Evaluation of consensus method for the culture of Burkholderia pseudomallei in soil samples from Laos

David A.B Dance,Viengmon Davong,Sabine Dittrich,Sayan Langla,Vanaporn Wuthiekanun,Manivanh Vongsouvath,Joy Silisouk,Michael Knappik,Alain Pierret,Premjit Amornchai,Sayaphet Rattanavong,Direk Limmathurotsakul,Paul N Newton

doi:10.12688/wellcomeopenres.14851.1

Abstract

Background: We have previously shown that PCR following enrichment culture is the most sensitive method to detect Burkholderia pseudomallei in environmental samples. Here we report an evaluation of the published consensus method for the culture of B. pseudomallei from Lao soil in comparison with our conventional culture method and with PCR with or without prior broth enrichment. Methods: One hundred soil samples were collected from a field known to contain B. pseudomallei and processed by: (i) the conventional method, (ii-iii) the consensus method using media prepared in either Laos or Thailand, and (iv) the consensus method performed in Thailand, as well as by (v) PCR following direct extraction of DNA from soil and (vi) PCR following broth pre-enrichment. Results: The numbers of samples in which B. pseudomallei was detected were 42, 10, 7, 6, 6 and 84, respectively. However, two samples were positive by the consensus method but negative by conventional culture, and one sample was negative by PCR following enrichment although B. pseudomallei was isolated by the conventional culture method. Conclusions/Discussion: The results show that no single method will detect all environmental samples that contain B. pseudomallei. People conducting environmental surveys for this organism should be aware of the possibility of false-negative results using the consensus culture method. An approach that entails screening using PCR after enrichment, followed by the evaluation of a range of different culture methods on PCR-positive samples to determine which works best in each setting, is recommended.

Highlights

Melioidosis, or infection with Burkholderia pseudomallei, is an important but under-recognised public health problem throughout the tropics[1]
During studies of the seasonal variation of the distribution of B. pseudomallei in a rice paddy in northern Laos, we noticed that enrichment cultures often failed to isolate B. pseudomallei even when it was isolated from the same sample on solid media[34]
The sensitivity of the consensus method conducted in Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit (LOMWRU) using locally made media (12% [10/85]; 95%CI 5.8-20.6%) or media prepared in MORU (8% [7/85]; 95%CI 3.4-16.2) was slightly higher than the sensitivity of the consensus method in MORU this did not achieve significance (p=0.29 and p>0.99, respectively)

Summary

Introduction

Melioidosis, or infection with Burkholderia pseudomallei, is an important but under-recognised public health problem throughout the tropics[1]. During studies of the seasonal variation of the distribution of B. pseudomallei in a rice paddy in northern Laos, we noticed that enrichment cultures often failed to isolate B. pseudomallei even when it was isolated from the same sample on solid media[34]. This led to a formal comparison of the consensus method on Lao soil with other culture and molecular methods. The results of the comparison of the molecular methods with culture on Lao soil and water samples have already been published[13], and this paper will focus on a comparison of the performance of the consensus soil method with other methods

Objectives

Methods

Results