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.

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%.

Lymphatic tissue changes in AIDS and other retrovirus infections: tools and insights.

Based on immunohistochemistry, in situ hybridization, and electron microscopy, lymphatic tissue changes in human immunodeficiency virus (HIV) and other retroviral infections represent different stages of a dynamic process progressing from hyperplasia to atrophy. The germinal centers (GC) function early as a virus reservoir in both HIV and feline leukemia virus infection, which also produces lymphadenopathy, severe immune impairment, and death from opportunistic infections. Core proteins of HIV can be detected on the surface of follicular dendritic cells, electron microscopy reveals cell-free HIV and virus replication in the same location, and in situ hybridization shows that the majority of cells with mRNA of HIV can be found in germinal centers (GC). Double immunohistochemical labeling of lymphocyte populations suggests that one of the most important events in HIV lymphadenitis with explosive follicular hyperplasia is the accumulation of CD8+CD45R0+ lymphocytes in the lymph nodes. Their clustering in the vicinity of the FDC network could play a key role in disintegration of GC and lymphocyte depletion as the disease progresses.