Case Report: Acquired Disseminated BCG in the Context of a Delayed Immune Reconstitution After Hematological Malignancy

Context Disseminated infections due to Mycobacterium bovis Bacillus Calmette-Guérin (BCG) are unusual and occur mostly in patients with inborn error of immunity (IEI) or acquired immunodeficiency. However, cases of secondary BCGosis due to intravesical BCG instillation have been described. Herein, we present a case of severe BCGosis occurring in an unusual situation. Case Description We report one case of severe disseminated BCG disease occurring after hematological malignancy in a 48-year-old man without BCG instillation and previously vaccinated in infancy with no complication. Laboratory investigations demonstrated that he was not affected by any known or candidate gene of IEI or intrinsic cellular defect involving IFNγ pathway. Whole genome sequencing of the BCG strain showed that it was most closely related to the M. bovis BCG Tice strain, suggesting an unexpected relationship between the secondary immunodeficiency of the patient and the acquired BCG infection. Conclusion This case highlights the fact that, in addition to the IEI, physicians, as well as microbiologists and pharmacists should be aware of possible acquired disseminated BCG disease in secondary immunocompromised patients treated in centers that administrate BCG for bladder cancers.


Patients and healthy donors
The study was conducted in accordance with the principles of Helsinki declaration. Written informed consents were obtained from the patient as well as from age and gender matched healthy donors (HD).

Phagocytosis assays
The investigation of phagocytosis and of oxidative burst activity were performed at the hospital laboratory (CE/IVD methods PHAGOTEST™, PHAGOBURST™, Celonic) according to the manufacturer's protocols.

PBMC stimulation and cytokine detection
Heparinized blood samples were collected from HD. Peripheral blood mononuclear cells (PBMC) were separated by density gradient centrifugation. Cells were plated at 10 6 cells/well in a 24 well-plate containing RPMI supplemented with 10% Fetal Bovine Serum (FBS) and stimulated for 48 hours with phorbol myristate acetate (PMA; 100ng/mL; Invivogen) and ionomycin (1µg/mL; Invivogen), IL12 (100ng/mL; Preprotech), or heat-inactivated patient's strain of Mycobacterium Bovis bacillus Calmette-Guérin (1 CFU/cell). Culture supernatants were collected from the stimulated PBMCs after 48 hours, and cryopreserved. Later, thawed supernatant was used to measure IFNγ levels by ELISA following the manufacturer's instructions (Invitrogen) Intracellular cytokines detection: Protein transport inhibitor cocktail (eBioscience) was added during the last 6 hours of incubation. Surface staining was then performed during 15 minutes at +4°C. Fixation and permeabilization were performed with BD Cytofix/Cytoperm (BD) according to the manufacturer's recommendations. Samples were incubated with anti-cytokines antibodies for 30 minutes at +4°C. Cell viability was assessed by initial incubation with Fixable Viability Dye eFluor 780 (eBioscience) following the manufacturer's protocol.

Phospho-STAT1 (pSTAT1) assessment
Fresh EDTA peripheral blood samples were immediately subjected to density gradient centrifugation to get mononuclear cells suspensions. PBMCs were starved during 16 hours in serum free medium (at 37°C). 0.5*10 6 PBMC were then stimulated for 15 minutes with IFNγ (1000UI/mL (50 ng/mL); Preprotech) or 25% (v/v) HD, patient serum (from different time points). If indicated (i) PBMCs were preincubated 30 minutes at room temperature in the presence of 25% (v/v) HD or patient serum (from different time points) and washed before IFNγ stimulation, or (ii) PBMC were stimulated with a solution containing IFNγ preincubated 30 minutes at room temperature with 25% (v/v) HD or patient serum (from time points). Cells were then labeled for pSTAT-1(Y720) and surface markers using BD Phosflow™ Fix Buffer I and BD Phosflow™ Perm Buffer III (BD Biosciences) following the manufacturer's recommendations. The cells were acquired on a Gallios flow cytometer (Beckman Coulter) and data analysis was performed using Kaluza software (Beckman Coulter) The following monoclonal anti-human antibodies, purchased from BD Biosciences, were used: CD3 (UCHT1), CD4 (RPA-T4), CD14 (MɸP9), STAT1-pY701 (4a).

Whole Genome Sequencing (WGS) of M. bovis BCG strain and phylogenomic analysis
The M. bovis BCG strain was sent to the French National Center for Mycobacteria (NRC-MyRMA) for expertise. Total genomic DNA was extracted using the geneLEAD VIII The WGS raw data were analyzed with BioNumerics 7.6® (Applied Maths) and on the Galaxy server. The quality of paired end reads was checked with FastQC (version 0.11.7) The genomes of the vaccinal M. bovis BCG strains, M. bovis reference genome AF2122/97 were downloaded from PATRIC (https://www.patricbrc.org/) for genomic comparison and phylogenetic analysis. Alignment of protein sequences was performed with MUSCLE and CDS nucleotide sequences (1000 genes) with BioPython (Codon_align function). The Bootstrapping analysis was realized with RAxML (x100). The phylogenetic tree was generated according to the Maximum Likelihood method and visualized and annotated on iTOL (Interactive Tree of life) (https://itol.embl.de).

Whole Exome Sequencing
As previously described (1), genomic DNA was isolated from patient's and parents' whole peripheral blood using standard protocols. Exome sequencing libraries were prepared with the Twist Library preparation kit and captured with Human Core Exome probes extended by Twist Human RefSeq Panel (Twist Bioscience, San Francisco, CA) following the manufacturer's recommendations. Paired-end (2 × 75 bp) sequencing was performed on a NextSeq500 sequencer (Illumina, San Diego, CA). Before any processing, quality control was performed using FastQC. The raw reads data were next mapped using the Burrows-Wheeler Alignment (BWA) tool. Average target read coverage was at least 60-fold. After read mapping, further quality indicators were calculated from the resulting BAM file using SAMtools, Qualimap.