Angela M. Caliendo

Updated International Consensus Guidelines on the Management of Cytomegalovirus in Solid-Organ Transplantation

Cytomegalovirus (CMV) remains one of the most common infections after solid organ transplantation, resulting in significant morbidity, graft loss, and occasional mortality. Management of CMV varies considerably among transplant centers. A panel of experts on CMV and solid organ transplant was convened by The Infectious Diseases Section of The Transplantation Society to develop evidence and expert opinion-based consensus guidelines on CMV management including diagnostics, immunology, prevention, treatment, drug resistance, and pediatric issues.

Better Tests, Better Care: Improved Diagnostics for Infectious Diseases

Abstract In this IDSA policy paper, we review the current diagnostic landscape, including unmet needs and emerging technologies, and assess the challenges to the development and clinical integration of improved tests. To fulfill the promise of emerging diagnostics, IDSA presents recommendations that address a host of identified barriers. Achieving these goals will require the engagement and coordination of a number of stakeholders, including Congress, funding and regulatory bodies, public health agencies, the diagnostics industry, healthcare systems, professional societies, and individual clinicians.

Clinical utility of quantitative cytomegalovirus viral load determination for predicting cytomegalovirus disease in liver transplant recipients

BACKGROUND The early detection of cytomegalovirus (CMV) after liver transplantation may form the basis of a preemptive strategy for prevention of active CMV disease. METHODS We prospectively analyzed the clinical use of weekly quantitative polymerase chain reaction-(PCR) based plasma viral load determinations and the antigenemia assay for predicting the development of active CMV disease in 97 consecutive liver transplant recipients. RESULTS CMV disease occurred in 21/97 patients. Using a positive cut-off of >400 copies/ml plasma, PCR had a sensitivity of 100%, specificity 47.4%, positive predictive value 34.4% and negative predictive value 100% for prediction of CMV disease. Respective values for a positive antigenemia (>0 positive cells/slide) were 95.2, 55.3, 37.0, and 97.7%. Different cut-off points for a positive test were analyzed using receiver-operating characteristic (ROC) curves. The optimal cut-off for viral load was in the range of 2000-5000 copies/ml (sensitivity 85.7%, specificity 86.8%, PPV 64.3%, NPV 95.7% for >5000 copies/ml). The optimal cut-off for antigenemia was in the range of four to six positive cells/slide. Mean peak viral load in symptomatic patients was 73,715 copies per/ml versus 3615 copies/ml in patients with asymptomatic CMV reactivation (P<0.001). In a multivariate logistic regression analysis of risk factors for CMV disease (CMV serostatus, acute rejection, and induction immunosuppression), peak viral load and peak antigenemia emerged as the only significant independent predictors of CMV disease (for PCR, odds ratio=1.40/1000 copy/ml increase in viral load, P=0.0001; for antigenemia odds ratio=1.17/1 positive cell/slide). CONCLUSIONS Plasma viral load by quantitative PCR is useful for predicting CMV disease and could be used in a preemptive strategy.

Interlaboratory Comparison of Cytomegalovirus Viral Load Assays

To assess interlaboratory variability in qualitative and quantitative cytomegalovirus (CMV) viral load (VL) testing, we distributed a panel of samples to 33 laboratories in the USA, Canada and Europe who performed testing using commercial reagents (n = 17) or laboratory‐developed assays (n = 18). The panel included two negatives, seven samples constructed from purified CMV nucleocapsids in plasma (2.0–6.0 log10 copies/mL) and three clinical plasma samples. Interlaboratory variation was observed in both actual (range, 2.0–4.0 log10 copies/mL) and self‐reported lower limits of detection (range, 1.0–4.0 log10 copies/mL). Variation observed in reported results for individual samples ranged from 2.0 log10 (minimum) to 4.3 log10 (maximum). Variation was greatest at low VLs. Assuming ± 0.5 log10 relative to the expected result represents an acceptable result, 57.6% of results fell within this range. Use of commercially available reagents and procedures was associated with less variability compared with laboratory‐developed assays. Interlaboratory variability on replicate samples was significantly greater than intralaboratory variability (p < 0.0001). The significant interlaboratory variability in CMV VL observed may be impacting patient care and limiting interinstitutional comparisons. The creation of an international reference standard for CMV VL assay calibration would be an important step in quality improvement of this laboratory tool.

Comparison of Droplet Digital PCR to Real-Time PCR for Quantitative Detection of Cytomegalovirus

ABSTRACT Quantitative real-time PCR (QRT-PCR) has been widely implemented for clinical viral load testing, but a lack of standardization and relatively poor precision have hindered its usefulness. Digital PCR offers highly precise, direct quantification without requiring a calibration curve. Performance characteristics of real-time PCR were compared to those of droplet digital PCR (ddPCR) for cytomegalovirus (CMV) load testing. Tenfold serial dilutions of the World Health Organization (WHO) and the National Institute of Standards and Technology (NIST) CMV quantitative standards were tested, together with the AcroMetrix CMV tc panel (Life Technologies, Carlsbad, CA) and 50 human plasma specimens. Each method was evaluated using all three standards for quantitative linearity, lower limit of detection (LOD), and accuracy. Quantitative correlation, mean viral load, and variability were compared. Real-time PCR showed somewhat higher sensitivity than ddPCR (LODs, 3 log10 versus 4 log10 copies/ml and IU/ml for NIST and WHO standards, respectively). Both methods showed a high degree of linearity and quantitative correlation for standards (R 2 ≥ 0.98 in each of 6 regression models) and clinical samples (R 2 = 0.93) across their detectable ranges. For higher concentrations, ddPCR showed less variability than QRT-PCR for the WHO standards and AcroMetrix standards (P < 0.05). QRT-PCR showed less variability and greater sensitivity than did ddPCR in clinical samples. Both digital and real-time PCR provide accurate CMV load data over a wide linear dynamic range. Digital PCR may provide an opportunity to reduce the quantitative variability currently seen using real-time PCR, but methods need to be further optimized to match the sensitivity of real-time PCR.

Effect of highly active antiretroviral therapy on cervicovaginal HIV-1 RNA.

ObjectivesTo determine the frequency of cervicovaginal lavage and plasma HIV-1 RNA levels that are below detectable levels (< 400 copies/ml) among women on highly active antiretroviral therapy (HAART), non-HAART and on no therapy. To compare the effect of initiating HAART on the timing of HIV-1 RNA suppression in the blood plasma and genital tract among antiretroviral-naïve women. MethodsData were obtained from 205 HIV-infected women with paired plasma and cervicovaginal lavage viral load measurements. Seven antiretroviral-naïve women starting HAART had viral load measurements performed daily for one week, at 2 weeks and at 1 month after initiating therapy. Viral load quantification was carried out by nucleic acid sequence-based amplification assay. The lower limit of detection was 400 copies/ml. ResultsPlasma and cervicovaginal HIV-1 RNA was detectable in 71 and 26% of the women, respectively. Among women with plasma viral loads less than 400, 400–9999, and 10 000 copies/ml or over, genital tract HIV-1 RNA was detected in 3, 17 and 48%, respectively (P < 0.001). Fifty-one per cent of the women with CD4 cell counts of less than 200/mm3 had detectable cervicovaginal viral loads compared with 18% among women with CD4 cell counts of 200/mm3 or over (P < 0.001). Cervicovaginal HIV-1 RNA was less than 400 copies/ml in 85% of those on HAART, 69% of those on non-HAART and 69% of those on no therapy (P < 0.045). In seven antiretroviral-naïve women initiating HAART, cervicovaginal HIV-1 RNA decreased by 0.7–2.1 log10 within 1–14 days of starting therapy. ConclusionThe cervicovaginal HIV-1 RNA level was positively correlated with plasma HIV-1 RNA and negatively with the CD4 cell count. The use of HAART was significantly associated with below-detectable levels of HIV-1 RNA in both plasma and the genital tract. HIV-1 RNA suppression in the genital tract may occur rapidly after initiating therapy.

Association between bacterial vaginosis and expression of human immunodeficiency virus type 1 RNA in the female genital tract.

We assessed the effect of lower genital tract infections on human immunodeficiency virus type 1 (HIV-1) RNA shedding in the female genital tract. Bacterial vaginosis was significantly associated with HIV-1 RNA expression in the female genital tract of HIV-infected women.

Multiplex PCR and Emerging Technologies for the Detection of Respiratory Pathogens

Abstract Molecular methods are becoming more widely used for the detection of respiratory pathogens, in part because of their superior sensitivity, relatively rapid turnaround time, and ability to identify pathogens that are slow growing or difficult to culture. The recent novel H1N1 influenza A pandemic served to underscore how quickly new molecular tests can become available for clinical use. Over the years PCR has been the dominant amplification method. Recently, modifications of this technology have emerged, some of which allow for the rapid detection of multiple pathogens in a single test. This review will focus on emerging multiplex molecular technologies and their clinical utility for the detection of respiratory pathogens.

Cytomegalovirus (CMV) Virus Load Kinetics to Predict Recurrent Disease in Solid-Organ Transplant Patients with CMV Disease

Despite standard therapy, cytomegalovirus (CMV) disease recurs in a significant proportion of organ transplant recipients. The kinetics of CMV load in response to therapy may allow early prediction of recurrence. CMV loads were obtained at regular intervals after starting ganciclovir therapy in 52 transplant recipients with CMV disease. Virus load kinetics were analyzed using decay curves to assess viral dynamics, including half-life and time to viral clearance. Recurrent CMV disease occurred in 12 (23%) of 50 patients. The time period to viral clearance was longer among patients with recurrence of CMV disease (P=.002), and lack of clearance was also associated among patients with disease recurrence (P<.001). Viral kinetics followed a logarithmic decay curve expressed by the equation y=y(0)e(-ax). Virus load half-life was 8.8 days among patients with recurrence versus 3.17 days among patients without recurrence (P=.001). This was not explainable by detectable ganciclovir resistance. CMV load kinetics are useful for identifying, at a very early stage, patients who are more likely to have recurrence.

Molecular and Nonmolecular Diagnostic Methods for Invasive Fungal Infections

SUMMARY Invasive fungal infections constitute a serious threat to an ever-growing population of immunocompromised individuals and other individuals at risk. Traditional diagnostic methods, such as histopathology and culture, which are still considered the gold standards, have low sensitivity, which underscores the need for the development of new means of detecting fungal infectious agents. Indeed, novel serologic and molecular techniques have been developed and are currently under clinical evaluation. Tests like the galactomannan antigen test for aspergillosis and the β-glucan test for invasive Candida spp. and molds, as well as other antigen and antibody tests, for Cryptococcus spp., Pneumocystis spp., and dimorphic fungi, have already been established as important diagnostic approaches and are implemented in routine clinical practice. On the other hand, PCR and other molecular approaches, such as matrix-assisted laser desorption ionization (MALDI) and fluorescence in situ hybridization (FISH), have proved promising in clinical trials but still need to undergo standardization before their clinical use can become widespread. The purpose of this review is to highlight the different diagnostic approaches that are currently utilized or under development for invasive fungal infections and to identify their performance characteristics and the challenges associated with their use.