Clinicopathological features and outcomes of pythiosis

International Journal of Infectious Diseases 71 (2018) 33–41

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Clinicopathological features and outcomes of pythiosis Maria Nina Chitasombata,* , Noppadol Larbcharoensubb , Ariya Chindampornc , Theerapong Krajaejunb a Division of Infectious Disease, Department of Medicine, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, 270 Rama VI Road, Ratchathewi, Bangkok 10400, Thailand b Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Ratchathewi, Bangkok, Thailand c Mycology Unit, Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand

A R T I C L E I N F O

A B S T R A C T

Article history: Received 14 February 2018 Received in revised form 29 March 2018 Accepted 30 March 2018 Corresponding Editor: Eskild Petersen, Aarhus, Denmark

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 nonthalassemic 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. © 2018 The Author(s). Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/).

Keywords: Pythiosis Pythium insidiosum Arteritis

Introduction Pythiosis is an emerging life-threatening disease that is endemic in Thailand and occurs in tropical and subtropical areas of the world (Krajaejun et al., 2006b). Pythiosis is caused by Pythium insidiosum, an oomycete of the kingdom Straminipila, phylum Oomycota, and was first known as an aquatic fungus (Krajaejun et al., 2006b). The clinical presentation of pythiosis includes skin, subcutaneous tissue, cornea, vascular, and disseminated forms (Krajaejun et al., 2006b; Sathapatayavongs et al., 1989). Known risk factors for vascular pythiosis include thalassemia, hemoglobinopathy, paroxysmal nocturnal hemoglobinuria, aplastic anemia, and leukemia (Krajaejun et al., 2006b). However,

* Corresponding author. E-mail addresses: [email protected], [email protected] (M.N. Chitasombat), [email protected] (N. Larbcharoensub), [email protected] (A. Chindamporn), [email protected] (T. Krajaejun).

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 et al., 2015; Narkwiboonwong et al., 2011; Shenep et al., 1998). Furthermore, the rarity of the disease has led to underrecognition, under-diagnosis, and delays in diagnosis, and this has contributed to the occurrence of advanced disease, which affects survival (Permpalung et al., 2015; Reanpang et al., 2015; Sermsathanasawadi et al., 2016). Historically, the histopathological diagnosis has often been revealed after the surgical procedure (Krajaejun et al., 2006b). The gold standard for diagnosis requires fungal culture, which is a time-consuming method (Chaiprasert et al., 1990; Intaramat et al., 2016; Krajaejun et al., 2009, 2006a). Recent advances made over the past decade include serodiagnosis, e.g., immunodiffusion (Pracharktam et al., 1991), ELISA (Krajaejun et al., 2002), Western blot analysis, the hemagglutination test (Jindayok et al. 2009), and the immunochromatographic test (Chareonsirisuthigul et al., 2013). The early recognition of pythiosis and confirmation by

https://doi.org/10.1016/j.ijid.2018.03.021 1201-9712/© 2018 The Author(s). Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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M.N. Chitasombat et al. / International Journal of Infectious Diseases 71 (2018) 33–41

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 et al., 2016). Patients with unresectable disease, e.g., those with suprainguinal lesions or aortic involvement, often die (Krajaejun et al., 2006b; Permpalung et al., 2015; Sermsathanasawadi et al., 2016). Antifungal therapy with itraconazole and terbinafine alone has rarely been successful (Krajaejun et al., 2006b; Shenep et al., 1998). Immunotherapeutic vaccination with P. insidiosum antigen (PIA) has been used as an adjunctive treatment. However, the efficacy of this therapy remains inconclusive (Permpalung et al., 2015; Thitithanyanont et al., 1998; Wanachiwanawin et al., 2004). 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 et al., 1990); (2) detection of serum anti-P. insidiosum antibodies by established serodiagnostic tests (immunodiffusion testing, immunochromatography test (ICT), hemagglutination method (HA), Western blotting) (Chareonsirisuthigul et al., 2013; Jindayok et al., 2009; Krajaejun et al., 2002; Pracharktam et al., 1991); and (3) presence of clinical and pathological features typical of P. insidiosum infection (Chareonsirisuthigul et al., 2013; Jindayok et al., 2009; Krajaejun et al., 2006b). Immunohistochemical staining for P. insidiosum was confirmed in tissue pathology using the method described in a previous report (Inkomlue et al., 2016). The Institutional Review Board Ethics Committee approved this study (No. 05-60-77).

Table 1 Baseline characteristics of patients with vascular pythiosis (N = 13). Parameter

Median (range)

Age (years) Sex, male, n (%) Occupation, agriculture, n (%)

52 (13–75) 11 (84.6) 11 (84.6)

Underlying disease, n (%) Hematological disease Thalassemia Duration of symptoms (months) Temperature ( C) White blood cell count (109/l) Hematocrit (%) Platelet count (109/l) Albumin (g/l) Total bilirubin (mg/dl) Alanine aminotransferase (U/l) Creatinine (mg/dl) Ferritin (ng/ml) (n = 8/13)

11 (84.6) 8 (61.5) 4 (0–11) 38 (36.2–40) 11.40 (2.69–15.10) 23.2 (14–36) 162.5 (21.0–527.0) 25.5 (15.8–41) 2.2 (0.3–3.1) 45 (22–125) 0.54 (0.3–1.8) 3493 (920–19 238)

Treatment, n (%) Amputation Disease-free surgical margins

10 (77) 5 (38.4)

Medical therapy, n (%) Combination of itraconazole and terbinafine Itraconazole Terbinafine SSKI Othera Duration of antifungals (days) Immunotherapeutic vaccine, n (%) Doses (no., range)

11 (84.6) 13 (100) 11 (84.6) 5 (38.4) 3 (23) 181 (3–2231) 11 (84.6) 3.5 (0–7)

Outcome Died, n (%) Duration of follow-up after presentation (days) Duration of follow-up among survivors (days)

4 (31) 184 (6–2481) 384 (50–2481)

SSKI, potassium iodide oral solution. a Voriconazole, amphotericin B deoxycholate, caspofungin (one of each).

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 et al., 2015). 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. 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-yearold 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 et al. (2015) (18). Unusual presentations with carotid involvement occurred in two nonthalassemic 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.

Table 2 Clinical presentation of patients with vascular pythiosis (N = 13). Underlying Age (years)/ disease sex

Clinical manifestations (duration, months)

Vascular involvement (side)

1

54/M

HCV cirrhosis HbCS trait reported by Khunchet et al. (18)

Right radial a., ulnar a., interosseous a. Serology (ICT) (day 11) Debridement Abscess isolation of P. Above elbow amputation (day 32) insidiosum from culture (day 21) Pathology

2

75/M

HbH

3

55/M

CSA2A Bart’s H

4

56/M

bThal/HbE

Multiple subcutaneous nodules in forearm (4) Thumb gangrene (day 7 prior to admission (PTA)) Thenar abscess Muscle necrosis Chronic ulcer (4) Gangrene of toe (day 2 PTA) Chronic foot ulcer (6) Pain, pallor of leg (day 3 PTA) Chronic foot ulcer (2)

5

48/M

Thal

Chronic ulcer of Right superficial femoral a. leg and foot (6)

6

29/F

HbH CS, AIHA, HBV

Chronic leg ulcer (5)

Right femoral a.

7

38/M

Thal

Chronic leg ulcer (4)

Left superficial femoral a.

8

28/M

Thal

Leg 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

9

52/M

bThal

Chronic 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

Right superficial femoral a., popliteal a., tibial a., peroneal a.

Right femoral a., popliteal a., posterior tibial a.

Right popliteal a., tibial a., peroneal a.

Diagnosis (duration, Surgery (duration, days after initial days after initial visit) visit)

Serology (ICT) (day 0) Vessel isolation of P. insidiosum from culture Serology (ICT) day 0 Vessel isolation of P. insidiosum from culture Serology (ICT) day 0 Vessel isolation of P. insidiosum from culture Pathology Serology (ICT) day 1 Vessel isolation of P. insidiosum from culture Pathology Pathology Serology (HA) day 20 after surgery Serology (ICT) day 1 Vessel isolation of P. insidiosum from culture Pathology NA Serology (ICT) day 2 before second surgery Vessel isolation of P. insidiosum from culture Pathology NA Serology (Western blot, immunodiffusion) day 10 (after surgery) (ICT)

Margins— Treatment drugs (duration, days) vascular, soft tissue

PIA vaccine— number of doses and schedule

Outcome and duration of survival (days)

Survived (lost to F/U after discharge)

Free, free

Two: days 0 Itraconazole, terbinafine, SSKI (46 and 6 days)

Above knee amputation (day 4)

Free, No


Free, free

Terbinafine, SSKI (27, 32 days) Discontinuation due to DRESS syndrome Itraconazole, terbinafine (262 days)

Six: days 0, 14, 28,42, 84, 168


Free, free

Itraconazole, terbinafine (383 days)

Seven: days 0, Survived (day 105 14, 28, 40, 82, after last dose of vaccine) 166, 208


Free, free


Four: days 0, 13, 27, 75

Survived (lost to F/U, day 105 after last dose of vaccine)

Above knee amputation (OSH)

No, no

None

Survived (day 145 off Rx)


No, no

Itraconazole, Terbinafine (368 days) SSKI Terbinafine, itraconazole (122 days)

Below knee amputation (OSH) Aneurysmectomy with axillofemoral bypass, superficial femoral a. resection, high above knee amputation (day 2) Debridement abdomen with mesh graft Above knee amputation Aortofemoral and femoral dorsalis pedis bypass graft (left) day 2

No, no

Terbinafine, itraconazole (18 days)

Three: days 0, Died (day 22) 8, 15

No, no

Terbinafine, itraconazole (2231 days, 6 years)

Three: days 0, Survived (lost to F/U, 17, 70 day 85 after last dose of vaccine)

Seven: days 0, Survived (day 704 off Rx; day 340 after 14, 46, 58, 105, 196, 392 last dose of vaccine) Survived (day 365 off Rx; day 453 after last dose of vaccine)

Three: days 0, Survived (lost to F/U, 7, 22 day 99 after last dose of vaccine)


No.

35

36

Table 2 (Continued) Vascular involvement (side)

Diagnosis (duration, Surgery (duration, days after initial days after initial visit) visit)

Underlying Age (years)/ disease sex

Clinical manifestations (duration, months)

10

48/M

Aplastic anemia

11

73/M

MDS

Serology (ICT) day 1 Vessel/skin tissue isolation of P. insidiosum from culture Pathology Left common iliac a., external iliac a., Pathology Acute limb ischemia (day 3 internal iliac a., superficial femoral a. Serology (ICT) day 4 (after surgery) Right common iliac a. PTA)

12

13/F

AML

Fever, headache Right hemiparesis, facial palsy (2)

Left distal supraclinoid internal carotid a., middle cerebral a., anterior cerebral a., common/internal/external carotid a.

13

57/M

Anemia of chronic disease, alcoholism, HT

Swelling of the neck (1)

Left common/internal/external carotid a., cerebral a.

Chronic leg wound (10)

Right external iliac a., common femoral a., femoral a., popliteal a., anterior tibial a.

Abscess isolation of Aspergillus spp and P. insidiosum Pathology Serology (HA: weakly positive) day 22 (after surgery) Vessel isolation of P. insidiosum Serology (ICT) day 4 (after surgery) Pathology

Margins— Treatment drugs (duration, days) vascular, soft tissue

High above knee amputation (day 2) No, no Skin biopsy (day 3) Common femoral a., profunda a. excision, flank skin excision (day 6)

Thrombectomy, balloon angioplasty No, no with stenting (day 1)

Craniectomy with removal of the abscess (day 1, 7, 22, 168)

No, no

Resection of left external carotid artery aneurysm, ligation part of internal and external carotid artery (day 1) Surgical debridement (day 9)

No, no

Terbinafine, itraconazole (26 days) SSKI (7 days) Caspofungin (13 days) Amphotericin B deoxycholate, itraconazole, terbinafine (3 days) Voriconazole (184 days) Terbinafine (42 days)


PIA vaccine— number of doses and schedule

Outcome and duration of survival (days)

Two: days 0, 14

Died (day 28)

None

Died (day 6)

Five: days 0, Survived (lost to F/U, 6, 19, 145, 154 day 298; day 13 after last dose of vaccine)

Four: days 0, 13, 24, 41

Died (day 81; day 36 after last dose of vaccine)

a, artery; AIHA, autoimmune hemolytic anemia; AML, acute myeloid leukemia; bThal, b-thalassemia; bThal/HbE, b-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.


No.


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. 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 et al., 2015). 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

37

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. Discussion Human pythiosis remains a life-threatening disease with challenges in diagnosis and treatment (Krajaejun et al., 2006b). 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 et al., 2009). This study identified malnourishment and immunosuppression as additional risk factors to hemoglobinopathy (Krajaejun et al., 2006b). Iron overload among patients with MDS and thalassemia predisposed the patient to infection (Krajaejun et al., 2006b). P.

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Table 3 Pathology (N = 13). No.

Underlying Age (years)/ disease sex

Organ

Procedure

1

54/M

HCV cirrhosis HbCS trait reported by Khunchet et al. (18)

Subcutaneous nodule Thenar abscess Thenar abscess Vessel: hand, forearm

2

75/M

HbH

Leg (vessel) Leg (soft tissue)

3

55/M

CSA2A Bart’s H

Leg (vessel) Leg (soft tissue)

4

56/M

bThal/HbE

Leg (vessel)

5

48/M

Thal

Leg (vessel)

6

29/F

Leg (vessel)

7

38/M

HbH CS, AIHA, HBV Thal

8

28/M

Thal

Leg (vessel) Leg (soft tissue) Aorta

9

52/M

Thal

Leg (vessel)

10

48/M

Aplastic anemia

Leg (vessel) Intramuscular abscesses Osteomyelitis of the lower extremities Abdominal wall

11

73/M

MDS

Leg (vessel)

12

13/F

AML

Brain Carotid artery Cerebral artery

13

57/M

Anemia of chronic disease, Alcoholism, HT

Carotid artery aneurysm

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 Suppurative granulomatous inflammation of the tibial, peroneal, Above knee amputation popliteal, superficial femoral, ilioperoneal a. GMS stain and IHC stain for P. insidiosum were positive Dry gangrene with acute and chronic inflammation Necrotizing granulomatous inflammation of vascular wall Above knee amputation GMS stain and IHC stain for P. insidiosum were positive Gangrenous necrosis involved dorsal skin and subcutaneous tissue of foot Above knee amputation Acute 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 Above knee amputation Neutrophilic infiltration, fungus invading vascular wall, GMS and IHC stain for P. insidiosum were positive Above knee amputation Necrotizing suppurative arteritis, short fungal fragment, GMS stain and periodic acid-Schiff stain were positive Above knee amputation Fungal vasculitis with thrombosis, presence of fungal organism at vascular margin, IHC stain for P. insidiosum was positive Below knee amputation NA Aneurysmectomy with axillofemoral bypass, Acute infective arteritis (neutrophilic infiltration) of popliteal a., removal of superficial femoral a., high above femoral a., iliac a., and aorta, with aneurysmal dilatation of aorta, knee amputation presence of fungi with vascular wall invasion, GMS stain and IHC stain for P. insidiosum were positive NA Above knee amputation NA Aortofemoral and femoral dorsalis pedis bypass graft High above knee amputation Necrotizing arteritis with intraluminal thrombus involving Skin biopsy popliteal a., anterior tibial a., femoral a, and external iliac a., Common femoral a., profunda a. excision, irregular branch hyphae in thrombus with vascular wall invasion, flank skin excision 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 Thrombectomy, balloon angioplasty stenting Thrombus with fungal hyphae, IHC stain for P. insidiosum was positive Mini craniectomy, abscess removal Acute inflammatory cell infiltrates with necrotic tissue, branching Craniectomy with abscess removal septate hyphae, GMS stain and IHC stain for P. insidiosum were Craniectomy with abscess removal, dura positive Acute and chronic inflammation with granulation tissue, foreign repair 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 Acute suppurative inflammation, GMS stain and IHC stain for P. Resection of external carotid artery aneurysm, ligation of part of the internal and insidiosum were positive external carotid artery

Leg (vessel)

Pathology

Skin biopsy Skin biopsy; debridement Debridement; biopsy Above elbow amputation

a, artery; AIHA, autoimmune hemolytic anemia; AML, acute myeloid leukemia; bThal, b-thalassemia; bThal/HbE, b-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.

insidiosum carries a gene encoding a ferrochelatase, which plays a role in its virulence (Krajaejun et al., 2011). 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 et al., 2013). 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 et al., 2013). Baseline ferritin levels


39

Table 4 Radiographic findings after surgery (N = 13). No.

Age (years)/ sex

Underlying disease

Imaging findings after surgery (duration after surgery)

1

54/M

NA

2

75/M

HCV cirrhosis HbCS trait HbH

3

55/M

CSA2A Bart’s H

4 5 6

56/M 48/M 29/F

bThal/HbE

7 8

38/M 28/M

Thal Thal

9

52/M

Thal

10

48/M

Aplastic anemia

11 12

73/M 13/F

MDS AML

13

57/M

Anemia of chronic disease, alcoholism, HT

Thal HbH CS, AIHA, HBV

Day 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 Week 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 NA NA Month 3, CTA aorta: soft tissue thickening of proximal SFA thrombosis and occlusion of proximal SFA at level 7 cm above the stump Month 5, postoperative, CTA aorta: AKA with irregular wall, patent SFA, no evidence of arterial occlusion or stenosis 1st 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 1st 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 Day 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 NA 1st 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 1st 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; bThal, b-thalassemia; bThal/HbE, b-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.

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 et al., 1998; Krajaejun et al., 2006b; Narkwiboonwong et al., 2011; Kirzhner et al., 2015; Shenep et al. 2011). Dissemination has been shown to occur among patients with leukemia; e.g., gastrointestinal involvement (Krajaejun et al., 2006a,b). 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 et al., 2006b). A literature review revealed that the natural history of immunosuppressed patients differs from that of thalassemia patients in many aspects (Hilton et al., 2016; Hoffman et al., 2011; Pan et al., 2014; Wanachiwanawin et al., 2004): 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 et al., 2004). 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

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oral voriconazole for 9 months (Hoffman et al., 2011). The neutropenic patient showed a rapid deterioration within days after the onset of symptoms (Hilton et al., 2016; Hoffman et al., 2011). 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 et al., 1998). The inflammatory response among thalassemia patients revealed a granulomatous reaction or chronic inflammation, as has been reported previously (Imwidthaya, 1994; Krajaejun et al., 2006b; Prasertwitayakij et al., 2003). 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 et al., 2006b; Thianprasit et al., 1996). 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 et al. (2015). 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 et al., 2016). IHC stain of P. insidiosum was useful for differentiating fungal pathogens on tissue pathology, with a specificity of 100% (Inkomlue et al., 2016), 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 et al., 2016). Mortality in this study was 31%, similar to the rate reported previously (Krajaejun et al., 2006b; Permpalung et al., 2015; Sermsathanasawadi et al., 2016). 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 et al., 2016). Antifungal therapy There were few successful outcomes with antifungal treatment alone, particularly in those with unresectable disease (Krajaejun et al., 2006b). Most antifungals are not active against P. insidiosum due to its lack of ergosterol (Lerksuthirat et al., 2017). 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 mg/ml and 0.03–4 mg/ml, respectively, against clinical isolates of P. insidiosum (Kirzhner et al., 2015; Permpalung et al., 2015; Shenep et al., 1998) with evidence of in vitro synergistic

activity (Shenep et al., 1998). However, recent data from Thai clinical isolates showed no evidence of synergy (Permpalung et al., 2015). Moreover, in vitro data showed that P. insidiosum isolates were sensitive to the antifungal agents only at high concentrations (Lerksuthirat et al., 2017). 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 et al., 2014). Terbinafine is more easily tolerated than itraconazole, with fewer side effects and drug interactions (Dolton et al., 2014). Future studies should evaluate the use of high-dose terbinafine for the treatment of pythiosis. The new antifungal caspofungin, which inhibits b-d-glucan found in the cell wall of Pythium spp has demonstrated in vitro synergistic activity in combination with terbinafine (Cavalheiro et al., 2009). 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, 1994; Sathapatayavongs et al., 1989). 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 et al., 2017; Permpalung et al., 2015). The duration of antifungal use varied from 1 to 2 years after surgery due to the lack of supporting data (Sermsathanasawadi et al., 2016). Although antifungals have been associated with clinical resistance in the treatment of human pythiosis (Lerksuthirat et al., 2017), prolonged therapy (>1 year) with itraconazole and terbinafine successfully cured unresectable disease in one patient (Shenep et al., 1998). 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 and Newton, 2005; Wanachiwanawin et al., 2004). 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 et al., 2004). The PIA vaccine successfully cured the first human case treated with immunotherapy with inoperable vascular pythiosis (Thitithanyanont et al., 1998). The efficacy of the Pythium vaccine remains inconclusive (Permpalung et al., 2015; Wanachiwanawin et al., 2004). 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 et al., 2006b; Sermsathanasawadi et al., 2016). 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. References Cavalheiro AS, Maboni G, de Azevedo MI, Argenta JS, Pereira DI, Spader TB, et al. In Vitro activity of terbinafine combined with caspofungin and azoles against Pythium insidiosum. Antimicrob Agents Chemother 2009;53(5):2136–8. Chaiprasert A, Samerpitak K, Wanachiwanawin W, Thasnakorn P. Induction of zoospore formation in Thai isolates of Pythium insidiosum. Mycoses 1990;33 (6):317–23. Chareonsirisuthigul T, Khositnithikul R, Intaramat A, Inkomlue R, Sriwanichrak K, Piromsontikorn, et al. Performance comparison of immunodiffusion, enzymelinked immunosorbent assay, immunochromatography and hemagglutination for serodiagnosis of human pythiosis. Diagn Microbiol Infect Dis 2013;76(1):42– 5. Dolton MJ, Perera V, Pont LG, McLachlan AJ. Terbinafine in combination with other antifungal agents for treatment of resistant or refractory mycoses: investigating optimal dosing regimens using a physiologically based pharmacokinetic model. Antimicrob Agents Chemother 2014;58(1):48–54. Hilton RE, Tepedino K, Glenn CJ, Merkel KL. Swamp cancer: a case of human pythiosis and review of the literature. Br J Dermatol 2016;175(2):394–7. Hoffman MA, Cornish NE, Simonsen KA. A painful thigh lesion in an immunocompromised 11-year-old boy. Pediatr Infect Dis J 2011;30(11):1017–8. Imwidthaya P. Human pythiosis in Thailand. Postgrad Med J 1994;70(826):558–60. 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. J Clin Microbiol 2016;54(1):43–8. 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. Med Mycol 2016;54(6):641–7. Jindayok T, Piromsontikorn S, Srimuang S, Khupulsup K, Krajaejun T. Hemagglutination test for rapid serodiagnosis of human pythiosis. Clin Vaccine Immunol 2009;16(7):1047–51.

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