Quantitation of cytomegalovirus in the blood of liver transplant recipients

Abstract

An assay for quantitation of cytomegalovirus (CMV) has been developed. The assay combines DNA amplification and enzyme-linked immunosorbent assay (ELISA) detection. In this study, the assay has been used to examine sequential buffycoats from 32 consecutive liver transplant recipients. In a febrile patient, CMV titres in excess of lo4 copies per 150,000 cells strongly suggest a diagnosis of symptomatic CMV infection. Antiviral therapy causes a rapid decline in viral titre. Viral titres are seen to rise presymptomatically in nfection with cytomegalovims (CMV) remains an I important cause of morbidity in immunosuppressed transplant recipients. The association of symptomatic infection with virus isolation from buQ-coat is well e~tab1ished.l.~ Recently, the polymerase chain reaction (PCR) has been proposed as a potentially useful test for the detection of CMV in this ~e t t i n g .~. '~ Assay sensitivity depends on (1) method of substrate preparation, (2) primer design, (33 thermal cycling conditions, and (4) post-PCR detection method. When performance of the assay is optimized, CMV can be detected in blood of immunocompetent blood donor^.'^,'^ Such a sensitive assay would not provide useful information for the management of immunosuppressed patients. Most investigators agree that PCR has excellent sensitivity and negative predictive value (for the diagnosis of symptomatic infection), but that specificity and positive predictive value are poor. Discordant conclusions concerning the clinical utility of PCR detection may result from differences in assay DNA detection sensitivity, which in turn result from interlaboratory methodological differences.For any given substrate and conditions, the efficiency of the PCR reaction varies. Problems of interpretation arising from interassay and interlaboratory variation in PCR efficiency might be overcome by the inclusion of an internal standard DNA sequence in each r e a c t i~n . '~.~~ We have developed a quantitative assay for CMV detection that combines a single PCR reaction with enzyme-linked immunosorbent assay (ELISA) detection.Sequential bu&-coats Materials and MethodsThirtytwo consecutive liver transplant recipients were studied for 3 months after transplantation. Prospective virologcal surveillance was not performed. Sera were examined retrospectively for CMV immunoglobulin (19, M, and for the purposes of this report CMV infection is defined by the presence of CMV IgM. Symptomatic CMV infection ("CMV disease") is defined by the following criteria. Fever (>38"C on at least 2 consecutive days) and serum IgM positivity, with either leukopenia (count < 3 x 10y/l), and/or histological evidence of invasive infection.Patients did not receive antiviral prophylaxis. When symptomatic CMV infection was diagnosed, treatment included reduction of immunosuppression and administration of ganciclovir ( I 0 mg/kg/d adjusted for renal dysfunction).Blood was sampled weekly during the inpatient stay, and then at each outpatient attendance for 3 months. Serum was separated from clotted bloo...

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

“…Quantitative PCR of CMV DNA has been attempted previously [34][35][36][37][38][39][40][41] and successfully discriminated between asymptomatic and symptomatic CMV infection in renal 37 and liver 38 transplant recipients. Most attempts have been made using buVy coat.…”

New Strategies for Prevention and Therapy of Cytomegalovirus Infection and Disease in Solid-Organ Transplant Recipients

“…CMV infections occurring early in the posttransplantation period, that is, within 2 months of transplantation, appear to have higher viral loads, most probably because immunosuppression is more intense, and are usually associated with disease (278). Viral titers rise presymptomatically in some cases (314). In addition, the maximum CMV DNA level during infection is significantly higher in patients who are experiencing primary infection compared to those who have asymptomatic and/or reactivation CMV infection (148,278,314,378,399,470).…”

“…Viral titers rise presymptomatically in some cases (314). In addition, the maximum CMV DNA level during infection is significantly higher in patients who are experiencing primary infection compared to those who have asymptomatic and/or reactivation CMV infection (148,278,314,378,399,470). After the initiation of antiviral therapy, CMV DNA levels in blood fall rapidly, correlating with the disappearance of clinical symptoms.…”

“…Significant decreases in viral loads are observed during ganciclovir administration (213,314,470). The discontinuation of treatment when CMV PCR is positive in bronchoalveolar lavage specimens of lung transplant recipients (448) and in blood specimens of kidney/kidney-pancreas transplant recipients (213) has been associated with recurrence of CMV infection.…”

Cytomegalovirus (CMV) DNA Load Predicts Relapsing CMV Infection after Solid Organ Transplantation

“…led to the correlation of high viral loads with more severe forms of CMV disease [6][7][8][9]. Because solid organ transplant patients fulfill many of the epidemiologic risk factors that predispose to relapsing CMV disease, it is likely that viral load could be used as a marker of relapsing CMV disease.…”