Supplementary online-only material
Voriconazole for chronic pulmonary aspergillosis: a prospective multicenter trial
Journal name: European Journal of Clinical Microbiology and Infectious Diseases
Jacques Cadranel, Bruno Philippe, Christophe Hennequin, Anne Bergeron, Emmanuel Bergot, Arnaud Bourdin, Vincent Cottin, Thierry Jeanfaivre, Cendrine Godet, Marc Pineau, and Patrick Germaud
Appendix 1
Methods
Participating institutions
The work was performed at the following institutions: Hôpital Tenon, Paris, France; Hôpital St Louis, Paris, France; Hôpital de la Côte de Nacre, Caen, France; Hôpital René Pleven, Dinan, France; Hôpital Arnaud de Villeneuve, Montpellier, France; Hôpital Louis Pradel, Lyon, France; Hôpital Cavale Blanche, Brest, France; Hôpital Bichat, Paris, France; Centre Medical de Bligny, Bris sous Forges, France; Hôpital Germont, Rouen, France; Hôpital Laennec, Nantes, France; Hôpital la Miletrie, Poitiers, France; Hôpital d’Angers, Angers, France; Hôpital Maison Blanche, Reims, France; Hôpital René Dubos, Pontoise, France; Hôpital A. Michallon, Grenoble, France; Hôpital A. Calmette, Lille, France; Hôpital Avicenne, Bobigny, France.
Ethics Committee
The protocol was approved by the French ethics committee of Robert Ballanger hospital: Comité Consultatif de Protection des Personnes dans la Recherche Biomédicale, Hôpital Robert Ballanger, 93602 Aulnay-sous-Bois Cedex, France.
Determination of study population size
The trial was designed in order to positively reject weak drug efficacy corresponding to the null hypothesis (pp0 defined as a response rate <15%). The alternative hypothesis (ppA) indicated a response rate >30%. To reduce the chance of incorrectly rejecting the null hypothesis to 5% (a=5%) and incorrectly rejecting the alternate hypothesis to 20% (b=20%), a sample of 48 patients was required [1].
Inclusion criteria
Performance status 0–2 [2] and proven CPA based on: 1) a compatible chest computed tomography scan and/or a photographically confirmed endoscopic lesion sourced by a photograph; 2) mycologic confirmation of Aspergillus infection from a bronchopulmonary specimen (sputum or endoscopy): either positive direct microscopy and culture, or at least two Aspergillus-positive cultures; and 3) a positive serologic test for Aspergillus antibodies (detected using any screening method and further confirmed by positive [i.e., ≥2 lines] counterimmunoelectrophoresis).
Exclusion criteria
Pregnancy, breast-feeding, or women of childbearing potential not using an effective contraceptive; threatening hemoptysis; systemic antifungal drug therapy within the previous 6 months; other form of aspergillosis (simple aspergilloma, allergic bronchopulmonary aspergillosis, or invasive aspergillosis); cystic fibrosis; immunocompromised condition or immunosuppression except administration of low-dose corticosteroids; other bronchopulmonary infection that could interfere with the evaluation of the disease under study (including tuberculosis in progress, mycobacteriosis in progress, and Pseudomonas infection); azole allergy or intolerance; elevated hepatic enzymes (5 ´ upper limit of normal); risk factors of cardiac arrhythmia, symptomatic arrhythmia, or prolongation of QTc interval; abnormal liver function or renal function; potential for drug–drug interaction with azoles; inability to take oral voriconazole.
Definition of radiologic and mycologic response
Assessment of the radiologic response was carried out by a data review committee using tomodensitometry results with or without contrast as follows: 1) for each patient and at each time point, description of every single lesion was done by location (right, left, upper, median, or lower lobe) and type (cavity, mycetoma, nodule, infiltrate, pleural thickening); 2) size in millimeters was recorded for measurable lesions, including nodules and cavity (as a product of orthogonal diameters) and pleural thickening; and 3) change in non-measurable lesions (e.g., infiltrates) was assessed by disappearance or persistence of the lesion.
The radiologic response was defined as follows: 1) complete: resolution of all radiographic and/or bronchoscopic abnormalities attributable to aspergillosis present at baseline; 2) partial: ³50% reduction in the sum of all measurable lesions on chest tomodensitometry, or a regression of endoscopic lesions without any new lesion; 3) stabilization: minor (<50%) or no improvement; or 4) failure: deterioration.
The mycologic response was categorized as follows: 1) eradication: absence of Aspergillus spp. in sputum, bronchial aspirates, or bronchoalveolar lavage (negative direct examination and negative culture) and negative histologic examination, where available; 2) presumed eradication: no culture data available for a patient with clinical improvement, no sputum production, and a partial or complete radiologic response; or 3) persistence: presence of Aspergillus spp. in sputum, bronchial aspirates, or bronchoalveolar lavage (positive direct examination and/or positive culture), and/or histologic evidence.
Data review committee procedure
Data review committee members could enroll patients but, according to predefined committee procedures, a member could not play a role in assessing any patients from his or her own study site. Although the committee was not blinded, its members performed an independent review of the study data, based on the above predefined objective measurements.
Mycologic diagnosis
All Aspergillus strains isolated from bronchopulmonary specimens were sent to a centralized mycology laboratory for confirmation of species identification. There, strains were cultured onto Sabouraud agar with added chloramphenicol and gentamicin (Bio Rad, Marnes la Coquette, France) and incubated for 15 days at 37°C. Strains were then subcultured on malt agar plates and identified according to the macroscopic appearance of the colonies and microscopic observation of fungal structures [3].
Serologic assessment and response
All serum samples collected from each patient at study entry and at each study visit were also sent to the centralized mycology laboratory where anti-Aspergillus antibodies were detected using an immunoelectrophoresis method. Ready-to-use agarose gels (Hydragel; Sebia, Inc., Norcross, GA) were used for the migration of antigens and sera components. Two microliters each of somatic and culture filtrate A. fumigatus antigen (FKS1; Microgen Bioproducts, Surrey, UK) and 80 µL of serum were deposited in pre-cut wells, sera being on the anodic side. Following a 30-min run under 100 V constant current, the gels were incubated in barbital buffer pH 9.2 (barbital 2.45%, sodium barbital 13.73%, NaN3 0.13%) for 48 h at room temperature. Following two washes in distilled water, gels were dried for 10 min using the Paragon system (Beckman Coulter, Inc., Brea, CA). Gels were then stained for 5 min in amidoschwartz solution (amidoschwartz 4 g/L, ethylene-glycol 6.7%), discolored with citric acid solution (5 g/L), washed, and then dried before reading. Positive and negative controls were used for each run. Gels were then read under blinded conditions by two technicians in order to determine the number of the precipitin lines. Sequential serum samples for each specific patient were all tested within the same run, thus allowing for more accurate comparison of the precipitin lines count.
Serologic response was assessed at 3, 6, 9, and 12 months and at end of treatment in comparison with baseline values. Results were assessed as: 1) normalization: return to normal values (≤1 line); 2) partial response: significant decrease but not complete (decrease of ³2 lines compared with baseline); 3) stabilization: no significant change (³1 line compared with baseline); and 4) failure: significant increase (³2 lines compared with baseline).
Results
Table 1 Assessment of quality of life using the St. George’s Hospital Respiratory Questionnaire
Change from baselineMean change (95% CI)
6 Months / End of study
Symptoms component / –3.3 (–14.0 to 7.4) / –6.8 (–17.0 to 3.3)
Activity component / –9.4 (–16.1 to –2.7)a / –8.1 (–14.6 to –1.5)a
Impact component / –8.4 (–15.2 to –1.6)a / –7.9 (–13.5 to –2.4)a
Total score / –8.4 (–14.0 to 2.8)a / –8.2 (–12.4 to –4.0)a
aSignificant decrease at this time point, since the upper limit of the 95% CI remained below 0
Table 2 Mean number of Aspergillus precipitin lines at baseline and at 6 months in all patientsa and those with global success at 6 months
(All patients*) / Month 6
(Patientsa with global success at month 6)
CNPA / 4.79
(n=19) / 2.73
(n=15) / 2.90
(n=10)
CCPAb / 4.95
(n=21) / 3.86
(n=14) / 4.67
(n=3)
Total / 4.88
(n=40) / 3.28
(n=29) / 3.31
(n=13)
CNPA: chronic necrotizing pulmonary aspergillosis; CCPA: chronic cavitary pulmonary aspergillosis
aThose patients from the modified intent-to-treat population who had a serologic assessment at 6 months;
bOne patient with CCPA did not have a centrally conducted serologic test at baseline
References
1. A’Hern RP (2001) Sample size tables for exact single-stage phase II designs. Stat Med 20:859–866
2. Oken MM, Creech RH, Tormey DC, Horton J, Davis TE, McFadden ET et al (1982) Toxicity and response criteria of the Eastern Cooperative Oncology Group. Am J Clin Oncol 5:649–655
3. De Hoog GS, Guarro J, Gene J, Figueras MJ (2001) Atlas of Clinical Fungi. 2nd ed. ASM Press, Washington, DC
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