Excision of pre-ulcerative forms of Buruli ulcer disease: a curative treatment?

BACKGROUND: Previous investigations have revealed that Mycobacterium ulcerans is extensively distributed spatially throughout ulcerative lesions, including in the margins of excised tissue. In contrast, bacilli in pre-ulcerative lesions are assumed to be concentrated in the center of the lesion. In order to assess the extent to which the surgical excision of pre-ulcerative lesions is capable of removing all infected tissue, we subjected the excision margins of pre-ulcerative lesions to laboratory analysis. PATIENTS AND METHODS: Eleven patients with laboratory-confirmed pre-ulcerative lesions were included in the study. The diameter of the lesion and excised tissue and the "surgical distance" between the border of the lesion and excision margin were measured. The entire excision margin was cut into segments and subjected to IS2404 PCR. RESULTS: The results from the PCR analysis on the samples of excision margins were highly significantly associated with the surgical distance (p < 0.001). The margin samples of nodules were significantly more often PCR positive than the plaques (p = 0.025). The size of the lesion and the size of the excised tissue did not significantly influence the PCR results. Statistically, a surgical distance of more than 9 mm was found to reduce the risk of remaining infected tissue to less than 10%, that of 13 mm to reduce the risk to less than 5%, and that of 25 mm to reduce the risk to nearly 0%. CONCLUSION: The results of this study show that in preulcerative Buruli ulcer disease, bacilli may extend beyond the actual size of the lesion and that there is a strong correlation between the presence of M. ulcerans in the margin samples and the surgical distance. Excision with a surgical distance of 25 mm avoided the risk of remaining mycobacteria in this study. However, no recurrences occurred in the patients with M. ulcerans-positive excision margins. The need of postoperative antimycobacterial treatment in these patients remains to be determined.

A stepwise approach to the laboratory diagnosis of Buruli ulcer disease.

OBJECTIVE: In view of technical and financial limitations in areas of endemicity, the current practice and recommendations for the laboratory diagnosis of Buruli ulcer disease (BUD) may have to be reconsidered. We reviewed diagnostic results in order to explore options for a modified, more practicable, cost-effective and timely approach to the laboratory diagnosis of BUD. METHODS: Diagnostic specimens from 161 clinically diagnosed BUD patients from four different treatment centres in Ghana were subjected to laboratory analysis. The positivity rates of the laboratory assays were compared. RESULTS: The number of laboratory-confirmed clinically diagnosed BUD cases with one positive confirmative test was 20% higher than that with two positive confirmative tests. The specificity of microscopy (MIC) and PCR was 96.6% and 100%, respectively. Subsequent analysis of specimens from surgically excised pre-ulcerative tissue-by-tissue MIC and tissue PCR rendered 65% laboratory-confirmed BUD cases. Subsequent analysis of diagnostic swabs from ulcerative lesions by swab smear MIC and swab PCR rendered 70% of laboratory-confirmed BUD cases. CONCLUSIONS: The specificity of the diagnostic tests used in this study suggests that one positive diagnostic test may be considered sufficient for the laboratory confirmation of BUD. Subsequent application of different diagnostic tests rendered a laboratory confirmation of 65% pre-ulcerative and of 70% ulcerative lesions. Implementation of a stepwise, subsequent analysis of diagnostic specimens will result in considerable cost saving compared with simultaneous testing of specimens by several diagnostic assays.

External quality assurance for the laboratory diagnosis of Buruli ulcer disease in Ghana.

OBJECTIVE: To assure the quality of the laboratory diagnosis of Buruli ulcer disease; microscopy and PCR were subjected to external quality assurance (EQA). METHODS: Slides were read by test laboratory staff, followed by blinded re-reading by the controller. Parallel testing of PCR specimens was carried out at the local and external reference laboratory. Slides and PCR specimens with discordant results were subjected to a second reading/testing by the controller to determine the final result. For training purposes, slides and PCR specimens with discrepant results were subsequently re-read/re-tested under supervision at the test laboratory. RESULTS: Microscopy. First reading: concordance rate 82.9%, discordance rate 17.1%, percentage false negatives 27.1% (sensitivity 72.9%), percentage false positives 10.1% (specificity 89.9%). Second reading: concordance rate 97.9%, discordance rate 2.1%, percentage false negatives 4.2% (sensitivity 95.8%), percentage false positives 0.6% (specificity 99.4%). PCR. First testing: concordance rate 87.9%, discordance rate 12.1%, percentage false negatives 8.2% (sensitivity 91.8%), percentage false positives 19.1% (specificity 80.9%). Second testing: concordance rate 96.2%, discordance rate 3.8%, percentage false negatives 4.7% (sensitivity 95.3%), percentage false-positives 2.1% (specificity 97.9%). CONCLUSIONS: EQA identified deficiencies in the laboratory performance. Corrective action consisted in on-site training and reduced the number of false-negative and false-positive microscopy and PCR results.

Post-surgical assessment of excised tissue from patients with Buruli ulcer disease: progression of infection in macroscopically healthy tissue.

OBJECTIVE: The current standard of treatment of Buruli ulcer disease (BUD) is surgical excision of lesions. Excision size is determined macroscopically assuming the complete removal of all infected tissue. However, dissemination of infection beyond the excision margins into apparently healthy tissue, possibly associated with recurrences, cannot be excluded in this way. To assess the central to peripheral progression of Mycobacterium ulcerans infection and the mycobacterial infiltration of excision margins, excised tissue was examined for signs of infection. METHODS: 20 BUD lesions were excised in general anaesthesia including all necrotic and subcutaneous adipose tissue down to the fascia and at an average of 40 mm into the macroscopically unaffected tissue beyond the border of the lesion. Tissue samples were subjected to PCR and histopathology. RESULTS: Although the bacillary load decreased from central to peripheral, M. ulcerans infection was detected throughout all examined tissue specimens including the peripheral segments as well as excision margins of all patients. During the post-operative hospitalization period (averaging 2 months) no local recurrences were observed. CONCLUSION: Available data suggest a correlation of surgical techniques with local recurrences. The results of this study indicate the unnoticed early progression of mycobacterial infection into macroscopically healthy tissue. Thus, the removal of all infected tissue cannot always be verified visually by the surgeon. Provided that long-term follow up of patients with positive excision margins will establish the clinical relevance of these findings, on-site laboratory assessment of excised tissue in combination with follow up may contribute to reduce recurrence rates.

Dry-reagent-based PCR as a novel tool for laboratory confirmation of clinically diagnosed Mycobacterium ulcerans-associated disease in areas in the tropics where M. ulcerans is endemic.

After tuberculosis and leprosy, Buruli ulcer (BU), caused by Mycobacterium ulcerans, is the third most common mycobacterial disease in immunocompetent humans. The disease occurs in tropical countries, with foci in West Africa, Central Africa, and the western Pacific. BU is defined as an infectious disease involving the skin and the subcutaneous adipose tissue characterized by a painless nodule, papule, plaque, or edema, evolving into a painless ulcer with undermined edges and often leading to invalidating sequelae. Due to the fundamental lack of understanding of modes of transmission, disease control in endemic countries is limited to early case detection through improved active surveillance and surgical treatment. The laboratory confirmation of BU is complicated by the absence of a diagnostic "gold standard." Therefore, misclassification and delayed diagnosis of BU may occur frequently, causing a considerable socioeconomic impact in terms of treatment costs due to prolonged hospitalization. In order to respond to the urgent need to develop reliable tools for early case detection and to overcome technical difficulties accompanying the implementation of diagnostic PCR procedures in tropical countries, a dry-reagent-based PCR formulation for the detection of M. ulcerans in diagnostic specimens has been developed at the Bernhard Nocht Institute for Tropical Medicine. Following technical and clinical validation, the assay has been successfully installed and field tested at the Kumasi Centre for Collaborative Research in Tropical Medicine, Kumasi, Ghana. Preliminary results show an excellent diagnostic sensitivity of >95%.

Primary in vivo T cell reactivity of NZB grafts in H-2 identical allogenic hosts.

By means of the Simonson GVH-assay and the popliteal lymph node (PLN) assay, the T-cell reactivity of NZB mice against H-2 identical allogenic cells was investigated in vivo and compared to that of normal mice. None of the normal mice did react, but a highly significant NZB response could be demonstrated, which did not depend on differences in Mls antigens. These in vivo results extend previous findings of a T-cell hyperreactivity of NZB mice in primary in vitro reactions. They favour the possibility that the T-cell hyperreactivity might be relevant in vivo in facilitating autoimmune responses.