Application of Real-time PCR to Recognize Atypical Mycobacteria in Archival Skin Biopsies

Abstract

Atypical mycobacterial skin infections are difficult to diagnose owing to their aspecific histopathologic presentations and to the presence of few bacteria. Therefore, these infections are often not recognized. Molecular detection of mycobacterial DNA has proven to be useful in clinical samples. The aim of this study was to investigate the incidence of mycobacterial involvement in skin biopsies showing granulomatous inflammation, using real-time polymerase chain reaction (PCR). Real-time PCR specific for the genus Mycobacterium and the species Mycobacterium avium and Mycobacterium haemophilum was performed on formalin-fixed/paraffin-embedded biopsies from patients with granulomatous inflammation of the skin, from the period 1984 to 2004. A control group was assembled from patients with proven basal cell carcinoma. Amplicons of all positive reactions were sequenced to confirm or identify the mycobacterial species. Of 30 patients, 13 (43%) were found to be positive for mycobacterial infection, of whom only 5 patients had been previously diagnosed with a mycobacterial disease. M. haemophilum was identified as the most common species (n=7). The other identified species were Mycobacterium malmoense, Mycobacterium gordonae, and Mycobacterium marinum. The results show that real-time PCR is useful in detecting mycobacterial infections in undiagnosed formalin-fixed/paraffin-embedded skin samples and that the application of molecular approaches would improve the diagnoses of mycobacterial skin infections.

Clinical Manifestations, Diagnosis, and Treatment of Mycobacterium haemophilum Infections

“…This assay was also applied to formalin-fixed/paraffin-embedded biopsy specimens from patients with granulomatous inflammation of the skin, which were stored between 1984 and 2004 (15). Of 30 patient materials tested, 13 (43%) were found to contain mycobacterial DNA.…”

“…Because culture for M. haemophilum is less sensitive than the real-time PCR assay described above (14), molecular diagnosis should also be attempted, preferably using genus-specific detection and M. haemophilum-specific detection. Molecular detection also enables biopsy specimens and other histopathological materials to be examined for the presence of mycobacterial DNA when culturing is not possible due to tissue fixation (15). This approach offers an excellent opportunity to investigate the presence of newly identified Mycobacterium species in stored patient materials.…”

Cutaneous Mycobacterium haemophilum infection in iatrogenically immunocompromised patients without transplantation

“…15 Likewise, the clinical spectrum of cutaneous M haemophilum infection is varied and may mimic cellulitis, vasculitis, panniculitis, sarcoidosis, mycosis, or chronic granulomatous inflammation. 12 Acknowledging the various clinical contexts in which cutaneous M haemophilum may manifest, as seen in our patients and others listed above, biopsy of lesional tissues remains critical for initial diagnosis. Histopathologic examination of lesional tissue generally exhibits mixed granulomatous inflammation with varying degrees of tissue necrosis, findings of which were seen in the biopsy specimens of our two patients.…”

“…8 These requirements, however, may account for many cases of culture-negative, acid-fast bacilli reported previously, [9][10][11] a hypothesis buttressed by recent research indicating a high incidence of M haemophilum in archival skin biopsy specimens retrospectively detected by real-time polymerase chain reaction. 12 M haemophilum is now recognized as an important cause of cutaneous pathology in immunocompromised patients, although infrequent cases of extracutaneous manifestations have also been described. 4,13 To date, more than 100 cases of cutaneous M haemophilum infection have been reported in the literature; however, very few cases have been described in HIV-negative patients who are taking immunosuppressive medications for medical conditions other than transplantation.…”

Fatal skin and soft tissue infection of multidrug resistant Acinetobacter baumannii: A case report

“…Therefore, these infections are often not recognized. There is data to suggest that real-time PCR is useful in detecting mycobacterial infections in undiagnosed formalin-fixed/paraffin-embedded skin samples and that the application of molecular approaches would improve the diagnoses of mycobacterial skin infections 19 . Atypical mycobacterial infections should be suspected in patients who develop late-onset skin and soft tissue infection after cutaneous injury, injection, and surgical intervention, particularly if they do not respond to conventional antibiotic treatment.…”