Melioidosis In Thailand Research Articles

Optimization and prospective evaluation of sensitive real-time PCR assays with an internal control for the diagnosis of melioidosis in Thailand.

Melioidosis is a severe infectious disease caused by Burkholderia pseudomallei. Effective treatment of melioidosis requires rapid detection of B. pseudomallei infection. Previous studies demonstrated that real-time PCR for the diagnosis of melioidosis in blood samples was insensitive. We optimized the DNA extraction method and evaluated real-time PCRs with an internal control for the detection of B. pseudomallei in plasma samples and compared the performance of real-time PCRs based on six target genes. The lower limit of detection (LOD) of TTS1-orf2, BPSS0745, BPSS1187, and BPSS1498 real-time PCRs was ≤10 genome equivalent (GE)/reaction, while the LOD of BPSS0087 and BPSS1492 was 100 GE/reaction. In the initial phase, we evaluated four targets (TTS1-orf2, BPSS0745, BPSS1187, BPSS1498) for the diagnosis of melioidosis using 87 plasma samples from 38 bacteremic-melioidosis patients and 49 other infection patients at a hospital in Northeast Thailand. Among these, BPSS1187 exhibited the highest sensitivity of 76.3% and specificity of 100%. A second evaluation phase was conducted at two hospitals in Northeast Thailand to compare two real-time PCRs between BPSS1187 and widely used TTS1-orf2 targets. A total of 705 clinical samples were collected from 421 suspected melioidosis cases; 77 of them were subsequently confirmed as melioidosis by culture using one or more specimens. The sensitivities of BPSS1187 and TTS1-orf2 were 89.6% and 79.2%, respectively (P = 0.004), and the specificities were 96% and 99%, respectively. The sensitivities of these two assays in plasma samples were 84.6% and 67.7%, respectively (P = 0.007). Therefore, the BPSS1187 real-time PCR was sensitive and specific for early diagnosis of melioidosis. IMPORTANCE Melioidosis is a serious infectious disease caused by Burkholderia pseudomallei, an environmental Gram-negative bacterium. Early detection of B. pseudomallei infection is crucial for successful antibiotic treatment and reducing mortality rates associated with melioidosis. Bacteria culture is currently used to identify B. pseudomallei in clinical samples, but the method is slow. Therefore, there is a need for more accurate and sensitive molecular-based diagnostic methods that can detect B. pseudomallei in all sample types, including samples from blood. We developed an optimal DNA extraction method for B. pseudomallei from plasma samples and used an internal control for real-time PCR. We evaluated six PCR target genes and identified the most effective target for the early detection of B. pseudomallei infection in patients. To prevent delays in the treatment of melioidosis that can lead to fatal outcomes, we recommend implementing this new approach for routine early detection of B. pseudomallei in clinical settings.

Role of Burkholderia pseudomallei-Specific IgG2 in Adults with Acute Melioidosis, Thailand.

Melioidosis is a life-threatening infectious disease caused by the gram-negative bacillus Burkholderia pseudomallei. An effective vaccine is needed, but data on protective immune responses in human melioidosis are lacking. We used ELISA and an antibody-dependent cellular phagocytosis assay to identify the major features of protective antibodies in patients with acute melioidosis in Thailand. We found that high levels of B. pseudomallei–specific IgG2 are associated with protection against death in a multivariable logistic regression analysis adjusting for age, diabetes, renal disease, and neutrophil count. Serum from melioidosis survivors enhanced bacteria uptake into human monocytes expressing FcγRIIa-H/R131, an intermediate-affinity IgG2-receptor, compared with serum from nonsurvivors. We did not find this enhancement when using monocytes carrying the low IgG2–affinity FcγRIIa-R131 allele. The findings indicate the importance of IgG2 in protection against death in human melioidosis, a crucial finding for antibody-based therapeutics and vaccine development.

Immune responses in beta-thalassaemia: heme oxygenase 1 reduces cytokine production and bactericidal activity of human leucocytes

Patients with beta-thalassaemia increase the risk of bacterial infections, particularly Burkholderia pseudomallei (Bp), the causative agent of melioidosis in Thailand. Impaired immune cell functions may be the cause of this susceptibility, but detailed mechanisms have not been defined. In this study, we observed impaired production of IFN-gamma and IL-10 by whole blood from beta-thalassaemia patients upon stimulation with a range of bacteria-derived stimuli. In contrast, IFN-gamma response via TCR and plasma IgG specific for Bp were still intact. Importantly, mRNA expression of heme oxygenase 1 (HO-1), a potential modulator of immune function, was increased in whole blood from beta-thalassaemia patients, either with or without stimulation with Bp in vitro. Induction of HO-1 by hemin or CoPP in vitro reduced production of IFN-gamma and IL-10 from healthy human PBMCs and decreased bacterial clearance activity of whole blood from healthy controls and beta-thalassaemia, while inhibition of HO-1 by SnPP enhanced both functions in healthy controls. These results were confirmed to some extent in purified human monocytes of healthy controls. Our results suggest a mechanism that excess hemin of beta-thalassaemia patients is a significant cause of immune suppression via HO-1 induction and may underlie the susceptibility of these individuals to severe bacterial infection.

Clinical Utility of Platelet Count as a Prognostic Marker for Melioidosis.

Thromobocytopenia predicts mortality in patients with melioidosis in Thailand. We analyzed platelet counts in two large cohorts of melioidosis patients in tropical northern Australia to assess utility in a different clinical setting. Admission platelet counts were compared between subgroups of patients with different clinical outcomes. Patients with more severe disease (indicated by bacteremia, septic shock, and death) had significantly lower platelet counts than those with less severe disease. Logistic regression analysis was carried out for potential predictors of mortality among various clinical parameters, and platelet count was shown to be an independent predictor of mortality. Furthermore, in patients critically ill with melioidosis, an increasing platelet count after admission was associated with a significantly greater chance of survival. However, given that most patients with severe disease still had platelet counts within the normal range, platelet count is not a useful biomarker for predicting the severity of melioidosis in a clinical context.

Melioidosis in Thailand: Present and Future.

A recent modelling study estimated that there are 2800 deaths due to melioidosis in Thailand yearly. The Thailand Melioidosis Network (formed in 2012) has been working closely with the Ministry of Public Health (MoPH) to investigate and reduce the burden of this disease. Based on updated data, the incidence of melioidosis is still high in Northeast Thailand. More than 2000 culture-confirmed cases of melioidosis are diagnosed in general hospitals with microbiology laboratories in this region each year. The mortality rate is around 35%. Melioidosis is endemic throughout Thailand, but it is still not uncommon that microbiological facilities misidentify Burkholderia pseudomallei as a contaminant or another organism. Disease awareness is low, and people in rural areas neither wear boots nor boil water before drinking to protect themselves from acquiring B. pseudomallei. Previously, about 10 melioidosis deaths were formally reported to the National Notifiable Disease Surveillance System (Report 506) each year, thus limiting priority setting by the MoPH. In 2015, the formally reported number of melioidosis deaths rose to 112, solely because Sunpasithiprasong Hospital, Ubon Ratchathani province, reported its own data (n = 107). Melioidosis is truly an important cause of death in Thailand, and currently reported cases (Report 506) and cases diagnosed at research centers reflect the tip of the iceberg. Laboratory training and communication between clinicians and laboratory personnel are required to improve diagnosis and treatment of melioidosis countrywide. Implementation of rapid diagnostic tests, such as a lateral flow antigen detection assay, with high accuracy even in melioidosis-endemic countries such as Thailand, is critically needed. Reporting of all culture-confirmed melioidosis cases from every hospital with a microbiology laboratory, together with final outcome data, is mandated under the Communicable Diseases Act B.E.2558. By enforcing this legislation, the MoPH could raise the priority of this disease, and should consider implementing a campaign to raise awareness and melioidosis prevention countrywide.

Protection against Experimental Melioidosis with a Synthetic manno-Heptopyranose Hexasaccharide Glycoconjugate.

Melioidosis is an emerging infectious disease caused by Burkholderia pseudomallei and is associated with high morbidity and mortality rates in endemic areas. Antibiotic treatment is protracted and not always successful; even with appropriate therapy, up to 40% of individuals presenting with melioidosis in Thailand succumb to infection. In these circumstances, an effective vaccine has the potential to have a dramatic impact on both the scale and the severity of disease. Currently, no vaccines are licensed for human use. A leading vaccine candidate is the capsular polysaccharide consisting of a homopolymer of unbranched 1→3 linked 2-O-acetyl-6-deoxy-β-d-manno-heptopyranose. Here, we present the chemical synthesis of this challenging antigen using a novel modular disaccharide assembly approach. The resulting hexasaccharide was coupled to the nontoxic Hc domain of tetanus toxin as a carrier protein to promote recruitment of T-cell help and provide a scaffold for antigen display. Mice immunized with the glycoconjugate developed IgM and IgG responses capable of recognizing native capsule, and were protected against infection with over 120 × LD50 of B. pseudomallei strain K96243. This is the first report of the chemical synthesis of an immunologically relevant and protective hexasaccharide fragment of the capsular polysaccharide of B. pseudomallei and serves as the rational starting point for the development of an effective licensed vaccine for this emerging infectious disease.

Cost-effectiveness analysis of parenteral antimicrobials for acute melioidosis in Thailand.

BackgroundMelioidosis is a common community-acquired infectious disease in northeast Thailand associated with overall mortality of approximately 40% in hospitalized patients, and over 70% in severe cases. Ceftazidime is recommended for parenteral treatment in patients with suspected melioidosis. Meropenem is increasingly used but evidence to support this is lacking.MethodsA decision tree was used to estimate the cost-effectiveness of treating non-severe and severe suspected acute melioidosis cases with either ceftazidime or meropenem.ResultsEmpirical treatment with meropenem is likely to be cost-effective providing meropenem reduces mortality in severe cases by at least 9% and the proportion with subsequent culture-confirmed melioidosis is over 20%.ConclusionsIn this context, treatment of severe cases with meropenem is likely to be cost-effective, while the evidence to support the use of meropenem in non-severe suspected melioidosis is not yet available.

Public awareness of melioidosis in Thailand and potential use of video clips as educational tools.

BackgroundMelioidosis causes more than 1,000 deaths in Thailand each year. Infection occurs via inoculation, ingestion or inhalation of the causative organism (Burkholderia pseuodmallei) present in soil and water. Here, we evaluated public awareness of melioidosis using a combination of population-based questionnaire, a public engagement campaign to obtain video clips made by the public, and viewpoints on these video clips as potential educational tools about the disease and its prevention.MethodsA questionnaire was developed to evaluate public awareness of melioidosis, and knowledge about its prevention. From 1 March to 31 April 2012, the questionnaire was delivered to five randomly selected adults in each of 928 districts in Thailand. A video clip contest entitled “Melioidosis, an infectious disease that Thais must know” was run between May and October 2012. The best 12 video clips judged by a contest committee were shown to 71 people at risk from melioidosis (diabetics). Focus group interviews were used to evaluate their perceptions of the video clips.ResultsOf 4,203 Thais who completed our study questionnaire, 74% had never heard of melioidosis, and 19% had heard of the disease but had no further knowledge. Most participants in all focus group sessions felt that video clips were beneficial and could positively influence them to increase adherence to recommended preventive behaviours, including drinking boiled water and wearing protective gear if in contact with soil or environmental water. Participants suggested that video clips should be presented in the local dialect with simple words rather than medical terms, in a serious manner, with a doctor as the one presenting the facts, and having detailed pictures of each recommended prevention method.ConclusionsIn summary, public awareness of melioidosis in Thailand is very low, and video clips could serve as a useful medium to educate people and promote disease prevention.Presented in PartWorld Melioidosis Congress 2013, Bangkok, Thailand, 18–20 September 2013 (abstract OS VII-04).

Incidence of Bacteremic Melioidosis in Eastern and Northeastern Thailand

Burkholderia pseudomallei, the causative agent of melioidosis, is endemic in northeastern Thailand. Population-based disease burden estimates are lacking and limited data on melioidosis exist from other regions of the country. Using active, population-based surveillance, we measured the incidence of bacteremic melioidosis in the provinces of Sa Kaeo (eastern Thailand) and Nakhon Phanom (northeastern Thailand) during 2006-2008. The average annual incidence in Sa Kaeo and Nakhon Phanom per 100,000 persons was 4.9 (95% confidence interval [CI] = 3.9-6.1) and 14.9 (95% CI = 13.3-16.6). The respective population mortality rates were 1.9 (95% CI = 1.3-2.8) and 4.4 (95% CI = 3.6-5.3) per 100,000. The case-fatality proportion was 36% among those with known outcome. Our findings document a high incidence and case fatality proportion of bacteremic melioidosis in Thailand, including a region not traditionally considered highly endemic, and have potential implications for clinical management and health policy.

Prospective Clinical Evaluation of the Accuracy of 16S rRNA Real-Time PCR Assay for the Diagnosis of Melioidosis

The accuracy of a Burkholderia pseudomallei 16s rRNA real-time PCR assay was evaluated against culture for the diagnosis of melioidosis in Thailand. A total of 846 samples were obtained from 383 patients with suspected melioidosis. One or more specimens were PCR positive for 47 of 77 patients with culture-proven melioidosis (sensitivity 61.0%, 95% CI: 49.2-72.0%). PCR was negative for all 306 patients who were culture negative for B. pseudomallei (specificity 100%, 95% CI: 98.8-100%). Diagnostic sensitivity of PCR was 22.7% for patients who were culture positive for blood only, compared with 79.4% for patients who were culture positive for samples other than blood. The median (interquartile range) B. pseudomallei colony count in blood for 44 of 77 patients with positive blood cultures was 2.4 CFU/ml (0.2-13.5 CFU/ml); this may explain the low sensitivity of PCR for this specimen. The PCR assay described here is not sufficiently sensitive to replace culture in our clinical setting.

Lenograstim "is not associated with a clinically significant effect on mortality in severely septic patients suspected of having melioidosis in Thailand",

Lenograstim "is not associated with a clinically significant effect on mortality in severely septic patients suspected of having melioidosis in Thailand",

A Randomized Controlled Trial of Granulocyte Colony-Stimulating Factor for the Treatment of Severe Sepsis Due to Melioidosis in Thailand

Melioidosis is a tropical infectious disease associated with significant mortality. Most deaths occur early and are caused by fulminant sepsis. In this randomized, placebo-controlled trial, we assessed the efficacy of lenograstim (granulocyte colony-stimulating factor [G-CSF], 263 mu g per day administered intravenously) in ceftazidime-treated patients with severe sepsis caused by suspected melioidosis in Thailand. Over a 27-month period, 60 patients were enrolled to receive either G-CSF (30 patients, 18 of whom had culture-confirmed melioidosis) or placebo (30 patients, 23 of whom had culture-confirmed melioidosis). Mortality rates were similar in both groups (G-CSF group, 70%; placebo group, 87%; risk ratio, 0.81; 95% confidence interval, 0.61-1.06; P=.2), including among patients with confirmed melioidosis (83% vs. 96%; P=.3). The duration of survival was longer for patients who received G-CSF than for patients who received placebo (33 h vs. 18.6 h; hazard ratio, 0.56; 95% confidence interval, 0.31-1.00; P=.05). Receipt of G-CSF is associated with a longer duration of survival but is not associated with a mortality benefit in patients with severe sepsis who are suspected of having melioidosis in Thailand. We hypothesize that G-CSF may "buy time" for severely septic patients, but survival is more likely to be improved by management of associated metabolic abnormalities and organ dysfunction associated with severe sepsis.

Melioidosis in Southeast Asia

After the first report of human melioidosis in Burma by Whitmore and Krishnaswami (1912), it took approximately 50 years for the establishment of its endemicity in Southeast Asian countries such as Malaysia, Singapore and Thailand. In other Southeast Asian countries, little is known about the epidemiology although melioidosis is gradually being recognized as an emerging infectious disease in Vietnam (Weber et al., 1969). The septicemic or bacteremic form is the best indicator of its presence since blood isolates generally receive greater attention for proper identification than isolates from other sites. In Thailand, the first case report appeared in 1955 (Chittivej et al., 1955). However, melioidosis was not widely recognized until the Vietnam war when the death of American soldiers due to acute pneumonitis provoked great interest in the disease (Weber et al., 1969). At that time little evidence could be found to substantiate the existence of melioidosis in Thailand. The microorganism could be isolated from soil and water collected from various parts of the country (Finkelstein et al., 1967). The prevalence of a positive serological test was found in 29% of Thai military personnel (Nigg, 1963) but strangely enough, clinical cases could not be identified. In 1975, Sompone Punyagupta and colleagues reported ten cultureproven cases of melioidosis at the meeting of the Infectious Disease Group of Thailand (Punyagupta et al., 1976). Since then clinicians and laboratory personnel in medical schools have been alert to look for the disease. The knowledge and expertise gained from a handful of cases diagnosed at medical schools was passed on to various provincial hospitals. The first national workshop on melioidosis was held 10 years later. It compiled more than 700 reported cases that led to research activities on various aspects of melioidosis. At present, 2000–3000 cases of clinical melioidosis are estimated to occur each year in Thailand with a population of 60 000 000. The incidence rate in highly endemic areas was calculated to be 3.6–5.5 cases per 100 000 population and is seasonal (Suputtamongkol et al., 1994). A multicenter study on the etiology of acute undifferentiated febrile illness from 1991 to 1992 revealed one blood isolate per 1033 cases and 11 (0.9%) cases with a positive serological test among the 1218 cases. Since there are approximately 400 000 cases of acute undifferentiated febrile illness reported to the Division of Epidemiology, Ministry of Public Health each year, 387 cases of bacteremic melioidosis and another 3612 cases with positive serology could be derived according to calculation using this information. In a laboratory survey, total numbers of the isolates were 1131 strains in 1994 and 1165 strains in 1995 (Leelarasamee et al., 1997). When the site of isolation and the annual number of isolates were plotted on a map, it was seen that Burkholderia * Tel.: +66-2-4197785. E-mail address: siall@mahidol.ac.th (A. Leelarasamee)

Genome fingerprinting by pulsed-field gel electrophoresis of isolates of Burkholderia pseudomallei from patients with melioidosis in Thailand

A total of 95 isolates of Burkholderia pseudomallei from 53 sporadic cases in Thailand were examined by pulsed-field gel electrophoresis. Digestion of genomic DNA of all isolates by NcoI generated a macrorestriction pattern similar to that of B. pseudomallei which cannot assimilate l-arabinose. Analysis using restriction enzymes SpeI and AvrII demonstrated greater sensitivity than NcoI digestion in the differentiation of B. pseudomallei and could be used for epidemiological groupings. Four cluster groups were evident among 37 isolates tested and the majority of isolates within each cluster displayed more than 65% similarity. Furthermore, multiple isolates from 18 and 35 patients with single and recurrent episodes of melioidosis, respectively, were examined. All patients with a single episode yielded genetically identical isolates and four of 35 patients with recurrent episodes were infected with strains of different genotypic patterns from the primary isolate(s). Hence, most repeated episodes of infection in melioidosis are as a result of the original infecting strains.

Aminoglycoside and Macrolide Resistance in Burkholderia pseudomallei

We were very interested in the recent paper by Moore et al. (3) in which the authors used transposon mutants to demonstrate efflux-mediated resistance to aminoglycosides and macrolides in Burkholderia (formerly Pseudomonas) pseudomallei. Melioidosis is very difficult to treat because of intrinsic resistance to many antimicrobial agents, particularly the aminoglycosides. Aminoglycoside–β-lactam combinations are used empirically to treat suspected community-acquired sepsis in many areas of the world, but they are ineffective in melioidosis. We have been conducting clinical and laboratory studies of melioidosis in Thailand since 1986. During this time we have collected over 3,700 clinical isolates of B. pseudomallei from more than 1,800 patients with melioidosis admitted to Sappasitprasong Hospital, Ubon Ratchathani, northeast Thailand. We have performed susceptibility testing using the disk diffusion method on most of these isolates, according to the guidelines of the National Committee for Clinical Laboratory Standards (4), for ceftazidime, co-amoxiclav, chloramphenicol, doxycycline, ciprofloxacin, gentamicin, polymyxin B, and, more recently, imipenem. MIC determinations, using an agar incorporation method (5), have been performed for representative isolates. During this time we have isolated three strains of B. pseudomallei, from two patients, which were susceptible to gentamicin by disk testing. The isolates were from a splenic aspirate of one patient (708a) (2) and from blood and knee aspirate cultures of the second (2188a and b). All other isolates were resistant to gentamicin by disk testing (disk content, 10 μg). B. pseudomallei is usually highly resistant to aminoglycosides, with a gentamicin MIC at which 50% of the isolates are inhibited of 128 mg/liter (1), and also to the macrolide antibiotics. The MIC results for 100 clinical isolates of B. pseudomallei, collected during 1998, for the macrolides erythromycin, clarithromycin, and azithromycin are listed in Table ​Table1.1. In contrast, the three gentamicin-susceptible isolates were also susceptible to these macrolide antibiotics (Table ​(Table1).1). TABLE 1 MICsa for isolates of B. pseudomallei For all three isolates the MICs of ceftazidime, imipenem, chloramphenicol, ciprofloxacin, and doxycycline were within the expected ranges for each agent. All three were resistant to polymyxin B. Thus, there are very rare wild strains of B. pseudomallei which exhibit antibiotic susceptibility patterns similar to those of the transposon mutants created by Moore et al. (3), i.e., with linked loss of resistance to aminoglycosides and macrolides. By definition the three clinical isolates described here are virulent. This is consistent with the evidence for retention of virulence in hamsters by the mutant strains. Whether these findings have therapeutic implications remains to be seen. This study was part of the Wellcome-Mahidol University Oxford Tropical Medicine Research Programme, funded by the Wellcome Trust of Great Britain.

Fatal melioidosis in a tourist returning from Thailand

Severe infections with Pseudomonas pseudomallei, the causative agent of melioidosis, is a common cause of community acquired septicaemia in South East Asia and parts of Northern Australia, but infection in travellers returning to the United Kingdom is extremely rare. We describe the case of a tourist who acquired melioidosis in Thailand. Despite intensive intravenous therapy with antibiotics to which the organism was sensitive, the patient's infection proved fatal. Ps. pseudomallei was isolated from blood cultures at a late stage in his illness by use of a commercial blood culture system that included an antibiotic removal device (Bac/TAlert, Organon-Technica).

Application of indirect hemagglutination test and indirect fluorescent antibody test for IgM antibody for diagnosis of melioidosis in Thailand.

In hyperendemic areas such as Thailand, rapid diagnosis of melioidosis depends upon both bacteriological culture and serological methods. However, interpretation of indirect hemagglutination (IHA) for melioidosis which is the only test available, is seriously hampered by increased IHA titers present in one-third to one-half of the population. In order to get the best results from the available tests, IHA and indirect fluorescent antibody for IgM (IFA-IgM) were evaluated in controls and patients in Thailand. IHA titers of greater than or equal to 1:40 were considered remote or recent exposure to P. pseudomallei. IHA titers of this level were found in 47.1% of 227 blood donors and 29.5% of 210 sera submitted for other tests, while IFA-IgM was positive in only one donor who had an IHA titer of 1:1,280. IHA was positive in eight out of nine patients with melioidosis with IHA titers of less than 1:20 to 1:2,560. IFA-IgM was positive in six out of seven melioidosis patients whose sera were available for this test including a serum with IHA titer of less than 1:20. Six patients were predisposed by diabetes mellitus. Among sera serologically tested for melioidosis, 33 had IHA titers of 1:80-1:1,280, 10 of which were positive for IFA-IgM. This study demonstrates high background IHA titers among IHA titers among Thai people which greatly limits its use for serodiagnosis of melioidosis. In sharp contrast, serodiagnosis by IFA-IgM was more successful. Positive IFA-IgM among healthy Thais did exist indicating that serologic tests for melioidosis at best are only supplementary to bacteriological culture and clinical awareness.