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.

Development and Evaluation of a Novel Loop-Mediated Isothermal Amplification Assay for Diagnosis of Cutaneous and Visceral Leishmaniasis.

A novel pan-Leishmania loop-mediated isothermal amplification (LAMP) assay for the diagnosis of cutaneous and visceral leishmaniasis (CL and VL) that can be used in near-patient settings was developed. Primers were designed based on the 18S ribosomal DNA (rDNA) and the conserved region of minicircle kinetoplast DNA (kDNA), selected on the basis of high copy number. LAMP assays were evaluated for CL diagnosis in a prospective cohort trial of 105 patients in southwest Colombia. Lesion swab samples from CL suspects were collected and were tested using the LAMP assay, and the results were compared to those of a composite reference of microscopy and/or culture in order to calculate diagnostic accuracy. LAMP assays were tested on samples (including whole blood, peripheral blood mononuclear cells, and buffy coat) from 50 suspected VL patients from Ethiopia. Diagnostic accuracy was calculated against a reference standard of microscopy of splenic or bone marrow aspirates. To calculate analytical specificity, 100 clinical samples and isolates from fever-causing pathogens, including malaria parasites, arboviruses, and bacteria, were tested. We found that the LAMP assay had a sensitivity of 95% (95% confidence interval [CI], 87.2% to 98.5%) and a specificity of 86% (95% CI, 67.3% to 95.9%) for the diagnosis of CL. With VL suspects, the sensitivity of the LAMP assay was 92% (95% CI, 74.9% to 99.1%) and its specificity was 100% (95% CI, 85.8% to 100%) in whole blood. For CL, the LAMP assay is a sensitive tool for diagnosis and requires less equipment, time, and expertise than alternative CL diagnostics. For VL, the LAMP assay using a minimally invasive sample is more sensitive than the gold standard. Analytical specificity was 100%.

Quantitative analysis of Cryptosporidium growth in in vitro culture--the impact of parasite density on the success of infection.

Cryptosporidium is an important waterborne pathogen for which no treatment or vaccination is available. This study set out to quantify DNA replication of Cryptosporidium parvum in vitro. Cryptosporidium DNA could be detected at up to 60 % of input level in both host-cell-free and host cell containing cultures 6 days after infection with living sporozoites, but was lost within 2 days in cultures inoculated with UV-inactivated sporozoites. Total DNA increased between days 2 and 6, evidence of successful DNA replication in both cell-free and host-cell-containing cultures. Overall however, only a small fraction (up to 5 %) of parasite DNA could be found associated with host cells or bound to plastic of the cell-free cultures, and the majority of parasite DNA was present in the cell culture medium, separable by simple decantation. After 2 days, in host-cell-containing cultures, the parasite DNA could be concentrated by slow centrifugation, suggesting that it was associated with intact parasite cells, but at 6 days, the majority could not be centrifuged and is therefore thought to have represented copies associated with dead and degraded parasites. In cell-free cultures and in larger plates, the majority of DNA was in this form. Performance of the parasite was best in small culture plates, and least in the largest plate sizes. We interpret these results as suggesting that Cryptosporidium sporozoites first bind to the host cell monolayer or to the plasticware, but then by 2 days, there has been a substantial release of parasites back into the medium. Host-cell-free cultures also supported modest replication and may have represented DNA synthesis in cells beginning merogony. The role of the host cells is unclear, as so much of the parasite DNA is released into the medium. Host cells may provide a feeder role, conditioning the medium for Cryptosporidium development.

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.

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.

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.

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.

Comparison between quantitative nucleic acid sequence-based amplification, real-time reverse transcriptase PCR, and real-time PCR for quantification of Leishmania parasites.

DNA or RNA amplification methods for detection of Leishmania parasites have advantages regarding sensitivity and potential quantitative characteristics in comparison with conventional diagnostic methods but are often still not routinely applied. However, the use and application of molecular assays are increasing, but comparative studies on the performance of these different assays are lacking. The aim of this study was to compare three molecular assays for detection and quantification of Leishmania parasites in serial dilutions of parasites and in skin biopsies collected from cutaneous leishmaniasis (CL) patients in Manaus, Brazil. A serial dilution of promastigotes spiked in blood was tested in triplicate in three different runs by quantitative nucleic acid sequence-based amplification (QT-NASBA), quantitative real-time reverse transcriptase PCR (qRT-PCR), and quantitative real-time PCR (qPCR). In addition, the costs, durations, and numbers of handling steps were compared, and 84 skin biopsies from patients with suspected CL were tested. Both QT-NASBA and qRT-PCR had a detection limit of 100 parasites/ml of blood, while qPCR detected 1,000 parasites/ml. QT-NASBA had the lowest range of intra-assay variation (coefficients of variation [CV], 0.5% to 3.3%), while qPCR had the lowest range of interassay variation (CV, 0.4% to 5.3%). Furthermore, qRT-PCR had higher r2 values and amplification efficiencies than qPCR, and qPCR and qRT-PCR had faster procedures than QT-NASBA. All assays performed equally well with patient samples, with significant correlations between parasite counts. Overall, qRT-PCR is preferred over QT-NASBA and qPCR as the most optimal diagnostic assay for quantification of Leishmania parasites, since it was highly sensitive and reproducible and the procedure was relatively fast.

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.

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.].

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.

Application of direct agglutination test (DAT) and fast agglutination screening test (FAST) for sero-diagnosis of visceral leishmaniasis in endemic area of Minas Gerais, Brazil.

BACKGROUND: The direct agglutination test (DAT) has proved to be a very important sero-diagnostic tool combining high levels of intrinsic validity and ease of performance. Otherwise, fast agglutination screening test (FAST) utilises only one serum dilution making the test very suitable for the screening of large populations. RESULTS: We have tested FAST and DAT for the detection anti-Leishmania antibodies in serum samples from patients with American visceral (AVL) and cutaneous leishmaniases (ACL) in Minas Gerais State, Brazil. The DAT on serum and blood samples of confirmed AVL patients found all samples positive at a serum dilution of > or = 1:800. This dilution was subsequently used as cut off value in the study. The blood and serum samples of these confirmed patients could also be clearly read in FAST using a 1:100 dilution with the same high sensitivity. DAT and FAST were not able to detect significant amounts of antibodies in samples from ACL patients and are not suitable for the diagnosis of this manifestation of the disease. CONCLUSION: We suggest that both DAT and FAST are very practical diagnostic tools for the sero-diagnosis of AVL under rural conditions as both serological tests do not require sophisticated equipment, a cold chain and are very simple to perform.

Quantification of Plasmodium falciparum gametocytes in differential stages of development by quantitative nucleic acid sequence-based amplification.

Two quantitative nucleic acid sequence-based amplification assays (QT-NASBA) based on Pfs16 and Pfs25, have been developed to quantify sexual stage commitment and mature gametocytes of Plasmodium falciparum. Pfs16 mRNA is expressed in all sexual forms including sexually committed ring stages while expression of Pfs25 mRNA is restricted to late stage gametocytes. Both assays showed a sensitivity of one sexual stage parasite/microl of blood. Blood samples from experimentally infected non-immune human volunteers were tested for Plasmodium falciparum by standard microscopy, a previously developed asexual 18S rRNA QT-NASBA, Pfs16 and Pfs25 mRNA QT-NASBA. Pfs16 QT-NASBA was positive in 9 out of 10 volunteers within 48 h after first detection of 18S rRNA, mostly before or at the day of positive microscopy. In contrast, the Pfs25 mRNA QT-NASBA was negative during the 28 days of follow-up, but consistently positive in gametocyte samples from naturally infected Kenyan patients. These data suggest that sexual stage commitment can occur early in the blood-stage infection without successful maturation into infectious gametocytes. In conclusion, Pfs16 and Pfs25 QT-NASBA assays in combination with a previously developed asexual stage QT-NASBA allow for the separate quantification of all developmental stages present in the circulation. The application of sexual stage QT-NASBA assays may contribute to a better understanding of the biology and epidemiology of malaria transmission.

Sero-epidemiological study of canine Leishmania spp. infection in the municipality of Alijó (Alto Douro, Portugal).

Visceral leishmaniosis caused by Leishmania infantum is a zoonotic disease in the Mediterranean basin. We report an epidemiological survey carried out in dogs from the municipality of Alijó in the endemic region of Alto Douro (north Portugal). Performance of the direct agglutination test (DAT) was assessed in 205 matching samples of blood collected on filter paper and serum. A high degree of agreement (97.6%; k = 0.83) was found between the results obtained from both types of samples. DAT was then used to test more blood on filter paper (B-FP) samples from other dogs of the same municipality. The detected sero-prevalence was 18.7% (288/1540), with values ranging from 0.0 to 81.1% in each of the 19 parishes of Alijó. Three distinct geographical zones of mean sero-prevalence could be defined: northwestern (2.5%), intermediate (11.4%) and southern (49.9%). No statistically significant difference was observed between male (19.1%) and female (17.8%) sero-prevalences (P = 0.560). Dogs of 9-11 years of age showed the highest sero-prevalence (28.4%), but all the other age-intervals (0-2, 3-5, 6-8 and 12-17 years) presented values (15.0-22.3%) not significantly different from the mean of the whole study population. Risk factors for canine Leishmania infection were age and geographical zone. Only 5.9% of the sero-positive animals had clinical signs of canine leishmaniosis and the overall prevalence of disease was 1.1%. This study validates the use of B-FP samples and confirms DAT as a simple and sensitive serological test to evaluate the level of canine Leishmania infection in areas of high sero-prevalence.

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.

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.

Leishmaniasis direct agglutination test: using pictorials as training materials to reduce inter-reader variability and improve accuracy.

BACKGROUND: The Direct Agglutination Test (DAT) has a high diagnostic accuracy and remains, in some geographical areas, part of the diagnostic algorithm for Visceral Leishmaniasis (VL). However, subjective interpretation of results introduces potential for inter-reader variation. We report an assessment of inter-laboratory agreement and propose a pictorial-based approach to standardize reading of the DAT. METHODOLOGY: In preparation for a comparative evaluation of immunochromatographic diagnostics for VL, a proficiency panel of 15 well-characterized sera, DAT-antigen from a single batch and common protocol was sent to nine laboratories in Latin-America, East-Africa and Asia. Agreement (i.e., equal titre or within 1 titer) with the reading by the reference laboratory was computed. Due to significant inter-laboratory disagreement on-site refresher training was provided to all technicians performing DAT. Photos of training plates were made, and end-titres agreed upon by experienced users of DAT within the Visceral-Leishmaniasis Laboratory-Network (VL-LN). RESULTS: Pre-training, concordance in DAT results with reference laboratories was only 50%, although agreement on negative sera was high (94%). After refresher training concordance increased to 84%; agreement on negative controls increased to 98%. Variance in readings significantly decreased after training from 3.3 titres to an average of 1.0 titre (two-sample Wilcoxon rank-sum (Mann-Whitney) test (z = -3,624 and p = 0.0003)). CONCLUSION: The most probable explanation for disagreement was subjective endpoint reading. Using pictorials as training materials may be a useful tool to reduce disparity in results and promote more standardized reading of DAT, without compromising diagnostic sensitivity.