Burkholderia pseudomallei: Its Detection in Soil and Seroprevalence in Bangladesh

Abstract

Background: Melioidosis, caused by Burkholderia pseudomallei, is an endemic disease in Bangladesh. No systematic study has yet been done to detect the environmental source of the organism and its true extent in Bangladesh. The present study attempted to isolate B. pseudomallei in soil samples and to determine its seroprevalence in several districts in Bangladesh.

Methodology and results: Soil samples were collected from rural areas of four districts of Bangladesh from where culture confirmed melioidosis cases were detected earlier. Multiple soil samples, collected from 5-7 sampling points of 3-5 sites of each district, were cultured in Ashdown selective media. Suspected colonies of B. pseudomallei were identified by biochemical and serological test, and by polymerase chain reaction (PCR) using 16s rRNA specific primers. Blood samples were collected from 940 healthy individuals of four districts to determine anti- B. pseudomallei IgG antibody levels by indirect enzyme linked immunosorbent assay (ELISA) using sonicated crude antigen. Out of 179 soil samples, B. pseudomallei was isolated from two samples of Gazipur district which is located 58 km north of capital Dhaka city. Both the isolates were phenotypically identical, arabinose negative and showed specific 550bp band in PCR. Out of 940 blood samples, anti- B. pseudomallei IgG antibody, higher than the cut-off value (>0.8), was detected in 21.5% individuals. Seropositivity rate was 22.6%-30.8% in three districts from where melioidosis cases were detected earlier, compared to 9.8% in a district where no melioidosis case was either detected or reported (p<0.01). Seropositivity increased with the advancement of age from 5.3% to 30.4% among individuals aged 1-10 years and > 50 years respectively. The seropositivity rates were 26.0% and 20.6% in male and female respectively, while it was 20-27% among different occupational groups. No significant association was observed with gender (χ2 = 3.441, p = 0.064) or any occupational group (χ2 = 3.835, p = 0.280).

Conclusion: This is the first study demonstrating the presence of B. pseudomallei in the environmental (soil) samples of Bangladesh. It also suggested that a large proportion of people, residing in these districts, were exposed to the organism.

Fig 1. Map of Bangladesh showing collection sites (districts) of soil and blood samples.

Shaded areas indicate districts with known melioidosis cases. M = Mymensingh, G = Gazipur, K = Kishoregange, S = Sylhet, N = Narayangange. The distance of Mymensingh, Gazipur, Kishoregange, Sylhet and Narayangange from capital Dhaka is 115, 40, 89, 198 and 14 km respectively. ■ = Site of soil collection; ▲ = Site of blood collection; ● = Capital Dhaka. Source of the map: [9].

Fig 2. The effect of adsorption with whole cell P. aeruginosa and B. pseudomallei on the anti- B. pseudomallei IgG antibodies.

Before Ad = Before adsorption; Ps Ad = Adsorption with P. aeruginosa; Bps Ad = Adsorption with B. pseudomallei. n = 24 seropositive sera in each group.

Fig 3. PCR analysis of B. pseudomallei isolates from Gazipur district.

Lane 1- 100bp Marker, Lane 2- Positive control (USM strain of B. pseudomallei, Lane 3- Negative control (distilled water), Lane 4- K23 (Gazipur soil isolate) and Lane 5- K35 (Gazipur soil isolate).