Hyperammonemia syndrome post-lung transplantation: Case series and systematic review of literature.

BACKGROUND: Hyperammonemia syndrome (HS) is a rare post-transplant complication associated with high morbidity and mortality. Its incidence appears to be higher in lung transplant recipients and its pathophysiology is not well understood. In addition to underlying metabolic abnormalities, it is postulated that HS may be associated with Ureaplasma or Mycoplasma spp. lung infections. Management of this condition is not standardized and may include preemptive antimicrobials, renal replacement, nitrogen scavenging, and bowel decontamination therapies, as well as dietary modifications. METHODS: In this case series, we describe seven HS cases, five of whom had metabolic deficiencies ruled out. In addition, a literature review was performed by searching PubMed following PRISMA-P guidelines. Articles containing the terms "hyperammonemia" and "lung" were reviewed from 1 January 1997 to 31 October 2021. RESULTS: All HS cases described in our center had positive airway samples for Mycoplasmataceae, neurologic abnormalities and high ammonia levels post-transplant. Mortality in our group (57%) was similar to that published in previous cases. The literature review supported that HS is an early complication post-transplant, associated with Ureaplasma spp. and Mycoplasma hominis infections and of worse prognosis in patients presenting cerebral edema and seizures. CONCLUSION: This review highlights the need for rapid testing for Ureaplasma spp. and M. hominis after lung transplant, as well as the necessity for future studies to explore potential therapies that may improve outcomes in these patients.

Donor Cytomegalovirus Transmission Patterns in Solid Organ Transplant Recipients With Primary Infection.

BACKGROUND: The epidemiology of single versus multiple cytomegalovirus (CMV) strain transmission from donor (D+) to seronegative solid organ transplant (SOT) recipients (R-) is uncertain, as is whether "relapsing" recipient infection represents changing strain predominance when multiple strains are transmitted. Here we characterized CMV strain transmission patterns in D+/R- SOT recipients. METHODS: We studied pairs or groups of D+/R- SOT recipients who received organs from a common donor (group A) and recipients who experienced ≥2 waves of CMV DNAemia (group B). CMV in plasma was characterized by genotype-specific real-time PCR for genes gB and gH. RESULTS: Single concordant genotypes were identified in 12 of 18 recipient pairs/group sharing a common donor (group A); at least 6 of 18 (33%) donors transmitted > 1 strain. A single CMV strain was detected in 14 of 15 recipients in group B; only 1 recipient had coinfection. A shift in CMV strain predominance occurred after the first posttransplant year in at least 4 recipients with coinfection. CONCLUSIONS: Using a common donor approach, we confirmed that multiple CMV strain transmission from donors to R- SOT recipients is not uncommon. D+/R- SOT recipients with CMV coinfection can undergo changes in strain predominance in late waves of CMV DNAemia.

Association between Mycoplasma and Ureaplasma airway positivity, ammonia levels, and outcomes post-lung transplantation: A prospective surveillance study.

Hyperammonemia syndrome (HS) is a rare complication with high mortality described after lung transplantation. Its pathophysiology is still unclear, but previous studies, including murine models, have linked the identification of Mycoplasmataceae in airway specimens with HS occurrence. This study explores the association between Mycoplasmataceae polymerase chain reaction (PCR) positivity, ammonia levels, HS, and mortality post-lung transplant. Adults who underwent lung transplantation between July 2017 and August 2019 had prospective surveillance testing for Mycoplasma and Ureaplasma using PCR on post-operative bronchoscopy samples. One hundred and fifty-nine patients underwent lung transplantation during the study period. Mean age was 54 (±13) years; baseline diseases were predominantly pulmonary fibrosis (37.7%) and chronic obstructive pulmonary disease (35.8%). Mycoplasma and/or Ureaplasma airway positivity was found in 42 (26.4%) of tested patients, represented mostly by M. salivarium (26/43; 60.4%), U. parvum (7/43; 16.2%), and U. urealyticum (5/43; 11.6%). Median peak ammonia levels were higher in those with Ureaplasma colonization compared to uncolonized patients (p = .04), however, only three patients developed HS. Recipient airway Ureaplasma positivity was independently associated with younger (aOR 0.94, 95% CI 0.88-0.99, p = .04) and female donors (aOR 4.29; 95% CI 1.01-18.2, p = .05).

Post-transplant lymphoproliferative disorders, Epstein-Barr virus infection, and disease in solid organ transplantation: Guidelines from the American Society of Transplantation Infectious Diseases Community of Practice.

These updated guidelines from the American Society of Transplantation Infectious Diseases Community of Practice review the diagnosis, management, and prevention of post-transplant lymphoproliferative disorders (PTLD) and other Epstein-Barr virus (EBV) syndromes after solid organ transplantation. PTLD are a heterogeneous spectrum of predominantly B-cell disorders, often extra-nodal, with complex distinct pathogeneses and variable clinical presentations determined by pathologic subtype. Recent epidemiologic studies report a decrease in early EBV-positive (+) PTLD and an increase in late EBV-negative (-) PTLD. Pre-transplant EBV-seronegativity and primary EBV infection, often from donor-transmitted infection, are an important risk factors for EBV syndromes and early EBV + PTLD. Low-quality evidence supports preemptive prevention strategies for early EBV + PTLD in EBV-seronegative recipients that involve EBV DNA measurement in peripheral blood using assays requiring further result harmonization, combined with interventions to lower viral load. Reduction in immunosuppression (RIS) is the best validated intervention. WHO pathology classification of a tissue biopsy remains the gold standard for PTLD diagnosis; optimal staging procedures are uncertain. Treatment of CD20+ PTLD with the response-dependent sequential use of RIS, rituximab, and cytotoxic chemotherapy is recommended. Evidence gaps requiring future research and alternate treatment strategies including immunotherapy are highlighted.

Impact of donor and recipient cytomegalovirus serology on long-term survival of heart transplant recipients.

BACKGROUND: Some studies have shown that pre-transplant cytomegalovirus (CMV) serostatus is associated with heart transplant patient survival while others have not. We analyzed the relationship between pre-transplant donor/recipient CMV serostatus and long-term mortality in a retrospective cohort of heart transplant recipients at our center. METHODS: Adult (Age >17 years) heart recipients transplanted between July 1985-December 2015 were analyzed. Variables included age, sex, pre-transplant donor (D)/recipient (R) serostatus [D-/R-, D-/R+, D+/R+, D+/R-], CMV infection within 2 years of transplant and transplant eras divided by changes in CMV prevention strategies: Era 1 (Pre-ganciclovir, July 1985-April 1998), Era 2 (Oral ganciclovir, May 1998-December 2004), Era 3 (Valganciclovir, January 2005-December 2015). Survival analysis and Cox regression were performed at 10 years. RESULTS: A total of 620 heart transplants were included in our analysis; 20% were CMV mismatched pre-transplant. Thirty-eight percent of patients were infected with CMV within the first two post-transplant years. Survival analysis showed D/R CMV serostatus did not significantly impact survival of heart recipients at 10 years (P = 0.11). Survival was significantly different across eras for D-/R+, D+/R+, and D+/R+ (P = 0.043) but not D-/R- patients (P = 0.8). Cox regression revealed that patients transplanted in the valganciclovir era have an estimated 29% reduced risk of death (P = 0.047) compared to patients transplanted in the pre-ganciclovir era after controlling for age at transplantation, D/R CMV serostatus and CMV infection. CONCLUSION: Our review of the impact of CMV managed differently across eras suggests in heart transplantation there is no influence of D/R CMV serostatus on 10 year survival.

Epstein-Barr virus associated smooth muscle tumors in solid organ transplant recipients: Incidence over 31 years at a single institution and review of the literature.

INTRODUCTION: Epstein-Barr virus (EBV) associated smooth muscle tumors (EBV-SMT) are a rare complication of solid organ transplantation (SOT). Incidence data related to this EBV-SMT are limited. EBV DNA is universally present in these tumors. How these cells get infected with EBV, whether this is a result of primary EBV infection vs reactivation, and how persistent active EBV infection post-transplant influences EBV-SMT pathogenesis remains unknown. METHODS: Among 5006 SOT recipients (474 pediatric, 4532 adult) receiving SOT at our center between Jan 1984 and Dec 2015, three cases of post-transplant EBV-SMT were identified. RESULTS: All cases were pediatric heart transplants who were EBV seronegative prior to transplant, and experienced primary EBV infection with persistently elevated EBV viral loads, despite antiviral therapy. Two are deceased at 3.2 and 0.9 years post-diagnosis, while one remains alive 6.2 years post diagnosis. The overall local incidence of post-transplant EBV-SMT at our institution was 0.7 (95% CI, 0.2-1.7) per 1000 patient years, and 2.6 (95% CI, 0.6-6.7) per 1000 patient years in pediatric heart transplants. A literature review identified 36 pediatric and 51 adult cases of post-transplant EBV-SMT. CONCLUSIONS: We hypothesize that pre-transplant EBV seronegativity, followed by primary EBV infection and persistently high EBV viral loads, represents a unique risk factor for post-transplant EBV-SMT. Pediatric heart transplant recipients were found to be disproportionately affected by post-transplant EBV-SMT at our institution.

Assigning Cytomegalovirus Status in Children Awaiting Organ Transplant: Viral Shedding, CMV-Specific T Cells, and CD27-CD28-CD4+ T Cells.

Passive antibodies, maternal or transfusion-acquired, make serologic determination of pretransplant cytomegalovirus (CMV) status unreliable. We evaluated 3 assays unaffected by passive antibodies, in assignment of CMV infection status in children awaiting solid organ transplant and in controls: (1) CMV nucleic acid amplification testing (NAAT), (2) quantification of CMV-specific CD4+ T cells, and (3) quantification of CD27-CD28-CD4+ T cells. Our results highlight that CMV NAAT, from urine and oropharynx, is useful in confirming positive CMV status. Detection of CMV-specific CD4+ T cells was sensitive and specific in children >18 months but was less sensitive in children <12 months. CD27-CD28-CD4+ T cells are not likely useful in CMV risk stratification in children.

Assignment of cytomegalovirus infection status in infants awaiting solid organ transplant: Viral detection methods as adjuncts to serology.

Assignment of CMV infection status in infants awaiting SOT is challenging as passive maternal antibody can lead to false-positive serology. Since 2000, our protocol has recommended sending throat and urine samples for CMV viral detection, culture, or NAAT, for CMV-seropositive infants <18 months awaiting SOT. We reviewed pretransplant CMV serology for 152 infants and, for CMV seropositives, examined relationships between CMV IgG OD values, age, and CMV viral detection to explore time to clearance of maternal CMV IgG and evaluate viral detection in assignment of pretransplant CMV infection status. The proportion of CMV-seropositive infants decreased from 52% in infants 0-6 months of age to 28% in those 12-18 months. Among CMV-seropositive infants, median OD was significantly higher in the 6- to 12- and 12- to 18-month groups compared to the 0- to 6-month group. Distribution of OD by age group suggested that maternal antibody cleared before 12 months. Of 59 eligible CMV-seropositive infants, 49 (83%) had CMV viral detection studies and 18 of 49 (36.7%) had detectable CMV: 9 of 30 (30.0%) infants 0-6 months, 7 of 15 (46.7%) infants 6-12 months, and 2 of 4 (50.0%) infants 12-18 months. CMV viral detection studies are useful to confirm positive CMV infection status in CMV-seropositive infants awaiting SOT. Maternal CMV IgG likely clears before 12 months.

Determination of the Biological Form of Human Cytomegalovirus DNA in the Plasma of Solid-Organ Transplant Recipients.

Background: Whether cytomegalovirus (CMV) DNA exists in plasma as virion-associated or free DNA is uncertain. Methods: An assay combining DNase I digestion and CMV quantitative polymerase chain reaction (DNase-CMV-qPCR) was developed to differentiate free naked DNA from virion DNA. One hundred three frozen and 10 fresh CMV DNA-positive plasma samples from solid-organ transplant recipients (SOTRs) were tested. Three sets of paired qPCR (P-qPCR) assays with amplicons of variable length were used to study CMV DNA fragmentation in 20 SOTR plasma samples, viral stocks (Towne, Merlin, AD169) and the first World Health Organization (WHO) international standard (IS) for CMV DNA. Results: In all plasma samples, 98.8%-100% of CMV DNA was free DNA; this was the only form in 93 of 103 (90.3%) frozen and all 10 fresh samples tested using DNase-CMV-qPCR. Low levels of virion CMV DNA were found in 10 of 103 (9.7%) samples with higher total DNA load. Cytomegalovirus DNA results were highly reproducible for 3 CMV virus stocks and WHO IS (P > .80), tested by three sets of paired q-PCR. However, for the 20 SOTR plasma samples, the smaller amplicon assay result was 2.6-fold, 3.4-fold, and 6.5-fold higher than the longer amplicion result (P < .001). Conclusions: Cytomegalovirus DNA in SOTR plasma is almost exclusively free DNA, highly fragmented, and not virion associated.

Are We There Yet? Impact of the First International Standard for Cytomegalovirus DNA on the Harmonization of Results Reported on Plasma Samples.

BACKGROUND: Interassay harmonization of cytomegalovirus (CMV) DNA measurement is important for infection management. Uncertainty exists regarding the result harmonization achievable in patient plasma samples using quantitative polymerase chain reaction (qPCR) assays with calibrators now traceable to the First World Health Organization International Standard (IS) for CMV DNA. METHOD: Serial dilutions of the IS and a blinded panel of 40 genotyped CMV DNA-positive pooled plasma samples and 10 negative plasma samples were tested by 6 laboratories using 10 qPCR assays calibrated to the IS. Each clinical sample was constructed using plasma from a single unique transplant recipient. RESULTS: The variance for individual CMV DNA-positive samples was greater for clinical samples (median, 1.50 [range, 1.22-2.82] log10 IU/mL) than for IS dilutions (median, 0.94 [range, 0.69-1.35] log10 IU/mL) (P < .001); 58.9% of all clinical sample results and 93.6% of IS dilution results fell within ±0.5 log10 IU/mL of the mean viral load of each sample. Result variability was not impacted by either genotype or quantitative levels of CMV DNA. Testing procedure differences can significantly influence results, even when analyte-specific reagents are identical. For clinical samples, all assays demonstrated result bias (P < .008). Assays with amplicon sizes ≤86 bp had significantly higher results compared to assays with larger amplicon sizes (≥105 bp) (P < .001). CONCLUSIONS: The variability in CMV DNA results reported on individual samples has been reduced by the IS, but ongoing clinically relevant variability persists, preventing meaningful interassay result comparison.

A next generation sequencing-based method to study the intra-host genetic diversity of norovirus in patients with acute and chronic infection.

BACKGROUND: Immunocompromised individuals with chronic norovirus (NoV) infection and elderly patients are hypothesized to be reservoirs where NoV might accumulate mutations and evolve into pandemic strains. Next generation sequencing (NGS) methods can monitor the intra-host diversity of NoV and its evolution but low abundance of viral RNA results in sub-optimal efficiency. In this study, we: 1) established a next generation sequencing-based method for NoV using bacterial rRNA depletion as a viral RNA enrichment strategy, and 2) measured the intra-host genetic diversity of NoV in specimens of patients with acute NoV infection (n = 4) and in longitudinal specimens of an immunocompromised patient with chronic NoV infection (n = 2). RESULTS: A single Illumina MiSeq dataset resulted in near full-length genome sequences for 5 out of 6 multiplexed samples. Experimental depletion of bacterial rRNA in stool RNA provided up to 1.9 % of NoV reads. The intra-host viral population in patients with acute NoV infection was homogenous and no single nucleotide variants (SNVs) were detected. In contrast, the NoV population from the immunocompromised patient was highly diverse and accumulated SNVs over time (51 SNVs in the first sample and 122 SNVs in the second sample collected 4 months later). The percentages of SNVs causing non-synonymous mutations were 27.5 % and 20.5 % for the first and second samples, respectively. The majority of non-synonymous mutations occurred, in increasing order of frequency, in p22, the major capsid (VP1) and minor capsid (VP2) genes. CONCLUSIONS: The results provide data useful for the selection and improvement of NoV RNA enrichment strategies for NGS. Whole genome analysis using next generation sequencing confirmed that the within-host population of NoV in an immunocompromised individual with chronic NoV infection was more diverse compared to that in individuals with acute infection. We also observed an accumulation of non-synonymous mutations at the minor capsid gene that has not been reported in previous studies and might have a role in NoV adaptation.

A risk-stratified approach to cytomegalovirus prevention in pediatric solid organ transplant recipients.

Optimal strategies to prevent cytomegalovirus (CMV) disease following pediatric solid organ transplantation remain controversial. The purpose of this study was to review the outcomes of a risk-stratified strategy that uses a hybrid or prophylactic strategy for donor (D)+ recipient (R)- patients, a preemptive strategy for D+R+/D-R+, and clinical follow-up alone for D-R+ patients. A retrospective chart review was undertaken at the Stollery Children's Hospital in Edmonton, Alberta for pediatric solid organ transplants 2004 through 2010. Transplants were risk-stratified according to D/R CMV serostatus, organ group, and type of induction or rejection immunosuppression. The incidence of DNAemia and CMV disease and adverse effects from prophylaxis were analyzed. The study included 197 recipients. CMV DNAemia was detected in 49 of 197 recipients (24.8%), and CMV disease occurred in eight of 197 (4%) of which all but one were D+R-. All recovered. Seventeen of 142 recipients who received prophylaxis (12%) had hematologic toxicity. No other toxicities were identified. In conclusion, A risk-stratified approach resulted in very low rates of CMV disease with minimal adverse effects. Lowering the dosage rather than stopping antivirals in the face of neutropenia has the potential to further lower the rate of CMV disease.

Enhanced enteric virus detection in sporadic gastroenteritis using a multi-target real-time PCR panel: a one-year study.

Viral gastroenteritis causes significant mortality and morbidity worldwide. Identifying the etiology of viral gastroenteritis is a challenge as most enteric viruses (EVs) are non-culturable. This study is to develop an EV testing panel using real-time PCR (EVPrtPCR) to simultaneously detect rotavirus, norovirus, sapovirus, astrovirus, and enteric adenovirus in stool samples. EVPrtPCR using universal amplification conditions was run in a single instrument run. EVPrtPCR was used to test 2,486 sporadic gastroenteritis samples submitted for EV testing using electron microscopy (EM) between July 2008 and July 2009. Retesting spiked negative stool samples and Salmon DNA as internal control were used to evaluate inhibition. EVPrtPCR detected viruses in significantly more samples: 748 (34%) as compared to 94 (3.8%) by EM. EM did not detect any norovirus, sapovirus, and mixed infection, and detected only 39% of rotavirus and 38.2% of enteric adenovirus positive samples. Four samples that tested positive for rotavirus and two for adenovirus and for small-round-structured viruses by EM were negative by EVPrtPCR. Norovirus was the most common virus detected (17.6%) with 92.4% as genogroup II, followed by rotavirus (6.8%), sapovirus (4.2%), astrovirus (2.0%), and enteric adenovirus (1.4%) with 9% samples positive for mixed infection. Overall, EV identification followed a U-shaped age distribution; positive samples were more common in children ≤5 years old and adults >60 years old. Norovirus, sapovirus and astrovirus showed winter predominance and rotavirus peaked in the spring. No inhibition was observed. Molecular technology significantly enhanced the identification of EV causing sporadic gastroenteritis in Alberta.

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.

Emergence of a new norovirus GII.4 variant and changes in the historical biennial pattern of norovirus outbreak activity in Alberta, Canada, from 2008 to 2013.

The public health impact of the emergence of new norovirus (NoV) strains is uncertain. A biennial pattern of alternating quiescent and epidemic levels of NoV outbreak activity associated with the emergence of new GII.4 variants was observed in Alberta, Canada, between July 2000 and June 2008. In this study, NoV genogroup I (GI) and GII strains isolated from 710 outbreak specimens in Alberta between July 2008 and January 2013 were characterized to update historical data. The seasonality and annual variation in NoV outbreak burden were analyzed over a 10-year period (July 2002 to June 2012). We found that GII.4-2006b had persisted as the predominant variant over three observation periods (July 2006 to June 2009) during which the biennial NoV outbreak pattern continued. The emergence of GII.4-2010 (winter 2009) was not associated with increased outbreak activity, and outbreak activity between July 2009 and June 2012 when GII.4-2010 predominated (67.5 to 97.7%) did not follow a biennial pattern. GII.4-2012 first emerged in Alberta in September 2011 and became predominant in observation period July 2012 to June 2013. NoV GI, relatively rare in past years, had a higher activity level (37.3%) as represented by GI.6 and GI.7 in the winter of 2012 to 2013. A higher proportion of GI outbreaks occurred in non-health care facility settings compared to GII. Our study suggests that factors other than new variants emergence contribute to the levels of NoV outbreak activity in Alberta.

Use of quantitative HIV RNA detection for early diagnosis of HIV infection in infants and acute HIV infections in Alberta, Canada.

Quantitative HIV RNA viral load (QVL) assays (Roche Diagnostics) were sensitive and specific when used to diagnose HIV infection in (i) HIV-exposed infants (sensitivity of 100% [63.1 to 100%] and specificity of 100% [97.9 to 100%]) and (ii) suspected acute HIV infection patients with a negative/indeterminate Western blot (sensitivity of 97.6% [91.6 to 99.7%] and specificity of 100% [96.1 to 100%]). No false-positive QVL results were identified.

Tumor Infiltrating Lymphocytes Predict Survival in Solid Organ Transplant Recipients With Monomorphic Post-transplant Lymphoproliferative Disorders.

INTRODUCTION: The tumor microenvironment (TME) in post-transplant lymphoproliferative disorders (PTLDs) remains unexplored. Tumor infiltrating lymphocytes (TILs) are prognostic in other lymphomas. We assessed the prognostic impact of TILs in monomorphic B-cell PTLD. METHODS: TIL density (CD3+ cells/mm2) was determined by CD3 immunohistochemistry in archived diagnostic biopsies from patients diagnosed with monomorphic B-cell PTLD. RESULTS: Amongst monomorphic PTLDs (N = 107), low TIL-count was associated with inferior 2-year progression-free survival (PFS) (41% versus 86%, P = .003) and 2-year overall survival (OS) (52% versus 93%, P = .003) by Kaplan-Meier analysis. Low TIL-count was significant on Cox univariate regression for inferior PFS (HR 4.5, 95% CI 2.0-9.9, P < .001) and OS (HR 4.6, 95% CI 1.8-11.8, P < .001). Multivariate analysis with clinical variables (age ≥60 years, high LDH, stage III/IV, CNS involvement) and TIL-count showed significance for PFS (HR 3.3, 95% CI 1.3-8.3, P = .010) and a non-significant trend for OS (HR 2.6, 95% CI 0.9-7.3, P = .064). A composite score including TILs and clinical variables (age ≥60 years, high LDH, stage III/IV, CNS involvement) effectively stratified monomorphic PTLD patients by PFS and OS (2-year OS: low-risk 93%, intermediate-risk 61%, high-risk 23%, P < .001). CONCLUSIONS: The TME and TILs are prognostically relevant in monomorphic PTLD. Prognostic models including measures of the TME may improve risk stratification for patients with monomorphic PTLDs.

Impact of genetic polymorphisms in cytomegalovirus glycoprotein B on outcomes in solid-organ transplant recipients with cytomegalovirus disease.

BACKGROUND: It is unknown whether specific viral polymorphisms affect in vivo therapeutic response in patients with cytomegalovirus (CMV) disease. Polymorphisms in the CMV glycoprotein B (gB) gene allow discrimination of 4 distinct genotypes (gB1-gB4). We assessed the influence of gB genotypes on the clinical and virologic outcome of CMV disease. METHODS: Solid-organ transplant recipients enrolled in a multicenter trial of CMV disease treatment (VICTOR study) were included in this study. CMV gB genotyping was performed using quantitative real-time polymerase chain reaction at day 0 (start of antiviral therapy). RESULTS: Among 239 patients with CMV disease, the prevalence of gB strain types was 26% for gB1, 10% for gB2, 10% for gB3, and 5% for gB4, whereas mixed infections were present in 49%. Donor-seropositive/recipient-seropositive patients were more likely to have mixed gB infection than donor-seropositive/recipient-seronegative patients (40% vs. 12%; P = .001). Median baseline viral loads were higher and time to viral eradication was longer ( P = .006 and P = .026 , respectively) for mixed infection versus infection with a single genotype. In a multivariate model, mixed gB infection was a significant predictor of failure to eradicate virus by day 21 (mixed vs single genotype; odds ratio, 2.66; 95% confidence interval, 1.31-5.38; P = .007 ) after controlling for baseline viral load, CMV serostatus at baseline, ganciclovir resistance, and antiviral treatment. No effect of gB genotype was seen on virologic or clinical CMV recurrence. CONCLUSIONS: No specific gB genotype appears to confer a specific CMV virulence advantage. However, mixed gB genotype infections are associated with higher viral loads and delayed viral clearance.

Design and validation of real-time reverse transcription-PCR assays for detection of pandemic (H1N1) 2009 virus.

Tracking novel influenza viruses which have the potential to cause pandemics, such as the pandemic (H1N1) 2009 virus, is a public health priority. Pandemic (H1N1) 2009 virus was first identified in Mexico in April 2009 and spread worldwide over a short period of time. Well-validated diagnostic tools that are rapid, sensitive, and specific for the detection and tracking of this virus are needed. Three real-time reverse transcription PCR (RT-PCR) assays for the amplification and detection of pandemic (H1N1) 2009 virus were developed, and their performance characteristics were compared with those of other published diagnostic assays. Thirty-nine samples confirmed to be positive for pandemic (H1N1) 2009 virus from Alberta, Canada, and six additional samples that were positive for influenza A virus but that were not typeable by using published seasonal influenza H1/H3 virus assays were available for this validation. Amplification and direct sequencing of the products was considered the "gold standard" for case identification. The new assays were sensitive and able to reproducibly detect virus in a 10(-6) dilution of 4 x 10(6) 50% tissue culture infective doses/ml when 5 microl was used as the template. They showed 100% specificity and did not cross-react with other respiratory viruses or seasonal influenza A virus subtypes. The coefficient of variation in crossing cycle threshold values for the detection of different template concentrations of pandemic (H1N1) 2009 virus was < or =3.13%, showing good reproducibility. The assays had a wide dynamic range for the detection of pandemic (H1N1) 2009 virus and utilized testing platforms appropriate for high diagnostic throughput with rapid turnaround times. We developed and validated these real-time PCR procedures with the goal that they will be useful for diagnosis and surveillance of pandemic (H1N1) 2009 virus. These findings will contribute to the informed management of this novel virus.

CMV-specific T-cells and CD27-CD28-CD4+ T-cells for assignment of cytomegalovirus (CMV) status in adults awaiting organ transplant.

BACKGROUND/OBJECTIVES: Determination of Cytomegalovirus (CMV) status in solid organ transplant (SOT) candidates is essential to stratify risk of post-transplant CMV disease. Passive transfusion-acquired antibodies can make serologic determination of CMV status unreliable. We evaluated 3 assays, not affected by passive antibodies (PA), in assignment of CMV status: quantification of CMV-specific CD4 + T-cells (CMV-TC) and exhausted CD27-CD28- CD4 + T-cells, and detection of CMV DNA with Nucleic Acid Amplification Testing (NAAT). STUDY DESIGN: We enrolled 50 adults awaiting SOT and 50 immunocompetent age-matched controls, and collected a throat swab, urine, saliva and blood sample on each. Using flow cytometry CD4 + T-cells were phenotypically analyzed for expression of CD27 and CD28 and CMV-specific CD4 + T-cells were identified by CD69 expression and intracellular IFN-γ quantification after stimulation with CMV-antigen lysate. CMV NAAT was performed on all specimens using real-time PCR. CMV serology (CMV IgG) was determined by enzyme immunoassay. Subjects were considered to have potential PA if they received blood products within 2 months of collection. RESULTS: The CMV-TC assay discriminated between CMV-seropositive and seronegative SOT candidates without PA well (sensitivity 79%, specificity 93%) while the CD27-CD28-CD4 + T-cell assay had good sensitivity (86%) but specificity of 74%. Detection of CMV DNA was uncommon in CMV-seropositive SOT candidates (2/21). CONCLUSIONS: Given its high specificity, the CMV-TC assay is valuable in confirming true-positive CMV status in seropositive SOT candidates with PA, while use of CD27-CD28-CD4 + T-cell analysis is limited by moderate specificity. Detection of CMV DNA is of limited value in assignment of CMV status in adults.