[Show abstract][Hide abstract] ABSTRACT: Objective To compare the use of a generic molecular assay to 'standard' investigations used to assist the diagnosis of late onset bacterial sepsis in very low birth weight infants (VLBW, <1500g). Methods VLBW infants, greater than 48 hours of age, who were clinically suspected to have sepsis were investigated using standard tests (full blood count, C-reactive protein (at presentation) and blood culture), in addition, blood was taken for a universal molecular assay (16S rRNA reverse transcriptase PCR) for comparison. Clinical data were recorded during the suspected infection episode. A validated sepsis score (NEO-KISS) was used to retrospectively determine the presence of sepsis (independent of blood culture). The performance of each of the tests were compared by sensitivity, specificity, positive/negative likihood ratios (+/-LR) and postive/negative predictive values (PPV/NPV).
PLoS ONE 08/2015; 10(8):e0136472. DOI:10.1371/journal.pone.0136472 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Gardnerella vaginalis is a Gram variable anaerobic bacterium present in 100% of women with bacterial vaginosis (BV). BV is a complex polymicrobial condition with no single causative agent. The current laboratory detection method for BV relies on a Gram stain Nugent score to estimate the quantity of different bacterial morphotypes in the vaginal micro-flora. While the Nugent score can distinguish between women with and without BV, a significant proportion categorise as intermediate, which fails to differentiate a normal from an abnormal vaginal micro-flora. A singleplex G.vaginalis TaqMan® real-time quantitative Polymerase Chain Reaction (qPCR) assay was developed compared against the 'gold standard' Nugent score. Detection and quantification of G.vaginalis was performed on vaginal specimens with positive, negative and intermediate Nugent scores. The G.vaginalis assay demonstrated high analytical specificity against a broad microbial panel and analytical sensitivity down to 3.1x104 copies per ml. There was a significantly higher G.vaginalis load in women with BV compared to intermediate and non-BV women (p value=5.1x10-14). All Nugent scores in keeping with BV had qPCR loads of ≥ 107 copies per ml. Amongst the 24 undefined women (11.8%) in the study with an intermediate flora, 14 (58.3%) had a G.vaginalis load ≥107 copies per ml. In this study a threshold of 107 copies per ml had positive and negative predictive values of 57.1% and 100% for BV; the high qPCR loads amongst the Intermediate Nugent scores suggest the need for a new approach in classifying BV and the potential for qPCR to play a role.
Journal of Medical Microbiology 06/2015; DOI:10.1099/jmm.0.000118 · 2.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background: Approximately 5-6% of all infective episodes in neonatal intensive care unit (NICU) are of viral origin. Previous studies suggest that human parechovirus (HPeV) infection presents most commonly in term infants, as a sepsis-like syndrome in which meningoencephalitis is prominent. Our aim was to study the infection rate and associated features of HPeV. Methods: Blood samples were taken from NICU babies older than 48 hours, who were being investigated for late onset sepsis. Clinical and laboratory data were collected at the time of the suspected sepsis episode. Samples were tested using universal primers and probe directed at the 5'-untranslated region of the HPeV genome by reverse transcriptase polymerase chain reaction (RT-PCR). Results were confirmed by electrophoresis and DNA sequencing. Results: HPeV was detected in 11 of 84 samples (13%). These infants had a mean [interquartile range (IQR)] gestational age of 28.9 (26.9-30.6) weeks and mean birth weight of 1.26 (SD = 0.72) kg. The median day of presentation was 16 (IQR: 11-27). These characteristics were similar to the infants without positive viral detection. Six infants presented with respiratory signs. One infant presented with signs of meningitis. Six of the 11 episodes of HPeV infection occurred during the winter months (December to February). No HPeV positive infants had abnormal findings on their 28-day cranial ultrasound examination. Conclusions: We found an HPeV infection rate of 13% in infants being tested for late onset sepsis. HPeV should be considered as a possible cause of sepsis-like symptoms in preterm infants.
[Show abstract][Hide abstract] ABSTRACT: Norovirus (NoV) gastroenteritis occurs in all age groups and is the most common cause of gastroenteritis in the community. However, detection methods and rates vary widely, and few data are available to compare these, particularly in Ireland. Detection of noroviruses through antigen and molecular-based strategies was carried out on 135 suspected NoV-positive samples, collected over the course of three NoV outbreaks, from 2002 to 2006, in the southern region of Ireland. A commercially available ELISA and a panel of six primer sets were evaluated to determine their suitability for NoV detection in Irish clinical samples. The key findings of this study were the detection of both GGI and GGII noroviruses by ELISA, but the detection of only GGII noroviruses by RT-PCR. In addition to this, a variation in the levels of detection from 9.4 % to 17.3 % was observed for conventional PCR assays, while a detection rate of 46.3 % was observed for the real-time PCR assay. A proportion (17.8 %) of samples were found to be negative by all detection strategies, suggesting the possibility of reporting false positives for these samples or low-copy positives that do not often repeat. Sequencing information from selected samples also revealed nucleotide polymorphisms, compromising efficient primer binding in the case of one primer pairing. Phylogenetic analysis of the partial polymerase gene identified NoV GII.4 as the dominant genotype, in accordance with previous NoV studies in Ireland. Investigating the NoV diversity of the circulating strains and the dynamics of strain replacement is important to better assess the efficacy of future NoV vaccines and to facilitate the early detection of changes in circulating NoV strains.
Archives of Virology 01/2014; 159(7). DOI:10.1007/s00705-014-1987-5 · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Respiratory syncytial virus (RSV) is a major pathogen that primarily infects airway epithelium. Most infants suffer mild upper respiratory tract (URT) symptoms, while approximately one third progress to lower respiratory tract (LRT) involvement. Despite the ubiquity of URT infection, little is known about the relative cytopathogenesis of RSV infection in infant URT and LRT.
This study aimed to compare RSV cytopathogenesis in nasal- and bronchial-derived epithelium from the same individuals using novel models derived from well-differentiated primary pediatric nasal (WD-PNECs) and bronchial epithelial cells (WD-PBECs).
WD-PNECs and WD-PBECs were generated from nasal and bronchial brushes, respectively, and mock-infected or infected with RSV BT2a. RSV tropism, infectivity, cytopathology, growth kinetics, cell sloughing, apoptosis, and cytokine/chemokine responses were determined.
RSV infection in both cultures was restricted to apical ciliated cells and occasional non-ciliated cells but not goblet cells. It did not cause obvious cytopathology. Infection resulted in apical release of progeny virus, increased apical cell sloughing, apoptosis and occasional syncytia. RSV growth kinetics and peak titers were higher in WD-PBECs, coincident with higher ciliated cell contents, cell sloughing and slightly compromised tight junctions. However, pro-inflammatory chemokine responses were similar for both cultures. Also, lambda interferons, especially IL-29, were induced by RSV infection.
RSV induced remarkably similar, albeit quantitatively lower, cytopathogenesis and pro-inflammatory responses in WD-PNECs compared to WD-PBECs that reproduce many hallmarks of RSV pathogenesis in infants. WD-PNECs may provide an authentic surrogate model with which to study RSV cytopathogenesis in infant airway epithelium.
American Journal of Respiratory and Critical Care Medicine 08/2013; 188(7). DOI:10.1164/rccm.201304-0750OC · 11.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: P jirovecii causes Pneumocystis pneumonia (PCP), a severe opportunistic infection in immunosuppressed patients with both person to person airborne transmission and environmental transmission important routes of infection. An increasing incidence of P. jirovecii in Northern Ireland prompted a detailed epidemiological and molecular review including enhanced surveillance on all lower respiratory specimens. Genotyping of these P. jirovecii positive specimens was undertaken using Multiple Locus Sequence Typing (MLST) targeting known variable regions of the P. jirovecii genome (Hauser et al., 1997). Multiple circulating types were found amongst all patient categories. However a predominance of one MLST type was found in a P. jirovecii outbreak amongst the renal transplant population. Our results demonstrate the diversity of P. jirovecii strains among the local immunosuppressed cohort and highlight the importance of genotyping in the investigation of common sources of P. jirovecii amongst immunosuppressed patients.
Journal of Medical Microbiology 05/2013; 62(Pt_8). DOI:10.1099/jmm.0.057794-0 · 2.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: ABSTRACT BACKGROUND: Paper-based diaries and self-report of symptom worsening in COPD studies may lead to under-detection of exacerbations. Epidemiologically, COPD exacerbations exhibit seasonal patterns peaking at year-end. We examined whether use of a BlackBerry-based daily symptom diary would detect 95% or more of exacerbations and enable characterization of seasonal differences between them. METHODS: Fifty participants with GOLD stage l to lV COPD began a community-based study in December 2007. Another 30 began in December 2008. Participants transmitted daily symptom diaries using a BlackBerry. Alerts were triggered when symptom changes, missed diary transmissions or medical care for a respiratory problem occurred. Participant encounters were initiated if COPD exacerbations were suspected. Participants reported returns to normal breathing using their BlackBerry. RESULTS: Participants transmitted 99.9% of 28,514 possible daily diaries. All 191 (2.5/participant-year) COPD exacerbations meeting Anthonisen criteria were detected. During 148/191 exacerbations (78%; 1.97/participant-year) patients were hospitalized and/or ordered prednisone, an antibiotic or both. Respiratory viruses were detected in 78/191 (41%) of exacerbations. Those coinciding with a respiratory viral infection averaged 12.0 days, those without averaged 8.9 days (P <.04), with no difference in Anthonisen score. Respiratory symptom scores before exacerbations and after normal breathing return showed no differences. Exacerbations were more frequent during the Christmas period than the rest of the year but not than the rest of winter alone. CONCLUSIONS: Smartphone-based collection of COPD symptom diaries enables near complete identification of exacerbations at inception. Exacerbation rates in the Christmas season do not reach levels that necessitate changes in disease management.
[Show abstract][Hide abstract] ABSTRACT: In Europe fetal loss due to parvovirus B19 (B19V) is under-reported and a poorly addressed occupational risk to pregnant women. This is exemplified internationally, where it was unmentioned in the last 2 ECDC annual surveillance reports or its 2009 special report on infections in pregnancy. To assess this potential for under estimating B19V fetal loss in pregnancy, we undertook a systematic review of practice in Northern Ireland in the management and reporting of B19V infections over a 12 month period of heightened transmission, 1 of 6 observed in a span of 9 years. Pregnant and non-pregnant women presented with symptomatic infection in 24% and 93% of confirmed B19V infections respectively, with no difference in viral loads. There was under investigation of viral causes of fetal loss, with only 143/2739 (5%) tested for B19V, and a failure to follow-up most non-immune women tested following rash contact. Occupational exposure was recorded in 31/60 (51.6%) pregnancies audited following rash exposure, the majority teachers or day care workers. Against a background seroprevalence of 66.5% immunity in women of child-bearing years, two patterns of infection were identified. Firstly, pregnant women investigated for a rash or exposure to slapped cheek syndrome, where an infection incidence of 18% was observed, resulted in 42 confirmed infections, all proceeding to healthy term deliveries. Secondly, pregnant women with unsuspected infection had 6 cases of confirmed B19V fetal loss, including 4 of 22 (18%) diagnosed at autopsy, of which 3 were non-hydropic. While many studies have reported B19V fetal loss in pregnancy, there are no robust public health surveillance figures to draw on. That all 6 confirmed fetal losses came from the small number of miscarriages / stillbirths investigated, 143 out of 2739, suggests inadequate follow-up of those pregnancies where B19V related fetal loss may be most common and supports the need for enhanced surveillance pilots to address this significant gap in public health knowledge.
Journal of Medical Microbiology 09/2012; 62(Pt_1). DOI:10.1099/jmm.0.046714-0 · 2.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nucleic acid amplification methods such as the PCR have had a major impact on the diagnosis of viral infections, often achieving greater sensitivities and shorter turnaround times than conventional assays and an ability to detect viruses refractory to conventional isolation methods. Their effectiveness is, however, significantly influenced by assay target sequence variability due to natural diversity and rapid sequence changes in viruses that prevent effective binding of primers and probes. This was investigated for a diverse range of enteroviruses (EVs; species A to D), human rhinoviruses (HRVs; species A to C), and human parechovirus (HPeV) in a multicenter assay evaluation using a series of full-length prequantified RNA transcripts. RNA concentrations were quantified by absorption (NanoDrop) and fluorescence methods (RiboGreen) prior to dilution in buffer supplemented with RNase inhibitors and carrier RNA. RNA transcripts were extremely stable, showing minimal degradation after prolonged storage at temperatures between ambient and -20°C and after multiple freeze-thaw cycles. Transcript dilutions distributed to six referral laboratories were screened by real-time reverse transcriptase PCR assays using different primers and probes. All of the laboratories reported high assay sensitivities for EV and HPeV transcripts approaching single copies and similar amplification kinetics for all four EV species. HRV detection sensitivities were more variable, often with substantially impaired detection of HRV species C. This could be accounted for in part by the placement of primers and probes to genetically variable target regions. Transcripts developed in this study provide reagents for the ongoing development of effective diagnostics that accommodate increasing knowledge of genetic heterogeneity of diagnostic targets.
[Show abstract][Hide abstract] ABSTRACT: Against a background of point-source outbreaks of Pneumocystis pneumonia (PCP) in renal transplant units in Europe, we undertook a retrospective 3 year observational review of PCP in Northern Ireland. This showed an unexpected increase in incidence, with a mortality rate of 30 %. Fifty-one cases were confirmed compared to 10 cases confirmed in the preceding 7 years. Where undiagnosed HIV infection had previously been the main risk factor for PCP, this was now equally matched by chemotherapy for haematological and non-haematological malignancy and immune suppression for a range of autoimmune conditions. Congenital immunodeficiency and transplantation were less common predisposing factors, but renal grafts also showed a rising incidence. Asymptomatic carriage was uncommon. At presentation both upper and lower respiratory samples were of equal use in establishing the diagnosis, and treatment resulted in rapid clearance. These data suggest the need for considering PCP in at-risk patients, reviewing its mode of acquisition and whether iatrogenic colonization is a treatable pre-condition.
Journal of Medical Microbiology 04/2012; 61(Pt 7):1009-15. DOI:10.1099/jmm.0.043984-0 · 2.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Respiratory syncytial virus (RSV) is the major viral cause of severe pulmonary disease in young infants worldwide. However, the mechanisms by which RSV causes disease in humans remain poorly understood. To help bridge this gap, we developed an ex vivo/in vitro model of RSV infection based on well-differentiated primary pediatric bronchial epithelial cells (WD-PBECs), the primary targets of RSV infection in vivo. Our RSV/WD-PBEC model demonstrated remarkable similarities to hallmarks of RSV infection in infant lungs. These hallmarks included restriction of infection to noncontiguous or small clumps of apical ciliated and occasional nonciliated epithelial cells, apoptosis and sloughing of apical epithelial cells, occasional syncytium formation, goblet cell hyperplasia/metaplasia, and mucus hypersecretion. RSV was shed exclusively from the apical surface at titers consistent with those in airway aspirates from hospitalized infants. Furthermore, secretion of proinflammatory chemokines such as CXCL10, CCL5, IL-6, and CXCL8 reflected those chemokines present in airway aspirates. Interestingly, a recent RSV clinical isolate induced more cytopathogenesis than the prototypic A2 strain. Our findings indicate that this RSV/WD-PBEC model provides an authentic surrogate for RSV infection of airway epithelium in vivo. As such, this model may provide insights into RSV pathogenesis in humans that ultimately lead to successful RSV vaccines or therapeutics.
Proceedings of the National Academy of Sciences 03/2012; 109(13):5040-5. DOI:10.1073/pnas.1110203109 · 9.81 Impact Factor