Surasakdi Wongratanacheewin

Increasing Incidence of Human Melioidosis in Northeast Thailand

Melioidosis is a serious community-acquired infectious disease caused by the Gram-negative environmental bacterium Burkholderia pseudomallei. A prospective cohort study identified 2,243 patients admitted to Sappasithiprasong Hospital in northeast Thailand with culture-confirmed melioidosis between 1997 and 2006. These data were used to calculate an average incidence rate for the province of 12.7 cases of melioidosis per 100,000 people per year. Incidence increased incrementally from 8.0 (95% confidence interval [CI] = 7.2–10.0) in 2000 to 21.3 (95% CI = 19.2–23.6) in 2006 (P < 0.001; χ2 test for trend). Male sex, age ≥ 45 years, and either known or undiagnosed diabetes were independent risk factors for melioidosis. The average mortality rate from melioidosis over the study period was 42.6%. The minimum estimated population mortality rate from melioidosis in 2006 was 8.63 per 100,000 people (95% CI = 7.33–10.11), the third most common cause of death from infectious diseases in northeast Thailand after human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) and tuberculosis.

Biological Relevance of Colony Morphology and Phenotypic Switching by Burkholderia pseudomallei

Melioidosis is a notoriously protracted illness and is difficult to cure. We hypothesize that the causative organism, Burkholderia pseudomallei, undergoes a process of adaptation involving altered expression of surface determinants which facilitates persistence in vivo and that this is reflected by changes in colony morphology. A colony morphotyping scheme and typing algorithm were developed using clinical B. pseudomallei isolates. Morphotypes were divided into seven types (denoted I to VII). Type I gave rise to other morphotypes (most commonly type II or III) by a process of switching in response to environmental stress, including starvation, iron limitation, and growth at 42 degrees C. Switching was associated with complex shifts in phenotype, one of which (type I to type II) was associated with a marked increase in production of factors putatively associated with in vivo concealment. Isogenic types II and III, derived from type I, were examined using several experimental models. Switching between isogenic morphotypes occurred in a mouse model, where type II appeared to become adapted for persistence in a low-virulence state. Isogenic type II demonstrated a significant increase in intracellular replication fitness compared with parental type I after uptake by epithelial cells in vitro. Isogenic type III demonstrated a higher replication fitness following uptake by macrophages in vitro, which was associated with a switch to type II. Mixed B. pseudomallei morphologies were common in individual clinical specimens and were significantly more frequent in samples of blood, pus, and respiratory secretions than in urine and surface swabs. These findings have major implications for therapeutics and vaccine development.

Development of a PCR-based method for the detection of Opisthorchis viverrini in experimentally infected hamsters

Opisthorchis viverrini infection is an endemic disease that causes a serious public health problem in southeast Asia, especially in northeast Thailand. We have developed a PCR method using a pair of primers named OV-6F/OV-6R for detecting O. viverrini eggs in stool samples and compared it with Stolls egg-count method. The primers were designed based on the pOV-A6 specific DNA probe sequence which gave a 330 base pair product. The PCR method can detect a single egg in artificially inoculated faeces or as little as 2 x 10(-17) ng of O. viverrini genomic DNA. The method gave 100% sensitivity in all hamster groups except in animals exposed to the lowest intensity of infection (1 metacercaria/hamster). In the first month of infection, the PCR method was more sensitive than using the egg-count method in all infected groups especially in the light infections. The PCR method was also successfully used in monitoring a therapeutic study. Since the PCR method showed no cross-reaction with Heterophyid flukes, it can be useful for specific identification of O. viverrini eggs in stool samples without the risk of false positives. It also has great potential for application in clinical epidemiological studies.

Detection of Opisthorchis viverrini in Human Stool Specimens by PCR

Opisthorchiasis is a liver fluke infection, found mainly in Southeast Asia (8) but increasingly in developed countries due to an influx of Asian immigrants (7). The diagnostic methods are based on the demonstration of eggs in stools, although there are still difficulties in distinguishing eggs from heterophyid and lecithodendriid parasites (2). We have successfully developed a PCR-based method for detection of Opisthorchis viverrini in experimentally infected hamsters (6). The aim of the present study was to determine the value of PCR for detection of O. viverrini eggs in human feces. Stool specimens from 85 parasite-infected individuals, as confirmed by the formalin-ether technique, were collected from Srinagarind Hospital, Faculty of Medicine, Khon Kaen University. The specimens were composed of 40 O. viverrini parasites, 10 minute intestinal flukes, 10 hookworms, 10 echinostomes, 7 Taenia spp. parasites, 3 Strongyloides stercolaris parasites, 2 Entamoeba coli parasites, and 3 specimens from individuals with mixed infections. To evaluate the sensitivity of the method, normal stool specimens were spiked with either 1, 10, 25, 50, 100, 200, 500, and 1,000 O. viverrini eggs or 0.02, 0.2, 2, 20, and 200 pg and 2 ng of genomic DNA, respectively. The sensitivity of the detection for stool specimens was compared with that for Stolls egg count technique (5). To extract the DNA from stool specimens, 100 mg of feces in 1 ml of sterile distilled water was extracted with 200 μl of ether and centrifuged at 6,500 × g for 2 min. The pellet was mixed with 500 μl of 0.5 N NaOH and left for 1 h at room temperature. After being autoclaved, the supernatant was collected and invertedly mixed with 250 μl of 3 M sodium acetate and 500 μl of absolute ethanol before being cleaned up with the QIAamp DNA Mini kit (Qiagen, Hilden, Germany), and 10 μl was used in the PCR (6). The sensitivity test in the spiking experiment demonstrated that the limit of our PCR detection is 200 eggs or 200 pg of genomic DNA. The expected 330-bp product and its doublet of 660 bp were in 32 of the 40 fecal specimens (80.0%) from patients with O. viverrini (Table ​(Table1).1). Compared with that of Stolls egg count, the sensitivity of the PCR was found to be 100, 68.2, and 50% in the fecal specimens containing >1,000, 200 to 1,000, and <200 eggs per g of feces, respectively. Our method gave one false positive (97.8% specificity) in the specimen containing eggs of echinostomes (Table ​(Table1).1). The positive and negative predictive values are 96.9 and 84.6%, respectively. TABLE 1. Results of PCR and Stolls egg count compared to formalin-ether technique Our PCR method is sensitive and highly specific, with no amplification occurring when samples contained small intestinal flukes or other parasitic eggs. It was more sensitive than the previous method using a DNA probe as a diagnostic tool (3). The detection limit of 200 eggs can be assumed to be produced by one adult fluke, since an adult worm can release 50 to 200 eggs per g of feces (1, 4). The sensitivity value of PCR is somewhat lower than those of the formalin-ether and Stoll egg count techniques. This may be caused by the use of low amounts of stool sample (0.1 g for PCR and 2 and 1 g for formalin-ether and Stolls egg count, respectively). However, mild infection (200 to 1,000 eggs per g of feces) could be easily missed by Stolls dilution technique (4), whereas our technique gave a 68.2% positive rate. The false-positive result occurring for a specimen containing eggs of echinostomes may not have been an actual positive result, since the specimen was the only one positive among the 10 stool samples containing echinostomes. Thus, the results in this study suggest that our PCR method is a suitable tool for detection of O. viverrini infection, especially in a large number of samples at one time. With some improvement in sample preparation, it will be very useful for sensitive and specific diagnosis and epidemiological studies and offers a potential means for detection of the parasite in the intermediate hosts.

Opisthorchis viverrini : relationships between egg counts, worms recovered and antibody levels within an endemic community in Northeast Thailand

Three techniques for estimating the intensity of Opisthorchis viverrini infection in individuals from a Northeast Thai community are compared. Egg counts were determined using a quantitative formalin/ethyl acetate technique, worm burdens were estimated by expulsion chemotherapy and antibody levels were measured by ELISA. Log-transformed worm and egg counts were closely correlated (r = 0.80), suggesting that both measurements provide good assessments of relative intensity of infection. However, no Opisthorchis worms were recovered from 34 people with high egg counts; probably due to problems with the expulsion technique in some individuals. Examination of egg production per fluke indicated that each fluke contributed an average of 180 eggs per gram (epg) of faeces and fecundity was negatively associated with total worm burden. Serum IgG levels correlated significantly with Opisthorchis egg count (r = 0.61) at two independent assessments. Although significant associations were observed between antibody levels and echinostome infection, analysis suggested that these reflected independent associations between these two variables and Opisthorchis infection and age. We conclude that all three measurements are useful for epidemiological studies.

Recent developments in laboratory diagnosis of melioidosis.

a Laboratory of Immunology, Chulabhorn Research Institute, Bangkok 10210, Thailand b Department of Microbiology, Faculty of Science, Mahidol Uni6ersity, Rama VI Road, Bangkok 10400, Thailand c Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol Uni6ersity, Bangkok, Thailand d Department of Microbiology, Faculty of Medicine, Khon Kaen Uni6ersity, Khon Kaen, Thailand e National Institute of Health, Department of Medical Ser6ices, Ministry of Public Health, Nonthaburi, Thailand f Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol Uni6ersity, Bangkok, Thailand g Department of Internal Medicine, Faculty of Medicine Siriraj Hospital, Mahidol Uni6ersity, Bangkok, Thailand

Immune responsiveness and parasite-specific antibody levels in human hepatobiliary disease associated with Opisthorchis viverrini infection.

Opisthorchis viverrini infection is associated with human hepatobiliary disease and cholangiocarcinoma, but the role of the immune response in the pathogenesis of infection is unclear. Here ultrasonography was used to examine the biliary tracts of residents from an endemic community. Delayed‐type hypersensitivity responses to unrelated antigens, and fluke‐specific IgG and IgA levels in serum of this group were also examined. Relationships between immunological parameters, intensity of infection and radiologically measured variables are reported. Immune responsiveness to unrelated antigens did not vary with intensity of parasite infection or disease status. Of all the variables, IgG levels were most markedly elevated in disease cases compared with normal subjects and were closely associated with gall bladder size and dysfunction. This is consistent with the hypothesis that an immunopathologic mechanism is involved in opisthorchiasis and suggests that antibody levels may be useful in screening populations for fluke‐associated hepatobiliary disease.

Growing Burkholderia pseudomallei in Biofilm Stimulating Conditions Significantly Induces Antimicrobial Resistance

Background Burkholderia pseudomallei, a Gram-negative bacterium that causes melioidosis, was reported to produce biofilm. As the disease causes high relapse rate when compared to other bacterial infections, it therefore might be due to the reactivation of the biofilm forming bacteria which also provided resistance to antimicrobial agents. However, the mechanism on how biofilm can provide tolerance to antimicrobials is still unclear. Methodology/Principal Findings The change in resistance of B. pseudomallei to doxycycline, ceftazidime, imipenem, and trimethoprim/sulfamethoxazole during biofilm formation were measured as minimum biofilm elimination concentration (MBEC) in 50 soil and clinical isolates and also in capsule, flagellin, LPS and biofilm mutants. Almost all planktonic isolates were susceptible to all agents studied. In contrast, when they were grown in the condition that induced biofilm formation, they were markedly resistant to all antimicrobial agents even though the amount of biofilm production was not the same. The capsule and O-side chains of LPS mutants had no effect on biofilm formation whereas the flagellin-defective mutant markedly reduced in biofilm production. No alteration of LPS profiles was observed when susceptible form was changed to resistance. The higher amount of N-acyl homoserine lactones (AHLs) was detected in the high biofilm-producing isolates. Interestingly, the biofilm mutant which produced a very low amount of biofilm and was sensitive to antimicrobial agents significantly resisted those agents when grown in biofilm inducing condition. Conclusions/Significance The possible drug resistance mechanism of biofilm mutants and other isolates is not by having biofilm but rather from some factors that up-regulated when biofilm formation genes were stimulated. The understanding of genes related to this situation may lead us to prevent B. pseudomallei biofilms leading to the relapse of melioidosis.

Soil physicochemical properties related to the presence of Burkholderia pseudomallei.

The incidence of Burkholderia pseudomallei in the soil from north-east Thailand is estimated to be 20-fold higher than that from central Thailand, and is associated with a 10-fold higher incidence of melioidosis in the region than in central Thailand. This study investigated the presence of B. pseudomallei in relation to the physicochemical properties of soil from Khon Kaen province, north-east Thailand. Thirteen districts (54.2%) were positive for B. pseudomallei. From a selected district, B. pseudomallei was cultured from 19 of 50 sites (38%). The soil in this area was predominantly sandy. From the positive sites, the organism was found mainly at a depth of 30 cm (43/68, 63% of isolates) and was significantly associated with certain soil physicochemical parameters, including a pH of 5.0-6.0, a moisture content >10%, and higher chemical oxygen demand and total nitrogen than negative sites (P<0.05). Burkholderia pseudomallei is unevenly distributed in this area, with the pH of the soil being the major determinant of the presence of the organism. The sandy soil type of north-east Thailand can support the survival of B. pseudomallei and allow it to move freely with water flow, and thus readily come in contact with people during the rainy season.

Accuracy of Burkholderia pseudomallei Identification Using the API 20NE System and a Latex Agglutination Test

ABSTRACT In an evaluation of the API 20NE for the identification of Burkholderia spp., 792/800 (99%) Burkholderia pseudomallei and 17/19 (89%) B. cepacia isolates were correctly identified but 10 B. mallei and 98 B. thailandensis isolates were not correctly identified. A latex agglutination test was positive for 796/800 (99.5%) B. pseudomallei isolates and negative for 120 other oxidase-positive gram-negative bacilli.