Treatments for Metronidazole-Resistant Giardiasis A Review

Treatments for Metronidazole-Resistant Giardiasis A Review Nicole Wagner Abstract Giardiasis is a protozoal infection and a health issue in many parts of the world for both humans and animals. Giardia spp. Are responsible for diarrheal diseases, and current treatments are not consistently effective due to the development of drug resistance. The common drugs used to treat the parasite often have undesirable side effects. New drugs, drugs previously used for other conditions, and natural remedies are all being investigated for use in treating giardiasis and reducing Giardia numbers. The mode-of-action and potency of these alternative treatments give insight into new therapies, although more research is needed. Introduction Protozoal Giardia spp. Occur in two forms, a motile, flagellated trophozoite, and a resistant cyst. The motile trophozoite adheres to the intestinal wall of its host, while the cyst form is passed in the hosts feces then re-ingested in another host (Hendrix and Robinson 2012). It is now suspected that Giardia is species specific and the human forms are included in the Giardia lamblia assemblage and include Giardia intestinalis and Giardia duodenalis (Hendrix and Robinson 2012). Giardiasis is caused by a protozoal parasite that is responsible for health issues worldwide in humans and animals. In humans, the parasite is responsible for approximately 184 million cases of giardiasis each year (Hart et al. 2015). In animals, it has been found that in North America some herds of dairy calves have the parasite in 100% of the animals, and in Australia, the most common enteric parasite of dogs is a species of Giardia (Thompson. 2000). Signs and symptoms of giardiasis include chronic and acute diarrhea and vomiting (Hart et al. 2015). These significant intestinal diseases can decrease appetite, cause malabsorption, malnutrition and even death (El-Taweed 2015). And because this parasite is found in domesticated animals, there is a significant zoonotic transmission potential (Thompson. 2000). A calf can shed 105 to 106 cysts per gram of feces, and re-infection can occur with ingesting as few as 10 cysts. As the Giardia cysts survive well in water there is a potential f or contamination of untreated water supply as well (Thompson 2000 and Watkins and Eckmann 2014). There is no vaccine available for Giardia, so the treatment of the disease involves drug therapy, like the antibiotic metronidazole, a 5-nitroimadazole class drug (Watkins and Eckmann 2014 and Hart et al. 2017). Other drugs of this class are also used to treat giardiasis with varying degrees of success. However, like many antibiotics some drug resistance has been found (Watkins and Eckmann 2014). Several studies demonstrate Giardia spp. resistance to the 5-nitroimadazole class of drugs, including one that recorded 22% of patients in a clinic in Spain receiving treatment for giardiasis did not respond to metronidazole (El-Taweed 2015). Perhaps contributing to the problem of emerging drug resistance, are the undesirable side effects of common antiprotozoal drugs. Some of these side effects include intestinal discomfort, flatulence, nausea, vomiting, and the metallic taste of the drugs (Sahib et al. 2014). It is human nature to stop using a drug with these side effects as soon as it seems to be working, and this leads to the potential for an increase in 5-nitroimidazole drug resistance over time. Also, the use of the 5-nitromadazole class drugs at lower doses for treating gingivitis and pre-surgical colorectal cases, may allow for the growth of drug-resistant forms of Giardia (El-Taweed et al. 2015). Research into how Giardia become resistant to metronidazole and the active components of other successful drugs is important to the success of treating giardiasis in humans and animals. The investigations into different drugs and herbal remedies involve identifying the mode-of-action, how the active component is effectiv e against this protozoan in both trophozoites and cyst form (Galeh et al. 2016). As an alternative to metronidazole, older antibiotic drugs, new antibiotic drugs and plant extracts and essential oils are being considered by many researchers. Newer drugs are being considered to overcome Giardia resistance like auranofin, a drug currently prescribed for rheumatoid arthritis (Watkins and Eckmann 2014.). Researchers are also considering plants and plant extracts for a new anti-giardia agent. These extracts have been used in traditional medicine to treat diarrhea in places in South America, India, Iran, and Turkey where Giardia is endemic (Watkins and Eckmann 2014). Research has been done investigating compounds from garlic (Mikaili et al. 2014), ginger and cinnamon (Mahmoud et al. 2014), pomegranate (Al-Megrin 2017), and native plants from Brazil (Martins et al. 2015), the Yucatan (Sergio et al. 2005) and their effectiveness against Giardia. Identifying the compounds in the plants that are most effective against the protozoa will provide another option for treatment of giardiasis. Drug Therapies As previously mentioned, the number of cases of Metronidazole-resistant giardiasis is growing. Other 5-nitroimadazole and nitro-class drugs have been tested against Giardia like nitazoxanide and furazolidone (Watkins and Eckmann 2014). These drugs work by reducing the nitrogen group on the drug compounds to activate toxic free radicals. The advantages of the new drugs are shorter length of treatment and reduced cellular damage to the liver and kidneys (Jarrad et al 2016). Side effects are seen in the use of these drugs, as in the case of furazolidone, where some hemolysis was observed in some patients (Watkins and Eckmann 2014) and in 5-nitroimadazole drugs side effects like nausea, vomiting and headaches are reported (Jarrad et al. 2016). When developing new drug treatments, researchers investigate how the parasite is developing resistance to current treatments. In the case of 5-nitroimodazole drugs, the potency of a substance against Giardia species is due to the activation of the drug by an enzyme pyruvate ferredoxin oxidoreductase (PFOR). The drugs nitro group is reduced by the protozoans PFOR enzyme, producing toxic free radicals which bond to target molecules in the microbe, inactivating them and killing the parasite (Watkins and Eckmann 2014). The resistant protozoans have down-regulated the PFOR enzyme (Jarrad et al. 2016 and Galeh et al. 2016). To treat giardiasis, research has focused on this nitro group and the formation of toxic free radicals. Other drugs are being developed that incorporate a benzene ring, instead of (or in addition to) the nitro groups. These drugs act on the Giardia cellular vesicles, causing swelling, and are more effective than nitazoxanide and metronidazole (Watkins and Eckmann 2014) . Not all research has resulted in support for the development of new drugs in the 5-nitromiadazole class. In a study conducted in Iran, researchers tested the theory in human clinical studies, utilizing PCR to identify genetic mutations that appeared in metronidazole-resistant Giardia lamblia. In the metronidazole-resistant G. lamblia, the protozoa did not show down regulation of PFOR and it is suspected that other predisposing factors were responsible for the drugs ineffectiveness(Galeh et al. 2016). Further research is being done to test the new drugs like nitroimidazole and furazolidone for cytotoxicity towards human liver, kidney, and intestinal cells (Watkins and Eckmann 2014). These side effects can be serious in immune-compromised subjects and children. Older antibiotic drugs like albendazole and mebendazole, benzimidazole class drugs, have been shown to be effective against Giardia as well. These drugs are currently used to treat roundworm infections. The benzimidazole class drugs act by binding to tubulin and interfering with the protozoan cytoskeleton (Watkins and Eckmann, 2014). Research into the effectiveness of metronidazole and albendazole in mice showed that some Giardia isolates developed resistance to one or the other drug and in some cases both drugs (Lemà ©e et al. 2000). In the case of albendazole, some Giardia duodenalis resistance developed in a mouse model (Lemà ©e et al. 2000). In another study in Bolivia when albendazole was used to reduce the hookworm infection, the number of Giardia infections increased showing some (Watkins and Eckmann 2014). Drug research of existing drug libraries has also revealed some drugs that previously were not used as antimicrobials are effective against Giardia. A drug used for rheumatoid arthritis, auranofin, inhibits the growth of metronidazole-resistant Giardia. It works by inhibiting the thioredoxin-glutathione reductase enzyme (Watkins and Eckmann 2014). A drug used for obesity treatment, orlistat, which is poorly absorbed, is active in the intestine. Orlistat works by inhibiting lipases in Giardia isolates, preventing lipid metabolism (Watkins and Eckmann 2014). Ginger and Cinnamon Current research into metronidazole-resistant Giardia infections has gone beyond investigating new drugs or existing drug libraries. Diarrheal treatments around the world have shown promise in providing avenues for new giardiasis therapies and treatments. Some of this research involves the investigation into anti-nausea spices, ginger and cinnamon. Research has been conducted using ginger extracts in both in vivo and in vitro studies. In an in vitro study (Abdel-Hafeez et al. 2016), a comparison was made between gingers and nitazoxanides effectiveness against Giardia lamblia trophozoites. Ginger was chosen to contrast with the drug because of its historical use as an anti-nausea and anti-diarrheal treatment. Ginger extract is an antioxidant with alkaloids, saponins, tannins, and flavonoids. It is suspected that either the antioxidant or flavonoids have a negative effect on the trophozoites (Mahmoud et al. 2014). A study investigated curcumin, an antioxidant, also found in Zingiber officinale and other pungent spices (Perrez-Arriaga et al. 2006) showed that in the presence of curcumin at concentrations similar to metronidazole, significant swelling was seen in the Giardia trophozoites, indicating possible cell membrane interference (Perrez-Arriaga et al. 2006). In the research conducted by Abdel-Hafeez et al. (2016), cultured fecal samples treated with ginger extract at 20mg/mL resulted in similar reduction of the number of viable trophozoites in the culture as nitazoxanide. The findings were based on counts of viable trophozoites (pear-shaped, mobile, and non-refractory quality) under a light microscope. Verification of findings may use in vivo studies with mice or rats, and improved counting and staining techniques are indicated for future research. Meanwhile, ginger continues to be used for a variety of intestinal infections in Thailand, India, and Egypt (Abdel-Hafeez et al. 2016). In the in vivo study by Mahmoud, et al (2014)., cinnamon was also evaluated against Giardia trophozoites and cysts. As stated previously, the cyst is found in fecal samples as a transmissible form. The researchers This study involved using Giardia lamblia cyst infected rats and measured doses of ginger at 10 and 20 mg/kg/day and cinnamon at 10 and 20 mg/kg/day. As Perrez-Arriaga et al. (2006) previously researched, active compounds in ginger may affect the protozoans cell membrane. In another study, Proanthocyanidins were identified as an active compound in cinnamon (Williams et al. 2015). Proanthocyanidins, also found in berries, disrupt protozoans adhesive ability which is necessary for the trophozoite to maintain its position in the intestine (Anthony et al. 2007). In the Mahmoud study (2014) the rats were euthanized and their intestines washed for a measurement of trophozoites and to determine intestinal damage (Anthony et al. 2007). Their stools were also collected for three day s prior to euthanasia to perform a count of cysts excreted (Mahmoud et al. 2014). According to the results of this study, cinnamon given to the rats at the 20 mg/kg dose resulted in a 100% reduction in the number of cysts found in their fecals, and a 34% reduction in the number of trophozoites. When rats were given a dose of 20mg/kg of ginger the number of cysts found in their stool was reduced by 90.1% and the number of trophozoites in the intestinal wash was reduced by 75.45%. So, the cinnamon was more active against cysts and the ginger was more active against the trophozoites in the rats (Mahmoud et al. 2014). The study also utilized electron microscopy to count and identify Giardia cysts and trophozoites. By utilizing this technology, and the researchers were able to show that not only was the infection reduced with cinnamon, but the intestinal mucosa was healthier in the samples taken as compared to the ginger-dosed rats (Mahmoud et al. 2014). This study, however did not utili ze a positive control group to measure the difference (if any) between using ginger and cinnamon extracts compared with using metronidazole or other drug treatment for giardiasis. Additional research may determine whether the intestinal mucosa would improve, with the drug treatment as occurred in the study with cinnamon and ginger (Mahmoud et al. 2014). Garlic and Shallot One of the most promising and researched herbal extract is allicin, a component of plants in the garlic and onion family. Garlic and shallot plants have been used in traditional medicine in various parts of the world for hundreds of years (Mikaili et al. 2013). Sulfur-based components like allicin (diallyl dithiosulfinate), diallyl disulfide, and S-allylcystein of these plants are of interest to research for use in pharmacological studies (Mikaili et al. 2013). In an in vitro study investigating whole garlic extract (Harris et al. 2000), garlic was shown to be effective against Giardia intestinalis at a concentration of 0.3mg/mL. The researchers went on to examine how the compounds in garlic were acting on the Giardia and whether allicin was the compound of interest. Allicin has a very short half-life in vivo, but the thiosulfates that result from its breakdown are bioavailable longer to act against Giardia trophozoites (Harris et al. 2000). In this study the researchers investigated the anti-parasitic activity of these compounds, and they showed that diallyl disulphide was particularly effective in reducing the number of trophozoites. Another component, allyl alcohol, damaged the trophozoites by causing cellular swelling and immobility (Harris et al. 2000). In another study focusing on diallyl trisulfide (DAT) from garlic, a concentration of 300 ug/mL was effective against Giardia lamblia (Lun et al. 1994). The researchers used an in vitro test to determine the IC50 for DAT, and the result was 8.5-14 ug/mL, using the same tests that determined the IC50 of metronidazole. The use of DAT in China for treatment of other parasitic infections is not uncommon (Lun et al. 1994), but it will be necessary to determine how DAT is effective against the parasite in vivo in future research. One avenue that research may pursue is in studying how DAT affects tubulin (Hosono et al. 2005), which would indicate that this substance has a similar effect as the benzimidazole drugs like albendazole. Further research into obtaining effective concentrations of these components of garlic and onions is necessary. Herbal Extracts Other herbal extracts have been investigated for their biologically active components and effectiveness as a treatment for giardiasis. Three of these show promise in recent studies. Peppermint (Mentha x piperta L.) is used as an herbal remedy for stomach discomfort (Vidal et al. 2007). Dill is also used in some areas of the world to treat children with diarrhea (Sahib et al. 2014). Pomegranate peel contains some substances that prove active against Giardia (Al-Megrin 2016). Mentha x piperta (peppermint) is known to have a relaxation effect on gastrointestinal smooth muscle, and this is suspected to be the result of the menthol affecting calcium channels (Kiefer et al. 2008). In one study methanolic extract from Mentha x piperta was tested against Giardia lamblia (Vidal et al. 2007). This study measures IC50, which is the calculation of the amount of a substance necessary to inhibit or kill one-half of the microbes. The IC50 after 48 hours of exposure was 0.8 ug/mL, similar to the IC50 of metronidazole and furazalidone after 24 hours (Vidal et al. 2007). The study tested different concentrations of the dichloromethane (DCM) from Mentha x piperta and found that a dose of 100 ug/mL after 48 hours almost eliminated the presence of trophozoites in the culture media (Vidal et al. 2007). The study also examined how DCM was altering the morphology of trophozoites utilizing electron microscopy, and found changes to plasma membranes (Vidal et al. 2007). This sugg ests the need for further research into why the protozoal membranes were altered, what biologically active components of peppermint were involved, and whether this alteration would also affect intestinal cells in mammals (measuring potential toxicity). In a study that researched the extracts from the Dill plant (Anethum graveolens), researchers conducted a clinical trial with children Giardia trophozoites and cysts, or if the dill acted on the muscle cells in the intestine, reducing smooth muscle contraction. Further research needs to be conducted into these issues. Pomegranate peel extract was also tested for anti-Giardia activity in another study (Al-Megrin 2016). Researchers studied mice that were infected with the Giardia cysts, and given an extract of pomegranate peel daily. Then the number of cysts produced in the mouse stool and the antigen presence for Giardia were counted and measured respectively. There was a reduction in the number of cysts counted in the groups of mice that had received the pomegranate, however there was also a natural reduction in the number of cysts in the studys control group (Al-Megrin 2016). The researchers also reported that the rate of detection of the Giardia antigen in the groups that were treated was significantly (P Native Plants Ethnopharmacology is becoming one method for identifying plant-based sources for new pharmacological treatments. Ethnopharmacology studies involve interviewing people who are native to specific areas to identify historical and current plant species used to treat disease. In the case of Giardia treatment, researchers identified diarrhea as the key symptom in 90% of human cases (Neiva et al. 2014). Although people reporting to clinics or hospitals with diarrhea did not necessarily identify causative agents, in some cases they were self-treating with plants from the area or from their own gardens (Neiva, et al. 2014). The identification of plants used by people suffering from diarrhea allowed researchers to focus on specific plant species in anti-giardiasis studies. In one study by Neiva et al. (2014) the researchers focused on an area around Sao Luis, Brazil. From interviewing and collecting plant samples that people were using to treat diarrhea and dysentery symptoms, the researchers identified five potential plants to test for anti-Giardia effectiveness. The researchers also identified the part of the plants and the preparation of the plants used for treatment of diarrhea by these patients of a healthcare facility and private specialty institution (Neiva et al. 2014). The five species of plants selected to investigate were Anacardium occidentale L., Chenopodium ambrosioides L., Passiflora edulis Sims., Psiddiumguajava L., and Stachytarpheta cayennesis (Rich) Vahl. (Neiva et al. 2014). It was found that all the plants had some giardicidal activity, but Passiflora was most effective at IC50 In another similar ethnopharmacology study, researchers investigated plants native to the Yucatan peninsula in Mexico that were used to treat diarrhea (Paraza-Sanchez et al. 2005). This study was conducted in vitro using 10 methanol extracts from native plants. In this study Tridax procumens as a whole plant was used (air-dried and powdered into methanol, then evaporated, and added to DMSO) (Paraza-Sanchez et al. 2005). The researchers identified from other studies that the plant contains hydrocarbons, fatty acids, flavonoids, bis-bithiophene. Flavonoids may contain polyphenols and inhibit Giardia from adhering to the intestinal wall (Anthony et al. 2007) In other plants tested, C. dentata, D. cahagenesis, and B. cressifolia have not had their chemical components analyzed, although they all had giardicidal activity (Paraza-Sanchez et al. 2005). It is unknown at this time why these native plants are effective against Giardia spp. (Paraza-Sanchez et al. 2005). Research has been conducted on the Rubus liebmanii medicinal plant native to Mexico. The antiprotozoal active compounds were identified as epicatechin and catechin (polyphenols), Nigaichigoside F1, beta-sitosterol, squalene, and 3,4 hydroxybenzoic acid (Jimà ©nez-Arellanes et al. 2012). In this study, extracts from the plant were fractionated to identify specific compounds, an extract of R. liebomanii was tested against G. lamblia with a negative control and a metronidazole infused positive control, and the research continued using guinea pigs and mice as test subjects. The results indicated that the Nigaichigoside F1 had an IC50 of 2.17 ug/mL as compared with metronidazole which had an IC50 of 0.5 ug/mL in cultures of G. lamblia (Jimà ©nez-Arellanes et al. 2012). When the potential toxicity of the plant was tested in male rats, the pure extract of R. liebmanii was not toxic, even at 1000mg/kg dosages (Jimà ©nez-Arellanes et al. 2012). Further research into these plants may involv e identifying how the active components are working against the protozoans and identify more effective treatments, which will need to be tested in vivo. Solanum lycoparum is also a native plant of Brazil. In research published in 2015 (Gilmarcio et al. 2015), this native species was investigated for its anti-Giardia potential. The fruit of this plant is used in traditional medicine (Gilmarcio et al. 2015). Two glycoalkaloids, solamargin (Sg) and solasonine (Sn) have been identified as having potential as anti-Giardia treatments (Gilmarcio et al. 2015). Both compounds were effective against Giardia lamblia, with Sg having an IC50 of 120.3 ug/mL and Sn having an IC50 of 103.7 ug/mL. However, when both compounds were mixed as they would be in the fruit of S. lycoparum, their IC50 was 13.23 ug/mL, much lower (Gilmarcio et al. 2015). This demonstrates a synergistic effect of the compounds. The researchers also used an index of selectivity calculation to measure effectiveness of the compound as compared to toxicity against macrophages, and the combined glycoalkaloids had a relatively high index (Gilmarcio et al. 2015). This index was neces sary because glycoalkaloids can be toxic. In vitro studies of the combined Sn+Sg treatment would provide additional research opportunities and determine the toxic effects of the glycoalkaloid while treating Giardia lamblia. Glycoalkaloids affect permeability of mammalian intestinal cells (Gee et al. 1996), however the glycoalkaloids found and studied from the S. lycoparum are not the most toxic. It is likely; however, no research could be found in the current literature searches, that the effect of glycoalkaloids on the Giardia trophozoites cell membrane did cause cellular swelling and changes to the vesicles and flagella. Essential Oils Clove oil, an essential oil from Syzgium aromaticum, has been used to treat digestive disorders and diarrhea (Machado et al. 2011). Research into essential oils has shown they are effective against many bacterial and fungal infections, but little research has been done to discover how the oil works (Machado et al. 2011). Eugenol is a major of several essential oils including S. aromaticum, and in this research the eugenol comprised 85% of the essential oil tested (Machado et al. 2011).In a study testing the effect of clove oil and eugenol on Giardia lamblia it was shown that S. aromaticum had an IC50 value of 134 ug/mL and eugenol had an IC50 value of 101 ug/mL (Machado et al. 2011). The study also utilized scanning and transmission electron microscopy to measure morphological changes in the Giardia that were incubated with the S. aromaticum essential oil. It was observed that the adherence of the Giardia was inhibited in the presence of eugenol (Machado et al. 2011). Giardia normall y attach to the intestinal wall to maintain position, obtain nutrients, and reproduce. The essential oil contained eugenol, and although it affected adherence, it did not cause the Giardia trophozoites to lyse, so the results in this study indicated that other components of the S. aromaticum oil were responsible for the cell death (Machado et al. 2011). Other essential oils are used to treat digestive issues. One, from Ocimum basilicum of the basil family, was studied for its anti-Giardia effects (de Almeida et al. 2007). In this study the researchers again isolated components of the essential oil and tested for antigiardial activity (de Almeida et al. 2007). This study also investigated eugenol as one of those components, and found the eugenol was effective against the Giardia trophozoites (de Almeida et al. 2007). The study included testing the linalool, which makes up 69.33% of the essential oil in addition to eugenol. Linalool was shown to be even more effective at reducing the number of viable Giardia lamblia trophozoites (de Almeida et al. 2007). To verify potential toxicity of the essential oil and its components, the researchers tested mouse macrophages in the same concentrations of Ocimum basilicum, eugenol and linalool and found that there was little to no effect on the cells (de Almeida et al. 2007). The researchers also identified an inhibitory effect of the oil and it components to a group of cysteine peptidases enzymes that are commonly found in these protozoans (de Almeida et al. 2007). The inhibition of cysteine peptidases has been shown to have a lethal effect on trophozoites of another protozoal species, Entamoeba histolytica (Ankri et al. 1997). Ozone In an interesting study (Boland-Nazar et al. 2016), olive oil injected with ozone was tested as a treatment for giardiasis. In an in vitro study, the tubes of Giardia were combined with different concentrations of ozonated olive oil, and this proved effective against Giardia cysts after 100 hours of incubation (Boland-Nazar et al. 2016). It is suspected by the researchers that the extra oxygen atom acts a free radical, like the immunological response of macrophages and neutrophils (Boland-Nazar et al. 2016). Olive oil is effective at stabilizing a delivery system for ozone without losing its durability (Boland-Nazar et al. 2016), and the higher the concentration of ozone in the oil, the more effective the treatment is against Giardia cysts. The researchers suggest conducting an in vivo study with this substance to verify their hypothesis (Boland-Nazar et al. 2016). Discussion Research into treating giardiasis will continue as standard treatments like metronidazole go up against greater drug resistance. The mode of action of different therapies has been investigated and includes inhibiting PFOR, cysteine proteinases, inhibiting adherence (Anthony et al. 2007), tubulin and cytoskeleton interference, and cell membrane interference. Table 1 shows some of the different treatments dosage requirements and active compounds. The 5nitroimadazole drugs are the most commonly prescribed treatments for giardiasis world-wide. New drugs are being developed from within this class, altering the nitro-group or adding benzene rings to the molecules to improve effectiveness considering growing metronidazole resistance (Watkins and Eckmann 2014). Other drugs that have been prescribed for anti-parasitic treatment or other purposes entirely are also being investigated. This includes anthelminthic drugs as well as drugs that were initially developed to treat obesity and rheumatoid arthritis (Watkins and Eckmann 2014). Finally, herbal, spice, and plant-based extracts are being investigated for their antiprotozoal activity. Although Table1 does not show an IC50 for each of the compounds discussed in this paper, it does highlight the active components. Table 1: Effectiveness of different substances in inhibiting Giardia IC50 Active compound Reference Metronidazole 0.8 ug/mL 5-nitroimadazole Jarrad et al. 2016, Watkins and Eckmann, 2014, Vidal et al 2007 Albendazole 52.4 ug/mL Benzimidazole Jarrad et al. 2016, Watkins and Eckmann, 2014, Lemee et al. 2000 Furazolidone 0.65 ug/mL 5-nitrofurans Jarrad et al. 2016, Watkins and Eckmann, 2014, Vidal et al 2007 Garlic 14 ug/mL Diallyl trisulfide Lun