Plasma Polymerase Chain Reaction Research Articles

Clinical Significance of Epstein-Barr Virus in Peripheral T-Cell Lymphoma, Not Otherwise Specified

Introduction: The prognostic implication of Epstein-Barr virus (EBV) in patients with peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) is unclear. This study aimed to identify its clinical impact on survival outcomes in newly diagnosed patients with PTCL-NOS. Methods: Between January 2007 and March 2020, 145 patients were newly diagnosed with PTCL-NOS at Asan Medical Center, Seoul, Korea. A total of 131 patients who underwent baseline blood real-time polymerase chain reaction (PCR) for EBV-DNA were included in this analysis. Real-time blood PCR for EBV-DNA was performed using whole blood (WB) or plasma samples prior to treatments. All patients were tested for WB EBV-DNA, of which 52 were also tested for plasma EBV-DNA. In addition, the presence of EBV was assessed by EBV-encoded small RNA (EBER) in situ hybridization (ISH) in tissue samples from 121 patients. Progression-free survival (PFS) was defined as time from the date of diagnosis to disease progression or death of any cause, and overall survival (OS) was defined as time from the date of diagnosis to death of any cause. Survival curves were estimated by Kaplan-Meier method and compared using log-rank test. Results: The median age at diagnosis was 60 years (range, 19-86 years), 84 patients (64.1%) were male, and 108 patients (82.4%) had advanced stage diseases (stage III or IV). A total of 55 patients (42.0%) were positive for WB EBV-DNA in the entire cohort (n=131), 9 patients (17.3%, 9/52) were positive for plasma EBV-DNA, and 59 patients (48.8%, 59/121) were positive for EBER ISH. Among 52 patients who were tested for both WB and plasma PCR, 16 patients (30.8%) showed discordant results, all of them with positive results for WB PCR but negative results for plasma PCR. There was no significant difference in baseline characteristics between EBV positive and negative groups according to WB EBV-DNA (n=131) and EBER ISH (n=121), while higher proportion of patients with positive plasma EBV-DNA had bone marrow involvement (77.8% vs. 23.3%, p = 0.003) and thrombocytopenia (37.5% vs. 7.5%, p = 0.050) compared to patients with negative plasma EBV-DNA. With a median follow-up duration of 80.5 months (95% confidence interval [CI], 63.8-130), the median PFS and OS of the entire cohort were 8.1 months (95% CI, 5.8-10.2) and 18.6 months (95% CI, 13.1-28.0), respectively. Patients with positive WB EBV-DNA showed worse PFS compared to those with negative WB EBV-DNA (median, 5.0 vs. 9.6 months, p = 0.046), although there was no significant difference in OS (positive vs. negative: median, 13.0 vs. 20.8 months, p = 0.337). There was no significant difference in survival outcomes according to positivity of EBER ISH (positive vs. negative: median PFS, 6.7 vs. 9.7 months, p = 0.389; median OS, 15.7 vs. 21.2 months, p = 0.565) or plasma EBV-DNA (positive vs. negative: median PFS, 5.4 vs. 9.3 months, p = 0.268; median OS, 13.3 vs. 30.5 months, p = 0.325). In the multivariate analysis, positive WB EBV-DNA showed a trend towards worse PFS with hazard ratio of 1.54 (95% CI, 0.99-2.38, p = 0.055), but there was no association with OS (p = 0.713). Conclusion: Our study demonstrated that EBV positivity as measured by EBER-ISH, WB, or plasma PCR in PTCL-NOS was not significantly associated with survival. Nevertheless, there was a trend towards worse PFS in patients who were positive for WB EBV-DNA. Thus, further investigations are warranted in larger cohorts to determine its clinical implications.

210.7: Undetectable Recipient COVID-19 Plasma PCR Following COVID-19 Positive Donor Kidney Transplant: Proof of Concept Analysis

Introduction: With one-third of the United States still not fully vaccinated for COVID-19 and almost one-half unvaccinated in Arkansas, it is common to see patients presenting with non-survivable diagnosis, incidentally testing COVID-19+ or with recent history of mild COVID-19 infection. In the absence of COVID-19, such patients could become organ donors. Limited case series have demonstrated no COVID-19 syndrome following transplantation from such donors. However, hematogenous asymptomatic transmission of the COVID-19 virus following transplant remains unclear. Aim of this study was to assess COVID-19 virus presence in the recipient’s bloodstream at the early post-transplant period following COVID-19+ organ transplantation. Methods: An institutional protocol was drafted for the use of COVID-19+ donor organs. Eligible donors included those test COVID-19+, however with cause of death not related to COVID-19, and no evidence of moderate to severe COVID-19. Eligible recipients included those who tested negative for COVID-19 at the time of transplant, had been fully vaccinated against COVID-19, and consented to receive such organs. A protocol deviation occurred when a COVID-19+ organ was transplanted into a non-vaccinated recipient with recent history of resolved COVID-19 infection. Plasma was drawn to test for COVID-19 polymerase chain reaction (PCR) prior to hospital discharge post-transplant. All recipients received tixagevimab-cilgavimab (Evusheld) after plasma COVID-19 PCR testing. Results: Six recipients underwent kidney transplantation from COVID-19+ donors. One donor had reported mild symptoms of COVID-19 prior to hospitalization. Two donors tested Hepatitis C positive by nucleic amplification testing. 83.3% (N=5) of recipients were fully vaccinated prior to transplant (Table 1). An additional recipient had not been vaccinated but had history of recent COVID-19 infection. None of the recipients developed COVID-19 symptoms post-transplant. All had undetectable serum plasma COVID-19 PCR prior to discharge (testing range post-operative day 2 to 5). Post-transplant median length of stay was 3 days. Median follow-up was 39 days (33, 62). Two recipients had delayed renal allograft function. One patient developed antibody-mediated rejection diagnosed on post-operative day 30. No recipients required post-transplant testing for suspected COVID-19 symptoms. There was no mortality (Table 2). Discussion: This case series provides proof of concept that there is no evidence of hematogenous transfer of COVID-19 from donor positive organs to recipients immune to the virus. Therefore, otherwise transplantable organs from donors incidentally diagnosed with COVID-19 at the time of donation, with no indication of severe COVID-19 disease at the time of donation, should be considered for non-thoracic organ transplantation to suitable recipients, after appropriate consenting.

Investigation of Cytomegalovirus in Intestinal Tissue in a Country With High CMV Seroprevalence.

In Turkey, cytomegalovirus (CMV) seropositivity has been reported to be high, between 85 and 100%. CMV has been responsible for disease exacerbation in inflammatory bowel disease (IBD). We aimed to evaluate the presence of CMV in intestinal tissue by immunohistochemical staining in IBD and non-IBD patient groups, in a country with high CMV seroprevalence. In this prospective cross-sectional study, the presence of intestinal CMV was investigated with tissue immunohistochemistry (IHC) staining, which is accepted as the gold standard method, and with polymerase chain reaction (PCR) in tissue and blood. Patients (≥18 years old, n = 189) who had a colonoscopic biopsy between January and May 2017 were included in the study at our hospital. Clinical, laboratory, endoscopic, and histopathological data of patients were assessed by dividing them into IBD (n = 34) and non-IBD (n = 155) groups. In this study, 567 colonic biopsy samples from 189 patients were evaluated. Tissue IHC staining was positive for 3 (1.58%) non-IBD patients. One of them was diagnosed as CMV ileitis. CMV DNA was also detected in 14 plasma (7.40%, <80-469 copies/mL) and 20 tissue samples (10.69%, 7-15 289 copies/mL). Tissue IHC staining is accepted as the gold standard for CMV ileitis, and the sensitivity and specificity of tissue PCR was 33% and 89.67%, while the sensitivity and specificity of plasma PCR was 66.66% and 93.54%, respectively. Although CMV seroprevalence is high in Turkey, CMV ileitis was diagnosed in only one non-IBD patient (0.53%). Compared to tissue IHC staining, the sensitivity of tissue and blood CMV PCR was low while their specificity was higher.

2569. High Incidence of Enterovirus, HHV6, Parechovirus and Adenovirus Blood Viremia in Children 0 to 3 Years Old Presenting With Fever Without Source

BackgroundFever without source (FWS) is defined as a fever in which an extensive history and clinical examination fail to identify a cause. Although the vast majority of children with FWS have a self-limited viral infection, up to 10–25% have a serious bacterial infection (SBI). Therefore, many children require invasive diagnostic tests, hospital admission, and empirical administration of broad-spectrum antibiotics. The aim of this study was to assess the respective role of Human enterovirus (HEV), human parechovirus (HPeV), adenovirus (ADV) and herpesvirus type 6 (HHV6) viremia in children <3 years old presenting with FWS.MethodsProspective monocentric diagnostic study. Between November 2015 to December 2017, children <3 year olds with FWS had, in addition to the standardized institutional work-up for FWS, plasma tested by real-time (reverse-transcription) polymerase chain reaction (PCR) for ADV, HHV6, HEV, and HPeV. Specimens with cycle threshold values <40 were considered positive. Quantification was performed on positive specimens for HEV, ADV, and HHV6 specimens when volume permitted.ResultsOne hundred thirty-five patients had plasma PCR for ADV, HHV6, HEV, and HPeV. Male:female ratio was 1.45:1 and median age was 2.4 months (interquartile range 1.3–9.7). Among those, 47/135 (34.8%) had at least 1 virus detected in the plasma. More specifically, HEV was detected in 19 patients (14.1%), HHV6 in 15 (11.1%), HPeV in 8 (5.9%), and ADV in 7 (5.2%). Co-infection with 2 viruses was detected in 2 patients (ADV/HEV and ADV/HPeV). No patient with positive plasma PCR had a positive blood or CSF culture. Two patients with positive plasma PCR fulfilled American Academy of Pediatrics criteria for urinary tract infection. The first was HEV+ in plasma and CSF, midstream urine was positive for leukocytes and grew E. coli 106 CFU/mL, whereas the second was HHV6+ in plasma and catheter urine was positive leukocytes/nitrites and grew P. mirabilis 105 CFU/mL.ConclusionThis epidemiological study highlights the frequent detection of active enteroviral, adenoviral, and HHV6 infections in plasma of children with FWS. Virus–virus and virus–bacteria co-infections are rare. Further studies are needed to establish causality between FWS and viremia.Disclosures A. G. L’Huillier, Gertrude Von Meissner foundation: Investigator, Research grant. Ernst and Lucie Schmidheiny foundation: Investigator, Research grant. Research and Development Grant, Geneva University Hospitals: Investigator, Research grant. L. Kaiser, Swiss National Funds: Investigator, Research grant. A. Galetto-Lacour, Gertrude Von Meissner foundation: Investigator, Research grant. Ernst and Lucie Schmidheiny foundation: Investigator, Research grant. Research and Development Grant, Geneva University Hospitals: Investigator, Research grant.

Parvovirus B19 Infection in Solid Organ Transplantation: Report of A Case and A Review

Infection with parvovirus B19 is common, with up to 55% of adults showing seropositive evidence of prior infection. Clinical disease can occur due to acute infection, secondary infection, or reactivation. The clinical presentation of primary infection depends on age, the presence of a hematologic condition, and immune status. It is an uncommon infection in solid organ transplant (SOT) recipients that has atypical features, usually presenting with pure red cell aplasia which is refractory to erythropoietin (EPO) and which can be transfusion dependent. Plasma polymerase chain reaction (PCR) measurement of DNA, both qualitative and quantitative, can be very helpful for diagnosis, but a negative test does not exclude infection. In those cases, bone marrow biopsy may be needed to document the viral infection. Loss of or inability to produce neutralizing antibodies is the immune deficit leading to chronic parvovirus disease, in which serologic diagnosis is unreliable. An even more unusual manifestation of parvovirus infection is end organ disease, though more common in immunocompromised individuals. Intravenous immunoglobulin (IVIG) replacement therapy appears to be most effective treatment, although there may also be a role for reduction in immune suppression. The ability to prevent this unusual infection is limited, as patients who are asymptomatic may transmit the disease.

RNase H2-Dependent Polymerase Chain Reaction and Elimination of Confounders in Sample Collection, Storage, and Analysis Strengthen Evidence That microRNAs in Bovine Milk Are Bioavailable in Humans

Evidence suggests that dietary microRNAs (miRs) are bioavailable and regulate gene expression across species boundaries. Concerns were raised that the detection of dietary miRs in plasma might have been due to sample contamination or lack of assay specificity. Objectives: The objectives of this study were to assess potential confounders of plasma miR analysis and to detect miRs from bovine milk in human plasma. Potential confounders of plasma miR analysis (circadian rhythm, sample collection and storage, calibration, and erythrocyte hemolysis) were assessed by quantitative reverse transcriptase polymerase chain reaction (PCR) by using blood from healthy adults (7 men, 6 women; aged 23-57 y). Bovine miRs were analyzed by RNase H2-dependent PCR (rhPCR) in plasma collected from a subcohort of 11 participants before and 6 h after consumption of 1.0 L of 1%-fat bovine milk. The use of heparin tubes for blood collection resulted in a complete loss of miRs. Circadian variations did not affect the concentrations of 8 select miRs. Erythrocyte hemolysis caused artifacts for some miRs if plasma absorbance at 414 nm was >0.300. The stability of plasma miRs depended greatly on the matrix in which the miRs were stored and whether the plasma was frozen before analysis. Purified miR-16, miR-200c, and cel-miR-39 were stable for ≤24 h at room temperature, whereas losses equaled ≤80% if plasma was frozen, thawed, and stored at room temperature for as little as 4 h. rhPCR distinguished between bovine and human miRs with small variations in the nucleotide sequence; plasma concentrations of Bos taurus (bta)-miR-21-5p and bta-miR-30a-5p were >100% higher 6 h after milk consumption than before milk consumption. Confounders in plasma miR analysis include the use of heparin tubes, erythrocyte hemolysis, and storage of thawed plasma at room temperature. rhPCR is a useful tool to detect dietary miRs.

First case of imported Zika virus infection in Spain

We report a case of Zika virus (ZIKV) infection in a patient with diarrhea, fever, synovitis, non-purulent conjunctivitis, and with discreet retro-orbital pain, after returning from Colombia in January 2016. The patient referred several mosquito bites. Presence of ZIKV was detected by PCR (polymerase chain reaction) in plasma. Rapid microbiological diagnosis of ZIKV infection is needed in European countries with circulation of its vector, in order to avoid autochthonous circulation. The recent association of ZIKV infection with abortion and microcephaly, and a Guillain-Barré syndrome highlights the need for laboratory differentiation of ZIKV from other virus infection. Women with potential risk for Zika virus infection who are pregnant or planning to become pregnant must mention that fact during prenatal visits in order to be evaluated and properly monitored.

A Scheduled Program of Molecular Screening for Epstein-Barr Virus Decreases the Incidence of Post-transplantation Lymphoproliferative Disease in Pediatric Liver Transplantation

This is a cohort, retrospective, comparative study of all liver transplant recipients from a single center, from May 1998 to July 2015. Patients were divided into two groups according to the type of Epstein-Barr viral load monitoring. For group I (1998–2007), polymerase chain reaction (PCR) was not available or it was only qualitative with limited access. For group II (2008–2015), we used periodically scheduled quantitative PCR in plasma and leukocytes, with aggressive tapering of immunosuppression as soon as viral replication was detected. Ninety-eight recipients were included, 41 (41.8%) were Epstein-Barr virus (EBV) − seronegative before liver transplantation (LT). EBV replication was confirmed in 74 patients (75.5%), being more frequent in seronegative (87.8%) than seropositive patients (66.6%). Eight recipients (8.1%) developed post-transplantation lymphoproliferative disorder (PTLD) on average at 14.3 months post-LT, seven of eight were <3 years at LT, four of eight were D+/R− for EBV, and all had post-LT EBV replication confirmed by PCR. PTLD was classified as lymphoma (n = 4), polymorphic polyclonal (n = 3), and lymphoid hyperplasia (n = 1). Five patients died, and three cleared PTLD after immunosuppression tapering or interruption. There were no significant differences in the etiology, age at LT (5.6 vs. 7.3 years, P = .069), patients <4 years (53.2% vs. 35.3%, P = .103), or EBV seronegative recipients (44.7% vs. 37.3%, P = .54); however, the incidence of PTLD decreased from 14.9% to 1.9% (P = .026), and graft rejection from 51.1% to 29.4% (P = .039). One- and 5-year patient survival rates were 94.7% and 85%, respectively, with no differences between groups. This strategy dramatically decreased the incidence of PTLD (14.9% vs. 1.9%), without increasing the incidence of rejection; therefore, we recommend that it should be used in the follow-up of all pediatric LT recipients.

Quantitative polymerase chain reaction for detection of human herpesvirus-7 infection in umbilical cord blood donors.

Umbilical cord blood (UCB) has been a reasonable alternative to granulocyte colony-stimulating factor-mobilized peripheral blood or bone marrow, as a source of hematopoietic stem cells with a lower risk of graft-versus-host disease. In immunocompromised hosts after transplantation, the risk of viral infection in adults, especially with beta-herpesviruses such as human herpesvirus-7 (HHV-7), may be increased. This virus in immunocompromised patients can be reactivated from latency and converted to an active phase. Therefore, light-upon-extension real-time polymerase chain reaction (PCR) was developed to assess the prevalence and load of HHV-7 in the plasma and buffy coat of donors. About 825 UCB samples under standard protocol from donors were collected. Then, DNA from plasma and buffy coat was extracted and quantitative real-time PCR was performed with light-upon-extension primers. Overall, HHV-7 was detected in 3.64% (30/825) of UCB donors. HHV-7 DNA was detected in 26 (3.2%) buffy coat samples (latent infection), and only 4 (0.48%) of them were positive for HHV-7 DNA in plasma samples (active infection); the mean HHV-7 viral load was 1.31×10(1) copies/mL in latent infection, and 1.94×10(5) copies/mL in active infection. We suggest that real-time PCR in plasma and buffy coat could be a useful method to detect active and latent HHV-7 infection in UCB donors and determine its role in subsequent transmission events.

Prospective study on CMV-reactivations under preemptive strategy in CMV-seropositive adult liver transplant recipients

BackgroundCytomegalovirus (CMV) is a significant infectious agent after liver transplantation. To prevent CMV, most centres use prophylaxis for high-risk CMV-seronegative recipient/seropositive donor and many even for all seropositive recipients. However, pre-emptive therapy is commonly used for seropositive patients. ObjectivesA prospective, long-term follow-up of CMV-seropositive adult liver-transplant patients under pre-emptive strategy was investigated. Study designCMV-seropositive liver recipients were monitored for CMV by real-time quantitative plasma polymerase chain reaction (PCR) and received ganciclovir/valganciclovir pre-emptive therapy. The 161 patients with follow-up of >4 years were included in the study. ResultsNo CMV was detected in most cases 98/161 (61%), but 63/161 (39%) developed CMV-DNAaemia mean 49 days (7–183 days) after transplantation. Only 25/63 reactivations exceeded 5000copies/ml, which was considered as cut-off for the pre-emptive treatment by the method used (median 21,500, range 5100–813300copies/ml) and most were self-limiting, low-level DNAaemias (median 850, range 234–4000copies/ml). Thus, low-level temporal CMV viraemia occurred in 38/161 patients (23.5%) and only 25/161 (15.5%) demonstrated significant viral loads. Recurrent CMV appeared in one patient with low-level and in 11/25 with high-level DNAaemia, only 5/11 exceeding 5000copies/ml. CMV infections were successfully treated with ganciclovir/valganciclovir. Four patients with low and three with high DNAaemia have been retransplanted. Five patients with low and two with high DNAaemia have died subsequently. No patient or graft was lost due to CMV. ConclusionsMost CMV-seropositive liver recipients did not develop CMV reactivation, and if reactivations occurred, most were temporal, low-level DNAaemias. Significant CMV infections were successfully treated and recurrences were rare.

Detection of BK polyomavirus after kidney transplantation: a comparison of urine electron microscopy with plasma polymerase chain reaction

BK polyomavirus (BKV) infection continues to be a significant source of allograft dysfunction in kidney transplant recipients. The optimal screening method to detect BKV remains undetermined. In this retrospective analysis of 347 consecutive kidney transplant recipients, we compare the diagnostic and screening performance of urine electron microscopy (EM) with plasma polymerase chain reaction (PCR) in testing for BKV, using biopsy-proved polyomavirus-associated nephropathy (PVAN) as the gold standard. Sixty-nine of 347 recipients had a positive screening test for BKV infection. Twenty-nine patients underwent biopsy, and 11 were diagnosed with PVAN. Sensitivity rates of urine EM and plasma PCR were 88% and 100%, respectively. Specificity rates of urine EM and plasma PCR were 91% and 78%. There was no statistical difference in the operating characteristics of the two tests. The majority of both plasma PCR and urine EM tests were positive in the six months prior to a diagnostic biopsy confirming PVAN. In those patients who had evidence of BKV infection but did not have PVAN, the percentage of positive screening tests decreased with aggressive lowering of immunosuppression. We conclude that urine EM and plasma PCR both function well in screening for BKV infection and in the diagnosis of PVAN. There is an opportunity to detect viral replication, lower immunosuppression, and to prevent PVAN in this population.

Risk Assessment for Polyomavirus Nephropathy Using Urine Cytology and the Detection of Decoy Cells

Risk Assessment for Polyomavirus Nephropathy Using Urine Cytology and the Detection of Decoy Cells

Performance of Candida Real-time Polymerase Chain Reaction, -D-Glucan Assay, and Blood Cultures in the Diagnosis of Invasive Candidiasis

The sensitivity of blood cultures for diagnosing invasive candidiasis (IC) is poor. We performed a validated Candida real-time polymerase chain reaction (PCR) and the Fungitell 1,3-β-D-glucan (BDG) assay on blood samples collected from prospectively identified patients with IC (n = 55) and hospitalized controls (n = 73). Patients with IC had candidemia (n = 17), deep-seated candidiasis (n = 33), or both (n = 5). Controls had mucosal candidiasis (n = 5), Candida colonization (n = 48), or no known Candida colonization (n = 20). PCR using plasma or sera was more sensitive than whole blood for diagnosing IC (P = .008). Plasma or sera PCR was more sensitive than BDG in diagnosing IC (80% vs 56%; P = .03), with comparable specificity (70% vs 73%; P = .31). The tests were similar in diagnosing candidemia (59% vs 68%; P = .77), but PCR was more sensitive for deep-seated candidiasis (89% vs 53%; P = .004). PCR and BDG were more sensitive than blood cultures among patients with deep-seated candidiasis (88% and 62% vs 17%; P = .0005 and .003, respectively). PCR and culture identified the same Candida species in 82% of patients. The sensitivity of blood cultures combined with PCR or BDG among patients with IC was 98% and 79%, respectively. Candida PCR and, to a lesser extent, BDG testing significantly enhanced the ability of blood cultures to diagnose IC.

Clinical Usefulness of Plasma Quantitative Polymerase Chain Reaction Assay: Diagnosis of Cytomegalovirus Infection in Kidney Transplant Recipients

Background Preemptive therapy is used to prevent cytomegalovirus (CMV) disease in transplant recipients. The CMV antigenemia assay, which has been commonly used as a predictive marker for preemptive therapy, requires intensive labor and immediate processing. We compared the cutoff value of plasma CMV polymerase chain reaction (PCR) with CMV antigenemia in kidney transplant recipients. Methods We compared two diagnostic methods for CMV infection in kidney transplant recipients: quantitative PCR (qPCR) versus antigenemia. We evaluated the optimal cutoff value of plasma CMV qPCR by using receiver-operating characteristic curves for specific antigenemia values. All kidney transplant recipients from January 2004 to January 2005 were enrolled and followed with CMV antigenemia and plasma CMV qPCR. Results The analyses were performed on 899 samples collected from 111 patients in the early posttransplant period, matching 84.1% of patients for the results of CMV antigenemia and plasma CMV qPCR. For patients with symptomatic CMV infection and disease, who showed ≥25 positive cells in the antigenemia assay, the cutoff value for qPCR was 17.8 copies/μL with a sensitivity of 97.1%, a specificity of 89.1%, and a positive predictive value of 26.6%. Conclusions Diagnostic assays for CMV such as CMV antigenemia and quantitative plasma PCR, showed similar diagnostic values. They are the methods of choice for the diagnosis and monitoring of active CMV infection after kidney transplantation. However, because of the relatively low positive predictive value of qPCR, this test may lead to unnecessary preemptive treatment in kidney transplant recipients.

The Clinical Utility of Whole Blood Versus Plasma Cytomegalovirus Viral Load Assays for Monitoring Therapeutic Response

In patients with cytomegalovirus (CMV) disease, regular monitoring of viral loads and treatment until negative are recommended. However, with more sensitive polymerase chain reaction (PCR) assays and cellular peripheral sample types, detection of low-level viremia is achievable. We compared a whole blood real-time PCR with a plasma PCR assay for monitoring therapeutic response. Patients enrolled in a trial to treat CMV disease for 21 days had regular viral load monitoring. The results of a plasma-based PCR assay were compared with a real-time PCR assay of whole blood and assessed for their ability to predict recurrence. In 219 evaluable patients, viral loads in plasma versus whole blood demonstrated good correlation but significant difference in absolute value and clearance kinetics. Virus was still detectable by day 21 in 154 of 219 (70.3%) patients with the whole blood versus 105 of 219 (52.1%; P<0.001) patients with the plasma assay. The positive predictive value of persistent plasma viremia at day 21 for virologic recurrence was 41.9% vs. 36.3% for the whole blood assay. In the subset of patients with a negative plasma but positive whole blood at day 21 (n = 49), the incidence of virologic recurrence was similar to that of all patients with a negative plasma assay (23.1% vs. 23.6%). When treating CMV disease, enhanced detection of residual viremia using a whole blood real-time PCR does not seem to offer significant clinical advantages nor allows for better prediction of recurrence of CMV viremia or disease. The treat-to-negative paradigm may not hold true when such assays are used.

Hemophagocytic syndrome caused by primary herpes simplex virus 1 infection: report of a first case

Hemophagocytic syndrome represents a severe hyperinflammatory condition by activated macrophages. Leading viral triggering agents are Epstein-Barr virus (EBV), cytomegalovirus (CMV), and adenovirus. We present a patient with Wegener's granulomatosis on azathioprine and prednisone medication, who developed a life-threatening hemophagocytic syndrome. Positive plasma polymerase chain reaction (PCR) with negative serology revealed a primary, disseminated infection with herpes simplex virus-1 as the triggering pathogen. After treatment with acyclovir, high-dose steroids, immunoglobulins, and etoposide, the patient recovered. Early diagnosis of potentially underlying infections of hemophagocytic syndrome influences the therapeutic approach. It is important to consider a variety of infectious agents, particularly in immunosuppressed individuals. The reported case emphasizes the importance of screening for herpes simplex virus 1.

Real-Time PCR Assay Compared With Antigenemia Assay for Detecting Cytomegalovirus Infection in Kidney Transplant Recipients

Human cytomegalovirus (CMV) infection is a major cause of morbidity and mortality among kidney transplant recipients. The CMVpp65 antigenemia assay has been used for preemptive therapy. Real-time polymerase chain reaction (PCR) technology for CMV DNA quantification in blood has demonstrated a good correlation with the currently employed CMV antigenemia assay. In this study, 90 renal transplant recipients were prospectively enrolled from July 2004 and May 2005. Monitoring of CMV infection was routinely performed with CMV antigenemia and real-time PCR assays. Real-time plasma PCR and CMV antigenemia assays were assessed on 797 samples. CMV antigenemia correlated with a positive CMV PCR (χ 2 = 78.05; P < .0001). Not only the positive rate but also the number of positive cells correlated with the number of PCR DNA copies (F = 26.07, r 2 = .25, P < .0001). To define an optimal cutoff value of CMV DNA load to initiate treatment in kidney transplant patients, we considered a CMV antigenemia titer of >50 positive cells per 400,000 leukocytes as the gold standard in our previous study. The optimal cutoff value for the quantitative real-time PCR assay was predicted to be 86 copies/μL. Thus, we observed that CMV real-time PCR assay would not completely replace antigenemia assay in kidney transplant recipients, but can be used complementarily to screen antigenemia and monitor preemptive therapy.

CMV monitoring using blood cells and plasma: a comparison of apples with oranges?

Quantitative cytomegalovirus (CMV) monitoring is still far from being standardized between transplant centers. In the present study, we compared assays for quantitative CMV monitoring using blood cells and plasma. Four hundred and thirty-five consecutive samples from 29 patients with active CMV infection after allogeneic T-cell-depleted hemopoietic stem cell transplantation were tested in parallel using pp65 antigenemia and quantitative CMV polymerase chain reaction (PCR) in blood cells and plasma (COBAS AMPLICOR CMV MONITOR). Although only 142 (53.1%) of 253 positive samples were concordantly identified by all three assays, the number of positive samples detected by each assay was not different and the quantitative values were correlated, provided that nucleic acid (NA) in plasma was isolated by COBAS AmpliPrep and not by the manual protocol. Six (18%) of 34 episodes with active CMV infection were not detected using CMV PCR in plasma; whereas in times of white blood cell aplasia or blast crisis of leukemia, samples with active CMV infection in plasma could not be detected using blood cells. We conclude that CMV monitoring in whole blood could be favorable compared with assays using plasma or blood cells alone. Automated NA isolation could become an attractive tool for a more sensitive and better standardized molecular diagnostics.

PCR in the investigation of canine American tegumentary leishmaniasis in northwestern Paraná State, Brazil

American tegumentary leishmaniasis (ATL) was studied in 143 dogs in a rural area in the county of Mariluz, northwestern Paraná State, Brazil, using direct parasite search, indirect immunofluorescence (IIF), and polymerase chain reaction (PCR). Thirty-nine dogs (27.3%) presented lesions suggestive of the disease, 5 (12.8%) of which were positive in direct parasite search and PCR (lesion), and of these 5, 4 were also positive by IIF. Of the 34 dogs with negative direct parasite search, 12 (35.3%) had PCR-positive lesions, and of these, 5 were also IIF-positive. One hundred and four dogs had no lesions, but 17/101 (16.8%) were IIF-positive. PCR in blood was positive in 10/38 (26.3%) of the dogs with lesions and in 16/104 (15.4%) of dogs without lesions. The association between PCR (lesion or blood), direct parasite search, and IIF detected 24/39 (61.5%) positive results among symptomatic dogs and 31/104 (29.8%) among asymptomatic animals. PCR was useful for diagnosing ATL, but there was no correlation between lesions, serology, and plasma PCR. Furthermore, detection of parasite DNA in the blood may indicate hematogenous parasite dissemination.

Initial experience with oral valganciclovir for pre-emptive cytomegalovirus therapy after lung transplantation

The most common opportunistic viral pathogen after lung transplantation is cytomegalovirus (CMV). Oral valganciclovir, a prodrug of ganciclovir, has been introduced as a potential drug for prophylaxis and treatment of CMV infection and disease in lung transplantation. The goal of this study was to describe our initial experience with oral valganciclovir for pre-emptive treatment of CMV infections after lung transplantation. We summarize our experience with 19 patients who underwent lung transplantation and received pre-emptive oral valganciclovir therapy in the situation of positive CMV polymerase chain reaction (PCR) in either plasma or bronchoalveolar lavage. None of the patients presented with manifest CMV disease. Treatment dosage of valganciclovir was 450 mg to 1800 mg daily, depending on renal function and white blood count. Treatment was continued until the CMV PCR became negative, in any case for a period of at least 14 days. Three patients received two courses of pre-emptive oral valganciclovir; 16 patients were treated once. Eleven patients (57.9%) were treated because of a positive plasma CMV PCR; in eight patients (42.1%) the PCR was positive only in bronchoalveolar lavage. Therapy was initiated 896 +/- 1186 days (range, 108-3911) after transplantation with a mean CMV PCR of 45,536 +/- 149,294 copies (range, 426-706,000). In all cases the PCR fell below detectability (<400 copies) after a period of 22 +/- 10 days of treatment (range, 7-50 days). Mild to moderate leucopenia was observed in seven patients (36.8%) during treatment. None of the patients developed new onset of other potentially drug-related disorders such as neutropenia, anemia, deterioration of renal function or gastrointestinal disorder. Pre-emptive therapy with oral valganciclovir for CMV infections detected by PCR in either plasma or bronchoalveolar lavage after lung transplantation seems to be efficacious and safe. However, regular blood counts should be performed to detect developing leucopenia.