Development of a PCR assay for rapid diagnosis of Mycobacterium ulcerans infection.
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ABSTRACT:
SUBMITTER: Ross BC
PROVIDER: S-EPMC229824 | biostudies-other | 1997 Jul
REPOSITORIES: biostudies-other
Publications
Development of a PCR assay for rapid diagnosis of Mycobacterium ulcerans infection.
Ross B C BC Marino L L Oppedisano F F Edwards R R Robins-Browne R M RM Johnson P D PD
Journal of clinical microbiology 19970701 7
The diagnosis of Mycobacterium ulcerans infection is hampered by the slow growth of the bacterium in culture, resulting in a delay of several months before a specific diagnosis can be obtained. In addition, M. ulcerans cannot be isolated from water even when there is convincing epidemiological evidence implicating this as the source of infection. The aim of the present study was to develop a PCR assay to circumvent the problems of delayed diagnosis and insensitivity of standard bacterial culture ...[more]
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Project description:Buruli ulcer, an infection caused by Mycobacterium ulcerans, is, after tuberculosis and leprosy, the third most common mycobacterial disease. The mode of transmission of M. ulcerans is not exactly known, but since Buruli ulcer often occurs in focalized swampy areas, it is assumed that there is a reservoir of the pathogen in stagnant water. Buruli ulcer usually starts as a painless nodule and can lead to massive destruction of skin, subcutaneous tissue, and eventually muscle and bone. Currently the only recommended treatment is wide surgical excision. In this report we describe the development of a real-time PCR method for the quantification of M. ulcerans DNA (IS2404 TaqMan). The highly specific assay is based on the detection of the M. ulcerans specific insertion sequence IS2404. The IS2404 TaqMan assay turned out to be about 10 times more sensitive than the available conventional PCR-based diagnostic test. It is demonstrated that the IS2404 TaqMan assay is suitable for the quantitative assessment of the dissemination of the mycobacteria in Buruli ulcer lesions. Prototype results obtained with excised tissue from a patient with a late preulcerative Buruli ulcer lesion reconfirmed earlier histopathological findings indicating that tissue damage occurs far beyond the regions in which large numbers of mycobacteria are detectable. The IS2404 TaqMan assay should be a useful tool for both diagnosis and research into the pathology and mode of transmission of this still inadequately investigated mycobacterial disease.
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Project description:BACKGROUND:Access to an accurate diagnostic test for Buruli ulcer (BU) is a research priority according to the World Health Organization. Nucleic acid amplification of insertion sequence IS2404 by polymerase chain reaction (PCR) is the most sensitive and specific method to detect Mycobacterium ulcerans (M. ulcerans), the causative agent of BU. However, PCR is not always available in endemic communities in Africa due to its cost and technological sophistication. Isothermal DNA amplification systems such as the recombinase polymerase amplification (RPA) have emerged as a molecular diagnostic tool with similar accuracy to PCR but having the advantage of amplifying a template DNA at a constant lower temperature in a shorter time. The aim of this study was to develop RPA for the detection of M. ulcerans and evaluate its use in Buruli ulcer disease. METHODOLOGY AND PRINCIPAL FINDINGS:A specific fragment of IS2404 of M. ulcerans was amplified within 15 minutes at a constant 42°C using RPA method. The detection limit was 45 copies of IS2404 molecular DNA standard per reaction. The assay was highly specific as all 7 strains of M. ulcerans tested were detected, and no cross reactivity was observed to other mycobacteria or clinically relevant bacteria species. The clinical performance of the M. ulcerans (Mu-RPA) assay was evaluated using DNA extracted from fine needle aspirates or swabs taken from 67 patients in whom BU was suspected and 12 patients with clinically confirmed non-BU lesions. All results were compared to a highly sensitive real-time PCR. The clinical specificity of the Mu-RPA assay was 100% (95% CI, 84-100), whiles the sensitivity was 88% (95% CI, 77-95). CONCLUSION:The Mu-RPA assay represents an alternative to PCR, especially in areas with limited infrastructure.
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Project description:BackgroundBuruli ulcer is a neglected tropical disease caused by Mycobacterium ulcerans. This skin disease is the third most common mycobacterial disease and its rapid diagnosis and treatment are necessary. Polymerase chain reaction (PCR) is considered to be the most sensitive method for the laboratory confirmation of Buruli ulcer. However, PCR remains expensive and involves reagents unsuitable for use in tropical countries with poor storage conditions, hindering the development of reliable quantitative PCR (qPCR) diagnosis. We aimed to overcome this problem by developing a ready-to-use dry qPCR mix for the diagnosis of M. ulcerans infection.Methodology/principal findingsWe compared the efficiency of three different dry qPCR mixes, lyophilized with various concentrations of cryoprotectants, with that of a freshly prepared mixture, for the detection of a standard range of M. ulcerans DNA concentrations. We evaluated the heat resistance of the dry mixes, comparing them with the fresh mix after heating. We also evaluated one of the dry mixes in field conditions, by analyzing 93 specimens from patients with suspected Buruli ulcers. The dry mix was (i) highly resistant to heat; (ii) of similar sensitivity and efficiency to the fresh mix and (iii) easier to use than the fresh mix.ConclusionsDry qPCR mixes are suitable for use in the diagnosis of M. ulcerans infection in endemic countries. The user-friendly format of this mix makes it possible for untrained staff to perform diagnostic tests with a limited risk of contamination. The possibility of using this mix in either vial or strip form provides considerable flexibility for the management of small or large amounts of sample. Thus, dry-mix qPCR could be used as a reliable tool for the diagnosis of Buruli ulcer in the field.
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Project description:Mycobacterium ulcerans (M. ulcerans), the causative agent of the devastating skin disease Buruli ulcer (BU), is characterized by an extremely low level of genetic diversity. Recently, we have reported the first discrimination of closely related M. ulcerans variants in the BU endemic Densu River Valley of Ghana. In the study real-time PCR-based single nucleotide polymorphism (SNP) typing at 89 predefined loci revealed the presence of ten M. ulcerans haplotypes circulating in the BU endemic region. Here we describe the development of temperature-switch PCR (TSP) assays that allow distinguishing these haplotypes by conventional agarose gel-based analysis of the PCR products. After validation of the accuracy of typing results, the TSP assays were successfully established in a reference laboratory in Ghana. Development of the cost-effective and rapid TSP-based genetic fingerprinting method will thus allow investigating the spread of M. ulcerans clones by regular genetic monitoring in BU endemic countries.
| S-EPMC3499370 | biostudies-literature
Project description:BACKGROUND: Buruli ulcer (BU), a neglected tropical skin disease caused by Mycobacterium ulcerans, has been reported in over 30 countries worldwide and is highly endemic in rural West and Central Africa. The mode of transmission remains unknown and treatment is the only alternative to disease control. Early and effective treatment to prevent the morbid effects of the disease depends on early diagnosis; however, current diagnosis based on clinical presentation and microscopy has to be confirmed by PCR and other tests in reference laboratories. As such confirmed BU diagnosis is either late, inefficient, time consuming or very expensive, and there is the need for an early diagnosis tool at point of care facilities. In this paper we report on a simple, quick and inexpensive diagnostic test that could be used at point of care facilities, in resource-poor settings. METHODS: The methodology employed is based on the loop mediated isothermal amplification (LAMP) technique. Four sets of Primers, targeting the mycolactone encoding plasmid genome sequence of M. ulcerans were designed. The BU-LAMP assay was developed and tested on five M. ulcerans strains from patients in Ghana and two American Type Culture Control (ATCC) reference isolates; Ghana #970321 (D19F9) and Benin #990826 (D27D14). We also tested the assay on other closely related, mycolactone-producing mycobacterial strains; M. marinum 1218, M. marinum DL240490, M. liflandii and M. pseudoshotsii, as well as experimentally infected laboratory animal and clinical samples. RESULTS: The results revealed a high specificity of the BU-LAMP assay for selectively detecting M. ulcerans. Compared to the conventional IS-2404 PCR, the new assay is cheaper and simpler and ten times more sensitive. Test results can be obtained within 1 hour. CONCLUSIONS: This study indicates that the BU-LAMP assay could be suitable for early disease diagnosis and application in low-resource health facilities.
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Project description:BackgroundBuruli ulcer (BU) caused by Mycobacterium ulcerans is the world's third most common mycobacterial infection. There is no vaccine against BU and surgery is needed for patients with large ulcers. Although recent experience indicates combination chemotherapy with streptomycin and rifampin improves cure rates, the utility of this regimen is limited by the 2-month duration of therapy, potential toxicity and required parenteral administration of streptomycin, and drug-drug interactions caused by rifampin. Discovery and development of drugs for BU is greatly hampered by the slow growth rate of M. ulcerans, requiring up to 3 months of incubation on solid media to produce colonies. Surrogate markers for evaluating antimicrobial activity in real-time which can be measured serially and non-invasively in infected footpads of live mice would accelerate pre-clinical evaluation of new drugs to treat BU. Previously, we developed bioluminescent M. ulcerans strains, demonstrating proof of concept for measuring luminescence as a surrogate marker for viable M. ulcerans in vitro and in vivo. However, the requirement of exogenous substrate limited the utility of such strains, especially for in vivo experiments.Methodology/principal findingFor this study, we engineered M. ulcerans strains that express the entire luxCDABE operon and therefore are autoluminescent due to endogenous substrate production. The selected reporter strain displayed a growth rate and virulence similar to the wild-type parent strain and enabled rapid, real-time monitoring of in vitro and in vivo drug activity, including serial, non-invasive assessments in live mice, producing results which correlated closely with colony-forming unit (CFU) counts for a panel of drugs with various mechanisms of action.Conclusions/significanceOur results indicate that autoluminescent reporter strains of M. ulcerans are exceptional tools for pre-clinical evaluation of new drugs to treat BU due to their potential to drastically reduce the time, effort, animals, compound, and costs required to evaluate drug activity.
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Project description: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.
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Project description:BackgroundAccess to laboratory diagnosis can be a challenge for individuals suspected of Buruli Ulcer (BU). Our objective was to develop a clinical score to assist clinicians working in resource-limited settings for BU diagnosis.Methododology/principal findingsBetween 2011 and 2013, individuals presenting at Akonolinga District Hospital, Cameroon, were enrolled consecutively. Clinical data were collected prospectively. Based on a latent class model using laboratory test results (ZN, PCR, culture), patients were categorized into high, or low BU likelihood. Variables associated with a high BU likelihood in a multivariate logistic model were included in the Buruli score. Score cut-offs were chosen based on calculated predictive values. Of 325 patients with an ulcerative lesion, 51 (15.7%) had a high BU likelihood. The variables identified for the Buruli score were: characteristic smell (+3 points), yellow color (+2), female gender (+2), undermining (+1), green color (+1), lesion hyposensitivity (+1), pain at rest (-1), size >5cm (-1), locoregional adenopathy (-2), age above 20 up to 40 years (-3), or above 40 (-5). This score had AUC of 0.86 (95%CI 0.82-0.89), indicating good discrimination between infected and non-infected individuals. The cut-off to reasonably exclude BU was set at scores <0 (NPV 96.5%; 95%CI 93.0-98.6). The treatment threshold was set at a cut-off ?4 (PPV 69.0%; 95%CI 49.2-84.7). Patients with intermediate BU probability needed to be tested by PCR.Conclusions/significanceWe developed a decisional algorithm based on a clinical score assessing BU probability. The Buruli score still requires further validation before it can be recommended for wide use.
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