Increased Detection of Mycobacterium tuberculosis Disease Using a Tissue-Based Laboratory-Developed Polymerase Chain Reaction Assay Compared to Standard Diagnostics.

Standard diagnostics for Mycobacterium tuberculosis (MTB) including acid-fast bacilli (AFB) smear and culture, and Xpert™ MTB/RIF real-time Polymerase Chain Reaction (RT-PCR; Xpert) have variable sensitivity and/or long turnaround times. We describe the clinical performance of a laboratory-developed tissue-based MTB PCR compared with AFB culture and Xpert using a composite reference standard (CRS). Over an 8-year period, MTB PCR was performed on pulmonary, pleural, or lymph node specimens for 36 patients. Of these, 11 met criteria for confirmed/probable MTB using CRS. MTB PCR was positive in 100% (11/11), AFB cultures were positive in 73% (8/11), and Xpert in 0% (0/4). MTB PCR was negative in 25 cases of "No MTB" (100% specific). The MTB PCR assay resulted faster than positive AFB culture (mean time 4.3 versus 21.2 days). Tissue-based MTB PCR was associated with increased and rapid detection of MTB, improving clinical sensitivity in strongly suspected MTB cases.

A unique case of Fusobacterium nucleatum spondylodiscitis communicating with a pleural empyema through a fistula.

Species (spp.) belonging to the genus Fusobacterium are anaerobic commensals colonizing the upper respiratory tract, the gastrointestinal tract, and the genitals. Infections with Fusobacterium spp. have been reported at many anatomical sites, including pneumonias and pleural empyemas; however, there are very few published cases of Fusobacterium spp. causing spondylodiscitis or fistulas. Bone infections with Fusobacterium can spread directly to surrounding muscular tissue or by hematogenous transmission to any other tissue including pleurae and lungs. Similarly, pleural infections can spread Fusobacterium spp. to any other tissue including fistulas and bone. Concomitant pleural empyema and spondylodiscitis are rare; however, there are a few published cases with concomitant disease, although none caused by Fusobacterium spp. A 77-year-old female patient was assessed using computed tomography (CT) scanning of the thorax and abdomen, as well as analyses of fluid drained from the region affected by the pleural empyema. A diagnosis of Fusobacterium empyema, fistula, bacteremia, and spondylodiscitis was made, and the patient's condition improved significantly after drainage of the pleural empyema and relevant long-term antibiotic treatment. We describe the first confirmed case with concomitant infection with Fusobacterium nucleatum as spondylodiscitis and pleural empyema connected by a fistula.

Clinical utility of 16S rRNA PCR in pleural infection.

Pleural infections cause major morbidity and mortality, particularly amongst paediatric and elderly populations. The aetiology is broad, but pleural culture fails to yield a causative pathogen in approximately 40 % of cases. Alternative pathogen identification methods are therefore required. The aim of the study was to investigate the yield from and impact on patient care when performing 16S rRNA PCR on culture-negative pleural fluid specimens and to determine whether any individual laboratory parameters were associated with a positive 16S rRNA PCR result. We conducted a study on 90 patients with suspected pleural infection, who had a culture-negative pleural fluid specimen, which underwent 16S rRNA PCR analysis between August 2017 and June 2019. This study was undertaken at a large NHS Trust in London, UK. Thirty-one per cent of culture-negative pleural fluid specimens tested by 16S rRNA PCR yielded a positive PCR result. Our data demonstrated that 16S rRNA PCR detected a significantly higher proportion of Streptococcus pneumoniae (P<0.0001) and fastidious, slow-growing and anaerobic pathogens (P=0.0025) compared with culture-based methods. Of the 25 16S rRNA PCR results that were positive for a causative pathogen, 76 % had a direct impact on clinical management. No single laboratory variable was found to be associated with a positive 16S rRNA PCR result. The findings from our real-world evaluation highlight the importance of 16S rRNA PCR in confirming pleural infection when the aetiology is unknown, and its direct, positive impact on clinical management.

Endothelial Cell Protein C Receptor Deficiency Attenuates Streptococcus pneumoniae-induced Pleural Fibrosis.

Streptococcus pneumoniae is the leading cause of hospital community-acquired pneumonia. Patients with pneumococcal pneumonia may develop complicated parapneumonic effusions or empyema that can lead to pleural organization and subsequent fibrosis. The pathogenesis of pleural organization and scarification involves complex interactions between the components of the immune system, coagulation, and fibrinolysis. EPCR (endothelial protein C receptor) is a critical component of the protein C anticoagulant pathway. The present study was performed to evaluate the role of EPCR in the pathogenesis of S. pneumoniae infection-induced pleural thickening and fibrosis. Our studies show that the pleural mesothelium expresses EPCR. Intrapleural instillation of S. pneumoniae impairs lung compliance and lung volume in wild-type and EPCR-overexpressing mice but not in EPCR-deficient mice. Intrapleural S. pneumoniae infection induces pleural thickening in wild-type mice. Pleural thickening is more pronounced in EPCR-overexpressing mice, whereas it is reduced in EPCR-deficient mice. Markers of mesomesenchymal transition are increased in the visceral pleura of S. pneumoniae-infected wild-type and EPCR-overexpressing mice but not in EPCR-deficient mice. The lungs of wild-type and EPCR-overexpressing mice administered intrapleural S. pneumoniae showed increased infiltration of macrophages and neutrophils, which was significantly reduced in EPCR-deficient mice. An analysis of bacterial burden in the pleural lavage, the lungs, and blood revealed a significantly lower bacterial burden in EPCR-deficient mice compared with wild-type and EPCR-overexpressing mice. Overall, our data provide strong evidence that EPCR deficiency protects against S. pneumoniae infection-induced impairment of lung function and pleural remodeling.

NOX1 Promotes Mesothelial-Mesenchymal Transition through Modulation of Reactive Oxygen Species-mediated Signaling.

Pleural organization may occur after empyema or complicated parapneumonic effusion and can result in restrictive lung disease with pleural fibrosis (PF). Pleural mesothelial cells (PMCs) may contribute to PF through acquisition of a profibrotic phenotype, mesothelial-mesenchymal transition (MesoMT), which is characterized by increased expression of α-SMA (α-smooth muscle actin) and other myofibroblast markers. Although MesoMT has been implicated in the pathogenesis of PF, the role of the reactive oxygen species and the NOX (nicotinamide adenine dinucleotide phosphate oxidase) family in pleural remodeling remains unclear. Here, we show that NOX1 expression is enhanced in nonspecific human pleuritis and is induced in PMCs by THB (thrombin). 4-Hydroxy-2-nonenal, an indicator of reactive oxygen species damage, was likewise increased in our mouse model of pleural injury. NOX1 downregulation blocked THB- and Xa (factor Xa)-mediated MesoMT, as did pharmacologic inhibition of NOX1 with ML-171. NOX1 inhibition also reduced phosphorylation of Akt, p65, and tyrosine 216-GSK-3ß, signaling molecules previously shown to be implicated in MesoMT. Conversely, ML-171 did not reverse established MesoMT. NOX4 downregulation attenuated TGF-ß- and THB-mediated MesoMT. However, NOX1 downregulation did not affect NOX4 expression. NOX1- and NOX4-deficient mice were also protected in our mouse model of Streptococcus pneumoniae-mediated PF. These data show that NOX1 and NOX4 are critical determinants of MesoMT.

Open pleural decortication for the late stage empyema thoracis in children: a retrospective observational study from a tertiary hospital of eastern Nepal.

The management of late-stage empyema thoracis requires surgical intervention. We performed a retrospective descriptive analysis of open pleural decortication for late stage empyema thoracis in 55 children (age ≤ 15 years; median age = 6 years; age range = 1-15 years; 40 [72.7%] boys) over 42 months. The median time to thoracotomy from the onset of symptoms was 24 days, and the median duration of hospital stay before and after surgery was 15 and 4 days, respectively. Three (5.5%) patients had necrotising pneumonia, requiring debridement; 4 (7.3%) patients had superficial surgical site infection; 12 (21.8%) patients had perioperative pus culture positive for bacteria; and 3 (5.5%) patients had tubercular aetiology. There was no operative mortality. At median follow-up of 18 months, all patients are in good general health. Open pleural decortication leads to rapid resolution of symptoms and reduces hospital stay in paediatric late-stage empyema thoracis.

Streptococcus caledonicus sp. nov., isolated from sheep.

Five strains of an unidentified Gram-positive, catalase-negative, chain-forming coccus-shaped organism recovered from sheep in Scotland were characterized using phenotypic and molecular taxonomic methods. Based on morphological and biochemical criteria, the strains were tentatively identified as streptococci but they did not appear to correspond to any recognised species of the genus. Comparative 16S rRNA gene sequencing showed the strains were highly related to each other and confirmed their placement in the genus Streptococcus, with a maximum nucleotide identity of around 97 % to extant species. Best matches were with Streptococcus hillyeri followed by Streptococcus porci. Average nucleotide identity and in silico DNA-DNA hybridization values determined from whole-genome sequence were also consistent with the group representing a novel species. Best matches, again seen to S. hillyeri, followed by S. porci and S. plurextorum, were below accepted cut-off values for species delineation. Based on biochemical criteria and molecular genetic evidence, it is proposed that the unknown isolates from sheep be assigned to a new species of the genus Streptococcus as Streptococcus caledonicus sp. nov. The type strain of Streptococcus caledonicus is S784/96/1T=CCUG 73951T=NCTC 14363T.

Gastric mucormycosis complicated by a gastropleural fistula: A case report and review of the literature.

RATIONALE: Mucormycosis is a rare opportunistic fungal infection with poor prognosis. The incidence of mucormycosis has been increasing, and it is a threat to immunocompromised hosts. We present a case of gastric mucormycosis complicated by a gastropleural fistula during immunosuppressive treatment for adult-onset Still disease (AOSD). PATIENT CONCERNS: An 82-year-old woman diagnosed with AOSD who developed gastric ulcers during the administration of an immunosuppressive therapy with corticosteroids, cyclosporine, and tocilizumab complained of melena and epigastralgia. Esophagogastroduodenoscopy showed multiple ulcers covered with grayish or greenish exudates. DIAGNOSES: The patient diagnosed with mucormycosis based on culture and biopsy of the ulcers, which showed nonseptate hyphae branching at wide angles. Mucor indicus was identified using polymerase chain reaction. INTERVENTIONS AND OUTCOMES: Although liposomal amphotericin B was administered, gastric mucormycosis was found to be complicated by a gastropleural fistula. The patient died because of pneumonia due to cytomegalovirus infection, and autopsy revealed the presence of Mucorales around the fistula connecting the stomach and diaphragm. LESSONS: Gastric mucormycosis is refractory to treatment and fatal. Surgical resection, if possible, along with antifungal drugs can result in better outcomes.

Sputum culture for the diagnosis of tuberculous pleural effusion: analysis of absolute and incremental yields.

INTRODUCTION: Pleural fluid culture yield in tuberculous pleural effusion (TPE) is disappointing in immunocompetent hosts. Herein, we attempt to define the role of serial sputum cultures in the diagnosis of TPE. MATERIAL AND METHODS: We identified cases diagnosed with TPE over a16-year period in ahigh-prevalence US hospital. Absolute yields of one, two, and three sputa were calculated as well as the incremental yield of adding second and third sputa. These calculations were then performed separately for expectorated and induced sputum and for patients with and without infiltrates on chest X-ray. RESULTS: Sixty sputum collections were performed in 46 patients with TPE. The per-patient sensitivity of sputum culture was 45.6%. On aper-sputum collection basis, the overall yield of the first sputum was 30%, of two sputa 39%, and of three sputa 54%. The corresponding incremental yields were 9% and 15%, respectively. The three-sputum yields of expectorated and induced collections were similar. The three-sputum yield in patients with infiltrates on X-ray was 11% lower than that in those without infiltrates. CONCLUSIONS: Serial sputum collection of three specimens can be expected to produce ayield of > 50% in cases of suspected TPE regardless of whether obtained by expectoration or induction, and the yield increases incrementally.

Diagnostic value of polymerase chain reaction/acid-fast bacilli in conjunction with computed tomography-guided pleural biopsy in tuberculous pleurisy: A diagnostic accuracy study.

Patients with tuberculous pleurisy often remain undiagnosed even after blind thoracentesis and closed pleural biopsy (PB). In this study, we assessed the value of computed tomography (CT)-guided core needle biopsy of pleural lesion and evaluated the diagnostic accuracy of polymerase chain reaction (PCR)/staining for acid-fast bacilli (AFB) in suspicious tuberculous pleurisy undiagnosed in blind thoracentesis.Patients with exudative pleural effusion (PE) without specific etiology after blind thoracentesis and closed PB were enrolled in this study. PB specimens were obtained through CT-guided core needle biopsy of pleural lesion, then underwent PCR, AFB, histopathological examination, and some routine tests. Diagnostic values were evaluated through sensitivity, specificity, negative predictive value, positive predictive value, and accuracy.A total of 261 participants (TB group: 241, non-TB group: 20) were recruited. In this cohort, the sensitivity, specificity, and accuracy were 56.0%, 95.0%, and 59.0%, respectively for PCR, whereas 57.3%, 95.0%, and 60.2%, respectively for AFB. Their parallel test achieved an improved sensitivity (76.8%) and accuracy (77.8%), with a slight decrease in specificity (90.0%). In histopathological examination, granuloma was the most common finding in TB group (88.4%, 213/241), but also observed in non-TB group (10.0%, 2/20). In addition, pleural lymphocyte percentage in TB group was significantly higher than that of non-TB group (92% vs 61%, respectively; P = .003). However, no significant differences were found for other biomarkers.CT-guided core needle PB is essential for patients with exudative PE but undiagnosed after blind thoracentesis. Combining with PCR and AFB, it strongly improves the diagnosis of tuberculous pleurisy.

Pleural infection: a closer look at the etiopathogenesis, microbiology and role of antibiotics.

INTRODUCTION: Pleural infection is a condition that continues to pose a significant challenge to respiratory physicians. We hypothesize that the main barriers to progress include limited understanding of the etiopathogenesis, microbiology,and role of antibiotics in the pleural space. Areas covered: PubMed was searched for articles related to adult pleural infection using the terms 'pleural infection', 'empyema' and 'parapneumonic'. The search focused on relevant literature within the last 10 years, with any older citations used only to display context or lack of progress. Tuberculous pleural infection was excluded. We chose to give specific attention to the etiopathogenesis of pleural infection, including recent advances in diagnostics and biomarkers. We discuss our understanding of the pleural microbiome and rationalize the current use of antibiotics in treating this condition. Expert commentary: Understanding of key events in the development of this condition remains limited. The microbiology is unique compared to the lung, and highly variable. Higher culture yields from pleural biopsy may add new insights into the etiopathogenesis. There is little evidence into achievable effective antibiotic concentration within the pleura. Research into issues including the relevance of biofilm formation and significance of pleural thickening is necessary for treatment progress.

Recent developments in the management of pleural infection: A comprehensive review.

OBJECTIVES: Pleural infection is a condition commonly encountered by the respiratory physician. This review aims to provide the reader with an update on the most recent data regarding the epidemiology, microbiology, and the management of pleural infection. DATA SOURCE: Medline was searched for articles related to pleural infection using the terms "pleural infection," "empyema," and "parapneumonic." The search was limited to the years 1997-2017. Only human studies and reports in English were included. RESULTS: A rise in the incidence of pleural infection is seen worldwide. Despite the improvement in healthcare practices, the mortality from pleural infection remains high. The role of oral microflora in the etiology of pleural infection is firmly established. A concise review of the recent insights on the pathogenesis of pleural infections is presented. A particular focus is made on the role of tPA, DNAse and similar substances and their interaction with inflammatory cells and how this affects the pathogenesis and treatment of pleural infection. CONCLUSION: Pleural infection is a common disease with significant morbidity and mortality, as well as a considerable economic burden. The role of medical management is expanding thanks to the widespread use of newer treatments.

Streptococcus pneumoniae potently induces cell death in mesothelial cells.

Pleural infection/empyema is common and its incidence continues to rise. Streptococcus pneumoniae is the commonest bacterial cause of empyema in children and among the commonest in adults. The mesothelium represents the first line of defense against invading microorganisms, but mesothelial cell responses to common empyema pathogens, including S. pneumoniae, have seldom been studied. We assessed mesothelial cell viability in vitro following exposure to common empyema pathogens. Clinical isolates of S. pneumoniae from 25 patients with invasive pneumococcal disease and three reference strains were tested. All potently induced death of cultured mesothelial cells (MeT-5A) in a dose- and time-dependent manner (>90% at 107 CFU/mL after 24 hours). No significant mesothelial cell killing was observed when cells were co-cultured with Staphylococcus aureus, Streptococcus sanguinis and Streptococcus milleri group bacteria. S. pneumoniae induced mesothelial cell death via secretory product(s) as cytotoxicity could be: i) reproduced using conditioned media derived from S. pneumoniae and ii) in transwell studies when the bacteria and mesothelial cells were separated. No excess cell death was seen when heat-killed S. pneumoniae were used. Pneumolysin, a cytolytic S. pneumoniae toxin, induced cell death in a time- and dose-dependent manner. S. pneumoniae lacking the pneumolysin gene (D39 ΔPLY strain) failed to kill mesothelial cells compared to wild type (D39) controls, confirming the necessity of pneumolysin in D39-induced mesothelial cell death. However, pneumolysin gene mutation in other S. pneumoniae strains (TIGR4, ST3 and ST23F) only partly abolished their cytotoxic effects, suggesting different strains may induce cell death via different mechanisms.

Are intra-pleural bacterial products associated with longer survival in adults with malignant pleural effusions? A systematic review.

BACKGROUND: Intra-pleural bacteria are effective pleurodesis agents in malignant pleural effusions. However, their relationship with survival is unclear. OBJECTIVES: We undertook a comprehensive, structured evaluation of survival outcomes in adults with malignant pleural effusions treated with intra-pleural bacterial products. DATA SOURCES: Medline, Embase, Cochrane library, Clinical Trials Registers and Open Grey. STUDY ELIGIBILITY CRITERIA, PARTICIPANTS, AND INTERVENTIONS: Randomised controlled trials and non-randomised comparative studies were included, if the population included adults with malignant pleural effusions. Interventions of interest were any intra-pleural bacterial product, compared with placebo, alternative intra-pleural drug, or no treatment. Survival outcomes were collected. STUDY APPRAISAL AND SYNTHESIS METHODS: Two reviewers independently screened studies for eligibility, assessed papers for risk of bias and extracted data. Narrative synthesis was performed as high heterogeneity between studies precluded meta-analysis. RESULTS: 631 studies were identified, of which 14 were included. All were at high or unclear risk of bias in at least one domain. Six studies reported a survival benefit associated with intra-pleural bacterial products, whilst 8 reported no difference. Non-randomised studies and studies published prior to 2000 were more likely to report survival benefits. LIMITATIONS: There was high heterogeneity between studies, which limited the generalisability of findings. Publication bias may have affected the review as five full-text papers were unobtainable, and survival outcomes were missing in a further five. CONCLUSIONS: There is a lack of high quality evidence regarding the relationship between intra-pleural bacterial products and survival. Implications of key findings: Well-designed, prospective randomised trials are needed, to determine whether intra-pleural bacterial products can improve survival in pleural malignancy. PROSPERO REGISTRATION NUMBER: CRD42017058067.

Factors Affecting Postoperative Lung Expansion in Patients with Pyogenic Empyema.

BACKGROUND: In patients with parapneumonic empyema, decortication is usually preferred to ensure functional lung re-expansion. However, there could be patients exhibiting incomplete postoperative lung expansion and inadequate drainage despite decortication. Therefore, we evaluated factors affecting postoperative lung expansion in patients undergoing decortication. METHODS: A total of 221 patients with pyogenic empyema who underwent video-assisted thoracoscopic surgery (VATS) between January and October 2016 in our hospital were reviewed in terms of surgical success. The following factors were evaluated: age; the time between identification of a localized effusion and surgical referral; chest tube drainage durations; any underlying morbidity preoperative blood culture data; and the thickness of the visceral pleura. RESULTS: Several factors that significantly prolonged the postoperative time to lung expansion were evident in patients with diabetes mellitus (DM) and bacteremia; postoperative chest tube drainage was significantly longer in those with DM (p = 0.009) and bacteremia (p = 0.01); and postoperative hospitalization time was significantly longer in patients with bacteremia (p = 0.01). The thickness of the visceral pleura was strongly correlated with postoperative chest tube drainage duration and postoperative hospitalization time (Pearson correlation coefficient, r = 0.245, p = 0.00). CONCLUSIONS: In patients with DM, bacteremia, or thickened pleura, the time to lung expansion after operation was longer. Therefore, stricter pre- and post-operative control of blood-sugar levels and adequate antibiotics are required to facilitate postoperative lung re-expansion. In patients with thickened pleurae, prolonged chest tube placement is unavoidable.

Thoracoscopic pleural biopsy improves yield of Xpert MTB/RIF for diagnosis of pleural tuberculosis.

BACKGROUND AND OBJECTIVE: Extrapulmonary tuberculosis (EPTB) accounts for ~15% of all TB patients, and TB pleural effusion is the second most common site of EPTB. The diagnosis of pleural TB is challenging due to the pauci-bacillary nature of the disease. Histopathology of thoracoscopically obtained pleural biopsy provides the highest diagnostic yield. The Xpert MTB/RIF assay (Xpert) is a PCR test that can identify both Mycobacterium tuberculosis (MTB) and rifampicin resistance. Currently, there is a lack of clarity regarding the value of Xpert on pleural tissue. We report our experience of using Xpert on thoracoscopic pleural biopsy samples. METHODS: We retrospectively reviewed the records of patients who underwent thoracoscopy in our institution over a 1-year period. Relevant clinical details; indications; and results of tests on pleural tissue and fluid, including histopathology, mycobacterial cultures and Xpert, were extracted. RESULTS: Of the 156 patients who underwent thoracoscopy, 73 (47%) had TB, 66 (42%) malignancy and 17 (11%) other conditions. Histopathology was diagnostic in all the 73 TB patients (100%). The yields of the microbiological tests against histopathology on thoracoscopic biopsy sample and pleural fluid were: pleural tissue Xpert 45%, pleural tissue culture 39%, pleural fluid culture 17% and pleural fluid Xpert 14%. Pleural tissue provided higher yields than fluid in both Xpert and culture (P < 0.05). Pleural tissue Xpert provided a higher yield than culture and substantially improved yield compared with closed pleural biopsy as we previously reported. CONCLUSION: Thoracoscopic pleural biopsy results in increased sensitivity on Xpert testing.

The Utility of Ultrasound to Diagnose Tunnel-Tract Infection Related to Indwelling Pleural Catheters.

Indwelling pleural catheter (IPC) infections lead to increased morbidity and treatment failure in patients with chronic recurrent pleural effusions. Ultrasonography is a readily available diagnostic tool used by pulmonologists on a daily basis. Ultrasonography has been used to identify the etiology of indwelling peritoneal catheter obstruction, including infection of the exit site and tunnel tract. The use of ultrasonography to identify tunnel-tract infection involving IPC has not been reported. We describe the ultrasonographic characteristics of 3 cases of confirmed tunnel-tract infection and compared them with noninfected chronic IPCs. Ultrasonographic evaluation of the soft tissue tunnel tract can accurately identify fluid collections around the catheter and cuff, which is highly suggestive of tunnel-tract infection.