Human cytomegalovirus (HCMV) late-mRNA detection in peripheral blood of AIDS patients: diagnostic value for HCMV disease compared with those of viral culture and HCMV DNA detection
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A reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect a human cytomegalovirus (HCMV) late mRNA in peripheral blood leukocytes (PBL) of 102 human immunodeficiency virus-infected individuals. The clinical value of this new technique for the diagnosis of acute HCMV disease was evaluated in comparison with viral culture and direct amplification of viral DNA (PCR). The sensitivity of the RT-PCR was slightly lower than that of the two other methods, but its specificity was 94%, compared to 55 and 32% for culture and PCR, respectively. Transcription of this late mRNA is linked to viral replication, and its detection in PBL confirms that these cells can support a complete viral cycle. The relationship between complete replicative cycles and HCMV disease makes RT-PCR a useful clinical tool.Keywords:
Viral culture
Betaherpesvirinae
Cytomegalovirus
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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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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A multiplex, single-step PCR protocol for the detection of human cytomegalovirus (HCMV) DNA is described. The protocol amplifies regions of the viral LA and IE genes and employs elevated temperatures for both reagent mixing and primer annealing together with product detection by silver staining on polyacrylamide gels. This assay detects one to five HCMV genomes in clinical samples containing up to 100 ng of human DNA, a level of sensitivity equivalent to that of more complex assays involving either nested PCR or postamplification hybridization. As well as being of importance in clinical situations where high-sensitivity qualitative diagnosis is required, this assay is also applicable to the monitoring of HCMV infection in renal transplant recipients. Due to its multiplex format the assay provides quantitative information, in that samples from which a single target is amplified contain on average sevenfold fewer viral genomes per 10(6) leukocytes than those from which both targets are amplified. When weekly blood leukocyte DNA preparations from renal transplant patients were assayed, findings of three consecutive tests in which both HCMV targets were amplified were highly indicative of patients who had developed very high loads of HCMV (100% sensitivity, 88% specificity). We thus show that the same simple PCR assay which permits highly sensitive HCMV diagnosis can also be used for the efficient identification of transplant recipients at risk of clinically significant infection.
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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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Abstract In two studies comparing detection of human cytomegalovirus (HCMV) in 118 patients (93 of whom were immunocompromised) by the polymerase chain reaction (PCR) and virus isolation using either early antigen detection by culture‐immunofluorescence or conventional cytopathic effect, DNA‐PCR was found to be the most sensitive, followed by culture‐immunof luorescence, then by cytopathic effect. Urine was inhibitory to the action of Taq polymerase; this was overcome by concentration of HCMV with PEG 6000 prior to gene amplification. Without PEG treatment, HCMV‐DNA in 6 of the 11 specimens positive by culture‐immunofluorescence was not detectable by PCR. In healthy seropositive individuals, HCMV‐DNA was not detected i n leucocytes. However, in immunocompromised patients with AIDS or transplants, and therefore at high risk of HCMV infection or reactivation, blood leucocytes were usually positive for HCMV‐DNA (19/20), some for as long as 20 weeks after initial detection and persisting for long after culture‐immunofluorescence became negative. Neither HCMV‐RNA nor infectious HCMV were detected in the follow‐up blood leucocycte specimens from immunocompromised patients who had detectable HCMV‐DNA in these cells. These data suggest that persistence of HCMV‐DNA in blood leucocytes of immunocom‐promised patients after reactivation or primary infection may be due to persistence of non‐viable virus, residual HCMV genomic DNA, or latent HCMV‐DNA. © 1992 Wiley‐Liss, Inc.
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Members of the Herpesviridae family have been implicated in a number of tumours in humans. At least 75% of the human population has had contact with cytomegalovirus (HCMV). In this work, we screened 75 Brazilian glioma biopsies for the presence of HCMV DNA sequences. HCMV DNA was detected in 36% (27/75) of the biopsies. It is possible that HCMV could be a co-factor in the evolution of brain tumours.
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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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Abstract Eight patients were referred for prenatal diagnosis for suspected fetal cytomegalovirus infection (CMV): six for documented first‐trimester infection and two for abnormal ultrasound evaluation suggestive of fetal infection. Three methods of diagnosis were employed: (1) amniotic fluid viral cultures and CMV‐specific IgM in fetal serum; (2) amniotic fluid cultures and detection by polymerase chain reaction amplification of CMV‐specific DNA in chorionic villi; and (3) detection of CMV‐specific DNA in villus samples only. Amniotic fluid cultures detected all cases of infection, but CMV‐specific IgM was not a reliable indicator of infection in any case. DNA analysis correlated well with both culture results and clinical outcome.
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