Virus isolation: Isolation of CMV from various clinical samples like urine, saliva, blood, amniotic fluid, etc. is done on fibroblast cell lines. The conventional culture is time consuming and also poorly sensitive. This has been replaced with shell vial culture where sample is inoculated with low-speed centrifugation onto the cells to enhance the infection. Viral immediate early proteins are expressed within 24 hours in the infected cells, which are then identified by immunofluorescence using monoclonal antibodies. Diploid cell lines like MRC5 or WI 38 are the commonly used cell lines for the growth of HCMV.

CMV inclusion body: CMV produces enlargement of infected cells with production of intranuclear inclusion body with peri-nuclear halo. This cytopathic effect is typically known as owl’s eye appearance (Fig. 1a). Cytomegalic cells bearing intranuclear inclusion body with owl’s eye appearance can be demonstrated in the tissue of the affected organ which indicates tissue invasion and thus CMV disease. Presence of these cells in the exfoliated urinary epithelial cells in neonates can be used to diagnose congenital CMV infection.

Detection of viral antigen: Among the various CMV antigens, detection of pp65 (CMV phosphoprotein 65) antigen in polymorphonuclear leukocytes is most commonly used. Detection is done by indirect immunofluorescence test using monoclonal antibody to pp65 antigen. The pp65 antigen positive cells show intranuclear apple green fluorescence (Fig. 1b).

Presence of CMV pp65 antigen positive cell in blood sample (antigenemia) indicates active infection. Quantitation of antigenemia helps in differentiating CMV infection and disease. This test is of more importance in transplant recipients where antibody response may not be helpful for diagnosis because of immunosuppression. Number of positive cells over total number of cells counted is determined.

Advantage of the test: Rapidity

 Limitations:

 a. Subjectivity

 b. Sample needs to be processed rapidly

c. Limited value in leukopenic patients

Fig1. (a) Microphotograph showing intranuclear inclusion with perinuclear halo giving owl’s eye appearance of CMV (H&E ×40). (Photograph courtesy: Prof Uma N. Saikia, Histopathology, PGIMER, Chandigarh); (b) Microphotograph showing CMV pp65 antigen positivity in polymorphs by IIF using monoclonal antibody (IF ×20).

Detection of viral genome: Viral genome can be detected by conventional PCR from various clinical samples. CMV gB gene or immediate early gene are the most commonly targeted genes by PCR.

Qualitative detection of CMV virus DNA in blood of neonate is done to diagnose congenital infection.

In adults, detection of viral genome by conventional PCR can be possible due to active infection or presence of latent virus. Therefore, viral load estimation is important in order to differentiate between CMV infection and CMV disease. High viral load indicates CMV disease.

Serology: Several serological methods are available for detection of antibodies, however, ELISA is most widely employed test.

CMV IgG antibody: CMV IgG antibody once appears during primary infection persists for life. Positive CMV IgG in serum sample in a previously negative individual indicates seroconversion which acts as marker of primary infection.

CMV IgG positivity in a single sample indicates past infection.

 CMV IgM antibody: Several types of ELISA are available for detection of CMV IgM anti body; however, capture ELISA and recombinant ELISA methods are preferred because of their high sensitivity and specificity.

Positive IgM antibody in immunocompetent pregnant women with rapid fall in titer within months is generally considered as primary infection. Whereas, in immunocompromised individuals, IgM antibody usually persists for a long time and can give positive result in recurrent infection also. As CMV IgM antibodies can persist from a few months to up to one year after subsidence of the acute phase of infection. The positivity can, therefore, be due to acute infection, convalescent phase of primary infection or due to persistence of antibody. Thus, IgM antibody status in a single sample should be interpreted with caution.

CMV IgG avidity ELISA: It is based on the principle that virus specific IgG antibody of lower avidity is produced during the initial phase of infection which becomes of high avidity with recurrent infection:

• High avidity IgG antibody indicates remote or recurrent infection.

• Low avidity indicates recent infection.

The mean avidity index during the first few months of primary infection has been reported to be of 21% as compared to 78% from patients with remote infection.

Presence of virus specific IgM antibody along with low avidity IgG antibody indicates recent or primary infection, whereas IgM antibody along with high avidity IgG is suggestive of past infection.

Serology has limited role in diagnosis in immunocompromised individuals.

Diagnosis of CMV infection is important in the following clinical settings

 • To diagnose the type of infection during pregnancy: Primary or recurrent.

 • To diagnose congenital infection.

• To determine the viral load in congenitally infected neonates as a prognostic indicator.

 • To diagnose CMV disease in immunocompromised patients.

Diagnosis of CMV infection during pregnancy: It is now understood that diagnosis of primary infection during pregnancy is important. Serology is the mainstay to distinguish between primary and recurrent infection.

Primary CMV infection in pregnancy is determined by:

• Seroconversion: Most authentic marker of primary infection.

 • Positive IgM antibody with low avidity IgG antibody: More reliable than only IgM positivity.

Diagnosis of congenital infection: Detection of CMV (virus/DNA) within first three weeks of birth is considered as congenital infection. Detection of virus beyond three weeks cannot differentiate between congenital, intranatal and postnatal infections.

Criteria of diagnosis:

 • Isolation of virus from urine, saliva or blood sample by shell vial assay.

• Detection of viral genome by conventional or real-time PCR.

• Serum sample positive for IgM antibody.

• Demonstration of four-fold rise of IgG antibody titer.

Prenatal diagnosis is made by detection of virus or viral genome in amniotic fluid at 20 or more weeks of gestation by culture and PCR, respectively.

Diagnosis in immunocompromised host: In immunocompromised patients, reactivation of latent virus is the most common cause of CMV associated disease. However, reactivation and active replication of virus can occur without causing any clinical manifestation. Therefore, mere detection of virus/DNA in blood does not implicate the association of CMV with clinical disease. Hence, it has no clinical relevance.

Presence of virus in the affected organ is the definitive method to associate with the disease. This requires the demonstration of virus/antigen or its inclusion body in the tissue specimen. However, as collection of tissue biopsy is an invasive procedure, it is not feasible in most of the cases. This makes the quantitation or viral load estimation in blood of the patient important which helps (i) to associate with the ongoing clinical disease, (ii) start pre-emptive therapy in post-trans plant cases, (iii) to monitor the treatment response.

Methods of CMV quantitation/viral load estimation: This is often done from the blood sample (whole blood or plasma). Quantitation of CMV viral load is done by CMV antigenemia or CMV viral load. There is no consensus regarding the cut of value for viral antigen or DNA load in different types of transplant patients or different groups of immunocompromised patients. In general, lower viral load (antigenemia/DNA) is associated with disease in HSCT patients as compared to SOT patients. Similarly, the cut-off value for preemptive therapy also has been set up at different level in different clinical settings. Between the two methods, viral DNA is better as it can be detected earlier than antigen and also more sensitive.

1. Quantitation of CMV antigenemia: Presence of CMV pp65 positive cells is an indicator of active infection. Quantitation is done by counting the number of positive cells over total number of counted cells. Quantitative estimation helps in differentiating between CMV infection and disease, however, as compared to PCR, it is less sensitive and appears later than PCR.

2. CMV DNA/mRNA load: This is determined by real-time PCR or NASBA respectively. Detection of CMV mRNA is considered as more specific as this reflects the replicative stage of the virus. However, as it degrades rapidly, the sensitivity of mRNA is less than DNA. CMV DNA in blood is often used to determine the time of pre-emptive therapy initiation and also to monitor the course of the disease. Various PCR platforms are available commercially. One of the problem with CMV PCR is different PCR system is standardized to express the result in different units. Therefore, the result should be normalized as per WHO’s reference International standard and should be reported in IU/ml. It is also recommended to use same specimen type, same extraction and PCR assay for serial testing.

Diagnosis of gastrointestinal CMV disease: Detection of antigenemia or PCR from blood sample in gastrointestinal CMV disease is often negative at the time of diagnosis and hence unreliable. This is possibly because, initially it is a local tissue event and thus the CMV load in plasma or whole blood does not reflect properly the CMV replication in gastric mucosa. Histopathology and immunohistochemistry of gastric mucosal biopsy is the gold standard to confirm CMV GI disease. However, some of the recent studies using fresh endoscopic biopsy have shown promising result with quantitative PCR as compared to IHC.

Diagnosis of CMV pneumonia: CMV pneumonia posseses challenge to conform its diagnosis because lung biopsy is not a preferred specimen as it involves invasive procedure. Virus cultivation from bronchoalveolar lavage (BAL) sample does not differentiate between CMV pneumonia and asymptomatic viral shedding. Quantitative PCR in BAL in both HCT and SOT has shown wide variation in viral load cut-off to diagnose CMV pneumonia in various studies (500 IU/ml to 30000 IU/mL in HCT and 3.2 × 10⁵ IU/ml to 1.8 × 10⁷/mL) in SOT respectively.

Though exact cutoff of viral load has not been determined to diagnose any particular type of CMV disease or for initiation of preemptive therapy, it is proved beyond doubt that the role of viral load acts as a surrogate marker for disease as evident from below mentioned findings:

1. Association of high viral load with disease: In a meta-analysis, 7-fold higher viral load has been shown in CMV disease as compared to asymptomatic infection when studies using one type of PCR platform and >9-fold when all types of studies are included.

 2. Relation of viral load kinetics with development of CMV disease: More rapid increase in viral load is positively correlated with development of disease.

3. Rate of viremia during prophylaxis: The incidence of viremia is significantly less during prophylaxis as compared to the entire period; 3.2% vs 34.3%.

4. Incidence of CMV disease during prophylaxis: The pooled incidence of CMV disease during prophylaxis is 0.8% as compared to 13% during the entire period.

5. Decrease in viral load and symptom resolution: Correlation between decrease in viral load due to treatment with resolution of symptoms was observed. Patients with viral load <18000 IU/mL showed faster resolution. Patients in whom faster decrease in viral load was found, showed faster clinical resolution.

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شعبة الخطابة النسوية تعقد اجتماعًا تحضيريًّا للمتطوعات خلال زيارة الأربعين

ملاكات الحزام الأخضر الجنوبي تشرع بجني التمور والمحاصيل الخضرية من واحاته

المجمع العلمي يقيم أنشطة قرآنية متنوعة في محافظات عدة

قسم المشاريع ينجز صبّ أكثر من 200 م³ من الخرسانة في المساحات الإضافية لصحن أم البنين (عليها السلام)

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المزيد

مواضيع ذات صلة

Treatment of Cytomegalovirus

Clinical Manifestations of Cytomegalovirus

The Viral Genome

Human Immunodeficiency Virus

Viral Pathogenesis

VIRUSES

Human Cytomegalovirus

Influenza Virus Infections in Humans

Varicella-Zoster Virus

Adenovirus Coinfection with SARS-CoV-2

Genetic Polymorphisms susceptibility to SARS-CoV-2

Acute viral hepatitis