Detection of Recently Discovered Human Polyomaviruses in a Longitudinal Kidney Transplant Cohort.

A large number of human polyomaviruses have been discovered in the last 7 years. However, little is known about the clinical impact on vulnerable immunosuppressed patient populations. Blood, urine, and respiratory swabs collected from a prospective, longitudinal adult kidney transplant cohort (n = 167) generally pre-operatively, at day 4, months 1, 3, and 6 posttransplant, and at BK viremic episodes within the first year were screened for 12 human polyomaviruses using real-time polymerase chain reaction. Newly discovered polyomaviruses were most commonly detected in the respiratory tract, with persistent shedding seen for up to 6 months posttransplant. Merkel cell polyomavirus was the most common detection, but was not associated with clinical symptoms or subsequent development of skin cancer or other skin abnormalities. In contrast, KI polyomavirus was associated with respiratory disease in a subset of patients. Human polyomavirus 9, Malawi polyomavirus, and human polyomavirus 12 were not detected in any patient samples.

Prevalence, codetection and seasonal distribution of upper airway viruses and bacteria in children with acute respiratory illnesses with cough as a symptom.

Most studies exploring the role of upper airway viruses and bacteria in paediatric acute respiratory infections (ARI) focus on specific clinical diagnoses and/or do not account for virus-bacteria interactions. We aimed to describe the frequency and predictors of virus and bacteria codetection in children with ARI and cough, irrespective of clinical diagnosis. Bilateral nasal swabs, demographic, clinical and risk factor data were collected at enrollment in children aged <15 years presenting to an emergency department with an ARI and where cough was a symptom. Swabs were tested by polymerase chain reaction for 17 respiratory viruses and seven respiratory bacteria. Logistic regression was used to investigate associations between child characteristics and codetection of the organisms of interest. Between December 2011 and August 2014, swabs were collected from 817 (93.3%) of 876 enrolled children, median age 27.7 months (interquartile range 13.9-60.3 months). Overall, 740 (90.6%) of 817 specimens were positive for any organism. Both viruses and bacteria were detected in 423 specimens (51.8%). Factors associated with codetection were age (adjusted odds ratio (aOR) for age <12 months = 4.9, 95% confidence interval (CI) 3.0, 7.9; age 12 to <24 months = 6.0, 95% CI 3.7, 9.8; age 24 to <60 months = 2.4, 95% CI 1.5, 3.9), male gender (aOR 1.46; 95% CI 1.1, 2.0), child care attendance (aOR 2.0; 95% CI 1.4, 2.8) and winter enrollment (aOR 2.0; 95% CI 1.3, 3.0). Haemophilus influenzae dominated the virus-bacteria pairs. Virus-H. influenzae interactions in ARI should be investigated further, especially as the contribution of nontypeable H. influenzae to acute and chronic respiratory diseases is being increasingly recognized.

Comparison of test specificities of commercial antigen-based assays and in-house PCR methods for detection of rotavirus in stool specimens.

Seven commercial rotavirus antigen assays were compared with in-house PCR methods for detecting rotavirus in stool specimens. The assay sensitivities were 80% to 100%, while the specificities were 54.3% for one commercial immunochromatographic (ICT) method and 99.4% to 100% for other assays. Thus, except for one commercial ICT, all the assays were generally reliable for rotavirus detection.

Feasibility of parental collected nasal swabs for virus detection in young children with cystic fibrosis.

BACKGROUND: The detrimental role of viruses has been well described in CF, although the pattern of virus infections has not been investigated in a longitudinal study. The primary aim was to determine the feasibility of fortnightly parent collected swabs in young children with CF. METHODS: Children under three years with CF were recruited. Nasal swabs were collected by parents every fortnight and during periods of symptoms over 12 months. Nasal swabs were posted and virus detected using real-time PCR. RESULTS: Only 27% of the patients completed the study to 10 months, although 98% of the swabs returned were adequate for analysis. Mould was observed growing on 23% of the returned swabs. There was no evidence to demonstrate relationships with symptoms and viruses, prolonged symptoms, prolonged shedding or patterns of virus infections. CONCLUSIONS: This study highlights the need to further investigate the role of viruses in children with CF using a robust method of frequent collection in children for a longitudinal study, with appropriate storage and shipping techniques to avoid mould growth or other potential contaminants.

Potential animal and environmental sources of Q fever infection for humans in Queensland.

Q fever is a vaccine-preventable disease; despite this, high annual notification numbers are still recorded in Australia. We have previously shown seroprevalence in Queensland metropolitan regions is approaching that of rural areas. This study investigated the presence of nucleic acid from Coxiella burnetii, the agent responsible for Q fever, in a number of animal and environmental samples collected throughout Queensland, to identify potential sources of human infection. Samples were collected from 129 geographical locations and included urine, faeces and whole blood from 22 different animal species; 45 ticks were removed from two species, canines and possums; 151 soil samples; 72 atmospheric dust samples collected from two locations and 50 dust swabs collected from domestic vacuum cleaners. PCR testing was performed targeting the IS1111 and COM1 genes for the specific detection of C. burnetii DNA. There were 85 detections from 1318 animal samples, giving a detection rate for each sample type ranging from 2.1 to 6.8%. Equine samples produced a detection rate of 11.9%, whilst feline and canine samples showed detection rates of 7.8% and 5.2%, respectively. Native animals had varying detection rates: pooled urines from flying foxes had 7.8%, whilst koalas had 5.1%, and 6.7% of ticks screened were positive. The soil and dust samples showed the presence of C. burnetii DNA ranging from 2.0 to 6.9%, respectively. These data show that specimens from a variety of animal species and the general environment provide a number of potential sources for C. burnetii infections of humans living in Queensland. These previously unrecognized sources may account for the high seroprevalence rates seen in putative low-risk communities, including Q fever patients with no direct animal contact and those subjects living in a low-risk urban environment.

Polymerase chain reaction-based screening for the ceftriaxone-resistant Neisseria gonorrhoeae F89 strain.

Emergence and spread of Neisseria gonorrhoeae resistant to extended spectrum cephalosporins is a major problem threatening treatment of gonorrhoea and is further highlighted by the recent report of a second ceftriaxone-resistant N. gonorrhoeae strain (F89) in Europe, initially observed in France and subsequently identified in Spain. N. gonorrhoeae antimicrobial resistance (AMR) surveillance has acquired new importance and molecular tools have the potential to enhance bacterial culture-based methods. In this study, we established a polymerase chain reaction (PCR) protocol for direct detection of the F89 strain. A key component of this screening protocol was the development of a hybridisation probe-based melting curve analysis assay (mosaic501-hybPCR) to detect the presence of an A501P substitution on the N. gonorrhoeae mosaic penicillin binding protein 2 (PBP2) sequence, an important characteristic of the F89 strain. The mosaic501-hybPCR was evaluated using plasmid-derived positive controls (n=3) and characterised gonococcal (n=33) and non-gonococcal (n=58) isolates. The protocol was then applied to 159 clinical specimens from Sydney, Australia, collected during the first half of the year 2012 that were N. gonorrhoeae PCR-positive. Overall, the results indicate that the PCR-based protocol is suitable for direct detection of the N. gonorrhoeae F89 strain in non-cultured clinical samples. It therefore provides an additional tool to aid investigations into the potential spread of F89 strain throughout Europe and elsewhere.

Association of micropapillary urothelial carcinoma of the bladder and BK viruria in kidney transplant recipients.

INTRODUCTION: BK virus (BKV) is an ubiquitous human polyomavirus that establishes latency in urothelium. BKV is known to re-activate in immunosuppressed individuals, and is an increasingly important cause of nephropathy and graft loss in kidney transplant recipients. Animal studies have demonstrated BKV has a potential role as a tumor virus. However, its role in precipitating or facilitating oncogenesis in humans is still debated. REPORT: We report 2 cases of aggressive micropapillary urothelial carcinoma of the bladder in kidney transplant recipients with persistent BK viruria and preserved graft function. RESULTS: In both cases, polyomavirus immunohistochemistry performed on the tumor specimens was strongly positive, and limited to the malignant tissue. BKV DNA, viral protein 1, and large T antigen mRNA were detected in the tumor; however, no viral particles were seen on electron microscopy. CONCLUSION: In one of the cases, BKV integration into the host genome was identified, leading to the truncation of the major viral capsid gene. This finding raises the concern that persisting BK viruria may be a risk factor for this aggressive form of bladder cancer. Further studies to determine screening and management strategies are required.

A novel duplex real-time PCR for HPIV-4 detects co-circulation of both viral subtypes among ill children during 2008.

The two subtypes of the human parainfluenzavirus type 4 (HPIV-4) are rarely sought in testing for acute respiratory illness (ARI) and this may be confounding our understanding of its role. This study presents a novel duplex real-time RT-PCR assay targeting the P gene that can detect and differentiate the two subtypes in a single reaction. Subtype-specific synthetic RNA positive controls were prepared and used to determine an analytical sensitivity of 10 copies per reaction with an 8log(10) dynamic range. The assays were validated using 1140 clinical specimens mostly nasopharyngeal aspirates collected from children during 2008. These included specimens previously determined to be positive for all commonly considered respiratory viruses. The novel assay did not cross-reaction with any other virus. Fourteen HPIV-4 positives, ten detected in the absence of any co-detections (four with rhinovirus), were identified in 2008 and their subtype confirmed by conventional RT-PCR and sequencing of P gene fragments. Most detections were in children two years of age or younger. Our assay proved suitably sensitive and specific for inclusion in future studies seeking to better understand the role HPIV-4 and other respiratory viruses in children with ARI.

False-negative results using Neisseria gonorrhoeae porA pseudogene PCR - a clinical gonococcal isolate with an N. meningitidis porA sequence, Australia, March 2011.

The gonococcal porA pseudogene is a popular target for in-house Neisseria gonorrhoeae PCR methods. With this study we present two novel findings: the first case of an N. gonorrhoeae porA pseudogene PCR false-negative result caused by sequence variation, and in the same organism, the first description of a clinical N. gonorrhoeae strain harbouring an N. meningitidis porA sequence.

Comparison of a multiplexed MassARRAY system with real-time allele-specific PCR technology for genotyping of methicillin-resistant Staphylococcus aureus.

The Sequenom MassARRAY iPLEX single-nucleotide polymorphism (SNP) typing platform uses matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) coupled with single-base extension PCR for high-throughput multiplex SNP detection. In this study, we investigated the use of iPLEX MassARRAY technology for methicillin-resistant Staphylococcus aureus (MRSA) genotyping. A 16-plex MassARRAY iPLEX GOLD assay (MRSA-iPLEX) was developed that targets a set of informative SNPs and binary genes for MRSA characterization. The method was evaluated with 147 MRSA isolates, and the results were compared with those of an established SYBR Green-based real-time PCR system utilizing the same SNP-binary markers. A total of 2352 markers belonging to 44 SNP-binary profiles were analysed by both real-time PCR and MRSA-iPLEX. With real-time PCR as the reference standard, MRSA-iPLEX correctly assigned 2298 of the 2352 (97.7%) markers. Sequence variation in the MRSA-iPLEX primer targets accounted for the majority of MRSA-iPLEX erroneous results, highlighting the importance of primer target selection. MRSA-iPLEX provided optimal throughput for MRSA genotyping, and was, on a reagent basis, more cost-effective than the real-time PCR methods. The 16-plex MRSA-iPLEX is a suitable alternative to SYBR Green-based real-time PCR typing of major sequence types and clonal complexes of MRSA.

Q fever seroprevalence in metropolitan samples is similar to rural/remote samples in Queensland, Australia.

Q fever is a vaccine preventable disease; however, despite this, high notification numbers are still recorded annually in Australia. We investigated the seroprevalence of Coxiella burnetii, the Q fever agent, in a Queensland sample population. Notification data (N = 6425) from 1984-2008 were collated, identifying high risk areas of Q fever exposure. Of these 177 were recorded in children. Serum samples were collected from Queensland and screened using both an immunoflourescence assay at 1:10 dilution and a commercially available ELISA kit. Results were collated based on age, geographical location and sex. From 1988 Queensland samples screened, 103 were identified as Q fever IgG-positive, giving a seroprevalence of 5.2% (95% CI 4.3-6.2%). Seroprevalence in the rural/remote population was 5.3% (95% CI 4.6-6.6%), and the metropolitan Brisbane population, which is considered not at risk, was 5.0% (95% CI 3.7-6.7%). Sixty-three seropositive males and 40 females were identified, along with an increase in seropositivity with increasing age. The seropositivity of children was 1.3% (95% CI 0.7-2.3%) from 844 samples. We have shown that both metropolitan and paediatric populations which are considered low risk of Coxiella exposure have surprisingly high seropositivity. These emerging groups require further investigation and consideration for the introduction of preventive measures.

Evaluation of a clinical scoring system and directed laboratory testing for respiratory virus infection in hematopoietic stem cell transplant recipients.

A simple clinical screening (CS) tool for respiratory virus (RV) infection was introduced and evaluated in a single hematology ward, as part of a strategy to reduce nosocomial RV infection. Up to 6 clinical symptoms or signs were scored and a predefined threshold score of ≥ 2 prompted paired nose/throat swab (NTS) collection for RV testing. The criterion standard for RV infection was positive immunofluorescence (IF) or polymerase chain reaction (PCR) for 7 and 15 viruses, respectively. The tool was shown to be most beneficial at excluding infection at a threshold score of 1 (negative predictive value [NPV] 89%, [95% confidence interval 78-96%], sensitivity 85% [70-94%], specificity 35% [27-43%]), compared with a score of 2 (NPV 85% [76-91%], sensitivity 63% [46-77%], specificity 57% [48-65%]) at a prevalence of 22%. The tool's ability to diagnose infection was limited (positive predictive value 27% and 29% at thresholds 1 and 2). The sensitivity of IF compared with PCR was 45% for the 7 viruses common to both, and 23% for the extended virus panel detected by PCR. An algorithm incorporating CS, paired NTS collection at a threshold of 1 symptom or sign, and sensitive testing including PCR can guide infection control measures in hospitalized hematopoietic stem cell transplant recipients.

Rapid single-nucleotide polymorphism-based identification of clonal Pseudomonas aeruginosa isolates from patients with cystic fibrosis by the use of real-time PCR and high-resolution melting curve analysis.

Pseudomonas aeruginosa genotyping relies mainly upon DNA fingerprinting methods, which can be subjective, expensive and time-consuming. The detection of at least three different clonal P. aeruginosa strains in patients attending two cystic fibrosis (CF) centres in a single Australian city prompted the design of a non-gel-based PCR method to enable clinical microbiology laboratories to readily identify these clonal strains. We designed a detection method utilizing heat-denatured P. aeruginosa isolates and a ten-single-nucleotide polymorphism (SNP) profile. Strain differences were detected by SYBR Green-based real-time PCR and high-resolution melting curve analysis (HRM10SNP assay). Overall, 106 P. aeruginosa sputum isolates collected from 74 patients with CF, as well as five reference strains, were analysed with the HRM10SNP assay, and the results were compared with those obtained by pulsed-field gel electrophoresis (PFGE). The HRM10SNP assay accurately identified all 45 isolates as members of one of the three major clonal strains characterized by PFGE in two Brisbane CF centres (Australian epidemic strain-1, Australian epidemic strain-2 and P42) from 61 other P. aeruginosa strains from Australian CF patients and two representative overseas epidemic strain isolates. The HRM10SNP method is simple, is relatively inexpensive and can be completed in <3 h. In our setting, it could be made easily available for clinical microbiology laboratories to screen for local P. aeruginosa strains and to guide infection control policies. Further studies are needed to determine whether the HRM10SNP assay can also be modified to detect additional clonal strains that are prevalent in other CF centres.

Molecular characterization and distinguishing features of a novel human rhinovirus (HRV) C, HRVC-QCE, detected in children with fever, cough and wheeze during 2003.

BACKGROUND: Human rhinoviruses (HRVs) are associated with more acute respiratory tract infections than any other viral group yet we know little about viral diversity, epidemiology or clinical outcome resulting from infection by strains, in particular the recently identified HRVs. OBJECTIVES: To determine whether HRVC-QCE was a distinct HRV-C strain, by determining its genome and prevalence, by cataloguing genomic features for strain discrimination and by observing clinical features in positive patients. STUDY DESIGN: Novel real-time RT-PCRs and retrospective chart reviews were used to investigate a well-defined population of 1247 specimen extracts to observe the prevalence and the clinical features of each HRV-QCE positive case from an in- and out-patient pediatric, hospital-based population during 2003. An objective illness severity score was determined for each HRVC-QCE positive patient. RESULTS: Differences in overall polyprotein and VP1 binding pocket residues and the predicted presence of a cis-acting replication element in 1B defined HRVC-QCE as a novel HRV-C strain. Twelve additional HRVC-QCE detections (1.0% prevalence) occurred among infants and toddlers (1-24 months) suffering mild to moderate illness, including fever and cough, who were often hospitalized. HRVC-QCE was frequently detected in the absence of another virus and was the only virus detected in three (23% of HRVC-QCE positives) children with asthma exacerbation and in two (15%) toddlers with febrile convulsion. CONCLUSIONS: HRVC-QCE is a newly identified, genetically distinct HRV strain detected in hospitalized children with a range of clinical features. HRV strains should be independently considered to ensure we do not overestimate the HRVs in asymptomatic illness.

Cough-generated aerosols of Pseudomonas aeruginosa and other Gram-negative bacteria from patients with cystic fibrosis.

BACKGROUND: Pseudomonas aeruginosa is the most common bacterial pathogen in patients with cystic fibrosis (CF). Current infection control guidelines aim to prevent transmission via contact and respiratory droplet routes and do not consider the possibility of airborne transmission. It was hypothesised that subjects with CF produce viable respirable bacterial aerosols with coughing. METHODS: A cross-sectional study was undertaken of 15 children and 13 adults with CF, 26 chronically infected with P aeruginosa. A cough aerosol sampling system enabled fractioning of respiratory particles of different sizes and culture of viable Gram-negative non-fermentative bacteria. Cough aerosols were collected during 5 min of voluntary coughing and during a sputum induction procedure when tolerated. Standardised quantitative culture and genotyping techniques were used. RESULTS: P aeruginosa was isolated in cough aerosols of 25 subjects (89%), 22 of whom produced sputum samples. P aeruginosa from sputum and paired cough aerosols were indistinguishable by molecular typing. In four cases the same genotype was isolated from ambient room air. Approximately 70% of viable aerosols collected during voluntary coughing were of particles

Do rhinoviruses reduce the probability of viral co-detection during acute respiratory tract infections?

BACKGROUND: Human rhinoviruses (HRVs) are often concurrently detected with other viruses found in the respiratory tract because of the high total number of HRV infections occurring throughout the year. This feature has previously relegated HRVs to being considered passengers in acute respiratory infections. HRVs remain poorly characterized and are seldom included as a target in diagnostic panels despite their pathogenic potential, infection-associated healthcare expenditure and relatively unmoderated elicitation of an antiviral state. OBJECTIVES: To test the hypothesis that respiratory viruses are proportionately more or less likely to co-occur, particularly the HRVs. STUDY DESIGN: Retrospective PCR-based analyses of 1247 specimens for 17 viruses, including HRV strains, identified 131 specimens containing two or more targets. We investigated the proportions of co-detections and compared the proportion of upper vs. lower respiratory tract presentations in the HRV positive group. Both univariate contingency table and multivariate logistic regression analyses were conducted to identify trends of association among the viruses present in co-detections. RESULTS: Many of the co-detections occurred in patterns. In particular, HRV detection was associated with a reduced probability of detecting human adenoviruses, coronaviruses, bocavirus, metapneumovirus, respiratory syncytial virus, parainfluenza virus, influenza A virus, and the polyomaviruses KIPyV and WUPyV (p < or = 0.05). No single HRV species nor cluster of particular strains predominated. CONCLUSIONS: HRVs were proportionately under-represented among viral co-detections. For some period, HRVs may render the host less likely to be infected by other viruses.

A novel gel-based method for self-collection and ambient temperature postal transport of urine for PCR detection of Chlamydia trachomatis.

OBJECTIVES: The aim of this study was to develop a novel urine transport method to be used in self-collection-based screening for Chlamydia trachomatis. The method needed to be suitable for C trachomatis PCR detection, be economical and suitable for transport by standard envelope mailing. METHODS: An anhydrous gel composed of super-absorbent polymer and buffering agent was used to desiccate urine into a dry granulous state, which could subsequently be reconstituted upon arrival at a laboratory. DNA was then extracted from the reconstituted solution using the Roche MagNA Pure protocol for the detection of C trachomatis by PCR. Collections of urine specimens from three populations with widely differing chlamydia prevalence (100%,n = 56; 47%, n = 70; 3%, n = 97) were used. We determined the gel method's impact on C trachomatis PCR sensitivity and specificity using neat and gel-processed urine specimens. An equine herpes virus PCR was used to test for assay inhibition. RESULTS: Overall, the sensitivity of the gel-based method ranged from 94.6-100% compared with neat urine, with a specificity of 100%. No PCR inhibition or decrease in analytical sensitivity was observed using the gel-processed extracts. CONCLUSIONS: The gel-based method was found to be suitable for the detection of C trachomatis by PCR. In addition, its ease of use, effectiveness at ambient temperature and low cost makes it well-suited for self-collection kits used in population-based C trachomatis screening, particularly for geographically and socially isolated individuals.

False-negative results in nucleic acid amplification tests-do we need to routinely use two genetic targets in all assays to overcome problems caused by sequence variation?

Nucleic acid amplification tests (NAATs) have numerous advantages over traditional diagnostic techniques and so are now widely used by diagnostic laboratories for routine detection of infectious agents. However, there is some concern over the increasing numbers of reports of NAAT false-negative results caused by sequence variation. Highly conserved NAAT target sequences have been reported for many organisms, yet sequence-related problems continue to be observed in commercial and in-house assays targeting a broad range of microbial pathogens. In light of these ongoing problems, it may be time to consider the use of two genetic targets in NAAT methods to reduce the potential for sequence-related false-negative results.