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Clinicopathological features and outcomes of pythiosis

Open AccessPublished:April 10, 2018DOI:https://doi.org/10.1016/j.ijid.2018.03.021

      Highlights

      • Serum immunochromatography tests are useful for the rapid diagnosis of vascularpythiosis.
      • Non-thalassemic patients had an unusual presentation, with brain infection.
      • Vascular pythiosis is a rapidly fatal disease in neutropenic patients.
      • Itraconazole and terbinafine prolonged survival in patients with residual disease.

      Abstract

      Objectives

      Vascular pythiosis is a life-threatening infection caused by the oomycete Pythium insidiosum. This article reports the clinical presentation, serodiagnosis, pathology, and outcomes seen at the authors’ institution.

      Methods

      The cases of patients with proven vascular pythiosis at Ramathibodi Hospital, Mahidol University, Bangkok, Thailand from January 2006 to December 2016 were analyzed retrospectively.

      Results

      Thirteen patients were analyzed, eight of whom had underlying thalassemias. Of the remaining five patients, one had aplastic anemia, one had myelodysplasia, one had acute leukemia, one had cirrhosis, and one had alcoholism. Neutropenic patients showed a rapid clinical deterioration. Atypical presentations including carotid arteritis, aneurysm, brain abscess, and stroke occurred in the non-thalassemic patients. Serology yielded positive results in all cases, with a rapid turnaround time. Serology has the advantage of providing a presurgical diagnosis, which allows prompt surgery and clinical cure to be achieved. Pathology revealed a neutrophilic response in the acute phase and a later shift to granuloma. Immunotherapy in combination with itraconazole and terbinafine was given. The amputation rate was 77%, and disease-free surgical margins were achieved in five cases (38%). The mortality rate was 31%.

      Conclusions

      This study highlights new aspects of pythiosis, such as the unusual host, clinical presentation, serology as a marker for rapid diagnosis, histopathology, and outcomes. Early recognition of the disease with prompt multimodality treatment may improve survival.

      Keywords

      Introduction

      Pythiosis is an emerging life-threatening disease that is endemic in Thailand and occurs in tropical and subtropical areas of the world (
      • Krajaejun T.
      • Sathapatayavongs B.
      • Pracharktam R.
      • Nitiyanant P.
      • Leelachaikul P.
      • Wanachiwanawin W.
      • et al.
      Clinical and epidemiological analyses of human pythiosis in Thailand.
      ). Pythiosis is caused by Pythium insidiosum, an oomycete of the kingdom Straminipila, phylum Oomycota, and was first known as an aquatic fungus (
      • Krajaejun T.
      • Sathapatayavongs B.
      • Pracharktam R.
      • Nitiyanant P.
      • Leelachaikul P.
      • Wanachiwanawin W.
      • et al.
      Clinical and epidemiological analyses of human pythiosis in Thailand.
      ). The clinical presentation of pythiosis includes skin, subcutaneous tissue, cornea, vascular, and disseminated forms (
      • Krajaejun T.
      • Sathapatayavongs B.
      • Pracharktam R.
      • Nitiyanant P.
      • Leelachaikul P.
      • Wanachiwanawin W.
      • et al.
      Clinical and epidemiological analyses of human pythiosis in Thailand.
      ,
      • Sathapatayavongs B.
      • Leelachaikul P.
      • Prachaktam R.
      • Atichartakarn V.
      • Sriphojanart S.
      • Trairatvorakul
      • et al.
      Human pythiosis associated with thalassemia hemoglobinopathy syndrome.
      ). Known risk factors for vascular pythiosis include thalassemia, hemoglobinopathy, paroxysmal nocturnal hemoglobinuria, aplastic anemia, and leukemia (
      • Krajaejun T.
      • Sathapatayavongs B.
      • Pracharktam R.
      • Nitiyanant P.
      • Leelachaikul P.
      • Wanachiwanawin W.
      • et al.
      Clinical and epidemiological analyses of human pythiosis in Thailand.
      ). However, the pattern of the disease has been changing and it has been found to affect individuals without risk factors, who have presented with unusual features, e.g., necrotizing cellulitis and brain abscesses (
      • Kirzhner M.
      • Arnold S.R.
      • Lyle C.
      • Mendoza L.L.
      • Fleming J.C.
      Pythium insidiosum: a rare necrotizing orbital and facial infection.
      ,
      • Narkwiboonwong T.
      • T.K
      • Watanakijthavonkul K.
      • Paocharern P.
      • Singsakul A.
      • Wongsa A.
      • Woracharoensri N.
      • et al.
      Cerebral Pythiosis: a case report of Pythium insidiosum infection presented with brain abscess.
      ,
      • Shenep J.L.
      • English B.K.
      • Kaufman L.
      • Pearson T.A.
      • Thompson J.W.
      • Kaufman R.A.
      • et al.
      Successful medical therapy for deeply invasive facial infection due to Pythium insidiosum in a child.
      ). Furthermore, the rarity of the disease has led to under-recognition, under-diagnosis, and delays in diagnosis, and this has contributed to the occurrence of advanced disease, which affects survival (
      • Permpalung N.
      • Worasilchai N.
      • Plongla R.
      • Upala S.
      • Sanguankeo A.
      • Paitoonpong L.
      • et al.
      Treatment outcomes of surgery, antifungal therapy and immunotherapy in ocular and vascular human pythiosis: a retrospective study of 18 patients.
      ,
      • Reanpang T.
      • Orrapin S.
      • Orrapin S.
      • Arworn S.
      • Kattipatanapong T.
      • Srisuwan T.
      • et al.
      Vascular pythiosis of the lower extremity in Northern Thailand: ten years’ experience.
      ,
      • Sermsathanasawadi N.
      • Praditsuktavorn B.
      • Hongku K.
      • Wongwanit C.
      • Chinsakchai K.
      • Ruangsetakit
      • et al.
      Outcomes and factors influencing prognosis in patients with vascular pythiosis.
      ).
      Historically, the histopathological diagnosis has often been revealed after the surgical procedure (
      • Krajaejun T.
      • Sathapatayavongs B.
      • Pracharktam R.
      • Nitiyanant P.
      • Leelachaikul P.
      • Wanachiwanawin W.
      • et al.
      Clinical and epidemiological analyses of human pythiosis in Thailand.
      ). The gold standard for diagnosis requires fungal culture, which is a time-consuming method (
      • Chaiprasert A.
      • Samerpitak K.
      • Wanachiwanawin W.
      • Thasnakorn P.
      Induction of zoospore formation in Thai isolates of Pythium insidiosum.
      ,
      • Intaramat A.
      • Sornprachum T.
      • Chantrathonkul B.
      • Chaisuriya P.
      • Lohnoo T.
      • Yingyong W.
      • et al.
      Protein A/G-based immunochromatographic test for serodiagnosis of pythiosis in human and animal subjects from Asia and Americas.
      ,
      • Krajaejun T.
      • Imkhieo S.
      • Intaramat A.
      • Ratanabanangkoon K.
      Development of an immunochromatographic test for rapid serodiagnosis of human pythiosis.
      ,
      • Krajaejun T.
      • Kunakorn M.
      • Pracharktam R.
      • Chongtrakool P.
      • Sathapatayavongs B.
      • Chaiprasert A.
      • et al.
      Identification of a novel 74-kiloDalton immunodominant antigen of Pythium insidiosum recognized by sera from human patients with pythiosis.
      ). Recent advances made over the past decade include serodiagnosis, e.g., immunodiffusion (
      • Pracharktam R.
      • Changtrakool P.
      • Sathapatayavongs B.
      • Jayanetra P.
      • Ajello L.
      Immunodiffusion test for diagnosis and monitoring of human pythiosis insidiosi.
      ), ELISA (
      • Krajaejun T.
      • Kunakorn M.
      • Niemhom S.
      • Chongtrakool P.
      • Pracharktam R.
      Development and evaluation of an in-house enzyme-linked immunosorbent assay for early diagnosis and monitoring of human pythiosis.
      ), Western blot analysis, the hemagglutination test (
      • Jindayok T.
      • Piromsontikorn S.
      • Srimuang S.
      • Khupulsup K.
      • Krajaejun T.
      Hemagglutination test for rapid serodiagnosis of human pythiosis.
      ), and the immunochromatographic test (
      • Chareonsirisuthigul T.
      • Khositnithikul R.
      • Intaramat A.
      • Inkomlue R.
      • Sriwanichrak K.
      • Piromsontikorn
      • et al.
      Performance comparison of immunodiffusion, enzyme-linked immunosorbent assay, immunochromatography and hemagglutination for serodiagnosis of human pythiosis.
      ). The early recognition of pythiosis and confirmation by rapid serological tests could improve the diagnosis and allow the prompt management of this life-threatening disease. Surgery to achieve organism-free surgical margins is the mainstay of successful treatment of vascular pythiosis, which often leads to amputation (
      • Sermsathanasawadi N.
      • Praditsuktavorn B.
      • Hongku K.
      • Wongwanit C.
      • Chinsakchai K.
      • Ruangsetakit
      • et al.
      Outcomes and factors influencing prognosis in patients with vascular pythiosis.
      ). Patients with unresectable disease, e.g., those with suprainguinal lesions or aortic involvement, often die (
      • Krajaejun T.
      • Sathapatayavongs B.
      • Pracharktam R.
      • Nitiyanant P.
      • Leelachaikul P.
      • Wanachiwanawin W.
      • et al.
      Clinical and epidemiological analyses of human pythiosis in Thailand.
      ,
      • Permpalung N.
      • Worasilchai N.
      • Plongla R.
      • Upala S.
      • Sanguankeo A.
      • Paitoonpong L.
      • et al.
      Treatment outcomes of surgery, antifungal therapy and immunotherapy in ocular and vascular human pythiosis: a retrospective study of 18 patients.
      ,
      • Sermsathanasawadi N.
      • Praditsuktavorn B.
      • Hongku K.
      • Wongwanit C.
      • Chinsakchai K.
      • Ruangsetakit
      • et al.
      Outcomes and factors influencing prognosis in patients with vascular pythiosis.
      ).
      Antifungal therapy with itraconazole and terbinafine alone has rarely been successful (
      • Krajaejun T.
      • Sathapatayavongs B.
      • Pracharktam R.
      • Nitiyanant P.
      • Leelachaikul P.
      • Wanachiwanawin W.
      • et al.
      Clinical and epidemiological analyses of human pythiosis in Thailand.
      ,
      • Shenep J.L.
      • English B.K.
      • Kaufman L.
      • Pearson T.A.
      • Thompson J.W.
      • Kaufman R.A.
      • et al.
      Successful medical therapy for deeply invasive facial infection due to Pythium insidiosum in a child.
      ). Immunotherapeutic vaccination with P. insidiosum antigen (PIA) has been used as an adjunctive treatment. However, the efficacy of this therapy remains inconclusive (
      • Permpalung N.
      • Worasilchai N.
      • Plongla R.
      • Upala S.
      • Sanguankeo A.
      • Paitoonpong L.
      • et al.
      Treatment outcomes of surgery, antifungal therapy and immunotherapy in ocular and vascular human pythiosis: a retrospective study of 18 patients.
      ,
      • Thitithanyanont A.
      • Mendoza L.
      • Chuansumrit A.
      • Pracharktam R.
      • Laothamatas J.
      • Sathapatayavongs B.
      • et al.
      Use of an immunotherapeutic vaccine to treat a life-threatening human arteritic infection caused by Pythium insidiosum.
      ,
      • Wanachiwanawin W.
      • Mendoza L.
      • Visuthisakchai S.
      • Mutsikapan P.
      • Sathapatayavongs B.
      • Chaiprasert
      • et al.
      Efficacy of immunotherapy using antigens of Pythium insidiosum in the treatment of vascular pythiosis in humans.
      ).

      Methods

      A retrospective analysis of patients with vascular pythiosis seen at Ramathibodi Hospital, Mahidol University, Bangkok, Thailand from January 2006 to December 2016 was conducted. The diagnosis was confirmed by the presence of one or more of the following: (1) isolation of P. insidiosum from infected tissue confirmed by induction and the identification of zoospores (
      • Chaiprasert A.
      • Samerpitak K.
      • Wanachiwanawin W.
      • Thasnakorn P.
      Induction of zoospore formation in Thai isolates of Pythium insidiosum.
      ); (2) detection of serum anti-P. insidiosum antibodies by established serodiagnostic tests (immunodiffusion testing, immunochromatography test (ICT), hemagglutination method (HA), Western blotting) (
      • Chareonsirisuthigul T.
      • Khositnithikul R.
      • Intaramat A.
      • Inkomlue R.
      • Sriwanichrak K.
      • Piromsontikorn
      • et al.
      Performance comparison of immunodiffusion, enzyme-linked immunosorbent assay, immunochromatography and hemagglutination for serodiagnosis of human pythiosis.
      ,
      • Jindayok T.
      • Piromsontikorn S.
      • Srimuang S.
      • Khupulsup K.
      • Krajaejun T.
      Hemagglutination test for rapid serodiagnosis of human pythiosis.
      ,
      • Krajaejun T.
      • Kunakorn M.
      • Niemhom S.
      • Chongtrakool P.
      • Pracharktam R.
      Development and evaluation of an in-house enzyme-linked immunosorbent assay for early diagnosis and monitoring of human pythiosis.
      ,
      • Pracharktam R.
      • Changtrakool P.
      • Sathapatayavongs B.
      • Jayanetra P.
      • Ajello L.
      Immunodiffusion test for diagnosis and monitoring of human pythiosis insidiosi.
      ); and (3) presence of clinical and pathological features typical of P. insidiosum infection (
      • Chareonsirisuthigul T.
      • Khositnithikul R.
      • Intaramat A.
      • Inkomlue R.
      • Sriwanichrak K.
      • Piromsontikorn
      • et al.
      Performance comparison of immunodiffusion, enzyme-linked immunosorbent assay, immunochromatography and hemagglutination for serodiagnosis of human pythiosis.
      ,
      • Jindayok T.
      • Piromsontikorn S.
      • Srimuang S.
      • Khupulsup K.
      • Krajaejun T.
      Hemagglutination test for rapid serodiagnosis of human pythiosis.
      ,
      • Krajaejun T.
      • Sathapatayavongs B.
      • Pracharktam R.
      • Nitiyanant P.
      • Leelachaikul P.
      • Wanachiwanawin W.
      • et al.
      Clinical and epidemiological analyses of human pythiosis in Thailand.
      ). Immunohistochemical staining for P. insidiosum was confirmed in tissue pathology using the method described in a previous report (
      • Inkomlue R.
      • Larbcharoensub N.
      • Karnsombut P.
      • Lerksuthirat T.
      • Aroonroch R.
      • Lohnoo T.
      • et al.
      Development of an anti-elicitin antibody-based immunohistochemical assay for diagnosis of pythiosis.
      ).
      The Institutional Review Board Ethics Committee approved this study (No. 05-60-77).

      Data collection

      Patient characteristics including age, sex, occupation, residential province, history of trauma, history of contact with contaminated water, underlying disease, clinical presentation, and duration of symptoms were collected. Laboratory data recorded included the complete blood count, liver function tests, blood urea nitrogen, serum creatinine, fasting glucose, ferritin level, hemoglobin typing, serum anti-Pythium antibody, radiological findings, microbiological data, and histopathological data. Treatment data, including the time from disease onset to first surgery, type of surgery, type and duration of antifungal treatment, PIA vaccine use, and outcome were collected.
      The PIA vaccine given in this study has been described in detail in a previous report (
      • Permpalung N.
      • Worasilchai N.
      • Plongla R.
      • Upala S.
      • Sanguankeo A.
      • Paitoonpong L.
      • et al.
      Treatment outcomes of surgery, antifungal therapy and immunotherapy in ocular and vascular human pythiosis: a retrospective study of 18 patients.
      ). The PIA concentration was 2.0 mg/ml. PIA was administered subcutaneously. This vaccine was provided by Dr. Ariya Chindamporn. All patients were made aware that the PIA vaccine is still under investigation and informed consent was obtained.

      Results

      Thirteen patients were included in this study. Most of the patients were male with an occupation involving agriculture; only two were female (Table 1). Most patients had an underlying thalassemia; only half had been diagnosed previously. Most patients had anemia and hypoalbuminemia. Eight patients, seven with thalassemia and one with myelodysplasia (MDS), had an elevated ferritin level. However, few patients had a prior diagnosis of hemochromatosis and had received an iron chelator. Details of the underlying diseases, clinical presentation, diagnosis, treatment, and outcomes are shown in Table 2.
      Table 1Baseline characteristics of patients with vascular pythiosis (N = 13).
      ParameterMedian (range)
      Age (years)52 (13–75)
      Sex, male, n (%)11 (84.6)
      Occupation, agriculture, n (%)11 (84.6)
      Underlying disease, n (%)
       Hematological disease11 (84.6)
       Thalassemia8 (61.5)
      Duration of symptoms (months)4 (0–11)
      Temperature (°C)38 (36.2–40)
      White blood cell count (×109/l)11.40 (2.69–15.10)
      Hematocrit (%)23.2 (14–36)
      Platelet count (×109/l)162.5 (21.0–527.0)
      Albumin (g/l)25.5 (15.8–41)
      Total bilirubin (mg/dl)2.2 (0.3–3.1)
      Alanine aminotransferase (U/l)45 (22–125)
      Creatinine (mg/dl)0.54 (0.3–1.8)
      Ferritin (ng/ml) (n = 8/13)3493 (920–19 238)
      Treatment, n (%)
       Amputation10 (77)
       Disease-free surgical margins5 (38.4)
      Medical therapy, n (%)
       Combination of itraconazole and terbinafine11 (84.6)
       Itraconazole13 (100)
       Terbinafine11 (84.6)
       SSKI5 (38.4)
       Other
      Voriconazole, amphotericin B deoxycholate, caspofungin (one of each).
      3 (23)
      Duration of antifungals (days)181 (3–2231)
      Immunotherapeutic vaccine, n (%)11 (84.6)
       Doses (no., range)3.5 (0–7)
      Outcome
       Died, n (%)4 (31)
      Duration of follow-up after presentation (days)184 (6–2481)
      Duration of follow-up among survivors (days)384 (50–2481)
      SSKI, potassium iodide oral solution.
      a Voriconazole, amphotericin B deoxycholate, caspofungin (one of each).
      Table 2Clinical presentation of patients with vascular pythiosis (N = 13).
      No.Age (years)/sexUnderlying diseaseClinical manifestations (duration, months)Vascular involvement (side)Diagnosis (duration, days after initial visit)Surgery (duration, days after initial visit)Margins—vascular, soft tissueTreatment drugs (duration, days)PIA vaccine—number of doses and scheduleOutcome and duration of survival (days)
      154/MHCV cirrhosis

      HbCS trait reported by Khunchet et al. (18)
      Multiple subcutaneous nodules in forearm (4)

      Thumb gangrene (day 7 prior to admission (PTA))

      Thenar abscess

      Muscle necrosis
      Right radial a., ulnar a., interosseous a.Serology (ICT) (day 11)

      Abscess isolation of P. insidiosum from culture (day 21)

      Pathology
      Debridement

      Above elbow amputation (day 32)
      Free, freeItraconazole, terbinafine, SSKI (46 days)Two: days 0 and 6Survived (lost to F/U after discharge)
      275/MHbHChronic ulcer (4)

      Gangrene of toe (day 2 PTA)
      Right superficial femoral a., popliteal a., tibial a., peroneal a.Serology (ICT) (day 0)

      Vessel isolation of P. insidiosum from culture
      Above knee amputation (day 4)Free, NoTerbinafine, SSKI (27, 32 days)

      Discontinuation due to DRESS syndrome
      Seven: days 0, 14, 46, 58, 105, 196, 392Survived (day 704 off Rx; day 340 after last dose of vaccine)
      355/MCSA2A Bart’s HChronic foot ulcer (6)

      Pain, pallor of leg (day 3 PTA)
      Right femoral a., popliteal a., posterior tibial a.Serology (ICT) day 0

      Vessel isolation of P. insidiosum from culture
      Above knee amputation (day 1)Free, freeItraconazole, terbinafine (262 days)Six: days 0, 14, 28,42, 84, 168Survived (day 365 off Rx; day 453 after last dose of vaccine)
      456/MβThal/HbEChronic foot ulcer (2)Right popliteal a., tibial a., peroneal a.Serology (ICT) day 0

      Vessel isolation of P. insidiosum from culture

      Pathology
      Above knee amputation (day 1)Free, freeItraconazole, terbinafine (383 days)Seven: days 0, 14, 28, 40, 82, 166, 208Survived (day 105 after last dose of vaccine)
      548/MThalChronic ulcer of leg and foot (6)Right superficial femoral a.Serology (ICT) day 1

      Vessel isolation of P. insidiosum from culture

      Pathology
      Above knee amputation (day 1)Free, freeItraconazole, terbinafine (181 days)Four: days 0, 13, 27, 75Survived (lost to F/U, day 105 after last dose of vaccine)
      629/FHbH CS, AIHA, HBVChronic leg ulcer (5)Right femoral a.Pathology

      Serology (HA) day 20 after surgery
      Above knee amputation (OSH)No, noItraconazole, Terbinafine (368 days)NoneSurvived (day 145 off Rx)
      738/MThalChronic leg ulcer (4)Left superficial femoral a.Serology (ICT) day 1

      Vessel isolation of P. insidiosum from culture

      Pathology
      Above knee amputation (day 3)No, noSSKI Terbinafine, itraconazole (122 days)Three: days 0, 7, 22Survived (lost to F/U, day 99 after last dose of vaccine)
      828/MThalLeg pain (1)

      Pulsatile groin mass (3)
      Right popliteal/tibial/peroneal a.

      Right popliteal a., femoral a., common iliac/internal/external iliac a., infrarenal aortic aneurysm at bifurcation
      NA

      Serology (ICT) day 2 before second surgery

      Vessel isolation of P. insidiosum from culture

      Pathology
      Below knee amputation (OSH)

      Aneurysmectomy with axillofemoral bypass, superficial femoral a. resection, high above knee amputation (day 2)

      Debridement abdomen with mesh graft
      No, noTerbinafine, itraconazole (18 days)Three: days 0, 8, 15Died (day 22)
      952/MβThalChronic leg ulcer (1)

      Chronic stump ulcer/abscess (10)

      Toe gangrene (day 3 PTA)
      Right femoral a.

      Right common iliac a, femoral a. tibial/peroneal a.

      Left common iliac a., external iliac a., femoral a., tibial/peroneal a., abdominal aorta
      NA

      Serology (Western blot, immunodiffusion) day 10 (after surgery) (ICT)
      Above knee amputation

      Aortofemoral and femoral dorsalis pedis bypass graft (left) day 2
      No, noTerbinafine, itraconazole (2231 days, 6 years)Three: days 0, 17, 70Survived (lost to F/U, day 85 after last dose of vaccine)
      1048/MAplastic anemiaChronic leg wound (10)Right external iliac a., common femoral a., femoral a., popliteal a., anterior tibial a.Serology (ICT) day 1

      Vessel/skin tissue isolation of P. insidiosum from culture

      Pathology
      High above knee amputation (day 2)

      Skin biopsy (day 3)

      Common femoral a., profunda a. excision, flank skin excision (day 6)
      No, noTerbinafine, itraconazole (26 days)

      SSKI (7 days)

      Caspofungin (13 days)
      Two: days 0, 14Died (day 28)
      1173/MMDSAcute limb ischemia (day 3 PTA)Left common iliac a., external iliac a., internal iliac a., superficial femoral a.

      Right common iliac a.
      Pathology

      Serology (ICT) day 4 (after surgery)
      Thrombectomy, balloon angioplasty with stenting (day 1)No, noAmphotericin B deoxycholate, itraconazole, terbinafine (3 days)NoneDied (day 6)
      1213/FAMLFever, headache

      Right hemiparesis, facial palsy (2)
      Left distal supraclinoid internal carotid a., middle cerebral a., anterior cerebral a., common/internal/external carotid a.Abscess isolation of Aspergillus spp and P. insidiosum

      Pathology

      Serology (HA: weakly positive) day 22 (after surgery)
      Craniectomy with removal of the abscess (day 1, 7, 22, 168)No, noVoriconazole (184 days)

      Terbinafine (42 days)
      Five: days 0, 6, 19, 145, 154Survived (lost to F/U, day 298; day 13 after last dose of vaccine)
      1357/MAnemia of chronic disease, alcoholism, HTSwelling of the neck (1)Left common/internal/external carotid a., cerebral a.Vessel isolation of P. insidiosum

      Serology (ICT) day 4 (after surgery)

      Pathology
      Resection of left external carotid artery aneurysm, ligation part of internal and external carotid artery (day 1)

      Surgical debridement (day 9)
      No, noItraconazole, terbinafine (77 days)Four: days 0, 13, 24, 41Died (day 81; day 36 after last dose of vaccine)
      a, artery; AIHA, autoimmune hemolytic anemia; AML, acute myeloid leukemia; βThal, β-thalassemia; βThal/HbE, β-thalassemia hemoglobin E disease; CSA2A Bart’s H, hemoglobin H disease with Hb constant spring; DRESS, drug rash with eosinophilia and systemic symptoms; F, female; F/U, follow up; HA, hemagglutination; HbCS, hemoglobin constant spring; HbH, hemoglobin H disease; HbH CS, hemoglobin H constant spring disease; HBV, hepatitis B virus infection; HCV, hepatitis C virus infection; HT, hypertension; ICT, immunochromatography test; M, male; MDS, myelodysplasia; NA, not available; OSH, outside hospital; PIA, Pythium insidiosum antigen; Rx, therapy; SSKI, saturated solution of potassium iodine; Thal, thalassemia.
      The clinical presentations of vascular pythiosis in this study included skin and soft tissue infections after a minor injury, which led to a chronic ulcer lasting for a period of several months. Those with rapid progression presented with painful gangrene of the toe/foot/digit which developed after vascular thrombosis in the distal limb. Nine patients had a typical vascular presentation of the lower extremities with chronic symptoms. Only one patient – a 73-year-old male with MDS (patient 11) – had abrupt onset of lower limb ischemia. There was upper extremity involvement in a 54-year-old male with hepatitis C virus (HCV) cirrhosis, who presented with a subcutaneous nodule and a condition mimicking vasculitis, which led to a thenar abscess and eventually required above elbow amputation reported case by
      • Khunkhet S.
      • Rattanakaemakorn P.
      • Rajatanavin N.
      Pythiosis presenting with digital gangrene and subcutaneous nodules mimicking medium vessel vasculitis.
      (18). Unusual presentations with carotid involvement occurred in two non-thalassemic patients, a 13-year-old female with acute myeloid leukemia (patient 12) who presented with a brain abscess and stroke, and a 57-year-old male with alcoholism (patient 13) who had a pulsatile neck mass due to concealed rupture of carotid artery aneurysm, and subsequently suffered a stroke. The patients with leukopenia deteriorated rapidly and died within 4 and 27 days of admission, respectively: patient 11 with MDS died due to the delay in diagnosis, sepsis, and acute kidney injury; patient 10 with aplastic anemia developed rapidly progressive subcutaneous disease within 24 h after a high above the knee amputation and later died due to sepsis.

      Diagnosis

      The diagnostic modalities used, along with the sequential order of the tests, are shown in Table 2. Serology was positive in all cases. The ICT used at the authors’ center, which has a turnaround time of approximately 30 min, was helpful for the management of pythiosis. Twelve patients had an ICT done, five before surgery which led to urgent surgery (within 4 days of the test result), and clinical cure was achieved in three. The ICT yielded weakly positive results in two immunosuppressed patients. The first was the patient with HCV cirrhosis (patient 1), who received intravenous dexamethasone and cyclophosphamide as treatment for a presumed giant cell arteritis prior to the diagnosis of vascular pythiosis of the radial and ulnar arteries; serology was performed on day 11 and fungal culture on day 21 of hospitalization. The second was the 13-year-old female with acute myeloid leukemia (patient 12), who had received chemotherapy 2 months before the onset of symptoms; she later developed a neutropenic fever with headache and subsequent brain abscesses/hemiparesis. She received corticosteroids to reduce the brain swelling before surgery. Serology was tested on day 22 of hospitalization, after surgical drainage of the abscess with evidence of residual disease. Interestingly, the etiology of the brain abscesses was Aspergillus spp and Pythium spp, both confirmed by fungal culture and IHC stain.
      Among all of the samples tested during the study period, none of the ICT showed false-positive results (data not shown). Serology was used as a follow-up biomarker in conjunction with clinical and radiographic assessment in three patients with residual disease.

      Pathology

      Pathology data are shown in Table 3.
      Table 3Pathology (N = 13).
      No.Age (years)/sexUnderlying diseaseOrganProcedurePathology
      154/MHCV cirrhosis

      HbCS trait reported by Khunchet et al. (18)
      Subcutaneous nodule

      Thenar abscess

      Thenar abscess

      Vessel: hand, forearm
      Skin biopsy

      Skin biopsy; debridement

      Debridement; biopsy

      Above elbow amputation
      Dense infiltration of mixed inflammatory cells (eosinophils, lymphocytes, plasma cells, histiocytes, and multiple giant cells)

      Suppurative granuloma

      Acute and chronic inflammation with panniculitis

      Granulation tissue

      Granulation tissue, multiple broad ribbon-like hyphae in tissue, unremarkable a., no evidence of vasculitis

      Large thrombi containing broad ribbon-like hyphae occluded within the lumen of radial and ulnar a., vascular walls severely destroyed, small thrombi containing hyphae within the lumen of brachial a., proximal margin of brachial a. free margin
      275/MHbHLeg (vessel)

      Leg (soft tissue)
      Above knee amputationSuppurative granulomatous inflammation of the tibial, peroneal, popliteal, superficial femoral, ilioperoneal a.

      GMS stain and IHC stain for P. insidiosum were positive

      Dry gangrene with acute and chronic inflammation
      355/MCSA2A Bart’s HLeg (vessel)

      Leg (soft tissue)
      Above knee amputationNecrotizing granulomatous inflammation of vascular wall

      GMS stain and IHC stain for P. insidiosum were positive

      Gangrenous necrosis involved dorsal skin and subcutaneous tissue of foot
      456/MβThal/HbELeg (vessel)Above knee amputationAcute necrotizing vasculitis with focal granulomatous formation involving posterior tibial a., dorsalis pedis a., and small a. of foot, gangrene of foot, abscess of leg (granulomatous response), GMS stain was positive
      548/MThalLeg (vessel)Above knee amputationNeutrophilic infiltration, fungus invading vascular wall, GMS and IHC stain for P. insidiosum were positive
      629/FHbH CS, AIHA, HBVLeg (vessel)Above knee amputationNecrotizing suppurative arteritis, short fungal fragment, GMS stain and periodic acid-Schiff stain were positive
      738/MThalLeg (vessel)Above knee amputationFungal vasculitis with thrombosis, presence of fungal organism at vascular margin, IHC stain for P. insidiosum was positive
      828/MThalLeg (vessel)

      Leg (soft tissue)

      Aorta
      Below knee amputation

      Aneurysmectomy with axillofemoral bypass, removal of superficial femoral a., high above knee amputation
      NA

      Acute infective arteritis (neutrophilic infiltration) of popliteal a., femoral a., iliac a., and aorta, with aneurysmal dilatation of aorta, presence of fungi with vascular wall invasion, GMS stain and IHC stain for P. insidiosum were positive
      952/MThalLeg (vessel)Above knee amputation

      Aortofemoral and femoral dorsalis pedis bypass graft
      NA

      NA
      1048/MAplastic anemiaLeg (vessel)

      Intramuscular abscesses

      Osteomyelitis of the lower extremities

      Abdominal wall
      High above knee amputation

      Skin biopsy

      Common femoral a., profunda a. excision, flank skin excision
      Necrotizing arteritis with intraluminal thrombus involving popliteal a., anterior tibial a., femoral a, and external iliac a., irregular branch hyphae in thrombus with vascular wall invasion, IHC stain for P. insidiosum was positive, proximal and distal margins of external iliac a. were free, multiple skin ulcers with granuloma in the soft tissue, irregular branched hyphae, viable soft tissue margin

      Microabscesses (diffuse infiltrates with eosinophils and neutrophils)

      Acute suppurative granulomatous inflammation, GMS stain was positive
      1173/MMDSLeg (vessel)Thrombectomy, balloon angioplasty stentingThrombus with fungal hyphae, IHC stain for P. insidiosum was positive
      1213/FAMLBrain

      Carotid artery

      Cerebral artery
      Mini craniectomy, abscess removal

      Craniectomy with abscess removal

      Craniectomy with abscess removal, dura repair
      Acute inflammatory cell infiltrates with necrotic tissue, branching septate hyphae, GMS stain and IHC stain for P. insidiosum were positive

      Acute and chronic inflammation with granulation tissue, foreign body multinucleated giant cell reaction, foamy histiocytic aggregation and necrotic tissue, branching septate hyphae, GMS stain and IHC stain for P. insidiosum were positive

      Suppurative granuloma with multiple brain abscesses
      1357/MAnemia of chronic disease, Alcoholism, HTCarotid artery aneurysmResection of external carotid artery aneurysm, ligation of part of the internal and external carotid arteryAcute suppurative inflammation, GMS stain and IHC stain for P. insidiosum were positive
      a, artery; AIHA, autoimmune hemolytic anemia; AML, acute myeloid leukemia; βThal, β-thalassemia; βThal/HbE, β-thalassemia hemoglobin E disease; CSA2A Bart’s H, hemoglobin H disease with Hb constant spring; F, female; GMS, Gomori methenamine silver; HbCS, hemoglobin constant spring; HbH, hemoglobin H disease; HbH CS, hemoglobin H constant spring disease; HBV, hepatitis B virus infection; HCV, hepatitis C virus infection; HT, hypertension; IHC, immunohistochemistry; M, male; NA, not available; Thal, thalassemia.
      Among the patients with the vascular form who had thalassemia and chronic symptoms, pathology showed suppurative or necrotizing inflammation and granuloma with evidence of fungal thrombus. For the non-thalassemic patients with abscesses (patients 12 and 13), pathology revealed acute suppurative inflammation with necrotic tissue. Histopathology of the brain abscess in patient 12 demonstrated a neutrophilic reaction, which shifted to a multinucleated giant cell reaction 7 days later; six months after this, there was a shift to suppurative granuloma with abscess. Of those with skin and subcutaneous tissue involvement, patient 1 had a preceding history of subcutaneous nodules and presented a condition mimicking vasculitis. Histopathology demonstrated mixed inflammatory cells, suppurative granuloma, and panniculitis (
      • Khunkhet S.
      • Rattanakaemakorn P.
      • Rajatanavin N.
      Pythiosis presenting with digital gangrene and subcutaneous nodules mimicking medium vessel vasculitis.
      ).
      The patient with aplastic anemia (patient 10) presented an acute onset, and histopathology revealed eosinophilic and neutrophilic infiltrates.

      Management

      Radical surgery aiming to achieve organism-free margins was successful only in five patients due to the delay in diagnosis, most patients had a history of chronic ulcer/skin lesions with a median duration of 4 months before the hospital visit. Inadequate assessment of the surgical free margins of a limb at the time of amputation led to aortic involvement in two patients (patients 8 and 9).
      Regarding antifungal therapy (Table 1), 11 patients (84.6%) received a combination of itraconazole and terbinafine. Adjunctive amphotericin B deoxycholate was used as an empirical regimen in a patient with MDS (patient 11) with a history of scrotal phaeohyphomycosis. Caspofungin was briefly added to the regimen in a patient with aplastic anemia (13 days). Prolonged combination therapy of itraconazole and terbinafine (for 1 year and 4.5 years, respectively) stopped the disease progression in two patients with unresectable disease (patients 6 and 9). In vitro susceptibility data were not available.

      Outcomes

      The morbidity rate of vascular pythiosis was high: the amputation rate was 77% and the mortality rate was 31%. However, the loss to follow-up rate was high due to referral to a local hospital and loss of contact. The duration of follow-up is shown in Table 1.
      Clinical cure was achieved in three patients; there was no evidence of relapse at 45, 365, and 704 days after antifungal discontinuation. Only two patients with thalassemia and residual vascular disease survived. The first patient (patient 6) had superficial femoral artery thrombosis at a level 7 cm above the surgical stump. Serum hemagglutination became negative after 8 months of antifungal treatment without the PIA vaccine (time between the positive and negative test was 129 days). This patient received 1 year of itraconazole and terbinafine in combination. The patient did well up to 145 days after the cessation of medication; she was then referred to the local hospital. The second patient (patient 9) had residual disease (suprainguinal lesions, aorta) treated with the PIA vaccine and a combination of itraconazole and terbinafine. The ICT became negative after 4.5 years of treatment involving PIA vaccine (three doses within the first 3 months) and prolonged antifungal treatment (time between the positive and negative test was 227 days). He had stable disease. He survived for 7 years after initial symptoms before developing renal failure caused by aneurysm compression of the ureter and was then lost to follow-up.

      Radiography

      Ten patients had post-surgical radiographic imaging performed; data are shown in Table 4. Intramuscular abscess (stump site), myositis, and ascending abnormalities of the vessel walls were seen in the patients with residual disease. Reactive lymphadenitis resolved after therapy. The radiographic assessment of vascular pythiosis after surgery was difficult to interpret, especially among elderly patients with atherosclerosis as a comorbidity; for example, patient 2 (75-year-old male) had femoral artery thrombosis without evidence of vascular wall enhancement/aneurysm formation postoperatively at the site of the stump and follow-up imaging showed spontaneous resolution.
      Table 4Radiographic findings after surgery (N = 13).
      No.Age (years)/sexUnderlying diseaseImaging findings after surgery (duration after surgery)
      154/MHCV cirrhosis

      HbCS trait
      NA
      275/MHbHDay 14, CTA aorta: right AKA with newly seen occlusion of mid to distal SFA, 1.8 cm intramuscular abscess, several enlarged bilateral inguinal nodes 0.6–1.4 cm

      Day 28, CTA aorta: right AKA with a few focal calcifications, mild stenosis, no pseudoaneurysm at SFA, enlarged groin, iliac, paraaortic lymph nodes; aorta: no aneurysm
      355/MCSA2A Bart’s HWeek 7, CTA aorta: occlusion of SFA (no aneurysm, no wall enhancement), a few enlarged groin and iliac lymph nodes

      Month 9, CTA aorta: occlusion of SFA (no aneurysm, no wall enhancement), a few enlarged groin and iliac lymph nodes, mild atherosclerotic change of aorta
      456/MβThal/HbENA
      548/MThalNA
      629/FHbH CS, AIHA, HBVMonth 3, CTA aorta: soft tissue thickening of proximal SFA thrombosis and occlusion of proximal SFA at level 7 cm above the stump
      738/MThalMonth 5, postoperative, CTA aorta: AKA with irregular wall, patent SFA, no evidence of arterial occlusion or stenosis
      828/MThal1st surgery; month 3, CTA aorta: infrarenal aortic aneurysm, right common iliac/internal iliac aneurysm, thrombosis of external iliac a., femoral a. and branches

      2nd surgery; day 12 CTA aorta: patent graft, no leakage, newly seen rim enhancing lesion at pubic bone consistent with abscess, small liver abscess
      952/MThal1st surgery; month 7, angiogram: severe stenosis of common iliac a., occlusion of tibioperoneal trunk

      2nd surgery; month 3, CTA aorta: complete occlusion of abdominal aorta, both common/external iliac a., left internal iliac a., right femoral a., popliteal a., patent axillofemoral bypass graft with occlusion of popliteal graft, intramuscular abscess at stump site

      2nd surgery; month 7, CTA aorta: resolution of circumferential wall thickening with enhancement of vessels, resolution of intramuscular abscess at the stump, right common iliac aneurysm compressing right kidney causing severe hydroureter and hydronephrosis, stable findings of complete occlusion of abdominal aorta, both common/external iliac a., left internal iliac a., right femoral a., popliteal a., patent axillofemoral bypass graft with occlusion of popliteal graft

      2nd surgery; year 2, CTA aorta: stable findings, except increased hydroureter and hydronephrosis

      2nd surgery; year 4, ultrasound abdomen: marked right hydroureter and hydronephrosis; aorta: stable findings
      1048/MAplastic anemiaDay 19, CTA aorta: total arterial occlusion from right iliac bifurcation down to FA with diffuse thickened, enhanced wall and surrounding fat stranding, extensive acute deep vein thrombosis of right leg, myositis of right thigh, two intramuscular abscesses involving right iliacus and quadriceps muscle, subcutaneous swelling along right side abdominal wall extending to proximal thigh and stump
      1173/MMDSNA
      1213/FAML1st surgery; day 7, CT brain: residual brain abscess size 2.8 × 4.2 cm, leptomeningeal enhancement

      2nd surgery; month 5, CT brain: brain abscess size 1.2 cm

      3rd surgery; month 1: multiple small brain abscesses, leptomeningeal enhancement
      1357/MAnemia of chronic disease, alcoholism, HT1st surgery; MRI and MRA of the brain revealed pseudoaneurysm at left carotid–parapharyngeal spaces (2.8 × 2.0 × 3.1 cm) associated with extensive inflammation of the surrounding soft tissue resulting in mild narrowing of the upper airway. Left common carotid artery was occluded along the origin to the cavernous part of the left internal carotid artery with evidence of wall enhancement. Multifocal cerebritis consistent with cerebral septic emboli and leptomeningeal enhancement at the left cerebral hemisphere
      a., artery; AKA, above knee amputation; AIHA, autoimmune hemolytic anemia; AML, acute myeloid leukemia; βThal, β-thalassemia; βThal/HbE, β-thalassemia hemoglobin E disease; CSA2A Bart’s H, hemoglobin H disease with Hb constant spring; CT, computed tomography; CTA, computed tomography angiography; F, female; FA, femoral artery; HbCS, hemoglobin constant spring; HbH, hemoglobin H disease; HbH CS, hemoglobin H constant spring disease; HBV, hepatitis B virus infection; HCV, hepatitis C virus infection; HT, hypertension; M, male; MDS, myelodysplasia; MRA, magnetic resonance angiography; MRI, magnetic resonance imaging; NA, not available; SFA, superficial femoral artery; Thal, thalassemia.

      Discussion

      Human pythiosis remains a life-threatening disease with challenges in diagnosis and treatment (
      • Krajaejun T.
      • Sathapatayavongs B.
      • Pracharktam R.
      • Nitiyanant P.
      • Leelachaikul P.
      • Wanachiwanawin W.
      • et al.
      Clinical and epidemiological analyses of human pythiosis in Thailand.
      ). Most patients experienced a delay in diagnosis for multiple reasons: first, a lack of awareness of the disease by the physician; second, tissue diagnosis was not obtained from the ulcer/skin lesions/amputated limb; third, specimens were not sent for fungal culture from rural areas. The results of histopathology and fungal culture require time, which could also be the reason for the delay in diagnosis. Serological diagnostic methods such as the rapid ICT have become useful tests for the presurgical diagnosis of vascular pythiosis, leading to more prompt treatment.
      Immunosuppression affected anti-P. insidiosum antibody production, as seen in two patients with weakly positive results. Serum hemagglutination has been proposed as a helpful tool for monitoring the response to treatment (
      • Jindayok T.
      • Piromsontikorn S.
      • Srimuang S.
      • Khupulsup K.
      • Krajaejun T.
      Hemagglutination test for rapid serodiagnosis of human pythiosis.
      ). This study identified malnourishment and immunosuppression as additional risk factors to hemoglobinopathy (
      • Krajaejun T.
      • Sathapatayavongs B.
      • Pracharktam R.
      • Nitiyanant P.
      • Leelachaikul P.
      • Wanachiwanawin W.
      • et al.
      Clinical and epidemiological analyses of human pythiosis in Thailand.
      ). Iron overload among patients with MDS and thalassemia predisposed the patient to infection (
      • Krajaejun T.
      • Sathapatayavongs B.
      • Pracharktam R.
      • Nitiyanant P.
      • Leelachaikul P.
      • Wanachiwanawin W.
      • et al.
      Clinical and epidemiological analyses of human pythiosis in Thailand.
      ). P. insidiosum carries a gene encoding a ferrochelatase, which plays a role in its virulence (
      • Krajaejun T.
      • Khositnithikul R.
      • Lerksuthirat T.
      • Lowhnoo T.
      • Rujirawat T.
      • Petchthong T.
      • et al.
      Expressed sequence tags reveal genetic diversity and putative virulence factors of the pathogenic oomycete Pythium insidiosum.
      ). An experimental model of rabbits infected with P. insidiosum revealed an increase in total iron binding capacity levels and a decrease in transferrin saturation levels during infection (
      • Zanette R.A.
      • Bitencourt P.E.R.
      • Alves S.H.
      • Fighera R.A.
      • Flores M.M.
      • Wolkmer P.
      • et al.
      Insights into the pathophysiology of iron metabolism in Pythium insidiosum infections.
      ). The ferritin level, a marker of iron status used in this study, has certain limitations: ferritin itself is an acute-phase reactant molecule which increases during inflammation (
      • Zanette R.A.
      • Bitencourt P.E.R.
      • Alves S.H.
      • Fighera R.A.
      • Flores M.M.
      • Wolkmer P.
      • et al.
      Insights into the pathophysiology of iron metabolism in Pythium insidiosum infections.
      ). Baseline ferritin levels prior to illness were not available. Furthermore, serum iron, transferrin levels, and total iron binding capacity were not available in this study.
      The natural history and histopathology of the disease differ among thalassemia and non-thalassemia patients; an example is the unusual sites of infection: carotid involvement, brain abscess, and orbital cellulitis (
      • Thitithanyanont A.
      • Mendoza L.
      • Chuansumrit A.
      • Pracharktam R.
      • Laothamatas J.
      • Sathapatayavongs B.
      • et al.
      Use of an immunotherapeutic vaccine to treat a life-threatening human arteritic infection caused by Pythium insidiosum.
      ,
      • Krajaejun T.
      • Sathapatayavongs B.
      • Pracharktam R.
      • Nitiyanant P.
      • Leelachaikul P.
      • Wanachiwanawin W.
      • et al.
      Clinical and epidemiological analyses of human pythiosis in Thailand.
      ,
      • Narkwiboonwong T.
      • T.K
      • Watanakijthavonkul K.
      • Paocharern P.
      • Singsakul A.
      • Wongsa A.
      • Woracharoensri N.
      • et al.
      Cerebral Pythiosis: a case report of Pythium insidiosum infection presented with brain abscess.
      ,
      • Kirzhner M.
      • Arnold S.R.
      • Lyle C.
      • Mendoza L.L.
      • Fleming J.C.
      Pythium insidiosum: a rare necrotizing orbital and facial infection.
      ). Dissemination has been shown to occur among patients with leukemia; e.g., gastrointestinal involvement (
      • Krajaejun T.
      • Kunakorn M.
      • Pracharktam R.
      • Chongtrakool P.
      • Sathapatayavongs B.
      • Chaiprasert A.
      • et al.
      Identification of a novel 74-kiloDalton immunodominant antigen of Pythium insidiosum recognized by sera from human patients with pythiosis.
      ,
      • Krajaejun T.
      • Sathapatayavongs B.
      • Pracharktam R.
      • Nitiyanant P.
      • Leelachaikul P.
      • Wanachiwanawin W.
      • et al.
      Clinical and epidemiological analyses of human pythiosis in Thailand.
      ). Two immunosuppressed patients in this study died and one was lost to follow-up. However, data are scarce among the immunosuppressed population; the largest case series of 102 cases from Thailand included only two patients with acute leukemia (
      • Krajaejun T.
      • Sathapatayavongs B.
      • Pracharktam R.
      • Nitiyanant P.
      • Leelachaikul P.
      • Wanachiwanawin W.
      • et al.
      Clinical and epidemiological analyses of human pythiosis in Thailand.
      ).
      A literature review revealed that the natural history of immunosuppressed patients differs from that of thalassemia patients in many aspects (
      • Hilton R.E.
      • Tepedino K.
      • Glenn C.J.
      • Merkel K.L.
      Swamp cancer: a case of human pythiosis and review of the literature.
      ,
      • Hoffman M.A.
      • Cornish N.E.
      • Simonsen K.A.
      A painful thigh lesion in an immunocompromised 11-year-old boy.
      ,
      • Pan J.H.
      • Kerkar S.P.
      • Siegenthaler M.P.
      • Hughes M.
      • Pandalai P.K.
      A complicated case of vascular Pythium insidiosum infection treated with limb-sparing surgery.
      ,
      • Wanachiwanawin W.
      • Mendoza L.
      • Visuthisakchai S.
      • Mutsikapan P.
      • Sathapatayavongs B.
      • Chaiprasert
      • et al.
      Efficacy of immunotherapy using antigens of Pythium insidiosum in the treatment of vascular pythiosis in humans.
      ): first, the abrupt onset after exposure to an aquatic habitat (although some did not have a history of exposure); in such cases, the patient often presented with necrotic skin lesions/cellulitis, which progressed to vascular infection shortly afterwards (within days or weeks). Second, the clinical course of rapid deterioration occurred within a few days, similar to the present study patients with MDS and aplastic anemia. Third, the delay in diagnosis: none of the cases in the literature had a presurgical diagnosis. Histopathology and fungal culture obtained from the surgery often revealed the diagnosis only shortly before the patient died or postmortem. Fourth, the response to PIA vaccine was reduced, as demonstrated by minimal local inflammatory reactions and cytokine assay, which correlated with no clinical response in two patients with aplastic anemia (
      • Wanachiwanawin W.
      • Mendoza L.
      • Visuthisakchai S.
      • Mutsikapan P.
      • Sathapatayavongs B.
      • Chaiprasert
      • et al.
      Efficacy of immunotherapy using antigens of Pythium insidiosum in the treatment of vascular pythiosis in humans.
      ). Fifth, the mortality rate was exceedingly high at >70% vs. 30% among thalassemia patients, as dissemination occurred rapidly and there was a delay in diagnosis. Only a cancer patient with pythiosis survived: an 11-year-old boy with relapsed Ewing sarcoma and pulmonary metastases developed neutropenic fever with a necrotic painful thigh lesion that progressed within 24 h after onset. He underwent surgical debridement and was treated with oral voriconazole for 9 months (
      • Hoffman M.A.
      • Cornish N.E.
      • Simonsen K.A.
      A painful thigh lesion in an immunocompromised 11-year-old boy.
      ). The neutropenic patient showed a rapid deterioration within days after the onset of symptoms (
      • Hilton R.E.
      • Tepedino K.
      • Glenn C.J.
      • Merkel K.L.
      Swamp cancer: a case of human pythiosis and review of the literature.
      ,
      • Hoffman M.A.
      • Cornish N.E.
      • Simonsen K.A.
      A painful thigh lesion in an immunocompromised 11-year-old boy.
      ). This observation reflected the importance of neutrophils in containing the disease, as is known for other fungal infections such as aspergillosis and mucormycosis. Prompt diagnosis is crucial for the immunosuppressed patient with skin/subcutaneous disease or vascular presentation with a history of exposure to an aquatic habitat, as pythiosis is fatal among this population. A rapid ICT or HA should be used for early detection; however, the sensitivity of these tests should be evaluated further in this population.
      The host’s immune response reflected by histopathology demonstrated that acute inflammatory cells consisted of neutrophils, lymphocytes, plasma cells. The subcutaneous tissue revealed microabscesses, necrosis, and a mixture of acute and chronic inflammatory cell infiltrates, similar to previous reports (
      • Shenep J.L.
      • English B.K.
      • Kaufman L.
      • Pearson T.A.
      • Thompson J.W.
      • Kaufman R.A.
      • et al.
      Successful medical therapy for deeply invasive facial infection due to Pythium insidiosum in a child.
      ). The inflammatory response among thalassemia patients revealed a granulomatous reaction or chronic inflammation, as has been reported previously (
      • Imwidthaya P.
      Human pythiosis in Thailand.
      ,
      • Krajaejun T.
      • Sathapatayavongs B.
      • Pracharktam R.
      • Nitiyanant P.
      • Leelachaikul P.
      • Wanachiwanawin W.
      • et al.
      Clinical and epidemiological analyses of human pythiosis in Thailand.
      ,
      • Prasertwitayakij N.
      • Louthrenoo W.
      • Kasitanon N.
      • Thamprasert K.
      • Vanittanakom N.
      Human pythiosis, a rare cause of arteritis: case report and literature review.
      ).
      A host Th2 helper reaction with eosinophil response including Splendore–Hoeppli phenomenon, which has been found to be predominant in animal and some human cases, was not found in this study (
      • Krajaejun T.
      • Sathapatayavongs B.
      • Pracharktam R.
      • Nitiyanant P.
      • Leelachaikul P.
      • Wanachiwanawin W.
      • et al.
      Clinical and epidemiological analyses of human pythiosis in Thailand.
      ,
      • Thianprasit M.
      • C. A
      • Imwidthaya P.
      Human pythiosis.
      ). The exception was the one patient with HCV cirrhosis, who had eosinophilic infiltration and panniculitis associated with the formation of granulation tissue and presented a condition mimicking vasculitis reported by
      • Khunkhet S.
      • Rattanakaemakorn P.
      • Rajatanavin N.
      Pythiosis presenting with digital gangrene and subcutaneous nodules mimicking medium vessel vasculitis.
      . The inflammatory response among neutropenic patients has revealed a dense perivascular inflammatory infiltrate with vessel wall damage and necrosis, similar to the present study findings (
      • Hilton R.E.
      • Tepedino K.
      • Glenn C.J.
      • Merkel K.L.
      Swamp cancer: a case of human pythiosis and review of the literature.
      ). IHC stain of P. insidiosum was useful for differentiating fungal pathogens on tissue pathology, with a specificity of 100% (
      • Inkomlue R.
      • Larbcharoensub N.
      • Karnsombut P.
      • Lerksuthirat T.
      • Aroonroch R.
      • Lohnoo T.
      • et al.
      Development of an anti-elicitin antibody-based immunohistochemical assay for diagnosis of pythiosis.
      ), particularly among patients with multiple infections or in the absence of fungal culture.
      Three of the study patients had a stump abscess along with evidence of an ascending vascular infection (thrombosis, aneurysm). Soft tissue abscess, myositis, and lymphadenopathy should be assessed carefully with computed tomography or magnetic resonance imaging before surgery to evaluate the soft tissue margins. Inadequate intraoperative assessment of vascular and soft tissue margins will lead to progression of the disease (
      • Sermsathanasawadi N.
      • Praditsuktavorn B.
      • Hongku K.
      • Wongwanit C.
      • Chinsakchai K.
      • Ruangsetakit
      • et al.
      Outcomes and factors influencing prognosis in patients with vascular pythiosis.
      ).
      Mortality in this study was 31%, similar to the rate reported previously (
      • Krajaejun T.
      • Sathapatayavongs B.
      • Pracharktam R.
      • Nitiyanant P.
      • Leelachaikul P.
      • Wanachiwanawin W.
      • et al.
      Clinical and epidemiological analyses of human pythiosis in Thailand.
      ,
      • Permpalung N.
      • Worasilchai N.
      • Plongla R.
      • Upala S.
      • Sanguankeo A.
      • Paitoonpong L.
      • et al.
      Treatment outcomes of surgery, antifungal therapy and immunotherapy in ocular and vascular human pythiosis: a retrospective study of 18 patients.
      ,
      • Sermsathanasawadi N.
      • Praditsuktavorn B.
      • Hongku K.
      • Wongwanit C.
      • Chinsakchai K.
      • Ruangsetakit
      • et al.
      Outcomes and factors influencing prognosis in patients with vascular pythiosis.
      ). However, due to the large proportion of patients who were lost to follow-up, statistical analyses were not conducted. The main predictor of survival was the microscopic demonstration of an organism-free surgical margin (vascular and soft tissue), which was not feasible in the study patient with carotid and intracranial vessel involvement (
      • Sermsathanasawadi N.
      • Praditsuktavorn B.
      • Hongku K.
      • Wongwanit C.
      • Chinsakchai K.
      • Ruangsetakit
      • et al.
      Outcomes and factors influencing prognosis in patients with vascular pythiosis.
      ).

      Antifungal therapy

      There were few successful outcomes with antifungal treatment alone, particularly in those with unresectable disease (
      • Krajaejun T.
      • Sathapatayavongs B.
      • Pracharktam R.
      • Nitiyanant P.
      • Leelachaikul P.
      • Wanachiwanawin W.
      • et al.
      Clinical and epidemiological analyses of human pythiosis in Thailand.
      ). Most antifungals are not active against P. insidiosum due to its lack of ergosterol (
      • Lerksuthirat T.
      • Sangcakul A.
      • Lohnoo T.
      • Yingyong W.
      • Rujirawat T.
      • Krajaejun T.
      Evolution of the sterol biosynthetic pathway of Pythium insidiosum and related oomycetes contributes to antifungal drug resistance.
      ). Antifungals were given except in one patient who developed a severe drug hypersensitivity reaction to terbinafine after 27 days of therapy. The combination of itraconazole and terbinafine was commonly used, owing to their low minimum inhibitory concentrations of 0.125–4 μg/ml and 0.03–4 μg/ml, respectively, against clinical isolates of P. insidiosum (
      • Kirzhner M.
      • Arnold S.R.
      • Lyle C.
      • Mendoza L.L.
      • Fleming J.C.
      Pythium insidiosum: a rare necrotizing orbital and facial infection.
      ,
      • Permpalung N.
      • Worasilchai N.
      • Plongla R.
      • Upala S.
      • Sanguankeo A.
      • Paitoonpong L.
      • et al.
      Treatment outcomes of surgery, antifungal therapy and immunotherapy in ocular and vascular human pythiosis: a retrospective study of 18 patients.
      ,
      • Shenep J.L.
      • English B.K.
      • Kaufman L.
      • Pearson T.A.
      • Thompson J.W.
      • Kaufman R.A.
      • et al.
      Successful medical therapy for deeply invasive facial infection due to Pythium insidiosum in a child.
      ) with evidence of in vitro synergistic activity (
      • Shenep J.L.
      • English B.K.
      • Kaufman L.
      • Pearson T.A.
      • Thompson J.W.
      • Kaufman R.A.
      • et al.
      Successful medical therapy for deeply invasive facial infection due to Pythium insidiosum in a child.
      ). However, recent data from Thai clinical isolates showed no evidence of synergy (
      • Permpalung N.
      • Worasilchai N.
      • Plongla R.
      • Upala S.
      • Sanguankeo A.
      • Paitoonpong L.
      • et al.
      Treatment outcomes of surgery, antifungal therapy and immunotherapy in ocular and vascular human pythiosis: a retrospective study of 18 patients.
      ). Moreover, in vitro data showed that P. insidiosum isolates were sensitive to the antifungal agents only at high concentrations (
      • Lerksuthirat T.
      • Sangcakul A.
      • Lohnoo T.
      • Yingyong W.
      • Rujirawat T.
      • Krajaejun T.
      Evolution of the sterol biosynthetic pathway of Pythium insidiosum and related oomycetes contributes to antifungal drug resistance.
      ).
      The dose of terbinafine used in the study patients varied widely (data not shown), with higher doses used for patients with residual unresectable disease. Terbinafine 500 mg twice daily is the highest dose reported in the literature for use in the combination treatment of refractory fungal infection (
      • Dolton M.J.
      • Perera V.
      • Pont L.G.
      • McLachlan A.J.
      Terbinafine in combination with other antifungal agents for treatment of resistant or refractory mycoses: investigating optimal dosing regimens using a physiologically based pharmacokinetic model.
      ). Terbinafine is more easily tolerated than itraconazole, with fewer side effects and drug interactions (
      • Dolton M.J.
      • Perera V.
      • Pont L.G.
      • McLachlan A.J.
      Terbinafine in combination with other antifungal agents for treatment of resistant or refractory mycoses: investigating optimal dosing regimens using a physiologically based pharmacokinetic model.
      ). Future studies should evaluate the use of high-dose terbinafine for the treatment of pythiosis.
      The new antifungal caspofungin, which inhibits β-d-glucan found in the cell wall of Pythium spp has demonstrated in vitro synergistic activity in combination with terbinafine (
      • Cavalheiro A.S.
      • Maboni G.
      • de Azevedo M.I.
      • Argenta J.S.
      • Pereira D.I.
      • Spader T.B.
      • et al.
      In Vitro activity of terbinafine combined with caspofungin and azoles against Pythium insidiosum.
      ). However, no clinical studies have reported the successful use of caspofungin. Saturated solution of potassium iodine has successfully cured cutaneous and subcutaneous disease and has been used in a few patients (
      • Imwidthaya P.
      Human pythiosis in Thailand.
      ,
      • Sathapatayavongs B.
      • Leelachaikul P.
      • Prachaktam R.
      • Atichartakarn V.
      • Sriphojanart S.
      • Trairatvorakul
      • et al.
      Human pythiosis associated with thalassemia hemoglobinopathy syndrome.
      ).
      In the present study, in vitro susceptibility testing was not performed due to the lack of a standardized assay including several different methods of antifungal susceptibility testing (
      • Lerksuthirat T.
      • Sangcakul A.
      • Lohnoo T.
      • Yingyong W.
      • Rujirawat T.
      • Krajaejun T.
      Evolution of the sterol biosynthetic pathway of Pythium insidiosum and related oomycetes contributes to antifungal drug resistance.
      ,
      • Permpalung N.
      • Worasilchai N.
      • Plongla R.
      • Upala S.
      • Sanguankeo A.
      • Paitoonpong L.
      • et al.
      Treatment outcomes of surgery, antifungal therapy and immunotherapy in ocular and vascular human pythiosis: a retrospective study of 18 patients.
      ). The duration of antifungal use varied from 1 to 2 years after surgery due to the lack of supporting data (
      • Sermsathanasawadi N.
      • Praditsuktavorn B.
      • Hongku K.
      • Wongwanit C.
      • Chinsakchai K.
      • Ruangsetakit
      • et al.
      Outcomes and factors influencing prognosis in patients with vascular pythiosis.
      ). Although antifungals have been associated with clinical resistance in the treatment of human pythiosis (
      • Lerksuthirat T.
      • Sangcakul A.
      • Lohnoo T.
      • Yingyong W.
      • Rujirawat T.
      • Krajaejun T.
      Evolution of the sterol biosynthetic pathway of Pythium insidiosum and related oomycetes contributes to antifungal drug resistance.
      ), prolonged therapy (>1 year) with itraconazole and terbinafine successfully cured unresectable disease in one patient (
      • Shenep J.L.
      • English B.K.
      • Kaufman L.
      • Pearson T.A.
      • Thompson J.W.
      • Kaufman R.A.
      • et al.
      Successful medical therapy for deeply invasive facial infection due to Pythium insidiosum in a child.
      ). As unresectable residual disease is associated with a high mortality rate, use of the PIA vaccine and prolonged therapy with itraconazole and terbinafine may be a suitable option for the patient to prolong survival. In this study, two patients had a successful response to antifungals despite the lack microscopic demonstration of organism-free surgical margins, and they achieved stable disease.
      The two types of PIA vaccine used in this study have demonstrated a similar safety profile (data not shown). The goal of the PIA vaccine is to stimulate a shift in Th2 response to a Th1 reaction to trigger the release of T-cytotoxic lymphocytes, natural killer cells, and activated macrophages at the infection site (
      • Mendoza L.
      • Newton J.C.
      Immunology and immunotherapy of the infections caused by Pythium insidiosum.
      ,
      • Wanachiwanawin W.
      • Mendoza L.
      • Visuthisakchai S.
      • Mutsikapan P.
      • Sathapatayavongs B.
      • Chaiprasert
      • et al.
      Efficacy of immunotherapy using antigens of Pythium insidiosum in the treatment of vascular pythiosis in humans.
      ). No predictor of a response to the vaccine, such as a local inflammatory reaction at the injection site, was seen in any of the study patients; however different preparations of the PIA vaccine were used (
      • Wanachiwanawin W.
      • Mendoza L.
      • Visuthisakchai S.
      • Mutsikapan P.
      • Sathapatayavongs B.
      • Chaiprasert
      • et al.
      Efficacy of immunotherapy using antigens of Pythium insidiosum in the treatment of vascular pythiosis in humans.
      ). The PIA vaccine successfully cured the first human case treated with immunotherapy with inoperable vascular pythiosis (
      • Thitithanyanont A.
      • Mendoza L.
      • Chuansumrit A.
      • Pracharktam R.
      • Laothamatas J.
      • Sathapatayavongs B.
      • et al.
      Use of an immunotherapeutic vaccine to treat a life-threatening human arteritic infection caused by Pythium insidiosum.
      ). The efficacy of the Pythium vaccine remains inconclusive (
      • Permpalung N.
      • Worasilchai N.
      • Plongla R.
      • Upala S.
      • Sanguankeo A.
      • Paitoonpong L.
      • et al.
      Treatment outcomes of surgery, antifungal therapy and immunotherapy in ocular and vascular human pythiosis: a retrospective study of 18 patients.
      ,
      • Wanachiwanawin W.
      • Mendoza L.
      • Visuthisakchai S.
      • Mutsikapan P.
      • Sathapatayavongs B.
      • Chaiprasert
      • et al.
      Efficacy of immunotherapy using antigens of Pythium insidiosum in the treatment of vascular pythiosis in humans.
      ). Further assessment is required through multicenter study collaboration in order to have a sufficiently large sample size. There have been variations in vaccine schedule depending on the institutional protocol and physician decision.
      Radiographic clues for vascular pythiosis include thickening of the vessel wall with enhancement, thrombosis, and aneurysmal dilation, which should raise the suspicion of arteritis (
      • Krajaejun T.
      • Sathapatayavongs B.
      • Pracharktam R.
      • Nitiyanant P.
      • Leelachaikul P.
      • Wanachiwanawin W.
      • et al.
      Clinical and epidemiological analyses of human pythiosis in Thailand.
      ,
      • Sermsathanasawadi N.
      • Praditsuktavorn B.
      • Hongku K.
      • Wongwanit C.
      • Chinsakchai K.
      • Ruangsetakit
      • et al.
      Outcomes and factors influencing prognosis in patients with vascular pythiosis.
      ).
      A prospective study with a larger sample size is needed to assess the usefulness of rapid serodiagnosis for the patient with subcutaneous disease or limb ischemia, and for treatment monitoring. There remains the need for a new antimicrobial agent with potent fungicidal activity.
      In conclusion, pythiosis is a life-threatening disease with rapid progression among immunosuppressed neutropenic patients. Early diagnosis and surgery to achieve organism-free margins, along with adjunctive antifungal therapy and use of an immunotherapeutic vaccine remain the primary treatment for the disease, which is associated with high morbidity and mortality. This study highlighted new aspects of pythiosis, including the unusual clinical presentation, risk factors (e.g., malnourished state, immunosuppression), serology as a marker for rapid diagnosis and follow-up, histopathology, and outcomes.

      Author contributions

      MNC and TK contributed to the study design. MNC, NL, and TK analyzed the data and took responsibility for the accuracy of the data analysis. All authors participated in the writing of the manuscript and approved the final version of the manuscript.

      Funding

      This study was partially supported by the Faculty of Medicine, Ramathibodi Hospital, Mahidol University (CF60001), and Thailand Research Fund (grant number, G5980009).

      Acknowledgements

      We thank Tassanee Lohnoo, Wanta Yingyong, and Sureewan Kitiwanwanich for the serological diagnosis.

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