Michael Forman

Cytomegalovirus Retinitis and Viral Resistance: Ganciclovir Resistance

Cytomegalovirus (CMV) retinitis is among the most common opportunistic infections in patients with AIDS and a substantial cause of visual loss. With long-term therapy, resistant CMV may develop. In a prospective study of 108 patients with CMV retinitis, 80.6% of patients were found to have either a positive blood culture or positive urine culture for CMV at the diagnosis of retinitis. At diagnosis of retinitis, 0.9% and 2.7% of patients had a ganciclovir-resistant blood culture isolate and urine culture isolate, respectively. Of 76 patients initially treated with ganciclovir, 11.4% had a resistant blood or urine culture isolate by 6 months of treatment and 27.5% by 9 months. The development of ganciclovir resistance during follow-up correlated with the occurrence of CMV retinitis in the contralateral eye (odds ratio = 9.06, P = .003).

Prediction of severe disseminated adenovirus infection by serum PCR

Adenoviruses are increasingly recognised as viral pathogens that can cause fatal infections in immunocompromised patients, particularly recipients of haematopoietic stem-cell grafts. Adenovirus infections are not easily diagnosed and the development of a severe infection cannot be predicted by standard culture techniques. In a pilot study, we investigated the value of adenovirus DNA detection in serum as a marker of disseminated disease in 14 patients with defined patterns of adenovirus infections. The results show that the appearance of adenoviral DNA in serum preceded the development of a severe or fatal adenovirus infection. Because proper management is dependent on early diagnosis and differentiation from other conditions, this test may be a valuable tool in the management of adenovirus infection.

Mutations Conferring Ganciclovir Resistance in a Cohort of Patients with Acquired Immunodeficiency Syndrome and Cytomegalovirus Retinitis

Cytomegalovirus (CMV) retinitis is among the most common opportunistic infections in patients with acquired immunodeficiency syndrome. In a prospective study of 210 patients with CMV retinitis, 26 were identified as having either a phenotypic or a genotypic ganciclovir-resistant isolate from either blood or urine cultures. For blood culture isolates with an IC(50) >6.0 microm for ganciclovir, the sensitivity and specificity for detecting a UL97 mutation were 95% and 98%, respectively, whereas for an IC(50) >8.0 microM they were 79% and 99%, respectively. Although there were trade-offs between the 2 thresholds for blood culture isolates, for urine culture isolates an IC(50) >8.0 microM appeared to be better at identifying genotypic resistance. UL97 mutations identified in both the blood and urine cultures of individual patients were identical in 87.5% of cases. High-level ganciclovir resistance (IC(50), >30 microM) typically, but not invariably, was associated with a mutation in both the UL97 and UL54 genes.

Incidence of Foscarnet Resistance and Cidofovir Resistance in Patients Treated for Cytomegalovirus Retinitis

ABSTRACT Cytomegalovirus (CMV) retinitis is a common opportunistic infection in patients with AIDS. With long-term therapy for CMV retinitis, resistant CMV may develop. In a prospective study of 122 patients with CMV retinitis, 2.4 and 0.8% of patients had foscarnet-resistant blood culture isolates (50% inhibitory concentration [IC50], >400 μM) and urine culture isolates, respectively, at diagnosis of CMV retinitis prior to treatment, whereas 4.1 and 6.6% had cidofovir-resistant (IC50, >2 μM) blood and urine culture isolates, respectively. Patients were treated according to best medical judgement. Of 44 foscarnet-treated patients, 26% had a resistant blood or urine culture isolate by 6 months of treatment and 37% had a resistant isolate by 9 months; of 13 cidofovir-treated patients, 29% had a resistant blood or urine culture isolate by 3 months of therapy. The probabilities of developing foscarnet resistance while on foscarnet and developing cidofovir resistance while on cidofovir were not significantly different from that for developing ganciclovir resistance while on ganciclovir (odds ratios, 1.87 [P = 0.19] and 2.28 [P = 0.15], respectively).

Comparison of Automated and Manual Nucleic Acid Extraction Methods for Detection of Enterovirus RNA

ABSTRACT Automated nucleic acid extraction is an attractive alternative to labor-intensive manual methods. We compared two automated methods, the BioRobot M48 instrument (Qiagen, Inc.) and MagNA Pure (Roche Applied Sciences) methods, to two manual methods, the QIAamp Viral RNA Mini kit (Qiagen) and TRIzol (Invitrogen), for the extraction of enterovirus RNA. Analytical sensitivity was assessed by dilution analysis of poliovirus type 2 Sabin in cerebrospinal fluid. The sensitivity of PCR was equivalent after RNA extraction with QIAamp, BioRobot M48, and MagNA Pure. All 18 replicates of 100 PFU/ml were detected after extraction by the four methods. Fewer replicates of each successive dilution were detected after extraction by each method. At 10−1 PFU/ml, 17 of 18 replicates were positive by QIAamp, 15 of 18 replicates were positive by BioRobot M48, and 12 of 18 replicates were positive by MagNA Pure; at 10−2 PFU/ml, 4 of 17 replicates were positive by QIAamp, 2 of 18 replicates were positive by BioRobot M48, and 0 of 18 replicates were positive by MagNA Pure. At 10−3 PFU/ml, no replicates were detected. Evaluation of TRIzol was discontinued after nine replicates due to a trend of lower sensitivity (at 10−3 PFU/ml eight of nine replicates were positive at 100 PFU/ml, four of nine replicates were positive at 10−1 PFU/ml, and zero of nine replicates were positive at 10−2 PFU/ml). Concordant results were obtained in 24 of 28 clinical specimens after extraction by all methods. No evidence of contamination was observed after extraction by automated instruments. The data indicate that the sensitivity of enterovirus PCR is largely similar after extraction by QIAamp, BioRobot M48, and MagNA Pure; a trend of decreased sensitivity was observed after TRIzol extraction. However, the results of enterovirus PCR were largely concordant in patient samples, indicating that the four extraction methods are suitable for detection of enteroviruses in clinical specimens.

Multicenter Comparison of Different Real-Time PCR Assays for Quantitative Detection of Epstein-Barr Virus

ABSTRACT Quantification of Epstein-Barr virus (EBV) in peripheral blood is important for the diagnosis and management of serious EBV diseases, including posttransplant lymphoproliferative disorder. A variety of PCR-based methods are currently in use; however, there is little information on their comparability. This study assessed the relative performance of different quantitative assays. A multicenter comparative study was performed at eight sites using three panels consisting of serial dilutions of quantified EBV DNA and extracts from a total of 19 whole-blood specimens. Samples were distributed and tested blindly. Instrumentation, probe chemistries, amplification targets, and other test-related aspects varied considerably between laboratories. Each laboratorys calibration curve indicated strong evidence of a consistent log-linear relationship between viral load and cycle threshold, suggesting that intralaboratory tracking of a given patient would yield similar relative quantitative trends among the participating test sites. There was strong concordance among laboratories with respect to qualitative test results; however, marked quantitative discordance was seen. For most samples, the across-laboratory interquartile range of the reported viral load (in copies/μl) was roughly 0.6 log-units, and for one sample the overall range was approximately 4.2 log-units. While intralaboratory tracking of patients may yield similar results, these data indicate a need for caution when attempting to compare clinical results obtained at different institutions and suggest the potential value to be gained by more standardized testing methodology.

Reducing the rate of nosocomially transmitted respiratory syncytial virus

BACKGROUND A large number (17) of nosocomial respiratory syncytial virus cases led to the development of control measures to prevent transmission of respiratory syncytial virus (RSV) within the Johns Hopkins Hospitals Childrens Center. METHODS The control plan is based on a 2-stage process. In stage 1, the staff are notified that RSV is in the community, and information is distributed through a communication tree. Stage 2 requires that nasopharyngeal aspirates be obtained from all children <3 years of age who have respiratory symptoms. The aspirates are tested directly for RSV antigen and cultured for RSV. The children are placed on pediatric droplet precautions pending those results. RESULTS The proportion of nosocomial RSV cases dropped from 16.5% before the use of RSV control measures to 7.2% after the initiation of the control program. A case of RSV identified in the hospital was 2.6 times more likely to be nosocomially acquired before the intervention compared with after the intervention. Approximately 14 cases of RSV are prevented each year, which results in a savings of 56 hospital-days and more than

Detecting respiratory viruses in asymptomatic children

84,000 in direct hospital-related charges alone. CONCLUSIONS The nosocomial spread of RSV can be reduced by a specific and feasible control plan that includes early identification and rapid isolation of potential RSV cases.

Cytomegalovirus resistance to ganciclovir and clinical outcomes of patients with cytomegalovirus retinitis

Background: Viral respiratory infections are among the most common reasons for hospitalization of children in the United States. Our objective was to compare molecular and conventional methods in a cohort of hospitalized children with and without symptoms of respiratory viral illness (RVI). Methods: We conducted a retrospective cohort study of infants and toddlers hospitalized between December 2007 and March 2008 at Johns Hopkins Hospital. Five hundred sixty-nine of 641 patient visits (89%) were tested on admission. Conventional tests (immunochromatography, direct fluorescent antibody, shell vial and tube culture) were performed on all patients and nucleic acid tests (NATs) were performed on available samples (n = 306). Viruses were grouped into those routinely (group 1) and those not routinely (group 2) detected by conventional methods. Results: In children with RVI symptoms (n = 148), NATs identified a virus in 83% of specimens compared with 49% by conventional methods (P < 0.001), but detected a similar percentage of specimens with group 1 viruses (48.6% and 55.4%; P = 0.13) compared with conventional tests. In children without RVI symptoms (n = 158), NATs identified a virus in 41.7% of specimens compared with 4.4% by conventional tests (P < 0.001) and identified more group 1 viruses (9.5% and 4.4%; P = 0.03) compared with conventional tests. Group 2 viruses were identified by NATs in a similar percentage of symptomatic and asymptomatic patients (25% and 32.3%; P = 0.20). Conclusions: Molecular assays may have several advantages over conventional methods for detecting respiratory viruses, including improved sensitivity and rapid detection, but given the high prevalence of positive results in children without RVI symptoms, results should be interpreted cautiously.

Viral Sensitivity Testing in Patients With Cytomegalovirus Retinitis Clinically Resistant to Foscarnet or Ganciclovir

PURPOSE To evaluate whether cytomegalovirus resistant to ganciclovir, detected in either the blood or urine, correlates with adverse ocular outcomes. DESIGN Prospective cohort study. METHODS Patients with cytomegalovirus and AIDS were enrolled in a study of the occurrence and clinical correlates of resistant cytomegalovirus. Blood and urine cultures for cytomegalovirus were performed at the time of diagnosis of retinitis, 1 and 3 months after the initiation of therapy, and every 3 months thereafter. Patients were seen monthly, at which time fundus photographs were obtained and forwarded to the Fundus Photograph Reading Center for evaluation of retinitis progression (movement of a border of a cytomegalovirus lesion > or = 750 microm, or the occurrence of a new lesion > or = 0.25 disk area in size) and the amount of retinal area affected by cytomegalovirus retinitis. Visual acuity was measured using logarithmic visual acuity charts. Phenotypic resistance to ganciclovir was defined as an IC50 > 6.0 micromol/l, and genotypic resistance to ganciclovir was defined as the occurrence of a cytomegalovirus UL97 gene mutation known to confer ganciclovir resistance. Time-dependent analyses were performed and included viral resistance, highly active antiretroviral therapy, and treatment variables as predictors of clinical outcomes. RESULTS One hundred ninety-seven patients received ganciclovir therapy. Nineteen patients developed phenotypic resistance to ganciclovir, and 18 developed genotypic resistance. The detection of cytomegalovirus resistant to ganciclovir was associated with a 4.17- to 5.61-fold increase in the odds of retinitis progression (P values all < or = .0002), depending upon the definition of resistance and the culture sources analyzed. Resistance was associated with a greater increase in retinal area involved by cytomegalovirus by 3-month interval (1.10% vs 0.05% to 0.10%), which was significant for phenotypic resistance and for genotypic resistance in the blood or urine (P =.012 to.021). There was a suggestion that resistance was associated with a greater loss of visual acuity (P =.009 to.096). Highly active antiretroviral therapy was associated with an approximate 50% reduction in the odds of retinitis progression, and the ganciclovir implant was associated with an approximate 60% reduction. CONCLUSIONS The detection of cytomegalovirus resistant to ganciclovir in either the blood or urine of a patient with cytomegalovirus retinitis is associated with an increased risk of adverse ocular outcomes.