In T2‐mediated severe asthma, biologic therapies, such as mepolizumab, are increasingly used to control disease. Current biomarkers can indicate adequate suppression of T2 inflammation, but it is unclear whether they provide information about airway microbial composition. We investigated the relationships between current T2 biomarkers and microbial profiles, characteristics associated with a ProteobacteriaHIGH microbial profile and the effects of mepolizumab on airway ecology.

Background Clinical trials with mepolizumab, a humanised monoclonal antibody against interleukin-5, show a 50% reduction in severe asthma exacerbations in people with severe eosinophilic asthma. Exacerbations in patients treated with mepolizumab seem to be different to exacerbations in those given placebo, as patients treated with mepolizumab report fewer symptoms, have a lower sputum eosinophil count, and smaller fall in peak expiratory flow. We aimed to investigate the inflammatory phenotype and physiological characteristics of exacerbation events in patients with severe eosinophilic asthma who were treated with mepolizumab. Methods This multicentre, prospective, observational cohort study was carried out at four UK specialist severe asthma centres. Participants were aged 18–80 years, with severe eosinophilic asthma (Global Initiative for Asthma steps 4 and 5), and were eligible for mepolizumab therapy. All participants received mepolizumab 100 mg subcutaneously every 4 weeks, had a scheduled study visit when stable on mepolizumab (≥3 months on treatment), and measured daily peak flow and completed symptoms diaries throughout the course of the study. Participants attended their study centre for unscheduled exacerbation assessment when symptoms worsened outside of their normal daily variation and before commencing rescue treatment. If a participant was unable to attend their study centre for exacerbation or had initiated rescue treatment before the study visit, clinical details of the missed exacerbation were collected by clinical staff. In this exploratory study, the endpoint was 100 clinical assessments at exacerbation completed across all sites for participants on mepolizumab before initiation of rescue treatment. Characteristics of those who had exacerbations on mepolizumab were compared with those who did not, peak flow and symptoms diaries were compared for assessed versus missed exacerbations, and exacerbation phenotypes defined by sputum eosinophil cell count were compared. The utility of fractional exhaled nitric oxide (FeNO) and C-reactive protein in determining exacerbation phenotype on mepolizumab treatment were also assessed. This study is registered with ClinicalTrials.gov, NCT03324230. Findings Between Nov 30, 2017, and May 29, 2019, 145 participants were enrolled and treated with mepolizumab, five were excluded from the analysis. 172 exacerbations occurred, with 96 (56%) assessed before commencing rescue treatment. Compared with patients who did not exacerbate, patients who exacerbated had a higher exacerbation rate and more emergency department attendances in the year before commencing mepolizumab. The change in peak expiratory flow at nadir in the assessed exacerbation group was mean −40·5 L/min (SD 76·3) versus mean −37·0 L/min (93·0; p=0·84) in the missed exacerbation group, and there was no difference in reported symptom burden. When comparing exacerbations with a high sputum eosinophil count (≥2%; SEHIGH) with exacerbations with a low sputum eosinophil count (<2%; SELOW), the SEHIGH exacerbations were FeNO high (median difference 33 parts per billion [ppb; 95% CI 8 to 87]; p=0·0004), with lower FEV1 percent predicted (mean difference −15·9% [–27·0 to −4·8]; p=0·0075), lower FEV1 to forced vital capacity ratio (mean difference −10·3 [–17·0 to −3·6]; p=0·0043), and higher blood eosinophil counts (median difference 40 cells per μL [20 to 70]; p=0·0009). By contrast, SELOW exacerbations had higher C-reactive protein concentrations (median difference 12·7 mg/L [3·5 to 18·5]; p<0·0001), higher sputum neutrophil counts (median difference 52·7% [34·5 to 59·2]; p<0·0001), and were more likely to be treated with antibiotics (p=0·031). FeNO (≤20 or ≥50 ppb) was the most useful discriminator of inflammatory phenotype at exacerbation. The most common adverse event was hospital admission due to asthma exacerbation (17 [50%] of 34 events), none of the adverse events were study procedure related. Interpretation Exacerbations on mepolizumab are two distinct entities, which can largely be differentiated using FeNO: non-eosinophilic events are driven by infection with a low FeNO and high C-reactive protein concentration, whereas eosinophilic exacerbations are FeNO high. The results of the MEX study challenge the routine use of oral corticosteroids for the treatment of all asthma exacerbation events on mepolizumab, as well as the switching of biological therapies for treatment failure without profiling the inflammatory phenotype of ongoing asthma exacerbations. The results highlight clinically available tools to enable profiling of these residual exacerbations in patients treated with mepolizumab. Funding UK Medical Research council.

Background: Clinical trials with anti-IL5 therapies show a 50% reduction in severe asthma exacerbations; exacerbations on mepolizumab appear different from placebo questioning their inflammatory phenotype and physiological characteristics. Methods: Observational study in mepolizumab treated patients (n=145) at 4 UK Severe Asthma Specialist clinics. Patients attended study centre for exacerbation assessment pre-treatment. Results: 172 exacerbations with 96 assessed pre-treatment; peak flow & symptoms diaries showed no difference in assessed & missed exacerbations. At initial exacerbation/participant, 45/69 (65%) produced sputum of whom 47% were sputum eosinophil (SE) high ≥2% & 53% were SE low <2%. SE≥2% were FeNO high (57[30,111] vs 24[16,45] ppb, p<0.001), had low FEV1% predicted (56(15) vs 72(21), p=0.008), obstructive spirometry (FEV1/FVC% 58(14) vs 68(9), p=0.004), higher blood eosinophils (70[50,90] vs 30[10,50]cells/µL, p<0.001) and median SE 10%[5,21] vs 0.4%[0.2, 0.8], p<0.001. In contrast, SE<2% exacerbations were FeNO low, CRP high (15[5,24] vs 2.3[2,5] mg/L, p <0.001), with sputum neutrophilia (90%[72, 95] vs 37%[29,54], p<0.001) and 50% receiving antibiotics (v 19% in SE≥2%, p0.03). FeNO (<20 or ≥50ppb) was a useful discriminator. Conclusion: Exacerbations on mepolizumab are 2 distinct entities; non-eosinophilic events are driven by infection & FeNO low, while eosinophilic exacerbations can be differentiated by high FENO.

Background Mycoplasma genitalium (MG) can cause urethritis in men and pelvic inflammatory disease in women. Despite the increasing rate of MG resistance to first-line macrolide treatment due to 23S rRNA gene mutations, the availability of testing remains limited in UK laboratories. This study obtained preliminary data on the rate of macrolide resistant strains circulating in N. Ireland. Methods Clinical specimens from 1052 patients attending a Genitourinary Medicine (GUM) clinic in Belfast from April 2017 to April 2018 were screened using an in-house MG PCR assay. MG positive samples were subsequently tested using Resistance Plus™ MG assay (Speedx, Australia) to detect 23S rRNA gene mutations associated with macrolide resistance. Results Amongst all study samples, 3.9 % (n=41) tested positive for MG. Out of these 41.5 % (n=17) had a 23S rRNA mutation. The rate of MG was highest in rectal swabs (8.13 %) followed by vaginal swabs (4.96 %) and then male urines (2.85 %). The rate of 23 S rRNA mutations associated with macrolide resistance in positive vaginal swabs was 21.4 % whereas the rate amongst urine and rectal swabs was at least 50 %. Conclusion Data from this study adds to the evidence base to perform MG testing in risk groups in N. Ireland to improve patient outcomes and reduce antimicrobial resistance.

Enterovirus D68 (EV-D68) detection has recently been associated with severe neurological symptoms in adults and children in European countries. The spectrum of disease by EV-D68 ranges from asymptomatic to acute respiratory symptoms, hospitalisation, and sporadically to neurological symptoms, including acute flaccid paralysis (AFP) and acute flaccid myelitis (AFM), and death. This has led to increased vigilance for detection of enterovirus D68, especially in cases that present with the more severe clinical syndromes. In normal cases, enterovirus is transmitted by faecal-oral routes and/or respiratory routes, however in the case of EV-D68 it is almost exclusively passed through respiratory transmission. An infection of EV-D68 is generally distinguished by its rapid onset of disease. The incubation period of the disease between 3 to 5 days, which is unlike the other enteroviruses which usually have an incubation period of around 10 days. This study describes a retrospective analysis of 150 non-invasive respiratory specimens (nose/throat or throat swabs) collected from paediatric outpatients presenting to Childrens Hospital Emergency Department with respiratory and/or CNS symptoms including fever and seizure. Specimens have been collected from November 2017- November 2018. All specimens tested by four RT-PCR assays for Pan-Enterovirus, EV-D68 specific, human Rhinovirus and RNaseP (quality control). Results on prevalence of EV-D68, as single or co-infection, are presented. Results indicate low prevalence of EV-D68 in the Northern Ireland symptomatic paediatric population. As EV-D68 is an emerging infection it is critical to remain vigilant particularly in the case of neurological presentation.

Galactokinase catalyses the ATP-dependent phosphorylation of galactose. A galactokinase-like sequence was identified in a Fasciola hepatica EST library. Recombinant expression of the corresponding protein in Escherichia coli resulted in a protein of approximately 50 kDa. The protein is monomeric, like galactokinases from higher animals, yeasts and some bacteria. The protein has no detectable enzymatic activity with galactose or N-acetylgalactosamine as a substrate. However, it does bind to ATP. Molecular modelling predicted that the protein adopts a similar fold to galactokinase and other GHMP kinases. However, a key loop in the active site was identified which may influence the lack of activity. Sequence analysis strongly suggested that this protein (and other proteins annotated as “galactokinase” in the trematodes Schistosoma mansoni and Clonorchis sinensis) are closer to N-acetylgalactosamine kinases. No other galactokinase-like sequences appear to be present in the genomes of these three species. This raises the intriguing possibility that these (and possibly other) trematodes are unable to catabolise galactose through the Leloir pathway due to the lack of a functional galactokinase.

Purpose. Neonatal sepsis caused by Streptococcus agalactiae [group B streptococcus (GBS)] is a life-threatening condition, which is preventable if colonized mothers are identified and given antibiotic prophylaxis during labour. Conventional culture is time consuming and unreliable, and many available non-culture diagnostics are too complex to implement routinely at point of care. Loop-mediated isothermal amplification (LAMP) is a method that, enables the rapid and specific detection of target nucleic acid sequences in clinical materials without the requirement for extensive sample preparation.Methodology. A prototype LAMP assay targeting GBS sip gene is described.Results. The assay was 100% specific for GBS, with a limit of detection of 14 genome copies per reaction. The clinical utility of the LAMP assay for rapid direct molecular detection of GBS was determined by testing a total of 157 vaginal swabs with minimal sample processing using a rapid lysis solution. Compared to a reference quantitative real-time PCR assay, the direct LAMP protocol had a sensitivity and specificity of 95.4 and 100 %, respectively, with positive and negative predictive values of 100 and 98.3 %, respectively. Positive and negative likelihood ratios were infinity and 0.05, respectively. The direct LAMP method required a mean time of 45 min from the receipt of a swab to generation of a confirmed result, compared to 2 h 30 min for the reference quantitative real-time PCR test.Conclusion. The direct LAMP protocol described is easy to perform, facilitating rapid and accurate detection of GBS in vaginal swabs. This test has a potential for use at point of care.

The numbers of rectal sexually transmitted infections are on the rise especially among men who have sex with men. Males from men who have sex with men population are encouraged to send a rectal swab to the laboratory for sexually transmitted infection screening at their visit to the Genitourinary Medicine Clinic. In healthy asymptomatic males, the range of pathogens tested is limited therefore other pathogens may be left untreated allowing infections to persist among sexual partners. Molecular techniques have revolutionarised sexually transmitted infection testing enabling the detection of previously difficult-to-culture pathogens in extra-genital sites and have increased the evidence base for their clinical significance. The present study tests 107 rectal swabs from men who have sex with men negative for Chlamydia trachomatis and Neisseria gonorrhoeae against quantitative polymerase chain reaction (qPCR) assays targeting five common sexually transmitted bacteria which include Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma urealyticum, Ureaplasma parvum and Gardnerella vaginalis. The pathogenic role of these five bacteria in men who have sex with men is currently unknown. Amongst the 107 patients, a positive qPCR was obtained respectively for G. vaginalis 89 (83.2%); U. urealyticum 26 (24.3%); M. hominis 26 (24.3%); M. genitalium 10 (9.3%) and U. parvum 5 (4.7%). Bacterial loads in single and co-infections were compared for each organism. G. vaginalis and M. hominis loads were significantly ( p = 0.007 and p = 0.005, respectively) higher when co-infecting with at least one other organism. Amongst co-infections, the loads of each organism were assessed to determine possible synergies. G. vaginalis and M. hominis displayed a synergistic pattern ( r = 0.51; p = 0.02) which is in keeping with a similar synergy detected previously in the vagina of women with bacterial vaginosis. This study outlines that potential significant infections are being missed in men who have sex with men population; however, further research is warranted to confirm a pathogenesis in the rectal mucosa before routine screening can be introduced to clinical settings.

Gardnerella vaginalis plays an important role in bacterial vaginosis (BV,) while the role of genital Mollicutes is less obvious. The diagnosis of BV by use of the current Gram stain Nugent score is also suboptimal for defining the role of Mollicutes that lack a cell wall. Since bacterial load and diversity is an important prerequisite for BV, real-time quantitative polymerase chain reaction (qPCR) assays enable these to be assessed. The purpose of this study was to define the role of genital Mollicutes and potential patterns of synergy with G. vaginalis in women with BV. Vaginal swabs from 130 women categorised by Nugent score as BV (n = 28), intermediate (n = 22) and non-BV (n = 80) were tested against four qPCR TaqMan assays targeting G. vaginalis, Mycoplasma hominis, M. genitalium, Ureaplasma urealyticum and U. parvum. Statistical analyses were used to compare bacterial prevalence and load between the three groups of women. Mycoplasma hominis and G. vaginalis co-infection was significantly more common in BV (60.7 %) compared to intermediate (36.4 %) and non-BV (8.8 %) Nugent scores (p < 0.001). Significantly higher loads of M. hominis (p = 0.001) and G. vaginalis (p < 0.001) were detected in women with BV and the respective loads in M. hominis and G. vaginalis co-infections displayed a significant positive correlation (p < 0.001; r = 0.60). No significant associations were seen with the other Mollicutes. The findings strengthen the evidence of a role for M. hominis in BV and a potential synergy with G. vaginalis. This synergy could be an important trigger of the condition and sexual contact the conduit for the transmission of an otherwise commensal bacterium that could initiate it.

OBJECTIVE:To assess the association of vaginal commensal and low-grade pathogenic bacteria including Ureaplasma parvum, Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium, Group B streptococcus (GBS), and Gardnerella vaginalis, in women who delivered preterm at less than 37-week gestation in the presence or absence of inflammation of the chorioamnionitic membranes. METHODS:A case control study involving women who delivered before 37-week gestation with and without inflammation of chorioamnionitic membranes. A total of 57 placental samples were histologically examined for polymorphonuclear leukocyte infiltration of placental tissue for evidence of chorioamnionitis, and by type-specific nucleic acid amplification for evidence of infection with one or more of the target bacteria. Demographic data were collected for each mother. RESULTS:Among the 57 placental samples, 42.1% had chorioamnionitis and 24.6% delivered in the second trimester of pregnancy; U. parvum, U. urealyticum, G. vaginalis, and GBS were all detected in the study with respective prevalence of 19.3%, 3.5%, 17.5%, and 15.8%; M. genitalium and M. hominis were not detected. U. parvum was significantly associated with chorioamnionitis (p = 0.02; OR 5.0; (95% CI 1.2-21.5) and was more common in women who delivered in the second (35.7%) compared to the third trimester of pregnancy (13.9%). None of the other bacteria were associated with chorioamnionitis or earlier delivery, and all G. vaginalis-positive women delivered in the third trimester of pregnancy (p = 0.04). CONCLUSIONS:The detection of U. parvum in placental tissue was significantly associated with acute chorioamnionitis in women presenting in extreme preterm labor.

Sub-optimal recovery of bacterial DNA from whole blood samples can limit the sensitivity of molecular assays to detect pathogenic bacteria. We compared 3 different pre-lysis protocols (none, mechanical pre-lysis and achromopeptidase pre-lysis) and 5 commercially available DNA extraction platforms for direct detection of Group B Streptococcus (GBS) in spiked whole blood samples, without enrichment culture. DNA was extracted using the QIAamp Blood Mini kit (Qiagen), UCP Pathogen Mini kit (Qiagen), QuickGene DNA Whole Blood kit S (Fuji), Speed Xtract Nucleic Acid Kit 200 (Qiagen) and MagNA Pure Compact Nucleic Acid Isolation Kit I (Roche Diagnostics Corp). Mechanical pre-lysis increased yields of bacterial genomic DNA by 51.3 fold (95% confidence interval; 31.6–85.1, p<0.001) and pre-lysis with achromopeptidase by 6.1 fold (95% CI; 4.2–8.9, p<0.001), compared with no pre-lysis. Differences in yield due to pre-lysis were 2–3 fold larger than differences in yield between extraction methods. Including a pre-lysis step can improve the limits of detection of GBS using PCR or other molecular methods without need for culture.

Summary Inflammation of the urethra defined by an excess of polymorphonuclear leukocytes in the absence of sexually transmitted Chlamydia trachomatis and Neisseria gonorrhoeae is called non-chlamydial non-gonococcal urethritis (NCNGU). Although Mycoplasma genitalium is now recognised as causing a sexually transmitted infection, the clinical significance of the other Mollicute species is less clear. This study used specific real-time quantitative polymerase chain reaction assays to detect and quantify four Mollicute species, M. genitalium, M. hominis, Ureaplasma urealyticum and U. parvum, in urine specimens from men with and without NCNGU. A total of 165 urine specimens from male patients attending a genitourinary medicine clinic were eligible for the study, with microscopy-confirmed (≥5 polymorphonuclear leukocytes in urethral swab) NCNGU in 75 (45.5%) and non-confirmed NCNGU in 90 (54.5%). Chi-squared statistical analysis indicated a significantly higher prevalence of U. parvum (17.3% vs. 5.6%; p = 0.03) and M. genitalium (12% vs. 0%; p < 0.001) in NCNGU. In a subset analysis, M. genitalium was also significantly (p = 0.03) higher in men who have sex with men (MSM; 13.5%) compared to non-MSM (3.1%). No significant associations were reported for U. urealyticum and M. hominis. In conclusion, this study supports a clinically significant role in NGNCU for both U. parvum and M. genitalium.

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. Whilst the Nugent score can distinguish between women with and without BV, a significant proportion are categorized as intermediate, which fails to differentiate a normal from an abnormal vaginal micro flora. A singleplex G. vaginalis TaqMan real-time quantitative PCR (qPCR) assay was developed and compared with 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 qPCR assay demonstrated high analytical specificity against a broad microbial panel and analytical sensitivity down to 3.1 × 10(4) copies ml(-1). There was a significantly higher G. vaginalis load in women with BV compared with intermediate and non-BV women (P value = 5.1 × 10(-14)). All Nugent scores in keeping with BV had qPCR loads of ≥ 10(7) copies ml(-1). Among the 24 undefined women (11.8%) in the study with an intermediate flora, 14 (58.3%) had a G. vaginalis load of ≥ 10(7) copies ml(-1). In this study a threshold of 107 copies ml(-1) had positive and negative predictive values of 57.1 and 100% for BV; the high qPCR loads among the intermediate Nugent scores suggest the need for a new approach in classifying BV and the potential for qPCR to play a role.

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.