Rapid detection and quantification of CMV DNA in urine using LightCycler™-based real-time PCR

doi:10.1016/S1386-6532(01)00240-2

Journal of Clinical Virology

Volume 24, Issues 1–2, February 2002, Pages 131-134

https://doi.org/10.1016/S1386-6532(01)00240-2 Get rights and content

Abstract

A real-time quantitative PCR-hybridisation assay was developed for the detection of human cytomegalovirus DNA in clinical material. The assay is based on a LightCycler (LC) and provides both rapid results (<1 h) and quantification over a broad dynamic range (2×10³–5×10⁸ CMV DNA copies/ml). Given that the assay showed a 3-fold increase in sensitivity compared to detection of early antigen fluorescent foci (DEAFF) testing of urine samples, we investigated the practicality of testing surveillance such specimens from immunocompromised patients at risk of CMV disease. Over a 12-month period, CMV DNA was detected in 81 (7%) of 1154 urine samples examined. A total of 28 patients tested positive; urine viral loads were higher in 13 infants being investigated for suspected congenital infection (median 1.6×10⁵ copies/ml) compared with 15 transplant recipients (median 9×10³ copies/ml). Urine samples could be tested directly without processing such that results were available in <1h. Real-time PCR provided information on the quantification of CMV DNA in urine and proved a reliable method for the surveillance of immunocompromised patients at risk of CMV disease. This approach should facilitate a better understanding of the epidemiology and natural history of CMV disease. Moreover, LC-based quantitative PCR is a potentially valuable tool for the management of CMV disease; assisting with the prompt initiation of treatment and assessing therapeutic response.

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Development of a real-time quantitative polymerase chain reaction assay for the detection of congenital human cytomegalovirus infection in urine samples
2016, Molecular and Cellular Probes
Citation Excerpt :
These results are in according to previous studies, in which shell vial viral culture was compared with HCMV DNA detection. In fact, shell vial viral culture showed a sensitivity ranging from 61.5% to 100% assuming real-time qPCR as a reference method [12,15,23–25]. Discordant results were observed for only a single sample, in which classical viral culture failed to detect the HCMV target.
A TaqMan real-time quantitative PCR (qPCR), based on amplification of HCMV UL54 gene specific sequence, was developed and compared with shell vial viral culture assay, the gold standard technique for the diagnosis of congenital HCMV infection, using urine samples collected from 110 newborns. The results indicate that this qPCR is slightly more sensitive than shell vial assay suggesting that qPCR may be considered a useful alternative for diagnosing congenital HCMV infection.
Immunotherapy with Donor T Cells Sensitized with Overlapping Pentadecapeptides for Treatment of Persistent Cytomegalovirus Infection or Viremia
2015, Biology of Blood and Marrow Transplantation
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Patients were sequentially monitored for toxicities using the Common Terminology Criteria for Adverse Events (version 4, 2009) and for acute GVHD as graded by the Center for International Blood and Marrow Transplant Research Consensus [27]. CMV in the blood was measured by quantitation of CMV antigenemia in the initial 8 patients and by CMV polymerase chain reaction thereafter, using methods previously described [28-31]. CMV levels were monitored before the T cell infusion, at weekly intervals for 6 weeks thereafter and monthly until clearing or clinical progression.
We conducted a phase I trial of allogeneic T cells sensitized in vitro against a pool of pentadecapeptides (15-mer peptides) spanning the sequence of CMVpp65 for adoptive therapy of 17 allogeneic hematopoietic cell transplant recipients with cytomegalovirus (CMV) viremia or clinical infection persisting despite prolonged treatment with antiviral drugs. All but 3 of the patients had received T cell–depleted transplants without graft-versus-host disease (GVHD) prophylaxis with immunosuppressive drugs after transplantation. The CMVpp65-specific T cells (CMVpp65CTLs) generated were oligoclonal and specific for only 1 to 3 epitopes, presented by a limited set of HLA class I or II alleles. T cell infusions were well tolerated without toxicity or GVHD. Of 17 patients treated with transplant donor (n = 16) or third-party (n = 1) CMVpp65CTLs, 15 cleared viremia, including 3 of 5 with overt disease. In responding patients, the CMVpp65CTLs infused consistently proliferated and could be detected by T cell receptor V_β usage in CMVpp65/HLA tetramer + populations for period of 120 days to up to 2 years after infusion. Thus, CMVpp65CTLs generated in response to synthetic 15-mer peptides of CMVpp65 are safe and can clear persistent CMV infections in the post-transplantation period.
Usefulness of a real-time quantitative polymerase-chain reaction (PCR) assay for the diagnosis of congenital and postnatal cytomegalovirus infection
2014, Anales de Pediatria
El citomegalovirus (CMV) es principal virus causante de infecciones congénitas y posnatales en la población pediátrica. El objetivo de este estudio es evaluar la utilidad de una PCR cuantitativa en tiempo real en el diagnóstico de estas infecciones utilizando la orina como única muestra.
Se estudiaron todas aquellas muestras de orina de recién nacidos (< 7 días) con sospecha de infección congénita y las orinas de pacientes con sospecha de infección posnatal (orina al nacer negativa). Las orinas se han estudiado de forma simultánea mediante cultivo celular, PCR cualitativa (PCRc) y PCR cuantitativa en tiempo real (PCRq).
Se han analizado 332 orinas (270 para descartar infección congénita y 62 infección posnatal). De las primeras 22, fueron positivas en la PCRq, 19 en la PCRc y 17 en el cultivo. Al comparar el cultivo con el resto de técnicas, la PCRq presentó una sensibilidad del 100%. Si se utiliza la PCRq como referencia, el cultivo presentó una sensibilidad del 77,2% y la PCRc del 86,3%. En los casos de infección posnatal, la PCRq detectó 16 positivas, la PCRc 12 y el cultivo celular 10 orinas como positivas. Las orinas presentaron unas cargas virales que oscilaban entre 2.178 y 116.641 copias/ml.
La técnica de amplificación genómica PCRq en tiempo real se ha mostrado más sensible que las otras técnicas analizadas. Esta técnica debería ser considerada como de referencia (gold standard), dejando al cultivo celular como técnica secundaria. El bajo coste y la automatización de la PCRq permitirían realizar el cribado de infección por CMV a grandes poblaciones neonatales y posnatales.
Cytomegalovirus (CMV) is the main virus causing congenital and postnatal infections in the pediatric population. The aim of this study is to evaluate the usefulness of a quantitative real-time PCR in the diagnosis of these infections using urine as a single sample.
We studied all the urine samples of newborns (< 7 days) with suspected congenital infection, and urine of patients with suspected postnatal infection (urine negative at birth). Urines were simultaneously studied by cell culture, qualitative PCR (PCRc), and quantitative real-time PCR (PCRq).
We analyzed 332 urine samples (270 to rule out congenital infection and 62 postnatal infections). Of the first, 22 were positive in the PCRq, 19 in the PCRc, and 17 in the culture. PCRq had a sensitivity of 100%, on comparing the culture with the rest of the techniques. Using the PCRq as a reference method, culture had a sensitivity of 77.2%, and PCRc 86.3%. In cases of postnatal infection, PCRq detected 16 positive urines, the PCRq 12, and the cell culture 10. The urines showed viral loads ranging from 2,178 to 116,641 copies/ml.
The genomic amplification technique PCRq in real time was more sensitive than the other techniques evaluated. This technique should be considered as a reference (gold standard), leaving the cell culture as a second diagnostic level. The low cost and the automation of PCRq would enable the screening for CMV infection in large neonatal and postnatal populations.
Real-time PCR versus viral culture on urine as a gold standard in the diagnosis of congenital cytomegalovirus infection
2012, Journal of Clinical Virology
Citation Excerpt :
These discrepant test results theoretically can be attributed to either false negative viral culture results, or false positive real-time PCR results. False negative viral culture results have been described both in experimental setting9 and in clinical setting, testing urine samples of (immunocompromised) patients.13–15,24 Loss of viable CMV particles implicated in false negative culture results may be caused by transport at room temperature25 and antiviral therapy.
Cytomegalovirus (CMV) infection is the most common cause of congenital infection. Whereas CMV PCR has replaced viral culture and antigen detection in immunocompromised patients because of higher sensitivity, viral culture of neonatal urine is still referred to as the gold standard in the diagnosis of congenital CMV infection.
To compare real-time CMV PCR with shell vial culture on urine in the diagnosis of congenital CMV, in a multicenter design.
A series of neonatal urines (n = 340), received for congenital CMV diagnostics and routinely assessed with shell vial CMV culture, was retrospectively tested by real-time CMV PCR.
The proportion of newborns found to be congenitally infected by real-time CMV PCR was 8.2% (28/340, 95%CI 5.6–11.8%), and 7.4% (25/340, 95%CI 4.9–10.8%) by rapid culture. When considering rapid culture as reference, real-time PCR was highly sensitive (100%), whereas sensitivity of rapid culture was 89.3% when considering real-time PCR as reference.
Our results, supported by analytical and clinical data on CMV DNA detection in neonatal urine, suggest enhanced sensitivity of recent PCR techniques when compared to viral culture. There is considerable rationale to favor real-time CMV PCR as a gold standard in the diagnosis of congenital CMV infection. A large-scale study combining both laboratory and clinical data is required to determine the exact time frame for sampling of neonatal urine when using real-time PCR.
Results of a phase I/II British Society of Bone Marrow Transplantation study on PCR-based pre-emptive therapy with valganciclovir or ganciclovir for active CMV infection following alemtuzumab-based reduced intensity allogeneic stem cell transplantation
2009, Leukemia Research
Citation Excerpt :
All centres participating in this study performed weekly monitoring post-allograft for CMV reactivation by quantitative PCR for CMV DNA. The majority of samples in the study were analysed using a LightCycler real-time PCR, with primers against the gB region of CMV genome used to quantify CMV DNA [7]. If a positive result (>1000 copies/ml) was detected a repeat sample was obtained as soon as possible.
This multi-centre randomized study assessed the bioavailability of ganciclovir in patients undergoing alemtuzumab-based reduced intensity conditioning (RIC) haematopoietic stem cell transplantation (HSCT) after oral administration of valganciclovir. Patients were randomized to 2 groups receiving either oral valganciclovir (900 mg twice daily) or intravenous ganciclovir (5 mg/kg twice daily) for 14 days. Twenty-seven patients were recruited and 18 patients (67%) completed allocated treatment resulting in clearance of cytomegolovirus (CMV) DNA load at a median of 14 days. The bioavailability of ganciclovir from valganciclovir was 73% (95% CI: 34–112%). The average exposure in the valganciclovir group (36.9 ± 14.9 μg h/ml) was higher than the ganciclovir cohort (27.9 ± 7.5 μg h/ml). When compared with intravenous ganciclovir, oral valganciclovir had high bioavailability in patients undergoing alemtuzumab-based RIC HSCT.
Cytomegalovirus Ileocolitis and Kaposi's Sarcoma in Aids
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Rapid detection and quantification of CMV DNA in urine using LightCycler™-based real-time PCR

Abstract

Anal. Biochem.

Lancet

Lancet

J. Virol. Methods

J. Clin. Virol.

Use of molecular assays in diagnosis and monitoring of cytomegalovirus disease following renal transplantation

J. Clin. Microbiol.