فایل ورد کامل شناسایی، کمیت سنجی و تعیین نوع واژینوز باکتریال در نمونه های واژنی بالینی غیرکشت شده توسط PCR کمّی

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تعداد صفحات این فایل: ۲۷ صفحه

بخشی از مقاله انگلیسیعنوان انگلیسی:Identification, quantification and subtyping of Gardnerella vaginalis in noncultured clinical vaginal samples by quantitative PCR~~en~~

Abstract

Gardnerella vaginalis is an important component of the human vaginal microflora. It is proposed to play a key role in the pathogenesis of bacterial vaginosis (BV), the most common vaginal condition. Here we describe the development, validation and comparative analysis of a novel molecular approach capable of G. vaginalis identification, quantification and subtyping in noncultured vaginal specimens. Using two quantitative PCR (qPCR) assays, we analysed G. vaginalis bacterial loads and clade distribution in 60 clinical vaginal-swab samples. A very high pathogen prevalence was revealed by species-specific qPCR not only among BV patients (100 %), but also in healthy women (97 %), although the G. vaginalis concentration was significantly lower in non-BV samples. G. vaginalis clades identified in vaginal specimens by subtyping multiplex qPCR, which targets four clade-specific genetic markers, had frequencies of 53 % for clade 1, 25 % for clade 2, 32 % for clade 3 and 83 % for clade 4. Multiple clades were found in 70 % of samples. Single G. vaginalis clades were represented by clade 1 and clade 4 in 28 % of specimens. A positive association with BV was shown for clade 1 and clade 3, while clade 2 was positively associated with intermediate vaginal microflora, but not with BV. Clade 4 demonstrated no correlation with the disorder. The presence of multiple clades had a high positive association with BV, whereas G. vaginalis identified as a single clade was negatively linked with the condition. Polyclonal G. vaginalis infection may be a risk factor for BV.

۱ Introduction

Gardnerella vaginalis is a facultatively anaerobic, catalaseand oxidase-negative bacterium. G. vaginalis cells are pleomorphic, Gram-negative to Gram-variable, nonencapsulated and non-motile rods with a mean size of 0.5 to 1.5 mm (Greenwood & Pickett, 1980). Initially named Haemophilus vaginalis by discoverers, the microorganism was later referred to as Corynebacterium vaginale and taxonomically assigned as G. vaginalis (Gardner & Dukes, 1955; Piot et al., 1980). Currently it is the only species within the genus Gardnerella (List of Prokaryotic Names with Standing in Nomenclature – www.bacterio. net). G. vaginalis is mainly considered a part of the lower female genital tract microflora (Catlin, 1992). It can also be routinely isolated from the male urogenital tract as was reported in the first publication on this micro-organism and a number of other studies (Briselden & Hillier, 1990; Eren et al., 2011; Leopold, 1953; Swidsinski et al., 2010). The presence of G. vaginalis in the oral cavity and anal samples has also been described (El Aila et al., 2011; Holst, 1990; Marrazzo et al., 2012). Along with disorders in the urinary and genital tracts, G. vaginalis has been identified as a causative agent of bacteraemia, septicaemia with infective endocarditis, vertebral osteomyelitis, acute hip arthritis and retinal vasculitis (Graham et al., 2009; Lagace´- Wiens et al., 2008; Neri et al., 2009; Sivadon-Tardy et al., 2009; Yoon et al., 2010).

Since the moment of discovery G. vaginalis has been linked with bacterial vaginosis (BV), the most common vaginal condition. BV is characterized by the elevation of vaginal pH and clinical symptoms, such as malodorous vaginal discharge and the presence of clue cells visualized on wet mount, although a large percentage of women with BV can be asymptomatic (Amsel et al., 1983; Schwebke & Desmond, 2007). The disorder is associated with a dramatic change in vaginal microflora, when protective Lactobacillus species are depleted and replaced by fastidious anaerobic bacteria including Prevotella, Atopobium, Megasphaera, Sneathia and G. vaginalis (Eschenbach, 1993; Forsum et al., 2005; Nugent et al., 1991). Described as ‘a single etiological agent’ of BV by the discoverers, G. vaginalis, however, failed to fulfil Koch’s postulates for microbial infection in multiple subsequent studies (Catlin, 1992; Holst, 1990; Menard et al., 2008; Zozaya-Hinchliffe et al., 2010). The complex polymicrobial nature of BV and the presence of G. vaginalis in the vaginal milieu of healthy individuals argue against the definition of G. vaginalis as a sole disease-causative species. The micro-organism, nevertheless, remains a prime suspect in the pathogenesis of BV since it is present in 95–۱۰۰ % of BV patients, although the association of G. vaginalis with Atopobium or Megasphaera has been shown to display better predictive value for this condition (Fredricks et al., 2009; Menard et al., 2008; Muzny & Schwebke, 2013; Schellenberg et al., 2011). It was proposed that G. vaginalis might serve as an initial colonizer critical for the development of BV (Patterson et al., 2010). Some studies suggest that G. vaginalis dominance in the vaginal microflora can represent a distinct transitional or intermediate state between healthy microflora occupied by lactobacilli and BV-type microflora populated with fastidious anaerobes (Schellenberg et al., 2011). After more than half a century the role of G. vaginalis in such an ‘enigmatic’ condition as BV remains obscure.

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