Real-time PCR for detection of Trypanosoma brucei in human blood samples

Becker, Sven; Franco, José R.; Stich, August; Abel, Paulo M.; Steverding, Dietmar

doi:10.1016/j.diagmicrobio.2004.07.001

Cited by 97 publications

(72 citation statements)

References 24 publications

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“…On the other hand the use of species specific PCR is recommended as a more specific tool for identification of the Trypanosoma members when comparing to ITS-PCR [27]. Different types of PCR have also been used to detect trypanosomes in tsetse flies [9,11,36,[42][43][44] and in human blood [45].…”

Section: Villagementioning

confidence: 99%

Molecular Identification of Trypanosome Species in Cattle of the Mikumi Human/Livestock/Wildlife Interface Areas, Tanzania

Nhamitambo¹

2017

J Infect Dis Epidemiol

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Trypanosomosis is a major neglected disease of animals and man that causes great negative socio-economic impact in many African countries. It is caused by protozoan parasites of the blood from the genus Trypanosoma. Previous studies have investigated the prevalence and risk factors of trypanosomosis in Tanzania, but none has been done in the human/ livestock/wildlife interface areas of Mikumi National Park. The present study determined prevalence of trypanosomosis in cattle blood sampled in five villages of the Mikumi interface areas. Trypanosome species were identified using the nested ITS-PCR and SRA-LAMP. Acquired biodata and spatial information were used for the analysis of risk factors based on the proximity from the park. Data analysis for categorical variables was performed using Chi-square, Fishers exact test, Odds and Risk ratios. Maps were created using the ArcMap ™ version 10.1 GIS software (ESRI 2012). Overall prevalence was 51.47%. Infection significantly varied among the 5 villages (p = 0.0022). The study identified 7 different species of trypanosomes. Trypanosoma simiae had the highest rate of infection 48.15%, followed by T. theileri 26.67%, T. vivax 16.30%, T. brucei brucei 4.44%, T. congolense forest 2.22%, T. simiae tsavo 1.48% and T. congolense kilifi 0.74%. No zoonotic species were identified. High density vegetation (p = 0.0001) and human activity (livestock market and cattle movement) (p = 0.0001) were identified as strong risk factors for infection. Areas with charco dams (p = 0.0001) and those close to tsetse screens and traps (p = 0.0006) had significantly lower infection prevalence. Identifying risk factors, quantifying the risk and doing spatial analysis is essential to determine the control measures to be used in the affected rural communities.

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Section: Villagementioning

confidence: 99%

Molecular Identification of Trypanosome Species in Cattle of the Mikumi Human/Livestock/Wildlife Interface Areas, Tanzania

Nhamitambo¹

2017

J Infect Dis Epidemiol

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“…Deoxyribonucleic acid (DNA) was extracted and eluted from FTA ® sample discs according to a protocol previously described by Becker et al (2004). Briefly, each FTA ® card was placed on a supporting base (Whatman BioScience Ltd) and from each of the individual samples five discs were punched out using a Harris 3.0-mm Micro Punch (Whatman BioScience Ltd) and discharged into 1.5-mL Eppendorf tubes.…”

Section: Dna Extractionmentioning

confidence: 99%

“…After drying for 45 min at 37 °C or at room temperature overnight, the test sample or control discs were boiled at 90 °C for 30 min in DNA Engine Dyad ® Cycler PTC-0221 (Bio-Rad Laboratories Inc., Hercules, USA) in 100 μL of 5% (w/v) aqueous suspension of Chelex 100 resin (Sodium form, 50-100 Dry mesh; Bio-Rad Laboratories Inc.). Eluted DNA samples were kept at -20 °C for PCR analyses or 4 °C if they were to be analysed within a few days after extraction (Ahmed et al 2011;Becker et al 2004;Muhanguzi et al 2014;Picozzi, Carrington & Welburn 2008).…”

Section: Dna Extractionmentioning

confidence: 99%

Bovine trypanosome species prevalence and farmers’ trypanosomiasis control methods in south-western Uganda

Alingu¹,

Muhanguzi²,

MacLeod³

et al. 2014

J S Afr Vet Assoc

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A cross-sectional study was conducted in Mbarara district, south-western Uganda in May 2012 to determine the burden of African animal trypanosomosis (AAT) in the semi-intensive dairy production systems where pyrethroid acaricides are frequently used in the control of tick-borne diseases (TBDs). A total of 295 cattle blood samples were taken and analysed using a single pair of primers previously designed to amplify internal transcribed spacer (ITS1) of trypanosome ribosomal deoxyribonucleic acid (rDNA). A structured questionnaire was administered to 55 participating livestock farmers to generate data on acaricide and trypanocidal drug usage. The overall prevalence of trypanosome species was 2.4% (95% CI; 1.0% – 4.8%); Trypanosoma vivax was the most predominant species (2.0%; 95% CI; 0.7% – 4.4%). A single mixed infection of T. vivax and Trypanosoma brucei s.l. was detected. All the participating farmers used acaricides for tsetse and TBD control; 89.1% of the acaricides used were pyrethroids. About half of the farmers used trypanocidal drugs, mainly diminazene formulations (Berenil®). Low prevalence of trypanosomes in examined samples is most likely related to the frequent use of pyrethroid insecticides, trypanocides and restricted grazing (paddocking and tethering). These rigorous management practices are geared towards optimising production of exotic dairy breeds kept in this region that are highly susceptible to TBDs and AAT.

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“…Amplicons are normally identified using UV transillumination after being electrophoresed in the presence of ethidium bromide (a carcinogen). Alternative methods for PCR product detection, such as realtime PCR, PCR-enzyme-linked immunosorbent assay, or mass spectrometry, have been developed (3,10,21) but are complex, expensive, and equipment, recourse, and personnel hungry. There is a demand for a simplified method of amplification and product detection which would make these tools usable in the facilities available in regional sleeping sickness diagnostic laboratories.…”

Section: Human African Trypanosomiasis (Hat) Is a Complex Of Protozoamentioning

confidence: 99%

Molecular Dipstick Test for Diagnosis of Sleeping Sickness

et al. 2006

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Human African trypanosomiasis (HAT) or sleeping sickness is a neglected disease that affects poor rural populations across sub-Saharan Africa. Confirmation of diagnosis is based on detection of parasites in either blood or lymph by microscopy. Here we present the development and the first-phase evaluation of a simple and rapid test (HAT-PCR-OC [human African trypanosomiasis-PCR-oligochromatography]) for detection of amplified Trypanosoma brucei DNA. PCR products are visualized on a dipstick through hybridization with a gold-conjugated probe (oligochromatography). Visualization is straightforward and takes only 5 min. Controls both for the PCR and for DNA migration are incorporated into the assay. The lower detection limit of the test is 5 fg of pure T. brucei DNA. One parasite in 180 l of blood is still detectable. Sensitivity and specificity for T. brucei were calculated at 100% when tested on blood samples from 26 confirmed sleeping sickness patients, 18 negative controls (nonendemic region), and 50 negative control blood samples from an endemic region. HAT-PCR-OC is a promising new tool for diagnosis of sleeping sickness in laboratory settings, and the diagnostic format described here may have wider application for other infectious diseases.

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Real-time PCR for detection of Trypanosoma brucei in human blood samples

Cited by 97 publications

References 24 publications

Molecular Identification of Trypanosome Species in Cattle of the Mikumi Human/Livestock/Wildlife Interface Areas, Tanzania

Molecular Identification of Trypanosome Species in Cattle of the Mikumi Human/Livestock/Wildlife Interface Areas, Tanzania

Bovine trypanosome species prevalence and farmers’ trypanosomiasis control methods in south-western Uganda

Molecular Dipstick Test for Diagnosis of Sleeping Sickness

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