Detection Of Cytomegalovirus DNA Research Articles

Detection of Cytomegalovirus DNA in Serum Correlates with Clinical Cytomegalovirus Retinitis in AIDS

The high sensitivity of nested polymerase chain reaction (PCR) offers the possibility of rapid detection of cytomegalovirus (CMV) DNA in serum. Five consecutive serum samples were examined from 52 human immunodeficiency virus (HIV)-seropositive patients (19 of whom had clinically presumed diagnosis of CMV chorioretinitis). Presence of CMV DNA in serum was shown to precede development of clinical disease. Eleven patients who developed chorioretinitis were positive for CMV DNA in serum samples obtained 3 months before clinical disease, and 3 retinitis patients who initially were negative for CMV DNA became positive with the onset of clinical retinitis. In contrast, 29 of 33 HIV-seropositive subjects without clinical CMV chorioretinitis and matched with respect to age and CD4 T cell numbers were negative for CMV DNA in all 5 serum samples. Thus, the presence of CMV DNA in serum analyzed by PCR is a good predictive marker of CMV retinitis in HIV-seropositive subjects. A positive PCR results supports the clinical diagnosis and may be useful for monitoring response to antiviral treatment.

Detection of Cytomegalovirus DNA in Urine from Newborns in NICU Using a Polymerase Chain Reaction.

To investigate the natural course of viral shedding during the newborn period, the presence of Cytomegalovirus (CMV) DNA in specimens at 3, 7, 14, 21 and 28 days of life was examined using the polymerase chain reaction (PCR) method. At the 3rd day of life, the viral DNA positive rate in the urine was 7% (4/60), at the 7th day 7% (3/46), at the 14th day 10% (2/20), at the 21st day 10% (1/10), and at the 28th day 25% (1/5). CMV was also detected in samples co-cultivated with HeLa 229 cells and this positive rate was 5% (3/60). The viral positive rate in newborns did not correlate with the gestational age, body weight, or serum IgM level. Six congenital infection cases were identified; two of which were small-for-date babies (SFD) and three of which were born with premature rupture of membranes (PROM). They had no complications during the six months after birth.

Vitreous Fluid Sampling and Viral Genome Detection for the Diagnosis of Viral Retinitis in Patients with AIDS

Cytomegalovirus (CMV) causes severe necrotizing retinitis in patients with the acquired immune deficiency syndrome (AIDS) and other herpesviruses have been implicated in the acute retinal necrosis syndrome (ARN), seen in both the immunocompetent and the immunosuppressed. At present the diagnosis of viral retinitis relies solely on clinical appearances. In order to assess whether the detection of herpesvirus-specific DNA in cell-free vitreous biopsy samples could be useful in the early diagnosis of viral retinitis, vitreous fluid samples were taken from 100 patients. Fifty patients had AIDS as defined by the Centers for Disease Control, (MMWR 36 (suppl 1S):1S-15S, 1987) and retinal disease. The remainder were not known to be HIV infected and had no clinical evidence of retinal infection. Each sample was tested for the presence of CMV, herpes simplex virus 1 (HSV-1), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), and human herpesvirus 6 (HHV6), by amplification of viral DNA using a sensitive and specific nested polymerase chain reaction (PCR). The presence of detectable CMV or VZV DNA was clearly associated with clinical disease whereas the presence of HSV-1, EBV, and HHV6 sequences were not. Clinical discrimination between CMV- and VZV-associated retinitis was greatly enhanced when the PCR results were taken into consideration.

Rapid Detection of Cytomegalovirus DNA in Sera Using the Polymerase Chain Reaction: Relationship with Clinical Diagnosis of Cytomegalovirus Infection after Renal Transplantation

A 21-year-old man had chronic renal failure and received a renal transplant. We used the polymerase chain reaction technique to detect cytomegalovirus (CMV) DNA in serum, blood, and urine for the diagnosis of CMV infection after the renal transplantation. CMV DNA in the serum was detected by polymerase chain reaction only during the active stage, while CMV DNA in urine and blood was detected even during the silent stage of CMV infection. It is suggested that the use of the polymerase chain reaction in serum would be useful for the rapid diagnosis of active CMV infection.

Detection Of Cytomegalovirus Dna In Serum Correlates With Clinical Cytomegalovirus Retinitis in AIDS

The high sensitivity of nested polymerase chain reaction (PCR) offers the possibility of rapid detection of cytomegalovirus (CMV) DNA in serum. Five consecutive serum samples were examined from 52 human immunodeficiency virus (HIV)-seropositive patients (19 of whom had clinically presumed diagnosis of CMV chorioretinitis). Presence of CMV DNA in serum was shown to precede development of clinical disease. Eleven patients who developed chorioretinitis were positive for CMV DNA in serum samples obtained 3 months before clinical disease, and 3 retinitis patients who initially were negative for CMV DNA became positive with the onset of clinical retinitis. In contrast, 29 of 33 HIV-seropositive subjects without clinical CMV chorioretinitis and matched with respect to age and CD4 T cell numbers were negative for CMV DNA in all 5 serum samples. Thus, the presence of CMV DNA in serum analyzed by PCR is a good predictive marker of CMV retinitis in HIV-seropositive subjects. A positive PCR results supports the clinical diagnosis and may be useful for monitoring response to antiviral treatment.

Rapid diagnosis of cytomegalovirus encephalitis in patients with AIDS using in situ hybridisation.

To evaluate the presence of cytomegalovirus (CMV) DNA in the cerebrospinal fluid of patients with AIDS and suspected viral encephalitis using an in situ hybridisation assay with digoxigenin labelled CMV DNA probes. The presence of CMV DNA was evaluated in cerebrospinal fluid cells of 10 patients with AIDS using in situ hybridisation. The positivity of CMV DNA was confirmed by the presence of CMV induced antigens in the same specimens. The presence of CMV DNA and CMV induced antigens was also analysed in peripheral blood leucocytes. The time required to perform the in situ hybridisation assay was about eight hours. The in situ hybridisation assay was sensitive, specific, and provided good resolution. Six patients proved positive for the presence of CMV DNA in CSF cells and all six also proved positive for CMV DNA in blood leucocytes. Of the six CMV positive patients, five were treated with specific antiviral drugs: of these, one died during the treatment while four clinically recovered after one month of treatment. The in situ hybridisation assay using digoxigenin labelled CMV DNA probes can be used as a valuable diagnostic test for the detection of CMV DNA in the cerebrospinal fluid cells of patients with suspected CMV encephalitis and can therefore prompt adequate antiviral therapeutic intervention.

Polymerase chain reaction to detect cytomegalovirus DNA in the cerebrospinal fluid of neonates with congenital infection.

To determine if cytomegalovirus (CMV) DNA could be detected in the cerebrospinal fluid (CSF) of infants with congenital infection with CMV, polymerase chain reaction (PCR) was done on CSF samples from 13 infants (10 with confirmed, 1 with possible, and 2 with asymptomatic congenital CMV infection) and on CSF samples from 100 control patients with alternative diagnoses. By use of two sets of primers that targeted different areas of the CMV genome, CMV DNA was amplified in the CSF of 6 of 10 symptomatic infants, 0 of 3 infants with asymptomatic CMV infection or possible congenital CMV disease, and 2 control patients (one primer set only). A positive CSF PCR result at birth correlated with a poor neurodevelopmental outcome (P = .048; two-tailed Fisher's exact test).

Association of cytomegalovirus infection with post‐transplantation cardiac rejection as studied using the polymerase chain reaction

The relationship between cytomegalovirus (CMV) infection and cardiac allograft rejection is controversial, some authors reporting a significant association, others not, on the basis of the results of conventional virological diagnosis by culture or serology. This problem was reinvestigated in 88 patients using a semi-quantitative nest polymerase chain reaction (PCR) procedure for detecting CMV DNA in endomyocardial biopsy specimens. Significantly more positive biopsies were obtained from patients with moderate (grade 2; P = 0.02) or severe (grade 3a-4; P = 0.03) rejection than with no or mild (grade 0-1b) rejection, whereas there was no significant association between rejection and CMV as diagnosed by virus isolation from urine, throat or blood, or by the detection of CMV-IgM. PCR-positive biopsies originated most frequently from CMV-antibody positive recipients (R+) of hearts from seropositive donors (D+), in association with moderate or severe rejection rather than with mild or no rejection The detection of CMV in the heart thus seemed to be related more to R+D+ serological status than to severity of rejection, that is, to circumstances that favoured co-infection with strains of CMV from both donor and recipient. Studies on sequential biopsy specimens from selected patients also provided evidence that CMV infection and rejection were not always related events. The PCR was able to differentiate latent from active CMV infection; moreover, some seronegative individuals gave repeatedly positive biopsies, thereby supporting the work of others that some patients undergo CMV infection without mounting a detectable antibody response.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of cytomegalovirus DNA in peripheral blood of allogeneic bone marrow transplant patients by the nested polymerase chain reaction.

In order to investigate the contribution of transfusion to CMV infection, 20 CMV-seropositive and 3 seronegative patients undergoing allogeneic bone marrow transplantation were studied for their CMV infection. All the patients received cellular blood component transfusion through a leukocyte depletion filter. CMV infection is defined as positive results with a nested double polymerase chain reaction (PCR) and/or IgM antibody testing. Blood samples of 10 healthy marrow donors and 10 pretransplant patients were available for PCR examination. No CMV-DNA was demonstrated. In contrast, among a total of 122 peripheral blood specimens from the 23 posttransplant patients, 51 from 14 patients were positive for CMV-DNA. Of 20 seropositive patients, 15 were transfused with CMV-seronegative blood which is consisted of 14.3% of blood donors and 5 were transfused with CMV-unscreened blood. Four (80%) of the latter 5 seropositive patients showed CMV infection. Of 15 seropositive patients transfused with CMV-seronegative blood, 11 (73.3%) exhibited CMV infection. There was no significant difference in the incidence of CMV infection between the two seropositive patient groups (Fisher's exact probability test). In most cases, CMV infection occurred after 30 days post BMT. Interstitial pneumonia developed in two seropositive patients who had received CMV-seronegative blood products, and had positive results on both PCR and IgM testing. Regardless of transfusing CMV-unscreened or seronegative blood, none of the seronegative patients were infected with CMV. Our present observations suggest that the CMV-seronegative blood component contributes little if any to prevent seropositive patients from CMV infection.

Rapid and sensitive diagnosis of cytomegalovirus and Pneumocystis carinii pneumonia in patients with haematological neoplasia by using capillary polymerase chain reaction.

We attempted the simultaneous detection of cytomegalovirus DNA (CMV-DNA) and Pneumocystis carinii (carinii-DNA) in sputum samples obtained from 20 patients with haematological neoplasm with pneumonia, using rapid cycle DNA amplification (capillary PCR). We used a thermal cycler for capillary PCR which featured recirculation of hot air for rapid temperature control of 10 microliters reaction samples in thin glass capillary tubes. We extracted DNA from patients' sputa using a simple method. A comparison of the results obtained using the phenol extraction-ethanol precipitation method and those obtained using our simple method was made, and demonstrated complete agreement between the two. For detection of CMV-DNA and P. carinii-DNA with capillary PCR it was not necessary to vary temperature setting based on the primers used. Therefore, capillary PCR was used for the simultaneous detection of CMV and P. carinii. After amplification, the total time required for which was 20 min, amplified products were electrophoresed on agarose gels and visualized with ethidium bromide. Product sensitivity was higher with capillary PCR than with conventional PCR. We conclude that capillary PCR amplification is a valuable tool for rapid and simple diagnosis of CMV and P. carinii pneumonias.

DEMONSTRATION OF CYTOMEGALOVIRUS BYPOLYMERASE CHAIN REACTION AFTER LIVER TRANSPLANTATION

The polymerase chain reaction (PCR) is a highly sensitive and specific technique for detection of cytomegalovirus DNA. With this method we prospectively analyzed buffy coat leukocytes at weekly intervals over 3 months in 60 patients after liver transplantation (LTX). The PCR results were correlated with the pretransplant donor/recipient CMV antibody status and with the occurrence of CMV-induced disease. Thirty-three of 60 (55%) patients became PCR-positive during their posttransplant course. None of the 27 patients with permanent negative PCRs developed CMV disease. Of 33 patients with positive PCRs, 13 (39%) became ill. CMV disease developed in 9 of 22 (41%) antibody-negative recipients but only in 4 of 38 (10%) seropositive graft recipients. The incidence of CMV disease was 75% (9 of 12 patients) in seronegative recipients who converted to positive PCR results and 19% (4 of 21 patients) in seropositive patients with positive PCR findings. The predictive value of a positive PCR was 75% in seronegative patients but it was low (19%) in seropositive recipients. The predictive value of a negative PCR is 100%. Thus, PCR determinations are useful in identifying patients who will not develop CMV disease and in narrowing down the number of individuals who will become sick. Further, PCR is a helpful tool in the follow-up of patients under antiviral treatment.

Detection of cytomegalovirus in blood donors by the polymerase chain reaction

A polymerase chain reaction (PCR) assay was developed and optimized to detect cytomegalovirus (CMV) DNA in the blood of 86 normal donors who had originally tested seropositive for CMV. Evidence of previous or current infection with CMV was determined by rescreening of the blood for CMV antibodies and by detecting the presence of infectious virus in the white cells by cell culture. DNA was extracted from the blood of donors by a manual or an automated method and amplified by PCR using primers from the major immediate early gene of CMV DNA. The amplified product was detected by visualization of a fluorescent 435-base pair DNA band in an electrophoretic agarose gel after ethidium bromide staining and confirmed by slot-blot DNA hybridization using an oligonucleotide probe with complementarity for the major immediate early gene. Seven (8%) of the 86 donors were positive for CMV DNA in both fluorescence and hybridization studies. These donors were also antibody positive. While 74 (86%) of the 86 donors were positive for the presence of CMV antibodies in enzyme-linked immunosorbent assay, none was positive for virus in cell culture. PCR has the potential to be an effective and reliable procedure for the detection of CMV DNA in donor blood, but further study is required for this technique to be used for diagnostic or routine screening purposes.

Polymerase chain reaction detection of cytomegalovirus DNA in peripheral blood leukocytes as a predictor of cytomegalovirus disease in HIV-infected patients

To describe and evaluate a polymerase chain reaction (PCR) method for early diagnosis and prompt management of cytomegalovirus (CMV) retinitis in HIV-infected patients. A total of 110 HIV-infected patients (Centers for Disease Control and Prevention stages II to IV) were sampled sequentially for isolation of CMV from peripheral blood leukocytes (PBL; n = 560) and for amplification of CMV DNA in PBL. Semiquantitative analysis of the PCR product was performed and each PCR-positive specimen was assigned a score between 1+ and 4+ (corresponding to four points on a standard curve of dilutions: 80, 800, 8000 and 80,000 CMV genome copies). Levels of CMV DNA in blood increased with HIV infection stage. We focused on eight patients who developed one or more episodes of retinitis during longitudinal follow-up, in whom we found a strong correlation between viraemia, high PCR signal (3+ or 4+) (P < 0.0001) and clinical symptoms. Relapse was preceded by an increase in CMV DNA and resolution correlated with clearance of CMV DNA from blood. Persistent high PCR levels always preceded virus isolation and may be the first indication of organ involvement and thus early treatment. PCR scores were consistently useful as indicators of drug efficacy and for monitoring of treatment.

Rapid diagnosis of cytomegalovirus and Pneumocystis carinii pneumonia by using the capillary polymerase chain reaction.

We attempted to detect cytomegalovirus DNA (CMV-DNA) and Pneumocystis carinii DNA (P. carinii-DNA) in sputum samples of 18 hematological neoplasm patients with pneumonia, using rapid cycle DNA amplification. A thermal cycler based on recirculating hot air was used for rapid temperature control of 10-microliters samples in this glass capillary tubes. After a total amplification time of 15 min, the amplified products were electrophoresed on agarose gels and visualized with ethidium bromide. In three cases, CMV-DNA was detected at about the time the pneumonia occurred. These patients were successfully treated with ganciclovir in the early stages of infection and CMV was not detected by the virus culture method. In four other cases, P. carinii-DNA was detected in their sputum samples but not detected by Grocott staining. These four cases of P. carinii were successfully treated with sulfamethoxazole-trimethoprim. For detection of CMV-DNA and P. carinii-DNA using the capillary polymerase chain reaction (PCR), a different temperature setting base on the primer difference was not necessary. Therefore, capillary PCR was performed at the same time for detection of CMV and P. carinii. We conclude that capillary PCR amplification is a valuable tool for rapid diagnosis and early treatment of pneumonia due to CMV and P. carinii.

Rapid detection of cytomegalovirus DNA in cerebrospinal fluid of AIDS patients with neurologic disorders.

A polymerase chain reaction (PCR)-based method was used to detect cytomegalovirus (CMV) DNA in 82 cerebrospinal fluid (CSF) samples from 67 patients infected by human immunodeficiency virus (HIV). The test was positive for 14 patients, 8 of whom had CMV-related neurologic disease proven by viral culture of CSF or histologic examination. Encephalitis was the most frequent manifestation in patients with positive PCR results, but CMV DNA was also present in some patients with peripheral neuropathy or polyradiculomyelitis. All patients with proven CMV neurologic disease were positive by PCR. In contrast, viral culture was negative for 4 of the 8 patients and pathologic studies were available only for 5. The specificity of the PCR-based assay could not be assessed precisely because of the lack of a reference standard, but the results correlated well with clinical course and results of the other methods. These findings suggest that the PCR-based method may be a useful noninvasive tool for the rapid diagnosis of CMV-related neurologic disease.

Une nouvelle technique de diagnostic des infections actives à cytomégalovirus: la « nested-Polymerase-Chain-Reaction

We evaluated a new technique, the « nested-Polymerase-Chain-Reactionto diagnose Cytomegalovirus (CMV) infection. This method appears to be highly sensitive and specific to detect CMV DNA in peripheral blood, especially in mononuclear cells, during active infection in immuno-compromised patients.

Failure to detect human cytomegalovirus DNA in peripheral blood leukocytes of healthy blood donors by the polymerase chain reaction.

The transmission of cytomegalovirus (CMV) by blood transfusion may have a major effect on certain immunocompromised patients. To protect susceptible blood recipients from infection, it is advisable to use blood components from CMV-seronegative donors. However, serologic tests are not capable of indicating which blood component actually harbors infectious virus and can transfer it to the recipient. Therefore, a sensitive method is needed for the detection of the virus itself. There have been three reports on the detection of CMV in healthy volunteer blood donors by the polymerase chain reaction (PCR). CMV DNA was found in all seropositive and most seronegative blood donors. However, many other authors have failed to confirm these data. A highly sensitive and specific PCR assay was developed for the detection of CMV DNA in peripheral blood leukocytes. With this protocol, blood samples from 116 volunteer blood donors were investigated. None of these samples proved to be positive for CMV DNA. In contrast, CMV DNA was detected in 10 of 10 renal transplant patients early in the course of active CMV infection. It can be concluded that the CMV genome copy number in the peripheral blood leukocytes of healthy individuals is beyond the detection limit of current PCR technology.

Cytomegalovirus DNA detection in sera from patients with active cytomegalovirus infections.

Using a specific and sensitive polymerase chain reaction method, we detected reliably the presence of human cytomegalovirus (HCMV) DNA directly in serum samples collected at an early stage of HCMV infection, even before immunoglobulin M (IgM) antibodies were measurable. HCMV DNA was detected in serum from all patients with active HCMV infection; in 91% of these patients, HCMV DNA was found in the acute-phase serum. In 13 of 44 patients, HCMV DNA was found in serum before HCMV-specific IgM. For four kidney transplant recipients, the occurrence of HCMV DNA in serum, virus isolation from urine and leukocytes, and HCMV IgG and IgM serology were determined. We found a correlation between HCMV DNA in serum and positive virus isolation from leukocytes. In three of five congenitally infected infants, HCMV DNA and HCMV IgM were detected in the same sample. Two other infants were HCMV DNA positive, although no HCMV IgM antibodies were measurable. HCMV was found in urine from these infants either by virus isolation or with the polymerase chain reaction. Serum from one of the 22 healthy HCMV-seropositive blood donors was HCMV polymerase chain reaction positive.

A survey of cytomegalovirus (CMV) DNA in primary sclerosing cholangitis (PSC) liver tissues using a sensitive polymerase chain reaction (PCR) based assay

Reactivation of cytomegalovirus (CMV) has been implicated as a possible etiological agent in primary sclerosing cholangitis (PSC) partly because of the ability of CMV infection to cause hepatobiliary damage, and further because of the recent recognition of a PSC-like syndrome in AIDS patients, many of whom have hepatobiliary infection with CMV. Direct evidence of CMV infection in PSC has come from a study detecting CMV DNA in 7/7 PSC livers, but only 5/20 controls. We have developed an assay for CMV-DNA by amplification of the immediate early region of CMV using the polymerase chain reaction, followed by Southern blotting and 32P oligoprobing of the amplification product. This system has an average sensitivity of at least 25 copies of CMV-DNA per 5000 formalin-fixed paraffin-embedded cells. 37 PSC and 19 control samples of formalin-fixed paraffin-embedded hepatobiliary tissues were studied. Amplification for the β-globin in each sample was used as an amplification control, and fetal lung with known CMV infection as the CMV-positive control. 37/37 PSC tissues amplified for β-globin, and one of these was positive for CMV-DNA. All 19 controls amplified for β-globin, with none being positive for CMV. The lack of CMV-DNA in 35/36 PSC samples at a level of 25 copies per 5000 cells, we believe, rules out any significant CMV reactivation in these tissues, and suggests that CMV replication and re-activation is not responsible for the progression of PSC.

Polymerase chain reaction in detection of CMV DNA in renal allograft recipients.

This study investigates the use of polymerase chain reaction (PCR) in comparison with viral culture and serology for monitoring of cytomegalovirus (CMV) infection in 21 consecutive renal allograft recipients treated with a quadruple immunosuppression protocol. In addition, an attempt is made to explore the significance of quantitation of CMV signals obtained from peripheral blood leucocytes. CMV infection developed in 16 patients with seven of these patients having organ involvement. All of these 16 patients had a fourfold rise in antibody titres as well as positive identification of CMV DNA in peripheral blood leucocytes by PCR. Blood viral cultures were negative in two of these patients. All five patients who remained PCR negative also remained culture negative with no antibody change. PCR detected CMV infection on average 15 days and 20 days earlier than viral culture and serology respectively. All except one of the patients with CMV organ involvement had an initial peak of CMV DNA followed by prolonged carriage of detectable CMV. The majority of patients with fever only or asymptomatic CMV infection had a transient peak of CMV DNA. A high incidence of CMV disease with organ involvement occurred in seronegative recipients of kidneys from seropositive donors (3/5) and in seropositive recipients of kidneys from seronegative donors (3/7). OKT3 was associated with a higher incidence of CMV organ involvement compared to Antilymphocytic globulin (3/5 v 4/16) but there was a higher incidence of CMV mismatched patients in the OKT3 treated group.(ABSTRACT TRUNCATED AT 250 WORDS)