Primer-mediated enzymatic amplification of cytomegalovirus (CMV) DNA. Application to the early diagnosis of CMV infection in marrow transplant recipients.
S CassolM C PoonR PalMatthew J. NaylorJ Culver-JamesTom BowenJoanne RussellStephen A. KrawetzRichard T. PonDI Hoar
105
Citation
36
Reference
10
Related Paper
Citation Trend
Abstract:
A nucleic acid amplification procedure, the polymerase chain reaction (PCR), has been used to establish a diagnostic assay for the identification of cytomegalovirus (CMV) immediate-early sequences in clinical specimens. Preliminary testing against virus-infected cell cultures indicated that the PCR assay was highly CMV-specific, recognizing both wild-type and laboratory strains of CMV. There was no cross-reactivity with human DNA or with DNA from other herpes viruses. The sensitivity of the assay, using cloned CMV AD169 Eco RI fragment-J as template, was 1 viral genome per 40,000 cells. In a prospective study of CMV infection in bone marrow transplant recipients, the PCR assay correctly identified four patients with confirmed CMV infection. In three of these patients who were followed longitudinally, correlation of DNA reactivity with CMV culture and CMV antibody status over time indicated that DNA was the most sensitive marker for the diagnosis of CMV infection.Keywords:
Betaherpesvirinae
Cytomegalovirus
Primer (cosmetics)
Abstract A novel nested quantitative‐competitive polymerase chain reaction (nQC‐PCR) assay was developed to quantify as few as ten copies per tube of human cytomegalovirus DNA with an overall dynamic range of 10–10 5 copies per tube. This nQC‐PCR assay is based on co‐amplification of a mimic DNA and it was evaluated with 26 cerebrospinal fluid (CSF) specimens and 44 serum specimens from 70 CMV‐infected AIDS patients, 35 of them were diagnosed of CMV retinitis. An excellent correlation was found between nQC‐PCR assay and the commercially available Cobas Amplicor CMV Monitor™ (CACM) assay ( R = 0.9999; P < 0.001; n = 42). Moreover, 13 serum samples with CMV viral loads undetectable with the CACM were successfully quantified by nQC‐PCR. CMV viral load was significantly higher in patients with CMV retinitis ( P = 0.003). The nQC‐PCR assay described below is a very sensitive test for accurate quantitative detection of CMV DNA in different clinical specimens that avoids the need for high‐cost instrumentation. J. Med. Virol. 72:249–256, 2004. © 2004 Wiley‐Liss, Inc.
Betaherpesvirinae
Cytomegalovirus
Cite
Citations (6)
ABSTRACT Applying nested-PCRs, we frequently detected DNA of Epstein-Barr virus and cytomegalovirus but not JC virus in cerebrospinal fluid samples from 140 human immunodeficiency virus-infected patients with central nervous system symptoms in northern Thailand. Despite the low incidence of primary central nervous system lymphoma or cytomegalovirus encephalitis among Thai AIDS patients, Epstein-Barr virus and cytomegalovirus infections in the central nervous system are common.
Cytomegalovirus
Betaherpesvirinae
Cite
Citations (9)
Detection of cytomegalovirus (CMV) DNA by the polymerase chain reaction (PCR) in samples of cerebrospinal fluid (CSF) has been shown to be a sensitive method of diagnosing CMV disease in the central nervous system. Since CMV causes latent infection in white blood cells, an unanswered question is whether detection of latent CMV DNA in the cell fraction of CSF samples by PCR is possible in seropositive patients. In a prospective study, the finding of CMV DNA in CSF of CMV seropositive patients with suspected viral infection of the central nervous system (CNS) was evaluated clinically. Fractionation of 64 CSF samples from seropositive patients was carried out before analysing the samples for CMV DNA by PCR. In four of the five patients who had CMV DNA in the cell pellet and/or supernatant, the clinical data suggested CMV-associated neurological disease. The remaining 59 samples were negative in both pellet and supernatant. In addition, 11 CSF samples with high cell counts from patients with bacterial meningitis were examined for CMV DNA and found to be negative in 10 patients and positive in 1. One hundred thirty two uncentrifuged CSF samples were used as negative controls. The results of the study indicate that detection of CMV DNA in CSF samples by PCR correlated well with disease and was not due to latent CMV infection.
Cytomegalovirus
Betaherpesvirinae
Cite
Citations (43)
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.
Cytomegalovirus
Betaherpesvirinae
Cite
Citations (105)
The polymerase chain reaction (PCR) and viral culture techniques were prospectively compared for the detection of cytomegalovirus (CMV) in blood samples from 24 liver transplant recipients. Nine patients had one or more episodes of viremia, seven of which were clinically symptomatic infections. All samples in which CMV was isolated by culture were positive by the PCR. However, the PCR result was also positive for one or more samples from 11 patients who never developed CMV-related symptoms. Although the PCR is a very sensitive technique for CMV detection in blood samples from liver transplant recipients, it is not useful as a marker of symptomatic CMV disease.
Viremia
Cytomegalovirus
Betaherpesvirinae
Liver disease
Viral culture
Cite
Citations (113)
Cytomegalovirus (CMV) disease is a serious complication after bone marrow transplantation (BMT). Recently, semi-quantitative polymerase chain reaction (SQ-PCR) analysis for CMV-DNA has been developed. We compared the time course of SQ-PCR and that of antigenaemia assay after BMT. Our findings suggested that SQ-PCR is more sensitive than antigenaemia assay in the detection of CMV infection and that quantitation of CMV-DNA could discriminate patients with positive PCR but without the risk for CMV disease. We considered that SQ-PCR could be a useful guide for starting pre-emptive therapy against CMV disease.
Cytomegalovirus
Betaherpesvirinae
Cite
Citations (23)
We used the polymerase chain reaction (PCR) to demonstrate cytomegalovirus (CMV) DNA in the CSF of a patient with the CMV radiculomyelopathy syndrome. To investigate the significance of this finding, we also performed PCR for CMV on CSF samples from 30 patients with human immunodeficiency virus (HIV) infection and neurologic disease, four patients with solid organ transplants including three with active CMV infection, and 10 patients with no clinical suspicion of HIV or CMV infection. There was CMV DNA only in patients with HIV, and it was present more often in patients with evidence of spinal cord dysfunction. Our results suggest that PCR may be useful in the rapid diagnosis of CMV infection of the CNS in patients with HIV and that the radiculomyelopathy syndrome may represent only part of a spectrum of CMV-induced spinal cord dysfunction in these patients.
Cytomegalovirus
Betaherpesvirinae
Cite
Citations (50)
The nested Polymerase Chaain Reaction (PCR) was compared with virus isolation and serology to establish which is the best method for the diagnosis of active cytomegalovirus (CMV) infection. Samples of blood leucocytes, urine and throat washings from immunosuppressed patients and patients with congenitally acquired CMV infection, and well as from healthy persons were examined with PCR. CMV DNA was detected in all samples from which CMV could be isolated, but not from any sample from healthy adults, whether CMV seropositive or CMV seronegative. In contrast to the findings in healthy persons, CMV genomes were frequently detected in urine and throat washings from immunosuppressed, CMV-seropositive patients without symptoms of CMV infection. The appearance of CMV genomes in blood cells in immunosuppressed CMV-seronegative patients may be the first sign of primary CMV infection. Congenital CMV infection could be rapidly and safely diagnosed when urine samples were examined by PCR. Nested PCR is a valuable tool for the diagnosis of active CMV infection, when selected materials are used.
Cytomegalovirus
Betaherpesvirinae
Throat
Cite
Citations (32)
Cytomegalovirus
Betaherpesvirinae
Cite
Citations (2)
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.
Betaherpesvirinae
Cytomegalovirus
Buffy coat
Cite
Citations (18)