Successful Management of Sequential Pulmonary Infections in a Cardiac Transplant Recipient

A case of a cardiac allograft recipient who had an initial combined pulmonary infection with cytomegalovirus, Aspergillus fumigatus and Nocardia asteroides, successfully treated with liposomal amphotericin B and sulfisoxazole and followed by an episode of respiratory syncytial virus pneumonitis, is presented. This case illustrates the role of computed tomographic imaging in the recognition, diagnosis and monitoring of complex opportunistic pulmonary infections and the benefits of liposomal amphotericin B in the treatment of aspergillosis

Influence of Novel Norovirus GII.4 Variants on Gastroenteritis Outbreak Dynamics in Alberta and the Northern Territories, Canada between 2000 and 2008

BACKGROUND: Norovirus GII.4 is the predominant genotype circulating worldwide over the last decade causing 80% of all norovirus outbreaks with new GII.4 variants reported in parallel with periodic epidemic waves of norovirus outbreaks. The circulating new GII.4 variants and the epidemiology of norovirus outbreaks in Alberta, Canada have not been described. Our hypothesis is that the periodic epidemic norovirus outbreak activity in Alberta was driven by new GII.4 variants evolving by genetic drift. METHODOLOGY/PRINCIPAL FINDINGS: The Alberta Provincial Public Health Laboratory performed norovirus testing using RT-PCR for suspected norovirus outbreaks in the province and the northern Territories between 2000 and 2008. At least one norovirus strain from 707 out of 1,057 (66.9%) confirmed norovirus outbreaks were successfully sequenced. Phylogenetic analysis was performed using BioNumerics and 617 (91.1%) outbreaks were characterized as caused by GII.4 with 598 assigned as novel variants including: GII.4-1996, GII.4-2002, GII.4-2004, GII.4-2006a, GII.4-2006b, GII.4-2008a and GII.4-2008b. Defining July to June of the following year as the yearly observation period, there was clear biannual pattern of low and high outbreak activity in Alberta. Within this biannual pattern, high outbreak activity followed the emergence of novel GII.4 variants. The two variants that emerged in 2006 had wider geographic distribution and resulted in higher outbreak activity compared to other variants. The outbreak settings were analyzed. Community-based group residence was the most common for both GII.4 variants and non-GII.4 variants. GII.4 variants were more commonly associated with outbreaks in acute care hospitals while outbreaks associated non-GII.4 variants were more commonly seen in school and community social events settings (p<0.01). CONCLUSIONS/SIGNIFICANCE: The emergence of new norovirus GII.4 variants resulted in an increased norovirus outbreak activity in the following season in a unique biannual pattern in Alberta over an eight year period. The association between antigenic drift of GII.4 strains and epidemic norovirus outbreak activity could be due to changes in host immunity, viral receptor binding efficiency or virulence factors in the new variants. Early detection of novel GII.4 variants provides vital information that could be used to forecast the norovirus outbreak burden, enhance public health preparedness and allocate appropriate resources for outbreak management

Concurrent Genotyping and Quantitation of Cytomegalovirus gB Genotypes in Solid-Organ-Transplant Recipients by Use of a Real-Time PCR Assay▿

We have developed a real-time genotyping and quantitative PCR (RT-GQ-PCR) assay to genotype cytomegalovirus (CMV) and quantify viral loads simultaneously in solid organ transplant (SOT) recipients. Special minor-groove DNA-binding probes were designed based on sequence polymorphism in the gB gene to increase genotyping specificity for gB1 to gB4. For validation, 28 samples with known genotypes determined by restriction fragment analysis (RFA) and 121 with unknown genotypes were tested. All samples were from SOT patients with CMV viremia. A 100% concordance for genotyping was achieved by using the RT-GQ-PCR with known genotypes determined by RFA. The RT-GQ-PCR identified more cases of CMV infections with mixed genotypes than RFA did. No cross-reaction between genotypes was observed. All four gB genotypes were detected in the 121 samples of unknown genotype. gB1 was the predominant single genotype (n = 61, 50.4%), followed by gB2 (n = 26, 21.0%), gB3, (n = 11, 9.1%), and gB4 (n = 3, 2.5%). Mixed-genotype infections were detected in 17% (20/121) of the samples. Patients with mixed-genotype infections had significantly higher CMV viral loads than those with single-genotype infections (P = 0.019). The RT-GQ-PCR assay was found to be highly sensitive and specific, with a wide dynamic range (2.7 to 10.7 log10 copies/ml) and very good precision (coefficient of variation, ∼1.78%). With the prominent feature of concurrent CMV gB genotyping and quantitation in a single reaction, the new assay provides a rapid and cost-effective method for monitoring CMV infection in SOT recipients

Evaluation of a Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR)-Based Microneutralization Assay for Assessing Clinical Human Cytomegalovirus-Neutralizing Antibody Activity

Development of a vaccine for human cytomegalovirus (hCMV) is critical because of the severe consequences of infection in congenitally infected newborns and immunocompromised patients. The assessment of hCMV-neutralizing antibody activity is crucial for vaccine development. This study evaluated a RT-qPCR assay targeting the immediate-early gene transcript of hCMV for determining microneutralizing antibody activity. The assay was evaluated for sensitivity, specificity, and precision using endotheliotropic clinical isolate VR1814 that infects fibroblasts, epithelial, and endothelial cells. The RT-qPCR-based neutralization assay was compared with an immunostaining-based neutralization assay using virions present in hCMV-positive urine, saliva, and breast-milk samples. Our results showed that hCMV replication was detectable at 20 h post-infection with a limit of detection of 1 infectious units (IU)/reaction. The RT-qPCR assay had a dynamic range of 1 to 1.0 × 104 IU/reaction, with coefficients of variation ranging from 0.94% to 15.08%. The RT-qPCR results were in high agreement with the immunostaining assay for hCMV-antibody neutralization assessment. Overall, the RT-qPCR neutralization assay is a reliable, rapid, efficient, and sensitive alternative method for evaluating hCMV-neutralizing activity in vitro

Successful Management of Sequential Pulmonary Infections in a Cardiac Transplant Recipient

A case of a cardiac allograft recipient who had an initial combined pulmonary infection with cytomegalovirus, Aspergillus fumigatus and Nocardia asteroides, successfully treated with liposomal amphotericin B and sulfisoxazole and followed by an episode of respiratory syncytial virus pneumonitis, is presented. This case illustrates the role of computed tomographic imaging in the recognition, diagnosis and monitoring of complex opportunistic pulmonary infections and the benefits of liposomal amphotericin B in the treatment of aspergillosis

Evaluation and Validation of Real-Time Reverse Transcription-PCR Assay Using the LightCycler System for Detection and Quantitation of Norovirus

We developed an assay for the detection and quantitation of norovirus with the LightCycler SYBR Green-based real-time reverse transcription-PCR (real-time LC RT-PCR) and previously published primers in the capsid and the polymerase gene. One hundred thirty-two stool specimens from the Provincial Laboratory for Public Health (Microbiology), Alberta, Canada, and the Centers for Disease Control and Prevention, Atlanta, Ga., were used to validate the new assay. The samples were collected from patients involved in outbreaks of acute gastroenteritis or children who presented with sporadic gastroenteritis. The real-time LC RT-PCR assay detected norovirus strains from three genogroup I (G-I) clusters (G-I/1, G-I/2, and G-I/3) and 10 genogroup II (G-II) clusters (G-II/1, G-II/2, G-II/3, G-II/4, G-II/6, G-II/7, G-II/10, G-II/12, G-II/15, and G-II/16). There was 100% concordance with the results from 58 stool specimens which tested positive by conventional RT-PCR assays. By dilution analysis, the real-time LC RT-PCR was 10,000 times more sensitive than the conventional RT-PCR. The new assay increased the number of samples in which noroviruses were detected by 19%. The real-time LC RT-PCR had a wide dynamic range, detecting from 5 to 5 × 10(6) copies of RNA per reaction, resulting in a theoretical lower limit of detection of 25,000 copies of RNA per g of stool. No cross-reactions were found with specimens containing sapovirus, rotavirus, astrovirus, and adenovirus. Because of the high sensitivity and specificity of the assay with a relatively rapid and simple procedure, the real-time LC RT-PCR will be useful as a routine assay for the clinical diagnosis of norovirus infection

Comparison of LightCycler-Based PCR, COBAS Amplicor CMV Monitor, and pp65 Antigenemia Assays for Quantitative Measurement of Cytomegalovirus Viral Load in Peripheral Blood Specimens from Patients after Solid Organ Transplantation

In order to evaluate the LightCycler-based PCR (LC-PCR) as a diagnostic assay technique, a classical pp65 antigenemia assay and the commercially available COBAS Amplicor CMV Monitor (CACM) assay were compared to the LC-PCR assay for the detection and quantitation of cytomegalovirus (CMV) load in 404 parallel specimens of peripheral blood from 66 patients after solid organ transplantation. A good correlation existed among these three assays (r ≅ 0.6, P < 0.0001). The LC-PCR assay was the most sensitive (54% of specimens positive) compared to the CACM (48.6%) and the pp65 antigenemia (26%) assays. The LC-PCR assay detected all samples found positive by using both the CMV pp65 antigenemia assay and the CACM assay. The LC-PCR also had the widest dynamic range (from 250 to 10(7) DNA copies/ml of plasma). No cross-reactions were found among CMV and Epstein-Barr virus, varicella-zoster virus, or herpes simplex virus in the LC-PCR by using amplification with specifically designed primer pairs. Precision, expressed as the coefficient of variation, was <3% with standard DNA from cell cultures and between 6.55 and 14.1% with clinical specimens in repeat LC-PCR runs. One run of the LC-PCR took half of the time required for the semiautomated CACM procedure. Because of its sensitivity, specificity, cost-effectiveness, and simplicity, the LC-PCR assay could replace the pp65 antigenemia and the CACM assays as the preferred technique for the surveillance, diagnosis, and monitoring of response of CMV diseases in high-risk populations

Maculopapular rash and tremor are associated with West Nile fever and neurological syndromes

The clinical features associated with West Nile virus (WNV) infections are described based on data collected from history forms submitted with samples during a province‐wide WNV testing programme. Age 40–59 years (OR 1.7, p<0.008), residence in the southeast of Alberta (OR 4.2, p<0.001), maculopapular rash (OR 8.6, p<0.001) or tremor (OR 3.6, p<0.001) were independently associated with WNV infection

Monitoring of Polyomavirus BK Virus Viruria and Viremia in Renal Allograft Recipients by Use of a Quantitative Real-Time PCR Assay: One-Year Prospective Study▿

We have developed a real-time quantitative PCR (rt-QPCR) assay to detect and kinetically monitor BK virus viruria and viremia in renal transplant recipients (RTRs). A total of 607 urine and 223 plasma samples were collected from 203 individuals including those with BK virus-associated nephropathy (BKVAN) (n = 8), those undergoing routine posttransplant surveillance (SV) (n = 155), those with nontransplant chronic kidney disease (NT-CKD) (n = 20), and healthy living kidney donors (LD) (n = 20). The rt-QPCR assay was found to be highly sensitive and specific, with a wide dynamic range (2.4 to 11 log10 copies/ml) and very good precision (coefficient of variation, ∼5.9%). There was a significant difference in the prevalences of viruria and viremia between the BKVAN (100% and 100%) and SV (23% and 3.9%) groups (P < 0.001). No viruria or viremia was detected in LD or in NT-CKD patients. The median (range) peak levels of BK virus viruria and viremia, in log10 copies/ml, were 10.26 (9.04 to 10.83) and 4.83 (3.65 to 5.86) for the BKVAN group versus 0 (0 to 10.83) and 0 (0 to 5.65) for the SV group, respectively (P < 0.001). When the BK virus load in the urine was <7.0 log10 copies/ml, no BK virus viremia was detected. When the BK virus load in the urine reached 7.0, 8.0, 9.0, and ≥10.0 log10 copies/ml, the corresponding detection of BK virus viremia increased to 20, 33, 50, and 100%, respectively. We propose monitoring of BK virus viruria in RTRs, with plasma BK virus load testing reserved for those with viruria levels of ≥7.0 log10 copies/ml