David F. Bibby

Molecular Investigations of an Outbreak of Parainfluenza Virus Type 3 and Respiratory Syncytial Virus Infections in a Hematology Unit

ABSTRACT A large simultaneous outbreak of respiratory syncytial virus (RSV) and parainfluenza type 3 (PIV-3) infections occurred on an adult hematology unit. Implementation of enhanced infection control was complicated by cocirculation of the two different viruses, with prolonged viral shedding from infected patients, and placed great pressure on health care staff; of 27 infected hematopoietic stem cell transplant patients, 9 died, and the unit was closed for 2 months. Retrospective molecular investigation of the virus strains involved in the outbreak was performed by analyzing part of the fusion gene of PIV-3 and part of the glycoprotein gene of RSV. Reverse transcription-PCR on nasopharyngeal aspirates from patients infected before and during the simultaneous outbreak generated amplicons for sequence analysis. A single strain of RSV and a single strain of PIV-3 had spread from person to person within the unit; 7 patients were infected with RSV, 22 were infected with PIV-3, and 4 were infected with both viruses. The PIV-3 outbreak had started at the beginning of August 3 months before the RSV outbreak; it had arisen when PIV-3 was introduced from the community by a patient and passed to another patient, who became chronically infected with the identical strain and, in spite of being nursed in isolation, was most likely the source from which widespread infection occurred in November. Had these early cases been linked to a common PIV-3 strain at the time of diagnosis, enhanced infection control precautions might have prevented the eventual extensive spread of PIV-3, making it much easier to deal with the later RSV outbreak.

A MIQE-Compliant Real-Time PCR Assay for Aspergillus Detection

The polymerase chain reaction (PCR) is widely used as a diagnostic tool in clinical laboratories and is particularly effective for detecting and identifying infectious agents for which routine culture and microscopy methods are inadequate. Invasive fungal disease (IFD) is a major cause of morbidity and mortality in immunosuppressed patients, and optimal diagnostic criteria are contentious. Although PCR-based methods have long been used for the diagnosis of invasive aspergillosis (IA), variable performance in clinical practice has limited their value. This shortcoming is a consequence of differing sample selection, collection and preparation protocols coupled with a lack of standardisation of the PCR itself. Furthermore, it has become clear that the performance of PCR-based assays in general is compromised by the inadequacy of experimental controls, insufficient optimisation of assay performance as well as lack of transparency in reporting experimental details. The recently published “Minimum Information for the publication of real-time Quantitative PCR Experiments” (MIQE) guidelines provide a blueprint for good PCR assay design and unambiguous reporting of experimental detail and results. We report the first real-time quantitative PCR (qPCR) assay targeting Aspergillus species that has been designed, optimised and validated in strict compliance with the MIQE guidelines. The hydrolysis probe-based assay, designed to target the 18S rRNA DNA sequence of Aspergillus species, has an efficiency of 100% (range 95–107%), a dynamic range of at least six orders of magnitude and limits of quantification and detection of 6 and 0.6 Aspergillus fumigatus genomes, respectively. It does not amplify Candida, Scedosporium, Fusarium or Rhizopus species and its clinical sensitivity is demonstrated in histological material from proven IA cases, as well as concordant PCR and galactomannan data in matched broncho-alveolar lavage and blood samples. The robustness, specificity and sensitivity of this assay make it an ideal molecular diagnostic tool for clinical use.

Comparative evaluation of the seegene seeplex RV15 and real‐time PCR for respiratory virus detection

Respiratory virus infections contribute substantially both to hospitalizations of young children, and to morbidity in immunocompromised patients such as those with hematological malignancies. Their rapid and accurate diagnosis is essential to patient management. To evaluate the prospective utility of Seeplex® DPO technology in respiratory virus diagnosis, a panel of 99 respiratory samples positive by real‐time RT‐PCR for one or more viruses was assayed by the Seegene Seeplex® RV12 system. As well as being able to detect all 10 viruses in the real‐time RT‐PCR system with the exception of enteroviruses, RV12 can also distinguish between the two subgroups of RSV and detect two subgroups of coronaviruses. Seven of the nine viruses in common with the RT‐PCR were detected reliably by RV12. Eleven samples RT‐PCR‐positive for Metapneumovirus and five samples positive for influenza B were not detected by RV12. Seegene developed a second‐generation system, RV15, which not only allowed detection of three additional viruses, but also addressed the potential problems with RV12 specificity. To address these concerns, 84 respiratory samples positive for a range of viruses by real‐time PCR were assayed with RV15. The results of this evaluation improved significantly upon those seen with RV12. The high throughput capabilities and potential lower technical requirements afforded by the Seeplex® system may offer an alternative to real‐time RT‐PCR systems. J. Med. Virol. 83:1469–1475, 2011.

Comparison of Next-Generation Sequencing Technologies for Comprehensive Assessment of Full-Length Hepatitis C Viral Genomes.

ABSTRACT Affordable next-generation sequencing (NGS) technologies for hepatitis C virus (HCV) may potentially identify both viral genotype and resistance genetic motifs in the era of directly acting antiviral (DAA) therapies. This study compared the ability of high-throughput NGS methods to generate full-length, deep, HCV sequence data sets and evaluated their utility for diagnostics and clinical assessment. NGS methods using (i) unselected HCV RNA (metagenomics), (ii) preenrichment of HCV RNA by probe capture, and (iii) HCV preamplification by PCR implemented in four United Kingdom centers were compared. Metrics of sequence coverage and depth, quasispecies diversity, and detection of DAA resistance-associated variants (RAVs), mixed HCV genotypes, and other coinfections were compared using a panel of samples with different viral loads, genotypes, and mixed HCV genotypes/subtypes [geno(sub)types]. Each NGS method generated near-complete genome sequences from more than 90% of samples. Enrichment methods and PCR preamplification generated greater sequence depth and were more effective for samples with low viral loads. All NGS methodologies accurately identified mixed HCV genotype infections. Consensus sequences generated by different NGS methods were generally concordant, and majority RAVs were consistently detected. However, methods differed in their ability to detect minor populations of RAVs. Metagenomic methods identified human pegivirus coinfections. NGS provided a rapid, inexpensive method for generating whole HCV genomes to define infecting genotypes, RAVs, comprehensive viral strain analysis, and quasispecies diversity. Enrichment methods are particularly suited for high-throughput analysis while providing the genotype and information on potential DAA resistance.

The role of TLR2 and 4 in Behcet's disease pathogenesis

TLRs are PRRs that play a pivotal role in sensing exogenous pathogens and endogenous danger signals. Their role in the pathogenesis of inflammatory and immune-related diseases is gradually being unravelled. TLR2 and TLR4 are capable of sensing the oral microbial community, which is considered a potential trigger for Behçet’s disease (BD). This study aimed to investigate the expression and function of TLR2 and TLR4 in the oral mucosa of BD. A total of 87 patients was included: 55 BD, 24 healthy controls and eight recurrent aphthous stomatitis. Total RNA was purified from non-lesional oral mucosal brush biopsies and analysed for the presence of TLR2 and TLR4 mRNA, along with their splice variants. The response of peripheral blood mononuclear cells to classical TLR2 and TLR4 agonists was also investigated. TLR2b, TLR2d, TLR2e, TLR4.3 and TLR4.4 were significantly elevated in relapsed BD. A significant defect in the response to cognate agonists of TLR1/2 heterodimer and TLR4 was also observed in BD. The expression of unusual splice variants of TLR2 and TLR4 might explain the observed defect in these receptors’ function in BD.

First Reported Outbreak of Diarrhea Due to Adenovirus Infection in a Hematology Unit for Adults

ABSTRACT Numerous outbreaks of adenovirus infection from different types of health care settings, except a hematology unit, have been reported. This is the first report describing an outbreak of adenovirus infection causing diarrhea among adult hematopoietic stem cell transplant recipients. Six of 21 patients from the outbreak cohort were affected with diarrhea. Electron microscopy, cell culture, and direct DNA sequencing of amplicons generated from stool and blood samples were used to investigate this outbreak. Electron microscopy and cell culture detected adenovirus in stools from symptomatic patients. DNA sequencing of amplicons generated from stool samples confirmed nosocomial transmission of infection from a single index case. The outbreak strain was also detected in plasma of four of these patients, suggesting systemic infection. The outbreak strain was identified as type 12. Standard infection control measures were effective to control this outbreak.

Sustained Immunological Responses to Highly Active Antiretroviral Therapy at 36 Months in a Ghanaian HIV Cohort

Two hundred thirty-seven Ghanaian human immunodeficiency virus-infected patients who were starting antiretroviral therapy underwent clinical and immunological monitoring for 3 years. Seventy-eight percent of patients had disease classified as World Health Organization stage III or IV. The mean increase in the CD4 cell count was 395 cells/mm(3), 13% of patients experienced immunological failure, and 8% of patients switched treatment to a second-line regimen. However, two-thirds of patients who experienced immunological failure did not switch treatment, and 31% of all patients were lost to follow-up.

Disease Progression in HIV-1–Infected Viremic Controllers

Background:The mechanism of CD4+ T-cell decline in HIV-1 infection is unclear, but the association with plasma viral RNA load suggests viral replication is involved. Indeed, viremic controller patients with low viral RNA loads typically maintain high CD4+ T-cell counts. Within a local cohort of 86 viremic controllers, we identify a subgroup (18 “discord controllers”) with low CD4+ T-cell counts that present clinical uncertainty. The underlying mechanism accounting for CD4+ T-cell decline in the face of low or undetectable plasma (RNA) viral load remains unresolved. The objective of this study was to investigate the viral and host immune system dynamics in discord controllers by measuring cellular HIV-1 DNA load, T-cell populations, and T-cell activation markers. Methods:We compared discord controllers (viral RNA load <2000 copies/mL, <450 CD4+ T-cells/mm3) with typical controllers (viral RNA load <2000 copies/mL, >450 CD4+ T-cells/mm3) and progressors (viral RNA load >10,000 copies/mL, <450 CD4+ T-cells/mm3). We quantified CD4+/CD8+ naive/central memory/effector memory subsets (CD45RA/RO ± CD62L), activation levels (CD38+HLA-DR+), and HIV-1 DNA load. Results:Discord controllers resembled progressors showing high viral DNA load, depletion of naive CD4+ T-cells, and higher activation in all CD4+ T-cell subsets, compared with typical controllers. They were similar to typical controllers with lower CD8+ T-cell activation compared with progressors. Conclusions:Our data are consistent with a relationship between CD4+ T-cell activation and disease progression. HIV-1 DNA load may be a better marker of viral replication and disease progression than viral RNA load. Lower level CD8+ T-cell activation correlates with low viral RNA load but not with disease progression or viral DNA load.

Inadvertent non-nucleoside reverse transcriptase inhibitor (NNRTI)-based antiretroviral therapy in dual HIV-1/2 and HIV-2 seropositive West Africans: a retrospective study.

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Nosocomial transmission of parainfluenza 3 virus in hematological patients characterized by molecular epidemiology

A.V. Lee, D.F. Bibby, H. Oakervee, A. Rohatiner, I. Ushiro‐Lumb, D.A. Clark, F.M. Mattes. Nosocomial transmission of parainfluenza 3 virus in hematological patients characterized by molecular epidemiology.
Transpl Infect Dis 2011: 13: 433–437. All rights reserved