Literature review > Issue 7 > Review on Palmer et al. 

The study by Palmer et al. compares detection of T. pallidum by a multiplex DNA PCR in ulcerative lesions in early syphilis with serology, dark field microscopy, and clinical diagnosis. The currently available diagnostic procedures have limitations, and the sensitivity and specificity vary according to the specific method. As correctly stated by the authors, serologic testing may fail to detect a significant proportion of primary syphilis. It is particularly insensitive for early primary lesions and cannot differentiate current from past infection. Darkfield microscopy and immunostaining, useful for new primary and secondary lesions, requires infrastructure and trained personnel, clearly not available in the majority of settings both in industrialized and in developing countries. Because this spirochete cannot be cultured in vitro, nucleic acid amplification tests (NAATs) are particularly useful in the detection of this organism in genital ulcers. Although in this study clinical diagnosis agreed with the PCR results in 95 out of 98 patients, it is uncommon that genital ulcers caused by T. pallidum can be readily distinguished from ulcerative lesions caused by the other two most common etiologies of GUD (herpes simplex virus and Haemophilus ducreyi) based upon typical clinical appearance. Several studies have shown that these three conditions cannot be distinguished clinically from each other in many cases. The important aspect of this study is, however, that it points out the usefulness of sensitive PCR tests to detect T. pallidum in genital ulcers and highlights in the discussion the potential of multiplex PCR (M-PCR) assays for the simultaneous detection of other GUD causative agents.

In the last 15 years, several DNA PCR techniques have been developed for the detection of T. pallidum [1,2,3,4,5]. The M-PCR developed by Orle et al. has been used in several countries for detecting T. pallidum, showing excellent sensitivity and specificity compared to darkfield microscopy and serology with resolved sensitivities and specificities of 91-100% and nearly 100%, respectively [4]. Published studies for the different PCR assays report limits of detection of 1 to 130 treponemes. The PCR detection limit in this study is 800 treponemes, as determined by serial dilutions of purified genomic DNA, which by inference indicates the presence of a high number of treponemes in the ulcerative lesions. In this study, there was one case of primary and two cases of secondary syphilis that were PCR negative. This could be explained by the presence of low number of treponemes in the samples (below the detection limit of this assay of 800 organisms per reaction) in resolving lesions or by the presence of PCR inhibitors. Although the authors included inhibition controls, these would detect only strong inhibitory effects because of the high number of treponemes used in the inhibition control tubes (8 x 10⁴ treponeme DNA equivalents). In our experience, we can routinely detect an amplicon for the tp47 gene from as few as 10 treponemes, and spiking samples with 100 treponeme DNA equivalents provides a good control for inhibitory effects. The lower detection limit of the amplification in this study may not affect the overall performance of the assay, probably due to the presence of large number of treponemes in the majority of lesions at the time of diagnosis.

The M-PCR assay was shown to be very useful for detection of T. pallidum in GUD. Unfortunately, there are no commercially available kits for thermocyclers or real-time PCR for the detection of T. pallidum. Commercial kits have the advantage of addressing most of the technical issues involving sample collection, sample processing, optimization of reagents, and controls. More recently, the availability of fully integrated platforms such as the GeneXpert® System, which purifies, concentrates, detects, and identifies nucleic acid sequences in less than 30 minutes, makes the potential use of real-time M-PCR more appealing. Its compact design would simplify and facilitate the use of these tests in different settings. In industrialized countries, they would allow a rapid and specific diagnosis at the point-of-care, reducing the risk of transmission, over-treatment or mistreatment. Because of its sensitivity, PCR can be used on samples obtained by non-invasive methods such as self-collected swabs from ulcers without reduction in test performance. The collection of samples by non-invasive methods will facilitate surveillance and clinical diagnosis of syphilis and other common GUDs in routine care as well as in community-based settings. For developing countries, such systems will be useful for the evaluation of the spectrum of GUD pathogens and their geographical and temporal patterns of distribution.

Real-time PCR offers significant advantages in that nucleic acid detection is rapid and specificity of the amplification is confirmed in a closed system by melting curve analysis. Although, we could argue that all diagnostic laboratories in industrialized and in developing countries do not have thermocyclers or real-time PCR capabilities and that these systems are not cost effective, it is difficult to imagine that such systems will not be available everywhere in the near future. But until such diagnostic systems are widely disseminated, clinical examination, serology, and/or dark field microscopy, and in some cases PCR detection, will continue to be the cornerstone of GUD diagnosis.

References:

1. Burstain, J. M., E. Grimprel, S. A. Lukehart, M. V. Norgard, and J. D. Radolf. 1991. Sensitive detection of Treponema pallidum by using the polymerase chain reaction. J. Clin. Microbiol. 29:62-69.

2. Hay, P. E., J. R. Clarke, R. A. Strugnell, D. Taylor-Robinson, and D. Goldmeier. 1990. Use of polymerase chain reaction to detect DNA sequences specific to pathogenic treponemes in cerebrospinal fluid. FEMS Microbiol. Lett. 68:233-238.

3. Noordhoek, G. T., E. C. Wolters, M. E. J. de Jonge, and J. D. A. van Embden. 1991. Detection by polymerase chain reaction of Treponema pallidum DNA in cerebrospinal fluid from neurosyphilis patients before and after antibiotic treatment. J. Clin. Microbiol. 29:1976-1984.

4. Orle, K. A., C. A. Gates, D. H. Martin, B. A. Body, and J. B. Weiss. 1996. Simultaneous PCR detection of Haemophilus ducreyi, Treponema pallidum, and herpes simplex virus types 1 and 2 from genital ulcers. J. Clin. Microbiol. 34:49-54

5. Centurion-Lara A., Castro C., Shaffer J.M., Van Voorhis W.C., Marra C.M., and Lukehart S.A.Detection of Treponema pallidum by a sensitive reverse transcriptase PCR. J Clin Microbiol. 1997. 35:1348-52

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