Sensitivity of PCR targeting the IS2404 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 AFB-negative cases only, in order to minimize costs. Histology and culture remain important as quality control tests, particularly in studies of treatment efficacy.

FIG. 1.

(A) The sensitivity of PCR for M. ulcerans was demonstrated by spiking normal skin tissue with 5 μl of a solution containing 10³ bacteria/ml (5 bacteria; lanes 1 to 3), 5 μl of 10⁴ bacteria/ml (50 bacteria; lanes 4 to 6), or no bacteria (lanes 7 to 9). Lanes 3, 6, and 9 were also spiked with 5 fg of M. ulcerans DNA before PCR to control for inhibition. Lane M, a 100-bp molecular ladder size marker. (B) Lane 1, 100 fg of M. ulcerans DNA (approximately 5,000 copies, based on 249 copies of the IS2404 insertion element per genome of M. ulcerans); lanes 2 and 3, 10 fg of M. ulcerans DNA (approximately 500 copies); lanes 4 and 5, 1 fg of M. ulcerans DNA (approximately 50 copies); lanes 6 and 7, TE control; lane M, a 100-bp molecular ladder size marker. (C) UNG in the reaction mixture breaks down dUTP-containing amplified PCR products, preventing false-positive results, as demonstrated with a dUTP-containing amplified product prepared with 10 pg M. ulcerans DNA solution and primer pair PU4F and PU7Rbio, which resulted in a 154-bp product. This amplified product was diluted 10⁵, 10⁶, and 10⁷ times with TE and was further amplified in the absence of UNG (lanes 1 to 5) or in the presence of 0.01 unit UNG as the template in the PCR (lanes 6 to 10). Lane M, a 100-bp molecular ladder size marker.