Sunday, October 13, 2013

Laboratory Diagnosis of Cytomegalovirus

Laboratory diagnosis of HCMV infection
Various samples for detection of HCMV infection include body fluids, blood, polymorphnuclear leucocytes, tissues, etc. They should be rapidly transported to laboratory under sterile and aseptic conditions.

• Viral isolation

Human Diploid Fibroblast (HDF)
Cell cultures are obtained from the foreskin or from the embryonic lung tissues, have been used for conventional and shell vial methods for the isolation of HCMV. In conventional viral isolation methods, commonly used in earlier times, determination of viral replication is based on typical cytopathic effects (CPE) produced by HCMV. The time required for the development of CPE usually varies from 2 to 4 weeks, even up to six weeks.

Shell vial assay
One of the most commonly used rapid methods is the shell vial culture. In this method, for the improvement of absorption of the virus, the specimen is centrifuged onto the cell culture. Fibroblasts monolayers cultured in vials containing coverslips are used. These shell vial culture methods utilize indirect immunofluorescence to detect the immediate-early (IE) viral antigen after incubation of culture for one to three days
The spin amplification shell vial assay

The spin amplification shell vial assay has gained wide acceptance, as it is based on amplification of the virus in cell cultures after low speed centrifugation and detect viral antigen produced in the early replication of HCMV before appearance of CPE.

The isolation of HCMV from the blood or target organ specimen by cell culture methods has a high correlation with disease. However, it is relatively low sensitive in detection of the virus from blood samples compared to nucleic acid-based and antigenemia methods.

• Histological methods
Traditionally the recognition of cytomegalic inclusion bodies in histological specimens has been used for the diagnosis. In organ specific HCMV infection, such as HCMV pneumonitis or hepatitis, characteristic viral inclusions may be seen. The large inclusions are intranuclear and have a characteristic owl-eye appearance in haematoxylin and eosin stained tissue specimens. The positive results correlate well with active HCMV infection of the organ, but the sensitivity of the histopathological finding is relatively low.

• Immunofluoresense methods
Immunofluoresense staining with specific polyclonal or monoclonal antibodies against HCMV antigens has increased the sensitivity of the method compared to conventional staining. However, false-negative results may occur because of the focal and scarce distribution of HCMV positive cells in tissue samples.
Immunofluorescense (IF) either direct or indirect (IF) is a rapid diagnostic tool for viral diseases. The results are available within several hours after tissues obtained. The sensitivity of IF is improved when mixitures of monoclonal antibodies are used for detection of early and late antigens.
• Electron microscopy:
It could be used in detection of HCMV in urine, oral and other specimens. Positive results with almost all specimens that have infectivity titre >104 copies / ml.

• Serological methods

HCMV antibodies (IgM, Ig G)

Human cytomegalovirus infection is manifested by the production of IgG and IgM antibodies. Thus a diagnosis of HCMV infection can be obtained indirectly through serology. A variety of laboratory tests with different degrees of sensitivity have been described for the measurement of HCMV antibodies in human sera. The methods include complement fixation, indirect hemagglutination, latex agglutination, radioimmunoassay, immunofluorescence and enzyme immunoassay.

In enzyme-linked immunosorbent (ELISA) assays many different antigens have successfully been used as targets for detecting specific antibody production. The rise in serum antibody levels is an insensitive sign of actual HCMV infection in transplant patients.

The seroprevalence is high and the presence of IgG antibodies is only informative of the patient’s past history regarding HCMV infection. Furthermore, there is a time lag between primary infection and IgM antibody production (IgM level can remain undetectable because of delayed seroconversion owing to immunosupressive agents), and IgM antibodies can also persist for a long time after infection in some healthy individuals.

HCMV low avidity IgG

Primary HCMV infection can be detected by low avidity IgG. Also, in a study on renal transplant recipients there was an agreement between HCMV low avidity IgG and both direct Ag detection and polymerase chain reaction (PCR) in diagnosis of primary HCMV infection.

• Antigen detection(Antigenemia test)

The antigenemia assay, first described by professor The and his associates had a major advance in the diagnosis of HCMV infection in transplant patients. In this test, monoclonal antibodies to pp65 (the lower matrix phosphoprotein 65) are used for the direct immunostaining of blood polymorphonuclear leukocytes (PMNL).

It has been shown that the pp65 antigen in PMNLs is not a direct indication of virus replication in vivo, since the virus and viral material detected in PMNLs are transferred from other infected cells, e.g. endothelial cells, mainly by microfusion events.
The pp65 antigenemia assay consists of numerous steps, including isolation of PMNL, fixation, immunostaining, and microscopic evaluation and quantitation.

The disadvantage of the antigenemia assay is that the blood samples should be processed within a certain time, preferably within six hours, for optimal results.

It is still quite time-consuming and laborious, at least with large specimen numbers. The automation of the test is also difficult and the subjective evaluation of the infected leukocytes. Although there have been attempts to standardize the assay.

There are a number of various in-house and commercial modifications available. This makes comparison of the results between different centers difficult. The clinically significant threshold of the number of positive leukocytes seems to vary also among different types of transplant populations.

• Molecular methods

Nucleic acid amplification methods

Qualitative PCR has been proven to be more sensitive than antigenemia test or cell culture assay on the detection of HCMV infection.
Despite extreme sensitivity and specificity in detecting HCMV DNA, qualitative assays usually have a low positive predictive value for symptomatic infection, at least when peripheral blood leukocytes have been used as a specimen.
The sensitivity and specificity in detecting HCMV DNA is due to the capacity of these tests to detect HCMV DNA even in the case of latent infection. It has also been reported that the demonstration of HCMV DNA in plasma may correlate more closely with disease, and also with the antigenemia test, than that in leukocytes or whole blood.

The development and the availability of automated real-time instruments, have further simplified quantitative PCR assays and reduced the turnaround time needed for the test performance.

In real-time PCR the accumulation of the PCR products is monitored continuously during the PCR run, compared with the end-point measurements that quantitate the final PCR product. At present, several instruments are available for real-time PCR, in which the accumulation of the product is monitored by measuring the fluorescence in each cycle. The measured fluorescence is plotted against the cycle number.
There are various methods for the detection of PCR products during real-time PCR are available. These can be classified into amplicon sequence specific or non-specific detection methods. The most commonly used detection methods in the virus diagnostic assays are based on the use of specific fluorogenic oligoprobes .
These methods rely upon fluorescence resonance energy transfer (FRET), which is the interaction of two fluorescent dyes. TaqMan probes also called 5´ nuclease or hydrolysis oligoprobes, were the first ones used in special real-time instruments.
• Other nucleic acid amplification methods
Nucleic acid based sequence amplification (NASBA) is an isothermal amplification process which involves the coordinated activities of three different enzymes: ribonuclease H, reverse transcriptase and DNA-dependent RNA polymerase.

The final product of the process is a specific single stranded RNA, instead of the dsDNA of PCR. The high sensitivity in detecting HCMV infection has been shown by the NASBA assay with the (immediate early 1) IE-1 target.
There are many applications of the NASBA technique based on the detection of pp67 mRNA have been developed and the test application is also commercially available.

HCMV DNA has also been detected by a signal amplification method, Hybrid capture system, from blood samples of transplant patients. It is a solution hybridization assay that involves amplified detection instead of amplification of the desired nucleic acid fragment.

Prevention and treatment of HCMV disease

The pharmacologic agents used for HCMV prevention have evolved, from the use of acyclovir , immunoglobulins and IV and oral ganciclovir and valganciclovir.
Antiviral prophylaxis
One major strategy is antiviral prophylaxis, where antiviral drugs such as valganciclovir or oral ganciclovir are given to patients for at least 3 months after liver transplantation. However, antiviral prophylaxis is associated with delayed-onset HCMV disease, which typically occurs soon after completion of prophylaxis.

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