Card Agglutination Test For Trypanosomiasis Research Articles

Investigation of Trypanosoma evansi in Sumatran Elephant (Elephas maximus sumatranus) in Indonesia Using Various Methods

Trypanosoma evansi is a widespread hemoprotozoa that causes trypanosomiasis in both livestock and wildlife. One of the susceptible animals is the Sumatran elephant, which is included in the endangered species category. The infection of this parasite often develops into chronic and sub-clinical forms in elephants, therefore it may become unnoticed and hard to diagnose. This study aimed to analyze and evaluate the infection of T. evansi in semi-captive Sumatran elephants in Way Kambas National Park, Indonesia, using various diagnostic tests. The prevalence of T. evansi in a total of 53 Sumatran elephants was estimated using a card agglutination test for trypanosomiasis (CATT) in 2016. A longitudinal study was later conducted in 2019 using Giemsa stained blood smear (GSBS) and polymerase chain reaction (PCR). The results showed that 26.4% of samples (14/53) were positively detected by both CATT in 2016 and PCR in 2019, while GSBS was unable to detect the parasites in all samples. Furthermore, four individuals were confirmed to have persistent infections. This study concluded that the ability of CATT and PCR were more convincing over GSBS for the diagnosis of sub-clinical trypanosomiasis in Sumatran elephants. However, it is recommended to use a combination of CATT as a screening tool and PCR as a confirmatory test for reliable results.

Parasitological and serological detection of Trypanosoma evansi on Bali cattle at the Pesanggaran slaughterhouse, Denpasar, Indonesia using hematological profile

Abstract. Apsari IAP, Suratma NA, Swacita IBN, Soma IG, Sari TK, Putra IPC, Sudipa PH. 2024. Parasitological and serological detection of Trypanosoma evansi on Bali cattle at the Pesanggaran slaughterhouse, Denpasar, Indonesia using hematological profile. Biodiversitas 25: 1057-1062. Bali cattle (Bos sondaicus Blyth, 1842 syn. Bos javanicus domesticus Wilckens, 1905) are vulnerable to blood protozoan infections like Trypanosoma evansi. Indonesia's warm and moist environment provides ideal conditions for blood-sucking flies such as Stomoxys calcitrans, Haematobia irritans, and Hipobosca sp. to thrive and spread. These flies act as carriers of T. evansi, which can then infect Bali cattle. This study aims to detect the presence of T. evansi in Bali cattle. Blood samples were collected from 275 Bali cattle at the Pesanggaran slaughterhouse, Denpasar City, Bali, Indonesia. The samples were examined using the Giemsa-Staining Blood Smear (GSBS) method and hematology profile examination using Hematology Analyzer RT-7600 for Vet. Serum samples were also collected for serologic examination using the Card Agglutination Test for Trypanosomiasis (CATT) method. The hematological profile data obtained were analyzed descriptively, and the Mann-Whitney U test was used to determine the significance of hematological values between female and male cattle seropositive to T. evansi. The results showed that the T. evansi parasite was not detected in the blood smear examination. However, the serological detection with CATT revealed that 6.54% (18/275) of Bali cattle were positive for T. evansi. The hematological profiles of T. evansi seropositive cattle remained within the normal range, except for neutrophil percentage. White Blood Cell (WBC), Hemoglobin (Hb) levels, and hematocrit (PCV) values of male cattle were significantly (P?0.05) lower than those of females. Therefore, based on serological examination, it can be concluded that the protozoa T. evansi is present in 6.54% of Bali cattle at the Pesanggaran slaughterhouse.

Conducting active screening for human African trypanosomiasis with rapid diagnostic tests: The Guinean experience (2016-2021).

Strategies to detect Human African Trypanosomiasis (HAT) cases rely on serological screening of populations exposed to trypanosomes. In Guinea, mass medical screening surveys performed with the Card Agglutination Test for Trypanosomiasis have been progressively replaced by door-to-door approaches using Rapid Diagnostic Tests (RDTs) since 2016. However, RDTs availability represents a major concern and medical teams must often adapt, even in the absence of prior RDT performance evaluation. For the last 5 years, the Guinean HAT National Control Program had to combine three different RDTs according to their availability and price: the SD Bioline HAT (not available anymore), the HAT Sero-K-SeT (most expensive), and recently the Abbott Bioline HAT 2.0 (limited field evaluation). Here, we assess the performance of these RDTs, alone or in different combinations, through the analysis of both prospective and retrospective data. A parallel assessment showed a higher positivity rate of Abbott Bioline HAT 2.0 (6.0%, n = 2,250) as compared to HAT Sero-K-SeT (1.9%), with a combined positive predictive value (PPV) of 20.0%. However, an evaluation of Abbott Bioline HAT 2.0 alone revealed a low PPV of 3.9% (n = 6,930) which was surpassed when using Abbott Bioline HAT 2.0 in first line and HAT Sero-K-SeT as a secondary test before confirmation, with a combined PPV reaching 44.4%. A retrospective evaluation of all 3 RDTs was then conducted on 189 plasma samples from the HAT-NCP biobank, confirming the higher sensitivity (94.0% [85.6-97.7%]) and lower specificity (83.6% [76.0-89.1%]) of Abbott Bioline HAT 2.0 as compared to SD Bioline HAT (Se 64.2% [52.2-74.6%]-Sp 98.4% [94.2-99.5%]) and HAT Sero-K-SeT (Se 88.1% [78.2-93.8%]-Sp 98.4% [94.2-99.5%]). A comparison of Abbott Bioline HAT 2.0 and malaria-RDT positivity rates on 479 subjects living in HAT-free malaria-endemic areas further revealed that a significantly higher proportion of subjects positive in Abbott Bioline HAT 2.0 were also positive in malaria-RDT, suggesting a possible cross-reaction of Abbott Bioline HAT 2.0 with malaria-related biological factors in about 10% of malaria cases. This would explain, at least in part, the limited specificity of Abbott Bioline HAT 2.0. Overall, Abbott Bioline HAT 2.0 seems suitable as first line RDT in combination with a second HAT RDT to prevent confirmatory lab overload and loss of suspects during referral for confirmation. A state-of-the-art prospective comparative study is further required for comparing all current and future HAT RDTs to propose an optimal combination of RDTs for door-to-door active screening.

Risk factors and prevalence of human African trypanosomiasis in individuals living in remote areas of the republic of Congo

BackgroundHuman African trypanosomiasis (HAT) is one of the world’s classical neglected tropical diseases representing a major public health threat in sub-Saharan Africa. Although the parasitic disease is in decline in the Republic of Congo, the better understanding of the epidemiological situation of active foci is required to reduce the risk of disease resurgence which could impede progress registered so far. The aim of this study was to determine the prevalence of HAT and the associated risk factors in individuals living in remote areas of the Republic of Congo.MethodsA cross-sectional survey was carried out in volunteers living in rural settings from June 2020 to January 2021. Socio-demographic and Clinical parameters of the participants were recorded. The presence of HAT-specific antibodies was assessed in whole blood, and then confirmed in serial diluted plasma samples using Card-Agglutination Trypanosomiasis Test (CATT)/T.b. gambiense CATT. The Capillary Tube Centrifugation (CTC) and Lymph nodes (LN) examination were done for detecting trypanosome parasites in CATT-serum positive cases. The staging of positive participants was determined by cerebrospinal fluid (CSF) examination.ResultsOut of 8556 enrolled participants, 48.5% were more than 15 years old, 57.7% were unschooled and 67.2% practiced peasant activities. The prevalence of HAT infection was 0.3% with the predominance of patients at stage 1 of the disease (84.0%). The districts of Mindouli (OR: 25.9 (5.2–468); p = 0.0016) and Mpouya (OR: 13.3 (2.5–246); p = 0.0140) was revealed as the foci of high risk of HAT infection. Several factors were associated with an increased risk of HAT infection mainly including the non-schooling (OR: 5.1 (1.2–21.9); p = 0.0268), the life in couple or married (OR: 3.3 (1.0–11.3); p = 0.0545) and the practice of peasant activities (OR: 6.9 (2.4–29.3); p = 0.0017).ConclusionThis study highlights the need of revising and strengthening the strategies of HAT control in Republic of Congo, using an approach which will take into account the education level, the marital status and the occupation of the population at risk.

Performance of the SD Bioline rapid diagnostic test as a good alternative to the detection of human African trypanosomiasis in Cameroon.

BackgroundCase detection is essential for the management of human African trypanosomiasis (HAT), which is caused by Trypanosoma brucei gambiense. Prior to parasitological confirmation, routine screening using the card agglutination test for trypanosomiasis (CATT) is essential. Recently, individual rapid diagnostic tests (RDTs) for the serodiagnosis of HAT have been developed.ObjectiveThe purpose of this study was to evaluate the contribution of SD Bioline HAT to the serological screening of human African trypanosomiasis in Cameroonian foci.Methods. Between June 2014 and January 2015, blood samples were collected during surveys in the foci of Campo, Yokadouma, and Fontem. The sensitivity (Se) and specificity (Sp) of SD Bioline HAT were determined using the CATT as the gold standard for the detection of specific antibodies against Trypanosoma brucei gambiense.ResultsA total of 88 samples were tested: 59.1% (n=52) in Campo, 31.8% (n=28) in Yokadouma, and 9.1% (n=8) in Fontem. There were 61.4% (n=54) males and 38.4% (n=34) females, and the average age was 35.4 19.0 years. In probed foci, the overall seroprevalence was 11.4% (95% confidence interval: 6.3-19.7) with the CATT method and 18.2% (95% confidence interval: 11.5-27.2%) with the SD Bioline HAT RDT method. The SD Bioline HAT’s Se and Sp were 80.0% and 89.7%, respectively.ConclusionsThis study demonstrated that the overall performance of the SD Bioline HAT was comparable to that of the CATT, with high specificity in the serological detection of HAT.

Microscopic and serological survey of Trypanosoma evansi infection in Tunisian dromedary camels (Camelus dromedarius).

Camels are the most adapted species to the harsh ecosystems of the African continent where they represent 80% of world's population. However, they can be susceptible to many pathogens such as Trypanosoma evansi. This parasite is widely distributed in Africa and represents a real threat to animal health and production. In Tunisia, despite its importance, this disease is underestimated and only a few data are available. The present study aims to investigate the prevalence of the disease by combining microscopic and serological tests. A cross-sectional study was conducted on 307 dromedary camels randomly selected from Southern Tunisia. Collected blood samples were microscopically examined and tested with Card Agglutination Trypanosomiasis Test to detect anti-T. evansi antibodies. The microscopic and serological prevalence were estimated at 2.93 and 62.54%, respectively. Microscopic examination showed morphological features of T. evansi. All positives samples were collected from aged males (>5years old). Seroprevalence was similar for both sexes but it was higher in dry season and in the southwest of Tunisia. The present study revealed high prevalence of T. evansi infection among dromedary camels. This highlights the need for adequate control measures based on the detection and treatment of infected animals and vector control. We recommend investigating the prevalence of infection in other domestic animals living in the same environment.

Unusual neurological presentation of second stage African trypanosomiasis in a young boy: a case report

BackgroundIn South Sudan, sleeping sickness is a frequent condition caused by human African trypanosomiasis. There are two stages that are well-known. When the CNS is affected, especially with Trypanosoma gambiense infection, the early hemolymphatic stage and the late encephalitic stage have been observed, including mental, motor, and sensory symptoms. In this case, second-stage African trypanosomiasis manifested itself in an atypical neurological manner.Case presentationA 16-year-old boy from South Sudan referred to Sudan National Centre for Neurological Sciences, Khartoum, Sudan suffering from non-convulsive status epilepticus, mental deterioration and behavioral changes for the last nine months. He was conscious but disorientated. Low hemoglobin concentration, elevated ESR, enlarged spleen and positive card agglutination test for trypanosomiasis was found in this patient. Electro-encephalogram (EEG) found an on-going generalized seizure activity. The patient showed improvement after management with carbamazepine and tonic.ConclusionOur case highlights that late second stage African trypanosomiasis with neurological complications such as non-convulsive status epilepticus should be suspected in any patient who developed progressive cognitive decline and behavioral changes following long standing history of African Trypanosomiasis and routine Electro-encephalogram EEG is the best tool to diagnose non convulsive status epilepticus.

Accelerating elimination of sleeping sickness from the Guinean littoral through enhanced screening in the post-Ebola context: A retrospective analysis.

BackgroundActivities to control human African trypanosomiasis (HAT) in Guinea were severely hampered by the Ebola epidemic that hit this country between 2014 and 2016. Active screening was completely interrupted and passive screening could only be maintained in a few health facilities. At the end of the epidemic, medical interventions were progressively intensified to mitigate the risk of HAT resurgence and progress towards disease elimination.Methodology/Principal findingsA retrospective analysis was performed to evaluate the medical activities that were implemented in the three most endemic prefectures of Guinea (Boffa, Dubreka and Forecariah) between January 2016 and December 2018. Passive screening using rapid diagnostic tests (RDTs) was progressively resumed in one hundred and one health facilities, and active screening was intensified by visiting individual households and performing RDTs, and by conducting mass screening in villages by mobile teams using the Card Agglutination Test for Trypanosomiasis. A total of 1885, 4897 and 8023 clinical suspects were tested in passive, while 5743, 14442 and 21093 people were actively screened in 2016, 2017 and 2018, respectively. The number of HAT cases that were diagnosed first went up from 107 in 2016 to 140 in 2017, then subsequently decreased to only 73 in 2018. A progressive decrease in disease prevalence was observed in the populations that were tested in active and in passive between 2016 and 2018.Conclusions/SignificanceIntensified medical interventions in the post-Ebola context first resulted in an increase in the number of HAT cases, confirming the fear that the disease could resurge as a result of impaired control activities during the Ebola epidemic. On the other hand, the decrease in disease prevalence that was observed between 2016 and 2018 is encouraging, as it suggests that the current strategy combining enhanced diagnosis, treatment and vector control is appropriate to progress towards elimination of HAT in Guinea.

Seroprevalence and risk factors for Trypanosoma evansi, the causative agent of surra, in the dromedary camel (Camelus dromedarius) population in Southeastern Algeria.

Surra, caused by Trypanosoma evansi, is a re-emerging animal trypanosomosis, which is of special concern for camel-rearing regions of Africa and Asia. Surra decreases milk yield, lessens animal body condition score and reduces market value of exported animals resulting in substantial economic losses. A cross-sectional seroprevalence study of dromedary camels was conducted in Algeria, and major risk factors associated with infection were identified by collecting data on animal characteristics and herd management practices. The seroprevalence of T. evansi infection was determined in sera of 865 camels from 82 herds located in eastern Algeria using an antibody test (card agglutination test for Trypanosomiasis – CATT/T. evansi). Individual and herd seroprevalence were 49.5% and 73.2%, respectively, indicating substantial exposure of camels to T. evansi in the four districts studied. Five significant risk factors for T. evansi hemoparasite infection were identified: geographical area, herd size, husbandry system, accessibility to natural water sources and type of watering. There was no association between breed, sex or age with T. evansi infection. Results of this study provide baseline information that will be useful for launching control programmes in the region and potentially elsewhere.

Highly Localized Enrichment of Trypanosoma brucei Parasites Using Dielectrophoresis.

Human African trypanosomiasis (HAT), also known as sleeping sickness, is a vector-borne neglected tropical disease endemic to rural sub-Saharan Africa. Current methods of early detection in the affected rural communities generally begin with general screening using the card agglutination test for trypanosomiasis (CATT), a serological test. However, the gold standard for confirmation of trypanosomiasis remains the direct observation of the causative parasite, Trypanosoma brucei. Here, we present the use of dielectrophoresis (DEP) to enrich T. brucei parasites in specific locations to facilitate their identification in a future diagnostic assay. DEP refers to physical movement that can be selectively induced on the parasites when exposing them to electric field gradients of specific magnitude, phase and frequency. The long-term goal of our work is to use DEP to selectively trap and enrich T. brucei in specific locations while eluting all other cells in a sample. This would allow for a diagnostic test that enables the user to characterize the presence of parasites in specific locations determined a priori instead of relying on scanning a sample. In the work presented here, we report the characterization of the conditions that lead to high enrichment, 780% in 50 s, of the parasite in specific locations using an array of titanium microelectrodes.

The separation of trypanosomes from blood by anion exchange chromatography: From Sheila Lanham's discovery 50 years ago to a gold standard for sleeping sickness diagnosis.

Human African trypanosomiasis (HAT), or sleeping sickness, is a neglected tropical disease that is fatal if untreated, caused by Trypanosoma brucei gambiense and T. brucei rhodesiense. In its 2012 roadmap, WHO targeted HAT for elimination as a public health problem in 2020 and for zero transmission in 2030. Diagnosis of HAT is a multistep procedure comprising of clinical suspicion, confirmation, and stage determination. Suspects are identified on clinical signs and/or on screening for specific antibodies. Parasitological confirmation of suspects remains mandatory to avoid unnecessary toxic drug administration. The positive predictive value of the antibody detection tests is low. Simple parasite detection techniques, microscopic examination of lymph node aspirate, or stained thick blood films lack sensitivity, whereas in T. brucei gambiense patients, the number of blood trypanosomes may be very low. Parasite concentration techniques are therefore indispensable. Half a century ago, Sheila Lanham discovered a technique to separate trypanosomes from the blood of infected rodents, based on anion exchange chromatography with diethyl amino ethyl (DEAE) cellulose, a weak anion exchanger. Between pH 6−9, trypanosome surface is less negatively charged than that of blood cells. When blood is poured on top of a DEAE cellulose column, blood cells are retained, whereas parasites pass the column together with the elution buffer. The result is a pure suspension of trypanosomes that retain their morphology and infectivity. Because cell surface charges vary among trypanosome and mammal species, the optimal buffer pH and ionic strength conditions for different combinations of host and trypanosome species were established. Lanham's technique revolutionized the diagnosis of HAT. It is indispensable in the production of the Card Agglutination Test for Trypanosomiasis (CATT), the most used field test for screening in T. brucei gambiense HAT foci and essential to confirm the diagnosis in suspected people. Lumsden and colleagues developed the mini anion exchange centrifugation technique (mAECT). After adaptation for field conditions, its superior diagnostic and analytical sensitivity compared to another concentration technique was demonstrated. It was recommended as the most sensitive test for demonstrating trypanosomes in human blood. At the beginning of the 21st century, the mAECT was redesigned, allowing examination of a larger volume of blood, up to 0.35 ml with whole blood and up to 10 ml with buffy coat. The plastic collector tube in the new kit is also used for detection of trypanosomes in the cerebrospinal fluid. Unfortunately, mAECT also has some disadvantages, including its price, the need to centrifuge the collector tube, and the fact that it is manufactured on a noncommercial basis at only two research institutes. In conclusion, 50 years after Sheila Lanham's discovery, CATT and mAECT have become essential elements in the elimination of HAT.

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.

Identification of human African Trypanosomiasis foci using school-going children in post-conflict era in Nwoya District, Northern Uganda: A cross-sectional study

Background: Human African Trypanosomiasis (HAT) is fatal if untreated; the drugs to treat it are toxic making its management difficult and diagnosis complex. Nwoya district has a long history of sleeping-sickness dating back to pre-colonial times. The civil war of 1986-2008 displaced many who upon return complained of cattle and dogs dying of unknown causes alongside increased tsetse flies infestation hence, the needs for the study. Methods: We enrolled local 3,040 pupils and recorded their social-demographic characteristics and access to different domesticated animals/fowls in their homes. Screening for HAT using the card agglutination test for trypanosomiasis (CATT) was performed; positive individuals had their titres determined, followed by microscopy and loop mediated isothermal amplification analysis (LAMP). R was used for analysis where associations were sought between dependent and independent variables. Any factor with P-value <0.05 was taken as statistically significant. Results: HAT serological prevalence of 1.2% (95% CI 0.8-1.6) was obtained, 58.3% being boys while 41.7% were girls with titres ranging from 1:2 - 1:16. Two schools alone, constituted 47% of the CATT positive cases. Pupils who came from homes with dogs were more likely to be CATT/ Trypanosoma brucei gambiense positive; (adjusted odds ratio = 3.12, 95% CI 1.41-6.99 & p=0.005). Conclusions: Though no parasites were detected, with prevalence of CATT positive at 1.2%, active surveillance in the district is still recommended. CATT positive cases needs follow-ups were immune trypanolysis test done to ascertain their exposure.

Identification of human African Trypanosomiasis foci using school-going children in post-conflict era in Nwoya District, Northern Uganda: A cross-sectional study.

Background: Human African Trypanosomiasis (HAT) is fatal if untreated; the drugs to treat it are toxic making its management difficult and diagnosis complex. Nwoya district has a long history of sleeping-sickness dating back to pre-colonial times. The civil war of 1986-2008 displaced many who upon return complained of cattle and dogs dying of unknown causes alongside increased tsetse flies infestation hence, the needs for the study. Methods: We enrolled local 3,040 pupils and recorded their social-demographic characteristics and access to different domesticated animals/fowls in their homes. Screening for HAT using the card agglutination test for trypanosomiasis (CATT) was performed; positive individuals had their titres determined, followed by microscopy and loop mediated isothermal amplification analysis (LAMP). R was used for analysis where associations were sought between dependent and independent variables. Any factor with P-value <0.05 was taken as statistically significant. Results: HAT serological prevalence of 1.2% (95% CI 0.8-1.6) was obtained, 58.3% being boys while 41.7% were girls with titres ranging from 1:2 - 1:16. Two schools alone, constituted 47% of the CATT positive cases. Pupils who came from homes with dogs were more likely to be CATT/ Trypanosoma brucei gambiense positive; (adjusted odds ratio = 3.12, 95% CI 1.41-6.99 & p=0.005). Conclusions: Though no parasites were detected, with prevalence of CATT positive at 1.2%, active surveillance in the district is still recommended.CATT positive cases needs follow-ups were immune trypanolysis test done to ascertain their exposure.

Carbon-Electrode Dielectrophoresis to Concentrate Trypanosoma Brucei

Trypanosoma brucei (T. brucei) is a parasite that causes human African Trypanosomiasis (HAT), commonly known as sleeping sickness. HAT is spread via bite from a Tsetse fly or congenitally, affecting an estimated 60 million people in Sub-Saharan Africa (Chappuis et al., 2005). This translates to a disease burden of 1.78 million disability-adjusted life years (DALY) (Fèvre et al., 2008). Depending on which parasite strain is contracted, T. b. gambiense or T. b. rhodesiense, patients can live 6-7 years or die within 6 months of infection (Centers for Disease Control and Prevention, 2012). Analysis of cerebrospinal fluid and lymph nodes are conclusive diagnostic tests, but are unaffordable and inaccessible in rural, impoverished areas. In these cases, the most relevant form of screening for HAT is the Card Agglutination Trypanosomiasis Test (CATT) (Chappius et al., 2004). This serological test is inexpensive, fast, portable, easy to use, and easy to read. However, current CATTs are limited in sensitivity and specificity: it can only identify T. b. gambiense and produces false positives for other diseases like schistosomiasis and malaria in 5-20% of readings (Inojosa, 2006). The tests also must be stored at low temperatures, which may be a logistical challenge in rural Africa. Hence, there is a need for a test with improved specificity, sensitivity, and that is easily stored while retaining the low cost advantage of the CATT. We propose Dielectrophoresis (DEP) as a rapid way to agglutinate targeted T. brucei strains. DEP is an electric-field technique that can induce motion on only selected targets, which will increase the specificity of the test. We use DEP to rapidly segregate T. brucei from its surroundings and agglutinate it on specific spots to facilitate its detection. To this end, we use microelectrodes made of either titanium or carbon. In particular, we postulate the use of carbon electrodes to enable DEP of T. brucei and thus inexpensive, rapid and specific agglutination assays. The experimental setup includes a microfluidics chip featuring multiple rows of intercalated carbon or titanium electrodes (Martinez-Duarte, 2011). Experimental samples of the animal strain T. brucei brucei were pipetted onto a single electrode array, which was polarized using sinusoidal signals with frequencies between 1kHz to 10 MHz at 1-10 Vpp. The array was monitored to characterize the movement of the parasite around the electrodes. Images taken of the experiments were analyzed to assess the velocity of attachment to the electrode array. Our initial results with both titanium and carbon electrode arrays showed parasite attachment in the range 750 kHz to 2 MHz. By implementing numerical modeling of the electric field of the device, the electric field gradient was simulated as strongest along the curve of the semicircle. This simulation corresponds to the observed location of attraction. Ongoing work is to further characterize specificity of T. brucei movement and to investigate new carbon electrode geometries that will increase the speed of parasitic attachment. Our goal is to eventually create a DEP device with an operational protocol similar to that currently used. We aim to create an inexpensive DEP device that only require batteries for operation. References Chappius, F, et al. “Card agglutination test for trypanosomiasis (CATT) end-Dilution titer and cerebrospinal fluid cell count as predictors of human African Trypanosomiasis (Trypanosoma brucei gambiense) among serologically suspected individuals in southern Sudan.” The American Journal of Tropical Medicine and Hygiene, vol. 71, no. 3, 2004, 313–317. Chappuis, F., et al. “Options for Field Diagnosis of Human African Trypanosomiasis.” Clinical Microbiology Reviews, vol. 18, no. 1, Jan. 2005, pp. 133–146., doi:10.1128/cmr.18.1.133-146.2005. Fèvre, Eric M., et al. “The Burden of Human African Trypanosomiasis.” PLoS Neglected Tropical Diseases, vol. 2, no. 12, 2008, doi:10.1371/journal.pntd.0000333. Inojosa, W. O. “Diagnosing human African trypanosomiasis in Angola using a card agglutination test: observational study of active and passive case finding strategies.” BMJ, vol. 332, 7556, 2006, pp. 1479–1484., doi:10.1136/bmj.38859.531354.7c. Martinez-Duarte, Rodrigo, et al. “A novel approach to dielectrophoresis using carbon electrodes.” Electrophoresis, vol. 32, no. 17, 2011, pp. 2385–2392., doi:10.1002/elps.201100059. “Parasites - African Trypanosomiasis” Centers for Disease Control and Prevention, Centers for Disease Control and Prevention, 29 Aug. 2012, www.cdc.gov/parasites/sleepingsickness/disease.html. Accessed 15 Nov. 2017.

Epidemiological Situation of the Human African Trypanosomiasis in the Bilolo Municipality of Central African Republic

The Human African Trypanosomiasis (HAT) is a disabling and fatal disease caused by a vector-borne parasite still impacting in residual hotbeds. The aim of our study was to update the epidemiological data of HAT in one of Central African Republic foci after the (2012-2014) period of conflict. The survey was carried out in 24 villages in the Bilolo's municipality where 4788 persons were examined by the CATT (Card Agglutination Trypanosomiasis Test) technique. The identified cases were submitted to a serological titration test followed by parasitological testing for confirmation. The cytorachia technique was used for clinical phase determination. The CATT has identified 221 persons, of which 54 were confirmed by serological titration test, giving a seroprevalence of 1.12%. Parasitological confirmation was obtained for 15 patients. Our study showed the abundance of Trypanosoma parasites in the Bilolo municipality. However, since 2007, no case of HAT had been diagnosed in this focus after screening, treatment and vector control.

Mass Survey of Human African Trypanosomiasis in Ukwani and Ethiope East Local Government Areas, Delta State, Nigeria

This study was intended to update the epidemiological status of Human African Trypanosomiasis (HAT), also known as sleeping sickness in Ukwuani and Ethope East Local Government Areas (LGAs) Delta State, Nigeria, and to establish a HAT control programme. In 2012, 1979 people from 26 villages were serologically screened for Trypanosomabruceigambiense, the causative agent of HAT. In 2012, surveys were carried out in villages where HAT cases had been identified. 119individuals were screened using the card agglutination Trypanosomiasis test (CATT), and then parasitologically examined for the presence of the parasite. CATT- positive individuals in whom the presence of the parasite could not be confirmed were further tested with the CATT using serum dilutions, and those with a positive antibody titer of 1 – in – 4or above were followed-up. Patients with 10 white cells and no trypanosomes in their cerebrospinal fluid (CSF) were classified as being in the second stage of the disease. Vector control was also considered necessary.In this survey, of the 1979 people screened, consisting of 7(0.57%) out of 1220 females and 4(0.53%) out of 759 males with no difference in sex – specificincidences. The positive cases occurred in Umutu and Umuebu communities in Ukuwani LGA, and in Ovu and Okpara inland communities in Ethiope- East LGA.

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.

Carbon-Electrode Dielectrophoresis for Concentrating Trypanosoma Brucei

Trypanosoma brucei (T. brucei) is a parasite that causes African Trypanosomiasis, or African sleeping sickness. This disease is spread through bite from a Tsetse fly or congenitally. Current tests used to properly diagnose the disease, such as cerebrospinal fluid testing (1) and card agglutination tests (2), are unaffordable in developing countries. For example, impoverished areas in Africa suffering from the disease do not have proper resources to house and maintain equipment needed for these tests. Hence, the goal of this research is to find a more cost-efficient way to identify the disease in rural settings. Dielectrophoresis (DEP) is a separation technique that utilizes the characteristic response of different microorganisms to a non-uniform electric field to segregate a specimen of interest from its background (3). In particular, positive DEP enables the concentration of the targeted organism in regions of high electric field gradient. Here, we postulate the use of inexpensive carbon electrodes to implement positive DEP of T. brucei. We aim at attracting and concentrating T. brucei parasites around the electrodes to facilitate and ease Trypanosomiasis detection by untrained personnel, thus reducing costs and enabling faster treatment of African Trypanosomiasis. The experimental setup used here included a microfluidics chip featuring multiple rows of intercalated carbon microelectrodes as detailed elsewhere (4). The experimental sample was prepared by re-suspending T. brucei parasites in a sugar solution with a conductivity of 500 μS/cm. The sample was flowed through the polarized electrode array at a flow rate of 0.2 μL/min for 2 minutes. After this time the field was turned off and the parasites that may had been trapped were released for elution. The electrode array was polarized with a sinusoidal signal at different frequencies, ranging from 100 kHz to 10 MHz at 100 kHz intervals, but at an optimized constant amplitude of 2 Vpp. The array was monitored to characterize the movement of the parasite around the electrodes and sequential images were taken at the end of the array to characterize the parasite elution pattern once the the field was turned off. These images were analyzed for each experiment to assess the difference in parasite attachment before and after turning the field off. Preliminary results indicate that the use of a voltage value higher than 2 Vpp leads to parasite death. At such polarizing voltage, attraction of the parasite around the electrodes was observed in the frequency range 500-900 kHz. However, trapping is predominantly around the electrically-insulating SU-8 structure around the electrodes. The attraction to this part of the chip, rather than to the carbon electrodes where it was expected, indicates a possible affinity to topography but most likely the possibility of a distorted electric field that surrounds the insulator and generates a high field gradient. Such distortion is the primary hypothesis for the attraction witnessed because we do not see this behavior at other frequencies outside the range 500-900 kHz. While some trapping was observed at experiments run at 10 Vpp, many parasites died when exposed to a high voltage. Shear stress was also examined as a possible cause of parasite death and the flow rate used lead to continued parasite viability. Ongoing work includes investigating the attraction of the parasites to the SU-8 layer, and whether this is caused by the non-uniform electric field around the curvature of the insulator layer. To this end, we are implementing numerical modeling of the electric field in the device to further identify the magnitude of the gradient that causes trapping. Once this is identified, the effect of sample conductivity on the trapping will be addressed to further understand the DEP behavior of T. brucei and facilitate its detection. References Chappuis, F., Loutan, L., Simarro, P., Lejon, V., & Büscher, P. (2005). Options for field diagnosis of human African trypanosomiasis. Clinical Microbiology Reviews, 18(1), 133-146. Chappuis, F., Stivanello, E., Adams, K., Kidane, S., Pittet, A., & Bovier, P. A. (2004). Card agglutination test for trypanosomiasis (CATT) end-dilution titer and cerebrospinal fluid cell count as predictors of human African Trypanosomiasis (Trypanosoma brucei gambiense) among serologically suspected individuals in southern Sudan. The American Journal of Tropical Medicine and Hygiene, 71(3), 313-317. Menachery, M., Kremer, C., Wong, P., Carlsson, A., Neale, S., Barrett M., Cooper, J. (2012). Counterflow Dielectrophoresis for Trypanosome Enrichment and Detection in Blood. Scientific Reports, 775(2), 1-5. Martinez-Duarte, R., Renaud, P., Madou, M. (2011). A Novel Approach to Dielectrophoresis Using Carbon Electrodes. Electrophoresis, 32(17), 2385-2392. Figure 1

Studying the Behavior of T. Brucei Under Electric Field Gradients Implemented Using Optoelectronic Tweezers

Here we present a study of the response of Trypanosoma brucei (T. brucei) to electric field gradients. T. brucei is a parasitic protozoan that causes trypanosomiasis or African Sleeping Sickness. Unfortunately, accurate detection of the parasite is now costly, requiring considerable technical training as in the case of cerebrospinal fluid testing (1) and card agglutination tests (2), which hinders the diagnosis and treatment of the disease in patients living in developing countries. Further characterization of T.brucei is thus necessary to create more affordable and accessible diagnostic techniques. The long term objective of this research is to develop alternatives to detecting T. brucei by aggregating the parasites to a specific area which will facilitate their observation. The work presented here is aimed at characterizing the response of T. brucei to electric field gradients towards assessing the potential of dielectrophoresis as a tool for parasite concentration. To do this, we implemented an Optoelectronic tweezer (OET) setup to create an electric field gradient by simple illumination of specific areas of a photoconductive layer (3). Briefly, an OET setup features two plates of transparent indium tin oxide (ITO) polarized by a function generator and spaced apart a specific distance to enclose a volume filled with media. A photoconductive layer must be deposited on one of the ITO plates such that electrical coupling between the plates only occurs on those areas that are illuminated by visible light. Fabrication of such devices only requires film coatings, which makes production scalable and relatively inexpensive. Here we generate the light pattern using a general purpose DLP-based projector fitted with reduction optics. Hence, by changing this light pattern we are able to efficiently introduce electric field gradients in the fluidic volume. We utilize this capability to expose T. brucei to gradients of varying magnitude and frequency. We seek those parameters under which T. brucei shows a clear migration towards the gradient that will enable its concentration. To run OET experiments on T. brucei, the parasite was centrifuged twice and resuspended in a sugar solution with its conductivity adjusted to values from 500 μS to 1,500 μS using Phosphate Buffered Saline (PBS). 10 μL of such sample were then introduced in the OET setup. A powerpoint slide with a black background and white, square rings was used to illuminate the photoconductive layer. The ITO plates were polarized using a sinusoidal signal with varying voltage and frequency, in the range 3.5 - 10V and 100kHz- 20MHz respectively. The parasite behavior was monitored throughout the experiment to assess its response to the light-induced electric field gradient. Initial results when probing the frequency range mentioned above showed attraction of the parasite to the illuminated square only at 18 and 19 MHz across the complete range of tested signal amplitudes (3.5 Vpp, 5 Vpp, 6 Vpp, 7.5 Vpp and 10 Vpp) with a conductivity of 500 μS/cm. When there was no attraction, the parasites did not move towards the light, or passed through the light, showing no restriction of movement from an attractive force. By increasing the voltage at a frequency that causes attraction, the speed of movement towards the light increases proportionally. Lowering the electrical conductivity of the media also causes a stronger attraction of the parasite. The use of conductivity values below 500 μS and amplitudes above 10 V renders the parasite non viable regardless of the frequency value. The focus of ongoing work includes validating the frequencies where attraction was seen, 18-19 MHz, and further optimizing the voltage to maximize attraction of the parasite while keeping them viable. Future work includes experimentation with different shapes and colors of the incident light to assess their importance in the rapid concentration of the parasite working towards an inexpensive and accurate diagnostic test for T. brucei. References Chappuis, F., Loutan, L., Simarro, P., Lejon, V., & Büscher, P. (2005). Options for field diagnosis of human African trypanosomiasis. Clinical Microbiology Reviews, 18(1), 133-146.Chappuis, F., Stivanello, E., Adams, K., Kidane, S., Pittet, A., & Bovier, P. A. (2004). Card agglutination test for trypanosomiasis (CATT) end-dilution titer and cerebrospinal fluid cell count as predictors of human African Trypanosomiasis (Trypanosoma brucei gambiense) among serologically suspected individuals in southern Sudan. The American Journal of Tropical Medicine and Hygiene, 71(3), 313-317.Kremer, C., Neale, S., Menachery, A., Barrett, M., Cooper, M. (2012). Optoelectronic Tweezers for Medical Diagnostics. Frontiers in Biological Detection: From Nanosensors to Systems IV, 8(212).