Duplex PCR for differential identification of Mycobacterium bovis, M. avium, and M. avium subsp. paratuberculosis in formalin- fixed paraffin-embedded tissues from cattle.

We previously isolated and sequenced two genomic segments of Mycobacterium avium subsp. paratuberculosis, namely, f57, a species-specific sequence, and the p34 gene, coding for a 34-kDa antigenic protein. Comparison of sequences upstream of the p34 open reading frame (us-p34) from M. avium subsp. paratuberculosis and M. tuberculosis showed a 79-base deletion in M. tuberculosis. Sequence analysis of the p34 genes in another two species, M. bovis (strain BCG) and M. avium (strain D4), confirmed the differences observed between tuberculous and nontuberculous species. A duplex diagnostic PCR strategy based on coamplification of nonhomologous us-p34 and species-specific f57 sequences was therefore developed. Duplex PCR yielded three different patterns, specific either for tuberculous bacilli (M. tuberculosis, M. bovis, and M. africanum), for both nontuberculous mycobacteria M. avium and M. intracellulare, or for M. avium subsp. paratuberculosis. The specificity of this single-step DNA-based assay was assessed on DNA from cultured mycobacterial strains, as well as on a panel of formalin-fixed and paraffin-embedded tissues from cattle. Molecular assay results from tissular DNA were compared to conventional bacteriological and histological test results, including those obtained by Ziehl-Neelsen staining on tissue biopsy specimens. Molecular discrimination was successful and confirmed the value of duplex us-p34 and f57 sequence amplification for differential diagnosis of tuberculosis, paratuberculosis, or infections caused by other members of the M. avium complex.

The fate of the oblique abdominal muscles after free TRAM flap surgery.

During recent years, clinical research on the donor site morbidity after free or pedicled transverse rectus abdominis myocutaneous (TRAM) flap surgery has been focusing on the reduced flexion capacity of the abdominal wall. However, the rectus abdominis muscles have close interactions with their synergists and antagonists and collaborate with their neighbouring muscles. The purpose of this study was to examine the consequences of partially resecting the rectus abdominis muscle on the different muscle groups of the abdominal wall. Twenty free TRAM flap patients, 12-61 months (mean 32.1 months) after surgery, were clinically examined, evaluated for curl-up performance and underwent isokinetic dynamometry for flexion, extension and rotation. The patients were compared with 20 non-operated controls. Nineteen patients answered a questionnaire. Abdominal wall abnormalities occurred in 10 patients: umbilical asymmetry (n = 3), abdominal wall asymmetry (n = 4), lower abdominal bulging (n = 2) and hernia (n = 1). Curl-up performance was less in the TRAM flap patients (P = 0.001, Mann-Whitney). Isokinetic flexion, extension and rotation were also less in the TRAM flap patients (Fisher's exact test). This study indicates that what has been believed to be 'limited' surgical damage to the abdominal wall leads to an important reduction in flexion strength but to an even more important reduction of rotation strength due to bilateral displacement and damage of the insertion of the oblique muscles. Partial compensation by synergists is variable and unpredictable on an individual basis. These functional disorders can potentially lead to important changes in activities of daily life.

The donor site morbidity of free DIEP flaps and free TRAM flaps for breast reconstruction.

This study was undertaken to demonstrate that the deep inferior epigastric perforator (DIEP) flap can provide the well-known advantages of autologous breast reconstruction with lower abdominal tissue while avoiding the abdominal wall complications of the transverse rectus abdominis myocutaneous (TRAM) flap. Eighteen unilateral free DIEP flap breast reconstruction patients were assessed 12-30 months (mean 17.8 months) after surgery. Clinical examination, physical exercises and isokinetic dynamometry were performed preoperatively and two months and one year postoperatively. Intraoperative segmental nerve stimulation, visual evaluation and postoperative CT scans were also used to quantify the damage to the rectus muscle. The 18 patients were then compared with 20 free TRAM flap patients and 20 non-operated controls. Two DIEP flap patients presented with abdominal asymmetry. A limited decrease of trunk flexing strength was noticed but rotatory function was intact. Ten of the TRAM flap patients had umbilical or abdominal asymmetry, bulging or hernias. TRAM flap patients showed a statistically significant reduction in strength to flex and to rotate the upper trunk compared to both the one year postoperative DIEP flap group and the control group. The answers to a questionnaire revealed impairment of activities of daily living for some TRAM flap patients while the activities of all DIEP flap patients were unaffected. Our data demonstrate that the free DIEP flap can limit the surgical damage to the rectus abdominis and oblique muscles to an absolute minimum. We believe it is worthwhile to spend extra operative time, the main disadvantage of this technique, to limit late postoperative weakness of the lower abdominal wall.