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 non-significant 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

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

Curr. Issues Mol. Biol. 9: 87–102. Online journal at www.cimb.org 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

Sequence-Based Classification Scheme for the Genus Legionella Targeting the mip Gene

The identification and speciation of strains of Legionella is often difficult, and even the more successful chromatographic classification techniques have struggled to discriminate newly described species. A sequence-based genotypic classification scheme is reported, targeting approximately 700 nucleotide bases of the mip gene and utilizing gene amplification and direct amplicon sequencing. With the exception of Legionella geestiana, for which an amplicon was not produced, the scheme clearly and unambiguously discriminated among the remaining 39 Legionella species and correctly grouped 26 additional serogroup and reference strains within those species. Additionally, the genotypic classification of approximately 150 wild strains from several continents was consistent with their phenotypic classification, with the exception of a few strains where serological cross-reactivity was complex, potentially confusing the latter classification. Strains thought to represent currently uncharacterized species were also found to be genotypically unique. The scheme is technically simple for a laboratory with even basic molecular capabilities and equipment, if access to a sequencing laboratory is available

Genome similarity plot of HBoV3-W471 against HBoV (black line, DQ000495) and HBoV2-W153 (grey line) showing percentage similarity along the aligned genomes (SimPlot 3.2, 2-parameter (Kimura) distance model, window size is 300 bp, step size is 30 bp).

<p>Approximate recombination sites are indicated by the crossover of the sequence most similar to HBoV3-W471 at positions 200 and 3,000 nucleotides, just upstream of NS1 and VP1/2 ORFs; highlighted by the two arrows. Note the high level of conservation between all three genomes toward the 3′ terminus of NS1 and the 5′ terminus of VP1/2 ORFs, presumably the result of functional constraint.</p

Univariate analysis of viruses detected in matched case control pairs enrolled during 2001.

<p>Two-tailed univariate analysis by McNemar's test for correlated proportions comparing matched case-control pairs concordant and discordant for exposure to an individual virus.</p>*<p>Viruses detected from 186 cases and 186 age-matched paired controls in which recognized bacterial pathogens were not detected.</p>†<p>OR, odds ratio.</p>‡<p>CI, 95% confidence interval.</p>§<p>Multiple viruses in 20 different combinations.</p>∥<p>Multiple viruses in 7 different combinations.</p

Phylogenetic analysis of parvoviruses: Maximum likelihood dendrogram (bootstrapped, 1,000 replicates) based on full-length parvovirus genome sequence (nucleotide) alignments.

<p>Approximately 100–200 nucleotides are absent from each termini of the HBoV2, HBoV3, and HBoV sequences.</p

Conditional logistic regression of all viruses associated with gastroenteritis with P value<0.2 in the univariate analysis (Table 2, pooled samples from cases).

*<p>Viruses detected from 186 cases and 186 age-matched paired controls in which recognized bacterial pathogens were not detected.</p>†<p>OR, odds ratio.</p>‡<p>CI, 95% confidence interval.</p

Comparison of the open reading frames for HBoV2-W153, HBoV2-W208, HBoV3-W471, and HBoV st1.

*<p>Number of amino acids encoded for each protein.</p>†<p>Pairwise percent dissimilarity at both the nucleotide and amino acid (parentheses) levels.</p