Molecular diagnostics in infectious skin diseases

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  • 50 2013 The Authors | DDG Blackwell Verlag GmbH, Berlin | JDDG | 11 (Suppl. 4), 5058

    DOI: 10.1111/ddg.12069

    Molecular diagnostics in infectious skin diseases

    Summary

    The identification of pathogens is of vital importance for the adequate treatment of infections. Compared to classic detection procedures, molecular biology methods allow for swifter identification of pathogens with high sensitivity and specificity. In dermatopathology, PCR-based procedures are employed for the detection of bacte-ria (Borrelia, Treponema pallidum , mycobacteria), viruses (among others herpesvi-ruses, Merkel cell polyomavirus), fungi (dermatophytes, molds), and parasites (e.g., Leishmania). When interpreting molecular biology findings, the peculiarities of each pathogen have to be taken into account. This especially includes their epidemiology, the type of infection (replicative-productive vs. latent), their cellular reservoirs, and the expected number of pathogens dependent on disease duration. Correlating these findings with clinical and histologic results is pivotal. The present review discusses the significance of molecular biology in the diagnosis of infectious skin diseases. It descri-bes the indications, sensitivity, and limitations of such methods for the detection of pathogens in skin specimens compared to other detection techniques.

    Werner Kempf 1,2 , Michael J. Flaig 3 , Heinz Kutzner 4

    (1) Kempf and Pfaltz , Histologic Diagnostics , Zurich , Switzerland (2) Department of Dermatology , University of Zurich , Zurich , Switzerland (3) Department of Dermatology and Allergology , Ludwig-Maximilians University , Munich , Germany (4) Dermatopathology Friedrichshafen , Friedrichshafen , Germany .

    Introduction

    The identifi cation of pathogens is of vital importance for the adequate treatment of infections. In many cases, the classic detection of pathogens in tissue through special stains and pathogen cultures constitutes the diagnostic gold standard. The sensitivity of these methods, however, is limited, parti-cularly in infections with low pathogen numbers or the ina-bility to grow the pathogen in culture. Due to target sequence amplifi cation of infectious pathogens, polymerase chain re-action (PCR) and related procedures show a high sensitivity and specifi city. They may be performed in DNA and RNA extracts of fresh as well as formalin-fi xed tissue. Molecular biology is therefore pivotal in the diagnosis of infectious skin diseases (Table 1 ). In addition, real-time PCR allows for the quantifi cation of pathogen numbers within the tissue samp-le, thus enabling the differentiation between infection and contamination. Moreover, PCR-based procedures aid in the analysis of virulence and resistance factors.

    PCR-based methods facilitate swifter pathogen detec-tion than culturing. Their use is primarily limited by their comparatively high technical complexity and costs. Further-more, DNA and RNA sequence amplifi cation is subject to potential sample contamination in the lab, potentially resul-ting in false positive results. Adequate prevention measures have been described as so-called good molecular diagnostic practices (GMDP) [ 1 ] . As formalin fi xation results in frag-mentation of DNA and RNA, sequence lengths should not exceed 250400 bp. Sensitivity may be enhanced by nested PCR involving two consecutive amplifi cation protocols and particularly suited for infections with a low pathogen count.

    In dermatology and dermatophathology, PCR-based procedures have been employed in the detection of bacteria (Borrelia, Treponema pallidum , mycobacteria, Staphylococ-cus aureus , Pseudomonas aeruginosa ), viruses (herpesviru-ses, Merkel cell polyomavirus, human T-lymphotropic virus), fungi (dermatophytes, molds), and parasites (Leishmania). This review discusses the indications, sensitivity, and

  • Molecular diagnostics in infectious skin diseases

    51 2013 The Authors | DDG Blackwell Verlag GmbH, Berlin | JDDG | 11 (Suppl. 4), 5058

    limitations of molecular biology methods for the detection of pathogens in skin specimens compared to other detection techniques.

    Molecular biology detection of bacteria

    In dermatopathology, molecular biology methods are pri-marily used for the detection of S. aureus , Pseudomonas aeruginosa , mycobacteria, and spirochetes (Borrelia, T. pal-lidum ). All bacterial species feature 16S ribosomal DNA, which is not present in eukaryotes. Detection of bacterial DNA may therefore be performed using so-called eubacterial PCR with universal primers amplifying 16S ribosomal DNA. Subsequent sequencing enables assignment of the amplifi ed DNA to a certain bacterial species. Alternatively, species-spe-cifi c PCR protocols may be used. The mere detection of bac-terial DNA, however, does not allow for the differentiation between intact replicating bacteria and already dead bacteria or remnants thereof. DNA-based methods are therefore only suitable for follow-up, if the amplifi ed DNA sequences are quantifi ed by quantitative or real-time PCR. As the number of bacterial rRNA sequences is substantially greater than the amount of bacterial DNA, RNA-based detection methods present an alternative method, usually with higher sensitivity. PCR-based techniques are of particular signifi cance for the

    identifi cation of virulence and resistance factors in methicil-lin-resistant strains and also S. aureus featuring the Panton Valentine Leucocidin (PVL) gene. Due to their high virulence, these types are potentially associated with a more severe clinical course.

    Serology is the gold standard in the diagnosis of secondary and tertiary syphilis . At the primary stage (primary sore), antibodies may initially not be found and direct pathogen detection by dark-fi eld microscopy or PCR becomes crucial. Pathogens may also be identifi ed in biopsy tissue by immunohistochemical methods. At the secondary stage of the infection, characterized by broad clinical and histologic variety, serology presents the diagnostic gold stan-dard. Compared to serology, the sensitivity for pathogen detection by Warthin-Starry staining is 3177 %, and thus even lower than for immunohistochemistry (7195 %) [ 2 ] . Data on the sensitivity of PCR in stage 2 disease has been very heterogeneous and ranges from 42 % to 100 % [ 3, 4 ] . In a recent study, T. pallidum was immunohistochemically de-tected in 67 % (primary stage), 55 % (secondary stage), and 13 % (tertiary stage), whereas PCR-based analysis showed a higher sensitivity (primary stage: 100 %; secondary stage: 76 %; tertiary stage: 14 %) [ 5 ] . In comparison, focus-fl oating microscopy yielded higher detection rates of 100 % (primary stage), 97 % (secondary stage), and 87 % (tertiary stage).

    Table 1 Sensitivity for pathogen detection by polymerase chain reaction compared to immunohistochemistry, special stains and culture in bacterial and parasitic infections

    Pathogen PCR Immunohistochemistry Special stains and culture

    Borrelia burgdorferi sensu lato

    EM: 6771 % ACA: 5071 % BL: 68 %

    Treponema pallidum

    Stage 1: 100 % Stage 2: 42100 % Stage 3: 14 %

    Stage 1: 67 % * Stage 2: 7195 % * Stage 3: 13 % *

    Stage 2: WS 3177 %

    Mycobacterium tuberculosis Lupus vulgaris: 5573 %

    Mycobacterium leprae

    5561 % (AFB neg.) 100 % (AFB pos.)

    Mycobacterium marinum

    3067 %

    ZN: 1331 % Culture: 330 %

    Mycobacterium chelonae complex 60 %

    ZN: 27 % Culture: 7090 %

    Cutaneous Leishmaniasis 93100 % 5169 % 1450 %

    * Focus-floating microscopy shows a higher sensitivity. Abbr.: AFB: Detection of acid-fast rods by Ziehl-Neelsen stain; EM: Erythema migrans; ACA: Acrodermatitis chronica atrophicans; BL: Borrelial lymphocytoma; WS: Warthin-Starry stain; ZN: Ziehl-Neelsen stain

  • Molecular diagnostics in infectious skin diseases

    52 2013 The Authors | DDG Blackwell Verlag GmbH, Berlin | JDDG | 11 (Suppl. 4), 5058

    Spirochetes of the genus Treponema physiologically occur in the oral cavity as saprophytes or commensals. As immuno-histochemistry harbors the risk for false positive results due to cross-reactivity with commensal spirochetes, PCR-based detection of T. pallidum -specifi c DNA plays a crucial role when analyzing oral samples.

    Just like T. pallidum , Borrelia belongs to the class of spirochetes. The dermatologically relevant Borrelia burg-dorferi sensu lato comprises B. burgdorferi s. stricto , B. garinii , and B. afzelii . PCR protocols enable detection of Borrelia burgdorferi sensu lato DNA (e. g. p66 gene). Using formalin-fi xed paraffi n-embedded tissue, amplifi -cation sequences of 250 to 400 bp are recommended [ 6 ] . Following antibiotic therapy, Borrelia DNA can usually no longer be identifi ed in tissue, while serum IgG (and partly also IgM) antibodies remain detectable for quite some time. Detection rates of 6771 % have been reported for erythema migrans (EM), 5071 % for acrodermatitis chronica atro-phicans (ACA), and 68 % for borrelial lymphocytoma (BL) (syn. lymphadenosis cutis benigna) [ 79 ] . Serologic fi ndings may initially be negative in up to 26 % of patients with EM. Since plasma cells as potential disease indicators may histologically also be missing at fi rst [ 10, 11 ] , PCR presents an invaluable diagnostic tool for pathogen detection in EM, espe-cially in clinically and/or histologically ambiguous cases. The sensitivity of PCR may be enhanced by using unfi xed, frozen tissue or nested PCR [ 10 ] . Due to the low pathogen count in ACA, the sensitivity of cultures and PCR-based detect

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