Relationship between inflammatory status and microbial composition in severe asthma and during exacerbation.

BACKGROUND: 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. METHODS: Microbiota sequencing was performed on sputum samples obtained at stable and exacerbation state from 140 subjects with severe asthma participating in two clinical trials. Inflammatory subgroups were compared on the basis of biomarkers, including FeNO and sputum and blood eosinophils. ProteobacteriaHIGH subjects were identified by Proteobacteria to Firmicutes ratio ≥0.485. Where paired sputum from stable visits was available, we compared microbial composition at baseline and following ≥12 weeks of mepolizumab. RESULTS: Microbial composition was not related to inflammatory subgroup based on sputum or blood eosinophils. FeNO ≥50 ppb when stable and at exacerbation indicated a group with less dispersed microbial profiles characterised by high alpha-diversity and low Proteobacteria. ProteobacteriaHIGH subjects were neutrophilic and had a longer time from asthma diagnosis than ProteobacteriaLOW subjects. In those studied, mepolizumab did not alter airway bacterial load or lead to increased Proteobacteria. CONCLUSION: High FeNO could indicate a subgroup of severe asthma less likely to benefit from antimicrobial strategies at exacerbation or in the context of poor control. Where FeNO is <50 ppb, biomarkers of microbial composition are required to identify those likely to respond to microbiome-directed strategies. We found no evidence that mepolizumab alters airway microbial composition.

The inflammatory profile of exacerbations in patients with severe refractory eosinophilic asthma receiving mepolizumab (the MEX study): a prospective observational study.

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.

A galactokinase-like protein from the liver fluke Fasciola hepatica.

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.

Loop-mediated isothermal amplification assay for rapid detection of Streptococcus agalactiae (group B streptococcus) in vaginal swabs - a proof of concept study.

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.

Gardnerella vaginalis and Mollicute detection in rectal swabs from men who have sex with men.

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.

A comparison of different pre-lysis methods and extraction kits for recovery of Streptococcus agalacticae (Lancefield group B Streptococcus) DNA from whole blood.

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.

The common vaginal commensal bacterium Ureaplasma parvum is associated with chorioamnionitis in extreme preterm labor.

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.

Ureaplasma parvum and Mycoplasma genitalium are found to be significantly associated with microscopy-confirmed urethritis in a routine genitourinary medicine setting.

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.

New assay for Gardnerella vaginalis loads correlates with Nugent scores and has potential in the diagnosis of bacterial vaginosis.

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.

Rising incidence of Pneumocystis jirovecii pneumonia suggests iatrogenic exposure of immune-compromised patients may be becoming a significant problem.

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.