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

Westerman, Laurens J.; Stel, H. V.; Schipper, Marguérite E.I.; Bakker, Leendert J.; Neefjes-Borst, E. Andra; Brande, Jan H. M. van den; Boel, Edwin; Seldenrijk, Kees A.; Siersema, Peter D.; Bonten, Marc J. M.; Kusters, Johannes G.

doi:10.1371/journal.pone.0052281

Cited by 25 publications

(36 citation statements)

References 43 publications

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“…Four samples were positive for both B. aalborgi and "B. hominis" (two cases and two controls). B. pilosicoli was not detected in any of the included samples, although this species was responsible for 15.4% of HIS cases in a previous study in the Netherlands using similar methods (3). No statistically significant association (P Ͻ 0.05) was found regarding the presence of Brachyspira species in cases and controls or regarding age, gender, age, coinfections, urbanization, seasons, year of study, or duration of symptoms.…”

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confidence: 55%

“…Three Brachyspira species have been reported to colonize humans: Brachyspira aalborgi, Brachyspira pilosicoli, and the provisionally named "Brachyspira hominis" (3)(4)(5)(6)(7). Although it is unclear whether HIS is associated with clinical symptoms, there is evidence suggesting an association between B. pilosicoli and diarrhea in humans (3,(8)(9)(10)(11). However, due to the obligate anaerobic culture requirements for these bacteria, most studies on HIS are based on histopathology without species differentiation, as histopathology requires additional techniques to differentiate between species (3,12).…”

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confidence: 99%

“…Although it is unclear whether HIS is associated with clinical symptoms, there is evidence suggesting an association between B. pilosicoli and diarrhea in humans (3,(8)(9)(10)(11). However, due to the obligate anaerobic culture requirements for these bacteria, most studies on HIS are based on histopathology without species differentiation, as histopathology requires additional techniques to differentiate between species (3,12). As a result, the clinical significance of HIS has remained controversial for decades (1,(13)(14)(15)(16).…”

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Brachyspira Species and Gastroenteritis in Humans

Westerman

Boer²,

Roelfsema

et al. 2013

J Clin Microbiol

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e Brachyspira species have been implicated as a potential cause of gastroenteritis in humans; this is, however, controversial. In 733 gastroenteritis cases and 464 controls, we found 29 samples positive for Brachyspira species (2.3% of cases and 2.6% of controls; P ‫؍‬ 0.77). Brachyspira species were not associated with gastroenteritis in humans.H uman intestinal spirochetosis (HIS) is caused by spirochetes from the genus Brachyspira (1) and is histologically characterized by the apical attachment of these bacteria to the mucosa of the colon (2). Three Brachyspira species have been reported to colonize humans: Brachyspira aalborgi, Brachyspira pilosicoli, and the provisionally named "Brachyspira hominis" (3-7). Although it is unclear whether HIS is associated with clinical symptoms, there is evidence suggesting an association between B. pilosicoli and diarrhea in humans (3,(8)(9)(10)(11). However, due to the obligate anaerobic culture requirements for these bacteria, most studies on HIS are based on histopathology without species differentiation, as histopathology requires additional techniques to differentiate between species (3, 12). As a result, the clinical significance of HIS has remained controversial for decades (1,(13)(14)(15)(16). Several studies have investigated the prevalence of Brachyspira species in patients with diarrhea, and various incidences have been reported, depending on the investigated population (high-versus low-income countries) and techniques used (culture versus histopathology). In this study, we investigated the prevalence of Brachyspira species in Dutch primary care patients with and those without symptoms of gastroenteritis. For this purpose, we used fecal samples stored from a case-control study reported in 2001 (17). DNA was extracted as previously described (18), directly from the fecal samples without selenite enrichment. Amplification was performed by using a previously described real-time PCR amplifying a 142-bp region of the 16S rRNA of all Brachyspira species (3), with some minor modifications. The modifications consisted of three species-specific probes targeting B. pilosicoli, B. aalborgi, and "B. hominis" and another reverse primer. These probes were validated against a known set of 66 Brachyspira-positive samples (cultured isolates and histopathologically positive samples) and 26 Brachyspira-negative samples (3). The PCR protocol and primer and probe sequences are supplied in Table 1. PCR inhibition was defined as a PhHV cycle threshold value greater than the average of all PCRs plus 2 times the standard deviation. Samples exceeding this value and all Brachyspira-positive samples were reextracted from the original fecal samples. Samples were excluded from analysis if the internal control exceeded the cutoff again. Statistical analyses were performed by using IBM SPSS Statistics version 20 (release 20.0.0) for Macintosh OS X.Samples from 736 cases and 465 controls were available. Four samples (three cases and one control) had to be excluded due to inhibition of the PCR. T...

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Brachyspira Species and Gastroenteritis in Humans

Westerman

Boer²,

Roelfsema

et al. 2013

J Clin Microbiol

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“…Many members of the genus Brachyspira are important veterinary pathogens but occasionally have been reported to be associated with human pathology (108)(109)(110). Spirochaetes are often not visible by Gram staining, requiring dark-field microscopy or special coloration (111).…”

Section: Spirochetesmentioning

confidence: 99%

Current and Past Strategies for Bacterial Culture in Clinical Microbiology

et al. 2015

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SUMMARY A pure bacterial culture remains essential for the study of its virulence, its antibiotic susceptibility, and its genome sequence in order to facilitate the understanding and treatment of caused diseases. The first culture conditions empirically varied incubation time, nutrients, atmosphere, and temperature; culture was then gradually abandoned in favor of molecular methods. The rebirth of culture in clinical microbiology was prompted by microbiologists specializing in intracellular bacteria. The shell vial procedure allowed the culture of new species of Rickettsia . The design of axenic media for growing fastidious bacteria such as Tropheryma whipplei and Coxiella burnetii and the ability of amoebal coculture to discover new bacteria constituted major advances. Strong efforts associating optimized culture media, detection methods, and a microaerophilic atmosphere allowed a dramatic decrease of the time of Mycobacterium tuberculosis culture. The use of a new versatile medium allowed an extension of the repertoire of archaea. Finally, to optimize the culture of anaerobes in routine bacteriology laboratories, the addition of antioxidants in culture media under an aerobic atmosphere allowed the growth of strictly anaerobic species. Nevertheless, among usual bacterial pathogens, the development of axenic media for the culture of Treponema pallidum or Mycobacterium leprae remains an important challenge that the patience and innovations of cultivators will enable them to overcome.

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“…48 In addition, B. pilosicoli is a potentially zoonotic pathogen. 34,70 Therefore, it is important not only to detect Brachyspira spp in diagnostic specimens but also to identify them accurately to species level. Both tasks are difficult phenotypically, since culture of Brachyspira requires strict anaerobic conditions and is time-consuming and labor-intensive.…”

Section: Pcr Assaysmentioning

confidence: 99%

Nonculture Molecular Techniques for Diagnosis of Bacterial Disease in Animals

2014

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The past decade has seen remarkable technical advances in infectious disease diagnosis, and the pace of innovation is likely to continue. Many of these techniques are well suited to pathogen identification directly from pathologic or clinical samples, which is the focus of this review. Polymerase chain reaction (PCR) and gene sequencing are now routinely performed on frozen or fixed tissues for diagnosis of bacterial infections of animals. These assays are most useful for pathogens that are difficult to culture or identify phenotypically, when propagation poses a biosafety hazard, or when suitable fresh tissue is not available. Multiplex PCR assays, DNA microarrays, in situ hybridization, massive parallel DNA sequencing, microbiome profiling, molecular typing of pathogens, identification of antimicrobial resistance genes, and mass spectrometry are additional emerging technologies for the diagnosis of bacterial infections from pathologic and clinical samples in animals. These technical advances come, however, with 2 caveats. First, in the age of molecular diagnosis, quality control has become more important than ever to identify and control for the presence of inhibitors, cross-contamination, inadequate templates from diagnostic specimens, and other causes of erroneous microbial identifications. Second, the attraction of these technologic advances can obscure the reality that medical diagnoses cannot be made on the basis of molecular testing alone but instead through integrated consideration of clinical, pathologic, and laboratory findings. Proper validation of the method is required. It is critical that veterinary diagnosticians understand not only the value but also the limitations of these technical advances for routine diagnosis of infectious disease.Keywords bacterial infections, mass spectrometry, molecular diagnostic techniques, fluorescent in situ hybridization, quality control, realtime polymerase chain reaction Traditionally, pathogen identification for infectious disease was mostly based on isolation in culture, and this was also critical for fulfilling Koch's postulates. 24 Molecular identification methods have been used for infectious disease diagnosis since the 1980s, and ''molecular Koch's postulates'' were applied to bacterial pathogenicity at the gene rather than the wholeorganism level. 21,22 Molecular guidelines for establishing microbial disease causation were described as a reconsideration of Koch's postulates. 24 Among the nonculture methods, polymerase chain reaction (PCR) and DNA sequencing have been widely used to identify infectious pathogens. Recently, massive parallel DNA sequencing has rapidly developed for the identification of both described and new viral pathogens. The technology will likely revolutionize bacterial disease diagnosis in the near future. Not only have many nonculturable or difficult-to-culture bacterial pathogens become detectable, but many readily isolated bacterial infections can now be identified with nonculture methods. Molecular identification methods ca...

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

Cited by 25 publications

References 43 publications

Brachyspira Species and Gastroenteritis in Humans

Brachyspira Species and Gastroenteritis in Humans

Current and Past Strategies for Bacterial Culture in Clinical Microbiology

Nonculture Molecular Techniques for Diagnosis of Bacterial Disease in Animals

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