[show abstract][hide abstract] ABSTRACT: This work reports the design and evaluation of a rapid loop-mediated isothermal amplification test for detecting Mycobacterium ulcerans DNA based on the multicopy insertion sequence IS2404. The test is robust and specific with a detection limit equivalent to 20 copies of the target sequence (0.01 to 0.1 genome). The test has potential for the diagnosis of Buruli ulcer under field conditions.
Journal of clinical microbiology 02/2012; 50(5):1737-41. · 4.16 Impact Factor
[show abstract][hide abstract] ABSTRACT: Human African trypanosomiasis is a debilitating disease prevalent in rural sub-Saharan Africa. Control of this disease almost exclusively relies on chemotherapy that should be driven by accurate diagnosis, given the unacceptable toxicity of the few available drugs. Unfortunately, the available diagnostics are characterised by low sensitivities due to the inherent low parasitaemia in natural infections. Demonstration of the trypanosomes in body fluids, which is a prerequisite before treatment, often follows complex algorithms. In this paper, we review the available diagnostics and explore recent advances towards development of novel point-of-care diagnostic tests.
Journal of Tropical Medicine 01/2012; 2012:340538.
[show abstract][hide abstract] ABSTRACT: Trypanosoma vivax is major cause of animal trypanosomiasis and responsible for enormous economic burden in Africa and South America animal industry. T. vivax infections mostly run low parasitaemia with no apparent clinical symptoms, making diagnosis a challenge. This work reports the design and evaluation of a loop-mediated isothermal amplification (LAMP) test for detecting T. vivax DNA based on the nuclear satellite repeat sequence. The assay is rapid with results obtained within 35 min. The analytical sensitivity is ∼ 1 trypanosome/ml while that of the classical PCR tests ranged from 10 to 10(3)trypanosomes/ml. The T. vivax LAMP test reported here is simple, robust and has future potential in diagnosis of animal trypanosomiasis in the field.
[show abstract][hide abstract] ABSTRACT: The mobile genetic element PCR (MGE-PCR) is a simple and sensitive technique that can be used to detect genetic variability in Trypanosoma brucei ssp. To investigate the reliability of MGE-PCR in genotyping Trypanosoma evansi, stocks that were isolated directly from camels and after their respective passage in mice were analyzed. Construction of a dendrogram using the MGE-PCR banding profiles revealed a clear distinction between T. evansi and T. brucei, as well as discriminating the T. evansi strains (T. evansi with minicircle types B and A). A minor host-dependent clustering shows a genetic difference of <15%. Changes in the banding profiles were observed after serial passage of T. evansi type B in mice, while those of T. evansi type A were identical. It is apparent that significant random insertion mobile element positional variation occurs when T. evansi isolates are introduced into a new host, a factor that needs to be considered when MGE-PCR is used to determine genetic variation in T. evansi isolates that have different host origins.
Parasitology Research 02/2011; 108(6):1583-7. · 2.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Journal of clinical microbiology 02/2011; 49(4):1530-6. · 4.16 Impact Factor
[show abstract][hide abstract] ABSTRACT: Camel Trypanosomiasis (Surra) is mainly caused by Trypanosoma evansi strains that express variable surface glycoprotein (VSG) RoTat 1.2. However, in Kenya a second causative strain that does not express RoTat 1.2 VSG (T. evansi type B) has been identified. The prevalence of T. evansi type B largely remains unknown due to inadequate diagnostic assay. This work reports the development of a sensitive and specific diagnostic assay capable of detecting T. evansi type B based on the strategy of Loop-mediated Isothermal Amplification (LAMP) of DNA. The test is rapid and amplification is achieved within 20-25min at 63 degrees C using a real time PCR machine. Restriction enzyme AluI digestion of the amplicon gave the predicted 83bp and 89bp sized bands and the LAMP product melt curves showed consistent melting temperature (T(m)) of approximately 89 degrees C. The assay analytical sensitivity is approximately 0.1tryps/ml while that of classical PCR test targeting the same gene is approximately 10tryps/ml. There was a 100% agreement in detection of the LAMP amplification product in real time, gel electrophoresis, on addition of SYBR Green I, and when using chromatographic Lateral Flow Dipstick (LFD) format. The use of the LAMP test revealed nine more T. evansi type B DNA samples that were not initially detected through PCR. The robustness and higher sensitivity of the T. evansi type B LAMP assay coupled with the visual detection of the amplification product indicate that the technique has strong potential as a point-of-use test in surra endemic areas.
[show abstract][hide abstract] ABSTRACT: Coyotes from southern Alberta and Saskatchewan, Canada, were examined for the presence of Giardia and Cryptosporidium and cohabiting helminths. Toxascaris was present in over 90% of the 70 animals examined, and Taenia sp. in 6.5-25% of the two groups of animals studied. Giardia (12.5-21.7%) and Cryptosporidium (0-17.4%) were also common and molecular characterisation revealed both zoonotic and host-adapted genotypes of Giardia, whereas the Cryptosporidium proved to be a variant of the canine species C. canis. The seasonal variation observed in the occurrence of Cryptosporidium may be related to stress-induced shedding of the parasite.
[show abstract][hide abstract] ABSTRACT: 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.
International Journal for Parasitology 05/2008; 38(5):589-99. · 3.64 Impact Factor
[show abstract][hide abstract] ABSTRACT: To investigate the population genetic structure of Trypanosoma evansi from domesticated animals, we have analysed 112 stocks from camels, buffaloes, cattle and horses using the tandemly repeated coding sequence (MORF2) and minisatellite markers 292 and cysteine-rich acidic integral membrane protein (CRAM). We recorded a total of six alleles at the MORF2 locus, seven at 292 and 12 at the CRAM loci. Nei's genetic distance showed reduced allelic diversity between buffaloes and cattle stocks (1.2) as compared to the diversity between camels and buffaloes (3.75) and camels and cattle stock (1.69). The mean index of association (IA=0.92) significantly deviated from zero, and the average number of multilocus genotypes (G/N ratio) was 0.21. Twenty-four multilocus genotypes were defined from the combination of alleles at the three loci. The Kenyan sub-populations showed Fst=0.28 and analysis of molecular variance showed significant divergence (22.7%) between the Laikipia, Kulal and Galana regions. The regional and host distribution of multi-locus genotypes significant population differentiation and high Nei's genetic distances suggest existence of genetic sub-structuring within T. evansi stocks while the few multi-locus genotypes and deviation of association index from zero indicate the lack of recombination. In conclusion, this study reveals that some genetic sub-structuring does occur within T. evansi, which has a clonal population structure.
Parasitology Research 11/2007; 101(5):1215-24. · 2.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: Studies on genetic variability in Trypanosoma evansi have been limited by a lack of high-resolution techniques. In this study, we have investigated the use of inter-simple sequence repeats (ISSR) and microsatellites in revealing polymorphism among T. evansi isolates. Twelve ISSR primers and five microsatellite loci were used to generate polymorphic bands and alleles, respectively, to investigate the genetic variability among T. evansi isolates from Africa and Asia. Seven of the twelve ISSR primers showed variability between isolates with a total of 71 fragments of which 49(69%) were polymorphic. Microsatellite analysis revealed a total of 60 alleles. On average the ISSR markers revealed a higher genetic diversity (23%) than microsatellites (21.1%). The two techniques showed a strong agreement of r=0.95 for Dice and r=0.91 for Jaccard indices in estimating the genetic distances between isolates. The distance UPGMA tree revealed two major clusters of T. evansi which correlate with the minicircle classification of subtype A and B. The cophenetic correlation coefficient between Dice and Jaccard based matrices were r=0.79 for microsatellites and r=0.73 for ISSR indicating a strong agreement between dendrograms. The results suggest that both ISSR and microsatellites markers are useful in detecting genetic variability within T. evansi.
[show abstract][hide abstract] ABSTRACT: A distinctive feature of Trypanosoma evansi is the possession of a kinetoplast that contains homogeneous DNA minicircles, but lacks DNA maxicircles. Two major sequence variants of the minicircle have been described and here we have sequenced the type B variant and designed a specific PCR test to distinguish it from type A. Further a test based on maxicircles to distinguish T. brucei brucei from T. evansi was designed and evaluated. Using the designed PCR tests, we detected three type B isolates from camel blood samples collected in northern Kenya, more than 20 years after the first isolation of type B. Comparison of minicircle sequences from all four type B isolates shows >96% identity within the group, and 50-60% identity to type A minicircles. Phylogenetic analysis based on minicircle sequences reveals two clusters, one comprising isolates of type A and one of type B, while random amplification of polymorphic DNA show slight polymorphic bands within type B. Most T. evansi isolates analysed were heterozygous at a repetitive coding locus (MORF2). All type B isolates had one genotype designated 3/5 based on the alleles present. Three camel isolates, which had homogenous type A minicircles, lacked the RoTat 1.2 gene, while another five isolates were T. b. brucei, based on the heterogeneity of their minicircles and presence of maxicircles as demonstrated by PCR amplification of the gene for cytochrome oxidase subunit 1. Our results confirm the existence of T. evansi type B isolates, T. b. brucei and existence of T. evansi type A without RoTat 1.2 gene in Kenyan isolates.
Infection Genetics and Evolution 08/2006; 6(4):292-300. · 2.77 Impact Factor
[show abstract][hide abstract] ABSTRACT: There are 11 different pathogenic trypanosomes in trypanosomiasis endemic regions of Africa. Their detection and characterisation by molecular methods relies on species-specific primers; consequently several PCR tests have to be made on each sample. Primers ITS1 CF and ITS1 BR, previously designed to amplify the internal transcribed spacer (ITS1) of rDNA, have been evaluated for use in a universal diagnostic test for all pathogenic trypanosomes. Blood was collected from 373 cattle and 185 camels. The primers gave constant PCR products with the stocks of each taxon tested. Members of subgenus Trypanozoon (T. brucei brucei, T. evansi, T. b. rhodesiense and T. b. gambiense) gave a constant product of approximately 480 bp; T. congolense, savannah 700 bp, T. congolense kilifi 620 bp and T. congolense forest 710 bp: T. simiae 400 bp, T. simiae tsavo 370 bp, T. godfreyi 300 bp and T. vivax 250 bp. The sensitivity of the test ranged from 10 pg for Trypanozoon, T. congolense clade and T. vivax to 100 pg for T. simiae and T. godfreyi. The primers detected cases of multi-taxa samples, although the sensitivity was reduced with an increase in the combinations. A better detection rate of trypanosome DNA was recorded with buffy coats than from direct blood. With the field samples, the diagnostic sensitivity was close to the sensitivity obtained using single reactions with species-specific primers for Trypanozoon 38/40 (95%) and T. congolense savannah 30/33 (90.9%) but was lower with T. vivax 25/31 (77.4%). The primers offer promise as a routine diagnostic tool through the use of a single PCR; however, further evaluation is recommended.
Parasitology Research 03/2005; 95(3):186-92. · 2.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: Camel trypanosomosis (Surra) causes high morbidity and is an impediment to the camel husbandry in Kenya. The lack of a sensitive diagnostic test has hindered the collection of accurate epidemiological data and institution of control programmes. A cross-sectional study was conducted in three districts of Kenya to estimate the prevalence of Trypanosoma evansi (T. evansi) and to compare four diagnostic tests: polymerase chain reaction (PCR), card agglutination test (CATT/T. evansi), microhaematocrit centrifugation technique (MHCT) and mouse inoculation (MI). A total of 549 camels were randomly sampled. The overall prevalence of Surra was 5.3% using MHCT, 26.6% using PCR and 45.9% using CATT/T.evansi. There was a significant difference (P < 0.001) between PCR and CATT/T.evansi test, MHCT and MI in detection of T. evansi. The prevalence of T. evansi was 39.8% in Samburu, 24.7% in Nanyuki and 14.4% in Isiolo districts using PCR. A male camel was 2.6 times more likely to be infected with T. evansi compared to a female camel (OR = 3.0% CI: 1.6, 4.1), while an adult camel was 2.2 times more likely to be infected compared to non-adults (OR = 2.2; 95% CI: 1.2, 5.0). There was a poor association between the presence of the published clinical signs and seropositivity (kappa = 0.12), PCR (kappa = 0.11) and MHCT (kappa = 0.05). However, there was a higher agreement between farmers' classification of disease with the PCR test (kappa = 0.5, n = 61). The mean PCV varied with age, presence of infection, locality and gender, with the lowest mean PCV being recorded in MHCT-positive animals (20.97 +/- 0.5) and from infected calves (19.5 +/- 1.2). This study shows that PCR was more sensitive in detecting T. evansi than other tests used. Further, the prevalence of T. evansi in the camel herds sampled is higher than that previously reported in Kenya, and that the judgment by camel keepers may be a reliable "pen-side" diagnostic test for Surra. Considering the low sensitivity of parasitological techniques in detection of chronic T. evansi infection and high cost of PCR, development of a sensitive pen side diagnostic test, with a low cost is still a priority.
[show abstract][hide abstract] ABSTRACT: The human serum resistance associated (SRA) gene has been found exclusively in Trypanosoma brucei rhodesiense, allowing the unequivocal detection of this pathogen in reservoir hosts and the tsetse vector without recourse to laborious strain characterisation procedures. We investigated the presence of the SRA gene in 264 T. brucei ssp. isolates from humans, domestic animals and Glossina pallidipes from foci of human trypanosomiasis in Kenya and Uganda. The SRA gene was present in all isolates that were resistant to human serum, and absent from all serum sensitive isolates tested. Further, the gene was present in all isolates that had previously been shown to be identical to human infective trypanosomes by isoenzyme characterisation. The SRA gene was detected in isolates from cattle, sheep, pigs, dog, reedbuck, hyena and G. pallidipes from sleeping sickness foci, but was not found in Trypanosoma evansi or in Trypanosoma brucei gambiense isolates. The present study indicates that the SRA gene may be invaluable in detecting and differentiating T. brucei rhodesiense from other T. brucei ssp. in reservoir hosts and tsetse.
[show abstract][hide abstract] ABSTRACT: The polymerase chain reaction (PCR) was used to identify trypanosomes in Glossina pallidipes and G. longipennis caught in Kenya. Of 3826 flies dissected, 188 (4.9%) were parasitologically positive overall. The infection rate in G. pallidipes was 5.7% (187 of 3301 flies), but only one of 525 G. longipennis was infected (infection rate 0.2%). There was a higher infection rate in female G. pallidipes flies than male flies (chi(2) = 18.5, P < 0.001) and odds ratio = 2.5 (95% 1.6, 3.7). The infected flies were analysed by PCR using 10 sets of primers specific for species and subgroups within the subgenera Nannomonas, Trypanozoon and Duttonella. Of 188 parasitologically positive samples, PCR identified 137 (72.9%), leaving 51 (27.1%) non-identified. We recorded infection rates of 47.2% for Trypanosoma congolense savannah, forest and kilifi subgroups, 20.9% for T. simiae/T. simiae tsavo/T. godfreyi, 14.9% for T. brucei ssp. and 13.8% for T. vivax. Thirty-nine (26.7%) flies had mixed infections, with a minor association between T. congolense savannah/T. simiae tsavo/T. godfreyi (chi(2) = 6.93, d.f. = 1, P < 0.05). The relative proportion of each trypanosome species or subgroup varied between fly belts with T. congolense (all subgroups) being the most abundant and T. godfreyi the least. Statistical analysis showed that dissection method and PCR test classified infections independently (chi(2) = 10.5, d.f. = 1, P < 0.05 and kappa = 0.38). This study shows that pathogenic trypanosomes are widespread in all sampled testes fly belts with G. pallidipes as the main vector. Further, PCR test is more reliable in detecting and identifying trypanosomes than dissection method.
Infection Genetics and Evolution 03/2004; 4(1):29-35. · 2.77 Impact Factor
[show abstract][hide abstract] ABSTRACT: The accurate identification of trypanosome species and subspecies remains a challenging task in the epidemiology of human and animal trypanosomiasis in tropical Africa. Currently, there are specific PCR tests to identify about 10 different species, subspecies or subgroups of African tsetse-transmitted trypanosomes. These PCR tests have been used here to identify trypanosomes in four species of tsetse (Glossina brevipalpis, G. pallidipes, G. swynnertoni, G. morsitans morsitans) from two areas of Tanzania. PCR using species-specific primers was performed on 1041 dissection-positive proboscides, giving an overall positive identification in 254 (24%). Of these, 61 proboscides (24%) contained two or more trypanosomes. The trypanosome with the greatest overall prevalence at both field sites was Trypanosoma simiae Tsavo, which was identified in a total of 118 infected tsetse proboscides (46%). At Pangani, T. godfreyi was found in G. pallidipes but not in G. brevipalpis, suggesting that these flies might have different susceptibility to this trypanosome or might have fed on a different range of hosts. A high proportion (about 75%) of trypanosome infections remained unidentified. To investigate the identity of these unidentified samples, we used primers complementary to the conserved regions of trypanosomal small subunit ribosomal RNA (ssu rRNA) genes to amplify variable segments of the gene. Amplified DNA fragments were cloned, sequenced and compared with ssu rRNA genes on database of known trypanosome species. In this way, we have tentatively identified two new trypanosomes: a trypanosome related to Trypanosoma vivax and a trypanosome related to T. godfreyi. The T. godfreyi-related trypanosome occurred frequently in the Tanzanian field samples and appears to be widespread. Molecular identification of these two new trypanosomes should now facilitate their isolation and full biological characterisation.
Infection Genetics and Evolution 12/2003; 3(4):271-9. · 2.77 Impact Factor
[show abstract][hide abstract] ABSTRACT: Camel trypanosomosis (Surra) is one of the most important diseases affecting camel calves. It presents itself as an acute form and is usually fatal if treatment is not carried out. A study was initiated at Mogwooni ranch in Laikipia district of Kenya to survey the prevalence of trypanosomosis in camel calves of mixed breeds, and to evaluate the microhaematocrit centrifugation technique (MHCT), monoclonal antibody based card latex agglutination test (Suratex®), wet smear and mouse inoculation (MI) in the diagnosis of the disease in camels. The tests were assessed for a period of 16 months. The mean Trypanosoma evansi prevalence ranged from 4.5% as determined by the wet smear, 11.1% by MHCT, 14.6% by MI, to 28.3% by Suratex®. Young calf death rate due to trypanosomosis was 12.3% while overall mortality rate was 15.0%. The cost of veterinary care (anti-helminthics, acaricides and trypanocides) was on average US$ 4.6 per calf per year. It is thus recommended that diagnosis accompanied by proper treatment be carried out routinely for the survival of camel calves in trypanosomosis endemic areas.