Molecular Diagnosis of Skin-Associated Infectious Agents FREE

Molecular pathology can be applied to 5 areas of diagnosis: (1) hematology and/or oncology, (2) solid tumors, (3) genetics, (4) pharmacogenetics, and (5) infectious diseases. Based on test volume, the molecular pathology application most used today is for the detection and characterization of infectious diseases, and we project this application to remain its strongest contribution for the next several years.

This review will focus on the methods used to detect common infectious agents of cutaneous and mucosal epithelium. Molecular methods used for detecting infectious agents have several advantages over classic microbiology approaches. (One exception is the use of molecular methods to detect parasites, which has not been well developed for clinical use.) Molecular methods are highly sensitive and therefore can detect minute amounts of infectious agents. Since these methods generally do not require growth in culture media, various bacteria, viruses, and fungi that are difficult or impossible to culture can be readily identified using the molecular approach (Table 1). In addition, the sensitivity of these methods can allow the analysis of nonviable infectious agents (ie, it permits the analysis of archived formalin-fixed tissue). The viral load and genotype of certain infectious agents can also be determined, which can facilitate the development of treatment protocols. The 2 approaches used to detect infectious agents are target-based amplification and signal-based amplification.

Target amplification is the most commonly used molecular method for diagnosis and is accomplished using several technologies. It increases the amount of the infectious agent's nucleic acid in a test tube by using an enzymatic in vitro replication step. Examples of target amplification techniques are the polymerase chain reaction (PCR), transcription-mediated amplification (TMA), nucleic acid sequence–based amplification (NASBA), and ligase chain reaction (LCR). All of these methods use a polymerase or ligase and short synthetic oligonucleotides known as primers. The primers specifically bind to complementary sequences found in the infectious agent. In most of the commercially available kits, an additional level of specificity is added to the assay with a third probe that is complementary to the amplified target sequence. The probe is used to detect the amplicon.

Figure 1 shows how these target amplification methods compare with one another. Note that TMA and NASBA applications use the same or similar temperatures. Thus these methods are referred to as isothermal applications. The most common nucleic acid targets for these techniques are ribosomal RNA (rRNA) sequences that exist in numbers exceeding 10 000 copies per organism. Targeting these nucleic acid sequences takes advantage of the naturally occurring biological amplification of these sequences.

The process begins with the synthetic manufacture of a customized primer with a binding sequence for an RNA polymerase and a region that is complementary to the target rRNA. The region of this primer that is complementary to the rRNA sequence on hybridization acts as a primer for generating a complementary DNA molecule using a reverse transcriptase enzyme. RNAse H activity degrades the rRNA target leaving the single strand of complementary DNA. The addition of a DNA polymerase and another complementary DNA primer generates the second strand. The final product results in a double-stranded DNA copy of the original rRNA sequence plus an RNA polymerase binding region. The addition of an RNA polymerase causes this double-stranded DNA to act as a template for the generation of numerous RNA molecules via transcription (hence the name transcription-mediated amplification). By targeting a high-copy rRNA molecule rather than a DNA sequence that is present at 1 to 2 copies per organism, every 10 000 rRNA molecules will be converted to a transcriptional active DNA template capable of producing numerous copies of RNA. Finally, a probe complementary to the amplicon is used to detect the amplified sequence. While rRNA sequences are most often targeted, this approach can be used for other RNA targets and, with minor adjustments, DNA targets as well.

In multithermal target amplification (specifically PCR), a high temperature is used to denature or "unzip" the double-stranded DNA target. Then primers complementary to 2 different regions of the target (known colloquially as forward and reverse primers) bind to their specific targets. This step is referred to as the annealing step and can vary depending on the ratio of guanine/cytosine to thymine/adenine bases in the regions complementary to the primer sequences. Since guanine forms 3 hydrogen bonds with cytosine vs the 2 hydrogen bonds formed between thymine and adenine, primers with high G-C ratios are more stable at higher annealing temperatures. Higher annealing temperatures also result in greater specificity. The last step in the PCR reaction takes place at the optimal temperature for the DNA polymerase so that elongation or extension can proceed from the primers. Since PCR requires multiple cycles through all 3 phases of the process, a thermostable enzyme capable of withstanding temperatures of 95°C and higher without becoming denatured or inactive is required. Polymerase chain reaction results in the logarithmic amplification of a target sequence.

The amplicon can be detected as a discrete band that appears after fractionation by gel electrophoresis following staining with dyes capable of intercalating or inserting themselves into the double-stranded DNA amplicon. An increasingly common method for detecting amplicons is to use either a fluorescent probe or fluorescent intercalating dye that fluoresces on binding to the amplicon. Rather than fractionating the product following PCR, the technician can monitor the number of PCR products in real time as the products are produced. In this way, fractionation or additional end point probe detection can be eliminated. This requires that fluoroflore excitation and emission filters be linked to the tube containing the PCR reaction so that the number of PCR products can be detected within a closed tube. When the data are processed using the appropriate software, they create a curve, as in Figure 1. Note that the number of PCR products increases with the number of cycles until a plateau is reached. The advantages of real-time PCR monitoring for clinical applications are (1) the closed tube system decreases the risk for contamination; (2) the turnaround time is decreased because a fractionating step is not necessary; and (3) quantitative data can also be obtained based on the number of cycles required to reach the plateau phase of the reaction.

The main difference between the PCR/LCR method and the TMA/NASBA method is that the PCR/LCR approach uses only 1 enzyme with multiple temperatures to denature the amplified target and allows logarithmic amplification. The TMA/NASBA method uses multiple enzymes and only 1 temperature. Thus, the term isothermal is applied to the TMA/NASBA method, from the Latin iso meaning the same or similar.

Numerous commercially available kits are available to detect common infectious agents. Roche Molecular Systems, Pleasanton, Calif, has PCR kits for detecting Chlamydia trachomatis and gonorrhea cocci, Mycobacterium tuberculosis, the human immunodeficiency virus (HIV), and the hepatitis C virus. Gen-Probe Inc, San Diego, Calif, also has commercially available TMA kits for the detection of GC/CT, M tuberculosis, Blastomyces dermatitidis, Histoplasma capsulatum, and Coccidioides immitis. Abbott Laboratories, Abbot Park, Ill, offers kits available for detecting these organisms by LCR.

Commercially available kits are not available to detect all infectious agents associated with cutaneous and mucosal tissues. For these less common agents, also known as esoteric agents, clinical laboratories must develop and validate in-house molecular assays. Borrowing terminology from the amateur beer maker's art, laboratory personnel refer to these tests informally as home brew tests. Home brew assays for esoteric testing most often use PCR technology, possibly because of the higher prevalence of PCR equipment and greater PCR experience among the personnel in clinical laboratories. However, another explanation for the predominance of PCR use could be the relative ease with which PCR allows the technicians to optimize this assay.¹ Using 1 enzyme rather than the 3 needed for isothermal amplification methods is obviously easier. In addition, the use of thermostable enzymes allows for a higher hybridization or annealing temperature for the primers than would be possible using isothermal enzymes. Higher annealing temperature results in greater specificity.² The amplification product can be visualized using relatively inexpensive agarose gel electrophoresis methods and DNA-intercalating dyes. Isothermal methods are more dependent on probe detection because of the need to design primers that bind at relatively low temperatures (42°C).

Several infectious agents are difficult to culture because of the need for specialized media and protracted incubation times. Some of these infectious agents (a subset of which are considered to cause emerging infections by the World Health Organization) that are associated with cutaneous manifestations are Mycobacterium species, Borrelia species, Bartonella species, Rickettsia species, Ehrlichia species, and other infectious agents.^{3 - 4} Polymerase chain reaction has been used for diagnosing mycobacterial infections such as Mycobacterium leprae (Hansen disease),^{5 - 7} M tuberculosis,⁸ and Mycobacterium ulcerans infections associated with Buruli ulcers.⁹ It was initially used to identify M tuberculosis as the causal agent of a disseminated infection associated with cutaneous manifestations.¹⁰

While specificity is the ideal for definitively identifying infectious agents, PCR directed toward conserved regions of viral, fungal, or bacterial genomes allows the identification of novel, unculturable infectious entities. Because these PCR reactions are designed to be capable of amplifying and identifying groups of different bacteria, viruses, and fungi, sequencing techniques or probes are required to specifically identify these organisms. In bacteria, the DNA sequence that encodes the 16s ribosomal RNA has regions that are highly conserved and referred to as the ribosomal DNA (rDNA). The entire 1541–base pair (bp) sequence can be generated using 1 pair of PCR primers, and sequenced with 12 different sequencing primers using the Microseq Microbial Identification Systems (Applied Biosystems, Foster City, Calif). For routine identification, only 500 bases need be sequenced. With this approach, different bacterial strains or biotypes have been identified for Streptococcus species, Mycobacterium species, coryneform bacterial isolates,^{11 - 13} and other bacterial species.¹⁴

The rDNA sequence most amenable to identification by amplification and subsequent sequencing for fungi is the large subunit. Within this sequence are 12 domains; domain 2 is the largest at 200 to 500 bp. For routine identification, only domain 2 can be used with the Microseq system. Strain identification of Trichophyton rubrum and the sinus cavity–associated Schizophyllum commune has been assisted by the use of conserved PCR primers followed by sequencing.^{15 - 16} Using this approach, Candida dubliniensis, which is often incorrectly identified as Candida albicans, has been classified as an emerging yeast pathogen.¹⁷

Sequencing is not always required for the identification of fungal isolates. The information gained through sequencing can be adapted to a simpler approach. A line probe assay where unique probes are affixed to a solid support and hybridized with a labeled PCR product has been used to identify C albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Candida krusei, C dubliniensis, Cryptococccus neoformans, Aspergillus fumigatus, Aspergillus versicolor, Aspergillus nidulans, and Aspergillus flavus.¹⁸

Primers designed in this manner can detect more than 80 different types of human papillomaviruses (HPVs) .^{19 - 20} While most interest in HPV-associated cancers has been with the types associated with cervical dysplasia, identifying cutaneous types associated with epidermodysplasia verruciformis is useful to confirm the clinical diagnosis. In addition, this approach can be used to detect various herpesviruses associated with lymphomatoid papulosis.²¹

All amplification assays require the use of enzymes to facilitate the increase in the number of target molecules. Inhibitors are often identified in clinical specimens. Internal standards are necessary to rule out the presence of inhibitors that could lead to false-negative results.²² Another problem is the possibility of amplicon contamination. Since small amounts of amplicons can result in widespread contamination, clinical laboratories must implement numerous contamination control protocols such as dedicated pipettes; aerosol-resistant pipette tips; different work areas for master mix preparation, specimen extraction, and amplicon detection; and unidirectional work flow. Some commercial kits contain reagents that control contamination by making the amplicon sensitive to enzymatic degradation (Amp Erase, Roche Molecular Systems). While closed-tube systems such as real-time PCR decrease the risk for contamination by physically separating the amplified product from the environment, precautions must still be taken to avoid inadvertently contaminating the reagent preparation area and specimen extraction area with positive specimens or control material. Since target amplification methods require the use of short primers complementary to the organisms' genome for initiating the amplification process, mutations in the organisms' sequences can result in false-negative results. Inaccurate quantification can also result from variations in the sequences complementary to the assay primers, and has been demonstrated in studies with HIV.²³ Incorporation of erroneous bases during the in vitro replication process can hinder end point probe–based detection if the misincorporated base is introduced early (within the first 3 cycles) in the reaction. In this case, the resulting amplicon will not reflect the actual target sequence. If the error is present in the sequence complementary to the probe, the probe may not hybridize due to this mismatch.

Signal amplification is not prone to the problems of amplicon contamination because the target is not amplified. Signal amplification methods have been available in serology for many years and are characterized by increases in the capacity for detection of a target molecule rather than the molecule itself. Similar principles are used to detect various nucleic acid targets. In signal amplification, the target is immobilized using complementary capture probes. Ultimately, the hybrid molecule is detected using molecules labeled with alkaline phosphatase to generate either a colorimetric reaction or a chemiluminescence reaction, as seen in Figure 1B. Several commercial kits are available to detect various infectious agents associated with cutaneous diseases.

The hybrid capture kits from Digene Inc (Gaithersburg, Md) can be used to detect HPV and herpes simplex virus. Full-length RNA probes are used to detect the HPVs and to determine whether they are high risk or low risk in oncogenic potential. Full-length probes negate the potential for false-negative results since single-nucleotide polymorphisms do not inhibit hybridization. The hybrid that is formed consists of the DNA virus with the RNA probe. The hybrid is captured to the side of a well containing an antibody specific to the DNA/RNA hybrid. In this way, only the hybrid is retained. The hybrid is detected using another antibody specific to DNA/RNA molecules, but this molecule is conjugated to alkaline phosphatase. Following addition of a chemiluminescence substrate, the light units are measured using a luminometer. An advantage to this particular method is that specimens obtained using the AutoCyte PREP system (Tripath Imaging, Burlington, NC) during routine pelvic examination can be used later to identify infectious agents in specimens determined by cytopathologists to be suggestive or indeterminant.^{24 - 25} For example, many Papanicolaou smear findings are not clearly normal or dysplastic/neoplastic, but are designated ASCUS (atypical squamous cells of unknown significance). Without viral typing, the clinical significance of ASCUS is not clear. Using the hybrid capture assay, however, a reading of ASCUS in the absence of HPV DNA or with HPV DNA from a nononcogenic type would be viewed as benign. If oncogenic HPV DNA is found, however, the patient would be more likely treated and/or observed as if her cytopathologic results were dysplastic. Unfortunately, there are no universal kits adaptable for home brew assays using the Digene system. However, using currently available reagents and user-supplied probes, a home brew assay can be developed, as is illustrated by the development of an in-house parvovirus B19 test.²⁶

Bayer Diagnostics, Norwood, Mass, uses branch-chain DNA to detect HIV and hepatitis C virus using numerous subtype-specific probes to capture the target molecule (Figure 1B). The use of numerous subtype-specific probes avoids the occurrence of genetic variations that could result in false-negative findings or inaccurate quantification (as has occurred in various target amplification methods).²¹ Synthetic oligomers that are complementary to the target molecule and to an adaptamer are then hybridized to the captured target. Adaptamers are then hybridized to this bridging molecule, and these are extended in a branch formation using additional alkaline-conjugated oligomers. All of these reactions are carried out using different hybridization conditions to ensure appropriate branching reactions from the initial target. Ultimately, a substrate is added to generate a colorimetric reaction product. While Bayer does not have a kit specifically designed for detecting cutaneous infectious agents, it does have a QuantiGene kit that can be adapted by the user to develop home brew assays. The limiting factor for setting up home brew tests using this system is that the equipment for the various incubations is not readily available in most clinical laboratories.

In situ hybridization is the only method that allows the identification of infectious agents in the context of the tissue's morphologic characteristics. While both target and signal amplification methods can be used for in situ hybridization, signal amplification using the tyramide amplification method (DAKO Corporation, Carpinteria, Calif) is frequently cited. Epstein-Barr virus has been detected in subcutaneous panniculitis and in hypersensitivity reactions to mosquito bites, and has been specifically localized to the infiltrating lymphocytes.^{27 - 28} The use of in situ hybridization is also useful to definitively identify HPV-containing cells adjacent to Molluscum contagiosum bodies.²⁹ Localizing infectious agents to the site of inflammation provides pathologists with the context of the infection that no other method provides. Often infectious agents can be present as a contaminant but not associated with the actual infection. This problem is addressed by in situ hybridization.

In conclusion, molecular methods are increasingly being used to assist in the diagnosis of various cutaneous diseases associated with infectious agents. The use of these methods improves patient care by decreasing test result turnaround times (relative to culture or serologic testing) and confirming diagnoses based on clinical observations. In many cases, molecular techniques allow identification of organisms that are not culturable. Technology is rapidly advancing to decrease the time required for these assays as well as their costs. The range of molecular pathology will eventually extend beyond nucleic acid–based detection systems and evolve to use information derived from the proteome as well as provide information on optimizing treatment to eradicate these infectious agents.

Accepted for publication May 24, 2001.

Corresponding author and reprints: Stephen K. Tyring, MD, PhD, The University of Texas Medical Branch, Route 1070, UTMB Galveston, TX 77555.