Invasive pulmonary aspergillosis is associated with adverse clinical outcomes in critically ill patients receiving veno-venous extracorporeal membrane oxygenation.

To identify the incidence, risk factors and impact on long-term survival of invasive pulmonary aspergillosis (IPA) and Aspergillus colonisation in patients receiving vv-extracorporeal membrane oxygenation (ECMO). A retrospective evaluation was performed of patients receiving vv-ECMO at a tertiary hospital in Manchester (UK) between January 2012 and December 2016. Data collected included epidemiological data, microbiological cultures, radiographic findings and outcomes. Cases were classified as proven IPA, putative IPA or Aspergillus colonisation according to a validated clinical algorithm. One hundred thirty-four patients were supported with vv-ECMO, median age of 45.5 years (range 16.4-73.4). Ten (7%) patients had putative IPA and nine (7%) had Aspergillus colonisation. Half of the patients with putative IPA lacked classical host risk factors for IPA. The median number of days on ECMO prior to Aspergillus isolation was 5 days. Immunosuppression and influenza A infection were significantly associated with developing IPA in a logistic regression model. Cox regression model demonstrates a three times greater hazard of death associated with IPA. Overall 6-month mortality rate was 38%. Patients with putative IPA and colonised patients had a 6-month mortality rate of 80 and 11%, respectively. Immunosuppression and influenza A infection are independent risk factors for IPA. IPA, but not Aspergillus colonisation, is associated with high long-term mortality in patients supported with vv-ECMO.

Impact of self-reported environmental mould exposure on COPD outcomes.

BACKGROUND: Indoor and outdoor mould exposure can affect respiratory symptoms, but its contribution to COPD outcomes such as exacerbation rates or antibiotics courses is not well defined. Some patients with COPD develop chronic pulmonary aspergillosis (CPA), but the contribution of environmental exposure is not known. METHODS: We correlated activities or exposures related to mould with COPD outcomes in patients with COPD with or without CPA using a questionnaire. RESULTS: One hundred and forty patients were included and 60 had CPA in addition to COPD. Seventy-six were male and mean age was 66.9 years (range 40-87). Thirty-nine (28%) were active cigarette smokers. On multivariate analysis, occupational contact with agricultural resources (p = 0.017), vacuuming once weekly or more often (p = 0.026) and not asking visitors to remove shoes on home entry (p = 0.035) were significantly more common in participants reporting ≥ 4 office visits for COPD symptoms in the last year. Living within one mile of industrial composting sites (p = 0.013), vacuuming once weekly or more often (p = 0.016) and not asking visitors to remove shoes on home entry (p = 0.028) were significantly more common in participants reporting ≥4 antibiotics courses in the last year. Patients with CPA showed a trend for residence within one mile of farms or agricultural areas (P = 0.088, OR 2, 95% CI 0.9-4.4). CONCLUSION: Activities potentially leading to mould exposure were common in a population with COPD with or without CPA and were associated with adverse COPD outcomes. Environmental mould exposure may play a role in the development of CPA in patients with COPD.

High-volume culture and quantitative real-time PCR for the detection of Aspergillus in sputum.

OBJECTIVES: Sputum culture is an insensitive method for the diagnosis of pulmonary aspergillosis. Growth of the organism allows identification of the causative species and susceptibility testing, both of which can inform treatment choices. The current practice is to culture an aliquot of diluted sputum. We assessed the value of culturing large volumes of unprocessed sputum, a method that we have termed high-volume culture (HVC). METHODS: Specimens were processed by conventional culture (using an aliquot of homogenized, diluted sputum on Sabouraud agar at 37°C and 45°C for up to 5 days) and HVC (using undiluted sputum on Sabouraud agar at 30°C for up to 14 days). A separate specimen was tested by quantitative real-time PCR. Antifungal susceptibility testing was performed by the EUCAST standard. RESULTS: We obtained sputum specimens from 229 individuals with the following conditions: chronic pulmonary aspergillosis (66.8%, 153/229), allergic bronchopulmonary aspergillosis (25.3%, 58/229) and Aspergillus bronchitis (7.9%, 18/229). Individuals with invasive pulmonary aspergillosis were not included. The positivity rate of conventional culture was 15.7% (36/229, 95% CI 11.6%-21.0%) and that of HVC was 54.2% (124/229, 95% CI 47.7%-60.5%) (p < 0.001). The higher positivity rate of HVC was demonstrated regardless of administration of antifungal treatment. Quantitive real-time PCR had an overall positivity rate of 49.2% (65/132, 95% CI 40.9%-57.7%), comparable to that of HVC. CONCLUSION: Detection of Aspergillus spp. in sputum is greatly enhanced by HVC. HVC allows for detection of azole-resistant isolates that would have been missed by conventional culture. This method can be performed in any microbiology laboratory without the need for additional equipment.

A retrospective TBNET assessment of linezolid safety, tolerability and efficacy in multidrug-resistant tuberculosis.

Linezolid is used to treat patients with multidrug-resistant (MDR)/extensively drug-resistant (XDR)-tuberculosis (TB) cases, although clinical data on its safety, tolerability and efficacy are lacking. We performed a retrospective, nonrandomised, unblinded observational study evaluating the safety and tolerability of linezolid at 600 mg q.d. or b.i.d. in MDR/XDR-TB treatment in four European countries. Efficacy evaluation compared end-points of 45 linezolid-treated against 110 linezolid-nontreated cases. Out of 195 MDR/XDR-TB patients, 85 were treated with linezolid for a mean of 221 days. Of these, 35 (41.2%) out of 85 experienced major side-effects attributed to linezolid (anaemia, thrombocytopenia and/or polyneuropathy), requiring discontinuation in 27 (77%) cases. Most side-effects occurred after 60 days of treatment. Twice-daily administration produced more major side-effects than once-daily dosing (p = 0.0004), with no difference in efficacy found. Outcomes were similar in patients treated with/without linezolid (p = 0.8), although linezolid-treated cases had more first-line (p = 0.002) and second-line (p = 0.02) drug resistance and a higher number of previous treatment regimens (4.5 versus 2.3; p = 0.07). Linezolid 600 mg q.d. added to an individualised multidrug regimen may improve the chance of bacteriological conversion, providing a better chance of treatment success in only the most complicated MDR/XDR-TB cases. Its safety profile does not warrant use in cases for which there are other, safer, alternatives.

Epidemiology and clinical management of XDR-TB: a systematic review by TBNET.

Extensively drug-resistant tuberculosis (XDR-TB) is present in all regions and poses serious challenges for public health and clinical management. Laboratory diagnosis is difficult and little evidence exists to guide clinicians in treating people with XDR-TB effectively. To summarise the available data on diagnosis and treatment, the current authors performed a systematic review on 13 recent studies of the epidemiology and clinical management of XDR-TB. Studies that met inclusion criteria were reviewed, in order to assess methodology, treatment regimens and treatment outcomes. Meta-analysis of currently available data is not possible because of inconsistent definitions and methodologies. Data show that XDR-TB can be successfully treated in up to 65% of patients, particularly those who are not co-infected with HIV. However, treatment duration is longer and outcomes are in general poorer than for non-XDR TB patients. To strengthen the evidence for extensively drug-resistant tuberculosis diagnosis, treatment and prevention, future studies should: 1) be prospective in design; 2) adopt standardised, internationally accepted definitions; 3) use quality-assured laboratory testing for all first- and second-line drugs; and 4) collect data on an agreed-upon set of standard variables, allowing for comparisons across studies. Early diagnosis and aggressive management of extensively drug-resistant tuberculosis provide the best chance of positive outcome, but prevention is still paramount.

Successfully Engaging Private Providers to Improve Diagnosis, Notification, and Treatment of TB and Drug-Resistant TB: The EQUIP Public-Private Model in Chennai, India.

BACKGROUND: Private physicians in India see and treat more than half of all people with tuberculosis (TB) each year and thus have potential to make significant contributions to TB control. The EQUIP project was designed as a prospective cohort study to assess the potential of private providers to diagnose and appropriately treat drug-resistant TB (DR-TB) in the Central and South districts of Chennai, India. METHODS: The private-sector engagement model consisted of free access to rapid diagnostics; choice of free daily or thrice-weekly treatment regimens; support for notification of patients; and patient support including directly observed therapy through EQUIP centers staffed by a community-based interface agency. Data were collected on provider participation; referral results; treatment regimens prescribed; and treatment outcomes. RESULTS: From October 2015 through June 2017, 227 of the 466 (48.7%) private providers approached referred at least 1 patient to an EQUIP center for evaluation. A total of 2,621 patients received testing and 1,232 (47.0%) were diagnosed with TB. Of those, 727 (59.0%) were bacteriologically confirmed, including 694 (56.3%) using GeneXpert and 33 (2.7%) using smear microscopy. A total of 26 (3.7% of GeneXpert diagnosed) patients were confirmed as rifampicin-resistant cases. EQUIP-related notifications comprised approximately 10% of TB and DR-TB notifications in Chennai during the project period. The project initiated 1,167 (96.8%) drug-sensitive TB patients on treatment. Of those, 691 (59.2%) received standard daily regimens with EQUIP support and 288 (24.7%) received standard intermittent regimens. At the time of writing, 89.4% of 868 drug-susceptible TB patients receiving EQUIP support had treatment success. Of the 26 rifampicin-resistant TB cases notified, 20 (77%) started and continued on second-line treatment; 2 died and 4 were lost to follow-up prior to treatment initiation. CONCLUSION: Private providers can make a substantial contribution to detection and appropriate treatment of patients with TB and DR-TB in India when provided with access to rapid diagnostics, support for notification and patient treatment through interface agencies, and free, quality anti-TB drugs.

Resistance to second-line injectables and treatment outcomes in multidrug-resistant and extensively drug-resistant tuberculosis cases.

No information is currently available on the influence of injectable second-line drugs on treatment outcomes of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) patients. To investigate this issue, a large series of MDR- and XDR-TB cases diagnosed in Estonia, Germany, Italy and the Russian Federation (Archangels Oblast) between 1999 and 2006 were analysed. All study sites performed drug susceptibility testing for first- and second-line anti-TB drugs, laboratory quality assurance and treatment delivery according to World Health Organization recommendations. Out of 4,583 culture-confirmed cases, 240 MDR- and 48 XDR-TB cases had a definitive outcome recorded (treatment success, death, failure). Among MDR- and XDR-TB cases, capreomycin resistance yielded a higher proportion of failure and death than capreomycin-susceptible cases. Resistance to capreomycin was independently associated with unfavourable outcome (logistic regression analysis: odds ratio 3.51). In the treatment of patients with multidrug-resistant and extensively drug-resistant tuberculosis, resistance to the injectable drug capreomycin was an independent predictor for therapy failure in this cohort. As Mycobacterium tuberculosis drug resistance is increasing worldwide, there is an urgent need for novel interventions in the fight against tuberculosis.

Chronic pulmonary aspergillosis as a cause of smear-negative TB and/or TB treatment failure in Nigerians.

OBJECTIVE: To evaluate chronic pulmonary aspergillosis (CPA) as an alternative diagnosis of smear-negative tuberculosis (TB) and treatment failure in TB patients in Nigeria. METHODS: We conducted a cross-sectional multicentre survey in human immunodeficiency virus (HIV) positive and negative adult patients at the end of their TB treatment in clinics in Lagos and Ilorin states. All were assessed using clinical examination, chest X-ray (CXR) and aspergillus immunoglobulin G (IgG) serology, and some for sputum fungal culture. CPA was defined as a positive Aspergillus fumigatus IgG titre with compatible CXR or a positive sputum culture of Aspergillus with a visible fungal ball on CXR with symptoms of underlying lung disease. RESULTS: Of 208 patients recruited between June 2014 and May 2015, 153 (73.6%) were HIV-positive. The mean age was 39.8 years, 124 (59.6%) were female and 39 (18.8%) were unable to work. The median CD4 count was 169.5 cells/ml (range 4-593) in HIV-infected patients with positive Aspergillus IgG. Overall, 109 (52.4%) had documented TB, 140 (67.3%) had a productive cough and 50 had haemoptysis. CPA prevalence was 8.7%; 10 (6.5%) had HIV infection and 8 (14.5%) were HIV-negative (Fisher's exact P = 0.092). CONCLUSION: CPA is a neglected disease in Nigeria, and most cases match the World Health Organization diagnostic criteria for smear-negative TB.

Beta(2)-adrenergic and several other G protein-coupled receptors in human atrial membranes activate both G(s) and G(i).

Cardiac G protein-coupled receptors that couple to Galpha(s) and stimulate cAMP formation (eg, beta-adrenergic, histamine, serotonin, and glucagon receptors) play a key role in cardiac inotropy. Recent studies in rodent cardiac myocytes and transfected cells have revealed that one of these receptors, the beta(2)-adrenergic receptor (AR), also couples to the inhibitory G protein Galpha(i) (activation of which inhibits cAMP formation). If beta(2)ARs could be shown to couple to Galpha(i) in the human heart, it would have important ramifications, because levels of Galpha(i) increase with age and in failing human heart. Therefore, we investigated whether beta(2)ARs in the human heart activate Galpha(i). By photoaffinity labeling human atrial membranes with [(32)P]azidoanilido-GTP, followed by immunoprecipitation with antibodies specific for Galpha(i), we found that Galpha(i) is activated by stimulation of beta(2)ARs but not of beta(1)ARs. In addition, we found that other Galpha(s)-coupled receptors also couple to Galpha(i), including histamine, serotonin, and glucagon. When coupling of these receptors to Galpha(i) is disrupted by pertussis toxin, their ability to stimulate adenylyl cyclase is enhanced. These data provide the first evidence that beta(2)AR and many other Galpha(s)-coupled receptors in human atrium also couple to Galpha(i) and that abolishing the coupling of these receptors to Galpha(i) increases the receptor-mediated adenylyl cyclase activity.

Diagnosis and prevention of fungal infection in the immunocompromized patient.

Profound and prolonged neutropenia following chemotherapy is a major risk factor for systemic fungal infections. Mortality associated with disseminated fungal infection is high and treatment with conventional amphotericin B is complicated by renal toxicity. Candida and Aspergillus are among the major pathogens in this patient population. Many patients remaining neutropenic over a prolonged period of time will receive empirical antifungal therapy. The clinical and laboratory diagnosis of these infections are neither sensitive nor specific and are generally limited in the early detection of invasive fungal infection. However, several new approaches to diagnosis are being developed which should be translated into routine practice. These include antigen detection and PCR. It is still unclear how effective the various measures that are currently being used are in preventing serious fungal infection. Refinements in the prophylactic use of fluconazole, itraconazole and aerosolized amphoteric in B, and the introduction of new formulations of existing antifungals may reduce the incidence of systemic fungal infections in some patient groups. Patients with presumed fungal infection require more intense and accurate monitoring for signs of disseminated infection. Early diagnosis may guide appropriate treatment and prevent mortality.

Matrix deposition by a calcifying human osteogenic sarcoma cell line (SAOS-2).

Procollagen and proteoglycan biosynthesis was defined in long-term culture of a human osteogenic sarcoma cell line, SAOS-2. An osteoblast phenotype was maintained by these cells up to 40 days post-confluent in the presence of ascorbic acid. Under these conditions, cells deposited around them an extensive collagenous matrix that was able to mineralize in the presence of an exogenous phosphate donor (beta-glycerophosphate). The collagenous matrix was comprised predominantly of collagen type I with significant amounts of collagen type V, and greater than 80% of the collagen in the matrix was involved in covalent crosslinkages. With increasing time in culture there was a decrease in the collagen synthetic rate, although the collagenous matrix was maintained. The proteoglycans synthesized by nonmineralizing and mineralizing cultures were purified after biosynthetic labeling with [35S]sulfate and [3H]glucosamine. Two major species were apparent: a large chondroitin sulfate proteoglycan (CSPG), and a small chondroitin sulfate proteoglycan, decorin. In nonmineralizing cultures, decorin partitioned equally between the cell layer and culture medium, whereas the large CSPG species partitioned exclusively into the cell layer-associated matrix. In the presence of extensive mineral deposition, greater than 90% of the newly synthesized proteoglycans were secreted into the medium. Northern blot hybridization indicated that SAOS-2 cells express mRNA encoding a range of bone proteins, including decorin, osteonectin, and bone sialoprotein.

Antigen and antibody attachment in ELISA for Aspergillus fumigatus IgG antibodies.

Four commercial antigen extracts of Aspergillus fumigatus were evaluated for use in a rapid enzyme-linked immunosorbent assay (ELISA) for anti-A. fumigatus IgG. Initial binding of both somatic and culture filtrate preparations to a polyvinyl chloride solid phase was concentration dependent and increased with incubation time. Antigen binding to the solid phase was reproducible. Binding of A. fumigatus precipitin-positive serum to bound antigen was rapid. All four A. fumigatus antigens demonstrated similar dose-response curves when tested against pooled sera containing a high titre of A. fumigatus antibodies. Detectable activity in precipitin test-negative sera decreased rapidly with dilution. All the antigen preparations were found to be suitable for ELISA procedures and permit the rapid determination of IgG antibodies to A. fumigatus.

Differentiation of extracellular from ingested Candida albicans blastospores in phagocytosis tests by staining with fluorescein-labelled concanavalin A.

In phagocytosis tests with human polymorphonuclear (PMN) cells, extracellular blastospores of Candida albicans could be distinguished microscopically from ingested blastospores mor easily than with methods used previously by staining phagocyte monolayers with fluorescent-labelled concanavalin A. This stain reacted with the cell wall mannan of extracellular blastospores but seemed unable to reach that of the ingested blastospores. This technique both improves determination of phagocytic indices and checks that extracellular blastospores have been washed away before assessment of intracellular survival and growth.

The occurrence and treatment of false positive reactions in enzyme-linked immunosorbent assays (ELISA) for the presence of fungal antigens in clinical samples.

Non-specific positive reactions have been revealed in enzyme-linked immunosorbent assays (ELISA) of sera for the presence of fungal antigen. These false positives were recognized by their occurrence in tests for both Candida albicans and Aspergillus fumigatus antigens and by their response to dithiothreitol, combined with their reaction with non-immune rabbit globulin. A scheme is proposed which differentiates between true and false positive reactions. Use of fractionated anti-fungal globulin in conjugates reduced the incidence of false positive results in sera from hospitalized patients and eliminated them from sera of healthy subjects. The test scheme was applied to two panels of sera containing samples from patients with (a) invasive candidosis and (b) invasive aspergillosis. The relevance of ELISA tests for the detection of fungal antigen in human serum is discussed.

Computer-assisted rapid enzyme-linked immunosorbent assay (ELISA) in the serological diagnosis of aspergillosis.

A computer-assisted method for the positive/negative discrimination of ELISA data is described. This method was applied to a rapid ELISA procedure for IgG class antibodies to Aspergillus fumigatus in which the performance time of the test was reduced to 1 h. The method gives results which compare well with those by agar gel double diffusion (AGDD). The computer-assisted reading, calculation and tabulation of ELISA results from one microtitre plate is performed in less than 2 min and permits analysis of large numbers of specimens.

Measurement of serum arabinitol by gas-liquid chromatography: limitations for detection of systemic candida infections.

The measurement of serum arabinitol in the diagnosis of systemic candidosis was evaluated using a gas-liquid chromatography technique in a cohort of at risk patients. The prevalence of seropositivity was low and did not correlate with evidence of infection. This technique is unlikely to achieve acceptance because it does not discriminate between patients with and without infection; it requires specialised equipment and it is expensive.

Rapid enzyme-linked immunosorbent assay (ELISA) for Aspergillus fumigatus antibodies.

A rapid enzyme-linked immunosorbent assay (ELISA) where component incubation periods were shortened to one hour, was compared with agar gel double diffusion (AGDD) and a standard ELISA procedure for detecting antibodies to Aspergillus fumigatus in 28 asthmatic patients with suspected allergic aspergillosis. Using two A fumigatus antigens the rapid ELISA compared well with AGDD and the standard ELISA method. Eleven sera that reacted with both antigens in AGDD were all positive against antigen 1 in both forms of ELISA, but two failed to react with antigen 2 in the standard ELISA and three failed to react with this antigen in the rapid method. Thirteen AGDD-negative sera were also negative in both forms of ELISA. The rapid ELISA provides a sensitive and reproducible test for routine serological investigation of allergic aspergillosis.

Interaction of Candida krusei with human neutrophils in vitro.

In human neutrophil monolayer assays the percentage phagocytosis of Candida krusei by neutrophils was found to be significantly lower (9%) than that for C. albicans (37%). Both organisms required opsonisation with complement products for ingestion. The number of competent neutrophils phagocytosing C. krusei was increased with: antisera specific to C. albicans (49%); the neutrophil chemo-attractant formyl-methionyl-leucyl-phenylalanine (fmlp) (49%); mannan extracted from the cell wall of C. albicans (72%); and a crude cell extract from C. krusei (61%). In the case of C. albicans all but one of these methods increased the proportion of phagocytosing neutrophils per slide. The data provide evidence for differences in quantitative phagocytosis of C. krusei and C. albicans and suggest that C. krusei is resistant to phagocytosis in vitro.

GM-CSF-modulated phagocytosis of Trichosporon beigelii by human neutrophils.

Trichosporon beigelii has emerged as a lethal opportunist pathogen in granulocytopenic and corticosteroid-treated patients. Little is known of the host defence mechanisms against this yeast. The interaction between human neutrophils and serum-opsonised T. beigelii and the effect of GM-CSF on binding and ingestion of the yeast by neutrophils were investigated by a microscopic analysis of neutrophil monolayers stained with FITC-Concanavalin A. Positive staining with FITC-Concanavalin A distinguished between intracellular and extracellular yeast cells. Binding of T. beigelii to neutrophils was an energy- and complement-dependent process involving movement of actin in the neutrophil cytoskeleton. The mean percentage binding of T. beigelii was 37.5% and the mean binding index (BI) was 1.30 whereas the mean percentage ingestion was 3.5% and the mean phagocytic index (PI) was 1.34. GM-CSF increased percentage ingestion of T. beigelli from 2.8% to 30.5% and the PI was increased from 1.3 to 1.86. The percentage binding was 36.8% and the mean BI was 1.3 in control experiments compared with 49.3% and 1.6, respectively, in the presence of GM-CSF. In conclusion, GM-CSF significantly increased percentage ingestion of opsonised T. beigelii by neutrophils, but its effect on percentage binding of the yeast was not statistically significant.

Non-opsonic phagocytosis of Trichophyton mentagrophytes arthroconidia by human neutrophils in vitro.

A non-opsonic mechanism of binding and phagocytosis by human neutrophils of Trichophyton mentagrophytes arthroconidia is described. This was in direct contrast to the complement dependency of Candida albicans phagocytosis. Both serum complement and specific antibody to T. mentagrophytes promoted maximal phagocytosis (61% and 40% of neutrophils, respectively, contained arthroconidia). Increasing the ratio of arthroconidia to neutrophils did not increase non-opsonic phagocytosis (18-26%). Phagocytosis of arthroconidia exposed to trypsin in the absence of opsonin was not affected (18%). However, proteinase and chitinase reduced the level of non-opsonic and opsonic phagocytosis to negligible levels (6.3% and 4.5%, respectively). When mannose was added to neutrophils, mannose receptors on the phagocyte membrane were partially blocked when arthroconidia were opsonised, but this did not reduce the level of non-opsonic phagocytosis. The non-opsonic mechanism proposed here may have direct relevance in skin sites poor in opsonins.