Detection of cytomegalovirus using PCR in serum from renal transplant recipients.

AIMS--To develop a polymerase chain reaction (PCR) assay for the detection of cytomegalovirus (CMV) DNA in serum and leucocytes of renal transplant recipients and compare this assay with CMV culture and serodiagnosis. METHODS--Monthly specimens were obtained from 12 patients starting immediately before transplant. CMV infection was monitored by IgM enzyme linked immunosorbent assay, virus culture and PCR on serum and leucocytes. RESULTS--Two of four IgG positive patients had reactivation of CMV disease confirmed by culture, three of eight seronegative patients had a primary infection, one confirmed by serology and two by culture. PCR was positive earlier than conventional methods in three cases and concurrently in two. No positive PCR reactions occurred in the seven patients who remained negative by culture and serology. CONCLUSIONS--CMV DNA is detectable in serum; serum may be positive before virus is detectable by buffy coat culture; and PCR may be useful as an early indication of potential CMV disease in renal transplant recipients.     

Cytomegaloviru; antibody in the urine of renal transplant patients.

Small amounts of cytomegalovirus (CMV) antibody were detected in the urine of renal transplant patients excreting the virus. The antibody was probably produced locally, as a result of active CMV infection of the urinary tract.     

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 reinvesti-gated 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 0-1 b) 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 seroneg-ative individuals gave repeatedly positive biopsies, thereby supporting the work of others that some patients undergo CMV infection without mounting a detectable antibody response. The use of the PCR provided additional and more definitive information linking CMV and post-transplantation cardiac rejection when compared to conventional methods of viral diagnosis which were less suitable as correlates of deep seated focal infection. © 1994 Wiley-Liss, inc.     

Qualitative and semiquantitative polymerase chain reaction testing for cytomegalovirus DNA in serum allows prediction of CMV related disease in liver transplant recipients

AIM: To identify cytomegalovirus (CMV) infection in liver transplant recipients by polymerase chain reaction (PCR) techniques and to separate the cases in which CMV related disease will occur, for whom treatment is indicated, from those in whom infection will remain innocuous. METHODS: The combination of qualitative and semiquantitative PCR of serum and urine was assessed to determine whether these assays can identify those at risk of CMV related disease and compared their performance with conventional approaches to diagnosis. RESULTS: Qualitative PCR of serum had superior specificity, sensitivity, and positive and negative predictive values compared with urine DEAFF (detection of early antigen fluorescent foci) and PCR of urine. All episodes of CMV related disease were associated with the presence of CMV DNA by PCR in serum or urine; CMV was detected before clinical onset in 70% and 60% of cases, respectively. The period over which CMV DNA could be detected was not correlated with CMV related disease. Both peak viral load and cumulative viral load estimated using a semiquantitative PCR method on serum samples positive by the qualitative method could be used to distinguish asymptomatic infection from CMV related disease with 100% specificity and sensitivity. In contrast semiquantitative PCR of urine was of little value. CONCLUSIONS: An approach based on PCR testing with a combination of qualitative and subsequently semiquantitative serum samples would improve the diagnosis of CMV infection and aid identification of those patients at risk of CMV related disease, allowing treatment to be targeted specifically.     

High prevalence of renal transplant recipients infected with more than one cytomegalovirus glycoprotein B genotype

A prospective analysis of cytomegalovirus (CMV) glycoprotein B (gB) genotypes was conducted on 34 renal transplant recipients using peripheral blood leukocytes (PBLs) and urine specimens. The CMV gB genotypes were analyzed by polymerase chain reaction (PCR) followed by enzyme digestion. PBLs and urine samples showed almost equal proportions of the 4 known gB genotypes, as well as equal proportions of gB genotype mixtures. The gB genotypes 1, 2 and 3 were equally distributed in the patients. Twenty-four (70.6%) patients had more than one gB genotype during follow-up. There was no association of gB genotypes with the development of symptomatic CMV infection.     

Monitoring levels of human cytomegalovirus DNA in blood after liver transplantation.

We evaluated a semiquantitative PCR assay prospectively in 40 liver transplant recipients as an aid in making a prompt diagnosis of cytomegalovirus (CMV) infection. For 2 months after transplantation, clinical specimens from patients were tested weekly by PCR, virus isolation from peripheral blood and urine, and CMV serology. The incidence of active CMV infection was 70%. The levels of CMV DNA determined by hybridization of PCR samples and densitometric scanning of blots were assigned a score of 1 to 4 by comparison with four external standards amplified in parallel and corresponding to a range of 80 to 80,000 genomes. The first detection of CMV in blood by PCR occurred at a mean of 15 days, and high-level PCR scores of 3 or 4 were obtained 21 days after transplantation, whereas viremia occurred 33 days after transplantation. Significantly higher levels of CMV DNA were seen in patients with CMV disease (P < 0.05) than in asymptomatic patients. The prevalence of symptomatic CMV infection was 30%. The positive predictive value of PCR was 48%, while the negative predictive value was 100%. After treatment, the clearance of CMV DNA was always observed and the disappearance of symptoms occurred concomitantly with undetectable PCR signals.     

Immunoglobulin M to cytomegalovirus in primary and reactivation infections in renal transplant recipients.

Two commercially available enzyme immunoassays and one assembled in house were used to measure immunoglobulin M (IgM) antibody to cytomegalovirus (CMV) in a total of 220 serum specimens from 104 renal transplant recipients. All assays included a step in which interfering IgG antibody was removed or complexed. Concordance of results between pairs of assays ranged from 84 to 96%. All sera from patients with recent seroconversion (primary CMV infection) had measurable anti-CMV IgM. Among those already seropositive to CMV when transplanted, 26 to 55% had IgM antibody posttransplant, depending on the assay. This was observed regardless of the CMV serologic status of the kidney donor, indicating that reactivation of endogenous CMV, as well as reinfection, can induce this antibody in transplant recipients. Four cadaver donors known to transmit CMV to eight recipients did not have measurable IgM antibody to CMV.     

Early diagnosis of cytomegalovirus infection in renal transplant and dialysis patients by DNA-DNA hybridisation assay

One hundred forty-eight urine specimens were collected from 47 renal transplant and dialysis patients and screened for the detection of cytomegalovirus (CMV). Diagnosis of CMV infection was suggested in 17 out of 47 patients (36.2%) by more than one of the five methods used. DNA hybridisation assay (DNA HA) using 32P-labelled probe detected CMV DNA in 15 (31.9%) of 47 patients, whereas virus isolation on conventional tube cell cultures (CTC), immunofluorescence incorporating monoclonal antibodies on centrifugation vial cultures (IF), complement fixation test (CFT), and electron microscopy (EM) yielded positive results in only nine (19.2%), 12 (25.2%), 11 (23.4%), and one (2.1%) of 47 patients, respectively. The significance of these results obtained by DNA HA lies not only in the apparent increase in number of patients diagnosed, but also in both early and rapid detection of CMV DNA. More importantly, the DNA HA is highly specific in that it correlates accurately with clinical and laboratory data characteristic of CMV disease. In respect of clinically manifest CMV disease, the specificity of DNA HA, CTC, IF, CFT, and EM was 87.5, 43.7, 56.3, 43.7, and 6.3%, respectively. These advantages of DNA HA make it the test of choice for early diagnosis of CMV infections in immunosuppressed patients.     

Detection of antibodies to pre-early nuclear antigen and immediate-early antigens in patients immunized with cytomegalovirus vaccine.

To define the role of antibody to immediate early antigens of human cytomegalovirus (CMV) in diagnosing acute infection, we studied antibody responses of adult volunteers and renal transplant candidates receiving CMV vaccine. In addition, CMV antibodies in healthy adults were determined. Antibody to pre-early nuclear antigen, one of the immediate early antigens, did not develop in all of the volunteers or renal transplant candidates receiving the vaccine. In those vaccinees who developed an antibody response, the duration of the response was variable. Antibody to pre-early nuclear antigen and immediate early antigens was also detected in some healthy adults with serological evidence of past CMV infection, but without clinical evidence of recent infection. In view of these results, antibody to immediate early antigens and specifically to pre-early nuclear antigen does not appear to be suitable for rapid diagnosis of acute CMV infection.     

Detection of virus-specific IgA antibodies in serum of kidney transplant patients with recurrent cytomegalovirus infection by enzymeimmuno and radioimmunoassay techniques.

The feasibility of using human cytomegalovirus (CMV)-specific IgA antibody determinations as a signal for early detection of recurrent CMV infections in eight renal transplant recipients was analyzed. Solid phase radioimmunoassay (RIA), enzyme-liked immunosorbent assay (ELISA) and immunoperoxidase assay (IPA) techniques were used for IgA antibody determinations. In parallel, IgG antibodies to CMV were studied by immunoperoxidase assay. A significant rise of CMV-specific IgG antibody titre was observed in all of these patients between 5 and 53 weeks post-transplantation. CMV-specific IgA antibody production was detected close to the time a rise in CMV IgG antibody was observed in seven out of eight patients studied by RIA and ELISA, and in six out of eight patients studied by IPA. In two patients specific CMV IgA antibodies were detected by all three methods before a significant rise of CMV IgG antibody titre was demonstrated. In these patients CMV IgA was detected by RIA earlier than by ELISA and IPA. The potential application of CMV-specific IgA antibody determination for early detection of recurrent CMV infection in renal transplant patients is discussed.     

Usefulness of blood and urine samples collected on filter paper in detecting cytomegalovirus by the polymerase chain reaction technique

A rapid test for the diagnosis of congenital CMV infection is still needed. This study evaluated the usefulness of dried blood and urine samples collected on filter paper for detecting cytomegalovirus (CMV) by the polymerase chain reaction (PCR) assay compared with the use of liquid urine. Samples were obtained from 332 infants aged 1-7 days. Liquid urine samples were collected into bags, cultured in human fibroblasts, and processed using a multiplex PCR technique. Dried urine samples were obtained by placing a piece of filter paper in contact with the infant's genitals. The heels of neonates were punctured and capillary blood was blotted onto filter paper and dried. Dried blood and urine specimens were analyzed by multiplex PCR and nested-PCR assays. A diagnosis of congenital CMV infection was established by isolating the virus, and by detecting viral DNA in the liquid urine. Of the 332 liquid urine samples collected from 332 neonates, seven (2.1%) were positive for CMV and 325 were negative, by both cell culture and PCR assay. In dried samples, CMV DNA was detectable only with a nested PCR assay. Compared with known CMV infection status, 5/7 (71.4%) neonates were positive for congenital CMV infection using dried blood samples. All 325 uninfected neonates were negative. In the dried urine samples, 4/4 CMV-infected infants gave positive tests, and all 262 uninfected infants were negative. Although further improvements in sample collection and/or processing are still needed, PCR testing on dried urine or blood collected on filter paper is a promising approach in the diagnosis of neonatal CMV infection.     

HLA mismatching and cytomegalovirus infection as risk factors for transplant failure in cyclosporin-treated renal allograft recipients

In a study of the effects on renal allograft survival of HLA mismatching, mismatching for cytomegalovirus (CMV) antibody status, and post-transplant CMV infection, 148 cyclosporin-treated renal transplant recipients were given kidneys optimally matched for HLA-A, -B, and -DR antigens but not matched for CMV antibody status. Mismatching for HLA-B and -DR antigens was associated with a greater number of rejection episodes and a lower graft survival, but mismatching for CMV antibody status and posttransplant primary or recurrent CMV infection exerted no effect on graft survival. The role of matching of renal transplant recipients and donors for CMV antibody status in preference to HLA matching (proposed as a means of reducing the mortality associated with primary CMV infection) is discussed.     

Comparison of quantitative cytomegalovirus (CMV) PCR in plasma and CMV antigenemia assay: clinical utility of the prototype AMPLICOR CMV MONITOR test in transplant recipients

The correlation between the prototype AMPLICOR CMV MONITOR test (Roche Molecular Systems), a quantitative PCR assay, and the cytomegalovirus (CMV) pp65 antigenemia assay was evaluated in transplant recipients. Sequential blood specimens were collected on 29 patients (491 specimens), the leukocyte fraction was tested by CMV antigenemia, and quantitative PCR was performed on plasma specimens. None of the 15 patients (242 specimens) who were antigenemia negative were positive for CMV DNA by PCR, and none of these patients developed active CMV disease. There were 14 antigenemia-positive patients, 8 of whom developed active CMV disease. In all patients, there was a good association between the antigenemia and PCR assays. Ganciclovir-resistant virus was isolated from three patients with active CMV disease. These three patients had persistently elevated levels of antigenemia and CMV DNA by PCR when resistance to ganciclovir developed. This standardized, quantitative CMV PCR assay on plasma has clinical utility for the diagnosis of active disease and in monitoring the response to antiviral therapy in transplant recipients.     

Diagnosis of cytomegalovirus infection in cyclosporin-treated renal allograft recipients

The relative merits of antibody response and virus shedding as markers of cytomegalovirus (CMV) infection among cyclosporin-treated renal allograft recipients were analysed. CMV-specific antibody was assayed by IgG-specific radioimmunosorbent test (RIST) and by complement fixation test (CFT). CMV shedding was assayed by virus isolation and by the rapid test immediate early nuclear antigen detection (IENAD). RIST and CFT detected seroconversion in similar numbers of patients, but the former test was the more sensitive when CMV antibody was sought in pretransplant sera to differentiate primary from recurrent infection. IENAD detected or excluded CMV shedding for more urine specimens than virus isolation (462/515 [90%] vs. 366/515 [71%]), but the reverse applied to saliva specimens (33/57 [58%] vs. 54/57 [95%]). The high specificity of IENAD allowed positive results by IENAD to be accepted when virus isolation failed to provide a result. IENAD was, however, less sensitive than virus isolation even when specimens yielding CMV by IENAD, but no result by virus isolation, were included in the analysis (27/44 [61%] vs. 38/44 [86%]). Assays of both antibody response and virus shedding were required to maximise the diagnosis of recurrent CMV infections, but the former assay detected all primary CMV infections. The diagnostic implications of these results are discussed.     

Comparison of PCR and pp65 antigenemia assay with quantitative shell vial culture for detection of cytomegalovirus in blood leukocytes from solid-organ transplant recipients.

This study compared PCR and an assay for cytomegalovirus (CMV) pp65 antigenemia (CMV-vue; INCSTAR Corp.) with a quantitative shell vial culture (QSVC) technique for the detection of CMV in serial blood specimens from 46 solid-organ transplant recipients. In a comparison based on 535 specimens tested by PCR and QSVC, CMV was detected by PCR in 41 and by QSVC in 37 of 43 recipients at risk of CMV infection. The mean number of days after transplantation of initial detection of CMV was 29.9 for PCR and 34.0 for QSVC (P = 0.01). The antigenemia assay was performed on 395 specimens, including 304 of those also tested by PCR. In these specimens, CMV was detected by the antigenemia assay, QSVC, and PCR in 30, 32, and 35 (respectively) of 38 patients at risk, with no statistically significant difference in the time to detection. Each of the assays detected CMV in similar proportions of patients with and without clinically significant CMV infection. PCR stayed positive longer after transplantation than the other assays but frequently returned to negative when more than 6 months had elapsed after transplantation. The antigenemia assay and PCR stayed positive longer after institution of antiviral therapy than QSVC. PCR can provide highly sensitive detection of CMV viremia, but a PCR assay for CMV is not yet available in kit form. The pp65 antigenemia assay and shell vial culture are quantifiable and comparable in sensitivity. Either is recommended for rapid detection of CMV in blood specimens from solid-organ transplant recipients.     

Detection of cytomegalovirus DNA in sera of liver transplant recipients.

We prospectively studied the utility of the amplification of cytomegalovirus (CMV) DNA in the sera of liver transplant recipients in order to predict symptomatic CMV infection, thus enabling preemptive therapy with antiviral agents. Serum samples obtained at biweekly intervals from 20 sequential liver transplant recipients for at least 8 weeks following transplantation were tested by the PCR amplification procedure. Results were correlated with blood and urine cultures, histopathological findings from infected organs, and clinical manifestations. Six patients (30%) developed symptomatic CMV infection; in five (83%) of these patients, CMV DNA was detected prior to symptomatic CMV infection, and in one (17%) of these patients, CMV DNA was detected at the time of symptomatic CMV infection. CMV DNA was detected a mean of 13 days (range, 0 to 23 days) prior to the onset of symptomatic CMV infection. In addition, CMV DNA was detected in the sera of four of five patients with asymptomatic viremia and two patients with asymptomatic viruria. Lastly, the PCR was negative for sera from seven patients with no evidence of CMV infection. We found that PCR was able to detect the presence of CMV DNA in the sera of liver transplant recipients at a sensitivity of 92% and a specificity of 100% for CMV infection, while the sensitivity and specificity for symptomatic infection were 100 and 57%, respectively.     

Antibodies to cytomegalovirus-induced pre-early nuclear antigen in the anticomplement-immunofluorescent test in comparison to IgG and IgM antibodies in the indirect and direct enzyme-linked immunosorbent assay in diagnosing cytomegalovirus infections

Summary Development of antibody to pre-early nuclear antigen (anti-PENA) in persons with primary cytomegalovirus (CMV) infection was tested in serial serum specimens of four renal transplant patients and four patients undergoing open heart surgery using the anticomplement-immunofluorescent test (ACIF). In patients undergoing open heart surgery seroconversion of anti-PENA was mostly concomitant with the rise of IgG or IgM antibodies determined in the indirect or direct enzyme-linked immunosorbent assay (ELISA) whereas in all renal transplant patients developing anti-PENA a delayed rise of this antibody compared to IgG and IgM antibodies was observed. Significant rise of anti-PENA accompanied by an increase of IgG and IgM antibodies in indirect and direct ELISA was also found in three patients undergoing open heart surgery with recurrent CMV-infection. Anti-PENA was shown to persist longer than IgM antibody. Moreover, anti-PENA was present in the serum of nearly two-thirds of 30 persons with IgG antibody but without IgM antibody.It is concluded that antibody determination to PENA can serve as an additional means of diagnosing primary and recurrent CMV infections. Because of its long persistence only seroconversion or significant rise of this antibody may be considered evidence of infection. In some patients a delayed development of anti-PENA must be taken into consideration.     

Screening of bone marrow and kidney transplant donors and recipients for cytomegalovirus antibody by commercial latex agglutination or competitive enzyme-linked immunosorbent assays

A commercial competitive enzyme-linked immunosorbent assay and a sensitive in-house radioimmunosorbent test detected cytomegalovirus (CMV) antibody in similar numbers of serum specimens (66 and 67 of 152, respectively) obtained from bone marrow and renal transplant donors and recipients. In contrast a commercial latex agglutination assay for CMV antibody apparently gave false positive results when testing serum specimens obtained from organ donors. The implications for CMV screening of organ transplant donors and recipients are discussed.     

Comparison of two quantitative CMV PCR tests, Cobas Amplicor CMV Monitor and TaqMan assay, and pp65-antigenemia assay in the determination of viral loads from peripheral blood of organ transplant patients.

BACKGROUND: Quantitative PCR assays have become the most common methods in the determination of viral load during cytomegalovirus (CMV) infection of transplant patients. However, usually these tests are still quite time-consuming and labor-intensive which diminishes their utility of these tests in routine diagnostic laboratories. Objectives: The objective of this study was to develop a quantitative CMV PCR test which is time-saving and easy to perform for the detection and monitoring of CMV infection of transplant patients. STUDY DESIGN: The quantitative real time CMV PCR assay using TaqMan chemistry and an automated sample preparation system, MagNA Pure LC, was developed. The designed quantitative CMV test was compared to commercial quantitative PCR test, Cobas Amplicor Monitor, in the determination of CMV DNA loads in plasma samples of liver and kidney transplant patients. The results were also correlated with the CMV pp65-antigenemia test. The clinical material of 270 blood specimens of transplant patients were tested using these two PCR methods and pp65-antigenemia test in parallel. Plasma samples were used for PCR assays and leucocytes for the antigenemia test. RESULTS: The TaqMan assay described was easy to perform, it was rapid (3-4 h) and hands-on time needed for performing the test was short. The detection limit of the assay was 250 copies/ml (cps/ml) plasma and the linear range up to 25,000,000 cps/ml. TaqMan assay was the most sensitive test detecting 92% of the CMV positive findings. Cobas Monitor detected 80% and pp65 test 88% of the positive findings. The correlations between TaqMan and antigenemia assays, and between Cobas Amplicor and antigenemia were statistically significant and high, R = 0.84 (P < 0.0001) and R = 0.80 (P < 0.0001), respectively. Also correlation between two PCR tests was statistically significant (R = 0.64, P < 0.0001). Of the 27 patient studied, 19 demonstrated CMV antigenemia and DNAemia in their blood during the post transplant monitoring. Thirteen of these patients developed a symptomatic CMV infection and were treated with ganciclovir. The peak viral loads of symptomatic patients were statistically higher by all three methods than those of asymptomatic patients. CONCLUSIONS: The developed real time TaqMan assay was rapid and easily performed and could be the best alternative for the diagnosis of CMV infection and monitoring of liver and kidney transplant patients.     

Detection of CMV DNA: is EDTA whole blood superior to EDTA plasma?

The early diagnosis of human cytomegalovirus (CMV) infection in immunosuppressed patients has been improved by molecular detection of CMV DNA. In this study, corresponding EDTA whole blood and EDTA plasma specimens were obtained from 42 bone marrow transplant recipients. For detection of CMV DNA, two commercially available assays, the CMV HHV6,7,8 R-gene (ARGENE), and the artus CMV LC PCR Kit (QIAGEN), were employed. The linearity of both assays was determined by using a clinical EDTA whole blood sample with high CMV DNA load. With the CMV HHV6,7,8 R-gene test, CMV DNA was detected in 40 EDTA whole blood and in 19 EDTA plasma samples, while the artus CMV LC PCR Kit test detected CMV DNA in 27 EDTA whole blood and in 30 EDTA plasma samples. In conclusion, EDTA whole blood samples were found to be the superior material when using the CMV HHV6,7,8 R-gene test. However, this benefit may not exist when employing alternative assays.