Prototheca Wickerhamii Algaemia: an Emerging Infection in Solid Organ Transplant Recipients

Transplant Infectious Disease, ISSN 1398-2273 Case report Prototheca wickerhamii algaemia: an emerging infection in solid organ transplant recipients

1 T.D. Bandaranayake, A. Paniz Mondolfi, D.R. Peaper, M.F. Malinis. T.D. Bandaranayake , A. Paniz 2 2 1 Prototheca wickerhamii algaemia: an emerging infection in solid Mondolfi , D.R. Peaper , M.F. Malinis organ transplant recipients. 1Section of Infectious Diseases, Yale School of Medicine, Transpl Infect Dis 2015: 17: 599–604. All rights reserved New Haven, Connecticut, USA, 2Department of Laboratory Medicine, Yale School of Medicine, New Haven, Abstract: Prototheca wickerhamii is an alga that rarely causes Connecticut, USA human disease but has been reported increasingly among Prototheca wickerhamii immunocompromised individuals. We report a fatal case of Key words: ; algaemia; solid organ transplant; MALDI-TOF P. wickerhamii in a renal transplant recipient who presented with a cutaneous lesion that led to disseminated disease despite treatment Correspondence to: with voriconazole. We reviewed previous cases of protothecosis Thilinie D. Bandaranayake, Yale School of involving solid organ transplant recipients in the literature and Medicine, TAC Building Room S140, 300 Cedar discussed the value of newer microbiology platforms, i.e., matrix- Street, New Haven, CT 06519, USA assisted laser desorption ionization time-of-flight mass spectrometry Tel: (203) 737-4279 (MALDI-TOF), to achieve early diagnosis and impact outcomes. Fax: (203) 785-6815 E-mail: [email protected]

Received 12 March 2015, revised 25 April 2015, accepted for publication 14 May 2015

DOI: 10.1111/tid.12407 Transpl Infect Dis 2015: 17: 599–604

Prototheca wickerhamii is an achlorophyl algae ubiqui- tously found in environmental sources such as tree Case report slime, grass, fruits, vegetables, salt or fresh water, and in animals including deer, cattle, and dogs (1, 2). A 59-year-old man underwent deceased donor kidney Human disease caused by Prototheca is infrequent. The transplant because of end-stage renal disease second- first reported case was a soft tissue infection in 1964 ary to hypertension and diabetes mellitus. The imme- (3). To date, the most commonly reported species diate post-transplant course was complicated by causing human disease are P. wickerhamii and Proto- delayed graft function owing to focal segmental glom- theca zopfii (4, 5). Although the majority of described erulosclerosis, which required return to hemodialysis, cases have cutaneous involvement, gastrointestinal, after plasmapharesis and intravenous immunoglobulin respiratory, and urinary tract infections have also been (IVIG) treatment failed. His immunosuppression was reported (2). A significantly high mortality from limited to prednisone 5 mg/day after discontinuation of P. wickerhamii algaemia has been observed. Antifungal mycophenolate mofetil, azathioprine, and tacrolimus agents, such as amphotericin B (AmB) and voricona- 2 months post transplant after primary graft failure. zole, were used for treatment in reported cases based Four months after transplantation, he was admitted on its in vitro susceptibility (6). Herein, we report a fatal to a community hospital for altered mental status. An case of P. wickerhamii cutaneous disease in an immu- Escherichia coli urinary tract infection with bacteremia nosuppressed patient that led to dissemination despite was diagnosed and treated with cefepime. This admis- treatment with voriconazole. sion was also complicated by Clostridium difficile colitis.

599 Bandaranayake et al: Protothecosis in solid organ transplant

After a 12 hospital-day course, he was transferred to our Voriconazole was switched to anidulafungin. A Trans- institution because of a 3.4 9 2.8 9 4.8 cm perinephric plant Infectious Disease service consult recommended fluid collection adjacent to the graft, evident on com- switching the anti-infective from anidulafungin to lipo- puted tomography (CT) scan of the abdomen. The fluid somal AmB, because of concern for disseminated collection was drained by catheter and sent for culture, protothecosis, while awaiting follow-up cultures. Fol- which isolated E. coli. Treatment was modified to low-up blood culture drawn on postoperative day ciprofloxacin. In addition, he had cytomegalovirus (POD) 2 revealed no growth. viremia (8048 copies/mL) requiring valganciclovir. The patient had no evidence of endophthalmitis on During this hospitalization, he developed a draining eye exam, and no vegetation on transthoracic echocar- right upper extremity abscess. He denied prior trau- diogram. He remained hemodynamically stable on matic skin injury, despite appearance of poor skin treatment. Based on available microbiology data, anti- integrity. Wound debridement was performed. Isolation fungal therapy was switched back from anidulafungin of P. wickerhamii in tissue culture prompted the Trans- to voriconazole for treatment of cutaneous prototheco- plant Infectious Disease consult. CT scan of the right sis and possible candidemia. upper extremity revealed no deep-seated infection. His Additional testing was performed in the microbiology blood cultures revealed no growth. The patient was laboratory following incubation. A wet mount from a prescribed oral voriconazole 200 mg twice a day for colony of the Saboraud’s culture stained with lacto- 14 days, for cutaneous infection without evidence of phenol cotton blue revealed the distinctive endosporu- disseminated disease. He was discharged to an lating sporangia “morula forms” suggestive of extended care facility where he continued his treatment. Prototheca species, as did the periodic acid–Schiff stain Eight days after discharge, he presented to the (Fig. 1). Identification of the organism as P. wicker- emergency room (ER) with altered mental status. He hamii was confirmed by biochemical testing (VITEK 2; was hypotensive, with blood pressure (BP) of 62/ bioMerieux, Durham, North Carolina, USA). 31mmHg, but afebrile. The base of the right forearm The initial MALDI-TOF identification was attempted wound was clean. His abdomen was notably distended. using a direct formic acid-acetronile method, but Laboratory data were remarkable for leukocytosis Prototheca organisms possess a rigid thick wall. There- (24,000 cells/lL) with 55% bands and mildly elevated fore, the isolate was retested using manufacturer- lactate (2.5 mmol/L; normal = 0.4–2.2). CT of the abdo- recommended protocols for mycobacterial inactivation men and pelvis revealed a perforated anterior wall of the and protein extraction. MALDI-TOF MS analysis using sigmoid colon, with a 13 9 12 9 12 cm fluid collection the MicroFlex LT mass spectrometer (Bruker Dalton- and intraperitoneal and retroperitoneal free air. ics Inc., Billerica, Massachusetts, USA) confirmed the The patient underwent an emergency exploratory identification (1.686 score ID) as P. wickerhamii, fol- laparotomy, which revealed matted small intestinal lowed by Candida haemulonii (1.214) and Aspergillus loops and a large pelvic abscess. Drainage of the terreus (1.202) as the second and third best matches. abscess, and end descending loop colostomy, and colon The VITEK MS (bioMerieux) also matched (59.70%) resection were performed, and the patient was trans- the protein spectral fingerprint of the isolate as ferred to the surgical intensive care unit. P. wickerhamii using the reference spectral database. Cultures from the intra-abdominal abscess grew Although the score cutoff values were lower than those mixed flora without evidence of yeast or Prototheca. proposed by the manufacturers, correct identification Piperacillin-tazobactam was initiated and voriconazole was achieved by both methods. was continued. Blood culture obtained in the ER grew On POD 8, the isolated yeast was confirmed as multiple and variable-sized round and oval-shaped Prototheca. Because of the microbiological and clinical gram-positive cells, only in the aerobic bottle, which failure of voriconazole, treatment was changed to con- were reported as yeast. Plates were inoculated and, ventional AmB 0.5 mg/kg body weight. Nevertheless, after 16 h of incubation, small smooth, moist, dull white the next day, the patient was found lethargic and colonies were seen. Matrix-assisted laser desorption hypotensive (BP 84/59mmHg) with leukocytosis ionization time-of-flight mass spectrometry (MALDI- (19,000 cells/lL), elevated lactate (6.6 mmol/L), and TOF MS) was performed, yielding an identification of elevated bilirubin (11 mg/dL). Chest x-ray revealed Candida dublinensis with an 83.5% score. Because of the pulmonary edema with bilateral pleural effusions. CT of age of the colonies and concerns about discordance chest, abdomen, and pelvis revealed diffuse anasarca. between morphology and identification, further incu- On POD 10, he was found unresponsive and pulseless, bation was undertaken, and this result was not released with systolic BP in the 30s. Advanced cardiac life support to the chart. was performed, but, unfortunately, the patient died.

600 Transplant Infectious Disease 2015: 17: 599–604 Bandaranayake et al: Protothecosis in solid organ transplant

A C

Fig. 1. Histochemical stains of Prototheca wickerhamii. (A) Gram-positive spherical cells mimicking Candida yeast forms (Gram stain 4009). (B) Lactophenol cotton blue wet mount, depicting variably sized organisms with typical morula forms (4009). (C) Periodic acid-Schiff stain showing characteristic “cartwheel” and “honeycomb” appearance of morula-like structures (6009). Bar 10 lm.

Antifungal susceptibility testing was performed post- infection, and 1 (our present case) had cutaneous mortem by gradient diffusion, and the minimum inhib- infection that disseminated. Of note, 8 of 9 patients itory concentrations were AmB = 0.5 lg/mL, and with known outcomes died. Thus, mortality for voriconazole = 6 lg/mL. Testing was also done for protothecosis is estimated at 89% among SOT caspofungin and fluconazole; however, as no interpre- recipients. tive criteria are available for Prototheca, the usefulness Immunosuppression is a known risk factor for poor of these values is questionable. outcomes, despite low virulence of Prototheca species (17, 18). Infections caused by bacteria, virus, and fungus were often reported to precede or occur simultaneously Discussion with Prototheca infection, indicative of the individual’s net state of immunosuppression (19). Neutrophils, P. wickerhamii is an algae that may infect humans natural killer cells, and cell-mediated and humoral through multiple environmental contact sources such immunity are implicated to have a significant role in as water or soil (7). The pathogenesis, as well as many infection control (2, 18). Humoral immunity has been biological aspects of human protothecosis, remain implicated as an important host defense against Proto- largely unknown. Cutaneous and osteoarticular dis- theca (18). Immunoglobulin-G antibody specific for eases can result from compromised skin integrity P. wickerhamii showed independent killing activity in secondary to trauma or postoperative wounds (3), an in vitro study (20). As hypogammaglobulinemia is which can disseminate in immunosuppressed individu- common after transplantation, intravenous immunoglo- als with an associated mortality rate of 62.5% (6). In our bulin G (IVIG) could be a possible adjunct treatment for patient, the initial Prototheca cutaneous infection could protothecosis in SOT recipients. The exact pathogenesis be from inoculation by either traumatic injury or line- of protothecosis and role of host factors are unknown; related procedure. therefore, more studies are needed to understand this Ten solid organ transplant (SOT) recipients with disease. In our patient, cytomegalovirus viremia, E. coli protothecosis have been reported in the literature to sepsis, and dialysis status could have contributed to his date, including our patient (Table 1) (8–16). Four had immune dysfunction, and hence, the increased risk for primary cutaneous infection only, 5 had bloodstream disseminated disease.

Transplant Infectious Disease 2015: 17: 599–604 601 Bandaranayake et al: Protothecosis in solid organ transplant

Reported cases of protothecosis in solid organ transplant recipients

Age/ Prototheca Organ Gender Immunosuppression species Presentation Co-infection Treatment Outcome Reference

Kidney 30/M AZT, prednisone, P. wickerhamii Cutaneous Klebsiella Tetracycline Died1 (10) ALG pneumoniae, Proteus, Candida albicans Kidney NA NA P. wickerhamii Cutaneous NA NA NA (11) Kidney 44/M NA P. wickerhamii Cutaneous NA Amputation of ﬁnger Cured (12) and local debridement, AmB Kidney 44/M NA P. wickerhamii Cutaneous NA Local excision, Died2 (13) tetracycline Lung 59/F NA P. zopﬁi BSI CMV NA Died (14) Liver 61/M MMF, prednisone, P. wickerhamii BSI VRE, Escherichia AmB Died (15) TAC coli, CMV Heart 78/F Prednisone MMF, P. wickerhamii BSI VRE LAmB Died (9) CSA Kidney 61/M NA P. wickerhamii BSI NA None Died (16) Heart 69/F Prednisone, MMF, P. wickerhamii BSI Candida glabrata, Caspofungin Died (8) CSA CMV Kidney 59/M Prednisone P. wickerhamii Cutaneous, CMV viremia Debridement, Died Present BSI voriconazole, case then AmB

1Died of Klebsiella septicemia and shock. 2Died of acute necrotizing pancreatitis. M, male; AZT, azathioprine; ALG, antilymphocyte globulin; NA, not available; AmB, amphotericin B deoxycholate; F, female; BSI, bloodstream infection; CMV, cytomegalovirus; MMF, mycophenolate mofetil; TAC, tacrolimus, VRE, vancomycin-resistant Enterococcus faecium. CSA, cyclosporine; LAmB, liposomal amphotericin.

Table 1

Because of the rarity of protothecosis, treatment is AmB had a reported success rate of 77% in 26 treated not clearly established. Case reports have described immunocompromised and immunocompetent hosts use of certain antifungal agents. Prototheca species (17). Among SOT with protothecosis, only 1 of the 3 have in vitro susceptibility to AmB and voriconazole, patients who received AmB as initial treatment survived explained by the presence of ergosterol in the algae’s (Table 1), which could be explained by limited disease cell membrane similar to fungi (6, 17). Voriconazole and aggressive surgical intervention compared with the has achieved treatment success in 3 of 4 reported 2 cases with disseminated disease. Even with limited cutaneous cases (17). Treatment failure in our patient data, AmB might be a better option for immunocom- could be explained by the high minimum inhibitory promised hosts, regardless of disease presentation. concentration. Experts emphasize that azole in vitro Other medications, such as tetracycline, aminoglyco- susceptibility does not necessarily predict favorable side, echinocandin, terbinafine, and polymyxin, used clinical outcome; thus, it remains debatable how the alone or in combination, had mixed results (17). in vitro susceptibility results should be interpreted (21). Miltefosine, an alkylphosphocholine drug used for In retrospect, drug level monitoring could have been leishmaniasis and salvage therapy for fungal infection, implemented to ensure therapeutic levels. A subthera- has in vitro activity against P. zopfii; hence, it could be a peutic voriconazole level could be a plausible reason for potential treatment option for protothecosis but treatment failure. Given our clinical experience, caution remains to be evaluated in clinical practice (22). should be exercised with use of empiric voriconazole, High mortality associated with disseminated proto- despite previous suggestions (6). thecosis is often a result of delayed identification.

602 Transplant Infectious Disease 2015: 17: 599–604 Bandaranayake et al: Protothecosis in solid organ transplant

Prototheca can be easily misdiagnosed as a yeast, and mortality. Future studies on other treatment because of morphologic similarity (8, 9, 23), as we have modalities, such as IVIG and miltefosine, are needed. experienced (Fig. 1A). If clinical suspicion is high, an experienced microbiologist is required to reach the correct diagnosis. Suggested tests to confirm diagnosis Acknowledgements: include histochemical stains (lactophenol cotton blue, periodic acid-Schiff, Gram, and trichrome), histopathol- Author contributions: T.D.B.: Drafting of the article, data ogy, and advanced techniques including VITEK and collection, analysis and interpretation of data, and MALDI-TOF (2). approval of the article. A.P.M.: Drafting of the article, MALDI-TOF has dramatically impacted the tradi- analysis and interpretation of data, critical revision of tional workflow for identification of clinical isolates by the article, and approval of the article. D.R.P: Analysis providing rapid and reliable identifications with low and interpretation of data, critical revision of the article, cost per identification. However, Prototheca species and approval of the article. M.F.M.: Drafting of the remain rarely isolated pathogens and the profiles have article, analysis and interpretation of data, critical been included in reference databases only recently. revision of the article, and approval of the article. Manufacturers continue to expand the reference databases, improving the limitations of this technology (24). The initial failed identification in our case, and in a prior References report (22), likely reflects the absence of representative reference spectra for certain species isolates, protein 1. Huerre M, Ravisse P, Solomon H, et al. [Human protothecosis variability among strains, or the nature of the organism, and environment]. Bull Soc Pathol Exotique 1993; 86 (5 Pt 2): and the extraction method used to reveal its protein 484–488. milieu. The forces required to produce efficient break- 2. Lass-Florl C, Mayr A. Human protothecosis. Clin Microbiol Rev age of the organisms may be comparable to those 2007; 20 (2): 230–242. required for organisms with lipid-rich waxy cell wall, 3. Davies RR, Spencer H, Wakelin PO. A case of human protothecosis. Transact Roy Soc Trop Med Hyg 1964; 58: such as Mycobacteria, because Prototheca species con- 448–451. tain a rigid wall with high tensile strength and several 4. Tubaki K, Soneda M. Cultural and toxonomical studies on thick-walled autospores (2, 25). The addition of a Proteotheca. Mycol J Nagao Instit Tokyo 1959; 6: 25–34. sonication step could increase the identification scores 5. Kruger WK. Characteristics of some microorganisms in (26). Thus, we believe that, as with Mycobacteria, deciduous tree sap. Hedwigia 1894; 33: 241–266. 6. Linares MJ, Solis F, Casal M. In vitro activity of voriconazole mechanical disruption of cell walls by bead beating against Prototheca wickerhamii: comparative evaluation of aided in increasing the yield of protein extraction and sensititre and NCCLS M27-A2 methods of detection. J Clin favored a more accurate recording of the protein Microbiol 2005; 43 (5): 2520–2522. spectra in this case. Despite accurate identification at 7. Wirth FA, Passalacqua JA, Kao G. Disseminated cutaneous a genus and species level, the scores obtained for our protothecosis in an immunocompromised host: a case report and literature review. Cutis 1999; 63 (3): 185–188. isolate were lower than the cutoff values established by 8. Nwanguma V, Cleveland K, Baselski V. Fatal Prototheca the manufacturer for identification of gram-negative wickerhamii bloodstream infection in a cardiac allograft recipient. bacilli and gram-positive cocci, but still within accept- J Clin Microbiol 2011; 49 (11): 4024; author reply 4025. able validated ranges for yeast and dermatophytes (27). 9. McMullan B, Muthiah K, Stark D, Lee L, Marriott D. Prototheca wickerhamii mimicking yeast: a cautionary tale. J Clin Microbiol 2011; 49 (8): 3078–3081. 10. Wolfe ID, Sacks HG, Samorodin CS, Robinson HM. Cutaneous Conclusion protothecosis in a patient receiving immunosuppressive therapy. Arch Dermatol 1976; 112 (6): 829–832. Protothecosis is an emerging fatal infection among SOT 11. Mezger E, Eisses JF, Smith MJ. Protothecal cellulitis in a renal recipients. Early recognition of Prototheca is important transplant patient. Lab Invest (abstract) 1981; 44: 8IA. 12. Wolfson JS, Sober AJ, Rubin RH. Dermatologic manifestations of to help clinicians institute prompt therapeutic interven- infections in immunocompromised patients. Medicine tion. In our case, MALDI-TOF, an emerging identifica- (Baltimore) 1985; 64 (2): 115–133. tion method, was demonstrated to be a valuable 13. Tejada E, Parker CM. Cutaneous erythematous nodular lesion in adjunctive diagnostic test. Treatment options for proto- a crab fisherman. Protothecosis. Arch Dermatol 1994; 130 (2): thecosis are limited. Voriconazole has unpredictable 244–245, 247–248. 14. Kwok N, Schwartz SN. Prototheca sepsis in a lung transplant in vitro susceptibility. Therefore, AmB should be patient. Clin Microbiol Newslet 1996; 18: 183–184. considered as first-line treatment, even in limited 15. Narita M, Muder RR, Cacciarelli TV, Singh N. Protothecosis after disease in SOT recipients, to prevent dissemination liver transplantation. Liver Transplant 2008; 14 (8): 1211–1215.

Transplant Infectious Disease 2015: 17: 599–604 603 Bandaranayake et al: Protothecosis in solid organ transplant

16. Mohd Tap R, Sabaratnam P, Salleh MA, Abd Razak MF, Ahmad review of previous cases. Transpl Infect Dis 2014; 16 (3): N. Characterization of Prototheca wickerhamii isolated from 490–495. disseminated algaemia of kidney transplant patient from 23. Min Z, Moser SA, Pappas PG. Prototheca wickerhamii algaemia Malaysia. Mycopathologia 2012; 173 (2–3): 173–178. presenting as cholestatic hepatitis in a patient with systemic lupus 17. Todd JR, King JW, Oberle A, et al. Protothecosis: report of a case erythematosus: a case report and literature review. Med Mycol with 20-year follow-up, and review of previously published cases. Case Rep 2012; 2: 19–22. Med Mycol 2012; 50 (7): 673–689. 24. Murugaiyan J, Ahrholdt J, Kowbel V, Roesler U. Establishment of 18. Kwong JC, Ward PB, Johnson PD. Cutaneous protothecosis in a a matrix-assisted laser desorption ionization time-of-flight mass patient with hypogammaglobulinemia. Med Mycol Case Rep spectrometry database for rapid identification of infectious 2013; 2: 132–133. achlorophyllous green micro-algae of the genus Prototheca. Clin 19. Yeh CT, Li MC, Chuang YC, et al. Prototheca algaemia: a rare but Microbiol Infect 2012; 18 (5): 461–467. fatal opportunistic infection among immunocompromised 25. Lloyd D, Turner G. The cell wall of Prototheca zopfii. J Gen individuals. Japan J Infect Dis 2013; 66 (6): 523–525. Microbiol 1968; 50 (3): 421–427. 20. Phair JP, Williams JE, Bassaris HP, Zeiss CR, Morlock BA. 26. Ahrholdt J, Murugaiyan J, Straubinger RK, Jagielski T, Roesler U. Phagocytosis and algicidal activity of human polymorphonuclear Epidemiological analysis of worldwide bovine, canine and neutrophils against Prototheca wickerhamii. J Infect Dis 1981; human clinical Prototheca isolates by PCR genotyping and 144 (1): 72–77. MALDI-TOF mass spectrometry proteomic phenotyping. Med 21. Leimann BC, Monteiro PC, Lazera M, Candanoza ER, Wanke B. Mycol 2012; 50 (3): 234–243. Protothecosis. Med Mycol 2004; 42 (2): 95–106. 27. Theel ES. Matrix-assisted laser desorption ionization-time of flight 22. Macesic N, Fleming S, Kidd S, et al. Protothecosis in mass spectrometry for the identification of bacterial and fungal hematopoietic stem cell transplantation: case report and isolates. Clin Microbiol Newslet 2013; 35 (19): 155–161.

604 Transplant Infectious Disease 2015: 17: 599–604