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.

Respiratory viruses in exacerbations of non-cystic fibrosis bronchiectasis in children.

BACKGROUND: Respiratory viral infections precipitate exacerbations of chronic respiratory diseases such as asthma and chronic obstructive pulmonary disease though similar data in non-cystic fibrosis (CF) bronchiectasis are missing. Our study aimed to determine the point prevalence of viruses associated with exacerbations and evaluate clinical and investigational differences between virus-positive and -negative exacerbations in children with bronchiectasis. METHODS: A cohort of 69 children (median age 7 years) with non-CF bronchiectasis was prospectively followed for 900 child-months. PCR for 16 respiratory viruses was performed on nasopharyngeal aspirates collected during 77 paediatric pulmonologist-defined exacerbations. Clinical data, systemic (C reactive protein (CRP), IL-6, procalcitonin, amyloid-A, fibrinogen) and lung function parameters were also collected. FINDINGS: Respiratory viruses were detected during 37 (48%) exacerbations: human rhinovirus (HRV) in 20; an enterovirus or bocavirus in four each; adenoviruses, metapneumovirus, influenza A virus, respiratory syncytial virus, parainfluenza virus 3 or 4 in two each; coronavirus or parainfluenza virus 1 and 2 in one each. Viral codetections occurred in 6 (8%) exacerbations. HRV-As (n=9) were more likely to be present than HRV-Cs (n=2). Children with virus-positive exacerbations were more likely to require hospitalisation (59% vs 32.5% (p=0.02)) and have fever (OR 3.1, 95% CI 1.2 to 11.1), hypoxia (OR 25.5, 95% CI 2.0 to 322.6), chest signs (OR 3.3, 95% CI 1.1 to 10.2) and raised CRP (OR 4.7, 95% CI 1.7 to 13.1) when compared with virus-negative exacerbations. INTERPRETATION: Respiratory viruses are commonly detected during pulmonary exacerbations of children with bronchiectasis. HRV-As were the most frequently detected viruses with viral codetection being rare. Time-sequenced cohort studies are needed to determine the role of viral-bacterial interactions in exacerbations of bronchiectasis.

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.

Prospective characterization of protracted bacterial bronchitis in children.

BACKGROUND: Prior studies on protracted bacterial bronchitis (PBB) in children have been retrospective or based on small cohorts. As PBB shares common features with other pediatric conditions, further characterization is needed to improve diagnostic accuracy among clinicians. In this study, we aim to further delineate the clinical and laboratory features of PBB in a larger cohort, with a specific focus on concurrent viral detection. METHODS: Children with and without PBB (control subjects) undergoing flexible bronchoscopy were prospectively recruited. Basic immune function testing and lymphocyte subset analyses were performed. BAL specimens were processed for cellularity and microbiology. Viruses were identified using polymerase chain reaction (PCR) and bacteria were identified via culture. RESULTS: The median age of the 104 children (69% male) with PBB was 19 months (interquartile range [IQR], 12-30 mo). Compared with control subjects, children with PBB were more likely to have attended childcare (OR, 8.43; 95% CI, 2.34-30.46). High rates of wheeze were present in both groups, and tracheobronchomalacia was common. Children with PBB had significantly elevated percentages of neutrophils in the lower airways compared with control subjects, and adenovirus was more likely to be detected in BAL specimens in those with PBB (OR, 6.69; 95% CI, 1.50-29.80). Median CD56 and CD16 natural killer (NK) cell levels in blood were elevated for age in children with PBB (0.7 × 109/L; IQR, 0.5-0.9 cells/L). CONCLUSIONS: Children with PBB are, typically, very young boys with prolonged wet cough and parent-reported wheeze who have attended childcare. Coupled with elevated NK-cell levels, the association between adenovirus and PBB suggests a likely role of viruses in PBB pathogenesis.

Respiratory virus detection in nasopharyngeal aspirate versus bronchoalveolar lavage is dependent on virus type in children with chronic respiratory symptoms.

BACKGROUND: The comparative yield of respiratory virus detection from nasopharyngeal aspirate (NPA) versus bronchoalveolar lavage (BAL) is uncertain. Furthermore, the significance of virus detection and its relationship to lower airway neutrophilic inflammation is poorly studied. OBJECTIVES: To evaluate the sensitivity, specificity and predictive values of NPA for detecting respiratory viruses in BAL; and to determine the relationship between viruses and lower airway neutrophilia in children with non-acute respiratory illness. STUDY DESIGN: 150 paired NPA and BAL samples were obtained from 75 children aged <18 years undergoing flexible bronchoscopy for investigation of chronic respiratory symptoms. Viral studies were performed using polymerase chain reaction (PCR). Cellularity studies were performed on BALs. Diagnostic parameters of NPA compared to BAL and associations between viruses and lower airway %neutrophils were evaluated. RESULTS: NPA had a higher yield than BAL for detection of any respiratory virus (52 versus 38, respectively). NPA had a high sensitivity (92%) and low specificity (57%) for detecting HRV in BAL with poor kappa agreement value of 0.398 (95% CI 0.218-0.578, p<0.001). NPA had a fair sensitivity (69%) and good specificity (90.3%) for detecting HAdV on BAL, kappa agreement was 0.561 (95% CI 0.321-0.801, p<0.001). HAdV positivity on NPA, compared to negativity, was independently associated with heightened airway neutrophilia [mean difference (95% CI): 18 (1,35); p=0.042]. CONCLUSIONS: NPA has a higher yield for respiratory virus detection than BAL, however its diagnostic accuracy is dependent on viral species. Adenovirus positivity is associated with significantly heightened lower airway neutrophilia in children with chronic respiratory symptoms.

Bronchiectasis exacerbation study on azithromycin and amoxycillin-clavulanate for respiratory exacerbations in children (BEST-2): study protocol for a randomized controlled trial.

BACKGROUND: Bronchiectasis unrelated to cystic fibrosis (CF) is being increasingly recognized in children and adults globally, both in resource-poor and in affluent countries. However, high-quality evidence to inform management is scarce. Oral amoxycillin-clavulanate is often the first antibiotic chosen for non-severe respiratory exacerbations, because of the antibiotic-susceptibility patterns detected in the respiratory pathogens commonly associated with bronchiectasis. Azithromycin has a prolonged half-life, and with its unique anti-bacterial, immunomodulatory, and anti-inflammatory properties, presents an attractive alternative. Our proposed study will test the hypothesis that oral azithromycin is non-inferior (within a 20% margin) to amoxycillin-clavulanate at achieving resolution of non-severe respiratory exacerbations by day 21 of treatment in children with non-CF bronchiectasis. METHODS: This will be a multicenter, randomized, double-blind, double-dummy, placebo-controlled, parallel group trial involving six Australian and New Zealand centers. In total, 170 eligible children will be stratified by site and bronchiectasis etiology, and randomized (allocation concealed) to receive: 1) azithromycin (5 mg/kg daily) with placebo amoxycillin-clavulanate or 2) amoxycillin-clavulanate (22.5 mg/kg twice daily) with placebo azithromycin for 21 days as treatment for non-severe respiratory exacerbations. Clinical data and a parent-proxy cough-specific quality of life (PC-QOL) score will be obtained at baseline, at the start and resolution of exacerbations, and on day 21. In most children, blood and deep-nasal swabs will also be collected at the same time points. The primary outcome is the proportion of children whose exacerbations have resolved at day 21. The main secondary outcome is the PC-QOL score. Other outcomes are: time to next exacerbation; requirement for hospitalization; duration of exacerbation, and spirometry data. Descriptive viral and bacteriological data from nasal samples and blood inflammatory markers will be reported where available. DISCUSSION: Currently, there are no published randomized controlled trials (RCT) to underpin effective, evidence-based management of acute respiratory exacerbations in children with non-CF bronchiectasis. To help address this information gap, we are conducting two RCTs. The first (bronchiectasis exacerbation study; BEST-1) evaluates the efficacy of azithromycin and amoxycillin-clavulanate compared with placebo, and the second RCT (BEST-2), described here, is designed to determine if azithromycin is non-inferior to amoxycillin-clavulanate in achieving symptom resolution by day 21 of treatment in children with acute respiratory exacerbations. TRIAL REGISTRATION: Australia and New Zealand Clinical Trials Register (ANZCTR) number http://ACTRN12612000010897. http://www.anzctr.org.au/trial_view.aspx?id=347879.

Human rhinovirus C in adult haematopoietic stem cell transplant recipients with respiratory illness.

BACKGROUND: A previously unidentified species of human rhinovirus, HRV-C, was described in 2006 in association with lower respiratory tract infection (LRTI). Features of infection in immunosuppressed adults are poorly characterised. OBJECTIVES: This study aims to determine the epidemiology of HRV-C in haematopoietic stem cell transplant (HSCT) recipients in a single centre. STUDY DESIGN: A prospective cohort study of all HSCT recipients admitted to Westmead Hospital, Westmead, Australia from 1 July 2005 to 30 September 2007 was undertaken. Nose/throat samples were collected from all patients at the time of admission and patients developing pre-defined symptoms and/or signs of respiratory infection during the admission. Samples were processed and tested for rhinoviruses and 14 other respiratory viruses using nucleic acid-based methods, immunofluorescence and culture. HRV genotyping was performed by sequencing a region of the rhinovirus 5' untranslated region (UTR). Clinical data on each episode were collected prospectively. RESULTS: HRVs were identified in 24 episodes: 8% of 299 episodes of clinically- defined respiratory infections and 39% of 61 episodes in which respiratory viruses were detected. HRV-C was most frequent (HRV-C: nine, HRV-A: eight and HRV-B: two). Seven episodes of HRV-C, five with pneumonia, occurred within 100 days of HSCT. Co-pathogens were frequent. CONCLUSIONS: The newly described HRV-C was the most common rhinovirus group detected in HSCT recipients with respiratory infection, with co-pathogens being frequent. Further research is required to understand the activity and pathogenicity of this virus in HSCT recipients.

Antibiotics for bronchiectasis exacerbations in children: rationale and study protocol for a randomised placebo-controlled trial.

BACKGROUND: Despite bronchiectasis being increasingly recognised as an important cause of chronic respiratory morbidity in both indigenous and non-indigenous settings globally, high quality evidence to inform management is scarce. It is assumed that antibiotics are efficacious for all bronchiectasis exacerbations, but not all practitioners agree. Inadequately treated exacerbations may risk lung function deterioration. Our study tests the hypothesis that both oral azithromycin and amoxicillin-clavulanic acid are superior to placebo at improving resolution rates of respiratory exacerbations by day 14 in children with bronchiectasis unrelated to cystic fibrosis. METHODS: We are conducting a bronchiectasis exacerbation study (BEST), which is a multicentre, randomised, double-blind, double-dummy, placebo-controlled, parallel group trial, in five centres (Brisbane, Perth, Darwin, Melbourne, Auckland). In the component of BEST presented here, 189 children fulfilling inclusion criteria are randomised (allocation-concealed) to receive amoxicillin-clavulanic acid (22.5 mg/kg twice daily) with placebo-azithromycin; azithromycin (5 mg/kg daily) with placebo-amoxicillin-clavulanic acid; or placebo-azithromycin with placebo-amoxicillin-clavulanic acid for 14 days. Clinical data and a paediatric cough-specific quality of life score are obtained at baseline, at the start and resolution of exacerbations, and at day 14. In most children, blood and deep nasal swabs are also collected at the same time points. The primary outcome is the proportion of children whose exacerbations have resolved at day 14. The main secondary outcome is the paediatric cough-specific quality of life score. Other outcomes are time to next exacerbation; requirement for hospitalisation; duration of exacerbation; and spirometry data. Descriptive viral and bacteriological data from nasal samples and blood markers will also be reported. DISCUSSION: Effective, evidence-based management of exacerbations in people with bronchiectasis is clinically important. Yet, there are few randomised controlled trials (RCTs) in the neglected area of non-cystic fibrosis bronchiectasis. Indeed, no published RCTs addressing the treatment of bronchiectasis exacerbations in children exist. Our multicentre, double-blind RCT is designed to determine if azithromycin and amoxicillin-clavulanic acid, compared with placebo, improve symptom resolution on day 14 in children with acute respiratory exacerbations. Our planned assessment of the predictors of antibiotic response, the role of antibiotic-resistant respiratory pathogens, and whether early treatment with antibiotics affects duration and time to the next exacerbation, are also all novel. TRIAL REGISTRATION: Australia and New Zealand Clinical Trials Register (ANZCTR) number ACTRN12612000011886.

Protocol for the use of enzyme-linked hybridization assays for genital ulcer disease.

Etiologic agents of genital ulcer disease include herpes simplex 1 and 2, Treponema pallidum pallidum, Haemophilus ducreyi, and Klebsiella granulomatis. The advent of PCR has allowed for more rapid and sensitive detection of microbial pathogens. In this protocol, we describe the simultaneous detection of these five pathogens and an internal control using a single-tube multiplex PCR and colorimetric enzyme-linked amplicon hybridization assay.

Distinguishing molecular features and clinical characteristics of a putative new rhinovirus species, human rhinovirus C (HRV C).

BACKGROUND: Human rhinoviruses (HRVs) are the most frequently detected pathogens in acute respiratory tract infections (ARTIs) and yet little is known about the prevalence, recurrence, structure and clinical impact of individual members. During 2007, the complete coding sequences of six previously unknown and highly divergent HRV strains were reported. To catalogue the molecular and clinical features distinguishing the divergent HRV strains, we undertook, for the first time, in silico analyses of all available polyprotein sequences and performed retrospective reviews of the medical records of cases in which variants of the prototype strain, HRV-QPM, had been detected. METHODOLOGY/PRINCIPLE FINDINGS: Genomic analyses revealed that the six divergent strains, residing within a clade we previously called HRV A2, had the shortest polyprotein of all picornaviruses investigated. Structure-based amino acid alignments identified conserved motifs shared among members of the genus Rhinovirus as well as substantive deletions and insertions unique to the divergent strains. Deletions mostly affected regions encoding proteins traditionally involved in antigenicity and serving as HRV and HEV receptor footprints. Because the HRV A2 strains cannot yet be cultured, we created homology models of predicted HRV-QPM structural proteins. In silico comparisons confirmed that HRV-QPM was most closely related to the major group HRVs. HRV-QPM was most frequently detected in infants with expiratory wheezing or persistent cough who had been admitted to hospital and required supplemental oxygen. It was the only virus detected in 65% of positive individuals. These observations contributed to an objective clinical impact ranging from mild to severe. CONCLUSIONS: The divergent strains did not meet classification requirements for any existing species of the genus Rhinovirus or Enterovirus. HRV A2 strains should be partitioned into at least one new species, putatively called Human rhinovirus C, populated by members detected with high frequency, from individuals with respiratory symptoms requiring hospital admission.

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.

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.

Frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections.

Viruses are the major cause of pediatric acute respiratory tract infection (ARTI) and yet many suspected cases of infection remain uncharacterized. We employed 17 PCR assays and retrospectively screened 315 specimens selected by season from a predominantly pediatric hospital-based population. Before the Brisbane respiratory virus research study commenced, one or more predominantly viral pathogens had been detected in 15.2% (n = 48) of all specimens. The Brisbane study made an additional 206 viral detections, resulting in the identification of a microbe in 67.0% of specimens. After our study, the majority of microbes detected were RNA viruses (89.9%). Overall, human rhinoviruses (HRVs) were the most frequently identified target (n = 140) followed by human adenoviruses (HAdVs; n = 25), human metapneumovirus (HMPV; n = 18), human bocavirus (HBoV; n = 15), human respiratory syncytial virus (HRSV; n = 12), human coronaviruses (HCoVs; n = 11), and human herpesvirus-6 (n = 11). HRVs were the sole microbe detected in 37.8% (n = 31) of patients with suspected lower respiratory tract infection (LRTI). Genotyping of the HRV VP4/VP2 region resulted in a proposed subdivision of HRV type A into sublineages A1 and A2. Most of the genotyped HAdV strains were found to be type C. This study describes the high microbial burden imposed by HRVs, HMPV, HRSV, HCoVs, and the newly identified virus, HBoV on a predominantly paediatric hospital population with suspected acute respiratory tract infections and proposes a new formulation of viral targets for future diagnostic research studies.

Detection and discrimination of herpes simplex viruses, Haemophilus ducreyi, Treponema pallidum, and Calymmatobacterium (Klebsiella) granulomatis from genital ulcers.

BACKGROUND: Genital ulcer disease (GUD) is commonly caused by pathogens for which suitable therapies exist, but clinical and laboratory diagnoses may be problematic. This collaborative project was undertaken to address the need for a rapid, economical, and sensitive approach to the detection and diagnosis of GUD using noninvasive techniques to sample genital ulcers. METHODS: The genital ulcer disease multiplex polymerase chain reaction (GUMP) was developed as an inhouse nucleic acid amplification technique targeting serious causes of GUD, namely, herpes simplex viruses (HSVs), H. ducreyi, Treponema pallidum, and Klebsiella species. In addition, the GUMP assay included an endogenous internal control. Amplification products from GUMP were detected by enzyme linked amplicon hybridization assay (ELAHA). RESULTS: GUMP-ELAHA was sensitive and specific in detecting a target microbe in 34.3% of specimens, including 1 detection of HSV-1, three detections of HSV-2, and 18 detections of T. pallidum. No H. ducreyi has been detected in Australia since 1998, and none was detected here. No Calymmatobacterium (Klebsiella) granulomatis was detected in the study, but there were 3 detections during ongoing diagnostic use of GUMP-ELAHA in 2004 and 2005. The presence of C. granulomatis was confirmed by restriction enzyme digestion and nucleotide sequencing of the 16S rRNA gene for phylogenetic analysis. CONCLUSIONS: GUMP-ELAHA permitted comprehensive detection of common and rare causes of GUD and incorporated noninvasive sampling techniques. Data obtained by using GUMP-ELAHA will aid specific treatment of GUD and better define the prevalence of each microbe among at-risk populations with a view to the eradication of chancroid and donovanosis in Australia.

A sensitive, specific, and cost-effective multiplex reverse transcriptase-PCR assay for the detection of seven common respiratory viruses in respiratory samples.

Cell culture and direct fluorescent antibody (DFA) assays have been traditionally used for the laboratory diagnosis of respiratory viral infections. Multiplex reverse transcriptase polymerase chain reaction (m-RT-PCR) is a sensitive, specific, and rapid method for detecting several DNA and RNA viruses in a single specimen. We developed a m-RT-PCR assay that utilizes multiple virus-specific primer pairs in a single reaction mix combined with an enzyme-linked amplicon hybridization assay (ELAHA) using virus-specific probes targeting unique gene sequences for each virus. Using this m-RT-PCR-ELAHA, we examined the presence of seven respiratory viruses in 598 nasopharyngeal aspirate (NPA) samples from patients with suspected respiratory infection. The specificity of each assay was 100%. The sensitivity of the DFA was 79.7% and the combined DFA/culture amplified-DFA (CA-DFA) was 88.6% when compared to the m-RT-PCR-ELAHA. Of the 598 NPA specimens screened by m-RT-PCR-ELAHA, 3% were positive for adenovirus (ADV), 2% for influenza A (Flu A) virus, 0.3% for influenza B (Flu B) virus, 1% for parainfluenza type 1 virus (PIV1), 1% for parainfluenza type 2 virus (PIV2), 5.5% for parainfluenza type 3 virus (PIV3), and 21% for respiratory syncytial virus (RSV). The enhanced sensitivity, specificity, rapid result turnaround time and reduced expense of the m-RT-PCR-ELAHA compared to DFA and CA-DFA, suggests that this assay would be a significant improvement over traditional assays for the detection of respiratory viruses in a clinical laboratory.

Detection and differentiation of herpes simplex virus types 1 and 2 by a duplex LightCycler PCR that incorporates an internal control PCR reaction.

BACKGROUND: In recent years polymerase chain reaction (PCR) has proven to be a highly sensitive and specific method for the diagnosis of herpes simplex virus (HSV) infections. The advent of real-time HSV PCR protocols now enables rapid result turnaround times with minimal hands-on time. OBJECTIVES: In this study, we developed a real-time duplex PCR assay (HSVgD-dPCR) comprising of HSV and internal control PCR reactions. STUDY DESIGN: Using the LightCycler, the HSVgD-dPCR targeted the HSV glycoprotein D gene and HSV typing was performed by melting curve analysis. The internal control PCR reaction targeted sequences of the DNA of the human endogenous retrovirus (ERV-3). In total, 300 swab specimens, from patients with suspected HSV infection, were tested by the HSVgD-dPCR assay. The results were then compared to the results obtained by another HSV LightCycler assay, which utilized published primer and probe sequences targeting the HSV DNA polymerase gene (Dpol-HSV-LCPCR). RESULTS: Overall, 91 (30.3%) specimens were positive and 204 (68.0%) specimens were negative for HSV by both LightCycler assays. In addition, four (1.3%) specimens were positive by Dpol-HSV-LCPCR and negative by HSVgD-dPCR, whereas one (0.3%) specimen was positive by HSVgD-dPCR and negative by Dpol-HSV-LCPCR. The presence of HSV in these five specimens was confirmed by conventional PCR. Melting curve analysis by the HSVgD-dPCR assay enabled all HSV positive specimens to be typed, whereas sequence variation prevented three HSV positive specimens from being typed by the Dpol-HSV-LCPCR. Using the ERV-3 PCR, 5% specimens were found to contain inhibitory substances. CONCLUSIONS: By developing the HSVgD-dPCR we have enhanced the diagnostic utility of real-time detection of HSV by incorporating an internal control reaction and by accurately typing a greater proportion of HSV positive specimens.

Simultaneous detection and differentiation of human polyomaviruses JC and BK by a rapid and sensitive PCR-ELAHA assay and a survey of the JCV subtypes within an Australian population.

Human polyomaviruses JCV and BKV can cause several clinical manifestations in immunocompromised hosts, including progressive multifocal leukoencephalopathy (PML) and haemorrhagic cystitis. Molecular detection by polymerase chain reaction (PCR) is recognised as a sensitive and specific method for detecting human polyomaviruses in clinical samples. In this study, we developed a PCR assay using a single primer pair to amplify a segment of the VP1 gene of JCV and BKV. An enzyme linked amplicon hybridisation assay (ELAHA) using species-specific biotinylated oligonucleotide probes was used to differentiate between JCV and BKV. This assay (VP1-PCR-ELAHA) was evaluated and compared to a PCR assay targeting the human polyomavirus T antigen gene (pol-PCR). DNA sequencing was used to confirm the polyomavirus species identified by the VP1-PCR-ELAHA and to determine the subtype of each JCV isolate. A total of 297 urine specimens were tested and human polyomavirus was detected in 105 specimens (35.4%) by both PCR assays. The differentiation of JCV and BKV by the VP1-PCR-ELAHA showed good agreement with the results of DNA sequencing. Further, DNA sequencing of the JCV positive specimens showed the most prevalent JCV subtype in our cohort was 2a (27%) followed by 1b (20%), 1a (15%), 2c (14%), 4 (14%) and 2b (10%). The results of this study show that the VP1-PCR-ELAHA is a sensitive, specific and rapid method for detecting and differentiating human polyomaviruses JC and BK and is highly suitable for routine use in the clinical laboratory.