Tom Kotsimbos

Comparison of a Multiplex Reverse Transcription-PCR-Enzyme Hybridization Assay with Conventional Viral Culture and Immunofluorescence Techniques for the Detection of Seven Viral Respiratory Pathogens

ABSTRACT A multiplex reverse transcription-PCR-enzyme hybridization assay (RT-PCR-EHA; Hexaplex; Prodesse Inc., Waukesha, Wis.) was used for the simultaneous detection of human parainfluenza virus types 1, 2, and 3, influenza virus types A and B, and respiratory syncytial virus types A and B. One hundred forty-three respiratory specimens from 126 patients were analyzed by RT-PCR-EHA, and the results were compared to those obtained by conventional viral culture and immunofluorescence (IF) methods. RT-PCR-EHA proved to be positive for 17 of 143 (11.9%) specimens, whereas 8 of 143 (5.6%) samples were positive by viral culture and/or IF. Eight samples were positive by both RT-PCR-EHA and conventional methods, while nine samples were RT-PCR-EHA positive and viral culture and IF negative. Eight of the nine samples with discordant results were then independently tested by a different multiplex RT-PCR assay for influenza virus types A and B, and all eight proved to be positive. In comparison to viral culture and IF methods, RT-PCR-EHA gave a sensitivity and a specificity of 100 and 93%, respectively. Since RT-PCR-EHA was able to detect more positive samples, which would otherwise have been missed by routine methods, we suggest that this multiplex RT-PCR-EHA provides a highly sensitive and specific means of diagnostic detection of major respiratory viruses.

Airway neutrophilia in stable and bronchiolitis obliterans syndrome patients following lung transplantation

BACKGROUND The bronchiolitis obliterans syndrome (BOS) remains the major constraint on the long term success of lung transplantation. Neutrophils have been associated with fibrosing lung conditions and have been noted to be increased in the bronchoalveolar lavage (BAL) fluid of patients with BOS. METHODS This study was undertaken to examine neutrophil accumulation in the BAL fluid, airway wall and lung parenchyma, as well as levels of interleukin (IL)-8 in the BAL fluid, in normal controls and lung transplant recipients with and without BOS. Bronchoscopic examination included endobronchial biopsy (EBB), BAL fluid, and transbronchial biopsy (TBB) sampling. Tissue neutrophils were identified by neutrophil elastase staining on 3 μm paraffin biopsy sections and quantified by computerised image analyser. IL-8 levels were measured in unconcentrated BAL fluid by ELISA. RESULTS Compared with controls, airway wall neutrophilia was increased in both stable lung transplant recipients and those with BOS (p<0.05). BAL neutrophils and IL-8 levels were also increased in both groups of transplant recipients compared with controls (p<0.01), the levels being significantly higher in the BOS group (p<0.01). Neutrophil numbers in the lung parenchyma were not significantly different between the two groups of lung transplant recipients. CONCLUSION Increased levels of neutrophils are present in the airway wall and BAL fluid of lung transplant recipients with and without BOS. BAL fluid levels of IL-8 are also increased, raising the possibility that neutrophils and/or IL-8 may play a part in the pathogenesis of BOS following lung transplantation.

Antifungal Prophylaxis in Lung Transplantation— A World-wide Survey

While variations in antifungal prophylaxis have been previously reported in lung transplant (LTx) recipients, recent clinical practice is unknown. Our aim was to determine current antifungal prophylactic practice in LTx centers world‐wide. One nominated LTx clinician from each active center was invited by e‐mail to participate in a web‐based survey between September 2009 and January 2010. Fifty‐seven percent (58/102) responded. The majority of responses were from medical directors of LTx centers (72.4%), and from the United States (44.8%). Within the first 6 months post‐LTx, most centers (58.6%) employed universal prophylaxis, with 97.1% targeting Aspergillus species. Voriconazole alone, and in combination with inhaled amphotericin B (AmB), were the preferred first‐line agents. Intolerance to side effects of voriconazole (69.2%) was the main reason for switching to alternatives. Beyond 6 months post‐LTx, most (51.8%) did not employ antifungal prophylaxis. Fifteen centers (26.0%) conducted routine antifungal therapeutic drug monitoring during prophylactic period. There are differences in strategies employed between U.S. and European centers. Most respondents indicated a need for antifungal prophylactic guidelines. In comparison to earlier findings, there was a major shift toward prophylaxis with voriconazole and an increased use of echinocandins, posaconazole and inhaled lipid formulation AmB.

Reduced bone density in cystic fibrosis: ΔF508 mutation is an independent risk factor

The aim of this cross-sectional study was to determine the prevalence and identify determinants of reduced bone mineral density (BMD) in adults with cystic fibrosis (CF). Adults (88) with CF (mean±sd age 29.9±7.7 yrs; forced expiratory volume in one second (FEV1) 58.2±21.5% of the predicted value) were studied. BMD at the lumbar spine (LS) and femoral neck (FN) and body composition were measured using dual-energy X-ray absorptiometry. Blood and urine were analysed for hormones, bone turnover markers, and the cytokines tumour necrosis factor-α, and interleukin-6 and -1β. FEV1 (% pred); CF genotype; malnutrition; history of growth, development or weight gain delays; and corticosteroid use were analysed. BMD Z-scores were −0.58±1.30 (mean±sd) at the LS and −0.24±1.19 at the FN. Z-scores of <−2.0 were found in 17% of subjects. Subjects who were homozygous or heterozygous for the ΔF508 mutation exhibited significantly lower Z-scores than those with no ΔF508 allele. Multiple linear regression showed that the ΔF508 genotype and male sex were independently associated with lower BMD at both sites. Other factors also independently associated with lower BMD included malnutrition, lower 25-hydroxyvitamin D level, lower fat-free mass and lower FEV1 (% pred). In conclusion, reduced bone mineral density in cystic fibrosis is associated with a number of factors, including ΔF508 genotype, male sex, greater lung disease severity and malnutrition.

Randomised placebo-controlled trial of non-invasive ventilation for hypercapnia in cystic fibrosis

Background: The clinical benefits of domiciliary non-invasive positive pressure ventilation (NIV) have not been established in cystic fibrosis (CF). We studied the effects of nocturnal NIV on quality of life (QoL), functional and physiological outcomes in CF subjects with awake hypercapnia (arterial carbon dioxide pressure Paco2>45 mm Hg). Methods: In a randomised, placebo controlled, crossover study, eight subjects with CF, mean (SD) age 37 (8) years, body mass index 21.1 (2.6) kg/m2, forced expiratory volume in 1 s 35 (8)% predicted and Paco2 52 (4) mm Hg received 6 weeks of nocturnal (1) air (placebo), (2) oxygen and (3) NIV. The primary outcome measures were CF specific QoL, daytime sleepiness and exertional dyspnoea. Secondary outcome measures were awake and asleep gas exchange, sleep architecture, lung function and peak exercise capacity. Results: Compared with air, NIV improved the chest symptom score in the CF QoL Questionnaire (mean difference 10; 95% CI 5 to 16; p = 0.002) and the transitional dyspnoea index score (mean difference 3.1; 95% CI 1.2–5.0; p = 0.01). It reduced maximum nocturnal pressure of transcutaneous CO2 (Ptcco2 mean difference −17 mm Hg; 95% CI −7 to −28 mm Hg; p = 0.005) and increased exercise performance on the Modified Shuttle Test (mean difference 83 m; 95% CI 21 to 144 m; p = 0.02). NIV did not improve sleep architecture, lung function or awake Paco2. Conclusion: 6 weeks of nocturnal NIV improves chest symptoms, exertional dyspnoea, nocturnal hypoventilation and peak exercise capacity in adult patients with stable CF with awake hypercapnia. Further studies are required to determine whether or not NIV can improve survival.

Linking CMV Serostatus to Episodes of CMV Reactivation Following Lung Transplantation by Measuring CMV-Specific CD8+ T-Cell Immunity

CMV‐specific immunity was assessed in a longitudinal cohort of 39 lung transplant recipients (LTR) who were followed prospectively from the time of transplant using a novel assay. At the time of surveillance bronchoscopy, CMV‐specific CD8+ T‐cell responses were assessed in the peripheral blood, using the QuantiFERON®‐CMV assay, which measures IFN‐γ‐secreting T cells following stimulation with CMV peptides. In total, 297 samples were collected from 39 LTR (CMV D+/R−, n = 8; D+/R+, n = 18; D−/R+, n = 6; D−/R−, n = 7). CMV‐specific T‐cell immunity was not detected in any of the CMV D−/R− LTR. In CMV seropositive LTR levels of CMV immunity were lowest early posttransplant and increased thereafter. While levels of CMV‐specific immunity varied between LTR, measurements at any one time point did not predict episodes of CMV reactivation. In CMV mismatched (D+/R−) LTR, primary CMV immunity was not observed during the period of antiviral prophylaxis, but typically developed during episodes of CMV reactivation. Measuring CMV‐specific CD8+ T‐cell function with the QuantiFERON®‐CMV assay provides insights into the interrelationship between CMV immunity and CMV reactivation in individual LTR. A better understanding of these dynamics may allow the opportunity to individualize antiviral prophylaxis in the future.

Influenza vaccine effectiveness against hospitalisation with confirmed influenza in the 2010-11 seasons: a test-negative observational study.

Immunisation programs are designed to reduce serious morbidity and mortality from influenza, but most evidence supporting the effectiveness of this intervention has focused on disease in the community or in primary care settings. We aimed to examine the effectiveness of influenza vaccination against hospitalisation with confirmed influenza. We compared influenza vaccination status in patients hospitalised with PCR-confirmed influenza with patients hospitalised with influenza-negative respiratory infections in an Australian sentinel surveillance system. Vaccine effectiveness was estimated from the odds ratio of vaccination in cases and controls. We performed both simple multivariate regression and a stratified analysis based on propensity score of vaccination. Vaccination status was ascertained in 333 of 598 patients with confirmed influenza and 785 of 1384 test-negative patients. Overall estimated crude vaccine effectiveness was 57% (41%, 68%). After adjusting for age, chronic comorbidities and pregnancy status, the estimated vaccine effectiveness was 37% (95% CI: 12%, 55%). In an analysis accounting for a propensity score for vaccination, the estimated vaccine effectiveness was 48.3% (95% CI: 30.0, 61.8%). Influenza vaccination was moderately protective against hospitalisation with influenza in the 2010 and 2011 seasons.

Cytomegalovirus replication within the lung allograft is associated with bronchiolitis obliterans syndrome

Early studies reported cytomegalovirus (CMV) pneumonitis as a risk factor for development of bronchiolitis obliterans syndrome (BOS) following lung transplantation. While improvements in antiviral prophylaxis have resulted in a decreased incidence of CMV pneumonitis, molecular diagnostic techniques allow diagnosis of subclinical CMV replication in the allograft. We hypothesized that this subclinical CMV replication was associated with development of BOS. We retrospectively evaluated 192 lung transplant recipients (LTR) from a single center between 2001 and 2009. Quantitative (PCR) analysis of CMV viral load and histological evidence of CMV pneumonitis and acute cellular rejection was determined on 1749 bronchoalveolar lavage (BAL) specimens and 1536 transbronchial biopsies. CMV was detected in the BAL of 41% of LTR and was significantly associated with the development of BOS (HR 1.8 [1.1–2.8], p = 0.02). This association persisted when CMV was considered more accurately as a time‐dependent variable (HR 2.1 [1.3–3.3], p = 0.003) and after adjustment for significant covariates in a multivariate model. CMV replication in the lung allograft is common following lung transplantation and is associated with increased risk of BOS. As antiviral prophylaxis adequately suppresses CMV longer prophylactic strategies may improve long‐term outcome in lung transplantation.

Addition of inhaled corticosteroids to systemic immunosuppression after lung transplantation: a double-blind, placebo-controlled trial.

Background. It is postulated that bronchiolitis obliterans syndrome (BOS) is preceded by airway inflammation that has been described even in stable lung transplant recipients. Airway inflammation is known to be suppressed by inhaled steroids in other chronic inflammatory lung diseases, e.g., asthma and chronic obstructive pulmonary disease. BOS is the major cause of morbidity and mortality after lung transplantation. Objective. To examine the effect of inhaled corticosteroids on the development of BOS in lung transplant recipients. Methods. Thirty patients were recruited and randomized in a double-blind fashion to receive either 750 &mgr;g of fluticasone propionate (FP) or an identical-appearing placebo twice daily for 3 months; 20 of this group continued until 2 years posttransplantation. Detailed spirometry was performed regularly throughout the study. Results. In the short-term study no differences were found in any examined parameters. In the long-term component of the study no differences were found in the development of neither BOS nor survival. There were minor differences in bronchoalveolar lavage (BAL) lymphocyte percentages. Conclusions. FP is ineffective for the prevention of BOS after lung transplantation despite the airway inflammation that characterizes this condition. Inadequate local delivery, timing of the therapy relative to transplantation and inherent steroid resistance of this condition may explain the negative finding of this study.

C3d and C4d Deposition Early After Lung Transplantation

BACKGROUND Complement staining as a predictor of antibody-mediated rejection (AMR) after lung transplantation continues to be debated. METHODS In a cohort of 33 lung transplant recipients (LTRs) we assessed early post-transplant (<or=3 months) graft deposition of the complement factors C3d and C4d and correlated staining with clinical outcome. A retrospective analysis of allograft C3d and C4d deposition was performed by an experienced histopathologist blinded to clinical outcomes. Biopsies were graded 0 to 3 based on extent of septal capillary complement staining. RESULTS Significant C3d and C4d staining (i.e., Grade >or=2 on more than one occasion) was observed in 20 and 11 LTRs, respectively. Complement staining was increased in LTRs with severe primary graft dysfunction or airway infection, but was not associated with acute cellular or chronic rejection, or with morphologic features of AMR. In a sub-group analysis we identified 9 LTRs who developed early bronchiolitis obliterans syndrome (BOS) in the absence of acute cellular rejection or cytomegalovirus reactivation, but they had significant lung allograft C3d/C4d deposition along with corroborative light-microscopic features suggestive of AMR. CONCLUSIONS Complement activation, as judged by lung allograft deposition of C3d/C4d, is common early post-lung transplant and may be triggered by primary graft dysfunction and/or airway infection, and may play a role in the development of early BOS.