Detection Of Trypanosomes Research Articles

Challenges in the Diagnostic Performance of Parasitological and Molecular Tests in the Surveillance of African Trypanosomiasis in Eastern Zambia

African animal trypanosomiasis (AAT) control programs rely on active case detection through the screening of animals reared in disease endemic areas. This study compared the application of the polymerase chain reaction (PCR) and microscopy in the detection of trypanosomes in cattle blood in Mambwe, a rural district in eastern Zambia. Blood samples were collected from 227 cattle and tested for infection with trypanosomes using microscopy and Ribosomal RNA Internal Transcribed Spacers (ITS)-PCR. Microscopy on the buffy coat detected 17 cases, whilst thin and thick smears detected 26 cases and 28 cases, respectively. In total, microscopy detected 40 cases. ITS-PCR-filter paper (FP) on blood spots stored on FP detected 47 cases, and ITS-PCR-FTA on blood spots stored on Whatman FTA Classic cards detected 83 cases. Using microscopy as the gold standard, ITS-PCR-FTA had a better specificity (SP) and sensitivity (SE) (SP = 72.2%; SE = 77.5%; kappa = 0.35) than ITS-PCR-FP (SP = 88%; SE = 60%; kappa = 0.45). The prevalence of Trypanosoma brucei s.l. was higher on ITS-PCR-FTA (19/227) than on ITS-PCR-FP (0/227). Our results illustrate the complexities around trypanosomiasis surveillance in rural Africa and provide evidence of the impact that field conditions and staff training can have on diagnostic results, which in turn impact the success of tsetse and trypanosomiasis control programs in the region.

Vector-borne haemoparasitic and rickettsial diseases of dogs in South west, Nigeria: An eleven years retrospective study

Adequate knowledge of the occurrence and seasonal variation in the distribution of parasitic and rickettsial diseases affecting dogs is required for formulation of effective disease control measures. Hence, this study assessed the prevalence of vector borne haemoparasites and rickettsial of dogs between 2009 and 2019. Clinical records of dogs presented for clinical examination and confirmed by microscopic screening of their blood were assessed and analysed statistically. Overall, 1483 dogs were presented to the Veterinary Teaching Hospital (VTH). This included 641 (43.2%) male, 842 (56.8%) female, 967 (65.2%) young and 516 (34.8%) adult dogs. 33 (2.2%) and 1450 (97.8%) were mongrel and exotic breeds, respectively. 511 (34.5%) were infected with either haemoparasites or rickettsial. This comprised 34.0% (218/641) and 34.8% (293/842) male and female, 317 (32.8%) and 194 (37.6%) young and adult dogs, respectively. There was no significant (p =0.05) difference in the prevalence among sexes and age groups. Seasonal prevalence was 35.88% (315/878) and 32.40% (196/605) in wet and dry seasons, respectively. Six haemoparasites/rickettsials were reported from 511 dogs, these included single infections of Babesia species 477 (93.5%), Ehrlichia species 8 (1.6%), Hepatozoon canis 2(0.39%), Mycoplasma haemocanis 7 (1.4%), Trypanosoma species 16 (3.1%) and mixed infection of Babesia and Hepatozoon canis 1 (0.2%). The mean PCV values were significantly (t = 2.45; P = 0.014) different between infected and non-infected dogs. This study suggests that Babesia spp. isthe most prevalentin the dogs presented to the VTH in the study area and the detection of Trypanosoma spp. in some dogssuggeststhat dogs could be reservoir of trypanosomesfor both animals and human.
 Keywords: Dogs, haemoparasites, prevalence, retrospective-study

Risk factors for triatominae infestation in a municipality of Colombia.

Background:Identifying risk factors for Triatominae infestation is essential for the development of vector control interventions.Methods:To determine the intra- and peridomiciliary risk factors associated with triatomine infestation, a cross-sectional analytical study was carried out with random cluster sampling in two stages, which included the identification of risk factors by survey and direct observation, as well as the search and capture of triatomines. The detection of trypanosomes in triatomines was carried out by observing the rectal content and then by conventional polymerase chain reaction (PCR).Results:In 21 of the 207 houses inspected, 13 specimens of R. colombiensis and 19 specimens of P. geniculatus were found. Entomological indices included: dispersion 36%, infestation 10%, infection 65%, colonization 4.7%, density 15%, and concentration 152%. An association was found between the presence of Triatominae and the existence of branches and fissures in the floors, as well as with the presence of accumulated objects and with knowledge about Chagas disease. The risk of having triatomines in urban homes is 5.7 times higher than the risk in rural areas [confidence interval (CI) 0.508–67.567]; 6.6 times in houses with cracked soil (CI 0.555–81.994), 6 times in houses located near caneys (CI 0.820–44.781), and 6.16 times with accumulated objects (CI 1.542–39.238).Conclusion:Chagas disease is a complex problem that requires control based on the vector’s elimination or surveillance, which implies identifying species and their distribution, generating alerts, knowledge, and awareness in the population. It is necessary to intensify surveillance activities for the event, especially in changing aspects of Chagas diseases’ transmission dynamics, such as urbanization and the type of housing associated with the vector’s presence.

Trypanosomosis prevalence in natural field-based infection: insights into systematic review and meta-analysis of studies from 1980 to 2018 on The Gambian ruminants with special emphasis on cattle.

Consistent quantification of trypanosomes, the parasite responsible for African animal trypanosomosis, is important for effective surveillance, control, and eradication strategies. Here, we used a rigorously predefined protocol to search and select eligible publications that utilized either microscopy, serology, or molecular methods to investigate prevalence of trypanosomosis based on the presence of any of three most common Trypanosoma spp. (T. congolense, T. vivax, and T. brucei) in the field-based naturally grazed Gambian cattle, sheep, and goats. To combine results of studies on cattle through meta-analysis, sensitivity and subgroup analyses were carried out with the random effects model, and prevalence estimates of each study with 95% confidence intervals (CI) were presented with a forest plot. All the eligible studies utilized the buffy coat technique (BCT) to detect trypanosomes in the blood samples, while the more sensitive serological and molecular detection methods are yet to be widely exploited. Heterogeneity among the studies on cattle was moderate (I2 = 55%), and the pooled trypanosomosis prevalence based on the BCT was 5.2% (95% CI: 4.0-6.4). Meanwhile, estimated prevalence varied according to the trypanosome detection methods, study locations, types of publication, year, and length of observations. We could not pool the trypanosomosis prevalence in sheep and goats through meta-analysis due to small number of studies. The prevalence estimates based on the BCT ranged from 3.2 to 8.1% in goats and 2.8 to 10.6% in sheep. Even though there seems to be a slight decrease in trypanosomosis prevalence in cattle in one of the Gambian districts, there was no consistent trend across the years. It is thought that the literature search and formatting procedures presented in this study contribute to doing systematic reviews on the investigated subject and can be adapted for similar cases.

Prevalence and hematological parameters of bovine Trypanosomosis in Nsukka municipal abattoir, Southeastern Nigeria

The prevalence and hematological parameters of trypanosomosis in slaughter cattle in Nsukka, Southeastern Nigeria, were investigated. Blood samples from 200 randomly selected cattle were collected following slaughter into labeled sample bottles with anticoagulant. The sex and breed of the sampled cattle were properly noted. Standard trypanosome detection techniques involving wet blood film examination, buffy coat technique, examination of Giemsa-stained blood smears, and mice inoculation were used to detect and identify the trypanosome species present. The hematological parameters—packed cell volume (PCV), hemoglobin concentration (HbC), total erythrocyte count (TEC), total leucocyte count (TLC), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC)—were determined following standard procedures. A prevalence rate of 1.5% (95% CI = 0.005–0.043) was obtained for bovine trypanosomosis in Nsukka with 1% (95% CI = 0.003–0.036) and 0.5% (95% CI = 0.001–0.028) prevalence rates in female and male cattle, respectively. Whereas a prevalence rate of 1.5% (95% CI = 0.005–0.043) was recorded in the White Fulani breed, the Sokoto Gudali had 0% prevalence rate. The prevalence recorded in this study was not associated (P ˃ 0.05) with sex or breed. The mean PCV, HbC, TEC, MCV, MCH, MCHC, and TLC values of the infected cattle differed (P < 0.05) from that of the uninfected cattle. In conclusion, trypanosomosis is prevalent in cattle slaughtered in Nsukka, Southeastern Nigeria, with associated hematological changes. Hence, the need for the adoption and implementation of national trypanosomosis integrated control strategies.

Molecular Detection of &lt;i&gt;Trypanosoma species&lt;/i&gt; in Cattle Slaughtered at Zango and Yantukwane Abattoirs in Zaria, Kaduna State, Nigeria

African animal trypanosomoses constitute the most important vector-borne cattle diseases in Sub-Saharan Africa. A total of170 blood samples were collected from cattle in Zango abattoir and Yantukwane abattoir in Zaria, Kaduna State. BloodSamples collected were examined for the presence of trypanosomes using the buffy coat technique and Giemsa thin bloodsmears which shows a single 1(0.58%; 1/170) infection using microscope. Positive and negative samples were subjected tofurther analysis using nested PCR reaction with nested primers for detection of trypanosomes, and 9 of the samples showedpositive bands after gel electrophoresis at 998bp (lane 1-9) indicating the presence of Trypanosoma theileri. Percentage, Chisquareand Odds ratio were used to analyse the data. A total of 9(5.29%; 9/170) animals were found infected with the parasiteout of which 4(4.65%; 4/85) were found in Zango abattoir, while 5 (5.95%) were found in Yantukwane abattoir. The resultswere not statistically (P&gt;0.05) location dependent. The infection in male (6.45%) cattle was higher than the female (4.63%)with no infection in young animals. No infection was observed in Sokoto Gudali breed, while White Fulani breed had 5.77%.From this study, it is clear that trypanosomosis is still a major obstacle to livestock production in Zaria and its Environs andthe incidence is majorly in adult cattle. In addition, nested PCR reaction showed more sensitivity than microscopy. Hence,there is need for proper diagnosis and treatment of animals to promote more beef and milk production for humanconsumption. Further study is necessary to determine the pathogenicity of T. theileri in other domestic animals.&#x0D; Keywords: Trypanosomosis, White Fulani, Sokoto Gudali, breed, infection.

Parasitological and molecular detection of Trypanosoma spp. in cattle, goats and sheep in Somalia.

African animal trypanosomiasis (AAT) affects the livestock of 12.3 million Somalis and constrains their development and wellbeing. There is missing data on AAT in the country after the civil war of the 1990s. Therefore, this study has aimed to assess the prevalence of Trypanosoma spp. in 614 blood samples from cattle (n = 202), goats (n = 206) and sheep (n = 206) in Afgoye and Jowhar districts, Somalia using parasitological and molecular methods. Twenty-one out of 614 (3.4%; 95% CI: 2.1-5.2%) and 101/614 (16.4%; 95% CI: 13.6-19.6%) ruminants were positive for Trypanosoma spp. by buffy coat technique (BCT) and internal transcribed spacer 1 (ITS1)-polymerase chain reaction (PCR), respectively. Using ITS1-PCR, the highest prevalence was observed in cattle (23.8%; 95% CI: 18.4-30.1%) followed by goats (17.5%; 95% CI: 12.9-23.3%) and sheep (8.3%; 95% CI: 5.1-12.9%). A total of 74/101 (73.3%; 95% CI: 63.5-81.6%) ruminants were shown coinfection with at least two Trypanosome species. The four T. brucei-positive samples have tested negative for T. b. rhodesiense, by the human-serum-resistance-associated-PCR. Trypanosoma evansi, T. godfreyi, T. vivax, T. brucei, T. simiae and T. congolense were the Trypanosoma species found in this study. This is the first study on the molecular detection of Trypanosoma sp. in ruminants in Somalia. Further investigations and control measures are needed to manage Trypanosomiasis spreading in the country. Studies should also focus on the detection of T. b. rhodesiense in the country.

Prevalence and Associated Risk Factors of Trypanosoma evansi in Camels in Ethiopia Based on Parasitological Examinations.

Camel trypanosomosis (surra), caused by Trypanosoma evansi , is a life-threatening disease with negative impacts on health, production, and working efficiency of camels in different camel-rearing areas of the world, including Ethiopia. A cross-sectional study was carried out from November 2016 to May 2017 to determine the prevalence of camel trypanosomosis (surra) and assess the associated potential risk factors in Dubti and Asayita districts of Afar region, Ethiopia. Blood samples were collected from 200 camels. Wet film and Giemsa-stained blood smears were used for the detection of trypanosomes. Out of 200 examined, 9(4.5%) were positive for Trypanosoma evansi. The higher prevalence of the disease was observed in Dubti (6.97%) than in Asayita (2.63%) districts. Highest infection was observed in the age group >4 years old camels (7.47%), followed by <4 years old camels (1.07%). Previously aborted camels were found at higher risk (P=0.03; OR = 5.11, 95% CI = 1.174–22.317) than camels without an abortion history. There was no statistically significant difference in between body condition categories and herd size of camels with the occurrence of the disease (X2 = 3.839; P=0.147 and X2 = 0.718; P=0.698), respectively. The result of the current study revealed that camel trypanosomosis is substantially prevalent in the study area, indicating the need for designing control and prevention strategies.

Bovine trypanosomiasis and vectors in Lom and Djerem Division of East-Cameroon

Bovine trypanosomiasis is a major threat to the livestock sector of Cameroon. The main objective of this study was to determine the prevalence of bovine trypanosomes and vectors in the SODEPA Ranch of Ndokayo. A cross sectional parasitological survey was carried out in the late dry season, where blood was collected through the jugular vein of 390 cattle. The trypanosome detection in cattle blood was carried out using the buffy coat technique (BCT). An entomological prospection was carried out in the late dry season and early rainy season using two unbaited trap types notably Nzi (n = 4) and Vavoua (n = 4). The overall bovine trypanosomiasis prevalence was 2.82% and species specific prevalence included: Trypanosoma congolense (63.6%), Trypanosoma brucei (18.2%) and mixed species infections (T. congolense + T. brucei and T. brucei + T. vivax) (18.2%). Sixteen (16) species of flies were identified and grouped under six genera: Stomoxys, Ancala, Tabanus, Chrysops, Hematopota and Glossina. The overall trap apparent density (ADT) was 3.92 flies/trap/day, with a higher ADT in early rainy season than in the late dry season. The highest species richness was noticed with the genus Tabanus, while the highest ADT was found in Stomoxyini. The Nzi trap recorded a higher mean ADT for most fly vectors than Vavoua, except for Stomoxyini that rather recorded a higher ADT with the Vavoua trap. The present findings will instruct fly control authorities of Cameroon to better manage.

Trypanosome prevalence in pigs and tsetse flies from selected areas of Jomoro district of the western region of Ghana.

Detection of trypanosomes in tsetse or domestic livestock is a basic requirement for epidemiological studies as well as for planning and implementing control measures against tsetse and trypanosomiasis. This epidemiological study aimed at assessing the prevalence of trypanosomes in pigs and tsetse flies in the Jomoro district of the western region of Ghana using molecular techniques. Blood was collected from pigs and biconical traps were used to collect tsetse flies. DNA was isolated from 300 pig blood samples and 300 flies for trypanosome detection and identification by PCR. Packed Cell Volume (PCV) of blood samples from 300 pigs was measured using a micro-haematocrit reader. Glossina palpalis palpalis was the only tsetse species found in the area with fly apparent density of 18.4 fly/trap/day. An overall prevalence of trypanosomes in the study area was 4.3% and 0.8% in pigs and tsetse flies respectively. Mixed infection with Trypanosoma (T.) congolense forest and T. vivax was most prevalent 46.2% followed by single infection of T. vivax 15.4%, T. congolense and a mixed infection of T. congolense, T. vivax and T. brucei sl. were the least with 7.7% each. There were no significant differences in trypanosome prevalence among different age groups and between both sexes of the studied pigs (p>0.05). Trypanosome prevalence was lower in healthy looking 1.9% than the sick looking 20%, pigs (P<0.05). Mean PCV of parasitaemic pigs 29.3% was significantly lower than that of aparasitaemic pigs 37.8%. Two out of the five species-specific primers used could not identify any trypanosome species from the total blood samples examined. This could possibly mean that those species are not found in the present study area. These results provide useful background information for further study and justification to extend tsetse control to the Jomoro district.

Molecular identification of diminazene aceturate resistant trypanosomes in tsetse flies from Yoko in the Centre region of Cameroon and its epidemiological implications

African animal trypanosomiases are caused by trypanosomes cyclically or mechanically transmitted by tsetse and other biting flies. Although molecular tools have been developed to identify drug-resistant trypanosomes in mammals, little or no investigation on drug-resistance has been undertaken on trypanosomes harbored by tsetse flies. Moreover, no data on mechanical vectors of African trypanosomes is available in most endemic areas of Cameroon. This study was designed to update our knowledge on the cyclical and mechanical vectors of African trypanosomes, and using molecular tools to identify different trypanosome species as well as diminazene aceturate resistant trypanosomes in tsetse flies trapped at Yoko in the Centre region of Cameroon.For this study, traps were used to catch tsetse and mechanical vectors of African trypanosomes. The flies trapped were counted and identified by sex and species. DNA was extracted from tsetse and species-specific primers were used to identify different trypanosome species. PCR-RFLP was used to detect diminazene aceturate resistant strains of Trypanosoma congolense.In all, 454 flies comprising 168 (37%) Tabanus spp., 71 (15.6%) Stomoxys spp. and 215 (47.4%) tsetse fly (i.e. 107 (49.8%) Glossina fusca congolensis, 71 (33%) Glossina fusca fusca and 37 (17.2%) Glossina palpalis palpalis) were trapped. Trypanosome infections were identified in 12.6% (27/215) of tsetse flies: 13 in G. f. congolensis, 6 in G. p. palpalis and 5 in G. f. fusca. From 24 T. congolense positive samples, PCR-RFLP was successful on 37.5% of the samples. Four samples (16.2%) harbored T. congolense strains that were resistant to diminazene aceturate while the remaining samples had drug-sensitive strains.These results show for the first time the applicability of molecular tools for the identification of drug-resistant trypanosomes in tsetse. They revealed the existence of diminazene aceturate resistant strains of T. congolense in the tsetse-infested area of Yoko in the Centre region of Cameroon. Detection of drug-resistant trypanosomes in tsetse may enable scientists to map with accuracy specific areas where these parasites are transmitted. With such mapping, control strategies against African trypanosomiases could be improved by adapting control measures according to drug resistance distribution.

Parasitological, serological and molecular survey of camel trypanosomiasis in Somalia

BackgroundCamel trypanosomiasis or surra is of great concern in Somalia, since the country possesses the largest one-humped camel (Camelus dromedarius) population in the world. Civil war in Somalia has resulted in the destruction of educational, research, economic and social structures, making the country scores very low for most humanitarian indicators. Previous studies on detection of Trypanosoma species in Somali camels have only been performed during the 1990s using standard trypanosome detection methods (STDM). Considering the lack of state-of-the-art knowledge on camel trypanosomiasis in Somalia, the present study aimed to assess the prevalence of Trypanosoma spp. in three districts of Somalia.MethodsA total of 182 blood samples from C. dromedarius from nomadic and dairy farms were evaluated using STDM, serological (CATT/T. evansi) and molecular (ITS1-PCR) methods.ResultsAll samples were negative for Trypanosoma spp. by STDM. A total of 125/182 (68.7%, 95% CI: 61.4–75.3%) camels were seropositive for T. evansi by CATT/T. evansi. Camels reared in nomadic system were more likely to be seropositive for T. evansi than those under dairy production system (OR: 5.6, 95% CI: 2.1–15.2, P = 0.0001). Five out of 182 (2.7%, 95% CI: 0.9–6.3%) camels tested positive for Trypanosoma sp. by ITS1-PCR. Sequencing of the ITS1 region of the Trypanosoma species detected herein revealed that camels were infected with T. evansi and T. simiae.ConclusionsTrypanosoma evansi is highly prevalent in camels from the Banadir region of Somalia, particularly in nomadic herds. To our knowledge, this is the first study to confirm infections with T. evansi and T. simiae in Somali camels through DNA sequencing. Our data highlight the need for implementation of adequate control measures aiming to reduce the impact on camel production in the country.

Molecular detection of Megatrypanum trypanosomes intabanid flies.

Trypanosomes of the subgenus Megatrypanum have been isolated from many mammalian hosts around the world. They are usually non-pathogenic, although they may confuse the parasitological diagnosis of trypanosomosis. Additionally, Trypanosoma theileri has been associated with disease in cattle. Megatrypanum trypanosomes are considered to be transmitted by different arthropods, including tabanids. However, little is known about the potential vectors of Megatrypanum trypanosomes in different parts of the world. The present study reports on the detection of Megatrypanum trypanosomes in Heamatopota pluvialis, Tabanus bromius, Tabanus maculicornis and Tabanus distinguendus in Poland. It also discusses the possible role of these tabanids in the transmission of Megatrypanum trypanosomes.

Using detergent-enhanced LAMP for African trypanosome detection in human cerebrospinal fluid and implications for disease staging.

ObjectiveWhere human African trypanosomiasis (HAT) patients are seen, failure to microscopically diagnose infections by Trypanosoma brucei gambiense in blood smears and/or cerebrospinal fluid (CSF) in the critical early stages of the disease is the single most important factor in treatment failure, a result of delayed treatment onset or its absence. We hypothesized that the enhanced sensitivity of detergent-enhanced loop-mediated isothermal amplification (LAMP) will allow for point of care (POC) detection of African trypanosomes in the CSF of HAT patients where the probability for detecting a single parasite or parasite DNA molecule in 1 μL of CSF sample is negligible by current methods.MethodologyWe used LAMP targeting the multicopy pan-T. brucei repetitive insertion mobile element (RIME LAMP) and the Trypanosoma brucei gambiense 5.8S rRNA-internal transcribed spacer 2 gene (TBG1 LAMP). We tested 1 μL out of 20 μL sham or Triton X-100 treated CSFs from 73 stage-1 and 77 stage-2 HAT patients from the Central African Republic and 100 CSF negative controls.ResultsUnder sham conditions, parasite DNA was detected by RIME and TBG1 LAMP in 1.4% of the stage-1 and stage-2 gambiense HAT CSF samples tested. After sample incubation with detergent, the number of LAMP parasite positive stage-2 CSF’s increased to 26%, a value which included the 2 of the 4 CSF samples where trypanosomes were identified microscopically. Unexpected was the 41% increase in parasite positive stage-1 CSF’s detected by LAMP. Cohen’s kappa coefficients for RIME versus TBG1 LAMP of 0.92 (95%CI: 0.82–1.00) for stage-1 and 0.90 (95%CI: 0.80–1.00) for stage-2 reflected a high level of agreement between the data sets indicating that the results were not due to amplicon contamination, data confirmed in χ2 tests (p<0.001) and Fisher’s exact probability test (p = 4.7e-13).ConclusionThis study detected genomic trypanosome DNA in the CSF independent of the HAT stage and may be consistent with early CNS entry and other scenarios that identify critical knowledge gaps for future studies. Detergent-enhanced LAMP could be applicable for non-invasive African trypanosome detection in human skin and saliva or as an epidemiologic tool for the determination of human (or animal) African trypanosome prevalence in areas where chronically low parasitemias are present.

Purification of Extracellular Trypanosomes, Including African, from Blood by Anion-Exchangers (Diethylaminoethyl-cellulose Columns).

This method allows the separation of trypanosomes, parasites responsible for animal and human African trypanosomiasis (HAT), from infected blood. This is the best method for diagnosis of first stage HAT and furthermore this parasite purification method permits serological and research investigations. HAT is caused by Tsetse fly transmitted Trypanosoma brucei gambiense and T. b. rhodesiense. Related trypanosomes are the causative agents of animal trypanosomiasis. Trypanosome detection is essential for HAT diagnosis, treatment and follow-up. The technique described here is the most sensitive parasite detection technique, adapted to field conditions for the diagnosis of T. b. gambiense HAT and can be completed within one hour. Blood is layered onto an anion-exchanger column (DEAE cellulose) previously adjusted to pH 8, and elution buffer is added. Highly negatively charged blood cells are adsorbed onto the column whereas the less negatively charged trypanosomes pass through. Collected trypanosomes are pelleted by centrifugation and observed by microscopy. Moreover, parasites are prepared without cellular damage whilst maintaining their infectivity. Purified trypanosomes are required for immunological testing; they are used in the trypanolysis assay, the gold standard in HAT serology. Stained parasites are utilized in the card agglutination test (CATT) for field serology. Antigens from purified trypanosomes, such as variant surface glycoprotein, exoantigens, are also used in various immunoassays. The procedure described here is designed for African trypanosomes; consequently, chromatography conditions have to be adapted to each trypanosome strain, and more generally, to the blood of each species of host mammal. These fascinating pathogens are easily purified and available to use in biochemical, molecular and cell biology studies including co-culture with host cells to investigate host-parasite relationships at the level of membrane receptors, signaling, and gene expression; drug testing in vitro; investigation of gene deletion, mutation, or overexpression on metabolic processes, cytoskeletal biogenesis and parasite survival.

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.

A single test approach for accurate and sensitive detection and taxonomic characterization of Trypanosomes by comprehensive analysis of internal transcribed spacer 1 amplicons.

To improve our knowledge on the epidemiological status of African trypanosomiasis, better tools are required to monitor Trypanosome genotypes circulating in both mammalian hosts and tsetse fly vectors. This is important in determining the diversity of Trypanosomes and understanding how environmental factors and control efforts affect Trypanosome evolution. We present a single test approach for molecular detection of different Trypanosome species and subspecies using newly designed primers to amplify the Internal Transcribed Spacer 1 region of ribosomal RNA genes, coupled to Illumina sequencing of the amplicons. The protocol is based on Illumina’s widely used 16s bacterial metagenomic analysis procedure that makes use of multiplex PCR and dual indexing. Results from analysis of wild tsetse flies collected from Zambia and Zimbabwe show that conventional methods for Trypanosome species detection based on band size comparisons on gels is not always able to accurately distinguish between T. vivax and T. godfreyi. Additionally, this approach shows increased sensitivity in the detection of Trypanosomes at species level with the exception of the Trypanozoon subgenus. We identified subspecies of T. congolense, T. simiae, T. vivax, and T. godfreyi without the need for additional tests. Results show T. congolense Kilifi subspecies is more closely related to T. simiae than to other T. congolense subspecies. This agrees with previous studies using satellite DNA and 18s RNA analysis. While current classification does not list any subspecies for T. godfreyi, we observed two distinct clusters for these species. Interestingly, sequences matching T. congolense Tsavo (now classified as T. simiae Tsavo) clusters distinctly from other T. simiae Tsavo sequences suggesting the Nannomonas group is more divergent than currently thought thus the need for better classification criteria. This method presents a simple but comprehensive way of identification of Trypanosome species and subspecies-specific using one PCR assay for molecular epidemiology of trypanosomes.

Detection of Trypanosoma spp. in Bandicota indica from the Thai-Myanmar border area, Mae Sot District Tak Province, Thailand

Objective: To investigate the prevalence of trypanosome infection and their phylogeny in Bandicota indica rats from the cadmium-contaminated area of Mae Sot and the Myanmar border. Methods: Blood samples were taken from 100 animals, and parasite infection was examined by light microscopy observation and polymerase chain reaction (PCR) studies. Results: Trypanosoma spp. infection was found in 20% of the thin blood smear samples. PCR showed positive 623 bp DNA bands in 21 samples (21%). The sequencing analysis showed that all of the samples (100%) had the Trypanasoma lewisi 18S ribosomal RNA gene. Phylogenetic analysis confirmed that these 16 isolates of Trypanosoma spp. were closely related to Trypanasoma lewisi. Conclusions: Molecular detection using PCR is as effective as conventional light microscopy analysis. This study confirms that trypanosomal infection in rodents is still high; therefore, fleas as their vectors need to be controlled in order to prevent transmission to humans.

Microscopy Underestimates the Prevalence of Trypanosomes’ Infection in Asymptomatic Cattle and Sheep in a Lowland Area within the Kenyan Rift Valley

Animal trypanosomosis continues to impede animal production in sub-Saharan Africa mostly in locations where tsetse flies are endemic. This has ended up devastating many livelihoods where majority of the people depend on livestock farming as source of food and income generation. The true picture on prevalence and identity of trypanosome species is scanty or unknown in most areas where tsetse flies are present. This study sought to investigate the prevalence of trypanosomes’ infection in cattle and sheep using microscopy and polymerase chain reaction (PCR) methods. The use of PCR for detection and identification of trypanosomes has increased sensitivity of diagnostic method compared to conventional microscopy. Ninety asymptomatic free range grazed animals including 72 cattle and 18 sheep randomly sampled from farmers in Kerio Valley of Elgeyo-Marakwet County, Kenya were used in the present study. Blood samples (5 ml) obtained from each of the animals were used for trypanosomes’ detection by microscopy and PCR assay methods. Microscopy results showed that only 2 cattle (2.8%) were positive for trypanosomosis infection. The microscopy results for the sheep showed zero prevalence. On the other hand, PCR results reported 26 trypanosomosis positive cattle (36.1%) and 3 (16.7%) trypanosomosis positive sheep. The PCR method was further used for trypanosomes’ species identification and the results showed that the 26 infected cattle were positive for T. congolense (12) and T. brucei (14) while the three sheep were all positive for T. brucei. The findings of the present study show that microscopy underestimates trypanosomosis detection and therefore cannot be relied upon as a tool for diagnosis. Besides, the method is weak in reporting species differentiation in a case where the morphological differences have only minor details or where the species are very close morphologically. This study recommends routine use of molecular biology-based technique for trypanosomosis detection in the Kenyan Rift Valley lowland areas.

Motility-based label-free detection of parasites in bodily fluids using holographic speckle analysis and deep learning

Parasitic infections constitute a major global public health issue. Existing screening methods that are based on manual microscopic examination often struggle to provide sufficient volumetric throughput and sensitivity to facilitate early diagnosis. Here, we demonstrate a motility-based label-free computational imaging platform to rapidly detect motile parasites in optically dense bodily fluids by utilizing the locomotion of the parasites as a specific biomarker and endogenous contrast mechanism. Based on this principle, a cost-effective and mobile instrument, which rapidly screens ~3.2 mL of fluid sample in three dimensions, was built to automatically detect and count motile microorganisms using their holographic time-lapse speckle patterns. We demonstrate the capabilities of our platform by detecting trypanosomes, which are motile protozoan parasites, with various species that cause deadly diseases affecting millions of people worldwide. Using a holographic speckle analysis algorithm combined with deep learning-based classification, we demonstrate sensitive and label-free detection of trypanosomes within spiked whole blood and artificial cerebrospinal fluid (CSF) samples, achieving a limit of detection of ten trypanosomes per mL of whole blood (~five-fold better than the current state-of-the-art parasitological method) and three trypanosomes per mL of CSF. We further demonstrate that this platform can be applied to detect other motile parasites by imaging Trichomonas vaginalis, the causative agent of trichomoniasis, which affects 275 million people worldwide. With its cost-effective, portable design and rapid screening time, this unique platform has the potential to be applied for sensitive and timely diagnosis of neglected tropical diseases caused by motile parasites and other parasitic infections in resource-limited regions.