Bacteria and viruses in the nasopharynx immediately prior to onset of acute lower respiratory infections in Indigenous Australian children.

Acute lower respiratory infection (ALRI) is a major cause of hospitalization for Indigenous children in remote regions of Australia. The associated microbiology remains unclear. Our aim was to determine whether the microbes present in the nasopharynx before an ALRI were associated with its onset. A retrospective case-control/crossover study among Indigenous children aged up to 2 years. ALRI cases identified by medical note review were eligible where nasopharyngeal swabs were available: (1) 0-21 days before ALRI onset (case); (2) 90-180 days before ALRI onset (same child controls); and (3) from time and age-matched children without ALRI (different child controls). PCR assays determined the presence and/or load of selected respiratory pathogens. Among 104 children (182 recorded ALRI episodes), 120 case-same child control and 170 case-different child control swab pairs were identified. Human adenoviruses (HAdV) were more prevalent in cases compared to same child controls (18 vs 7%; OR = 3.08, 95% CI 1.22-7.76, p = 0.017), but this association was not significant in cases versus different child controls (15 vs 10%; OR = 1.93, 95% CI 0.97-3.87 (p = 0.063). No other microbes were more prevalent in cases compared to controls. Streptococcus pneumoniae (74%), Haemophilus influenzae (75%) and Moraxella catarrhalis (88%) were commonly identified across all swabs. In a pediatric population with a high detection rate of nasopharyngeal microbes, HAdV was the only pathogen detected in the period before illness presentation that was significantly associated with ALRI onset. Detection of other potential ALRI pathogens was similar between cases and controls.

Detection of Toscana virus from an adult traveler returning to Australia with encephalitis.

Toscana virus (TOSV) is identified in sandflies, animals, and humans around the Mediterranean Sea. TOSV has not been reported in Australia. During investigations of cerebrospinal fluid samples from patients with encephalitis, TOSV genetic sequences were identified in a traveler returning to Australia from Europe. TOSV should be considered, especially during May to October, in travelers to Australia who embarked in countries in and around the Mediterranean Sea and who subsequently present for medical care because of neurological symptoms.

Heterogeneous and Dynamic Prevalence of Asymptomatic Influenza Virus Infections.

Influenza infection manifests in a wide spectrum of severity, including symptomless pathogen carriers. We conducted a systematic review and meta-analysis of 55 studies to elucidate the proportional representation of these asymptomatic infected persons. We observed extensive heterogeneity among these studies. The prevalence of asymptomatic carriage (total absence of symptoms) ranged from 5.2% to 35.5% and subclinical cases (illness that did not meet the criteria for acute respiratory or influenza-like illness) from 25.4% to 61.8%. Statistical analysis showed that the heterogeneity could not be explained by the type of influenza, the laboratory tests used to detect the virus, the year of the study, or the location of the study. Projections of infection spread and strategies for disease control require that we identify the proportional representation of these insidious spreaders early on in the emergence of new influenza subtypes or strains and track how this rate evolves over time and space.

MERS coronavirus: diagnostics, epidemiology and transmission.

The first known cases of Middle East respiratory syndrome (MERS), associated with infection by a novel coronavirus (CoV), occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia (KSA). Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels (DC). MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract (LRT) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20% to 40% of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome (SARS), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control (IPC) in healthcare settings, with approximately 20% of all virus detections reported among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.

Adenovirus species C is associated with chronic suppurative lung diseases in children.

BACKGROUND: The role of human adenoviruses (HAdVs) in chronic respiratory disease pathogenesis is recognized. However, no studies have performed molecular sequencing of HAdVs from the lower airways of children with chronic endobronchial suppuration. We thus examined the major HAdV genotypes/species, and relationships to bacterial coinfection, in children with protracted bacterial bronchitis (PBB) and mild bronchiectasis (BE). METHODS: Bronchoalveolar lavage (BAL) samples of 245 children with PBB or mild (cylindrical) BE were included in this prospective cohort study. HAdVs were genotyped (when possible) in those whose BAL had HAdV detected (HAdV(+)). Presence of bacterial infection (defined as ≥10(4) colony-forming units/mL) was compared between BAL HAdV(+) and HAdV negative (HAdV(-)) groups. Immune function tests were performed including blood lymphocyte subsets in a random subgroup. RESULTS: Species C HAdVs were identified in 23 of 24 (96%) HAdV(+) children; 13 (57%) were HAdV-1 and 10 (43%) were HAdV-2. An HAdV(+) BAL was significantly associated with bacterial coinfection with Haemophilus influenzae, Moraxella catarrhalis, or Streptococcus pneumoniae (odds ratio [OR], 3.27; 95% confidence interval, 1.38-7.75; P = .007) and negatively associated with Staphylococcus aureus infection (P = .03). Young age was related to increased rates of HAdV(+). Blood CD16 and CD56 natural killer cells were significantly more likely to be elevated in those with HAdV (80%) compared with those without (56.1%) (P = .027). CONCLUSIONS: HAdV-C is the major HAdV species detected in the lower airways of children with PBB and BE. Younger age appears to be an important risk factor for HAdV(+) of the lower airways and influences the likelihood of bacterial coinfection.

Community-wide, contemporaneous circulation of a broad spectrum of human rhinoviruses in healthy Australian preschool-aged children during a 12-month period.

Human rhinovirus (HRV) replication triggers exacerbation of asthma and causes most acute respiratory illnesses (ARIs), which may manifest as influenza-like illness. The recent assignment of 60 previously unknown HRV types to a third HRV species, Human rhinovirus C, raised questions about the prevalence of these picornavirus types in the community, the extent of HRV diversity at a single site, and whether the HRVs have an equally diverse clinical impact on their hosts. We quantified HRV diversity, and there was no clinical impact attributable to HRV species and genotypes among a community population of preschool-aged children with ARI who provided respiratory samples during 2003. All HRV species were represented among 138 children with ARI, and 74 distinct HRV types were cocirculating. Fever accompanied 32.8% of HRV-positive ARI cases. HRVs were less likely than DNA viruses to be codetected with another virus, suggesting virus interference at the community level, demonstrated by the inverse correlation between influenza virus detection and HRV detection.

Usefulness of published PCR primers in detecting human rhinovirus infection.

We conducted a preliminary comparison of the relative sensitivity of a cross-section of published human rhinovirus (HRV)-specific PCR primer pairs, varying the oligonucleotides and annealing temperature. None of the pairs could detect all HRVs in 2 panels of genotyped clinical specimens; >1 PCR is required for accurate description of HRV epidemiology.

Newly identified human rhinoviruses: molecular methods heat up the cold viruses.

Human rhinovirus (HRV) infections cause at least 70% of virus-related wheezing exacerbations and cold and flu-like illnesses. They are associated with otitis media, sinusitis and pneumonia. Annually, the economic impact of HRV infections costs billions in healthcare and lost productivity. Since 1987, 100 officially recognised HRV serotypes reside in two genetically distinct species; HRV A and HRV B, within the genus Enterovirus, family Picornaviridae. Sequencing of their approximately 7kb genomes was finalised in 2009. Since 1999, many globally circulating, molecularly-defined 'strains', perhaps equivalent to novel serotypes, have been discovered but remain uncharacterised. Many of these currently unculturable strains have been assigned to a proposed new species, HRV C although confusion exists over the membership of the species. There has not been sufficient sampling to ensure the identification of all strains and no consensus criteria exist to define whether clinical HRV detections are best described as a distinct strain or a closely related variant of a previously identified strain (or serotype). We cannot yet robustly identify patterns in the circulation of newly identified HRVs (niHRVs) or the full range of associated illnesses and more data are required. Many questions arise from this new found diversity: what drives the development of so many distinct viruses compared to other species of RNA viruses? What role does recombination play in generating this diversity? Are there species- or strain-specific circulation patterns and clinical outcomes? Are divergent strains sensitive to existing capsid-binding antivirals? This update reviews the findings that trigger these and other questions arising during the current cycle of intense rhinovirus discovery.

Proposals for the classification of human rhinovirus species C into genotypically assigned types.

Human rhinoviruses (HRVs) are common respiratory pathogens associated with mild upper respiratory tract infections, but also increasingly recognized in the aetiology of severe lower respiratory tract disease. Wider use of molecular diagnostics has led to a recent reappraisal of HRV genetic diversity, including the discovery of HRV species C (HRV-C), which is refractory to traditional virus isolation procedures. Although it is heterogeneous genetically, there has to date been no attempt to classify HRV-C into types analogous to the multiple serotypes identified for HRV-A and -B and among human enteroviruses. Direct investigation of cross-neutralization properties of HRV-C is precluded by the lack of methods for in vitro culture, but sequences from the capsid genes (VP1 and partial VP4/VP2) show evidence for marked phylogenetic clustering, suggesting the possibility of a genetically based system comparable to that used for the assignment of new enterovirus types. We propose a threshold of 13% divergence for VP1 nucleotide sequences for type assignment, a level that classifies the current dataset of 86 HRV-C VP1 sequences into a total of 33 types. We recognize, however, that most HRV-C sequence data have been collected in the VP4/VP2 region (currently 701 sequences between positions 615 and 1043). We propose a subsidiary classification of variants showing > 10% divergence in VP4/VP2, but lacking VP1 sequences, to 28 provisionally assigned types (subject to confirmation once VP1 sequences are determined). These proposals will assist in future epidemiological and clinical studies of HRV-C conducted by different groups worldwide, and provide the foundation for future exploration of type-associated differences in clinical presentations and biological properties.

Newly identified respiratory viruses in children with asthma exacerbation not requiring admission to hospital.

There are few data describing the comprehensive identification in and influence of newly identified respiratory viruses on asthma exacerbations. Most studies focus on inpatients. In this preliminary study, the point prevalence and the associations of picornavirus species described recently and human bocavirus (HBoV) with the recovery from exacerbations in non-hospitalized asthmatic children (median age 5.1 years) were examined. Human rhinoviruses (HRVs) were present in 52.6% of specimens, HBoV-1 was in 7.7%. Viral co-detections occurred in 25.6% of children and were associated (P = 0.04) with lower asthma quality of life scores upon presentation than were single viral detections. The undifferentiated presence or absence of virus did not influence the severity of asthma or recovery however when virus species were examined individually, specific clinical associations emerged. HRV species C (HRV-Cs) were the viruses most frequently detected as single virus detections. Among 41 genotyped HRVs, more HRV-Cs (n = 23) were identified than HRV-As (n = 16) however HRV-A detection was associated (P = 0.01) with worse asthma symptoms and cough for longer than was HRV-C detection. Larger, PCR-based studies are required to elucidate further the true impact of HRV species in childhood asthma exacerbations of both hospitalized and non-hospitalized cohorts.

Human bocavirus: passenger or pathogen in acute respiratory tract infections?

Human bocavirus (HBoV) is a newly identified virus tentatively assigned to the family Parvoviridae, subfamily Parvovirinae, genus Bocavirus. HBoV was first described in 2005 and has since been detected in respiratory tract secretions worldwide. Herein we review the literature on HBoV and discuss the biology and potential clinical impact of this virus. Most studies have been PCR based and performed on patients with acute respiratory symptoms, from whom HBoV was detected in 2 to 19% of the samples. HBoV-positive samples have been derived mainly from infants and young children. HBoV DNA has also been detected in the blood of patients with respiratory tract infection and in fecal samples of patients with diarrhea with or without concomitant respiratory symptoms. A characteristic feature of HBoV studies is the high frequency of coinciding detections, or codetections, with other viruses. Available data nevertheless indicate a statistical association between HBoV and acute respiratory tract disease. We present a model incorporating these somewhat contradictory findings and suggest that primary HBoV infection causes respiratory tract symptoms which can be followed by prolonged low-level virus shedding in the respiratory tract. Detection of the virus in this phase will be facilitated by other infections, either simply via increased sample cell count or via reactivation of HBoV, leading to an increased detection frequency of HBoV during other virus infections. We conclude that the majority of available HBoV studies are limited by the sole use of PCR diagnostics on respiratory tract secretions, addressing virus prevalence but not disease association. The ability to detect primary infection through the development of improved diagnostic methods will be of great importance for future studies seeking to assign a role for HBoV in causing respiratory illnesses.

Genotypic diversity, circulation patterns and co-detections among rhinoviruses in Queensland, 2001.

PURPOSE: Rhinoviruses (RVs) occur more frequently than other viruses and more often in people displaying symptoms than in those without. We sought to estimate the spectrum of RV diversity, RV species seasonality and to analyse RV involvement in respiratory virus co-detections. METHODOLOGY: A convenience collection of 1179 airway sample extracts from patients with suspected respiratory infections, collected during 2001, was subjected to comprehensive molecular testing. RESULTS: RVs were the most common virus detected. We were able to genotype ~90 % of RV detections, identifying 70 distinct RVs, spanning all three species. RV-Bs were under-represented. We found RV species co-circulated at times, although one species usually dominated. Each species displayed a bimodal distribution. CONCLUSION: Notably, RVs and influenza A viruses (IFAV) seldom co-occurred, supporting their roles as primary pathogens of the airway among acutely ill infants. Whether RV circulation has a moderating or controlling effect on the IFAV season or is controlled by it cannot be determined from these data. Despite the frequent perception that RVs commonly co-occur with another virus, our findings indicated this was not always the case. Nearly 80 % of RV detections occurred alone. Understanding more about population-level interference between viruses may allow us to harness aspects of it to generate a non-specific antiviral intervention that mimics a putative protective effect. For routine respiratory virus screening to best serve the patient, RV testing should be a principal component of any acute respiratory illness testing algorithm throughout the year.

Laboratory methods supporting measles surveillance in Queensland, Australia, 2010-2017.

PURPOSE: Australia was officially recognised as having eliminated endemic measles transmission in 2014. Maintaining laboratory support for surveillance of vaccine-preventable diseases, such as measles, is an essential component of reaching and maintaining transmission-free status. METHODOLOGY: Real-time and conventional PCR-based tools were used to detect, differentiate from measles vaccine virus (MeVV), and sequence fragments of measles viruses (MeV) identified from specimens collected in Queensland. Specimens were mostly from travellers who had visited or returned to Queensland from international or interstate sites or been in contact with a case from either group. RESULTS: Between 2010 and 2017, 13 678 specimens were tested in our laboratory using real-time RT-PCR (RT-rPCR), identifying 533 positives. Most specimens were swabs (70.98 %) and urines (25.56 %). A MeVV RT-rPCR was used on request and identified 154 instances of MeVV. MeV-positive extracts were genotyped as required. Genotypes identified among sequenced specimens included B3, D4, D8, D9, G3, and H1 as well as members of clade A as expected from the detection of MeV among virus introductions due to global travel and vaccination. CONCLUSION: We describe the workflow employed and results from our laboratory between 2010 and 2017 for the sensitive detection of MeV infection, supporting high-quality surveillance to ensure the maintenance of Australia's measles-free status.

Identification of a novel polyomavirus from patients with acute respiratory tract infections.

We report the identification of a novel polyomavirus present in respiratory secretions from human patients with symptoms of acute respiratory tract infection. The virus was initially detected in a nasopharyngeal aspirate from a 3-year-old child from Australia diagnosed with pneumonia. A random library was generated from nucleic acids extracted from the nasopharyngeal aspirate and analyzed by high throughput DNA sequencing. Multiple DNA fragments were cloned that possessed limited homology to known polyomaviruses. We subsequently sequenced the entire virus genome of 5,229 bp, henceforth referred to as WU virus, and found it to have genomic features characteristic of the family Polyomaviridae. The genome was predicted to encode small T antigen, large T antigen, and three capsid proteins: VP1, VP2, and VP3. Phylogenetic analysis clearly revealed that the WU virus was divergent from all known polyomaviruses. Screening of 2,135 patients with acute respiratory tract infections in Brisbane, Queensland, Australia, and St. Louis, Missouri, United States, using WU virus-specific PCR primers resulted in the detection of 43 additional specimens that contained WU virus. The presence of multiple instances of the virus in two continents suggests that this virus is geographically widespread in the human population and raises the possibility that the WU virus may be a human pathogen.

Measles Vaccine Virus RNA in Children More Than 100 Days after Vaccination.

Measles vaccines have been in use since the 1960s with excellent safety and effectiveness profiles. Limited data are available on detection of measles vaccine virus (MeVV) RNA in human subjects following vaccination. Available evidence suggests MeVV RNA can be identified up to 14 days after vaccination, with detection beyond this rare. In routine diagnostic testing, we used two real-time reverse transcription-polymerase chain reaction (RT-rPCR) assays targeting M and F genes to identify measles virus (MeV) and MeVV RNA. Confirmatory testing was performed with an N gene RT-rPCR, followed by sequence confirmation of RT-rPCR positives by semi-nested conventional RT-PCR assays targeting portions of the N, H, and L genes. We report detection and confirmation of MeVV RNA from the respiratory tract of 11 children between 100 and 800 days after most recent receipt of measles-containing vaccine. These novel findings emphasize the importance of genotyping all MeV detections and highlight the need for further work to assess whether persistent MeVV RNA represents viable virus and if transmission to close contacts can occur.

Erratum: Pyke, A.T. et al. On the Home Front: Specialized Reference Testing for Dengue in the Australasian Region. Trop. Med. Infect. Dis. 2018, 3, 75.

The authors wish to make the following correction to Table 1 of this paper [...].

Malaria surveillance from both ends: concurrent detection of Plasmodium falciparum in saliva and excreta harvested from Anopheles mosquitoes.

BACKGROUND: Malaria is the most important vector-borne disease in the world. Epidemiological and ecological studies of malaria traditionally utilize detection of Plasmodium sporozoites in whole mosquitoes or salivary glands by microscopy or serological or molecular assays. However, these methods are labor-intensive, and can over- or underestimate mosquito transmission potential. To overcome these limitations, alternative sample types have been evaluated for the study of malaria. It was recently shown that Plasmodium could be detected in saliva expectorated on honey-soaked cards by Anopheles stephensi, providing a better estimate of transmission risk. We evaluated whether excretion of Plasmodium falciparum nucleic acid by An. stephensi correlates with expectoration of parasites in saliva, thus providing an additional sample type for estimating transmission potential. Mosquitoes were exposed to infectious blood meals containing cultured gametocytes, and excreta collected at different time points post-exposure. Saliva was collected on honey-soaked filter paper cards, and salivary glands were dissected and examined microscopically for sporozoites. Excreta and saliva samples were tested by real time polymerase chain reaction (RT-rtPCR). RESULTS: Plasmodium falciparum RNA was detected in mosquito excreta as early as four days after ingesting a bloodmeal containing gametocytes. Once sporogony (the development of sporozoites) occurred, P. falciparum RNA was detected concurrently in both excreta and saliva samples. In the majority of cases, no difference was observed between the Ct values obtained from matched excreta and saliva samples, suggesting that both samples provide equally sensitive results. A positive association was observed between the molecular detection of the parasites in both samples and the proportion of mosquitoes with sporozoites in their salivary glands from each container. No distinguishable parasites were observed when excreta samples were stained and microscopically analyzed. CONCLUSIONS: Mosquito saliva and excreta are easily collected and are promising for surveillance of malaria-causing parasites, especially in low transmission settings or in places where arboviruses co-circulate.

Orthopoxvirus detection in environmental specimens during suspected bioterror attacks: inhibitory influences of common household products.

After terrorists attacked the United States in 2001, the appearance of letters and other objects containing powdery substances with unknown potentials for biological threat focused attention on the speed, sensitivity, and reliability of diagnostic methods. This study summarizes the abilities and limitations of real-time PCR, electron microscopy (EM), and virus isolation when used to detect potential bioweapons. In particular, we investigated the inhibitory influences of different common household products present in environmental specimens on PCR yield, EM detection, and virus isolation. We used vaccinia virus as a model for orthopoxviruses by spiking it into specimens. In the second part of the study, we describe modifications of diagnostic methods to overcome inhibitory effects. A variety of PCR amplification enhancers, DNA extraction protocols, and applications of internal controls were evaluated to improve diagnostic simplicity, speed, and reliability. As a result, we strongly recommend using at least two different frontline techniques in parallel, e.g., EM and PCR. A positive result obtained by any one of these techniques should be followed by a biological method to confirm the putative diagnosis. Confirmatory methods include virus isolation followed by an agent-specific immunofluorescence assay to confirm the presence of replication-competent particles.

Human rhinoviruses: the cold wars resume.

BACKGROUND: Human rhinoviruses (HRVs) are the most common cause of viral illness worldwide but today, less than half the strains have been sequenced and only a handful examined structurally. This viral super-group, known for decades, has still to face the full force of a molecular biology onslaught. However, newly identified viruses (NIVs) including human metapneumovirus and bocavirus and emergent viruses including SARS-CoV have already been exhaustively scrutinized. The clinical impact of most respiratory NIVs is attributable to one or two major strains but there are 100+ distinct HRVs and, because we have never sought them independently, we must arbitrarily divide the literature's clinical impact findings among them. Early findings from infection studies and use of inefficient detection methods have shaped the way we think of 'common cold' viruses today. OBJECTIVES: To review past HRV-related studies in order to put recent HRV discoveries into context. RESULTS: HRV infections result in undue antibiotic prescriptions, sizable healthcare-related expenditure and exacerbation of expiratory wheezing associated with hospital admission. CONCLUSION: The finding of many divergent and previously unrecognized HRV strains has drawn attention and resources back to the most widespread and frequent infectious agent of humans; providing us the chance to seize the advantage in a decades-long cold war.

HPeV-3 predominated among Parechovirus A positive infants during an outbreak in 2013-2014 in Queensland, Australia.

BACKGROUND: Parechoviruses (HPeV) are endemic seasonal pathogens detected from the respiratory tract, gut, blood and central nervous system (CNS) of children and adults, sometimes in conjunction with a range of acute illnesses. HPeV CNS infection may lead to neurodevelopmental sequelae, especially following infection by HPeV-3, hence screening and genotyping are important to inform epidemiology, aetiology and prognosis. OBJECTIVES: To identify and characterise HPeVs circulating during an outbreak between November 2013 and April 2014 in Queensland, Australia. STUDY DESIGN: To perform PCR-based screening and comparative nucleotide sequence analysis on samples from children with clinically suspected infections submitted to a research laboratory for HPeV investigations. RESULTS: HPeVs were detected among 25/62 samples, identified as HPeV-3 from 23 that could be genotyped. These variants closely matched those which have occurred worldwide and in other States of Australia. CONCLUSIONS: The inclusion of PCR-based HPeV testing is not systematically applied but should be considered essential for children under 3 months of age with CNS symptoms as should long-term follow-up of severe sepsis-like cases.