Serologic Testing

Field: Medicine (Health Sciences)

Syphilis. French P. BMJ. 2007;334:143–147Diagnostic Evaluation of Mononucleosis-like Illnesses. Hurt C, Tammaro D. Am J Med. 2007;120:911.e1–8Notice to Readers: Caution Regarding Testing for Lyme Disease.MMWR Morbid Mortal Wkly Rep. 2005;54:125Newborn Screening for Congenital Infectious Diseases. Neto EC, Rubin R, Schulte J, Giugliani R. Emerg Infect Dis. 2004;10:1069–1073Diagnosing infectious diseases involves the same skills as police and detectives use in their work. The investigator must identify the culprit, based on evidence at the scene, to bring the case to a successful close. Unfortunately, aficionados of mystery novels and television crime shows know that the sleuth rarely finds the criminal at the scene of the crime, with weapon in hand. Instead, solving the crime usually requires the tedious collection of specific evidence that links the suspect to the deed.In medicine, we have the ability to culture many organisms, giving definitive proof of the presence of a pathogen at the time of an illness. However, this technique is not available for many microorganisms that cause human disease. Many bacteria, of several different categories, are difficult to culture in the microbiology laboratory. Among them are spirochetes, such as Borrelia burgdorferi (Lyme disease), B recurrentis (relapsing fever), Leptospira sp (leptospirosis), and Treponema pallidum (syphilis, pinta, yaws, bejel). Another group includes intracellular pathogens such as Brucella sp (brucellosis), Bartonella sp (cat-scratch disease), and Mycoplasma sp (walking pneumonia), as well as rickettsiae, which are obligate parasites and cause spotted fevers (Rocky Mountain spotted fever and rickettsial pox), typhus, and scrub typhus. Finally, some bacterial species are not isolated by routine techniques in the microbiology laboratory, such as Legionella sp (legionellosis), Francisella tularensis (tularemia), and Bordetella sp (pertussis, parapertussis).Similarly, most microbiology laboratories are not able to culture viruses routinely, necessitating the use of other assays. Viruses that commonly infect children and are especially difficult to isolate, even by using sophisticated cell culture techniques, include Epstein-Barr virus (EBV); hepatitis A, B, and C viruses; parvovirus B19; and many arthropod-borne viruses widespread throughout the world.Furthermore, even as newer antigen- and molecular-based assays have been developed, many organisms remain difficult to identify by using these techniques. Some spirochetes, such as Treponema sp, have very few outer surface proteins to serve as antigens. As a side note, although they are sensitive tests, the Venereal Disease Research Laboratory Test, rapid plasma reagin test, and microhemagglutinin assay are not serologic tests for syphilis. Rather, they are nontreponemal tests, which actually measure cardiolipin antibodies that rise as Treponema incorporate cardiolipin into their cell walls. Similarly, the heterophile or monospot tests do not measure anti-EBV antibodies directly. Tests of the serum and extracellular milieu may fail to detect or interact with intracellular organisms and viruses. Therefore, clinicians must rely on an alternative approach, using more circumstantial evidence, namely, serologic testing.The basis of serologic testing is the measurement of antibodies, which the immune system develops in response to the presence of a pathogen. If a reliable immunoglobulin (Ig) M assay is available for a given pathogen, a single sample may be diagnostic. Otherwise, a paired convalescent titer drawn at a later date is needed to document an increase in specific IgG, which limits the usefulness of such testing in evaluating an acute illness or its initial ability to distinguish acute infection from past exposure. Because this methodology is an indirect method of identifying a pathogen, the strength of a serologic test depends primarily on how accurate it is. Statistically, this accuracy is indicated by the test's sensitivity (the probability of a positive test in patients who have the disease) and specificity (the probability of a negative test in patients who do not have the disease). To continue the crime analogy, accuracy is the equivalent of the reliability and sobriety of the witness.Additional problems limit the applicability of serologic testing. For some localized infections, such as herpes simplex virus type 2 (HSV-2), there may be no significant humoral antibody response. B burgdorferi can cause clinical Lyme disease before measurable antibodies appear, and antibiotic treatment may blunt the normal antibody response. Cross-reactivity between antigens from related organisms such as rickettsiae, HSV, and varicella-zoster virus as well as Japanese B encephalitis virus and dengue virus limits specificity. Some infections, such as those caused by EBV, cytomegalovirus, and Mycoplasma, cause oligoclonal stimulation of the immune system, creating an increase in a broad range of antibodies and, therefore, multiple false-positive results. For patients who have EBV infection and autoimmune and connective tissue diseases, nonspecific reactions such as rheumatoid factor can cause false-positive IgM reactions, and the passive transfer of antibodies in transfused blood products can result in false-positive serologic tests. When serologic tests can detect only the presence of antibodies without distinguishing IgM from IgG, paired convalescent titers must be drawn weeks apart to document acute rather than past infection, delaying the diagnosis. Finally, if certain antibodies have a high seroprevalence in the general population, such as for Lyme disease in some geographic areas, the relevance of a positive serologic test can be difficult to determine for a given illness. It always is important for clinicians to account for local seroprevalences when interpreting the clinical relevance of a positive serologic test in patients.Despite these limitations, one of the most useful and commonly occurring situations in which serologic testing is important is the evaluation of a newborn suspected of having a congenital infection. A pregnant woman can infect her unborn baby if she develops certain infections that can be transmitted through the placenta. In utero infections that occur early in the pregnancy often have the most profound impact on the fetus because the damaging effects occur at a time when the fetus is growing rapidly and organ systems are developing.Customarily referred to as the TORCH group, common agents of congenital infection include Toxoplasma, rubella, cytomegalovirus, and HSV 1 and 2. The “O” represents “other” and can include a significant number of other pathogens: hepatitis B and C viruses, EBV, human immunodeficiency virus, enteroviruses, Bartonella, parvovirus B19, T pallidum, varicella-zoster virus, Listeria, lymphocytic choriomeningitis virus, the trypanosomiases that cause Chagas disease, Coxsackieviruses, and adenoviruses. In most cases, the primary mode of diagnosis for such infections is serologic analysis.When a congenital infection is suspected, prompt testing of serum from both the mother and newborn for pathogen-specific antibodies is imperative, particularly when considering diseases such as syphilis and toxoplasmosis, in which early recognition and treatment can prevent serious sequelae in the developing child. IgM to a specific organism from the neonate (obtained from cord or peripheral blood) is particularly helpful because maternal IgM is too large to cross the placenta; hence, any IgM detected must have arisen endogenously from the child. IgG does cross the placenta, and because it usually takes 2 to 3 weeks after infection to mount an IgG response, IgG found in the newborn usually reflects passive acquisition of maternal antibodies.However, there are limitations to serologic testing for congenital infections. Most maternal IgG is transferred to the fetus after the 35th week of gestation, affecting results from preterm infants. IgM typically is detectable 1 to 2 weeks after infection and persists for 2 to 3 months. Depending on the timing of the congenital infection, false-negative results can occur readily. False-negative IgM results also can occur in infants who have maternal IgG and fetal IgM directed against the same antigens. To clarify these results, follow-up antibody titers (preferably IgG and IgM if available for the particular pathogen) can be performed on serum collected 2 to 4 weeks later to confirm the previous findings. In infants younger than 12 to 15 months of age, such follow-up titers may distinguish transplacentally acquired IgG (which decreases over time because IgG has a half-life of about 1 month) from response to acute infection in the infant (in whom titers should increase by fourfold or more).For certain diseases, serologic testing is the only tool for diagnosis. However, it is important to remember that it is just a tool, and although it may be the only option, such testing may not be optimal. By understanding its limitations and benefits, clinicians can use serologic testing appropriately for diagnosis and treatment.By chance, the week I wrote this comment, Morbidity and Mortality Weekly Reports (2008;57:603–605) published a report on the public health consequences of a false-positive serologic test for brucellosis. Apparently, the cell wall lipopolysaccharide of Brucella is antigenically similar to that of other gram-negative rods, so cross-reactivity occurs. As rare as brucellosis is in the United States, the likelihood of a positive serology being false is significant, and a definitive diagnosis cannot be made without careful confirmation. To pick up on Drs Lee and Krilov's crime detection analogy, Brucella was exonerated in this case, but only after an extensive, expensive, and ultimately needless epidemiologic investigation. We can hope that just as DNA evidence has revolutionized criminal investigation, newer technologies such as polymerase chain reaction will give us a better chance of pinning the pathogenic culprit directly to the spot rather than having to depend on circumstantial serologic evidence. Notice that, as a pediatrician, I refuse to use the image of a smoking gun.