Search

Introduction: Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae are pathogens of significant public health interest for which new antibiotics are urgently needed. Aim: To determine the prevalence of ESBLs in E. coli and K. pneumoniae isolates from patients attending the Tamale Teaching Hospital (TTH) in Ghana. Methodology: The study was a cross-sectional study involving convenience sampling of E. coli and K. pneumoniae isolates from consenting patients' clinical specimens, between April and June 2015. Antimicrobial susceptibility test was performed, and ESBL-producer phenotypes were further screened for BlaTEM, BlaSHV, and BlaCTX-M genes. Patients' clinical data were additionally collected using a structured questionnaire. Results: Of the 150 non-duplicate E. coli and K. pneumoniae isolates identified, 140 were confirmed as E. coli (84%, n = 117) and K. pneumoniae (16%, n = 23). Of these, sixty-two (44%) [E. coli (84%; n = 52); K. pneumoniae (16%; n = 10)] phenotypically expressed ESBLs. The proportion of ESBL-producing isolates was higher in adults (15–65 years) than in neonates (< 28 days) (p = 0.14). Most of the isolates showed a high percentage resistance to ampicillin (96%) and tetracycline (89%), but a relatively lower resistance to amikacin (36%). No isolate was resistant to meropenem. More ESBL producers were multidrug resistant compared to non-ESBL-producers [23% (14/62) versus 18% (14/78); p = 0.573]. Overall, 74% (n = 46) of the ESBL genotypes expressed BlaCTX-M-1 genes, followed by 63% (n = 39) BlaTEM, and 16% (n = 10) BlaSHV. The study showed a high prevalence of ESBL-positive E. coli and K. pneumoniae, mostly CTX-M-1 producers at TTH. Conclusion: Routine laboratory ESBL screening is warranted to inform patient management. [ABSTRACT FROM AUTHOR]

Buruli ulcer is an emerging chronic infectious skin disease caused by Mycobacterium ulcerans. Mycolactone, an exotoxin produced by the bacterium, is the only identified virulence factor so far, but the functions of this toxin and the mechanisms of disease progression remain unclear. By interfering Sec61 translocon, mycolactone inhibits the Sec61-dependent co-translational translocation of newly synthesized proteins, such as induced cytokines and immune cell receptors, into the endoplasmic reticulum. However, in regard to IL-1β, which is secreted by a Sec61-independent mechanism, mycolactone has been shown to induce IL-1β secretion via activation of inflammasomes. In this study, we clarified that cytokine induction, including that of IL-1β, in infected macrophages was suppressed by mycolactone produced by M. ulcerans subsp. shinshuense, despite the activation of caspase-1 through the inflammasome activation triggered in a manner independent of mycolactone. Intriguingly, mycolactone suppressed the expression of proIL-1β as well as TNF-α at the transcriptional level, suggesting that mycolactone of M. ulcerans subsp. shinshuense may exert additional inhibitory effect on proIL-1β expression. Remarkably, constitutively produced IL-18 was cleaved and mature IL-18 was actually released from macrophages infected with the causative mycobacterium. IL-18-deficient mice infected subcutaneously with M. ulcerans exhibited exacerbated skin inflammation during the course of disease progression. On the other hand, IL-1β controls bacterial multiplication in skin tissues. These results provide information regarding the mechanisms and functions of the induced cytokines in the pathology of Buruli ulcer. Author summary: Buruli ulcer, caused by Mycobacterium ulcerans, is an emerging chronic disease of the skin, characterized by massive skin ulceration. Mycolactone, an exotoxin produced by M. ulcerans is the sole identified virulence factor, but its functions are still unclear. By interfering Sec61 translocon, mycolactone inhibits Sec61-dependent co-translational translocation of newly synthesized proteins, such as induced cytokines and immune cell receptors, into the endoplasmic reticulum. However, in regard to IL-1β, which is secreted by a Sec61-independent mechanism, mycolactone has been shown to induce IL-1β secretion. In this study, we analyzed the activation of inflammasomes in infected macrophages using wild-type and toxin-negative strains of M. ulcerans subsp. shinshuense, and found that mycolactone inhibited IL-1β secretion, but not IL-18, resulting in the exclusive induction of IL-18 by the bacterial infection. Intriguingly, mycolactone suppressed the expression of proIL-1β as well as TNF-α at the transcriptional level, suggesting that mycolactone of M. ulcerans subsp. shinshuense may exert additional inhibitory effect on proIL-1β expression. Inflammasome activation accompanied by caspase-1 activation in cases of Buruli ulcer appears to be triggered in a manner largely independent of mycolactone. Using a mouse model of Buruli ulcer, we further demonstrated that IL-18 attenuates the progression of Buruli ulcer by controlling accumulation of neutrophils in the infected skin lesions. On the other hand, IL-1β controls bacterial multiplication in the skin. Our results shed light on the etiology of Buruli ulcer and provide insight into the relationship between the activation of inflammasomes and functions of cytokines. [ABSTRACT FROM AUTHOR]

Background: A recent study detected cutaneous leishmaniasis (CL) in 31.9% of persons with skin ulcers in the Oti Region of Ghana, resulting in a need to investigate other potential causes of the unexplained skin ulcers. Methodology/Principal findings: A community based cross-sectional study was conducted in the Oti region to investigate skin ulcers of undetermined aetiologies. To confirm a diagnosis of cutaneous leishmaniasis, Buruli ulcer, Haemophilus ducreyi ulcers, or yaws, DNA obtained from each patient skin ulcer sample was systematically subjected to polymerase chain reaction (PCR) for Leishmania spp., Mycobacterium ulcerans, Haemophilus ducreyi, and Treponema pallidum sub species pertenue. A total of 101 skin ulcer samples were obtained from 101 persons. Co-infection of more than one organism was observed in 68.3% of the samples. Forty (39.6%) participants had a positive result for Leishmania spp., 68 (67.3%) for Treponema pallidum sub. Sp. pertenue, and 74 (73.3%) for H. ducreyi. Twenty (19.8%) of the patient ulcers were simultaneously infected with Leishmania spp., Treponema pallidum sub. Sp. pertenue, and H. ducreyi. None of the patients' lesions yielded a positive result for Mycobacterium ulcerans. Conclusions/Significance: This study detected single and mixed occurrence of the causative organisms of CL, yaws, and H. ducreyi cutaneous ulcers in CL endemic communities of the Oti Region in Ghana. These findings emphasize the importance of integrating multiple skin diseases on a common research platform and calls for the development of a comprehensive guideline for diagnosing and treating tropical ulcers in the study areas. [ABSTRACT FROM AUTHOR]

The gold standard for detection of Mycobacterium ulcerans is PCR due to its high accuracy in confirmation of suspected cases. But the available PCR assays are designed for standard size thermocyclers which are immobile and suited for reference laboratories often located long distances from endemic communities. This makes it a challenge to obtain immediate results for patient management. We validated and evaluated a dried reagent-based PCR assay adapted for a handheld, battery-operated, portable thermocycler with the potential to extend diagnostics to endemic communities with limited infrastructure. The diagnostic accuracy of the assay following a multi-center evaluation by three Buruli ulcer reference laboratories with over 300 clinical samples showed sensitivity and specificity of 100–97% and 100–94%, respectively using centralized IS2404 quantitative PCR platform as a reference standard. This assay coupled with a field-friendly extraction method fulfill almost all the target product profiles of Buruli ulcer for decentralized testing at the district, health center and community levels; a key critical action for achieving the NTD Road Map 2030 target for Buruli ulcer. Author summary: Early diagnosis of Buruli ulcer remains a major problem in many endemic countries particularly in sub-Saharan Africa which continues to report large wounds as a result. Though reference laboratories with capacity to confirm diagnosis of the disease by recommended PCR method are available in most endemic countries, they are often located long distances from endemic communities and treatment centres. This makes it a challenge to obtain immediate results for patient management. There is therefore the need to have innovative diagnostic tools that can be fully implemented in local health facilities but not compromising accuracy. We have evaluated a diagnostic tool with accuracy like the recommended method with the potential to be used in health facilities closer to the patients. [ABSTRACT FROM AUTHOR]

Background: Buruli ulcer is a tissue necrosis infection caused by an environmental mycobacterium called Mycobacterium ulcerans (MU). The disease is most prevalent in rural areas with the highest rates in West and Central African countries. The bacterium produces a toxin called mycolactone which can lead to the destruction of the skin, resulting in incapacitating deformities with an enormous economic and social burden on patients and their caregivers. Even though there is an effective antibiotic treatment for BU, the control and management rely on early case detection and rapid diagnosis to avert morbidities. The diagnosis of Mycobacterium ulcerans relies on smear microscopy, culture histopathology, and PCR. Unfortunately, all the current laboratory diagnostics have various limitations and are not available in endemic communities. Consequently, there is a need for a rapid diagnostic tool for use at the community health centre level to enable diagnosis and confirmation of suspected cases for early treatment. The present study corroborated the diagnostic performance and utility of fluorescent-thin layer chromatography (f-TLC) for the diagnosis of Buruli ulcer. Methodology/Principal findings: The f-TLC method was evaluated for the diagnosis of Buruli ulcer in larger clinical samples than previously reported in an earlier preliminary study Wadagni et al. (2015). A total of 449 patients suspected of BU were included in the final data analysis out of which 122 (27.2%) were positive by f-TLC and 128 (28.5%) by PCR. Using a composite reference method generated from the two diagnostic methods, 85 (18.9%) patients were found to be truly infected with M. ulcerans, 284 (63.3%) were uninfected, while 80 (17.8%) were misidentified as infected or noninfected by the two methods. The data obtained was used to determine the discriminatory accuracy of the f-TLC against the gold standard IS2404 PCR through the analysis of its sensitivity, specificity, positive (+LR), and negative (–LR) likelihood ratio. The positive (PPV) and negative (NPV) predictive values, area under the receiver operating characteristic curve Azevedo et al. (2014), and diagnostic odds ratio were used to assess the predictive accuracy of the f-TLC method. The sensitivity of f-TLC was 66.4% (85/128), specificity was 88.5% (284/321), while the diagnostic accuracy was 82.2% (369/449). The AUC stood at 0.774 while the PPV, NPV, +LR, and–LR were 69.7% (85/122), 86.9% (284/327), 5.76, and 0.38, respectively. The use of the rule-of-thumb interpretation of diagnostic tests suggests that the method is good for use as a diagnostic tool. Conclusions/Significance: Larger clinical samples than previously reported had been used to evaluate the f-TLC method for the diagnosis of Buruli ulcer. A sensitivity of 66.4%, a specificity of 88.5%, and diagnostic accuracy of 82.2% were obtained. The method is good for diagnosis and will help in making early clinical decisions about the patients as well as patient management and facilitating treatment decisions. However, it requires a slight modification to address the challenge of background interference and lack of automatic readout to become an excellent diagnostic tool. [ABSTRACT FROM AUTHOR]

Global dissemination of New Delhi metallo-β-lactamase (NDM)-producing bacteria has become a major health threat. However, there are few reports regarding the identification and characterisation of NDM-producing bacteria from West Africa, including Ghana. An Escherichia coli strain with resistance to meropenem was isolated from the Tamale Teaching Hospital in Ghana. Its identification and determination of antibiotic susceptibility profile were carried out using commercial systems. The antibiotic resistance mechanism was analysed by phenotypic detection kits, PCR, and DNA sequencing. Conjugation experiments, S1 nuclease pulsed field gel electrophoresis, and Southern blotting were performed. Finally, the NDM-1-harbouring plasmid was characterised using next-generation sequencing and phylogenetic analysis. The meropenem-resistant Escherichia coli strain EC2189 harboured blaNDM-1 and belonged to sequence type 410. blaNDM-1 was located on the IncHI type transferrable plasmid p2189-NDM (248,807 bp long), which co-carried multiple resistance genes, such as blaCTX-M-15, aadA1, aac(6')-Ib, sul3, dfrA12, and cmlA1. p2189-NDM phylogenetically differed from previously identified blaNDM-1-positive IncHI type plasmids. A truncated Tn125 containing blaNDM-1 was bracketed by an ISSm-1-like insertion sequence upstream and by a site-specific integrase downstream. To the best of our knowledge, we have, for the first time identified and molecularly characterised an NDM-1-producing Enterobacteriaceae strain in Ghana with blaNDM-1 that had a novel genetic structure. Our findings indicate a possibility of NDM-1 dissemination in Ghana and underscore the need for constant monitoring of carbapenemase-producing bacteria. [ABSTRACT FROM AUTHOR]

Background: The environmental pathogen, Mycobacterium ulcerans (MU) can infect both humans and animals and cause Buruli ulcer (BU) disease. However, its mode(s) of transmission from the colonized environment to human/animal hosts remain unclear. In Australia, MU can infect both wildlife and domestic mammals. Till date, BU-like lesions have only been reported in wildlife in Africa. This warrants a thorough assessment of possible MU in domestic animals in Africa. Here, we screened roaming domesticated animals that share the human microhabitat in two different BU endemic sites, Sedje-Denou in Benin and Akonolinga in Cameroon, for MU lesions. Methodology/Principal findings: We screened roaming mammals and birds across 3 endemic villages of Sedje-Denou in Southern Benin and 6 endemic villages of Akonolinga in Cameroon. After approval from relevant authorities, specimens (wound swabs and tissue fragments) were collected from animals with open or active lesion and systematically screened to detect the presence of MU though the diagnostic DNA targets IS2404, IS2606 and KR-B. Out of 397 animals surveyed in Akonolinga, 44 (11.08%) carried skin lesions and all were negative for MU DNA. For Sedje-Denou, only 25 (6.93%) out of 361 animals surveyed carried external skin lesions of which 2 (8%) were positive for MU DNA targets. These MU infected lesions were found in two different villages on a goat (abdominal part) and on a dog (nape area of the neck). Source-tracking of MU isolates within infected animal lesions was performed using VNTR genotyping and further confirmed with sequencing. One MU VNTR genotype (Z) was successfully typed from the goat lesion. The evolutionary history inferred from sequenced data revealed a clustering of animal MU isolates within isolates from human lesions. Conclusion/Significance: This study describes the first report of two MU infected lesions in domestic animals in Africa. Their DNA sequence analyses show close relationship to isolates from human cases. It suggests that MU infection should be suspected in domestic hosts and these could play a role in transmission. The findings further support the hypothesis that MU is a ubiquitous environmental pathogen found in endemic areas, and probably involved in a multiple transmission pathway. [ABSTRACT FROM AUTHOR]

Background: In developing countries, hospitalized burn victims are at high risk of nosocomial infections caused by Staphylococcus aureus. Risk factors include poor infection control practices, prolonged hospitalisation and limited capacity for laboratory microbiological analyses. These problems are compounded by widespread use of antibiotics that drives the spread of multi-drug resistant bacteria. Methods: During the study period (November 2014-June 2015), nasal and invasive S. aureus isolates were collected consecutively from patients and healthcare workers (HCWs) within the burn unit of the Reconstructive Plastic Surgery and Burn Center of Korle Bu Teaching Hospital in Ghana. Antibiotic prescription, antibiotic susceptibility and bacterial typing were used to assess antibiotic pressure, antibiotic resistance, and possible transmission events among patients and HCWs. Results: Eighty S. aureus isolates were obtained from 37 of the 62 included burn patients and 13 of the 29 HCWs. At admission, 50% of patients carried or were infected with S. aureus including methicillin resistant S. aureus (MRSA). Antibiotic use per 100 days of hospitalization was high (91.2 days), indicating high selective pressure for resistant pathogens. MRSA isolates obtained from 11 patients and one HCW belonged to the same spa-type t928 and multi-locus sequence type 250, implying possible transmission events. A mortality rate of 24% was recorded over the time of admission in the burn unit. Conclusion: This study revealed a high potential for MRSA outbreaks and emergence of resistant pathogens amongst burn patients due to lack of patient screening and extended empirical use of antibiotics. Our observations underscore the need to implement a system of antibiotic stewardship and infection prevention where microbiological diagnostics results are made available to physicians for timely and appropriate patient treatment. [ABSTRACT FROM AUTHOR]

Introduction Mycobacterium ulcerans infection, known as Buruli ulcer, is a disease of the skin and subcutaneous tissues which is an important but neglected tropical disease with its major impact in rural parts of West and Central Africa where facilities for diagnosis and management are poorly developed. We evaluated fluorescent thin layer chromatography (f-TLC) for detection of mycolactone in the laboratory using samples from patients with Buruli ulcer and patients with similar lesions that gave a negative result on PCR for the IS2404 repeat sequence of M. ulcerans. Methodology/Principal findings Mycolactone and DNA extracts from fine needle aspiration (FNA), swabs and biopsy specimen were used to determine the sensitivity and specificity of f-TLC when compared with PCR for the IS2404. For 71 IS2404 PCR positive and 28 PCR negative samples the sensitivity was 73.2% and specificity of 85.7%for f-TLC. The sensitivity was similar for swabs (73%), FNAs (75%) and biopsies (70%). Conclusions We have shown that mycolactone can be detected from M. ulcerans infected skin tissue by f-TLC technique. The technique is simple, easy to perform and read with minimal costs. In this study it was undertaken by a member of the group from each endemic country. It is a potentially implementable tool at the district level after evaluation in larger field studies. [ABSTRACT FROM AUTHOR]

Background: Buruli ulcer (BU) is a skin infection caused by Mycobacterium ulcerans. The wounds of most BU patients are colonized with different microorganisms, including Staphylococcus aureus. Methodology: This study investigated possible patient-to-patient transmission events of S. aureus during wound care in a health care center. S. aureus isolates from different BU patients with overlapping visits to the clinic were whole-genome sequenced and analyzed by a gene-by-gene approach using SeqSphere+ software. In addition, sequence data were screened for the presence of genes that conferred antibiotic resistance. Principal Findings: SeqSphere+ analysis of whole-genome sequence data confirmed transmission of methicillin resistant S. aureus (MRSA) and methicillin susceptible S. aureus among patients that took place during wound care. Interestingly, our sequence data show that the investigated MRSA isolates carry a novel allele of the fexB gene conferring chloramphenicol resistance, which had thus far not been observed in S. aureus. [ABSTRACT FROM AUTHOR]

Efforts to control the spread of Buruli ulcer – an emerging ulcerative skin infection caused by Mycobacterium ulcerans - have been hampered by our poor understanding of reservoirs and transmission. To help address this issue, we compared whole genomes from 18 clinical M. ulcerans isolates from a 30km2 region within the Asante Akim North District, Ashanti region, Ghana, with 15 other M. ulcerans isolates from elsewhere in Ghana and the surrounding countries of Ivory Coast, Togo, Benin and Nigeria. Contrary to our expectations of finding minor DNA sequence variations among isolates representing a single M. ulcerans circulating genotype, we found instead two distinct genotypes. One genotype was closely related to isolates from neighbouring regions of Amansie West and Densu, consistent with the predicted local endemic clone, but the second genotype (separated by 138 single nucleotide polymorphisms [SNPs] from other Ghanaian strains) most closely matched M. ulcerans from Nigeria, suggesting another introduction of M. ulcerans to Ghana, perhaps from that country. Both the exotic genotype and the local Ghanaian genotype displayed highly restricted intra-strain genetic variation, with less than 50 SNP differences across a 5.2Mbp core genome within each genotype. Interestingly, there was no discernible spatial clustering of genotypes at the local village scale. Interviews revealed no obvious epidemiological links among BU patients who had been infected with identical M. ulcerans genotypes but lived in geographically separate villages. We conclude that M. ulcerans is spread widely across the region, with multiple genotypes present in any one area. These data give us new perspectives on the behaviour of possible reservoirs and subsequent transmission mechanisms of M. ulcerans. These observations also show for the first time that M. ulcerans can be mobilized, introduced to a new area and then spread within a population. Potential reservoirs of M. ulcerans thus might include humans, or perhaps M. ulcerans-infected animals such as livestock that move regularly between countries. Author Summary: In this study we use the power of whole genome sequence comparisons to track the spread of Mycobacterium ulcerans, the causative agent of Buruli ulcer, through several villages in the Ashanti region of Ghana, providing new insights on the behaviour of this enigmatic and emerging pathogen. [ABSTRACT FROM AUTHOR]

Background: Buruli ulcer (BU) is a necrotizing skin disease caused by Mycobacterium ulcerans. Previous studies have shown that wounds of BU patients are colonized with M. ulcerans and several other microorganisms, including Staphylococcus aureus, which may interfere with wound healing. The present study was therefore aimed at investigating the diversity and topography of S. aureus colonizing BU patients during treatment. Methodology: We investigated the presence, diversity, and spatio-temporal distribution of S. aureus in 30 confirmed BU patients from Ghana during treatment. S. aureus was isolated from nose and wound swabs, and by replica plating of wound dressings collected bi-weekly from patients. S. aureus isolates were characterized by multiple-locus variable number tandem repeat fingerprinting (MLVF) and spa-typing, and antibiotic susceptibility was tested. Principal Findings: Nineteen (63%) of the 30 BU patients tested positive for S. aureus at least once during the sampling period, yielding 407 S. aureus isolates. Detailed analysis of 91 isolates grouped these isolates into 13 MLVF clusters and 13 spa-types. Five (26%) S. aureus-positive BU patients carried the same S. aureus genotype in their anterior nares and wounds. S. aureus isolates from the wounds of seven (37%) patients were distributed over two different MLVF clusters. Wounds of three (16%) patients were colonized with isolates belonging to two different genotypes at the same time, and five (26%) patients were colonized with different S. aureus types over time. Five (17%) of the 30 included BU patients tested positive for methicillin-resistant S. aureus (MRSA). Conclusion/Significance: The present study showed that the wounds of many BU patients were contaminated with S. aureus, and that many BU patients from the different communities carried the same S. aureus genotype during treatment. This calls for improved wound care and hygiene. Author Summary: Buruli ulcer (BU) is a disease of the skin and soft tissue caused by Mycobacterium ulcerans. The resulting skin lesions provide a niche for survival of other microorganisms such as Staphylococcus aureus, which may cause delayed wound healing. This study investigated the presence, diversity, and spatio-temporal distribution of S. aureus in BU patients from Ghana during treatment by isolating the bacteria from nose and wound swabs or wound dressings. S. aureus isolates were subsequently characterized by two complementary DNA typing approaches. This showed that 19 (63%) of the 30 investigated BU patients carried S. aureus. Five (26%) of these 19 BU patients carried the same S. aureus type in their anterior nares and wounds. Seven (37%) patients carried the same S. aureus type in their wounds, which is indicative of transmission. Three of them (16%) carried at least two different S. aureus types at the same time in their wounds, while five (26%) carried different S. aureus types over time. Notably, five (17%) BU patients tested positive for methicillin-resistant S. aureus (MRSA). These findings imply that the spatio-temporal diversity of S. aureus in BU is most likely related to factors such as antibiotic pressure, and insufficient wound care and hygiene. [ABSTRACT FROM AUTHOR]

Background: The reservoir and mode of transmission of Mycobacterium ulcerans, the causative agent of Buruli ulcer, still remain a mystery. It has been suggested that M. ulcerans persists with difficulty as a free-living organism due to its natural fragility and inability to withstand exposure to direct sunlight, and thus probably persists within a protective host environment. Methodology/Principal Findings: We investigated the role of free-living amoebae as a reservoir of M. ulcerans by screening the bacterium in free-living amoebae (FLA) cultures isolated from environmental specimens using real-time PCR. We also followed the survival of M. ulcerans expressing green fluorescence protein (GFP) in Acanthameoba castellanii by flow cytometry and observed the infected cells using confocal and transmission electron microscopy for four weeks in vitro. IS2404 was detected by quantitative PCR in 4.64% of FLA cultures isolated from water, biofilms, detritus and aerosols. While we could not isolate M. ulcerans, 23 other species of mycobacteria were cultivated from inside FLA and/or other phagocytic microorganisms. Laboratory experiments with GFP-expressing M. ulcerans in A. castellani trophozoites for 28 days indicated the bacteria did not replicate inside amoebae, but they could remain viable at low levels in cysts. Transmission electron microscopy of infected A. castellani confirmed the presence of bacteria within both trophozoite vacuoles and cysts. There was no correlation of BU notification rate with detection of the IS2404 in FLA (r = 0.07, n = 539, p = 0.127). Conclusion/Significance: This study shows that FLA in the environment are positive for the M. ulcerans insertion sequence IS2404. However, the detection frequency and signal strength of IS2404 positive amoabae was low and no link with the occurrence of BU was observed. We conclude that FLA may host M. ulcerans at low levels in the environment without being directly involved in the transmission to humans. Author Summary: Mycobacterium ulcerans, the causative agent of Buruli ulcer (BU) is an environmental pathogen known to reside in aquatic habitat. However, the reservoir and modes of transmission to humans still remain unknown. M. ulcerans can probably not live freely due to its natural fragility and inability to withstand exposure to direct sunlight. This study investigated the hypothesis that free-living amoebae (FLA) can serve as a reservoir of M. ulcerans by testing for its presence in amoebae isolated from water bodies in BU endemic and non-endemic communities and whether the pathogen can remain viable when experimentally infected in amoebae in the laboratory. We detected only one (IS2404) of the three (IS2606 and KRB) targets for the presence of M. ulcerans in amoebae cultures and found no correlation between its presence in the environment and BU notification rate. M. ulcerans remained viable at low levels in amoebae for 28 days in vitro. We therefore conclude that FLA may host M. ulcerans at low levels in the environment without being directly involved in the transmission to humans. [ABSTRACT FROM AUTHOR]

Background: The reservoir and mode of transmission of Mycobacterium ulcerans, the causative agent of Buruli ulcer, remain unknown. Ecological, genetic and epidemiological information nonetheless suggests that M. ulcerans may reside in aquatic protozoa. Methodology/Principal Findings: We experimentally infected Acanthamoeba polyphaga with M. ulcerans and found that the bacilli were phagocytised, not digested and remained viable for the duration of the experiment. Furthermore, we collected 13 water, 90 biofilm and 45 detritus samples in both Buruli ulcer endemic and non-endemic communities in Ghana, from which we cultivated amoeboid protozoa and mycobacteria. M. ulcerans was not isolated, but other mycobacteria were as frequently isolated from intracellular as from extracellular sources, suggesting that they commonly infect amoebae in nature. We screened the samples as well as the amoeba cultures for the M. ulcerans markers IS2404, IS2606 and KR-B. IS2404 was detected in 2% of the environmental samples and in 4% of the amoeba cultures. The IS2404 positive amoeba cultures included up to 5 different protozoan species, and originated both from Buruli ulcer endemic and non-endemic communities. Conclusions/Significance: This is the first report of experimental infection of amoebae with M. ulcerans and of the detection of the marker IS2404 in amoeba cultures isolated from the environment. We conclude that amoeba are potential natural hosts for M. ulcerans, yet remain sceptical about their implication in the transmission of M. ulcerans to humans and their importance in the epidemiology of Buruli ulcer. Author Summary: Buruli ulcer (BU) is a devastating skin disease caused by Mycobacterium ulcerans, an environmental bacterium that is probably linked to slow-running water. It is unlikely to occur free-living, but even though M. ulcerans DNA has been detected in quite a few different organisms (with most studies focusing on insects), it is still not clear what its real reservoir is. Amoeboid protozoa, inhabitants of biofilms in slow flowing water, are good candidates since all previously tested mycobacteria are resistant to the digestion by these macrophage-like organisms. In this paper we demonstrate that M. ulcerans can indeed infect Acanthamoeba polyphaga in the lab, and remain viable intracellularly. We also collected water, biofilm and detritus samples in BU endemic and non-endemic regions in Ghana. We found that several mycobacteria species commonly occur intracellularly in protozoa in these environments. Amoebae were isolated from almost all samples, and an M. ulcerans marker (IS2404) was detected in 4% of the amoeba cultures. We conclude that amoebae are potential hosts for M. ulcerans. However, because these IS2404 positive amoebae originated from both BU endemic and non-endemic areas, we remain sceptical about their implication in the transmission of M. ulcerans to humans. [ABSTRACT FROM AUTHOR]

Background: Buruli ulcer (BU) caused by Mycobacterium ulcerans (M. ulcerans) has emerged as an important public health problem in several rural communities in sub-Saharan Africa. Early diagnosis and prompt treatment are important in preventing disfiguring complications associated with late stages of the disease progression. Presently there is no simple and rapid test that is appropriate for early diagnosis and use in the low-resource settings where M. ulcerans is most prevalent. Methodology: We compared conventional and pocket warmer loop mediated isothermal amplification (LAMP) methods (using a heat block and a pocket warmer respectively as heat source for amplification reaction) for the detection of M. ulcerans in clinical specimens. The effect of purified and crude DNA preparations on the detection rate of the LAMP assays were also investigated and compared with that of IS2404 PCR, a reference assay for the detection of M. ulcerans. Thirty clinical specimens from suspected BU cases were examined by LAMP and IS2404 PCR. Principal Findings: The lower detection limit of both LAMP methods at 60°C was 300 copies of IS2404 and 30 copies of IS2404 for the conventional LAMP at 65°C. When purified DNA extracts were used, both the conventional LAMP and IS2404 PCR concordantly detected 21 positive cases, while the pocket warmer LAMP detected 19 cases. Nine of 30 samples were positive by both the LAMP assays as well as IS2404 PCR when crude extracts of clinical specimens were used. Conclusion/Significance: The LAMP method can be used as a simple and rapid test for the detection of M. ulcerans in clinical specimens. However, obtaining purified DNA, as well as generating isothermal conditions, remains a major challenge for the use of the LAMP method under field conditions. With further improvement in DNA extraction and amplification conditions, the pwLAMP could be used as a point of care diagnostic test for BU Author Summary: In order to develop a simple and rapid test that can be used to diagnose Buruli ulcer under field conditions, we modified the conventional LAMP assay by using a disposable pocket warmer as a heating device for generating a constant temperature for the test reaction and employed the use of crude sample preparations consisting of boiled and unboiled extracts of the clinical specimen instead of using purified DNA as the diagnostic specimen. Thirty clinical specimens from suspected Buruli ulcer patients were investigated by the modified LAMP (or pocket warmer LAMP) and the conventional LAMP, as well as IS2404 PCR, a reference method for the detection of Mycobacterium ulcerans. There was no significant difference in the detection rate (63–70%) in all of the methods when purified samples were used for the tests. On the other hand the use of crude specimen preparation resulted in a drop in detection rate (30–40%). This study demonstrates that the LAMP test can be used for rapid detection of M. ulcerans when purified DNA preparations are used. With further improvements in the sample reaction, as well as in specimen purification, the pocket warmer LAMP may provide a simple and rapid diagnostic test for Buruli ulcer. [ABSTRACT FROM AUTHOR]

This document is a correction notice for an article on the distribution of Mycobacterium ulcerans genotypes in a region of Ghana affected by Buruli ulcer. It corrects an error in Table 1 of the article, specifically in the "ENA" column. The corrected table is provided. The document also includes a list of individuals and their corresponding data, collected as part of a study, from various locations in Ghana and Cote d'Ivoire. Additionally, it provides a table of information about different strains of bacteria collected from various locations in Africa, including Nigeria, Democratic Republic of the Congo, and Ghana. This information can be valuable for researchers studying bacterial strains and their geographical distribution in Africa. [Extracted from the article]