Changing Incidence and Characteristics of Nontuberculous Mycobacterial Infections in Scotland and Comparison With Mycobacterium tuberculosis Complex Incidence (2011 to 2019).

Background: An increase in infections with nontuberculous mycobacteria (NTM) has been noted globally, and their incidence has overtaken that of Mycobacterium tuberculosis complex (MTBc) in many countries. Using data from a national reference laboratory, we aimed to determine if this trend could be observed in Scotland. Methods: We undertook a retrospective review of all NTM isolates received by the Scottish Mycobacteria Reference Laboratory (SMRL) over 9 years from 2011 to 2019 inclusive. Clinical episodes were defined as per 2017 British Thoracic Society and 2020 American Thoracic Society/European Respiratory Society/European Society of Clinical Microbiology and Infectious Diseases/Infectious Diseases Society of America NTM guidelines. These rates were compared with Scottish tuberculosis rates over the same period. Results: Of 8552 NTM isolates from 4586 patients in 2011 to 2019, 7739 (90.5%) were considered clinically relevant. These represented 2409 episodes of NTM infection, with M. avium, M. intracellulare, and M. abscessus complex being most common. A total of 1953 (81.1%) were pulmonary NTM infection episodes from 1470 patients and 456 extrapulmonary episodes from 370 patients. We estimated a rise in incidence from 3.4 to 6.5 per 100 000 person-years (2011-2019 inclusive), with an increase in NTM incidence over MTBc incidence in Scotland by 2017. Conclusions: The incidence of NTM infection in Scotland has overtaken MTBc incidence. NTM infection leads to a costly health care burden, possibly as much as UK£1.47 million (US$ and €1.73 million) annually. We recommend standardization of isolate referral with clinical surveillance and implementation of agreed standards of care delivered through multidisciplinary teams. This would improve diagnosis and patient management as well as assessment of diagnostics and novel treatments through clinical trials.

Heterogeneity in Staphylococcus aureus Bacteraemia Clinical Trials Complicates Interpretation of Findings.

We systematically evaluated randomized-controlled trials (RCTs) for Staphylococcus aureus bacteremia (SAB). There was intertrial heterogeneity in cohort characteristics, including bacteremia source, complicated SAB, and comorbidities. Reporting of cohort characteristics was itself variable, including bacteremia source and illness severity. Selection bias was introduced by exclusion criteria relating to comorbidities, illness severity, infection types, and source control. Mortality was lower in RCT control arms compared with observational cohorts. Differences in outcome definitions impedes meta-analysis. These issues complicate the interpretation and application of SAB RCT results. The value of these trials should be maximized by a standardized approach to recruitment, definitions, and reporting.

False detection of rifampicin resistance using Xpert® MTB/RIF Ultra assay due to an A451V mutation in Mycobacterium tuberculosis.

BACKGROUND: In a 12 month period, three Irish-born adult cases with pulmonary TB were initially diagnosed by Xpert® MTB/RIF Ultra assay, which detected a rifampicin resistance-conferring mutation prompting treatment as potential MDR cases. METHODS: Further laboratory investigations on the cultured isolates included GenoType MTBDRplus assay, phenotypic drug susceptibility tests using the BD BACTEC MGIT culture system and MIC broth microdilution tests. Sequencing of the rpoB gene was performed using Sanger sequencing and WGS. RESULTS: Phenotypic drug susceptibility tests determined the isolates to be rifampicin susceptible. Molecular investigations identified an A451V (codon 532) mutation in the Mycobacterium tuberculosis rpoB gene that has not previously been found to cause rifampicin resistance. Genome sequencing revealed that the three isolates' genomes differed by ≤5 SNPs, indicating a high likelihood of recent transmission events. Furthermore, a cluster of six related M. tuberculosis isolates from our in-house typing database showed four were highly related; all were rifampicin susceptible and lacked this mutation. CONCLUSIONS: False detection of rifampicin resistance, albeit rare, should be considered possible with Xpert® MTB/RIF Ultra assay, particularly in low TB incidence settings. Confirmatory sequencing methods should be performed to prevent the unnecessary use of second-line anti-tuberculous drugs.

A Subgroup of Patients With Hospital-acquired Pneumonia Do Not Require Broad-spectrum Gram-negative Antimicrobial Coverage.

Among 200 patients developing hospital-acquired pneumonia (HAP) outside the intensive care unit, 61% were treated empirically without broad-spectrum Gram-negative coverage, with clinical cure in 69.7%. Lower disease severity markers (systemic inflammatory response syndrome, hypoxia, tachypnoea, neutrophilia) and the absence of diabetes mellitus and prior doxycycline treatment (but not the time to HAP onset) identified patients not requiring broad-spectrum Gram-negative coverage.

Sensitivity of RT-PCR testing of upper respiratory tract samples for SARS-CoV-2 in hospitalised patients: a retrospective cohort study.

Background: This study aimed to determine the sensitivity and specificity of reverse transcription PCR (RT-PCR) testing of upper respiratory tract (URT) samples from hospitalised patients with coronavirus disease 2019 (COVID-19), compared to the gold standard of a clinical diagnosis. Methods: All URT RT-PCR testing for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in NHS Lothian, Scotland, United Kingdom between the 7 th of February and 19 th April 2020 (inclusive) was reviewed, and hospitalised patients were identified. All URT RT-PCR tests were analysed for each patient to determine the sequence of negative and positive results. For those who were tested twice or more but never received a positive result, case records were reviewed, and a clinical diagnosis of COVID-19 allocated based on clinical features, discharge diagnosis, and radiology and haematology results. For those who had a negative RT-PCR test but a clinical diagnosis of COVID-19, respiratory samples were retested using a multiplex respiratory panel, a second SARS-CoV-2 RT-PCR assay, and a human RNase P control. Results: Compared to the gold standard of a clinical diagnosis of COVID-19, the sensitivity of a single upper respiratory tract RT-PCR for COVID-19 was 82.2% (95% confidence interval 79.0-85.1%).   The sensitivity of two upper respiratory tract RT-PCR tests increased sensitivity to 90.6% (CI 88.0-92.7%). A further 2.2% and 0.9% of patients who received a clinical diagnosis of COVID-19 were positive on a third and fourth test; this may be an underestimate of the value of further testing as the majority of patients 93.0% (2999/3226) only had one or two URT RT-PCR tests. Conclusions: The sensitivity of a single RT-PCR test of URT samples in hospitalised patients is 82.2%. Sensitivity increases to 90.6% when patients are tested twice.  A proportion of cases with clinically defined COVID-19 never test positive on URT RT-PCR despite repeat testing.

Phenotypic and molecular detection methods for carbapenemase-producing organisms and their clinical significance at two Scottish tertiary care hospitals.

PURPOSE: This study evaluated in-house PCR testing for local identification of bacteria carrying the major carbapenemase genes (blaOXA-48-like, blaVIM, blaNDM, blaKPC and blaIMP). METHODOLOGY: Carbapenemase-producing organisms (CPOs) isolated from patients managed in two tertiary care hospitals in Scotland from September 2014-January 2017 were investigated. A combination of chromogenic screening agar (ChromID CARBA SMART), a carbapenem hydrolysis test (Rapidec Carba NP) and in-house real-time PCR for the blaOXA-48-like, blaVIM, blaNDM, blaKPC and blaIMP genes were utilized. All isolates were sent to the AMRHAI reference unit for confirmatory testing. RESULTS: During the 29-month study period 39 CPO were isolated from 34 patients. The average turnaround time for a workflow involving phenotypic and molecular testing was 4.2 days. PCR had a sensitivity and specificity of 100 %. The most common carbapenemase genes were blaOXA-48-like (31%), blaVIM (23%) and blaNDM (20%). Resistance to antimicrobials other than beta-lactams was common; the most active agents were colistin, amikacin and fosfomycin. Twenty-seven patients were considered to be colonized (although CPO detection influenced empiric antimicrobials in five) and a CPO was implicated in infection in seven patients (bacteraemia in immunocompromised patients, n=2; surgical site infections, n=2; osteomyelitis in a patient with diabetes mellitus, n=1; and urinary tract infections, n=2). All patients survived infection. CONCLUSION: In a lowincidence setting we demonstrate the efficacy of a combined local laboratory workflow for rapid detection of CPOs, incorporating phenotypic and molecular testing. In 7/34 patients the CPO was implicated as a pathogen and detection influenced antimicrobial decision-making in five colonized patients.

Tractable targets for meropenem-sparing antimicrobial stewardship interventions.

BACKGROUND: As meropenem is a restricted antimicrobial, lessons learned from its real-life usage will be applicable to antimicrobial stewardship (AMS) more generally. OBJECTIVES: To retrospectively evaluate meropenem usage at our institution to identify targets for AMS interventions. METHODS: Patients receiving meropenem documented with an 'alert antimicrobial' form at two tertiary care UK hospitals were identified retrospectively. Clinical records and microbiology results were reviewed. RESULTS: A total of 107 adult inpatients receiving meropenem were identified. This was first-line in 47% and escalation therapy in 53%. Source control was required in 28% of cases after escalation, for predictable reasons. Those ultimately requiring source control had received more prior antimicrobial agents than those who did not (P = 0.03). Meropenem was rationalized in 24% of cases (after median 4 days). Positive microbiology enabled rationalization (OR 12.3, 95% CI 2.7-55.5, P = 0.001) but rates of appropriate sampling varied. In cases with positive microbiology where meropenem was not rationalized, continuation was retrospectively considered clinically and microbiologically necessary in 8/40 cases (0/17 empirical first-line usage). Rationalization was more likely when meropenem susceptibility was not released on the microbiology report (OR 5.2, 95% CI 1.3-20.2, P = 0.02). Input from an infection specialist was associated with a reduced duration of meropenem therapy (P < 0.0001). Early review by an infection specialist has the potential to further facilitate rationalization. CONCLUSIONS: In real-life clinical practice, core aspects of infection management remain tractable targets for AMS interventions: microbiological sampling, source control and infection specialist input. Further targets include supporting rationalization to less familiar carbapenem-sparing antimicrobials, restricting first-line meropenem usage and selectively reporting meropenem susceptibility.

Three year evaluation of Xpert MTB/RIF in a low prevalence tuberculosis setting: A Scottish perspective.

OBJECTIVES: Xpert MTB/RIF (Cepheid) is a rapid molecular assay shown to be sensitive and specific for pulmonary tuberculosis (TB) diagnosis in highly endemic countries. We evaluated its diagnostic performance in a low TB prevalence setting, examined rifampicin resistance detection and quantitative capabilities predicting graded auramine microscopy and time to positivity (TTP) of culture. METHODS: Xpert MTB/RIF was used to test respiratory samples over a 3 year period. Samples underwent graded auramine microscopy, solid/liquid culture, in-house IS6110 real-time PCR, and GenoType MTBDRplus (HAIN Lifescience) to determine rifampicin and/or isoniazid resistance. RESULTS: A total of 2103 Xpert MTB/RIF tests were performed. Compared to culture sensitivity was 95.8%, specificity 99.5%, positive predictive value (PPV) 82.1%, and negative predictive value (NPV) 99.9%. A positive correlation was found between auramine microscopy grade and Xpert MTB/RIF assay load. We found a clear reduction in the median TTP as Xpert MTB/RIF assay load increased. Rifampicin resistance was detected. CONCLUSIONS: Xpert MTB/RIF was rapid and accurate in diagnosing pulmonary TB in a low prevalence area. Rapid results will influence infection prevention and control and treatment measures. The excellent NPV obtained suggests further work should be carried out to assess its role in replacing microscopy.

British Thoracic Society Guideline for the management of non-tuberculous mycobacterial pulmonary disease (NTM-PD).

The full guideline for the management of non-tuberculous mycobacterial pulmonary disease is published in Thorax. The following is a summary of the recommendations and good practice points. The sections referred to in the summary refer to the full guideline.

Pulmonary Mycobacterium marinum infection: 'fish tank granuloma' of the lung.

A 65-year-old man presented with a six-month history of lethargy, weight loss and dry cough. He had a background of mild chronic obstructive pulmonary disease. Chest radiograph showed new right upper lobe cavitary opacification. Sputum cultures were acid-fast bacilli smear positive and yielded Mycobacterium marinum - a non-tuberculous mycobacterium (NTM) often found in aquatic environments and rarely associated with respiratory disease. The suspected source was silent aspiration of contaminated water, likely due to his initiating the siphon of his fish-tank by mouth. He completed a one-year course of rifampicin, ethambutol and clarithromycin, with negative repeat sputum mycobacteria cultures and radiological improvement. This case report demonstrates a successful approach to investigation and further management of Mycobacterium marinum pulmonary disease - a rare condition, particularly in immunocompetent individuals, with limited treatment guidelines.

Comprehensive Molecular Testing for Respiratory Pathogens in Community-Acquired Pneumonia.

BACKGROUND: The frequent lack of a microbiological diagnosis in community-acquired pneumonia (CAP) impairs pathogen-directed antimicrobial therapy. This study assessed the use of comprehensive multibacterial, multiviral molecular testing, including quantification, in adults hospitalized with CAP. METHODS: Clinical and laboratory data were collected for 323 adults with radiologically-confirmed CAP admitted to 2 UK tertiary care hospitals. Sputum (96%) or endotracheal aspirate (4%) specimens were cultured as per routine practice and also tested with fast multiplex real-time polymerase-chain reaction (PCR) assays for 26 respiratory bacteria and viruses. Bacterial loads were also calculated for 8 bacterial pathogens. Appropriate pathogen-directed therapy was retrospectively assessed using national guidelines adapted for local antimicrobial susceptibility patterns. RESULTS: Comprehensive molecular testing of single lower respiratory tract (LRT) specimens achieved pathogen detection in 87% of CAP patients compared with 39% with culture-based methods. Haemophilus influenzae and Streptococcus pneumoniae were the main agents detected, along with a wide variety of typical and atypical pathogens. Viruses were present in 30% of cases; 82% of these were codetections with bacteria. Most (85%) patients had received antimicrobials in the 72 hours before admission. Of these, 78% had a bacterial pathogen detected by PCR but only 32% were culture-positive (P < .0001). Molecular testing had the potential to enable de-escalation in number and/or spectrum of antimicrobials in 77% of patients. CONCLUSIONS: Comprehensive molecular testing significantly improves pathogen detection in CAP, particularly in antimicrobial-exposed patients, and requires only a single LRT specimen. It also has the potential to enable early de-escalation from broad-spectrum empirical antimicrobials to pathogen-directed therapy.

Genotypic assessment of drug-resistant tuberculosis in Baghdad and other Iraqi provinces using low-cost and low-density DNA microarrays.

We report on a molecular investigation carried out to ascertain the prevalence of drug-resistant tuberculosis (TB) and the specific gene mutations responsible for resistance to rifampicin (RIF) and/or isoniazid (INH) in Iraq. In total, 110 clinical isolates from category II TB cases from Baghdad (58%) and several Iraqi provinces (42%) were analysed using colorimetric, low-cost and low-density (LCD) microarrays (MYCO-Direct and MYCO-Resist LCD array kits) to identify the point mutations responsible for resistance in Mycobacterium tuberculosis isolates. We found 76 patients (69.1%) had resistant strains, of which 40 (36%) were multidrug-resistant (MDR)-TB. Where mono-resistance was identified, it was found to be predominantly to RIF (83%). The most common mutations were rpoB S531L (50%), inhA C15T (25%) and katG S315T (15%). The most common MDR-TB genotypes were rpoB S531L with inhA C15T (60%) and rpoB S531L with katG S315T (20%). Where phenotypic analysis of clinical isolates was also performed, genotypic data were found to show excellent correlation with phenotypic results. Correlation was found between the MYCO-Resist LCD array and GenoType MTBDRplus for detection of resistance to RIF. Our study shows MDR-TB in 36% of category II TB cases in Baghdad and surrounding Iraqi provinces, which reflects the World Health Organization findings based on phenotypic studies. Diagnosis of TB and MDR-TB using culture-based tests is a significant impediment to global TB control. The LCD arrays investigated herein are easy to use, sensitive and specific molecular tools for TB resistance profiling in resource-limited laboratory settings.

Diagnostic accuracy of pulmonary host inflammatory mediators in the exclusion of ventilator-acquired pneumonia.

BACKGROUND: Excessive use of empirical antibiotics is common in critically ill patients. Rapid biomarker-based exclusion of infection may improve antibiotic stewardship in ventilator-acquired pneumonia (VAP). However, successful validation of the usefulness of potential markers in this setting is exceptionally rare. OBJECTIVES: We sought to validate the capacity for specific host inflammatory mediators to exclude pneumonia in patients with suspected VAP. METHODS: A prospective, multicentre, validation study of patients with suspected VAP was conducted in 12 intensive care units. VAP was confirmed following bronchoscopy by culture of a potential pathogen in bronchoalveolar lavage fluid (BALF) at >10(4) colony forming units per millilitre (cfu/mL). Interleukin-1 beta (IL-1ß), IL-8, matrix metalloproteinase-8 (MMP-8), MMP-9 and human neutrophil elastase (HNE) were quantified in BALF. Diagnostic utility was determined for biomarkers individually and in combination. RESULTS: Paired BALF culture and biomarker results were available for 150 patients. 53 patients (35%) had VAP and 97 (65%) patients formed the non-VAP group. All biomarkers were significantly higher in the VAP group (p<0.001). The area under the receiver operator characteristic curve for IL-1ß was 0.81; IL-8, 0.74; MMP-8, 0.76; MMP-9, 0.79 and HNE, 0.78. A combination of IL-1ß and IL-8, at the optimal cut-point, excluded VAP with a sensitivity of 100%, a specificity of 44.3% and a post-test probability of 0% (95% CI 0% to 9.2%). CONCLUSIONS: Low BALF IL-1ß in combination with IL-8 confidently excludes VAP and could form a rapid biomarker-based rule-out test, with the potential to improve antibiotic stewardship.

Rapid real-time PCR for detection of Mycobacterium tuberculosis complex DNA in formalin-fixed paraffin embedded tissues: 16% of histological 'sarcoid' may contain such DNA.

AIMS: To investigate the diagnostic accuracy of IS6110 real-time PCR for detection of Mycobacterium tuberculosis complex (MTBC) in DNA extracted from formalin-fixed paraffin embedded (FFPE) tissues using two different methods. In the absence of material submitted for tuberculosis (TB) culture, MTBC detection in FFPE tissue can be an important aid to diagnosis. METHODS: We collected 144 FFPE tissue blocks (lung and lymph node) for IS6110 real-time PCR. Two DNA extraction methods (QIAamp FFPE tissue kit and NucliSENS easyMAG) were assessed within a general laboratory setting. PCR results were compared with histology and culture. RESULTS: In the histological MTBC and culture MTBC (TB-positive) groups, 72.4% were IS6110-positive and 27.6% negative. IS6110-negative results were obtained from 98%, 61.5% and 84% of the histologically MTBC-negative (TB-negative) group, histologically TB/no culture group and sarcoidosis group, respectively. Review of 19 IS6110-positive patients in the latter three groups showed that 15 had clinical TB. Thirteen of 15 (86.7%) IS6110-positive patients in the histological TB/no culture group and 2 of 4 (50%) IS6110-positive patients in the sarcoidosis group were clinically diagnosed with TB which highlights the difficulty of a pathological diagnosis. CONCLUSIONS: IS6110 real-time PCR using easyMAG extracted DNA is a moderately sensitive, specific and rapid method for MTBC detection in FFPE material, but must be interpreted in the overall clinical context. PCR results can be available in around 5 h from FFPE specimen receipt, with minimal hands-on time.

Assessment of Mycobacterium tuberculosis transmission in Oxfordshire, UK, 2007-12, with whole pathogen genome sequences: an observational study.

BACKGROUND: Patients born outside the UK have contributed to a 20% rise in the UK's tuberculosis incidence since 2000, but their effect on domestic transmission is not known. Here we use whole-genome sequencing to investigate the epidemiology of tuberculosis transmission in an unselected population over 6 years. METHODS: We identified all residents with Oxfordshire postcodes with a Mycobacterium tuberculosis culture or a clinical diagnosis of tuberculosis between Jan 1, 2007, and Dec 31, 2012, using local databases and checking against the national Enhanced Tuberculosis Surveillance database. We used Illumina technology to sequence all available M tuberculosis cultures from identified cases. Sequences were clustered by genetic relatedness and compared retrospectively with contact investigations. The first patient diagnosed in each cluster was defined as the index case, with links to subsequent cases assigned first by use of any epidemiological linkage, then by genetic distance, and then by timing of diagnosis. FINDINGS: Although we identified 384 patients with a diagnosis of tuberculosis, country of birth was known for 380 and we sequenced isolates from 247 of 269 cases with culture-confirmed disease. 39 cases were genomically linked within 13 clusters, implying 26 local transmission events. Only 11 of 26 possible transmissions had been previously identified through contact tracing. Of seven genomically confirmed household clusters, five contained additional genomic links to epidemiologically unidentified non-household members. 255 (67%) patients were born in a country with high tuberculosis incidence, conferring a local incidence of 109 cases per 100,000 population per year in Oxfordshire, compared with 3·5 cases per 100,000 per year for those born in low-incidence countries. However, patients born in the low-incidence countries, predominantly UK, were more likely to have pulmonary disease (adjusted odds ratio 1·8 [95% CI 1·2-2·9]; p=0·009), social risk factors (4·4 [2·0-9·4]; p<0·0001), and be part of a local transmission cluster (4·8 [1·6-14·8]; p=0·006). INTERPRETATION: Although inward migration has contributed to the overall tuberculosis incidence, our findings suggest that most patients born in high-incidence countries reactivate latent infection acquired abroad and are not involved in local onward transmission. Systematic screening of new entrants could further improve tuberculosis control, but it is important that health care remains accessible to all individuals, especially high-risk groups, if tuberculosis control is not to be jeopardised. FUNDING: UK Clinical Research Collaboration (Wellcome Trust, Medical Research Council, National Institute for Health Research [NIHR]), and NIHR Oxford Biomedical Research Centre.

Adjunctive rifampicin may improve outcomes in Staphylococcus aureus bacteraemia: a systematic review.

Staphylococcus aureus bacteraemia (SAB) is associated with substantial morbidity and mortality. By surviving within leukocytes, S. aureus can evade both immunological defences and antimicrobial drugs, thus facilitating haematogenous dissemination. We performed a systematic review to determine whether antimicrobials with intracellular activity improve outcomes in SAB when used as an adjunct to ß-lactam or glycopeptide monotherapy. The Pubmed/MEDLINE, Embase and Cochrane databases were systematically searched for eligible studies that reported on the use of first-line antimicrobials plus a single additional antimicrobial of interest in patients with SAB (any cause). Six relevant studies were identified, all reporting on rifampicin use. Four studies (three randomized controlled trials and one cohort) reported on adults with SAB, including 54 patients treated with adjunctive rifampicin and 44 standard-therapy controls. Estimated across all of these studies, adjunctive rifampicin was associated with trends towards reduced all-cause mortality and reduced clinical or bacteriological failure. The fifth study indicated that adjunctive rifampicin accelerates the resolution of persistent SAB in neonates. Data from the sixth study were considered flawed owing to differences in co-morbidities between groups. Limited data suggest that rifampicin-induced hepatitis is not clinically significant but that drug interactions are. In conclusion, adjunctive rifampicin may improve outcomes in SAB when used as an adjunct to ß-lactam or glycopeptide monotherapy.

Non-tuberculous mycobacteria: a retrospective review of Scottish isolates from 2000 to 2010.

There is growing recognition of the clinical importance of non-tuberculous mycobacteria (NTM), a group of versatile opportunistic bacterial pathogens. We describe the characteristics of NTM isolates in Scotland over an 11-year period using data held by the Scottish Mycobacteria Reference Laboratory. American Thoracic Society microbiological criteria were used to evaluate the clinical significance of isolates. Data presented include analysis of trends across time, species/body site associations, gender and age differences, geographical variations and the association between cystic fibrosis and Mycobacterium abscessus. We emphasise the need for standardised reporting criteria for NTM isolates to ensure optimal surveillance of NTM disease.