Tissue-Resident Memory-Like CD8+ T Cells Exhibit Heterogeneous Characteristics in Tuberculous Pleural Effusion.

Tissue-resident memory T (TRM) cells are well known to play critical roles in peripheral tissues during virus infection and tumor immunology. Our previous studies indicated that CD69+CD4+ and CD69+CD8+ T cells in tuberculous pleural effusion (TPE) were antigen-specific memory T cells. However, the phenotypical and functional characteristics of CD8+ TRM cells in tuberculosis remain unknown. We found that CD103+CD8+ T cells were the predominant subset of CD103+ lymphocytes in TPE; both CD103 and CD69 expressed on memory CD8+ T cells from TPE were significantly increased compared with those from paired peripheral blood. Phenotypically, CD103+CD69+ and CD103+CD69-CD8+ T cells expressed higher levels of CD45RO than CD103-CD69+CD8+ T cells did; CD103+CD69-CD8+ T cells highly expressed CD27, CD127, and CD62L and some chemokine receptors. We further compared the functional differences among the four distinct CD45RO+CD8+ T subsets identified by CD103 and CD69 expression. In consist with our published results, CD69+CD8+ T cells, but not CD103+CD8+, produced high levels of IFN-γ after treatment with BCG in the presence of BFA. Nevertheless, CD103-CD69+ and CD103+CD69+ memory CD8+ T cells expressed higher levels of Granzyme B, while CD103+CD69- memory CD8+ T cells were characterized as a possibly immunosuppressive subset by highly expressing CTLA-4, CD25, and FoxP3. Furthermore, TGF-ß extremely increased CD103 expression but not CD69 in vitro. Together, CD103+CD8+ T cells form the predominant subset of CD103+ lymphocytes in TPE; CD103 and CD69 expression defines distinct CD8+ TRM-like subsets exhibiting phenotypical and functional heterogeneity. Our findings provide an important theoretical basis to optimize and evaluate new tuberculosis vaccines.

Predictors of Indwelling Pleural Catheter Removal and Infection: A Single-center Experience With 336 Procedures.

BACKGROUND: Indwelling pleural catheters (IPCs) offer ambulatory management of symptomatic persistent pleural effusions, but their widespread use is somewhat hampered by the risk of pleural infection and the inconvenience of carrying a catheter for a prolonged period of time. Factors associated with these 2 limitations were analyzed in this study. METHODS: Retrospective review of consecutive patients who had undergone IPC placement over a 5 ½-year period. Time to IPC removal was analyzed with the Fine and Gray competing risks survival model, with competing risk being death. A binary logistic regression method was used to evaluate factors influencing IPC-related pleural infections. RESULTS: A total of 336 IPCs were placed in 308 patients, mostly because of malignant effusions (83%). IPC removal secondary to pleurodesis was achieved in 170 (51%) procedures at a median time of 52 days. Higher rates of IPC removal were associated with an Eastern Cooperative Oncology Group (ECOG) grade of 0 to 2 [subhazard ratio (SHR)=2.22], an expandable lung (SHR=1.93), and development of a multiseptated pleural space (SHR=1.37). IPC-related pleural infections occurred in 8% of the cases, and were more often seen in hepatic hydrothoraces [odds ratio (OR)=4.75] and pleural fluids with a C-reactive protein <15 mg/L before the IPC insertion (OR=4.42). CONCLUSION: IPC removal is more likely to occur in patients with good performance status whose lungs fully expand after drainage. Hepatic hydrothorax is the most significant predictor of IPC-related infections.

Comparison of Killing Activity of Micafungin Against Six Candida Species Isolated from Peritoneal and Pleural Cavities in RPMI-1640, 10 and 30% Serum.

Currently echinocandins are recommended in Candida peritonitis and pleuritis. We determined micafungin killing rates (k values) at therapeutic concentrations (0.25-2 mg/L) in RPMI-1640 with and without 10 and 30% serum mimicking in vivo conditions against six Candida species isolated from peritoneal and pleural fluid. In RPMI-1640, micafungin was fungicidal against C. glabrata, C. krusei and C. kefyr within 2.27 ± 10.68, 2.69 ± 10.29 and 3.10 ± 4.41 h, respectively, while was fungistatic against C. albicans, C. tropicalis and C. parapsilosis. In 10% serum, ≥ 0.25, ≥ 0.5, ≥ 0.5 and ≥ 1 mg/L micafungin produced positive k values (killing) for all C. albicans, C. glabrata, C. kefyr and C. krusei, respectively. In 30% serum, 2 mg/L micafungin produced killing against all C. albicans, C. glabrata and C. kefyr isolates, but was ineffective against C. krusei, C. parapsilosis and 2 of 3 C. tropicalis. Micafungin exposure should be increased against non-albicans species to eradicate fungi from peritoneal and pleural cavities.

Loculated empyema due to tuberculosis in a child.

A 9-year-old girl from black ethnic origin presented with a history of fever, cough, loss of weight and right-sided chest wall pain for 2 weeks. Chest X-ray demonstrated an effusion, which was shown to be loculated on chest CT scan. She was not responding to medical treatment and at thoracotomy loculated pus was drained. Mycobacterium tuberculosis (TB) was cultured from the pus. TB is a rare cause of loculated empyema with an overlapping clinical and radiological picture with pyogenic infections.

Evaluation of the eazyplex MRSA assay for the rapid detection of Staphylococcus aureus in pleural and synovial fluid.

OBJECTIVE: Loop-mediated isothermal amplification (LAMP) is a simple and sensitive technique for rapid microbiological diagnosis. The aim of this study was to evaluate the analytical and diagnostic performance of the LAMP eazyplex MRSA test for the direct detection and differentiation of methicillin-susceptible (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) in synovial/pleural fluid. METHODS: Analytical validation included the determination of the limit of detection (LoD) and analytical specificity of the eazyplex MRSA test. A diagnostic evaluation of the eazyplex test against bacterial culture was performed on routine pleural/synovial samples collected prospectively from patients aged less than 18 years with complicated pneumonia with empyema or arthritis admitted to the Children's Hospital Sant Joan de Déu in Barcelona, Spain, between April 2015 and May 2016. RESULTS: The new system appropriately detected a quality control panel of clinical samples with DNA of MSSA, MRSA, and other pathogens. The LoD was established at 6.4×103 CFU/ml for S. aureus and 1.0×104 CFU/ml for MRSA. Diagnostic validation of the eazyplex MRSA assay was performed on 51 prospective clinical invasive samples, resulting in sensitivity and specificity values of 83.3% and 97.8%, respectively, for S. aureus detection. The mean turnaround time was 70min. CONCLUSIONS: The eazyplex MRSA assay was found to be a useful test for the rapid detection of S. aureus in invasive samples such as pleural/synovial fluid.

Paradoxical results of two automated real-time PCR assays in the diagnosis of pleural tuberculosis.

Tuberculosis (TB) is a major cause of worldwide mortality. We report the case of a non-HIV-infected woman with clinical suspicion of pleural tuberculosis and contradictory results between Xpert® MTB/RIF and Abbott RealTime MTB assays from pleural fluid specimen. Liquid and solid cultures for tuberculosis were performed with negative results. The patient received treatment, and clinical improvement was observed. Both techniques detect Mycobacterium tuberculosis complex, but they have different targets and limits of detection. Abbott RealTime MTB results correlated well with the clinical findings of the patient.

Clinical and Laboratory Differences between Lymphocyte- and Neutrophil-Predominant Pleural Tuberculosis.

Pleural tuberculosis (TB), a form of extrapulmonary TB, can be difficult to diagnose. High numbers of lymphocytes in pleural fluid have been considered part of the diagnostic criteria for pleural TB; however, in many cases, neutrophils rather than lymphocytes are the predominant cell type in pleural effusions, making diagnosis more complicated. Additionally, there is limited information on the clinical and laboratory characteristics of neutrophil-predominant pleural effusions caused by Mycobacterium tuberculosis (MTB). To investigate clinical and laboratory differences between lymphocyte- and neutrophil-predominant pleural TB, we retrospectively analyzed 200 patients with the two types of pleural TB. Of these patients, 9.5% had neutrophil-predominant pleural TB. Patients with lymphocyte-predominant and neutrophil-predominant pleural TB showed similar clinical signs and symptoms. However, neutrophil-predominant pleural TB was associated with significantly higher inflammatory serum markers, such as white blood cell count (P = 0.001) and C-reactive protein (P = 0.001). Moreover, MTB was more frequently detected in the pleural fluid from patients in the neutrophil-predominant group than the lymphocyte-predominant group, with the former group exhibiting significantly higher rates of positive results for acid-fast bacilli in sputum (36.8 versus 9.4%, P = 0.003), diagnostic yield of MTB culture (78.9% versus 22.7%, P < 0.001) and MTB detected by polymerase chain reaction (31.6% versus 5.0%, P = 0.001). Four of seven patients with repeated pleural fluid analyses revealed persistent neutrophil-predominant features, which does not support the traditional viewpoint that neutrophil-predominant pleural TB is a temporary form that rapidly develops into lymphocyte-predominant pleural TB. In conclusion, neutrophil-predominant pleural TB showed a more intense inflammatory response and a higher positive rate in microbiological testing compared to lymphocyte-predominant pleural TB. Pleural TB should be considered in neutrophil-predominant pleural effusions, and microbiological tests are warranted.

[ANALYSIS OF MICROFLORA OF PLEURAL CAVITY IN PLEURAL EMPYEMA].

In the pleural empyema (PE) treatment, not depending on introduction of multiple operative procedures and the medicinal preparations application, some issues remain unsolved, including the infection agents verification, the most rapid bronchial fistula elimination and the lung volume restoration. The EP infection agents spectrum, their sensitivity to preparations were revealed, as well as the enhanced rate of the methicillin-resistant stamms (MRSA) and the microorganisms associations verification. A reduction of the infection agents sensitivity towards "simple" antibacterial preparations was established, so the physicians, treating PE, must prescribe "hard" antibiotics, what enhances its cost.

Impact of fibrinolytics on the outcome of empyema in South African children.

BACKGROUND: Childhood pneumonia is common in all countries, and empyema is one of the commonest complications. The role of routine intrapleural fibrinolytics in the management of childhood empyema is not well established in low- and middle-income countries. METHODS: We did a prospective observational study of children sequentially hospitalised with empyema between December 2006 and December 2011 in South Africa (SA). Intrapleural tissue plasminogen activator (TPA), administered according to a standard protocol, was introduced in September 2009. Outcomes in children treated with TPA after 2009 were compared with the historical cohort not treated with TPA who met the treatment criteria. RESULTS: One hundred and forty-two children with empyema, median age 17 months (interquartile range 8-43), were admitted during the study period. Excluding children who did not have a chest tube inserted and those in whom fibrinolysis was contraindicated, there were 99 patients, 52 of whom received fibrinolytics. Clinical characteristics and empyema aetiology were similar in those who received fibrinolysis and those who did not. Eighteen children (38.3%) not treated with TPA required surgery v. 5 (9.6%) treated with TPA (relative risk 0.25; 95% confidence interval 0.1-0.6). The median duration of hospitalisation was similar in both groups. Complications occurred rarely and with a similar incidence in both groups. In-hospital mortality was low, with two deaths in each group. CONCLUSION: Intrapleural TPA resulted in a four-fold reduction in surgery. Fibrinolytics should be used for management of empyema in children in SA.

Diagnostic performance of different pleural fluid biomarkers in tuberculous pleurisy.

Due to the paucibacillary nature of tuberculous pleural effusion the diagnosis of pleural tuberculosis is challenging. This prospective study was undertaken to evaluate the diagnostic performance of ten different pleural fluid biomarkers in the differentiation between tuberculous and non-tuberculous pleural effusions. Two hundred and three patients with pleural effusion (117 men and 86 women, median age 65 years) were enrolled. Routine diagnostic work-up, including thoracentesis and pleural fluid analysis, was performed to determine the cause of pleural effusion. The following biomarkers were measured in pleural fluid: adenosine deaminase (ADA), interferon gamma (IFN-γ), interleukin 2 soluble receptor (IL-2sRα), sub-unit p40 of interleukin 12b (IL-12p40), interleukin 18 (IL-18), interleukin 23 (IL-23), IFN-γ induced protein 10 kDa (IP-10), Fas-ligand, human macrophage-derived chemokine (MDC) and tumor necrosis factor alfa (TNF-α). There were 44 (21.7%) patients with tuberculous pleural effusion, 88 (43.3%) patients with malignant pleural effusion, 35 (17.2%) with parapneumonic effusion/pleural empyema, 30 (14.8%) with pleural transudates, and 6 (3%) with miscellaneous underlying diseases. Pleural fluid IFN-γ was found the most accurate marker differentiating tuberculous from non-tuberculous pleural effusion, with sensitivity, specificity, PPV, NPV, and AUC 97%, 98%, 95.5%, 99.4%, and 0.99, respectively. Two other biomarkers (IP-10 and Fas ligand) also showed very high diagnostic accuracy with AUC≥0.95. AUC for ADA was 0.92. We conclude that IFN-γ, IP-10, and Fas-ligand in pleural fluid are highly accurate biomarkers differentiating tuberculous from non-tuberculous pleural effusion.

Development of primary invasive pneumococcal disease caused by serotype 1 pneumococci is driven by early increased type I interferon response in the lung.

The pneumococcus is the world's foremost respiratory pathogen, but the mechanisms allowing this pathogen to proceed from initial asymptomatic colonization to invasive disease are poorly understood. We have examined the early stages of invasive pneumococcal disease (IPD) by comparing host transcriptional responses to an invasive strain and a noninvasive strain of serotype 1 Streptococcus pneumoniae in the mouse lung. While the two strains were present in equal numbers in the lung 6 h after intranasal challenge, only the invasive strain (strain 1861) had invaded the pleural cavity at that time point; this correlated with subsequent development of bacteremia in mice challenged with strain 1861 but not the noninvasive strain (strain 1). Progression beyond the lung was associated with stronger induction of the type I interferon (IFN-I) response in the lung at 6 h. Suppression of the IFN-I response through administration of neutralizing antibody to IFNAR1 (the receptor for type I interferons) led to significantly reduced invasion of the pleural cavity by strain 1861 at 6 h postchallenge. Our data suggest that strong induction of the IFN-I response is a key factor in early progression of invasive serotype 1 strain 1861 beyond the lung during development of IPD.

Empyema necessitans: very late complication of pulmonary tuberculosis.

Empyema necessitans is a rare clinical finding nowadays. We report the case of a patient admitted in our ward for investigation of an unknown onset anterior chest wall mass, with no accompanying signs or symptoms. It is noteworthy that the patient had had pulmonary tuberculosis submitted to thoracoplasty more than 60 years before. Thoracic MRI showed a large heterogeneous mass, with a thick wall and internal septations located at the right anterior chest wall, as well as a heterogeneous content inside the right pleural cavity, with direct communication between both. An aspirative puncture of both masses was performed, with positive cultures for Mycobacterium tuberculosis, thus leading to the diagnosis of pleural tuberculosis with anterior chest wall empyema necessitans. A drain was inserted and antibiotics started. This case draws our attention to a very rare complication of pulmonary tuberculosis and its surgical treatment, though it aroused many decades after primary infection.

Aspergillus fumigatus fungus ball in the pleural cavity.

OBJECTIVE: To report the cases of 6 patients with fungus ball caused by Aspergillus fumigatus (aspergilloma) in the pleural cavity. METHODS: Between 1980 and 2009, 391 patients were diagnosed with aspergilloma at the Santa Casa Hospital Complex in Porto Alegre, Brazil. The diagnosis of aspergilloma in the pleural cavity was made through imaging tests revealing effusion and pleural thickening with air-fluid level; direct mycological examination revealing septate hyphae, consistent with Aspergillus sp.; and positive culture for A. fumigatus in the surgical specimen from the pleural cavity. RESULTS: Of the 391 patients studied, 6 (2%) met the established diagnostic criteria. The mean age of those 6 patients was 48 years (range, 29-66 years), and 5 (83%) were male. The most common complaints were cough, expectoration, and hemoptysis. Four patients (67%) had a history of tuberculosis that had been clinically cured. All of the patients were submitted to surgical removal of the aspergilloma, followed by intrapleural instillation of amphotericin B, in 4; and 2 received systemic antifungal treatment p.o. There was clinical improvement in 5 patients, and 1 died after the surgery. CONCLUSIONS: In adult patients with a history of cavitary lung disease or pleural fistula, a careful investigation should be carried out and fungal infection, especially aspergilloma, should be taken into consideration. In such cases, laboratory testing represents the most efficient use of the resources available to elucidate the diagnosis.

Bola fúngica por Aspergillus fumigatus em cavidade pleural / Aspergillus fumigatus fungus ball in the pleural cavity

OBJETIVO: Relatar os casos de 6 pacientes com bola fúngica (BF) na cavidade pleural por Aspergillus fumigatus. MÉTODOS: Entre 1980 e 2009, foram diagnosticados 391 pacientes com BF aspergilar no Complexo Hospitalar Santa Casa de Porto Alegre (RS). O diagnóstico de BF na cavidade pleural foi definido com exames de imagem demonstrando derrame e espessamento pleural com nível líquido; exame micológico direto demonstrando hifas septadas, consistentes com Aspergillus sp.; e cultura positiva para A. fumigatus no espécime cirúrgico da cavidade pleural. RESULTADOS: Dos 391 pacientes estudados, 6 (2 por cento) preencheram os critérios diagnósticos estabelecidos. A média de idade desses 6 pacientes foi de 48 anos (variação, 29-66 anos), e 5 (83 por cento) eram do sexo masculino. As queixas mais frequentes dos pacientes foram tosse, expectoração e hemoptise. Quatro (67 por cento) dos pacientes tinham tuberculose curada. Todos os pacientes realizaram remoção cirúrgica da colonização fúngica, e houve infusão intrapleural com anfotericina B em 4; e 2 pacientes receberam tratamento antifúngico sistêmico v.o. Cinco pacientes melhoraram clinicamente, e um foi a óbito após a cirurgia. CONCLUSÕES: Em pacientes adultos com história de doença pulmonar cavitária ou fístula pleural, deve-se realizar uma investigação criteriosa levando em consideração a infecção fúngica, principalmente BF pulmonar. Portanto, a exploração laboratorial torna-se mais eficiente em relação aos recursos disponíveis para elucidação diagnóstica.

OBJECTIVE: To report the cases of 6 patients with fungus ball caused by Aspergillus fumigatus (aspergilloma) in the pleural cavity. METHODS: Between 1980 and 2009, 391 patients were diagnosed with aspergilloma at the Santa Casa Hospital Complex in Porto Alegre, Brazil. The diagnosis of aspergilloma in the pleural cavity was made through imaging tests revealing effusion and pleural thickening with air-fluid level; direct mycological examination revealing septate hyphae, consistent with Aspergillus sp.; and positive culture for A. fumigatus in the surgical specimen from the pleural cavity. RESULTS: Of the 391 patients studied, 6 (2 percent) met the established diagnostic criteria. The mean age of those 6 patients was 48 years (range, 29-66 years), and 5 (83 percent) were male. The most common complaints were cough, expectoration, and hemoptysis. Four patients (67 percent) had a history of tuberculosis that had been clinically cured. All of the patients were submitted to surgical removal of the aspergilloma, followed by intrapleural instillation of amphotericin B, in 4; and 2 received systemic antifungal treatment p.o. There was clinical improvement in 5 patients, and 1 died after the surgery. CONCLUSIONS: In adult patients with a history of cavitary lung disease or pleural fistula, a careful investigation should be carried out and fungal infection, especially aspergilloma, should be taken into consideration. In such cases, laboratory testing represents the most efficient use of the resources available to elucidate the diagnosis.

Steady antibiotic release from biodegradable beads in the pleural cavity: an in vitro and in vivo study.

BACKGROUND: Inadequate localized drug concentrations and systemic adverse effects are among the concerns when regional infections are treated with systemic antibiotics. We designed and fabricated a poly(D,L)-lactide-co-glycolide (PLGA)-based biodegradable drug delivery system and evaluated the release of antibiotics both in vitro and in vivo. METHODS: PLGA copolymer and penicillin G sodium were mixed, compressed, and sintered to fabricate biodegradable antibiotic beads. The beads were placed in phosphate-buffered saline to test the characteristics of in vitro drug release. The beads then were introduced into the pleural cavities through chest tubes of six New Zealand white rabbits. Daily pleural effusion was collected to measure the antibiotic concentration and bacterial inhibitory characteristics. RESULTS: Forty percent of the penicillin was released in the first day in the in vitro study. The rest of the antibiotic was then gradually released in the following 30 days. All six animals survived the experiment. The initial surge of drug release was less significant in the pleural cavity than in the phosphate-buffered saline. The drug concentrations were well above the minimum inhibitory concentration breakpoint for penicillin susceptibility throughout the study period in both in vitro (30 days) and in vivo (14 days) studies. CONCLUSIONS: These preliminary findings demonstrated that the biodegradable PLGA antibiotic beads could achieve a fairly steady antibiotic release in the pleural cavity for at least 2 weeks. This drug delivery system may have the potential to serve as an adjuvant treatment of pleural cavity infection.