Development and Clinical Evaluation of A PCR Assay Targeting the Metalloprotease Gene (mprA) of B. pseudomallei

Introduction

Melioidosis is an infectious disease of animals and humans caused by Burkholderia pseudomallei. Burkholderia pseudomallei is a motile, gram-negative, oxidase-positive rod genetically closely related to B. mallei the causative agent of glanders in solipeds (Yabuuchi et al., 1992). Its genetic information is encoded by two chromosomes, 4.03 and 3.17 Mbp in size (Holden et al., 2004). Melioidosis occurs in tropical areas between latitudes 20°N and 20°S, predominantly in South-east Asia and Northern Australia. Burkholderia pseudomallei is a saprophyte that can be regularly isolated from soil and muddy water in endemic areas. It has a high natural resistance to antibiotics, disinfectants and environmental living conditions, such as a soil pH value as low as pH 4, minimal humidity of 10–15% and temperatures ranging from 4 to 42°C (Nachiangmai et al., 1985; Tong et al., 1996; Cheng et al., 2003). It is commonly accepted that importation of the bacterium occurs with (subclinically) infected animals, which start to excrete bacteria with faeces or through abscesses due to stress caused by travelling, climate or poor confinement (Ketterer et al., 1975, 1986; Dodin, 1992; Currie et al., 2000). Thus, new (timely limited) endemic foci may establish even in the cooler European climate (Dodin, 1992). The spreading of B. pseudomallei clearly profits from globalization of animal trade, modern mass production involving crowding and the use of enormous amounts of potable water (Sprague and Neubauer, 2004). Consequently, cases in animals have not only been reported from Australia, Papua New Guinea, Malaysia, Thailand, Viet Nam, Singapore and Taiwan, but also from China, the Indian subcontinent, Turkey, Iran, Saudi Arabia, United Arab Emirates, Madagascar, Egypt, Chad, Niger, South Africa, Aruba, USA, Brazil, UK, France and Spain (Sprague and Neubauer, 2004). Occasional infection in a multitude of warm- and cold-blooded animals can occur by inhalation, ingestion, via skin wounds in contact with contaminated water or dust particles (Thomas et al., 1988). The clinical picture can present itself in various forms: acute fulminate septicaemia, local infection, subacute illness, chronic infection and subclinical disease. Acute melioidosis is often a fulminate infection with sepsis and high mortality, especially in young animals. The incubation period for naturally occurring infection in animals is not known. In solipeds and camelids melioidosis can easily be mistaken for glanders (Sprague and Neubauer, 2004).

Although B. pseudomallei is not a fastidious bacterium, isolation and identification of the agent are often unsuccessful. No optimized technique or media for enrichment and cultivation exists (Ashdown and Clarke, 1992; Brooks et al., 1997). Using biochemical identification by conventional and automated identification systems such as API^TM stripes, Vitek^TM cards (both BioMérieux, Nurtingen, Germany) or the Phoenix^TM (Becton Dickinson, Heidelberg, Germany), B. pseudomallei is often misdiagnosed as B. cepacia, Chromobacterium violaceum or not identified at all (Sprague and Neubauer, 2004; Glass and Popovic, 2005). Assays for a sensitive and specific detection of antigens are not commonly available. The development of specific PCR assays is hampered by the fact that B. pseudomallei and B. mallei are genetically closely related on the basis of their genetic information (Yabuuchi et al., 1992; Godoy et al., 2003; Holden et al., 2004; Nierman et al., 2004). Therefore, various PCR and real-time PCR systems have been described with a low detection limit but not capable of discriminating both pathovars e.g. flagellin C gene specific assays (Hagen et al., 2002; Sonthayanon et al., 2002; Sprague et al., 2002; Tomaso et al., 2004, 2005). Recently, strategies for the detection of B. pseudomallei from pure cultures have been proposed using real-time PCR and multiplex PCR technology (Thibault et al., 2004; Lee et al., 2005). However, the assays developed are highly sophisticated.

In this article, we describe the development and evaluation of a simple single target PCR assay with a low detection limit for the detection of B. pseudomallei DNA in pure cultures and lung tissue of a naturally infected dromedary.

Materials and Methods

Results

The comparison of the available sequences of the metalloprotease gene (mprA) of B. pseudomallei K96243 (Lee and Liu, 2000) and B. mallei ATCC 23344^T revealed that this open reading frame was destroyed by an insertion event involving parts of the EPS regulon and an IS407A element in the B. mallei genome. Sequence identity of mprA was only 92% when compared with the corresponding sequence in B. thailandensis. This genetic difference in B. pseudomallei, B. mallei and B. thailandensis was used to design specific primers for the detection of B. pseudomallei by PCR. In subsequent PCR analyses, template DNA of all B. pseudomallei strains investigated were amplified, whereas other bacteria including B. mallei and B. thailandensis tested negative (Tables 1 and 2).

The detection limit of the assay was 10 fg for purified B. pseudomallei DNA or two genome equivalents respectively (Fig. 1). To demonstrate the applicability of the test for clinical application lung samples of a culture-proven B. pseudomallei-infected camel (Wernery et al., 1997) were tested. In the PCR analysis, the B. pseudomallei specific DNA fragment was amplified from each tissue (Fig. 1) and the integrity of the amplicons was proven by subsequent DNA sequencing (data not shown).

Discussion and conclusions

Burkholderia pseudomallei, the causative agent of melioidosis in man and animals, is a saprophyte originating from South-east Asia. Because of the importation of clinically and subclinically infected animals and its extraordinary resistance, it has spread to regions on all continents with appropriate environmental conditions (Sprague and Neubauer, 2004). Nowadays, the regions of endemicity of B. pseudomallei (melioidosis) and B. mallei (glanders) overlap in Asia (Krishna et al., 1992; Bazargani et al., 1996; Al-Ani et al., 1998; Muhammad et al., 1998; Arun et al., 1999), America (Mota et al., 2000), and probably Eastern Europe. The clinical presentation of melioidosis in horses can easily be confused with glanders. Serological and molecular diagnostic tools are not discriminatory (Zysk et al., 2000). The reliable diagnosis is also hampered by the low sensitivity of bacterial cultivation and animal inoculation in clinical cases of both agents. In contrast to equine melioidosis, B. mallei infections in solipeds are notifiable to the Organisation Mondiale de la Santé Animal (OIE) and can cause severe restrictions in trading. The specific identification of B. pseudomallei and the exclusion of a B. mallei infection, especially in regions where both agents are endemic, are extremely important for the well timed onset of counter measures. The aim of this study was, therefore, to develop a robust but specific and sensitive PCR assay with a low detection limit to detect and to identify B. pseudomallei in pure cultures and clinical samples.

The mprA PCR assay had a sensitivity and specificity of 100%. We therefore assume that the metalloprotease gene is a stable genetic marker as it was detected in each of the B. pseudomallei strains examined and absent in all B. mallei strains we had at hand. The strains tested originated from environmental samples, i.e. soil and from warm-blooded animals including monkeys and humans. Neither the geographical origin (Europe, Asia, Africa, America or Australia) nor the time of isolation influenced the specificity of the assay as the strains were collected over a time period of more than 60 years. Moreover, the recombination which disrupted the metalloprotease gene in B. mallei was unlikely to revert spontaneously as it was present in all B. mallei strains investigated, including the oldest strain of our collection dating back to 1920, London (NCTC 00120) and a recent isolate from an outbreak in the United Arab Emirates in 2004.

Additionally, by using a point of recombination it is unlikely that mprA will be re-arranged in a B. mallei clone in such a way that an amplicon as in B. pseudomallei is produced. We believe that a specific conventional PCR assay targeting a single gene is superior to costly real-time PCR technology and complex multiplex PCR systems as PCR technology is also regularly available in many developing countries, i.e. the endemic region of melioidosis.

Despite the well known low bacterial load in infected tissues, the amplification was successful, demonstrating that the problems of cultivation and animal inoculation could be overcome by our PCR assay. The developed PCR assay can therefore be used to confirm a presumptive clinical diagnosis. That B. pseudomallei can even cause disease in unexpected and unusual geographical regions is furthermore demonstrated by the case described in this report. The climate in the UAE is extremely dry and sunny with a high UV light fraction and as such extremely adverse for B. pseudomallei. A case of melioidosis in this location is thus highly unlikely. In contrast to melioidosis, glanders in camelids has caused severe outbreaks in the past, as camelids are highly susceptible to B. mallei and are easily infected through the contact with glanderous horses (Sprague and Neubauer, 2004). However, the phenotypic diagnosis of B. pseudomallei was retrospectively confirmed by means of the developed PCR. By investigative epidemiology the probable source of infection was ascertained. The dromedary had been infected by contaminated alfalfa cultivated in Dubai on wet cow dung which had previously been imported from Pakistan. Alfalfa is an important diet of valuable racing camels. Additionally, it was also a very wet season with more rain.

In summary, the developed PCR assay provides a simple tool for the detection of B. pseudomallei in samples with low bacterial load as found in fresh clinical samples and formalin-fixed tissues. We therefore recommend the use of this assay for the diagnosis of melioidosis in natural outbreaks, especially in B. mallei-endemic regions.