BK viremia surveillance after kidney transplant: single-center experience during a change from cyclosporine-to lower-dose tacrolimus-based primary immunosuppression regimen.

BACKGROUND: The aim was to report our experience of BK viremia surveillance after kidney transplant during a period of change from cyclosporine (CyA)-to lower-dose tacrolimus (Tac)-based primary immunosuppression regimens. METHODS: In a prospective single-center observational cohort study, 68 consecutive patients received renal transplant during the period when we used a CyA-based primary immunosuppression regimen and 66 after we changed to a lower-dose Tac-based regimen. Testing for BK viremia by quantitative polymerase chain reaction assay was performed at least monthly for a minimum of 1 year. RESULTS: Thirty-nine (29.1%) patients developed BK viremia and 2 (1.5%) developed BK nephropathy. The actuarial time to BK viremia was shorter in patients receiving CyA/mycophenolate mofetil (MMF)/prednisolone (Pred) compared with Tac/MMF/Pred (P=0.04) and primary immunosuppression with CyA/MMF/Pred was the only independent predictor of BK viremia (hazard ratio 1.95; P=0.047). Comparing patients who experienced BK viremia and those who did not, there was no difference in incidence of acute rejection (20.5% vs. 25.3%; P=0.56) or estimated glomerular filtration rate at 12 months (48.8 vs. 49.9 mL/min/1.73 m(2)), but the incidence of ureteric stenosis was higher (10.3% vs. 1.1%; P=0.01). CONCLUSIONS: Our data demonstrate a lower incidence of BK viremia in patients on lower-dose Tac compared with CyA-based primary immunosuppression in contrast to previous studies, and provide further support for the association between BK virus and ureteric complications.

Influenza A(H1N1)2009 antibody seroprevalence in Scotland following the 2010/11 influenza season.

Following the 2010/11 influenza season, we determined the age- and location-specific seroprevalence of antibodies against the influenza A(H1N1)2009 virus in Scotland. Samples were analysed by microneutralisation assay. Age/seropositivity profiles varied significantly between cities. The increases in seroprevalence relative to the previous influenza season (2009/10) were similar across age groups and geographic locations. However, the increased seropositivity in older adults appeared to be driven by exposure to vaccination, indicating significantly lower levels of infection than in younger age groups.

Hepatitis B virus: origin and evolution.

The pathogenesis of hepatitis B virus (HBV) is complex and it appears that molecular variants play a role in this process. HBV undergoes numerous rounds of error prone production within an infected host. The resulting quasispecies are heterogeneous and in the absence of archaeological records of past infection, the evolution of HBV can only be inferred indirectly from its epidemiology and by genetic analysis. This review gathered the controversies about the HBV origin and factors influencing its quasispecies. Also, it provided some evidence on how HBV genotypes correlated with human history and patterns of migration. It is our belief that this topic deserves further attention and thus it is likely that more critical research work will be performed to elucidate the unknown mechanisms and processes in this area.

Prevalence of chronic viral hepatitis in people of south Asian ethnicity living in England: the prevalence cannot necessarily be predicted from the prevalence in the country of origin.

The prevalence of hepatitis B and hepatitis C in immigrant communities is unknown. Immigrants from south Asia are common in England and elsewhere, and the burden of viral hepatitis in these communities is unknown. We aimed to determine the prevalence of viral hepatitis in immigrants from south Asia living in England, and we therefore undertook a community-based testing project in such people at five sites in England. A total of 4998 people attending community centres were screened for viral hepatitis using oral fluid testing. The overall prevalence of anti-hepatitis C virus (HCV) in people of south Asian origin was 1.6% but varied by country of birth being 0.4%, 0.2%, 0.6% and 2.7% in people of this ethnic group born in the UK, India, Bangladesh and Pakistan, respectively. The prevalence of hepatitis B surface antigen was 1.2%-0.2%, 0.1%, 1.5% and 1.8% in people of this ethnic group born in the UK, India, Bangladesh and Pakistan, respectively. Analysis of risk factors for HCV infection shows that people from the Pakistani Punjab and those who have immigrated recently are at increased risk of infection. Our study suggests that migrants from Pakistan are at highest risk of viral hepatitis, with those from India at low risk. As prevalence varies both by country and region of origin and over time, the prevalence in migrant communities living in western countries cannot be easily predicted from studies in the country of origin.

2009 pandemic influenza A(H1N1) virus in Scotland: geographically variable immunity in Spring 2010, following the winter outbreak.

We determined the age- and location-specific seroprevalence of antibodies against 2009 pandemic influenza A(H1N1) virus in Scotland following the first two waves of infection. Serum samples collected following the winter outbreak were analysed by microneutralisation assay. The proportion of positive sera varied significantly between cities and, in the case of Inverness, between age groups (with younger adults more likely to be positive than older individuals). This study demonstrates that older people are no longer more likely to have antibodies against the virus than younger adults.

Simultaneous detection and quantitation of cytomegalovirus, Epstein-Barr virus, and adenovirus by use of real-time PCR and pooled standards.

Quantitative real-time PCR has become the most widely used preemptive approach for managing cytomegalovirus (CMV), Epstein-Barr virus (EBV), and adenovirus infections in immunosuppressed patients. These three assays are normally available as separate tests, each using five quantitation standards that are tested in duplicate. We have developed an adenovirus-CMV-EBV triplex assay that uses one set of five pooled quantitative standards, tested singly rather than in duplicate. This test demonstrated a sensitivity and an accuracy of quantitation equivalent to those of our previous single tests and was shown to be able to detect mixed infections with no loss in sensitivity. This assay is now in routine use in our laboratory and has considerably simplified the work flow of the laboratory, with a resultant improvement in sample turnaround time and significantly reduced costs.

Using multiplex real time PCR in order to streamline a routine diagnostic service.

An increasing number of virology laboratories are now utilising in house real time PCR assays as the frontline diagnostic tests. As the number of tests on offer increases the natural progression from this will be to rationalise their service via multiplexing. Since 2003 we have introduced a large number of qualitative and quantitative multiplex real time PCR assays into our routine testing service. This paper describes the development of the multiplex assays, the problems encountered and the resultant benefits to the routine service.

Practical experience of high throughput real time PCR in the routine diagnostic virology setting.

The advent of PCR has transformed the utility of the virus diagnostic laboratory. In comparison to traditional gel based PCR assays, real time PCR offers increased sensitivity and specificity in a rapid format. Over the past 4 years, we have introduced a number of qualitative and quantitative real time PCR assays into our routine testing service. During this period, we have gained substantial experience relating to the development and implementation of real-time assays. Furthermore, we have developed strategies that have allowed us to increase our sample throughput while maintaining or even reducing turn around times. The issues resulting from this experience (some of it bad) are discussed in detail with the aim of informing laboratories that are only just beginning to investigate the potential of this technology.

A multi-centre pilot proficiency programme to assess the quality of molecular detection of respiratory viruses.

OBJECTIVES: To assess the quality of molecular detection of respiratory viruses in clinical diagnostic laboratories. STUDY DESIGN: Respiratory virus proficiency panels were produced from diluted stocks of respiratory viruses provided and tested by four reference laboratories. The panels consisted of strong positive, positive, low positive and negative samples for influenza viruses A and B, respiratory syncytial virus, parainfluenza viruses 1 and 3, adenovirus serotypes 4 and 7, human rhinovirus serotypes 16, 72 and 90, human coronaviruses OC43 and 229E. The panels were sent to 17 participants; results and information on methodology was collected. RESULTS: All laboratories returned results, of which five submitted complete data sets. So, for analysis all results were combined. Samples were correctly identified by participants in 93.75%, 76.75% and 47.03% for the high positive, positive and low positive samples, respectively. One false positive was reported for all data sets (1.1%). The overall score for all assays using different methodologies was 78.8%. Laboratory performance was not dependant on methodology as all in-house methodologies could achieve optimal results, but dependant on careful optimisation and procedures specific to the laboratory. CONCLUSIONS: The first proficiency panel showed that in general all participants performed well. Although, it also highlights areas for improvement for all participants in order to generate robust results for use in clinical diagnostics.

Contamination with PCR detectable virus in a virus isolation quality assurance panel.

External quality control schemes are an essential part of the quality assurance of all diagnostic virology laboratories. There are a few providers of nucleic acid detection quality control panels. Consequently, diagnostic laboratories may test panels that are developed specifically for virus isolation by nucleic acid detection methods. Here, we report on a recent simulated eye swab panel from a National external quality assessment service, which was meant for virus isolation but which we tested by polymerase chain reactions assays. The results suggest that the samples had been contaminated at source leading to difficulty in interpretation of the panel and a poor score.

Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections in health care workers (HCWs): guidelines for prevention of transmission of HBV and HCV from HCW to patients.

The transmission of viral hepatitis from health care workers (HCW) to patients is of worldwide concern. Since the introduction of serologic testing in the 1970s there have been over 45 reports of hepatitis B virus (HBV) transmission from HCW to patients, which have resulted in more than 400 infected patients. In addition there are six published reports of transmissions of hepatitis C virus (HCV) from HCW to patients resulting in the infection of 14 patients. Additional HCV cases are known of in the US and UK, but unpublished. At present the guidelines for preventing HCW to patient transmission of viral hepatitis vary greatly between countries. It was our aim to reach a Europe-wide consensus on this issue. In order to do this, experts in blood-borne infection, from 16 countries, were questioned on their national protocols. The replies given by participating countries formed the basis of a discussion document. This paper was then discussed at a meeting with each of the participating countries in order to reach a Europe-wide consensus on the identification of infected HCWs, protection of susceptible HCWs, management and treatment options for the infected HCW. The results of that process are discussed and recommendations formed. The guidelines produced aim to reduce the risk of transmission from infected HCWs to patients. The document is designed to complement existing guidelines or form the basis for the development of new guidelines. This guidance is applicable to all HCWs who perform EPP, whether newly appointed or already in post.

Age related prevalence of hepatitis G virus in South Africans.

AIM: To investigate the age related prevalence of hepatitis G virus (HGV) infection and its mode of transmission in relation to hepatitis B (HBV) and C (HCV) co-infection in South African blacks. METHODS: Reverse transcriptase polymerase chain reaction was performed to detect active infection, using primers for the 5'-NCR, NS5a, and NS3 regions. Antibodies to HGV envelope-2 protein (anti-E2), which measures past infection, were also sought. RESULTS: The overall prevalence of active infection was 116/580 (20%). A higher prevalence was noted in HBsAg carriers (28/106; 26.4%) and HCV positive subjects (25/82; 30.5%). In contrast to developed countries, active and past infection was seen in 12.9% and 12.1% of the general population, respectively (subjects negative for HBsAg and anti-HCV markers and with normal alanine aminotransferase values), with a total prevalence of 21.1% (52/248). Viraemia and anti-E2 were almost mutually exclusive. The distribution of viraemia by age was: < or = 15 years, 20/223 (9.0%); 16-35 years, 42/147 (28.6%); > or = 36 years, 37/151 (24.5%), with a significant difference (p = 0.001) in age related prevalence. A similar trend was observed for the prevalence of past infection in the general population. CONCLUSIONS: HGV infection begins in childhood and increases with age in South Africa, but transmission is largely independent of HBV and HCV. No association was found between HGV viraemia and hepatitis, or with co-infection with either HBV or HCV.

Use of PCR in resolving diagnostic difficulties potentially caused by genetic variation of hepatitis B virus.

AIMS: To assess the relevance of genetic variants of hepatitis B virus (HBV) and to demonstrate the usefulness of the polymerase chain reaction (PCR) in cases of HBV diagnostic difficulty. METHODS: Five serum samples from patients that presented diagnostic difficulty in routine laboratories were sent to a research laboratory for PCR, and if appropriate, S gene sequencing, in vitro expression, and antigenic analysis. RESULTS: The demonstration of HBV in serum by PCR allowed a definitive diagnosis of current infection. One serum sample with poor reactivity in a diagnostic assay had a minor hepatitis B surface antigen (HBsAg) variant and another with very poor reactivity had multiple variants of HBsAg. Transient HBsAg reactivity was observed in a recently vaccinated patient. A hepatitis Be antigen (HBeAg) false positive reaction was noted in a patient from a well defined risk group for HBV. One patient who was strongly HBsAg/HBeAg positive, but anti-hepatitis B core antibody negative, was viraemic. CONCLUSIONS: PCR may become the gold standard for the diagnosis of current HBV infection. HBV variants are responsible for a proportion of diagnostically difficult cases. Modification of commercial assays is necessary to increase the sensitivity of detection of such variants.

An assessment of optimal conditions for amplification of HIV cDNA using Thermus aquaticus polymerase.

Optimal conditions for in-vitro enzymatic DNA amplification using Thermus aquaticus polymerase were defined using env and gag sequences of human immunodeficiency virus type I as templates. It was found that specific amplification of the target sequence occurred when the primer annealing temperature was above 55 degrees C. Polymerase added once at the beginning of the procedure was sufficient for good results. Deoxynucleotide and primer concentrations and chain elongation time were not found to be important as long as they were kept above a critical level. Differences were noted between the efficiency of specific amplification from purified plasmid and genomic DNA. A rapid and simple method without the use of radioactive reagents for confirmation of a suggestive positive result on gel electrophoresis using restriction enzyme digestion of the amplified products is described. Some possible drawbacks of the technique particularly if used as a diagnostic tool are discussed.

Reverse transcription and subsequent DNA amplification of rubella virus RNA.

A method is described whereby rubella virus RNA was reverse transcribed and the resulting cDNA enzymatically amplified using Taq polymerase. The reactions were carried out in a single reaction vessel, with only minor modifications to the buffer conditions between the reverse transcription and the subsequent amplification step. Using an oligonucleotide probe to the E1 glycoprotein region and limited restriction endonuclease mapping, the resulting amplified products were shown to be specific for rubella virus. This method was also successfully applied to crude cell lysates, without the need for RNA purification. The possible applications of the polymerase chain reaction as applied to RNA sequences are discussed.

Molecular variants of hepatitis B virus.

Variation in the genome of hepatitis B virus has been associated with a number of clinical parameters, but there is a paucity of functional data to show a causal relationship. For example, variants of the core and X genes have been linked with the pathogenesis of chronic hepatitis, viral replication efficiency, and serologically negative infections, among other observations. Variants of the surface antigen are linked with infection after vaccination, infection of liver grafts in patients undergoing immunotherapy, and samples that react poorly in diagnostic assays.

Influenza diagnosis: from dark isolation into the molecular light. West of Scotland Respiratory Virus Study Group.

OBJECTIVES: To compare the conventional virus isolation method for diagnosis of influenza infection with reverse-transcription polymerase chain reaction (RT-PCR) in prospectively collected nose and throat swabs from elderly patients during the winter influenza season. The use of a denaturing buffer as an alternative to viral transport medium (VTM) for submission of combined nose and throat swabs to the laboratory for PCR was then investigated in a second study. METHODS: Virus was cultured in microtitre plates using two different cell lines and detected using monoclonal antibody staining. A multiplex, matrix gene PCR assay was optimized to increase the sensitivity and specificity of detection of influenza A (H3 and H1) and B nucleic acid. RESULTS: The multiplex assay detected all viruses with equal sensitivity to individual assays. In a large, multicentre field study PCR detected twice as many influenza infections compared with virus isolation. No positive culture was missed. PCR has a rapid turn around time (< 36 h) vs. a minimum of 7 days for virus isolation. Greater sensitivity and specificity in the PCR were achieved using a 'hot-start' method. Although the numbers were small, the detection rate using PCR was greater for swabs submitted in denaturing buffer than in VTM. CONCLUSIONS: PCR significantly increased the sensitivity and clinical utility of influenza A (H3 and H1) and B diagnosis. There were a number of advantages in using denaturing buffer for submission of samples, including high sensitivity, rapidity, ease of use and no requirement for the virus to be viable on arrival at the laboratory. Therefore, PCR is a rapid, sensitive and user-friendly alternative for influenza diagnosis. Virus isolation technology should be limited to referral centres for further epidemiological characterization.