Molecular techniques can be helpful in the diagnosis of atypical herpes virus infections. Conventional microscopic findings in these cases may not be specific and immunohistochemical studies depend on the presence of infected cells in the sections examined.

ISH techniques have been used for the identification of varicella zoster virus in FFPE tissue [28]. Routine PCR-based methods performed on biopsies [29] and swab samples are being increasingly used to detect herpes virus DNA [30]. A proposed algorithm is to use immunofluorescent tests—which are cheaper and faster—at first screening and then run PCR on negative samples [31]. The use of internal controls is emphasized in order to rule out false negative results [32].

Multiplex reverse transcriptase PCR (RT-PCR) has been applied to detect herpesvirus-derived transcripts, [33] even in dried crusts or scales [34]. This method has the advantage of detecting nuclear RNAs or mRNAs, ensuring the pathogenic role of the virus in the disease [34].

Epstein–Barr virus (EBV, human herpesvirus 4) cutaneous infection is associated with multiple disorders, ranging from acute syndromes such as infectious mononucleosis, Gianotti–Crosti syndrome, and Kikuchi disease to chronic infections such as hydroa vacciniforme, hypersensitivity to mosquito bites, and oral hairy leukoplakia [35]. B-, T-, and NK-lymphoproliferative disorders and lymphomas are also associated with EBV infection.

Immunohistochemical studies for detection of EBV viral proteins (EBNA1, EBNA2, LMP1, LMP2, among others) are used in some instances. The interpretation of these stains should always be made in the context of the histological findings, since in some EBV-related conditions the expression of these proteins may be only focal or even undetectable by immunohistochemistry.

EBV-encoded RNAs (EBER1 and EBER2) are transcripts associated with latent infection that are expressed in very high levels in virtually all infected cells [36]. EBER ISH is regarded as the gold standard assay for defining a lesion as EBV-related [36] (Fig. 5.2). False positive interpretations have been related to nonspecific staining and cross-reactivity with other materials [37]. False negative results due to RNA degradation can be avoided by running a parallel internal control [38]. It is important to consider that rare conditions, such as oral hairy leukoplakia shows downregulation of EBER; [39] again, interpretation of the test should be done correlating the results with the histopathological findings.

PCR amplification of latent EBV transcripts using RNA extracted from crusts of hydroa vacciniforme patients has been reported [40]. qPCR can be applied to identify EBV DNA; [41] the quantification of EBV DNA using qPCR on biopsies has been used for determination of viral load [42]. Detection of EBV DNA by PCR methods can be advantageous over EBER ISH in cases in which there is poor quality RNA or the EBV infection lacks EBER transcripts [36].

EBV episomal DNA, which is passed from infected cells to their daughters, is used as a marker for tumor clonality. Southern blot assays of the viral terminal repeat fragment (unique for every infecting EBV virion) will show a single band in cases of malignant neoplasms, thus demonstrating a monoclonal proliferation [43].

Immunohistochemical studies using monoclonal antibodies directed against human herpesvirus 8 (HHV-8) latent nuclear antigen-1 have been demonstrated to be 99 % sensitive and 100 % specific for Kaposi sarcoma lesions [44, 45].

PCR assays are both highly sensitive and specific for the detection of HHV-8 and permit quantitation of viral load [46, 47]. One caveat in the interpretation of these molecular tests is the detection of HHV-8 DNA in skin lesions due to concurrent viremia and not due to viral replication within lesional cells [48]. Correlation with histological and immunohistochemical findings is needed for an adequate interpretation of molecular tests results .

Dermatophytoses are common superficial fungal infections limited to the keratinized integument, that is, the nail, stratum corneum of skin and hair. They are caused primarily by fungi of the genera Trichophyton, Epidermophyton, and Microsporum. Regardless of the causative species, dermatophytoses are characteristically either pruritic or scaly eruptions, although more protean and widespread presentations may be seen in the immunocompromised host. Whereas dermatophytes have low pathogenicity, persistent infection has adverse effects on the quality of life [104]. Furthermore, dermatophyte infection frequently results in a compromised skin barrier creating a portal of entry for other pathogens, with tinea pedis strongly associated with bacterial cellulitis of the lower limb [105]. It can be particularly difficult to identify the causative microorganism in nail fungal infections which are exquisitely recalcitrant to topical therapy. Traditional methods for detection include light microscopic analysis of wet preparations of involved tissue, such as nail clippings, incubated with potassium hydroxide to digest keratin, leaving the fungal chitinous wall intact. Artifacts are difficult to distinguish from fungal elements complicating interpretation, with a sensitivity between 83 and 88 % and specificity of 84–95 % in some studies [106, 107]. Another popular method is to expose nail clippings treated with KOH, or biopsy specimens stained with the PAS to the calcofluor white or blankophor fluorescent stains which bind to the fungal wall polysaccharide chitin, made of glucosamine residues, and enhance the contrast between fungal walls and artifactual debris. The reported sensitivity and specificity with calcofluor white with these methods ranges from 80 to 92 % and 72 to 95 % respectively [106–109]. A pitfall is that protozoa are also highlighted by calcofluor white, although morphology is helpful in distinguishing the two entities [110]. Culture remains the gold standard for fungal speciation. The problem with culture as a gold standard is the possibility of fungal contaminants during culture, fastidious and slow growing fungi resulting in false negatives (as high as 30 %), and delayed results following laborious culture protocols [111]. The correct identification of the causative microorganism in onychomycosis, for example, is of importance, as Trichophyton rubrum is susceptible to oral terbinafine, whereas Candida spp. are not. Fluconazole is efficacious for candidal onychomycosis and itraconazole is recommended for onychomycosis due to nondermatophyte molds such as Scytalidium spp. [112].

Deep fungal infections can be categorized as primary pathogens that are inherently virulent, and secondary or opportunistic pathogens that are pathogenic in the context of an immunocompromised host. Timely diagnosis is crucial and targeted therapy based on species identification may be life saving. Concomitant culture and culture independent assays, such as galactomannan and (1→3)-β-D-glucan antigen testing, of blood (unfortunately frequently negative in invasive fungal infection) and other accessible compartments in conjunction with biopsy of involved skin are key, with the goal being rapid detection and source control. For evaluation of cutaneous infection, whether primary or secondary, the conventional diagnostic approaches are tissue culture and histopathological examination. The same limitations identified in superficial fungal infections regarding sensitivity, specificity, interobserver variability, and lead time to diagnosis apply when evaluating deep fungal infections [118]. Successful identification on histopathology alone often depends on the abundance of organisms and the stage of infection. Additionally, a fungal organism may be visible on histopathology, but its identification may be limited if fresh tissue was not submitted for culture simultaneously. In cases where there was no isolation of organism on culture or there are significant discrepancies between the organism growing in culture and that seen on histopathology, doubts may rise about the certainty of the actual pathogen. Molecular techniques that can be evaluated in conjunction with skin biopsies are thus attractive .

A great example of the utility of molecular techniques in diagnosing infectious diseases applies to leishmaniasis, the third most common vector transmitted disease worldwide. When dealing with tegumentary leishmaniasis, one has to confront a variety of clinical presentations, some of them easily confused with various other processes, infectious or not. To construct a solid diagnosis of tegumentary leishmaniasis it is required to visualize the parasite, either by direct exam of a smear, isolation in culture or by conventional histology in biopsy material. Indirect techniques, such as the intradermal test or Montenegro reaction, are significant when dealing with patients that are foreign to the endemic areas, but not in natives. The interpretation of smear preparations requires expertise, something expected in ­laboratory workers of endemic areas but unlikely in other locations. This last situation is far from being exceptional due to the increasing number of cutaneous leishmaniasis seen in travelers, such as tourists or army personnel, thus the increasing importance of molecular methods for diagnosis.

The molecular technique most commonly used is PCR amplification of specific primers targeting either the kinetoplast or rRNA gene (Fig. 5.3), ITS, mini-exon genes, and specific gene sequences (e.g., glycoproteins, heat shock proteins, and cysteine proteinases) [135]. Multiple publications have corroborated the validity of the method for diagnostic purposes [136, 137]. The technique has been simplified to make it more accessible to the caregivers in primary care settings [138–141]. qPCR can provide additional information, such as parasitic load or determination of species, contributing to the treatment strategies as well as to epidemiology studies on this subject [142, 143]. As the response to medical treatment may vary depending on the species of Leishmania involved, the utility of molecular biology becomes more manifest when assisting such species identification [144, 145].

Filariasis is caused by nematodes (round worms) which parasitize humans, residing in the lymphatics and subcutaneous tissue with resultant lymphedema and cutaneous lesions. Over 120 million people worldwide are affected by filariasis which can be extremely disfiguring and disabling [146]. Early institution of diethylcarbamazine during the microfilarial phase can effectively eradicate the nematode Loa Loa, whereas treatment with diethylcarbazine can cause life threatening systemic collapse when the infecting nematode is onchocerca, an adverse reaction known as the Mazotti reaction [147]. Thus, speciation of the causative nematode in filariasis is key for administration of the correct treatment. Traditional diagnosis of nematodes depends heavily on morphology, specifically, by recognition of microfilaria on skin biopsies and thick blood smears. These methods are labor intensive, time consuming, and require experienced microscopists who can discern between the multiple species present in a region and between coinfecting nematodes in the same individual, making accuracy challenging [148, 149]. Antibody testing has its pitfalls, for example, an enzyme linked immunosorbent assay (ELISA) was unsuccessful in Brazil due to cross reactivity between Onchocerca volvulus and Mansonella ozzardi [150]. A nested PCR protocol tested on skin biopsies and whole blood samples holds promise. In this protocol, four PCR primers with partial sequences for 18S (small subunit rRNA), ITS-1, 5.8S and ITS-2 are used, thus amplifying the ITS of filarial species [151, 152]. Differentiation between species and detection of coinfection is possible as the ITS regions vary between species and thus yields amplicons of different sizes on gel electrophoresis, for example, O. volvulus

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