Evaluation of different DNA extraction methods and loop-mediated isothermal amplification primers for the detection of Mycobacterium ulcerans in clinical specimens.

BACKGROUND: Early diagnosis and treatment of Buruli ulcer is critical in order to avoid the debilitating effects of the disease. In this regard, the development of new diagnostic and point of care tools is encouraged. The loop-mediated isothermal amplification for the detection of Mycobacterium ulcerans represents one of the new tools with a good potential of being developed into a point of care test. There is however the need to standardize the assays, reduce sample preparation times, improve the detection/visualization system and optimize them for high-throughput screening, adaptable to low resourced laboratories. METHODS: In this study, we assessed two DNA extraction protocols (modified Boom and EasyNAT methods), three previously published LAMP primer sets (BURULI, MU 2404 and BU-LAMP), and compared the sensitivity and specificity of LAMP assays on three DNA amplification platforms. RESULTS: Our results show that Buruli ulcer diagnosis using primers targeting IS2404 for the LAMP method is sensitive (73.75-91.49%), depending on the DNA extraction method used. Even though the modified Boom DNA extraction method provided the best results, its instrumentation requirement prevent it from being field applicable. The EasyNAT method on the other hand is simpler and may represent the best method for DNA extraction in less resourced settings. CONCLUSIONS: For further work on the development and use of LAMP tests for Buruli diagnosis, it is recommended that the BURULI sets of primers be used, as these yielded the best results in terms of sensitivity (87.50-91.49%) and specificity (89.23-100%), depending on the DNA extraction methods used.

Trypanosoma cruzi loop-mediated isothermal amplification (Trypanosoma cruzi Loopamp) kit for detection of congenital, acute and Chagas disease reactivation.

A Trypanosoma cruzi Loopamp kit was recently developed as a ready-to-use diagnostic method requiring minimal laboratory facilities. We evaluated its diagnostic accuracy for detection of acute Chagas disease (CD) in different epidemiological and clinical scenarios. In this retrospective study, a convenience series of clinical samples (venous blood treated with EDTA or different stabilizer agents, heel-prick blood in filter paper or cerebrospinal fluid samples (CSF)) from 30 infants born to seropositive mothers (13 with congenital CD and 17 noninfected), four recipients of organs from CD donors, six orally-infected cases after consumption of contaminated guava juice and six CD patients coinfected with HIV at risk of CD reactivation (N = 46 patients, 46 blood samples and 1 CSF sample) were tested by T. cruzi Loopamp kit (Tc LAMP) and standardized quantitative real-time PCR (qPCR). T. cruzi Loopamp accuracy was estimated using the case definition in the different groups as a reference. Cohen's kappa coefficient (κ) was applied to measure the agreement between Tc LAMP (index test) and qPCR (reference test). Sensitivity and specificity of T. cruzi Loopamp kit in blood samples from the pooled clinical groups was 93% (95% CI: 77-99) and 100% (95% CI: 80-100) respectively. The agreement between Tc LAMP and qPCR was almost perfect (κ = 0.92, 95% CI: 0.62-1.00). The T. cruzi Loopamp kit was sensitive and specific for detection of T. cruzi infection. It was carried out from DNA extracted from peripheral blood samples (via frozen EDTA blood, guanidine hydrochloride-EDTA blood, DNAgard blood and dried blood spots), as well as in CSF specimens infected with TcI or TcII/V/VI parasite populations. The T. cruzi Loopamp kit appears potentially useful for rapid detection of T. cruzi infection in congenital, acute and CD reactivation due to HIV infection.

Performance evaluation of a prototype rapid diagnostic test for combined detection of gambiense human African trypanosomiasis and malaria.

BACKGROUND: Malaria is endemic in all regions where gambiense or rhodesiense human African trypanosomiasis (HAT) is reported, and both diseases have similarities in their symptomatology. A combined test could be useful for both diseases and would facilitate integration of the screening for gambiense HAT (gHAT) and malaria diagnosis. This study aimed to evaluate a combined prototype rapid diagnostic test (RDT) for gHAT and malaria. METHODS: Blood samples were collected in the Democratic Republic of the Congo and in Uganda to evaluate the performance of a prototype HAT/Malaria Combined RDT in comparison to an individual malaria RDT based on Plasmodium falciparum (P.f.) Histidine Rich Protein II (HRP-II or HRP2) antigen (SD BIOLINE Malaria Ag P.f. RDT) for malaria detection and an individual gHAT RDT based on recombinant antigens, the SD BIOLINE HAT 2.0 RDT for HAT screening. Due to the current low prevalence of gHAT in endemic regions, the set of blood samples that were collected was used to evaluate the specificity of the RDTs for gHAT, and additional archived plasma samples were used to complete the evaluation of the HAT/Malaria Combined RDT in comparison to the HAT 2.0 RDT. RESULTS: Frozen whole blood samples from a total of 486 malaria cases and 239 non-malaria controls, as well as archived plasma samples from 246 gHAT positive and 246 gHAT negative individuals were tested. For malaria, the sensitivity and specificity of the malaria band in the HAT/Malaria Combined RDT were 96.9% (95% CI: 95.0-98.3) and 97.1% (95% CI: 94.1-98.8) respectively. The sensitivity and specificity of the SD BIOLINE malaria Ag P.f. RDT were 97.3% (95% CI: 95.5-98.6) and 97.1% (95% CI: 94.1-98.8) respectively. For gHAT, using archived plasma samples, the sensitivity and specificity were respectively 89% (95% CI: 84.4-92.6) and 93.5% (95% CI: 89.7-96.2) with the HAT/Malaria Combined RDT, and 88.2% (95% CI: 83.5-92) and 94.7% (95% CI: 91.1-97.2) with the HAT 2.0 RDT. Using the whole blood samples that were collected during the study, the specificity of the HAT/Malaria Combined RDT for gHAT was 95.8% (95% CI: 94.3-97.0). CONCLUSION: The HAT/Malaria Combined prototype RDT was as accurate as the individual malaria or gHAT RDTs. The HAT/Malaria Combined prototype RDT is therefore suitable for both malaria diagnosis and gHAT screening. However, there is a need to assess its accuracy using fresh samples in prospective clinical trials.

Assessing the impact of aggregating disease stage data in model predictions of human African trypanosomiasis transmission and control activities in Bandundu province (DRC).

Since the turn of the century, the global community has made great progress towards the elimination of gambiense human African trypanosomiasis (HAT). Elimination programs, primarily relying on screening and treatment campaigns, have also created a rich database of HAT epidemiology. Mathematical models calibrated with these data can help to fill remaining gaps in our understanding of HAT transmission dynamics, including key operational research questions such as whether integrating vector control with current intervention strategies is needed to achieve HAT elimination. Here we explore, via an ensemble of models and simulation studies, how including or not disease stage data, or using more updated data sets affect model predictions of future control strategies.

Neopterin and CXCL-13 in Diagnosis and Follow-Up of Trypanosoma brucei gambiense Sleeping Sickness: Lessons from the Field in Angola.

Human African Trypanosomiasis may become manageable in the next decade with fexinidazole. However, currently stage diagnosis remains difficult to implement in the field and requires a lumbar puncture. Our study of an Angolan cohort of T. b. gambiense-infected patients used other staging criteria than those recommended by the WHO. We compared WHO criteria (cell count and parasite identification in the CSF) with two biomarkers (neopterin and CXCL-13) which have proven potential to diagnose disease stage or relapse. Biological, clinical, and neurological data were analysed from a cohort of 83 patients. A neopterin concentration below 15.5 nmol/L in the CSF denoted patients with stage 1 disease, and a concentration above 60.31 nmol/L characterized patients with advanced stage 2 (trypanosomes in CSF and/or cytorachia higher than 20 cells) disease. CXCL-13 levels below 91.208 pg/mL denoted patients with stage 1 disease, and levels of CXCL-13 above 395.45 pg/mL denoted patients with advanced stage 2 disease. Values between these cut-offs may represent patients with intermediate stage disease. Our work supports the existence of an intermediate stage in HAT, and CXCL-13 and neopterin levels may help to characterize it.

Target Product Profile for a point-of-care diagnostic test for dermal leishmaniases.

OBJECTIVES: Localized cutaneous leishmaniasis and its evolving forms diffuse cutaneous leishmaniasis, mucosal leishmaniasis and cutaneous leishmaniasis recidivans, together with the visceral leishmaniasis sequelae post-kala azar dermal leishmaniasis account for about one million dermal leishmaniases cases per year worldwide. Although not lethal, the dermal leishmaniases cause chronic and disfiguring skin lesions, which are an important cause of morbidity and stigma.Microscopy remains the reference test for diagnosis of dermal leishmaniasis; however, it has low and variable sensitivity and requires well trained personnel. The technical complexity and cost of the more sensitive molecular techniques (e.g. PCR) limits their application in routine diagnosis in endemic areas. Point-of-care (POC) tests for early diagnosis are much needed in order to benefit both patients and communities, by reducing the risk of both sequelae and Leishmania transmission. To this end we developed a Target Product Profile (TPP) for a POC test for dermal leishmaniases. METHODS: The TPP was defined through several rounds of discussions and by consensus with stakeholders and experts in dermal leishmaniases from different type of organizations and endemic regions. RESULTS AND CONCLUSIONS: A rapid, simple and robust test that can be implemented in resource-limited settings, enabling decentralized diagnosis and treatment of dermal leishmaniasis should be developed. Ideally it should enable the diagnosis of all forms of dermal leishmaniasis, but the minimally accepted target would be localized cutaneous leishmaniasis. A minimum sensitivity of 95% and specificity of 90% would be required. The consensus was that the POC test should target Leishmania antigens.

Knowledge, attitudes and practices about human African trypanosomiasis and their implications in designing intervention strategies for Yei county, South Sudan.

BACKGROUND: A clear understanding of the knowledge, attitudes and practices (KAP) of a particular community is necessary in order to improve control of human African trypanosomiasis (HAT).New screening and diagnostic tools and strategies were introduced into South Sudan, as part of integrated delivery of primary healthcare. Knowledge and awareness on HAT, its new/improved screening and diagnostic tools, the places and processes of getting a confirmatory diagnosis and treatment are crucial to the success of this strategy. METHODOLOGY: A KAP survey was carried out in Yei County, South Sudan, to identify gaps in community KAP and determine the preferred channels and sources of information on the disease. The cross-sectional KAP survey utilized questionnaires, complemented with key informant interviews and a focus group discussion to elicit communal as well as individual KAP on HAT. FINDINGS: Most (90%) of the respondents had general knowledge on HAT. Lower levels of education, gender and geographic locations without a history of HAT interventions were associated with incorrect knowledge and/or negative perceptions about the treatability of HAT. Symptoms appearing in the late stage were best known. A majority (97.2%) would seek treatment for HAT only in a health centre. However, qualitative data indicates that existing myths circulating in the popular imagination could influence people's practices. Seventy-one percent of the respondents said they would offer social support to patients with HAT but qualitative data highlights that stigma still exists. Misconceptions and stigma can negatively influence the health seeking behaviour of HAT cases. In relation to communication, the top preferred and effective source of communication was radio (24%). CONCLUSION: Gaps in relation to KAP on HAT still exist in the community. Perceptions on HAT, specifically myths and stigma, were key gaps that need to be bridged through effective education and communication strategies for HAT control alongside other interventions.

Cost-effectiveness of using a rapid diagnostic test to screen for human African trypanosomiasis in the Democratic Republic of the Congo.

New rapid diagnostic tests (RDTs) for screening human African trypanosomiasis (HAT) have been introduced as alternatives to the card agglutination test for trypanosomiasis (CATT). One brand of RDT, the SD BIOLINE HAT RDT has been shown to have lower specificity but higher sensitivity than CATT, so to make a rational choice between screening strategies, a cost-effectiveness analysis is a key element. In this paper we estimate the relative cost-effectiveness of CATT and the RDT when implemented in the Democratic Republic of the Congo (DRC). Data on the epidemiological parameters and costs were collected as part of a larger study. These data were used to model three different diagnostic algorithms in mobile teams and fixed health facilities, and the relative cost-effectiveness was measured as the average cost per case diagnosed. In both fixed facilities and mobile teams, screening of participants using the SD BIOLINE HAT RDT followed by parasitological confirmation had a lower cost-effectiveness ratio than in algorithms using CATT. Algorithms using the RDT were cheaper by 112.54 (33.2%) and 88.54 (32.92%) US dollars per case diagnosed in mobile teams and fixed health facilities respectively, when compared with algorithms using CATT. Sensitivity analysis demonstrated that these conclusions were robust to a number of assumptions, and that the results can be scaled to smaller or larger facilities, and a range of prevalences. The RDT was the most cost-effective screening test in all realistic scenarios and detected more cases than CATT. Thus, on this basis, the SD BIOLINE HAT RDT could be considered as the most cost-effective option for use in routine screening for HAT in the DRC.

Assessing Strategies Against Gambiense Sleeping Sickness Through Mathematical Modeling.

Background: Control of gambiense sleeping sickness relies predominantly on passive and active screening of people, followed by treatment. Methods: Mathematical modeling explores the potential of 3 complementary interventions in high- and low-transmission settings. Results: Intervention strategies that included vector control are predicted to halt transmission most quickly. Targeted active screening, with better and more focused coverage, and enhanced passive surveillance, with improved access to diagnosis and treatment, are both estimated to avert many new infections but, when used alone, are unlikely to halt transmission before 2030 in high-risk settings. Conclusions: There was general model consensus in the ranking of the 3 complementary interventions studied, although with discrepancies between the quantitative predictions due to differing epidemiological assumptions within the models. While these predictions provide generic insights into improving control, the most effective strategy in any situation depends on the specific epidemiology in the region and the associated costs.

Sensitive and less invasive confirmatory diagnosis of visceral leishmaniasis in Sudan using loop-mediated isothermal amplification (LAMP).

BACKGROUND: Confirmatory diagnosis of visceral leishmaniasis (VL), as well as diagnosis of relapses and test of cure, usually requires examination by microscopy of samples collected by invasive means, such as splenic, bone marrow or lymph node aspirates. This causes discomfort to patients, with risks of bleeding and iatrogenic infections, and requires technical expertise. Molecular tests have great potential for diagnosis of VL using peripheral blood, but require well-equipped facilities and trained personnel. More user-friendly, and field-amenable options are therefore needed. One method that could meet these requirements is loop-mediated isothermal amplification (LAMP) using the Loopamp Leishmania Detection Kit, which comes as dried down reagents that can be stored at room temperature, and allows simple visualization of results. METHODOLOGY/PRINCIPAL FINDINGS: The Loopamp Leishmania Detection Kit (Eiken Chemical Co., Japan), was evaluated in the diagnosis of VL in Sudan. A total of 198 VL suspects were tested by microscopy of lymph node aspirates (the reference test), direct agglutination test-DAT (in house production) and rK28 antigen-based rapid diagnostic test (OnSite Leishmania rK39-Plus, CTK Biotech, USA). LAMP was performed on peripheral blood (whole blood and buffy coat) previously processed by: i) a direct boil and spin method, and ii) the QIAamp DNA Mini Kit (QIAgen). Ninety seven of the VL suspects were confirmed as cases by microscopy of lymph node aspirates. The sensitivity and specificity for each of the tests were: rK28 RDT 98.81% and 100%; DAT 88.10% and 78.22%; LAMP-boil and spin 97.65% and 99.01%; LAMP-QIAgen 100% and 99.01%. CONCLUSIONS/SIGNIFICANCE: Due to its simplicity and high sensitivity, rK28 RDT can be used first in the diagnostic algorithm for primary VL diagnosis, the excellent performance of LAMP using peripheral blood indicates that it can be also included in the algorithm for diagnosis of VL as a simple test when parasitological confirmatory diagnosis is required in settings that are lower than the reference laboratory, avoiding the need for invasive lymph node aspiration.

Performance of the SD BIOLINE® HAT rapid test in various diagnostic algorithms for gambiense human African trypanosomiasis in the Democratic Republic of the Congo.

We carried out a study to compare the performance, in terms of sensitivity and specificity, of the new SD BIOLINE® HAT rapid diagnostic test (RDT) with the card agglutination test for trypanosomiasis (CATT) for diagnosis of human African trypanosomiasis (HAT) in the Democratic Republic of the Congo (DRC). Participants were enrolled actively by four mobile teams, and passively at four health facilities in three provinces. Consenting participants were tested concurrently with the RDT and CATT on whole blood. Those found positive by either test were tested with CATT on serial dilutions of plasma, and with a parasitological composite reference standard (CRS). Cases were only the individuals found positive by the CRS, while controls were negative by both CATT and RDT, as well as those that were positive by CATT or RDT, but were negative by the CRS, and had no history of HAT. Over five months, 131 cases and 13,527 controls were enrolled. The sensitivity of the RDT was 92.0% (95% confidence interval (CI) = 86.1-95.5), which was significantly higher than CATT (sensitivity 69.1%; 95% CI = 60.7-76.4). The sensitivity of CATT on plasma at a dilution of 1:8 was 59.0% (95% CI = 50.2-67.2). The specificity of the RDT was 97.1% (95% CIs = 96.8-97.4) while that of CATT was 98.0% (95% CIs = 97.8, 98.2) and specificities of algorithms involving CATT at 1:8 dilution were 99.6% (95% CI = 99.5-99.7). Reproducibility of results was excellent. We concluded that an algorithm in which the SD BIOLINE® HAT RDT is used for screening is optimal for case detection in both passive and active screening settings. However, the lower specificity of the RDT compared to that of CATT would result in a larger number of false positive individuals undergoing confirmatory testing.

Analytical sensitivity and specificity of a loop-mediated isothermal amplification (LAMP) kit prototype for detection of Trypanosoma cruzi DNA in human blood samples.

This study aimed to assess analytical parameters of a prototype LAMP kit that was designed for detection of Trypanosoma cruzi DNA in human blood. The prototype is based on the amplification of the highly repetitive satellite sequence of T.cruzi in microtubes containing dried reagents on the inside of the caps. The reaction is carried out at 65°C during 40 minutes. Calcein allows direct detection of amplified products with the naked eye. Inclusivity and selectivity were tested in purified DNA from Trypanosoma cruzi stocks belonging to the six discrete typing units (DTUs), in DNA from other protozoan parasites and in human DNA. Analytical sensitivity was estimated in serial dilutions of DNA samples from Sylvio X10 (Tc I) and CL Brener (Tc VI) stocks, as well as from EDTA-treated or heparinized blood samples spiked with known amounts of cultured epimastigotes (CL Brener). LAMP sensitivity was compared after DNA extraction using commercial fiberglass columns or after "Boil & Spin" rapid preparation. Moreover, the same DNA and EDTA-blood spiked samples were subjected to standardized qPCR based on the satellite DNA sequence for comparative purposes. A panel of peripheral blood specimens belonging to Chagas disease patients, including acute, congenital, chronic and reactivated cases (N = 23), as well as seronegative controls (N = 10) were evaluated by LAMP in comparison to qPCR. LAMP was able to amplify DNAs from T. cruzi stocks representative of the six DTUs, whereas it did not amplify DNAs from Leishmania sp, T. brucei sp, T. rangeli KPN+ and KPN-, P. falciparum and non-infected human DNA. Analytical sensitivity was 1x10-2 fg/µL of both CL Brener and Sylvio X10 DNAs, whereas qPCR detected up to 1x 10-1 fg/µL of CL Brener DNA and 1 fg/µl of Sylvio X10 DNA. LAMP detected 1x10-2 parasite equivalents/mL in spiked EDTA blood and 1x10-1 par.eq/mL in spiked heparinized blood using fiberglass columns for DNA extraction, whereas qPCR detected 1x10-2 par.eq./mL in EDTA blood. Boil & Spin extraction allowed detection of 1x10-2 par.eq /mL in spiked EDTA blood and 1 par.eq/ml in heparinized blood. LAMP was able to detect T.cruzi infection in peripheral blood samples collected from well-characterised seropositive patients, including acute, congenital, chronic and reactivated Chagas disease. To our knowledge, this is the first report of a prototype LAMP kit with appropriate analytical sensitivity for diagnosis of Chagas disease patients, and potentially useful for monitoring treatment response.

Metabolomics Identifies Multiple Candidate Biomarkers to Diagnose and Stage Human African Trypanosomiasis.

Treatment for human African trypanosomiasis is dependent on the species of trypanosome causing the disease and the stage of the disease (stage 1 defined by parasites being present in blood and lymphatics whilst for stage 2, parasites are found beyond the blood-brain barrier in the cerebrospinal fluid (CSF)). Currently, staging relies upon detecting the very low number of parasites or elevated white blood cell numbers in CSF. Improved staging is desirable, as is the elimination of the need for lumbar puncture. Here we use metabolomics to probe samples of CSF, plasma and urine from 40 Angolan patients infected with Trypanosoma brucei gambiense, at different disease stages. Urine samples provided no robust markers indicative of infection or stage of infection due to inherent variability in urine concentrations. Biomarkers in CSF were able to distinguish patients at stage 1 or advanced stage 2 with absolute specificity. Eleven metabolites clearly distinguished the stage in most patients and two of these (neopterin and 5-hydroxytryptophan) showed 100% specificity and sensitivity between our stage 1 and advanced stage 2 samples. Neopterin is an inflammatory biomarker previously shown in CSF of stage 2 but not stage 1 patients. 5-hydroxytryptophan is an important metabolite in the serotonin synthetic pathway, the key pathway in determining somnolence, thus offering a possible link to the eponymous symptoms of "sleeping sickness". Plasma also yielded several biomarkers clearly indicative of the presence (87% sensitivity and 95% specificity) and stage of disease (92% sensitivity and 81% specificity). A logistic regression model including these metabolites showed clear separation of patients being either at stage 1 or advanced stage 2 or indeed diseased (both stages) versus control.

Sensitivity and Specificity of a Prototype Rapid Diagnostic Test for the Detection of Trypanosoma brucei gambiense Infection: A Multi-centric Prospective Study.

BACKGROUND: A major challenge in the control of human African trypanosomiasis (HAT) is lack of reliable diagnostic tests that are rapid and easy to use in remote areas where the disease occurs. In Trypanosoma brucei gambiense HAT, the Card Agglutination Test for Trypanosomiasis (CATT) has been the reference screening test since 1978, usually on whole blood, but also in a 1/8 dilution (CATT 1/8) to enhance specificity. However, the CATT is not available in a single format, requires a cold chain for storage, and uses equipment that requires electricity. A solution to these challenges has been provided by rapid diagnostic tests (RDT), which have recently become available. A prototype immunochromatographic test, the SD BIOLINE HAT, based on two native trypanosomal antigens (VSG LiTat 1.3 and VSG LiTat 1.5) has been developed. We carried out a non-inferiority study comparing this prototype to the CATT 1/8 in field settings. METHODOLOGY/PRINCIPAL FINDINGS: The prototype SD BIOLINE HAT, the CATT Whole Blood and CATT 1/8 were systematically applied on fresh blood samples obtained from 14,818 subjects, who were prospectively enrolled through active and passive screening in clinical studies in three endemic countries of central Africa: Angola, the Democratic Republic of the Congo and the Central African Republic. One hundred and forty nine HAT cases were confirmed by parasitology. The sensitivity and specificity of the prototype SD BIOLINE HAT was 89.26% (95% confidence interval (CI) = 83.27-93.28) and 94.58% (95% CI = 94.20-94.94) respectively. The sensitivity and specificity of the CATT on whole blood were 93.96% (95% CI = 88.92-96.79) and 95.91% (95% CI = 95.58-96.22), and of the CATT 1/8 were 89.26% (95% CI = 83.27-93.28) and 98.88% (95% CI = 98.70-99.04) respectively. CONCLUSION/SIGNIFICANCE: After further optimization, the prototype SD BIOLINE HAT could become an alternative to current screening methods in primary healthcare settings in remote, resource-limited regions where HAT typically occurs.

Illuminating the Prevalence of Trypanosoma brucei s.l. in Glossina Using LAMP as a Tool for Xenomonitoring.

BACKGROUND: As the reality of eliminating human African trypanosomiasis (HAT) by 2020 draws closer, the need to detect and identify the remaining areas of transmission increases. Here, we have explored the feasibility of using commercially available LAMP kits, designed to detect the Trypanozoon group of trypanosomes, as a xenomonitoring tool to screen tsetse flies for trypanosomes to be used in future epidemiological surveys. METHODS AND FINDINGS: The DNA extraction method was simplified and worked with the LAMP kits to detect a single positive fly when pooled with 19 negative flies, and the absolute lowest limit of detection that the kits were able to work at was the equivalent of 0.1 trypanosome per ml. The DNA from Trypanosoma brucei brucei could be detected six days after the fly had taken a blood meal containing dead trypanosomes, and when confronted with a range of non-target species, from both laboratory-reared flies and wild-caught flies, the kits showed no evidence of cross-reacting. CONCLUSION: We have shown that it is possible to use a simplified DNA extraction method in conjunction with the pooling of tsetse flies to decrease the time it would take to screen large numbers of flies for the presence of Trypanozoon trypanosomes. The use of commercially-available LAMP kits provides a reliable and highly sensitive tool for xenomonitoring and identifying potential sleeping sickness transmission sites.

Evaluation of the diagnostic accuracy of prototype rapid tests for human African trypanosomiasis.

BACKGROUND: Diagnosis of human African trypanosomiasis (HAT) remains a challenge both for active screening, which is critical in control of the disease, and in the point-of-care scenario where early and accurate diagnosis is essential. Recently, the first field deployment of a lateral flow rapid diagnostic test (RDT) for HAT, "SD BIOLINE HAT" has taken place. In this study, we evaluated the performance of "SD BIOLINE HAT" and two new prototype RDTs. METHODOLOGY/PRINCIPAL FINDINGS: The performance of "SD BIOLINE HAT" and 2 prototype RDTs was tested using archived plasma from 250 Trypanosoma brucei gambiense patients, and 250 endemic controls. As well as comparison of the sensitivity and specificity of each device, the performance of individual antigens was assessed and the hypothetical performance of novel antigen combinations extrapolated. Neither of the prototype devices were inferior in sensitivity or specificity to "SD BIOLINE HAT" (sensitivity 0.82±0.01, specificity 0.97±0.01, 95% CI) at the 5% margins, while one of the devices (BBI) had significantly superior sensitivity (0.88±0.03). Analysis of the performance of individual antigens was used to model new antigen combinations to be explored in development of the next generation of HAT RDTs. The modelling showed that an RDT using two recombinant antigens (rLiTat1.5 and rISG65) would give a performance similar to the best devices in this study, and would also offer the most robust performance under deteriorating field conditions. CONCLUSIONS/SIGNIFICANCE: Both "SD BIOLINE HAT" and the prototype devices performed comparably well to one another and also to the published performance range of the card agglutination test for trypanosomiasis in sensitivity and specificity. The performance of individual antigens enabled us to predict that an all-recombinant antigen RDT can be developed with an accuracy equivalent to " SD BIOLINE HAT." Such an RDT would have advantages in simplified manufacture, lower unit cost and assured reproducibility.

Matrix metalloproteinase-9 and intercellular adhesion molecule 1 are powerful staging markers for human African trypanosomiasis.

OBJECTIVES: A critical step before treatment of human African trypanosomiasis (HAT) is the correct staging of the disease. As late stage is established when trypanosomes cross the blood-brain barrier and invade the central nervous system, we hypothesized that matrix metalloproteinases and cell adhesion molecules could indicate, alone or in combination, the disease progression from the first to the second stage of HAT. METHODS: We measured the levels of MMP-2, MMP-9, ICAM-1, VCAM-1 and E-selectin in the cerebrospinal fluid (CSF) of 63 Trypanosoma brucei gambiense-infected patients (15 stage 1 and 48 stage 2). Staging was based on counting of white blood cells (WBC) and/or parasite detection in CSF. Concentrations were obtained either by ELISA or multiplex bead suspension assays, and results were compared with three known HAT staging markers (CXCL10, CXCL8 and H-FABP). RESULTS: ICAM-1 and MMP-9 accurately discriminated between stage 1 and stage 2 patients with HAT with 95% sensitivity (SE) for 100% specificity (SP), which was better than CXCL10 (93% SE for 100% SP), one of the most promising known markers. Combination of ICAM-1 and MMP-9 with H-FABP provided a panel that resulted in 100% of SE and SP for staging HAT. CONCLUSIONS: ICAM-1 and MMP-9, alone or in combination, appeared as powerful CSF staging markers of HAT. Final validation of all newly discovered staging markers on a large multi-centric cohort including both forms of the disease as well as patients with others infections should be performed.

Isolation and propagation of Trypanosoma brucei gambiense from sleeping sickness patients in south Sudan.

This study aimed at isolating Trypanosoma brucei gambiense from human African trypanosomiasis (HAT) patients from south Sudan. Fifty HAT patients identified during active screening surveys were recruited, most of whom (49/50) were in second-stage disease. Blood and cerebrospinal fluid samples collected from the patients were cryopreserved using Triladyl as the cryomedium. The samples were stored at -150 degrees C in liquid nitrogen vapour in a dry shipper. Eighteen patient stabilates could be propagated in immunosuppressed Mastomys natalensis and/or SCID mice. Parasitaemia was highest in SCID mice. Further subpassages in M. natalensis increased the virulence of the trypanosomes and all 18 isolates recovered from M. natalensis or SCID mice became infective to other immunosuppressed mouse breeds. A comparison of immunosuppressed M. natalensis and Swiss White, C57/BL and BALB/c mice demonstrated that all rodent breeds were susceptible after the second subpassage and developed a parasitaemia >10(6)/ml by Day 5 post infection. The highest parasitaemias were achieved in C57/BL and BALB/c mice. These results indicate that propagation of T. b. gambiense isolates after initial isolation in immunosuppressed M. natalensis or SCID mice can be done in a range of immunosuppressed rodents.

Immunization of rabbits with Glossina pallidipes tsetse fly midgut proteins: effects on the fly and trypanosome transmission.

Proteins isolated from the midgut of Glossina pallidipes were used to immunize rabbits and their efficacy as vaccine candidate(s) against the fly, and their potential to block transmission of Trypanosoma brucei rhodesiense assessed. Two fractions, detergent (DET) and aqueous (AQ) fractions were separated using a non-ionic detergent (Triton X-114) and a series of bioassay experiments carried out using serum obtained from rabbits immunized with either of the two fractions. The mortality rates of tsetse flies fed on serum from rabbits immunized with DET and AQ was 56 and 35%, respectively, as compared to 20% mortality in controls. The DET antigen(s) caused considerably higher mortality (chi(2)=1.194, P<0.05) than that on controls. These findings suggest that midgut proteins contain antigens that are lethal to tsetse flies, and are potential candidates for the development of anti-tsetse vaccine. When flies fed on serum derived from DET immunized rabbits were fed on T. b. rhodesiense infected blood, only 20% of them picked the infection. Very few flies (20%) fed on serum derived from DET immunized rabbits had infection of T. b. rhodesiense. In the control flies 45% of them had infection in the midgut with a higher and actively motile parasite load. Assessment of fecundity indicated significantly higher (chi(2)=2.117, P<0.05) larviposition for the control flies when compared to the AQ group of flies (chi(2)=1.054, P<0.05). Significant differences in abortions and pupal weights were also observed. These results suggest that midgut proteins contain antigens with potential for use in development of vaccine to block transmission of trypanosomes through tsetse.