Testing the performance of a prototype lateral flow device using bronchoalveolar lavage fluid for the diagnosis of invasive pulmonary aspergillosis in high‐risk patients

The diagnosis of invasive pulmonary aspergillosis (IPA) increasingly relies on non‐culture‐based biomarkers in bronchoalveolar lavage (BAL) fluid. The Aspergillus lateral flow device (LFD) is a rapid immunoassay that uses a novel Aspergillus monoclonal antibody to gain specificity. The objective of the study is to compare specificity and sensitivity of the prototype LFD and the galactomannan (GM) enzyme immunoassay in BAL fluid in high‐risk patients. A total of 114 BAL samples from 106 patients at high risk for IPA were studied: 8 patients had proven/probable IPA, 16 had possible IPA and 82 did not have IPA. In patients with proven/probable IPA, specificity of LFD was 94% and GM was 89%; sensitivity of LFD was 38% and GM was 75%. Negative predictive value (NPV) for LFD was 94% and for GM was 98%; positive predictive value (PPV) was 38% for both tests. The use of anti‐mould prophylaxis did not affect specificity but resulted in decreased NPV of both LFD and GM. Union and intersection analysis showed no improvement in the performance by using both tests. Among patients at risk for IPA, the diagnostic performance of LFD and GM in BAL fluid appears comparable; specificity is high, but sensitivity of both LFD and GM is poor.     

Utility of galactomannan antigen detection in bronchoalveolar lavage fluid in immunocompromised patients

Diagnosis of invasive pulmonary aspergillosis (IPA) is a challenging process in immunocompromised patients. Galactomannan (GM) antigen detection in bronchoalveolar lavage (BAL) fluid is a method to detect IPA with improved sensitivity over conventional studies. We sought to determine the diagnostic yield of BAL GM assay in a diverse population of immunocompromised patients. A retrospective review of 150 fiberoptic bronchoscopy (FOB) with BAL for newly diagnosed pulmonary infiltrate in immunocompromised patients was performed. Patient information, procedural details and laboratory studies were collected. BAL and serum samples were evaluated for GM using enzyme‐linked immunoassay. Of 150 separate FOB with BAL, BAL GM was obtained in 143 samples. There were 31 positive BAL GM assays. In those 31 positive tests, 13 were confirmed as IPA, giving a positive predictive value of 41.9%. There was one false negative BAL GM. Of the 18 false positive BAL GM, 4 were receiving piperacillin–tazobactam and 11 were receiving an alternative beta‐lactam antibiotic. BAL GM assay shows excellent sensitivity for diagnosing IPA. There was a significant number of false positive BAL GM assays and several of those patients were receiving beta‐lactam antibiotics at the time of bronchoscopy.     

Clinical evaluation of the newly formatted lateral‐flow device for invasive pulmonary aspergillosis

The study evaluated the newly formatted Aspergillus‐specific lateral‐flow‐device (LFD), and compared its performance to the original prototype “old” LFD test using BALF samples from 28 patients (14 patients with probable/proven invasive pulmonary aspergillosis [IPA] and 14 patients with no evidence for IPA). A total of 10/14 (71%) of BALF samples from patients with probable/proven IPA resulted positive with the new LFD, including 8/9 with true‐positive and 2/5 with false‐negative results with the old LFD. All 14 samples from patients without IPA resulted negative with the new LFD; specificity of the new LFD was significantly improved compared to the old LFD.     

Detection of (1→3)‐β‐D‐glucan in same‐day urine and serum samples obtained from patients with haematological malignancies

Serum 1,3‐beta‐d ‐glucan (BDG) testing is an established diagnostic marker for invasive fungal infections (IFI) among patients with haematological malignancies. In contrast limited data exist regarding the application of urine BDG testing. Same‐day midstream urine and serum screening samples were collected in adult patients with underlying haematological malignancies. A total of 80 urine samples from 46 patients were investigated: Twenty‐six had positive corresponding serum BDG >120 pg ml^?1, 27 intermediate (60–80 pg ml^?1), and 27 negative serum BDG (<25 pg ml^?1). A significant positive correlation between BDG in serum and urine samples was observed (P = 0.025; r = 0.252). Sensitivity, specificity, positive predictive value and negative predictive value (compared with same‐day serum results) were: 42%, 76%, 46%, 73% when using an 80 pg ml^?1 urine cut‐off, and 35%, 96%, 82%, 75% for a 250 pg ml^?1 cut‐off. Urine BDG seemed to be higher in samples obtained from patients with probable IFI (n = 13, median 145, IQR 22–253) compared to those from patients without IFI (n = 56, median 24, IQR 15–88) but the difference was not significant (P = 0.069). Overall correlation of same‐day urine BDG and serum BDG was moderate. However, urine BDG testing may warrant further investigation in larger studies, as high‐positive urine results correlated with high‐positive corresponding serum levels and clinical performance was comparable to serum BDG.     

Efficacy and safety of combination antifungal therapy in Korean haematological patients with invasive aspergillosis

This randomised, double‐blind, placebo‐controlled trial assessed the efficacy, safety and tolerability of voriconazole+anidulafungin (combination) or voriconazole+placebo (monotherapy) for invasive aspergillosis (IA; NCT00531479). We present a post hoc analysis of Korean and non‐Korean patients with IA (including baseline positive serum galactomannan [GM]). Immunocompromised patients ≥ 16 years with IA were randomised 1:1, combination or monotherapy, for ≥ 2 weeks’ treatment. The primary endpoint was 6‐ and 12‐week all‐cause mortality (Korean modified intent‐to‐treat [mITT] population). Overall, 454 patients enrolled (Koreans: 56 [combination: 28, monotherapy: 28], non‐Koreans: 398 [combination: 200, monotherapy: 198]). The mITT population comprised 40 Koreans (combination: 23; monotherapy: 17) and 237 non‐Koreans (combination: 112; monotherapy: 125). Week 6 treatment difference in mortality rate between combination and monotherapy was ?6.4% in non‐Koreans. This reduction was more marked in Koreans (?22.4%). Week 12 difference in all‐cause mortality between combination and monotherapy was ?17.7% (Koreans) and ?20.2% at Week 6 (Koreans; positive baseline GM). Week 6 mortality (Koreans [mITT]; baseline GM >0.5‐2.0) was 0/13 (combination) and 2/6 (monotherapy). Serious adverse events were numerically higher for combination than monotherapy (Koreans: 57.1%, 46.4%; non‐Koreans: 49.5%, 46.0%). In Koreans, combination therapy was associated with marginally better outcomes than monotherapy and more so than in non‐Koreans.     

Prospective evaluation of a combination of fungal biomarkers for the diagnosis of invasive fungal disease in high‐risk haematology patients

We prospectively evaluated a combination of fungal biomarkers in adult haematology patients with focus on their clinical utility at different time points during the course of infection. In total, 135 patients were monitored once to twice weekly for serum (1‐3)‐ß‐d ‐glucan (BG), galactomannan (GM), bis‐methyl‐gliotoxin and urinary d ‐arabinitol/l ‐arabinitol ratio. In all, 13 cases with proven or probable invasive fungal disease (IFD) were identified. The sensitivity of BG and GM at the time of diagnosis (TOD) was low, but within 2 weeks from the TOD the sensitivity of BG was 92%. BG >800 pg/mL was highly specific for IFD. At a pre‐test probability of 12%, both BG and GM had negative predictive values (NPV) >0.9 but low positive predictive values (PPV). In a subgroup analysis of patients with clinically suspected IFD (pre‐test probability of 35%), the NPV was lower, but the PPV for BG was 0.86 at cut‐off 160 pg/mL. Among IFD patients, 91% had patterns of consecutively positive and increasing BG levels. Bis‐methyl‐gliotoxin was undetectable in 15 patients with proven, probable and possible IA. To conclude, BG was the superior fungal marker for IFD diagnosis. Quantification above the limit of detection and graphical evaluation of the pattern of dynamics are warranted in the interpretation of BG results.     

Non-value of Aspergillus antigen detection in bronchoalveolar lavage fluids of patients undergoing bone marrow transplantation

Summary. A commercially available antigen assay (Pastorex Aspergillus ) was used to detect Aspergillus antigen in serial bronchoalveolar lavage (BAL) fluids and sera of patients undergoing bone marrow transplantation (six patients with autopsy-proven aspcrgillosis and 10 control patients without evidence of fungal infection). Aspergillus antigen was not detected in 17 BAL fluids of the six patients with proven aspergillosis. In two of the six patients the assay gave positive results in serum specimens. Three of the 10 control patients showed reactive BAL fluids. It is concluded that the latex agglutination assay of BAL fluids has no value in the diagnosis of invasive (pulmonary) aspergillosis in patients undergoing bone marrow transplantation.
Zusammenfassung. Ein kommerziell erhältlicher Test zum Nachweis von Aspergillus -Antigen (Pastorex Aspergillus ) wurde verwendet, um Aspergillus -Antigen in bronchoalveolären Lavage (BAL)-Flüssigkeiten und Seren von Patienten nach Knochenmarktransplantation (sechs Patienten mit autoptisch gesicherter Aspergillose und zehn Kontroll-Patienten ohne Hinweise auf eine Pilz-Infektion) nachzuweisen. Aspergillus -Antigen wurde in 17 BAL-Flüssigkeiten von sechs Patienten mit histologisch gesicherter Aspergillose nicht nachge-wiesen. Bei zwei der sechs Patienten ergab die Untersuchung von Seren positive Resultate. BAL-Flüssigkeiten von drei der zehn Kontroll-Patienten zeigten ein positives Resultat. Der Nachweis von Aspergillus -Antigen in BAL-Flüssigkeiten hat somit keine diagnostische Relevanz für eine invasive (pulmonale) Aspergillose bei Patienten nach Knochenmarktransplantation.     

支气管肺泡灌洗液联合TCT在周围型肺癌诊断中的应用

目的:探讨支气管肺泡灌洗液联合TCT在周围型肺癌诊断中的应用价值.方法:纳入2014年12月至2015年12月收治的123例周围型肺癌患者,所有患者支气管镜检查未发现病灶,遂同时获得支气管肺泡灌洗液和支气管镜刷片标本,同时采用TCT和直接涂片法检测,比较四种方法的敏感度.并对支气管镜检查结果与术后病理结果进行比较,分析支气管镜检查结果的特异性.结果:支气管肺泡灌洗液联合TCT的敏感度显著高于支气管肺泡灌洗液直接涂片法检测的敏感度(36.6% vs 17.9%,P<0.05);支气管镜刷片联合TCT的敏感度亦显著高于支气管镜刷片直接涂片法检测的敏感度(14.6% vs 8.1%,P<0.05);而且四种方法中支气管肺泡灌洗液联合TCT的敏感度最高(P<0.05).支气管镜检查结果与术后手术组织病理结果比较的特异性为100%.支气管肺泡灌洗液联合TCT的分类诊断和手术病理组织学诊断的符合率为84.4%.结论:支气管肺泡灌洗液联合TCT能提高周围型肺癌的检出诊断率,支气管肺泡灌洗液联合TCT可推广应用于临床.     

Assessment of the lightcycler PCR assay for diagnosis of invasive aspergillosis in paediatric patients with onco-haematological diseases

A reliable diagnosis of invasive aspergillosis (IA) is hampered by the difficulty in obtaining suitable tissue samples. To evaluate the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the LightCycler PCR for the diagnosis of IA, 536 blood samples were collected over a 22-month period from 62 paediatric patients (median age 10 years, range 1-18) considered at risk of IA. The galactomannan antigen (GM) and fungal DNA were assessed on serial blood samples. IA was diagnosed in eight of 62 patients (13%): proven, five, probable, three. Sensitivity, specificity, PPV and NPV of LightCycler PCR varied according to the number of positive samples used to define positivity: 88%; 37%; 17% and 95% for single sample positivity; and 63%, 81%, 33% and 94% for serial sample positivity respectively. The concordance between positivity of LightCycler PCR assay and the diagnosis of IA was 79%. The single positivity of LightCycler PCR assay showed a good sensitivity for the diagnosis of IA in paediatric patients. The high NPV makes LightCycler PCR a promising tool in addition to GM testing to design a strategy of pre-emptive antifungal therapy, although further validation studies are needed.     

Diagnostic value of serum galactomannan, (1,3)‐β‐d‐glucan,and Aspergillus fumigatus‐specific IgA and IgG assays for invasive pulmonary aspergillosis in non‐neutropenic patients

Detection of serum galactomannan (GM) and (1,3)‐β‐d ‐glucan (BG) is considered useful for non‐culture diagnosis of invasive pulmonary aspergillosis (IPA) in neutropenic patients. Only few studies evaluated these seromarkers in non‐neutropenic patients suspected of having IPA. The aim of this study was to evaluate both tests together with the Aspergillus fumigatus‐specific serum IgG and IgA (IgAG) test for serological IPA diagnosis in non‐neutropenic patients. Sera from 87 patients suspected of having IPA were retrospectively analysed. Patients were categorised into groups of proven IPA (n = 10), putative IPA (n = 31) and non‐IPA colonisation (n = 46). When the GM, BG and IgAG assays were used for patients included in the study, the sensitivity/specificity/positive predictive value (PPV)/negative predictive value (NPV) were 48.8%/91.3%/83.3%/66.7%, 82.9%/73.9%/73.9%/82.9% and 75.6%/95.7%/93.9%/81.5%, respectively. Thus, the highest specificity and PPV were confirmed for the IgAG assay. Improvements in the sensitivity and NPV were achieved by “at least one positive” analysis with the GM and BG assays, with the sensitivity/specificity/PPV/NPV values being 85.0%/69.6%/71.4%/84.2%. Nevertheless, the highest sensitivity and NPV were achieved by the “at least one positive” analysis combining the GM, BG and IgAG tests (97.6% and 96.8%, respectively). The involvement of the IgAG assay could improve IPA diagnosis in non‐neutropenic patients by increasing the sensitivity and NPV when combined with the GM or BG assays. Furthermore, improvement was achieved by combining the GM, BG and IgAG assays using the “at least one positive test” strategy, especially if doubt exists.     

Performance of lateral flow device and galactomannan for the detection of Aspergillus species in bronchoalveolar fluid of patients at risk for invasive pulmonary aspergillosis

Early diagnosis of invasive pulmonary aspergillosis (IPA) remains difficult due to the variable performance of the tests used. We compared the performance characteristics of Aspergillus lateral flow device (LFD) in bronchoalveolar lavage (BAL) vs. BAL‐galactomannan (GM), for the diagnosis of IPA. 311 BAL specimens were prospectively collected from patients who underwent bronchoscopy from January to May 2013. Patients at risk for IPA were divided into haematological malignancy (HEM) and non‐HEM groups: solid organ transplants (SOT) (lung transplant (LT) and non‐LT SOT); chronic steroid use (CSU); solid tumour (STU) and others. We identified 96 patients at risk for IPA; 89 patients (93%) were in the non‐HEM groups: SOT 57 (LT, 46, non‐LT SOT, 11); CSU 21; STU 6, other 5. Only three patients met criteria for IA (two probable; one possible). Overall sensitivity (SS) was 66% for both and specificity (SP) was 94% vs. 52% for LFD and GM respectively. LFD and GM performance was similar in the HEM group (SS 100% for both and SP 83% vs. 100% respectively). LFD performance was better than GM among non‐HEM SOT patients (P = 0.02). Most false‐positive GM results occurred in the SOT group (50.8%), especially among LT patients (56.5%). LFD performance was superior with an overall SP of 95.6% in SOT (P < 0.002) and 97% in LT patients (P = 0.0008). LFD is a rapid and simple test that can be performed on BAL to rule out IPA.     

Levels of interleukin (IL)‐6 and IL‐8 are elevated in serum and bronchoalveolar lavage fluid of haematological patients with invasive pulmonary aspergillosis

Aspergillus spp. have been shown to induce T‐helper cell (Th) 1 and Th17 subsets resulting in elevated levels of several cytokines. The objective of this study was to analyse a bundle of cytokines in serum and bronchoalveolar lavage fluid (BALF) in patients with and without invasive pulmonary aspergillosis (IPA). This nested case‐control analysis included 10 patients with probable/proven IPA and 20 matched controls without evidence of IPA, out of a pool of prospectively enrolled (2014‐2017) adult cases with underlying haematological malignancies and suspected pulmonary infection. Serum samples were collected within 24 hours of BALF sampling. All samples were stored at ?70°C for retrospective determination of cytokines. IL‐6 and IL‐8 were significantly associated with IPA in both serum (P = .011 and P = .028) and BALF (P = .006 and P = .012, respectively), and a trend was observed for serum IL‐10 (P = .059). In multivariate conditional logistic regression analysis, IL‐10 remained a significant predictor of IPA in serum and IL‐8 among BALF cytokines. In conclusion, levels of IL‐6 and IL‐8 were significantly associated with probable/proven IPA, and a similar trend was observed for serum IL‐10. Future cohort studies should determine the diagnostic potential of these cytokines for IPA, and evaluate combinations with other IPA biomarkers/diagnostic tests.     

The role of surgery in the treatment of invasive fungal infection in paediatric haematology patients: a retrospective single‐centre survey

Surgery may improve the control of fungal disease and patient survival. The aim of this study was to report a single‐centre experience in using surgery for the treatment of paediatric invasive fungal infection (IFI). From 2001 to 2009, 18 paediatric onco‐haematology patients underwent 24 surgical procedures as treatment of IFI. At surgery, severe thrombocytopenia and neutropenia were present in four and one episodes respectively. Complications were one pleural effusion, one pleural effusion and surgical wound infection, one pneumothorax with wound dehiscence and one wound dehiscence. None of them required repeat surgery. The median duration of hospitalisation for four complicated procedures was 11 days, range 3–16, and 7 days, range 2–13, for the 20 uncomplicated procedures. No surgery‐related deaths occurred. Fourteen patients resumed chemotherapy after a median of 26 days, range 9–77, whereas nine patients underwent hematopoietic stem cell transplantation after a median of 42 days, range 27–110. At 3 months from IFI, 17 patients were alive (94%) and one patient (6%) died from mycosis; the 3‐month overall survival (OS) being 94.4%, CI 66.6–99.2. After a median follow‐up of 7.1 years (CI 2.8–7.5), the OS was 54.5%, CI 29.2–74.2. Surgery is a feasible and valuable option in paediatric patients because it is associated with a low incidence of complications and an acceptable delay in resuming the chemotherapeutic plan.     

Need to re‐look cut‐off of Aspergillus‐specific IgE levels in children with ABPA

The cut‐offs for total and specific IgE used for diagnosing ABPA in children have been adopted from adult literature and have not been validated in the paediatric population. To establish the ideal cut‐offs of total IgE and Aspergillus‐specific IgE for the diagnosis of ABPA in children. This study was a prospective observational case‐control study, conducted in a tertiary care hospital in North India, enrolling 140 children with partly controlled and uncontrolled asthma. Seventy children had ABPA based on the Rosenberg‐Patterson Criteria (Cases) whereas 70 children were without ABPA (Controls). All children were subjected to clinical examination and investigations such as absolute eosinophil count, total IgE, Aspergillus‐specific IgE, Aspergillus skin prick test and radiological tests. ROC curve analysis was done to determine the ideal cut‐offs of total and specific IgE to diagnose ABPA. The ROC curve analysis determined 1204IU/L as the cut‐off value of total IgE with a sensitivity of 79.7% (95%CI 68.31% to 88.44%) and specificity of 53.1% (95%CI 40.23 to 65.7). The ROC analysis of specific IgE levels of children with ABPA determined the cut‐off value of 0.49 KAU/L with a sensitivity of 94.03% (95%CI 85.41 to 98.35) and specificity of 88.89% (95%CI 75.94% to 96.29%). We propose that the cut‐offs of total and specific IgE need to be relooked in the paediatric population. Cut‐offs of total IgE as 1204 IU/L and for Aspergillus‐specific IgE as 0.49KAU/L seem appropriate. Large multicentric studies should be conducted to determine the ideal values for diagnosing paediatric ABPA.     

Diagnostic cut‐off of Aspergillus fumigatus‐specific IgG in the diagnosis of chronic pulmonary aspergillosis

Aspergillus fumigatus‐specific IgG is pivotal in making the diagnosis of chronic pulmonary aspergillosis (CPA). However, the cut‐off value for A. fumigatus‐specific IgG remains unknown. We included consecutive treatment‐naïve subjects with chronic cavitary pulmonary aspergillosis (CCPA, cases). The controls were subjects with treated pulmonary tuberculosis, who had residual radiological abnormality and minimal symptoms. The diagnosis of CCPA was based on consistent clinicoradiological features along with demonstration of Aspergillus infection (growth of Aspergillus in sputum or bronchoalveolar lavage fluid [BALF] culture; serum or BALF galactomannan index >0.5 and >1, respectively). For determining the cut‐off of A. fumigatus‐specific IgG (Phadia), subjects were randomly classified as derivation (two‐thirds) and validation (one‐third) cohort. One hundred and thirty‐seven cases and 50 controls were included. The best cut‐off value for A. fumigatus‐specific IgG (derivation cohort) was 27.3 mgA/L (AUROC, 0.976) at a sensitivity and specificity of 95.6% and 100%, respectively. Using a cut‐off of 27 mgA/L, the sensitivity and specificity in the validation cohort was 91.3% and 100%, respectively. In contrast, the sensitivity of Aspergillus precipitins was only 25.5%. At a cut‐off value of 27 mgA/L, A. fumigatus‐specific IgG is a reliable test with high sensitivity and specificity in the diagnosis of CPA. More studies are required to confirm our findings.     

Role of bi‐weekly serum galactomannan screening for the diagnosis of invasive aspergillosis in haematological cancer patients

Invasive aspergillosis (IA) is a life‐threatening infection affecting haematological cancer patients with chemotherapy‐induced neutropenia. The diagnosis of IA often relies on the detection of galactomannan (GM) in serum or bronchoalveolar lavage fluid (BAL). Bi‐weekly serum GM screening has been proposed for a pre‐emptive therapeutic approach of IA in patients not receiving mold‐active prophylaxis. We have analysed all IA cases among patients with haematological malignancies and prolonged chemotherapy‐induced neutropenia (>14 days) in our institution over a 10‐year period (2007‐2017). Serum GM was measured twice weekly and mold‐active prophylaxis was not routinely administered. Thirty IA cases were observed and a positive serum GM was the first indicator of IA in 10 (33%) of them, which represents a need of approximately 500 GM tests for the detection of a single IA case. In the other 20 (67%) cases, suggestive chest CT lesion was the first sign of IA and bronchoscopy was required in 15 (50%) cases with negative serum GM for establishing the diagnosis of probable/proven IA. A positive serum GM was associated with a worse prognosis (57% 12‐week survival vs 100% among serum GM‐negative patients, P = .006), irrespective of the timing of GM positivity compared to CT. We concluded that bi‐weekly serum GM screening demonstrated limited benefit in this population.     

Real‐world challenges and unmet needs in the diagnosis and treatment of suspected invasive pulmonary aspergillosis in patients with haematological diseases: An illustrative case study

Recent years have seen important advances in the diagnosis of invasive pulmonary aspergillosis (IPA), complemented by the introduction of new therapies. Despite this, IPA remains a major cause of infection‐related mortality in patients with haematological diseases. There are two main reasons for this. First, diagnosis of IPA remains a challenge, since risk factors and the clinical, radiological and mycological presentations vary not only by fungal disease stage, but also by patient group (eg neutropenic vs non‐neutropenic patients). Diagnosis is particularly challenging in patients receiving mould‐active prophylactic or empirical treatment, which reduces the sensitivity of all diagnostic tests for IPA. Second, treatment of IPA is complex due to unpredictable pharmacokinetic profiles of antifungal agents, small therapeutic window in terms of exposure and adverse events, and multiple drug‐drug interactions through the CYP450 system. Here we report a case of a 23‐year‐old male with severe aplastic anaemia and subpleural nodules. Diagnostic tests for IPA obtained during ongoing mould‐active empirical treatment were negative. Intravenous voriconazole was stopped after visual disturbances and hallucinations. The patient then had an anaphylactic reaction to liposomal amphotericin B and was switched to intravenous posaconazole, which had to be discontinued due to a significant increase in transaminase levels. He was treated with oral isavuconazole with reduced dosage, triggered by increasing transaminases under the standard dosage. Even under reduced dosage, blood concentrations of isavuconazole were high and treatment was successful. The case illustrates real‐world challenges and unmet needs in the diagnosis and treatment of IPA in patients with haematological diseases.