Apophysomyces Variabilis Infection in Transplant Recipients due to Unrecognized Infection in an Intravenous Drug-Using Donor

We report donor-derived infection due to a Mucorales fungus which caused fatal infection in 2 organ recipients despite early aggressive antifungal therapy, misdiagnosed as systemic autoimmune vasculitis. In mid-2019, a man in his 20’s with no history of travel presented with abdominal pain, fever, headache, and photophobia. Urinalysis showed hematuria, pyuria and moderate proteinuria. Computed tomography (CT) of the abdomen showed bilateral hypodense areas in the kidney. Core biopsy of the kidney was culture negative and showed severe, acute necrosis of glomeruli, tubules and interstitium without vasculitis, immunological deposition, or inflammatory response. Serum autoimmune and thrombophilia screen were negative. Initial noncontrast CT of the head was unremarkable, lumbar puncture <1 × 106 white cells/L, 3 × 106 red cells/L, protein 0.25 mg/L, glucose 4.2 mmol/L. Blood, cerebrospinal fluid, urine, and renal biopsy bacterial cultures were negative. The patient was treated for presumed vasculitis with methylprednisolone, following which he developed intracranial hemorrhage diagnosed by noncontrast CT head, thought to be due to autoimmune cerebral vasculitis. He became an organ donor day 9 of admission (just prior he admitted to recent intravenous drug use [IVDU]). Transthoracic echocardiogram showed no valvular abnormalities and a small pericardial effusion. CT chest showed 2 small lung nodules with inflammatory appearance. Bronchoalveolar lavage (BAL) was performed. Organ donation proceeded and the liver, heart and both lungs were transplanted. As the kidneys were necrotic they were not utilized. At procurement of the liver, purulent peritoneal fluid was sampled. Day 1 after transplantation, donor peritoneal fluid, and BAL grew Apophysomyces variabilis. Minimum inhibitory concentrations (MICs) by the YeastOne Y10 method (Thermo Fisher Scientific, United States) were posaconazole 0.5 mg/L, amphotericin 2.0 mg/L, itraconazole 2.0 mg/L, and voriconazole >8.0 mg/L. Echinocandin minimum effective concentrations were >8 mg/L. Isavuconazole MIC was 1.5 mg/L by MIC strip. The liver recipient (recipient 1) commenced intravenous liposomal amphotericin 5 mg/kg/d Day 2 (D+2) posttransplant. He had massive upper gastrointestinal bleeding from the gastroduodenal artery D+14. Subsequent small bowel biopsy taken at upper gastrointestinal endoscopy grew A. variabilis, and the patient later developed deep wound infection which grew A. variabilis. CT abdomen showed an occluded hepatic artery and hypodense areas in the liver, likely due to ischemia. Despite liposomal amphotericin at 10 mg/kg/d and anidulafungin 100 mg/d, he died D+36. The bilateral lung recipient (recipient 2) was commenced on liposomal amphotericin 5 mg/kg/d D+1 posttransplant for 14 d followed by posaconazole. At D+58, he reported chest pain, productive cough, fever, and headache. Investigation revealed disseminated disease involving the thyroid, lung parenchyma, pleura, adrenal gland, kidney, and muscles. Despite liposomal amphotericin 5 mg/kg/d, anidulafungin 100 mg/d, posaconazole (300 mg mane and 200 mg nocte, trough levels 2.8–3.5 mg/L) for 12 wk, hyperbaric oxygen therapy for 6 wk, a trial of isavuconazole with high dose terbinafine, aggressive thyroid debridement, positron emission tomography scan D+152 showed new uptake at the C2 vertebrae, a costochondral junction and the tibia with progressive cervical spine and leptomeningeal involvement D+203. He received palliative treatment and died D+237. He grew A. variabilis from the D+0 posttransplant BAL and from the tibia D+182. The cardiac recipient continues indefinite posaconazole with no evidence of invasive fungal disease to the last clinical review D+511. Phylogenetic analysis using whole-genome sequencing (WGS) was performed as previously described1 with de novo raw sequence read assembly using SPAdes v3.12,2 and reduction of assembled contigs to homozygous chromosomes with the Redundans pipeline v0.13c,3 using A. variabilis NCCPF 102052 (accession no. PRJNA379216) as reference genome. The donor and recipient A. variabilis strains were closely clustered compared to nonlinked strains. Single-nucleotide polymorphisms (SNPs) ranged from 112 to 4563 in the transplant cluster or 0.000003-0.00013 as proportions of nucleotide difference to the whole genome, 330-1865 SNPs between donor strains, 329-2771 SNPs between individual recipient strains, and 275-2796 SNPs between strains from linked donor and recipient sites. This compared with 172889 to 176727 SNPs or 0.0046 to 0.0047 as proportions of nucleotide difference to the whole genome between non-related A. variabilis strains and transplant cluster strains (Figure 1). SNPs between linked isolates may be due to a rapid mutation rate of Mucorales fungi, in contrast to a case of donor-derived mold infection where 5/6 WGS-typed Microascus alveolaris strains were identical (our unpublished data). Within-host strain diversity is a possible explanation, demonstrated in a WGS study of a Mucor circinelloides outbreak in a burns unit, where a significant number of SNPs were recorded between isolates taken from the same patient.4 WGS of strains from mucormycosis outbreaks have shown thousands of SNP differences.4,5FIGURE 1.: Phylogenetic tree of the donor and recipient fungal strains rooted with the reference strain of Apophysomyces variabilis. Isolates are donor peritoneal fluid isolate (donor site A, D0, and donor site A replicate extracted separately); donor bronchoalveolar lavage (BAL; donor site B, D-1); liver recipient deep wound infection tissue isolate (recipient 1 site A, D+16) and small bowel isolate (recipient 1 site B, D+16); lung recipient bronchial washing isolates (recipient 2 site A, D0 and recipient 2 site C, D0) and tibia isolate (recipient 2 site B, D+178); four nonrelated case isolates of A. variabilis; and a reference strain of A. variabilis NCCPF 1012052. For species identification using the combination nucleotide sequences of histone 3 gene, internal transcribed spacer region of the rRNA, D1 and D2 of the 28s, the donor and recipient isolates were most similar to A. variabilis with a sequence similarity of >95%. Genome sizes of 35.89–37.90 Mb and G+C% 41.65%–41.84% were comparable to the reference genome (39.39 Mb, G+C% 41.65%). N50 was 36899-73898.The donor had disseminated A. variabilis infection evident by growth from BAL and peritoneal fluid, causing kidney necrosis and intracranial hemorrhage through renal and cerebral vascular involvement, respectively. Transplanted lungs and liver were clearly infected as infection manifested in recipients of these organs. There were barriers to making the diagnosis antemortem in the donor due to the rare diagnosis, poor sensitivity of culture samples, and his initial denial of intravenous drug use. The lack of inflammatory response and lack of immunoglobulin or complement deposition in the kidney biopsy were unexpected findings for systemic autoimmune vasculitis and might have prompted further investigation, and urgent microscopy of purulent peritoneal fluid found at organ procurement may have shown fungal elements. Mold infections in IVDU are very uncommon. Roden et al6 documented 45 cases of mucormycosis in IVDU, of which 62% were manifest by cerebral involvement and 5% were disseminated. In conclusion, we have described disseminated A. variabilis infection in an IVDU organ donor misdiagnosed as systemic vasculitis, with infection transmitted to the lung and liver recipients with fatal outcome despite early aggressive antifungal therapy. Donors need very careful evaluation with specialist input when there are unusual clinical or pathological features which do not align with the proffered diagnosis. ACKNOWLEDGMENTS Thanks to Ian Arthur, PathWest Laboratory Medicine WA, QEII medical centre, for supplying the liver recipient and nonrelated A. variabilis strains. Availability of data and materials: Genomic data are available at SRA, accession numbers: SAMN18579195 (nonrelated case 1); SAMN18579196 (recipient 2, site B [tibia] D+178); SAMN18579197 (donor site B [BAL] D-1); SAMN18579198 (recipient 2, site A [bronchial washing] D0); SAMN18579199 (donor site A [peritoneal fluid] D0); SAMN18579200 (recipient 1 site B [small bowel] D+16); SAMN18579201 (nonrelated case 3); SAMN18579202 (nonrelated case 2); SAMN18579203 (nonrelated case 4); SAMN18579204 (recipient 1 site A [deep wound] D+16); SAMN18579205 (recipient 2 site C [bronchial washing] D0); and SAMN18579206 (donor site A [peritoneal fluid] D0).