Circulating human papillomavirus DNA detection in Barrett's dysplasia and esophageal adenocarcinoma.

There is evidence to suggest that human papillomaviruses (HPV) are associated with Barrett's dysplasia and esophageal adenocarcinoma. In other HPV-linked cancers such as cervical and oropharyngeal cancer, circulating HPV DNA is a potential biomarker to assist in tumor diagnosis and management. This study aimed to determine whether circulating HPV DNA was detectable in patients with Barrett's dysplasia and esophageal adenocarcinoma, and if so, whether there is any correlation with esophageal tissue HPV status. Plasma from 138 patients representing esophageal adenocarcinoma (N = 41), Barrett's dysplasia (N = 48) and hospital controls (N = 49) were analyzed for the presence of circulating HPV DNA using droplet-digital PCR targeting the E7 gene of HPV types 16 and 18. Circulating HPV DNA was detected in 11/138 (8.0%) study subjects including 1/49 (2.0%) hospital controls, 4/48 (8.3%) Barrett's dysplasia patients, and 6/41 (14.6%) esophageal adenocarcinoma patients. Detection of circulating HPV DNA was higher in patients with HPV-positive esophageal tissue (6/35, 17.1%) compared to those with HPV-negative specimens (5/103; 4.9%) (OR = 4.06; 95% CI 1.15-14.25; P = 0.020). The highest rates of detection occurred in esophageal adenocarcinoma patients, particularly those with invasive tumors that had breached the esophageal submucosa, had regional lymph node involvement or metastatic disease. Circulating HPV DNA was detectable in a subset of Barrett's dysplasia and esophageal adenocarcinoma patients. Detection was associated with tissue HPV positivity and possibly disease severity.

Virological diagnosis of Ebolavirus infection.

Ebolaviruses, and the other viral causes of haemorrhagic fevers (VHF) have always posed special problems for diagnostic laboratories. These arise from the rarity of human infections, minimal documented experience with test delivery and interpretation, the paucity of established commercial or in-house assays, the lack of clinical material for test development and validation, the high level containment required for handling live virus, the ongoing evolution of the viruses, and the high personal and public health requirements for accurate diagnosis. This article addresses the current situation and the ongoing challenges associated with delivering timely, high quality and safe testing within Australia for people exposed as part of the current major outbreak of Ebolavirus disease (EVD) in Western Africa. The members of the Public Health Laboratory Network have developed deliverable and reliable nucleic acid detection tests, and also have the laboratory capacity to handle the live viruses if necessary. However delivering and maintaining these services necessitates high levels of experience in developing and applying tests for exotic and emerging infections, strong national and international links and collaborations, ongoing monitoring and reassessment of test design and performance, innovative approaches to generation of positive control material, and a regular quality assurance program.

Ex vivo model of congenital cytomegalovirus infection and new combination therapies.

INTRODUCTION: Congenital human cytomegalovirus (HCMV) infection is a major public health problem due to severe sequelae in the fetus and newborns. Currently, due to their toxicity anti-CMV treatments cannot be administered to pregnant women. We thus developed an ex vivo model of 1(st) trimester placental CMV infection to observe the route of infection across the placenta and to test the efficacy of various new drugs targeting different stages of viral cycle. METHODS: After validation of the viability of floating villi explants by ELISA ß-HCG, the kinetics of placental infection were determined by immunochemistry and qPCR in this ex vivo model. Antiviral susceptibility was determined in vitro using focus reduction assay and by qPCR in the ex vivo model. RESULTS: The ex vivo model showed viral infection in trophoblasts and mesenchymal space of floating villi. In vitro, antiviral combinations of maribavir with baïcalein or artesunate inhibited viral infection by more than 90%. On the other hand, in ex vivo model, infection was reduced by 40% in presence of maribavir and artesunate. The synergistic effect observed in vitro was not observed ex vivo. DISCUSSION: This model allowed us to understand the CMV spread in 1(st) trimester floating villi better and to analyze the anti-CMV efficacy and toxicity of new drugs that could be administered to pregnant women, either alone or in combination. CONCLUSIONS: Such an ex vivo model could be applied to other viruses such as rubella or parvovirus B19 and in new drug development.

Discordant hepatitis C serological testing in Australia and the implications for organ transplant programs.

BACKGROUND: Discordant and equivocal hepatitis C (HCV) serology testing is problematic for making decisions regarding deceased organ donor (DOD) transplant allocation based on allograft infection status. OBJECTIVES: This study aimed to analyse the prevalence and follow-up testing of discordant HCV tested patients from an Australian population at increased risk of HCV infection, with prevalence modelling for the Australian DOD population. STUDY DESIGN: De-identified patient discordant HCV serology results (primary chemiluminescent microparticle immunoassay and secondary Bio-Rad MonoLisa HCV Ag/Ab Ultra assay) were retrospectively identified in a general referral laboratory between May 2008 and August 2011. Prior and follow-up serology testing was reviewed. Discordant result prevalencewas calculated using Bayes' theorem for the DOD population using Australian DOD rates and HCV seroprevalence. RESULTS: The tested population had a 6.6% HCV seroprevalence. The rate of discordant serotesting was 0.54%, with no cases identified as having definite HCV infection at follow-up. Two patients had evidence of definite HCV seropositivity before the index discordant test. Modelling for the Australian DOD population of 337 per year estimated a discordant test prevalence of 1.8 per year. CONCLUSIONS: Discordant HCV serotesting may occur for 1 of 185 patients tested in higher risk populations. The majority of such tests represent falsely reactive tests although a small number may reflect partial seroreversion. Amongst Australian DOD, this represents 1 or 2 discordant cases per year. It is likely that if this discordant sample were from a donor with no blood borne virus risk factors, and was concurrently RNA negative, that HCV infectious risk would be extremely low.

Clinical aspects of cytomegalovirus antiviral resistance in solid organ transplant recipients.

Despite advances in the prophylaxis and acute treatment of cytomegalovirus (CMV), it remains an important pathogen affecting the short- and long-term clinical outcome of solid organ transplant. The emergence of CMV resistance in a patient reduces the clinical efficacy of antiviral therapy, complicates therapeutic and clinical management decisions, and in some cases results in loss of the allograft and/or death of the patient. There is increasing use of antiviral prophylaxis after transplant with little expansion in the range of antiviral agents effective in treatment of CMV. Further understanding is needed of the risk factors for development of CMV antiviral resistance and of therapeutic strategies for treating patients infected with resistant viruses. We review the current status of CMV resistance in solid organ transplant recipients, and provide diagnostic and therapeutic suggestions for the clinician in managing antiviral resistance.

Organ donor screening using parallel nucleic acid testing allows assessment of transmission risk and assay results in real time.

Expansion of the donor pool may lead to utilization of donors with risk factors for viral infections. Donor laboratory screening relies on serological and nucleic acid testing (NAT). The increased sensitivity of NAT in low prevalence populations may result in false-positive results (FPR) and may cause unnecessary discard of organs.We developed a screening algorithm to deal, in real time, with potential FPR. Three NAT assays: COBAS AmpliScreen assay (CAS), AmpliPrep Total Nucleic Acid Isolation/CAS, and AmpliPrep/TaqMan assays, were validated and used in parallel for prospective screening of increased-risk donors (IRD), and the probability of FPR was calculated. The lower limit of detection of this algorithm was 9.79, 21.02, and 4.31 IU/mL for human immunodeficiency virus-1, hepatitis C virus, and hepatitis B virus, respectively, with an average turn-around-time of 7.67 h from sample receipt to result reporting. The probability that a donor is potentially infectious with two NAT concordant results was >90%. NAT screening of 35 IRD within 18 months resulted in transplantation of 102 additional organs that without screening would either not be used or used with restrictions in Australia. Using a parallel testing algorithm, real-time confirmation of seropositive donors allows use of organs from IRD and safer expansion of the donor pool.

Viral factors influencing the outcome of human cytomegalovirus infection in liver transplant recipients.

BACKGROUND: Cytomegalovirus (CMV) remains the leading viral cause of disease following orthotopic liver transplantation (OLT) despite the availability of antiviral agents for prophylaxis and therapy. OBJECTIVE: Examine the viral factors that influence the outcome of CMV infection following valganciclovir prophylaxis or laboratory-guided preemptive therapy in OLT recipients. STUDY DESIGN: The value of valganciclovir prophylaxis and laboratory-guided preemptive therapy for the prevention of CMV infection and disease was observed in 64 OLT recipients. Prophylaxis was given to all CMV seronegative recipients receiving a liver from a seropositive donor (D+R-; n=15), and all other recipients were randomised to receive either prophylaxis (n=24) or laboratory-guided preemptive therapy (n=25). Recipients were monitored for CMV DNAemia, viral load, emergence of antiviral resistant strains and co-infections. RESULTS: CMV end-organ disease and antiviral resistant strains only occurred in D+R- recipients despite the use of prophylaxis in these patients. The D+R- recipients commencing prophylaxis immediately following transplantation had better outcomes compared to those for whom prophylaxis was delayed due to renal impairment. Prophylaxis reduced the incidence of CMV DNAemia, persistent infection, and high viral loads for CMV seropositive (D-R+and D+R+) recipients, but laboratory-guided preemptive therapy effectively controlled CMV infection and prevented disease in these OLT recipients. CONCLUSION: Delaying the commencement of valganciclovir prophylaxis may be associated with worse outcomes for high-risk OLT recipients. Laboratory-guided pre-emptive therapy remains an alternative approach for seropositive recipients at lower risk of CMV disease.

Transmission of hepatitis C virus to recipients of parenteral vitamin therapy in a primary care facility.

BACKGROUND AND OBJECTIVES: The Australian prevalence of hepatitis C virus (HCV) is approximately 1%, with the majority of cases acquired through injecting drug use. However, occasionally HCV infection occurs in healthcare settings. Three new HCV infections were identified amongst patients attending a general practice in Sydney, Australia, specialising in parenteral vitamin therapy. STUDY DESIGN: An investigation was conducted to identify the source of infection and mechanism of transmission. Molecular analysis was conducted by sequencing the HCV NS5A, Core and NS5B regions. RESULTS: Two sources were identified using molecular epidemiology - a genotype 3a case was the source for a case acquired in late 2004 and a genotype 1b case the source for one case acquired in late 2006 and another in early 2007. The common risk factor was parenteral vitamin C therapy. CONCLUSIONS: Inadequate infection control was apparent and likely to have resulted in blood contamination of the healthcare workers, their equipment, the clinic environment and parenteral medications. Molecular and clinical epidemiology clearly identified parenteral transmission of HCV, highlighting the risks of blood contamination of parenteral equipment and use of multi-dose flasks on more than one patient.

Diversity of antiviral-resistant human cytomegalovirus in heart and lung transplant recipients.

Immunocompromised transplant recipients are at high risk for human cytomegalovirus (CMV)-related infection and disease. Antiviral prophylaxis and treatment have reduced CMV morbidity and mortality, but at times promote development of antiviral-resistant CMV strains that can significantly contribute to adverse clinical outcomes in transplant recipients. We have investigated CMV genotypes in transplant recipients (bone marrow, stem cell, kidney, heart, lung, and liver) receiving antiviral prophylaxis or preemptive therapy or treatment, to determine the viral characteristics and clinical impact of antiviral-resistant CMV in these different groups. Antiviral-resistant CMV strains were detected by polymerase chain reaction sequencing of the CMV protein kinase (UL97) and viral DNA polymerase (UL54) genes from clinical specimens. A trend toward more frequent detection of multidrug resistance and co-circulation of multiple resistant strains was seen in heart and lung transplant recipients compared with other transplantation types. A greater diversity and number of UL97 and UL54 mutations were observed in heart and lung transplant recipients; whereas antiviral-resistant CMV infections in other transplant recipients were predominantly the result of a single mutant genotype. Furthermore, 43% (6/14) of CMV-positive heart and lung transplant recipients were infected with CMV strains containing UL54 mutations conferring multidrug resistance compared with only 6% (1/18) of CMV-positive recipients of other transplanted organs or stem cells. Emergence of CMV strains containing previously unrecognized UL54 mutations (F412S and D485N) also occurred in 1 lung and 1 heart transplant recipient. The development of these mutations under antiviral selective pressure, and clinical outcome of infection suggests these mutations are likely to confer antiviral resistance. Emergence of CMV antiviral resistance remains a significant issue in immunocompromised patients treated with antiviral agents, and emphasizes the relevance of regular antiviral resistance testing when designing optimal patient-management strategies.

Mycoplasma genitalium is associated with cervicitis and HIV infection in an urban Australian STI clinic population.

OBJECTIVES: To investigate the prevalence of the genital mollicutes, Mycoplasma genitalium (MG), Mycoplasma hominis (MH), Ureaplasma urealyticum (UU) and Ureaplasma parvum (UP), and their associations with cervicitis in a sexually transmitted infection (STI) clinic population. Clinical correlates of MG infection were also assessed. METHODS: 527 women were enrolled in a cross-sectional study at two STI clinics in Sydney between June 2006 and January 2010. Genital mollicutes were detected by multiplex PCR testing of cervical swabs, and associations with cervicitis were analysed. Cervicitis was defined as >30 polymorphonuclear cells per high-power field in at least three non-adjacent fields of cervical mucus on Gram stain. RESULTS: MG was found in 4.0% of women, MH in 17.1%, UU in 14.1%, and UP in 51.8%. MG was the only mollicute associated with cervicitis (unadjusted prevalence ratio (PR) 1.85, 95% CI 1.52 to 2.26, p<0.0001), and this association remained after adjustment for Chlamydia trachomatis (CT) infection (adjusted PR 1.24 (95% CI 1.04 to 1.48), p=0.02). MG was significantly associated with women being HIV positive (p=0.03), but not with age, vaginal discharge, commercial sex work, being of culturally and linguistically diverse background, or concurrent CT infection. Two of the 21 women with MG had ectopic pregnancies. CONCLUSIONS: The authors recommend wider application of PCR testing for MG in STI services, particularly in high-risk women and those with cervicitis or HIV infection.

A modern miasma hypothesis and back-to-school asthma exacerbations.

A sudden increase in the rate of asthma exacerbations has been observed among young children in many countries 2-3 weeks after their return-to-school following the summer holidays. These exacerbations are frequently associated with human rhinovirus (hRV) infections, with possible interactions with allergen sensitisation, allergen exposure and medication use. It was originally proposed that the sudden increase resulted from new strains of respiratory viruses acquired during the holidays spreading rapidly on return to school. While there is compelling evidence implicating hRV in these exacerbations, recent observations on virus transmission, infection patterns and immune responses to both viruses and allergens have led us to propose an additional hypothesis for this increase in exacerbations. We propose that classrooms typically provide persistent exposure to a mixture of airborne viruses, viral proteins, endotoxin, community allergens and other human-derived aerosols - a modern miasma. During the preceding school term, this exposure establishes and maintains a level of immune tolerance and herd immunity, which then declines during the two-month holidays due to lack of such exposure, creating a transitory window of susceptibility to viral infections and asthma. The return to school re-establishes exposure to these aerosols resulting in an acceleration of exacerbations, until the tolerance and herd immunity are re-established. Thus, the peak in return-to-school asthma is more a function of a transitory increase in susceptibility due to a temporary lack of this complex exposure, than it is to novel, locally endemic strains of hRV.

Early IL-10 predominant responses are associated with progression to chronic hepatitis C virus infection in injecting drug users.

The critical events in clearance or persistence of hepatitis C virus (HCV) infection are unknown but likely to be determined early in acute infection. Type 1 and type 2 cytokine production was assessed by HCV peptide ELISpot and multiplex in vitro cytokine production assays in longitudinally collected samples from 20 untreated participants enrolled in the Australian Trial in Acute Hepatitis C (ATAHC); a prospective cohort of acute HCV infection (77% injecting drug users, IDU). Significantly higher interleukin-10 (IL-10) production (P = 0.048), in the relative absence of interferon-gamma (IFN-γ) and IL-2 production, was present early in HCV infection in those who progressed to chronic infection. In contrast, viral clearance was associated with a greater magnitude and broader specificity of IFN-γ (magnitude P < 0.001, breadth P = 0.004) and IL-2 responses, in the relative absence of IL-10. Early IL-10 production was correlated with higher HCV RNA level at baseline (P = 0.046) and week 12 (P = 0.018), while IFN-γ and IL-2 production was inversely correlated with HCV RNA level at baseline (IFN-γ P = 0.020, IL-2 P = 0.050) and week 48 (IFN-γ P = 0.045, IL-2 P = 0.026). Intracellular staining (ICS) indicated the HCV-specific IFN-γ response was primarily from CD8(+) T cells and NK cells, whereas IL-10 production was predominantly from monocytes, with a subset of IL-10 producing CD8(+) T cells present only in those who progressed to chronic infection. IL-10, an immunoregulatory cytokine, appears to play a key role in progression to chronic HCV infection.

Viruses and other infections in stillbirth: what is the evidence and what should we be doing?

In Australia, as in other developed countries, approximately 40-50% of stillbirths are of unknown aetiology. Emerging evidence suggests stillbirths are often multifactorial. The absence of a known cause leads to uncertainty regarding the risk of recurrence, which can cause extreme anguish for parents that may manifest as guilt, anger or bewilderment. Further, clinical endeavours to prevent recurrences in future pregnancies are impaired by lack of a defined aetiology. Therefore, efforts to provide an aetiological diagnosis of stillbirth impact upon all aspects of care of the mother, and inform many parts of clinical decision making. Despite the magnitude of the problem, that is 7 stillbirths per 1000 births in Australia, diagnostic efforts to discover viral aetiologies are often minimal. Viruses and other difficult to culture organisms have been postulated as the aetiology of a number of obstetric and paediatric conditions of unknown cause, including stillbirth. Reasons forwarded for testing stillbirth cases for infectious agents are non-medical factors, including addressing all parents' need for diagnostic closure, identifying infectious agents as a sporadic cause of stillbirth to reassure parents and clinicians regarding risk for future pregnancies, and to reduce unnecessary testing. It is clear that viral agents including rubella, human cytomegalovirus (CMV), parvovirus B19, herpes simplex virus (HSV), lymphocytic choriomeningitis virus (LCMV), and varicella zoster virus (VZV) may cause intrauterine deaths. Evidence for many other agents is that minimal or asymptomatic infections also occur, so improved markers of adverse outcomes are needed. The role of other viruses and difficult-to-culture organisms in stillbirth is uncertain, and needs more research. However, testing stillborn babies for some viral agents remains a useful adjunct to histopathological and other examinations at autopsy. Modern molecular techniques such as multiplex PCR, allow searches for multiple agents. Now that such testing is available, it is important to assess the clinical usefulness of such testing.

Presence of mouse mammary tumour-like virus gene sequences may be associated with morphology of specific human breast cancer.

BACKGROUND: Mouse mammary tumour virus (MMTV) has a proven role in breast carcinogenesis in wild mice and genetically susceptible in-bred mice. MMTV-like env gene sequences, which indicate the presence of a replication-competent MMTV-like virus, have been identified in some human breast cancers, but rarely in normal breast tissues. However, no evidence for a causal role of an MMTV-like virus in human breast cancer has emerged, although there are precedents for associations between specific histological characteristics of human cancers and the presence of oncogenic viruses. AIM: To investigate the possibility of an association between breast cancer and MMTV-like viruses. METHODS: Histological characteristics of invasive ductal human breast cancer specimens were compared with archival MMTV-associated mammary tumours from C3H experimental mice. The presence of MMTV-like env DNA sequences in the human breast cancer specimens was determined by polymerase chain reaction and confirmed by Southern hybridisation. RESULTS: MMTV-like env gene sequences were identified in 22 of 59 (37.3%) human breast cancer specimens. Seventeen of 43 (39.5%) invasive ductal carcinoma breast cancer specimens and 4 of 16 (25%) ductal carcinoma in situ specimens had some histological characteristics, which were similar to MMTV-associated mouse mammary tumours. However, these similarities were not associated with the presence or absence of MMTV-like gene sequences in the human breast tumour specimens. A significant (p = 0.05) correlation was found between the grade of the human breast cancer and similarity to the mouse mammary tumours. The lower the grade, the greater the similarity. CONCLUSION: Some human breast cancer specimens, in which MMTV-like env DNA sequences have been identified, were shown to have histological characteristics (morphology) similar to MMTV-associated mouse mammary tumours. These observations are compatible with, but not conclusive of, an association between the presence of MMTV-like env DNA sequences and some human breast cancers.

Correlates of placental infection with cytomegalovirus, parvovirus B19 or human herpes virus 7.

Vertical transmission of viruses is an important cause of morbidity in the fetus and neonate. Placental viral infection indicates risk of vertical transmission, but not always transmission to, or disease of the fetus. Specimens from mothers and babies from three groups-two prospective and one retrospective cohort-were tested for pathogens of teratogenic potential using multiplex PCR. Placental infection was present in 13% of the 105 samples collected. Assessment of the prospective cohorts showed cytomegalovirus (CMV) detected in 4% of placentae from unselected women, parvovirus B19 in 1% and Ureaplasma parvum in 1% of placentae. In a retrospective cohort of women at high risk of transmitting congenital infection due to seroconversion during pregnancy, miscarriage or stillbirth, CMV was detected in 64% and human herpes virus type 7 in 9% of placentae. Of 14 PCR-positive placentae, two were associated with the birth of a living symptomatic infant, two with stillbirth, one with miscarriage, and two with elective terminations of pregnancy. Directed laboratory assessment of women at high risk of transmitting congenital infection, on the basis of clinical or laboratory markers, is important for accurate diagnosis of adverse outcomes of pregnancy. However, routine screening for viruses in the placentae from women with a low-risk serological profile for transmitting congenital infection is unlikely to result in significant numbers of additional diagnoses and is confounded by inadequacy of current diagnostic methods. The major pathogen detected in all cases of placental infection associated with fetal death was human CMV.

Development of multiplex PCRs for detection of common viral pathogens and agents of congenital infections.

Potential causes of congenital infection include Toxoplasma gondii and viruses such as cytomegalovirus (CMV), enterovirus, hepatitis C virus, herpes simplex virus types 1 and 2 (HSV-1 and -2), human herpesvirus types 6, 7, and 8, lymphocytic choriomeningitis virus, parvovirus, rubella virus, and varicella-zoster virus. Testing for each of these agents using nucleic acid tests is time consuming and the availability of clinical samples such as amniotic fluid or neonatal blood is often limited. The aim of this study was to develop multiplex PCRs (mPCRs) for detection of DNA and RNA agents in the investigation of congenital infection and an mPCR for the viruses most commonly requested in a diagnostic virology laboratory (CMV, Epstein-Barr virus, enterovirus, HSV-1, HSV-2, and varicella-zoster virus). The assays were assessed using known pathogen-positive tissues (cultures, placentae, plasma, and amniotic fluid) and limits of detection were determined for all the agents studied using serial dilutions of plasmid targets. Nested PCR was performed as the most sensitive assay currently available, and detection of the amplicons using hybridization to labeled probes and enzyme-linked immunosorbent assay detection was incorporated into three of the four assays. This allowed detection of 10 to 10(2) copies of each agent in the samples processed. In several patients, an unexpected infection was diagnosed, including a case of encephalitis where HSV was the initial clinical suspicion but CMV was detected. In the majority of these cases the alternative agent could be confirmed using reference culture, serology, or fluorescence methods and was of relevance to clinical care of the patient. The methods described here provide useful techniques for diagnosing congenital infections and a paradigm for assessment of new multiplex PCRs for use in the diagnostic laboratory.

Diagnosis of and screening for cytomegalovirus infection in pregnant women.

No single diagnostic test for cytomegalovirus (CMV) infection is currently available for pregnant women at all stages of gestation. Improved accuracy in estimating the timing of primary infections can be used to identify women at higher risk of giving birth to congenitally infected infants. A diagnostic algorithm utilizing immunoglobulin G (IgG), IgM, and IgG avidity was used to prospectively screen serum from 600 pregnant women enrolled from two groups: < or =20 weeks gestation (n = 396) or >20 weeks gestation (n = 204). PCR testing of urine and/or blood was performed on all seropositive women (n = 341). The majority (56.8%) of women were CMV IgG seropositive, with 5.5% being also CMV IgM positive. In the IgM-positive women, 1.2% had a low-avidity IgG, indicating a primary CMV infection and a high risk of intrauterine transmission. Two infants with asymptomatic CMV infection were born of mothers who had seroconverted in the second trimester of pregnancy. Baseline, age-stratified CMV serostatus was established from 1,018 blood donors. Baseline seropositivity from a blood donor population increased with age from 34.9% seroprevalence at less than 20 years of age to 72% seroprevalence at 50 years of age. Women at high risk of intrauterine transmission of CMV were identified at all stages of gestation. Women infected with CMV during late gestation may be more likely to transmit the virus, so failure to detect seroconversions in late gestation may result in failure to detect infected neonates.

Murine cytomegalovirus resistant to antivirals has genetic correlates with human cytomegalovirus.

Human cytomegalovirus (HCMV) resistance to antivirals is a significant clinical problem. Murine cytomegalovirus (MCMV) infection of mice is a well-described animal model for in vivo studies of CMV pathogenesis, although the mechanisms of MCMV antiviral susceptibility need elucidation. Mutants resistant to nucleoside analogues aciclovir, adefovir, cidofovir, ganciclovir, penciclovir and valaciclovir, and the pyrophosphate analogue foscarnet were generated by in vitro passage of MCMV (Smith) in increasing concentrations of antiviral. All MCMV antiviral resistant mutants contained DNA polymerase mutations identical or similar to HCMV DNA polymerase mutations known to confer antiviral resistance. Mapping of the mutations onto an MCMV DNA polymerase three-dimensional model generated using the Thermococcus gorgonarius Tgo polymerase crystal structure showed that the DNA polymerase mutations potentially confer resistance through changes in regions surrounding a catalytic aspartate triad. The ganciclovir-, penciclovir- and valaciclovir-resistant isolates also contained mutations within MCMV M97 identical or similar to recognized GCV-resistant mutations of HCMV UL97 protein kinase, and demonstrated cross-resistance to antivirals of the same class. This strongly suggests that MCMV M97 has a similar role to HCMV UL97 in the phosphorylation of nucleoside analogue antivirals. All MCMV mutants demonstrated replication-impaired phenotypes, with the lowest titre and plaque size observed for isolates containing mutations in both DNA polymerase and M97. These findings indicate DNA polymerase and protein kinase regions of potential importance for antiviral susceptibility and replication. The similarities between MCMV and HCMV mutations that arise under antiviral selective pressure increase the utility of MCMV as a model for in vivo studies of CMV antiviral resistance.

BK viral infection in an Australian pediatric renal transplant population.

BK virus (BKV) is recognized as a significant cause of renal allograft dysfunction in adults, and there is growing awareness of its importance in the pediatric population. Eighteen pediatric renal transplant recipients and 18 age-matched controls were prospectively studied. Anti-BKV immunoglobulin G (IgG) and IgM titres were assayed in all subjects at entry to the study. Polymerase chain reaction (PCR) for BKV DNA was performed on urine and serum at entry, and prospectively tested again at 4, 8 and 12 months. Mean age +/- s.d. of transplant recipients and controls was 14.6 +/- 3.3 and 13.9 +/- 0.33 yr respectively [not significant (NS)]. Transplant patients were studied at a mean time of 5.6 +/- 4.2 yr post-transplant. 56% of transplant patients and 39% of controls were seropositive (+ve BKV IgG) (NS). Plasma BKV PCR was positive in one transplant patient (who also had positive urine PCR) and in none of the controls. The prevalence of positive urine PCR in transplant patients was greater than in controls (33% vs. 0%, p = 0.02). Positive urine BKV PCR was more commonly found in patients treated with mycophenolate than azathioprine (p = 0.04). We conclude that the prevalence of BKV seropositivity and viral activation in this Australian pediatric renal transplant population is similar to that reported in adult and pediatric populations in other countries. BK viruria was more common in children with greater immunosuppression, suggesting that this group is at higher risk of BKV induced nephropathy.

Cytomegalovirus antiviral resistance associated with treatment induced UL97 (protein kinase) and UL54 (DNA polymerase) mutations.

HCMV-related illness due to infections with antiviral resistant virus was verified by phenotypic and genotypic assays in 17% (8/47) of high-risk immunocompromised Australian patients. Selective PCR-sequencing of UL97 (protein kinase; PK) and UL54 (DNA polymerase; DNApol) regions important for antiviral sensitivity, identified the majority (6/8) of resistant strains through detection of mutations known to confer antiviral resistance. Additionally, eight UL54 (DNApol) mutations (N408K, T691S, A692V, S695T, L737M, A834P, V955I, and A972V) of unknown phenotype were identified in six specimens from patients with clinical evidence of antiviral resistant infections. One isolate was resistant to ganciclovir (GCV) and another resistant to PFA on phenotypic testing where mutations in UL97 (PK) or UL54 (DNApol) were not detected, suggesting a loss of correlation between phenotype and genotype. Selective PCR-sequencing of UL97 (PK) and UL54 (DNApol) provided rapid and comprehensive results, but missed some resistance detected by phenotypic assays. A combination of phenotypic and genotypic assays is recommended for complete analysis of CMV antiviral resistance, as well as further definition of the clinical relationship between novel UL54 (DNApol) mutations and antiviral resistance.