The Microbial Etiology of Community-Acquired Pneumonia in Adults: from Classical Bacteriology to Host Transcriptional Signatures.

All modern advances notwithstanding, pneumonia remains a common infection with substantial morbidity and mortality. Understanding of the etiology of pneumonia continues to evolve as new techniques enable identification of already known organisms and as new organisms emerge. We now review the etiology of pneumonia (at present often called "community-acquired pneumonia") beginning with classic bacteriologic techniques, which identified Streptococcus pneumoniae as the overwhelmingly common cause, to more modern bacteriologic studies, which emphasize Haemophilus influenzae, Staphylococcus aureus, Moraxella catarrhalis, Enterobacteriaceae, Pseudomonas, and normal respiratory flora. Urine antigen detection is useful in identifying Legionella and pneumococcus. The low yield of bacteria in recent studies is due to the failure to obtain valid sputum samples before antibiotics are administered. The use of high-quality sputum specimens enables identification of recognized ("typical") bacterial pathogens as well as a role for commensal bacteria ("normal respiratory flora"). Nucleic acid amplification technology for viruses has revolutionized diagnosis, showing the importance of viral pneumonia leading to hospitalization with or without coinfecting bacterial organisms. Quantitative PCR study of sputum is in its early stages of application, but regular detection of high counts of bacterial DNA from organisms that are not seen on Gram stain or grown in quantitative culture presents a therapeutic dilemma. This finding may reflect the host microbiome of the respiratory tract, in which case treatment may not need to be given for them. Finally, host transcriptional signatures might enable clinicians to distinguish between viral and bacterial pneumonia, an important practical consideration.

A prospective surveillance study to determine the prevalence of 16S rRNA methyltransferase-producing Gram-negative bacteria in the UK.

OBJECTIVES: To determine the prevalence of 16S rRNA methyltransferase- (16S RMTase-) producing Gram-negative bacteria in patients in the UK and to identify potential risk factors for their acquisition. METHODS: A 6 month prospective surveillance study was conducted from 1 May to 31 October 2016, wherein 14 hospital laboratories submitted Acinetobacter baumannii, Enterobacterales and Pseudomonas aeruginosa isolates that displayed high-level amikacin resistance according to their testing methods, e.g. no zone of inhibition with amikacin discs. Isolates were linked to patient travel history, medical care abroad, and previous antibiotic exposure using a surveillance questionnaire. In the reference laboratory, isolates confirmed to grow on Mueller-Hinton agar supplemented with 256 mg/L amikacin were screened by PCR for 16S RMTase genes armA, rmtA-rmtH and npmA, and carbapenemase genes (blaKPC, blaNDM, blaOXA-48-like and blaVIM). STs and total antibiotic resistance gene complement were determined via WGS. Prevalence was determined using denominators for each bacterial species provided by participating hospital laboratories. RESULTS: Eighty-four isolates (44.7%), among 188 submitted isolates, exhibited high-level amikacin resistance (MIC >256 mg/L), and 79 (94.0%) of these harboured 16S RMTase genes. armA (54.4%, 43/79) was the most common, followed by rmtB (17.7%, 14/79), rmtF (13.9%, 11/79), rmtC (12.7%, 10/79) and armA + rmtF (1.3%, 1/79). The overall period prevalence of 16S RMTase-producing Gram-negative bacteria was 0.1% (79/71 063). Potential risk factors identified through multivariate statistical analysis included being male and polymyxin use. CONCLUSIONS: The UK prevalence of 16S RMTase-producing Gram-negative bacteria is low, but continued surveillance is needed to monitor their spread and inform intervention strategies.

Two cases of imported respiratory diphtheria in Edinburgh, Scotland, October 2019.

We report two cases of respiratory toxigenic Corynebacterium diphtheriae infection in fully vaccinated UK born adults following travel to Tunisia in October 2019. Both patients were successfully treated with antibiotics and neither received diphtheria antitoxin. Contact tracing was performed following a risk assessment but no additional cases were identified. This report highlights the importance of maintaining a high index of suspicion for re-emerging infections in patients with a history of travel to high-risk areas outside Europe.

Comparison of Unyvero P55 Pneumonia Cartridge, in-house PCR and culture for the identification of respiratory pathogens and antibiotic resistance in bronchoalveolar lavage fluids in the critical care setting.

Faster respiratory pathogen detection and antibiotic resistance identification are important in critical care due to the severity of illness, significant prior antibiotic exposure and infection control implications. Our objective was to compare the performance of the commercial Unyvero P55 Pneumonia Cartridge (Curetis AG) with routine bacterial culture methods and in-house bacterial multiplex real-time PCR assays. Seventy-four bronchoalveolar lavage specimens from patients admitted to a Scottish intensive care unit (ICU) over a 33-month period were tested prospectively by routine culture and viral PCR and retrospectively by Unyvero P55 and in-house bacterial PCR. Sensitivity/specificity was 56.9%/58.5% and 63.2%/54.8% for the Unyvero P55 and in-house bacterial PCR panels respectively; sensitivity for in-panel targets was 63.5 and 83.7% respectively. Additional organisms were detected by Unyvero P55 and in-house bacterial PCR panels in 16.2% specimens. Antibiotics were changed on the basis of routine test results in 48.3% cases; of these, true-positive or true-negative results would have been obtained earlier by Unyvero P55 or in-house bacterial PCR panel in 15 (53.6%) and 17 (60.7%) cases respectively. However, a false-negative molecular test result may have been acted upon in six (21.4%) cases with either assay. Sensitivity/specificity of Unyvero P55 antibiotic resistance detection was 18.8%/94.9% respectively. Molecular testing identified a number of respiratory pathogens in this patient cohort that were not grown in culture, but resistance detection was not a reliable tool for faster antibiotic modification. In their current set-up, molecular tests may only have benefit as additional tests in the ICU pneumonia setting.

16S pan-bacterial PCR can accurately identify patients with ventilator-associated pneumonia.

Ventilator-associated pneumonia (VAP) remains a challenge to intensive care units, with secure diagnosis relying on microbiological cultures that take up to 72 hours to provide a result. We sought to derive and validate a novel, real-time 16S rRNA gene PCR for rapid exclusion of VAP. Bronchoalveolar lavage (BAL) was obtained from two independent cohorts of patients with suspected VAP. Patients were recruited in a 2-centre derivation cohort and a 12-centre confirmation cohort. Confirmed VAP was defined as growth of >104 colony forming units/ml on semiquantitative culture and compared with a 16S PCR assay. Samples were tested from 67 patients in the derivation cohort, 10 (15%) of whom had confirmed VAP. Using cycles to cross threshold (Ct) values as the result of the 16S PCR test, the area under the receiver operating characteristic (ROC) curve (AUROC) was 0.94 (95% CI 0.86 to 1.0, p<0.0001). Samples from 92 patients were available from the confirmation cohort, 26 (28%) of whom had confirmed VAP. The AUROC for Ct in this cohort was 0.89 (95% CI 0.83 to 0.95, p<0.0001). This study has derived and assessed the diagnostic accuracy of a novel application for 16S PCR. This suggests that 16S PCR in BAL could be used as a rapid test in suspected VAP and may allow better stewardship of antibiotics. TRIAL REGISTRATION NUMBER: VAPRAPID trial ref NCT01972425.

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.

Molecular diagnosis of Legionella infections--Clinical utility of front-line screening as part of a pneumonia diagnostic algorithm.

OBJECTIVES: Urinary antigen testing for Legionella pneumophila serogroup 1 is the leading rapid diagnostic test for Legionnaires' Disease (LD); however other Legionella species and serogroups can also cause LD. The aim was to determine the utility of front-line L. pneumophila and Legionella species PCR in a severe respiratory infection algorithm. METHODS: L. pneumophila and Legionella species duplex real-time PCR was carried out on 1944 specimens from hospitalised patients over a 4 year period in Edinburgh, UK. RESULTS: L. pneumophila was detected by PCR in 49 (2.7%) specimens from 36 patients. During a LD outbreak, combined L. pneumophila respiratory PCR and urinary antigen testing had optimal sensitivity and specificity (92.6% and 98.3% respectively) for the detection of confirmed cases. Legionella species was detected by PCR in 16 (0.9%) specimens from 10 patients. The 5 confirmed and 1 probable cases of Legionella longbeachae LD were both PCR and antibody positive. CONCLUSIONS: Front-line L. pneumophila and Legionella species PCR is a valuable addition to urinary antigen testing as part of a well-defined algorithm. Cases of LD due to L. longbeachae might be considered laboratory-confirmed when there is a positive Legionella species PCR result and detection of L. longbeachae specific antibody response.

Comparative evaluation of the Diagenode multiplex PCR assay on the BD max system versus a routine in-house assay for detection of Bordetella pertussis.

This study looked at 128 nasopharyngeal aspirates (NPA) and 162 throat swabs (TS) tested with the Diagenode multiplex assay on the BD Max system versus our in-house Bordetella pertussis PCR. Sensitivity and specificity were 97.3% and 100% for NPA and 88.3% and 98% for TS, respectively. Of positive NPA, 42.1% were coinfected with respiratory viruses.

Clinical outcomes and macrolide resistance in Mycoplasma pneumoniae infection in Scotland, UK.

Mycoplasma pneumoniae has a cyclical, epidemic pattern of infection and the most recent epidemic occurred in Europe in 2011. Macrolides are recommended for the treatment of M. pneumoniae respiratory tract infection, but macrolide resistance has been reported at low levels in Europe. The aim of the study was to examine the clinical impact of the recent M. pneumoniae epidemic in a hospital setting in Scotland and to determine whether macrolide-resistant strains are present. Data were analysed retrospectively for 307 patients with M. pneumoniae respiratory infection diagnosed in 2010 and 2011 in Edinburgh, UK. Genotypic macrolide resistance testing was also carried out in 32 patients in whom resistance was considered most likely, based on their clinical picture. We found that 175 patients (59 %) were admitted to hospital, 20 (7 %) were admitted to critical care and 97 (38 %) required oxygen. All 48 adult patients (100 %) were admitted to hospital, compared with 127 children (51 %). Adults were also more likely to require oxygen [odds ratio (OR) 4.964, P<0.001, 95 % confidence interval (CI) 2.129-11.803] and to be admitted to critical care (OR 4.909, P = 0.001, 95 % CI 1.735-13.829), compared with children. Macrolide resistance conferred by the 23S rRNA gene mutation was found in samples from 6 out of 32 patients (19 %) in the subset tested. The results suggest that the recent M. pneumoniae epidemic was associated with a significant burden of hospital admission locally. The study also describes the first case series of macrolide-resistant M. pneumoniae in the UK, indicating that macrolide resistance surveillance is warranted in preparation for the next epidemic.

Comparison of the Luminex Respiratory Virus Panel fast assay with in-house real-time PCR for respiratory viral infection diagnosis.

The Luminex xTAG Respiratory Virus Panel (RVP) assay has been shown to offer improved diagnostic sensitivity over traditional viral culture methods and to have a sensitivity comparable to those of individual real-time nucleic acid tests for respiratory viruses. The objective of this retrospective study was to test a new, streamlined version of this assay, the RVP Fast assay, which requires considerably less run time and operator involvement. The study compared the performance of the RVP Fast assay with those of viral culture, a direct fluorescent assay (DFA), and a panel of single and multiplex real-time PCRs in the testing of 286 respiratory specimens submitted to the Edinburgh Specialist Virology Centre for routine diagnosis of viral infection between December 2007 and February 2009. At least one respiratory viral infection was detected in 13.6% of specimens by culture and DFA combined, in 49.7% by real-time PCR, and in 46.2% by the RVP Fast assay. The sensitivity and specificity of the RVP Fast assay compared to the results of real-time PCR as the gold standard were 78.8% and 99.6%, respectively. Real-time PCR-positive specimens missed by the RVP Fast assay generally had low viral loads or were positive for adenovirus. Additionally, a small number of specimens were positive by the RVP Fast assay but were not detected by real-time PCR. For some viral targets, only a small number of positive results were found in our sample set using either method; therefore, the sensitivity of detection of the RVP Fast assay for individual targets could be investigated further with a greater number of virus-positive specimens.

A study on pathogenicity and mosquito transmission success in the rodent malaria parasite Plasmodium chabaudi adami.

We investigated the parasitology, pathogenicity (virulence) and infectivity to mosquitoes of blood infections in mice, of two strains, DS and DK, of the rodent malaria parasite Plasmodium chabaudi adami. Blood infections of DS were found to be highly pathogenic; the asexual parasites in these infections were fast-growing and showed no evidence of selectivity in their infection of host erythrocytes. In contrast to DS, blood infections of DK were much less pathogenic; the asexual parasites were slower-growing and showed a moderate degree of selectivity to a subset of erythrocytes which were not reticulocytes. In both DS and DK infections, infectivity to mosquitoes was highest before the peak of asexual parasitaemia had occurred; usually this did not coincide with the time when gametocyte numbers in the blood were highest. Infections with the pathogenic DS strain in CBA mice produced fewer gametocytes than did the less pathogenic DK strain. The DS strain infections in both CBA and C57 mice were also significantly much less infective to mosquitoes than the DK strain. Investigations by others on the related rodent malaria parasite subspecies, Plasmodium chabaudi chabaudi, have indicated that the mosquito infectivity of blood infections in mice tended to be higher in the more pathogenic (virulent) and lower in the less pathogenic strains of this parasite subspecies. This is the converse of the finding of the present investigation of blood infections of P. c. adami in mice in which a more pathogenic, or virulent, strain (DS) of these parasites was significantly much less infective to mosquitoes than was a less pathogenic strain (DK).