article: Burkholderia pseudomallei, a Tier 1 Select Agent and the cause of melioidosis, is a Gram-negative bacillus present in the environment in many tropical countries. Defining the global pattern of B. pseudomallei distribution underpins efforts to prevent infection, and is dependent upon robust environmental sampling methodology. Our objective was to review the literature on the detection of environmental B. pseudomallei, update the risk map for melioidosis, and propose international consensus guidelines for soil sampling. An international working party (Detection of Environmental Burkholderia pseudomallei Working Party (DEBWorP) ) was formed during the VIth World Melioidosis Congress in 2010. PubMed (January 1912 to December 2011) was searched using the following MeSH terms: pseudomallei or melioidosis. Bibliographies were hand-searched for secondary references. The reported geographical distribution of B. pseudomallei in the environment was mapped and categorized as definite, probable, or possible. The methodology used for detecting environmental B. pseudomallei was extracted and collated. We found that global coverage was patchy, with a lack of studies in many areas where melioidosis is suspected to occur. The sampling strategies and bacterial identification methods used were highly variable, and not all were robust. We developed consensus guidelines with the goals of reducing the probability of false-negative results, and the provision of affordable and ‘low-tech’ methodology that is applicable in both developed and developing countries. The proposed consensus guidelines provide the basis for the development of an accurate and comprehensive global map of environmental B. pseudomallei.
  Melioidosis, a community-acquired infectious disease caused by the environmental Gram-negative bacillus Burkholderia pseudomallei, was first described in Burma in 1912 [1]. To date, most cases have been reported from northeast Thailand where it is the third most common cause of death due to infectious diseases after HIV/AIDS and tuberculosis [2], and from Darwin in northern Australia where it has been the commonest cause of fatal community-acquired bacteremic pneumonia [3]. Melioidosis is also being increasingly reported from many countries across south and east Asia as well as parts of South America, Papua New Guinea and the Caribbean. It is apparently rare in Africa [4], although infection may pass unrecognized because diagnostic confirmation relies on microbiological culture, which is often unavailable in resource-restricted regions of the world. Even with such facilities, B. pseudomallei may be dismissed as a culture contaminant [5], or misidentified by standard identification methods including API20NE and automated bacterial identification systems [6], [7]. Humans acquire melioidosis following contact with B. pseudomallei in the environment. A number of epidemiological and animal studies have indicated that melioidosis is not contagious, and that disease is acquired following skin inoculation, inhalation or ingestion of B. pseudomallei [8]. Defining the global distribution of environmental B. pseudomallei is important for the development of a risk map for melioidosis, since this provides the geographical setting for preventive measures as well as raising awareness of this disease among healthcare workers in affected areas. Environmental sampling can be used to identify areas where people are at risk even before cases are recognized. For example, the first environmental survey around Vientiane City (the capital of Lao PDR) in 1998 demonstrated the presence of B. pseudomallei prior to the recognition of human disease [9]. This drove an effort to identify B. pseudomallei from clinical specimens, with the first case of melioidosis being identified in 1999 [10], which has been followed by the identification of more than 560 culture-positive melioidosis patients in the past 12 years. Environmental surveys have provided evidence for the presence of environmental B. pseudomallei in geographically defined regions within numerous countries in southeast Asia, Australia, Papua New Guinea, parts of South America and elsewhere [4]. Although this has provided valuable information, these studies lacked standardization in almost all aspects of study design and conduct. Whilst methodological variability has no effect in the event that the result is positive, poor sampling methods may give rise to false negative results and inappropriate reassurances regarding the absence of risk [11]. The information generated to date has also been piecemeal and provides a very incomplete global risk map, with vast regions of the world completely unmapped, including Indonesia, India, Africa, North America and most of South America. In addition, questions extending beyond risk, such as B. pseudomallei persistence and bacterial load in soil over time, during different weather conditions and in neighboring regions of the same or adjacent countries cannot be addressed unless the methodology is standardized. Ideally, the sampling technique should be relatively simple and detection of B. pseudomallei performed at low cost across the world. However, no protocol or standard operating procedure (SOP) is currently available for investigators to download and use. Recognising these problems, our objectives were to form a working party of individuals with experience in the detection of environmental B. pseudomallei, to use this body to develop consensus guidelines on sampling study design and conduct, to make these freely available to the scientific community, and to facilitate their uptake worldwide by ensuring affordability and simplicity of methodology.
  The search terms used identified 2,218 articles, 62 of which remained after screening of titles and abstracts (Figure 1). These were retrieved and the full text reviewed. An additional 10 articles were identified from the bibliography of the 62 articles which had been missed during the primary search either because they did not have an informative title or abstract (n = 4) [14], [19], [24], [25], or were not listed on PubMed (n = 6) [26]–[31]. Three review articles without additional information on primary environmental sampling were excluded [32]–[34]. Eight articles described more information on previous environmental sampling studies and were included [11], [29], [30], [35]–[39]. Therefore, 69 articles reporting 61 environmental studies for the presence of B. pseudomallei published between 1912 and 2011 were included in the review (Table 1 and Table S1). A total of 50/61 (82%) environmental studies reported the detection of environmental B. pseudomallei identified using culture and/or a PCR specific for B. pseudomallei (Table S1). Strains collected in France [14], Burkina Faso [40], Madagascar [14], and Niger [40] were later confirmed as B. pseudomallei by genotyping [41]. Another 7/61 studies reported the detection of environmental B. pseudomallei using culture and/or PCR, but did not exclude the possibility that isolates were other, non-pathogenic environmental Burkholderia spp. [13], [15]–[18], [20], [21], [38]. Only 3/61 studies (one from Kenya and two from Australia) reported negative environmental surveys for B. pseudomallei [25], [42], [43], and a study from the USA in 1977 identified a B. pseudomallei-like organism which was later identified as B. oklahomensis [44], [45]. All 16 members of DEBWorP agreed that the first recommendations would focus on soil sampling alone, and that there was not enough evidence for recommendations to be made on the detection of B. pseudomallei in water and air. All members completed the original version of the questionnaire about variations in study design and methodology for the qualitative detection of B. pseudomallei in soil (Text S2). A second iteration was developed after identifying additional issues that could not be resolved without further consultation. All 16 members completed the second version, after which consensus recommendations were developed, sent to all members for comments, and agreed upon. Specific recommendations are shown in Table 2–4, the basis for which is described below.
summary: Melioidosis is a serious infectious disease caused by the Tier 1 selected agent and Gram-negative environmental saprophyte, Burkholderia pseudomallei. The organism is commonly found in soil and water in melioidosis endemic areas. Infection in humans occurs following bacterial inoculation, inhalation or ingestion. There is a striking lack of accurate information on the global risk of melioidosis, something that could be determined from the global distribution of environmental B. pseudomallei. Soil sampling to detect the presence of B. pseudomallei has been ad hoc, poorly standardized, and the available information poorly collated. Negative studies are almost never reported, and there is no published review on this topic. We responded to this problem during the VIth World Melioidosis Congress held in Townsville, Australia in December 2010 by forming the ‘Detection of Environmental Burkholderia pseudomallei Working Party (DEBWorP) ’. We have since worked together to undertake a systematic review, map the available information, and reach a consensus on low cost methods for the detection of environmental B. pseudomallei. Our goal is to promote the use of these consensus methods and encourage people worldwide to participate in an effort to produce a comprehensive global map of environmental B. pseudomallei.
keywords: medicine
  bacterial diseases
  infectious diseases
  melioidosis
  burkholderia infection
title: Systematic Review and Consensus Guidelines for Environmental Sampling of Burkholderia pseudomallei