SERIAL NO. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

CONTENT Introduction Morphology of CMV Epidemiology Transmission Immune response Clinical features Clinical manifestation Diagnostic method Objective of work Study design HCMV viral load assay by RT-PCR pp65 Antigenemia assay IL-6 EASIA Human TGF- ELISA Human IFN- ELISA Reference

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INTRODUCTION: Cytomegalovirus or CMV is a DNA virus member of the Betaherpesvirinae family and subfamily Herpesviridae (1). Its classification as a herpesvirus is based on its tendency to infect nucleated cells and on its molecular phylogenetic relationship to other herpes viruses. Its classification as a betaherpesvirus is based on its long replication cycle, cytopathology and restricted host range, which are all characteristic of the betaherpesviruses. It is ubiquitously obtained. In developing countries most of the people are infected by this virus by the early childhood. In case of immunocompetent individuals, CMV infection does not usually show any symptoms. However, in case of immunocompromised individuals or whose immunity is suppressed by some immunosuppressive drugs as in the case of organ transplantation, CMV can cause severe disease. In case of fetus also where the immune system is immature, vertical transmission of CMV may have serious implications. CMV is species specific and has been isolated from many animal species. The most studied is human CMV (HCMV), which is also known as human herpesvirus-5 (HHV-5). Other primate CMV species include chimpanzee CMV (CCMV) that infects chimpanzees and orangutans, and simian CMV (SCCMV) and rhesus CMV (RhCMV) that infect macaques etc. Smith in 1956, Rowe and co-workers in 1956, and Weller et al in 1957 independently isolated human CMV strains. In 1960, Weller and co-workers proposed the term "cytomegalovirus" and subsequently isolated CMV from the urine of infants with generalized disease (2). The term Cytomegalovirus is from a Greek word cyto-, "cell", and -megalo-, "large. CMV has now become one of the most common opportunistic pathogens encountered in patients immunocompromised from congenital or acquired causes such as AIDS or transplantation procedures. Synthesis of viral DNA and assembly of capsid occur in the nucleus where infective progeny are released by budding through the nuclear envelope. MORPHOLOGY: From the electron micrograph studies performed, a typical herpes virus like appearance of CMV is revealed. The central DNA-containing core is surrounded by a protein capsid. The capsid is an icosahedron composed of 162 capsomeres. The capsid is surrounded by a ill-defined tegument or matrix- the capsid is 5% larger than that of HSV resulting in 17% increased volume(3-5). It is 100nm in diameter. Of the 162 capsomeres, 150 are hexamers and 12 are pentamers. Each of the capsid is a hollow hexagon in cross-section. The capsid in turn is surrounded by the tegument, which is a poorly demarcated area. This tegument is again surrounded by a loosely applied lipoprotein envelope. Glycoproteins complexes are embeded in the lipid envelope. (6) HCMV infected cells may contain several forms of the capsid- A,B and C capsids (7). Type A capsids lack the viral DNA and they accumulate because of failure in stably packing the viral genome. Type B capsids are found in predominantly in the nucleus as precursors of the mature capsids that lack DNA but contain the viral assembly proteins (3). Type C capsids are however, fully mature virion nucleocapsids.

FIG 1. Diagrammatic representation of CMV Genome Organisation: The icosahedral capsid of CMV of Ad169 strain encases 235 kb double stranded linear DNA (8). The genome of all the charecterised CMVs are linear DNA molecules and the size ranges from 200-240 kbp. A more compact DNA exists- interhelix DNA packing distance is 26Ao vs 30Ao that of HSV. The size of the genome of the CMVs is considerably larger than that of the other herpesviruses. The following table (TABLE 1) lists the genome size of some of the CMVs:

Virus DNA Size (kbp) Ref. Human CMV 230-235 9 Simian CMV 209-220 10 Rat (Maastricht) CMV 224 11 Rat (England) CMV 204 12 Murine CMV 230 9 Guinea Pig CMV 230 13 The HCMV genome can be divided into two unique regions termed unique short (US) and unique long (UL). HCMV genome exhibits a pattern of terminal and inverted repeats that vary in size depending upon the strain of virus. The long and short unique sequences are bounded terminally by repeated segments. The US is bounded by an inverted and a terminal repeat sequence, designated as IRS and TRS. The U L is flanked by two repeats termed as IRL and TRL (1). The polarity of the genetic information in either of the unique region can be inverted. This thus leads to 4 isomeric forms of virion DNA. In CMV-infected cells these 4 isomers are prevalent in equal amounts. However, no pathologic significance is attributed to them so far. The entire genome of Ad169 strain of HCMV has been sequenced and has been published in and is available on EMBL and GenBank databases (9). It is predicted to have more than 208 ORFs. The HCMV genome is G+C rich and contains a significant number of direct and inverted repeats and thus it exhibits a greater number of genome repeats than other herpes viruses (10). Virion Proteins: Capsid proteins: The CMV produces approximately 178 unique proteins, the molecular weight of these proteins range from 20-over 300 kD (11). The capsid is composed of seven types of proteins. Each of these is homologous to HSV. The corresponding genes for this set of capsid proteins are present on the genome within the herpes virus set of genes. The table (TABLE 2) below lists the total seven Capsid proteins and their corresponding genes (21): Viral Protein Major Capsid Protein (MCP) Minor Capsid Protein (mCP) Minor Capsid Binding Protein (mC-BP) Smallest Capsid Protein (SCP) 3 distinct Assembly Proteins (AP) Corresponding Genes UL86 UL85 UL46 UL48.5 (UL48/49) UL80, UL80a,UL80.5

Envelope proteins: The envelope consists of two protein complexes which are formed of herpesvirus- conserved glycoproteins (12). One of the complexes is formed of covalently linked dimers of glycoprotein B which is encoded by UL55. The other being composed of the complexes of gH,gL and gO. These are encoded by UL75, UL115 and UL74 genes (B2 224,514). These glycoprotein complexes play a very critical role for the entry of the virus in the host body (B2 91). gB encoded by UL55 is the major envelope protein (22). Tegument proteins: At 25 proteins are located in the tegument layer of CMV which lies between the capsid and the envelope (13).the ORFs for the corresponding proteins are all conserved in betaherpes viruses but only a few are common to herpes viruses (14). The most common of these proteins as found in the tegument layer are pp150 and pp65 (15). Of them again, pp65 is the most abundant of the tegument proteins. pp65 is a lower matrix protein of molecular weight 65-68 kD. It is composed of 561 amoniacids and is encoded by UL83. pp65 is the target antigen in antigenemia assays that are used for early determination of CMV infection from clinical samples. pp150 is a basic phosphoprotein encoded by UL32. pp150 is estimated to make up to about 20% of the mass of CMV (16).

Other proteins: gpUL18 encodes the protein which has stricking similarity with Human HLA class I. It may thus happen that this protein may sequester with 2 microglobulin (2m) in the cytoplasm of the infected host (17) and with endogenous peptides derived from cytoplasmic proteins that resemble those bound to host class I proteins (18) and thus mimic the host MHC I (19). Despite sharing only 25% sequence identity, UL18's structure and peptide binding are surprisingly similar to host MHC I (19). This mimicking of HCMV may play a very important role in the process of infecting the host by preventing proper functioning/ weakening the host immune system. CMV down-regulates cell surface expression of host class I MHC molecules, thereby allowing HCMV-infected cells to avoid recognition by virus-specific cytotoxic T cells. However, cells that lack surface class I MHC molecules are susceptible to lysis by natural killer (NK) cells. By mimicking, the MHC I protein, the virus thus prevents any NK cell activity to lyse the virus infected cell (19). However, the role of UL18 in NK cell recognition is controversial; it has been reported to inhibit NK-cell-mediated lysis in some experimental conditions but not others (20). EPIDEMIOLOGY: Seroepidemiological studies confirm that CMV is universally distributed amongst human population from the most developed of human population to the least or isolated of human populations. Amongst the different populations studied reveal that the rate of CMV infection increases with age. But the age of primary infection with CMV greatly varies with the living conditions of the individuals. In general, CMV infection is found to be greater in case of developing countries than that in developed countries. CMV infection may occur both vertically and horizontally. It can be transmitted in either route during primary infection, reinfection or reactivation. Transmission to others even during asymptomatic stage of CMV infection may occur frequently through saliva, semen, cervicovaginal secretions etc. 2 to 10% of the infants throughout the world are infected by CMV by the age of 12 months. However, in case of non-infected individuals close contact with different infected persons or living in crowded place in unhygienic areas develops the chances of developing CMV infection by early childhood. Child to child transmission in different play groups and from there to their uninfected parents is often documented. This thus creates a potential threat for future pregnancies. 40% of the adolescents living in good social circumstances are found to be CMV seropositive and this figure is increasing by 1% annually (21). As expected, in case of adults, the figure rises dramatically to 70% in case of adults living in good social circumstances and further increase to a staggering 90% for those living in poor conditions. Following primary infection, in case of immunocompetent individuals CMV persists in a latent form. Occasional reactivations are almost always asymptomatic but this allows horizontal CMV transmission as well as vertical CMV transmission. TRANSMISSION OF HCMV: Transmission of CMV takes place on frequent contact with body fluids of persons excreting the virus. Following CMV infection, infectious virus in present in urine, saliva, tears , semen and cervical secretions for months and years. Thus transmission of CMV is a common event from an infected sex partner to the other, among child in day-care centers etc.CMV infection is thus very much a common affair in case of people having more than one sex partner or for those suffering from sexually transmitted diseases (22). Routes of Primary Transmission Intrauterine It usually follows due to maternal viremia and associated placental infection. Intrauterine infection occurs in approximately 40% of the pregnant women with primary infection. Approximately 1% of the CMV seropositive individuals have their uterine infected with CMV but there is still now no laboratory marker to identify those at highest risk. Perinatal Perinatal CMV infection is acquired from infected maternal genital secreations or breast milk. Perinatal infection was found to be common where breastfeeding was done but absent in case of bottle feeds to infants. Virus shedding toddlers Cohort studies and restriction enzyme analysis of CMV strains indicate that children in day care centers frequently transmit the virus to each other. Day care workers have markedly increased rates of CMV infection ranging from 7 to 20% which is greater than the expected rate amongst US adults of only 2% per year. That, CMV is transmitted from infected child is evident from the fact that, a high rate of seroconvertion of 45% per year was noted in parents of toddlers who shed CMV as compared to 0% in case of parents of toddlers who do not shed CMV (23). Via sexual transmission CMV seroprevalence is very common amongst the patients visiting the clinics for sexually transmitted diseases and amongst the male homosexuals. Following CMV infection, infectious virus is present in semen and cervical secretions for months and years and also body fluids. Thus transmission of CMV is a common event from an infected sex partner to the other.CMV infection is thus very much a common affair in case of people having more than one sex partner or for those suffering from sexually transmitted diseases (22). Blood transfusion The introduction of extracorporeal blood perfusion in the 1960s led to a syndrome of leukopenia, pyrexia etc. caused by HCMV. But, HCMV transmission via blood transfusion is not a very common event and occurring in only about 1 to 5% of seronegative individuals exposed to seropositive blood. Organ transplantation Solid organ transplant recipients are at risk of infection from cytomegalovirus (CMV). A wide range of disease is associated with CMV infection. Cytomegalovirus (CMV) disease continues to be a common problem in solid organ transplant (SOT) recipients. CMV may result in viral syndromes or tissue invasive disease and may have indirect effects on graft function. One of the most important risk factors for CMV disease is the pre-transplant donor (D) and recipient (R) serostatus. Patients who are CMV D+/R are at the highest risk for CMV disease post-transplant. Other recognized risk factors include the use of more potent

immunosuppression, and specifically anti-lymphocyte antibody preparations. In the absence of prophylaxis, CMV disease may occur in up to 60% and 80% of D+/R kidney and liver transplants recipients, respectively (24). It is also possible to transmit CMV in other types of organ transplantation such as the bone marrow, heart, heart and lung, and the liver. Although in the case of the liver, 50 - 100 units of blood is commonly given which carries a far greater risk of transmission. Often, particularly in the case of bone marrow transplantation, the virus causing disease after the transplantation came from the recipient rather than the donor. Also the serological evidence for the transmission of CMV in the case for bone marrow transplantation is not as strong as that for renal transplantation. Mechanism of Virus Recurrence In the general adult population, recurrent CMV infection is uncommon since very few seropositive individuals seem to be excreting virus from the saliva or urine at any time. Where multiple isolates have been obtained and typed by restriction endonuclease analysis, they usually appear to be the same strain which suggests reactivation of the latent virus, though reinfection by the same strain cannot be ruled out. Where the strains are different, reinfection may have occurred or although 2 latent strains of CMV may be reactivated at the time. Immunosuppression produces as increased state of CMV isolation which is usually due to reactivation. Recurrent CMV infections in adults seem to be age dependent. Studies of pregnant and non-pregnant women as well as male homosexuals have shown that up to 10% of seropositive individuals may be excreting CMV form the saliva, urine or the cervix in the case of women. Excretion rates are however, very low after the age of 30 years, suggesting that a more mature host response for suppression may have developed at this age. The table below lists the incidence of CMV infection in various groups: The Rate as referred in the table below (Table 3) is the percent per year who seroconverted themselves from CMV antibody seronegative to CMV antibody seropositive. GROUP Blood donors Hospital employees Richmond, VA Birmingham, AL RATE 1.57 Ref. 46

2 2.2

39

Women in STD clinics 37 The table below (TABLE 4) lists the different routes of transmission of HCMV infection in Infants Route Transplacental Maternal situation Seropositive before conception Primary gestational infection 20-40% Intrapartium Seropositive Virus in genital tract at term 5% 50% Rate of CMV infected infant 0.2-2.2% 48

Breast milk

Seropositive mother Virolactia

25% 65-70%

IMMUNE RESPONSES: During the primary infection CMV specific IgM antibodies are produced. The CMV specific IgM antibodies thus produced persists for 3 or 4 months, but are not produced in recurrent infections in immunocompetent individuals. However, CMV specific IgM may not be produced with primary infection in case of immunocompromised individuals and only 1/3rd of these immunocompromised patients have detectable amounts of IgM in their serum with recurrent infection. CMV IgG antibodies are produced at the time of primary infection and persist lifelong. On reactivation of latent CMV infection, additional IgM are produced. To provide long-term protection IgG antibodies are produced by the body several weeks after the initial CMV infection. Levels of IgG rise during the active infection then stabilize as the CMV infection resolves and the virus becomes inactive. With intrauterine infections, both IgM and IgG are produced by the foetus but the foetal IgG response can only be detected as the passively acquired IgG from the mother is catabolized. Although there is evidence to suggest that the humoral response may be beneficial, eg. Immunocompromised patients who fail to develop IgM run a high risk of developing disseminated infection, the exact role of the humoral response remain uncertain.

CMV antibody testing may be performed to determine immunity to CMV in pregnant women, in patients prior to organ or bone marrow transplantation, and in a person diagnosed with HIV/AIDS. Since CMV infection is widespread and causes few problems to those with intact immune systems, general population screening is rarely done. IgM and IgG antibody testing and viral CMV detection may be used to help diagnose primary CMV infection in young adults, pregnant women, and some immune-compromised patients with flu- or mononucleosis-like symptoms. By comparing the absence or presence of both IgG and IgM in the same sample, the doctor can distinguish between primary, latent, and reactivated CMV in symptomatic patients. CLINICAL FEATURES: The vast majority of people infected with CMV have no symptoms. A few infected people feel ill and have a fever. CMV infection in adolescents and young adults can cause an illness with symptoms of fever and fatigue that resembles infectious mononucleosis. If a person receives a transfusion of blood containing CMV, fever and sometimes liver inflammation may develop 2 to 4 weeks later. If a person with a severely weakened immune system becomes infected with CMV, the infection may be severe, sometimes resulting in serious disease or death. In people with AIDS, CMV infection is a common viral complication. The virus tends to infect the retina of the eye. This infection (CMV retinitis) can cause blindness. Infection of the brain (encephalitis), pneumonia, or painful ulcers of the intestine or esophagus may also develop. If a pregnant woman transmits CMV to the fetus, miscarriage, stillbirth, or death of the newborn may result. Death is caused by bleeding, anemia, or extensive damage to the liver or brain. Newborns who survive may have hearing loss and mental retardation. Congenital Infection: CMV may be isolated from the saliva or urine within 3 weeks of birth, after that, the virus may be transferred from the mother to the infant via breast milk. In the neonate CMV is now the commonest cause of congenital infection and affects around 0.3% - 1% of all live births. 5 - 10% of congenitally infected infants have symptoms at birth. Fatal disease occurs in 20% of these infants. 90% of the symptomatic survivors have sequelae and up to 15% of the asymptomatic survivors have sequelae. CMV is now the second commonest cause of mental retardation after Down's syndrome and causes more cases of congenital damage than rubella. U.S.A. U.K. No. of live births p.a. 3,000,000 700,000 Rate of congenital CMV 1% 0.3% No. of infected infants 30,000 2100 Symptomatic at birth (5 - 10%) 1,500 - 3,000 105 Fatal disease (~20%) 300 - 600 22 No. with sequelae (90% of survivors) 1080 - 2160 83 Asymptomatic (90 - 95% ) 27000 1995 No. with late sequelae 1350 - 4550 315 In the UK, 5% of congenitally infected babies are born with symptoms of "cytomegalic inclusion disease" and their prognosis is poor. The remaining 95% appear to be normal at birth but a proportion develops sequelae later on in life. The classical presentation of cytomegalic inclusion disease is Intra Uterine Growth Restriction (IUGR), jaundice, hepatosplenomegaly, thrombocytopenia and encephalitis with or without microcephaly. It is often difficult to differentiate on clinical grounds between several agents that cause intrauterine infection. The severe thrombocytopenia, hepatitis, pneumonitis and myocarditis may be life threatening. CNS involvement may lead to Epilepsy (seizures), focal neurological signs and mental retardation. Unlike rubella, there is no evidence that CMV is teratogenic. Most of the damage is caused by the destruction of target cells once they have been formed and unlike rubella, the foetus can be damaged by infection during any stage of pregnancy. In 20% of cases (1% of those who are congenitally infected), the infection is so severe that they die during infancy. The rest are likely to sustain serious abnormalities for the rest of their lives. Brain damage is by far the most common abnormality on followup and this may manifest as microcephaly, mental retardation, seizures, cerebral palsy. The spectrum of brain damage may vary from mild to profound. Optic atrophy, deafness and blindness may also be present. The following is a list of the effects of congenital CMV infection:1. CNS abnormalities - microcephaly, mental retardation, spasticity, epilepsy, periventricular calcification. 2. Eye - choroidoretinitis and optic atrophy 3. Ear - sensorineural deafness 4. Liver - hepatosplenomegaly and jaundice which is due to hepatitis. 5. Lung - pneumonitis 6. Heart - myocarditis 7.Thrombocytopenic purpura 8.Haemolytic anaemia 9. Late sequelae - damage to the enamel forming organ of the teeth resulting in yellow discoloration of the teeth and brittleness. This occurs in 40% of such infants. It has become apparent that late sequelae are seen in congenitally infected individuals who are asymptomatic at birth. 15% of such individuals are likely to have hearing defects and reduced intelligence compared to normal people. Like congenital rubella, progressive sensorineural deafness is seen. Prenatal Infection: Cases of infantile pneumonitis have been reported in case of CMV infected infants. But, despite the fact that continued excretion of high titres of virus for many months, the vast majority of prenatally infected infants do not develop acute symptoms although. This appears to be an exceedingly rare event. Postnatal Infection:

4 - 8 weeks is the incubation period for CMV. Primary CMV infection in the postnatal period is usually mild or asymptomatic. Syndrome of infectious mononucleosis with atypical lymphocytosis occasionally accompanies primary infection. This is similar to the syndrome produced by EBV except that lymphadenopathy is uncommon and the Paul-Bunnel test is negative. The post perfusion syndrome is essentially CMV mononucleosis acquired by blood transfusion. Sometimes, the hepatitis picture predominates so that a diagnosis of non-A, non-B hepatitis is made. Immunocompromised (HIV/AIDS) patients: Primary infection in immunocompromised individuals is far less likely to be asymptomatic. The following may be seen;A. The general clinical features are : 1. These patients develop spiking pyrexia which resolves within a few days. Some may develop a viremia with a septicaemia like syndrome in the presence or absence of hepatitis. 2. Pneumonitis may develop which carries a grave prognosis, with 80 - 90% mortality. 3. The virus may disseminate to involve the retina, causing a CMV retinitis. 4. CMV may disseminate to the gut, where it may cause an asymptomatic infection or ulceration or haemorrhage by the erosion of nearby blood vessels. 5. CMV induced immunosuppressive syndrome - The patient becomes unable to deal with opportunistic infections such as pseudomonas. In this instance, the underlying nature of CMV infection is not recognized by the clinical staff who are naturally more concern with the opportunistic infection. B. In addition to these general clinical features other features may be found in patients with AIDS or renal transplant recipients. AIDS patients: Progressive loss of immune function and in particular loss of cell-mediated immunity in patients with AIDS, permits CMV reactivation and replication to begin; asymptomatic excretion of CMV in urine can be detected in approximately 50% of HIVinfected individuals with a CD4 lymphocyte count <100 cells/L.(25) In tissue necrosis in association with nonspecific inflammation cell-to-cell transmission of CMV occurs. Occurrence of transient episodes of CMV viremia can also observe. Viremia probably result in dissemination of CMV to other organs (eg, the retina), thereby setting the stage for subsequent endorgan disease although the clinical significance is uncertain. In the United States >90% of patients with AIDS had evidence of disseminated CMV infection at autopsy prior to the availability of effective ART. At least a partial return of CMV immune responses in most patients and to substantial decreases in CMV titers in blood is possible by ART CMV retinitis is usually seen in those who have not begun or cannot tolerate ART , in settings where ART is in widespread use. In patients with HIV viral loads that increase despite ART, CMV retinitis is relatively rare. A prolonged lag time between failure of ART and impairment of CMV immune responses to an extent allows CMV retinitis to develop. A critical level of immune impairment is required for CMV replication to develop CMV disease, and as long as the CMV load can be kept below this critical level, there is protection against retinitis and other end-organ manifestations. Large atypical cells with intranuclear and intracytoplasmic inclusion bodies is the result of CMV infection .Generally 2 to 4 times larger than normal cells, these cells have an eccentrically displaced nucleus resulting in an owl-like appearance. CLINICAL MANIFESTATIONS: Chorioretinitis: 80% to 90% of CMV disease in patients with AIDS with CD4 lymphocyte counts <50 cells/L suffer from Chorioretinitis. Decreased visual acuity, the perception of floaters or visual field loss are the common presenting symptoms. Ophthalmologic screening of patients with a CD4 lymphocyte count <50 cells/L can detect asymptomatic retinitis. Large creamy to yellowish-white granular areas with perivascular exudates and haemorrhages appear on Ophthalmologic examination of patients with CMV retinitis. These lesions may occur at either the periphery or center of the fundus. Lesions generally progress within 2 to 3 weeks and if left untreated can result in blindness. Retinitis often begins unilaterally, but progression to bilateral disease is common. Systemic CMV disease involving other viscera may also be present. Of at least 90% of HIV-related infectious retinopathies CMV is the causative agent. Differentiating cotton wool spots from suspected CMV retinitis is essential. Cotton wool spots appear as small, fluffy white lesions with indistinct margins and are not associated with exudates or haemorrhages. These lesions do not progress and often undergo spontaneous regression. Toxoplasmosis is the second most common opportunistic infection of the eye followed by CMV, but it can be distinguished from CMV retinitis as it does not cause haemorrhage and is associated in patients with cerebral toxoplasmosis. Herpes Simplex Virus, Varicella-Zoster Virus, Syphilis and Tuberculosis are other infections that may rarely involve the retina. Beginning of prompt treatment for patients with confirmed CMV chorioretinitis should be done. A variety of systemically administered agents as well as local, intravitreal therapies have demonstrated efficacy in delaying time to progression of retinitis. The choice of initial treatment should be based on several factors, including location of lesion (sight threatening or not), the patient's antiretroviral history (treatment naive vs. failing therapy), concomitant medication (with potentially overlapping toxicity), clinical status (especially underlying myelosuppression or renal impairment), and patient preference (especially regarding placement of an indwelling intravenous catheter). Before the availability of ART, CMV retinitis regularly progressed once therapy was discontinued, so maintenance treatment was considered necessary; and even with maintenance therapy, reactivation of retinitis and/or development of new lesions would commonly occur. It is possible to stop maintenance therapy if ART is successful in restoring CD4 counts to at least 100 cells/L (although to 150-200 cells/L is stands better). This should not be attempted until a patient's CD4 count has remained above the levels mentioned for at least 3 months because the lymphocyte response requires time to reach full effectiveness. Patients should be followed carefully for clinical or laboratory evidence of drug failure if maintenance therapy is discontinued. Other factors to consider when deciding to discontinue maintenance therapy include patient adherence to ART medications, location of the retinal lesion, and the patient's vision in the unaffected eye.

Gastrointestinal Disease: Prior to the availability of effective ART in the U.S., CMV enterocolitis occurred in 5% to 10% of patients with AIDS having CD4 lymphocyte counts below 50 cells/L. Diarrhoea, weight loss, abdominal pain, anorexia, and fever are frequently present in patients with CMV enterocolitis. Diseases due to other gastrointestinal pathogens, including Cryptosporidium, Giardia, Mycobacterium, Shigella, Campylobacter, and Strongyloides stercoralis and Entamoeba histolytica as well as involvement by lymphoma or Kaposi sarcoma are also diagnosed. Although a grossly normal-appearing mucosa may be encountered in up to 10% of those individuals with histologic evidence of CMV colitis, endoscopy usually reveals diffuse submucosal hemorrhages and mucosal ulcerations. Vasculitis, neutrophilic infiltration, and nonspecific inflammation is revealed by biopsy; confirmation of the diagnosis is done by the presence of characteristic CMV inclusions and the absence of other pathogens. In patients with AIDS, esophagitis is most commonly due to either Candida albicans or herpes simplex virus, but may also be caused by CMV. Esophagitis patients with CMV often have pain on swallowing in association with a large distal ulceration. Endoscopic examination and tissue biopsy should be performed to establish the diagnosis as in colitis. Patients with symptomatic esophagitis or enterocolitis having no other pathogens detected by endoscopy, histology, or culture and have been confirmed tissue invasion by CMV should receive treatment until clinical improvement occurs. In treating CMV colitis and esophagitis Ganciclovir and foscarnet have demonstrated equivalent efficacy, but the benefits are not dramatic.(26)

Pneumonitis: From lung biopsy specimens or lavage fluid of patients with pulmonary disease due to Pneumocystis jiroveci (formerly carinii) CMV often have been isolated. Responding to therapy directed at P. jiroveci pneumonia alone among many of these patients, indicates that CMV is not a pathogen in such cases. The syndrome is an interstitial pneumonia when CMV causes pulmonary disease in patients with AIDS. Shortness of breath, dyspnea on exertion, and a dry, non-productive cough are often complained by patients. The heart and respiratory rates are elevated, but auscultation of the lungs often reveals minimal findings with no evidence of consolidation. Diffuse interstitial infiltrates showed by Chest radiography, and hypoxemia is usually present. Invasive CMV pneumonia may be present in lung tissues of Patients with interstitial pneumonia and positive CMV. After the exclusion of other pathogens Anti-CMV therapy should be instituted in patients with a progressive, deteriorating clinical course. The addition of CMV immune globulin to antiviral therapy improves outcome in transplant patients with CMV pneumonia, but no such benefit has been observed in patients with AIDS.

Central Nervous System Disease: Radiculopathy, a spinal cord syndrome which is characterized by lower extremity pain and weakness, areflexia, urinary retention, spasticity and hypoesthesia- a distinct neurologic syndrome is caused by CMV in patients with AIDS. The findings from CSF of infected patients are atypical for viral infection and they include polymorphonuclear pleocytosis and a moderately low glucose concentration. Antigen or DNA assays are usually positive- however, culture of CSF may be negative. There are reports of CMV has been isolated from brain tissue or CSF in cases of subacute encephalitis. Subacute encephalitis from other pathogens is comparable to clinical manifestations of CMV encephalitis in patients with AIDS. Personality changes, difficulty concentrating, headaches and somnolence are frequently present. The diagnosis can be confirmed by brain biopsy, with evidence of periventricular necrosis, giant cells, intranuclear and intracytoplasmic inclusions, and positive CMV culture or by identification of CMV by antigen staining or DNA assay in brain tissue or CSF. A study was carried out by Andrea Kovacs et al (1999) (27) where they included 440 infants as their study subjects.75 of the study subject were HIV-1infected and 365 of whom were not. Their CMV status was known and was followed prospectively. The progression of HIV-1 disease was defined as the presence of class C symptoms (according to the criteria of the Centers for Disease Control and Prevention [CDC]) or CD4 counts of less than 750 cells per cubic millimeter by 1 year of age and less than 500 cells per cubic millimeter by 18 months of age. But a significant difference in the rates of CMV infection among the groups of infants studied was observed. CMV infection was found to be more prevalent in the HIV-1 infected infants at six months of age with respect to the non-HIV infected ones - (39.9 percent vs. 15.3 percent, P=0.001). This trend seemed to be persistent and a higher rate of CMV infection was thus recorded in case of four years of age children also (P=0.04). In case of 18 months age infants with both infections ie. CMV coinfection with HIV showed higher rates of HIV-1 disease progression (70.0 percent vs. 30.4 percent, P=0.001), CDC class C symptoms or death (52.5 percent vs. 21.7 percent, P=0.008), and impaired brain growth or progressive motor deficits (35.6 percent vs. 8.7 percent, P=0.005) than infants infected only with HIV-1. Increased risk of HIV-1 disease progression was found to be more associated with CMV infection (relative risk, 2.59; 95 percent confidence interval, 1.13 to 5.95).(28) DIAGNOSTIC METHODS FOR CMV DISEASE: By isolating the virus by culture, histology of biopsies, serological methods, measurement of pp65 antigen in leukocytes and detection of viral DNA using molecular methods, particularly the PCR are used to diagnose CMV infection and CMV associated disease . In predicting development of CMV disease up to several months prior to clinical disease detection of antigen pp65 in peripheral blood polymorphonuclear leukocytes and PCR are useful. Quantitative plasma CMV DNA has high sensitivity (89% ) and good specificity (75% ) for predicting development of CMV disease; against a lower -matrix phosphoprotein (pp65) of the CMV, monoclonal antibodies are used by pp65 antigenemia. CMV DNA has been detected by the PCR test a median of 46 days before the onset of disease, earlier than 34 days median time for antigenemia test and a median of 1 day for CMV blood cultures. Superiority of PCR method to other methods can be showed by multivariate analysis (odds ratio: CMV PCR 10.0, antigenemia test

4.4 and CMV cultures 4.3). In one study, comparison of the results of culturing CMV from plasma and urine with that of determining the plasma PCR in 99 patients showed that the plasma PCR was superior to culture for identification of AIDS patients at risk for CMV disease, and that quantitation of plasma DNA further identified high- risk persons.(29) Development of the branched DNA signal amplification for CMV viral load is performed. Earlier results show that CMV can be quantified very accurately, but in the lower range it lacks sensitivity. The most commonly available serologic test for measuring antibody to CMV is the Enzyme Linked Immunosorbent Assay(ELISA) .This recombinant antigen microtiter ELISA is more sensitive than a viral antigen microtiter ELISA and was able to detect the presence of CMV- specific IgM in response to CMV disease, before the detection of viral proteins by the pp65 antigenemia as say in some patients . A variety of different tests can revealed serum IgM to CMV infection, but ELISA is most commonly used. CMV retinitis is usually diagnosed based on its distinct clinical appearance of retinal necrosis. Most commonly manifestation of these results as a whitish opacification of the retina with exudates and variable amounts of haemorrhage. There is variability in appearance of this lesion depending upon the localization and rate of disease progression. It is neither necessary nor sufficient to make a diagnosis of CMV retinitis by the presence of positive blood cultures for CMV. In a study, only 15 out of 24 (63% ) had positive blood cultures though all 24 patients had positive urine cultures for CMV at the time of diagnosis of CMV retinitis.(29) The histopathological evidence of CMV with an inflammatory response in the appropriate clinical setting helps for diagnosis of CMV esophagitis. The classical sign of the disease is the presence of extensive, large, shallow mucosal ulcers in the distal esophagus. The nonspecific radiographic manifestation of CMV colitis may mimic the findings of other inflammatory bowel conditions, including ulcerative colitis. Flexible sigmoidoscopy or colonoscopy with biopsy and culture should be included for the evaluation in this group of patients. In the person with HIV, CMV involvement of the liver and biliary tract is often noted only at autopsy, hepatitis proven clinically to be secondary to CMV is rare. Histological identification of multinucleated CMV inclusion bodies in lung tissues should be used for diagnosis of CMV pneumonitis. The pathological findings such as typical inclusion bodies or by immunohistochemical or in situ hybridization techniques are used to diagnose CMV neurological diseases. With the prevalence of encephalitis increasing to 75% if the retinitis was adjacent to or involved the optic nerve, a recent autopsy study showed that 42% of patients with CMV retinitis had CMV encephalitis. And 91% of those with CMV encephalitis had CMV retinitis.(30) Therefore, an ophthalmologic evaluation is indicated in AIDS patients with CMV encephalitis and may be of diagnostic value in AIDS patients with neurological symptoms . For documenting CMV infection of the CNS in patients with AIDS,PCR appears to be more useful than clinical and neuroradiologic findings.(31) OBJECTIVE OF WORK: Here we detect the CMV infection in patients from metropolis hospitals by both qualitative and quantitative method. The quantitative test was done by RT-PCR (Shanghai ZJ Bio-Tech Co Ltd kit was used) for viral load assay. The qualitative assay was performed by the indirect immunofluorescence method (US Biological kit was used) for the detection of most abundant pp65 protein of CMV. The pp65 assay was considered as the most sensitive assay for the detection for CMV. Human Interleukin 6 (IL-6) acts as both a pro-inflammatory and anti-inflammatory cytokine and is secreted by T cells and macrophages to stimulate immune response. However, at molecular level, IL6 can induce HIV production thus enhancing the HIV multiplication by a post-transcriptional mechanism.(1) TGF beta1 is a cytokine which is involved with the regulation of different aspects of host defence responses to foreign invaders. Over expression of TGF beta1 can lead to the conversion of its protective functions to pathogenetic manifestations. TGF beta1 promotes HIV replication and thus help in spreading the virus in infected monocytes and peripheral blood mononuclear cells. Through its profound and broad inhibitory effects on different antiviral defence mechanisms, it acts as an immunosuppressant- it facilitates more rapid progression of HIV infection and increases susceptibility to opportunistic infections and malignancies. (2) IFN-gamma, which is a cytokine mediates enhancement of HIV Replication. (3) AIDS patients are under tremendous immunesupression. Under this condition, HCMV reactivation is very common .We are thus interested to study the effect of HCMV infection on the serum levels of these 3 cytokines in these HCMV infected AIDS patients and compare them with the non-HCMV infected ones and to the healthy controls. STUDY DESIGN: Blood was collected from 100 AIDS patients showing symptoms of active HCMV infection with varying CD4 counts (CD4 count from 10-200) and compared with 100 healthy non-HIV controls The serum levels of the cytokines-IL6, IFN gamma and TGF Beta1 in each case of these HCMV infected AIDS patients and healthy volunteers were determined using specific ELISA kits. The serum levels of IL6 and IFN gamma were measured by using ELISA kits from Invitrogen (USA). The serum levels of TGF Beta1 were measured by using ELISA kits from Abcam (USA). HCMV VIRAL LOAD ASSAY BY REAL-TIME PCR In molecular biology, real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction (QPCR/qPCR/qrt-PCR) or kinetic polymerase chain reaction (KPCR), is a laboratory technique based on the PCR, which is used to amplify and simultaneously quantify a targeted DNA molecule. It enables both detection and quantification (as absolute number of copies or relative amount when normalized to DNA input or additional normalizing genes) of one or more specific sequences in a DNA sample. The procedure follows the general principle of polymerase chain reaction; its key feature is that the amplified DNA is detected as the reaction progresses in real time, a new approach compared to standard PCR, where the product of the reaction is detected at its end. Two common methods for detection of products in real-time PCR are: (1) non-specific fluorescent dyes that intercalate with any double-stranded DNA, and (2) sequence-specific DNA probes consisting of oligonucleotides that are labelled with fluorescent reporter which permits detection only after hybridization of the probe with its complementary DNA target. RATIONALE:

Fluorescent reporter probe method: Fluorescent reporter probes detect only the DNA containing the probe sequence; therefore, use of the reporter probe significantly increases specificity, and enables quantification even in the presence of non-specific DNA amplification. Fluorescent probes can be used in multiplex assaysfor detection of several genes in the same reactionbased on specific probes with differentcoloured labels, provided that all targeted genes are amplified with similar efficiency. The specificity of fluorescent reporter probes also prevents interference of measurements caused by primer dimers, which are undesirable potential by-products in PCR. However, fluorescent reporter probes do not prevent the inhibitory effect of the primer dimers, which may depress accumulation of the desired products in the reaction. The method relies on a DNA-based probe with a fluorescent reporter at one end and a quencher of fluorescence at the opposite end of the probe. The close proximity of the reporter to the quencher prevents detection of its fluorescence; breakdown of the probe by the 5' to 3' exonuclease activity of the Taq polymerase breaks the reporter-quencher proximity and thus allows unquenched emission of fluorescence, which can be detected after excitation with a laser. An increase in the product targeted by the reporter probe at each PCR cycle therefore causes a proportional increase in fluorescence due to the breakdown of the probe and release of the reporter. 1. The PCR is prepared as usual, and the reporter probe is added. 2. As the reaction commences, during the annealing stage of the PCR both probe and primers anneal to the DNA target. 3. Polymerisation of a new DNA strand is initiated from the primers, and once the polymerase reaches the probe, its 5'-3'exonuclease degrades the probe, physically separating the fluorescent reporter from the quencher, resulting in an increase in fluorescence. 4. Fluorescence is detected and measured in the Real-time PCR thermocycler, and its geometric increase corresponding to exponential increase of the product is used to determine the threshold cycle (CT) in each reaction. Fig: Fluorescence reporter probe method. Quantification Relative concentrations of DNA present during the exponential phase of the reaction are determined by plotting fluorescence against cycle number on a logarithmic scale (so an exponentially increasing quantity will give a straight line). A threshold for detection of fluorescence above background is determined. The cycle at which the fluorescence from a sample crosses the threshold is called the cycle threshold, Ct. The quantity of DNA theoretically doubles every cycle during the exponential phase and relative amounts of DNA can be calculated, e.g. a sample whose Ct is 3 cycles earlier than another's has 23 = 8 times more template. Since all sets of primers don't work equally well, one has to calculate the reaction efficiency first. Thus, by using this as the base and the cycle difference C(t) as the exponent, the precise difference in starting template can be calculated (in previous example, if efficiency was 1.96, then the sample would have 7.53 times more template). Amounts of RNA or DNA are then determined by comparing the results to a standard curve produced by real-time PCR of serial dilutions (e.g. undiluted, 1:4, 1:16, and 1:64) of a known amount of RNA or DNA. Real-time PCR can be used to quantify nucleic acids by two strategies - Relative quantification and Absolute quantification. Relative quantification measures the fold-difference (2X, 3X etc.) in the target amount. Absolute quantification gives the exact number of target molecules present by comparing with known standards. The quality of Standard is important for accurate quantification. Applications of real-time polymerase chain reaction: There are numerous applications for real-time polymerase chain reaction in the laboratory. It is commonly used for both diagnostic and basic research. Diagnostic real-time PCR is applied to rapidly detect nucleic acids that are diagnostic of, for example, infectious diseases, cancer and genetic abnormalities. The introduction of real-time PCR assays to the clinical microbiology laboratory has significantly improved the diagnosis of infectious diseases, and is deployed as a tool to detect newly emerging diseases, such as flu, in diagnostic tests. MATERIALS REQUIRED:

1. DNA Extraction Buffer 1 vial, 1.8 ml 2. HCMV Reaction mix 1vial, 950 l 3. PCR Enzyme mix 1 vial, 12l 4. Molecular Grade Water 1 vial, 400l 5. Internal Control 1 vial, 30l 6. HCMV Positive Control (1107 copies /ml) 1vial, 30l 7. Biological Cabinet 8. Real Time PCR system (ABI Prism) 9. Desktop microcentrifuge for eppendorf type tubes (RCF max 16,000 x g) 10. Vortex mixture

11. Real Time PCR tubes 12. Cryo-container 13. Pipettes (0.5l 1000 l) 14. Sterile filter tips for micro pipettes 15. Sterile micro tubes 16. Disposable Gloves 17. Biohazard waste container 18. Refrigerator and freezer 19. Tube racks.
SAMPLE COLLECTION: The clinically selected patients from Calcutta Medical and Hospital, School of Tropical Medicine and S.S.K.M Hospital were subjected to routine medical as well as haematological check up with proper history recording. Written consent was obtained from the selected patients for performing collection of sample. Venous blood was drawn after taking adequate precaution and absolute sterilization was maintained. Proper marking of the container was done. The blood sample was transported in a container with EDTA so that coagulation does not occur. At least 5 ml of venous blood were drawn from each subject included in the study. PROCEDURE: DNA EXTRACTION: 1) At first 50l serum or plasma was pipet out to a 0.5 ml tube .Then 50 l DNA extraction buffer was added. Then the tube was closed and vortexed for 10 seconds. Then the tube was spun down briefly in a table centrifuge. 2) Then it was incubate for the 10 min at 1000 C. 3) Then the tube was centrifuge at 13,000 rpm for 13 min. The supernatant contains the DNA was extracted and used for template of PCR. INTERNAL CONTROL AND POSITIVE CONTROL: 1) At first the Internal Control was diluted 10 times by added the molecular grade water provided with the kit. 2) Then it was added and the result was got in the HEX/VIC/JOE channel. It is necessary to add internal control(IC) in the reaction mix .Internal Control allow us to determine and control the possibility of PCR inhibition. QUANTITATION: For the quantitative analysis the standard dilution was prepared as follows. Molecular grade water was used for dilution. 1) At first Positive control(1x 107 copies/ml) was taken, which is provided with the kit, as starting high standard in the first tube. 2) Then pipette 36 l of Molecular Grade water into next three tubes. 3) Then 4 l positive contol was added to the first tubes. 4) Then from the first tube 4 l was pipette out and added to the second tube. 5) Then from the second tube the 4 l was pipette out and added to the next or third tube. 6) Which means concentration of first tube was 1x106 copies/ml. Concentration of the second tube was 1x 105 copies/ml and the concentration of third tube was 1x104 copies/ml.

PCR PROTOCOL: The Master mix for each reaction was pipetted as follows, 35 l Reaction mix + 0.1 l Enzyme mix+ 1 l Internal control

36.4 l Master mix

4 l Extraction DNA

36 l Master mix

Reaction tube

PCR Instrument

1) The volumes of Reaction mix and Enzyme mix per reaction was multiplied with the number of samples, which include the number of controls, standards and sample prepared. Molecular Grade water was used as the negative control. For reasons of imprecise pipetting, always an extra virtual sample was added. Mixed completely then spin down briefly in a centrifuge. 2) 36 l Master mix was pipette out with micropipette of sterile filter tips to each of the Real Time PCR reaction tubes. 4 l DNA sample separately added, positive and negative control to different reaction tubes. Tubes were immediately closed to avoid the contamination. 3) Then it was spun down briefly to in order to collect the Master mix in the bottom of the reaction tubes. 4) Then the following protocol was performed in the instrument: (370 C for 2 min,;940 C for 2 min)1cycle; followed by (93 0 C for 15 sec, 600 C for 60 sec) for 40 cycles. Fluorescence was measured at 600 C; FAM and HEX/VIC/JOE channels were chosen. BASELINE SETTING: It was set at just above the maximum level of Molecular Grade water. OBSERVATIONS FROM THE EXPERIMENTS: When the HCMV symptomatic infection present in the AIDS patients, their blood samples contained more than 600 HCMV DNA copies /ml blood. Below this concentration the virus may not show any physiological effects. pp65 ANTIGENEMIA ASSAY The Cytomegalovirus (CMV) pp65 Antigenemia, Human, Immunofluorescence Kit is intended for qualitative detection and identification of lower matrix protein pp65 of CMV in isolated peripheral blood leukocytes. It is a rapid, sensitive method for detection of CMV in isolated leukocytes. pp65: It is lower matrix protein of CMV. It encoded by the unique long region of CMV genome and by open reading frame 83. The protein is component of dense bodies and a major target for class 1 restricted cytotoxic T lymphocytes (CTL). It is also found in polymorphonuclear leukocytes and endothelial cells during CMV viremia in immunocompromised patients. TEST PRINCIPLE: The assay utilizes an indirect immunofluorescence technique for identifying the lower matrix proteinpp65 of human CMV in cytospin preparation of peripheral blood leukocytes. The blend of monoclonal antibodies provided is bind to CMV pp65 antigen present in formalin fixed leukocytes. Unbound monoclonal antibodies are removed by washing with phosphate buffered saline (PBS). Fluorescein isothiocynate (FITC) conjugated antibody is bind to antigen antibody complex. Unbound conjugate is removed by washing with phosphate buffered saline (PBS). FITC exhibits an apple green fluorescence when excited by ultraviolet light allowing visualization of complex by fluorescence microscopy. Nuclear fluorescence indicates a positive specimen. Uninfected cells counterstain dull red due to presences of Evans Blue in FITC conjugated antibody reagent. MATERIALS: 1. CMV pp65 Mab, 1x5 ml: contains one dropper vial of monoclonal antibody blend against CMV pp65 antigen, protein stabilizer, 0.05%Tween20, and 0.1%sodium azide. 2. IgG (FITC) Pab, 1x10 ml: contains one dropper vial containing anti-mouse IgG (FITC) Pab, 0.02% Evans blue, protein stabilizer and 0.1% sodium azide. 3. Separation Solution, 1x125 ml: contains PBS, Dextarn,0.1% sodium azide. 4. Lyse Stop Solution, 1x60ml: contains PBS, 0.5%sodium azide. 5. Fixation Solution 5X, 1x220ml: contains PBS, formalin, sucrose, 0.5% sodium azide. 6. Permeabilization Solution 5X, 1x220ml: contains PBS, Nonidet P-40, sucrose, protein stabilizer and 0.5% sodium azide. 7. PBS, 1x3 packets. 8. Wash Solution 100x, 1x30 ml: contains protein solution, 0.1% sodium azide. 9. Mounting Fluid, 1x10ml: contains Tris buffered glycerine, a fluorescence enhancer and 0.1 % sodium azide. 10. Cytocentrifuge (Shandon Lipshaw, Cytospin 4), cytocentrifuge slides, holders, funnels and filter cards. 11. Laboratory centrifuge 12. Fluorescence microscope with appropriate filter combination for FITC (excitation=490 nm, emission= 515 nm) with 100x, 200x and 400x magnification (dry objective) 13. Hemocytometer. 14. Microscope slide covers lips. 15. Sodium hypochlorite solution, 0.05% 16. Humid chamber. 17. Incubator (37o c). 18. Coplin staining jars. 19. Distilled water. SPECIMEN COLLECTION: 3 ml of whole blood was collected by venipuncture in EDTA vacuitainer sufficient for CMV pp65 detection. Specimen was transported and kept in an ice box until separation of leukocytes was performed. Sample may be stored up to 24 hours with constant gentle mixing. REAGENT PREPARATION: Prior to specimen processing the required volume of PBS, Fixation Solution, Permeabilization Solution and Wash Solution was prepared as follows: 1. PBS: The content of PBS packet was dissolved in 950 ml of ddH2O.It was mixed thoroughly and the volume was adjusted to 1L with ddH2 O. 2. Fixation solution 5X: it was shaked well before each use ensuring that the stock solution was completely mixed. It was diluted to 1:5 in ddH2 O and mixed thoroughly.

Permeabilization solution 5X: it was shaked well before each use ensuring that the stock solution was completely mixed. It was diluted to 1:5 in ddH2 O and mixed thoroughly. 4. Wash solution: It was ensured that the wash supplement was completely mixed. It was diluted to1:100 in PBS and mixed thoroughly. 5. All other reagent was provided with kit were ready to use. SPECIMEN PROCESSING: 1. Leukocyte separation: Separation solution was directly added to the blood specimen at a ratio of 4:1(blood: separation solution) and mixed thoroughly. Then it was incubated at 37o c for 20 min. Then leukocyte containing supernatant was transferred to a 15 ml conical centrifuge tube and centrifuged at ~ 300x g for 10 min. 2. Erythrocyte lysing: Then the supernatant was discarded. Then the cell pellet was resuspended in ~4 ml of ddH2O, vortex and incubated for 10 to 30 seconds. Then 1 ml of Lyse Stop solution was added, mixed thoroughly and centrifuged at ~ 300 x g for 10 min. Then the supernatant was discarded. The following process was repeated until the heavy RBC contamination was gone. 3. Slide Preparation: The cell pellet was resuspended in ~1 ml of PBS. The cell concentration was determined by using a Hemocytometer. The cell concentration was adjusted to 1x10e6 cells/ml in PBS. The 200l (2x10e5 cells) of cell suspension onto a glass cytocentrifuge slide was centrifuged by using a cytocentrifuge at 600-900 rpm for 3-4 min. Then the slides were removed from the centrifuge and allowed the slides to air dry, after that the slides were immediately fixed. 4. Fixation: The slides were fixed in Fixative solution for 10 min at room temperature. Then the slides were transferred to a Coplin jar contained Wash solution. The wash procedure was repeated using fresh Wash solution. 5. Permeabilization: The cells were permeabilized by immersing the fixed slides in Permeabilization solution for 5 min at RT. Then the slides were placed in fresh Wash solution then it was rinsed in distilled water. Then the slides were rapidly air dried. Then slides were stored desiccated at <-70o C for 3 days. 6. Control Slide production: Control slides were prepared from known positive and negative blood sample followed by the procedure outlined in above section. Control slide were also stored desiccated at <-70o C for 3days. Isolated leukocytes from positive samples, identified by antigenemia testing, was diluted and used to produce positive control slides dilution was targeted 10-15 positive cells per cell spot. Known negative samples were used to produce negative control slides. STAINING PROCEDURE: 1. The test slides and reagents were allowed to equilibrate at RT. 2. Then the slides were placed in a Coplin jar contained PBS for 3-5 min. 3. Then the excess buffer was shaked from slides and carefully area surrounding the cell spot was dried with a cotton swab. The cell spot of test samples were overlaid with sufficient CMV pp65 Monoclonal Antibody to cover with fluid(~40l or one drop).The cell spot of the cell control slides were overlaid with PBS(one drop). 4. Then the slides were incubated at 370 C for 30 min in a humid chamber. 5. Then the antibody was shaked from the slides. Then the slides was washed in a Coplin jar contained PBS.Then the slides were repeatedly washed in fresh PBS. 6. Then the excess buffer was shaked from slides and carefully the area surrounding the cell spot was dried with a cotton swab. 7. Then IgG (FITC) Secondary ab was added to cover each cell spot. 8. Then the slides were incubated at 370 C for 30 min in a humid chamber. 9. Then the antibody was shaked from the slides. Then the slides was washed in a Coplin jar contained PBS. Then the slides were repeatedly washed in fresh PBS. 10. Then the slides were rinsed by dipping in a Coplin jar contained distilled water. 11. Then the excess water was shaked from the slides. The cell spot surrounding area was dried with a cotton swab. Then the cover slip was mounted using the Mounting fluid which was supplied. Then the excess fluid from the edges of the slides was wiped with a cotton swab. 12. Then the slides were examined with a fluorescence microscope at 200 to 400 x, for cell exhibited the apple green fluorescence of FITC. The detailed examination was carried out at 400 x. OBSERVATION AND INFERENCE: Here the negative slide produced no green fluorescence but exhibited the dull red colour of Evans blue counter stain. The positive slide showed the apple green fluorescence due to FITC as it bound to pp65 antigen of CMV.

3.

Figure: negative slide

Figure: positive slide

IL-6 EASIA An immune enzymatic assay for the quantitative measurement of human interleukin-6(IL-6) in serum, plasma, cell culture medium or other biological fluids. IL-6: Human Interleukin-6 (IL-6) is a 184 amino acids polypeptide with potential O and N-glycosylation sites, and a significant homology with G-CSF. It is produced by various cells, including T and B cells, monocytes, fibroblasts, keratinocytes, endothelial cells, bone marrow stroma cells and several tumor cells. It regulates the growth and differentiation of various cell types with major activities on the immnuno system, heamatopoiesis and inflammation. IL-6 induces final maturation of B-cells into antibody producing cells and is a potent growth factors for myeloma/plasmacytoma cells. It stimulates T cell growth and cytotoxic T cell differentiation. IL-6 is also major inducer of the acute phase reaction in response to inflammation or tissue injury. Although all normal controls have undetectable level of IL-6 in their serum huge quantities of IL-6 are detected in severe inflammatory infection such as septicemia. TEST PRINCIPLE: The IL-6 EASIA is a solid phase Enzyme Amplified Sensitivity Immunoassay(EASIA) performed on microtiter plate. The assay is based on an oligoclonal system in which a blend of monoclonal antibodies (MAbs) directed against distinct epitopes of IL-6 is used. Standards or samples containing IL-6 react with captured MAbs1 coated on the microtiter well. After incubation the occasional excess of antigen is removed by washing. MAb2, the Horse Radish Peroxidase(HRP) lebelled-antibody is added. After an incubation period allowing the formation of a sandwitched:coated MAbs1 IL-6-MAb2- HRP, the microtiter plate is washed to remove unbound labelled antibodies. Bound enzyme labelled antibodies are through chromgenic(TMB+H2O2) reaction. The reaction is stopped with the addition of Stop Solution (H2SO4 ) and the microtiter plate is then read at appropriate wavelength. MATERIALS: Microtiter plates with 96 anti-IL-6 coated wells. Standard 0-5 in human plasma with preservatives. Solution A (human plasma with preservatives) for cell culture or urine. Solution B (buffer with preservatives) for serum/plasma. Anti-IL-6- RP conjugate in buffer solution with protein and preservatives. Controls 1 and 2 in human plasma with preservatives. Washing solution concentrate (buffer with preservatives). Chromogen: TMB Stop Solution High quality distilled water Precision pipettes : 50 l, 100 l, 200 l, 1 ml, 10 ml Horizontal microtiter plate shaker capable of 700 rpm. Microtiter plate reader capable of reading at 450 nm and 490 nm Microtiter plate washer. SPECIMEN COLLECTION AND PREPARATION: Blood samples were collected using venipuncture technique and serum was removed from the coagulated or packed cells within 60 minutes after collection. Those specimens which could not assay within 24 hours of collection were frozen at -80C. ASSAY PROCEDURE: Required no. of stripes for the run were selected. 50 l of solution B was pipetted into the appropriate wells. 50 l of each standards and controls or samples were pipetted into the appropriate wells Incubated for 1 hour at room temperature. Liquid from each well was aspirated.

 The plate was washed 3 times by dispensing of 0.4 ml of wash solution into each well and aspirated the contents of each well. 100 l of anti-IL6 conjugate was pipetted into all the wells 50 l of solution A was pipette into each well. Incubated for 1 hour at room temperature. The liquid was aspirated from each well. The plate was washed 3 times by dispensing of 0.4 ml of wash solution into each well and aspirated the contents of each well. 200 l of chromogen was pipette into each well within 15 minutes following the washing step. Incubated for 15 minutes at room temperature. 100 l of stop solution was pipette into each well. The plate was observed at 450 nm within 3 hours. CALCULATION AND RESULT: The mean absorbance value (OD 450) for each set of reference standards, controls and samples were calculated. A standard curve was constructed by ploting the mean absorbance obtained for each reference standard against its concentration in mg/l on graph paper, with absorbance on the vertical (y) axis and concentration on the horizontal (x) axis. Using the mean absorbance value for each sample, the corresponding concentration of IL-6 (mg/l) was determined from the standard curve. The obtained values of the patient samples and control sera should be multiplied by the dilution factor of 100 to obtain IL-6 results in mg/l. HUMAN TGF- ELISA The Invitrogen Multispecies Transforming Growth Factor-beta 1 (Multispecies) ELISA is to be used for the quantitative determination of TGF-1 in human, mouse, rat and swine serum, plasma, buffered solution, or cell culture medium. The assay will recognize both natural and recombinant TGF-1. TGF-1: Transforming growth factor-alpha (TGF-) and transforming growth factor-beta (TGF-) have been implicated in diverse physiologic and pathophysiologic functions including immunological, inflammatory, and neoplastic processes. TGF- is one of the most potent immunoregulatory molecules known. It is a 25,000-dalton homodimeric protein, with three known isoforms in man, TGF-1, TGF-2 and TGF-3. TGF-s are synthesized and secreted by various transformed and normal cells including lymphocytes and monocytes. Differential expression of the isoforms of TGF- is controlled both in vivo and in vitro. Selective regulation of expression of the TGF-s is under the control of several factors, including oncogenes and tumor suppressor genes. In addition to transcriptional control, TGF-s appear to be regulated posttranscriptionally. TGF-1 has a special importance in immunoregulation. Further evidence for the differential expression of TGF- isoforms comes from studies of osteosarcoma. In osteosarcoma, the expression of TGF-3, but not TGF-2 or TGF-1, is highly correlated with disease progression TEST PRINCIPLE: The Invitrogen Multispecies TGF-1 kit is a solid phase sandwich Enzyme Linked -Immuno-Sorbent Assay (ELISA). A monoclonal antibody specific for TGF-1 has been coated onto the wells of the microtiter strips provided. Samples, including standards of known TGF-1 content, control specimens, and extracted unknowns, are pipetted into these wells, followed by the addition of a biotinylated second antibody. During the first incubation, TGF-1 antibody binds simultaneously to the immobilized (capture) antibody on one site, and to the solution phase biotinylated antibody on a second site. After removal of excess detection antibody, Streptavidin-Peroxidase (enzyme) is added. This binds to the biotinylated antibody to complete the four-member sandwich. After a second incubation and washing to remove all the unbound enzyme, a substrate solution is added, which is acted upon by the bound enzyme to produce color. The intensity of this colored product is directly proportional to the concentration of TGF-1 present in the original specimen. MATERIALS: TGF-1 Standard, recombinant human TGF-1. Refer to vial label for quantity and reconstitution volume. Standard Diluent Buffer. Contains 8 mM sodium azide; 25 mL per bottle. Antibody Coated Wells; 96 wells per plate. 1 plate Extraction Solution; 25 mL per bottle. TGF-1 Biotin Conjugate, (Biotin-labeled anti-TGF-1). Contains 8 mM sodium azide; 5.5 mL per bottle. Streptavidin-Peroxidase (HRP) (100x) concentrate. Contains 3.3 mM thymol; 0.125 mL per vial. Streptavidin-Peroxidase (HRP) Diluent. Contains 0.05% Proclin 300; 25 mL per bottle. Wash Buffer Concentrate (25X). 100 mL per bottle. Stabilized Chromogen, Tetramethylbenzidine (TMB). 25 mL per bottle. Stop Solution; 25 mL per bottle. Plate Covers, adhesive strips. 1. Microtiter plate reader capable of measurement at or near 450 nm. 2. Calibrated adjustable precision pipettes, preferably with disposable plastic tips. (A manifold multi-channel pipette is desirable for large assays.) 3. Distilled or deionized water. 4. Plate washer: automated or manual (squirt bottle, manifold dispenser, etc.).

 5. Data analysis and graphing software. Graph paper: linear (Cartesian), log-log, or semi-log, as desired. 6. Polypropylene test tubes for standard dilution and sample extraction. 7. Absorbent p aper t owels. 8. Calibrated beakers and graduated cylinders in various sizes SAMPLE PREPARATION: A. Cell culture media: Centrifuge at 1000 x g for 10 minutes, before extraction step to eliminate any particulates. B. Serum: Collect blood by venipuncture, taking care to avoid hemolysis. Separate the serum from the cells within 1 hour. Centrifuge at 1000 x g for 10 minutes at 4C. Collect the serum. C. Plasma: It is very important to prevent platelet degranulation during the plasma collection because platelets constitute one of the main sources of TGF-1. Heparin may be used as anticoagulant. Separate the plasma from the cells within 1 hour. Centrifuge at 1000 x g for 10 minutes, at 4C. Collect the plasma. Centrifuge the plasma at 3000 x g for 10 minutes (4C) to eliminate platelets. Collect platelet-depleted plasma. D. Storage of samples: If samples (cell culture medium, serum, heparin plasma) are not assayed immediately, but within one month following collection, is stored frozen at 20C. For longer storage, it is freezed at 70C. Avoid repetitive freeze-thaw cycles. SAMPLE EXTRACTION: Serum and plasma: This step allows for the release of TGF-1. In a polypropylene tube, add: 0.1 mL of each serum or plasma sample. 0.3 mL of Extraction Solution, vortexed. Incubate 30 minutes at room temperature with vigorous continuous shaking. Centrifuge at 1000 x g for 10 minutes. Dilute the supernatant 10-fold with Standard Diluent Buffer, in polypropylene tubes. ASSAY PROCEDURE: The number of 8-well strips needed for the assay was determined 200 L of the Standard Diluent Buffer was added to zero wells. Well(s) reserved for chromogen blank should be left empty 200 L of standards, samples (after extraction) or controls were added to the appropriate microtiter wells 50 L of biotinylated anti-TGF-1 (Biotin Conjugate) solution was pipette into each well except the chromogen blank(s). Tap gently on the side of the plate to mix thoroughly Plate was covered with plate cover and incubated for 3 hours at room temperature Thoroughly aspirated or decanted solution from wells and discard the liquid. Washed wells 4 times 100 L Streptavidin-HRP Working Solution was added to each well except the chromogen blank The plate was covered with the plate cover and incubates for 30 minutes at room temperature Thoroughly aspirated or decanted solution from wells and discard the liquid. Washed wells 4 times 100 L of Stabilized Chromogen was added to each well. The liquid in the wells will begin to turn blue Incubated for 30 minutes at room temperature and in the dark 100 L of Stop Solution was added to each well. Tap side of plate gently to mix. The solution in the wells should change from blue to yellow. Read the absorbance of each well at 450 nm having blanked the plate reader against a chromogen blank composed of 100 L each of Stabilized Chromogen and Stop Solution. Read the plate within 2 hours after adding the Stop Solution. CALCULATION AND RESULT: The mean absorbance value (OD 450) for each set of reference standards, controls and samples were calculated. A standard curve was constructed by ploting the mean absorbance obtained for each reference standard against its concentration in mg/l on graph paper, with absorbance on the vertical (y) axis and concentration on the horizontal (x) axis. Using the mean absorbance value for each sample, the corresponding concentration of TGF- (mg/l) was determined from the standard curve. The obtained values of the patient samples and control sera should be multiplied by the dilution factor of 100 to obtain TGF- results in mg/l. HUMAN IFN- ELISA An immunoenzymometric assay for the quantitative measurement of human interferon gamma (IFN-) in serum, plasma, cell culture medium or other biological fluids. IFN-: IFN- (type 2, immune IFN) is structurally and functionally distinct from type 1 (alpha/beta) interferons and acts on a separate receptor. Only one IFN- gene has been identified, coding for a 146 AA protein that is post-translationally processed into two glycosylated species of 20 and 25 Kd. Native IFN- is pH2 -labile, highly basic, and can aggregate to form dimers that are biologically active. IFN- is a real lymphokine produced by activated T (and NK) cells. Despite its clear antiviral and cellular growth regulating activities, its immunomodulatory properties are believed to be the most important. IFN- is the principal activator of macrophage function (Macrophage Activating Factor, MAF), and it also regulates the pathway of differentiation of myeloid cells. It plays an important role in the growth and differentiation of cytotoxic (and possibly suppressor) T cells, activates

NK cells and acts as a B cell maturation factor. It regulates Ig isotype production and inhibits IgE responses. One of the modes of action of IFN- is to induce the expression of membrane proteins, such as class 1 and class 2 MHC antigens and adhesion molecules on various cell types, high affinity Fc receptors for IgG on myelomonocytic cells, etc. Integrated in the cytokine network, IFN- interacts with other cytokines, in either a synergistic (e.g. TNF) or antagonistic TEST PRINCIPLE: The Invitrogen IFN- kit is a solid phase sandwich Enzyme Linked -Immuno-Sorbent Assay (ELISA). The assay is based on an oligoclonal system in which a blend of monoclonal antibodies (MAbs 1) directed against distinct epitopes of IFN- are used. The use of a number of distinct MAbs avoids hyperspecificity and allows high sensitive assays with extended standard range and short incubation time. The oligoclonal antibody has been coated onto the wells of the microtiter strips provided. During the first incubation, standards of known IFN- content, controls, and unknown samples are pipetted into the wells together with a monoclonal antibody (MAb 2) labelled with horseradish peroxidase (HRP). An incubation period allows the formation of a sandwich: coated MAbs 1 - IFN- - MAb 2 HRP. After washing to remove all the unbound enzyme, a substrate solution is added, which is acted upon by the bound enzyme to produce color. The intensity of this colored product is directly proportional to the concentration of IFN- present in the original specimen. MATERIALS: Storage Store at 2 to 8C. Reagents Provided Standard 0 in human serum, with benzamidin and thymol(Lyophilized) Standards 1 - 5 in human serum, with benzamidin and thymol. (Lyophilized). Refer to vial label for concentration and reconstitution volume. 1 U of the standard preparation is equivalent to 1 IU NIBSC 87/586. Controls 1 and 2 in human serum, with benzamidin and thymol. (Lyophilized) Refer to vial label for reconstitution volume and range. IFN- Antibody-Coated Wells, 96 wells per plate. Anti-IFN--HRP Conjugate in Tris-Maleate Buffer with BSA and thymol; 6 mL per bottle. Wash Buffer Concentrate (200x); 10 mL per bottle. Concentrated Chromogen, Tetramethylbenzidine (TMB) in DMF, 1 mL per vial Substrate Buffer: H202 in acetate/citrate buffer; 21 mL per botttle Stop Solution, 1.8 N H2SO4; 6 mL per bottle Microtiter plate reader (at or near 450 nm) with software Horizontal microtiter plate shaker capable of 700 rpm 100 rpm Calibrated adjustable precision pipettes Distilled or deionized water Plate washer: automated or manual (squirt bottle, manifold dispenser, etc.) Glass or plastic tubes for diluting solutions Absorbent paper towels Calibrated beakers and graduated cylinders SAMPLE PREPARATION: The IFN- kit may be used to measure IFN- in serum, plasma, cell culture supernatant as well as other biological fluids. Isolation and culture of peripheral blood mononuclear cells may be realized by usual methods. However, one should avoid an unintentional stimulation of the cells by the procedure. The use of pyrogen-free reagents and adequate controls are mandatory. Sampling conditions can affect values measured in serum or plasma, therefore, strict precautions have to be taken during sampling to avoid impurities contained in sampling materials that would stimulate IFN- production by blood cells and thus falsely increase plasma IFN- values. Serum must be removed as soon as possible from the clot of red cells after clotting and centrifugation, and kept at 4C. Collection tubes must be pyrogen-free. Plasma can be collected on sterile EDTA or heparin tubes (at 4C) and rapidly separated after centrifugation. However, as batches of heparin are often contaminated with pyrogen, it is recommended to test each batch of heparin to avoid unintentional stimulation of blood cells. Other substances in the tube must be also pyrogen-free. These recommendations are also valuable for other biological fluids (urine, etc.). Sample Dilution: If samples generate values higher than the highest standard, dilute the sample with the Standard 0 (Diluent) and repeat the assay. Storage: Serum/plasma samples must be kept at -20C for maximum 2 months, and for longer storage (maximum one year) at - 70C. Samples with low protein levels (e.g. cell culture medium, urine, etc.) should be stored at -70C (maximum one year).

ASSAY PROCEDURE: The number of 8-well strips needed for the assay was determined 50 L of each Standard, Control, or Sample was pipette into the appropriate wells. 50 L of anti-IFN- HRP Conjugate was pipette into all the wells. Incubated for 2 hours at room temperature on a horizontal shaker set at Thoroughly aspirated or decanted solution from wells and discard the liquid. Washed wells 4 times. Pipette 200 L of freshly prepared Chromogen Solution into each well within 15 min.

 Incubate the plate for 15 min. at room temperature on an horizontal shaker set at 700 100 rpm, avoiding direct sunlight 50 L of Stop Solution was pipette into each well. The solution in the wells should change from blue to yellow Read absorbance at 450 nm and 490 nm (reference filter: 630 or 650 nm) within 3 hours. CALCULATION AND RESULT: The mean absorbance value (OD 450) for each set of reference standards, controls and samples were calculated. A standard curve was constructed by ploting the mean absorbance obtained for each reference standard against its concentration in mg/l on graph paper, with absorbance on the vertical (y) axis and concentration on the horizontal (x) axis. Using the mean absorbance value for each sample, the corresponding concentration of IFN- (mg/l) was determined from the standard curve. The obtained values of the patient samples and control sera should be multiplied by the dilution factor of 100 to obtain IFN- results in mg/l.