Clinical performance of the GenMark Dx ePlex respiratory pathogen panels for upper and lower respiratory tract infections.

The GenMark Dx ePlex Respiratory Pathogen Panel (RP) is a multiplexed nucleic acid test for the qualitative detection of common viral and a few bacterial causes of respiratory tract infections. The ePlex RP has received FDA clearance for nasopharyngeal swab (NPS) specimens collected in viral transport media. In this study, we evaluated the performance of the ePlex RP panel in comparison to the NxTAG Respiratory Pathogen Panel (NxTAG-RPP) from Luminex in use in our laboratory, not only for NPS but also for bronchoalveolar lavage specimens (BAL). We also evaluated the impact of implementing the ePlex RP on the test turn-around time (TAT). The newest panel from GenMark Dx, the ePlex Respiratory Pathogen Panel 2 (RP2), which added the SARS-CoV-2 target to the RP was also evaluated for NPS. Verification of the performance of the ePlex RP for both NPS and BAL showed 93.3 % and 84.9 % total agreement with the NxTAG-RPP respectively. An overall comparison of the TAT after implementing the ePlex RP as compared to the NxTAG-RPP assay showed an average decrease of almost seven-fold.

Outcomes of transplant recipients treated with cidofovir for resistant or refractory cytomegalovirus infection.

BACKGROUND: Treatment of ganciclovir-resistant (GCV-R)/refractory cytomegalovirus (CMV) infections in blood/marrow transplant (BMT) and solid organ transplant (SOT) recipients remains suboptimal. Cidofovir (CDV), a nucleotide analogue with anti-CMV activity, is nephrotoxic and oculotoxic. METHODS: We retrospectively evaluated the outcomes of SOT and BMT patients with GCV-R/refractory CMV treated with CDV between 1/1/2008 and 12/31/2017. DATA COLLECTED: baseline demographics, CMV serostatus, clinical and virologic presentations and outcomes, UL97 and UL54 genotype mutations, drug toxicities, and cause of death. Descriptive statistics were used. RESULTS: 16 patients received CDV for treatment of CMV: six BMT and 10 SOT. Seven (47%) of the patients had high-risk donor/recipient serostatus: six (60%) SOT were D+/R-; one (16.7%) BMT was D-/R+. Median time to CMV DNAemia was 131 days post-transplant (IQR, 37.5-230.3). Proven tissue invasive disease was present in three patients (18.8%). Twelve (75%) had genotype testing; 10 (83.3%) of those had antiviral resistance mutations. While on CDV, six (37.5%) developed nephrotoxicity, and four (25%) developed uveitis (two had both uveitis and nephrotoxicity). Eight (50%) had failure to clear CMV DNAemia despite CDV treatment. Eight (50%) of the patients died; median time to death, after initiation of CDV, was 33.5 days [IQR22-988]. CONCLUSIONS: In the absence of good therapeutic alternatives, CDV is used in GCV-R/refractory CMV infection. However, it is associated with a substantial risk of toxicity and failure to clear CMV DNAemia, highlighting the need for development of newer and less toxic therapies. The high mortality in this group of patients underscores the severity of illness in this population.

Probable transmission of hepatitis E virus (HEV) via transfusion in the United States.

BACKGROUND: Hepatitis E virus (HEV) can inapparently infect blood donors. To assess transfusion transmission of HEV in the United States, which has not been documented, a donor-recipient repository was evaluated. STUDY DESIGN AND METHODS: To identify donations that contained HEV RNA and were linked to patient-recipients with antibody evidence of HEV exposure, we assayed samples from the Retrovirus Epidemiology Donor Study (REDS) Allogeneic Donor and Recipient repository that represents 13,201 linked donations and 3384 transfused patients. Posttransfusion samples, determined to contain IgG anti-HEV by enzyme-linked immunosorbent assay, were reassayed along with corresponding pretransfusion samples for seroconversion (incident exposure) or at least fourfold IgG anti-HEV increase (reexposure). HEV-exposed patients were linked to donations in which HEV RNA was then detected by reverse-transcription quantitative polymerase chain reaction, confirmed by transcription-mediated amplification, and phylogenetically analyzed as subgenomic cDNA sequences. RESULTS: Among all patients, 19 of 1036 (1.8%) who had IgG anti-HEV before transfusion were reexposed; 40 of 2348 (1.7%) without pretransfusion IgG anti-HEV seroconverted. These 59 patients were linked to 257 donations, 1 of which was positive by reverse-transcription quantitative polymerase chain reaction and transcription-mediated amplification. Plasma from this donation contained 5.5 log IU/mL of HEV RNA that grouped with HEV genotype 3, clade 3abchij. The patient-recipient of RBCs from this donation had a greater than eightfold IgG increase; however, clinical data are unavailable. CONCLUSIONS: This is the first report of probable HEV transmission via transfusion in the United States, although it has been frequently observed in Europe and Japan. Additional data on the magnitude of the risk in the United States are needed.

Comparison of Hepatitis B Virus Infection in HIV-Infected and HIV-Uninfected Participants Enrolled in a Multinational Clinical Trial: HPTN 052.

OBJECTIVE: Data comparing hepatitis B virus (HBV) infection in HIV-infected [HIV(+)], and HIV-uninfected [HIV(-)] individuals recruited into the same study are limited. HBV infection status and chronic hepatitis B (cHB) were characterized in a multinational clinical trial: HIV Prevention Trials Network (HPTN 052). METHOD: HBV infection status at enrollment was compared between HIV(+) (N = 1241) and HIV(-) (N = 1232) from 7 HBV-endemic countries. Hepatitis B e antigen and plasma HBV DNA were determined in cHB. Median CD4, median plasma HIV RNA, and prevalence of transaminase elevation were compared in HIV(+) with and without cHB. Significance was assessed with χ, Fisher exact, and median tests. RESULTS: Among all participants, 33.6% had HBV exposure without cHB (8.9% isolated HBV core antibody, "HBcAb"; 24.7% HBcAb and anti-HB surface antibody positive, "recovered"), 4.3% had cHB, 8.9% were vaccinated, and 53.5% were uninfected. Data were similar among HIV(+) and HIV(-) except for isolated HBcAb, which was more prevalent in HIV(+) than HIV(-) [10.1% vs. 7.7%, P = 0.046]. Median HBV DNA trended higher in HIV(+) than in HIV(-). In HIV(+) with cHB versus those without cHB, transaminase elevations were more prevalent (alanine aminotransferase ≤ grade 2, 12% vs. 5.2%, P = 0.037; aspartate aminotransferase ≤ grade 2, 26% vs. 6.0%, P < 0.001), CD4 trended lower, and HIV RNA was similar. CONCLUSIONS: HBV infection status did not differ by HIV infection status. HIV co-infection was associated with isolated HBcAb and a trend of increased HBV DNA. In HIV, cHB was associated with mild transaminase elevations and a trend toward lower CD4.

Cytomegalovirus Kinetics Following Primary Infection in Healthy Women.

The kinetics of cytomegalovirus (CMV) DNA in infected asymptomatic hosts are largely unknown. We measured viral load (VL) in 124 fluid samples (oral, urine, vaginal, blood) collected from 21 women who acquired CMV. A quantitative real-time polymerase chain reaction assay of US17, which correlated with clinical assays, was used. VL decreased following primary infection in all fluids. The geometric mean VL of vaginal fluid was significantly higher than that of other sources: oral (3.89; 95% confidence interval [CI], 1.43-10.57), urine (6.36; 95% CI, 2.48-16.32), and whole blood (11.88; 95% CI, 4.12-34.20). Vaginal CMV shedding may provide a route for sexual and possibly perinatal transmission.

Absence of Cytomegalovirus in Glioblastoma and Other High-grade Gliomas by Real-time PCR, Immunohistochemistry, and In Situ Hybridization.

Purpose: Reports of cytomegalovirus (CMV) detection in high-grade gliomas (HGG)/glioblastoma have been conflicting. We undertook a comprehensive approach to determine the presence or absence of CMV in tissue, plasma, and serum of HGG patients.Experimental Design: In a retrospective arm, 25 fresh frozen tissues from glioblastoma patients were tested for CMV by real-time PCR. Tissue microarrays from 70 HGG patients were tested by IHC and 20 formalin-fixed paraffin-embedded (FFPE) glioblastoma tissues by IHC and chromogenic in situ hybridization (CISH), targeting CMV-encoded IE1/2 and pp65. In a prospective arm, 18 patients with newly diagnosed HGG provided tissue and blood samples.Results: All retrospectively collected tissues were negative for CMV by all methods. In the prospective cohort, 18 patients with newly diagnosed HGG provided blood samples at the time of diagnosis and during follow-up. Of 38 plasma specimens, CMV DNA was detected in 3 of 18 samples at baseline and 1 of 20 follow-up samples. Serum CMV IgG was positive in 8 of 15 (53%) of patients. Among the FFPE samples tested in the prospective arm, all were negative for CMV by IHC, CISH, and PCR.Conclusions: Utilizing 6 highly sensitive assays with three orthogonal technologies on multiple specimens and specimen types, no evidence for CMV in glioblastoma tissues was found. Our findings call for multicenter blinded analyses of samples collected from different geographical areas with agreed upon study designs and determination of causality or lack thereof of CMV in HGG/glioblastoma for future guidance on the necessary antiviral and/or CMV-based therapies. Clin Cancer Res; 23(12); 3150-7. ©2016 AACR.

Variability between credit units dedicated to dental and clinical sciences in dental schools across the USA.

PURPOSE: The Commission of Dental Accreditation (CODA) does not set minimum standards for clock hours of training in Dental and Clinical sciences. The purpose of this evaluation was to compare United States (US) dental schools for variability in clock hours. The current paper utilizes the American Dental Association's survey of clock hours of all US dental schools which is publicly available data. Clock hours survey from 2010 to 2011 was utilized and the analysis tool, JMP, was utilized to visualize and report variability. PERSPECTIVE: The current paper highlights the large variation in clock hours of training among core clinical subjects in accredited dental schools around the United States. For example, teaching Physical Evaluations; Oral and Maxillofacial; and Oral Diagnosis and Treatment Planning were 97.0; 126.6; and 74.4 h. Moreover, upper limit for hours of Operative Dentistry teaching was 1410 h and lower limit was 129 h. Various other fields of education do enforce strict requirements on educational clock hours. For instance, Massachusetts' General Law states that both private and public schools must have 900 and 990 h in a school year for elementary and secondary schools, respectively. However, no such stipulation exists in the field of Dental Education. CODA's mission is "to serve the oral health care needs of the public" and CODA must consider if the average dental patient would consider a dentist who attended the school delivering 1410 h of Operative Dentistry to be the same standard as a graduate of the school delivering 129 h.

The Accuracy of Clinical Diagnosis of Oral Lesions and Patient-Specific Risk Factors that Affect Diagnosis.

PURPOSE: To examine the rate of discrepancy between clinical impression and histologic diagnosis of oral lesions in patients undergoing biopsy examination and to determine whether there are patient-specific variables associated with a higher rate of discrepancy. MATERIALS AND METHODS: The authors designed and implemented a retrospective cohort study that consisted of patients who underwent biopsy examination of oral lesions from 2005 through 2013 by oral and maxillofacial surgeons at the Massachusetts General Hospital. Accuracy was determined by comparing the clinical impression with the final histologic diagnosis. Clinical and histologic diagnoses were categorized as premalignant or malignant (group 1) or benign (group 2). The primary outcome variable was concordance (yes vs no) between clinical impression and histopathologic diagnosis. The effect of individual predictor variables (age, gender, duration, American Society of Anesthesiology status, cancer history, radiation therapy history, medications, alcohol abuse, and tobacco history) on outcome also was evaluated through univariate and multivariate regression analyses. RESULTS: The study sample was composed of 1,003 oral lesions (74 pathologically confirmed premalignant or malignant and 929 benign) from patients with a mean age of 44.8 years. Of the lesions evaluated, concordance between exact clinical and histologic diagnoses was found in 61% of cases. Overall, the clinical impression, reported as benign versus premalignant or malignant, was 48.6% sensitive and 98.1% specific. Clinicians accurately identified lesions as benign in 95.9% of cases. The most common of these were fibromas (positive predictive value [PPV], 99.2%), mucoceles (PPV, 98.1%), and squamous papillomas (PPV, 96.3%). Several independent risk factors were associated with discrepancy: radiation therapy history (P = .0102), male gender (P = .0381), and patient age (P = .0468). CONCLUSION: The results of this study suggest that the clinical impression, although highly accurate for common benign conditions, is not an acceptable alternative to definitive biopsy findings in other cases, particularly in cases of premalignancy or malignancy. In addition, patients with identified independent risk factors (age, gender, and radiation therapy) should receive timely biopsy examination.

Diagnostic performance of two highly multiplexed respiratory virus assays in a pediatric cohort.

BACKGROUND: Rapid detection of respiratory viruses is important for management and infection control in hospitalized patients. Multiplex nucleic acid tests (NATs) have begun to replace conventional methods as gold standards for respiratory virus detection. OBJECTIVE: To compare the performance of two large multiplex NATS, ResPlex II (RPII) and Respiratory Virus Surveillance kit with electrospray ionization mass spectrometry (RVS/MS) using nasopharyngeal aspirates (NPAs) from hospitalized children who had been tested previously with conventional methods. STUDY DESIGN: Stored residual NPAs (N=306) were tested concomitantly by RPII and RVS/MS. Alternate NATs were used to adjudicate discordant results. RESULTS: More viruses were detected with multiplex NATs (RPII, 110; RVS/MS, 109) than conventional assays (86); diagnostic gain was primarily for fastidious viruses (coronaviruses and enteroviruses [EVs]/human rhinoviruses [HRVs]). Total positive and negative agreement between the multiplex NATs for all viruses detected was quite high (86% positive agreement, 99% negative agreement). Most individual viruses were detected with fairly equivalent accuracy by the multiplex NATs, except for adenoviruses (RPII sensitivity 40%) and human metapneumovirus (RVS/MS sensitivity 42%). RPII had the advantage of detecting EVs and HRVs, however, it demonstrated considerable EV/HRV cross-reactivity (29 HRV-positive specimens by real-time PCR were positive for EV by RPII and 21 specimens positive for HRV only by RT-PCR were dual positive for EV/HRV by RPII). RPII also had reduced sensitivity for HRV detection (in 36 specimens, HRV was detected by RT-PCR but not by RPII). CONCLUSIONS: Both multiplex NATs were promising, but had notable limitations.

Detection of a single identical cytomegalovirus (CMV) strain in recently seroconverted young women.

BACKGROUND: Infection with multiple CMV strains is common in immunocompromised hosts, but its occurrence in normal hosts has not been well-studied. METHODS: We analyzed CMV strains longitudinally in women who acquired CMV while enrolled in a CMV glycoprotein B (gB) vaccine trial. Sequencing of four variable genes was performed in samples collected from seroconversion and up to 34 months thereafter. RESULTS: 199 cultured isolates from 53 women and 65 original fluids from a subset of 19 women were sequenced. 51 women were infected with one strain each without evidence for genetic drift; only two women shed multiple strains. Genetic variability among strains increased with the number of sequenced genetic loci. Nevertheless, 13 of 53 women proved to be infected with an identical CMV strain based on sequencing at all four variable genes. CMV vaccine did not alter the degree of genetic diversity amongst strains. CONCLUSIONS: Primary CMV infection in healthy women nearly always involves shedding of one strain that remains stable over time. Immunization with CMVgB-1 vaccine strain is not selective against specific strains. Although 75% of women harbored their unique strain, or a strain shared with only one other woman, 25% shared a single common strain, suggesting that this predominant strain with a particular combination of genetic loci is advantageous in this large urban area.

Gangliosides block Aggregatibacter Actinomycetemcomitans leukotoxin (LtxA)-mediated hemolysis.

Aggregatibacter actinomycetemcomitans is an oral pathogen and etiologic agent of localized aggressive periodontitis. The bacterium is also a cardiovascular pathogen causing infective endocarditis. A. actinomycetemcomitans produces leukotoxin (LtxA), an important virulence factor that targets white blood cells (WBCs) and plays a role in immune evasion during disease. The functional receptor for LtxA on WBCs is leukocyte function antigen-1 (LFA-1), a ß-2 integrin that is modified with N-linked carbohydrates. Interaction between toxin and receptor leads to cell death. We recently discovered that LtxA can also lyse red blood cells (RBCs) and hemolysis may be important for pathogenesis of A. actinomycetemcomitans. In this study, we further investigated how LtxA might recognize and lyse RBCs. We found that, in contrast to a related toxin, E. coli α-hemolysin, LtxA does not recognize glycophorin on RBCs. However, gangliosides were able to completely block LtxA-mediated hemolysis. Furthermore, LtxA did not show a preference for any individual ganglioside. LtxA also bound to ganglioside-rich C6 rat glioma cells, but did not kill them. Interaction between LtxA and C6 cells could be blocked by gangliosides with no apparent specificity. Gangliosides were only partially effective at preventing LtxA-mediated cytotoxicity of WBCs, and the effect was only observed when a high ratio of ganglioside:LtxA was used over a short incubation period. Based on the results presented here, we suggest that because of the similarity between N-linked sugars on LFA-1 and the structures of gangliosides, LtxA may have acquired the ability to lyse RBCs.

Mortality associated with resistant cytomegalovirus among patients with cytomegalovirus retinitis and AIDS.

OBJECTIVE: To evaluate the effect of drug-resistant cytomegalovirus (CMV) on survival among patients with CMV retinitis. DESIGN: Prospective cohort study during 1993 to 2003. PARTICIPANTS: We included 266 patients with AIDS and newly diagnosed CMV retinitis treated with either ganciclovir or foscarnet. METHODS: Data on ganciclovir and foscarnet resistance were obtained from blood and urine specimens collected at regular, predetermined intervals. The effect of resistant CMV on mortality was evaluated with a time-dependent Cox proportional hazard model. MAIN OUTCOME MEASURES: Mortality. RESULTS: The median survival of the entire cohort was 12.6 months. Analysis of risk factors for mortality demonstrated that resistant CMV was associated with an increased mortality (hazard ratio, 1.65; 95% confidence interval, 1.05-2.56; P = 0.032). Among the other parameters tested, only time since AIDS diagnosis was associated significantly with mortality, with a hazard ratio of 1.10 per year since AIDS diagnosis (P = 0.001). CONCLUSIONS: Resistant CMV is associated with increased mortality among patients with AIDS being treated for CMV retinitis. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.

Evaluation of an automated, highly sensitive, real-time PCR-based assay (COBAS Ampliprep/COBAS TaqMan) for quantification of HCV RNA.

BACKGROUND: Diagnosis of hepatitis C virus (HCV) infection and its therapy is based on qualitative and quantitative measurement of HCV RNA. OBJECTIVES: A new assay that employs automated specimen extraction and real-time RT-PCR (COBAS Ampliprep/COBAS TaqMan, "CAP/CTM", Roche Diagnostics, Pleasanton, USA) was designed for linear quantification and highly sensitive detection of HCV RNA. STUDY DESIGN: The performance characteristics of CAP/CTM were compared to standard RT-PCR-based COBAS Amplicor Monitor 2.0 (CAM) assay in a multicenter study. RESULTS: The limit of detection of CAP/CTM was 7.4 IU/ml (95% CI 6.2-10.6) and clinical specificity was 99%. The linear range of HCV RNA quantification by CAP/CTM was between 28 and 1.4 x 10(7) IU/ml, with a correlation coefficient between expected and observed results of >0.99. A fivefold dilution of serum- or plasma-samples showed a linear correlation of HCV RNA levels in undiluted and diluted samples. Analyses of the mean intra- and inter-assay imprecision within the linear range of quantification showed a coefficient of variation of 3% and 3%, respectively. HCV genotypes 1a/b, 2b, 3a, 4, 5 and 6 were equally quantified by the CAP/CTM and CAM assay with mean deviations ranging from -0.29log(10) to 0.32log(10) IU/ml. HCV RNA quantification by CAP/CTM and CAM was highly concordant (correlation coefficient of 0.96). CONCLUSIONS: The CAP/CTM assay is a reliable and robust assay for highly sensitive detection and quantification of HCV RNA within a broad linear range.

Performance characteristics of a quantitative hepatitis C virus RNA assay using COBAS AmpliPrep total nucleic acid isolation and COBAS taqman hepatitis C virus analyte-specific reagent.

Performance characteristics of a hepatitis C virus (HCV) RNA quantification assay comprised automated specimen extraction [COBAS AmpliPrep (CAP) using total nucleic acid isolation reagents (TNAI)], and real-time polymerase chain reaction [COBAS TaqMan 48 HCV with analyte-specific reagents (CTM48)] were determined. CAP TNAI/CTM48 performed linearly from approximately 2.0 to at least 6.7 log10 IU/ml for HCV genotypes (Gts) 1, 2, and 3. The limit of detection for the World Health Organization International Standard was 23 IU/ml. Variabilities ranged from 1.3 to 2.1%. Excellent quantitative agreement was observed in clinical samples using CTM48 and two different methods for HCV RNA extraction (CAP TNAI and BioRobot M48; regression line slope, 0.98; y-intercept, 0.11; R2, 0.98; mean difference, 0.003). Good agreement was also observed between CAP TNAI/CTM48 and COBAS Amplicor Monitor (regression line slope, 0.94; y-intercept, 0.08; R2, 0.96), although HCV RNA concentrations were on average greater by COBAS Amplicor Monitor (mean difference -0.27 log10 IU/ml). Better overall agreement was observed for Gt 1 than non-Gt 1 specimens when comparing extraction and quantification methods; however, no consistent genotype-dependent quantification bias was observed. These data suggest that CAP TNAI/CTM48 offers an alternative method for the quantification of HCV in plasma samples.

Change over time in incidence of ganciclovir resistance in patients with cytomegalovirus retinitis.

BACKGROUND: In the mid-1990s, the incidence of cytomegalovirus (CMV) resistance to ganciclovir was estimated to be approximately 25% by 1 year after diagnosis of retinitis in patients with acquired immunodeficiency syndrome. METHODS: Two hundred fifty-seven patients with CMV retinitis were enrolled in a prospective observational study during 1993-2003 and were treated with ganciclovir. Demographic characteristics and data on CMV disease, antiretroviral therapy, and ganciclovir resistance were recorded for all patients. Human immunodeficiency virus (HIV) load and CMV load were measured for patients enrolled in 1996 or later. Kaplan-Meier and Cox proportional hazards regression methods were used to examine incidence of resistance. RESULTS: The 2-year incidence of resistance was 28% among patients enrolled before 1996 and 9% among those enrolled in or after 1996 (P=.001). All cases of resistance occurred among patients with CD4+ T cell counts <50 cells/microL, and positive CMV culture results at baseline were associated with a approximately 4-fold increase in resistance. Among patients whose CMV and HIV loads were measured, a detectable CMV load at baseline and during follow-up was associated with increased risk of resistance, but a detectable HIV load was not. CONCLUSIONS: Rates of resistance have decreased from the high levels seen in the pre-HAART era. Better control of CMV replication may have contributed to this decrease.

Detection of ganciclovir resistance in patients with AIDS and cytomegalovirus retinitis: correlation of genotypic methods with viral phenotype and clinical outcome.

BACKGROUND: The cytomegalovirus (CMV) UL97 gene can be sequenced either from blood specimens directly amplified by polymerase chain reaction (PCR) or from culture isolates, to detect resistance to ganciclovir. METHODS: A prospective epidemiological study was conducted in which paired specimens were routinely obtained for sequencing of the UL97 gene from blood specimens (i.e., plasma and leukocytes) directly amplified by PCR and from CMV culture isolates. The specimens then were compared with each other and in terms of results of susceptibility testing and their association with progression of retinitis. RESULTS: A total of 845 paired specimens were obtained from 165 patients with AIDS and CMV retinitis. There typically was >90% agreement between the UL97 gene sequences from blood specimens directly amplified by PCR and those from culture isolates. The agreement between phenotypic resistance and the detection of UL97 mutations was >92% for PCR-amplified blood specimens and >97% for culture isolates. Plasma and leukocytes performed similarly. Progression of retinitis was correlated with the detection of UL97 mutations in PCR-amplified blood specimens, with adjusted odds ratios of 7.02 (P=.002) for leukocytes, 9.11 (P=.02) for plasma, and 17.6 for culture isolates (P<.0001). CONCLUSIONS: Because blood specimens directly amplified by PCR can be analyzed more rapidly than can cultures (< or =48 h vs. > or =4 weeks), sequencing the CMV UL97 gene from blood specimens directly amplified by PCR may be useful clinically.

Cytomegalovirus (CMV) blood DNA load, CMV retinitis progression, and occurrence of resistant CMV in patients with CMV retinitis.

BACKGROUND: The amount of cytomegalovirus (CMV) DNA in the blood (CMV load) may be a marker for detection of resistant CMV.METHODS. A total of 165 patients with AIDS and CMV retinitis had CMV load measurements (plasma and leukocyte) and cultures performed every 3 months; these measurements were correlated with CMV resistance to antiviral drugs and CMV retinitis progression (from masked readings of retinal photographs).RESULTS. Detectable plasma and leukocyte CMV loads were associated with CMV retinitis progression (odds ratios [OR], 6.3; P<.0001 and OR, 6.6; P<.0001, respectively), phenotypic resistance (OR, 6.1; P<.0001 and OR, 23.4; P=.0002, respectively), and genotypic resistance (OR, 17.5; P<.0001 and OR, 51.6; P=.0004, respectively). The sensitivity, specificity, and positive and negative predictive values of plasma CMV loads were 0.47, 0.86, 0.36, and 0.91, respectively, for progression and 0.59, 0.81, 0.07, and 0.99, respectively, for resistance; those of leukocyte CMV loads were 0.52, 0.83, 0.35, and 0.91, respectively, for progression and 0.82, 0.78, 0.08, and 0.99, respectively, for resistance. A detectable plasma CMV load at the time of diagnosis of CMV retinitis was associated with mortality (median survival time, 13.6 vs. 29.7 months; P=.007).CONCLUSIONS. CMV load has limited clinical utility, because of its low positive predictive value. Its high negative predictive value for occurrence of resistant CMV suggests that it may have utility as a screening tool to exclude resistance.

Verification of an assay for quantification of hepatitis C virus RNA by use of an analyte-specific reagent and two different extraction methods.

Protocols were designed for quantification and detection of hepatitis C virus (HCV) RNA by the use of an analyte-specific reagent (ASR) (Roche COBAS TaqMan48 [CTM48] HCV) after manual and automated RNA extraction. The purposes were to determine (i) assay performance characteristics using manual and automated RNA extraction methods, (ii) whether measurable range and limit of detection (LOD) of the ASR assay were influenced by genotype, and (iii) correlation of quantification by CTM48 HCV ASR and COBAS Monitor HCV v. 2.0. For HCV genotype 1 (Gt1), the lower limits of quantification after manual extraction were slightly lower than those for automated extraction (1.0 versus 1.5 log(10) IU/ml). Results were linear up to the highest concentration tested after extraction by both methods (manual, 6.1 log(10); automated, 6.4 log(10)). Similar results were obtained for Gt2 (1.8 to 6.8 log(10) IU/ml) and Gt3 (1.6 to 6.8 log(10) IU/ml) after automated extraction. The LOD of Gt1 virus was 10 IU/ml after manual extraction and between 25 and 37.5 IU/ml after automated extraction. Results with Gt2 and Gt3 viruses were similar after automated extraction (Gt2, between 25 and 50 IU/ml; Gt3, 25 IU/ml). Variability (intrarun and interrun, at concentrations throughout the range of quantification) was