Clinical Immunology & Serology CLS 311 PDF

Clinical Immunology & Serology CLS 311 Serology of Viral Infections Mr. Abdullah Abdali CLS Lecturer MS.c in Immunology and Immunotherapy  Background: Viruses Submicroscopic particles, size is measured in nanometers. Basic structure consists of: – Cor...

Clinical Immunology & Serology CLS 311 Serology of Viral Infections Mr. Abdullah Abdali CLS Lecturer MS.c in Immunology and Immunotherapy  Background: Viruses Submicroscopic particles, size is measured in nanometers. Basic structure consists of: – Core of DNA or RNA packaged into a protein coat, or capsid. – some viruses, the capsid is surrounded by an outer envelope of glycolipids and proteins derived from the host cell membrane. Obligate intracellular pathogens that rely on the host cell for their replication and survival. Background: Viruses (cont’d) Background: Viruses (cont’d) Viruses infect their host cells by: – Attaching to specific receptors on the cell surface; – Penetrating the host cell membrane – Then, releasing their nucleic acid, which then directs the host cell’s machinery to produce more viral nucleic acid and proteins. These components assemble to form intact viruses that are released by lysis of the cell or by budding off the cell’s surface. can then infect neighboring host cells and begin new replication cycles. The free virions promote dissemination of the infection. A complete virus particle with all its parts that can infect a host organism is called a virion.  Response of immune system to viral infection  Innate Immune response: Provides the first line of protection against viral pathogens. Two important nonspecific defenses against viruses involve type I interferons and NK cells. Virus-infected cells are stimulated to produce IFN- α and IFN- ᵦ following recognition of viral RNA/DNA by Toll-like receptors (TLR). These interferons inhibit viral replication by inducing the transcription of several genes that code for proteins with antiviral activity – For example, a ribonuclease enzyme that degrades viral RNA. IFN- α and IFN- ᵦ also enhance the activity of NK cells, which bind to virus infected cells and release cytotoxic proteins like perforin and granzymes, which cause the cells to die and release their virus particles. These cell-free virions are now accessible to antibody molecules. Response of immune system to viral infection (cont’d) When innate defenses are insufficient in preventing viral infection, specific humoral and cell-mediated defenses are activated. Phagocytes have the widest variety and greatest numbers of PRR o E.g. Toll-like Receptor (TLR), NOD-like Receptor (NLR). Role of NK cells In Viral infection Response of immune system to viral infection (cont’d)  Adaptive Immune Response: A. Humoral Immune response: Virus-specific antibodies are produced by B cells and plasma cells and can attack free virus particles in several ways. – Antibodies play a key role in preventing the spread of a viral infection through neutralization. In this process, antibodies specific for a component of the virus that binds to a receptor on the host cell membrane will bind to the virus and prevent it from attaching to and penetrating the cell.  Response of immune system to viral infection (cont’d) Secretory IgA antibodies play an especially important role in this process (neutralization), because they neutralize viruses in the mucosal surfaces (e.g., respiratory and digestive tracts). Meanwhile, IgM and IgG antibodies can bind to viruses in the bloodstream and inhibit dissemination of the infection. – IgG; promote phagocytosis of viruses through their opsonizing activity and promote destruction of viruses through antibody-dependent cell-mediated cytotoxicity (ADCC). – IgG and IgM also activate complement, which can participate in opsonization via C3b or lysis of enveloped viruses by the membrane attack complex. Response of immune system to viral infection (cont’d) B. Cell-mediated immunity: Elimination of intracellular viruses requires the action of cell-mediated immunity. Cytotoxic T cells (CTL) play a key role in this mechanism of defense. – Upon activation of CD4 T helper cells and cytokines, CD8 CTL become programmed to expand in number and attack the virus-infected cells. – To recognize the virus-infected host cell, the T-cell receptor on the CTL must bind to a viral antigen complexed with MHC class I on the surface of the virus- infected host cell Response of immune system to viral infection (cont’d) Upon the activation of CTL: It to release a pore-forming protein called perforin, which produces pores in the membrane of the infected host cell, and proteases called granzymes, which enter the pores. These enzymes activate apoptosis of the host cell, resulting in interruption of the viral- replication cycle and release of assembled infectious virions. The free virions can then be bound by antibodies. Response of immune system to viral infection (cont’d) CD4+ T cells help antiviral CD8+ T cells in two main ways: They maximize CD8+ T cell population expansion during a primary immune response and also facilitate the generation of virus-specific memory CD8+ T cell populations. In addition to their helper functions, CD4+ T cells contribute directly to viral clearance. Strategy of Viruses to evade immune system First, they are rapidly dividing agents that undergo frequent genetic mutations producing new viral antigens which is common in the influenza virus. Antigenic variation is also employed commonly by other viruses, such as rhinoviruses, which cause the common cold, and human immunodeficiency virus (HIV), which causes AIDS. Second, some viruses can evade the action of components of the immune system such as interferons, complement proteins, or the lysosomal enzymes in phagocytic cells. For example, the hepatitis C virus can block the degradation of viral RNA induced by the interferons. Third, viruses can evade the host’s defense by suppressing the immune system. Some viruses, like the cytomegalovirus (CMV), rubeola, and HIV, accomplish this by reducing the expression of MHC molecules on the surface of virus-infected cells, making them less likely to be recognized by T lymphocytes. Strategy of Viruses to evade immune system Other viruses can alter the function of certain cells of the immune system after directly infecting them. For example, the Epstein-Barr virus (EBV) causes polyclonal activation of the B lymphocytes it infects, and HIV suppresses the function of the CD4 T helper cells. EBV can also suppress the immune system by causing a cytokine imbalance. Finally, some viruses, such as CMV, varicella-zoster virus, and HIV, can remain in a latent state by integrating their nucleic acid into the genome of the host cells they infect. VIRAL HEPATITIS Hepatitis is a general term that means inflammation of the liver. It can be caused by several viruses and by noninfectious agents, including ionizing radiation, chemicals, and autoimmune processes. The primary hepatitis viruses affect mainly the liver. Other viruses, such as cytomegalovirus, Epstein-Barr virus, and herpes simplex virus, can also produce liver inflammation, but it is secondary to other disease processes. VIRAL HEPATITIS  VIRAL HEPATITIS Viral Hepatitis Method of Transmission Hepatitis A virus (HAV) Primarily by the fecal-oral route Hepatitis E virus (HEV) Hepatitis B virus (HBV) Mainly by the parenteral route (i.e., through contact with blood Hepatitis D virus (HCV) and other body fluids). Hepatitis C virus (HEV) All may produce similar clinical manifestations. The early, or acute, stages of hepatitis are characterized by general flulike symptoms such as fatigue, fever, myalgia, loss of appetite, nausea, vomiting, diarrhea or constipation, and mild to moderate pain in the right upper quadrant of the abdomen. Progression of the disease leads to liver enlargement (hepatomegaly) and tenderness, jaundice, dark urine, and light feces. Initial laboratory findings typically include elevations in bilirubin and in the liver enzymes, most notably alanine aminotransferase (ALT). These findings are nonspecific indicators of liver inflammation and must be followed by specific serological or molecular tests to identify the cause of hepatitis more definitively.  Hepatitis A Virus HAV is a non-enveloped, single-stranded ribonucleic acid (RNA) virus that belongs to the Hepatovirus genus of the Picornaviridae family. HAV is transmitted primarily by the fecal-oral route, by close person-to-person contact, or by ingestion of contaminated food or water. Conditions of poor personal hygiene, poor sanitation, and overcrowding facilitate transmission. Rarely, transmission through transfusion of contaminated blood has been reported and may occur during a short period within the acute stage of infection when a high number of viral particles can be found in the source blood. Hepatitis A Virus Average incubation period of 28 days, then, the virus produces symptoms of acute hepatitis in the majority of infected adults; however, most infections in children are asymptomatic. The infection does not progress to a chronic state and is usually self-limiting, with symptoms typically resolving within 2 months. Massive hepatic necrosis resulting in fulminant hepatitis and death are rare and occur mainly in those patients with underlying liver disease or advanced age. Diagnosis of Hepatitis A Virus HAV antigens are shed in the feces of infected individuals during the incubation period and the early acute stage of infection. Acute hepatitis A is routinely diagnosed in symptomatic patients by demonstrating the presence of IgM antibodies to HAV. – Are most commonly detected by a solid-phase antibody-capture enzyme immunoassay (EIA) in which IgM in the patient serum is bound to anti- antibodies on a solid phase and detected after the addition of HAV antigen, followed by an enzyme-conjugated anti-HAV IgG. – IgM anti-HAV is usually detectable at the onset of clinical symptoms and declines to undetectable levels within 6 to 12 months. – Tests for total HAV antibodies detect predominantly IgG and are available in a competitive inhibition EIA format. – IgG antibodies persist for life, and a positive total anti-HAV in the context of a negative IgM anti-HAV indicates that the patient has developed immunity to the virus, either through natural infection or vaccination. Diagnosis of Hepatitis A Virus Diagnosis of Hepatitis A Virus Several molecular methods have also been developed to detect HAV RNA, the most common format being reverse transcriptase polymerase chain reaction (RT- PCR). Source: https://www.cdc.gov/hepatitis/statistics/surveillanceguidance/HepatitisA.htm Vaccine Against HAV. A vaccine consisting of formalin-killed HAV was licensed in the mid-1990s to prevent hepatitis A. – Resulted in a significant decrease in the number of HAV infections. While the vaccine was originally recommended for only high-risk individuals, it is now recommended for routine immunization of children aged 12 to 23 months. – It is also recommended for persons traveling to geographical areas where hepatitis A is endemic, users of illicit drugs, for persons working with HAV- infected primates or with HAV in a research laboratory Hepatitis E Virus (HEV) HEV is a non-enveloped, single-stranded ribonucleic acid (RNA) virus that belongs to the genus Hepevirus, in the family Hepeviridae. Like HAV, it is transmitted by the fecal- oral route. Most HEV infections are related to consumption of fecally contaminated drinking water in developing regions of Africa, the Middle East, Southeast Asia, and Central Asia, all of which have poor sanitation conditions. Incubation period of 3 to 8 weeks, HEV causes an acute, self-limiting hepatitis that lasts 1 to 4 weeks in most people who become infected. Hepatitis E Virus (HEV) Fulminant hepatitis, associated with rapidly progressing disease and a high mortality rate, occurs more commonly in pregnant women. Like HAV, the infection does not progress to a chronic carrier state. Cases are commonly seen in severely immunocompromised individuals, including organ transplant recipients, patients undergoing chemotherapy, and those with acquired immunodeficiency syndrome and concomitant HEV infection Transmission of HEV  Diagnosis of HEV Diagnosis of it relies on serology. Acute infection is indicated by the presence of IgM anti-HEV, which is detectable at clinical onset but declines rapidly in the early recovery period. These antibodies are typically identified by: – Highly sensitive enzyme immunoassays that use recombinant and synthetic HEV antigens – Specificity of the assays may be increased by testing for IgA anti-HEV along with the IgM assays. – In patients who are suspected of having hepatitis E but who yield a negative IgM test, molecular testing for HEV RNA can be performed (RT-PCR). Performed on stool samples collected within 3 weeks of clinical onset. – Immunoassays for IgG anti-HEV, which persists longer, may be performed to determine previous exposure and sero-prevalence of the infection. Diagnosis of HEV (Zhao and Wang, 2016)  Hepatitis B Virus (HBV) Hepatitis B is a major cause of morbidity and mortality throughout the world. The WHO estimates that HBV has infected 2 billion people worldwide, causing 360 million chronic infections and between 500,000 and 1.2 million deaths each year due to liver disease. HBV is transmitted through the parenteral route by intimate contact with HBV- contaminated blood or other body fluids, most notably semen, vaginal secretions, and saliva. Transmission of HBV may also occur via the perinatal route, from infected mother to infant, most likely during delivery. Hepatitis B Virus (HBV) Several measures have been introduced to prevent HBV infection including: – Screening of blood donors, – Treating plasma-derived products to inactivate HBV, – Implementing infection-control measures, – Most importantly, immunizing with a hepatitis B vaccine The first vaccine, licensed in 1982, was composed of an antigen called HBsAg that was purified from inactivated virus particles in plasma from HBV-infected donors. The current vaccines, consisting of recombinant HBsAg produced from genetically engineered yeast, are some of the most widely used vaccines throughout the world.  Hepatitis B Virus (HBV) In Saudi Arabia, hepatitis B vaccine is administered to infants and children as part of their routine immunization schedule and is recommended for high-risk individuals such as health-care workers and hemodialysis patients. The vaccine can also be administered to individuals thought to be exposed to the virus, along with HBIG (hepatitis B immune globulin), a preparation derived from donor plasma with high concentrations of antibodies to HBV, administered as a means of passive immunization to provide temporary protection. An average incubation period of 45 to 90 days, followed by a clinical course that varies in different age groups. Most HBV-infected adults recover within 6 months and develop immunity to the virus, but about 1 percent develop fulminant liver disease with hepatic necrosis, which has a high rate of fatality.  Shape of HBV The virus responsible for hepatitis B, HBV, is a DNA virus belonging to the Hepadnaviridae family. Eight genotypes, designated A through H, have been identified. The intact virion is a 42 nm sphere consisting of a nucleocapsid core surrounded by an outer envelope of lipoprotein. The core of the virus contains: – Circular partially double-stranded DNA; – A DNA-dependent DNA polymerase enzyme; – Two proteins, the hepatitis B core antigen and the hepatitis B e antigen (HBeAg). A protein called the hepatitis B surface antigen (HBsAg) is found in the outer envelope of the virus. HBsAg is produced in excess and is found in noninfectious spherical and tubular particles that lack viral DNA and circulate freely in the blood. Shape of HBV These antigens (HBeAg, HBsAg, and HBcAg) , or antibodies to them, serve as: – Serological markers for hepatitis B – Have been used in differential diagnosis of HBV infection; In monitoring the course of infection in patients, In assessing immunity to the virus In screening blood products for infectivity. The levels of these markers vary with the amount of viral replication and the host’s immune response.  Diagnosis of HBV Serological markers for hepatitis B are most commonly detected by commercial immunoassays. These are available in a variety of formats, such as enzyme immunoassay and chemiluminescent immunoassay. An example of an immunoassay (chemiluminescent Microparticle) for detecting HBsAg is shown in the Figure below: Because false-positive results can occur, any initial positive results should be verified by – Repeated testing of the same specimen in duplicate, – Followed by confirmation with an additional assay, such as an HBsAg neutralization test or by a molecular test that detects HBV DNA. Molecular test for HBV Several molecular methods have been developed to detect HBV DNA in serum or plasma and are based on target amplification, branched DNA signal amplification, – Hybridization, or real-time PCR. The most sensitive of these is real-time PCR, which can detect as few as 10 copies of HBV DNA per ml. – HBV DNA can be detected in the serum about 21 days before HBsAg and may be a useful adjunct in detecting acute HBV infection in certain situations, such as when HBsAg test results are equivocal or in cases of occupational exposures.  HBV Markers HBsAg HBeAg HBcAg Is the first marker to appear, becoming Appears shortly after HBsAg and disappears The HBcAg is not detectable in serum, because detectable 2 to 10 weeks after exposure to HBV shortly before HBsAg in recovering patients the viral envelope masks it. Serum HBsAg usually becomes undetectable by 4 It may be elevated during chronic infection. it acts as a marker of viral replication to 6 months after the onset of symptoms in patients with acute hepatitis B. In patients with chronic HBV infection, HBsAg This marker is present during periods of active remains elevated for 6 months or more. replication of the virus and indicates a high degree of infectivity. an indicator of active infection and is an important marker in detecting initial infection, monitoring the course of infection and progression to chronic disease, and screening of donor blood Antibodies against HBV Protective titers of the antibody are considered to be 10 mIU/mL of serum or higher. Anti-HBs are not produced during chronic HBV infection, in which immunity fails to develop. Diagnosis of HBV Typical serological markers in acute hepatitis B  Hepatitis D Virus (HDV) Hepatitis D, also known as delta hepatitis, is a parenterally transmitted infection that can occur only in the presence of hepatitis B. HDV is a defective virus that requires the help of HBV for its replication and expression. HDV consists of a circular RNA genome and a single structural protein called hepatitis delta antigen within its core, surrounded by a viral envelope that is of HBV origin and contains the HBsAg. (Lee et al., https://www.mdpi.com/2075-1729/13/7/1527) Hepatitis D Virus (HDV) The number of new infections appears to be declining, most likely due to the implementation of the hepatitis B vaccine. Hepatitis D can either occur as a co-infection with hepatitis B, or as a superinfection: – Co-infection with hepatitis B: in which infection of HDV and HBV occurs simultaneously – Superinfection: in which HDV infects individuals who are already chronic HBV carriers. Result in a greater risk of developing fulminant hepatitis or chronic liver disease with an accelerated progression toward cirrhosis, liver decompensation, and hepatocellular carcinoma. Diagnosis of HDV HDV RNA is detected by reverse-transcriptase PCR assays, which are highly sensitive, specific, and quantitative. These assays can be used not only to detect HDV infection, but also to monitor patients during antiviral therapy. Hepatitis D infection is also indicated by the presence of anti-HDV in the patient’s serum, which can be detected by immunoassays employing hepatitis D antigen. – IgM anti-HDV may be used to detect acute hepatitis D infections, its appearance may be delayed, it may persist for only a short period of time, and it may be missed. – High titers of IgM and IgG antibodies are associated with chronic infection.  Hepatitis C Virus (HCV) Hepatitis C is a major public health problem, having infected over 170 million people worldwide. It is the cause of the majority of infections previously classified as “non-A-non-B” before the discovery of HCV in 1989. Hepatitis C is transmitted mainly by exposure to contaminated blood, with intravenous drug use being the main source of infection. Blood transfusion was also a major source of infection before 1992. Sexual transmission of HCV is thought to be less common but is higher in those who have had a history of sexually transmitted diseases. Perinatal transmission has been estimated to occur at a rate of about 6 percent. HCV has an average incubation period of 7 weeks (range is 2 to 30 weeks Hepatitis C Virus (HCV) The majority of infections are asymptomatic. – Because about 85 percent of persons develop chronic infection, which leads to cirrhosis in about 20 percent of these individuals. – Cirrhosis develops slowly, over 20 to 25 years, causing damage to the liver and posing an increased risk of developing hepatocellular carcinoma. – End stage liver disease related to HCV is now the leading cause for liver transplantation. Early detection of HCV would help in preventing these complications but occurs infrequently, due to the asymptomatic nature of the infection in most individuals Shape of HCV HCV is an enveloped, single-stranded, positive-sense RNA virus belonging to the family Flaviviridae and the genus Hepacivirus. HCV has a high mutation rate, which allows it to escape the immune response and persist in the host. This has also created difficulty in developing an effective vaccine. Diagnosis of HCV Both serological tests and molecular tests have been developed to identify persons with HCV infection. Serological tests are used in the screening of blood and organ donors for HCV infection and in the initial diagnosis of symptomatic patients. – This testing involves detection of HCV IgG antibody by third-generation enzyme immunoassays or chemiluminescent immunoassay methods, which use recombinant and synthetic antigens developed from the conserved domains of the C, NS3, NS4, and NS5 proteins. – Improvements in the serological assays for anti-HCV, enabled antibodies to be detected earlier than previous methods—about 4 to 6 weeks after infection.  Any positive results from an anti-HCV screening test should be confirmed. – The traditional confirmatory method was the recombinant immunoblot assay (RIBA), which detects antibodies to different HCV antigens that have been immobilized onto a nitrocellulose strip by a colorimetric reaction. – However, RIBA has been replaced in many laboratories by molecular methods, which are more sensitive and less labor-intensive. – The most commonly used qualitative method is reverse transcriptase polymerase chain reaction (RT-PCR), but a highly sensitive transcription mediated amplification (TMA) test has recently been developed as well. These can detect HCV infection within 1 to 3 weeks after exposure—much earlier than serological methods. – Quantitative tests are performed by RT-PCR, real-time PCR, or branched DNA amplification (bDNA). Used to monitor the amount of HCV RNA, or “viral load  Herpes Virus Complex DNA viruses that are surrounded by a protein capsid, an amorphous tegument, and an outer envelope. These viruses are all capable of establishing a latent infection with lifelong persistence in the host. The Herpesviridae family includes eight viruses that can cause disease in humans: – The herpes simplex viruses (HSV-1 and HSV-2) – Varicella-zoster (also known as human herpes virus-3 or HHV-3); – the Epstein-Barr virus (HHV-4); – Cytomegalovirus (HHV-5); and – The human herpes viruses 6, 7, and 8 (HHV-6, HHV-7, and HHV-8), the latter of which has been associated with Kaposi’s sarcoma. Epstein-Barr Virus (EBV) Causes a wide spectrum of diseases, including infectious mononucleosis, lymphoproliferative disease, and several malignancies. EBV infections most commonly result from intimate contact with salivary secretions from an infected individual. EBV infections usually occur during early childhood, However, by adulthood, more than 90 percent of individuals have been infected, as evidenced by the presence of EBV antibodies in their serum. EBV selectively infects host cells that are positive for the CD21 molecule, which serves as a receptor for both the virus and the C3d protein of complement.  Epstein-Barr Virus (EBV) The virions also infect B lymphocytes, which spread the virus throughout the lymphoreticular system. The virus-infected B cells become – Polyclonally activated – Proliferating – Secreting a number of antibodies, including: EBV-specific antibodies; Heterophile antibodies; Autoantibodies such as cold agglutinins, rheumatoid factor, and antinuclear antibodies. In healthy individuals, this process is kept in check by the immune response of natural killer cells and specific cytotoxic T cells. EBV can persist in the body indefinitely in a small percentage of B cells, in which it establishes a latent infection. Epstein-Barr Virus (EBV) Primary infections in healthy adolescents or adults commonly result in infectious mononucleosis (IM). More than half of patients with IM present with three classic symptoms: fever, lymphadenopathy, and sore throat. Symptoms usually last for 2 to 4 weeks, but fatigue, myalgias, and need for sleep can persist for months. These symptoms are essential in diagnosing IM, they can also be caused by many other infectious agents, so laboratory testing plays an important role in differentiating IM from other infections. Serological findings include presence of a heterophile antibody and antibodies to certain EBV antigens. Antibodies against EBV The heterophile antibodies associated with IM are IgM antibodies produced as a result of polyclonal B-cell activation and are capable of reacting with horse red blood cells, sheep red blood cells, and bovine red blood cells. – Produced by 40 percent of patients with IM during the first week of clinical illness and by up to 90 percent of patients by the fourth week. – They disappear in most patients by 3 months after the onset symptoms. – The heterophile antibody is present in most patients during the acute phase of illness (screening test for IM). Antibodies against EBV IgM antibody to the VCA is the most useful marker for acute IM, because it usually appears at the onset of clinical symptoms and disappears by 3 months. IgG anti-VCA is also present at the onset of IM but persists for life and can thus indicate a past infection. Antibodies to EA-D are also seen during acute IM, and anti-EBNA appears during convalescence. Antigen of EBV EA-D, which has a diffuse distribution in the nucleus and cytoplasm. EA-R, which is restricted to the cytoplasm only. The late antigens of EBV are those that appear during the period of the lytic cycle following viral DNA synthesis (VCAs - in the protein capsid and the membrane antigens in the viral envelope). Methods of Diagnosis EBV The heterophile antibodies are most often detected by indirect immunofluorescence assays (IFA) using EBV infected cells or ELISA techniques using recombinant or synthetic EBV proteins. While both methods have a high level of sensitivity (95 to 99 percent), – IFA tests have a higher level of specificity and are considered the “gold standard” of EBV serology methods. – However, many laboratories prefer ELISA tests, because they are less time- consuming and easier to interpret. More recently, methods employing chemiluminescence-based detection of the antibodies have also become available. Molecular testing of EBV Methods to detect EBV DNA in blood and tissue samples. Molecular tests may be more reliable than serology in immunocompromised patients who may not demonstrate a good humoral response, and they are also useful in monitoring viral load in patients with EBV-related malignancies who are undergoing therapy.  Cytomegalovirus (CMV) Is a ubiquitous virus with worldwide distribution. Nearly all persons have been exposed by their elderly years. CMV is spread through close, prolonged contact with infectious body secretions (saliva, urine, semen, and breast milk); intimate sexual contact; blood transfusions; solid organ transplants An immune response against the virus is stimulated, but the virus persists in a latent state in monocytes, dendritic cells, and myeloid progenitor cells The clinical consequences of CMV infection are much more serious in the immunocompromised host, most notably organ-transplant recipients and patients with AIDS.  CMV is the most important infectious agent associated with organ transplantation, with infections resulting from reactivation of CMV in the recipient or transmission of CMV from the donor. CMV is also the most common cause of congenital infections, occurring in approximately 1 percent of all neonates. Transmission of the virus may occur through the placenta.  Laboratory Diagnosis of CMV Several laboratory methods have been developed to detect CMV infection, including viral culture, viral antigen assays, molecular assays, and serology. – Isolation of the virus in culture, or demonstration of CMV antigens or DNA from appropriate clinical specimens, are the preferred diagnostic methods. – The standard reference method for detecting congenital CMV infection is to isolate the virus from the urine or saliva of the neonate within 3 weeks of birth. Immunofluorescence method that uses monoclonal antibodies to detect immediate early CMV antigens in infected cells grown on coverslips in shell vials. Laboratory Diagnosis of CMV PCR amplification of CMV DNA has been extremely useful for detecting CNS infections in immunodeficient hosts, for detecting CMV in amniotic fluid, and for establishing the diagnosis of CMV infection in transplant récipients. Quantitative PCR, which detects CMV copy number in the peripheral blood, is used to monitor the effectiveness of antiviral treatment in immunocompromised hosts and to identify patients at risk for developing disseminated CMV disease. Serology tests for CMV have been commercially available for many years, their clinical utility is limited. – Assays for IgM CMV antibodies have been developed but are limited in value because of the potential for false-negative results in newborns and immunocompromised patients and for false-positive results due to other infections or the presence of rheumatoid factor.