key: cord-0036213-7wdcfe1d
authors: Ye, Fei; Cui, Miao; Khasawneh, Rame H.; Shibata, Robert; Wu, Josephine; Sharaan, Mona; Zhang, David Y.
title: Molecular Virology
date: 2012-12-28
journal: Molecular Genetic Pathology
DOI: 10.1007/978-1-4614-4800-6_25
sha: c8af978501073fa6082806873a4180fb6481948e
doc_id: 36213
cord_uid: 7wdcfe1d

This chapter describes common viral pathogens in humans with emphasis on the molecular diagnosis. Each section includes molecular characteristics, clinical presentation, and commonly used diagnostic methods (both conventional and molecular). Commercially available molecular diagnostic kits are preferentially described. Recommendations and guidelines (if available) for result interpretation and clinical approach are also included. For detailed methods, please refer to the chapter “Methodology and Instrumentation” in this book.

• Limitation and pitfalls -In the past decade, molecular methods for detection and quantification of virus infections have replaced many traditional viral culture and serological methods -The molecular tests significantly improved clinical turnaround time and reduced handson time in addition to increase the diagnostic sensitivity and specificity -Many commercial assay kits and automatic instruments are available which allow many clinical microbiology laboratories to offer molecular tests -However, the routine implementation of nucleic acid (both DNA and RNA) amplification and hybridization methodologies in clinical laboratories is still associated with a number of limitations • Increased cost/test due to expensive instrumentation and reagents • Amplification carryover contamination • Standardization of positive, negative, and quantitative controls • Integrated coamplified internal DNA control to demonstrate absence of polymerase chain reaction (PCR) inhibitors and amplification • Prevention of false-positive and falsenegative reports due to antigenic and pathogen nucleic acid sequence drift and accurate interpretation of data and software analyses • Specimens -Collection of adequate specimens is important for molecular diagnosis of virus. These are specimen types commonly used for molecular diagnosis -Whole blood: 3-5 mls collected in an EDTA (lavender top) tube. Store at 4-25 C. Do not freeze -Plasma: Collect 7-10 mls of whole blood in EDTA, ACD solution A, or PPT sterile tube. Store whole blood at room temperature (18-30 C) for no more than 4 h. Remove plasma from cells within 4 h of collection by centrifugation at 1,000 Â g for 10-15 min. Do not clarify by filtration or further centrifugation. Store plasma at -60 to -80 C within 30 min of separation. Plasma may also be stored at -20 C in nonfrost-free freezer for up to 72 h if colder freezer is not available. Ship on dry ice for overnight delivery. The minimum volume of specimen is 2 mls of plasma -Urine: first 10-20 mls of voided urine collected in a sterile urinalysis container (15 ml sterile screw cap tube preferred). Store at 4-25 C for less than 24 h or store at -70 C for long term -Bronchial lavage/tracheal aspirate: 1-4 mls, collected in a sterile tube. Store at 4-25 C for less than 24 h -Bone marrow: 1-2 mls, collected in EDTA tube. Store at 4-25 C. Do not freeze -Tissue: $0.5-cm tissue block collected in a sterile screw-top container, add small amount of saline to keep it moist. Avoid the use of viral transport media to avoid potential inhibition of PCR. Fresh tissues should be stored at -72 C immediately to preserve the nucleic acids • Paraffin-embedded tissue is acceptable. Usually 5-10 sections (5 mm thickness) are sufficient for PCR analysis. The tissue sections must be deparaffinized with xylene before DNA extraction -Fecal: sterile swab (plastic shaft only) or very small fecal sample placed in 1-2 mls sterile saline in a container with tight fitting lid. Do not use viral transport media to avoid potential inhibition of PCR -Swab: sterile swab (plastic shaft only) placed in 1-2 mls sterile saline. Do not use viral transport media to avoid potential inhibition of PCR -Cerebrospinal fluid (CSF): 1-1.5 mls fluid, submitted in a sterile, leakproof tube, store at 4-25 C for less than 24 h or store at -70 C for long term • Assay performance analysis -Analytical performance • Analytical sensitivity: to determine the lowest number of targets that can be detected by the assay • Cross-reactivity (specificity): to determine if the assay can produce falsepositive results in the presence of high concentration of other similar or unrelated pathogens (bacteria, yeast, and virus) • Linearity: to evaluate the log differences from the expected concentration; this difference should be within AE0.1 log (or a ratio of observed mean quantitation to expected concentration within 95%) • Quantitative range: the measured concentrations within the linear range with a good reproducibility -Clinical performance • Limits of detection: the lowest concentration of target nucleic acids that can be detected (at or above the detection cutoff in 95% of replicates, usually 10 replicates) • Detection cutoff: the point on the assay quantitation scale such that 95% of negative specimens produce results below this cutoff with 95% confidence • Limits of quantification: the lowest concentration of target nucleic acids that can be quantified in 95% of replicates • Reproducibility: The reproducibility of the test is usually established by testing three to six sample panels with known concentrations of target in triplicate or quadruplicate. A commercial panel should be used to establish this parameter, if available. Reproducibility is expressed as percent correlation coefficient. For quantitative assays, the CVs range from 10% to 50% • Precision: the reproducibility of a test result (e.g., inter-and intratechnologist and inter-and intra-assay) • Sensitivity: true positive samples, % of true positive samples above the limits of detection • Specificity: true negative samples, % of true negative samples below the limits of detection • Quality controls: For quantitative assay, additional quality control procedures should be performed, including calibration and calibration verification -Calibration is the set of operations that establish, under specified conditions, the relationship between reagent system/ instrument response and the corresponding concentration/activity values of an analyte. Calibration procedures are typically specified by a method manufacturer, but may also be established by the laboratory -Calibration verification denotes the process of confirming that the current calibration settings remain valid for a method -Recalibration or calibration verification and analytical measurement range validation must be performed at least once every 6 months -For each run, sensitivity controls should be included in addition to positive and negative controls. It is recommended that two levels of controls (high and low) should be included

• Human immunodeficiency virus (HIV) is an RNA retrovirus belonging to the lentivirus family. HIV1 and HIV2 are genetically different; HIV2 shares 40% nucleotide homology with HIV1. HIV2 is more related to SIV than to HIV1. Both types appear to cause clinically indistinguishable AIDS. However, it seems that HIV2 is less easily transmitted, and the period between initial infection and illness is longer in the case of HIV2. Worldwide, the predominant virus is HIV1, and generally, when people refer to HIV without specifying the type of virus, they will be referring to HIV1. The relatively uncommon HIV2 is concentrated in West Africa and is rarely found elsewhere • Structure of HIV virion ( Fig. 25.1) -HIV virus consists of a spherical viral particle encased in a lipid bilayer derived from host cell covered by protruding peg-like structures composed of gp41 and gp120 glycoproteins -The virus core nucleocapsid contains the major capsid protein, p24; two copies of genomic RNA; and three viral enzymes (protease, reverse transcriptase, and integrase) • Viral replication ( Fig. 25 .2) -The first step of infection is entry into the host cell, which requires binding of the gp120 molecule on the virus to CD4 molecules on the host cell's surface, and is mediated by the gp41 molecule. Two surface molecules CCR5 and CXCR4, chemokine receptors for beta-chemokines and alpha-chemokines are also required for entry -Once bound, the viral envelope fuses with the cell membrane and the virus' RNA and enzymes enter the cytoplasm -Reverse transcriptase catalyzes, first, the synthesis of a DNA copy of the viral RNA and, second, the synthesis of a second DNA strand complementary to the first one. Therefore, a double-stranded DNA (dsDNA) is generated -Integrase then facilitates the integration of viral DNA into the cellular chromosome when the cell divides and provides latency enabling the virus to effectively evade host responses -Transcription of the DNA results in the production of RNA. This RNA can serve as the genome for new viruses and can be translated to produce viral proteins. Viral proteins are facilitated by protease and assembled into viral particles using the host cell's protein-making machinery -Complete HIV particles are assembled.

In macrophages, HIV buds out of the cell without rupturing the cell, and the cycle begins again. In T cells, HIV exits the cell by rupturing it, effectively killing the cell • The gag, pol, and env genes encode for structural proteins for new virus particles.

The other six genes, tat, rev, nef, vif, vpr, and vpu, regulate the synthesis and assembly of viral particles • The phylogenic analysis of the nucleotide sequences of the env gene has enabled classification of HIV1 into three groups: M (major), N (non-M), and O (outlier). The group M of HIV1 infection has been classified into nine different genetic subtypes A-K. More than 90% of HIV1 infections belong to HIV1 group M. Subtype/class B is the most prevalent in the developed world • HIV is transmitted via sexual contact, blood (via transfusion, blood products, or contaminated needles), or passage from mother to child (in utero, during birth, or ingestion of breast milk). Although saliva can contain small quantities of the virus, the virus cannot be spread by kissing. HIV is not spread by the fecal-oral route, aerosols, insects, or casual contact

• HIV is the causative agent of acquired immunodeficiency syndrome (AIDS), the leading cause of death in humans between the ages of 25-44 years • Two main targets of HIV: immune system and central nervous system. HIV targets CD4+ T cells, monocytes/macrophages, and Langerhans cells/dendritic cells causing severe immunosuppression and neuropathologic symptoms such as dementia, meningitis, and encephalopathy in the host • Common opportunistic infections: Pneumocystis carinii, candidiasis, tuberculosis, Cryptococcus, cytomegaloretinitis • Common malignancies: Kaposi sarcoma, lymphoma (non-Hodgkin and brain primary), and uterine carcinoma • Individuals who have HIV face a long challenging road. The disease has a steady natural history, starting with an asymptomatic state and progressing toward AIDS. Natural history includes three phases -Early-stage HIV infection is defined as the presence of HIV with a CD4 count greater than 500. Early stage develops 3-6 weeks after initial exposure with self-limited flulike symptoms resolving 2-4 weeks later in 50-60% of patients by high level of viral production, viremia, and widespread seeding of lymphoid tissues -Chronic phase HIV infection occurs when the CD4 count is between 200 and 500. Chronic phase is associated with a period of latency in which the immune system is intact, but there is continuous HIV replication that may last for years. Patients are either asymptomatic or develop persistent lymphadenopathy with minor opportunistic infections, such as candidiasis or herpes zoster -When the CD4 count drops below 200, the HIV infection has entered the crisis phase. This is when certain infections that are easily handled by an intact immune system take advantage of this immunocompromised state (opportunistic infections). Certain cancers may also appear for the same reason. When a patient has a CD4 count less than 200 and at least one opportunistic infection or cancer specifically seen in crisis phase HIV, he or she is officially designated as having AIDS

• Specimens -whole blood, serum, and plasma (Table 25 .1) • Conventional tests and problems -Lymphocyte count • D4 cells (also called T cells or T-helper cells) are the primary targets of the HIV virus. Quantitation of CD4 cells was the first effective predictor of HIV progression. The CD4 count is one of many factors (including clinical status, HIV viral load, and medication adherence) that should be assessed before starting or changing antiretroviral (ARV) treatment • The CD4 cell count (<200 cells/mm 3 ) is important in determining the staging of HIV disease and for indicating the need for prophylaxis against opportunistic pathogens • Most laboratories report the CD4 count as part of a list of several types of lymphocytes, as both an absolute count and a relative percentage. Measurement and trending of CD4 percentage in addition to absolute count must be performed prior to initiation or adjustment of ARV treatment management decisions • The CD4 percentage sometimes is used in coordination with the absolute value to assess the significance of changes in the absolute CD4 count. The absolute CD4 count can fluctuate as overall lymphocyte counts vary, but the CD4 percentage often remains stable during insignificant CD4 fluctuations. CD8 cell (or cytotoxic T cell) counts do not appear to predict clinical outcomes • For monitoring purposes, the CD4 count should be repeated approximately every 3-4 months both in stable untreated patients and in patients on stable ART. The CD4 count should be checked more frequently according to the clinical situation -Viral culture • Although very specific, single positive culture must be confirmed with a second specimen • Rarely used due to high cost, laborintensive, and less sensitivity than antibody testing • Negative culture may be caused by technical problems, a defective virus, or the inability of the virus to replicate in culture -Serological studies • p24 antigen -Early developed assay to detect HIV infection and screen donated blood for HIV -Advantage is to detect HIV infection prior to development of antibodies -Disadvantage is limited utility due to the short window of time and should only be used when other tests are unavailable Whole blood should be stored at 2-25 C for no longer than 6 h. Plasma must be separated within 6 h of collection by centrifugation at 800-1,600x g for 20 min at room temperature and transferred to a polypropylene tube to prevent viral degradation Plasma may be stored at 2-8 C for up to 5 days or frozen at -70 C Specimens should be stored in 600-700 ul aliquots in sterile, 2 ml polypropylene tubes. Freeze-thaw studies have shown that specimens may be tested for up to three freeze-thaw cycles without loss of viral RNA HIV genotyping Plasma specimens anticoagulated with EDTA. Specimens must not be anticoagulated with heparin Whole blood should be stored at 2-25 C for no longer than 2 h. Plasma should be separated within 30 mins, but no later than 120 min by centrifugation at 1,000-2,000x g for 15 min at 15-25 C and transferred to a polypropylene tube Plasma may be stored frozen at -65-80 C for up to 6 months Samples may be tested up to two freeze-thaw cycles. Plasma specimens containing the following have been shown to interfere with results: lipids up to 30 mg/ml bilirubin up to 0.6 mg/ml hemoglobin up to 5 mg/ml • Antibody screening assays (qualitative) -Detection of antibodies to HIV is the most common way to diagnose HIV infection in adults and children >18 months old -These antibodies are usually detectable within 3-6 weeks after infection -Most individuals seroconvert by 12 weeks, although may not be detectable for months or years -The window period is the time between infection and the development of antibodies. When a person is infected with HIV, it takes a few weeks for the body to make antibodies to the virus. Most people develop antibodies within a month of infection, although some people can take up to 3 months. In very rare cases, it can take 6 months of the test to be positive. In general, repeat testing for HIV should occur at 3 months if there is a significant concern of recent HIV infection. Additionally, because the level of virus in blood is high during the window period, people can more easily transmit HIV -Serologic HIV antibody screening testing is highly sensitive (ELISA, rapid test, or home test), but requires followup of preliminary positive specimens with a highly specific HIV antibody confirmatory assay (Western blot) ( Fig. 25 .3) -ELISA method is most common and earliest developed antibody screening assay -Home Access HIV1 test system analyzes a dried-blood spot from finger stick collected on filter paper at home and sent to a testing facility -Rapid tests for HIV are assays that detect antibodies to HIV within minutes. The rapid test is highly specific: negative means negative except during window period; and the test is also highly sensitive:

positive means most likely has HIV, but must be confirmed using Western blot for HIV diagnosis • Confirmatory antibody assays: Western blot -Gold standard for HIV diagnostic testing -The virus is disrupted, and the individual proteins are separated by molecular weight via differential migration on a polyacrylamide gel and blotted onto a membrane support. HIV serum antibodies from the patient are allowed to bind to the proteins in the membrane support, and patterns of reactivity can be visibly read -Detects three major proteins/viral bands: p24 core protein and two envelope proteins, gp41 and gp120/160 -Reactive WB demonstrates antibody to two of the three major bands; nonreactive WB will have no detectable viral bands ( Fig. 25 .4) -Repeated reactivity by ELISA and reactivity by the confirmatory assay are reported as positive for antibody to HIV1 -Nonreactive specimens by ELISA or repeatedly reactive by ELISA and nonreactive by the confirmatory assay are negative for antibody to HIV1 -WB in which serum antibodies bind to any other combination of viral bands is considered indeterminate; followup blood specimen should be obtained 1 month later for repeat HIV antibody testing -Individuals with repeat indeterminate results should undergo further testing using molecular assays, such as PCR -At least as sensitive as and more specific than screening assays, although they are not as sensitive in the detection of early seroconversion -Disadvantages: more laborintensive, more prone to subjective interpretation, and more costly than screening assays • Alternative antibody screening assay (qualitative) -US Food and Drug Administration (FDA) has approved assays that test body fluids other than blood to detect HIV1 antibodies, although sensitivity and specificity are less reliable -Utilizes same testing algorithm as serum (ELISA followed by WB) Serum samples that do not produce a reaction in the ELISA are considered negative.

Report absence of HIV antibodies.

The patient is not HIV infected.

Retest in 6 months after the most recent risk event. transcriptase regions of the HIV1 genome using RT-PCR -The amplified DNA is then sequenced to yield to the nucleotide profile of the virus using a sequencing gel -Once the sequence has been generated, it is compared to the wild-type HIV1 sequence and any differences that confer drug resistance are highlighted • Another FDA-approved assay for DNA sequencing is ViroSeq™ HIV1 genotyping system, Celera Diagnostics, Alameda, CA (distributed by Abbott Laboratories, Abbott Park, Il) -It is a two-step procedure which first amplifies the protease and reverse transcriptase regions of the HIV1 genome using RT-PCR and cycling sequencing -The amplified DNA is then sequenced to yield to the nucleotide profile of the virus using a capillary electrophoresis -The minimum input of viral RNA to the assay should be 1,000 copies/ml when using 1 ml of plasma to be successful in genotyping • Pitfalls of genotyping -Genotypic variants comprising less than 20-30% of the sample may not be detected as genotyping results reflect the predominate subtype -Interpretation of genotyping results is based on the HIV1 clade B, the most prevalent clade in the developed world. However, other subtypes and recombinants of HIV1 may be undetected -Assessing HIV1 resistance is complicated by the replication kinetics of resistant mutants. Resistant mutants are often less fit than wild-type virus and may become undetectable with selective drugs. Nevertheless, these mutants persist in the patient and when the selective drug pressure is reapplied, the mutants replicate and a resistant population quickly predominates

• Plasma HIV RNA is a surrogate marker of HIV disease progression that is used to guide and monitor therapy and management • ARV therapy should be implemented in patients with any of the following clinical findings: symptomatic HIV infection or AIDS-defining condition, CD4 count 350 cells/mm3 or viral load !100,000 copies/ml (pregnant mothers: !1,000 copies/ml) • The initial highly active antiretroviral therapy (HAART) goal in the ARV therapy-naïve patient should be to attain a viral load of <50 copies/mL and should include the rational sequencing of ARV agents to achieve the maximum possible viral replication suppression • In ARV treatment-naïve patients or patients who are on a successful treatment regimen, monitoring of viral loads should be measured at baseline, every 2-4 weeks after initiation and every 3-4 months once maximal suppression is attained, although patients with CD4 counts >500 cells/mm 3 may require less frequent viral load monitoring • Typically, in patients beginning therapy or in those changing therapy as a result of virologic failure, viral load measured 2-4 weeks after therapy initiation. A decrease by at least 1 log (10-fold) indicates effective therapy. Most patients reach the goal of <50 copies/mL within 6 months. An absent or incomplete response of the viral load to ARV therapy should raise concerns about poor patient adherence to therapy and/or viral resistance • If significant increase (3-fold increase or more) in viral load without clear explanation, viral load should be repeated to confirm virologic failure • Genotypic resistance testing should be performed prior to initiating treatment in ARV therapy-naïve patients and in patients with >1,000 copies/ml, or nonresponsive to ARV • Genotypic resistance testing is not recommended in patients with 500-1,000 copies/mL or less and has discontinued ARV therapy for more than 1 year

• Hepatitis C virus (HCV) was first recognized in 1974 as a non-A, non-B hepatitis virus (NANBH) and first identified in 1989 using molecular methods. HCV is the major cause of non-A, non-B hepatitis (91%) affecting about 3% of the world's population • The most common route of transmission is via blood and blood products, i.e., immune globulin, surgery, and intravenous drug abuse which has significantly reduced with the advent of routine blood screenings. Sexual transmission as well as vertically from mother to infant occurs; the rate of vertical transmission of HCV is 6% • HCV is a positive sense, single-stranded RNA virus that represents the third genus of the family Flaviviridae. The genome encodes for a single open reading frame coding structural (one core and two envelopes) proteins as well as a series of nonstructural proteins ( Fig. 25 .10) -5 0 untranslated region: most constant, used for HCV RNA assays and genotyping -Core region: constant, used in some genotype assays, core protein assay, PCR-RFLP, and RIBA tests -Envelope region: hypervariable region, associated with high rate of mutation in quasispecies -NS2 region: codes for protease -NS3 region: codes for protease/helicase, RIBA tests found in this region -NS4 region: c100p antigen used in anti-HCV, RIBA tests targeted this region -NS5a region: codes for interferon response element -NS5b region: codes for RNA polymerase, NS5 antigen used in anti-HCV, RIBA tests target this area • HCV consists of a heterogeneous group of genotypes based on the sequence homology of 5 0 untranslated region. Currently, there are 6 types and over 90 subtypes. Types 1, 2, and 3 distributed worldwide, with types 1a and 1b responsible for approximately 60% of infections. Type 4 occurs primarily in the Middle East, type 5 in South Africa, and type 6 in Hong Kong. In the United States (US), approximately 72% of people infected with HCV have genotype 1, and most others are types 2 or 3 (genotypes 4, 5, and 6 are not common in the US)

• There is little difference in the mode of transmission or natural history of infection among the different genotypes • Cure rates with antiviral therapy are notably higher with genotypes 2 and 3, and the duration of HCV therapy is shorter for these genotypes • Infection with HCV is curable by therapy, with the current standard treatment based on -Rapid separation of serum or plasma from cells is recommended by centrifugation within 1 h of collection -Unseparated EDTA plasma is stable at room temperature up to 24 h after collection -Separated serum or plasma is stable at room temperature for up to 3 days, at refrigerator temperatures for up to 1 week, and frozen at -70 C for years • Conventional tests and problems -Serological studies • Enzyme immunoassay (EIA) -The detection of HCV antibodies is recommended as the initial test for the identification of HCV and is useful for screening at risk populations -EIA is comparatively inexpensive, reproducible, and carries a high sensitivity (99%) and specificity (99%) -EIA can detect antibodies average 2-10 weeks after infection. However, during this "window period," a patient will have detectable viral RNA, but have undetectable levels of antibodies -A negative enzyme immunoassay is usually sufficient to exclude the diagnosis of HCV infection in immunocompetent patients -However, the test can be falsely negative in those with immunodeficiencies or end-stage renal disease -Conversely, false-positive EIAs may occur in patients with autoimmune disorders. In these patients, an assay for HCV RNA is necessary for diagnosis of chronic infection -Once patients seroconvert, they usually remain positive for HCV antibody. Thus, the presence of HCV antibody may reflect remote or recent infection -The immunoblot assay is still useful as a supplemental assay for persons screened in nonclinical settings and in persons with a positive EIA who test negative for HCV RNA -Other HCV antibody assays include anti-HCV IgM assays and avidity tests. The significance of the presence of anti-HCV IgM during HCV infection is unclear. Anti-HCV IgMs have been reported in 50-93% of patients with acute HCV and 50-70% of patients with chronic HCV -Therefore, anti-HCV IgM cannot be used as a reliable marker of acute HCV infection and, so far, IgM assays have not been used in clinical practice. However, increasing serial measurements of anti-HCV IgM titers early after the onset of the symptoms may help to identify patients with acute HCV. Alternatively, an increase in the anti-HCV IgG avidity index within a week after the onset of clinical symptoms has also been reported to indicate acute HCV infection. Both parameters could be used together before anti-HCV seroconversion occurs, or when no baseline sample is available to confirm the diagnosis of acute infection • A total HCV core antigen enzyme-linked immunosorbent assay (ELISA HCV 3.0) and ORTHO ® trak-C™ assay (Ortho Diagnostics) for detection and quantification of total core antigen in blood -The HCV core protein is highly antigenic, induces specific cellular and humoral responses, and probably plays a pivotal role in the pathogenesis of HCV infection. The availability of an anticore monoclonal antibody allowed the development of an ELISA to detect HCV core Ag in peripheral blood of patients with HCV -It tests positive for anti-HCV antibodies and for prospective low-risk population screening -Total HCV core antigen ELISA (quantitative, Ortho Clinical Diagnostics) has sensitivity close to PCR assays in diagnosing acute HCV infection in windows period (before HCV antibodies developed) -It is also used in monitoring response to antiviral treatment -The ELISA HCV 3.0 uses three recombinant antigens (c22-3, c200, and NS5) originating from four regions of the viral genome (core, NS3, NS4, and NS5) -ORTHO ® trak-C™ assay is a quantitative immunoassay for total (both free and antibody bound) HCV nucleocapsid core antigen. The assay uses a capture ELISA format. The difference between the two assays (ELISA HCV 3.0 and ORTHO ® trak-C™ assay) is a step in this assay that disrupts immune complexes present in the sample. The assay's intended use is testing for HCV core antigen either during preseroconversion (acute) or postseroconversion (chronic) phases of HCV infection -Less expensive and less prone to carryover than PCR testing and can be used as a rough screen as it correlates with HCV RNA load. However, it is less sensitive than PCR-based assays and individual variation is higher -Similar technology is used by the Architect HCV assay by Abbot Laboratories. The analytical sensitivity of the recently developed Architect HCV assay (Abbott Laboratories, Abbott Park, IL) varies according to the HCV genotype from 500 to 3,000 international units of HCV RNA per milliliter (IU/mL) -A drawback of the total HCV core Ag ELISA is its lower limit of detection, and HCV-positive patients on dialysis often have low HCV RNA levels • Recombinant immunoblot assay (RIBA) -RIBA has been developed for the simultaneous detection of anti-HCV antibodies and HCV core antigen -RIBA was used to confirm EIA results since the early generation. It had a high rate of false positives -Use of centrifugation or Ultracolumn (QIAGEN) to process a large volume (1 ml), the LOD can be further improved -Quantitative (Table 25.3) • On average 1-2 log10 units/ml less sensitive than qualitative tests • Used to establish baseline viral load (prior to therapy) and to monitor changes in viral load during therapy • PCR -Two real-time PCR platforms are currently available for the detection and quantification of HCV RNA: the COBAS TaqMan platform, which can be used together with automated sample preparation with the COBAS AmpliPrep system (CAP-CTM; Roche Molecular System, Pleasanton, CA), and the Abbott platform (Abbott Diagnostic, Chicago, IL), which uses the m2000RT amplification platform together with the m2000SP device for sample preparation (ART). The lower limits of detection were 12 IU/ml for ART and 15 IU/ml for CAP/CTM • VERSANT HCV RNA 3.0, quantiplex assay (bDNA) (Bayer) -Signal amplification directed to the 5 0 NC region and core regions of the HCV genome -Microwell plate format -Equivalent detection of genotypes 1-6 -LOD: 3200 HCV RNA copies/mL (5.2 HCV RNA copies/IU) -Broad dynamic range (615-7,690,000 IU/mL) -Comparative evaluations between Bayer bDNA and Roche PCR viral load assays demonstrated that PCR type-specific oligonucleotide probes attached to nitrocellulose strips to detect sequence variations found in the 5 0 NC region of HCV -The biotin-labeled PCR product is hybridized to the probes on the strip under stringent conditions. After hybridization and washing, streptavidin-labeled alkaline phosphatase is added; followed by incubation with a chromogen, which results in the development of a purple-brown precipitate when there is a match between the probe and the biotinylated PCR product -Hybridization of the amplicon with one or more lines on the strip allows the classification of six major genotypes and their most common subtypes • Third Wave Technologies' Invader assay (Third Wave Technologies, Madison, Wisconsin) is a new DNAscanning method application, which has been termed cleavase fragment length polymorphism (CFLP) -Relies on formation of unique secondary structure that results when DNA is allowed to cool following brief heat denaturation and serves as substrates for structure-specific cleavase I enzyme generating a set of cleavage products -Formation of secondary structures is sensitive to nucleotide sequences -The presence of sequence polymorphisms results in the generation of unique collections of cleavage products or structural fingerprints -It targets the well-conserved 5 0 NCR of HCV • Determination of HCV genotype is needed before the initiation of therapy with pegylated IFNa and ribavirin because it determines both the dose of ribavirin and the treatment duration required -HCV resistance testing

• Several amino acid substitutions that confer resistance to directly acting antiviral molecules, such as protease inhibitors, have been identified. In case of a failure of the triple combination of pegylated IFNa, ribavirin, and either telaprevir or boceprevir, HCV variants that are resistant to these compounds are selected • Direct sequence analysis or reverse hybridization methods can be used to identify amino acid substitutions that confer resistance to antiviral drugs • Ultradeep sequencing methods, such as pyrosequencing, can detect minor resistant populations down to <1% • HCV GenoSure NS3/4A represents the first in a series of HCV drug resistance assays that have been developed at Monogram Biosciences to support the clinical evaluation of HCV direct-acting antiviral (DAA) agents and their use in the management of HCV infection. HCV GenoSure NS3/4A analyzes the genetic sequence for the nonstructural proteins NS3 and NS4A of HCV genotypes 1a and 1b that encode for an enzyme essential to viral replication. The assay detects mutations in NS3 and NS4A and specifically identifies those associated with boceprevir and telaprevir resistance • Pitfalls -It is important to note that a "genotype bias" is possible for all HCV molecular assays because of the extensive genetic heterogeneity of the virus -False-positive results due to contamination (detected by negative control) -False-negative results due to amplification inhibition (detected by internal control) or due to a loss of bacteria during specimen preparation -"Home brew" PCR assays are not standardized and variations in sample handling and laboratory methods can affect the sensitivity of the assay

• HCV tests should be used in high-risk patients, such as intravenous drug users, children born to HCV-positive mothers, and HIV-positive patients. Figure 25 .12 shows the algorithm of HCV testing • Patients suspected of having chronic HCV infection should be tested for HCV antibodies. Patients suspected of having an acute infection should be tested for both HCV antibodies and also HCV RNA with a real-time PCR analysis. HCV RNA should be repeated as the patient is undergoing treatment, to better adjust the use of therapeutic agents • HCV RNA testing should be performed in -Patients with a positive anti-HCV test -Patients for whom antiviral treatment is being considered, using a quantitative assay -Patients with unexplained liver disease whose anti-HCV test is negative and who are immune compromised or suspected of having acute HCV infection • HCV genotype should be determined in all HCV-infected individuals prior to treatment in order to determine the duration of therapy and likelihood of response. Genotypic analysis has shown some amino acid substitutions to correlate with resistance to therapy. infection, other cell types may be affected to a lesser extent. The life cycle of HBV begins when it attaches to the cell surface. In the cytoplasm, the DNA is still in the core but then capsid is removed and DNA passes into nucleus, where it forms a covalently closed circular DNA (cccDNA) • HBV uses the host transcription machinery to replicate its genes and uses RNA polymerase II of the host. The (-) strand of the cccDNA will act as the template for this transcription. After transcription, the mRNAs are translated by the host's protein synthesis machinery to form viral proteins in the endoplasmic reticulum. The proteins are then assembled into virions that are secreted • HBV is recognized as endemic in China and other parts of Asia. Over one-third of the world's population has been or is actively infected by HBV • HBV strains are classified into at least 10 HBV genotypes (A to J) and several subtypes and based on the nucleotide homology of the surface gene. Except for the newly identified genotypes I and J, the geographic and ethnic distributions of HBV genotypes and subtypes are well characterized. Genotype A is mainly found in Northwestern Europe (subtype A2), North America, and Africa (subtype A1 or A3), whereas genotypes B and C have been described in Southeastern Asian populations. At present, genotype B is divided into B1-B6 subtypes. Among them, B1 is isolated in Japan, B2-5 are found in East Asia, and B6 is found in indigenous populations living in the Arctic, such as Alaska, Northern Canada, and Greenland. Genotype C, including subtypes C1-C5, mainly exists in East and Southeast Asia. Genotypes E and F are seen in East Africa and the New World, respectively. Genotype D is most often found in southern Europe, parts of Central Asia, India, Africa, and the Middle East. Genotype G is a recently determined genotype in France, America, and Germany while genotype H has been reported in patients from Central America. Recently, genotype I, a novel intergenotypic recombination among genotypes A, C, and G, was isolated in Vietnam and Laos. The newest HBV genotype, J, was identified in the Ryukyu islands in Japan, and this genotype has a close relationship with gibbon/orangutan genotypes and human genotype C • Acute infection with genotypes A and D results in higher rates of chronicity than genotypes B and C. Compared to genotype A and B cases, patients with genotypes C and D have lower rates of spontaneous HBV e antigen (HBeAg) seroconversion; when this occurs, it tends to be delayed. Hepatitis genotypes C and D have lower response rates than genotypes A and B. Genotype C is also more associated with severe liver disease, including to hepatocellular carcinoma • The rate of new HBV infections has declined by approximately 82% since 1991, when a national strategy to eliminate HBV infection was implemented in the US. The decline has been greatest among children born since 1991, when routine vaccination of children was first recommended

• Transmitted parenterally and sexually by contaminating open cuts or mucous membranes and has a long incubation period (45-120 days) ( Fig. 25 .14). Asian patients are more likely to be vertically infected (mother to child) than African or Western patients • Majority of affected patients recover from the illness, characterized by -Anorexia, nausea, vomiting, headache, fever, abdominal pain, dark urine, and sometimes jaundice -Elevated transaminases, hyperbilirubinemia, and elevated alkaline phosphatase may also occur -Extrahepatic manifestations include arthralgias, arthritis, nephritis, and dermatitis • 10% of patients continue to carry the virus or markers of the active viral infection greater than 6 months after initial infection -Small percentage may develop chronic persistent hepatitis with sequence fibrosis and cirrhosis -Incidence of HCC is increased with the viral genome found integrated in the cellular DNA in 75% of cases -May be associated with polyarteritis and cryoglobulinemia • Recently, several clinical scoring systems, or nomograms, consisting of previously confirmed independent risk predictors such as sex, age, family history of HCC, alcohol consumption, serum alanine aminotransferase (ALT) level, HBeAg status, serum HBV DNA level, and/or HBV genotype have been introduced. These easy-to-use nomograms are based on noninvasive clinical characteristics and have been found to accurately predict HCC risk in either community-or hospitalbased HBV-infected persons -It detects HBV DNA in human plasma -It is intended to be used to screen donors for HBV DNA -Detection limit is 100 copies/ml -It targets the S gene -Quantitative

• Used to establish baseline viral load (prior to therapy) and to monitor changes in viral load during therapy • Digene HBV DNA hybrid capture II -Detection and quantitation of HBV DNA in serum -Limit of detection: 4,700 HBV DNA copies/ml -Quantitative range: 1.4 Â 10 5 and 1.7 Â 10 9 HBV copies/ml • PCR -Amplicor HBV Monitor and its semiautomated COBAS HBV Amplicor Monitor test (Roche) -Detection and quantitation of HBV DNA in serum or plasma -Use the primers HBV-104UB and HBV-104D to amplify a 104-bp sequence within the highly conserved precore/core region of the HBV genome -Amplify genotypes A to E equally and reduced amplification of genotypes F and G -Limit of detection: 200 copies/ml -Quantitative range: 10 3 -4 Â 10 7 copies/mL • Real-time PCR LightCycler/FRET hybridization probes -It targets 259-bp fragment of S gene, -Quantitative range: 250-5 Â 10 8 copies/ml • Real-time PCR -Roche TaqMan Assay -Utilizes FRET technology and probes based on the detection of amplicon during temperature cycling -It targets S gene -Limit of detection: 50 copies/ml -Quantitative range: 5 to 2 Â 10 8 HBV IU/mL (30-10 7 copies/ml; 1 IU ¼ 5.82 copies) • bDNA assay -(VERSANT Hepatitis B Virus DNA 3.0 Assay) (Bayer Corporation) -Signal amplification directed to the 5 0 NC region and core regions of the HCV genome -Microwell plate format -Limit of detection: 2,000 copies/ml -Quantitative range: 2.0 Â 10 3 to 1.0 Â 10 8 HBV DNA copies/ml -Equivalent detection of genotypes A through F -Genotyping and mutation analysiscurrently used mainly for epidemiological purposes, rarely needed for clinical purposes • Line probe assay-LiPA; INNO-LiPA HBV Genotyping assay, Innogenetics N.V., Ghent, Belgium -This method is based on the reverse hybridization principle, such that biotinylated amplicons hybridize to specific oligonucleotide probes that are immobilized as parallel lines on membrane-based strips.

The amplified region analyzed overlaps the sequence encoding the major hydrophilic region of HbsAg -Sequencing and phylogenetic analysis of the pre-S1/pre-S2 region of the HBV genome -Identifies HBV genotype, drug resistance mutations, and anti-HBs escape mutations based on comparison of DNA sequence • Pitfalls -The analytical sensitivity and specificity of current real-time PCR assays allow for accurate quantification over a range of approximately 7-8 logs. They are not sufficient to quantify the very high HBV DNA levels that can be found in certain HBV-infected patients, which necessitates retesting these samples after dilution, a factor of quantification errors -Equal quantification of all HBV genotypes and robustness of quantification in case of nucleotide polymorphisms has not been validated for the current commercial real-time PCR assays -There is currently no uniform tendency to report HBV DNA levels in standardized units (such as copies/ml or genome equivalents/ml or IU/ml) -Not all assays are currently registered for use with plasma and serum -No precise thresholds of HBV DNA have been established that could guide medical decisions

• Viral load testing is used for the assessment and monitoring of responses to therapy in HBV infection (Fig. 25 .17) • In HBV carriers with active liver disease, HBV DNA loads are measured not only to assess patients regarding the need for either interferon alpha or lamivudine (a DNA polymerase inhibitor) antiviral therapy but also to monitor their effectiveness • An increase in HBV viral load is also used as a marker of the emergence of lamivudineresistant viral mutants • Active chronic infections with HBV treated with lamivudine require surveillance for the emergence of lamivudine-resistant viral mutants. During lamivudine monotherapy, point mutations at the active site of the polymerase gene (YMDD variants) occur with a frequency of 14-32% after 1 year in phase III studies, and in 42% and 52% of Asian patients after 2 and 3 years of therapy, respectively. The emergence of lamivudine resistance is detected by a rise in HBV viral load and confirmed by sequencing of the active site of the DNA polymerase gene than 80% concordance with CMV antigen test results, but also has the added benefits of increased specimen stability, smaller required specimen volume, and with the ability to be performed in patients with depressed white blood cell counts results -Pitfalls

• False-positive results due to contamination (detected by negative control) • False-negative results due to amplification inhibition (detected by internal control) or due to a loss of bacteria during specimen preparation • There is a lack of standardization of this process, with different techniques and assays, different quantitation methods, and different tissues and blood compartments being tested, thus making interpretation of results across studies challenging. Quantitative PCR is more sensitive than qualitative PCR, and CMV DNA values obtained with "in-house" quantitative assays are 3-to 10-fold higher than the commercial assay • Different quantitation methods include reporting results as genomic copies/mL, copies/mL, copies/ microgram of total DNA, copies/106 leukocytes, and copies/2 Â 10 5 leukocytes; such varied quantitation methods can make comparison of results almost impossible • No clearly defined cutoff values for determining CMV disease, this method has low specificity and low positive predictive value. Most of CMV cutoff on plasma ranging from 400 to 10,000 copies/mL of CMV DNA • Antiviral susceptibility testing of CMV isolates -General • Resistance of CMV to antivirals was a major clinical problem in patients with AIDS. Currently marketed anti-CMV drugs, namely, ganciclovir (GCV), its oral prodrug valganciclovir (vGCV), foscarnet (FOS), and cidofovir (CDV), all target the viral DNA polymerase • CMV infection remains a major problem in transplantation, and resistance to antivirals is encountered in all forms of transplantation • In general, it takes weeks to months for CMV to develop resistance to antivirals.

In patients with AIDS, some studies showed a 10% prevalence of resistance to ganciclovir by 3 months of therapy; similar time courses were found for foscarnet and cidofovir -Phenotypic methods

• Plaque reduction assay -The gold standard for antiviral susceptibility testing of CMV is plaque reduction assay -In this assay, a standardized inoculum of a stock virus is inoculated into cultures and incubated in the presence of the antiviral agent -The cultures are then observed for the presence of viral plaques -The IC50 of the agent for the isolate is defined as the concentration of agent causing a 50% reduction in the number of plaques produced -Plaque reduction assays are laborintensive -Plaque reduction assays are limited by the excessive time required completing the assay (4-6 weeks) and the lack of a standardized method validated across different laboratories -In addition, repeated passage of isolates to prepare viral stocks may influence the results of assays by selecting CMV strains that are not representative of the original population of the viruses • DNA hybridization assay -Whole genomic DNA is extracted and transferred by capillary action onto negatively charged nylon membranes after incubation with a specific agent -The membranes are hybridized to a 125I-labeled human CMV probe (Diagnostic Hybrids, Athens, Ohio), rinsed, washed, and counted in a gamma counter -Mean hybridization values (in counts per minute [cpm]) for each concentration of antiviral agent are calculated and expressed as a percentage of the cpm in control cultures -The IC50 is defined as the concentration of antiviral agent resulting in a 50% reduction in viral nucleic acid hybridization values (i.e., DNA synthesis) compared with the hybridization values of controls -Disadvantage of DNA hybridization assays is that they require the use of radiolabeled probes -DNA hybridization assays have the advantage over plaque reduction assays of eliminating the variation due to subjective errors resulting from plaque counting by different individuals • Viral load assays (e.g., antigenemia or quantitative DNA) -CMV viral load assay may rise as an indicator of antiviral resistance, but other factors (including compliance and declining immune function) may be responsible -The assay can measure viral DNA concentration exposed to a range of drug concentrations and is grown for 4 days in the presence or absence of drug then the IC50 is determined -Quantitative antigenemia assays are less exact than quantitative polymerase chain reaction -In many patients, certain CMV diseases (e.g., gastrointestinal disease or retinitis) are not always associated with measurable viral loads • Other phenotypic methods: Viral production is measured by using internal competitor DNA (ssDNA) that is confirmed against a known number of Namalwa cells (B cell lymphoma cell line containing two integrated copies of the EBV genome per cell) • Four separate PCR reaction tubes each containing internal competitor DNA (8 copies/ml, 40 copies/ml, 200 copies/ ml, or 1,000 copies/ml) are placed in competition with EBV-specific primers for amplification of patient DNA • PCR amplicons are examined by electrophoresis through a 2% agarose gel and visualized using a gel imaging documentation system. The band densities are quantitatively measured using Bio-Rad Quantity One software and used to calculate EBV copies • Although highly accurate and reproducible, such assays are rather laborious and require intensive post-PCR handling. Each sample has to be spiked with different amounts of internal standard to achieve precise quantification -Real-time PCR

• LightCycler ® EBV quantitative kit (Roche) -Detection of LMP gene in EBV viral genome -EBV is amplified with specific primers in a PCR reaction. The amplicon is detected by fluorescence using a specific pair of hybridization probes -A melting curve analysis is performed after the PCR run to differentiate positive samples from non-EBV species; i.e., other herpes virus family -The internal control is a synthetic double-stranded DNA molecule with primer binding sites identical to the EBV target sequence, comprising a unique hybridization probe binding region that differentiates the internal control from the target-specific amplicon. It is added already to the lysed sample before the purification step and copurified/amplified with the EBV DNA from the specimen in the same PCR reaction (dual color detection) -The kit allows quantification in a range of 10 2 -106 copies per reaction. The lower detection limit of the kit is 10 copies per reaction (95% confidence interval; probit analysis) • Other commercial assays and reagents -Other commercial assays and reagents included Nanogen EBV Q-PCR • Real-time PCR using SYBR Green I dye -To maximize detection rates and reduce false-negative results, two primer sets targeting the highly conserved EBV regions, (1) Epstein-Barr nuclear antigen 1 (EBNA1) and

(2) BamHI fragment H rightward open reading frame 1 (BHRF1), used to detect and measure absolute EBV DNA load in clinical settings with different EBV-associated diseases. Two separate real-time quantitative PCR assays using SYBR Green I dye and a single quantification standard containing the two EBV genes -PCR products analyzed by an amplification curve, melt analyses, and amplification efficiency -The lower limit of detection for both EBV regions was 2.0 Â 10 3 copies/ml -Sensitive and cost-effective • Pitfalls -Quantitative PCR requires analysis of absolute lymphocyte count, which inversely affects viremia; real-time PCR does not -Important to note that real-time PCR assay requires sequential analysis of run data prior to result reporting to prevent false positives (i.e., pseudoamplification and amplification of non-EBV species) and false negatives (i.e., shifted melting curve for EBV variants) -PCR assays are not standardized and variations in sample handling and laboratory methods can affect the sensitivity of the assay

• Serial viral load testing can be used to monitor disease burden and assess efficacy of immunosuppressive therapy in posttransplant patients 

• Varicella (chicken pox) -Chicken pox, which is caused by the VZV, is one of the most contagious childhood diseases. Nearly every unvaccinated child becomes infected with it -Mild self-limited illness common in school-aged children with fever followed by vesicular eruption on skin and mucous membranes -Spreads by respiratory secretions with a 10-14 days incubation period -More severe in adults, pneumonia common • Herpes zoster (shingles) -Recurrent infection, usually in adults that may be activated by trauma, neoplasm, or immunosuppression -Virus remains latent in sensory ganglia of spinal or cranial nerves causing dermatomal pain and vesicular eruptions, fever, and malaise. Commonly occurs in trunk, but may affect any dermatome -Associated with encephalitis and delayed cerebral vasculitis • Zoster sine herpete occurs in the event of recurrence in the absence of vesicle formation • Post herpetic neuralgia: pain lasting longer than 1 month after an episode, occurs in as many as 14% of affected individuals, particularly those over 60 years of age. Most neuralgias resolve within one year with 50% experiencing resolution within 2 months • Ramsay Hunt syndrome: combination of cutaneous involvement of herpes zoster infection of external auditory canal and ipsilateral facial and auditory nerve. Syndrome can cause facial paralysis, hearing deficits, and vertigo

• Specimens -Skin vesicle fluid, cerebrospinal fluid, nasopharyngeal secretion, bronchial washings, blood, amniocentesis fluid, and urine • Conventional tests and problems (Table 25.7) -Viral culture • Conventional (especially 16, 18, 31, 45, and 58, but also 33, 35, 39, 51, 52, 56, 59, 68, 73, 82) , is recognized as a necessary factor for the development of cervical cancer • The genome HPV virus is circular (Fig. 25.22) . The genome has eight open reading frames that encode ten proteins. The genes for these are divided into an early region that are expressed in the skin's infected basal cells that have yet to differentiate, and a late region with two genes whose protein products exist only in cells after cell differentiation • The E5 (changes the cellular responses to programmed cell death or apoptosis), E6 (binds to tumor suppressor protein, p53), and E7 (binds and inactivates retinoblastoma protein, Rb) proteins are early viral proteins expressed upon infection and cause destabilization of the infected cell and induces replication • As the cell differentiates, it migrates upward and induces expression of the E1, E2, and E4 genes; E1 and E2 cause viral replication and E4 destabilizes the cytoskeleton and prevents cellular differentiation • In the upper epithelial cell layers, the late viral proteins L1 (major capsid protein) and L2 (minor capsid protein) are expressed. They bind the viral DNA and autoassemble, giving rise to the complete virions, ready for a new infection that is released as the keratinocytes desquamate • The most common mode of transmission is via contact; i.e., sexual or autoinoculation

• HPV is by far the most common sexually transmitted disease. An estimated 80% of sexually active adults have been infected with one or more genital HPV strains. The vast majority of infected adults experience transient infectivity and are unaware of the condition; however, they may be able to infect others • However, most women infected with high-risk HPV, especially women under 30 years of age, do not develop cervical cancer. Their immune system effectively clears the infection over the course of several months • Specific factors that determine which HPV infections persist and develop into squamous intraepithelial lesions currently are unknown.

Cigarette smoking, ultraviolet radiation, pregnancy, folate deficiency, and immune suppression have been implicated as possible cofactors • Low-risk HPV types (6, 11, 42, 43, and 44) produce benign epithelial tumors of the skin and mucous membranes. Infection with certain types of HPV (high risk) can also increase the risk of developing cervical and other cancer types. Conditions associated with HPV -Verruca vulgaris (common wart) -associated with HPV2, HPV4, and HPV40. Highly contagious and can spread to other sites of skin or mucous membranes via autoinoculation -Condyloma acuminatum (venereal wart)associated with HPV6, HPV11, HPV16, and HPV18 and is considered a sexually transmitted disease with lesions occurring in sites of sexual contact or trauma (i.e., mucous membranes of genitalia, perianal region, oral cavity, and larynx) -Flat warts are most commonly found on the face or forehead and are most common in children and teens -Plantar warts are found on the soles of the feet -Subungual and periungual warts form under the fingernail (subungual) and around the fingernail or on the cuticle (periungual) and are a subtype of the common skin wart. They may be more difficult to cure than warts in other locations -Butcher warts are caused by HPV7 and occur in people handling meat, poultry, and fish -Focal epithelial hyperplasia (Heck disease) is caused by HPV13 (and possibly HPV32) and commonly occurs in Native American and Inuit populations. A childhood condition characterized by multiple soft, nontender flat papules and plaques of the oral mucous membrane -Laryngeal papillomatosis -frequently recurs and may require repetitive surgery when interferes with breathing. Rare cases can progress to laryngeal cancer (HPV30 and HPV40) -HIV-associated papillomatosis -HPV7 and immunocompromised states -Cervical cancer -History of HPV (highrisk types) infection is strongly associated with development of cervical cancer. However, most HPV infections do not progress to cervical cancer. Because the progression of transforming normal cervical into cancerous cells is a slow process, cancer occurs in people who have been infected with HPV for a long time, usually over a decade. High-risk HPV types 16 and 18 are together responsible for over 70% of cervical cancer cases; type 16 alone causes 41-54% of cervical cancers -Anal/rectal cancer -Although rare, anal/ rectal cancer is becoming more prevalent in the US. Similar to cervical cancer, the main cause of anal cancer is HPV and is most commonly acquired through anal intercourse. However, anal cancer can also be acquired from other genital areas that are infected with HPV, particularly from the vulva or penis. High-risk HPV types (16, 18, 31, 33 and 35) are associated with anal squamous intraepithelial lesions and account for approximately 80% of cervical and anal cancers -Head and neck squamous cell cancer -HPV16 is the most common type detected in head and neck squamous cell cancers (HNSCCs). HPV16 accounts for 78.6-100% of HPV-positive oropharyngeal cases. HPV type 18 accounts for 1% of oropharyngeal, 8% of oral cavity, and 4% of laryngeal HPV-positive SCCs. HPV16 and HPV18 genotypes are associated with a better prognosis in squamous cell carcinomas of the head and neck. Furthermore, the HPV-positive tumors are also more sensitive to radiation and chemotherapy. Coinfections are possible and most frequently include HPV16. HPV33 is often reported and has been identified in up to 10% of HPV-positive HNSCCs. Numerous other HPV types have been rarely detected in HNSCCs and include types 6, 11, 35, 45, 51, 52, 56, 58, 59, 18, and 31) -Use tissue sections, liquid-based cytology specimens, and cervical smears -On slide detection of high-and lowrisk HPV genotypes -16 probe cocktail for high-risk HPV genotypes 16, 18, 31, 33, 35, 39, 51, 52, 56, 58 , and 66 -6 probe cocktail for low-risk HPV genotypes 6 and 11 -Nucleic acid hybridization • Digene Hybrid Capture II (Digene Corporation, Gaithersburg, MD) -Method utilizes an RNA probe mix for the detection of the L1 gene of HPV. Assay can identify HR HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 . In addition, a kit detecting low-risk virus (6, 11, 42, 43, 44) is also available -Manual, semiautomated (rapid capture), and automated (utilizing QIAGEN's QiaSymphony extraction system) platforms available -Signal amplification is based on immunocapture of DNA/RNA hybrids that are immobilized on a 96-well microplate, reacted with alkaline phosphatase-conjugated antibodies specific for the RNA: DNA hybrids and detected with a chemiluminescent substrate -Can detect 5,000 viral copies per sample, or one picogram of HPV DNA per sample -Signal amplification • Invader Assay Cervista HPV assays (Hologic, Madison, WI) -The Invader assay is an isothermal linear signal amplification using structure-specific oligonucleotide cleavage and has been applied to DNA-based genotyping. This method uses two types of isothermal reactions: a primary reaction that occurs on the targeted DNA sequence and a secondary reaction that produces a fluorescent signal -Invader utilizes an internal control for human HIST2HBE to assure DNA quality and quantity in each reaction -Two diagnostic qualitative assay formats that utilize the Invader chemistry are available: Cervista HPV HR (detects pool of 13 HPV genotypes) and Cervista HPV16/18 (detects HPV16 and/or HPV18) -Cervista HPV HR uses isothermal signal amplification to detect 13 h HPV types utilizing three probe pools based on phylogenic relatedness. 3 probe pools include A5/A6 [51, 56] , A7 [18, 39, 45, 59, 68] , and A9 pool [16, 31, 33, 35, 52, 58] . Assay cannot determine the specific HPV genotype present -The Cervista HPV16/18 test is a diagnostic test to genotype HPV16 and HPV18 in cervical specimens -Can detect 1,250-5,000 viral copies per specimen -Target amplification • TMA -APTIMA HPV assay (Gen-Probe; San Diego, CA) -The APTIMA HPV assay is an in vitro nucleic acid amplification test for the qualitative detection of E6/E7 viral messenger RNA (mRNA) from 14 high-risk types of HPV in cervical specimens -Detects HPV subtypes: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 , and 68, but does not distinguish between the 14 high-risk types -The assay is used with the TIGRIS DTS system, one of the first diagnostic instruments to truly automate nucleic acid testing (NAT) from start to finish by from sample preparation, amplification, and detection to reporting results -The TIGRIS system can process approximately 450 samples in an 8h shift, and up to 1,000 samples in approximately 13.5 h -The APTIMA HPV assay involves three main steps, mainly: target capture, target amplification by transcription-mediated amplification (TMA), and detection of the amplification products (amplicon) by the hybridization protection assay (HPA) that measures the emitted relative light units (RLU) in a luminometer -Can detect 100-300 viral copies per sample -Real-time PCR -COBAS ® HPV test (Roche Diagnostics, Indianapolis, IN) • The COBAS HPV test is a clinical diagnostic qualitative assay to detect HPV in patient samples using the COBAS 4800 system that automates specimen extraction, amplification, and detection. The assay utilizes amplification of HPV DNA by real-time PCR and nucleic acid hybridization to detect 14 high-risk HPV genotypes in a single reaction tube that target the polymorphic L1 region of the HPV genome • The assay specifically genotypes HPV16 and HPV18 while concurrently detecting the other high-risk genotypes in a pooled fashion (31, 33, 35, 39, 45, 51, 51, 56, 58, 59, 66 , 68) • The COBAS HPV test is based on two major processes: (1) automated specimen preparation to simultaneously extract HPV and cellular DNA and (2) PCR amplification of target DNA sequences • Using both HPV-and b-globin-specific complementary primer pairs and real-time detection of cleaved fluorescent-labeled HPV-and b-globinspecific oligonucleotide detection probes • The master mix reagent for the COBAS HPV test contains primer pairs and probes specific for the 14 high-risk HPV types and b-globin DNA • The detection of amplified DNA (amplicon) is performed during thermal cycling using oligonucleotide probes labeled with four different fluorescent dyes. The amplified signal from 12 high-risk HPV types (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) is detected using the same fluorescent dye, while HPV16, HPV18, and b-globin signals are each detected with their own dedicated fluorescent dye • An additional primer pair and probe target the human b-globin gene (330-bp amplicon) to provide a process control • Can detect 300-1,200 viral copies/ml -Genotyping • Roche linear array (Fig. 25.23 ) -Qualitative test that utilizes amplification of HPV target DNA by PCR and nucleic acid hybridization bases on four major steps: (1) sample preparation, (2) PCR amplification of target DNA using HPV-specific complementary primers, (3) hybridization of the amplified products to oligonucleotide probes, and (4) colorimetric detection of the probe-bound amplified products -Uses a pool of biotinylated primers to define a sequence of nucleotides for the L1 region of the HPV genome designed to amplify HPV DNA from 37 HPV genotypes, including 13 high-risk genotypes (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 , and 68) -b-globulin gene is concurrently isolated and ensures adequacy of cellularity, extraction, and amplification for each processed sample • Sensitivity and specificity -Overall, the sensitivity for cytology for detecting HGSIL ranges from 50% to 70% and specificity of 86-98% -Overall, the sensitivity of HPV DNA test for detecting HGSIL is about 80-98% and specificity of 64-95% -Overall, the sensitivity of COBAS HPV assay for detecting HGSIL is about 69-71% and specificity of 90-94% -Overall, the sensitivity of Cervista HPV assay for detecting HGSIL is about 93-100% and specificity of 43-44% -Overall, the sensitivity of APTIMA HPV test for detecting HGSIL is about 87-93% and specificity of 60-63% -However, the sensitivity and specificity are influenced by the age and prevalence (Table 25 .8) • Pitfalls -Presently available assays provide only qualitative results and do not correlate the magnitude of the positive assay signal to meaningful quantitative results -The effects of other potential variables such as vaginal discharge, use of tampons, douching, personal lubricants, topical medicaments, and specimen collection variables may affect the performance of the assay -A negative HPV result does not exclude the possibility of present or future cytologic abnormalities -COBAS HPV assay Fig. 25 .23 Roche linear array HPV genotyping assay with reference guide utilized for interpretation. P positive control, N negative control, ßgL ß globulin low, ßgH ß globulin high Pap smear results to determine the need for referral to colposcopy. The results of this test are not intended to prevent women from proceeding to colposcopy (Table 25 .9) • In women 30 years and older, the hc2 high-risk HPV DNA test can be used with Pap smear to adjunctively screen to assess the presence or absence of high-risk HPV types. This information, together with the physician's assessment of cytology history, other risk factors, and professional guidelines, may be used to guide patient management (Table 25 .9) • Recently, a new test scheme was proposed ( Fig. 25.24) 25.10 Influenza A, B, and C Viruses

• Influenza is part of the Orthomyxoviridae family and can be classified into three basic types, influenza A, B, or C (Table 25 .10). Each influenza virus type is an enveloped single-stranded RNA virus that shares 

• Specimens (Table 25 .11) -Nasopharyngeal aspirate/swab/washing, tracheal aspirate, or bronchoalveolar lavage -Transport

• Culture/DFA -3 mL (minimum 1 ml) of respiratory sample in viral transport media (Microtest M4) or in sterile leakproof container at 2-8 C • Serologic -1 mL (minimum 0.5 ml) serum in an SST tube at 2-8 C -Unacceptable specimens: dry swabs or wood and calcium alginate swabs that may inactivate the virus for culture. Plasma or hemolyzed, lipemic, icteric, turbid, bacterially contaminated, or heat-inactivated serum is inadequate for serological test • Influenza viruses that infect birds are called avian influenza viruses, and commonly known as "bird flu." Only influenza A viruses and subtypes infect birds • There are substantial genetic differences between the subtypes that typically infect both people and birds. Within subtypes of avian influenza A viruses, there also are different strains • The incubation period of avian influenza A virus is typically 2-5 days but can be as long as 8-17 days • These influenza viruses occur naturally among birds -Wild birds worldwide carry the viruses in their intestines, but usually do not get sick from them -However, avian influenza is very contagious among birds and can make some domesticated birds, including chickens, ducks, and turkeys, very sick and kill them • There are many different subtypes of type A influenza viruses (Fig. 25 .26) -These subtypes differ because of changes in certain proteins on the surface of the influenza A virus (hemagglutinin and neuraminidase proteins) -There are 16 known hemagglutinin subtypes and 9 known neuraminidase subtypes of influenza A viruses -Many different combinations of hemagglutinin and neuraminidase proteins are possible. Each combination represents a different subtype -Avian influenza A H5 and H7 viruses can be distinguished as "low pathogenic" and "high pathogenic" forms on the basis of genetic features of the virus and the severity of the illness they cause in poultry; influenza H9 virus has been identified only in a "low pathogenicity" form -Each of these three avian influenza A viruses (H5, H7, and H9) theoretically can be partnered with any one of nine neuraminidase surface proteins; thus, there are potentially nine different forms of each subtype (e.g., H5N1, H5N2, H5N3, H5N9)

• The reported signs and symptoms of avian influenza in humans have ranged from eye infections (conjunctivitis) to influenza-like illness symptoms (e.g., fever, cough, sore throat, muscle aches) to severe respiratory illness (e.g., pneumonia, acute respiratory distress, -Sensitivity of commercial assays ranged from 36% to 80% and specificity ranged from 80% to 100% -The absolute sensitivity of the RT-PCR assays ranged from 10 to 100 genome equivalents per reaction • Pitfalls -When present, SARS antibodies can be detected in serum at any point during the course of the disease. However, most patients do not seroconvert until after the second week, highlighting the importance of an RT-PCR assay for early diagnosis of the virus -Positive results must be confirmed by repeat testing using an aliquot of the original specimen and/or another laboratory before reporting. Alternatively, testing of a second gene region may be helpful. Furthermore, testing of one sample from a single source does not rule out the presence of SARS-associated coronavirus -A negative result does not rule out SARS as the presence of PCR inhibitors in the patient specimen, poor RNA quality, or nucleic acid concentrations below the level of detection of the assay may occur

• During the first week, serum and plasma are preferred for RT-PCR. Between 1 and 3 weeks, these sample types are less effective; stool and respiratory samples are the preferred types. After 3 weeks, stool is the preferred sample type for RT-PCR. Viral load in the upper respiratory tract and feces is low during the first 4 days of infections and peaks at approximately the 10th day of illness • During the 10th-15th day of illness, high viral loads are independent predictors of poor clinical outcomes

• Enteroviruses represent one of the most common human viruses, affecting an estimate 50 million individuals in the US and potentially one billion worldwide. Enterovirus infections most commonly occur in temperate zones during the summer and early fall • Enteroviruses are a diverse group of small, nonenveloped ssRNA viruses of 6-7 kb that are transmitted by the fecal-oral route. Enteroviruses comprise a group of human viruses that includes polioviruses, echoviruses, coxsackie A viruses, coxsackie B viruses, and various enterovirus subtypes. The original classification of human enteroviruses has been substituted by a taxonomic scheme based on molecular and biological properties of the viruses. This revised classification recognizes at least 90 subtypes and separates them into four species • Several modes of transmission exist for these viruses, including fecal-oral, respiratory, transplacental, perinatal, and self-inoculation modes, but the majority are fecal-oral • Although enteroviruses undergo rapid replication in the GI tract, they rarely cause significant GI disease. Instead, they travel via the bloodstream to target organs where they further replicate and induce pathologic alteration • Most infections are subclinical, although may cause a variety of acute and chronic diseases -Acute: mild upper respiratory illness (common cold), febrile rash (hand, foot, and mouth disease and herpangina), aseptic meningitis, pleurodynia, encephalitis, acute flaccid paralysis, and neonatal sepsis-like disease -Chronic: myocarditis, cardiomyopathy, type 1 diabetes mellitus, and neuromuscular disease • The highest incidence of enterovirus infection is in infants and young children

• Specimens -Non sterile sites: nasal/throat swabs, and feces where the presence of the virus might merely indicate coincidental carriage -Sterile sites: vesicular fluid, CSF, serum, urine, or gathered at autopsy, are more reliable -Samples transported in viral transport media, were either transported directly to the laboratory or were stored at 4 C for a maximum of 24 • Conventional tests and problems -Viral culture and shell vial culture • Gold standard to detect enterovirus • Time-consuming methods and insensitive methods, relying on the presence of viable virus • Inability to fully characterize some enterovirus strains associated with late inadequate collection, handling and processing of samples, or because of intrinsic insensitivity to cell lines used • It can take up to 8 days for CPE to appear when virus is present in low titers (e.g., in CSF specimens), and some type A coxsackievirus do not grow in cell culture • Although shell vial culture using monoclonal antibodies has decreased the culture time compared with that for tube culture, it is less sensitive than conventional culture -Serology

• Serotype is usually irrelevant to individual management • The absence of a widely shared antigen has hampered the development of immunoassays for the enterovirus • Reports of monoclonal antibodies that cross-react with multiple enterovirus serotypes are promising, but further testing is required to determine the clinical relevance of those observations • Molecular methods -Real time RT-PCR -ABI Prism (Applied Biosystems, Foster City, Calif.) • Improved speed and accuracy using TaqMan assay platform • Targets conserve sequences of the 5 0 NTR and VP 1 and 2 (capsid protein). The 5 0 NTR is the most highly conserved region and is involved in viral protein translation (Fig. 25 .29) • An enterovirus real-time TaqMan PCR analysis of serum or plasma may be a good alternative for the enterovirus culture of feces, particularly in neonates with sepsis protein, P polypeptide, NT nontranslational region). PCR primers usually design to target to 5 0 NT region -AnDiaTec ® Enterovirus real time RT-PCR Kit • The kit is a screening assay for the detection of enteroviruses (coxsackie A, coxsackie B, and echovirus) in the capillary system of the LightCycler (Roche) • Targets conserve sequences of the 5 0 NTR -Cepheid SmartCycler ® System • It detects a 115-bp region of the 5 0 NTR -NASBA

• NASBA-electrochemiluminescently (ECL) and NASBA-beacon are not significantly different in sensitivity and specificity -Targets conserve sequences of the 5 0 NTR • NASBA-ECL -NucliSens basic kit has proved of equal or greater sensitivity for detection of enteroviruses -In the NucliSens basic kit, amplified RNA products are detected by hybridization using ECL-labeled probes, a highly sensitive methodology • NASBA-beacon -NucliSens EasyQ Enterovirus Test (bioMerieux, Durham, NC), which utilizes real-time molecular beacons as probes (NASBA-beacon) -Real-time RT-PCR using TaqMan to shorten both technical hands-on time and time to result -Enterovirus consensus, Argene Biosoft (for research use only in the US) • One-step RT-PCR of all enterovirus serotypes in one single reaction tube • Amplified region is in the 5 0 NCR of the genome • Detection is performed with a biotinylated enterovirus generic probe • Sensitivity and specificity -NASBA-ECL and NASBA-beacon were similar in sensitivity, 100% and 94.5%, respectively -RT-PCR sensitivity is 97%, while culture sensitivity is 54.5% JC and BK after the initials of the patients in which they were first discovered. JCV was isolated from the brain tissue of a patient with progressive multifocal leukoencephalopathy (PML); BKV was isolated from the urine of a renal transplant patient who developed ureteral stenosis postoperatively • BKV and JCV share 75% homology at the level of nucleotide sequence. Each is 70% homologous to simian virus 40 (SV40), which belongs to polyomaviruses and was introduced in the human population, between 1955 and 1963, by contaminated polio vaccines produced in SV40-infected monkey cells • The two are not cross-reactive serologically, and serologic tests for antibodies are able to distinguish between BKV and JCV • More than 70% of the adult population has antibodies to BKV and JCV, with primary infections typically occurring in childhood • After an initial infection, polyomaviruses establish latency in various tissues. The primary sites of latency are uroepithelial cells for BK virus and B lymphocytes and renal tissue for JCV. Additional sites of latency for both viruses include the ureters, brain, and spleen

• Both BKV and JCV have been associated with human tumors. The recent evidence that SV40 may be a cofactor in the etiology of specific human tumor types has raised again the interest on the two human polyomaviruses as possible agents involved in human oncogenesis • In immunocompetent individuals, primary BKV infections usually cause a mild respiratory illness and, rarely, cystitis, whereas primary JCV infections are typically asymptomatic. BKV seroprevalence peaks at $90% at age 5-9 years old • Reactivation of latent as well as primary BKV and JCV infections may occur in immunocompromised individuals, i.e., organ transplantation, AIDS, and leukemia. BKV infections can lead to interstitial nephritis, tubulitis, hemorrhagic cystitis, and kidney allograft rejection • JCV is responsible for progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease of the central nervous system seen in up to 70% of AIDS patients

• Specimens: urine, plasma, CSF, and tissue biopsy • Conventional tests and problems -Cytology and immunohistocytochemistry for urine sample • It can be used to confirm BKV reactivation diagnosis • The urine sample can be stained with JC/BK and SV40 monoclonal antibodies -Viral culture

• JC virus is difficult to culture • The most sensitive cell type for JCV is primary human fetal glial cells, which is not an easy reagent to acquire • BK virus will grow in common cell lines, such as human diploid fibroblasts, but several days and weeks are required before CPE is evident -Serologic studies • Hemagglutination inhibition or enzymelinked immunosorbent assay methods can measure titers of antibodies to JCV and BKV • Serological tests of blood and CSF for anti-JCV and BKV antibodies are not useful in the diagnosis of PML and immunosuppressed individuals because antibodies to JCV and BKV are common and many patients with PML or immunosuppressed patients fail to develop a significant rise in antiviral antibody titers in serum or CSF • Molecular methods (Fig. 25.30) -PCR, quantitative • Seminested PCR to measure serum BKV DNA has been shown to have a higher specificity of 88% (50% positive predictive value) to detect BKV nephropathy. It was also shown to have a sensitivity of 100% for detecting BKV nephropathy • Serial quantification of BKV DNA levels through PCR can aid in managing BKV nephritis by measuring increasing or decreasing activity levels noninvasively

• PCR analysis of JCV DNA in spinal fluid is a noninvasive method of detecting active JCV infection which is 95% specific and 80% sensitive • JC/BK Consensus Complete kit, Argene Biosoft (US: For research use only). The kit is for the detection and typing of JC and BK viruses by PCR and hybridization on microwell plates, and offers high sensitivity, up to 1 copy per PCR for BKV and 10 copies per PCR for JCV primers/ probe product is designed to amplify JCV/ BKV using 5 0 nuclease real-time assay. The targeted sequence corresponds to a fragment of 197/198 bp located in the gene of large T antigen • Real-time TaqMan PCR and LightCycler Probes (Homebrew) targets highly conserved sequence of JCV/ BKV genomes (VP2 gene) • Sensitivity and specificity -Analytical specificity: no cross-reactivity with HSV family viruses, simian virus, adenovirus, and HIV. Absolute sensitivity: 10 JC/BK virus detection -PCR has been able to detect JCV in CSF in 80-90% of PML patients -The specificity of diagnosis is influenced by the choice of primers and extraction methods but can approach 100% • Pitfalls -Sequence variation of polyomavirus genome and within various JC/BK subtypes that may cause difficulty in primer and probe design -Competition between JC and BK viruses due to sensitivity may lead to falsenegative PCR result

• Detection of the virus by PCR may be indicative of an active infection. Therefore, the identification of viral DNA may warrant the institution of antiviral therapies and/or a decrease of immunosuppressive therapies • Determination of viral DNA presence or concentration in transplant patients is useful in establishing the cause of allograft rejection. Viral load may also be useful in immunocompromised patients • BKV nephropathy is associated with BK viremia of >5,000 copies/mL (plasma) and BK viremia >107 copies/mL and is seen in approximately 8% of kidney transplant recipients • Though latency is typically associated with the absence of viremia, low levels (<2,000 copies/mL plasma) are seen in some asymptomatic individuals 

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