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Fatal Melioidosis in Goats in Bangkok, Thailand
brief-report
Author(s):
Walaiporn Tonpitak ,
Chulabha Sornklien ,
Mongkol Chawanit ,
Suvarin Pavasutthipaisit ,
Vanaporn Wuthiekanun ,
Viriya Hantrakun ,
Premjit Amornchai ,
Janjira Thaipadungpanit ,
Nicholas P. J. Day ,
Samuel Yingst ,
Sharon J. Peacock ,
Direk Limmathurotsakul
*
Publication date PMC-release:
06 August 2014
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Abstract
Bangkok, Thailand, is a city considered to be at low risk for melioidosis. We describe 10 goats that died of melioidosis in Bangkok. Half of them were born and reared in the city. Multilocus sequence typing ruled out an outbreak. This finding challenges the assumption that melioidosis is rarely acquired in central Thailand.
Related collections
Most cited references13
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Multilocus sequence typing and evolutionary relationships among the causative agents of melioidosis and glanders, Burkholderia pseudomallei and Burkholderia mallei.
A collection of 147 isolates of Burkholderia pseudomallei, B. mallei, and B. thailandensis was characterized by multilocus sequence typing (MLST). The 128 isolates of B. pseudomallei, the causative agent of melioidosis, were obtained from diverse geographic locations, from humans and animals with disease, and from the environment and were resolved into 71 sequence types. The utility of the MLST scheme for epidemiological investigations was established by analyzing isolates from captive marine mammals and birds and from humans in Hong Kong with melioidosis. MLST gave a level of resolution similar to that given by pulsed-field gel electrophoresis and identified the same three clones causing disease in animals, each of which was also associated with disease in humans. The average divergence between the alleles of B. thailandensis and B. pseudomallei was 3.2%, and there was no sharing of alleles between these species. Trees constructed from differences in the allelic profiles of the isolates and from the concatenated sequences of the seven loci showed that the B. pseudomallei isolates formed a cluster of closely related lineages that were fully resolved from the cluster of B. thailandensis isolates, confirming their separate species status. However, isolates of B. mallei, the causative agent of glanders, recovered from three continents over a 30-year period had identical allelic profiles, and the B. mallei isolates clustered within the B. pseudomallei group of isolates. Alleles at six of the seven loci in B. mallei were also present within B. pseudomallei isolates, and B. mallei is a clone of B. pseudomallei that, on population genetics grounds, should not be given separate species status.
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Activities of Daily Living Associated with Acquisition of Melioidosis in Northeast Thailand: A Matched Case-Control Study
Introduction Burkholderia pseudomallei is a Category B select agent and the cause of naturally acquired melioidosis in South and East Asia, Northern Australia, the Indian subcontinent and areas of South America [1]–[3]. Northeast Thailand is a hotspot for this infection, with an annual incidence of 21.0 per 100,000 population and a crude mortality rate of 40% [4]. This rate is comparable to that for deaths from tuberculosis in this region, where melioidosis is the third most common cause of death from infectious diseases [4]. Visitors to areas where melioidosis is endemic are also at risk of acquiring this infection. Melioidosis is readily misdiagnosed in returning travelers because of a lack of familiarity with the clinical and microbiological features, compounded by a highly variable incubation period that may extend to many decades [5], [6]. The largest transient population to have been affected in living memory was US combatants in the conflict with Vietnam, when the disease acquired the nickname ‘Vietnamese time bomb’ [7]. B. pseudomallei is present in soil and surface water in areas where melioidosis is endemic, and most cases are thought to result from bacterial inoculation [8]. This is based on the observations that people at high risk of melioidosis such as agricultural workers in Thailand and indigenous people in Australia are regularly exposed to soil and water without protective clothing and may suffer repeated minor injuries [9], [10]. The role of other routes of infection is uncertain. Inhalation may have been a route of infection for US combatants during the Vietnam conflict [11], and several studies from northern Australia have reported a shift towards a higher frequency of pneumonia and severe disease during the rainy season or following heavy monsoon rains and winds [12]–[14]. Recent evidence also suggests that ingestion might be an important route of B. pseudomallei infection. West et al. showed that gastric inoculation of B. pseudomallei led to melioidosis in an experimental mouse model [15]. Several clusters of melioidosis cases have been reported from Australia in which a strain of B. pseudomallei isolated from a common water source was a genetic match for the strain causing disease in the cluster [16], [17], although it is not clear whether these cases were infected through ingestion rather than inoculation. Melioidosis is potentially preventable, but developing prevention guidelines is hampered by a lack of evidence on which to base them. Advice in Northern Australia is based on common sense and includes avoidance of direct contact with soil and standing water and washing after exposure [18]. There are no recommendations to prevent melioidosis via inhalation or ingestion. No advice is given in Asia or other places where melioidosis is endemic, and no advice is given to tourists despite the steady trickle of cases in returning travelers. Here, we describe a matched case-control study in which we identify activities associated with an increased risk of disease acquisition, define the importance of three routes of melioidosis infection, and describe the first evidence-based guidelines for the prevention of melioidosis. Methods Setting and study design A prospective 1∶2 matched case-control study was performed at Sappasithiprasong Hospital between Jul 2010 and Dec 2011. This 1,100-bed hospital is situated in the provincial town of Ubon Ratchathani in northeast Thailand, 70 km west of Laos and 95 km north of Cambodia, and serves around 2 million people. Cases were initially identified through daily contact with the hospital diagnostic microbiology laboratory, and were defined as patients aged ≥18 years with culture-proven melioidosis (isolation of B. pseudomallei from any clinical sample and compatible clinical features). Controls were identified through the hospital computerized admission records, and were defined as patients admitted with non-infectious conditions during the same period (+/−2 weeks, and therefore season), matched for gender, age (+/−5 years), and presence or absence of diabetes mellitus. Patients admitted with infectious conditions were not eligible as controls, as the sensitivity of culture for the diagnosis of melioidosis is not perfect [19]. As a result, culture-negative melioidosis patients were not enrolled as controls. Matching was performed for known predisposing factors (diabetes, gender, age and time of presentation) to control for confounding. Target enrollment numbers were at least 250 cases and 500 controls, which would allow the detection of an approximate odds ratio of 2.0 with 90% power using a two-sided 1% test [19]. Each case and control was interviewed and information collected on specified activities of daily living during the 30 days preceding the onset of symptoms using a standardized study form. Relatives were interviewed if patients were not capable of answering questions. Patients with melioidosis are often severely unwell, and complete data capture via relatives was considered important to avoid the bias associated with exclusion of this group (Text S1). Trained study staff administered the questionnaire. The study was approved by the research ethics committees of Sappasithiprasong Hospital, and the Faculty of Tropical Medicine, Mahidol University. Written informed consent was obtained from all participants. Further details of the study design, definitions of cases and controls, assessment of exposure and statistical methods are provided in the supporting information. Sampling and culture of drinking water A home visit was performed for case and control patients who resided within 100 km of the hospital, a distance limit imposed by the feasibility of travel, sample and data collection in the course of a single day. Five liters was collected from each source of drinking water, and tap water regardless of consumption. If the water was filtered or boiled by the householder before consumption, samples of these were collected for culture. Water samples were transported on the same day to our research laboratory at Sappasithiprasong Hospital and cultured for the presence of B. pseudomallei. In brief, for each 5 liter sample, 1 liter was passed through two 0.45 µm filters and 4 liters was passed through 2.5 g of sterile diatomaceous earth (Celite, World Minerals, USA) [20]. Filters were cultured on Ashdown agar to provide a quantitative bacterial count, and diatomaceous earth was cultured in selective broth (TBSS-C50) [21] to provide a sensitive, qualitative method. Broth was incubated at 40°C in air for 48 hours, after which 10 µl of the upper layer was streaked onto an Ashdown agar plate to achieve single colonies, incubated at 40°C in air and examined every 24 hours for 7 days. In the event that enrichment broth was positive but filters on Ashdown agar were negative, the quantitative count was defined as <1 CFU/L. Identification of bacterial colonies was performed as described previously [22]. Statistical analysis Univariable and multivariable conditional logistic regression analyses were performed. All variables that were either statistically significant in univariable analyses (with 0.25 significance level) or that were selected a priori based on current knowledge were included in multivariable analyses. The final multivariable model was developed using a purposeful selection method [23]. Data were analyzed using Stata12.0 (StataCorp, Texas, US). Conditional odds ratios are presented, and all p-values are two-tailed. Results Study subjects A total of 414 patients presenting to Sappasithiprasong Hospital with culture confirmed melioidosis between July 2010 and December 2012 were assessed for eligibility (Figure 1). Of these, 84 patients were excluded because they were less than 18 years of age (n = 50), had recurrent melioidosis (n = 33), or declined to participate (n = 1). 10.1371/journal.pntd.0002072.g001 Figure 1 Study flow diagram. Of the 330 cases enrolled into the study, two matched controls were identified for each of 226 cases (69%) and one matched control for 61 cases (18%). No matched control could be identified for the remaining 43 cases (13%) who were excluded from further analysis, giving a total of 287 cases and 513 controls. A history of activities of daily living prior to the onset of infective symptoms was obtained from relatives for a total of 92 cases (32%) and 26 controls (5%). A diagnosis of diabetes was more common in patients with melioidosis who were excluded because of failure to find a control, compared with those enrolled as cases (72% v.s. 42%, Table S1). The median age of cases was 54 years (interquartile range 46–64 years, range 18–88 years), 181 (63%) were male, 120 (42%) were diabetic, and 100 (35%) died within 28 days of the admission date. The 513 controls were enrolled from various departments including surgery, orthopedics, general internal medicine and ophthalmology (Table S2). Common causes of illness were cancer (n = 58), bone fracture (n = 35), corneal ulcer (n = 26), cerebrovascular diseases (n = 20), cataract (n = 17), glaucoma (n = 17), calculus formation in the kidney or ureter (n = 16), and intervertebral disc disorder (n = 15). Activities of daily living Working in a rice field in the month prior to the onset of infective symptoms was reported in 72% of cases and 48% of controls (Table S3), and almost tripled the odds of acquiring melioidosis (conditional odds ratio [cOR] 2.9, 95% confidence interval [CI] 2.1–4.0). The odds of having melioidosis increased by approximately 10% for each 10 working hours/week increase (cOR 1.1, 95%CI 1.0–1.1), and by approximately 20% for each 10 centimeter increase in depth that the legs were submerged in soil or water (cOR 1.2, 95%CI 1.0–1.3). Conversely, there was a decreased risk associated with wearing long trousers or rubber boots. There was no significant reduction in risk associated with wearing cloth gloves, and wearing rubber gloves was not reported. Washing after working in the rice field was associated with a decreased risk, but washing with water pooled in the rice field was associated with an increased risk of melioidosis (Table S3). Other activities leading to exposure to soil or water were also strongly associated with a risk of melioidosis (cOR 1.8, 95%CI 1.3–2.5 and cOR 2.3, 95%CI 1.7–3.3, respectively). People who walked barefoot everyday had nearly 2.5 times the odds of developing melioidosis compared to those who never walked barefoot (cOR 2.4, 95%CI 1.1–5.5). People who bathed in pond water had 11 times the odds of having melioidosis (cOR 11.1, 95%CI 1.3–92.5). Having an open wound was strongly associated with risk (cOR 2.4, 95%CI 1.4–4.1), and the risk increased if herbal remedies or an organic substance was applied directly onto an open wound (cOR 2.9, 95%CI 1.6–5.3). Eating food contaminated with soil or dust was reported by 40% of cases and 22% of controls (cOR 2.4, 95%CI 1.7–3.3). Table S4 shows the water drinking habits of the 800 participants. Overall, 17% filtered and 13% boiled water before drinking. Following inspection of filtration machines, these were not considered to represent adequate treatment because of poor machine maintenance. Therefore, only bottled and boiled water was considered treated, while unboiled water from wells, boreholes, collected rainwater and tap water was considered untreated. Drinking untreated water was reported by 85% of cases and 72% of controls, and was associated with a doubling in the odds of acquiring melioidosis (cOR 2.3, 95%CI 1.5–3.3). Outdoor exposure to a dust cloud or rain was associated with increased risk (cOR 1.6, 95%CI 1.2–2.2 and cOR 2.9, 95%CI 2.0–4.1, respectively). The use of a protective item (a mask or umbrella) was associated with a lower risk of infection although this did not reach significance (Table S4). Water inhalation of untreated water (accidental choking during drinking or swimming, associated with vigorous coughing) was reported by 23% of cases compared with 9% of controls (cOR 3.0, 95%CI 2.0–4.5). Being an active smoker was associated with increased risk (cOR 2.2, 95%CI 1.4–3.7), but being an ex-smoker was not (Table S4). Consumption of any oral steroid medication was associated with a cOR of 3.2 (95%CI 1.6–6.3). Education beyond primary school was reported by 15% of cases and 26% of controls. A monthly income of greater than 5,000 baht per month was reported by 24% of cases and 37% of controls. Both factors were associated with halving the odds of acquiring melioidosis in the univariable model (Table S4). Multivariable conditional logistic regression The final multivariable conditional logistic regression model included 286 cases and 512 controls (1 case and 1 control were excluded because of missing values). The findings of this analysis indicated that activities associated with an increased risk of melioidosis involved all three routes of acquisition. Working in a rice field, other activities leading to exposure to soil or water, eating contaminated food, drinking untreated water, outdoor exposure to rain, an open wound, water inhalation and taking steroids were independent risk factors in the final model (Table 1). There was borderline evidence that active smoking was associated with acquiring melioidosis (cOR 1.5, 95%CI 1.0–2.3, p = 0.069). 10.1371/journal.pntd.0002072.t001 Table 1 Multivariable analysis of risk factors for melioidosis. Activities Conditional OR (95%CI) P value Activities related to skin inoculation No activities involving exposure to soil or water 1.0 0.003 Working in a rice field 2.1 (1.4–3.3) Other activities involving exposure to soil or water 1.4 (0.8–2.6) Open wound 2.0 (1.2–3.3) 0.005 Activities related to ingestion Eating food contaminated with soil or dust 1.5 (1.0–2.2) 0.045 Drinking untreated water 1.7 (1.1–2.6) 0.03 Activities related to inhalation Outdoor exposure to dust cloud 1.3 (0.9–1.8) 0.23 Outdoor exposure to rain 2.1 (1.4–3.2) <0.001 History of water inhalation 2.4 (1.5–3.9) <0.001 Other risk factors Current smoker 1.5 (1.0–2.3) 0.069 Taking oral steroids 3.1 (1.4–6.9) 0.006 Estimated odds ratios (OR) are conditional on the matching variables (gender, age, admission date (+/−2 weeks), and diagnosis of diabetes mellitus) and adjusted for the other risk factors included in the model. B. pseudomallei in drinking water Home visits and sampling of drinking water was performed in 142/287 cases (49%) and 228/513 controls (44%) who resided within 100 kilometers of the hospital. B. pseudomallei was detected in 12% (10/84) of borehole water samples, 12% (32/273) of tap water samples, and 4% (1/27) of well water samples. B. pseudomallei was not detected in rain water (which is collected into a closed earthenware containers), or bottled water (0/160 and 0/32, respectively) (Table S5). The median quantitative count of B. pseudomallei in culture-positive samples was 1 CFU/L (interquartile range [IQR] <1 to 13; range <1 to 65 CFU/L). Two out of 53 samples of water that had been treated by a household member using filtration were culture positive for B. pseudomallei. Combining the results from the interview and microbiological data, we found that 7% (10/142) of cases and 3% (7/228) of controls drank water from sources that were demonstrated to contain B. pseudomallei (cOR 2.2, 95%CI 0.8–5.8). Guidelines for the prevention of melioidosis On the basis of our findings, we propose that protection is required against all three routes of B. pseudomallei acquisition. We recommend that residents and visitors to melioidosis-endemic areas avoid direct contact with soil and water, outdoor exposure to heavy rain or dust clouds, do not consume untreated water, and wash food to be eaten raw using boiled or bottled water (Table 2). If direct contact with soil or water is necessary, we recommend that protective gear such as rubber gloves and boots or waders should be worn. We encourage cessation of smoking (particularly in those with underlying conditions such as diabetes that are known predisposing factors for melioidosis), and discourage the application of herbal remedies or organic substances to wounds. 10.1371/journal.pntd.0002072.t002 Table 2 Recommendations for the prevention of melioidosis. 1. Avoid direct contact with soil or environmental water. 2. If contact with soil or environmental water is necessary, wear protective gear including rubber gloves, boots or waders, and wash with soap and clean water immediately after exposure. 3. In the event of an injury involving contamination with soil or environmental water, immediately clean the wound with soap and clean water. 4. Keep open wounds covered and avoid contact with soil or water until completely healed. Do not apply any herbal remedies or other substances to the wound. In the event that the wound comes into contact with soil or environmental water, clean the wound thoroughly with soap and clean water. 5. Always wear shoes. Do not walk bare foot. 6. Only drink bottled or boiled water. Do not drink any untreated water. 7. Do not eat food contaminated with soil or dust. If food is to be eaten without cooking, wash thoroughly using clean water. Use clean eating utensils, and wash these in clean water. 8. When outside, avoid heavy rain or dust clouds. If caught in a dust cloud, cover mouth and nose. Use an umbrella to protect yourself from the rain. 9. Do not smoke. 10. Be aware that you are at greater risk of melioidosis if you have certain conditions, including diabetes, chronic kidney disease, and diseases that require steroid therapy or medications that suppress the immune system. Discussion This study has provided evidence to indicate that ingestion and inhalation, together with inoculation, are important routes for the development of melioidosis in Thailand. A range of activities were found to be independently associated with melioidosis, including presumed inoculation during unprotected occupational exposure to soil or environmental water, ingestion by eating contaminated food or drinking untreated water, and inhalation by outdoor exposure to rain. We also confirmed the presence of B. pseudomallei in water obtained from wells and boreholes and from piped water supplies, and recorded that a number of cases had consumed untreated water from these sources prior to presentation with melioidosis. This is the first study to show that ingestion is an important route of human B. pseudomallei infection. Based on data obtained from The Provincial Waterworks Authority of Ubon Ratchathani province, only people living in the town of Ubon Ratchathani (3% of the provincial population) receive piped chlorinated water [24]. Tap water quality control does not include assessment for the presence of B. pseudomallei, which we found in 12% of tap water samples (32/273) and which a number of cases had consumed without adequate treatment. Unlike observations made in Hong Kong (14), none of the collected rainwater samples tested positive for B. pseudomallei. It is customary for the drinking rain water to be collected in large earthenware pots situated close to the house, the water in which can reach temperatures in excess of 40°C. This may explain the negative culture results in our setting, although it is also possible that the bacterial count was below the level of detection of our methodology. We recommend that all non-bottled water should be boiled prior to consumption. Although filtration is an alternative method of water purification, we observed that filters were poorly maintained and detected B. pseudomallei in some filtered water samples. In view of this, we do not recommend the use of filtration. This is also the first evidence to indicate that exposure to rain is an independent risk factor for melioidosis. Exposure to dust clouds was a significant risk on univariable but not multivariable analysis. This is the first study to identify that smoking may be associated with acquiring melioidosis. Smoking could decrease the effectiveness of the local inflammatory response and increase the risk of infection by inhalation. Although microbiological confirmation of aerosolized B. pseudomallei has not been published, this could be due to poor sensitivity of the techniques used or a very low bacterial concentration. In experimental mice, inhalation of only 5 CFU can result in death within a few days [25]. Our study has several limitations. Relatives were asked about activities of daily living when cases or controls were not capable of providing this information, and it is possible that they were not aware of the full spectrum of activities undertaken. It is also possible that cases who were aware of their diagnosis of melioidosis might mention risk factors more readily than controls. This would only be the case if people were knowledgeable about melioidosis, but in a recent survey most Thai people (72%) had not heard of melioidosis, and the remainder had heard of the word but did not know what it meant (unpublished data). The education about melioidosis was given to all participants after the interview. There may be other factors associated with a risk of melioidosis that we failed to examine, and we cannot evaluate the relative risk of a matched variable. The study was not powered to identify risk factors with a relative risk less than 2.0. The criteria specified for matching were stringent and we were unable to find controls for some patients. A diagnosis of diabetes was more common in patients with melioidosis who were excluded because of failure to find a control, compared with those enrolled as cases (72% v.s. 42%). This is because the prevalence of diabetes in patients admitted to the hospital with non-infectious conditions (potential controls) was low, and finding matched controls for diabetic cases was more difficult than that for non-diabetic cases. However, diabetes is the strongest risk factor for melioidosis [8], and matching for diabetes is very important to control the possible confounding effect. We consider it likely that our findings are applicable to similar settings in neighboring Asia but may be less applicable to more distant geographical settings including Australia. Current efforts are being directed toward increasing public awareness and implementing preventive measures for melioidosis in endemic areas, particularly Thailand. A vaccine that protects against B. pseudomallei infection is not available and there is no prospect of one being developed and ready for use in the near future [26]. There is, therefore, every reason to look for alterative solutions to prevent melioidosis, both in people living in regions of the world that are endemic for melioidosis, and visitors (e.g. travelers and military personnel) to these regions. The Ministry of Public Health in Thailand has included melioidosis on a priority list of emerging diseases in Thailand, and public health campaigns for melioidosis prevention based on the knowledge of this work are being developed. These actions will include the implementation of an education programme based on the recommendations provided here, the improvement of infrastructure relating to effective treatment of public water supplies and access to protective clothing, tractors and other machinery to reduce contact time of farmers with soil and environmental water. Further studies are required to model the cost-benefit of guidelines for the prevention of melioidosis, together with their acceptability, up-take and impact on rates of melioidosis. Supporting Information Table S1 Characteristics of patients with culture confirmed melioidosis. (DOC) Click here for additional data file. Table S2 Characteristics of matched controls. (DOC) Click here for additional data file. Table S3 Activities associated with melioidosis acquisition by inoculation in the 30 days before onset of symptoms. (DOC) Click here for additional data file. Table S4 Activities associated with melioidosis acquisition by ingestion and inhalation in the 30 days before onset of symptoms, and other risk factors. (DOC) Click here for additional data file. Table S5 Sources of water consumed and presence of B. pseudomallei in drinking water. (DOC) Click here for additional data file. Text S1 Supplementary methods. (DOC) Click here for additional data file. Text S2 STROBE checklist. (DOCX) Click here for additional data file.
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Systematic Review and Consensus Guidelines for Environmental Sampling of Burkholderia pseudomallei
Introduction 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. Methods Literature Review Search strategy and study selection PubMed (January 1912 to January 2011) was searched using the following MeSH terms: melioidosis and pseudomallei. The search was limited to studies published in English and French. The predetermined eligibility criterion for inclusion was a study conducted to detect B. pseudomallei in the environment. Titles and abstracts were screened for relevance, and bibliographies from selected studies hand-searched for secondary references. Database searching was performed and selected by DL and reviewed by DABD and SJP. Data extraction A data extraction form to record the methodology used to detect environmental B. pseudomallei and study findings was developed and piloted with a subset of the first 20 eligible studies prior to development of a final version (Text S1). In brief, the data extracted related to geographical location, study design, type of sample taken (soil or water), depth of sampling (for soil sampling), amount of soil (in gram) or water (in ml) collected, number of samples collected, the proportion of positive samples, and the methods used to detect and identify B. pseudomallei. Data from all studies included in the final review were extracted by DL, reviewed by DABD and SJP, and any disagreement resolved by discussion. Definitions The presence of environmental B. pseudomallei in each country was categorized as being (i) definite, (ii) probable, or (iii) possible (Table 1). ‘Definite’ was defined by the detection of B. pseudomallei from the environment using culture or a specific PCR for B. pseudomallei with or without evidence of melioidosis having been acquired in that country. ‘Probable’ was defined when no reports were identified in the published literature of environmental sampling but clinical reports indicated in-country disease acquisition. This drew on data from the most recent reviews of the distribution of human melioidosis [4], [12]. ‘Possible’ was defined as the detection of B. pseudomallei from the environment using culture or PCR methodology that did not include a confirmatory test for B. pseudomallei in a setting that lacked evidence of melioidosis having been acquired in that area/country. This included several countries where the detection of environmental B. pseudomallei was reported prior to the description of the highly related Burkholderia thailandensis as a separate species in 1998 [13]–[21]. Prior to this, B. thailandensis was referred to as ‘non-pathogenic’ or ‘arabinose-positive’ B. pseudomallei [22]. B. pseudomallei and B. thailandensis are indistinguishable on the basis of colony morphology, antimicrobial susceptibility pattern and many biochemical tests (arabinose assimilation being an important exception) [22], [23]. A few early studies inoculated suspected B. pseudomallei colonies or environmental samples into an animal model to isolate the organism or determine virulence. This would be predicted to distinguish between B. pseudomallei and non-virulent Burkholderia spp. [22], and was accepted as ‘definite’ evidence of B. pseudomallei. The global map showing the distribution of B. pseudomallei was generated by ArcGIS (10.0, Redlands, CA) 10.1371/journal.pntd.0002105.t001 Table 1 Global distribution of environmental B. pseudomallei. Level of evidence Definition Countries Definite (1) Organism isolated from soil or water with adequate identification by culture or a B. pseudomallei-specific PCR, and (2) Evidence for melioidosis having been acquired in that country Asia (Cambodia [98], China [31], [48], [49], Iran [50], Lao PDR [9], [63], Malaysia [28], [58], [68], [77], Singapore [78], [99], Sri Lanka [51], Taiwan [52]–[55], Thailand [11], [15], [16], [37], [39], [57], [60], [64]–[66], [84], [85], [100], [101] and Vietnam [19], [26], [102], [103]), Oceania (Australia, [17], [18], [20], [24], [25], [27], [38], [42], [59], [61], [62], [71]–[75], [104] and Papua New Guinea [67]), Africa (Burkina Faso [40], Madagascar,[14], Niger [40]), Europe (France [14], [29], [30])*, and, South America (Brazil [14], [46], [47]) Probable (1) No report identified of B. pseudomallei isolation from soil or water, and (2) Evidence for melioidosis having been acquired in that country Asia (Bangladesh, Brunei, Egypt, India, Indonesia, Myanmar, Pakistan, Philippines and Saudi Arabia), Ocenia (Fiji), Africa (Chad, Gambia, Kenya, Nigeria, Sierra Leone, South Africa and Uganda), Central America (Costa Rica, El Salvador, Honduras, Mexico and Panama), South America (Colombia, Ecuador, Puerto Rico and Venezuela), Europe (Turkey), and Others (Aruba, Guadeloupe, Guam, Mauritius, Martinique, New Caledonia, Puerto Rico) [4], [12] Possible (1) Organism isolated from soil or water that was considered to be B. pseudomallei, but (2) identification process not sufficient to exclude other, non-pathogenic environmental Burkholderia spp. such as B. thailandensis, and 2) No evidence for melioidosis having been acquired in that country Côte d'Ivoire [14], Haiti [14], Italy [21] and Peru [14] * In France, soil culture positive for B. pseudomallei was initially reported in the ‘Jardin des Plantes’ in Paris after an outbreak of animal melioidosis which was thought to have originated from a panda imported from China, and the organism was subsequently reported to have been detected in soil throughout the country [14], [29], [30]. There is no evidence to suggest its continuing presence. Recommendations Forming the working party The Detection of Environmental Burkholderia pseudomallei Working Party (DEBWorP) was formed during the VIth World Melioidosis Congress held in Townsville, Australia in December 2010. Following an announcement of the initiative, interested individuals were identified, the consortium formed, and email used to communicate with its members. Development of consensus on the detection of B. pseudomallei in soil A questionnaire was formulated by four investigators (DL, DABD, BC and SJP) based on areas of variation in practice relating to study design and methodology for the detection of B. pseudomallei in soil (Text S2). This was sent to all members of DEBWorP. Answers and comments were collated, and areas of common and variant practice identified. A second questionnaire was developed to cover areas of variant practice, which was again sent to all members. Recommendations on best practice were reached based on a combination of information from both questionnaires, and circulated to the working party members for final approval. The recommendations did not include study design and methodology for detection of B. pseudomallei in air or water, or quantitation of B. pseudomallei in soil. Results and Discussion Literature Review 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). 10.1371/journal.pntd.0002105.g001 Figure 1 Flow diagram showing study selection. 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]. Global distribution of environmental B. pseudomallei There was ‘definite’ evidence for the presence of environmental B. pseudomallei in 17 countries (Table 1 & Figure 2). Eight were either in southeast Asia (Cambodia, Lao PDR, Malaysia, Singapore, Thailand and Vietnam) or Oceania (Australia and Papua New Guinea), with the remainder (n = 9) being Brazil [46], [47], Burkina Faso [40], China [31], [48], [49], France [14], Iran [50], Madagascar [14], Niger [40], Sri Lanka [51] and Taiwan [52]–[55]. The area sampled within each country was nearly always limited (Table S1). In France, soil culture positive for B. pseudomallei were initially reported in the ‘Jardin des Plantes’ in Paris after an outbreak of animal melioidosis, which was thought to have originated from a panda imported from China, but the organism was subsequently reported to have been detected in soil throughout the country [14], [29], [30]. Although one clinical and one environmental strain isolated in France were later confirmed as B. pseudomallei by genotyping [41], there was insufficient information given about the identification of B. pseudomallei isolated from multiple soil samples collected from across France to be entirely sure of their identity, and they are not available for further testing [14], [29], [30]. Importation followed by environmental treatment to eradicate B. pseudomallei will result in a change in classification, but it is unclear from the literature whether B. pseudomallei has been eradicated in France. A further 34 countries were assigned to the ‘probable’ category based on clinical evidence of indigenous melioidosis but lack of environmental studies. Two studies described the molecular identification or genotyping of environmental B. pseudomallei isolates from Ecuador, Kenya and Venezuela [41], [56], but no environmental sampling studies positive for B. pseudomallei were identified for these countries in the published literature. A total of 4 countries including Côte d'Ivoire [14], Haiti [14], Italy [21] and Peru [14] were assigned to the ‘possible’ category (Table 1 & Figure 2) based on inadequate bacterial confirmation of putative environmental B. pseudomallei combined with a lack of evidence for indigenous melioidosis. 10.1371/journal.pntd.0002105.g002 Figure 2 Global map showing the distribution of B. pseudomallei. Definitions of definite, probable and possible presence of environmental B. pseudomallei are described in Table 1. 1 represents ‘Jardin des Plantes’ in Paris where soil cultures positive for B. pseudomallei were initially reported after an outbreak of melioidosis, which was thought to have originated from a panda imported from China [14]. 2 represents Bologna, Italy, where B. pseudomallei in tap water (6 out of 85 specimens) was reported in 2000 [21]. However, confirmation of B. pseudomallei by specific identification methods was not reported. 3 represents Chittering, southwest Western Australia, where B. pseudomallei was isolated and confirmed from a single soil specimen in 1980, following the outbreak of melioidosis in animals [17], [38]. There has been no evidence of environmental B. pseudomallei in southwest Western Australia since then. Sampling strategies used for the detection of environmental B. pseudomallei Published sampling strategies for the detection of environmental B. pseudomallei are shown in Table 2. Sampling was performed in both dry and wet seasons, and sampling duration ranged from 1 day to 3 years [57]. A consistent difference in positivity rates between the wet and dry season was not established. Three studies found a higher positivity rate in the wet season [35], [58], [59], and two studies reported a higher positivity rate in the dry season [20], [60]. A recent study found that, in a given region, most areas had higher positivity in the wet season but some had a higher positivity in the dry season, which suggested that other factors such as the presence of animals or land use also contribute to differences in positivity rates between wet and dry seasons [61]. 10.1371/journal.pntd.0002105.t002 Table 2 Published and recommended sampling strategies for the isolation of B. pseudomallei from soil. Sampling strategy Published sampling strategy Consensus guideline Sample size calculation Not stated and often low sample size Sample size calculation should be presented and should correspond with the aims of the study Sampling site selection Variable, including random site selection and practical considerations (e.g. sampling at points along a main road) For pilot studies that are conducted to identify environmental B. pseudomallei in areas where sampling has not been done previously, choose sites most likely to be positive based on available information such as areas around households or working fields of melioidosis patients. If such information is unavailable, use the GIS program to randomly select sampling sites For large environmental surveys in areas where B. pseudomallei is known to be present in the environment, use the GIS program to randomly select sites across the designated region Sampling points per site Ranged from 2 to 100 points per field Use a fixed interval sampling grid To determine presence of B. pseudomallei in one field (around 50×50 sq meters), 100 points per site To determine presence or distribution of B. pseudomallei in a wider area, number of points per site and number of sites should be calculated based on geo-statistical sample size calculation which should provide the confidence level required Distance between sampling point within a sampling site 1 to 5 meters, or not reported If no prior information available for B. pseudomallei distribution in test area, take samples at a distance of 2.5 to 5 meters apart If prior information is available, samples should be taken at an optimal distance based on geo-statistical sample size calculation Soil sampling depth Ranged from 0 to 90 cm of depth 30 cm depth Weight of soil sample per sampling point Ranged from 2 to 1,000 gram of soil 10 gram of soil (put into universal tube) Temperature during transportation of sample to laboratory Variable, including room temperature and refrigerated temperature At ambient temperature and away from direct sunlight or heat source Process soil samples as soon as possible Of 61 studies, 55 evaluated the presence of B. pseudomallei in soil, and 35 in water. The majority of studies chose sampling sites on an ad hoc basis. Of 54 studies with information about land use for the sampling site, 20 were conducted in rice fields and 35 in other areas including animal pens, residential areas around the homes of cases, forests, scrubland, and agricultural fields containing other crops. Most studies collected a low number of samples (2 to 7) per study site, and did not provide a detailed description of the sampling design or strategy, sample size calculation or distance between sampling points within each site. Three articles described random selection of the study site in a given area using GPS, and provided a detailed sampling strategy [61]–[63]. The largest number of samples collected from a single site was 100, in which samples were collected using a fixed interval grid [63]–[65]. Soil sampling depth ranged from surface to 90 cm. The weight of each soil sample collected ranged from 2 to 1,000 grams [18], [43]. Methods of B. pseudomallei detection in soil The methodology used to detect B. pseudomallei in soil samples has 2 main stages: (i) bacterial extraction, and (ii) detection methods using culture or PCR (or historically, animal inoculation) (Table 3). The process of bacterial extraction involves the addition of a solution to the soil, mixing with various degrees of homogenization, and a period of settling prior to removal of the supernatant. The solution used has varied between distilled water, normal saline, detergent solution [66], or enrichment media, with a variable soil to solution ratio (wt/vol) ranging from 2∶1 to 1∶10 [49], [57]. The method used to mix the soil and solution has varied between manual shaking, vortexing or use of an orbital shaker. The time period used to mix the solution has varied from less than 1 minute to 48 hours [61], [67], and the time for soil sedimentation after mixing from 5 minutes to 24 hours [66], [67]. The volume of fluid used for culture has varied from 0.5 to 10 ml of supernatant [60], [67], or the spun deposit of 80 ml of supernatant [35]. The volume used for DNA extraction prior to PCR has varied from 3 ml of supernatant [53], the deposit of 20 ml of supernatant [61], [62], or direct extraction from different weight of soil [39], [52], [54]. The volume used for guinea-pig or hamster inoculation has varied from 1 to 2 ml [57], [68]. 10.1371/journal.pntd.0002105.t003 Table 3 Published and recommended methodologies for the isolation of B. pseudomallei from soil. Methodologies Published methods Consensus guideline B. pseudomallei extraction solution Distilled water, normal saline, detergents or enrichment media Threonine-basal salt plus colistin 50 mg/L (TBSS-C50 broth) Ashdown broth containing colistin and crystal violet is an alternative Ratio of soil and extraction solution (wt/wt) Ranged from 2∶1 to 1∶10 1∶1 (10 gram of soil to 10 ml of TBSS-C50 or Ashdown broth) Extraction method Manual shaking, vortexing or orbital shaker Vortexing for 30 seconds Manual mixing of soil is an alternative option, and may be required if sample is compacted Techniques for detection of B. pseudomallei Culture, PCR or animal inoculation Culture (PCR could be added as an additional technique if available) Protocol for culture Variable, including direct culture on solid media and quantitation, and qualitative methods relying on broth enrichment Incubate the specimen (universal tube with 10 gram of soil plus 10 ml TBSS-C50 or Ashdown broth) for 48 hours Temperature of incubator Variable, ranged from 37 to 42°C 40°C is recommended, and 37–42°C is an alternative option Protocol for sub-culture Variable Subculture 10 µL of supernatant onto an Ashdown agar plate, and streak to achieve single colonies Incubate plate and examine every 24 hours for 7 days Identification of B. pseudomallei Variable, including basic microbiological tests (which include typical colony morphology, Gram stain, positive oxidase test, inability to assimilate arabinose, resistance to gentamicin and colistin with susceptibility to co-amoxiclav) and biochemical kits (including API20NE [105] and Vitek) with or without additional confirmatory tests (specific latex agglutination test [89], or a specific PCR assay [62], [75], [91], [93]) Basic microbiological tests (which include typical colony morphology, Gram stain, positive oxidase test, inability to assimilate arabinose, resistance to gentamicin and colistin with susceptibility to co-amoxiclav) is mandatory plus at least one confirmatory test (API20NE, Vitek system, specific latex agglutination test [89] or a specific PCR assay [62], [75], [91], [93], unless latex test or PCR assay was used during screening) Specific latex agglutination test [89], or a specific PCR assay [62], [75], [91], [93] can be used a screening test The most common detection method has been culture using selective media (n = 46). Most protocols used a selective enrichment broth (n = 44), with a variable specimen to medium ratio (vol/vol) ranging from 1∶1 to 1∶20 [67]. The broth used varied and included tryptone soya broth plus crystal violet (5 mg/l) and colistin (20 or 50 mg/l) (CVCB or Ashdown broth) [69], and L-threonine buffered salt solution (TBSS or Galimand and Dodin broth) [14] with or without colistin (20 or 50 mg/l). Culture of bacterial extraction solution on selective agar plates was described in 15 studies, and Ashdown agar was commonly used [69]. The volume of fluid inoculated onto each agar plate varied from 10 to 400 µl [9], [64]. Temperature of incubation varied between 30 and 42°C [19], [67]. The overall efficiency of different techniques at each stage has not been adequately compared. In eight studies using both culture and PCR, the positivity rate for B. pseudomallei was higher by PCR than by culture [20], [39], [51]–[55], [62], [64], [70]. Methods used to detect B. pseudomallei in water The methodology used to detect B. pseudomallei in water samples has 2 main stages: (i) bacterial concentration, and (ii) detection methods using culture or animal inoculation. The volume of each water sample collected ranged from 1 to 5,000 ml [24], [59], [71]–[74]. The method used for bacterial concentration has varied between filtration, centrifugation [43], or precipitation with potassium alum [27]. Filter pore size has varied from 0.20, 0.22 or 0.45 µm [18], [21], [25], [42], [73]–[76]. The volume of fluid used for direct culture was 50 ml, from which the bacteria were extracted either by centrifugation [43], or using potassium alum [27]. The volume used for guinea-pig or hamster inoculation has varied from 1 to 2 ml [24], [58], [68], [71], [77], [78]. The first evidence of B. pseudomallei in water came from a study published in 1937 which involved immersion of a guinea pig in water following scarification of its abdomen, following which B. pseudomallei was isolated from its blood [26]. The relative sensitivity of detection using culture versus animal inoculation has not been reported. Methods used to detect B. pseudomallei in air There are no studies in PubMED that report air sampling for B. pseudomallei. An MSc thesis written by Kinoshita contains details of the culture of B. pseudomallei from air at the Hong Kong oceanarium in 1989, 1993 and 1995 [31]. The sampling technique used was to hold an agar plate at about shoulder level to oncoming winds during a typhoon. Kinoshita repeated air sampling by collecting 171 typhoon samples between 1999 and 2002, but all were culture negative for B. pseudomallei [31]. Recommendations on the Detection of B. pseudomallei in Soil 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. 10.1371/journal.pntd.0002105.t004 Table 4 Publishing the findings of studies conducted to isolate B. pseudomallei from soil. Reporting the findings Published findings Consensus guideline After publication, deposit raw data to website Variably reported After publication, raw data can be deposited to website www.melioidosis.info at the discretion of PI and sponsor of each study GPS location of study site Variably reported After publication, GPS data can be deposited to website www.melioidosis.info at the discretion of PI and sponsor of each study taking account of issues of anonymity. Positivity rate in each study site and pattern of positivity in each study site Variably reported Describe in the manuscript if available. After publication, details of results can be deposited to website www.melioidosis.info at the discretion of PI and sponsor of each study Soil type and history of land use Variably reported Describe the current land use in the manuscript, together with the history of land use if available Describe the soil texture using previously described method such as ribbon test [97]. After publication, details of results can be deposited to website www.melioidosis.info at the discretion of PI and sponsor of each study Sampling time and weather at sampling time point (e.g. rainfall, season) Variably reported Describe in the manuscript. After publication, details of results can be deposited to website www.melioidosis.info at the discretion of PI and sponsor of each study Choice of sampling site and strategy The most appropriate sampling strategy will depend on the objectives of the study, and whether any information is already available for the geographical area to be sampled (Table 2). For pilot studies that are conducted to identify B. pseudomallei in the environment in areas where sampling has not been performed previously, investigators should gather any available information about possible or definite melioidosis cases in the locality, and sampling site selection should target their residence or work place. In the absence of such information, a less targeted approach will be required in which GIS (geographic information system) software is used to support the random identification of several pilot locations in a given region or country. For large environmental surveys in areas where B. pseudomallei is known to be present in the environment, selection of sampling sites using GIS software is also recommended. Within a given location (study site), we recommend the use of a fixed interval grid based on its simplicity and the need for standardization. Number of samples Taking an insufficient number of soil samples from a designated sampling site runs the risk of a false negative result [11]. This may be due to insensitive detection methods, or because saprophytic bacteria exist in aggregates and can give rise to hot spots and intervening areas that are negative for a specific bacterium. This has been shown to be the case for B. pseudomallei [11]. Because of this, random sampling methods using a low sample size may be associated with a low power of detection and a high false negative (type II error) rate [79]. This can be avoided by increasing the number of samples taken [11]. Based on statistical considerations, to determine the presence of B. pseudomallei in an area of around 50×50 sq meters, a minimum of 100 sampling points is suggested. This is strongly supported by a recent study in Lao PDR in which one field was deemed positive based on only 1 out of 100 positive sampling points [63]. If a region is already known or highly suspected to be positive for B. pseudomallei, an alternative approach is to use adaptive sampling in which a pilot study is performed in a defined experimental area in which a number of random points (e.g. 20) are sampled. If any are positive for B. pseudomallei, this confirms the presence of the organism and is sufficient to define this as an area of risk for humans and livestock. If all samples are negative, a second round of sampling is done in which 100 samples are taken from the same site using a fixed interval grid. To determine the presence or distribution of B. pseudomallei in a wider area, the number of samples taken per site and the number of sites investigated could be calculated based on a geo-statistical sample size calculation [80], [81]. Distance between samples The presence of hot spots for a specific bacterium in the environment leads to an effect described by the term ‘spatial autocorrelation’, which influences the distance required between each sampling point. What this means in practice is that sampling points adjacent to each other are more likely to yield the same result (e.g. a sample next to a negative sample is likely to be negative) [11]. The distance over which counts of a given environmental bacterium are related (range of spatial autocorrelation) can be defined using a geostatistical tool called the semivariogram [80]. Ideally, the effect of spatial autocorrelation would be factored in to the sampling strategy for B. pseudomallei, but this value is likely to be influenced by physicochemical soil parameters and vegetation [82], and vary between and possibly within countries. Therefore, it is not practical to define this prior to formal sampling in most settings. Studies in Thailand suggest that the distance between samples should be between 2.5 and 5 m apart [11], although it is uncertain whether this applies elsewhere. Given the paucity of data on the optimal distance between samples we suggest that sampling be performed 2.5 to 5 m apart, accepting that this is somewhat arbitrary. The optimal sampling distance specific to the study region could be subsequently estimated based on the results of pilot study data for 100 sampling points for one or more sites [80]. Soil sampling: quantity, sampling depth and transport to the laboratory We recommend a depth for soil sampling of 30 cm. This is based on published evidence that the proportion of samples that are culture positive for B. pseudomallei is higher at 30 cm than at a shallower depth, but comparable to samples taken deeper than 30 cm [35], [47], [53], [60], [62], [83], [84]. The quantity of soil collected per sample has varied markedly in published studies, and there is no evidence that collecting a greater weight of soil is associated with a higher sensitivity. We suggest taking a weight of 10 grams per sample based on practicality and ease of methodology [85]. As there is evidence showing that survival of B. pseudomallei is decreased at low temperatures [86], soil samples should be kept at ambient temperature (24 to 32°C) and away from direct sunlight or heat source during transportation to the laboratory. The specimen should be processed as soon as possible. Extraction of bacteria from soil, and detection and identification of B. pseudomallei We recommend the use of culture as the standard method for environmental B. pseudomallei detection in the context of global mapping efforts on the basis of simplicity, specificity and low cost (Table 3). The optimal ratio of soil to extraction solution, mixing technique and sedimentation time are not known. Selective broths have been compared in both laboratory [87] and field settings [20], [60]. We proposed that each 10 gram soil sample be placed into a universal tube, mixed with 10 ml of enrichment medium (either TBSS with colistin 50 mg/l (TBSS-C50) or Ashdown broth), vortexed for 30 seconds, and incubated at 40°C in air for 48 hours. Based on scientific evidence and agreement of the working party, TBSS-C50 is recommended as the primary enrichment medium with Ashdown broth as an alternative. A volume of 10 µl of the upper layer of enrichment medium should be streaked to achieve single colonies onto a whole Ashdown agar plate, incubated at 40°C in air and examined every 24 hours for 7 days. This incubation temperature was chosen based on evidence that it allows growth of B. pseudomallei [88], but is inhibitory to some other soil flora (personal observation by DABD and VW). However, incubation at 37°C is acceptable in the event that resources are not available to incubate at 40°C. Subculture of 10 µl is based on experience in Thailand and represents a balance between detection of B. pseudomallei and limiting the bioburden of other flora that grow on the agar plate. Subculture of higher volumes (100 µl) may be associated with a higher yield although there currently is no published evidence to support this. Several steps of the method recommended here (direct culture of 10 gram of soil in 10 ml of TBSS-C50 and subculture onto Ashdown agar) are based on methods in widespread use in Australia [62], [75]. Furthermore, the sensitivity of our recommended method was recently compared to a more laborious method which has been used extensively in Thailand [85]. The latter involves collection of 100 gram of soil which is mixed with 100 ml of distilled water, left to settle overnight, and the upper layer of water removed for culture on Ashdown agar and in TBSS-C50. In the comparative study, 94 out of 200 soil samples were culture positive for B. pseudomallei [85]. Yield was not different between the two methods (70/94 vs. 79/94 respectively; p = 0.15), supporting the use of our currently recommended method. Identification of B. pseudomallei Any colony with a colony morphology suggestive of B. pseudomallei can be tested by basic microbiological tests (typical colony morphology on Ashdown agar, Gram stain, positive oxidase test, inability to assimilate arabinose, resistant to gentamicin and colistin, susceptible to co-amoxiclav) followed by confirmatory tests (specific latex agglutination test [89], a specific PCR assay [53], [55], [62], [75], [90]–[95], or validated identification kits such as API20NE or Vitek system). The API20NE database does not include a profile of B. thailandensis, which give results that are similar to those for B. pseudomallei except that B. thailandensis is positive for arabinose assimilation. For rapid evaluation, a specific latex agglutination [89], [96] or PCR assay [53], [55], [62], [75], [90]–[95] could be used as a screening test, followed by basic microbiological tests to complete the identification process. Data presentation and data sharing We propose that publication of studies on environmental detection of B. pseudomallei include the positivity rate and pattern of positivity over 100 sampling points, history of land use, date of sampling, weather conditions and soil texture (%sand, loam and clay) using the methodology described previously [97] (Table 4). DEBworP is in the process of developing a website (www.melioidosis.info) where complete data from sampling studies can be deposited with the assistance of a curator (DL), and at the discretion of the principal investigator and sponsor of each study. This will be used to build an interactive global map of the distribution of environmental B. pseudomallei, as well as those places where melioidosis has been acquired in humans and animals. The website will also provide downloadable protocols describing methodology for soil sampling and culture, including details of each reagent and test used (Text S3). The recommended protocols have been successfully used in Thailand [85], although further evaluation of these is required in different countries. Although the methodology presented here aims to reduce the risk of false negative sampling surveys, this is unlikely to be perfect. As a result, a single negative sampling survey does not represent definite evidence that the site is free of B. pseudomallei, although it would be predicted to reflect a region of much lower risk compared with a positive site. The need to undertake further sampling requires consideration of risk-benefit. There is also considerable scope to improve on the methodology described here, including improvement in the sensitivity of culture which could include the development of media that are even more selective for B. pseudomallei in soil, and ultimately the development of easy-to-use and accurate diagnostic kits for environmental sampling. Our recommendations will be updated in the future as and when new information or knowledge becomes available. Concluding comments Our knowledge of the global distribution of B. pseudomallei is incomplete, and the methodology to determine the presence of this organism in the environment has not been standardized and is liable to false negativity (if insufficient samples are taken or inappropriate techniques are used), and false positivity (if methods are not adequate to exclude related Burkholderia species). We have provided consensus guidelines on strategies and methodologies to determine the presence of B. pseudomallei in soil that are simple and applicable in settings with limited resources. To develop a complete risk map of melioidosis, our working party aims to support and promote environmental studies on a global scale, supported by a website (www.melioidosis.info) with downloadable protocols and a mechanism for data collection and sharing. Supporting Information Table S1 Characteristics of studies included in the review. (DOC) Click here for additional data file. Table S2 PRISMA checklist. (DOC) Click here for additional data file. Text S1 Data extraction form for studies that determined the presence of Burkholderia pseudomallei in the environment. (DOC) Click here for additional data file. Text S2 Questionnaire on the detection of environmental Burkholderia pseudomallei. (DOC) Click here for additional data file. Text S3 Standard Operating Procedure (SOP): simplified method for the isolation of Burkholderia pseudomallei from soil. (DOC) Click here for additional data file.
Author and article information
Journal
Journal ID (nlm-ta): Am J Trop Med Hyg
Journal ID (iso-abbrev): Am. J. Trop. Med. Hyg
Journal ID (publisher-id): tpmd
Title:
The American Journal of Tropical Medicine and Hygiene
Publisher:
The American Society of Tropical Medicine and Hygiene
ISSN
(Print):
0002-9637
ISSN
(Electronic):
1476-1645
Publication date
(Print):
06
August
2014
Publication date PMC-release: 06
August
2014
Volume: 91
Issue: 2
Pages: 287-290
Affiliations
Department of Microbiology, Ruminant Clinic and Department of Pathology, Faculty of
Veterinary Medicine, Mahanakorn University of Technology, Bangkok, Thailand; Mahidol-Oxford
Tropical Medicine Research Unit, Department of Microbiology and Immunology and Department
of Tropical Hygiene, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand;
Center for Clinical Vaccinology and Tropical Medicine, Nuffield Department of Clinical
Medicine, University of Oxford, United Kingdom; Department of Epidemiology and Diseases
Surveillance, Armed Force Research Institute of Medical Science, Bangkok, Thailand;
Department of Medicine, University of Cambridge, Cambridge, United Kingdom
Author notes
*Address correspondence to Direk Limmathurotsakul, Mahidol-Oxford Tropical Medicine
Research Unit, Faculty of Tropical Medicine, Mahidol University, 420/6 Rajvithee Road,
Bangkok 10400, Thailand. E-mail:
direk@
123456tropmedres.ac
Article
DOI: 10.4269/ajtmh.14-0115
PMC ID: 4125250
PubMed ID: 24891468
SO-VID: acf65e3b-95b4-4c2c-a38c-6bdb0e121203
Copyright © ©The American Society of Tropical Medicine and Hygiene
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History
Date
received
: 23
February
2014
Date
accepted
: 14
April
2014
Categories
Subject:
Short Report
Subject:
Articles
ScienceOpen disciplines: Infectious disease & Microbiology
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ScienceOpen disciplines: Infectious disease & Microbiology
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