Quantitative Cytomegalovirus PCR Research Articles

Evaluation of CMV T-cell Mediated Immunity in Children with CMV DNAemia After Allogeneic Hematopoietic Cell Transplantation

Abstract Background Cytomegalovirus (CMV) is among the most common infectious complications following allogeneic hematopoietic cell transplantation (allo-HCT). A commercially available CMV T-cell immune panel (CMV-TCIP, Viracor®), reported as percentages of CMV-specific interferon gamma releasing CD4 and CD8 cells (%CMV-CD4 or %CMV-CD8 IFN-γ), may be used to assess CMV cell-mediated immunity (CMV-CMI). However, the utility of this assay among pediatric allo-HCT recipients remains unknown. Methods A retrospective analysis among pediatric allo-HCT recipients (age ≤18 years) from 1/2020 to 11/2023 who developed CMV DNAemia and had CMV-TCIP testing was performed. Per institutional protocol, weekly quantitative plasma CMV PCR was performed until day +100. Clinically significant CMV infection (csCMVi) was defined as CMV viral load (VL) ≥500IU/ml and pre-emptive therapy (PET) was provided for csCMVi. The most frequent indications for CMV-TCIP testing were to evaluate CMV-CMI to inform the need of CMV virus-specific T-cell therapy (CMV-VST) in the setting of resistant/refractory CMV, to monitor CMV-CMI as a proxy of VST expansion post CMV-VST, or to evaluate the need of ongoing secondary antiviral prophylaxis. A %CMV-CD4 or %CMV-CD8 IFN-γ ≤0.2% was categorized as a poor response, whereas a result >0.2% was classified as an adequate response. Specimens with insufficient quality, high background, or discrepant results from %CMV-CD4 or %CMV-CD8 were classified as a discordant response. Demographics, underlying condition, HCT type, conditioning regimen, antiviral agents and use of VST, VL, and CMV-TCIP results were recorded. Descriptive and nonparametric statistics (median, interquartile range [IQR]) were used for analysis. Results Twenty-seven CMV-TCIP results from 10 unique allo-HCT recipients were analyzed. The median age at transplant was 8.5 years (IQR 2.75-14.3), predominantly for hematologic malignancy (70%). CMV donor/recipient (D/R) serostatus were D+/R+ (60%), D-/R+ (30%) and D+/R- (10%). CMV DNAemia developed post-HCT in 80% (8/10) with median onset of 4 days post-HCT (IQR 3-40) and 2 subjects had known DNAemia prior to HCT. Nine (90%) subjects were started on PET for csCMVi, whereas 1 (10%) received antiviral therapy when VL was <500IU/ml. The first CMV-TCIP was obtained at median of 112 days (IQR 42-130) post-HCT. Despite appropriate antiviral therapy, patients with a peak VL ≥500 IU/mL (obtained within 1 week before and after CMV-TCIP testing) demonstrated significantly lower absolute CD4 (median 12 vs. 198 cells/uL, p=0.02) and CD8 (median 79 vs. 443 cells/uL, p=0.04) counts and %CMV-CD4 IFN-γ (0.04% vs. 0.36%, p=0.02) compared to those with a peak VL <500 IU/mL. Conclusion Our results suggest that CMV-CMI, particularly %CMV-CD4 IFN-γ >0.2%, was observed concurrently with downward trending VL, and may serve as a proxy of VST expansion in patients receiving CMV-VSTs. Figure 1A. The distribution of peak VLs over time based on %CMV-CD4 and %CMV-CD8 IFN-γ are demonstrated in Figure 1B. CMV peak VLs were lower with TCIP testing results reported as adequate combined responses compared to those with poor combined responses. In five subjects (50%) who received CMV-VSTs as part of CMV therapy, increased %CMV-CD4 and %CMV-CD8 IFN-γ were observed post-infusion with an observed downward trend in VL.

Increased frequency and quantity of mucosal and plasma cytomegalovirus replication among Ugandan Adults Living with HIV.

Co-infection with HIV can result in impaired control of cytomegalovirus (CMV) replication, increasing the likelihood of disease and onward transmission. The objective of this analysis was to measure the impact of HIV on CMV replication in an intensively-sampled cohort in Kampala, Uganda. CMV seropositive men and women aged 18-65, with or without HIV co-infection, were followed for one month. Daily oral swabs and weekly anogenital swabs and plasma were collected. Quantitative CMV PCR was performed on all samples. Eighty-five participants were enrolled and provided ≥1 oral swab; 43 (51%) were HIV-seropositive. People living with HIV (PLWH; median CD4 count 439 cells/mm3; none on antiretrovirals) had 2-4 times greater risk of CMV detection at each anatomical site assessed. At the oral site, 773 of 1272 (61%) of samples from PLWH had CMV detected, compared to 214 of 1349 (16%) among people without HIV. Similarly, the mean CMV quantity was higher among PLWH at all anatomical sites, with the largest difference seen for oral swabs (mean difference 1.63 log/mL; 95% CI 1.13-2.13). Among PLWH, absolute quantity of CD4+ T-cells was not associated with risk of CMV detection. HIV plasma RNA quantity was positively correlated with oral CMV shedding frequency, but not detection at other sites. Mucosal and systemic CMV replication occurs at higher levels in PLWH than people without HIV, particularly oral shedding, which is a major mode of CMV transmission. Increased CMV replication despite relatively preserved CD4+ T-cell counts suggests that additional interventions are required to improve CMV control in PLWH.

376. Testing for Cytomegalovirus Viremia in People with HIV/AIDS- An Opportunity for Diagnostic Stewardship

Abstract Background Guidelines recommend against testing for cytomegalovirus (CMV) viremia as a screening tool for CMV end-organ disease (EOD) in people with HIV/AIDS (PWHA) due to low positive predictive value. Additionally, a negative result does not exclude EOD. In this study, we aim to assess the clinical utility of plasma CMV quantitative PCR (qPCR) for the diagnosis of CMV EOD and to determine if positive results lead to further testing for EOD or earlier initiation of anti-CMV therapy. Methods We retrospectively identified PWHA who had a plasma CMV qPCR result or who were diagnosed with CMV EOD between 2014-2021. qPCR results that were undetectable or less than the minimum quantifiable value (300 IU/mL) were considered negative and results >300 IU/mL were considered positive. EOD was confirmed via tissue biopsy, ophthalmic exam, or cerebrospinal fluid analysis. We compared CMV qPCR results for participants with proven EOD and those without proven EOD. For participants diagnosed with CMV EOD, we evaluated if a positive qPCR prompted further workup for EOD diagnosis or triggered anti-CMV therapy. Results We identified 138 PWHA with a CMV qPCR result and 17 PWHA with CMV EOD. Participant characteristics are shown in Table 1. Among participants with a qPCR result, 27 (19.6%) were positive; 13 (9.42%) of those with a qPCR results had EOD. 4 participants with EOD did not have qPCR. Participants with a positive qPCR were 4.55 (95% CI 2.63-7.90) times more likely to have EOD than those with a negative result. Table 2 shows qPCR results among participants with and without EOD. Table 3 shows the performance of qPCR for the diagnosis of EOD. 9 (69.2%) of 13 participants with EOD had a positive qPCR. 7 of them were diagnosed with EOD and started on treatment before the qPCR resulted. In 1 case, the qPCR resulted as positive on the same day ophthalmology diagnosed CMV retinitis. In only 1 case did a positive qPCR result and initiation of therapy precede the diagnosis of EOD. Conclusion Contrary to guidelines, clinicians often order plasma CMV qPCR on PWHA. Although PWHA who have CMV EOD are more likely to a have a positive qPCR than those without EOD, our data confirm CMV qPCR should not be used as a screening tool for EOD. Instead, clinical assessment should guide diagnostic investigation. Disclosures All Authors: No reported disclosures.

#45 Antibiotic Exposure and Risk of Cytomegalovirus Viremia Among Pediatric Hematopoietic Stem Cell Transplant Recipients at a Single Institution

Abstract Background Cytomegalovirus (CMV) causes substantial morbidity and mortality after hematopoietic stem cell transplantation (HCT), and antibiotic exposure after HCT is increasingly recognized as a risk for negative clinical outcomes. Recent studies identified an association between receipt of antibiotics with an anaerobic spectrum of activity and infection with cytomegalovirus (CMV) after HCT in adults. We sought to evaluate the association between anaerobic antibiotic exposure and CMV viremia in a well-characterized cohort of pediatric HCT recipients. Method We conducted a single-center retrospective cohort study of HCT recipients <18 years of age who underwent HCT at Duke University between 2000 and 2016. Routine pre-emptive surveillance testing for CMV was performed; CMV viremia was defined as a positive whole blood hybrid capture CMV DNA assay or quantitative plasma CMV PCR. We used log-logistic regression to evaluate associations between anaerobic antibiotic exposure (vancomycin, metronidazole, meropenem, piperacillin-tazobactam) and CMV viremia-free survival at 100 days after HCT. The first model considered anaerobic antibiotic exposure in the first 14 days after HCT as a binary variable. The second model considered anaerobic antibiotic exposure in the first 100 days as a time-varying variable; once a subject was exposed to a specific antibiotic, they were considered exposed for the remainder of the study period. Both models adjusted for year of HCT, age, race, HCT type and donor cell source, underlying diagnosis, conditioning regimen, CMV serostatus, and treatment for graft-versus-host-disease. Results Among 966 children, median (interquartile range) age was 6.5 (3.1, 11.5) years. Hematologic malignancy was the indication for transplantation in 417 (43%) subjects. Of 770 allogeneic HCT, 566 (74%) were from an umbilical cord blood donor and 204 (26%) were from a bone marrow donor. The majority of HCT were recipient CMV-seronegative and donor CMV-seronegative (R-/D-; 537, 56%), with 24 (2%) R-/D+, 270 (28%) R+/D-, and 135 (14%) R+/D+ transplants. Within the first 100 days after HCT, 692 (72%) children received vancomycin, 363 (38%) received metronidazole, 231 (24%) received meropenem, and 121 (13%) received piperacillin-tazobactam. Exposure to vancomycin (OR=0.86; 95% CI: 0.59, 1.26), metronidazole (OR=0.76; 95% CI: 0.53, 1.10), meropenem (OR=1.19; 95% CI: 0.74, 1.90), or piperacillin-tazobactam (OR=1.03; 95% CI: 0.55, 1.92) in the first 14 days after HCT was not associated with the risk of CMV viremia-free survival at 100 days. Similarly, we found no increased risk of CMV viremia with anaerobic antibiotic exposure in the first 100 days after HCT (vancomycin OR=0.90; 95% CI: 0.60, 1.34; metronidazole OR=0.80; 95% CI: 0.53, 1.21; meropenem OR=0.98; 95% CI: 0.60, 1.62; piperacillin-tazobactam OR=0.69; 95% CI: 0.38, 1.23). Conclusion In this study of pediatric HCT recipients, we did not find evidence of an association between anaerobic antibiotic exposures and CMV viremia risk. However, future pediatric and adult studies are warranted to evaluate this previously reported association in other populations, including studies evaluating for a possible role of the gut microbiome in modifying CMV viremia risk after HCT.

SUCCESSFUL CLEARANCE OF CYTOMEGALOVIRUS COLITIS IN ACUTE SEVERE ULCERATIVE COLITIS DURING INDUCTION OF OZANIMOD

Cytomegalovirus (CMV) colitis, commonly observed in patients with acute severe ulcerative colitis (UC), increases the risk for colectomy and treatment resistance. Unfortunately, corticosteroids and immunomodulators increase CMV reactivation and infection. The use of certain biologics such as anti-TNF mAb increase the risk for CMV colitis, while experience with newer biologics is limited leaving clinicians with few validated options. Ozanimod, an oral sphingosine-1-phosphate receptor modulator, was recently approved for induction and maintenance therapy for UC. Experience with ozanimod in the setting of UC complicated by CMV infection has not been reported to this date. We report here on a severe UC patient with tissue proven CMV ulceration that cleared the viral infection during ozanimod induction. A 21-year-old male with UC treated with tofacitinib presented with five days of diffuse abdominal pain and 10-12 episodes of bloody diarrhea per day with CRP 341mg/L (<8.0mg/L) and calprotectin >3000μg/g (<49μg/g). Computed topography of the abdomen and pelvis showed diffuse wall thickening and mucosal enhancement throughout the entire colon and rectum. He was started on methylprednisolone 20mg TID for acute severe UC. He previously failed infliximab, adalimumab, ustekinumab, and vedolizumab. Colonoscopy showed pancolitis (Mayo score 3) with deep ulceration in the cecum. Random colon biopsies were consistent with active colitis with an area of CMV infection in an area of cecal ulceration (Figure 1). Serum CMV Quantitative PCR was 410IU/ml (normal <50IU/ml). The patient refused colectomy and was started on IV ganciclovir 5mg/kg q12h while transitioning to prednisone 40mg/day. Three weeks after initial presentation and eleven days after starting ganciclovir, the patient began ozanimod. He remained on ganciclovir for three weeks then transitioned to oral valganciclovir 900mg BID. The patient’s symptoms improved, CRP normalized, and calprotectin levels dropped (1200μg/g). Repeat colonoscopy performed eighteen days after ozanimod initiation revealed moderately-improved pancolitis (Mayo 2) with multiple healing cecal ulcers. Analysis of cecal ulceration IHC and peripheral blood PCR indicated clearing of the infection from the colon (Figure 2). Cytomegalovirus colitis remains a common complication in severe UC, while avoidance of colectomy with viral clearance remains a challenge. Ozanimod and ganciclovir were chosen by this patient after refusing surgical resection. The improvement in clinical biomarkers of colitis and viral clearance suggest that ozanimod given with ganciclovir may be an exciting rescue therapy in these patients. Future studies will be needed to ascertain the rates of ozanimod response and CMV clearance in a larger patient cohort.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

P028 High cytomegalovirus DNA load in mucosal biopsies predicts steroid failure as well as colectomy in acute severe ulcerative colitis

BACKGROUND: Cytomegalovirus (CMV) reactivation may be responsible for steroid refractory acute severe colitis (ASC), which requires rescue therapy in form of surgery or advanced immunosuppression. The optimum technique for diagnosing CMV colitis in this setting remains unclear. We investigated the role of CMV Quantitative PCR for diagnosing CMV colitis and for predicting of steroid-failure in ASC. METHODS: Consecutive patients with ASC satisfying Truelove and Witts' criteria, hospitalized at a single centre from May 2016 to November 2017, were included. The primary outcome measure was steroid-failure defined as colectomy and/or rescue therapy with ciclosporin or infliximab during admission. Oxford criteria, ulcerative colitis index of severity (UCEIS) at day 1 and fecal calprotectin (FCP) at day 3 were used to predict steroid response. Immunohistochemistry (IHC) and quantitative PCR for CMV was done on mucosal biopsies and the results were compared between steroid responders and non-responders. RESULTS: Of 37 patients (Mean age: 35 ± 12 years, 70% males), 14 (38%) failed iv corticosteroids and 8 (25%) required surgery. Although IHC for CMV was not different between steroid failures and responders (29% vs 17%, P = 0.40), patients with steroid failure had a significantly higher median level of mucosal CMV DNA (7,840 [0–2,700,000] vs 112 [0–34,459] copies/mg, P = 0.03). Significantly greater number of patients with steroid failure had CMV DNA count &gt;1,000 copies/mg (71% vs 26%, P = 0.007). CMV DNA count &gt;1,000 copies/mg (odds ratio 6.5 [95% confidence interval 1.3–33, P = 0.03]) and positive oxford criterion on day 3 of iv corticosteroids (OR 6 [95% CI 1.2–30, P = 0.03]) were independent predictors of steroid-failure and need for rescue therapy/colectomy. CONCLUSION(S): CMV DNA quantification in mucosal biopsy can detect CMV colitis and predict steroid failure in acute severe colitis with reasonable accuracy.

A retrospective regional audit of Cytomegalovirus (CMV) laboratory diagnostics in Crohn’s/Colitis patients in Northern Ireland – ‘towards a diagnostic algorithm’

Introduction Concurrent cytomegalovirus (CMV) in inflammatory bowel disease (IBD) related colitis, (Ulcerative Colitis (UC) or Crohn’s Disease (CD) is an important yet complex scenario associated with high rates of colectomy and other morbidity. This regional audit aimed to identify baseline standards for a virological diagnostic-based approach for the identification of those at risk of CMV reactivation (flare) in the setting of IBD. Methods Retrospective, cross-sectional study over a three year time-period, January 2010–March 2013, involving all five Northern Ireland HSC Trusts. Sample cohort n=277 IBD patients of which n=106 further grouped as SRC ‘severe acute colitis and/or steroid refractory colitis’. Seven audit standards were assessed including specimen(s) submission for CMV diagnostic analysis, antiviral therapy, ascertainment of colectomy rate, and result communication protocol. Findings Audit primarily found a need to better define test requesting protocols (optimal sample required for optimal serology and molecular diagnostics), and a need to work towards timeliness in both test requesting and result reporting. This audit also identified the high risk patient group (SRC) when test positive for CMV (PCR and/or histopathology) three times more likely to undergo colectomy (OR 3.16, 95 % CI (0.74, 13.21; P=0.06). Conclusions Diagnostic-based patient identification is optimal to identify those most at risk of CMV reactivation in the IBD setting. The serological IgG profile is key in risk assessment of potential reactivation, together with quantitative CMV PCR in colonic tissue, with/without supportive histopathology, as the most sensitive and timely means of identify those high risk patients.

Toxicities of CMV Preemptive Therapy in the First 100 Days after Allogeneic Hematopoietic Cell Transplantation (HCT): A Real-World Study at Memorial Sloan Kettering Cancer Center (MSKCC)

Background Cytomegalovirus (CMV) is the most common viral infection after HCT. Current antivirals used for preemptive therapy (PET) effectively prevent CMV disease, but their use is limited by toxicities. We examined real-world utilization patterns of PET and associated toxicities in HCT recipients at high risk (HR) and low risk (LR) for CMV through day (D) 100 post HCT. Methods Adult, CMV-seropositive first bone-marrow or peripheral blood HCT recipients from January 1, 2015 through December 31, 2017, routinely monitored by quantitative CMV PCR (Taqman, Roche) were analyzed. PET utilization including types and sequence of antivirals, dose and duration, and reasons for discontinuation through D 100 were extracted from medical records. HR included T-cell depleted HCT or conventional mismatched or haploidentical HCT. All other were LR. Descriptive statistics are reported. Results Of 232 patients (pts), 121 (52%) received PET including 40 LR and 81 HR. PET started at a median 35 D post HCT [HR vs. LR: 32 vs. 39, P Conclusions Valganciclovir was the preferred first line PET agent. HR pts had earlier initiation and longer PET duration. 1st PET discontinuation/change due to clinical or laboratory defined toxicity occurred commonly with similar frequency between HR and LR. Our findings support the need for more effective and safer PET alternatives for HCT recipients.

Cytomegalovirus resistance in CD34+ -selected hematopoietic cell transplant recipients.

Cytomegalovirus (CMV) viremia after CD34+ -selected hematopoietic stem cell transplant (HCT) often requires prolonged antiviral therapy. We report rates and outcomes of resistant CMV in a contemporary cohort of CD34+ -selected HCT recipients managed preemptively. We retrospectively reviewed 220 consecutive, CMV-seropositive recipients (R+), who received CD34+ -selected HCT at Memorial Sloan Kettering Cancer Center between June 2010 and December 2014. Patients were monitored by quantitative CMV PCR and were treated preemptively. CMV resistance was tested by a genotypic assay. One hundred and sixty-one (73%) patients developed CMV viremia and 47 (29% of viremic and 21% of total patients) had CMV resistance testing by one-year from HCT. CMV resistance was confirmed in 19 (12% of viremic and 9% of total) patients and was identified >3months from HCT in 90% of patients. Twelve patients had mutations in UL97 only; the remaining 7 patients had mutations in UL54 only or UL54 and UL97. By 1 year from HCT, 11 of 19 (58%) patients with mutations had CMV end-organ disease. CMV-related mortality in patients with resistance was 42%. Nine percent of CMV R+, CD34+ -selected HCT recipients had resistant CMV by 1 year from HCT. Of 19 patients with resistant CMV, 58% had CMV end-organ disease and 42% died of CMV. Effective strategies for CMV prevention and restoration of CMV immunity are needed for CD34+ -selected HCT.

546 - Cytomegalovirus (CMV) Infection and Disease Incidence and Risk Factors Across Diverse Hematopoietic Cell Transplantation (HCT) Platforms Using a Standardized Monitoring and Treatment Approach: A Comprehensive Evaluation From a Single Institution

CMV remains a major complication of allogeneic HCT, with over 40% of at risk recipients (donor (D) and/or recipient (R) seropositive) experiencing infection in the first 100 days post-HCT. At the NIH, all HCT recipients are monitored weekly from day 0-100 with blood CMV quantitative PCR (qPCR) with a uniform approach to preemptive therapy across HCT protocols. This offers a unique opportunity to comprehensively evaluate the cumulative incidence (CI) of and risk factors for post-HCT CMV infection and disease across different T cell manipulation approaches (posttransplantation cyclophosphamide (PTCy), proximal serotherapy, ex vivo T cell depletion (TCD), and calcineurin/mTOR inhibitor (CNI/mTORi) based) and different graft sources (marrow (BM), peripheral blood (PBSC), and cord blood (CB)). This is a retrospective study of 364 consecutive patients transplanted between Aug 10, 2011 and Feb 10, 2017. CMV infection was defined as 2 qPCRs between 3.08-4.11 log10 IU/mL in 1 week, 1 qPCR value >4.11 log10 IU/mL, or sufficient clinical suspicion for CMV disease to prompt therapy. Competing risks (CR) included death, relapse, and graft failure, with analyses including and not including systemic steroids for GVHD or engraftment syndrome as an additional CR. Recipient/donor/HCT characteristics are shown in Figure 1. Among at risk recipients, the 100 day CI of CMV infection was 46% (34% when systemic steroid use was included as a CR). The CI of CMV infection varied mainly by recipient CMV serostatus (Figure 2, P < .0001), with 100 day estimates of 2% (D/R −/−), 6% (+/−), 38% (−/+), and 40% (+/+). With regard to graft T cell manipulation, the 100 day CI of CMV infection, with (Figure 3, P = .029) and without steroids (p = NS) as a CR, respectively, was 25% and 42% (CNI/mTORi), 37% and 42% (serotherapy), 40% and 54% (PTCy), and 50% and 60% (TCD). CB was associated with 100 day CI of CMV infection of 64%, higher than BM at 39% and PBSCs at 30%, P = .002. There was a tendency toward lower 100 day CI of CMV infection for recipients of higher T cell doses when systemic steroid use was included as a CR, P = .07. Among CMV seropositive recipients, CMV therapy duration was longer with CMV seronegative donors (median 28 days) compared to CMV seropositive (median 20 days), P = .03. The median duration of CMV therapy was longer at 36 days for CB HCT, compared to PBSC (21 days) and BM (29 days), P = .03. CMV disease developed in 12 patients in the 100 days post HCT at a median of 27 days: 75% in the setting of GVHD, 83% in seropositive recipients, 75% involving gut, and occurring in .5% of PTCy, .4% of CNI/mTORi, .3% of serotherapy, and 0% of CB and TCD HCTs. In conclusion, CMV infection incidence varies based on recipient CMV serostatus, T cell manipulation strategy, and graft source, with longer treatment duration with seronegative donors and/or CB recipients. CMV disease rates before day 100 are similar across platforms and occur predominantly in the setting of GVHD.Figure 2Cumulative incidence of CMV infection in the first 100 days after HCT, by donor (D) and recipient (R) CMV serostatus.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Cumulative incidence of CMV infection in the first 100 days after HCT, by T cell manipulation strategy, with comparison using Gray's method of post-transplantation cyclophosphamide (PTCy), serotherapy (proximal ATG or alemtuzumab), ex vivo T cell depletion (TCD), and calcineurin inhibitor/mTOR inhibitor (CNI/mTOR)-based approaches.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Cytomegalovirus Management in Adult Hematopoietic Stem Cell Transplant Patients with Pre-Engraftment Viremia: A Single-Center, Retrospective, Descriptive Study

BackgroundScant data exist regarding cytomegalovirus (CMV) viremia in hematopoietic stem cell transplant (HSCT) recipients during the pre-engraftment period. The goal of this study was to describe management of CMV in neutropenic adult HSCT patients at our institution, and to assess the possible impact of different quantitative CMV PCR tests (QPCRs).MethodsPost-HSCT monitoring at this center includes weekly CMV QPCR from plasma. Three different QPCR assays were used sequentially during the study period (1/2010–12/2015): two with lower limits of quantification (LLOQ) of 300 and 100 copies/mL through 4/2013, and after that the FDA-approved assay with LLOQ of 137 IU/mL. Medical records of first-time HSCT patients were reviewed. Pre-/peri-engraftment CMV was defined as detectable CMV DNA with [ANC] < 1000 cells/mm3. Information collected included demographics, donor/recipient CMV serostatus, conditioning regimen, CMV QPCR and ANC results, dates of CMV treatment, CMV disease within 100 days, and death within 6 months of HSCT. Data were analyzed with STATA v14.ResultsOf 1151 total HSCT, 76 patients had a positive CMV QPCR when ANC < 1000 cells/mm3. CMV was first detected a median of 12 days (0–48) post-transplant, and was above LLOQ at a median of 28 days (0–49). 71/76 (93%) were treated at a median of 33 days post-transplant (range 4–105 days), most with valganciclovir (40) or ganciclovir (30); 1 received foscarnet initially. 5 patients with low-level viremia were monitored without treatment. At initiation of therapy, median CMV level was 1471 (range 159–22,900) copies or IU/mL and ANC was 1202 (range 28–9680) cells/mm3. Median treatment duration was 34 days (range 9–392). Only 2 patients had possible tissue-invasive CMV disease.ConclusionGanciclovir and valganciclovir were used to treat most pre- and peri-engraftment CMV viremia, despite potential bone marrow toxicity. The LLOQ of different CMV QPCR tests did not affect the viral threshold for starting treatment. The time between first CMV DNA detection (median day +12) and initiation of treatment (median day +33) suggests clinicians waited for CMV DNA and/or ANC to rise before treating. With this deferred-treatment approach, the proportion of patients with tissue-invasive disease remained low.Disclosures All authors: No reported disclosures.

Pneumopathie congénitale à cytomégalovirus chez un nouveau-né à terme né de mère séropositive pour le VIH

Cytomegalovirus (CMV) is one of the most common intrauterine infections, affecting approximately 1% of all live births. There are few reports on congenital CMV infections manifesting as isolated pneumonitis. We report a case of congenital CMV with neonatal respiratory distress affecting an HIV-exposed uninfected infant. This infant required noninvasive ventilation beginning within the first 15min of life. The initial chest X-ray showed diffuse bilateral ground-glass opacifications. Bacterial infection, meconium aspiration and hyaline membrane disease were excluded. Salivary quantitative CMV PCR was positive (2,342,261IU/mL) and serum viral load for CMV was low (476IU/mL). Bronchoalveolar lavage (BAL) performed on day 12 for quantitative CMV PCR was significantly positive (1,045,942IU/mL). Intravenous ganciclovir treatment was started on day 14 (7.5mg/kg/12h) for 2weeks and oral valganciclovir (15mg/kg/12h) was given for 4weeks afterwards. Ventilatory support was stopped on day18. HIV serum viral load was negative on day30. Congenital CMV infection can present as isolated pneumonitis with persistent neonatal respiratory symptoms, emphysematous lung disease, or persistent pulmonary hypertension. If this diagnosis is suspected, and even if CMV viremia remains low, BAL with quantitative CMV PCR must be performed to ascertain the diagnosis and indicate antiviral treatment. HIV-exposed uninfected infants have higher rates of congenital CMV infection when the mother's CD4 rate is<200/mm3. Most cases of CMV transmission in HIV-exposed uninfected infants have occurred by maternal endogenous reactivation or reinfection.

Cytomegalovirus Reactivation Does Not Increase Subsequent Risk for Acute Graft-Versus-Host Disease, Malignant Disease Relapse, or Infection Following Allogeneic Hematopoietic Cell Transplantation

Background: Identifying factors that influence donor-derived immune response may ultimately enable its therapeutic redirection, lessening risk for complications following allogeneic hematopoietic cell transplantation (alloHCT) like acute graft-versus-host disease (aGvHD) and promoting protection against infection and malignant disease relapse. Viral reactivation seems poised to influence donor-derived immune response, potentially disrupting the balance between immune surveillance in eradicating malignancy and infection and immune tolerance in preventing aGvHD. Cytomegalovirus (CMV) is a clinically-significant virus with immunomodulatory capabilities. However, studies interrogating such effects are limited in the modern transplant era. The primary study aim was to estimate the cumulative incidence of initial CMV reactivation (RA) and aGvHD in alloHCT patients and to assess reciprocal influence between CMV RA and aGvHD. The secondary study aim was to define whether CMV RA predisposed alloHCT patients to infection or increased relapse risk.Methods: Consecutive adult patients (n=324) with acute lymphoblastic leukemia, acute myelogenous leukemia or myelodysplasia whom received initial matched sibling or unrelated donor (MUD) bone marrow (n=33), peripheral blood stem cell (PBSC, n=253) or umbilical cord blood (n=38) grafts from January 2010 through December 2014 at The Ohio State University Comprehensive Cancer Center comprised the study cohort. Patient-, transplant-, and infection-related data were retrospectively analyzed (Table 1). Initial CMV RA was defined as plasma quantitative CMV PCR≥1000 viral copies/ml for which CMV-directed antiviral therapy was started. Microbiologically-documented infections were recorded for the first year after alloHCT and categorized as bacterial blood stream infection, invasive fungal infection, human herpes virus 6 viremia, and respiratory viral infection.Cumulative incidences of CMV RA and aGvHD were estimated accounting for competing risks (death from any cause). Association between CMV RA and incidence of aGvHD was evaluated in a proportional sub-distribution hazards model, where CMV RA was treated as a time-dependent covariate with competing risk as death from any cause. Similarly, influence of aGvHD on incidence of CMV RA was evaluated where development of aGvHD was treated as the time-dependent covariate and CMVR RA as the end point of interest. Associations between CMV RA and subsequent infection or disease relapse were similarly analyzed. To evaluate impact of CMV and aGvHD on long-term outcomes, landmark analysis (LMA) at D100 and D365 were compared among four distinct patient groups: (1) No CMV RA, no aGvHD; (2) CMV RA, no aGvHD; (3) No CMV RA, aGvHD; and (4) CMV RA and aGvHD.Results: Most transplant patients had AML in CR1, received MUD PBSC grafts following myeloablative conditioning, and were given methotrexate and calcineurin-based GvHD prophylaxis. Patients who developed aGvHD grades 2 (HR=1.93, 95% CI 1.15-3.23, p=0.013) or grades 3 and 4 (HR=3.36, 95% CI 1.76-6.45, p<0.001) had higher risk for developing subsequent CMV RA. In contrast, patients with initial CMV RA did not have increased risk for developing future aGvHD. Similarly, CMV RA did not have a significant impact on future infection, regardless of infection type, nor impact subsequent risk of malignant disease relapse.Overall survival (OS) at 1, 2, and 3 years post-transplant were 63%, 53%, and 45%, respectively. Cumulative incidence rates for non-relapse mortality (NRM) at 1, 2, and 3 years were 16%, 19%, and 23%, respectively; for infection-related mortality (IRM), cumulative incidence rates were 10%, 13%, and 16%, respectively. OS, NRM and IRM were stratified by initial CMV RA and aGvHD status at D100 and D365 (Table 2). Among patients who were alive, patients whom had initial CMV RA and aGvHD by D100 were at higher risk for NRM (HR=2.85, 95% CI 1.24-6.56, p=0.014) and IRM (HR=3.65, 95% CI 1.39-9.57, p=0.008) than patients who experienced neither CMV RA nor aGvHD. D365 LMA did not reveal any statistically significant differences in OS, NRM, and IRM between groups.Conclusion: aGvHD associated with increased risk for CMV RA, but initial CMV RA did not associate with subsequent aGvHD risk. Furthermore, initial CMV RA did not associate with increased risk for disease relapse or infection, but did increase NRM and IRM, particularly in combination with aGvHD. [Display omitted] [Display omitted] DisclosuresAuletta:Shire Pharmaceuticals: Consultancy. Lozanski:Genentech: Research Funding; Stemline Therapeutics Inc.: Research Funding; Boehringer Ingelheim: Research Funding; Beckman Coulter: Research Funding. Hofmeister:Janssen: Pharmaceutical Companies of Johnson & Johnson: Research Funding; Karyopharm Therapeutics: Research Funding; Signal Genetics, Inc.: Membership on an entity’s Board of Directors or advisory committees; Incyte, Corp: Membership on an entity’s Board of Directors or advisory committees; Celgene: Research Funding; Arno Therapeutics, Inc.: Research Funding; Takeda Pharmaceutical Company: Research Funding; Teva: Membership on an entity’s Board of Directors or advisory committees. Andritsos:Hairy Cell Leukemia Foundation: Research Funding.

Cytomegalovirus Infection after CD34+-Selected Hematopoietic Cell Transplantation

The effectiveness of preemptive treatment (PET) for cytomegalovirus (CMV) in recipients of ex vivo T cell–depleted (TCD) hematopoietic cell transplantation (HCT) by CD34+ selection is not well defined. We analyzed 213 adults who received TCD-HCT at our institution from June 2010 through May 2014. Patients were monitored by a CMV quantitative PCR assay if recipient (R) or donor (D) were CMV seropositive. CMV viremia occurred early (median, 27 days after HCT) in 91 of 213 (42.7%) patients for a 180-day cumulative incidence of 84.5%, 61.8%, and 0 for R+/D+, R+/D−, and R−/D+ patients, respectively. CMV disease occurred in 5% of patients. In Cox regression analysis, R+/D+ status was associated with increased risk for CMV viremia compared with R+/D− (hazard ratio [HR], 1.79, 95% confidence interval [CI], 1.16 to 2.76, P = .01), whereas matched unrelated donor allograft was associated with decreased risk (HR, .62; 95% CI, .39 to .97, P = .04). Of 91 patients with CMV viremia, 52 (57%) had persistent viremia (>28 days duration). Time lag from detection of CMV viremia to PET was associated with incremental risk for persistent viremia (HR, 1.09; 95% CI, 1.01 to 1.18; P = .03). Overall, 166 of 213 (77.9%) patients were alive 1 year after HCT, with no difference between patients with and without CMV viremia or among the different CMV serostatus pairs (P = not significant). CMV viremia occurred in 70% of R + TCD-HCT. Delay in PET initiation was associated with persistent viremia. With PET, CMV R/D serostatus did not adversely impact survival in TCD-HCT on 1-year survival in the present cohort.

Fever of Unknown Origin in a Kidney Transplant Recipient

A 27-year-old African-American man presented with fever for two days and diarrhea for five days. He had end-stage renal disease due to chronic glomerulonephritis and was 18 months post-deceased donor kidney transplant. He received induction therapy with rabbit anti-thymocyte globulin, and his maintenance immunosuppression consisted of mycophenolate mofetil (MMF), tacrolimus (FK) and prednisone at 500 mg twice a day, 3 mg twice a day, and 7.5 mg once a day, respectively. His FK levels ranged from 6-9 ng/ml in the previous six months. His posttransplant course was complicated by mild acute cellular rejection one week posttransplant treated with pulse steroid therapy. He also had cytomegalovirus (CMV) viremia one year posttransplant, at a low level of 600 copies/ml of blood that disappeared with reduction in MMF dose. He also had a prior living related renal transplant that failed after four years due to chronic rejection. He had lived all his life in Cleveland, OH, and was living with his girlfriend. He denied recent travel out of the state of Ohio. His other medications included sulfamethoxazole-trimethoprim. On review of systems, he admitted to having dry cough that resolved spontaneously within a couple of days. On exam, he appeared to be in mild distress. His chest exam was unremarkable, and his abdominal exam did not reveal any tenderness over the allograft. He did not have any peripheral lymphadenopathy. His white blood cell count on admission was mildly decreased at 4000/mm3, with lymphopenia and normal neutrophil count. A chest X-ray on admission was unremarkable. Blood and urine cultures, CMV quantitative PCR in plasma, ultrasound examination of the transplant kidney, upper gastrointestinal endoscopy, and a CT scan of the abdomen and pelvis were unrevealing. Diarrhea resolved within one day of conversion of MMF to azathioprine, and was followed by constipation. We presumed it was from MMF toxicity. The patient remained febrile for at least 7 days after hospitalization. Empiric antibiotics were begun, including ciprofloxacin, metronidazole, and ganciclovir. Three weeks after initial presentation, a PET/CT scan of the chest was performed to evaluate for any lymphoproliferative disorders. It revealed ground-glass bilateral infiltrates and mediastinal lymphadenopathy (Figure 1 and Figure 2). Flexible bronchoscopy was performed, and the pathology of mediastinal lymph node aspirate is shown in Figure 3.Figure 2PET scan showing FDG-avid areas of activity in both lungs and mediastinal lymph nodes. FDG, fluorodeoxyglucoseView Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Small granular structures in the macrophages of the lymph node (Quick-Diff stain).View Large Image Figure ViewerDownload Hi-res image Download (PPT) 1.The term fever of unknown origin (FUO) in transplant patients should be used only for prolonged fevers with no obvious cause after three consecutive outpatient visits or after three days of hospital evaluation, provided that commonly used imaging and microbiologic tests are reported negative by that time. What is the most likely cause of FUO in our case?a.CMV pneumoniab.Tuberculosis pneumoniac.Posttransplant lymphoproliferative disorderd.Histoplasma pneumoniae.Lung cancer2.Based on Figure 3, what would be the most appropriate special stain to identify the organism?a.Acid-fast stainb.Brown–Hopps gram stainc.Grocott’s methenamine silver staind.Warthin–Starry staine.Dieterle method3.Which of the following features of the patient is NOT a risk factor for development of opportunistic infectious pneumonia?a.Episode of rejectionb.CMV viremiac.T cell depletion inductiond.Young age4.The fi ndings of fluorodeoxyglucose (FDG)-avid mediastinal lymphadenopathy in Figure 1 would NOT be seen in:a.Lung cancerb.Mycobacterium tuberculosis infectionc.Histoplasma infectiond.Posttransplant lymphomae.Drug fever5.Which of the following diagnostic tests has the highest sensitivity for the diagnosis of disseminated histoplasmosis?a.Histopathologyb.Culturec.Serum Histoplasma antigend.Serum Histoplasma antibody6.Which of the following is the best initial choice of treatment for disseminated histoplasmosis?a.Amphotericin Bb.Fluconazolec.Ketoconazoled.Itraconazole

Cytomegalovirus: Why Viral Dynamics Matter

Contrary to bacterial replicating kinetics, where the doubling time varies considerably but often is measured in minutes or hours, viral replication kinetics is calculated in days. A new study by Isabelle Lodding and colleagues in E-Biomedicine attempts to calculate the doubling time of cytomegalovirus (CMV) in a cohort of solid organ and stem cell recipients (Lodding et al., 2015). Why does the doubling time of CMV matter? This number is of great interest to the transplant specialist, as the preemptive treatment approach relies on the detection of replicating CMV in blood before disease occurs. Regular determination of quantitative CMV PCR testing allows selecting those patients with reactivation of CMV in need of a preemptive treatment. Often, a quantitative CMV copy threshold in blood is used to start antiviral treatment. Neither the optimal frequency of CMV testing nor the ideal source of CMV PCR (whole blood, or plasma), nor the optimal threshold has been established. Thus, protocols between transplant centers vary. The recently published updated international consensus guidelines on the management of cytomegalovirus in solid-organ transplantation recommended weekly testing for 3–4 months, with moderate evidence (Kotton et al., 2013). For most centers, however, it would be very difficult to adhere to such a tight schedule, in particular later after transplantation. Therefore, a precise knowledge of the doubling time would allow to safely widen the interval between CMV PCR testing, without an increase in CMV disease episodes. The main finding of the study by Lodding and colleagues is a CMV doubling time of 4.3 days (median, IQR 2.5–7.8), which in contrast to earlier studies is considerably longer. Neither the donor–recipient CMV sero-constellation nor the type of transplant did influence these results. Earlier studies by V. Emery and P. Griffiths in bone marrow transplant patients estimated a shorter doubling time of CMV of 1.5 days (median, range 0.38–4.7) (Emery et al., 1999). Similar doubling times were calculated in a cohort of liver transplant recipients (2 days (median, 0.1–69)) (Nebbia et al., 2007). Many factors may have influenced these different estimates, including the intensity of immunosuppression, the type of sample used for CMV PCR detection, frequency of measurement, type of transplantation, or percentage of patients at high risk for CMV reactivation. While some are less likely than other to play a role, data on the influence of these factors on the doubling time are conflicting. Interestingly, in their simulation model, Lodding and colleagues were able to predict their real rate of recipients with a high CMV viral load (1.4%, arbitrarily set at > 18,200 IU/mL) with the assumption of their lower doubling time of 4.3 days. Using a shorter doubling time of 1.3 days, the proportion of patient with a high viral load would have been 11%. The authors suggest that in cohorts with comparable doubling times, the screening interval can be safely extended. While this may decrease the cost burden due to less visits and screening costs, it should be emphasized that the most important goal is to reduce symptomatic CMV episodes in these vulnerable patients. We and others have shown that many factors including as strict adherence to a guideline with timely start of antiviral therapy in case of reactivation contribute to the success of the preemptive approach (Greiner et al., 2012). Of note, the number of patients with CMV disease was rather high and reached 31% during the observation time in the study by Lodding and colleagues. The basis of any informed decision on how to adapt the screening schedule is a precise knowledge of the rate of CMV disease episodes in its own institution. Such information is crucial to recognize a relevant increase in the rate of CMV disease once changes of the preemptive protocol have been implemented. Any change in clinical routine should be accompanied by a careful evaluation of its effect on the pertinent endpoints. The importance of the role of carefully maintained cohorts such as the one built and used to by Lodding and colleagues to answer such questions in a real-life environment cannot be overstated.

Detection of cytomegalovirus (CMV) by real-time PCR in fecal samples for the non-invasive diagnosis of CMV intestinal disease

BackgroundCytomegalovirus (CMV) infection of the gastrointestinal tract can cause CMV intestinal disease (CMV-ID), a severe complication in immunocompromised patients. Current gold standard for diagnosing CMV-ID requires the analysis of colon biopsies. Testing of fecal samples by CMV PCR might be a non-invasive diagnostic alternative, but data on this method is scarce. ObjectivesTo evaluate the use of quantitative CMV real-time PCR in fecal samples for diagnosing CMV-ID. Study designFecal samples and lower intestinal tract biopsies from 66 patients were analyzed by quantitative CMV PCR. To evaluate the diagnostic significance of CMV detection by PCR in fecal samples, patients were classified according to the etiology of their intestinal disease (based on results of endoscopy, histopathology and quantitative CMV DNA detection in biopsies) into three groups: “CMV-ID”, “non-CMV-ID”, and “equivocal”. Results10/66 fecal samples were tested positive by quantitative CMV PCR, but CMV DNA loads were low (range <1000–11,000copies/ml). CMV detection by PCR in fecal samples was positive in 8/12 patients of the CMV-ID group, resulting in a sensitivity of 67% for diagnosing CMV-ID. With two exceptions, fecal CMV PCR was negative in the non-CMV-ID group (45/47) indicating a good specificity (96%). Moreover, CMV DNA detection in feces was associated with high CMV DNA levels in intestinal biopsies. ConclusionsNegative CMV PCR results from fecal samples cannot exclude CMV-ID and thus have to be confirmed by analyzing intestinal biopsies. However, positive fecal PCR results are diagnostically useful and might help to circumvent invasive diagnostic procedures as endoscopy.

Development of a high-throughput human cytomegalovirus quantitative PCR cell-based assay

This report describes the development and optimization of a quantitative real-time PCR assay for evaluating human cytomegalovirus (CMV) replication in vitro and susceptibility to antiviral drugs. This assay measures the level of intracellular CMV DNA in both 96- and 384-well microplate formats. Normalization of CMV levels using mitochondrial DNA enhanced the robustness of the assay and minimized variability. The assay throughput was further enhanced by eliminating several wash steps and by lysing the cells directly in the presence of cell culture media, both of which had no impact on the assay metrics. The assay was validated using several known CMV antiviral compounds. The CMV quantitative PCR (qPCR) assay represents a rapid, reliable and reproducible method that can be used with both CMV laboratory strains and clinical isolates.

Multicenter Evaluation of a Commercial Cytomegalovirus Quantitative Standard: Effects of Commutability on Interlaboratory Concordance

Commutability of quantitative reference materials has proven important for reliable and accurate results in clinical chemistry. As international reference standards and commercially produced calibration material have become available to address the variability of viral load assays, the degree to which such materials are commutable and the effect of commutability on assay concordance have been questioned. To investigate this, 60 archived clinical plasma samples, which previously tested positive for cytomegalovirus (CMV), were retested by five different laboratories, each using a different quantitative CMV PCR assay. Results from each laboratory were calibrated both with lab-specific quantitative CMV standards ("lab standards") and with common, commercially available standards ("CMV panel"). Pairwise analyses among laboratories were performed using mean results from each clinical sample, calibrated first with lab standards and then with the CMV panel. Commutability of the CMV panel was determined based on difference plots for each laboratory pair showing plotted values of standards that were within the 95% prediction intervals for the clinical specimens. Commutability was demonstrated for 6 of 10 laboratory pairs using the CMV panel. In half of these pairs, use of the CMV panel improved quantitative agreement compared to use of lab standards. Two of four laboratory pairs for which the CMV panel was noncommutable showed reduced quantitative agreement when that panel was used as a common calibrator. Commutability of calibration material varies across different quantitative PCR methods. Use of a common, commutable quantitative standard can improve agreement across different assays; use of a noncommutable calibrator can reduce agreement among laboratories.

CMV Valganciclovir Prophylaxis Versus Preemptive Therapy after Renal Transplantation: Three Year Results of a Randomized Clinical Trial

Background: Prophylaxis and preemptive therapy are competitive approaches to prevent cytomegalovirus (CMV) infection after renal transplantation. Several prospective randomized studies show that in high risk (D+/R-) patients, prophylaxis is the best option for preventing CMV, and that D+/R+ patients may also benefit from prophylaxis. Methods: We performed a randomized clinical trial to compare rates of CMV infection and disease of R+ renal transplant recipients receiving primary CMV prophylaxis to those treated preemptively; and whether this correlates with a higher rate of long-term graft and patient survival. Prophylaxis consisted of 900mg valganciclovir/day adjusted for renal function for 100 days post-transplantation. Patients were monitored with a quantitative CMV PCR test (Cobas® Amplicor® CMV-Monitor) and positive patients (≥ 400 copies/ml) received 1800mg/day valganciclovir adjusted for renal function followed by secondary prophylaxis with 900mg/day valganciclovir for 28 days. Three year data are summarized in this abstract. Results: 296 patients were randomized, 146 receiving prophylaxis and 150 preemptive therapy. At 3 years there were 79 acute rejections in 41 patients vs. 69 acute rejections in 44 patients in the preemptive and prophylaxis groups respectively (p=0.5940). As there were no new active CMV infections after the second year, their percentage remained significantly higher with pre-emptive therapy (38.7% vs. 11.6%, p< 0.0001). 16 of the 17 CMV infections in the prophylaxis group and 6 of the 58 in the preemptive group occurred after 100 days post transplantation. Most CMV infections and highest rate of CMV disease were seen for D+/R+ patients receiving preemptive therapy (53.9% vs. 16.7%, p< 0.0001 for CMV infections and 21.8% vs. 5.6%, p=0.0019 for CMV disease). Two additional graft losses occurred in the preemptive group (10 at 3 years) and none in the prophylaxis group (4 at 3 years) after the second year. Uncensored graft losses were twice as many in the preemptive group (n=17, 11.3%) as in the prophylaxis group (n=8, 5.5%, p=0.0702). In preemptive group patients with active CMV infection death and/or graft loss increased fourfold to 20.7% compared to patients without active CMV infection. A similar increase was not observed in the prophylaxis group (Table 1).[Table 1: Uncensored graft loss at 3 years after Tx]Conclusion: At 3 years post-transplantation, the incidence of active CMV infection in both arms did not increase, preserving the significant benefit for the prophylactic group observed at 2 years. There were twice as many graft losses and/or deaths in patients treated preemptively compared with patients who received valganciclovir prophylaxis. The ongoing study aims to determine if prophylaxis is associated with long-term benefits for R+ patients.