Detection of Group 1 Trypanosoma brucei gambiense by loop-mediated isothermal amplification.

Trypanosoma brucei gambiense group 1 is the major causative agent of the Gambian human African trypanosomiasis (HAT). Accurate diagnosis of Gambian HAT is still challenged by lack of precise diagnostic methods, low and fluctuating parasitemia, and generally poor services in the areas of endemicity. In this study, we designed a rapid loop-mediated isothermal amplification (LAMP) test for T. b. gambiense based on the 3' end of the T. b. gambiense-specific glycoprotein (TgsGP) gene. The test is specific and amplifies DNA from T. b. gambiense isolates and clinical samples at 62°C within 40 min using a normal water bath. The analytical sensitivity of the TgsGP LAMP was equivalent to 10 trypanosomes/ml using purified DNA and ∼1 trypanosome/ml using supernatant prepared from boiled blood, while those of classical PCR tests ranged from 10 to 10(3) trypanosomes/ml. There was 100% agreement in the detection of the LAMP product by real-time gel electrophoresis and the DNA-intercalating dye SYBR green I. The LAMP amplicons were unequivocally confirmed through sequencing and analysis of melting curves. The assay was able to amplify parasite DNA from native cerebrospinal fluid (CSF) and double-centrifuged supernatant prepared from boiled buffy coat and bone marrow aspirate. The robustness, superior sensitivity, and ability to inspect results visually through color change indicate the potential of TgsGP LAMP as a future point-of-care test.

African trypanosomiasis: sensitive and rapid detection of the sub-genus Trypanozoon by loop-mediated isothermal amplification (LAMP) of parasite DNA.

Control of human African trypanosomiasis (HAT) is dependent on accurate diagnosis and treatment of infected patients. However, sensitivities of tests in routine use are unsatisfactory, due to the characteristically low parasitaemias in naturally infected individuals. We have identified a conserved sequence in the repetitive insertion mobile element (RIME) of the sub-genus Trypanozoon and used it to design primers for a highly specific loop-mediated isothermal amplification (LAMP) test. The test was used to analyse Trypanozoon isolates and clinical samples from HAT patients. The RIME LAMP assay was performed at 62 degrees C using real-time PCR and a water bath. DNA amplification was detectable within 25min. All positive samples detected by gel electrophoresis or in real-time using SYTO-9 fluorescence dye could also be detected visually by addition of SYBR Green I to the product. The amplicon was unequivocally confirmed through restriction enzyme NdeI digestion, analysis of melt curves and sequencing. The analytical sensitivity of the RIME LAMP assay was equivalent to 0.001 trypanosomes/ml while that of classical PCR tests ranged from 0.1 to 1000 trypanosomes/ml. LAMP detected all 75 Trypanozoon isolates while TBR1 and two primers (specific for sub-genus Trypanozoon) showed a sensitivity of 86.9%. The SRA gene PCR detected 21 out of 40 Trypanosoma brucei rhodesiense isolates while Trypanosoma gambiense-specific glycoprotein primers (TgsGP) detected 11 out of 13 T. b. gambiense isolates. Using clinical samples, the LAMP test detected parasite DNA in 18 out of 20 samples which included using supernatant prepared from boiled blood, CSF and direct native serum. The sensitivity and reproducibility of the LAMP assay coupled with the ability to detect the results visually without the need for sophisticated equipment indicate that the technique has strong potential for detection of HAT in clinical settings. Since the LAMP test shows a high tolerance to different biological substances, determination of the appropriate protocols for processing the template to make it a user-friendly technique, prior to large scale evaluation, is needed.

Strengthening human genetics research in Africa: report of the 9th meeting of the African Society of Human Genetics in Dakar in May 2016.

The 9th meeting of the African Society of Human Genetics, in partnership with the Senegalese Cancer Research and Study Group and the Human Heredity and Health in Africa (H3Africa) Consortium, was held in Dakar, Senegal. The theme was Strengthening Human Genetics Research in Africa. The 210 delegates came from 21 African countries and from France, Switzerland, UK, UAE, Canada and the USA. The goal was to highlight genetic and genomic science across the African continent with the ultimate goal of improving the health of Africans and those across the globe, and to promote the careers of young African scientists in the field. A session on the sustainability of genomic research in Africa brought to light innovative and practical approaches to supporting research in resource-limited settings and the importance of promoting genetics in academic, research funding, governmental and private sectors. This meeting led to the formation of the Senegalese Society for Human Genetics.

Le 9ème congrès de la Société Africaine de Génétique Humaine, en partenariat avec le Groupe d'Etude et de Recherche sur le Cancer (GERC) et le Consortium H3Africa, s'est tenu à Dakar, au Sénégal. Le thème était «Renforcer la recherche en Génétique Humaine en Afrique¼. Les 210 participants sont venus de 21 pays africains et de six non africains. L'objectif était de valoriser la génétique et la génomique à travers l'Afrique avec comme but ultime d'améliorer la santé des populations, et de promouvoir les carrières des jeunes chercheurs Africains. Une session sur la pérennité de la recherche génomique a révélé des approches innovantes et pratiques supportant la recherche dans des contextes de ressources limitées et l'importance de promouvoir la formation universitaire en génétique, le financement de la recherche par les gouvernements et le privé. Ce congrès conduisit à la création de la Société Sénégalaise de Génétique Humaine.

Role of cytokines in Trypanosoma brucei-induced anaemia: A review of the literature.

BACKGROUND: Anaemia is an important complication of trypanosomiasis. The mechanisms through which trypanosomal infection leads to anaemia are poorly defined. A number of studies have implicated inflammatory cytokines, but these data are limited and inconsistent. In this article, we reviewed the published literature on cytokines associated with Trypanosoma brucei infections and their role in the immunopathology leading to anaemia. METHODOLOGY: Articles were searched in PubMed through screening of titles and abstracts with no limitation on date of publishing and study design. Articles in English were searched using keywords "African trypanosomiasis", "sleeping sickness", "Trypanosoma brucei", in all possible combinations with "anaemia" and/or "cytokines". RESULTS: Twelve articles examining cytokines and their role in trypanosomeinduced anaemia were identified out of 1095 originally retrieved from PubMed. None of the articles identified were from human-based studies. A total of eight cytokines were implicated, with four cytokines (IFN-γ, IL-10, TNF-α, IL-12) showing an association with anaemia. These articles reported that mice lacking TNF-α were able to control anaemia, and that IFN-γ was linked to severe anaemia given its capacity to suppress erythropoiesis, while IL-10 was shown to regulate IFN-γ and TNF-α, providing a balance that was associated with severity of anaemia. IFN-γ and TNF-α have also been reported to work in concert with other factors such as nitric oxide and iron in order to induce anaemia. CONCLUSION: IFN-γ, IL-10, and TNF-α were the three major cytokines identified to be heavily involved in anaemia caused by Trypanosoma brucei infection. The anti-inflammatory cytokine, IL-10, was shown to counter the effects of proinflammatory cytokines in order to balance the severity of anaemia. The mechanism of anaemia is multifactorial and therefore requires further, more elaborate research. Data from human subjects would also shed more light.

Drug resistance in Trypanosoma brucei spp., the causative agents of sleeping sickness in man and nagana in cattle.

Drug resistance in pathogenic trypanosomes threatens successful control of fatal sleeping sickness in man and hinders economic livestock production in sub-Saharan Africa. We report on the occurrence and development of drug resistance, and discuss the genetic basis of such resistance in Trypanosoma brucei. Understanding these mechanisms at the molecular level will enable improved management of existing drugs and provide valuable clues to the development of new trypanocides.

Genetic variants of the TbAT1 adenosine transporter from African trypanosomes in relapse infections following melarsoprol therapy.

We have analyzed the TbAT1 gene, which codes for the P2 adenosine transporter, from Trypanosoma brucei field isolates to investigate a possible link between the presence of mutations in this gene and melarsoprol treatment failure. Of 65 T. b. gambiense isolates analyzed from a focus in north-western Uganda with high treatment failure rates following melarsoprol therapy, 38 had a mutated TbAT1. Unexpectedly, all individual isolates contained the same set of nine mutations in their TbAT1 genes. Of these, five point mutations resulted in amino acid substitutions, one resulted in the deletion of an entire codon, and three were silent point mutations. Eight of these mutations had previously been reported in a laboratory-derived Cymelarsan-resistant T. b. brucei clone. Identical sets of mutations were also found in a drug-resistant T.b.rhodesiense isolate from south-eastern Uganda and in a T.b.gambiense isolate from a relapsing patient from northern Angola. A deletion of the TbAT1 gene was found in a single T. b. gambiense isolate from a relapsing patient from northern Angola. The data presented demonstrate the surprising finding that trypanosomes from individual relapse patients of one area, as well as from geographically distant localities, contain an identical set of point mutations in the transporter gene TbAT1. They further demonstrate that many isolates from relapse patients contained the wild-type TbAT1 genes, suggesting that melarsoprol refractoriness is not solely due to a mutational inactivation of TbAT1.

Response of a T. b. rhodesiense stock with reduced drug susceptibility in vitro to treatment in mice and cattle.

In vivo drug susceptibility tests involving treatment of infected mice and cattle were performed on two trypanosome stocks, a T. brucei brucei and a T.b. rhodesiense, isolated in South Eastern Uganda. The T. b. rhodesiense stock had expressed reduced susceptibility to diminazene aceturate and isometamidium chloride in vitro, while the other, a T. b. brucei stock was susceptible. Diminazene aceturate at 14 mg/kg was not sufficient to cure all T. b. rhodesiense infected mice. Similarly, in the case of isometamidium chloride, 33% of infected mice treated with 2.0 mg/kg drug were not cured. In contrast, mice infected with the susceptible T. b. brucei and treated similarly with either drug were all cured. However, when cattle infected with the T. b. rhodesiense stock, or the susceptible T. b. brucei stock, or a 1:1 mixture of the two were treated with 7 mg/kg diminazene aceturate, they were all cured. Use of diagnostic PCR employing T. brucei specific primers confirmed that although the cattle had acquired infection pre-treatment, no trypanosome DNA amplification signal was demonstrated in the samples collected 60 days post-treatment. The reduced susceptibility of this T. b. rhodesiense, which could be demonstrated in mice as well as in culture, may indicate the existing potential for evolution of resistance in South Eastern Uganda.

Trypanosomosis agglutination card test for Trypanosoma brucei rhodesiense sleeping sickness.

OBJECTIVE: To develop a simple field test for diagnosis of Trypanosoma brucei rhodesiense in man. DESIGN: Trypanosomosis Agglutination Card Test (TACT) was developed for the diagnosis of sleeping sickness due to Trypanosoma brucei rhodesiense infection, based on stabilised procyclic forms derived from Utat 4.1. Procyclics were fixed in buffered formalin at 4 degrees for 24 hours and further stabilised in acid/alcohol mixture for 30 minutes. The fixed antigen was stained with Coomassie blue and suspended in 0.1 M PBS/sodium azide buffer pH 7.2 at a concentration of 1 x 10(8) trypanosomes/ml and kept at room temperature. This antigen was used to screen 100 sera from rabbits infected with T. b. rhodesiense, eight from normal rabbits, and 220 only sera 60 of which were from sleeping sickness patients, 50 from normal persons and 110 from other parasitic infections. SETTING: Laboratory testing of the antigen types against the rabbit and human sera infected with cloned variable antigen types of T. b. rhodesiense, was routinely carried on test cards under room temperature. SUBJECTS/PARTICIPANTS: Serum samples from normal and infected rabbits and human subjects. RESULTS: All sera from infected rabbits and 59 from sleeping sickness patients reacted strongly with the antigen showing agglutination reaction which ranged from 1:4 to 1:1024 serum dilution. There was minimal cross reaction with other parasitic infections as follows: one out of 20 malaria patients none of the 20 hookworm patients, one out of 30 for schistosomiasis patients, none of the 10 amoebiasis patients and one out of 20 for filariasis patients. Agglutination titres from all these non-sleeping sickness patients were below 1:16. Based on rabbit positive and negative sera, TACT gave a sensitivity and specificity of 100% and 80% while for human sera a sensitivity of 98.3% and specificity of 96% were observed. CONCLUSION: These preliminary results show that TACT could be a promising screening field test for T. b. rhodesiense sleeping sickness.

Innate lack of susceptibility of Ugandan Trypanosoma brucei rhodesiense to DL-alpha-difluoromethylornithine (DFMO).

Trypanosoma brucei rhodesiense isolates from South East Uganda were characterized for susceptibility to the drugs suramin, nifurtimox, melarsoprol and DL-alpha-difluoromethylornithine (DFMO). Two different assays were used to determine the drug susceptibility of the field isolates: the [3H]hypoxanthine incorporation assay (24 hours) and the long term viability assay (10 days). All trypanosome stocks were susceptible to suramin and nifurtimox. Differences in the susceptibility to melarsoprol were observed in the [3H]hypoxanthine incorporation assay, but could not be confirmed in the long term viability assay. All T. b. rhodesiense stocks were found in vitro to have innate tolerance to DFMO, under conditions where T. b. gambiense stocks from West Africa were susceptible to the drug. Ugandan T. b. rhodesiense stocks did respond to 25-100 micrograms/ml after 10 days of drug exposure, but the DFMO level reached in cerebrospinal fluid during treatment is only 16.3 +/- 7.8 micrograms/ml. Therefore, DFMO is not an appropriate alternative or backup drug for treatment of Rhodesian sleeping sickness in Uganda.

Detection of trypanosomes in suspected sleeping sickness patients in Uganda using the polymerase chain reaction.

Diagnosis of sleeping sickness (trypanosomiasis) is difficult because of the fluctuating levels of parasitaemia encountered in patients. In the present study we found that the polymerase chain reaction (PCR) demonstrated trypanosome infection in 20 out of 35 (57.1%) blood samples and in 21 out of 34 (61.7%) cerebrospinal fluid (CSF) samples collected from an area endemic for sleeping sickness in north-west Uganda. A total of 14 blood samples and 13 CSF samples that were positive for trypanosomes by double centrifugation were also positive by PCR, demonstrating good concordance between the two methods. However, 6 (28.6%) of the 21 blood samples that were parasitologically negative were positive by PCR, while 8 (38.0%) out of 21 CSF samples that were negative by double centrifugation were positive by PCR. These 14 negative samples could therefore be from sleeping sickness cases even though a positive PCR test is not evidence for the presence of trypanosomes. Furthermore, of these 8 CSF samples, 4 had been designated as early cases, based on the absence of trypanosomes and on a count of < or = 5 white blood cells (WBC) per microliter. This suggests that some late-stage cases could potentially be missed according to the present criteria, and it is therefore important to perform clinical trials to determine whether these cases could be treated successfully with the first-stage drug alone. The remaining four CSF samples had been classified as late-stage cases, based on a count of > 6 WBC per microliter, even though trypanosomes could not be detected in these samples by either double centrifugation or PCR. A cut-off point of 5 WBC per microliter, which is used as a rule of thumb to stage sleeping sickness patients, seems to leave some late-stage cases undetected since trypanosomes were detected in four CSF samples from suspected cases with < 5 WBC per microliter.

PIP: This study evaluates the effectiveness of polymerase chain reaction (PCR) in detecting trypanosomes in the blood and cerebrospinal fluid (CSF) of suspected sleeping sickness patients in Uganda. A total of 35 blood samples and 34 CSF samples were analyzed. Trypanosomes were detected in 20 of 35 (57.1%) blood samples, and in 21 of 34 (61.7%) CSF samples by PCR. However, 6 (28.6%) of the 20 blood samples that were parasitologically negative were positive by PCR, while 8 (38.0%) out of 21 CSF samples that were negative by double centrifugation were positive by PCR. These 14 negative samples could therefore be from sleeping sickness cases even though a positive PCR test is not an evidence for the presence of trypanosomes. Furthermore, of these 8 CSF samples, 4 had been designated as early cases, based on the absence of trypanosomes and on a count of 5 or fewer white blood cells (WBCs) per microliter. The remaining 4 CSF samples had been classified as late-stage cases, based on a count of 6 WBCs per microliter, even though trypanosomes could not be detected in these samples by either double centrifugation or PCR. Usually, 5 WBCs per microliter is considered to be the cut-off point in the staging and treatment of sleeping sickness patients. In conclusion, it is imperative to carry out a detailed clinical study on the use of PCR for trypanosomiasis diagnosis and staging of patients in order to demonstrate the relation between PCR and the outcome of treatment.

Melarsoprol refractory T. b. gambiense from Omugo, north-western Uganda.

Culture adapted T. b. gambiense isolated from Northwest Uganda were exposed to 0.001-0.14 microg/ml melarsoprol or 1.56-100 microg/ml DL-alpha-difluoromethylornithine (DFMO). Minimum inhibitory concentrations (MICs) of each drug were scored for each isolate after a period of 10 days drug exposure. The results indicate that T. b. gambiense isolates from Northwest Uganda had elevated MIC values for melarsoprol ranging from 0.009 to 0.072 microg/ml as compared with T. b. gambiense isolates from Cote d'Ivoire with MIC values ranging from 0.001 to 0.018 microg/ml or with T. b. rhodesiense from Southeast Uganda with MIC values from 0.001 to 0.009 microg/ml. All MIC values obtained fell below expected peak melarsoprol concentrations in serum of treated patients. However, it may not be possible to maintain constant drug concentrations in serum of patients as was the case in our in vitro experiments. Importantly, the MIC of 0.072 microg/ml exhibited by one of the isolates from Northwest Uganda was above levels attainable in CSF indicating that this isolate would probably not be eliminated from CSF of treated patients. PCR amplification of the gene encoding the P2-like adenosine transporter followed by restriction digestion with Sfa NI enzyme revealed presence of fragments previously observed in a trypanosome clone with laboratory-induced arsenic resistance. From our findings it appears that reduced drug susceptibility may be one factor for the frequent relapses of sleeping sickness after melarsoprol treatment occurring in Northwest Uganda.

Susceptibility of Ugandan Trypanosoma brucei rhodesiense isolated from man and animal reservoirs to diminazene, isometamidium and melarsoprol.

Thirty-six Trypanosoma brucei spp. stocks isolated from man and domestic animals in south-east Uganda were studied for susceptibility to diminazene, isometamidium and melarsoprol in vitro. All stocks were susceptible to melarsoprol. One T.b. rhodesiense stock isolated from a sleeping sickness patient showed reduced susceptibility to the veterinary drugs diminazene and isometamidium. More than 100 ng/ml diminazene or 0.78 ng/ml isometamidium were required to eliminate that stock during 10 days drug exposure. In contrast, the remaining stocks were eliminated by 0.8-6.3 ng/ml diminazene and 0.01-0.20 ng/ml isometamidium. Clones derived from the resistant T. b. rhodesiense stock showed reduced susceptibility to isometamidium and diminazene comparable to the parental population. Control of sleeping sickness by treatment of the animal reservoir could, therefore, face serious problems since drug-resistant stocks as reported here would most likely not be eliminated by recommended doses of diminazene and isometamidium.

Evidence for the occurrence of Trypanosoma brucei rhodesiense sleeping sickness outside the traditional focus in south-eastern Uganda.

The occurrence of Trypanosoma brucei rhodesiense west of the River Nile, in Masindi district in the mid-western part of Uganda, is confirmed. Masindi borders the traditional belt of T. b. gambiense infection in the north-west, Gulu in the north and the Democratic Republic of Congo in the west. Of the 702 persons tested for sleeping sickness in Masindi, 113 (16%) were positive by the card agglutination test for trypanosomiasis (CATT). Trypanosomes were observed in samples of cerebrospinal fluid (CSF) from two (0.3%) of the subjects: a 7-year-old girl, who had been ill for 2 weeks and yet was in good general condition, with three white blood cells (WBC)/microliter CSF; and a 47-year-old woman who had been ill for 8 months, looked sickly, had seven WBC/microliter CSF, but was still able to dig in her gardens. Rats and mice inoculated with blood from the two parasitologically confirmed cases became parasitaemic on day 3 post-inoculation, indicating that the parasites were T. b. rhodesiense. Isoenzyme analysis revealed that the parasites isolated from one of these confirmed cases belonged to a zymodeme (449) which has not been previously observed among isolates from south-eastern or north-western Uganda. Although the isolate shared PGM2 and ICD3 patterns with T. b. gambiense and T. b. rhodesiense, respectively, it did not have the SOD3:5 pattern characteristic of T. b. gambiense. The spread of T. b. rhodesiense beyond its traditional focus and the development of areas where this subspecies and T. b. gambiense are co-endemic will complicate the control of sleeping sickness in Uganda; although the CATT is very useful for the mass screening of populations for T. b. gambiense area, it is not applicable in the detection of T. b. rhodesiense.

Parasitological detection of Trypanosoma brucei gambiense in serologically negative sleeping-sickness suspects from north-western Uganda.

Forty-five parasitologically confirmed cases of sleeping sickness were diagnosed in north-western Uganda using a combination of two or three techniques. Forty of the cases were positive by the card agglutination test for trypanosomiasis (CATT), four were negative and one was not screened by the CATT. Trypanosomes isolated from the four CATT-negative but parasitologically positive cases were propagated for detailed biochemical genetic analysis. The aim was to demonstrate whether these four stocks lacked the LiTat 1.3 gene which encodes the antigen on which the CATT is based. All the DNA extracts isolated from these CATT-negative stocks and from six CATT-positive stocks of Trypanosoma brucei gambiense were targeted for amplification by the three variable-surface-glycoprotein genes thought to be ubiquitous in T. b. gambiense. The LiTat 1.3 gene was shown to be present in all 10 stocks. Trypanosome carriers may be CATT-negative because the CATT is not sensitive enough, because their parasites lack the LiTat 1.3 gene, or because their parasites have this gene but do not express it. The four sleeping-sickness cases who gave negative CATT results in the present study have very important implications in the diagnosis of T. b. gambiense infections using the CATT. Following treatment of the CATT-positive cases, the CATT-negative carriers of the trypanosomes remain as human reservoir hosts for continuous infection of the population. Because CATT-negative individuals are rarely examined further, the general prevalence of parasitologically positive but CATT-negative cases is unclear. This study demonstrates the value of co-ordinated use of serological and parasitological techniques in the diagnosis of Gambian sleeping sickness.