Reinhard B. Raggam

Performance of Galactomannan, Beta-d-Glucan, Aspergillus Lateral-Flow Device, Conventional Culture, and PCR Tests with Bronchoalveolar Lavage Fluid for Diagnosis of Invasive Pulmonary Aspergillosis

ABSTRACT Galactomannan detection in bronchoalveolar lavage (BAL) fluid samples (GM test) is currently considered the gold standard test for diagnosing invasive pulmonary aspergillosis (IPA). The limitations, however, are the various turnaround times and availability of testing. We compared the performance of GM testing with that of conventional culture, an Aspergillus lateral-flow-device (LFD) test, a beta-d-glucan (BDG) test, and an Aspergillus PCR assay by using BAL fluid samples from immunocompromised patients. A total of 78 BAL fluid samples from 78 patients at risk for IPA (74 samples from Graz and 4 from Mannheim) collected between December 2012 and May 2013 at two university hospitals in Austria and Germany were included. Three patients had proven IPA, 14 probable IPA, and 17 possible IPA, and 44 patients had no IPA. The diagnostic accuracies of the different methods for probable/proven IPA were evaluated. The diagnostic odds ratios were the highest for the GM, PCR, and LFD tests. The sensitivities for the four methods (except culture) were between 70 and 88%. The combination of the GM (cutoff optical density index [ODI], >1.0) and LFD tests increased the sensitivity to 94%, while the combination of the GM test (>1.0) and PCR resulted in 100% sensitivity (specificity for probable/proven IPA, 95 to 98%). The performance of conventional culture was limited by low sensitivity, while that of the BDG test was limited by low specificity. We evaluated established and novel diagnostic methods for IPA and found that the Aspergillus PCR, LFD, and GM tests were the most useful methods for diagnosing the disease by using BAL fluid samples. In particular, the combination of the GM test and PCR or, if PCR is not available, the LFD test, allows for sensitive and specific diagnosis of IPA.

Novel Tests for Diagnosis of Invasive Aspergillosis in Patients with Underlying Respiratory Diseases

RATIONALE Invasive pulmonary aspergillosis has been increasingly reported in nonneutropenic patients, including those with underlying respiratory diseases. OBJECTIVES We compared the diagnostic performances of galactomannan, 1,3-β-D-glucan, and Aspergillus-specific lateral-flow device tests with that of conventional culture by using bronchoalveolar lavage fluid samples from patients with underlying respiratory diseases. METHODS We analyzed 268 bronchoalveolar lavage samples from 221 patients with underlying respiratory diseases (and without hematologic malignancy or previous solid organ transplantation) that were collected for routine microbiological workup between February 2012 and May 2014 at the University Hospital of Graz, Austria. Invasive pulmonary aspergillosis was defined according to European Organization of Research and Treatment of Cancer/Mycoses Study Group criteria modified for patients with respiratory diseases. MEASUREMENTS AND MAIN RESULTS Thirty-one patients (14%) had probable or proven, 25 possible, and the remaining 165 patients no invasive pulmonary aspergillosis. Probable/proven aspergillosis was associated with a significantly higher (P = 0.034) 30-day mortality rate of 32%. Sensitivities, specificities, and diagnostic odd ratios differed markedly between galactomannan (cut-off 0.5: optical density index, 0.97, 0.81, 124.4; cut-off 1.0: 0.97, 0.93, 422.1; cut-off 3.0: 0.61, 0.99, 109.8), β-D-glucan (cut-off 80 pg/ml: 0.90, 0.42, 6.57; cut-off 200 pg/ml: 0.70, 0.61, 3.7), lateral-flow device tests (0.77, 0.92, 41.8), and mycological culture (0.29, 0.97, 14). CONCLUSIONS Probable or proven invasive pulmonary aspergillosis was diagnosed in 14% of our study population and associated with significantly higher 30-day mortality rates. Although the performance of β-D-glucan was limited by low specificity and that of mycological culture by low sensitivity, the Aspergillus lateral-flow device seems to be a promising alternative to galactomannan testing, which remains the diagnostic gold standard for aspergillosis. Clinical trial registered with www.clinicaltrials.gov (NCT 02058316).

Posaconazole plasma concentrations and invasive mould infections in patients with haematological malignancies

Posaconazole (PCZ) is a triazole antifungal agent that has broad activity against pathogenic fungi and is increasingly used for prophylaxis and treatment of invasive mould infections (IMIs). PCZ is only available as an oral formulation, with varying absorption from the gastrointestinal tract. However, reports correlating PCZ plasma concentrations (PPCs) with breakthrough IMIs are rare. In this study, PPCs were analysed in a prospective, observational, single-centre study and the correlation of PPCs with breakthrough IMIs in patients with haematological malignancies was evaluated. Risk factors associated with low PPCs were further evaluated. A total of 109 PPCs were measured in 34 cases receiving PCZ prophylaxis (n=31) or treatment (n=3). Levels below the target of 0.5 μg/mL were detected in 24 (71%) of the 34 cases; in 15 (63%) of these 24 cases concentrations were found to be <0.20 μg/mL. Three patients receiving PCZ prophylaxis met the criteria of breakthrough infection. Notably, prior to development of IMI, PPCs were below the target in all three individuals. Associated risk factors for insufficient PPCs varied from previous reports. In conclusion, these data demonstrate that therapeutic drug monitoring of PCZ is mandatory in all patients with haematological malignancies as low PPCs are common and may be associated with development of IMIs.

Single-Run, Parallel Detection of DNA from Three Pneumonia-Producing Bacteria by Real-Time Polymerase Chain Reaction

A molecular assay for parallel detection of three bacteria, Chlamydia (C.) pneumoniae, Legionella (L.) spp., and Mycoplasma (M.) pneumoniae, in clinical specimens by a set of real-time polymerase chain reactions (PCRs) in a single run was evaluated. Bacterial DNAs were extracted by an automated DNA extraction protocol on the MagNA Pure LC System. Amplification and detection were done by real-time PCR on the LightCycler (LC) instrument. For amplification, specific oligonucleotides derived from the 16s rRNA genes of C. pneumoniae, L. spp., and M. pneumoniae were used. The three assays were complemented with an internal control (IC), a specially designed DNA fragment which contains the specific primer binding sites for the three PCRs. The IC was added to the samples, co-extracted, and co-amplified. Primers and hybridization probes were designed to suit one LC PCR program. LC PCRs were established, detection limits were determined, and clinical samples were tested. The detection limits were found between 5.0 and 0.5 IFU/CFU per PCR reaction for each of the bacteria. A total number of 100 clinical specimens were tested for validation of the molecular assay. Tested samples included 63 bronchoalveolar lavages (BALs) and 37 induced sputa specimens. The internal control was detected in all negative and low-positive samples; no inhibition was found throughout the whole study. Additionally, samples underwent testing by culture for L. spp., and M. pneumoniae; for C. pneumoniae, the serological microimmunofluorescence (MIF) test was used. In conclusion, the developed set of LC PCR assays permits parallel detection of C. pneumoniae, L. spp., and M. pneumoniae in a single LC run. This molecular assay may lead to accurate and early diagnosis of pneumonia produced by these three types of bacteria. The assay proved to be suitable for the high-throughput routine diagnostic laboratory.

Diagnostic accuracy of soluble urokinase plasminogen activator receptor (suPAR) for prediction of bacteremia in patients with systemic inflammatory response syndrome.

OBJECTIVES Soluble urokinase plasminogen activator receptor (suPAR) serum concentrations have recently been described to reflect the severity status of systemic inflammation. In this study, the diagnostic accuracy of suPAR, C-reactive protein (CRP), procalcitonin (PCT), and interleukin-6 (IL-6) to predict bacteremia in patients with systemic inflammatory response syndrome (SIRS) was compared. METHODS A total of 132 patients with SIRS were included. In 55 patients blood cultures had resulted positive (study group 1, Gram positive bacteria: Staphylococcus aureus and Streptococcus spp., n=15; study group 2, Gram-negative bacteria, n=40) and 77 patients had negative blood culture results (control group, n=77). Simultaneously with blood cultures suPAR, CRP, PCT, IL-6 and white blood count (WBC) were determined. RESULTS SuPAR values were significantly higher in study group 1 (median 8.11; IQR 5.78-15.53; p=0.006) and study group 2 (median 9.62; IQR 6.52-11.74; p<0.001) when compared with the control group (median 5.65; IQR 4.30-7.83). ROC curve analysis revealed an AUC of 0.726 for suPAR in differentiating SIRS patients with bacteremia from those without. The biomarkers PCT and IL-6 showed comparable results. Regarding combinations of biomarkers multiplying suPAR, PCT and IL-6 was most promising and resulted in an AUC value of 0.804. Initial suPAR serum concentrations were significantly higher (p=0.028) in patients who died within 28 days than in those who survived. No significant difference was seen for PCT, IL-6 and CRP. CONCLUSION In conclusion, suPAR, IL-6 and PCT may contribute to predicting bacteremia in SIRS patients.

European Organization for the Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) host factors and invasive fungal infections in patients with haematological malignancies

OBJECTIVES Fulfilment of host factors defined by the revised European Organization for the Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) criteria is required for establishing the diagnosis of possible or probable invasive fungal infection (IFI). This case-control study evaluates EORTC/MSG host factors among patients with haematological malignancies. METHODS Fifty-eight patients with haematological malignancies who developed probable (n = 38) or proven (n = 20) IFI over a 5 year period were retrospectively evaluated regarding EORTC/MSG host factors. Results were compared with those obtained from patients with haematological malignancies who did not develop IFI (116 patients who received systemic antifungal prophylaxis or empirical therapy and 116 patients who did not; all data collected in 2010). RESULTS Fourteen patients had invasive yeast infection and 44 patients had invasive mould infection (IMI). Prolonged neutropenia (35/58, 60% versus 29/116, 25%), prolonged systemic corticosteroid (cut-off 21 days: 13/58, 22% versus 6/116, 5%; cut-off 14 days: 18/58, 31% versus 9/116, 8%) and T cell suppressive therapy (35/44, 80% versus 69/116, 59%) were significantly associated with development of IFI/IMI in our cohort. Previous allogeneic stem cell transplantation (SCT; >6 months prior to episode) was not significantly associated with development of IMI (8/44, 18% versus 22/116, 19%), while recent SCT (<6 months prior to episode) was (11/44, 25% versus 12/116, 10%). CONCLUSIONS We conclude that host factors according to revised EORTC/MSG criteria were significantly associated with the development of IFI/IMI in our patients. Previous allogeneic SCT was not a predisposing host factor for the development of IMI. Concerning prolonged corticosteroid treatment, a cut-off of 14 days seems preferable to the proposed cut-off.

Link between leptin and interleukin-6 levels in the initial phase of obesity related inflammation

The mechanisms underlying the pathogenesis of obesity-related atherosclerosis remain to be clarified. To investigate the preclinical phase, interleukin-6 (IL-6) plasma levels were analyzed together with clinical, anthropometric, inflammatory, and metabolic variables in a well-defined cohort of 677 young and middle-aged overweight/obese and normal-weight subjects. In the juvenile and adult overweight/obese study group, IL-6 levels were increased significantly compared with normal-weight, age-matched controls (P < 0.001). In both juveniles and adults, higher levels of IL-6 were observed in obese compared with overweight participants. Subjects with metabolic syndrome (MS) had significantly higher IL-6 levels than those without MS. In juveniles, leptin, and in adults, the waist-to-height ratio, turned out to be the best predictor of IL-6 plasma levels in a multiple stepwise regression model. Taken together, in every age group, interleukin-6 is associated positively with the grade of overweight. Interestingly, leptin, which is the best known adipokine, is associated predictively with interleukin-6 plasma levels only in juveniles, which may indicate an important role of this molecule in the initiation of obesity-related inflammation.

Potential Factors for Inadequate Voriconazole Plasma Concentrations in Intensive Care Unit Patients and Patients with Hematological Malignancies

ABSTRACT Voriconazole plasma concentrations (VPCs) vary widely, and concentrations outside the therapeutic range are associated with either worse outcome in invasive aspergillosis (IA) or increased toxicity. The primary goal of this cohort study conducted in a real-life setting was to identify potential factors associated with inadequate VPCs in ICU patients and patients with hematological malignancies. Within a period of 12 months, trough VPCs were obtained and analyzed with high-performance liquid chromatography, and the adequate range was defined as 1.5 to 5.5 mg/liter. VPCs of <1.5 mg/liter were defined as low, whereas VPCs of >5.5 mg/liter were defined as potentially toxic. A total of 221 trough VPCs were obtained in 61 patients receiving voriconazole, and 124/221 VPCs (56%) were found to be low. Multivariate analysis revealed that low VPCs were significantly associated with clinical failure of voriconazole, prophylactic use, younger age, underlying hematological malignancy, concomitant proton pump inhibitor (PPI) (pantoprazole was used in 88% of the patients), and absence of side effects. Low VPCs remained an independent predictor of clinical failure of voriconazole. The defined adequate range was reached in 79/221 (36%) VPCs. In 18 samples (8%), potentially toxic levels were measured. Multivariate analysis revealed higher body mass index (BMI), absence of hematological malignancy, therapeutic application, and diarrhea as factors associated with potentially toxic VPCs. Neurotoxic adverse events occurred in six patients and were mostly associated with VPCs in the upper quartile of our defined adequate range. In conclusion, potential factors like younger age, prophylaxis, underlying hematological malignancy, BMI, and concomitant PPI should be considered within the algorithm of voriconazole treatment.

Bronchoalveolar lavage lateral-flow device test for invasive pulmonary aspergillosis in solid organ transplant patients: a semiprospective multicenter study.

Background Invasive pulmonary aspergillosis (IPA) remains an important cause of morbidity and mortality among patients undergoing solid organ transplantation (SOT). Because of the crude mortality of 80% to 90% in the absence of adequate treatment, timely diagnosis and early intervention with antifungal drugs are key factors in the successful treatment of IPA. Diagnosis, however, remains difficult. Therefore, new diagnostic tests are urgently needed. The Lateral-Flow Device (LFD) test is a rapid (15 min) single-sample point-of-care test that is based on the detection of an Aspergillus extracellular glycoprotein antigen by monoclonal antibody JF5. Methods This semiprospective multicenter study evaluated the LFD test for IPA diagnosis (established by galactomannan and culture results) by using bronchoalveolar lavage (BAL) samples from patients after SOT. Participating centers were the three Austrian Medical Universities of Innsbruck, Vienna, and Graz. Results Forty-seven BAL samples from 47 SOT patients were included (26 patients had undergone lung transplantation, 13 liver, 6 kidney, and 2 heart transplantation; 11 probable or proven IPA, 11 possible IPA, 25 no IPA) at the three Austrian Medical Universities of Innsbruck, Vienna, and Graz. Sensitivity and specificity, positive and negative predictive values, as well as diagnostic odds ratio of BAL LFD tests for probable IPA were 91%, 83%, 63%, 97%, and 50% (95% confidence interval, 5.4%–467%), respectively. Conclusion To conclude, the LFD test of BAL specimens is performed easily and provides accurate and rapidly available results in patients after SOT. Therefore, this new point-of-care test may be a promising diagnostic approach for detecting IPA using BAL specimens from SOT patients.

Impact of galactomannan testing on the prevalence of invasive aspergillosis in patients with hematological malignancies

Galactomannan (GM) is a polysaccharide component of the cell wall of Aspergillus spp. and is released into the hosts circulation by growing hyphae. GM testing of patients with hematological malignancies has been rarely considered in recent epidemiologic studies of invasive mould infections (IMIs). The aim of the investigation was to analyze the impact of GM testing on the reported prevalence of IMI by comparing detection rates of IMI before and after the introduction of this diagnostic procedure. Prevalence of IMI was assessed by conducting a prospective single-centre study over seven months in 2010. Results obtained were then compared to those obtained with a representative collection of patients assessed by the same investigators at the same institution over seven months in 2007, i.e., prior to the introduction of GM testing. We found that, in general, detection rates of invasive aspergillosis (IA) and invasive mould infections increased significantly after the introduction of GM analysis. This study may therefore indicate that GM testing has a significant impact on the reported prevalence of IMI. Broad usage of such testing in patients with hematological malignancies may be able to produce a realistic picture of IMI rates when current diagnostic criteria are applied.