Repopulation of ganciclovir-resistant cytomegalovirus by wild-type virus.

We report a patient in whom ganciclovir (GCV)-resistant cytomegalovirus (CMV) was replaced by wild-type virus after discontinuation of GCV/valganciclovir and review other similar cases. Repopulation by wild-type virus may occur soon after discontinuation and may be fostered by discontinuing GCV altogether rather than continuing it in combination with foscarnet when treating patients with GCV-resistant CMV disease.

Cytomegalovirus resistance testing: pitfalls and problems for the clinician.

In this review, I describe the available assays for detecting resistance of cytomegalovirus (CMV) to antivirals and emphasize the practical utility of the genotypic approach. The indications for such testing and the contraindications are discussed, as are the interpretation of results and the evidence of cross-resistance between antivirals.

Pandemic (H1N1) 2009 infection in patients with hematologic malignancy.

To assess outcomes of patients with hematologic malignancy and pandemic (H1N1) 2009 infection, we reviewed cases during June-December 2009 at the University of California San Francisco Medical Center. Seventeen (63%) and 10 (37%) patients had upper respiratory tract infection (URTI) and lower respiratory tract infection (LRTI), respectively. Cough (85%) and fever (70%) were the most common signs; 19% of patients had nausea, vomiting, or diarrhea. Sixty-five percent of URTI patients were outpatients; 35% recovered without antiviral therapy. All LRTI patients were hospitalized; half required intensive care unit admission. Complications included acute respiratory distress syndrome, pneumomediastinum, myocarditis, and development of oseltamivir-resistant virus; 3 patients died. Of the 3 patients with nosocomial pandemic (H1N1) 2009, 2 died. Pandemic (H1N1) 2009 may cause serious illness in patients with hematologic malignancy, primarily those with LRTI. Rigorous infection control, improved techniques for diagnosing respiratory disease, and early antiviral therapy can prevent nosocomial transmission and optimize patient care.

Highly quantitative serological detection of anti-cytomegalovirus (CMV) antibodies.

BACKGROUND: Human cytomegalovirus infection is associated with a variety of pathological conditions including retinitis, pneumonia, hepatitis and encephalitis that may be transmitted congenitally, horizontally and parenterally and occurs both as a primary infection and as reactivation in immunocompromised individuals. Currently, there is a need for improved quantitative serological tests to document seropositivity with high sensitivity and specificity. METHODS: Here we investigated whether luciferase immunoprecipitation systems (LIPS) would provide a more quantitative and sensitive method for detecting anti-CMV antibodies. Four protein fragments of immunodominant regions of CMV antigens pp150 and pp65 were generated as Renilla luciferase (Ruc) fusion proteins and used in LIPS with two cohorts of CMV positive and negative sera samples previously tested by ELISA. RESULTS: Analysis of the antibody responses to two of these antigen fragments, pp150-d1 and pp150-d2, revealed geometric mean antibody titers in the first cohort that were 100-1000 fold higher in the CMV positive sera compared to the CMV negative samples (p < 0.0001) and infection status exactly matched the ELISA results for the 46 samples of the first cohort (100% sensitivity and 100% specificity). Two additional antigen fragments, pp65-d1 and pp65-d2 also showed robust antibody titers in some CMV-infected sera and yielded 50% and 96% sensitivity, respectively. Analysis of a second cohort of 70 samples using a mixture of the 4 antigens, which simplifies data collection and analysis, yielded values which correlated well with the sum of the values from the 4 separate tests (rs = 0.93, p < 0.00001). While comparison of the LIPS results from this second cohort with ELISA showed 100% sensitivity, LIPS detected six additional CMV positive samples that were not detected by ELISA. Heat map analysis revealed that several of the LIPS positive/ELISA negative samples had positive LIPS immunoreactivity with 3-4 of the CMV antigens. CONCLUSION: These results suggest that LIPS provides a highly robust and quantitative method for studying anti-CMV antibodies and has the potential to more accurately document CMV infection than standard ELISA.

Active cytomegalovirus retinitis after the start of antiretroviral therapy.

Patients with AIDS-related cytomegalovirus (CMV) retinitis receiving combined antiretroviral therapy (cART), but not specific anti-CMV therapy, consistently showed active retinitis for several months. Delayed diagnosis and treatment of CMV retinitis may have severe consequences. Patients first entering care with advanced HIV infection and vulnerability to reactivation of latent CMV infection should be screened immediately for CMV retinitis by dilated indirect ophthalmoscopy and treated with specific anti-CMV therapy without delay, in addition to cART.

Utility of DNA microarrays for detection of viruses in acute respiratory tract infections in children.

OBJECTIVE: To assess the utility of a panviral DNA microarray platform (Virochip) in the detection of viruses associated with pediatric respiratory tract infections (RTIs). STUDY DESIGN: The Virochip was compared with conventional direct fluorescent antibody (DFA)- and polymerase chain reaction (PCR)-based testing for the detection of respiratory viruses in 278 consecutive nasopharyngeal aspirate samples from 222 children. RESULTS: The Virochip was superior in performance to DFA, showing a 19% increase in the detection of 7 respiratory viruses included in standard DFA panels, and was similar to virus-specific PCR (sensitivity, 85% to 90%; specificity, >/=99%; positive predictive value, 94% to 96%; negative predictive value, 97% to 98%) in the detection of respiratory syncytial virus, influenza A, and rhinoviruses/enteroviruses. The Virochip also detected viruses not routinely tested for or missed by DFA and PCR, as well as double infections and infections in critically ill patients that DFA failed to detect. CONCLUSIONS: Given its favorable sensitivity and specificity profile and expanded spectrum for detection, microarray-based viral testing holds promise for clinical diagnosis of pediatric RTIs.

Development of new cytomegalovirus UL97 and DNA polymerase mutations conferring drug resistance after valganciclovir therapy in allogeneic stem cell recipients.

BACKGROUND: We report on two allogeneic stem cell transplant recipients who developed cytomegalovirus disease associated with new viral mutations that conferred antiviral drug resistance. METHODS: Blood specimens obtained during symptomatic disease were analyzed for mutations in the CMV UL97 and DNA polymerase genes and new mutations were assessed by recombinant phenotyping. RESULTS: Rising cytomegalovirus (CMV) antigenemia occurred after 4-5 months of preemptive valganciclovir therapy, followed by symptomatic CMV disease including fatal pneumonia in one case. In one case, a new viral UL97 mutation (deletion of codons 601-603) was found which conferred 15-fold increased ganciclovir resistance. In the other case, a known UL97 resistance mutation M460V and a new DNA polymerase (pol) mutation D413A were found. D413A conferred ganciclovir and cidofovir resistance. CONCLUSIONS: Known and newly discovered drug resistance mutations arising during preemptive therapy may complicate post-transplant CMV disease in stem cell recipients. Improved recombinant phenotyping methods enable the rapid quantitation of the resistance conferred by newly identified UL97 and pol mutations.

Is combination antiviral therapy for CMV superior to monotherapy?

BACKGROUND: Many clinicians are under the impression that the combination of ganciclovir (GCV) and foscarnet is synergistic versus cytomegalovirus (CMV) and/or that combination therapy might prevent the emergence of resistance to one or both antivirals. The combination is frequently used when resistance to either drug is suspected. OBJECTIVE: To review in vitro and clinical data regarding the activity of ganciclovir plus foscarnet and evidence of synergy between the two drugs. STUDY DESIGN: We reviewed two in vitro studies of synergy between ganciclovir and foscarnet followed by reviewing all clinical studies utilizing series of patients. RESULTS: The combination of ganciclovir and foscarnet was synergistic against three clinical isolates, the Towne strain and one laboratory derived strain moderately resistant to GCV but synergy was not demonstrated against laboratory derived strains highly resistant to GCV or foscarnet. AD169, susceptible to both drugs, was not inhibited synergistically by the combination in one study but was in the second study. In the only carefully controlled in vivo study of combination versus monotherapy for GCV susceptible viremia superiority of the combination was not demonstrated. In treating clinically resistant CMV retinitis, the combination was superior to continued or alternative monotherapy. CONCLUSION: There is suggestive but inconclusive evidence of in vitro synergy for the combination of GCV and foscarnet versus CMV with very limited data versus GCV resistant virus. The in vivo data for synergy is even less convincing. Additional in vitro and in vivo data is needed, especially to prevent or treat CMV resistance.

Maribavir sensitivity of cytomegalovirus isolates resistant to ganciclovir, cidofovir or foscarnet.

BACKGROUND: The cytomegalovirus (CMV) UL97 inhibitor drug maribavir (MBV) is undergoing clinical antiviral trials. OBJECTIVES: To assess the MBV sensitivity of CMV strains and isolates containing mutations that confer resistance to current antiviral drugs ganciclovir, cidofovir or foscarnet. STUDY DESIGN: Resistant clinical isolates and laboratory strains containing UL97 and or UL54 DNA polymerase mutations were tested for sensitivity to all four drugs by standard plaque reduction assay and a reporter-based yield reduction assay. Sensitive control strains were also tested. RESULTS: Eleven CMV strains or isolates resistant to GCV, four resistant to FOS and two resistant to CDV, were all sensitive to MBV. These viruses represent four UL97 mutations and three UL54 DNA polymerase mutations. The laboratory derived UL97 L397R mutant was highly MBV-resistant but remained sensitive to the other three drugs. CONCLUSIONS: No cross-resistance has been detected between viruses resistant to MBV and those resistant to one or more of the current CMV antiviral drugs, consistent with differences in their mechanisms of action.

The role of photochemical treatment with amotosalen and UV-A light in the prevention of transfusion-transmitted cytomegalovirus infections.

Primary cytomegalovirus (CMV) infection is usually asymptomatic in immunocompetent patients but can cause serious life-threatening complications in immunocompromised CMV-seronegative patients, including patients receiving a bone marrow or peripheral blood stem cell transplant, recipients of some solid-organ transplants, and low-birth-weight neonates. Current recommendations for preventing transfusion-transmitted CMV (TT-CMV) infection in these patients include exclusive use of CMV-seronegative and/or leukoreduced cellular blood components (red blood cells and platelets) for transfusion. However, breakthrough cases of TT-CMV still occur. Despite improving the safety of blood components, testing remains a reactive approach to blood safety. In contrast, pathogen inactivation technologies offer a proactive approach with the potential to further improve blood safety. To reduce the risks associated with platelet transfusions, a photochemical treatment (PCT) process using a combination of the psoralen amotosalen HCl and long-wavelength UV light has been developed and introduced into clinical practice in Europe. PCT has been shown to result in greater than 5.9-log reductions in infectivity of human CMV in platelet concentrates and to prevent the transfusion transmission of murine CMV in a mouse transfusion model. Thus, PCT pathogen inactivation may play a role in further reducing the incidence of TT-CMV infection in patients who are at risk for serious CMV disease. Because PCT is a technology that targets nucleic acids, it also offers a proactive process for the inactivation of a broad range of viral, bacterial, and protozoan pathogens in addition to CMV.

Characterization of viral agents causing acute respiratory infection in a San Francisco University Medical Center Clinic during the influenza season.

BACKGROUND: With use of polymerase chain reaction (PCR) and a centrifugation-enhanced viral culture method, we characterized the viruses causing acute respiratory infection in adults during an influenza season. METHODS: During January-March 2002, nasopharyngeal wash specimens from previously healthy adults presenting with respiratory symptoms were evaluated for viral pathogens with centrifugation-enhanced viral culture and PCR. RESULTS: The diagnoses in 266 cases included unspecified upper respiratory infection (in 142 [54%] of the cases), acute bronchitis (42 [16%]), sinusitis (23 [9%]), pharyngitis (22 [8%]), and pneumonia (17 [6%]). The use of a shell vial assay and PCR identified a pathogen in 103 (39%) of the patients, including influenza A or B in 54, picornavirus in 28 (including rhinovirus in 24), respiratory syncytial virus (RSV) in 12, human metapneumovirus in 4, human coronavirus OC43 in 2, adenovirus in 2, parainfluenza virus type 1 in 1, and coinfection with influenza and parainfluenza virus type 1 in 2. CONCLUSION: Our findings demonstrate that, even during the influenza season, rhinovirus and RSV are prevalent and must be considered in the differential diagnosis of adult acute respiratory infection before prescribing antiviral medication. Human coronavirus and human metapneumovirus did not play a substantial role. PCR was an especially useful tool in the identification of influenza and other viral pathogens not easily detected by traditional testing methods.

Performance characteristics of clinical diagnosis, a clinical decision rule, and a rapid influenza test in the detection of influenza infection in a community sample of adults.

STUDY OBJECTIVE: The accurate diagnosis of influenza remains a diagnostic dilemma. We examine the performance of various strategies for diagnosing influenza infection in an unselected sample of adults during influenza season. METHODS: Consecutive adults presenting to a university emergency department or urgent care clinic between January and March 2002 with acute respiratory complaints were eligible for this prospective observational study. The performance of clinician judgment, a rapid influenza test, and a clinical prediction rule in predicting influenza infection was evaluated using referent standard of reverse transcriptase polymerase chain reaction. Statistical significance was assessed using McNemar's test of proportions. RESULTS: Fifty-three of 258 (21%) patients had a positive influenza reverse transcriptase polymerase chain reaction test. Overall, clinician judgment showed sensitivity of 29% (95% confidence interval [CI] 18% to 43%) and specificity of 92% (95% CI 87% to 95%). The rapid influenza test showed a sensitivity of 33% (95% CI 22% to 47%) and specificity of 98% (95% CI 96% to 99%). The clinical prediction rule showed a sensitivity of 40% (95% CI 27% to 54%) and specificity of 92% (95% CI 87% to 95%). Clinician judgment when patients presented within 48 hours showed a sensitivity of 67% (95% CI 39% to 86%) and specificity of 96% (95% CI 81% to 99%). Neither the rapid influenza test (P=.10) nor the clinical prediction rule (P=.42) was superior to clinician judgment alone in the diagnosis of influenza. CONCLUSION: The suggestion that a clinical decision rule or a rapid influenza test is better than clinical judgment alone for the diagnosis of influenza in an unselected patient population is not supported by this study.

Performance of a bedside C-reactive protein test in the diagnosis of community-acquired pneumonia in adults with acute cough.

PURPOSE: To evaluate the performance of a rapid, bedside whole blood C-reactive protein test as a diagnostic test for pneumonia in adults. METHODS: We enrolled consecutive adults who presented with acute cough (duration < or =3 weeks). A fingerstick blood specimen for C-reactive protein level was obtained. Patients also provided information about demographic characteristics and symptoms. Physical examination findings, diagnoses, and treatments were abstracted from the medical record; illness duration and subsequent office visits were determined with follow-up telephone calls. A clinical prediction rule for pneumonia was calculated for each patient and compared with C-reactive protein levels. RESULTS: Twenty (12%) of the 168 patients in the study had radiographic evidence of pneumonia. Median C-reactive protein levels were significantly higher for patients with pneumonia than in the remaining patients (60 mg/L vs. 9 mg/L, P <0.0001). The area under the receiver operating characteristic (ROC) curve for C-reactive protein level as a predictor of pneumonia was 0.83. C-reactive protein level and the clinical prediction rule were independently associated with pneumonia, yielding a combined area under the ROC curve of 0.93. C-reactive protein level was not associated with hospitalization or resolution of symptoms. CONCLUSION: C-reactive protein levels could be a valuable addition to clinical prediction rules for pneumonia. A C-reactive protein level > or =100 mg/L might be a useful indication for chest radiography or empiric antibiotic therapy when the diagnosis of pneumonia is in doubt.

High dose oral ganciclovir treatment for cytomegalovirus retinitis.

BACKGROUND: The oral formulation of ganciclovir is approved at a dose of 3.0 g/day for maintenance treatment of cytomegalovirus (CMV) retinitis following an initial induction course of intravenous (IV) anti-CMV therapy. Median time to progression of CMV retinitis is 12-20 days shorter with oral compared to IV ganciclovir maintenance, likely due to the limited oral bioavailability of ganciclovir. OBJECTIVES: We hypothesized that higher systemic drug exposures associated with increased doses of oral ganciclovir would be associated with increased efficacy. STUDY DESIGN: Maintenance treatment of CMV retinitis with higher than standard doses of oral ganciclovir (>3.0 g/day) was studied in 281 AIDS patients with previously treated, stable retinitis randomized to 3.0, 4.5 or 6.0 g/day oral, or 5 m/kg/day IV ganciclovir. Graders unaware of treatment assignments determined retinitis progression using fundus photographs. Vision, other ophthalmic measures and safety were assessed open-label. RESULTS: Median days to photographic progression were 41, 50, 57 and 70, respectively (P=0.052; 3.0 g vs. IV). Hazard ratios for progression relative to IV were 1.66, 1.28 and 1.19 (P=0.016 for 3.0 g). NONMEM-modeled estimates of average serum ganciclovir concentration area under the curve (AUC(0-24)) correlated best with time to progression (P=0.0019). Six grams per day oral ganciclovir was most similar in efficacy to IV, although broad confidence intervals prevented a conclusive comparison. Patients receiving oral ganciclovir had a lower frequency of sepsis and IV catheter events. CONCLUSIONS: This study suggests that the efficacy of ganciclovir for the maintenance treatment of CMV retinitis improves with increasing total drug exposure (measured as average serum concentration AUC(0-24)). All four regimens of ganciclovir were reasonably well tolerated, with safety profiles similar to what has been reported in prior work.

CMV retinitis screening and treatment in a resource-poor setting: three-year experience from a primary care HIV/AIDS programme in Myanmar.

BACKGROUND: Cytomegalovirus retinitis is a neglected disease in resource-poor settings, in part because of the perceived complexity of care and because ophthalmologists are rarely accessible. In this paper, we describe a pilot programme of CMV retinitis management by non-ophthalmologists. The programme consists of systematic screening of all high-risk patients (CD4 <100 cells/mm3) by AIDS clinicians using indirect ophthalmoscopy, and treatment of all patients with active retinitis by intravitreal injection of ganciclovir. Prior to this programme, CMV retinitis was not routinely examined for, or treated, in Myanmar. METHODS: This is a retrospective descriptive study. Between November 2006 and July 2009, 17 primary care AIDS clinicians were trained in indirect ophthalmoscopy and diagnosis of CMV retinitis; eight were also trained in intravitreal injection. Evaluation of training by a variety of methods documented high clinical competence. Systematic screening of all high-risk patients (CD4 <100 cells/mm3) was carried out at five separate AIDS clinics throughout Myanmar. RESULTS: A total of 891 new patients (1782 eyes) were screened in the primary area (Yangon); the majority of patients were male (64.3%), median age was 32 years, and median CD4 cell count was 38 cells/mm3. CMV retinitis was diagnosed in 24% (211/891) of these patients. Bilateral disease was present in 36% of patients. Patients with active retinitis were treated with weekly intravitreal injection of ganciclovir, with patients typically receiving five to seven injections per eye. A total of 1296 injections were administered. CONCLUSIONS: A strategy of management of CMV retinitis at the primary care level is feasible in resource-poor settings. With appropriate training and support, CMV retinitis can be diagnosed and treated by AIDS clinicians (non-ophthalmologists), just like other major opportunistic infections.

Laboratory diagnosis of cytomegalovirus infection and disease in immunocompromised patients.

PURPOSE OF REVIEW: To review new developments in PCR technology as they apply to detecting cytomegalovirus viremia and pneumonia, recent advances in detecting CMV resistance to antivirals and assays of specific CMV lymphocyte function. RECENT FINDINGS: This review summarizes the attempts to use real time PCR for cytomegalovirus deoxyribonucleic acidemia and to compare it to conventional PCR and antigenemia, it also reviews the use of quantitative PCR on bronchoalveolar lavage to assist in the diagnosis of CMV pneumonia. Phenotypic assays of susceptibility in tissue culture are much too slow to assist clinical decisions, taking weeks for completion. Genotypic assays may be performed directly on clinical samples such as blood, and cerebrospinal fluid and can be done by sequencing in a very few days.Finally, assays of lymphocytic functional responsiveness to cytomegalovirus can be used to identify transplant recipients at continuing risk for cytomegalovirus disease. SUMMARY: Assays for CMV DNA or antigen in blood are superior to culture for documenting viremia and pneumonia. Genotypic assays have largely replaced phenotypic assays for CMV resistance to antivirals. Lymphocyte responses to CMV antigen(s) may identify patients at risk for CMV disease.