Loop-Mediated Isothermal Amplification for Laboratory Confirmation of Buruli Ulcer Disease—Towards a Point-of-Care Test

Background

As the major burden of Buruli ulcer disease (BUD) occurs in remote rural areas, development of point-of-care (POC) tests is considered a research priority to bring diagnostic services closer to the patients. Loop-mediated isothermal amplification (LAMP), a simple, robust and cost-effective technology, has been selected as a promising POC test candidate. Three BUD-specific LAMP assays are available to date, but various technical challenges still hamper decentralized application. To overcome the requirement of cold-chains for transport and storage of reagents, the aim of this study was to establish a dry-reagent-based LAMP assay (DRB-LAMP) employing lyophilized reagents.

Methodology/Principal Findings

Following the design of an IS2404 based conventional LAMP (cLAMP) assay suitable to apply lyophilized reagents, a lyophylization protocol for the DRB-LAMP format was developed. Clinical performance of cLAMP was validated through testing of 140 clinical samples from 91 suspected BUD cases by routine assays, i.e. IS2404 dry-reagent-based (DRB) PCR, conventional IS2404 PCR (cPCR), IS2404 qPCR, compared to cLAMP. Whereas qPCR rendered an additional 10% of confirmed cases and samples respectively, case confirmation and positivity rates of DRB-PCR or cPCR (64.84% and 56.43%; 100% concordant results in both assays) and cLAMP (62.64% and 52.86%) were comparable and there was no significant difference between the sensitivity of the assays (DRB PCR and cPCR, 86.76%; cLAMP, 83.82%). Likewise, sensitivity of cLAMP (95.83%) and DRB-LAMP (91.67%) were comparable as determined on a set of 24 samples tested positive in all routine assays.

Conclusions/Significance

Both LAMP formats constitute equivalent alternatives to conventional PCR techniques. Provided the envisaged availability of field friendly DNA extraction formats, both assays are suitable for decentralized laboratory confirmation of BUD, whereby DRB-LAMP scores with the additional advantage of not requiring cold-chains. As validation of the assays was conducted in a third-level laboratory environment, field based evaluation trials are necessary to determine the clinical performance at peripheral health care level.     

Laboratory confirmation of Buruli ulcer disease in Togo, 2007-2010

Background

Since the early 1990s more than 1,800 patients with lesions suspicious for Buruli ulcer disease (BUD) have been reported from Togo. However, less than five percent of these were laboratory confirmed. Since 2007, the Togolese National Buruli Ulcer Control Program has been supported by the German Leprosy and Tuberculosis Relief Association (DAHW). Collaboration with the Department for Infectious Diseases and Tropical Medicine (DITM), University Hospital, Munich, Germany, allowed IS2404 PCR analysis of diagnostic samples from patients with suspected BUD during a study period of three years.

Methodology/Principal Findings

The DAHW integrated active BUD case finding in the existing network of TB/Leprosy Controllers and organized regular training and outreach activities to identify BUD cases at community level. Clinically suspected cases were referred to health facilities for diagnosis and treatment. Microscopy was carried out locally, external quality assurance (EQA) at DITM. Diagnostic samples from 202 patients with suspected BUD were shipped to DITM, 109 BUD patients (54%) were confirmed by PCR, 43 (29.9%) by microscopy. All patients originated from Maritime Region. EQA for microscopy resulted in 62% concordant results.

Conclusions/Significance

This study presents a retrospective analysis of the first cohort of clinically suspected BUD cases from Togo subjected to systematic laboratory analysis over a period of three years and confirms the prevalence of BUD in Maritime Region. Intensified training in the field of case finding and sample collection increased the PCR case confirmation rate from initially less than 50% to 70%. With a PCR case confirmation rate of 54% for the entire study period the WHO standards (case confirmation rate ≥50%) have been met. EQA for microscopy suggests the need for intensified supervision and training. In January 2011 the National Hygiene Institute, Lomé, has assumed the role of a National Reference Laboratory for PCR confirmation and microscopy.     

Effectiveness of Routine BCG Vaccination on Buruli Ulcer Disease: A Case-Control Study in the Democratic Republic of Congo,Ghana and Togo

Background

The only available vaccine that could be potentially beneficial against mycobacterial diseases contains live attenuated bovine tuberculosis bacillus (Mycobacterium bovis) also called Bacillus Calmette-Guérin (BCG). Even though the BCG vaccine is still widely used, results on its effectiveness in preventing mycobacterial diseases are partially contradictory, especially regarding Buruli Ulcer Disease (BUD). The aim of this case-control study is to evaluate the possible protective effect of BCG vaccination on BUD.

Methodology

The present study was performed in three different countries and sites where BUD is endemic: in the Democratic Republic of the Congo, Ghana, and Togo from 2010 through 2013. The large study population was comprised of 401 cases with laboratory confirmed BUD and 826 controls, mostly family members or neighbors.

Principal Findings

After stratification by the three countries, two sexes and four age groups, no significant correlation was found between the presence of BCG scar and BUD status of individuals. Multivariate analysis has shown that the independent variables country (p = 0.31), sex (p = 0.24), age (p = 0.96), and presence of a BCG scar (p = 0.07) did not significantly influence the development of BUD category I or category II/III. Furthermore, the status of BCG vaccination was also not significantly related to duration of BUD or time to healing of lesions.

Conclusions

In our study, we did not observe significant evidence of a protective effect of routine BCG vaccination on the risk of developing either BUD or severe forms of BUD. Since accurate data on BCG strains used in these three countries were not available, no final conclusion can be drawn on the effectiveness of BCG strain in protecting against BUD. As has been suggested for tuberculosis and leprosy, well-designed prospective studies on different existing BCG vaccine strains are needed also for BUD.     

Treatment Outcome of Patients with Buruli Ulcer Disease in Togo

Background

Following introduction of antimycobacterial treatment of Buruli ulcer disease (BUD), several clinical studies evaluated treatment outcomes of BUD patients, in particular healing times, secondary lesions and functional limitations. Whereas recurrences were rarely observed, paradoxical reactions and functional limitations frequently occurred. Although systematic BUD control in Togo was established as early as 2007, treatment outcome has not been reviewed to date. Therefore, a pilot project on post-treatment follow-up of BUD patients in Togo aimed to evaluate treatment outcomes and to provide recommendations for optimization of treatment success.

Methodology/Principal Findings

Out of 199 laboratory confirmed BUD patients, 129 could be enrolled in the study. The lesions of 109 patients (84.5%) were completely healed without any complications, 5 patients (3.9%) had secondary lesions and 15 patients (11.6%) had functional limitations. Edema, category III ulcers >15cm, healing times >180 days and a limitation of movement at time of discharge constituted the main risk factors significantly associated with BUD related functional limitations (P<0.01). Review of all BUD related documentation revealed major shortcomings, in particular concerning medical records on adjuvant surgical and physiotherapeutic treatment.

Conclusions/Significance

This study presents the first systematic analysis of treatment outcome of BUD patients from Togo. Median times to healing and the absence of recurrences were in line with findings reported by other investigators. The percentage of functional limitations of 11.6% was lower than in other studies, and edema, category III ulcers, healing time >180 days and limitation of movement at discharge constituted the main risk factors for functional limitations in Togolese BUD patients. Standardized treatment plans, patient assessment and follow-up, as well as improved management of medical records are recommended to allow for intensified monitoring of disease progression and healing process, to facilitate implementation of therapeutic measures and to optimize treatment success.