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50 © 2013 The Authors | DDG © Blackwell Verlag GmbH, Berlin | JDDG | 11 (Suppl. 4), 50–58

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 250–400 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


51© 2013 The Authors | DDG © Blackwell Verlag GmbH, Berlin | JDDG | 11 (Suppl. 4), 50–58

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 31–77 %, and thus even lower than for immunohistochemistry (71–95 %) [ 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: 67–71 % ACA: 50–71 % BL: 68 %

Treponema pallidum

Stage 1: 100 % Stage 2: 42–100 % Stage 3: 14 %

Stage 1: 67 % * Stage 2: 71–95 % * Stage 3: 13 % *

Stage 2: WS 31–77 %

Mycobacterium tuberculosis Lupus vulgaris: 55–73 %

Mycobacterium leprae

55–61 % (AFB neg.) 100 % (AFB pos.)

Mycobacterium marinum

30–67 %

ZN: 13–31 % Culture: 3–30 %

Mycobacterium chelonae complex 60 %

ZN: 27 % Culture: 70–90 %

Cutaneous Leishmaniasis 93–100 % 51–69 % 14–50 %

* 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



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 67–71 % have been reported for erythema migrans (EM), 50–71 % for acrodermatitis chronica atro-phicans (ACA), and 68 % for borrelial lymphocytoma (BL) (syn. lymphadenosis cutis benigna) [ 7–9 ] . 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 detection of Borrelia DNA in ACA is considerably limited and inferior to se-rologic methods [ 10 ] . Rare manifestations are interstitial gra-nulomatous dermatitis (IGD), juxta-articular fi broid nodules, and morphea-like lesions (Figure 1 ). Borrelia-associated IGD histologically resembles interstitial anular granuloma and frequently shows so-called histiocytic pseudorosettes (Figure 2 ). In some cases of IGD, Borrelia burgdorferi DNA may only be identifi ed by means of a very sensitive PCR-ELISA procedure [ 12 ] . Immunohistochemical detection of Borrelia antigens using focus-fl oating microscopy (FFM) suggested an association of Borrelia species with anular granuloma, morphea, and lichen sclerosus et atrophicus [ 13, 14 ] . PCR-based studies by our group could not confi rm this association [ 15, 16 ] . Geographic differences in epidemiology and disease spectrum also play an essential part in Borrelia infections and may thus account for these inconsistent results [ 15 ] .

In summary, PCR displays a high sensitivity at the early pathogen-rich stage of borreliosis, yet only low sensitivity in advanced or late stages of the disease [ 10 ] . At any rate, mo-lecular biology fi ndings have to be correlated with serology and clinical presentation.

Mycobacterial infections comprise tuberculosis, aty-pical mycobacterial infections, and leprosy. PCR detection rates for M. tuberculosis using the target sequence IS6110

have been reported to be 73 %. Pathogen-scarce forms of cu-taneous tuberculosis like lupus vulgaris, commonly showing no acid-fast rods in Ziehl-Neelson staining, reveal DNA of M. tuberculosis in only 55 % of biopsies [ 17 ] . In pathogen-rich forms like scrofuloderma, PCR primarily serves to con-fi rm the diagnosis. Its high specifi city, however, also enables differentiation against infections by atypical mycobacteria that may show identical histologic fi ndings [ 18 ] .

In leprosy, PCR is performed both to confi rm the diagnosis and for therapeutic follow-up [ 19 ] . The sensitivi-ty of PCR detection for M. leprae in biopsies that did not reveal any acid-fast rods by Fite-Faraco staining is about 56–61 %. There is no signifi cant difference between fresh and

Figure 1 Juxta-articular nodule in borreliosis. Detection of Borrelia DNA in biopsy tissue by nested PCR.

Figure 2 Interstitial granulomatous dermatitis in borreliosis: infiltrates with histiocytic pseudorosettes around collagen bundles.



Figure 4 Mycobacterium chelonae infection with ulcerations in a patient with giant cell arteritis (temporal arteritis) treated with interleukin 6 and systemic steroids.

formalin-fi xed tissue [ 20 ] . Atypical mycobacterioses exhibit a large clinical as well as histologic spectrum. The histology of M. marinum is characterized by granulomas, pseudoepithe-liomatous hyperplasia of the epidermis, and frequently ulcera-tion (Figure 3 a, b). Acid-fast rods can be detected in 13–31 % of biopsies. Pathogen cultures are successful in only 3–30 % of cases, which is why its identifi cation by PCR is of great diagnostic value. Here, detection rates of 30–67 % have been reported [ 21 ] . Cutaneous and subcutaneous infections with M. abscessus and M. chelonae complex often occur in immunodefi cient patients. Histology is marked by nodu-lar or diffuse granulomatous infi ltrates and sometimes also abscesses [ 22, 23 ] (Figure 4 ). Applying Ziehl-Neelsen stai-ning, acid-fast rods were only found in 27 % of cutaneous infections with M. abscessus [ 22 ] . PCR-based identifi cation of

M. abscessus and M. chelonae complex shows a sensitivity of 60 % and is thus slightly lower than detection rates of 70–90 % for tissue cultures [ 21, 22, 24 ] . Although tissue cultures take 2–8 weeks, both procedures are commonly employed in the diagnosis of atypical mycobacteria, as cultures also facilitate additional resistance testing.

Parasites

Leishmaniasis is a vector-borne infection characterized by cutaneous, mucocutaneous, and visceral involvement. It is caused by strictly intracellular parasites of the genus Leishmania, which are generally prevalent in the tropics, Asia, East Africa, and the Mediterranean region. Travel habits, however, have spread the disease to other parts of the world as well. Cutaneous leishmaniasis features localized papules that usually progress to ulcers. L. tropica major , L. tropica minor , L. tropica infantum , and L. tropica aethiopica as well as L. mexicana and brasiliensis are known pathogens

Figure 3 Atypical mycobacteriosis with Mycobacterium marinum : dense dermal, granulomatous and lymphocytic infiltrates (a) with detection of occasional acid-fast rods in Ziehl-Neelsen staining (b). Identification of M. marinum by nested PCR and sequencing.



of cutaneous leishmaniasis. The two latter species are also found in the mucocutaneous variant. The various Leishma-nia species also differ in their respective therapeutic respon-se. The sensitivity for the detection of amastigotes in biopsies is approximately 14–50 % in the cutanteous form and even lower in the mucocutaneous variant [ 25 ] . Immunohistoche-mical identifi cation reveals a higher sensitivity of 51 to 69 %, but does not enable exact pathogen specifi cation [ 26, 27 ] . Most PCR protocols amplify small subunit ribosomal DNA (SSU-rDNA). Sequencing of amplifi ed regions or use of po-lymorphism-specifi c PCR protocols allow for classifi cation on the species and subspecies level, which is essential for selecting the appropriate therapy [ 28, 29 ] . In most studies, PCR-based pathogen detection has shown a high sensitivity (90–98 %) and specifi city (93–100 %) [ 27, 30 ] . The highest sensitivity may be attained by combined use of PCR-based and immunohistochemical procedures [ 27 ] .

Viruses

Not only does the identifi cation of viral sequences play a role in the diagnosis of neoplasms (Merkel cell carcinoma, Kaposi sarcoma), but also in infectious disorders (among them milker’s nodule, herpesvirus infections, parvovirus B19 infections).

Human papilloma viruses (HPV) (family: papovavi-ruses) are ubiquitous non-enveloped DNA viruses. Various phylogenetic groups may be distinguished among the more than 150 known HPV types. Group A HPV types, occurring on genitals and mucous membranes, and cutaneous group B beta HPV, also associated with epidermodysplasia verruci-formis (EV), are of greatest signifi cance to dermatologists. The diagnosis of HPV-associated benign and malignant epi-thelial lesions is predominantly based on clinical features and histology. With only few exceptions (HPV type 4 and 63 with fi lamentous inclusion bodies), histology does not grant exact specifi cation of the HPV type involved. Immunohistochemi-cal detection of the HPV L1 capsid antigen is indicative of replicative HPV infections. This method does not facilitate identifi cation of the virus type involved, either. HPV anti-gens are detected in 64 % of acanthopapillomas with koilo-cytes, but not in any lesions without histologic evidence for koilocytes [ 31 ] , thus indicating that immunohistochemistry is only of limited value. Showing a sensitivity of 87 %, in situ hybridization allows for type-specifi c identifi cation in genital acanthopapillomas with histologic evidence for HPV infection [ 32 ] . PCR-based methods display a slightly higher sensitivity. Besides type-specifi c protocols, multiplex PCR and consensus PCR may also be employed, the latter enab-ling DNA amplifi cation of multiple HPV types from a single group (A or B). The use of nested PCR applying MY and GP primers has been described for the detection of genital and

mucosal HPV types [ 33 ] . After consensus PCR, HPV typing is achieved by either sequencing PCR products, restriction fragment length polymorphisms (RFLP), or by hybridization of PCR products with type specifi c probes. With respect to cutaneous and EV-associated HPV types, various consensus primer-based PCR protocols have been depicted, which vary in sensitivity and their spectrum of HPV types detected. With these procedures, beta/EV-HPV types were identifi ed in 28–77 % of malignant non-melanocytic skin tumors [ 34 ] . None of the Consensus PCR protocol allow detection of all HPV types. It is, however, important to keep in mind that the mere detection of HPV DNA does not result in a defi nitive diagno-sis, as the same HPV type may be found in various disorders. Moreover, using PCR, DNA of beta/EV-HPV may be detec-ted in up to 60 % of healthy skin samples and epilated hairs [ 35 ] . Detection of HPV DNA is not suited for therapeutic fol-low-up, either, since HPV DNA may persist after resolution of clinical manifestations [ 36 ] . Clinical features and histologic fi ndings therefore always have to be taken into account when interpreting results obtained from PCR-based HPV detection.

In 2008, the Merkel cell polyomavirus (MCPyV) was fi rst discovered in Merkel cell carcinomas (MCC) by means of digital transcriptome subtraction. It belongs to the human polyomavirus group and has been identifi ed in 60–80 % of MCC worldwide [ 37 ] , featuring clonal integration of viral DNA into the host genome. PCR-based detection of MCPyV serves as useful diagnostic marker in differentiating MCC from histologically similar tumors, especially metastases of other neuroendocrine tumors [ 38 ] . Several studies have shown that MCPyV DNA may be identifi ed by PCR in up to 70 % of non-melanocytic cutaneous neoplasms in immu-nocompetent and immunosuppressed patients. However, the spectrum of MCPyV-positive tumors and respective de-tection rates vary greatly [ 37, 39, 40 ] . Unlike MCC, though, these skin tumors exhibit no immunohistochemical evidence for intracellular viral antigens. Infl ammatory cells may have transported viral DNA into peritumoral infi ltrates as sugge-sted by the fact that infl ammatory CD14 + CD16 − monocytes constitute a reservoir for MCPyV [ 41 ] . When interpreting MCPyV detection, it is therefore essential to consider mole-cular biology and immunohistochemical results.

The Epstein-Barr virus (EBV) and the human herpesvirus type 8 (HHV-8) are representatives of oncogenic gamma her-pesviruses. HHV-8 has been identifi ed as the triggering agent in Kaposi sarcoma, in some cases of multicentric Castleman disease, and in plasmablastic lymphomas in immunosup-pressed patients. Unlike EBV, detection of viral DNA and RNA may be used as diagnostic marker, since HHV-8 DNA is almost exclusively present in the aforementioned disor-ders [ 42 ] . Nevertheless, additional immunohistochemical detection of latent nuclear antigen 1 (LANA) of HHV-8 is desirable. As immunohistochemical procedures show a high



Figure 6 Herpes incognito: Superficial and deep lymphocytic infiltrates (a, b) with detection of varizella zoster virus DNA by nested PCR in P1 and P2 (c). Figure legend 6 c: P1 to P5: Skin biopsies, NK: negative control, PK: positive control (224 bp).

sensitivity and specifi city and correlate very well with mole-cular biology fi ndings, they have largely replaced PCR-based methods in the diagnosis of Kaposi sarcoma [ 43 ] .

EBV displays a very high seroprevalence and is characte-rized by its latent persistence in immune cells. The mere de-tection of EBV DNA therefore lacks diagnostic relevance. By identifying EBV-associated RNA transcripts (EBER), in situ hybridization facilitates viral detection with high sensitivity and specifi city. Furthermore, it allows for the visualization of viral RNA in the nucleus of tumor cells. The expression of EBV antigens, particularly the latent membrane protein 1 (LMP 1) and nuclear antigens (EBNA 1 & 2), differs as a function of latency pattern [ 44 ] .

Infections with the alpha herpesviruses herpes simplex virus (HSV) and varicella zoster virus (VZV) show patho-gnomonic histologic fi ndings with ballooning degeneration and syncytial cell formation of keratinocytes. Chronic alpha herpesvirus infections, primarily seen in immunosuppressed patients, may present with lichenoid, verrucous, or plasma cell-rich dense infi ltrates lacking epithelial changes typical for alpha herpesviruses. Apart from necroses of the follicu-lar epithelium (Figure 5 ), adnexotropic superfi cial and deep lymphocytic, partly pseudolymphomatous infi ltrates, edema, and partly atypical lymphocytes may all be indicative of an alpha herpesvirus infection. So-called herpes incognito is fre-quently a manifestation of VZV infection [ 45 ] (Figure 6 a–c). In these cases, detection of viral DNA by PCR and integrati-on of clinical and pathologic fi ndings are of vital diagnostic and therapeutic signifi cance [ 46 ] . Infections with the human herpesviruses type 6 and type 7, along with cytomegaly virus (CMV) all beta herpesviruses, are very common and

show a high seroprevalence. Exanthema subitum is caused by HHV-6. Reactivation of viral replication may be seen in drug reactions, e. g. DRESS syndrome. HHV-7 has been pathogenetically linked to pityriasis rosea, but available data

Figure 5 Necrotizing folliculitis in disseminated zoster.



are controversial [ 47 ] . As infl ammatory cells (lymphocytes, monocytes) present a reservoir for latent HHV-6 and HHV-7 infections and DNA of both viruses may be found in normal skin, PCR-based viral DNA detection has to be interpreted with caution. In order to diagnose a replicative infection, immunohistochemical or electron microscopic virus identi-fi cation is required.

The cowpox virus as pathogenic agent of milker’s no-dules shows typical epithelial changes. Detection of cowpox virus DNA by PCR frequently merely confi rms the clinical diagnosis, but may serve as diagnostic alternative to electron microscopy in atypical manifestations [ 48 ] .

Fungi

Molecular biology procedures for the detection of fungi are more time-consuming and costlier than special stains and direct microscopy, but also facilitate swifter pathogen identifi cation. Thus, PCR-based fungi detection presents an interesting and much faster alternative to fungal cultures. Multiplex and con-sensus PCR methods are used for the identifi cation of ribosomal DNA (18S or ITS rDNA), since ribosomal DNA is present in large amounts and shows highly conserved sequences [ 49 ] . The use of PCR-based techniques is particularly useful in immuno-suppressed patients, in whom rare fungal species may lead to infections with subsequent systemic dissemination (Figure 7 ).

Summary

Detection of pathogens using molecular biology techniques like PCR-based procedures on formalin-fi xed tissue has

become an integral part of modern dermatopathologic dia-gnostics. When interpreting molecular biology fi ndings, the peculiarities of each pathogen have to be taken into account. This especially includes their epidemiology, the type of in-fection (replicative-productive vs. latent), their cellular reser-voirs, and the expected number of pathogens dependent on disease duration. High sensitivity and specifi city are promi-nent advantages of PCR-based methods. On the other hand, the amount of time and money required as well as the risk for contamination with subsequent false-positive fi ndings are obvious disadvantages when compared to immunohistoche-mistry or in situ hybridization. Especially when dealing with pathogens prone to causing latent infections in infl ammatory cells, direct visualization of the pathogen is desirable in addi-tion to molecular biology techniques. Correlating molecular biology fi ndings with clinical and histologic results is pivotal.

Acknowledgements

We would like to thank Dr. med. N. Hilty, dermatologist, Schaan Furstentum Liechtenstein, for fi gure 1, Mrs. Dr. med. D. Jakob-Spasojevic, internist and rheumatologist, Bad Ragaz, Switzerland for fi gure 4, and Prof. Dr. St. Lautenschlager, department for dermatology at the Triemli Hospital, Zurich, Switzerland, for consulting us with regard to the biopsy in fi gure 7.

Conflicts of Interest

The authors have no confl icts of interests to declare.

Correspondence to

Prof. Dr. med. Werner KempfKempf und PfaltzHistologische DiagnostikSeminarstrasse 18042 Zürich, Switzerland

Dermatologische KlinikUniversitätsspital Zürich8091 Zürich, Switzerland

E-Mail: [email protected]

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