coupled to oligochromatography (OC) to develop a simplified detection method for the diagnosis of leishmaniasis. NASBA-OC, detecting Leishmania RNA, was evaluated using clinical samples from visceral leishmaniasis patients from East Africa (n = 30) and cutaneous leishmaniasis from South America (n = 70) and appropriate control samples. RESULTS: Analytical sensitivity was 10 parasites/ml of spiked blood, and 1 parasite/ml of culture. Diagnostic sensitivity of NASBA-OC was 93.3% (95% CI: 76.5%-98.8%) and specificity was 100% (95% CI: 91.1%-100%) on blood samples, while sensitivity and specificity on skin biopsy samples was 98.6% (95% CI: 91.2%-99.9%) and 100% (95% CI: 46.3%-100%), respectively. CONCLUSION: The NASBA-OC format brings implementation of molecular diagnosis of leishmaniasis in resource poor countries one step closer.

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.

Detection of Trypanosoma brucei parasites in blood samples using real-time nucleic acid sequence-based amplification.

Currently, the conventional diagnosis of human African trypanosomiasis (HAT) is by microscopic demonstration of trypomastigotes in blood, lymph, and/or cerebrospinal fluid. However, microscopic diagnosis of HAT is not sensitive enough and may give false-negative results, thus, denying the patient the necessary treatment of the otherwise fatal disease. For this reason, a highly sensitive technique needs to be developed to enhance case findings. In this study, the real-time nucleic acid sequence-based amplification assay described is based on amplification and concurrent detection of small subunit rRNA (18S rRNA) of Trypanosoma brucei. The sensitivity of the assay was evaluated on nucleic acid from in vitro cultured parasites and blood spiked with various parasites quantities. The assay detected 10 parasites/mL using cultured parasites as well as spiked blood. A sensitive assay such as the one developed in this study may become an alternative tool to confirm diagnosis of human African trypanosomiasis.

Didelphis marsupialis (common opossum): a potential reservoir host for zoonotic leishmaniasis in the metropolitan region of Belo Horizonte (Minas Gerais, Brazil).

Identification of the zoonotic reservoir is important for leishmaniasis control program. A number of (wild) animal species may serve as reservoir hosts, including the opossum Didelphis marsupialis. A survey carried out in Didelphis specimens (n = 111) from the metropolitan region of Belo Horizonte, an important focus of human leishmaniasis in Brazil, is reported. All animals were serologically tested with indirect fluorescence antibody test (IFAT) and direct agglutination tests (DAT) based on L. (L.) donovani or L. (V.) braziliensis antigen. A sub-population (n = 20) was analyzed with polymerase chain reaction (PCR) for the presence of Leishmania-specific DNA. For species identification, PCR-positive samples were subjected to restriction enzyme fragment polymorphism (RFLP) analysis. Depending on the sero-diagnostic test employed, the sero-prevalence varied between 8.1% (9/111 animals positive with DAT test based on L. braziliensis antigen) and 21.6% (24/111 animals positive with IFAT). Five out of 20 samples analyzed with PCR tested positive for the presence of Leishmania-specific DNA. RFLP analysis revealed that two samples contained L. braziliensis complex DNA, one contained L. donovani complex DNA, and two samples could not be typed with the methodology used. These data suggest a potential role for the opossum as a reservoir host for zoonotic leishmaniasis in the region.

Detection and identification of human Plasmodium species with real-time quantitative nucleic acid sequence-based amplification.

BACKGROUND: Decisions concerning malaria treatment depend on species identification causing disease. Microscopy is most frequently used, but at low parasitaemia (<20 parasites/mul) the technique becomes less sensitive and time consuming. Rapid diagnostic tests based on Plasmodium antigen detection do often not allow for species discrimination as microscopy does, but also become insensitive at <100 parasites/microl. METHODS: This paper reports the development of a sensitive and specific real-time Quantitative Nucleic Acid Sequence Based Amplification (real-time QT-NASBA) assays, based on the small-subunit 18S rRNA gene, to identify the four human Plasmodium species. RESULTS: The lower detection limit of the assay is 100-1000 molecules in vitro RNA for all species, which corresponds to 0.01-0.1 parasite per diagnostic sample (i.e. 50 microl of processed blood). The real-time QT-NASBA was further evaluated using 79 clinical samples from malaria patients: i.e. 11 Plasmodium. falciparum, 37 Plasmodium vivax, seven Plasmodium malariae, four Plasmodium ovale and 20 mixed infections. The initial diagnosis of 69 out of the 79 samples was confirmed with the developed real-time QT-NASBA. Re-analysis of seven available original slides resolved five mismatches. Three of those were initially identified as P. malariae mono-infection, but after re-reading the slides P. falciparum was found, confirming the real-time QT-NASBA result. The other two slides were of poor quality not allowing true species identification. The remaining five discordant results could not be explained by microscopy, but may be due to extreme low numbers of parasites present in the samples. In addition, 12 Plasmodium berghei isolates from mice and 20 blood samples from healthy donors did not show any reaction in the assay. CONCLUSION: Real-time QT-NASBA is a very sensitive and specific technique with a detection limit of 0.1 Plasmodium parasite per diagnostic sample (50 microl of blood) and can be used for the detection, identification and quantitative measurement of low parasitaemia of Plasmodium species, thus making it an effective tool for diagnostic purposes and useful for epidemiological and drug studies.