Quantitative CMV-PCR Research Articles

#50: Cytomegalovirus Retinitis Among Pediatric Hematopoietic Stem Cell and Solid Organ Transplantation Recipients: A Contemporary Review

Abstract Background Cytomegalovirus infection can cause significant morbidity and mortality after transplantation. Cytomegalovirus retinitis (CMVR) can be a major complication of tissue invasive CMV disease in transplant recipients. There are little data regarding the contemporary incidence and outcomes of CMVR among pediatric transplant recipients. Methods We performed a retrospective cohort analysis of allogeneic hematopoietic (allo-HCT) and solid organ transplant (SOT) recipients who received medical care at Nationwide Children’s Hospital (NCH) from 1/1/10–11/30/20 and had a diagnosis of CMVR to describe the incidence, timing post-transplant, ocular manifestations, management, and outcomes. Basic demographic and clinical data, pre-transplant donor/recipient CMV serostatus, and post-transplant CMV infection data were collected. Graft-specific CMV surveillance and prophylaxis strategies were performed according to NCH hospital guidelines. During the study period, quantitative plasma CMV PCR assays included copies/mL (2010–2013) and WHO international standard IU/mL (2013–2020). Results During the 10-year study period, 347 patients underwent allo-HCT (N=214) or SOT (N=133; heart N=46, liver N=36, kidney N= 52; 1 patient had concomitant kidney and liver transplantation); median age was 8.6 years [range 0.1–25.9] and 198 (56.5%) were male. In total, 98 patients had CMV DNAemia post-transplant, and CMVR was diagnosed in 3 (HCT=2, SOT=1), for an overall CMVR incidence of 0.86% among all transplant recipients and occurring in 3% of patients with CMV DNAemia. No CMVR was diagnosed in patients without CMV DNAemia. An ophthalmologic examination was performed in 58 (59%) of patients with any DNAemia compared with 67 (27%) of patients without DNAemia. CMVR was diagnosed on funduscopic examination a median of 183 days [range 34–512] post-transplant based on visual symptomatology (N=2) or by routine eye screening prompted by DNAemia (N=1). CMV DNAemia at the time of CMVR was 1,463 copies/mL, 3,000, and 14,204 IU/mL; all patients had prolonged (>3 months) CMV detection before CMVR. One SOT recipient had concomitant pathology confirming CMV gastrointestinal tract disease. Two of the 3 CMVR cases occurred in the setting of genotypic UL94 CMV resistance mutations (A594V, H520Q)(Figure 1). Two patients were already receiving CMV antiviral therapy (maribavir N=1; valganciclovir=1) at the time of CMVR diagnosis. After the CMVR diagnosis, all patients were prescribed systemic foscarnet. Adjunctive therapies included concurrent intravitreal antiviral therapy (ganciclovir N=1; foscarnet N=1) and adoptive immunotherapy with CMV antiviral specific T-cells (N=1, HCT). Improvement of ocular symptoms occurred in 2 patients at 15 and 20 days after starting targeted CMV antiviral therapy; one patient developed permanently impaired vision. Conclusion CMVR remains as an important complication in transplant recipients. Although CMVR incidence was low in our cohort of transplant recipients, it occurred most frequently in cases of CMV resistance, prolonged DNAemia, and led to permanent visual impairment in 1 of 3 patients. The development of resistant CMV may have a role in the occurrence, progression, and severity of CMVR. Additional pediatric-specific data are needed to better characterize CMVR and inform optimal prevention strategies.

PF766 T CELL EXHAUSTION PROFILE (CD4+/CD28/PER+/GRA+ CD8+/CD28/PER+/GRA+) IN STEM CELL TRANSPLANTATION, CMV RE ACTIVATIONS AND RESPIRATORY VIRUS INFECTION.CELLULAR DYNAMICS IN THE FIRST 100 DAYS POST SCT

Background:Recipients of allogeneic stem cells grafts have clonally expanded CD8+/CD28‐ T lymphocytes during the early period after SCT, this cellular dynamic is associated with the acquisition of a cytoxic phenotype. This scenario predisposes to continuous inflammation. On the other hand, respiratory viruses (RV) are recognized as the predominant leading to pneumonia after SCT, and CMV re activations were associated with morbi‐mortality in SCT patients.RVI and CMV re‐activations might contribute to the emergence of exhausted T cells phenotype.Aims:The present study aimed to evaluate the dynamics of T cell exhaustion profile during the +100d after SCT(auto, allo/patients with RVI and CMV re activations)Methods:Informed consent was obtained from all patients. PBs were obtained previous SCT and at day+100 post SCT from 37 patients (11 auto, 26 allo). To evaluate the expression of CD4+/CD28Per+/Gran+, CD8+/CD28Per+/Gran+. Flow cytometry analysis was performed: 100 μL of PB was labeled, with a panel of 8 AbMos: PERFORIN FITC, GRANZIME PE, CD4PerCP, CD28 APC, CD8 APCH7, CD16/56 V450 and CD45 V500. The molecular detection of RV was tested with theCLART® Pneumovir assay (Clinical Array Technology, Genomica, Spain). CMV detection was performed by quantitative CMV PCR (COBAS®Amplicor® CMV MonitorTM test) (qPCR CT). Statistical analysis was performed with IBM SPSS v24Results:Thirty seven patients were evaluated from November 2016 to December 2017 at the University Hospital of La Princesa, Madrid Spain. Patient characteristics:Table 1.The median percentages of baseline CD8+/CD28‐ cell line was 8,81%(range, 0,6–49,5) and the median percentages of CD8+/CD28‐ cellline at +100d were 29,2%(range, 1,2–60,8).Likewise, significant differences were found(p = 0.001).The group of patients who had an RVI and the group without RVI: in the CD8+/CD28+100d cell line, the median percentages of the RVI patients were 58.43%(range, 42.7–68.4)and the median percentages in the uninfected were 29.26%(range, 13.2–38.9)(p = 0.002). CMV re activations,the median percentages at +100d of CD8+/CD28‐ cell line with CMV‐ was 5,7%(range, 0,6–20,6)and the median percentage of CD8+/CD28‐with CMV+ at +100d was 36,7%(range, 13,5–68,7)p = 0,001.CMV reactivations and RVI, the median percentages at +100d of CD8+/CD28‐cell line with CMV/RVI was 6,82%(range, 0,6–20,6)and the medianpercentage of CD8+/CD28‐ with CMV+/RVI+ at +100d was 34,6%(range, 1,4–49,8)p = 0,012.Summary/Conclusion:We show a dynamic change of the T cell exhausted phenotype CD8+/CD28‐throughout the SCT, an also ID interactions (RVI, CMV re activations).There is a statistically significant difference when analyzing patients with RVI+ vs RVI‐ (baseline vs +100d), also might exist a synergic effect RVI+/CMV+.The CD8+/CD28‐Tcell population has been shown to contain virus specific memory CTL that respond to CMV and to other antigens.Thus, clonally expanded CD8+CD28‐ Tcells following STC may be derived from the T cells recognizing antigens that persistently exist in the host (CMV) or to the exposure to certain virus antigens in the first 100d after STC (synergic effect), this may contribute to the appearance of populations of exhausted T cells CD8+/CD28‐ favoring an inflammatory environment and triggering certain immunological complications (immunedysregulation).We need to follow up these patients and correlate with other clinical epidemiological variables in SCT(aGVHD,cGVHD,pulmonary functional tests,ID complications)image

Management and prevention of cytomegalovirus infection in paediatric hematopoietic stem cell transplant (HSCT) recipients: A binational survey.

CMV infection is an important cause of morbidity and mortality among HSCT recipients. Optimal strategies for prevention and management of CMV disease following haematopoietic stem cell transplantation remain uncertain. We conducted an online survey of Australasian paediatric allogeneic HSCT centres on management and prevention of CMV disease in this patient group. We asked for one response from a representative of the HSCT team and one from a representative of the ID team at each centre. All Australasian paediatric HSCT centres responded to our survey. Management of CMV in pre-transplant setting was consistent between centres. All centres used a pre-emptive strategy to prevent CMV disease, guided by quantitative CMV PCR. In the post-transplant post engraftment setting, all centres recommended using ganciclovir (5mg/kg/dose twice daily) as a first-line therapy for CMV reactivation or disease, with treatment duration of 14days, provided declining CMV quantitative PCR. There was substantial variability of practice between centres in post-transplant management of CMV reactivation, especially during the pre-engraftment phase. Similarly, there was lack of uniformity in indication, dosing and duration of maintenance therapy. Divergence was noted between responses from HSCT and ID physicians within centres. This study identifies areas of uniformity and others of great variability in prevention and management strategies for CMV in paediatric HSCT. Data on CMV infection and management in HSCT patients should be routinely collected as part of prospective trials to inform guidelines and improve prevention and treatment of this important complication.

The effect of cytomegalovirus infection on acute rejection in kidney transplanted patients.

Introduction: It is known that cytomegalovirus (CMV) infection is a common problem among kidney transplant patients. This infection can be increased morbidity and decreased graft survival. This problem has been associated with acute rejection too. Patients and Methods: One hundred and thirty renal transplant patients were included in a prospective, case-control study. The renal transplant patients were divided into two groups; patients group with CMV infection and control group without CMV infection. Serum CMV-IgG in all patients was positive (donor and recipients). None of patients had received anti-thymocyte-globulin and thymoglobulin. CMV infection was diagnosed by quantitative CMV-PCR (polymerase chain reaction) test (more than 500 copies/μg). Rejection episode was defined by kidney isotope scan or biopsy. Results: In the group of 66 CMV infection patients (41 male [62.1%] and 25 female [37.9%]) the incidence of graft rejection was 36%, however in the group of 64 control patients the incidence of graft rejection was 9.4 % (P < 0.005). Conclusion: CMV infection is important predisposing factor for acute allograft rejection after kidney transplantation. The results of this study suggests that the control of CMV infection could decrease episodes of acute kidney rejection.

Coexisting cytomegalovirus infection in immunocompetent patients with Clostridium difficile colitis.

Cytomegalovirus (CMV) colitis usually occurs in immunocompromised patients with human immunodeficiency virus infection, organ transplantation, and malignancy receiving chemotherapy or ulcerative colitis receiving immunosuppressive agents. However, CMV colitis is increasingly recognized in immunocompetent hosts. Notably, CMV colitis coexisting with Clostridium difficile infection (CDI) in apparently healthy individuals has been published in recent years, which could result in high morbidity and mortality. CMV colitis is a rare but possible differential diagnosis in immunocompetent patients with abdominal pain, watery, or especially bloody diarrhea, which could be refractory to standard treatment for CDI. As a characteristic of CDI, however, pseudomembranous colitis may be only caused by CMV infection. Real-time CMV-polymerase chain reaction (PCR) for blood and stool samples may be a useful and noninvasive diagnostic strategy to identify CMV infection when treatment of CDI eventually fails to show significant benefits. Quantitative CMV-PCR in mucosal biopsies may increase the diagnostic yield of traditional histopathology. CMV colitis is potentially life-threatening if severe complications occur, such as sepsis secondary to colitis, massive colorectal bleeding, toxic megacolon, and colonic perforation, so that may necessitate pre-emptive antiviral treatment for those who are positive for CMV-PCR in blood and/or stool samples while pending histological diagnosis.

Splenic Infarction Due to CMV Infection

Purpose: Acute CMV infection is a rare cause of splenic infarction in immunocompetent patients. Similarly, thrombotic events are rare complications of CMV, reported in occasional cases of pulmonary embolism, deep vein thrombophlebitis, portal vein thrombosis, and exceptionally as splenic infarction. We report a case of unexplained fever and abdominal pain due to this unusual association. Case Report: A 22 year-old woman presented with 2 weeks of daily fever to 1030 F, rash, and worsening diffuse, crampy abdominal pain. She also reported fatigue, sore throat and nausea. Two weeks prior she was seen in the ER for mild abdominal pain. Abdominal CT at that time was unremarkable and she received acetaminophen and azithromycin. She denied bowel complaints, easy bruising or bleeding. No oral contraceptive or illicit drug use. No family history of clotting disorders. Admission physical exam: Temp 101.2 F. Cardiac: normal. Abdomen: non-tender, hepatosplenomegaly. Skin: mild erythematous rash on upper arms and right thigh; no cutaneous stigmata of endocarditis. Lab: WBC 7,300/mm3 with 19% atypical lymphocytes on smear; Hgb 12.5 g/dL with MCV 84; platelets 174,000; INR 1.2, PTT 28.8; AST 98, ALT 167 (mildly elevated); HIV, EBV and hepatitis serologies: negative. Abdominal CT: wedge-shaped defect in the upper pole indicative of splenic infarction. Transesophageal echocardiogram: no vegetations or mural thrombi. Blood cultures: negative. Urine pregnancy: negative. Unremarkable hypercoagulability evaluation, except anti-cardiolipin (aCL) IgM elevated at 57.2 units (normal <40). CMV IgM: elevated (7.48 units), indicating acute infection, qualitative CMV PCR: positive; quantitative CMV PCR (Viracor) 3,100 copies/mL. Broad-spectrum antibiotics and valganciclovir were begun but discontinued by the patient. She was discharged and at follow-up improved without further treatment. Discussion: CMV may precipitate vascular thrombosis by (1) enhancing platelet adhesion to infected endothelial cells; (2) producing vascular endothelial inflammation and vasculitis with subsequent ischemia; (3) inducing transient anti-phospholipid AB production as we postulate in this patient. Presentation may be subtle without pain; tenderness on exam is reported in as few as one-third of patients. Our patient had no evidence of other causes of splenic infarct, including endocarditis, other viral infection, vasculitis, sickle cell disease, inherited hypercoagulability, malignancy or contiguous inflammation. Patients with unexplained fever and thrombotic events should be screened for CMV. Transient markers of hypercoagulability, such as aCL antibodies and lupus anti-coagulant, are infrequently present.

Comparison of HCMV loads using in-house quantitative CMV PCR, Artus CMV TM PCR and COBAS Amplicor CMV monitor test.

A real-time PCR assay based on highly conserved cytomegalovirus glycoprotein B (CMV-gB) gene was developed to quantify the CMV genome loads in human plasma obtained from immunocompromised patients. The sequence comparison from several strains resulted in the best possible set of primers and a probe that directed against the most highly conserved region. The detection limit of In-house TaqMan polymerase chain reaction assay for CMV-DNA was 2.3 log10 copies/ml and the linear measure interval was 2.3 to 8.3 log10 copies/ml (R > 0.99). The In-house TaqMan assay was compared to commercial quantitative PCR test, COBAS Amplicor CMV monitor test and Artus CMV TM PCR test, in the determination of CMV DNA load in plasma samples. The correlation between In-house TaqMan and COBAS assays was statistically significant, R = 0.915 (P < 0.05; n = 27). Also correlation between In-house TaqMan and Artus CMV assays was statistically significant and higher (R = 0.998; P < 0.05; n = 8). When the In-house TaqMan system was evaluated with the Nucleospin ® RNA virus kit and QIAamp blood minikit, no significant difference was found at the 98.2% confidence interval. Preliminary results indicate that the In-house TaqMan real-time PCR with the designed primer and probe set can be an alternative tool for quantification of CMV loads in clinical routine laboratories.

A prospective study comparing quantitative Cytomegalovirus (CMV) polymerase chain reaction in plasma and pp65 antigenemia assay in monitoring patients after allogeneic stem cell transplantation

BackgroundLow levels of Cytomegalovirus (CMV) viral load are frequently detected following allogeneic stem cell transplantation (SCT) and CMV disease may still develop in some allogeneic SCT patients who have negative pp65-antigenemia (pp65-Ag) or undetectable DNA. Pp65Ag is a sensitive method to diagnose CMV infection. Quantitative CMV-DNA PCR assay in plasma has been proposed to monitor CMV infection in SCT patients. We evaluated the clinical utility of pp65Ag and PCR assay in plasma of SCT recipients.MethodsIn a prospective longitudinal study, 38 consecutive patients at risk of CMV infection (donor and/or recipient CMV seropositive) were weekly monitored for CMV infection by both quantitative CMV-PCR in plasma (COBAS AMPLICOR CMV MONITOR) and pp65 Ag, during the first 100 days after SCT.ResultsA total of 534 blood samples were simultaneously analysed for pp65Ag and PCR. Overall, 28/38 patients (74%) had active CMV infection within 100 days from SCT. In 16 patients, CMV was first detected by pp65 Ag alone; in 5 patients by both methods and in 6 by PCR assay alone; one patient had CMV biopsy-proven intestinal disease without pp65Ag and PCR assays positivity before CMV disease. Overall, three patients developed intestinal CMV disease (7.9%): one had negative both pp65Ag and PCR assays before CMV disease, one had disease and concomitant positivity of both methods, while in the remaining patient, only pp65Ag was positive before CMV disease.ConclusionPlasma PCR(COBAS AMPLICOR CMV MONITOR) and pp65Ag assays were effective in detecting CMV infection, however, discordance between both methods were frequently observed. Plasma PCR and pp65Ag assays may be complementary for diagnosis and management of CMV infection.

Comparison of Two Commercially Available pp65 Antigenemia Tests and COBAS Amplicor CMV Monitor for Early Detection and Quantification of Episodes of Human CMV-Viremia in Transplant Recipients

The performance of two commercially available CMV pp65 antigenemia (AG) assays (CINA kit, Argene Biosoft, CK, and CMV Brite kit, IQ Products, CBK), and a quantitative PCR test (COBAS Amplicor CMV Monitor Test, Roche Diagnostics, CACM) was evaluated with 667 blood samples from 215 transplant recipients. The diagnostic value of the three tests was defined by their ability to detect episodes of systemic CMV activity. A test score was defined, assigning 7 virtual units (VU) for any episode detected first, 5 VU for any episode detected second, and three VU for any episode detected last. Within the evaluated collective, the following overall scores could be determined for the three assays: 45 VU for the CK, 39 for CACM and 31 for the CBK assay. We conclude from our results that (1) the CK is superior to the CBK assay in detecting episodes of CMV antigenemia, and (2) quantitative serum CMV PCR with CACM is not generally superior to pp65 antigenemia testing.

Once Daily Ganciclovir (ODG) as Initial Pre-Emptive Therapy (PT) Delayed until Threshold Viral Load ≥10,000 Copies/ml: A Safe and Effective Strategy for Post-Allogeneic Stem Cell Transplant (ASCT) Patients.

CMV infection is a major cause of morbidity and mortality in patients who have undergone ASCT. We previously evaluated the efficacy of ODG (5mg/kg/day) for 21 days as pre-emptive treatment for CMV viremia and found it to be effective and better tolerated than twice daily doses. We now report data evaluating ODG and PT using a quantitative CMV PCR strategy. At our institution, the detection limit of whole blood CMV PCR (WBP) is 200 copies/ml and the lower limit of quantitation 2000 copies/ml. No criteria for a standard treatment threshold currently exist for quantitative CMV PCR in plasma or whole blood. We elected to initiate treatment with ODG using a threshold WBP ≥10,000 copies/ml in clinically stable patients. Dose escalation was permitted for increasing WBP on ODG treatment. From 4/03 to 3/04, 98 patients underwent ASCT (44 related donors and 54 unrelated donors). 69/98 patients were at risk for CMV reactivation (donor/recipient CMV seropositive). Median follow-up was 318 (120–478) days post transplant. 48/69 (69.6%) patients had 1 or more episodes of CMV viremia (WBP >200 copies/ml). In 5/48 (10.4%) patients the WBP remained <10,000 copies/ml. All 5 patients cleared their viremia without treatment. 43/69 (62.3%) patients developed WBP ≥10,000 (11,324–53,000) copies/ml. 15/43 (34.9%) patients initiated treatment with BID ganciclovir (5mg/kg/BID) or foscarnet at the discretion of the treating physician. Most of these patients were acutely ill at the time of initial viremia. Two of these patients developed CMV pneumonitis which contributed to their deaths. 28/43 (65.1%) patients initiated treatment with ODG. Time to onset of viremia was a mean of 37.3 (19–62) days (>200 copies/ml) and 51.7 (27–196) days (≥10,000 copies/ml) post transplant. 10/28 patients increased ganciclovir to 5mg/kg/BID a mean of 12 (4–19) days after initiation of therapy due to increasing WBP (30,200–231,000 copies/ml). One patient developed neutropenia and was changed to foscarnet. In patients for whom WBP were available for the time points indicated, time to clearance of viremia (≤2000 copies/ml) for the 28 patients was 1/21 (5%) week 1, 8/25 (32%) week 2, 14/26 (54%) week 3, 22/27 (82%) week 4 and 26/28 (93%) week 5. Two patients did not clear their viremia at 5 weeks. One developed CMV colitis which was successfully treated. 17/28 patients remained on ODG for the 21 day treatment period. Time to clearance of viremia in this group was 1/10 (10%) week 1, 8/14 (57%) week 2, 12/15 (80%) week 3, 15/16 (94%) week 4, and 17/17 (100%) week 5. Recurrent viremia (WBP ≥10,000 copies/ml) occurred in 12/28 (43%) patients a mean of 109 (54–247) days post transplant. 2/28 (7%) patients developed CMV disease (colitis), 1 during the initial episode of viremia and 1 during a recurrence. Both were successfully treated. There were no deaths due to CMV disease in patients initiating therapy with ODG. Conclusion: Delaying treatment with ODG as initial PT until WBP ≥10,000 copies/ml is a safe and effective treatment strategy for CMV viremia post ASCT. This strategy reduces over treatment of patients and neutropenia.

Decreased Incidence of Cytomegalovirus Infection in Thymoglobulin-Treated Transplant Patients with 6 Months of Valganciclovir Prophylaxis

To the Editor: Cytomegalovirus (CMV) infection is still the most common opportunistic infection following solid organ transplantation despite the introduction of newer anti-viral medications for prophylaxis, such as ganciclovir, valacyclovir, and valganciclovir (1). We recently reported that the overall incidence of CMV disease at 1 year post transplant was 14% (4% tissue-invasive, 10% non-invasive) in 129 kidney or pancreas transplant recipients with 3 months of ganciclovir or valganciclovir prophylaxis (2). However, of the 18 patients who developed CMV disease, 83% (n = 15) had received thymoglobulin induction treatment, and 25% of all thymoglobulin-treated patients developed CMV disease. Four of 15 (27%) patients had tissue-invasive CMV disease (two CMV colitis, one CMV gastritis, and one CMV nephritis). In light of these findings, we extended CMV prophylaxis with valganciclovir to 6 months in thymoglobulin-treated patients and prospectively investigated the incidence, time-course, and clinical patterns of CMV disease. Thirty-seven consecutive thymoglobulin-treated patients, transplanted between February and December 2002, received 6 months of CMV prophylaxis with valganciclovir 450 mg p.o. q.d. There were 20 males and 17 females; 29 of them were Caucasian or Hispanic, and 8 African-American; ages were 29–67 years. Thirty-two patients received kidney-, and five pancreas transplantation. Median follow-up is 13 months (range 8–19 months). In terms of CMV antibody status, 21 were Donor (D)+/Recipient (R)+, eight were D–/R+, four were D+/R–, and four were D–/R–. All patients received quadruple immunosuppression with thymoglobulin 1.5 mg/kg for 5 days after transplantation, corticosteroids, mycophenolate mofetil (MMF), and microemulsion cyclosporin or tacrolimus. All patients received valganciclovir prophylaxis regardless of donor/recipient CMV antibody status. No patient developed tissue-invasive CMV disease. Only three patients (8%) developed non-invasive CMV disease, characterized by fever, leukopenia (less than 3000) and CMV viremia, as detected by qualitative blood PCR (with Southern blot confirmation). CMV-PCR titers were 4590, 21 900, and over 100 000 in those three patients by quantitative CMV-PCR (Cobas amplicor CMV monitor). Two were D+/R+ and one D–/R–. Patients developed CMV viremia 5–6 weeks after completion of 6 months of CMV prophylaxis. All three patients responded to valganciclovir treatment, and the control CMV-PCR titers were undetectable. A recent phase III clinical trial in D+/R– solid-organ recipients, using valganciclovir at 900 mg daily, demonstrated similar incidences of CMV disease in valganciclovir- or oral ganciclovir-treated patients (3). We preferred to extend the course of valganciclovir treatment to 6 months instead of increasing the dose to 900 mg daily for 3 reasons: A 450-mg dose of prophylactic valganciclovir prevented CMV viremia in all but one of the patients during prophylaxis. Half of our valganciclovir-treated patients required the MMF and/or valganciclovir doses to be decreased or held due to neutropenia at our previous experience (2). Two of them had prolonged neutropenia requiring granulocyte colony stimulating factor. Using higher doses of valganciclovir might have further increased the incidence of neutropenia. Patients with creatinine clearance of less than 60 mL/min are recommended to take 450 mg daily (3, 4). It is unlikely that a kidney transplant recipient will have a better creatinine clearance early after transplantation. These results indicate that 6 months of valganciclovir prophylaxis significantly decreases CMV incidence (from 25% to 8%) in thymoglobulin-treated patients, and abrogates the development of tissue-invasive CMV disease. The expected number of patients with CMV disease in 37 thymoglobulin-treated patients with 3 months of valganciclovir prophylaxis would be nine (three tissue-invasive, six non-invasive). Six months of valganciclovir prophylaxis prevented the development of CMV disease in six patients. To extend the prophylaxis to 6 months is cost effective by decreasing the incidence of CMV disease, to avoid not only the cost of CMV treatment but also the indirect effects of CMV disease, such as chronic rejection, thrombotic microangiopathy, and decreased graft survival.

CMV infection of the renal allograft is much more common than the pathology indicates: a retrospective analysis of qualitative and quantitative buffy coat CMV-PCR, renal biopsy pathology and tissue CMV-PCR.

Quantitative blood polymerase chain reaction (PCR) for cytomegalovirus (CMV) is used to direct therapy in kidney transplant patients, but cytomegalic inclusions are rarely found in allograft renal biopsies even with an elevated serum creatinine and apparent CMV disease. The relationship between quantitative blood CMV and renal allograft pathology is unknown. Thirteen biopsy samples were available for analysis from patients suspected of CMV disease, who had a buffy coat CMV-PCR drawn within 2-5 days of a renal allograft biopsy for an elevated creatinine. All were evaluated for CMV pathologically, by light microscopy, immunohistochemistry, in situ hybridization and tissue PCR. Qualitative and quantitative buffy coat CMV-PCR were positive in 10/13 (77%) patients. Tissue CMV-PCR was positive in five (50%) biopsies, including two with CMV inclusions and three with no inclusions. Quantitative buffy coat CMV-PCR levels did not correlate with detection of CMV inclusions in renal tissue. Paradoxically, quantitative buffy coat CMV-PCR was low (239 and 538 copies/microg of DNA) when CMV inclusions were detected. All five biopsies with acute rejection were associated with CMV viraemia and two of the five with allograft CMV inclusions. A quantitative buffy coat CMV-PCR of <100 copies/microg of DNA ruled out disease with CMV inclusions. CMV nephropathy is much more common than previously reported when sensitive techniques are used for detection in tissue. Acute rejection and CMV viraemia occur commonly together in patients at risk for CMV. Quantitative buffy coat CMV-PCR does not correlate with the presence of CMV inclusions. These findings have implications for management of patients who have elevated serum creatinine and are at risk for CMV disease.

Comparison of quantitative PCR to antigenemia for CMV surveillance after lung transplantation

Background: Prophylactic antiviral therapy(AVT) may contribute to CMV drug resistance, present in 5-9% of lung transplant recipients (LTR). Serial CMV screening with preemptive treatment provides an alternative to prophylaxis, but the optimal screening test has not been defined. We compared two quantitative assays for CMV surveillance in LTR. Methods: Between 6/15/01-6/15/02, all new LTR underwent screening for CMV in the first 4 post-LT mo. Screening was performed weekly or with each clinic visit. CMV antigenemia(AG) was compared to a quantitative(QNT) CMV PCR assay, the COBAS Amplicor CMV Monitor Test (Roche Diagnostics, Indianapolis, IN). AVT was initiated during treatment of acute rejection episodes, for clinical suspicion of CMV disease(dz), or positive(pos) CMV assay result. Results: A total of 113 specimens in 10 LTR were tested by both methods. 16/113 samples were both AG and QNT pos. There was 1 AG pos/QNT neg, and 16 AG neg/QNT pos samples. Using AG as the “gold standard”, the QNT had a sensitivity of 94% and a specificity of 83%. The CMV D/R status of the 10 LTR was / and -/ in 3 each, and -/and /in 2 each. Over the course of the study, 3 LTR had no evidence of CMV. One LTR, on acyclovir, was treated for clinical suspicion of CMV dz despite neg AG and QNT. Five LTR had a pos QNT a mean of 30d after LT, (mean viral count 22356 cpu, range 580-56200 cpu) with 3/5 also having pos AG (mean 20 cells/slide, range 8-71 cells/slide). One LTR developed pos AG and QNT 115d post-LT. Of note, the QNT converted in the 2 CMV D-/RLTR. Overall, 3/6 LTR with a pos QNT developed CMV dz, while the remaining 3 LTR were asymptomatic. The AG never converted or converted after the QNT in 2/3 LTR with clinical dz. The highest QNT counts were found in LTR with clinical CMV dz. Conclusions: Compared to the AG test, QNT is more predictive of CMV dz. CMV D-/Rstatus does not predict freedom from CMV infection.

Cytomegalovirus DNA concentration in plasma predicts development of cytomegalovirus disease in kidney transplant recipients

The clinical significance of cytomegalovirus (CMV) DNA detection in post-kidney transplantation infection surveillance was examined by comparing the performance of three assays for detection of CMV in blood: the test for CMV-pp65-antigen in leukocytes, which is routinely employed in our laboratory, the quantitative plasma CMV-DNA-polymerase chain reaction (PCR; Cobas Amplicor CMV Monitor test) and the qualitative plasma CMV-DNA-PCR (Amplicor CMV test). Thirteen kidney transplant recipients were monitored with serial samples taken over a period of 3 months following transplantation. The quantitative CMV-PCR was the test with highest sensitivity, 95.9%, vs. 88.9% and 76.9% for the CMV-pp65 antigen assay and qualitative CMV-PCR, respectively. The virus load in the first positive specimens, assessed as DNA-copies/mL, was significantly associated with CMV disease because five of the six patients who developed disease, but only one of the seven who did not develop disease, had more than 3000 CMV-DNA-copies/mL. The number of CMV-pp65 antigen-positive cells in the first positive specimens did not have predictive value for development of CMV disease. Assessment of CMV in plasma by the quantitative CMV-PCR is especially useful since it has a high sensitivity and the amount of CMV DNA in plasma is a good predictor of CMV disease.

One year analysis of CMV-PCR surveillance data in lung transplant recipients

The optimal strategy for preventing CMV disease post transplantation (Tx) is unknown. In January 2000 we introduced quantitative CMV-PCR (QPCR) monitoring of CMV-IgG +ve and CMV-status mismatched patients (CMV-MM) post-Tx. All patients received aciclovir prophylaxis and CMV-MM also received oral ganciclovir (GCV) prophylaxis for 3 months.