Diagnosis of Lung Complication in Allogeneic Stem Cell Transplantation By Combined Use of Multiple Methods through Bronchoscopic Alveolar Lavage

Background: High morbidity and mortality in patient receiving hematopoietic Stem Cell Transplantation (HSCT) are resulted from lung complications. Diagnosis of the etiology affects the choice of treatment. The bronchoalveolar lavage (BAL) analysis is safe and widely accepted, which provide valuable information for pathogen identification in pulmonary infections diagnosis. Aims: The aim of this study was to evaluate the diagnostic yield of multiple diagnostic methods in BAL fluid (BALF) microbiological analysis from HSCT patients, and determine clinically relevant cutoffs of (1→3)-β-D-glucan (BDG) level and CMV virus load(VL) for pneumonia diagnosis. Methods: This retrospective study enrolled 104 patients with pulmonary infiltrate between January 2016 and June 2019 at the Department of Hematology of the Shanghai General hospital(Table 1). This study was approved by the ethics committee of the Shanghai general hospital Institute. BAL sampling was routinely performed by trained physicians by fiberoptic bronchoscope. BALF samples were analyzed by microbiological culture, flow cytometry, PCR and mNGS.Data were analyzed by the Statistical Package of SPSS version 15.0 (SPSS, Inc., Chicago, IL, USA) for statistical analyses.Continuous variables were compared with Student's t-test.ROC curves were constructed to assess BDG for Pneumocystis jirovecii pneumonia (PJP),CMVDNA and the percent of CD4+T lymphocyte for CMV pneumonia Results: All cases were examined by diverse methodology, including microbiological, radiologic, molecular and cytological methods. A total of 130 episodes of lung complication were identified in 104 patients. A specific diagnosis was obtained in 108 of the 130 episodes evaluated (83%)(Table 2).By microbiological method, The percentage of bacteria cultured from BALF was 19.2%, which was similar with that from sputum(17.7%).BALF culture produced better diagnostic yield of fungi than sputum (18.5% VS 8.5%, P=.028).In 95 episodes receiving GM test, 5 episodes were diagnosed as probable IA and 41 episodes as possible IA.At a cutoff optical density index (ODI) value of ≥0.7, the sensitivity of BALF GM detection was much higher than that of serum GM detection (71.7% versus 21.7%; P=0.001), but there was no significant difference between their specificities (91.8% versus 100%; P=0.68). BDG level were highest in patient with PJP than other fungi and non-fungi pneumonia(Figure 1).For the detection of suspected PJP, the sensitivity, specificity, the cutoff (pg/mL) of BDG was 71.43%, 88.89% and 161.2pg/ml in BAL fluid and 71.43%, 83.33% and 317.4pg/ml in serum(Figure 2). Radiologic results show that not well circumscribed or diffuse consolidations were common seen in bacterial infection(66.7%) and fungul infection(94.1%) and mixed infection(>50%), while interstitial lung abnormalities were seen in viral infection (62.5%) and IPS (90%) and mixed fungal and viral infection(62.5%)(Table 3). As target test of molecular diagnostic method, PCR analysis of BALF produced better diagnosis yield than serum and the sensitivity, specificity, CMVDNA cutoff (pg/mL) of PCR in BAL fluid was 96.3%, 92.31% and 8610 copy/ml(Figure 3). As unbias molecular diagnositic method, metagenomics (mNGS) results from 42 patients show positive agreement of 28.6% (12/42) and unique respiratory pathogens found of 14.3% (6/42) when compared with traditional culture and PCR testing method. In 28.6% (12/42) cases, the results of mNGS agreed with traditional culture and PCR, while pathogens were only detected by mNGS in 6 cases (14.3% ) . The false negative rate of mNGS was 22.2%, and the specificity was 54.5%. Flow cytometry results show that CD4+/CD8+ T cells in BALF were significantly different between infectious disease and idiopathic pneumonia. CD4+ T cells in BALF were lowest in CMV pneumonia when compared with bacteria and fungi infection and IPS(Figure 4). For the detection of suspected CMV pneumonia, the sensitivity, specificity and percentage cutoff of CD4+T cells in BALF were 88.89%, 80.49% and 13.74%(Figure 5). Conclusion: Each method applied in this study shows unique advantage in diagnosis of lung complication following HSCT with BAL based analysis. mNGS cannot replace either culture or PCR, but it is useful in culture-negative patients. Combination of multiple examination is required. Cytology analysis and flow cytometry of BALF facilitated the etiology diagnosis in HSCT patients. Disclosures No relevant conflicts of interest to declare.

Galactomannan Is a Biomarker of Fosmanogepix (APX001) Efficacy in Treating Experimental Invasive Pulmonary Aspergillosis

ABSTRACT Galactomannan (GM) detection in biological samples has been shown to predict therapeutic response by azoles and polyenes. In a murine invasive pulmonary aspergillosis model, fosmanogepix or posaconazole treatment resulted in an ∼6- to 7-log reduction in conidial equivalents (CE)/g lung tissue after 96 h versus placebo. Changes in GM levels in BAL fluid and serum mirrored reductions in lung CE, with significant decreases seen after 96 h or 72 h for fosmanogepix or posaconazole, respectively (P < 0.02).

Diagnostic Value of Galactomannan Antigen Test in Serum and Bronchoalveolar Lavage Fluid Samples from Patients with Nonneutropenic Invasive Pulmonary Aspergillosis

ABSTRACT The objective of this study was to compare the diagnostic value of galactomannan (GM) detection in bronchoalveolar lavage fluid (BALF) and serum samples from nonneutropenic patients with invasive pulmonary aspergillosis (IPA) and determine the optimal BALF GM cutoff value for pulmonary aspergillosis. GM detection in BALF and serum samples was performed by enzyme-linked immunosorbent assay (ELISA) in 128 patients with clinically suspected nonneutropenic pulmonary aspergillosis between June 2014 and June 2016. On the basis of the clinical and pathological diagnoses, 8 patients were excluded because their diagnosis was uncertain. The remaining 120 patients were diagnosed with either IPA ( n = 37), community-acquired pneumonia (CAP; n = 59), noninfectious diseases ( n = 19), or tuberculosis ( n = 5). At a cutoff optical density index (ODI) value of ≥0.5, the sensitivity of BALF GM detection was much higher than that of serum GM detection (75.68% versus 37.84%; P = 0.001), but there was no significant difference between their specificities (80.72% versus 87.14%; P = 0.286). At a cutoff value of ≥1.0, the sensitivity of BALF GM detection was still much higher than that of serum GM detection (64.86% versus 24.32%; P < 0.001), and their specificities were similar (90.36% versus 95.71%; P = 0.202). Receiver operating characteristic (ROC) curve analysis showed that when the BALF GM detection cutoff value was 0.7, its diagnostic value for pulmonary aspergillosis was optimized, and the sensitivity and specificity reached 72.97% and 89.16%, respectively. BALF GM detection was valuable for the diagnosis of IPA in nonneutropenic patients, and its diagnostic value was superior to that of serum GM detection. The optimal BALF GM cutoff value was 0.7.

Galactomannan in Bronchoalveolar Lavage Fluid for Diagnosis of Invasive Pulmonary Aspergillosis with Nonneutropenic Patients

Background. We evaluated the utility of galactomannan (GM) in bronchoalveolar lavage fluid (BALF) for the diagnosis of invasive pulmonary aspergillosis (IPA) in nonneutropenic patients. Methods. A total of 183 patients were included in the final analysis. Bronchoscopies and the detection of GM in BALF were all performed on them. Results. Ten cases of IPA were diagnosed. ROC data demonstrated that, for diagnosing IPA, an optimal cutoff value for GM in BALF of 0.76 yielded a sensitivity of 100.0% and a specificity of 76.2%. Symptoms and radiological findings had no significant difference between proven or probable IPA group and non-IPA group. In our case-control analysis, although nine patients with false-positive results received treatment with Piperacillin/tazobactam, there was no significant difference between case and control group. Conclusions. BALF GM detection is a valuable adjunctive diagnostic tool. Our retrospective study suggests that the optimal value of GM detection in BALF is 0.76 in nonneutropenic patients.

Application of Mass Spectrometry Technology to Early Diagnosis of Invasive Fungal Infections

We recently developed a mass spectrometry (MS) procedure based on the detection of a serum disaccharide (MS-DS) in patients with invasive candidiasis (IC). Here, we compare the performance of MS-DS for the diagnosis of IC, invasive aspergillosis (IA), and mucormycosis (MM) with those of commercially available antigen detection tests. This retrospective study included 48 patients (23 IC patients [74 serum samples], 15 IA patients [40 serum samples], and 10 MM patients [15 serum samples]) and 49 appropriate controls (102 serum samples). MS-DS, mannan (Mnn), galactomannan (GM), and (1,3)-β-d-glucan (BDG) were detected by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) MS, Platelia, and Fungitell assays, respectively. For IC, the sensitivity and specificity of the MS-DS index, BDG detection, and Mnn detection were 62% and 84%, 82% and 60%, and 33% and 94% per serum sample and 83% and 69%, 96% and 31%, and 39% and 86% per patient, respectively. For IA, the corresponding values in comparison to BDG and GM detection were 83% and 81%, 62% and 95%, and 62% and 100% per serum sample and 93% and 76%, 87% and 90%, and 93% and 100% per patient, respectively. Nine of the 10 MM patients had a positive MS-DS result. MS-DS gave an early diagnosis in IC (73% positivity before blood culture), IA (positive before GM detection in six patients), and MM (positivity mainly preceded the date of diagnosis) patients. For IC, persisting MS-DS was associated with a poor prognosis. The different biomarkers were rarely detected simultaneously, suggesting different kinetics of release and clearance. For IA, MS-DS provided better complementation to GM monitoring than BDG monitoring. MS-DS detects panfungal molecules circulating during invasive fungal infections. The performance of MS-DS compared favorably with those of biological tests currently recommended for monitoring at-risk patients. Further validation of this test in multicenter studies is required.

Visual and Real-Time Event-Specific Loop-Mediated Isothermal Amplification Based Detection Assays for Bt Cotton Events MON531 and MON15985

Bt cotton events MON531 and MON15985 are authorized for commercial cultivation in more than 18 countries. In India, four Bt cotton events have been commercialized; more than 95% of total area under genetically modified (GM) cotton cultivation comprises events MON531 and MON15985. The present study reports on the development of efficient event-specific visual and real-time loop-mediated isothermal amplification (LAMP) assays for detection and identification of cotton events MON531 and MON15985. Efficiency of LAMP assays was compared with conventional and real-time PCR assays. Real-time LAMP assay was found time-efficient and most sensitive, detecting up to two target copies within 35 min. The developed real-time LAMP assays, when combined with efficient DNA extraction kit/protocol, may facilitate onsite GM detection to check authenticity of Bt cotton seeds.

INVASIVE ASPERGILLOSIS IN INTENSIVE CARE UNIT PATIENTS IN IRAN

We assessed the intensive care unit (ICU) patients for Invasive aspergillosis (IA) with culture and non-culture based diagnostic methods from Iran. Thirty-six ICU patients with underlying predisposing conditions for IA were enrolled in the study. Sixty eight Bronchoalveolar lavage (BAL) samples were collected by bronchoscope twice a weekly. BAL samples were analyzed by microscopic examination, fungal culture and galactomannan (GM) detection. The Platelia Aspergillus GM EIA was used to quantify GM indices. Samples with a BAL GM index ≥1 were considered as positive for GM. Patients were classified as having probable or possible IA. Out of 36 suspected patients to IA, 36.1% of cases showed IA which were categorized as: 4 cases of possible IA and 9 of probable IA. 76.2% of BAL samples were positive for GM. From 13 patients with IA, 11 (84.6%) had at least one positive BAL GM index. Of these patients, 9 (81.8%) showed probable IA. The main underlying predisposing conditions were neutropenia (53.8%) and COPD (30.8%). Our study has indicated that COPD must be considered as one of the main predisposing condition for occurrence of aspergillosis in ICU patients. Our data have also revealed that GM detection in BAL samples play a significant role to IA diagnosis.

Comparison of Galactomannan Detection, PCR-Enzyme-Linked Immunosorbent Assay, and Real-Time PCR for Diagnosis of Invasive Aspergillosis in a Neutropenic Rat Model and Effect of Caspofungin Acetate

ABSTRACT The performance of different in vitro diagnostic tests for the diagnosis of invasive aspergillosis (IA) was investigated in a transiently neutropenic rat model. Rats were immunosuppressed with cyclophosphamide and then inoculated intravenously with 1.5 × 104 CFU Aspergillus fumigatus spores. Animals were then either treated with caspofungin acetate, 1 mg/kg/day for 7 days, or not treated. PCR-enzyme-linked immunosorbent assay (ELISA), real-time PCR, and galactomannan (GM) detection were performed on postmortem blood samples, along with culture of liver, lung, and kidney homogenate. Caspofungin-treated animals showed a decrease in residual tissue burden of A. fumigatus from organ homogenate compared to untreated animals (P < 0.002). PCR-ELISA returned positive results for 11/17 animals treated with antifungal agents and for 10/17 untreated animals. Galactomannan was positive in 8/17 caspofungin-treated animals and 4/17 untreated animals. Real-time PCR was positive in 2/17 treated and 3/17 untreated animals. This study demonstrates that PCR-ELISA is a more sensitive test than either GM detection (P = 0.052) or real-time PCR (P < 0.01) for diagnosis of IA but that any of the three tests may return false-negative results in cases of histologically proven disease. Galactomannan indices from animals treated with antifungal agents showed a trend (P = 0.1) towards higher levels than those of untreated animals, but no effect was observed with PCR-ELISA indices (P = 0.29). GM detection, as previously described, may be enhanced by the administration of caspofungin, but PCR-ELISA appears not to be affected in the same way. We conclude that PCR-ELISA is a more sensitive and reliable method for laboratory diagnosis of IA.