Loop-mediated isothermal amplification (LAMP) method for rapid detection of Trypanosoma brucei rhodesiense

Loop-mediated isothermal amplification (LAMP) of DNA is a novel technique that rapidly amplifies target DNA under isothermal conditions. In the present study, a LAMP test was designed from the serum resistance-associated (SRA) gene of Trypanosoma brucei rhodesiense, the cause of the acute form of African sleeping sickness, and used to detect parasite DNA from processed and heat-treated infected blood samples. The SRA gene is specific to T. b. rhodesiense and has been shown to confer resistance to lysis by normal human serum. The assay was performed at 62°C for 1 h, using six primers that recognised eight targets. The template was varying concentrations of trypanosome DNA and supernatant from heat-treated infected blood samples. The resulting amplicons were detected using SYTO-9 fluorescence dye in a real-time thermocycler, visual observation after the addition of SYBR Green I, and gel electrophoresis. DNA amplification was detected within 35 min. The SRA LAMP test had an unequivocal detection limit of one pg of purified DNA (equivalent to 10 trypanosomes/ml) and 0.1 pg (1 trypanosome/ml) using heat-treated buffy coat, while the detection limit for conventional SRA PCR was ∼1,000 trypanosomes/ml. The expected LAMP amplicon was confirmed through restriction enzyme RsaI digestion, identical melt curves, and sequence analysis. The reproducibility of the SRA LAMP assay using water bath and heat-processed template, and the ease in results readout show great potential for the diagnosis of T. b. rhodesiense in endemic regions

Development of a Real-Time PCR for Identification of Brachyspira Species in Human Colonic Biopsies

Background: Brachyspira species are fastidious anaerobic microorganisms, that infect the colon of various animals. The genus contains both important pathogens of livestock as well as commensals. Two species are known to infect humans: B. aalborgi and B. pilosicoli. There is some evidence suggesting that the veterinary pathogenic B. pilosicoli is a potential zoonotic agent, however, since diagnosis in humans is based on histopathology of colon biopsies, species identification is not routinely performed in human materials. Methods: The study population comprised 57 patients with microscopic evidence of Brachyspira infection and 26 patients with no histopathological evidence of Brachyspira infection. Concomitant faecal samples were available from three infected patients. Based on publically available 16S rDNA gene sequences of all Brachyspira species, species-specific primer sets were designed. DNA was extracted and tested by real-time PCR and 16S rDNA was sequenced. Results: Sensitivity and specificity for identification of Brachyspira species in colon biopsies was 100% and 87.7% respectively. Sequencing revealed B. pilosicoli in 15.4% of patients, B. aalborgi in 76.9% and a third species, tentatively named ‘‘Brachyspira hominis’’, in 26.2%. Ten patients (12.3%) had a double and two (3.1%) a triple infection. The presence of Brachyspira pilosicoli was significantly associated with inflammatory changes in the colon-biopsy (p = 0.028). Conclusions: This newly designed PCR allows for sub-differentiation of Brachyspira species in patient material and thus allows large-scaled surveillance studies to elucidate the pathogenicity of human Brachyspira infections. One-third of affected patients appeared to be infected with a novel species

Differential and integrated cross sections for excitation to the 3s, 3p, and 3d states of atomic hydrogen by electron impact below the n=4 threshold

Integrated cross sections for the electron-impact excitation of ground-state hydrogen to the 3s, 3p, and 3d final states have been calculated using propagating exterior complex scaling and convergent close-coupling methods at energies between the n=3 and 4 excitation thresholds. The calculations are in excellent agreement and demonstrate that exterior complex scaling methods can accurately reproduce the resonance structure and magnitude of the excitation cross sections below the ionization threshold. Measurements of the separate 3s, 3p, and 3d differential cross sections were made at 12.24 eV, and are consistent with both calculations within a total experimental uncertainty of about 35%

Carriage of intestinal spirochaetes by humans: epidemiological data from Western Australia.

The purpose of this study was to investigate carriage of intestinal spirochaetes by selected population groups in Western Australia. Stool specimens from 293 rural patients with gastrointestinal disorders, and from 227 healthy migrants from developing countries were cultured. Spirochaete isolates were identified using PCR, and typed by pulsed field gel electrophoresis (PFGE). Brachyspira aalborgi was not isolated. Brachyspira pilosicoli was recovered from 15 rural patients, all Aboriginal. Prevalence was 9.9% in 151 Aboriginals and 0% in 142 non-Aboriginals. Carriage of B. pilosicoli amongst migrants was 10.6% (24/227). Carriage was significantly increased in Aboriginal children aged 2-5 years (P = 0.0027) and in migrant individuals from the Middle East and Africa (P = 0.0034). Carriage was significantly associated with detection of faecal protozoa in both Aboriginals (P = 0.0021) and migrants (P = 0.012). PFGE results indicated that the B. pilosicoli strains were genetically diverse

Colonization and risk factors for Brachyspira aalborgi and Brachyspira pilosicoli in humans and dogs on tea estates in Assam, India.

The prevalence of colonization with the anaerobic intestinal spirochaetes Brachyspira aalborgi and Brachyspira pilosicoli was investigated in humans (n = 316) and dogs (n = 101) living on three tea estates in Assam, India. Colonization was detected using PCR on DNA from faeces. Nineteen (6%) human faecal samples contained B. aalborgi DNA, 80 (25.3%) contained B. pilosicoli DNA, and 10 (3.2%) contained DNA from both species. One canine sample contained DNA from B. pilosicoli. Significant factors for B. aalborgi colonization in logistic regression were: infection of family members with B. aalborgi (P < 0.001), being a resident of Balipara (P = 0.03), and use of water treatment (P = 0.03). For B. pilosicoli, significant factors were: other family members being positive for B. pilosicoli (P < 0.001), water obtained from a well (P = 0.006), water treatment (P = 0.03), and not having visited a doctor in the previous 12 months (P = 0.03)