Bangladesh J Med Microbiol 2010; 04 (02): 39-44

Bangladesh Society of Medical Microbiologists

Review Article

Laboratory Diagnosis of CMV Infection: A Review

Munira Jahan1

1 Department of Virology, BSMMU;

A diagnosis of CMV disease is much more difficult to infection in high-risk patients prior to the onset of clinical
1,2
establish, as patients may excrete the virus in urine, semen, or disease is preferred .
cervical secretions for years following its acquisition. Thus, a Since rapid and sensitive technique for diagnosis of CMV
positive cultures from these sites does not, by itself, prove infection is of vital importance for the management of
that CMV is the cause of the patient's current symptoms. immunocompromised patients, a number of rapid and
sensitive virus detection methods have been developed. These
Although recovery of the virus or detection of CMV antigen 3
from the blood or tissues is suggestive of active disease due includes DNA probe techniques , Polymerase chain reaction
4,5
to CMV. The diagnosis of CMV infection can be (PCR) , CMV antigen detection in biopsies and
substantiated by one or more of the following : 1) electron 6,7
bronchoalveolar lavage , and immunofluorescence
microscopic detection of typical CMV virion, 2) histologic or technique for detection of CMV early antigens in cell
7-,10
.
Cytologic detection of typical CMV cytopathology, 3) Moreover, an assay has recently been developed for CMV
Isolation of Virus, 4) detection of CMV antigen in blood and antigenemia based on the detection of CMV immediate early
tissues, 5) detection of CMV genome in tissues, 6) DNA 65
antigen (pp ) in circulating leucocytes
1,10-12
.
amplification, 7) serology
Electron microscopy
Inroduction Electron microscopy has been used to identify CMV in urine
It is difficult to diagnose CMV infection in and is 25 %-95% as sensitive as viral culture in congenitally
13,14 14
immunocompromised patients as it requires not only infected infants . Monplaisir (1972) used
13
detection of virus but also determining whether CMV is ultracentrifugation, whereas Lee et al (1978) used a
causing disease. CMV shedding and viremia are common in simpler, pseudoreplica technique. This method was 95%
patients with impaired cellular immunity even when disease 4 13
sensitive when the viral infectivity titer was 10 /ml .
due to CMV is not present. In addition, preventive and Below this titer the sensitivity of the technique was only 25%.
therapeutic options are few and not completely effective. The Since many infants >6 months old and adults (especially
severity of CMV infection are roughly parallel with the 4
those who are immunocompetent) have titer > 10 / ml, the
degree of immunosuppression. Although use of electron microscopy is limited. Electron microscopy is
immunocompromised patients are at risk for morbidity due to now rarely employed to identify CMV in clinical virology
wide variety of pathogens, few, if any of these are capable of laboratories.
producing such widspread disease as CMV. CMV-related
morbidity follow a progressive, relentless course in the
absence of effective therapeutic intervention. Thus, rapid
diagnosis of active CMV infection is of great importance to
avoid over treatment with immunosuppressive drugs and to
guide antiviral therapy. In recent years, treatment of CMV

* Correspondence: Dr.
Munira Jahan Assistant
Professor Department of
Virology
BSMMU
Tel No: 8617099, 0171527700
E-mail : mjahan1970@yahoo.com
Figure 1: Electron micrograph of Cytomegalovirus

39
Lab Diagnosis a CMV Infection: A Review Jahan M

Cytology/ Histology 18,19

appearance of identifiable cytopathology . Unlike
Cytologic technique may be applied in an attempt to find conventional cultures, which are usually performed in tubes
characteristics intranuclear inclusions in specimens. containing fibroblast monolayer, cultures intended for
Inclusion-bearing cells may be found in saliva, milk. cervical monoclonal antibody staining are performed on flat
and tracheal secretions, and in touch preparations from biopsy monolayers, either on cover slips in shell vials or in 24-well
or necropsy tissues. The microscopic hallmark of CMV cluster plates. Centrifugation of monolayers greatly assists
infection is the large (cytoplasmic) 25-35 µm cell containing absorption of virus, thereby apparently increasing four fold
a large, central, basophilic intranuclear inclusion is referred to 18
infectivity of the viral inoculums . Urine and
as "owl's eye" because it is separated from the nuclear bronchoalveolar lavage specimens have given the best results
membrane by a hallow. These inclusions are seen well with with the rapid culture techniques. The sensitivity has been
Papanicolaou or hematoxylin-eosin stains. Clusters of small reported to exceed that of conventional culture, probably
intracytoplasmic inclusions may also be seen in CMV because of the enhancement of infectivity provided by
infected cells. These inclusions are best seen with Wright- 18,19
centrifugation . Antibodies to early and late CMV
15
Giemsa Stains . Sensitivity of the standard Cytologic antigens are also useful for direct visualization of infected
techniques is low relative to virus isolation, irrespective of the cells in clinical specimens. This has been demonstrated with
type of specimen. Only 50% of the urine samples from 7 6
bronchoalveolar lavage cells , biopsy specimens , and
infants with symptomatic congenital CMV infection are 10,12
positive. Even with the use of monoclonal antibody, leucocytes .
16
successful identification of infected neonates is only 50% .
In addition , the presence of these cells in urine sediment is
not pathognomonic of CMV. Histologic examination of a
small piece of tissue obtained, e.g., by bronchoscopic lung
biopsy is prone to sampling errors and to false-negative
results and was only 75% as sensitive as
6
immunofluorescence .
Isolation of Virus
Isolation of CMV from a clinical specimen remains a
definitive diagnostic procedure against which all newer
assays must be compared. Traditionally, urine, blood, and
throat specimens have been cultured, but during active
infection a wide variety of specimens yield viruses. In most
infected individuals urine contains moderate to large amounts Figure 2: Cytopathic effect of CMV
of infectious virus particles and is a convenient and reliable
specimen for culture. However , viruria often represents
asymptomatic infection and can be prolonged (for months to
years). Viremia, demonstrated by culture of leukocytes,
correlates better with active disease, although many viremic
17
individuals are also asymptomatic .
Since CMV grows only in human diploids fibroblast cell
cultures, specimens for isolation must be inoculated into cell
lines such as WI-38, MA-184, or Flow 2000. The
fundamental limitation of traditional isolation of virus is the
prolonged interval required for development of visible
cytopathology in cell cultures, which on average, takes 1-2
weeks. However when little virus is present in the specimen, Figure 3: CMV centrifugation culture fixed and stained 16 hr
recovery of virus can sometime take 6 weeks. after inocculation showing viral protein in nuclei of infected
Important advances resulted from the development of human fibroblast cells
monoclonal antibodies to the 72-KDa major immediate-early
protein of CMV . These antibodies enable the detection of a Nucleic acid hybridization
nuclear antigen in fibroblasts within hours of infection , thus Methods that utilize the detection of CMV DNA directly or
permitting the evaluation of cultures long before the indirectly are now used regularly for diagnosis of CMV

Bangladesh J Med Microbiol 40 Volume 4: Number 2 July, 2010

Lab Diagnosis a CMV Infection: A Review Jahan M

infection. In situ cytohybridization has been available for sensitivity can detect low levels of CMV that are not always
more than a decade and is currently able to detect single 5
predictive of disease . That is the increased analytical
infected cells. Nevertheless in situ cytohybridization sensitivity of the PCR assay leads to a lower clinical
technology remains labour intensive, which limits its use specificity.
except in the research setting.The use of DNA-DNA
hybridization to detect CMV in clinical samples directly was Antigen detection
20
first described by Chou and Merigan in 1983 . They used PP-65 antigenemia test in which specific monoclonal
ultra centrifuged urine, immobilized the sample on antibodies are used to detect, a CMV matrix phosphoprotein
32 known as pp-65 in leukocytes,. Since its initial description
nitrocellulose filters, and hybridized with a P-labeled
10 65
cloned probe. This cloned probe was prepared by digestion of , the pp antigenemia assay has represented a major
CMV strain AD-169 by the restriction enzyme EcoRI. breakthrough for the diagnosis of CMV infections. The test
Although the test could be performed in 24 hours, sensitivity has several advantages from the clinical perspective, and also
was very low. Out of 48 culture-positive sample 14 (29%) in terms of laboratory practices. Method of detection of
were negative. Virtually all samples with titers <500cfu/ml 65
pp is very fast, allowing viral detection after 4-5 hour of
were negative. No false positive results were obtained. blood sampling. CMV antigen positive blood cells appeared 1-
Investigators in other laboratories have reported improved 3 weeks on average nine days before serologic signs of active
sensitivity of nucleic acid hybridization on urine samples( 92%) 12 65
infection . Thus detection of CMV pp antigens
but have also reported occasional positive results (3%- appeared to be earlier indicator of active infection than
3 10,12,23
12%) in specimens that were culture negative . On the basis CMV IgM antibody .
of additional clinical and laboratory data , it was suggested
that the cultures for the patients were false negative rather This assay is sensitive and specific and yields result within 5
3 10
than that the hybridization assays were false positive . hours . In addition, the antigenemia assay is quantitative
and has been useful for estimating the likelihood of disease
Polymerase chain reaction progression, as well as for monitoring the response to therapy
CMV DNA PCR (Polymerase Chain Reaction) is a highly 1,23.
sensitive, rapid (~ 6 hrs) technique based on selective
amplification of specific nucleic acid sequences. The PCR In clinical practice this could be useful for the early
assay is more versatile and can be used either qualitatively diagnosis and early institution of antiviral therapy.
(diagnostic PCR) or quantitatively to measure the viral load, Furthermore the declining phase of CMV antigenemia
which is proportional to the level of CMV DNA. It was first indicating good prognosis is to guide safe discontinuation
developed as a sensitive methods for CMV detection. But 1
because its ability to distinguish small DNA sequence of antiviral drugs which may have severe side effects .
differences, it can be used to differentiate strains of CMV by 65
selective amplification of hypervariable regions of the viral So pp antigen test is a valuable tool for the early
genome. PCR has the advantage that it can be used to detect diagnosis and monitoring of active CMV disease in
CMV DNA in samples other than White blood cell (WBC), immunocompromised patients. The quantitative nature of
the antigenemia assay may give an estimate of viral load,
such as whole blood, plasma, cerebrospinal fluid, or
4,21
and this may be useful for monitoring patients before,
bronchoalveolar lavage fluid . CMV DNA in plasma 24 65
during, and after therapy . Sequential pp antigenemia
correlates with presence of CMV infection in BAL samples.
assay in the CMV antibody negative patients will allow
CMV DNAemia is correlated with risk and severity of CMV early detection of primary CMV infection and in the CMV
4,5 22
disease in immunocompromised patients . Rasmussen IgG antibody positive patients early reactivation of CMV
(1999) demonstrated a direct relationship between the viral and may allow the detection of important changes in the
load as estimated by PCR and the risk for subsequent antigenemia level. Early positive or rising antigenemia
development of CMVD. With the PCR method CMV levels may signal the onset of active CMV disease and
infection may be detected as early as two weeks before the allow early preemptive therapy to be initiated, particularly
24
onset of symptomatic CMVD. This advantage has given in transplant recipients . Transplant recipients with
impetus to the preemptive therapy strategy for the prevention clinical CMV infection treated with ganciclovir showed a
of CMVD in high-risk patients. More recently the rapid decline in antigenemia levels which paralleled clinical
quantitative plasma assay by real time PCR methods has been 25
improvement . A persistently high or rising level of
described and are becoming essential in the management of antigenemia despite appropriate therapy may signal
transplant recipients by defining the population at high risk progressive CMV disease or the development of viral
for CMV disease that requires pre- emptive antiviral 25,26
resistance .
treatment. The major disadvantage of PCR is that its great

Bangladesh J Med Microbiol 41 Volume 4: Number 2 July, 2010

Lab Diagnosis a CMV Infection: A Review Jahan M

Culture of lavage fluid , with overnight detection of

immediate-early antigen, has proven to be more reliable than
the culture of tissue obtained at biopsy in identifying CMV
35
infection . Support for a diagnosis of CMV hepatitis can be
conveniently obtained by in situ hybridization with biopsy
36
specimens . Evidence of CMV infection has been obtained
by in situ hybridization or antigen detection for a variety of
37
other organs, including gastrointestinal mucosa , and
38
numerous other tissue specimens in the disseminated CMV
infection. CMV central nervous system disease can be
21
diagnosed by DNA amplification .
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