Role of PCR in the diagnosis and management of CMV in solid organ recipients: What is the predictive value for development of disease and should PCR be used to guide antiviral therapy?
Michaël AbécassisAlan J. KoffronMarguerite BuckinghamDixon B. KaufmanJonathan P. FryerJ StuartFrank P. Stuart
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Cytomegalovirus
Betaherpesvirinae
Antiviral Therapy
Valganciclovir
Peripheral blood samples (n= 1240), obtained at variable intervals from 483 heart transplantation (HTx patients under immunosuppressive therapy, and blood samples (n=1013) obtained upon blood donation from 1013 healthy anti-human cytomegalovirus (HCMV) positive blood donors, were tested for HCMV DNA by nested polymerase chain reaction (PCR). The detection limit of the nested PCR was determined to be less than 10 copies of the plasmid pRR 47, containing the HCMV immediate early gene. HCMV DNA was detected in 79 of 483 HTx patient (17%). To the contrary' HCMV DNA was only detected in 1 of 1013 anti-HCMV positive, healthy blood donors (0.1%). This PCR positive donor had recently contracted a primary HCMV infection. The rate of HCMV PCR positive immunosuppressed HTx patients in our study was lower than the rate of HCMV PCR positive healthy blood donors in previous reports in the literature. Blood samples (269 from 117 HTx patients) were assayed for HCMV DNA in peripheral blood leukocytes, HCMV DNA in plasma, and HCMV tegument protein 65 kDa (pp 65 antigen). Three laboratory diagnostic patterns were observed and related to clinical findings: (1) HCMV DNA only in leukocytes was observed in 26 patients, 7 of whom had HCMV disease, 5 of whom had graft rejection, and 14 of whom had no specific symptoms; (2) HCMV DNA both in leukocytes and in plasma (viremia) was observed in 3 patients, who were all symptomatic with HCMV disease; (3) HCMV DNA in leukocytes and in plasma (viremia) and pp 65 antigen were observed in 13 patients, all of whom were symptomatic (10 patients had HCMV disease, and 3 patients had graft rejection). A similar sequence of diagnostic patterns was observed in all symptomatic HCMV infections and reactivations in this study: HCMV DNA appeared first in peripheral blood leukocytes, then also in plasma, followed by pp 65 antigen detectable in peripheral blood leukocytes. Upon clinical recovery, these findings disappeared in reverse order. However, HCMV DNA remained detectable in peripheral blood leukocytes for several weeks. The detection of HCMV DNA in the peripheral blood is an exception, not the rule, even in severely immunosuppressed HTx patients. It indicates a pathological condition, albeit without clinical symptoms in some patients, and it is the earliest signal of HCMV replication. Of 42 patients in whom HCMV DNA was initially detected only in peripheral blood leukocytes, 16 patients progressed into viremia. Thus, HCMV-specific PCR performed on nucleic acid extracts from lysed peripheral blood is an appropriate method for the monitoring of HCMV infections in immunosuppressed HTx patients.
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The ratio of human cytomegalovirus (HCMV) genomes per cellular genomes in serial peripheral blood leukocyte (PBL) extracts of renal allograft recipients was quantitated by competitive nested polymerase chain reaction (PCR). Patients were also monitored for the development of acute HCMV infection by detection of HCMV pp65 antigenemia, HCMV IgM antibodies, and viruria. Compared to qualitative nested HCMV PCR, the frequency of positive PCR results in renal allograft recipients without further evidence of acute HCMV infection was significantly reduced by quantitative HCMV PCR. HCMV DNA levels > or = 1,000 copies HCMV/10(6) copies beta-globin were found to be highly indicative for the development of a clinically symptomatic HCMV infection following renal allograft transplantation. In patients treated with ganciclovir, quantitation of HCMV target sequences allowed the assessment of the efficacy of antiviral therapy.
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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.
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We developed a rapid, sensitive, and specific PCR-based assay for human cytomegalovirus (HCMV). The assay includes primer and probe sequences derived from conserved HCMV nucleotide sequences and nonradioactive hybridization-confirmation. The assay detected between 10 and 100 viral genomes. All HCMV clinical isolates tested (39 of 39) gave positive reactions.
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The possible correlation between cytomegalovirus, human herpesvirus types 6, 7 and cytomegalovirus-related clinical symptoms was studied in kidney transplant patients in Kuwait. Cytomegalovirus infection was diagnosed using the pp65 antigenemia assay. DNA of cytomegalovirus was detected by nested polymerase chain reaction (nested-PCR). PCR was also used to amplify the genes coding for structural proteins of human herpesvirus-6 (240 bp) and human herpesvirus-7 (186 bp). Glycoprotein B genotypes of cytomegalovirus were determined by restriction fragment length polymorphism. The average number of cells positive for cytomegalovirus pp65 antigen showed a steady increase with the severity of the cytomegalovirus-related symptoms. Furthermore, cytomegalovirus pp65 antigen positivity was significantly more frequent among recipients of cadaver kidney (45.5%) than among those who received live related kidneys (22.6%). Cytomegalovirus gB genotype 1 was detected more frequently (P<0.036) in recipients with live related donor kidney (38%) than in patients of cadaver kidney (13%). The genome of human herpesvirus-6 was detected at the same rate in patients with or without cytomegalovirus-related symptoms. However, the genome of human herpesvirus-7 was detected significantly more frequently (P<0.0001) in asymptomatic patients (41.7%) than in recipients with symptomatic cytomegalovirus infection (17%). We conclude that cytomegalovirus gB genotypes are not associated with the outcome of a cytomegalovirus infection in kidney transplant patients, that human herpesvirus-6 does not play a role in cytomegalovirus pathogenesis and that the role of human herpesvirus-7 in cytomegalovirus-related morbidity in kidney recipients remains unclear.
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Abstract Human cytomegalovirus (HCMV) is one of the major pathogens causing neurologic disease in the immunocompromised host. A competitive nested polymerase chain reaction (PCR) was used to determine DNA load, distribution, and sequence variability of HCMV genomes in the brain of AIDS patients with and without HCMV encephalitis confirmed by histology and immunocytochemistry. By quantitative PCR, HCMV genomes were found to be distributed diffusely in the central nervous system (CNS) of all five patients with histologically proven HCMV encephalitis, but also in the brain of five of eight AIDS patients without neuropathological evidence of HCMV encephalitis. The viral DNA load in cases with HCMV encephalitis was increased 10‐ to 1, 000‐fold as compared to patients without evidence of encephalitis. A viral load above 6, 000 copies HCMV/106 copies β‐globin was found to be highly suggestive for HCMV encephalitis. Characterization of PCR products by temperature gradient gel electrophoresis (TGGE) and direct sequencing allowed us to detect a sequence variability of the amplified fragment of HCMV glycoprotein B (gB) among different patients, but also among different HCMV foci within the same patient. Furthermore, two of five AIDS patients with HCMV encephalitis most likely experienced double infections with different HCMV strains. The experimental procedure described in this study should also be applicable to the detection of significant HCMV DNA levels in biopsy samples. © 1995 Wiley‐Liss, Inc.
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Human herpesvirus (HHV)-6 is a beta-herpesvirus-like human cytomegalovirus (HCMV) with the potential to reactivate in immunocompromised persons. HHV-6 and HCMV were assessed in the peripheral blood leukocytes of 26 lung transplant recipients and of 37 human immunodeficiency virus (HIV)-infected patients receiving highly active antiretroviral therapy, to determine the degree of concordance between HHV-6 and HCMV reactivation in different biologic settings. In the lung transplant recipients (145 samples), HHV-6 was not detected, even though 44 (30%) of 145 samples were from 9 HCMV DNA-positive patients (13 episodes of HCMV pneumonitis). Among the HIV-infected patients (172 samples), HCMV DNA was detected in 29 (17%) of 172 samples from 10 patients (4 episodes of HCMV disease). HHV-6 DNA was detected in 2 HIV-infected patients who did not have HCMV detected at that time. These findings suggest that the pathobiologic control mechanisms for these 2 beta-herpesviruses may be significantly different.
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Fifty human cytomegalovirus (HCMV) isolates were recovered from different clinical specimens (buffy coat, throat washing and urine) obtained from fifty patients (23 AIDS patients, 20 heart transplant recipients, 1 bone marrow transplant recipient, 2 newborns with congenital HCMV infection and 4 immunocompetent individuals with acute HCMV infection). The isolates were previously identified by immunological methods and then examined for identification by the polymerase chain reaction. In parallel, reference HCMV strains as well as other human members of the Herpesviridae family (reference and wild strains) were examined as controls. Two pairs of primers relevant to the immediate-early and late regions of HCMV DNA, respectively, were used. The DNA amplification product was highly specific; in addition, all fifty HCMV isolates were amplified by both pairs of primers and thus identified as HCMV. These preliminary results show that selected pairs of primers are able to amplify DNA regions conserved enough to allow virus identification among a large number of HCMV strains. In addition, they are highly promising in view of the use of PCR for direct detection of HCMV DNA in clinical samples.
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