Multitarget Quantitative PCR Improves Detection and Predicts Cultivability of the Pathogen Burkholderia pseudomallei.

Burkholderia pseudomallei is present in the environment in many parts of the world and causes the often-fatal disease melioidosis. The sensitive detection and quantification of B. pseudomallei in the environment are a prerequisite for assessing the risk of infection. We recently reported the direct detection of B. pseudomallei in soil samples using a quantitative PCR (qPCR) targeting a single type three secretion system 1 (TTSS1) gene. Here, we extend the qPCR-based analysis of B. pseudomallei in soil by validating novel qPCR gene targets selected from a comparative genomic analysis. Two hundred soil samples from two rice paddies in northeast Thailand were evaluated, of which 47% (94/200) were B. pseudomallei culture positive. The TTSS1 qPCR and two novel qPCR assays that targeted open reading frames (ORFs) BPSS0087 and BPSS0745 exhibited detection rates of 76.5% (153/200), 34.5% (69/200), and 74.5% (150/200), respectively. The combination of TTSS1 and BPSS0745 qPCR increased the detection rate to 90% (180/200). Combining the results of the three qPCR assays and the BPSS1187 nested PCR previously published, all 200 samples were positive by at least one PCR assay. Samples positive by either TTSS1 (n = 153) or BPSS0745 (n = 150) qPCR were more likely to be direct-culture positive, with odds ratios of 4.0 (95% confidence interval [CI], 1.7 to 9.5; P < 0.001) and 9.0 (95% CI, 3.1 to 26.4; P < 0.001), respectively. High B. pseudomallei genome equivalents correlated with high CFU counts by culture. In conclusion, multitarget qPCR improved the B. pseudomallei detection rate in soil samples and predicted culture positivity. This approach has the potential for use as a sensitive environmental screening method for B. pseudomalleiIMPORTANCE The worldwide environmental distribution of the soil bacterium Burkholderia pseudomallei remains to be determined. So far, most environmental studies have relied on culture-based approaches to detect this pathogen. Since current culture methods are laborious, are time consuming, and have limited sensitivity, culture-independent and more sensitive methods are needed. In this study, we show that a B. pseudomallei-specific qPCR approach can detect significantly higher numbers of B. pseudomallei-positive soil samples from areas where it is endemic compared with that from culture. The use of multiple independent B. pseudomallei-specific qPCR targets further increased the detection rate of B. pseudomallei compared with that from single targets. Samples with a high molecular B. pseudomallei load were more likely to be culture positive. We conclude that our quantitative multitarget approach might be useful in defining areas where there is a risk of B. pseudomallei infections in different parts of the world.

Highly sensitive direct detection and quantification of Burkholderia pseudomallei bacteria in environmental soil samples by using real-time PCR.

The soil bacterium and potential biothreat agent Burkholderia pseudomallei causes the infectious disease melioidosis, which is naturally acquired through environmental contact with the bacterium. Environmental detection of B. pseudomallei represents the basis for the development of a geographical risk map for humans and livestock. The aim of the present study was to develop a highly sensitive, culture-independent, DNA-based method that allows direct quantification of B. pseudomallei from soil. We established a protocol for B. pseudomallei soil DNA isolation, purification, and quantification by quantitative PCR (qPCR) targeting a type three secretion system 1 single-copy gene. This assay was validated using 40 soil samples from Northeast Thailand that underwent parallel bacteriological culture. All 26 samples that were B. pseudomallei positive by direct culture were B. pseudomallei qPCR positive, with a median of 1.84 × 10(4) genome equivalents (range, 3.65 × 10(2) to 7.85 × 10(5)) per gram of soil, assuming complete recovery of DNA. This was 10.6-fold (geometric mean; range, 1.1- to 151.3-fold) higher than the bacterial count defined by direct culture. Moreover, the qPCR detected B. pseudomallei in seven samples (median, 36.9 genome equivalents per g of soil; range, 9.4 to 47.3) which were negative by direct culture. These seven positive results were reproduced using a nested PCR targeting a second, independent B. pseudomallei-specific sequence. Two samples were direct culture and qPCR negative but nested PCR positive. Five samples were negative by both PCR methods and culture. In conclusion, our PCR-based system provides a highly specific and sensitive tool for the quantitative environmental surveillance of B. pseudomallei.

Distribution of Serological Response to Burkholderia pseudomallei in Swine from Three Provinces of Vietnam.

(1) Background: Burkholderia pseudomallei is an environmentally mediated saprophytic pathogen that can cause severe disease in humans. It is well known that B. pseudomallei survives in tropical moist soil environments worldwide, but melioidosis is gaining recognition as a public and veterinary health issue in Vietnam. The contribution of animals to human disease is unknown, necessitating further investigation. (2) Methods: Swine sera were collected from two populations, one grazing and one commercially farmed, from three provinces in Vietnam. ELISAs utilizing B. pseudomallei capsular polysaccharide (CPS), outer polysaccharide (OPS), and Hcp1 protein were used to screen serum samples. Positive samples were mapped to the commune level. Seroprevalence calculations and pig population estimates were used to approximate number of swine exposures per commune. (3) Results: Grazing pigs from Hoa Binh had significantly higher seropositivity levels (11.4%, 95% CI: 9.7-13.1) compared to farmed pigs from Ha Tinh and Nghe An (4%, 95% CI: 3.3-4.7). Average swine seropositivity rates were ~6.3% (95% CI: 5-7.9), higher than previously identified in Vietnam (~0.88%). (4) Conclusions: Initial serological sampling identified a significant number of seropositive and potential melioidosis infections occurring in swine in Vietnam. This work is a critical step in understanding the role swine may play in the epidemiology of human melioidosis in Vietnam.

Improved culture-based detection and quantification of Burkholderia pseudomallei from soil.

Environmental surveillance of the Gram-negative soil bacterium Burkholderia pseudomallei, the aetiological agent of melioidosis, is important in order to define human populations and livestock at risk of acquiring the infection. This study aimed to develop a more sensitive method for the detection of B. pseudomallei from soil samples in endemic areas compared with the currently used culture method based on soil dispersion in water. We report the development of a new protocol that involves soil dispersion in a polyethylene glycol (PEG) and sodium deoxycholate (DOC) solution to increase the yield of viable B. pseudomallei from soil samples. Comparative testing of soil samples from Northeast Thailand covering a wide range of B. pseudomallei concentrations demonstrated a significantly higher recovery (P<0.0001) of B. pseudomallei colony-forming units by the new method compared with the conventional method. The data indicate that using the detergents PEG and DOC not only results in a higher recovery of viable B. pseudomallei but also results in a shift in the bacterial species recovered from soil samples. Future studies on the geographical distribution and population structure of B. pseudomallei in soil are likely to benefit from the new protocol described here.

Clinical and microbiological features of melioidosis in northern Vietnam.

Sporadic cases of melioidosis have been reported from Vietnam for decades, but clinical and epidemiological data for the indigenous population are still scarce. In this study, we reviewed clinical and demographic data of patients with culture-proven melioidosis diagnosed at a single large referral hospital in Hanoi between November 1997 and December 2005. We found that the clinical manifestations of melioidosis (with fatal septicaemia as the most common presentation), a high rate of underlying diseases, and a peak of cases admitted during the wet season, were similar to studies from other endemic areas. The geographical origin of patients with melioidosis showed that melioidosis existed in at least 18 northern provinces. The characterization of clinical Burkholderia pseudomallei strains by multilocus sequence typing identified 17 different sequence types (STs), 11 of which have (as yet) not been found outside Vietnam. Several of these STs presumably were generated through recent evolutionary events in this rapidly diversifying bacterial species, and thus, restricted geographic distribution may be a consequence of limited time passed since emergence. To our knowledge, this is the first report on a series of cases describing clinical and epidemiological features of melioidosis and corresponding B. pseudomallei strains from northern Vietnam.