Publications by authors named "Richard Buller"

55 Publications

Predictive Accuracy and Densitometric Analysis of Point-of-Care Immunoassay for Adenoviral Conjunctivitis.

Transl Vis Sci Technol 2021 08;10(9):30

Ohio State University, Columbus, OH, USA.

Purpose: Accurate diagnosis of adenoviral conjunctivitis (Ad-Cs) is important for timely and appropriate patient management to reduce disease transmission. This study assessed the diagnostic accuracy of a commercially available point-of-care adenovirus immunoassay and determined whether its predictive accuracy is influenced by signal intensities of test result bands.

Methods: Point-of-care immunoassay (AdenoPlus) testing and quantitative polymerase chain reaction (qPCR) testing was performed on conjunctival swab samples obtained from eyes of 186 eligible adult participants with presumed infectious conjunctivitis and symptoms of ≤4 days. Masked observers assessed signal intensities of the immunoassay test and control bands using densitometry.

Results: Ad-Cs was confirmed by qPCR in 28 of the 56 eyes that tested positive on the AdenoPlus, a 50% positive predictive value (95% confidence interval [CI] = 36.9, 63.1). No adenovirus was detected by qPCR in 128 of 130 eyes that tested negative on AdenoPlus, a 98.5% negative predictive value (CI = 96.3, 100). Sensitivity and specificity were 93% (CI = 84.4, 100) and 82% (CI = 76.0, 88.1), respectively. Viral titers significantly correlated with ratio of test band signal intensities (R2 = 0.32, P = 0.002). Higher positive predictive value was associated with higher densitometry ratios (receiver operating characteristic [ROC] area = 0.71; 95% CI = 0.59, 0.83).

Conclusions: Densitometric analyses suggest that the diagnostic accuracy of AdenoPlus is influenced by the signal intensity of the test result bands. Visual comparison of the test band intensities by clinicians could reduce the false positive rate of point-of-care immunoassays and aid in the diagnosis of viral infections.

Translational Relevance: Ratiometric densitometry of point-of-care immunoassays could aid clinicians' decision making in diagnosing infectious diseases, including Ad-Cs.
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http://dx.doi.org/10.1167/tvst.10.9.30DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8399540PMC
August 2021

Epidemiology and persistence of rhinovirus in pediatric lung transplantation.

Transpl Infect Dis 2020 Dec 2;22(6):e13422. Epub 2020 Aug 2.

Cincinnati Children's Hospital Medical Center, University of Cincinnati, Cincinnati, Ohio, USA.

Background: Infection with rhinovirus (HRV) occurs following pediatric lung transplantation. Prospective studies documenting frequencies, persistence, and progression of HRV in this at-risk population are lacking.

Methods: In the Clinical Trials in Organ Transplant in Children prospective observational study, we followed 61 lung transplant recipients for 2 years. We quantified molecular subtypes of HRV in serially collected nasopharyngeal (NP) and bronchoalveolar lavage (BAL) samples and correlated them with clinical characteristics.

Results: We identified 135 community-acquired respiratory infections (CARV) from 397 BAL and 480 NP samples. We detected 93 HRV events in 42 (68.8%) patients, 22 of which (23.4%) were symptomatic. HRV events were contiguous with different genotypes identified in 23 cases, but symptoms were not preferentially associated with any particular species. Nine (9.7%) HRV events persisted over multiple successive samples for a median of 36 days (range 18-408 days). Three persistent HRV were symptomatic. When we serially measured forced expiratory volume in one second (FEV1) in 23 subjects with events, we did not observe significant decreases in lung function over 12 months post-HRV.

Conclusion: In conjunction with our previous reports, our prospectively collected data indicate that molecularly heterogeneous HRV infections occur commonly following pediatric lung transplantation, but these infections do not negatively impact clinical outcomes.
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http://dx.doi.org/10.1111/tid.13422DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7900771PMC
December 2020

Detection of Viruses in Clinical Samples by Use of Metagenomic Sequencing and Targeted Sequence Capture.

J Clin Microbiol 2018 12 27;56(12). Epub 2018 Nov 27.

Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri, USA.

Metagenomic shotgun sequencing (MSS) is a revolutionary approach to viral diagnostic testing that allows simultaneous detection of a broad range of viruses, detailed taxonomic assignment, and detection of mutations associated with antiviral drug resistance. To enhance sensitivity for virus detection, we previously developed ViroCap, a targeted sequence capture panel designed to enrich nucleic acid from a comprehensive set of eukaryotic viruses prior to sequencing. To demonstrate the utility of MSS with targeted sequence capture for detecting clinically important viruses and characterizing clinically important viral features, we used ViroCap to analyze clinical samples from a diagnostic virology laboratory containing a broad range of medically relevant viruses. From 26 samples, MSS with ViroCap detected all of the expected viruses and 30 additional viruses. Comparing sequencing after capture enrichment with standard MSS, we detected 13 viruses only with capture enrichment and observed a consistent increase in the number and percentage of viral sequence reads as well as the breadth and depth of coverage of the viral genomes. Compared with clinical testing, MSS enhanced taxonomic assignment for 15 viruses, and codons associated with antiviral drug resistance in influenza A virus, herpes simplex virus (HSV), human immunodeficiency virus (HIV), and hepatitis C virus (HCV) could be analyzed. Overall, in clinical samples, MSS with targeted sequence capture provides enhanced virus detection and information of clinical and epidemiologic relevance compared with clinical testing and MSS without targeted sequence capture.
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http://dx.doi.org/10.1128/JCM.01123-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6258860PMC
December 2018

Heartland Virus and Hemophagocytic Lymphohistiocytosis in Immunocompromised Patient, Missouri, USA.

Emerg Infect Dis 2018 05;24(5):893-897

Heartland virus is a suspected tickborne pathogen in the United States. We describe a case of hemophagocytic lymphohistiocytosis, then death, in an immunosuppressed elderly man in Missouri, USA, who was infected with Heartland virus.
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http://dx.doi.org/10.3201/eid2405.171802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5938783PMC
May 2018

Epstein-Barr viral loads do not predict post-transplant lymphoproliferative disorder in pediatric lung transplant recipients: A multicenter prospective cohort study.

Pediatr Transplant 2017 Sep 21;21(6). Epub 2017 Jun 21.

Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA.

Prediction of PTLD after pediatric lung transplant remains difficult. Use of EBV VL in WB has been poorly predictive, while measurement of VL in BAL fluid has been suggested to have enhanced utility. The NIH-sponsored Clinical Trials in Organ Transplantation in Children (CTOTC-03) prospectively obtained serial quantitative measurements of EBV PCR in both WB and BAL fluid after pediatric lung transplantation. Descriptive statistics, contingency analyses, and Kaplan-Meier analyses evaluated possible association between EBV and PTLD. Of 61 patients, 34 (56%) had an EBV+PCR (at least once in WB or BAL). EBV donor (D)+patients more often had a positive PCR (D+/recipient (R)-: 13/18; D+/R+: 14/23) compared to EBV D- patients (6/17). Several D-/R- (5/12) patients developed EBV, but none developed PTLD. All four PTLD patients were D+/R- with EBV+PCR. Neither the time to first EBV+PCR nor the CT for PCR positivity in BAL or WB was statistically different between those with and without PTLD. Having an EBV-seropositive donor was associated with increased risk of EBV+PCR in WB. EBV load in BAL was not predictive of PTLD.
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http://dx.doi.org/10.1111/petr.13011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5568922PMC
September 2017

Multicenter Clinical Evaluation of the Luminex Aries Flu A/B & RSV Assay for Pediatric and Adult Respiratory Tract Specimens.

J Clin Microbiol 2017 08 24;55(8):2431-2438. Epub 2017 May 24.

Scott and White Medical Center, Baylor Scott and White Healthcare, Temple, Texas, USA.

Influenza A and B viruses and respiratory syncytial virus (RSV) are three common viruses implicated in seasonal respiratory tract infections and are a major cause of morbidity and mortality in adults and children worldwide. In recent years, an increasing number of commercial molecular tests have become available to diagnose respiratory viral infections. The Luminex Aries Flu A/B & RSV assay is a fully automated sample-to-answer molecular diagnostic assay for the detection of influenza A, influenza B, and RSV. The clinical performance of the Aries Flu A/B & RSV assay was prospectively evaluated in comparison to that of the Luminex xTAG respiratory viral panel (RVP) at four North American clinical institutions over a 2-year period. Of the 2,479 eligible nasopharyngeal swab specimens included in the prospective study, 2,371 gave concordant results between the assays. One hundred eight specimens generated results that were discordant with those from the xTAG RVP and were further analyzed by bidirectional sequencing. Final clinical sensitivity values of the Aries Flu A/B & RSV assay were 98.1% for influenza A virus, 98.0% for influenza B virus, and 97.7% for RSV. Final clinical specificities for all three pathogens ranged from 98.6% to 99.8%. Due to the low prevalence of influenza B, an additional 40 banked influenza B-positive specimens were tested at the participating clinical laboratories and were all accurately detected by the Aries Flu A/B & RSV assay. This study demonstrates that the Aries Flu A/B & RSV assay is a suitable method for rapid and accurate identification of these causative pathogens in respiratory infections.
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http://dx.doi.org/10.1128/JCM.00318-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5527421PMC
August 2017

Tissue polymerase chain reaction for the diagnosis of cytomegalovirus disease after allogeneic hematopoietic cell transplantation.

Am J Hematol 2017 02;92(2):E19-E20

Bone Marrow Transplantation and Leukemia Section, Division of Oncology, Washington University School of Medicine, St. Louis, Missouri.

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http://dx.doi.org/10.1002/ajh.24609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8211556PMC
February 2017

Epidemiology, Co-Infections, and Outcomes of Viral Pneumonia in Adults: An Observational Cohort Study.

Medicine (Baltimore) 2015 Dec;94(50):e2332

From the Pharmacy St. Louis College of Pharmacy (STLCOP) (MPC); STLCOP and Dept of Pharmacy, Barnes-Jewish Hospital (DJR); STLCOP (SM); STLCOP and Dept of Pharmacy, Barnes-Jewish Hospital (STM); Center for Clinical Excellence, BJC Healthcare (NH); Department of Pediatrics, Washington University School of Medicine (RSB, GAS); and Division of Pulmonary and Critical Care Medicine, Washington University School of Medicine (MHK).

Advanced technologies using polymerase-chain reaction have allowed for increased recognition of viral respiratory infections including pneumonia. Co-infections have been described for several respiratory viruses, especially with influenza. Outcomes of viral pneumonia, including cases with co-infections, have not been well described. This was observational cohort study conducted to describe hospitalized patients with viral pneumonia including co-infections, clinical outcomes, and predictors of mortality. Patients admitted from March 2013 to November 2014 with a positive respiratory virus panel (RVP) and radiographic findings of pneumonia within 48  h of the index RVP were included. Co-respiratory infection (CRI) was defined as any organism identification from a respiratory specimen within 3 days of the index RVP. Predictors of in-hospital mortality on univariate analysis were evaluated in a multivariate model. Of 284 patients with viral pneumonia, a majority (51.8%) were immunocompromised. A total of 84 patients (29.6%) were found to have a CRI with 48 (57.6%) having a bacterial CRI. Viral CRI with HSV, CMV, or both occurred in 28 patients (33.3%). Fungal (16.7%) and other CRIs (7.1%) were less common. Many patients required mechanical ventilation (54%) and vasopressor support (36%). Overall in-hospital mortality was high (23.2%) and readmissions were common with several patients re-hospitalized within 30 (21.1%) and 90 days (36.7%) of discharge. Predictors of in-hospital mortality on multivariate regression included severity of illness factors, stem-cell transplant, and identification of multiple respiratory viruses. In conclusion, hospital mortality is high among adult patients with viral pneumonia and patients with multiple respiratory viruses identified may be at a higher risk.
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http://dx.doi.org/10.1097/MD.0000000000002332DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5058945PMC
December 2015

A case for the use of receiver operating characteristic analysis of potential clinical efficacy biomarkers in advanced renal cell carcinoma.

Future Oncol 2016 Jan 17;12(2):175-82. Epub 2015 Dec 17.

Pfizer Oncology, 10646 Science Center Drive, San Diego, CA 92121, USA.

Aim: Assess patient-level utility of suggested pretreatment biomarkers of sunitinib in advanced renal cell carcinoma.

Patients & Methods: Kaplan-Meier analysis of data from a randomized, Phase II study (n = 292) suggested baseline predictive value for circulating soluble Ang-2 and MMP-2 and HIF-1α percentage of tumor expression. Using this dataset, the sensitivity, specificity and area under the curve (AUC) were calculated, using receiver operating characteristic (ROC) curves.

Results: Based on a ROC (sensitivity vs 1 - specificity) threshold AUC value of >0.8, neither Ang-2 (0.67) nor MMP-2 (0.65), nor HIF-1α percentage of tumor expression (0.65), performed appropriately from a patient-selection standpoint.

Conclusion: To properly assess potential biomarkers, sensitivity and specificity characteristics should be obtained by ROC analysis.
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http://dx.doi.org/10.2217/fon.15.290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5549778PMC
January 2016

Impact of antibacterials on subsequent resistance and clinical outcomes in adult patients with viral pneumonia: an opportunity for stewardship.

Crit Care 2015 Nov 18;19:404. Epub 2015 Nov 18.

St. Louis College of Pharmacy, 4588 Parkview Place, St. Louis, MO, 63110, USA.

Introduction: Respiratory viruses are increasingly recognized as significant etiologies of pneumonia among hospitalized patients. Advanced technologies using multiplex molecular assays and polymerase-chain reaction increase the ability to identify viral pathogens and may ultimately impact antibacterial use.

Method: This was a single-center retrospective cohort study to evaluate the impact of antibacterials in viral pneumonia on clinical outcomes and subsequent multidrug-resistant organism (MDRO) infections/colonization. Patients admitted from March 2013 to November 2014 with positive respiratory viral panels (RVP) and radiographic findings of pneumonia were included. Patients transferred from an outside hospital or not still hospitalized 72 hours after the RVP report date were excluded. Patients were categorized based on exposure to systemic antibacterials: less than 3 days representing short-course therapy and 3 to 10 days being long-course therapy.

Results: A total of 174 patients (long-course, n = 67; short-course, n = 28; mixed bacterial-viral infection, n = 79) were included with most being immunocompromised (56.3 %) with active malignancy the primary etiology (69.4 %). Rhinovirus/Enterovirus (23 %), Influenza (19 %), and Parainfluenza (15.5 %) were the viruses most commonly identified. A total of 13 different systemic antibacterials were used as empiric therapy in the 95 patients with pure viral infection for a total of 466 days-of-therapy. Vancomycin (50.7 %), cefepime (40.3 %), azithromycin (40.3 %), meropenem (23.9 %), and linezolid (20.9 %) were most frequently used. In-hospital mortality did not differ between patients with viral pneumonia in the short-course and long-course groups. Subsequent infection/colonization with a MDRO was more frequent in the long-course group compared to the short-course group (53.2 vs 21.1 %; P = 0.027).

Conclusion: This study found that long-course antibacterial use in the setting of viral pneumonia had no impact on clinical outcomes but increased the incidence of subsequent MDRO infection/colonization.
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http://dx.doi.org/10.1186/s13054-015-1120-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4650137PMC
November 2015

Does azithromycin modify viral load during severe respiratory syncytial virus bronchiolitis?

J Allergy Clin Immunol 2015 Oct 26;136(4):1129-31. Epub 2015 Jul 26.

Department of Pediatrics, Washington University School of Medicine and St Louis Children's Hospital, St Louis, Mo.

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http://dx.doi.org/10.1016/j.jaci.2015.06.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4600448PMC
October 2015

Development and Evaluation of an Enterovirus D68 Real-Time Reverse Transcriptase PCR Assay.

J Clin Microbiol 2015 Aug 10;53(8):2641-7. Epub 2015 Jun 10.

Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri, USA

We have developed and evaluated a real-time reverse transcriptase PCR (RT-PCR) assay for the detection of human enterovirus D68 (EV-D68) in clinical specimens. This assay was developed in response to the unprecedented 2014 nationwide EV-D68 outbreak in the United States associated with severe respiratory illness. As part of our evaluation of the outbreak, we sequenced and published the genome sequence of the EV-D68 virus circulating in St. Louis, MO. This sequence, along with other GenBank sequences from past EV-D68 occurrences, was used to computationally select a region of EV-D68 appropriate for targeting in a strain-specific RT-PCR assay. The RT-PCR assay amplifies a segment of the VP1 gene, with an analytic limit of detection of 4 copies per reaction, and it was more sensitive than commercially available assays that detect enteroviruses and rhinoviruses without distinguishing between the two, including three multiplex respiratory panels approved for clinical use by the FDA. The assay did not detect any other enteroviruses or rhinoviruses tested and did detect divergent strains of EV-D68, including the first EV-D68 strain (Fermon) identified in California in 1962. This assay should be useful for identifying and studying current and future outbreaks of EV-D68 viruses.
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http://dx.doi.org/10.1128/JCM.00923-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4508392PMC
August 2015

A simplified interventional mapping system (SIMS) for the selection of combinations of targeted treatments in non-small cell lung cancer.

Oncotarget 2015 Jun;6(16):14139-52

Karolinska Institutet, Stockholm, Sweden.

Non-small cell lung cancer (NSCLC) is a leading cause of death worldwide. Targeted monotherapies produce high regression rates, albeit for limited patient subgroups, who inevitably succumb. We present a novel strategy for identifying customized combinations of triplets of targeted agents, utilizing a simplified interventional mapping system (SIMS) that merges knowledge about existent drugs and their impact on the hallmarks of cancer. Based on interrogation of matched lung tumor and normal tissue using targeted genomic sequencing, copy number variation, transcriptomics, and miRNA expression, the activation status of 24 interventional nodes was elucidated. An algorithm was developed to create a scoring system that enables ranking of the activated interventional nodes for each patient. Based on the trends of co-activation at interventional points, combinations of drug triplets were defined in order to overcome resistance. This methodology will inform a prospective trial to be conducted by the WIN consortium, aiming to significantly impact survival in metastatic NSCLC and other malignancies.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4546456PMC
http://dx.doi.org/10.18632/oncotarget.3741DOI Listing
June 2015

Multicenter comparison of laboratory performance in cytomegalovirus and Epstein-Barr virus viral load testing using international standards.

Clin Transplant 2014 Dec 13;28(12):1416-23. Epub 2014 Nov 13.

Clinical Microbiology Laboratory, Massachusetts General Hospital, Boston, MA, USA.

Background: Infections with cytomegalovirus (CMV) and Epstein-Barr virus (EBV) remain important in solid organ transplantation. Quantitative viral nucleic acid testing is a major advance to patient management. These assays are limited by a lack of standardization, resulting in viral load measurements that differ among clinical laboratories. The variability in viral load measurements makes interpretation of multicenter clinical trials data difficult. This study compares the current practices in CMV and EBV viral load testing at four large transplant centers participating in multicenter Clinical Trials in Organ Transplantation and the Clinical Trials in Organ Transplantation in Children (CTOT and CTOTC).

Methods: Viral load testing was performed on well-defined viral preparations according to standard operating procedures at each site.

Results: Among centers, CMV viral load testing was accurate compared to WHO International Standards and within acceptable variation for this testing method. Epstein-Barr virus viral load data were more variable and less accurate despite the use of international standards.

Conclusions: These data suggest that comparison of CMV, but not EBV, viral load measurements at these sites is possible using current assays and control standards. Standardization of these assays is facilitated using the WHO International Standards and will allow comparison of viral load results among transplant centers. Assay standardization must be performed prior to initiation of multicenter trials.
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http://dx.doi.org/10.1111/ctr.12473DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4481866PMC
December 2014

National Working Group Meeting on ALK diagnostics in lung cancer.

Asia Pac J Clin Oncol 2014 Apr;10 Suppl 2:11-7

Tissue Pathology and Diagnostic Oncology, Royal Prince Alfred Hospital, Sydney, New South Wales, Australia; Sydney Medical School, University of Sydney, Sydney, New South Wales, Australia; School of Medicine, University of Western Sydney, Sydney, New South Wales, Australia.

The global landscape of molecular testing is rapidly changing, with the recent publication of the International Association for the Study of Lung Cancer (IASLC)/College of American Pathologists (CAP) guidelines and the ALK Atlas. The IASLC/CAP guidelines recommend that tumors from patients with non-small cell lung cancer (NSCLC) be tested for ALK rearrangements in addition to epidermal growth factor receptor (EGFR) mutations. The spur for this recommendation is the availability of novel therapies that target these rearrangements. This article is based on coverage of a Pfizer-sponsored National Working Group Meeting on ALK Diagnostics in Lung Cancer, held around the 15th World Lung Cancer Conference, in Sydney on October 31, 2013. It is based on the presentations given by the authors at the meeting and the discussion that ensued. The content for this article was discussed and agreed on by the authors.
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http://dx.doi.org/10.1111/ajco.12190DOI Listing
April 2014

Considerations for the successful co-development of targeted cancer therapies and companion diagnostics.

Nat Rev Drug Discov 2013 10 6;12(10):743-55. Epub 2013 Sep 6.

Genentech, 1 DNA Way, MS 441b, South San Francisco, California 94080, USA.

As diagnostic tests become increasingly important for optimizing the use of drugs to treat cancers, the co-development of a targeted therapy and its companion diagnostic test is becoming more prevalent and necessary. In July 2011, the US Food and Drug Administration released a draft guidance that gave the agency's formal definition of companion diagnostics and introduced a drug-diagnostic co-development process for gaining regulatory approval. Here, we identify areas of drug-diagnostic co-development that were either not covered by the guidance or that would benefit from increased granularity, including how to determine when clinical studies should be limited to biomarker-positive patients, defining the diagnostically selected patient population in which to use a companion diagnostic, and defining and clinically validating a biomarker signature for assays that use more than one biomarker. We propose potential approaches that sponsors could use to deal with these challenges and provide strategies to help guide the future co-development of drugs and diagnostics.
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http://dx.doi.org/10.1038/nrd4101DOI Listing
October 2013

Molecular detection of respiratory viruses.

Authors:
Richard S Buller

Clin Lab Med 2013 Sep;33(3):439-60

Department of Pediatrics, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA.

Over the past several years a wide variety of molecular assays for the detection of respiratory viruses has reached the market. The tests described herein range from kits containing primers and probes detecting specific groups of viruses, to self-contained systems requiring specialized instruments that extract nucleic acids and perform the polymerase chain reaction with little operator input. Some of the tests target just the viruses involved in large yearly epidemics such as influenza, or specific groups of viruses such as the adenoviruses or parainfluenza viruses; others can detect most of the known respiratory viruses and some bacterial agents.
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http://dx.doi.org/10.1016/j.cll.2013.03.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7126104PMC
September 2013

Detection of viruses in young children with fever without an apparent source.

Pediatrics 2012 Dec 5;130(6):e1455-62. Epub 2012 Nov 5.

Department of Pediatrics, Washington University School of Medicine, St Louis, MO 63110, USA.

Objective: Fever without an apparent source is common in young children. Currently in the United States, serious bacterial infection is unusual. Our objective was to determine specific viruses that might be responsible.

Methods: We enrolled children aged 2 to 36 months with temperature of 38°C or greater without an apparent source or with definite or probable bacterial infection being evaluated in the St Louis Children's Hospital Emergency Department and afebrile children having ambulatory surgery. Blood and nasopharyngeal swab samples were tested with an extensive battery of virus-specific polymerase chain reaction assays.

Results: One or more viruses were detected in 76% of 75 children with fever without an apparent source, 40% of 15 children with fever and a definite or probable bacterial infection, and 35% of 116 afebrile children (P < .001). Four viruses (adenovirus, human herpesvirus 6, enterovirus, and parechovirus) were predominant, being detected in 57% of children with fever without a source, 13% of children with fever and definite or probable bacterial infection, and 7% of afebrile children (P < .001). Thirty-four percent of 146 viral infections were detected only by polymerase chain reaction performed on blood. Fifty-one percent of children with viral infections and no evidence of bacterial infection were treated with antibiotics.

Conclusions: Viral infections are frequent in children with fever without an apparent source. Testing of blood in addition to nasopharyngeal secretions expanded the range of viruses detected. Future studies should explore the utility of testing for the implicated viruses. Better recognition of viruses that cause undifferentiated fever in young children may help limit unnecessary antibiotic use.
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http://dx.doi.org/10.1542/peds.2012-1391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3507256PMC
December 2012

Rising rates of macrolide-resistant Mycoplasma pneumoniae in the central United States.

Pediatr Infect Dis J 2012 Apr;31(4):409-0

Department of Pediatrics, Washington University School of Medicine, St. Louis, MO, USA.

Macrolide-resistant Mycoplasma pneumoniae is widespread in Asia, and severe cases of pneumonia have been described in children. Little information is available about the resistance pattern in the United States. We collected respiratory samples from 49 patients with Mycoplasma infection in the central United States between 2007 and 2010. We found a macrolide resistance rate of 8.2%. Resistance should be considered when patients with M. pneumoniae infection do not have a satisfactory response to macrolides. Alternative antibiotics include tetracyclines or fluoroquinolones.
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http://dx.doi.org/10.1097/INF.0b013e318247f3e0DOI Listing
April 2012

Comparison of the Eragen Multi-Code Respiratory Virus Panel with conventional viral testing and real-time multiplex PCR assays for detection of respiratory viruses.

J Clin Microbiol 2010 Jul 19;48(7):2387-95. Epub 2010 May 19.

Department of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110, USA.

High-throughput multiplex assays for respiratory viruses are an important step forward in diagnostic virology. We compared one such assay, the PLx Multi-Code Respiratory Virus Panel (PLx-RVP), manufactured by Eragen Biosciences, Inc. (Madison, WI), with conventional virologic testing, consisting of fluorescent-antibody staining plus testing with the R-mix system and fibroblast tube cultures. The test set consisted of 410 archived respiratory specimens, mostly nasopharyngeal swabs, including 210 that had been positive by conventional testing for a balanced selection of common respiratory viruses. Specimens yielding discrepant results were evaluated using a panel of respiratory virus PCR assays developed, characterized, and validated with clinical specimens. PLx-RVP increased the total rate of detection of viruses by 35.8%, and there was a 25.7% increase in the rate of detection of positive specimens. Reference PCR assay results corroborated the PLx-RVP result for 54 (82%) of 66 discrepancies with conventional testing. Of the 12 specimens with discrepancies between PLx-RVp and the reference PCRs, 6 were positive for rhinovirus by PLx-RVP and the presence of rhinovirus was confirmed by nucleotide sequencing. The remaining six specimens included five in which the PLx-RVP failed to detect parainfluenza virus and one in which the detection of influenza A virus by PLx-RVP could not be confirmed by the reference PCR. Taking the results of the reference PCR assay results into account, the sensitivities of the PLx-RVP for individual viruses ranged from 94 to 100% and the specificities ranged from 99 to 100%. We conclude that PLx-RVP is a highly accurate system for the detection of respiratory viruses and significantly improves the rate of detection of these viruses compared to that by conventional virologic testing.
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http://dx.doi.org/10.1128/JCM.00220-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2897522PMC
July 2010

KI polyomavirus detected in respiratory tract specimens from patients in St. Louis, Missouri.

Pediatr Infect Dis J 2010 Apr;29(4):329-33

Department of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110, USA.

Background: Studies have reported the presence of KI polyomavirus (KIPyV) and WU polyomavirus (WUPyV) in respiratory secretions of young patients. So far, evidence has not supported a link between infections with either virus and respiratory tract disease; however, there has not been a large comparison of KIPyV-infected patients to age-matched patient groups.

Methods: A retrospective study comparing clinical aspects of KIPyV-positive patients with respiratory syncytial virus (RSV)-positive, WUPyV-positive, and respiratory-virus negative patients. Using real-time polymerase chain reaction, 2599 respiratory samples from patients ranging from 1 day to 88 years of age were tested for KIPyV. Electronic medical records were reviewed for 65 cases, for a comparison group consisting of 195 patients negative for common respiratory viruses, and for 56 WUPyV-positive patients drawn from the same population. Twelve patients testing positive for KIPyV as the sole pathogen were matched to 36 RSV-positive patients and clinical features of both groups were compared.

Results: Seventy-two (2.8%) respiratory samples were positive for KIPyV. Another virus was detected in 71% of the KIPyV-positive samples. Analysis showed no statistically significant differences in clinical manifestations between KIPyV-positive patients and patients negative for common respiratory viruses, however, clinical characteristics of KIPyV-positive patients were less severe than those of patients positive for RSV. KIPyVpositive patients >or=3 years of age were usually immunocompromised in contrast to the younger children with KIPyV.

Conclusions: This study did not demonstrate a link between KIPyV infection and symptomatic respiratory disease. Patients positive for KIPyV exhibited less severe clinical symptoms than patients positive for RSV.
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http://dx.doi.org/10.1097/INF.0b013e3181c1795cDOI Listing
April 2010

Clinical and epidemiologic characterization of WU polyomavirus infection, St. Louis, Missouri.

Emerg Infect Dis 2007 Dec;13(12):1936-8

Washington University School of Medicine, St. Louis, Missouri, USA.

WU polyomavirus is a recently described polyomavirus found in patients with respiratory infections. Of 2,637 respiratory samples tested in St. Louis, Missouri, 2.7% were positive for WU polyomavirus by PCR, and 71% were coinfected with other respiratory viruses. Persistent human infection with WU polyomavirus is described.
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http://dx.doi.org/10.3201/eid1312.070977DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2876771PMC
December 2007

MultiCode-PLx system for multiplexed detection of seventeen respiratory viruses.

J Clin Microbiol 2007 Sep 27;45(9):2779-86. Epub 2007 Jun 27.

Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia, USA.

The MultiCode-PLx system (EraGen Biosciences, Inc., Madison, WI) for the detection of respiratory viruses uses an expanded genetic alphabet, multiplex PCR chemistry, and microsphere flow cytometry to rapidly detect and specifically identify 17 different respiratory viruses directly in clinical specimens. The MultiCode-PLx system was tested in parallel with direct fluorescent-antibody (DFA) staining and rapid shell vial culture (R-mix cells; Diagnostic Hybrids, Inc. Athens, OH) with 354 respiratory specimens from adult patients that were submitted to the clinical virology laboratory at the Emory University Hospital. Single-target PCRs were performed with retained samples to confirm the positive results obtained with the MultiCode-PLx system for viruses not covered by DFA and R-mix culture (metapneumovirus, coronaviruses [CoV], parainfluenza viruses 4a and 4b, and rhinoviruses) and to resolve any discrepancies between the DFA and R-mix culture and the MultiCode-PLx results for viruses common to both systems. Respiratory viruses were detected in 77 (21.8%) and 116 (32.7%) specimens by DFA and R-mix culture and with the MultiCode-PLx system, respectively. Among the viruses common to both systems, the MultiCode-PLx system detected significantly more influenza A viruses (P = 0.0026). An additional increased diagnostic yield with the MultiCode-PLx system resulted from the detection of human metapneumovirus (HMPV) in 9 specimens, human CoV (HCoV) in 3 specimens, and human rhinovirus (HRV) in 16 specimens. Also, two mixed viral infections were detected by the MultiCode-PLx system (HCoV OC43 and HRV infections and HMPV and HRV infections), but none were detected by DFA and R-mix culture. Single-target PCRs verified the results obtained with the MultiCode-PLx system for 73 of 81 (90.1%) specimens that had discordant results or that were not covered by DFA and R-mix culture. The MultiCode-PLx system provides clinical laboratories with a practical, rapid, and sensitive means for the massively multiplexed molecular detection of common respiratory viruses.
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http://dx.doi.org/10.1128/JCM.00669-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2045250PMC
September 2007

Panton-Valentine Leukocidin-positive methicillin-resistant Staphylococcus aureus lung infection in patients with cystic fibrosis.

Chest 2007 Jun 30;131(6):1718-25. Epub 2007 Mar 30.

Department of Pediatrics, Washington University School of Medicine, Campus Box 8116, 660 S Euclid Ave, Saint Louis, MO 63110, USA.

Background: Panton-Valentine Leukocidin-expressing (PVL+) methicillin-resistant Staphylococcus aureus (MRSA) is an emerging pathogen worldwide causing fatal necrotizing pneumonias in otherwise healthy individuals but has not been described in patients with cystic fibrosis (CF). Following two cases of patients with CF admitted with lung abscesses in association with PVL+ MRSA, we examined the incidence and the clinical characteristics of MRSA acquisition in our CF patient population.

Methods: Newly acquired MRSA isolates from patients with CF followed up at St. Louis Children's Hospital were analyzed for the presence of Panton-Valentine leukocidin coding region, clindamycin susceptibility, staphylococcal cassette chromosome (SCC) mec type, and multilocus sequence type. Medical records and pulmonary function studies at the time of MRSA isolation were reviewed.

Results: MRSA isolates from 40 CF patients were available for analysis. Six children (15%) had PVL+ MRSA infection. All PVL+ organisms were clindamycin susceptible. Patients who acquired a PVL+ organism were more likely to have a focal pulmonary infiltrate on chest radiograph, including cavitary lung lesions in two patients (p = 0.04), a markedly greater decline in FEV1 at the time of MRSA detection (p = 0.01), and a significantly higher WBC count (p = 0.04) and absolute neutrophil count (p = 0.04). These patients were more likely to be admitted for IV antibiotic therapy for respiratory illnesses (p < 0.01).

Conclusions: We describe the emergence of PVL+ MRSA in our CF population in association with development of invasive lung infections including lung abscesses. Early identification and treatment of CF patients with newly acquired PVL+ MRSA may be crucial.
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http://dx.doi.org/10.1378/chest.06-2756DOI Listing
June 2007

Roles of KLF6 and KLF6-SV1 in ovarian cancer progression and intraperitoneal dissemination.

Clin Cancer Res 2006 Jun;12(12):3730-9

Department of Human Genetics, Mount Sinai School of Medicine, 1425 Madison Avenue, New York, New York 10029, USA.

Purpose: We investigated the role of the KLF6 tumor suppressor gene and its alternatively spliced isoform KLF6-SV1 in epithelial ovarian cancer (EOC).

Experimental Design: We first analyzed tumors from 68 females with EOC for KLF6 gene inactivation using fluorescent loss of heterozygosity (LOH) analysis and direct DNA sequencing and then defined changes in KLF6 and KLF6-SV1 expression levels by quantitative real-time PCR. We then directly tested the effect of KLF6 and KLF6-SV1 inhibition in SKOV-3 stable cell lines on cellular invasion and proliferation in culture and tumor growth, i.p. dissemination, ascites production, and angiogenesis in vivo using BALB/c nu/nu mice. All statistical tests were two sided.

Results: LOH was present in 59% of samples in a cell type-specific manner, highest in serous (72%) and endometrioid (75%) subtypes, but absent in clear cell tumors. LOH was significantly correlated with tumor stage and grade. In addition, KLF6 expression was decreased in tumors when compared with ovarian surface epithelial cells. In contrast, KLF6-SV1 expression was increased approximately 5-fold and was associated with increased tumor grade regardless of LOH status. Consistent with these findings, KLF6 silencing increased cellular and tumor growth, angiogenesis, and vascular endothelial growth factor expression, i.p. dissemination, and ascites production. Conversely, KLF6-SV1 down-regulation decreased cell proliferation and invasion and completely suppressed in vivo tumor formation.

Conclusion: Our results show that KLF6 and KLF6-SV1 are associated with key clinical features of EOC and suggest that their therapeutic targeting may alter ovarian cancer growth, progression, and dissemination.
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http://dx.doi.org/10.1158/1078-0432.CCR-06-0054DOI Listing
June 2006

Blinded comparison of repetitive-sequence PCR and multilocus sequence typing for genotyping methicillin-resistant Staphylococcus aureus isolates from a children's hospital in St. Louis, Missouri.

J Clin Microbiol 2006 Jun;44(6):2254-7

Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110, USA.

We performed a blinded study to compare repetitive-sequence PCR and multilocus sequence typing for genotyping hospital- and community-acquired methicillin-resistant Staphylococcus aureus (MRSA). The MRSA strains that were sequence type 8 (ST8), staphylococcal cassette chromosome mec (SCCmec) type IV, and Panton-Valentine leukocidin-positive clustered separately from those that were ST5 and SCCmec type II.
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http://dx.doi.org/10.1128/JCM.00690-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1489435PMC
June 2006

Topotecan in the treatment of elderly patients with relapsed small-cell lung cancer.

Clin Lung Cancer 2005 Nov;7(3):190-6

Department of Medicine, Oncology, Duke Comprehensive Cancer Center, Durham, NC 27710, USA.

Background: Almost 70% of all patients with lung cancer in the United States are>65 years of age, and the incidence of small-cell lung cancer (SCLC) increases with age until the eighth decade of life. However, elderly patients are underrepresented in clinical trials and are often suboptimally treated. The validity of age as a prognostic factor for toxicity or survival remains controversial.

Patients And Methods: To investigate the safety and efficacy of topotecan (an approved treatment for relapsed SCLC) in older patients, we performed a retrospective analysis in patients >or= 65 years of age versus patients < 65 years of age from 5 large topotecan trials. In all 5 trials, patients received topotecan 1.5 mg/m2 per day via a 30-minute intravenous infusion on days 1 through 5 of a 21-day cycle. Efficacy and tolerability outcomes were assessed for both age groups.

Results: Topotecan was similarly tolerated in both age groups, with generally manageable hematologic toxicity. The incidence, duration, and onset of severe hematologic toxicities did not vary significantly with age. In the<65 age group, grade 4 neutropenia and leukopenia were reported in 72% and 32% of patients, respectively; in the >or= 65 age group, grade 4 neutropenia and leukopenia were reported in 77% and 31% of patients, respectively. Grade 4 thrombocytopenia was less common in the<65 age group. Nonhematologic toxicities, median time to progression, and overall survival were comparable between groups.

Conclusion: This is the first demonstration of the safety and efficacy of topotecan in older patients with recurrent SCLC. Future studies are needed to fully characterize the role of topotecan in the treatment of older patients.
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http://dx.doi.org/10.3816/CLC.2005.n.035DOI Listing
November 2005

Neoadjuvant chemotherapy in vulvar cancer: avoiding primary exenteration.

Gynecol Oncol 2006 Jan 27;100(1):53-7. Epub 2005 Oct 27.

Indiana Women's Oncology, University of Iowa-Holden Comprehensive Cancer Center, Division of Gynecologic Oncology, St. Vincent Hospitals, 8301 Harcourt Road, Suite 201, Indianapolis, IN 46260, USA.

Objective: To determine whether neoadjuvant cisplatin and 5-fluorouracil chemotherapy can be used to preserve the anal sphincter and/or urethra in patients with advanced vulvar cancer involving these sites.

Methods: Fourteen patients with advanced vulvar cancer (1997-2003) involving the anal sphincter and/or urethra were given 3-4 cycles of neoadjuvant chemotherapy to attempt preservation of these pelvic structures rather than undergoing a primary pelvic exenteration. Following 3 cycles, a radical vulvectomy and groin lymph node dissection were planned. All patients had lesion size documented by measurement and photograph prior to and following chemotherapy.

Results: The median age was 63 years (range 39-88). Thirteen patients received a median of 3 cycles (range 2-4) of neoadjuvant chemotherapy. Ten patients received cisplatin and 5-fluorouracil, while three received cisplatin alone. The median time from diagnosis to surgery was 77 days (range 54-143). All patients with cisplatin and 5-fluorouracil chemotherapy underwent surgery except one patient who had a synchronous renal cell carcinoma and died prior to surgery. Patients receiving cisplatin alone showed no measurable response, while all patients receiving cisplatin and 5-fluorouracil demonstrated at least a partial response. Two patients had no residual invasive carcinoma on final pathology. All patients receiving cisplatin and 5-fluorouracil followed by surgery are disease-free, while two of three receiving cisplatin have progressive disease. The anal sphincter and urethra were conserved in all patients receiving cisplatin and 5-fluorouracil.

Conclusion: Neoadjuvant cisplatin and 5-fluorouracil in advanced vulvar cancer demonstrated a response rate of 100%. The anal sphincter and urethra were conserved in all patients receiving cisplatin and 5-fluorouracil. Responders are disease-free at this time. This response rate demonstrates superior activity of 5-fluorouracil in vulvar cancer and spares these patients the morbidity of exenteration or radiation.
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http://dx.doi.org/10.1016/j.ygyno.2005.06.068DOI Listing
January 2006

Critical function of the CD40 pathway in parvovirus B19 infection revealed by a hypomorphic CD40 ligand mutation.

Clin Immunol 2005 Dec 5;117(3):231-7. Epub 2005 Oct 5.

Institute of Transfusion Medicine, University of Leipzig, Leipzig, Germany.

Parvovirus B19-induced chronic anemia has been associated with failure to mount an effective neutralizing antibody response. We describe an adolescent male with a 13-year history of parvovirus B19-induced anemia as the primary manifestation of X-linked hyper IgM immunodeficiency (XHIM). This patient, whose serum IgG concentration was at the low end of the normal range and who mounted IgG antibody responses to T cell-dependent antigens, suffered from a nonsense mutation (R11 --> X) in the CD40 ligand (CD40L) gene. This resulted in low-level expression of a mutant CD40L predicted to lack the cytoplasmic domain. Intravenous immunoglobulin therapy alone or in combination with interferon gamma, given in the context of impaired Th1 cytokine production, suppressed but did not eradicate the infection. These results highlight the critical function of the CD40/CD40L pathway in parvovirus B19 infection and suggest that subtle defects in this pathway may underlie cases of chronic parvovirus B19 infection atypical of XHIM.
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http://dx.doi.org/10.1016/j.clim.2005.08.005DOI Listing
December 2005
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