Sensitivity of PCR Targeting the IS 2404 Insertion Sequence of Mycobacterium ulcerans in an Assay Using Punch Biopsy Specimens for Diagnosis of Buruli Ulcer

Abstract

Punch biopsy specimens from Mycobacterium ulcerans disease lesions were used to compare the sensitivities and specificities of direct smear, culture, PCR, and histopathology in making a diagnosis of M. ulcerans disease in a field setting. PCR for the insertion element IS2404 was modified to include uracil-N-glycosylase and deoxyuridine triphosphate instead of deoxythymidine triphosphate to reduce the risk of cross contamination. The "gold standard" for confirmation of clinically diagnosed Buruli ulcer was a definite histological diagnosis, a positive culture for M. ulcerans, or a smear positive for acid-fast bacilli (AFB), together with a possible histological diagnosis. For 70 clinically diagnosed cases of M. ulcerans disease, the modified PCR was 98% sensitive and gave a rapid result. The sensitivities of microscopy, culture, and histology were 42%, 49%, and 82%, respectively. The use of a 4-mm punch biopsy specimen was preferred to a 6-mm punch biopsy specimen since the wound was less likely to bleed and to need stitching. Given adequate technical expertise and the use of controls, the PCR was viable in a teaching hospital setting in Ghana; and in routine practice, we would recommend the use of Ziehl-Neelsen staining of biopsy specimens to detect AFB, followed by PCR, in AFBnegative cases only, in order to minimize costs. Histology and culture remain important as quality control tests, particularly in studies of treatment efficacy.Mycobacterium ulcerans disease (Buruli ulcer) manifests as a nodule, papule, plaque, or edematous lesion prior to ulceration (5). The disease has been reported in more than 31 countries, mostly countries with a tropical climate (2). The recommended treatment has been surgical excision, but areas of endemicity in tropical countries are often rural, with unmade roads and relatively poor health care facilities (5).The clinical diagnosis of Buruli ulcer is relatively easy when a child from an area of endemicity presents looking well with a painless ulcer eroding into subcutaneous fat and the ulcer has undermined edges (2); but the disease can affect people of any age, and other ulcers can be mistaken for Buruli ulcer, particularly when they are around the ankles, for example, venous ulcer, cutaneous leishmaniasis, neurogenic ulcer, yaws, tropical ulcer, fungal lesions, and squamous cell carcinoma (7). Diagnosis is more difficult in the case of nodules, and the sensitivity of clinical diagnosis in experienced hands was 48% to 52% in one study (5). Confirmation of the clinical diagnosis of Buruli ulcer is becoming increasingly important now that there is growing evidence of a beneficial effect from treatment with antibiotics (4), which is beginning to replace excision surgery as the standard treatment. In the case of an ulcer, the diagnostic technique most often available in areas of endemicity is ZiehlNeelsen (ZN) staining for acid-fast bacilli (AFB) of a smear from a swab taken from below the undermined edge of the ulcer base, but its sensitivity is low (40%) (16) and the technique cann...

Persistence of Mycobacterium ulcerans Disease (Buruli Ulcer) in the Historical Focus of Kasongo Territory, the Democratic Republic of Congo

“…The contamination rate could probably have been reduced if the appropriate resources for (punch) biopsies and/or fine needle aspiration had been present. 3,36 Based on the results of clinical and laboratory diagnosis, our study confirmed that the Kasongo Territory is still (or again) an active BU focus.…”

Use of Fine-Needle Aspiration for Diagnosis of Mycobacterium ulcerans Infection

“…Therefore, the sensitivity of the diagnosis from FNA samples was calculated to be 93.4% (57/ 61). The rate of positive smears from ulcerative and nonulcerative lesions in our study was lower than those obtained with other sampling methods (1,8,10). Thus, M. ulcerans DNA not associated with bacilli may be detected in aspirate liquid.…”

Diabetic Foot Infections: Microbiology Made Modern?

“…With PCR methods, it is possible to detect most species of pathogens in a wound in a matter of hours rather than days. PCR has already shown promise in various clinical situations, including identifying antibiotic-resistant gram-negative organisms (20), bacteria that elaborate neurotoxins (21) or other virulence factors (22), and slow-growing organisms like mycobacteria (23). Additionally, previous antibiotic therapy would be much less likely to cause false-negative results than with a standard culture.…”