Cytomegalovirus infection in organ-transplant recipients: diagnostic value of pp65 antigen test, qualitative polymerase chain reaction (PCR) and quantitative Taqman PCR

BACKGROUND: The human cytomegalovirus (CMV) is a major cause of morbidity and mortality in transplant patients. In this study, we compared the diagnostic value of pp65 antigen test, qualitative nested polymerase chain reaction (PCR), and quantitative Taqman PCR in predicting the clinical outcome of CMV infection. METHODS: A total of 169 samples derived from 59 organ-transplant recipients (kidney n= 46, liver n= 11, kidney and pancreas n= 2) were analyzed. Peripheral blood leukocytes (PBL) were isolated using dextran gradient centrifugation, and 2 x 10 cells were analyzed for pp65 antigen by immunofluorescence. A crude DNA extract obtained from the same number of cells was used for qualitative nested PCR and quantitative Taqman PCR analysis. RESULTS.: The correlation coefficient of pp65 antigen test and Taqman PCR was R= 0.699 (P = 0.001). With cut-off values for pp65 antigen test set at greater than 10 positive nuclei per 2 x 10 PBL, sensitivity was 91%, and positive predictive value (PPV) was 70%. When the corresponding cut-off value for Taqman PCR was applied (>125000 genome copies per 2 x 10 PBL), a sensitivity of 83% and a PPV of 68% were found. Both assays allowed for the monitoring of successful antiviral therapy. Although qualitative nested PCR was highly sensitive (95%), it was less useful in predicting CMV disease (PPV 47%) and in therapy control. CONCLUSION: Our data show that pp65 antigen test and Taqman PCR are almost equivalent in the monitoring of CMV infection and disease when identical cell numbers are used for both assays.     

Evaluation of a new method for early detection of active cytomegalovirus infections. A study in kidney transplant recipients

Abstract Early detection of active cytomegalovirus (CMV) infection after organ transplantation is necessary to start effective antiviral treatment. In the present study, blood specimens of kidney transplant recipients ( n = 38) were monitored for the expression of CMV immediate early (IE) and late (L) mRNA using nucleic acid sequence-based amplification (NASBA). Results were compared with virus isolation, pp65 antigenemia and serology. In patients developing active CMV infection, pp65 antigen and L mRNA were detected simultaneously. At the same time, positive cell culture results could be reported to the clinic. CMV was detected significantly earlier with IE NASBA than with the other assays. However, the specificity of IE NASBA is lower than that of antigenemia, late NASBA and cell culture. Early detection of IE mRNA is especially useful for patients at high risk of developing symptomatic CMV infection in order that early, adequate antiviral therapy may be started. Late NASBA can be used to monitor further development of CMV infection, comparable to antigenemia.     

Monitoring of human cytomegalovirus infections in heart transplant recipients by pp65 antigenemia

BACKGROUND: Rapid diagnostic techniques offer the opportunity of early diagnosis of human cytomegalovirus (CMV) infection in immunocompromized patients at risk of developing CMV disease and syndrome. The use of CMV pp65 antigenemia as a predictor of CMV syndrome and disease in heart transplant recipient after induction therapy was studied retrospectively. METHODS: One hundred and nineteen consecutive heart transplant recipients treated with induction therapy who survived more then 14 d were monitored for CMV infection. Ninety-four recipients were seropositive for CMV. Twenty-five recipients were seronegative for CMV and received grafts from seropositive donors. Pre-emptive therapy was used in seropositive patients when CMV pp65 antigenemia was greater than 50 antigen-positive cells per 2 x 10(5) peripheral blood leukocytes (PBL); prophylactic therapy was done only in seronegative recipient matched with seropositive donor. RESULTS: High-level CMV pp65 antigenemia (50 antigen-positive cells 2 x 10(5) PBL) occurred in 34% (41 of 119) of patients at a median of 44 d following transplantation. In seropositive recipients, 16% (15 of 94) of patients developed CMV invasive disease or syndrome, and in seronegative recipients 20% (5 of 25) of patients developed CMV disease or syndrome. Sixty-six per cent (62 of 94) of CMV seropositive patients were identified as not requiring pre-emptive therapy. In seropositive and seronegative recipients, the sensibility and negative predictive value of the cut-off level of 50 antigen positive cell for CMV disease and syndrome was 100%. The specificity was 79% and positive predictive value was 49%. CONCLUSION: Because of the excellent sensibility and negative predictive value of the cut-off level of 50 antigen positive cell per 2 x 10(5) PBL, application of pre-emptive therapy guided by high level of CMV pp65 antigenemia in the context of induction therapy allow to omit antiviral therapy in many at risk patients. In the context of pre-emptive and prophylactic therapy, the cut-off level of 50 antigen positive cell do not allow to predict with accuracy the development of CMV disease or syndrome.     

A prospective multicenter observational study of cell‐mediated immunity as a predictor for cytomegalovirus infection in kidney transplant recipients

T cell immunity is essential for the control of cytomegalovirus (CMV) infection after transplantation. We evaluated a CMV‐specific peptide‐based enzyme‐linked immunosorbent spot (ELISPOT) assay to determine whether assay results could predict subsequent CMV events. Adult kidney transplant recipients at 43 centers underwent ELISPOT testing to enumerate interferon gamma (IFN‐γ) binding spot‐forming units (sfu) after stimulation of cells with an overlapping peptide pool of CMV phosphoprotein 65 (pp65) and immediate early‐1 (IE‐1) protein at the end of antiviral prophylaxis (EOP) and various time points thereafter. The primary outcome was a CMV event in the first posttransplant year. In 583 kidney transplant recipients (260 seropositive donor [D+]/seronegative recipient [R?] and 277 R+), CMV events occurred in 44 of 368 eligible patients (11.8%) at a median of 227 days (range 92‐360) posttransplant. A cutoff value of >40 sfu/2.5 × 10⁵ cells for either IE‐1 or pp65 was derived as a threshold for positivity, with a negative predictive value of >97% for CMV events. CMV events were significantly lower in assay positive vs assay negative patients (3.0% vs 19.5%, P < .0001 for pp65). Time to CMV event post‐EOP was significantly greater in those with sfu >40 at EOP (P < .0001). In this large, multicenter trial of kidney transplant recipients, we show that an assessment of CMV‐specific immunity using a novel ELISPOT assay is able to predict protection from CMV infection.     

Cytomegalovirus‐Responsive CD8+ T Cells Expand After Solid Organ Transplantation in the Absence of CMV Disease

Cytomegalovirus (CMV) is a major cause of morbidity and mortality in solid organ transplant recipients. Approximately 60% of adults are CMV seropositive, indicating previous exposure. Following resolution of the primary infection, CMV remains in a latent state. Reactivation is controlled by memory T cells in healthy individuals; transplant recipients have reduced memory T cell function due to chronic immunosuppressive therapies. In this study, CD8⁺ T cell responses to CMV polypeptides immediate‐early‐1 and pp65 were analyzed in 16 CMV‐seropositive kidney and heart transplant recipients longitudinally pretransplantation and posttransplantation. All patients received standard of care maintenance immunosuppression, antiviral prophylaxis, and CMV viral load monitoring, with approximately half receiving T cell–depleting induction therapy. The frequency of CMV‐responsive CD8⁺ T cells, defined by the production of effector molecules in response to CMV peptides, increased during the course of 1 year posttransplantation. The increase commenced after the completion of antiviral prophylaxis, and these T cells tended to be terminally differentiated effector cells. Based on this small cohort, these data suggest that even in the absence of disease, antigenic exposure may continually shape the CMV‐responsive T cell population posttransplantation.     

实时定量PCR方法监测肾移植患者血浆巨细胞病毒DNA载量及其意义

目的 采用实时定量荧光PCR方法检测巨细胞病毒(CMV)DNA拷贝数,探讨病毒载量变化在诊断CMV活动性感染中的意义和指导抗病毒治疗中的作用.方法 对40例采用预排空治疗策略预防CMV病的肾移植受者同时进行CMV抗原血症(pp65)分析和病毒载量监测并进行随访,观察CMV-DNA载量和pp65在判断肾移植受者CMV活动性感染和指导抗病毒治疗中的价值.结果 264份样本中逆转录-聚合酶链反应(RT-PCR)检测57份阳性(21.59%),病毒载量水平2.648×101～5.273×105copy/ml;抗原血症分析检测48份阳性(18.18%),抗原指数1～18/5万WBC;Cohen,s-Kappa检验结果表明两种检测方法一致性良好(Kappa=0.762).25例(62.5%)患者PCR方法检测阳性,19例(47.5%)抗原血症分析阳性.病毒载量首次出现阳性时间为(13.22±14.78)d,pp65首次出现时间为(26.72±20.61)d,差异有统计学意义(P＜0.05).40例患者中4例出现CMV病,发病时间133～179 d,3例发病前pp65和CMV-DNA均为阳性,1例仅CMV-DNA阳性.结论 血浆CMV载量检测与抗原血症分析一致性良好,而敏感性较高,更准确反映肾移植术后CMV活动性,更适合用于肾移植术后预防CMV感染的预排空治疗策略.     

Acyclovir plus CMV immunoglobulin prophylaxis and early therapy with ganciclovir are effective and safe in CMV high-risk renal transplant pediatric recipients

Abstract Cytomegalovirus (CMV) infection is still a major cause of morbidity in high-risk renal transplant recipients. In the present report, we have reviewed our records of renal transplant pediatric recipients (RTPR; mean age 14.1 ± 4.9 years) since 1991, when we started a policy of CMV prophylaxis constituting high-dose oral acyclovir plus CMV hyperimmune immunoglobulins (Hlg) followed by early i.v. ganciclovir therapy in high-risk patients (i.e., CMV donor +/ recipient -). Four patients received a kidney from a living relative (LR), 2 patients had one previous transplant, and 1 had a combined liver -kidney transplant. Thirty-three patients who were negative for CMV antibodies (ab) before transplantation received a kidney from CMV ab positive donors. The immunosuppressive regimen included cyclosporin A and steroids, with the addition of azathioprine in the 4 patients who received an LR kidney. Serial assessments for CMV antigenemia (pp 65) were routinely performed for 6 months after transplantation to define CMV infection. Among the 33 CMV seronegative recipients (R -) who received the graft from a CMV seropositive donor (D +), 18 (54.5 %) experienced CMV infection, whereas among the 28 CMV R +, who received a graft from a CMV D +, 11 (39.3 %) experienced CMV infection. With regard to CMV - related symptoms, only 2 patients suffered from a CMV syndrome (fever and leukopenia in 1 patient, fever and arthralgia in the other). In no case did the spectrum of CMV disease occur; only minor symptoms were present in 7 of the remaining CMV-infected patients (fever in 6 and leukopenia in 1). Rejection episodes and renal function did not differ between CMV-infected and non-CMV-infected patients. Our experiences support the use of prophylactic acyclovir plus CMV HIg followed by early therapy with i.v. ganciclovir to combat the risk of increased morbidity in high risk RTPR.     

The use of flow cytometry for the detection of CMV-specific antigen (pp65) in leukocytes of kidney recipients

Flow cytometric assay (FCA) was used to detect a cytomegalovirus (CMV) specific antigen (pp65) in CMV-infected fibroblast cells and in leukocytes of kidney recipients. FCA distinguished clearly between the infected and non-infected fibroblast cells. Regarding transplant patients, the FCA was positive when the number of antigenemia assay (AA) positive cells was five or more per 5 x 10(4). Moreover, the percentage of antigenemia-positive cells by FCA correlated well with symptomatic CMV infections.     

Antigenemia for cytomegalovirus in renal transplantation: choosing a cutoff for the diagnosis criteria in cytomegalovirus disease

Cytomegalovirus (CMV) infection is a frequent complication in transplant recipients, causing a high level of morbidity and mortality. We studied 203 consecutive renal transplant recipients performed between January 2000 and December 2001. Patients underwent weekly measurements of CMV pp65 antigen to assess CMV activity from the 4th to the 12th week posttransplantation. The results were reported as number of cells positive for the pp65 antigen among 10(5) granulocytes. In order to define a best cutoff to diagnose CMV disease with desirable sensitivity and specificity, we used a receiver operator characteristics (ROC) curve. The cutoff of four positive cells corresponded to a sensitivity of 93% and specificity of 60% (AUC = 0.87) for the diagnosis of CMV disease. The chosen cutoff for starting antiviral treatment was 10 cells, since this was associated with a sensitivity of 92% and specificity of 70% (AUC = 0.90). In conclusion, the highly sensitive cutoff points for the diagnosis of antigenemia was four cells and 10 cells for initiation of antiviral therapy.     

Therapeutic implication of quantitative pp65 antigen assay in living renal transplant in a high seroendemic population

Various methods have been used to diagnose cytomegalovirus (CMV) infection/disease; however, pp65 antigenemia assay has emerged as a good marker for CMV disease in a high seroendemic population. We studied the role of quantitative pp65 antigen assay in live related renal transplant recipients in a high seroendemic population. Between November 1998 and May 2003, a total of 350 blood samples from 250 symptomatic patients were tested by quantitative pp65 antigen assay; 14% of the patients tested positive. There were 5 (14%) low-positive and 30 (86%) high-positive patients. All high-positive patients had CMV disease. The response to antiviral therapy monitored by the assay was dramatic, and one low-positive patient responded to reduction in immunosuppression. In conclusion, pp65 antigen assay is a good test for diagnosing CMV disease and monitoring response to antiviral therapy in a high seroendemic population.     

Development of intrapancreatic abscess - a consequence of CMV pancreatitis?

Abstract. Cytomegalovirus (CMV) pancreatitis was diagnosed in eight out of 124 pancreatic transplant recipients. Five of the eight patients developed intrapancreatic abscesses and four of the grafts were lost, but one is still functioning. In the three additional cases of pancreatitis, antiviral treatment with foscarnet or ganciclovir was given as soon as signs of CMV pancreatitis were detected. No such grafts were lost during the acute phase. CMV infection was diagnosed in cells from pancreatic juice, by virus isolation, detection of CMV antigen in cells from pancreatic juice or by CMV serology. The signs and symptoms of CMV pancreatitis included fever, general malaise, abdominal pain, diarrhoea, localized peritonitis, hyperamylasaemia, leukopenia and hyperglycaemia. It is recommended that rapid diagnostic procedures for CMV should be carried out when early signs of pancreatitis develop in pancreatic graft recipients. Antiviral treatment should be given when CMV pancreatitis is suspected or diagnosed in order to prevent the development of intrapancreatic abscesses and graft loss.     

Development of intrapancreatic abscess — a consequence of CMV pancreatitis?

Cytomegalovirus (CMV) pancreatitis was diagnosed in eight out of 124 pancreatic transplant recipients. Five of the eight patients developed intrapancreatic abscesses and four of the grafts were lost, but one is still functioning. In the three additional cases of pancreatitis, antiviral treatment with foscarnet or ganciclovir was given as soon as signs of CMV pancreatitis were detected. No such grafts were lost during the acute phase. CMV infection was diagnosed in cells from pancreatic juice, by virus isolation, detection of CMV antigen in cells from pancreatic juice or by CMV serology. The signs and symptoms of CMV pancreatitis included fever, general malaise, abdominal pain, diarrhoea, localized peritonitis, hyperamylasaemia, leukopenia and hyperglycaemia. It is recommended that rapid diagnostic procedures for CMV should be carried out when early signs of pancreatitis develop in pancreatic graft recipients. Antiviral treatment should be given when CMV pancreatitis is suspected or diagnosed in order to prevent the development of intrapancreatic abscesses and graft loss.     

用RT-PCR法检测肾移植患者巨细胞病毒感染的初步研究

采用逆转录聚合酶链反应法检测人类巨细胞病毒主要直接早期抗原ＤＮＡ和ｍＲＮＡ。抽取２０名患者３７份血标本，结果１１份ｍＲＮＡ阳性，其巨细胞病毒ＤＮＡ均为阳性，其中７人临床伴发热，白细胞减少，血小板减少，３人血肌酐〉１７７μｍｏｌ．Ｌ＾１，２例丙氨酸转氨酶轻度升高。     

HHV‐6 reactivation is often associated with CMV infection in liver transplant patients

Abstract Human herpesvirus 6 (HHV‐6) infection has been recently reported in liver transplant patients. HHV‐6 is closely related to cytomegalo‐virus (CMV), and some interaction between the viruses has been suggested. In this study, the post‐transplant HHV‐6 antigenemia was investigated in relation to symptomatic CMV infections in adult liver transplant patients. CMV infections were diagnosed by the pp65 antigenemia test and by viral cultures. HHV‐6 infections were demonstrated by the HHV‐6 antigenemia test and by serology. Significant symptomatic CMV infection was diagnosed in 42 of 75 patients during the first 6 months after transplantation. All CMV infections were successfully treated with ganciclovir. Concurrent HHV‐6 antigenemia was detected in 21 (50%) of 42 patients with CMV infection. All HHV‐6 infections were reactivations. HHV‐6 also responded to the antiviral treatment, but with less clear effect. In conclusion, HHV‐6 reactivation is often associated with CMV infection in liver transplant patients. The results support the suggestion that CMV and HHV‐6 may have interactions.     

Symptomatic cytomegalovirus (CMV) infections identified by image cytometry and other parameters for CMV infection

Abstract. Thirty-eight renal transplant recipients were followed during the first 3 months after transplantation. Once weekly, cultures of urine and buffy coat for cytomegalovirus (CMV) were taken and an immunocytochemical assay for immediate early antigens of CMV (IEA assay) was performed. Thirty patients had evidence of a CMV infection and 11 had a symptomatic CMV infection. All symptomatic patients had one or more positive urine cultures or a positive IEA assay. However, 15 patients with positive urine cultures and 12 patients with a positive IEA assay lacked any signs of symptomatic CMV disease. Moreover, 6 out of 15 patients with positive buffy coat cultures for CMV did not have symptomatic CMV disease. Using a computerized system to quantify IEA-positive granulocytes, we show that the absolute number of positive cells per million correlates very well with the occurrence of symptomatic CMV disease.     

Comparison between RFLP-PCR and antigenemia for pp65 antigen for diagnosis of cytomegalovirus disease after kidney transplantation

Cytomegalovirus (CMV) infection is the single most frequent infectious complication in renal transplant recipients. The purpose of this study was to analyze the diagnostic efficacy of PCR-RFLP compared to antigenemia for CMV disease (CMVD) in kidney transplant recipients. From November 2001 to February 2002, 19 renal adult transplant recipients were followed with weekly measurements of CMV pp65 antigen to monitor the activity of CMV from the week 4 to 12 posttransplantation. Only 4 (21.1%) patients did not develop viremia during the first 12 posttransplantation weeks. Active infection was observed in 15 patients (78.9%): asymptomatic viremia in 6 (31.6%) and CMVD in 9 (47%). All patients who developed CMVD showed positivity in both methods during the observation period. The number of positive cells ranged from 11 to 292 cells in patients with CMVD and one to eight cells among those with asymptomatic viremia. Both methods revealed 100% sensitivity for CMVD diagnosis. The specificity was 60% for antigenemia and 70% for PCR, with positive predictive values of 60% and 75%, respectively.     

Symptomatic cytomegalovirus (CMV) infections identified by image cytometry and other parameters for CMV infection

Thirty-eight renal transplant recipients were followed during the first 3 months after transplantation. Once weekly, cultures of urine and buffy coat for cytomegalovirus (CMV) were taken and an immunocytochemical assay for immediate early antigens of CMV (IEA assay) was performed. Thirty patients had evidence of a CMV infection and 11 had a symptomatic CMV infection. All symptomatic patients had one or more positive urine cultures or a positive IEA assay. However, 15 patients with positive urine cultures and 12 patients with a positive IEA assay lacked any signs of symptomatic CMV disease. Moreover, 6 out of 15 patients with positive buffy coat cultures for CMV did not have symptomatic CMV disease. Using a computerized system to quantify IEA-positive granulocytes, we show that the absolute number of positive cells per million correlates very well with the occurrence of symptomatic CMV disease.     

Clinical validation of a novel enzyme‐linked immunosorbent spot assay‐based in vitro diagnostic assay to monitor cytomegalovirus‐specific cell‐mediated immunity in kidney transplant recipients: a multicenter,longitudinal, prospective,observational study

Impaired cytomegalovirus (CMV)‐specific cell‐mediated immunity (CMV‐CMI) is a major cause of CMV reactivation and associated complications in solid‐organ transplantation. Reliably assessing CMV‐CMI is desirable to individually adjust antiviral and immunosuppressive therapy. This study aimed to evaluate the suitability of T‐Track^® CMV, a novel IFN‐γ ELISpot assay based on the stimulation of peripheral blood mononuclear cells with pp65 and IE‐I CMV proteins, to monitor CMV‐CMI following kidney transplantation. A prospective longitudinal multicenter study was conducted in 86 intermediate‐risk renal transplant recipients. CMV‐CMI, CMV viral load, and clinical complications were monitored over 6 months post‐transplantation. Ninety‐five percent and 88–92% ELISpot assays were positive pre‐ and post‐transplantation, respectively. CMV‐specific response was reduced following immunosuppressive treatment and increased in patients with graft rejection, indicating the ability of the ELISpot assay to monitor patients' immunosuppressive state. Interestingly, median pp65‐specific response was ninefold higher in patients with self‐clearing viral load compared to antivirally treated patients prior to first viral load detection (P < 0.001), suggesting that reactivity to pp65 represents a potential immunocompetence marker. Altogether, T‐Track^® CMV is a highly sensitive IFN‐γ ELISpot assay, suitable for the immunomonitoring of CMV‐seropositive renal transplant recipients, and with a potential use for the risk assessment of CMV‐related clinical complications (ClinicalTrials.gov Identifier: NCT02083042).     

Natural Killer Cell Receptor Repertoire and Their Ligands,and the Risk of CMV Infection After Kidney Transplantation

Cytomegalovirus (CMV) infection is the most common viral complication after solid organ transplantation (SOT). Whilst current immunosuppression is known to impair antiviral‐specific T‐cell immunity in SOT, a potential role for natural killer (NK) cells not affected by immunosuppressive therapy remains to be determined. To address this, we compared the genotype of the NK immunoglobulin‐like receptor (KIR) genes and their HLA cognate ligands to the rate of CMV infection in 196 kidney transplant recipients. We have shown that the absence of the HLA‐C ligand for inhibitory KIR and the presence of activating KIR genes in the recipients were both associated with a lower rate of CMV infection after transplantation. In a cohort of 17 recipients with acute CMV infection, NK cells were phenotyped over a period of time after diagnosis by their expression profile of C‐type lectin receptors and capacity to secrete IFN‐γ. The increased expression of the activating C‐type lectin receptors NKG2C and NKG2D was paralleled by the decreased IFN‐γ secretion during the early phase of CMV infection. In conclusion, our findings suggest that KIR/HLA genotype and expression of NKG2C and NKG2D might play a significant role in regulating NK cell function and anti‐CMV immunity after kidney transplantation.     

CMV‐infected kidney grafts drive the expansion of blood‐borne CMV‐specific T cells restricted by shared class I HLA molecules via presentation on donor cells

We aimed to determine the role of cytomegalovirus (CMV)‐infected donor cells in the development of a CMV‐specific immune response in kidney transplant recipients. We assessed the CMV pp65‐specific immune response by using interferon‐? ELISPOT and dextramers in peripheral blood mononuclear cells from 115 recipients (D+R? 31, D+R + 44, D?R + 40) late after transplantation (mean 59 ± 42 months). Receiving a kidney from a D+ donor resulted in a higher number of IFN‐?‐producing anti‐CMV T cells (P = .004). This effect disappeared with the absence of shared HLA class I specificities between donors and recipients (P = .430). To confirm the role of donor cells in stimulating the expansion of newly developed CMV‐specific CD8⁺ T cells after transplantation, we compared the number of HLA‐A2–restricted CMV‐specific CD8⁺ T cells in primo‐infected recipients who received an HLA‐A2 or non–HLA‐A2 graft. The median of anti‐CMV pp65 T cells restricted by HLA‐A2 was very low for patients who received a non–HLA‐A2 graft vs an HLA‐A2 graft (300 [0‐14638] vs. 17972 [222‐85594] anti‐CMV pp65 CD8⁺ T cells/million CD8⁺ T cells, P = .001). This adds new evidence that CMV‐infected kidney donor cells present CMV peptides and drive an inflation of memory CMV‐specific CD8⁺ T cells, likely because of frequent CMV replications within the graft.