Human pythiosis is a life-threatening human disease caused by Pythium insidiosum. In Thailand, vascular pythiosis is the most common form and carries a mortality rate of 10 to 40%, despite aggressive treatment with radical surgery, antifungal agents, and immunotherapy. Itraconazole and terbinafine have been the mainstay of treatment, until recently, based on case report data showing potential synergistic effects against Brazilian P. insidiosum isolates. However, the synergistic effects of itraconazole and terbinafine against Thai P. insidiosum isolates were not observed.
KEYWORDS: Pythium insidiosum, susceptibility profile, antibacterial agents
ABSTRACT
Human pythiosis is a life-threatening human disease caused by Pythium insidiosum. In Thailand, vascular pythiosis is the most common form and carries a mortality rate of 10 to 40%, despite aggressive treatment with radical surgery, antifungal agents, and immunotherapy. Itraconazole and terbinafine have been the mainstay of treatment, until recently, based on case report data showing potential synergistic effects against Brazilian P. insidiosum isolates. However, the synergistic effects of itraconazole and terbinafine against Thai P. insidiosum isolates were not observed. This study tested the in vitro susceptibilities of 27 Thai human P. insidiosum isolates (clade II, n = 17; clade IV, n = 10), 12 Thai environmental P. insidiosum isolates (clade II, n = 4; clade IV, n = 8), and 11 non-Thai animal P. insidiosum isolates (clade I, n = 9; clade II, n = 2) to antibiotics in eight antibacterial classes to evaluate alternative effective treatments. Tetracycline and macrolide antibiotics demonstrated in vitro activity against Thai P. insidiosum isolates, with doxycycline MICs (1 to 16 μg/ml), minocycline MICs (1 to 4 μg/ml), tigecycline MICs (1 to 4 μg/ml), azithromycin MICs (1 to 16 μg/ml), and clarithromycin MICs (0.125 to 8 μg/ml) being the lowest, on average. Synergistic effects of tetracyclines and macrolides were also observed.
INTRODUCTION
Human pythiosis is caused by an oomycete, Pythium insidiosum. Oomycota or oomycetes are a group of fungus-like stramenopiles. P. insidiosum is currently classified into four clades, clades I, II, III, and IV, based on the phylogenetic distribution of the internal transcribed spacer (ITS) region and cytochrome oxidase II (COX2) gene. Clade I isolates have been identified in the United States, and clade II isolates are mainly from Australia, India, Japan, New Zealand, Taiwan, and Thailand. Clade III isolates are from the United States, and clade IV comprises isolates from the Asia region and the Middle East (1, 2). This oomycete naturally inhabits soil, swampy areas, and stagnant freshwater. Accordingly, most observed opportunistic infections in Thailand tend to occur during the rainy seasons (3, 4). Human pythiosis has four clinical manifestations: vascular, ocular, skin and soft tissue, and disseminated infections (5). Vascular pythiosis is the most common form in Thailand, followed by ocular pythiosis. These infections result in devastating outcomes, with mortality rates being 10 to 40% in individuals with vascular pythiosis and an eye loss rate of 50% in those with ocular pythiosis (3, 4, 6).
Combination therapy, including radical surgery, itraconazole, terbinafine, and immunotherapy, had been the mainstay of treatment, according to the King Chulalongkorn Memorial Hospital (KCMH) research protocols, until December 2018. Itraconazole and terbinafine were used on the basis of their synergistic effects against Brazilian animal P. insidiosum isolates and a single case study of a patient with deep facial tissue infection who was effectively treated (7–9). However, synergistic effects of itraconazole and terbinafine were not observed against Thai clinical P. insidiosum isolates (3, 4, 6).
Recently, data on the in vitro and in vivo susceptibilities of Brazilian P. insidiosum isolates to antibacterial agents have shown that the MICs are more favorable than those of antifungal agents (7, 10–14). A prospective study from India likewise demonstrated the efficacy of topical linezolid, topical azithromycin, and oral azithromycin in treating Pythium keratitis. In that study, none of the patients required evisceration or enucleation; 33% were successfully treated with medications alone (15). In contrast, the most recent study of ocular pythiosis from Thailand using oral itraconazole and terbinafine in conjunction with various topical antifungal agents demonstrated an eye loss rate of 50%, and all patients required ocular surgery (3).
Antibacterial agents were first used in Thailand in 2019 as adjunctive therapy in two relapsed vascular pythiosis patients after itraconazole and immunotherapy failed (16). This study was conducted to better understand the susceptibility patterns of antibacterial classes against Thai human, Thai environmental, and non-Thai animal P. insidiosum isolates in different clades.
RESULTS
Tetracyclines (doxycycline [DOX], minocycline [MIN], tigecycline [TIG]), macrolides (azithromycin [AZM], clarithromycin [CLR]), and oxazolidinones (linezolid) had the lowest MIC values compared to those of the other antibacterial classes for all clades of Thai human, Thai environmental, and non-Thai animal P. insidiosum isolates (Tables 1 to 3). The geometric mean (GM) MICs of MIN, TIG, and CLR ranged from 1.00 to 2.18 μg/ml, followed by those for DOX and AZM, which had GM MICs of 2.72 to 5.28 μg/ml, and linezolid, which had GM MICs of 5.44 to 9.51 μg/ml. The other antibiotic agents had high GM MIC values (range, 10.77 to >32 μg/ml).
TABLE 1.
In vitro susceptibility of Thai human P. insidiosum isolates to eight classes of antibacterial antibioticsa
| Antibiotic class | Clade II (n = 17) |
Clade IV (n = 10) |
|||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| No. of isolates with the following MIC (μg/ml): |
GM MIC (μg/ml) | No. of isolates with the following MIC (μg/ml): |
GM MIC (μg/ml) | ||||||||||||||||||||
| Agent | 0.125 | 0.25 | 0.50 | 1 | 2 | 4 | 8 | 16 | 32 | >32 | 0.125 | 0.25 | 0.50 | 1 | 2 | 4 | 8 | 16 | 32 | >32 | |||
| Tetracyclines | Doxycycline | 1 | 4 | 9 | 2 | 1 | 3.69 | 2 | 5 | 3 | 4.29 | ||||||||||||
| Minocycline | 6 | 10 | 1 | 1.63 | 2 | 7 | 1 | 1.87 | |||||||||||||||
| Tigecycline | 7 | 9 | 1 | 1.57 | 8 | 2 | 1.15 | ||||||||||||||||
| Macrolides | Azithromycin | 1 | 7 | 9 | 3.13 | 2 | 3 | 4 | 1 | 5.28 | |||||||||||||
| Clarithromycin | 1 | 1 | 1 | 5 | 6 | 2 | 1 | 1.33 | 4 | 4 | 2 | 1.74 | |||||||||||
| Beta-lactams | Cefazolin | 17 | >32 | 10 | >32 | ||||||||||||||||||
| Ceftriaxone | 17 | >32 | 10 | >32 | |||||||||||||||||||
| Ceftazidime | 17 | >32 | 10 | >32 | |||||||||||||||||||
| Meropenem | 1 | 16 | 32.00 | 1 | 9 | 32.00 | |||||||||||||||||
| Oxazolidinone | Linezolid | 4 | 9 | 3 | 1 | 8.33 | 1 | 6 | 3 | 9.19 | |||||||||||||
| Glycopeptide | Vancomycin | 17 | >32 | 10 | >32 | ||||||||||||||||||
| Aminoglycosides | Amikacin | 17 | >32 | 10 | >32 | ||||||||||||||||||
| Gentamicin | 17 | >32 | 10 | >32 | |||||||||||||||||||
| Neomycin | 1 | 16 | 32.00 | 10 | >32 | ||||||||||||||||||
| Streptomycin | 1 | 1 | 15 | 22.63 | 10 | >32 | |||||||||||||||||
| Tobramycin | 17 | >32 | 10 | >32 | |||||||||||||||||||
| Quinolones | Ciprofloxacin | 17 | >32 | 10 | >32 | ||||||||||||||||||
| Levofloxacin | 17 | >32 | 10 | >32 | |||||||||||||||||||
| Moxifloxacin | 2 | 1 | 14 | 20.16 | 1 | 1 | 8 | 22.63 | |||||||||||||||
| Polymyxins | Colistin (polymyxin E) | 1 | 2 | 14 | 25.40 | 1 | 9 | 32.00 | |||||||||||||||
| Polymyxin B | 17 | >32 | 10 | >32 | |||||||||||||||||||
The MIC of each agent was determined by 100% inhibition of the mycelium by visual observation compared to the inhibition in the control well (no antibiotics). GM, geometric mean.
TABLE 2.
In vitro susceptibility of Thai environmental P. insidiosum isolates against eight classes of antibacterial antibioticsa
| Antibiotic class | Agent | Clade II (n = 4) |
Clade IV (n = 8) |
||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| No. of isolates with the following MIC (μg/ml): |
GM MIC (μg/ml) | No. of isolates with the following MIC (μg/ml): |
GM MIC (μg/ml) | ||||||||||||||||||||
| 0.125 | 0.25 | 0.50 | 1 | 2 | 4 | 8 | 16 | 32 | >32 | 0.125 | 0.25 | 0.50 | 1 | 2 | 4 | 8 | 16 | 32 | >32 | ||||
| Tetracyclines | Doxycycline | 3 | 1 | 4.76 | 3 | 3 | 1 | 1 | 4.00 | ||||||||||||||
| Minocycline | 4 | 2.00 | 7 | 1 | 2.18 | ||||||||||||||||||
| Tigecycline | 4 | 2.00 | 7 | 1 | 2.18 | ||||||||||||||||||
| Macrolides | Azithromycin | 1 | 2 | 1 | 4.76 | 2 | 4 | 2 | 4.00 | ||||||||||||||
| Clarithromycin | 4 | 2.00 | 2 | 5 | 1 | 1.83 | |||||||||||||||||
| Beta-lactams | Cefazolin | 4 | >32 | 8 | >32 | ||||||||||||||||||
| Ceftriaxone | 4 | >32 | 8 | >32 | |||||||||||||||||||
| Ceftazidime | 4 | >32 | 8 | >32 | |||||||||||||||||||
| Meropenem | 1 | 3 | 32.00 | 8 | >32 | ||||||||||||||||||
| Oxazolidinone | Linezolid | 1 | 1 | 2 | 9.51 | 6 | 2 | 9.51 | |||||||||||||||
| Glycopeptide | Vancomycin | 4 | >32 | 8 | >32 | ||||||||||||||||||
| Aminoglycosides | Amikacin | 4 | >32 | 8 | >32 | ||||||||||||||||||
| Gentamicin | 4 | >32 | 8 | >32 | |||||||||||||||||||
| Neomycin | 4 | >32 | 8 | >32 | |||||||||||||||||||
| Streptomycin | 4 | >32 | 8 | >32 | |||||||||||||||||||
| Tobramycin | 4 | >32 | 8 | >32 | |||||||||||||||||||
| Quinolones | Ciprofloxacin | 4 | >32 | 8 | >32 | ||||||||||||||||||
| Levofloxacin | 4 | >32 | 8 | >32 | |||||||||||||||||||
| Moxifloxacin | 1 | 1 | 2 | 22.63 | 8 | >32 | |||||||||||||||||
| Polymyxins | Colistin (polymyxin E) | 1 | 1 | 2 | 22.63 | 1 | 7 | 16.00 | |||||||||||||||
| Polymyxin B | 4 | >32 | 8 | >32 | |||||||||||||||||||
The MIC of each agent was determined by 100% inhibition of the mycelium by visual observation compared to the inhibition in the control well (no antibiotics). GM, geometric mean.
TABLE 3.
In vitro susceptibility of non-Thai animal P. insidiosum isolates against eight classes of antibacterial antibioticsa
| Antibiotic class | Agent | Clade I (n = 9) |
Clade II (n = 2) |
||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| No. of isolates with the following MIC (μg/ml): |
GM MIC (μg/ml) | No. of isolates with the following MIC (μg/ml): |
GM MIC (μg/ml) | ||||||||||||||||||||
| 0.125 | 0.25 | 0.5 | 1 | 2 | 4 | 8 | 16 | 32 | >32 | 0.125 | 0.25 | 0.5 | 1 | 2 | 4 | 8 | 16 | 32 | >32 | ||||
| Tetracyclines | Doxycycline | 1 | 2 | 5 | 1 | 3.43 | 2 | 4.00 | |||||||||||||||
| Minocycline | 1 | 1 | 5 | 2 | 1.08 | 2 | 2.00 | ||||||||||||||||
| Tigecycline | 1 | 6 | 2 | 1.08 | 2 | 2.00 | |||||||||||||||||
| Macrolides | Azithromycin | 6 | 2 | 1 | 2.72 | 1 | 1 | 2.83 | |||||||||||||||
| Clarithromycin | 1 | 5 | 3 | 1.00 | 1 | 1 | 1.41 | ||||||||||||||||
| Beta-lactams | Cefazolin | 1 | 8 | >32 | 2 | >32 | |||||||||||||||||
| Ceftriaxone | 9 | >32 | 2 | >32 | |||||||||||||||||||
| Ceftazidime | 9 | >32 | 2 | >32 | |||||||||||||||||||
| Meropenem | 5 | 4 | 32.00 | 2 | >32 | ||||||||||||||||||
| Oxazolidinone | Linezolid | 5 | 4 | 5.44 | 2 | 8.00 | |||||||||||||||||
| Glycopeptide | Vancomycin | 9 | >32 | 2 | >32 | ||||||||||||||||||
| Aminoglycosides | Amikacin | 9 | >32 | 2 | >32 | ||||||||||||||||||
| Gentamicin | 1 | 3 | 5 | 26.91 | 2 | >32 | |||||||||||||||||
| Neomycin | 2 | 7 | 32.00 | 2 | >32 | ||||||||||||||||||
| Streptomycin | 1 | 3 | 5 | 26.91 | 2 | >32 | |||||||||||||||||
| Tobramycin | 9 | >32 | 2 | >32 | |||||||||||||||||||
| Quinolones | Ciprofloxacin | 1 | 8 | 32.00 | 2 | >32 | |||||||||||||||||
| Levofloxacin | 9 | >32 | 2 | >32 | |||||||||||||||||||
| Moxifloxacin | 1 | 2 | 3 | 3 | 20.16 | 2 | >32 | ||||||||||||||||
| Polymyxins | Colistin (polymyxin E) | 4 | 3 | 2 | 10.77 | 1 | 1 | 16.00 | |||||||||||||||
| Polymyxin B | 9 | >32 | 2 | >32 | |||||||||||||||||||
The MIC of each agent was determined by 100% inhibition of the mycelium by visual observation compared to the inhibition in the control well (no antibiotics). GM, geometric mean.
MIN, TIG, and CLR showed the lowest MIC values in this study. Among Thai human P. insidiosum isolates, MIN MICs ranged from 1 to 4 μg/ml (GM, 1.63 μg/ml) for clade II isolates and 1 to 4 μg/ml (GM, 1.87 μg/ml) for clade IV isolates; TIG MICs ranged from 1 to 4 μg/ml (GM, 1.57 μg/ml) for clade II isolates and 1 to 2 μg/ml (GM, 1.15 μg/ml) for clade IV isolates; CLR MICs ranged from 0.125 to 8 μg/ml (GM, 1.33 μg/ml) for clade II isolates and 1 to 4 μg/ml (GM, 1.74 μg/ml) for clade IV isolates. Among Thai environmental isolates, all clade II isolates had MIN, TIG, and CLR MICs of 2 μg/ml (GM, 2.00 μg/ml); P. insidiosum clade IV isolates had MIN, TIG, and CLR MICs of 2 to 4 μg/ml (GM, 2.18 μg/ml), 2 to 4 μg/ml (GM, 2.18 μg/ml), and 1 to 4 μg/ml (GM, 1.83 μg/ml), respectively. Among clade I and clade II non-Thai animal P. insidiosum isolates, MIN MICs were 0.25 to 4 μg/ml (GM, 1.08 μg/ml) for clade I isolates and 2 μg/ml (GM, 2.00 μg/ml) for clade II isolates; TIG MICs were 0.5 to 2 μg/ml (GM, 1.08 μg/ml) for clade I isolates and 2 μg/ml (GM, 2.00 μg/ml) for clade II isolates; CLR MICs ranged from 0.125 to 2 μg/ml (GM, 1.00 μg/ml) for clade I isolates and 1 to 2 μg/ml (GM, 1.41 μg/ml) for clade II isolates.
DOX and AZM had the second lowest MIC values. Among Thai human P. insidiosum isolates, DOX MICs ranged from 1 to 16 μg/ml (GM, 3.69 μg/ml) for clade II isolates and 2 to 8 μg/ml (GM, 4.29 μg/ml) for clade IV isolates; AZM MICs ranged from 1 to 4 μg/ml (GM, 3.13 μg/ml) for clade II isolates and 2 to 16 μg/ml (GM, 5.28 μg/ml) for clade IV isolates. Among Thai environmental isolates, DOX MICs ranged from 4 to 8 μg/ml (GM, 4.76 μg/ml) for clade II isolates and 2 to 16 μg/ml (GM, 4.00 μg/ml) for clade IV isolates; AZM MICs ranged from 2 to 16 μg/ml (GM, 4.76 μg/ml) for clade II isolates and 2 to 8 μg/ml (GM, 4.00 μg/ml) for clade IV isolates. For the other group, non-Thai animal P. insidiosum isolates, DOX MICs were 1 to 16 μg/ml (GM, 3.43 μg/ml) for clade I isolates and 4 μg/ml (GM, 4.00 μg/ml) for clade II isolates; AZM MICs ranged from 2 to 8 μg/ml (GM, 2.72 μg/ml) for clade I isolates and 2 to 4 μg/ml (GM, 2.83 μg/ml) for clade II isolates.
Linezolid MICs ranged from 4 to 32 μg/ml (GM, 8.33 μg/ml) for the clade II isolates and 4 to 16 μg/ml (GM, 9.19 μg/ml) for the clade IV isolates among Thai human P. insidiosum isolates and from 4 to 16 μg/ml (GM, 9.51 μg/ml) for the clade II isolates and 8 to 16 μg/ml (GM, 9.51 μg/ml) for the clade IV isolates among Thai environmental P. insidiosum isolates. However, among non-Thai animal P. insidiosum isolates, linezolid MICs were 4 to 8 μg/ml (GM, 5.44 μg/ml) for clade I isolates and 8 μg/ml (GM, 8.00 μg/ml) for clade II isolates. Mycelium growth of all isolates was not inhibited by beta-lactams, glycopeptides, aminoglycosides, quinolones, or polymyxins (Tables 1 to 3).
The in vitro activities of the tetracyclines combined with the macrolides are displayed in Tables 4 to 6. Synergistic effects were observed in more than 90% of the Thai human P. insidiosum isolates (94.85% of clade II isolates and 93.75% of clade IV isolates) and Thai environmental P. insidiosum isolates (90.63% of clade II isolates and 90.63% of clade IV isolates) and more than 85% of non-Thai animal P. insidiosum isolates (97.22% of clade I isolates and 87.50% of clade II isolates). Antagonistic interactions were not observed in this study.
TABLE 4.
In vitro activity of combinations of azithromycin, clarithromycin, minocycline, doxycycline, and tigecycline against Thai human P. insidiosum isolatesa
| Drug combination | Interpretation of activity | Clade II (n = 17) |
Clade IV (n = 10) |
||
|---|---|---|---|---|---|
| No. of isolates | FICI range (GM) | No. of isolates | FICI range (GM) | ||
| AZM-MIN | Synergism | 13 | 0.19–0.56 (0.31) | 9 | 0.09–0.38 (0.23) |
| Indifference | 4 | 0.56–0.75 (0.60) | 1 | 0.75 (0.75) | |
| AZM-TIG | Synergism | 14 | 0.09–0.38 (0.24) | 10 | 0.25–0.38 (0.31) |
| Indifference | 3 | 0.56–0.63 (0.59) | |||
| CLR-MIN | Synergism | 17 | 0.09–0.50 (0.30) | 10 | 0.09–0.31 (0.15) |
| Indifference | |||||
| CLR-TIG | Synergism | 17 | 0.15–0.38 (0.24) | 10 | 0.19–0.49 (0.31) |
| Indifference | |||||
| MIN-TIG | Synergism | 17 | 0.19–0.38 (0.28) | 9 | 0.19–0.38 (0.28) |
| Indifference | 1 | 0.63 (0.63) | |||
| DOX-AZM | Synergism | 17 | 0.19–0.42 (0.27) | 8 | 0.19–0.50 (0.31) |
| Indifference | 2 | 0.75 (0.75) | |||
| DOX-CLR | Synergism | 17 | 0.25–0.50 (0.28) | 9 | 0.25–0.50 (0.35) |
| Indifference | 1 | 0.56 (0.56) | |||
| DOX-TIG | Synergism | 17 | 0.19–0.50 (0.32) | 10 | 0.18–0.50 (0.28) |
| Indifference | |||||
AZM, azithromycin; CLR, clarithromycin; MIN, minocycline; TIG, tigecycline; DOX, doxycycline; FICI, fractional inhibitory concentration index. Interpretations of activity were as follows: antagonism, FICI > 4; indifference, 0.5 < FICI < 4; synergism, FICI ≤ 0.5.
TABLE 5.
In vitro activity of combinations of azithromycin, clarithromycin, minocycline, doxycycline, and tigecycline against Thai environmental P. insidiosum isolatesa
| Drug combination | Interpretation of activity | Clade II (n = 4) |
Clade IV (n = 8) |
||
|---|---|---|---|---|---|
| No. of isolates | FICI range (GM) | No. of isolates | FICI range (GM) | ||
| AZM-MIN | Synergism | 3 | 0.08–0.50 (0.23) | 6 | 0.12–0.25 (0.21) |
| Indifference | 1 | 0.75 (0.75) | 2 | 0.63–0.75 (0.68) | |
| AZM-TIG | Synergism | 3 | 0.19–0.31 (0.24) | 7 | 0.19–0.38 (0.28) |
| Indifference | 1 | 0.63 (0.63) | 1 | 0.56 (0.56) | |
| CLR-MIN | Synergism | 4 | 0.08–0.31 (0.17) | 8 | 0.09–0.38 (0.19) |
| Indifference | |||||
| CLR-TIG | Synergism | 4 | 0.19–0.37 (0.26) | 8 | 0.16–0.38 (0.25) |
| Indifference | |||||
| MIN-TIG | Synergism | 3 | 0.25 (0.25) | 8 | 0.18–0.38 (0.23) |
| Indifference | 1 | 0.63 (0.63) | |||
| DOX-AZM | Synergism | 4 | 0.25–0.38 (0.31) | 7 | 0.25–0.38 (0.34) |
| Indifference | 1 | 0.56 (0.56) | |||
| DOX-CLR | Synergism | 4 | 0.19–0.31 (0.26) | 6 | 0.25–0.38 (0.30) |
| Indifference | 2 | 0.56–0.75 (0.65) | |||
| DOX-TIG | Synergism | 4 | 0.19–0.31 (0.25) | 8 | 0.18–0.31 (0.25) |
| Indifference | |||||
AZM, azithromycin; CLR, clarithromycin; MIN, minocycline; TIG, tigecycline; DOX, doxycycline; FICI, fractional inhibitory concentration index. Interpretations of activity were as follows: antagonism, FICI > 4; indifference, 0.5 < FICI < 4; synergism, FICI ≤ 0.5.
TABLE 6.
In vitro activity of combinations of azithromycin, clarithromycin, minocycline, doxycycline, and tigecycline against non-Thai animal P. insidiosum isolatesa
| Drug combination | Interpretation | Clade I (n = 9) |
Clade II (n = 2) |
||
|---|---|---|---|---|---|
| No. of isolates | FICI range (GM) | No. of isolates | FICI range (GM) | ||
| AZM-MIN | Synergism | 8 | 0.14–0.38 (0.22) | 2 | 0.19–0.29 (0.23) |
| Indifference | 1 | 1.13 (1.13) | |||
| AZM-TIG | Synergism | 8 | 0.25–0.38 (0.33) | 1 | 0.38 (0.38) |
| Indifference | 1 | 0.75 (0.75) | 1 | 0.67 (0.67) | |
| CLR-MIN | Synergism | 9 | 0.27–0.38 (0.32) | 2 | 0.31–0.38 (0.34) |
| Indifference | |||||
| CLR-TIG | Synergism | 9 | 0.27–0.49 (0.35) | 2 | 0.37 (0.37) |
| Indifference | |||||
| MIN/TIG | Synergism | 9 | 0.25–0.49 (0.31) | 2 | 0.28–0.31 (0.29) |
| Indifference | |||||
| DOX-AZM | Synergism | 9 | 0.28–0.50 (0.34) | 1 | 0.29 (0.29) |
| Indifference | 1 | 0.63 (0.63) | |||
| DOX-CLR | Synergism | 9 | 0.19-038 (0.33) | 2 | 0.50 (0.50) |
| Indifference | |||||
| DOX-TIG | Synergism | 9 | 0.25–0.50 (0.42) | 2 | 0.37–0.38 (0.37) |
| Indifference | |||||
AZM, azithromycin; CLR, clarithromycin; MIN, minocycline; TIG, tigecycline; DOX, doxycycline; FICI, fractional inhibitory concentration index. Interpretations of activity were as follows: antagonism, FICI > 4; indifference, 0.5 < FICI < 4; synergism, FICI ≤ 0.5.
DISCUSSION
This is the largest in vitro susceptibility study of human, environmental, and animal P. insidiosum isolates to date examining their antibacterial susceptibility patterns and evaluating new treatment options for the devastating disease caused by this organism. This study reveals that tetracyclines and macrolides have MICs 10 to 100 times lower than those of antifungal agents determined in previous studies (3, 6). These findings correlate with published data from Brazil (10, 11, 13). More importantly, synergistic effects of antimicrobial agents against Thai P. insidiosum isolates were observed, highlighting new potential treatment options for human pythiosis in Thailand.
Until recently in Thailand, itraconazole and terbinafine were used to treat all pythiosis patients, despite their unfavorable MICs and lack of synergy (4). Pragmatically, it has been very challenging to maintain therapeutic serum itraconazole levels throughout 6 to 12 months of treatment (recognizing that there are no standard guidelines for ideal therapeutic drug levels or an MIC breakpoint interpretation for P. insidiosum). Approximately 25% of patients cannot receive terbinafine because it is unavailable in rural Thailand (6). However, these patients can generally receive azithromycin, clarithromycin, and doxycycline without geographic limitations or restrictions from health insurance. With the significantly lower MICs of tetracycline and macrolide classes, there is a reasonable probability that patients’ blood or tissue drug concentrations would be above the MICs. For instance, oral doxycycline at 200 mg and clarithromycin at 500 mg can result in serum drug concentrations of 2 to 4 μg/ml and 2 to 3 μg/ml, respectively (11). In fact, treatment by the adjunctive use of antibacterial agents (azithromycin-doxycycline and clarithromycin-doxycycline), guided by in vitro susceptibility results, was successful in two patients in Thailand with relapses of vascular pythiosis (16).
The mechanisms of action of tetracyclines and macrolides against P. insidiosum remain unclear. However, it has been hypothesized that the potential mechanisms could include inhibition of protein synthesis, cell wall synthesis, and/or amino acid transport (11). We observed that clade I of non-Thai animal P. insidiosum isolates tended to have the lowest MICs. Broader MIC ranges of clarithromycin were found for clade II Thai human isolates and clade I non-Thai animal isolates. These findings imply that clinical in vitro susceptibility testing should be performed, if feasible, to determine individual treatment options. The necessity of susceptibility testing is also supported by the findings of McMeekin et al., who studied the growth of a Thai human P. insidiosum isolate and found that it was stimulated by streptomycin, while the growth of other isolates was inhibited by the same concentration of streptomycin (17). This result raises the concern that some antibiotics might have unpredictable effects on the inhibition of P. insidiosum.
Limitations of this study include the limited number of isolates tested and the underrepresentation of all clades among all isolate sources. There were no Thai human or environmental clade I or clade III isolates in this study, although this likely represents an accurate sampling, as P. insidiosum clade I isolates have not been reported in Thai patients or the Thai environmental P. insidiosum clade III isolates are also not commonly found in the Thai environment (1, 2). Among non-Thai animal P. insidiosum isolates, clade II isolates were not examined in this study due to its commercial unavailability during the study period.
With those limitations, we are still hopeful that tetracyclines (doxycycline, minocycline, and tigecycline) and macrolides (azithromycin and clarithromycin) may offer new treatment options for human pythiosis in Thailand. Further clinical trials are needed to evaluate the clinical efficacy of these and other antibacterial agents.
MATERIALS AND METHODS
The study was approved by the Chulalongkorn University Institutional Review Board (IRB) (certificate of authenticity; COA no. 099/2019, IRB no. 760/61).
A total of 50 P. insidiosum isolates were tested, including Thai human (n = 27), Thai environmental (n = 12), and non-Thai animal (n = 11) P. insidiosum isolates. The Thai human isolates were derived from patients treated according to the KCMH research protocol (July 2002 to June 2019) (n = 22), and another 5 isolates were received from the Dutch Centraalbureau voor Schimmelcultures (CBS Bank) (Table 7).
TABLE 7.
Characteristics of the 50 isolates of Pythium insidiosum used in this study
| Isolate no. | ITS GenBank accession no. or CBS no.a | Isolate source | Country | Clade |
|---|---|---|---|---|
| 1. | KX389263 | Human, cerebral | Thailand | II |
| 2. | CBS 119454 | Human, cerebral | Thailand | IV |
| 3. | AY151173 | Human, vascular | Thailand | II |
| 4. | GU137329 | Human, vascular | Thailand | II |
| 5. | FJ917395 | Human, vascular | Thailand | II |
| 6. | JQ409330 | Human, vascular | Thailand | II |
| 7. | KX371893 | Human, vascular | Thailand | II |
| 8. | KX371894 | Human, vascular | Thailand | II |
| 9. | KX371895 | Human, vascular | Thailand | II |
| 10. | FJ917393 | Human, vascular | Thailand | II |
| 11. | GQ260121 | Human, vascular | Thailand | II |
| 12. | GQ260123 | Human, vascular | Thailand | II |
| 13. | CBS 119452 | Human, vascular | Thailand | II |
| 14. | CBS 119453 | Human, vascular | Thailand | II |
| 15. | GU812440 | Human, vascular | Thailand | IV |
| 16. | GQ260120 | Human, vascular | Thailand | IV |
| 17. | GQ260122 | Human, vascular | Thailand | IV |
| 18. | FJ917390 | Human, vascular | Thailand | IV |
| 19. | CBS 673.85 | Human, vascular | Thailand | IV |
| 20. | JQ409332 | Human, ocular | Thailand | II |
| 21. | GQ260119 | Human, ocular | Thailand | II |
| 22. | GQ260118 | Human, ocular | Thailand | II |
| 23. | CBS 119455 | Human, ocular | Thailand | II |
| 24. | FJ917389 | Human, ocular | Thailand | IV |
| 25. | GQ475491 | Human, ocular | Thailand | IV |
| 26. | GQ260125 | Human, ocular | Thailand | IV |
| 27. | GQ260124 | Human, ocular | Thailand | IV |
| 28. | EF016908 | Environment, rice irrigation in Chiang Rai | Thailand | II |
| 29. | EF016902 | Environment, reservoir in Chiang Rai | Thailand | II |
| 30. | EF016910 | Environment, reservoir in Chiang Rai | Thailand | II |
| 31. | EF016885 | Environment, reservoir in Lumpang | Thailand | II |
| 32. | EF016883 | Environment, reservoir in Lumphun | Thailand | IV |
| 33. | EF016870 | Environment, reservoir in Lumphun | Thailand | IV |
| 34. | EF016866 | Environment, irrigation channel in Lumphun | Thailand | IV |
| 35. | EF016878 | Environment, reservoir in Lumphun | Thailand | IV |
| 36. | EF016895 | Environment, reservoir in Lumpang | Thailand | IV |
| 37. | EF016879 | Environment, reservoir in Lumphun | Thailand | IV |
| 38. | FJ917392 | Environment, reservoir in Lopburi | Thailand | IV |
| 39. | EF016875 | Environment, reservoir in Lumphun | Thailand | IV |
| 40. | CBS 573.85 | Equine | Costa Rica, USA | I |
| 41. | CBS 574.85 | Equine | Costa Rica, USA | I |
| 42. | CBS 575.85 | Equine | Costa Rica, USA | I |
| 43. | CBS 576.85 | Equine | Costa Rica, USA | I |
| 44. | CBS 577.85 | Equine | Costa Rica, USA | I |
| 45. | CBS 578.85 | Equine | Costa Rica, USA | I |
| 46. | CBS 579.85 | Equine | Costa Rica, USA | I |
| 47. | CBS 580.85 | Equine | Costa Rica, USA | I |
| 48. | CBS 10155 | Equine | Brazil | I |
| 49. | CBS 702.83 | Equine | Japan | II |
| 50. | CBS 777.84 | Mosquito larva | India | II |
ITS, internal transcribed spacer; CBS, Centraalbureau voor Schimmelcultures.
Among the 27 Thai human P. insidiosum isolates, 2 were from cerebral lesions, 17 were from arterial clots, and 8 were from corneas. Among those 27 Thai human P. insidiosum isolates, 17 were clade II and 10 were clade IV. P. insidiosum environmental isolates were obtained from a water reservoir (n = 1) and from the Faculty of Medicine, Chiang Mai University (n = 11). Among the 12 Thai environmental P. insidiosum isolates, 4 were in clade II and 8 were in clade IV. Of the animal isolates from other countries, 8 were equine isolates from the United States, 1 was an equine isolate from Brazil, 1 was an equine isolate from Japan, and 1 was a mosquito larva isolate from India. Among the 11 non-Thai animal P. insidiosum isolates, both clades I (n = 9) and II (n = 2) were represented (Table 7). The genus and species of the Thai P. insidiosum isolates were confirmed using a PCR-based assay of the ITS region and/or COX2 gene, whereas the clades were classified according to DNA sequencing of the ITS region (18–20).
Broth microdilution was performed according to Clinical and Laboratory Standards Institute (CLSI) document M38-A2 guidelines for filamentous fungi (21), modified for P. insidiosum against eight classes of antibacterial antibiotics (Tables 1 to 3). All standard antimicrobial powders were commercially obtained from Sigma-Aldrich (St. Louis, MO, USA). Briefly, zoospores were obtained by zoosporogenesis induction (22). The inoculum (2 × 103 to 3 × 103 zoospores/ml) was counted by use of a Neubauer chamber and diluted in RPMI 1640 broth, pH 7.0 (with glucose and l-glutamine). The agents were prepared by 2-fold dilution in RPMI 1640 over a concentration range of 0.125 to 32 μg/ml. All assays were performed in triplicate. The MICs of each agent were determined by 100% inhibition of mycelium growth after 24 and 48 h of incubation at 37°C by visual observation (10, 11, 13, 14). The in vitro synergy of the tetracyclines and macrolides was determined according to the checkerboard technique (16).
ACKNOWLEDGMENTS
This study was funded by a Ratchadapiseksompotch Award from the Faculty of Medicine, Chulalongkorn University (grant number RA62/113).
The funder had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
We thank Nongnuch Vanittanakom, Professor of Microbiology, Faculty of Medicine, Chiang Mai University, for P. insidiosum isolates.
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