The emergence and evolution of the novel epidemic norovirus GII.4 variant Sydney 2012.

Norovirus is the leading cause of acute gastroenteritis with most infections caused by GII.4 variants. To understand the evolutionary processes that contribute to the emergence of GII.4 variants, we examined the molecular epidemiology of norovirus-associated acute gastroenteritis in Australia and New Zealand from 893 outbreaks between 2009 and 2012. Throughout the study GII.4 New Orleans 2009 was predominant; however, during 2012 it was replaced by an emergent GII.4 variant, Sydney 2012. An evolutionary analysis of capsid gene sequences was performed to determine the origins and selective pressures driving the emergence of these recently circulating GII.4 variants. This revealed that both New Orleans 2009 and Sydney 2012 share a common ancestor with GII.4 Apeldoorn 2007. Furthermore, pre-epidemic ancestral variants of each virus were identified up to two years before their pandemic emergence. Adaptive changes at known blockade epitopes in the viral capsid were also identified that likely contributed to their emergence.

HIV non-B subtype distribution: emerging trends and risk factors for imported and local infections newly diagnosed in South Australia.

Monitoring HIV subtype distribution is important for understanding transmission dynamics. Subtype B has historically been dominant in Australia, but in recent years new clades have appeared. Since 2000, clade data have been collected as part of HIV surveillance in South Australia. The aim of this study was to evaluate the prevalence of and risk factors for HIV-1 non-B subtypes. The study population was composed of newly diagnosed, genotyped HIV subjects in South Australia between 2000 and 2010. We analyzed time trends and subtype patterns in this cohort; notification data were aggregated into three time periods (2000-2003, 2004-2006, and 2007-2010). Main outcome measures were number of new non-B infections by year, exposure route, and other demographic characteristics. There were 513 new HIV diagnoses; 425 had information on subtype. The majority (262/425) were in men who have sex with men (MSM), predominantly subtype B and acquired in Australia. Infections acquired in Australia decreased from 77% (2000-2003) to 64% (2007-2010) (p=0.007) and correspondingly the proportion of subtype B declined from 85% to 68% (p=0.002). Non-B infections were predominantly (83%) heterosexual contacts, mostly acquired overseas (74%). The majority (68%) of non-B patients were born outside of Australia. There was a nonsignificant increase from 1.6% to 4.2% in the proportion of locally transmitted non-B cases (p=0.3). Three non-B subtypes and two circulating recombinant forms (CRFs) were identified: CRF_AE (n=41), C (n=36), CRF_AG (n=13), A (n=9), and D (n=2). There has been a substantial increase over the past decade in diagnosed non-B infections, primarily through cases acquired overseas.

Sequence-based identification of legionella.

Legionella strains are considered biologically inert with respect to traditional identification schemes. Various phenotypic alternatives have been attempted but all have lacked resolution as additional species have been added to what is proving to be a large genus. Only sequence-based schemes have the required resolution to confidently speciate or recognize potentially novel strains. The mip gene target is the most comprehensive currently available, with the added advantage of a Web-based analysis tool. Other gene targets are available for most if not all species, the best of which target 16S rRNA, rpoB, rnpB, or proA genes. One or several of these should be used to confirm important strains or clarify apparent novelness. The increased resolution of these sequence-based schemes has recognized many new species, and many more remain to be characterized. I provide a mip analysis of 44 such strains along with the recognized species, and a SplitsTree network analysis of recognized species and 20 novel strains for which sequence for the five targets is available.

Legionella steelei sp. nov., isolated from human respiratory specimens in California, USA, and South Australia.

Legionella-like bacteria were isolated from the respiratory tract of two patients in California, USA, and South Australia, but were not thought to cause disease. These bacteria, strains F2632 and IMVS-3376(T), were found to have identical Legionella macrophage infectivity potentiator (mip) gene sequences and were therefore further characterized to determine their genetic and phenotypic relatedness and properties. Both of these Gram-negative-staining bacterial strains grew on buffered charcoal yeast extract medium, were cysteine auxotrophs and made a characteristic diffusible bright yellow fluorescent pigment, with one strain making a late appearing colony-bound blue-white fluorescent pigment. The optimal in vitro growth temperature was 35 °C, with very poor growth at 37 °C in broth or on solid media. There was no growth in human A549 cells at either 35 or 37 °C, but excellent growth in Acanthamoeba castellani at 30 °C and poorer growth at 35 °C. Phylogenetic analysis of these bacteria was performed by sequence analysis of 16S rRNA, mip, ribonuclease P, ribosomal polymerase B and zinc metalloprotease genes. These studies confirmed that the new strains represented a single novel species of the genus Legionella for which the name Legionella steelei sp. nov. is proposed. The type strain is IMVS-3376(T) ( = IMVS 3113(T) = ATCC BAA-2169(T)).

Legionella nagasakiensis sp. nov., isolated from water samples and from a patient with pneumonia.

A novel Legionella species was identified based on analysis of 16S rRNA and mip (macrophage infectivity potentiator) gene sequences, cellular fatty acids, isoprenoid quinones, biochemical reactions, antigens and quantitative DNA-DNA hybridization. Strain CDC-1796-JAP-E(T) was isolated from well water at the Nagasaki Municipal Medical Center, Japan. Two strains, CDC-3041-AUS-E and CDC-3558-AUS-E, were isolated from water samples during an outbreak of legionellosis in South Australia. Strain CDC-5427-OH-H was isolated from a 66-year-old female patient diagnosed with Legionnaires' disease in the US. Cells from these four strains were gram-negative, non-fluorescent, rod-shaped, and positive for alkaline phosphatase, esterase, leucine arylamidase, catalase, gelatinase, ß-lactamase and tyrosine browning assay. Phylogenetic analysis of 16S rRNA and mip genes revealed that the four strains formed a distinct cluster within the genus Legionella. The bacteria contained branched-chain fatty acids and quinones that are typical of members of the genus Legionella. Slide agglutination tests demonstrated no cross-reaction with 52 previously described members of the Legionellaceae. DNA-DNA hybridization studies indicated that DNAs from the four strains were highly related (78-84 %) but they showed 29 % relatedness to Legionella oakridgensis ATCC 33761(T) and less than 10 % to strains of other Legionella species tested. These characterizations suggest that the isolates represent a novel species, for which the name Legionella nagasakiensis sp. nov. is proposed; the type strain is CDC-1796-JAP-E(T) ( = ATCC BAA-1557(T) = JCM 15315(T)).

Redescriptions of two species of microcotylid monogeneans from three arripid hosts in southern Australian waters.

Microcotyle arripis Sandars, 1945 is redescribed from Arripis georgianus from four localities: Spencer Gulf, Gulf St. Vincent, off Kangaroo Island and Coffin Bay, South Australia, Australia. Kahawaia truttae (Dillon & Hargis, 1965) Lebedev, 1969 is reported from A. trutta off Bermagui, New South Wales and is redescribed from a new host, A. truttaceus, from four localities in South Australian waters: Spencer Gulf, Gulf St. Vincent, off Kangaroo Island and Coffin Bay. Phylogenetic analysis of the partial 28S ribosomal RNA gene (28S rRNA) nucleotide sequences for both microcotylid species and comparison with other available sequence data for microcotylid species across four genera contributes to our understanding of relationships in this monogenean family.

Norovirus illness is a global problem: emergence and spread of norovirus GII.4 variants, 2001-2007.

BACKGROUND: Noroviruses (NoVs) are the most common cause of viral gastroenteritis. Their high incidence and importance in health care facilities result in a great impact on public health. Studies from around the world describing increasing prevalence have been difficult to compare because of differing nomenclatures for variants of the dominant genotype, GII.4. We studied the global patterns of GII.4 epidemiology in relation to its genetic diversity. METHODS: Data from NoV outbreaks with dates of onset from January 2001 through March 2007 were collected from 15 institutions on 5 continents. Partial genome sequences (n=775) were collected, allowing phylogenetic comparison of data from different countries. RESULTS: The 15 institutions reported 3098 GII.4 outbreaks, 62% of all reported NoV outbreaks. Eight GII.4 variants were identified. Four had a global distribution--the 1996, 2002, 2004, and 2006b variants. The 2003Asia and 2006a variants caused epidemics, but they were geographically limited. Finally, the 2001 Japan and 2001 Henry variants were found across the world but at low frequencies. CONCLUSIONS: NoV epidemics resulted from the global spread of GII.4 strains that evolved under the influence of population immunity. Lineages show notable (and currently unexplained) differences in geographic prevalence. Establishing a global NoV network by which data on strains with the potential to cause pandemics can be rapidly exchanged may lead to improved prevention and intervention strategies.

A novel bocavirus associated with acute gastroenteritis in Australian children.

Acute gastroenteritis (AGE) is a common illness affecting all age groups worldwide, causing an estimated three million deaths annually. Viruses such as rotavirus, adenovirus, and caliciviruses are a major cause of AGE, but in many patients a causal agent cannot be found despite extensive diagnostic testing. Proposing that novel viruses are the reason for this diagnostic gap, we used molecular screening to investigate a cluster of undiagnosed cases that were part of a larger case control study into the etiology of pediatric AGE. Degenerate oligonucleotide primed (DOP) PCR was used to non-specifically amplify viral DNA from fecal specimens. The amplified DNA was then cloned and sequenced for analysis. A novel virus was detected. Elucidation and analysis of the genome indicates it is a member of the Bocavirus genus of the Parvovirinae, 23% variant at the nucleotide level from its closest formally recognized relative, the Human Bocavirus (HBoV), and similar to the very recently proposed second species of Bocavirus (HBoV2). Fecal samples collected from case control pairs during 2001 for the AGE study were tested with a bocavirus-specific PCR, and HBoV2 (sequence confirmed) was detected in 32 of 186 cases with AGE (prevalence 17.2%) compared with only 15 controls (8.1%). In this same group of children, HBoV2 prevalence was exceeded only by rotavirus (39.2%) and astrovirus (21.5%) and was more prevalent than norovirus genogroup 2 (13.4%) and adenovirus (4.8%). In a univariate analysis of the matched pairs (McNemar's Test), the odds ratio for the association of AGE with HBoV2 infection was 2.6 (95% confidence interval 1.2-5.7); P = 0.007. During the course of this screening, a second novel bocavirus was detected which we have designated HBoV species 3 (HBoV3). The prevalence of HBoV3 was low (2.7%), and it was not associated with AGE. HBoV2 and HBoV3 are newly discovered bocaviruses, of which HBoV2 is the thirdmost-prevalent virus, after rotavirus and astrovirus, associated with pediatric AGE in this study.

Identifying the fundamental units of bacterial diversity: a paradigm shift to incorporate ecology into bacterial systematics.

The central questions of bacterial ecology and evolution require a method to consistently demarcate, from the vast and diverse set of bacterial cells within a natural community, the groups playing ecologically distinct roles (ecotypes). Because of a lack of theory-based guidelines, current methods in bacterial systematics fail to divide the bacterial domain of life into meaningful units of ecology and evolution. We introduce a sequence-based approach ("ecotype simulation") to model the evolutionary dynamics of bacterial populations and to identify ecotypes within a natural community, focusing here on two Bacillus clades surveyed from the "Evolution Canyons" of Israel. This approach has identified multiple ecotypes within traditional species, with each predicted to be an ecologically distinct lineage; many such ecotypes were confirmed to be ecologically distinct, with specialization to different canyon slopes with different solar exposures. Ecotype simulation provides a long-needed natural foundation for microbial ecology and systematics.

Molecular diagnosis of medical viruses.

The diagnosis of infectious diseases has been revolutionized by the development of molecular techniques, foremost with the applications of the polymerase chain reaction (PCR). The achievable high sensitivity and ease with which the method can be used to detect any known genetic sequence have led to its wide application in the life sciences. More recently, real-time PCR assays have provided additional major contributions, with the inclusion of an additional fluorescent probe detection system resulting in an increase in sensitivity over conventional PCR, the ability to confirm the amplification product and to quantitate the target concentration. Further, nucleotide sequence analysis of the amplification products has facilitated epidemiological studies of infectious disease outbreaks, and the monitoring of treatment outcomes for infections, in particular with viruses which mutate at high frequency. This review discusses the applications of qualitative and quantitative real-time PCR, nested PCR, multiplex PCR, nucleotide sequence analysis of amplified products and quality assurance with nucleic acid testing (NAT) in diagnostic laboratories.

Legionella drancourtii sp. nov., a strictly intracellular amoebal pathogen.

A Legionella-like amoebal pathogen (LLAP), formerly named LLAP12(T), was characterized on the basis of microscopic appearance, staining characteristics, growth in Acanthamoeba polyphaga at different temperatures, DNA G+C content, serological cross-reactivity and 16S rRNA and macrophage infectivity potentiator (mip) gene sequence analysis. LLAP12(T) was found to be a motile, Gram-negative bacterium that grew within cytoplasmic vacuoles in infected amoebae. The infecting bacteria induced lysis of their amoebal hosts and time taken to do so was dependent on incubation temperature. Recovery of LLAP12(T) from amoebae onto axenic media could not be achieved. Phylogenetic analysis of LLAP12(T), based on 16S rRNA and mip gene sequence analysis, indicated that it lay within the radiation of the Legionellaceae and that it clustered specifically with Legionella lytica and Legionella rowbothamii. The divergence observed between LLAP12(T) and these two species was of a degree equal to, or greater than, that observed between other members of the family. In support of this delineation, LLAP12(T) was found not to cross-react serologically with any other Legionella species. The mip and 16S rRNA gene sequence-based analyses also indicated that LLAP12(T) was related very closely to two other previously identified LLAP isolates, LLAP4 and LLAP11. Taken together, these results support the proposal of LLAP12(T) as the type strain of Legionella drancourtii sp. nov.

Legionella pneumophila mip gene sequencing to investigate a cluster of pneumonia cases.

AIMS: To use fluorescent antibody (FA) and PCR studies on fixed lung tissue to investigate whether Legionella pneumophila was the cause of pneumonia in a cluster of three haematology patients. METHODS: Cut sections of paraffin blocks of lung tissue were examined by direct FA (DFA) using fluorescently labelled antibody to serogroup 1 and Pontiac strains of L. pneumophila. In addition, a single tube 'hanging drop' nested PCR targeting the mip gene of Legionella was performed on DNA extracted from the lung sections. Products were sequenced using dye terminator chemistry. RESULTS: Numerous fluorescing bacteria were seen on staining with both antibodies in lung tissue from two of the patients. Identical L. pneumophila mip gene sequences were amplified from both DFA-positive lung sections. Two differing L. pneumophila mip sequences were obtained on three separate occasions from the tissue sections from the third patient negative by DFA. These sequences differed slightly from those obtained from the two DFA positive lung tissues. CONCLUSIONS: There is good epidemiological evidence to link the first two cases who had been treated in the same ward prior to development of fever within two days of each other. The significance of results is controversial for the third patient.

Aureobacterium masquerading as 'Corynebacterium aquaticum' infection: case report and review of the literature.

A gram-positive bacillus was isolated repeatedly from blood taken through the lumina of a central venous catheter of a patient with multiple myeloma who developed febrile neutropenia following chemotherapy. The bacterium was identified by the API CORYNE system as 'Corynebacterium aquaticum'. Gene analysis targeting the 16S rRNA indicated that the organism had a 99.5% identity with Aureobacterium liquefaciens although there were two phenotypic characteristics at variance with the description of this species. Problems remain with the routine identification of 'C. aquaticum' and Aureobacterium species. The few clinical reports on patients infected with 'C. aquaticum' and A. liquefaciens indicate that these are rare infections often associated with immunocompromise.