Melissa Ayers

Clinical Impact of Community‐Acquired Respiratory Viruses on Bronchiolitis Obliterans After Lung Transplant

Community‐acquired viral respiratory tract infections (RTI) in lung transplant recipients may have a high rate of progression to pneumonia and can be a trigger for immunologically mediated detrimental effects on lung function. A cohort of 100 patients was enrolled from 2001 to 2003 in which 50 patients had clinically diagnosed viral RTI and 50 were asymptomatic. All patients had nasopharyngeal and throat swabs taken for respiratory virus antigen detection, culture and RT‐PCR. All patients had pulmonary function tests at regular intervals for 12 months. Rates of rejection, decline in forced expiratory volume (L) in 1 s (FEV‐1) and bacterial and fungal superinfection were compared at the 3‐month primary endpoint. In the 50 patients with RTI, a microbial etiology was identified in 33 of 50 (66%) and included rhinovirus (9), coronavirus (8), RSV (6), influenza A (5), parainfluenza (4) and human metapneumovirus (1). During the 3‐month primary endpoint, 8 of 50 (16%) RTI patients had acute rejection versus 0 of 50 non‐RTI patients (p = 0.006). The number of patients experiencing a 20% or more decline in FEV‐1 by 3 months was 9 of 50 (18%) RTI versus 0 of 50 non‐RTI (0%) (p = 0.003). In six of these nine patients, the decline in FEV‐1 was sustained over a 1‐year period consistent with bronchiolitis obliterans syndrome (BOS). Community‐acquired respiratory viruses may be associated with the development of acute rejection and BOS.

Hepatitis B virus DNA prediction rules for hepatitis B e antigen–negative chronic hepatitis B

After hepatitis B e antigen (HBeAg) seroconversion, hepatitis B may become inactive or progress to HBeAg‐negative hepatitis with persistent or intermittent alanine aminotransferase (ALT) elevation. The aim of this study was to prospectively identify factors predictive of the clinical course in HBeAg‐negative chronic hepatitis B (CHB). Patients were stratified by ALT and HBeAg status and followed every 3 months for up to 5 years. Kaplan‐Meier and Cox regression analysis using the change from normal ALT to elevated ALT as endpoints were performed to determine factors associated with ALT elevation/normalization. Seventy‐four HBeAg‐negative and 32 HBeAg‐positive patients were prospectively evaluated. For HBeAg‐negative patients, hepatitis B virus (HBV) DNA was predictive of future ALT. Only 1 patient with normal ALT and an HBV DNA value lower than 10,000 copies/mL developed an elevated ALT within the subsequent year, whereas 67% with an HBV DNA value greater than 100,000 copies/mL had a rise in ALT above normal within 1 year. Patients with a previous history of ALT elevation and longer follow‐up at all levels of HBV DNA were more likely to experience ALT elevations. For HBeAg‐negative patients with elevated ALT and all HBeAg‐positive patients, HBV DNA did not predict future ALT. Other viral and host factors were not predictive of future ALT. Conclusion: HBeAg‐negative CHB has a fluctuating course. HBV DNA values lower than 10,000 copies/mL predict persistently normal ALT for at least 1 year. Patients with HBV DNA values between 10,000 and 100,000 copies/mL can safely be followed at 6 monthly intervals, whereas HBV DNA values greater than 100,000 copies/mL are highly predictive of future ALT elevation and should prompt regular follow‐up. (HEPATOLOGY 2007.)

Detection and identification of Bartonella species pathogenic for humans by PCR amplification targeting the riboflavin synthase gene (ribC).

ABSTRACT Several Bartonella species have now been implicated as human pathogens. The recovery of these fastidious organisms in the clinical microbiology laboratory remains difficult, and current methods are still relatively insensitive. Thus, the bartonellae are good candidates for detection by PCR. We have developed a PCR assay which uses a single primer pair targeting the riboflavin synthase gene (ribC) and detected six Bartonella species that have been implicated in human disease, B. henselae, B. quintana, B. bacilliformis, B. clarridgeiae, B. elizabethae, and B. vinsonii subsp. berkhoffii. Species identification is achieved simply by restriction enzyme digestion of the amplicon. This PCR assay appears to be specific for the Bartonella genus because it failed to amplify DNA from several other bacterial species.

A single tube RT-PCR assay for the detection of mosquito-borne flaviviruses

Abstract Mosquito-borne flaviviruses include several important agents of human disease and have provided striking examples of emerging infections. In this study we present the design and validation of a single tube RT-PCR assay using a pair of consensus primers for the detection of mosquito-borne flaviviruses. Sequencing of the amplicons permits the species identification. The assay was validated using RNA from the yellow fever virus vaccine strain and from representative strains of dengue viruses 1, 2, 3 and 4, West Nile virus, Kunjin virus (a clade of West Nile virus), and St. Louis encephalitis virus.

Comprehensive detection and identification of human coronaviruses, including the SARS-associated coronavirus, with a single RT-PCR assay

Abstract The SARS-associated human coronavirus (SARS-HCoV) is a newly described, emerging virus conclusively established as the etiologic agent of the severe acute respiratory syndrome (SARS). This study presents a single-tube RT-PCR assay that can detect with high analytical sensitivity the SARS-HCoV, as well as several other coronaviruses including other known human respiratory coronaviruses (HCoV-OC43 and HCoV-229E). Species identification is provided by sequencing the amplicon, although a rapid screening test by restriction enzyme analysis has proved to be very useful for the analysis of samples obtained during the SARS outbreak in Toronto, Canada.

Monitoring of Hepatitis C Virus Quasispecies in Chronic Infection by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Mutation Detection

ABSTRACT Using both a mass spectrometry-based method and the classical method of cloning and sequencing, we demonstrated weekly changes in the hypervariable region 1 quasispecies of a chimpanzee infected with an infectious clone, coinciding with neutralizing antibody emergence. We also used the mass spectrometry method in the clinical follow-up of a chronically infected patient over a 5-year period.

Amplification by long RT-PCR of near full-length norovirus genomes

A long RT-PCR method was developed to amplify the norovirus genome. Starting from RNA extracted directly from clinical samples and using broadly reactive primers, it can generate near full-length amplicons that allow for easy determination of the near complete genomic sequence. Two norovirus isolates from Toronto, Canada, in 2002 and 2005 were sequenced. This approach will facilitate molecular epidemiology studies of noroviruses.

Characterization of Hepatitis C Virus Quasispecies by Matrix-Assisted Laser Desorption Ionization-Time of Flight (Mass Spectrometry) Mutation Detection

ABSTRACT Hepatitis C virus (HCV), the causative agent of hepatitis C, frequently causes chronic infection. The mechanisms of viral persistence continue to be the object of investigation. An important aspect of HCV chronic infection is the quasispecies nature of the viral population, which has been particularly well documented in the hypervariable region 1 of the E2 glycoprotein. Recent studies show that characterization of the quasispecies diversity at the amino acid level can help to predict the outcome of HCV infection. Currently the accurate characterization of HCV quasispecies requires the cloning of PCR products, followed by the sequencing of many clones. In this study we present a new method to characterize HCV quasispecies, based on in vitro translation of the amplicons, followed by mass spectrometry analysis of the resulting peptide mix. The assay was used on reference HCV samples and on clinical samples. In principle, this method could be applied to other chronic viral infections in which quasispecies play a role.

Detection and species-level identification of primate herpesviruses with a comprehensive PCR test for human herpesviruses.

ABSTRACT A comprehensive assay for the identification of all eight human herpesviruses has been previously reported. This assay was extended to the detection and species-level identification of herpes B virus (Cercopithecine herpesvirus 1) and African green monkey cytomegalovirus (Cercopithecine herpesvirus 5), two herpesviruses of relevance to the clinical virology laboratory.