Joseph Mathu Ndung’u

Cerebrospinal fluid neopterin as marker of the meningo-encephalitic stage of Trypanosoma brucei gambiense sleeping sickness.

Background Sleeping sickness, or human African trypanosomiasis (HAT), is a protozoan disease that affects rural communities in sub-Saharan Africa. Determination of the disease stage, essential for correct treatment, represents a key issue in the management of patients. In the present study we evaluated the potential of CXCL10, CXCL13, ICAM-1, VCAM-1, MMP-9, B2MG, neopterin and IgM to complement current methods for staging Trypanosoma brucei gambiense patients. Methods and Findings Five hundred and twelve T. b. gambiense HAT patients originated from Angola, Chad and the Democratic Republic of the Congo (D.R.C.). Their classification as stage 2 (S2) was based on the number of white blood cells (WBC) (>5/µL) or presence of parasites in the cerebrospinal fluid (CSF). The CSF concentration of the eight markers was first measured on a training cohort encompassing 100 patients (44 S1 and 56 S2). IgM and neopterin were the best in discriminating between the two stages of disease with 86.4% and 84.1% specificity respectively, at 100% sensitivity. When a validation cohort (412 patients) was tested, neopterin (14.3 nmol/L) correctly classified 88% of S1 and S2 patients, confirming its high staging power. On this second cohort, neopterin also predicted both the presence of parasites, and of neurological signs, with the same ability as IgM and WBC, the current reference for staging. Conclusions This study has demonstrated that neopterin is an excellent biomarker for staging T. b. gambiense HAT patients. A rapid diagnostic test for detecting this metabolite in CSF could help in more accurate stage determination.

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.

Neopterin is a cerebrospinal fluid marker for treatment outcome evaluation in patients affected by Trypanosoma brucei gambiense sleeping sickness.

Background Post-therapeutic follow-up is essential to confirm cure and to detect early treatment failures in patients affected by sleeping sickness (HAT). Current methods, based on finding of parasites in blood and cerebrospinal fluid (CSF) and counting of white blood cells (WBC) in CSF, are imperfect. New markers for treatment outcome evaluation are needed. We hypothesized that alternative CSF markers, able to diagnose the meningo-encephalitic stage of the disease, could also be useful for the evaluation of treatment outcome. Methodology/Principal findings Cerebrospinal fluid from patients affected by Trypanosoma brucei gambiense HAT and followed for two years after treatment was investigated. The population comprised stage 2 (S2) patients either cured or experiencing treatment failure during the follow-up. IgM, neopterin, B2MG, MMP-9, ICAM-1, VCAM-1, CXCL10 and CXCL13 were first screened on a small number of HAT patients (n = 97). Neopterin and CXCL13 showed the highest accuracy in discriminating between S2 cured and S2 relapsed patients (AUC 99% and 94%, respectively). When verified on a larger cohort (n = 242), neopterin resulted to be the most efficient predictor of outcome. High levels of this molecule before treatment were already associated with an increased risk of treatment failure. At six months after treatment, neopterin discriminated between cured and relapsed S2 patients with 87% specificity and 92% sensitivity, showing a higher accuracy than white blood cell numbers. Conclusions/Significance In the present study, neopterin was highlighted as a useful marker for the evaluation of the post-therapeutic outcome in patients suffering from sleeping sickness. Detectable levels of this marker in the CSF have the potential to shorten the follow-up for HAT patients to six months after the end of the treatment.

Improved detection of Trypanosoma brucei by lysis of red blood cells, concentration and LED fluorescence microscopy.

Confirmatory diagnosis of African trypanosomiasis relies on demonstration of parasites in body fluids by bright field microscopy. The parasitaemia in infected patients and animals is usually low, and concentration methods are used to try and increase the chances of seeing parasites. Recently, fluorescence microscopes using light-emitting diodes (LED) have been developed. Since they emit strong light, their use does not require a dark room, making field application a possibility. We have combined LED fluorescence microscopy with lysis of red blood cells (RBC) to improve the sensitivity and speed of detecting trypanosomes. In studies conducted at four centers in Uganda and the Democratic Republic of the Congo, parasitaemic blood was serially diluted and the RBCs lysed using commercial buffer. Samples were then concentrated by centrifugation, and different volumes of the sediment used to make thin and thick smears. Next, these were stained with acridine orange or Giemsa, and examined using an LED microscope under fluorescence or bright light, respectively. Detection of parasites was significantly improved by RBC lysis and concentration, regardless of the staining and microscopy method used. Further improvements were made when smears were prepared using larger volumes of sediment. The best results were obtained with thin smears prepared using 20 μl of sediment and stained with acridine orange. The time taken to see the first parasite was dramatically reduced when smears were examined by LED fluorescence microscopy, compared to bright light. LED fluorescence microscopy was found to be easier and requiring less visual effort than bright field microscopy. These studies demonstrate the potential for incremental improvement in detection of Trypanosoma brucei by combining LED fluorescence microscopy with RBC lysis and concentration. The lysis and concentration method may also be useful in sample preparation for other diagnostic tests for trypanosomiasis.

Enabling the Development and Deployment of Next Generation Point-of-Care Diagnostics

A major goal of the 1st International Point-of-Care Diagnostic Workshop in Nairobi, Kenya was to provide a forum for open dialog concerning current challenges in, and potential solutions for, the development of the next generation of POC diagnostics. The focus was not solely on descriptions of new technologies in development but also included demonstrations of their use in a field setting. The resulting conversations identified a number of obstacles to the successful translation of prototypes into field-deployable tools. These obstacles superseded those typically encountered in research; changes must be implemented at the institutional and governmental level to enable equitable collaborations between Western and African partners, and proper funding mechanisms must be established to support these collaborations. Additionally, this workshop showcased emerging technologies for POC tests and fostered new partnerships between technology developers and African research laboratories. Equitable partnerships are critical for the successful implementation of new POC technology. The attendees agreed that the most effective methods to effect change require improved communication of needs, ideas and abilities, and a conduit for the sharing of experiences and information. We plan to implement many of the changes that are suggested here in our own research programs and to use future conferences and workshops to guide the development of both technologies and partnerships. Our successes and failures will serve as models for those scientists striving to develop technological and biomedical solutions to similar problems in global health.

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.

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.

Enhanced passive screening and diagnosis for gambiense human African trypanosomiasis in north-western Uganda – Moving towards elimination

Introduction The incidence of gambiense human African trypanosomiasis (gHAT) in Uganda has been declining, from 198 cases in 2008, to only 20 in 2012. Interruption of transmission of the disease by early diagnosis and treatment is core to the control and eventual elimination of gHAT. Until recently, the format of available screening tests had restricted screening and diagnosis to central health facilities (passive screening). We describe a novel strategy that is contributing to elimination of gHAT in Uganda through expansion of passive screening to the entire population at risk. Methodology / Principal findings In this strategy, patients who are clinically suspected of having gHAT at primary health facilities are screened using a rapid diagnostic test (RDT), followed by parasitological confirmation at strategically located microscopy centres. For patients who are positive with the RDT and negative by microscopy, blood samples undergo further testing using loop-mediated isothermal amplification (LAMP), a molecular test that detects parasite DNA. LAMP positive patients are considered strong suspects, and are re-evaluated by microscopy. Location and upgrading of facilities to perform microscopy and LAMP was informed by results of georeferencing and characterization of all public healthcare facilities in the 7 gHAT endemic districts in Uganda. Three facilities were upgraded to perform RDTs, microscopy and LAMP, 9 to perform RDTs and microscopy, and 200 to screen patients with RDTs. This reduced the distance that a sick person must travel to be screened for gHAT to a median distance of 2.5km compared to 23km previously. In this strategy, 9 gHAT cases were diagnosed in 2014, and 4 in 2015. Conclusions This enhanced passive screening strategy for gHAT has enabled full coverage of the population at risk, and is being replicated in other gHAT endemic countries. The improvement in case detection is making elimination of the disease in Uganda an imminent possibility.

Prospective evaluation of a rapid diagnostic test for Trypanosoma brucei gambiense infection developed using recombinant antigens

Background Diagnosis and treatment are central elements of strategies to control Trypanosoma brucei gambiense human African trypanosomiasis (HAT). Serological screening is a key entry point in diagnostic algorithms. The Card Agglutination Test for Trypanosomiasis (CATT) has been the most widely used screening test for decades, despite a number of practical limitations that were partially addressed by the introduction of rapid diagnostic tests (RDTs). However, current RDTs are manufactured using native antigens, which are challenging to produce. Methodology/Principal findings The objective of this study was to evaluate the accuracy of a new RDT developed using recombinant antigens (SD BIOLINE HAT 2.0), in comparison with an RDT produced using native antigens (SD BIOLINE HAT) and CATT. A total of 57,632 individuals were screened in the Democratic Republic of the Congo, either passively at 10 health centres, or actively by 5 mobile teams, and 260 HAT cases were confirmed by parasitology. The highest sensitivity was achieved with the SD BIOLINE HAT 2.0 (71.2%), followed by CATT (62.5%) and the SD BIOLINE HAT (59.0%). The most specific test was CATT (99.2%), while the specificity of the SD BIOLINE HAT and SD BIOLINE HAT 2.0 were 98.9% and 98.1%, respectively. Sensitivity of the tests was lower than previously reported, as they identified cases from partially overlapping sub-populations. All three tests were significantly more sensitive in passive than in active screening. Combining two or three tests resulted in a markedly increased sensitivity: When the SD BIOLINE HAT was combined with the SD BIOLINE HAT 2.0, sensitivity reached 98.4% in passive and 83.0% in active screening. Conclusions/Significance The recombinant antigen-based RDT was more sensitive than, and as specific as, the SD BIOLINE HAT. It was as sensitive as, but slightly less specific than CATT. While the practicality and cost-effectiveness of algorithms including several screening tests would need to be investigated, using two or more tests appears to enhance sensitivity of diagnostic algorithms, although some decrease in specificity is observed as well.