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The immuassay handbook parte90

  1. 1. 919© 2013 David G. Wild. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/B978-0-08-097037-0.00073-7 Viral Diseases1 Carey-Ann D. Burnham (cburnham@path.wustl.edu) Christopher Doern (christopher.doern@childrens.com) Steven R Binder (steve_binder@bio-rad.com) 1 This chapter contains a few passages from chapters in the third edition of The Immunoassay Handbook, written by Bruce J. Dille, Alan S. Armstrong, Isa K. Mushahwar and John W. Safford Jr. Cytomegalovirus (CMV) ETIOLOGIC AGENT AND PATHOGENESIS Human cytomegalovirus (CMV), a member of the Herpes- virus family, is extremely complex, containing a linear dou- ble-stranded DNA of about 230kb and over 30 structural proteins. The slowly replicating virus causes characteristic cell enlargement and intranuclear inclusions. CMV is endemic to populations throughout the world, and 50–80% of the adults in developed nations are sero- positive for this virus. The virus is transmitted from person to person, primarily via oropharyngeal secretions under conditions of close personal contact, but can also be trans- mitted vertically by intrauterine transmission, by blood transfusion, and by bone marrow and solid organ transplan- tation. For instance, children in day care centers may acquire the virus and spread it to their mothers and other family members. In the healthy child or adult, primary infection with CMV is usually subclinical or results in a very mild disease which resembles infectious mononucleosis. CMV infection can cause serious disease in a number of situations in which the host is immunocompromised or receives a large viral inoculum. Active maternal infection during pregnancy can lead to infection of the fetus with devastating effects such as deafness and developmental delay. Both primary and reactivated latent infections dur- ing pregnancy can lead to the infection of the fetus with CMV. The rate of infection of the primary infection is much greater, at 40%, than the 0.2%–1.8% occurring dur- ing reactivation. CMV is the most common cause of con- genital infection in the United States with an estimated occurrence rate of 0.5%–2.2% of all live births. Immuno- suppressed allograft recipients and patients with acquired immunodeficiency syndrome (AIDS) frequently develop severe CMV infections. CMV can be transmitted by blood transfusion and organ grafts. Seronegative blood should be used for seronegative, pregnant patients, seronegative, low birth weight infants, and seronegative recipients of organs from seronegative donors. DIAGNOSIS AND ASSAY TECHNOLOGY Cell culture is the ‘gold standard’ for diagnosis of CMV disease. It detects virus in samples such as urine, blood, throat washings, and bronchoalveolar lavage. Using the shell vial tissue culture technique allows results of CMV culture to be reported within 18–48h. Enzyme immunoas- says are available to detect CMV pp65 antigen in serum and urine as evidence of active infection, but this approach is being rapidly replaced by polymerase chain reaction (PCR) viral load testing, in plasma or serum, to correlate with the extent of the disease. Serological assays for IgG, IgM, or total (IgG, IgM, and IgA) antibody to CMV are useful in screening, determining susceptibility to primary infection, providing serological evidence of recent infection, and, in certain situations, dif- ferentiating between primary and reactivated infection or reinfection. Latex agglutination tests are simple and quick to perform but they are scored subjectively. Enzyme-linked immunosorbent assays (ELISAs) and bead-based immuno- assays are widely used due to speed, simplicity, and objec- tivity. The principal challenge for serological testing is the identification of active infection, since IgM levels can be persistent, and can also be intermittently positive during viral reactivation. Outside of the USA, avidity testing is commonly performed as a follow-up to a positive IgM result during pregnancy. However avidity tests do not show good concordance with each other and cannot be viewed as a reference method. A recent study performed by the Cen- ter for Disease Control (CDC) documented 58% preva- lence of IgG, 3.0% prevalence of IgM, and 2.0% prevalence of low avidity IgG. While IgG prevalence increased strongly with age, low avidity IgG decreased sharply with age, and was observed mainly in IgM positive sera. In view of the conflicting information available from these tests, both IgM and avidity testing should be used together in the assess- ment of pregnant women. PCR testing of amniotic fluid has also been evaluated but the method is not widely used and may not be sensitive enough to rule out infection. Suggested Reading Adler, S.P. Screening for cytomegalovirus during pregnancy, Infect. Dis. Obstet. Gynecol. (2011). Article ID 194937. Dal Monte, P., Lazzarotto, T., Ripalti, A. and Landini, M. Human cytomegalovirus infection: a complex diagnostic problem in which molecular biology has induced a rapid evolution. Intervirol. 39, 193–203 (1996). Dollard, S.C., Staras, S.A., Amin, M.M., Schmid, D.S. and Cannon, M.J. National prevalence estimates for cytomegalovirus IgM and IgG avidity and association between high IgM antibody titer and low IgG avidity. Clin. Vaccine Immunol. 18, 1895–1899 (2011). Gabbay-Ben Ziv, R., Yogev, Y., Peled, Y., Amir, J. and Pardo, J. Congenital cyto- megalovirus infection following antenatal negative diagnostic amniotic fluid analysis – a single center experience, J. Matern. Fetal Neonatal. Med. (2012). [Epub ahead of print]. Lazzarotto, T., Guerra, B., Lanari, M., Gabrielli, L. and Landini, M.P. New advances in the diagnosis of congenital cytomegalovirus infection. J. Clin. Virol. 41, 192–197 (2008). Mullier, F., Kabamba-Mukadi, B., Bodéus, M. and Goubau, P. Definition of clini- cal threshold for CMV real-time PCR after comparison with PP65 antigen- aemia and clinical data. Acta Clin. Belg. 64, 477–482 (2009). Nelson, C. and Demmler, G. Cytomegalovirus infection in the pregnant mother, fetus, and newborn infant. Infect. Perinatol. 24, 151–160 (1997). Revello, M.G., Genini, E., Gorini, G., Klersy, C., Piralla, A. and Gerna, G. Comparative evaluation of eight commercial human cytomegalovirus IgG avid- ity assays. J. Clin. Virol. 48, 255–259 (2010). C H A P T E R 9.19
  2. 2. 920 The Immunoassay Handbook Epstein-Barr Virus (EBV) ETIOLOGIC AGENT AND CLINICAL MANIFESTATIONS Epstein-Barr virus (EBV) was discovered in 1964 and is a member of the herpesvirus family. Over 95% of the world’s population has been infected with EBV, making it the most ubiquitous virus known. Most people are infected as infants and children in whom the infection may cause a mild sore throat and minor fever for a few days. Transmission of the virus requires direct contact with a person shedding virus, which can be found in saliva. If an individual escapes child- hood without getting infected (generally only in developed countries), the primary infection as an adolescent or adult can be more severe. Infectious mononucleosis (IM) was first recognized as a distinct clinical condition in 1888, but it was not until 1969 that EBV was identified as the cause. About one third of primary infections in adults result in IM, with clinically apparent IM occurring with a frequency of about 45 per 100,000. Symptoms include fever, malaise, swollen lymph nodes, and sometimes splenomegaly. The situation is very different in individuals who have genetic or acquired immunosuppression (transplant recipients, AIDS, X-linked lymphoproliferative syndrome). Some patients who are apparently immunocompetent may develop chronic EBV infections, a condition associated with high viral loads, clonal expansion of EBV-infected T or Natural killer (NK) cells, and increased morbidity. EBV is closely associated with several malignancies. Burkitt lymphoma occurs in about 10 per 100,000 chil- dren in equatorial Africa and New Guinea. Nasopharyngeal carcinoma (NPC) constitutes 20% of all cancers in south- east China. Following receipt of an organ graft, patients may develop post-transplant lymphoproliferative disor- der (PTLD), an aggressive neoplasm that almost always harbors EBV DNA within the neoplastic lymphocytes, and it is often fatal if not recognized and treated promptly. DIAGNOSIS AND ASSAY TECHNOLOGY IM must be distinguished from other more serious condi- tions that produce similar symptoms. These conditions include streptococcal tonsillitis, infectious hepatitis, CMV mononucleosis, Lyme disease, toxoplasmosis, and chronic lymphatic diseases such as Hodgkin’s disease, lymphoma, and leukemia. Traditionally, a first step in the diagnosis of IM is hema- tology. Lymphocytosis occurs with an increase in lympho- cytes and monocytes to 50% or more and atypical lymphocytes represent 10–20% of the lymphocyte and monocyte population. In 1932, Paul and Bunnell discovered that heterophile antibodies in serum from IM patients caused sheep red blood cells to agglutinate. Inclusion of an absorption step using guinea pig kidney a few years later improved speci- ficity. Even though heterophile antibodies have nothing to do with EBV, the cause of IM, heterophile antibody tests remain the principal method to diagnose IM and per- haps the earliest serological test to find routine use in the near-patient setting. Numerous versions of heterophile antibody tests are available commercially, most using agglutination of red cells as the end point. Latex aggluti- nation versions and a few ELISAs are also available. Analysis of the proteins produced by EBV has led to a variety of serological tests for the antibodies produced at various stages of the infection (see Figure 2 in the chapter DETECTION OF ANTIBODIES RELEVANT TO INFECTIOUS DIS- EASE). These tests can be used to differentiate acute disease from prior infection. Tests based on indirect fluorescence (IFA) have been largely replaced by ELISAs in most labo- ratories; these tests use recombinant antigens and offer better reproducibility as well as automation. It is now recognized that the heterophile antibody test is indicative of acute disease, but sensitivity is 60–70% at best. Detection of IgM antibody to the Viral Capsid Anti- gen (VCA) is the most useful test for acute infection, and the IgG antibody for VCA is also seen within a few weeks of the onset of clinical symptoms and typically persists for life. On the other hand, the IgG antibody against Epstein- Barr Nuclear Antigen-1 (EBNA-1) is not seen until 2–3 months after infection and is indicative of convalescence or prior disease. Antibodies against early antigen (EA) have also been measured; it is proposed that re-appearance of IgG antibodies against EA may indicate reinfection. Evaluation of ELISA tests and heterophile antibody meth- ods have been reported in numerous studies. One evaluation in 2000 of 12 commercial tests demonstrated discordance for individual antibody results for numerous samples, although it is generally possible to distinguish acute from past infection when a panel of multiple tests is used. Sensitivity of the indi- vidual tests were >95%. In a 2010 evaluation of three meth- ods used for IgM testing, with immunoblot as a reference method, sensitivity of 84–89% and specificity of 96–98% was reported. In the last decade, automated multiplex tests have been established; the availability of multiple antibody results simplifies the interpretation of a panel of IgM and IgG tests. In situations where the clinical status is unclear, avidity test- ing of anti-VCA IgG or anti-EBNA-1 IgG is often helpful. In areas where nasopharyngeal carcinoma is common, the measurement of IgA antibodies to VCA and EBNA-1 has been very helpful to establish that diagnosis. Virus detection is difficult and rarely done and has little diagnostic value. Because previously infected people can periodically shed virus, it is impossible to determine from the presence of virus alone whether the patient’s symptoms are due to a primary EBV infection or another cause. PCR testing is important for immunocompromised patients but is not used for routine screening. Large changes in viral load may be indicative of reactivation. Also, PCR testing can be used to detect EBV in cerebrospinal fluid (CSF), and diag- nose and monitor patients with PTLD. Suggested Reading Berth, M. and Bosmans, E. Comparison of three automated immunoassay methods for the determination of Epstein-Barr virus-specific immunoglobulin M. Clin. Vaccine Immunol. 17, 559–563 (2010). Bruu, A.L., Hjetland, R., Holter, E., Mortensen, L., Natås, O., Petterson, W., Skar, A.G., Skarpaas, T., Tjade, T. and Asjø, B. Evaluation of 12 commercial tests for detection of Epstein-Barr virus-specific and heterophile antibodies. Clin. Diagn. Lab. Immunol. 7, 451–456 (2000). Germi, R., Lupo, J., Semenova, T., Larrat, S., Magnat, N., Grossi, L., Seigneurin, J.M. and Morand, P. Comparison of commercial extraction systems and PCR assays for quantification of Epstein-Barr virus DNA load in whole blood. J. Clin. Microbiol. 50, 1384–1389 (2012).
  3. 3. 921CHAPTER 9.19 Viral Diseases Gulley, M.L. and Tang, W. Using Epstein-Barr viral load assays to diagnose, monitor, and prevent post-transplant lymphoproliferative disorder. Clin. Microbiol. Rev. 23, 350–366 (2010). Hess, R.D. Routine Epstein-Barr virus diagnostics from the laboratory perspective: still challenging after 35 years. J. Clin. Microbiol. 42, 3381–3387 (2004). Kimura, H., Hoshino, Y., Kanegane, H., Tsuge, I., Okamura, T., Kawa, K. and Morishima, T. Clinical and virologic characteristics of chronic active Epstein- Barr virus infection. Blood 98, 280–286 (2011). Klutts, J.S., Ford, B.A., Perez, N.R. and Gronowski, A.M. Evidence-based approach for interpretation of Epstein-Barr virus serological patterns. J. Clin. Microbiol. 47, 3204–3210 (2009). Odumade, O.A., Hogquist, K.A. and Balfour, Jr. H.H. Progress and problems in understanding and managing primary Epstein-Barr virus infections. Clin. Microbiol. Rev. 24, 193–209 (2011). Okano, M., Thiele, G.M., Davis, J.R., Grierson, H.L. and Purtilo, D.T. Epstein- Barr virus and human diseases: recent advances in diagnosis. Clin. Microbiol. Rev. 1, 300–312 (1988). Paramita, D.K., Fachiroh, J., Haryana, S.M. and Middeldorp, J.M. Two-step Epstein-Barr virus immunoglobulin A enzyme-linked immunosorbent assay system for serological screening and confirmation of nasopharyngeal carci- noma. Clin. Vaccine Immunol. 16, 706–711 (2009). Paul, J.R. and Bunnell, W.W. The presence of heterophile antibodies in infectious mononucleosis. Am. J. Med. Sci. 183, 90–104 (1932). Rea, T.D., Ashley, R.L., Russo, J.E. and Buchwald, D.S. A systematic study of Epstein-Barr virus serologic assays following acute infection. Am. J. Clin. Pathol. 117, 156–161 (2002). Vilibic-Cavlek, T., Ljubin-Sternak, S., Kos, L. and Mlinaric-Galinovic, G. The role of IgG avidity determination in diagnosis of Epstein-Barr virus infection in immunocompetent and immunocompromised patients. Acta Microbiol. Immunol. Hung. 58, 351–357 (2011). Herpes Simplex Virus (HSV) ETIOLOGIC AGENT AND PATHOGENESIS Herpes simplex virus-1 (HSV-1) and HSV-2 are two of the eight known members of the human herpesvirus family. The DNAs of HSV-1 and HSV-2 have considerable homology, and most of the polypeptides expressed by one are also expressed by the other. The identification of specific glyco- protein G (gG) sequences in the 1990s permitted serological differentiation of these two viruses. At one time HSV-1 was considered essentially an orolabial infection and HSV-2 was considered a genital infection but these distinctions are less useful today. HSV-1 infection rates rise steadily through childhood and more than 90% of the population has acquired the infection by adulthood. HSV-2 infection rates are closer to 25% but recurrence is much more common. Prior HSV-1 infection does not offer protection against HSV-2. HSV-2 is acquired through direct contact with virus shed from another person. Infections acquired in the absence of HSV-1 antibody are usually more severe. The onset of symptoms occur 7–10 days after exposure and the symp- toms generally last 7–14 days. Reactivation can be triggered by stress, hormone changes, trauma to the lesion site etc. Asymptomatic genital infections are a significant problem. In addition, 2–5% of all infected individuals are shedding virus asymptomatically at any given time. It is estimated that 70% of HSV-2 transmission occurs during periods of asymptomatic shedding. The most serious consequence of genital herpes infection is to the newborn at the time of delivery. Untreated neonatal herpes has a very high mortality rate (80%) with less than 10% of the survivors developing normally. If the risk for infection is known from the history of the mother, and infec- tion is identified early and treated promptly with acyclovir, the prognosis for the newborn is significantly improved. HSV testing is also required for investigations of central nervous system (CNS) infections, where all herpes viruses may be involved but most commonly HSV or varicella zoster virus (VZV). Herpes simplex encephalitis is the most common cause of sporadic fatal encephalitis in the West- ern world; more than 90% of the cases are due to HSV-1. DIAGNOSIS AND ASSAY TECHNOLOGY Cell culture is the historic ‘gold standard’ for HSV diagnosis, with high sensitivity and specificity. However, turnaround time even with shell vial assays is 2–3 days. Immunoassays were developed to provide faster turn- around times and increased automation. Tests that do not distinguish HSV-1 and HSV-2 were developed in the 1990s and are still offered commercially, but the use of type-specific methods has been recommended by all experts for the past 10 years. The advantage of HSV immunology testing is the ability to detect past infection, in the absence of visible lesions or other symptoms. IgM tests have been offered but do not add much to sensitivity, since the IgG response occurs rapidly and persists indefi- nitely. Point-of-care devices have also been introduced for HSV-2 testing. Multiplex bead-based tests are available to support simultaneous detection and differentiation of HSV-1 and HSV-2. Western blot testing has been widely used as a reference method for IgG testing and shows good concordance with current methods. PCR tests were developed in the early 2000s for diagno- sis of CNS infections, and also for HSV detection from swabs from lesions, because of the rapid turnaround time compared to culture, as well as high sensitivity and speci- ficity. This method is now available for the diagnosis of active infection at a variety of sites, and commercial kits are widely distributed. Suggested Reading Ashley, R.L. Performance and use of HSV type-specific serology test kits. Herpes 9, 38–45 (2002). Binnicker, M.J., Jespersen, D.J. and Harring, J.A. Evaluation of three multiplex flow immunoassays compared to an enzyme immunoassay for the detection and differentiation of IgG class antibodies to herpes simplex virus types 1 and 2. Clin. Vaccine Immunol. 17, 253–257 (2010). Boivin, G. Diagnosis of herpesvirus infections of the central nervous system. Herpes 11 (Suppl 2), 48A–56A (2004). Kimberlin, D.W. Neonatal herpes simplex infection. Clin. Microbiol. Rev. 17, 1–13 (2004). Laderman, E.I., Whitworth, E., Dumaual, E., Jones, M., Hudak, A., Hogrefe, W., Carney, J. and Groen, J. Rapid, sensitive, and specific lateral-flow immuno- chromatographic point-of-care device for detection of herpes simplex virus type 2-specific immunoglobulin G antibodies in serum and whole blood. Clin. Vaccine Immunol. 15, 159–163 (2008). Roett, M.A., Mayor, M.T. and Uduhiri, K.A. Diagnosis and management of genital ulcers. Am. Fam. Physician. 85, 254–262 (2012). Strick, L. and Wald, A. Type-specific testing for herpes simplex virus. Expert Rev. Mol. Diagn. 4, 443–453 (2004). Strick, L.B. and Wald, A. Diagnostics for herpes simplex virus: is PCR the new gold standard? Mol. Diagn. Ther. 10, 17–28 (2006). Dengue ETIOLOGIC AGENT AND PATHOGENESIS The four serotypes of dengue virus are members of the Fla- viviridae family. They have type-common epitopes present on the viral glycoproteins, making accurate serologic diag- nosis difficult. Infection with one serotype does provide lifelong immunity to reinfection with that same serotype,
  4. 4. 922 The Immunoassay Handbook but provides only partial cross-protective immunity to the other three serotypes. The dengue viruses can be transmitted to humans via a number of mosquito species in the genus Aedes. The prin- cipal vector is Aedes aegypti, a small, highly domesticated tropical mosquito that lays its eggs in artificial containers found in and around homes. If the geographic distribution of the secondary vector Aedes albopictus is included, nearly two thirds of the world’s population is at risk for infection. The mobility of the human population has led to the introduction of new serotypes into new regions of the world. During the 1960s, for example, only dengue virus types 2 and 3 were present in America. Dengue type 1 was intro- duced in 1977, it rapidly spread and became endemic in the region. These hyperendemic regions, where multiple strains of dengue virus are circulating, have led to the emergence and spread of dengue hemorrhagic fever (DHF). Uncomplicated, classic dengue fever develops 3–14 days after being bitten by an infected mosquito. Primary dengue infections are always self-limiting and the disease resolves within two weeks of onset. Dengue fever is primarily observed in older children and adults. DHF and dengue shock syndrome (DSS), in con- trast, are predominantly infections seen in children less than 15 years of age. The acute phase of DHF is almost indistinguishable from the acute phase of dengue fever, or the acute phase of any number of illnesses common in dengue endemic regions. In contrast to dengue fever, DHF and DSS can progress rapidly, resulting in death within 8–24h of onset of symptoms. With early recogni- tion of infection and aggressive supportive therapy, par- ticularly with fluid and electrolyte replacement, starting with saline then on to plasma and plasma expanders in more severe cases, the fatality rate can be reduced to less than 1%. Once the shock is overcome, recovery is rapid, usually within 2–3 days. The majority of cases of DHF appear to be the result of a secondary infection with a dengue serotype different than that which caused the primary dengue fever infec- tion. While protective antibody was made to the first virus, cross-reactive antibodies do not neutralize other strains of dengue, thus permitting infection of the second strain. The cross-reactive antibody binds to the virus enhancing uptake into leukocytes through normal Fc receptor bind- ing and uptake functions. This phenomenon is called the Dengue Antibody-Dependent Enhancement. DIAGNOSIS AND ASSAY TECHNOLOGY Traditional serologic methods of hemagglutination inhi- bition (HI), complement fixation, and neutralization have been largely supplanted by a variety of enzyme immunoas- say (ELISA) formats. One of the most widely used formats is an IgM capture ELISA. In primary infection, IgM is present in 80% of patients’ sera by day 5 of illness, increas- ing to 93% by day 10 and 99% by day 20. For this assay, IgM from the patient sera is captured on the solid support by anti-IgM antibodies coated to the support. Once cap- tured, dengue antigen is added with enzyme-labeled monoclonal antibody to the antigen. ELISAs to detect IgG have also been developed and are best used to assess previous exposure to dengue virus. IgG testing alone is not useful for assessing acute disease unless both an acute and a convalescent serum sample are avail- able, to demonstrate a significant increase in antibody titer. Ratios of IgM over IgG are often used to differentiate pri- mary and secondary dengue infection. IgG avidity tests can also be used to distinguish active from past infection, but this approach is not widely performed. In addition to the standard microtiter formats, rapid immunochromatographic assays have been developed to simplify testing in poor resource settings. For this for- mat, a drop of patient serum or blood migrates along a nitrocellulose strip and the IgG and IgM are captured on separate bands where anti-human IgG or anti-IgM anti- bodies have been applied. Diagnosis of a primary dengue infection is made if a band appears for the IgM line, but not the IgG line. Secondary dengue infection is defined by the presence of IgG with or without IgM. There are certain limitations to this approach. The sample during primary infection must be late enough after infection to have detectable IgM, but early enough not to have detect- able IgG, a window of about 5–10 days. The presence of IgG must also be viewed cautiously. The IgG is indica- tive of previous exposure to dengue virus, but its pres- ence does not necessarily prove that the set of symptoms the patient is presenting with is a secondary dengue infection. In the past decade, tests for the NS1 antigen of dengue have been introduced. This antigen, present at high con- centrations and readily detected at the initial onset of fever, can be used to diagnose dengue more quickly than other protein markers. Both sandwich ELISA and strip tests are available to detect NS1. Some studies have suggested that NS1 antigen is the most sensitive marker of disease, with up to 92% sensitivity. However, after the early acute phase, NS1 concentration decreases and the test is less useful for the detection of acute Dengue fever. A combination of NS1 and IgM detection has therefore been recommended to cover a wider range of patients, especially when the time since disease onset may not be known. Use of NS1, IgM, and IgG testing together may also be useful in differentiat- ing acute secondary infection. Reverse transcriptase- polymerase chain reaction (RT-PCR) methods are available but have not been as sensitive as immunological tests in published evaluations and they are not as easily performed in endemic regions. Suggested Reading Blacksell, S.D., Jarman, R.G., Gibbons, R.V., Tanganuchitcharnchai, A., Mammen, Jr. M.P., Nisalak, A., Kalayanarooj, S., Bailey, M.S., Premaratna, R., de Silva, H.J., Day, N.P. and Lalloo, D.G. Comparison of seven commercial antigen and antibody enzyme-linked immunosorbent assays for detection of acute dengue infection. Clin. Vaccine Immunol. 19, 804–810 (2012). Chua, K.B., Mustafa, B., Abdul Wahab, A.H., Chem, Y.K., Khairul, A.H., Kumarasamy, V., Mariam, M., Nurhasmimi, H. and Abdul Rasid, K. A com- parative evaluation of dengue diagnostic tests based on single acute serum samples for laboratory confirmation of acute dengue. Malays. J. Pathol. 33, 13–20 (2011). Gubler, D.J. Dengue and dengue hemorrhagic fever. Clin. Microbiol. Rev. 11, 480– 496 (1998). Guzman, M.G., Halstead, S.B., Artsob, H., Buchy, P., Farrar, J., Gubler, D.J., Hunsperger, E., Kroeger, A., Margolis, H.S., Martínez, E., Nathan, M.B., Pelegrino, J.L., Simmons, C., Yoksan, S. and Peeling, R.W. Dengue: a continu- ing global threat, Nat. Rev. Microbiol. 8 (Suppl) (2010). S7–16. Halstead, S. Pathogenesis of dengue: challenges to molecular biology. Science 239, 476–481 (1988).
  5. 5. 923CHAPTER 9.19 Viral Diseases Peeling, R.W., Artsob, H., Pelegrino, J.L., Buchy, P., Cardosa, M.J., Devi, S., Enria, D.A., Farrar, J., Gubler, D.J., Guzman, M.G., Halstead, S.B., Hunsperger, E., Kliks, S., Margolis, H.S., Nathanson, C.M., Nguyen, V.C., Rizzo, N., Vázquez, S. and Yoksan, S. Evaluation of diagnostic tests: dengue. Nat. Rev. Microbiol. 8 (Suppl), S30–S38 (2010). Whitehorn, J. and Simmons, C.P. The pathogenesis of dengue. Vaccine 23, 7221– 7228 (2011). Rubella ETIOLOGIC AGENT AND PATHOGENESIS Rubella virus (also known as German measles) is the sole member of the genus Rubivirus within the Togaviridae family. Although experimental infections can be induced in a variety of laboratory animals, man is the only known natural reservoir for rubella virus. Postnatal primary infections, acquired by inhalation of aerosols, are nearly always mild. Within a week after exposure, virus can be found in both the blood and naso- pharynx; the latter being responsible for the spread of the virus from one individual to another. Symptoms of pri- mary rubella infection may include a rash (the hallmark of clinical rubella), low-grade fever, lymphadenopathy, sore throat, conjunctivitis, and arthralgia. Joint involve- ment is very common in adults, particularly among women. Serious sequelae are rare but CNS involvement and thrombocytopenia have been reported. Primary rubella infection during pregnancy can be devas- tating. Viremia associated with primary infection leads to infection of the placenta and fetus, which can lead to fetal death. Those infants surviving fetal infection may exhibit one or more of a variety of symptoms known collectively as congenital rubella syndrome (CRS), which include low birth weight, deafness, eye disease, mental retardation, car- diac abnormalities, hepatomegaly, splenomegaly, and throm- bocytopenia. Virus may be isolated from virtually every organ and is shed in urine and nasopharyngeal secretions; a few infants will continue to shed virus for well over a year. Infants born without apparent symptoms may develop late-onset disease, months to years later, which includes hearing loss, mental retardation, retinopathy, and diabetes mellitus. The risk of CRS or late-onset disease is as high as 85% if infection occurs during the first trimester, falls to 10–24% during the 13th to 16th weeks and approaches 0% beyond the 20th week of gestation. The introduction of a vaccine in 1969 has greatly reduced the risk of rubella dur- ing pregnancy in developed countries, but it is remains a significant problem in other areas. DIAGNOSIS AND ASSAY TECHNOLOGY Serology plays a critical role in the diagnosis of rubella for two main reasons. First, 30% of primary infections are subclinical; and second, other rash-inducing illnesses may confuse a diagnosis of acute, primary rubella infection. Infection with parvovirus B19 is frequently impossible to distinguish from rubella; it is nonteratogenic but is associ- ated with a high incidence of miscarriage. HHV 6 can also cause rash and fever in children. In many parts of the trop- ics, alpha viruses and flaviviruses, including Dengue, may induce rubella-like illness. A primary immune response, consisting both of IgG and IgM antibodies, can be detected at about the same time as the rash. IgM antibody usually falls to undetectable or very low levels within 4–6 months; however, in some cases, it may remain at detectable levels for a year or more. IgG antibody may eventually decline to a low level but lasts indefinitely. Diagnosis of rubella infection and the determination of an individual’s immune status are accomplished with sero- logical assays detecting antibody in serum samples. Hem- agglutination inhibition (HAI) has largely been replaced by passive enzyme-immunoassays. Some ELISAs provide quantitative results expressed in international units (IU) of antibody per mL referenced to the World Health Organi- zation International Standard for Anti-Rubella Serum. Use of the appropriate assay provides data required in the three principal areas of rubella serodiagnosis: immune status screening, detection of recent primary infection, and diagnosis of congenital rubella syndrome. Immune Status Screening It is generally agreed that levels of antibody detectable by HAI, about 10–15IU/mL, are protective. Antibodies at or above these levels are sufficient to protect an individual from clinical reinfection; levels of antibody below 10–15IU/mL may not prevent clinical rubella after exposure. CRS in babies born to mothers with detectable antibody prior to conception has been reported but is very rare. Today IgG tests are widely used to establish immune status. Sensitivity is typically 99%. Specificity is harder to define, since low levels that were below the detection limit of HAI are observed, with considerable variability, by the more sensitive modern methods. Serodiagnosis of Recent Primary Infection Historically, primary infection was established using two serum samples. The first sample was drawn from the patient during the acute stage of the disease and a second, the con- valescent sample, about 10–14 days later. A fourfold, or greater, rise in HAI titer indicated recent exposure. IgG- specific ELISAs available today may be used in a similar manner if the equivalent of a fourfold rise in HAI titer for that assay has been defined. However, paired sera testing only differentiates primary from secondary rubella infec- tions if the acute sample is seronegative and the convalescent sample is positive, demonstrating seroconversion. Primary infection induces both an IgG and a significant IgM antibody response. The secondary immune response induced by secondary rubella infection is characterized by rising IgG antibody in the absence of significant IgM anti- body. The presence of clinically significant levels of IgM antibody is serodiagnostic evidence of recent, primary rubella infection. Because of the relatively short duration of IgM antibody, the time at which a sample is drawn after exposure is critical. IgG avidity has also been reported as an aid to differentiate primary and secondary infections and is available in a commercial kit. Serodiagnosis of Congenital Rubella Syndrome Maternal IgM antibody induced by primary rubella infec- tion does not cross the intact placenta as does maternal
  6. 6. 924 The Immunoassay Handbook IgG. The infected fetus develops IgM antibody to the virus and detection of that antibody in the neonate aids in the diagnosis of CRS. A sample should be drawn from the neonate as soon as possible following parturition. Com- parison of IgG antibody in the neonate at the time of birth and 6 months later also aids in the diagnosis of CRS. A significant drop in antibody over that time interval sug- gests decreasing maternal antibody in the absence of peri- natal or prenatal infection. Pre- or perinatal infection should be suspected if stable or increasing IgG antibody levels are detected. Little data are available comparing Rubella RNA detec- tion by PCR with IgM detection, since outbreaks are rare. One 2008 study in Peru reported that IgM testing is more sensitive for samples collected at the time of rash onset but gave equivalent performance after 3–4 days. Since virus levels in blood drop rapidly after rash onset, PCR may be less useful to establish this diagnosis. Suggested Reading Abernathy, E., Cabezas, C., Sun, H., Zheng, Q., Chen, M.H., Castillo-Solorzano, C., Ortiz, A.C., Osores, F., Oliveira, L., Whittembury, A., Andrus, J.K., Helfand, R.F. and Icenogle, J. Confirmation of rubella within 4 days of rash onset: comparison of rubella virus RNA detection in oral fluid with immunoglobulin M detection in serum or oral fluid. J. Clin. Microbiol. 47, 182–188 (2009). Banatvala, J.E. and Brown, D.W. Rubella. Lancet 363, 1127–1137 (2004). Dimech, W., Panagiotopoulos, L., Francis, B., Laven, N., Marler, J., Dickeson, D., Panayotou, T., Wilson, K., Wootten, R. and Dax, E.M. Evaluation of eight anti-rubella virus immunoglobulin g immunoassays that report results in inter- national units per milliliter. J. Clin. Microbiol. 46, 1955–1960 (2008). Morice, A., Ulloa-Gutierrez, R. and Avila-Agüero, M.L. Congenital rubella syn- drome: progress and future challenges. Expert Rev. Vaccines 8, 323–331 (2009). Vauloup-Fellous, C., Ursulet-Diser, J. and Grangeot-Keros, L. Development of a rapid and convenient method for determination of rubella virus-specific immu- noglobulin G avidity. Clin. Vaccine Immunol. 14, 1416–1419 (2007). Measles, Mumps, and Varicella Mumps, measles, and varicella-zoster virus infections are typically clinical diagnoses and laboratory tests are rarely required. Although at one time nearly eradicated, mumps and measles outbreaks now regularly occur in developed countries, as many families choose to decline vaccination. IgG testing is important to establish immunity, especially for healthcare workers; ELISAs and multiplex methods (which also measure rubella) are widely available for this purpose. IgM testing is available by ELISA but cannot be used for screening since the incidence of these diseases is so low and the positive predictive value is poor. IgM can be useful in tracking an outbreak and IgG avidity testing has been used to identify vaccination failures. PCR testing is also available for identifying active infections but is not widely employed. Suggested Reading Arvin, A.M. Varicella-zoster virus. Clin. Microbiol. Rev. 9, 361–381 (1996). Hviid, A., Rubin, S. and Mühlemann, K. Mumps. Lancet 371, 932–944 (2008). Mosquera, M.M., de Ory, F., Gallardo, V., Cuenca, L., Morales, M., Sánchez-Yedra, W., Cabezas, T., Hernández, J.M. and Echevarría, J.E. Evaluation of diagnostic markers for measles virus infection in the context of an outbreak in Spain. J. Clin. Microbiol. 43, 5117–5121 (2005). Moss, W.J. and Griffin, D.E. Measles. Lancet 379, 153–164 (2012). Park, D.W., Nam, M.H., Kim, J.Y., Kim, H.J., Sohn, J.W., Cho, Y., Song, K.J. and Kim, M.J. Mumps outbreak in a highly vaccinated school population: assess- ment of secondary vaccine failure using IgG avidity measurements. Vaccine 25, 4665–4670 (2007). Tipples, G. and Hiebert, J. Detection of measles, mumps, and rubella viruses. Methods Mol. Biol. 665, 183–193 (2011). Human T-Cell Leukemia Virus (HTLV) Human T-cell leukemia virus (HTLV-1) was the first human retrovirus discovered, and shortly thereafter, HTLV-2 was described. It is estimated that 20million people worldwide are infected with HTLV-1, although 90% are asymptomatic. HTLV-1 infection can result in subclinical immunosuppression such that infected individ- uals are at increased risk for development of opportunistic coinfections, including but not limited to strongyloidiasis and tuberculosis. Approximately 5% of infected individu- als will go on to develop HTLV-1 associated manifesta- tions, typically following an incubation period of 10–40 years. Adult T-cell leukemia/lymphoma is an aggressive lymphoma of the skin and most viscera, including the liver, spleen, and lymph nodes. The other major clinical mani- festation is HTLV-1 associated myelopathy (HAM) or tropical spastic paraparesis (TSP) which is a chronic neurological disorder due to progressive demyelination in the spinal cord. HTLV-1 uveitis and dermatological con- ditions have also been reported. HTLV-2 causes hairy T-cell leukemia, although this is rare. Japan, Africa, Central and South America, and the Caribbean islands are the areas with the highest prevalence of HTLV-1. The most common routes of HTLV-1 trans- mission are sexual transmission, intravenous drug abuse, and blood transfusion. Mother to child transmission via breastfeeding is also considered an important route of transmission. The risk of HTLV-1 transmission during blood trans- fusion varies with the prevalence of the virus in the specific blood donor population, but prevention of transfusion- associated HTLV-1 transmission has been a major focus of public health efforts. Programs throughout the world have been initiated to screen blood donors to prevent transmission of HTLV-1 via blood transfusion. The Cen- ters for Disease Control and Prevention recommended screening for HTLV-1 in 1988, and several countries fol- lowed with similar recommendations in subsequent years. In the United States, all blood donors have been routinely screened for both HTLV-1 and HTLV-2 antibodies since 1997. As a result of widespread donor screening, the resid- ual risk of transfusion-associated HTLV-1 infection in low prevalence countries is now minimal. The most common screening tests for HTLV are enzyme immunoassays (EIA) and particle agglutination (PA) assays performed on serum or plasma. Typically the EIA assays detect HTLV-1 and -2, while the PA is HTLV-1 specific. PA assays are somewhat more subjective as the interpretation is a visual interpretation by the test operator. In addition, there are no FDA licensed PA assays, so this test is not used in the United States. Modern EIA assays use either HTLV-1 and -2 infected cell lysates or recombinant antigens, and have excellent sensitivity and
  7. 7. 925CHAPTER 9.19 Viral Diseases specificity. Analysis of commercially available screening assays for HTLV-1 and HTLV-2 suggests that sensitivity ranges from 98 to 100% and specificity ranges from 90 to 100%. A positive screening test should be followed with a more specific confirmation test; the assays most commonly used for this purpose are western blot or immunofluores- cence assays. These assays are also used to differentiate HTLV-1 from HTLV-2. The most commonly used ver- sion of the western blot is a “second generation” western blot. Specimens that react with p19gag, r21e, and recom- binant gp461 are typical for HTLV-1, and specimens that are positive for reactivity with p24 gag, r21e, and gp46II are consistent with HTLV-2 infection. Specimens without reactivity to any of the western blot antigens are consid- ered to be negative for HTLV, consistent with a false- positive EIA screening assay. If indeterminate results are obtained during confirma- tory testing, these are most commonly due to the individual being in the “window period” early in infection before fully seroconverting, or a false-positive screening EIA result due to a non-specific reaction of the patient’s serum to viral antigens. These indeterminate results are typically resolved using PCR for the detection of HTLV-1 proviral DNA. Suggested Reading Andersson, S., Thorstensson, R., Ramirez, K.G., Krook, A., von Sydow, M., Dians, F. and Biberfeld, G. Comparative evaluation of 14 immunoassays for detection of antibodies to the human T-lymphotropic virus types I and II using panels of sera from Sweden and West Africa. Transfusion 39, 845–851 (1999). Goncalves, D.U., Proiette, F.A., Ramos Ribas, J.O.G., Grossi, M., Pinheiro, S.R., Guedies, A.C. and Carneiro-Proiettei, A.B.F. Epidemiology, Treatment, and Prevention of Human T-Cell Leukemia Virus 1-associated diseases. Clin. Micro. Rev. 23, 577–589 (2010). Guidance for Industry Donor Screening for Antibodies to HTLV-2 http://www.fda.gov/ downloads/BiologicsBloodVaccines/GuidanceComplianceRegulatory Information/Guidances/Blood/UCM170916.pdf (1997) . Qiu, X., Hodges, S., Lukaszewka, T., Hino, S., Arai, H., Yamaguchi, J., Swanson, P., Schochetman, G. and Davare, S.G. Evaluation of a new, fully automated immunoassay for detection of HTLV-1 and HTLV-2 antibodies. J. Med. Virol. 80, 484–493 (2008). Thorstensson, R., Albert, J. and Anderson, S. Strategies for diagnosis of HTLV-1 and -2. Transfusion 42, 780–791 (2002). Watanabe, T. Current Status of HTLV infection. Int. J. Hematol. 94, 430–434 (2011). Parvovirus B19 Parvovirus B19 is a single-stranded, nonenveloped Eryth- rovirus. The virus is so named because of its capacity to autonomously replicate in erythroid precursor cells. B19 is found worldwide and has been associated with a late winter, spring, and early summer seasonality. The virus is spread from person to person by droplets containing viral particles which are produced during a cough or a sneeze. Parvovirus B19 disease manifests in several different ways depending on the immune status of the host. The most common manifestation of disease is erythema infec- tosum (EI) or Fifth’s Disease which primarily affects immunocompetent school-aged children. Parvovirus B19 also causes EI in adults but has also been associated with joint manifestations in these patients. Only a minority of adults experiences the facial exanthema (rash) seen in chil- dren. Parvovirus can also cause transient aplastic crisis in patients with low erythrocyte counts. This can lead to a potentially life-threatening drop in hemoglobin. Immuno- compromised patients lacking functional humoral immu- nity can be persistently infected with parvovirus B19, leading to bone marrow suppression and chronic anemia. Lastly, parvovirus can cause fetal hydrops when nonim- mune pregnant women get their primary infection in the first or second trimester. Molecular and serological methodologies exist for diag- nosing parvovirus infection, and their respective utilities are dependentonthespecificconditionbeingdiagnosed.Nucleic acid amplification techniques can be either qualitative or quantitative and detect viral DNA. Serologic tests detect B19-specific IgM and IgG antibody directed against viral capsid antigen. In the United States, these assays are most commonly EIA, but IFA and western blot assays do exist. PCR is useful in patients with aplastic crisis because high levels of circulating virus are usually present. How- ever, PCR is rarely positive in children with EI and serol- ogy is the preferred method of diagnosis. EI patients will generally have detectable IgM within 7–10 days of expo- sure, which remains detectable for 2–3 months but can sometimes be found out to 6 months postinfection. There- fore, presence of IgM is suggestive but not absolutely diag- nostic for acute infection. Demonstrating a ≥4-fold rise in IgG titer can also be used to diagnose acute infection. In immunocompromised patients with chronic infection and sustained erythropenia, parvovirus IgM and IgG are typically not detectable and PCR is preferred. It is impor- tant to note that levels of viremia can be low in these patients so PCR sensitivity varies. The most important role for parvovirus serology is in screening pregnant women. Parvovirus naive women who acquire their primary parvovirus B19 infection have about a 30% risk of vertical transmission to the baby. The risk appears to be greatest if the infection occurs during the first or second trimester. Pregnant women who are exposed develop signs and symptoms of parvovirus B19 infection, and IgG and IgM tests should be carried out for evidence of parvovirus infection. IgG positive, IgM negative results suggest past infection and that the mother is immune to future parvovirus infection. IgG negative, IgM positive may suggest acute infection or a falsely positive IgM result. During acute infection, there is only a short window of time when IgM is positive and IgG is negative. Thus, this is a relatively rare finding. Pregnant women should have repeat titers drawn in 1–2 weeks, and if IgG seroconver- sion occurs, this is suggestive of an acute infection. If both IgG and IgM are negative, the IgM was likely a false- positive. Nucleic acid amplification assays performed on amniotic fluid are also occasionally employed in this situa- tion to help resolve serological findings. Suggested Reading Dijkmans, A.C., de Jong, E.P., Dijkmans, B.A., Loprior, E., Vossen, A., Walther, F.J. and Oepkes, D. Parvovirus B19 in pregnancy: prenatal diagnosis and man- agement of fetal complications. Curr. Opin. Obstet. Gynecol. 24, 95–101 (2012). De Jong, E.P., de Haan, T.R., Kroes, A.C., Beersma, M.R., Oepkes, D. and Wlahter, F.J. Parvovirus B19 infection in pregnancy. J. Clin. Virol. 36, 1–7 (2006). De Jong, E.P., Wlather, F.J., Kroes, A.C. and Oepkes, D. Parvovirus B19 infection in pregnancy: new insights and management. Prenat. Diagn. 31, 419–425 (2011).
  8. 8. 926 The Immunoassay Handbook West Nile Virus West Nile Virus (WNV) is a member of the Japanese encephalitis serogroup in the family Flaviviridae. It is an enveloped, single-stranded, positive-sense RNA virus. WNV is transmitted primarily between birds and mosquitoes, and while it can infect a wide variety of bird species, the Passeri- formes (which includes crows, blackbirds, finches, and spar- rows amongst others) appear to be a particularly important reservoir for disease. In contrast to birds, infection in humans results in a much lower viremia and therefore, disease is not normally transmitted from humans. Based on epidemiological surveys, most infections are asymptomatic in humans. The incubation period for WNV disease is 2–14 days, after which time symptomatic indi- viduals will develop some or all of the following symptoms: high fever with headache, myalgias, arthralgias, and eye- pain. Symptoms present acutely and may also include a rash, hepatomegaly, and splenomegaly. Patients who will go on to develop neurologic symptoms, experience a febrile prodrome period of 1–7 days prior to onset of neurologic symptoms. WNV neurologic symptoms can manifest as either encephalitis (~66%) or meningitis (~33%). WNV infection is primarily diagnosed using serologic methods. Direct detection via PCR of blood or cerebrospi- nal fluid is usually negative because viral loads are low by the time patients develop symptoms. One study estimated that PCR-based detection only captured 55% of infections. The diagnostic test of choice is the detection of WNV-specific IgM antibody in either the serum or CSF. IgM detected in the CSF is diagnostic for neurologic infection. Many patients will have positive serology at the time of admission (~50%) and nearly all will be positive 7 days post-admission. The preferred method of detecting West Nile specific IgM is an IgM-specific ELISA otherwise referred to as the MAC ELISA (MAC: IgM Antibody Capture). This assay is more than 95% sensitive if done on samples collected between 7 and 10 days after the onset of illness. In cases of suspected encephalitis it is important to analyze both the serum and CSF, as CSF IgM antibodies may develop earlier. Although sensitivity for infection is very high, IgM antibodies to WNV can linger in the serum for up 16 months, which lim- its specificity. This assay can cross-react with other flavivi- ruses and so the CDC recommends that positive results be confirmed with a neutralization test. The plaque reduction and neutralization test (PRNT) is a specific test that allows for identification of virus specificity. Suggested Reading Solomon, T., Ooi, M.H., Beasley, D.W. and Mallewa, M. West Nile encephalitis. BMJ 326, 865–869 (2003). Centers for Disease Control and Prevention. Epidemic/Epizootic West Nile Virus in the United States: Guidelines for Surveillance, Prevention and Control. (2003) 3rd Revision. De Filette, M., Ulbert, S., Diamond, M. and Sanders, N.N. Recent progress in West Nile virus diagnosis and vaccination. Vet. Res. 43 (2012). Rotavirus Rotavirus is a double capsid and double-stranded segmented RNA virus in the family Reoviridae, with at least 7 distinct antigenic groups (A through G). Group A are the major cause of rotavirus diarrhea worldwide. Infection with rotavi- rus causes nonbloody diarrhea, often preceded or accompa- nied by fevers, and symptoms generally last 3–8 days. Rehydration therapy may be required for patients with severe infection. The most common patient population affected is children less than 5 years of age. While mortality due to Rotavirus is very low in developed nations, in devel- oping countries Rotavirus is a major cause of mortality. The route of infection is fecal–oral; infection typically results from direct or indirect contact with infected people. Rota- virus is present at a high titer in stools of infected patients and can persist in the stool for as long as 21 days after the onset of symptoms in immunocompetent hosts (longer in immunocompromised). Rotavirus can be found on toys and hard surfaces in day care centers; spread within families and institutions is common. In temperate climates, Rotavirus is most prevalent during the cooler months. Seasonal varia- tion is less pronounced in tropical climates. No specific antiviral therapy exists for rotavirus. Fluid replacement can be given to prevent dehydration, especially in infants. The monovalent rotavirus vaccine (RV1; Rotarix) and pentavalent rotavirus vaccine (RV5; RotaTeq) have been approved for use in many countries. Widespread vaccina- tion has dramatically altered the epidemiology of rotavirus in areas where vaccination has occurred. Prior to the vac- cine, virtually all children were infected by 3 years of age. Historically, electron microscopy was used to identify Rotavirus in stool. Today EIA and latex agglutination assays for the detection of rotavirus antigen in stool are commercially available and commonly used for detecting rotavirus infection. In addition to improved sensitivity (close to 100%) these immunoassays are much easier to perform than electron microscopy, result in improved turnaround time, and are considered the Rotavirus diag- nostic test of choice. The specificity of these assays is not perfect (80–99%) and Rotavirus does exhibit seasonal vari- ation in temperate countries, with a typical winter–spring peak. Therefore, the positive predictive value of Rotavirus EIA testing drops dramatically when these tests are per- formed on stool samples outside the Rotavirus season. This is important for clinical laboratories to consider when designing testing algorithms for gastroenteritis. In addi- tion, Rotavirus EIA assays can remain positive for many days following resolution of symptoms. Nucleic acid amplification tests are also in development for Rotavirus diagnostics, and some labs are using labora- tory-developed tests for this purpose, sometimes as part of a multiplex panel to detect multiple agents of gastroenteritis simultaneously. Suggested Reading Bernstein, D.P. Rotavirus overview. Pediatr. Infect. Dis. J. 28, S50–S53 (2009). Bodo, R., Guenter, E., Horst, M.A., Baumeister, G. and Kuhn, J.E. Evaluation of two enzyme immunoassays for detection of Human Rotaviruses in fecal speci- mens. J. Clin. Microbiol. 39, 4532–4534 (2001). Dennehy, P.H. Effects of vaccine on rotavirus disease in the pediatric population. Curr. Opin. Pediatr. 24, 76–84 (2012). Greenberg, H.B. and Estes, M.K. Rotaviruses: from pathogenesis to vaccination. Gastroenteriol. 136, 1939–1951 (2009). Kenswick, B.H., Hejkalk, T.W., DuPonta, H.L. and Pickeringa, L.K. Evaluation of a commercial enzyme immunoassay kit for rotavirus detection. Diag. Microbiol. Infect. Dis. 1, 111–115 (1983).
  9. 9. 927CHAPTER 9.19 Viral Diseases Wolffs, P.F., Bruggeman, C.A., vanWell, G.T. and van Loo, I.H. Replacing tradi- tional diagnostics of fecal viral pathogens by a comprehensive panel of real- time PCRs. J. Clin. Microbiol. 49, 1926–1931 (2011). Yolken, R.H. and Leister, F. Rapid Multiple-Determinant Enzyme Immunoassay for the detection of Human Rotavirus. J. Infect. Dis. 146, 43–46 (1982). Adenovirus Human adenoviruses are members of the Adenoviridae family. Adenoviruses are large, nonenveloped icosahedral viruses that are 70–90nm in diameter. Each particle con- sists of a single linear, double-stranded DNA molecule. There are seven recognized species (A through G) based on immunologic and genetic properties. Within each spe- cies there are many serotypes, and about half of the known serotypes cause disease. Adenovirus infections are com- mon and ubiquitous, and this virus can result in a wide variety of clinical manifestations, including gastroenteritis, ocular infections, and, less commonly, infections of the urinary tract, CNS, and liver. Adenovirus is an important cause of respiratory infections in both immunocompro- mised and immunocompetent individuals. The specific manifestations and clinical course of adenovirus infection vary depending on the age and immune status of the patient. In immunocompromised hosts (such as stem cell transplant or solid organ transplant patients) disseminated adenovirus can result in serious systemic infections with disease manifestations including hemorrhagic cystitis, pneumonia, hepatitis, viremia, and disseminated disease. Adenoviruses are highly resistant to inactivation by chemi- cal and physical treatment; most serotypes are stable at 35°C for a week, room temperature for several weeks, and for several months at 4°C. It has been estimated that adenoviruses account for 5–15% of diarrheal illnesses in children; specifically, ade- noviruses 40 and 41 are an important cause of gastroen- teritis in children less than 2 years of age. The stool is usually watery and nonbloody, lacks fecal leukocytes, and diarrhea lasts a mean of 10 days. Vomiting, mild fever, and abdominal pain may also be present. The disease is typi- cally self-limiting and resolves without sequelae. Although the gold standard for adenovirus detection is usually con- sidered to be viral culture, the enteric adenoviruses (40 and 41) do not grow well in culture. Therefore, if a laboratory confirmation of adenovirus gastroenteritis is required, EIA for adenovirus 40/41 antigen detection can be used to detect this virus directly in stool samples; these assays are commercially available. Compared to electron micros- copy, the sensitivity and specificity of these assays is thought to be >90% and >97%, respectively. In immuno- competent individuals, viral shedding is not typically pro- longed after symptoms resolve and a positive adenovirus EIA is suggestive of acute infection. “Generic” lateral-flow immunoassays that are thought to detect most types of adenovirus are also commercially available; these tests can be performed on ocular swabs, respiratory secretions, and urine. The rapid turnaround time and ease of use makes this assay format an attractive option, but the performance characteristics of these assays are suboptimal. Namely, these assays lack sensitivity, with values ranging from 55 to 85% reported. The sensitivity of these tests is degraded even further if ocular swabs are placed into viral transport medium prior to analysis. Therefore these assays have not been widely adopted. Typical signs and symptoms of adenoviral respiratory tract infection include fever, nasal congestion, cervical ade- nopathy, pharyngitis, and cough. Direct fluorescent anti- body (DFA) reagents are widely used for the detection of adenoviral antigens in respiratory specimens, such as nasal washes, nasal aspirates, nasopharyngeal swabs, and bron- chial alveolar lavage, and tracheal aspirates. The reported sensitivity for DFA of adenovirus in respiratory specimens is approximately 40–60% compared to viral culture, which is lower than the DFA sensitivity for most other respiratory viruses. Sensitivity is higher in specimens collected from pediatric patients (who typically shed higher concentrations of virus) compared to specimens from adults. PCR is emerging as an additional diagnostic mode for adenovirus gastroenteritis, respiratory infections, and dis- seminated adenoviral disease, but challenges exist in designing PCR assays that are efficient at amplification of all serotypes that could be implicated in infection from a particular specimen type. In addition, quantitative adeno- virus PCR can be used in disseminated infections in immunocompromised patients to assess viral load and response to therapy. It is likely in the future that PCR will be the mainstay of adenovirus detection in both stool and respiratory specimens. Suggested Reading Adenovirus infections. In: Red Book: Report of the Committee on Infectious Diseases, 28th edn, (eds Pickering, L.K., Baker, C.J., Kimberlin, D.W. and Long, S.S.), 204–206 (American Academy of Pediatrics, Elk Grove Village, Illinois, USA, 2009). August, M.J. and Warford, A.L. Evaluation of a commercial monoclonal antibody for detection of adenovirus antigen. J. Clin. Microbiol. 25, 2233–2253 (1987). Damen, M., Minnaar, R. and Glasius, P. Real-time PCR with an internal control for detection of all known human adenovirus serotypes. J. Clin. Microbiol. 46, 3997 (2008). Herrmann, J.E., Perron-Henry, D.M. and Blacklow, N.R. Antigen detection with monoclonal antibodies for the diagnosis of adenovirus gastroenteritis. J. Infect. Dis 155, 1167–1171 (1987). LaSala, P.R., Bufton, K.K., Ismail, N. and Smith, M.B. Prospective comparison of R-mix shell vial system with direct antigen tests and conventional culture for respiratory virus detection. J. Clin. Virol 38, 210–216 (2007). Levent, F., Greer, J.M., Snider, M. and Demmler-Harrison, G.J. Performance of a new immunochromatographic assay for detection of adenoviruses in children. J. Clin. Virol. 44, 173 (2009). Mahafazah, A.M. and Landry, M.L. Evaluation of immunofluorescent reagents, centrifugation, and conventional cultures for the diagnosis of adenovirus infec- tion. Diagn. Microbiol. Infect. Dis. 12, 407–411 (1989). Russell, W.C. Update on adenovirus and its vectors. J. Gen. Virol. 81, 2573–2604 (2000). Christenson, M.L. Human viral gastroenteritis. Clin. Micro. Rev. 2, 51–89 (1989). Sambursky, R., Tauber, S., Schirra, F., Kozich, K., Davidson, R. and Cohen, E.J. The RPS adeno detector for diagnosing adenoviral conjunctivitis. Ophthalmology 113, 1758–1764 (2006).

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