Comparison of molecular and microscopic techniques for detection of Treponema pallidum in genital ulcers

Abstract

We compared the ability of direct immunofluorescent staining (DFA) and the PCR to detect Treponema pallidum in specimens from patients with genital ulcer disease. Touch preparations from 156 patients with genital lesions were fixed in acetone and stained with a fluorescein-labeled monoclonal antibody specific for the 37-kDa antigen of T. pallidum. After microscopic examination, the smear was removed from the slide with a swab. DNA was extracted with phenol-chloroform and precipitated with isopropanol. Ten microliters of the extracted DNA was amplified by PCR using primers for the gene encoding the 47-kDa protein of T. pallidum and hybridized to an internal probe. Twenty-two of 156 specimens were positive for T. pallidum by DFA and PCR, while 127 were negative by both methods, yielding a concordance of 95.5% ( ‫؍‬ 0.84). Four specimens were positive by PCR and negative by DFA, while three specimens were negative by PCR and positive by DFA. The DFA-negative, PCR-positive specimens may have resulted from the presence of large numbers of leukocytes on the slides, obscuring visualization of treponemes. The DFA-positive, PCR-negative results were not due to inhibition of the PCR since purified T. pallidum DNA was amplified when added to aliquots of these specimens. Negative results in these specimens were most likely due to inefficient recovery of their DNA. These data suggest that DFA and PCR are equivalent methods for detection of T. pallidum on touch preparations of genital lesions. Further refinements of the PCR assay are necessary for it to significantly improve the detection of T. pallidum in genital lesions.

Simultaneous PCR detection of Haemophilus ducreyi, Treponema pallidum, and herpes simplex virus types 1 and 2 from genital ulcers

“…The results for the seven specimens that were falsely negative by PCR may have been due to sampling error or a lower specificity of dark-field microscopy because of the presence of commensal spirochetes in the specimen. A monoclonal antibody immunostaining test can be used to increase the specificity of microscopic diagnosis of syphilis (15,28).…”

“…Currently, serologic tests are useful for the confirmation of a diagnosis based on clinical presentation and for follow-up treatment, but sensitivities as low as 30% have been reported for the early stages of syphilis (21). Direct examination methods, including dark-field microscopy and immunofluorescent-antibody staining, are limited by low degrees of sensitivity and specificity (15). The accurate diagnosis of H. ducreyi depends on the ability to culture the organism; specimens must be cultured on solid medium within 4 to 6 h after collection from the patient.…”

Detection of infection or infectious agents by use of cytologic and histologic stains

“…Specific antibodies for immunofluorescence staining are commercially available for a few bacteria: Francisella tularensis, Bordetella pertussis, Treponema pallidum, Legionella pneumophila, and other Legionella species (50,51,60,81,85,86). For detection of B. pertussis, a smear of nasopharyngeal cells obtained by swab or aspirate is prepared.…”

Comparative analysis of immunohistochemistry, polymerase chain reaction and focus-floating microscopy for the detection of Treponema pallidum in mucocutaneous lesions of primary, secondary and tertiary syphilis

“…Dark‐field microscopic observation is a sensitive but nonspecific technique that requires a very well trained examiner 6 . Silver stain is an ancillary method that is limited by low degrees of sensitivity and specificity 7 . In addition, this technique is characterized by marked background artefacts, it is not specific to T. pallidum , and it may be inapplicable in nontreponemal spirochaete‐contaminated tissue sites (e.g.…”