Research Article: Mycobacterium ulcerans DNA Not Detected in Faecal Samples from Buruli Ulcer Patients: Results of a Pilot Study

Date Published: May 4, 2011

Publisher: Public Library of Science

Author(s): Fred S. Sarfo, Caroline J. Lavender, Janet A. M. Fyfe, Paul D. R. Johnson, Timothy P. Stinear, Richard O. Phillips, Pere-Joan Cardona.

Abstract: It has recently been shown that in a Buruli ulcer (BU) endemic region of southeastern Australia, significant numbers of possums (native tree-dwelling marsupials) have clinical BU disease. Furthermore, based on quantitative PCR (qPCR) analysis, animals with BU lesions (and some without) shed M. ulcerans DNA in their faeces, indicative of bacterial loads of up to 108 organisms/gram. These findings led us to propose that humans might also harbour M. ulcerans in their gastrointestinal tract and shed the bacterium in their faeces. We conducted a pilot study and collected faecal swabs from 26 patients with confirmed BU and 31 healthy household controls. Faecal samples were also collected from 10 healthy controls from non-endemic regions in Ghana. All 67 specimens were negative when tested by IS2404 PCR. The detection sensitivity of this method was ≥104 bacteria per gram (wet-weight) of human faecal material. We conclude that the human gastrointestinal tract is unlikely to be a significant reservoir of M. ulcerans.

Partial Text: Efforts to control the spread of Buruli ulcer (BU) will be significantly improved if we understand the ecology of the causative agent, Mycobacterium ulcerans. Recent surveys of the environment in BU endemic regions near Melbourne, Australia, have revealed that over 40% of possums (small tree-dwelling animals native to Australia with a body temperature like humans of ∼36°C) are shedding high levels of M. ulcerans DNA in their faeces and may be acting as reservoirs of M. ulcerans[1]. A transmission model is proposed where contaminated possum excreta enters mosquito breeding habitats (drains and roof gutters near houses) where the insects may acquire the bacterium and then transmit it to humans during biting [2], [3]. There are no possums in Africa and a recent survey of small animals in Benin did not identify any species with M. ulcerans in their organs or faeces [4]. Nevertheless, the presence of M. ulcerans in the gastrointestinal tract of warm-blooded, terrestrial animals has led to a reconsideration of where M. ulcerans is located in the environment, and raises the possibility of a terrestrial animal reservoir for the bacterium in African BU endemic regions. We therefore speculated that humans were acting like the possums – as both a disease-susceptible host and possible reservoir. To test this hypothesis we used IS2404 qPCR to screen faecal specimens from confirmed BU patients, household contacts, and control samples from BU non-endemic regions in Ghana for the presence of M. ulcerans DNA.

Prior to PCR screening of human faecal material we established the detection sensitivity of DNA extraction and IS2404 qPCR analysis method. Spiking experiments with serial 10-fold dilutions of M. ulcerans in human faeces showed that the smallest inoculum of M. ulcerans that was readily detected by IS2404 qPCR was 1.2×103 bacterial cells per swab (Fig. 1), which represents an overall minimum detection limit ≥3×104M. ulcerans per gram wet faecal material. PCR inhibitors were effectively removed by this DNA extraction method as the IPC was readily detected in DNA extracted from all dilutions of spiked faecal material (Figure 1).

The data collected in this pilot study suggest that, unlike possums in a BU endemic area (which may shed up to 108 organisms per gram of faeces), humans do not shed detectable levels of M. ulcerans in their faeces. Nevertheless, even though humans do not appear to carry M. ulcerans in their gastrointestinal tracts, they may act as a susceptible host and a reservoir of M. ulcerans. With the exception of possums in Australia, human BU lesions represent the highest concentration of M. ulcerans cells in any source identified so far. Bacteria from human BU lesions may be shed into the environment by various means, such as household fomite contamination, shedding into waterways during bathing of lesions and/or disseminated by insects. High resolution molecular epidemiological analysis of strains from the Densu River Basin in Ghana suggest that transmission of M. ulcerans occurs at a very local level, consistent with a local reservoir of the bacterium [6]. Furthermore, a recent study in Benin of the seroepidemiological potential of M. ulcerans antigens suggested that there is widespread exposure to M. ulcerans in BU endemic areas, again consistent with a significant local reservoir of the bacterium in close contact with humans [7].