Andrew S. J. Mikosza

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.

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

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°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.

The prevalence of Serpulina pilosicoli in humans and domestic animals in the Eastern Highlands of Papua New Guinea

In a survey of five villages in the Eastern Highlands of Papua New Guinea, Serpulina pilosicoli was isolated from rectal swabs from 113 of 496 individuals (22.8%). Colonization rates ranged from 22.6-30.1% in four of the villages but was only 8.6% in the other village. In comparison colonization was demonstrated in only 5 of 54 indigenous people (9.3%) and none of 76 non-indigenous people living in an urban environment in the same region. Colonization did not relate to reported occurrence of diarrhoea, age, sex, or length of time resident in a village. A second set of 94 faecal specimens was collected from 1 village 6 weeks after the first set. S. pilosicoli was isolated from 27 of 29 individuals (93.1%) who were positive on the first sampling and from 7 of 65 individuals (10.8%) who previously were negative. In this case, isolates were significantly more common in watery stools than in normal stools. The annual incidence of infection in the village was calculated as 93.6%, with an average duration of infection of 117 days. S. pilosicoli could not be isolated from any village pig (n = 126) despite its confirmed presence in 17 of 50 commercial pigs (34.0%) sampled at a local piggery. Four of 76 village dogs (5.3%) and 1 of 2 village ducks were colonized with S. pilosicoli, suggesting the possibility of cross transmission between humans and animals.

Population genetic analysis of Serpulina pilosicoli and its molecular epidemiology in villages in the Eastern Highlands of Papua New Guinea

The population genetics of Serpulina pilosicoli and its molecular epidemiology in villages in the Eastern Highlands province of Papua New Guinea were investigated. Multilocus enzyme electrophoresis (MLEE) was used to analyse 164 isolates from humans and animals. These were divided into 33 electrophoretic types (ETs), four of which contained 65% of the isolates. The mean genetic diversity (n = number of ETs) for 145 human isolates was 0.18, and the mean number of alleles at five polymorphic loci was 2.6. The species appeared to be recombinant, as there was a lack of linkage disequilibrium, and 25% of all the possible combinations of alleles was present in the population. PFGE analysis using the enzymes M/ul and Sa/l divided 157 of the isolates into 99 PFGE types, demonstrating the existence of considerable strain diversity in a geographically restricted area. The two techniques were in excellent agreement; however, PFGE was more discriminatory for strain typing than was MLEE. Nine out of 19 (47.4%) culture-positive individuals were colonized by the same PFGE type of S. pilosicoli when retested after 6 weeks. For three individuals, the PFGE profiles of the second isolate differed from the first in only one or two DNA bands, while the other seven individuals were colonized with distinct PFGE types on each occasion. In two cases, strains with the same PFGE pattern were isolated from humans and dogs, suggesting that cross-species transmission of S. pilosicoli may occur naturally and that the infection can be zoonotic.

Comparative Prevalences of Brachyspira aalborgi and Brachyspira (Serpulina) pilosicoli as Etiologic Agents of Histologically Identified Intestinal Spirochetosis in Australia

ABSTRACT DNA from gastrointestinal biopsy specimens from 28 Australian patients with histologic evidence of intestinal spirochetosis (IS) was subjected to PCRs to amplify segments of the 16S rRNA and NADH oxidase genes of Brachyspira aalborgi and Brachyspira (Serpulina) pilosicoli. B. aalborgi was identified in specimens from 24 (85.7%) patients and B. pilosicoli in those from 4 (14.3%) patients (2 of whom were also positive forB. aalborgi). For two patients, no product was amplified. This study demonstrates that B. aalborgi is much more commonly involved in histologically identified IS in Australian patients than is B. pilosicoli. This is the first report of amplification of B. pilosicoli DNA from humans with IS.

Human intestinal spirochetosis: Brachyspira aalborgi and/or Brachyspira pilosicoli?

Abstract Intestinal spirochetosis in humans (HIS) is a condition defined by the presence of a layer of spirochetes attached by one cell end to the colorectal epithelium. The pathologic significance of HIS is uncertain, but it has been linked to chronic diarrhea and other abdominal complaints. Two anaerobic intestinal spirochete species have been associated with HIS, namely Brachyspira pilosicoli and Brachyspira aalborgi. Brachyspira pilosicoli, which colonizes many animal species, is common (~30%) in the feces of people from developing countries, including Australian Aborigines, and in HIV+ patients and male homosexuals in Western societies. It is also commonly seen attached to the rectal mucosa of homosexual males. In other groups in Western societies both the presence of B. pilosicoli in feces and histologic HIS are uncommon (~1.5%). Brachyspira aalborgi is an extremely slow growing and fastidious spirochete, which previously had been isolated from an HIS patient in Denmark. Recent studies using polymerase chain reaction amplification of DNA from intestinal biopsies from a series of cases of HIS in the general Western population demonstrated that B. aalborgi, rather than B. pilosicoli, was the main spirochete species involved in these patients. This review outlines recent developments in the study of HIS and the two spirochete species, and identifies priorities for future research.

Identification of the gene encoding BmpB, a 30 kDa outer envelope lipoprotein of Brachyspira (Serpulina) hyodysenteriae, and immunogenicity of recombinant BmpB in mice and pigs.

A gene encoding a 30kDa outer envelope protein of the intestinal spirochaete Brachyspira (Serpulina) hyodysenteriae, was cloned and expressed in Escherichia coli strain XLOLR. Five phagemids containing DNA inserts encoding the protein were established and one clone (pSHA) was sequenced. An 816bp hypothetical open reading frame (ORF) was identified, with a potential ribosome binding site (AGGAG), and putative -10 (TATAAT) and -35 (TTGAAA) promoter regions upstream from the ATG start of the ORF. A 12bp inverted repeat sequence, possibly serving as a transcription terminator, was identified downstream from the TAA stop codon. Analysis of the amino acid sequence identified a 19 residue hydrophobic signal peptide, incorporating a potential signal peptidase cleavage site and membrane lipoprotein lipid attachment site. Further analysis of the amino acid usage of this lipoprotein, designated BmpB, showed its possible outer membrane localisation. Comparison of the gene encoding the lipoprotein, bmpB, with GenBank nucleotide sequences showed that it has homology with the gene (plp3) encoding Plp3, an outer membrane lipoprotein of Pasteurella haemolytica (54% identity in 735bp). Comparison of the deduced amino acid sequence with the SWISS-PROT amino acid database revealed greatest homology with the outer membrane lipoproteins (Plp1, 2, 3) of P. haemolytica (34% identity in 242 aa, 37% identity in 250 aa, and 39% identity in 272 aa, respectively), and lipoproteins (rcsF and lipoprotein-28) of E. coli (40% identity in 267 aa and 36% identity in 263 aa, respectively). Three of the recombinant E. coli clones (pSHA, pSHD, and pSHE) were formalinised and used to immunise mice. A bacterin preparation of one recombinant E. coli clone (pSHA) was used to immunise pigs. Sera from these mice and pigs recognised the 30kDa lipoprotein in outer membrane preparations of B. hyodysenteriae, indicating the immunogenicity of recombinant BmpB. Sera from pigs naturally infected with B. hyodysenteriae also reacted with recombinant BmpB expressed in E. coli.

Detection by PCR and Isolation Assays of the Anaerobic Intestinal Spirochete Brachyspira aalborgi from the Feces of Captive Nonhuman Primates

ABSTRACT The purpose of this study was to investigate the presence of the anaerobic intestinal spirochetes Brachyspira aalborgi and Brachyspira pilosicoli in the feces of captive nonhuman primates (n = 35) from 19 species housed at the Zoological Gardens, Perth, Western Australia. Both spirochete species are known to infect human beings. DNA was extracted from freshly collected feces with a commercially available QIAamp DNA stool minikit and subjected to PCR protocols amplifying portions of the 16S rRNA genes of the two spirochete species. The feces were also subjected to selective culture for the spirochetes. Subsequently, feces from 62 other captive animals or birds representing 39 species at the zoo were examined by PCR to determine whether they were reservoirs of infection. Six fecal samples from individuals from four primate species (two vervet monkeys, two Tonkean macaques, one Japanese macaque, and one hamadryas baboon) tested positive in the B. aalborgi PCR. B. aalborgi was not detected by PCR in any of the other animal or bird species tested, and B. pilosicoli was not detected in the primates or any of the other animals or birds. B. aalborgi was isolated from both PCR-positive vervet monkeys. This is the first time that B. aalborgi has been isolated from nonhuman primates and the first time that it has been isolated from the feces of any species.

Brachyspira aalborgi infection in four Australian children

Aim: The clinical presentation of four children and adolescents (two males and two females with a mean age of 12.4 years; range 9–16 years) with colorectal spirochetosis is discussed.

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).