Burkholderia pseudomallei is a Gram-negative bacterium found in soil and water in many tropical countries. It causes melioidosis, a potentially fatal infection first described in 1911 in Myanmar. Melioidosis is a common cause of sepsis and death in South and South-east Asia, but it is rarely diagnosed in Myanmar. We conducted a nationwide soil study to identify areas where B. pseudomallei is present.
We collected soil samples from 387 locations in all 15 states and regions of Myanmar between September 2017 and June 2019. At each site, three samples were taken at each of three different depths (30, 60 and 90 cm) and were cultured for B. pseudomallei separately, along with a pooled sample from each site (i.e. 10 cultures per site). We used a negative binomial regression model to assess associations between isolation of B. pseudomallei and environmental factors (season, soil depth, soil type, land use and climate zones). B. pseudomallei was isolated in 7 of 15 states and regions. Of the 387 sites, 31 (8%) had one or more positive samples and of the 3,870 samples cultured, 103 (2.7%) tested positive for B. pseudomallei. B. pseudomallei was isolated more frequently during the monsoon season [RR-2.28 (95% CI: 0.70–7.38)] and less in the hot dry season [RR-0.70 (95% CI: 0.19–2.56)] compared to the cool dry season, and in the tropical monsoon climate zone [RR-2.26; 95% CI (0.21–6.21)] compared to the tropical dry winter climate zone. However, these associations were not statistically significant. B. pseudomallei was detected at all three depths and from various soil types (clay, silt and sand). Isolation was higher in agricultural land (2.2%), pasture land (8.5%) and disused land (5.8%) than in residential land (0.4%), but these differences were also not significant.
Melioidosis is caused by a soil dwelling bacterium called Burkholderia pseudomallei. It is a potentially fatal disease even if appropriate antibiotics are given. The disease was first described in Rangoon (now Yangon) in 1911 but it has rarely been reported in Myanmar over the past century even though it is now known to be a common cause of community-acquired infection in neighbouring countries in South and Southeast Asia. The true melioidosis burden in Myanmar is obscured by a lack of awareness among clinicians and limited diagnostic facilities to identify the causative organism. To explore this issue, we conducted a nationwide study to assess the presence of B. pseudomallei in soil samples collected from all fifteen states and regions of Myanmar. Our study revealed a widespread distribution of the organism in the Myanmar environment. This finding contributes to the understanding of the global distribution of B. pseudomallei and should alert clinicians throughout Myanmar to consider melioidosis as a potential cause of fever, sepsis, pneumonia and abscesses.
Citation: Swe MMM, Win MM, Cohen J, Phyo AP, Lin HN, Soe K, et al. (2021) Geographical distribution of Burkholderia pseudomallei in soil in Myanmar. PLoS Negl Trop Dis 15(5): e0009372. https://doi.org/10.1371/journal.pntd.0009372
Editor: Alfredo G. Torres, University of Texas Medical Branch, UNITED STATES
Received: October 6, 2020; Accepted: April 7, 2021; Published: May 24, 2021
Copyright: © 2021 Swe et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This project was supported by the Planet Wheeler foundation (http://www.planetwheeler.org/, MAM2017-19) awarded to F.S. The Myanmar Oxford Clinical Research Unit is part of the Mahidol Oxford Tropical Medicine Research Unit (MORU) network and was funded by the Wellcome Trust (https://wellcome.org/, 106698) awarded to FS, MMMS, APP. This research was funded in whole, or in part, by the Wellcome Trust (Grant number 220211). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Melioidosis is a serious tropical infection caused by the Gram-negative bacterium Burkholderia pseudomallei. It is endemic in tropical areas of South and South-east Asia and Northern Australia and is increasingly being detected in many other tropical regions. It often presents with life threatening sepsis, pneumonia or abscesses in the internal organs, [2,3] but the presentation can also be uncharacteristic making it very difficult to differentiate from other diseases clinically, hence its nickname “the remarkable imitator”.[4–10] In addition, laboratory diagnosis is not straightforward and the organism is often dismissed as a contaminant or misidentified.[11,12]
B. pseudomallei is intrinsically resistant to a wide range of antibiotics (penicillin, ampicillin, first- and second-generation cephalosporins, aminoglycosides, macrolides and polymyxins) and routine treatment protocols for sepsis and pneumonia frequently do not cover B. pseudomallei. Treatment usually comprises at least 10 days of parenteral antibiotics (ceftazidime or a carbapenem) followed by oral ‘eradication’ antimicrobials for 3 to 5 months. Even with effective antimicrobial therapy, case fatality rates still reach up to 50%. In 2016, it was estimated that 165,000 (95% credible interval 68,000–412,000) cases of melioidosis occurred annually with 89,000 (36,000–227,000) deaths worldwide , representing a higher disease burden than that associated with many infections classified as Neglected Tropical Diseases.
B. pseudomallei can be found in soil and surface water of endemic regions [16–19] and infection is acquired by direct inoculation with contaminated soils, ingestion or aspiration of contaminated water or inhalation of dusts or aerosols. It occurs commonly in farmers or other people who are regularly exposed to soil and surface water. People with underlying health conditions such as diabetes mellitus or chronic renal disease and patients on treatment with immunosuppressive drugs are at particular risk of contracting the disease.[20,21]
Melioidosis was first recognised in Rangoon (now Yangon), Myanmar in 1911. Since then it has become recognised as an important public health problem in some countries, mainly in north Australia  and South East Asia.[23,24] However, a lack of awareness amongst clinicians and laboratory staff, and the poor availability of diagnostic facilities means that there is significant under-recognition.
In Myanmar only 298 melioidosis cases have been reported since its first discovery in 1911 and most of these were identified before 1949. However, it is unlikely that the bacterium has disappeared from the environment and the incidence is probably significantly underestimated. Limmathurotsakul et al. suggested that the burden of melioidosis in Myanmar was more than 6,000 cases and 3,000 deaths per year.
Recently two small studies confirmed the presence of B. pseudomallei in the soil in three regions (Yangon, Ayeyarwaddy and Bago) [26,27] but the national distribution in Myanmar remains unknown. We therefore carried out a nationwide study to identify the geographical distribution of B. pseudomallei in soil in Myanmar. This information can be used as a basis for further investigation into melioidosis as a public health problem in Myanmar.
Materials and methods
The study was approved by Ethics Review Committee, Department of Medical Research, Ministry of Health and Sports, Myanmar (Approval number: Ethics/DMR/2017/087).
Myanmar is a lower middle income country situated in Southeast Asia with a population of approximately 54 million, of whom 70% live in rural areas. The country has a diverse landscape with central lowland areas surrounded by mountains in the north, east and west, and coastline along the south. The climate is largely tropical, with a temperate climate in some areas in the North and a dry steppe climate in a small area in the centre of the country (Fig 1). The monsoon season is from mid-May to October, followed by a cool, dry season (November to February) and a hot, dry season (March to mid-May) .
A. Map of the states and regions of Myanmar with corresponding population density (person per square km). B. Map of Myanmar climate zones based on the Koppen Geiger climate classification (Source: http://koeppen-geiger.vu-wien.ac.at/present.htm). Black dots represent B. pseudomallei-positive sites (at least one sample positive for B. pseudomallei) and white dots represent B. pseudomallei-negative sites).
Sample site selection
We collected soil samples from all 15 states and regions of Myanmar; Kachin, Kayar, Kayin, Mon, Rakhine, Shan, Chin, Sagaing, Mandalay, Magway, Bago, Ayeyawady, Yangon, Tanintharyi and Naypyitaw. Eight hundred locations were generated by random sampling using a Geographic Information System (QGIS version 3.4.9 https://qgis.org/). These points were subsequently evaluated and 413 sites were excluded based on geographic characteristics (uninhabited forest, mountains or lakes), safety (armed conflict areas), and extreme remoteness (far away from accessible roads). We collected soil samples from the remaining 387 sites. At these sites, we selected locations purposively based on factors believed to be associated with the presence of B. pseudomallei and the presence of people, such as moist soil, animal grazing land and disused land within the proximity of a village. We classified the type of current land use into four categories; agricultural land used for rice and other plantation, pasture land (livestock grazing area), residential area and disused land. The soil type was classified by physical texture as sand, silt or clay. We selected more sites in the states and regions where population density is high (Ayeyawady, Bago, and Yangon), and in the states and regions with a larger geographic area (Sagaing and Shan). Overall, we collected soil samples from 161 of the 330 townships of Myanmar.
The consensus guidelines for environmental sampling of B. pseudomallei recommends collecting 100 samples per site. However, as we wanted to explore a very large geographical area, we chose to take fewer samples per site (10) in favour of a greater number of sites. At each selected site, a fixed interval sampling grid method was used to make nine holes (each hole five meters from its neighbours) at three different depths; three at 30cm, three at 60cm and three at 90cm, since the proportion of B. pseudomallei-positive samples at these different depths has varied in other studies. We used a mechanical auger to dig the holes at each selected site. The auger and shovel were cleaned with domestic water and 70% alcohol in between sites to avoid cross-contamination. Two samples (10 grams each) were collected from each hole. One sample was kept for culture while the second samples from each hole were pooled (weight 90 grams) and mixed on site before being sent for culture. We pooled samples in order to determine the sensitivity of pooled samples in comparison to individual samples as an attempt to simplify sampling procedures for detecting B. pseudomallei in soil. The 10 samples were kept in separate sealed plastic bags and stored at ambient temperature and away from direct sunlight. The samples were transported in a cool box and cultured within 48 hours at the Department of Medical Research in Yangon. Geographic coordinates (latitude and longitude), soil type (clay, silt, sand), and land use status were recorded at each sampling site.
Isolation of B. pseudomallei
Ten grams of individual soil samples were mixed with 10 ml of threonine-basal salt solution plus colistin 50mg/L (TBSS-C50) in a universal tube. The pooled 90 gram soil samples were mixed with 90 ml of TBSS-C50 solution. The tubes were vortexed for 30 seconds before incubating at 40°C for 48 hours. After that, 10 μl of the supernatant liquid was sub-cultured onto Ashdown agar and streaked to achieve single colonies. The plates were incubated at 40°C in air and checked daily for bacterial growth for the next 4 days. Colonies suspected of being B. pseudomallei were selected based on their typical appearance (purple colour, metallic sheen and rough/wrinkled surface). All such colonies underwent an oxidase test and a three disc (co-amoxiclav/colistin/gentamicin) screening test. Colonies that had a positive oxidase test and a compatible antibiogram (resistance to gentamicin and colistin with susceptibility to co-amoxiclav) were confirmed as B. pseudomallei by a specific latex agglutination test and API 20NE.[32,33] Isolates with a compatible colonial appearance and antibiogram that were negative for latex agglutination and positive for arabinose assimilation in the Analytical Profile Index (API) 20NE were identified as Burkholderia thailandensis. The identity of a subset of seventeen isolates was confirmed by polymerase chain reaction (PCR) targeting the Type III Secretion System of B. pseudomallei using the method adapted and validated at Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit from a previously published assay [34,35] and seven isolates were also confirmed by Whole Genome Sequencing (WGS).
We categorised the 15 states and regions into 3 climate zones based on the Koppen Geiger climate classification of Myanmar; the tropical monsoon zone along the coastal regions, the tropical dry winter/dry steppe zone in the central part of the country and the temperate dry winter zone in the north (Fig 1). To investigate the association between environmental factors and isolation of B. pseudomallei, we used negative binomial regression to analyse the data. The outcome was the total number of positive B. pseudomallei samples across all depths at each site without clustering at hole level. We fitted univariable negative binomial regression model for the variables; climate zones (tropical monsoon, tropical dry winter/dry steppe and temperate dry winter), seasons (hot dry, monsoon and cool dry), soil type (clay, silt and sand), current land use (residential, agriculture, pasture land and disused land that did not vary at site level. To explore association between soil depth (three different depths at each site) and B. pseudomallei positivity, we analysed the outcome as the number of positive samples at site-depth level and fitted the site as random effect in the model to account for clustering of depths within each site. We did not perform multivariable regression because there were too few sites with positive samples (n = 31) to reliably estimate adjusted associations. We also fitted the model for a subset analyses between soil depth and season.
We collected 3870 soil samples from 387 sites in 161 townships across all 15 states and regions of Myanmar between September 2017 and June 2019. B. pseudomallei was isolated from 103 (3%) samples at 31 (8%) sites in 21 townships in 7 of the 15 states and regions (Tables 1 and S1). Ayeyawady, Kayin, Bago and Rakhine states and regions had the highest site positivity rates (defined as the percentage of sites with B. pseudomallei isolated from at least one of the 10 samples) at 9 of 36 (25%), 5 of 24 (22%), 6 of 37 (16%) and 2 of 13 (15%) sites respectively. The number of positive samples from each positive site ranged from a minimum of 1 to a maximum of 9 and the median (interquartile ranges) was 3 (1–4). Rakhine had the highest sample positivity rate with 15 of 130 (12%) samples, followed by Ayeyawady at 34 of 360 (9%), Bago at 18 of 370 (5%) and Kayin at 11 of 240 (5%). Although most B. pseudomallei-positive sites were located in the lower half of Myanmar, we did not detect B. pseudomallei in Tanintharyi province, located in the southernmost part of the country (Fig 1).
Details of soil sample culture results related to the climate zone, season of sample collection, soil type, current land use and soil depth are presented in Fig 2 and S2 Table. The results of univariable negative binomial regression analysis are presented in Table 2. We also fitted the model for subset analyses between soil depth and season, and the results are presented in Table 3. In terms of climate zones, 1700 (43.9%) and 1400 (36.2%) of samples were collected from tropical dry winter zone, largely along the main rivers in the central region, and the tropical monsoon zone in the coastal region, while 770 (19.9%) of samples were collected from the temperate dry winter zone in the northern regions (S2 Table).
Bar-plot showing percentage of sample positive for B. pseudomallei and B. thailandensis in different categories of (a) Climate zone (b) Season (c) Soil depth (d) Soil type and (e) Land use. The numbers below each category represent the total number of samples collected in the corresponding category.
We identified B. pseudomallei in 67 (4.8%) samples collected in the tropical monsoon zone and 36 (2.1%) samples in the tropical winter dry zone (Fig 2 and S2 Table). We did not identify B. pseudomallei in any samples collected from the temperate dry winter zone. The tropical monsoon zone had a higher proportion of B. pseudomallei-positive samples [RR-2.26; 95% CI (0.21–6.21), p = 0.11] than the tropical winter dry zone but this was not statistically significant.
B. pseudomallei was isolated from 4.6% (57/1250) of samples collected in the monsoon season compared to 2.0% (31/1550) in the cool dry season and 1.4% (15/1070) in the hot dry season (Fig 2 and S2 Table). Soil samples positive for B. pseudomallei were more common when collected during the monsoon season [RR-2.28; 95% CI (0.70–7.38), p = 0.17] and, less common, in the hot dry season compared to those collected in the cool dry season [RR-0.70, 95% CI (0.19–2.56), p = 0.59] although these associations were not statistically significant (Table 2).
The most common soil type was silt [2590 samples (66.9%)], followed by clay [1200 samples (31.0%)] and sand [80 samples (2.1%)]. The proportion of samples positive for B. pseudomallei was higher among sandy soil [5/80 samples (6.3%)] compared to clay type (36/1200 samples [3.0%]) and silt type [62/2590 samples (2.4%)]. Soil samples positive for B. pseudomallei were more common in sandy soil [RR- 2.61, 95% CI (0.08–86.86), p = 0.59] and in clay type of soil [RR-1.25, 95% CI (0.41–3.84), p = 0.69] compared to the silt type but these associations were not statistically significant.
Of 3870 samples, 3150 (81.4%) were collected from agricultural land, 360 (9.3%) from disused land, 230 (5.8%) from residential areas and 130 (3.4%) from pasture land. Most B. pseudomallei positive cultures came from samples collected from agricultural land (70 samples (2.2%)), but the proportion of B. pseudomallei-positive samples was higher from pasture land (11 samples (8.5%)) and disused land (21 samples (5.8%)), whereas the proportion of positive samples from residential land was lower (1 sample (0.4%)). B. pseudomallei was isolated more often in soil samples collected from pasture land [RR-19.46; 95% CI (0.46–816.13), p = 0.12], disused land [RR-13.42; 95% CI (0.59–307.30), p = 0.10] and rice and agriculture land [RR-5.11; 95% CI (0.32–81.69), p = 0.25] compared to those collected from residential areas but these associations were not statistically significant. The B. pseudomallei isolation rate was 2.4% (28/1161) for both 30 and 60 cm and 2.6% (30/1161) for 90 cm depths (Fig 2). However, the effect of depth on isolation of B. pseudomallei varied between seasons (Table 3). In the cool dry season B. pseudomallei was isolated more often in soil samples collected at 60 cm [RR-2.51; 95% CI (0.64–9.83), p = 0.19] and 90 cm [RR-3.30; 95% CI (0.86–12.65), p = 0.08] compared to those collected at 30 cm but the associations were not statistically significant. In contrast, in the monsoon season B. pseudomallei was isolated less often in soil samples collected at 60 cm [RR-0.66; 95% CI (0.25–1.76), p = 0.41] and 90 cm [RR-0.67; 95% CI (0.26–1.75), p = 0.42] compared to those collected at 30 cm although the associations were not statistically significant. Pooled samples had a significantly higher overall positivity rate than individual samples (p = 0.02). Of the 387 pooled samples, 17 were positive. Thus of 31 sites from which B. pseudomallei was isolated, only half of the pooled samples 54.8% (17/31) had a positive culture. In one site, however, B. pseudomallei was isolated only in the pooled sample but not in any of the individual samples. We also identified B. thailandensis, a close relative of B. pseudomallei, in 72 of 387 (19%) sites and 135 out of 3870 (4%) samples in 13 states and regions (S3 Table). Both B. pseudomallei and B. thailandensis were identified in 4 of the 387 sites.
This study, the first of its kind in terms of coverage and sample size in Myanmar, revealed a widespread distribution of B. pseudomallei in soil. Our main interest was whether B. pseudomallei was present in the soil near human settlements. Therefore, we only collected soil samples from anthropogenically modified areas close to human settlements and native pristine soils were not included. We isolated B. pseudomallei in 103 samples from 31 locations across seven states and regions. All positive samples were located in the more densely populated central and southern coastal areas of the country. We did not isolate B. pseudomallei in samples from the northern and southernmost parts of the country. This was unexpected, as these locations have been predicted to be environmentally suitable for B. pseudomallei  and we are aware of a confirmed case of melioidosis most likely acquired in Kachin in the north of Myanmar (Tun Tun Win pers. comm. 2018). In addition, although we did not isolate B. pseudomallei in the southernmost province, Tanintharyi, which is located in the coastal area between the Andaman Sea and Thailand, a number of culture-confirmed human and animal cases of melioidosis have been reported in neighbouring provinces in western Thailand in recent years. Possible explanations include the relative insensitivity of culture for detecting environmental B. pseudomallei, the uneven distribution of the organism in the environment, [39,40] and the relatively low number of samples tested in the sparsely populated South. It is thus quite possible that B. pseudomallei is also present in the northern and southern parts of Myanmar, but that we simply did not detect it in our study.
Most B. pseudomallei were isolated in the coastal regions of Myanmar, which corresponds to the tropical monsoon climate zone having a high annual rainfall between 2500 and 5000 mm (S4 Table). In the central plain region, which corresponds to the tropical dry winter zone with an annual rainfall between 500 and 1000 mm of annual rainfall,  isolation of B. pseudomallei was less common. In the temperate dry winter zone, in the northern part of the country, we did not isolate any B. pseudomallei. Rivers have been suggested as potential carriers and sentinels for B. pseudomallei  and the higher positivity rate for B. pseudomallei in the river delta of Ayeyawady region might be related to erosion of the soil, which harbour the organisms, during heavy rainfall, which is then transported by the main rivers and subsequently deposited downstream and in the coastal regions.
Although we isolated B. pseudomallei in all three seasons, a higher isolation rate was found in the rainy monsoon season. The monsoon season is also the time that most patients with melioidosis are diagnosed in other countries in the region including India, Nepal, Bangladesh, Thailand, Laos and Cambodia.[43–46]
B. pseudomallei was isolated in all three types of soils; sand, silt and clay. Although isolation was more frequent in sandy and clay type of soil compared to silt, the association was not statistically significant in our study. Previous studies have also reported a positive association of B. pseudomallei with sandy soil compared to clay.[18,47,48]
We found a higher proportion of B. pseudomallei-positive soil samples collected from agricultural land, pasture land and disused land compared with those collected from residential areas. The presence of B. pseudomallei in agricultural land including rice fields has frequently been described [17,19,49] and this is where most contact between soil and people is expected. Rice farmers are known to be at high risk for melioidosis. In Myanmar, farmers use cattle and buffalo as draft animals throughout the country and these animals are typically grazed on the pasture land nearby agriculture land. The occurrence of B. pseudomallei in land disturbed by livestock animals has also been reported in an earlier study carried out in northern Australia.
Samples collected at three different depths (30 cm, 60 cm and 90 cm) yielded a similar overall prevalence of B. pseudomallei in this study. The isolation of B. pseudomallei at the three soil depths varied between the seasons. We isolated B. pseudomallei more often at deeper depths (60 cm, 90 cm) during the cool dry season but more often at the shallower depth (30 cm) during the monsoon season (Table 3). This is in line with an earlier study in Australia. but differs from a study carried out in Laos where higher isolation rates were found at soil depths greater than 30 cm. However, based on these results we believe that it remains good practice to collect samples at all 3 different depths. It might be advisable to collect samples from depths beyond 30 cm in the hot dry and cool dry seasons but this may vary from place to place and needs further confirmation.
As part of this study, we explored the potential of pooling multiple soil samples to simplify the procedures for isolation of B. pseudomallei, but this method was only partially successful. Pooled samples had a significantly higher overall positivity rate than individual samples, but failed to isolate B. pseudomallei from 14 of 31 sites where B. pseudomallei was isolated from one or more individual samples. On the other hand, there was one site where the pooled sample was positive for B. pseudomallei whereas the nine individual samples were all negative. This also highlights the difficulty of isolating B. pseudomallei from environmental samples and the advisability of using multiple samples and methods before regarding any site as negative. We did not identify both B. pseudomallei and B. thailandensis in the same sample in our study, suggesting there might have been competition between the species as suggested by a previous study. However, other studies have found the two species present in the same samples  and, since the ability to detect this is likely to be highly method dependent and our study was designed primarily to detect the presence of B. pseudomallei, it would be inappropriate to draw and conclusions on this point. Soil pH was not tested in our study, and therefore we could not investigate the effect of soil pH on the occurrence of B. pseudomallei in the soil samples. However, recent data from an agricultural company (S1 Fig) indicates that the soil pH in the southern part of the country, where most B. pseudomallei was identified, was generally within the range of 3.7 to 4.8, while soil in the central part of Myanmar, where only a few samples with B. pseudomallei were identified, has a higher pH, above 5.4.
Advantage and limitations
The failure to isolate B. pseudomallei in certain areas does not mean that it was not present and the results of this study probably present an underestimate of the true distribution of B. pseudomallei across Myanmar for several reasons. First, we only collected samples from 9 points in each site compared to 100 points per site as suggested in the consensus guideline. With limited resources available, we chose to select more geographically dispersed sites with fewer samples in order to obtain a better overview of the geographical distribution nationally. Secondly, we omitted a number of areas because of limited access or armed conflict, potentially missing positive locations. Third, as samples were only collected at one time point for each province, each province was only sampled in a single season, which may have reduced our ability to detect B. pseudomallei. Fourth, the culture method used in the study, although based on a consensus of international experts on melioidosis, is known to have a lower sensitivity for the detection of B. pseudomallei in soil when compared with molecular methods such as PCR. And finally, the fact that pooled samples often tested negative while one or more of the individual samples of the same sites tested positives, and a pooled sample tested positive while all individual samples tested negative, shows clearly the ‘hit and miss’ sensitivity of the culture method. Negative findings are therefore never an assurance that B. pseudomallei is not present in the area.
The widespread distribution of B. pseudomallei is a worrying finding given the rarity with which melioidosis is diagnosed in the country, and suggests that many cases of melioidosis are going undiagnosed. This is particularly likely amongst agricultural workers, who comprise 65% percent of the population in Myanmar with rice being the main cultivated crop.[54,55] In addition, diabetes mellitus is the major predisposing factor for melioidosis and the prevalence of diabetes is rapidly increasing in Myanmar. The incidence of melioidosis is associated with heavy rainfall in the monsoon season [45,46] and people engaging in agricultural work during the monsoon season are at an increased risk. Ayeyawady province has the highest proportion of paddy fields and the highest proportion of B. pseudomallei-positive sites. In this regard it is worthy of note that a recent serological study carried out in 124 febrile patients in the delta area of Ayeyawady province observed a 3.2% positivity rate consistent with possible B. pseudomallei infection. In addition to its widespread distribution, a recent whole genome sequencing (WGS) study demonstrated the genetic diversity of B. pseudomallei isolates from Myanmar suggesting long-term endemicity of B. pseudomallei in the country.
The fact that B. pseudomallei is widely distributed in the soil in Myanmar is not surprising. The environmental suitability for the organism is high in large areas of south and south-east Asia, and the predicted burden of melioidosis in 2015 was high for the entire region; India (52,506 cases/31,425 deaths), Bangladesh (16,931/9,454), Laos (420/260), Thailand (7,572/2,838): the corresponding figures for Myanmar were 6,247 cases and 3,687 deaths.
How can these patients be identified? It is difficult to diagnose melioidosis clinically as it has no pathognomonic clinical signs and symptoms. It has been nicknamed the ‘great imitator’ because of its variable presentation and the wide differential diagnosis. Laboratory confirmation is therefore essential, but in most rural areas of Myanmar where melioidosis is expected to be most common, microbiology laboratories are not available. And even in laboratories where cultures are performed, B. pseudomallei is often misidentified as a contaminant or other species, especially by laboratory staff who are not familiar with the organism and where selective media and specific identification tests are not in-use.[12,58]
In Thailand and Laos, two neighbouring countries, melioidosis was only recognized as a major cause of community acquired septicaemia after researchers actively looked for the disease by establishing laboratory facilities to culture and identify B. pseudomallei.[24,38,59,60] In northeast Thailand melioidosis is now the second most common cause of community-acquired bacteraemia (19.3%) after E. coli (23.1%), and the number of deaths caused by melioidosis is comparable to deaths related to tuberculosis and higher than that caused by other common infectious diseases such as malaria, dengue, measles and leptospirosis.  The number of deaths from melioidosis in Thailand has, however, been significantly under-estimated by the National Notifiable Diseases Surveillance System until recently. In Laos, the number of melioidosis cases detected annually also increased dramatically after 1999 when researchers began specifically looking for it. It is very likely that the situation in Myanmar is similar and that the number of patients diagnosed with melioidosis will increase substantially when clinicians start actively looking for the disease, diagnostic microbiology services are strengthened, and laboratory staff gain experience in the detection and identification of B. pseudomallei. Recent training initiatives in Cambodia and Laos have been undertaken in order to raise awareness of this disease and similar events should be considered in Myanmar.[63,64]
Melioidosis is a neglected disease in many tropical and sub-tropical regions. This is the first environmental survey for B. pseudomallei conducted in all states and regions of Myanmar. The findings confirm that B. pseudomallei is widely distributed in central parts of Myanmar. Due to limited diagnostic facilities, and the lack of awareness among many clinical staff, particularly in rural areas of Myanmar where the disease is thought to be most common, it is very likely that melioidosis is substantially under-diagnosed and under-reported in Myanmar.
These findings should alert clinicians in Myanmar to consider melioidosis as one of the potential causes of fever, sepsis, pneumonia and abscesses, in particular during the rainy season in patients in regular contact with soil and those with underlying risk factors like diabetes. Clinical awareness should be accompanied with the provision of diagnostic facilities. In settings without microbiology facilities, a rapid diagnostic test for melioidosis has the potential to improve the accuracy and timeliness of diagnosis and treatment of melioidosis and thus save lives.[65,66]
In addition, further clinical studies to explore the epidemiology of melioidosis in Myanmar, prioritising regions where B. pseudomallei has been found in soil, should be initiated.
S1 Table. Burkholderia pseudomallei positive and negative townships in 15 states and regions of Myanmar.
S2 Table. Details of culture sample result and seasons, soil depths, soil types, current land use and climate zones.
S3 Table. Positivity of Burkholderia thailandensis in 15 states and regions of Myanmar.
S4 Table. Positivity of Burkholderia pseudomallei in 15 states and regions with annual rainfall data.
We would like to express our thanks to Tin Tin Htwe and Su Nyein Aye from the Department of Medical Research in Yangon, Myanmar, involved in the laboratory cultures, Wiphada Sripongsut Srinark from Shoklo Malaria Research Unit in Maesot and staff from Menzies School of Health, Darwin for helping in real time PCR assay and WGS. We also thank Alistair McLean for providing statistical advice and Direk Limmathurotsakul from Mahidol Oxford Tropical Medicine Research Unit for supporting practical training in the isolation and identification of B. pseudomallei in soil samples at the Department of Medical Research in Yangon. We would also like to thank the soil sample collection team, Saw Kan Sint Tun and staff from Medical Action Myanmar for their fieldwork.
- 1. Limmathurotsakul D, Golding N, Dance DA, Messina JP, Pigott DM, Moyes CL, et al. Predicted global distribution of Burkholderia pseudomallei and burden of melioidosis. Nature microbiology. 2016;1(1). Epub 2016/02/16. pmid:26877885; PubMed Central PMCID: PMC4746747.
- 2. White NJ. Melioidosis. Lancet (London, England). 2003;361(9370):1715–22. Epub 2003/05/28. pmid:12767750.
- 3. Gassiep I, Armstrong M, Norton R. Human Melioidosis. Clinical microbiology reviews. 2020;33(2). Epub 2020/03/13. pmid:32161067; PubMed Central PMCID: PMC7067580.
- 4. Pande A, Nambi PS, Pandian S, Subramanian S, Ghosh S. Melioidosis mimicking tuberculous vertebral osteitis: Case report and review of literature. Neurology India. 2018;66(4):1100–5. Epub 2018/07/25. pmid:30038101.
- 5. Shrestha N, Adhikari M, Pant V, Baral S, Shrestha A, Basnyat B, et al. Melioidosis: misdiagnosed in Nepal. BMC infectious diseases. 2019;19(1):176. Epub 2019/02/21. pmid:30782129; PubMed Central PMCID: PMC6381653.
- 6. Nernsai P, Sophonsritsuk A, Lertvikool S, Jinawath A, Chitasombat MN. A case report of Tubo-ovarian abscess caused by Burkholderia pseudomallei. BMC infectious diseases. 2018;18(1):73. Epub 2018/02/10. pmid:29422033; PubMed Central PMCID: PMC5806353.
- 7. Demar M, Ferroni A, Dupont B, Eliaszewicz M, Bouree P. Suppurative epididymo-orchitis and chronic prostatitis caused by Burkholderia pseudomallei: a case report and review. Journal of travel medicine. 2005;12(2):108–12. Epub 2005/07/06. pmid:15996456.
- 8. Koshy M, Sadanshiv P, Sathyendra S. Genitourinary melioidosis: a descriptive study. Tropical doctor. 2019;49(2):104–7. Epub 2018/12/19. pmid:30558480.
- 9. Garg R, Shaw T, Bhat SN, Mukhopadhyay C. Melioidosis: the great mimicker presenting as spondylodiscitis. BMJ case reports. 2018;2018. Epub 2018/02/14. pmid:29437812; PubMed Central PMCID: PMC5836698.
- 10. Kundangar RS, Bhat SN, Mohanty SP. Melioidosis mimicking tubercular cold abscess. BMJ case reports. 2017;2017. Epub 2017/09/21. pmid:28928256; PubMed Central PMCID: PMC5747799.
- 11. Lau SKP, Sridhar S, Ho C-C, Chow W-N, Lee K-C, Lam C-W, et al. Laboratory diagnosis of melioidosis: past, present and future. Exp Biol Med (Maywood). 2015;240(6):742–51. Epub 04/22. pmid:25908634.
- 12. Greer RC, Wangrangsimakul T, Amornchai P, Wuthiekanun V, Laongnualpanich A, Dance DAB, et al. Misidentification of Burkholderia pseudomallei as Acinetobacter species in northern Thailand. Transactions of the Royal Society of Tropical Medicine and Hygiene. 2019;113(1):48–51. Epub 2018/10/09. pmid:30295891; PubMed Central PMCID: PMC6314150.
- 13. Wiersinga WJ, Virk HS, Torres AG, Currie BJ, Peacock SJ, Dance DAB, et al. Melioidosis. Nature reviews Disease primers. 2018;4:17107. Epub 2018/02/02. pmid:29388572; PubMed Central PMCID: PMC6456913.
- 14. Currie BJ. Melioidosis: evolving concepts in epidemiology, pathogenesis, and treatment. Seminars in respiratory and critical care medicine. 2015;36(1):111–25. Epub 2015/02/03. pmid:25643275.
- 15. Birnie E, Virk HS, Savelkoel J, Spijker R, Bertherat E, Dance DAB, et al. Global burden of melioidosis in 2015: a systematic review and data synthesis. The Lancet Infectious diseases. 2019;19(8):892–902. Epub 2019/07/10. pmid:31285144.
- 16. Crowe A, McMahon N, Currie BJ, Baird RW. Current antimicrobial susceptibility of first-episode melioidosis Burkholderia pseudomallei isolates from the Northern Territory, Australia. International journal of antimicrobial agents. 2014;44(2):160–2. Epub 2014/06/14. pmid:24924662.
- 17. Rattanavong S, Wuthiekanun V, Langla S, Amornchai P, Sirisouk J, Phetsouvanh R, et al. Randomized Soil Survey of the Distribution of Burkholderia pseudomallei in Rice Fields in Laos. Applied and Environmental Microbiology. 2011;77(2):532–6. PMC3020526. pmid:21075883
- 18. Hantrakun V, Rongkard P, Oyuchua M, Amornchai P, Lim C, Wuthiekanun V, et al. Soil Nutrient Depletion Is Associated with the Presence of Burkholderia pseudomallei. Applied and Environmental Microbiology. 2016;82(24):7086–92. PMC5118919. pmid:27694236
- 19. Ong CEL, Wongsuvan G, Chew JSW, Kim TY, Teng LH, Amornchai P, et al. Presence of Burkholderia pseudomallei in Soil and Paddy Rice Water in a Rice Field in Northeast Thailand, but Not in Air and Rainwater. The American journal of tropical medicine and hygiene. 2017;97(6):1702–5. PMC5805070. pmid:29016340
- 20. Suputtamongkol Y, Hall AJ, Dance DA, Chaowagul W, Rajchanuvong A, Smith MD, et al. The epidemiology of melioidosis in Ubon Ratchatani, northeast Thailand. International journal of epidemiology. 1994;23(5):1082–90. Epub 1994/10/01. pmid:7860160.
- 21. Currie BJ, Fisher DA, Howard DM, Burrow JN, Lo D, Selva-Nayagam S, et al. Endemic melioidosis in tropical northern Australia: a 10-year prospective study and review of the literature. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America. 2000;31(4):981–6. Epub 2000/10/26. pmid:11049780.
- 22. Whitmore A, Krishnaswami CS. A Hitherto Undescribed Infective Disease in Rangoon. The Indian medical gazette. 1912;47(7):262–7. Epub 1912/07/01. pmid:29005374; PubMed Central PMCID: PMC5168169.
- 23. Punyagupta S. Review of 686 cases and presentation of a new clinical classification: In Melioidosis: Punyagupta S, Sirisanthana T, Stapatayavong B, eds. Melioidosis Bangkok: Bangkok Medical Publisher. 1989;1989:217–29.
- 24. Chaowagul W, White NJ, Dance DA, Wattanagoon Y, Naigowit P, Davis TM, et al. Melioidosis: a major cause of community-acquired septicemia in northeastern Thailand. Journal of Infectious Diseases. 1989;159(5):890–9.
- 25. Win MM, Ashley EA, Zin KN, Aung MT, Swe MMM, Ling CL, et al. Melioidosis in Myanmar. Tropical Medicine and Infectious Disease. 2018;3(1):28.
- 26. Win TT, Su KK, Than AM, Htut ZM, Pyar KP, Ashley EA, et al. Presence of Burkholderia pseudomallei in the ’Granary of Myanmar’. Trop Med Infect Dis. 2019;4(1). Epub 2019/01/10. pmid:30621177; PubMed Central PMCID: PMC6473437.
- 27. Win MM, Hla T, Phyu KP, Aung WW, Nyein Win KK, Aye SN, et al. A Study of Burkholderia pseudomallei in the Environment of Farms in Thanlyin and Hmawbi Townships, Myanmar. The American journal of tropical medicine and hygiene. 2019;100(5):1082–4. Epub 2019/02/23. pmid:30793685; PubMed Central PMCID: PMC6493951.
- 28. Myanmar Population Ministry of Labour, Immigration and Population, The Republic of the Union of Myanmar; 2020 [1.07.2020]. Available from: http://www.dop.gov.mm/en.
- 29. Aung L, Zin E, Theingi P, Elevera N, Aung P, Han T, et al. Myanmar Climate Report. Department of Meteorology and Hydrology, Ministry of Transport and Communications, Government of Myanmar. 2017.
- 30. Limmathurotsakul D, Dance DA, Wuthiekanun V, Kaestli M, Mayo M, Warner J, et al. Systematic review and consensus guidelines for environmental sampling of Burkholderia pseudomallei. PLoS neglected tropical diseases. 2013;7(3):e2105. Epub 2013/04/05. pmid:23556010; PubMed Central PMCID: PMC3605150.
- 31. Trinh TT, Hoang TS, Tran DA, Trinh VT, Göhler A, Nguyen TT, et al. A simple laboratory algorithm for diagnosis of melioidosis in resource-constrained areas: a study from north-central Vietnam. Clinical microbiology and infection: the official publication of the European Society of Clinical Microbiology and Infectious Diseases. 2018;24(1):84.e1–.e4. Epub 2017/08/07. pmid:28780059.
- 32. Duval BD, Elrod MG, Gee JE, Chantratita N, Tandhavanant S, Limmathurotsakul D, et al. Evaluation of a latex agglutination assay for the identification of Burkholderia pseudomallei and Burkholderia mallei. The American journal of tropical medicine and hygiene. 2014;90(6):1043–6. Epub 2014/04/09. pmid:24710616; PubMed Central PMCID: PMC4047727.
- 33. Dance DA, Wuthiekanun V, Naigowit P, White NJ. Identification of Pseudomonas pseudomallei in clinical practice: use of simple screening tests and API 20NE. Journal of clinical pathology. 1989;42(6):645–8. Epub 1989/06/01. pmid:2472432; PubMed Central PMCID: PMC1141995.
- 34. Novak RT, Glass MB, Gee JE, Gal D, Mayo MJ, Currie BJ, et al. Development and evaluation of a real-time PCR assay targeting the type III secretion system of Burkholderia pseudomallei. Journal of clinical microbiology. 2006;44(1):85–90. Epub 2006/01/05. pmid:16390953; PubMed Central PMCID: PMC1351940.
- 35. Knappik M, Dance DA, Rattanavong S, Pierret A, Ribolzi O, Davong V, et al. Evaluation of Molecular Methods To Improve the Detection of Burkholderia pseudomallei in Soil and Water Samples from Laos. Appl Environ Microbiol. 2015;81(11):3722–7. Epub 2015/03/31. pmid:25819969; PubMed Central PMCID: PMC4421066.
- 36. Webb J, Win MM, Zin K, Win KK, Wah T, Ashley EA, et al. Myanmar Burkholderia pseudomallei strains are genetically diverse and originate from Asia with phylogenetic evidence of reintroductions from neighbouring countries. Scientific Reports (in press).
- 37. Kottek M, Grieser J, Beck C, Rudolf B, Rubel F. World map of the Köppen-Geiger climate classification updated. Meteorol Z: 259–263. 2006.
- 38. Hinjoy S, Hantrakun V, Kongyu S, Kaewrakmuk J, Wangrangsimakul T, Jitsuronk S, et al. Melioidosis in Thailand: Present and Future. Trop Med Infect Dis. 2018;3(2):38. Epub 2018/05/05. pmid:29725623; PubMed Central PMCID: PMC5928800.
- 39. Limmathurotsakul D, Wuthiekanun V, Chantratita N, Wongsuvan G, Amornchai P, Day NPJ, et al. Burkholderia pseudomallei Is Spatially Distributed in Soil in Northeast Thailand. PLoS neglected tropical diseases. 2010;4(6):e694. pmid:20532233
- 40. Dance DAB, Knappik M, Dittrich S, Davong V, Silisouk J, Vongsouvath M, et al. Evaluation of consensus method for the culture of Burkholderia pseudomallei in soil samples from Laos. Wellcome open research. 2018;3:132. Epub 2018/12/26. pmid:30569022; PubMed Central PMCID: PMC6283377.2.
- 41. Hadden RL. The geology of Burma (Myanmar): An annotated bibliography of Burma’s geology, geography and earth science. GEOSPATIAL INFORMATION LIBRARY (GIL) ALEXANDRIA VA, 2008.
- 42. Zimmermann RE, Ribolzi O, Pierret A, Rattanavong S, Robinson MT, Newton PN, et al. Rivers as carriers and potential sentinels for Burkholderia pseudomallei in Laos. Scientific reports. 2018;8(1):8674. Epub 2018/06/08. pmid:29875361; PubMed Central PMCID: PMC5989208.
- 43. Chowdhury FR, Jilani MSA, Barai L, Rahman T, Saha MR, Amin MR, et al. Melioidosis in Bangladesh: A Clinical and Epidemiological Analysis of Culture-Confirmed Cases. Trop Med Infect Dis. 2018;3(2). Epub 2018/10/03. pmid:30274436; PubMed Central PMCID: PMC6073520.
- 44. Jatapai A, Gregory CJ, Thamthitiwat S, Tanwisaid K, Bhengsri S, Baggett HC, et al. Hospitalized Bacteremic Melioidosis in Rural Thailand: 2009–2013. The American journal of tropical medicine and hygiene. 2018;98(6):1585–91. Epub 2018/04/04. pmid:29611505.
- 45. Bulterys PL, Bulterys MA, Phommasone K, Luangraj M, Mayxay M, Kloprogge S, et al. Climatic drivers of melioidosis in Laos and Cambodia: a 16-year case series analysis. The Lancet Planetary health. 2018;2(8):e334–e43. Epub 2018/08/08. pmid:30082048; PubMed Central PMCID: PMC6076299.
- 46. Mukhopadhyay C, Shaw T, Varghese GM, Dance DAB. Melioidosis in South Asia (India, Nepal, Pakistan, Bhutan and Afghanistan). Trop Med Infect Dis. 2018;3(2). Epub 2018/10/03. pmid:30274447; PubMed Central PMCID: PMC6073985.
- 47. Palasatien S, Lertsirivorakul R, Royros P, Wongratanacheewin S, Sermswan RW. Soil physicochemical properties related to the presence of Burkholderia pseudomallei. Transactions of the Royal Society of Tropical Medicine and Hygiene. 2008;102 Suppl 1:S5–9. Epub 2009/01/16. pmid:19121688.
- 48. Baker AL, Ezzahir J, Gardiner C, Shipton W, Warner JM. Environmental Attributes Influencing the Distribution of Burkholderia pseudomallei in Northern Australia. PLoS One. 2015;10(9):e0138953. pmid:26398904
- 49. Manivanh L, Pierret A, Rattanavong S, Kounnavongsa O, Buisson Y, Elliott I, et al. Burkholderia pseudomallei in a lowland rice paddy: seasonal changes and influence of soil depth and physico-chemical properties. Scientific reports. 2017;7(1):3031. Epub 2017/06/10. pmid:28596557; PubMed Central PMCID: PMC5465195.
- 50. Kaestli M, Mayo M, Harrington G, Ward L, Watt F, Hill JV, et al. Landscape changes influence the occurrence of the melioidosis bacterium Burkholderia pseudomallei in soil in northern Australia. PLoS neglected tropical diseases. 2009;3(1):e364. Epub 2009/01/22. pmid:19156200; PubMed Central PMCID: PMC2617783.
- 51. Thomas AD, Forbes-Faulkner J, Parker M. Isolation of Pseudomonas pseudomallei from clay layers at defined depths. American journal of epidemiology. 1979;110(4):515–21. Epub 1979/10/01. pmid:507042.
- 52. Ngamdee W, Tandhavanant S, Wikraiphat C, Reamtong O, Wuthiekanun V, Salje J, et al. Competition between Burkholderia pseudomallei and B. thailandensis. BMC microbiology. 2015;15:56. Epub 2015/04/17. pmid:25879538; PubMed Central PMCID: PMC4365494.
- 53. Hantrakun V, Thaipadungpanit J, Rongkard P, Srilohasin P, Amornchai P, Langla S, et al. Presence of B. thailandensis and B. thailandensis expressing B. pseudomallei-like capsular polysaccharide in Thailand, and their associations with serological response to B. pseudomallei. PLoS neglected tropical diseases. 2018;12(1):e0006193. Epub 2018/01/25. pmid:29364892; PubMed Central PMCID: PMC5809093.
- 54. FAO. Food and Agriculture Organization of the United Nations: Rice Information. FAO, December 2002. Report No.
- 55. Myint T. Myanmar Rice Industry and Policies Towards Value Addition and Export. Food and Fertilizer Technology Center (FFTC) Policy Policy Articles. 2018.
- 56. Latt TS, Zaw KK, Ko K, Hlaing MM, Ohnmar M, Oo ES, et al. Measurement of diabetes, prediabetes and their associated risk factors in Myanmar 2014. Diabetes, metabolic syndrome and obesity: targets and therapy. 2019;12:291–8. Epub 2019/03/19. pmid:30881072; PubMed Central PMCID: PMC6407671.
- 57. Win ZZ, Phokrai P, Aung Z, Zaw T, Burtnick MN, Chantratita N, et al. Use of Rapid Enzyme-Linked Immunosorbent Assays for Serological Screening of Melioidosis in Myanmar. The American journal of tropical medicine and hygiene. 2018;98(5):1300–2. Epub 2018/03/21. pmid:29557332; PubMed Central PMCID: PMC5953378.
- 58. Hoffmaster AR, AuCoin D, Baccam P, Baggett HC, Baird R, Bhengsri S, et al. Melioidosis diagnostic workshop, 2013. Emerging infectious diseases. 2015;21(2). pmid:25626057
- 59. Dance DAB, Luangraj M, Rattanavong S, Sithivong N, Vongnalaysane O, Vongsouvath M, et al. Melioidosis in the Lao People’s Democratic Republic. Trop Med Infect Dis. 2018;3(1). Epub 2018/10/03. pmid:30274419; PubMed Central PMCID: PMC6136615.
- 60. Dance DA, Limmathurotsakul D. Global Burden and Challenges of Melioidosis. Trop Med Infect Dis. 2018;3(1). Epub 2018/10/03. pmid:30274411; PubMed Central PMCID: PMC6136634.
- 61. Melioidosis in Thailand [cited 2020 6 May]. Available from: https://www.melioidosis.info/info.aspx?pageID=104&contentID=1040103.
- 62. Hantrakun V, Kongyu S, Klaytong P, Rongsumlee S, Day NPJ, Peacock SJ, et al. Clinical Epidemiology of 7126 Melioidosis Patients in Thailand and the Implications for a National Notifiable Diseases Surveillance System. Open forum infect. 2019;6(12):ofz498. Epub 2020/02/23. pmid:32083145; PubMed Central PMCID: PMC7020769.
- 63. Bory S, Daily F, Khim G, Letchford J, Sok S, Kol H, et al. A Report from the Cambodia Training Event for Awareness of Melioidosis (C-TEAM), October 2017. Trop Med Infect Dis. 2018;3(1). Epub 2018/10/03. pmid:30274421; PubMed Central PMCID: PMC6136626.
- 64. Laos Training Event for Awareness of Melioidosis (L-TEAM) [cited 2020 3 Sep 2020].
- 65. Woods KL, Boutthasavong L, NicFhogartaigh C, Lee SJ, Davong V, AuCoin DP, et al. Evaluation of a Rapid Diagnostic Test for Detection of Burkholderia pseudomallei in the Lao People’s Democratic Republic. Journal of clinical microbiology. 2018;56(7). Epub 2018/05/04. pmid:29720430; PubMed Central PMCID: PMC6018328.
- 66. Rizzi MC, Rattanavong S, Bouthasavong L, Seubsanith A, Vongsouvath M, Davong V, et al. Evaluation of the Active Melioidosis Detect test as a point-of-care tool for the early diagnosis of melioidosis: a comparison with culture in Laos. Transactions of the Royal Society of Tropical Medicine and Hygiene. 2019;113(12):757–63. Epub 2019/10/23. pmid:31638152; PubMed Central PMCID: PMC6907004.