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Diagnostic Virology: Special Section: Medical Microbiology
Diagnostic Virology: Special Section: Medical Microbiology
Diagnostic Virology
Diagnostic virology has now entered the mainstream of medical practice. Multiple methods
are used for the laboratory diagnosis of viral infections, including viral culture, antigen de-
tection, nucleic acid detection, and serology. The role of culture is diminishing as new im-
munologic and molecular tests are developed that provide more rapid results and are able to
detect a larger number of viruses. This review provides specific recommendations for the
diagnostic approach to clinically important viral infections.
Diagnostic virology is rapidly moving into the mainstream sive (and in some cases potentially toxic) has created an obvious
of clinical medicine as a result of the convergence of several need for specific viral diagnosis.
independent developments. First, dramatic progress in antiviral In some cases, establishing a specific viral diagnosis limits
therapeutics has increased the need for specific viral diagnoses. other diagnostic procedures and may allow discontinuation of
Second, technological developments, particularly in the area of antibiotic therapy [1]. Likewise, in some cases, confirmation of
nucleic acid chemistry, have provided important new tools for a specific viral diagnosis helps in determining prognosis. A re-
viral diagnosis. Third, the number of patients at risk for op- cent study of diagnostic testing for respiratory syncytial virus
portunistic viral infections has expanded greatly as a result of (RSV) documented that physicians usually believed that rapid
the HIV/AIDS epidemic. Finally, modern management of HIV test results influenced their management of cases [2]. Rapid
infection and hepatitis C is providing a new paradigm for the RSV testing has also been used as the basis for patient place-
integration of molecular techniques into management of ment to limit nosocomial transmission [3]. Finally, viral diag-
chronic viral infections. These developments are not only in- nosis may be important for public health purposes. For ex-
creasing the use of diagnostic virology but are reshaping the ample, laboratory documentation of cases of rubella or rubeola
field. The purpose of this article is to review the field of di- can set in motion extensive vaccination campaigns.
agnostic virology at the beginning of the 21st century, to provide
guidance about current use of the tools of diagnostic virology,
and to provide a glimpse of important future developments. Methods Used in Diagnostic Virology
Table 1. Techniques used in diagnostic virology. fected into indicator cell lines to direct insertion of viral receptors
Cell culture on the surface of the cell and/or to direct expression of promoters
Antigen detection that respond to a specific viral protein present in the specimen.
Fluorescent antibody staining Activation of the promoter triggers a reporter enzyme such as b-
Immunoperoxidase antibody staining
galactosidase that acts on a substrate to indicate the presence of
Enzyme immunoassay
Nucleic acid detection the virus being sought. This approach has been most widely used
for HSV [8] and HIV [9].
plification assays or signal amplification assays. Examples of target of any antiviral antibodies is always (HIV) or usually (HCV) in-
amplification assays in addition to PCR include the ligase chain dicative of current infection. Finally, serology is uniquely useful
reaction, which has been used for detection of sexually transmitted for defining specific antiviral immunity. Viruses for which definition
disease agents [17], and the transcription-mediated amplification of immune status by serology is useful include VZV, CMV, EBV,
assay [18]. In signal amplification assays, the target itself is not HSV, measles and rubella viruses, parvovirus B19, hepatitis A (total
amplified; rather, amplification is of a chemical signal used to detect antibodies), and hepatitis B (antibodies to the hepatitis B surface
hybridization of a probe with the target nucleic acid. Examples
Table 3. Laboratory diagnosis of viral infections of the respiratory A and B viruses. This assay had a sensitivity comparable to
tract. that of culture in one evaluation [30]. Zstatflu (ZymeTx,
Virus Antigen detection Culture Oklahoma City, OK) is based on detection of influenza neu-
Respiratory syncytial Yes Yes raminidase activity and also detects but does not distinguish
Influenza Yes Yes between influenza A and B viruses. This assay had a sensitivity
Parainfluenza Yes Yes
Adenovirus Yes Yes (in comparison with culture) of 76% for influenza A virus and
Table 5. Laboratory diagnosis by PCR analysis of opportunistic HSV infection of the CNS. We also recommend that a negative
viral infections of the CNS. result can be used as the basis for discontinuing therapy with
PCR analysis acyclovir if the clinical suspicion for that diagnosis is low. How-
Sensitivity, Specificity, ever, when the clinical suspicion is high, acyclovir should be
Virus Syndrome % % continued even when the HSV PCR analysis is negative, be-
Epstein-Barr Primary CNS lymphoma 97 98 cause the sensitivity of the test is !100%. In these cases, it may
Table 7. Preferred test methods for diagnosis of cytomegalovirus used for the laboratory diagnosis of viral gastroenteritis are
infection.
shown in table 6.
a
Diagnostic need Preferred test(s) Rotavirus is the most important cause of gastroenteritis in
Immune status Serology (IgG) young children. Because large amounts of rotavirus are shed
Congenital infection, postpartum Culture of urine in the stool during acute rotavirus infection, antigen detection
Congenital infection, in utero Culture or PCR analysis of amniotic fluid
Mononucleosis, normal host Serology (IgM) tests performed on stool specimens are very sensitive for es-
a relationship between the level of CMV in blood and the like- value [68]. PCR analysis performed on plasma may be more
lihood of present or future symptomatic disease [62–66]. specific [69], but further clinical evaluation is required. Local-
The choice of test begins with the purpose of the testing. ized CMV infection such as pneumonitis or gastrointestinal
Important considerations include the clinical syndrome under tract disease is best diagnosed by histologic examination of
investigation and whether the patient is immunologically nor- biopsy tissue, supplemented when necessary with
mal or immunocompromised. Test methods for different di- immunohistochemistry.
agnostic needs related to CMV are shown in table 7. In general, Preemptive therapy is now a widely used approach for the
active infection is best diagnosed by direct tests for the presence control of CMV after solid organ or bone marrow transplan-
of the virus, although an exception is the diagnosis of CMV tation. This approach is based upon the use of a laboratory
mononucleosis in otherwise normal hosts, in which testing for marker that signals the onset of CMV infection before signif-
CMV-specific IgM antibodies is useful [67]. A caveat in using icant clinical manifestations have occurred. For this purpose,
this test is that for some patients with acute EBV infection, leukocyte or plasma PCR analysis, the pp65 antigenemia assay,
tests are false-positive for CMV-specific IgM antibodies [67], and the hybrid capture assay are all appropriate. PCR analysis
suggesting that testing for acute EBV infection should be per- is preferred for bone marrow transplantation patients because
formed simultaneously. Congenital infection can be conven- of its greater sensitivity [70, 71]. In AIDS patients who are
iently diagnosed by urine culture, which must be performed asymptomatic, detection of CMV by any of a variety of meth-
within the first week of life to distinguish congenital infection ods signifies an increased risk of future symptomatic disease
acquired in utero from neonatal infection occurred during or [72], but such a finding is not currently used as an indication
shortly after birth. for starting therapy with anti-CMV drugs.
For the diagnosis of systemic CMV infection in immuno- Epstein-Barr virus. EBV is the cause of most cases of in-
compromised patients, the pp65 antigenemia assay [59] prob- fectious mononucleosis. It is also associated with primary CNS
ably has the best combination of sensitivity and specificity lymphoma in patients with AIDS and posttransplantation lym-
among widely available test methods. An additional advantage phoproliferative syndrome in recipients of solid organ and bone
is that it provides a quantitative estimate of the level of CMV marrow transplants. The diagnostic approach differs for each
viremia. Qualitative PCR analysis performed on peripheral of these clinical syndromes. Because cultures for EBV are too
blood leukocytes is too sensitive for the diagnosis of clinically slow and cumbersome to be carried out in clinical laboratories,
significant systemic infection, having a low positive predictive other diagnostic methods must be used. Serological methods
746 Storch CID 2000;31 (September)
are used to diagnose infectious mononucleosis and other man- impossible to culture, and hence all current laboratory testing
ifestations in immunologically normal individuals, while mo- involves detection of specific antigens, antibodies, or nucleic
lecular methods are used in immunocompromised individuals. acids. Although no molecular tests are currently licensed by
The diagnosis of infectious mononucleosis can be made in the FDA, molecular testing for HCV is widely used by clinicians
most cases on the basis of characteristic clinical findings, the caring for patients with HCV, and molecular testing for hep-
presence of atypical lymphocytes, and the demonstration of atitis B virus is coming into wider use as well. Blood banks are
transplant recipients, and can also be useful for monitoring Diagnosis of congenital infection HIV DNA PCR analysis, plasma HIV
a
RNA analysis
response to therapy [76]. Quantitative testing is required be-
Prognosis Plasma HIV RNA analysis
cause many transplant recipients have EBV detectable in pe- Response to therapy Plasma HIV RNA analysis
ripheral blood mononuclear cells after transplantation, and Blood donor screening EIA/western blot, p24 antigen assay,
plasma HIV RNA analysis
measurement of the level of DNA is required to distinguish
Resistance to antiretroviral drugs Genotypic or phenotypic susceptibility
those with posttransplantation lymphoproliferative disorder. assay
Viral hepatitis. Five organisms, hepatitis viruses A–E, are a
A positive test for plasma HIV RNA should not be used as the sole basis
currently known to cause hepatitis. All are either difficult or for diagnosis, because there have been false-positive test results [79].
CID 2000;31 (September) Diagnostic Virology 747
this purpose and allows determination of HIV infection status Table 11. Laboratory diagnosis of miscellaneous viral infections.
within the first few months of life [83]. Assays for HIV RNA Virus; indication for testing Test(s)
(see below) can also be used [84]. Measles; acute infection Measles IgM antibody, culture
Assays for plasma HIV RNA, often referred to as viral load Rubella
Acute infection Rubella IgM antibody, culture
assays, are now an essential component of the management of
Congenital infection, in utero Culture or reverse transcriptase PCR
care for patients infected with HIV [85]. The level of HIV RNA analysis of amniotic fluid
Laboratory diagnosis of hantavirus infection can be achieved the result of a limited number of mutations in a specific gene
by detection of hantavirus-specific IgM antibodies [96] or by designated UL97, and these mutations can be detected by a
detection of hantavirus RNA by means of RT-PCR analysis PCR-based assay [102–104]. This assay can be carried out either
performed on peripheral blood leukocytes [97]. Some state pub- on an isolate recovered from a patient or directly in a patient
lic health laboratories perform hantavirus tests, and others for- specimen if the level of virus is sufficiently high to allow effective
ward specimens to the Centers for Disease Control and Pre- PCR amplification.
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