An V. Le 6 , Hao V. Nguyen 7 , 8 , Karoline Assig  9 , Sabine  Lichtenegger  9 , Gabriel E. Wagner 9 , Cuong D. Do 10 and Ivo Steinmetz 9 , 11  ID

1      Institute of Microbiology and Biotechnology, Vietnam National University, Hanoi  100000, Vietnam

2      Department of Academic Affairs, Phan  Chau  Trinh University, Quang Nam  560000, Vietnam;

[email protected]

3      Department of Medical  Microbiology, Hanoi  Medical  University, Hanoi  100000, Vietnam;

nguyen.vu.tr[email protected]

4      National Hospital for Tropical  Diseases,  Hanoi  100000, Vietnam

5      Department of Infectious Diseases,  Hue University of Medicine and Pharmacy, Hue 530000, Vietnam;

[email protected]

6      Department of Medical  Microbiology, Hue University of Medicine and Pharmacy, Hue 530000, Vietnam;

[email protected]

7      Department of Infectious Diseases,  University of Medicine and Pharmacy, Ho Chi Minh 700000, Vietnam;

[email protected]

8      Hospital for Tropical  Diseases,  Ho Chi Minh 700000, Vietnam

9      Institute of Hygiene, Microbiology and Environmental Medicine, Medical  University of Graz,

8010 Graz, Austria; kar[email protected] (K.A.); [email protected] (S.L.);

gabriel.wagner[email protected] (G.E.W.); [email protected] (I.S.)

10     Department of Infectious Diseases,  Bach Mai Hospital, Hanoi  100000, Vietnam;  doduy[email protected]

11     Friedrich Loeffler Institute of Medical  Microbiology, University Medicine Greifswald,

17475 Greifswald, Germany

*     Correspondence: tttr[email protected]; Tel.: +84-243-7548747

Abstract:  The  first  cases  of human melioidosis were  described in Vietnam in the  1920s, almost a century  ago. It was in Vietnam  in the thirties  that the saprophytic nature of B. pseudomallei was first recognized. Although a significant number of French  and U.S. soldiers acquired the disease during the Vietnam wars,  indigenous cases in the Vietnamese population were only sporadically reported over many decades. After reunification in 1975, only two retrospective studies reported relatively small numbers of indigenous cases from single tertiary care hospitals located  in the biggest  cities in the South and the North,  respectively. Studies from provincial hospitals throughout the country were missing until the Research Network on Melioidosis  and Burkholderia pseudomallei (RENOMAB) project started in 2014. From then on seminars, workshops, and national  scientific conferences on melioidosis have  been  conducted to raise  awareness among physicians and  clinical  laboratory staff.  This led to the recognition of a significant number of cases in at least 36 hospitals in 26 provinces and  cities throughout Vietnam.  Although a widespread distribution of melioidosis has now been documented, there  are  still  challenges to understand the  true  epidemiology of the  disease.  Establishment of national guidelines for diagnosis, management, and  reporting of the disease together with  more investigations on animal melioidosis, genomic diversity of B. pseudomallei and  its environmental distribution are required.

Keywords:  melioidosis; Burkholderia pseudomallei; Vietnam;  public  awareness; animal;  environment

1. Introduction

2. Review of Melioidosis in Vietnam

2.1. Human Melioidosis

2.1.1. 1920s to 1950s

The first case of human melioidosis in Vietnam was detected in 1925 in an ill-nourished female patient, pregnant in the  fifth month, living  at Thu  Duc,  close to Saigon  (now  Ho  Chi Minh  City). Six days after onset of symptoms, the patient  had a miscarriage and, after 14 days of illness, the patient died  [3]. B. pseudomallei was isolated from the blood  at the Pasteur Institute of Ho Chi Minh City and the identification was confirmed at the Institute for Medical  Research,  Malaysia [4].

Subsequently cases were reported by Menard in 1928 from Ho Chi Minh City and  Tonkin  (now Hanoi in the  North), as cited  by Pons  (1930) [5].  Further culture-confirmed cases  of melioidosis, including cases related  to traffic accidents (see below Section 3) were then reported at Hue city (Central Vietnam) and from the North [1]. In 1947, 28 cases of acute,  sub-acute, and chronic  melioidosis were described from various hospitals in the South [6]. Of those, only nine cases were Vietnamese patients. The others were 15 Caucasians, 2 black patients, and 1 patient  of Chinese and 1 of south Asian descent. Fatal outcomes were reported in 19 out of those 28 cases, although the outcome was not documented for all patients. From 1951 to 1953, five pulmonary cases of melioidosis were diagnosed at hospitals in both southern Vietnam  and France. All of the cases were French citizens with a history  of residence or station in the region of Vietnam, Laos, and Cambodia [7]. A case of chronic pulmonary melioidosis was also described in a soldier suffering from a chest wound caused by a bullet in Vietnam [8]. Between 1948 and 1954, approximately 100 cases were reported among 400,000 French  forces stationed in Vietnam, Laos, and Cambodia, as cited by Sanford  (1978) [9]. In 1956, a fatal case of septicemic  melioidosis was diagnosed in a 40-year-old south Vietnamese soldier  admitted to Cong Hoa Hospital [10].

2.1.2. 1960s to 1980s

It is interesting to note  that  already in 1967, Mo and  Duong stated that  the disease was  most likely underdiagnosed in hospitals of southern Vietnam and that cases of melioidosis were probably misdiagnosed as  tuberculosis or  disseminated fungus infections.  The  authors also  mentioned several cases detected annually in different hospitals of the South and described the development of melioidosis in a young  soldier  after a military operation in a swampy area of the South [11]. With the deployment of the U.S. Armed  Forces in Vietnam, melioidosis started to be detected in the U.S. military personnel. As cited by Diamond and Pastore (1967), through February 1967, 35 cases were diagnosed in U.S. troops stationed in Southeast Asia,  with  8 fatal  cases  reported [12].  Those  patients were diagnosed because diseased or wounded soldiers were referred  to modern hospitals of either Republic of South  Vietnam or United States, where microbiological laboratories were  available for diagnosis of melioidosis as well  as for other infectious diseases. Within the  year  of 1966, a series  of 9 cases of pulmonary melioidosis in U.S. soldiers was described [13]. Another nine  cases of melioidosis in U.S. soldiers, including four  fatal cases, were  diagnosed in Vietnam in Long  Binh (now  Dong  Nai province  in the South) [14]. Since more cases of melioidosis were subsequently reported in U.S. soldiers, melioidosis became  one of six major tropical infectious diseases for which  U.S. physicians needed to maintain a high index  of suspicion in febrile soldiers returning from Vietnam [15]. From April  1965 to December 1969, 187 cases with  13 deaths were  reported in the U.S. Army personnel stationed in Vietnam.  Until the U.S. withdrawal in 1972, approximately two to three culture-confirmed cases were detected among the U.S. soldiers every  month [9,16,17]. Serology using indirect hemagglutination (IHA) showed that 8.9% of the sera from the Vietnam veterans had titers of 1:40 or greater [18]. Based on such  titers  it was  estimated by Clayton et al.  that  approximately 250,000 among three  million U.S. Army personnel got infected with  B. pseudomallei when serving in Vietnam [18,19]. Although these estimates might be interpreted with some cautions considering the limited specificity  and sensitivity of the non-standardized IHA test, this study indicated a potential reservoir of latent B. pseudomallei infection among personnel returning from Vietnam. Indeed, it became obvious that  latent infection and reactivation, sometimes after many years, does occur [20]. This phenomenon led to the melioidosis nickname ‘Vietnam Time Bomb’ [21]. A possible human-to-human transmission via sexual contact was  suggested in the  wife  of a returning Vietnam veteran with  prostatitis due  to B. pseudomallei infection [22]. Melioidosis was  also diagnosed in a newborn whose father had  served in Vietnam, although the source of a possible transmission remained unclear [23]. Despite this large  number of melioidosis cases related to Vietnam during this period of time, very little information on the disease was reported for the indigenous population (Table 1).

Table 1. Number of indigenous Vietnamese patients with  melioidosis and  the respective outcome reported over time in medical journals.

a Recovery  during the study.

2.1.3. 1990s until Present

In 1991, a medical thesis  defended at Hanoi Medical University described 16 cases detected in hospitals of Hanoi over a 10-year period from 1980 to 1990, with  seven  fatal outcomes [29]. A study by Phung et al. (1993) using an IHA test revealed seropositivity in populations living  in suburban communities of Hanoi ranging from  6.4% to 31.8%.  This  study also  observed an  association of seropositivity with  rice  farming [30].  In a study on  cellular lipid  and  fatty  acid  composition of B. pseudomallei, seven  patients with  melioidosis were  listed  from  whom strains were  isolated in Vietnam between 1981 and  1991 [24]. In 1999, a retrospective study in the South  reported only nine culture-confirmed cases with melioidosis from 3653 blood  cultures of febrile patients admitted to the largest  hospital for tropical diseases  in Ho Chi Minh City from 1992 to 1998 [26]. Antibiotic treatment and  patient outcomes were  not  reported in this  study. In 2008, another retrospective study in the largest general hospital in the  North reported 55 culture-confirmed cases  observed in a period of time  from  1997 to 2005. Analysis of clinical  data  showed that  septicemia with  pneumonia was  the most common clinical presentation and diabetes was the most common risk factor.  Seventeen out of 40 septicemic patients died,  with  nine  deaths occurring within 48 h after  admission. Based  on the residential addresses of the patients, it was concluded that melioidosis is widely distributed and occurs in at least 18 of 25 northern provinces [27]. In the context of the Research Network on Melioidosis and  Burkholderia pseudomallei (RENOMAB;  see below  Section  5) that  started in 2014, a recent  study reported 70 cases detected within seven  months at five hospitals in North Central Vietnam. During the  study period, the  detection rate  of B. pseudomallei ranged from  3.4% to 10.2% among positive blood  cultures in those hospitals. Fifty-eight patients had septicemia. Of the 36 patients with known outcome, 18 patients died,  with 6 deaths occurring within 48 h after admission [28]. However, as for other  regions, the true  burden of melioidosis in North Central Vietnam still needs to be determined (see below Section 7). The few epidemiological data available for Vietnam  imply  that rice farmers are at particular risk to acquire a B. pseudomallei infection  [26–28].

2.2. Animal Melioidosis

2.2.1. 1930s to 1960s

At the time  when the first human case of melioidosis was  detected in Vietnam, the disease was considered to be a zoonosis and  animals a reservoir for B. pseudomallei. This assumption was based on the observation that  B. pseudomallei could infect laboratory rodents and  its virulence was comparable to Yesinia pestis, the causative agent of plague. However, a large  microbiological study of more  than

20,500 rats collected in southern Vietnam found only one rat to be culture-positive for B. pseudomallei as cited by Luong (1956) [31]. None  of the 560 wild  rats caught in Hanoi were  positive for B. pseudomallei by culture [1]. Serological studies showed that serum collected from pigs contained antibodies against B. pseudomallei, as cited by Luong (1956) [31]. In 1954, melioidosis was detected in rabbits and  guinea pigs at the Pasteur Institute of Ho Chi Minh City, as cited by Luong (1961) [32]. In 1955, Luong described culture-confirmed melioidosis cases in pigs at a pig farm near a rubber plantation in Thu Dau Mot (now Ho Chi Minh City), with the isolation of B. pseudomallei from creamy pus in lung and spleen abscesses [31]. Using serology and culture methods, several  deaths of pigs were confirmed to be caused by melioidosis in the farms at Tan Son Nhat  and Gia Dinh (now both are Ho Chi Minh City) [32].

2.2.2. 1970s until Present

In 1971, cases  of melioidosis were  reported in the  U.S. Army dogs  in the  Republic of South Vietnam. During a six-month period, 31 working dogs  died,  with  four confirmed melioidosis cases. B. pseudomallei was cultured from the dogs’ lungs  and various organs. The dogs came from different units in diverse locations in Vietnam. All of the dogs  showed lesions in the lungs, epididymides, and  testes  [33]. The author stated that  melioidosis remained underdiagnosed because bacteriologic cultures  were not routinely performed at necropsy  of the U.S. Army working dogs and that the disease does not seem to be uncommon in dogs. A serologic surveillance on 64 healthy  U.S. military  scout and tracker dogs after service in the Republic  of South Vietnam  showed that 12 (19%) dogs had developed antibodies against B. pseudomallei, with  IHA titers  higher than  1:80 [34]. After reunification in 1975, no further information about  melioidosis in pigs or other livestock, as well as dogs, has been reported in Vietnam.  Only recently, in the context of RENOMAB, a study  carried out by a group at the National Institute of Veterinary Research reported a culture-confirmed melioidosis case of a pig in a farm  at Nghe  An province in North Central Vietnam [35].

3. B. pseudomallei  in the Environment

The first experimental indication of the saprophytic nature of B. pseudomallei and its environmental reservoir was provided in Vietnam by Vaucel in 1937 [1]. His studies were triggered by the observation that individuals who developed melioidosis after traffic accidents were either immersed for a prolonged time  in water of a pond or had  a skull  wound contaminated with  mud. To test the hypothesis that B. pseudomallei exists in the environment, Vaucel  submerged the scratched abdomen of guinea pigs into water of a pond collected in the North. Five days  later,  a moribund animal was sacrificed and B. pseudomallei was isolated from  pus  of the liver and  spleen and  other sites on solid  media in pure culture [1].  About 20 years  later  in 1955, Chambon provided final  evidence by directly isolating seven  B. pseudomallei strains from five environmental samples including pond muds, rice field water, and a sample of pond water collected in the South  [2]. In 1961, Luong  provided further evidence for environmental B. pseudomallei, when  two strains  were cultured from water  samples  of a water  spinach plantation from southern Vietnam [32]. Apart from  this early  environmental work,  there  are only a few more  recent  studies sampling rice fields for B. pseudomallei. In 1991, a study reported the isolation of B. pseudomallei from  4 out of 240 soil samples, and  1 out of 190 surface water samples collected in rice fields at four communities surrounding Hanoi was positive for B. pseudomallei [30]. Between 1992 and  1998 soil samples were collected from 137 rice fields around Ho Chi Minh City in southern Vietnam and nine fields were found to be positive [26]. The low B. pseudomallei detection rate from soil in those sampling studies is likely to be the result of the current culture protocols for environmental B. pseudomallei which have a limited sensitivity. It has been  shown recently, that  a multitarget quantitative PCR approach improves the detection rate  and  can predict cultivability of B. pseudomallei [36]. By using this  multitarget qPCR, B. pseudomallei was detected in 35 (83.3%) out of 42 soil samples collected  at 28 rice fields in southern Vietnam. From those  samples, B. pseudomallei strains could be isolated from six (14.3%) samples by using  conventional culture methods [36]. A recent  prediction of the global environmental presence of B. pseudomallei at 5 × 5 km2  spatial resolution suggested that  predominant parts of Vietnam are highly suitable for the environmental occurrence of B. pseudomallei [37]. However, mountainous areas near the border with Laos and China, especially in the  Northwest region, some  regions in the  Central Highlands, and  a zone  involving the  lower  South  Central Coast  were  found to be less  suitable.  It will  be most  important to test these  predictions in future environmental studies and  also to investigate the environmental factors involved in creating a habitat for B. pseudomallei at a high  spatial resolution. The country harbors an enormous diversity of habitats ranging from  tropical rain forest  to dry  forest,  natural grassland, and agricultural land  such as rice paddies to wetland habitats including rivers  and lakes and coastal wetlands. Apart  from the limited  studies on rice fields, there is no information on the potential role of such different  habitats as environmental reservoirs for B. pseudomallei. Since Vietnam  has an enormous north–south expansion, including humid subtropical climate in the north,  tropical  monsoon climate in the center, and tropical wet and dry climate  in the south,  the potential influence of climate  factors on the environmental presence of B. pseudomallei needs to be investigated. For the development of any preventive strategies or environmental countermeasures it will be important to define  risk areas  for infection  and the role of different habitats and climate  factors more precisely. 

When the first B. pseudomallei strain  was isolated from clinical specimens in 1925, the occurrence of different morphotypes, in this case rugose and  ultra-rugose colonies  on agar,  were  described [4]. As we now know various morphotypes represent a frequent characteristic of this species. Subsequent work demonstrated virulence of clinical  and  environmental B. pseudomallei strains in experimental infections and  described antibiotic susceptibility to drugs available at that  time  [4,8,11,38]. A more recent  study with  25 strains from  northern Vietnam reported an  antibiotic resistance profile  that is also found in other endemic areas,  with  susceptibility to currently-recommended antibiotics for treatment of melioidosis such as ceftazidime, imipenem, meropenem, and co-trimoxazole. Resistance and intermediate resistance to tetracycline were noted for one strain and three strains, respectively [27].

In a study describing physiological and  biochemical characteristics of 15 environmental and clinical  B. pseudomallei strains from  northern Vietnam, all  strains shared typical B. pseudomallei characteristics with   respect to  motility, salt  tolerance, growth temperature,  sugar assimilation, cytochrome c oxidase and  acid  production, and  colony  morphology on  different routine agar media [24].   The  composition of  cellular lipids and  fatty  acids  were  similar among the  tested strains [24]. Biochemical and  antigenic characteristics typical  for B. pseudomallei were  also described by Phuong et al. (2008) using the API 20 NE system and  through agglutination of those  strains with a B. pseudomallei-specific monoclonal antibody [27].

The Burkholderia pseudomallei ‘Multi-Locus Sequence Typing’  (MLST) database is a rich resource to assess the genomic population structure based on seven  housekeeping genes (/ [39]. Since the development of the B. pseudomallei MLST scheme in 2003 [40], 104 isolates from Vietnam have been deposited in the database (5309 isolates  overall)  as of 14 February 2018 [27,41]. A total  of 61 sequence types (STs) were  identified, emphasizing the  great  genotypic diversity of the sampled population in this region. Of those, 31 STs were uniquely reported in Vietnam so far and most of the remaining STs are shared with neighboring countries like Thailand or Cambodia. The co-occurrence of the latter and the concomitant lack of regional specificity  have already been noticed for other STs [41]. As already implied by Phuong et al. (2008), analysis of current B. pseudomallei MLST data  showed the Vietnamese strains to mainly cluster  with other Asian isolates as opposed to the Australian STs.

However, special   care  must be  taken when analyzing B. pseudomallei MLST  data.     Its  high recombination rate complicates the inferences of phylogenetic relationships of sequence types (STs) [42,43]. Nevertheless, the unprecedented amount of deposited data and the comparatively cheap prices render it a valuable tool, despite the emergence of next generation sequencing. With the advent of next generation sequencing (NGS), whole genome sequencing (WGS) has proven a powerful tool to study population genetics at a much higher resolution. This was recently shown by De Smet et al. (2015), reporting two strains, which shared one ST due to homoplasy, but could be distinguished by WGS [44].

Phylogentic studies based on single nucleotide polymorphisms (SNP) present  strong evidence  for an Australian B. pseudomallei origin  [43,45] and  a single transmission event  to Southeast Asia [43,46]. In a recent study by Chewapreecha et al. (2017) on the global evolution and prevalence of B. pseudomallei,

19 strains from Vietnam (spanning a time period from 1947 to 2011) were subjected to whole  genome sequencing and  placed in a global  context (469 isolates) [46]. A SNP-based phylogenetic analysis of these genomes clearly resolved Australian, Asian, and African/American clusters.

Strains from Vietnam appeared in 7 of 17 Asian subgroups, with most of the other members of the subgroup being from Thailand and Cambodia and belonging to the ‘Mekong  sub-region’. The other Asian subgroups contained mainly isolates from Malaysia  and Singapore (‘Malay cluster ’). It could be shown that the transitions of B. pseudomallei between these two sub-regions was less than  within the sub-region. As noted by the authors, this observation might be linked to trading networks and cultural links. Furthermore, the results  indicate  that the Mekong  sub-region might  have been a hotspot for the evolution of B. pseudomallei in Southeast Asia. Unfortunately, in contrast to other Asian clusters, no time could be estimated for the emergence of the most recent common ancestor of the Vietnam-containing subgroups. Besides,  the study also demonstrates the potential of WGS to account for geographical differences in disease outcome, by screening for region-specific (virulence) loci [46].  The insights, based  on just 19 Vietnamese isolates, already imply  the wealth  of information that can presumably be gained from such a high level of resolution. Should more rigorous clinical and environmental sampling schemes be applied and a higher number of strains analyzed, this will likely increase  our knowledge of B. pseudomallei phylogeny and virulence tremendously. There is no doubt that these are interesting times for studying the genomic diversity of B. pseudomallei in Vietnam.

5. Activities to Raise Awareness of Melioidosis in Vietnam  and Major Achievements

As in many parts of the  world, neither melioidosis nor  characteristics of B. pseudomallei are mandatory components of curricula at  medical universities in  Vietnam.  This  leads  to  a lack  of knowledge among many doctors and  other health care  professionals about how  to diagnose and manage the disease.  As outlined above, this limited clinical awareness together with limited laboratory resources result in significant underdiagnosis. This has been very obvious in the central part of Vietnam. Although it is located  in the same geographical belt and is close to highly  endemic areas of Laos and northeast Thailand, cases of melioidosis were not reported from there until recently.

In 2014, a bilateral project called Research  Network on Melioidosis and Burkholderia pseudomallei (RENOMAB),  sponsored by the German Ministry for Education and  Research and  the Vietnamese Ministry of Science  and  Technology, was  started.  The  project  involved 40 national and  regional hospitals in 27 provinces and  cities throughout the country. By organizing a series of workshops on diagnosis of melioidosis for laboratory staff, hundreds of culture-confirmed cases have recently been detected [28].  Two national scientific  conferences on melioidosis were  organized in order to raise awareness of the disease among  infectious  disease physicians, microbiological laboratory staff, medical teachers, researchers, and health-care managers.

Active  melioidosis case-finding reports were  presented at national scientific  conferences for infectious and respiratory diseases [47,48]. Scientific papers on melioidosis have started to appear in national journals [49]. The existence of the disease was  also broadcasted in a wide  range of public media  such as hospital web portals, newspapers and television programs. A private Facebook  group named Research Network on  Melioidosis in Vietnam (Hoi  Nghien cuu  Melioidosis tai  Vietnam) was  created and  consists of approximately 600 members from  the  healthcare system interested in melioidosis diagnostics.  At the  time  of writing, 36 hospitals in 26 provinces and  cities  reported culture-confirmed melioidosis cases (Figure 1). Of these,  28 hospitals detected the first cases of the disease after joining case-finding  activities within  RENOMAB. This project has shown  that melioidosis is widely distributed throughout the country with a potential area of high endemicity in North  Central Vietnam [28]. The organization of the 9th World Melioidosis  Congress in 2019 in Hanoi will be a golden opportunity to further raise awareness for melioidosis and to promote research on different aspects  of epidemiology, diagnosis, and treatment in Vietnam.

Figure 1. Location of hospitals taking  part in the RENOMAB project and reporting culture-confirmed melioidosis cases. All of the B. pseudomallei strains  were sent to the reference  laboratory at the Institute of Microbiology and Biotechnology, Vietnam  National University, Hanoi.  B. pseudomallei identification of bacterial strains was confirmed by using either recA sequence analysis or B. pseudomallei-specific TTSS1 real-time PCR assay [28]. Border lines of provinces or cities are shown. The color indicates the number of hospitals within the province or city that reported cases of melioidosis. Geographic map was constructed by the MapInfo 7.8 (MapInfo, Troy, NY, USA).

6. Current Recommendations for Diagnosis and Treatment and for Reporting  and Prevention

6.1. Diagnosis and Treatment

Currently, there  are no official guidelines for diagnosis and  treatment of melioidosis issued by the Vietnamese Ministry of Health, and  B. pseudomallei has not been  yet on the list of any  national surveillance programs for infectious agents.  The current recommendations that have been distributed via seminars, workshops, and  national conferences, are based on studies and  guidelines from  the international literature.

In well-equipped laboratories commercially-available biochemical tests  and  automated identification systems are  routinely used.   However, even  under such  circumstances, a lack  of awareness and of additional diagnostic tools can lead to unreliable diagnoses, since misidentification of B. pseudomallei has been well documented for such systems [50]. In remote areas where laboratory facilities and resources for consumables are still limited, the identification of bacterial pathogens mostly relies  on some  basic microbiological tests,  and  the identification of Gram-negative non-fermenting bacterial species  is even  more  challenging.  We therefore recently introduced a simple laboratory algorithm for identification of B. pseudomallei from clinical specimens under such resource-constrained conditions. The algorithm makes use of the inherent resistance of B. pseudomallei to gentamicin and colistin and the susceptibility to amoxicillin-clavulanic acid [28].

Although the sensitivity and specificity  of this algorithm have not been validated yet, compared to more sophisticated identification procedures including selective media  such as Ashdown’s agar for non-sterile sites, it proved to be an effective  and  inexpensive procedure leading to the diagnosis of a significant number of melioidosis patients at provincial general hospitals in North  Central Vietnam in a short period of time after introduction [28]. We therefore currently recommend the implementation of this simple algorithm in clinical  laboratories for a presumptive diagnosis of B. pseudomallei from clinical materials. Confirmation of the identification can be obtained in reference laboratories using specific type three secretion system 1 (TTSS1) real-time  PCR assays and sequencing of the recA gene as reliable  target  [28].

6.2. Surveillance and Prevention

Melioidosis is not a notifiable disease in Vietnam. There is neither a formal  surveillance system for human nor  for animal melioidosis in Vietnam. There  are also no official recommendations on prevention. The limited epidemiological data  available in Vietnam indicate that  rice farmers are at a particular risk, similar to other  parts of Southeast Asia. At present, it seems  plausible to primarily target  the rice farming population in terms of better diagnostics and evaluation of possible  preventive measures. The majority of them  are living  in remote areas  where the primary healthcare system of the country does not cover microbiological investigations. It is therefore most likely that fatal cases of melioidosis in remote areas are still grossly  under recognized.

7. Current and Future Challenges

Although some progress has recently  been made  in Vietnam  to increase awareness of melioidosis and to enhance laboratory skills for identification of B. pseudomallei from clinical specimens, there are still significant efforts needed to further improve capacity  building in different regions  of Vietnam.

Apart from further improving the identification capacity in the laboratories via diagnostic workshops and scientific conferences it will be crucial to generally  increase the number of clinical specimens—such as urine, throat swabs, sputum, pus,  etc.—to be sent to the laboratory for microbiological investigations. Since bacteremic melioidosis is a common clinical  presentation and  also associated with  a high  case fatality rate, increasing the number of blood cultures in patients with prolonged fever has a high priority. There are financial barriers and insurance issues that hinder an appropriate use of blood cultures in the diagnosis not only for melioidosis, but also for other  systemic infections, which need  to be addressed. Epidemiological studies are needed to determine the true  incidence and  prevalence of melioidosis in the various regions. The same is true  for the veterinary field, where information on melioidosis is still very scarce.

In parallel to capacity building in the clinical laboratory, studies on the environmental distribution of B. pseudomallei in Vietnam  will be important to define risk areas for the indigenous population more precisely. This information can be used  to further target clinical microbiology activities. In addition, serological screening of the  indigenous population using newly-developed devices might help  to detect  exposure to B. pseudomallei and to identify possible  endemic hot spots.

Acknowledgments: We would  like to thank Minh N. Nguyen, at the Faculty of Environmental Science, University of Science, Vietnam National University, Hanoi, for his help in constructing the geographic map.  We would like to thank the Vietnamese Ministry of Science and  Technology and  the German Federal Ministry of Education and Research  for co-funding the collaborative RENOMAB project (reference 01DP13007). The National Conferences on  Melioidosis were  made possible by  support from  the  Vietnam National University, Hanoi; the  World Health Organization representative in  Vietnam; the  Cooperative Biological  Engagement Program, Defense Threat  Reduction Agency,  US Department of Defence,  USA; and the US Agency  for International Development in  Vietnam.   The  authors and  conference organizers are  extremely grateful to  the  valuable contributions of international melioidosis experts, including David Dance  (Lao-Oxford-Mahosot Hospital Wellcome Trust Research Unit, Vientiane,  Laos), Ploenchan Chetchotisakd (Faculty of Medicine, Khon Kaen University, Thailand), Daniel Altmann (Department of Medicine, Imperial College London,  United  Kingdom), Direk Limmathurotsakul (Mahidol-Oxford Tropical  Medicine Research  Unit, Faculty of Tropical  Medicine, Mahidol University, Thailand), Wuthiekanun Vanaporn (Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Thailand), and Bart Currie  (Menzies School of Health  Research, Charles Darwin  University and Royal Darwin  Hospital, Australia).  We acknowledge all of the hospitals and partners involved in the RENOMAB project.

Author Contributions: T.T.T., I.S., T.V.N., C.D.D., C.X.T., A.V.L. and  H.V.N.  participated in activities related to RENOMAB  and  conceived and  designed the overall structure of the manuscript. L.D.N.N. and  I.S. compiled information from French  articles.  K.A., S.L. and G.E.W. contributed to drafting Sections 3 and 4 and editing the manuscript. T.T.T., G.E.W. and I.S. wrote the manuscript. All authors approved the final version of the manuscript.

Conflicts of Interest: The authors declare  no conflict of interest.

References

1.      Vaucel, M. Présence probable du bacille de Whitmore dans  l’eau de mare au Tonkin.  Bull. Soc. Pathol. Exot.

1937, 30, 10–15.

2.      Chambon, L. Isolement du  bacille  de Whitmore a partir du  milieu extérieur. Ann.  Inst.  Pasteur 1955, 89,

229–235.

3.      Pons, R.; Advier, M. Melioidosis in Cochin China.  J. Hyg. (Lond.) 1927, 26, 28–30. [] []

4.      Stanton, A.T.; Fletcher, W. Melioidosis:  Notes on a culture  of B. whitmori from Saigon. J. Hyg. (Lond.) 1927, 26,

31–32. [] []

5.      Pons, R. La Mélioïdose maladie commune à I’homme, aux rongeurs et aux équidés. Bull. Soc. Pathol. Exot.

1930, 23, 210–215.

6.      Alain, M.; Saint-Etienne,  J.; Reynes, V. La mélioïdose;  considérations étiologiques cliniques  et pathogèniques

à propos de 28 cas. Med. Trop. 1949, 9, 119–142.

7.      Duroux, A. La mélioïdose pulmonaire. Poumon Coeur 1965, 21, 355–372. []

8.       Guillermand, J.; Barrié,  J.; Morilleau, R.; Couderc, P. Mélioïdose pulmonaire  chronique (Observation anatomoclinique). Poumon Couer 1964, 20, 269–285.

9.      Sanford,  J.P. Melioidosis: Forgotten but not gone. Trans. Am. Clin. Climatol. Assoc. 1978, 89, 201–205. []

10.    Stein, W.; Duc, N.H.; Low, F.J. Acute melioidosis. USA Armed Forces Med. J. 1958, 9, 273–276.

11.    Mo, H.D.; Cuong,  T.H. A typical  case of melioidosis in South Vietnam. Mil. Med. 1967, 132, 98–100.

12.    Diamond, H.S.; Pastore, R. Septic arthritis due  to Pseudomonas pseudomallei. Arthritis  Rheumatol. 1967, 10,

459–466. []

13.     Spotnitz, M.; Rudnitzky, J.; Rambaud, J.J. Melioidosis pneumonitis. Analysis of nine cases of a benign  form of melioidosis. JAMA 1967, 202, 950–954. [] []

14.     Weber, D.R.; Douglass,  L.E.; Brundage, W.G.; Stallkamp, T.C. Acute varieties  of melioidosis occurring in U.S. soldiers in Vietnam. Am. J. Med. 1969, 46, 234–244. []

15.    Deller, J.J. Diseases  from Vietnam. Calif. Med. 1969, 111, 461–466.

16.    Sanford, J.P.; Moore, W.L., Jr. Recrudescent melioidosis:  A Southeast Asian legacy. Am. Rev. Respir. Dis. 1971,

104, 452–453. [] []

17.    Everett, E.D.; Nelson, R.A. Pulmonary melioidosis. Observations in thirty-nine cases. Am. Rev. Respir. Dis.

1975, 112, 331–340. []

18.    Clayton, A.J.; Lisella, R.S.; Martin, D.G. Melioidosis: A serological survey in military personnel. Mil. Med.

1973, 138, 24–26. [] []

965–967. []

20.    Koponen, M.A.; Zlock, D.; Palmer, D.L.; Merlin, T.L. Melioidosis.  Forgotten, but not gone! Arch. Intern. Med.

1991, 151, 605–608. [] []

21.     Time Magazine. Diseases:  Vietnam’s Time Bomb, 10 February 1967. Available online:  //content.time.(accessed  on 12 March 2018).

22.     McCormick, J.B.; Sexton,  D.J.; McMurray, J.G.; Carey,  E.; Hayes, P.; Feldman, R.A. Human-to-human transmission of Pseudomonas pseudomallei. Ann. Intern. Med. 1975, 83, 512–513. [] []

23.    Osteraas, G.R.; Hardman, J.M.; Bass, J.W.; Wilson,  C. Neonatal melioidosis. Am. J. Dis.  Child. 1971, 122,

446–448. [] []

24.    Phung, L.V.; Tran,  T.B.; Hotta, H.;  Yabuuchi, E.; Yano,  I. Cellular lipid  and  fatty  acid  compositions of

Burkholderia pseudomallei strains  isolated  from human and environment in Vietnam.  Microbiol. Immunol. 1995,

39, 105–116. [] []

25.    Worthington, M.G.; McEniry, D.W. Chronic  melioidosis in a Vietnamese  immigrant. Rev. Infect. Dis. 1990, 12,

966. [] []

26.     Parry, C.M.; Wuthiekanun, V.; Hoa,  N.T.; Diep,  T.S.; Thao, L.T.; Loc, P.V.; Wills, B.A.; Wain,  J.; Hien,  T.T.; White,  N.J.; et al.  Melioidosis in Southern Vietnam: Clinical  surveillance and  environmental sampling. Clin. Infect. Dis. 1999, 29, 1323–1326. [] []

27.     Phuong, D.M.; Trung, T.T.; Breitbach, K.; Tuan,  N.Q.; Nubel, U.; Flunker, G.; Khang, D.D.; Quang, N.X.; Steinmetz, I. Clinical  and  microbiological features of melioidosis in northern Vietnam. Trans. R. Soc. Trop. Med. Hyg. 2008, 102, S30–S36. []

28.    Trinh,  T.T.; Hoang, T.S.; Tran,  D.A.;  Trinh,  V.T.; Gohler, A.;  Nguyen, T.T.; Hoang, S.N.;  Krumkamp, R.; Nguyen, L.T.N.; May, J.; et al. A simple laboratory algorithm for diagnosis of melioidosis in resource-constrained areas: A study from north-central Vietnam. Clin. Microbiol. Infect. 2018, 24, 84.e1–84.e4. [] []

29.    Lam,  N.T.  Clinical  Features and  Outcomes of Melioidosis Patients.  Bachelor ’s Thesis,  Hanoi Medical

University, Hanoi,  Vietnam, 1991.

30.     Phung, L.V.; Quynh, H.T.; Yabuuchi, E.; Dance, D.A. Pilot study of exposure to Pseudomonas pseudomallei in northern Vietnam. Trans. R. Soc. Trop. Med. Hyg. 1993, 87, 416. []

31.    Luong, N.B. A propos d’une  épizootie porcine de mélioidose dans  une province méridionale du Vietnam.

Bull. Soc. Pathol. Exot. 1956, 49, 25–31.

32.    Luong,  N.B.; Kim, N.T. La mélioidose porcine au Vietnam. Off. Int. Epizoot. 1961, 56, 944–976.

33.    Stedham, M.A. Melioidosis in dogs in Vietnam. J. Am. Vet. Med. Assoc. 1971, 158, 1948–1950. []

34.     Alexander, A.D.; Huxsoll, D.L.; Warner, A.R., Jr.; Shepler, V.; Dorsey, A. Serological diagnosis of human melioidosis with indirect  hemagglutination and complement fixation tests. Appl. Microbiol. 1970, 20, 825–833. []

35.     Hang, N.T.T.; Hang, T.T.T.; Trung, T.T. Initial  investigation of pig with  melioidosis caused by Burkholderia pseudomallei at Nghe  An province. Vietnam Vet. Assoc. Vet. Sci. Tech. 2018, 25, 48–55.

36.     Gohler, A.; Trung, T.T.; Hopf, V.; Kohler, C.; Hartleib, J.; Wuthiekanun, V.; Peacock, S.J.; Limmathurotsakul, D.; Tuanyok, A.; Steinmetz, I. Multitarget quantitative PCR improves detection and predicts cultivability of the pathogen Burkholderia pseudomallei. Appl. Environ. Microbiol. 2017, 83, e03212–e03216. [] []

37.     Limmathurotsakul, D.; Golding, N.; Dance,  D.A.; Messina, J.P.; Pigott,  D.M.;  Moyes,  C.L.; Rolim,  D.B.; Bertherat, E.; Day, N.P.; Peacock,  S.J.; et al. Predicted global  distribution of Burkholderia pseudomallei and burden of melioidosis. Nat. Microbiol. 2016, 1, 15008. [] []

38.    Borchardt, K.A.; Stansifer,  P.; Albano,  P.M. Osteomyelitis due to Pseudomonas pseudomallei. JAMA 1966, 196,

660–662. [] []

39.    Jolley, K.A.; Maiden, M.C. BIGSdb: Scalable analysis of bacterial genome variation at the population level.

BMC Bioinf. 2010, 11, 595. [] []

40.     Godoy, D.; Randle, G.; Simpson, A.J.; Aanensen, D.M.; Pitt,  T.L.; Kinoshita, R.; Spratt, B.G. Multilocus sequence typing and  evolutionary relationships among the causative agents of melioidosis and  glanders, Burkholderia pseudomallei and Burkholderia mallei. J. Clin. Microbiol. 2003, 41, 2068–2079. [] []

41.     McCombie, R.L.; Finkelstein, R.A.;  Woods, D.E. Multilocus sequence typing of historical Burkholderia pseudomallei isolates collected in Southeast Asia from 1964 to 1967 provides insight  into the epidemiology of melioidosis. J. Clin. Microbiol. 2006, 44, 2951–2962. [] []