Detection and genotyping of enteroviruses in cerebrospinal fluid in patients in Victoria, Australia, 2007-2013.

Genotyping by VP1 fragment polymerase chain reaction (PCR) and nucleic acid sequencing to detect enterovirus (EV) genotypes was performed directly on 729 EV PCR positive cerebrospinal fluid (CSF) samples collected between 2007 and 2012 from Victorian hospital inpatients. The overall genotype identification rate from CSF-positive material was 43%. The four most common genotypes identified were Echovirus 6 (24%), Echovirus 30 (17%), Echovirus 25 (10%), and Coxsackievirus A9 (10%), together comprising 61% of all EVs typed. The seasonal distribution of all EVs identified followed the recognized pattern of mainly summer epidemics. Three of the four predominant genotypes were present in each of the 6 years in which the study was conducted, with 20 other EV genotypes also detected, often in only a single year. Genotyping of EVs directly in CSF is faster, simpler and more sensitive than traditional virus neutralization assays performed on EV positive samples.

Evaluation of swabs, transport media, and specimen transport conditions for optimal detection of viruses by PCR.

Depletion of swabs and viral transport medium during epidemics may prompt the use of unvalidated alternatives. Swabs collected and transported dry or in saline were compared to commercially available swab/medium combinations for PCR detection of influenza, enterovirus, herpes simplex virus, and adenovirus. Each was detected at an ambient temperature (22°C) and 4°C for 7 days. Detection of influenza on dry or saline swabs is important because of its capacity to cause outbreaks involving large numbers of cases.

Respiratory virus laboratory pandemic planning and surveillance in central Viet Nam, 2008-2010.

INTRODUCTION: Laboratory capacity is needed in central Viet Nam to provide early warning to public health authorities of respiratory outbreaks of importance to human health, for example the outbreak of influenza A(H1N1) pandemic in 2009. Polymerase chain reaction (PCR) procedures established as part of a capacity-building process were used to conduct prospective respiratory surveillance in a region where few previous studies have been undertaken. METHODS: Between October 2008 and September 2010, nose and throat swabs from adults and children (approximately 20 per week) presenting with an acute respiratory illness to the Ninh Hoa General Hospital were collected. Same-day PCR testing and result reporting for 13 respiratory viruses were carried out by locally trained scientists. RESULTS: Of 2144 surveillance samples tested, 1235 (57.6%) were positive for at least one virus. The most common were influenza A strains (17.9%), with pandemic influenza A(H1N1) 2009 and seasonal H3N2 strain accounting for 52% and 43% of these, respectively. Other virus detections included: rhinovirus (12.4%), enterovirus (8.9%), influenza B (8.3%), adenovirus (5.3%), parainfluenza (4.7%), respiratory syncytial virus (RSV) (3.9%), human coronavirus (3.0%) and human metapneumovirus (0.3%). The detection rate was greatest in the 0-5 year age group. Viral co-infections were identified in 148 (6.9%) cases. DISCUSSION: The outbreak in 2009 of the influenza A(H1N1) pandemic strain provided a practical test of the laboratory's pandemic plan. This study shows that the availability of appropriate equipment and molecular-based testing can contribute to important individual and public health outcomes in geographical locations susceptible to emerging infections.

HIV transmissions during seroconversion contribute significantly to new infections in men who have sex with men in Australia.

Transmission of HIV from recently infected individuals contributes to the number of new cases of infection, but the extent to which it occurs from those who are unaware of their infection is not known. Phylogenetic analysis was performed on 209 cases of acute HIV subtype B infection detected between January 2005 and September 2010, most of whom (88%) were men who have sex with men. Only new cases with an evolving Western blot profile confirmed by detection of HIV RNA were included. Subjects whose known dates of seroconversion were within 1 month of at least one other phylogenetically linked case identified during the 6 years of the study were then examined to estimate the prevalence of onward transmission. Almost 30% of cases could have acquired their infection from another person undergoing seroconversion within the same month. Temporal increases in the number of phylogenetically related strains within several clusters were demonstrated during the study, although the rate of increase varied. Transmission of HIV from individuals undergoing seroconversion is an important contributor to the number of new infections identified every year and very likely occurs before they have knowledge of their infection. Clusters of related HIV strains can grow at a disconcerting rate, demonstrating that more focused public health efforts are required to minimize further HIV transmissions within sexual networks.

Increasing diversity of Human Immunodeficiency Virus type 1 subtypes circulating in Australia.

Characterization of HIV subtypes can provide a more comprehensive understanding of the epidemic within a distinct region, and when combined with notification data, may also be helpful in enhancing current HIV prevention strategies. In this study, we characterized 1056 HIV-positive individuals (948 males and 108 females) living in Victoria and whose infection was detected for the first time between 2005 and 2010 inclusive. HIV-1 strains were subtyped based on pol gene sequence. Phylogenetic analysis was performed on all non-B subtype sequences identified. Of the 1056 sequences analyzed, 825 were subtype B and 231 were non-B. Overall 6 HIV-1 subtypes, 6 circulating recombinant forms (CRFs), and 12 unique recombinant forms (URFs) were identified. Regardless of gender, the majority of individuals were infected with a subtype B virus (78%). Subtype B was dominant in males (n=806, 85%). In contrast, the majority of females were infected with non-B subtypes (n=89, 82%), in particular subtype C (n=48, 45%). Phylogenetic analysis of the non-B subtypes revealed that the majority of clustering, and thereby transmission, occurred with CRF01_AE strains. Despite the relatively high numbers identified in females there was very little clustering of subtype C viruses. Subtypes C and A1 both historically associated with heterosexual transmission, and CRF01_AE often associated with IVDU, were also associated with transmission within the MSM population, demonstrating the potential for non-B subtypes to expand into the MSM population. The observation of increasing numbers of females and heterosexual males infected with non-subtype B viruses, the majority imported through migration and travel to countries where there is a high prevalence of HIV, suggests a targeted public health message may be required to prevent further increases within these two groups.

Extremely prolonged HIV seroconversion associated with an MHC haplotype carrying disease susceptibility genes for antibody deficiency disorders.

Severe immunodeficiency during primary human immunodeficiency virus (HIV) infection is unusual. Here, we characterized viral and immunological parameters in a subject presenting with Pneumocystis jirovecii pneumonia in the setting of prolonged primary HIV illness and delayed seroconversion. HIV antibody was only detected by enzyme-linked immunosorbent assay 12 months after presentation, and Western blot profiles remain indeterminate. Isolated virus was of R5 phenotype, exhibited poor viral fitness, but was otherwise unremarkable. Analysis of HIV antibody isotypes showed failure to mount a detectable HIV IgG response over nearly 2 years of infection, in particular IgG(1)- and IgG(3)-specific responses, despite normal responses to common infections and vaccines. Genetic analysis demonstrated homozygosity for part of an MHC haplotype containing susceptibility genes for common variable immunodeficiency (CVID) syndrome and other antibody deficiency disorders. Thus, a primary disorder of specific antibody production may explain exceptionally slow antibody development in an otherwise severe seroconversion illness. This highlights the need for multiparameter testing, in particular use of a fourth generation HIV test, for confirming HIV infection and underscores the importance of host factors in HIV pathogenesis.

A case of apparent resistance to all available antiretroviral drugs in a HIV-positive patient.

We describe a case of multidrug and class resistant HIV in a patient with psychological issues affecting his adherence. The complexities involved in subsequent treatment decisions when resistance interpretation algorithms are discordant are discussed.

Hospitalised adult patients with pandemic (H1N1) 2009 influenza in Melbourne, Australia.

OBJECTIVE: To describe the case characteristics and outcomes of patients hospitalised with pandemic (H1N1) 2009 influenza infection during the first 2 months of the epidemic. DESIGN, PARTICIPANTS AND SETTING: Prospective case series of 112 patients admitted to seven hospitals in Melbourne with laboratory-confirmed pandemic (H1N1) 2009 influenza between 1 May and 17 July 2009. MAIN OUTCOME MEASURES: Details of case characteristics, risk factors for severe disease, treatment and clinical course. RESULTS: Of 112 hospitalised patients, most presented with cough (88%) and/or fever (82%), but several (4%) had neither symptom. A quarter of female patients (15) were pregnant or in the post-partum period. Patients presenting with multifocal changes on chest x-ray had significantly longer hospital lengths of stay, and were more likely to require intensive care unit admission. Thirty patients required admission to an intensive care unit, and three died during their acute illness. The median length of intensive care admission was 10.5 days (interquartile range, 5-16 days). CONCLUSIONS: This study highlights risk factors for severe disease, particularly pregnancy. Clinical and public health planning for upcoming influenza seasons should take into account the spectrum and severity of clinical infection demonstrated in this report, and the need to concentrate resources effectively in high-risk patient groups.

Prevalence of transmitted HIV drug resistance since the availability of highly active antiretroviral therapy.

We investigated the prevalence of HIV-1-associated transmitted drug resistance (TDR) in Victoria from the time of first availability of highly active antiretroviral therapy. Drug resistance genotyping was performed on virus present in blood samples collected from individuals with serologically confirmed primary infection, between 1996 and 2007. The significance of any mutations detected was interpreted according to a standardised list of drug resistance mutations. The main outcomes measured were the prevalence by year of TDR to any antiretroviral drug class, the numbers of infected individuals with TDR involving multiple drug classes, and the resistance mutations implicated in all cases. There was an average annual prevalence of TDR of 16%, predominantly associated with nucleoside and non-nucleoside reverse transcriptase (RT) inhibitors and most commonly occurring at codons 41, 103 and 215 in the RT. The prevalence of thymidine-associated mutations remained high throughout the period of study. While mutations known to cause resistance to protease inhibitors were uncommon, they were present in several individuals infected with virus resistant to multiple drug classes. The prevalence of TDR in Victoria is similar to geographical locations outside Australia where HIV-specific drug treatment is widely available. Primary infection with drug resistant HIV is a future treatment issue for the individual patient and for the wider population at risk of infection. At this time TDR shows no sign of waning and our data support recent treatment guidelines recommending baseline testing for TDR before therapy is initiated.

Evaluation of twenty rapid antigen tests for the detection of human influenza A H5N1, H3N2, H1N1, and B viruses.

Twenty rapid antigen assays were compared for their ability to detect influenza using dilutions of virus culture supernatants from human isolates of influenza A H5N1 (clade 1 and 2 strains), H3N2 and H1N1 viruses, and influenza B. There was variation amongst the rapid antigen assays in their ability to detect different influenza viruses. Six of the 12 assays labeled as distinguishing between influenza A and B had comparable analytical sensitivities for detecting both influenza A H5N1 strains, although their ability to detect influenza A H3N2 and H1N1 strains varied. The two assays claiming H5 specificity did not detect either influenza A H5N1 strains, and the two avian influenza-specific assays detected influenza A H5N1, but missed some influenza A H3N2 virus supernatants. Clinical trials of rapid antigen tests for influenza A H5N1 are limited. For use in a pandemic where novel influenza strains are circulating (such as the current novel influenza A H1N1 09 virus), rapid antigen tests should ideally have comparable sensitivity and specificity for the new strains as for co-circulating seasonal influenza strains.

Comparison of Bayesian and maximum-likelihood phylogenetic approaches in two legal cases involving accusations of transmission of HIV.

We investigated two cases of alleged criminal transmission of HIV-1 using Bayesian and maximum-likelihood phylogenetic approaches to determine whether the inference method used influenced the outcome in these cases. In the first case, Bayesian methods were used to reexamine gag and env sequences from an earlier investigation in which the HIV-1 strains infecting one of several contacts could not be linked phylogenetically to that of the accused despite strongly suggestive epidemiological evidence. In the second case, maximum-likelihood and Bayesian inference methods were used to investigate the relatedness of gag and env sequences from HIV-1 strains infecting a man accused of intentionally transmitting the virus to several contacts. Bayesian analysis of HIV-1 strains from the first case confirmed earlier results obtained by maximum-likelihood analysis. A monophyletic cluster linking viruses from the accused and three of his direct and indirect contacts was supported, but a linkage between these viruses and a fourth epidemiologically linked contact could not be demonstrated. In the second case, a strong virological link between the accused and two of his contacts, and the absence of links with four other contacts, was confirmed by both maximum-likelihood and Bayesian inference methods. It is important that phylogenetic programs applied in a legal setting are conservative in their outcome. Although Bayesian methods offer computational tractability for large data sets and complex evolutionary models, this study demonstrates they do not assist when clear linkages between viruses are demonstrated using maximum-likelihood methods.

Emergence and spread of oseltamivir-resistant A(H1N1) influenza viruses in Oceania, South East Asia and South Africa.

The neuraminidase inhibitors (NAIs) are an effective class of antiviral drugs for the treatment of influenza A and B infections. Until recently, only a low prevalence of NAI resistance (<1%) had been detected in circulating viruses. However, surveillance in Europe in late 2007 revealed significant numbers of A(H1N1) influenza strains with a H274Y neuraminidase mutation that were highly resistant to the NAI oseltamivir. We examined 264 A(H1N1) viruses collected in 2008 from South Africa, Oceania and SE Asia for their susceptibility to NAIs oseltamivir, zanamivir and peramivir in a fluorescence-based neuraminidase inhibition assay. Viruses with reduced oseltamivir susceptibility were further analysed by pyrosequencing assay. The frequency of the oseltamivir-resistant H274Y mutant increased significantly after May 2008, resulting in an overall proportion of 64% (168/264) resistance among A(H1N1) strains, although this subtype represented only 11.6% of all isolates received during 2008. H274Y mutant viruses demonstrated on average a 1466-fold reduction in oseltamivir susceptibility and 527-fold reduction in peramivir sensitivity compared to wild-type A(H1N1) viruses. The mutation had no impact on zanamivir susceptibility. Ongoing surveillance is essential to monitor how these strains may spread or persist in the future and to evaluate the effectiveness of treatments against them.

Relocation requires specialist skills.

Chris Birch, business development manager, Medical Services, at Romac Technical Services, examines the key steps that need to be followed to ensure successful removal, installation, or relocation of the wide range of equipment, machinery and associated items used in hospitals and other healthcare facilities.

Phylogenetic investigation of transmission pathways of drug-resistant HIV-1 utilizing pol sequences derived from resistance genotyping.

OBJECTIVES: To investigate the nature of transmission links existing between patients recently infected with HIV strains containing transmitted drug resistance (TDR) mutations. METHODS: Virus from 63 individuals recently infected with HIV-1 containing TDR mutations was analyzed phylogenetically to determine virological links. Phylogenetic trees were reconstructed using maximum likelihood and distance-based methods. Monophyletic clusters detected on the basis of pol sequences were confirmed using env and gag sequences. Potential bias caused by the presence of drug resistance mutations was assessed by reanalyzing the pol sequence set after the omission of 16 drug resistance codons identified in the TDR population. RESULTS: Phylogenetic analysis revealed 9 apparent transmission clusters involving 24 of the 63 (38%) TDR patients. Each cluster was supported by high bootstrap values and low intracluster genetic distances. The 9 transmission clusters were confirmed in separate analyses using env and gag sequences and in pol sequences after the removal of codons associated with drug resistance. CONCLUSIONS: Pol sequences generated during baseline resistance genotyping for newly HIV-infected patients provide the opportunity for real-time phylogenetics to identify sources of multiple HIV transmission events. This study demonstrated the existence of several distinct clusters of patients whose TDR strains were linked. Several discrete clusters involving transmission of K103N- and/or M41L-resistant virus to multiple recipients were detected, suggesting that multiple transmission pathways can exist for viruses with the same resistance mutations.

A new arenavirus in a cluster of fatal transplant-associated diseases.

BACKGROUND: Three patients who received visceral-organ transplants from a single donor on the same day died of a febrile illness 4 to 6 weeks after transplantation. Culture, polymerase-chain-reaction (PCR) and serologic assays, and oligonucleotide microarray analysis for a wide range of infectious agents were not informative. METHODS: We evaluated RNA obtained from the liver and kidney transplant recipients. Unbiased high-throughput sequencing was used to identify microbial sequences not found by means of other methods. The specificity of sequences for a new candidate pathogen was confirmed by means of culture and by means of PCR, immunohistochemical, and serologic analyses. RESULTS: High-throughput sequencing yielded 103,632 sequences, of which 14 represented an Old World arenavirus. Additional sequence analysis showed that this new arenavirus was related to lymphocytic choriomeningitis viruses. Specific PCR assays based on a unique sequence confirmed the presence of the virus in the kidneys, liver, blood, and cerebrospinal fluid of the recipients. Immunohistochemical analysis revealed arenavirus antigen in the liver and kidney transplants in the recipients. IgM and IgG antiviral antibodies were detected in the serum of the donor. Seroconversion was evident in serum specimens obtained from one recipient at two time points. CONCLUSIONS: Unbiased high-throughput sequencing is a powerful tool for the discovery of pathogens. The use of this method during an outbreak of disease facilitated the identification of a new arenavirus transmitted through solid-organ transplantation.

Chikungunya virus infection in travellers to Australia.

We report eight recent cases of Chikungunya virus infection in travellers to Australia. Patients presented with fevers, rigors, headaches, arthralgia, and rash. The current Indian Ocean epidemic and Italian outbreak have featured prominently on Internet infectious disease bulletins, and Chikungunya virus infection had been anticipated in travellers from the outbreak areas. Diagnosis was by a generic alphavirus reverse transcriptase polymerase chain reaction with confirmatory sequencing. Prompt diagnosis of Chikungunya virus infections is of public health significance as the mosquito vectors for transmission exist in Australia. There is potential for this infection to spread in the largely naïve Australian population.

Community epidemiology of human metapneumovirus, human coronavirus NL63, and other respiratory viruses in healthy preschool-aged children using parent-collected specimens.

OBJECTIVES: The purpose of this work was to assess the impact of recently described human metapneumovirus and human coronavirus NL63 compared with other respiratory viruses by using sensitive molecular techniques in a cohort of healthy preschool-aged children. We also aimed to assess the use of parent collection to obtain an adequate respiratory specimen from acutely unwell children in the community. PATIENTS AND METHODS: The community epidemiology and burden of human metapneumovirus and other respiratory viruses (influenza A, influenza B, respiratory syncytial virus, parainfluenza viruses, adenoviruses, and picornaviruses) were examined in a cohort of 234 preschool-aged children from Melbourne, Australia, over a 12-month period by using polymerase chain reaction testing. Parents collected a daily symptom diary for the duration of the study and were taught to collect a combined nose-throat swab and complete an impact diary when the study child had an acute respiratory illness. RESULTS: The average incidence of acute respiratory illness was 0.48 per child-month for the duration of the study, with a winter peak. Of 543 illnesses with > or = 1 specimen returned, 33 were positive for human metapneumovirus (6.1%) and 18 for human coronavirus NL63 (3.3%). Of all of the viruses for which we tested, human metapneumovirus and human coronavirus NL63 were most strongly linked to child care attendance, occurring in 82% and 78% of infected children, respectively. Picornaviruses were the most commonly identified virus group (269 [49.5%]). Influenza virus and adenovirus illnesses had the greatest impact, with fever in more than three quarters and requiring, on average, > 1 local doctor visit per illness. CONCLUSIONS: Recently identified human metapneumovirus and human coronavirus NL63 are important pathogens in community-based illness in children, particularly in those who attend child care. Picornaviruses were detected in half of the nose-throat swabs collected during acute respiratory illness in children but resulted in milder illnesses; influenza and adenovirus caused the highest-impact illnesses. The use of parent-collected specimens should be considered for additional community-based epidemiologic studies and vaccine trials.

Virological fitness of HIV in patients with resistance to enfuvirtide.

Resistance to the HIV fusion inhibitor enfuvirtide is associated with mutations in the first heptad repeat region of gp41, but little is known of their impact on replicative fitness in vivo. We followed seven patients undergoing salvage therapy that included enfuvirtide in order to document the temporal generation of genotypic and phenotypic resistance in parallel with replicative fitness. Resistance to enfuvirtide was not associated with decreased replicative fitness of HIV strains infecting these patients.

The nonstructural protein 8 (nsp8) of the SARS coronavirus interacts with its ORF6 accessory protein.

Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) caused a severe outbreak in several regions of the world in 2003. The SARS-CoV genome is predicted to contain 14 functional open reading frames (ORFs). The first ORF (1a and 1b) encodes a large polyprotein that is cleaved into nonstructural proteins (nsp). The other ORFs encode for four structural proteins (spike, membrane, nucleocapsid and envelope) as well as eight SARS-CoV-specific accessory proteins (3a, 3b, 6, 7a, 7b, 8a, 8b and 9b). In this report we have cloned the predicted nsp8 gene and the ORF6 gene of the SARS-CoV and studied their abilities to interact with each other. We expressed the two proteins as fusion proteins in the yeast two-hybrid system to demonstrate protein-protein interactions and tested the same using a yeast genetic cross. Further the strength of the interaction was measured by challenging growth of the positive interaction clones on increasing gradients of 2-amino trizole. The interaction was then verified by expressing both proteins separately in-vitro in a coupled-transcription translation system and by coimmunoprecipitation in mammalian cells. Finally, colocalization experiments were performed in SARS-CoV infected Vero E6 mammalian cells to confirm the nsp8-ORF6 interaction. To the best of our knowledge, this is the first report of the interaction between a SARS-CoV accessory protein and nsp8 and our findings suggest that ORF6 protein may play a role in virus replication.