We found both resazurin and XTT assays to be suitable for high-throughputanalysis of drug susceptibility in Blastocystis isolates. TheHTS parameters (a Z' factor of >0.5 and a Cv of <10%)provide a highly conservative estimate of the sensitivity ofan assay (Li et al., 2009; Zhang et al., 1999). The high Z' factor value, low Cv, and reproducibilityof both resazurin and XTT assays suggest that they are robustand suitable for HTS. The option of semiquantitative visualevaluation of color gives
these assays the flexibility to beapplied in the field without the need for
sophisticated equipment.The suppression of metabolic activity observed in these redoxassays was also found to be associated with morphological signsof cell death (Wu et al., 2010), i.e., nuclear incorporation of PI and annexinV binding to the cell membrane, further validating these assaysin determining drug susceptibilities.
Considering the largenumber of variant Blastocystis isolates and the predominance of the parasite in developing countries (Tan et al., 2010) with limited research funding, these assays will be particularly useful due to theirlow cost and high yield.
In addition to the use of resazurin in assessment of Blastocystis susceptibility to antibiotics, in the next Chapter it will also be used to compare the susceptibility of Blastocystis isolates to potential host antimicrobial defense mechanisms.
Subtype-7 isolates were shown to be resistant to Mz and cross-resistantto Tz, the 1- position-substituted 5-NI of choice to treat awide variety of anaerobic organisms (Bassily S, 1987; Harder, 2002). This is consistentwith previous reports of cross- resistance between the two drugsin Trichomonas (Crowell et al., 2003; Lửfmark et al., 2010) and Giardia (Boreham, 1988). In these organisms,resistance is proposed to be due to downregulation of the enzymesPFOR (Upcroft and Upcroft, 2001) and thioredoxin oxidoreductase (Leitsch et al., 2010), which in conjunctionwith the electron acceptor ferredoxin are believed to activatethe 5-NI prodrugs to the toxic radical states inside the parasite (Leitsch et al., 2010; Upcroft and Upcroft, 2001).
However, this mechanism of activation has not beenshown for Blastocystis, although
et al., 2008). The subtype-4 isolatesshowed no convincing uniformity in
susceptibility to Mz andTz, indicating that new, unknown mechanisms of activation and/orresistance may be involved.
All isolates were similarly susceptible to another 1-position5-NI, Oz. Compared to Mz, the drug has significantly higherefficacy against Mzr isolates of Blastocystis (P <
0.01),as observed in other parasites (Chintana et al., 1986; Upcroft et al., 1999) and also reported forBlastocystis previously (Dunn and Boreham, 1991). However, its superior efficacyagainst Mzs isolates is not as obvious, again suggesting new, unknown mechanisms of activation and/or resistance to 1-position5-NIs in the parasite. Oz is frequently used to treat amoebiasisin India (Güven, 2003). Although the IC50s of Oz against all four isolatestested here (4.9 to 6.44 àM) were higher than the MICof the drug against Entamoeba (0.25 àM) (Chintana et al., 1986), its
effectivenessagainst both Mzr and Mzs isolates suggests the drug would bea useful alternative to Mz to treat Blastocystis infections.
Similarly to Oz, 2-position 5-NIs, the commercially availablepoultry drug Rz and the experimental drug C-17, were uniformlyeffective against the isolates of both
subtypes tested. These2-position 5-NIs exhibited significantly higher efficacy againstMzr isolates than 1-position 5-NIs (P < 0.01), as observedin Giardia and Trichomonas (Upcroft et al., 2006). Again, the improved efficacyof 2-position 5-NIs against Mzs subtype-4 isolates is not asobvious, suggesting a different mechanism of action in Blastocystisthan in other organisms (Upcroft et al., 2006). The IC90 of C-17 against Giardiawas recently reported to be 0.5 àM (Dunn et al., 2010), whereas
againstTrichomonas it exhibited a MIC of 6.3 àM (Upcroft et al., 2006). In thisstudy, the IC50 of C-17 against Blastocystis ranged from 0.89to 1.54 àM, suggesting the potential of the drug as abroad-spectrum antiprotozoal agent against Mzr parasites.
Two-position5-NIs may prove to be effective alternatives to treat Blastocystis infections in cases of Mz treatment failure.
The susceptibility of the Mzr subtype-7 isolates to NTZ andthe reduced susceptibility to the Mzs subtype-4 isolates arealso evidence for different
mechanisms of action of NTZ in Blastocystisthan in Giardia and Trichomonas, where cross-resistance betweenMz and NTZ is apparent (Adagu et al., 2002). These data suggest that Mz treatmentfailures in blastocystosis may well respond to NTZ, as in thecase of Cryptosporidium parvum infections. C. parvum infectionsdo not respond well to Mz (Gargala, 2008), and NTZ is the treatment ofchoice, with in-vitro IC50s of
<10 àg/ml (Theodos et al., 1998), similarto the IC50s of the drug against both Mzr and Mzs isolates ofBlastocystis in this study. Recent in-vitro (Vdovenko and
Williams, 2000) and clinicaldata (Stensvold et al., 2010) also suggest the usefulness of the drug in Blastocystisinfections.
Another alternative to treat Mzr Blastocystis isolates is FUR,which was equally effective against all isolates in this study.FUR is a nitrofuran commonly used to treat giardiasis (Quiros-Buelna, 1989).It is activated inside the cell by NADH oxidase and generatestoxic products that interfere with DNA processes in the parasite (Brown
The prophylactic antimalarial MQ and a closely related drug,QC, were also found to be more effective against Mzr subtype-7 isolates than Mzs subtype-4 isolates. These findings are surprisingbecause in Giardia, cross-resistance against QC has been observedbetween Mzr (Brasseur and Favennec, 1995) and Tzr (Upcroft et al., 1996) strains, suggesting a differentmode of action of the drug in Blastocystis. The exact mechanismsof action of these drugs against luminal parasites are not known, although they have been suggested to act on protozoan cell membranes (Upcroft J, 1996). The activity of QC against Blastocystis has been reportedpreviously (Dunn and Boreham, 1991; Vdovenko and Williams, 2000), but the current study is the first to reportthe potential usefulness of MQ as an anti-Blastocystis drug.
EM is an effective antiamoebic agent with unpleasant side effects.It targets
ribosomes and limits protein synthesis (Orozco, 2009). Thein-vitro activity of EM against Blastocystis has been evaluatedin two previous studies. While both studies suggested its effectivenessagainst Blastocystis, Zierdt et al. reported strain-to-strain variation in the susceptibility of the parasite to the drug (Zierdt et al., 1983). The multidrug resistance (MDR) phenotype of Entamoebahistolytica exhibits resistance to a wide range of drugs, includingEM, while responding to Mz (Orozco, 2009), but no such MDR phenotypeshave been reported in Blastocystis spp. Our study
describesthe existence of EM resistance in Mzs isolates of Blastocystis,suggesting that MDR phenotypes might be present in the parasite.Clinically, however, EM has limited use because of its severeside effects (Marino et al., 1990; Sugie et al., 1984).
TMP and SMZ are often prescribed in combination at a 1:5 ratioas an alternative to Mz in Blastocystis infections. Clinicalstudies suggest that this drug combination successfully eradicatesBlastocystis infections in 95% to 100% of cases (Stensvold et al., 2008; Stensvold et al., 2009b). Thereare no reports of the effectiveness of a 1:2 combination againstBlastocystis. Our findings suggest the superiority of a 1:2 combination over a 1:5 combination with no subtype-dependentvariation in
susceptibility. We suggest that the 1:2 combinationis likely to be more effective than the 1:5 combination in treatmentof clinical infections of Blastocystis.
Cysteine proteases play an important role in the cell cycleand pathophysiology of protozoan parasites. Blastocystis cysteineproteases have been reported to cleave human secretory IgAs (Tan et al., 2010) and to induce upregulation of
proinflammatory cytokines (Puthia et al., 2008a). A prosurvival role of legumain, a cysteine protease, hasalso been reported recently for Blastocystis (Wu et al., 2010).
Accumulatingdata in recent years suggest the therapeutic potential of protease inhibitors in parasitic infections (Abdulla et al., 2007; Olson et al., 1999). Several cysteineprotease inhibitors are being investigated as potential chemotherapeutic agents against parasites as diverse as Plasmodium (Olson et al., 1999; Parikh et al., 2005; Rosenthal et al., 1993),trypanosomes (Engel et al., 1999), and schistosomes (Wasilewski et al., 1996). In this study, wefound all four isolates to be highly susceptible to IA, a cysteineprotease inhibitor, irrespective of their susceptibility to Mz. These findings suggest a potential role of cysteine proteaseinhibitors as a
therapeutic option for Blastocystis isolatesresistant to conventional antiprotozoal agents.
PAR is a broad-spectrum aminoglycoside (Davidson et al., 2009). Although clinical studies suggest its effectiveness in the treatment of Blastocystisinfections (Armentia et al., 1993; Pasqui et al., 2004; Valsecchi et al., 2004), in-vitro data are equivocal (Vdovenko and Williams, 2000; Yakoob et al., 2004).In this study, PAR was found to be ineffective against the isolatesof both subtypes tested. The high clinical efficacy of the drugagainst Blastocystis could be due to its broad-spectrum antibioticactivity (Davidson et al., 2009). Although predominantly used for parasitic infections,PAR is also bactericidal (Donald et al., 2000). It might act by destruction ofthe gut bacterial flora essential for Blastocystis survival (Tan, 2008).
All four isolates tested were found to be nonsensitive to severalother broad- spectrum antibiotics, PAR, PYR, CQ, DOX, and AMP.This feature could be exploited for the isolation and axenizationof Blastocystis from clinical samples.
Clinical (Stensvold et al., 2009b) and animal infection (Hussein et al., 2008b) studies, as well asin-vitro data (Mirza and Tan, 2009), suggest a subtype-dependent
variation inthe pathobiology of Blastocystis. Although strain-to-strainvariation in parasite susceptibilities to drugs has been reportedpreviously, subtype-dependent variation in parasite responsesto chemotherapeutic agents has not been described before. Tothe best of our knowledge, this is the first study of its kindsuggesting a variation in parasite susceptibilities to six commonantiparasitic agents between
isolates of two subtypes knownto infect humans (Stensvold et al., 2009b). It will be interesting to conduct a moreextensive evaluation analyzing variability in the drug responsesof different isolates across all 11 subtypes of the parasite.
Although the vacuolar form is the most commonly reported formof the parasite, Blastocystis is also known to exist in amoeboid,granular, and cyst forms. Blastocystis cysts have been reportedto be Mzr, suggesting that different forms might respond differentlyto drug pressure (Zaman and Zaki, 1996). Since there are no
standardized methodsavailable for maintaining axenic cultures of other Blastocystis forms, only vacuolar forms were evaluated in this study, limitingthe application of our findings across different life cyclestages of the parasite. Despite this limitation, this is thefirst study suggesting subtype-dependent variation in the parasite
response to chemotherapeutic pressure.
In conclusion, Blastocystis spp. subtypes 4 and 7 exhibit extensive variation in morphology and protease activity. Considerable variations in cell size distribution were observed for Blastocystis sp. subtype-7. Cell size distribution and protease activity of different isolates vary over time and all isolates exhibit peaks in cysteine protease activity as well as cell size. The generation time of various isolates also differ significantly. There appears to be an association between cell size
distributions, protease activity and generation time of Blastocystis cultures.
Furthermore, this study describes two cost-effective assaysfor high-throughput
Blastocystis to six different antiprotozoal agents. We identified4 new potential therapeutic options against Blastocystis, namely,MQ, TMP-SMZ (1:2), Oz, and FUR.
Furthermore, we confirmed theantiprotozoal activities of 10 compounds already reported tobe effective against Blastocystis. We also demonstrated in-vitroMz and EM resistance in Blastocystis. By assessing the susceptibilityof the parasite to different 5-NIs, we also demonstrated that5-NI resistance could be overcome in Blastocystis with moreeffective 5-NI compounds. Based on our findings, there is clearlya need to reevaluate currently established treatment regimensfor
Blastocystis infections.Extensive intra- and inter-subtype variations in antibiotic susceptibility observed in this study appears to be related to variation in parasite biology. Later Chapters in this study will compare the pathological effects of these subtypes on colonic epithelial cells and investigate the role of cysteine proteases in mediating these effects.
CHAPTER 3: