Cytomegalovirus DNA Levels Research Articles

Validation of Preamplification to Improve Quantification of Cytomegalovirus DNA Using Droplet Digital Polymerase Chain Reaction.

Subclinical cytomegalovirus (CMV) replication is associated with strong cellular immune response and chronic inflammation, which could contribute to aging-related conditions such as cardiovascular disease and frailty. However, because of very low levels of CMV DNA present in people with chronic CMV infection, it has been difficult to explore the virologic and immunologic mechanisms of chronic low-level CMV infection and a sensitive method to monitor CMV replication is needed. Droplet digital PCR (ddPCR) has been shown to have higher precision and reproducibility than real-time quantitative PCR (qPCR) in quantifying low levels of CMV DNA, but it is not always sensitive enough for this purpose. Through rigorous validation experiments, we demonstrated that sensitivity and precision of quantification of very low levels of CMV DNA by ddPCR can be significantly increased by preamplification of samples with 10-20 cycles of conventional PCR, especially when testing CMV DNA in the presence of cellular DNA. With preamplification, we could reliably quantify down to two copies of CMV DNA, as opposed to five copies without preamplification. Further studies are needed to determine if ddPCR with preamplification can facilitate mechanistic studies of the characteristics and consequences of chronic CMV infection in aging adults.

Prevalence and correlates of plasma cytomegalovirus viremia among newly reported HIV/AIDS patients in Taizhou city, 2017-2018

Objective: To study the prevalence and correlates of plasma cytomegalovirus (CMV) viremia among newly reported antiretroviral therapy (ART)-naive HIV/AIDS patients in Taizhou during 2017-2018. Methods: CMV DNA was measured in plasma specimens of newly reported ART-naive HIV/AIDS patients by quantitative PCR. Both univariable and multivariable logistic regression analyses were carried out to evaluate CMV viremia correlations among the individuals. Results: Of 612 HIV/AIDS patients, 480 (78.4%) were male, 125 (20.4%) were over 60 years old, 177 (28.9%) were infected via homosexual transmission, and 430 (70.3%) via heterosexual transmission. The prevalence of CMV viremia among HIV/AIDS patients was 13.4% (82/612). Multivariable logistic regression analysis showed that the risk of CMV viremia in CD4+ lymphocyte cells counts (CD4+) ≤200 cells/μl group was higher than CD4 counts >500 cells/μl (OR=5.10, 95%CI:1.74-14.96, P=0.003); The median CMV DNA level (log10) of 82 viremic patients was 1.57 (P25,P75:1.04,2.13); Viremic patients with CD4 counts ≤200 cells/μl had the highest CMV viral load (P<0.01). Conclusions: Among ART-naive HIV/AIDS patients, the prevalence of CMV viremia was significantly associated with immunodeficiency status. Further research is needed to evaluate the association between CMV viremia and the course of HIV infection.

Role of antenatal plasma cytomegalovirus DNA levels on pregnancy outcome and HIV-1 vertical transmission among mothers in the University of Zimbabwe birth cohort study (UZBCS)

IntroductionDespite being a leading infectious cause of childhood disability globally, testing for cytomegalovirus (CMV) infections in pregnancy is generally not done in Sub-Sahara Africa (SSA), where breastfeeding practice is almost universal. Whilst CMV and human immunodeficiency virus (HIV) are both endemic in SSA, the relationship between antenatal plasma CMV-DNA, HIV-1-RNA levels and HIV-1-mother to child transmission (MTCT) including pregnancy outcomes remains poorly described.MethodsPregnant women at least 20 weeks’ gestational age at enrolment were recruited from relatively poor high-density suburbs in Harare, Zimbabwe. Mother-infant dyads were followed up until 6 months postpartum. In a case–control study design, we tested antenatal plasma CMV-DNA levels in all 11 HIV-1 transmitting mothers, as well as randomly selected HIV-infected but non-transmitting mothers and HIV-uninfected controls.CMV-DNA was detected and quantified using polymerase chain reaction (PCR) technique. Antenatal plasma HIV-1-RNA load was quantified by reverse transcriptase PCR. Infants’ HIV-1 infection was detected using qualitative proviral DNA-PCR. Predictive value of antenatal plasma CMV-DNAemia (CMV-DNA of > 50 copies/mL) for HIV-1-MTCT was analyzed in univariate and multivariate regression analyses. Associations of CMV-DNAemia with HIV-1-RNA levels and pregnancy outcomes were also explored.ResultsCMV-DNAemia data were available for 11 HIV-1 transmitting mothers, 120 HIV-infected but non-transmitting controls and 46 HIV-uninfected mothers. In a multivariate logistic regression model, we found a significant association between CMV-DNAemia of > 50 copies/mL and HIV-1 vertical transmission (p = 0.035). There was no difference in frequencies of detectable CMV-DNAemia between HIV-infected and -uninfected pregnant women (p = 0.841). However, CMV-DNA levels were higher in immunosuppressed HIV-infected pregnant women, CD4 < 200 cells/µL (p = 0.018). Non-significant associations of more preterm births (< 37 weeks, p = 0.063), and generally lower birth weights (< 2500 g, p = 0.450) were observed in infants born of HIV-infected mothers with CMV-DNAemia. Furthermore, in a multivariate analysis of HIV-infected but non-transmitting mothers, CMV-DNAemia of > 50 copies/mL correlated significantly with antenatal plasma HIV-1-RNA load (p = 0.002).ConclusionAntenatal plasma CMV-DNA of > 50 copies/mL may be an independent risk factor for HIV-1-MTCT and higher plasma HIV-1-RNA load, raising the possibility that controlling antenatal CMV-DNAemia might improve infant health outcomes. Further studies with larger sample sizes are warranted to confirm our findings.

Investigation of the effect of chemotherapy on cytomegalovirus reactivity in patients with solid organ tumors

Objectives: Chemotherapy induces an immunosuppressive state in patients with solid organ tumors. Cytomegalovirus (CMV) reactivation as a result of immunosuppression causes a severe clinical manifestation. However, in this group, CMV infections developing due to reactivation were not adequately discussed in the literature. The aims of this study were to determine the incidence of CMV reactivation after chemotherapy, to evaluate the contribution of chemotherapy to reactivation, to determine the incidence of asymptomatic and symptomatic infections and to investigate the results of the treatment.Methods: A total of 93 patients with solid tumors were included in the study. Weekly blood samples were collected from the patients for three weeks before and after chemotherapy. Quantitative analysis of DNA was detected using CMV PCR kit (GeneProof CMV PCR kit, Bruno, Czech Republic). Diagnosis and treatment of patients were retrospectively reviewed.Results: Of the patients, 65.6% were female and 34.4% were male. The mean age was 55 ± 12 years. The most common cancer types among the patients were breast cancer in 45.2%, lung cancer in 15.1%, and colon cancer in 12.9%. The mean leukocyte count of the patients was 7,647/mm3. CMV DNA was not detected in any patient. According to this result, none of the patients had CMV reactivation after chemotherapy. Conclusions: In this study including patients with solid organ tumors with mild to moderate level of immunosuppression CMV DNA was not detected in any patient. Based on this finding no standard prophylaxis was required for CMV in this group of patients.

Cervical cytomegalovirus reactivation, cytokines and spontaneous preterm birth in Kenyan women.

Genital cytomegalovirus (CMV) reactivation is common during the third trimester of pregnancy. We hypothesized that cervical CMV shedding may increase risk of spontaneous preterm birth (sPTB) through the release of inflammatory cytokines in the cervix. We conducted a nested case-control analysis to determine the relationship between CMV shedding and sPTB using data and samples from a prospective cohort study in western Kenya. Women who delivered between 28+0 and 33+6weeks gestation were matched by gestational age at sample collection to controls who delivered ≥37+0weeks. Levels of CMV DNA and interleukin (IL)-1 beta (β), IL-6, IL-8 and tumor necrosis factor (TNF)-α were measured in cervical swabs. We used conditional logistic regression to assess relationships between CMV shedding, cervical cytokine levels and sPTB. Among 86 cases and 86 matched controls, cervical CMV levels were not significantly associated with sPTB [odds ratio (OR)=1·23, 95% confidence interval (CI)=0·59-2·56], but were significantly associated with higher levels of cervical IL-6 (β=0·15, 95% CI=0·02-0·29) and TNF-α (β=0·14, 95% CI=0·01-0·27). In univariate analysis, higher odds of sPTB was associated with higher cervical IL-6 levels (OR=1·54, 95% CI =1·00-2·38), but not with other cervical cytokines. In this cohort of Kenyan women, we did not find a significant association between cervical CMV shedding and sPTB before 34 weeks.

S2852 Tissue Invasive Cytomegalovirus Enteritis With Gastric Outlet Obstruction in an Immunocompetent Patient

INTRODUCTION: Cytomegalovirus (CMV) usually causes asymptomatic or mononucleosis-like syndrome in immunocompetent individuals. We present a case of severe tissue invasive gastrointestinal CMV in an immunocompetent individual complicated by gastric outlet obstruction. CASE DESCRIPTION/METHODS: A 72-year-old man with a history of hypertension presented with vomiting, postprandial abdominal pain, and hematochezia for one week. He had also lost 40lbs in three months. Physical examination was normal except for epigastric tenderness. The previous colonoscopy done 5 years ago was normal. No personal or family history of inflammatory bowel disease, gastrointestinal malignancy, or lymphoma. He had never had immunosuppressive therapy, organ transplantation, or any other biological agent. Initial blood work revealed Hemoglobin 10.9 g/dl, AST 64 IU/l, ALT 83 IU/l, and ALP 350 IU/l. His HIV and Tuberculosis screening were negative. Ultrasound and CT scan of the abdomen and pelvis were normal except for increased gallbladder wall thickness (3mm). An Esophagogastroduodenoscopy (EGD) revealed esophagitis, pyloric channel ulcer, and duodenal ulcer. Colonoscopy revealed an ulcer at the ileocecal valve. The biopsy of all the ulcers revealed ulcerated mucosa, architectural distortion, and reactive epithelial atypia suspicious of CMV infection which was later confirmed by immunohistochemical staining (IHC) [Figure 1]. Quantitative CMV DNA PCR analysis showed levels of 1739 IU/ml. He was started on intravenous ganciclovir while inpatient and sent home on oral valganciclovir for 21 days. His symptoms improved significantly after treatment. CMV DNA levels decreased to 1230 IU/ml post-treatment. The patient returned with symptoms of gastric outlet obstruction a few months later which were getting worse. His barium X-ray revealed a delayed gastric emptying time with pyloric stenosis and partial malrotation of the small bowel [Figure 2]. A repeat EGD revealed pyloric stenosis with an inner diameter of 11 mm, which was dilated endoscopically. The biopsy taken from an antral ulcer was negative for CMV on IHC staining. There were no other signs of prior CMV infection on the EGD. DISCUSSION: The gastric outlet obstruction in the patient is likely a long term sequela of CMV. Due to the undertreatment and delay in diagnosis, CMV infections can lead to long term and potentially life-threatening complications. Therefore, gastrointestinal CMV should be considered in immunocompetent patients and aggressively treated.Figure 1.: Medium power (100×) Immunohistochemical staining for CMV with hematoxylin counter-stain. A representative high power view demonstrating strong focal CMV immunoreactivity.Figure 2.: Barium Xray reveals pyloric stenosis (black arrow) and malrotation with the first loop of jejunum to the right.

Metagenomic analysis and identification of emerging pathogens in blood from healthy donors

Emerging infectious pathogens that threaten blood transfusions are known to be present in blood samples from healthy/qualified donors. The objective of this study was to investigate the microbiome of blood from healthy donors from the Luzhou area in southwestern China. Potential pathogens and cytomegalovirus (CMV) infection in the donor blood were identified. Total plasma nucleic acids were extracted from one pool of 5734 samples and were constructed for metagenomics analysis using Illumina sequencing. The microbiome and potential emerging/re-emerging pathogens were identified using bioinformatics analysis. Moreover, CMV antigen was measured via an enzyme-linked immunosorbent assay, and the CMV DNA level was assessed by quantitative RT-PCR. A total of 132 bacterial reads, 65 viral reads and 165 parasitic reads were obtained. The most frequent bacterium was Escherichia coli (95/132, 72%) with 95 reads in 132 bacterial reads, and the most prevalent parasite was Toxoplasma gondii (131/165, 79%). Among the viruses, cytomegalovirus (44/65, 68%) accounted for the highest frequency, followed by Hepatitis E Virus (10/65, 15%). Moreover, the positive rate of CMV-IgG was 46.25% (2652/5734), and the positive rate of CMV-IgM was 5.82% (334/5734). The positive rate of dual positive (IgG+ and IgM+) CMV was 0.07% (4/5734). Twenty-one (0.37%) specimens from 5734 donated blood samples were positive for CMV DNA. The CMV DNA levels ranged from 7.56 × 102 to 3.58 × 103 copies/mL. The current study elucidated the microbiome structure in blood from healthy/qualified donors in the Luzhou area and identified emerging/re-emerging pathogens. This preliminary study contributes to information regarding blood transfusion safety in China.

Prognostic factors of cytomegalovirus retinitis after hematopoietic stem cell transplantation.

To identify the visual prognostic factors in patients with cytomegalovirus (CMV) retinitis after hematopoietic stem cell transplantation (HSCT). This retrospective cohort study included 4241 patients who underwent HSCT from April 1, 2010 to March 31, 2019 at Seoul St. Mary's Hospital. Of them, 1063 patients presented CMV viremia, and 67 patients (93 eyes) were diagnosed with CMV retinitis. We enrolled 66 patients (91 eyes). The main outcomes included the initial best-corrected visual acuity (BCVA), BCVA at the diagnosis of retinitis and last visit, involved retinal zone, peak CMV DNA levels in the peripheral blood and aqueous humor, time between HSCT and the diagnosis of retinitis, time between the diagnosis of viremia and retinitis, complications, recurrence, survival, and so on. The mean BCVA (logarithm of the minimum angle of resolution) values before HSCT, at the time of retinitis diagnosis, and at the last visit were 0.041 ± 0.076, 0.262 ± 0.529, and 0.309 ± 0.547, respectively. Multiple regression analysis revealed that the involved zone (P = 0.001), time between HSCT and retinitis diagnosis (P = 0.019), and survival status (P = 0.001) were associated with the final visual acuity. The final visual prognosis was worse in patients with greater invasion of the central retinal zone, those with a longer interval between HSCT and the diagnosis of retinitis, and those who died. Prompt diagnosis of CMV retinitis through periodic fundus examinations of patients with CMV viremia can prevent severe vision loss. Once CMV viremia is confirmed, we recommend fundus examinations to be immediately performed and repeated every 2 weeks for at least 2 months, even if the CMV DNA titer in the peripheral blood becomes negative.

Cytomegalovirus DNA load monitoring in stool specimens for anticipating the occurrence of intestinal acute graft-versus-host disease following allogeneic hematopoietic stem cell transplantation: Is it of any value?

Data have been published suggesting a bidirectional interaction between cytomegalovirus (CMV) infection and acute graft-versus-host disease (aGvHD) in allogeneic hematopoietic stem cell transplant (allo-HSCT) recipients. Here, we hypothesized that prospective CMV DNA monitoring in stool specimens may be useful for predicting subsequent occurrence of intestinal aGvHD (IaGvHD). This two-center study enrolled 121 consecutive adult patients undergoing any modality of allo-HSCT. A total of 1,009 stool specimens were collected (a median of 7 specimens/patient; range, 1-18). CMV DNA monitoring in stools and plasma was performed using real-time PCR assays. CMV DNA was detected in stools in 20 patients (cumulative incidence, 16.9%; 95% CI, 6.3%-31.8%). Median CMV DNA level in stool specimens was 1,258IU/0.1g (range, 210-4,087IU/0.1g). All these patients and their donors were CMV seropositive, and 16 of the 20 patients also had CMV DNAemia, while 4 patients had CMV DNA detected in stools without CMV DNAemia. No correlation was found between CMV DNA loads in plasma and stools (P=.40). Prior CMV DNAemia, aGvHD, or IaGvHD were not associated with presence of CMV DNA in feces. IaGvHD was present in 30 patients, in 5 of whom CMV DNA was detected in stools. Neither detection of CMV DNA in feces nor in plasma was associated with subsequent IaGvHD (OR, 0.67; 95% CI, 0.18-2.52; P=.55 and OR, 0.86; 95% CI, 0.38-1.96; P=.71, respectively). No patient in this cohort had CMV end-organ disease within the study period. Our study failed to provide evidence pointing to a reciprocal interaction between GI CMV infection and IaGvHD. CMV DNA monitoring in stools seems of no value to anticipate occurrence of IaGvHD.

Immune Monitoring Using QuantiFERON®-CMV Assay in Congenital Cytomegalovirus Infection: Correlation With Clinical Presentation and CMV DNA Load.

Cytomegalovirus (CMV)-specific CD8 + T-cell responses can be detected early in fetal life, but their role in the manifestations of congenital CMV (cCMV) infection remains largely unknown. CMV-specific CD8 + T-cell responses were assessed in neonates with cCMV using QuantiFERON®-CMV assay, within day 14 of life (T0) and during the second month of life (T1). Detection and quantification of CMV DNA in whole blood and urine samples were performed at both time points. QuantiFERON®-CMV results were evaluated in relation to timing of maternal infection, clinical manifestations of cCMV and CMV DNA levels. Thirty neonates were enrolled (10/30 [33%] symptomatic; 20/30 [67%] asymptomatic). At T0 16/30 (53%) subjects had a reactive QuantiFERON®-CMV result and 16/16 (100%) were asymptomatic, whereas 14/30 (47%) had a nonreactive or indeterminate QuantiFERON®-CMV result and 4/14 (29%) were asymptomatic. At T1, 17/29 (59%) subjects had a reactive QuantiFERON®-CMV result, and 17/17 (100%) were asymptomatic, whereas 12/29 (41%) had a nonreactive or indeterminate result and 3/12 (25%) were asymptomatic. At both T0 and T1 reactive QuantiFERON®-CMV results correlated with lack of symptoms (P = .0001). At T1 median CMV DNAemia was lower in subjects with reactive QuantiFERON®-CMV results as compared with subjects with nonreactive or indeterminate results (1.82 log IU/mL [1.82-2.89] vs 2.55 log IU/mL [1.82-4.42], P = .009). No correlation was found between QuantiFERON®-CMV results and gestational age at maternal infection nor with urine CMV DNA levels. A detectable CMV-specific CD8 + T-cell response, evaluated using the QuantiFERON®-CMV assay, correlates with the lack of CMV-related symptoms and the control of CMV DNAemia.

Regime for cytomegalovirus retinitis based on aqueous virology and inflammatory cytokine determination

Objective To observe the safety and efficacy of regime that based on aqueous cytomegalovirus-DNA (CMV-DNA) load and IL-8 determination for therapeutic monitoring and local treatment cessation of cytomegalovirus retinitis (CMVR) patients after allogeneic hematopoietic stem cell transplantation (HSCT). Methods A prospective case series study. A total of 14 CMVR patients (22 eyes) after allogeneic HSCT diagnosed in Ophthalmology Department of Peking University People's Hospital between January 2016 and December 2018 were involved in this study. All patients were CMV-DNA seronegative at baseline and were treated with intravitreous injection of ganciclovir (IVG, 3 mg in 0.05 ml) twice per week for 4 times in the induction stage and once a week in the maintenance stage. Aqueous humor sample was collected during the first time of IVG every week. CMV-DNA and the level of IL-8 were measured by real time quantitative PCR and ELISA, respectively. During follow-up, negative CMV-DNA (<103/ml) or level of IL-8<30 pg/ml in aqueous sample was set as local treatment cessation. Then patients were followed every 2 weeks for at least 6 months. BCVA, intraocular pressure and fundus examination were taken for each visit. The BCVA examination was performed using the international standard visual acuity chart, which was converted into logMAR visual acuity. BCVA and intraocular pressure at the baseline and the last follow-up were compared by the Student t matching test. Results Of the 14 CMVR patients (22 eyes) after allogeneic HSCT, 8 patients (16 eyes) were bilateral, 6 patients (6 eyes) were unilateral. At the baseline, the mean logMAR BCVA was 0.814±0.563, the intraocular pressure was 17.2±7.8 mmHg (1 mmHg=0.133 kPa), the mean aqueous CMV-DNA load was (3.43±4.96)×105/ml, the mean level of IL-8 was 518±541 pg/ml. At cessation of local treatment, the median number of intravitreal injections was 5 times. Nine eyes showed negative CMV-DNA in aqueous humor, of which, 7 eyes showed negative IL-8 in aqueous. CMV-DNA could still be detected in 13 eyes, while IL-8 was negative. Only one eye’s retinal lesion was completely quiet. Six months after local treatment cessation, the mean logMAR BCVA was 0.812±0.691, the intraocular pressure was 14.8± 5.4 mmHg; which was not significantly different from baseline (t=-0.107, 1.517; P=0.916, 0.137). Recurrence of CMVR happened in only 1 eye because of systemic EB virus infection. Retinal lesions progressively improved and became completely quiet in all the remaining 20 eyes. In 22 eyes, iatrogenic vitreous hemorrhage occurred due to low platelet count during treatment (<30×109/ml) in 4 eyes. When the treatment was terminated for 6 months, the fundus of hematoma absorption was clearly visible. At the time of CMVR diagnosis, there were 2 eyes (9%) with posterior subcapsular opacity, which may be caused by systemic glucocorticoid therapy after allogeneic HSCT. Conclusion Aqueous CMV-DNA load and level of IL-8 could be used as quantitative variables for monitoring the therapeutic effect and determining time for local treatment cessation for CMVR after HSCT safely and efficiently. Key words: Cytomegalovirus retinitis; Aqueous humor; Virology; Intereukin-8

A Comparison of Different Valgancyclovir Formulations in the Universal 6-Month Prophylaxis Against CMV Infection in Renal Transplant Recipients: A Randomized Single-Centre Study.

Cytomegalovirus (CMV) is the most common opportunistic infective pathogen in kidney transplant recipients. Valganciclovir (VAL) is commonly used for prophylaxis, especially in high-risk recipients. Generic VAL formulations have become available, but the data about their safety and efficacy are lacking. Consecutive de novo kidney transplant patients were randomized to generic VAL Valganciklovir Teva® (VT group)(24 patients) or Alvanocyte® (A group), Alvogen (19 patients) or to Valcyte® (V group), Roche (23 patients) in a 18-month open-label study. Universal prophylaxis was used for 6 months after the transplantation. CMV DNA levels were measured at 1,3,6,9,12 and 18 months after the transplantation. All positive measurements of CMV DNA were recorded. Groups did not differ regarding the clinical characteristics or the risk for developing CMV infection in the post-transplant period. CMV replications were most common at 9 months after the transplantation with rates of 9% for the V, 13% for the VT and 26% for the A group (p=0.26). At 12 months, positive CMV DNA was recorded in 22%, 8% and 11 % of patients taking V, VT and A, respectively (p=0.37). Rates of biopsy-proven acute rejection, adverse events, and serious adverse events were similar for all formulations. Lymphocele occurred most commonly in the V group (35%) compared to 17% in VT and 17% in the A group (p=0.23). One patient from each of the A and VT groups developed CMV disease. Additionally, they were the only two patients with CMV DNA copies above 656 IU/ml. Glomerular filtration rates were similar in all groups at all time points, while proteinuria was significantly higher at 12 months in patients who received V 0.32 g/day (0.18 - 0.42), compared to patients on VT 0.2 (0.1 - 0.2), or A 0.2 (0.2 - 0.3), p=0.04. Valgancyclovir efficacy and safety in this limited data set is similar with early administration of V, VT and A after kidney transplantation. Additional studies aimed at elucidating the effectiveness of this treatment regimen in patients who are at high risk for developing CMV infection are necessary to draw further conclusions.

The use of CMVIg rescue therapy in cardiothoracic transplantation: A single‐center experience over 6 years (2011‐2017)

Cytomegalovirus (CMV) is the most important infectious agent in solid organ transplant recipients and has a major impact on morbidity and mortality. Most cases are well managed with antiviral agents, but CMV hyperimmune globulin (CMVIg) can be used alongside antiviral therapy for prophylaxis in high-risk thoracic organ recipients and to treat life-threatening CMV infection or disease. CMVIg may also improve antiviral host defences when genetic resistance to antivirals or unwanted side effects occur. In this single-center, retrospective study, we reviewed the CMVIg use to supplement antiviral therapy as a "rescue therapy" in cardiothoracic transplant recipients. These comprised 12 single lung, 11 double lung, and 12 heart transplant recipients. Patients received a median of 2 doses of CMVIg, most often in combination with ganciclovir or valganciclovir, and reduced immunosuppression. One week after rescue therapy was initiated, CMV DNA levels were significantly reduced, and after four weeks, CMV DNA was undetectable in 73% patients. Only one patient died as a result of CMV-related disease. No significant adverse effects were observed. We conclude that CMVIg rescue therapy is safe, well tolerated, and effective at controlling viral replication in cardiothoracic transplant recipients.

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.

PS1270 HIGH INCIDENCE OF CYTOMEGALOVIRUS (CMV) REACTIVATION IN HEMATOLOGICAL PATIENTS TREATED WITH BENDAMUSTINE BASED CHEMOTERAPY REGIMENS

Background:Bendamustine (BENDA) alone or in combination with other drugs is being increasingly used in the treatment of chronic lymphocytic leukaemia (CLL), non‐Hodgkin lymphomas (NHL), and multiple myeloma (MM) due to its safety and efficacy. Variable infection rates both in BENDA monotherapy and in BENDA‐containing regimens have been documented. Limited data are available on cytomegalovirus (CMV) reactivations and infections in patients with hematologic malignancies undergoing BENDA therapy.Aims:We retrospectively analysed for CMV reactivation 120 consecutive patients who underwent therapy with BENDA alone (6%), combined with rituximab (81.5%) and combined with rituximab and dexamethasone. Our cohort included 95 B‐NHL (49 indolent e 46 aggressive B‐LNH), 5 T‐NHL, 18 CLL and 2 HL with a median age of 72 years (range 47–88) and a female to male ratio of 0.67 (female/male: 48/72). BENDA was used as first line therapy in 94 patients (78%) and as a salvage therapy in 26 patients (22%).Methods:CMV monitoring was performed on plasma samples by a real‐time TaqMan CMV‐DNA polymerase chain reaction (PCR) assay, according to the manufacturers’ recommendations (Roche). Quantitative CMV DNA was detected every 3 weeks before starting the next BENDA cycle, while CMV DNA monitoring was performed once a week in cases of CMV reactivation defined by plasma CMV DNA levels higher than 137 IU/ml.Results:CMV reactivation was detected in 29 patients (24%) among the 120 patients analysed for CMV‐DNA, with a median CMV peak viral load of 1835 UI/ml. Reactivation occurred after 55 days (range: 11–313 days) since the first BENDA administration in 27 B‐LNH (10 aggressive and 17 indolent B‐LNH), 1 T‐LNH and 1 CLL.Reactivation rates were different in each BENDA protocols: 1/7 (14.3%) in patients treated with BENDA, 21/98 (28.6%) in patients treated with R‐BENDA and 7/15 (46.7%) in patients treated with RD‐BENDA. All patients showed a deep lymphocytopenia at the time of CMV reactivation with a median of 345 lymphocytes/mm3.We furthermore analyzed potential risk factors for CMV reactivation during a BENDA containing treatment such as: age of the patients, diagnosis, type of BENDA regimen used, CMV‐specific IgG levels, nadir of total lymphocytes and total IgG during treatment, T CD4+ lymphocytes levels.The nadir of total lymphocytes and total IgG resulted significantly associated to CMV reactivation in a multivariate analysis (P‐value = 0.0001 and P‐value = 0.0008, respectively).Summary/Conclusion:Using these variables, we built a risk score for CMV reactivation which can help the physician to assess the risk of CMV reactivation in each patient and eventually start CMV‐DNA monitoring, prophylaxis or preemptive therapy.This single‐center study provides further evidence that BENDA regimens for lymphoproliferative diseases are associated with a high CMV reactivation rate and therefore a strict surveillance for CMV is required during their use.

Cytomegalovirus Infection May Trigger Adult-Onset Still's Disease Onset or Relapses.

Previous studies have revealed that several micro-organisms, especially DNA viruses, have been associated with adult-onset Still's disease (AOSD). However, there are no studies on the relationship between the presence of viral infections in AOSD patients with disease occurrence and reactivation. In the present study, we aimed to investigate the presence of antibodies against virus, virus DNA load and nucleic acid sensors in AOSD patients. Anti-viral antibodies were measured by enzyme-linked immunosorbent assay (ELISA) in plasma samples from 100 AOSD patients and 70 healthy controls (HCs). The copy number of cytomegalovirus (CMV) DNA in 100 AOSD patients was detected by PCR. The expression levels of nucleic acid sensors interferon gamma-inducible protein 16 (IFI16) and absent in melanoma 2 (AIM2) in peripheral blood mononuclear cell (PBMC) and skin from AOSD patients and HCs were analyzed by PCR and immunohistochemistry. The levels of antibodies against CMV were significantly higher in AOSD patients compared to HCs. Moreover, the level of anti-CMV IgM antibody was significantly increased in patients with fever, sore throat, arthralgia and rash. CMV DNA was found in plasma of AOSD patients with disease new-onset and relapse. Furthermore, the copy number of CMV DNA significantly increased in patients with fever, sore throat, arthralgia and rash. And the significant associations of the CMV DNA level with the levels of leukocytes, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) and tumor necrosis factor-α (TNF-α) were observed. Moreover, we found an upregulation of cytoplasmic DNA-sensing receptor IFI16 and AIM2 in PBMC and skin from AOSD patients. In conclusion, our results showed that CMV infection may play a role in the initiation or amplification of inflammatory responses in AOSD.

Association of Cytomegalovirus DNA and Immunologic Markers of Cardiovascular Disease.

BackgroundPersons living with human immunodeficiency virus (HIV) (PLWH) with high cytomegalovirus (CMV)–specific interferon (IFN) γ response have increased numbers of endothelium homing receptor (CX3CR1)+–expressing cells that are associated with cardiovascular disease. The current study was performed to investigate the effect of cellular levels of CMV DNA on these markers.MethodsEighty paired peripheral blood mononuclear cell samples were collected ≥12 months apart from 40 CMV-seropositive PLWH with suppressed HIV RNA, who started antiretroviral therapy at median of 3-months of infection. The samples were assessed for CMV-specific IFN-γ response by means of enzyme-linked immunospot assay, and participants were classified as low responders (LRs) or high responders (HRs) based on IFN-γ production (≤100 or >100 spot-forming units [SFUs]/105 cells).ResultsOf the 40 participants, 26 (65%) were HRs and 14 (35%) LRs at baseline, which did not change over time or by CMV levels (median at first/second time points, 383/308 SFUs/106 cells for HRs vs 21/41 SFUs/106 for LRs). A decrease in IFN-γ over time was associated with higher CMV DNA levels (P < .01). High CMV response was also associated with increased CD28+CD27−CD4+ T cells expressing CX3CR1 (P < .001). Similarly, increased IFN-γ production was associated with increased CMV-specific CX3CR1+CD28+CD27−CD4+ and CD8+ T cells (P < .001).ConclusionsThese findings demonstrate that levels of CMV-specific IFN-γ response in PLWH are stable over time, and that HRs have increased circulating T cells expressing CX3CR1 that may put them at increased risk of cardiovascular disease and other inflammatory diseases.

Evaluation of rapid and sensitive DNA extraction methods for detection of cytomegalovirus in dried blood spots

BackgroundDried blood spots (DBS), collected universally from newborns in the U.S., could be used as a matrix for the detection of cytomegalovirus (CMV) infection in infants. However, sensitivity to detect CMV in DBS as compared to saliva and urine is variable across studies largely due to the DNA extraction method. Thermal shock, a widely used DNA extraction method, is highly sensitive for the detection of CMV in DBS, however, the processing time required is not practical for high-throughput testing. ObjectiveTo determine if rapid and cost-effective DNA extraction methods amenable to newborn screening (NBS) could achieve the same sensitivity as the thermal shock method. Study designDBS were prepared from CMV positive blood samples from 20 organ transplant recipients. Three DNA extraction methods were compared for relative yield and sensitivity of detection of CMV DNA: thermal shock, KOH Tris buffer, and DNA Extract All. CMV DNA was detected by real-time quantitative polymerase chain reaction (qPCR). ResultsThe KOH Tris and DNA Extract All methods gave higher yields and sensitivity of CMV detection in DBS than thermal shock, which were significantly greater when viral loads were ≤ 10,000 copies/ml blood. Both methods gave faster turnaround times than thermal shock and would be better suited for NBS. ConclusionsThe choice of DNA extraction method greatly influences the ability to detect low levels of CMV DNA in DBS. Moreover, development of highly sensitive yet rapid methods for CMV detection could help facilitate future newborn screening of CMV in DBS.

Repurposing Nilotinib for Cytomegalovirus Infection Prophylaxis after Allogeneic Hematopoietic Stem Cell Transplantation: A Single-Arm, Phase II Trial

Platelet-derived growth factor receptor-alpha (PDGFRa) is a critical receptor for cytomegalovirus (CMV) entry into cells, leading to subsequent infection. This trial tested whether PDGFRa inhibition by nilotinib could prevent CMV infection in patients after allogeneic stem cell transplantation (allo-HSCT). Nilotinib (200 mg/day) was given continuously after engraftment, and plasma CMV DNA levels were monitored weekly. The primary endpoint was successful prophylaxis of CMV infection, defined as plasma CMV DNA copies less than 10,000 copies/mL, no anti-CMV treatment initiated, and no clinical CMV disease by day 100. All 37 enrolled recipients and their donors were CMV seropositive. Thirty patients received matched sibling transplants, 15 received nonmyeloablative conditioning regimens, and 15 received antithymocyte globulin as a part of graft-versus-host disease prophylaxis. The median interval from transplantation to nilotinib treatment was 23 days, and the median duration of administration was 76 days. None of the 31 assessable patients had nilotinib-associated grade 3/4 adverse events or nilotinib discontinuation. Twenty-five of 31 assessable patients (80.6%) fulfilled the predefined criteria for successful CMV prophylaxis, and none of them had clinical CMV disease. Only 1 of 6 failed patients developed CMV colitis. Nilotinib is well tolerated in allo-HSCT recipients, and its preliminary efficacy results suggest that blocking CMV entry to prevent CMV infection may warrant further exploration. (ClinicalTrials.gov identifier: NCT01252017.)

Cytomegalovirus and Epstein-Barr Virus DNA Kinetics in Whole Blood and Plasma of Allogeneic Hematopoietic Stem Cell Transplantation Recipients

Currently, no consensus has been reached on the optimal blood compartment to be used for surveillance of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) DNAemia. Although several comparative studies have been performed correlating CMV and EBV DNA loads in whole blood (WB) versus plasma, to our knowledge, no studies to date have analyzed the kinetics of both viruses in the 2 blood compartments. In this retrospective noninterventional multicenter cohort study, the kinetics of CMV and EBV DNA in 121 hematopoietic stem cell transplantation (HSCT) recipients were investigated by analyzing in parallel 569 and 351 paired samples from 80 and 58 sequential episodes of CMV and EBV DNAemia, respectively. Unlike previous studies, this study used a single automated molecular method that was CE-marked and Food and Drug Administration-approved for use in quantifying CMV and EBV DNA in both plasma and WB. Furthermore, the complete viral replication kinetics of all episodes (including both the ascending and the descending phases of the active infection) was examined in each patient. The previously observed overall correlation between CMV DNA levels in WB and plasma was confirmed (Spearman's ρ = .85; P < .001). However, although WB and plasma CMV DNAemia reached peak levels simultaneously, in the ascending phase, the median CMV DNA levels in plasma were approximately 1 log10 lower than WB. Furthermore, in patients who received preemptive therapy, CMV DNA showed a delayed decrease in plasma compared with WB. A lower correlation between EBV DNA levels in plasma versus WB was found (Spearman's ρ = .61; P < .001). EBV DNA kinetics was not consistent in the 2 blood compartments, mostly due to the lower positivity in plasma. Indeed, in 19% of episodes, EBV DNA was negative at the time of the EBV DNA peak in WB. Our results suggest a preferential use of WB for surveillance of CMV and EBV infection in HSCT recipients.