Publications by authors named "Françoise Portaels"

167 Publications

Mycobacterium ulcerans Population Genomics To Inform on the Spread of Buruli Ulcer across Central Africa.

mSphere 2019 02 6;4(1). Epub 2019 Feb 6.

Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium.

Buruli ulcer is a neglected tropical disease of skin and subcutaneous tissue caused by infection with the pathogen Many critical issues for disease control, such as understanding the mode of transmission and identifying source reservoirs of , are still largely unknown. Here, we used genomics to reconstruct in detail the evolutionary trajectory and dynamics of populations at a central African scale and at smaller geographical village scales. Whole-genome sequencing (WGS) data were analyzed from 179 strains isolated from all Buruli ulcer foci in the Democratic Republic of the Congo, The Republic of Congo, and Angola that have ever yielded positive cultures. We used both temporal associations and the study of the mycobacterial demographic history to estimate the contribution of humans as a reservoir in Buruli ulcer transmission. Our phylogeographic analysis revealed one almost exclusively predominant sublineage of that arose in Central Africa and proliferated in its different regions of endemicity during the Age of Discovery. We observed how the best sampled endemic hot spot, the Songololo territory, became an area of endemicity while the region was being colonized by Belgium (1880s). We furthermore identified temporal parallels between the observed past population fluxes of from the Songololo territory and the timing of health policy changes toward control of the Buruli ulcer epidemic in that region. These findings suggest that an intervention based on detecting and treating human cases in an area of endemicity might be sufficient to break disease transmission chains, irrespective of other reservoirs of the bacterium. Buruli ulcer is a destructive skin and soft tissue infection caused by The disease is characterized by progressive skin ulceration, which can lead to permanent disfigurement and long-term disability. Currently, the major hurdles facing disease control are incomplete understandings of both the mode of transmission and environmental reservoirs of As decades of spasmodic environmental sampling surveys have not brought us much closer to overcoming these hurdles, the Buruli ulcer research community has recently switched to using comparative genomics. The significance of our research is in how we used both temporal associations and the study of the mycobacterial demographic history to estimate the contribution of humans as a reservoir in Buruli ulcer transmission. Our approach shows that it might be possible to use bacterial population genomics to assess the impact of health interventions, providing valuable feedback for managers of disease control programs in areas where health surveillance infrastructure is poor.
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http://dx.doi.org/10.1128/mSphere.00472-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6365612PMC
February 2019

Improving clinical and epidemiological predictors of Buruli ulcer.

PLoS Negl Trop Dis 2018 08 6;12(8):e0006713. Epub 2018 Aug 6.

Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium.

Background: Buruli ulcer (BU) is a chronic necrotizing infectious skin disease caused by Mycobacterium ulcerans. The treatment with BU-specific antibiotics is initiated after clinical suspicion based on the WHO clinical and epidemiological criteria. This study aimed to estimate the predictive values of these criteria and how they could be improved.

Methodology/principal Findings: A total of 224 consecutive patients presenting with skin and soft tissue lesions that could be compatible with BU, including those recognized as unlikely BU by experienced clinicians, were recruited in two BU treatment centers in southern Benin between March 2012 and March 2015. For each participant, the WHO and four additional epidemiological and clinical diagnostic criteria were recorded. For microbiological confirmation, direct smear examination and IS2404 PCR were performed. We fitted a logistic regression model with PCR positivity for BU confirmation as outcome variable. On univariate analysis, most of the clinical and epidemiological WHO criteria were associated with a positive PCR result. However, lesions on the lower limbs and WHO category 3 lesions were rather associated with a negative PCR result (respectively OR: 0.4, 95%CI: 0.3-0.8; OR: 0.5, 95%IC: 0.3-0.9). Among the additional characteristics studied, the characteristic smell of BU was strongest associated with a positive PCR result (OR = 16.4; 95%CI = 7.5-35.6).

Conclusion/significance: The WHO diagnostic criteria could be improved upon by differentiating between lesions on the upper and lower limbs and by including lesion size and the characteristic smell recognized by experienced clinicians.
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http://dx.doi.org/10.1371/journal.pntd.0006713DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6095624PMC
August 2018

Subcutaneous Granulomatous Inflammation due to Basidiobolomycosis: Case Reports of 3 Patients in Buruli Ulcer Endemic Areas in Benin.

Case Rep Pathol 2018 10;2018:1351694. Epub 2018 Jan 10.

Institute of Tropical Medicine, Nationalestraat 155, 2000 Antwerpen, Belgium.

Background: Basidiobolomycosis is a rare subcutaneous mycosis, which can be mistaken for several other diseases, such as soft tissue tumors, lymphoma, or Buruli ulcer in the preulcerative stage. Microbiological confirmation by PCR for and culture yield the most specific diagnosis, yet they are not widely available in endemic areas and with varying sensitivity. A combination of histopathological findings, namely, granulomatous inflammation with giant cells, septate hyphal fragments, and the Splendore-Hoeppli phenomenon, can confirm basidiobolomycosis in patients presenting with painless, hard induration of soft tissue.

Case Presentations: We report on three patients misdiagnosed as suffering from Buruli ulcer, who did not respond to Buruli treatment. Histopathological review of the tissue sections from these patients suggests basidiobolomycosis. All patients had been lost to follow-up, and none received antifungal therapy. On visiting the patients at their homes, two were reported to have died of unknown causes. The third patient was found alive and well and had experienced local spontaneous healing.

Conclusion: Basidiobolomycosis is a rare subcutaneous fungal disease mimicking preulcerative Buruli ulcer. We stress the importance of the early recognition by clinicians and pathologists of this treatable disease, so patients can timely receive antifungal therapy.
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http://dx.doi.org/10.1155/2018/1351694DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5818906PMC
January 2018

Diagnostic Accuracy of Clinical and Microbiological Signs in Patients With Skin Lesions Resembling Buruli Ulcer in an Endemic Region.

Clin Infect Dis 2018 08;67(6):827-834

Mycobacteriology Unit, Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium.

Background: The diagnosis of the neglected tropical skin and soft tissue disease Buruli ulcer (BU) is made on clinical and epidemiological grounds, after which treatment with BU-specific antibiotics is initiated empirically. Given the current decline in BU incidence, clinical expertise in the recognition of BU is likely to wane and laboratory confirmation of BU becomes increasingly important. We therefore aimed to determine the diagnostic accuracy of clinical signs and microbiological tests in patients presenting with lesions clinically compatible with BU.

Methods: A total of 227 consecutive patients were recruited in southern Benin and evaluated by clinical diagnosis, direct smear examination (DSE), polymerase chain reaction (PCR), culture, and histopathology. In the absence of a gold standard, the final diagnosis in each patient was made using an expert panel approach. We estimated the accuracy of each test in comparison to the final diagnosis and evaluated the performance of 3 diagnostic algorithms.

Results: Among the 205 patients with complete data, the attending clinicians recognized BU with a sensitivity of 92% (95% confidence interval [CI], 85%-96%), which was higher than the sensitivity of any of the laboratory tests. However, 14% (95% CI, 7%-24%) of patients not suspected to have BU at diagnosis were classified as BU by the expert panel. The specificities of all diagnostics were high (≥91%). All diagnostic algorithms had similar performances.

Conclusions: A broader clinical suspicion should be recommended to reduce missed BU diagnoses. Taking into consideration diagnostic accuracy, time to results, cost-effectiveness, and clinical generalizability, a stepwise diagnostic approach reserving PCR to DSE-negative patients performed best.
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http://dx.doi.org/10.1093/cid/ciy197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6117443PMC
August 2018

Bacterial diversity in Buruli ulcer skin lesions: Challenges in the clinical microbiome analysis of a skin disease.

PLoS One 2017 27;12(7):e0181994. Epub 2017 Jul 27.

Mycobacteriology unit, Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium.

Background: Buruli ulcer (BU) is an infectious disease caused by Mycobacterium ulcerans and considered the third most prevalent mycobacterial disease in humans. Secondary bacterial infections in open BU lesions are the main cause of pain, delayed healing and systemic illness, resulting in prolonged hospital stay. Thus, understanding the diversity of bacteria, termed the microbiome, in these open lesions is important for proper treatment. However, adequately studying the human microbiome in a clinical setting can prove difficult when investigating a neglected tropical skin disease due to its rarity and the setting.

Methodology/principal Findings: Using 16S rRNA sequencing, we determined the microbial composition of 5 BU lesions, 3 non-BU lesions and 3 healthy skin samples. Although no significant differences in diversity were found between BU and non-BU lesions, the former were characterized by an increase of Bacteroidetes compared to the non-BU wounds and the BU lesions also contained significantly more obligate anaerobes. With this molecular-based study, we were also able to detect bacteria that were missed by culture-based methods in previous BU studies.

Conclusions/significance: Our study suggests that BU may lead to changes in the skin bacterial community within the lesions. However, in order to determine if such changes hold true across all BU cases and are either a cause or consequence of a specific wound environment, further microbiome studies are necessary. Such skin microbiome analysis requires large sample sizes and lesions from the same body site in many patients, both of which can be difficult for a rare disease. Our study proposes a pipeline for such studies and highlights several drawbacks that must be considered if microbiome analysis is to be utilized for neglected tropical diseases.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0181994PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5531519PMC
September 2017

Characterization of -Like Strains by Comparative Genomics.

Front Microbiol 2017 8;8:789. Epub 2017 May 8.

Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São PauloSão Paulo, Brazil.

Isolates of the - complex are subdivided into four clusters (CHI to CHIV) in the INNO-LiPA® spp DNA strip assay. A considerable phenotypic variability was observed among isolates of the CHII cluster. In this study, we examined the diversity of 26 CHII cluster isolates by phenotypic analysis, drug susceptibility testing, whole genome sequencing and single-gene analysis. Pairwise genome comparisons were performed using several approaches, including average nucleotide identity (ANI) and genome-to-genome distance (GGD) among others. Based on ANI and GGD the isolates were identified as (14 isolates), (2 isolates) and (1 isolate). The remaining 9 isolates were subdivided into three novel putative genomospecies. Phenotypic analyses including drug susceptibility testing, as well as whole genome comparison by TETRA and delta differences, were not helpful in separating the groups revealed by ANI and GGD. The analysis of standard four conserved genomic regions showed that alone and the concatenated sequences clearly distinguished the taxonomic groups delimited by whole genome analyses. In conclusion, the CHII INNO-LiPa is not a homogeneous cluster; on the contrary, it is composed of closely related different species belonging to the complex and also several unidentified isolates. The detection of these isolates, putatively novel species, indicates a wider inner variability than the presently known in this complex.
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http://dx.doi.org/10.3389/fmicb.2017.00789DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5420552PMC
May 2017

Multiple Introductions and Recent Spread of the Emerging Human Pathogen Mycobacterium ulcerans across Africa.

Genome Biol Evol 2017 03;9(3):414-426

Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium.

Buruli ulcer (BU) is an insidious neglected tropical disease. Cases are reported around the world but the rural regions of West and Central Africa are most affected. How BU is transmitted and spreads has remained a mystery, even though the causative agent, Mycobacterium ulcerans, has been known for more than 70 years. Here, using the tools of population genomics, we reconstruct the evolutionary history of M. ulcerans by comparing 165 isolates spanning 48 years and representing 11 endemic countries across Africa. The genetic diversity of African M. ulcerans was found to be restricted due to the bacterium's slow substitution rate coupled with its relatively recent origin. We identified two specific M. ulcerans lineages within the African continent, and inferred that M. ulcerans lineage Mu_A1 existed in Africa for several hundreds of years, unlike lineage Mu_A2, which was introduced much more recently, approximately during the 19th century. Additionally, we observed that specific M. ulcerans epidemic Mu_A1 clones were introduced during the same time period in the three hydrological basins that were well covered in our panel. The estimated time span of the introduction events coincides with the Neo-imperialism period, during which time the European colonial powers divided the African continent among themselves. Using this temporal association, and in the absence of a known BU reservoir or-vector on the continent, we postulate that the so-called "Scramble for Africa" played a significant role in the spread of the disease across the continent.
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http://dx.doi.org/10.1093/gbe/evx003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5381664PMC
March 2017

Twenty Years of Global Surveillance of Antituberculosis-Drug Resistance.

N Engl J Med 2016 Sep;375(11):1081-9

From the World Health Organization, Geneva (M.Z., A.S.D., D.F., W.G., A.W., K.W., E.J., P.N., K.F., M.C.R.); Institute of Tropical Medicine, Antwerp, Belgium (A.D., F.P.); Laboratory Centre for Disease Control, Ottawa (A.L.); Pan American Health Organization-World Health Organization (M.A.E.) and the U.S. Agency for International Development (A.P.-M., A.B.), Washington, DC; and the World Health Organization, Cairo (M.A.A.).

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http://dx.doi.org/10.1056/NEJMsr1512438DOI Listing
September 2016

A Genomic Approach to Resolving Relapse versus Reinfection among Four Cases of Buruli Ulcer.

PLoS Negl Trop Dis 2015 Nov 30;9(11):e0004158. Epub 2015 Nov 30.

Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium.

Background: Increased availability of Next Generation Sequencing (NGS) techniques allows, for the first time, to distinguish relapses from reinfections in patients with multiple Buruli ulcer (BU) episodes.

Methodology: We compared the number and location of single nucleotide polymorphisms (SNPs) identified by genomic screening between four pairs of Mycobacterium ulcerans isolates collected at the time of first diagnosis and at recurrence, derived from a collection of almost 5000 well characterized clinical samples from one BU treatment center in Benin.

Principal Findings: The findings suggest that after surgical treatment-without antibiotics-the second episodes were due to relapse rather than reinfection. Since specific antibiotics were introduced for the treatment of BU, the one patient with a culture available from both disease episodes had M. ulcerans isolates with a genomic distance of 20 SNPs, suggesting the patient was most likely reinfected rather than having a relapse.

Conclusions: To our knowledge, this study is the first to study recurrences in M. ulcerans using NGS, and to identify exogenous reinfection as causing a recurrence of BU. The occurrence of reinfection highlights the contribution of ongoing exposure to M. ulcerans to disease recurrence, and has implications for vaccine development.
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http://dx.doi.org/10.1371/journal.pntd.0004158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4664471PMC
November 2015

Beninese Medicinal Plants as a Source of Antimycobacterial Agents: Bioguided Fractionation and In Vitro Activity of Alkaloids Isolated from Holarrhena floribunda Used in Traditional Treatment of Buruli Ulcer.

Biomed Res Int 2015 28;2015:835767. Epub 2015 May 28.

Pharmacognosy Research Group, Louvain Drug Research Institute (LDRI), Université Catholique de Louvain (UCL), B1 7203 Avenue E. Mounier 72, 1200 Bruxelles, Belgium.

Buruli ulcer (BU) imposes a serious economic burden on affected households and on health systems that are involved in diagnosing the disease and treating patients. Research is needed to find cost-effective therapies for this costly disease. Plants have always been an important source of new pharmacologically active molecules. Consequently we decided to undertake the study of plants used in traditional treatment of BU in Benin and investigate their antimycobacterial activity as well as their chemical composition. Extracts from forty-four (44) plant species were selected on account of reported traditional uses for the treatment of BU in Benin and were assayed for antimycobacterial activities. Crude hydroethanolic extract from aerial parts of Holarrhena floribunda (G. Don) T. Durand and Schinz was found to have significant antimycobacterial activity against M. ulcerans (MIC = 125 µg/mL). We describe here the identification of four steroidal alkaloids from Mycobacterium ulcerans growth-inhibiting fractions of the alkaloidal extract of the aerial parts of Holarrhena floribunda. Holadysamine was purified in sufficient amount to allow the determination of its MCI (=50 µg/mL). These results give some support to the use of this plant in traditional medicine.
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http://dx.doi.org/10.1155/2015/835767DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4477427PMC
April 2016

Whole genome comparisons suggest random distribution of Mycobacterium ulcerans genotypes in a Buruli ulcer endemic region of Ghana.

PLoS Negl Trop Dis 2015 Mar 31;9(3):e0003681. Epub 2015 Mar 31.

Department of Microbiology and Immunology, University of Melbourne, Parkville, Australia.

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 30 km2 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 5.2 Mbp 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.
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http://dx.doi.org/10.1371/journal.pntd.0003681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4380315PMC
March 2015

Buruli ulcer in traveler from Suriname, South America, to the Netherlands.

Emerg Infect Dis 2015 Mar;21(3):497-9

We report Buruli ulcer in a man in the Netherlands. Phenotyping of samples indicate the Buruli pathogen was acquired in Suriname and activated by trauma on return to the Netherlands. Awareness of this disease by clinicians in non-Buruli ulcer-endemic areas is critical for identification.
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http://dx.doi.org/10.3201/eid2103.141237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4344276PMC
March 2015

Investigating the role of free-living amoebae as a reservoir for Mycobacterium ulcerans.

PLoS Negl Trop Dis 2014 Sep 4;8(9):e3148. Epub 2014 Sep 4.

Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium.

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.
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http://dx.doi.org/10.1371/journal.pntd.0003148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4154674PMC
September 2014

Multicenter external quality assessment program for PCR detection of Mycobacterium ulcerans in clinical and environmental specimens.

PLoS One 2014 21;9(2):e89407. Epub 2014 Feb 21.

Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium.

Background: Mycobacterium ulcerans is the causative agent of Buruli ulcer (BU), a necrotizing disease of the skin, soft tissue and bone. PCR is increasingly used in the diagnosis of BU and in research on the mode of transmission and environmental reservoir of M. ulcerans.

Methodology/principal Findings: The aim of this study was to evaluate the performance of laboratories in detecting M. ulcerans using molecular tests in clinical and environmental samples by implementing sequential multicenter external quality assessment (EQA) programs. The second round of the clinical EQA program revealed somewhat improved performance.

Conclusions/significance: Ongoing EQA programs remain essential and continued participation in future EQA programs by laboratories involved in the molecular testing of clinical and environmental samples for M. ulcerans for diagnostic and research purposes is strongly encouraged. Broad participation in such EQA programs also benefits the harmonization of quality in the BU research community and enhances the credibility of advances made in solving the transmission enigma of M. ulcerans.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0089407PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3931755PMC
January 2015

Burden of Mycobacterium ulcerans disease (Buruli ulcer) and the underreporting ratio in the territory of Songololo, Democratic Republic of Congo.

PLoS Negl Trop Dis 2013 5;7(12):e2563. Epub 2013 Dec 5.

General Reference Hospital of Kimpese, Institut Médical Evangélique, Kimpese, Bas-Congo, Democratic Republic of Congo ; Department of Public Health, Unit of Epidemiology and Disease Control, Institute of Tropical Medicine, Antwerp, Belgium.

Background: Cutaneous infection by Mycobacterium ulcerans, also known as Buruli ulcer (BU), represents the third most common mycobacterial disease in the world after tuberculosis and leprosy. Data on the burden of BU disease in the Democratic Republic of Congo are scanty. This study aimed to estimate the prevalence rate and the distribution of BU in the Songololo Territory, and to assess the coverage of the existing hospital-based reporting system.

Methods: We conducted a cross-sectional survey (July-August 2008) using the door-to-door method simultaneously in the two rural health zones (RHZ) of the Songololo Territory (RHZ of Kimpese and Nsona-Mpangu), each containing twenty health areas. Cases were defined clinically as active BU and inactive BU in accordance with WHO-case definitions.

Results: We detected 775 BU patients (259 active and 516 inactive) in a total population of 237,418 inhabitants. The overall prevalence of BU in Songololo Territory was 3.3/1000 inhabitants, varying from 0 to 27.5/1000 between health areas. Of the 259 patients with active BU, 18 (7%) had been reported in the hospital-based reporting system at Kimpese in the 6-8 months prior to the survey.

Conclusion: The survey demonstrated a huge variation of prevalence between health areas in Songololo Territory and gross underreporting of BU cases in the hospital-based reporting system. Data obtained may contribute to better targeted and improved BU control interventions, and serve as a baseline for future assessments of the control program.
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http://dx.doi.org/10.1371/journal.pntd.0002563DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3855042PMC
July 2014

Insertion sequence element single nucleotide polymorphism typing provides insights into the population structure and evolution of Mycobacterium ulcerans across Africa.

Appl Environ Microbiol 2014 Feb 2;80(3):1197-209. Epub 2013 Dec 2.

Mycobacteriology Unit, Microbiology Group, Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium.

Buruli ulcer is an indolent, slowly progressing necrotizing disease of the skin caused by infection with Mycobacterium ulcerans. In the present study, we applied a redesigned technique to a vast panel of M. ulcerans disease isolates and clinical samples originating from multiple African disease foci in order to (i) gain fundamental insights into the population structure and evolutionary history of the pathogen and (ii) disentangle the phylogeographic relationships within the genetically conserved cluster of African M. ulcerans. Our analyses identified 23 different African insertion sequence element single nucleotide polymorphism (ISE-SNP) types that dominate in different areas where Buruli ulcer is endemic. These ISE-SNP types appear to be the initial stages of clonal diversification from a common, possibly ancestral ISE-SNP type. ISE-SNP types were found unevenly distributed over the greater West African hydrological drainage basins. Our findings suggest that geographical barriers bordering the basins to some extent prevented bacterial gene flow between basins and that this resulted in independent focal transmission clusters associated with the hydrological drainage areas. Different phylogenetic methods yielded two well-supported sister clades within the African ISE-SNP types. The ISE-SNP types from the "pan-African clade" were found to be widespread throughout Africa, while the ISE-SNP types of the "Gabonese/Cameroonian clade" were much rarer and found in a more restricted area, which suggested that the latter clade evolved more recently. Additionally, the Gabonese/Cameroonian clade was found to form a strongly supported monophyletic group with Papua New Guinean ISE-SNP type 8, which is unrelated to other Southeast Asian ISE-SNP types.
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http://dx.doi.org/10.1128/AEM.02774-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3911215PMC
February 2014

Genetic diversity and population structure of Mycobacterium marinum: new insights into host and environmental specificities.

J Clin Microbiol 2012 Nov 5;50(11):3627-34. Epub 2012 Sep 5.

INSERM U1058 Infection by HIV and by agents with mucocutaneous tropism: from pathogenesis to prevention, Montpellier, France.

Mycobacterium marinum causes a systemic tuberculosis-like disease in fish and skin infections in humans that can spread to deeper structures, resulting in tenosynovitis, arthritis, and osteomyelitis. However, little information is available concerning (i) the intraspecific genetic diversity of M. marinum isolated from humans and animals; (ii) M. marinum genotype circulation in the different ecosystems, and (iii) the link between M. marinum genetic diversity and hosts (humans and fish). Here, we conducted a genetic study on 89 M. marinum isolates from humans (n = 68) and fish (n = 21) by using mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) typing. The results show that the M. marinum population is genetically structured not only according to the host but also according to the ecosystem as well as to tissue tropism in humans. This suggests the existence of different genetic pools in the function of the biological and ecological compartments. Moreover, the presence of only certain M. marinum genotypes in humans suggests a different zoonotic potential of the M. marinum genotypes. Considering that the infection is linked to aquarium activity, a significant genetic difference was also detected when the human tissue tropism of M. marinum was taken into consideration, with a higher genetic polymorphism in strains isolated from patients with cutaneous forms than from individuals with deeper-structure infection. It appears that only few genotypes can produce deeper infections in humans, suggesting that the immune system might play a filtering role.
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http://dx.doi.org/10.1128/JCM.01274-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3486196PMC
November 2012

Amoebae as potential environmental hosts for Mycobacterium ulcerans and other mycobacteria, but doubtful actors in Buruli ulcer epidemiology.

PLoS Negl Trop Dis 2012 7;6(8):e1764. Epub 2012 Aug 7.

Evolutionary Ecology Group, University of Antwerp, Antwerp, Belgium.

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.
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http://dx.doi.org/10.1371/journal.pntd.0001764DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3413716PMC
January 2013

On the origin of Mycobacterium ulcerans, the causative agent of Buruli ulcer.

BMC Genomics 2012 Jun 19;13:258. Epub 2012 Jun 19.

Department of Microbiology and Immunology, University of Melbourne, Parkville, Australia.

Background: Mycobacterium ulcerans is an unusual bacterial pathogen with elusive origins. While closely related to the aquatic dwelling M. marinum, M. ulcerans has evolved the ability to produce the immunosuppressive polyketide toxin mycolactone and cause the neglected tropical disease Buruli ulcer. Other mycolactone-producing mycobacteria (MPM) have been identified in fish and frogs and given distinct species designations (M. pseudoshottsii, M. shinshuense, M. liflandii and M. marinum), however the evolution of M. ulcerans and its relationship to other MPM has not been defined. Here we report the comparative analysis of whole genome sequences from 30 MPM and five M. marinum.

Results: A high-resolution phylogeny based on genome-wide single nucleotide polymorphisms (SNPs) showed that M. ulcerans and all other MPM represent a single clonal group that evolved from a common M. marinum progenitor. The emergence of the MPM was driven by the acquisition of the pMUM plasmid encoding genes for the biosynthesis of mycolactones. This change was accompanied by the loss of at least 185 genes, with a significant overrepresentation of genes associated with cell wall functions. Cell wall associated genes also showed evidence of substantial adaptive selection, suggesting cell wall remodeling has been critical for the survival of MPM. Fine-grain analysis of the MPM complex revealed at least three distinct lineages, one of which comprised a highly clonal group, responsible for Buruli ulcer in Africa and Australia. This indicates relatively recent transfer of M. ulcerans between these continents, which represent the vast majority of the global Buruli ulcer burden. Our data provide SNPs and gene sequences that can differentiate M. ulcerans lineages, suitable for use in the diagnosis and surveillance of Buruli ulcer.

Conclusions: M. ulcerans and all mycolactone-producing mycobacteria are specialized variants of a common Mycobacterium marinum progenitor that have adapted to live in restricted environments. Examination of genes lost or retained and now under selective pressure suggests these environments might be aerobic, and extracellular, where slow growth, production of an immune suppressor, cell wall remodeling, loss or modification of cell wall antigens, and biofilm-forming ability provide a survival advantage. These insights will guide our efforts to find the elusive reservoir(s) of M. ulcerans and to understand transmission of Buruli ulcer.
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http://dx.doi.org/10.1186/1471-2164-13-258DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3434033PMC
June 2012

A pivotal registration phase III, multicenter, randomized tuberculosis controlled trial: design issues and lessons learnt from the Gatifloxacin for TB (OFLOTUB) project.

Trials 2012 May 18;13:61. Epub 2012 May 18.

Faculty of Epidemiology and Population Health, Tropical Epidemiological Group, London School of Hygiene and Tropical Medicine, Keppel Street, London, WC1E 7HT, UK.

Background: There have been no major advances in tuberculosis (TB) drug development since the first East African/British Medical Research Council short course chemotherapy trial 35 years ago. Since then, the landscape for conducting TB clinical trials has profoundly changed with the emergence of HIV infection, the spread of resistant TB bacilli strains, recent advances in mycobacteriological capacity, and drug discovery. As a consequence questions have arisen on the most appropriate approach to design and conduct current TB trials. To highlight key issues discussed: Is a superiority, equivalence, or non-inferiority design most appropriate? What should be the primary efficacy outcome? How to consider re-infections in the definition of the outcome? What is the optimal length of patient follow-up? Is blinding appropriate when treatment duration in test arm is shorter? What are the appropriate assumptions for sample size calculation?

Methods: Various drugs are currently in the development pipeline. We are presenting in this paper the design of the most recently completed phase III TB trial, the OFLOTUB project, which is the pivotal trial of a registration portfolio for a gatifloxacin-containing TB regimen. It is a randomized, open-label, multicenter, controlled trial aiming to evaluate the efficacy and safety of a gatifloxacin-containing 4-month regimen (trial registration: ClinicalTrial.gov database: NCT00216385).

Results: In the light of the recent scientific and regulatory discussions, we discuss some of the design issues in TB clinical trials and more specifically the reasons that guided our choices, in order to best answer the trial objectives, while at the same time satisfying regulatory authority requirements.

Conclusion: When shortening TB treatment, we are advocating for a non-inferiority, non-blinded design, with a composite unfavorable endpoint assessed 12 months post treatment completion, and added trial procedures specifically aiming to: (1) minimize endpoint unavailability; and (2) distinguish between relapse and re-infection.
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http://dx.doi.org/10.1186/1745-6215-13-61DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3528451PMC
May 2012

Cellular immunity confers transient protection in experimental Buruli ulcer following BCG or mycolactone-negative Mycobacterium ulcerans vaccination.

PLoS One 2012 8;7(3):e33406. Epub 2012 Mar 8.

Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal.

Background: Buruli ulcer (BU) is an emerging infectious disease caused by Mycobacterium ulcerans that can result in extensive necrotizing cutaneous lesions due to the cytotoxic exotoxin mycolactone. There is no specific vaccine against BU but reports show some degree of cross-reactive protection conferred by M. bovis BCG immunization. Alternatively, an M. ulcerans-specific immunization could be a better preventive strategy.

Methodology/principal Findings: In this study, we used the mouse model to characterize the histological and cytokine profiles triggered by vaccination with either BCG or mycolactone-negative M. ulcerans, followed by footpad infection with virulent M. ulcerans. We observed that BCG vaccination significantly delayed the onset of M. ulcerans growth and footpad swelling through the induction of an earlier and sustained IFN-γ T cell response in the draining lymph node (DLN). BCG vaccination also resulted in cell-mediated immunity (CMI) in M. ulcerans-infected footpads, given the predominance of a chronic mononuclear infiltrate positive for iNOS, as well as increased and sustained levels of IFN-γ and TNF. No significant IL-4, IL-17 or IL-10 responses were detected in the footpad or the DLN, in either infected or vaccinated mice. Despite this protective Th1 response, BCG vaccination did not avoid the later progression of M. ulcerans infection, regardless of challenge dose. Immunization with mycolactone-deficient M. ulcerans also significantly delayed the progression of footpad infection, swelling and ulceration, but ultimately M. ulcerans pathogenic mechanisms prevailed.

Conclusions/significance: The delay in the emergence of pathology observed in vaccinated mice emphasizes the relevance of protective Th1 recall responses against M. ulcerans. In future studies it will be important to determine how the transient CMI induced by vaccination is compromised.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0033406PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3297633PMC
August 2012

Effects of decontamination, DNA extraction, and amplification procedures on the molecular diagnosis of Mycobacterium ulcerans disease (Buruli ulcer).

J Clin Microbiol 2012 Apr 18;50(4):1195-8. Epub 2012 Jan 18.

Laboratoire de Référence des Mycobactéries, Cotonou, Benin.

We compared two DNA extraction methods (a semiautomated method using a Maxwell kit and a modified Boom method) and three amplification procedures (a single-step PCR, a nested PCR, and a real-time quantitative PCR) on 74 surgical tissue specimens from patients with clinically suspected Buruli ulcer. All of these procedures were compared before and after decontamination. We observed that, among the procedures tested, real-time PCR after the modified Boom extraction method or a single-run PCR assay after the Maxwell 16 extraction method, performed on nondecontaminated suspensions, are the best for the molecular diagnosis of Mycobacterium ulcerans disease.
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http://dx.doi.org/10.1128/JCM.05592-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3318535PMC
April 2012

hsp65 PCR-restriction analysis (PRA) with capillary electrophoresis for species identification and differentiation of Mycobacterium kansasii and Mycobacterium chelonae-Mycobacterium abscessus group.

Int J Infect Dis 2012 Mar 11;16(3):e193-7. Epub 2012 Jan 11.

Mycobacteriology Unit, Department of Microbiology, Institute of Tropical Medicine, Nationalestraat 155, B-2000 Antwerp, Belgium.

Objectives: The aim of the present study was to identify and differentiate Mycobacterium kansasii and Mycobacterium chelonae-Mycobacterium abscessus group strains isolated from clinical and environmental sources in different countries.

Methods: PCR-restriction analysis of the hsp65 gene (PRA) with automated capillary electrophoresis was applied to the isolates previously identified by conventional biochemical testing and the molecular INNO-LiPA MYCOBACTERIA assay.

Results: PRA performed very well in comparison with the two other methods (96.4% concordance). Among 27 M. kansasii isolates, this method detected five genetic types, of which type 1 represented the most common clinical isolate, as it is worldwide. PRA differentiated 29 M. chelonae-M. abscessus group isolates into Mycobacterium immunogenum type 2 (n=13), M. chelonae (n=12), and M. abscessus types 1 (n=1) and 2 (n=1). M. immunogenum was the most frequent (69%) isolate from humans, but only one of 11 cases was clinically significant. M. chelonae was the most commonly (83%) recovered from water. PRA also identified two isolates with hsp65 alleles representing previously unreported patterns.

Conclusions: PRA based on automated capillary electrophoresis is a rapid, simple, and reliable method for the identification and differentiation of both clinically relevant and environmental isolates of M. kansasii and M. chelonae-M. abscessus group.
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http://dx.doi.org/10.1016/j.ijid.2011.11.011DOI Listing
March 2012

Effect of a control project on clinical profiles and outcomes in buruli ulcer: a before/after study in Bas-Congo, Democratic Republic of Congo.

PLoS Negl Trop Dis 2011 Dec 27;5(12):e1402. Epub 2011 Dec 27.

General Reference Hospital of Kimpese, Institut Médical Evangélique, Kimpese, Bas-Congo, Democratic Republic of Congo.

Background: Buruli ulcer (BU) is a necrotizing bacterial infection of skin, subcutaneous tissue and bone caused by Mycobacterium ulcerans. Although the functional impairment caused by BU results in severe suffering and in socio-economic problems, the disease remains largely neglected in Africa. The province of Bas-Congo in Democratic Republic of Congo contains one of the most important BU foci of the country, i.e. the Songololo Territory in the District of Cataractes. This study aims to assess the impact of a BU control project launched in 2004 in the Songololo Territory.

Methods: We used a comparative non-randomized study design, comparing clinical profiles and outcomes of the group of patients admitted at the General Reference Hospital (GRH) of the "Institut Médical Evangélique" (IME) of Kimpese 3 years before the start of the project (2002-2004) with those admitted during the 3 years after the start of the project (2005-2007).

Results: The BU control project was associated with a strong increase in the number of admitted BU cases at the GRH of IME/Kimpese and a fundamental change in the profile of those patients; more female patients presented with BU, the proportion of relapse cases amongst all admissions reduced, the proportion of early lesions and simple ulcerative forms increased, more patients healed without complications and the case fatality rate decreased substantially. The median duration since the onset of first symptoms however remained high, as well as the proportion of patients with osteomyelitis or limitations of joint movement, suggesting that the diagnostic delay remains substantial.

Conclusion: Implementing a specialized program for BU may be effective in improving clinical profiles and outcomes in BU. Despite these encouraging results, our study highlights the need of considering new strategies to better improve BU control in a low resources setting.
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http://dx.doi.org/10.1371/journal.pntd.0001402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3246436PMC
December 2011

Mycobacterium ulcerans infection (Buruli ulcer) on the face: a comparative analysis of 13 clinically suspected cases from the Democratic Republic of Congo.

Am J Trop Med Hyg 2011 Dec;85(6):1100-5

Institut Médical Evangélique, Kimpese Hospital, Kimpese, Bas-Congo, Democratic Republic of Congo.

We report our experience in managing 13 consecutive clinically suspected cases of Buruli ulcer on the face treated at the hospital of the Institut Médical Evangélique at Kimpese, Democratic Republic of Congo diagnosed during 2003-2007. During specific antibiotherapy, facial edema diminished, thus minimizing the subsequent extent of surgery and severe disfigurations. The following complications were observed: 1) lagophthalmos from scarring in four patients and associated ectropion in three of them; 2) blindness in one eye in one patient; 3) disfiguring exposure of teeth and gums resulting from excision of the left labial commissure that affected speech, drinking, and eating in one patient; and 4) dissemination of Mycobacterium ulcerans infection in three patients. Our study highlights the importance of this clinical presentation of Buruli ulcer, and the need for health workers in disease-endemic areas to be aware of the special challenges management of Buruli ulcer on the face presents.
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http://dx.doi.org/10.4269/ajtmh.2011.10-0530DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3225160PMC
December 2011

Situation of bovine tuberculosis in Ecuador.

Rev Panam Salud Publica 2011 Sep;30(3):279-86

International Center for Zoonoses, Central University of Ecuador, Quito.

Bovine tuberculosis (BTB) is a chronic and contagious disease that affects domestic animals, wildlife, and humans. Caused by Mycobacterium bovis, BTB causes major economic losses and poses a serious constraint to international livestock trade. Moreover, in developing countries where BTB controls are lacking, M. bovis is a public health concern. In most developing countries, the prevalence of BTB in livestock is unknown because the information is either not reported or not available. In Ecuador, there is no national BTB control program. This article reviews the BTB situation in Ecuador by examining exhaustive data from tuberculin testing surveys and slaughterhouse surveillance studies conducted in 1972-2008 in a variety of the country's geographic areas. In Ecuador, several factors, including the dairy industry's expansion (preempted by the high demand for milk and its by-products), intensified efforts to increase the cattle population, the presence of M. bovis, and a lack of BTB controls, have caused a rise in BTB prevalence, and consequently, a growing push for the implementation of a national BTB control program.
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http://dx.doi.org/10.1590/s1020-49892011000900013DOI Listing
September 2011

Mycobacteria in terrestrial small mammals on cattle farms in Tanzania.

Vet Med Int 2011 8;2011:495074. Epub 2011 Jun 8.

Evolutionary Ecology Group, Department of Biology, University of Antwerp, 2020 Antwerp, Belgium.

The control of bovine tuberculosis and atypical mycobacterioses in cattle in developing countries is important but difficult because of the existence of wildlife reservoirs. In cattle farms in Tanzania, mycobacteria were detected in 7.3% of 645 small mammals and in cow's milk. The cattle farms were divided into "reacting" and "nonreacting" farms, based on tuberculin tests, and more mycobacteria were present in insectivores collected in reacting farms as compared to nonreacting farms. More mycobacteria were also present in insectivores as compared to rodents. All mycobacteria detected by culture and PCR in the small mammals were atypical mycobacteria. Analysis of the presence of mycobacteria in relation to the reactor status of the cattle farms does not exclude transmission between small mammals and cattle but indicates that transmission to cattle from another source of infection is more likely. However, because of the high prevalence of mycobacteria in some small mammal species, these infected animals can pose a risk to humans, especially in areas with a high HIV-prevalence as is the case in Tanzania.
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http://dx.doi.org/10.4061/2011/495074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3139188PMC
November 2011

Post-mortem examination and laboratory-based analysis for the diagnosis of bovine tuberculosis among dairy cattle in Ecuador.

Prev Vet Med 2011 Aug 8;101(1-2):65-72. Epub 2011 Jun 8.

International Centre for Zoonoses, Central University of Ecuador, Quito, Ecuador.

Veterinary inspection in slaughterhouses allows for the detection of macroscopic lesions reminiscent of bovine tuberculosis, but the presence of Mycobacterium bovis must be confirmed by laboratory methods. This study aimed at comparing the performances of the standard diagnostic tools used to identify M. bovis in tissue specimens sampled from suspicious animals. During a two years period, 1390 cattle were inspected at the Machachi abattoir in the Mejia canton - Ecuador. A total of 33 animals with granulomatous lesions were detected, representing 2.33% (16/687) and 2.42% (17/703) animals examined in 2007 and 2008, respectively. Ninety-four tissue specimens were sampled and screened for the presence of mycobacteria. Acid-fast bacilli were identified in one third of the suspicious cattle (11/33) and suggestive microscopic lesions in 27.3% (9/33) of the samples examined by direct microscopy and histopathology, respectively. Culturing on Stonebrink medium and 16S-rRNA-based polymerase chain reaction (PCR) yielded 36.4% (12/33) and 27.3% (9/33) of positives, respectively. Compared to culture, other diagnostic procedures displayed a lower sensitivity, with 56.5% for PCR, and 43.5% for direct microscopy and histopathology; however, the specificity was higher (94.4% for PCR and microscopy, and 97.2% for histopathology). We conclude that reliable post-mortem laboratory testing either requires the combination of a set of available diagnostic tools or necessitates the development of improved new-generation tools with better sensitivity and specificity characteristics.
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http://dx.doi.org/10.1016/j.prevetmed.2011.04.018DOI Listing
August 2011
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